Piezo-type mechanosensitive ion channel component 1 (PIEZO1) variants and uses thereof

Abstract

Provided herein are methods for treating patients with varicose veins, methods for identifying subjects at high risk for developing varicose veins, and methods for diagnosing varicose veins in a human subject, comprising detecting the presence of a Piezo-type mechanosensitive ion channel component 1 (PIEZO1) predicted loss-of-function variant nucleic acid molecule and polypeptide in a biological sample derived from the patient or subject.

Description

[Technical field]

[0001] The present disclosure provides methods of treating patients with varicose veins, methods of identifying subjects at high risk for developing varicose veins, and methods of diagnosing varicose veins in a human subject, comprising detecting the presence of PIEZO1 predicted loss-of-function variant nucleic acid molecules and polypeptides in a biological sample derived from the patient or subject.

[0002] Sequence Listing Reference This application contains a Sequence Listing, which has been submitted electronically in a text file of 132 kilobytes in size, entitled 18923802502SEQ, created on February 5, 2020. This Sequence Listing is incorporated herein by reference. [Background technology]

[0003] Varicose veins are a common multifactorial disease, the genetic factors of which are largely unknown, and are often found in patients with chronic venous insufficiency, and are classified as chronic venous disease. Dysfunction of venous valves is associated with varicose veins, venous hypertension, and thrombosis. Several processes, such as altered hemodynamics, endothelial activation, inflammation, hypoxia, and dysregulation of matrix metalloproteinases and their tissue inhibitors, are associated with the development of varicose veins. Risk factors for varicose veins include increasing age, female gender, number of pregnancies, obesity, history of deep vein thrombosis, and standing occupation. Varicose veins are also associated with poor lymphatic drainage and chronic venous insufficiency. In addition, several genome-wide association studies (GWAS) have shown a heritability of varicose veins of about 18.5%.

[0004] PIEZO1 is encoded by a 70 kb gene located at 16q24.3 and exists in five potential isoforms. The PIEZO1 protein is 2,521 amino acids in length and is a 286 kDa transmembrane protein that contains 38 transmembrane domains and functions as a homotetramer. PIEZO1 encodes an evolutionarily conserved endothelial mechanosensitive cation channel that generates a current characterized by a linear current-voltage relationship in response to ruthenium red and gadolinium. PIEZO1 is ubiquitously expressed and plays a role in epithelial cell adhesion by maintaining integrin activation, possibly through recruitment of R-Ras to the endoplasmic reticulum, its activated state, and subsequent stimulation of calpain signaling. In the vasculature, PIEZO1 is involved in endothelial cell migration and angiogenic sprouting. Specifically, PIEZO1 functions as a sensor of shear stress associated with blood flow and organizes endothelial cells and aligns them along the blood flow to ensure proper vessel formation, remodeling, and maturation. PIEZO1 is also required for the formation of lymphatic valves. Other reported functions include blood pressure regulation, urinary osmolality, red blood cell integrity, pressure sensing, and regulation of collecting duct osmolality. Summary of the Invention [Means for solving the problem]

[0005] The present disclosure provides a method for identifying a human subject at high risk for developing varicose veins, the method comprising determining, or having determined, the presence or absence, in a biological sample obtained from the subject, of: a piezoelectric mechanosensitive ion channel component 1 (PIEZO1) predicted loss-of-function variant genomic nucleic acid molecule; a PIEZO1 predicted loss-of-function variant mRNA molecule; a PIEZO1 loss-of-function variant cDNA molecule generated from the mRNA molecule; or a PIEZO1 predicted loss-of-function variant polypeptide; the absence of a PIEZO1 predicted loss-of-function variant genomic nucleic acid molecule, mRNA molecule, cDNA molecule, or polypeptide indicates that the subject is not at high risk for developing varicose veins; and the presence of a PIEZO1 predicted loss-of-function variant genomic nucleic acid molecule, mRNA molecule, cDNA molecule, or polypeptide indicates that the subject is at high risk for developing varicose veins.

[0006] The disclosure also provides a method of diagnosing varicose veins in a human subject, comprising detecting the presence or absence, in a sample obtained from the subject, of: a piezoelectric mechanosensitive ion channel component 1 (PIEZO1) predicted loss-of-function variant genomic nucleic acid molecule; a PIEZO1 predicted loss-of-function variant mRNA molecule; a PIEZO1 loss-of-function variant cDNA molecule generated from the mRNA molecule; or a PIEZO1 predicted loss-of-function variant polypeptide; and if the subject has a PIEZO1 predicted loss-of-function variant genomic nucleic acid molecule, mRNA molecule, cDNA molecule, or polypeptide and has one or more symptoms of varicose veins, the subject is diagnosed as having varicose veins.

[0007] The disclosure also provides a method of treating a patient with a therapeutic agent to treat or inhibit varicose veins, the patient suffering from or at high risk of developing varicose veins, the method comprising: obtaining or having obtained a biological sample from the patient; and determining whether the patient has a Piezo-type mechanosensitive ion channel component 1 (PIEZO1) predicted loss-of-function variant nucleic acid molecule encoding a human PIEZO1 polypeptide by performing or having performed a genotyping assay on the biological sample to determine whether the patient has a genotype that includes a Piezo-type mechanosensitive ion channel component 1 (PIEZO1) predicted loss-of-function variant genomic nucleic acid molecule. determining whether the patient has a PIEZO1 association; and if the patient is PIEZO1-associated, administering or continuing to administer to the patient a standard dosage of a therapeutic agent for treating or inhibiting varicose veins; and if the patient is heterozygous or homozygous for the PIEZO1 predicted loss-of-function variant nucleic acid molecule, administering or continuing to administer to the patient an amount equal to or greater than the standard dosage of a therapeutic agent for treating or inhibiting varicose veins; the presence of a genotype having a PIEZO1 predicted loss-of-function variant nucleic acid molecule encoding a human PIEZO1 polypeptide indicates that the patient is at increased risk for developing varicose veins.

[0008] The accompanying drawings, which are incorporated in and constitute a part of this specification, illustrate several aspects and, when read in conjunction with the detailed description, provide a further understanding of the principles of the disclosure. [Brief description of the drawings]

[0009] [Figure 1] Representative distribution of shared IBD for individual pairs in UKB 50k WES; estimated proportion of WES genotypes with homoeologous IBD without any alleles and WES genotypes with one allele IBD across all pairs of UKB 50k exome participants. [Diagram 2]Site frequency spectra (SFS) are shown for all autosomal variants and by functional prediction; the UKB 50k exome was randomly downsampled for the number of individuals shown on the horizontal axis; the number of genes containing at least the number of explicit LOFs AAF<1% as indicated in the legend is plotted on the vertical axis; the maximum number of autosomal genes is 18,272. [Figure 3-1] UK Biobank 500k and 50k continental ancestry; Principal Components 1 and 2 for n=488,377 available from the UK Biobank Data Showcase; three regions of predefined plots represent ancestry in Africa (blue), East Asia (green), and Europe (red). [Figure 3-2] UK Biobank 500k and 50k continental ancestry; Principal Components 1 and 2 for n=488,377 available from the UK Biobank Data Showcase; three regions of predefined plots represent ancestry in Africa (blue), East Asia (green), and Europe (red). [Figure 4] Single point and aggregate results for 65 pLOF variants in PIEZO1 are shown. [Diagram 5] LD evaluation for PIEZO1 is shown. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0010] Various terms relating to the aspects of the present disclosure are used throughout the specification and claims. Such terms are to be given their ordinary meaning in the art unless otherwise specified. Other specifically defined terms are to be interpreted in a manner consistent with the definition provided herein.

[0011] Unless otherwise specified, it is not intended that any method or aspect described herein be construed as requiring that its steps be performed in a particular order. Thus, unless a method claim is specifically recited in the claims or detailed description to be limited to a particular order of steps, no order is intended to be inferred in any respect. This applies to issues of logic regarding the arrangement of steps or operational flow, the apparent meaning derived from grammatical construction or punctuation, or possible imprecise criteria for interpretation, including the number or type of aspects described herein.

[0012] As used herein, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise. As used herein, the terms "subject" and "patient" are used interchangeably. A subject can include any animal, including mammals. Mammals include, but are not limited to, farm animals (e.g., horses, cows, pigs, etc.), pet animals (e.g., dogs, cats), laboratory animals (e.g., mice, rats, rabbits), and non-human primates. In some embodiments, the subject is a human.

[0013] As used herein, "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 single strand of a nucleic acid also refers to its complement.

[0014] As used herein, the term "comprising" may be interchanged with "consisting of" or "consisting essentially of" in certain embodiments, as appropriate.

[0015] As used herein, the phrase "corresponding to" or grammatical variations thereof, when used in the context of numbering a particular amino acid or nucleotide sequence or position, refers to the numbering in the particular reference sequence when comparing the particular amino acid or nucleotide sequence to a reference sequence (e.g., the reference sequence herein is a nucleic acid molecule or polypeptide of (wild type) PIEZO1). In other words, the number of residues (e.g., amino acids or nucleotides) or the position of the residues (e.g., amino acids or nucleotides) of a particular polymer is specified with respect to the reference sequence, not the actual numerical position of the residue within the particular amino acid or nucleic acid sequence. For example, a particular amino acid sequence can be aligned to a reference sequence by introducing gaps to optimize the residue matching between the two sequences. In these cases, the numbering of the residues in the particular amino acid or nucleic acid sequence is performed with respect to the reference sequence to which it is aligned, even if gaps exist.

