Treatment of varicose veins with piezoelectric mechanosensitive ion channel component 1 (PIEZO1) agonists

Abstract

The present disclosure provides methods of treating a subject having or at risk of developing varicose veins, methods of identifying a subject having an increased risk of developing varicose veins, and methods of detecting Piezo-type mechanosensitive ion channel component 1 (PIEZO1) variant nucleic acid molecules and variant polypeptides.

Description

[Technical field]

[0001] Reference to sequence listing This application contains a Sequence Listing that has been submitted electronically as a text file entitled 18923808402SEQ, created on July 2, 2022, having a size of 542 kilobytes. The Sequence Listing is incorporated herein by reference.

[0002] The present disclosure relates generally to treatment of subjects having varicose veins or at risk of developing varicose veins with piezoelectric mechanosensitive ion channel component 1 (PIEZO1) agonists, and methods of identifying subjects at increased risk of developing varicose veins. [Background technology]

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

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

[0005] The present disclosure provides a method of treating a subject having or at risk of developing varicose veins, the method comprising administering to the subject a PIEZO1 agonist. The present disclosure also provides a method of treating a subject having or at risk of developing saphenous varicose veins, the method comprising administering to the subject a PIEZO1 agonist.

[0006] The present disclosure also provides a method of treating a subject having or at risk of developing retinal varices, the method comprising administering to the subject a PIEZO1 agonist.

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

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

[0009] The disclosure also provides a method of treating a subject with a therapeutic agent for treating or preventing varicose veins, the subject having varicose veins or at risk of developing varicose veins, the method comprising determining whether the subject has a PIEZO1 variant nucleic acid molecule encoding a predicted gain-of-function polypeptide of PIEZO1 by obtaining or obtaining a biological sample from the subject; and performing or performing a sequence analysis on the biological sample to determine whether the subject has a genotype that includes a PIEZO1 variant nucleic acid molecule encoding a predicted gain-of-function polypeptide of PIEZO1; and determining whether the subject has a genotype that includes a PIEZO1 variant nucleic acid molecule encoding a predicted gain-of-function polypeptide of PIEZO1; and administering or continuing administration of a therapeutic agent for treating or preventing varicose veins at a standard dosage to a subject, and / or administering a PIEZO1 agonist to the subject; and administering or continuing administration of a therapeutic agent for treating or preventing varicose veins at the same or a lower standard dosage to a subject who is heterozygous for a PIEZO1 variant nucleic acid molecule, and / or administering a PIEZO1 agonist to the subject, wherein the presence of a genotype having a PIEZO1 variant nucleic acid molecule encoding a predicted gain-of-function polypeptide of PIEZO1 indicates that the subject has a reduced risk of developing varicose veins.

[0010] 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 predicted gain-of-function polypeptide of PIEZO1 in a biological sample obtained from the subject; if the subject is a PIEZO1 reference, the subject has an increased risk of developing varicose veins; if the subject is heterozygous or homozygous for a PIEZO1 variant nucleic acid molecule encoding a predicted gain-of-function polypeptide of PIEZO1, the subject has a decreased risk of developing varicose veins.

[0011] The present disclosure also provides a therapeutic agent for treating or preventing varicose veins, comprising: i) a genomic nucleic acid molecule or a complement thereof encoding a predicted gain-of-function polypeptide of PIEZO1, the genomic nucleic acid molecule having a nucleotide sequence including a thymine at a position corresponding to position 69,579 according to SEQ ID NO:2 or a complement thereof; or ii) an mRNA molecule or a complement thereof encoding a predicted gain-of-function polypeptide of PIEZO1, the mRNA molecule having a nucleotide sequence including a thymine at a position corresponding to position 7,785 according to SEQ ID NO:13 or a complement thereof. uracil at a position corresponding to position 1,095 according to SEQ ID NO:14 or its complement; uracil at a position corresponding to position 6,073 according to SEQ ID NO:15 or its complement; uracil at a position corresponding to position 1,336 according to SEQ ID NO:16 or its complement; uracil at a position corresponding to position 6,073 according to SEQ ID NO:17 or its complement; uracil at a position corresponding to position 6,236 according to SEQ ID NO:18 or its complement; 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 predicted gain-of-function polypeptide of PIEZO1, wherein the cDNA molecule has a nucleotide sequence comprising: a thymine at the corresponding position; a thymine at the position corresponding to position 1,095 according to SEQ ID NO:34 or its complement; a thymine at the position corresponding to position 6,073 according to SEQ ID NO:35 or its complement; a thymine at the position corresponding to position 1,336 according to SEQ ID NO:36 or its complement; a thymine at the position corresponding to position 6,073 according to SEQ ID NO:37 or its complement; a thymine at the position corresponding to position 6,236 according to SEQ ID NO:38 or its complement; a thymine at the position corresponding to position 7,526 according to SEQ ID NO:39 or its complement;Provided is a therapeutic agent for use in treating or preventing varicose veins in a subject identified as having a cDNA molecule, or a complement thereof, having a nucleotide sequence including 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;

[0012] The present disclosure also provides a PIEZO1 agonist, comprising: a) a PIEZO1 genomic nucleic acid molecule, a PIEZO1 mRNA molecule, or a PIEZO1 a subject which is a reference for the cDNA molecule; or b) a subject which is heterozygous for: i) a genomic nucleic acid molecule or its complement encoding a predicted gain-of-function polypeptide of PIEZO1, the genomic nucleic acid molecule having a nucleotide sequence that includes 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 predicted gain-of-function polypeptide of PIEZO1, the mRNA molecule having 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 predicted gain-of-function polypeptide of PIEZO1, wherein the cDNA molecule comprises 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;Provided is a PIEZO1 agonist for use in treating or preventing varicose veins in a subject who is heterozygous for a cDNA molecule having a nucleotide sequence comprising: 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. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0013] Various terms relating to aspects of the present disclosure are used throughout the specification and claims. Such terms are to be given their ordinary meaning in the art unless otherwise indicated. Other specifically defined terms are to be interpreted in a manner consistent with the definitions provided herein.

[0014] Unless expressly stated otherwise, any method or embodiment described herein is not intended to be construed as requiring that its steps be performed in a particular order. Thus, unless a method claim specifically states in the claims or specification that the steps are to be limited to a particular order, no order is intended to be implied in any respect. This also applies to any possible unstated criteria for interpretation, including questions of logic regarding the arrangement of steps or operational flow, the plain meaning derived from grammatical construction or punctuation, or the number or type of embodiments described herein.

[0015] As used herein, the singular forms "a," "an," and "the" include plural referents unless the context clearly indicates otherwise. As used herein, the term "about" means that the numerical value described is approximate and that small variations will not significantly affect the practice of the disclosed embodiments. When numerical values ​​are used, unless otherwise indicated by the context, the term "about" means the numerical value may vary by ±10% and remain within the scope of the disclosed embodiments.

[0016] As used herein, the term "comprising" may in certain embodiments be optionally replaced with "consisting of" or "consisting essentially of." As used herein, the term "isolated" in reference to a nucleic acid molecule or polypeptide means that the nucleic acid molecule or polypeptide is in a state other than its native environment, e.g., apart from blood and / or other tissues. In some embodiments, an isolated nucleic acid molecule or polypeptide is substantially free of other nucleic acid molecules or other polypeptides, particularly other nucleic acid molecules or polypeptides of animal origin. In some embodiments, the nucleic acid molecule or polypeptide may be in a highly purified form, i.e., more than 95% pure or more than 99% pure. When used in this context, the term "isolated" does not exclude the presence of the same nucleic acid molecule or polypeptide in alternative physical forms, such as dimers or alternatively phosphorylated or derivatized forms.

[0017] As used herein, the terms "nucleic acid," "nucleic acid molecule," "nucleic acid sequence," "polynucleotide," or "oligonucleotide" can include a polymeric form of nucleotides of any length, can include DNA and / or RNA, and can be single-stranded, double-stranded, or multistranded. A strand of a nucleic acid also refers to its complement.

[0018] As used herein, the term "subject" includes any animal, including mammals. Mammals include, but are not limited to, farm animals (e.g., horses, cows, pigs, etc.), companion animals (e.g., dogs, cats, etc.), laboratory animals (e.g., mice, rats, rabbits, etc.), and non-human primates (e.g., apes and monkeys, etc.). In some embodiments, the subject is a human. In some embodiments, the subject is a patient under the care of a physician.

[0019] The present disclosure identifies a putative gain-of-function (GOF) missense variant in the PIEZO1 gene that is associated with a reduced risk of developing varicose veins in humans. Specifically, it has been observed that a genetic change that changes the cytosine at position 69,579 in the PIEZO1 reference genomic nucleic acid molecule (see SEQ ID NO: 1) to thymine indicates that subjects with such a change may have a reduced risk of developing varicose veins. In summary, the genetic analysis described herein surprisingly shows that the PIEZO1 gene, and in particular the pGOF of the PIEZO1 gene, is associated with a reduced risk of developing varicose veins. Therefore, subjects with PIEZO1 references who have an increased risk of developing varicose veins, such as saphenous varicose veins, reticular varicose veins, spider veins, or pregnancy-related varicose veins, can be treated to prevent varicose veins, reduce symptoms, and / or inhibit progression of symptoms. Thus, the present disclosure provides methods to utilize the identification of such variants in a subject to identify or stratify the risk in such a subject of developing varicose veins, such as saphenous, reticular, spider, or pregnancy-associated varicose veins, or to diagnose a subject as having an increased risk of developing varicose veins, such as saphenous, reticular, spider, or pregnancy-associated varicose veins, such that at-risk subjects or subjects with active disease can be treated appropriately.

[0020] The present disclosure further observes that PIEZO1 variant nucleic acid molecules encoding predicted gain-of-function polypeptides of PIEZO1 (whether these variations are homozygous or heterozygous in a particular subject) are associated with a reduced risk of developing varicose veins. Furthermore, the present disclosure's identification of the association of additional variants with the gene burden masks indicates that PIEZO1 itself (rather than linkage disequilibrium with variants of another gene) is responsible for the protective effect in varicose veins.

[0021] For the purpose of this disclosure, any particular subject may be classified as having one of three PIEZO1 genotypes: i) PIEZO1 reference; ii) heterozygous for PIEZO1 variant nucleic acid molecule encoding predicted gain-of-function polypeptide of PIEZO1; or iii) homozygous for PIEZO1 variant nucleic acid molecule encoding predicted gain-of-function polypeptide of PIEZO1. If the subject does not have a copy of PIEZO1 variant nucleic acid molecule encoding predicted gain-of-function polypeptide of PIEZO1, the subject is PIEZO1 reference. If the subject has a single copy of PIEZO1 variant nucleic acid molecule, the subject is heterozygous for PIEZO1 variant nucleic acid molecule encoding predicted gain-of-function polypeptide of PIEZO1. As used herein, PIEZO1 variant nucleic acid molecule is any PIEZO1 nucleic acid molecule (e.g., genomic nucleic acid molecule, mRNA molecule, or cDNA molecule) that encodes a PIEZO1 polypeptide with 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 predicted gain-of-function polypeptide of PIEZO1 with partial gain-of-function (or predicted partial gain-of-function) is hypomorphic for PIEZO1. The PIEZO1 variant nucleic acid molecule encoding a predicted gain-of-function polypeptide of PIEZO1 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. If the subject has two copies of a PIEZO1 variant nucleic acid molecule encoding a predicted gain-of-function polypeptide of PIEZO1, the subject is homozygous for the PIEZO1 variant nucleic acid molecule encoding a predicted gain-of-function polypeptide of PIEZO1.

[0022] For subjects genotyped or determined to be PIEZO1 reference, such subjects have an increased risk of developing varicose veins, such as saphenous varicose veins, reticular veins, spider veins, or pregnancy-associated varicose veins. For subjects genotyped or determined to be either PIEZO1 reference or heterozygous for a PIEZO1 variant nucleic acid molecule encoding a predicted gain-of-function polypeptide of PIEZO1, such subjects can be treated with a PIEZO1 agonist.

[0023] In any of the embodiments described throughout this disclosure, the PIEZO1 variant nucleic acid molecule can be any PIEZO1 nucleic acid molecule (e.g., a genomic nucleic acid molecule, an mRNA molecule, or a cDNA molecule, etc.) that encodes a PIEZO1 polypeptide with 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.

[0024] In any of the embodiments described throughout this disclosure, the predicted gain-of-function polypeptide of PIEZO1 can be any PIEZO1 polypeptide with partial gain-of-function, full gain-of-function, predicted partial gain-of-function, or predicted full gain-of-function. In any of the embodiments described throughout this disclosure, the predicted gain-of-function polypeptide of PIEZO1 can be any of the PIEZO1 polypeptides described herein, including, for example, PIEZO1 Pro2,510Leu, Pro2,024Leu, Pro2,079Leu, or Pro92Leu. In any of the embodiments described throughout this disclosure, the predicted gain-of-function polypeptide of PIEZO1 can be PIEZO1 Pro2,510Leu.

[0025] In any of the embodiments described throughout this disclosure, the varicose veins are saphenous, reticular, spider, or pregnancy-related varicose veins. In any of the embodiments described throughout this disclosure, the varicose veins are saphenous. In any of the embodiments described throughout this disclosure, the varicose veins are reticular. In any of the embodiments described throughout this disclosure, the varicose veins are spider veins. In any of the embodiments described throughout this disclosure, the varicose veins are pregnancy-related varicose veins.

[0026] Symptoms of varicose veins include, but are not limited to, leg fatigue, the appearance of spider veins in the affected legs (telangiectasia), swelling of the ankles (especially at night), a brown to yellow shiny discoloration of the skin near the affected veins, red, dry, and itchy areas of the skin (called stasis dermatitis or venous eczema), muscle cramps (especially when performing sudden movements such as standing up), minor bruising in the affected areas, bleeding more than normal or taking a long time to heal, shrinkage of the skin above the ankles (lipodermatosclerosis), restless leg syndrome, white, irregular, scar-like patches on the ankles (white atrophy), or any combination thereof.

[0027] The present disclosure provides a method of treating a subject having or at risk of developing varicose veins, the method comprising administering to the subject a PIEZO1 agonist. The present disclosure also provides a method of treating a subject having or at risk of developing saphenous varicose veins, the method comprising administering to the subject a PIEZO1 agonist.

[0028] The present disclosure also provides a method of treating a subject having or at risk of developing retinal varices, the method comprising administering to the subject a PIEZO1 agonist.

[0029] The present disclosure also provides a method of treating a subject having spider veins or at risk of developing spider veins, the method comprising administering to the subject a PIEZO1 agonist.

[0030] The present disclosure also provides a method of treating a subject having or at risk of developing pregnancy-associated varicose veins, the method comprising administering to the subject a PIEZO1 agonist.

[0031] In some embodiments, the treatment method further comprises detecting the presence or absence of a PIEZO1 variant nucleic acid molecule encoding a predicted gain-of-function polypeptide of PIEZO1 in a biological sample obtained from the subject. As used throughout this disclosure, a "PIEZO1 variant nucleic acid molecule" is any PIEZO1 nucleic acid molecule (e.g., a genomic nucleic acid molecule, an mRNA molecule, or a cDNA molecule, etc.) that encodes a PIEZO1 polypeptide having a partial gain-of-function, a full gain-of-function, a predicted partial gain-of-function, or a predicted full gain-of-function.

[0032] The present disclosure also provides a method of treating a subject with a therapeutic agent for treating or preventing 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 method includes determining whether the subject has a PIEZO1 variant nucleic acid molecule encoding a predicted gain-of-function polypeptide of PIEZO1 by obtaining or obtaining a biological sample from the subject and performing or performing sequence analysis on the biological sample to determine whether the subject has a genotype that includes the PIEZO1 variant nucleic acid molecule. If the subject is a PIEZO1 reference, the therapeutic agent for treating or preventing varicose veins is administered or continues to be administered to the subject at a standard dosage, and / or a PIEZO1 agonist is administered to the subject. If the subject is heterozygous for the PIEZO1 variant, the therapeutic agent for treating or preventing varicose veins is administered or continues to be administered to the subject at a standard dosage or less, and / or a PIEZO1 agonist is administered to the subject. The presence of a genotype having a PIEZO1 variant nucleic acid molecule encoding a predicted gain-of-function polypeptide of PIEZO1 indicates that the subject has a reduced risk of developing varicose veins. In some embodiments, the subject is PIEZO1 reference. In some embodiments, the subject is heterozygous for a PIEZO1 variant nucleic acid molecule encoding a predicted gain-of-function polypeptide of PIEZO1.

[0033] For subjects genotyped or determined to be either heterozygous for a PIEZO1 reference or a PIEZO1 variant nucleic acid molecule encoding a predicted gain-of-function polypeptide of PIEZO1, such subjects can be treated with a PIEZO1 agonist as described herein.

[0034] Detecting the presence or absence of a PIEZO1 variant nucleic acid molecule encoding a predicted gain-of-function polypeptide of PIEZO1 in a biological sample obtained from a subject and / or determining whether a subject has a PIEZO1 variant nucleic acid molecule encoding a predicted gain-of-function polypeptide of PIEZO1 may be performed by any of the methods described herein. In some embodiments, these methods may be performed in vitro. In some embodiments, these methods may be performed in situ. In some embodiments, these methods may be performed in vivo. In any of these embodiments, a PIEZO1 variant nucleic acid molecule encoding a predicted gain-of-function polypeptide of PIEZO1 may be present in a cell obtained from the subject.

[0035] In some embodiments, if the subject is a PIEZO1 reference, the subject is administered a standard dosage of a therapeutic agent for treating or preventing varicose veins. In some embodiments, if the subject is heterozygous for a PIEZO1 variant nucleic acid molecule encoding a predicted gain-of-function polypeptide of PIEZO1, the subject is administered a standard dosage of a therapeutic agent for treating or preventing varicose veins.

[0036] In some embodiments, the treatment method further comprises detecting the presence or absence of a predicted gain-of-function polypeptide of PIEZO1 in a biological sample obtained from the subject. In some embodiments, if the subject does not have a predicted gain-of-function polypeptide of PIEZO1, the subject is administered a therapeutic agent for treating or preventing varicose veins at a standard dosage. In some embodiments, if the subject has a predicted gain-of-function polypeptide of PIEZO1, the subject is administered a therapeutic agent for treating or preventing varicose veins at a dosage equal to or less than the standard dosage.

[0037] The present disclosure also provides a method of treating a subject with a therapeutic agent for treating or preventing 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 method includes determining whether the subject has a predicted gain-of-function polypeptide of PIEZO1 by obtaining or obtaining a biological sample from the subject and performing or performing an assay on the biological sample to determine whether the subject has a predicted gain-of-function polypeptide of PIEZO1. If the subject does not have a predicted gain-of-function polypeptide of PIEZO1, the therapeutic agent for treating or preventing varicose veins is administered or continues to be administered to the subject at a standard dosage, and / or a PIEZO1 agonist is administered to the subject. If the subject has a predicted gain-of-function polypeptide of PIEZO1, the therapeutic agent for treating or preventing varicose veins is administered or continues to be administered to the subject at a standard dosage or less, and / or a PIEZO1 agonist is administered to the subject. The presence of a predicted gain-of-function polypeptide of PIEZO1 indicates that the subject has a reduced risk of developing varicose veins. In some embodiments, the subject has a predicted gain-of-function polypeptide of PIEZO1. In some embodiments, the subject does not have a predicted gain-of-function polypeptide of PIEZO1.

[0038] Detecting the presence or absence of a predicted gain-of-function PIEZO1 polypeptide in a biological sample obtained from a subject and / or determining whether a subject has a predicted gain-of-function PIEZO1 polypeptide may be performed by any of the methods described herein. In some embodiments, these methods may be performed in vitro. In some embodiments, these methods may be performed in situ. In some embodiments, these methods may be performed in vivo. In any of these embodiments, the predicted gain-of-function PIEZO1 polypeptide may be present in a cell obtained from the subject.

