Treatment of hypertension with solute carrier family 9 isoform A3 regulator 2 (SLC9A3R2) inhibitors

Abstract

The present disclosure provides methods for treating subjects having or at risk of developing hypertension, coronary heart disease and / or atrial fibrillation, methods for identifying subjects at high risk for developing hypertension, coronary heart disease and / or atrial fibrillation, and methods for detecting solute carrier family 9 isoform A3 regulator 2 (SLC9A3R2) 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 a text file of 225 kilobytes in size under the title 18923807902SEQ, created on June 25, 2022. This Sequence Listing is incorporated herein by reference.

[0002] The present disclosure relates generally to the treatment of subjects having or at risk of developing hypertension, coronary heart disease and / or atrial fibrillation with solute carrier family 9 isoform A3 regulator 2 (SLC9A3R2) inhibitors, as well as methods of identifying subjects at high risk for developing hypertension, coronary heart disease and / or atrial fibrillation. [Background technology]

[0003] Hypertension is the most common of all cardiovascular diseases, affecting approximately 10%-20% of the adult population. Hypertension and its resulting diseases (arteriosclerosis, myocardial infarction, stroke, cardiac hypertrophy, and heart failure) are the most frequent cause of illness and death in all Western developed countries, even surpassing malignant degenerative diseases (cancer). Hypertension results from excessive constriction of blood vessels or insufficient excretion of fluids by the kidneys. Numerous central nervous mechanisms and hormonal systems are involved in regulating the tone of smooth muscle tissue in blood vessels, and thus vascular width, and also in fluid excretion by the kidneys. The above mechanisms and hormonal systems control and regulate blood pressure, which is physiologically elevated in association with physical labor, fear, stress, excitement, etc. Disturbances in one or more of these systems ultimately result in an increase in blood pressure. In many cases, the true cause of hypertension is unknown (essential hypertension). However, genetic predisposition resulting from mutations in genes that code for proteins involved in the blood pressure regulation system is likely to occur only occasionally and only in combination with external factors (stress, smoking, overweight, lack of exercise, unhealthy diet).

[0004] SLC9A3R2 regulates the function of the PDZ (PSD-95 / DLG / ZO-1) scaffolding protein Na + -H + SLC9A3R2 is a member of the NHERF family of exchange regulators. These proteins mediate many cellular processes by binding to membrane receptors and transport proteins and regulating their membrane expression and protein-protein interactions. SLC9A3R2 is expressed in the kidney and serves to connect plasma membrane proteins with members of the ezrin / moesin / radixin family, thereby linking them to the actin cytoskeleton and regulating their surface expression. SLC9A3R2 is also involved in intestinal sodium absorption by regulating the activity of sodium / hydrogen exchanger 3 and can also regulate the cystic fibrosis transmembrane conductance regulator (CFTR) ion channel, which is required for phosphorylation by cAMP and inhibition of SLC9A3. Summary of the Invention

[0005] The present disclosure provides a method of treating a subject having or at risk of developing hypertension, the method comprising administering to the subject an SLC9A3R2 inhibitor. The disclosure also provides a method of treating a subject having or at risk of developing primary hypertension, the method comprising administering to the subject an SLC9A3R2 inhibitor.

[0006] The disclosure also provides a method of treating a subject having or at risk of developing secondary hypertension, the method comprising administering to the subject an SLC9A3R2 inhibitor.

[0007] The disclosure also provides a method of treating a subject having or at risk of developing resistant hypertension, the method comprising administering to the subject an SLC9A3R2 inhibitor.

[0008] The disclosure also provides a method of treating a subject having or at risk of developing malignant hypertension, the method comprising administering to the subject an SLC9A3R2 inhibitor.

[0009] The disclosure also provides a method of treating a subject having or at risk of developing coronary heart disease, the method comprising administering to the subject an SLC9A3R2 inhibitor.

[0010] The disclosure also provides a method of treating a subject having or at risk of developing atrial fibrillation, the method comprising administering to the subject an SLC9A3R2 inhibitor. The disclosure also provides a method of treating a subject with a therapeutic agent for treating or preventing hypertension, coronary heart disease and / or atrial fibrillation, wherein the subject has or is at risk for developing hypertension, coronary heart disease and / or atrial fibrillation, the method comprising determining whether the subject has a SLC9A3R2 missense variant nucleic acid molecule encoding a loss-of-function polypeptide predicted in SLC9A3R2 by obtaining or obtaining a biological sample from the subject; performing or having performed sequence analysis on the biological sample to determine whether the subject has a genotype that includes a SLC9A3R2 missense variant nucleic acid molecule encoding a loss-of-function polypeptide predicted in SLC9A3R2; administering or continuing to administer a therapeutic agent for treating or preventing hypertension, coronary heart disease and / or atrial fibrillation at a standard dosage, and / or administering an SLC9A3R2 inhibitor to the subject; administering or continuing to administer a therapeutic agent for treating or preventing hypertension, coronary heart disease and / or atrial fibrillation at the same or a lower standard dosage, to a subject who is heterozygous for an SLC9A3R2 missense variant nucleic acid molecule, and / or administering an SLC9A3R2 inhibitor to the subject; wherein the presence of a genotype having an SLC9A3R2 missense variant nucleic acid molecule encoding a loss-of-function polypeptide predicted by SLC9A3R2 indicates that the subject is at low risk for developing hypertension, coronary heart disease and / or atrial fibrillation.

[0011] The present disclosure also provides a method for identifying a subject at high risk of developing hypertension, coronary heart disease and / or atrial fibrillation, the method comprising determining or having determined the presence or absence of an SLC9A3R2 missense variant nucleic acid molecule encoding a loss-of-function polypeptide predicted in SLC9A3R2 in a biological sample obtained from the subject; wherein if the subject is SLC9A3R2-based, the subject has an increased risk of developing hypertension, coronary heart disease and / or atrial fibrillation; and if the subject is heterozygous or homozygous for an SLC9A3R2 missense variant nucleic acid molecule encoding a loss-of-function polypeptide predicted in SLC9A3R2, the subject has a reduced risk of developing hypertension, coronary heart disease and / or atrial fibrillation.

[0012] The present disclosure also provides a therapeutic agent for treating or preventing hypertension, coronary heart disease, and / or atrial fibrillation, comprising: i) a genomic nucleic acid molecule encoding a predicted loss-of-function polypeptide of SLC9A3R2, having a nucleotide sequence including a thymine or its complement at a position corresponding to position 9,519 set forth in SEQ ID NO:2, or a complement thereof; ii) a uracil or its complement at a position corresponding to position 615 set forth in SEQ ID NO:22; a uracil or its complement at a position corresponding to position 589 set forth in SEQ ID NO:23; 4; uracil or its complement at a position corresponding to position 230 set forth in SEQ ID NO:25; uracil or its complement at a position corresponding to position 236 set forth in SEQ ID NO:26; uracil or its complement at a position corresponding to position 236 set forth in SEQ ID NO:27; uracil or its complement at a position corresponding to position 604 set forth in SEQ ID NO:28; or uracil or its complement at a position corresponding to position 126 set forth in SEQ ID NO:29. or iii) a thymine or its complement at a position corresponding to position 615 set forth in SEQ ID NO:59; a thymine or its complement at a position corresponding to position 589 set forth in SEQ ID NO:60; a thymine or its complement at a position corresponding to position 353 set forth in SEQ ID NO:61; a thymine or its complement at a position corresponding to position 230 set forth in SEQ ID NO:62; a thymine or its complement at a position corresponding to position 236 set forth in SEQ ID NO:63; a thymine or its complement at a position corresponding to position 236 set forth in SEQ ID NO:64; a thymine or its complement at a position corresponding to position 604 set forth in SEQ ID NO:65; or a thymine or its complement at a position corresponding to position 126 set forth in SEQ ID NO:66.

[0013] The present disclosure also provides an SLC9A3R2 inhibitor, comprising: a) an SLC9A3R2 genomic nucleic acid molecule, an SLC9A3R2 mRNA molecule, or an SLC9A3R2 a genomic nucleic acid molecule encoding a predicted loss-of-function polypeptide for SLC9A3R2, having a nucleotide sequence that includes i) a thymine or its complement at a position corresponding to position 9,519 as set forth in SEQ ID NO:2, or a complement thereof; ii) a uracil or its complement at a position corresponding to position 615 as set forth in SEQ ID NO:22; a uracil or its complement at a position corresponding to position 589 as set forth in SEQ ID NO:23; a uracil or its complement at a position corresponding to position 353 as set forth in SEQ ID NO:24; a uracil or its complement at a position corresponding to position 230 as set forth in SEQ ID NO:25; a uracil or its complement at a position corresponding to position 236 as set forth in SEQ ID NO:26; a uracil or its complement at a position corresponding to position 236 as set forth in SEQ ID NO:27; a uracil or its complement at a position corresponding to position 604 as set forth in SEQ ID NO:28; or a uracil or its complement at a position corresponding to position 126 as set forth in SEQ ID NO:29; or iii) a cDNA molecule encoding a SLC9A3R2 predicted loss-of-function polypeptide, or its complement, having a nucleotide sequence comprising a thymine or its complement at a position corresponding to position 615 as set forth in SEQ ID NO:59; a thymine or its complement at a position corresponding to position 589 as set forth in SEQ ID NO:60; a thymine or its complement at a position corresponding to position 353 as set forth in SEQ ID NO:61; a thymine or its complement at a position corresponding to position 230 as set forth in SEQ ID NO:62; a thymine or its complement at a position corresponding to position 236 as set forth in SEQ ID NO:63; a thymine or its complement at a position corresponding to position 236 as set forth in SEQ ID NO:64; a thymine or its complement at a position corresponding to position 604 as set forth in SEQ ID NO:65; or a thymine or its complement at a position corresponding to position 126 as set forth in SEQ ID NO:66. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0014] Various terms relating to the 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.

[0015] Unless expressly stated otherwise, no method or embodiment set forth herein is intended to be construed as requiring that its steps be performed in a particular order. Thus, unless a method claim specifically specifies in the claim or description that the steps are to be limited to a particular order, it is not intended to dictate order in any respect. This includes any possible implicit criteria of interpretation, including logical matters regarding the arrangement of steps or workflow, general meanings derived from grammatical construction or punctuation, or the number or type of embodiments described herein.

[0016] 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 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.

[0017] As used herein, the term "comprising" may in certain embodiments be replaced with "consisting" or "consisting essentially of," as desired.

[0018] As used herein, with respect to a nucleic acid molecule or polypeptide, the term "isolated" means that the nucleic acid molecule or polypeptide is in a state other than its native environment, e.g., away from blood and / or other tissues. 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 can be in a highly purified form, i.e., greater than 95% pure or greater 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.

[0019] As used herein, the terms "nucleic acid," "nucleic acid molecule," "nucleic acid sequence," "polynucleotide," or "oligonucleotide" can include polymeric forms 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 complement.

[0020] 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 (e.g., apes and monkeys). In some embodiments, the subject is a human. In some embodiments, the subject is a patient under the care of a physician.

[0021] The burden of rare putative loss of function (LOF) and harmful missense variants of the SLC9A3R2 gene associated with a reduced risk of developing hypertension in humans has been identified according to the present disclosure.For example, it has been observed that a genetic variant that changes cytosine to thymine at position 9,519 of the SLC9A3R2 reference genomic nucleic acid molecule (see SEQ ID NO: 1) indicates that subjects with such variants may have a lower risk of developing hypertension.Variants of the SLC9A3R2 gene or protein are not believed to have a known association with hypertension.In summary, the genetic analysis described herein surprisingly shows that the SLC9A3R2 gene, and in particular pLOF and harmful missense variants of the SLC9A3R2 gene, are associated with a reduced risk of developing hypertension. Thus, subjects with SLC9A3R2 criteria at high risk of developing hypertension, such as primary hypertension, secondary hypertension, resistant hypertension, or malignant hypertension, coronary heart disease, and / or atrial fibrillation may be treated such that hypertension, coronary heart disease, and / or atrial fibrillation is prevented, symptoms thereof are alleviated, and / or onset of symptoms is inhibited. Thus, the present disclosure provides methods utilizing the identification of such variants in a subject to identify or stratify the risk in such a subject of developing hypertension, such as primary hypertension, secondary hypertension, resistant hypertension, or malignant hypertension, coronary heart disease, and / or atrial fibrillation, or to diagnose a subject as having an elevated risk of developing hypertension, such as primary hypertension, secondary hypertension, resistant hypertension, or malignant hypertension, coronary heart disease, and / or atrial fibrillation, so that subjects at risk or with active disease may be treated accordingly.

[0022] In accordance with the present disclosure, it has further been observed that SLC9A3R2 missense variant nucleic acid molecules encoding SLC9A3R2 predicted loss-of-function polypeptides (whether these variants are homozygous or heterozygous in a particular subject) are associated with a reduced risk of developing hypertension. Furthermore, the identification of the association of additional variants with the genetic burden masks according to the present disclosure indicates that SLC9A3R2 itself (rather than linkage disequilibrium with variants of another gene) is responsible for the protective effect in hypertension.

[0023] For the purpose of this disclosure, any particular subject can be classified as having one of three SLC9A3R2 genotypes: i) SLC9A3R2 standard; ii) heterozygous for SLC9A3R2 missense variant nucleic acid molecule encoding loss-of-function polypeptide predicted by SLC9A3R2; or iii) homozygous for SLC9A3R2 missense variant nucleic acid molecule encoding loss-of-function polypeptide predicted by SLC9A3R2. If the subject does not have a copy of SLC9A3R2 missense variant nucleic acid molecule encoding loss-of-function polypeptide predicted by SLC9A3R2, the subject is SLC9A3R2 standard. If the subject has a single copy of SLC9A3R2 missense variant nucleic acid molecule, the subject is heterozygous for SLC9A3R2 missense variant nucleic acid molecule encoding loss-of-function polypeptide predicted by SLC9A3R2. As used herein, a SLC9A3R2 missense variant nucleic acid molecule is any SLC9A3R2 nucleic acid molecule (e.g., genomic nucleic acid molecule, mRNA molecule, or cDNA molecule) that encodes a SLC9A3R2 polypeptide with partial loss of function, complete loss of function, predicted partial loss of function, or predicted complete loss of function. A subject that has a SLC9A3R2 missense variant nucleic acid molecule that encodes a predicted SLC9A3R2 loss of function polypeptide with partial loss of function (or predicted partial loss of function) is hypomorphic for SLC9A3R2. A SLC9A3R2 missense variant nucleic acid molecule that encodes a predicted SLC9A3R2 loss of function polypeptide can be any nucleic acid molecule that encodes SLC9A3R2 Arg171Trp-Long, Arg171Trp-Short, Arg65Trp, Arg58Trp, Arg60Trp-Short, Arg60Trp-Long, or Arg170Trp. In some embodiments, the SLC9A3R2 missense variant nucleic acid molecule encodes SLC9A3R2 Arg171Trp-Long or Arg171Trp-Short.If a subject has two copies of an SLC9A3R2 missense variant nucleic acid molecule encoding a loss-of-function polypeptide predicted by SLC9A3R2, then the subject is homozygous for an SLC9A3R2 missense variant nucleic acid molecule encoding a loss-of-function polypeptide predicted by SLC9A3R2.

[0024] For subjects who are genotyped or determined to be SLC9A3R2 based, such subjects are at increased risk of developing hypertension, such as primary hypertension, secondary hypertension, resistant hypertension, or malignant hypertension, coronary heart disease, and / or atrial fibrillation. For subjects who are genotyped or determined to be SLC9A3R2 based or heterozygous for a SLC9A3R2 missense variant nucleic acid molecule encoding a SLC9A3R2 predicted loss-of-function polypeptide, such subjects can be treated with a SLC9A3R2 inhibitor.

[0025] In any of the embodiments described throughout this disclosure, the SLC9A3R2 missense variant nucleic acid molecule can be any SLC9A3R2 nucleic acid molecule (e.g., genomic nucleic acid molecule, mRNA molecule, or cDNA molecule) that encodes a SLC9A3R2 polypeptide with partial loss of function, complete loss of function, predicted partial loss of function, or predicted complete loss of function.For example, the SLC9A3R2 missense variant nucleic acid molecule can be any nucleic acid molecule that encodes SLC9A3R2 Arg171Trp-Long, Arg171Trp-Short, Arg65Trp, Arg58Trp, Arg60Trp-Short, Arg60Trp-Long, or Arg170Trp.In some embodiments, the SLC9A3R2 missense variant nucleic acid molecule encodes SLC9A3R2 Arg171Trp-Long or Arg171Trp-Short.

[0026] In any of the embodiments described throughout this disclosure, the SLC9A3R2 predicted loss-of-function polypeptide can be any SLC9A3R2 polypeptide with partial loss-of-function, complete loss-of-function, predicted partial loss-of-function, or predicted complete loss-of-function. In any of the embodiments described throughout this disclosure, the SLC9A3R2 predicted loss-of-function polypeptide can be any of the SLC9A3R2 polypeptides described herein, including, for example, SLC9A3R2 Arg171Trp-Long, Arg171Trp-Short, Arg65Trp, Arg58Trp, Arg60Trp-Short, Arg60Trp-Long, or Arg170Trp. In some embodiments, the SLC9A3R2 predicted loss-of-function polypeptide is SLC9A3R2 Arg171Trp-Long or Arg171Trp-Short.

[0027] Any one or more (i.e., any combination) of SLC9A3R2 missense variant nucleic acid molecules encoding SLC9A3R2 predicted loss-of-function polypeptides can be used within any of the methods described herein to determine whether a subject has an increased risk of developing hypertension, coronary heart disease, and / or atrial fibrillation. Particular variant combinations can form a mask that is used to statistically analyze a particular correlation between SLC9A3R2 and a reduced risk of developing hypertension, coronary heart disease, and / or atrial fibrillation.

[0028] In any of the embodiments described throughout this disclosure, the hypertension is primary hypertension, secondary hypertension, resistant hypertension, or malignant hypertension. In any of the embodiments described throughout this disclosure, the hypertension is primary hypertension. In any of the embodiments described throughout this disclosure, the hypertension is secondary hypertension. In any of the embodiments described throughout this disclosure, the hypertension is resistant hypertension. In any of the embodiments described throughout this disclosure, the hypertension is malignant hypertension.

[0029] Symptoms of hypertension include, but are not limited to, elevated blood pressure, headache, shortness of breath, nosebleeds, flushing, dizziness, chest pain, visual changes, and / or blood in the urine. The present disclosure provides a method of treating a subject having or at risk of developing hypertension, the method comprising administering to the subject an SLC9A3R2 inhibitor.

[0030] The disclosure also provides a method of treating a subject having or at risk of developing primary hypertension, the method comprising administering to the subject an SLC9A3R2 inhibitor.

[0031] The disclosure also provides a method of treating a subject having or at risk of developing secondary hypertension, the method comprising administering to the subject an SLC9A3R2 inhibitor.

[0032] The disclosure also provides a method of treating a subject having or at risk of developing resistant hypertension, the method comprising administering to the subject an SLC9A3R2 inhibitor.

[0033] The disclosure also provides a method of treating a subject having or at risk of developing malignant hypertension, the method comprising administering to the subject an SLC9A3R2 inhibitor.

[0034] The disclosure also provides a method of treating a subject having or at risk of developing coronary heart disease, the method comprising administering to the subject an SLC9A3R2 inhibitor.

[0035] The disclosure also provides a method of treating a subject having or at risk of developing atrial fibrillation, the method comprising administering to the subject an SLC9A3R2 inhibitor. In some embodiments, the SLC9A3R2 inhibitor comprises an inhibitory nucleic acid molecule. In some embodiments, the inhibitory nucleic acid molecule comprises an antisense molecule, a small interfering RNA (siRNA) molecule, or a short hairpin RNA (shRNA) molecule. In some embodiments, the inhibitory nucleic acid molecule comprises an antisense molecule. In some embodiments, the inhibitory nucleic acid molecule comprises an siRNA molecule. In some embodiments, the inhibitory nucleic acid molecule comprises an shRNA molecule. Such inhibitory nucleic acid molecules can be designed to target any region of a SLC9A3R2 nucleic acid molecule, such as an mRNA molecule. In some embodiments, the inhibitory nucleic acid molecule hybridizes with a sequence within a SLC9A3R2 genomic nucleic acid molecule or an mRNA molecule, and reduces the expression of a SLC9A3R2 polypeptide in a cell of a subject. In some embodiments, the SLC9A3R2 inhibitor comprises an antisense RNA that hybridizes with a SLC9A3R2 genomic nucleic acid molecule or an mRNA molecule, and reduces the expression of a SLC9A3R2 polypeptide in a cell of a subject. In some embodiments, the SLC9A3R2 inhibitor comprises an siRNA that hybridizes with an SLC9A3R2 genomic nucleic acid molecule or an mRNA molecule and reduces the expression of an SLC9A3R2 polypeptide in a cell of a subject. In some embodiments, the SLC9A3R2 inhibitor comprises an shRNA that hybridizes with an SLC9A3R2 genomic nucleic acid molecule or an mRNA molecule and reduces the expression of an SLC9A3R2 polypeptide in a cell of a subject.

[0036] The inhibitory nucleic acid molecule can comprise RNA, DNA, or both RNA and DNA. The inhibitory nucleic acid molecule can also be linked or fused to a heterologous nucleic acid sequence, for example in a vector, or a heterologous label. For example, the inhibitory nucleic acid molecule can be present as an exogenous donor sequence in or containing a vector that contains the inhibitory nucleic acid molecule and the heterologous nucleic acid sequence. The inhibitory nucleic acid molecule can also be linked or fused to a heterologous label. The label can be directly detectable (e.g., a fluorophore) or indirectly detectable (e.g., a hapten, an enzyme, or a fluorophore quencher). Such labels can be detectable by spectroscopic, photochemical, biochemical, immunochemical, or chemical means. Such labels include, for example, radioactive labels, pigments, dyes, chromogens, spin labels, and fluorescent labels. The label can also be, for example, a chemiluminescent substance; a metal-containing substance; or an enzyme, whereby an enzyme-dependent secondary generation of a signal occurs. The term "label" can also refer to a "tag" or hapten that can be selectively attached to a binding molecule such that the binding molecule is subsequently added with a substrate and used to generate a detectable signal. For example, biotin can be used as a tag together with an avidin or streptavidin conjugate of horseradish peroxidase (HRP) to bind to the tag and probed using a colorimetric (e.g., tetramethylbenzidine (TMB)) or fluorogenic substrate to detect the presence of HRP. Exemplary labels that can be used as tags to facilitate purification include, but are not limited to, myc, HA, FLAG or 3xFLAG, 6xHis or polyhistidine, glutathione-S-transferase (GST), maltose binding protein, epitope tags, or the Fc portion of an immunoglobulin. Numerous labels include, for example, particles, fluorophores, haptens, enzymes and their colorimetric, fluorescent and chemiluminescent substrates, as well as other labels.

[0037] Inhibitory nucleic acid molecules can include, for example, nucleotides, or non-natural or modified nucleotides, such as, for example, nucleotide analogs or nucleotide substitutes. Such nucleotides include nucleotides that contain modified bases, sugars, or phosphate groups, or nucleotides that incorporate non-natural moieties into their structure. Examples of non-natural nucleotides include, but are not limited to, dideoxynucleotides, biotinylated nucleotides, aminated nucleotides, deaminated nucleotides, alkylated nucleotides, benzylated nucleotides, and fluorophore-labeled nucleotides.

[0038] The inhibitory nucleic acid molecule can also include one or more nucleotide analogs or nucleotide substitutes. A nucleotide analog is a nucleotide that contains a modification to either the base, sugar, or phosphate moiety. Modifications to the base moiety include, but are not limited to, natural and synthetic modifications of A, C, G, and T / U, as well as various purine or pyrimidine bases, such as, for example, pseudouridine, uracil-5-yl, hypoxanthin-9-yl (I), and 2-aminoadenin-9-yl. Modified bases include 5-methylcytosine (5-me-C), 5-hydroxymethylcytosine, xanthine, hypoxanthine, 2-aminoadenine, 6-methyl and other alkyl derivatives of adenine and guanine, 2-propyl and other alkyl derivatives of adenine and guanine, 2-thiouracil, 2-thiothymine and 2-thiocytosine, 5-halouracil and cytosine, 5-propynyluracil and cytosine, 6-azouracil, cytosine and thymine, 5-uracil (cytosine), and thymine. Examples of uracils and cytosines include, but are not limited to, 4-isopropyl uracil, 4-isopropyl uracil, 8 ...

[0039] Nucleotide analogs can also include modifications of the sugar moiety. Modifications to the sugar moiety include, but are not limited to, natural modifications of ribose and deoxyribose, as well as synthetic modifications. Sugar modifications include, but are not limited to, the following modifications at the 2' position: OH; F; O-, S-, or N-alkyl; O-, S-, or N-alkenyl; O-, S-, or N-alkynyl; or O-alkyl-O-alkyl, where alkyl, alkenyl, and alkynyl are substituted or unsubstituted C-alkyl groups. 1~10 Alkyl or C 2~10 Alkenyl, and C 2~10 Exemplary 2' sugar modifications include -O[(CH2) n O] m CH3, -O(CH2) n OCH3, -O(CH2) n NH2, -O(CH2) n CH3, -O(CH2) n -ONH2 and -O(CH2) n ON [(CH2) n Other modifications at the 2' position include, but are not limited to, C 1~10Examples of suitable substituents include, but are not limited to, alkyl, substituted lower alkyl, alkaryl, aralkyl, O-alkaryl or O-aralkyl, SH, SCH3, OCN, Cl, Br, CN, CF3, OCF3, SOCH3, SO2CH3, ONO2, NO2, N3, NH2, heterocycloalkyl, heterocycloalkaryl, aminoalkylamino, polyalkylamino, substituted silyl, RNA cleaving groups, reporter groups, intercalators, groups for improving the pharmacokinetic properties of oligonucleotides, or groups for improving the pharmacodynamic properties of oligonucleotides, and other substituents with similar properties. Similar modifications may be made at other positions on the sugar, particularly the 3' position of the sugar in the 3' terminal nucleotide or 2'-5' linked oligonucleotides, and the 5' position of the 5' terminal nucleotide. Modified sugars can also include those containing modifications at the bridging ring oxygen, such as CH2 and S. Nucleotide sugar analogs can also have sugar mimetics such as cyclobutyl moieties in place of the pentofuranosyl sugar.

[0040] Nucleotide analogs can also be modified at the phosphate moiety. Modified phosphate moieties include, but are not limited to, those in which the linkage between two nucleotides can be modified to contain phosphorothioates, chiral phosphorothioates, phosphorodithioates, phosphotriesters, aminoalkyl phosphotriesters, methyl phosphonates and other alkyl phosphonates including 3'-alkylene phosphonates and chiral phosphonates, phosphinates, phosphoramidates including 3'-amino phosphoramidates and aminoalkyl phosphoramidates, thionophosphoramidates, thionoalkyl phosphonates, thionoalkyl phosphotriesters, and boranophosphates. Such phosphate or modified phosphate linkages between two nucleotides can be via 3'-5' or 2'-5' linkages, and the linkages can contain reverse polarity such as 3'-5' to 5'-3' or 2'-5' to 5'-2'. Various salts, mixed salts, and free acid forms are also included. Nucleotide substitutes also include peptide nucleic acids (PNAs).

[0041] In some embodiments, the antisense nucleic acid molecule is a gapmer, whereby the first 1-7 nucleotides of the 5'-end and the 3'-end, respectively, have a 2'-methoxyethyl (2'-MOE) modification. In some embodiments, the first 5 nucleotides of the 5'-end and the 3'-end, respectively, have a 2'-MOE modification. In some embodiments, the first 1-7 nucleotides of the 5'-end and the 3'-end are RNA nucleotides. In some embodiments, the first 5 nucleotides of the 5'-end and the 3'-end are RNA nucleotides. In some embodiments, each of the internucleotide backbone linkages is a phosphorothioate linkage.

[0042] In some embodiments, the siRNA molecule has terminal modification.In some embodiments, the 5'-end of the antisense strand is phosphorylated.In some embodiments, a 5'-phosphate analog that cannot be hydrolyzed, such as 5'-(E)-vinyl-phosphonate, is used.