[0016] In accordance with the present disclosure, it has been found that certain alterations in PIEZO1 are associated with the risk of developing varicose veins. It is believed that variants of the PIEZO1 gene or protein do not have any known association with varicose veins in humans. Thus, human subjects with PIEZO1 alterations associated with varicose veins may be treated to inhibit varicose veins, reduce symptoms, and / or prevent the onset of symptoms. Thus, the present disclosure provides a method of identifying or stratifying the risk of developing varicose veins in such subjects, or diagnosing subjects with varicose veins, so that subjects at risk or with active disease can be treated.

[0017] For the purposes of this disclosure, a particular human can be classified as having one of three PIEZO1 genotypes: i) PIEZO1-related; ii) heterozygosity for a PIEZO1 predicted loss-of-function variant; and iii) homozygosity for a PIEZO1 predicted loss-of-function variant. If a human does not have a copy of a PIEZO1 predicted loss-of-function variant nucleic acid molecule, the human is PIEZO1-related. If a human has a single copy of a PIEZO1 predicted loss-of-function variant nucleic acid molecule, the human is heterozygous for a PIEZO1 predicted loss-of-function variant. A PIEZO1 predicted loss-of-function variant nucleic acid molecule is any PIEZO1 nucleic acid molecule (e.g., a genomic nucleic acid molecule, an mRNA molecule, or a cDNA molecule) that encodes a PIEZO1 polypeptide with partial loss-of-function, complete loss-of-function, partial predicted loss-of-function, or complete predicted loss-of-function. A human with a PIEZO1 polypeptide with partial loss-of-function (or partial predicted loss-of-function) is hypomorphic for PIEZO1. The PIEZO1 predicted loss-of-function variant nucleic acid molecule can be any variant nucleic acid molecule described herein. If a human has two copies of any of the PIEZO1 predicted loss-of-function variant nucleic acid molecules, the human is homozygous for the PIEZO1 predicted loss-of-function variant.

[0018] In the case of a human subject or patient who has been genotyped or determined to be heterozygous or homozygous for a PIEZO1 predicted loss-of-function variant nucleic acid molecule, such a human subject or patient is at high risk of developing varicose veins.In the case of a human subject or patient who has been genotyped or determined to be heterozygous or homozygous for a PIEZO1 predicted loss-of-function variant nucleic acid molecule, such a human subject or patient can be treated with an agent effective in treating varicose veins.

[0019] The present disclosure provides a method for identifying a human subject at high risk for developing varicose veins, the method comprising determining, or having determined, the presence or absence of a PIEZO1 predicted loss-of-function variant nucleic acid molecule (such as a genomic nucleic acid molecule, an mRNA molecule, and / or a cDNA molecule) or polypeptide in a biological sample obtained from the subject; the absence of the PIEZO1 predicted loss-of-function variant nucleic acid molecule or polypeptide indicates that the subject is not at high risk for developing varicose veins; and the presence of a PIEZO1 predicted loss-of-function variant genomic nucleic acid molecule, mRNA molecule, cDNA molecule, or polypeptide indicates that the subject is at high risk for developing varicose veins.

[0020] The disclosure also provides a method for identifying a human subject at increased risk for developing varicose veins, the method comprising determining, or having determined, the presence or absence in a biological sample obtained from the subject: i) a PIEZO1 predicted loss-of-function variant genomic nucleic acid molecule; ii) a PIEZO1 predicted loss-of-function variant mRNA molecule; iii) a PIEZO1 predicted loss-of-function variant cDNA molecule generated from the mRNA molecule, or iv) a PIEZO1 predicted loss-of-function variant polypeptide; wherein the absence of the PIEZO1 predicted loss-of-function variant genomic nucleic acid molecule, mRNA molecule, cDNA molecule, or polypeptide indicates that the subject is not at increased risk for developing varicose veins; and the presence of the PIEZO1 predicted loss-of-function variant genomic nucleic acid molecule, mRNA molecule, cDNA molecule, or polypeptide indicates that the subject is at increased risk for developing varicose veins.

[0021] The disclosure also provides a method for identifying a human subject at high risk for developing varicose veins, the method comprising determining, or having determined, the presence or absence of a PIEZO1 predicted loss-of-function variant nucleic acid molecule (such as a genomic nucleic acid molecule, an mRNA molecule, and / or a cDNA molecule) encoding a human PIEZO1 polypeptide in a biological sample obtained from the subject; i) if the human subject does not have the PIEZO1 predicted loss-of-function variant nucleic acid molecule (i.e., the human subject is genotypically classified as PIEZO1-related), the human subject is not at high risk for developing varicose veins; and ii) if the human subject has the PIEZO1 predicted loss-of-function variant nucleic acid molecule (i.e., the human subject is classified as heterozygous for the PIEZO1 predicted loss-of-function variant or is classified as homozygous for the PIEZO1 predicted loss-of-function variant), the human subject is at high risk for developing varicose veins.

[0022] For any of the embodiments described herein, the PIEZO1 predicted loss-of-function variant nucleic acid molecule can be any PIEZO1 nucleic acid molecule (e.g., a genomic nucleic acid molecule, an mRNA molecule, or a cDNA molecule, etc.) that encodes a PIEZO1 polypeptide having partial loss-of-function, complete loss-of-function, partial predicted loss-of-function, or complete predicted loss-of-function. For example, the PIEZO1 predicted loss-of-function variant nucleic acid molecule can be any of the PIEZO1 variant nucleic acid molecules described herein.

[0023] Determining whether a human subject has a PIEZO1 predicted loss-of-function variant nucleic acid molecule in a biological sample can be performed by any of the methods described herein. In some embodiments, these methods can be performed in vitro. In some embodiments, these methods can be performed in situ. In some embodiments, these methods can be performed in vivo. In any of these embodiments, the nucleic acid molecule can be present in a cell obtained from the human subject.

[0024] For any of the embodiments described herein, the varicose veins can be early stage varicose veins (e.g., C0 according to the CEAP (Clinical, Etiological, Anatomical, and Pathophysiological) classification). In some embodiments, the varicose veins can be late stage varicose veins (e.g., C6 according to the CEAP classification). In some embodiments, the varicose veins can be of any stage (e.g., C0-C6 according to the CEAP classification). In some embodiments, the human subject is female.

[0025] In some embodiments, if a human subject is identified as being at high risk of developing varicose veins, the human subject is further treated with a therapeutic agent for treating or inhibiting varicose veins as described herein. For example, if a human subject is heterozygous or homozygous for a PIEZO1 predicted loss-of-function variant nucleic acid molecule and is therefore at high risk of developing varicose veins, the human subject is administered a therapeutic agent for treating or inhibiting varicose veins. In some embodiments, if a patient is homozygous for a PIEZO1 predicted loss-of-function variant nucleic acid molecule, the patient is administered a therapeutic agent for treating or inhibiting varicose veins in an amount equal to or greater than the standard dose administered to a patient who is heterozygous for a PIEZO1 predicted loss-of-function variant nucleic acid molecule. In some embodiments, the patient is heterozygous for a PIEZO1 predicted loss-of-function variant nucleic acid molecule. In some embodiments, the patient is homozygous for a PIEZO1 predicted loss-of-function variant nucleic acid molecule.

[0026] The present disclosure provides a method of diagnosing varicose veins in a human subject, the method comprising detecting the presence or absence of a PIEZO1 predicted loss-of-function variant nucleic acid molecule (such as a genomic nucleic acid molecule, an mRNA molecule, and / or a cDNA molecule) or polypeptide in a sample obtained from the subject; if the subject has a PIEZO1 predicted loss-of-function variant genomic nucleic acid molecule, mRNA molecule, cDNA molecule, or polypeptide and exhibits one or more symptoms of varicose veins, the subject is diagnosed as having varicose veins.

[0027] The disclosure also provides a method of diagnosing varicose veins in a human subject, the method comprising detecting the presence or absence, in a sample obtained from the subject, of: i) a PIEZO1 predicted loss-of-function variant genomic nucleic acid molecule; ii) a PIEZO1 predicted loss-of-function variant mRNA molecule; iii) a PIEZO1 predicted loss-of-function variant cDNA molecule generated from the mRNA molecule, or iv) a PIEZO1 predicted loss-of-function variant polypeptide; and if the subject has a PIEZO1 predicted loss-of-function variant genomic nucleic acid molecule, mRNA molecule, cDNA molecule, or polypeptide and exhibits one or more symptoms of varicose veins, the subject is diagnosed as having varicose veins.

[0028] The disclosure also provides a method of diagnosing varicose veins in a human subject, the method comprising detecting the presence or absence, in a sample obtained from the subject, of a PIEZO1 predicted loss-of-function variant nucleic acid molecule (such as a genomic nucleic acid molecule, an mRNA molecule, and / or a cDNA molecule) encoding a human PIEZO1 polypeptide; and diagnosing the subject as having varicose veins if the subject has the PIEZO1 predicted loss-of-function variant genomic nucleic acid molecule, mRNA molecule, cDNA molecule, or polypeptide (i.e., the human subject is classified as heterozygous or homozygous for the PIEZO1 predicted loss-of-function variant nucleic acid molecule) and exhibits one or more symptoms of varicose veins.