[0039] Examples of therapeutic agents that treat or inhibit varicose veins include, but are not limited to, flavonoids such as diosmin or hesperidin, and anti-inflammatory agents such as ibuprofen and aspirin.

[0040] Examples of PIEZO1 agonists include, but are not limited to, Yodal, Jedil, and Jedi2. In some embodiments, the PIEZO1 agonist comprises a small molecule.

[0041] In some embodiments, the dosage of a therapeutic agent and / or PIEZO1 agonist for treating or preventing varicose veins may 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) for a subject who is heterozygous for a PIEZO1 variant nucleic acid molecule encoding a predicted gain-of-function polypeptide of PIEZO1, compared to a subject who is a PIEZO1 reference (which may receive a standard dosage). In some embodiments, the dosage of a therapeutic agent and / or PIEZO1 agonist for treating or preventing varicose veins may be reduced by about 10%, about 20%, about 30%, about 40%, or about 50%. Also, the dosage of a therapeutic agent and / or PIEZO1 agonist for treating or preventing varicose veins in a subject who is heterozygous for a PIEZO1 variant nucleic acid molecule encoding a predicted gain-of-function polypeptide of PIEZO1 may be administered less frequently compared to a subject who is a PIEZO1 reference.

[0042] The administration of the therapeutic agent and / or PIEZO1 agonist for treating or preventing varicose veins can be repeated, for example, after 1 day, 2 days, 3 days, 5 days, 1 week, 2 weeks, 3 weeks, 1 month, 5 weeks, 6 weeks, 7 weeks, 8 weeks, 2 months, or 3 months. The repeated administration can be the same dose or a different dose. The administration can be repeated 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 times, or more. For example, according to a given dosing regimen, the subject can undergo therapy for an extended period of time, such as, for example, 6 months, 1 year, or more. The therapeutic agent and / or PIEZO1 agonist for treating or preventing varicose veins can also be administered sequentially or simultaneously. The therapeutic agent and / or PIEZO1 agonist for treating or preventing varicose veins can also be administered in separate compositions, or together in the same composition.

[0043] The administration of the therapeutic agent and / or PIEZO1 agonist for treating or preventing varicose veins can be by any suitable route, including but not limited to parenteral, intravenous, oral, subcutaneous, intraarterial, intracranial, intrathecal, intraperitoneal, topical, intranasal, or intramuscular. The pharmaceutical composition for administration is desirably sterile, substantially isotonic, and manufactured under GMP conditions. The pharmaceutical composition can be provided in unit dosage form (i.e., dosage for single administration). The pharmaceutical composition can be formulated using one or more physiologically and pharmacologic acceptable carriers, diluents, excipients, or adjuvants. The formulation depends on the route of administration selected. The term "pharmaceutical acceptable" means that the carrier, diluent, excipient, or adjuvant is compatible with the other ingredients of the formulation and is not substantially harmful to the recipient thereof.

[0044] The terms "treat," "treating," and "treatment," as well as "prevent," "preventing," and "prevention," as used herein, refer to eliciting a desired biological response, e.g., therapeutic and prophylactic effects, respectively. In some embodiments, the therapeutic effect comprises one or more of: lowering / reducing varicose veins, lowering / reducing the severity of varicose veins (e.g., reducing or inhibiting the onset of varicose veins), lowering / reducing symptoms and varicose vein related effects, delaying the onset of symptoms and varicose vein related effects, reducing the severity of symptoms of varicose vein related effects, reducing the severity of acute attacks, reducing the number of symptoms and varicose vein related effects, reducing the latency of symptoms and varicose vein related effects, ameliorating symptoms and varicose vein related effects, reducing secondary symptoms, reducing secondary infections, preventing varicose vein flare-ups, reducing the number or frequency of flare-up attacks, increasing the latency between symptomatic attacks, increasing the time to sustained progression, hastening remission, inducing remission, enhancing remission, accelerating recovery, or increasing the effectiveness of or reducing resistance to alternative therapeutic agents, and / or increased survival time of an affected host animal following administration of the agent or a composition comprising the agent. A prophylactic effect may include complete or partial avoidance and / or inhibition or delay (e.g., complete or partial avoidance / inhibition or delay, etc.) of varicose vein development / progression following administration of a therapeutic protocol, and increased survival time of an affected host animal. Treatment of varicose veins includes treatment of subjects already diagnosed as having some form of varicose veins at any clinical stage or manifestation, delaying the onset or progression or aggravation or worsening of symptoms or signs of varicose veins, and / or prevention of varicose veins and / or reducing their severity.

[0045] The present disclosure also provides a method for identifying a subject with an increased risk of developing varicose veins. In some embodiments, the method comprises determining or having determined the presence or absence of a PIEZO1 variant nucleic acid molecule (e.g., a genomic nucleic acid molecule, an mRNA molecule, and / or a cDNA molecule) encoding a predicted gain-of-function polypeptide of PIEZO1 in a biological sample obtained from the subject. If the subject lacks a PIEZO1 variant nucleic acid molecule encoding a predicted gain-of-function polypeptide of PIEZO1 (i.e., the subject is genotypically classified as a PIEZO1 reference), the subject has an increased risk of developing varicose veins. If the subject has a PIEZO1 variant nucleic acid molecule encoding a predicted gain-of-function polypeptide of PIEZO1 (i.e., the subject is heterozygous or homozygous for a PIEZO1 variant nucleic acid molecule), the subject has a reduced risk of developing varicose veins compared to a subject who is a PIEZO1 reference.

[0046] Having a single copy of a PIEZO1 variant nucleic acid molecule encoding a predicted gain-of-function polypeptide of PIEZO1 is more protective for a subject to develop varicose veins than not having a copy of a PIEZO1 variant nucleic acid molecule encoding a predicted gain-of-function polypeptide of PIEZO1. Without intending to be limited to 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 a PIEZO1 variant nucleic acid molecule) is protective for a subject to develop varicose veins, and it is believed that having two copies of a PIEZO1 variant nucleic acid molecule encoding a predicted gain-of-function polypeptide of PIEZO1 (i.e., homozygous for a PIEZO1 variant nucleic acid molecule) may be more protective for a subject to develop varicose veins than a subject having a single copy. Thus, in some embodiments, a single copy of a PIEZO1 variant nucleic acid molecule encoding a predicted gain-of-function polypeptide of PIEZO1 may not be completely protective, but instead may be partially or incompletely protective for a subject to develop varicose veins. Without wishing to be bound by any particular theory, it is possible that less than complete protection from the development of varicose veins may result from additional factors or molecules involved in the development of varicose veins that are still present in subjects having a single copy of a PIEZO1 variant nucleic acid molecule encoding a predicted gain-of-function polypeptide of PIEZO1.

[0047] Detecting the presence or absence of a PIEZO1 variant nucleic acid molecule encoding a predicted gain-of-function polypeptide of PIEZO1 in a biological sample obtained from a subject and / or determining whether a subject has a PIEZO1 variant nucleic acid molecule encoding a predicted gain-of-function polypeptide of PIEZO1 may be performed by any of the methods described herein. In some embodiments, these methods may be performed in vitro. In some embodiments, these methods may be performed in situ. In some embodiments, these methods may be performed in vivo. In any of these embodiments, a PIEZO1 variant nucleic acid molecule encoding a predicted gain-of-function polypeptide of PIEZO1 may be present in a cell obtained from the subject.

[0048] Any one or more (i.e., any combination) of PIEZO1 variant nucleic acid molecules encoding predicted gain-of-function polypeptides of PIEZO1 can be used in any of the methods described herein to determine whether a subject has an increased risk of developing varicose veins. A particular combination of variants can form a mask that is used to statistically analyze a particular correlation between PIEZO1 and a reduced risk of developing varicose veins.

[0049] In some embodiments, once a subject is identified as having an increased risk of developing varicose veins, the subject is further treated with a therapeutic agent for treating or preventing varicose veins and / or a PIEZO1 agonist as described herein. For example, if a subject is a PIEZO1 reference 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 for treating or preventing varicose veins. In some embodiments, if a subject is heterozygous for a PIEZO1 variant nucleic acid molecule encoding a predicted gain-of-function polypeptide of PIEZO1, the subject is administered a therapeutic agent for treating or preventing varicose veins at a dosage equal to or less than the standard dosage and / or a PIEZO1 agonist. In some embodiments, the subject is a PIEZO1 reference. In some embodiments, the subject is heterozygous for a PIEZO1 variant nucleic acid molecule encoding a predicted gain-of-function polypeptide of PIEZO1.

[0050] In some embodiments, once a subject is identified as having an increased risk of developing varicose veins, the subject is further administered a therapeutic agent for treating or preventing varicose veins and / or a PIEZO1 agonist as described herein. For example, if a subject is a PIEZO1 reference 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 for treating or preventing varicose veins. In some embodiments, if a subject is heterozygous for a PIEZO1 variant nucleic acid molecule encoding a predicted gain-of-function polypeptide of PIEZO1, the subject is administered a therapeutic agent for treating or preventing varicose veins at a dosage equal to or less than the standard dosage and / or a PIEZO1 agonist. In some embodiments, the subject is a PIEZO1 reference. In some embodiments, the subject is heterozygous for a PIEZO1 variant nucleic acid molecule encoding a predicted gain-of-function polypeptide of PIEZO1.

[0051] In some embodiments, any of the methods described herein may further comprise determining the total burden of the subject having a PIEZO1 variant nucleic acid molecule encoding a predicted gain-of-function polypeptide of PIEZO1 and / or a predicted gain-of-function variant polypeptide of PIEZO1 associated with a reduced risk of developing varicose veins. The total burden is the sum of all variants of the PIEZO1 gene, which may be performed in an association analysis with varicose veins. In some embodiments, the subject is homozygous for one or more PIEZO1 variant nucleic acid molecules encoding a predicted gain-of-function polypeptide of PIEZO1 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 a predicted gain-of-function polypeptide of PIEZO1 associated with a reduced risk of developing varicose veins. The results of the association analysis suggest that the PIEZO1 variant nucleic acid molecule encoding a predicted gain-of-function polypeptide of PIEZO1 is associated with a reduced risk of developing varicose veins. If the subject has a lower total load, the subject is at higher risk of developing varicose veins, and the subject is administered or continues to be administered with the therapeutic agent for treating or preventing varicose veins and / or the PIEZO1 agonist in standard dosage.If the subject has a higher total load, the subject is at lower risk of developing varicose veins, and the subject is administered or continues to be administered with the therapeutic agent for treating or preventing varicose veins in the same or lower amount than the standard dosage.The higher the total load, the lower the risk of developing varicose veins.

[0052] In some embodiments, the total burden of a subject having any one or more PIEZO1 variant nucleic acid molecules encoding predicted gain-of-function polypeptides of PIEZO1 represents the weighted sum of any multiple of the PIEZO1 variant nucleic acid molecules encoding predicted gain-of-function polypeptides of PIEZO1. In some embodiments, the total burden is calculated using at least about 2, at least about 3, at least about 4, at least about 5, at least about 10, at least about 20, at least about 30, at least about 40, at least about 50, at least about 60, at least about 70, at least about 80, at least about 100, at least about 120, at least about 150, at least about 200, at least about 250, at least about 300, at least about 400, at least about 500, at least about 1,000, at least about 10,000, at least about 100,000, or at least about 1,000,000 or more genetic variants present in or surrounding (within 10 Mb) the PIEZO1 gene, where the genetic burden is the number of alleles multiplied by the estimated association with varicose veins or a related outcome for each allele (e.g., a weighted polygenic burden score). This may include any genetic variants in the vicinity of the PIEZO1 gene (within 10Mb around the gene) that show non-zero association with varicose veins-related traits in genetic association analysis, regardless of genome annotation.In some embodiments, if a subject has a total load higher than a desired threshold score, the subject has a reduced risk of developing varicose veins.In some embodiments, if a subject has a total load lower than a desired threshold score, the subject has an increased risk of developing varicose veins.

[0053] In some embodiments, the total burden can be divided into quintiles, for example, top quintile, middle quintile, and bottom quintile, with the top quintile of total burden corresponding to the lowest risk group and the bottom quintile of total burden corresponding to the highest risk group. In some embodiments, subjects with a larger total burden include the highest weighted total burden, including but not limited to the top 10%, top 20%, top 30%, top 40%, or top 50% total burden from the subject population. In some embodiments, the genetic variants include genetic variants that have an association with varicose veins in the top 10%, top 20%, top 30%, top 40%, or top 50% of the p-value range of the association. In some embodiments, each of the identified genetic variants has a p-value of about 10 -2 Below, about 10 -3 Below, about 10 -4 Below, about 10 -5 Below, about 10 -6 Below, about 10 -7 Below, about 10 -8 Below, about 10 -9 Below, about 10 -10 Below, about 10 -11 Below, about 10 -12 Below, about 10 -13 Below, about 10 -14 Less than or equal to 10 -15 In some embodiments, the identified genetic variants have an association with varicose veins with a p-value of 5×10 -8In some embodiments, the identified genetic variants include genetic variants having an association with varicose veins in high risk subjects compared to the remainder of a reference population with an odds ratio (OR) of about 1.5 or more, about 1.75 or more, about 2.0 or more, or about 2.25 or more for the top 20% of the distribution, or about 1.5 or more, about 1.75 or more, about 2.0 or more, about 2.25 or more, about 2.5 or more, or about 2.75 or more. In some embodiments, the odds ratio (OR) may range from about 1.0 to about 1.5, about 1.5 to about 2.0, about 2.0 to about 2.5, about 2.5 to about 3.0, about 3.0 to about 3.5, about 3.5 to about 4.0, about 4.0 to about 4.5, about 4.5 to about 5.0, about 5.0 to about 5.5, about 5.5 to about 6.0, about 6.0 to about 6.5, about 6.5 to about 7.0, or may be greater than 7.0. In some embodiments, high-risk subjects include subjects with a total burden in the bottom decile, quintile, or tertile in the reference population. The total burden threshold is determined based on the nature of the intended practical application and the risk difference that is considered meaningful for that practical application.

[0054] In some embodiments, once a subject is identified as having an increased risk of developing varicose veins, the subject is further administered a therapeutic agent for treating or preventing varicose veins and / or a PIEZO1 agonist as described herein. For example, if a subject is a PIEZO1 reference 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 for treating or preventing varicose veins. In some embodiments, if a subject is heterozygous for a PIEZO1 variant nucleic acid molecule encoding a predicted gain-of-function polypeptide of PIEZO1, the subject is administered a therapeutic agent for treating or preventing varicose veins at a dosage equal to or less than the standard dosage and / or a PIEZO1 agonist. In some embodiments, the subject is a PIEZO1 reference. In some embodiments, the subject is heterozygous for a PIEZO1 variant nucleic acid molecule encoding a predicted gain-of-function polypeptide of PIEZO1. Furthermore, if a subject has a lower total burden of having PIEZO1 variant nucleic acid molecules encoding predicted gain-of-function polypeptides of PIEZO1 and therefore has an increased risk of developing varicose veins, the subject is administered a therapeutic agent for treating or preventing varicose veins. In some embodiments, if a subject has a lower total burden of having PIEZO1 variant nucleic acid molecules encoding predicted gain-of-function polypeptides of PIEZO1, the subject is administered a therapeutic agent for treating or preventing varicose veins at a dosage equal to or greater than the standard dosage administered to a subject with a higher total burden of having PIEZO1 variant nucleic acid molecules encoding predicted gain-of-function polypeptides of PIEZO1.

[0055] The present disclosure also provides a method for detecting the presence or absence of a PIEZO1 variant genomic nucleic acid molecule encoding a predicted gain-of-function polypeptide of PIEZO1 in a biological sample obtained from a subject, and / or a PIEZO1 variant mRNA molecule encoding a predicted gain-of-function polypeptide of PIEZO1 in a biological sample obtained from a subject, and / or a PIEZO1 variant cDNA molecule encoding a predicted gain-of-function polypeptide of PIEZO1 generated from an mRNA molecule in a biological sample obtained from a subject. It is understood that gene sequences within a population, and the mRNA molecules encoded by such genes, may differ due to polymorphisms, such as single nucleotide polymorphisms (SNPs). The sequences provided herein for the PIEZO1 variant genomic nucleic acid molecule, the PIEZO1 variant mRNA molecule, and the PIEZO1 variant cDNA molecule are merely exemplary sequences. Other sequences for the PIEZO1 variant genomic nucleic acid molecule, the variant mRNA molecule, and the variant cDNA molecule are also possible.

[0056] The biological sample may be derived from any cell, tissue, or biological fluid from a subject. The biological sample may include any clinically relevant tissue, such as 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, the biological sample includes a buccal swab. The biological sample used in the methods disclosed herein may vary based on the assay format, the nature of the detection method, and the tissue, cell, or extract used as the sample. The biological sample may be processed differently depending on the assay being used. For example, when detecting PIEZO1 variant nucleic acid molecules, a pretreatment designed to isolate or enrich the biological sample for PIEZO1 variant nucleic acid molecules may be used. A variety of techniques may be used for this purpose. When detecting the level of PIEZO1 variant mRNA molecules, a variety of techniques may be used to enrich the biological sample with mRNA molecules. A variety of methods may be used to detect the presence or level of mRNA molecules or the presence of a particular variant genomic DNA locus.

[0057] The present disclosure also provides a method for detecting a PIEZO1 variant nucleic acid molecule or its complement encoding a predicted gain-of-function polypeptide of PIEZO1 in a subject, the method comprising assaying a biological sample obtained from the subject to determine whether a nucleic acid molecule in the biological sample is a PIEZO1 variant nucleic acid molecule encoding a predicted gain-of-function polypeptide of PIEZO1.

[0058] In some embodiments, a PIEZO1 variant nucleic acid molecule, or its complement, encoding a predicted gain-of-function polypeptide of PIEZO1 is a genomic nucleic acid molecule having a nucleotide sequence that includes a thymine at a position corresponding to position 69,579 according to SEQ ID NO:2, or its complement.

[0059] In some embodiments, a PIEZO1 variant nucleic acid molecule or its complement encoding a predicted gain-of-function polypeptide of PIEZO1 contains 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 6,111 according to SEQ ID NO:17 or its complement; a uracil at a position corresponding to position 073 according to SEQ ID NO:18 or its complement; a uracil at a position corresponding to position 6,236 according to SEQ ID NO:19 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.

[0060] In some embodiments, a PIEZO1 variant nucleic acid molecule encoding a predicted gain-of-function polypeptide of PIEZO1, or its complement, contains 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.

[0061] In some embodiments, a PIEZO1 variant nucleic acid molecule has a nucleotide sequence that includes a thymine at a position corresponding to position 69,579 according to SEQ ID NO:2 or its complement (for a genomic nucleic acid molecule); a uracil at a position corresponding to position 7,785 according to SEQ ID NO:13 or its complement (for an mRNA molecule); or a thymine at a position corresponding to position 7,785 according to SEQ ID NO:33 or its complement (for a cDNA molecule derived from an mRNA molecule).

[0062] In some embodiments, the biological sample comprises cells or cell lysates. Such methods may, for example, comprise obtaining a biological sample from a subject that comprises PIEZO1 genomic nucleic acid molecule or mRNA molecule, and, in the case of mRNA, optionally reverse transcribe the mRNA into cDNA. Such assays may, for example, comprise determining the identity of these positions of a particular PIEZO1 nucleic acid molecule. In some embodiments, the methods are in vitro methods.