[0043] In some embodiments, the siRNA molecule has a backbone modification. In some embodiments, modified phosphodiester groups linking consecutive ribose nucleosides have been shown to increase the stability and bioavailability of siRNA in vivo. Non-ester groups (-OH, =O) of the phosphodiester bond can be replaced with sulfur, boron, or acetate to obtain phosphorothioate, boranophosphate, and phosphonoacetate linkages. In addition, the phosphodiester group can be replaced with a phosphotriester to facilitate cellular uptake of the siRNA and retention in serum components by removing its negative charge. In some embodiments, the siRNA molecule has a sugar modification. In some embodiments, the sugar is deprotonated (a reaction catalyzed by exonucleases and endonucleases), allowing the 2'-hydroxyl to act as a nucleophile and attack the adjacent phosphorus of the phosphodiester bond. Such alternatives include 2'-O-methyl, 2'-O-methoxyethyl, and 2'-fluoro modifications.

[0044] In some embodiments, the siRNA molecule has base modifications, in some embodiments, the bases may be replaced with modified bases such as pseudouridine, 5'-methylcytidine, N6-methyladenosine, inosine, and N7-methylguanosine.

[0045] In some embodiments, siRNA molecules are bound to lipid.Lipid can be bound to 5'-end or 3'-end of siRNA, and can improve their bioavailability in vivo by associating with serum lipoprotein.Representative lipids include, but are not limited to, cholesterol and vitamin E, and fatty acids such as palmitic acid and tocopherol.

[0046] In some embodiments, an exemplary siRNA has the following formula: Sense: mN*mN* / i2FN / mN / i2FN / mN / i2FN / mN / i2FN / mN / i2FN / mN / i2FN / mN / i2FN / mN / i2FN / *mN* / 32FN / Antisense: / 52FN / * / i2FN / *mN / i2FN / mN / i2FN / mN / i2FN / mN / i2FN / mN / i2FN / mN / i2FN / mN / i2FN / mN / i2FN / mN*N*N where "N" is a base; "2F" is a 2'-F modification; "m" is a 2'-O-methyl modification; "I" is an internal base; and "*" is a phosphorothioate backbone linkage.

[0047] The present disclosure also provides a vector comprising any one or more of the inhibitory nucleic acid molecules. In some embodiments, the vector comprises any one or more of the inhibitory nucleic acid molecules and a heterologous nucleic acid. The vector can be a viral vector or a non-viral vector capable of transporting the nucleic acid molecule. In some embodiments, the vector is a plasmid or a cosmid (such as a circular double-stranded DNA to which additional DNA segments can be ligated). In some embodiments, the vector is a viral vector to which additional DNA segments can be ligated into the viral genome. Expression vectors include, but are not limited to, plasmids, cosmids, retroviruses, adenoviruses, adeno-associated viruses (AAV), plant viruses such as cauliflower mosaic virus and tobacco mosaic virus, yeast artificial chromosomes (YACs), Epstein-Barr (EBV) derived episomes, and other expression vectors known in the art.

[0048] The present disclosure also provides compositions comprising any one or more of the inhibitory nucleic acid molecules. In some embodiments, the composition is a pharmaceutical composition. In some embodiments, the composition comprises a carrier and / or excipient. Examples of carriers include, but are not limited to, poly(lactic acid) (PLA) microspheres, poly(D,L-lactic-co-glycolic acid) (PLGA) microspheres, liposomes, micelles, reverse micelles, lipid cochleates, and lipid microtubules. The carrier may comprise a buffered salt solution such as PBS, HBSS, and the like.

[0049] In some embodiments, the SLC9A3R2 inhibitor comprises a nuclease agent that induces one or more nicks or double-strand breaks in the recognition sequence(s) in the SLC9A3R2 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 SLC9A3R2 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 SLC9A3R2 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 cleavage by these nuclease agents can result in the deletion of the coding region between the two nuclease recognition sequences.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.

[0050] 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 to about 36 bp for zinc finger proteins or ZFN pairs, about 15 to about 18 bp for each ZFN, about 36 bp for TALE proteins or TALEN, and about 20 bp for CRISPR / Cas guide RNA.

[0051] In some embodiments, the CRISPR / Cas system can be used to modify the SLC9A3R2 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 SLC9A3R2 nucleic acid molecule.

[0052] Cas proteins generally contain at least one RNA recognition domain or RNA binding domain that can interact with gRNA. Cas proteins can also contain nuclease domains (e.g., 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 (e.g., FnCpf1). Cas proteins can have full cleavage activity to create double-stranded breaks in SLC9A3R2 genomic nucleic acid molecules, or can be nickases to create single-stranded breaks in SLC9A3R2 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), Cas proteins include, but are not limited to, Cse4 (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, as well as homologs or variants thereof. 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 transcriptional activation domain, or a transcriptional 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.

[0053] In some embodiments, targeted genetic modification of an SLC9A3R2 genomic nucleic acid molecule can be generated by contacting a cell with a Cas protein and one or more gRNAs that hybridize to one or more gRNA recognition sequences within a target genomic locus in the SLC9A3R2 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 9,519 of SEQ ID NO:1. For example, the gRNA recognition sequence can be located 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 position 9,519 of SEQ ID NO:1. The gRNA recognition sequence can include or be adjacent to a start codon or a stop codon of an SLC9A3R2 genomic nucleic acid molecule. For example, the gRNA 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 away from the start codon or the stop codon.

[0054] The gRNA recognition sequence in the target genomic locus in the SLC9A3R2 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 to about 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 base pairs, 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.

[0055] gRNA is an RNA molecule that binds to Cas protein and targets Cas protein to a specific position in SLC9A3R2 genomic nucleic acid molecule. An exemplary gRNA is an effective gRNA for inducing Cas enzyme to bind to or cleave SLC9A3R2 genomic nucleic acid molecule, wherein the gRNA comprises a DNA targeting segment that hybridizes with a gRNA recognition sequence in SLC9A3R2 genomic nucleic acid molecule that includes or is adjacent to the position corresponding to position 9,519 of SEQ ID NO:1. For example, the gRNA can be selected to hybridize with a gRNA recognition sequence that is 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 the position corresponding to position 9,519 of SEQ ID NO:1. Other exemplary gRNA comprises a DNA targeting segment that hybridizes with the gRNA recognition sequence present in the SLC9A3R2 genomic nucleic acid molecule that includes or is adjacent to the start codon or stop codon.For example, gRNA can be selected to hybridize with the 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 the start codon, or with the 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 the stop codon. A suitable gRNA can comprise 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 comprises 20 nucleotides.

[0056] Examples of suitable gRNA recognition sequences located within the SLC9A3R2 reference gene are set forth in Table 1 as SEQ ID NOs: 93-112.

[0057] [Table 1]

[0058] The Cas protein and gRNA form a complex, and the Cas protein cuts the target SLC9A3R2 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 SLC9A3R2 genomic nucleic acid molecule to which the DNA targeting segment of the gRNA binds. For example, the formation of a CRISPR complex (including the gRNA hybridized with the gRNA recognition sequence and complexed with the Cas protein) can cause one or both strand cuts within or near (e.g., within 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, or 50 or more base pairs) the nucleic acid sequence present in the SLC9A3R2 genomic nucleic acid molecule to which the DNA targeting segment of the gRNA binds.

[0059] Such a method can produce a SLC9A3R2 genomic nucleic acid molecule in which, for example, a region of SEQ ID NO:1 is destroyed, the start codon is destroyed, the stop codon is destroyed, or the coding sequence is destroyed or deleted. Optionally, the cell can be further contacted with one or more additional gRNAs that hybridize with additional gRNA recognition sequences in the target genomic locus of the SLC9A3R2 genomic nucleic acid molecule. By contacting the cell with one or more additional gRNAs (e.g., a second gRNA that hybridizes with a second gRNA recognition sequence), the cleavage by Cas protein can create two or more double-strand breaks or two or more single-strand breaks.

[0060] In some embodiments, the SLC9A3R2 inhibitor comprises a small molecule. In some embodiments, the method of treatment further comprises detecting the presence or absence of a SLC9A3R2 missense variant nucleic acid molecule encoding a predicted loss-of-function polypeptide of SLC9A3R2 in a biological sample obtained from the subject. As used throughout this disclosure, a "SLC9A3R2 missense variant nucleic acid molecule" is any SLC9A3R2 nucleic acid molecule (e.g., a genomic nucleic acid molecule, an mRNA molecule, or a cDNA molecule) that encodes a SLC9A3R2 polypeptide having partial loss-of-function, complete loss-of-function, predicted partial loss-of-function, or predicted complete loss-of-function.

[0061] The disclosure also provides methods of treating a subject with a therapeutic agent to treat or prevent hypertension, coronary heart disease and / or atrial fibrillation. In some embodiments, the subject has hypertension. In some embodiments, the subject is at risk for developing hypertension. In some embodiments, the subject has coronary heart disease. In some embodiments, the subject is at risk for developing coronary heart disease. In some embodiments, the subject has atrial fibrillation. In some embodiments, the subject is at risk for developing atrial fibrillation. In some embodiments, the method includes determining whether the subject has a SLC9A3R2 missense variant nucleic acid molecule encoding a SLC9A3R2 predicted loss-of-function polypeptide by obtaining or obtaining a biological sample from the subject and performing or performing sequence analysis on the biological sample to determine whether the subject has a genotype that includes a SLC9A3R2 missense variant nucleic acid molecule. If the subject is SLC9A3R2-based, the subject is administered or continues to be administered a standard dose of a therapeutic agent for treating or preventing hypertension, coronary heart disease and / or atrial fibrillation, and / or an SLC9A3R2 inhibitor is administered to the subject. If the subject is heterozygous for a SLC9A3R2 missense variant, the subject is administered or continues to be administered a standard dose of a therapeutic agent for treating or preventing hypertension, coronary heart disease and / or atrial fibrillation, and / or an SLC9A3R2 inhibitor is administered to the subject. The presence of a genotype having a SLC9A3R2 missense variant nucleic acid molecule encoding a loss-of-function polypeptide predicted by SLC9A3R2 indicates that the subject is at a low risk of developing hypertension, coronary heart disease and / or atrial fibrillation. In some embodiments, the subject is SLC9A3R2-based. In some embodiments, the subject is heterozygous for a SLC9A3R2 missense variant nucleic acid molecule that encodes a SLC9A3R2 predicted loss-of-function polypeptide.

[0062] For subjects who have been genotyped or determined to be heterozygous for a SLC9A3R2 missense variant nucleic acid molecule encoding a SLC9A3R2-based or SLC9A3R2-predicted loss-of-function polypeptide, such subjects can be treated with a SLC9A3R2 inhibitor as described herein.

[0063] Detecting the presence or absence of a SLC9A3R2 missense variant nucleic acid molecule encoding a predicted loss-of-function polypeptide in a biological sample obtained from a subject and / or determining whether a subject has a SLC9A3R2 missense variant nucleic acid molecule encoding a predicted loss-of-function polypeptide in SLC9A3R2 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 SLC9A3R2 missense variant nucleic acid molecule encoding a predicted loss-of-function polypeptide in SLC9A3R2 can be present in a cell obtained from the subject.

[0064] In some embodiments, if the subject is SLC9A3R2 normative, the subject is also administered a therapeutic agent for treating or preventing hypertension, coronary heart disease and / or atrial fibrillation at a standard dosage. In some embodiments, if the subject is heterozygous for a SLC9A3R2 missense variant nucleic acid molecule encoding a SLC9A3R2 predicted loss-of-function polypeptide, the subject is administered a therapeutic agent for treating or preventing hypertension, coronary heart disease and / or atrial fibrillation at a dosage equal to or less than the standard dosage.

[0065] In some embodiments, the method of treatment further comprises detecting the presence or absence of a SLC9A3R2 predicted loss-of-function polypeptide in a biological sample obtained from the subject. In some embodiments, if the subject does not have a SLC9A3R2 predicted loss-of-function polypeptide, the subject is administered a standard dose of a therapeutic agent for treating or preventing hypertension, coronary heart disease and / or atrial fibrillation. In some embodiments, if the subject has a SLC9A3R2 predicted loss-of-function polypeptide, the subject is administered a standard dose of a therapeutic agent for treating or preventing hypertension, coronary heart disease and / or atrial fibrillation.

[0066] The disclosure also provides methods of treating a subject with a therapeutic agent to treat or prevent hypertension, coronary heart disease and / or atrial fibrillation. In some embodiments, the subject has hypertension. In some embodiments, the subject is at risk for developing hypertension. In some embodiments, the subject has coronary heart disease. In some embodiments, the subject is at risk for developing coronary heart disease. In some embodiments, the subject has atrial fibrillation. In some embodiments, the subject is at risk for developing atrial fibrillation. In some embodiments, the method includes determining whether the subject has a loss-of-function polypeptide predicted in SLC9A3R2 by obtaining or obtaining a biological sample from the subject and performing or performing an assay on the biological sample to determine whether the subject has a loss-of-function polypeptide predicted in SLC9A3R2. If the subject does not have a loss-of-function polypeptide predicted by SLC9A3R2, the therapeutic agent for treating or preventing hypertension, coronary heart disease and / or atrial fibrillation is administered or continues to be administered to the subject at a standard dose, and / or an SLC9A3R2 inhibitor is administered to the subject. If the subject has a loss-of-function polypeptide predicted by SLC9A3R2, the therapeutic agent for treating or preventing hypertension, coronary heart disease and / or atrial fibrillation is administered or continues to be administered to the subject at a standard dose or less, and / or an SLC9A3R2 inhibitor is administered to the subject. The presence of a loss-of-function polypeptide predicted by SLC9A3R2 indicates that the subject is at a low risk of developing hypertension, coronary heart disease and / or atrial fibrillation. In some embodiments, the subject has a loss-of-function polypeptide predicted by SLC9A3R2. In some embodiments, the subject does not have a SLC9A3R2 predicted loss-of-function polypeptide.

[0067] Detecting the presence or absence of a SLC9A3R2 predicted loss-of-function polypeptide in a biological sample obtained from a subject and / or determining whether a subject has a SLC9A3R2 predicted loss-of-function 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 SLC9A3R2 predicted loss-of-function polypeptide can be present in a cell obtained from the subject.

[0068] Examples of therapeutic agents for treating or preventing hypertension include thiazide diuretics (e.g., chlorthalidone, chlorothiazide, hydrochlorothiazide, indapamide, or metolazone); potassium-sparing diuretics (e.g., amiloride, spironolactone, or triamterene); loop diuretics (e.g., bumetanide, furosemide, or torsemide); beta-blockers (e.g., acebutolol, atenolol, betaxolol, bisoprolol, bisoprolol / hydrochlorothiazide, metoprolol tartrate, metoprolol succinate, nadolol, pindolol, propranolol, solotol, or timolol); angiotensin-converting enzyme (ACE) inhibitors (e.g., benazepril, captopril, enalapril, fosinopril, lisinopril, moexipril, perindopril, quinapril, ramipril, or angiotensin II receptor blockers (ARBs) (e.g., candesartan, eprosartan, irbesartan, losartan, telmisartan, or valsartan); calcium channel blockers (e.g., amlodipine, diltiazem, felodipine, isradipine, nicardipine, nifedipine, nisoldipine, or verapamil); alpha blockers (e.g., doxazosin, prazosin, or terazosin); alpha beta blockers (e.g., carvedilol or labetalol); central agonists (e.g., methyldopa, clonidine, or guanfacine); vasodilators (e.g., hydralazine or minoxidil); aldosterone receptor antagonists (e.g., eplerenone or spironolactone), and renin inhibitors (e.g., aliskiren).

[0069] In some embodiments, the therapeutic agent for treating or preventing hypertension is a thiazide diuretic, a potassium-sparing diuretic, a loop diuretic, a beta blocker, an ACE inhibitor, an ARB, a calcium channel blocker, an alpha blocker, an alpha-beta blocker, a central agonist, a vasodilator, an aldosterone receptor antagonist, or a renin inhibitor. In some embodiments, the thiazide diuretic is chlorthalidone, chlorothiazide, hydrochlorothiazide, indapamide, or metolazone. In some embodiments, the potassium-sparing diuretic is amiloride, spironolactone, or triamterene. In some embodiments, the loop diuretic is bumetanide, furosemide, or torsemide. In some embodiments, the beta blocker is acebutolol, atenolol, betaxolol, bisoprolol, bisoprolol / hydrochlorothiazide, metoprolol tartrate, metoprolol succinate, nadolol, pindolol, propranolol, solotol, or timolol. In some embodiments, the ACE inhibitor is benazepril, captopril, enalapril, fosinopril, lisinopril, moexipril, perindopril, quinapril, ramipril, or trandolapril. In some embodiments, the ARB is candesartan, eprosartan, irbesartan, losartan, telmisartan, or valsartan. In some embodiments, the calcium channel blocker is amlodipine, diltiazem, felodipine, isradipine, nicardipine, nifedipine, nisoldipine, or verapamil. In some embodiments, the alpha blocker is doxazosin, prazosin, or terazosin. In some embodiments, the alpha beta blocker is carvedilol or labetalol. In some embodiments, the central agonist is methyldopa, clonidine, or guanfacine. In some embodiments, the vasodilator is hydralazine or minoxidil. In some embodiments, the aldosterone receptor antagonist is eplerenone or spironolactone. In some embodiments, the renin inhibitor is aliskiren.

[0070] In some embodiments, the dosage of a therapeutic agent for treating or preventing hypertension, coronary heart disease, and / or atrial fibrillation may be reduced by about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, or about 90% for a subject who is heterozygous for a SLC9A3R2 missense variant nucleic acid molecule encoding a SLC9A3R2 predicted loss-of-function polypeptide (i.e., receiving less than the standard dosage) compared to a subject who is SLC9A3R2 normative (which may receive the standard dosage). In some embodiments, the dosage of a therapeutic agent for treating or preventing hypertension, coronary heart disease, and / or atrial fibrillation may be reduced by about 10%, about 20%, about 30%, about 40%, or about 50%. In addition, the dose of a therapeutic agent for treating or preventing hypertension, coronary heart disease and / or atrial fibrillation in a subject who is heterozygous for an SLC9A3R2 missense variant nucleic acid molecule encoding a loss-of-function polypeptide predicted by SLC9A3R2 can be administered less frequently compared to a subject who is SLC9A3R2 normative.

[0071] Administration of the therapeutic agent and / or SLC9A3R2 inhibitor for treating or preventing hypertension, coronary heart disease and / or atrial fibrillation 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. 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, a subject can be treated for an extended period of time, such as, for example, 6 months, 1 year, or more. Furthermore, the therapeutic agent and / or SLC9A3R2 inhibitor for treating or preventing hypertension, coronary heart disease and / or atrial fibrillation can be administered sequentially or simultaneously. Additionally, therapeutic agents treating or preventing hypertension, coronary heart disease and / or atrial fibrillation and / or SLC9A3R2 inhibitors may be administered in separate compositions or together in the same composition.

[0072] Administration of the therapeutic agent and / or SLC9A3R2 inhibitor for treating or preventing hypertension, coronary heart disease and / or atrial fibrillation can occur 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 administration dose). Pharmaceutical compositions can be formulated using one or more physiologically and pharmacologic 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.

[0073] As used herein, the terms "treat", "treating", and "treatment" and "prevent", "prevention", 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: reducing / alleviating hypertension, reducing / alleviating the severity of hypertension (e.g., reducing or inhibiting the onset of hypertension), reducing / alleviating symptoms and hypertension-related effects, delaying the onset of symptoms and hypertension-related effects, reducing the severity of symptoms of hypertension-related effects, reducing the severity of acute episodes, reducing the number of symptoms and hypertension-related effects, reducing the latency period of symptoms and hypertension-related effects, ameliorating symptoms and hypertension-related effects, reducing secondary symptoms, reducing secondary infections, preventing recurrence of hypertension, reducing the number or frequency of recurrent episodes, increasing the latency period between symptomatic episodes, increasing the time to sustained progression, promoting remission, inducing remission, enhancing remission, accelerating recovery, or increasing the effectiveness or reducing resistance to alternative therapeutic agents, and / or increasing the survival time of the affected host animal. A prophylactic effect may include complete or partial avoidance / suppression or delay (e.g., complete or partial avoidance / suppression or delay) of the onset / progression of hypertension following administration of a treatment protocol, and increasing the survival time of an affected host animal. Treatment of hypertension includes treatment of a subject already diagnosed with some form of hypertension, either at a clinical stage or clinical symptoms, delaying the onset or progression or aggravation or worsening of symptoms or signs of hypertension, and / or preventing and / or reducing the severity of hypertension.

[0074] The present disclosure also provides methods for identifying subjects at high risk of developing hypertension, coronary heart disease and / or atrial fibrillation. In some embodiments, the methods include determining or having determined the presence or absence of a SLC9A3R2 missense variant nucleic acid molecule (e.g., a genomic nucleic acid molecule, an mRNA molecule and / or a cDNA molecule) encoding a loss-of-function polypeptide predicted by SLC9A3R2 in a biological sample obtained from the subject. If the subject lacks a SLC9A3R2 missense variant nucleic acid molecule encoding a loss-of-function polypeptide predicted by SLC9A3R2 (i.e., the subject is classified as SLC9A3R2-based upon genotyping), the subject has a high risk of developing hypertension, coronary heart disease and / or atrial fibrillation. If a subject has an SLC9A3R2 missense variant nucleic acid molecule encoding a loss-of-function polypeptide predicted by SLC9A3R2 (i.e., the subject is heterozygous or homozygous for the SLC9A3R2 missense variant nucleic acid molecule), the subject has a lower risk of developing hypertension, coronary heart disease and / or atrial fibrillation compared to subjects who are SLC9A3R2 normative.

[0075] Having a single copy of an SLC9A3R2 missense variant nucleic acid molecule encoding a loss-of-function polypeptide predicted by SLC9A3R2 further protects a subject from developing hypertension, coronary heart disease and / or atrial fibrillation than not having a copy of an SLC9A3R2 missense variant nucleic acid molecule encoding a loss-of-function polypeptide predicted by SLC9A3R2. Without intending to be limited to a particular theory or mechanism of action, it is believed that a single copy of a SLC9A3R2 missense variant nucleic acid molecule (i.e., heterozygous for the SLC9A3R2 missense variant nucleic acid molecule) protects a subject from developing hypertension, coronary heart disease and / or atrial fibrillation, and it is also believed that having two copies of a SLC9A3R2 missense variant nucleic acid molecule (i.e., homozygous for the SLC9A3R2 missense variant nucleic acid molecule) encoding a loss-of-function polypeptide predicted in SLC9A3R2 may further protect a subject from developing hypertension, coronary heart disease and / or atrial fibrillation compared to a subject having a single copy. Thus, in some embodiments, a single copy of a SLC9A3R2 missense variant nucleic acid molecule encoding a loss-of-function polypeptide predicted in SLC9A3R2 may not completely protect a subject from developing hypertension, coronary heart disease and / or atrial fibrillation, but may instead provide partial or incomplete protection. Without wishing to be bound by any particular theory, there may be additional factors or molecules involved in the development of hypertension, coronary heart disease and / or atrial fibrillation that are still present in subjects having a single copy of an SLC9A3R2 missense variant nucleic acid molecule encoding a loss-of-function polypeptide predicted for SLC9A3R2, which may result in less than complete protection from the development of hypertension, coronary heart disease and / or atrial fibrillation.

[0076] Detecting the presence or absence of a SLC9A3R2 missense variant nucleic acid molecule encoding a predicted loss-of-function polypeptide in a biological sample obtained from a subject and / or determining whether a subject has a SLC9A3R2 missense variant nucleic acid molecule encoding a predicted loss-of-function polypeptide in SLC9A3R2 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 SLC9A3R2 missense variant nucleic acid molecule encoding a predicted loss-of-function polypeptide in SLC9A3R2 can be present in a cell obtained from the subject.

[0077] In some embodiments, once a subject is identified as having a high risk of developing hypertension, coronary heart disease and / or atrial fibrillation, the subject is further treated with a therapeutic agent and / or SLC9A3R2 inhibitor that treats or prevents hypertension, coronary heart disease and / or atrial fibrillation, as described herein. For example, if a subject has an SLC9A3R2 criteria and is therefore at high risk of developing hypertension, coronary heart disease and / or atrial fibrillation, the subject is administered an SLC9A3R2 inhibitor. In some embodiments, such a subject is also administered a therapeutic agent that treats or prevents hypertension, coronary heart disease and / or atrial fibrillation. In some embodiments, if the subject is heterozygous for a SLC9A3R2 missense variant nucleic acid molecule encoding a loss-of-function polypeptide predicted by SLC9A3R2, the subject is administered a therapeutic agent for treating or preventing hypertension, coronary heart disease and / or atrial fibrillation at a dose equal to or less than the standard dose, and / or is administered an SLC9A3R2 inhibitor. In some embodiments, the subject is SLC9A3R2 norm. In some embodiments, the subject is heterozygous for a SLC9A3R2 missense variant nucleic acid molecule encoding a loss-of-function polypeptide predicted by SLC9A3R2.

[0078] In some embodiments, any of the methods described herein may further comprise determining the total burden of the subject having SLC9A3R2 missense variant nucleic acid molecules encoding SLC9A3R2 predicted loss-of-function polypeptides and / or SLC9A3R2 predicted loss-of-function variant polypeptides associated with a reduced risk of developing hypertension, coronary heart disease, and / or atrial fibrillation. The total burden is the sum of all variants in the SLC9A3R2 gene, which may be performed in an association analysis with hypertension, coronary heart disease, and / or atrial fibrillation. In some embodiments, the subject is homozygous for one or more SLC9A3R2 missense variant nucleic acid molecules encoding SLC9A3R2 predicted loss-of-function polypeptides associated with a reduced risk of developing hypertension, coronary heart disease, and / or atrial fibrillation. In some embodiments, the subject is heterozygous for one or more SLC9A3R2 missense variant nucleic acid molecules encoding loss-of-function polypeptides predicted by SLC9A3R2 associated with a reduced risk of developing hypertension, coronary heart disease and / or atrial fibrillation. The results of the association analysis suggest that the SLC9A3R2 missense variant nucleic acid molecules encoding loss-of-function polypeptides predicted by SLC9A3R2 are associated with a reduced risk of developing hypertension, coronary heart disease and / or atrial fibrillation. If the subject has a lower total load, the subject is at a higher risk of developing hypertension, coronary heart disease and / or atrial fibrillation, and the subject is administered or continues to be administered a standard dose of a therapeutic agent for treating or preventing hypertension, coronary heart disease and / or atrial fibrillation, and / or an SLC9A3R2 inhibitor. If a subject has a higher total load, the subject is at lower risk of developing hypertension, coronary heart disease and / or atrial fibrillation, and the subject is administered or continues to be administered a standard or lower dose of a therapeutic agent for treating or preventing hypertension, coronary heart disease and / or atrial fibrillation. The higher the total load, the lower the risk of developing hypertension, coronary heart disease and / or atrial fibrillation.

[0079] SLC9A3R2 variants that can be used in the total loading analysis include any one or more or any combination of the following:

[0080] [Table 2-1]

[0081] [Table 2-2]

[0082] [Table 2-3]

[0083] [Table 2-4]

[0084] [Table 2-5]

[0085] [Table 2-6]

[0086] [Table 2-7]

[0087] [Table 2-8]

[0088] [Table 2-9]

[0089] [Table 2-10]

[0090]

Table 2-11

[0091]

Table 2-12

[0092]

Table 2-13

[0093]

Table 2-14

[0094]

Table 2-15

[0095]

Table 2-16

[0096]

Table 2-17

[0097]

Table 2-18

[0098]

Table 2-19

[0099]

Table 2-20

[0100]

Table 2-21

[0101]

Table 2-22

[0102]

Table 2-23

[0103]

Table 2-24

[0104]

Table 2-25

[0105]

Table 2-26

[0106] In some embodiments, the total burden of subjects having any one or more SLC9A3R2 missense variant nucleic acid molecules encoding loss-of-function polypeptides predicted by SLC9A3R2 represents a weighted sum of any multiple SLC9A3R2 missense variant nucleic acid molecules encoding loss-of-function polypeptides predicted by SLC9A3R2. In some embodiments, the total burden is calculated using at least about 2, at least about 3, at least about 4, at least about 5, at least about 10, at least about 20, at least about 30, at least about 40, at least about 50, at least about 60, at least about 70, at least about 80, at least about 100, at least about 120, at least about 150, at least about 200, at least about 250, at least about 300, at least about 400, at least about 500, at least about 1,000, at least about 10,000, at least about 100,000, or at least about 1,000,000 or more genetic variants present in or surrounding the SLC9A3R2 gene (up to 10 Mb), where the genetic burden is the number of alleles multiplied by the estimated association for each allele with hypertension, coronary heart disease and / or atrial fibrillation or related outcomes (e.g., a weighted polygenic burden score). This may include any genetic variants close to the SLC9A3R2 gene (up to 10Mb around the gene) that show non-zero association with hypertension-related traits in gene association analysis, regardless of genome annotation. In some embodiments, if a subject has a total burden higher than the desired threshold score, the subject has a low risk of developing hypertension, coronary heart disease and / or atrial fibrillation. In some embodiments, if a subject has a total burden lower than the desired threshold score, the subject has a high risk of developing hypertension, coronary heart disease and / or atrial fibrillation.

