Treatment of varicosity with piezoelectric mechanically sensitive ion channel component 1 (PIEZO1) agonists

Abstract

The present disclosure provides methods of treating subjects having or at risk of developing varicosity, methods of identifying subjects at increased risk of developing varicosity, and methods of detecting piezoelectric mechanically sensitive ion channel component 1 (PIEZO1) variant nucleic acid molecules and variant polypeptides.

Description

[0001] Incorporation by Reference of the Sequence Listing

[0002] This application includes a Sequence Listing submitted electronically as a text file via EFS-Web. The name of the text file containing the Sequence Listing is 18923808402SEQ, created on July 2, 2022, and is 542 kilobytes in size. The Sequence Listing is incorporated by reference herein. TECHNICAL FIELD

[0003] The present disclosure relates generally to treating a subject having varicose veins or at risk of developing varicose veins with a Piezoelectric Mechanosensitive Ion Channel Component 1 (PIEZO1) agonist, and to methods of identifying a subject at increased risk of developing varicose veins. BACKGROUND

[0004] Varicose veins are a common multifactorial disease with largely unknown genetic drivers, often seen in patients with chronic venous insufficiency, collectively known as chronic venous disease. Valvular dysfunction in veins is associated with varicose veins, venous hypertension, and thrombosis. Several processes, such as changes in hemodynamics, endothelial activation, inflammation, hypoxia, and dysregulation of matrix metalloproteinases and their tissue agonists, have been linked to varicose vein development. Risk factors for varicose veins include increasing age, female sex, number of pregnancies, obesity, history of deep vein thrombosis, and standing occupation. Varicose veins have also been linked to insufficient lymphatic drainage and chronic venous insufficiency. In addition, several genome-wide association studies (GWAS) have shown a genetic rate of approximately 18.5% for varicose veins.

[0005] 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 long and is a 286 kDa transmembrane protein containing 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, which is sensitive to ruthenium red and gadolinium. PIEZO1 is ubiquitously expressed and recruited to the endoplasmic reticulum (most likely in its activated state) through R-Ras and subsequently stimulates calpain signaling to maintain integrin activation, playing a role in epithelial cell adhesion. In the vasculature, PIEZO1 is involved in endothelial cell migration and sprouting angiogenesis. Specifically, PIEZO1 acts as a sensor of blood flow-related shear stress and promotes endothelial cell organization and alignment along the direction of blood flow, ensuring proper vascular formation, remodeling, and maturation. PIEZO1 also appears to be required for lymphatic valve formation. Other reported functions include blood pressure regulation, urine osmolarity, red blood cell integrity, pressure sensing, and collecting duct permeability regulation. SUMMARY

[0006] The present disclosure provides methods of treating a subject having varicose veins or at risk of developing varicose veins, the methods comprising administering to the subject a PIEZO1 agonist.

[0007] The present disclosure also provides methods of treating a subject having saphenous varicose veins or at risk of developing saphenous varicose veins, the methods comprising administering to the subject a PIEZO1 agonist.

[0008] The present disclosure also provides methods of treating a subject having veticular varicose veins or at risk of developing veticular varicose veins, the methods comprising administering to the subject a PIEZO1 agonist.

[0009] The present disclosure also provides methods of treating a subject having spider veins or at risk of developing spider veins, the methods comprising administering to the subject a PIEZO1 agonist.

[0010] The present disclosure also provides methods of treating a subject having pregnancy-related varicose veins or at risk of developing pregnancy-related varicose veins, the methods comprising administering to the subject a PIEZO1 agonist.

[0011] The present disclosure also provides methods of treating a subject with a therapeutic agent that treats or prevents varicose veins, wherein the subject has varicose veins or is at risk of developing varicose veins, the methods comprising: determining whether the subject has a PIEZO1 variant nucleic acid molecule encoding a PIEZO1 predicted gain-of-function polypeptide by: obtaining or having obtained a biological sample from the subject; and performing or having performed sequence analysis on the biological sample to determine whether the subject has a genotype comprising the PIEZO1 variant nucleic acid molecule encoding the PIEZO1 predicted gain-of-function polypeptide; and administering or continuing to administer to the subject the therapeutic agent that treats or prevents varicose veins at a standard dosage, and / or administering to the subject a PIEZO1 agonist; and administering or continuing to administer to the subject the therapeutic agent that treats or prevents varicose veins in an amount that is equal to or less than a standard dosage, and / or administering to the subject a PIEZO1 agonist, to a subject that is heterozygous for the PIEZO1 variant nucleic acid molecule; wherein the presence of the genotype having the PIEZO1 variant nucleic acid molecule encoding a PIEZO1 predicted gain-of-function polypeptide indicates a reduced risk of the subject developing varicose veins.

[0012] The present disclosure also provides a method of identifying a subject having an increased risk of developing varicose veins, the method comprising: determining or having determined the presence or absence of a PIEZO1 variant nucleic acid molecule encoding a PIEZO1 predicted loss-of-function polypeptide in a biological sample obtained from the subject; wherein: when the subject is a PIEZO1 reference type, then the subject has an increased risk of developing varicose veins; and when the subject is heterozygous or homozygous for a PIEZO1 variant nucleic acid molecule encoding the PIEZO1 predicted loss-of-function polypeptide, then the subject has a decreased risk of developing varicose veins.

[0013] The present disclosure also provides a therapeutic agent for treating or preventing varicose veins for use in treating or preventing varicose veins in a subject identified as having: i) a genomic nucleic acid molecule encoding a PIEZO1 predicted gain-of-function polypeptide, or its complement, wherein the genomic nucleic acid molecule has a nucleotide sequence comprising: i) a thymine at a position corresponding to position 69,579 according to SEQ ID NO: 2, or its complement; ii) an mRNA molecule encoding a PIEZO1 predicted gain-of-function polypeptide, or its complement, wherein the mRNA molecule has a nucleotide sequence comprising: a uracil at a position corresponding to position 7,785 according to SEQ ID NO: 13, or its complement; a uracil at a position corresponding to position 1,095 according to SEQ ID NO: 14, or its complement; a uracil at a position corresponding to position 6,073 according to SEQ ID NO: 15, or its complement; a uracil at a position corresponding to position 1,336 according to SEQ ID NO: 16, or its complement; a uracil at a position corresponding to position 6,073 according to SEQ ID NO: 17, or its complement; a uracil at a position corresponding to position 6,236 according to SEQ ID NO: 18, or its complement; a uracil at a position corresponding to position 7,526 according to SEQ ID NO: 19, or its complement; a uracil at a position corresponding to position 1,336 according to SEQ ID NO: 20, or its complement; a uracil at a position corresponding to position 361 according to SEQ ID NO: 21, or its complement; or a uracil at a position corresponding to position 7,776 according to SEQ ID NO: 22, or its complement; or iii) a cDNA molecule encoding a PIEZO1 predicted gain-of-function polypeptide, or its complement, wherein the cDNA molecule has a nucleotide sequence comprising: a thymine at a position corresponding to position 7,785 according to SEQ ID NO: 33, or its complement; a thymine at a position corresponding to position 1,095 according to SEQ ID NO: 34, or its complement; a thymine at a position corresponding to position 6,073 according to SEQ ID NO: 35, or its complement; an adenine at a position corresponding to position 1,336 according to SEQ ID NO: 36, or its complement; an adenine at a position corresponding to position 6,073 according to SEQ ID NO: 37, or its complement; an adenine at a position corresponding to position 6,236 according to SEQ ID NO: 38, or its complement; an adenine at a position corresponding to position 7,526 according to SEQ ID NO: 39, or its complement; an adenine at a position corresponding to position 1,336 according to SEQ ID NO: 40, or its complement;an adenine at a position corresponding to position 361 according to SEQ ID NO: 41, or the complement thereof; or an adenine at a position corresponding to position 7,776 according to SEQ ID NO: 42, or the complement thereof.

[0014] The present disclosure also provides a PIEZO 1 agonist for use in treating or preventing varicose veins in a subject who: a) is of reference type for a PIEZO 1 genomic nucleic acid molecule, a PIEZO 1 mRNA molecule, or a PIEZO 1 cDNA molecule; or b) is heterozygous for: i) a genomic nucleic acid molecule, or its complement, encoding a PIEZO 1 predicted gain-of-function polypeptide, wherein the genomic nucleic acid molecule has a nucleotide sequence comprising a thymine at a position corresponding to position 69,579 according to SEQ ID NO:2, or its complement; ii) an mRNA molecule, or its complement, encoding a PIEZO 1 predicted gain-of-function polypeptide, wherein the mRNA molecule has a nucleotide sequence comprising: a uracil at a position corresponding to position 7,785 according to SEQ ID NO: 13, or its complement; a uracil at a position corresponding to position 1,095 according to SEQ ID NO: 14, or its complement; a uracil at a position corresponding to position 6,073 according to SEQ ID NO: 15, or its complement; a uracil at a position corresponding to position 1,336 according to SEQ ID NO: 16, or its complement; a uracil at a position corresponding to position 6,073 according to SEQ ID NO: 17, or its complement; a uracil at a position corresponding to position 6,236 according to SEQ ID NO: 18, or its complement; a uracil at a position corresponding to position 7,526 according to SEQ ID NO: 19, or its complement; a uracil at a position corresponding to position 1,336 according to SEQ ID NO: 20, or its complement; a uracil at a position corresponding to position 361 according to SEQ ID NO: 21, or its complement; or a uracil at a position corresponding to position 7,776 according to SEQ ID NO: 22, or its complement; or iii) a cDNA molecule, or its complement, encoding a PIEZO 1 predicted gain-of-function polypeptide, wherein the cDNA molecule has a nucleotide sequence comprising: a thymine at a position corresponding to position 7,785 according to SEQ ID NO: 33, or its complement; a thymine at a position corresponding to position 1,095 according to SEQ ID NO: 34, or its complement; a thymine at a position corresponding to position 6,073 according to SEQ ID NO: 35, or its complement; a thymine at a position corresponding to position 1,336 according to SEQ ID NO: 36, or its complement; a thymine at a position corresponding to position 6,073 according to SEQ ID NO: 37, or its complement; a thymine at a position corresponding to position 6,236 according to SEQ ID NO: 38, or its complement.a thymine at a position corresponding to position 7,526 according to SEQ ID NO: 39, or the complement thereof; a thymine at a position corresponding to position 1,336 according to SEQ ID NO: 40, or the complement thereof; a thymine at a position corresponding to position 361 according to SEQ ID NO: 41, or the complement thereof; or a thymine at a position corresponding to position 7,776 according to SEQ ID NO: 42, or the complement thereof. DETAILED DESCRIPTION

[0015] Throughout the specification and claims, various terms related to aspects of the present disclosure are used. Unless otherwise indicated, such terms are to be given their ordinary meaning in the art. Other specifically defined terms are to be construed in a manner consistent with the definition provided herein.

[0016] Unless specifically stated otherwise, and otherwise apparent from the context, nothing herein is intended to be construed as requiring its steps to be performed in a particular order. Thus, in claims or descriptions or both in this or any document, the terms "first," "second," "adjiacent," "adjacent," "above," "below," and the like, where

[0017] As used herein, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise.

[0018] As used herein, the term "about" means that the recited numerical values are approximations and slight variations do not materially affect the practice of the disclosed embodiments. In the case of numerical values, the term "about" means that the numerical value can vary ±10% and still be within the scope of the disclosed embodiments, unless the context clearly indicates otherwise.

[0019] As used herein, the term "comprising" can be replaced with "consisting of" or "consisting essentially of" as desired in a particular embodiment.

[0020] As used herein, the term "isolated" with respect to a nucleic acid molecule or polypeptide means that the nucleic acid molecule or polypeptide is in a condition other than its natural environment, such as away from blood and / or other tissues. In some embodiments, an isolated nucleic acid molecule or polypeptide is essentially free of other nucleic acid molecules or other polypeptides, particularly other nucleic acid molecules or polypeptides of animal origin. In some embodiments, a nucleic acid molecule or polypeptide can be in highly purified form, i.e., greater than 95% pure or greater than 99% pure. The term "isolated" when used in this context does not exclude the presence of the same nucleic acid molecule or polypeptide in an alternative physical form, e.g., a dimer or alternatively phosphorylated or derivatized form.

[0021] As used herein, the term "nucleic acid," "nucleic acid molecule," "nucleic acid sequence," "polynucleotide," or "oligonucleotide" can include polymeric forms of nucleotides of any length, can include both DNA and / or RNA, and can be single-stranded, double-stranded, or multi-stranded. A strand of nucleic acid also refers to its complementary strand.

[0022] As used herein, the term "subject" includes any animal, including mammals. Mammals include, but are not limited to, farm animals (such as, for example, horses, cows, pigs), companion animals (such as, for example, dogs, cats), laboratory animals (such as, for example, mice, rats, rabbits), and non-human primates (such as, for example, apes and monkeys). In some embodiments, the subject is a human. In some embodiments, the subject is a patient under medical care.

[0023] According to the present disclosure, a putative gain-of-function (GOF) missense variant in the PIEZO1 gene has been identified that is associated with a reduced risk of developing varicose veins in humans. Specifically, it has been observed that a genetic alteration changing the cytosine to a thymine at position 69,579 in the PIEZO1 reference genome nucleic acid molecule (see SEQ ID NO: 1) indicates that a subject having such an alteration can have a reduced risk of developing varicose veins. In sum, the genetic analyses described herein surprisingly indicate that the PIEZO1 gene and, in particular, pGOF in the PIEZO1 gene, is associated with a reduced risk of developing varicose veins. Accordingly, subjects who are at increased risk of developing varicose veins (such as saphenous varicose veins, varicose veins of the lower extremities, spider veins, or pregnancy-related varicose veins) that are of the PIEZO1 reference type can be treated, thereby preventing varicose veins, alleviating symptoms thereof, and / or arresting development of symptoms. Accordingly, the present disclosure provides methods of identifying or stratifying a risk of developing varicose veins (such as saphenous varicose veins, varicose veins of the lower extremities, spider veins, or pregnancy-related varicose veins) in a subject, or diagnosing a subject as having an increased risk of developing varicose veins (such as saphenous varicose veins, varicose veins of the lower extremities, spider veins, or pregnancy-related varicose veins), using identification of such a variant in the subject, so that subjects at risk or subjects with active disease can be treated accordingly.

[0024] According to the present disclosure, it is also observed that PIEZO1 variant nucleic acid molecules encoding PIEZO1 predicted gain-of-function polypeptides (whether these variants are homozygous or heterozygous in a particular subject) are associated with a reduced risk of developing varicose veins. Moreover, the identification of the association between additional variants and the genetic burden mask by the present disclosure indicates that PIEZO1 itself (rather than linkage disequilibrium with a variant in another gene) is responsible for the protective effect in varicose veins.

[0025] For purposes of the present disclosure, any particular subject can be categorized as having one of the following three PIEZO1 genotypes: i) a PIEZO1 reference type; ii) heterozygous for a PIEZO1 variant nucleic acid molecule encoding a PIEZO1 predicted gain-of-function polypeptide; or iii) homozygous for a PIEZO1 variant nucleic acid molecule encoding a PIEZO1 predicted gain-of-function polypeptide. A subject is a PIEZO1 reference type when the subject does not have a copy of a PIEZO1 variant nucleic acid molecule encoding a PIEZO1 predicted gain-of-function polypeptide. A subject is heterozygous for a PIEZO1 variant nucleic acid molecule encoding a PIEZO1 predicted gain-of-function polypeptide when the subject has a single copy of a PIEZO1 variant nucleic acid molecule. As used herein, a PIEZO1 variant nucleic acid molecule is any PIEZO1 nucleic acid molecule (such as a genomic nucleic acid molecule, an mRNA molecule, or a cDNA molecule) that encodes a PIEZO1 polypeptide having partial gain-of-function, complete gain-of-function, predicted partial gain-of-function, or predicted complete gain-of-function. A subject having a PIEZO1 variant nucleic acid molecule encoding a PIEZO1 predicted gain-of-function polypeptide having partial gain-of-function (or predicted partial gain-of-function) is hypomorphic for PIEZO1. A PIEZO1 variant nucleic acid molecule encoding a PIEZO1 predicted gain-of-function polypeptide can be any nucleic acid molecule encoding PIEZO1 Pro2,510Leu, Pro2,024Leu, Pro2,079Leu, or Pro92Leu. In some embodiments, the PIEZO1 variant nucleic acid molecule encodes PIEZO1 Pro2,510Leu. A subject is homozygous for a PIEZO1 variant nucleic acid molecule encoding a PIEZO1 predicted gain-of-function polypeptide when the subject has two copies of a PIEZO1 variant nucleic acid molecule encoding a PIEZO1 predicted gain-of-function polypeptide.

[0026] For subjects genotyped or determined to be a PIEZO1 reference type, such subjects have an increased risk of developing varicose veins, such as varices of the saphenous vein, varices of the venous plexus of the skin, spider veins, or pregnancy-related varicose veins. For subjects genotyped or determined to be a PIEZO1 reference type or for subjects that are heterozygous for a PIEZO1 variant nucleic acid molecule encoding a PIEZO1 predicted gain-of-function polypeptide, such subjects can be treated with a PIEZO1 agonist.

[0027] In any of the embodiments described throughout the present disclosure, the PIEZO1 variant nucleic acid molecule can be any PIEZO1 nucleic acid molecule (such as a genomic nucleic acid molecule, an mRNA molecule, or a cDNA molecule) that encodes a PIEZO1 polypeptide having partial gain-of-function, complete gain-of-function, predicted partial gain-of-function, or predicted complete gain-of-function. For example, the PIEZO1 variant nucleic acid molecule can be any nucleic acid molecule that encodes PIEZO1 Pro2,510Leu, Pro2,024Leu, Pro2,079Leu, or Pro92Leu. In some embodiments, the PIEZO1 variant nucleic acid molecule encodes PIEZO1 Pro2,510Leu.

[0028] In any of the embodiments described throughout the present disclosure, the PIEZO1 predicted gain-of-function polypeptide can be any PIEZO1 polypeptide having partial gain-of-function, complete gain-of-function, predicted partial gain-of-function, or predicted complete gain-of-function. In any of the embodiments described throughout the present disclosure, the PIEZO1 predicted gain-of-function polypeptide can be any PIEZO1 polypeptide described herein, including, for example, PIEZO1 Pro2,510Leu, Pro2,024Leu, Pro2,079Leu, or Pro92Leu. In any of the embodiments described throughout the present disclosure, the PIEZO1 predicted gain-of-function polypeptide can be PIEZO1 Pro2,510Leu.

[0029] In any of the embodiments described throughout the present disclosure, the varicose vein is a hidden varicose vein, a varicosis varicose vein, a spider vein, or a pregnancy-related varicose vein. In any of the embodiments described throughout the present disclosure, the varicose vein is a hidden varicose vein. In any of the embodiments described throughout the present disclosure, the varicose vein is a varicosis varicose vein. In any of the embodiments described throughout the present disclosure, the varicose vein is a spider vein. In any of the embodiments described throughout the present disclosure, the varicose vein is a pregnancy-related varicose vein.

[0030] Symptoms of varicose veins include, but are not limited to, leg heaviness, spider veins (telangiectasia) appearing on the affected leg, ankle swelling (especially at night), discoloration of the brownish-golden shiny skin near the affected veins, red, dry, and itchy skin in the area (called stasis dermatitis or venous eczema), cramping (especially when developing upon sudden movement such as standing), minor injury to the affected area, bleeding more than normal or taking a long time to heal, skin atrophy above the ankle (lipodermatosclerosis), restless leg syndrome, appearance of irregular patches of white scars on the ankle (white atrophy), or any combination thereof.

[0031] The present disclosure provides methods of treating a subject having a varicose vein or at risk of developing a varicose vein, the methods comprising administering to the subject a PIEZO1 agonist.

[0032] The present disclosure also provides methods of treating a subject having varicose veins or at risk of developing varicose veins, the methods comprising administering to the subject a PIEZO1 agonist.

[0033] The present disclosure also provides methods of treating a subject having varicose veins or at risk of developing varicose veins, the methods comprising administering to the subject a PIEZO1 agonist.

[0034] The present disclosure also provides methods of treating a subject having varicose veins or at risk of developing varicose veins, the methods comprising administering to the subject a PIEZO1 agonist.

[0035] The present disclosure also provides methods of treating a subject having varicose veins or at risk of developing varicose veins, the methods comprising administering to the subject a PIEZO1 agonist.

[0036] In some embodiments, these methods of treatment further comprise detecting the presence or absence of a PIEZO1 variant nucleic acid molecule encoding a PIEZO1 predicted gain-of-function polypeptide in a biological sample obtained from the subject. As used throughout the present disclosure, a “PIEZO1 variant nucleic acid molecule” is any PIEZO1 nucleic acid molecule (such as a genomic nucleic acid molecule, an mRNA molecule, or a cDNA molecule) that encodes a PIEZO1 polypeptide having partial gain-of-function, complete gain-of-function, predicted partial gain-of-function, or predicted complete gain-of-function.

[0037] The present disclosure also provides methods of treating a subject with a therapeutic agent to treat or prevent varicose veins. In some embodiments, the subject has varicose veins. In some embodiments, the subject is at risk of developing varicose veins. In some embodiments, the methods comprise determining whether the subject has a PIEZO1 variant nucleic acid molecule encoding a PIEZO1 predicted gain-of-function polypeptide by obtaining or having obtained a biological sample obtained from the subject and performing or having performed sequence analysis on the biological sample to determine whether the subject has a genotype comprising a PIEZO1 variant nucleic acid molecule. When the subject is a PIEZO1 reference type, the subject is administered or continues to be administered a therapeutic agent to treat or prevent varicose veins at a standard dose, and / or the subject is administered a PIEZO1 agonist. When the subject is heterozygous for a PIEZO1 variant, the subject is administered or continues to be administered a therapeutic agent to treat or prevent varicose veins in an amount that is equal to or less than a standard dose, and / or the subject is administered a PIEZO1 agonist. The presence of a genotype having the PIEZO1 variant nucleic acid molecule encoding the PIEZO1 predicted gain-of-function polypeptide indicates a reduced risk of the subject developing varicose veins. In some embodiments, the subject is a PIEZO1 reference type. In some embodiments, the subject is heterozygous for a PIEZO1 variant nucleic acid molecule encoding a PIEZO1 predicted gain-of-function polypeptide.

[0038] For subjects who are genotyped or determined to be a PIEZO1 reference type or who are heterozygous for a PIEZO1 variant nucleic acid molecule encoding a PIEZO1 predicted gain-of-function polypeptide, such subjects can be treated with a PIEZO1 agonist, as described herein.

[0039] Detecting the presence or absence of a PIEZO1 variant nucleic acid molecule encoding a PIEZO1 predicted gain-of-function polypeptide in a biological sample obtained from a subject and / or determining whether a subject has a PIEZO1 variant nucleic acid molecule encoding a PIEZO1 predicted gain-of-function polypeptide can be performed by any of the methods described herein. In some embodiments, the methods can be performed in vitro. In some embodiments, the methods can be performed in situ. In some embodiments, the methods can be performed in vivo. In any of these embodiments, the PIEZO1 variant nucleic acid molecule encoding a PIEZO1 predicted gain-of-function polypeptide can be present in a cell obtained from the subject.

[0040] In some embodiments, when the subject is a PIEZO1 reference type, the subject is also administered a therapeutic agent to treat or prevent varicose veins at a standard dose. In some embodiments, when the subject is heterozygous for a PIEZO1 variant nucleic acid molecule encoding a PIEZO1 predicted gain-of-function polypeptide, the subject is administered a therapeutic agent to treat or prevent varicose veins at a dose that is equal to or less than a standard dose.

[0041] In some embodiments, the method of treatment further comprises detecting the presence or absence of a PIEZO1 predicted gain-of-function polypeptide in a biological sample obtained from the subject. In some embodiments, when the subject does not have a PIEZO1 predicted gain-of-function polypeptide, the subject is administered a therapeutic or prophylactic varicose vein therapeutic at a standard dose. In some embodiments, when the subject has a PIEZO1 predicted gain-of-function polypeptide, the subject is administered a therapeutic or prophylactic varicose vein therapeutic at a dose that is equal to or less than the standard dose.

[0042] The present disclosure also provides methods of treating a subject with a therapeutic or prophylactic varicose vein therapeutic. In some embodiments, the subject has varicose veins. In some embodiments, the subject is at risk of developing varicose veins. In some embodiments, the method comprises determining whether the subject has a PIEZO1 predicted gain-of-function polypeptide by obtaining or having obtained a biological sample from the subject and performing or having performed an assay on the biological sample to determine whether the subject has a PIEZO1 predicted gain-of-function polypeptide. When the subject does not have a PIEZO1 predicted gain-of-function polypeptide, the subject is administered or continues to be administered a therapeutic or prophylactic varicose vein therapeutic at a standard dose, and / or the subject is administered a PIEZO1 agonist. When the subject has a PIEZO1 predicted gain-of-function polypeptide, the subject is administered or continues to be administered a therapeutic or prophylactic varicose vein therapeutic at an amount that is equal to or less than the standard dose, and / or the subject is administered a PIEZO1 agonist. The presence of a PIEZO1 predicted gain-of-function polypeptide indicates that the subject is at a reduced risk of developing varicose veins. In some embodiments, the subject has a PIEZO1 predicted gain-of-function polypeptide. In some embodiments, the subject does not have a PIEZO1 predicted gain-of-function polypeptide.

[0043] Detecting the presence or absence of a PIEZO1 predicted gain-of-function polypeptide in a biological sample obtained from a subject and / or determining whether a subject has a PIEZO1 predicted gain-of-function polypeptide can be performed by any of the methods described herein. In some embodiments, the methods can be performed in vitro. In some embodiments, the methods can be performed in situ. In some embodiments, the methods can be performed in vivo. In any of these embodiments, the PIEZO1 predicted gain-of-function polypeptide can be present in a cell obtained from the subject.

[0044] Examples of therapeutics 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.

[0045] Examples of PIEZO1 agonists include, but are not limited to, Yoda1, Jedi1, and Jedi2. In some embodiments, the PIEZO1 agonist comprises a small molecule.

[0046] In some embodiments, the dosage of the therapeutic agent for treating or preventing varicose veins and / or the PIEZO1 agonist can be reduced by about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, or about 90% (i.e., less than the standard dosage) in a subject that is heterozygous for a PIEZO1 variant nucleic acid molecule encoding a PIEZO1 predicted gain-of-function polypeptide as compared to a subject that is a PIEZO1 reference type (receiving the standard dosage). In some embodiments, the dosage of the therapeutic agent for treating or preventing varicose veins and / or the PIEZO1 agonist can be reduced by about 10%, about 20%, about 30%, about 40%, or about 50% in a subject that is heterozygous for a PIEZO1 variant nucleic acid molecule encoding a PIEZO1 predicted gain-of-function polypeptide as compared to a subject that is a PIEZO1 reference type. Furthermore, the dosage of the therapeutic agent for treating or preventing varicose veins and / or the PIEZO1 agonist can be administered less frequently in a subject that is heterozygous for a PIEZO1 variant nucleic acid molecule encoding a PIEZO1 predicted gain-of-function polypeptide as compared to a subject that is a PIEZO1 reference type.

[0047] The therapeutic agent for treating or preventing varicose veins and / or the PIEZO1 agonist can be administered repeatedly, e.g., after one day, two days, three days, five days, one week, two weeks, three weeks, one month, five weeks, six weeks, seven weeks, eight weeks, two months, or three months. The repeated administration can be at the same dosage or at a different dosage. The administration can be repeated once, twice, three times, four times, five times, six times, seven times, eight times, nine times, ten times, or more. For example, according to certain dosage regimens, a subject can receive treatment for a longer period of time, e.g., like 6 months, 1 year, or more. Furthermore, the therapeutic agent for treating or preventing varicose veins and / or the PIEZO1 agonist can be administered sequentially or simultaneously. Furthermore, the therapeutic agent for treating or preventing varicose veins and / or the PIEZO1 agonist can be administered in separate compositions, or can be administered together in the same composition.

[0048] The therapeutic agent for treating or preventing varicose veins and / or the PIEZO1 agonist can be administered by any suitable route, including, but not limited to, parenteral, intravenous, oral, subcutaneous, intra-arterial, intracranial, intrathecal, intraperitoneal, topical, intranasal, or intramuscular. The pharmaceutical composition for administration is desirably sterile and substantially isotonic, and is manufactured under GMP conditions. The pharmaceutical composition can be provided in unit dosage form, i.e., the dosage for a single administration. The pharmaceutical composition can be formulated using one or more physiologically and pharmaceutically acceptable carriers, diluents, excipients or auxiliaries. The formulation depends on the selected route of administration. The term “pharmaceutically acceptable” means that the carrier, diluent, excipient or auxiliary is compatible with the other ingredients of the formulation and in no way deleterious to the recipient thereof.

[0049] 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 effect and a prophylactic effect, respectively. In some embodiments, a therapeutic effect includes one or more of the following: reduction / lessening of varicose veins, reduction / lessening of varicose vein severity (e.g., reduction or inhibition of varicose vein development), reduction / lessening of symptoms and varicose vein-related effects, delaying the onset of symptoms and varicose vein-related effects, lessening the severity of symptoms of varicose vein-related effects, lessening the severity of acute episodes, reducing the number and varicose vein-related effects, reducing the latency of symptoms and varicose vein-related effects, amelioration of symptoms and varicose vein-related effects, lessening of secondary symptoms, lessening of secondary infections, preventing recurrence of varicose veins, reducing the number or frequency of recurrences, increasing the latency between episodes of symptoms, increasing the time of sustained progression, accelerating remission, inducing remission, increasing remission, accelerating recovery, or increasing the efficacy of or reducing the tolerance to alternative therapies, and / or increasing the survival time of the affected host animal. A prophylactic effect can include completely or partially avoiding / inhibiting or delaying varicose vein development / progression (such as, for example, completely or partially avoiding / inhibiting or delaying), as well as increasing the survival time of the affected host animal, following administration of a therapeutic regimen. Treatment of varicose veins encompasses treating a subject who has been diagnosed with varicose veins in any clinical stage or manifestation, delaying the onset or evolution or aggravation or worsening of symptoms or signs of varicose veins, and / or preventing and / or lessening the severity of varicose veins.

[0050] The present disclosure also provides methods of identifying a subject at increased risk of developing varicose veins. In some embodiments, the methods include determining or having determined the presence or absence of a PIEZO1 variant nucleic acid molecule (such as a genomic nucleic acid molecule, an mRNA molecule, and / or a cDNA molecule) encoding a PIEZO1 predicted gain-of-function polypeptide in a biological sample obtained from the subject. When the subject lacks a PIEZO1 variant nucleic acid molecule encoding a PIEZO1 predicted gain-of-function polypeptide (i.e., the subject is genotypically classified as a PIEZO1 reference type), then the subject is at increased risk of developing varicose veins. When the subject has a PIEZO1 variant nucleic acid molecule encoding a PIEZO1 predicted gain-of-function polypeptide (i.e., the subject is heterozygous or homozygous for the PIEZO1 variant nucleic acid molecule), then the subject is at decreased risk of developing varicose veins compared to a subject that is a PIEZO1 reference type.

[0051] A single copy of a PIEZO1 variant nucleic acid molecule encoding a PIEZO1 predicted gain-of-function polypeptide protects a subject from developing varicose veins compared to a subject having no copies of a PIEZO1 variant nucleic acid molecule encoding a PIEZO1 predicted gain-of-function polypeptide. Without intending to be bound by any particular theory or mechanism of action, it is believed that a single copy of a PIEZO1 variant nucleic acid molecule (i.e., heterozygous for the PIEZO1 variant nucleic acid molecule) protects a subject from developing varicose veins and it is further believed that two copies of a PIEZO1 variant nucleic acid molecule encoding a PIEZO1 predicted gain-of-function polypeptide (i.e., homozygous for the PIEZO1 variant nucleic acid molecule) can protect a subject from developing varicose veins more than a subject having a single copy. Thus, in some embodiments, a single copy of a PIEZO1 variant nucleic acid molecule encoding a PIEZO1 predicted gain-of-function polypeptide can not be fully protective and can partially or incompletely protect a subject from developing varicose veins. While not wishing to be bound by any particular theory, there can be other factors or molecules involved in developing varicose veins that are still present in a subject having a single copy of a PIEZO1 variant nucleic acid molecule encoding a PIEZO1 predicted gain-of-function polypeptide, resulting in not being fully protected from developing varicose veins.

[0052] Detecting the presence or absence of a PIEZO1 variant nucleic acid molecule encoding a PIEZO1 predicted gain-of-function polypeptide in a biological sample obtained from a subject and / or determining whether a subject has a PIEZO1 variant nucleic acid molecule encoding a PIEZO1 predicted gain-of-function polypeptide can be performed by any of the methods described herein. In some embodiments, the methods can be performed in vitro. In some embodiments, the methods can be performed in situ. In some embodiments, the methods can be performed in vivo. In any of these embodiments, the PIEZO1 variant nucleic acid molecule encoding a PIEZO1 predicted gain-of-function polypeptide can be present in a cell obtained from the subject.

[0053] Any one or more (i.e., any combination) of PIEZO1 variant nucleic acid molecules encoding a PIEZO1 predicted gain-of-function polypeptide can be used in any of the methods described herein to determine whether a subject has an increased risk of developing varicose veins. Combinations of particular variants can form a mask for statistical analysis of the particular correlation of PIEZO1 to a reduced risk of developing varicose veins

[0054] In some embodiments, when a subject is identified as having an increased risk of developing varicose veins, the subject is further treated with a therapeutic agent that treats or prevents varicose veins and / or a PIEZO 1 agonist, as described herein. For example, when a subject is a PIEZO 1 reference type and therefore has an increased risk of developing varicose veins, the subject is administered a PIEZO 1 agonist. In some embodiments, such a subject is also administered a therapeutic agent that treats or prevents varicose veins. In some embodiments, when a subject is heterozygous for a PIEZO 1 variant nucleic acid molecule that encodes a PIEZO 1 predicted gain-of-function polypeptide, the subject is administered a therapeutic agent that treats or prevents varicose veins at a dose that is equal to or less than a standard dose, and / or is administered a PIEZO 1 agonist. In some embodiments, the subject is a PIEZO 1 reference type. In some embodiments, the subject is heterozygous for a PIEZO 1 variant nucleic acid molecule that encodes a PIEZO 1 predicted gain-of-function polypeptide.