[0029] For any of the embodiments described herein, the PIEZO1 predicted loss-of-function variant nucleic acid molecule can be any PIEZO1 nucleic acid molecule (e.g., a genomic nucleic acid molecule, an mRNA molecule, or a cDNA molecule, etc.) that encodes a PIEZO1 polypeptide having partial loss-of-function, complete loss-of-function, partial predicted loss-of-function, or complete predicted loss-of-function. For example, the PIEZO1 predicted loss-of-function variant nucleic acid molecule can be any of the PIEZO1 variant nucleic acid molecules described herein.

[0030] Detection of the presence or absence of a PIEZO1 predicted loss-of-function variant nucleic acid molecule in a sample obtained from a subject can be performed by any of the methods described herein. In some embodiments, these methods can be performed in vitro. In some embodiments, these methods can be performed in situ. In some embodiments, these methods can be performed in vivo. In any of these embodiments, the nucleic acid molecule can be present in a cell obtained from a human subject.

[0031] For any of the embodiments described herein, the varicose veins can be early stage varicose veins (e.g., C0 according to the CEAP (Clinical, Etiological, Anatomical, and Pathophysiological) classification). In some embodiments, the varicose veins can be late stage varicose veins (e.g., C6 according to the CEAP classification). In some embodiments, the varicose veins can be of any stage (e.g., C0-C6 according to the CEAP classification). In some embodiments, the human subject is female.

[0032] In some embodiments, if a human subject is diagnosed as having varicose veins, the human subject is further treated with a therapeutic agent for treating or inhibiting varicose veins as described herein. For example, if a human subject is determined to be heterozygous or homozygous for a PIEZO1 predicted loss-of-function variant nucleic acid molecule and exhibits one or more symptoms of varicose veins, the human subject is administered a therapeutic agent for treating or inhibiting varicose veins. In some embodiments, if a patient is homozygous for a PIEZO1 predicted loss-of-function variant nucleic acid molecule, the patient is administered a therapeutic agent for treating or inhibiting varicose veins in an amount equal to or greater than the standard dosage administered to a patient who is heterozygous for a PIEZO1 predicted loss-of-function variant nucleic acid molecule. In some embodiments, the patient is heterozygous for a PIEZO1 predicted loss-of-function variant nucleic acid molecule. In some embodiments, the patient is homozygous for a PIEZO1 predicted loss-of-function variant nucleic acid molecule.

[0033] The disclosure also provides a method of treating a patient with a therapeutic agent to treat or inhibit varicose veins, the patient having varicose veins or at high risk of developing varicose veins, the method comprising: obtaining or having obtained a biological sample from the patient; and determining whether the patient has a PIEZO1 predicted loss-of-function variant nucleic acid molecule encoding a human PIEZO1 polypeptide by performing or having performed a genotyping assay on the biological sample to determine whether the patient has a genotype that includes a PIEZO1 predicted loss-of-function variant nucleic acid molecule. and if the patient has a PIEZO1 association, administer or continue to administer a standard dose of a therapeutic agent for treating or inhibiting varicose veins to the patient; and if the patient is heterozygous or homozygous for the PIEZO1 predicted loss-of-function variant nucleic acid molecule, administer or continue to administer a standard dose of a therapeutic agent for treating or inhibiting varicose veins to the patient; the presence of a genotype having a PIEZO1 predicted loss-of-function variant nucleic acid molecule encoding a human PIEZO1 polypeptide indicates that the patient is at high risk for developing varicose veins. In some embodiments, the patient is heterozygous for the PIEZO1 predicted loss-of-function variant nucleic acid molecule. In some embodiments, the patient is homozygous for the PIEZO1 predicted loss-of-function variant nucleic acid molecule.

[0034] For any of the embodiments described herein, the PIEZO1 predicted loss-of-function variant nucleic acid molecule can be any PIEZO1 nucleic acid molecule (e.g., a genomic nucleic acid molecule, an mRNA molecule, or a cDNA molecule, etc.) that encodes a PIEZO1 polypeptide having partial loss-of-function, complete loss-of-function, partial predicted loss-of-function, or complete predicted loss-of-function. For example, the PIEZO1 predicted loss-of-function variant nucleic acid molecule can be any of the PIEZO1 variant nucleic acid molecules described herein.

[0035] Genotyping assays to determine whether a patient has a PIEZO1 predicted loss-of-function variant nucleic acid molecule encoding a human PIEZO1 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 nucleic acid molecule can be present in a cell obtained from a human subject.

[0036] In some embodiments, if the patient is homozygous for the PIEZO1 predicted loss-of-function variant nucleic acid molecule, the patient is administered an amount of a therapeutic agent for treating or inhibiting varicose veins that is equal to or exceeds a standard dose administered to a patient who is heterozygous for the PIEZO1 predicted loss-of-function variant nucleic acid molecule.

[0037] The disclosure also provides a method of treating a patient with a therapeutic agent for treating or inhibiting varicose veins, the patient suffering from varicose veins or at high risk of developing varicose veins, the method comprising: obtaining or having obtained a biological sample from the patient; and determining whether the patient has a PIEZO1 predicted loss-of-function variant polypeptide by performing or having performed a genotyping assay on the biological sample to determine whether the patient has a PIEZO1 predicted loss-of-function variant polypeptide; and if the patient does not have a PIEZO1 predicted loss-of-function variant polypeptide, administering or continuing to administer a therapeutic agent for treating or inhibiting varicose veins to the patient at a standard dosage; and if the patient has a PIEZO1 predicted loss-of-function variant polypeptide, administering or continuing to administer a therapeutic agent for treating or inhibiting varicose veins to the patient at an amount equal to or greater than the standard dosage; the presence of a PIEZO1 predicted loss-of-function variant polypeptide indicates that the patient is at high risk of developing varicose veins. In some embodiments, the patient has a PIEZO1 predicted loss-of-function variant polypeptide. In some embodiments, the patient does not have a PIEZO1 predicted loss-of-function variant polypeptide.

[0038] Assays for determining whether a patient has a PIEZO1 predicted loss-of-function variant polypeptide can be performed by any of the methods described herein. In some embodiments, these methods can be performed in vitro. In some embodiments, these methods can be performed in situ. In some embodiments, these methods can be performed in vivo. In any of these embodiments, the polypeptide can be present in a cell obtained from a human subject.

[0039] For any of the embodiments described herein, the PIEZO1 predicted loss-of-function variant polypeptide can be a PIEZO1 polypeptide having a partial loss of function, a complete loss of function, a partial predicted loss of function, or a complete predicted loss of function. For example, the PIEZO1 predicted loss-of-function variant polypeptide can be any of the PIEZO1 variant polypeptides described herein.

[0040] For any of the embodiments described herein, the varicose veins can be early stage varicose veins (e.g., C0 according to the CEAP (Clinical, Etiological, Anatomical, and Pathophysiological) classification). In some embodiments, the varicose veins can be late stage varicose veins (e.g., C6 according to the CEAP classification). In some embodiments, the varicose veins can be of any stage (e.g., C0-C6 according to the CEAP classification). In some embodiments, the human subject is female.

[0041] Symptoms of varicose veins include, but are not limited to, heaviness in the legs, spider veins (telangiectasia) in the affected areas of the legs, swelling of the ankles, especially in the evening, shiny yellow-brown discoloration of the skin near the affected veins, redness, dryness, and itching of the skin (also known as stasis dermatitis, or varicose eczema), cramping especially with sudden movements such as standing up, minor bruising in the affected areas, more bleeding than usual or longer healing times, atrophy of the skin above the ankles (lipodermatosclerosis), restless legs syndrome, irregular white bruise-like spots on the ankles (albinoatrophy), or any combination thereof.

[0042] Examples of therapeutic agents that treat or inhibit varicose veins include, but are not limited to, flavonoids, such as diosmin, or hesperidin, and anti-inflammatory agents, such as ibuprofen and aspirin.

[0043] In some embodiments, the dose of a therapeutic agent for treating or inhibiting varicose veins can be reduced (i.e., less than a standard dose) in a patient or human subject that is heterozygous for a PIEZO1 predicted loss-of-function variant nucleic acid molecule compared to a patient or human subject that is homozygous for a PIEZO1 predicted loss-of-function variant nucleic acid molecule (which can receive a standard dose). In some embodiments, the dose of a therapeutic agent for treating or inhibiting varicose veins can 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 inhibiting varicose veins can be administered less frequently in a patient or human subject that is heterozygous for a PIEZO1 predicted loss-of-function variant nucleic acid molecule compared to a patient or human subject that is homozygous for a PIEZO1 predicted loss-of-function variant nucleic acid molecule.

[0044] The administration of the therapeutic agent for treating or inhibiting varicose veins 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 at the same dose or at a different dose. The administration can be repeated 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more times. For example, according to a particular dosing regimen, a patient can be treated for an extended period of time, for example, 6 months, 1 year, or more.