[0107] In some embodiments, the total burden can be divided into quintiles, for example, top quintile, middle quintile, and bottom quintile, with the top quintile of total burden corresponding to the lowest risk group and the bottom quintile of total burden corresponding to the highest risk group. In some embodiments, subjects with a larger total burden include those with the highest weighted total burden, including but not limited to the top 10%, top 20%, top 30%, top 40%, or top 50% total burden from the subject population. In some embodiments, the genetic variants include genetic variants that have an association with hypertension, coronary heart disease, and / or atrial fibrillation in the top 10%, top 20%, top 30%, top 40%, or top 50% of the p-value range for the association. In some embodiments, each of the identified genetic variants is about 10 -2 , about 10 -3 , about 10 -4 , about 10 -5 , about 10 -6 , about 10 -7 , about 10 -8 , about 10 -9 , about 10 -10 , about 10 -11 , about 10 -12 , about 10 -13 , about 10 -14 , or about 10 -15 In some embodiments, the identified genetic variants include those having an association with hypertension, coronary heart disease and / or atrial fibrillation with a p-value of 5×10 -8In some embodiments, the identified genetic variants include genetic variants that have an association with hypertension, coronary heart disease, and / or atrial fibrillation in subjects at high risk relative to the remainder of a reference population with an odds ratio (OR) of about 1.5 or more, about 1.75 or more, about 2.0 or more, or about 2.25 or more for the top 20% of the distribution; or about 1.5 or more, about 1.75 or more, about 2.0 or more, about 2.25 or more, about 2.5 or more, or about 2.75 or more. In some embodiments, the odds ratio (OR) may range from about 1.0 to about 1.5, about 1.5 to about 2.0, about 2.0 to about 2.5, about 2.5 to about 3.0, about 3.0 to about 3.5, about 3.5 to about 4.0, about 4.0 to about 4.5, about 4.5 to about 5.0, about 5.0 to about 5.5, about 5.5 to about 6.0, about 6.0 to about 6.5, about 6.5 to about 7.0, or may be greater than 7.0. In some embodiments, high-risk subjects include subjects with a total burden in the bottom decile, quintile, or tertile in the reference population. The total burden threshold is determined based on the nature of the intended practical application and the risk difference that is considered meaningful for that practical application.

[0108] In some embodiments, once a subject is identified as having a high risk of developing hypertension, coronary heart disease and / or atrial fibrillation, the subject is further administered a therapeutic agent and / or an SLC9A3R2 inhibitor for treating or preventing hypertension, coronary heart disease and / or atrial fibrillation, as described herein. For example, if a subject has an SLC9A3R2 criteria and is therefore at high risk of developing hypertension, coronary heart disease and / or atrial fibrillation, the subject is administered an SLC9A3R2 inhibitor. In some embodiments, such a subject is also administered a therapeutic agent for treating or preventing hypertension, coronary heart disease and / or atrial fibrillation. In some embodiments, if the subject is heterozygous for a SLC9A3R2 missense variant nucleic acid molecule encoding a loss-of-function polypeptide predicted in SLC9A3R2, the subject is administered a therapeutic agent for treating or preventing hypertension, coronary heart disease and / or atrial fibrillation at a dose equal to or less than the standard dose, and / or is administered an SLC9A3R2 inhibitor. In some embodiments, the subject is SLC9A3R2 norm. In some embodiments, the subject is heterozygous for a SLC9A3R2 missense variant nucleic acid molecule encoding a loss-of-function polypeptide predicted in SLC9A3R2. Furthermore, if the subject has a lower overall burden of having a SLC9A3R2 missense variant nucleic acid molecule encoding a loss-of-function polypeptide predicted in SLC9A3R2, and therefore has a higher risk of developing hypertension, coronary heart disease and / or atrial fibrillation, the subject is administered a therapeutic agent for treating or preventing hypertension, coronary heart disease and / or atrial fibrillation. In some embodiments, if a subject has a lower overall burden for having SLC9A3R2 missense variant nucleic acid molecules encoding loss-of-function polypeptides predicted by SLC9A3R2, the subject is administered a therapeutic agent for treating or preventing hypertension, coronary heart disease and / or atrial fibrillation at a dosage that is the same as or greater than the standard dosage administered to a subject with a greater overall burden for having SLC9A3R2 missense variant nucleic acid molecules encoding loss-of-function polypeptides predicted by SLC9A3R2.

[0109] The present disclosure also provides a method for detecting the presence or absence of SLC9A3R2 missense variant genomic nucleic acid molecules encoding loss-of-function polypeptides predicted by SLC9A3R2 in a biological sample obtained from a subject, and / or SLC9A3R2 missense variant mRNA molecules encoding loss-of-function polypeptides predicted by SLC9A3R2 in a biological sample obtained from a subject, and / or SLC9A3R2 missense variant cDNA molecules encoding loss-of-function polypeptides predicted by SLC9A3R2 generated from mRNA molecules in a biological sample obtained from a subject. 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 (SNPs). The sequences provided herein for SLC9A3R2 variant genomic nucleic acid molecules, SLC9A3R2 variant mRNA molecules, and SLC9A3R2 variant cDNA molecules are merely exemplary sequences. Other sequences for SLC9A3R2 variant genomic nucleic acid molecules, variant mRNA molecules, and variant cDNA molecules are also possible.

[0110] The biological sample can be derived from any cell, tissue, or biological fluid of a subject. The biological sample may include any clinically relevant tissue, such as, for example, a bone marrow sample, a tumor biopsy, a fine needle aspirate, or a sample of a bodily fluid, such as, for example, blood, gingival crevicular fluid, plasma, serum, lymph, ascites, cyst fluid, or urine. In some embodiments, the biological 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 SLC9A3R2 variant nucleic acid molecules, a pretreatment designed to isolate or enrich the biological sample for SLC9A3R2 variant nucleic acid molecules may be employed. For this purpose, various techniques may be used. When detecting the level of SLC9A3R2 variant mRNA molecules, various techniques may be used to enrich the biological sample with mRNA molecules. Various methods may be used to detect the presence or level of mRNA molecules, or the presence of a particular variant genomic DNA locus.

[0111] The present disclosure also provides a method for detecting an SLC9A3R2 missense variant nucleic acid molecule or its complement encoding a predicted loss-of-function polypeptide in a subject, the method comprising assaying a biological sample obtained from the subject to determine whether a nucleic acid molecule in the biological sample is an SLC9A3R2 missense variant nucleic acid molecule encoding a predicted loss-of-function polypeptide in SLC9A3R2.

[0112] In some embodiments, an SLC9A3R2 missense mutant nucleic acid molecule or its complement encoding a predicted loss-of-function polypeptide of SLC9A3R2 is a genomic nucleic acid molecule having a nucleotide sequence comprising a thymine or its complement at a position corresponding to position 9,519 of SEQ ID NO:2.

[0113] In some embodiments, an SLC9A3R2 missense mutant nucleic acid molecule encoding a predicted loss-of-function polypeptide of SLC9A3R2, or its complement, is an mRNA molecule having a nucleotide sequence comprising uracil or its complement at a position corresponding to position 615 set forth in SEQ ID NO:22; uracil or its complement at a position corresponding to position 589 set forth in SEQ ID NO:23; uracil or its complement at a position corresponding to position 353 set forth in SEQ ID NO:24; uracil or its complement at a position corresponding to position 230 set forth in SEQ ID NO:25; uracil or its complement at a position corresponding to position 236 set forth in SEQ ID NO:26; uracil or its complement at a position corresponding to position 236 set forth in SEQ ID NO:27; uracil or its complement at a position corresponding to position 604 set forth in SEQ ID NO:28; or uracil or its complement at a position corresponding to position 126 set forth in SEQ ID NO:29.

[0114] In some embodiments, an SLC9A3R2 missense variant nucleic acid molecule encoding a predicted loss-of-function polypeptide of SLC9A3R2, or its complement, is a cDNA molecule generated from an mRNA molecule in a biological sample, having a nucleotide sequence comprising: a thymine or its complement at a position corresponding to position 615 set forth in SEQ ID NO:59; a thymine or its complement at a position corresponding to position 589 set forth in SEQ ID NO:60; a thymine or its complement at a position corresponding to position 353 set forth in SEQ ID NO:61; a thymine or its complement at a position corresponding to position 230 set forth in SEQ ID NO:62; a thymine or its complement at a position corresponding to position 236 set forth in SEQ ID NO:63; a thymine or its complement at a position corresponding to position 236 set forth in SEQ ID NO:64; a thymine or its complement at a position corresponding to position 604 set forth in SEQ ID NO:65; or a thymine or its complement at a position corresponding to position 126 set forth in SEQ ID NO:66.

[0115] In some embodiments, an SLC9A3R2 missense variant nucleic acid molecule has a nucleotide sequence that includes a thymine or its complement at a position corresponding to position 9,519 set forth in SEQ ID NO:2 (for a genomic nucleic acid molecule); a uracil or its complement at a position corresponding to position 615 set forth in SEQ ID NO:22 (for an mRNA molecule); or a thymine or its complement at a position corresponding to position 615 set forth in SEQ ID NO:59 (for a cDNA molecule derived from an mRNA molecule).

[0116] In some embodiments, the biological sample comprises cells or cell lysates. Such methods can further comprise, for example, obtaining a biological sample from a subject that comprises SLC9A3R2 genomic nucleic acid molecules or mRNA molecules, and, if mRNA, optionally reverse transcribing the mRNA into cDNA. Such assays can comprise, for example, determining the identity of these positions of a particular SLC9A3R2 nucleic acid molecule. In some embodiments, the methods are in vitro methods.

[0117] In some embodiments, the assay comprises sequencing at least a portion of the nucleotide sequence of a SLC9A3R2 nucleic acid molecule or its complement in the biological sample. In some embodiments, the assay comprises sequencing at least a portion of the nucleotide sequence of a SLC9A3R2 genomic nucleic acid molecule in the biological sample, where the sequenced portion comprises a position corresponding to position 9,519 set forth in SEQ ID NO:2 or its complement; sequencing at least a portion of the nucleotide sequence of a SLC9A3R2 mRNA molecule in the biological sample, where the sequenced portion comprises a position corresponding to position 615 set forth in SEQ ID NO:22 or its complement; and / or sequencing at least a portion of the nucleotide sequence of a SLC9A3R2 cDNA molecule generated from mRNA in the biological sample, where the sequenced portion comprises a position corresponding to position 615 set forth in SEQ ID NO:59 or its complement. If the sequenced portion of the SLC9A3R2 nucleic acid molecule in the biological sample contains a thymine or its complement at a position corresponding to position 9,519 set forth in SEQ ID NO:2, a uracil or its complement at a position corresponding to position 615 set forth in SEQ ID NO:22, or a thymine or its complement at a position corresponding to position 615 set forth in SEQ ID NO:59, then the SLC9A3R2 nucleic acid molecule in the biological sample is an SLC9A3R2 missense mutant nucleic acid molecule that encodes a loss-of-function polypeptide predicted for SLC9A3R2.

[0118] In some embodiments, the assay comprises sequencing at least a portion of the nucleotide sequence of a SLC9A3R2 genomic nucleic acid molecule or its complement in the biological sample, wherein the sequenced portion comprises a position corresponding to position 9,519 set forth in SEQ ID NO: 2 or its complement. If the sequenced portion of the SLC9A3R2 genomic nucleic acid molecule in the biological sample comprises a thymine or its complement at a position corresponding to position 9,519 set forth in SEQ ID NO: 2, then the SLC9A3R2 genomic nucleic acid molecule in the biological sample is a SLC9A3R2 missense variant genomic nucleic acid molecule that encodes a loss-of-function polypeptide predicted for SLC9A3R2.

[0119] In some embodiments, the assay comprises sequencing at least a portion of the nucleotide sequence of an SLC9A3R2 mRNA molecule in a biological sample, wherein the sequenced portion comprises a position corresponding to position 615 or its complement set forth in SEQ ID NO:22; position 589 or its complement set forth in SEQ ID NO:23; position 353 or its complement set forth in SEQ ID NO:24; position 230 or its complement set forth in SEQ ID NO:25; position 236 or its complement set forth in SEQ ID NO:26; position 236 or its complement set forth in SEQ ID NO:27; position 604 or its complement set forth in SEQ ID NO:28; or position 126 or its complement set forth in SEQ ID NO:29. If the sequenced portion of the SLC9A3R2 mRNA molecule in the biological sample comprises uracil or its complement at a position corresponding to position 615 in SEQ ID NO:22; uracil or its complement at a position corresponding to position 589 in SEQ ID NO:23; uracil or its complement at a position corresponding to position 353 in SEQ ID NO:24; uracil or its complement at a position corresponding to position 230 in SEQ ID NO:25; uracil or its complement at a position corresponding to position 236 in SEQ ID NO:26; uracil or its complement at a position corresponding to position 236 in SEQ ID NO:27; uracil or its complement at a position corresponding to position 604 in SEQ ID NO:28; or uracil or its complement at a position corresponding to position 126 in SEQ ID NO:29, then the SLC9A3R2 mRNA molecule in the biological sample is an SLC9A3R2 missense mutant mRNA molecule that encodes a loss-of-function polypeptide predicted for SLC9A3R2.

[0120] In some embodiments, the assay comprises sequencing at least a portion of the nucleotide sequence of an SLC9A3R2 cDNA molecule generated from an mRNA molecule in a biological sample, wherein the sequenced portion comprises a position corresponding to position 615 or its complement set forth in SEQ ID NO:59; position 589 or its complement set forth in SEQ ID NO:60; position 353 or its complement set forth in SEQ ID NO:61; position 230 or its complement set forth in SEQ ID NO:62; position 236 or its complement set forth in SEQ ID NO:63; position 236 or its complement set forth in SEQ ID NO:64; position 604 or its complement set forth in SEQ ID NO:65; or position 126 or its complement set forth in SEQ ID NO:66. If the sequenced portion of the SLC9A3R2 cDNA molecule in the biological sample comprises a thymine or its complement at a position corresponding to position 615 in SEQ ID NO:59; a thymine or its complement at a position corresponding to position 589 in SEQ ID NO:60; a thymine or its complement at a position corresponding to position 353 in SEQ ID NO:61; a thymine or its complement at a position corresponding to position 230 in SEQ ID NO:62; a thymine or its complement at a position corresponding to position 236 in SEQ ID NO:63; a thymine or its complement at a position corresponding to position 236 in SEQ ID NO:64; a thymine or its complement at a position corresponding to position 604 in SEQ ID NO:65; or a thymine or its complement at a position corresponding to position 126 in SEQ ID NO:66, then the SLC9A3R2 cDNA molecule generated from the mRNA molecule in the biological sample is an SLC9A3R2 missense variant cDNA molecule that encodes a loss-of-function polypeptide predicted for SLC9A3R2.

[0121] In some embodiments, the assay comprises: a) contacting a biological sample with a primer that hybridizes to a portion of the nucleotide sequence of a SLC9A3R2 genomic nucleic acid molecule or its complement adjacent to a position corresponding to position 9,519 set forth in SEQ ID NO:2 or its complement; a SLC9A3R2 mRNA molecule or its complement adjacent to a position corresponding to position 615 set forth in SEQ ID NO:22 or its complement; and / or a SLC9A3R2 cDNA molecule or its complement adjacent to a position corresponding to position 615 set forth in SEQ ID NO:59 or its complement; and b) contacting a biological sample with a primer that hybridizes to a portion of the nucleotide sequence of a SLC9A3R2 genomic nucleic acid molecule or its complement corresponding to position 9,519 set forth in SEQ ID NO:2 or its complement; a SLC9A3R2 mRNA molecule or its complement corresponding to position 615 set forth in SEQ ID NO:22 or its complement; and / or a SLC9A3R2 cDNA molecule or its complement adjacent to a position corresponding to position 615 set forth in SEQ ID NO:59 or its complement. and c) determining whether an extension product of the primer comprises: a thymine or its complement at a position corresponding to position 9,519 set forth in SEQ ID NO:2; a uracil or its complement at a position corresponding to position 615 set forth in SEQ ID NO:22; and / or a thymine or its complement at a position corresponding to position 615 set forth in SEQ ID NO:59.

[0122] In some embodiments, the assay comprises: a) contacting a biological sample with a primer that hybridizes to a portion of the nucleotide sequence of an SLC9A3R2 mRNA molecule or its complement adjacent to a position corresponding to position 589 set forth in SEQ ID NO:23 or its complement; and / or an SLC9A3R2 cDNA molecule or its complement adjacent to a position corresponding to position 589 set forth in SEQ ID NO:60 or its complement; b) extending the primer at least through a position in the nucleotide sequence of an SLC9A3R2 mRNA molecule or its complement corresponding to position 589 set forth in SEQ ID NO:23 or its complement; and / or an SLC9A3R2 cDNA molecule or its complement corresponding to position 589 set forth in SEQ ID NO:60 or its complement; and c) determining whether an extension product of the primer comprises: a uracil or its complement at the position corresponding to position 589 set forth in SEQ ID NO:23; and / or a thymine or its complement at the position corresponding to position 589 set forth in SEQ ID NO:60.

[0123] In some embodiments, the assay comprises: a) contacting a biological sample with a primer that hybridizes to a portion of the nucleotide sequence of an SLC9A3R2 mRNA molecule or its complement adjacent to a position corresponding to position 353 set forth in SEQ ID NO:24 or its complement; and / or an SLC9A3R2 cDNA molecule or its complement adjacent to a position corresponding to position 353 set forth in SEQ ID NO:61 or its complement; b) extending the primer at least through a position in the nucleotide sequence of an SLC9A3R2 mRNA molecule or its complement corresponding to position 353 set forth in SEQ ID NO:24 or its complement; and / or an SLC9A3R2 cDNA molecule or its complement corresponding to position 353 set forth in SEQ ID NO:61 or its complement; and c) determining whether an extension product of the primer comprises: uracil or its complement at the position corresponding to position 353 set forth in SEQ ID NO:24; and / or thymine or its complement at the position corresponding to position 353 set forth in SEQ ID NO:61.

[0124] In some embodiments, the assay comprises: a) contacting a biological sample with a primer that hybridizes to a portion of the nucleotide sequence of an SLC9A3R2 mRNA molecule or its complement adjacent to a position corresponding to position 230 set forth in SEQ ID NO:25 or its complement; and / or an SLC9A3R2 cDNA molecule or its complement adjacent to a position corresponding to position 230 set forth in SEQ ID NO:62 or its complement; b) extending the primer at least through a position in the nucleotide sequence of an SLC9A3R2 mRNA molecule or its complement corresponding to position 230 set forth in SEQ ID NO:25 or its complement; and / or an SLC9A3R2 cDNA molecule or its complement corresponding to position 230 set forth in SEQ ID NO:62 or its complement; and c) determining whether an extension product of the primer comprises: uracil or its complement at the position corresponding to position 230 set forth in SEQ ID NO:25; and / or thymine or its complement at the position corresponding to position 230 set forth in SEQ ID NO:62.

[0125] In some embodiments, the assay comprises: a) contacting a biological sample with a primer that hybridizes to a portion of the nucleotide sequence of an SLC9A3R2 mRNA molecule or its complement adjacent to a position corresponding to position 236 set forth in SEQ ID NO:26 or its complement; and / or an SLC9A3R2 cDNA molecule or its complement adjacent to a position corresponding to position 236 set forth in SEQ ID NO:63 or its complement; b) extending the primer at least through a position in the nucleotide sequence of an SLC9A3R2 mRNA molecule or its complement corresponding to position 236 set forth in SEQ ID NO:26 or its complement; and / or an SLC9A3R2 cDNA molecule or its complement corresponding to position 236 set forth in SEQ ID NO:63 or its complement; and c) determining whether an extension product of the primer comprises: uracil or its complement at the position corresponding to position 236 set forth in SEQ ID NO:26; and / or thymine or its complement at the position corresponding to position 236 set forth in SEQ ID NO:63.

[0126] In some embodiments, the assay comprises: a) contacting a biological sample with a primer that hybridizes to a portion of the nucleotide sequence of an SLC9A3R2 mRNA molecule or its complement adjacent to a position corresponding to position 236 set forth in SEQ ID NO:27 or its complement; and / or an SLC9A3R2 cDNA molecule or its complement adjacent to a position corresponding to position 236 set forth in SEQ ID NO:64 or its complement; b) extending the primer at least through a position in the nucleotide sequence of an SLC9A3R2 mRNA molecule or its complement corresponding to position 236 set forth in SEQ ID NO:27 or its complement; and / or an SLC9A3R2 cDNA molecule or its complement corresponding to position 236 set forth in SEQ ID NO:64 or its complement; and c) determining whether an extension product of the primer comprises: uracil or its complement at the position corresponding to position 236 set forth in SEQ ID NO:27; and / or thymine or its complement at the position corresponding to position 236 set forth in SEQ ID NO:64.

[0127] In some embodiments, the assay comprises: a) contacting a biological sample with a primer that hybridizes to a portion of the nucleotide sequence of an SLC9A3R2 mRNA molecule or its complement adjacent to a position corresponding to position 604 set forth in SEQ ID NO:28 or its complement; and / or an SLC9A3R2 cDNA molecule or its complement adjacent to a position corresponding to position 604 set forth in SEQ ID NO:65 or its complement; b) extending the primer at least through a position in the nucleotide sequence of an SLC9A3R2 mRNA molecule or its complement corresponding to position 604 set forth in SEQ ID NO:28 or its complement; and / or an SLC9A3R2 cDNA molecule or its complement corresponding to position 604 set forth in SEQ ID NO:65 or its complement; and c) determining whether an extension product of the primer comprises: a uracil or its complement at the position corresponding to position 604 set forth in SEQ ID NO:28; and / or a thymine or its complement at the position corresponding to position 604 set forth in SEQ ID NO:65.

[0128] In some embodiments, the assay comprises: a) contacting a biological sample with a primer that hybridizes to a portion of the nucleotide sequence of an SLC9A3R2 mRNA molecule or its complement adjacent to a position corresponding to position 126 set forth in SEQ ID NO:29 or its complement; and / or an SLC9A3R2 cDNA molecule or its complement adjacent to a position corresponding to position 126 set forth in SEQ ID NO:66 or its complement; b) extending the primer at least through a position in the nucleotide sequence of an SLC9A3R2 mRNA molecule or its complement corresponding to position 126 set forth in SEQ ID NO:29 or its complement; and / or an SLC9A3R2 cDNA molecule or its complement corresponding to position 126 set forth in SEQ ID NO:66 or its complement; and c) determining whether an extension product of the primer comprises: uracil or its complement at the position corresponding to position 126 set forth in SEQ ID NO:29; and / or thymine or its complement at the position corresponding to position 126 set forth in SEQ ID NO:66.

[0129] In some embodiments, the assay comprises: a) contacting a biological sample with a primer that hybridizes to a portion of the nucleotide sequence of an SLC9A3R2 genomic nucleic acid molecule or its complement adjacent to a position corresponding to position 9,519 set forth in SEQ ID NO:2 or its complement; b) extending the primer at least through a position in the nucleotide sequence of an SLC9A3R2 genomic nucleic acid molecule or its complement corresponding to position 9,519 set forth in SEQ ID NO:2 or its complement; and c) determining whether the extension product of the primer comprises a thymine at a position corresponding to position 9,519 set forth in SEQ ID NO:2.

[0130] In some embodiments, the assay involves: a) detecting an SLC9A3R2 region adjacent to a position corresponding to: position 615 set forth in SEQ ID NO:22, or its complement; position 589 set forth in SEQ ID NO:23, or its complement; position 353 set forth in SEQ ID NO:24, or its complement; position 230 set forth in SEQ ID NO:25, or its complement; position 236 set forth in SEQ ID NO:26, or its complement; position 236 set forth in SEQ ID NO:27, or its complement; position 604 set forth in SEQ ID NO:28, or its complement; or position 126 set forth in SEQ ID NO:29, or its complement. contacting the biological sample with a primer that hybridizes to a portion of the nucleotide sequence of the mRNA molecule or its complement; and b) detecting an SLC9A3R2 corresponding to position 615 or its complement as set forth in SEQ ID NO:22; position 589 or its complement as set forth in SEQ ID NO:23; position 353 or its complement as set forth in SEQ ID NO:24; position 230 or its complement as set forth in SEQ ID NO:25; position 236 or its complement as set forth in SEQ ID NO:26; position 236 or its complement as set forth in SEQ ID NO:27; position 604 or its complement as set forth in SEQ ID NO:28; or position 126 or its complement as set forth in SEQ ID NO:29. extending the primer at least through a position in the nucleotide sequence of the mRNA molecule or its complement; and c) determining whether the extension product of the primer comprises: uracil or its complement at a position corresponding to position 615 set forth in SEQ ID NO:22; uracil or its complement at a position corresponding to position 589 set forth in SEQ ID NO:23; uracil or its complement at a position corresponding to position 353 set forth in SEQ ID NO:24; uracil or its complement at a position corresponding to position 230 set forth in SEQ ID NO:25; uracil or its complement at a position corresponding to position 236 set forth in SEQ ID NO:26; uracil or its complement at a position corresponding to position 236 set forth in SEQ ID NO:27; uracil or its complement at a position corresponding to position 604 set forth in SEQ ID NO:28; or uracil or its complement at a position corresponding to position 126 set forth in SEQ ID NO:29.

[0131] In some embodiments, the assay involves: a) detecting an SLC9A3R2 region adjacent to a position corresponding to: position 615 set forth in SEQ ID NO:59, or its complement; position 589 set forth in SEQ ID NO:60, or its complement; position 353 set forth in SEQ ID NO:61, or its complement; position 230 set forth in SEQ ID NO:62, or its complement; position 236 set forth in SEQ ID NO:63, or its complement; position 236 set forth in SEQ ID NO:64, or its complement; position 604 set forth in SEQ ID NO:65, or its complement; or position 126 set forth in SEQ ID NO:66, or its complement. contacting the biological sample with a primer that hybridizes to a portion of the nucleotide sequence of the cDNA molecule or its complement; and b) detecting an SLC9A3R2 corresponding to position 615 set forth in SEQ ID NO:59 or its complement; position 589 set forth in SEQ ID NO:60 or its complement; position 353 set forth in SEQ ID NO:61 or its complement; position 230 set forth in SEQ ID NO:62 or its complement; position 236 set forth in SEQ ID NO:63 or its complement; position 236 set forth in SEQ ID NO:64 or its complement; position 604 set forth in SEQ ID NO:65 or its complement; or position 126 set forth in SEQ ID NO:66 or its complement. extending the primer at least through a position in the nucleotide sequence of the cDNA molecule or its complement; and c) determining whether the extension product of the primer comprises: a thymine or its complement at a position corresponding to position 615 set forth in SEQ ID NO:59; a thymine or its complement at a position corresponding to position 589 set forth in SEQ ID NO:60; a thymine or its complement at a position corresponding to position 353 set forth in SEQ ID NO:61; a thymine or its complement at a position corresponding to position 230 set forth in SEQ ID NO:62; a thymine or its complement at a position corresponding to position 236 set forth in SEQ ID NO:63; a thymine or its complement at a position corresponding to position 236 set forth in SEQ ID NO:64; a thymine or its complement at a position corresponding to position 604 set forth in SEQ ID NO:65; or a thymine or its complement at a position corresponding to position 126 set forth in SEQ ID NO:66.

