Treatment of glaucoma with Rho guanine nucleotide exchange factor 12 (ARHGEF12) inhibitors

Abstract

The present disclosure provides methods for treating a subject suffering from glaucoma or elevated intraocular pressure (IOP), methods for identifying a subject at high risk for developing glaucoma or elevated IOP, methods for detecting human Rho guanine nucleotide exchange factor 12 (ARHGEF12) variant nucleic acid molecules and variant polypeptides, and ARHGEF12 variant nucleic acid molecules and variant polypeptides.

Description

[Technical field]

[0001] Reference to sequence listing This application contains a Sequence Listing that has been submitted electronically as an XML file entitled 381203616SEQ, created on Sep. 23, 2022, and is 2,000 kilobytes in size. This Sequence Listing is incorporated herein by reference.

[0002] The present disclosure relates generally to the treatment of subjects with glaucoma with Rho guanine nucleotide exchange factor 12 (ARHGEF12) inhibitors, methods for identifying subjects at increased risk for developing glaucoma, methods for detecting ARHGEF12 variant nucleic acid molecules and variant polypeptides, and ARHGEF12 variant nucleic acid molecules and ARHGEF12 variant polypeptides. [Background technology]

[0003] Glaucoma is a group of disorders that damage the optic nerve of the eye and can result in partial vision loss and blindness. There are several types of glaucoma, and in the primary type, open-angle glaucoma, fluid accumulates in the eye, increasing pressure inside the eye to a level that can damage the optic nerve. In low- or normal-pressure glaucoma, optic nerve damage and lateral visual field constriction occur in people with normal eye pressure. In angle-closure glaucoma, fluid in the front of the eye cannot drain properly, which can cause a sudden increase in eye pressure. In congenital glaucoma, children are born with an eye defect that delays the normal drainage of fluid. Glaucoma treatments include medications, laser trabeculoplasty, and conventional surgery. These treatments can preserve residual vision, but do not improve vision already lost to glaucoma.

[0004] Rho guanine nucleotide exchange factor 12 (ARHGEF12) is a member of the Rho GTPase family of signaling proteins that play fundamental roles in numerous cellular processes initiated by extracellular stimuli acting through G protein-coupled receptors. ARHGEF12 is transduced by heteromeric G proteins (Gα 12 / 13 and Gα qARHGEF12 is activated by ARHGEF11 (through interaction with ARHGEF12), which then binds to RhoA and promotes the GDP / GTP exchange reaction. This interaction activates the ROCK pathway, leading to cytoskeletal reorganization. ARHGEF12 can form homodimers or heterodimers with ARHGEF11, which can regulate the activity of ARHGEF12. Summary of the Invention

[0005] The disclosure provides a method of treating a subject having glaucoma, the method comprising administering to the subject an ARHGEF12 inhibitor. The disclosure also provides a method of treating a subject having primary open angle glaucoma (POAG), the method comprising administering to the subject an ARHGEF12 inhibitor.

[0006] The disclosure also provides a method of treating a subject having elevated intraocular pressure (IOP), the method comprising administering to the subject an ARHGEF12 inhibitor. The disclosure also provides a method of treating a subject with a therapeutic agent that treats or inhibits glaucoma or elevated IOP, the subject suffering from glaucoma or elevated IOP, the method comprising obtaining or obtaining a biological sample from the subject, determining whether the subject has a genotype that includes a predicted loss-of-function variant nucleic acid molecule of ARHGEF12 or a predicted gain-of-function variant nucleic acid molecule of ARHGEF12, by determining whether the subject has a genotype that includes a predicted loss-of-function variant nucleic acid molecule of ARHGEF12 or a predicted gain-of-function variant nucleic acid molecule of ARHGEF12, and if the subject is of the ARHGEF12 reference type, administering or continuing to administer a standard dose of a therapeutic agent that treats or inhibits glaucoma or elevated IOP to the subject, administering an ARHGEF12 inhibitor to the subject, and determining whether the subject is genotyped for the predicted loss-of-function variant of ARHGEF12. the presence of a genotype having a predicted loss-of-function variant nucleic acid molecule of ARHGEF12 encoding a human ARHGEF12 polypeptide indicates that the subject has a low risk of developing glaucoma or elevated IOP, and the presence of a genotype having a predicted gain-of-function variant nucleic acid molecule of ARHGEF12 encoding a human ARHGEF12 polypeptide indicates that the subject has a high risk of developing glaucoma or elevated IOP.

[0007] The disclosure also provides a method of identifying a subject at increased risk of developing glaucoma or elevated IOP, the method comprising determining or having determined the presence or absence of a predicted gain-of-function variant nucleic acid molecule of ARHGEF12 encoding a human ARHGEF12 polypeptide or a predicted loss-of-function variant nucleic acid molecule of ARHGEF12 encoding a human ARHGEF12 polypeptide in a biological sample obtained from the subject, where if the subject is an ARHGEF12 reference type, the subject has an increased risk of developing glaucoma, if the subject is heterozygous or homozygous for the predicted loss-of-function variant nucleic acid molecule of ARHGEF12, the subject has a reduced risk of developing glaucoma or elevated IOP, and if the subject is heterozygous or homozygous for the predicted gain-of-function variant nucleic acid molecule of ARHGEF12, the subject has an increased risk of developing glaucoma or elevated IOP.

[0008] The disclosure also provides a method for detecting a human ARHGEF12 variant nucleic acid molecule in a subject, comprising assaying a sample obtained from the subject to determine whether a nucleic acid molecule in the sample is a genomic nucleic acid molecule comprising a nucleotide sequence comprising a thymine at a position corresponding to position 141,048 of SEQ ID NO:4 or its complement, or an adenine at a position corresponding to position 73,039 of SEQ ID NO:5 or its complement, a thymine at a position corresponding to position 4,297 of SEQ ID NO:31 or its complement, a thymine at a position corresponding to position 4,297 of SEQ ID NO:32 or its complement, a thymine at a position corresponding to position 4,297 of SEQ ID NO:33 or its complement, a thymine at a position corresponding to position 4,297 of SEQ ID NO:34 or its complement, a thymine at a position corresponding to position 4,297 of SEQ ID NO:35 or its complement, a thymine at a position corresponding to position 5,120 of SEQ ID NO:36 or its complement, a thymine at a position corresponding to position 5,120 of SEQ ID NO:37 or its complement, or a thymine at a position corresponding to position 5,120 of SEQ ID NO:38 or its complement. and determining whether an mRNA molecule has a nucleotide sequence that comprises uracil at a position corresponding to position 3,163 of SEQ ID NO:38 or its complement, or a cDNA molecule generated from the mRNA molecule, where the cDNA molecule has a nucleotide sequence that comprises a thymine at a position corresponding to position 4,297 of SEQ ID NO:62 or its complement, position 3,379 of SEQ ID NO:63 or its complement, position 3,627 of SEQ ID NO:64 or its complement, position 4,240 of SEQ ID NO:65 or its complement, position 3,594 of SEQ ID NO:66 or its complement, position 3,473 of SEQ ID NO:67 or its complement, position 3,602 of SEQ ID NO:68 or its complement, or position 3,163 of SEQ ID NO:69 or its complement.

[0009] The present disclosure also provides a method for detecting the presence of a human ARHGEF12 Glu1156STOP variant polypeptide, the method comprising performing an assay on a sample taken from a subject to determine whether ARHGEF12 protein in the sample terminates at a position corresponding to position 1,155 set forth in SEQ ID NO:81.

[0010] The disclosure also provides an isolated variant-specific probe or variant-specific primer comprising at least about 15 nucleotides, the variant-specific probe or variant-specific primer comprising a nucleotide sequence complementary to a portion of a nucleotide sequence encoding a human ARHGEF12 polypeptide, the portion being at position 141,048 of SEQ ID NO:4 or its complement, position 4,297 of SEQ ID NO:31 or its complement, position 3,739 of SEQ ID NO:32 or its complement, position 3,627 of SEQ ID NO:33 or its complement, position 4,240 of SEQ ID NO:34 or its complement, position 3,594 of SEQ ID NO:35 or its complement, position 3,627 of SEQ ID NO:36 or its complement, position 3,636 of SEQ ID NO:37 or its complement, position 3,640 of SEQ ID NO:38 or its complement, position 3,771 of SEQ ID NO:39 or its complement, position 3,811 of SEQ ID NO:40 or its complement, position 3,821 of SEQ ID NO:41 or its complement, position 3,831 of SEQ ID NO:42 or its complement, position 3,821 of SEQ ID NO:43 or its complement, position 3,831 of SEQ ID NO:44 or its complement, position 3,821 of SEQ ID NO:45 or its complement, position 3,831 of SEQ ID NO:46 or its complement, position 3,831 of SEQ ID NO:47 or its complement, position 3,831 of SEQ ID NO:48 or its complement, position 3,831 of SEQ ID NO:49 or its complement, position 3,921 of SEQ ID NO:50 or its complement, position 3,921 of SEQ ID NO:51 or its complement, position 3,921 of SEQ ID NO:52 or its complement, position 3,921 of or a position corresponding to position 3,473 of SEQ ID NO:37 or its complement, position 3,602 of SEQ ID NO:37 or its complement, position 3,163 of SEQ ID NO:38 or its complement, position 4,297 of SEQ ID NO:62 or its complement, position 3,379 of SEQ ID NO:63 or its complement, position 3,627 of SEQ ID NO:64 or its complement, position 4,240 of SEQ ID NO:65 or its complement, position 3,594 of SEQ ID NO:66 or its complement, position 3,473 of SEQ ID NO:67 or its complement, position 3,602 of SEQ ID NO:68 or its complement, position 3,163 of SEQ ID NO:69 or its complement, or position 73,039 of SEQ ID NO:5 or its complement.

[0011] The present disclosure also provides a molecular complex comprising a variant-specific primer or variant-specific probe hybridized to a genomic nucleic acid molecule comprising a nucleotide sequence encoding a human ARHGEF12 polypeptide, where the variant-specific primer or variant-specific probe hybridizes to a thymine at a position corresponding to position 141,048 of SEQ ID NO:4 or its complement, or an adenine at a position corresponding to position 73,039 of SEQ ID NO:5 or its complement.

[0012] The present disclosure also provides a molecular complex comprising a variant-specific primer or variant-specific probe hybridized to an mRNA molecule comprising a nucleotide sequence encoding a human ARHGEF12 polypeptide, wherein the variant-specific primer or variant-specific probe hybridizes to a uracil at a position corresponding to position 4,297 of SEQ ID NO:31 or its complement, position 3,739 of SEQ ID NO:32 or its complement, position 3,627 of SEQ ID NO:33 or its complement, position 4,240 of SEQ ID NO:34 or its complement, position 3,594 of SEQ ID NO:35 or its complement, position 3,473 of SEQ ID NO:36 or its complement, position 3,602 of SEQ ID NO:37 or its complement, or position 3,163 of SEQ ID NO:38 or its complement.

[0013] The disclosure also provides a molecular complex comprising a variant-specific primer or variant-specific probe hybridized to a cDNA molecule comprising a nucleotide sequence encoding a human ARHGEF12 polypeptide, wherein the variant-specific primer or variant-specific probe hybridizes to a thymine at a position corresponding to position 4,297 of SEQ ID NO:62 or its complement, position 3,379 of SEQ ID NO:63 or its complement, position 3,627 of SEQ ID NO:64 or its complement, position 4,240 of SEQ ID NO:65 or its complement, position 3,594 of SEQ ID NO:66 or its complement, position 3,473 of SEQ ID NO:67 or its complement, position 3,602 of SEQ ID NO:68 or its complement, or position 3,163 of SEQ ID NO:69 or its complement.

[0014] The present disclosure also provides an isolated nucleic acid molecule comprising a nucleotide sequence encoding a human ARHGEF12 polypeptide or a complement thereof, wherein the polypeptide terminates at a position corresponding to position 1,155 of SEQ ID NO:81, position 1,136 of SEQ ID NO:82, or position 1,052 of SEQ ID NO:83.

[0015] The present disclosure also provides an isolated genomic nucleic acid molecule comprising a nucleotide sequence encoding a human ARHGEF12 polypeptide, the nucleotide sequence comprising a thymine at a position corresponding to position 141,048 of SEQ ID NO:4, or its complement, or an adenine at a position corresponding to position 73,039 of SEQ ID NO:5, or its complement.

[0016] The present disclosure also provides an isolated mRNA molecule comprising a nucleotide sequence encoding a human ARHGEF12 polypeptide, wherein the nucleotide sequence comprises a uracil at a position corresponding to position 4,297 of SEQ ID NO:31 or its complement, position 3,739 of SEQ ID NO:32 or its complement, position 3,627 of SEQ ID NO:33 or its complement, position 4,240 of SEQ ID NO:34 or its complement, position 3,594 of SEQ ID NO:35 or its complement, position 3,473 of SEQ ID NO:36 or its complement, position 3,602 of SEQ ID NO:37 or its complement, or position 3,163 of SEQ ID NO:38 or its complement.

[0017] The present disclosure also provides an isolated cDNA molecule comprising a nucleotide sequence encoding a human ARHGEF12 polypeptide, wherein the nucleotide sequence comprises a thymine at a position corresponding to position 4,297 of SEQ ID NO:62 or its complement, position 3,379 of SEQ ID NO:63 or its complement, position 3,627 of SEQ ID NO:64 or its complement, position 4,240 of SEQ ID NO:65 or its complement, position 3,594 of SEQ ID NO:66 or its complement, position 3,473 of SEQ ID NO:67 or its complement, position 3,602 of SEQ ID NO:68 or its complement, or position 3,163 of SEQ ID NO:69 or its complement.

[0018] The present disclosure also provides an isolated human ARHGEF12 polypeptide, having an amino acid sequence that is at least about 90% identical to SEQ ID NO:81 (wherein the polypeptide ends at a position corresponding to position 1,155 of SEQ ID NO:81), at least about 90% identical to SEQ ID NO:82 (wherein the polypeptide ends at a position corresponding to position 1,136 of SEQ ID NO:82), or at least about 90% identical to SEQ ID NO:83 (wherein the polypeptide ends at a position corresponding to position 1,052 of SEQ ID NO:83).

[0019] The disclosure also provides: i) a genomic nucleic acid molecule having a nucleotide sequence encoding a human ARHGEF12 polypeptide, the nucleotide sequence comprising a thymine at a position corresponding to position 132,939 of SEQ ID NO:2 or its complement, a guanine at a position corresponding to position 143,698 of SEQ ID NO:3 or its complement, a thymine at a position corresponding to position 141,048 of SEQ ID NO:4 or its complement, an adenine at a position corresponding to position 73,039 of SEQ ID NO:5 or its complement, a cytosine at a position corresponding to position 121,307 of SEQ ID NO:6 or its complement, or a cytosine at position 141,978 of SEQ ID NO:7 or its complement; ii) an mRNA molecule having a nucleotide sequence encoding a human ARHGEF12 polypeptide, the nucleotide sequence comprising: a uracil at position 3,749 of SEQ ID NO:16 or its complement, a uracil at position 3,191 of SEQ ID NO:17 or its complement, a uracil at position 3,079 of SEQ ID NO:18 or its complement, a uracil at position 3,692 of SEQ ID NO:19 or its complement, a uracil at the corresponding position, a uracil at the position corresponding to position 3,046 of SEQ ID NO:20 or its complement, a uracil at the position corresponding to position 2,925 of SEQ ID NO:21 or its complement, a uracil at the position corresponding to position 3,054 of SEQ ID NO:22 or its complement, a uracil at the position corresponding to position 2,615 of SEQ ID NO:23 or its complement, a guanine at the position corresponding to position 4,748 of SEQ ID NO:24 or its complement, a guanine at the position corresponding to position 4,190 of SEQ ID NO:25 or its complement, a guanine at the position corresponding to position 4,07 of SEQ ID NO:26 or its complement, a guanine at position corresponding to position 8 of SEQ ID NO:27 or its complement; a guanine at position corresponding to position 4,691 of SEQ ID NO:27 or its complement; a guanine at position corresponding to position 4,045 of SEQ ID NO:28 or its complement; a guanine at position corresponding to position 3,924 of SEQ ID NO:29 or its complement; a guanine at position corresponding to position 3,614 of SEQ ID NO:30 or its complement; a uracil at position corresponding to position 4,297 of SEQ ID NO:31 or its complement; a uracil at position corresponding to position 3,739 of SEQ ID NO:32 or its complement;or iii) a nucleotide sequence encoding a human ARHGEF12 polypeptide. a thymine at a position corresponding to position 3,749 of SEQ ID NO:47 or its complement; a thymine at a position corresponding to position 3,191 of SEQ ID NO:48 or its complement; a thymine at a position corresponding to position 3,079 of SEQ ID NO:49 or its complement; a thymine at a position corresponding to position 3,692 of SEQ ID NO:50 or its complement; a thymine at a position corresponding to position 3,046 of SEQ ID NO:51 or its complement; a thymine at a position corresponding to position 2,925 of SEQ ID NO:52 or its complement; a thymine at a position corresponding to position 3,749 of SEQ ID NO:47 or its complement; a thymine at a position corresponding to position 3,191 of SEQ ID NO:48 or its complement; a thymine at a position corresponding to position 3,079 of SEQ ID NO:49 or its complement; a thymine at a position corresponding to position 3,054 of SEQ ID NO:54 or its complement, a thymine at a position corresponding to position 2,615 of SEQ ID NO:54 or its complement, a guanine at a position corresponding to position 4,748 of SEQ ID NO:55 or its complement, a guanine at a position corresponding to position 4,190 of SEQ ID NO:56 or its complement, a guanine at a position corresponding to position 4,078 of SEQ ID NO:57 or its complement, a guanine at a position corresponding to position 4,691 of SEQ ID NO:58 or its complement, a guanine at a position corresponding to position 4,045 of SEQ ID NO:59 or its complement, SEQ ID NO:60 or its complement a guanine at position 3,924 of SEQ ID NO:61 or its complement, a guanine at position 3,614 of SEQ ID NO:62 or its complement, a thymine at position 4,297 of SEQ ID NO:62 or its complement, a thymine at position 3,379 of SEQ ID NO:63 or its complement, a thymine at position 3,627 of SEQ ID NO:64 or its complement, a thymine at position 4,240 of SEQ ID NO:65 or its complement, a thymine at position 3,594 of SEQ ID NO:66 or its complement, a thymine at position 3,626 of SEQ ID NO:67 or its complement,The present invention provides a therapeutic agent for treating or inhibiting glaucoma or elevated IOP for use in treating glaucoma or elevated IOP in a subject having a cDNA molecule comprising a thymine at a position corresponding to position 473, a thymine at a position corresponding to position 3,602 of SEQ ID NO:68 or its complement, or a thymine at a position corresponding to position 3,163 of SEQ ID NO:69 or its complement.

[0020] The disclosure also provides: i) a genomic nucleic acid molecule having a nucleotide sequence encoding a human ARHGEF12 polypeptide, the nucleotide sequence comprising a thymine at a position corresponding to position 132,939 of SEQ ID NO:2 or its complement, a guanine at a position corresponding to position 143,698 of SEQ ID NO:3 or its complement, a thymine at a position corresponding to position 141,048 of SEQ ID NO:4 or its complement, an adenine at a position corresponding to position 73,039 of SEQ ID NO:5 or its complement, a cytosine at a position corresponding to position 121,307 of SEQ ID NO:6 or its complement, or a cytosine at position 141,978 of SEQ ID NO:7 or its complement; ii) an mRNA molecule having a nucleotide sequence encoding a human ARHGEF12 polypeptide, the nucleotide sequence comprising: a uracil at position 3,749 of SEQ ID NO:16 or its complement, a uracil at position 3,191 of SEQ ID NO:17 or its complement, a uracil at position 3,079 of SEQ ID NO:18 or its complement, a uracil at position 3,692 of SEQ ID NO:19 or its complement, a uracil at the corresponding position, a uracil at the position corresponding to position 3,046 of SEQ ID NO:20 or its complement, a uracil at the position corresponding to position 2,925 of SEQ ID NO:21 or its complement, a uracil at the position corresponding to position 3,054 of SEQ ID NO:22 or its complement, a uracil at the position corresponding to position 2,615 of SEQ ID NO:23 or its complement, a guanine at the position corresponding to position 4,748 of SEQ ID NO:24 or its complement, a guanine at the position corresponding to position 4,190 of SEQ ID NO:25 or its complement, a guanine at the position corresponding to position 4,07 of SEQ ID NO:26 or its complement, a guanine at position corresponding to position 8 of SEQ ID NO:27 or its complement; a guanine at position corresponding to position 4,691 of SEQ ID NO:27 or its complement; a guanine at position corresponding to position 4,045 of SEQ ID NO:28 or its complement; a guanine at position corresponding to position 3,924 of SEQ ID NO:29 or its complement; a guanine at position corresponding to position 3,614 of SEQ ID NO:30 or its complement; a uracil at position corresponding to position 4,297 of SEQ ID NO:31 or its complement; a uracil at position corresponding to position 3,739 of SEQ ID NO:32 or its complement;or iii) a nucleotide sequence encoding a human ARHGEF12 polypeptide. a thymine at a position corresponding to position 3,749 of SEQ ID NO:47 or its complement; a thymine at a position corresponding to position 3,191 of SEQ ID NO:48 or its complement; a thymine at a position corresponding to position 3,079 of SEQ ID NO:49 or its complement; a thymine at a position corresponding to position 3,692 of SEQ ID NO:50 or its complement; a thymine at a position corresponding to position 3,046 of SEQ ID NO:51 or its complement; a thymine at a position corresponding to position 2,925 of SEQ ID NO:52 or its complement; a thymine at a position corresponding to position 3,749 of SEQ ID NO:47 or its complement; a thymine at a position corresponding to position 3,191 of SEQ ID NO:48 or its complement; a thymine at a position corresponding to position 3,079 of SEQ ID NO:49 or its complement; a thymine at a position corresponding to position 3,054 of SEQ ID NO:54 or its complement, a thymine at a position corresponding to position 2,615 of SEQ ID NO:54 or its complement, a guanine at a position corresponding to position 4,748 of SEQ ID NO:55 or its complement, a guanine at a position corresponding to position 4,190 of SEQ ID NO:56 or its complement, a guanine at a position corresponding to position 4,078 of SEQ ID NO:57 or its complement, a guanine at a position corresponding to position 4,691 of SEQ ID NO:58 or its complement, a guanine at a position corresponding to position 4,045 of SEQ ID NO:59 or its complement, SEQ ID NO:60 or its complement a guanine at position 3,924 of SEQ ID NO:61 or its complement, a guanine at position 3,614 of SEQ ID NO:62 or its complement, a thymine at position 4,297 of SEQ ID NO:62 or its complement, a thymine at position 3,379 of SEQ ID NO:63 or its complement, a thymine at position 3,627 of SEQ ID NO:64 or its complement, a thymine at position 4,240 of SEQ ID NO:65 or its complement, a thymine at position 3,594 of SEQ ID NO:66 or its complement, a thymine at position 3,626 of SEQ ID NO:67 or its complement,The present invention provides an ARHGEF12 inhibitor for use in treating glaucoma or elevated IOP in a human subject having a cDNA molecule comprising a thymine at a position corresponding to position 473, a thymine at a position corresponding to position 3,602 of SEQ ID NO:68 or its complement, or a thymine at a position corresponding to position 3,163 of SEQ ID NO:69 or its complement.

[0021] The accompanying drawings, which are incorporated in and constitute a part of this specification, illustrate several features of the present disclosure. The patent or application file contains at least one drawing executed in color. Copies of this patent or patent application publication with color drawing(s) will be provided by the Office upon request and payment of the necessary fee. [Brief description of the drawings]

[0022] [Figure 1A] A missense variant (Tyr973Phe) in ARHGEF12 associated with increased IOP is shown. [Figure 1B] A missense variant (Tyr973Phe) in ARHGEF12 is associated with an increased risk of glaucoma. [Diagram 2] A set of predicted loss-of-function variants in ARHGEF12 associated with lower IOP is shown. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0023] Various terms relating to aspects of the present disclosure are used throughout the specification and claims. Unless otherwise indicated, such terms are to be given their ordinary meaning in the art. Other terms that are specifically defined are to be interpreted in a manner consistent with the definitions set forth herein.

[0024] Unless expressly stated otherwise, any method or embodiment set forth herein is in no way intended to be construed as requiring that its steps be performed in a particular order. Thus, unless specifically stated by a method claim in the claims or description that the steps should be limited to a particular order, no order is intended to be imposed in any respect. This also applies to any possible implicit criteria of interpretation, including logical matters regarding the arrangement of steps or work flow, general meanings derived from grammatical construction or punctuation, or the number or type of embodiments described herein.

[0025] As used herein, the singular forms "a," "an," and "the" include plural referents unless the context clearly indicates otherwise. As used herein, the term "about" means that a cited numerical value is approximate and that small variations do not significantly affect the practice of the disclosed embodiments. When a numerical value is used, unless otherwise indicated by context, the term "about" means that the numerical value can vary by ±10% and remain within the range of the disclosed embodiments.

[0026] As used herein, the term "comprising" may, in certain embodiments, be replaced with "consisting of" or "consisting essentially of," as appropriate.

[0027] As used herein, the term "isolated" refers to a nucleic acid molecule or polypeptide, meaning that the nucleic acid molecule or polypeptide is in a state other than its natural environment, e.g., apart from blood and / or animal tissue. In some embodiments, an isolated nucleic acid molecule or polypeptide is substantially free of other nucleic acid molecules or other polypeptides, particularly other nucleic acid molecules or polypeptides of animal origin. In some embodiments, the nucleic acid molecule or polypeptide may be in a highly purified form, i.e., more than 95% pure or more than 99% pure. When used in this context, the term "isolated" does not exclude the presence of the same nucleic acid molecule or polypeptide in alternative physical forms, such as dimers or alternatively phosphorylated or derivatized forms.

[0028] As used herein, the terms "nucleic acid," "nucleic acid molecule," "nucleic acid sequence," "polynucleotide," or "oligonucleotide" can include a polymeric form of nucleotides of any length, can include DNA and / or RNA, and can be single-stranded, double-stranded, or multistranded. A strand of a nucleic acid also refers to its complementary strand.

[0029] As used herein, the term "subject" includes any animal, including mammals. Mammals include, but are not limited to, farm animals (e.g., horses, cows, pigs), pet animals (e.g., dogs, cats), laboratory animals (e.g., mice, rats, rabbits), and non-human primates. In some embodiments, the subject is a human. In some embodiments, the subject is a patient under the care of a physician.

[0030] According to the present disclosure, the rare variant of ARHGEF12 gene is identified, which is associated with the increased risk of developing glaucoma and elevated IOP in subjects.For example, it has been observed that the genetic mutation that changes the adenine nucleotide at position 132,939 of human ARHGEF12 reference genomic nucleic acid molecule (see SEQ ID NO: 1) to thymine can show that the risk of developing glaucoma and elevated IOP in people with such mutation.

[0031] In accordance with the present disclosure, another rare variant in the ARHGEF12 gene has been identified that is associated with a reduced risk of developing glaucoma and elevated IOP in a subject. For example, genetic modifications that change the adenine nucleotide at position 143,698 of the human ARHGEF12 reference genomic nucleic acid molecule (see SEQ ID NO:1) to guanine, or the guanine nucleotide at position 141,048 of the human ARHGEF12 reference genomic nucleic acid molecule (see SEQ ID NO:1) to thymine, or the guanine nucleotide at position 73,039 of the human ARHGEF12 reference genomic nucleic acid molecule (see SEQ ID NO:1) to adenine, or the adenine nucleotide at position 121,307 of the human ARHGEF12 reference genomic nucleic acid molecule (see SEQ ID NO:1) to cytosine, or the guanine nucleotide at position 141,978 of the human ARHGEF12 reference genomic nucleic acid molecule (see SEQ ID NO:1) to cytosine have been observed, indicating that humans carrying such modifications may be at reduced risk of developing glaucoma and elevated IOP.

[0032] ARHGEF12 predicted loss-of-function variants are not believed to be known to be associated with lowering IOP. Overall, the genetic analysis described herein surprisingly shows that the ARHGEF12 gene, and in particular variants of the ARHGEF12 gene, are associated with increased or decreased risk of developing glaucoma and elevated IOP. Thus, subjects with ARHGEF12 reference types at high risk of developing glaucoma and elevated IOP may be treated to prevent, alleviate symptoms of, and / or inhibit the onset of symptoms of glaucoma and / or elevated IOP. Thus, the present disclosure provides a method of identifying such variants in subjects to identify or stratify the risk of developing glaucoma and elevated IOP in such subjects, or to diagnose subjects as at high risk of developing glaucoma and elevated IOP, so that subjects at risk or with active disease may be treated accordingly. The present disclosure also provides isolated ARHGEF12 variant genomic nucleic acid molecules, variant mRNA molecules, and variant cDNA molecules.

[0033] For the purposes of this disclosure, any particular human can be classified as having one of five ARHGEF12 genotypes: i) ARHGEF12 reference type, ii) heterozygous for predicted loss-of-function variants of ARHGEF12, iii) homozygous for predicted loss-of-function variants of ARHGEF12, iv) heterozygous for predicted gain-of-function variants of ARHGEF12, or v) homozygous for predicted gain-of-function variants of ARHGEF12. If the human does not have a copy of a predicted loss-of-function variant nucleic acid molecule of ARHGEF12 or a predicted gain-of-function variant molecule of ARHGEF12, the human is an ARHGEF12 reference type. If the human has a single copy of a predicted loss-of-function variant nucleic acid molecule of ARHGEF12, the human is a heterozygous for predicted loss-of-function variant of ARHGEF12. If a person has a single copy of a predicted gain-of-function variant nucleic acid molecule of ARHGEF12, the person is heterozygous for a predicted gain-of-function variant of ARHGEF12. As used herein, a predicted loss-of-function variant nucleic acid molecule of ARHGEF12 is any ARHGEF12 nucleic acid molecule (such as a genomic nucleic acid molecule, an mRNA molecule, or a cDNA molecule) that encodes an ARHGEF12 polypeptide with partial loss-of-function, complete loss-of-function, predicted partial loss-of-function, or predicted complete loss-of-function. As used herein, a predicted gain-of-function variant nucleic acid molecule of ARHGEF12 is any ARHGEF12 nucleic acid molecule (such as a genomic nucleic acid molecule, an mRNA molecule, or a cDNA molecule) that encodes an ARHGEF12 polypeptide with enhanced activity, be it constitutive activity, increased ability to cause disease, decreased ability to resist disease, resistance to inhibitors, or resistance to therapeutic intervention. A human having an ARHGEF12 polypeptide with a partial loss of function (or a predicted partial loss of function) is ARHGEF12 hypomorphic. If a human has two copies of an ARHGEF12 predicted loss-of-function variant nucleic acid molecule, the human is homozygous for a predicted loss-of-function variant in ARHGEF12.If a human has two copies of the ARHGEF12 predicted gain-of-function variant nucleic acid molecule, then the human is homozygous for a predicted gain-of-function variant in ARHGEF12.

[0034] For subjects who are genotyped or determined to be ARHGEF12 reference type, such subjects are at high risk of developing glaucoma and / or elevated IOP. Such subjects can be administered or continue to be administered a standard dose of a therapeutic agent to treat or inhibit glaucoma or elevated IOP, and / or can be administered an ARHGEF12 inhibitor.

[0035] For subjects who are genotyped or determined to be heterozygous for a predicted gain-of-function variant in ARHGEF12, such subjects are at increased risk of developing glaucoma and / or elevated IOP. Such subjects can be administered or continue to be administered a standard or higher dose of a therapeutic agent to treat or inhibit glaucoma or elevated IOP, and / or can be administered an ARHGEF12 inhibitor.

[0036] For subjects who are genotyped or determined to be heterozygous for a predicted loss-of-function variant in ARHGEF12, such subjects are at low risk of developing glaucoma and / or elevated IOP. Such subjects can be administered or continue to be administered a standard or lower dose of a therapeutic agent to treat or inhibit glaucoma or elevated IOP, and / or can be administered an ARHGEF12 inhibitor.

[0037] In any of the embodiments described herein, the predicted loss-of-function variant nucleic acid molecule of ARHGEF12 is any ARHGEF12 nucleic acid molecule (e.g., a genomic nucleic acid molecule, an mRNA molecule, or a cDNA molecule, etc.) that encodes an ARHGEF12 polypeptide with partial loss-of-function, complete loss-of-function, predicted partial loss-of-function, or predicted complete loss-of-function. In some embodiments, the predicted loss-of-function variant nucleic acid molecule of ARHGEF12 is a nucleic acid molecule that encodes ARHGEF12 Tyr1306Cys, Tyr1287Cys, Tyr1203Cys, Glu1156STOP, Glu1137STOP, or Glu1053STOP. In some embodiments, the predicted loss-of-function variant nucleic acid molecule of ARHGEF12 is a nucleic acid molecule that encodes ARHGEF12 Tyr1306Cys. In some embodiments, the predicted loss-of-function variant nucleic acid molecule of ARHGEF12 is a nucleic acid molecule that encodes ARHGEF12 Tyr1287Cys. In some embodiments, the predicted loss-of-function variant nucleic acid molecule of ARHGEF12 is a nucleic acid molecule encoding ARHGEF12 Tyr1203Cys. In some embodiments, the predicted loss-of-function variant nucleic acid molecule of ARHGEF12 is a nucleic acid molecule encoding ARHGEF12 Glu1156STOP. In some embodiments, the predicted loss-of-function variant nucleic acid molecule of ARHGEF12 is a nucleic acid molecule encoding ARHGEF12 Glu1137STOP. In some embodiments, the predicted loss-of-function variant nucleic acid molecule of ARHGEF12 is a nucleic acid molecule encoding ARHGEF12 Glu1053STOP.

[0038] In any of the embodiments described herein, the predicted gain-of-function variant nucleic acid molecule of ARHGEF12 can be any ARHGEF12 nucleic acid molecule (e.g., a genomic nucleic acid molecule, an mRNA molecule, or a cDNA molecule, etc.) that encodes an ARHGEF12 polypeptide having a gain of function. In some embodiments, the predicted gain-of-function variant nucleic acid molecule of ARHGEF12 is a nucleic acid molecule encoding ARHGEF12 Tyr973Phe, Tyr954Phe, or Tyr870Phe. In some embodiments, the predicted gain-of-function variant nucleic acid molecule of ARHGEF12 is a nucleic acid molecule encoding ARHGEF12 Tyr973Phe. In some embodiments, the predicted gain-of-function variant nucleic acid molecule of ARHGEF12 is a nucleic acid molecule encoding ARHGEF12 Tyr954Phe. In some embodiments, the predicted gain-of-function variant nucleic acid molecule of ARHGEF12 is a nucleic acid molecule encoding ARHGEF12 Tyr870Phe.

[0039] In any of the embodiments described herein, the predicted loss-of-function polypeptide of ARHGEF12 can be any ARHGEF12 polypeptide having partial loss-of-function, complete loss-of-function, predicted partial loss-of-function, or predicted complete loss-of-function. In some embodiments, the predicted loss-of-function polypeptide of ARHGEF12 is ARHGEF12 Tyr1306Cys, Tyr1287Cys, Tyr1203Cys, Glu1156STOP, Glu1137STOP, or Glu1053STOP. In some embodiments, the predicted loss-of-function polypeptide of ARHGEF12 is ARHGEF12 Tyr1306Cys. In some embodiments, the predicted loss-of-function polypeptide of ARHGEF12 is ARHGEF12 Tyr1287Cys. In some embodiments, the predicted loss-of-function polypeptide of ARHGEF12 is ARHGEF12 Tyr1203Cys. In some embodiments, the predicted loss-of-function polypeptide of ARHGEF12 is ARHGEF12 Glu1156STOP. In some embodiments, the predicted loss-of-function polypeptide of ARHGEF12 is ARHGEF12 Glu1137STOP. In some embodiments, the predicted loss-of-function polypeptide of ARHGEF12 is ARHGEF12 Glu1053STOP.

[0040] In any of the embodiments described herein, the predicted gain-of-function polypeptide of ARHGEF12 can be any ARHGEF12 polypeptide having a gain of function. In some embodiments, the predicted gain-of-function polypeptide of ARHGEF12 is ARHGEF12 Tyr973Phe, Tyr954Phe, or Tyr870Phe. In some embodiments, the predicted gain-of-function polypeptide of ARHGEF12 is ARHGEF12 Tyr973Phe. In some embodiments, the predicted gain-of-function polypeptide of ARHGEF12 is ARHGEF12 Tyr954Phe. In some embodiments, the predicted gain-of-function polypeptide of ARHGEF12 is ARHGEF12 Tyr870Phe.

[0041] In any of the embodiments described herein, the subject may have glaucoma. In any of the embodiments described herein, the glaucoma may be primary open-angle glaucoma (POAG). In any of the embodiments described herein, the subject may have elevated IOP.

[0042] Symptoms of glaucoma include, but are not limited to, severe headaches, eye pain, nausea and vomiting, blurred vision, halos around lights, and bloodshot eyes. The disclosure provides a method of treating a subject having glaucoma, the method comprising administering to the subject an ARHGEF12 inhibitor.

[0043] The disclosure also provides a method of treating a subject having primary open angle glaucoma (POAG), the method comprising administering to the subject an ARHGEF12 inhibitor. The disclosure also provides a method of treating a subject having elevated IOP, the method comprising administering to the subject an ARHGEF12 inhibitor.

[0044] In some embodiments, the ARHGEF12 inhibitor comprises an inhibitory nucleic acid molecule. In some embodiments, the inhibitory nucleic acid molecule comprises an antisense molecule. In some embodiments, the inhibitory nucleic acid molecule comprises a small interfering RNA (siRNA) molecule. In some embodiments, the inhibitory nucleic acid molecule comprises a short hairpin RNA (shRNA) molecule. Such inhibitory nucleic acid molecules can be designed to target any region of ARHGEF12 mRNA. In some embodiments, the inhibitory nucleic acid molecule hybridizes with a sequence in an ARHGEF12 genomic nucleic acid molecule or mRNA molecule and reduces the expression of an ARHGEF12 polypeptide in a cell of a subject. In some embodiments, the ARHGEF12 inhibitor comprises an antisense RNA that hybridizes with an ARHGEF12 genomic nucleic acid molecule or mRNA molecule and reduces the expression of an ARHGEF12 polypeptide in a cell of a subject. In some embodiments, the ARHGEF12 inhibitor comprises an siRNA that hybridizes with an ARHGEF12 genomic nucleic acid molecule or mRNA molecule and reduces the expression of an ARHGEF12 polypeptide in a cell of a subject. In some embodiments, an ARHGEF12 inhibitor comprises an shRNA that hybridizes to a genomic nucleic acid molecule or an mRNA molecule of ARHGEF12 and reduces expression of an ARHGEF12 polypeptide in cells of a subject.