[0045] Administration of the therapeutic agent for treating or inhibiting varicose veins can be by any suitable route, including, but not limited to, parenteral, intravenous, oral, subcutaneous, intraarterial, intracranial, intrathecal, intraperitoneal, topical, intranasal, or intramuscular. Pharmaceutical compositions for administration are desirably sterile and substantially isotonic and prepared under GMP conditions. Pharmaceutical compositions can be provided in unit dosage form (i.e., a single dosage for administration). Pharmaceutical compositions can be formulated with one or more physiologically and pharma- ceutical acceptable carriers, diluents, excipients, or adjuvants. The formulation depends on the route of administration chosen. The term "pharma-ceutical 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.

[0046] As used herein, the terms "treat," "treating," and "treatment" and "prevent," "preventing," and "prevention" refer to eliciting a desired biological response, such as a therapeutic or prophylactic effect, respectively. In some embodiments, a therapeutic effect includes one or more of the following observed following administration of a therapeutic agent or a composition comprising the therapeutic agent: reduction / inhibition of varicose veins, reduction / inhibition of the severity of varicose veins (e.g., reduction or inhibition of the onset of varicose veins), reduction / inhibition of symptoms of varicose vein related effects, delay in onset of symptoms of varicose vein related effects, reduction in the severity of symptoms of varicose vein related effects, reduction in the severity of acute attacks, reduction in the number of symptoms of varicose vein related effects, reduction in the latency of symptoms of varicose vein related effects, relief of symptoms of varicose vein related effects, reduction in secondary symptoms, reduction in secondary infections, prevention of recurrence of varicose veins, reduction in the number or frequency of recurrent attacks, longer time between symptomatic attacks, longer sustained progression, quicker recovery, and / or improved efficiency of alternative therapies, or reduced resistance. A prophylactic effect may include complete or partial avoidance / inhibition or delay in onset / progression of varicose veins (e.g., complete or partial avoidance / inhibition or delay, etc.) observed following administration of a therapeutic protocol. Treatment of varicose veins includes treatment of patients who have been diagnosed with any form of varicose veins, at any clinical stage or presentation, delaying the onset or progression or severity or worsening of symptoms or signs of varicose veins, and / or preventing and / or inhibiting the progression of varicose veins.

[0047] The present disclosure also provides for the detection or determination of the presence of PIEZO1 predicted loss-of-function variant genomic nucleic acid molecules, PIEZO1 predicted loss-of-function variant mRNA molecules, and / or PIEZO1 predicted loss-of-function variant cDNA molecules in biological samples obtained from human subjects in any of the embodiments described herein. It should be understood that gene sequences within a population, and the mRNA molecules encoded by such genes, may vary due to polymorphisms, such as single nucleotide polymorphisms. The sequences provided herein for the PIEZO1 variant nucleic acid molecules disclosed herein are merely exemplary sequences. Other sequences of PIEZO1 variant nucleic acid molecules are possible.

[0048] The biological sample can be derived from any cell, tissue, or bodily fluid from a subject. The sample can include any clinically relevant tissue, such as a bone marrow sample, a tumor biopsy, a fine needle aspirate, or a sample of bodily fluids, such as blood, gingival crevicular fluid, plasma, serum, lymph, ascites, cyst, or urine. In some cases, the sample includes a buccal swab. The sample used in the methods disclosed herein varies depending on the assay format, the nature of the detection method, and the tissue, cell, or extract used as the sample. The biological sample can be treated in various ways depending on the assay used. For example, when detecting PIEZO1 variant nucleic acid molecules, a pretreatment designed to isolate or enrich the sample for genomic DNA can be performed. For this purpose, various known techniques can be used. When detecting the level of PIEZO1 variant mRNA, various techniques can be used to enrich the biological sample containing mRNA. Various methods can be used to detect the presence or level of mRNA, or the presence of a specific variant genomic DNA locus.

[0049] In some embodiments, detection of a human PIEZO1 predicted loss-of-function variant nucleic acid molecule in a human subject involves assaying or genotyping a biological sample obtained from the human subject to determine whether a PIEZO1 genomic nucleic acid molecule, a PIEZO1 mRNA molecule, or a PIEZO1 cDNA molecule generated from the mRNA molecule in the biological sample contains one or more alterations that cause or are predicted to cause a loss of function (partial or complete).

[0050] In some embodiments, a method for detecting the presence or absence of a PIEZO1 predicted loss-of-function variant nucleic acid molecule (e.g., a genomic nucleic acid molecule, an mRNA molecule, and / or a cDNA molecule) in a human subject includes: performing an assay on a biological sample obtained from the human subject, where the assay determines whether a nucleic acid molecule in the biological sample comprises a particular nucleotide sequence.

[0051] In some embodiments, the biological sample comprises cells or cell lysates.Such a method can further comprise, for example, obtaining a biological sample from a subject that comprises PIEZO1 genomic nucleic acid molecule or mRNA molecule, and if it is mRNA, optionally reverse transcribing the mRNA into cDNA, and such an assay can comprise, for example, determining the identity of these positions of a particular PIEZO1 nucleic acid molecule.In some embodiments, this method is an in vitro method.

[0052] In some embodiments, the determining step, detecting step, or genotyping assay comprises sequencing at least a portion of the nucleotide sequence of a PIEZO1 genomic nucleic acid molecule, a PIEZO1 mRNA molecule, or a PIEZO1 cDNA molecule generated from the mRNA molecule in a biological sample, wherein the sequenced portion contains one or more alterations that cause or are predicted to cause a loss of function (partial or complete).

[0053] In any of the methods described herein, the determining step, detecting step, or genotyping assay includes sequencing at least a portion of the nucleotide sequence of a PIEZO1 nucleic acid molecule in the biological sample, wherein the sequenced portion includes a position corresponding to a predicted loss-of-function variant position, and if a variant nucleotide at the predicted loss-of-function variant position is detected, the PIEZO1 nucleic acid molecule in the biological sample is a PIEZO1 predicted loss-of-function variant nucleic acid molecule.

[0054] In some embodiments, the determining step, detecting step, or genotyping assay comprises: a) contacting the biological sample with a primer that hybridizes to a portion of the nucleotide sequence of the PIEZO1 nucleic acid molecule adjacent to the predicted loss-of-function variant position; b) extending the primer to at least the position of the predicted loss-of-function variant; and c) determining whether the extension product of the primer comprises a variant nucleotide at the predicted loss-of-function variant position.

[0055] In some embodiments, the assay comprises sequencing the entire nucleic acid molecule. In some embodiments, only the PIEZO1 genomic nucleic acid molecule is analyzed. In some embodiments, only the PIEZO1 mRNA is analyzed. In some embodiments, only the PIEZO1 cDNA derived from the PIEZO1 mRNA is analyzed.

[0056] In some embodiments, the determining step, detecting step, or genotyping assay comprises: a) amplifying at least a portion of a PIEZO1 nucleic acid molecule encoding a human PIEZO1 polypeptide, the portion including the predicted loss-of-function variant position; b) labeling the amplified nucleic acid molecule with a detectable label; c) contacting the labeled nucleic acid molecule with a support comprising a variant-specific probe, the variant-specific probe including a nucleotide sequence that hybridizes to the predicted loss-of-function variant position under stringent conditions; and d) detecting the detectable label.

[0057] In some embodiments, the nucleic acid molecule is mRNA and the determining step further comprises reverse transcribing the mRNA into cDNA prior to the amplifying step. In some embodiments, the determining step, detecting step, or genotyping assay comprises contacting a nucleic acid molecule in a biological sample with a variant-specific probe comprising a detectable label, the variant-specific probe comprising a nucleotide sequence that hybridizes to the predicted loss-of-function variant position under stringent conditions; and detecting the detectable label.

[0058] The variant-specific probe or variant-specific primer described herein comprises a nucleic acid sequence that is complementary and / or hybridizes or specifically hybridizes to a PIEZO1 predicted loss-of-function variant nucleic acid molecule or its complement. In some embodiments, the variant-specific probe or variant-specific primer comprises or consists 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, at least about 25, at least about 30, at least about 35, at least about 40, at least about 45, or at least about 50 nucleotides. In some embodiments, the variant-specific probe or variant-specific primer comprises or consists of at least 15 nucleotides. In some embodiments, the variant-specific probe or variant-specific primer comprises or consists of at least 15 nucleotides to at least about 35 nucleotides. In some embodiments, the variant-specific probe or variant-specific primer hybridizes under stringent conditions to a PIEZO1 predicted loss-of-function variant genomic nucleic acid molecule, a PIEZO1 predicted loss-of-function variant mRNA molecule, and / or a PIEZO1 predicted loss-of-function variant cDNA molecule.

[0059] Variation-specific polymerase chain reaction techniques can be used to detect mutations, such as SNPs, in nucleic acid sequences. Variation-specific primers can be used because DNA polymerase will not extend if there is a mismatch with the template.

[0060] In some embodiments, the nucleic acid molecule contained in the sample is mRNA, and the mRNA is reverse transcribed into cDNA prior to the amplification step. In some embodiments, the nucleic acid molecule is present in a cell obtained from a human subject.

[0061] For any of the embodiments described herein, the PIEZO1 predicted loss-of-function variant nucleic acid molecule can be any PIEZO1 nucleic acid molecule (e.g., a genomic nucleic acid molecule, an mRNA molecule, or a cDNA molecule, etc.) that encodes a PIEZO1 polypeptide having a partial loss-of-function, a complete loss-of-function, a predicted partial loss-of-function, or a predicted complete loss-of-function. For example, the PIEZO1 predicted loss-of-function variant nucleic acid molecule can be any of the PIEZO1 variant nucleic acid molecules described herein.