[0055] In some embodiments, when a subject is identified as having an increased risk of developing varicose veins, the subject is further administered a therapeutic agent that treats or prevents varicose veins and / or a PIEZO 1 agonist, as described herein. For example, when a subject is a PIEZO 1 reference type and therefore has an increased risk of developing varicose veins, the subject is administered a PIEZO 1 agonist. In some embodiments, such a subject is also administered a therapeutic agent that treats or prevents varicose veins. In some embodiments, when a subject is heterozygous for a PIEZO 1 variant nucleic acid molecule that encodes a PIEZO 1 predicted gain-of-function polypeptide, the subject is administered a therapeutic agent that treats or prevents varicose veins at a dose that is equal to or less than a standard dose, and / or is administered a PIEZO 1 agonist. In some embodiments, the subject is a PIEZO 1 reference type. In some embodiments, the subject is heterozygous for a PIEZO 1 variant nucleic acid molecule that encodes a PIEZO 1 predicted gain-of-function polypeptide.

[0056] In some embodiments, any of the methods described herein can further comprise determining that the subject has a total burden of PIEZO1 variant nucleic acid molecules encoding PIEZO1 predicted loss-of-function polypeptides and / or PIEZO1 predicted loss-of-function variant polypeptides associated with a reduced risk of developing varicose veins. The total burden is the sum of all variants in the PIEZO1 gene, which can be analyzed in association with varicose veins. In some embodiments, the subject is homozygous for one or more PIEZO1 variant nucleic acid molecules encoding PIEZO1 predicted loss-of-function polypeptides associated with a reduced risk of developing varicose veins. In some embodiments, the subject is heterozygous for one or more PIEZO1 variant nucleic acid molecules encoding PIEZO1 predicted loss-of-function polypeptides associated with a reduced risk of developing varicose veins. The results of the association analysis indicate that PIEZO1 variant nucleic acid molecules encoding PIEZO1 predicted loss-of-function polypeptides are associated with a reduced risk of developing varicose veins. When the subject has a lower total burden, the subject is at a higher risk of developing varicose veins, and the subject is administered or continues to be administered a therapeutic or prophylactic varicose vein therapeutic agent, and / or a PIEZO1 agonist, at a standard dose. When the subject has a higher total burden, the subject is at a lower risk of developing varicose veins, and the subject is administered or continues to be administered a therapeutic or prophylactic varicose vein therapeutic agent in an amount equal to or less than a standard dose. The higher the total burden, the lower the risk of developing varicose veins.

[0057] In some embodiments, the total burden of any one or more PIEZO1 variant nucleic acid molecules encoding a PIEZO1 predicted gain-of-function polypeptide in a subject represents a weighted sum of any PIEZO1 variant nucleic acid molecules encoding a PIEZO1 predicted gain-of-function polypeptide. 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 or more than 1,000,000 genetic variants present in or around (up to 10 Mb) the PIEZO1 gene, where the genetic burden is the number of alleles multiplied by the association estimate or the associated result per allele (e.g., a weighted polygenic burden score) to varicosis. This can include any genetic variant in proximity to the PIEZO1 gene (up to 10 Mb around the gene), regardless of its genomic annotation, that shows a non-zero association to a varicosis-associated trait in a genetic association analysis. In some embodiments, a subject has a reduced risk of developing varicosis when the subject’s total burden is above a desired threshold score. In some embodiments, a subject has an increased risk of developing varicosis when the subject’s total burden is below a desired threshold score.

[0058] In some embodiments, the total burden can be divided into quintiles, such as a top quintile, a middle quintile, and a bottom quintile, where the top quintile of total burden corresponds to the lowest risk group, and the bottom quintile of total burden corresponds to the highest risk group. In some embodiments, a subject with a greater total burden includes the top weighted total burden, including but not limited to the top 10%, top 20%, top 30%, top 40%, or top 50% of total burden of a subject population. In some embodiments, the genetic variants include genetic variants with an association to varicosis in the top 10%, top 20%, top 30%, top 40%, or top 50% of a range of associated p-values. In some embodiments, each of the identified genetic variants includes a genetic variant associated with varicosis with a p-value no greater than 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 about 10 -15 In some embodiments, the identified genetic variants include genetic variants associated with varices having a p-value less than 5 x 10 -8 In some embodiments, the identified genetic variants include genetic variants associated with varices in high risk subjects having an odds ratio (OR) of about 1.5 or greater, about 1.75 or greater, about 2.0 or greater, or about 2.25 or greater for the top 20% of the distribution; or about 1.5 or greater, about 1.75 or greater, about 2.0 or greater, about 2.25 or greater, about 2.5 or greater, or about 2.75 or greater compared to the remainder of the reference population. In some embodiments, the odds ratio (OR) can 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 greater than 7.0. In some embodiments, high risk subjects include subjects having the lowest decile, quintile, or tertile of total burden in the reference population. The threshold for total burden is determined according to the nature of the intended practical application and the risk differential that would be considered meaningful for that practical application.

[0059] In some embodiments, when a subject is identified as having an increased risk of developing varicose veins, the subject is further administered a therapeutic agent that treats or prevents varicose veins and / or a PIEZO1 agonist, as described herein. For example, when a subject is a PIEZO1 reference type and therefore has an increased risk of developing varicose veins, the subject is administered a PIEZO1 agonist. In some embodiments, such a subject is also administered a therapeutic agent that treats or prevents varicose veins. In some embodiments, when a subject is heterozygous for a PIEZO1 variant nucleic acid molecule encoding a PIEZO1 predicted gain-of-function polypeptide, the subject is administered a therapeutic agent that treats or prevents varicose veins at a dose equal to or less than a standard dose, and / or is administered a PIEZO1 agonist. In some embodiments, the subject is a PIEZO1 reference type. In some embodiments, the subject is heterozygous for a PIEZO1 variant nucleic acid molecule encoding a PIEZO1 predicted gain-of-function polypeptide. Further, when a subject has a lower total burden of a PIEZO1 variant nucleic acid molecule encoding a PIEZO1 predicted gain-of-function polypeptide and therefore has an increased risk of developing varicose veins, the subject is administered a therapeutic agent that treats or prevents varicose veins. In some embodiments, when a subject has a lower total burden of a PIEZO1 variant nucleic acid molecule encoding a PIEZO1 predicted gain-of-function polypeptide, the subject is administered a therapeutic agent that treats or prevents varicose veins at a dose equal to or less than a standard dose administered to a subject having a higher total burden of a PIEZO1 variant nucleic acid molecule encoding a PIEZO1 predicted gain-of-function polypeptide.

[0060] The present disclosure also provides methods of detecting the presence or absence of a PIEZO1 variant genomic nucleic acid molecule encoding a PIEZO1 predicted gain-of-function polypeptide in a biological sample obtained from a subject, and / or the presence or absence of a PIEZO1 variant mRNA molecule encoding a PIEZO1 predicted gain-of-function polypeptide in a biological sample obtained from a subject, and / or the presence or absence of a PIEZO1 variant cDNA molecule encoding a PIEZO1 predicted gain-of-function polypeptide produced from an mRNA molecule in a biological sample obtained from a subject. It is understood that genetic sequences within a population and mRNA molecules encoded by such genes can vary due to polymorphisms, such as single nucleotide polymorphisms (SNPs). The sequences of the PIEZO1 variant genomic nucleic acid molecules, PIEZO1 variant mRNA molecules, and PIEZO1 variant cDNA molecules provided herein are merely exemplary sequences. Other sequences of PIEZO1 variant genomic nucleic acid molecules, variant mRNA molecules, and variant cDNA molecules are possible.

[0061] A biological sample can be derived from any cell, tissue, or biological fluid from a subject. A biological sample can include any clinically relevant tissue, such as a bone marrow sample, a tumor biopsy, a fine needle aspirate, or a sample of a bodily fluid, such as blood, gingival crevicular fluid, plasma, serum, lymph, ascites, cyst fluid, or urine. In some embodiments, a biological sample includes a buccal swab. The biological sample used in the methods disclosed herein can 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 can be treated differently depending on the assay employed. For example, when detecting any PIEZO1 variant nucleic acid molecule, a preliminary treatment designed to isolate or enrich the biological sample for PIEZO1 variant nucleic acid molecules can be employed. A variety of techniques can be used for this purpose. When detecting the level of any PIEZO1 variant mRNA molecule, different techniques can be employed to enrich the biological sample for mRNA molecules. Various methods can be used to detect the presence or level of mRNA molecules or the presence of specific variant genomic DNA sites.

[0062] The present disclosure also provides a method of detecting a PIEZO1 variant nucleic acid molecule, or a complement thereof, encoding a PIEZO1 predicted gain-of-function polypeptide in a subject. The method comprises assaying a biological sample obtained from the subject to determine whether a nucleic acid molecule in the biological sample is a PIEZO1 variant nucleic acid molecule encoding a PIEZO1 predicted gain-of-function polypeptide.

[0063] In some embodiments, the PIEZO1 variant nucleic acid molecule, or a complement thereof, encoding a PIEZO1 predicted gain-of-function polypeptide is a genomic nucleic acid molecule having a nucleotide sequence comprising a thymine, or a complement thereof, at a position corresponding to position 69,579 according to SEQ ID NO: 2.

[0064] In some embodiments, the PIEZO1 variant nucleic acid molecule encoding a PIEZO1 predicted gain-of-function polypeptide, or its complement, is an mRNA molecule having a nucleotide sequence comprising: a uracil at a position corresponding to position 7,785 according to SEQ ID NO: 13, or its complement; a uracil at a position corresponding to position 1,095 according to SEQ ID NO: 14, or its complement; a uracil at a position corresponding to position 6,073 according to SEQ ID NO: 15, or its complement; a uracil at a position corresponding to position 1,336 according to SEQ ID NO: 16, or its complement; a uracil at a position corresponding to position 6,073 according to SEQ ID NO: 17, or its complement; a uracil at a position corresponding to position 6,236 according to SEQ ID NO: 18, or its complement; a uracil at a position corresponding to position 7,526 according to SEQ ID NO: 19, or its complement; a uracil at a position corresponding to position 1,336 according to SEQ ID NO: 20, or its complement; a uracil at a position corresponding to position 361 according to SEQ ID NO: 21, or its complement; or a uracil at a position corresponding to position 7,776 according to SEQ ID NO: 22, or its complement.

[0065] In some embodiments, the PIEZO1 variant nucleic acid molecule encoding a PIEZO1 predicted gain-of-function polypeptide, or its complement, is a cDNA molecule having a nucleotide sequence comprising: a thymine at a position corresponding to position 7,785 according to SEQ ID NO: 33, or its complement; a thymine at a position corresponding to position 1,095 according to SEQ ID NO: 34, or its complement; a thymine at a position corresponding to position 6,073 according to SEQ ID NO: 35, or its complement; a thymine at a position corresponding to position 1,336 according to SEQ ID NO: 36, or its complement; a thymine at a position corresponding to position 6,073 according to SEQ ID NO: 37, or its complement; a thymine at a position corresponding to position 6,236 according to SEQ ID NO: 38, or its complement; a thymine at a position corresponding to position 7,526 according to SEQ ID NO: 39, or its complement; a thymine at a position corresponding to position 1,336 according to SEQ ID NO: 40, or its complement; a thymine at a position corresponding to position 361 according to SEQ ID NO: 41, or its complement; or a thymine at a position corresponding to position 7,776 according to SEQ ID NO: 42, or its complement.

[0066] In some embodiments, the PIEZO1 variant nucleic acid molecule has a nucleotide sequence comprising a thymine at a position corresponding to position 69,579 according to SEQ ID NO: 2, or a complement thereof (for genomic nucleic acid molecules); a uracil at a position corresponding to position 7,785 according to SEQ ID NO: 13, or a complement thereof (for mRNA molecules); or a thymine at a position corresponding to position 7,785 according to SEQ ID NO: 33, or a complement thereof (for cDNA molecules obtained from mRNA molecules).

[0067] In some embodiments, the biological sample comprises cells or cell lysates. Such methods can further comprise, for example, obtaining a biological sample comprising a PIEZO1 genomic nucleic acid molecule or mRNA molecule from a subject, and, if mRNA, optionally reverse transcribing the mRNA to cDNA. Such assays can comprise, for example, determining the identity of these positions for a particular PIEZO1 nucleic acid molecule. In some embodiments, the method is an in vitro method.

[0068] In some embodiments, the assay comprises sequencing at least a portion of the nucleotide sequence of a PIEZO1 nucleic acid molecule, or a complement thereof, in a biological sample. In some embodiments, the assay comprises sequencing at least a portion of the nucleotide sequence of: a PIEZO1 genomic nucleic acid molecule in a biological sample, wherein the sequenced portion comprises a position corresponding to position 69,579 according to SEQ ID NO: 2, or a complement thereof; a PIEZO1 mRNA molecule in a biological sample, wherein the sequenced portion comprises a position corresponding to position 7,785 according to SEQ ID NO: 22, or a complement thereof; and / or a PIEZO1 cDNA molecule in a biological sample produced from mRNA, wherein the sequenced portion comprises a position corresponding to position 7,785 according to SEQ ID NO: 33, or a complement thereof. When the sequenced portion of the PIEZO1 nucleic acid molecule in a biological sample comprises: a thymine at a position corresponding to position 69,579 according to SEQ ID NO: 2, or a complement thereof; a uracil at a position corresponding to position 7,785 according to SEQ ID NO: 13, or a complement thereof; or a thymine at a position corresponding to position 7,785 according to SEQ ID NO: 33, or a complement thereof; then the PIEZO1 nucleic acid molecule in the biological sample is a PIEZO1 variant nucleic acid molecule that encodes a PIEZO1 predicted gain-of-function polypeptide.

[0069] In some embodiments, the assay comprises sequencing at least a portion of the nucleotide sequence of a PIEZO 1 genomic nucleic acid molecule, or the complement thereof, in a biological sample, wherein the sequenced portion comprises a position corresponding to position 69,579 according to SEQ ID NO:2, or the complement thereof. When the sequenced portion of the PIEZO 1 genomic nucleic acid molecule in the biological sample comprises a thymine at a position corresponding to position 69,579 according to SEQ ID NO:2, or the complement thereof, then the PIEZO 1 genomic nucleic acid molecule in the biological sample is a PIEZO 1 variant genomic nucleic acid molecule that encodes a PIEZO 1 predicted loss-of-function polypeptide.

[0070] In some embodiments, the assay comprises sequencing at least a portion of the nucleotide sequence of a PIEZOl mRNA molecule in the biological sample, wherein the sequenced portion comprises a position corresponding to position: 7,785 according to SEQ ID NO: 13, or the complement thereof; 1,095 according to SEQ ID NO: 14, or the complement thereof; 6,073 according to SEQ ID NO: 15, or the complement thereof; 1,336 according to SEQ ID NO: 16, or the complement thereof; 6,073 according to SEQ ID NO: 17, or the complement thereof; 6,236 according to SEQ ID NO: 18, or the complement thereof; 7,526 according to SEQ ID NO: 19, or the complement thereof; 1,336 according to SEQ ID NO: 20, or the complement thereof; a position corresponding to position 361 according to SEQ ID NO: 21, or the complement thereof; or 7,776 according to SEQ ID NO: 22, or the complement thereof. When the sequenced portion of the PIEZOl mRNA molecule in the biological sample comprises: a uracil at a position corresponding to position 7,785 according to SEQ ID NO: 13, or the complement thereof; a uracil at a position corresponding to position 1,095 according to SEQ ID NO: 14, or the complement thereof; a uracil at a position corresponding to position 6,073 according to SEQ ID NO: 15, or the complement thereof; a uracil at a position corresponding to position 1,336 according to SEQ ID NO: 16, or the complement thereof; a uracil at a position corresponding to position 6,073 according to SEQ ID NO: 17, or the complement thereof; a uracil at a position corresponding to position 6,236 according to SEQ ID NO: 18, or the complement thereof; a uracil at a position corresponding to position 7,526 according to SEQ ID NO: 19, or the complement thereof; a uracil at a position corresponding to position 1,336 according to SEQ ID NO: 20, or the complement thereof; a uracil at a position corresponding to position 361 according to SEQ ID NO: 21, or the complement thereof; or a uracil at a position corresponding to position 7,776 according to SEQ ID NO: 22, or the complement thereof, then the PIEZOl mRNA molecule in the biological sample is a PIEZOl variant mRNA molecule encoding a PIEZOl predicted gain-of-function polypeptide.

[0071] In some embodiments, the assay comprises sequencing at least a portion of the nucleotide sequence of the PIEZO 1 cDNA molecule produced from an mRNA molecule in the biological sample, wherein the sequenced portion comprises a position corresponding to position: 7,785 according to SEQ ID NO:33, or the complement thereof; 1,095 according to SEQ ID NO:34, or the complement thereof; 6,073 according to SEQ ID NO:35, or the complement thereof; 1,336 according to SEQ ID NO:36, or the complement thereof; 6,073 according to SEQ ID NO:37, or the complement thereof; 6,236 according to SEQ ID NO:38, or the complement thereof; 7,526 according to SEQ ID NO:39, or the complement thereof; 1,336 according to SEQ ID NO:40, or the complement thereof; 361 according to SEQ ID NO:41, or the complement thereof; or 7,776 according to SEQ ID NO:42, or the complement thereof. When the sequenced portion of the PIEZO 1 cDNA molecule in the biological sample comprises: a thymine at a position corresponding to position 7,785 according to SEQ ID NO:33, or the complement thereof; a thymine at a position corresponding to position 1,095 according to SEQ ID NO:34, or the complement thereof; a thymine at a position corresponding to position 6,073 according to SEQ ID NO:35, or the complement thereof; a thymine at a position corresponding to position 1,336 according to SEQ ID NO:36, or the complement thereof; a thymine at a position corresponding to position 6,073 according to SEQ ID NO:37, or the complement thereof; a thymine at a position corresponding to position 6,236 according to SEQ ID NO:38, or the complement thereof; a thymine at a position corresponding to position 7,526 according to SEQ ID NO:39, or the complement thereof; a thymine at a position corresponding to position 1,336 according to SEQ ID NO:40, or the complement thereof; a thymine at a position corresponding to position 361 according to SEQ ID NO:41, or the complement thereof; or a thymine at a position corresponding to position 7,776 according to SEQ ID NO:42, or the complement thereof; then the PIEZO 1 cDNA molecule produced from an mRNA molecule in the biological sample is a PIEZO 1 variant cDNA molecule encoding a PIEZO 1 predicted loss-of-function polypeptide.

[0072] In some embodiments, the assay comprises: a) contacting a biological sample with a primer that hybridizes to a portion of a nucleotide sequence of a PIEZOl genomic nucleic acid molecule, or a complement thereof, proximal to a position corresponding to position 69,579 according to SEQ ID NO:2, or a complement thereof; a portion of a nucleotide sequence of a PIEZOl mRNA molecule, or a complement thereof, proximal to a position corresponding to position 7,785 according to SEQ ID NO: 13, or a complement thereof; and / or a portion of a nucleotide sequence of a PIEZOl cDNA molecule, or a complement thereof, proximal to a position corresponding to position 7,785 according to SEQ ID NO:33, or a complement thereof; b) extending the primer at least through: a position of a nucleotide sequence of a PIEZOl genomic nucleic acid molecule, or a complement thereof, corresponding to position 69,579 according to SEQ ID NO:2, or a complement thereof; a position of a nucleotide sequence of a PIEZOl mRNA molecule, or a complement thereof, corresponding to position 7,785 according to SEQ ID NO: 13, or a complement thereof; and / or a position of a nucleotide sequence of a PIEZOl cDNA molecule, or a complement thereof, corresponding to position 7,785 according to SEQ ID NO:33, or a complement thereof; and c) determining whether the extension product of the primer comprises: a thymine at a position corresponding to position 69,579 according to SEQ ID NO:2, or a complement thereof; a uracil at a position corresponding to position 7,785 according to SEQ ID NO: 13, or a complement thereof; and / or a thymine at a position corresponding to position 7,785 according to SEQ ID NO:33, or a complement thereof.

[0073] 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 PIEZOl mRNA molecule or the complement thereof proximal to a position corresponding to position 1,095 according to SEQ ID NO: 14, or the complement thereof; and / or a portion of the nucleotide sequence of a PIEZOl cDNA molecule or the complement thereof proximal to a position corresponding to position 1,095 according to SEQ ID NO: 34, or the complement thereof; b) extending the primer at least through: a position of the nucleotide sequence of a PIEZOl mRNA molecule or the complement thereof corresponding to position 1,095 according to SEQ ID NO: 14, or the complement thereof; and / or a position of the nucleotide sequence of a PIEZOl cDNA molecule or the complement thereof corresponding to position 1,095 according to SEQ ID NO: 34, or the complement thereof; and c) determining whether the extension product of the primer comprises: a uracil at a position corresponding to position 1,095 according to SEQ ID NO: 14, or the complement thereof; and / or a thymine at a position corresponding to position 1,095 according to SEQ ID NO: 34, or the complement thereof.

[0074] 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 PIEZOl mRNA molecule or the complement thereof proximal to a position corresponding to position 6,073 according to SEQ ID NO: 15, or the complement thereof; and / or a portion of the nucleotide sequence of a PIEZOl cDNA molecule or the complement thereof proximal to a position corresponding to position 6,073 according to SEQ ID NO: 35, or the complement thereof; b) extending the primer at least through: a position of the nucleotide sequence of a PIEZOl mRNA molecule or the complement thereof corresponding to position 6,073 according to SEQ ID NO: 15, or the complement thereof; and / or a position of the nucleotide sequence of a PIEZOl cDNA molecule or the complement thereof corresponding to position 6,073 according to SEQ ID NO: 35, or the complement thereof; and c) determining whether the extension product of the primer comprises: a uracil at a position corresponding to position 6,073 according to SEQ ID NO: 15, or the complement thereof; and / or a thymine at a position corresponding to position 6,073 according to SEQ ID NO: 35, or the complement thereof.

[0075] 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 PIEZOl mRNA molecule or the complement thereof proximal to a position corresponding to position 1,336 according to SEQ ID NO: 16, or the complement thereof; and / or a portion of the nucleotide sequence of a PIEZOl cDNA molecule or the complement thereof proximal to a position corresponding to position 1,336 according to SEQ ID NO: 36, or the complement thereof; b) extending the primer at least through: a position of the nucleotide sequence of a PIEZOl mRNA molecule or the complement thereof corresponding to position 1,336 according to SEQ ID NO: 16, or the complement thereof; and / or a position of the nucleotide sequence of a PIEZOl cDNA molecule or the complement thereof corresponding to position 1,336 according to SEQ ID NO: 36, or the complement thereof; and c) determining whether the extension product of the primer comprises: a uracil at a position corresponding to position 1,336 according to SEQ ID NO: 16, or the complement thereof; and / or a thymine at a position corresponding to position 1,336 according to SEQ ID NO: 36, or the complement thereof.

[0076] 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 PIEZOl mRNA molecule or the complement thereof proximal to a position corresponding to position 6,073 according to SEQ ID NO: 17, or the complement thereof; and / or a portion of the nucleotide sequence of a PIEZOl cDNA molecule or the complement thereof proximal to a position corresponding to position 6,073 according to SEQ ID NO: 37, or the complement thereof; b) extending the primer at least through: a position of the nucleotide sequence of a PIEZOl mRNA molecule or the complement thereof corresponding to position 6,073 according to SEQ ID NO: 17, or the complement thereof; and / or a position of the nucleotide sequence of a PIEZOl cDNA molecule or the complement thereof corresponding to position 6,073 according to SEQ ID NO: 37, or the complement thereof; and c) determining whether the extension product of the primer comprises: a uracil at a position corresponding to position 6,073 according to SEQ ID NO: 17, or the complement thereof; and / or a thymine at a position corresponding to position 6,073 according to SEQ ID NO: 37, or the complement thereof.

[0077] In some embodiments, the assay comprises: a) contacting a biological sample with a primer that hybridizes to a portion of a nucleotide sequence of a PIEZOl mRNA molecule, or the complement thereof, proximal to a position corresponding to position 6,236 according to SEQ ID NO: 18, or the complement thereof; and / or a nucleotide sequence of a PIEZOl cDNA molecule, or the complement thereof, proximal to a position corresponding to position 6,236 according to SEQ ID NO: 38, or the complement thereof; b) extending the primer at least through: a position of the nucleotide sequence of the PIEZOl mRNA molecule, or the complement thereof, corresponding to position 6,236 according to SEQ ID NO: 18, or the complement thereof; and / or a position of the nucleotide sequence of the PIEZOl cDNA molecule, or the complement thereof, corresponding to position 6,236 according to SEQ ID NO: 38, or the complement thereof; and c) determining whether the extension product of the primer comprises: a uracil at a position corresponding to position 6,236 according to SEQ ID NO: 18, or the complement thereof; and / or a thymine at a position corresponding to position 6,236 according to SEQ ID NO: 38, or the complement thereof.

[0078] In some embodiments, the assay comprises: a) contacting a biological sample with a primer that hybridizes to a portion of a nucleotide sequence of a PIEZOl mRNA molecule, or the complement thereof, proximal to a position corresponding to position 7,526 according to SEQ ID NO: 19, or the complement thereof; and / or a nucleotide sequence of a PIEZOl cDNA molecule, or the complement thereof, proximal to a position corresponding to position 7,526 according to SEQ ID NO: 39, or the complement thereof; b) extending the primer at least through: a position of the nucleotide sequence of the PIEZOl mRNA molecule, or the complement thereof, corresponding to position 7,526 according to SEQ ID NO: 19, or the complement thereof; and / or a position of the nucleotide sequence of the PIEZOl cDNA molecule, or the complement thereof, corresponding to position 7,526 according to SEQ ID NO: 39, or the complement thereof; and c) determining whether the extension product of the primer comprises: a uracil at a position corresponding to position 7,526 according to SEQ ID NO: 19, or the complement thereof; and / or a thymine at a position corresponding to position 7,526 according to SEQ ID NO: 39, or the complement thereof.

[0079] In some embodiments, the assay comprises: a) contacting a biological sample with a primer that hybridizes to a portion of a nucleotide sequence of a PIEZOl mRNA molecule, or the complement thereof, proximal to a position corresponding to position 1,336 according to SEQ ID NO:20, or the complement thereof; and / or a nucleotide sequence of a PIEZOl cDNA molecule, or the complement thereof, proximal to a position corresponding to position 1,336 according to SEQ ID NO:40, or the complement thereof; b) extending the primer at least through: a position of the nucleotide sequence of the PIEZOl mRNA molecule, or the complement thereof, corresponding to position 1,336 according to SEQ ID NO:20, or the complement thereof; and / or a position of the nucleotide sequence of the PIEZOl cDNA molecule, or the complement thereof, corresponding to position 1,336 according to SEQ ID NO:40, or the complement thereof; and c) determining whether the extension product of the primer comprises: a uracil at a position corresponding to position 1,336 according to SEQ ID NO:20, or the complement thereof; and / or a thymine at a position corresponding to position 1,336 according to SEQ ID NO:40, or the complement thereof.

[0080] In some embodiments, the assay comprises: a) contacting a biological sample with a primer that hybridizes to a portion of a nucleotide sequence of a PIEZOl mRNA molecule, or the complement thereof, proximal to a position corresponding to position 361 according to SEQ ID NO:21, or the complement thereof; and / or a nucleotide sequence of a PIEZOl cDNA molecule, or the complement thereof, proximal to a position corresponding to position 361 according to SEQ ID NO:41, or the complement thereof; b) extending the primer at least through: a position of the nucleotide sequence of the PIEZOl mRNA molecule, or the complement thereof, corresponding to position 361 according to SEQ ID NO:21, or the complement thereof; and / or a position of the nucleotide sequence of the PIEZOl cDNA molecule, or the complement thereof, corresponding to position 361 according to SEQ ID NO:41, or the complement thereof; and c) determining whether the extension product of the primer comprises: a uracil at a position corresponding to position 361 according to SEQ ID NO:21, or the complement thereof; and / or a thymine at a position corresponding to position 361 according to SEQ ID NO:41, or the complement thereof.

[0081] 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 PIEZOl mRNA molecule, or the complement thereof, proximal to a position corresponding to position 7,776 according to SEQ ID NO: 22, or the complement thereof; and / or a PIEZOl cDNA molecule, or the complement thereof, proximal to a position corresponding to position 7,776 according to SEQ ID NO: 42, or the complement thereof; b) extending the primer at least through a position of the nucleotide sequence of a PIEZOl mRNA molecule, or the complement thereof, corresponding to position 7,776 according to SEQ ID NO: 22, or the complement thereof; and / or a PIEZOl cDNA molecule, or the complement thereof, corresponding to position 7,776 according to SEQ ID NO: 42, or the complement thereof; and c) determining whether the extension product of the primer comprises: a Uracil at a position corresponding to position 7,776 according to SEQ ID NO: 22, or the complement thereof; and / or a Thymine at a position corresponding to position 7,776 according to SEQ ID NO: 42, or the complement thereof.

[0082] 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 PIEZOl genomic nucleic acid molecule, or the complement thereof, proximal to a position corresponding to position 69,579 according to SEQ ID NO: 2, or the complement thereof; b) extending the primer at least through a position of the nucleotide sequence of a PIEZOl genomic nucleic acid molecule, or the complement thereof, corresponding to position 69,579 according to SEQ ID NO: 2, or the complement thereof; and c) determining whether the extension product of the primer comprises a Thymine at a position corresponding to position 69,579 according to SEQ ID NO: 2, or the complement thereof.

[0083] In some embodiments, the assay comprises: a) contacting a biological sample with a primer that hybridizes to a portion of a nucleotide sequence of a PIEZOl mRNA molecule, or a complement thereof, proximal to a position corresponding to: position 7,785 according to SEQ ID NO: 13, or a complement thereof; position 1,095 according to SEQ ID NO: 14, or a complement thereof; position 6,073 according to SEQ ID NO: 15, or a complement thereof; position 1,336 according to SEQ ID NO: 16, or a complement thereof; position 6,073 according to SEQ ID NO: 17, or a complement thereof; position 6,236 according to SEQ ID NO: 18, or a complement thereof; position 7,526 according to SEQ ID NO: 19, or a complement thereof; position 1,336 according to SEQ ID NO: 20, or a complement thereof; a position corresponding to position 361 according to SEQ ID NO: 21, or a complement thereof; or position 7,776 according to SEQ ID NO: 22, or a complement thereof; b) extending the primer at least through a position of the nucleotide sequence of the PIEZOl mRNA molecule, or a complement thereof, corresponding to: position 7,785 according to SEQ ID NO: 13, or a complement thereof; position 1,095 according to SEQ ID NO: 14, or a complement thereof; position 6,073 according to SEQ ID NO: 15, or a complement thereof; position 1,336 according to SEQ ID NO: 16, or a complement thereof; position 6,073 according to SEQ ID NO: 17, or a complement thereof; position 6,236 according to SEQ ID NO: 18, or a complement thereof; position 7,526 according to SEQ ID NO: 19, or a complement thereof; position 1,336 according to SEQ ID NO: 20, or a complement thereof; position 361 according to SEQ ID NO: 21, or a complement thereof; or position 7,776 according to SEQ ID NO: 22, or a complement thereof; and c) determining whether the extension product of the primer comprises: a uracil at a position corresponding to position 7,785 according to SEQ ID NO: 13, or a complement thereof; a uracil at a position corresponding to position 1,095 according to SEQ ID NO: 14, or a complement thereof; a uracil at a position corresponding to position 6,073 according to SEQ ID NO: 15, or a complement thereof; a uracil at a position corresponding to position 1,336 according to SEQ ID NO: 16, or a complement thereof; a uracil at a position corresponding to position 6,073 according to SEQ ID NO: 17, or a complement thereof; a uracil at a position corresponding to position 6,236 according to SEQ ID NO: 18, or a complement thereof;a uracil at a position corresponding to position 7,526 according to SEQ ID NO: 19, or a complement thereof; a uracil at a position corresponding to position 1,336 according to SEQ ID NO: 20, or a complement thereof; a uracil at a position corresponding to position 361 according to SEQ ID NO: 21, or a complement thereof; or a uracil at a position corresponding to position 7,776 according to SEQ ID NO: 22, or a complement thereof.

[0084] In some embodiments, the assay comprises: a) contacting a biological sample with a primer that hybridizes to a portion of a nucleotide sequence of a PIEZOl cDNA molecule, or the complement thereof, proximal to a position corresponding to: position 7,785 according to SEQ ID NO:33, or the complement thereof; position 1,095 according to SEQ ID NO:34, or the complement thereof; position 6,073 according to SEQ ID NO:35, or the complement thereof; position 1,336 according to SEQ ID NO:36, or the complement thereof; position 6,073 according to SEQ ID NO:37, or the complement thereof; position 6,236 according to SEQ ID NO:38, or the complement thereof; position 7,526 according to SEQ ID NO:39, or the complement thereof; position 1,336 according to SEQ ID NO:40, or the complement thereof; position 361 according to SEQ ID NO:41, or the complement thereof; or position 7,776 according to SEQ ID NO:42, or the complement thereof; b) extending the primer at least through a position of the nucleotide sequence of the PIEZOl cDNA molecule, or the complement thereof, corresponding to: position 7,785 according to SEQ ID NO:33, or the complement thereof; position 1,095 according to SEQ ID NO:34, or the complement thereof; position 6,073 according to SEQ ID NO:35, or the complement thereof; position 1,336 according to SEQ ID NO:36, or the complement thereof; position 6,073 according to SEQ ID NO:37, or the complement thereof; position 6,236 according to SEQ ID NO:38, or the complement thereof; position 7,526 according to SEQ ID NO:39, or the complement thereof; position 1,336 according to SEQ ID NO:40, or the complement thereof; position 361 according to SEQ ID NO:41, or the complement thereof; or position 7,776 according to SEQ ID NO:42, or the complement thereof; and c) determining whether the extension product of the primer comprises: a thymine at a position corresponding to position 7,785 according to SEQ ID NO:33, or the complement thereof; a thymine at a position corresponding to position 1,095 according to SEQ ID NO:34, or the complement thereof; a thymine at a position corresponding to position 6,073 according to SEQ ID NO:35, or the complement thereof; a thymine at a position corresponding to position 1,336 according to SEQ ID NO:36, or the complement thereof; a thymine at a position corresponding to position 6,073 according to SEQ ID NO:37, or the complement thereof; a thymine at a position corresponding to position 6,236 according to SEQ ID NO:38, or the complement thereof;a thymine at a position corresponding to position 7,526 according to SEQ ID NO: 39, or a complement thereof; a thymine at a position corresponding to position 1,336 according to SEQ ID NO: 40, or a complement thereof; a thymine at a position corresponding to position 361 according to SEQ ID NO: 41, or a complement thereof; or a thymine at a position corresponding to position 7,776 according to SEQ ID NO: 42, or a complement thereof.

[0085] In some embodiments, the assay comprises sequencing the entire nucleic acid molecule. In some embodiments, only PIEZO1 genomic nucleic acid molecules are analyzed. In some embodiments, only PIEZO1 mRNA is analyzed. In some embodiments, only PIEZO1 cDNA obtained from PIEZO1 mRNA is analyzed.