[0045] In some embodiments, the ARHGEF12 inhibitor comprises a nuclease agent that induces one or more nicks or double-strand breaks in the recognition sequence(s) in the ARHGEF12 genomic nucleic acid molecule or a DNA binding protein that binds to the recognition sequence. The recognition sequence can be located in the coding region of the ARHGEF12 gene or in a regulatory region that affects the expression of the gene. The recognition sequence of the DNA binding protein or nuclease agent can be located in an intron, exon, promoter, enhancer, regulatory region, or any non-protein coding region. The recognition sequence can include or be adjacent to the start codon of the ARHGEF12 gene. For example, the recognition sequence can be located about 10, about 20, about 30, about 40, about 50, about 100, about 200, about 300, about 400, about 500, or about 1,000 nucleotides from the start codon. As another example, two or more nuclease agents can be used, each of which targets a nuclease recognition sequence that includes or is adjacent to the start codon. As another example, two nuclease agents can be used, one targeting a nuclease recognition sequence containing or adjacent to a start codon, and the other targeting a nuclease recognition sequence containing or adjacent to a stop codon, and the coding region between the two nuclease recognition sequences can be deleted by cleavage with these nuclease agents.Any nuclease agent that induces a nick or double-strand break at the desired recognition sequence can be used in the methods and compositions disclosed herein.Any DNA binding protein that binds to the desired recognition sequence can be used in the methods and compositions disclosed herein.

[0046] Suitable nuclease agents and DNA binding proteins for use herein include, but are not limited to, zinc finger proteins or zinc finger nuclease (ZFN) pairs, transcription activator-like effector (TALE) proteins or transcription activator-like effector nucleases (TALEN), or clustered regularly interspaced short palindromic repeats (CRISPR) / CRISPR-associated (Cas) systems. The length of the recognition sequence can vary, but includes, for example, recognition sequences that are about 30-36 bp for zinc finger proteins or ZFN pairs, about 15-18 bp for each ZFN, about 36 bp for TALE proteins or TALEN, and about 20 bp for CRISPR / Cas guide RNA.

[0047] In some embodiments, the CRISPR / Cas system can be used to modify the ARHGEF12 genomic nucleic acid molecule in a cell. The methods and compositions disclosed herein can employ the CRISPR-Cas system by utilizing a CRISPR complex (comprising a guide RNA (gRNA) complexed with a Cas protein) for site-specific cleavage of the ARHGEF12 nucleic acid molecule.

[0048] Cas proteins generally contain at least one RNA recognition domain or RNA binding domain that can interact with gRNA. Cas proteins may also contain nuclease domains (such as DNase or RNase domains), DNA binding domains, helicase domains, protein-protein interaction domains, dimerization domains, and other domains. Suitable Cas proteins include, for example, wild-type Cas9 proteins and wild-type Cpf1 proteins (such as FnCpf1). Cas proteins can have full cleavage activity to create double-stranded breaks in ARHGEF12 genomic nucleic acid molecules, or can be nickases to create single-stranded breaks in ARHGEF12 genomic nucleic acid molecules. Additional examples of Cas proteins include Cas1, Cas1B, Cas2, Cas3, Cas4, Cas5, Cas5e (CasD), Cas6, Cas6e, Cas6f, Cas7, Cas8a1, Cas8a2, Cas8b, Cas8c, Cas9 (Csn1 or Csx12), Cas10, Cas10d, CasF, CasG, CasH, Csy1, Csy2, Csy3, Cse1 (CasA), Cse2 (CasB), Cse3 (CasE), Cse4 (CasF), Cse5 (CasF), Cse6 (CasF), Cse7 (CasF), Cse8 (CasF), Cse9 (CasF), Cse10 (CasF), Cse11 (CasF), Cse12 (CasF), Cse13 (CasF), Cse14 (CasF), Cse15 (CasF), Cse16 (CasF), Cse17 (CasF), Cse18 (CasF), Cse19 (CasF), Cse20 (CasF), Cse21 (CasF), Cse22 (CasF), Cse23 (CasF), Cse24 (CasF), Cse25 (CasF), Cse26 (CasF), Cse27 (CasF), Cse28 (CasF), Cse29 ...9 (CasF), Cse21 (CasF), Cse22 (CasF), Cse23 (CasF), Cse24 (CasF), Cse25 (CasF), Cse26 (CasF), Cse27 (Ca Cas proteins include, but are not limited to, 4 (CasC), Csc1, Csc2, Csa5, Csn2, Csm2, Csm3, Csm4, Csm5, Csm6, Cmr1, Cmr3, Cmr4, Cmr5, Cmr6, Csb1, Csb2, Csb3, Csx17, Csx14, Csx10, Csx16, CsaX, Csx3, Csx1, Csx15, Csf1, Csf2, Csf3, Csf4, and Cu1966, and their homologs or modified versions. Cas proteins can also be operably linked to heterologous polypeptides as fusion proteins. For example, Cas proteins can be fused to a cleavage domain, an epigenetic modification domain, a transcription activation domain, or a transcription repressor domain. Cas proteins can be provided in any form. For example, the Cas protein can be provided in the form of a protein, e.g., a Cas protein complexed with a gRNA.Alternatively, the Cas protein can be provided in the form of a nucleic acid molecule, e.g., RNA or DNA, encoding the Cas protein.

[0049] In some embodiments, targeted genetic modification of an ARHGEF12 genomic nucleic acid molecule can be generated by contacting a cell with a Cas protein and one or more gRNAs that hybridize with one or more gRNA recognition sequences within a target genomic locus in an ARHGEF12 genomic nucleic acid molecule. For example, the gRNA recognition sequence can be located within a region of SEQ ID NO: 1. The gRNA recognition sequence can also include or be adjacent to a position corresponding to position 132,939, 143,698, 141,048, 73,039, 121,307, or 141,978 of SEQ ID NO: 1. For example, the gRNA recognition sequence can be positioned about 1000, about 500, about 400, about 300, about 200, about 100, about 50, about 45, about 40, about 35, about 30, about 25, about 20, about 15, about 10, or about 5 nucleotides away from a position corresponding to positions 132,939, 143,698, 141,048, 73,039, 121,307, or 141,978 of SEQ ID NO: 1. The gRNA recognition sequence can include or be adjacent to the start codon of an ARHGEF12 genomic nucleic acid molecule or the stop codon of an ARHGEF12 genomic nucleic acid molecule. For example, the gRNA recognition sequence can be positioned about 10, about 20, about 30, about 40, about 50, about 100, about 200, about 300, about 400, about 500, or about 1,000 nucleotides away from the start or stop codon.

[0050] The gRNA recognition sequence in the target genomic locus in the ARHGEF12 genomic nucleic acid molecule is located near a protospacer adjacent motif (PAM) sequence, which is a 2-6 base pair DNA sequence that immediately follows the DNA sequence targeted by the Cas9 nuclease. A canonical PAM is the sequence 5'-NGG-3', where "N" is any nucleobase followed by two guanine ("G") nucleobases. The gRNA can transport Cas9 anywhere in the genome for gene editing, but cannot edit at sites other than the site where Cas9 recognizes the PAM. In addition, 5'-NGA-3' can be a highly efficient non-canonical PAM for human cells. Generally, the PAM is about 2-6 nucleotides downstream of the DNA sequence targeted by the gRNA. The PAM can be adjacent to the gRNA recognition sequence. In some embodiments, the gRNA recognition sequence can be adjacent to the PAM at the 3' end. In some embodiments, the gRNA recognition sequence can be adjacent to the PAM at the 5' end. For example, the cleavage site of the Cas protein can be about 1 to about 10, about 2 to about 5 base pairs, or 3 base pairs upstream or downstream of the PAM sequence. In some embodiments (e.g., when using Cas9 from S. pyogenes or a closely related Cas9), the PAM sequence of the non-complementary strand can be 5'-NGG-3', where N is any DNA nucleotide and is immediately 3' to the gRNA recognition sequence of the non-complementary strand of the target DNA. Thus, the PAM sequence of the complementary strand is 5'-CCN-3', where N is any DNA nucleotide and is immediately 5' to the gRNA recognition sequence of the complementary strand of the target DNA.

[0051] gRNA is an RNA molecule that binds to Cas protein and targets Cas protein to a specific position in ARHGEF12 genomic nucleic acid molecule.Exemplary gRNA is an effective gRNA that induces Cas enzyme to bind to or cleave ARHGEF12 genomic nucleic acid molecule, and gRNA comprises a DNA targeting segment that hybridizes with a gRNA recognition sequence in ARHGEF12 genomic nucleic acid molecule that includes or is adjacent to the position corresponding to position 132,939, 143,698, 141,048, 73,039, 121,307 or 141,978 of SEQ ID NO:1. For example, a gRNA can be selected to hybridize with a gRNA recognition sequence located about 5, about 10, about 15, about 20, about 25, about 30, about 35, about 40, about 45, about 50, about 100, about 200, about 300, about 400, about 500, or about 1,000 nucleotides from a position corresponding to 132,939, 143,698, 141,048, 73,039, 121,307, or 141,978 of SEQ ID NO: 1. Other exemplary gRNAs include a DNA targeting segment that hybridizes with a gRNA recognition sequence present in an ARHGEF12 genomic nucleic acid molecule that includes or is adjacent to a start codon or a stop codon. For example, a gRNA can be selected to hybridize with a gRNA recognition sequence located about 5, about 10, about 15, about 20, about 25, about 30, about 35, about 40, about 45, about 50, about 100, about 200, about 300, about 400, about 500, or about 1,000 nucleotides away from a start codon, or a gRNA recognition sequence located about 5, about 10, about 15, about 20, about 25, about 30, about 35, about 40, about 45, about 50, about 100, about 200, about 300, about 400, about 500, or about 1,000 nucleotides away from a stop codon. Suitable gRNAs can include about 17 to about 25 nucleotides, about 17 to about 23 nucleotides, about 18 to about 22 nucleotides, or about 19 to about 21 nucleotides. In some embodiments, the gRNA can include 20 nucleotides.

[0052] Examples of suitable gRNA recognition sequences located within the human ARHGEF12 reference type gene are listed in Table 1 as SEQ ID NOs: 84-110.

[0053] [Table 1]

[0054] The Cas protein and gRNA form a complex, and the Cas protein cuts the target ARHGEF12 genomic nucleic acid molecule. The Cas protein can cut the nucleic acid molecule at a site inside or outside the nucleic acid sequence present in the target ARHGEF12 genomic nucleic acid molecule to which the DNA targeting segment of the gRNA binds. For example, the formation of a CRISPR complex (comprising the gRNA hybridized to the gRNA recognition sequence and complexed with the Cas protein) can result in the cleavage of one or both strands within or near (e.g., within 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 50 or more base pairs from) the nucleic acid sequence present in the ARHGEF12 genomic nucleic acid molecule to which the DNA targeting segment of the gRNA binds.

[0055] Such a method may result in, for example, an ARHGEF12 genomic nucleic acid molecule in which the region of SEQ ID NO:1 is disrupted, the start codon is disrupted, the stop codon is disrupted, or the coding sequence is disrupted or deleted. Optionally, the cell may be further contacted with one or more additional gRNAs that hybridize with additional gRNA recognition sequences in the target genomic locus of the ARHGEF12 genomic nucleic acid molecule. By contacting the cell with one or more additional gRNAs (such as a second gRNA that hybridizes with a second gRNA recognition sequence), the cleavage by the Cas protein may generate two or more double-strand breaks or two or more single-strand breaks.

[0056] In some embodiments, the ARHGEF12 inhibitor comprises a small molecule. In some embodiments, the ARHGEF12 small molecule inhibitor is Y16, CCG-13528, CCG-14631, CCG-7167, CCG-12529, or RC-063.

[0057] In some embodiments, the method of treatment further comprises detecting the presence or absence of a predicted loss-of-function variant nucleic acid molecule of ARHGEF12 encoding an ARHGEF12 polypeptide in a biological sample from the subject. As used throughout this disclosure, a "predicted loss-of-function variant nucleic acid molecule of ARHGEF12" is any ARHGEF12 nucleic acid molecule (such as, for example, a genomic nucleic acid molecule, an mRNA molecule, or a cDNA molecule) that encodes an ARHGEF12 polypeptide having partial loss-of-function, complete loss-of-function, predicted partial loss-of-function, or predicted complete loss-of-function.

[0058] In some embodiments, the method of treatment further comprises detecting the presence or absence of a predicted gain-of-function variant nucleic acid molecule of ARHGEF12 encoding a human ARHGEF12 polypeptide in a biological sample from the subject. As used throughout this disclosure, a "predicted gain-of-function variant nucleic acid molecule of ARHGEF12" is any ARHGEF12 nucleic acid molecule (such as, for example, a genomic nucleic acid molecule, an mRNA molecule, or a cDNA molecule) that encodes an ARHGEF12 polypeptide that has enhanced activity, constitutive activity, increased ability to cause disease, decreased ability to resist disease, resistance to inhibitors, or resistance to therapeutic intervention.

[0059] The present disclosure also provides a method of treating a subject with a therapeutic agent that treats or suppresses glaucoma and / or elevated IOP, where the subject suffers from glaucoma and / or elevated IOP. In some embodiments, the method includes taking or having taken a biological sample from the subject, and determining whether the subject has a predicted loss-of-function variant nucleic acid molecule of ARHGEF12 encoding a human ARHGEF12 polypeptide by performing or having performed a genotyping assay in the biological sample to determine whether the subject has a genotype that includes a predicted loss-of-function variant nucleic acid molecule of ARHGEF12. If the subject is of the ARHGEF12 reference type, the subject is administered or continues to be administered a therapeutic agent that treats or suppresses glaucoma and / or elevated IOP at a standard dose, and an ARHGEF12 inhibitor can be administered to the subject. If the subject is heterozygous for the predicted loss-of-function variant of ARHGEF12, the subject can be administered or continue to be administered the therapeutic agent for treating or suppressing glaucoma and / or elevated IOP at the same or less than the standard dose, and can also be administered an ARHGEF12 inhibitor to the subject. If there is a genotype with a predicted loss-of-function variant nucleic acid molecule of ARHGEF12 encoding a human ARHGEF12 polypeptide, it indicates that the subject has a low risk of developing glaucoma and / or elevated IOP. In some embodiments, the subject is ARHGEF12 reference type. In some embodiments, the subject is heterozygous for the predicted loss-of-function variant of ARHGEF12.

[0060] The present disclosure also provides a method of treating a subject with a therapeutic agent that treats or suppresses glaucoma and / or elevated IOP, where the subject suffers from glaucoma and / or elevated IOP. In some embodiments, the method includes taking or having taken a biological sample from the subject, and determining whether the subject has a predicted gain-of-function variant nucleic acid molecule of ARHGEF12 encoding a human ARHGEF12 polypeptide by performing or having performed a genotyping assay in the biological sample to determine whether the subject has a genotype that includes a predicted gain-of-function variant nucleic acid molecule of ARHGEF12. If the subject is of the ARHGEF12 reference type, the subject is administered or continues to be administered a therapeutic agent that treats or suppresses glaucoma and / or elevated IOP at a standard dose, and an ARHGEF12 inhibitor can be administered to the subject. If the subject is heterozygous for a predicted gain-of-function variant of ARHGEF12, the subject can be administered or continue to be administered a therapeutic agent for treating or suppressing glaucoma and / or elevated IOP at a dose higher than standard dose, and can also be administered an ARHGEF12 inhibitor to the subject. If there is a genotype with a predicted gain-of-function variant nucleic acid molecule of ARHGEF12 encoding a human ARHGEF12 polypeptide, it indicates that the subject is at high risk of developing glaucoma and / or elevated IOP. In some embodiments, the subject is ARHGEF12 reference type. In some embodiments, the subject is heterozygous for a predicted gain-of-function variant of ARHGEF12.

[0061] For subjects who are genotyped or determined to be ARHGEF12 reference type, heterozygous for a predicted loss-of-function variant in ARHGEF12, or heterozygous or homozygous for a predicted gain-of-function variant in ARHGEF12, such subjects can be treated with an ARHGEF12 inhibitor as described herein.

[0062] Detecting the presence or absence of a predicted loss-of-function variant nucleic acid molecule of ARHGEF12 or a predicted gain-of-function variant nucleic acid molecule of ARHGEF12 in a biological sample from a subject and / or determining whether a subject has a predicted loss-of-function variant nucleic acid molecule of ARHGEF12 or a predicted gain-of-function variant nucleic acid molecule of ARHGEF12 can be performed by any of the methods described herein. In some embodiments, these methods can be performed in vitro. In some embodiments, these methods can be performed in situ. In some embodiments, these methods can be performed in vivo. In any of these embodiments, the nucleic acid molecule can be present in a cell obtained from the subject.

[0063] In some embodiments, if the subject is ARHGEF12 reference type, the subject is also administered a standard dose of a therapeutic agent for treating or inhibiting glaucoma and / or elevated IOP. In some embodiments, if the subject is heterozygous for a predicted loss-of-function variant in ARHGEF12, the subject is also administered a dose of a therapeutic agent for treating or inhibiting glaucoma and / or elevated IOP that is the same as or less than the standard dose. In some embodiments, if the subject is heterozygous for a predicted gain-of-function variant in ARHGEF12, the subject is administered or continues to be administered a therapeutic agent for treating or inhibiting glaucoma and / or elevated IOP that is greater than the standard dose.

[0064] In some embodiments, the method of treatment further comprises detecting the presence or absence of a predicted loss-of-function polypeptide of ARHGEF12 or a predicted gain-of-function polypeptide of ARHGEF12 in a biological sample from the subject. In some embodiments, if the subject does not have a predicted loss-of-function polypeptide of ARHGEF12, the subject is also administered a standard dose of a therapeutic agent for treating or suppressing glaucoma and / or elevated IOP. In some embodiments, if the subject has a predicted loss-of-function polypeptide of ARHGEF12, the subject is also administered a dose of a therapeutic agent for treating or suppressing glaucoma and / or elevated IOP that is equal to or less than the standard dose. In some embodiments, if the subject is heterozygous for a predicted gain-of-function polypeptide of ARHGEF12, the subject is also administered a dose of a therapeutic agent for treating or suppressing glaucoma and / or elevated IOP that is greater than the standard dose.

[0065] The present disclosure also provides a method of treating a subject with a therapeutic agent that treats or suppresses glaucoma and / or elevated IOP, where the subject suffers from glaucoma and / or elevated IOP. In some embodiments, the method includes taking or having taken a biological sample from the subject, and performing or having performed an assay in the biological sample to determine whether the subject has a predicted loss-of-function polypeptide of ARHGEF12, thereby determining whether the subject has a predicted loss-of-function polypeptide of ARHGEF12. If the subject does not have a predicted loss-of-function polypeptide of ARHGEF12, the therapeutic agent that treats or suppresses glaucoma and / or elevated IOP can be administered or continued to be administered to the subject at a standard dose, and an ARHGEF12 inhibitor can be administered to the subject. If the subject has a predicted loss-of-function polypeptide of ARHGEF12, the therapeutic agent that treats or suppresses glaucoma and / or elevated IOP can be administered or continued to be administered to the subject at a standard dose or less, and an ARHGEF12 inhibitor can be administered to the subject. The presence of a predicted loss-of-function polypeptide of ARHGEF12 indicates a low risk of developing glaucoma and / or elevated IOP in the subject. In some embodiments, the subject has a predicted loss-of-function polypeptide of ARHGEF12. In some embodiments, the subject does not have a predicted loss-of-function polypeptide of ARHGEF12.

[0066] In some embodiments, the method includes taking or having taken a biological sample from the subject, and performing or having performed an assay in the biological sample to determine whether the subject has a predicted gain-of-function polypeptide of ARHGEF12, thereby determining whether the subject has a predicted gain-of-function polypeptide of ARHGEF12. If the subject does not have a predicted gain-of-function polypeptide of ARHGEF12, the subject may be administered or continue to be administered a therapeutic agent for treating or suppressing glaucoma and / or elevated IOP at a standard dose, and an ARHGEF12 inhibitor may be administered to the subject. If the subject has a predicted gain-of-function polypeptide of ARHGEF12, the subject may be administered or continue to be administered a therapeutic agent for treating or suppressing glaucoma and / or elevated IOP at a higher than standard dose, and an ARHGEF12 inhibitor may be administered to the subject. If a predicted gain-of-function polypeptide of ARHGEF12 is present, it indicates that the subject is at high risk of developing glaucoma and / or elevated IOP. In some embodiments, the subject has a predicted gain-of-function polypeptide of ARHGEF12. In some embodiments, the subject does not have a predicted gain-of-function polypeptide of ARHGEF12.

[0067] Detecting the presence or absence of a predicted loss-of-function or gain-of-function ARHGEF12 polypeptide in a biological sample from a subject and / or determining whether a subject has a predicted loss-of-function or gain-of-function ARHGEF12 polypeptide can be performed by any of the methods described herein. In some embodiments, these methods can be performed in vitro. In some embodiments, these methods can be performed in situ. In some embodiments, these methods can be performed in vivo. In any of these embodiments, the polypeptide can be present in a cell taken from the subject.

[0068] Examples of therapeutic agents for treating or suppressing glaucoma and / or elevated IOP include, but are not limited to, prostaglandins, beta-blockers, alpha-adrenergic agonists, carbonic anhydrase inhibitors, rho kinase inhibitors, or miotics or cholinergic agonists. In some embodiments, the agent for treating or suppressing glaucoma and / or elevated IOP is a prostaglandin. In some embodiments, the agent for treating or suppressing glaucoma and / or elevated IOP is a beta-blocker. In some embodiments, the agent for treating or suppressing glaucoma and / or elevated IOP is an alpha-adrenergic agonist. In some embodiments, the agent for treating or suppressing glaucoma and / or elevated IOP is a carbonic anhydrase inhibitor. In some embodiments, the agent for treating or suppressing glaucoma and / or elevated IOP is a rho kinase inhibitor. In some embodiments, the agent for treating or suppressing glaucoma and / or elevated IOP is a miotic or cholinergic agonist.

[0069] In some embodiments, the prostaglandin is latanopros, travoprost, tafluprost, bimatoprost, or latanoprostene bunod. In some embodiments, the prostaglandin is latanopros. In some embodiments, the prostaglandin is travoprost. In some embodiments, the prostaglandin is tafluprost. In some embodiments, the prostaglandin is bimatoprost. In some embodiments, the prostaglandin is latanoprostene bunod. In some embodiments, the prostaglandin is XALATAN® (latanoprost), TRAVATAN Z® (travopros), ZIOPTAN® (tafluprost), LUMIGAN® (bimatoprost), or VYZULTA® (latanoprostene bunod). In some embodiments, the prostaglandin is XALATAN® (latanoprost). In some embodiments, the prostaglandin is TRAVATAN Z® (travoprost). In some embodiments, the prostaglandin is ZIOPTAN® (tafluprost). In some embodiments, the prostaglandin is LUMIGAN® (bimatoprost). In some embodiments, the prostaglandin is VYZULTA® (latanoprostene bunod).

[0070] In some embodiments, the beta blocker is timolol or betaxolol. In some embodiments, the beta blocker is timolol. In some embodiments, the beta blocker is betaxolol. In some embodiments, the beta blocker is BETIMOL®, ISTALOL®, or TIMOPTIC® (timolol) or BETOPTIC® (betaxolol). In some embodiments, the beta blocker is BETIMOL® (timolol). In some embodiments, the beta blocker is ISTALOL® (timolol). In some embodiments, the beta blocker is TIMOPTIC® (timolol). In some embodiments, the beta blocker is BETOPTIC® (betaxolol).

[0071] In some embodiments, the alpha-adrenergic agonist is apraclonidine or brimonidine. In some embodiments, the alpha-adrenergic agonist is apraclonidine. In some embodiments, the alpha-adrenergic agonist is brimonidine. In some embodiments, the alpha-adrenergic agonist is IOPIDINE® (apraclonidine) or ALPHAGAN® or QOLIANA® (brimonidine). In some embodiments, the alpha-adrenergic agonist is IOPIDINE® (apraclonidine). In some embodiments, the alpha-adrenergic agonist is ALPHAGAN® (brimonidine). In some embodiments, the alpha-adrenergic agonist is QOLIANA® (brimonidine).

[0072] In some embodiments, the carbonic anhydrase inhibitor is dorzolamide or brinzolamide. In some embodiments, the carbonic anhydrase inhibitor is dorzolamide. In some embodiments, the carbonic anhydrase inhibitor is brinzolamide. In some embodiments, the carbonic anhydrase inhibitor is TRUSOPT® (dorzolamide) or AZOPT® (brinzolamide). In some embodiments, the carbonic anhydrase inhibitor is TRUSOPT® (dorzolamide). In some embodiments, the carbonic anhydrase inhibitor is AZOPT® (brinzolamide).

[0073] In some embodiments, the rho kinase inhibitor is netarsudil. In some embodiments, the rho kinase inhibitor is RHOPRESSA® (netarsudil).

[0074] In some embodiments, the miotic or cholinergic agonist is pilocarpine. In some embodiments, the miotic or cholinergic agonist is ISOPTO® Carpine (pilocarpine).

[0075] In some embodiments, the dose of a therapeutic agent for treating or suppressing glaucoma and / or elevated IOP in a subject who is heterozygous or homozygous for a predicted loss-of-function variant in ARHGEF12 can be reduced (i.e., below a standard dose) by about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, or about 90% compared to a subject who is an ARHGEF12 reference type (wherein a standard dose can be administered). In some embodiments, the dose of a therapeutic agent for treating or suppressing glaucoma and / or elevated IOP can be reduced by about 10%, about 20%, about 30%, about 40%, or about 50%. Furthermore, the dose of a therapeutic agent for treating or suppressing glaucoma and / or elevated IOP in a subject who is heterozygous for a predicted loss-of-function variant in ARHGEF12 can be administered less frequently compared to a subject who is an ARHGEF12 reference type.

[0076] In some embodiments, the dose of a therapeutic agent for treating or suppressing glaucoma and / or elevated IOP in a subject who is heterozygous for a predicted gain-of-function variant in ARHGEF12 can be increased (i.e., above a standard dose) by about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, or about 90% compared to a subject who is an ARHGEF12 reference type (wherein a standard dose can be administered). In some embodiments, the dose of a therapeutic agent for treating or suppressing glaucoma and / or elevated IOP can be increased by about 10%, about 20%, about 30%, about 40%, or about 50%. Furthermore, the dose of a therapeutic agent for treating or suppressing glaucoma and / or elevated IOP in a subject who is heterozygous for a predicted gain-of-function variant in ARHGEF12 can be administered more frequently compared to a subject who is an ARHGEF12 reference type.

[0077] The administration of the therapeutic agent for treating or suppressing glaucoma and / or elevated IOP and / or the ARHGEF12 inhibitor can be repeated, for example, after 1 day, 2 days, 3 days, 5 days, 1 week, 2 weeks, 3 weeks, 1 month, 5 weeks, 6 weeks, 7 weeks, 8 weeks, 2 months, or 3 months. The repeated administration can be the same dose or different doses. The administration can be repeated 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more times. For example, according to a particular dosing regimen, the subject can be treated for an extended period of time, for example, 6 months, 1 year, or more.

[0078] Administration of the therapeutic agent for treating or suppressing glaucoma and / or elevated IOP and / or the ARHGEF12 inhibitor can be by any suitable route, including but not limited to parenteral, intravenous, oral, subcutaneous, intraarterial, intracranial, intrathecal, intraperitoneal, topical, intranasal, or intramuscular. Pharmaceutical compositions for administration are desirably sterile, substantially isotonic, and manufactured under GMP conditions. Pharmaceutical compositions can be provided in unit dosage form (i.e., a single dose for administration). Pharmaceutical compositions can be formulated using one or more physiologically and pharma- ceutical acceptable carriers, diluents, excipients, or adjuvants. The formulation depends on the route of administration selected. The term "pharmaceutical acceptable" means that the carrier, diluent, excipient, or adjuvant is compatible with the other ingredients of the formulation and is not substantially deleterious to the recipient thereof.

[0079] As used herein, the terms "treat," "treating," and "treatment," as well as "prevent," "preventing," and "prevention," refer to eliciting a desired biological response, such as a therapeutic effect and a prophylactic effect, respectively. In some embodiments, the therapeutic effect includes one or more of the following following administration of the agent or a composition comprising the agent: reduction / alleviation of glaucoma and / or elevated IOP, reduction / alleviation of the severity of glaucoma and / or elevated IOP (e.g., reduction or inhibition of the onset of glaucoma and / or elevated IOP), reduction / alleviation of glaucoma symptoms and effects associated with elevated IOP, delay in the onset of glaucoma symptoms and effects associated with elevated IOP, reduction in the severity of glaucoma symptoms and effects associated with elevated IOP, reduction in the severity of acute episodes, reduction in the number of glaucoma symptoms and effects associated with elevated IOP, reduction in the latency of glaucoma symptoms and effects associated with elevated IOP, amelioration of glaucoma symptoms and effects associated with elevated IOP, reduction in secondary symptoms, reduction in secondary infection, prevention of recurrence of glaucoma and / or elevated IOP, reduction in the number or frequency of recurrent episodes, increase in the latency between symptomatic episodes, increase in the time to sustained progression, accelerated recovery, and / or increased effectiveness or reduced resistance to alternative therapeutic agents. A prophylactic effect can include complete or partial avoidance / inhibition or delay (e.g., complete or partial avoidance / inhibition or delay, etc.) of the onset / progression of glaucoma and / or elevated IOP following administration of a treatment protocol. Treating glaucoma and / or elevated IOP includes treating a subject already diagnosed with any form of glaucoma and / or elevated IOP at any clinical stage or clinical manifestation, delaying the onset or progression or worsening or aggravation of symptoms or signs of glaucoma and / or elevated IOP, and / or preventing and / or reducing the severity of glaucoma and / or elevated IOP.

[0080] The present disclosure also provides a method for identifying a subject at high risk of developing glaucoma and / or elevated IOP. In some embodiments, the method includes determining or having determined the presence or absence of a predicted loss-of-function variant nucleic acid molecule (e.g., a genomic nucleic acid molecule, an mRNA molecule, and / or a cDNA molecule) of ARHGEF12 encoding a human ARHGEF12 polypeptide in a biological sample taken from the subject. If the subject lacks a predicted loss-of-function variant nucleic acid molecule of ARHGEF12 (e.g., the subject is genotypically categorized as an ARHGEF12 reference type), the subject is at high risk of developing glaucoma and / or elevated IOP. If the subject has a predicted loss-of-function variant nucleic acid molecule of ARHGEF12 (e.g., the subject is heterozygous or homozygous for a predicted loss-of-function variant of ARHGEF12), the subject is at low risk of developing glaucoma and / or elevated IOP.

[0081] By having a single copy of a predicted loss-of-function variant nucleic acid molecule of ARHGEF12, a subject is further protected from developing glaucoma and / or elevated IOP compared to a subject not having a copy of a predicted loss-of-function variant nucleic acid molecule of ARHGEF12. Without intending to be limited to a particular theory or mechanism of action, it is believed that a single copy of a predicted loss-of-function variant nucleic acid molecule of ARHGEF12 (i.e., heterozygous for a predicted loss-of-function variant of ARHGEF12) protects a subject from developing glaucoma and / or elevated IOP, and it is believed that having two copies of a predicted loss-of-function variant nucleic acid molecule of ARHGEF12 (i.e., homozygous for a predicted loss-of-function variant of ARHGEF12) may further protect a subject from developing glaucoma and / or elevated IOP compared to a subject having a single copy. Thus, in some embodiments, a single copy of a predicted loss-of-function variant nucleic acid molecule of ARHGEF12 may not completely protect a subject from developing glaucoma and / or elevated IOP, but may instead provide partial or incomplete protection. Without wishing to be bound by any particular theory, it is possible that there are additional factors or molecules involved in developing glaucoma and / or elevated IOP that are still present in a subject having a single copy of a predicted loss-of-function variant nucleic acid molecule of ARHGEF12, resulting in less than complete protection from developing glaucoma and / or elevated IOP.

[0082] In some embodiments, the method includes determining or having determined the presence or absence of a predicted gain-of-function variant nucleic acid molecule of ARHGEF12 (such as a genomic nucleic acid molecule, an mRNA molecule, and / or a cDNA molecule) encoding a human ARHGEF12 polypeptide in a biological sample obtained from the subject. If the subject lacks a predicted gain-of-function variant nucleic acid molecule of ARHGEF12 (e.g., the subject is genotypically categorized as an ARHGEF12 reference type), the subject is at low risk of developing glaucoma and / or elevated IOP. If the subject has a predicted gain-of-function variant nucleic acid molecule of ARHGEF12 (i.e., the subject is heterozygous or homozygous for a predicted gain-of-function variant of ARHGEF12), the subject is at high risk of developing glaucoma and / or elevated IOP.

[0083] By having a single copy of a predicted gain-of-function variant nucleic acid molecule of ARHGEF12, a subject is at a greater risk of developing glaucoma and / or elevated IOP than if the subject does not have a copy of the predicted gain-of-function variant nucleic acid molecule of ARHGEF12. Without intending to be limited to a particular theory or mechanism of action, a single copy of a predicted gain-of-function variant nucleic acid molecule of ARHGEF12 (i.e., a heterozygous form of a predicted gain-of-function variant of ARHGEF12) is believed to increase a subject's risk of developing glaucoma and / or elevated IOP, and having two copies of a predicted gain-of-function variant nucleic acid molecule of ARHGEF12 (i.e., a homozygous form of a predicted loss-of-function variant of ARHGEF12) is believed to further increase a subject's risk of developing glaucoma and / or elevated IOP compared to a subject having a single copy. Thus, in some embodiments, a single copy of a predicted gain-of-function variant nucleic acid molecule of ARHGEF12 may be a partial or incomplete risk of glaucoma and / or elevated IOP in a subject. Without wishing to be bound by any particular theory, it is possible that there are additional factors or molecules involved in the development of glaucoma and / or elevated IOP that are still present in subjects having a single copy of a predicted gain-of-function variant nucleic acid molecule of ARHGEF12, thereby increasing the subject's risk of developing glaucoma and / or elevated IOP.

[0084] Determining whether a subject has a predicted loss-of-function variant nucleic acid molecule of ARHGEF12 or a predicted gain-of-function variant nucleic acid molecule of ARHGEF12 in a biological sample from a subject and / or determining whether a subject has a predicted loss-of-function variant nucleic acid molecule of ARHGEF12 or a predicted gain-of-function variant nucleic acid molecule of ARHGEF12 can be performed by any of the methods described herein. In some embodiments, these methods can be performed in vitro. In some embodiments, these methods can be performed in situ. In some embodiments, these methods can be performed in vivo. In any of these embodiments, the nucleic acid molecule can be present in a cell obtained from the subject.

[0085] In some embodiments, if a subject is identified as being at high risk of developing glaucoma and / or elevated IOP, the subject is further treated with a therapeutic agent for treating or suppressing glaucoma and / or elevated IOP, and / or an ARHGEF12 inhibitor, as described herein. For example, if a subject is ARHGEF12 reference type and therefore at high risk of developing glaucoma and / or elevated IOP, the subject is administered an ARHGEF12 inhibitor. In some embodiments, such a subject is also administered a therapeutic agent for treating or suppressing glaucoma and / or elevated IOP. In some embodiments, if a subject is heterozygous for a predicted loss-of-function variant of ARHGEF12, the subject can be administered a therapeutic agent for treating or suppressing glaucoma and / or elevated IOP at a dose equal to or less than the standard dose, and can also be administered an ARHGEF12 inhibitor. In some embodiments, if the subject is heterozygous for a predicted gain-of-function variant in ARHGEF12, the subject can be administered a therapeutic agent that treats or inhibits glaucoma and / or elevated IOP at a dose equal to or greater than the standard dose, and can also be administered an ARHGEF12 inhibitor. In some embodiments, the subject is ARHGEF12 reference type. In some embodiments, the subject is heterozygous for a predicted loss-of-function variant in ARHGEF12.

[0086] The present disclosure also provides a method for detecting the presence or absence of a predicted loss-of-function variant genomic nucleic acid molecule of ARHGEF12 in a biological sample from a subject, and / or a predicted gain-of-function variant genomic nucleic acid molecule of ARHGEF12, and / or a predicted loss-of-function variant mRNA of ARHGEF12 in a biological sample from a subject, and / or a predicted gain-of-function variant mRNA of ARHGEF12 in a biological sample from a subject, and / or a predicted loss-of-function variant cDNA molecule of ARHGEF12 produced from an mRNA molecule in a biological sample from a subject, and / or a predicted gain-of-function variant cDNA molecule of ARHGEF12. It is understood that gene sequences within a population, and the mRNA molecules encoded by such genes, may differ due to polymorphisms, such as single nucleotide polymorphisms. The sequences provided herein for ARHGEF12 variant genomic nucleic acid molecules, ARHGEF12 variant mRNA molecules, and ARHGEF12 variant cDNA molecules are merely exemplary sequences. Other sequences for ARHGEF12 variant genomic nucleic acid molecules, variant mRNA molecules, and variant cDNA molecules are also possible.

[0087] The biological sample may be derived from any cell, tissue, or biological fluid from a subject. The biological sample may include any clinically relevant tissue, such as bone marrow sample, tumor biopsy, fine needle aspirate, or a sample of bodily fluid, such as blood, gingival crevicular fluid, plasma, serum, lymph, ascites, cyst fluid, or urine. In some cases, the sample includes a buccal swab. The biological sample used in the methods disclosed herein may vary based on the assay format, the nature of the detection method, and the tissue, cell, or extract used as the sample. The biological sample may be subjected to different treatments depending on the assay employed. For example, when detecting any ARHGEF12 variant nucleic acid molecule, a pretreatment designed to isolate or enrich the biological sample for genomic DNA may be employed. For this purpose, various techniques may be used. When detecting the level of ARHGEF12 variant mRNA molecules, various techniques may be used to enrich the biological sample containing mRNA molecules. Various methods may be used to detect the presence or level of mRNA molecules, or the presence of a specific variant genomic DNA locus.

[0088] In some embodiments, detecting a predicted loss-of-function variant nucleic acid molecule of human ARHGEF12 in a subject comprises assaying or genotyping a biological sample obtained from the subject to determine whether an ARHGEF12 genomic nucleic acid molecule in the biological sample, and / or an ARHGEF12 mRNA molecule in the biological sample, and / or an ARHGEF12 cDNA molecule generated from the mRNA molecule in the biological sample, contains one or more mutations that cause or are predicted to cause loss-of-function (partial or complete).

[0089] In some embodiments, detecting a predicted gain-of-function variant nucleic acid molecule of human ARHGEF12 in a subject comprises assaying or genotyping a biological sample obtained from the subject to determine whether an ARHGEF12 genomic nucleic acid molecule in the biological sample, and / or an ARHGEF12 mRNA molecule in the biological sample, and / or an ARHGEF12 cDNA molecule generated from an mRNA molecule in the biological sample contains one or more mutations that result in or are predicted to result in a gain-of-function.

[0090] In some embodiments, a method for detecting the presence or absence of a predicted loss-of-function variant nucleic acid molecule of ARHGEF12 or a predicted gain-of-function variant nucleic acid molecule of ARHGEF12 (e.g., a genomic nucleic acid molecule, an mRNA molecule, and / or a cDNA molecule generated from the mRNA) in a subject comprises performing an assay on a biological sample obtained from the subject, the assay determining whether a nucleic acid molecule in the biological sample comprises a particular nucleotide sequence.

[0091] In some embodiments, a nucleotide sequence encoding a predicted gain-of-function variant of ARHGEF12 comprises a thymine at a position corresponding to position 132,939 of SEQ ID NO:2 (for a genomic nucleic acid molecule), a uracil at a position corresponding to position 3,749 of SEQ ID NO:16, position 3,191 of SEQ ID NO:17, position 3,079 of SEQ ID NO:18, position 3,692 of SEQ ID NO:19, position 3,046 of SEQ ID NO:20, position 2,925 of SEQ ID NO:21, position 3,054 of SEQ ID NO:22, or position 2,615 of SEQ ID NO:23 (for an mRNA molecule), a thymine at a position corresponding to position 3,749 of SEQ ID NO:47, position 3,191 of SEQ ID NO:48, position 3,079 of SEQ ID NO:49, position 3,692 of SEQ ID NO:50, position 3,046 of SEQ ID NO:51, position 2,925 of SEQ ID NO:52, position 3,054 of SEQ ID NO:53, or position 2,615 of SEQ ID NO:54 (for a cDNA molecule).