[0132] In some embodiments, the assay comprises sequencing the entire nucleic acid molecule. In some embodiments, only the SLC9A3R2 genomic nucleic acid molecule is analyzed. In some embodiments, only the SLC9A3R2 mRNA is analyzed. In some embodiments, only the SLC9A3R2 cDNA obtained from the SLC9A3R2 mRNA is analyzed.

[0133] In some embodiments, the assay comprises: a) amplifying at least a portion of a SLC9A3R2 nucleic acid molecule or its complement in a biological sample, wherein the amplified portion comprises thymine or its complement at a position corresponding to position 9,519 set forth in SEQ ID NO:2; uracil or its complement at a position corresponding to position 615 set forth in SEQ ID NO:22; and / or thymine or its complement at a position corresponding to position 615 set forth in SEQ ID NO:59; b) labeling the amplified nucleic acid molecule with a detectable label; c) contacting the labeled nucleic acid molecule with a support comprising a mutation-specific probe, the mutation-specific probe comprising a nucleotide sequence that hybridizes under stringent conditions to a nucleotide sequence of the amplified nucleic acid molecule comprising thymine or its complement at a position corresponding to position 9,519 set forth in SEQ ID NO:2; uracil or its complement at a position corresponding to position 615 set forth in SEQ ID NO:22; and / or thymine or its complement at a position corresponding to position 615 set forth in SEQ ID NO:59; and d) detecting the detectable label.

[0134] In some embodiments, the assay comprises: a) amplifying at least a portion of a SLC9A3R2 nucleic acid molecule or its complement in a biological sample, wherein the amplified portion comprises uracil or its complement at a position corresponding to position 589 of SEQ ID NO:23; and / or thymine or its complement at a position corresponding to position 589 of SEQ ID NO:60; b) labeling the amplified nucleic acid molecule with a detectable label; c) contacting the labeled nucleic acid molecule with a support comprising a mutation-specific probe, the mutation-specific probe comprising a nucleotide sequence that hybridizes under stringent conditions to a nucleotide sequence of the amplified nucleic acid molecule comprising uracil or its complement at a position corresponding to position 589 of SEQ ID NO:23; and / or thymine or its complement at a position corresponding to position 589 of SEQ ID NO:60; and d) detecting the detectable label.

[0135] In some embodiments, the assay comprises: a) amplifying at least a portion of a SLC9A3R2 nucleic acid molecule or its complement in a biological sample, wherein the amplified portion comprises uracil or its complement at a position corresponding to position 353 set forth in SEQ ID NO:24; and / or thymine or its complement at a position corresponding to position 353 set forth in SEQ ID NO:61; b) labeling the amplified nucleic acid molecule with a detectable label; c) contacting the labeled nucleic acid molecule with a support comprising a mutation-specific probe, the mutation-specific probe comprising a nucleotide sequence that hybridizes under stringent conditions to a nucleotide sequence of the amplified nucleic acid molecule comprising uracil or its complement at a position corresponding to position 353 set forth in SEQ ID NO:24; and / or thymine or its complement at a position corresponding to position 353 set forth in SEQ ID NO:61; and d) detecting the detectable label.

[0136] In some embodiments, the assay comprises: a) amplifying at least a portion of a SLC9A3R2 nucleic acid molecule or its complement in a biological sample, wherein the amplified portion comprises uracil or its complement at a position corresponding to position 230 set forth in SEQ ID NO:25; and / or thymine or its complement at a position corresponding to position 230 set forth in SEQ ID NO:62; b) labeling the amplified nucleic acid molecule with a detectable label; c) contacting the labeled nucleic acid molecule with a support comprising a mutation-specific probe, the mutation-specific probe comprising a nucleotide sequence that hybridizes under stringent conditions to a nucleotide sequence of the amplified nucleic acid molecule comprising uracil or its complement at a position corresponding to position 230 set forth in SEQ ID NO:25; and / or thymine or its complement at a position corresponding to position 230 set forth in SEQ ID NO:62; and d) detecting the detectable label.

[0137] In some embodiments, the assay comprises: a) amplifying at least a portion of a SLC9A3R2 nucleic acid molecule or its complement in a biological sample, wherein the amplified portion comprises uracil or its complement at a position corresponding to position 236 set forth in SEQ ID NO:26; and / or thymine or its complement at a position corresponding to position 236 set forth in SEQ ID NO:63; b) labeling the amplified nucleic acid molecule with a detectable label; c) contacting the labeled nucleic acid molecule with a support comprising a mutation-specific probe, the mutation-specific probe comprising a nucleotide sequence that hybridizes under stringent conditions to a nucleotide sequence of the amplified nucleic acid molecule comprising uracil or its complement at a position corresponding to position 236 set forth in SEQ ID NO:26; and / or thymine or its complement at a position corresponding to position 236 set forth in SEQ ID NO:63; and d) detecting the detectable label.

[0138] In some embodiments, the assay comprises: a) amplifying at least a portion of a SLC9A3R2 nucleic acid molecule or its complement in a biological sample, wherein the amplified portion comprises uracil or its complement at a position corresponding to position 236 set forth in SEQ ID NO:27; and / or thymine or its complement at a position corresponding to position 236 set forth in SEQ ID NO:64; b) labeling the amplified nucleic acid molecule with a detectable label; c) contacting the labeled nucleic acid molecule with a support comprising a mutation-specific probe, the mutation-specific probe comprising a nucleotide sequence that hybridizes under stringent conditions to a nucleotide sequence of the amplified nucleic acid molecule comprising uracil or its complement at a position corresponding to position 236 set forth in SEQ ID NO:27; and / or thymine or its complement at a position corresponding to position 236 set forth in SEQ ID NO:64; and d) detecting the detectable label.

[0139] In some embodiments, the assay comprises: a) amplifying at least a portion of a SLC9A3R2 nucleic acid molecule or its complement in a biological sample, wherein the amplified portion comprises uracil or its complement at a position corresponding to 604 as set forth in SEQ ID NO:28; and / or thymine or its complement at a position corresponding to 604 as set forth in SEQ ID NO:65; b) labeling the amplified nucleic acid molecule with a detectable label; c) contacting the labeled nucleic acid molecule with a support comprising a mutation-specific probe, the mutation-specific probe comprising a nucleotide sequence that hybridizes under stringent conditions to a nucleotide sequence of the amplified nucleic acid molecule comprising uracil or its complement at a position corresponding to 604 as set forth in SEQ ID NO:28; and / or thymine or its complement at a position corresponding to 604 as set forth in SEQ ID NO:65; and d) detecting the detectable label.

[0140] In some embodiments, the assay comprises: a) amplifying at least a portion of a SLC9A3R2 nucleic acid molecule or its complement in a biological sample, wherein the amplified portion comprises uracil or its complement at a position corresponding to position 126 set forth in SEQ ID NO:29; and / or thymine or its complement at a position corresponding to position 126 set forth in SEQ ID NO:66; b) labeling the amplified nucleic acid molecule with a detectable label; c) contacting the labeled nucleic acid molecule with a support comprising a mutation-specific probe, the mutation-specific probe comprising a nucleotide sequence that hybridizes under stringent conditions to a nucleotide sequence of the amplified nucleic acid molecule comprising uracil or its complement at a position corresponding to position 126 set forth in SEQ ID NO:29; and / or thymine or its complement at a position corresponding to position 126 set forth in SEQ ID NO:66; and d) detecting the detectable label.

[0141] In some embodiments, the assay comprises: a) amplifying at least a portion of an SLC9A3R2 genomic nucleic acid molecule or its complement in a biological sample, the portion comprising a thymine or its complement at a position corresponding to position 9,519 of SEQ ID NO:2; and d) detecting a detectable label.

[0142] In some embodiments, the assay comprises: a) amplifying at least a portion of an SLC9A3R2 mRNA molecule or its complement in a biological sample, the portion comprising uracil or its complement at a position corresponding to position 615 set forth in SEQ ID NO:22; uracil or its complement at a position corresponding to position 589 set forth in SEQ ID NO:23; uracil or its complement at a position corresponding to position 353 set forth in SEQ ID NO:24; uracil or its complement at a position corresponding to position 230 set forth in SEQ ID NO:25; uracil or its complement at a position corresponding to position 236 set forth in SEQ ID NO:26; uracil or its complement at a position corresponding to position 236 set forth in SEQ ID NO:27; uracil or its complement at a position corresponding to position 604 set forth in SEQ ID NO:28; or uracil or its complement at a position corresponding to position 126 set forth in SEQ ID NO:29; b) labeling the amplified nucleic acid molecule with a detectable label; and c) detecting the presence of a uracil or its complement at a position corresponding to position 126 set forth in SEQ ID NO:29 using a mutation-specific probe. a uracil or its complement at a position corresponding to position 615 set forth in SEQ ID NO:22; a uracil or its complement at a position corresponding to position 589 set forth in SEQ ID NO:23; a uracil or its complement at a position corresponding to position 353 set forth in SEQ ID NO:24; a uracil or its complement at a position corresponding to position 230 set forth in SEQ ID NO:25; a uracil or its complement at a position corresponding to position 236 set forth in SEQ ID NO:26; a uracil or its complement at a position corresponding to position 236 set forth in SEQ ID NO:27; a uracil or its complement at a position corresponding to position 604 set forth in SEQ ID NO:28; or a uracil or its complement at a position corresponding to position 126 set forth in SEQ ID NO:29; and d) detecting the detectable label.

[0143] In some embodiments, the assay comprises: a) amplifying at least a portion of a SLC9A3R2 cDNA molecule or its complement generated from an mRNA molecule in a biological sample, the portion comprising a thymine or its complement at a position corresponding to position 615 set forth in SEQ ID NO:59; a thymine or its complement at a position corresponding to position 589 set forth in SEQ ID NO:60; a thymine or its complement at a position corresponding to position 353 set forth in SEQ ID NO:61; a thymine or its complement at a position corresponding to position 230 set forth in SEQ ID NO:62; a thymine or its complement at a position corresponding to position 236 set forth in SEQ ID NO:63; a thymine or its complement at a position corresponding to position 236 set forth in SEQ ID NO:64; a thymine or its complement at a position corresponding to position 604 set forth in SEQ ID NO:65; or a thymine or its complement at a position corresponding to position 126 set forth in SEQ ID NO:66; b) labeling the amplified nucleic acid molecule with a detectable label; and c) detecting the presence of a thymine or its complement at a position corresponding to position 126 set forth in SEQ ID NO:66 using a mutation-specific probe. a thymine or its complement at a position corresponding to position 615 set forth in SEQ ID NO:59; a thymine or its complement at a position corresponding to position 589 set forth in SEQ ID NO:60; a thymine or its complement at a position corresponding to position 353 set forth in SEQ ID NO:61; a thymine or its complement at a position corresponding to position 230 set forth in SEQ ID NO:62; a thymine or its complement at a position corresponding to position 236 set forth in SEQ ID NO:63; a thymine or its complement at a position corresponding to position 236 set forth in SEQ ID NO:64; a thymine or its complement at a position corresponding to position 604 set forth in SEQ ID NO:65; or a thymine or its complement at a position corresponding to position 126 set forth in SEQ ID NO:66; and d) detecting the detectable label.

[0144] 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 assay comprises contacting an SLC9A3R2 nucleic acid molecule or its complement in a biological sample with a mutation-specific probe comprising a detectable label, wherein the mutation-specific probe comprises a nucleotide sequence that hybridizes under stringent conditions to a nucleotide sequence of an SLC9A3R2 nucleic acid molecule or its complement comprising thymine or its complement at a position corresponding to position 9,519 set forth in SEQ ID NO:2; uracil or its complement at a position corresponding to position 615 set forth in SEQ ID NO:22; and / or thymine or its complement at a position corresponding to position 615 set forth in SEQ ID NO:59, and detecting the detectable label.

[0145] In some embodiments, the assay comprises contacting an SLC9A3R2 nucleic acid molecule or its complement in a biological sample with a mutation-specific probe comprising a detectable label, wherein the mutation-specific probe comprises a nucleotide sequence that hybridizes under stringent conditions to a nucleotide sequence of an SLC9A3R2 nucleic acid molecule or its complement comprising uracil or its complement at a position corresponding to position 589 set forth in SEQ ID NO: 23; and / or thymine or its complement at a position corresponding to position 589 set forth in SEQ ID NO: 60, and detecting the detectable label.

[0146] In some embodiments, the assay comprises contacting an SLC9A3R2 nucleic acid molecule or its complement in a biological sample with a mutation-specific probe comprising a detectable label, wherein the mutation-specific probe comprises a nucleotide sequence that hybridizes under stringent conditions to a nucleotide sequence of an SLC9A3R2 nucleic acid molecule or its complement comprising uracil or its complement at a position corresponding to position 353 set forth in SEQ ID NO:24; and / or thymine or its complement at a position corresponding to position 353 set forth in SEQ ID NO:61, and detecting the detectable label.

[0147] In some embodiments, the assay comprises contacting an SLC9A3R2 nucleic acid molecule or its complement in a biological sample with a mutation-specific probe comprising a detectable label, wherein the mutation-specific probe comprises a nucleotide sequence that hybridizes under stringent conditions to a nucleotide sequence of an SLC9A3R2 nucleic acid molecule or its complement comprising uracil or its complement at a position corresponding to position 230 set forth in SEQ ID NO:25; and / or thymine or its complement at a position corresponding to position 230 set forth in SEQ ID NO:62, and detecting the detectable label.

[0148] In some embodiments, the assay comprises contacting an SLC9A3R2 nucleic acid molecule or its complement in a biological sample with a mutation-specific probe comprising a detectable label, wherein the mutation-specific probe comprises a nucleotide sequence that hybridizes under stringent conditions to a nucleotide sequence of an SLC9A3R2 nucleic acid molecule or its complement comprising uracil or its complement at a position corresponding to position 236 set forth in SEQ ID NO:26; and / or thymine or its complement at a position corresponding to position 236 set forth in SEQ ID NO:63, and detecting the detectable label.

[0149] In some embodiments, the assay comprises contacting an SLC9A3R2 nucleic acid molecule or its complement in a biological sample with a mutation-specific probe comprising a detectable label, wherein the mutation-specific probe comprises a nucleotide sequence that hybridizes under stringent conditions to a nucleotide sequence of an SLC9A3R2 nucleic acid molecule or its complement comprising uracil or its complement at a position corresponding to position 236 set forth in SEQ ID NO:27; and / or thymine or its complement at a position corresponding to position 236 set forth in SEQ ID NO:64, and detecting the detectable label.

[0150] In some embodiments, the assay comprises contacting an SLC9A3R2 nucleic acid molecule or its complement in a biological sample with a mutation-specific probe comprising a detectable label, wherein the mutation-specific probe comprises a nucleotide sequence that hybridizes under stringent conditions to a nucleotide sequence of an SLC9A3R2 nucleic acid molecule or its complement comprising uracil or its complement at a position corresponding to position 604 set forth in SEQ ID NO:28; and / or thymine or its complement at a position corresponding to position 604 set forth in SEQ ID NO:65, and detecting the detectable label.

[0151] In some embodiments, the assay comprises contacting an SLC9A3R2 nucleic acid molecule or its complement in a biological sample with a mutation-specific probe comprising a detectable label, wherein the mutation-specific probe comprises a nucleotide sequence that hybridizes under stringent conditions to a nucleotide sequence of an SLC9A3R2 nucleic acid molecule or its complement comprising uracil or its complement at a position corresponding to position 126 set forth in SEQ ID NO:29; and / or thymine or its complement at a position corresponding to position 126 set forth in SEQ ID NO:66, and detecting the detectable label.

[0152] In some embodiments, the assay comprises contacting an SLC9A3R2 genomic nucleic acid molecule or its complement in a biological sample with a mutation-specific probe comprising a detectable label, wherein the mutation-specific probe comprises a nucleotide sequence that hybridizes under stringent conditions to a nucleotide sequence of the SLC9A3R2 genomic nucleic acid molecule or its complement comprising a thymine or its complement at a position corresponding to position 9,519 set forth in SEQ ID NO:2, and detecting the detectable label.

[0153] In some embodiments, the assay comprises contacting an SLC9A3R2 mRNA molecule or its complement in a biological sample with a mutation-specific probe comprising a detectable label, wherein the mutation-specific probe comprises a nucleotide sequence that hybridizes under stringent conditions to a nucleotide sequence of an SLC9A3R2 mRNA molecule or its complement comprising: uracil or its complement at a position corresponding to position 615 as set forth in SEQ ID NO:22; uracil or its complement at a position corresponding to position 589 as set forth in SEQ ID NO:23; uracil or its complement at a position corresponding to position 353 as set forth in SEQ ID NO:24; uracil or its complement at a position corresponding to position 230 as set forth in SEQ ID NO:25; uracil or its complement at a position corresponding to position 236 as set forth in SEQ ID NO:26; uracil or its complement at a position corresponding to position 236 as set forth in SEQ ID NO:27; uracil or its complement at a position corresponding to position 604 as set forth in SEQ ID NO:28; or uracil or its complement at a position corresponding to position 126 as set forth in SEQ ID NO:29; and detecting the detectable label.

[0154] In some embodiments, the assay comprises contacting a SLC9A3R2 cDNA molecule or its complement generated from an mRNA molecule in a biological sample with a mutation-specific probe comprising a detectable label, wherein the mutation-specific probe comprises a nucleotide sequence that hybridizes under stringent conditions to a nucleotide sequence of the SLC9A3R2 cDNA molecule or its complement comprising: a thymine or its complement at a position corresponding to position 615 set forth in SEQ ID NO:59; a thymine or its complement at a position corresponding to position 589 set forth in SEQ ID NO:60; a thymine or its complement at a position corresponding to position 353 set forth in SEQ ID NO:61; a thymine or its complement at a position corresponding to position 230 set forth in SEQ ID NO:62; a thymine or its complement at a position corresponding to position 236 set forth in SEQ ID NO:63; a thymine or its complement at a position corresponding to position 236 set forth in SEQ ID NO:64; a thymine or its complement at a position corresponding to position 604 set forth in SEQ ID NO:65; or a thymine or its complement at a position corresponding to position 126 set forth in SEQ ID NO:66; and detecting the detectable label.

[0155] In some embodiments, the SLC9A3R2 nucleic acid molecule is present in a cell obtained from the subject. Mutation-specific polymerase chain reaction techniques can be used to detect mutations such as SNPs in nucleotide sequences. Mutation-specific primers can be used because DNA polymerase will not extend if there is a mismatch with the template.

[0156] 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).

[0157] 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 identify polynucleotides comprising mutant genomic nucleic acid molecules, mutant mRNA molecules, or mutant cDNA molecules of SLC9A3R2. 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 the detection method of choice, 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 can have complete nucleotide sequence identity to consecutive nucleotides within the target nucleotide sequence, although probes that are different from the target nucleotide sequence and that retain the ability to specifically detect and / or identify the target nucleotide sequence can 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.

[0158] In some embodiments, to determine whether a SLC9A3R2 nucleic acid molecule (genomic nucleic acid molecule, mRNA molecule, or cDNA molecule) or its complement in a biological sample contains a nucleotide sequence that includes a thymine at a position corresponding to position 9,519 set forth in SEQ ID NO:2 (genomic nucleic acid molecule), a uracil at a position corresponding to position 615 set forth in SEQ ID NO:22 (mRNA molecule), or a thymine at a position corresponding to position 615 set forth in SEQ ID NO:59 (cDNA molecule), a thymine at a position corresponding to position 9,519 set forth in SEQ ID NO:2, a uracil at a position corresponding to position 615 set forth in SEQ ID NO:22, or a thymine at a position corresponding to position 615 set forth in SEQ ID NO:59 is detected. The biological sample can be subjected to an amplification method using a primer pair comprising a first primer derived from a 5' flanking sequence adjacent to a thymine and a second primer derived from a 3' flanking sequence adjacent to a thymine at a position corresponding to position 9,519 of SEQ ID NO:2, a uracil at a position corresponding to position 615 of SEQ ID NO:22, or a thymine at a position corresponding to position 615 of SEQ ID NO:59, to generate an amplicon indicative of the presence of a SNP at a position encoding a thymine at a position corresponding to position 9,519 of SEQ ID NO:2, a uracil at a position corresponding to position 615 of SEQ ID NO:22, or a thymine at a position corresponding to position 615 of SEQ ID NO:59. In some embodiments, the length of the amplicon can range from the combination of 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, or up to about 20,000 nucleotide base pairs. Optionally, the primer pair flanks a region that includes a thymine at a position corresponding to position 9,519 in SEQ ID NO:2, a uracil at a position corresponding to position 615 in SEQ ID NO:22, or a thymine at a position corresponding to position 615 in SEQ ID NO:59, 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 thymine at a position corresponding to position 9,519 in SEQ ID NO:2, a uracil at a position corresponding to position 615 in SEQ ID NO:22, or a thymine at a position corresponding to position 615 in SEQ ID NO:59.

[0159] In some embodiments, to determine whether an SLC9A3R2 nucleic acid molecule (genomic nucleic acid molecule, mRNA molecule, or cDNA molecule) or its complement in a biological sample contains a nucleotide sequence comprising uracil at a position corresponding to position 589 of SEQ ID NO:23 (mRNA molecule) or thymine at a position corresponding to position 589 of SEQ ID NO:60 (cDNA molecule), the biological sample can be subjected to an amplification method using a primer pair comprising a first primer derived from a 5' flanking sequence adjacent to the uracil at a position corresponding to position 589 of SEQ ID NO:23 or thymine at a position corresponding to position 589 of SEQ ID NO:60 and a second primer derived from a 3' flanking sequence adjacent to the uracil at a position corresponding to position 589 of SEQ ID NO:23 or thymine at a position corresponding to position 589 of SEQ ID NO:60 to generate an amplicon indicative of the presence of a SNP at a position encoding uracil at a position corresponding to position 589 of SEQ ID NO:23 or thymine at a position corresponding to position 589 of SEQ ID NO:60. In some embodiments, the length of the amplicon can range from the combination of the length of the primer pair plus one nucleotide base pair to any length of the amplicon that can be produced by the DNA amplification protocol. This distance can range from one nucleotide base pair to the limit of the amplification reaction, or up to about 20,000 nucleotide base pairs. Optionally, the primer pair flanks a region that includes a position that includes a uracil at position corresponding to position 589 of SEQ ID NO:23 or a thymine at position corresponding to position 589 of SEQ ID NO:60, 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 uracil at position corresponding to position 589 of SEQ ID NO:23 or a thymine at position corresponding to position 589 of SEQ ID NO:60.

[0160] In some embodiments, to determine whether an SLC9A3R2 nucleic acid molecule (genomic nucleic acid molecule, mRNA molecule, or cDNA molecule) or its complement in a biological sample contains a nucleotide sequence comprising uracil at a position corresponding to position 353 set forth in SEQ ID NO:24 (mRNA molecule) or thymine at a position corresponding to position 353 set forth in SEQ ID NO:61 (cDNA molecule), the biological sample can be subjected to an amplification method using a primer pair comprising a first primer derived from a 5' flanking sequence adjacent to the uracil at a position corresponding to position 353 set forth in SEQ ID NO:24 or thymine at a position corresponding to position 353 set forth in SEQ ID NO:61 and a second primer derived from a 3' flanking sequence adjacent to the uracil at a position corresponding to position 353 set forth in SEQ ID NO:24 or thymine at a position corresponding to position 353 set forth in SEQ ID NO:61 to generate an amplicon indicative of the presence of a SNP at a position encoding uracil at a position corresponding to position 353 set forth in SEQ ID NO:24 or thymine at a position corresponding to position 353 set forth in SEQ ID NO:61. In some embodiments, the length of the amplicon can range from the combination of the length of the primer pair plus one nucleotide base pair to any length of the amplicon that can be produced by the DNA amplification protocol. This distance can range from one nucleotide base pair to the limit of the amplification reaction, or up to about 20,000 nucleotide base pairs. Optionally, the primer pair flanks a region that includes a position that includes a uracil at position corresponding to position 353 of SEQ ID NO:24 or a thymine at position corresponding to position 353 of SEQ ID NO:61, 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 uracil at position corresponding to position 353 of SEQ ID NO:24 or a thymine at position corresponding to position 353 of SEQ ID NO:61.

[0161] In some embodiments, to determine whether an SLC9A3R2 nucleic acid molecule (genomic nucleic acid molecule, mRNA molecule, or cDNA molecule) or its complement in a biological sample contains a nucleotide sequence comprising uracil at a position corresponding to position 230 set forth in SEQ ID NO:25 (mRNA molecule) or thymine at a position corresponding to position 230 set forth in SEQ ID NO:62 (cDNA molecule), the biological sample can be subjected to an amplification method using a primer pair comprising a first primer derived from a 5'-flanking sequence adjacent to the uracil at a position corresponding to position 230 set forth in SEQ ID NO:25 or thymine at a position corresponding to position 230 set forth in SEQ ID NO:62 and a second primer derived from a 3'-flanking sequence adjacent to the uracil at a position corresponding to position 230 set forth in SEQ ID NO:25 or thymine at a position corresponding to position 230 set forth in SEQ ID NO:62 to generate an amplicon indicative of the presence of a SNP at a position encoding uracil at a position corresponding to position 230 set forth in SEQ ID NO:25 or thymine at a position corresponding to position 230 set forth in SEQ ID NO:62. In some embodiments, the length of the amplicon can range from the combination of the length of the primer pair plus one nucleotide base pair to any length of the amplicon that can be produced by the DNA amplification protocol. This distance can range from one nucleotide base pair to the limit of the amplification reaction, or up to about 20,000 nucleotide base pairs. Optionally, the primer pair flanks a region that includes a position that includes a uracil at position corresponding to position 230 of SEQ ID NO:25 or a thymine at position corresponding to position 230 of SEQ ID NO:62, 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 uracil at position corresponding to position 230 of SEQ ID NO:25 or a thymine at position corresponding to position 230 of SEQ ID NO:62.

[0162] In some embodiments, to determine whether a SLC9A3R2 nucleic acid molecule (genomic nucleic acid molecule, mRNA molecule, or cDNA molecule) or its complement in a biological sample comprises a nucleotide sequence, uracil at a position corresponding to position 236 set forth in SEQ ID NO:26 (mRNA molecule), or thymine at a position corresponding to position 236 set forth in SEQ ID NO:63 (cDNA molecule), the biological sample can be subjected to an amplification method using a primer pair comprising a first primer derived from a 5' flanking sequence adjacent to the uracil at a position corresponding to position 236 set forth in SEQ ID NO:26, or thymine at a position corresponding to position 236 set forth in SEQ ID NO:63, and a second primer derived from a 3' flanking sequence adjacent to the uracil at a position corresponding to position 236 set forth in SEQ ID NO:26, or thymine at a position corresponding to position 236 set forth in SEQ ID NO:63, to generate an amplicon indicative of the presence of a SNP at a position encoding the uracil at a position corresponding to position 236 set forth in SEQ ID NO:26, or thymine at a position corresponding to position 236 set forth in SEQ ID NO:63. In some embodiments, the length of the amplicon can range from the combination of the length of the primer pair plus one nucleotide base pair to any length of the amplicon that can be produced by the DNA amplification protocol. This distance can range from one nucleotide base pair to the limit of the amplification reaction, or up to about 20,000 nucleotide base pairs. Optionally, the primer pair flanks a region that includes a position that includes a uracil at position corresponding to position 236 of SEQ ID NO:26 or a thymine at position corresponding to position 236 of SEQ ID NO:63, 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 uracil at position corresponding to position 236 of SEQ ID NO:26 or a thymine at position corresponding to position 236 of SEQ ID NO:63.