[0086] In some embodiments, the assay comprises: a) amplifying at least a portion of a PIEZO1 nucleic acid molecule, or a complement thereof, in a biological sample, wherein the amplified portion comprises: a thymine at a position corresponding to position 69,579 according to SEQ ID NO: 2, or a complement thereof; a uracil at a position corresponding to position 7,785 according to SEQ ID NO: 13, or a complement thereof; and / or a thymine at a position corresponding to position 7,785 according to SEQ ID NO: 33, or a complement thereof; b) labeling the amplified nucleic acid molecule with a detectable label; c) contacting the labeled nucleic acid molecule with a support comprising an alteration-specific probe, wherein the alteration-specific probe comprises a nucleotide sequence that hybridizes under stringent conditions to a nucleotide sequence of the amplified nucleic acid molecule comprising: a thymine at a position corresponding to position 69,579 according to SEQ ID NO: 2, or a complement thereof; a uracil at a position corresponding to position 7,785 according to SEQ ID NO: 13, or a complement thereof; and / or a thymine at a position corresponding to position 7,785 according to SEQ ID NO: 33, or a complement thereof; and d) detecting the detectable label.

[0087] In some embodiments, the assay comprises: a) amplifying at least a portion of a PIEZOl nucleic acid molecule, or the complement thereof, in a biological sample, wherein the amplified portion comprises: a uracil at a position corresponding to position 1,095 according to SEQ ID NO: 14, or the complement thereof; and / or a thymine at a position corresponding to position 1,095 according to SEQ ID NO: 34, or the complement thereof; b) labeling the amplified nucleic acid molecule with a detectable label; c) contacting the labeled nucleic acid molecule with a support comprising an alteration-specific probe, wherein the alteration-specific probe comprises a nucleotide sequence that hybridizes under stringent conditions to a nucleotide sequence of the amplified nucleic acid molecule comprising: a uracil at a position corresponding to position 1,095 according to SEQ ID NO: 14, or the complement thereof; and / or a thymine at a position corresponding to position 1,095 according to SEQ ID NO: 34, or the complement thereof; and d) detecting the detectable label.

[0088] In some embodiments, the assay comprises: a) amplifying at least a portion of a PIEZOl nucleic acid molecule, or the complement thereof, in a biological sample, wherein the amplified portion comprises: a uracil at a position corresponding to position 6,073 according to SEQ ID NO: 15, or the complement thereof; and / or a thymine at a position corresponding to position 6,073 according to SEQ ID NO: 35, or the complement thereof; b) labeling the amplified nucleic acid molecule with a detectable label; c) contacting the labeled nucleic acid molecule with a support comprising an alteration-specific probe, wherein the alteration-specific probe comprises a nucleotide sequence that hybridizes under stringent conditions to a nucleotide sequence of the amplified nucleic acid molecule comprising: a uracil at a position corresponding to position 6,073 according to SEQ ID NO: 15, or the complement thereof; and / or a thymine at a position corresponding to position 6,073 according to SEQ ID NO: 35, or the complement thereof; and d) detecting the detectable label.

[0089] In some embodiments, the assay comprises: a) amplifying at least a portion of a PIEZOl nucleic acid molecule, or the complement thereof, in a biological sample, wherein the amplified portion comprises: a uracil at a position corresponding to position 1,336 according to SEQ ID NO: 16, or the complement thereof; and / or a thymine at a position corresponding to position 1,336 according to SEQ ID NO: 36, or the complement thereof; b) labeling the amplified nucleic acid molecule with a detectable label; c) contacting the labeled nucleic acid molecule with a support comprising an alteration-specific probe, wherein the alteration-specific probe comprises a nucleotide sequence that hybridizes under stringent conditions to a nucleotide sequence of the amplified nucleic acid molecule comprising: a uracil at a position corresponding to position 1,336 according to SEQ ID NO: 16, or the complement thereof; and / or a thymine at a position corresponding to position 1,336 according to SEQ ID NO: 36, or the complement thereof; and d) detecting the detectable label.

[0090] In some embodiments, the assay comprises: a) amplifying at least a portion of a PIEZOl nucleic acid molecule, or the complement thereof, in a biological sample, wherein the amplified portion comprises: a uracil at a position corresponding to position 6,073 according to SEQ ID NO: 17, or the complement thereof; and / or a thymine at a position corresponding to position 6,073 according to SEQ ID NO: 37, or the complement thereof; b) labeling the amplified nucleic acid molecule with a detectable label; c) contacting the labeled nucleic acid molecule with a support comprising an alteration-specific probe, wherein the alteration-specific probe comprises a nucleotide sequence that hybridizes under stringent conditions to a nucleotide sequence of the amplified nucleic acid molecule comprising: a uracil at a position corresponding to position 6,073 according to SEQ ID NO: 17, or the complement thereof; and / or a thymine at a position corresponding to position 6,073 according to SEQ ID NO: 37, or the complement thereof; and d) detecting the detectable label.

[0091] In some embodiments, the assay comprises: a) amplifying at least a portion of a PIEZOl nucleic acid molecule, or the complement thereof, in a biological sample, wherein the amplified portion comprises: a uracil at a position corresponding to position 6,236 according to SEQ ID NO: 18, or the complement thereof; and / or a thymine at a position corresponding to position 6,236 according to SEQ ID NO: 38, or the complement thereof; b) labeling the amplified nucleic acid molecule with a detectable label; c) contacting the labeled nucleic acid molecule with a support comprising an alteration-specific probe, wherein the alteration-specific probe comprises a nucleotide sequence that hybridizes under stringent conditions to a nucleotide sequence of the amplified nucleic acid molecule comprising: a uracil at a position corresponding to position 6,236 according to SEQ ID NO: 18, or the complement thereof; and / or a thymine at a position corresponding to position 6,236 according to SEQ ID NO: 38, or the complement thereof; and d) detecting the detectable label.

[0092] In some embodiments, the assay comprises: a) amplifying at least a portion of a PIEZOl nucleic acid molecule, or the complement thereof, in a biological sample, wherein the amplified portion comprises: a uracil at a position corresponding to position 7,526 according to SEQ ID NO: 19, or the complement thereof; and / or a thymine at a position corresponding to position 7,526 according to SEQ ID NO: 39, or the complement thereof; b) labeling the amplified nucleic acid molecule with a detectable label; c) contacting the labeled nucleic acid molecule with a support comprising an alteration-specific probe, wherein the alteration-specific probe comprises a nucleotide sequence that hybridizes under stringent conditions to a nucleotide sequence of the amplified nucleic acid molecule comprising: a uracil at a position corresponding to position 7,526 according to SEQ ID NO: 19, or the complement thereof; and / or a thymine at a position corresponding to position 7,526 according to SEQ ID NO: 39, or the complement thereof; and d) detecting the detectable label.

[0093] In some embodiments, the assay comprises: a) amplifying at least a portion of a PIEZO 1 nucleic acid molecule, or the complement thereof, in a biological sample, wherein the amplified portion comprises: a uracil at a position corresponding to position 1,336 according to SEQ ID NO: 20, or the complement thereof; and / or a thymine at a position corresponding to position 1,336 according to SEQ ID NO: 40, or the complement thereof; b) labeling the amplified nucleic acid molecule with a detectable label; c) contacting the labeled nucleic acid molecule with a support comprising an alteration-specific probe, wherein the alteration-specific probe comprises a nucleotide sequence that hybridizes under stringent conditions to a nucleotide sequence of the amplified nucleic acid molecule comprising: a uracil at a position corresponding to position 1,336 according to SEQ ID NO: 20, or the complement thereof; and / or a thymine at a position corresponding to position 1,336 according to SEQ ID NO: 40, or the complement thereof; and d) detecting the detectable label.

[0094] In some embodiments, the assay comprises: a) amplifying at least a portion of a PIEZO 1 nucleic acid molecule, or the complement thereof, in a biological sample, wherein the amplified portion comprises: a uracil at a position corresponding to position 361 according to SEQ ID NO: 21, or the complement thereof; and / or a thymine at a position corresponding to position 361 according to SEQ ID NO: 41, or the complement thereof; b) labeling the amplified nucleic acid molecule with a detectable label; c) contacting the labeled nucleic acid molecule with a support comprising an alteration-specific probe, wherein the alteration-specific probe comprises a nucleotide sequence that hybridizes under stringent conditions to a nucleotide sequence of the amplified nucleic acid molecule comprising: a uracil at a position corresponding to position 361 according to SEQ ID NO: 21, or the complement thereof; and / or a thymine at a position corresponding to position 361 according to SEQ ID NO: 41, or the complement thereof; and d) detecting the detectable label.

[0095] In some embodiments, the assay comprises: a) amplifying at least a portion of a PIEZO 1 nucleic acid molecule, or a complement thereof, in a biological sample, wherein the amplified portion comprises: a uracil at a position corresponding to position 7,776 according to SEQ ID NO: 22, or a complement thereof; and / or a thymine at a position corresponding to position 7,776 according to SEQ ID NO: 42, or a complement thereof; b) labeling the amplified nucleic acid molecule with a detectable label; c) contacting the labeled nucleic acid molecule with a support comprising an alteration-specific probe, wherein the alteration-specific probe comprises a nucleotide sequence that hybridizes under stringent conditions to a nucleotide sequence of the amplified nucleic acid molecule comprising: a uracil at a position corresponding to position 7,776 according to SEQ ID NO: 22, or a complement thereof; and / or a thymine at a position corresponding to position 7,776 according to SEQ ID NO: 42, or a complement thereof; and d) detecting the detectable label.

[0096] In some embodiments, the assay comprises: a) amplifying at least a portion of a PIEZO 1 genomic nucleic acid molecule, or a complement thereof, in a biological sample, wherein the portion comprises: a thymine at a position corresponding to position 69,579 according to SEQ ID NO: 2, or a complement thereof; and d) detecting the detectable label.

[0097] In some embodiments, the assay comprises: a) amplifying at least a portion of a PIEZOl mRNA molecule, or a complement thereof, in a biological sample, wherein the portion comprises: a uracil at a position corresponding to position 7,785 according to SEQ ID NO: 13, or a complement thereof; a uracil at a position corresponding to position 1,095 according to SEQ ID NO: 14, or a complement thereof; a uracil at a position corresponding to position 6,073 according to SEQ ID NO: 15, or a complement thereof; a uracil at a position corresponding to position 1,336 according to SEQ ID NO: 16, or a complement thereof; a uracil at a position corresponding to position 6,073 according to SEQ ID NO: 17, or a complement thereof; a uracil at a position corresponding to position 6,236 according to SEQ ID NO: 18, or a complement thereof; a uracil at a position corresponding to position 7,526 according to SEQ ID NO: 19, or a complement thereof; a uracil at a position corresponding to position 1,336 according to SEQ ID NO: 20, or a complement thereof; a uracil at a position corresponding to position 361 according to SEQ ID NO: 21, or a complement thereof; or a uracil at a position corresponding to position 7,776 according to SEQ ID NO: 22, or a complement thereof; b) labeling the amplified nucleic acid molecule with a detectable label; c) contacting the labeled nucleic acid molecule with a support comprising an alteration-specific probe, wherein the alteration-specific probe comprises a nucleotide sequence that hybridizes under stringent conditions to a nucleic acid sequence of the amplified nucleic acid molecule comprising: a uracil at a position corresponding to position 7,785 according to SEQ ID NO: 13, or a complement thereof; a uracil at a position corresponding to position 1,095 according to SEQ ID NO: 14, or a complement thereof; a uracil at a position corresponding to position 6,073 according to SEQ ID NO: 15, or a complement thereof; a uracil at a position corresponding to position 1,336 according to SEQ ID NO: 16, or a complement thereof; a uracil at a position corresponding to position 6,073 according to SEQ ID NO: 17, or a complement thereof; a uracil at a position corresponding to position 6,236 according to SEQ ID NO: 18, or a complement thereof; a uracil at a position corresponding to position 7,526 according to SEQ ID NO: 19, or a complement thereof; a uracil at a position corresponding to position 1,336 according to SEQ ID NO: 20, or a complement thereof; a uracil at a position corresponding to position 361 according to SEQ ID NO: 21, or a complement thereof; or a uracil at a position corresponding to position 7,776 according to SEQ ID NO: 22, or a complement thereof; and d) detecting the detectable label.

[0098] In some embodiments, the assay comprises: a) amplifying at least a portion of a PIEZOl cDNA molecule, or the complement thereof, produced from an mRNA molecule in a biological sample, wherein the portion comprises: a thymine at a position corresponding to position 7,785 according to SEQ ID NO:33, or the complement thereof; a thymine at a position corresponding to position 1,095 according to SEQ ID NO:34, or the complement thereof; a thymine at a position corresponding to position 6,073 according to SEQ ID NO:35, or the complement thereof; a thymine at a position corresponding to position 1,336 according to SEQ ID NO:36, or the complement thereof; a thymine at a position corresponding to position 6,073 according to SEQ ID NO:37, or the complement thereof; a thymine at a position corresponding to position 6,236 according to SEQ ID NO:38, or the complement thereof; a thymine at a position corresponding to position 7,526 according to SEQ ID NO:39, or the complement thereof; a thymine at a position corresponding to position 1,336 according to SEQ ID NO:40, or the complement thereof; a thymine at a position corresponding to position 361 according to SEQ ID NO:41, or the complement thereof; or a thymine at a position corresponding to position 7,776 according to SEQ ID NO:42, or the complement thereof; b) labeling the amplified nucleic acid molecule with a detectable label; c) contacting the labeled nucleic acid molecule with a support comprising an alteration-specific probe, wherein the alteration-specific probe comprises a nucleotide sequence that hybridizes under stringent conditions to a nucleic acid sequence of the amplified nucleic acid molecule comprising: a thymine at a position corresponding to position 7,785 according to SEQ ID NO:33, or the complement thereof; a thymine at a position corresponding to position 1,095 according to SEQ ID NO:34, or the complement thereof; a thymine at a position corresponding to position 6,073 according to SEQ ID NO:35, or the complement thereof; a thymine at a position corresponding to position 1,336 according to SEQ ID NO:36, or the complement thereof; a thymine at a position corresponding to position 6,073 according to SEQ ID NO:37, or the complement thereof; a thymine at a position corresponding to position 6,236 according to SEQ ID NO:38, or the complement thereof; a thymine at a position corresponding to position 7,526 according to SEQ ID NO:39, or the complement thereof; a thymine at a position corresponding to position 1,336 according to SEQ ID NO:40, or the complement thereof; a thymine at a position corresponding to position 361 according to SEQ ID NO:41, or the complement thereof;or a Thymine at a position corresponding to position 7,776, according to SEQ ID NO: 42, or a complement thereof; and d) detecting the detectable label.

[0099] In some embodiments, the nucleic acid molecules in the sample are mRNA, and the mRNA is reverse transcribed into cDNA prior to the amplifying step.

[0100] In some embodiments, the assay comprises: contacting a PIEZO1 nucleic acid molecule, or a complement thereof, in a biological sample with an alteration-specific probe comprising a detectable label, wherein the alteration-specific probe comprises a nucleotide sequence that hybridizes under stringent conditions to a nucleotide sequence of a PIEZO1 nucleic acid molecule, or a complement thereof, comprising: a Thymine at a position corresponding to position 69,579, according to SEQ ID NO: 2, or a complement thereof; a Uracil at a position corresponding to position 7,785, according to SEQ ID NO: 13, or a complement thereof; and / or a Thymine at a position corresponding to position 7,785, according to SEQ ID NO: 33, or a complement thereof; and detecting the detectable label.

[0101] In some embodiments, the assay comprises: contacting a PIEZO1 nucleic acid molecule, or a complement thereof, in a biological sample with an alteration-specific probe comprising a detectable label, wherein the alteration-specific probe comprises a nucleotide sequence that hybridizes under stringent conditions to a nucleotide sequence of a PIEZO1 nucleic acid molecule, or a complement thereof, comprising: a Uracil at a position corresponding to position 1,095, according to SEQ ID NO: 14, or a complement thereof; and / or a Thymine at a position corresponding to position 1,095, according to SEQ ID NO: 34, or a complement thereof; and detecting the detectable label.

[0102] In some embodiments, the assay comprises: contacting a PIEZO1 nucleic acid molecule, or a complement thereof, in a biological sample with an alteration-specific probe comprising a detectable label, wherein the alteration-specific probe comprises a nucleotide sequence that hybridizes under stringent conditions to a nucleotide sequence of a PIEZO1 nucleic acid molecule, or a complement thereof, comprising: a Uracil at a position corresponding to position 6,073, according to SEQ ID NO: 15, or a complement thereof; and / or a Thymine at a position corresponding to position 6,073, according to SEQ ID NO: 35, or a complement thereof; and detecting the detectable label.

[0103] In some embodiments, the assay comprises: contacting a PIEZO1 nucleic acid molecule, or a complement thereof, in a biological sample with an alteration-specific probe comprising a detectable label, wherein the alteration-specific probe comprises a nucleotide sequence that hybridizes, under stringent conditions, to a nucleotide sequence of a PIEZO1 nucleic acid molecule, or a complement thereof, comprising: a uracil at a position corresponding to position 1,336 according to SEQ ID NO: 16, or a complement thereof; and / or a thymine at a position corresponding to position 1,336 according to SEQ ID NO: 36, or a complement thereof; and detecting the detectable label.

[0104] In some embodiments, the assay comprises: contacting a PIEZO1 nucleic acid molecule, or a complement thereof, in a biological sample with an alteration-specific probe comprising a detectable label, wherein the alteration-specific probe comprises a nucleotide sequence that hybridizes, under stringent conditions, to a nucleotide sequence of a PIEZO1 nucleic acid molecule, or a complement thereof, comprising: a uracil at a position corresponding to position 6,073 according to SEQ ID NO: 17, or a complement thereof; and / or a thymine at a position corresponding to position 6,073 according to SEQ ID NO: 37, or a complement thereof; and detecting the detectable label.

[0105] In some embodiments, the assay comprises: contacting a PIEZO1 nucleic acid molecule, or a complement thereof, in a biological sample with an alteration-specific probe comprising a detectable label, wherein the alteration-specific probe comprises a nucleotide sequence that hybridizes, under stringent conditions, to a nucleotide sequence of a PIEZO1 nucleic acid molecule, or a complement thereof, comprising: a uracil at a position corresponding to position 6,236 according to SEQ ID NO: 18, or a complement thereof; and / or a thymine at a position corresponding to position 6,236 according to SEQ ID NO: 38, or a complement thereof; and detecting the detectable label.

[0106] In some embodiments, the assay comprises: contacting a PIEZO1 nucleic acid molecule, or a complement thereof, in a biological sample with an alteration-specific probe comprising a detectable label, wherein the alteration-specific probe comprises a nucleotide sequence that hybridizes, under stringent conditions, to a nucleotide sequence of a PIEZO1 nucleic acid molecule, or a complement thereof, comprising: a uracil at a position corresponding to position 7,526 according to SEQ ID NO: 19, or a complement thereof; and / or a thymine at a position corresponding to position 7,526 according to SEQ ID NO: 39, or a complement thereof; and detecting the detectable label.

[0107] In some embodiments, the assay comprises: contacting a PIEZO1 nucleic acid molecule, or a complement thereof, in a biological sample with an alteration-specific probe comprising a detectable label, wherein the alteration-specific probe comprises a nucleotide sequence that hybridizes, under stringent conditions, to a nucleotide sequence of a PIEZO1 nucleic acid molecule, or a complement thereof, comprising: a uracil at a position corresponding to position 1,336 according to SEQ ID NO: 20, or a complement thereof; and / or a thymine at a position corresponding to position 1,336 according to SEQ ID NO: 40, or a complement thereof; and detecting the detectable label.

[0108] In some embodiments, the assay comprises: contacting a PIEZO1 nucleic acid molecule, or a complement thereof, in a biological sample with an alteration-specific probe comprising a detectable label, wherein the alteration-specific probe comprises a nucleotide sequence that hybridizes, under stringent conditions, to a nucleotide sequence of a PIEZO1 nucleic acid molecule, or a complement thereof, comprising: a uracil at a position corresponding to position 361 according to SEQ ID NO: 21, or a complement thereof; and / or a thymine at a position corresponding to position 361 according to SEQ ID NO: 41, or a complement thereof; and detecting the detectable label.

[0109] In some embodiments, the assay comprises: contacting a PIEZO1 nucleic acid molecule, or a complement thereof, in a biological sample with an alteration-specific probe comprising a detectable label, wherein the alteration-specific probe comprises a nucleotide sequence that hybridizes, under stringent conditions, to a nucleotide sequence of a PIEZO1 nucleic acid molecule, or a complement thereof, comprising: a uracil at a position corresponding to position 7,776 according to SEQ ID NO: 22, or a complement thereof; and / or a thymine at a position corresponding to position 7,776 according to SEQ ID NO: 42, or a complement thereof; and detecting the detectable label.

[0110] In some embodiments, the assay comprises: contacting a PIEZO1 nucleic acid molecule, or a complement thereof, in a biological sample with an alteration-specific probe comprising a detectable label, wherein the alteration-specific probe comprises a nucleotide sequence that hybridizes, under stringent conditions, to a nucleotide sequence of the PIEZO1 nucleic acid molecule, or a complement thereof, comprising: a thymine at a position corresponding to position 69,579 according to SEQ ID NO: 2, or a complement thereof; and detecting the detectable label.

[0111] In some embodiments, the assay comprises: contacting a PIEZOl mRNA molecule, or a complement thereof, in a biological sample with an alteration-specific probe comprising a detectable label, wherein the alteration-specific probe comprises a nucleotide sequence that hybridizes under stringent conditions to a nucleotide sequence of a PIEZOl mRNA molecule, or a complement thereof, comprising: a uracil at a position corresponding to position 7,785 according to SEQ ID NO: 13, or a complement thereof; a uracil at a position corresponding to position 1,095 according to SEQ ID NO: 14, or a complement thereof; a uracil at a position corresponding to position 6,073 according to SEQ ID NO: 15, or a complement thereof; a uracil at a position corresponding to position 1,336 according to SEQ ID NO: 16, or a complement thereof; a uracil at a position corresponding to position 6,073 according to SEQ ID NO: 17, or a complement thereof; a uracil at a position corresponding to position 6,236 according to SEQ ID NO: 18, or a complement thereof; a uracil at a position corresponding to position 7,526 according to SEQ ID NO: 19, or a complement thereof; a uracil at a position corresponding to position 1,336 according to SEQ ID NO: 20, or a complement thereof; a uracil at a position corresponding to position 361 according to SEQ ID NO: 21, or a complement thereof; or a uracil at a position corresponding to position 7,776 according to SEQ ID NO: 22, or a complement thereof; and detecting the detectable label.

[0112] In some embodiments, the assay comprises: contacting a PIEZO1 cDNA molecule, or a complement thereof, produced from an mRNA molecule in the biological sample with an alteration-specific probe comprising a detectable label, wherein the alteration-specific probe comprises a nucleotide sequence that hybridizes under stringent conditions to a nucleotide sequence of the PIEZO1 cDNA molecule, or a complement thereof, comprising: a thymine at a position corresponding to position 7,785 according to SEQ ID NO: 33, or a complement thereof; a thymine at a position corresponding to position 1,095 according to SEQ ID NO: 34, or a complement thereof; a thymine at a position corresponding to position 6,073 according to SEQ ID NO: 35, or a complement thereof; a thymine at a position corresponding to position 1,336 according to SEQ ID NO: 36, or a complement thereof; a thymine at a position corresponding to position 6,073 according to SEQ ID NO: 37, or a complement thereof; a thymine at a position corresponding to position 6,236 according to SEQ ID NO: 38, or a complement thereof; a thymine at a position corresponding to position 7,526 according to SEQ ID NO: 39, or a complement thereof; a thymine at a position corresponding to position 1,336 according to SEQ ID NO: 40, or a complement thereof; a thymine at a position corresponding to position 361 according to SEQ ID NO: 41, or a complement thereof; or a thymine at a position corresponding to position 7,776 according to SEQ ID NO: 42, or a complement thereof; and detecting the detectable label.

[0113] In some embodiments, the PIEZO1 nucleic acid molecule is present within a cell obtained from the subject.

[0114] Alteration-specific polymerase chain reaction techniques can be used to detect mutations in nucleotide sequences, such as SNPs. Alteration-specific primers can be used because DNA polymerase will not extend when there is a mismatch to the template.

[0115] In some embodiments, the assay comprises RNA sequencing (RNA-Seq). In some embodiments, the assay further comprises reverse transcribing the mRNA into cDNA, such as by reverse transcriptase polymerase chain reaction (RT-PCR).

[0116] In some embodiments, the methods utilize probes and primers of sufficient nucleotide length to bind to a target nucleic acid sequence, and specifically detect and / or identify a polynucleotide comprising a PIEZOl variant genomic nucleic acid molecule, a variant mRNA molecule, or a variant cDNA molecule. Hybridization conditions or reaction conditions can be determined by the operator to achieve this result. The nucleotide length can be any length sufficient for the selected detection method, including any of the assays described or exemplified herein. Such probes and primers can specifically hybridize to a target nucleotide sequence under high stringency hybridization conditions. The probes and primers can have complete nucleotide sequence identity to contiguous nucleotides within the target nucleotide sequence, but can be designed differently from the target nucleotide sequence by routine methods and retain the ability to specifically detect and / or identify 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.

[0117] In some embodiments, to determine whether a PIEZOl nucleic acid molecule (genomic nucleic acid molecule, mRNA molecule, or cDNA molecule) or complement thereof in a biological sample comprises a nucleotide sequence comprising a thymine at a position corresponding to position 69,579 according to SEQ ID NO:2 (genomic nucleic acid molecule), a uracil at a position corresponding to position 7,785 according to SEQ ID NO: 13 (mRNA molecule), or a thymine at a position corresponding to position 7,785 according to SEQ ID NO:33 (cDNA molecule), the biological sample can be subjected to an amplification method using a primer pair comprising a first primer derived from the 5' flanking sequence adjacent to the thymine at a position corresponding to position 69,579 according to SEQ ID NO:2, the uracil at a position corresponding to position 7,785 according to SEQ ID NO: 13, or the thymine at a position corresponding to position 7,785 according to SEQ ID NO:33; and a second primer derived from the 3' flanking sequence adjacent to the thymine at a position corresponding to position 69,579 according to SEQ ID NO:2, the uracil at a position corresponding to position 7,785 according to SEQ ID NO: 13, or the thymine at a position corresponding to position 7,785 according to SEQ ID NO:33, to produce an amplicon indicative of the presence of the SNP at the position encoding the thymine at a position corresponding to position 69,579 according to SEQ ID NO:2, the uracil at a position corresponding to position 7,785 according to SEQ ID NO: 13, or the thymine at a position corresponding to position 7,785 according to SEQ ID NO:33. In some embodiments, the length of the amplicon can range from the combined length of the primer pair plus one nucleotide base pair to any length of amplicon that can be produced by a DNA amplification protocol. This range can be from one nucleotide base pair to the limit of the amplification reaction, or about twenty thousand nucleotide base pairs.Optionally, the primer pair flanks a region comprising: a thymine at a position corresponding to position 69,579 according to SEQ ID NO:2, a uracil at a position corresponding to position 7,785 according to SEQ ID NO: 13, or a thymine at a position corresponding to position 7,785 according to SEQ ID NO:33; and at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more nucleotides on each side of the position comprising: a thymine at a position corresponding to position 69,579 according to SEQ ID NO:2, a uracil at a position corresponding to position 7,785 according to SEQ ID NO: 13, or a thymine at a position corresponding to position 7,785 according to SEQ ID NO:33.

[0118] In some embodiments, to determine whether a PIEZOl nucleic acid molecule (genomic nucleic acid molecule, mRNA molecule, or cDNA molecule) or the complement thereof in a biological sample comprises a nucleotide sequence comprising a cytosine at a position corresponding to position 1, 095 according to SEQ ID NO: 14 (mRNA molecule), or a guanine at a position corresponding to position 1, 095 according to SEQ ID NO: 34 (cDNA molecule), the biological sample can be subjected to an amplification method using a primer pair comprising a first primer derived from the 5' flanking sequence adjacent immediately upstream of a cytosine at a position corresponding to position 1, 095 according to SEQ ID NO: 14, or a guanine at a position corresponding to position 1, 095 according to SEQ ID NO: 34; and a second primer derived from the 3' flanking sequence adjacent immediately downstream of a cytosine at a position corresponding to position 1, 095 according to SEQ ID NO: 14, or a guanine at a position corresponding to position 1, 095 according to SEQ ID NO: 34, to produce an amplicon indicative of the presence of the SNP at the position encoding a cytosine at a position corresponding to position 1, 095 according to SEQ ID NO: 14, or a guanine at a position corresponding to position 1, 095 according to SEQ ID NO: 34. In some embodiments, the length of the amplicon can range from the combined length of the primer pair plus one nucleotide base pair to any length of amplicon that can be produced by a DNA amplification protocol. This distance can range from one nucleotide base pair to the limit of the amplification reaction, or about twenty thousand nucleotide base pairs. Optionally, the primer pair is flanking a region comprising a position comprising a cytosine at a position corresponding to position 1, 095 according to SEQ ID NO: 14, or a guanine at a position corresponding to position 1, 095 according to SEQ ID NO: 34, and at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more nucleotides on each side of the position comprising a cytosine at a position corresponding to position 1, 095 according to SEQ ID NO: 14, or a guanine at a position corresponding to position 1, 095 according to SEQ ID NO: 34.

[0119] In some embodiments, to determine whether a PIEZOl nucleic acid molecule (genomic nucleic acid molecule, mRNA molecule, or cDNA molecule) or the complement thereof in a biological sample comprises a nucleotide sequence comprising a cytosine at a position corresponding to position 6,073 according to SEQ ID NO: 15 (mRNA molecule), or a guanine at a position corresponding to position 6,073 according to SEQ ID NO: 35 (cDNA molecule), the biological sample can be subjected to an amplification method using a primer pair comprising a first primer derived from the 5' flanking sequence adjacent immediately upstream of a cytosine at a position corresponding to position 6,073 according to SEQ ID NO: 15, or a guanine at a position corresponding to position 6,073 according to SEQ ID NO: 35; and a second primer derived from the 3' flanking sequence adjacent immediately downstream of a cytosine at a position corresponding to position 6,073 according to SEQ ID NO: 15, or a guanine at a position corresponding to position 6,073 according to SEQ ID NO: 35, to produce an amplicon indicative of the presence of the SNP at the position encoding a cytosine at a position corresponding to position 6,073 according to SEQ ID NO: 15, or a guanine at a position corresponding to position 6,073 according to SEQ ID NO: 35. In some embodiments, the length of the amplicon can range from the combined length of the primer pair plus one nucleotide base pair to any length of amplicon that can be produced by a DNA amplification protocol. This distance can range from one nucleotide base pair to the limit of the amplification reaction, or about twenty thousand nucleotide base pairs. Optionally, the primer pair is flanking a region comprising a position comprising a cytosine at a position corresponding to position 6,073 according to SEQ ID NO: 15, or a guanine at a position corresponding to position 6,073 according to SEQ ID NO: 35, and at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more nucleotides on each side of the position comprising a cytosine at a position corresponding to position 6,073 according to SEQ ID NO: 15, or a guanine at a position corresponding to position 6,073 according to SEQ ID NO: 35.

[0120] In some embodiments, to determine whether a PIEZOl nucleic acid molecule (genomic nucleic acid molecule, mRNA molecule, or cDNA molecule) or the complement thereof in a biological sample comprises a nucleotide sequence comprising a cytosine at a position corresponding to position 1,336 according to SEQ ID NO: 16 (mRNA molecule), or a guanine at a position corresponding to position 1,336 according to SEQ ID NO: 36 (cDNA molecule), the biological sample can be subjected to an amplification method using a primer pair comprising a first primer derived from the 5' flanking sequence adjacent immediately upstream of a cytosine at a position corresponding to position 1,336 according to SEQ ID NO: 16, or a guanine at a position corresponding to position 1,336 according to SEQ ID NO: 36, and a second primer derived from the 3' flanking sequence adjacent immediately downstream of a cytosine at a position corresponding to position 1,336 according to SEQ ID NO: 16, or a guanine at a position corresponding to position 1,336 according to SEQ ID NO: 36, to produce an amplicon indicative of the presence of the SNP at the position encoding a cytosine at a position corresponding to position 1,336 according to SEQ ID NO: 16, or a guanine at a position corresponding to position 1,336 according to SEQ ID NO: 36. In some embodiments, the length of the amplicon can range from the combined length of the primer pair plus one nucleotide base pair to any length of amplicon that can be produced by a DNA amplification protocol. This distance can range from one nucleotide base pair to the limit of the amplification reaction, or about twenty thousand nucleotide base pairs. Optionally, the primer pair is flanking a region comprising a position comprising a cytosine at a position corresponding to position 1,336 according to SEQ ID NO: 16, or a guanine at a position corresponding to position 1,336 according to SEQ ID NO: 36, and at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more nucleotides on each side of the position comprising a cytosine at a position corresponding to position 1,336 according to SEQ ID NO: 16, or a guanine at a position corresponding to position 1,336 according to SEQ ID NO: 36.

[0121] In some embodiments, to determine whether a PIEZOl nucleic acid molecule (genomic nucleic acid molecule, mRNA molecule, or cDNA molecule) or the complement thereof in a biological sample comprises a nucleotide sequence comprising a cytosine at a position corresponding to position 6,073 according to SEQ ID NO: 17 (mRNA molecule), or a guanine at a position corresponding to position 6,073 according to SEQ ID NO: 37 (cDNA molecule), the biological sample can be subjected to an amplification method using a primer pair comprising a first primer derived from the 5' flanking sequence adjacent immediately upstream of a cytosine at a position corresponding to position 6,073 according to SEQ ID NO: 17, or a guanine at a position corresponding to position 6,073 according to SEQ ID NO: 37, and a second primer derived from the 3' flanking sequence adjacent immediately downstream of a cytosine at a position corresponding to position 6,073 according to SEQ ID NO: 17, or a guanine at a position corresponding to position 6,073 according to SEQ ID NO: 37, to produce an amplicon indicative of the presence of the SNP at the position encoding a cytosine at a position corresponding to position 6,073 according to SEQ ID NO: 17, or a guanine at a position corresponding to position 6,073 according to SEQ ID NO: 37. In some embodiments, the length of the amplicon can range from the combined length of the primer pair plus one nucleotide base pair to any length of amplicon that can be produced by a DNA amplification protocol. This distance can range from one nucleotide base pair to the limit of the amplification reaction, or about twenty thousand nucleotide base pairs. Optionally, the primer pair is flanking a region comprising a position comprising a cytosine at a position corresponding to position 6,073 according to SEQ ID NO: 17, or a guanine at a position corresponding to position 6,073 according to SEQ ID NO: 37, and at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more nucleotides on each side of the position comprising a cytosine at a position corresponding to position 6,073 according to SEQ ID NO: 17, or a guanine at a position corresponding to position 6,073 according to SEQ ID NO: 37.