[0092] In some embodiments, a nucleotide sequence encoding a predicted loss-of-function variant of ARHGEF12 comprises a guanine at a position corresponding to position 143,698 of SEQ ID NO:3 (for a genomic nucleic acid molecule), a guanine at a position corresponding to position 4,748 of SEQ ID NO:24, position 4,190 of SEQ ID NO:25, position 4,078 of SEQ ID NO:26, position 4,691 of SEQ ID NO:27, position 4,045 of SEQ ID NO:28, position 3,924 of SEQ ID NO:29, or position 3,614 of SEQ ID NO:30, a guanine at a position corresponding to position 4,748 of SEQ ID NO:55, position 4,190 of SEQ ID NO:56, position 4,078 of SEQ ID NO:57, position 4,691 of SEQ ID NO:58, position 4,045 of SEQ ID NO:59, position 3,924 of SEQ ID NO:60, or position 3,614 of SEQ ID NO:61 (for a cDNA molecule).

[0093] In some embodiments, a nucleotide sequence encoding a predicted loss-of-function variant of ARHGEF12 comprises a thymine at a position corresponding to position 141,048 of SEQ ID NO:4 (for a genomic nucleic acid molecule), a uracil at a position corresponding to position 4,297 of SEQ ID NO:31, position 3,739 of SEQ ID NO:32, position 3,627 of SEQ ID NO:33, position 4,240 of SEQ ID NO:34, position 3,594 of SEQ ID NO:35, position 3,473 of SEQ ID NO:36, position 3,602 of SEQ ID NO:37, or position 3,163 of SEQ ID NO:38 (for an mRNA molecule), a thymine at a position corresponding to position 4,297 of SEQ ID NO:62, position 3,379 of SEQ ID NO:63, position 3,627 of SEQ ID NO:64, position 4,240 of SEQ ID NO:65, position 3,594 of SEQ ID NO:66, position 3,473 of SEQ ID NO:67, position 3,602 of SEQ ID NO:68, or position 3,163 of SEQ ID NO:69 (for a cDNA molecule).

[0094] In some embodiments, a nucleotide sequence encoding a predicted loss-of-function variant of ARHGEF12 comprises an adenine at a position corresponding to position 73,039 of SEQ ID NO:5 (in the case of a genomic nucleic acid molecule).

[0095] In some embodiments, a nucleotide sequence encoding a predicted loss-of-function variant of ARHGEF12 comprises a cytosine at a position corresponding to position 121,307 of SEQ ID NO:6 (in the case of a genomic nucleic acid molecule).

[0096] In some embodiments, a nucleotide sequence encoding a predicted loss-of-function variant of ARHGEF12 comprises a cytosine at a position corresponding to position 141,978 of SEQ ID NO:7 (in the case of a genomic nucleic acid molecule).

[0097] In some embodiments, the nucleotide sequence comprises a thymine at a position corresponding to position 132,939 of SEQ ID NO:2 or its complement, a guanine at a position corresponding to position 143,698 of SEQ ID NO:3 or its complement, a thymine at a position corresponding to position 141,048 of SEQ ID NO:4 or its complement, an adenine at a position corresponding to position 73,039 of SEQ ID NO:5 or its complement, a cytosine at a position corresponding to position 121,307 of SEQ ID NO:6 or its complement, or a cytosine at a position corresponding to position 141,978 of SEQ ID NO:7 or its complement.

[0098] In some embodiments, the nucleotide sequence of the ARHGEF12 variant mRNA molecule contains i) a uracil at a position corresponding to position 3,749 of SEQ ID NO:16 or its complement, position 3,191 of SEQ ID NO:17 or its complement, position 3,079 of SEQ ID NO:18 or its complement, position 3,692 of SEQ ID NO:19 or its complement, position 3,046 of SEQ ID NO:20 or its complement, position 2,925 of SEQ ID NO:21 or its complement, position 3,054 of SEQ ID NO:22 or its complement, or position 2,615 of SEQ ID NO:23 or its complement; ii) a uracil at a position corresponding to position 4,748 of SEQ ID NO:24 or its complement, position 4,190 of SEQ ID NO:25 or its complement, position 4,078 of SEQ ID NO:26 or its complement, position 4,193 of SEQ ID NO:27 or its complement, position 4,201 of SEQ ID NO:28 or its complement, position 4,216 of SEQ ID NO:29 or its complement, position 4,301 of SEQ ID NO:30 or its complement, position 4,315 of SEQ ID NO:31 or its complement, position 4,320 of SEQ ID NO:32 or its complement, position 4,330 of SEQ ID NO:33 or its complement, position 4,340 of SEQ ID NO:34 or its complement, position 4,350 of SEQ ID NO:35 or its complement, position 4,360 of SEQ ID NO:36 or its complement, position 4,370 of SEQ ID NO:37 or its complement, position 4,381 of SEQ ID NO:38 or its complement, position 4,392 of SEQ ID NO:39 or its complement, position 5,401 of SEQ ID a guanine at a position corresponding to position 4,691 of SEQ ID NO:27 or its complement, position 4,045 of SEQ ID NO:28 or its complement, position 3,924 of SEQ ID NO:29 or its complement, or position 3,614 of SEQ ID NO:30 or its complement; or iii) a uracil at a position corresponding to position 4,297 of SEQ ID NO:31 or its complement, position 3,739 of SEQ ID NO:32 or its complement, position 3,627 of SEQ ID NO:33 or its complement, position 4,240 of SEQ ID NO:34 or its complement, position 3,594 of SEQ ID NO:35 or its complement, position 3,473 of SEQ ID NO:36 or its complement, position 3,602 of SEQ ID NO:37 or its complement, or position 3,163 of SEQ ID NO:38 or its complement.

[0099] In some embodiments, the nucleotide sequence of the ARHGEF12 variant cDNA molecule contains i) a thymine at a position corresponding to position 3,749 of SEQ ID NO:47 or its complement, position 3,191 of SEQ ID NO:48 or its complement, position 3,079 of SEQ ID NO:49 or its complement, position 3,692 of SEQ ID NO:50 or its complement, position 3,046 of SEQ ID NO:51 or its complement, position 2,925 of SEQ ID NO:52 or its complement, position 3,054 of SEQ ID NO:53 or its complement, or position 2,615 of SEQ ID NO:54 or its complement; ii) a thymine at a position corresponding to position 4,748 of SEQ ID NO:55 or its complement, position 4,190 of SEQ ID NO:56 or its complement, position 4,078 of SEQ ID NO:57 or its complement. , a guanine at a position corresponding to position 4,691 of SEQ ID NO:58 or its complement, position 4,045 of SEQ ID NO:59 or its complement, position 3,924 of SEQ ID NO:60 or its complement, or position 3,614 of SEQ ID NO:61 or its complement; or iii) a thymine at a position corresponding to position 4,297 of SEQ ID NO:62 or its complement, position 3,379 of SEQ ID NO:63 or its complement, position 3,627 of SEQ ID NO:64 or its complement, position 4,240 of SEQ ID NO:65 or its complement, position 3,594 of SEQ ID NO:66 or its complement, position 3,473 of SEQ ID NO:67 or its complement, position 3,602 of SEQ ID NO:68 or its complement, or position 3,163 of SEQ ID NO:69 or its complement.

[0100] In some embodiments, the biological sample comprises cells or cell lysates.Such method may further comprise, for example, taking biological sample from subject that comprises ARHGEF12 genomic nucleic acid molecule or mRNA molecule, and in case of mRNA, optionally reverse transcribing mRNA into cDNA.Such assay may comprise, for example, determining the identity of these positions of specific ARHGEF12 nucleic acid molecule.In some embodiments, the method is an in vitro method.

[0101] In some embodiments, the determining, detecting, or genotyping assay comprises sequencing at least a portion of the nucleotide sequence of an ARHGEF12 genomic nucleic acid molecule, an ARHGEF12 mRNA molecule, or an ARHGEF12 cDNA molecule in a biological sample, where the portion to be sequenced contains one or more mutations that cause or are predicted to cause a loss of function (partial or complete), or that cause or are predicted to cause a gain of function (partial or complete).

[0102] In some embodiments, the determining, detecting or genotyping assay comprises determining a nucleotide sequence of an ARHGEF12 genomic nucleic acid molecule in a biological sample, the portion of which to be sequenced comprising a position corresponding to position 132,939 of SEQ ID NO:2 or its complement; ii) determining a nucleotide sequence of an ARHGEF12 mRNA molecule in a biological sample, the portion of which to be sequenced comprising a position corresponding to position 3,749 of SEQ ID NO:16 or its complement, position 3,191 of SEQ ID NO:17 or its complement, position 3,079 of SEQ ID NO:18 or its complement, position 3,692 of SEQ ID NO:19 or its complement, position 3,046 of SEQ ID NO:20 or its complement, position 2,925 of SEQ ID NO:21 or its complement, position 3,054 of SEQ ID NO:22 or its complement, or position 2,615 of SEQ ID NO:23 or its complement; and / or iii) determining a nucleotide sequence of an ARHGEF12 mRNA molecule in a biological sample, the portion of which to be sequenced comprising a position corresponding to position 3,749 of SEQ ID NO:16 or its complement, position 3,191 of SEQ ID NO:17 or its complement, position 3,079 of SEQ ID NO:18 or its complement, position 3,692 of SEQ ID NO:19 or its complement, position 3,046 of SEQ ID NO:20 or its complement, position 2,925 of SEQ ID NO:21 or its complement, position 3,054 of SEQ ID NO:22 or its complement, or position 2,615 of SEQ ID NO:23 or its complement; The method includes sequencing at least a portion of the nucleotide sequence of the ARHGEF12 cDNA molecule, wherein the nucleotide sequence of the cDNA molecule includes a position corresponding to position 3,749 of SEQ ID NO:47 or its complement, position 3,191 of SEQ ID NO:48 or its complement, position 3,079 of SEQ ID NO:49 or its complement, position 3,692 of SEQ ID NO:50 or its complement, position 3,046 of SEQ ID NO:51 or its complement, position 2,925 of SEQ ID NO:52 or its complement, position 3,054 of SEQ ID NO:53 or its complement, or position 2,615 of SEQ ID NO:54 or its complement.The portion of the ARHGEF12 nucleic acid molecule in the biological sample to be sequenced contains i) a thymine at a position corresponding to position 132,939 of SEQ ID NO:2, ii) a uracil at a position corresponding to position 3,749 of SEQ ID NO:16, position 3,191 of SEQ ID NO:17, position 3,079 of SEQ ID NO:18, position 3,692 of SEQ ID NO:19, position 3,046 of SEQ ID NO:20, position 2,925 of SEQ ID NO:21, position 3,054 of SEQ ID NO:22, or position 2,615 of SEQ ID NO:23, or or iii) a thymine at a position corresponding to position 3,749 of SEQ ID NO:47, position 3,191 of SEQ ID NO:48, position 3,079 of SEQ ID NO:49, position 3,692 of SEQ ID NO:50, position 3,046 of SEQ ID NO:51, position 2,925 of SEQ ID NO:52, position 3,054 of SEQ ID NO:53, or position 2,615 of SEQ ID NO:54, then the ARHGEF12 nucleic acid molecule in the biological sample is a predicted gain-of-function variant nucleic acid molecule of ARHGEF12.

[0103] In some embodiments, the determining, detecting or genotyping assay comprises determining the nucleotide sequence of: i) an ARHGEF12 genomic nucleic acid molecule in the biological sample, the portion of which to be sequenced comprises a position corresponding to position 143,698 of SEQ ID NO:3 or its complement; ii) an ARHGEF12 mRNA molecule in the biological sample, the portion of which to be sequenced comprises a position corresponding to position 4,748 of SEQ ID NO:24 or its complement, position 4,190 of SEQ ID NO:25 or its complement, position 4,078 of SEQ ID NO:26 or its complement, position 4,691 of SEQ ID NO:27 or its complement, position 4,045 of SEQ ID NO:28 or its complement, position 3,924 of SEQ ID NO:29 or its complement, or position 3,614 of SEQ ID NO:30 or its complement; and / or iii) an ARHGEF12 mRNA molecule generated from mRNA in the biological sample. The method includes sequencing at least a portion of the nucleotide sequence of the ARHGEF12 cDNA molecule, wherein the nucleotide sequence of the cDNA molecule includes a position corresponding to position 4,748 of SEQ ID NO:55 or its complement, position 4,190 of SEQ ID NO:56 or its complement, position 4,078 of SEQ ID NO:57 or its complement, position 4,691 of SEQ ID NO:58 or its complement, position 4,045 of SEQ ID NO:59 or its complement, position 3,924 of SEQ ID NO:60 or its complement, or position 3,614 of SEQ ID NO:61 or its complement.If the sequenced portion of the ARHGEF12 nucleic acid molecule in the biological sample comprises i) a guanine at a position corresponding to position 143,698 of SEQ ID NO:3, ii) a guanine at a position corresponding to position 4,748 of SEQ ID NO:24, position 4,190 of SEQ ID NO:25, position 4,078 of SEQ ID NO:26, position 4,691 of SEQ ID NO:27, position 4,045 of SEQ ID NO:28, position 3,924 of SEQ ID NO:29, or position 3,614 of SEQ ID NO:30, or iii) a guanine at a position corresponding to position 4,748 of SEQ ID NO:55, position 4,190 of SEQ ID NO:56, position 4,078 of SEQ ID NO:57, position 4,691 of SEQ ID NO:58, position 4,045 of SEQ ID NO:59, position 3,924 of SEQ ID NO:60, or position 3,614 of SEQ ID NO:61, then the ARHGEF12 nucleic acid molecule in the biological sample is a predicted loss-of-function variant nucleic acid molecule of ARHGEF12.

[0104] In some embodiments, the determining, detecting or genotyping assay comprises determining: i) the nucleotide sequence of an ARHGEF12 genomic nucleic acid molecule in the biological sample, the portion of which to be sequenced comprises a position corresponding to position 141,048 of SEQ ID NO:4 or its complement; ii) the nucleotide sequence of an ARHGEF12 mRNA molecule in the biological sample, the portion of which to be sequenced comprises a position corresponding to position 4,297 of SEQ ID NO:31 or its complement, position 3,739 of SEQ ID NO:32 or its complement, position 3,627 of SEQ ID NO:33 or its complement, position 4,240 of SEQ ID NO:34 or its complement, position 3,594 of SEQ ID NO:35 or its complement, position 3,473 of SEQ ID NO:36 or its complement, position 3,602 of SEQ ID NO:37 or its complement, or position 3,163 of SEQ ID NO:38 or its complement; and / or iii) the nucleotide sequence of an ARHGEF12 mRNA molecule generated from mRNA in the biological sample. The method includes sequencing at least a portion of the nucleotide sequence of the ARHGEF12 cDNA molecule, wherein the sequenced portion of the nucleotide sequence of the cDNA molecule includes a position corresponding to position 4,297 of SEQ ID NO:62 or its complement, position 3,379 of SEQ ID NO:63 or its complement, position 3,627 of SEQ ID NO:64 or its complement, position 4,240 of SEQ ID NO:65 or its complement, position 3,594 of SEQ ID NO:66 or its complement, position 3,473 of SEQ ID NO:67 or its complement, position 3,602 of SEQ ID NO:68 or its complement, or position 3,163 of SEQ ID NO:69 or its complement.The portion of the ARHGEF12 nucleic acid molecule in the biological sample to be sequenced contains i) a thymine at a position corresponding to position 141,048 of SEQ ID NO:4, ii) a uracil at a position corresponding to position 4,297 of SEQ ID NO:31, position 3,739 of SEQ ID NO:32, position 3,627 of SEQ ID NO:33, position 4,240 of SEQ ID NO:34, position 3,594 of SEQ ID NO:35, position 3,473 of SEQ ID NO:36, position 3,602 of SEQ ID NO:37, or position 3,163 of SEQ ID NO:38, or or iii) contains a thymine at a position corresponding to position 4,297 of SEQ ID NO:62, position 3,379 of SEQ ID NO:63, position 3,627 of SEQ ID NO:64, position 4,240 of SEQ ID NO:65, position 3,594 of SEQ ID NO:66, position 3,473 of SEQ ID NO:67, position 3,602 of SEQ ID NO:68, or position 3,163 of SEQ ID NO:69, then the ARHGEF12 nucleic acid molecule in the biological sample is a predicted loss-of-function variant nucleic acid molecule of ARHGEF12.

[0105] In some embodiments, the determining step, detecting step, or genotyping assay comprises sequencing at least a portion of a nucleotide sequence of an ARHGEF12 genomic nucleic acid molecule in the biological sample, where the sequenced portion comprises a position corresponding to position 73,039 of SEQ ID NO:5 or its complement. If the sequenced portion of the ARHGEF12 nucleic acid molecule in the biological sample comprises an adenine at a position corresponding to position 73,039 of SEQ ID NO:5, then the ARHGEF12 nucleic acid molecule in the biological sample is a predicted loss-of-function variant nucleic acid molecule of ARHGEF12.

[0106] In some embodiments, the determining step, detecting step, or genotyping assay comprises sequencing at least a portion of a nucleotide sequence of an ARHGEF12 genomic nucleic acid molecule in the biological sample, where the sequenced portion comprises a position corresponding to position 121,307 of SEQ ID NO:6 or its complement. If the sequenced portion of the ARHGEF12 nucleic acid molecule in the biological sample comprises a cytosine at a position corresponding to position 121,307 of SEQ ID NO:6, then the ARHGEF12 nucleic acid molecule in the biological sample is a predicted loss-of-function variant nucleic acid molecule of ARHGEF12.

[0107] In some embodiments, the determining step, detecting step, or genotyping assay comprises sequencing at least a portion of a nucleotide sequence of an ARHGEF12 genomic nucleic acid molecule in the biological sample, where the sequenced portion comprises a position corresponding to position 141,978 of SEQ ID NO:7 or its complement. If the sequenced portion of the ARHGEF12 nucleic acid molecule in the biological sample comprises a cytosine at a position corresponding to position 141,978 of SEQ ID NO:7, then the ARHGEF12 nucleic acid molecule in the biological sample is a predicted loss-of-function variant nucleic acid molecule of ARHGEF12.

[0108] In some embodiments, the determining step, detecting step, or genotyping assay comprises sequencing at least a portion of the nucleotide sequence of the ARHGEF12 genomic nucleic acid molecule in the biological sample, wherein the sequenced portion comprises a position corresponding to position 132,939 of SEQ ID NO:2 or its complement, a position corresponding to position 143,698 of SEQ ID NO:3 or its complement, a position corresponding to position 141,048 of SEQ ID NO:4 or its complement, a position corresponding to position 73,039 of SEQ ID NO:5 or its complement, a position corresponding to position 121,307 of SEQ ID NO:6 or its complement, or a position corresponding to position 141,978 of SEQ ID NO:7 or its complement. If the sequenced portion of the ARHGEF12 nucleic acid molecule in the biological sample comprises a thymine at the position corresponding to position 132,939 of SEQ ID NO:2, the ARHGEF12 nucleic acid molecule in the biological sample is a predicted gain-of-function variant nucleic acid molecule of ARHGEF12. If the sequenced portion of the ARHGEF12 nucleic acid molecule in the biological sample contains a guanine at a position corresponding to position 143,698 of SEQ ID NO:3, a thymine at a position corresponding to position 141,048 of SEQ ID NO:4, an adenine at a position corresponding to position 73,039 of SEQ ID NO:5, a cytosine at a position corresponding to position 121,307 of SEQ ID NO:6, or a cytosine at a position corresponding to position 141,978 of SEQ ID NO:7, the ARHGEF12 nucleic acid molecule in the biological sample is a predicted loss-of-function variant nucleic acid molecule of ARHGEF12.

[0109] In some embodiments, the determining, detecting, or genotyping assay comprises sequencing at least a portion of the nucleotide sequence of the ARHGEF12 mRNA molecule in the biological sample, wherein the portion to be sequenced is at position 3,749 of SEQ ID NO: 16 or its complement, position 3,191 of SEQ ID NO: 17 or its complement, position 3,079 of SEQ ID NO: 18 or its complement, position 3,692 of SEQ ID NO: 19 or its complement, position 3,046 of SEQ ID NO: 20 or its complement, position 2,925 of SEQ ID NO: 21 or its complement, position 3,054 of SEQ ID NO: 22 or its complement, position 2,615 of SEQ ID NO: 23 or its complement, position 4,748 of SEQ ID NO: 24 or its complement, position 4,190 of SEQ ID NO: 25 or its complement, position 4,200 of SEQ ID NO: 26 or its complement, position 4,078 of SEQ ID NO:27 or its complement, position 4,691 of SEQ ID NO:28 or its complement, position 4,045 of SEQ ID NO:29 or its complement, position 3,924 of SEQ ID NO:30 or its complement, position 4,297 of SEQ ID NO:31 or its complement, position 3,739 of SEQ ID NO:32 or its complement, position 3,627 of SEQ ID NO:33 or its complement, position 4,240 of SEQ ID NO:34 or its complement, position 3,594 of SEQ ID NO:35 or its complement, position 3,473 of SEQ ID NO:36 or its complement, position 3,602 of SEQ ID NO:37 or its complement, or position 3,163 of SEQ ID NO:38 or its complement. If the sequenced portion of the ARHGEF12 nucleic acid molecule in the biological sample contains uracil at a position corresponding to position 3,749 of SEQ ID NO:16, position 3,191 of SEQ ID NO:17, position 3,079 of SEQ ID NO:18, position 3,692 of SEQ ID NO:19, position 3,046 of SEQ ID NO:20, position 2,925 of SEQ ID NO:21, position 3,054 of SEQ ID NO:22, or position 2,615 of SEQ ID NO:23, then the ARHGEF12 nucleic acid molecule in the biological sample is a predicted gain-of-function variant nucleic acid molecule of ARHGEF12.If the sequenced portion of the ARHGEF12 nucleic acid molecule in the biological sample contains a guanine at a position corresponding to position 4,748 of SEQ ID NO:24, position 4,190 of SEQ ID NO:25, position 4,078 of SEQ ID NO:26, position 4,691 of SEQ ID NO:27, position 4,045 of SEQ ID NO:28, position 3,924 of SEQ ID NO:29, position 3,614 of SEQ ID NO:30, or position 4,297 of SEQ ID NO:31, or contains a uracil at a position corresponding to position 3,739 of SEQ ID NO:32, position 3,627 of SEQ ID NO:33, position 4,240 of SEQ ID NO:34, position 3,594 of SEQ ID NO:35, position 3,473 of SEQ ID NO:36, position 3,602 of SEQ ID NO:37, or position 3,163 of SEQ ID NO:38, then the ARHGEF12 nucleic acid molecule in the biological sample is a predicted loss-of-function variant nucleic acid molecule of ARHGEF12.

[0110] In some embodiments, the determining, detecting, or genotyping assay comprises sequencing at least a portion of the nucleotide sequence of an ARHGEF12 cDNA molecule generated from an mRNA molecule in a biological sample, wherein the portion to be sequenced is at position 3,749 of SEQ ID NO:47 or its complement, position 3,191 of SEQ ID NO:48 or its complement, position 3,079 of SEQ ID NO:49 or its complement, position 3,692 of SEQ ID NO:50 or its complement, position 3,046 of SEQ ID NO:51 or its complement, position 2,925 of SEQ ID NO:52 or its complement, position 3,054 of SEQ ID NO:53 or its complement, position 2,615 of SEQ ID NO:54 or its complement, position 4,748 of SEQ ID NO:55 or its complement, position 4,190 of SEQ ID NO:56 or its complement, position 4,200 of SEQ ID NO:57 or its complement, position 4,078 of SEQ ID NO:58 or its complement, position 4,691 of SEQ ID NO:59 or its complement, position 4,045 of SEQ ID NO:60 or its complement, position 3,924 of SEQ ID NO:60 or its complement, position 3,614 of SEQ ID NO:61 or its complement, position 4,297 of SEQ ID NO:62 or its complement, position 3,379 of SEQ ID NO:63 or its complement, position 3,627 of SEQ ID NO:64 or its complement, position 4,240 of SEQ ID NO:65 or its complement, position 3,594 of SEQ ID NO:66 or its complement, position 3,473 of SEQ ID NO:67 or its complement, position 3,602 of SEQ ID NO:68 or its complement, or position 3,163 of SEQ ID NO:69 or its complement. If the sequenced portion of the ARHGEF12 nucleic acid molecule in the biological sample contains a thymine at a position corresponding to position 3,749 of SEQ ID NO:47, position 3,191 of SEQ ID NO:48, position 3,079 of SEQ ID NO:49, position 3,692 of SEQ ID NO:50, position 3,046 of SEQ ID NO:51, position 2,925 of SEQ ID NO:52, position 3,054 of SEQ ID NO:53, or position 2,615 of SEQ ID NO:54, the ARHGEF12 nucleic acid molecule in the biological sample is a predicted gain-of-function variant nucleic acid molecule of ARHGEF12.If the sequenced portion of the ARHGEF12 nucleic acid molecule in the biological sample comprises a guanine at a position corresponding to position 4,748 of SEQ ID NO:55, position 4,190 of SEQ ID NO:56, position 4,078 of SEQ ID NO:57, position 4,691 of SEQ ID NO:58, position 4,045 of SEQ ID NO:59, position 3,924 of SEQ ID NO:60, or position 3,614 of SEQ ID NO:61, or a thymine at a position corresponding to position 4,297 of SEQ ID NO:62, position 3,379 of SEQ ID NO:63, position 3,627 of SEQ ID NO:64, position 4,240 of SEQ ID NO:65, position 3,594 of SEQ ID NO:66, position 3,473 of SEQ ID NO:67, position 3,602 of SEQ ID NO:68, or position 3,163 of SEQ ID NO:69, then the ARHGEF12 nucleic acid molecule in the biological sample is a predicted loss-of-function variant nucleic acid molecule of ARHGEF12.

[0111] In some embodiments, the determining, detecting, or genotyping assay comprises: a) subjecting a biological sample to a nucleotide sequence of ARHGEF12, i.e., i) a genomic nucleic acid molecule adjacent to a position corresponding to position 132,939 of SEQ ID NO:2; ii) an mRNA molecule adjacent to a position corresponding to position 3,749 of SEQ ID NO:16, position 3,191 of SEQ ID NO:17, position 3,079 of SEQ ID NO:18, position 3,692 of SEQ ID NO:19, position 3,046 of SEQ ID NO:20, position 2,925 of SEQ ID NO:21, position 3,054 of SEQ ID NO:22, or position 2,615 of SEQ ID NO:23; and / or ii) a genomic nucleic acid molecule adjacent to a position corresponding to position 3,749 of SEQ ID NO:16, position 3,191 of SEQ ID NO:17, position 3,079 of SEQ ID NO:18, position 3,692 of SEQ ID NO:19, position 3,046 of SEQ ID NO:20, position 2,925 of SEQ ID NO:21, position 3,054 of SEQ ID NO:22, or position 2,615 of SEQ ID NO:23. i) contacting a cDNA molecule with a primer that hybridizes to a portion of the cDNA molecule adjacent to a position corresponding to position 3,749 of SEQ ID NO:47, position 3,191 of SEQ ID NO:48, position 3,079 of SEQ ID NO:49, position 3,692 of SEQ ID NO:50, position 3,046 of SEQ ID NO:51, position 2,925 of SEQ ID NO:52, position 3,054 of SEQ ID NO:53, or position 2,615 of SEQ ID NO:54; and b) contacting a cDNA molecule with a primer that hybridizes to a portion of the cDNA molecule adjacent to a position corresponding to position 3,749 of SEQ ID NO:16, position 3,191 of SEQ ID NO:17, position 3,692 of SEQ ID NO:50, position 3,046 of SEQ ID NO:51, position 2,925 of SEQ ID NO:52, position 3,054 of SEQ ID NO:53, or position 2,615 of SEQ ID NO:54, and / or iii) an mRNA molecule corresponding to position 3,749 of SEQ ID NO:47, position 3,191 of SEQ ID NO:48, position 3,079 of SEQ ID NO:49, position 3,692 of SEQ ID NO:50, position 3,046 of SEQ ID NO:51, position 2,925 of SEQ ID NO:52, position 3,054 of SEQ ID NO:53, or position 2,615 of SEQ ID NO:54; and c) extending the primer at least through a position in a cDNA molecule corresponding to position 3,749 of SEQ ID NO:47, position 3,191 of SEQ ID NO:48, position 3,079 of SEQ ID NO:49, position 3,692 of SEQ ID NO:50, position 3,046 of SEQ ID NO:51, position 2,925 of SEQ ID NO:52, position 3,054 of SEQ ID NO:53, or position 2,615 of SEQ ID NO:54. the product has i) a thymine at a position corresponding to position 132,939 of SEQ ID NO:2, ii) a uracil at a position corresponding to position 3,749 of SEQ ID NO:16, position 3,191 of SEQ ID NO:17, position 3,079 of SEQ ID NO:18, position 3,692 of SEQ ID NO:19, position 3,046 of SEQ ID NO:20, position 2,925 of SEQ ID NO:21, position 3,054 of SEQ ID NO:22, or position 2,615 of SEQ ID NO:23, or iii) a uracil at a position corresponding to position 3,749 of SEQ ID NO:47, position 3,191 of SEQ ID NO:48, position 3,079 of SEQ ID NO:49, position 3,692 of SEQ ID NO:50, position 3,046 of SEQ ID NO:51, position 2,615 of SEQ ID NO:52, orThe method includes determining whether the nucleic acid sequence contains a thymine at position 925 of SEQ ID NO:53, position 3,054 of SEQ ID NO:53, or position 2,615 of SEQ ID NO:54.

[0112] In some embodiments, the determining, detecting, or genotyping assay comprises: a) subjecting a biological sample to a genomic nucleic acid molecule adjacent to a position corresponding to position 143,698 of SEQ ID NO:3; ii) an mRNA molecule adjacent to a position corresponding to position 4,748 of SEQ ID NO:24, position 4,190 of SEQ ID NO:25, position 4,078 of SEQ ID NO:26, position 4,691 of SEQ ID NO:27, position 4,045 of SEQ ID NO:28, position 3,924 of SEQ ID NO:29, or position 3,614 of SEQ ID NO:30; and / or iii) an mRNA molecule adjacent to a position corresponding to position 4,748 of SEQ ID NO:55, position 4,190 of SEQ ID NO:25, position 4,078 of SEQ ID NO:26, position 4,691 of SEQ ID NO:27, position 4,045 of SEQ ID NO:28, position 3,924 of SEQ ID NO:29, or position 3,614 of SEQ ID NO:30. 8 of SEQ ID NO:56, position 4,190 of SEQ ID NO:56, position 4,078 of SEQ ID NO:57, position 4,691 of SEQ ID NO:58, position 4,045 of SEQ ID NO:59, position 3,924 of SEQ ID NO:60, or position 3,614 of SEQ ID NO:61; and b) contacting a primer that hybridizes to a portion of the cDNA molecule adjacent to a position corresponding to a nucleotide sequence of ARHGEF12, i.e., i) a genomic nucleic acid molecule corresponding to position 143,698 of SEQ ID NO:3, ii) position 4,748 of SEQ ID NO:24, position 4,190 of SEQ ID NO:25, position 4,078 of SEQ ID NO:26, position 4,691 of SEQ ID NO:27, position 4,222 of SEQ ID NO:28, position 4,222 of SEQ ID NO:29, position 4,311 of SEQ ID NO:30, position 4,311 of SEQ ID NO:31, position 4,311 of SEQ ID NO:32, position 4,311 of SEQ ID NO:33, position 4,311 of SEQ ID NO:34, position 4,311 of SEQ ID NO:35, position 4,311 of SEQ ID NO:36, position 4,311 of SEQ ID NO:37, position 4,311 of SEQ ID NO:38, position 4,311 of SEQ ID NO:39, position 4,311 of SEQ ID NO:40, position 4,311 of SEQ ID NO:41, position 4,311 of SEQ ID NO:42, position 4,311 of SEQ ID NO:43, position 4,311 of SEQ ID and / or iii) a cDNA molecule corresponding to position 4,748 of SEQ ID NO:55, position 4,190 of SEQ ID NO:56, position 4,078 of SEQ ID NO:57, position 4,691 of SEQ ID NO:58, position 4,045 of SEQ ID NO:59, position 3,924 of SEQ ID NO:60, or position 3,614 of SEQ ID NO:61, and c) extending the primer at least through a position in the mRNA molecule corresponding to position 4,045 of SEQ ID NO:28, position 3,924 of SEQ ID NO:29, or position 3,614 of SEQ ID NO:30, and / or or iii) a guanine at a position corresponding to position 4,748 of SEQ ID NO:24, position 4,190 of SEQ ID NO:25, position 4,078 of SEQ ID NO:26, position 4,691 of SEQ ID NO:27, position 4,045 of SEQ ID NO:28, position 3,924 of SEQ ID NO:29, or position 3,614 of SEQ ID NO:30; or ii) a guanine at a position corresponding to position 4,748 of SEQ ID NO:55, position 4,190 of SEQ ID NO:56, position 4,078 of SEQ ID NO:57, position 4,691 of SEQ ID NO:58, position 4,045 of SEQ ID NO:59, position 3,924 of SEQ ID NO:60, or position 3,614 of SEQ ID NO:61.

[0113] In some embodiments, the determining, detecting, or genotyping assay comprises: a) subjecting a biological sample to a nucleotide sequence of ARHGEF12, i.e., i) a genomic nucleic acid molecule adjacent to a position corresponding to position 141,048 of SEQ ID NO:4; ii) an mRNA molecule adjacent to a position corresponding to position 4,297 of SEQ ID NO:31, position 3,739 of SEQ ID NO:32, position 3,627 of SEQ ID NO:33, position 4,240 of SEQ ID NO:34, position 3,594 of SEQ ID NO:35, position 3,473 of SEQ ID NO:36, position 3,602 of SEQ ID NO:37, or position 3,163 of SEQ ID NO:38; and / or ii) a genomic nucleic acid molecule adjacent to a position corresponding to position 4,297 of SEQ ID NO:31, position 3,739 of SEQ ID NO:32, position 3,627 of SEQ ID NO:33, position 4,240 of SEQ ID NO:34, position 3,594 of SEQ ID NO:35, position 3,473 of SEQ ID NO:36, position 3,602 of SEQ ID NO:37, or position 3,163 of SEQ ID NO:38. i) contacting a cDNA molecule with a primer that hybridizes to a portion of the cDNA molecule adjacent to a position corresponding to position 4,297 of SEQ ID NO:62, position 3,379 of SEQ ID NO:63, position 3,627 of SEQ ID NO:64, position 4,240 of SEQ ID NO:65, position 3,594 of SEQ ID NO:66, position 3,473 of SEQ ID NO:67, position 3,602 of SEQ ID NO:68, or position 3,163 of SEQ ID NO:69; and b) contacting a cDNA molecule with a primer that hybridizes to a portion of the cDNA molecule adjacent to a position corresponding to position 4,297 of SEQ ID NO:62, position 3,379 of SEQ ID NO:63, position 3,627 of SEQ ID NO:64, position 4,240 of SEQ ID NO:65, position 3,594 of SEQ ID NO:66, position 3,473 of SEQ ID NO:67, position 3,602 of SEQ ID NO:68, or position 3,163 of SEQ ID NO:69, and and / or iii) a mRNA molecule corresponding to position 4,297 of SEQ ID NO:62, position 3,379 of SEQ ID NO:63, position 3,627 of SEQ ID NO:64, position 4,240 of SEQ ID NO:65, position 3,594 of SEQ ID NO:66, position 3,473 of SEQ ID NO:67, position 3,602 of SEQ ID NO:68, or position 3,163 of SEQ ID NO:69. and c) elongating the primer, and the extension product of the primer has i) a thymine at a position corresponding to position 141,048 of SEQ ID NO:4, ii) a uracil at a position corresponding to position 4,297 of SEQ ID NO:31, position 3,739 of SEQ ID NO:32, position 3,627 of SEQ ID NO:33, position 4,240 of SEQ ID NO:34, position 3,594 of SEQ ID NO:35, position 3,473 of SEQ ID NO:36, position 3,602 of SEQ ID NO:37, or position 3,163 of SEQ ID NO:38, or iii) a uracil at a position corresponding to position 4,297 of SEQ ID NO:62, position 3,379 of SEQ ID NO:63, position 3,627 of SEQ ID NO:64, position 4,240 of SEQ ID NO:65, position 3,The method includes determining whether the nucleic acid sequence contains a thymine at position 594 of SEQ ID NO: 67, position 3,473 of SEQ ID NO: 67, position 3,602 of SEQ ID NO: 68, or position 3,163 of SEQ ID NO: 69.

[0114] In some embodiments, the determining step, detecting step, or genotyping assay includes: a) contacting the biological sample with a primer that hybridizes to a portion of the nucleotide sequence of an ARHGEF12 genomic nucleic acid molecule adjacent to a position corresponding to position 73,039 of SEQ ID NO:5; b) extending the primer at least through the position of the nucleotide sequence of an ARHGEF12 genomic nucleic acid molecule corresponding to position 73,039 of SEQ ID NO:5; and c) determining whether the extension product of the primer comprises an adenine at the position corresponding to position 73,039 of SEQ ID NO:5.

[0115] In some embodiments, the determining step, detecting step, or genotyping assay comprises: a) contacting the biological sample with a primer that hybridizes to a portion of the nucleotide sequence of an ARHGEF12 genomic nucleic acid molecule adjacent to a position corresponding to position 121,307 of SEQ ID NO:6; b) extending the primer at least through the position of the nucleotide sequence of an ARHGEF12 genomic nucleic acid molecule corresponding to position 121,307 of SEQ ID NO:6; and c) determining whether the extension product of the primer comprises a cytosine at the position corresponding to position 121,307 of SEQ ID NO:6.

[0116] In some embodiments, the determining step, detecting step, or genotyping assay comprises: a) contacting the biological sample with a primer that hybridizes to a portion of the nucleotide sequence of an ARHGEF12 genomic nucleic acid molecule adjacent to a position corresponding to position 141,978 of SEQ ID NO:7; b) extending the primer at least through the position of the nucleotide sequence of an ARHGEF12 genomic nucleic acid molecule corresponding to position 141,978 of SEQ ID NO:7; and c) determining whether the extension product of the primer comprises a cytosine at the position corresponding to position 141,978 of SEQ ID NO:7.