[0163] In some embodiments, to determine whether an SLC9A3R2 nucleic acid molecule (genomic nucleic acid molecule, mRNA molecule, or cDNA molecule) or its complement in a biological sample contains a nucleotide sequence comprising uracil at a position corresponding to position 236 set forth in SEQ ID NO:27 (mRNA molecule) or thymine at a position corresponding to position 236 set forth in SEQ ID NO:64 (cDNA molecule), the biological sample can be subjected to an amplification method using a primer pair comprising a first primer derived from a 5'-flanking sequence adjacent to the uracil at a position corresponding to position 236 set forth in SEQ ID NO:27 or thymine at a position corresponding to position 236 set forth in SEQ ID NO:64 and a second primer derived from a 3'-flanking sequence adjacent to the uracil at a position corresponding to position 236 set forth in SEQ ID NO:27 or thymine at a position corresponding to position 236 set forth in SEQ ID NO:64 to generate an amplicon indicative of the presence of a SNP at a position encoding uracil at a position corresponding to position 236 set forth in SEQ ID NO:27 or thymine at a position corresponding to position 236 set forth in SEQ ID NO:64. In some embodiments, the length of the amplicon can range from the combination of the length of the primer pair plus one nucleotide base pair to any length of the amplicon that can be produced by the DNA amplification protocol. This distance can range from one nucleotide base pair to the limit of the amplification reaction, or up to about 20,000 nucleotide base pairs. Optionally, the primer pair flanks a region that includes a position that includes a uracil at position corresponding to position 236 of SEQ ID NO:27 or a thymine at position corresponding to position 236 of SEQ ID NO:64, 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 uracil at position corresponding to position 236 of SEQ ID NO:27 or a thymine at position corresponding to position 236 of SEQ ID NO:64.

[0164] In some embodiments, to determine whether a SLC9A3R2 nucleic acid molecule (genomic nucleic acid molecule, mRNA molecule, or cDNA molecule) or its complement in a biological sample comprises a nucleotide sequence, uracil at a position corresponding to position 604 set forth in SEQ ID NO:28 (mRNA molecule), or thymine at a position corresponding to position 604 set forth in SEQ ID NO:65 (cDNA molecule), the biological sample can be subjected to an amplification method using a primer pair comprising a first primer derived from a 5' flanking sequence adjacent to the uracil at a position corresponding to position 604 set forth in SEQ ID NO:28, or thymine at a position corresponding to position 604 set forth in SEQ ID NO:65, and a second primer derived from a 3' flanking sequence adjacent to the uracil at a position corresponding to position 604 set forth in SEQ ID NO:28, or thymine at a position corresponding to position 604 set forth in SEQ ID NO:65, to generate an amplicon indicative of the presence of a SNP at a position encoding the uracil at a position corresponding to position 604 set forth in SEQ ID NO:28, or thymine at a position corresponding to position 604 set forth in SEQ ID NO:65. In some embodiments, the length of the amplicon can range from the combination of the length of the primer pair plus one nucleotide base pair to any length of the amplicon that can be produced by the DNA amplification protocol. This distance can range from one nucleotide base pair to the limit of the amplification reaction, or up to about 20,000 nucleotide base pairs. Optionally, the primer pair flanks a region that includes a position that includes a uracil at position corresponding to position 604 of SEQ ID NO:28 or a thymine at position corresponding to position 604 of SEQ ID NO:65, 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 uracil at position corresponding to position 604 of SEQ ID NO:28 or a thymine at position corresponding to position 604 of SEQ ID NO:65.

[0165] In some embodiments, to determine whether an SLC9A3R2 nucleic acid molecule (genomic nucleic acid molecule, mRNA molecule, or cDNA molecule) or its complement in a biological sample contains a nucleotide sequence comprising uracil at a position corresponding to position 126 set forth in SEQ ID NO:29 (mRNA molecule) or thymine at a position corresponding to position 126 set forth in SEQ ID NO:66 (cDNA molecule), the biological sample can be subjected to an amplification method using a primer pair comprising a first primer derived from a 5'-flanking sequence adjacent to the uracil at a position corresponding to position 126 set forth in SEQ ID NO:29 or thymine at a position corresponding to position 126 set forth in SEQ ID NO:66 and a second primer derived from a 3'-flanking sequence adjacent to the uracil at a position corresponding to position 126 set forth in SEQ ID NO:29 or thymine at a position corresponding to position 126 set forth in SEQ ID NO:66 to generate an amplicon indicative of the presence of a SNP at a position encoding uracil at a position corresponding to position 126 set forth in SEQ ID NO:29 or thymine at a position corresponding to position 126 set forth in SEQ ID NO:66. In some embodiments, the length of the amplicon can range from the combination of the length of the primer pair plus one nucleotide base pair to any length of the amplicon that can be produced by the DNA amplification protocol. This distance can range from one nucleotide base pair to the limit of the amplification reaction, or up to about 20,000 nucleotide base pairs. Optionally, the primer pair flanks a region that includes a position that includes a uracil at position corresponding to position 126 of SEQ ID NO:29 or a thymine at position corresponding to position 126 of SEQ ID NO:66, 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 uracil at position corresponding to position 126 of SEQ ID NO:29 or a thymine at position corresponding to position 126 of SEQ ID NO:66.

[0166] Similar amplicons can be generated from mRNA and / or cDNA sequences. PCR primer pairs can be derived from known sequences, for example, 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 inspected and primers manually specified using known guidelines.

[0167] 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).

[0168] Hybridization techniques can employ stringent conditions so that the probe or primer specifically hybridizes with its target. In some embodiments, a polynucleotide primer or probe under stringent conditions will hybridize to its target sequence detectably higher than other non-target sequences, for example, at least 2-fold, at least 3-fold, at least 4-fold or more above background, including more than 10-fold above background. In some embodiments, a polynucleotide primer or probe under stringent conditions will hybridize to its target nucleotide sequence detectably higher than other nucleotide sequences at least 2-fold. In some embodiments, a polynucleotide primer or probe under stringent conditions will hybridize to its target nucleotide sequence detectably higher than other nucleotide sequences at least 3-fold. In some embodiments, a polynucleotide primer or probe under stringent conditions will hybridize to its target nucleotide sequence detectably higher than other nucleotide sequences at least 4-fold. 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, more than 10-fold above background. Stringent conditions are sequence-dependent and will be different in different circumstances.

[0169] 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 + The conditions will be ionic concentration (or other salts) and 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 the addition of 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 the wash will be at least long enough to reach equilibrium.

[0170] The present disclosure also provides a method for detecting the presence of a predicted loss-of-function polypeptide in SLC9A3R2, comprising performing an assay on a biological sample obtained from a subject to determine whether the SLC9A3R2 polypeptide in the biological sample 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). The predicted loss-of-function polypeptide in SLC9A3R2 can be any of the predicted loss-of-function polypeptides in SLC9A3R2 described herein. In some embodiments, the method detects the presence of SLC9A3R2 Arg171Trp-Long, Arg171Trp-Short, Arg65Trp, Arg58Trp, Arg60Trp-Short, Arg60Trp-Long, or Arg170Trp. In some embodiments, the method detects the presence of SLC9A3R2 Arg171Trp-Long or Arg171Trp-Short.

[0171] In some embodiments, the method includes performing an assay on a biological sample obtained from the subject to determine whether an SLC9A3R2 polypeptide in the biological sample contains tryptophan at a position corresponding to position 171 set forth in SEQ ID NO: 86; tryptophan at a position corresponding to position 171 set forth in SEQ ID NO: 87; tryptophan at a position corresponding to position 65 set forth in SEQ ID NO: 88; tryptophan at a position corresponding to position 58 set forth in SEQ ID NO: 89; tryptophan at a position corresponding to position 60 set forth in SEQ ID NO: 90; tryptophan at a position corresponding to position 60 set forth in SEQ ID NO: 91; or tryptophan at a position corresponding to position 60 set forth in SEQ ID NO: 92.

[0172] In some embodiments, the assay comprises sequencing at least a portion of an SLC9A3R2 polypeptide comprising a position corresponding to position 171 set forth in SEQ ID NO:86 or SEQ ID NO:77; position 171 set forth in SEQ ID NO:87 or SEQ ID NO:78; position 65 set forth in SEQ ID NO:88 or SEQ ID NO:79; position 58 set forth in SEQ ID NO:89 or SEQ ID NO:80; position 60 set forth in SEQ ID NO:90 or SEQ ID NO:81; position 60 set forth in SEQ ID NO:91 or SEQ ID NO:82; or position 60 set forth in SEQ ID NO:92 or SEQ ID NO:83.

[0173] In some embodiments, the assay is an immunoassay that detects the presence of an SLC9A3R2 polypeptide comprising a position corresponding to position 171 set forth in SEQ ID NO:86 or SEQ ID NO:77; position 171 set forth in SEQ ID NO:87 or SEQ ID NO:78; position 65 set forth in SEQ ID NO:88 or SEQ ID NO:79; position 58 set forth in SEQ ID NO:89 or SEQ ID NO:80; position 60 set forth in SEQ ID NO:90 or SEQ ID NO:81; position 60 set forth in SEQ ID NO:91 or SEQ ID NO:82; or position 60 set forth in SEQ ID NO:92 or SEQ ID NO:83.

[0174] In some embodiments, if the subject does not have a SLC9A3R2 predicted loss-of-function polypeptide, the subject has an increased risk of developing hypertension, coronary heart disease and / or atrial fibrillation, or any of primary hypertension, secondary hypertension, resistant hypertension, or malignant hypertension. In some embodiments, if the subject has a SLC9A3R2 predicted loss-of-function polypeptide, the subject has a reduced risk of developing hypertension, coronary heart disease and / or atrial fibrillation, or any of primary hypertension, secondary hypertension, resistant hypertension, or malignant hypertension.

[0175] The present disclosure also provides isolated nucleic acid molecules that hybridize with SLC9A3R2 missense variant genomic nucleic acid molecules, SLC9A3R2 missense variant mRNA molecules, and / or SLC9A3R2 missense variant cDNA molecules (e.g., any of the genomic missense variant nucleic acid molecules, mRNA missense variant molecules, and cDNA missense variant molecules disclosed herein). In some embodiments, such isolated nucleic acid molecules hybridize with SLC9A3R2 missense variant nucleic acid molecules under stringent conditions. Such nucleic acid molecules can be used, for example, as probes, primers, mutation-specific probes, or mutation-specific primers as described or exemplified herein.

[0176] In some embodiments, the isolated nucleic acid molecule hybridizes to a portion of an SLC9A3R2 missense nucleic acid molecule that includes a position corresponding to position 9,519 set forth in SEQ ID NO:2, position 615 set forth in SEQ ID NO:22, or position 615 set forth in SEQ ID NO:59. In some embodiments, the isolated nucleic acid molecule hybridizes to a portion of an SLC9A3R2 missense nucleic acid molecule that includes a position corresponding to position 589 set forth in SEQ ID NO:23, or position 589 set forth in SEQ ID NO:60. In some embodiments, the isolated nucleic acid molecule hybridizes to a portion of an SLC9A3R2 missense nucleic acid molecule that includes a position corresponding to position 353 set forth in SEQ ID NO:24, or position 353 set forth in SEQ ID NO:61. In some embodiments, the isolated nucleic acid molecule hybridizes to a portion of an SLC9A3R2 missense nucleic acid molecule that includes a position corresponding to position 230 set forth in SEQ ID NO:25, or position 230 set forth in SEQ ID NO:62. In some embodiments, the isolated nucleic acid molecule hybridizes to a portion of an SLC9A3R2 missense nucleic acid molecule that includes position 236 as set forth in SEQ ID NO:26 or a position corresponding to position 236 as set forth in SEQ ID NO:63. In some embodiments, the isolated nucleic acid molecule hybridizes to a portion of an SLC9A3R2 missense nucleic acid molecule that includes position 236 as set forth in SEQ ID NO:27 or a position corresponding to position 236 as set forth in SEQ ID NO:64. In some embodiments, the isolated nucleic acid molecule hybridizes to a portion of an SLC9A3R2 missense nucleic acid molecule that includes position 604 as set forth in SEQ ID NO:28 or a position corresponding to position 604 as set forth in SEQ ID NO:65. In some embodiments, the isolated nucleic acid molecule hybridizes to a portion of an SLC9A3R2 missense nucleic acid molecule that includes position 126 as set forth in SEQ ID NO:29 or a position corresponding to position 126 as set forth in SEQ ID NO:66.

[0177] 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.

[0178] In some embodiments, the isolated mutation-specific probe or primer comprises at least about 15 nucleotides, wherein the mutation-specific probe or primer comprises a nucleotide sequence that is complementary to a nucleotide sequence of a portion of an SLC9A3R2 missense nucleic acid molecule encoding a predicted loss-of-function polypeptide of solute transporter family 9 isoform A3 regulator 2, or its complement. In some embodiments, the portion comprises a position corresponding to position 9,519 or its complement as set forth in SEQ ID NO:2; position 615 or its complement as set forth in SEQ ID NO:22; or position 615 or its complement as set forth in SEQ ID NO:59. In some embodiments, the portion comprises a position corresponding to position 589 or its complement as set forth in SEQ ID NO:23; or position 589 or its complement as set forth in SEQ ID NO:60. In some embodiments, the portion comprises a position corresponding to position 353 or its complement as set forth in SEQ ID NO:24; or position 353 or its complement as set forth in SEQ ID NO:61. In some embodiments, the portion includes a position corresponding to position 230 set forth in SEQ ID NO:25, or its complement; or to position 230 set forth in SEQ ID NO:62, or its complement. In some embodiments, the portion includes a position corresponding to position 236 set forth in SEQ ID NO:26, or its complement; or to position 236 set forth in SEQ ID NO:63, or its complement. In some embodiments, the portion includes a position corresponding to position 236 set forth in SEQ ID NO:27, or its complement; or to position 236 set forth in SEQ ID NO:64, or its complement. In some embodiments, the portion includes a position corresponding to position 604 set forth in SEQ ID NO:28, or its complement; or to position 604 set forth in SEQ ID NO:65, or its complement. In some embodiments, the portion includes a position corresponding to position 126 set forth in SEQ ID NO:29, or its complement; or to position 126 set forth in SEQ ID NO:66, or its complement.

[0179] 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 a SLC9A3R2 missense variant genomic nucleic acid molecule, a SLC9A3R2 missense variant mRNA molecule, and / or a SLC9A3R2 missense 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.

[0180] In some embodiments, the isolated mutation-specific probe or primer comprises at least about 15 nucleotides, wherein the mutation-specific probe or primer comprises a nucleotide sequence that is complementary to a portion of a nucleotide sequence of an SLC9A3R2 missense mutant nucleic acid molecule encoding a predicted loss-of-function polypeptide in SLC9A3R2, the portion comprising a position corresponding to position 9,519 or its complement as set forth in SEQ ID NO:2; position 615 or its complement as set forth in SEQ ID NO:22; or position 615 or its complement as set forth in SEQ ID NO:59. In some embodiments, the portion comprises positions 9,519-9,521 or their complement as set forth in SEQ ID NO:2; positions 615-617 or their complement as set forth in SEQ ID NO:22; and / or positions 615-617 or their complement as set forth in SEQ ID NO:59.

[0181] In some embodiments, the isolated mutation-specific probe or mutation-specific primer comprises at least about 15 nucleotides, wherein the mutation-specific probe or mutation-specific primer comprises a nucleotide sequence that is complementary to a portion of the nucleotide sequence of an SLC9A3R2 missense mutant nucleic acid molecule encoding a predicted loss-of-function polypeptide in SLC9A3R2, the portion comprising position 589 or its complement as set forth in SEQ ID NO:23; or a position corresponding to position 589 or its complement as set forth in SEQ ID NO:60. In some embodiments, the portion comprises positions 589-591 or their complement as set forth in SEQ ID NO:23; and / or positions corresponding to positions 589-591 or their complement as set forth in SEQ ID NO:60.

[0182] In some embodiments, the isolated mutation-specific probe or mutation-specific primer comprises at least about 15 nucleotides, wherein the mutation-specific probe or mutation-specific primer comprises a nucleotide sequence that is complementary to a portion of the nucleotide sequence of an SLC9A3R2 missense mutant nucleic acid molecule encoding a predicted loss-of-function polypeptide in SLC9A3R2, the portion comprising position 353 set forth in SEQ ID NO:24 or its complement; or a position corresponding to position 353 set forth in SEQ ID NO:61 or its complement. In some embodiments, the portion comprises positions 353-355 set forth in SEQ ID NO:24 or its complement; and / or positions corresponding to positions 353-355 set forth in SEQ ID NO:61 or its complement.

[0183] In some embodiments, the isolated mutation-specific probe or primer comprises at least about 15 nucleotides, wherein the mutation-specific probe or primer comprises a nucleotide sequence that is complementary to a portion of the nucleotide sequence of an SLC9A3R2 missense mutant nucleic acid molecule encoding a predicted loss-of-function polypeptide in SLC9A3R2, the portion comprising position 230 set forth in SEQ ID NO:25 or its complement; or a position corresponding to position 230 set forth in SEQ ID NO:62 or its complement. In some embodiments, the portion comprises positions 230-232 set forth in SEQ ID NO:25 or its complement; and / or positions corresponding to positions 230-232 set forth in SEQ ID NO:62 or its complement.

[0184] In some embodiments, the isolated mutation-specific probe or primer comprises at least about 15 nucleotides, wherein the mutation-specific probe or primer comprises a nucleotide sequence that is complementary to a portion of the nucleotide sequence of an SLC9A3R2 missense mutant nucleic acid molecule encoding a predicted loss-of-function polypeptide in SLC9A3R2, the portion comprising position 236 set forth in SEQ ID NO:26 or its complement; or a position corresponding to position 236 set forth in SEQ ID NO:63 or its complement. In some embodiments, the portion comprises positions 236-238 set forth in SEQ ID NO:26 or its complement; and / or positions corresponding to positions 236-238 set forth in SEQ ID NO:63 or its complement.

[0185] In some embodiments, the isolated mutation-specific probe or mutation-specific primer comprises at least about 15 nucleotides, wherein the mutation-specific probe or mutation-specific primer comprises a nucleotide sequence that is complementary to a portion of the nucleotide sequence of an SLC9A3R2 missense mutant nucleic acid molecule encoding a predicted loss-of-function polypeptide in SLC9A3R2, the portion comprising position 236 set forth in SEQ ID NO:27 or its complement; or a position corresponding to position 236 set forth in SEQ ID NO:64 or its complement. In some embodiments, the portion comprises positions 236-238 set forth in SEQ ID NO:27 or its complement; and / or positions corresponding to positions 236-238 set forth in SEQ ID NO:64 or its complement.

[0186] In some embodiments, the isolated mutation-specific probe or mutation-specific primer comprises at least about 15 nucleotides, wherein the mutation-specific probe or mutation-specific primer comprises a nucleotide sequence that is complementary to a portion of the nucleotide sequence of an SLC9A3R2 missense mutant nucleic acid molecule encoding a predicted loss-of-function polypeptide in SLC9A3R2, the portion comprising position 604 set forth in SEQ ID NO:28 or its complement; or a position corresponding to position 604 set forth in SEQ ID NO:65 or its complement. In some embodiments, the portion comprises positions 604-606 set forth in SEQ ID NO:28 or its complement; and / or positions 604-606 set forth in SEQ ID NO:65 or its complement.

[0187] In some embodiments, the isolated mutation-specific probe or mutation-specific primer comprises at least about 15 nucleotides, wherein the mutation-specific probe or mutation-specific primer comprises a nucleotide sequence that is complementary to a portion of the nucleotide sequence of an SLC9A3R2 missense mutant nucleic acid molecule encoding a predicted loss-of-function polypeptide in SLC9A3R2, the portion comprising position 126 set forth in SEQ ID NO:29 or its complement; or a position corresponding to position 126 set forth in SEQ ID NO:66 or its complement. In some embodiments, the portion comprises positions 126-128 set forth in SEQ ID NO:29 or its complement; and / or positions corresponding to positions 126-128 set forth in SEQ ID NO:66 or its complement.

[0188] In some embodiments, the isolated mutation-specific probe or mutation-specific primer comprises at least about 15 nucleotides, wherein the mutation-specific probe or mutation-specific primer comprises a nucleotide sequence that is complementary to a nucleotide sequence of a portion of a SLC9A3R2 missense nucleic acid molecule, or a complement thereof, that encodes a predicted loss-of-function polypeptide for solute transporter family 9 isoform A3 regulator 2. In some embodiments, the portion comprises a position corresponding to position 9,519 set forth in SEQ ID NO:2, or a complement thereof.

[0189] In some embodiments, the portion includes positions corresponding to positions 9,519 to 9,521 set forth in SEQ ID NO:2, or their complements. In some embodiments, the portion includes a position corresponding to position 615 set forth in SEQ ID NO:22, or its complement; position 589 set forth in SEQ ID NO:23, or its complement; position 353 set forth in SEQ ID NO:24, or its complement; position 230 set forth in SEQ ID NO:25, or its complement; position 236 set forth in SEQ ID NO:26, or its complement; position 236 set forth in SEQ ID NO:27, or its complement; position 604 set forth in SEQ ID NO:28, or its complement; or position 126 set forth in SEQ ID NO:29, or its complement.

[0190] In some embodiments, the portion includes a position corresponding to positions 615 to 617 set forth in SEQ ID NO:22, or their complement; positions 589 to 591 set forth in SEQ ID NO:23, or their complement; positions 353 to 355 set forth in SEQ ID NO:24, or their complement; positions 230 to 232 set forth in SEQ ID NO:25, or their complement; positions 236 to 238 set forth in SEQ ID NO:26, or their complement; positions 236 to 238 set forth in SEQ ID NO:27, or their complement; positions 604 to 606 set forth in SEQ ID NO:28, or their complement; or positions 126 to 128 set forth in SEQ ID NO:29, or their complement.

[0191] In some embodiments, the portion includes a position corresponding to position 615 set forth in SEQ ID NO:59, or its complement; position 589 set forth in SEQ ID NO:60, or its complement; position 353 set forth in SEQ ID NO:61, or its complement; position 230 set forth in SEQ ID NO:62, or its complement; position 236 set forth in SEQ ID NO:63, or its complement; position 236 set forth in SEQ ID NO:64, or its complement; position 604 set forth in SEQ ID NO:65, or its complement; or position 126 set forth in SEQ ID NO:66, or its complement.

[0192] In some embodiments, the portion includes positions corresponding to positions 615 to 617 set forth in SEQ ID NO:59, or their complement; positions 589 to 591 set forth in SEQ ID NO:60, or their complement; positions 353 to 355 set forth in SEQ ID NO:61, or their complement; positions 230 to 232 set forth in SEQ ID NO:62, or their complement; positions 236 to 238 set forth in SEQ ID NO:63, or their complement; positions 236 to 238 set forth in SEQ ID NO:64, or their complement; positions 604 to 606 set forth in SEQ ID NO:65, or their complement; or positions 126 to 128 set forth in SEQ ID NO:66, or their complement.

[0193] In some embodiments, the mutation-specific probe and the mutation-specific primer comprise DNA. In some embodiments, the mutation-specific probe and the mutation-specific primer comprise RNA.

[0194] In some embodiments, the probes and primers described herein (including mutation-specific probes and mutation-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.

[0195] In some embodiments, the primers can be used in second generation or high throughput sequencing, including mutation specific primers. Sometimes the primers can be modified, including mutation specific primers. In particular, the 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 several 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 typically 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 steps. Fluorescently labeled degenerate nonamer oligonucleotides are used in the detection step. The adapters can contain 5'-biotin tags for immobilizing the DNA library on streptavidin-coated beads.

[0196] The probes and primers described herein can be used to detect nucleotide variations within any of the SLC9A3R2 missense variant genomic nucleic acid molecules, SLC9A3R2 missense variant mRNA molecules, and / or SLC9A3R2 missense variant cDNA molecules disclosed herein. The primers described herein can be used to amplify SLC9A3R2 missense variant genomic nucleic acid molecules, SLC9A3R2 missense variant mRNA molecules, or SLC9A3R2 missense variant cDNA molecules, or fragments thereof.

[0197] The present disclosure also provides a pair of primers comprising any of the primers described above. For example, if one of the 3' ends of the primer hybridizes with cytosine (rather than thymine) at the position corresponding to position 9,519 of SEQ ID NO:1 in a particular SLC9A3R2 nucleic acid molecule, the presence of an amplified fragment indicates the presence of a SLC9A3R2 reference genomic nucleic acid molecule. Conversely, if one of the 3' ends of the primer hybridizes with thymine (rather than cytosine) at the position corresponding to position 9,519 of SEQ ID NO:2 in a particular SLC9A3R2 nucleic acid molecule, the presence of an amplified fragment indicates the presence of a SLC9A3R2 missense variant genomic nucleic acid molecule. In some embodiments, the nucleotide of the primer complementary to the thymine at the position corresponding to position 9,519 of SEQ ID NO:2 can be at the 3' end of the primer. In addition, if one of the 3' ends of the primer hybridizes to cytosine (rather than uracil) at a position corresponding to position 615 of SEQ ID NO:3 in a particular SLC9A3R2 nucleic acid molecule, the presence of the amplified fragment indicates the presence of an SLC9A3R2-based mRNA molecule. Conversely, if one of the 3' ends of the primer hybridizes to uracil (rather than cytosine) at a position corresponding to position 615 of SEQ ID NO:22 in a particular SLC9A3R2 mRNA molecule, the presence of the amplified fragment indicates the presence of an SLC9A3R2 missense variant mRNA molecule. In some embodiments, the nucleotide of the primer complementary to uracil at a position corresponding to position 615 of SEQ ID NO:22 can be at the 3' end of the primer. In addition, if one of the 3' ends of the primer hybridizes to cytosine (rather than thymine) at a position corresponding to position 615 of SEQ ID NO:40 in a particular SLC9A3R2 nucleic acid molecule, the presence of the amplified fragment indicates the presence of an SLC9A3R2-based cDNA molecule. Conversely, if one of the 3' ends of the primer hybridizes to thymine (but not cytosine) at a position corresponding to position 615 of SEQ ID NO:59 in a particular SLC9A3R2 cDNA molecule, the presence of an amplified fragment indicates the presence of a SLC9A3R2 missense mutant cDNA molecule.In some embodiments, the nucleotide of the primer complementary to the thymine at the position corresponding to position 615 set forth in SEQ ID NO:59 can be at the 3' end of the primer.

[0198] The present disclosure also provides a pair of primers comprising any of the primers described above. For example, if one of the 3' ends of the primer hybridizes with cytosine (rather than uracil) at a position corresponding to position 589 of SEQ ID NO: 4 in a particular SLC9A3R2 nucleic acid molecule, the presence of an amplified fragment indicates the presence of an SLC9A3R2 reference mRNA molecule. Conversely, if one of the 3' ends of the primer hybridizes with uracil (rather than cytosine) at a position corresponding to position 589 of SEQ ID NO: 23 in a particular SLC9A3R2 mRNA molecule, the presence of an amplified fragment indicates the presence of an SLC9A3R2 missense variant mRNA molecule. In some embodiments, the nucleotide of the primer complementary to uracil at a position corresponding to position 589 of SEQ ID NO: 23 can be at the 3' end of the primer. In addition, if one of the 3' ends of the primer hybridizes to a cytosine (rather than a thymine) at a position corresponding to position 589 of SEQ ID NO: 41 in a particular SLC9A3R2 nucleic acid molecule, the presence of an amplified fragment indicates the presence of a SLC9A3R2 reference cDNA molecule. Conversely, if one of the 3' ends of the primer hybridizes to a thymine (rather than a cytosine) at a position corresponding to position 589 of SEQ ID NO: 60 in a particular SLC9A3R2 cDNA molecule, the presence of an amplified fragment indicates the presence of a SLC9A3R2 missense variant cDNA molecule. In some embodiments, the nucleotide of the primer complementary to the thymine at the position corresponding to position 589 of SEQ ID NO: 60 can be at the 3' end of the primer.