[0122] In some embodiments, to determine whether a PIEZOl nucleic acid molecule (genomic nucleic acid molecule, mRNA molecule, or cDNA molecule) or the complement thereof in a biological sample comprises a nucleotide sequence comprising a cytosine at a position corresponding to position 6,236 according to SEQ ID NO: 18 (mRNA molecule), or a guanine at a position corresponding to position 6,236 according to SEQ ID NO: 38 (cDNA molecule), the biological sample can be subjected to an amplification method using a primer pair comprising a first primer derived from the 5' flanking sequence adjacent immediately upstream of a cytosine at a position corresponding to position 6,236 according to SEQ ID NO: 18, or a guanine at a position corresponding to position 6,236 according to SEQ ID NO: 38, and a second primer derived from the 3' flanking sequence adjacent immediately downstream of a cytosine at a position corresponding to position 6,236 according to SEQ ID NO: 18, or a guanine at a position corresponding to position 6,236 according to SEQ ID NO: 38, to produce an amplicon indicative of the presence of the SNP at the position encoding a cytosine at a position corresponding to position 6,236 according to SEQ ID NO: 18, or a guanine at a position corresponding to position 6,236 according to SEQ ID NO: 38. In some embodiments, the length of the amplicon can range from the combined length of the primer pair plus one nucleotide base pair to any length of amplicon that can be produced by a DNA amplification protocol. This distance can range from one nucleotide base pair to the limit of the amplification reaction, or about twenty thousand nucleotide base pairs. Optionally, the primer pair is flanking a region comprising a position comprising a cytosine at a position corresponding to position 6,236 according to SEQ ID NO: 18, or a guanine at a position corresponding to position 6,236 according to SEQ ID NO: 38, and at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more nucleotides on each side of the position comprising a cytosine at a position corresponding to position 6,236 according to SEQ ID NO: 18, or a guanine at a position corresponding to position 6,236 according to SEQ ID NO: 38.

[0123] In some embodiments, to determine whether a PIEZOl nucleic acid molecule (genomic nucleic acid molecule, mRNA molecule, or cDNA molecule) or the complement thereof in a biological sample comprises a nucleotide sequence comprising a cytosine at a position corresponding to position 7,526 according to SEQ ID NO: 19 (mRNA molecule), or a guanine at a position corresponding to position 7,526 according to SEQ ID NO: 39 (cDNA molecule), the biological sample can be subjected to an amplification method using a primer pair comprising a first primer derived from the 5' flanking sequence adjacent immediately upstream of a cytosine at a position corresponding to position 7,526 according to SEQ ID NO: 19, or a guanine at a position corresponding to position 7,526 according to SEQ ID NO: 39; and a second primer derived from the 3' flanking sequence adjacent immediately downstream of a cytosine at a position corresponding to position 7,526 according to SEQ ID NO: 19, or a guanine at a position corresponding to position 7,526 according to SEQ ID NO: 39, to produce an amplicon indicative of the presence of the SNP at the position encoding a cytosine at a position corresponding to position 7,526 according to SEQ ID NO: 19, or a guanine at a position corresponding to position 7,526 according to SEQ ID NO: 39. In some embodiments, the length of the amplicon can range from the combined length of the primer pair plus one nucleotide base pair, to any length of amplicon that can be produced by a DNA amplification protocol. This distance can range from one nucleotide base pair to the limit of the amplification reaction, or about twenty thousand nucleotide base pairs. Optionally, the primer pair is flanking a region comprising a position comprising a cytosine at a position corresponding to position 7,526 according to SEQ ID NO: 19, or a guanine at a position corresponding to position 7,526 according to SEQ ID NO: 39, and at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more nucleotides on each side of the position comprising a cytosine at a position corresponding to position 7,526 according to SEQ ID NO: 19, or a guanine at a position corresponding to position 7,526 according to SEQ ID NO: 39.

[0124] In some embodiments, to determine whether a PIEZOl nucleic acid molecule (genomic nucleic acid molecule, mRNA molecule, or cDNA molecule) or the complement thereof in a biological sample comprises a nucleotide sequence comprising a cytosine at a position corresponding to position 1,336 according to SEQ ID NO:20 (mRNA molecule), or a guanine at a position corresponding to position 1,336 according to SEQ ID NO:40 (cDNA molecule), the biological sample can be subjected to an amplification method using a primer pair comprising a first primer derived from the 5' flanking sequence adjacent immediately upstream of a cytosine at a position corresponding to position 1,336 according to SEQ ID NO:20, or a guanine at a position corresponding to position 1,336 according to SEQ ID NO:40, and a second primer derived from the 3' flanking sequence adjacent immediately downstream of a cytosine at a position corresponding to position 1,336 according to SEQ ID NO:20, or a guanine at a position corresponding to position 1,336 according to SEQ ID NO:40, to produce an amplicon indicative of the presence of the SNP at the position encoding a cytosine at a position corresponding to position 1,336 according to SEQ ID NO:20, or a guanine at a position corresponding to position 1,336 according to SEQ ID NO:40. In some embodiments, the length of the amplicon can range from the combined length of the primer pair plus one nucleotide base pair to any length of amplicon that can be produced by a DNA amplification protocol. This distance can range from one nucleotide base pair to the limit of the amplification reaction, or about twenty thousand nucleotide base pairs. Optionally, the primer pair is flanking a region comprising a position comprising a cytosine at a position corresponding to position 1,336 according to SEQ ID NO:20, or a guanine at a position corresponding to position 1,336 according to SEQ ID NO:40, and at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more nucleotides on each side of the position comprising a cytosine at a position corresponding to position 1,336 according to SEQ ID NO:20, or a guanine at a position corresponding to position 1,336 according to SEQ ID NO:40.

[0125] In some embodiments, to determine whether a PIEZOl nucleic acid molecule (genomic nucleic acid molecule, mRNA molecule, or cDNA molecule) or complement thereof in a biological sample comprises a nucleotide sequence comprising a cytosine at a position corresponding to position 361 according to SEQ ID NO: 21 (mRNA molecule) or a guanine at a position corresponding to position 361 according to SEQ ID NO: 41 (cDNA molecule), the biological sample can be subjected to an amplification method using a primer pair comprising a first primer derived from the 5' flanking sequence adjacent immediately upstream of a cytosine at a position corresponding to position 361 according to SEQ ID NO: 21 or a guanine at a position corresponding to position 361 according to SEQ ID NO: 41 and a second primer derived from the 3' flanking sequence adjacent immediately downstream of a cytosine at a position corresponding to position 361 according to SEQ ID NO: 21 or a guanine at a position corresponding to position 361 according to SEQ ID NO: 41 to produce an amplicon indicative of the presence of the SNP at the position encoding a cytosine at a position corresponding to position 361 according to SEQ ID NO: 21 or a guanine at a position corresponding to position 361 according to SEQ ID NO: 41. In some embodiments, the length of the amplicon can range from the combined length of the primer pair plus one nucleotide base pair to any length of amplicon that can be produced by a DNA amplification protocol. This range can be from one nucleotide base pair to the limit of the amplification reaction, or about twenty thousand nucleotide base pairs. Optionally, the primer pair is flanking a region comprising a position comprising a cytosine at a position corresponding to position 361 according to SEQ ID NO: 21 or a guanine at a position corresponding to position 361 according to SEQ ID NO: 41 and at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more nucleotides on each side of the position comprising a cytosine at a position corresponding to position 361 according to SEQ ID NO: 21 or a guanine at a position corresponding to position 361 according to SEQ ID NO: 41.

[0126] In some embodiments, to determine whether a PIEZOl nucleic acid molecule (genomic nucleic acid molecule, mRNA molecule, or cDNA molecule) or the complement thereof in a biological sample comprises a nucleotide sequence comprising a cytosine at a position corresponding to position 7,776 according to SEQ ID NO: 22 (mRNA molecule), or a guanine at a position corresponding to position 7,776 according to SEQ ID NO: 42 (cDNA molecule), the biological sample can be subjected to an amplification method using a primer pair comprising a first primer derived from the 5' flanking sequence adjacent immediately upstream of a cytosine at a position corresponding to position 7,776 according to SEQ ID NO: 22, or a guanine at a position corresponding to position 7,776 according to SEQ ID NO: 42, and a second primer derived from the 3' flanking sequence adjacent immediately downstream of a cytosine at a position corresponding to position 7,776 according to SEQ ID NO: 22, or a guanine at a position corresponding to position 7,776 according to SEQ ID NO: 42, to produce an amplicon indicative of the presence of the SNP at the position encoding a cytosine at a position corresponding to position 7,776 according to SEQ ID NO: 22, or a guanine at a position corresponding to position 7,776 according to SEQ ID NO: 42. In some embodiments, the length of the amplicon can range from the combined length of the primer pair plus one nucleotide base pair to any length of amplicon that can be produced by a DNA amplification protocol. This distance can range from one nucleotide base pair to the limit of the amplification reaction, or about twenty thousand nucleotide base pairs. Optionally, the primer pair is flanking a region comprising a position comprising a cytosine at a position corresponding to position 7,776 according to SEQ ID NO: 22, or a guanine at a position corresponding to position 7,776 according to SEQ ID NO: 42, and at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more nucleotides on each side of the position comprising a cytosine at a position corresponding to position 7,776 according to SEQ ID NO: 22, or a guanine at a position corresponding to position 7,776 according to SEQ ID NO: 42.

[0127] Similar amplicons can be generated from mRNA and / or cDNA sequences. PCR primer pairs can be derived from known sequences, for example, by using computer programs intended for this purpose, such as the PCR Primer Analysis tool of 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.). In addition, sequences can be visually scanned and primers identified manually using known guidelines.

[0128] Illustrative examples of nucleic acid sequencing techniques include, but are not limited to, chain terminator (Sanger) sequencing and dye terminator sequencing. Other methods involve nucleic acid hybridization methods other than sequencing, which include the use of labeled primers or probes against purified DNA, amplified DNA, and fixed cell preparations (fluorescence in situ hybridization (FISH)). In some methods, the target nucleic acid molecule can be amplified prior to or at the same time as 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 reaction (SDA), and nucleic acid sequence-based amplification reaction (NASBA). Other methods include, but are not limited to, ligase chain reaction, strand displacement amplification, and thermophilic SDA (tSDA).

[0129] In hybridization techniques, stringent conditions can be employed such that the probe or primer specifically hybridizes to its target. In some embodiments, a polynucleotide primer or probe under stringent conditions will hybridize to its target sequence to a detectably greater extent than to other non-target sequences, such as at least 2-fold, at least 3-fold, at least 4-fold or more (over background), including more than 10-fold (over background). In some embodiments, a polynucleotide primer or probe under stringent conditions will hybridize to its target nucleotide sequence to a detectably greater extent than to other nucleotide sequences by at least 2-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 to other nucleotide sequences by at least 3-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 to other nucleotide sequences by 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 to other nucleotide sequences by more than 10-fold (over background). Stringency conditions are sequence-dependent and will be different in different circumstances.

[0130] Appropriate stringency conditions (e.g., 6X sodium chloride / sodium citrate (SSC) at about 45°C, followed by a wash in 2X SSC at 50°C) to promote DNA hybridization are known and can be found in Current Protocols in Molecular Biology, John Wiley & Sons, N.Y. (1989), 6.3.1-6.3.6. In general, stringency conditions for hybridization and + ionic, typically about 0.01 to 1.0 M Na + concentration (or other salt), and the temperature is at least about 30°C for short probes (e.g., 10 to 50 nucleotides) and at least about 60°C for longer probes (e.g., greater than 50 nucleotides). Stringency conditions can also be achieved using the addition of destabilizing agents such as formamide. Optionally, the wash buffer can comprise about 0.1% to about 1% SDS. The duration of hybridization is typically less than about 24 hours, often about 4 to about 12 hours. The duration of the wash time will be at least long enough to achieve equilibrium.

[0131] The present disclosure also provides methods of detecting the presence of a PIEZO1 predicted gain-of-function polypeptide, the methods comprising performing an assay on a biological sample obtained from a subject to determine whether the PIEZO1 polypeptide in the biological sample contains one or more variations that cause the polypeptide to have a gain-of-function (partial or complete) or predicted gain-of-function (partial or complete). The PIEZO1 predicted gain-of-function polypeptide can be any of the PIEZO1 predicted gain-of-function polypeptides described herein. In some embodiments, the methods detect the presence of PIEZO1 Pro2,510Leu, Pro2,024Leu, Pro2,079Leu, or Pro92Leu. In some embodiments, the methods detect the presence of PIEZO1 Pro2,510Leu.

[0132] In some embodiments, the methods comprise performing an assay on a biological sample obtained from a subject to determine whether the PIEZO1 polypeptide in the biological sample comprises: a leucine at a position corresponding to position 2,510 according to SEQ ID NO: 47; a leucine at a position corresponding to position 2,024 according to SEQ ID NO: 48; a leucine at a position corresponding to position 2,079 according to SEQ ID NO: 49; or a leucine at a position corresponding to position 94 according to SEQ ID NO: 50.

[0133] In some embodiments, the assay comprises sequencing at least a portion of a PIEZO1 polypeptide, the portion comprising a position corresponding to position: 2,510 according to SEQ ID NO: 47; 2,024 according to SEQ ID NO: 48; 2,079 according to SEQ ID NO: 49; or 94 according to SEQ ID NO: 50.

[0134] In some embodiments, the assay is an immunoassay for detecting the presence of a PIEZO1 polypeptide comprising a position corresponding to position: 2,510 according to SEQ ID NO: 47; 2,024 according to SEQ ID NO: 48; 2,079 according to SEQ ID NO: 49; or 94 according to SEQ ID NO: 50.

[0135] In some embodiments, a subject has an increased risk of developing any of varicose veins or hidden varicose veins, varicosis vesiculosum, spider veins, or pregnancy-related varicose veins when the subject does not have a PIEZO1 predicted gain-of-function polypeptide. In some embodiments, a subject has a decreased risk of developing any of varicose veins or hidden varicose veins, varicosis vesiculosum, spider veins, or pregnancy-related varicose veins when the subject has a PIEZO1 predicted gain-of-function polypeptide.

[0136] The present disclosure also provides isolated nucleic acid molecules that hybridize to a PIEZO1 variant genomic nucleic acid molecule, a PIEZO1 variant mRNA molecule, and / or a PIEZO1 variant cDNA molecule, such as any of the genomic variant nucleic acid molecules, mRNA variant molecules, and cDNA variant molecules disclosed herein. In some embodiments, such isolated nucleic acid molecules hybridize to a PIEZO1 variant nucleic acid molecule under stringent conditions. Such nucleic acid molecules can be used, for example, as probes, primers, altered specificity probes, or altered specificity primers, as described or exemplified herein.

[0137] In some embodiments, the isolated nucleic acid molecule hybridizes to a portion of a PIEZOl nucleic acid molecule, the portion comprising positions corresponding to positions: 69,579 according to SEQ ID NO: 2, 7,785 according to SEQ ID NO: 13, or 7,785 according to SEQ ID NO: 33. In some embodiments, the isolated nucleic acid molecule hybridizes to a portion of a PIEZOl nucleic acid molecule, the portion comprising positions corresponding to positions: 1,095 according to SEQ ID NO: 14, or 1,095 according to SEQ ID NO: 34. In some embodiments, the isolated nucleic acid molecule hybridizes to a portion of a PIEZOl nucleic acid molecule, the portion comprising positions corresponding to positions: 6,073 according to SEQ ID NO: 15, or 6,073 according to SEQ ID NO: 35. In some embodiments, the isolated nucleic acid molecule hybridizes to a portion of a PIEZOl nucleic acid molecule, the portion comprising positions corresponding to positions: 1,336 according to SEQ ID NO: 16, or 1,336 according to SEQ ID NO: 36. In some embodiments, the isolated nucleic acid molecule hybridizes to a portion of a PIEZOl nucleic acid molecule, the portion comprising positions corresponding to positions: 6,073 according to SEQ ID NO: 17, or 6,073 according to SEQ ID NO: 37. In some embodiments, the isolated nucleic acid molecule hybridizes to a portion of a PIEZOl nucleic acid molecule, the portion comprising positions corresponding to positions: 6,236 according to SEQ ID NO: 18, or 6,236 according to SEQ ID NO: 38. In some embodiments, the isolated nucleic acid molecule hybridizes to a portion of a PIEZOl nucleic acid molecule, the portion comprising positions corresponding to positions: 7,526 according to SEQ ID NO: 19, or 7,526 according to SEQ ID NO: 39. In some embodiments, the isolated nucleic acid molecule hybridizes to a portion of a PIEZOl nucleic acid molecule, the portion comprising positions corresponding to positions: 1,336 according to SEQ ID NO: 20, or 1,336 according to SEQ ID NO: 20. In some embodiments, the isolated nucleic acid molecule hybridizes to a portion of a PIEZOl nucleic acid molecule, the portion comprising positions corresponding to positions: 361 according to SEQ ID NO: 21, or 361 according to SEQ ID NO: 21. In some embodiments, the isolated nucleic acid molecule hybridizes to a portion of a PIEZOl nucleic acid molecule, the portion comprising positions corresponding to positions: 7,776 according to SEQ ID NO: 22, or 7,776 according to SEQ ID NO: 42.

[0138] 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, 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.

[0139] In some embodiments, the isolated alteration-specific probe or alteration-specific primer comprises at least about 15 nucleotides, wherein the alteration-specific probe or alteration-specific primer comprises a sequence of nucleotides complementary to a portion of a PIEZOl nucleic acid molecule, or the complement thereof, that encodes a PIEZOl predicted loss-of-function polypeptide. In some embodiments, the portion comprises a position corresponding to position 69,579, or the complement thereof, according to SEQ ID NO: 2; position 7,785, or the complement thereof, according to SEQ ID NO: 13; or position 7,785, or the complement thereof, according to SEQ ID NO: 33. In some embodiments, the portion comprises a position corresponding to position 1,095, or the complement thereof, according to SEQ ID NO: 14; or position 1,095, or the complement thereof, according to SEQ ID NO: 34. In some embodiments, the portion comprises a position corresponding to position 6,073, or the complement thereof, according to SEQ ID NO: 15; or position 6,073, or the complement thereof, according to SEQ ID NO: 35. In some embodiments, the portion comprises a position corresponding to position 1,336, or the complement thereof, according to SEQ ID NO: 16; or position 1,336, or the complement thereof, according to SEQ ID NO: 36. In some embodiments, the portion comprises a position corresponding to position 6,073, or the complement thereof, according to SEQ ID NO: 17; or position 6,073, or the complement thereof, according to SEQ ID NO: 37. In some embodiments, the portion comprises a position corresponding to position 6,236, or the complement thereof, according to SEQ ID NO: 18; or position 6,236, or the complement thereof, according to SEQ ID NO: 38. In some embodiments, the portion comprises a position corresponding to position 7,526, or the complement thereof, according to SEQ ID NO: 19; or position 7,526, or the complement thereof, according to SEQ ID NO: 39. In some embodiments, the portion comprises a position corresponding to position 1,336, or the complement thereof, according to SEQ ID NO: 20; or position 1,336, or the complement thereof, according to SEQ ID NO: 20. In some embodiments, the portion comprises a position corresponding to position 361, or the complement thereof, according to SEQ ID NO: 21; or position 361, or the complement thereof, according to SEQ ID NO: 21. In some embodiments, the portion comprises a position corresponding to position 7,776, or the complement thereof, according to SEQ ID NO: 22; or position 7,776, or the complement thereof, according to SEQ ID NO: 42.

[0140] 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 PIEZOl variant genomic nucleic acid molecule, a PIEZOl variant mRNA molecule, and / or a PIEZOl variant cDNA molecule. In some embodiments, the isolated nucleic acid molecule comprises or consists of about 15 to about 100 nucleotides, or about 15 to about 35 nucleotides. In some embodiments, the isolated nucleic acid molecule comprises or consists of about 15 to about 100 nucleotides. In some embodiments, the isolated nucleic acid molecule comprises or consists of about 15 to about 35 nucleotides.

[0141] In some embodiments, the isolated alteration-specific probe or alteration-specific primer comprises at least about 15 nucleotides, wherein the alteration-specific probe or alteration-specific primer comprises a nucleotide sequence that is complementary to a portion of a nucleotide sequence of a PIEZOl variant nucleic acid molecule that encodes a PIEZOl predicted loss-of-function polypeptide, wherein the portion comprises positions corresponding to positions: 1,095 of SEQ ID NO: 14, or the complement thereof; or 1,095 of SEQ ID NO: 34, or the complement thereof. In some embodiments, the portion comprises positions corresponding to positions: 1,094-1,096 of SEQ ID NO: 14, or the complement thereof; or 1,094-1,096 of SEQ ID NO: 34, or the complement thereof.

[0142] In some embodiments, the isolated alteration-specific probe or alteration-specific primer comprises at least about 15 nucleotides, wherein the alteration-specific probe or alteration-specific primer comprises a nucleotide sequence that is complementary to a portion of a nucleotide sequence of a PIEZOl variant nucleic acid molecule that encodes a PIEZOl predicted loss-of-function polypeptide, wherein the portion comprises positions corresponding to positions: 1,095 of SEQ ID NO: 14, or the complement thereof; or 1,095 of SEQ ID NO: 34, or the complement thereof. In some embodiments, the portion comprises positions corresponding to positions: 1,094-1,096 of SEQ ID NO: 14, or the complement thereof; or 1,094-1,096 of SEQ ID NO: 34, or the complement thereof.

[0143] In some embodiments, the isolated alteration-specific probe or alteration-specific primer comprises at least about 15 nucleotides, wherein the alteration-specific probe or alteration-specific primer comprises a nucleotide sequence complementary to a portion of the nucleotide sequence of a PIEZO1 variant nucleic acid molecule that encodes a PIEZO1 predicted loss-of-function polypeptide, wherein the portion comprises positions corresponding to positions: 1,336 according to SEQ ID NO: 16, or the complement thereof; or 1,336 according to SEQ ID NO: 36, or the complement thereof. In some embodiments, the portion comprises positions corresponding to positions: 1,335-1,337 according to SEQ ID NO: 16, or the complement thereof; or 1,335-1,337 according to SEQ ID NO: 36, or the complement thereof.

[0144] In some embodiments, the isolated alteration-specific probe or alteration-specific primer comprises at least about 15 nucleotides, wherein the alteration-specific probe or alteration-specific primer comprises a nucleotide sequence complementary to a portion of the nucleotide sequence of a PIEZO1 variant nucleic acid molecule that encodes a PIEZO1 predicted loss-of-function polypeptide, wherein the portion comprises positions corresponding to positions: 1,336 according to SEQ ID NO: 16, or the complement thereof; or 1,336 according to SEQ ID NO: 36, or the complement thereof. In some embodiments, the portion comprises positions corresponding to positions: 1,335-1,337 according to SEQ ID NO: 16, or the complement thereof; or 1,335-1,337 according to SEQ ID NO: 36, or the complement thereof.

[0145] In some embodiments, the isolated alteration-specific probe or alteration-specific primer comprises at least about 15 nucleotides, wherein the alteration-specific probe or alteration-specific primer comprises a nucleotide sequence complementary to a portion of the nucleotide sequence of a PIEZO1 variant nucleic acid molecule that encodes a PIEZO1 predicted loss-of-function polypeptide, wherein the portion comprises positions corresponding to positions: 1,336 according to SEQ ID NO: 16, or the complement thereof; or 1,336 according to SEQ ID NO: 36, or the complement thereof. In some embodiments, the portion comprises positions corresponding to positions: 1,335-1,337 according to SEQ ID NO: 16, or the complement thereof; or 1,335-1,337 according to SEQ ID NO: 36, or the complement thereof.

[0146] In some embodiments, the isolated alteration-specific probe or alteration-specific primer comprises at least about 15 nucleotides, wherein the alteration-specific probe or alteration-specific primer comprises a nucleotide sequence complementary to a portion of the nucleotide sequence of a PIEZO1 variant nucleic acid molecule that encodes a PIEZO1 predicted loss-of-function polypeptide, wherein the portion comprises positions corresponding to positions: 1, 336-1, 338 of SEQ ID NO: 20, or the complement thereof; or 1, 336-1, 338 of SEQ ID NO: 40, or the complement thereof. In some embodiments, the portion comprises positions corresponding to positions: 1, 335-1, 339 of SEQ ID NO: 20, or the complement thereof; or 1, 335-1, 339 of SEQ ID NO: 40, or the complement thereof.

[0147] In some embodiments, the isolated alteration-specific probe or alteration-specific primer comprises at least about 15 nucleotides, wherein the alteration-specific probe or alteration-specific primer comprises a nucleotide sequence complementary to a portion of the nucleotide sequence of a PIEZO1 variant nucleic acid molecule that encodes a PIEZO1 predicted gain-of-function polypeptide, wherein the portion comprises positions corresponding to positions: 7, 525-7, 527 of SEQ ID NO: 19, or the complement thereof; or 7, 525-7, 527 of SEQ ID NO: 39, or the complement thereof. In some embodiments, the portion comprises positions corresponding to positions: 7, 524-7, 528 of SEQ ID NO: 19, or the complement thereof; or 7, 524-7, 528 of SEQ ID NO: 39, or the complement thereof.

[0148] In some embodiments, the isolated alteration-specific probe or alteration-specific primer comprises at least about 15 nucleotides, wherein the alteration-specific probe or alteration-specific primer comprises a nucleotide sequence complementary to a portion of the nucleotide sequence of a PIEZO1 variant nucleic acid molecule that encodes a PIEZO1 predicted gain-of-function polypeptide, wherein the portion comprises positions corresponding to positions: 7, 525-7, 527 of SEQ ID NO: 19, or the complement thereof; or 7, 525-7, 527 of SEQ ID NO: 39, or the complement thereof. In some embodiments, the portion comprises positions corresponding to positions: 7, 524-7, 528 of SEQ ID NO: 19, or the complement thereof; or 7, 524-7, 528 of SEQ ID NO: 39, or the complement thereof.

[0149] In some embodiments, the isolated alteration-specific probe or alteration-specific primer comprises at least about 15 nucleotides, wherein the alteration-specific probe or alteration-specific primer comprises a nucleotide sequence complementary to a portion of a nucleotide sequence of a PIEZO1 variant nucleic acid molecule that encodes a PIEZO1 predicted loss-of-function polypeptide, wherein the portion comprises positions corresponding to positions: 1,336 according to SEQ ID NO: 21, or a complement thereof; or 1,336 according to SEQ ID NO: 41, or a complement thereof. In some embodiments, the portion comprises positions corresponding to positions: 360-362 according to SEQ ID NO: 21, or a complement thereof; or 360-362 according to SEQ ID NO: 41, or a complement thereof.

[0150] In some embodiments, the isolated alteration-specific probe or alteration-specific primer comprises at least about 15 nucleotides, wherein the alteration-specific probe or alteration-specific primer comprises a nucleotide sequence complementary to a portion of a nucleotide sequence of a PIEZO1 variant nucleic acid molecule that encodes a PIEZO1 predicted loss-of-function polypeptide, wherein the portion comprises positions corresponding to positions: 7,776 according to SEQ ID NO: 22, or a complement thereof; or 7,776 according to SEQ ID NO: 42, or a complement thereof. In some embodiments, the portion comprises positions corresponding to positions: 7,775-7,777 according to SEQ ID NO: 22, or a complement thereof; or 7,775-7,777 according to SEQ ID NO: 42, or a complement thereof.

[0151] In some embodiments, the isolated alteration-specific probe or alteration-specific primer comprises at least about 15 nucleotides, wherein the alteration-specific probe or alteration-specific primer comprises a nucleotide sequence complementary to a portion of a PIEZO1 nucleic acid molecule that encodes a PIEZO1 predicted loss-of-function polypeptide, or a complement thereof. In some embodiments, the portion comprises positions corresponding to positions: 69,579 according to SEQ ID NO: 2, or a complement thereof.

[0152] In some embodiments, the portion comprises positions corresponding to positions: 69,578-69,580 according to SEQ ID NO: 2, or a complement thereof.

[0153] In some embodiments, the portion comprises a position corresponding to a position: position 7,785, or its complement, according to SEQ ID NO: 13; position 1,095, or its complement, according to SEQ ID NO: 14; position 6,073, or its complement, according to SEQ ID NO: 15; position 1,336, or its complement, according to SEQ ID NO: 16; position 6,073, or its complement, according to SEQ ID NO: 17; position 6,236, or its complement, according to SEQ ID NO: 18; position 7,526, or its complement, according to SEQ ID NO: 19; position 1,336, or its complement, according to SEQ ID NO: 20; a position corresponding to position 361, or its complement, according to SEQ ID NO: 21; or position 7,776, or its complement, according to SEQ ID NO: 22.

[0154] In some embodiments, the portion comprises a position corresponding to a position: position 7,784-7,786, or its complement, according to SEQ ID NO: 13; position 1,094-1,096, or its complement, according to SEQ ID NO: 14; position 6,072-6,074, or its complement, according to SEQ ID NO: 15; position 1,335-1,337, or its complement, according to SEQ ID NO: 16; position 6,072-6,074, or its complement, according to SEQ ID NO: 17; position 6,235-6,237, or its complement, according to SEQ ID NO: 18; position 7,525-7,527, or its complement, according to SEQ ID NO: 19; position 1,335-1,337, or its complement, according to SEQ ID NO: 20; position 360-362, or its complement, according to SEQ ID NO: 21; or position 7,775-7,777, or its complement, according to SEQ ID NO: 22.

[0155] In some embodiments, the portion comprises a position corresponding to position: position 7,785 according to SEQ ID NO: 33, or its complement; position 1,095 according to SEQ ID NO: 34, or its complement; position 6,073 according to SEQ ID NO: 35, or its complement; position 1,336 according to SEQ ID NO: 36, or its complement; position 6,073 according to SEQ ID NO: 37, or its complement; position 6,236 according to SEQ ID NO: 38, or its complement; position 7,526 according to SEQ ID NO: 39, or its complement; position 1,336 according to SEQ ID NO: 40, or its complement; position 361 according to SEQ ID NO: 41, or its complement; or position 7,776 according to SEQ ID NO: 42, or its complement.

[0156] In some embodiments, the portion comprises a position corresponding to position: position 7,784-7,786 according to SEQ ID NO: 33, or its complement; position 1,094-1,096 according to SEQ ID NO: 34, or its complement; position 6,072-6,074 according to SEQ ID NO: 35, or its complement; position 1,335-1,337 according to SEQ ID NO: 36, or its complement; position 6,072-6,074 according to SEQ ID NO: 37, or its complement; position 6,235-6,237 according to SEQ ID NO: 38, or its complement; position 7,525-7,527 according to SEQ ID NO: 39, or its complement; position 1,335-1,337 according to SEQ ID NO: 40, or its complement; position 360-362 according to SEQ ID NO: 41, or its complement; or position 7,775-7,777 according to SEQ ID NO: 42, or its complement.

[0157] In some embodiments, the altered-specificity probes and altered-specificity primers comprise DNA. In some embodiments, the altered-specificity probes and altered-specificity primers comprise RNA.

[0158] In some embodiments, the probes and primers described herein (including altered-specificity probes and altered-specificity primers) have a nucleotide sequence that specifically hybridizes to any of the nucleic acid molecules disclosed herein, or its complement. In some embodiments, the probes and primers specifically hybridize to any of the nucleic acid molecules disclosed herein under stringent conditions.

[0159] In some embodiments, primers, including altered specificity primers, can be used in second generation sequencing or high-throughput sequencing. In some cases, primers, including altered specificity primers, can be modified. In particular, primers can comprise various modifications used in different steps of, for example, massively parallel signature sequencing (MPSS), polony sequencing, and 454 pyrosequencing. Modified primers can be used in several steps of the process, including the use of biotinylated primers in the clonal step, and fluorescently labeled primers in the bead loading step and detection step. Polony sequencing is typically performed using a paired-end tag library, where each DNA template molecule is about 135 bp in length. Biotinylated primers are used in the bead loading step and emulsion PCR. Fluorescently labeled degenerate nonamer oligonucleotides are used in the detection step. Adapters can comprise a 5'-biotin tag for immobilization of the DNA library to streptavidin-coated beads.

[0160] The probes and primers described herein can be used to detect a nucleotide variation within any of the PIEZOl variant genomic nucleic acid molecules, PIEZOl variant mRNA molecules, and / or PIEZOl variant cDNA molecules disclosed herein. The primers described herein can be used to amplify a PIEZOl variant genomic nucleic acid molecule, a PIEZOl variant mRNA molecule, or a PIEZOl variant cDNA molecule, or a fragment thereof.

[0161] The present disclosure also provides primer pairs comprising any of the above primers. For example, if one of the 3 '-ends of the primer hybridizes to a cytosine (rather than a thymine) in a particular PIEZO1 nucleic acid molecule at a position corresponding to position 69,579 according to SEQ ID NO: 1, the presence of the amplified fragment would indicate the presence of a PIEZO1 reference genomic nucleic acid molecule. Conversely, if one of the 3 '-ends of the primer hybridizes to a thymine (rather than a cytosine) in a particular PIEZO1 nucleic acid molecule at a position corresponding to position 69,579 according to SEQ ID NO: 2, the presence of the amplified fragment would indicate the presence of a PIEZO1 variant genomic nucleic acid molecule. In some embodiments, the nucleotide of the primer complementary to a thymine at a position corresponding to position 69,579 according to SEQ ID NO: 2 can be at the 3 '-end of the primer. Furthermore, if one of the 3 '-ends of the primer hybridizes to a cytosine (rather than a uracil) in a particular PIEZO1 nucleic acid molecule at a position corresponding to position 7,785 according to SEQ ID NO: 3, the presence of the amplified fragment would indicate the presence of a PIEZO1 reference mRNA molecule. Conversely, if one of the 3 '-ends of the primer hybridizes to a uracil (rather than a cytosine) in a particular PIEZO1 mRNA molecule at a position corresponding to position 7,785 according to SEQ ID NO: 13, the presence of the amplified fragment would indicate the presence of a PIEZO1 variant mRNA molecule. In some embodiments, the nucleotide of the primer complementary to a uracil at a position corresponding to position 7,785 according to SEQ ID NO: 13 can be at the 3 '-end of the primer. Furthermore, if one of the 3 '-ends of the primer hybridizes to a cytosine (rather than a thymine) in a particular PIEZO1 nucleic acid molecule at a position corresponding to position 7,785 according to SEQ ID NO: 23, the presence of the amplified fragment would indicate the presence of a PIEZO1 reference cDNA molecule. Conversely, if one of the 3 '-ends of the primer hybridizes to a thymine (rather than a cytosine) in a particular PIEZO1 cDNA molecule at a position corresponding to position 7,785 according to SEQ ID NO: 33, the presence of the amplified fragment would indicate the presence of a PIEZO1 variant cDNA molecule. In some embodiments, the nucleotide of the primer complementary to a thymine at a position corresponding to position 7,785 according to SEQ ID NO: 33 can be at the 3 '-end of the primer.