[0062] In some embodiments, the assay involves contacting the biological sample with a primer or probe, such as a variant-specific primer or variant-specific probe, that specifically hybridizes to a PIEZO1 variant genomic sequence, variant mRNA sequence, or variant cDNA sequence under stringent conditions and does not hybridize to a corresponding PIEZO1-related sequence, and determining the presence or absence of hybridization.

[0063] In some embodiments, the assay comprises RNA sequencing (RNA-Seq). In some embodiments, the assay also comprises reverse transcription of mRNA into cDNA, such as by reverse transcriptase polymerase chain reaction (RT-PCR).

[0064] In some embodiments, the method utilizes probes and primers having a nucleotide length sufficient to bind to the target nucleotide sequence and specifically detect and / or identify the polynucleotide comprising the PIEZO1 variant genomic nucleic acid molecule, variant mRNA molecule, or variant cDNA molecule. Hybridization or reaction conditions can be determined by the operator to achieve this goal. The nucleotide length can be any length sufficient for use in the selected detection method, such as the assays described or exemplified herein. Such probes and primers can specifically hybridize to the target nucleotide sequence under high stringency hybridization conditions. The probes can be conventionally designed to be different from the target nucleotide sequence and retain the ability to specifically detect and / or identify the target nucleotide sequence, although the probes and primers can have complete nucleotide sequence identity to adjacent nucleotides in the target nucleotide sequence. 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.

[0065] 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, such as using labeled primers or probes on purified DNA, amplified DNA, and fixed cell preparations (fluorescence in situ hybridization (FISH)). In some methods, the target nucleic acid may be amplified prior to or simultaneously with detection. 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).

[0066] Hybridization technology can use stringent conditions that probe or primer specifically hybridize to its target.In some embodiments, polynucleotide primer or probe under stringent conditions hybridize to its target sequence with detectable intensity than other non-target sequences, for example, at least 2 times, at least 3 times, at least 4 times or more intensity than background, such as more than 10 times background.Stringent conditions vary according to sequence and situation.

[0067] Suitable stringent conditions for promoting 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 and are described in Current Protocols in Molecular Biology, John Wiley & Sons, NY (1989), 6.3.1-6.3.6. In general, 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 ion (or other salt) concentration and temperature are at least about 30°C for short probes (e.g., 10-50 nucleotides) and at least about 60°C for long probes (more than 50 nucleotides). Stringent conditions can also be achieved by adding destabilizing agents such as formamide. Optionally, the wash buffer can 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 length of the wash time is short but long enough to reach equilibrium.

[0068] The present disclosure also provides a molecular complex comprising any of the PIEZO1 nucleic acid molecules (genomic nucleic acid molecules, mRNA molecules, or cDNA molecules) described herein, or their complements, and any of the modification-specific primers or modification-specific probes described herein. In some embodiments, the PIEZO1 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 PIEZO1 nucleic acid molecule is any of the genomic nucleic acid molecules described herein. In some embodiments, the PIEZO1 nucleic acid molecule is any of the mRNA molecules described herein. In some embodiments, the PIEZO1 nucleic acid molecule is any of the cDNA molecules described herein. In some embodiments, the molecular complex comprises any of the PIEZO1 nucleic acid molecules (genomic nucleic acid molecules, mRNA molecules, or cDNA molecules) described herein, or their complements, and any of the modification-specific primers described herein. In some embodiments, the molecular complex comprises any of the PIEZO1 nucleic acid molecules (genomic nucleic acid molecules, mRNA molecules, or cDNA molecules) described herein, or their complements, and any of the modification-specific probes described herein. In some embodiments, the molecular complex comprises a non-human polymerase.

[0069] In some embodiments, detecting the presence of a human PIEZO1 predicted loss-of-function polypeptide comprises performing an assay on a sample obtained from a human subject to determine whether a PIEZO1 polypeptide in the subject comprises one or more variants that confer loss of function (partial or complete) or predicted loss of function (partial or complete) to the polypeptide. In some embodiments, the assay comprises sequencing at least a portion of the PIEZO1 polypeptide comprising the variant position. In some embodiments, the detecting step comprises sequencing the entire polypeptide. Identification of a variant amino acid at a variant position of the PIEZO1 polypeptide indicates that the PIEZO1 polypeptide is a PIEZO1 predicted loss-of-function polypeptide. In some embodiments, the assay comprises an immunoassay for detecting the presence of a polypeptide comprising a variant. Detection of a variant amino acid at a variant position of the PIEZO1 polypeptide indicates that the PIEZO1 polypeptide is a PIEZO1 predicted loss-of-function polypeptide.

[0070] The probes and / or primers described herein (including the modification-specific probes and the modification-specific primers) comprise or consist of about 15 to about 100, about 15 to about 35 nucleotides. In some embodiments, the modification-specific probes and the modification-specific primers comprise DNA. In some embodiments, the modification-specific probes and the modification-specific primers comprise RNA. In some embodiments, the probes and primers described herein (including the modification-specific probes and the modification-specific primers) have a nucleotide sequence that specifically hybridizes to any of the nucleic acid molecules disclosed herein, or to their complements. In some embodiments, the probes and primers (including the modification-specific probes and the modification-specific primers) specifically hybridize to any of the nucleic acid molecules disclosed herein under stringent conditions. In the context of the present disclosure, "specifically hybridize" means that the probe or primer (including the modification-specific probes and the modification-specific primers) does not hybridize to a nucleic acid sequence encoding a PIEZO1-related genomic nucleic acid molecule, a PIEZO1-related mRNA molecule, and / or a PIEZO1-related cDNA molecule. In some embodiments, the probe (such as, for example, a modification-specific probe) comprises a label. In some embodiments, the label is a fluorescent label, a radioactive label, or biotin.

[0071] The nucleotide sequence of the PIEZO1 reference genomic nucleic acid molecule is shown in SEQ ID NO: 1 and is 69,883 nucleotides in length. The first nucleotide set forth in SEQ ID NO: 1 corresponds to the nucleotide at position 88,715,338 of chromosome 16 (see hg38_knownGene_ENST00000301015.14).

[0072] PIEZO1 has numerous variant genomic nucleic acid molecules (using the human genome reference build GRch38): 16:88715629:G:A, 16:88715728:G:T, 16:88715767:G:A, 16:88715802:C:A, 16:88715822:D:4, 16:88715987:I:1, 16:88716359:A:G, 16:88716570:C:T, 16:88716874:G:A, 16:88717213:T:A, 16:88719588:G:A, 16:88719 722:C:G, 16:88719870:G:T, 16:88720068:D:2, 16:88720229:C:A, 16:887 20248:D:4, 16:88720394:C:T, 16:88720644:D:1, 16:88720698:D:1, 16:8 8720698:I:1, 16:88721165:C:A, 16:88721268:D:1, 16:88721307:G:A, 16 :88721586:G:C, 16:88721652:G:C, 16:88722217:C:T, 16:88722605:I:1, 16:88723005:I:7, 16:88723253:G:A, 16:88723311:C:T, 16:88725081:C: A, 16:88726282:G:A, 16:88726546:C:T, 16:88726619:G:A, 16:88726924: G:A, 16:88727038:C:T, 16:88727072:D:1, 16:88727163:G:A, 16:8873176 8:D:1, 16:88732334:C:G, 16:88732411:D:1, 16:88732720:D:1, 16:88733 326:G:C, 16:88733337:D:4, 16:88733587:C:A, 16:88733965:D:1, 16:887 34017:C:A, 16:88734042:I:1, 16:88734679:C:T, 16:88734909:I:1, 16:8 8736167:D:2, 16:88736324:G:A, 16:88736391:G:T, 16:88736409:C:T, 16 :88736671:G:A, 16:88737557:A:C, 16:88737727:C:G, 16:88737815:C:T,These variants include, but are not limited to, 16:88738283:G:C, 16:88738637:G:A, 16:88738735:D:1, 16:88741477:C:T, 16:88742306:D:1, 16:88749399:G:A, and 16:88784929:C:T. Thus, for example, using the reference genome nucleotide sequence of SEQ ID NO:1 as a basis (with the first nucleotide described therein shown as position 88,715,338), the first described variant (16:88715629:G:A) has a substitution of a guanine with an adenine (referred to as the "variant nucleotide") at position 88,715,629 (referred to as the "variant position"). Variants designated with a "D" followed by a number have a deletion of the indicated number of nucleotides. Variants designated with an "I" followed by a number have an insertion of the indicated number of nucleotides (any nucleotide). Any of these predicted PIEZO1 loss-of-function variant genomic nucleic acid molecules can be detected by any of the methods described herein.

[0073] The nucleotide sequence of the PIEZO1-related mRNA molecule is set forth in SEQ ID NO:2 (see NCBI Reference Sequence: NM_001142864.4) and is 8,089 nucleotides in length. The variant nucleotides at each variant position of the variant genomic nucleic acid molecules described herein also have corresponding variant nucleotides at each variant position of the variant mRNA molecules based on the PIEZO1-related mRNA sequence of SEQ ID NO:2. Any of these predicted PIEZO1 loss-of-function variant mRNA molecules can be detected by any of the methods described herein.