[0117] In some embodiments, the determining, detecting, or genotyping assay comprises: a) contacting the biological sample with a primer that hybridizes to a portion of a nucleotide sequence of an ARHGEF12 genomic nucleic acid molecule adjacent to a position corresponding to position 132,939 of SEQ ID NO:2, position 143,698 of SEQ ID NO:3, position 141,048 of SEQ ID NO:4, position 73,039 of SEQ ID NO:5, position 121,307 of SEQ ID NO:6, or position 141,978 of SEQ ID NO:7; and b) contacting the biological sample with a primer that hybridizes to a portion of a nucleotide sequence of an ARHGEF12 genomic nucleic acid molecule adjacent to a position corresponding to position 132,939 of SEQ ID NO:2, position 143,698 of SEQ ID NO:3, position 141,048 of SEQ ID NO:4, position 73,039 of SEQ ID NO:5, position 121,307 of SEQ ID NO:6, or position 141,978 of SEQ ID NO:7. 307, or a portion of the nucleotide sequence of the ARHGEF12 genomic nucleic acid molecule corresponding to position 141,978 of SEQ ID NO:7; and c) determining whether the extension product of the primer comprises a thymine at a position corresponding to position 132,939 of SEQ ID NO:2, a guanine at a position corresponding to position 143,698 of SEQ ID NO:3, a thymine at a position corresponding to position 141,048 of SEQ ID NO:4, an adenine at a position corresponding to position 73,039 of SEQ ID NO:5, a cytosine at a position corresponding to position 121,307 of SEQ ID NO:6, or a cytosine at a position corresponding to position 141,978 of SEQ ID NO:7.

[0118] In some embodiments, the determining step, detecting step, or genotyping assay comprises: a) subjecting a biological sample to a sequence encoding a nucleotide sequence corresponding to positions 3,749 of SEQ ID NO:16, 3,191 of SEQ ID NO:17, 3,079 of SEQ ID NO:18, 3,692 of SEQ ID NO:19, 3,046 of SEQ ID NO:20, 2,925 of SEQ ID NO:21, 3,054 of SEQ ID NO:22, 2,615 of SEQ ID NO:23, 4,748 of SEQ ID NO:24, 4,190 of SEQ ID NO:25, 4,200 of SEQ ID NO:26, 4,220 of SEQ ID NO:27, 4,360 of SEQ ID NO:28, 4,400 of SEQ ID NO:29, 4,500 of SEQ ID NO:30, 4,600 of SEQ ID NO:31, 4,700 of SEQ ID NO:32, 4,800 of SEQ ID NO:33, 4,820 of SEQ ID NO:34, 4,900 of SEQ ID NO:35, 4,100 of SEQ ID NO:36, 4,220 of SEQ ID NO:37, 4,300 of SEQ ID NO:38, 4,400 of SEQ ID NO:39, 4,500 of SEQ ID NO:40, 4,600 of SEQ ID NO:41, 4,700 of SEQ ID NO:42, 4,820 of SEQ ID NO:43, 4,300 of SEQ ID NO:44, 4,400 of SEQ ID NO:45, 4,500 of SEQ ID NO:46, 4,600 of S ARHGEF12 adjacent to a position corresponding to position 4,691 of SEQ ID NO:27, position 4,045 of SEQ ID NO:28, position 3,924 of SEQ ID NO:29, position 3,614 of SEQ ID NO:30, position 4,297 of SEQ ID NO:31, position 3,739 of SEQ ID NO:32, position 3,627 of SEQ ID NO:33, position 4,240 of SEQ ID NO:34, position 3,594 of SEQ ID NO:35, position 3,473 of SEQ ID NO:36, position 3,602 of SEQ ID NO:37, or position 3,163 of SEQ ID NO:38. and b) contacting the mRNA molecule with a primer that hybridizes to a portion of the nucleotide sequence of the mRNA molecule at position 3,749 of SEQ ID NO:16, position 3,191 of SEQ ID NO:17, position 3,079 of SEQ ID NO:18, position 3,692 of SEQ ID NO:19, position 3,046 of SEQ ID NO:20, position 2,925 of SEQ ID NO:21, position 3,054 of SEQ ID NO:22, position 2,615 of SEQ ID NO:23, position 4,748 of SEQ ID NO:24, position 4,190 of SEQ ID NO:25, position 4,200 of SEQ ID NO:26, ARHGEF12 corresponding to position 4,078, position 4,691 of SEQ ID NO:27, position 4,045 of SEQ ID NO:28, position 3,924 of SEQ ID NO:29, position 3,614 of SEQ ID NO:30, position 4,297 of SEQ ID NO:31, position 3,739 of SEQ ID NO:32, position 3,627 of SEQ ID NO:33, position 4,240 of SEQ ID NO:34, position 3,594 of SEQ ID NO:35, position 3,473 of SEQ ID NO:36, position 3,602 of SEQ ID NO:37, or position 3,163 of SEQ ID NO:38 c) extending the primer through at least a position in the nucleotide sequence of the mRNA molecule, and c) determining whether the extension product of the primer contains i) a uracil at a position corresponding to position 3,749 of SEQ ID NO:16, position 3,191 of SEQ ID NO:17, position 3,079 of SEQ ID NO:18, position 3,692 of SEQ ID NO:19, position 3,046 of SEQ ID NO:20, position 2,925 of SEQ ID NO:21, position 3,054 of SEQ ID NO:22, or position 2,615 of SEQ ID NO:23; ii) a uracil at a position corresponding to position 4,748 of SEQ ID NO:24, position 4,190 of SEQ ID NO:25, position 4,iii) a guanine at a position corresponding to position 4,297 of SEQ ID NO:31, position 3,739 of SEQ ID NO:32, position 3,627 of SEQ ID NO:33, position 4,240 of SEQ ID NO:34, position 3,594 of SEQ ID NO:35, position 3,473 of SEQ ID NO:36, position 3,602 of SEQ ID NO:37, or position 3,163 of SEQ ID NO:38.

[0119] In some embodiments, the determining step, detecting step, or genotyping assay comprises: a) subjecting a biological sample to a sequence encoding a nucleotide sequence corresponding to position 3,749 of SEQ ID NO:47, position 3,191 of SEQ ID NO:48, position 3,079 of SEQ ID NO:49, position 3,692 of SEQ ID NO:50, position 3,046 of SEQ ID NO:51, position 2,925 of SEQ ID NO:52, position 3,054 of SEQ ID NO:53, position 2,615 of SEQ ID NO:54, position 4,748 of SEQ ID NO:55, position 4,190 of SEQ ID NO:56, position 4,200 of SEQ ID NO:57, position 4,220 of SEQ ID NO:58, position 4,230 of SEQ ID NO:59, position 4,360 of SEQ ID NO:60, position 4,400 of SEQ ID NO:61, position 4,500 of SEQ ID NO:62, position 4,600 of SEQ ID NO:63, position 4,748 of SEQ ID NO:64, position 4,190 of SEQ ID NO:65, position 4,200 of SEQ ID NO:66, position 4,360 of SEQ ID NO:67, position 4,400 of SEQ ID NO:68, position 4,500 of SEQ ID NO:69, position 4,600 of SEQ ID NO:69, position 4,748 of SEQ ID NO:65, position 4,230 of SEQ ID NO:69, position 4,400 of SEQ ID NO:69, position 4,500 of SEQ ID NO:61, position 4,500 of SEQ ID NO:62, position 4,500 of SEQ ARHGEF12 adjacent to a position corresponding to position 3,078 of SEQ ID NO:58, position 4,691 of SEQ ID NO:58, position 4,045 of SEQ ID NO:59, position 3,924 of SEQ ID NO:60, position 3,614 of SEQ ID NO:61, position 4,297 of SEQ ID NO:62, position 3,379 of SEQ ID NO:63, position 3,627 of SEQ ID NO:64, position 4,240 of SEQ ID NO:65, position 3,594 of SEQ ID NO:66, position 3,473 of SEQ ID NO:67, position 3,602 of SEQ ID NO:68, or position 3,163 of SEQ ID NO:69 and b) contacting the cDNA molecule with a primer that hybridizes to a portion of the nucleotide sequence of the cDNA molecule at position 3,749 of SEQ ID NO:47, position 3,191 of SEQ ID NO:48, position 3,079 of SEQ ID NO:49, position 3,692 of SEQ ID NO:50, position 3,046 of SEQ ID NO:51, position 2,925 of SEQ ID NO:52, position 3,054 of SEQ ID NO:53, position 2,615 of SEQ ID NO:54, position 4,748 of SEQ ID NO:55, position 4,190 of SEQ ID NO:56, position 4,200 of SEQ ID NO:57, ARHGEF12 corresponding to position 4,078 of SEQ ID NO:58, position 4,691 of SEQ ID NO:58, position 4,045 of SEQ ID NO:59, position 3,924 of SEQ ID NO:60, position 3,614 of SEQ ID NO:61, position 4,297 of SEQ ID NO:62, position 3,379 of SEQ ID NO:63, position 3,627 of SEQ ID NO:64, position 4,240 of SEQ ID NO:65, position 3,594 of SEQ ID NO:66, position 3,473 of SEQ ID NO:67, position 3,602 of SEQ ID NO:68, or position 3,163 of SEQ ID NO:69 and c) extending the primer through at least one of the nucleotide sequences of the cDNA molecule, and c) determining whether the extension product of the primer contains a thymine at a position corresponding to: i) position 3,749 of SEQ ID NO:47, position 3,191 of SEQ ID NO:48, position 3,079 of SEQ ID NO:49, position 3,692 of SEQ ID NO:50, position 3,046 of SEQ ID NO:51, position 2,925 of SEQ ID NO:52, position 3,054 of SEQ ID NO:53, or position 2,615 of SEQ ID NO:54; ii) position 4,748 of SEQ ID NO:55, position 4,190 of SEQ ID NO:56, position 4,iii) a guanine at a position corresponding to position 4,297 of SEQ ID NO:62, position 3,379 of SEQ ID NO:63, position 3,627 of SEQ ID NO:64, position 4,240 of SEQ ID NO:65, position 3,594 of SEQ ID NO:66, position 3,473 of SEQ ID NO:67, position 3,602 of SEQ ID NO:68, or position 3,163 of SEQ ID NO:69.

[0120] In some embodiments, the assay comprises sequencing the entire nucleic acid molecule. In some embodiments, only the ARHGEF12 genomic nucleic acid molecule is analyzed. In some embodiments, only the ARHGEF12 mRNA is analyzed. In some embodiments, only the ARHGEF12 cDNA obtained from the ARHGEF12 mRNA is analyzed.

[0121] In some embodiments, the determining, detecting, or genotyping assay comprises a) amplifying at least a portion of a nucleic acid molecule encoding a human ARHGEF12 polypeptide, where the amplified portion comprises: i) a thymine at a position corresponding to position 132,939 of SEQ ID NO:2 or its complement; ii) a thymine at position 3,749 of SEQ ID NO:16 or its complement; iii) a thymine at position 3,191 of SEQ ID NO:17 or its complement; iv) a thymine at position 3,079 of SEQ ID NO:18 or its complement; iv) a thymine at position 3,692 of SEQ ID NO:19 or its complement; v) a thymine at position 3,046 of SEQ ID NO:20 or its complement; and / or iii) a uracil at a position corresponding to position 3,749 of SEQ ID NO:47 or its complement, position 3,191 of SEQ ID NO:48 or its complement, position 3,079 of SEQ ID NO:49 or its complement, position 3,692 of SEQ ID NO:50 or its complement, position 3,046 of SEQ ID NO:51 or its complement, position 2,925 of SEQ ID NO:52 or its complement, position 2,615 of SEQ ID NO:53 or its complement. or position 3,054 of SEQ ID NO:54 or its complement, or position 2,615 of SEQ ID NO:54 or its complement); b) labeling the amplified nucleic acid molecule with a detectable label; and c) contacting the labeled nucleic acid molecule with a support comprising an alteration-specific probe, wherein the alteration-specific probe comprises a nucleotide sequence that hybridizes under stringent conditions to a nucleic acid sequence of the amplified nucleic acid molecule, the nucleic acid sequence comprising i) a thymine at a position corresponding to position 132,939 of SEQ ID NO:2 or its complement, ii) a thymine at a position corresponding to position 3,749 of SEQ ID NO:16 or its complement, iii) a thymine at a position corresponding to position 2,615 of SEQ ID NO:54 or its complement, or position 3,054 of SEQ ID NO:54 or its complement, or position 2,615 of SEQ ID NO:54 or its complement; 3,191 of SEQ ID NO:17 or its complement, 3,079 of SEQ ID NO:18 or its complement, 3,692 of SEQ ID NO:19 or its complement, 3,046 of SEQ ID NO:20 or its complement, 2,925 of SEQ ID NO:21 or its complement, 3,054 of SEQ ID NO:22 or its complement, or 2,615 of SEQ ID NO:23 or its complement; and / or iii) a uracil at a position corresponding to position 3,749 of SEQ ID NO:47 or its complement, position 3,191 of SEQ ID NO:48 or its complement, position 3,079 of SEQ ID NO:50 or its complement, 3,692 of SEQ ID NO:51 or its complement, 3,046 of SEQ ID NO:51 or its complement, 2,925 of SEQ ID NO:52 or its complement, 3,054 of SEQ ID NO:53 or its complement, or 2,615 of SEQ ID NO:54 or its complement); and d) detecting the detectable label.

[0122] In some embodiments, the determining, detecting, or genotyping assay comprises a) amplifying at least a portion of a nucleic acid molecule encoding a human ARHGEF12 polypeptide, wherein the amplified portion comprises a guanine at a position corresponding to i) position 143,698 of SEQ ID NO:3 or its complement, ii) position 4,748 of SEQ ID NO:24 or its complement, 4,190 of SEQ ID NO:25 or its complement, 4,078 of SEQ ID NO:26 or its complement, 4,691 of SEQ ID NO:27 or its complement, 4,045 of SEQ ID NO:28 or its complement, or iii) a guanine at a position corresponding to position 143,698 of SEQ ID NO:3 or its complement. and / or iii) a guanine at a position corresponding to position 4,748 of SEQ ID NO:55 or its complement, position 4,190 of SEQ ID NO:56 or its complement, position 4,078 of SEQ ID NO:57 or its complement, position 4,691 of SEQ ID NO:58 or its complement, position 4,045 of SEQ ID NO:59 or its complement, position 3,924 of SEQ ID NO:60 or its complement, or position 3,614 of SEQ ID NO:61 or its complement. b) labeling the amplified nucleic acid molecule with a detectable label; and c) contacting the labeled nucleic acid molecule with a support comprising an alteration-specific probe, wherein the alteration-specific probe comprises a nucleotide sequence that hybridizes under stringent conditions to a nucleic acid sequence of the amplified nucleic acid molecule, the nucleic acid sequence comprising i) a guanine at a position corresponding to position 143,698 of SEQ ID NO:3 or its complement, ii) a guanine at position 4,748 of SEQ ID NO:24 or its complement, ii) a guanine at position 4,190 of SEQ ID NO:25 or its complement, iii) a guanine at position 4,078 of SEQ ID NO:26 or its complement, iv) a guanine at position 4,211 of SEQ ID NO:27 or its complement, iv) a guanine at position 4,181 of SEQ ID NO:29 or its complement, iv) a guanine at position 4,221 of SEQ ID NO:30 or its complement, iv) a guanine at position 4,182 of SEQ ID NO:31 or its complement, iv) a guanine at position 4,183 of SEQ ID NO:32 or its complement, iv) a guanine at position 4,184 of SEQ ID NO:33 or its complement, iv) a guanine at position 4,185 of SEQ ID NO:34 or its complement, iv) a guanine at position 4,231 of SEQ ID NO:35 or its complement, iv) a guanine at position 4,170 of SEQ ID NO:36 or its complement, iv) a guanine at position 4,171 of SEQ ID NO:37 or its complement, iv) a guanine at position 4,172 of SEQ ID NO:38 or its complement, iv) a guanine at position 4, and / or iii) a guanine at a position corresponding to position 4,691 of SEQ ID NO:27 or its complement, position 4,045 of SEQ ID NO:28 or its complement, position 3,924 of SEQ ID NO:29 or its complement, or position 3,614 of SEQ ID NO:30 or its complement, and / or iv) a guanine at a position corresponding to position 4,748 of SEQ ID NO:55 or its complement, position 4,190 of SEQ ID NO:56 or its complement, position 4,078 of SEQ ID NO:57 or its complement, position 4,691 of SEQ ID NO:58 or its complement, position 4,045 of SEQ ID NO:59 or its complement, position 3,614 of SEQ ID NO:60 or its complement.924, or a guanine at a position corresponding to position 3,614 of SEQ ID NO: 61 or its complement); and d) detecting the detectable label.

[0123] In some embodiments, the determining, detecting, or genotyping assay comprises a) amplifying at least a portion of a nucleic acid molecule encoding a human ARHGEF12 polypeptide, where the amplified portion comprises: i) a thymine at a position corresponding to position 141,048 of SEQ ID NO:4 or its complement; ii) a thymine at position 4,297 of SEQ ID NO:31 or its complement; iii) a thymine at position 3,739 of SEQ ID NO:32 or its complement; iv) a thymine at position 4,297 of SEQ ID NO:31 or its complement; iv) a thymine at position 3,739 of SEQ ID NO:32 or its complement; iv) a thymine at position 4,240 of SEQ ID NO:34 or its complement; iv) a thymine at position 4,250 of SEQ ID NO:35 or its complement; and / or iii) a uracil at a position corresponding to position 4,297 of SEQ ID NO:62 or its complement, position 3,379 of SEQ ID NO:63 or its complement, position 3,627 of SEQ ID NO:64 or its complement, position 4,240 of SEQ ID NO:65 or its complement, position 3,594 of SEQ ID NO:66 or its complement, position 3,473 of SEQ ID NO:67 or its complement, position 3,595 of SEQ ID NO:68 or its complement, position 3,602 of SEQ ID NO:69 or its complement, position 3,802 of SEQ ID NO:61 or its complement, position 3,163 of SEQ ID NO:62 or its complement, position 3,473 of SEQ ID NO:61 or its complement, position 3,473 of SEQ ID NO:62 or its complement, position 3,602 of SEQ ID NO:61 or its complement, or position 3,163 of SEQ ID NO:62 or its complement, or at position 3,602 of SEQ ID NO: 31 or its complement, or at position 3,163 of SEQ ID NO: 69 or its complement); b) labeling the amplified nucleic acid molecule with a detectable label; and c) contacting the labeled nucleic acid molecule with a support comprising an alteration-specific probe, wherein the alteration-specific probe comprises a nucleotide sequence that hybridizes under stringent conditions to a nucleic acid sequence of the amplified nucleic acid molecule, the nucleic acid sequence comprising i) a thymine at position corresponding to position 141,048 of SEQ ID NO: 4 or its complement, ii) a thymine at position 4,297 of SEQ ID NO: 31 or its complement, iii) a thymine at position 4,312 of SEQ ID NO: 31 or its complement, iv) a thymine at position 4,293 of SEQ ID NO: 31 or its complement, or at position 4,311 of SEQ ID NO: 31 or its complement; 3,739 of SEQ ID NO:32 or its complement, 3,627 of SEQ ID NO:33 or its complement, 4,240 of SEQ ID NO:34 or its complement, 3,594 of SEQ ID NO:35 or its complement, 3,473 of SEQ ID NO:36 or its complement, 3,602 of SEQ ID NO:37 or its complement, or 3,163 of SEQ ID NO:38 or its complement; and / or iii) a uracil at a position corresponding to position 4,297 of SEQ ID NO:62 or its complement, position 3,379 of SEQ ID NO:63 or its complement, position 3,402 of SEQ ID NO:64 or its complement, or position 3,594 of SEQ ID NO:35 or its complement, position 3,473 of SEQ ID NO:36 or its complement, position 3,602 of SEQ ID NO:37 or its complement, or position 3,163 of SEQ ID NO:38 or its complement.627 of SEQ ID NO: 65 or its complement, 4,240 of SEQ ID NO: 66 or its complement, 3,594 of SEQ ID NO: 67 or its complement, 3,473 of SEQ ID NO: 67 or its complement, 3,602 of SEQ ID NO: 68 or its complement, or 3,163 of SEQ ID NO: 69 or its complement); d) detecting the detectable label.

[0124] In some embodiments, the determining step, detecting step, or genotyping assay comprises: a) amplifying at least a portion of a nucleic acid molecule encoding a human ARHGEF12 polypeptide, where the amplified portion comprises an adenine at a position corresponding to position 73,039 of SEQ ID NO:5 or its complement; b) labeling the amplified nucleic acid molecule with a detectable label; c) contacting the labeled nucleic acid molecule with a support comprising a variant-specific probe, where the variant-specific probe comprises a nucleotide sequence that hybridizes under stringent conditions to a nucleic acid sequence of the amplified nucleic acid molecule comprising an adenine at a position corresponding to position 73,039 of SEQ ID NO:5 or its complement; and d) detecting the detectable label.

[0125] In some embodiments, the determining step, detecting step, or genotyping assay comprises: a) amplifying at least a portion of a nucleic acid molecule encoding a human ARHGEF12 polypeptide, where the amplified portion comprises a cytosine at a position corresponding to position 121,307 of SEQ ID NO:6 or its complement; b) labeling the amplified nucleic acid molecule with a detectable label; c) contacting the labeled nucleic acid molecule with a support comprising a variant-specific probe, where the variant-specific probe comprises a nucleotide sequence that hybridizes under stringent conditions to a nucleic acid sequence of the amplified nucleic acid molecule comprising a cytosine at a position corresponding to position 121,307 of SEQ ID NO:6 or its complement; and d) detecting the detectable label.

[0126] In some embodiments, the determining step, detecting step, or genotyping assay comprises: a) amplifying at least a portion of a nucleic acid molecule encoding a human ARHGEF12 polypeptide, where the amplified portion comprises a cytosine at a position corresponding to position 141,978 of SEQ ID NO:7 or its complement; b) labeling the amplified nucleic acid molecule with a detectable label; c) contacting the labeled nucleic acid molecule with a support comprising a variant-specific probe, where the variant-specific probe comprises a nucleotide sequence that hybridizes under stringent conditions to a nucleic acid sequence of the amplified nucleic acid molecule comprising a cytosine at a position corresponding to position 141,978 of SEQ ID NO:7 or its complement; and d) detecting the detectable label.

[0127] In some embodiments, the determining, detecting, or genotyping assay comprises: a) amplifying at least a portion of a genomic nucleic acid molecule encoding a human ARHGEF12 polypeptide, wherein the amplified portion comprises a thymine at a position corresponding to position 132,939 of SEQ ID NO:2 or its complement, a guanine at a position corresponding to position 143,698 of SEQ ID NO:3 or its complement, a thymine at a position corresponding to position 141,048 of SEQ ID NO:4 or its complement, an adenine at a position corresponding to position 73,039 of SEQ ID NO:5 or its complement, or a cytosine at a position corresponding to position 121,307 of SEQ ID NO:6 or its complement; b) labeling the amplified nucleic acid molecule with a detectable label; and c) detecting the modified nucleic acid molecule. with a support comprising a mutation-specific probe, where the mutation-specific probe comprises a nucleotide sequence that hybridizes under stringent conditions to a nucleic acid sequence of the amplified nucleic acid molecule comprising a thymine at a position corresponding to position 132,939 of SEQ ID NO:2 or its complement, a guanine at a position corresponding to position 143,698 of SEQ ID NO:3 or its complement, a thymine at a position corresponding to position 141,048 of SEQ ID NO:4 or its complement, an adenine at a position corresponding to position 73,039 of SEQ ID NO:5 or its complement, a cytosine at a position corresponding to position 121,307 of SEQ ID NO:6 or its complement, or a cytosine at a position corresponding to position 141,978 of SEQ ID NO:7 or its complement; and d) detecting the detectable label.

[0128] In some embodiments, the determining, detecting, or genotyping assay comprises a) amplifying at least a portion of an mRNA molecule encoding a human ARHGEF12 polypeptide, where the amplified portion is: i) at position 3,749 of SEQ ID NO: 16 or its complement, at position 3,191 of SEQ ID NO: 17 or its complement, at position 3,079 of SEQ ID NO: 18 or its complement, at position 3,692 of SEQ ID NO: 19 or its complement, at position 3,046 of SEQ ID NO: 20 or its complement, at position 2,925 of SEQ ID NO: 21 or its complement, at position 2,200 of SEQ ID NO: 22 or its complement, ii) a uracil at a position corresponding to position 4,748 of SEQ ID NO:24 or its complement, position 4,190 of SEQ ID NO:25 or its complement, position 4,078 of SEQ ID NO:26 or its complement, position 4,691 of SEQ ID NO:27 or its complement, position 4,045 of SEQ ID NO:28 or its complement, position 3,924 of SEQ ID NO:29 or its complement, or position 3,614 of SEQ ID NO:30 or its complement; or iii) a guanine at a position corresponding to position 4,748 of SEQ ID NO:24 or its complement, position 4,190 of SEQ ID NO:25 or its complement, position 4,078 of SEQ ID NO:26 or its complement, position 4,691 of SEQ ID NO:27 or its complement, position 4,045 of SEQ ID NO:28 or its complement, position 3,924 of SEQ ID NO:29 or its complement, or position 3,614 of SEQ ID NO:30 or its complement. position 4,297 of SEQ ID NO: 32 or its complement, position 3,739 of SEQ ID NO: 33 or its complement, position 3,627 of SEQ ID NO: 34 or its complement, position 4,240 of SEQ ID NO: 34 or its complement, position 3,594 of SEQ ID NO: 35 or its complement, position 3,473 of SEQ ID NO: 36 or its complement, position 3,602 of SEQ ID NO: 37 or its complement, or position 3,163 of SEQ ID NO: 38 or its complement); b) labeling the amplified nucleic acid molecule with a detectable label; and c) contacting the labeled nucleic acid molecule with a support comprising an alteration-specific probe, wherein The variant-specific probe comprises a nucleotide sequence that hybridizes under stringent conditions to a nucleic acid sequence of the amplified nucleic acid molecule, the nucleic acid sequence being selected from the group consisting of i) position 3,749 of SEQ ID NO: 16 or its complement, position 3,191 of SEQ ID NO: 17 or its complement, position 3,079 of SEQ ID NO: 18 or its complement, position 3,692 of SEQ ID NO: 19 or its complement, position 3,046 of SEQ ID NO: 20 or its complement, position 2,925 of SEQ ID NO: 21 or its complement, position 3,054 of SEQ ID NO: 22 or its complement, or position 2,461 of SEQ ID NO: 23 or its complement.ii) a uracil at a position corresponding to position 4,748 of SEQ ID NO:24 or its complement, position 4,190 of SEQ ID NO:25 or its complement, position 4,078 of SEQ ID NO:26 or its complement, position 4,691 of SEQ ID NO:27 or its complement, position 4,045 of SEQ ID NO:28 or its complement, position 3,924 of SEQ ID NO:29 or its complement, or position 3,614 of SEQ ID NO:30 or its complement; or iii) a guanine at a position corresponding to SEQ ID NO:31 or or its complement, position 3,739 of SEQ ID NO:32 or its complement, position 3,627 of SEQ ID NO:33 or its complement, position 4,240 of SEQ ID NO:34 or its complement, position 3,594 of SEQ ID NO:35 or its complement, position 3,473 of SEQ ID NO:36 or its complement, position 3,602 of SEQ ID NO:37 or its complement, or position 3,163 of SEQ ID NO:38 or its complement); d) detecting the detectable label.

[0129] In some embodiments, the determining, detecting, or genotyping assay comprises a) amplifying at least a portion of a cDNA molecule encoding a human ARHGEF12 polypeptide, wherein the amplified portion is: i) at position 3,749 of SEQ ID NO:47 or its complement, at position 3,191 of SEQ ID NO:48 or its complement, at position 3,079 of SEQ ID NO:49 or its complement, at position 3,692 of SEQ ID NO:50 or its complement, at position 3,046 of SEQ ID NO:51 or its complement, at position 2,925 of SEQ ID NO:52 or its complement, at position 2,251 of SEQ ID NO:53 or its complement, ii) a thymine at position 4,748 of SEQ ID NO:55 or its complement, position 4,190 of SEQ ID NO:56 or its complement, position 4,078 of SEQ ID NO:57 or its complement, position 4,691 of SEQ ID NO:58 or its complement, position 4,045 of SEQ ID NO:59 or its complement, position 3,924 of SEQ ID NO:60 or its complement, or position 3,614 of SEQ ID NO:61 or its complement; or iii) a guanine at position 4,748 of SEQ ID NO:55 or its complement, position 4,190 of SEQ ID NO:56 or its complement, position 4,078 of SEQ ID NO:57 or its complement, position 4,691 of SEQ ID NO:58 or its complement, position 4,045 of SEQ ID NO:59 or its complement, position 3,924 of SEQ ID NO:60 or its complement, or position 3,614 of SEQ ID NO:61 or its complement; position 4,297 of SEQ ID NO:63 or its complement, position 3,379 of SEQ ID NO:64 or its complement, position 3,627 of SEQ ID NO:65 or its complement, position 4,240 of SEQ ID NO:65 or its complement, position 3,594 of SEQ ID NO:66 or its complement, position 3,473 of SEQ ID NO:67 or its complement, position 3,602 of SEQ ID NO:68 or its complement, or position 3,163 of SEQ ID NO:69 or its complement); b) labeling the amplified nucleic acid molecule with a detectable label; and c) contacting the labeled nucleic acid molecule with a support comprising an alteration-specific probe, wherein The variant-specific probe comprises a nucleotide sequence that hybridizes under stringent conditions to a nucleic acid sequence of the amplified nucleic acid molecule, the nucleic acid sequence being selected from the group consisting of i) position 3,749 of SEQ ID NO: 47 or its complement, position 3,191 of SEQ ID NO: 48 or its complement, position 3,079 of SEQ ID NO: 49 or its complement, position 3,692 of SEQ ID NO: 50 or its complement, position 3,046 of SEQ ID NO: 51 or its complement, position 2,925 of SEQ ID NO: 52 or its complement, position 3,054 of SEQ ID NO: 53 or its complement, or position 2,926 of SEQ ID NO: 54 or its complement.or ii) a guanine at a position corresponding to position 4,748 of SEQ ID NO:55 or its complement, position 4,190 of SEQ ID NO:56 or its complement, position 4,078 of SEQ ID NO:57 or its complement, position 4,691 of SEQ ID NO:58 or its complement, position 4,045 of SEQ ID NO:59 or its complement, position 3,924 of SEQ ID NO:60 or its complement, or position 3,614 of SEQ ID NO:61 or its complement; or iii) a guanine at a position corresponding to SEQ ID NO:62 or its complement. or position 4,297 of SEQ ID NO:63 or its complement, position 3,379 of SEQ ID NO:64 or its complement, position 3,627 of SEQ ID NO:65 or its complement, position 4,240 of SEQ ID NO:65 or its complement, position 3,594 of SEQ ID NO:66 or its complement, position 3,473 of SEQ ID NO:67 or its complement, position 3,602 of SEQ ID NO:68 or its complement, or position 3,163 of SEQ ID NO:69 or its complement); d) detecting the detectable label.

[0130] In some embodiments, the nucleic acid molecule is mRNA and the determining step further comprises reverse transcribing the mRNA into cDNA prior to the amplifying step. In some embodiments, the determining step, detecting step, or genotyping assay includes contacting a nucleic acid molecule in a biological sample with a variant-specific probe comprising a detectable label, wherein the variant-specific probe comprises a nucleotide sequence that hybridizes under stringent conditions to a nucleotide sequence of the amplified nucleic acid molecule, the nucleic acid molecule comprising i) a thymine at a position corresponding to position 132,939 of SEQ ID NO:2 or its complement, ii) a thymine at position 3,749 of SEQ ID NO:16 or its complement, 3,191 of SEQ ID NO:17 or its complement, 3,079 of SEQ ID NO:18 or its complement, 3,692 of SEQ ID NO:19 or its complement, 3,046 of SEQ ID NO:20 or its complement, 3,200 of SEQ ID NO:21 or its complement, 3,220 of SEQ ID NO:22 or its complement, 3,230 of SEQ ID NO:23 or its complement, 3,240 of SEQ ID NO:24 or its complement, 3,250 of SEQ ID NO:25 or its complement, 3,260 of SEQ ID NO:26 or its complement, 3,270 of SEQ ID NO:27 or its complement, 3,280 of SEQ ID NO:28 or its complement, 3,300 of SEQ ID NO:29 or its complement, 3,400 of SEQ ID NO:30 or its complement, 3,400 of SEQ ID NO:31 or its complement, 3,400 of SEQ ID NO:32 or its complement, 3,400 of SEQ ID NO:33 or its complement, 3,400 of SEQ ID NO:34 or its complement, 3,400 of SEQ ID NO:35 or its complement, 3,400 of SEQ ID NO:36 or its complement, 3,400 of SEQ ID NO:37 or its iii) a uracil at a position corresponding to position 3,749 of SEQ ID NO:47 or its complement, position 3,191 of SEQ ID NO:48 or its complement, position 3,079 of SEQ ID NO:49 or its complement, position 3,692 of SEQ ID NO:50 or its complement, position 3,046 of SEQ ID NO:51 or its complement, position 2,925 of SEQ ID NO:52 or its complement, position 3,054 of SEQ ID NO:53 or its complement, or position 2,615 of SEQ ID NO:54 or its complement), detecting the detectable label.

[0131] In some embodiments, the determining step, detecting step, or genotyping assay comprises contacting a nucleic acid molecule in a biological sample with a variant-specific probe comprising a detectable label, wherein the variant-specific probe comprises a nucleotide sequence that hybridizes under stringent conditions to a nucleotide sequence of the amplified nucleic acid molecule, the nucleic acid molecule containing a guanine at a position corresponding to i) position 143,698 of SEQ ID NO:3 or its complement, ii) position 4,748 of SEQ ID NO:24 or its complement, 4,190 of SEQ ID NO:25 or its complement, 4,078 of SEQ ID NO:26 or its complement, 4,691 of SEQ ID NO:27 or its complement, iii) position 4,812 of SEQ ID NO:28 or its complement, iv) position 4,826 of SEQ ID NO:29 or its complement, iv) position 4,912 of SEQ ID NO:30 or its complement, iv) position 4,832 of SEQ ID NO:31 or its complement, iv) position 4,846 of SEQ ID NO:32 or its complement, iv) position 4,850 of SEQ ID NO:33 or its complement, iv) position 4,928 of SEQ ID NO:34 or its complement, iv) position 4,932 of SEQ ID NO:35 or its complement, iv) position 4,886 of SEQ ID NO:36 or its complement, iv) position 4,912 of SEQ ID NO:37 or its complement, iv) position 4,926 of SEQ ID NO:38 or its complement, iv) position 4,912 of SEQ ID NO:39 or its complement, iv) position 4,912 of SEQ ID NO:40 or its complement, iv) position 4,926 of SEQ ID NO:41 or its complement, iv) position 4,912 of SEQ ID NO:42 and / or iii) a guanine at a position corresponding to position 4,748 of SEQ ID NO:55 or its complement, position 4,190 of SEQ ID NO:56 or its complement, position 4,078 of SEQ ID NO:57 or its complement, position 4,691 of SEQ ID NO:58 or its complement, position 4,045 of SEQ ID NO:59 or its complement, position 3,924 of SEQ ID NO:60 or its complement, or position 3,614 of SEQ ID NO:61 or its complement), detecting the detectable label.

[0132] In some embodiments, the determining step, detecting step, or genotyping assay includes contacting a nucleic acid molecule in a biological sample with a variant-specific probe comprising a detectable label, wherein the variant-specific probe comprises a nucleotide sequence that hybridizes under stringent conditions to a nucleotide sequence of the amplified nucleic acid molecule, the nucleic acid molecule comprising i) a thymine at a position corresponding to position 141,048 of SEQ ID NO:4 or its complement, ii) a thymine at position 4,297 of SEQ ID NO:31 or its complement, 3,739 of SEQ ID NO:32 or its complement, 3,627 of SEQ ID NO:33 or its complement, 4,240 of SEQ ID NO:34 or its complement, 3,594 of SEQ ID NO:35 or its complement, iii) a thymine at position 4,297 of SEQ ID NO:31 or its complement, 4,371 of SEQ ID NO:32 or its complement, 4,370 of SEQ ID NO:33 or its complement, 4,242 of SEQ ID NO:34 or its complement, 4,373 of SEQ ID NO:35 or its complement, 4,374 of SEQ ID NO:35 or its complement, 4,375 of SEQ ID NO:35 or its complement, 4,376 of SEQ ID NO:35 or its complement, 4,377 of SEQ ID NO:35 or its complement, 4,378 of SEQ ID NO:35 or its complement, 4,379 of SEQ ID NO:35 or its complement, 5,400 of SEQ ID NO:35 or its complement, 5,400 of SEQ ID NO:35 or its complement, 6,400 of SEQ ID NO:35 or its complement, 7,400 of SEQ ID NO:35 or its complement, 8,400 of SEQ ID NO:35 or its complement, 9,400 of and / or iii) a thymine at a position corresponding to position 4,297 of SEQ ID NO:62 or its complement, position 3,379 of SEQ ID NO:63 or its complement, position 3,627 of SEQ ID NO:64 or its complement, position 4,240 of SEQ ID NO:65 or its complement, position 3,594 of SEQ ID NO:66 or its complement, position 3,473 of SEQ ID NO:67 or its complement, position 3,602 of SEQ ID NO:68 or its complement, or position 3,163 of SEQ ID NO:69 or its complement), detecting the detectable label.

[0133] In some embodiments, the determining step, detecting step, or genotyping assay comprises contacting a nucleic acid molecule in a biological sample with a variant-specific probe comprising a detectable label (wherein the variant-specific probe comprises a nucleotide sequence that hybridizes under stringent conditions to a nucleotide sequence of the amplified nucleic acid molecule that comprises an adenine at a position corresponding to position 73,039 of SEQ ID NO:5 or its complement), and detecting the detectable label.

[0134] In some embodiments, the determining step, detecting step, or genotyping assay comprises contacting a nucleic acid molecule in a biological sample with a variant-specific probe comprising a detectable label (wherein the variant-specific probe comprises a nucleotide sequence that hybridizes under stringent conditions to a nucleotide sequence of the amplified nucleic acid molecule that comprises a cytosine at a position corresponding to position 121,307 of SEQ ID NO:6 or its complement) and detecting the detectable label.

[0135] In some embodiments, the determining step, detecting step, or genotyping assay comprises contacting a nucleic acid molecule in a biological sample with a variant-specific probe comprising a detectable label (wherein the variant-specific probe comprises a nucleotide sequence that hybridizes under stringent conditions to a nucleotide sequence of the amplified nucleic acid molecule that comprises a cytosine at a position corresponding to position 141,978 of SEQ ID NO:7 or its complement) and detecting the detectable label.

[0136] In some embodiments, the determining step, detecting step, or genotyping assay comprises contacting the nucleic acid molecule in the biological sample with a variant-specific probe comprising a detectable label (wherein the variant-specific probe comprises a nucleotide sequence that hybridizes under stringent conditions to a nucleotide sequence of the amplified nucleic acid molecule comprising a thymine at a position corresponding to position 132,939 of SEQ ID NO:2 or its complement, a guanine at a position corresponding to position 143,698 of SEQ ID NO:3 or its complement, a thymine at a position corresponding to position 141,048 of SEQ ID NO:4 or its complement, an adenine at a position corresponding to position 73,039 of SEQ ID NO:5 or its complement, a cytosine at a position corresponding to position 121,307 of SEQ ID NO:6 or its complement, or a cytosine at a position corresponding to position 141,978 of SEQ ID NO:7 or its complement) and detecting the detectable label.