[0199] The present disclosure also provides a pair of primers comprising any of the primers described above. For example, if one of the 3' ends of the primer hybridizes with cytosine (rather than uracil) at a position corresponding to position 353 of SEQ ID NO:5 in a particular SLC9A3R2 nucleic acid molecule, the presence of an amplified fragment indicates the presence of an SLC9A3R2 reference mRNA molecule. Conversely, if one of the 3' ends of the primer hybridizes with uracil (rather than cytosine) at a position corresponding to position 353 of SEQ ID NO:24 in a particular SLC9A3R2 mRNA molecule, the presence of an amplified fragment indicates the presence of an SLC9A3R2 missense variant mRNA molecule. In some embodiments, the nucleotide of the primer complementary to uracil at a position corresponding to position 353 of SEQ ID NO:24 can be at the 3' end of the primer. In addition, if one of the 3' ends of the primer hybridizes to a cytosine (rather than a thymine) at a position corresponding to position 353 set forth in SEQ ID NO: 42 in a particular SLC9A3R2 nucleic acid molecule, the presence of an amplified fragment indicates the presence of a SLC9A3R2 reference cDNA molecule. Conversely, if one of the 3' ends of the primer hybridizes to a thymine (rather than a cytosine) at a position corresponding to position 353 set forth in SEQ ID NO: 61 in a particular SLC9A3R2 cDNA molecule, the presence of an amplified fragment indicates the presence of a SLC9A3R2 missense variant cDNA molecule. In some embodiments, the nucleotide of the primer complementary to the thymine at the position corresponding to position 353 set forth in SEQ ID NO: 61 can be at the 3' end of the primer.

[0200] The present disclosure also provides a pair of primers comprising any of the primers described above. For example, if one of the 3' ends of the primer hybridizes with cytosine (rather than uracil) at the position corresponding to position 230 of SEQ ID NO:6 in a particular SLC9A3R2 nucleic acid molecule, the presence of an amplified fragment indicates the presence of an SLC9A3R2 reference mRNA molecule. Conversely, if one of the 3' ends of the primer hybridizes with uracil (rather than cytosine) at the position corresponding to position 230 of SEQ ID NO:25 in a particular SLC9A3R2 mRNA molecule, the presence of an amplified fragment indicates the presence of an SLC9A3R2 missense variant mRNA molecule. In some embodiments, the nucleotide of the primer complementary to uracil at the position corresponding to position 230 of SEQ ID NO:25 can be at the 3' end of the primer. In addition, if one of the 3' ends of the primer hybridizes to a cytosine (rather than a thymine) at a position corresponding to position 230 set forth in SEQ ID NO: 43 in a particular SLC9A3R2 nucleic acid molecule, the presence of an amplified fragment indicates the presence of a SLC9A3R2 reference cDNA molecule. Conversely, if one of the 3' ends of the primer hybridizes to a thymine (rather than a cytosine) at a position corresponding to position 230 set forth in SEQ ID NO: 62 in a particular SLC9A3R2 cDNA molecule, the presence of an amplified fragment indicates the presence of a SLC9A3R2 missense variant cDNA molecule. In some embodiments, the nucleotide of the primer complementary to the thymine at the position corresponding to position 230 set forth in SEQ ID NO: 62 can be at the 3' end of the primer.

[0201] The present disclosure also provides a pair of primers comprising any of the primers described above. For example, if one of the 3' ends of the primer hybridizes with cytosine (rather than uracil) at the position corresponding to position 236 of SEQ ID NO:7 in a particular SLC9A3R2 nucleic acid molecule, the presence of an amplified fragment indicates the presence of an SLC9A3R2 reference mRNA molecule. Conversely, if one of the 3' ends of the primer hybridizes with uracil (rather than cytosine) at the position corresponding to position 236 of SEQ ID NO:26 in a particular SLC9A3R2 mRNA molecule, the presence of an amplified fragment indicates the presence of an SLC9A3R2 missense variant mRNA molecule. In some embodiments, the nucleotide of the primer complementary to uracil at the position corresponding to position 236 of SEQ ID NO:26 can be at the 3' end of the primer. In addition, if one of the 3' ends of the primer hybridizes to a cytosine (rather than a thymine) at a position corresponding to position 236 set forth in SEQ ID NO: 44 in a particular SLC9A3R2 nucleic acid molecule, the presence of an amplified fragment indicates the presence of a SLC9A3R2 reference cDNA molecule. Conversely, if one of the 3' ends of the primer hybridizes to a thymine (rather than a cytosine) at a position corresponding to position 236 set forth in SEQ ID NO: 63 in a particular SLC9A3R2 cDNA molecule, the presence of an amplified fragment indicates the presence of a SLC9A3R2 missense variant cDNA molecule. In some embodiments, the nucleotide of the primer complementary to the thymine at the position corresponding to position 236 set forth in SEQ ID NO: 63 can be at the 3' end of the primer.

[0202] The present disclosure also provides a pair of primers comprising any of the primers described above. For example, if one of the 3' ends of the primer hybridizes with cytosine (rather than uracil) at a position corresponding to position 236 of SEQ ID NO:8 in a particular SLC9A3R2 nucleic acid molecule, the presence of an amplified fragment indicates the presence of an SLC9A3R2 reference mRNA molecule. Conversely, if one of the 3' ends of the primer hybridizes with uracil (rather than cytosine) at a position corresponding to position 236 of SEQ ID NO:27 in a particular SLC9A3R2 mRNA molecule, the presence of an amplified fragment indicates the presence of an SLC9A3R2 missense variant mRNA molecule. In some embodiments, the nucleotide of the primer complementary to uracil at a position corresponding to position 236 of SEQ ID NO:27 can be at the 3' end of the primer. In addition, if one of the 3' ends of the primer hybridizes to a cytosine (rather than a thymine) at a position corresponding to position 236 set forth in SEQ ID NO: 45 in a particular SLC9A3R2 nucleic acid molecule, the presence of an amplified fragment indicates the presence of a SLC9A3R2 reference cDNA molecule. Conversely, if one of the 3' ends of the primer hybridizes to a thymine (rather than a cytosine) at a position corresponding to position 236 set forth in SEQ ID NO: 64 in a particular SLC9A3R2 cDNA molecule, the presence of an amplified fragment indicates the presence of a SLC9A3R2 missense variant cDNA molecule. In some embodiments, the nucleotide of the primer complementary to the thymine at the position corresponding to position 236 set forth in SEQ ID NO: 64 can be at the 3' end of the primer.

[0203] The present disclosure also provides a pair of primers comprising any of the primers described above. For example, if one of the 3' ends of the primer hybridizes with cytosine (rather than uracil) at a position corresponding to position 604 of SEQ ID NO:9 in a particular SLC9A3R2 nucleic acid molecule, the presence of an amplified fragment indicates the presence of an SLC9A3R2 reference mRNA molecule. Conversely, if one of the 3' ends of the primer hybridizes with uracil (rather than cytosine) at a position corresponding to position 604 of SEQ ID NO:28 in a particular SLC9A3R2 mRNA molecule, the presence of an amplified fragment indicates the presence of an SLC9A3R2 missense variant mRNA molecule. In some embodiments, the nucleotide of the primer complementary to uracil at a position corresponding to position 604 of SEQ ID NO:28 can be at the 3' end of the primer. In addition, if one of the 3' ends of the primer hybridizes to a cytosine (rather than a thymine) at a position corresponding to position 604 of SEQ ID NO: 46 in a particular SLC9A3R2 nucleic acid molecule, the presence of an amplified fragment indicates the presence of a SLC9A3R2 reference cDNA molecule. Conversely, if one of the 3' ends of the primer hybridizes to a thymine (rather than a cytosine) at a position corresponding to position 604 of SEQ ID NO: 65 in a particular SLC9A3R2 cDNA molecule, the presence of an amplified fragment indicates the presence of a SLC9A3R2 missense variant cDNA molecule. In some embodiments, the nucleotide of the primer complementary to the thymine at a position corresponding to position 604 of SEQ ID NO: 65 can be at the 3' end of the primer.

[0204] The present disclosure also provides a pair of primers comprising any of the primers described above. For example, if one of the 3' ends of the primer hybridizes with cytosine (rather than uracil) at the position corresponding to position 126 of SEQ ID NO: 10 in a particular SLC9A3R2 nucleic acid molecule, the presence of an amplified fragment indicates the presence of an SLC9A3R2 reference mRNA molecule. Conversely, if one of the 3' ends of the primer hybridizes with uracil (rather than cytosine) at the position corresponding to position 126 of SEQ ID NO: 29 in a particular SLC9A3R2 mRNA molecule, the presence of an amplified fragment indicates the presence of an SLC9A3R2 missense variant mRNA molecule. In some embodiments, the nucleotide of the primer that is complementary to uracil at the position corresponding to position 126 of SEQ ID NO: 29 can be at the 3' end of the primer. In addition, if one of the 3' ends of the primer hybridizes to a cytosine (rather than a thymine) at a position corresponding to position 126 of SEQ ID NO: 47 in a particular SLC9A3R2 nucleic acid molecule, the presence of an amplified fragment indicates the presence of a SLC9A3R2 reference cDNA molecule. Conversely, if one of the 3' ends of the primer hybridizes to a thymine (rather than a cytosine) at a position corresponding to position 126 of SEQ ID NO: 66 in a particular SLC9A3R2 cDNA molecule, the presence of an amplified fragment indicates the presence of a SLC9A3R2 missense variant cDNA molecule. In some embodiments, the nucleotide of the primer complementary to the thymine at the position corresponding to position 126 of SEQ ID NO: 66 can be at the 3' end of the primer.

[0205] In the context of the present disclosure, "specifically hybridizes" means that a probe or primer (e.g., a mutation-specific probe or mutation-specific primer) does not hybridize to a nucleotide sequence encoding a SLC9A3R2-based genomic nucleic acid molecule, a SLC9A3R2-based mRNA molecule, and / or a SLC9A3R2-based cDNA molecule.

[0206] In any of the embodiments described throughout this disclosure, the probe (e.g., the mutation-specific probe) can include a label. In some embodiments, the label is a fluorescent label, a radioactive label, or biotin.

[0207] 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 a molecule, such as any of the probes disclosed herein, can associate. 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, usually containing one array per well. In some embodiments, the support is a microarray.

[0208] The present disclosure also provides a molecular complex comprising or consisting of any of the SLC9A3R2 missense nucleic acid molecules (genomic nucleic acid molecules, mRNA molecules, or cDNA molecules) described herein, or their complements, and any of the mutation-specific primers or mutation-specific probes described herein. In some embodiments, the SLC9A3R2 missense nucleic acid molecules (genomic nucleic acid molecules, mRNA molecules, or cDNA molecules) or their complements in the molecular complex are single-stranded. In some embodiments, the SLC9A3R2 missense nucleic acid molecule is any of the genomic nucleic acid molecules described herein. In some embodiments, the SLC9A3R2 missense nucleic acid molecule is any of the mRNA molecules described herein. In some embodiments, the SLC9A3R2 missense nucleic acid molecule is any of the cDNA molecules described herein. In some embodiments, the molecular complex comprises or consists of any of the SLC9A3R2 missense nucleic acid molecules (genomic nucleic acid molecules, mRNA molecules, or cDNA molecules) described herein, or their complements, and any of the mutation-specific primers described herein. In some embodiments, the molecular complex comprises or consists of any of the SLC9A3R2 missense nucleic acid molecules (genomic nucleic acid molecules, mRNA molecules, or cDNA molecules) described herein, or their complements, and any of the mutation-specific probes described herein.

[0209] In some embodiments, the molecular complex comprises a mutation-specific primer or a mutation-specific probe hybridized to an SLC9A3R2 genomic nucleic acid molecule encoding a loss-of-function polypeptide predicted for SLC9A3R2, wherein the mutation-specific primer or the mutation-specific probe hybridizes to the SLC9A3R2 genomic nucleic acid molecule at a position corresponding to position 9,519 set forth in SEQ ID NO:2 or its complement.

[0210] In some embodiments, the mutation-specific primer or the mutation-specific probe of the molecular complex hybridizes to the TGG codon at a position corresponding to positions 9,519-9,521 of SEQ ID NO:2.

[0211] In some embodiments, the genomic nucleic acid molecule of the molecular complex comprises SEQ ID NO:2. In some embodiments, the molecular complex comprises a mutation-specific primer or a mutation-specific probe hybridized to an SLC9A3R2 mRNA molecule encoding a predicted loss-of-function polypeptide of SLC9A3R2, wherein the mutation-specific primer or the mutation-specific probe hybridizes to the SLC9A3R2 mRNA molecule at a position corresponding to position 615 or its complement set forth in SEQ ID NO:22; position 589 or its complement set forth in SEQ ID NO:23; position 353 or its complement set forth in SEQ ID NO:24; position 230 or its complement set forth in SEQ ID NO:25; position 236 or its complement set forth in SEQ ID NO:26; position 236 or its complement set forth in SEQ ID NO:27; position 604 or its complement set forth in SEQ ID NO:28; or position 126 or its complement set forth in SEQ ID NO:29.

[0212] In some embodiments, the mutation-specific primer or mutation-specific probe of the molecular complex hybridizes to a UGG codon at a position corresponding to positions 615-617 of SEQ ID NO:22, a UGG codon at a position corresponding to positions 589-591 of SEQ ID NO:23, a UGG codon at a position corresponding to positions 353-355 of SEQ ID NO:24, a UGG codon at a position corresponding to positions 230-232 of SEQ ID NO:25, a UGG codon at a position corresponding to positions 236-238 of SEQ ID NO:26, a UGG codon at a position corresponding to positions 236-238 of SEQ ID NO:27, a UGG codon at a position corresponding to positions 604-606 of SEQ ID NO:28, or a UGG codon at a position corresponding to positions 126-128 of SEQ ID NO:29.

[0213] In some embodiments, the mRNA molecule of the molecular complex comprises SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:28, or SEQ ID NO:29.

[0214] In some embodiments, the molecular complex comprises a mutation-specific primer or a mutation-specific probe hybridized to an SLC9A3R2 cDNA molecule encoding a predicted loss-of-function polypeptide of SLC9A3R2, wherein the mutation-specific primer or the mutation-specific probe hybridizes to the SLC9A3R2 cDNA molecule at a position corresponding to position 615 set forth in SEQ ID NO:59 or its complement; position 589 set forth in SEQ ID NO:60 or its complement; position 353 set forth in SEQ ID NO:61 or its complement; position 230 set forth in SEQ ID NO:62 or its complement; position 236 set forth in SEQ ID NO:63 or its complement; position 236 set forth in SEQ ID NO:64 or its complement; position 604 set forth in SEQ ID NO:65 or its complement; or position 126 set forth in SEQ ID NO:66 or its complement.

[0215] In some embodiments, the mutation-specific primer or mutation-specific probe of the molecular complex hybridizes to a TGG codon at a position corresponding to positions 615 to 617 of SEQ ID NO:59, a TGG codon at a position corresponding to positions 589 to 591 of SEQ ID NO:60, a TGG codon at a position corresponding to positions 353 to 355 of SEQ ID NO:61, a TGG codon at a position corresponding to positions 230 to 232 of SEQ ID NO:62, a TGG codon at a position corresponding to positions 236 to 238 of SEQ ID NO:63, a TGG codon at a position corresponding to positions 236 to 238 of SEQ ID NO:64, a TGG codon at a position corresponding to positions 604 to 606 of SEQ ID NO:65, or a TGG codon at a position corresponding to positions 126 to 128 of SEQ ID NO:66.

[0216] In some embodiments, the cDNA molecule of the molecular complex comprises SEQ ID NO:59, SEQ ID NO:60, SEQ ID NO:61, SEQ ID NO:62, SEQ ID NO:63, SEQ ID NO:64, SEQ ID NO:65, or SEQ ID NO:66.

[0217] In some embodiments, the molecular complex comprises a mutation-specific probe or a mutation-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.

[0218] The nucleotide sequence of the SLC9A3R2 reference genomic nucleic acid molecule is set forth in SEQ ID NO: 1 (ENSG00000065054.14 encompassing chr16:2,026,902-2,039,026 in the GRCh38 / hg38 human genome assembly). With reference to SEQ ID NO: 1, position 9,519 is a cytosine.

[0219] A SLC9A3R2 missense variant genomic nucleic acid molecule exists, in which the cytosine at position 9,519 is replaced with a thymine, and the nucleotide sequence of this SLC9A3R2 missense variant genomic nucleic acid molecule is set forth in SEQ ID NO:2.

[0220] The nucleotide sequence of an SLC9A3R2-based mRNA molecule is set forth in SEQ ID NO:3. With reference to SEQ ID NO:3, position 615 is a cytosine. The nucleotide sequence of another SLC9A3R2-based mRNA molecule is set forth in SEQ ID NO:4. With reference to SEQ ID NO:4, position 589 is a cytosine. The nucleotide sequence of another SLC9A3R2-based mRNA molecule is set forth in SEQ ID NO:5. With reference to SEQ ID NO:5, position 353 is a cytosine. The nucleotide sequence of another SLC9A3R2-based mRNA molecule is set forth in SEQ ID NO:6. With reference to SEQ ID NO:6, position 230 is a cytosine. The nucleotide sequence of another SLC9A3R2-based mRNA molecule is set forth in SEQ ID NO:7. With reference to SEQ ID NO:7, position 236 is a cytosine. The nucleotide sequence of another SLC9A3R2-based mRNA molecule is set forth in SEQ ID NO:7. With reference to SEQ ID NO:7, position 236 is a cytosine. The nucleotide sequence of another SLC9A3R2-based mRNA molecule is set forth in SEQ ID NO:8. With reference to SEQ ID NO:8, position 236 is a cytosine. The nucleotide sequence of another SLC9A3R2-based mRNA molecule is set forth in SEQ ID NO:9. With reference to SEQ ID NO:9, position 604 is a cytosine. The nucleotide sequence of another SLC9A3R2-based mRNA molecule is set forth in SEQ ID NO:10. With reference to SEQ ID NO:10, position 126 is a cytosine. The nucleotide sequence of another SLC9A3R2-based mRNA molecule is set forth in SEQ ID NO:11. The nucleotide sequence of another SLC9A3R2-based mRNA molecule is set forth in SEQ ID NO:12. With reference to SEQ ID NO:12, position 625 is a cytosine. The nucleotide sequence of another SLC9A3R2-based mRNA molecule is set forth in SEQ ID NO:13. With reference to SEQ ID NO:13, position 622 is a cytosine. The nucleotide sequence of another SLC9A3R2-based mRNA molecule is set forth in SEQ ID NO:14. With reference to SEQ ID NO: 14, position 618 is a cytosine. The nucleotide sequence of another SLC9A3R2-based mRNA molecule is set forth in SEQ ID NO: 15. With reference to SEQ ID NO: 15, position 527 is a cytosine. The nucleotide sequence of another SLC9A3R2-based mRNA molecule is set forth in SEQ ID NO: 16. With reference to SEQ ID NO: 16, position 511 is a cytosine.The nucleotide sequence of another SLC9A3R2-based mRNA molecule is set forth in SEQ ID NO: 17. With reference to SEQ ID NO: 17, position 649 is a cytosine. The nucleotide sequence of another SLC9A3R2-based mRNA molecule is set forth in SEQ ID NO: 18. With reference to SEQ ID NO: 18, position 615 is a cytosine. The nucleotide sequence of another SLC9A3R2-based mRNA molecule is set forth in SEQ ID NO: 19. With reference to SEQ ID NO: 19, position 602 is a cytosine. The nucleotide sequence of another SLC9A3R2-based mRNA molecule is set forth in SEQ ID NO: 20. With reference to SEQ ID NO: 20, position 260 is a cytosine. The nucleotide sequence of another SLC9A3R2-based mRNA molecule is set forth in SEQ ID NO: 21. With reference to SEQ ID NO: 21, position 259 is a cytosine.

[0221] A SLC9A3R2 missense variant mRNA molecule exists in which the cytosine at position 615 is replaced by uracil, the nucleotide sequence of which is set forth in SEQ ID NO:22.

[0222] Another SLC9A3R2 missense variant mRNA molecule exists in which the cytosine at position 589 is replaced by uracil, the nucleotide sequence of which is set forth in SEQ ID NO:23.

[0223] Another SLC9A3R2 missense variant mRNA molecule exists in which the cytosine at position 353 is replaced by uracil, the nucleotide sequence of which is set forth in SEQ ID NO:24.

[0224] Another SLC9A3R2 missense variant mRNA molecule exists in which the cytosine at position 230 is replaced by uracil, the nucleotide sequence of which is set forth in SEQ ID NO:25.

[0225] Another SLC9A3R2 missense variant mRNA molecule exists in which the cytosine at position 236 is replaced by uracil, the nucleotide sequence of which is set forth in SEQ ID NO:26.

[0226] Another SLC9A3R2 missense variant mRNA molecule exists in which the cytosine at position 236 is replaced by uracil, the nucleotide sequence of which is set forth in SEQ ID NO:27.

[0227] Another SLC9A3R2 missense variant mRNA molecule exists in which the cytosine at position 604 is replaced by uracil, the nucleotide sequence of which is set forth in SEQ ID NO:28.

[0228] Another SLC9A3R2 missense variant mRNA molecule exists in which the cytosine at position 126 is replaced by uracil, the nucleotide sequence of which is set forth in SEQ ID NO:29.

[0229] Another SLC9A3R2 missense variant mRNA molecule exists in which the cytosine at position 625 is replaced by uracil, the nucleotide sequence of which is set forth in SEQ ID NO:30.

[0230] Another SLC9A3R2 missense variant mRNA molecule exists in which the cytosine at position 622 is replaced by uracil, the nucleotide sequence of which is set forth in SEQ ID NO:31.

[0231] Another SLC9A3R2 missense variant mRNA molecule exists in which the cytosine at position 618 is replaced by uracil, the nucleotide sequence of which is set forth in SEQ ID NO:32.

[0232] Another SLC9A3R2 missense variant mRNA molecule exists in which the cytosine at position 527 is replaced by uracil, the nucleotide sequence of which is set forth in SEQ ID NO:33.

[0233] Another SLC9A3R2 missense variant mRNA molecule exists in which the cytosine at position 511 is replaced by uracil, the nucleotide sequence of which is set forth in SEQ ID NO:34.

[0234] Another SLC9A3R2 missense variant mRNA molecule exists in which the cytosine at position 649 is replaced by uracil, the nucleotide sequence of which is set forth in SEQ ID NO:35.

[0235] Another SLC9A3R2 missense variant mRNA molecule exists in which the cytosine at position 615 is replaced by uracil, the nucleotide sequence of which is set forth in SEQ ID NO:36.

[0236] Another SLC9A3R2 missense variant mRNA molecule exists in which the cytosine at position 602 is replaced by uracil, the nucleotide sequence of which is set forth in SEQ ID NO:37.

[0237] Another SLC9A3R2 missense variant mRNA molecule exists in which the cytosine at position 260 is replaced by uracil, the nucleotide sequence of which is set forth in SEQ ID NO:38.

[0238] Another SLC9A3R2 missense variant mRNA molecule exists in which the cytosine at position 259 is replaced by uracil, the nucleotide sequence of which is set forth in SEQ ID NO:39.

[0239] The nucleotide sequence of a cDNA molecule based on SLC9A3R2 is set forth in SEQ ID NO: 40. With reference to SEQ ID NO: 40, position 615 is a cytosine. The nucleotide sequence of another cDNA molecule based on SLC9A3R2 is set forth in SEQ ID NO: 41. With reference to SEQ ID NO: 41, position 589 is a cytosine. The nucleotide sequence of another cDNA molecule based on SLC9A3R2 is set forth in SEQ ID NO: 42. With reference to SEQ ID NO: 42, position 353 is a cytosine. The nucleotide sequence of another cDNA molecule based on SLC9A3R2 is set forth in SEQ ID NO: 43. With reference to SEQ ID NO: 43, position 230 is a cytosine. The nucleotide sequence of another cDNA molecule based on SLC9A3R2 is set forth in SEQ ID NO: 44. With reference to SEQ ID NO: 44, position 236 is a cytosine. The nucleotide sequence of another cDNA molecule based on SLC9A3R2 is set forth in SEQ ID NO: 45. With reference to SEQ ID NO: 45, position 236 is a cytosine. The nucleotide sequence of another SLC9A3R2-based cDNA molecule is set forth in SEQ ID NO: 46. With reference to SEQ ID NO: 46, position 604 is a cytosine. The nucleotide sequence of another SLC9A3R2-based cDNA molecule is set forth in SEQ ID NO: 47. With reference to SEQ ID NO: 47, position 126 is a cytosine. The nucleotide sequence of another SLC9A3R2-based cDNA molecule is set forth in SEQ ID NO: 48. The nucleotide sequence of another SLC9A3R2-based cDNA molecule is set forth in SEQ ID NO: 49. With reference to SEQ ID NO: 49, position 625 is a cytosine. The nucleotide sequence of another SLC9A3R2-based cDNA molecule is set forth in SEQ ID NO: 50. With reference to SEQ ID NO: 50, position 622 is a cytosine. The nucleotide sequence of another SLC9A3R2-based cDNA molecule is set forth in SEQ ID NO: 51. With reference to SEQ ID NO: 51, position 618 is a cytosine. The nucleotide sequence of another SLC9A3R2-based cDNA molecule is set forth in SEQ ID NO: 52. With reference to SEQ ID NO: 52, position 527 is a cytosine. The nucleotide sequence of another SLC9A3R2-based cDNA molecule is set forth in SEQ ID NO: 53. With reference to SEQ ID NO: 53, position 511 is a cytosine. The nucleotide sequence of another SLC9A3R2-based cDNA molecule is set forth in SEQ ID NO: 54.With reference to SEQ ID NO:54, position 649 is a cytosine. The nucleotide sequence of another SLC9A3R2-based cDNA molecule is set forth in SEQ ID NO:55. With reference to SEQ ID NO:55, position 615 is a cytosine. The nucleotide sequence of another SLC9A3R2-based cDNA molecule is set forth in SEQ ID NO:56. With reference to SEQ ID NO:56, position 602 is a cytosine. The nucleotide sequence of another SLC9A3R2-based cDNA molecule is set forth in SEQ ID NO:57. With reference to SEQ ID NO:57, position 260 is a cytosine. The nucleotide sequence of another SLC9A3R2-based cDNA molecule is set forth in SEQ ID NO:58. With reference to SEQ ID NO:58, position 259 is a cytosine.

[0240] A SLC9A3R2 missense variant cDNA molecule exists in which a cytosine at position 615 is replaced by a thymine, the nucleotide sequence of which is set forth in SEQ ID NO:59.

[0241] Another SLC9A3R2 missense variant cDNA molecule exists in which the cytosine at position 589 is replaced by a thymine, the nucleotide sequence of which is set forth in SEQ ID NO:60.

[0242] Another SLC9A3R2 missense variant cDNA molecule exists in which the cytosine at position 353 is replaced by a thymine, the nucleotide sequence of which is set forth in SEQ ID NO:61.

[0243] Another SLC9A3R2 missense variant cDNA molecule exists in which a cytosine at position 230 is replaced by a thymine, the nucleotide sequence of which is set forth in SEQ ID NO:62.

[0244] Another SLC9A3R2 missense variant cDNA molecule exists in which the cytosine at position 236 is replaced by a thymine, the nucleotide sequence of which is set forth in SEQ ID NO:63.

[0245] Another SLC9A3R2 missense variant cDNA molecule exists in which the cytosine at position 236 is replaced by a thymine, the nucleotide sequence of which is set forth in SEQ ID NO:64.

[0246] Another SLC9A3R2 missense variant cDNA molecule exists in which the cytosine at position 604 is replaced by a thymine, the nucleotide sequence of which is set forth in SEQ ID NO:65.

[0247] Another SLC9A3R2 missense variant cDNA molecule exists in which the cytosine at position 126 is replaced by a thymine, the nucleotide sequence of which is set forth in SEQ ID NO:66.

[0248] Another SLC9A3R2 missense variant cDNA molecule exists in which the cytosine at position 625 is replaced by a thymine, the nucleotide sequence of which is set forth in SEQ ID NO:67.

[0249] Another SLC9A3R2 missense variant cDNA molecule exists in which a cytosine at position 622 is replaced by a thymine, the nucleotide sequence of which is set forth in SEQ ID NO:68.

[0250] Another SLC9A3R2 missense variant cDNA molecule exists in which the cytosine at position 618 is replaced by a thymine, the nucleotide sequence of which is set forth in SEQ ID NO:69.

[0251] Another SLC9A3R2 missense variant cDNA molecule exists in which the cytosine at position 527 is replaced by a thymine, the nucleotide sequence of which is set forth in SEQ ID NO:70.