[0063] In some embodiments, the assay comprises sequencing at least a portion of the nucleotide sequence of the PIEZO1 nucleic acid molecule or its complement in the biological sample.In some embodiments, the assay comprises sequencing at least a portion of the nucleotide sequence of the PIEZO1 genomic nucleic acid molecule in the biological sample, the portion to be sequenced comprises the position corresponding to position 69,579 according to SEQ ID NO:2 or its complement; sequencing at least a portion of the nucleotide sequence of the PIEZO1 mRNA molecule in the biological sample, the portion to be sequenced comprises the position corresponding to position 7,785 according to SEQ ID NO:22 or its complement; and / or sequencing at least a portion of the nucleotide sequence of the PIEZO1 cDNA molecule generated from the mRNA in the biological sample, the portion to be sequenced comprises the position corresponding to position 7,785 according to SEQ ID NO:33 or its complement. If the sequenced portion of the PIEZO1 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 its complement; a uracil at a position corresponding to position 7,785 according to SEQ ID NO:13 or its complement; or a thymine at a position corresponding to position 7,785 according to SEQ ID NO:33 or its complement, then the PIEZO1 nucleic acid molecule in the biological sample is a PIEZO1 variant nucleic acid molecule that encodes a predicted gain-of-function polypeptide of PIEZO1.

[0064] In some embodiments, the assay comprises sequencing at least a portion of the nucleotide sequence of a PIEZO1 genomic nucleic acid molecule or its complement in a biological sample, wherein the sequenced portion comprises a position corresponding to position 69,579 according to SEQ ID NO:2 or its complement. If the sequenced portion of the PIEZO1 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 its complement, the PIEZO1 genomic nucleic acid molecule in the biological sample is a PIEZO1 variant genomic nucleic acid molecule that encodes a predicted gain-of-function polypeptide of PIEZO1.

[0065] In some embodiments, the assay comprises sequencing at least a portion of the nucleotide sequence of a PIEZO1 mRNA molecule in a biological sample, the portion sequenced comprising: position 7,785 according to SEQ ID NO:13 or its complement; position 1,095 according to SEQ ID NO:14 or its complement; position 6,073 according to SEQ ID NO:15 or its complement; position 1,336 according to SEQ ID NO:16 or its complement; position 6,073 according to SEQ ID NO:17 or its complement; position 6,236 according to SEQ ID NO:18 or its complement; position 7,526 according to SEQ ID NO:19 or its complement; position 1,336 according to SEQ ID NO:20 or its complement; position 361 according to SEQ ID NO:21 or its complement; or position 7,776 according to SEQ ID NO:22 or its complement. If the sequenced portion of the PIEZO1 mRNA molecule in the biological sample includes uracil at a position corresponding to position 7,785 according to SEQ ID NO: 13 or its complement; uracil at a position corresponding to position 1,095 according to SEQ ID NO: 14 or its complement; uracil at a position corresponding to position 6,073 according to SEQ ID NO: 15 or its complement; uracil at a position corresponding to position 1,336 according to SEQ ID NO: 16 or its complement; uracil at a position corresponding to position 6,073 according to SEQ ID NO: 17 or its complement; uracil at a position corresponding to position 6,236 according to SEQ ID NO: 18 or its complement; uracil at a position corresponding to position 7,526 according to SEQ ID NO: 19 or its complement; uracil at a position corresponding to position 1,336 according to SEQ ID NO: 20 or its complement; uracil at a position corresponding to position 361 according to SEQ ID NO: 21 or its complement; or uracil at a position corresponding to position 7,776 according to SEQ ID NO: 22 or its complement, the PIEZO1 mRNA molecule in the biological sample is detected. The mRNA molecule is a PIEZO1 variant mRNA molecule that encodes a predicted gain-of-function polypeptide of PIEZO1.

[0066] In some embodiments, the assay comprises sequencing at least a portion of the nucleotide sequence of a PIEZO1 cDNA molecule generated from an mRNA molecule in a biological sample, wherein the sequenced portion comprises a position corresponding to 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. If the sequenced portion of the PIEZO1 cDNA molecule in the biological sample includes 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, the PIEZO1 generated from the mRNA molecule in the biological sample is The cDNA molecule is a PIEZO1 variant cDNA molecule that encodes a predicted gain-of-function polypeptide of PIEZO1.

[0067] In some embodiments, the assay comprises contacting the biological sample with a primer that hybridizes to a portion of the nucleotide sequence of a PIEZO1 genomic nucleic acid molecule or its complement proximate to a position corresponding to position 69,579 of SEQ ID NO:2 or its complement; a portion of the nucleotide sequence of an mRNA molecule or its complement proximate to a position corresponding to position 7,785 of SEQ ID NO:13 or its complement; and / or a portion of the nucleotide sequence of a cDNA molecule or its complement proximate to a position corresponding to position 7,785 of SEQ ID NO:33 or its complement; and b) contacting the biological sample with a primer that hybridizes to a portion of the nucleotide sequence of a PIEZO1 genomic nucleic acid molecule or its complement proximate to a position corresponding to position 69,579 of SEQ ID NO:2 or its complement; a portion of the nucleotide sequence of an mRNA molecule or its complement proximate to a position corresponding to position 7,785 of SEQ ID NO:13 or its complement; and / or a position corresponding to position 7,785 according to SEQ ID NO:33 or its complement of the nucleotide sequence of the cDNA molecule or its complement; 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 its complement; a uracil at a position corresponding to position 7,785 according to SEQ ID NO:13 or its complement; and / or a thymine at a position corresponding to position 7,785 according to SEQ ID NO:33 or its complement of the nucleotide sequence of the cDNA molecule or its complement.

[0068] In some embodiments, the assay comprises: a) contacting the biological sample with a primer that hybridizes to a portion of the nucleotide sequence of a PIEZO1 mRNA molecule or its complement proximate to the position corresponding to position 1,095 according to SEQ ID NO: 14 or its complement; and / or to a portion of the nucleotide sequence of a cDNA molecule or its complement proximate to the position corresponding to position 1,095 according to SEQ ID NO: 34 or its complement; b) extending the primer at least through the position of the nucleotide sequence of a PIEZO1 mRNA molecule or its complement proximate to the position corresponding to position 1,095 according to SEQ ID NO: 14 or its complement; and / or the position of the nucleotide sequence of a cDNA molecule or its complement proximate to the position corresponding to position 1,095 according to SEQ ID NO: 34 or its complement; and c) determining whether the extension product of the primer comprises uracil at the position corresponding to position 1,095 according to SEQ ID NO: 14 or its complement; and / or thymine at the position corresponding to position 1,095 according to SEQ ID NO: 34 or its complement.

[0069] In some embodiments, the assay comprises: a) contacting the biological sample with a primer that hybridizes to a portion of the nucleotide sequence of a PIEZO1 mRNA molecule or its complement near the position corresponding to position 6,073 according to SEQ ID NO: 15 or its complement; and / or a portion of the nucleotide sequence of a cDNA molecule or its complement near the position corresponding to position 6,073 according to SEQ ID NO: 35 or its complement; b) extending the primer at least through the position of the nucleotide sequence of a PIEZO1 mRNA molecule or its complement near the position corresponding to position 6,073 according to SEQ ID NO: 15 or its complement; and / or the position of the nucleotide sequence of a cDNA molecule or its complement near the position corresponding to position 6,073 according to SEQ ID NO: 35 or its complement; and c) determining whether the extension product of the primer comprises uracil at the position corresponding to position 6,073 according to SEQ ID NO: 15 or its complement; and / or thymine at the position corresponding to position 6,073 according to SEQ ID NO: 35 or its complement.

[0070] In some embodiments, the assay comprises: a) contacting the biological sample with a primer that hybridizes to a portion of the nucleotide sequence of a PIEZO1 mRNA molecule or its complement proximate to a position corresponding to position 1,336 according to SEQ ID NO: 16 or its complement; and / or a portion of the nucleotide sequence of a cDNA molecule or its complement proximate to a position corresponding to position 1,336 according to SEQ ID NO: 36 or its complement; b) extending the primer at least through the position of the nucleotide sequence of a PIEZO1 mRNA molecule or its complement proximate to a position corresponding to position 1,336 according to SEQ ID NO: 16 or its complement; and / or the position of the nucleotide sequence of a cDNA molecule or its complement proximate to a position corresponding to position 1,336 according to SEQ ID NO: 36 or its complement; and c) determining whether the extension product of the primer comprises uracil at the position corresponding to position 1,336 according to SEQ ID NO: 16 or its complement; and / or thymine at the position corresponding to position 1,336 according to SEQ ID NO: 36 or its complement.

[0071] In some embodiments, the assay comprises: a) contacting the biological sample with a primer that hybridizes to a portion of the nucleotide sequence of a PIEZO1 mRNA molecule or its complement near the position corresponding to position 6,073 according to SEQ ID NO: 17 or its complement; and / or a portion of the nucleotide sequence of a PIEZO1 cDNA molecule or its complement near the position corresponding to position 6,073 according to SEQ ID NO: 37 or its complement; b) extending the primer at least through the position of the PIEZO1 mRNA molecule or its complement nucleotide sequence corresponding to position 6,073 according to SEQ ID NO: 17 or its complement; and / or the position of the PIEZO1 cDNA molecule or its complement nucleotide sequence corresponding to position 6,073 according to SEQ ID NO: 37 or its complement; and c) determining whether the extension product of the primer comprises uracil at the position corresponding to position 6,073 according to SEQ ID NO: 17 or its complement; and / or thymine at the position corresponding to position 6,073 according to SEQ ID NO: 37 or its complement.

[0072] In some embodiments, the assay comprises: a) contacting the biological sample with a primer that hybridizes to a portion of the nucleotide sequence of a PIEZO1 mRNA molecule or its complement near the position corresponding to position 6,236 according to SEQ ID NO: 18 or its complement; and / or to a portion of the nucleotide sequence of a cDNA molecule or its complement near the position corresponding to position 6,236 according to SEQ ID NO: 38 or its complement; b) extending the primer at least through the position of the nucleotide sequence of a PIEZO1 mRNA molecule or its complement near the position corresponding to position 6,236 according to SEQ ID NO: 18 or its complement; and / or the position of the nucleotide sequence of a cDNA molecule or its complement near the position corresponding to position 6,236 according to SEQ ID NO: 38 or its complement; and c) determining whether the extension product of the primer comprises uracil at the position corresponding to position 6,236 according to SEQ ID NO: 18 or its complement; and / or thymine at the position corresponding to position 6,236 according to SEQ ID NO: 38 or its complement.

[0073] In some embodiments, the assay comprises: a) contacting the biological sample with a primer that hybridizes to a portion of the nucleotide sequence of the mRNA molecule or its complement proximate to the position corresponding to position 7,526 according to SEQ ID NO: 19 or its complement; and / or to a portion of the nucleotide sequence of the cDNA molecule or its complement proximate to the position corresponding to position 7,526 according to SEQ ID NO: 39 or its complement; b) extending the primer at least through the position of the nucleotide sequence of the mRNA molecule or its complement proximate to the position corresponding to position 7,526 according to SEQ ID NO: 19 or its complement; and / or the position of the nucleotide sequence of the cDNA molecule or its complement; and c) determining whether the extension product of the primer comprises uracil at the position corresponding to position 7,526 according to SEQ ID NO: 19 or its complement; and / or thymine at the position corresponding to position 7,526 according to SEQ ID NO: 39 or its complement.

[0074] In some embodiments, the assay comprises: a) contacting the biological sample with a primer that hybridizes to a portion of the nucleotide sequence of a PIEZO1 mRNA molecule or its complement near the position corresponding to position 1,336 according to SEQ ID NO:20 or its complement; and / or to a portion of the nucleotide sequence of a cDNA molecule or its complement near the position corresponding to position 1,336 according to SEQ ID NO:40 or its complement; b) extending the primer at least through the position of the nucleotide sequence of a PIEZO1 mRNA molecule or its complement near the position corresponding to position 1,336 according to SEQ ID NO:20 or its complement; and / or the position of the nucleotide sequence of a cDNA molecule or its complement near the position corresponding to position 1,336 according to SEQ ID NO:40 or its complement; and c) determining whether the extension product of the primer comprises uracil at the position corresponding to position 1,336 according to SEQ ID NO:20 or its complement; and / or thymine at the position corresponding to position 1,336 according to SEQ ID NO:40 or its complement.

[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 PIEZO1 mRNA molecule or its complement near the position corresponding to position 361 according to SEQ ID NO:21 or its complement; and / or to a portion of the nucleotide sequence of a cDNA molecule or its complement near the position corresponding to position 361 according to SEQ ID NO:41 or its complement; b) extending the primer at least through the position of the nucleotide sequence of a PIEZO1 mRNA molecule or its complement near the position corresponding to position 361 according to SEQ ID NO:21 or its complement; and / or the position of the nucleotide sequence of a cDNA molecule or its complement near the position corresponding to position 361 according to SEQ ID NO:41 or its complement; and c) determining whether the extension product of the primer comprises uracil at the position corresponding to position 361 according to SEQ ID NO:21 or its complement; and / or thymine at the position corresponding to position 361 according to SEQ ID NO:41 or its complement.

[0076] In some embodiments, the assay comprises: a) contacting the biological sample with a primer that hybridizes to a portion of the nucleotide sequence of the mRNA molecule or its complement of PIEZO1 near the position corresponding to position 7,776 according to SEQ ID NO:22 or its complement; and / or to a portion of the nucleotide sequence of the cDNA molecule or its complement of PIEZO1 near the position corresponding to position 7,776 according to SEQ ID NO:42 or its complement; b) extending the primer at least through the position of the nucleotide sequence of the mRNA molecule or its complement of PIEZO1 near the position corresponding to position 7,776 according to SEQ ID NO:22 or its complement; and / or the position of the nucleotide sequence of the cDNA molecule or its complement of PIEZO1 near the position corresponding to position 7,776 according to SEQ ID NO:42 or its complement; and c) determining whether the extension product of the primer comprises uracil at the position corresponding to position 7,776 according to SEQ ID NO:22 or its complement; and / or thymine at the position corresponding to position 7,776 according to SEQ ID NO:42 or its complement.

[0077] In some embodiments, the assay comprises: a) contacting the biological sample with a primer that hybridizes to a portion of the nucleotide sequence of the PIEZO1 genomic nucleic acid molecule or its complement near the position corresponding to position 69,579 according to SEQ ID NO:2 or its complement; b) extending the primer at least through the position of the nucleotide sequence of the PIEZO1 genomic nucleic acid molecule or its complement corresponding to position 69,579 according to SEQ ID NO:2 or its complement; and c) determining whether the extension product of the primer comprises a thymine at the position corresponding to position 69,579 according to SEQ ID NO:2.

[0078] In some embodiments, the assay comprises: a) contacting the biological sample with a primer that hybridizes to a portion of the nucleotide sequence of a PIEZO1 mRNA molecule or its complement that is proximal to a position corresponding to: position 7,785 according to SEQ ID NO: 13 or its complement; position 1,095 according to SEQ ID NO: 14 or its complement; position 6,073 according to SEQ ID NO: 15 or its complement; position 1,336 according to SEQ ID NO: 16 or its complement; position 6,073 according to SEQ ID NO: 17 or its complement; position 6,236 according to SEQ ID NO: 18 or its complement; position 7,526 according to SEQ ID NO: 19 or its complement; position 1,336 according to SEQ ID NO: 20 or its complement; position 361 according to SEQ ID NO: 21 or its complement; or position 7,776 according to SEQ ID NO: 22 or its complement; b) hybridizing the PIEZO1 position 7,785 according to SEQ ID NO:13 or its complement; position 1,095 according to SEQ ID NO:14 or its complement; position 6,073 according to SEQ ID NO:15 or its complement; position 1,336 according to SEQ ID NO:16 or its complement; position 6,073 according to SEQ ID NO:17 or its complement; position 6,236 according to SEQ ID NO:18 or its complement; position 7,526 according to SEQ ID NO:19 or its complement; position 1,336 according to SEQ ID NO:20 or its complement; position 361 according to SEQ ID NO:21 or its complement; or position 7,776 according to SEQ ID NO:22 or its complement of the nucleotide sequence of the mRNA molecule or its complement. and c) extending the mer; and c) determining whether the extension product of the primer is uracil at a position corresponding to position 7,785 according to SEQ ID NO:13 or its complement; uracil at a position corresponding to position 1,095 according to SEQ ID NO:14 or its complement; uracil at a position corresponding to position 6,073 according to SEQ ID NO:15 or its complement; uracil at a position corresponding to position 1,336 according to SEQ ID NO:16 or its complement; uracil at a position corresponding to position 6,073 according to SEQ ID NO:17 or its complement; uracil at a position corresponding to position 6,236 according to SEQ ID NO:18 or its complement; uracil at a position corresponding to position 7,526 according to SEQ ID NO:19 or its complement;determining whether the nucleic acid sequence contains 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;

[0079] In some embodiments, the assay comprises: a) contacting the biological sample with a primer that hybridizes to a portion of the nucleotide sequence of a PIEZO1 cDNA molecule or its complement that is near a position corresponding to: 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; b) contacting the biological sample with a primer that hybridizes to a portion of the nucleotide sequence of a PIEZO1 cDNA molecule or its complement that is near a position corresponding to: 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; 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 of the nucleotide sequence of the cDNA molecule or its complement; and the extension product of has 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;or a thymine at a position corresponding to position 7,776 according to SEQ ID NO:42 or its complement;

[0080] In some embodiments, the assay comprises sequencing the entire nucleic acid molecule. In some embodiments, only the PIEZO1 genomic nucleic acid molecule is analyzed. In some embodiments, only the PIEZO1 mRNA is analyzed. In some embodiments, only the PIEZO1 cDNA obtained from the PIEZO1 mRNA is analyzed.

[0081] In some embodiments, the assay comprises: a) amplifying at least a portion of a PIEZO1 nucleic acid molecule or its complement in a biological sample, the amplified portion comprising a thymine at a position corresponding to position 69,579 according to SEQ ID NO:2 or its complement; a uracil at a position corresponding to position 7,785 according to SEQ ID NO:13 or its complement; and / or a thymine at a position corresponding to position 7,785 according to SEQ ID NO:33 or its complement; b) labeling the amplified nucleic acid molecule with a detectable label; c) contacting the labeled nucleic acid molecule with a support comprising a variation-specific probe, the variation-specific probe comprising 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 its complement; a uracil at a position corresponding to position 7,785 according to SEQ ID NO:13 or its complement; and / or a thymine at a position corresponding to position 7,785 according to SEQ ID NO:33 or its complement; and d) detecting the detectable label.

[0082] In some embodiments, the assay comprises: a) amplifying at least a portion of a PIEZO1 nucleic acid molecule or its complement 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 its complement; and / or a thymine at a position corresponding to position 1,095 according to SEQ ID NO: 34 or its complement; b) labeling the amplified nucleic acid molecule with a detectable label; c) contacting the labeled nucleic acid molecule with a support comprising a variation-specific probe, wherein the variation-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 its complement; and / or a thymine at a position corresponding to position 1,095 according to SEQ ID NO: 34 or its complement; and d) detecting the detectable label.

[0083] In some embodiments, the assay comprises: a) amplifying at least a portion of a PIEZO1 nucleic acid molecule or its complement 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 its complement; and / or a thymine at a position corresponding to position 6,073 according to SEQ ID NO: 35 or its complement; b) labeling the amplified nucleic acid molecule with a detectable label; c) contacting the labeled nucleic acid molecule with a support comprising a variation-specific probe, wherein the variation-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 its complement; and / or a thymine at a position corresponding to position 6,073 according to SEQ ID NO: 35 or its complement; and d) detecting the detectable label.