[0137] In some embodiments, the determining step, detecting step, or genotyping assay comprises contacting a nucleic acid molecule in a biological sample with a variant-specific probe comprising a detectable label, wherein the variant-specific probe contains i) a uracil at a position corresponding to position 3,749 of SEQ ID NO:16 or its complement, position 3,191 of SEQ ID NO:17 or its complement, position 3,079 of SEQ ID NO:18 or its complement, position 3,692 of SEQ ID NO:19 or its complement, position 3,046 of SEQ ID NO:20 or its complement, position 2,925 of SEQ ID NO:21 or its complement, position 3,054 of SEQ ID NO:22 or its complement, position 2,615 of SEQ ID NO:23 or its complement, or position 4,748 of SEQ ID NO:24 or its complement; ii) a uracil at a position corresponding to position 4,190 of SEQ ID NO:25 or its complement, position 4,078 of SEQ ID NO:26 or its complement, position 4,079 of SEQ ID NO:27 or its complement, or iii) a guanine at a position corresponding to position 3,739 of SEQ ID NO:32 or its complement, position 3,627 of SEQ ID NO:33 or its complement, position 4,240 of SEQ ID NO:34 or its complement, position 3,594 of SEQ ID NO:35 or its complement, position 3,473 of SEQ ID NO:36 or its complement, position 3,602 of SEQ ID NO:37 or its complement, or position 3,163 of SEQ ID NO:38 or its complement), detecting the detectable label.

[0138] In some embodiments, the determining step, detecting step, or genotyping assay comprises contacting a nucleic acid molecule in a biological sample with a variant-specific probe comprising a detectable label, wherein the variant-specific probe contains i) a thymine at a position corresponding to position 3,749 of SEQ ID NO:47 or its complement, position 3,191 of SEQ ID NO:48 or its complement, position 3,079 of SEQ ID NO:49 or its complement, position 3,692 of SEQ ID NO:50 or its complement, position 3,046 of SEQ ID NO:51 or its complement, position 2,925 of SEQ ID NO:52 or its complement, position 3,054 of SEQ ID NO:53 or its complement, or position 2,615 of SEQ ID NO:54 or its complement; ii) a thymine at a position corresponding to position 4,748 of SEQ ID NO:55 or its complement, position 4,190 of SEQ ID NO:56 or its complement, position 4,078 of SEQ ID NO:57 or its complement, position 4,191 of SEQ ID NO:58 or its complement, position 4,078 of SEQ ID NO:59 or its complement, position 4,200 of SEQ ID NO:59 or its complement, position 4,300 of SEQ ID NO:59 or its complement, position 5,000 of SEQ ID NO:59 or its complement, position 6,000 of SEQ ID NO:59 or its complement, position 7,000 of SEQ ID NO:59 or its complement, position 8,000 of SEQ ID NO:59 or its complement, position 9,000 of SEQ ID NO:59 or its complement, position 10,000 of SEQ ID NO:59 or its complement, position 11,000 of SEQ ID NO:59 or its complement, position 12,000 of SEQ ID NO:59 or its complement iii) a guanine at a position corresponding to position 4,691 of SEQ ID NO:59 or its complement, position 4,045 of SEQ ID NO:60 or its complement, position 3,924 of SEQ ID NO:60 or its complement, or position 3,614 of SEQ ID NO:61 or its complement; and iii) a thymine at a position corresponding to position 4,297 of SEQ ID NO:62 or its complement, position 3,379 of SEQ ID NO:63 or its complement, position 3,627 of SEQ ID NO:64 or its complement, position 4,240 of SEQ ID NO:65 or its complement, position 3,594 of SEQ ID NO:66 or its complement, position 3,473 of SEQ ID NO:67 or its complement, position 3,602 of SEQ ID NO:68 or its complement, or position 3,163 of SEQ ID NO:69 or its complement), detecting the detectable label.

[0139] Variation-specific polymerase chain reaction techniques can be used to detect mutations, such as SNPs, in nucleic acid sequences. Variation-specific primers can be used because DNA polymerase will not extend if there is a mismatch with the template.

[0140] In some embodiments, the nucleic acid molecule in the sample is mRNA, and the mRNA is reverse transcribed into cDNA prior to the amplification step. In some embodiments, the nucleic acid molecule is present in a cell obtained from the subject.

[0141] In some embodiments, the assay involves contacting the biological sample with a primer or probe, such as a variant-specific primer or variant-specific probe, that specifically hybridizes under stringent conditions to an ARHGEF12 variant genomic sequence, variant mRNA sequence, or variant cDNA sequence and does not hybridize to the corresponding ARHGEF12 reference sequence, and determining whether hybridization occurs.

[0142] In some embodiments, the assay involves RNA sequencing (RNA-Seq). In some embodiments, the assay also involves reverse transcribing mRNA into cDNA, for example, by reverse transcription polymerase chain reaction (RT-PCR).

[0143] In some embodiments, the method utilizes probes and primers of sufficient nucleotide length to bind to the target nucleotide sequence and specifically detect and / or distinguish polynucleotides including ARHGEF12 variant genomic nucleic acid molecules, variant mRNA molecules, or variant cDNA molecules. Hybridization or reaction conditions can be determined by the operator to achieve this result. The nucleotide length can be any length that is sufficient for use in an optimal detection method, including any assay described or exemplified herein. Such probes and primers can specifically hybridize to the target nucleotide sequence under highly stringent hybridization conditions. The probes and primers may have complete nucleotide sequence identity of consecutive nucleotides in the target nucleotide sequence, although probes that differ from the target nucleotide sequence but retain the ability to specifically detect and / or distinguish the target nucleotide sequence may be designed by conventional methods. The probes and primers can have about 80%, about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or 100% sequence identity or complementarity to the nucleotide sequence of the target nucleic acid molecule.

[0144] In some embodiments, the ARHGEF12 nucleic acid molecule (genomic nucleic acid molecule, mRNA molecule, or cDNA molecule) or its complement in the biological sample contains a thymine (for genomic nucleic acid molecules) at a position corresponding to position 132,939 of SEQ ID NO:2; i) a uracil (for mRNA molecules) at a position corresponding to position 3,749 of SEQ ID NO:16, position 3,191 of SEQ ID NO:17, position 3,079 of SEQ ID NO:18, position 3,692 of SEQ ID NO:19, position 3,046 of SEQ ID NO:20, position 2,925 of SEQ ID NO:21, position 3,054 of SEQ ID NO:22, or position 2,615 of SEQ ID NO:23; ii) a uracil (for mRNA molecules) at a position corresponding to position 3,749 of SEQ ID NO:16, position 3,191 of SEQ ID NO:17, position 3,079 of SEQ ID NO:18, position 3,692 of SEQ ID NO:19, position 3,046 of SEQ ID NO:20, position 2,925 of SEQ ID NO:21, position 3,054 of SEQ ID NO:22, or position 2,615 of SEQ ID NO:23; To determine whether a biological sample contains a nucleotide sequence containing a thymine (for a cDNA molecule) at a position corresponding to position 3,749 of sequence number 47, position 3,191 of SEQ ID NO:48, position 3,079 of SEQ ID NO:49, position 3,692 of SEQ ID NO:50, position 3,046 of SEQ ID NO:51, position 2,925 of SEQ ID NO:52, position 3,054 of SEQ ID NO:53, or position 2,615 of SEQ ID NO:54, the biological sample is subjected to a nucleotide sequence analysis using ... ii) a uracil at a position corresponding to position 3,749 of SEQ ID NO:47, position 3,191 of SEQ ID NO:48, position 3,079 of SEQ ID NO:49, position 3,692 of SEQ ID NO:50, position 3,046 of SEQ ID NO:51, position 2,925 of SEQ ID NO:52, position 3,054 of SEQ ID NO:53, or position 2,615 of SEQ ID NO:54, and a thymine at a position corresponding to position 132 of SEQ ID NO:20, position 3,046 of SEQ ID NO:20, position 2,925 of SEQ ID NO:21, position 3,054 of SEQ ID NO:22, or position 2,615 of SEQ ID NO:23; i) a thymine at a position corresponding to position 3,749 of SEQ ID NO:16, position 3,191 of SEQ ID NO:17, position 3,079 of SEQ ID NO:18, position 3,692 of SEQ ID NO:19, position 3,046 of SEQ ID NO:20, position 2,925 of SEQ ID NO:21, position 3,054 of SEQ ID NO:22, or position 2,615 of SEQ ID NO:23; or ii) a uracil at a position corresponding to position 3,749 of SEQ ID NO:47, position 3,191 of SEQ ID NO:48, position 3,079 of SEQ ID NO:49, position 3,692 of SEQ ID NO:50, position 3,046 of SEQ ID NO:51, position 2,925 of SEQ ID NO:52, position 3,and a second primer derived from a 3'-flanking sequence adjacent to a thymine at a position corresponding to position 132,939 of SEQ ID NO:2, i) position 3,749 of SEQ ID NO:16, position 3,191 of SEQ ID NO:17, position 3,079 of SEQ ID NO:18, position 3,692 of SEQ ID NO:19, position 3,046 of SEQ ID NO:20, position 2,925 of SEQ ID NO:21, position 2,615 of SEQ ID NO:22, 3,054 or uracil at position corresponding to position 2,615 of SEQ ID NO:23, or ii) thymine at position corresponding to position 3,749 of SEQ ID NO:47, position 3,191 of SEQ ID NO:48, position 3,079 of SEQ ID NO:49, position 3,692 of SEQ ID NO:50, position 3,046 of SEQ ID NO:51, position 2,925 of SEQ ID NO:52, position 3,054 of SEQ ID NO:53, or position 2,615 of SEQ ID NO:54. In some embodiments, the length of the amplicon can range from the primer pair plus one nucleotide base pair to any length of amplicon that can be generated by the DNA amplification protocol. This distance can range from one nucleotide base pair to the limit of the amplification reaction, i.e., about 20,000 nucleotide base pairs. Optionally, the primer pair is a primer that is complementary to a thymine at a position corresponding to position 132,939 of SEQ ID NO:2; i) a uracil at a position corresponding to position 3,749 of SEQ ID NO:16, position 3,191 of SEQ ID NO:17, position 3,079 of SEQ ID NO:18, position 3,692 of SEQ ID NO:19, position 3,046 of SEQ ID NO:20, position 2,925 of SEQ ID NO:21, position 3,054 of SEQ ID NO:22, or position 2,615 of SEQ ID NO:23; or ii) a uracil at a position corresponding to position 3,749 of SEQ ID NO:47, position 3,191 of SEQ ID NO:48, position 3,079 of SEQ ID NO:49, position 3,692 of SEQ ID NO:19, position 3,046 of SEQ ID NO:20, position 2,925 of SEQ ID NO:21, position 3,054 of SEQ ID NO:22, or position 2,615 of SEQ ID NO:23. i) a thymine at a position corresponding to position 3,692 of SEQ ID NO:51, position 3,046 of SEQ ID NO:51, position 2,925 of SEQ ID NO:52, position 3,054 of SEQ ID NO:53, or position 2,615 of SEQ ID NO:54, and a thymine at a position corresponding to position 132,939 of SEQ ID NO:2; i) a thymine at a position corresponding to position 3,749 of SEQ ID NO:16, position 3,191 of SEQ ID NO:17, position 3,079 of SEQ ID NO:18, position 3,692 of SEQ ID NO:19, position 3,046 of SEQ ID NO:20, position 2,925 of SEQ ID NO:21, position 3,054 of SEQ ID NO:22, or position 2,615 of SEQ ID NO:23or ii) a uracil at a position corresponding to position 615 of SEQ ID NO: 47, position 3,191 of SEQ ID NO: 48, position 3,079 of SEQ ID NO: 49, position 3,692 of SEQ ID NO: 50, position 3,046 of SEQ ID NO: 51, position 2,925 of SEQ ID NO: 52, position 3,054 of SEQ ID NO: 53, or position 2,615 of SEQ ID NO: 54.

[0145] In some embodiments, an ARHGEF12 nucleic acid molecule (genomic nucleic acid molecule, mRNA molecule, or cDNA molecule) or its complement in a biological sample contains a guanine at a position corresponding to position 143,698 of SEQ ID NO:3 (genomic nucleic acid molecule); i) a guanine at a position corresponding to position 4,748 of SEQ ID NO:24, position 4,190 of SEQ ID NO:25, position 4,078 of SEQ ID NO:26, position 4,691 of SEQ ID NO:27, position 4,045 of SEQ ID NO:28, position 3,924 of SEQ ID NO:29, or position 3,614 of SEQ ID NO:30 (for mRNA molecule); ii) To determine whether a biological sample contains a nucleotide sequence containing a guanine (in the case of a cDNA molecule) at a position corresponding to position 4,748 of SEQ ID NO:55, position 4,190 of SEQ ID NO:56, position 4,078 of SEQ ID NO:57, position 4,691 of SEQ ID NO:58, position 4,045 of SEQ ID NO:59, position 3,924 of SEQ ID NO:60, or position 3,614 of SEQ ID NO:61, the biological sample is subjected to a nucleotide sequence analysis using a nucleotide sequence containing a guanine at a position corresponding to position 143,698 of SEQ ID NO:3, i) position 4,748 of SEQ ID NO:24, position 4,190 of SEQ ID NO:25, position 4,078 of SEQ ID NO:26, position 4,190 of SEQ ID NO:27, position 4,078 of SEQ ID NO:28, position 4,222 of SEQ ID NO:29, position 4,311 of SEQ ID NO:30, position 4,223 of SEQ ID NO:31, position 4,311 of SEQ ID NO:32, position 4,311 of SEQ ID NO:33, position 4,311 of SEQ ID NO:34, position 4,311 of SEQ ID NO:35, position 4,311 of SEQ ID NO:36, position 4,311 of SEQ ID NO:37, position 4,311 of SEQ ID NO:38, position 4,311 of SEQ ID NO:39, position 4,311 of SEQ ID NO:40, position 4,311 of SEQ ID NO:41, position 4,311 of SEQ ID NO:42, position 4,311 of SEQ ID NO:43, or ii) a guanine at a position corresponding to position 4,691 of SEQ ID NO:55, position 4,045 of SEQ ID NO:28, position 3,924 of SEQ ID NO:29, or position 3,614 of SEQ ID NO:30; or ii) a guanine at a position corresponding to position 4,748 of SEQ ID NO:55, position 4,190 of SEQ ID NO:56, position 4,078 of SEQ ID NO:57, position 4,691 of SEQ ID NO:58, position 4,045 of SEQ ID NO:59, position 3,924 of SEQ ID NO:60, or position 3,614 of SEQ ID NO:61, i) a guanine at a position corresponding to position 4,748 of SEQ ID NO:24, position 4,190 of SEQ ID NO:25, position 4,078 of SEQ ID NO:26, position 4,691 of SEQ ID NO:27, position 4,045 of SEQ ID NO:28, position 3,924 of SEQ ID NO:29, or position 3,614 of SEQ ID NO:30, or ii) a guanine at a position corresponding to position 4,748 of SEQ ID NO:55, position 4,190 of SEQ ID NO:56, position 4,078 of SEQ ID NO:57, position 4,691 of SEQ ID NO:58, position 4,045 of SEQ ID NO:59, position 3,924 of SEQ ID NO:60, or position 3,614 of SEQ ID NO:61.The primers may be subjected to an amplification method using a primer pair comprising a second primer derived from the 3' flanking sequence adjacent to the guanine at position corresponding to position 614 to generate an amplicon indicative of the presence of a SNP at a position encoding a guanine at a position corresponding to position 143,698 of SEQ ID NO:3; i) a guanine at a position corresponding to position 4,748 of SEQ ID NO:24, position 4,190 of SEQ ID NO:25, position 4,078 of SEQ ID NO:26, position 4,691 of SEQ ID NO:27, position 4,045 of SEQ ID NO:28, position 3,924 of SEQ ID NO:29, or position 3,614 of SEQ ID NO:30; or ii) a guanine at a position corresponding to position 4,748 of SEQ ID NO:55, position 4,190 of SEQ ID NO:56, position 4,078 of SEQ ID NO:57, position 4,691 of SEQ ID NO:58, position 4,045 of SEQ ID NO:59, position 3,924 of SEQ ID NO:60, or position 3,614 of SEQ ID NO:61. In some embodiments, the length of the amplicon can range from the primer pair plus one nucleotide base pair to any length of the amplicon that can be generated by the DNA amplification protocol. This distance can range from one nucleotide base pair to the limit of the amplification reaction, i.e., about 20,000 nucleotide base pairs. Optionally, the primer pair is a guanine at a position corresponding to position 143,698 of SEQ ID NO:3, i) a guanine at a position corresponding to position 4,748 of SEQ ID NO:24, position 4,190 of SEQ ID NO:25, position 4,078 of SEQ ID NO:26, position 4,691 of SEQ ID NO:27, position 4,045 of SEQ ID NO:28, position 3,924 of SEQ ID NO:29, or position 3,614 of SEQ ID NO:30, or ii) a guanine at a position corresponding to position 4,748 of SEQ ID NO:55, position 4,190 of SEQ ID NO:56, position 4,078 of SEQ ID NO:57, position 4,691 of SEQ ID NO:58, position 4,045 of SEQ ID NO:59, position 3,614 of SEQ ID NO:6 ...1, or ii) a guanine at a position corresponding to position 4,748 of SEQ ID NO:55, position 4,190 of SEQ ID NO:56, position 4,07 a position including a guanine at position 924, or at a position corresponding to position 3,614 of SEQ ID NO:61, and a guanine at a position corresponding to position 143,698 of SEQ ID NO:3; i) a guanine at a position corresponding to position 4,748 of SEQ ID NO:24, position 4,190 of SEQ ID NO:25, position 4,078 of SEQ ID NO:26, position 4,691 of SEQ ID NO:27, position 4,045 of SEQ ID NO:28, position 3,924 of SEQ ID NO:29, or position 3,614 of SEQ ID NO:30; or iii) a guanine at a position corresponding to position 4,748 of SEQ ID NO:55, position 4,190 of SEQ ID NO:56, position 4,078 of SEQ ID NO:57, position 4,691, 4,045 of SEQ ID NO:59, 3,924 of SEQ ID NO:60, or 3,614 of SEQ ID NO:61.

[0146] In some embodiments, the ARHGEF12 nucleic acid molecule (genomic nucleic acid molecule, mRNA molecule, or cDNA molecule) or its complement in the biological sample contains a thymine (for genomic nucleic acid molecules) at a position corresponding to position 141,048 of SEQ ID NO:4; i) a uracil (for mRNA molecules) at a position corresponding to position 4,297 of SEQ ID NO:31, position 3,739 of SEQ ID NO:32, position 3,627 of SEQ ID NO:33, position 4,240 of SEQ ID NO:34, position 3,594 of SEQ ID NO:35, position 3,473 of SEQ ID NO:36, position 3,602 of SEQ ID NO:37, or position 3,163 of SEQ ID NO:38; ii) a uracil (for mRNA molecules) at a position corresponding to position 4,297 of SEQ ID NO:31, position 3,739 of SEQ ID NO:32, position 3,627 of SEQ ID NO:33, position 4,240 of SEQ ID NO:34, position 3,594 of SEQ ID NO:35, position 3,473 of SEQ ID NO:36, position 3,602 of SEQ ID NO:37, or position 3,163 of SEQ ID NO:38; To determine whether a biological sample contains a nucleotide sequence containing a thymine (for a cDNA molecule) at a position corresponding to position 4,297 of sequence number 62, position 3,379 of SEQ ID NO:63, position 3,627 of SEQ ID NO:64, position 4,240 of SEQ ID NO:65, position 3,594 of SEQ ID NO:66, position 3,473 of SEQ ID NO:67, position 3,602 of SEQ ID NO:68, or position 3,163 of SEQ ID NO:69, the biological sample is subjected to a nucleotide sequence analysis using ... ii) a uracil at a position corresponding to position 4,297 of SEQ ID NO:62, position 3,379 of SEQ ID NO:63, position 3,627 of SEQ ID NO:64, position 4,240 of SEQ ID NO:65, position 3,594 of SEQ ID NO:66, position 3,473 of SEQ ID NO:67, position 3,602 of SEQ ID NO:68, or position 3,163 of SEQ ID NO:69, and a thymine at a position corresponding to position 141 of SEQ ID NO:4 i) a thymine at a position corresponding to position 4,297 of SEQ ID NO:31, position 3,739 of SEQ ID NO:32, position 3,627 of SEQ ID NO:33, position 4,240 of SEQ ID NO:34, position 3,594 of SEQ ID NO:35, position 3,473 of SEQ ID NO:36, position 3,602 of SEQ ID NO:37, or position 3,163 of SEQ ID NO:38, or ii) a uracil at a position corresponding to position 4,297 of SEQ ID NO:62, position 3,379 of SEQ ID NO:63, position 3,627 of SEQ ID NO:64, position 4,240 of SEQ ID NO:65, position 3,594 of SEQ ID NO:66, position 3,473 of SEQ ID NO:67, position 3,and a second primer derived from a 3'-flanking sequence adjacent to a thymine at position 602 or a position corresponding to position 3,163 of SEQ ID NO:69, i) a thymine at a position corresponding to position 141,048 of SEQ ID NO:4, i) position 4,297 of SEQ ID NO:31, position 3,739 of SEQ ID NO:32, position 3,627 of SEQ ID NO:33, position 4,240 of SEQ ID NO:34, position 3,594 of SEQ ID NO:35, position 3,473 of SEQ ID NO:36, position 3,592 of SEQ ID NO:37, 3,602, or 3,163 of SEQ ID NO:38, or ii) a thymine-encoding position corresponding to position 4,297 of SEQ ID NO:62, position 3,379 of SEQ ID NO:63, position 3,627 of SEQ ID NO:64, position 4,240 of SEQ ID NO:65, position 3,594 of SEQ ID NO:66, position 3,473 of SEQ ID NO:67, position 3,602 of SEQ ID NO:68, or position 3,163 of SEQ ID NO:69. In some embodiments, the length of the amplicon can range from the primer pair plus one nucleotide base pair to any length of amplicon that can be generated by the DNA amplification protocol. This distance can range from one nucleotide base pair to the limit of the amplification reaction, i.e., about 20,000 nucleotide base pairs. Optionally, the primer pair is a primer that is complementary to a thymine at a position corresponding to position 141,048 of SEQ ID NO:4; i) a uracil at a position corresponding to position 4,297 of SEQ ID NO:31, position 3,739 of SEQ ID NO:32, position 3,627 of SEQ ID NO:33, position 4,240 of SEQ ID NO:34, position 3,594 of SEQ ID NO:35, position 3,473 of SEQ ID NO:36, position 3,602 of SEQ ID NO:37, or position 3,163 of SEQ ID NO:38; or ii) a uracil at a position corresponding to position 4,297 of SEQ ID NO:62, position 3,379 of SEQ ID NO:63, position 3,627 of SEQ ID NO:64, position 3,592 of SEQ ID NO:65, or position 3,627 of SEQ ID NO:66. i) a thymine at a position corresponding to position 4,240 of SEQ ID NO:6, position 3,594 of SEQ ID NO:66, position 3,473 of SEQ ID NO:67, position 3,602 of SEQ ID NO:68, or position 3,163 of SEQ ID NO:69, and a thymine at a position corresponding to position 141,048 of SEQ ID NO:4; i) a thymine at position 4,297 of SEQ ID NO:31, position 3,739 of SEQ ID NO:32, position 3,627 of SEQ ID NO:33, position 4,240 of SEQ ID NO:34, position 3,594 of SEQ ID NO:35, position 3,473 of SEQ ID NO:36, position 3,602 of SEQ ID NO:37, or position 3,uracil at a position corresponding to position 163, or ii) a thymine at a position corresponding to position 4,297 of SEQ ID NO:62, position 3,379 of SEQ ID NO:63, position 3,627 of SEQ ID NO:64, position 4,240 of SEQ ID NO:65, position 3,594 of SEQ ID NO:66, position 3,473 of SEQ ID NO:67, position 3,602 of SEQ ID NO:68, or position 3,163 of SEQ ID NO:69.

[0147] In some embodiments, to determine whether an ARHGEF12 nucleic acid molecule (genomic nucleic acid molecule, mRNA molecule, or cDNA molecule) in a biological sample, or its complement, contains a nucleotide sequence that contains an adenine at a position corresponding to position 73,039 of SEQ ID NO:5 (genomic nucleic acid molecule), the biological sample can be subjected to an amplification method using a primer pair that includes a first primer derived from the 5' flanking sequence adjacent to the cytosine at a position corresponding to position 73,039 of SEQ ID NO:5 and a second primer derived from the 3' flanking sequence adjacent to the cytosine at a position corresponding to position 73,039 of SEQ ID NO:5 to generate an amplicon indicative of the presence of a SNP at a position that encodes a cytosine at a position corresponding to position 73,039 of SEQ ID NO:5. In some embodiments, the length of the amplicon can range from the length of the primer pair plus one nucleotide base pair to any length of an amplicon that can be generated by a DNA amplification protocol. This distance can range from one nucleotide base pair to the limit of the amplification reaction, i.e., about 20,000 nucleotide base pairs. Optionally, the primer pair flanks a region that includes an adenine at a position corresponding to position 73,039 of SEQ ID NO:5 and at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more nucleotides on each side of the position that includes an adenine at a position corresponding to position 73,039 of SEQ ID NO:5.

[0148] In some embodiments, to determine whether an ARHGEF12 nucleic acid molecule (genomic nucleic acid molecule, mRNA molecule, or cDNA molecule) in a biological sample, or its complement, contains a nucleotide sequence that contains a cytosine at a position corresponding to position 121,307 of SEQ ID NO:6 (genomic nucleic acid molecule), the biological sample can be subjected to an amplification method using a primer pair that includes a first primer derived from the 5' flanking sequence adjacent to the cytosine at a position corresponding to position 121,307 of SEQ ID NO:6 and a second primer derived from the 3' flanking sequence adjacent to the cytosine at a position corresponding to position 121,307 of SEQ ID NO:6 to generate an amplicon indicative of the presence of a SNP at a position that encodes a cytosine at a position corresponding to position 121,307 of SEQ ID NO:6. In some embodiments, the length of the amplicon can range from the length of the primer pair plus one nucleotide base pair to any length of an amplicon that can be generated by a DNA amplification protocol. This distance can range from one nucleotide base pair to the limit of the amplification reaction, i.e., about 20,000 nucleotide base pairs. Optionally, the primer pair flanks a region that includes a cytosine at a position corresponding to position 121,307 of SEQ ID NO:6 and at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more nucleotides on each side of the position that includes a cytosine at a position corresponding to position 121,307 of SEQ ID NO:6.

[0149] In some embodiments, to determine whether an ARHGEF12 nucleic acid molecule (genomic nucleic acid molecule, mRNA molecule, or cDNA molecule) in a biological sample, or its complement, contains a nucleotide sequence that contains a cytosine at a position corresponding to position 141,978 of SEQ ID NO:7 (genomic nucleic acid molecule), the biological sample can be subjected to an amplification method using a primer pair that includes a first primer derived from the 5' flanking sequence adjacent to the cytosine at a position corresponding to position 141,978 of SEQ ID NO:7 and a second primer derived from the 3' flanking sequence adjacent to the cytosine at a position corresponding to position 141,978 of SEQ ID NO:7 to generate an amplicon indicative of the presence of a SNP at a position that encodes a cytosine at a position corresponding to position 141,978 of SEQ ID NO:7. In some embodiments, the length of the amplicon can range from the length of the primer pair plus one nucleotide base pair to any length of an amplicon that can be generated by a DNA amplification protocol. This distance can range from one nucleotide base pair to the limit of the amplification reaction, i.e., about 20,000 nucleotide base pairs. Optionally, the primer pair flanks a region that includes a cytosine at a position corresponding to position 141,978 of SEQ ID NO:7 and at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more nucleotides on each side of the position that includes a cytosine at a position corresponding to position 141,978 of SEQ ID NO:7.

[0150] Similar amplicons can be generated from mRNA and / or cDNA sequences. PCR primer pairs can be obtained from known sequences by using computer programs designed for that purpose, such as the PCR primer analysis tools in Vector NTI version 10 (Informax Inc., Bethesda Md.), PrimerSelect (DNASTAR Inc., Madison, Wis.), and Primer3 (Version 0.4.0.COPYRGT., 1991, Whitehead Institute for Biomedical Research, Cambridge, Mass.). Additionally, sequences can be visually scanned and primers manually identified using known guidelines.

[0151] Illustrative examples of nucleic acid sequencing techniques include, but are not limited to, chain terminator (Sanger) sequencing and dye terminator sequencing. Other methods include nucleic acid hybridization methods other than sequencing (fluorescence in situ hybridization (FISH)), including the use of labeled primers or labeled probes on purified DNA, amplified DNA, and fixed cell preparations. In some methods, the target nucleic acid molecule may be amplified prior to or simultaneously with detection. Illustrative examples of nucleic acid amplification techniques include, but are not limited to, polymerase chain reaction (PCR), ligase chain reaction (LCR), strand displacement amplification (SDA), and nucleic acid sequence-based amplification (NASBA). Other methods include, but are not limited to, ligase chain reaction, strand displacement amplification, and thermophilic SDA (tSDA).

[0152] In hybridization techniques, stringent conditions can be employed so that the probe or primer specifically hybridizes with its target. In some embodiments, a polynucleotide primer or probe under stringent conditions hybridizes with its target sequence to a detectably higher degree than other non-target sequences, for example, at least 2 times, at least 3 times, at least 4 times, or more than background, including more than 10 times background. In some embodiments, a polynucleotide primer or probe under stringent conditions hybridizes with its target nucleotide sequence to a detectably higher degree than other nucleotide sequences at least 2 times. In some embodiments, a polynucleotide primer or probe under stringent conditions hybridizes with its target nucleotide sequence to a detectably higher degree than other nucleotide sequences at least 3 times. In some embodiments, a polynucleotide primer or probe under stringent conditions hybridizes with its target nucleotide sequence to a detectably higher degree than other nucleotide sequences at least 4 times. In some embodiments, a polynucleotide primer or probe under stringent conditions will hybridize to its target nucleotide sequence to a detectably greater extent than other nucleotide sequences at more than 10-fold background. Stringent conditions are sequence-dependent and will be different in different circumstances.

[0153] Suitable stringent conditions that promote DNA hybridization, such as 6x sodium chloride / sodium citrate (SSC) at about 45°C, followed by a 2x SSC wash at 50°C, are known or can be found in Current Protocols in Molecular Biology, John Wiley & Sons, NY (1989), 6.3.1-6.3.6. Typically, stringent conditions for hybridization and detection include conditions in which the salt concentration is less than about 1.5 M NaCl at pH 7.0-8.3. + ion, usually about 0.01 to 1.0 M Na +ionic concentration (or other salts) and a temperature of at least about 30°C for short probes (e.g., 10-50 nucleotides) and at least about 60°C for longer probes (e.g., more than 50 nucleotides). Stringent conditions may be achieved by adding destabilizing agents such as formamide. Optionally, the wash buffer may contain about 0.1% to about 1% SDS. The duration of hybridization is generally less than about 24 hours, usually about 4 to about 12 hours. The duration of washing is at least long enough to reach equilibrium.

[0154] The present disclosure also provides a method for detecting the presence of a predicted loss-of-function polypeptide of human ARHGEF12 or a predicted gain-of-function polypeptide of human ARHGEF12, comprising performing an assay on a biological sample taken from a subject to determine whether an ARHGEF12 polypeptide in the subject contains one or more mutations that cause the polypeptide to have a loss of function (partial or complete) or a predicted loss of function (partial or complete) or a gain of function (partial or complete) or a predicted gain of function (partial or complete). The predicted gain-of-function polypeptide of ARHGEF12 can be any of the ARHGEF12 variant polypeptides described herein. In some embodiments, the method detects the presence of ARHGEF12 Tyr973Phe, Tyr954Phe, or Tyr870Phe. In some embodiments, the method detects the presence of ARHGEF12 Tyr973Phe. The predicted loss-of-function polypeptide of ARHGEF12 can be any of the ARHGEF12 variant polypeptides described herein. In some embodiments, the method detects the presence of ARHGEF12 Tyr1306Cys, Tyr1287Cys, Tyr1203Cys, Glu1156STOP, Glu1137STOP, or Glu1053STOP. In some embodiments, the method detects the presence of ARHGEF12 Tyr1306Cys.

[0155] In some embodiments, the method comprises performing an assay on a sample taken from the subject to determine whether an ARHGEF12 polypeptide in the sample comprises a phenylalanine at a position corresponding to position 973 of SEQ ID NO: 74, position 954 of SEQ ID NO: 75, position 870 of SEQ ID NO: 76, or position 870 of SEQ ID NO: 77. In some embodiments, the method comprises performing an assay on a sample taken from the subject to determine whether an ARHGEF12 polypeptide in the sample comprises a cysteine ​​at a position corresponding to position 1,306 of SEQ ID NO: 78, position 1,287 of SEQ ID NO: 79, or position 1,203 of SEQ ID NO: 80. In some embodiments, the method comprises performing an assay on a sample taken from the subject to determine whether an ARHGEF12 polypeptide in the sample terminates at a position corresponding to position 1,155 of SEQ ID NO: 81, position 1,136 of SEQ ID NO: 82, or position 1,052 of SEQ ID NO: 83.

[0156] In some embodiments, the detecting step comprises sequencing at least a portion of the polypeptide comprising a position corresponding to position 973 of SEQ ID NO:74 or SEQ ID NO:70, position 954 of SEQ ID NO:75 or SEQ ID NO:71, position 870 of SEQ ID NO:76 or SEQ ID NO:72, or position 870 of SEQ ID NO:77 or SEQ ID NO:73. In some embodiments, the detecting step comprises sequencing at least a portion of the polypeptide comprising a position corresponding to position 1,306 of SEQ ID NO:78 or SEQ ID NO:70, position 1,287 of SEQ ID NO:79 or SEQ ID NO:71, or position 1,203 of SEQ ID NO:80. In some embodiments, the detecting step comprises sequencing at least a portion of the polypeptide comprising a position corresponding to position 1,156 of SEQ ID NO:81 or SEQ ID NO:70, position 1,137 of SEQ ID NO:82 or SEQ ID NO:71, or position 1,053 of SEQ ID NO:83 or SEQ ID NO:72.

[0157] In some embodiments, the detecting step comprises an immunoassay for detecting the presence of a polypeptide comprising a position corresponding to position 973 of SEQ ID NO:74 or SEQ ID NO:70, position 954 of SEQ ID NO:75 or SEQ ID NO:71, position 870 of SEQ ID NO:76 or SEQ ID NO:72, or position 870 of SEQ ID NO:77 or SEQ ID NO:74. In some embodiments, the detecting step comprises an immunoassay for detecting the presence of a polypeptide comprising a position corresponding to position 1,306 of SEQ ID NO:78 or SEQ ID NO:70, position 1,287 of SEQ ID NO:79 or SEQ ID NO:71, or position 1,203 of SEQ ID NO:80 or SEQ ID NO:72. In some embodiments, the detecting step comprises an immunoassay for detecting the presence of a polypeptide comprising a position corresponding to position 1,156 of SEQ ID NO:81 or SEQ ID NO:70, position 1,137 of SEQ ID NO:82 or SEQ ID NO:71, or position 1,053 of SEQ ID NO:83 or SEQ ID NO:72.

[0158] In some embodiments, if the subject does not have a predicted loss-of-function polypeptide of ARHGEF12, the subject is at increased risk of developing glaucoma and / or elevated IOP. In some embodiments, if the subject has a predicted loss-of-function polypeptide of ARHGEF12, the subject is at decreased risk of developing glaucoma and / or elevated IOP. In some embodiments, if the subject has a predicted gain-of-function polypeptide of ARHGEF12, the subject is at increased risk of developing glaucoma and / or elevated IOP.

[0159] The present disclosure also provides isolated nucleic acid molecules that hybridize to an ARHGEF12 variant genomic nucleic acid molecule, an ARHGEF12 variant mRNA molecule, and / or an ARHGEF12 variant cDNA molecule (e.g., any of the genomic variant nucleic acid molecules, mRNA variant molecules, and cDNA variant molecules disclosed herein). In some embodiments, the isolated nucleic acid molecule hybridizes to a portion of an ARHGEF12 nucleic acid molecule including a position corresponding to position 132,939 of SEQ ID NO:2, position 3,749 of SEQ ID NO:16, position 3,191 of SEQ ID NO:17, position 3,079 of SEQ ID NO:18, position 3,692 of SEQ ID NO:19, position 3,046 of SEQ ID NO:20, position 2,925 of SEQ ID NO:21, position 3,054 of SEQ ID NO:22, position 2,615 of SEQ ID NO:23, position 3,749 of SEQ ID NO:47, position 3,191 of SEQ ID NO:48, position 3,079 of SEQ ID NO:49, position 3,692 of SEQ ID NO:50, position 3,046 of SEQ ID NO:51, position 2,925 of SEQ ID NO:52, position 3,054 of SEQ ID NO:53, or position 2,615 of SEQ ID NO:54.

[0160] In some embodiments, the isolated nucleic acid molecule hybridizes to a portion of an ARHGEF12 nucleic acid molecule including a position corresponding to position 143,698 of SEQ ID NO:3, position 4,748 of SEQ ID NO:24, position 4,190 of SEQ ID NO:25, position 4,078 of SEQ ID NO:26, position 4,691 of SEQ ID NO:27, position 4,045 of SEQ ID NO:28, position 3,924 of SEQ ID NO:29, position 3,614 of SEQ ID NO:30, position 4,748 of SEQ ID NO:55, position 4,190 of SEQ ID NO:56, position 4,078 of SEQ ID NO:57, position 4,691 of SEQ ID NO:58, position 4,045 of SEQ ID NO:59, position 3,924 of SEQ ID NO:60, or position 3,614 of SEQ ID NO:61.

[0161] In some embodiments, the isolated nucleic acid molecule hybridizes to a portion of an ARHGEF12 nucleic acid molecule including a position corresponding to position 141,048 of SEQ ID NO:4, position 4,297 of SEQ ID NO:31, position 3,739 of SEQ ID NO:32, position 3,627 of SEQ ID NO:33, position 4,240 of SEQ ID NO:34, position 3,594 of SEQ ID NO:35, position 3,473 of SEQ ID NO:36, position 3,602 of SEQ ID NO:37, position 3,163 of SEQ ID NO:38, position 4,297 of SEQ ID NO:62, position 3,379 of SEQ ID NO:63, position 3,627 of SEQ ID NO:64, position 4,240 of SEQ ID NO:65, position 3,594 of SEQ ID NO:66, position 3,473 of SEQ ID NO:67, position 3,602 of SEQ ID NO:68, or position 3,163 of SEQ ID NO:69.

[0162] In some embodiments, the isolated nucleic acid molecule hybridizes to a portion of the ARHGEF12 nucleic acid molecule that includes a position corresponding to position 73,039 of SEQ ID NO:5. In some embodiments, the isolated nucleic acid molecule hybridizes to a portion of the ARHGEF12 nucleic acid molecule that includes a position corresponding to position 121,307 of SEQ ID NO:6.