[0074] The nucleotide sequence of the PIEZO1-related cDNA molecule is set forth in SEQ ID NO: 3 (based on NCBI Reference Sequence: NM_001142864.4) and is 8,089 nucleotides in length. The variant nucleotides at each variant position of the variant genomic nucleic acid molecules described herein also have corresponding variant nucleotides at each variant position of the variant cDNA molecules based on the PIEZO1-related cDNA sequence of SEQ ID NO: 3. Any of these predicted PIEZO1 loss-of-function variant cDNA molecules can be detected by any of the methods described herein.

[0075] The amino acid sequence of the PIEZO1-related polypeptide is set forth in SEQ ID NO: 4 (see UniProt Accession No. Q92508.4 and NCBI RefSeq Accession No. NM_001142864.4) and is 2,521 amino acids in length. Using the translated nucleotide sequence of either the PIEZO1 mRNA or cDNA molecule, a PIEZO1 variant polypeptide having a translated variant amino acid corresponding to the variant position (codon) can be detected by any of the methods described herein. Any of these PIEZO1 predicted loss-of-function variant polypeptides can be detected by any of the methods described herein.

[0076] The nucleotide and amino acid sequences set forth in the accompanying sequence listing are presented using standard letter abbreviations for nucleotide bases and three letter codes for amino acids. The nucleotide sequences follow the standard convention of beginning at the 5'-terminus of the sequence and working forward (i.e., from left to right on each line) to the 3'-terminus. Only one strand of each nucleotide sequence is shown, but the complementary strand should be understood to be in any relationship with the strand shown. The amino acid sequences follow the standard convention of beginning at the amino-terminus of the sequence and working forward (i.e., from left to right on each line) to the carboxy-terminus.

[0077] As used herein, "corresponding to" or grammatical variations thereof is used to number a particular nucleotide or nucleotide sequence or position, and when a particular nucleotide or nucleotide sequence is compared to a reference sequence, it refers to the numbering of the particular reference sequence. In other words, the number of residues (e.g., nucleotides or amino acids, etc.) of a particular polymer or the position of the residues (e.g., nucleotides or amino acids, etc.) is specified with respect to the reference sequence, not the actual numerical position of the residue in a particular nucleotide or nucleotide sequence. For example, a particular nucleotide sequence can be aligned with a reference sequence by introducing gaps to optimize the residue matching between the two sequences. In these cases, even if gaps exist, the numbering of the residues in a particular nucleotide or nucleotide sequence is performed with respect to the reference sequence that is aligned. There are various computational algorithms that can be used to perform sequence alignment, and can identify the nucleotide or amino acid position in one polymer molecule that corresponds to the nucleotide or amino acid position in another polymer molecule. For example, sequence alignment can be performed using the NCBI BLAST algorithm (Altschul et al., Nucleic Acids Res., 1997, 25, 3389-3402) or the CLUSTALW software (Sievers and Higgins, Methods Mol. Biol., 2014, 1079, 105-116). Of course, sequences can also be aligned manually.

[0078] 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 incorporated by reference. Where different versions of a sequence are associated with an accession number at different times, it refers to the version associated with the accession number as of the effective filing date of this application. Effective filing date, if applicable, refers to the earlier of the actual filing date or the filing date of the priority application that references the accession number. Similarly, where different versions of a publication, website, etc. are published at different times, it refers to the version published closest to the effective filing date of the application, unless otherwise noted. 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 noted. 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 made within the scope of the appended claims.

[0079] The following examples are provided to further describe the embodiments. The examples are intended to be illustrative and not limiting of the claimed embodiments. The following examples provide one of ordinary skill in the art with disclosure and explanation of how to make and evaluate the compounds, compositions, articles, devices, and / or methods described herein, and are intended to be purely illustrative and in no way intended to limit the scope of the claims. Efforts have been made to ensure accuracy with respect to numbers (e.g., amounts, temperature, etc.), but some errors and deviations should be accounted for. Unless otherwise specified, parts are parts by weight, temperature is in °C or is at ambient temperature, and pressure is at or near atmospheric pressure. EXAMPLES

[0080] Example 1: Materials and Methods WES sample preparation and sequencing Genomic DNA samples normalized to approximately 16ng / μl in 0.5ml 2D matrix tubes (Thermo Fisher Scientific) were delivered to us from the UK Biobank and stored at -80°C in an automated sample biobank (LiCONiC Instruments) prior to sample preparation. One sample was insufficient for DNA sequencing. Exome capture was completed using a high-throughput fully automated method developed in-house. Briefly, 100ng of genomic DNA was enzymatically sheared to an average fragment size of 200 base pairs using a custom NEBNext Ultra II FS DNA library preparation kit (New England Biolabs) to create DNA libraries, and universal Y-shaped adapters (Integrated DNA Technologies) were ligated to all DNA libraries. Unique, asymmetric 10-base pair barcodes were added to DNA fragments during library amplification using KAPA HiFi polymerase (KAPA Biosystems) to facilitate multiplexed exome capture and sequencing. Equal amounts of samples were pooled before performing an overnight, approximately 16-hour exome capture using a slightly modified version of IDT's xGen probe library; supplemental probes were added to capture regions of the genome that were not well covered by the standard xGen probes, but were well covered by the previous capture reagent (NimbleGen VCRome). In total, the target regions had n=38,997,831 bases. Captured fragments were bound to streptavidin-conjugated DYNABEADS® (Thermo Fisher Scientific), and nonspecific DNA fragments were removed by a series of stringent washes using the xGen Hybridization and Wash kit according to the manufacturer's recommended protocol (Integrated DNA Technologies).Captured DNA was PCR amplified on a KAPA HiFi and quantified by qPCR using the KAPA Library Quantification Kit (KAPA Biosystems). Multiplexed samples were pooled and then sequenced on an Illumina NOVASEQ® 6000 platform using an S2 flow cell, using 75 base pair paired-end reads with two 10 base pair index reads.

[0081] Sequence alignment, variant identification, and genotype assignment Upon completion of sequencing, the raw data from each Illumina NOVASEQ® run was stored in local buffer storage and uploaded to the DNAnexus platform for automated analysis. After uploading was completed, analysis began with converting the CBCL file data into FASTQ format and assigning samples via specific barcodes using bcl2fastq conversion software (Illumina Inc., San Diego, CA). The sample-specific FASTQ file containing all data obtained for that sample was then aligned to the GRCh38 genome reference using BWA-mem. The binary alignment file (BAM) for each sample result contained the genomic coordinates of the mapped data, quality information, and the degree of difference between the specific data and the reference at the mapped position. The alignment data present in the BAM file was then evaluated to identify and flag duplicate data using the Picard MarkDuplicates tool (global web, “picard.sourceforge.net”), generating an alignment file (duplicatesMarked.BAM) containing all potential duplicate data identified for exclusion in downstream analyses.

[0082] GVCF files containing variant calls were then generated for each individual sample from Genomics PLC using the WeCall variant caller (global web "github.com / Genomicsplc / wecall") to identify both SNVs and INDELs compared to the reference. In addition, each GVCF file stored the zygosity of each variant, data counts for both the reference and alternative alleles, genotype properties indicating the confidence of the genotype call, and the overall properties of the variant call at those positions.

[0083] Once variant calling was completed, individual sample BAM files were converted to fully lossless CRAM files using samtools. Metric statistics for each sample were obtained and capture, alignment status, insert size, and quality of variant calls were assessed using Picard (global web at picard.sourceforge.net), bcftools (global web at samtools.github.io / bcftools), and FastQC (global web at bioinformatics.babraham.ac.uk / projects / fastqc).

[0084] After the sequencing of samples was completed, we excluded samples that showed discordance between genetically determined sex and reported sex (n=15), samples with high percentage of heterozygosity / opacity (D-stat>0.4) (n=7), samples with low sequencing coverage (<85% of target bases achieving 20X coverage) (n=1), or genetically identified sample replicates (n=14), and WES variants discordant with genotyping chips (n=9). Thirty-eight individuals were excluded because six samples could not successfully maintain quality in multiple categories. The remaining 49,960 samples were then used to assemble a project-level VCF (PVCF) for downstream analysis. The GLnexus joint genotyping tool was used to generate the PVCF. Care was taken to capture all homozygous associated genotypes, heterozygous genotypes, homozygous alternative genotypes, and non-called genotypes in the project-level VCF. A filtered PVCF, "Goldilocks", was also generated. In the filtered Goldilocks PVCF, samples with SNP variant calls in the single sample pipeline or with a DP<7 were converted to "No-Call". After applying the DP filter, all remaining samples were heterozygous and all samples were excluded where AB<85% reference / 15% alternate. Samples with INDEL variant calls in the single sample pipeline with a DP<10 were converted to "No-Call". After applying the DP filter, all remaining samples were heterozygous and all samples were excluded where AB<80% reference / 20% alternate. Multi-allelic variant positions in the PVCF file were normalized with the left alignment and shown as bi-allelic.