[0084] In some embodiments, the assay comprises: a) amplifying at least a portion of a PIEZO1 nucleic acid molecule or its complement 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 its complement; and / or a thymine at a position corresponding to position 1,336 according to SEQ ID NO: 36 or its complement; b) labeling the amplified nucleic acid molecule with a detectable label; c) contacting the labeled nucleic acid molecule with a support comprising a variation-specific probe, wherein the variation-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 its complement; and / or a thymine at a position corresponding to position 1,336 according to SEQ ID NO: 36 or its complement; and d) detecting the detectable label.

[0085] In some embodiments, the assay comprises: a) amplifying at least a portion of a PIEZO1 nucleic acid molecule or its complement 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 its complement; and / or a thymine at a position corresponding to position 6,073 according to SEQ ID NO: 37 or its complement; b) labeling the amplified nucleic acid molecule with a detectable label; c) contacting the labeled nucleic acid molecule with a support comprising a variation-specific probe, wherein the variation-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 its complement; and / or a thymine at a position corresponding to position 6,073 according to SEQ ID NO: 37 or its complement; and d) detecting the detectable label.

[0086] In some embodiments, the assay comprises: a) amplifying at least a portion of a PIEZO1 nucleic acid molecule or its complement in a biological sample, the amplified portion comprising a uracil at a position corresponding to position 6,236 according to SEQ ID NO: 18 or its complement; and / or a thymine at a position corresponding to position 6,236 according to SEQ ID NO: 38 or its complement; b) labeling the amplified nucleic acid molecule with a detectable label; c) contacting the labeled nucleic acid molecule with a support comprising a variation-specific probe, the variation-specific probe comprising 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 its complement; and / or a thymine at a position corresponding to position 6,236 according to SEQ ID NO: 38 or its complement; and d) detecting the detectable label.

[0087] In some embodiments, the assay comprises: a) amplifying at least a portion of a PIEZO1 nucleic acid molecule or its complement 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 its complement; and / or a thymine at a position corresponding to position 7,526 according to SEQ ID NO: 39 or its complement; b) labeling the amplified nucleic acid molecule with a detectable label; c) contacting the labeled nucleic acid molecule with a support comprising a variation-specific probe, wherein the variation-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 its complement; and / or a thymine at a position corresponding to position 7,526 according to SEQ ID NO: 39 or its complement; and d) detecting the detectable label.

[0088] In some embodiments, the assay comprises: a) amplifying at least a portion of a PIEZO1 nucleic acid molecule or its complement 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 its complement; and / or a thymine at a position corresponding to position 1,336 according to SEQ ID NO:40 or its complement; b) labeling the amplified nucleic acid molecule with a detectable label; c) contacting the labeled nucleic acid molecule with a support comprising a variation-specific probe, wherein the variation-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 its complement; and / or a thymine at a position corresponding to position 1,336 according to SEQ ID NO:40 or its complement; and d) detecting the detectable label.

[0089] In some embodiments, the assay comprises: a) amplifying at least a portion of a PIEZO1 nucleic acid molecule or its complement in a biological sample, the amplified portion comprising a uracil at a position corresponding to position 361 according to SEQ ID NO:21 or its complement; and / or a thymine at a position corresponding to position 361 according to SEQ ID NO:41 or its complement; b) labeling the amplified nucleic acid molecule with a detectable label; c) contacting the labeled nucleic acid molecule with a support comprising a variation-specific probe, the variation-specific probe comprising 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 its complement; and / or a thymine at a position corresponding to position 361 according to SEQ ID NO:41 or its complement; and d) detecting the detectable label.

[0090] In some embodiments, the assay comprises: a) amplifying at least a portion of a PIEZO1 nucleic acid molecule or its complement in a biological sample, the amplified portion comprising a uracil at a position corresponding to position 7,776 according to SEQ ID NO:22 or its complement; and / or a thymine at a position corresponding to position 7,776 according to SEQ ID NO:42 or its complement; b) labeling the amplified nucleic acid molecule with a detectable label; c) contacting the labeled nucleic acid molecule with a support comprising a variation-specific probe, the variation-specific probe comprising 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 its complement; and / or a thymine at a position corresponding to position 7,776 according to SEQ ID NO:42 or its complement; and d) detecting the detectable label.

[0091] In some embodiments, the assay comprises: a) amplifying at least a portion of a PIEZO1 genomic nucleic acid molecule or a complement thereof in a biological sample, the portion comprising 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.

[0092] In some embodiments, the assay comprises: a) detecting PIEZO1 in a biological sample; amplifying at least a portion of an mRNA molecule or its complement, the portion comprising uracil at a position corresponding to position 7,785 according to SEQ ID NO:13 or its complement; uracil at a position corresponding to position 1,095 according to SEQ ID NO:14 or its complement; uracil at a position corresponding to position 6,073 according to SEQ ID NO:15 or its complement; uracil at a position corresponding to position 1,336 according to SEQ ID NO:16 or its complement; uracil at a position corresponding to position 6,073 according to SEQ ID NO:17 or its complement; uracil at a position corresponding to position 6,236 according to SEQ ID NO:18 or its complement; uracil at a position corresponding to position 7,526 according to SEQ ID NO:19 or its complement; uracil at a position corresponding to position 1,336 according to SEQ ID NO:20 or its complement; uracil at a position corresponding to position 361 according to SEQ ID NO:21 or its complement; or uracil at a position corresponding to position 7,776 according to SEQ ID NO:22 or its complement. a) amplifying the nucleic acid molecule; b) labeling the amplified nucleic acid molecule with a detectable label; c) contacting the labeled nucleic acid molecule with a support comprising a variation-specific probe, the variation-specific probe being selected from the group consisting of uracil at a position corresponding to position 7,785 according to SEQ ID NO:13 or its complement; uracil at a position corresponding to position 1,095 according to SEQ ID NO:14 or its complement; uracil at a position corresponding to position 6,073 according to SEQ ID NO:15 or its complement; uracil at a position corresponding to position 1,336 according to SEQ ID NO:16 or its complement; uracil at a position corresponding to position 6,073 according to SEQ ID NO:17 or its complement; uracil at a position corresponding to position 6,236 according to SEQ ID NO:18 or its complement; uracil at a position corresponding to position 7,526 according to SEQ ID NO:19 or its complement; uracil at a position corresponding to position 1,336 according to SEQ ID NO:20 or its complement; uracil at a position corresponding to position 361 according to SEQ ID NO:21 or its complement;or a nucleotide sequence that hybridizes under stringent conditions to a nucleotide sequence of the amplified nucleic acid molecule that contains a uracil at a position corresponding to position 7,776 according to SEQ ID NO: 22 or its complement; and d) detecting the detectable label.

[0093] In some embodiments, the assay comprises: a) detecting PIEZO1 mRNA produced from an mRNA molecule in a biological sample; amplifying at least a portion of a cDNA molecule or its complement, the portion 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. amplifying; b) labeling the amplified nucleic acid molecule with a detectable label; c) contacting the labeled nucleic acid molecule with a support comprising a variation-specific probe, the variation-specific probe being selected from the group consisting of 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 nucleotide sequence that hybridizes under stringent conditions to a nucleotide sequence of the amplified nucleic acid molecule that contains a thymine at a position corresponding to position 7,776 according to SEQ ID NO: 42 or its complement; and d) detecting the detectable label;

[0094] In some embodiments, the nucleic acid molecule in the sample is mRNA, and the mRNA is reverse transcribed into cDNA prior to the amplification step. In some embodiments, the assay comprises contacting a PIEZO1 nucleic acid molecule or its complement in a biological sample with a variation-specific probe comprising a detectable label, wherein the variation-specific probe comprises a nucleotide sequence that hybridizes under stringent conditions to a nucleotide sequence of a PIEZO1 nucleic acid molecule or its complement comprising: a thymine at a position corresponding to position 69,579 according to SEQ ID NO:2 or its complement; a uracil at a position corresponding to position 7,785 according to SEQ ID NO:13 or its complement; and / or a thymine at a position corresponding to position 7,785 according to SEQ ID NO:33 or its complement; and detecting the detectable label.

[0095] In some embodiments, the assay includes contacting a PIEZO1 nucleic acid molecule or its complement in a biological sample with a mutation-specific probe comprising a detectable label, wherein the mutation-specific probe comprises a nucleotide sequence that hybridizes under stringent conditions to a nucleotide sequence of the PIEZO1 nucleic acid molecule or its complement comprising uracil at a position corresponding to position 1,095 according to SEQ ID NO: 14 or its complement; and / or thymine at a position corresponding to position 1,095 according to SEQ ID NO: 34 or its complement; and detecting the detectable label.

[0096] In some embodiments, the assay comprises contacting a PIEZO1 nucleic acid molecule or its complement in a biological sample with a mutation-specific probe comprising a detectable label, wherein the mutation-specific probe comprises a nucleotide sequence that hybridizes under stringent conditions to a nucleotide sequence of the PIEZO1 nucleic acid molecule or its complement comprising uracil at a position corresponding to position 6,073 according to SEQ ID NO: 15 or its complement; and / or thymine at a position corresponding to position 6,073 according to SEQ ID NO: 35 or its complement; and detecting the detectable label.

[0097] In some embodiments, the assay comprises contacting a PIEZO1 nucleic acid molecule or its complement in a biological sample with a mutation-specific probe comprising a detectable label, wherein the mutation-specific probe comprises a nucleotide sequence that hybridizes under stringent conditions to a nucleotide sequence of the PIEZO1 nucleic acid molecule or its complement comprising uracil at a position corresponding to position 1,336 according to SEQ ID NO: 16 or its complement; and / or thymine at a position corresponding to position 1,336 according to SEQ ID NO: 36 or its complement; and detecting the detectable label.

[0098] In some embodiments, the assay includes contacting a PIEZO1 nucleic acid molecule or its complement in a biological sample with a mutation-specific probe comprising a detectable label, wherein the mutation-specific probe comprises a nucleotide sequence that hybridizes under stringent conditions to a nucleotide sequence of the PIEZO1 nucleic acid molecule or its complement comprising uracil at a position corresponding to position 6,073 according to SEQ ID NO: 17 or its complement; and / or thymine at a position corresponding to position 6,073 according to SEQ ID NO: 37 or its complement; and detecting the detectable label.

[0099] In some embodiments, the assay comprises contacting a PIEZO1 nucleic acid molecule or its complement in a biological sample with a mutation-specific probe comprising a detectable label, wherein the mutation-specific probe comprises a nucleotide sequence that hybridizes under stringent conditions to a nucleotide sequence of the PIEZO1 nucleic acid molecule or its complement comprising uracil at a position corresponding to position 6,236 according to SEQ ID NO: 18 or its complement; and / or thymine at a position corresponding to position 6,236 according to SEQ ID NO: 38 or its complement; and detecting the detectable label.

[0100] In some embodiments, the assay comprises contacting a PIEZO1 nucleic acid molecule or its complement in a biological sample with a mutation-specific probe comprising a detectable label, wherein the mutation-specific probe comprises a nucleotide sequence that hybridizes under stringent conditions to a nucleotide sequence of the PIEZO1 nucleic acid molecule or its complement comprising uracil at a position corresponding to position 7,526 according to SEQ ID NO: 19 or its complement; and / or thymine at a position corresponding to position 7,526 according to SEQ ID NO: 39 or its complement; and detecting the detectable label.

[0101] In some embodiments, the assay includes contacting a PIEZO1 nucleic acid molecule or its complement in a biological sample with a mutation-specific probe comprising a detectable label, wherein the mutation-specific probe comprises a nucleotide sequence that hybridizes under stringent conditions to a nucleotide sequence of the PIEZO1 nucleic acid molecule or its complement comprising uracil at a position corresponding to position 1,336 according to SEQ ID NO: 20 or its complement; and / or thymine at a position corresponding to position 1,336 according to SEQ ID NO: 40 or its complement; and detecting the detectable label.

[0102] In some embodiments, the assay comprises contacting a PIEZO1 nucleic acid molecule or its complement in a biological sample with a mutation-specific probe comprising a detectable label, wherein the mutation-specific probe comprises a nucleotide sequence that hybridizes under stringent conditions to a nucleotide sequence of the PIEZO1 nucleic acid molecule or its complement comprising uracil at a position corresponding to position 361 according to SEQ ID NO:21 or its complement; and / or thymine at a position corresponding to position 361 according to SEQ ID NO:41 or its complement; and detecting the detectable label.

[0103] In some embodiments, the assay comprises contacting a PIEZO1 nucleic acid molecule or its complement in a biological sample with a mutation-specific probe comprising a detectable label, wherein the mutation-specific probe comprises a nucleotide sequence that hybridizes under stringent conditions to a nucleotide sequence of the PIEZO1 nucleic acid molecule or its complement comprising uracil at a position corresponding to position 7,776 according to SEQ ID NO: 22 or its complement; and / or thymine at a position corresponding to position 7,776 according to SEQ ID NO: 42 or its complement; and detecting the detectable label.

[0104] In some embodiments, the assay comprises contacting a PIEZO1 genomic nucleic acid molecule or its complement in a biological sample with a mutation-specific probe comprising a detectable label, wherein the mutation-specific probe comprises a nucleotide sequence that hybridizes under stringent conditions to a nucleotide sequence of the PIEZO1 genomic nucleic acid molecule or its complement comprising a thymine at a position corresponding to position 69,579 according to SEQ ID NO:2 or its complement; and detecting the detectable label.

[0105] In some embodiments, the assay comprises contacting a PIEZO1 mRNA molecule or its complement in a biological sample with a variation-specific probe comprising a detectable label, the variation-specific probe 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 1,336 according to SEQ ID NO:17 or its complement. the nucleotide sequence of a PIEZO1 mRNA molecule or its complement comprising: uracil at a position corresponding to position 6,073 according to SEQ ID NO:18 or its complement; uracil at a position corresponding to position 6,236 according to SEQ ID NO:19 or its complement; uracil at a position corresponding to position 1,336 according to SEQ ID NO:20 or its complement; uracil at a position corresponding to position 361 according to SEQ ID NO:21 or its complement; or uracil at a position corresponding to position 7,776 according to SEQ ID NO:22 or its complement; and detecting the detectable label.

[0106] In some embodiments, the assay comprises assaying for PIEZO1 generated from mRNA molecules in a biological sample. contacting the cDNA molecule or its complement with a variation-specific probe comprising a detectable label, the variation-specific probe comprising a PIEZO1 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; contacting a cDNA molecule comprising a nucleotide sequence that hybridizes under stringent conditions to a nucleotide sequence of the cDNA molecule or its complement; and detecting the detectable label.

[0107] In some embodiments, the PIEZO1 nucleic acid molecule is present in a cell obtained from the subject. Variation-specific polymerase chain reaction techniques can be used to detect mutations such as SNPs in nucleotide sequences. Variation-specific primers can be used because DNA polymerase will not extend if there is a mismatch with the template.

[0108] In some embodiments, the assay comprises RNA sequencing (RNA-Seq). In some embodiments, the assay also comprises reverse transcribing the mRNA into cDNA, for example, by reverse transcriptase polymerase chain reaction (RT-PCR).

[0109] In some embodiments, the method utilizes probes and primers of sufficient nucleotide length to bind to the target nucleotide sequence and specifically detect and / or identify polynucleotides including PIEZO1 variant genomic nucleic acid molecules, variant mRNA molecules, or variant cDNA molecules. Hybridization or reaction conditions can be determined by the operator to achieve this result. The nucleotide length can be any length sufficient for use in the selected detection method, including any assay described or exemplified herein. Such probes and primers can specifically hybridize to the target nucleotide sequence under high stringency hybridization conditions. Probes and primers can have a perfect nucleotide sequence identity of consecutive nucleotides in the target nucleotide sequence, but probes that are different from the target nucleotide sequence and retain the ability to specifically detect and / or identify the target nucleotide sequence can be designed by conventional methods. The probes and primers can have about 80%, about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or 100% sequence identity or complementarity to the nucleotide sequence of the target nucleic acid molecule.

[0110] In some embodiments, to determine whether a PIEZO1 nucleic acid molecule (genomic nucleic acid molecule, mRNA molecule, or cDNA molecule) in a biological sample, or a complement thereof, contains a nucleotide sequence that includes 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 is subjected to a nucleotide sequence analysis that includes 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 a second primer derived from a 3' flanking sequence adjacent to 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, to generate an amplicon indicating the presence of a SNP at a position encoding 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. In some embodiments, the amplicon can range in length from the combination of the primer pair and one nucleotide base pair to any length of an amplicon that can be generated by a DNA amplification protocol. This distance can range from one nucleotide base pair to the limit of the amplification reaction, or up to about 20,000 nucleotide base pairs.Optionally, the primer pair flanks a region comprising 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 a 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.

[0111] In some embodiments, to determine whether a PIEZO1 nucleic acid molecule (genomic nucleic acid molecule, mRNA molecule, or cDNA molecule), or its complement, in a biological sample contains a nucleotide sequence that includes uracil at a position corresponding to position 1,095 according to SEQ ID NO: 14 (mRNA molecule), or thymine 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 that includes a first primer derived from a 5' flanking sequence adjacent to uracil at a position corresponding to position 1,095 according to SEQ ID NO: 14, or thymine at a position corresponding to position 1,095 according to SEQ ID NO: 34, and a second primer derived from a 3' flanking sequence adjacent to uracil at a position corresponding to position 1,095 according to SEQ ID NO: 14, or thymine at a position corresponding to position 1,095 according to SEQ ID NO: 34, to generate an amplicon indicative of the presence of a SNP at a position that codes for uracil at a position corresponding to position 1,095 according to SEQ ID NO: 14, or thymine at a position corresponding to position 1,095 according to SEQ ID NO: 34. In some embodiments, the amplicon can range in length from the combination of the primer pair and one nucleotide base pair to any length of the amplicon that can be generated by the DNA amplification protocol. This distance can range from one nucleotide base pair to the limit of the amplification reaction, or up to about 20,000 nucleotide base pairs. Optionally, the primer pair flanks a region that includes a uracil at a position corresponding to position 1,095 according to SEQ ID NO: 14, or a thymine 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 uracil at a position corresponding to position 1,095 according to SEQ ID NO: 14, or a thymine at a position corresponding to position 1,095 according to SEQ ID NO: 34.

[0112] In some embodiments, to determine whether a PIEZO1 nucleic acid molecule (genomic nucleic acid molecule, mRNA molecule, or cDNA molecule), or its complement, in a biological sample contains a nucleotide sequence that includes uracil at a position corresponding to position 6,073 according to SEQ ID NO: 15 (mRNA molecule), or thymine 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 that includes a first primer derived from a 5' flanking sequence adjacent to uracil at a position corresponding to position 6,073 according to SEQ ID NO: 15, or thymine at a position corresponding to position 6,073 according to SEQ ID NO: 35, and a second primer derived from a 3' flanking sequence adjacent to uracil at a position corresponding to position 6,073 according to SEQ ID NO: 15, or thymine at a position corresponding to position 6,073 according to SEQ ID NO: 35, to generate an amplicon indicative of the presence of a SNP at a position that codes for uracil at a position corresponding to position 6,073 according to SEQ ID NO: 15, or thymine at a position corresponding to position 6,073 according to SEQ ID NO: 35. In some embodiments, the amplicon can range in length from the combination of the primer pair and one nucleotide base pair to any length of the amplicon that can be generated by the DNA amplification protocol. This distance can range from one nucleotide base pair to the limit of the amplification reaction, or up to about 20,000 nucleotide base pairs. Optionally, the primer pair flanks a region that includes a uracil at a position corresponding to position 6,073 according to SEQ ID NO: 15, or a thymine 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 uracil at a position corresponding to position 6,073 according to SEQ ID NO: 15, or a thymine at a position corresponding to position 6,073 according to SEQ ID NO: 35.