[0162] The present disclosure also provides primer pairs comprising any of the above primers. For example, if one of the 3 '-ends of the primer hybridizes to a cytosine (rather than a uracil) at a position corresponding to position 1,095 according to SEQ ID NO: 4 in a particular PIEZO1 nucleic acid molecule, the presence of the amplified fragment would indicate the presence of a PIEZO1 reference mRNA molecule. Conversely, if one of the 3 '-ends of the primer hybridizes to a uracil (rather than a cytosine) at a position corresponding to position 1,095 according to SEQ ID NO: 14 in a particular PIEZO1 mRNA molecule, the presence of the amplified fragment would indicate the presence of a PIEZO1 variant mRNA molecule. In some embodiments, the nucleotide of the primer complementary to a uracil at a position corresponding to position 1,095 according to SEQ ID NO: 14 can be at the 3 '-end of the primer. Furthermore, if one of the 3 '-ends of the primer hybridizes to a cytosine (rather than a thymine) at a position corresponding to position 1,095 according to SEQ ID NO: 24 in a particular PIEZO1 nucleic acid molecule, the presence of the amplified fragment would indicate the presence of a PIEZO1 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 1,095 according to SEQ ID NO: 34 in a particular PIEZO1 cDNA molecule, the presence of the amplified fragment would indicate the presence of a PIEZO1 variant cDNA molecule. In some embodiments, the nucleotide of the primer complementary to a thymine at a position corresponding to position 1,095 according to SEQ ID NO: 34 can be at the 3 '-end of the primer.

[0163] The present disclosure also provides primer pairs comprising any of the above primers. For example, if one of the 3 '-ends of the primer hybridizes to a cytosine (rather than a uracil) at a position corresponding to position 6,073 according to SEQ ID NO: 5 in a particular PIEZO1 nucleic acid molecule, the presence of the amplified fragment would indicate the presence of a PIEZO1 reference mRNA molecule. Conversely, if one of the 3 '-ends of the primer hybridizes to a uracil (rather than a cytosine) at a position corresponding to position 6,073 according to SEQ ID NO: 15 in a particular PIEZO1 mRNA molecule, the presence of the amplified fragment would indicate the presence of a PIEZO1 variant mRNA molecule. In some embodiments, the nucleotide of the primer complementary to a uracil at a position corresponding to position 6,073 according to SEQ ID NO: 15 can be at the 3 '-end of the primer. Furthermore, if one of the 3 '-ends of the primer hybridizes to a cytosine (rather than a thymine) at a position corresponding to position 6,073 according to SEQ ID NO: 25 in a particular PIEZO1 nucleic acid molecule, the presence of the amplified fragment would indicate the presence of a PIEZO1 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 6,073 according to SEQ ID NO: 35 in a particular PIEZO1 cDNA molecule, the presence of the amplified fragment would indicate the presence of a PIEZO1 variant cDNA molecule. In some embodiments, the nucleotide of the primer complementary to a thymine at a position corresponding to position 6,073 according to SEQ ID NO: 35 can be at the 3 '-end of the primer.

[0164] The present disclosure also provides primer pairs comprising any of the above primers. For example, if one of the 3 '-ends of the primer hybridizes to a cytosine (rather than a uracil) at a position corresponding to position 1,336 according to SEQ ID NO: 6 in a particular PIEZO1 nucleic acid molecule, the presence of the amplified fragment would indicate the presence of a PIEZO1 reference mRNA molecule. Conversely, if one of the 3 '-ends of the primer hybridizes to a uracil (rather than a cytosine) at a position corresponding to position 1,336 according to SEQ ID NO: 16 in a particular PIEZO1 mRNA molecule, the presence of the amplified fragment would indicate the presence of a PIEZO1 variant mRNA molecule. In some embodiments, the nucleotide of the primer complementary to a uracil at a position corresponding to position 1,336 according to SEQ ID NO: 16 can be at the 3 '-end of the primer. Furthermore, if one of the 3 '-ends of the primer hybridizes to a cytosine (rather than a thymine) at a position corresponding to position 1,336 according to SEQ ID NO: 26 in a particular PIEZO1 nucleic acid molecule, the presence of the amplified fragment would indicate the presence of a PIEZO1 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 1,336 according to SEQ ID NO: 36 in a particular PIEZO1 cDNA molecule, the presence of the amplified fragment would indicate the presence of a PIEZO1 variant cDNA molecule. In some embodiments, the nucleotide of the primer complementary to a thymine at a position corresponding to position 1,336 according to SEQ ID NO: 36 can be at the 3 '-end of the primer.

[0165] The present disclosure also provides primer pairs comprising any of the above primers. For example, if one of the 3 '-ends of the primer hybridizes to a cytosine (rather than a uracil) at a position corresponding to position 6,073 according to SEQ ID NO: 7 in a particular PIEZO1 nucleic acid molecule, the presence of the amplified fragment would indicate the presence of a PIEZO1 reference mRNA molecule. Conversely, if one of the 3 '-ends of the primer hybridizes to a uracil (rather than a cytosine) at a position corresponding to position 6,073 according to SEQ ID NO: 17 in a particular PIEZO1 mRNA molecule, the presence of the amplified fragment would indicate the presence of a PIEZO1 variant mRNA molecule. In some embodiments, the nucleotide of the primer complementary to a uracil at a position corresponding to position 6,073 according to SEQ ID NO: 17 can be at the 3 '-end of the primer. Furthermore, if one of the 3 '-ends of the primer hybridizes to a cytosine (rather than a thymine) at a position corresponding to position 6,073 according to SEQ ID NO: 27 in a particular PIEZO1 nucleic acid molecule, the presence of the amplified fragment would indicate the presence of a PIEZO1 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 6,073 according to SEQ ID NO: 37 in a particular PIEZO1 cDNA molecule, the presence of the amplified fragment would indicate the presence of a PIEZO1 variant cDNA molecule. In some embodiments, the nucleotide of the primer complementary to a thymine at a position corresponding to position 6,073 according to SEQ ID NO: 37 can be at the 3 '-end of the primer.

[0166] The present disclosure also provides primer pairs comprising any of the above primers. For example, if one of the 3 '-ends of the primer hybridizes to a cytosine (rather than a uracil) at a position corresponding to position 6,236 according to SEQ ID NO: 8 in a particular PIEZO1 nucleic acid molecule, the presence of the amplified fragment would indicate the presence of a PIEZO1 reference mRNA molecule. Conversely, if one of the 3 '-ends of the primer hybridizes to a uracil (rather than a cytosine) at a position corresponding to position 6,236 according to SEQ ID NO: 18 in a particular PIEZO1 mRNA molecule, the presence of the amplified fragment would indicate the presence of a PIEZO1 variant mRNA molecule. In some embodiments, the nucleotide of the primer complementary to a uracil at a position corresponding to position 6,236 according to SEQ ID NO: 18 can be at the 3 '-end of the primer. Furthermore, if one of the 3 '-ends of the primer hybridizes to a cytosine (rather than a thymine) at a position corresponding to position 6,236 according to SEQ ID NO: 28 in a particular PIEZO1 nucleic acid molecule, the presence of the amplified fragment would indicate the presence of a PIEZO1 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 6,236 according to SEQ ID NO: 38 in a particular PIEZO1 cDNA molecule, the presence of the amplified fragment would indicate the presence of a PIEZO1 variant cDNA molecule. In some embodiments, the nucleotide of the primer complementary to a thymine at a position corresponding to position 6,236 according to SEQ ID NO: 38 can be at the 3 '-end of the primer.

[0167] The present disclosure also provides primer pairs comprising any of the above primers. For example, if one of the 3 '-ends of the primer hybridizes to a cytosine (rather than a uracil) at a position corresponding to position 7,526 according to SEQ ID NO: 9 in a particular PIEZO1 nucleic acid molecule, the presence of the amplified fragment would indicate the presence of a PIEZO1 reference mRNA molecule. Conversely, if one of the 3 '-ends of the primer hybridizes to a uracil (rather than a cytosine) at a position corresponding to position 7,526 according to SEQ ID NO: 19 in a particular PIEZO1 mRNA molecule, the presence of the amplified fragment would indicate the presence of a PIEZO1 variant mRNA molecule. In some embodiments, the nucleotide of the primer complementary to a uracil at a position corresponding to position 7,526 according to SEQ ID NO: 19 can be at the 3 '-end of the primer. Furthermore, if one of the 3 '-ends of the primer hybridizes to a cytosine (rather than a thymine) at a position corresponding to position 7,526 according to SEQ ID NO: 29 in a particular PIEZO1 nucleic acid molecule, the presence of the amplified fragment would indicate the presence of a PIEZO1 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 7,526 according to SEQ ID NO: 39 in a particular PIEZO1 cDNA molecule, the presence of the amplified fragment would indicate the presence of a PIEZO1 variant cDNA molecule. In some embodiments, the nucleotide of the primer complementary to a thymine at a position corresponding to position 7,526 according to SEQ ID NO: 39 can be at the 3 '-end of the primer.

[0168] The present disclosure also provides primer pairs comprising any of the above primers. For example, if one of the 3 '-ends of the primer hybridizes to a cytosine (rather than a uracil) at a position corresponding to position 1,336 according to SEQ ID NO: 10 in a particular PIEZO1 nucleic acid molecule, the presence of the amplified fragment would indicate the presence of a PIEZO1 reference mRNA molecule. Conversely, if one of the 3 '-ends of the primer hybridizes to a uracil (rather than a cytosine) at a position corresponding to position 1,336 according to SEQ ID NO: 20 in a particular PIEZO1 mRNA molecule, the presence of the amplified fragment would indicate the presence of a PIEZO1 variant mRNA molecule. In some embodiments, the nucleotide of the primer complementary to a uracil at a position corresponding to position 1,336 according to SEQ ID NO: 20 can be at the 3 '-end of the primer. Furthermore, if one of the 3 '-ends of the primer hybridizes to a cytosine (rather than a thymine) at a position corresponding to position 1,336 according to SEQ ID NO: 30 in a particular PIEZO1 nucleic acid molecule, the presence of the amplified fragment would indicate the presence of a PIEZO1 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 1,336 according to SEQ ID NO: 40 in a particular PIEZO1 cDNA molecule, the presence of the amplified fragment would indicate the presence of a PIEZO1 variant cDNA molecule. In some embodiments, the nucleotide of the primer complementary to a thymine at a position corresponding to position 1,336 according to SEQ ID NO: 40 can be at the 3 '-end of the primer.

[0169] The present disclosure also provides primer pairs comprising any of the above primers. Furthermore, if one of the 3 '-ends of the primers hybridizes to a cytosine (rather than a uracil) at a position corresponding to position 361 according to SEQ ID NO: 11 in a particular PIEZO1 nucleic acid molecule, the presence of the amplified fragment will indicate the presence of a PIEZO1 reference mRNA molecule. Conversely, if one of the 3 '-ends of the primers hybridizes to a uracil (rather than a cytosine) at a position corresponding to position 361 according to SEQ ID NO: 21 in a particular PIEZO1 mRNA molecule, the presence of the amplified fragment will indicate the presence of a PIEZO1 variant mRNA molecule. In some embodiments, the nucleotide of the primer complementary to a uracil at a position corresponding to position 361 according to SEQ ID NO: 21 can be at the 3 '-end of the primer. Furthermore, if one of the 3 '-ends of the primers hybridizes to a cytosine (rather than a thymine) at a position corresponding to position 361 according to SEQ ID NO: 31 in a particular PIEZO1 nucleic acid molecule, the presence of the amplified fragment will indicate the presence of a PIEZO1 reference cDNA molecule. Conversely, if one of the 3 '-ends of the primers hybridizes to a thymine (rather than a cytosine) at a position corresponding to position 361 according to SEQ ID NO: 41 in a particular PIEZO1 cDNA molecule, the presence of the amplified fragment will indicate the presence of a PIEZO1 variant cDNA molecule. In some embodiments, the nucleotide of the primer complementary to a thymine at a position corresponding to position 361 according to SEQ ID NO: 41 can be at the 3 '-end of the primer.

[0170] The present disclosure also provides primer pairs comprising any of the above primers. For example, if one of the 3 '-ends of the primer hybridizes to a cytosine (rather than a uracil) at a position corresponding to position 7,776 according to SEQ ID NO: 12 in a particular PIEZO1 nucleic acid molecule, the presence of the amplified fragment would indicate the presence of a PIEZO1 reference mRNA molecule. Conversely, if one of the 3 '-ends of the primer hybridizes to a uracil (rather than a cytosine) at a position corresponding to position 7,776 according to SEQ ID NO: 22 in a particular PIEZO1 mRNA molecule, the presence of the amplified fragment would indicate the presence of a PIEZO1 variant mRNA molecule. In some embodiments, the nucleotide of the primer complementary to a uracil at a position corresponding to position 7,776 according to SEQ ID NO: 22 can be at the 3 '-end of the primer. Furthermore, if one of the 3 '-ends of the primer hybridizes to a cytosine (rather than a thymine) at a position corresponding to position 7,776 according to SEQ ID NO: 32 in a particular PIEZO1 nucleic acid molecule, the presence of the amplified fragment would indicate the presence of a PIEZO1 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 7,776 according to SEQ ID NO: 42 in a particular PIEZO1 cDNA molecule, the presence of the amplified fragment would indicate the presence of a PIEZO1 variant cDNA molecule. In some embodiments, the nucleotide of the primer complementary to a thymine at a position corresponding to position 7,776 according to SEQ ID NO: 42 can be at the 3 '-end of the primer.

[0171] In the context of the present disclosure, "specifically hybridizes" means that a probe or primer (such as an alteration-specific probe or alteration-specific primer) does not hybridize to the nucleotide sequence encoding a PIEZO1 reference genomic nucleic acid molecule, a PIEZO1 reference mRNA molecule, and / or a PIEZO1 reference cDNA molecule.

[0172] In any of the embodiments described throughout the present disclosure, a probe (e.g., an alteration-specific probe) can comprise a label. In some embodiments, the label is a fluorescent label, a radioactive label, or biotin.

[0173] The present disclosure also provides a support comprising a substrate to which is attached any one or more of the probes disclosed herein. A solid support is a solid state substrate or support to which a molecule, such as any of the probes disclosed herein, can be bound. One form of a solid support is an array. Another form of a solid support is an array detector. An array detector is a solid support to which a plurality of different probes are coupled in an array, grid, or other organized pattern. One form of a solid state substrate is a microtiter dish, such as a standard 96-well type. In some embodiments, a multi-well glass slide can be employed, typically containing an array in each well. In some embodiments, the support is a microarray.

[0174] The present disclosure also provides a molecular complex comprising or consisting of any one of the PIEZO1 nucleic acid molecules (genomic nucleic acid molecules, mRNA molecules, or cDNA molecules) described herein, or the complement thereof, and any one of the alteration-specific primers or alteration-specific probes described herein. In some embodiments, the PIEZO1 nucleic acid molecule (genomic nucleic acid molecule, mRNA molecule, or cDNA molecule) or the complement thereof in the molecular complex is single-stranded. In some embodiments, the PIEZO1 nucleic acid molecule is any one of the genomic nucleic acid molecules described herein. In some embodiments, the PIEZO1 nucleic acid molecule is any one of the mRNA molecules described herein. In some embodiments, the PIEZO1 nucleic acid molecule is any one of the cDNA molecules described herein. In some embodiments, the molecular complex comprises or consists of any one of the PIEZO1 nucleic acid molecules (genomic nucleic acid molecules, mRNA molecules, or cDNA molecules) described herein, or the complement thereof, and any one of the alteration-specific primers described herein. In some embodiments, the molecular complex comprises or consists of any one of the PIEZO1 nucleic acid molecules (genomic nucleic acid molecules, mRNA molecules, or cDNA molecules) described herein, or the complement thereof, and any one of the alteration-specific probes described herein.

[0175] In some embodiments, the molecular complex comprises an alteration-specific primer or alteration-specific probe that hybridizes to a PIEZO1 genomic nucleic acid molecule encoding a PIEZO1 predicted gain-of-function polypeptide, wherein the alteration-specific primer or the alteration-specific probe hybridizes to the PIEZO1 genomic nucleic acid molecule at a position corresponding to position 69,579, or the complement thereof, according to SEQ ID NO: 2.

[0176] In some embodiments, the alteration-specific primer or alteration-specific probe in the molecular complex hybridizes to a CTG codon at a position corresponding to positions 69,578-69,580 of SEQ ID NO: 2.

[0177] In some embodiments, the genomic nucleic acid molecule in the molecular complex comprises SEQ ID NO: 2.

[0178] In some embodiments, the molecular complex comprises an alteration-specific primer or alteration-specific probe that hybridizes to a PIEZO1 mRNA molecule encoding a PIEZO1 predicted gain-of-function polypeptide, wherein the alteration-specific primer or the alteration-specific probe hybridizes to the PIEZO1 mRNA molecule at a position corresponding to position 7,785 according to SEQ ID NO: 13, or the complement thereof; position 1,095 according to SEQ ID NO: 14, or the complement thereof; position 6,073 according to SEQ ID NO: 15, or the complement thereof; position 1,336 according to SEQ ID NO: 16, or the complement thereof; position 6,073 according to SEQ ID NO: 17, or the complement thereof; position 6,236 according to SEQ ID NO: 18, or the complement thereof; position 7,526 according to SEQ ID NO: 19, or the complement thereof; position 1,336 according to SEQ ID NO: 20, or the complement thereof; a position corresponding to position 361 according to SEQ ID NO: 21, or the complement thereof; or position 7,776 according to SEQ ID NO: 22, or the complement thereof.

[0179] In some embodiments, the alteration-specific primer or alteration-specific probe in the molecular complex hybridizes to a CUG codon at a position corresponding to positions 7,784-7,786 according to SEQ ID NO: 13, to a CUG codon at a position corresponding to positions 1,094-1,096 according to SEQ ID NO: 14, to a CUG codon at a position corresponding to positions 6,072-6,074 according to SEQ ID NO: 15, to a CUG codon at a position corresponding to positions 1,335-1,337 according to SEQ ID NO: 16, to a CUG codon at a position corresponding to positions 6,072-6,074 according to SEQ ID NO: 17, to a CUG codon at a position corresponding to positions 6,235-6,237 according to SEQ ID NO: 18, to a CUG codon at a position corresponding to positions 7,525-7,527 according to SEQ ID NO: 19, to a CUG codon at a position corresponding to positions 1,335-1,337 according to SEQ ID NO: 20, to a CUG codon at a position corresponding to positions 360-362 according to SEQ ID NO: 21, or to a CUG codon at a position corresponding to positions 7,775-7,777 according to SEQ ID NO: 22.

[0180] In some embodiments, the mRNA molecule in the molecular complex comprises SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21, or SEQ ID NO: 22.

[0181] In some embodiments, the molecular complex comprises an altered-specific primer or an altered-specific probe that hybridizes to a PIEZO1 cDNA molecule encoding a PIEZO1 predicted gain-of-function polypeptide, wherein the altered-specific primer or the altered-specific probe hybridizes to the PIEZO1 cDNA molecule at a position corresponding to position 7,785 according to SEQ ID NO: 33, or the complement thereof; position 1,095 according to SEQ ID NO: 34, or the complement thereof; position 6,073 according to SEQ ID NO: 35, or the complement thereof; position 1,336 according to SEQ ID NO: 36, or the complement thereof; position 6,073 according to SEQ ID NO: 37, or the complement thereof; position 6,236 according to SEQ ID NO: 38, or the complement thereof; position 7,526 according to SEQ ID NO: 39, or the complement thereof; position 1,336 according to SEQ ID NO: 40, or the complement thereof; position 361 according to SEQ ID NO: 41, or the complement thereof; or position 7,776 according to SEQ ID NO: 42, or the complement thereof.

[0182] In some embodiments, the altered-specificity primer or altered-specificity probe in the molecular complex hybridizes to a CTG codon at a position corresponding to positions 7,784-7,786 according to SEQ ID NO:33, hybridizes to a CTG codon at a position corresponding to positions 1,094-1,096 according to SEQ ID NO:34, hybridizes to a CTG codon at a position corresponding to positions 6,072-6,074 according to SEQ ID NO:35, hybridizes to a CTG codon at a position corresponding to positions 1,335-1,337 according to SEQ ID NO:36, hybridizes to a CTG codon at a position corresponding to positions 6,072-6,074 according to SEQ ID NO:37, hybridizes to a CTG codon at a position corresponding to positions 6,235-6,237 according to SEQ ID NO:38, hybridizes to a CTG codon at a position corresponding to positions 7,525-7,527 according to SEQ ID NO:39, hybridizes to a CTG codon at a position corresponding to positions 1,335-1,337 according to SEQ ID NO:40, hybridizes to a CTG codon at a position corresponding to positions 360-362 according to SEQ ID NO:41, or hybridizes to a CTG codon at a position corresponding to positions 7,775-7,777 according to SEQ ID NO:42.

[0183] In some embodiments, the cDNA molecule in the molecular complex comprises SEQ ID NO:33, SEQ ID NO:34, SEQ ID NO:35, SEQ ID NO:36, SEQ ID NO:37, SEQ ID NO:38, SEQ ID NO:39, SEQ ID NO:40, SEQ ID NO:41, or SEQ ID NO:42.

[0184] In some embodiments, the molecular complex comprises an altered-specificity probe or altered-specificity primer comprising 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.

[0185] The nucleotide sequence of the PIEZO1 reference genome nucleic acid molecule is set forth in SEQ ID NO: 1 (ENSG00000103335.22, comprising chr 16:88,715,338-88,785,220 in the GRCh38 / hg38 human genome assembly). See SEQ ID NO: 1, position 69,579 is cytosine.

[0186] There is a PIEZO1 variant genomic nucleic acid molecule in which the cytosine at position 69,579 is replaced by a thymine. The nucleotide sequence of this PIEZO1 variant genomic nucleic acid molecule is set forth in SEQ ID NO: 2.

[0187] The nucleotide sequence of a PIEZO1 reference mRNA molecule is set forth in SEQ ID NO: 3. See SEQ ID NO: 3, position 7,785 is a cytosine. The nucleotide sequence of another PIEZO1 reference mRNA molecule is set forth in SEQ ID NO: 4. See SEQ ID NO: 4, position 1,095 is a cytosine. The nucleotide sequence of another PIEZO1 reference mRNA molecule is set forth in SEQ ID NO: 5. See SEQ ID NO: 5, position 6,073 is a cytosine. The nucleotide sequence of another PIEZO1 reference mRNA molecule is set forth in SEQ ID NO: 6. See SEQ ID NO: 6, position 1,336 is a cytosine. The nucleotide sequence of another PIEZO1 reference mRNA molecule is set forth in SEQ ID NO: 7. See SEQ ID NO: 7, position 6,073 is a cytosine. The nucleotide sequence of another PIEZO1 reference mRNA molecule is set forth in SEQ ID NO: 8. See SEQ ID NO: 8, position 6,236 is a cytosine. The nucleotide sequence of another PIEZO1 reference mRNA molecule is set forth in SEQ ID NO: 9. See SEQ ID NO: 9, position 7,526 is a cytosine. The nucleotide sequence of another PIEZO1 reference mRNA molecule is set forth in SEQ ID NO: 10. See SEQ ID NO: 10, position 1,336 is a cytosine. The nucleotide sequence of another PIEZO1 reference mRNA molecule is set forth in SEQ ID NO: 11. See SEQ ID NO: 10, position 361 is a cytosine. The nucleotide sequence of another PIEZO1 reference mRNA molecule is set forth in SEQ ID NO: 12. See SEQ ID NO: 12, position 7,776 is a cytosine.

[0188] There is a PIEZO1 variant mRNA molecule in which the cytosine at position 7,785 is replaced by a uracil. The nucleotide sequence of this PIEZO1 variant mRNA molecule is set forth in SEQ ID NO: 13.

[0189] There is another PIEZO1 variant mRNA molecule in which the cytosine at position 1,095 is replaced by a uracil. The nucleotide sequence of this PIEZO1 variant mRNA molecule is set forth in SEQ ID NO: 14.

[0190] There is another PIEZO1 variant mRNA molecule in which the cytosine at position 6,073 is replaced by a uracil. The nucleotide sequence of this PIEZO1 variant mRNA molecule is set forth in SEQ ID NO: 15.

[0191] There is another PIEZO1 variant mRNA molecule in which the cytosine at position 1,336 is replaced by a uracil. The nucleotide sequence of this PIEZO1 variant mRNA molecule is set forth in SEQ ID NO: 16.

[0192] There is another PIEZO1 variant mRNA molecule in which the cytosine at position 6,073 is replaced by a uracil. The nucleotide sequence of this PIEZO1 variant mRNA molecule is set forth in SEQ ID NO: 17.

[0193] There is another PIEZO1 variant mRNA molecule in which the cytosine at position 6,236 is replaced by a uracil. The nucleotide sequence of this PIEZO1 variant mRNA molecule is set forth in SEQ ID NO: 18.

[0194] There is another PIEZO1 variant mRNA molecule in which the cytosine at position 7,526 is replaced by a uracil. The nucleotide sequence of this PIEZO1 variant mRNA molecule is set forth in SEQ ID NO: 19.

[0195] There is another PIEZO1 variant mRNA molecule in which the cytosine at position 1,336 is replaced by a uracil. The nucleotide sequence of this PIEZO1 variant mRNA molecule is set forth in SEQ ID NO: 20.

[0196] There is another PIEZO1 variant mRNA molecule in which the cytosine at position 361 is replaced by a uracil. The nucleotide sequence of this PIEZO1 variant mRNA molecule is set forth in SEQ ID NO: 21.

[0197] There is another PIEZO1 variant mRNA molecule in which the cytosine at position 7,776 is replaced by a uracil. The nucleotide sequence of this PIEZO1 variant mRNA molecule is set forth in SEQ ID NO: 22.

[0198] The nucleotide sequence of a PIEZO1 reference cDNA molecule is set forth in SEQ ID NO: 23. Referring to SEQ ID NO: 23, position 7,785 is a cytosine. The nucleotide sequence of another PIEZO1 reference cDNA molecule is set forth in SEQ ID NO: 24. Referring to SEQ ID NO: 24, position 1,095 is a cytosine. The nucleotide sequence of another PIEZO1 reference cDNA molecule is set forth in SEQ ID NO: 25. Referring to SEQ ID NO: 25, position 6,073 is a cytosine. The nucleotide sequence of another PIEZO1 reference cDNA molecule is set forth in SEQ ID NO: 26. Referring to SEQ ID NO: 26, position 1,336 is a cytosine. The nucleotide sequence of another PIEZO1 reference cDNA molecule is set forth in SEQ ID NO: 27. Referring to SEQ ID NO: 27, position 6,073 is a cytosine. The nucleotide sequence of another PIEZO1 reference cDNA molecule is set forth in SEQ ID NO: 28. Referring to SEQ ID NO: 28, position 6,236 is a cytosine. The nucleotide sequence of another PIEZO1 reference cDNA molecule is set forth in SEQ ID NO: 29. Referring to SEQ ID NO: 29, position 7,526 is a cytosine. The nucleotide sequence of another PIEZO1 reference cDNA molecule is set forth in SEQ ID NO: 30. Referring to SEQ ID NO: 30, position 1,336 is a cytosine. The nucleotide sequence of another PIEZO1 reference cDNA molecule is set forth in SEQ ID NO: 31. Referring to SEQ ID NO: 31, position 371 is a cytosine. The nucleotide sequence of another PIEZO1 reference cDNA molecule is set forth in SEQ ID NO: 32. Referring to SEQ ID NO: 32, position 7,776 is a cytosine.

[0199] There is a PIEZO1 variant cDNA molecule in which the cytosine at position 7,785 is replaced with a thymine. The nucleotide sequence of this PIEZO1 variant cDNA molecule is set forth in SEQ ID NO: 33.

[0200] There is another PIEZO1 variant cDNA molecule in which the cytosine at position 1,095 is replaced with a thymine. The nucleotide sequence of this PIEZO1 variant cDNA molecule is set forth in SEQ ID NO: 34.

[0201] There is another PIEZO1 variant cDNA molecule in which the cytosine at position 6,073 is replaced with a thymine. The nucleotide sequence of this PIEZO1 variant cDNA molecule is set forth in SEQ ID NO: 35.

[0202] There is another PIEZO1 variant cDNA molecule in which the cytosine at position 1,336 is replaced with a thymine. The nucleotide sequence of this PIEZO1 variant cDNA molecule is set forth in SEQ ID NO: 36.

[0203] There is another PIEZO1 variant cDNA molecule in which the cytosine at position 6,073 is replaced with a thymine. The nucleotide sequence of this PIEZO1 variant cDNA molecule is set forth in SEQ ID NO: 37.

[0204] There is another PIEZO1 variant cDNA molecule in which the cytosine at position 6,236 is replaced with a thymine. The nucleotide sequence of this PIEZO1 variant cDNA molecule is set forth in SEQ ID NO: 38.

[0205] There is another PIEZO1 variant cDNA molecule in which the cytosine at position 7,526 is replaced with a thymine. The nucleotide sequence of this PIEZO1 variant cDNA molecule is set forth in SEQ ID NO: 39.

[0206] There is another PIEZO1 variant cDNA molecule in which the cytosine at position 1,336 is replaced with a thymine. The nucleotide sequence of this PIEZO1 variant cDNA molecule is set forth in SEQ ID NO: 40.

[0207] There is another PIEZO1 variant cDNA molecule in which the cytosine at position 361 is replaced with a thymine. The nucleotide sequence of this PIEZO1 variant cDNA molecule is set forth in SEQ ID NO: 41.

[0208] There is another PIEZO1 variant cDNA molecule in which the cytosine at position 7,776 is replaced with a thymine. The nucleotide sequence of this PIEZO1 variant cDNA molecule is set forth in SEQ ID NO: 42.

[0209] Genomic nucleic acid molecules, mRNA molecules, and cDNA molecules can be from any organism. For example, genomic nucleic acid molecules, mRNA molecules, and cDNA molecules can be human or an ortholog from another organism, such as a non-human mammal, a rodent, a mouse, or a rat. It is understood that genetic sequences within a population can vary due to polymorphisms, such as single nucleotide polymorphisms. The examples provided herein are merely exemplary sequences. Other sequences are possible.

[0210] 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, agonists, modulators, and stimulators of specific activities possessed by target molecules, or functional polynucleotides can have novel activities independent of any other molecules.

[0211] 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 a heterologous nucleic acid sequence, such as in a vector, or a heterologous label. For example, the isolated nucleic acid molecules disclosed herein can be in a vector or as an exogenous donor sequence comprising the isolated nucleic acid molecule and a heterologous nucleic acid sequence. The isolated nucleic acid molecules can also be linked or fused to a heterologous label. The label can be directly detectable (e.g., like a fluorophore) or indirectly detectable (e.g., like a hapten, enzyme, or fluorophore quencher). Such labels can be detected 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 species; a metal-containing species; or an enzyme, where enzyme-dependent secondary signal generation occurs. The term "label" can also refer to a "tag" or hapten, which can selectively bind to a conjugate molecule such that the conjugate molecule, when added subsequently with a substrate, is used to generate a detectable signal. For example, biotin can be used as a tag to bind to an avidin or streptavidin conjugate of horseradish peroxidase (HRP) and examined using calorimetric substrates (e.g., like tetramethylbenzidine (TMB)) or fluorescent substrates 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 Fc portions of immunoglobulins. Many labels include, for example, particles, fluorophores, haptens, enzymes and calorimetric, fluorescent, and chemiluminescent substrates thereof, and other labels.

[0212] An isolated nucleic acid molecule or its complement can also be present in a host cell. In some embodiments, a host cell can comprise a vector comprising any of the nucleic acid molecules described herein or its complement. 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.

[0213] The disclosed nucleic acid molecules can comprise, for example, nucleotides or non-natural or modified nucleotides, such as nucleotide analogs or nucleotide substitutes. Such nucleotides include nucleotides containing modified bases, sugars, or phosphate groups, or nucleotides incorporating non-natural moieties in their structure. Examples of non-natural nucleotides include, but are not limited to, dideoxynucleotides, biotinylated, aminated, deaminated, alkylated, benzylated, and fluorophore-labeled nucleotides.

[0214] The nucleic acid molecules disclosed herein can also comprise one or more nucleotide analogs or substitutions. Nucleotide analogs are nucleotides containing modifications to the base, sugar, or phosphate moieties. Modifications to the base moiety include, but are not limited to, A, C, G, and T / U and natural and synthetic modifications of different purine or pyrimidine bases (e.g., like pseudouridine, uracil-5-yl, hypoxanthine-9-yl (I), and 2-aminoadenine-9-yl). Modified bases include, but are not limited to, 5-methylcytosine (5-me-C), 5-hydroxymethyl cytosine, 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-propynyl uracil and cytosine, 6-azo uracil, cytosine and thymine, 5-uracil (pseudouracil), 4-thiouracil, 8-halo, 8-amino, 8-thiol, 8-thioalkyl, 8-hydroxyl and other 8-substituted adenines and guanines, 5-halo (e.g., 5-bromo), 5-trifluoromethyl and other 5-substituted uracils and cytosines, 7-methylguanine, 7-methyladenine, 8-azaguanine, 8-azadenine, 7-deazaguanine, 7-deazaadenine, 3-deazaguanine and 3-deazaadenine.

[0215] Nucleotide analogs can also include modifications to the sugar moiety. Modifications to the sugar moiety include, but are not limited to, natural modifications of ribose and deoxyribose and 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, wherein alkyl, alkenyl and alkynyl can be substituted or unsubstituted C 1-10 alkyl or C 2-10 alkenyl and C 2-10 alkynyl. Exemplary 2' sugar modifications also include, but are not limited to, -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 CH3)]2, wherein n and m are independently 1 to about 10. Other modifications at the 2' position include, but are not limited to, C 1-10 alkyl, substituted lower alkyl, alkylaryl, arylalkyl, O-alkylaryl, or O-arylalkyl, SH, SCH3, OCN, Cl, Br, CN, CF3, OCF3, SOCH3, SO2CH3, ONO2, NO2, N3, NH2, heterocyclealkyl, heterocyclealkylaryl, aminoalkylamino, polyalkylamino, substituted silyl, an RNA cleaving group, a reporter group, an intercalator, a group for improving the pharmacokinetic properties of an oligonucleotide, or a group for improving the pharmacodynamic properties of an oligonucleotide, and other substituents having similar properties. A similar modification can also be made at other positions on the sugar, particularly the 3' position of the sugar on the 3' terminal nucleotide or in 2'-5' linked oligonucleotides and the 5' position of the 5' terminal nucleotide. Modified sugars can also include those containing a modification at the bridge oxygen such as CH2and S. Nucleotide sugar analogs can also have sugar mimics in place of the furanose pentose such as a cyclobutyl moiety.