[0163] In some embodiments, the isolated nucleic acid molecule hybridizes to a portion of the ARHGEF12 nucleic acid molecule that includes a position corresponding to position 141,978 of SEQ ID NO:7. In some embodiments, such isolated nucleic acid molecules comprise at least about 5, at least about 8, at least about 10, at least about 11, at least about 12, at least about 13, at least about 14, at least about 15, at least about 16, at least about 17, at least about 18, at least about 19, at least about 20, at least about 21, at least about 22, at least about 23, at least about 24, at least about 25, at least about 30, at least about 35, at least about 40, at least about 45, at least about 50, at least about 55 , at least about 60, at least about 65, at least about 70, at least about 75, at least about 80, at least about 85, at least about 90, at least about 95, at least about 100, at least about 200, at least about 300, at least about 400, at least about 500, at least about 600, at least about 700, at least about 800, at least about 900, at least about 1000, at least about 2000, at least about 3000, at least about 4000, or at least about 5000 nucleotides. In some embodiments, such isolated nucleic acid molecules comprise or consist of at least about 5, at least about 8, at least about 10, at least about 11, at least about 12, at least about 13, at least about 14, at least about 15, at least about 16, at least about 17, at least about 18, at least about 19, at least about 20, at least about 21, at least about 22, at least about 23, at least about 24, or at least about 25 nucleotides. In some embodiments, the isolated nucleic acid molecule comprises or consists of at least about 18 nucleotides. In some embodiments, the isolated nucleic acid molecule comprises or consists of at least about 15 nucleotides. In some embodiments, the isolated nucleic acid molecule comprises or consists of about 10 to about 35, about 10 to about 30, about 10 to about 25, about 12 to about 30, about 12 to about 28, about 12 to about 24, about 15 to about 30, about 15 to about 25, about 18 to about 30, about 18 to about 25, about 18 to about 24, or about 18 to about 22 nucleotides. In some embodiments, the isolated nucleic acid molecule comprises or consists of about 18 to about 30 nucleotides.In some embodiments, the isolated nucleic acid molecule comprises or consists of at least about 15 nucleotides to at least about 35 nucleotides.

[0164] In some embodiments, such isolated nucleic acid molecules hybridize under stringent conditions to ARHGEF12 variant nucleic acid molecules (e.g., genomic nucleic acid molecules, mRNA molecules, and / or cDNA molecules). Such nucleic acid molecules can be used, for example, as probes, primers, variant-specific probes, or variant-specific primers as described or exemplified herein, including, but not limited to, primers, probes, antisense RNA, shRNA, and siRNA, each of which is described in more detail elsewhere herein and can be used in any of the methods described herein.

[0165] In some embodiments, the isolated nucleic acid molecule hybridizes to at least about 15 contiguous nucleotides of a nucleic acid molecule that is at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% identical to an ARHGEF12 variant genomic nucleic acid molecule, an ARHGEF12 variant mRNA molecule, and / or an ARHGEF12 variant cDNA molecule. In some embodiments, the isolated nucleic acid molecule consists of or comprises about 15 to about 100 nucleotides, or about 15 to about 35 nucleotides. In some embodiments, the isolated nucleic acid molecule consists of or comprises about 15 to about 100 nucleotides. In some embodiments, the isolated nucleic acid molecule consists of or comprises about 15 to about 35 nucleotides.

[0166] In some embodiments, the isolated variant-specific probe or variant-specific primer comprises at least about 15 nucleotides, wherein the variant-specific probe or variant-specific primer comprises a nucleotide sequence complementary to a portion of a nucleotide sequence encoding a human ARHGEF12 polypeptide, the portion being selected from the group consisting of i) position 132,939 of SEQ ID NO:2 or its complement, ii) position 3,749 of SEQ ID NO:16 or its complement, 3,191 of SEQ ID NO:17 or its complement, 3,079 of SEQ ID NO:18 or its complement, 3,692 of SEQ ID NO:19 or its complement, 3,701 of SEQ ID NO:20 or its complement, 3,702 of SEQ ID NO:21 or its complement, 3,703 of SEQ ID NO:22 or its complement, 3,704 of SEQ ID NO:23 or its complement, 3,705 of SEQ ID NO:24 or its complement, 3,706 of SEQ ID NO:25 or its complement, 3,707 of SEQ ID NO:26 or its complement, 3,709 of SEQ ID NO:27 or its complement, 3,806 of SEQ ID NO:28 or its complement, 3,821 of SEQ ID NO:29 or its complement, 3,906 of SEQ ID NO:30 or its complement, 3,907 of SEQ ID NO:31 or its complement, 3,909 of SEQ ID NO:32 or its complement, 3,912 of SEQ ID NO:33 or its complement, 3,926 of SEQ ID NO:34 or its complement, 3,932 of SEQ ID NO:35 or its complement, 3,941 of SEQ ID NO:36 or its complement, 3,923 of SEQ ID NO:37 or its complement, 3,936 of SEQ ID NO:38 or its complement, iii) positions corresponding to position 3,749 of SEQ ID NO:47 or its complement, position 3,191 of SEQ ID NO:48 or its complement, position 3,079 of SEQ ID NO:49 or its complement, position 3,692 of SEQ ID NO:50 or its complement, position 3,046 of SEQ ID NO:51 or its complement, position 2,925 of SEQ ID NO:52 or its complement, position 3,054 of SEQ ID NO:53 or its complement, or position 2,615 of SEQ ID NO:54 or its complement.In some embodiments, the alteration-specific probe or alteration-specific primer is located at i) positions 132,938 to 132,940 of SEQ ID NO: 2 or its complement, ii) positions 3,748 to 3,750 of SEQ ID NO: 16 or its complement, positions 3,190 to 3,192 of SEQ ID NO: 17 or its complement, positions 3,078 to 3,080 of SEQ ID NO: 18 or its complement, positions 3,691 to 3,693 of SEQ ID NO: 19 or its complement, positions 3,045 to 3,047 of SEQ ID NO: 20 or its complement, positions 2,924 to 2,926 of SEQ ID NO: 21 or its complement, positions 3,053 to 3,055 of SEQ ID NO: 22 or its complement, or positions 3,056 to 3,058 of SEQ ID NO: 23 or its complement. and / or iii) a nucleotide sequence complementary to a portion of a nucleotide sequence including a position corresponding to positions 3,748 to 3,750 of SEQ ID NO: 47 or its complement, positions 3,190 to 3,192 of SEQ ID NO: 48 or its complement, positions 3,078 to 3,080 of SEQ ID NO: 49 or its complement, positions 3,691 to 3,693 of SEQ ID NO: 50 or its complement, positions 3,045 to 3,047 of SEQ ID NO: 51 or its complement, positions 2,924 to 2,926 of SEQ ID NO: 52 or its complement, positions 3,053 to 3,055 of SEQ ID NO: 53 or its complement, or positions 2,614 to 2,616 of SEQ ID NO: 54 or its complement.

[0167] In some embodiments, the isolated variant-specific probe or variant-specific primer comprises at least about 15 nucleotides, wherein the variant-specific probe or variant-specific primer comprises a nucleotide sequence complementary to a portion of a nucleotide sequence encoding a human ARHGEF12 polypeptide, the portion being selected from the group consisting of i) position 143,698 of SEQ ID NO:3 or its complement, ii) position 4,748 of SEQ ID NO:24 or its complement, 4,190 of SEQ ID NO:25 or its complement, 4,078 of SEQ ID NO:26 or its complement, 4,200 of SEQ ID NO:27 or its complement, 4,300 of SEQ ID NO:28 or its complement, 4,400 of SEQ ID NO:29 or its complement, 4,500 of SEQ ID NO:30 or its complement, 4,600 of SEQ ID NO:31 or its complement, 4,700 of SEQ ID NO:32 or its complement, 4,800 of SEQ ID NO:33 or its complement, 4,900 of SEQ ID NO:34 or its complement, 4,100 of SEQ ID NO:35 or its complement, 4,200 of SEQ ID NO:36 or its complement, 4,300 of SEQ ID NO:37 or its complement, 4,400 of SEQ ID NO:38 or its complement, 4,500 of SEQ ID NO:39 or its complement, 4,600 of SEQ ID NO:40 or its complement, 4,700 of SEQ ID NO:41 or its complement, 4,800 of SEQ ID NO:42 or its complement, 4,900 of SEQ ID NO:43 or its complement, 4,900 of SEQ ID NO:44 or its complement, 4,100 of SEQ ID NO:45 or its complement, 4,100 of SEQ ID NO:46 or its complement, and / or iii) a position corresponding to position 4,748 of SEQ ID NO:55 or its complement, position 4,190 of SEQ ID NO:56 or its complement, position 4,078 of SEQ ID NO:57 or its complement, position 4,691 of SEQ ID NO:58 or its complement, position 4,045 of SEQ ID NO:59 or its complement, position 3,924 of SEQ ID NO:60 or its complement, or position 3,614 of SEQ ID NO:61 or its complement.In some embodiments, the alteration-specific probe or alteration-specific primer is located at i) positions 143,697 to 143,699 of SEQ ID NO: 3 or its complement, ii) positions 4,747 to 4,749 of SEQ ID NO: 24 or its complement, positions 4,189 to 4,191 of SEQ ID NO: 25 or its complement, positions 4,077 to 4,079 of SEQ ID NO: 26 or its complement, positions 4,690 to 4,692 of SEQ ID NO: 27 or its complement, positions 4,044 to 4,046 of SEQ ID NO: 28 or its complement, positions 3,923 to 3,925 of SEQ ID NO: 29 or its complement, or ... and / or iii) a nucleotide sequence complementary to a portion of a nucleotide sequence including a position corresponding to positions 4,747 to 4,749 of SEQ ID NO: 55 or its complement, positions 4,189 to 4,191 of SEQ ID NO: 56 or its complement, positions 4,077 to 4,079 of SEQ ID NO: 57 or its complement, positions 4,690 to 4,692 of SEQ ID NO: 58 or its complement, positions 4,044 to 4,046 of SEQ ID NO: 59 or its complement, positions 3,923 to 3,925 of SEQ ID NO: 60 or its complement, or positions 3,613 to 3,615 of SEQ ID NO: 61 or its complement.

[0168] In some embodiments, the isolated variant-specific probe or variant-specific primer comprises at least about 15 nucleotides, and the variant-specific probe or variant-specific primer comprises a nucleotide sequence complementary to a portion of a nucleotide sequence encoding a human ARHGEF12 polypeptide, the portion being: i) position 141,048 of SEQ ID NO:4 or its complement; ii) position 4,297 of SEQ ID NO:31 or its complement; position 3,739 of SEQ ID NO:32 or its complement; position 3,627 of SEQ ID NO:33 or its complement; position 4,240 of SEQ ID NO:34 or its complement; position 3,590 of SEQ ID NO:35 or its complement; 4, position 3,473 of SEQ ID NO:36 or its complement, position 3,602 of SEQ ID NO:37 or its complement, or position 3,163 of SEQ ID NO:38 or its complement, and / or iii) a position corresponding to position 4,297 of SEQ ID NO:62 or its complement, position 3,379 of SEQ ID NO:63 or its complement, position 3,627 of SEQ ID NO:64 or its complement, position 4,240 of SEQ ID NO:65 or its complement, position 3,594 of SEQ ID NO:66 or its complement, position 3,473 of SEQ ID NO:67 or its complement, position 3,602 of SEQ ID NO:68 or its complement, or position 3,163 of SEQ ID NO:69 or its complement.In some embodiments, the alteration-specific probe or alteration-specific primer is located at i) positions 141,048 to 141,050 of SEQ ID NO: 4 or its complement, ii) positions 4,297 to 4,299 of SEQ ID NO: 31 or its complement, positions 3,739 to 3,741 of SEQ ID NO: 32 or its complement, positions 3,627 to 3,629 of SEQ ID NO: 33 or its complement, positions 4,240 to 4,242 of SEQ ID NO: 34 or its complement, positions 3,594 to 3,596 of SEQ ID NO: 35 or its complement, positions 3,473 to 3,475 of SEQ ID NO: 36 or its complement, positions 3,602 to 3,604 of SEQ ID NO: 37 or its complement, or positions 3,603 to 3,604 of SEQ ID NO: 38 or its complement. and / or iii) a nucleotide sequence complementary to a portion of a nucleotide sequence including a position corresponding to positions 4,297 to 4,299 of SEQ ID NO: 62 or its complement, positions 3,739 to 3,741 of SEQ ID NO: 63 or its complement, positions 3,627 to 3,629 of SEQ ID NO: 64 or its complement, positions 4,240 to 4,242 of SEQ ID NO: 65 or its complement, positions 3,594 to 3,596 of SEQ ID NO: 66 or its complement, positions 3,473 to 3,475 of SEQ ID NO: 67 or its complement, positions 3,602 to 3,604 of SEQ ID NO: 68 or its complement, or positions 3,163 to 3,165 of SEQ ID NO: 69 or its complement.

[0169] In some embodiments, the isolated variant-specific probe or variant-specific primer comprises at least about 15 nucleotides, and the variant-specific probe or variant-specific primer comprises a nucleotide sequence complementary to a portion of a nucleotide sequence encoding a human ARHGEF12 polypeptide, the portion comprising a position corresponding to position 73,039 of SEQ ID NO:5 or its complement.

[0170] In some embodiments, the isolated variant-specific probe or variant-specific primer comprises at least about 15 nucleotides, and the variant-specific probe or variant-specific primer comprises a nucleotide sequence complementary to a portion of a nucleotide sequence encoding a human ARHGEF12 polypeptide, the portion including a position corresponding to position 121,307 of SEQ ID NO:6 or its complement.

[0171] In some embodiments, the isolated variant-specific probe or variant-specific primer comprises at least about 15 nucleotides, and the variant-specific probe or variant-specific primer comprises a nucleotide sequence complementary to a portion of a nucleotide sequence encoding a human ARHGEF12 polypeptide, the portion including a position corresponding to position 141,978 of SEQ ID NO:7 or its complement.

[0172] In some embodiments, the variant-specific probe and the variant-specific primer comprise DNA. In some embodiments, the variant-specific probe and the variant-specific primer comprise RNA.

[0173] In some embodiments, the probes and primers described herein (including variant-specific probes and variant-specific primers) have nucleotide sequences that specifically hybridize to any of the nucleic acid molecules disclosed herein or their complements. In some embodiments, the probes and primers specifically hybridize to any of the nucleic acid molecules disclosed herein under stringent conditions.

[0174] In some embodiments, primers, including modification-specific primers, can be used in second generation or high throughput sequencing. In some examples, primers, including modification-specific primers, can be modified. In particular, primers can include various modifications used in various steps of, for example, Massive Parallel Signature Sequencing (MPSS), Polony sequencing, and 454 pyrosequencing. Modified primers can be used in some steps of the process, including biotinylated primers in the cloning step, and fluorescently labeled primers used in the bead loading and detection steps. Polony sequencing is generally performed using paired-end tag libraries, where each molecule of DNA template is about 135 bp in length. Biotinylated primers are used in the bead loading and emulsion PCR. Fluorescently labeled degenerate nonamer oligonucleotides are used in the detection step. Adapters can contain 5'-biotin tags for immobilizing DNA libraries on streptavidin-coated beads.

[0175] The probes and primers described herein can be used to detect nucleotide variations within any of the ARHGEF12 variant genomic nucleic acid molecules, ARHGEF12 variant mRNA molecules, and / or ARHGEF12 variant cDNA molecules disclosed herein. The primers described herein can be used to amplify an ARHGEF12 variant genomic nucleic acid molecule, an ARHGEF12 variant mRNA molecule, or an ARHGEF12 variant cDNA molecule, or a fragment thereof.

[0176] The present disclosure also provides a pair of primers comprising any of the above primers. For example, if the 3' end of one primer hybridizes with adenine (rather than thymine) at the position corresponding to position 132,939 of SEQ ID NO:1 in a specific ARHGEF12 nucleic acid molecule, the presence of an amplified fragment will indicate the presence of an ARHGEF12 reference genomic nucleic acid molecule. Conversely, if the 3' end of one primer hybridizes with thymine (rather than adenine) at the position corresponding to position 132,939 of SEQ ID NO:2 in a specific ARHGEF12 nucleic acid molecule, the presence of an amplified fragment will indicate the presence of an ARHGEF12 variant genomic nucleic acid molecule. In some embodiments, the nucleotide of the primer that is complementary to the thymine at the position corresponding to position 132,939 of SEQ ID NO:2 can be at the 3' end of the primer. Also, if the 3' end of one primer hybridizes to an adenine (rather than a uracil at position 3,749) in a particular ARHGEF12 nucleic acid molecule at a position corresponding to position 3,749 of SEQ ID NO:8, the presence of an amplified fragment will indicate the presence of an ARHGEF12 reference-type mRNA molecule. Conversely, if the 3' end of one primer hybridizes to an uracil (rather than an adenine) in a particular ARHGEF12 mRNA molecule at a position corresponding to position 3,749 of SEQ ID NO:16, the presence of an amplified fragment will indicate the presence of an ARHGEF12 variant mRNA molecule. In some embodiments, the nucleotide of the primer complementary to the uracil at position corresponding to position 3,749 of SEQ ID NO:16 may be at the 3' end of the primer. Also, if the 3' end of one primer hybridizes to an adenine (rather than a uracil at position 3,191) in a particular ARHGEF12 nucleic acid molecule at a position corresponding to position 3,191 of SEQ ID NO:9, the presence of an amplified fragment will indicate the presence of an ARHGEF12 reference-type mRNA molecule. Conversely, if the 3' end of one primer hybridizes to an uracil (rather than an adenine) in a particular ARHGEF12 mRNA molecule at a position corresponding to position 3,191 of SEQ ID NO:17, the presence of an amplified fragment will indicate the presence of an ARHGEF12 variant mRNA molecule.In some embodiments, the nucleotide of the primer complementary to the uracil at the position corresponding to position 3,191 of SEQ ID NO: 17 may be at the 3' end of the primer. Also, if the 3' end of one primer hybridizes to the adenine at the position corresponding to position 3,079 of SEQ ID NO: 10 in a particular ARHGEF12 nucleic acid molecule (rather than the uracil at position 3,079), the presence of the amplified fragment will indicate the presence of an ARHGEF12 reference-type mRNA molecule. Conversely, if the 3' end of one primer hybridizes to the uracil at the position corresponding to position 3,079 of SEQ ID NO: 18 in a particular ARHGEF12 mRNA molecule (rather than the adenine), the presence of the amplified fragment will indicate the presence of an ARHGEF12 variant mRNA molecule. In some embodiments, the nucleotide of the primer complementary to the uracil at the position corresponding to position 3,079 of SEQ ID NO: 18 may be at the 3' end of the primer. Also, if the 3' end of one primer hybridizes to an adenine (rather than a uracil at position 3,692) in a particular ARHGEF12 nucleic acid molecule at a position corresponding to position 3,692 of SEQ ID NO: 11, the presence of an amplified fragment will indicate the presence of an ARHGEF12 reference-type mRNA molecule. Conversely, if the 3' end of one primer hybridizes to an uracil (rather than an adenine) in a particular ARHGEF12 mRNA molecule at a position corresponding to position 3,692 of SEQ ID NO: 19, the presence of an amplified fragment will indicate the presence of an ARHGEF12 variant mRNA molecule. In some embodiments, the nucleotide of the primer complementary to the uracil at position corresponding to position 3,692 of SEQ ID NO: 19 may be at the 3' end of the primer. Furthermore, if the 3' end of one of the primers hybridizes to an adenine at a position corresponding to position 3,046 of SEQ ID NO:12 in a particular ARHGEF12 nucleic acid molecule (rather than to uracil at position 3,046), the presence of an amplified fragment will indicate the presence of an ARHGEF12 reference-type mRNA molecule.Conversely, if the 3' end of one primer hybridizes to uracil (rather than adenine) at a position corresponding to position 3,046 of SEQ ID NO:20 in a particular ARHGEF12 mRNA molecule, the presence of an amplified fragment will indicate the presence of an ARHGEF12 variant mRNA molecule. In some embodiments, the nucleotide of the primer complementary to uracil at a position corresponding to position 3,046 of SEQ ID NO:20 can be at the 3' end of the primer. Also, if the 3' end of one primer hybridizes to adenine (rather than uracil at position 2,925) at a position corresponding to position 2,925 of SEQ ID NO:13 in a particular ARHGEF12 nucleic acid molecule, the presence of an amplified fragment will indicate the presence of an ARHGEF12 reference-type mRNA molecule. Conversely, if the 3' end of one primer hybridizes to uracil (rather than adenine) at a position corresponding to position 2,925 of SEQ ID NO:21 in a particular ARHGEF12 mRNA molecule, the presence of an amplified fragment will indicate the presence of an ARHGEF12 variant mRNA molecule. In some embodiments, the nucleotide of the primer complementary to the uracil at the position corresponding to position 2,925 of SEQ ID NO:21 may be at the 3' end of the primer. Also, if the 3' end of one primer hybridizes to the adenine at the position corresponding to position 3,054 of SEQ ID NO:14 in a particular ARHGEF12 nucleic acid molecule (rather than the uracil at position 3,054), the presence of the amplified fragment will indicate the presence of an ARHGEF12 reference-type mRNA molecule. Conversely, if the 3' end of one primer hybridizes to the uracil at the position corresponding to position 3,054 of SEQ ID NO:22 in a particular ARHGEF12 mRNA molecule (rather than the adenine), the presence of the amplified fragment will indicate the presence of an ARHGEF12 variant mRNA molecule. In some embodiments, the nucleotide of the primer complementary to the uracil at the position corresponding to position 3,054 of SEQ ID NO:22 may be at the 3' end of the primer.Also, if the 3' end of one primer hybridizes to an adenine (rather than a uracil at position 2,615) in a particular ARHGEF12 nucleic acid molecule at a position corresponding to position 2,615 of SEQ ID NO: 15, the presence of an amplified fragment will indicate the presence of an ARHGEF12 reference-type mRNA molecule. Conversely, if the 3' end of one primer hybridizes to an uracil (rather than an adenine) in a particular ARHGEF12 mRNA molecule at a position corresponding to position 2,615 of SEQ ID NO: 23, the presence of an amplified fragment will indicate the presence of an ARHGEF12 variant mRNA molecule. In some embodiments, the nucleotide of the primer complementary to the uracil at position corresponding to position 2,615 of SEQ ID NO: 23 may be at the 3' end of the primer. Also, if the 3' end of one primer hybridizes to an adenine (rather than a thymine at position 3,749) in a particular ARHGEF12 nucleic acid molecule at a position corresponding to position 3,749 of SEQ ID NO: 39, the presence of an amplified fragment will indicate the presence of an ARHGEF12 reference-type cDNA molecule. Conversely, if the 3' end of one primer hybridizes to a thymine (rather than an adenine) in a particular ARHGEF12 cDNA molecule at a position corresponding to position 3,749 of SEQ ID NO: 47, the presence of an amplified fragment will indicate the presence of an ARHGEF12 variant cDNA molecule. In some embodiments, the nucleotide of the primer complementary to the thymine at position corresponding to position 3,749 of SEQ ID NO: 47 may be at the 3' end of the primer. Also, if the 3' end of one primer hybridizes to an adenine (rather than a thymine at position 3,191) in a particular ARHGEF12 nucleic acid molecule at a position corresponding to position 3,191 of SEQ ID NO:40, the presence of an amplified fragment will indicate the presence of an ARHGEF12 reference-type cDNA molecule. Conversely, if the 3' end of one primer hybridizes to a thymine (rather than an adenine) in a particular ARHGEF12 cDNA molecule at a position corresponding to position 3,191 of SEQ ID NO:48, the presence of an amplified fragment will indicate the presence of an ARHGEF12 variant cDNA molecule.In some embodiments, the nucleotide of the primer complementary to the thymine at the position corresponding to position 3,191 of SEQ ID NO:48 may be at the 3' end of the primer. Also, if the 3' end of one primer hybridizes to the adenine at the position corresponding to position 3,079 of SEQ ID NO:41 in a particular ARHGEF12 nucleic acid molecule (rather than the thymine at position 3,079), the presence of an amplified fragment will indicate the presence of an ARHGEF12 reference-type cDNA molecule. Conversely, if the 3' end of one primer hybridizes to the thymine at the position corresponding to position 3,079 of SEQ ID NO:49 in a particular ARHGEF12 cDNA molecule (rather than the adenine), the presence of an amplified fragment will indicate the presence of an ARHGEF12 variant cDNA molecule. In some embodiments, the nucleotide of the primer complementary to the thymine at the position corresponding to position 3,079 of SEQ ID NO:49 may be at the 3' end of the primer. Also, if the 3' end of one primer hybridizes to an adenine (rather than a thymine at position 3,692) in a particular ARHGEF12 nucleic acid molecule at a position corresponding to position 3,692 of SEQ ID NO: 42, the presence of an amplified fragment will indicate the presence of an ARHGEF12 reference-type cDNA molecule. Conversely, if the 3' end of one primer hybridizes to a thymine (rather than an adenine) in a particular ARHGEF12 cDNA molecule at a position corresponding to position 3,692 of SEQ ID NO: 50, the presence of an amplified fragment will indicate the presence of an ARHGEF12 variant cDNA molecule. In some embodiments, the nucleotide of the primer complementary to the thymine at position corresponding to position 3,692 of SEQ ID NO: 50 may be at the 3' end of the primer. Furthermore, if the 3' end of one of the primers hybridizes to an adenine at a position corresponding to position 3,046 of SEQ ID NO:43 in a particular ARHGEF12 nucleic acid molecule (rather than a thymine at position 3,046), the presence of an amplified fragment will indicate the presence of an ARHGEF12 reference-type cDNA molecule.Conversely, if the 3' end of one primer hybridizes to a thymine (rather than an adenine) at a position corresponding to position 3,046 of SEQ ID NO:51 in a particular ARHGEF12 cDNA molecule, the presence of an amplified fragment will indicate the presence of an ARHGEF12 variant cDNA molecule. In some embodiments, the nucleotide of the primer complementary to the thymine at a position corresponding to position 3,046 of SEQ ID NO:51 can be at the 3' end of the primer. Also, if the 3' end of one primer hybridizes to an adenine (rather than a thymine at position 2,925) at a position corresponding to position 2,925 of SEQ ID NO:44 in a particular ARHGEF12 nucleic acid molecule, the presence of an amplified fragment will indicate the presence of an ARHGEF12 reference-type cDNA molecule. Conversely, if the 3' end of one primer hybridizes to a thymine (rather than an adenine) at a position corresponding to position 2,925 of SEQ ID NO:52 in a particular ARHGEF12 cDNA molecule, the presence of an amplified fragment will indicate the presence of an ARHGEF12 variant cDNA molecule. In some embodiments, the nucleotide of the primer complementary to the thymine at the position corresponding to position 2,925 of SEQ ID NO:52 can be at the 3' end of the primer, and the 3' end of one primer can be at position 3,054 of SEQ ID NO:45 in a particular ARHGEF12 nucleic acid molecule. If the 3' end of one primer hybridizes to an adenine (rather than a thymine at position 3,054) in a particular ARHGEF12 cDNA molecule, the presence of the amplified fragment will indicate the presence of an ARHGEF12 reference-type cDNA molecule. Conversely, if the 3' end of one primer hybridizes to a thymine (rather than an adenine) in a position corresponding to position 3,054 of SEQ ID NO:53 in a particular ARHGEF12 cDNA molecule, the presence of the amplified fragment will indicate the presence of an ARHGEF12 variant cDNA molecule. In some embodiments, the nucleotide of the primer that is complementary to the thymine in a position corresponding to position 3,054 of SEQ ID NO:53 can be at the 3' end of the primer. Also, if the 3' end of one primer hybridizes to an adenine (rather than a thymine at position 2,615) in a particular ARHGEF12 nucleic acid molecule, the presence of the amplified fragment will indicate the presence of an ARHGEF12 reference-type cDNA molecule. Conversely, if the 3' end of one primer hybridizes to a thymine (rather than an adenine) at a position in a particular ARHGEF12 cDNA molecule corresponding to position 2,615 of SEQ ID NO: 54, the presence of an amplified fragment will indicate the presence of an ARHGEF12 variant cDNA molecule. In some embodiments, the nucleotide of the primer complementary to the thymine at the position corresponding to position 2,615 of SEQ ID NO: 54 may be at the 3' end of the primer.

[0177] The present disclosure also provides a pair of primers comprising any of the above primers. For example, if the 3' end of one primer hybridizes with adenine (rather than guanine) at a position corresponding to position 132,939 of SEQ ID NO:1 in a particular ARHGEF12 nucleic acid molecule, the presence of an amplified fragment will indicate the presence of an ARHGEF12 reference genomic nucleic acid molecule. Conversely, if the 3' end of one primer hybridizes with guanine (rather than adenine) at a position corresponding to position 143,698 of SEQ ID NO:3 in a particular ARHGEF12 nucleic acid molecule, the presence of an amplified fragment will indicate the presence of an ARHGEF12 variant genomic nucleic acid molecule. In some embodiments, the nucleotide of the primer that is complementary to the guanine at a position corresponding to position 143,698 of SEQ ID NO:3 can be at the 3' end of the primer. Also, if the 3' end of one primer hybridizes to an adenine (rather than a guanine at position 4,748) in a particular ARHGEF12 nucleic acid molecule at a position corresponding to position 4,748 of SEQ ID NO:8, the presence of an amplified fragment will indicate the presence of an ARHGEF12 reference-type mRNA molecule. Conversely, if the 3' end of one primer hybridizes to a guanine (rather than an adenine) in a particular ARHGEF12 mRNA molecule at a position corresponding to position 4,748 of SEQ ID NO:24, the presence of an amplified fragment will indicate the presence of an ARHGEF12 variant mRNA molecule. In some embodiments, the nucleotide of the primer that is complementary to the guanine at position corresponding to position 4,748 of SEQ ID NO:24 may be at the 3' end of the primer. Also, if the 3' end of one primer hybridizes to an adenine (rather than a guanine at position 4,190) in a particular ARHGEF12 nucleic acid molecule at a position corresponding to position 4,190 of SEQ ID NO:9, the presence of an amplified fragment will indicate the presence of an ARHGEF12 reference-type mRNA molecule. Conversely, if the 3' end of one of the primers hybridizes to a guanine (rather than an adenine) at a position corresponding to position 4,190 of SEQ ID NO:25 in a particular ARHGEF12 mRNA molecule, the presence of an amplified fragment will indicate the presence of an ARHGEF12 variant mRNA molecule.In some embodiments, the nucleotide of the primer complementary to the guanine at the position corresponding to position 4,190 of SEQ ID NO:25 may be at the 3' end of the primer. Also, if the 3' end of one primer hybridizes to the adenine at the position corresponding to position 4,078 of SEQ ID NO:10 in a particular ARHGEF12 nucleic acid molecule (rather than the guanine at position 4,078), the presence of the amplified fragment will indicate the presence of an ARHGEF12 reference-type mRNA molecule. Conversely, if the 3' end of one primer hybridizes to the guanine at the position corresponding to position 4,078 of SEQ ID NO:26 in a particular ARHGEF12 mRNA molecule (rather than the adenine), the presence of the amplified fragment will indicate the presence of an ARHGEF12 variant mRNA molecule. In some embodiments, the nucleotide of the primer complementary to the guanine at the position corresponding to position 4,078 of SEQ ID NO:26 may be at the 3' end of the primer. Also, if the 3' end of one primer hybridizes to an adenine (rather than a guanine at position 4,691) in a particular ARHGEF12 nucleic acid molecule at a position corresponding to position 4,691 of SEQ ID NO: 11, the presence of an amplified fragment will indicate the presence of an ARHGEF12 reference-type mRNA molecule. Conversely, if the 3' end of one primer hybridizes to a guanine (rather than an adenine) in a particular ARHGEF12 mRNA molecule at a position corresponding to position 4,691 of SEQ ID NO: 27, the presence of an amplified fragment will indicate the presence of an ARHGEF12 variant mRNA molecule. In some embodiments, the nucleotide of the primer complementary to the guanine at position corresponding to position 4,691 of SEQ ID NO: 27 may be at the 3' end of the primer. Furthermore, if the 3' end of one of the primers hybridizes to an adenine at a position corresponding to position 4,045 of SEQ ID NO:12 in a particular ARHGEF12 nucleic acid molecule (rather than a guanine at position 4,045), the presence of an amplified fragment will indicate the presence of an ARHGEF12 reference-type mRNA molecule.Conversely, if the 3' end of one primer hybridizes to a guanine (rather than an adenine) at a position corresponding to position 4,045 of SEQ ID NO:28 in a particular ARHGEF12 mRNA molecule, the presence of an amplified fragment will indicate the presence of an ARHGEF12 variant mRNA molecule. In some embodiments, the nucleotide of the primer complementary to the guanine at a position corresponding to position 4,045 of SEQ ID NO:28 can be at the 3' end of the primer. Also, if the 3' end of one primer hybridizes to an adenine (rather than a guanine at position 3,924) at a position corresponding to position 3,924 of SEQ ID NO:13 in a particular ARHGEF12 nucleic acid molecule, the presence of an amplified fragment will indicate the presence of an ARHGEF12 reference-type mRNA molecule. Conversely, if the 3' end of one primer hybridizes to a guanine (rather than an adenine) at a position corresponding to position 3,924 of SEQ ID NO:29 in a particular ARHGEF12 mRNA molecule, the presence of an amplified fragment will indicate the presence of an ARHGEF12 variant mRNA molecule. In some embodiments, the nucleotide of the primer complementary to the guanine at the position corresponding to position 3,924 of SEQ ID NO:29 may be at the 3' end of the primer. Also, if the 3' end of one primer hybridizes to the adenine at the position corresponding to position 3,614 of SEQ ID NO:15 in a particular ARHGEF12 nucleic acid molecule (rather than the guanine at position 3,614), the presence of the amplified fragment will indicate the presence of an ARHGEF12 reference-type mRNA molecule. Conversely, if the 3' end of one primer hybridizes to the guanine at the position corresponding to position 3,614 of SEQ ID NO:30 in a particular ARHGEF12 mRNA molecule (rather than the adenine), the presence of the amplified fragment will indicate the presence of an ARHGEF12 variant mRNA molecule. In some embodiments, the nucleotide of the primer complementary to the guanine at the position corresponding to position 3,614 of SEQ ID NO:30 may be at the 3' end of the primer.Also, if the 3' end of one primer hybridizes to an adenine (rather than a guanine at position 4,748) in a particular ARHGEF12 nucleic acid molecule at a position corresponding to position 4,748 of SEQ ID NO: 39, the presence of an amplified fragment will indicate the presence of an ARHGEF12 reference-type cDNA molecule. Conversely, if the 3' end of one primer hybridizes to a guanine (rather than an adenine) in a particular ARHGEF12 cDNA molecule at a position corresponding to position 4,748 of SEQ ID NO: 55, the presence of an amplified fragment will indicate the presence of an ARHGEF12 variant cDNA molecule. In some embodiments, the nucleotide of the primer complementary to the guanine at position corresponding to position 4,748 of SEQ ID NO: 55 may be at the 3' end of the primer. Also, if the 3' end of one primer hybridizes to an adenine (rather than a guanine at position 4,190) in a particular ARHGEF12 nucleic acid molecule at a position corresponding to position 4,190 of SEQ ID NO: 40, the presence of an amplified fragment will indicate the presence of an ARHGEF12 reference-type cDNA molecule. Conversely, if the 3' end of one primer hybridizes to a guanine (rather than an adenine) in a particular ARHGEF12 cDNA molecule at a position corresponding to position 4,190 of SEQ ID NO: 56, the presence of an amplified fragment will indicate the presence of an ARHGEF12 variant cDNA molecule. In some embodiments, the nucleotide of the primer that is complementary to the guanine at position corresponding to position 4,190 of SEQ ID NO: 56 may be at the 3' end of the primer. Also, if the 3' end of one primer hybridizes to an adenine (rather than a guanine at position 4,078) in a particular ARHGEF12 nucleic acid molecule at a position corresponding to position 4,078 of SEQ ID NO:41, the presence of an amplified fragment will indicate the presence of an ARHGEF12 reference-type cDNA molecule. Conversely, if the 3' end of one primer hybridizes to a guanine (rather than an adenine) in a particular ARHGEF12 cDNA molecule at a position corresponding to position 4,078 of SEQ ID NO:57, the presence of an amplified fragment will indicate the presence of an ARHGEF12 variant cDNA molecule.In some embodiments, the nucleotide of the primer complementary to the guanine at the position corresponding to position 4,078 of SEQ ID NO:57 may be at the 3' end of the primer. Also, if the 3' end of one primer hybridizes to the adenine at the position corresponding to position 4,691 of SEQ ID NO:42 in a particular ARHGEF12 nucleic acid molecule (rather than the guanine at position 4,691), the presence of an amplified fragment will indicate the presence of an ARHGEF12 reference-type cDNA molecule. Conversely, if the 3' end of one primer hybridizes to the guanine at the position corresponding to position 4,691 of SEQ ID NO:58 in a particular ARHGEF12 cDNA molecule (rather than the adenine), the presence of an amplified fragment will indicate the presence of an ARHGEF12 variant cDNA molecule. In some embodiments, the nucleotide of the primer complementary to the guanine at the position corresponding to position 4,691 of SEQ ID NO:58 may be at the 3' end of the primer. Also, if the 3' end of one primer hybridizes to an adenine (rather than a guanine at position 4,045) in a particular ARHGEF12 nucleic acid molecule at a position corresponding to position 4,045 of SEQ ID NO: 43, the presence of an amplified fragment will indicate the presence of an ARHGEF12 reference-type cDNA molecule. Conversely, if the 3' end of one primer hybridizes to a guanine (rather than an adenine) in a particular ARHGEF12 cDNA molecule at a position corresponding to position 4,045 of SEQ ID NO: 59, the presence of an amplified fragment will indicate the presence of an ARHGEF12 variant cDNA molecule. In some embodiments, the nucleotide of the primer complementary to the guanine at position corresponding to position 4,045 of SEQ ID NO: 59 may be at the 3' end of the primer. Furthermore, if the 3' end of one of the primers hybridizes to an adenine at a position corresponding to position 3,924 of SEQ ID NO:44 in a particular ARHGEF12 nucleic acid molecule (rather than a guanine at position 3,924), the presence of an amplified fragment will indicate the presence of an ARHGEF12 reference-type cDNA molecule.Conversely, if the 3' end of one primer hybridizes to a guanine (rather than an adenine) at a position corresponding to position 3,924 of SEQ ID NO:60 in a particular ARHGEF12 cDNA molecule, the presence of an amplified fragment will indicate the presence of an ARHGEF12 variant cDNA molecule. In some embodiments, the nucleotide of the primer complementary to the guanine at a position corresponding to position 3,924 of SEQ ID NO:60 can be at the 3' end of the primer. Also, if the 3' end of one primer hybridizes to an adenine (rather than a guanine at position 3,614) at a position corresponding to position 3,614 of SEQ ID NO:46 in a particular ARHGEF12 nucleic acid molecule, the presence of an amplified fragment will indicate the presence of an ARHGEF12 reference-type cDNA molecule. Conversely, if the 3' end of one primer hybridizes to a guanine (rather than an adenine) at a position corresponding to position 3,614 of SEQ ID NO:61 in a particular ARHGEF12 cDNA molecule, the presence of an amplified fragment will indicate the presence of an ARHGEF12 variant cDNA molecule. In some embodiments, the nucleotide of the primer complementary to the guanine at a position corresponding to position 3,614 of SEQ ID NO:61 may be at the 3' end of the primer.