[0113] In some embodiments, to determine whether a PIEZO1 nucleic acid molecule (genomic nucleic acid molecule, mRNA molecule, or cDNA molecule), or its complement, in a biological sample contains a nucleotide sequence that includes uracil at a position corresponding to position 1,336 according to SEQ ID NO: 16 (mRNA molecule) or thymine 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 including a first primer derived from a 5' flanking sequence adjacent to uracil at a position corresponding to position 1,336 according to SEQ ID NO: 16 or thymine at a position corresponding to position 1,336 according to SEQ ID NO: 36, and a second primer derived from a 3' flanking sequence adjacent to uracil at a position corresponding to position 1,336 according to SEQ ID NO: 16 or thymine at a position corresponding to position 1,336 according to SEQ ID NO: 36, to generate an amplicon indicative of the presence of a SNP at a position that codes for uracil at a position corresponding to position 1,336 according to SEQ ID NO: 16 or thymine at a position corresponding to position 1,336 according to SEQ ID NO: 36. In some embodiments, the amplicon can range in length from the combination of the primer pair and one nucleotide base pair to any length of the amplicon that can be generated by the DNA amplification protocol. This distance can range from one nucleotide base pair to the limit of the amplification reaction, or up to about 20,000 nucleotide base pairs. Optionally, the primer pair flanks a region that includes a uracil at a position corresponding to position 1,336 according to SEQ ID NO: 16, or a thymine 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 uracil at a position corresponding to position 1,336 according to SEQ ID NO: 16, or a thymine at a position corresponding to position 1,336 according to SEQ ID NO: 36.

[0114] In some embodiments, to determine whether a PIEZO1 nucleic acid molecule (genomic nucleic acid molecule, mRNA molecule, or cDNA molecule) in a biological sample, or its complement, contains a nucleotide sequence, uracil at a position corresponding to position 6,073 according to SEQ ID NO: 17 (mRNA molecule), or thymine 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 including a first primer derived from a 5' flanking sequence adjacent to the uracil at a position corresponding to position 6,073 according to SEQ ID NO: 17 or thymine at a position corresponding to position 6,073 according to SEQ ID NO: 37, and a second primer derived from a 3' flanking sequence adjacent to the uracil at a position corresponding to position 6,073 according to SEQ ID NO: 17 or thymine at a position corresponding to position 6,073 according to SEQ ID NO: 37, to generate an amplicon indicative of the presence of a SNP at a position encoding the uracil at a position corresponding to position 6,073 according to SEQ ID NO: 17 or thymine at a position corresponding to position 6,073 according to SEQ ID NO: 37. In some embodiments, the amplicon can range in length from the combination of the primer pair and one nucleotide base pair to any length of the amplicon that can be generated by the DNA amplification protocol. This distance can range from one nucleotide base pair to the limit of the amplification reaction, or up to about 20,000 nucleotide base pairs. Optionally, the primer pair flanks a region that includes a uracil at a position corresponding to position 6,073 according to SEQ ID NO: 17, or a thymine 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 uracil at a position corresponding to position 6,073 according to SEQ ID NO: 17, or a thymine at a position corresponding to position 6,073 according to SEQ ID NO: 37.

[0115] In some embodiments, to determine whether a PIEZO1 nucleic acid molecule (genomic nucleic acid molecule, mRNA molecule, or cDNA molecule), or its complement, in a biological sample contains a nucleotide sequence that includes uracil at a position corresponding to position 6,236 according to SEQ ID NO: 18 (mRNA molecule) or thymine 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 that includes a first primer derived from a 5' flanking sequence adjacent to uracil at a position corresponding to position 6,236 according to SEQ ID NO: 18 or thymine at a position corresponding to position 6,236 according to SEQ ID NO: 38, and a second primer derived from a 3' flanking sequence adjacent to uracil at a position corresponding to position 6,236 according to SEQ ID NO: 18 or thymine at a position corresponding to position 6,236 according to SEQ ID NO: 38, to generate an amplicon indicating the presence of a SNP at a position that codes for uracil at a position corresponding to position 6,236 according to SEQ ID NO: 18 or thymine at a position corresponding to position 6,236 according to SEQ ID NO: 38. In some embodiments, the amplicon can range in length from the combination of the primer pair and one nucleotide base pair to any length of the amplicon that can be generated by the DNA amplification protocol. This distance can range from one nucleotide base pair to the limit of the amplification reaction, or up to about 20,000 nucleotide base pairs. Optionally, the primer pair flanks a region that includes a uracil at a position corresponding to position 6,236 according to SEQ ID NO: 18, or a thymine 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 uracil at a position corresponding to position 6,236 according to SEQ ID NO: 18, or a thymine at a position corresponding to position 6,236 according to SEQ ID NO: 38.

[0116] In some embodiments, to determine whether a PIEZO1 nucleic acid molecule (genomic nucleic acid molecule, mRNA molecule, or cDNA molecule) in a biological sample, or its complement, contains a nucleotide sequence, uracil at a position corresponding to position 7,526 according to SEQ ID NO: 19 (mRNA molecule), or thymine 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 including a first primer derived from a 5' flanking sequence adjacent to the uracil at a position corresponding to position 7,526 according to SEQ ID NO: 19 or thymine at a position corresponding to position 7,526 according to SEQ ID NO: 39, and a second primer derived from a 3' flanking sequence adjacent to the uracil at a position corresponding to position 7,526 according to SEQ ID NO: 19 or thymine at a position corresponding to position 7,526 according to SEQ ID NO: 39, to generate an amplicon indicating the presence of a SNP at a position encoding the uracil at a position corresponding to position 7,526 according to SEQ ID NO: 19 or thymine at a position corresponding to position 7,526 according to SEQ ID NO: 39. In some embodiments, the amplicon can range in length from the combination of the primer pair and one nucleotide base pair to any length of the amplicon that can be generated by the DNA amplification protocol. This distance can range from one nucleotide base pair to the limit of the amplification reaction, or up to about 20,000 nucleotide base pairs. Optionally, the primer pair flanks a region that includes a uracil at a position corresponding to position 7,526 according to SEQ ID NO: 19, or a thymine 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 uracil at a position corresponding to position 7,526 according to SEQ ID NO: 19, or a thymine at a position corresponding to position 7,526 according to SEQ ID NO: 39.

[0117] In some embodiments, to determine whether a PIEZO1 nucleic acid molecule (genomic nucleic acid molecule, mRNA molecule, or cDNA molecule), or its complement, in a biological sample contains a nucleotide sequence, uracil at a position corresponding to position 1,336 according to SEQ ID NO:20 (mRNA molecule), or thymine 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 a 5' flanking sequence adjacent to the uracil at a position corresponding to position 1,336 according to SEQ ID NO:20, or thymine at a position corresponding to position 1,336 according to SEQ ID NO:40, and a second primer derived from a 3' flanking sequence adjacent to the uracil at a position corresponding to position 1,336 according to SEQ ID NO:20, or thymine at a position corresponding to position 1,336 according to SEQ ID NO:40, to generate an amplicon indicative of the presence of a SNP at a position encoding the uracil at a position corresponding to position 1,336 according to SEQ ID NO:20, or thymine at a position corresponding to position 1,336 according to SEQ ID NO:40. In some embodiments, the amplicon can range in length from the combination of the primer pair and one nucleotide base pair to any length of the amplicon that can be generated by the DNA amplification protocol. This distance can range from one nucleotide base pair to the limit of the amplification reaction, or up to about 20,000 nucleotide base pairs. Optionally, the primer pair flanks a region that includes a uracil at a position corresponding to position 1,336 according to SEQ ID NO:20, or a thymine 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 uracil at a position corresponding to position 1,336 according to SEQ ID NO:20, or a thymine at a position corresponding to position 1,336 according to SEQ ID NO:40.

[0118] In some embodiments, to determine whether a PIEZO1 nucleic acid molecule (genomic nucleic acid molecule, mRNA molecule, or cDNA molecule), or its complement, in a biological sample contains a nucleotide sequence, uracil at a position corresponding to position 361 according to SEQ ID NO:21 (mRNA molecule), or thymine 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 a 5' flanking sequence adjacent to the uracil at a position corresponding to position 361 according to SEQ ID NO:21, or thymine at a position corresponding to position 361 according to SEQ ID NO:41, and a second primer derived from a 3' flanking sequence adjacent to the uracil at a position corresponding to position 361 according to SEQ ID NO:21, or thymine at a position corresponding to position 361 according to SEQ ID NO:41, to generate an amplicon indicative of the presence of a SNP at a position encoding the uracil at a position corresponding to position 361 according to SEQ ID NO:21, or thymine at a position corresponding to position 361 according to SEQ ID NO:41. In some embodiments, the amplicon can range in length from the combination of the primer pair and one nucleotide base pair to any length of the amplicon that can be generated by the DNA amplification protocol. This distance can range from one nucleotide base pair to the limit of the amplification reaction, or up to about 20,000 nucleotide base pairs. Optionally, the primer pair flanks a region that includes a uracil at a position corresponding to position 361 according to SEQ ID NO:21, or a thymine 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 uracil at a position corresponding to position 361 according to SEQ ID NO:21, or a thymine at a position corresponding to position 361 according to SEQ ID NO:41.

[0119] In some embodiments, to determine whether a PIEZO1 nucleic acid molecule (genomic nucleic acid molecule, mRNA molecule, or cDNA molecule), or its complement, in a biological sample contains a nucleotide sequence that includes uracil at a position corresponding to position 7,776 according to SEQ ID NO:22 (mRNA molecule) or thymine 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 including a first primer derived from a 5' flanking sequence adjacent to uracil at a position corresponding to position 7,776 according to SEQ ID NO:22 or thymine at a position corresponding to position 7,776 according to SEQ ID NO:42 and a second primer derived from a 3' flanking sequence adjacent to uracil at a position corresponding to position 7,776 according to SEQ ID NO:22 or thymine at a position corresponding to position 7,776 according to SEQ ID NO:42 to generate an amplicon indicating the presence of a SNP at a position that codes for uracil at a position corresponding to position 7,776 according to SEQ ID NO:22 or thymine at a position corresponding to position 7,776 according to SEQ ID NO:42. In some embodiments, the amplicon can range in length from the combination of the primer pair and one nucleotide base pair to any length of the amplicon that can be generated by a DNA amplification protocol. The distance can range from one nucleotide base pair to the limit of the amplification reaction, or up to about 20,000 nucleotide base pairs. Optionally, the primer pair flanks a region that includes a uracil at a position corresponding to position 7,776 according to SEQ ID NO:22, or a thymine 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 uracil at a position corresponding to position 7,776 according to SEQ ID NO:22, or a thymine at a position corresponding to position 7,776 according to SEQ ID NO:42.

[0120] Similar amplicons can be generated from mRNA and / or cDNA sequences. PCR primer pairs can be generated from known sequences by using computer programs designed for that purpose, such as the PCR primer analysis tools in Vector NTI version 10 (Informax Inc., Bethesda Md.); PrimerSelect (DNASTAR Inc., Madison, Wis.); and Primer3 (Version 0.4.0.COPYRGT., 1991, Whitehead Institute for Biomedical Research, Cambridge, Mass.). Sequences can also be visually inspected and primers manually specified using known guidelines.

[0121] 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 (fluorescence in situ hybridization (FISH)), including using labeled primers or probes directed to purified DNA, amplified DNA, and fixed cell preparations. In some methods, the target nucleic acid molecule can be amplified prior to or simultaneously with detection. Illustrative examples of nucleic acid amplification techniques include, but are not limited to, polymerase chain reaction (PCR), ligase chain reaction (LCR), strand displacement amplification (SDA), and nucleic acid sequence-based amplification (NASBA). Other methods include, but are not limited to, ligase chain reaction, strand displacement amplification, and thermophilic SDA (tSDA).

[0122] Hybridization techniques may use stringent conditions so that the probe or primer hybridizes specifically to its target. In some embodiments, a polynucleotide primer or probe under stringent conditions will hybridize to its target sequence detectably higher than other non-target sequences, for example, at least 2-fold, at least 3-fold, at least 4-fold, or more than background (including more than 10-fold above background). In some embodiments, a polynucleotide primer or probe under stringent conditions will hybridize to its target sequence detectably higher than other nucleotide sequences at least 2-fold. In some embodiments, a polynucleotide primer or probe under stringent conditions will hybridize to its target sequence detectably higher than other nucleotide sequences at least 3-fold. In some embodiments, a polynucleotide primer or probe under stringent conditions will hybridize to its target sequence detectably higher than other nucleotide sequences at least 4-fold. In some embodiments, a polynucleotide primer or probe under stringent conditions will hybridize to its target nucleotide sequence to a detectably greater extent than other nucleotide sequences, more than 10-fold above background. Stringent conditions are sequence-dependent and will be different in different circumstances.

[0123] Suitable stringency conditions that promote DNA hybridization, such as 6× sodium chloride / sodium citrate (SSC) at about 45° C., followed by a 2× SSC wash at 50° C., are known or can be found in Current Protocols in Molecular Biology, John Wiley & Sons, NY (1989), 6.3.1-6.3.6. Typically, stringent conditions for hybridization and detection include conditions in which the salt concentration is less than about 1.5 M NaCl at pH 7.0-8.3. + ion, typically about 0.01 to 1.0 M Na+ The ionic concentration (or other salts) and the temperature will be at least about 30°C for short probes (e.g., 10-50 nucleotides, etc.) and at least about 60°C for longer probes (e.g., more than 50 nucleotides, etc.). Stringent conditions can also be achieved by the addition of destabilizing agents, such as formamide. Optionally, the wash buffer can contain about 0.1% to about 1% SDS. The duration of hybridization is generally less than about 24 hours, usually about 4 to about 12 hours. The duration of the wash period will be at least long enough to reach equilibrium.

[0124] The present disclosure also provides a method for detecting the presence of a predicted gain-of-function polypeptide of PIEZO1, 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 a predicted gain-of-function (partial or complete). The predicted gain-of-function polypeptide of PIEZO1 can be any of the predicted gain-of-function polypeptides of PIEZO1 described herein. In some embodiments, the method detects the presence of PIEZO1 Pro2,510Leu, Pro2,024Leu, Pro2,079Leu, or Pro92Leu. In some embodiments, the method detects the presence of PIEZO1 Pro2,510Leu.

[0125] In some embodiments, the method includes performing an assay on a biological sample obtained from the subject to determine whether a PIEZO1 polypeptide in the biological sample contains 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.

[0126] In some embodiments, the assay includes sequencing at least a portion of a PIEZO1 polypeptide including a position corresponding to position 2,510 according to SEQ ID NO:47, position 2,024 according to SEQ ID NO:48, position 2,079 according to SEQ ID NO:49, or position 94 according to SEQ ID NO:50.

[0127] In some embodiments, the assay is an immunoassay for detecting the presence of a PIEZO1 polypeptide including a position corresponding to position 2,510 according to SEQ ID NO:47, position 2,024 according to SEQ ID NO:48, position 2,079 according to SEQ ID NO:49, or position 94 according to SEQ ID NO:50.

[0128] In some embodiments, if a subject does not have a predicted gain-of-function polypeptide of PIEZO1, the subject has an increased risk of developing varicose veins or any of saphenous, reticular, spider, or pregnancy-associated varicose veins. In some embodiments, if a subject has a predicted gain-of-function polypeptide of PIEZO1, the subject has a decreased risk of developing varicose veins or any of saphenous, reticular, spider, or pregnancy-associated varicose veins.

[0129] The present disclosure also provides an isolated nucleic acid molecule that hybridizes to a PIEZO1 variant genomic nucleic acid molecule, a PIEZO1 variant mRNA molecule, and / or a PIEZO1 variant cDNA molecule (e.g., any of the genomic variant nucleic acid molecules, mRNA variant molecules, and cDNA variant molecules disclosed herein). In some embodiments, such an isolated nucleic acid molecule hybridizes to a PIEZO1 variant nucleic acid molecule under stringent conditions. Such a nucleic acid molecule can be used, for example, as a probe, primer, mutation-specific probe, or mutation-specific primer described or exemplified herein.

[0130] In some embodiments, the isolated nucleic acid molecule hybridizes to a portion of a PIEZO1 nucleic acid molecule that includes a position corresponding to position 69,579 according to SEQ ID NO:2, position 7,785 according to SEQ ID NO:13, or position 7,785 according to SEQ ID NO:33. In some embodiments, the isolated nucleic acid molecule hybridizes to a portion of a PIEZO1 nucleic acid molecule that includes a position corresponding to position 1,095 according to SEQ ID NO:14, or position 1,095 according to SEQ ID NO:34. In some embodiments, the isolated nucleic acid molecule hybridizes to a portion of a PIEZO1 nucleic acid molecule that includes a position corresponding to position 6,073 according to SEQ ID NO:15, or position 6,073 according to SEQ ID NO:35. In some embodiments, the isolated nucleic acid molecule hybridizes to a portion of a PIEZO1 nucleic acid molecule that includes a position corresponding to position 1,336 according to SEQ ID NO:16, or position 1,336 according to SEQ ID NO:36. In some embodiments, the isolated nucleic acid molecule hybridizes to a portion of the PIEZO1 nucleic acid molecule comprising a position corresponding to 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 the PIEZO1 nucleic acid molecule comprising a position corresponding to 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 the PIEZO1 nucleic acid molecule comprising a position corresponding to 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 the PIEZO1 nucleic acid molecule comprising a position corresponding to 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 the PIEZO1 nucleic acid molecule comprising a position corresponding to 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 PIEZO1 nucleic acid molecule that includes position 7,776 according to SEQ ID NO:22, or a position corresponding to position 7,776 according to SEQ ID NO:42.

[0131] In some embodiments, such isolated nucleic acid molecules comprise at least about 5, at least about 8, at least about 10, at least about 11, at least about 12, at least about 13, at least about 14, at least about 15, at least about 16, at least about 17, at least about 18, at least about 19, at least about 20, at least about 21, at least about 22, at least about 23, at least about 24, at least about 25, at least about 30, at least about 35, at least about 40, at least about 45, at least about 50, at least about 51, at least about 52, at least about 53, at least about 54, at least about 55, at least about 56, at least about 57, at least about 58, at least about 59, at least about 60, at least about 61, at least about 62, at least about 63, at least about 64, at least about 65, at least about 66, at least about 67, at least about 68, at least about 69, at least about 70, at least about 71, at least about 72, at least about 73, at least about 74, at least about 75, at least about 76, at least about 77, at least about 78, at least about 79, at least about 80, at least about 81, at least about 82, at least about 83, at least about 84, at least about 85, at least about 86, at least about 87, at least about 88, at least about 89, at least about 90, at least about 91, at least about 92, at least about 93, at least about 94, at least about 95, at least about 96, at least about 97, at least about 98, at least about 99 at least about 5, 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.