[0085] Phenotype definition ICD10-based cases required one or more of the following: a primary or secondary diagnosis in an inpatient Health Episode Statistics (HES) record. For ICD10-based cases, individuals with one or more primary or secondary diagnoses in the code range were excluded. Individuals who were not cases or escaped exclusion were defined as ICD10-based controls. Custom phenotype definitions included one or more of the following: an ICD-10 diagnosis, a self-reported illness during the interview, and a physician-diagnosed illness based on touchscreen information during online follow-up. Quantitative traits (e.g., physical measurements, blood counts, cognitive tests, and imaging-derived phenotypes) were downloaded from the UK Biobank (UKB) repository and accessed at least once. In total, 1,073 binary traits, including 50 or more cases and 669 quantitative traits, were tested in the WES association analysis.

[0086] Annotation of predicted loss-of-function (LOF) variants Variants were annotated using snpEff and gene models from Ensembl Release 85. A comprehensive, high-quality transcript set was obtained for protein-coding regions including all protein-coding transcripts with annotated start and stop codons derived from Ensembl gene models. Variants annotated with stop_gained, start_lost, splice_donor, splice_acceptor, stop_lost, and frameshift are considered as LOF variants.

[0087] A recent large-scale study of genetic variation in 141,456 individuals has provided a list of LOF variants. Direct comparison of this data is difficult due to many differences in exome sequencing capture platforms, variant calling algorithms, and annotations. In addition, the number of individuals and ascertained geographic distribution (and therefore genetic diversity) in the NFE subset of gnomAD may be more extensive than that reported in the UK Biobank using WES. Nevertheless, gnomAD exome sites tagged with "PASS" from gnomAD r2.1 were annotated using the annotation pipeline. Data from gnomAD were transferred to HG38 using Picard LiftoverVcf. The data is a subset of non-Finnish Europeans (NFE) (n=56,885 samples) and individuals were annotated using the MAF NFE Restricting to variants <1%, we found 261,309 LOFs across transcripts of 17,951 genes. Restricting to LOFs present across all transcripts, we found 175,162 LOFs across 16,462 genes. Across all transcripts of genes in UKB participants with WES of European ancestry, we found 134,745 LOFs.

[0088] LOF Burden Related Analysis Methodology An association Burden test was performed for rare LOFs in 49,960 individuals of European ancestry for each gene region defined in Ensembl. LOFs with MAF ≤ 0.01 were divided such that individuals who were heterozygous for at least one LOF in that gene region were considered heterozygous, and only individuals carrying two copies of the same LOF were considered homozygous. Because rare variants were not planned to be staged, compound heterozygotes were not considered in this analysis.

[0089] For each gene region, 668 strata-based cases with ≥5 individuals with non-missing phenotypic information were assessed with inverse normal transformed (RINT) quantitative measures (including all subjects and sex-stratified models) using additive mixed models implemented in BOLT-LMM v2. Before normalization, traits were first transformed (log10, squared) if necessary and then adjusted for a standard set of covariates, including age, sex, study center, the first four principal components of ancestry, and in some cases, BMI and / or smoking history. Data points beyond 5 median absolute deviations from the median were excluded as outliers before normalization. Generalized additive mixed models implemented in SAIGE were used to assess 1,073 discrete outcomes with ≥50 cases (including all subjects and sex-stratified models) using covariate adjustment for the first four principal components of age, sex, and ancestry. For each quantitative and discrete trait used in the analysis, only genetic regions with phenotypic and covariate information and with >3 LOF carriers present were evaluated.

[0090] Positive controls were systematically defined using a two-step approach. First, each gene for associated disease, trait, biological or functional evidence was annotated using publicly available publications such as OMIM, NCBIMedGen, and NHGRI-EBI GWAS catalogs. Second, genes with supporting evidence from at least one publication were manually curated using NCBI PubMed to validate the relationship between the trait and the LOF variant in the gene of interest. Genes for which the trait or associated phenotype(s) of interest in the GWAS catalogs are evident at the locus level but without evidence clearly supporting the LOF association are reported herein as novel LOF associations.

[0091] Methods for single variant LOF association analysis Single variant association analysis was performed using the same method as described in the Methods section for the Baden association analysis. p<10 -7For gene-trait associations, we calculated single variant association statistics with the relevant phenotype for all LOFs with ≥5 minor alleles in the Baden test in 49,960 individuals of European ancestry with WES. Association statistics for these variants are reported in Extended Data (ExtData_SingleVariantLOFs_Vl.x1sx).

[0092] P-VAL leave-one-out (LOO) burden is the p-value for a presence / absence test with the variant being tested left out. Delta P-Val burden is the ratio of p-values ​​for the leave-one-out analysis compared to the Burden test using all 65 variants. Burden summary statistics using all 65 variants in unrelated individuals: B=1.44, SE=0.26, p-value=3.12E-08, cMAF=0.00174, cMAC=152, cMAF_cases=0.0066, cMAC_cases=17, cMAF_controls=0.0016, cMAC_cases=135. Stepwise logistic regression selected 11 / 65 variants (AIC 11451). Burden summary statistic with 11 variants: B=3.71, SE=0.437, p-value<2e-16, cMAF=0.0003, cMAC=22, cMAF_cases=0.005, cMAC_cases=12, cMAF_controls=0.0001, cMAC_controls=10. Restricting the analysis to variants with MAC>1, stepwise logistic regression selects 5 / 13 variants (AIC 11484). Burden summary statistic with 5 variants: B=3.02, SE=0.53, p-value=8.92e-09.

[0093] LD (r2) across 11 pLOF variants selected by stepwise regression with PIEZO1 and a positive control reported in the literature for PIEZO1 (16:88835545_G_A; highlighted in green). None of these 12 variants were found in LD with any of them, with R2>0.01. When adjusting for the Burden test with respect to the variant rs2911463 (16:88835545_G_A) listed above, the Burden test p-value remained <2E-16 (AIC 11,444), indicating no association with the reported variant.

[0094] Example 2: Stratification and clinical characteristics of sequenced participants A total of 50,000 participants were selected, with priority given to individuals with more complete phenotypic data: whole-body MRI imaging data from the UK Biobank Imaging Study, in-depth baseline measurements, hospital case-emergence statistics (HES), and / or associated primary medical records (available to approved researchers shortly). Quality control lapses or participant withdrawals during data generation led to the exclusion of samples from 40 participants, resulting in a final set of 49,960 individuals. Overall, the sequenced samples represent 500,000 UKB participants (Table 1). No notable differences were noted between the sequenced samples and the overall study population in terms of age, sex, or ancestry. Sequenced participants were more likely to have HES diagnosis codes (84.2% of sequenced participants vs. 77.3% overall) and supplementary measurements (Table 1).

[0095] [Table 1-1]

[0096] [Table 1-2]

[0097] Participants with WES who were assigned at least one HES diagnosis code did not differ from non-sequenced participants in terms of median number of primary and secondary ICD10 codes or broad phenotypic distribution, except for asthma (ICD10 J45) and status asthmaticus (ICD10 J46), which were most abundant in sequenced samples, and senile cataract (ICD10 H25) and disease of unknown and unspecified etiology (ICD10 R69), which were least abundant in sequenced samples. The sequenced subset included 194 parent-child pairs, 613 same-parent sibling pairs, one monozygotic twin pair, and 195 second-degree relatives. The distribution of relatedness between pairs of individuals in the UKB WES is shown in Figure 1.

[0098] Example 3: Summary and characterization of coding variations from WES Protein coding regions and exon-intron splice sites of 19,467 genes were targeted. The number of autosomal variants across all individuals was recognized by type / function classification for overall and MAF frequencies <1%. All variants met QC criteria, with missing individuals and variants <10%, and Hardy Weinberg p-values ​​>10 -15Median and interquartile range (IQR) variant counts for all variants and for variants with a MAF <1%. The mean proportion of target bases (n=38,997,831) achieving at least 20-fold coverage in each sample was 94.6% (standard deviation 2.1%). After quality control, 10,028,025 single nucleotide and indel variants were identified, with 98.5% having a minor allele frequency (MAF) <1% (Table 2). Of the total variants, 3,995,785 were within the target regions. These variants included 2,431,680 nonsynonymous (98.9% with MAF <1%), 1,200,882 synonymous (97.8% with MAF <1%), and 205,867 predicted loss-of-function (pLOF) variants (loss of start codon, premature stop codon, splicing, and frameshift indel variants; 99.7% with MAF <1%) affecting at least one coding transcript (Figure 2). The median number of 9,403 synonymous (IQR 125), 8,369 nonsynonymous (IQR 132), and 161 pLOF variants (IQR 14) per individual is comparable to previous exome sequencing studies. When we restricted the analysis to pLOF variants affecting all transcripts of a gene, the number of pLOF variants decreased to 140,850 overall and 96 per individual (a decrease of approximately 31.6% and 40.4%, respectively), which is consistent with previous studies.

[0099] [Table 2]

[0100] Example 4: Phenotypic associations with LOF variation Combining WES with rich health information allows extensive investigation of the phenotypic consequences of human genetic variation. LOF variation can provide completely unexpected insights into gene function; however, most such variation cannot be discerned from the imputed dataset. WES is well-suited to identify LOF variants and assess their phenotypic associations. Genetic Burden studies of associations for rare (<1% AAF) pLOF variants (WES-identified pLOF variants for all genes with >3 pLOF variant carriers) were performed for 1,741 traits (1,073 individual traits with at least 50 cases defined by hospital case occurrence statistics and self-reported data, 668 quantitative body measurements, and blood characteristics) in n=46,979 individuals of predominantly European ancestry. For each genetic trait association, the magnitude of association for the pLOF genetic Burden study was also compared to the association results for each SNV included in the Burden study.