[0252] Another SLC9A3R2 missense variant cDNA molecule exists in which the cytosine at position 511 is replaced by a thymine, the nucleotide sequence of which is set forth in SEQ ID NO:71.

[0253] Another SLC9A3R2 missense variant cDNA molecule exists in which the cytosine at position 649 is replaced by a thymine, the nucleotide sequence of which is set forth in SEQ ID NO:72.

[0254] Another SLC9A3R2 missense variant cDNA molecule exists in which the cytosine at position 615 is replaced by a thymine, the nucleotide sequence of which is set forth in SEQ ID NO:73.

[0255] Another SLC9A3R2 missense variant cDNA molecule exists in which a cytosine at position 602 is replaced by a thymine, the nucleotide sequence of which is set forth in SEQ ID NO:74.

[0256] Another SLC9A3R2 missense variant cDNA molecule exists in which a cytosine at position 602 is replaced by a thymine, the nucleotide sequence of which is set forth in SEQ ID NO:75.

[0257] Another SLC9A3R2 missense variant cDNA molecule exists in which the cytosine at position 259 is replaced by a thymine, the nucleotide sequence of which is set forth in SEQ ID NO:76.

[0258] The genomic nucleic acid molecule, mRNA molecule, and cDNA molecule can be derived from any organism. For example, the genomic nucleic acid molecule, mRNA molecule, and cDNA molecule can be an ortholog from human or another organism (e.g., non-human mammal, rodent, mouse, or rat). It is understood that gene sequences within a population can differ due to polymorphisms, such as single nucleotide polymorphisms. The examples provided herein are only exemplary sequences. Other sequences are also possible.

[0259] Also provided herein are functional polynucleotides that can interact with the disclosed nucleic acid molecules. Examples of functional polynucleotides include, but are not limited to, antisense molecules, aptamers, ribozymes, triplex-forming molecules, and external guide sequences. Functional polynucleotides can act as effectors, inhibitors, modulators, and stimulators of the specific activity of target molecules, or functional polynucleotides can have de novo activity independent of any other molecules.

[0260] The isolated nucleic acid molecules disclosed herein can include RNA, DNA, or both RNA and DNA. The isolated nucleic acid molecules can also be linked or fused to heterologous nucleic acid sequences, for example in a vector, or heterologous labels. For example, the isolated nucleic acid molecules disclosed herein can be present as exogenous donor sequences in or containing a vector that includes the isolated nucleic acid molecule and a heterologous nucleic acid sequence. The isolated nucleic acid molecules can also be linked or fused to heterologous labels. The labels can be directly detectable (e.g., fluorophores) or indirectly detectable (e.g., haptens, enzymes, or fluorophore quenchers). Such labels can be detectable by spectroscopic, photochemical, biochemical, immunochemical, or chemical means. Such labels include, for example, radioactive labels, pigments, dyes, chromogens, spin labels, and fluorescent labels. The labels can also be, for example, chemiluminescent materials; metal-containing materials; or enzymes, where enzyme-dependent secondary generation of a signal occurs. The term "label" can also refer to a "tag" or hapten that can be selectively attached to a binding molecule such that the binding molecule is subsequently added with a substrate and used to generate a detectable signal. For example, biotin can be used as a tag together with an avidin or streptavidin conjugate of horseradish peroxidase (HRP) to bind to the tag and probed using a colorimetric (e.g., tetramethylbenzidine (TMB)) or fluorogenic substrate to detect the presence of HRP. Exemplary labels that can be used as tags to facilitate purification include, but are not limited to, myc, HA, FLAG or 3xFLAG, 6xHis or polyhistidine, glutathione-S-transferase (GST), maltose binding protein, epitope tags, or the Fc portion of an immunoglobulin. Numerous labels include, for example, particles, fluorophores, haptens, enzymes and their colorimetric, fluorescent and chemiluminescent substrates, as well as other labels.

[0261] The isolated nucleic acid molecule or its complement can be present in a host cell. In some embodiments, the host cell can comprise a vector comprising any of the nucleic acid molecules described herein or their complements. In some embodiments, the nucleic acid molecule is operably linked to a promoter active in the host cell. In some embodiments, the promoter is an exogenous promoter. In some embodiments, the promoter is an inducible promoter. In some embodiments, the host cell is a bacterial cell, a yeast cell, an insect cell, or a mammalian cell. In some embodiments, the host cell is a bacterial cell. In some embodiments, the host cell is a yeast cell. In some embodiments, the host cell is an insect cell. In some embodiments, the host cell is a mammalian cell.

[0262] The disclosed nucleic acid molecules can include, for example, nucleotides, or non-natural or modified nucleotides, such as, for example, nucleotide analogs or nucleotide substitutes. Such nucleotides include nucleotides that contain modified bases, sugars, or phosphate groups, or nucleotides that incorporate non-natural moieties into their structure. Examples of non-natural nucleotides include, but are not limited to, dideoxynucleotides, biotinylated nucleotides, aminated nucleotides, deaminated nucleotides, alkylated nucleotides, benzylated nucleotides, and fluorophore-labeled nucleotides.

[0263] The nucleic acid molecules disclosed herein may also include one or more nucleotide analogs or nucleotide substitutes. A nucleotide analog is a nucleotide that contains modifications to either the base, sugar, or phosphate moiety. Modifications to the base moiety include, but are not limited to, natural and synthetic modifications of A, C, G, and T / U, as well as various purine or pyrimidine bases, such as, for example, pseudouridine, uracil-5-yl, hypoxanthine-9-yl (I), and 2-aminoadenine-9-yl. Modified bases include 5-methylcytosine (5-me-C), 5-hydroxymethylcytosine, xanthine, hypoxanthine, 2-aminoadenine, 6-methyl and other alkyl derivatives of adenine and guanine, 2-propyl and other alkyl derivatives of adenine and guanine, 2-thiouracil, 2-thiothymine and 2-thiocytosine, 5-halouracil and cytosine, 5-propynyluracil and cytosine, 6-azouracil, cytosine and thymine, 5-uracil (cytosine), and thymine. Examples of uracils and cytosines include, but are not limited to, 4-isopropyl uracil, 4-isopropyl uracil, 8 ...

[0264] Nucleotide analogs can also include modifications of the sugar moiety. Modifications to the sugar moiety include, but are not limited to, natural modifications of ribose and deoxyribose, as well as synthetic modifications. Sugar modifications include, but are not limited to, the following modifications at the 2' position: OH; F; O-, S-, or N-alkyl; O-, S-, or N-alkenyl; O-, S-, or N-alkynyl; or O-alkyl-O-alkyl, where alkyl, alkenyl, and alkynyl are substituted or unsubstituted C-alkyl groups. 1~10 Alkyl or C 2~10 Alkenyl, and C 2~10Exemplary 2' sugar modifications include -O[(CH2) n O] m CH3, -O(CH2) n OCH3, -O(CH2) n NH2, -O(CH2) n CH3, -O(CH2) n -ONH2 and -O(CH2) n ON [(CH2) n Other modifications at the 2' position include, but are not limited to, C 1~10 Examples of suitable substituents include, but are not limited to, alkyl, substituted lower alkyl, alkaryl, aralkyl, O-alkaryl or O-aralkyl, SH, SCH3, OCN, Cl, Br, CN, CF3, OCF3, SOCH3, SO2CH3, ONO2, NO2, N3, NH2, heterocycloalkyl, heterocycloalkaryl, aminoalkylamino, polyalkylamino, substituted silyl, RNA cleaving groups, reporter groups, intercalators, groups for improving the pharmacokinetic properties of oligonucleotides, or groups for improving the pharmacodynamic properties of oligonucleotides, and other substituents with similar properties. Similar modifications may be made at other positions on the sugar, particularly the 3' position of the sugar in the 3' terminal nucleotide or 2'-5' linked oligonucleotides, and the 5' position of the 5' terminal nucleotide. Modified sugars can also include those containing modifications at the bridging ring oxygen, such as CH2 and S. Nucleotide sugar analogs can also have sugar mimetics such as cyclobutyl moieties in place of the pentofuranosyl sugar.

[0265] Nucleotide analogs can also be modified at the phosphate moiety. Modified phosphate moieties include, but are not limited to, those in which the linkage between two nucleotides can be modified to contain phosphorothioates, chiral phosphorothioates, phosphorodithioates, phosphotriesters, aminoalkyl phosphotriesters, methyl phosphonates and other alkyl phosphonates including 3'-alkylene phosphonates and chiral phosphonates, phosphinates, phosphoramidates including 3'-amino phosphoramidates and aminoalkyl phosphoramidates, thionophosphoramidates, thionoalkyl phosphonates, thionoalkyl phosphotriesters, and boranophosphates. Such phosphate or modified phosphate linkages between two nucleotides can be via 3'-5' or 2'-5' linkages, and the linkages can contain reverse polarity such as 3'-5' to 5'-3' or 2'-5' to 5'-2'. Various salts, mixed salts, and free acid forms are also included. Nucleotide substitutes also include peptide nucleic acids (PNAs).

[0266] The present disclosure also provides a vector comprising any one or more of the nucleic acid molecules disclosed herein. In some embodiments, the vector comprises any one or more of the nucleic acid molecules disclosed herein and a heterologous nucleic acid. The vector can be a viral vector or a non-viral vector capable of transporting the nucleic acid molecule. In some embodiments, the vector is a plasmid or a cosmid (such as a circular double stranded DNA into which additional DNA segments can be ligated). In some embodiments, the vector is a viral vector in which additional DNA segments can be ligated into the viral genome. Expression vectors include, but are not limited to, plasmids, cosmids, retroviruses, adenoviruses, adeno-associated viruses (AAV), plant viruses such as cauliflower mosaic virus and tobacco mosaic virus, yeast artificial chromosomes (YACs), Epstein-Barr (EBV) derived episomes, and other expression vectors known in the art.

[0267] Desirable regulatory sequences for mammalian host cell expression may include, for example, viral elements directing high levels of polypeptide expression in mammalian cells, such as retroviral LTRs, cytomegalovirus (CMV) (e.g., CMV promoter / enhancer), Simian Virus 40 (SV40) (e.g., SV40 promoter / enhancer), adenovirus (e.g., adenovirus major late promoter (AdMLP)), polyoma derived promoters and / or enhancers, as well as strong mammalian promoters, such as native immunoglobulin promoters and actin promoters. Methods for expressing polypeptides in bacterial or fungal cells, such as yeast cells, are also well known. The promoter may be, for example, a constitutively active promoter, a conditional promoter, an inducible promoter, a temporally restricted promoter (e.g., a developmentally regulated promoter), or a spatially restricted promoter (e.g., a cell-specific or tissue-specific promoter).

[0268] Percent identity (%) (or percent complementarity) between specific stretches of nucleotide sequences in nucleic acid molecules or amino acid sequences in polypeptides can be routinely determined using the BLAST program (basic sequence comparison search tool) and PowerBLAST program (Altschul et al., J. Mol. Biol., 1990, 215, 403-410; Zhang and Madden, Genome Res., 1997, 7, 649-656), or the Gap program (Wisconsin Sequence Analysis Package, Version 8 for Unix, Genetics Computer Group, University Research Park, Madison Wis.) using default settings that use the Smith and Waterman algorithm (Adv. Appl. Math., 1981, 2, 482-489). When referring to percent sequence identity in this specification, a higher percent sequence identity is preferred over a lower one.

[0269] The present disclosure also provides a composition comprising one or more of the isolated nucleic acid molecules, genomic nucleic acid molecules, mRNA molecules, and / or cDNA molecules disclosed herein, or a vector comprising the same. In some embodiments, the composition is a pharmaceutical composition. In some embodiments, the composition comprises a carrier and / or excipient. Examples of carriers include, but are not limited to, poly(lactic acid) (PLA) microspheres, poly(D,L-lactic-co-glycolic acid) (PLGA) microspheres, liposomes, micelles, reverse micelles, lipid cochleates, and lipid microtubules. The carrier may comprise a buffered salt solution, such as PBS, HBSS, and the like.

[0270] As used herein, the phrase "corresponding to" or grammatical variations thereof, when used in the context of numbering a particular nucleotide or sequence of nucleotides or position, refers to the numbering of the specified reference sequence when the particular nucleotide or sequence of nucleotides is compared to a reference sequence (e.g., SEQ ID NO:1, SEQ ID NO:3, or SEQ ID NO:40). In other words, the residue (e.g., nucleotide or amino acid) number or residue (e.g., nucleotide or amino acid) position of a particular polymer is specified with reference to the reference sequence, not by the actual position number of the residue within the particular nucleotide or nucleotide sequence. For example, a particular nucleotide sequence can be aligned to a reference sequence by introducing gaps to optimize residue matching between the two sequences. In these cases, although gaps exist, the numbering of the residues in the particular nucleotide or nucleotide sequence is done with reference to the reference sequence to which it is aligned.

[0271] For example, an SLC9A3R2 missense nucleic acid molecule comprising a nucleotide sequence encoding a predicted loss-of-function polypeptide for SLC9A3R2, the nucleotide sequence of which comprises a thymine at position corresponding to position 9,519 of SEQ ID NO:2, means that when the nucleotide sequence of the SLC9A3R2 genomic nucleic acid molecule is aligned against the sequence of SEQ ID NO:2, the sequence of SLC9A3R2 has a thymine residue at position corresponding to position 9,519 of SEQ ID NO:2. The same applies to an SLC9A3R2 mRNA molecule comprising a nucleotide sequence encoding a predicted loss-of-function polypeptide for SLC9A3R2, the nucleotide sequence of which comprises a uracil at position corresponding to position 615 of SEQ ID NO:22, and to an SLC9A3R2 cDNA molecule comprising a nucleotide sequence encoding a predicted loss-of-function polypeptide for SLC9A3R2, the nucleotide sequence of which comprises a thymine at position corresponding to position 615 of SEQ ID NO:59. These terms refer to SLC9A3R2 missense nucleic acid molecules encoding a loss-of-function polypeptide predicted for SLC9A3R2, where a genomic nucleic acid molecule has a nucleotide sequence that includes a thymine residue homologous to the thymine residue at position 9,519 of SEQ ID NO:2 (or an mRNA molecule has a nucleotide sequence that includes a uracil residue homologous to the uracil residue at position 615 of SEQ ID NO:22, or a cDNA molecule has a nucleotide sequence that includes a thymine residue homologous to the thymine residue at position 615 of SEQ ID NO:59). Such sequences are also referred to herein as "SLC9A3R2 sequences having an Arg171Trp-Long mutation" or "SLC9A3R2 sequences having an Arg171Trp-Long alteration."

[0272] As described herein, the position in the SLC9A3R2 missense genomic nucleic acid molecule corresponding to the 9,519th position of SEQ ID NO:2 can be identified, for example, by performing sequence comparison between the nucleotide sequence of a particular SLC9A3R2 nucleic acid molecule and the nucleotide sequence of SEQ ID NO:2. For example, there are various computer algorithms that can be used to perform sequence comparison to identify the position of the nucleotide corresponding to the 9,519th position of SEQ ID NO:2. For example, sequence comparison can be performed by using the NCBI BLAST algorithm (Altschul et al., Nucleic Acids Res., 1997, 25, 3389-3402) or CLUSTALW software (Sievers and Higgins, Methods Mol. Biol., 2014, 1079, 105-116). However, sequences can also be aligned manually.

[0273] The amino acid sequence of the SLC9A3R2 reference polypeptide is set forth in SEQ ID NO: 77. With reference to SEQ ID NO: 77, the SLC9A3R2 reference polypeptide is 337 amino acids in length. With reference to SEQ ID NO: 77, position 171 is an arginine.

[0274] Another SLC9A3R2 reference polypeptide amino acid sequence is set forth in SEQ ID NO: 78. With reference to SEQ ID NO: 78, the SLC9A3R2 reference polypeptide is 326 amino acids in length. With reference to SEQ ID NO: 78, position 171 is an arginine.

[0275] Another SLC9A3R2 reference polypeptide amino acid sequence is set forth in SEQ ID NO: 79. With reference to SEQ ID NO: 79, the SLC9A3R2 reference polypeptide is 231 amino acids in length. With reference to SEQ ID NO: 79, position 65 is an arginine.

[0276] Another SLC9A3R2 reference polypeptide amino acid sequence is set forth in SEQ ID NO: 80. With reference to SEQ ID NO: 80, the SLC9A3R2 reference polypeptide is 224 amino acids in length. With reference to SEQ ID NO: 80, position 58 is an arginine.

[0277] Another SLC9A3R2 reference polypeptide amino acid sequence is set forth in SEQ ID NO: 81. With reference to SEQ ID NO: 81, the SLC9A3R2 reference polypeptide is 215 amino acids in length. With reference to SEQ ID NO: 81, position 60 is an arginine.

[0278] Another SLC9A3R2 reference polypeptide amino acid sequence is set forth in SEQ ID NO: 82. With reference to SEQ ID NO: 82, the SLC9A3R2 reference polypeptide is 226 amino acids in length. With reference to SEQ ID NO: 82, position 60 is an arginine.

[0279] Another SLC9A3R2 reference polypeptide amino acid sequence is set forth in SEQ ID NO: 83. With reference to SEQ ID NO: 83, the SLC9A3R2 reference polypeptide is 450 amino acids in length. With reference to SEQ ID NO: 83, position 170 is an arginine.

[0280] Another SLC9A3R2 reference polypeptide amino acid sequence is set forth in SEQ ID NO: 84. With reference to SEQ ID NO: 84, the SLC9A3R2 reference polypeptide is 122 amino acids in length.

[0281] Another SLC9A3R2 reference polypeptide amino acid sequence is set forth in SEQ ID NO: 85. With reference to SEQ ID NO: 85, the SLC9A3R2 reference polypeptide is 151 amino acids in length.

[0282] There is a loss-of-function polypeptide predicted for SLC9A3R2 (Arg171Trp-Long), the amino acid sequence of which is set forth in SEQ ID NO: 86. With reference to SEQ ID NO: 86, the loss-of-function polypeptide predicted for SLC9A3R2 is 337 amino acids long. With reference to SEQ ID NO: 86, position 171 is tryptophan.

[0283] There is another SLC9A3R2 predicted loss-of-function polypeptide (Arg171Trp-Short), the amino acid sequence of which is set forth in SEQ ID NO: 87. With reference to SEQ ID NO: 87, the SLC9A3R2 predicted loss-of-function polypeptide is 326 amino acids long. With reference to SEQ ID NO: 87, position 171 is tryptophan.

[0284] There is another SLC9A3R2 predicted loss-of-function polypeptide (Arg65Trp), the amino acid sequence of which is set forth in SEQ ID NO: 88. With reference to SEQ ID NO: 88, the SLC9A3R2 predicted loss-of-function polypeptide is 231 amino acids in length. With reference to SEQ ID NO: 88, position 65 is a tryptophan.

[0285] There is another SLC9A3R2 predicted loss-of-function polypeptide (Arg58Trp), the amino acid sequence of which is set forth in SEQ ID NO: 89. With reference to SEQ ID NO: 89, the SLC9A3R2 predicted loss-of-function polypeptide is 224 amino acids in length. With reference to SEQ ID NO: 89, position 58 is a tryptophan.

[0286] There is another SLC9A3R2 predicted loss-of-function polypeptide (Arg60Trp-Short), the amino acid sequence of which is set forth in SEQ ID NO: 90. With reference to SEQ ID NO: 90, the SLC9A3R2 predicted loss-of-function polypeptide is 215 amino acids long. With reference to SEQ ID NO: 90, position 60 is tryptophan.

[0287] There is another SLC9A3R2 predicted loss-of-function polypeptide (Arg60Trp-Long), the amino acid sequence of which is set forth in SEQ ID NO: 91. With reference to SEQ ID NO: 91, the SLC9A3R2 predicted loss-of-function polypeptide is 226 amino acids long. With reference to SEQ ID NO: 91, position 60 is tryptophan.

[0288] There is another SLC9A3R2 predicted loss-of-function polypeptide (Arg170Trp), the amino acid sequence of which is set forth in SEQ ID NO: 92. With reference to SEQ ID NO: 92, the SLC9A3R2 predicted loss-of-function polypeptide is 450 amino acids in length. With reference to SEQ ID NO: 92, position 60 is a tryptophan.

[0289] The nucleotide and amino acid sequences listed in the accompanying sequence listing are shown using standard abbreviations for nucleotide bases and three-letter codes for amino acids. The nucleotide sequences follow the standard convention of beginning at the 5'-end of the sequence and proceeding toward the 3'-end (i.e., from left to right in each sequence). Only one strand of each nucleotide sequence is shown, but the complementary strand is understood to be included by reference to the shown strand. The amino acid sequences follow the standard convention of beginning at the amino-terminus of the sequence and proceeding toward the carboxy-terminus (i.e., from left to right in each sequence).

[0290] The present disclosure also provides a therapeutic agent for treating or preventing hypertension, coronary heart disease and / or atrial fibrillation for use in treating or preventing hypertension, coronary heart disease and / or atrial fibrillation in a subject (or for use in the preparation of a medicament for treating or preventing hypertension, coronary heart disease and / or atrial fibrillation), wherein the subject has any of the SLC9A3R2 missense variant genomic nucleic acid molecules, missense variant mRNA molecules, and / or missense variant cDNA molecules encoding a predicted loss-of-function polypeptide of SLC9A3R2 described herein. The therapeutic agent for treating or preventing hypertension, coronary heart disease and / or atrial fibrillation can be any of the therapeutic agents for treating or preventing hypertension, coronary heart disease and / or atrial fibrillation described herein. The hypertension can be any of primary hypertension, secondary hypertension, resistant hypertension, and malignant hypertension.

[0291] In some embodiments, the subject is identified as having a genomic nucleic acid molecule or its complement encoding a predicted loss-of-function polypeptide of SLC9A3R2 having a nucleotide sequence including a thymine or its complement at a position corresponding to position 9,519 of SEQ ID NO:2.

[0292] In some embodiments, the subject is identified as having an mRNA molecule encoding a loss-of-function polypeptide predicted for SLC9A3R2, having a nucleotide sequence comprising: uracil or its complement at a position corresponding to position 615 set forth in SEQ ID NO:22; uracil or its complement at a position corresponding to position 589 set forth in SEQ ID NO:23; uracil or its complement at a position corresponding to position 353 set forth in SEQ ID NO:24; uracil or its complement at a position corresponding to position 230 set forth in SEQ ID NO:25; uracil or its complement at a position corresponding to position 236 set forth in SEQ ID NO:26; uracil or its complement at a position corresponding to position 236 set forth in SEQ ID NO:27; uracil or its complement at a position corresponding to position 604 set forth in SEQ ID NO:28; or uracil or its complement at a position corresponding to position 126 set forth in SEQ ID NO:29, or a complement thereof.

[0293] In some embodiments, the subject is identified as having a cDNA molecule encoding a loss-of-function polypeptide predicted for SLC9A3R2, having a nucleotide sequence comprising: a thymine or its complement at a position corresponding to position 615 set forth in SEQ ID NO:59; a thymine or its complement at a position corresponding to position 589 set forth in SEQ ID NO:60; a thymine or its complement at a position corresponding to position 353 set forth in SEQ ID NO:61; a thymine or its complement at a position corresponding to position 230 set forth in SEQ ID NO:62; a thymine or its complement at a position corresponding to position 236 set forth in SEQ ID NO:63; a thymine or its complement at a position corresponding to position 236 set forth in SEQ ID NO:64; a thymine or its complement at a position corresponding to position 604 set forth in SEQ ID NO:65; or a thymine or its complement at a position corresponding to position 126 set forth in SEQ ID NO:66, or its complement.

[0294] In some embodiments, the subject is identified as having a genomic nucleic acid molecule having a nucleotide sequence encoding a loss-of-function polypeptide predicted for SLC9A3R2, the nucleotide sequence of which comprises a thymine or its complement at a position corresponding to position 9,519 set forth in SEQ ID NO:2; an mRNA molecule having a nucleotide sequence encoding a loss-of-function polypeptide predicted for SLC9A3R2, the nucleotide sequence of which comprises a uracil or its complement at a position corresponding to position 615 set forth in SEQ ID NO:22; a cDNA molecule having a nucleotide sequence encoding a loss-of-function polypeptide predicted for SLC9A3R2, the nucleotide sequence of which comprises a thymine or its complement at a position corresponding to position 615 set forth in SEQ ID NO:59; or a loss-of-function polypeptide predicted for SLC9A3R2 comprising a tryptophan at a position corresponding to position 171 set forth in SEQ ID NO:86.

[0295] In some embodiments, the subject is identified as having a genomic nucleic acid molecule having a nucleotide sequence encoding a loss-of-function polypeptide predicted in SLC9A3R2, wherein the nucleotide sequence includes an mRNA molecule having a nucleotide sequence encoding a loss-of-function polypeptide predicted in SLC9A3R2, the nucleotide sequence of which comprises uracil or its complement at a position corresponding to position 589 set forth in SEQ ID NO:23; a cDNA molecule having a nucleotide sequence encoding a loss-of-function polypeptide predicted in SLC9A3R2, the nucleotide sequence of which comprises thymine or its complement at a position corresponding to position 589 set forth in SEQ ID NO:60; or a loss-of-function polypeptide predicted in SLC9A3R2 comprising tryptophan at a position corresponding to position 171 set forth in SEQ ID NO:87.

[0296] In some embodiments, the subject is identified as having a genomic nucleic acid molecule having a nucleotide sequence encoding a loss-of-function polypeptide predicted in SLC9A3R2, wherein the nucleotide sequence includes an mRNA molecule having a nucleotide sequence encoding a loss-of-function polypeptide predicted in SLC9A3R2, the nucleotide sequence of which comprises uracil or its complement at a position corresponding to position 353 set forth in SEQ ID NO:24; a cDNA molecule having a nucleotide sequence encoding a loss-of-function polypeptide predicted in SLC9A3R2, the nucleotide sequence of which comprises thymine or its complement at a position corresponding to position 353 set forth in SEQ ID NO:61; or a loss-of-function polypeptide predicted in SLC9A3R2 comprising tryptophan at a position corresponding to position 65 set forth in SEQ ID NO:88.

[0297] In some embodiments, the subject is identified as having a genomic nucleic acid molecule having a nucleotide sequence encoding a loss-of-function polypeptide predicted in SLC9A3R2, wherein the nucleotide sequence includes an mRNA molecule having a nucleotide sequence encoding a loss-of-function polypeptide predicted in SLC9A3R2, the nucleotide sequence of which comprises uracil or its complement at a position corresponding to position 230 set forth in SEQ ID NO:25; a cDNA molecule having a nucleotide sequence encoding a loss-of-function polypeptide predicted in SLC9A3R2, the nucleotide sequence of which comprises thymine or its complement at a position corresponding to position 230 set forth in SEQ ID NO:62; or a loss-of-function polypeptide predicted in SLC9A3R2 comprising tryptophan at a position corresponding to position 58 set forth in SEQ ID NO:89.

[0298] In some embodiments, the subject is identified as having a genomic nucleic acid molecule having a nucleotide sequence encoding a loss-of-function polypeptide predicted in SLC9A3R2, wherein the nucleotide sequence includes an mRNA molecule having a nucleotide sequence encoding a loss-of-function polypeptide predicted in SLC9A3R2, the nucleotide sequence of which comprises uracil or its complement at a position corresponding to position 236 set forth in SEQ ID NO:26; a cDNA molecule having a nucleotide sequence encoding a loss-of-function polypeptide predicted in SLC9A3R2, the nucleotide sequence of which comprises thymine or its complement at a position corresponding to position 236 set forth in SEQ ID NO:63; or a loss-of-function polypeptide predicted in SLC9A3R2 comprising tryptophan at a position corresponding to position 60 set forth in SEQ ID NO:90.

[0299] In some embodiments, the subject is identified as having a genomic nucleic acid molecule having a nucleotide sequence encoding a loss-of-function polypeptide predicted in SLC9A3R2, wherein the nucleotide sequence includes an mRNA molecule having a nucleotide sequence encoding a loss-of-function polypeptide predicted in SLC9A3R2, the nucleotide sequence of which comprises uracil or its complement at a position corresponding to position 236 set forth in SEQ ID NO:27; a cDNA molecule having a nucleotide sequence encoding a loss-of-function polypeptide predicted in SLC9A3R2, the nucleotide sequence of which comprises thymine or its complement at a position corresponding to position 236 set forth in SEQ ID NO:64; or a loss-of-function polypeptide predicted in SLC9A3R2 comprising tryptophan at a position corresponding to position 60 set forth in SEQ ID NO:91.