[0132] In some embodiments, the isolated variation-specific probe or variation-specific primer comprises at least about 15 nucleotides, and the variation-specific probe or variation-specific primer comprises a nucleotide sequence that is complementary to the nucleotide sequence of a portion of a PIEZO1 nucleic acid molecule encoding a predicted gain-of-function polypeptide of PIEZO1, or its complement. In some embodiments, the portion comprises a position corresponding to position 69,579 according to SEQ ID NO:2 or its complement, position 7,785 according to SEQ ID NO:13 or its complement, or position 7,785 according to SEQ ID NO:33 or its complement. In some embodiments, the portion comprises a position corresponding to position 1,095 according to SEQ ID NO:14 or its complement, or position 1,095 according to SEQ ID NO:34 or its complement. In some embodiments, the portion comprises a position corresponding to position 6,073 according to SEQ ID NO:15 or its complement, or position 6,073 according to SEQ ID NO:35 or its complement. In some embodiments, the portion comprises a position corresponding to position 1,336 according to SEQ ID NO: 16 or its complement, or position 1,336 according to SEQ ID NO: 36 or its complement. In some embodiments, the portion comprises a position corresponding to position 6,073 according to SEQ ID NO: 17 or its complement, or position 6,073 according to SEQ ID NO: 37 or its complement. In some embodiments, the portion comprises a position corresponding to position 6,236 according to SEQ ID NO: 18 or its complement, or position 6,236 according to SEQ ID NO: 38 or its complement. In some embodiments, the portion comprises a position corresponding to position 7,526 according to SEQ ID NO: 19 or its complement, or position 7,526 according to SEQ ID NO: 39 or its complement. In some embodiments, the portion comprises a position corresponding to position 1,336 according to SEQ ID NO: 20 or its complement, or position 1,336 according to SEQ ID NO: 20 or its complement. In some embodiments, the portion includes position 361 according to SEQ ID NO:21 or its complement, or a position corresponding to position 361 according to SEQ ID NO:21 or its complement. In some embodiments, the portion includes position 7,776 according to SEQ ID NO:22 or its complement, or a position corresponding to position 7,776 according to SEQ ID NO:42 or its complement.

[0133] 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 PIEZO1 variant genomic nucleic acid molecule, a PIEZO1 variant mRNA molecule, and / or a PIEZO1 variant cDNA molecule. In some embodiments, the isolated nucleic acid molecule consists of or comprises about 15 to about 100 nucleotides, or about 15 to about 35 nucleotides. In some embodiments, the isolated nucleic acid molecule consists of or comprises about 15 to about 100 nucleotides. In some embodiments, the isolated nucleic acid molecule consists of or comprises about 15 to about 35 nucleotides.

[0134] In some embodiments, the isolated alteration-specific probe or alteration-specific primer comprises at least about 15 nucleotides, and the alteration-specific probe or alteration-specific primer comprises a nucleotide sequence that is complementary to a portion of a nucleotide sequence of a PIEZO1 variant nucleic acid molecule encoding a predicted gain-of-function polypeptide of PIEZO1, the portion including a position corresponding to position 69,579 according to SEQ ID NO:2 or its complement, position 7,785 according to SEQ ID NO:13 or its complement, or position 7,785 according to SEQ ID NO:33 or its complement. In some embodiments, the portion includes a position corresponding to positions 69,578-69,580 according to SEQ ID NO:2 or its complement, positions 7,784-7,786 according to SEQ ID NO:13 or its complement, and / or positions 7,784-7,786 according to SEQ ID NO:33 or its complement.

[0135] In some embodiments, the isolated alteration-specific probe or alteration-specific primer comprises at least about 15 nucleotides, and the alteration-specific probe or alteration-specific primer comprises a nucleotide sequence that is complementary to a portion of a nucleotide sequence of a PIEZO1 variant nucleic acid molecule encoding a predicted gain-of-function polypeptide of PIEZO1, the portion comprising a position corresponding to position 1,095 according to SEQ ID NO: 14 or its complement, or position 1,095 according to SEQ ID NO: 34 or its complement. In some embodiments, the portion comprises positions 1,094-1,096 according to SEQ ID NO: 14 or its complement, and / or positions 1,094-1,096 according to SEQ ID NO: 34 or its complement.

[0136] In some embodiments, the isolated alteration-specific probe or alteration-specific primer comprises at least about 15 nucleotides, and the alteration-specific probe or alteration-specific primer comprises a nucleotide sequence that is complementary to a portion of a nucleotide sequence of a PIEZO1 variant nucleic acid molecule encoding a predicted gain-of-function polypeptide of PIEZO1, the portion comprising a position corresponding to position 6,073 according to SEQ ID NO: 15 or its complement, or position 6,073 according to SEQ ID NO: 35 or its complement. In some embodiments, the portion comprises a position corresponding to positions 6,072-6,074 according to SEQ ID NO: 15 or its complement, and / or positions 6,072-6,074 according to SEQ ID NO: 35 or its complement.

[0137] In some embodiments, the isolated alteration-specific probe or alteration-specific primer comprises at least about 15 nucleotides, and the alteration-specific probe or alteration-specific primer comprises a nucleotide sequence that is complementary to a portion of a nucleotide sequence of a PIEZO1 variant nucleic acid molecule encoding a predicted gain-of-function polypeptide of PIEZO1, the portion comprising a position corresponding to position 1,336 according to SEQ ID NO: 16 or its complement, or position 1,336 according to SEQ ID NO: 36 or its complement. In some embodiments, the portion comprises positions 1,335-1,337 according to SEQ ID NO: 16 or its complement, and / or positions 1,335-1,337 according to SEQ ID NO: 36 or its complement.

[0138] In some embodiments, the isolated alteration-specific probe or alteration-specific primer comprises at least about 15 nucleotides, and the alteration-specific probe or alteration-specific primer comprises a nucleotide sequence that is complementary to a portion of a nucleotide sequence of a PIEZO1 variant nucleic acid molecule encoding a predicted gain-of-function polypeptide of PIEZO1, the portion comprising a position corresponding to position 6,073 according to SEQ ID NO: 17 or its complement, or position 6,073 according to SEQ ID NO: 37 or its complement. In some embodiments, the portion comprises a position corresponding to positions 6,072-6,074 according to SEQ ID NO: 17 or its complement, and / or positions 6,072-6,074 according to SEQ ID NO: 37 or its complement.

[0139] In some embodiments, the isolated alteration-specific probe or alteration-specific primer comprises at least about 15 nucleotides, and the alteration-specific probe or alteration-specific primer comprises a nucleotide sequence that is complementary to a portion of a nucleotide sequence of a PIEZO1 variant nucleic acid molecule encoding a predicted gain-of-function polypeptide of PIEZO1, the portion comprising a position corresponding to position 6,236 according to SEQ ID NO: 18 or its complement, or position 6,236 according to SEQ ID NO: 38 or its complement. In some embodiments, the portion comprises a position corresponding to positions 6,235-6,237 according to SEQ ID NO: 18 or its complement, and / or positions 6,235-6,237 according to SEQ ID NO: 38 or its complement.

[0140] In some embodiments, the isolated alteration-specific probe or alteration-specific primer comprises at least about 15 nucleotides, and the alteration-specific probe or alteration-specific primer comprises a nucleotide sequence that is complementary to a portion of a nucleotide sequence of a PIEZO1 variant nucleic acid molecule encoding a predicted gain-of-function polypeptide of PIEZO1, the portion comprising a position corresponding to position 7,526 according to SEQ ID NO: 19 or its complement, or position 7,526 according to SEQ ID NO: 39 or its complement. In some embodiments, the portion comprises a position corresponding to positions 7,525-7,527 according to SEQ ID NO: 19 or its complement, and / or positions 7,525-7,527 according to SEQ ID NO: 39 or its complement.

[0141] In some embodiments, the isolated alteration-specific probe or alteration-specific primer comprises at least about 15 nucleotides, and the alteration-specific probe or alteration-specific primer comprises a nucleotide sequence that is complementary to a portion of a nucleotide sequence of a PIEZO1 variant nucleic acid molecule encoding a predicted gain-of-function polypeptide of PIEZO1, the portion comprising a position corresponding to position 1,336 according to SEQ ID NO:20 or its complement, or position 1,336 according to SEQ ID NO:40 or its complement. In some embodiments, the portion comprises a position corresponding to positions 1,335-1,337 according to SEQ ID NO:20 or its complement, and / or positions 1,335-1,337 according to SEQ ID NO:40 or its complement.

[0142] In some embodiments, the isolated alteration-specific probe or alteration-specific primer comprises at least about 15 nucleotides, and the alteration-specific probe or alteration-specific primer comprises a nucleotide sequence that is complementary to a portion of a nucleotide sequence of a PIEZO1 variant nucleic acid molecule encoding a predicted gain-of-function polypeptide of PIEZO1, the portion comprising a position corresponding to position 1,336 according to SEQ ID NO:21 or its complement, or position 1,336 according to SEQ ID NO:41 or its complement. In some embodiments, the portion comprises a position corresponding to positions 360-362 according to SEQ ID NO:21 or its complement, and / or positions 360-362 according to SEQ ID NO:41 or its complement.

[0143] In some embodiments, the isolated alteration-specific probe or alteration-specific primer comprises at least about 15 nucleotides, and the alteration-specific probe or alteration-specific primer comprises a nucleotide sequence that is complementary to a portion of a nucleotide sequence of a PIEZO1 variant nucleic acid molecule encoding a predicted gain-of-function polypeptide of PIEZO1, the portion comprising a position corresponding to position 7,776 according to SEQ ID NO:22 or its complement, or position 7,776 according to SEQ ID NO:42 or its complement. In some embodiments, the portion comprises a position corresponding to positions 7,775-7,777 according to SEQ ID NO:22 or its complement, and / or positions 7,775-7,777 according to SEQ ID NO:42 or its complement.

[0144] In some embodiments, the isolated variation-specific probe or variation-specific primer comprises at least about 15 nucleotides, and the variation-specific probe or variation-specific primer comprises a nucleotide sequence that is complementary to the nucleotide sequence of a portion of a PIEZO1 nucleic acid molecule encoding a predicted gain-of-function polypeptide of PIEZO1, or a complement thereof. In some embodiments, the portion comprises a position corresponding to position 69,579 according to SEQ ID NO:2 or a complement thereof.

[0145] In some embodiments, the portion includes a position corresponding to positions 69,578-69,580 according to SEQ ID NO:2, or its complement. In some embodiments, the portion includes a position corresponding to position 7,785 according to SEQ ID NO:13 or its complement; position 1,095 according to SEQ ID NO:14 or its complement; position 6,073 according to SEQ ID NO:15 or its complement; position 1,336 according to SEQ ID NO:16 or its complement; position 6,073 according to SEQ ID NO:17 or its complement; position 6,236 according to SEQ ID NO:18 or its complement; position 7,526 according to SEQ ID NO:19 or its complement; position 1,336 according to SEQ ID NO:20 or its complement; position 361 according to SEQ ID NO:21 or its complement; or a position corresponding to position 7,776 according to SEQ ID NO:22 or its complement.

[0146] In some embodiments, the portion includes a position corresponding to positions 7,784 to 7,786 according to SEQ ID NO: 13 or its complement; positions 1,094 to 1,096 according to SEQ ID NO: 14 or its complement; positions 6,072 to 6,074 according to SEQ ID NO: 15 or its complement; positions 1,335 to 1,337 according to SEQ ID NO: 16 or its complement; positions 6,072 to 6,074 according to SEQ ID NO: 17 or its complement; positions 6,235 to 6,237 according to SEQ ID NO: 18 or its complement; positions 7,525 to 7,527 according to SEQ ID NO: 19 or its complement; or positions 1,335 to 1,337 according to SEQ ID NO: 20 or its complement; positions 360 to 362 according to SEQ ID NO: 21 or its complement; or positions 7,775 to 7,777 according to SEQ ID NO: 22 or its complement.

[0147] In some embodiments, the portion includes a position corresponding to 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.

[0148] In some embodiments, the portion includes a position corresponding to: position 7,784 to 7,786 according to SEQ ID NO:33 or its complement; position 1,094 to 1,096 according to SEQ ID NO:34 or its complement; position 6,072 to 6,074 according to SEQ ID NO:35 or its complement; position 1,335 to 1,337 according to SEQ ID NO:36 or its complement; position 6,072 to 6,074 according to SEQ ID NO:37 or its complement; position 6,235 to 6,237 according to SEQ ID NO:38 or its complement; position 7,525 to 7,527 according to SEQ ID NO:39 or its complement; position 1,335 to 1,337 according to SEQ ID NO:40 or its complement; position 360 to 362 according to SEQ ID NO:41 or its complement; or position 7,775 to 7,777 according to SEQ ID NO:42 or its complement.

[0149] In some embodiments, the variation-specific probe and the variation-specific primer comprise DNA. In some embodiments, the variation-specific probe and the variation-specific primer comprise RNA.

[0150] In some embodiments, the probes and primers described herein (including mutation-specific probes and mutation-specific primers) have nucleotide sequences that specifically hybridize to any of the nucleic acid molecules disclosed herein, or their complements. In some embodiments, the probes and primers specifically hybridize to any of the nucleic acid molecules disclosed herein under stringent conditions.

[0151] In some embodiments, primers including variation-specific primers can be used in second generation sequencing or high throughput sequencing. In some examples, primers including variation-specific primers can be modified. In particular, primers can include 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 biotinylated primers in the cloning step, and fluorescently labeled primers used in the bead loading and detection steps. Polony sequencing is usually performed using paired-end tag libraries, where each molecule of DNA template is about 135 bp in length. Biotinylated primers are used in the bead loading step and emulsion PCR. Fluorescently labeled degenerate nonamer oligonucleotides are used in the detection step. Adapters can contain 5'-biotin tags for immobilization of DNA libraries to streptavidin-coated beads.

[0152] The probes and primers described herein can be used to detect nucleotide variations in any of the PIEZO1 variant genomic nucleic acid molecules, PIEZO1 variant mRNA molecules, and / or PIEZO1 variant cDNA molecules disclosed herein. The primers described herein can be used to amplify a PIEZO1 variant genomic nucleic acid molecule, a PIEZO1 variant mRNA molecule, or a PIEZO1 variant cDNA molecule, or a fragment thereof.

[0153] The present disclosure also provides a pair of primers comprising any of the primers described above. For example, if the 3' end of one of the primers hybridizes to a cytosine (not a thymine) at a position corresponding to position 69,579 according to SEQ ID NO:1 in a specific PIEZO1 nucleic acid molecule, the presence of the amplified fragment indicates the presence of a PIEZO1 reference genomic nucleic acid molecule. Conversely, if the 3' end of one of the primers hybridizes to a thymine (not a cytosine) at a position corresponding to position 69,579 according to SEQ ID NO:2 in a specific PIEZO1 nucleic acid molecule, the presence of the amplified fragment indicates the presence of a PIEZO1 variant genomic nucleic acid molecule. In some embodiments, the nucleotide of the primer that is complementary to the thymine at a position corresponding to position 69,579 according to SEQ ID NO:2 can be at the 3' end of the primer. Also, if the 3' end of one of the primers hybridizes to a cytosine (not a uracil) at a position corresponding to position 7,785 according to SEQ ID NO:3 in a specific PIEZO1 nucleic acid molecule, the presence of the amplified fragment indicates the presence of a PIEZO1 reference mRNA molecule. Conversely, if the 3' end of one of the primers hybridizes to uracil (not cytosine) at the position corresponding to position 7,785 according to SEQ ID NO: 13 in a particular PIEZO1 mRNA molecule, the presence of the amplified fragment indicates the presence of a PIEZO1 variant mRNA molecule. In some embodiments, the nucleotide of the primer complementary to uracil at the position corresponding to position 7,785 according to SEQ ID NO: 13 can be at the 3' end of the primer. Also, if the 3' end of one of the primers hybridizes to cytosine (not thymine) at the position corresponding to position 7,785 according to SEQ ID NO: 23 in a particular PIEZO1 nucleic acid molecule, the presence of the amplified fragment indicates the presence of a PIEZO1 reference cDNA molecule. Conversely, if the 3' end of one of the primers hybridizes to thymine (not cytosine) at the position corresponding to position 7,785 according to SEQ ID NO: 33 in a particular PIEZO1 cDNA molecule, the presence of the amplified fragment indicates the presence of a PIEZO1 variant cDNA molecule.In some embodiments, the nucleotide of the primer complementary to the thymine at a position corresponding to position 7,785 according to SEQ ID NO:33 may be at the 3' end of the primer.

[0154] The present disclosure also provides a pair of primers comprising any of the primers described above. For example, if the 3' end of one of the primers hybridizes to a cytosine (not a uracil) at a position corresponding to position 1,095 according to SEQ ID NO: 4 in a specific PIEZO1 nucleic acid molecule, the presence of the amplified fragment indicates the presence of a PIEZO1 reference mRNA molecule. Conversely, if the 3' end of one of the primers hybridizes to a uracil (not a cytosine) at a position corresponding to position 1,095 according to SEQ ID NO: 14 in a specific PIEZO1 mRNA molecule, the presence of the amplified fragment indicates the presence of a PIEZO1 variant mRNA molecule. In some embodiments, the nucleotide of the primer that is complementary to the uracil at a position corresponding to position 1,095 according to SEQ ID NO: 14 can be at the 3' end of the primer. Also, if the 3' end of one of the primers hybridizes to a cytosine (not a thymine) at a position corresponding to position 1,095 according to SEQ ID NO: 24 in a specific PIEZO1 nucleic acid molecule, the presence of the amplified fragment indicates the presence of a PIEZO1 reference cDNA molecule. Conversely, if the 3' end of one of the primers hybridizes to a thymine (but not 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 an amplified fragment will indicate the presence of a PIEZO1 variant cDNA molecule. In some embodiments, the nucleotide of the primer complementary to the thymine at the position corresponding to position 1,095 according to SEQ ID NO: 34 can be at the 3' end of the primer.

[0155] The present disclosure also provides a pair of primers comprising any of the primers described above. For example, if the 3' end of one of the primers hybridizes to a cytosine (not a uracil) at a position corresponding to position 6,073 according to SEQ ID NO:5 in a specific PIEZO1 nucleic acid molecule, the presence of the amplified fragment indicates the presence of a PIEZO1 reference mRNA molecule. Conversely, if the 3' end of one of the primers hybridizes to a uracil (not a cytosine) at a position corresponding to position 6,073 according to SEQ ID NO:15 in a specific PIEZO1 mRNA molecule, the presence of the amplified fragment indicates the presence of a PIEZO1 variant mRNA molecule. In some embodiments, the nucleotide of the primer that is complementary to the uracil at a position corresponding to position 6,073 according to SEQ ID NO:15 can be at the 3' end of the primer. Also, if the 3' end of one of the primers hybridizes to a cytosine (not a thymine) at a position corresponding to position 6,073 according to SEQ ID NO:25 in a specific PIEZO1 nucleic acid molecule, the presence of the amplified fragment indicates the presence of a PIEZO1 reference cDNA molecule. Conversely, if the 3' end of one of the primers hybridizes to a thymine (but not 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 an amplified fragment will indicate the presence of a PIEZO1 variant cDNA molecule. In some embodiments, the nucleotide of the primer complementary to the thymine at the position corresponding to position 6,073 according to SEQ ID NO: 35 can be at the 3' end of the primer.

[0156] The present disclosure also provides a pair of primers comprising any of the primers described above. For example, if the 3' end of one of the primers hybridizes to a cytosine (not a uracil) at a position corresponding to position 1,336 according to SEQ ID NO:6 in a specific PIEZO1 nucleic acid molecule, the presence of the amplified fragment indicates the presence of a PIEZO1 reference mRNA molecule. Conversely, if the 3' end of one of the primers hybridizes to a uracil (not a cytosine) at a position corresponding to position 1,336 according to SEQ ID NO:16 in a specific PIEZO1 mRNA molecule, the presence of the amplified fragment indicates the presence of a PIEZO1 variant mRNA molecule. In some embodiments, the nucleotide of the primer that is complementary to the uracil at a position corresponding to position 1,336 according to SEQ ID NO:16 can be at the 3' end of the primer. Also, if the 3' end of one of the primers hybridizes to a cytosine (not a thymine) at a position corresponding to position 1,336 according to SEQ ID NO:26 in a specific PIEZO1 nucleic acid molecule, the presence of the amplified fragment indicates the presence of a PIEZO1 reference cDNA molecule. Conversely, if the 3' end of one of the primers hybridizes to a thymine (but not 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 an amplified fragment will indicate the presence of a PIEZO1 variant cDNA molecule. In some embodiments, the nucleotide of the primer complementary to the thymine at the position corresponding to position 1,336 according to SEQ ID NO: 36 can be at the 3' end of the primer.