[0101] Example 5: LOF association and novel gene discovery The pLOF gene Burden association analysis identified a novel association between PIEZO1 LOF (cumulative allele frequency = 0.2%) and a significantly increased risk of varicose veins. The results for PIEZO1 on the binary phenotype of asymptomatic varicose veins of the lower extremities in the UKB 50k exome and the UKB 150k exome are shown in Table 3.

[0102] [Table 3]

[0103] This finding was driven by the presence of rare LOF variants, with the most significant PIEZO1 single variant LOF association in the UKB 50k exome being 2.29 × 10 -3A leave-out analysis of the UKB 50k exome showed that no single variant explained the entire signal, and stepwise regression analysis showed that 11 distinct variants, five of which had a MAC >1, contributed to the overall Burden signal (Figure 4).

[0104] We replicated this finding in 2,953 varicose vein cases and 75,694 controls with previously exome sequenced (OR = 2.7, p = 1.86 × l0 -9 ). This region has previously been implicated in common noncoding variants with modest effects on disease risk, and rs2911463 and other common variants nearby on chromosome 16 have more recently been associated with varicose veins (frequency in GWAS of 408,455 genotyped UK Biobank participants = 0.69, OR = 0.996, p-value = 1.0 × 10 -27 The rs2911463 variant is not in LD with the major variants identified (Figure 5), and the Burden test remains relevant when adjusting for rs2911463.

Claims

1. 1. A method for identifying a human subject at high risk of developing varicose veins, the method comprising: In a biological sample obtained from the subject: Piezo-type mechanosensitive ion channel component 1 (PIEZO1) predicted loss-of-function variant genomic nucleic acid molecule; PIEZO1 predicted loss-of-function variant mRNA molecules; A PIEZO1 predicted loss-of-function variant cDNA molecule generated from said mRNA molecule; or PIEZO1 predicted loss-of-function variant polypeptides, determining whether or not the absence of the PIEZO1 predicted loss-of-function variant genomic nucleic acid molecule, mRNA molecule, cDNA molecule, or polypeptide indicates the subject is not at increased risk for developing varicose veins; the presence of the PIEZO1 predicted loss-of-function variant genomic nucleic acid molecule, mRNA molecule, cDNA molecule, or polypeptide indicates that the subject is at increased risk for developing varicose veins; The PIEZO1 prediction function loss variant is 16:88715629:G:A, 16:88715728:G:T, 16:88715767:G:A, 16:88715802:C:A, 16:88715822:D:4, 16:88715987:I:1, 16:88716359:A:G, 16:88716570:C:T, 16:88716874:G:A, 16:88717213:T:A, 16:88719588:G:A, 16:88719722:C:G, 16:88719870:G:T, 16:88720068:D:2, 16:88720229:C:A, 16:88720248:D:4, 16:88720394:C:T, 16:88720644:D:1, 16:88720698:D:1, 16:88720698:I:1, 16:88721165:C:A, 16:88721268:D:1, 16:88721307:G:A, 16:88721586:G:C, 16:88721652:G:C, 16:88722217:C:T, 16:88722605:I:1, 16:88723005:I:7, 16:88723253:G:A, 16:88723311:C:T, 16:88725081:C:A, 16:88726282:G:A, 16:88726546:C:T, 16:88726619:G:A, 16:88726924:G:A, 16:88727038:C:T, 16:88727072:D:1, 16:88727163:G:A, 16:88731768:D:1, 16:88732334:C:G, 16:88732411:D:1, 16:88732720:D:1, 16:88733326:G:C, 16:88733337:D:4, 16:88733587:C:A, 16:88733965:D:1, 16:88734017:C:A, 16:88734042:I:1, 16:88734679:C:T, 16:88734909:I:1, 16:88736167:D:2, 16:88736324:G:A, 16:88736391:G:T, 16:88736409:C:T, 16:88736671:G:A, 16:88737557:A:C, 16:88737727:C:G, 16:88737815:C:T, 16:88738283:G:C, 16:88738637:G:A, 16:88738735:D:1,16:88741477:C:T, 16:88742306:D:1, 16:88749399:G:A, or 16:88784929:C:T.

2. The method of claim 1 , wherein the determining is performed in vitro.

3. 3. The method of claim 1 or claim 2, wherein the determining comprises sequencing at least a portion of the nucleotide sequence of the PIEZO1 nucleic acid molecule in the biological sample, the sequenced portion comprising a position corresponding to a predicted loss-of-function variant position, and wherein if a variant nucleotide at the predicted loss-of-function variant position is detected, the PIEZO1 nucleic acid molecule in the biological sample is a PIEZO1 predicted loss-of-function variant nucleic acid molecule.

4. The determining comprises: a) contacting said biological sample with a primer that hybridizes to a portion of a nucleotide sequence of said PIEZO1 nucleic acid molecule adjacent to a predicted loss-of-function variant position; b) extending the primer to at least the predicted loss-of-function variant location; and c) determining whether an extension product of the primer contains a variant nucleotide at the predicted loss-of-function variant position; The method according to any one of claims 1 to 3, comprising:

5. The method of claim 3 or claim 4, wherein the determining comprises sequencing the entire nucleic acid molecule.

6. The determining comprises: a) amplifying at least a portion of the PIEZO1 nucleic acid molecule encoding the human PIEZO1 polypeptide, said portion including a predicted loss-of-function variant position; b) labeling the amplified nucleic acid molecules with a detectable label; c) contacting the labeled nucleic acid molecule with a support comprising a variant-specific probe, the variant-specific probe comprising a nucleotide sequence that hybridizes to the predicted loss-of-function variant position under stringent conditions; and d) detecting said detectable label; The method of claim 1 or claim 2, comprising:

7. 7. The method of claim 6, wherein the PIEZO1 nucleic acid molecule in the biological sample is mRNA, and the mRNA is reverse transcribed into cDNA prior to the amplifying step.

8. The determining comprises: contacting the PIEZO1 nucleic acid molecule in the biological sample with a variant-specific probe comprising a detectable label, the variant-specific probe comprising a nucleotide sequence that hybridizes to a predicted loss-of-function variant position under stringent conditions; and detecting said detectable label; The method of claim 6 or claim 7, comprising:

9. A therapeutic agent for treating or inhibiting varicose veins, for treating a patient suffering from or at high risk of developing varicose veins, comprising: The patient is obtaining or having obtained a biological sample from said patient; and performing a genotyping assay on the biological sample to determine whether the patient has a genotype that includes a piezoelectric mechanosensitive ion channel component 1 (PIEZO1) predicted loss-of-function variant nucleic acid molecule; It has been determined that the patient has a PIEZO1 predicted loss-of-function variant nucleic acid molecule encoding a human PIEZO1 polypeptide; the presence of a genotype having a PIEZO1 predicted loss-of-function variant nucleic acid molecule encoding the human PIEZO1 polypeptide indicates that the patient is at increased risk for developing varicose veins; and If the patient is determined to be heterozygous or homozygous for the PIEZO1 predicted loss-of-function variant nucleic acid molecule, the therapeutic agent is administered or continues to be administered to the patient in an amount equal to or greater than a standard dosage; The PIEZO1 prediction function loss variant is 16:88715629:G:A, 16:88715728:G:T, 16:88715767:G:A, 16:88715802:C:A, 16:88715822:D:4, 16:88715987:I:1, 16:88716359:A:G, 16:88716570:C:T, 16:88716874:G:A, 16:88717213:T:A, 16:88719588:G:A, 16:88719722:C:G, 16:88719870:G:T, 16:88720068:D:2, 16:88720229:C:A, 16:88720248:D:4, 16:88720394:C:T, 16:88720644:D:1, 16:88720698:D:1, 16:88720698:I:1, 16:88721165:C:A, 16:88721268:D:1, 16:88721307:G:A, 16:88721586:G:C, 16:88721652:G:C, 16:88722217:C:T, 16:88722605:I:1, 16:88723005:I:7, 16:88723253:G:A, 16:88723311:C:T, 16:88725081:C:A, 16:88726282:G:A, 16:88726546:C:T, 16:88726619:G:A, 16:88726924:G:A, 16:88727038:C:T, 16:88727072:D:1, 16:88727163:G:A, 16:88731768:D:1, 16:88732334:C:G, 16:88732411:D:1, 16:88732720:D:1, 16:88733326:G:C, 16:88733337:D:4, 16:88733587:C:A, 16:88733965:D:1, 16:88734017:C:A, 16:88734042:I:1, 16:88734679:C:T, 16:88734909:I:1, 16:88736167:D:2, 16:88736324:G:A, 16:88736391:G:T, 16:88736409:C:T, 16:88736671:G:A, 16:88737557:A:C, 16:88737727:C:G, 16:88737815:C:T, 16:88738283:G:C, 16:88738637:G:A, 16:88738735:D:1,The therapeutic agent is selected from 16:88741477:C:T, 16:88742306:D:1, 16:88749399:G:A, or 16:88784929:C:T.

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