[0216] Nucleotide analogs can also be modified at the phosphate moiety. Modified phosphate moieties include, but are not limited to, modified phosphate moieties which can be modified to contain a linkage between two nucleotides that contains a phosphorothioate, a chiral phosphorothioate, a phosphorodithioate, a phosphotriester, an aminoalkyl phosphotriester, a methyl and other alkyl phosphonates including 3'-alkylene phosphonates and chiral phosphonates, a phosphinothioate, an amino-phosphoramidate, a thioalkylphosphoramidate, a sulfide, a sulfamate, a boranophosphate, an alkyl- or alkylarylphosphonophosphate, and a phosphoramidate. These phosphate or modified phosphate linkages between two nucleotides can be either 3'-5' linkages or 2'-5' linkages, and the linkages can comprise inverted 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).

[0217] The present disclosure also provides vectors 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 or non-viral vector capable of transporting the nucleic acid molecule. In some embodiments, the vector is a plasmid or a cosmid (e.g., a circular double-stranded DNA like which can have additional DNA segments ligated into it). 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 virus (EBV)-derived episomes, and other expression vectors known in the art.

[0218] Desired regulatory sequences for mammalian host cell expression can include, for example, viral elements that direct high levels of polypeptide expression in mammalian cells, such as promoters derived from retroviral LTR, cytomegalovirus (CMV) (e.g., the CMV promoter / enhancer), simian virus 40 (SV40) (e.g., the SV40 promoter / enhancer), adenovirus (e.g., the adenovirus major late promoter (AdMLP)), polyoma virus, and mammalian strong promoters (e.g., the natural immunoglobulin and actin promoters). Methods for expressing polypeptides in bacterial cells or fungal cells (e.g., yeast cells) are also well known. The promoter can be, for example, a constitutive active promoter, a conditional promoter, an inducible promoter, a temporally limited promoter (e.g., a developmentally regulated promoter), or a spatially limited promoter (e.g., a cell-specific or tissue-specific promoter).

[0219] The percent identity (or percent complementarity) between a specified stretch of nucleotides within a nucleic acid molecule or a specified stretch of amino acids within a polypeptide can be routinely determined using the BLAST program (Basic Local Alignment Search Tool) and the PowerBLAST program (Altschul et al., J. Mol. Biol., 1990, 215, 403-410; Zhang and Madden, Genome Res., 1997, 7, 649-656) or by using the Gap program (Wisconsin Sequence Analysis Package, Version 8 for Unix, Genetics Computer Group, University Research Park, Madison Wis.) using default settings which use the algorithm of Smith and Waterman (Adv. Appl. Math., 1981, 2, 482-489). In this context, a higher percent sequence identity is preferred over a lower percent sequence identity, if reference is made to percent sequence identity.

[0220] The present disclosure also provides compositions comprising any one or more of the isolated nucleic acid molecules, genomic nucleic acid molecules, mRNA molecules, and / or cDNA molecules disclosed herein, or vectors comprising the same. In some embodiments, the composition is a pharmaceutical composition. In some embodiments, the composition comprises a carrier and / or an 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 helices, and lipid micropipes. The carrier can include a buffered salt solution, such as PBS, HBSS, and the like.

[0221] As used herein, the phrase "corresponds to" or grammatical variations thereof, when used in the context of numbering of a particular nucleotide or nucleotide sequence or position, refers to the numbering of the designated reference sequence (such as, for example, SEQ ID NO: 1, SEQ ID NO: 3, or SEQ ID NO: 40) when the particular nucleotide or nucleotide sequence is compared to the reference sequence. In other words, the residue (such as, for example, nucleotide or amino acid) numbering or residue (such as, for example, nucleotide or amino acid) position of a particular polymer is designated with respect to a reference sequence, rather than by the actual numerical position 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 matches between the two sequences. In these cases, while there are gaps, the numbering of the residues in the particular nucleotide or nucleotide sequence is made with respect to the reference sequence to which it is aligned.

[0222] For example, a PIEZO1 nucleic acid molecule comprising a nucleotide sequence encoding a PIEZO1 predicted gain-of-function polypeptide, wherein the nucleotide sequence comprises a thymine at a position corresponding to position 69,579 according to SEQ ID NO: 2, meaning that if the nucleotide sequence of the PIEZO1 genomic nucleic acid molecule is aligned with the sequence of SEQ ID NO: 2, the PIEZO1 sequence has a thymine residue at a position corresponding to position 69,579 according to SEQ ID NO: 2. The same applies to a PIEZO1 mRNA molecule comprising a nucleotide sequence encoding a PIEZO1 predicted gain-of-function polypeptide, wherein the nucleotide sequence comprises a uracil at a position corresponding to position 7,785 according to SEQ ID NO: 13; and a PIEZO1 cDNA molecule comprising a nucleotide sequence encoding a PIEZO1 predicted gain-of-function polypeptide, wherein the nucleotide sequence comprises a thymine at a position corresponding to position 7,785 according to SEQ ID NO: 33. These phrases refer to a PIEZO1 nucleic acid molecule encoding a PIEZO1 predicted gain-of-function polypeptide, wherein the genomic nucleic acid molecule has a nucleotide sequence comprising a thymine residue homologous to the thymine residue at position 69,579 of SEQ ID NO: 2 (or wherein the mRNA molecule has a nucleotide sequence comprising a uracil residue homologous to the uracil residue at position 7,785 of SEQ ID NO: 13, or wherein the cDNA molecule has a nucleotide sequence comprising a thymine residue homologous to the thymine residue at position 7,785 of SEQ ID NO: 33). In this context, such a sequence is also referred to as a “PIEZO1 sequence having a Pro2,510Leu alteration” or a “PIEZO1 sequence having a Pro2,510Leu variation”.

[0223] As described herein, the position within a PIEZO1 genomic nucleic acid molecule corresponding to position 69,579 according to SEQ ID NO: 2, for example, can be identified by performing a sequence alignment between the nucleotide sequence of a particular PIEZO1 nucleic acid molecule and the nucleotide sequence of SEQ ID NO: 2. There are various computational algorithms available for performing sequence alignments to identify a nucleotide position corresponding to, for example, position 69,579 in SEQ ID NO: 2. For example, sequence alignments can be performed by 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). However, sequences can also be aligned manually.

[0224] The amino acid sequence of another PIEZO1 reference polypeptide is set forth in SEQ ID NO: 44. Referring to SEQ ID NO: 44, the PIEZO1 reference polypeptide is 2,035 amino acids in length. Referring to SEQ ID NO: 44, position 2,024 is proline.

[0225] The amino acid sequence of another PIEZO1 reference polypeptide is set forth in SEQ ID NO: 44. Referring to SEQ ID NO: 44, the PIEZO1 reference polypeptide is 2,035 amino acids in length. Referring to SEQ ID NO: 44, position 2,024 is proline.

[0226] The amino acid sequence of another PIEZO1 reference polypeptide is set forth in SEQ ID NO: 44. Referring to SEQ ID NO: 44, the PIEZO1 reference polypeptide is 2,035 amino acids in length. Referring to SEQ ID NO: 44, position 2,024 is proline.

[0227] The amino acid sequence of another PIEZO1 reference polypeptide is set forth in SEQ ID NO: 44. Referring to SEQ ID NO: 44, the PIEZO1 reference polypeptide is 2,035 amino acids in length. Referring to SEQ ID NO: 44, position 2,024 is proline.

[0228] The amino acid sequence of another PIEZO1 reference polypeptide is set forth in SEQ ID NO: 44. Referring to SEQ ID NO: 44, the PIEZO1 reference polypeptide is 2,035 amino acids in length. Referring to SEQ ID NO: 44, position 2,024 is proline.

[0229] The amino acid sequence of another PIEZO1 reference polypeptide is set forth in SEQ ID NO: 44. Referring to SEQ ID NO: 44, the PIEZO1 reference polypeptide is 2,035 amino acids in length. Referring to SEQ ID NO: 44, position 2,024 is proline.

[0230] The amino acid sequence of another PIEZO1 reference polypeptide is set forth in SEQ ID NO: 44. Referring to SEQ ID NO: 44, the PIEZO1 reference polypeptide is 2,035 amino acids in length. Referring to SEQ ID NO: 44, position 2,024 is proline.

[0231] Another PIEZO1 predicted gain-of-function polypeptide (Pro94Leu) has an amino acid sequence set forth in SEQ ID NO: 50. See SEQ ID NO: 50, the PIEZO1 predicted gain-of-function polypeptide is 105 amino acids in length. See SEQ ID NO: 50, position 94 is a leucine.

[0232] The nucleotide and amino acid sequences listed in the accompanying sequence listing are shown using standard letter abbreviations for nucleotide bases and three letter code for amino acids. The nucleotide sequences follow the standard convention of starting at the 5' end of the sequence and proceeding forward (i.e., from left to right in each line) to the 3' end. Only one strand of each nucleotide sequence is shown, but the complementary strand is understood as included by any reference to the displayed strand. The amino acid sequences follow the standard convention of starting at the amino terminus of the sequence and proceeding forward (i.e., from left to right in each line) to the carboxy terminus.

[0233] The present disclosure also provides a therapeutic agent for treating or preventing varicose veins for use in (or for use in the manufacture of a medicament for) treating or preventing varicose veins in a subject, wherein the subject has any of the PIEZO1 variant genomic nucleic acid molecules, variant mRNA molecules, and / or variant cDNA molecules encoding a PIEZO1 predicted gain-of-function polypeptide described herein. The therapeutic agent for treating or preventing varicose veins can be any of the therapeutic agents for treating or preventing varicose veins described herein. The varicose veins can be any of varicose saphenous veins, varicose vesicular veins, spider veins, and pregnancy-related varicose veins.

[0234] In some embodiments, a subject is identified as having a genomic nucleic acid molecule encoding a PIEZO1 predicted gain-of-function polypeptide, or a complement thereof, wherein the genomic nucleic acid molecule has a nucleotide sequence comprising a thymine, or a complement thereof, at a position corresponding to position 69,579 according to SEQ ID NO: 2.

[0235] In some embodiments, the subject is identified as having a mRNA molecule, or a complement thereof, encoding a PIEZOl predicted gain-of-function polypeptide, wherein the mRNA molecule has a nucleotide sequence comprising: a uracil at a position corresponding to position 7,785 according to SEQ ID NO: 13, or a complement thereof; a uracil at a position corresponding to position 1,095 according to SEQ ID NO: 14, or a complement thereof; a uracil at a position corresponding to position 6,073 according to SEQ ID NO: 15, or a complement thereof; a uracil at a position corresponding to position 1,336 according to SEQ ID NO: 16, or a complement thereof; a uracil at a position corresponding to position 6,073 according to SEQ ID NO: 17, or a complement thereof; a uracil at a position corresponding to position 6,236 according to SEQ ID NO: 18, or a complement thereof; a uracil at a position corresponding to position 7,526 according to SEQ ID NO: 19, or a complement thereof; a uracil at a position corresponding to position 1,336 according to SEQ ID NO: 20, or a complement thereof; a uracil at a position corresponding to position 361 according to SEQ ID NO: 21, or a complement thereof; or a uracil at a position corresponding to position 7,776 according to SEQ ID NO: 22, or a complement thereof.

[0236] In some embodiments, the subject is identified as having a cDNA molecule, or a complement thereof, encoding a PIEZOl predicted gain-of-function polypeptide, wherein the cDNA molecule has a nucleotide sequence comprising: a thymine at a position corresponding to position 7,785 according to SEQ ID NO: 33, or a complement thereof; a thymine at a position corresponding to position 1,095 according to SEQ ID NO: 34, or a complement thereof; a thymine at a position corresponding to position 6,073 according to SEQ ID NO: 35, or a complement thereof; a thymine at a position corresponding to position 1,336 according to SEQ ID NO: 36, or a complement thereof; a thymine at a position corresponding to position 6,073 according to SEQ ID NO: 37, or a complement thereof; a thymine at a position corresponding to position 6,236 according to SEQ ID NO: 38, or a complement thereof; a thymine at a position corresponding to position 7,526 according to SEQ ID NO: 39, or a complement thereof; a thymine at a position corresponding to position 1,336 according to SEQ ID NO: 40, or a complement thereof; a thymine at a position corresponding to position 361 according to SEQ ID NO: 41, or a complement thereof; or a thymine at a position corresponding to position 7,776 according to SEQ ID NO: 42, or a complement thereof.

[0237] In some embodiments, the subject is identified as having: a genomic nucleic acid molecule having a nucleotide sequence encoding a PIEZOl predicted gain of function polypeptide, wherein the nucleotide sequence comprises a thymine at a position corresponding to position 69,579 according to SEQ ID NO:2, or the complement thereof; an mRNA molecule having a nucleotide sequence encoding a PIEZOl predicted gain of function polypeptide, wherein the nucleotide sequence comprises a uracil at a position corresponding to position 7,785 according to SEQ ID NO: 13, or the complement thereof; a cDNA molecule having a nucleotide sequence encoding a PIEZOl predicted gain of function polypeptide, wherein the nucleotide sequence comprises a thymine at a position corresponding to position 7,785 according to SEQ ID NO:33, or the complement thereof; or a PIEZOl predicted gain of function polypeptide comprising a leucine at a position corresponding to position 2,510 according to SEQ ID NO:47.

[0238] In some embodiments, the subject is identified as having: a genomic nucleic acid molecule having a nucleotide sequence encoding a PIEZOl predicted gain of function polypeptide, wherein the nucleotide sequence comprises an mRNA molecule having a nucleotide sequence encoding a PIEZOl predicted gain of function polypeptide, wherein the nucleotide sequence comprises a uracil at a position corresponding to position 1,095 according to SEQ ID NO: 14, or the complement thereof; a cDNA molecule having a nucleotide sequence encoding a PIEZOl predicted gain of function polypeptide, wherein the nucleotide sequence comprises a thymine at a position corresponding to position 1,095 according to SEQ ID NO:34, or the complement thereof; or a PIEZOl predicted gain of function polypeptide comprising a leucine at a position corresponding to position 2,024 according to SEQ ID NO:48.

[0239] In some embodiments, the subject is identified as having: a genomic nucleic acid molecule having a nucleotide sequence encoding a PIEZOl predicted gain of function polypeptide, wherein the nucleotide sequence comprises an mRNA molecule having a nucleotide sequence encoding a PIEZOl predicted gain of function polypeptide, wherein the nucleotide sequence comprises a uracil at a position corresponding to position 6,073 according to SEQ ID NO: 15, or the complement thereof; a cDNA molecule having a nucleotide sequence encoding a PIEZOl predicted gain of function polypeptide, wherein the nucleotide sequence comprises a thymine at a position corresponding to position 6,073 according to SEQ ID NO:35, or the complement thereof; or a PIEZOl predicted gain of function polypeptide comprising a leucine at a position corresponding to position 2,079 according to SEQ ID NO:49. In some embodiments, the subject is identified as having: a genomic nucleic acid molecule having a nucleotide sequence encoding a PIEZOl predicted gain of function polypeptide, wherein the nucleotide sequence comprises an mRNA molecule having a nucleotide sequence encoding a PIEZOl predicted gain of function polypeptide, wherein the nucleotide sequence comprises a uracil at a position corresponding to position 1,095 according to SEQ ID NO: 14, or the complement thereof; a cDNA molecule having a nucleotide sequence encoding a PIEZOl predicted gain of function polypeptide, wherein the nucleotide sequence comprises a thymine at a position corresponding to position 1,095 according to SEQ ID NO:34, or the complement thereof; or a PIEZOl predicted gain of function polypeptide comprising a leucine at a position corresponding to position 2,024 according to SEQ ID NO:48.

[0240] In some embodiments, the subject is identified as having: a genomic nucleic acid molecule having a nucleotide sequence encoding a PIEZOl predicted gain-of-function polypeptide, wherein the nucleotide sequence comprises an mRNA molecule having a nucleotide sequence encoding a PIEZOl predicted gain-of-function polypeptide, wherein the nucleotide sequence comprises a uracil at a position corresponding to position 1,336 according to SEQ ID NO: 16, or a complement thereof; a cDNA molecule having a nucleotide sequence encoding a PIEZOl predicted gain-of-function polypeptide, wherein the nucleotide sequence comprises a thymine at a position corresponding to position 1,336 according to SEQ ID NO: 36, or a complement thereof; or a PIEZOl predicted gain-of-function polypeptide comprising a leucine at a position corresponding to position 94 according to SEQ ID NO: 50.

[0241] In some embodiments, the subject is identified as having: a genomic nucleic acid molecule having a nucleotide sequence encoding a PIEZOl predicted gain-of-function polypeptide, wherein the nucleotide sequence comprises an mRNA molecule having a nucleotide sequence encoding a PIEZOl predicted gain-of-function polypeptide, wherein the nucleotide sequence comprises a uracil at a position corresponding to position 6,073 according to SEQ ID NO: 17, or a complement thereof; or a cDNA molecule having a nucleotide sequence encoding a PIEZOl predicted gain-of-function polypeptide, wherein the nucleotide sequence comprises a thymine at a position corresponding to position 6,073 according to SEQ ID NO: 37, or a complement thereof.

[0242] In some embodiments, the subject is identified as having: a genomic nucleic acid molecule having a nucleotide sequence encoding a PIEZOl predicted gain-of-function polypeptide, wherein the nucleotide sequence comprises an mRNA molecule having a nucleotide sequence encoding a PIEZOl predicted gain-of-function polypeptide, wherein the nucleotide sequence comprises a uracil at a position corresponding to position 6,236 according to SEQ ID NO: 18, or a complement thereof; or a cDNA molecule having a nucleotide sequence encoding a PIEZOl predicted gain-of-function polypeptide, wherein the nucleotide sequence comprises a thymine at a position corresponding to position 6,236 according to SEQ ID NO: 38, or a complement thereof.

[0243] In some embodiments, the subject is identified as having: a genomic nucleic acid molecule having a nucleotide sequence encoding a PIEZOl predicted loss of function polypeptide, wherein the nucleotide sequence comprises an mRNA molecule having a nucleotide sequence encoding a PIEZOl predicted loss of function polypeptide, wherein the nucleotide sequence comprises a cytosine at a position corresponding to position 1,336 according to SEQ ID NO: 20, or its complement; or a cDNA molecule having a nucleotide sequence encoding a PIEZOl predicted loss of function polypeptide, wherein the nucleotide sequence comprises a thymine at a position corresponding to position 1,336 according to SEQ ID NO: 40, or its complement.

[0244] In some embodiments, the subject is identified as having: a genomic nucleic acid molecule having a nucleotide sequence encoding a PIEZOl predicted loss of function polypeptide, wherein the nucleotide sequence comprises an mRNA molecule having a nucleotide sequence encoding a PIEZOl predicted loss of function polypeptide, wherein the nucleotide sequence comprises a cytosine at a position corresponding to position 1,336 according to SEQ ID NO: 20, or its complement; or a cDNA molecule having a nucleotide sequence encoding a PIEZOl predicted loss of function polypeptide, wherein the nucleotide sequence comprises a thymine at a position corresponding to position 1,336 according to SEQ ID NO: 40, or its complement.

[0245] In some embodiments, the subject is identified as having: a genomic nucleic acid molecule having a nucleotide sequence encoding a PIEZOl predicted loss of function polypeptide, wherein the nucleotide sequence comprises an mRNA molecule having a nucleotide sequence encoding a PIEZOl predicted loss of function polypeptide, wherein the nucleotide sequence comprises a cytosine at a position corresponding to position 1,336 according to SEQ ID NO: 20, or its complement; or a cDNA molecule having a nucleotide sequence encoding a PIEZOl predicted loss of function polypeptide, wherein the nucleotide sequence comprises a thymine at a position corresponding to position 1,336 according to SEQ ID NO: 40, or its complement.

[0246] In some embodiments, the subject is identified as having a genomic nucleic acid molecule having a nucleotide sequence encoding a PIEZO1 predicted loss of function polypeptide, wherein the nucleotide sequence comprises an mRNA molecule having a nucleotide sequence encoding a PIEZO1 predicted loss of function polypeptide, wherein the nucleotide sequence comprises a cytosine at a position corresponding to position 69,579 according to SEQ ID NO: 2, or a complement thereof; or a cDNA molecule having a nucleotide sequence encoding a PIEZO1 predicted loss of function polypeptide, wherein the nucleotide sequence comprises a guanine at a position corresponding to position 69,579 according to SEQ ID NO: 42, or a complement thereof.

[0247] The present disclosure also provides a PIEZO1 agonist for use in (or used in the manufacture of a medicament for) treating or preventing varicose veins in a subject, wherein the subject is heterozygous for any one of the PIEZO1 variant genomic nucleic acid molecules, variant mRNA molecules, and / or variant cDNA molecules encoding a PIEZO1 predicted loss of function polypeptide described herein, or wherein the subject is reference type for a PIEZO1 genomic nucleic acid molecule, mRNA molecule, or cDNA molecule. The PIEZO1 agonist can be any of the PIEZO1 agonists described herein. The varicose veins can be any one of a hidden varicose vein, a varicose vein with a blister, a spider vein, and a pregnancy-related varicose vein.

[0248] In some embodiments, the subject is reference type for a PIEZO1 genomic nucleic acid molecule, a PIEZO1 mRNA molecule, or a PIEZO1 cDNA molecule.

[0249] In some embodiments, the subject is heterozygous for a genomic nucleic acid molecule encoding a PIEZO1 predicted loss of function polypeptide, wherein the genomic nucleic acid molecule has a nucleotide sequence comprising a cytosine or a complement thereof at a position corresponding to position 69,579 according to SEQ ID NO: 2.

[0250] In some embodiments, the subject is heterozygous for a mRNA molecule, or complement thereof, encoding a PIEZOl predicted gain-of-function polypeptide, wherein the mRNA molecule has a nucleotide sequence comprising: a uracil at a position corresponding to position 7,785 according to SEQ ID NO: 13, or complement thereof; a uracil at a position corresponding to position 1,095 according to SEQ ID NO: 14, or complement thereof; a uracil at a position corresponding to position 6,073 according to SEQ ID NO: 15, or complement thereof; a uracil at a position corresponding to position 1,336 according to SEQ ID NO: 16, or complement thereof; a uracil at a position corresponding to position 6,073 according to SEQ ID NO: 17, or complement thereof; a uracil at a position corresponding to position 6,236 according to SEQ ID NO: 18, or complement thereof; a uracil at a position corresponding to position 7,526 according to SEQ ID NO: 19, or complement thereof; a uracil at a position corresponding to position 1,336 according to SEQ ID NO: 20, or complement thereof; a uracil at a position corresponding to position 361 according to SEQ ID NO: 21, or complement thereof; or a uracil at a position corresponding to position 7,776 according to SEQ ID NO: 22, or complement thereof.

[0251] In some embodiments, the subject is heterozygous for a cDNA molecule, or complement thereof, encoding a PIEZOl predicted gain-of-function polypeptide, wherein the cDNA molecule has a nucleotide sequence comprising: a thymine at a position corresponding to position 7,785 according to SEQ ID NO: 33, or complement thereof; a thymine at a position corresponding to position 1,095 according to SEQ ID NO: 34, or complement thereof; a thymine at a position corresponding to position 6,073 according to SEQ ID NO: 35, or complement thereof; a thymine at a position corresponding to position 1,336 according to SEQ ID NO: 36, or complement thereof; a thymine at a position corresponding to position 6,073 according to SEQ ID NO: 37, or complement thereof; a thymine at a position corresponding to position 6,236 according to SEQ ID NO: 38, or complement thereof; a thymine at a position corresponding to position 7,526 according to SEQ ID NO: 39, or complement thereof; a thymine at a position corresponding to position 1,336 according to SEQ ID NO: 40, or complement thereof; a thymine at a position corresponding to position 361 according to SEQ ID NO: 41, or complement thereof; or a thymine at a position corresponding to position 7,776 according to SEQ ID NO: 42, or complement thereof.

[0252] In some embodiments, the subject is identified as heterozygous for: a genomic nucleic acid molecule having a nucleotide sequence encoding a PIEZO1 predicted gain-of-function polypeptide, wherein the nucleotide sequence comprises a thymine at a position corresponding to position 69,579 according to SEQ ID NO:2, or the complement thereof; an mRNA molecule having a nucleotide sequence encoding a PIEZO1 predicted gain-of-function polypeptide, wherein the nucleotide sequence comprises a uracil at a position corresponding to position 7,785 according to SEQ ID NO: 13, or the complement thereof; a cDNA molecule having a nucleotide sequence encoding a PIEZO1 predicted gain-of-function polypeptide, wherein the nucleotide sequence comprises a thymine at a position corresponding to position 7,785 according to SEQ ID NO:33, or the complement thereof; or a PIEZO1 predicted gain-of-function polypeptide comprising a leucine at a position corresponding to position 2,510 according to SEQ ID NO:47. The PIEZO1 agonist can be any PIEZO1 agonist described herein.

[0253] In some embodiments, the subject is identified as heterozygous for: a genomic nucleic acid molecule having a nucleotide sequence encoding a PIEZO1 predicted gain-of-function polypeptide, wherein the nucleotide sequence comprises a mRNA molecule having a nucleotide sequence encoding a PIEZO1 predicted gain-of-function polypeptide, wherein the nucleotide sequence comprises a uracil at a position corresponding to position 1,095 according to SEQ ID NO: 14, or the complement thereof; a cDNA molecule having a nucleotide sequence encoding a PIEZO1 predicted gain-of-function polypeptide, wherein the nucleotide sequence comprises a thymine at a position corresponding to position 1,095 according to SEQ ID NO:34, or the complement thereof; or a PIEZO1 predicted gain-of-function polypeptide comprising a leucine at a position corresponding to position 2,024 according to SEQ ID NO:48. The PIEZO1 agonist can be any PIEZO1 agonist described herein.

[0254] In some embodiments, the subject is identified as heterozygous for a genomic nucleic acid molecule having a nucleotide sequence encoding a PIEZO1 predicted gain of function polypeptide, wherein the nucleotide sequence comprises an mRNA molecule having a nucleotide sequence encoding a PIEZO1 predicted gain of function polypeptide, wherein the nucleotide sequence comprises a uracil at a position corresponding to position 6,073 according to SEQ ID NO: 15, or the complement thereof; a cDNA molecule having a nucleotide sequence encoding a PIEZO1 predicted gain of function polypeptide, wherein the nucleotide sequence comprises a thymine at a position corresponding to position 6,073 according to SEQ ID NO: 35, or the complement thereof; or a PIEZO1 predicted gain of function polypeptide comprising a leucine at a position corresponding to position 2,079 according to SEQ ID NO: 49. The PIEZO1 agonist can be any PIEZO1 agonist described herein.

[0255] In some embodiments, the subject is identified as heterozygous for a genomic nucleic acid molecule having a nucleotide sequence encoding a PIEZO1 predicted gain of function polypeptide, wherein the nucleotide sequence comprises an mRNA molecule having a nucleotide sequence encoding a PIEZO1 predicted gain of function polypeptide, wherein the nucleotide sequence comprises a uracil at a position corresponding to position 1,336 according to SEQ ID NO: 16, or the complement thereof; a cDNA molecule having a nucleotide sequence encoding a PIEZO1 predicted gain of function polypeptide, wherein the nucleotide sequence comprises a thymine at a position corresponding to position 1,336 according to SEQ ID NO: 36, or the complement thereof; or a PIEZO1 predicted gain of function polypeptide comprising a leucine at a position corresponding to position 94 according to SEQ ID NO: 50. The PIEZO1 agonist can be any PIEZO1 agonist described herein.

[0256] In some embodiments, the subject is identified as heterozygous for a genomic nucleic acid molecule having a nucleotide sequence encoding a PIEZO1 predicted gain of function polypeptide, wherein the nucleotide sequence comprises an mRNA molecule having a nucleotide sequence encoding a PIEZO1 predicted gain of function polypeptide, wherein the nucleotide sequence comprises a uracil at a position corresponding to position 6,073 according to SEQ ID NO: 17, or the complement thereof; or a cDNA molecule having a nucleotide sequence encoding a PIEZO1 predicted gain of function polypeptide, wherein the nucleotide sequence comprises a thymine at a position corresponding to position 6,073 according to SEQ ID NO: 37, or the complement thereof. The PIEZO1 agonist can be any PIEZO1 agonist described herein.

[0257] In some embodiments, the subject is identified as heterozygous for a genomic nucleic acid molecule having a nucleotide sequence encoding a PIEZO1 predicted gain of function polypeptide, wherein the nucleotide sequence comprises an mRNA molecule having a nucleotide sequence encoding a PIEZO1 predicted gain of function polypeptide, wherein the nucleotide sequence comprises a uracil at a position corresponding to position 6,236 according to SEQ ID NO: 18, or its complement; or a cDNA molecule having a nucleotide sequence encoding a PIEZO1 predicted gain of function polypeptide, wherein the nucleotide sequence comprises a thymine at a position corresponding to position 6,236 according to SEQ ID NO: 38, or its complement. The PIEZO1 agonist can be any PIEZO1 agonist described herein.

[0258] In some embodiments, the subject is identified as heterozygous for a genomic nucleic acid molecule having a nucleotide sequence encoding a PIEZO1 predicted gain of function polypeptide, wherein the nucleotide sequence comprises an mRNA molecule having a nucleotide sequence encoding a PIEZO1 predicted gain of function polypeptide, wherein the nucleotide sequence comprises a uracil at a position corresponding to position 7,526 according to SEQ ID NO: 19, or its complement; or a cDNA molecule having a nucleotide sequence encoding a PIEZO1 predicted gain of function polypeptide, wherein the nucleotide sequence comprises a thymine at a position corresponding to position 7,526 according to SEQ ID NO: 39, or its complement. The PIEZO1 agonist can be any PIEZO1 agonist described herein.

[0259] In some embodiments, the subject is identified as heterozygous for a genomic nucleic acid molecule having a nucleotide sequence encoding a PIEZO1 predicted gain of function polypeptide, wherein the nucleotide sequence comprises an mRNA molecule having a nucleotide sequence encoding a PIEZO1 predicted gain of function polypeptide, wherein the nucleotide sequence comprises a uracil at a position corresponding to position 1,336 according to SEQ ID NO: 20, or its complement; or a cDNA molecule having a nucleotide sequence encoding a PIEZO1 predicted gain of function polypeptide, wherein the nucleotide sequence comprises a thymine at a position corresponding to position 1,336 according to SEQ ID NO: 40, or its complement.

[0260] In some embodiments, the subject is identified as heterozygous for a genomic nucleic acid molecule having a nucleotide sequence encoding a PIEZO1 predicted loss of function polypeptide, wherein the nucleotide sequence comprises an mRNA molecule having a nucleotide sequence encoding a PIEZO1 predicted loss of function polypeptide, wherein the nucleotide sequence comprises a cytosine at a position corresponding to position 7,776 according to SEQ ID NO: 22, or its complement; or a cDNA molecule having a nucleotide sequence encoding a PIEZO1 predicted loss of function polypeptide, wherein the nucleotide sequence comprises a thymine at a position corresponding to position 7,776 according to SEQ ID NO: 42, or its complement.

[0261] In some embodiments, the subject is identified as heterozygous for a genomic nucleic acid molecule having a nucleotide sequence encoding a PIEZO1 predicted loss of function polypeptide, wherein the nucleotide sequence comprises an mRNA molecule having a nucleotide sequence encoding a PIEZO1 predicted loss of function polypeptide, wherein the nucleotide sequence comprises a cytosine at a position corresponding to position 7,776 according to SEQ ID NO: 22, or its complement; or a cDNA molecule having a nucleotide sequence encoding a PIEZO1 predicted loss of function polypeptide, wherein the nucleotide sequence comprises a thymine at a position corresponding to position 7,776 according to SEQ ID NO: 42, or its complement.

[0262] In some embodiments, the subject is identified as having a PIEZO1 reference genomic nucleic acid molecule comprising SEQ ID NO: 1, a PIEZO1 reference mRNA molecule comprising one or more of SEQ ID NOs: 3-12, a PIEZO1 reference cDNA molecule comprising one or more of SEQ ID NOs: 23-32, or a PIEZO1 reference polypeptide comprising one or more of SEQ ID NOs: 43-46. The PIEZO1 agonist can be any PIEZO1 agonist described herein.

[0263] All patent documents, websites, other publications, accession numbers, and the like cited above or below are hereby incorporated by reference in their entirety for all purposes to the same extent as if each individual item were specifically and individually indicated to be incorporated by reference for its entirety. If different versions of a sequence are associated with different dates of accession numbers, the version associated with the accession number of the date of the effective filing date of the present application is intended. The 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. Likewise, if different versions of a publication, website, and the like are published at different times, the version published most recently as of the effective filing date of the present application is intended, unless otherwise noted. Unless specifically stated otherwise, any feature, step, element, embodiment or aspect of the disclosure can be used in combination with any other feature, step, element, embodiment or aspect. Although the disclosure has been described in detail for the purpose of clarity and understanding, it will be apparent that certain changes and modifications can be practiced within the scope of the appended claims.

[0264] The following examples are provided to describe the embodiments in greater detail. They are intended to illustrate but not limit the claimed embodiments. The following examples provide a disclosure and description of how the compounds, compositions, articles, devices, and / or methods described herein can be made and evaluated, and are intended to be purely exemplary and are not intended to limit the scope of any claim. Efforts have been made to ensure accuracy with respect to numbers (e.g., amounts, temperature, etc. ) but some errors and deviations can be contemplated. Unless otherwise indicated, parts are parts by weight, temperature is in °C or at ambient temperature, and pressure is at or near atmospheric.

[0265] Example

[0266] Example 1: Association of varicose veins with PIEZOl GOF variants

[0267] As previously described, exomes were sequenced for 454,787 UKB study participants, with 95.8% of targeted bases having a depth of coverage of 20x or greater (Szustakowski, Advancing Human Genetics Research and Drug Discovery through Exome Sequencing of the UK Biobank. bioRxiv, 2021; and Van Hout et al., Nature, 2020). One million and two hundred thousand variants were identified in 39 million base pairs of coding regions of 18,659 genes (data not shown). Of the identified variants, there were 3,375,252 (median of 10,260 per individual) synonymous variants, 7,689,495 (9,284 per individual) missense variants, and 889,957 (212 per individual) putatively loss-of-function (pLOF) variants (data not shown), with about half observed only once in this dataset (singletons; data not shown).

[0268] A missense variant in PIEZO1 (rs61745086:A, Pro2,510Leu, MAF=0.98%) was found to be associated with reduced risk of varicose veins (7,455 carriers; OR=0.69, 95% CI 0.61 to 0.70, P=2.61x10 -8 ). PIEZO1 encodes a mechanically sensitive cation channel that plays a key role in venous and lymphatic valve formation. Previously, rare pLOFs in this gene were shown to increase risk of asymptomatic varicose veins in the lower limbs by 4.9-fold in the first 50K exomes from UKB (162 carriers; 95% CI 2.8 to 8.6, P=3.2x10 -8 ), an association now estimated to be 2.04-fold, with data suggesting about 8-fold greater (1,302 carriers; OR=2.04, 95% CI 1.63 to 2.56, P=5.3x10 -10 ). The novel protective association with rs61745086:A was replicated in the GHS cohort (2,243 carriers; OR=0.66, 95% CI 0.47 to 0.93, P=0.017), suggesting that this missense variant can have a gain-of-function effect. This is important as it suggests that activation of PIEZO1 can provide a therapeutic avenue for a common condition for which there is no available pharmaceutical intervention.