[0157] The present disclosure also provides a pair of primers comprising any of the primers described above. For example, if the 3' end of one of the primers hybridizes to a cytosine (not a uracil) at a position corresponding to position 6,073 according to SEQ ID NO: 7 in a specific PIEZO1 nucleic acid molecule, the presence of the amplified fragment indicates the presence of a PIEZO1 reference mRNA molecule. Conversely, if the 3' end of one of the primers hybridizes to a uracil (not a cytosine) at a position corresponding to position 6,073 according to SEQ ID NO: 17 in a specific PIEZO1 mRNA molecule, the presence of the amplified fragment indicates the presence of a PIEZO1 variant mRNA molecule. In some embodiments, the nucleotide of the primer that is complementary to the uracil at a position corresponding to position 6,073 according to SEQ ID NO: 17 can be at the 3' end of the primer. Also, if the 3' end of one of the primers hybridizes to a cytosine (not a thymine) at a position corresponding to position 6,073 according to SEQ ID NO: 27 in a specific PIEZO1 nucleic acid molecule, the presence of the amplified fragment indicates the presence of a PIEZO1 reference cDNA molecule. Conversely, if the 3' end of one of the primers hybridizes to a thymine (but not 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 an amplified fragment will indicate the presence of a PIEZO1 variant cDNA molecule. In some embodiments, the nucleotide of the primer that is complementary to the thymine at the position corresponding to position 6,073 according to SEQ ID NO: 37 can be at the 3' end of the primer.

[0158] The present disclosure also provides a pair of primers comprising any of the primers described above. For example, if the 3' end of one of the primers hybridizes to a cytosine (not a uracil) at a position corresponding to position 6,236 according to SEQ ID NO:8 in a specific PIEZO1 nucleic acid molecule, the presence of the amplified fragment indicates the presence of a PIEZO1 reference mRNA molecule. Conversely, if the 3' end of one of the primers hybridizes to a uracil (not a cytosine) at a position corresponding to position 6,236 according to SEQ ID NO:18 in a specific PIEZO1 mRNA molecule, the presence of the amplified fragment indicates the presence of a PIEZO1 variant mRNA molecule. In some embodiments, the nucleotide of the primer that is complementary to the uracil at a position corresponding to position 6,236 according to SEQ ID NO:18 can be at the 3' end of the primer. Also, if the 3' end of one of the primers hybridizes to a cytosine (not a thymine) at a position corresponding to position 6,236 according to SEQ ID NO:28 in a specific PIEZO1 nucleic acid molecule, the presence of the amplified fragment indicates the presence of a PIEZO1 reference cDNA molecule. Conversely, if the 3' end of one of the primers hybridizes to a thymine (but not 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 an amplified fragment will indicate the presence of a PIEZO1 variant cDNA molecule. In some embodiments, the nucleotide of the primer complementary to the thymine at the position corresponding to position 6,236 according to SEQ ID NO: 38 can be at the 3' end of the primer.

[0159] The present disclosure also provides a pair of primers comprising any of the primers described above. For example, if the 3' end of one of the primers hybridizes to a cytosine (not a uracil) at a position corresponding to position 7,526 according to SEQ ID NO: 9 in a specific PIEZO1 nucleic acid molecule, the presence of the amplified fragment indicates the presence of a PIEZO1 reference mRNA molecule. Conversely, if the 3' end of one of the primers hybridizes to a uracil (not a cytosine) at a position corresponding to position 7,526 according to SEQ ID NO: 19 in a specific PIEZO1 mRNA molecule, the presence of the amplified fragment indicates the presence of a PIEZO1 variant mRNA molecule. In some embodiments, the nucleotide of the primer that is complementary to the uracil at a position corresponding to position 7,526 according to SEQ ID NO: 19 can be at the 3' end of the primer. Also, if the 3' end of one of the primers hybridizes to a cytosine (not a thymine) at a position corresponding to position 7,526 according to SEQ ID NO: 29 in a specific PIEZO1 nucleic acid molecule, the presence of the amplified fragment indicates the presence of a PIEZO1 reference cDNA molecule. Conversely, if the 3' end of one of the primers hybridizes to a thymine (but not 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 an amplified fragment will indicate the presence of a PIEZO1 variant cDNA molecule. In some embodiments, the nucleotide of the primer complementary to the thymine at the position corresponding to position 7,526 according to SEQ ID NO: 39 can be at the 3' end of the primer.

[0160] The present disclosure also provides a pair of primers comprising any of the primers described above. For example, if the 3' end of one of the primers hybridizes to a cytosine (not a uracil) at a position corresponding to position 1,336 according to SEQ ID NO: 10 in a specific PIEZO1 nucleic acid molecule, the presence of the amplified fragment indicates the presence of a PIEZO1 reference mRNA molecule. Conversely, if the 3' end of one of the primers hybridizes to a uracil (not a cytosine) at a position corresponding to position 1,336 according to SEQ ID NO: 20 in a specific PIEZO1 mRNA molecule, the presence of the amplified fragment indicates the presence of a PIEZO1 variant mRNA molecule. In some embodiments, the nucleotide of the primer that is complementary to the uracil at a position corresponding to position 1,336 according to SEQ ID NO: 20 can be at the 3' end of the primer. Also, if the 3' end of one of the primers hybridizes to a cytosine (not a thymine) at a position corresponding to position 1,336 according to SEQ ID NO: 30 in a specific PIEZO1 nucleic acid molecule, the presence of the amplified fragment indicates the presence of a PIEZO1 reference cDNA molecule. Conversely, if the 3' end of one of the primers hybridizes to a thymine (but not 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 an amplified fragment will indicate the presence of a PIEZO1 variant cDNA molecule. In some embodiments, the nucleotide of the primer complementary to the thymine at the position corresponding to position 1,336 according to SEQ ID NO: 40 can be at the 3' end of the primer.

[0161] The present disclosure also provides a pair of primers comprising any of the primers described above. For example, if the 3' end of one of the primers hybridizes to a cytosine (not a uracil) at a position corresponding to position 361 according to SEQ ID NO: 11 in a specific PIEZO1 nucleic acid molecule, the presence of the amplified fragment indicates the presence of a PIEZO1 reference mRNA molecule. Conversely, if the 3' end of one of the primers hybridizes to a uracil (not a cytosine) at a position corresponding to position 361 according to SEQ ID NO: 21 in a specific PIEZO1 mRNA molecule, the presence of the amplified fragment indicates the presence of a PIEZO1 variant mRNA molecule. In some embodiments, the nucleotide of the primer that is complementary to the uracil at a position corresponding to position 361 according to SEQ ID NO: 21 can be at the 3' end of the primer. Also, if the 3' end of one of the primers hybridizes to a cytosine (not a thymine) at a position corresponding to position 361 according to SEQ ID NO: 31 in a specific PIEZO1 nucleic acid molecule, the presence of the amplified fragment indicates the presence of a PIEZO1 reference cDNA molecule. Conversely, if the 3' end of one of the primers hybridizes to a thymine (but not a cytosine) at a position in a particular PIEZO1 cDNA molecule corresponding to position 361 according to SEQ ID NO: 41, the presence of an amplified fragment will indicate the presence of a PIEZO1 variant cDNA molecule. In some embodiments, the nucleotide of the primer complementary to the thymine at the position corresponding to position 361 according to SEQ ID NO: 41 can be at the 3' end of the primer.

[0162] The present disclosure also provides a pair of primers comprising any of the primers described above. For example, if the 3' end of one of the primers hybridizes to a cytosine (not a uracil) at a position corresponding to position 7,776 according to SEQ ID NO: 12 in a specific PIEZO1 nucleic acid molecule, the presence of the amplified fragment indicates the presence of a PIEZO1 reference mRNA molecule. Conversely, if the 3' end of one of the primers hybridizes to a uracil (not a cytosine) at a position corresponding to position 7,776 according to SEQ ID NO: 22 in a specific PIEZO1 mRNA molecule, the presence of the amplified fragment indicates the presence of a PIEZO1 variant mRNA molecule. In some embodiments, the nucleotide of the primer that is complementary to the uracil at a position corresponding to position 7,776 according to SEQ ID NO: 22 can be at the 3' end of the primer. Also, if the 3' end of one of the primers hybridizes to a cytosine (not a thymine) at a position corresponding to position 7,776 according to SEQ ID NO: 32 in a specific PIEZO1 nucleic acid molecule, the presence of the amplified fragment indicates the presence of a PIEZO1 reference cDNA molecule. Conversely, if the 3' end of one of the primers hybridizes to a thymine (but not 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 an amplified fragment will indicate the presence of a PIEZO1 variant cDNA molecule. In some embodiments, the nucleotide of the primer complementary to the thymine at the position corresponding to position 7,776 according to SEQ ID NO: 42 can be at the 3' end of the primer.

[0163] In the context of this disclosure, "specifically hybridizes" means that a probe or primer (e.g., a mutation-specific probe or mutation-specific primer, etc.) does not hybridize to a nucleotide sequence encoding a PIEZO1 reference genomic nucleic acid molecule, a PIEZO1 reference mRNA molecule, and / or a PIEZO1 reference cDNA molecule.

[0164] In any of the embodiments described throughout this disclosure, the probe (e.g., the mutation-specific probe, etc.) may include a label. In some embodiments, the label is a fluorescent label, a radioactive label, or biotin.

[0165] The present disclosure also provides a support comprising a substrate to which any one or more of the probes disclosed herein are attached. A solid support is a solid-state substrate or support to which molecules such as any of the probes disclosed herein can associate. One form of solid support is an array. Another form of solid support is an array detector. An array detector is a solid support to which a plurality of different probes are coupled in an array, grid, or other organized pattern. A form for a solid-state substrate is a microtiter dish, such as a standard 96-well type. In some embodiments, a multi-well glass slide, usually containing one array per well, can be used. In some embodiments, the support is a microarray.

[0166] The present disclosure also provides a molecular complex comprising or consisting of any of the PIEZO1 nucleic acid molecules (genomic nucleic acid molecules, mRNA molecules, or cDNA molecules) described herein, or their complements, and any of the change-specific primers or change-specific probes described herein. In some embodiments, the PIEZO1 nucleic acid molecule (genomic nucleic acid molecule, mRNA molecule, or cDNA molecule) or its complement in the molecular complex is single-stranded. In some embodiments, the PIEZO1 nucleic acid molecule is any of the genomic nucleic acid molecules described herein. In some embodiments, the PIEZO1 nucleic acid molecule is any of the mRNA molecules described herein. In some embodiments, the PIEZO1 nucleic acid molecule is any of the cDNA molecules described herein. In some embodiments, the molecular complex comprises or consists of any of the PIEZO1 nucleic acid molecules (genomic nucleic acid molecules, mRNA molecules, or cDNA molecules) described herein, or their complements, and any of the change-specific primers described herein. In some embodiments, the molecular complex comprises or consists of any of the PIEZO1 nucleic acid molecules (genomic nucleic acid molecules, mRNA molecules, or cDNA molecules) described herein, or their complements, and any of the change-specific probes described herein.

[0167] In some embodiments, the molecular complex comprises a mutation-specific primer or a mutation-specific probe hybridized to a PIEZO1 genomic nucleic acid molecule encoding a predicted gain-of-function polypeptide of PIEZO1, wherein the mutation-specific primer or the mutation-specific probe is hybridized to the PIEZO1 genomic nucleic acid molecule at a position corresponding to position 69,579 according to SEQ ID NO:2 or its complement.

[0168] In some embodiments, the variation-specific primer or variation-specific probe in the molecular complex hybridizes to a CTG codon at a position corresponding to positions 69,578 to 69,580 according to SEQ ID NO:2.

[0169] In some embodiments, the genomic nucleic acid molecule in the molecular complex comprises SEQ ID NO:2. In some embodiments, the molecular complex comprises a mutation-specific primer or a mutation-specific probe hybridized to a PIEZO1 mRNA molecule encoding a predicted gain-of-function polypeptide of PIEZO1, wherein the mutation-specific primer or the mutation-specific probe is hybridized to the PIEZO1 mRNA molecule at a position corresponding to position 7,785 according to SEQ ID NO: 13 or its complement; position 1,095 according to SEQ ID NO: 14 or its complement; position 6,073 according to SEQ ID NO: 15 or its complement; position 1,336 according to SEQ ID NO: 16 or its complement; position 6,073 according to SEQ ID NO: 17 or its complement; position 6,236 according to SEQ ID NO: 18 or its complement; position 7,526 according to SEQ ID NO: 19 or its complement; position 1,336 according to SEQ ID NO: 20 or its complement; position 361 according to SEQ ID NO: 21 or its complement; or position 7,776 according to SEQ ID NO: 22 or its complement.

[0170] In some embodiments, the variation-specific primer or variation-specific probe in the molecular complex comprises a CUG codon at a position corresponding to positions 7,784 to 7,786 according to SEQ ID NO: 13, a CUG codon at a position corresponding to positions 1,094 to 1,096 according to SEQ ID NO: 14, a CUG codon at a position corresponding to positions 6,072 to 6,074 according to SEQ ID NO: 15, a CUG codon at a position corresponding to positions 1,335 to 1,337 according to SEQ ID NO: 16, a CUG codon at a position corresponding to positions 6,072 to 6,074 according to SEQ ID NO: 17, It is hybridized to a CUG codon at a corresponding position, a CUG codon at a position corresponding to positions 6,235 to 6,237 according to SEQ ID NO: 18, a CUG codon at a position corresponding to positions 7,525 to 7,527 according to SEQ ID NO: 19, a CUG codon at a position corresponding to positions 1,335 to 1,337 according to SEQ ID NO: 20, a CUG codon at a position corresponding to positions 360 to 362 according to SEQ ID NO: 21, or a CUG codon at a position corresponding to positions 7,775 to 7,777 according to SEQ ID NO: 22.

[0171] 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.

[0172] In some embodiments, the molecular complex comprises a mutation-specific primer or a mutation-specific probe hybridized to a PIEZO1 cDNA molecule encoding a predicted gain-of-function polypeptide of PIEZO1, wherein the mutation-specific primer or the mutation-specific probe is hybridized to the PIEZO1 cDNA molecule at a position corresponding to: 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.

[0173] In some embodiments, the variation-specific primer or variation-specific probe in the molecular complex comprises a CTG codon at a position corresponding to positions 7,784 to 7,786 according to SEQ ID NO: 33, a CTG codon at a position corresponding to positions 1,094 to 1,096 according to SEQ ID NO: 34, a CTG codon at a position corresponding to positions 6,072 to 6,074 according to SEQ ID NO: 35, a CTG codon at a position corresponding to positions 1,335 to 1,337 according to SEQ ID NO: 36, a CTG codon at a position corresponding to positions 6,072 to 6,074 according to SEQ ID NO: 37, It is hybridized to a CTG codon at a corresponding position, a CTG codon at a position corresponding to positions 6,235 to 6,237 according to SEQ ID NO: 38, a CTG codon at a position corresponding to positions 7,525 to 7,527 according to SEQ ID NO: 39, a CTG codon at a position corresponding to positions 1,335 to 1,337 according to SEQ ID NO: 40, a CTG codon at a position corresponding to positions 360 to 362 according to SEQ ID NO: 41, or a CTG codon at a position corresponding to positions 7,775 to 7,777 according to SEQ ID NO: 42.

[0174] 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.

[0175] In some embodiments, the molecular complex comprises a variation-specific probe or a variation-specific primer that comprises a label. In some embodiments, the label is a fluorescent label, a radioactive label, or biotin. In some embodiments, the molecular complex further comprises a non-human polymerase.

[0176] The nucleotide sequence of the PIEZO1 reference genomic nucleic acid molecule is shown in SEQ ID NO: 1 (ENSG00000103335.22, encompassing chr16:88,715,338-88,785,220 in the GRCh38 / hg38 human genome assembly). With reference to SEQ ID NO: 1, position 69,579 is a cytosine.

[0177] There is a PIEZO1 variant genomic nucleic acid molecule in which the cytosine at position 69,579 is replaced with a thymine. The nucleotide sequence of this PIEZO1 variant genomic nucleic acid molecule is shown in SEQ ID NO:2.

[0178] The nucleotide sequence of the PIEZO1 reference mRNA molecule is shown in SEQ ID NO:3. With reference to SEQ ID NO:3, position 7,785 is a cytosine. The nucleotide sequence of another PIEZO1 reference mRNA molecule is shown in SEQ ID NO:4. With reference to SEQ ID NO:4, position 1,095 is a cytosine. The nucleotide sequence of another PIEZO1 reference mRNA molecule is shown in SEQ ID NO:5. With reference to SEQ ID NO:5, position 6,073 is a cytosine. The nucleotide sequence of another PIEZO1 reference mRNA molecule is shown in SEQ ID NO:6. With reference to SEQ ID NO:6, position 1,336 is a cytosine. The nucleotide sequence of another PIEZO1 reference mRNA molecule is shown in SEQ ID NO:7. With reference to SEQ ID NO:7, position 6,073 is a cytosine. The nucleotide sequence of another PIEZO1 reference mRNA molecule is shown in SEQ ID NO:8. With reference to SEQ ID NO:8, position 6,236 is a cytosine. The nucleotide sequence of another PIEZO1 reference mRNA molecule is shown in SEQ ID NO:9. With reference to SEQ ID NO:9, position 7,526 is a cytosine. The nucleotide sequence of another PIEZO1 reference mRNA molecule is shown in SEQ ID NO:10. With reference to SEQ ID NO:10, position 1,336 is a cytosine. The nucleotide sequence of another PIEZO1 reference mRNA molecule is shown in SEQ ID NO:11. With reference to SEQ ID NO:10, position 361 is a cytosine. The nucleotide sequence of another PIEZO1 reference mRNA molecule is shown in SEQ ID NO:12. With reference to SEQ ID NO:12, position 7,776 is a cytosine.

[0179] There is a PIEZO1 variant mRNA molecule in which the cytosine at position 7,785 is replaced with uracil, the nucleotide sequence of which is shown in SEQ ID NO:13.

[0180] There is another PIEZO1 variant mRNA molecule in which the cytosine at position 1,095 is replaced with uracil. The nucleotide sequence of this PIEZO1 variant mRNA molecule is shown in SEQ ID NO:14.

[0181] There is another PIEZO1 variant mRNA molecule in which the cytosine at position 6,073 is replaced by uracil, the nucleotide sequence of which is shown in SEQ ID NO:15.

[0182] There is another PIEZO1 variant mRNA molecule in which the cytosine at position 1,336 is replaced with uracil. The nucleotide sequence of this PIEZO1 variant mRNA molecule is shown in SEQ ID NO:16.

[0183] There is another PIEZO1 variant mRNA molecule in which the cytosine at position 6,073 is replaced with uracil, the nucleotide sequence of which is shown in SEQ ID NO:17.

[0184] There is another PIEZO1 variant mRNA molecule in which the cytosine at position 6,236 is replaced with uracil. The nucleotide sequence of this PIEZO1 variant mRNA molecule is shown in SEQ ID NO:18.

[0185] There is another PIEZO1 variant mRNA molecule in which the cytosine at position 7,526 is replaced with uracil, the nucleotide sequence of which is shown in SEQ ID NO:19.

[0186] There is another PIEZO1 variant mRNA molecule in which the cytosine at position 1,336 is replaced with uracil. The nucleotide sequence of this PIEZO1 variant mRNA molecule is shown in SEQ ID NO:20.

[0187] There is another PIEZO1 variant mRNA molecule in which the cytosine at position 361 is replaced by uracil. The nucleotide sequence of this PIEZO1 variant mRNA molecule is shown in SEQ ID NO:21.