[0178] The present disclosure also provides a pair of primers comprising any of the above primers. For example, if the 3' end of one primer hybridizes with adenine (rather than thymine) at a position corresponding to position 132,939 of SEQ ID NO:1 in a specific ARHGEF12 nucleic acid molecule, the presence of an amplified fragment will indicate the presence of an ARHGEF12 reference genomic nucleic acid molecule. Conversely, if the 3' end of one primer hybridizes with thymine (rather than adenine) at a position corresponding to position 141,048 of SEQ ID NO:4 in a specific ARHGEF12 nucleic acid molecule, the presence of an amplified fragment will indicate the presence of an ARHGEF12 variant genomic nucleic acid molecule. In some embodiments, the nucleotide of the primer that is complementary to the thymine at a position corresponding to position 141,048 of SEQ ID NO:4 can be at the 3' end of the primer. Also, if the 3' end of one primer hybridizes to a guanine (rather than a uracil at 4,297) in a particular ARHGEF12 nucleic acid molecule at a position corresponding to position 4,297 of SEQ ID NO:8, the presence of an amplified fragment will indicate the presence of an ARHGEF12 reference-type mRNA molecule. Conversely, if the 3' end of one primer hybridizes to a uracil (rather than a guanine) in a particular ARHGEF12 mRNA molecule at a position corresponding to position 4,297 of SEQ ID NO:31, the presence of an amplified fragment will indicate the presence of an ARHGEF12 variant mRNA molecule. In some embodiments, the nucleotide of the primer complementary to the uracil at a position corresponding to position 4,297 of SEQ ID NO:31 may be at the 3' end of the primer. Also, if the 3' end of one primer hybridizes to a guanine (rather than a uracil at position 3,739) in a particular ARHGEF12 nucleic acid molecule at a position corresponding to position 3,739 of SEQ ID NO:9, the presence of an amplified fragment will indicate the presence of an ARHGEF12 reference-type mRNA molecule. Conversely, if the 3' end of one primer hybridizes to a uracil (rather than a guanine) in a particular ARHGEF12 mRNA molecule at a position corresponding to position 3,739 of SEQ ID NO:32, the presence of an amplified fragment will indicate the presence of an ARHGEF12 variant mRNA molecule.In some embodiments, the nucleotide of the primer complementary to the uracil at the position corresponding to position 3,739 of SEQ ID NO:32 may be at the 3' end of the primer. Also, if the 3' end of one primer hybridizes to the guanine at the position corresponding to position 3,627 of SEQ ID NO:10 in a particular ARHGEF12 nucleic acid molecule (rather than the uracil at position 3,627), the presence of the amplified fragment will indicate the presence of an ARHGEF12 reference-type mRNA molecule. Conversely, if the 3' end of one primer hybridizes to the uracil at the position corresponding to position 3,627 of SEQ ID NO:33 in a particular ARHGEF12 mRNA molecule (rather than the guanine), the presence of the amplified fragment will indicate the presence of an ARHGEF12 variant mRNA molecule. In some embodiments, the nucleotide of the primer complementary to the uracil at the position corresponding to position 3,627 of SEQ ID NO:33 may be at the 3' end of the primer. Also, if the 3' end of one primer hybridizes to a guanine (rather than a uracil at position 4,240) in a particular ARHGEF12 nucleic acid molecule at a position corresponding to position 4,240 of SEQ ID NO: 11, the presence of an amplified fragment will indicate the presence of an ARHGEF12 reference-type mRNA molecule. Conversely, if the 3' end of one primer hybridizes to a uracil (rather than a guanine) in a particular ARHGEF12 mRNA molecule at a position corresponding to position 4,240 of SEQ ID NO: 34, the presence of an amplified fragment will indicate the presence of an ARHGEF12 variant mRNA molecule. In some embodiments, the nucleotide of the primer complementary to the uracil at position corresponding to position 4,240 of SEQ ID NO: 34 may be at the 3' end of the primer. Furthermore, if the 3' end of one of the primers hybridizes to a guanine at a position corresponding to position 3,594 of SEQ ID NO:12 in a particular ARHGEF12 nucleic acid molecule (rather than a uracil at position 3,594), the presence of an amplified fragment will indicate the presence of an ARHGEF12 reference-type mRNA molecule.Conversely, if the 3' end of one primer hybridizes to uracil (rather than guanine) at a position corresponding to position 3,594 of SEQ ID NO:35 in a particular ARHGEF12 mRNA molecule, the presence of an amplified fragment will indicate the presence of an ARHGEF12 variant mRNA molecule. In some embodiments, the nucleotide of the primer complementary to uracil at a position corresponding to position 3,594 of SEQ ID NO:35 can be at the 3' end of the primer. Also, if the 3' end of one primer hybridizes to guanine (rather than uracil at position 3,473) at a position corresponding to position 3,473 of SEQ ID NO:13 in a particular ARHGEF12 nucleic acid molecule, the presence of an amplified fragment will indicate the presence of an ARHGEF12 reference-type mRNA molecule. Conversely, if the 3' end of one primer hybridizes to uracil (rather than guanine) at a position corresponding to position 3,473 of SEQ ID NO:36 in a particular ARHGEF12 mRNA molecule, the presence of an amplified fragment will indicate the presence of an ARHGEF12 variant mRNA molecule. In some embodiments, the nucleotide of the primer complementary to the uracil at the position corresponding to position 3,473 of SEQ ID NO:36 may be at the 3' end of the primer. Also, if the 3' end of one primer hybridizes to the guanine at the position corresponding to position 3,602 of SEQ ID NO:14 in a particular ARHGEF12 nucleic acid molecule (rather than the uracil at position 3,602), the presence of the amplified fragment will indicate the presence of an ARHGEF12 reference-type mRNA molecule. Conversely, if the 3' end of one primer hybridizes to the uracil at the position corresponding to position 3,602 of SEQ ID NO:37 in a particular ARHGEF12 mRNA molecule (rather than the guanine), the presence of the amplified fragment will indicate the presence of an ARHGEF12 variant mRNA molecule. In some embodiments, the nucleotide of the primer complementary to the uracil at the position corresponding to position 3,602 of SEQ ID NO:37 may be at the 3' end of the primer.Also, if the 3' end of one primer hybridizes to a guanine (rather than a uracil at position 3,163) in a particular ARHGEF12 nucleic acid molecule at a position corresponding to position 3,163 of SEQ ID NO: 15, the presence of an amplified fragment will indicate the presence of an ARHGEF12 reference-type mRNA molecule. Conversely, if the 3' end of one primer hybridizes to a uracil (rather than a guanine) in a particular ARHGEF12 mRNA molecule at a position corresponding to position 3,163 of SEQ ID NO: 38, the presence of an amplified fragment will indicate the presence of an ARHGEF12 variant mRNA molecule. In some embodiments, the nucleotide of the primer complementary to the uracil at position corresponding to position 3,163 of SEQ ID NO: 38 may be at the 3' end of the primer. Also, if the 3' end of one primer hybridizes to a guanine (rather than a thymine at position 4,297) in a particular ARHGEF12 nucleic acid molecule at a position corresponding to position 4,297 of SEQ ID NO: 39, the presence of an amplified fragment would indicate the presence of an ARHGEF12 reference-type cDNA molecule. Conversely, if the 3' end of one primer hybridizes to a thymine (rather than a guanine) in a particular ARHGEF12 cDNA molecule at a position corresponding to position 4,297 of SEQ ID NO: 62, the presence of an amplified fragment would indicate the presence of an ARHGEF12 variant cDNA molecule. In some embodiments, the nucleotide of the primer that is complementary to the thymine at position corresponding to position 4,297 of SEQ ID NO: 62 may be at the 3' end of the primer. Also, if the 3' end of one primer hybridizes to a guanine (rather than a thymine at position 3,379) in a particular ARHGEF12 nucleic acid molecule at a position corresponding to position 3,379 of SEQ ID NO: 40, the presence of an amplified fragment would indicate the presence of an ARHGEF12 reference-type cDNA molecule. Conversely, if the 3' end of one of the primers hybridizes to a thymine (rather than a guanine) at a position corresponding to position 3,379 of SEQ ID NO:63 in a particular ARHGEF12 cDNA molecule, the presence of an amplified fragment will indicate the presence of an ARHGEF12 variant cDNA molecule.In some embodiments, the nucleotide of the primer complementary to the thymine at the position corresponding to position 3,379 of SEQ ID NO:63 may be at the 3' end of the primer. Also, if the 3' end of one primer hybridizes to the guanine at the position corresponding to position 3,627 of SEQ ID NO:41 in a particular ARHGEF12 nucleic acid molecule (rather than the thymine at position 3,627), the presence of an amplified fragment will indicate the presence of an ARHGEF12 reference-type cDNA molecule. Conversely, if the 3' end of one primer hybridizes to the thymine at the position corresponding to position 3,627 of SEQ ID NO:64 in a particular ARHGEF12 cDNA molecule (rather than the guanine), the presence of an amplified fragment will indicate the presence of an ARHGEF12 variant cDNA molecule. In some embodiments, the nucleotide of the primer complementary to the thymine at the position corresponding to position 3,627 of SEQ ID NO:64 may be at the 3' end of the primer. Also, if the 3' end of one primer hybridizes to a guanine (rather than a thymine at position 4,240) in a particular ARHGEF12 nucleic acid molecule at a position corresponding to position 4,240 of SEQ ID NO: 42, the presence of an amplified fragment will indicate the presence of an ARHGEF12 reference-type cDNA molecule. Conversely, if the 3' end of one primer hybridizes to a thymine (rather than a guanine) in a particular ARHGEF12 cDNA molecule at a position corresponding to position 4,240 of SEQ ID NO: 65, the presence of an amplified fragment will indicate the presence of an ARHGEF12 variant cDNA molecule. In some embodiments, the nucleotide of the primer complementary to the thymine at position corresponding to position 4,240 of SEQ ID NO: 65 may be at the 3' end of the primer. Furthermore, if the 3' end of one of the primers hybridizes to a guanine at a position corresponding to position 3,594 of SEQ ID NO:43 in a particular ARHGEF12 nucleic acid molecule (rather than a thymine at position 3,594), the presence of an amplified fragment will indicate the presence of an ARHGEF12 reference-type cDNA molecule.Conversely, if the 3' end of one primer hybridizes to a thymine (rather than a guanine) at a position corresponding to position 3,594 of SEQ ID NO:66 in a particular ARHGEF12 cDNA molecule, the presence of an amplified fragment will indicate the presence of an ARHGEF12 variant cDNA molecule. In some embodiments, the nucleotide of the primer complementary to the thymine at a position corresponding to position 3,594 of SEQ ID NO:66 can be at the 3' end of the primer. Also, if the 3' end of one primer hybridizes to a guanine (rather than a thymine at position 3,473) at a position corresponding to position 3,473 of SEQ ID NO:44 in a particular ARHGEF12 nucleic acid molecule, the presence of an amplified fragment will indicate the presence of an ARHGEF12 reference-type cDNA molecule. Conversely, if the 3' end of one primer hybridizes to a thymine (rather than a guanine) at a position corresponding to position 3,473 of SEQ ID NO:67 in a particular ARHGEF12 cDNA molecule, the presence of an amplified fragment will indicate the presence of an ARHGEF12 variant cDNA molecule. In some embodiments, the nucleotide of the primer complementary to the thymine at the position corresponding to position 3,473 of SEQ ID NO:67 can be at the 3' end of the primer, and the 3' end of one primer can be at position 3,602 of SEQ ID NO:45 in a particular ARHGEF12 nucleic acid molecule. If the 3' end of one primer hybridizes to a guanine (rather than a thymine at position 3,602) at the corresponding position in a particular ARHGEF12 cDNA molecule, the presence of the amplified fragment will indicate the presence of an ARHGEF12 reference-type cDNA molecule. Conversely, if the 3' end of one primer hybridizes to a thymine (rather than a guanine) at a position corresponding to position 3,602 of SEQ ID NO:68 in a particular ARHGEF12 cDNA molecule, the presence of the amplified fragment will indicate the presence of an ARHGEF12 variant cDNA molecule. In some embodiments, the nucleotide of the primer that is complementary to the thymine at the position corresponding to position 3,602 of SEQ ID NO:68 can be at the 3' end of the primer. Also, if the 3' end of one primer hybridizes to a guanine (rather than a thymine at position 3,163) at a position corresponding to position 3,163 of SEQ ID NO:46 in a particular ARHGEF12 nucleic acid molecule, the presence of the amplified fragment will indicate the presence of an ARHGEF12 reference-type cDNA molecule. Conversely, if the 3' end of one primer hybridizes to a thymine (rather than a guanine) at a position corresponding to position 3,163 of SEQ ID NO:69 in a particular ARHGEF12 cDNA molecule, the presence of an amplified fragment will indicate the presence of an ARHGEF12 variant cDNA molecule. In some embodiments, the nucleotide of the primer complementary to the thymine at the position corresponding to position 3,163 of SEQ ID NO:69 may be at the 3' end of the primer.

[0179] The present disclosure also provides a pair of primers comprising any of the above primers. For example, if the 3' end of one primer hybridizes with adenine (rather than adenine) at the position corresponding to position 132,939 of SEQ ID NO:1 in a specific ARHGEF12 nucleic acid molecule, the presence of an amplified fragment will indicate the presence of an ARHGEF12 reference genomic nucleic acid molecule. Conversely, if the 3' end of one primer hybridizes with adenine (rather than adenine) at the position corresponding to position 73,039 of SEQ ID NO:5 in a specific ARHGEF12 nucleic acid molecule, the presence of an amplified fragment will indicate the presence of an ARHGEF12 variant genomic nucleic acid molecule. In some embodiments, the nucleotide of the primer that is complementary to the adenine at the position corresponding to position 73,039 of SEQ ID NO:5 can be at the 3' end of the primer.

[0180] The present disclosure also provides a pair of primers comprising any of the above primers. For example, if the 3' end of one primer hybridizes with adenine (rather than cytosine) at the position corresponding to position 132,939 of SEQ ID NO:1 in a specific ARHGEF12 nucleic acid molecule, the presence of an amplified fragment will indicate the presence of an ARHGEF12 reference genomic nucleic acid molecule. Conversely, if the 3' end of one primer hybridizes with cytosine (rather than adenine) at the position corresponding to position 121,307 of SEQ ID NO:6 in a specific ARHGEF12 nucleic acid molecule, the presence of an amplified fragment will indicate the presence of an ARHGEF12 variant genomic nucleic acid molecule. In some embodiments, the nucleotide of the primer that is complementary to the cytosine at the position corresponding to position 121,307 of SEQ ID NO:6 can be at the 3' end of the primer.

[0181] The present disclosure also provides a pair of primers comprising any of the above primers.For example, if the 3' end of one primer hybridizes with adenine (rather than cytosine) at the position corresponding to position 132,939 of SEQ ID NO:1 in a specific ARHGEF12 nucleic acid molecule, the presence of an amplified fragment will indicate the presence of an ARHGEF12 reference genomic nucleic acid molecule.On the other hand, if the 3' end of one primer hybridizes with cytosine (rather than adenine) at the position corresponding to position 141,978 of SEQ ID NO:7 in a specific ARHGEF12 nucleic acid molecule, the presence of an amplified fragment will indicate the presence of an ARHGEF12 variant genomic nucleic acid molecule.

[0182] In the context of this disclosure, "specifically hybridizes" means that a probe or primer (e.g., a variant-specific probe or variant-specific primer) does not hybridize to a nucleic acid sequence encoding an ARHGEF12 reference-type genomic nucleic acid molecule, an ARHGEF12 reference-type mRNA molecule, and / or an ARHGEF12 reference-type cDNA molecule.

[0183] In some embodiments, the probe (such as, for example, a modification-specific probe) comprises a label. In some embodiments, the label is a fluorescent label, a radioactive label, or biotin. The present disclosure also provides a support comprising a substrate to which any one or more of the probes disclosed herein are attached. A solid support is a solid-state substrate or support to which molecules such as any of the probes disclosed herein can be associated. A form of solid support is an array. Another form of solid support is an array detector. An array detector is a solid support to which a plurality of different probes are attached in an array, grid, or other organized pattern. A form of solid-state substrate is a microtiter dish, such as a standard 96-well format. In some embodiments, a multi-well glass slide can be used, which usually contains one array per well.

[0184] The present disclosure also provides a molecular complex comprising or consisting of any of the ARHGEF12 nucleic acid molecules (genomic nucleic acid molecules, mRNA molecules, or cDNA molecules) described herein, or their complements, and any of the variant-specific primers or variant-specific probes described herein. In some embodiments, the ARHGEF12 nucleic acid molecule (genomic nucleic acid molecule, mRNA molecule, or cDNA molecule) or its complement in the molecular complex is single-stranded. In some embodiments, the ARHGEF12 nucleic acid molecule is any of the genomic nucleic acid molecules described herein. In some embodiments, the ARHGEF12 nucleic acid molecule is any of the mRNA molecules described herein. In some embodiments, the ARHGEF12 nucleic acid molecule is any of the cDNA molecules described herein. In some embodiments, the molecular complex comprises or consists of any of the ARHGEF12 nucleic acid molecules (genomic nucleic acid molecules, mRNA molecules, or cDNA molecules) described herein, or their complements, and any of the variant-specific primers described herein. In some embodiments, the molecular complex comprises or consists of any of the ARHGEF12 nucleic acid molecules (genomic nucleic acid molecules, mRNA molecules, or cDNA molecules) described herein, or their complements, and any of the variant-specific probes described herein.

[0185] In some embodiments, the molecular complex comprises or consists of a variant-specific primer or variant-specific probe that hybridizes to a genomic nucleic acid molecule comprising a nucleotide sequence encoding an ARHGEF12 polypeptide, and the variant-specific primer or variant-specific probe hybridizes to a thymine at a position corresponding to position 141,048 of SEQ ID NO:4, or its complement.

[0186] In some embodiments, the molecular complex comprises or consists of a variant-specific primer or variant-specific probe that hybridizes to a TAA codon at a position corresponding to positions 141,048 to 141,050 set forth in SEQ ID NO:4.

[0187] In some embodiments, the molecular complex comprises or consists of a genomic nucleic acid molecule comprising SEQ ID NO:4. In some embodiments, the molecular complex comprises or consists of a variant-specific primer or variant-specific probe hybridized to an mRNA molecule comprising a nucleotide sequence encoding a human ARHGEF12 polypeptide, where the variant-specific primer or variant-specific probe hybridizes to a uracil at a position corresponding to position 4,297 of SEQ ID NO:31 or its complement, position 3,739 of SEQ ID NO:32 or its complement, position 3,627 of SEQ ID NO:33 or its complement, position 4,240 of SEQ ID NO:34 or its complement, position 3,594 of SEQ ID NO:35 or its complement, position 3,473 of SEQ ID NO:36 or its complement, position 3,602 of SEQ ID NO:37 or its complement, or position 3,163 of SEQ ID NO:38 or its complement.

[0188] In some embodiments, the molecular complex comprises or consists of a variant-specific primer or variant-specific probe that hybridizes to a UAA codon at a position corresponding to positions 4,297 to 4,299 of SEQ ID NO:31, positions 3,739 to 3,741 of SEQ ID NO:32, positions 3,627 to 3,629 of SEQ ID NO:33, positions 4,240 to 4,242 of SEQ ID NO:34, positions 3,594 to 3,596 of SEQ ID NO:35, positions 3,473 to 3,475 of SEQ ID NO:36, positions 3,602 to 3,604 of SEQ ID NO:37, or positions 3,163 to 3,165 of SEQ ID NO:38.

[0189] In some embodiments, the molecular complex comprises or consists of an mRNA molecule comprising SEQ ID NO:31, SEQ ID NO:32, SEQ ID NO:33, SEQ ID NO:34, SEQ ID NO:35, SEQ ID NO:36, SEQ ID NO:37, or SEQ ID NO:38.

[0190] In some embodiments, the molecular complex comprises or consists of a variant-specific primer or variant-specific probe hybridized to a cDNA molecule comprising a nucleotide sequence encoding a human ARHGEF12 polypeptide, where the variant-specific primer or variant-specific probe hybridizes to a thymine at a position corresponding to position 4,297 of SEQ ID NO:62 or its complement, position 3,379 of SEQ ID NO:63 or its complement, position 3,627 of SEQ ID NO:64 or its complement, position 4,240 of SEQ ID NO:65 or its complement, position 3,594 of SEQ ID NO:66 or its complement, position 3,473 of SEQ ID NO:67 or its complement, position 3,602 of SEQ ID NO:68 or its complement, or position 3,163 of SEQ ID NO:69 or its complement.

[0191] In some embodiments, the molecular complex comprises or consists of a variant-specific primer or variant-specific probe that hybridizes to a TAA codon at a position corresponding to positions 4,297 to 4,299 of SEQ ID NO:62, positions 3,739 to 3,741 of SEQ ID NO:63, positions 3,627 to 3,629 of SEQ ID NO:64, positions 4,240 to 4,242 of SEQ ID NO:65, positions 3,594 to 3,596 of SEQ ID NO:66, positions 3,473 to 3,475 of SEQ ID NO:67, positions 3,602 to 3,604 of SEQ ID NO:68, or positions 3,163 to 3,165 of SEQ ID NO:69.

[0192] In some embodiments, the molecular complex comprises or consists of a cDNA molecule comprising SEQ ID NO:62, SEQ ID NO:63, SEQ ID NO:64, SEQ ID NO:65, SEQ ID NO:66, SEQ ID NO:67, SEQ ID NO:68, or SEQ ID NO:69.

[0193] In some embodiments, the molecular complex comprises a variant-specific probe or a variant-specific primer that comprises a label. In some embodiments, the label is a fluorescent label, a radioactive label, or biotin. In some embodiments, the molecular complex further comprises a non-human polymerase.

[0194] The present disclosure also provides an isolated nucleic acid molecule comprising a nucleotide sequence encoding a human ARHGEF12 variant polypeptide. In some embodiments, the ARHGEF12 variant polypeptide terminates at a position corresponding to position 1,155 of SEQ ID NO:81 or its complement. In some embodiments, the isolated nucleic acid molecule has at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to SEQ ID NO:81 and encodes an ARHGEF12 variant polypeptide having an amino acid sequence that terminates at a position corresponding to position 1,155 of SEQ ID NO:81. In some embodiments, the isolated nucleic acid molecule has at least about 90% sequence identity to SEQ ID NO:81 and encodes an ARHGEF12 variant polypeptide having an amino acid sequence that terminates at a position corresponding to position 1,155 of SEQ ID NO:81. In some embodiments, the isolated nucleic acid molecule encodes an ARHGEF12 variant polypeptide having an amino acid sequence that has at least about 92% sequence identity to SEQ ID NO:81 and terminates at a position corresponding to position 1,155 set forth in SEQ ID NO:81. In some embodiments, the isolated nucleic acid molecule encodes an ARHGEF12 variant polypeptide having an amino acid sequence that has at least about 94% sequence identity to SEQ ID NO:81 and terminates at a position corresponding to position 1,155 set forth in SEQ ID NO:81. In some embodiments, the isolated nucleic acid molecule encodes an ARHGEF12 variant polypeptide having an amino acid sequence that has at least about 96% sequence identity to SEQ ID NO:81 and terminates at a position corresponding to position 1,155 set forth in SEQ ID NO:81. In some embodiments, the isolated nucleic acid molecule encodes an ARHGEF12 variant polypeptide having an amino acid sequence that has at least about 98% sequence identity to SEQ ID NO:81 and terminates at a position corresponding to position 1,155 set forth in SEQ ID NO:81. In some embodiments, the nucleic acid molecule encodes an ARHGEF12 variant polypeptide comprising SEQ ID NO:81.In some embodiments, the nucleic acid molecule encodes an ARHGEF12 variant polypeptide consisting of SEQ ID NO:81.

[0195] In some embodiments, the ARHGEF12 variant polypeptide terminates at a position corresponding to position 1,136 of SEQ ID NO: 82 or its complement. In some embodiments, an isolated nucleic acid molecule has at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to SEQ ID NO: 82 and encodes an ARHGEF12 variant polypeptide having an amino acid sequence that terminates at a position corresponding to position 1,136 of SEQ ID NO: 82. In some embodiments, an isolated nucleic acid molecule has at least about 90% sequence identity to SEQ ID NO: 82 and encodes an ARHGEF12 variant polypeptide having an amino acid sequence that terminates at a position corresponding to position 1,136 of SEQ ID NO: 82. In some embodiments, the isolated nucleic acid molecule encodes an ARHGEF12 variant polypeptide having at least about 92% sequence identity to SEQ ID NO:82 and an amino acid sequence terminating at a position corresponding to position 1,136 set forth in SEQ ID NO:82. In some embodiments, the isolated nucleic acid molecule encodes an ARHGEF12 variant polypeptide having at least about 94% sequence identity to SEQ ID NO:82 and an amino acid sequence terminating at a position corresponding to position 1,136 set forth in SEQ ID NO:82. In some embodiments, the isolated nucleic acid molecule encodes an ARHGEF12 variant polypeptide having at least about 96% sequence identity to SEQ ID NO:82 and an amino acid sequence terminating at a position corresponding to position 1,136 set forth in SEQ ID NO:82. In some embodiments, the isolated nucleic acid molecule encodes an ARHGEF12 variant polypeptide having at least about 98% sequence identity to SEQ ID NO:82 and an amino acid sequence terminating at a position corresponding to position 1,136 set forth in SEQ ID NO:82. In some embodiments, the nucleic acid molecule encodes an ARHGEF12 variant polypeptide comprising SEQ ID NO:82. In some embodiments, the nucleic acid molecule encodes an ARHGEF12 variant polypeptide consisting of SEQ ID NO:82.

[0196] In some embodiments, the ARHGEF12 variant polypeptide terminates at a position corresponding to position 1,052 of SEQ ID NO: 83 or its complement. In some embodiments, the isolated nucleic acid molecule has at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to SEQ ID NO: 83 and encodes an ARHGEF12 variant polypeptide having an amino acid sequence that terminates at a position corresponding to position 1,052 of SEQ ID NO: 83. In some embodiments, the isolated nucleic acid molecule encodes an ARHGEF12 variant polypeptide having at least about 90% sequence identity to SEQ ID NO: 83 and encoding an amino acid sequence that terminates at a position corresponding to position 1,052 of SEQ ID NO: 83. In some embodiments, the isolated nucleic acid molecule encodes an ARHGEF12 variant polypeptide having an amino acid sequence that has at least about 92% sequence identity to SEQ ID NO: 83 and terminates at a position corresponding to position 1,052 set forth in SEQ ID NO: 83. In some embodiments, the isolated nucleic acid molecule encodes an ARHGEF12 variant polypeptide having an amino acid sequence that has at least about 94% sequence identity to SEQ ID NO: 83 and terminates at a position corresponding to position 1,052 set forth in SEQ ID NO: 83. In some embodiments, the isolated nucleic acid molecule encodes an ARHGEF12 variant polypeptide having an amino acid sequence that has at least about 96% sequence identity to SEQ ID NO: 83 and terminates at a position corresponding to position 1,052 set forth in SEQ ID NO: 83. In some embodiments, the isolated nucleic acid molecule encodes an ARHGEF12 variant polypeptide having an amino acid sequence that has at least about 98% sequence identity to SEQ ID NO: 83 and terminates at a position corresponding to position 1,052 set forth in SEQ ID NO: 83. In some embodiments, the nucleic acid molecule encodes an ARHGEF12 variant polypeptide comprising SEQ ID NO: 83. In some embodiments, the nucleic acid molecule encodes an ARHGEF12 variant polypeptide consisting of SEQ ID NO:83.

[0197] The nucleotide sequence of the ARHGEF12 reference genomic nucleic acid molecule is set forth in SEQ ID NO:1. As set forth in SEQ ID NO:1, position 132,939 is an adenine. As set forth in SEQ ID NO:1, position 143,698 is an adenine. As set forth in SEQ ID NO:1, position 141,048 is a guanine. As set forth in SEQ ID NO:1, position 73,039 is a guanine. As set forth in SEQ ID NO:1, position 121,307 is an adenine. As set forth in SEQ ID NO:1, position 141,978 is a guanine.

[0198] There is an ARHGEF12 variant genomic nucleic acid molecule in which the adenine at position 132,939 (as set forth in SEQ ID NO:1) is replaced with a thymine. The nucleotide sequence of this ARHGEF12 variant genomic nucleic acid molecule is set forth in SEQ ID NO:2.

[0199] Another ARHGEF12 variant genomic nucleic acid molecule exists in which the adenine at position 143,698 (as shown in SEQ ID NO:1) is replaced with a guanine. The nucleotide sequence of this ARHGEF12 variant genomic nucleic acid molecule is set forth in SEQ ID NO:3.

[0200] Another ARHGEF12 variant genomic nucleic acid molecule exists in which the guanine at position 141,048 (as shown in SEQ ID NO:1) is replaced with a thymine. The nucleotide sequence of this ARHGEF12 variant genomic nucleic acid molecule is set forth in SEQ ID NO:4.

[0201] Another ARHGEF12 variant genomic nucleic acid molecule exists in which the guanine at position 73,039 (as shown in SEQ ID NO:1) is replaced with an adenine. The nucleotide sequence of this ARHGEF12 variant genomic nucleic acid molecule is set forth in SEQ ID NO:5.

[0202] Another ARHGEF12 variant genomic nucleic acid molecule exists in which the adenine at position 121,307 (as shown in SEQ ID NO:1) is replaced with a cytosine. The nucleotide sequence of this ARHGEF12 variant genomic nucleic acid molecule is set forth in SEQ ID NO:6.

[0203] Another ARHGEF12 variant genomic nucleic acid molecule exists in which the guanine at position 141,978 (as shown in SEQ ID NO:1) is replaced with a cytosine. The nucleotide sequence of this ARHGEF12 variant genomic nucleic acid molecule is set forth in SEQ ID NO:7.

[0204] The present disclosure also provides an isolated genomic nucleic acid molecule comprising or consisting of a nucleotide sequence encoding a human ARHGEF12 polypeptide. In some embodiments, the isolated genomic nucleic acid molecule encodes an ARHGEF12 truncated variant polypeptide that terminates at a position corresponding to position 1,155 of SEQ ID NO:81. In some embodiments, the nucleotide sequence of the genomic nucleic acid molecule comprises a thymine at a position corresponding to position 141,048 of SEQ ID NO:4 or its complement. In some embodiments, the nucleotide sequence of the genomic nucleic acid molecule comprises a TAA codon at a position corresponding to positions 141,048-141,050 of SEQ ID NO:4.

[0205] In some embodiments, the nucleotide sequence of the genomic nucleic acid molecule comprises an adenine at a position corresponding to position 73,039 of SEQ ID NO:5, or its complement. In some embodiments, the isolated genomic nucleic acid molecule comprises or consists of a nucleotide sequence having at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to SEQ ID NO:4, and comprises a thymine at a position corresponding to position 141,048 of SEQ ID NO:4 or its complement. In some embodiments, the isolated genomic nucleic acid molecule comprises or consists of a nucleotide sequence having at least about 90% sequence identity to SEQ ID NO:4, and comprises a thymine at a position corresponding to position 141,048 of SEQ ID NO:4 or its complement. In some embodiments, the isolated genomic nucleic acid molecule comprises or consists of a nucleotide sequence having at least about 92% sequence identity to SEQ ID NO:4, and comprises a thymine at a position corresponding to position 141,048 of SEQ ID NO:4 or its complement. In some embodiments, the isolated genomic nucleic acid molecule comprises or consists of a nucleotide sequence having at least about 94% sequence identity to SEQ ID NO:4 and comprises a thymine at a position corresponding to position 141,048 of SEQ ID NO:4 or its complement. In some embodiments, the isolated genomic nucleic acid molecule comprises or consists of a nucleotide sequence having at least about 96% sequence identity to SEQ ID NO:4 and comprises a thymine at a position corresponding to position 141,048 of SEQ ID NO:4 or its complement. In some embodiments, the isolated genomic nucleic acid molecule comprises or consists of a nucleotide sequence having at least about 98% sequence identity to SEQ ID NO:4 and comprises a thymine at a position corresponding to position 141,048 of SEQ ID NO:4 or its complement.

[0206] In some embodiments, the isolated genomic nucleic acid molecule comprises or consists of a nucleotide sequence having at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to SEQ ID NO:5, and includes an adenine at a position corresponding to position 73,039 of SEQ ID NO:5 or its complement. In some embodiments, the isolated genomic nucleic acid molecule comprises or consists of a nucleotide sequence having at least about 90% sequence identity to SEQ ID NO:5, and includes an adenine at a position corresponding to position 73,039 of SEQ ID NO:5 or its complement. In some embodiments, the isolated genomic nucleic acid molecule comprises or consists of a nucleotide sequence having at least about 92% sequence identity to SEQ ID NO:5, and includes an adenine at a position corresponding to position 73,039 of SEQ ID NO:5 or its complement. In some embodiments, the isolated genomic nucleic acid molecule comprises or consists of a nucleotide sequence having at least about 94% sequence identity to SEQ ID NO:5 and comprises an adenine at a position corresponding to position 73,039 of SEQ ID NO:5 or its complement. In some embodiments, the isolated genomic nucleic acid molecule comprises or consists of a nucleotide sequence having at least about 96% sequence identity to SEQ ID NO:5 and comprises an adenine at a position corresponding to position 73,039 of SEQ ID NO:5 or its complement. In some embodiments, the isolated genomic nucleic acid molecule comprises or consists of a nucleotide sequence having at least about 98% sequence identity to SEQ ID NO:5 and comprises an adenine at a position corresponding to position 73,039 of SEQ ID NO:5 or its complement.

[0207] As used herein, when percentage of sequence identity is referred to, a higher percentage of sequence identity is preferred over a lower percentage of sequence identity. In some embodiments, the isolated genomic nucleic acid molecule comprises or consists of a nucleotide sequence having at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to SEQ ID NO:4, and comprises a TAA codon at a position corresponding to positions 141,048-141,050 of SEQ ID NO:4 or its complement. In some embodiments, the isolated genomic nucleic acid molecule comprises or consists of a nucleotide sequence having at least about 90% sequence identity to SEQ ID NO:4, and comprises a TAA codon at a position corresponding to positions 141,048-141,050 of SEQ ID NO:4 or its complement. In some embodiments, the isolated genomic nucleic acid molecule comprises or consists of a nucleotide sequence having at least about 92% sequence identity to SEQ ID NO:4, and comprises a TAA codon at a position corresponding to positions 141,048-141,050 of SEQ ID NO:4 or its complement. In some embodiments, the isolated genomic nucleic acid molecule comprises or consists of a nucleotide sequence having at least about 94% sequence identity to SEQ ID NO:4, and comprises a TAA codon at a position corresponding to positions 141,048-141,050 of SEQ ID NO:4 or its complement. In some embodiments, the isolated genomic nucleic acid molecule comprises or consists of a nucleotide sequence having at least about 96% sequence identity to SEQ ID NO:4, and comprises a TAA codon at a position corresponding to positions 141,048-141,050 of SEQ ID NO:4 or its complement. In some embodiments, the isolated genomic nucleic acid molecule comprises or consists of a nucleotide sequence having at least about 98% sequence identity to SEQ ID NO:4, and comprises a TAA codon at a position corresponding to positions 141,048-141,050 of SEQ ID NO:4 or its complement.

[0208] As used herein, when percentage of sequence identity is referred to, a higher percentage of sequence identity is preferred over a lower percentage of sequence identity. In some embodiments, the isolated genomic nucleic acid molecule comprises SEQ ID NO: 4. In some embodiments, the isolated genomic nucleic acid molecule consists of SEQ ID NO: 4. In some embodiments, the isolated genomic nucleic acid molecule comprises SEQ ID NO: 5. In some embodiments, the isolated genomic nucleic acid molecule consists of SEQ ID NO: 5.

[0209] In some embodiments, the isolated genomic nucleic acid molecule comprises less than the entire genomic DNA sequence. In some embodiments, the isolated genomic nucleic acid molecule comprises or consists of at least about 15, at least about 20, at least about 25, at least about 30, at least about 35, at least about 40, at least about 45, at least about 50, at least about 60, at least about 70, at least about 80, at least about 90, at least about 100, at least about 200, at least about 300, at least about 400, at least about 500, at least about 600, at least about 700, at least about 800, at least about 900, at least about 1000, at least about 2000, at least about 3000, at least about 4000, at least about 5000, at least about 6000, at least about 7000, at least about 8000, at least about 9000, or at least about 10000 consecutive nucleotides of any of the ARHGEF12 genomic nucleic acid molecules disclosed herein. In some embodiments, the isolated genomic nucleic acid molecules comprise or consist of at least about 1000 to at least about 2000 contiguous nucleotides of any of the ARHGEF12 genomic nucleic acid molecules disclosed herein. In some embodiments, these isolated genomic nucleic acid molecules comprise a thymine at a position corresponding to position 141,048 of SEQ ID NO:4. In some embodiments, these isolated genomic nucleic acid molecules comprise an adenine at a position corresponding to position 73,039 of SEQ ID NO:5.

[0210] The nucleotide sequence of the ARHGEF12 reference type mRNA molecule is set forth in SEQ ID NO: 8. As set forth in SEQ ID NO: 8, position 3,749 is an adenine. As set forth in SEQ ID NO: 8, position 4,748 is an adenine. As set forth in SEQ ID NO: 8, position 4,297 is a guanine.

[0211] The nucleotide sequence of another ARHGEF12 reference type mRNA molecule is set forth in SEQ ID NO:9. As set forth in SEQ ID NO:9, position 3,191 is an adenine. As set forth in SEQ ID NO:9, position 4,190 is an adenine. As set forth in SEQ ID NO:9, position 3,739 is a guanine.

[0212] The nucleotide sequence of another ARHGEF12 reference type mRNA molecule is set forth in SEQ ID NO: 10. As set forth in SEQ ID NO: 10, position 3,079 is an adenine. As set forth in SEQ ID NO: 10, position 4,078 is an adenine. As set forth in SEQ ID NO: 10, position 3,627 is a guanine.

[0213] The nucleotide sequence of another ARHGEF12 reference type mRNA molecule is set forth in SEQ ID NO: 11. As set forth in SEQ ID NO: 11, position 3,692 is an adenine. As set forth in SEQ ID NO: 11, position 4,691 is an adenine. As set forth in SEQ ID NO: 11, position 4,240 is a guanine.

[0214] The nucleotide sequence of another ARHGEF12 reference-type mRNA molecule is set forth in SEQ ID NO: 12. As set forth in SEQ ID NO: 12, position 3,046 is an adenine. As set forth in SEQ ID NO: 12, position 4,045 is an adenine. As set forth in SEQ ID NO: 12, position 3,594 is a guanine.

[0215] The nucleotide sequence of another ARHGEF12 reference type mRNA molecule is set forth in SEQ ID NO: 13. As set forth in SEQ ID NO: 13, position 2,925 is an adenine. As set forth in SEQ ID NO: 13, position 3,924 is an adenine. As set forth in SEQ ID NO: 13, position 3,473 is a guanine.

[0216] The nucleotide sequence of another ARHGEF12 reference-type mRNA molecule is set forth in SEQ ID NO: 14. As set forth in SEQ ID NO: 14, position 3,054 is an adenine. As set forth in SEQ ID NO: 14, position 3,602 is a guanine.

[0217] The nucleotide sequence of another ARHGEF12 reference type mRNA molecule is set forth in SEQ ID NO: 15. As set forth in SEQ ID NO: 15, position 2,615 is an adenine. As set forth in SEQ ID NO: 15, position 3,614 is an adenine. As set forth in SEQ ID NO: 15, position 3,163 is a guanine.

[0218] There is an ARHGEF12 variant mRNA molecule in which the adenine at position 3,749 (as shown in SEQ ID NO:8) is replaced with uracil. The nucleotide sequence of this ARHGEF12 variant mRNA molecule is shown in SEQ ID NO:16.

[0219] Another ARHGEF12 variant mRNA molecule exists in which the adenine at position 3,191 (as shown in SEQ ID NO:9) is replaced with uracil. The nucleotide sequence of this ARHGEF12 variant mRNA molecule is shown in SEQ ID NO:17.