[0300] In some embodiments, the subject is identified as having a genomic nucleic acid molecule having a nucleotide sequence encoding a loss-of-function polypeptide predicted in SLC9A3R2, wherein the nucleotide sequence includes an mRNA molecule having a nucleotide sequence encoding a loss-of-function polypeptide predicted in SLC9A3R2, the nucleotide sequence of which comprises uracil or its complement at a position corresponding to position 604 set forth in SEQ ID NO:28; a cDNA molecule having a nucleotide sequence encoding a loss-of-function polypeptide predicted in SLC9A3R2, the nucleotide sequence of which comprises thymine or its complement at a position corresponding to position 604 set forth in SEQ ID NO:65; or a loss-of-function polypeptide predicted in SLC9A3R2 comprising tryptophan at a position corresponding to position 60 set forth in SEQ ID NO:92.

[0301] In some embodiments, the subject is identified as having a genomic nucleic acid molecule having a nucleotide sequence encoding a loss-of-function polypeptide predicted in SLC9A3R2, wherein the nucleotide sequence comprises an mRNA molecule having a nucleotide sequence encoding a loss-of-function polypeptide predicted in SLC9A3R2, the nucleotide sequence of which comprises uracil or its complement at a position corresponding to position 126 set forth in SEQ ID NO:29; or a cDNA molecule having a nucleotide sequence encoding a loss-of-function polypeptide predicted in SLC9A3R2, the nucleotide sequence of which comprises thymine or its complement at a position corresponding to position 126 set forth in SEQ ID NO:66.

[0302] The present disclosure also provides an SLC9A3R2 inhibitor for use in treating or preventing hypertension, coronary heart disease and / or atrial fibrillation in a subject (or for use in preparing a medicament for treating or preventing hypertension, coronary heart disease and / or atrial fibrillation), wherein the subject is heterozygous for any of the SLC9A3R2 missense variant genomic nucleic acid molecules, missense variant mRNA molecules, and / or missense variant cDNA molecules encoding SLC9A3R2 predicted loss-of-function polypeptides described herein, or the subject is a reference for the SLC9A3R2 genomic nucleic acid molecules, mRNA molecules, or cDNA molecules. The SLC9A3R2 inhibitor can be any of the SLC9A3R2 inhibitors described herein. The hypertension can be any of primary hypertension, secondary hypertension, resistant hypertension, and malignant hypertension.

[0303] In some embodiments, the subject is a reference for a SLC9A3R2 genomic nucleic acid molecule, an SLC9A3R2 mRNA molecule, or an SLC9A3R2 cDNA molecule. In some embodiments, the subject is heterozygous for a genomic nucleic acid molecule encoding a predicted loss-of-function polypeptide of SLC9A3R2 having a nucleotide sequence including a thymine or its complement at a position corresponding to position 9,519 of SEQ ID NO:2, or its complement.

[0304] In some embodiments, the subject is heterozygous for an mRNA molecule encoding a loss-of-function polypeptide predicted for SLC9A3R2, having a nucleotide sequence comprising uracil or its complement at a position corresponding to position 615 set forth in SEQ ID NO:22; uracil or its complement at a position corresponding to position 589 set forth in SEQ ID NO:23; uracil or its complement at a position corresponding to position 353 set forth in SEQ ID NO:24; uracil or its complement at a position corresponding to position 230 set forth in SEQ ID NO:25; uracil or its complement at a position corresponding to position 236 set forth in SEQ ID NO:26; uracil or its complement at a position corresponding to position 236 set forth in SEQ ID NO:27; uracil or its complement at a position corresponding to position 604 set forth in SEQ ID NO:28; or uracil or its complement at a position corresponding to position 126 set forth in SEQ ID NO:29, or a complement thereof.

[0305] In some embodiments, the subject is heterozygous for a cDNA molecule encoding a loss-of-function polypeptide predicted for SLC9A3R2 having a nucleotide sequence comprising: a thymine or its complement at a position corresponding to position 615 set forth in SEQ ID NO:59; a thymine or its complement at a position corresponding to position 589 set forth in SEQ ID NO:60; a thymine or its complement at a position corresponding to position 353 set forth in SEQ ID NO:61; a thymine or its complement at a position corresponding to position 230 set forth in SEQ ID NO:62; a thymine or its complement at a position corresponding to position 236 set forth in SEQ ID NO:63; a thymine or its complement at a position corresponding to position 236 set forth in SEQ ID NO:64; a thymine or its complement at a position corresponding to position 604 set forth in SEQ ID NO:65; or a thymine or its complement at a position corresponding to position 126 set forth in SEQ ID NO:66, or its complement.

[0306] In some embodiments, the subject is identified as heterozygous for a genomic nucleic acid molecule having a nucleotide sequence encoding a SLC9A3R2 predicted loss-of-function polypeptide, the nucleotide sequence of which comprises a thymine or its complement at a position corresponding to position 9,519 as set forth in SEQ ID NO:2; an mRNA molecule having a nucleotide sequence encoding a SLC9A3R2 predicted loss-of-function polypeptide, the nucleotide sequence of which comprises a uracil or its complement at a position corresponding to position 615 as set forth in SEQ ID NO:22; a cDNA molecule having a nucleotide sequence encoding a SLC9A3R2 predicted loss-of-function polypeptide, the nucleotide sequence of which comprises a thymine or its complement at a position corresponding to position 615 as set forth in SEQ ID NO:59; or a SLC9A3R2 predicted loss-of-function polypeptide comprising a tryptophan at a position corresponding to position 171 as set forth in SEQ ID NO:86. The SLC9A3R2 inhibitor can be any of the SLC9A3R2 inhibitors described herein.

[0307] In some embodiments, the subject is identified as heterozygous for a genomic nucleic acid molecule having a nucleotide sequence encoding a SLC9A3R2 predicted loss-of-function polypeptide, wherein the nucleotide sequence comprises an mRNA molecule having a nucleotide sequence encoding a SLC9A3R2 predicted loss-of-function polypeptide, the nucleotide sequence of which comprises a uracil or its complement at a position corresponding to position 589 set forth in SEQ ID NO:23; a cDNA molecule having a nucleotide sequence encoding a SLC9A3R2 predicted loss-of-function polypeptide, the nucleotide sequence of which comprises a thymine or its complement at a position corresponding to position 589 set forth in SEQ ID NO:60; or a SLC9A3R2 predicted loss-of-function polypeptide comprising a tryptophan at a position corresponding to position 171 set forth in SEQ ID NO:87. The SLC9A3R2 inhibitor can be any of the SLC9A3R2 inhibitors described herein.

[0308] In some embodiments, the subject is identified as heterozygous for a genomic nucleic acid molecule having a nucleotide sequence encoding a SLC9A3R2 predicted loss-of-function polypeptide, wherein the nucleotide sequence comprises an mRNA molecule having a nucleotide sequence encoding a SLC9A3R2 predicted loss-of-function polypeptide, the nucleotide sequence of which comprises a uracil or its complement at a position corresponding to position 353 set forth in SEQ ID NO:24; a cDNA molecule having a nucleotide sequence encoding a SLC9A3R2 predicted loss-of-function polypeptide, the nucleotide sequence of which comprises a thymine or its complement at a position corresponding to position 353 set forth in SEQ ID NO:61; or a SLC9A3R2 predicted loss-of-function polypeptide comprising a tryptophan at a position corresponding to position 65 set forth in SEQ ID NO:88. The SLC9A3R2 inhibitor can be any of the SLC9A3R2 inhibitors described herein.

[0309] In some embodiments, the subject is identified as heterozygous for a genomic nucleic acid molecule having a nucleotide sequence encoding a SLC9A3R2 predicted loss-of-function polypeptide, wherein the nucleotide sequence comprises an mRNA molecule having a nucleotide sequence encoding a SLC9A3R2 predicted loss-of-function polypeptide, the nucleotide sequence of which comprises a uracil or its complement at a position corresponding to position 230 set forth in SEQ ID NO:25; a cDNA molecule having a nucleotide sequence encoding a SLC9A3R2 predicted loss-of-function polypeptide, the nucleotide sequence of which comprises a thymine or its complement at a position corresponding to position 230 set forth in SEQ ID NO:62; or a SLC9A3R2 predicted loss-of-function polypeptide comprising a tryptophan at a position corresponding to position 58 set forth in SEQ ID NO:89. The SLC9A3R2 inhibitor can be any of the SLC9A3R2 inhibitors described herein.

[0310] In some embodiments, the subject is identified as heterozygous for a genomic nucleic acid molecule having a nucleotide sequence encoding a SLC9A3R2 predicted loss-of-function polypeptide, wherein the nucleotide sequence comprises an mRNA molecule having a nucleotide sequence encoding a SLC9A3R2 predicted loss-of-function polypeptide, the nucleotide sequence of which comprises a uracil or its complement at a position corresponding to position 236 set forth in SEQ ID NO:26; a cDNA molecule having a nucleotide sequence encoding a SLC9A3R2 predicted loss-of-function polypeptide, the nucleotide sequence of which comprises a thymine or its complement at a position corresponding to position 236 set forth in SEQ ID NO:63; or a SLC9A3R2 predicted loss-of-function polypeptide comprising a tryptophan at a position corresponding to position 60 set forth in SEQ ID NO:90. The SLC9A3R2 inhibitor can be any of the SLC9A3R2 inhibitors described herein.

[0311] In some embodiments, the subject is identified as heterozygous for a genomic nucleic acid molecule having a nucleotide sequence encoding a SLC9A3R2 predicted loss-of-function polypeptide, wherein the nucleotide sequence comprises an mRNA molecule having a nucleotide sequence encoding a SLC9A3R2 predicted loss-of-function polypeptide, the nucleotide sequence of which comprises a uracil or its complement at a position corresponding to position 236 set forth in SEQ ID NO:27; a cDNA molecule having a nucleotide sequence encoding a SLC9A3R2 predicted loss-of-function polypeptide, the nucleotide sequence of which comprises a thymine or its complement at a position corresponding to position 236 set forth in SEQ ID NO:64; or a SLC9A3R2 predicted loss-of-function polypeptide comprising a tryptophan at a position corresponding to position 60 set forth in SEQ ID NO:91. The SLC9A3R2 inhibitor can be any of the SLC9A3R2 inhibitors described herein.

[0312] In some embodiments, the subject is identified as heterozygous for a genomic nucleic acid molecule having a nucleotide sequence encoding a SLC9A3R2 predicted loss-of-function polypeptide, wherein the nucleotide sequence comprises an mRNA molecule having a nucleotide sequence encoding a SLC9A3R2 predicted loss-of-function polypeptide, the nucleotide sequence of which comprises a uracil or its complement at a position corresponding to position 604 set forth in SEQ ID NO:28; a cDNA molecule having a nucleotide sequence encoding a SLC9A3R2 predicted loss-of-function polypeptide, the nucleotide sequence of which comprises a thymine or its complement at a position corresponding to position 604 set forth in SEQ ID NO:65; or a SLC9A3R2 predicted loss-of-function polypeptide comprising a tryptophan at a position corresponding to position 60 set forth in SEQ ID NO:92. The SLC9A3R2 inhibitor can be any of the SLC9A3R2 inhibitors described herein.

[0313] In some embodiments, the subject is identified as heterozygous for a genomic nucleic acid molecule having a nucleotide sequence encoding a loss-of-function polypeptide predicted in SLC9A3R2, wherein the nucleotide sequence comprises an mRNA molecule having a nucleotide sequence encoding a loss-of-function polypeptide predicted in SLC9A3R2, the nucleotide sequence of which comprises uracil or its complement at a position corresponding to position 126 set forth in SEQ ID NO:29; or a cDNA molecule having a nucleotide sequence encoding a loss-of-function polypeptide predicted in SLC9A3R2, the nucleotide sequence of which comprises thymine or its complement at a position corresponding to position 126 set forth in SEQ ID NO:66.

[0314] In some embodiments, the subject is identified as having a SLC9A3R2-referenced genomic nucleic acid molecule comprising SEQ ID NO:1, a SLC9A3R2-referenced mRNA molecule comprising one or more of SEQ ID NOs:3-21, a SLC9A3R2-referenced cDNA molecule comprising one or more of SEQ ID NOs:40-58, or a SLC9A3R2-referenced polypeptide comprising one or more of SEQ ID NOs:77-85. The SLC9A3R2 inhibitor can be any of the SLC9A3R2 inhibitors described herein.

[0315] All patent documents, websites, other publications, accession numbers, etc. cited above or below are incorporated by reference in their entirety for all purposes to the same extent as if each individual document was specifically and individually indicated to be so incorporated by reference. Where various versions of a sequence are associated with accession numbers at different times, the version associated with the accession number at the effective filing date of this application is meant. Effective filing date means the earlier of the actual filing date or the filing date of the priority application to which the accession number refers, if applicable. Similarly, where different versions of publications, websites, etc. have been published at different times, the version last published at the effective filing date of the application is meant unless otherwise indicated. Any feature, step, element, embodiment, or aspect of the present disclosure may be used in combination with any other feature, step, element, embodiment, or aspect, unless otherwise indicated. Although the present disclosure has been described in some detail by way of illustration and example for purposes of clarity and understanding, it will be apparent that certain changes and modifications may be practiced within the scope of the appended claims.

[0316] The following examples are provided to further illustrate the embodiments. They are intended to illustrate, not limit, the claimed embodiments. The following examples provide those skilled in the art with a disclosure and explanation of how the compounds, compositions, articles, devices and / or methods described herein are made and evaluated, and are intended to be merely illustrative and are not intended to limit the scope of any claims. Efforts have been made to ensure accuracy with respect to numbers (e.g., amounts, temperatures, etc.), but some error and deviation can be accounted for. Unless otherwise indicated, parts are parts by weight, temperature is °C or is at ambient temperature, and pressure is at or near atmospheric pressure. EXAMPLES

[0317] Example 1: Rare pLOF and deleterious missense variants in SLC9A3R2 are associated with reduced risk of hypertension The exomes of 454,787 UKB study participants were sequenced, covering 95.8% of the target bases at a depth of more than 20x, as previously described (Szustakowski, Advancing Human Genetics Research and Drug Discovery through Exome Sequencing of the UK Biobank. bioRxiv, 2021; and Van Hout et al., Nature, 2020). 12 million variants were identified in 39 million base pairs across the coding regions of 18,659 genes (data not shown). Among the variants identified, there were 3,375,252 (median 10,260 per individual) synonymous variants, 7,689,495 (9,284 per individual) missense variants, and 889,957 (212 per individual) putative loss-of-function (pLOF) variants (data not shown), of which approximately half were observed only once in this dataset (singleton variants; data not shown).

[0318] We identified a novel association between lower risk of hypertension and burden of rare pLOF and deleterious missense variants in SLC9A3R2 (5,873 carriers; OR = 0.81, 95% CI 0.76 to 0.87, P = 2.2 × 10 -10 In addition, lower systolic blood pressure (SBP; effect = -1.85 mmHg, 95% CI = -2.22 to -1.48, P = 2.0 × 10 -19 ) and lower diastolic blood pressure (effect = -1.01 mmHg, 95% CI = -1.31 to -0.80, P = 3.7 × 10 -18 ), and the association of SBP was replicated within the GHS cohort (1,517 carriers; effect = -0.077 SD units, 95% CI -0.118 to -0.0356, P = 2.6 × 10 -4 ).

[0319] A low-frequency missense variant in SLC9A3R2 (rs139491786, Arg171Trp, MAF=0.7%) was previously identified in a GWAS of blood pressure, but the signal was due to a nearby PKD1 gene variant (rs140869992, Arg2200Cys) (Giri et al., Nat Genet., 2019, 51, 51-62). The UKB WES demonstrated that rare pLOF and deleterious missense variants in SLC9A3R2 as well as a burden of Arg171Trp remained strongly associated with SBP, DBP and hypertension after conditioning with Arg2200Cys in PKD1 (Table 2). Overall, this signal is consistent with the established role of sodium balance in regulating blood pressure, suggesting that inhibiting SLC9A3R2 could be an attractive avenue for blood pressure management.

[0320] [Table 3-1]

[0321] [Table 3-2]

[0322] [Table 3-3]

[0323] In addition to those described herein, various modifications of the described subject matter will become apparent to those skilled in the art from the foregoing description. Such modifications are also intended to fall within the scope of the appended claims. Each reference cited in this application (including, but not limited to, journal articles, U.S. and non-U.S. patents, patent application publications, international patent application publications, gene bank accession numbers, etc.) is incorporated herein by reference in its entirety and for all purposes.

Claims

1. An in vitro method for identifying the susceptibility of a subject to the onset of hypertension, coronary artery disease, and / or atrial fibrillation, said method comprising: determining, or having determined, the presence or absence of an SLC9A3R2 missense mutant nucleic acid molecule encoding a loss-of-function polypeptide predicted by solute carrier family 9 isoform A3 regulator 2 (SLC9A3R2) in a biological sample obtained from said subject; said subject being SLC9A3R2 positive indicating that said subject has a high risk of onset of hypertension, coronary artery disease, and / or atrial fibrillation; said subject being heterozygous or homozygous for an SLC9A3R2 missense mutant nucleic acid molecule encoding a loss-of-function polypeptide predicted by SLC9A3R2 indicating that said subject has a low risk of onset of hypertension, coronary artery disease, and / or atrial fibrillation.

2. The method according to claim 1, wherein said SLC9A3R2 missense mutant nucleic acid molecule encodes Arg171Trp-Long, Arg171Trp-Short, Arg65Trp, Arg58Trp, Arg60Trp-Short, Arg60Trp-Long, or Arg170Trp.

3. The method according to claim 1, wherein said SLC9A3R2 missense mutant nucleic acid molecule encodes Arg171Trp-Long or Arg171Trp-Short.

4. The SLC9A3R2 missense mutant nucleic acid molecule is: a genomic nucleic acid molecule having a nucleotide sequence containing thymine at a position corresponding to position 9,519 set forth in SEQ ID NO: 2; An mRNA molecule having a nucleotide sequence comprising uracil at a position corresponding to position 615 set forth in SEQ ID NO: 22, uracil at a position corresponding to position 589 set forth in SEQ ID NO: 23, uracil at a position corresponding to position 353 set forth in SEQ ID NO: 24, uracil at a position corresponding to position 230 set forth in SEQ ID NO: 25, uracil at a position corresponding to position 236 set forth in SEQ ID NO: 26, uracil at a position corresponding to position 236 set forth in SEQ ID NO: 27, uracil at a position corresponding to position 604 set forth in SEQ ID NO: 28, or uracil at a position corresponding to position 126 set forth in SEQ ID NO: 29; or A cDNA molecule generated from an mRNA molecule having a nucleotide sequence comprising thymine at a position corresponding to position 615 set forth in SEQ ID NO: 59, thymine at a position corresponding to position 589 set forth in SEQ ID NO: 60, thymine at a position corresponding to position 353 set forth in SEQ ID NO: 61, thymine at a position corresponding to position 230 set forth in SEQ ID NO: 62, thymine at a position corresponding to position 236 set forth in SEQ ID NO: 63, thymine at a position corresponding to position 236 set forth in SEQ ID NO: 64, thymine at a position corresponding to position 604 set forth in SEQ ID NO: 65, or thymine at a position corresponding to position 126 set forth in SEQ ID NO: 66 The method according to claim 2.

5. The method according to any one of claims 1 to 4, wherein the hypertension includes essential hypertension, secondary hypertension, resistant hypertension, or malignant hypertension.

6. Use of a therapeutic agent for treating or preventing hypertension, coronary heart disease and / or atrial fibrillation in the preparation of a medicament for treating or preventing hypertension, coronary heart disease and / or atrial fibrillation in a subject, wherein the subject is heterozygous for a missense mutant nucleic acid molecule of solute carrier family 9 isoform A3 regulator 2 (SLC9A3R2) encoding Arg171Trp-Long, Arg171Trp-Short, Arg65Trp, Arg58Trp, Arg60Trp-Short, Arg60Trp-Long, or Arg170Trp.

7. The use according to claim 6, wherein the hypertension includes essential hypertension, secondary hypertension, resistant hypertension, or malignant hypertension.

8. The use according to claim 6 or 7, wherein the SLC9A3R2 missense mutant nucleic acid molecule encodes SLC9A3R2 Arg171Trp-Long or Arg171Trp-Short.

9. The SLC9A3R2 missense mutant nucleic acid molecule is a genomic nucleic acid molecule encoding a loss-of-function polypeptide predicted by solute carrier family 9 isoform A3 regulator 2 (SLC9A3R2) having a nucleotide sequence containing thymine or its complement at a position corresponding to position 9,519 described in SEQ ID NO: 2; or a uracil or its complement at a position corresponding to position 615 described in SEQ ID NO: 22; a uracil or its complement at a position corresponding to position 589 described in SEQ ID NO: 23; a uracil or its complement at a position corresponding to position 353 described in SEQ ID NO: 24; a uracil or its complement at a position corresponding to position 230 described in SEQ ID NO: 25; a uracil or its complement at a position corresponding to position 236 described in SEQ ID NO: 26; a uracil or its complement at a position corresponding to position 236 described in SEQ ID NO: 27; a uracil or its complement at a position corresponding to position 604 described in SEQ ID NO: 28; or a uracil or its complement at a position corresponding to position 126 described in SEQ ID NO: 29, and having a nucleotide sequence containing the same, an mRNA molecule encoding a loss-of-function polypeptide predicted by SLC9A3R2 or its complement The use according to claim 6 or 7.

10. The use according to claim 6, wherein the therapeutic agent for the treatment or prevention of hypertension, coronary heart disease and / or atrial fibrillation is a thiazide diuretic, a potassium-sparing diuretic, a loop diuretic, a beta blocker, an angiotensin-converting enzyme (ACE) inhibitor, an angiotensin II receptor blocker (ARB), a calcium channel blocker, an alpha blocker, an alpha-beta blocker, a central agonist, a vasodilator, an aldosterone receptor antagonist, or a renin inhibitor.

11. The thiazide diuretic includes chlorthalidone, chlorothiazide, hydrochlorothiazide, indapamide, or metolazone, the potassium-sparing diuretic includes amiloride, spironolactone, or triamterene, the loop diuretic includes bumetanide, furosemide, or torsemide, the beta blocker includes acebutolol, atenolol, betaxolol, bisoprolol, bisoprolol / hydrochlorothiazide, metoprolol tartrate, metoprolol succinate, nadolol, pindolol, propranolol, sotolol, or timolol, the ACE inhibitor includes benazepril, captopril, enalapril, fosinopril, lisinopril, moexipril, perindopril, quinapril, ramipril, ortrandolapril, the ARB includes candesartan, eprosartan, irbesartan, losartan, telmisartan, or valsartan, the calcium channel blocker includes amlodipine, diltiazem, felodipine, isradipine, nicardipine, nifedipine, nisoldipine, or verapamil, the alpha blocker includes doxazosin, prazosin, or terazosin, the alpha-beta blocker includes carvedilol or labetalol, the central agonist includes methyldopa, clonidine, or guanfacine, the vasodilator includes hydralazine or minoxidil, the aldosterone receptor antagonist includes eplerenone or spironolactone, the renin inhibitor includes aliskiren, The use according to claim 10.

12. Use of an inhibitor of solute carrier family 9 isoform A3 regulator 2 (SLC9A3R2) in the preparation of a medicament for the treatment or prevention of hypertension, coronary artery disease and / or atrial fibrillation in a subject, wherein the subject is a) a reference for an SLC9A3R2 genomic nucleic acid molecule or an SLC9A3R2 mRNA molecule, or b) heterozygous for an SLC9A3R2 missense variant nucleic acid molecule encoding SLC9A3R2 Arg171Trp-Long, Arg171Trp-Short, Arg65Trp, Arg58Trp, Arg60Trp-Short, Arg60Trp-Long, or Arg170Trp. **Claim 13** The use according to claim 12, wherein the SLC9A3R2 inhibitor is an inhibitory nucleic acid molecule. **Claim 14** The use according to claim 13, wherein the inhibitory nucleic acid molecule is an antisense nucleic acid molecule, a small interfering RNA (siRNA), or a short hairpin RNA (shRNA) that hybridizes to an SLC9A3R2 nucleic acid molecule. **Claim 15** The use according to claim 12, wherein the SLC9A3R2 inhibitor comprises a Cas protein and a guide RNA (gRNA) that hybridizes to a gRNA recognition sequence within the SLC9A3R2 genomic nucleic acid molecule. **Claim 16** The use according to claim 15, wherein the Cas protein is Cas9 or Cpf1. **Claim 17** The use according to claim 15, wherein the gRNA recognition sequence comprises or is proximate to position 9,519 as set forth in SEQ ID NO:

1. **Claim 18** The use according to claim 15, wherein the gRNA recognition sequence is located 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 the position corresponding to position 9,519 as set forth in SEQ ID NO:

1. **Claim 19** The use according to claim 15, wherein the protospacer adjacent motif (PAM) sequence is located about 2 to about 6 nucleotides downstream of the gRNA recognition sequence.

20. The use according to claim 15, wherein the gRNA comprises about 17 to about 23 nucleotides.

21. The use according to claim 15, wherein the gRNA recognition sequence comprises the nucleotide sequence set forth in any one of SEQ ID NOs: 93 to 112.

22. The use according to claim 12, wherein the hypertension comprises essential hypertension, secondary hypertension, resistant hypertension, or malignant hypertension.

23. The SLC9A3R2 missense variant nucleic acid molecule is a genomic nucleic acid molecule encoding a loss-of-function polypeptide predicted by solute carrier family 9 isoform A3 regulator 2 (SLC9A3R2) having a nucleotide sequence comprising thymine or its complement at a position corresponding to position 9,519 set forth in SEQ ID NO: 2; or uracil or its complement at a position corresponding to position 615 set forth in SEQ ID NO: 22; uracil or its complement at a position corresponding to position 589 set forth in SEQ ID NO: 23; uracil or its complement at a position corresponding to position 353 set forth in SEQ ID NO: 24; uracil or its complement at a position corresponding to position 230 set forth in SEQ ID NO: 25; uracil or its complement at a position corresponding to position 236 set forth in SEQ ID NO: 26; uracil or its complement at a position corresponding to position 236 set forth in SEQ ID NO: 27; uracil or its complement at a position corresponding to position 604 set forth in SEQ ID NO: 28; or uracil or its complement at a position corresponding to position 126 set forth in SEQ ID NO: 29, an mRNA molecule encoding a loss-of-function polypeptide predicted by SLC9A3R2 or its complement The use according to any one of claims 12 to 22.

24. A pharmaceutical composition for the treatment of hypertension, coronary heart disease and / or atrial fibrillation in a subject, The subject is heterozygous for a missense mutant nucleic acid molecule of solute carrier family 9 isoform A3 regulator 2 (SLC9A3R2) encoding Arg171Trp-Long, Arg171Trp-Short, Arg65Trp, Arg58Trp, Arg60Trp-Short, Arg60Trp-Long, or Arg170Trp, The pharmaceutical composition contains, as an active ingredient, a therapeutic agent for treating or preventing hypertension, coronary artery disease, and / or atrial fibrillation.

25. A pharmaceutical composition for treating hypertension, coronary artery disease, and / or atrial fibrillation in a subject, wherein the subject is a) a reference for a solute carrier family 9 isoform A3 regulator 2 (SLC9A3R2) genomic nucleic acid molecule or an SLC9A3R2 mRNA molecule, or b) heterozygous for a missense mutant nucleic acid molecule of SLC9A3R2 encoding Arg171Trp-Long, Arg171Trp-Short, Arg65Trp, Arg58Trp, Arg60Trp-Short, Arg60Trp-Long, or Arg170Trp, and the pharmaceutical composition contains an SLC9A3R2 inhibitor as an active ingredient.

26. The pharmaceutical composition according to claim 24 or 25, wherein the hypertension includes essential hypertension, secondary hypertension, resistant hypertension, or malignant hypertension.