[0269] Various modifications of the described subject matter can be made to the foregoing description and drawings disclosed herein. While specific embodiments of, and examples for, the subject disclosure are described herein for illustrative purposes only, various equivalent modifications are possible within the scope of the subject disclosure, as those skilled in the relevant art will recognize. Also, some of the features and benefits described herein can be employed i some embodiments without the corresponding description of some other features and benefits. Changes in, or substitutions of, elements from one embodiment to another are expressly contemplated as falling within the scope of the subject disclosure. In short, the present disclosure is not to be limited in scope by the embodiments disclosed herein, but is to be construed as broadly as reasonably permitted by law.

Claims

1. A method for treating a subject who has varicose veins or is at risk of developing varicose veins, the method comprising administering a piezoelectric mechanosensitive ion channel component 1 (PIEZO1) agonist to the subject.

2. A method for treating a subject with saphenous vein varicocele or at risk of developing saphenous vein varicocele, the method comprising administering a piezoelectric mechanosensitive ion channel component 1 (PIEZO1) agonist to the subject.

3. A method for treating a subject with vesicular varicose veins or at risk of developing vesicular varicose veins, the method comprising administering a piezoelectric mechanosensitive ion channel component 1 (PIEZO1) agonist to the subject.

4. A method of treating a subject with arachnoid veins or at risk of developing arachnoid veins, the method comprising administering a piezoelectric mechanosensitive ion channel component 1 (PIEZO1) agonist to the subject.

5. A method for treating a subject who has pregnancy-related varicose veins or is at risk of developing pregnancy-related varicose veins, the method comprising administering a piezoelectric mechanosensitive ion channel component 1 (PIEZO1) agonist to the subject.

6. The method according to any one of claims 1 to 5, wherein the PIEZO1 agonist comprises a small molecule.

7. The method according to claim 6, wherein the small molecule agonist is Yoda1.

8. The method according to claim 6, wherein the small molecule agonist is Jedi1.

9. The method according to claim 6, wherein the small molecule agonist is Jedi2.

10. The method according to any one of claims 1 to 9, further comprising detecting the presence or absence of a PIEZO1 variant nucleic acid molecule encoding a PIEZO1 predictive function gain polypeptide in a biological sample obtained from the subject.

11. The method of claim 10, further comprising administering a therapeutic agent for treating or preventing varicose veins to a subject at a standard dose, wherein the PIEZO1 variant nucleic acid molecule is not present in the biological sample.

12. The method of claim 10, further comprising administering a therapeutic agent for treating or preventing varicose veins to a subject who is heterozygous for the PIEZO1 variant nucleic acid molecule at a dose equal to or less than a standard dose.

13. The method according to any one of claims 10 to 12, wherein the PIEZO1 variant nucleic acid molecule encodes Pro2,510Leu, Pro2,024Leu, Pro2,079Leu, or Pro92Leu.

14. The method of claim 13, wherein the PIEZO1 variant nucleic acid molecule is: A genomic nucleic acid molecule having a nucleotide sequence containing thymine or its complement at position 69,579 according to SEQ ID NO:2; An mRNA molecule having the following nucleotide sequences: uracil at position 7,785 according to SEQ ID NO:13, or a complement thereof; uracil at position 1,095 according to SEQ ID NO:14, or a complement thereof; Uracil, or its complement, located at position 6,073 according to SEQ ID NO:15; Uracil, or its complement, located at position 1,336 according to SEQ ID NO:16; Uracil, or its complement, located at position 6,073 according to SEQ ID NO:17; Uracil, or its complement, located at positions 6,236 according to SEQ ID NO:18; Uracil, or its complement, located at position 7,526 according to SEQ ID NO:19; Uracil, or its complement, located at position 1,336 according to SEQ ID NO:20; Uracil, or its complement, located at position 361 according to SEQ ID NO: 21; or uracil, or its complement, located at position 7,776 according to SEQ ID NO: 22; or A cDNA molecule generated from an mRNA molecule, wherein the cDNA molecule has a nucleotide sequence comprising: thymine at position 7,785 according to SEQ ID NO:33, or a complement thereof; Thymine, or its complement thereof, is located at position 1,095 according to SEQ ID NO:34; Thymine, or its complement thereof, located at position 6,073 according to SEQ ID NO:35; Thymine, or its complement, is located at positions 1,336 according to SEQ ID NO:36; Thymine, or its complement thereof, is located at position 6,073 according to SEQ ID NO:37; Thymine, or its complement, located at position 6,236 according to SEQ ID NO:38; Thymine, or its complement, located at position 7,526 according to SEQ ID NO:39; Thymine, or its complement, located at position 1,336 according to SEQ ID NO:40; Thymine or its complement at position 361 according to SEQ ID NO:41; or thymine or its complement at position 7,776 according to SEQ ID NO:

42.

15. The method according to any one of claims 10 to 14, wherein the detection step is performed in vitro.

16. The method according to any one of claims 10 to 15, wherein the detection step comprises sequencing at least a portion of the nucleotide sequence of the PIEZO1 genomic nucleic acid molecule or its complement in the biological sample, wherein the sequenced portion comprises a position corresponding to position 69,579 according to SEQ ID NO:2, or its complement; Where the sequenced portion of the PIEZO1 genomic nucleic acid molecule in the biological sample contains thymine or its complement at position 69,579 according to SEQ ID NO:2, then the PIEZO1 genomic nucleic acid molecule in the biological sample is a PIEZO1 variant genomic nucleic acid molecule encoding a PIEZO1 predictive function gain polypeptide.

17. The method according to any one of claims 10 to 15, wherein the detection step comprises sequencing at least a portion of the nucleotide sequence of the PIEZO1 mRNA molecule in the biological sample, wherein the sequenced portion comprises a position corresponding to: position 7,785 of SEQ ID NO: 13, or its complement; position 1,095 of SEQ ID NO: 14, or its complement; position 6,073 of SEQ ID NO: 15, or its complement; position 1,336 of SEQ ID NO: 16, or its complement; position 6,073 of SEQ ID NO: 17, or its complement; position 6,236 of SEQ ID NO: 18, or its complement; position 7,526 of SEQ ID NO: 19, or its complement; position 1,336 of SEQ ID NO: 20, or its complement; position 361 of SEQ ID NO: 21, or its complement; or position 7,776 of SEQ ID NO: 22, or its complement. Wherein the sequenced portion of the PIEZO1 mRNA molecule in the biological sample comprises: uracil at position 7,785 according to SEQ ID NO: 13, or a complement thereof; and uracil at position 1,095 according to SEQ ID NO: 14, or a complement thereof. Uracil, or its complement, located at position 6,073 according to SEQ ID NO:15; Uracil, or its complement, located at position 1,336 according to SEQ ID NO:16; Uracil, or its complement, located at position 6,073 according to SEQ ID NO:17; Uracil, or its complement, located at positions 6,236 according to SEQ ID NO:18; Uracil, or its complement, located at position 7,526 according to SEQ ID NO:19; Uracil, or its complement, located at position 1,336 according to SEQ ID NO:20; When uracil or its complement is located at position 361 according to SEQ ID NO:21; or when uracil or its complement is located at position 7,776 according to SEQ ID NO:22, the PIEZO1 mRNA molecule in the biological sample is a PIEZO1 variant mRNA molecule encoding a PIEZO1 predictive function gain polypeptide.

18. The method according to any one of claims 10 to 15, wherein the detection step comprises sequencing at least a portion of the nucleotide sequence of the PIEZO1 cDNA molecule generated from the mRNA molecule in the biological sample, wherein the sequenced portion comprises a position corresponding to: position 7,785 of SEQ ID NO:33, or its complement; position 1,095 of SEQ ID NO:34, or its complement; position 6,073 of SEQ ID NO:35, or its complement; position 1,336 of SEQ ID NO:36, or its complement; position 6,073 of SEQ ID NO:37, or its complement; position 6,236 of SEQ ID NO:38, or its complement; position 7,526 of SEQ ID NO:39, or its complement; position 1,336 of SEQ ID NO:40, or its complement; position 361 of SEQ ID NO:41, or its complement; or position 7,776 of SEQ ID NO:42, or its complement. Wherein the sequenced portion of the PIEZO1 cDNA molecule in the biological sample comprises: thymine or a complement thereof at position 7,785 according to SEQ ID NO:33; and thymine or a complement thereof at position 1,095 according to SEQ ID NO:

34. Thymine, or its complement thereof, located at position 6,073 according to SEQ ID NO:35; Thymine, or its complement, is located at positions 1,336 according to SEQ ID NO:36; Thymine, or its complement thereof, is located at position 6,073 according to SEQ ID NO:37; Thymine, or its complement, located at position 6,236 according to SEQ ID NO:38; Thymine, or its complement, located at position 7,526 according to SEQ ID NO:39; Thymine, or its complement, located at position 1,336 according to SEQ ID NO:40; When thymine or its complement is present at position 361 according to SEQ ID NO:41; or at position 7,776 according to SEQ ID NO:42; the PIEZO1 cDNA molecule generated from the mRNA molecule in the biological sample is a PIEZO1 variant cDNA molecule encoding a PIEZO1 predictive function gain polypeptide.

19. The method according to any one of claims 10 to 15, wherein the detection step comprises: a) Contact the biological sample with primers that hybridize with a portion of the nucleotide sequence of the PIEZO1 genomic nucleic acid molecule or its complement, the portion being close to a position corresponding to position 69,579 of SEQ ID NO:2 or its complement; b) Extend the primer at least through the nucleotide sequence of the PIEZO1 genomic nucleic acid molecule or its complement corresponding to the following positions: according to position 69,579 of SEQ ID NO:2, or its complement; and c) Determine whether the primer extension product contains thymine, or its complement, at the position corresponding to position 69,579 according to SEQ ID NO:

2.

20. The method according to any one of claims 10 to 15, wherein the detection step comprises: a) Contact the biological sample with primers, which hybridize with a portion of the nucleotide sequence of the PIEZO1 mRNA molecule or its complement, said portion being close to the position corresponding to the following positions: position 7,785 of SEQ ID NO:13, or its complement; position 1,095 of SEQ ID NO:14, or its complement; position 6,073 of SEQ ID NO:15, or its complement; position 1,336 of SEQ ID NO:16, or its complement; position 6,073 of SEQ ID NO:17, or its complement; position 6,236 of SEQ ID NO:18, or its complement; position 7,526 of SEQ ID NO:19, or its complement; position 1,336 of SEQ ID NO:20, or its complement; position 361 of SEQ ID NO:21, or its complement; or position 7,776 of SEQ ID NO:22, or its complement. b) Extend the primer at least through the nucleotide sequence of the PIEZO1 mRNA molecule or its complement to the positions corresponding to the following: position 7,785 of SEQ ID NO:13, or its complement; position 1,095 of SEQ ID NO:14, or its complement; position 6,073 of SEQ ID NO:15, or its complement; position 1,336 of SEQ ID NO:16, or its complement; position 6,073 of SEQ ID NO:17, or its complement; position 6,236 of SEQ ID NO:18, or its complement; position 7,526 of SEQ ID NO:19, or its complement; position 1,336 of SEQ ID NO:20, or its complement; position 361 of SEQ ID NO:21, or its complement; or position 7,776 of SEQ ID NO:22, or its complement. and c) Determine whether the extended product of the primer contains: uracil at position 7,785 according to SEQ ID NO:13, or a complement thereof; Uracil, or its complement, located at position 1,095 according to SEQ ID NO:14; Uracil, or its complement, located at position 6,073 according to SEQ ID NO:15; Uracil, or its complement, located at position 1,336 according to SEQ ID NO:16; Uracil, or its complement, located at position 6,073 according to SEQ ID NO:17; Uracil, or its complement, located at positions 6,236 according to SEQ ID NO:18; Uracil, or its complement, located at position 7,526 according to SEQ ID NO:19; Uracil, or its complement, located at position 1,336 according to SEQ ID NO:20; Uracil, or a complement thereof, located at position 361 according to SEQ ID NO:21; or uracil, or a complement thereof, located at position 7,776 according to SEQ ID NO:

22.

21. The method according to any one of claims 10 to 15, wherein the detection step comprises: a) Contact the biological sample with primers, which hybridize with a portion of the nucleotide sequence of the PIEZO1 cDNA molecule or its complement, said portion being close to the position corresponding to the following positions: position 7,785 of SEQ ID NO:33, or its complement; position 1,095 of SEQ ID NO:34, or its complement; position 6,073 of SEQ ID NO:35, or its complement; position 1,336 of SEQ ID NO:36, or its complement; position 6,073 of SEQ ID NO:37, or its complement; position 6,236 of SEQ ID NO:38, or its complement; position 7,526 of SEQ ID NO:39, or its complement; position 1,336 of SEQ ID NO:40, or its complement; position 361 of SEQ ID NO:41, or its complement; or position 7,776 of SEQ ID NO:42, or its complement. b) Extend the primer at least through the nucleotide sequence of the PIEZO1 cDNA molecule or its complement to the positions corresponding to the following: position 7,785 of SEQ ID NO:33, or its complement; position 1,095 of SEQ ID NO:34, or its complement; position 6,073 of SEQ ID NO:35, or its complement; position 1,336 of SEQ ID NO:36, or its complement; position 6,073 of SEQ ID NO:37, or its complement; position 6,236 of SEQ ID NO:38, or its complement; position 7,526 of SEQ ID NO:39, or its complement; position 1,336 of SEQ ID NO:40, or its complement; position 361 of SEQ ID NO:41, or its complement; or position 7,776 of SEQ ID NO:42, or its complement. and c) Determine whether the extended product of the primer contains: thymine at position 7,785 according to SEQ ID NO:33, or a complement thereof; Thymine, or its complement thereof, is located at position 1,095 according to SEQ ID NO:34; Thymine, or its complement thereof, located at position 6,073 according to SEQ ID NO:35; Thymine, or its complement, is located at positions 1,336 according to SEQ ID NO:36; Thymine, or its complement thereof, is located at position 6,073 according to SEQ ID NO:37; Thymine, or its complement, located at position 6,236 according to SEQ ID NO:38; Thymine, or its complement, located at position 7,526 according to SEQ ID NO:39; Thymine, or its complement, located at position 1,336 according to SEQ ID NO:40; Thymine or its complement at position 361 according to SEQ ID NO:41; or thymine or its complement at position 7,776 according to SEQ ID NO:

42.

22. The method according to any one of claims 16 to 21, wherein the detection step comprises sequencing the entire nucleic acid molecule.

23. The method according to any one of claims 10 to 15, wherein the detection step comprises: a) Amplify at least a portion of the PIEZO1 genomic nucleic acid molecule or its complement in the biological sample, wherein the portion contains thymine or its complement at a position corresponding to position 69,579 according to SEQ ID NO:2; b) Label the amplified nucleic acid molecules with detectable markers; c) Contact the labeled nucleic acid molecule with a support containing a change-specific probe, wherein the change-specific probe contains a nucleotide sequence that hybridizes under stringent conditions to the nucleotide sequence of the amplified nucleic acid molecule, the nucleotide sequence of the amplified nucleic acid molecule containing thymine, or a complement thereof, at a position corresponding to position 69,579 according to SEQ ID NO:

2. and d) Detect the detectable marker.

24. The method according to any one of claims 10 to 15, wherein the detection step comprises: a) Amplify at least a portion of the PIEZO1 mRNA molecule or its complement in the biological sample, wherein the portion comprises: uracil at position 7,785 according to SEQ ID NO:13, or its complement; Uracil, or its complement, located at position 1,095 according to SEQ ID NO:14; Uracil, or its complement, located at position 6,073 according to SEQ ID NO: 15; Uracil, or its complement, located at position 1,336 according to SEQ ID NO:16; Uracil, or its complement, located at position 6,073 according to SEQ ID NO:17; Uracil, or its complement, located at positions 6,236 according to SEQ ID NO:18; Uracil, or its complement, located at position 7,526 according to SEQ ID NO:19; Uracil, or its complement, located at position 1,336 according to SEQ ID NO:20; Uracil at position 361 according to SEQ ID NO:21, or a complement thereof; or uracil at position 7,776 according to SEQ ID NO:22, or a complement thereof; b) Label the amplified nucleic acid molecules with detectable markers; c) Contact the labeled nucleic acid molecule with a support containing a change-specific probe, wherein the change-specific probe comprises a nucleotide sequence that hybridizes under stringent conditions to the nucleotide sequence of the amplified nucleic acid molecule, the nucleotide sequence of the amplified nucleic acid molecule comprising: uracil at position 7,785 according to SEQ ID NO:13, or a complement thereof; and uracil at position 1,095 according to SEQ ID NO:14, or a complement thereof. Uracil, or its complement, located at position 6,073 according to SEQ ID NO:15; Uracil, or its complementary compound, is located at positions 1,336 according to SEQ ID NO: 16; Uracil, or its complement, located at position 6,073 according to SEQ ID NO:17; Uracil, or its complement, located at positions 6,236 according to SEQ ID NO:18; Uracil, or its complement, located at position 7,526 according to SEQ ID NO:19; Uracil, or its complement, located at position 1,336 according to SEQ ID NO:20; Uracil at position 361 according to SEQ ID NO:21, or a complement thereof; or uracil at position 7,776 according to SEQ ID NO:22, or a complement thereof; and d) Detect the detectable marker.

25. The method according to any one of claims 10 to 15, wherein the detection step comprises: a) Amplify at least a portion of the PIEZO1 cDNA molecule or its complement generated from the mRNA molecule in the biological sample, wherein the portion comprises: thymine at a position corresponding to position 7,785 according to SEQ ID NO:33, or its complement; Thymine, or its complement thereof, is located at position 1,095 according to SEQ ID NO:34; Thymine, or its complement thereof, located at position 6,073 according to SEQ ID NO:35; Thymine, or its complement, is located at positions 1,336 according to SEQ ID NO:36; Thymine, or its complement thereof, is located at position 6,073 according to SEQ ID NO:37; Thymine, or its complement, located at position 6,236 according to SEQ ID NO:38; Thymine, or its complement, located at position 7,526 according to SEQ ID NO:39; Thymine, or its complement, located at position 1,336 according to SEQ ID NO:40; Thymine or its complement at position 361 according to SEQ ID NO:41; or thymine or its complement at position 7,776 according to SEQ ID NO:42; b) Label the amplified nucleic acid molecules with detectable markers; c) Contact the labeled nucleic acid molecule with a support containing a change-specific probe, wherein the change-specific probe comprises a nucleotide sequence that hybridizes under stringent conditions to the nucleotide sequence of the amplified nucleic acid molecule, the nucleotide sequence of the amplified nucleic acid molecule comprising: thymine at position 7,785 according to SEQ ID NO:33, or a complement thereof; and thymine at position 1,095 according to SEQ ID NO:34, or a complement thereof. Thymine, or its complement thereof, located at position 6,073 according to SEQ ID NO:35; Thymine, or its complement, is located at positions 1,336 according to SEQ ID NO:36; Thymine, or its complement thereof, is located at position 6,073 according to SEQ ID NO:37; Thymine, or its complement, located at position 6,236 according to SEQ ID NO:38; Thymine, or its complement, located at position 7,526 according to SEQ ID NO:39; Thymine, or its complement, located at position 1,336 according to SEQ ID NO:40; Thymine or its complement at position 361 according to SEQ ID NO:41; or thymine or its complement at position 7,776 according to SEQ ID NO:42; and d) Detect the detectable marker.

26. The method of claim 25, wherein the nucleic acid molecule in the sample is mRNA, and the mRNA is reverse transcribed into cDNA prior to the amplification step.

27. The method according to any one of claims 10 to 15, wherein the detection step comprises: The PIEZO1 genomic nucleic acid molecule or its complement in the biological sample is contacted with a change-specific probe containing a detectable label, wherein the change-specific probe contains a nucleotide sequence that hybridizes under stringent conditions with the nucleotide sequence of the PIEZO1 genomic nucleic acid molecule or its complement, and the nucleotide sequence of the PIEZO1 genomic nucleic acid molecule or its complement contains thymine, or its complement, at position 69,579 according to SEQ ID NO:2; and Detect the detectable marker.

28. The method according to any one of claims 10 to 15, wherein the detection step comprises: The PIEZO1 mRNA molecule or its complement in the biological sample is contacted with a change-specific probe containing a detectable label, wherein the change-specific probe contains a nucleotide sequence that hybridizes under stringent conditions to the nucleotide sequence of the PIEZO1 mRNA molecule or its complement, the nucleotide sequence of the PIEZO1 mRNA molecule or its complement containing: uracil at position 7,785 according to SEQ ID NO:13, or its complement; and uracil at position 1,095 according to SEQ ID NO:14, or its complement. Uracil, or its complement, located at position 6,073 according to SEQ ID NO:15; Uracil, or its complement, located at position 1,336 according to SEQ ID NO:16; Uracil, or its complement, located at position 6,073 according to SEQ ID NO:17; Uracil, or its complement, located at positions 6,236 according to SEQ ID NO:18; Uracil, or its complement, located at position 7,526 according to SEQ ID NO:19; Uracil, or its complement, located at position 1,336 according to SEQ ID NO:20; Uracil at position 361 according to SEQ ID NO:21, or a complement thereof; or uracil at position 7,776 according to SEQ ID NO:22, or a complement thereof; and Detect the detectable marker.

29. The method according to any one of claims 10 to 15, wherein the detection step comprises: The PIEZO1 cDNA molecule or its complement, generated from mRNA molecules in the biological sample, is contacted with a change-specific probe containing a detectable label, wherein the change-specific probe contains a nucleotide sequence that hybridizes under stringent conditions to the nucleotide sequence of the PIEZO1 cDNA molecule or its complement, the nucleotide sequence of the PIEZO1 cDNA molecule or its complement containing: thymine, or its complement, at position 7,785 according to SEQ ID NO:33; and thymine, or its complement, at position 1,095 according to SEQ ID NO:

34. Thymine, or its complement thereof, located at position 6,073 according to SEQ ID NO:35; Thymine, or its complement, is located at positions 1,336 according to SEQ ID NO:36; Thymine, or its complement thereof, is located at position 6,073 according to SEQ ID NO:37; Thymine, or its complement, located at position 6,236 according to SEQ ID NO:38; Thymine, or its complement, located at position 7,526 according to SEQ ID NO:39; Thymine, or its complement, located at position 1,336 according to SEQ ID NO:40; Thymine or its complement at position 361 according to SEQ ID NO:41; or thymine or its complement at position 7,776 according to SEQ ID NO:42; and Detect the detectable marker.

30. A method of treating a subject with a therapeutic agent for treating or preventing varicose veins, wherein the subject has varicose veins or is at risk of developing varicose veins, the method comprising: The PIEZO1 variant nucleic acid molecule was obtained to determine whether the subject possessed the predictive function of the piezoelectric mechanosensitive ion channel component 1 (PIEZO1): Biological samples obtained from or already obtained from the subject; and The biological sample was subjected to or has been subjected to sequence analysis to determine whether the subject has a genotype containing the PIEZO1 variant nucleic acid molecule encoding the PIEZO1 predictive function gain polypeptide; as well as Administering or continuing the treatment or prevention of varicose veins at a standard dose to a subject serving as a PIEZO1 reference type, and / or administering a PIEZO1 agonist to the subject; and Administer or continue the treatment or prevention of varicose veins to subjects who are heterozygous for the PIEZO1 variant nucleic acid molecule at an amount equal to or less than the standard dose, and / or administer a PIEZO1 agonist to the subjects; The presence of a genotype of the PIEZO1 variant nucleic acid molecule that encodes a polypeptide for predicting the PIEZO1 predictive function indicates a reduced risk of the subject developing varicose veins.

31. The method of claim 30, wherein the subject is a PIEZO1 reference type, and the subject is given or continues to be given the therapeutic agent for the treatment or prevention of varicose veins at a standard dose, and a PIEZO1 agonist is administered.

32. The method of claim 30, wherein the subject is heterozygous for the PIEZO1 variant nucleic acid molecule, and the therapeutic agent for the treatment or prevention of varicose veins is administered to or continued to the subject in an amount equal to or less than a standard dose, and a PIEZO1 agonist is administered.

33. The method according to any one of claims 30 to 32, wherein the PIEZO1 variant nucleic acid molecule encodes Pro2,510Leu, Pro2,024Leu, Pro2,079Leu, or Pro92Leu.

34. The method of claim 33, wherein the PIEZO1 variant nucleic acid molecule is: A genomic nucleic acid molecule having a nucleotide sequence containing thymine at positions corresponding to positions 69,579 according to SEQ ID NO:2; An mRNA molecule having the following nucleotide sequences: uracil at position 7,785 according to SEQ ID NO:13, uracil at position 1,095 according to SEQ ID NO:14, uracil at position 6,073 according to SEQ ID NO:15, uracil at position 1,336 according to SEQ ID NO:16, uracil at position 6,073 according to SEQ ID NO:17, uracil at position 6,236 according to SEQ ID NO:18, uracil at position 7,526 according to SEQ ID NO:19, uracil at position 1,336 according to SEQ ID NO:20, uracil at position 361 according to SEQ ID NO:21, or uracil at position 7,776 according to SEQ ID NO:22; or A cDNA molecule generated from an mRNA molecule, wherein the cDNA molecule has a nucleotide sequence comprising: thymine at position 7,785 according to SEQ ID NO:33, thymine at position 1,095 according to SEQ ID NO:34, thymine at position 6,073 according to SEQ ID NO:35, thymine at position 1,336 according to SEQ ID NO:36, thymine at position 6,073 according to SEQ ID NO:37, thymine at position 6,236 according to SEQ ID NO:38, thymine at position 7,526 according to SEQ ID NO:39, thymine at position 1,336 according to SEQ ID NO:40, thymine at position 361 according to SEQ ID NO:41, or thymine at position 7,776 according to SEQ ID NO:

42.

35. The method according to any one of claims 30 to 34, wherein the sequence analysis comprises sequencing at least a portion of the nucleotide sequence of the PIEZO1 genomic nucleic acid molecule or its complement in the biological sample, wherein the sequenced portion comprises a position corresponding to position 69,579 according to SEQ ID NO:2, or its complement; Wherein the sequenced portion of the PIEZO1 genomic nucleic acid molecule or its complement in the biological sample contains thymine or its complement at position 69,579 according to SEQ ID NO:2, then the PIEZO1 genomic nucleic acid molecule in the biological sample is a PIEZO1 variant genomic nucleic acid molecule encoding a PIEZO1 predictive function gain polypeptide.

36. The method according to any one of claims 30 to 34, wherein the sequence analysis comprises sequencing at least a portion of the nucleotide sequence of the PIEZO1 mRNA molecule or its complement in the biological sample, wherein the sequenced portion comprises a position corresponding to: position 7,785 of SEQ ID NO: 13, or its complement; position 1,095 of SEQ ID NO: 14, or its complement; position 6,073 of SEQ ID NO: 15, or its complement; position 1,336 of SEQ ID NO: 16, or its complement; position 6,073 of SEQ ID NO: 17, or its complement; position 6,236 of SEQ ID NO: 18, or its complement; position 7,526 of SEQ ID NO: 19, or its complement; position 1,336 of SEQ ID NO: 20, or its complement; position 361 of SEQ ID NO: 21, or its complement; or position 7,776 of SEQ ID NO: 22, or its complement. Wherein the sequenced portion of the PIEZO1 mRNA molecule in the biological sample comprises: uracil at position 7,785 according to SEQ ID NO: 13, or a complement thereof; and uracil at position 1,095 according to SEQ ID NO: 14, or a complement thereof. Uracil, or its complement, located at position 6,073 according to SEQ ID NO:15; Uracil, or its complement, located at position 1,336 according to SEQ ID NO:16; Uracil, or its complement, located at position 6,073 according to SEQ ID NO:17; Uracil, or its complement, located at positions 6,236 according to SEQ ID NO:18; Uracil, or its complement, located at position 7,526 according to SEQ ID NO:19; Uracil, or its complement, located at position 1,336 according to SEQ ID NO:20; When uracil or its complement is located at position 361 according to SEQ ID NO:21; or when uracil or its complement is located at position 7,776 according to SEQ ID NO:22, the PIEZO1 mRNA molecule in the biological sample is a PIEZO1 variant mRNA molecule encoding a PIEZO1 predictive function gain polypeptide.

37. The method according to any one of claims 30 to 34, wherein the sequence analysis comprises sequencing at least a portion of the nucleotide sequence of the PIEZO1 cDNA molecule or its complement generated from the mRNA molecule in the biological sample, wherein the sequenced portion comprises a position corresponding to: position 7,785 of SEQ ID NO:33, or its complement; position 1,095 of SEQ ID NO:34, or its complement; position 6,073 of SEQ ID NO:35, or its complement; position 1,336 of SEQ ID NO:36, or its complement; position 6,073 of SEQ ID NO:37, or its complement; position 6,236 of SEQ ID NO:38, or its complement; position 7,526 of SEQ ID NO:39, or its complement; position 1,336 of SEQ ID NO:40, or its complement; position 361 of SEQ ID NO:41, or its complement; or position 7,776 of SEQ ID NO:42, or its complement. Wherein the sequenced portion of the PIEZO1 cDNA molecule in the biological sample comprises: thymine or a complement thereof at position 7,785 according to SEQ ID NO:33; and thymine or a complement thereof at position 1,095 according to SEQ ID NO:

34. Thymine, or its complement thereof, located at position 6,073 according to SEQ ID NO:35; Thymine, or its complement, is located at positions 1,336 according to SEQ ID NO:36; Thymine, or its complement thereof, is located at position 6,073 according to SEQ ID NO:37; Thymine, or its complement, located at position 6,236 according to SEQ ID NO:38; Thymine, or its complement, located at position 7,526 according to SEQ ID NO:39; Thymine, or its complement, located at position 1,336 according to SEQ ID NO:40; When thymine or its complement is present at position 361 according to SEQ ID NO:41; or at position 7,776 according to SEQ ID NO:42; then the PIEZO1 cDNA molecule in the biological sample is a PIEZO1 cDNA molecule encoding a PIEZO1 predictive function gain polypeptide.

38. The method according to any one of claims 30 to 34, wherein the sequence analysis comprises: a) Contact the biological sample with primers that hybridize with a portion of the nucleotide sequence of the PIEZO1 genomic nucleic acid molecule or its complement, the portion being close to a position corresponding to position 69,579 of SEQ ID NO:2 or its complement; b) Extend the primer at least through the nucleotide sequence of the PIEZO1 genomic nucleic acid molecule or its complement corresponding to the following positions: according to position 69,579 of SEQ ID NO:2, or its complement; and c) Determine whether the primer extension product contains thymine, or its complement, at the position corresponding to position 69,579 according to SEQ ID NO:

2.

39. The method according to any one of claims 30 to 34, wherein the sequence analysis comprises: a) Contact the biological sample with primers, which hybridize with a portion of the nucleotide sequence of the PIEZO1 mRNA molecule or its complement, said portion being close to the position corresponding to the following positions: position 7,785 of SEQ ID NO:13, or its complement; position 1,095 of SEQ ID NO:14, or its complement; position 6,073 of SEQ ID NO:15, or its complement; position 1,336 of SEQ ID NO:16, or its complement; position 6,073 of SEQ ID NO:17, or its complement; position 6,236 of SEQ ID NO:18, or its complement; position 7,526 of SEQ ID NO:19, or its complement; position 1,336 of SEQ ID NO:20, or its complement; position 361 of SEQ ID NO:21, or its complement; or position 7,776 of SEQ ID NO:22, or its complement. b) Extend the primer at least through the nucleotide sequence of the PIEZO1 mRNA molecule or its complement to the positions corresponding to the following: position 7,785 of SEQ ID NO:13, or its complement; position 1,095 of SEQ ID NO:14, or its complement; position 6,073 of SEQ ID NO:15, or its complement; position 1,336 of SEQ ID NO:16, or its complement; position 6,073 of SEQ ID NO:17, or its complement; position 6,236 of SEQ ID NO:18, or its complement; position 7,526 of SEQ ID NO:19, or its complement; position 1,336 of SEQ ID NO:20, or its complement; position 361 of SEQ ID NO:21, or its complement; or position 7,776 of SEQ ID NO:22, or its complement. and c) Determine whether the extended product of the primer contains: uracil at position 7,785 according to SEQ ID NO:13, or a complement thereof; Uracil, or its complement, located at position 1,095 according to SEQ ID NO:14; Uracil, or its complement, located at position 6,073 according to SEQ ID NO:15; Uracil, or its complement, located at position 1,336 according to SEQ ID NO:16; Uracil, or its complement, located at position 6,073 according to SEQ ID NO:17; Uracil, or its complement, located at positions 6,236 according to SEQ ID NO:18; Uracil, or its complement, located at position 7,526 according to SEQ ID NO:19; Uracil, or its complement, located at position 1,336 according to SEQ ID NO:20; Uracil, or a complement thereof, located at position 361 according to SEQ ID NO:21; or uracil, or a complement thereof, located at position 7,776 according to SEQ ID NO:

22.

40. The method according to any one of claims 30 to 34, wherein the sequence analysis comprises: a) Contact the biological sample with primers, which hybridize with a portion of the nucleotide sequence of the PIEZO1 cDNA molecule or its complement, said portion being close to the position corresponding to the following positions: position 7,785 of SEQ ID NO:33, or its complement; position 1,095 of SEQ ID NO:34, or its complement; position 6,073 of SEQ ID NO:35, or its complement; position 1,336 of SEQ ID NO:36, or its complement; position 6,073 of SEQ ID NO:37, or its complement; position 6,236 of SEQ ID NO:38, or its complement; position 7,526 of SEQ ID NO:39, or its complement; position 1,336 of SEQ ID NO:40, or its complement; position 361 of SEQ ID NO:41, or its complement; or position 7,776 of SEQ ID NO:42, or its complement. b) Extend the primer at least through the nucleotide sequence of the PIEZO1 cDNA molecule or its complement to the positions corresponding to the following: position 7,785 of SEQ ID NO:33, or its complement; position 1,095 of SEQ ID NO:34, or its complement; position 6,073 of SEQ ID NO:35, or its complement; position 1,336 of SEQ ID NO:36, or its complement; position 6,073 of SEQ ID NO:37, or its complement; position 6,236 of SEQ ID NO:38, or its complement; position 7,526 of SEQ ID NO:39, or its complement; position 1,336 of SEQ ID NO:40, or its complement; position 361 of SEQ ID NO:41, or its complement; or position 7,776 of SEQ ID NO:42, or its complement. and c) Determine whether the extended product of the primer contains: thymine at position 7,785 according to SEQ ID NO:33, or a complement thereof; Thymine, or its complement thereof, is located at position 1,095 according to SEQ ID NO:34; Thymine, or its complement thereof, located at position 6,073 according to SEQ ID NO:35; Thymine, or its complement, is located at positions 1,336 according to SEQ ID NO:36; Thymine, or its complement thereof, is located at position 6,073 according to SEQ ID NO:37; Thymine, or its complement, located at position 6,236 according to SEQ ID NO:38; Thymine, or its complement, located at position 7,526 according to SEQ ID NO:39; Thymine, or its complement, located at position 1,336 according to SEQ ID NO:40; Thymine or its complement at position 361 according to SEQ ID NO:41; or thymine or its complement at position 7,776 according to SEQ ID NO:

42.