[0220] Another ARHGEF12 variant mRNA molecule exists in which the adenine at position 3,079 (as shown in SEQ ID NO:10) is replaced with uracil. The nucleotide sequence of this ARHGEF12 variant mRNA molecule is shown in SEQ ID NO:18.

[0221] Another ARHGEF12 variant mRNA molecule exists in which the adenine at position 3,692 (as shown in SEQ ID NO:11) is replaced with uracil. The nucleotide sequence of this ARHGEF12 variant mRNA molecule is shown in SEQ ID NO:19.

[0222] Another ARHGEF12 variant mRNA molecule exists in which the adenine at position 3,046 (as shown in SEQ ID NO:12) is replaced with uracil. The nucleotide sequence of this ARHGEF12 variant mRNA molecule is set forth in SEQ ID NO:20.

[0223] Another ARHGEF12 variant mRNA molecule exists in which the adenine at position 2,925 (as shown in SEQ ID NO: 13) is replaced with uracil. The nucleotide sequence of this ARHGEF12 variant mRNA molecule is set forth in SEQ ID NO:21.

[0224] Another ARHGEF12 variant mRNA molecule exists in which the adenine at position 3,054 (as set forth in SEQ ID NO:14) is replaced with uracil. The nucleotide sequence of this ARHGEF12 variant mRNA molecule is set forth in SEQ ID NO:22.

[0225] Another ARHGEF12 variant mRNA molecule exists in which the adenine at position 2,615 (as set forth in SEQ ID NO:15) is replaced with uracil. The nucleotide sequence of this ARHGEF12 variant mRNA molecule is set forth in SEQ ID NO:23.

[0226] There exists an ARHGEF12 variant mRNA molecule in which the adenine at position 4,748 (as set forth in SEQ ID NO:8) is replaced with a guanine. The nucleotide sequence of this ARHGEF12 variant mRNA molecule is set forth in SEQ ID NO:24.

[0227] Another ARHGEF12 variant mRNA molecule exists in which the adenine at position 4,190 (as set forth in SEQ ID NO:9) is replaced with a guanine. The nucleotide sequence of this ARHGEF12 variant mRNA molecule is set forth in SEQ ID NO:25.

[0228] Another ARHGEF12 variant mRNA molecule exists in which the adenine at position 4,078 (as set forth in SEQ ID NO:10) is replaced with a guanine. The nucleotide sequence of this ARHGEF12 variant mRNA molecule is set forth in SEQ ID NO:26.

[0229] Another ARHGEF12 variant mRNA molecule exists in which the adenine at position 4,691 (as set forth in SEQ ID NO:11) is replaced with a guanine. The nucleotide sequence of this ARHGEF12 variant mRNA molecule is set forth in SEQ ID NO:27.

[0230] Another ARHGEF12 variant mRNA molecule exists in which the adenine at position 4,045 (as shown in SEQ ID NO:12) is replaced with a guanine. The nucleotide sequence of this ARHGEF12 variant mRNA molecule is set forth in SEQ ID NO:28.

[0231] Another ARHGEF12 variant mRNA molecule exists in which the adenine at position 3,924 (as set forth in SEQ ID NO:13) is replaced with a guanine. The nucleotide sequence of this ARHGEF12 variant mRNA molecule is set forth in SEQ ID NO:29.

[0232] Another ARHGEF12 variant mRNA molecule exists in which the adenine at position 3,614 (as set forth in SEQ ID NO:15) is replaced with a guanine. The nucleotide sequence of this ARHGEF12 variant mRNA molecule is set forth in SEQ ID NO:30.

[0233] Another ARHGEF12 variant mRNA molecule exists in which the guanine at position 4,297 (as set forth in SEQ ID NO:8) is replaced with a uracil and the GAA codon at positions 4,297-4,299 of SEQ ID NO:8 is replaced with a stop codon UAA. The nucleotide sequence of this ARHGEF12 variant mRNA molecule is set forth in SEQ ID NO:31.

[0234] Another ARHGEF12 variant mRNA molecule exists in which the guanine at position 3,739 (as set forth in SEQ ID NO:9) is replaced with a uracil and the GAA codon at positions 3,739-3,741 of SEQ ID NO:9 is replaced with a stop codon UAA. The nucleotide sequence of this ARHGEF12 variant mRNA molecule is set forth in SEQ ID NO:32.

[0235] Another ARHGEF12 variant mRNA molecule exists in which the guanine at position 3,627 (as set forth in SEQ ID NO: 10) is replaced with a uracil and the GAA codon at positions 3,627-3,629 of SEQ ID NO: 10 is replaced with a stop codon UAA. The nucleotide sequence of this ARHGEF12 variant mRNA molecule is set forth in SEQ ID NO:33.

[0236] Another ARHGEF12 variant mRNA molecule exists in which the guanine at position 4,240 (as set forth in SEQ ID NO:11) is replaced by a uracil and the GAA codon at positions 4,240-4,242 of SEQ ID NO:11 is replaced by a stop codon UAA. The nucleotide sequence of this ARHGEF12 variant mRNA molecule is set forth in SEQ ID NO:34.

[0237] Another ARHGEF12 variant mRNA molecule exists in which the guanine at position 3,594 (as set forth in SEQ ID NO: 12) is replaced with a uracil and the GAA codon at positions 3,594-3,596 of SEQ ID NO: 12 is replaced with a stop codon UAA. The nucleotide sequence of this ARHGEF12 variant mRNA molecule is set forth in SEQ ID NO:35.

[0238] Another ARHGEF12 variant mRNA molecule exists in which the guanine at position 3,473 (as set forth in SEQ ID NO: 13) is replaced with a uracil and the GAA codon at positions 3,473-3,475 of SEQ ID NO: 13 is replaced with a stop codon UAA. The nucleotide sequence of this ARHGEF12 variant mRNA molecule is set forth in SEQ ID NO:36.

[0239] Another ARHGEF12 variant mRNA molecule exists in which the guanine at position 3,602 (as set forth in SEQ ID NO: 14) is replaced with a uracil and the GAA codon at positions 3,602-3,604 of SEQ ID NO: 14 is replaced with a stop codon UAA. The nucleotide sequence of this ARHGEF12 variant mRNA molecule is set forth in SEQ ID NO:37.

[0240] Another ARHGEF12 variant mRNA molecule exists in which the guanine at position 3,163 (as set forth in SEQ ID NO: 15) is replaced with a uracil and the GAA codon at positions 3,163-3,165 of SEQ ID NO: 15 is replaced with a stop codon UAA. The nucleotide sequence of this ARHGEF12 variant mRNA molecule is set forth in SEQ ID NO:38.

[0241] The disclosure also provides an isolated mRNA molecule comprising or consisting of a nucleotide sequence encoding a human ARHGEF12 polypeptide, the nucleotide sequence comprising a uracil at a position corresponding to position 4,297 of SEQ ID NO:31 or its complement. In some embodiments, the isolated mRNA molecule comprises or consists of a nucleotide sequence encoding a human ARHGEF12 polypeptide, the nucleotide sequence comprising a UAA codon at a position corresponding to positions 4,297-4,299 of SEQ ID NO:31.

[0242] The disclosure also provides an isolated mRNA molecule comprising or consisting of a nucleotide sequence encoding a human ARHGEF12 polypeptide, the nucleotide sequence comprising a uracil at a position corresponding to position 3,739 of SEQ ID NO:32 or its complement. In some embodiments, the isolated mRNA molecule comprises or consists of a nucleotide sequence encoding a human ARHGEF12 polypeptide, the nucleotide sequence comprising a UAA codon at a position corresponding to positions 3,739-3,741 of SEQ ID NO:32.

[0243] The disclosure also provides an isolated mRNA molecule comprising or consisting of a nucleotide sequence encoding a human ARHGEF12 polypeptide, the nucleotide sequence comprising a uracil at a position corresponding to position 3,627 of SEQ ID NO:33 or its complement. In some embodiments, the isolated mRNA molecule comprises or consists of a nucleotide sequence encoding a human ARHGEF12 polypeptide, the nucleotide sequence comprising a UAA codon at a position corresponding to positions 3,627-3,629 of SEQ ID NO:33.

[0244] The disclosure also provides an isolated mRNA molecule comprising or consisting of a nucleotide sequence encoding a human ARHGEF12 polypeptide, the nucleotide sequence comprising a uracil at a position corresponding to position 4,240 of SEQ ID NO:34 or its complement. In some embodiments, the isolated mRNA molecule comprises or consists of a nucleotide sequence encoding a human ARHGEF12 polypeptide, the nucleotide sequence comprising a UAA codon at a position corresponding to positions 4,240-4,242 of SEQ ID NO:34.

[0245] The disclosure also provides an isolated mRNA molecule comprising or consisting of a nucleotide sequence encoding a human ARHGEF12 polypeptide, the nucleotide sequence comprising a uracil at a position corresponding to position 3,594 of SEQ ID NO:35 or its complement. In some embodiments, the isolated mRNA molecule comprises or consists of a nucleotide sequence encoding a human ARHGEF12 polypeptide, the nucleotide sequence comprising a UAA codon at a position corresponding to positions 3,594-3,596 of SEQ ID NO:35.

[0246] The disclosure also provides an isolated mRNA molecule comprising or consisting of a nucleotide sequence encoding a human ARHGEF12 polypeptide, the nucleotide sequence comprising a uracil at a position corresponding to position 3,473 of SEQ ID NO:36 or its complement. In some embodiments, the isolated mRNA molecule comprises or consists of a nucleotide sequence encoding a human ARHGEF12 polypeptide, the nucleotide sequence comprising a UAA codon at a position corresponding to positions 3,473-3,475 of SEQ ID NO:36.

[0247] The disclosure also provides an isolated mRNA molecule comprising or consisting of a nucleotide sequence encoding a human ARHGEF12 polypeptide, the nucleotide sequence comprising a uracil at a position corresponding to position 3,602 of SEQ ID NO:37 or its complement. In some embodiments, the isolated mRNA molecule comprises or consists of a nucleotide sequence encoding a human ARHGEF12 polypeptide, the nucleotide sequence comprising a UAA codon at a position corresponding to positions 3,602-3,604 of SEQ ID NO:37.

[0248] The disclosure also provides an isolated mRNA molecule comprising or consisting of a nucleotide sequence encoding a human ARHGEF12 polypeptide, the nucleotide sequence comprising a uracil at a position corresponding to position 3,163 of SEQ ID NO:38 or its complement. In some embodiments, the isolated mRNA molecule comprises or consists of a nucleotide sequence encoding a human ARHGEF12 polypeptide, the nucleotide sequence comprising a UAA codon at a position corresponding to positions 3,163-3,165 of SEQ ID NO:38.

[0249] In some embodiments, the isolated mRNA molecule comprises or consists of a nucleotide sequence having at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to SEQ ID NO:31 and includes a uracil at a position corresponding to position 4,297 of SEQ ID NO:31 or its complement. In some embodiments, the isolated mRNA molecule comprises or consists of a nucleotide sequence having at least about 90% sequence identity to SEQ ID NO:31 and includes a uracil at a position corresponding to position 4,297 of SEQ ID NO:31 or its complement. In some embodiments, the isolated mRNA molecule comprises or consists of a nucleotide sequence having at least about 92% sequence identity to SEQ ID NO:31 and includes a uracil at a position corresponding to position 4,297 of SEQ ID NO:31 or its complement. In some embodiments, the isolated mRNA molecule comprises or consists of a nucleotide sequence having at least about 94% sequence identity to SEQ ID NO:31 and comprises a uracil at a position corresponding to position 4,297 of SEQ ID NO:31 or its complement. In some embodiments, the isolated mRNA molecule comprises or consists of a nucleotide sequence having at least about 96% sequence identity to SEQ ID NO:31 and comprises a uracil at a position corresponding to position 4,297 of SEQ ID NO:31 or its complement. In some embodiments, the isolated mRNA molecule comprises or consists of a nucleotide sequence having at least about 98% sequence identity to SEQ ID NO:31 and comprises a uracil at a position corresponding to position 4,297 of SEQ ID NO:31 or its complement.

[0250] In some embodiments, the isolated mRNA molecule comprises or consists of a nucleotide sequence having at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to SEQ ID NO:32 and includes a uracil at a position corresponding to position 3,739 of SEQ ID NO:32 or its complement. In some embodiments, the isolated mRNA molecule comprises or consists of a nucleotide sequence having at least about 90% sequence identity to SEQ ID NO:32 and includes a uracil at a position corresponding to position 3,739 of SEQ ID NO:32 or its complement. In some embodiments, the isolated mRNA molecule comprises or consists of a nucleotide sequence having at least about 92% sequence identity to SEQ ID NO:32 and includes a uracil at a position corresponding to position 3,739 of SEQ ID NO:32 or its complement. In some embodiments, the isolated mRNA molecule comprises or consists of a nucleotide sequence having at least about 94% sequence identity to SEQ ID NO:32 and comprises a uracil at a position corresponding to position 3,739 of SEQ ID NO:32 or its complement. In some embodiments, the isolated mRNA molecule comprises or consists of a nucleotide sequence having at least about 96% sequence identity to SEQ ID NO:32 and comprises a uracil at a position corresponding to position 3,739 of SEQ ID NO:32 or its complement. In some embodiments, the isolated mRNA molecule comprises or consists of a nucleotide sequence having at least about 98% sequence identity to SEQ ID NO:32 and comprises a uracil at a position corresponding to position 3,739 of SEQ ID NO:32 or its complement.

[0251] In some embodiments, the isolated mRNA molecule comprises or consists of a nucleotide sequence having at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to SEQ ID NO: 33 and includes a uracil at a position corresponding to position 3,627 of SEQ ID NO: 33 or its complement. In some embodiments, the isolated mRNA molecule comprises or consists of a nucleotide sequence having at least about 90% sequence identity to SEQ ID NO: 33 and includes a uracil at a position corresponding to position 3,627 of SEQ ID NO: 33 or its complement. In some embodiments, the isolated mRNA molecule comprises or consists of a nucleotide sequence having at least about 92% sequence identity to SEQ ID NO: 33 and includes a uracil at a position corresponding to position 3,627 of SEQ ID NO: 33 or its complement. In some embodiments, the isolated mRNA molecule comprises or consists of a nucleotide sequence having at least about 94% sequence identity to SEQ ID NO: 33 and comprises a uracil at a position corresponding to position 3,627 of SEQ ID NO: 33 or its complement. In some embodiments, the isolated mRNA molecule comprises or consists of a nucleotide sequence having at least about 96% sequence identity to SEQ ID NO: 33 and comprises a uracil at a position corresponding to position 3,627 of SEQ ID NO: 33 or its complement. In some embodiments, the isolated mRNA molecule comprises or consists of a nucleotide sequence having at least about 98% sequence identity to SEQ ID NO: 33 and comprises a uracil at a position corresponding to position 3,627 of SEQ ID NO: 33 or its complement.

[0252] In some embodiments, the isolated mRNA molecule comprises or consists of a nucleotide sequence having at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to SEQ ID NO: 34, and includes a uracil at a position corresponding to position 4,240 of SEQ ID NO: 34 or its complement. In some embodiments, the isolated mRNA molecule comprises or consists of a nucleotide sequence having at least about 90% sequence identity to SEQ ID NO: 34, and includes a uracil at a position corresponding to position 4,240 of SEQ ID NO: 34 or its complement. In some embodiments, the isolated mRNA molecule comprises or consists of a nucleotide sequence having at least about 92% sequence identity to SEQ ID NO: 34, and includes a uracil at a position corresponding to position 4,240 of SEQ ID NO: 34 or its complement. In some embodiments, the isolated mRNA molecule comprises or consists of a nucleotide sequence having at least about 94% sequence identity to SEQ ID NO: 34 and comprises a uracil at a position corresponding to position 4,240 of SEQ ID NO: 34 or its complement. In some embodiments, the isolated mRNA molecule comprises or consists of a nucleotide sequence having at least about 96% sequence identity to SEQ ID NO: 34 and comprises a uracil at a position corresponding to position 4,240 of SEQ ID NO: 34 or its complement. In some embodiments, the isolated mRNA molecule comprises or consists of a nucleotide sequence having at least about 98% sequence identity to SEQ ID NO: 34 and comprises a uracil at a position corresponding to position 4,240 of SEQ ID NO: 34 or its complement.

[0253] In some embodiments, the isolated mRNA molecule comprises or consists of a nucleotide sequence having at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to SEQ ID NO: 35, and includes a uracil at a position corresponding to position 3,594 of SEQ ID NO: 35 or its complement. In some embodiments, the isolated mRNA molecule comprises or consists of a nucleotide sequence having at least about 90% sequence identity to SEQ ID NO: 35, and includes a uracil at a position corresponding to position 3,594 of SEQ ID NO: 35 or its complement. In some embodiments, the isolated mRNA molecule comprises or consists of a nucleotide sequence having at least about 92% sequence identity to SEQ ID NO: 35, and includes a uracil at a position corresponding to position 3,594 of SEQ ID NO: 35 or its complement. In some embodiments, the isolated mRNA molecule comprises or consists of a nucleotide sequence having at least about 94% sequence identity to SEQ ID NO: 35 and comprises a uracil at a position corresponding to position 3,594 of SEQ ID NO: 35 or its complement. In some embodiments, the isolated mRNA molecule comprises or consists of a nucleotide sequence having at least about 96% sequence identity to SEQ ID NO: 35 and comprises a uracil at a position corresponding to position 3,594 of SEQ ID NO: 35 or its complement. In some embodiments, the isolated mRNA molecule comprises or consists of a nucleotide sequence having at least about 98% sequence identity to SEQ ID NO: 35 and comprises a uracil at a position corresponding to position 3,594 of SEQ ID NO: 35 or its complement.

[0254] In some embodiments, the isolated mRNA molecule comprises or...

Claims

1. A pharmaceutical composition for the treatment of a subject with glaucoma or elevated intraocular pressure (IOP), comprising a Rho guanine nucleotide exchange factor 12 (ARHGEF12) inhibitor.

2. The pharmaceutical composition described in claim 1, wherein the glaucoma is primary open-angle glaucoma (POAG).

3. 2. The pharmaceutical composition of claim 1, wherein the ARHGEF12 inhibitor comprises an antisense nucleic acid molecule, a small interfering RNA (siRNA), or a short hairpin RNA (shRNA) that hybridizes with ARHGEF12 mRNA.

4. 2. The pharmaceutical composition of claim 1, wherein the ARHGEF12 inhibitor comprises a Cas protein and a guide RNA (gRNA) that hybridizes to a gRNA recognition sequence within an ARHGEF12 genomic nucleic acid molecule.

5. 5. The pharmaceutical composition of claim 4, wherein the Cas protein is Cas9 or Cpf1.

6. A pharmaceutical composition described in any one of claims 1 to 5, further comprising a therapeutic agent for treating or suppressing glaucoma or elevated IOP.

7. A pharmaceutical composition for the treatment of a subject having glaucoma or elevated intraocular pressure (IOP), comprising a therapeutic agent that treats or inhibits glaucoma or elevated intraocular pressure (IOP), and / or a Rho guanine nucleotide exchange factor 12 (ARHGEF12) inhibitor; wherein the treatment determining whether the subject has a predicted loss-of-function variant nucleic acid molecule of ARHGEF12 encoding a human ARHGEF12 polypeptide or a predicted gain-of-function variant nucleic acid molecule of ARHGEF12 encoding a human ARHGEF12 polypeptide by performing or having performed a genotyping assay on a biological sample taken or taken from the subject, thereby determining whether the subject has a genotype comprising the predicted loss-of-function variant nucleic acid molecule of ARHGEF12 or the predicted gain-of-function variant nucleic acid molecule of ARHGEF12; if the subject is ARHGEF12 reference type, administering or continuing to administer to the subject a standard dose of a therapeutic agent that treats or inhibits glaucoma or elevated IOP, and further administering to the subject an ARHGEF12 inhibitor; if the subject is heterozygous for a predicted loss-of-function variant of ARHGEF12, administering or continuing to administer to the subject a therapeutic agent for treating or inhibiting glaucoma or elevated IOP at a standard dose or a lower dose, and further administering to the subject an ARHGEF12 inhibitor; if the subject is heterozygous for a predicted gain-of-function variant in ARHGEF12, administering or continuing to administer to the subject a therapeutic agent for treating or inhibiting glaucoma or elevated IOP at a dose equal to or greater than a standard dose, and further administering to the subject an ARHGEF12 inhibitor; the presence of a genotype having the predicted loss-of-function variant nucleic acid molecule of ARHGEF12 encoding the human ARHGEF12 polypeptide indicates a low risk of developing glaucoma or elevated IOP in the subject; A pharmaceutical composition, wherein the presence of a genotype having a predicted gain-of-function variant nucleic acid molecule of ARHGEF12 encoding the human ARHGEF12 polypeptide indicates an increased risk of developing glaucoma or elevated IOP in the subject.

8. 8. The pharmaceutical composition of claim 7, wherein the predicted gain-of-function variant nucleic acid molecule of ARHGEF12 is a nucleic acid molecule encoding Tyr973Phe, Tyr954Phe, or Tyr870Phe.

9. The pharmaceutical composition of claim 7, wherein the predicted loss-of-function variant nucleic acid molecule of ARHGEF12 is a nucleic acid molecule encoding Tyr1306Cys, Tyr1287Cys, Tyr1203Cys, Glu1156STOP, Glu1137STOP, or Glu1053STOP.

10. the predicted gain-of-function variant nucleic acid molecule of ARHGEF12 is a genomic nucleic acid molecule having a nucleotide sequence containing a thymine at a position corresponding to position 132,939 of SEQ ID NO:2; an mRNA molecule having a nucleotide sequence comprising uracil at a position corresponding to position 3,749 of SEQ ID NO:16, position 3,191 of SEQ ID NO:17, position 3,079 of SEQ ID NO:18, position 3,692 of SEQ ID NO:19, position 3,046 of SEQ ID NO:20, position 2,925 of SEQ ID NO:21, position 3,054 of SEQ ID NO:22, or position 2,615 of SEQ ID NO:23; or A cDNA molecule generated from an mRNA molecule, said cDNA molecule having a nucleotide sequence comprising a thymine at a position corresponding to position 3,749 of SEQ ID NO:47, position 3,191 of SEQ ID NO:48, position 3,079 of SEQ ID NO:49, position 3,692 of SEQ ID NO:50, position 3,046 of SEQ ID NO:51, position 2,925 of SEQ ID NO:52, position 3,054 of SEQ ID NO:53, or position 2,615 of SEQ ID NO:

54. and the predicted loss-of-function variant nucleic acid molecule of ARHGEF12 is a genomic nucleic acid molecule having a nucleotide sequence comprising a guanine at a position corresponding to position 143,698 of SEQ ID NO:3, a thymine at a position corresponding to position 141,048 of SEQ ID NO:4, an adenine at a position corresponding to position 73,039 of SEQ ID NO:5, a cytosine at a position corresponding to position 121,307 of SEQ ID NO:6, or a cytosine at a position corresponding to position 141,978 of SEQ ID NO:7; an mRNA molecule having a nucleotide sequence comprising a guanine at a position corresponding to position 4,748 of SEQ ID NO:24, a guanine at a position corresponding to position 4,190 of SEQ ID NO:25, a guanine at a position corresponding to position 4,078 of SEQ ID NO:26, a guanine at a position corresponding to position 4,691 of SEQ ID NO:27, a guanine at a position corresponding to position 4,045 of SEQ ID NO:28, a guanine at a position corresponding to position 3,924 of SEQ ID NO:29, a guanine at a position corresponding to position 3,614 of SEQ ID NO:30, a uracil at a position corresponding to position 4,297 of SEQ ID NO:31, a uracil at a position corresponding to position 3,739 of SEQ ID NO:32, a uracil at a position corresponding to position 3,627 of SEQ ID NO:33, a uracil at a position corresponding to position 4,240 of SEQ ID NO:34, a uracil at a position corresponding to position 3,594 of SEQ ID NO:35, a uracil at a position corresponding to position 3,473 of SEQ ID NO:36, a uracil at a position corresponding to position 3,602 of SEQ ID NO:37, or a uracil at a position corresponding to position 3,163 of SEQ ID NO:38; or 1. A cDNA molecule generated from an mRNA molecule, said cDNA molecule comprising a guanine at a position corresponding to position 4,748 of SEQ ID NO:55, a guanine at a position corresponding to position 4,190 of SEQ ID NO:56, a guanine at a position corresponding to position 4,078 of SEQ ID NO:57, a guanine at a position corresponding to position 4,691 of SEQ ID NO:58, a guanine at a position corresponding to position 4,045 of SEQ ID NO:59, a guanine at a position corresponding to position 3,924 of SEQ ID NO:60, a guanine at a position corresponding to position 3,614 of SEQ ID NO:61, a guanine at a position corresponding to position 3,615 of SEQ ID NO:62, a guanine at a position corresponding to position 3,626 of SEQ ID NO:63, a guanine at a position corresponding to position 3,630 of SEQ ID NO:64, a guanine at a position corresponding to position 3,640 of SEQ ID NO:65, a guanine at a position corresponding to position 3,652 of SEQ ID NO:66, a guanine at a position corresponding to position 3,656 of SEQ ID NO:67, a guanine at a position corresponding to position 3,660 of SEQ ID NO:68, a guanine at a position corresponding to position 3,670 of SEQ ID NO:69, a guanine at a position corresponding to position 3,682 of SEQ ID NO:69, a guanine at a position corresponding to position 4,691 of SEQ ID NO:60, a guanine at a position corresponding to position 4,691 of SEQ ID NO:61, a guanine at a position corresponding to position 4,691 of SEQ ID NO:62 the cDNA molecule having a nucleotide sequence comprising a thymine at a position corresponding to position 4,297 of SEQ ID NO:62, a thymine at a position corresponding to position 3,379 of SEQ ID NO:63, a thymine at a position corresponding to position 3,627 of SEQ ID NO:64, a thymine at a position corresponding to position 4,240 of SEQ ID NO:65, a thymine at a position corresponding to position 3,594 of SEQ ID NO:66, a thymine at a position corresponding to position 3,473 of SEQ ID NO:67, a thymine at a position corresponding to position 3,602 of SEQ ID NO:68, or a thymine at a position corresponding to position 3,163 of SEQ ID NO:

69. The pharmaceutical composition of claim 8, wherein

11. The pharmaceutical composition of any one of claims 7 to 10, wherein the ARHGEF12 inhibitor comprises an antisense nucleic acid molecule, a small interfering RNA (siRNA), or a short hairpin RNA (shRNA) that hybridizes with ARHGEF12 mRNA.

12. The pharmaceutical composition of any one of claims 7 to 10, wherein the ARHGEF12 inhibitor comprises a Cas protein and a guide RNA (gRNA) that hybridizes to a gRNA recognition sequence within an ARHGEF12 genomic nucleic acid molecule.

13. 13. The pharmaceutical composition of claim 12, wherein the Cas protein is Cas9 or Cpf1.

14. 1. An in vitro method for identifying a subject at high risk of developing glaucoma or elevated IOP, said method comprising: determining or having determined the presence or absence of a predicted gain-of-function variant nucleic acid molecule of Rho guanine nucleotide exchange factor 12 (ARHGEF12) encoding a human ARHGEF12 polypeptide, or a predicted loss-of-function variant nucleic acid molecule of ARHGEF12 encoding a human ARHGEF12 polypeptide, in a biological sample obtained from the subject; If the subject is of the ARHGEF12 reference type, the subject is at high risk of developing glaucoma; if the subject is heterozygous or homozygous for a predicted loss-of-function variant nucleic acid molecule of ARHGEF12, the subject is at low risk of developing glaucoma or elevated IOP; A method wherein the subject is at increased risk of developing glaucoma or elevated IOP if the subject is heterozygous or homozygous for a predicted gain-of-function variant nucleic acid molecule of ARHGEF12.

15. 15. The method of claim 14, wherein the predicted gain-of-function variant nucleic acid molecule of ARHGEF12 is a nucleic acid molecule encoding Tyr973Phe, Tyr954Phe, or Tyr870Phe.

16. The method of claim 14, wherein the predicted loss-of-function variant nucleic acid molecule of ARHGEF12 is a nucleic acid molecule encoding Tyr1306Cys, Tyr1287Cys, Tyr1203Cys, Glu1156STOP, Glu1137STOP, or Glu1053STOP.

17. the predicted gain-of-function variant nucleic acid molecule of ARHGEF12 is a genomic nucleic acid molecule having a nucleotide sequence containing a thymine at a position corresponding to position 132,939 of SEQ ID NO:2; an mRNA molecule having a nucleotide sequence comprising uracil at a position corresponding to position 3,749 of SEQ ID NO:16, position 3,191 of SEQ ID NO:17, position 3,079 of SEQ ID NO:18, position 3,692 of SEQ ID NO:19, position 3,046 of SEQ ID NO:20, position 2,925 of SEQ ID NO:21, position 3,054 of SEQ ID NO:22, or position 2,615 of SEQ ID NO:23; or A cDNA molecule generated from an mRNA molecule, said cDNA molecule having a nucleotide sequence comprising a thymine at a position corresponding to position 3,749 of SEQ ID NO:47, position 3,191 of SEQ ID NO:48, position 3,079 of SEQ ID NO:49, position 3,692 of SEQ ID NO:50, position 3,046 of SEQ ID NO:51, position 2,925 of SEQ ID NO:52, position 3,054 of SEQ ID NO:53, or position 2,615 of SEQ ID NO:

54. and the predicted loss-of-function variant nucleic acid molecule of ARHGEF12 is a genomic nucleic acid molecule comprising a guanine at a position corresponding to position 143,698 of SEQ ID NO:3, a thymine at a position corresponding to position 141,048 of SEQ ID NO:4, an adenine at a position corresponding to position 73,039 of SEQ ID NO:5, a cytosine at a position corresponding to position 121,307 of SEQ ID NO:6, or a cytosine at a position corresponding to position 141,978 of SEQ ID NO:7; an mRNA molecule having a nucleotide sequence comprising a guanine at a position corresponding to position 4,748 of SEQ ID NO:24, a guanine at a position corresponding to position 4,190 of SEQ ID NO:25, a guanine at a position corresponding to position 4,078 of SEQ ID NO:26, a guanine at a position corresponding to position 4,691 of SEQ ID NO:27, a guanine at a position corresponding to position 4,045 of SEQ ID NO:28, a guanine at a position corresponding to position 3,924 of SEQ ID NO:29, a guanine at a position corresponding to position 3,614 of SEQ ID NO:30, a uracil at a position corresponding to position 4,297 of SEQ ID NO:31, a uracil at a position corresponding to position 3,739 of SEQ ID NO:32, a uracil at a position corresponding to position 3,627 of SEQ ID NO:33, a uracil at a position corresponding to position 4,240 of SEQ ID NO:34, a uracil at a position corresponding to position 3,594 of SEQ ID NO:35, a uracil at a position corresponding to position 3,473 of SEQ ID NO:36, a uracil at a position corresponding to position 3,602 of SEQ ID NO:37, and a uracil at a position corresponding to position 3,163 of SEQ ID NO:38; or 1. A cDNA molecule generated from an mRNA molecule, said cDNA molecule comprising a guanine at a position corresponding to position 4,748 of SEQ ID NO:55, a guanine at a position corresponding to position 4,190 of SEQ ID NO:56, a guanine at a position corresponding to position 4,078 of SEQ ID NO:57, a guanine at a position corresponding to position 4,691 of SEQ ID NO:58, a guanine at a position corresponding to position 4,045 of SEQ ID NO:59, a guanine at a position corresponding to position 3,924 of SEQ ID NO:60, a guanine at a position corresponding to position 3,614 of SEQ ID NO:61, a guanine at a position corresponding to position 3,615 of SEQ ID NO:62, a guanine at a position corresponding to position 3,626 of SEQ ID NO:63, a guanine at a position corresponding to position 3,630 of SEQ ID NO:64, a guanine at a position corresponding to position 3,640 of SEQ ID NO:65, a guanine at a position corresponding to position 3,652 of SEQ ID NO:66, a guanine at a position corresponding to position 3,656 of SEQ ID NO:67, a guanine at a position corresponding to position 3,660 of SEQ ID NO:68, a guanine at a position corresponding to position 3,670 of SEQ ID NO:69, a guanine at a position corresponding to position 3,682 of SEQ ID NO:69, a guanine at a position corresponding to position 4,691 of SEQ ID NO:60, a guanine at a position corresponding to position 4,691 of SEQ ID NO:61, a guanine at a position corresponding to position 4,691 of SEQ ID NO:62 the cDNA molecule having a nucleotide sequence comprising a thymine at a position corresponding to position 4,297 of SEQ ID NO:62, a thymine at a position corresponding to position 3,379 of SEQ ID NO:63, a thymine at a position corresponding to position 3,627 of SEQ ID NO:64, a thymine at a position corresponding to position 4,240 of SEQ ID NO:65, a thymine at a position corresponding to position 3,594 of SEQ ID NO:66, a thymine at a position corresponding to position 3,473 of SEQ ID NO:67, a thymine at a position corresponding to position 3,602 of SEQ ID NO:68, or a thymine at a position corresponding to position 3,163 of SEQ ID NO:

69. The method of claim 14, wherein 18. A pharmaceutical composition for the treatment of glaucoma or elevated intraocular pressure (IOP) in a subject, comprising: a therapeutic agent for treating or inhibiting glaucoma or elevated IOP; The object is a genomic nucleic acid molecule having a nucleotide sequence encoding a human Rho guanine nucleotide exchange factor 12 (ARHGEF12) polypeptide, wherein said nucleotide sequence comprises a thymine at a position corresponding to position 132,939 of SEQ ID NO:2 or its complement, a guanine at a position corresponding to position 143,698 of SEQ ID NO:3 or its complement, a thymine at a position corresponding to position 141,048 of SEQ ID NO:4 or its complement, an adenine at a position corresponding to position 73,039 of SEQ ID NO:5 or its complement, a cytosine at a position corresponding to position 121,307 of SEQ ID NO:6 or its complement, or a cytosine at a position corresponding to position 141,978 of SEQ ID NO:7 or its complement; or 1. An mRNA molecule having a nucleotide sequence encoding a human ARHGEF12 polypeptide, wherein said nucleotide sequence contains a uracil at a position corresponding to position 3,749 of SEQ ID NO:16 or its complement, a uracil at a position corresponding to position 3,191 of SEQ ID NO:17 or its complement, a uracil at a position corresponding to position 3,079 of SEQ ID NO:18 or its complement, a uracil at a position corresponding to position 3,692 of SEQ ID NO:19 or its complement, a uracil at a position corresponding to position 3,046 of SEQ ID NO:20 or its complement, a uracil at a position corresponding to position 2,925 of SEQ ID NO:21 or its complement, a uracil at a position corresponding to position 3,054 of SEQ ID NO:22 or its complement, a uracil at a position corresponding to position 2,615 of SEQ ID NO:23 or its complement, a guanine at a position corresponding to position 4,748 of SEQ ID NO:24 or its complement, a guanine at a position corresponding to position 4,190 of SEQ ID NO:25 or its complement, a guanine at a position corresponding to position 4,078 of SEQ ID NO:26 or its complement. the mRNA molecule comprising: a guanine at a position corresponding to position 4,691 of SEQ ID NO:27 or its complement; a guanine at a position corresponding to position 4,045 of SEQ ID NO:28 or its complement; a guanine at a position corresponding to position 3,924 of SEQ ID NO:29 or its complement; a guanine at a position corresponding to position 3,614 of SEQ ID NO:30 or its complement; a uracil at a position corresponding to position 4,297 of SEQ ID NO:31 or its complement; a uracil at a position corresponding to position 3,739 of SEQ ID NO:32 or its complement; a uracil at a position corresponding to position 3,627 of SEQ ID NO:33 or its complement; a uracil at a position corresponding to position 4,240 of SEQ ID NO:34 or its complement; a uracil at a position corresponding to position 3,594 of SEQ ID NO:35 or its complement; a uracil at a position corresponding to position 3,473 of SEQ ID NO:36 or its complement; a uracil at a position corresponding to position 3,602 of SEQ ID NO:37 or its complement; or a uracil at a position corresponding to position 3,163 of SEQ ID NO:38 or its complement. A pharmaceutical composition comprising:

19. A pharmaceutical composition for the treatment of glaucoma or elevated intraocular pressure (IOP) in a human subject, comprising a Rho guanine nucleotide exchange factor 12 (ARHGEF12) inhibitor; The object is a genomic nucleic acid molecule having a nucleotide sequence encoding a human ARHGEF12 polypeptide, said nucleotide sequence comprising a thymine at a position corresponding to position 132,939 of SEQ ID NO:2 or its complement, a guanine at a position corresponding to position 143,698 of SEQ ID NO:3 or its complement, a thymine at a position corresponding to position 141,048 of SEQ ID NO:4 or its complement, an adenine at a position corresponding to position 73,039 of SEQ ID NO:5 or its complement, a cytosine at a position corresponding to position 121,307 of SEQ ID NO:6 or its complement, or a cytosine at a position corresponding to position 141,978 of SEQ ID NO:7 or its complement; or 1. An mRNA molecule having a nucleotide sequence encoding a human ARHGEF12 polypeptide, wherein said nucleotide sequence contains a uracil at a position corresponding to position 3,749 of SEQ ID NO:16 or its complement, a uracil at a position corresponding to position 3,191 of SEQ ID NO:17 or its complement, a uracil at a position corresponding to position 3,079 of SEQ ID NO:18 or its complement, a uracil at a position corresponding to position 3,692 of SEQ ID NO:19 or its complement, a uracil at a position corresponding to position 3,046 of SEQ ID NO:20 or its complement, a uracil at a position corresponding to position 2,925 of SEQ ID NO:21 or its complement, a uracil at a position corresponding to position 3,054 of SEQ ID NO:22 or its complement, a uracil at a position corresponding to position 2,615 of SEQ ID NO:23 or its complement, a guanine at a position corresponding to position 4,748 of SEQ ID NO:24 or its complement, a guanine at a position corresponding to position 4,190 of SEQ ID NO:25 or its complement, a guanine at a position corresponding to position 4,078 of SEQ ID NO:26 or its complement. the mRNA molecule comprising: a guanine at a position corresponding to position 4,691 of SEQ ID NO:27 or its complement; a guanine at a position corresponding to position 4,045 of SEQ ID NO:28 or its complement; a guanine at a position corresponding to position 3,924 of SEQ ID NO:29 or its complement; a guanine at a position corresponding to position 3,614 of SEQ ID NO:30 or its complement; a uracil at a position corresponding to position 4,297 of SEQ ID NO:31 or its complement; a uracil at a position corresponding to position 3,739 of SEQ ID NO:32 or its complement; a uracil at a position corresponding to position 3,627 of SEQ ID NO:33 or its complement; a uracil at a position corresponding to position 4,240 of SEQ ID NO:34 or its complement; a uracil at a position corresponding to position 3,594 of SEQ ID NO:35 or its complement; a uracil at a position corresponding to position 3,473 of SEQ ID NO:36 or its complement; a uracil at a position corresponding to position 3,602 of SEQ ID NO:37 or its complement; or a uracil at a position corresponding to position 3,163 of SEQ ID NO:38 or its complement. A pharmaceutical composition comprising:

20. The pharmaceutical composition of claim 19, wherein the ARHGEF12 inhibitor is an antisense nucleic acid molecule, a small interfering RNA (siRNA), or a short hairpin RNA (shRNA) that hybridizes with ARHGEF12 mRNA.

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