41. The method according to any one of claims 35 to 40, wherein the sequence analysis comprises sequencing the entire nucleic acid molecule.

42. The method according to any one of claims 30 to 34, wherein the sequence analysis comprises: a) Amplify at least a portion of the PIEZO1 genomic nucleic acid molecule or its complement in the biological sample, wherein the portion contains thymine or its complement at a position corresponding to position 69,579 according to SEQ ID NO:2; b) Label the amplified nucleic acid molecules with detectable markers; c) Contact the labeled nucleic acid molecule with a support containing a change-specific probe, wherein the change-specific probe contains a nucleotide sequence that hybridizes under stringent conditions to the nucleotide sequence of the amplified nucleic acid molecule, the nucleotide sequence of the amplified nucleic acid molecule containing thymine, or a complement thereof, at a position corresponding to position 69,579 according to SEQ ID NO:

2. and d) Detect the detectable marker.

43. The method according to any one of claims 30 to 34, wherein the sequence analysis comprises: a) Amplify at least a portion of the PIEZO1 mRNA molecule or its complement in the biological sample, wherein the portion comprises: uracil at position 7,785 according to SEQ ID NO:13, or its complement; Uracil, or its complement, located at position 1,095 according to SEQ ID NO:14; Uracil, or its complement, located at position 6,073 according to SEQ ID NO: 15; Uracil, or its complement, located at position 1,336 according to SEQ ID NO:16; Uracil, or its complement, located at position 6,073 according to SEQ ID NO:17; Uracil, or its complement, located at positions 6,236 according to SEQ ID NO:18; Uracil, or its complement, located at position 7,526 according to SEQ ID NO:19; Uracil, or its complement, located at position 1,336 according to SEQ ID NO:20; Uracil at position 361 according to SEQ ID NO:21, or a complement thereof; or uracil at position 7,776 according to SEQ ID NO:22, or a complement thereof; b) Label the amplified nucleic acid molecules with detectable markers; c) Contact the labeled nucleic acid molecule with a support containing a change-specific probe, wherein the change-specific probe comprises a nucleotide sequence that hybridizes under stringent conditions to the nucleotide sequence of the amplified nucleic acid molecule, the nucleotide sequence of the amplified nucleic acid molecule comprising: uracil at position 7,785 according to SEQ ID NO:13, or a complement thereof; and uracil at position 1,095 according to SEQ ID NO:14, or a complement thereof. Uracil, or its complement, located at position 6,073 according to SEQ ID NO:15; Uracil, or its complementary compound, is located at positions 1,336 according to SEQ ID NO: 16; Uracil, or its complement, located at position 6,073 according to SEQ ID NO:17; Uracil, or its complement, located at positions 6,236 according to SEQ ID NO:18; Uracil, or its complement, located at position 7,526 according to SEQ ID NO:19; Uracil, or its complement, located at position 1,336 according to SEQ ID NO:20; Uracil at position 361 according to SEQ ID NO:21, or a complement thereof; or uracil at position 7,776 according to SEQ ID NO:22, or a complement thereof; and d) Detect the detectable marker.

44. The method according to any one of claims 30 to 34, wherein the sequence analysis comprises: a) Amplify at least a portion of the PIEZO1 cDNA molecule or its complement generated from the mRNA molecule in the biological sample, wherein the portion comprises: thymine at a position corresponding to position 7,785 according to SEQ ID NO:33, or its complement; Thymine, or its complement thereof, is located at position 1,095 according to SEQ ID NO:34; Thymine, or its complement thereof, located at position 6,073 according to SEQ ID NO:35; Thymine, or its complement, is located at positions 1,336 according to SEQ ID NO:36; Thymine, or its complement thereof, is located at position 6,073 according to SEQ ID NO:37; Thymine, or its complement, located at position 6,236 according to SEQ ID NO:38; Thymine, or its complement, located at position 7,526 according to SEQ ID NO:39; Thymine, or its complement, located at position 1,336 according to SEQ ID NO:40; Thymine or its complement at position 361 according to SEQ ID NO:41; or thymine or its complement at position 7,776 according to SEQ ID NO:42; b) Label the amplified nucleic acid molecules with detectable markers; c) Contact the labeled nucleic acid molecule with a support containing a change-specific probe, wherein the change-specific probe comprises a nucleotide sequence that hybridizes under stringent conditions to the nucleotide sequence of the amplified nucleic acid molecule, the nucleotide sequence of the amplified nucleic acid molecule comprising: thymine at position 7,785 according to SEQ ID NO:33, or a complement thereof; and thymine at position 1,095 according to SEQ ID NO:34, or a complement thereof. Thymine, or its complement thereof, located at position 6,073 according to SEQ ID NO:35; Thymine, or its complement, is located at positions 1,336 according to SEQ ID NO:36; Thymine, or its complement thereof, is located at position 6,073 according to SEQ ID NO:37; Thymine, or its complement, located at position 6,236 according to SEQ ID NO:38; Thymine, or its complement, located at position 7,526 according to SEQ ID NO:39; Thymine, or its complement, located at position 1,336 according to SEQ ID NO:40; Thymine or its complement at position 361 according to SEQ ID NO:41; or thymine or its complement at position 7,776 according to SEQ ID NO:42; and d) Detect the detectable marker.

45. The method of claim 44, wherein the nucleic acid molecule in the sample is mRNA, and the mRNA is reverse transcribed into cDNA prior to the amplification step.

46. ​​The method according to any one of claims 30 to 34, wherein the sequence analysis comprises: The PIEZO1 genomic nucleic acid molecule or its complement in the biological sample is contacted with a change-specific probe containing a detectable label, wherein the change-specific probe contains a nucleotide sequence that hybridizes under stringent conditions with the nucleotide sequence of the PIEZO1 genomic nucleic acid molecule or its complement, and the nucleotide sequence of the PIEZO1 genomic nucleic acid molecule or its complement contains thymine, or its complement, at position 69,579 according to SEQ ID NO:2; and Detect the detectable marker.

47. The method according to any one of claims 30 to 34, wherein the sequence analysis comprises: The PIEZO1 mRNA molecule or its complement in the biological sample is contacted with a change-specific probe containing a detectable label, wherein the change-specific probe contains a nucleotide sequence that hybridizes under stringent conditions to the nucleotide sequence of the PIEZO1 mRNA molecule or its complement, the nucleotide sequence of the PIEZO1 mRNA molecule or its complement containing: uracil at position 7,785 according to SEQ ID NO:13, or its complement; and uracil at position 1,095 according to SEQ ID NO:14, or its complement. Uracil, or its complement, located at position 6,073 according to SEQ ID NO:15; Uracil, or its complement, located at position 1,336 according to SEQ ID NO:16; Uracil, or its complement, located at position 6,073 according to SEQ ID NO:17; Uracil, or its complement, located at positions 6,236 according to SEQ ID NO:18; Uracil, or its complement, located at position 7,526 according to SEQ ID NO:19; Uracil, or its complement, located at position 1,336 according to SEQ ID NO:20; Uracil at position 361 according to SEQ ID NO:21, or a complement thereof; or uracil at position 7,776 according to SEQ ID NO:22, or a complement thereof; and Detect the detectable marker.

48. The method according to any one of claims 30 to 34, wherein the sequence analysis comprises: The PIEZO1 cDNA molecule or its complement, generated from mRNA molecules in the biological sample, is contacted with a change-specific probe containing a detectable label, wherein the change-specific probe contains a nucleotide sequence that hybridizes under stringent conditions to the nucleotide sequence of the PIEZO1 cDNA molecule or its complement, the nucleotide sequence of the PIEZO1 cDNA molecule or its complement containing: thymine, or its complement, at position 7,785 according to SEQ ID NO:33; and thymine, or its complement, at position 1,095 according to SEQ ID NO:

34. Thymine, or its complement thereof, located at position 6,073 according to SEQ ID NO:35; Thymine, or its complement, is located at positions 1,336 according to SEQ ID NO:36; Thymine, or its complement thereof, is located at position 6,073 according to SEQ ID NO:37; Thymine, or its complement, located at position 6,236 according to SEQ ID NO:38; Thymine, or its complement, located at position 7,526 according to SEQ ID NO:39; Thymine, or its complement, located at position 1,336 according to SEQ ID NO:40; Thymine or its complement at position 361 according to SEQ ID NO:41; or thymine or its complement at position 7,776 according to SEQ ID NO:42; and Detect the detectable marker.

49. The method according to any one of claims 30 to 48, wherein the nucleic acid molecule is present in cells obtained from the subject.

50. The method according to any one of claims 30 to 49, wherein the PIEZO1 agonist comprises a small molecule.

51. The method of claim 50, wherein the small molecule agonist is Yoda1.

52. The method of claim 50, wherein the small molecule agonist is Jedi1.

53. The method of claim 50, wherein the small molecule agonist is Jedi2.

54. A method for identifying subjects at increased risk of developing varicose veins, the method comprising: The presence or absence of a PIEZO1 variant nucleic acid molecule encoding a peptide for predicting piezoelectric mechanosensitive ion channel component 1 (PIEZO1) was determined or has been determined in biological samples obtained from the subject; in: When the subject is a PIEZO1 reference type, the subject has an increased risk of developing varicose veins; and The risk of developing varicose veins is reduced when the subject is heterozygous or homozygous for the PIEZO1 variant nucleic acid molecule encoding the PIEZO1 predictive function gain peptide.

55. The method of claim 54, wherein the PIEZO1 variant nucleic acid molecule encodes Pro2,510Leu, Pro2,024Leu, Pro2,079Leu, or Pro92Leu.

56. The method of claim 55, wherein the PIEZO1 variant nucleic acid molecule is: A genomic nucleic acid molecule having a nucleotide sequence containing thymine at positions corresponding to positions 69,579 according to SEQ ID NO:2; An mRNA molecule having the following nucleotide sequences: uracil at position 7,785 according to SEQ ID NO:13; uracil at position 1,095 according to SEQ ID NO:14; and uracil at position 6,073 according to SEQ ID NO:

15. Uracil at positions 1,336 according to SEQ ID NO:16; Uracil at position 6,073 according to SEQ ID NO:17; uracil at position 6,236 according to SEQ ID NO:18; uracil at position 7,526 according to SEQ ID NO:19; and uracil at position 1,336 according to SEQ ID NO:

20. Uracil at position 361 according to SEQ ID NO: 21; or uracil at position 7,776 according to SEQ ID NO: 22; or A cDNA molecule generated from an mRNA molecule, wherein the cDNA molecule has a nucleotide sequence comprising: thymine at position 7,785 according to SEQ ID NO:33; thymine at position 1,095 according to SEQ ID NO:34; thymine at position 6,073 according to SEQ ID NO:35; thymine at position 1,336 according to SEQ ID NO:36; thymine at position 6,073 according to SEQ ID NO:37; thymine at position 6,236 according to SEQ ID NO:38; thymine at position 7,526 according to SEQ ID NO:39; thymine at position 1,336 according to SEQ ID NO:40; thymine at position 361 according to SEQ ID NO:41; or thymine at position 7,776 according to SEQ ID NO:

42.

57. The method according to any one of claims 54 to 56, wherein the determining step is performed in vitro.

58. The method according to any one of claims 54 to 57, wherein the determining step comprises sequencing at least a portion of the nucleotide sequence of the PIEZO1 genomic nucleic acid molecule or its complement in the biological sample, wherein the sequenced portion comprises a position corresponding to position 69,579 according to SEQ ID NO:2, or its complement; Where the sequenced portion of the PIEZO1 genomic nucleic acid molecule in the biological sample contains thymine or its complement at position 69,579 according to SEQ ID NO:2, then the PIEZO1 genomic nucleic acid molecule in the biological sample is a PIEZO1 variant genomic nucleic acid molecule encoding a PIEZO1 predictive function gain polypeptide.

59. The method according to any one of claims 54 to 57, wherein the determining step comprises sequencing at least a portion of the nucleotide sequence of the PIEZO1 mRNA molecule in the biological sample, wherein the sequenced portion comprises a position corresponding to: position 7,785 of SEQ ID NO: 13, or its complement; position 1,095 of SEQ ID NO: 14, or its complement; position 6,073 of SEQ ID NO: 15, or its complement; position 1,336 of SEQ ID NO: 16, or its complement; position 6,073 of SEQ ID NO: 17, or its complement; position 6,236 of SEQ ID NO: 18, or its complement; position 7,526 of SEQ ID NO: 19, or its complement; position 1,336 of SEQ ID NO: 20, or its complement; position 361 of SEQ ID NO: 21, or its complement; or position 7,776 of SEQ ID NO: 22, or its complement. Wherein the sequenced portion of the PIEZO1 mRNA molecule in the biological sample comprises: uracil at position 7,785 according to SEQ ID NO: 13, or a complement thereof; and uracil at position 1,095 according to SEQ ID NO: 14, or a complement thereof. Uracil, or its complement, located at position 6,073 according to SEQ ID NO:15; Uracil, or its complement, located at position 1,336 according to SEQ ID NO:16; Uracil, or its complement, located at position 6,073 according to SEQ ID NO:17; Uracil, or its complement, located at positions 6,236 according to SEQ ID NO:18; Uracil, or its complement, located at position 7,526 according to SEQ ID NO:19; Uracil, or its complement, located at position 1,336 according to SEQ ID NO:20; When uracil or its complement is located at position 361 according to SEQ ID NO:21; or when uracil or its complement is located at position 7,776 according to SEQ ID NO:22, the PIEZO1 mRNA molecule in the biological sample is a PIEZO1 variant mRNA molecule encoding a PIEZO1 predictive function gain polypeptide.

60. The method according to any one of claims 54 to 57, wherein the determining step comprises sequencing at least a portion of the nucleotide sequence of the PIEZO1 cDNA molecule generated from the mRNA molecule in the biological sample, wherein the sequenced portion comprises a position corresponding to: position 7,785 of SEQ ID NO:33, or its complement; position 1,095 of SEQ ID NO:34, or its complement; position 6,073 of SEQ ID NO:35, or its complement; position 1,336 of SEQ ID NO:36, or its complement; position 6,073 of SEQ ID NO:37, or its complement; position 6,236 of SEQ ID NO:38, or its complement; position 7,526 of SEQ ID NO:39, or its complement; position 1,336 of SEQ ID NO:40, or its complement; position 361 of SEQ ID NO:41, or its complement; or position 7,776 of SEQ ID NO:42, or its complement. Wherein the sequenced portion of the PIEZO1 cDNA molecule in the biological sample comprises: thymine or a complement thereof at position 7,785 according to SEQ ID NO:33; and thymine or a complement thereof at position 1,095 according to SEQ ID NO:

34. Thymine, or its complement thereof, located at position 6,073 according to SEQ ID NO:35; Thymine, or its complement, is located at positions 1,336 according to SEQ ID NO:36; Thymine, or its complement thereof, is located at position 6,073 according to SEQ ID NO:37; Thymine, or its complement, located at position 6,236 according to SEQ ID NO:38; Thymine, or its complement, located at position 7,526 according to SEQ ID NO:39; Thymine, or its complement, located at position 1,336 according to SEQ ID NO:40; When thymine or its complement is present at position 361 according to SEQ ID NO:41; or at position 7,776 according to SEQ ID NO:42; the PIEZO1 cDNA molecule generated from the mRNA molecule in the biological sample is a PIEZO1 variant cDNA molecule encoding a PIEZO1 predictive function gain polypeptide.

61. The method according to any one of claims 54 to 57, wherein the determining step comprises: a) Contact the biological sample with primers that hybridize with a portion of the nucleotide sequence of the PIEZO1 genomic nucleic acid molecule or its complement, the portion being close to a position corresponding to position 69,579 of SEQ ID NO:2 or its complement; b) Extend the primer at least through the nucleotide sequence of the PIEZO1 genomic nucleic acid molecule or its complement corresponding to the following positions: according to position 69,579 of SEQ ID NO:2, or its complement; and c) Determine whether the primer extension product contains thymine, or its complement, at the position corresponding to position 69,579 according to SEQ ID NO:

2.

62. The method according to any one of claims 54 to 57, wherein the determining step comprises: a) Contact the biological sample with primers, which hybridize with a portion of the nucleotide sequence of the PIEZO1 mRNA molecule or its complement, said portion being close to the position corresponding to the following positions: position 7,785 of SEQ ID NO:13, or its complement; position 1,095 of SEQ ID NO:14, or its complement; position 6,073 of SEQ ID NO:15, or its complement; position 1,336 of SEQ ID NO:16, or its complement; position 6,073 of SEQ ID NO:17, or its complement; position 6,236 of SEQ ID NO:18, or its complement; position 7,526 of SEQ ID NO:19, or its complement; position 1,336 of SEQ ID NO:20, or its complement; position 361 of SEQ ID NO:21, or its complement; or position 7,776 of SEQ ID NO:22, or its complement. b) Extend the primer at least through the nucleotide sequence of the PIEZO1 mRNA molecule or its complement to the positions corresponding to the following: position 7,785 of SEQ ID NO:13, or its complement; position 1,095 of SEQ ID NO:14, or its complement; position 6,073 of SEQ ID NO:15, or its complement; position 1,336 of SEQ ID NO:16, or its complement; position 6,073 of SEQ ID NO:17, or its complement; position 6,236 of SEQ ID NO:18, or its complement; position 7,526 of SEQ ID NO:19, or its complement; position 1,336 of SEQ ID NO:20, or its complement; position 361 of SEQ ID NO:21, or its complement; or position 7,776 of SEQ ID NO:22, or its complement. and c) Determine whether the extended product of the primer contains: uracil at position 7,785 according to SEQ ID NO:13, or a complement thereof; Uracil, or its complement, located at position 1,095 according to SEQ ID NO:14; Uracil, or its complement, located at position 6,073 according to SEQ ID NO:15; Uracil, or its complement, located at position 1,336 according to SEQ ID NO:16; Uracil, or its complement, located at position 6,073 according to SEQ ID NO:17; Uracil, or its complement, located at positions 6,236 according to SEQ ID NO:18; Uracil, or its complement, located at position 7,526 according to SEQ ID NO:19; Uracil, or its complement, located at position 1,336 according to SEQ ID NO:20; Uracil, or a complement thereof, located at position 361 according to SEQ ID NO: 21; or uracil, or a complement thereof, located at position 7,776 according to SEQ ID NO:

22.

63. The method according to any one of claims 54 to 57, wherein the determining step comprises: a) Contact the biological sample with primers, which hybridize with a portion of the nucleotide sequence of the PIEZO1 cDNA molecule or its complement, said portion being close to the position corresponding to the following positions: position 7,785 of SEQ ID NO:33, or its complement; position 1,095 of SEQ ID NO:34, or its complement; position 6,073 of SEQ ID NO:35, or its complement; position 1,336 of SEQ ID NO:36, or its complement; position 6,073 of SEQ ID NO:37, or its complement; position 6,236 of SEQ ID NO:38, or its complement; position 7,526 of SEQ ID NO:39, or its complement; position 1,336 of SEQ ID NO:40, or its complement; position 361 of SEQ ID NO:41, or its complement; or position 7,776 of SEQ ID NO:42, or its complement. b) Extend the primer at least through the nucleotide sequence of the PIEZO1 cDNA molecule or its complement to the positions corresponding to the following: position 7,785 of SEQ ID NO:33, or its complement; position 1,095 of SEQ ID NO:34, or its complement; position 6,073 of SEQ ID NO:35, or its complement; position 1,336 of SEQ ID NO:36, or its complement; position 6,073 of SEQ ID NO:37, or its complement; position 6,236 of SEQ ID NO:38, or its complement; position 7,526 of SEQ ID NO:39, or its complement; position 1,336 of SEQ ID NO:40, or its complement; position 361 of SEQ ID NO:41, or its complement; or position 7,776 of SEQ ID NO:42, or its complement. and c) Determine whether the extended product of the primer contains: thymine at position 7,785 according to SEQ ID NO:33, or a complement thereof; Thymine, or its complement thereof, is located at position 1,095 according to SEQ ID NO:34; Thymine, or its complement thereof, located at position 6,073 according to SEQ ID NO:35; Thymine, or its complement, is located at positions 1,336 according to SEQ ID NO:36; Thymine, or its complement thereof, is located at position 6,073 according to SEQ ID NO:37; Thymine, or its complement, located at position 6,236 according to SEQ ID NO:38; Thymine, or its complement, located at position 7,526 according to SEQ ID NO:39; Thymine, or its complement, located at position 1,336 according to SEQ ID NO:40; Thymine or its complement at position 361 according to SEQ ID NO:41; or thymine or its complement at position 7,776 according to SEQ ID NO:

42.

64. The method according to any one of claims 58 to 63, wherein the determining step comprises sequencing the entire nucleic acid molecule.

65. The method according to any one of claims 54 to 57, wherein the determining step comprises: a) Amplify at least a portion of the PIEZO1 genomic nucleic acid molecule or its complement in the biological sample, wherein the portion contains thymine or its complement at a position corresponding to position 69,579 according to SEQ ID NO:2; b) Label the amplified nucleic acid molecules with detectable markers; c) Contact the labeled nucleic acid molecule with a support containing a change-specific probe, wherein the change-specific probe contains a nucleotide sequence that hybridizes under stringent conditions to the nucleotide sequence of the amplified nucleic acid molecule, the nucleotide sequence of the amplified nucleic acid molecule containing thymine, or a complement thereof, at a position corresponding to position 69,579 according to SEQ ID NO:

2. and d) Detect the detectable marker.

66. The method according to any one of claims 54 to 57, wherein the determining step comprises: a) Amplify at least a portion of the PIEZO1 mRNA molecule or its complement in the biological sample, wherein the portion comprises: uracil at position 7,785 according to SEQ ID NO:13, or its complement; Uracil, or its complement, located at position 1,095 according to SEQ ID NO:14; Uracil, or its complement, located at position 6,073 according to SEQ ID NO: 15; Uracil, or its complement, located at position 1,336 according to SEQ ID NO:16; Uracil, or its complement, located at position 6,073 according to SEQ ID NO:17; Uracil, or its complement, located at positions 6,236 according to SEQ ID NO:18; Uracil, or its complement, located at position 7,526 according to SEQ ID NO:19; Uracil, or its complement, located at position 1,336 according to SEQ ID NO:20; Uracil at position 361 according to SEQ ID NO:21, or a complement thereof; or uracil at position 7,776 according to SEQ ID NO:22, or a complement thereof; b) Label the amplified nucleic acid molecules with detectable markers; c) Contact the labeled nucleic acid molecule with a support containing a change-specific probe, wherein the change-specific probe comprises a nucleotide sequence that hybridizes under stringent conditions to the nucleotide sequence of the amplified nucleic acid molecule, the nucleotide sequence of the amplified nucleic acid molecule comprising: uracil at position 7,785 according to SEQ ID NO:13, or a complement thereof; and uracil at position 1,095 according to SEQ ID NO:14, or a complement thereof. Uracil, or its complement, located at position 6,073 according to SEQ ID NO:15; Uracil, or its complementary compound, is located at positions 1,336 according to SEQ ID NO: 16; Uracil, or its complement, located at position 6,073 according to SEQ ID NO:17; Uracil, or its complement, located at positions 6,236 according to SEQ ID NO:18; Uracil, or its complement, located at position 7,526 according to SEQ ID NO:19; Uracil, or its complement, located at position 1,336 according to SEQ ID NO:20; Uracil at position 361 according to SEQ ID NO:21, or a complement thereof; or uracil at position 7,776 according to SEQ ID NO:22, or a complement thereof; and d) Detect the detectable marker.

67. The method according to any one of claims 54 to 57, wherein the determining step comprises: a) Amplify at least a portion of the PIEZO1 cDNA molecule or its complement generated from the mRNA molecule in the biological sample, wherein the portion comprises: thymine at a position corresponding to position 7,785 according to SEQ ID NO:33, or its complement; Thymine, or its complement thereof, is located at position 1,095 according to SEQ ID NO:34; Thymine, or its complement thereof, located at position 6,073 according to SEQ ID NO:35; Thymine, or its complement, is located at positions 1,336 according to SEQ ID NO:36; Thymine, or its complement thereof, is located at position 6,073 according to SEQ ID NO:37; Thymine, or its complement, located at position 6,236 according to SEQ ID NO:38; Thymine, or its complement, located at position 7,526 according to SEQ ID NO:39; Thymine, or its complement, located at position 1,336 according to SEQ ID NO:40; Thymine or its complement at position 361 according to SEQ ID NO:41; or thymine or its complement at position 7,776 according to SEQ ID NO:42; b) Label the amplified nucleic acid molecules with detectable markers; c) Contact the labeled nucleic acid molecule with a support containing a change-specific probe, wherein the change-specific probe comprises a nucleotide sequence that hybridizes under stringent conditions to the nucleotide sequence of the amplified nucleic acid molecule, the nucleotide sequence of the amplified nucleic acid molecule comprising: thymine at position 7,785 according to SEQ ID NO:33, or a complement thereof; and thymine at position 1,095 according to SEQ ID NO:34, or a complement thereof. Thymine, or its complement thereof, located at position 6,073 according to SEQ ID NO:35; Thymine, or its complement, is located at positions 1,336 according to SEQ ID NO:36; Thymine, or its complement thereof, is located at position 6,073 according to SEQ ID NO:37; Thymine, or its complement, located at position 6,236 according to SEQ ID NO:38; Thymine, or its complement, located at position 7,526 according to SEQ ID NO:39; Thymine, or its complement, located at position 1,336 according to SEQ ID NO:40; Thymine or its complement at position 361 according to SEQ ID NO:41; or thymine or its complement at position 7,776 according to SEQ ID NO:42; and d) Detect the detectable marker.

68. The method of claim 67, wherein the nucleic acid molecule in the sample is mRNA, and the mRNA is reverse transcribed into cDNA prior to the amplification step.

69. The method according to any one of claims 54 to 57, wherein the determining step comprises: The PIEZO1 genomic nucleic acid molecule or its complement in the biological sample is contacted with a change-specific probe containing a detectable label, wherein the change-specific probe contains a nucleotide sequence that hybridizes under stringent conditions with the nucleotide sequence of the PIEZO1 genomic nucleic acid molecule or its complement, and the nucleotide sequence of the PIEZO1 genomic nucleic acid molecule or its complement contains thymine, or its complement, at position 69,579 according to SEQ ID NO:2; and Detect the detectable marker.

70. The method according to any one of claims 54 to 57, wherein the determining step comprises: The PIEZO1 mRNA molecule or its complement in the biological sample is contacted with a change-specific probe containing a detectable label, wherein the change-specific probe contains a nucleotide sequence that hybridizes under stringent conditions to the nucleotide sequence of the PIEZO1 mRNA molecule or its complement, the nucleotide sequence of the PIEZO1 mRNA molecule or its complement containing: uracil at position 7,785 according to SEQ ID NO:13, or its complement; and uracil at position 1,095 according to SEQ ID NO:14, or its complement. Uracil, or its complement, located at position 6,073 according to SEQ ID NO:15; Uracil, or its complement, located at position 1,336 according to SEQ ID NO:16; Uracil, or its complement, located at position 6,073 according to SEQ ID NO:17; Uracil, or its complement, located at positions 6,236 according to SEQ ID NO:18; Uracil, or its complement, located at position 7,526 according to SEQ ID NO:19; Uracil, or its complement, located at position 1,336 according to SEQ ID NO:20; Uracil at position 361 according to SEQ ID NO:21, or a complement thereof; or uracil at position 7,776 according to SEQ ID NO:22, or a complement thereof; and Detect the detectable marker.

71. The method according to any one of claims 54 to 57, wherein the determining step comprises: The PIEZO1 cDNA molecule or its complement, generated from mRNA molecules in the biological sample, is contacted with a change-specific probe containing a detectable label, wherein the change-specific probe contains a nucleotide sequence that hybridizes under stringent conditions to the nucleotide sequence of the PIEZO1 cDNA molecule or its complement, the nucleotide sequence of the PIEZO1 cDNA molecule or its complement containing: thymine, or its complement, at position 7,785 according to SEQ ID NO:33; and thymine, or its complement, at position 1,095 according to SEQ ID NO:

34. Thymine, or its complement thereof, located at position 6,073 according to SEQ ID NO:35; Thymine, or its complement, is located at positions 1,336 according to SEQ ID NO:36; Thymine, or its complement thereof, is located at position 6,073 according to SEQ ID NO:37; Thymine, or its complement, located at position 6,236 according to SEQ ID NO:38; Thymine, or its complement, located at position 7,526 according to SEQ ID NO:39; Thymine, or its complement, located at position 1,336 according to SEQ ID NO:40; Thymine or its complement at position 361 according to SEQ ID NO:41; or thymine or its complement at position 7,776 according to SEQ ID NO:42; and Detect the detectable marker.

72. The method according to any one of claims 54 to 71, wherein the subject is a PIEZO1 reference type, and the method further comprises administering a therapeutic agent for treating or preventing varicose veins to the subject at a standard dose, and administering a PIEZO1 agonist to the subject.

73. The method according to any one of claims 54 to 71, wherein the subject is heterozygous for the PIEZO1 variant nucleic acid molecule, and the method further comprises administering to the subject a therapeutic agent for treating or preventing varicose veins in an amount equal to or less than a standard dose, and administering to the subject a PIEZO1 agonist.

74. A therapeutic agent for treating or preventing varicose veins in a subject, the subject being identified as having: A genomic nucleic acid molecule or its complement thereof encoding a piezoelectric mechanically sensitive ion channel component 1 (PIEZO1) predictive function to obtain a polypeptide, wherein the genomic nucleic acid molecule has a nucleotide sequence containing a thymine or its complement thereof at position 69,579 according to SEQ ID NO:

2. mRNA molecules or their complements that encode the PIEZO1 prediction function to obtain polypeptides, wherein the mRNA molecules have a nucleotide sequence comprising: uracil at position 7,785 according to SEQ ID NO:13, or its complement. Uracil, or its complement, located at position 1,095 according to SEQ ID NO:14; Uracil, or its complement, located at position 6,073 according to SEQ ID NO:15; Uracil, or its complement, located at position 1,336 according to SEQ ID NO:16; Uracil, or its complement, located at position 6,073 according to SEQ ID NO:17; Uracil, or its complement, located at positions 6,236 according to SEQ ID NO:18; Uracil, or its complement, located at position 7,526 according to SEQ ID NO:19; Uracil, or its complement, located at position 1,336 according to SEQ ID NO:20; Uracil at position 361 according to SEQ ID NO:21, or a complement thereof; or uracil at position 7,776 according to SEQ ID NO:22, or a complement thereof; or cDNA molecules or their complements that encode the PIEZO1 prediction function to obtain a polypeptide, wherein the cDNA molecule has a nucleotide sequence comprising: thymine at position 7,785 according to SEQ ID NO:33, or its complement. Thymine, or its complement thereof, is located at position 1,095 according to SEQ ID NO:34; Thymine, or its complement thereof, located at position 6,073 according to SEQ ID NO:35; Thymine, or its complement, is located at positions 1,336 according to SEQ ID NO:36; Thymine, or its complement thereof, is located at position 6,073 according to SEQ ID NO:37; Thymine, or its complement, located at position 6,236 according to SEQ ID NO:38; Thymine, or its complement, located at position 7,526 according to SEQ ID NO:39; Thymine, or its complement, located at position 1,336 according to SEQ ID NO:40; Thymine or its complement at position 361 according to SEQ ID NO:41; or thymine or its complement at position 7,776 according to SEQ ID NO:

42.

75. A piezoelectric mechanosensitive ion channel component 1 (PIEZO1) agonist for use in the treatment or prevention of varicose veins in a subject, wherein the subject: a) is a reference type for the PIEZO1 genomic nucleic acid molecule, PIEZO1 mRNA molecule, or PIEZO1 cDNA molecule; or b) For the following, it is heterogeneous: i) A genomic nucleic acid molecule or its complement encoding a polypeptide for predicting PIEZO1, wherein the genomic nucleic acid molecule has a nucleotide sequence comprising: thymine at a position corresponding to position 69,579 according to SEQ ID NO:2, or its complement. ii) an mRNA molecule or its complement encoding a PIEZO1 predicted loss-of-function polypeptide, wherein the mRNA molecule has a nucleotide sequence comprising uracil at position 7,785 according to SEQ ID NO:13, or its complement. Uracil, or its complement, located at position 1,095 according to SEQ ID NO:14; Uracil, or its complement, located at position 6,073 according to SEQ ID NO:15; Uracil, or its complement, located at position 1,336 according to SEQ ID NO:16; Uracil, or its complement, located at position 6,073 according to SEQ ID NO:17; Uracil, or its complement, located at positions 6,236 according to SEQ ID NO:18; Uracil at position 7,526 according to SEQ ID NO:19; uracil at position 361 according to SEQ ID NO:21, or a complement thereof; Uracil, or its complement or a complement thereof, located at position 361 according to SEQ ID NO:21; or uracil, or its complement thereof, located at positions 7,776 according to SEQ ID NO:22; or iii) a cDNA molecule or its complement encoding a peptide for predicting PIEZO1, wherein the cDNA molecule has a nucleotide sequence comprising thymine at position 7,785 according to SEQ ID NO:33, or its complement. Thymine, or its complement thereof, is located at position 1,095 according to SEQ ID NO:34; Thymine, or its complement thereof, located at position 6,073 according to SEQ ID NO:35; Thymine, or its complement, is located at positions 1,336 according to SEQ ID NO:36; Thymine, or its complement thereof, is located at position 6,073 according to SEQ ID NO:37; Thymine, or its complement, located at position 6,236 according to SEQ ID NO:38; Thymine, or its complement, located at position 7,526 according to SEQ ID NO:39; Thymine, or its complement, located at position 1,336 according to SEQ ID NO:40; Thymine or its complement at position 361 according to SEQ ID NO:41; or thymine or its complement at position 7,776 according to SEQ ID NO:

42.

76. The PIEZO1 agonist according to claim 75, wherein it is a small molecule agonist.

77. The PIEZO1 agonist according to claim 76, wherein the small molecule agonist is Yoda1.

78. The PIEZO1 agonist according to claim 76, wherein the small molecule agonist is Jedi1.

79. The PIEZO1 agonist according to claim 76, wherein the small molecule agonist is Jedi2.