[0188] There is another PIEZO1 variant mRNA molecule in which the cytosine at position 7,776 is replaced with uracil, the nucleotide sequence of which is shown in SEQ ID NO:22.

[0189] The nucleotide sequence of the PIEZO1 reference cDNA molecule is shown in SEQ ID NO:23. With reference to SEQ ID NO:23, position 7,785 is a cytosine. The nucleotide sequence of another PIEZO1 reference cDNA molecule is shown in SEQ ID NO:24. With reference to SEQ ID NO:24, position 1,095 is a cytosine. The nucleotide sequence of another PIEZO1 reference cDNA molecule is shown in SEQ ID NO:25. With reference to SEQ ID NO:25, position 6,073 is a cytosine. The nucleotide sequence of another PIEZO1 reference cDNA molecule is shown in SEQ ID NO:26. With reference to SEQ ID NO:26, position 1,336 is a cytosine. The nucleotide sequence of another PIEZO1 reference cDNA molecule is shown in SEQ ID NO:27. With reference to SEQ ID NO:27, position 6,073 is a cytosine. The nucleotide sequence of another PIEZO1 reference cDNA molecule is shown in SEQ ID NO:28. With reference to SEQ ID NO:28, position 6,236 is a cytosine. The nucleotide sequence of another PIEZO1 reference cDNA molecule is shown in SEQ ID NO:29. With reference to SEQ ID NO:29, position 7,526 is a cytosine. The nucleotide sequence of another PIEZO1 reference cDNA molecule is shown in SEQ ID NO:30. With reference to SEQ ID NO:30, position 1,336 is a cytosine. The nucleotide sequence of another PIEZO1 reference cDNA molecule is shown in SEQ ID NO:31. With reference to SEQ ID NO:31, position 371 is a cytosine. The nucleotide sequence of another PIEZO1 reference cDNA molecule is shown in SEQ ID NO:32. With reference to SEQ ID NO:32, position 7,776 is a cytosine.

[0190] There is a PIEZO1 variant cDNA molecule in which the cytosine at position 7,785 is replaced with a thymine, the nucleotide sequence of which is shown in SEQ ID NO:33.

[0191] 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 shown in SEQ ID NO:34.

[0192] There is another PIEZO1 variant cDNA molecule in which the cytosine at position 6,073 is replaced with a thymine, the nucleotide sequence of which is shown in SEQ ID NO:35.

[0193] There is another PIEZO1 variant cDNA molecule in which the cytosine at position 1,336 is replaced with a thymine, the nucleotide sequence of which is shown in SEQ ID NO:36.

[0194] There is another PIEZO1 variant cDNA molecule in which the cytosine at position 6,073 is replaced with a thymine, the nucleotide sequence of which is shown in SEQ ID NO:37.

[0195] There is another PIEZO1 variant cDNA molecule in which the cytosine at position 6,236 is replaced with a thymine, the nucleotide sequence of which is shown in SEQ ID NO:38.

[0196] There is another PIEZO1 variant cDNA molecule in which the cytosine at position 7,526 is replaced with a thymine, the nucleotide sequence of which is shown in SEQ ID NO:39.

[0197] 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 shown in SEQ ID NO:40.

[0198] Another PIEZO1 variant cDNA molecule exists in which the cytosine at position 361 is replaced by a thymine. The nucleotide sequence of this PIEZO1 variant cDNA molecule is shown in SEQ ID NO:41.

[0199] There is another PIEZO1 variant cDNA molecule in which the cytosine at position 7,776 is replaced with a thymine, the nucleotide sequence of which is shown in SEQ ID NO:42.

[0200] The genomic nucleic acid molecule, mRNA molecule, and cDNA molecule can be from any organism.For example, the genomic nucleic acid molecule, mRNA molecule, and cDNA molecule can be orthologs from human or another organism, such as non-human mammal, rodent, mouse, or rat.It is understood that gene sequences in a population can differ due to polymorphisms, such as single nucleotide polymorphisms.The examples provided herein are only exemplary sequences.Other sequences are also possible.

[0201] 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 the specific activity of target molecules, or functional polynucleotides can have de novo activity independent of any other molecule.

[0202] The isolated nucleic acid molecules disclosed herein may include RNA, DNA, or both RNA and DNA. The isolated nucleic acid molecules may also be linked or fused to heterologous nucleic acid sequences, such as in a vector, or to a heterologous label. For example, the isolated nucleic acid molecules disclosed herein may be present as exogenous donor sequences in or containing a vector that includes the isolated nucleic acid molecule and a heterologous nucleic acid sequence. The isolated nucleic acid molecules may also be linked or fused to a heterologous label. The label may be directly detectable (e.g., a fluorophore, etc.) or indirectly detectable (e.g., a hapten, enzyme, or fluorophore quencher, etc.). Such labels may be detectable by spectroscopic, photochemical, biochemical, immunochemical, or chemical means. Such labels include, for example, radioactive labels, pigments, dyes, chromogens, spin labels, and fluorescent labels. The label may also be, for example, a chemiluminescent material; a metal-containing material; or an enzyme, in which case an enzyme-dependent secondary generation of a signal occurs. The term "label" may also refer to a "tag" or hapten that can selectively bind to a conjugated molecule such that the conjugated molecule is subsequently added with a substrate and used to generate a detectable signal. For example, biotin can be used as a tag with an avidin or streptavidin conjugate of horseradish peroxidase (HRP) to bind to the tag and examined using a colorimetric substrate (e.g., tetramethylbenzidine (TMB) or the like) or a fluorogenic substrate to detect the presence of HRP. Exemplary labels that can be used as tags to facilitate purification include, but are not limited to, myc, HA, FLAG or 3XFLAG, 6XHis or polyhistidine, glutathione-S-transferase (GST), maltose binding protein, epitope tags, or the Fc portion of an immunoglobulin. Numerous labels include, for example, particles, fluorophores, haptens, enzymes and their calorimetric, fluorescent and chemiluminescent substrates and other labels.

[0203] The isolated nucleic acid molecule, or its complement, may also be present in a host cell. In some embodiments, the host cell may contain 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.

[0204] The disclosed nucleic acid molecules may, for example, contain nucleotides or non-natural or modified nucleotides, such as nucleotide analogs or nucleotide substitutes. Such nucleotides include nucleotides that contain modified bases, sugars, or phosphate groups, or incorporate non-natural sites in their structure. Examples of non-natural nucleotides include, but are not limited to, dideoxynucleotides, biotinylated, aminated, deaminated, alkylated, benzylated, and fluorophore-labeled nucleotides.

[0205] The nucleic acid molecules disclosed herein may also include one or more nucleotide analogs or substitutes. A nucleotide analog is a nucleotide that contains modifications to either the base, sugar, or phosphate moiety. Modifications to the base moiety include, but are not limited to, natural and synthetic modifications of A, C, G, and T / U, as well as different purine or pyrimidine bases, such as pseudouridine, uracil-5-yl, hypoxanthine-9-yl (I), and 2-aminoadenine-9-yl. Modified bases include 5-methylcytosine (5-me-C), 5-hydroxymethylcytosine, xanthine, hypoxanthine, 2-aminoadenine, 6-methyl and other alkyl derivatives of adenine and guanine, 2-propyl and other alkyl derivatives of adenine and guanine, 2-thiouracil, 2-thiothymine and 2-thiocytosine, 5-halouracil and cytosine, 5-propynyluracil and cytosine, 6-azouracil, cytosine and thymine, 5-uracil (sulfonyluracil), and 5-uracil (sulfonyluracil). These include, but are not limited to, 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-azaadenine, 7-deazaguanine, 7-deazaadenine, 3-deazaguanine, and 3-deazaadenine.

[0206] Nucleotide analogs can also include modifications at the sugar moiety. Modifications to the sugar moiety include, but are not limited to, natural and synthetic modifications of the ribose and deoxyribose. Sugar modifications include, but are not limited to, the following modifications at the 2' position: OH; F; O-, S-, or N-alkyl; O-, S-, or N-alkenyl; O-, S-, or N-alkynyl; or O-alkyl-O-alkyl, where alkyl, alkenyl, and alkynyl are substituted or unsubstituted C 1-10 Alkyl or C 2-10 Alkenyl, and C 2-10 Exemplary 2' sugar modifications also include -O[(CH2)n O] m CH3, -O(CH2) n OCH3, -O(CH2) n NH2, -O(CH2) n CH3, -O(CH2) n -ONH2 and -O(CH2) n ON [(CH2) n CH3)]2, where n and m are independently 1 to about 10. Other modifications at the 2' position include, but are not limited to, C 1-10 These include, but are not limited to, alkyl, substituted lower alkyl, alkaryl, aralkyl, O-alkaryl or O-aralkyl, SH, SCH3, OCN, Cl, Br, CN, CF3, OCF3, SOCH3, SO2CH3, ONO2, NO2, N3, NH2, heterocycloalkyl, heterocycloalkaryl, aminoalkylamino, polyalkylamino, substituted silyl, RNA cleaving groups, reporter groups, intercalators, groups for improving the pharmacokinetic properties of oligonucleotides, or groups for improving the pharmacodynamic properties of oligonucleotides, and other substituents with similar properties. Similar modifications 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 modifications at the bridging ring oxygen, such as CH2 and S. Nucleotide sugar analogs can also have sugar mimetics, such as a cyclobutyl moiety in place of the pentofuranosyl sugar.

[0207] Nucleotide analogs can also be modified at the phosphate site. Modified phosphate sites include, but are not limited to, those in which the bond between two nucleotides can be modified to contain phosphorothioates, chiral phosphorothioates, phosphorodithioates, phosphotriesters, aminoalkyl phosphotriesters, methyl phosphonates and other alkyl phosphonates (including 3'-alkylene phosphonates and chiral phosphonates), phosphinates, phosphoramidates (including 3'-amino phosphoramidates and aminoalkyl phosphoramidates), thionophosphoramidates, thionoalkyl phosphonates, thionoalkyl phosphotriesters, and boranophosphates. These phosphate or modified phosphate bonds between two nucleotides can be through 3'-5' or 2'-5' bonds, and the bonds can contain reverse polarity, such as 3'-5' to 5'-3' or 2'-5' to 5'-2'. Various salts, mixed salts, and free acid forms are also included. Nucleotide substitutes also include peptide nucleic acids (PNA).

[0208] The present disclosure also provides a vector comprising any one or more of the nucleic acid molecules disclosed herein. In some embodiments, the vector comprises any one or more of the nucleic acid molecules disclosed herein and a heterologous nucleic acid. The vector can be a viral or non-viral vector capable of transporting the nucleic acid molecule. In some embodiments, the vector is a plasmid or cosmid (e.g., a circular double-stranded DNA into which additional DNA segments can be ligated, etc.). In some embodiments, the vector is a viral vector into which additional DNA segments can be ligated into the viral genome. Expression vectors include, but are not limited to, plasmids, cosmids, retroviruses, adenoviruses, adeno-associated viruses (AAV), plant viruses such as cauliflower mosaic virus and tobacco mosaic virus, yeast artificial chromosomes (YACs), Epstein-Barr (EBV) derived episomes, and other expression vectors known in the art.

[0209] Desired regulatory sequences for mammalian host cell expression may include, for example, viral elements that induce high levels of polypeptide expression in mammalian cells, such as retroviral LTRs, cytomegalovirus (CMV) (e.g., CMV promoter / enhancer, etc.), Simian Virus 40 (SV40) (e.g., SV40 promoter / enhancer, etc.), adenovirus (e.g., adenovirus major late promoter (AdMLP), etc.), polyoma derived promoters and / or enhancers, as well as strong mammalian promoters, such as native immunoglobulin promoters and actin promoters. Methods for expressing polypeptides in bacterial or fungal cells (e.g., yeast cells, etc.) are also well known. The promoter may be, for example, a constitutively active promoter, a conditional promoter, an inducible promoter, a temporally restricted promoter (e.g., a developmentally regulated promoter, etc.), or a spatially restricted promoter (e.g., a cell-specific or tissue-specific promoter, etc.).

[0210] Percent identity (or complementarity) between particular stretches of nucleotide sequences in nucleic acid molecules or amino acid sequences in polypeptides can be routinely determined using the BLAST program (Basic Local Alignment Search Tool) and PowerBLAST programs (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 that employ the algorithm of Smith and Waterman (Adv. Appl. Math., 1981, 2, 482-489). When referring to percent sequence identity in this specification, a higher percentage of sequence identity is preferred over a lower one.

[0211] The present disclosure also provides a composition comprising one or more of the isolated nucleic acid molecules, genomic nucleic acid molecules, mRNA molecules, and / or cDNA molecules disclosed herein, or a vector comprising the same. In some embodiments, the composition is a pharmaceutical composition. In some embodiments, the composition comprises a carrier and / or excipient. Examples of carriers include, but are not limited to, poly(lactic acid) (PLA) microspheres, poly(D,L-lactic-co-glycolic acid) (PLGA) microspheres, liposomes, micelles, reverse micelles, lipid cochleates, and lipid microtubules. The carrier may comprise a buffered salt solution, such as PBS, HBSS, and the like.

[0212] As used herein, the phrase "corresponding to" or grammatical variations thereof, when used in the context of numbering a particular nucleotide or sequence of nucleotides or position, refers to the numbering of the specified reference sequence when that particular nucleotide or nucleotide sequence is compared to a reference sequence (e.g., SEQ ID NO:1, SEQ ID NO:3, or SEQ ID NO:40, etc.). In other words, the residue (e.g., nucleotide or amino acid) number or residue (e.g., nucleotide or amino acid) position of a particular polymer is specified with reference to the reference sequence, not by the actual position number of that residue within that particular nucleotide or nucleotide sequence. For example, a particular nucleotide sequence may be aligned to a reference sequence by introducing gaps to optimize residue matching between the two sequences. In these cases, although gaps are present, the numbering of the residues in a particular nucleotide or nucleotide sequence is done with respect to the reference sequence to which it is aligned.

[0213] For example, a PIEZO1 nucleic acid molecule comprising a nucleotide sequence encoding a predicted gain-of-function polypeptide of PIEZO1, the nucleotide sequence comprising a thymine at a position corresponding to position 69,579 according to SEQ ID NO:2, means that when the nucleotide sequence of a PIEZO1 genomic nucleic acid molecule is aligned to 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 predicted gain-of-function polypeptide of PIEZO1, the nucleotide sequence comprising 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 predicted gain-of-function polypeptide of PIEZO1, the nucleotide sequence comprising 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 predicted gain-of-function polypeptide of PIEZO1, in which a genomic nucleic acid molecule has a nucleotide sequence that includes a thymine residue that is homologous to the thymine residue at position 69,579 of SEQ ID NO:2 (or an mRNA molecule has a nucleotide sequence that includes a uracil residue that is homologous to the uracil residue at position 7,785 of SEQ ID NO:13, or a cDNA molecule has a nucleotide sequence that includes a thymine residue that is homologous to the thymine residue at position 7,785 of SEQ ID NO:33). Such sequences are also referred to herein as "PIEZO1 sequences having a Pro2,510Leu change" or "PIEZO1 sequences having a Pro2,510Leu variation."

[0214] As described herein, for example, the position in the PIEZO1 genomic nucleic acid molecule corresponding to position 69,579 according to SEQ ID NO:2 can be identified by performing sequence alignment between the nucleotide sequence of a particular PIEZO1 nucleic acid molecule and the nucleotide sequence of SEQ ID NO:2. For example, there are various computer algorithms that can be used to perform sequence alignment to identify the nucleotide position corresponding to position 69,579 in SEQ ID NO:2. For example, sequence alignment can be performed by using the NCBI BLAST algorithm (Altschul et al., Nucleic Acids Res., 1997, 25, 3389-3402) or CLUSTALW software (Sievers and Higgins, Methods Mol. Biol., 2014, 1079, 105-116). However, sequences can also be manually aligned.

[0215] The amino acid sequence of the PIEZO1 reference polypeptide is shown in SEQ ID NO: 43. With reference to SEQ ID NO: 43, the PIEZO1 reference polypeptide is 2,521 amino acids in length. With reference to SEQ ID NO: 43, position 2,510 is a proline.

[0216] The amino acid sequence of another PIEZO1 reference polypeptide is shown in SEQ ID NO: 44. With reference to SEQ ID NO: 44, the PIEZO1 reference polypeptide is 2,035 amino acids in length. With reference to SEQ ID NO: 44, position 2,024 is a proline.

[0217] The amino acid sequence of another PIEZO1 reference polypeptide is shown in SEQ ID NO: 45. With reference to SEQ ID NO: 45, the PIEZO1 reference polypeptide is 2,090 amino acids in length. With reference to SEQ ID NO: 45, position 2,079 is a proline.

[0218] The amino acid sequence of another PIEZO1 reference polypeptide is shown in SEQ ID NO: 46. With reference to SEQ ID NO: 46, the PIEZO1 reference polypeptide is 105 amino acids long. With reference to SEQ ID NO: 46, position 94 is a proline.

[0219] There is a predicted gain-of-function polypeptide of PIEZO1 (Pro2,510Leu), the amino acid sequence of which is shown in SEQ ID NO: 47. With reference to SEQ ID NO: 47, the predicted gain-of-function polypeptide of PIEZO1 is 2,521 amino acids long. With reference to SEQ ID NO: 86, position 2,510 is a leucine.

[0220] There is another predicted gain-of-function polypeptide of PIEZO1 (Pro2,024Leu), the amino acid sequence of which is shown in SEQ ID NO: 48. With reference to SEQ ID NO: 48, the predicted gain-of-function polypeptide of PIEZO1 is 2,035 amino acids long. With reference to SEQ ID NO: 48, position 2,024 is a leucine.

[0221] There is another predicted gain-of-function polypeptide of PIEZO1 (Pro2,079Leu), the amino acid sequence of which is shown in SEQ ID NO: 49. With reference to SEQ ID NO: 49, the predicted gain-of-function polypeptide of PIEZO1 is 2,090 amino acids long. With reference to SEQ ID NO: 49, position 2,079 is a leucine.

[0222] There is another predicted gain-of-function polypeptide of PIEZO1 (Pro94Leu), the amino acid sequence of which is shown in SEQ ID NO: 50. With reference to SEQ ID NO: 50, the predicted gain-of-function polypeptide of PIEZO1 is 105 amino acids in length. With reference to SEQ ID NO: 50, position 94 is a leucine.

[0223] The nucleotide and amino acid sequences listed in the accompanying sequence listing are shown using standard letter abbreviations for nucleotide bases and three letter codes for amino acids. The nucleotide sequences follow the standard convention of starting at the 5'-terminus of the sequence and proceeding forward to the 3'-terminus (i.e., from left to right on each line). Only one strand of each nucleotide sequence is shown, but it is understood that the complementary strand is encompassed by any reference to the presented strand. The amino acid sequences follow the standard convention of starting at the amino-terminus of the sequence and proceeding forward to the carboxy-terminus (i.e., from left to right on each sequence).

[0224] 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 (or for use in preparing a medicament for treating or preventing varicose veins), wherein the subject has any of the PIEZO1 variant genomic nucleic acid molecules, variant mRNA molecules, and / or variant cDNA molecules encoding the predicted gain-of-function polypeptides of PIEZO1 described herein. The therapeutic agent for treating or preventing varicose veins may be any of the therapeutic agents for treating or preventing varicose veins described herein. The varicose veins may be any of saphenous varicose veins, reticular varicose veins, spider veins, and pregnancy-related varicose veins.

[0225] In some embodiments, the subject is identified as having a genomic nucleic acid molecule, or a complement thereof, encoding a predicted gain-of-function polypeptide of PIEZO1, the genomic nucleic acid molecule having a nucleotide sequence that includes a thymine at a position corresponding to position 69,579 according to SEQ ID NO:2 or a complement thereof.

[0226] In some embodiments, the subject has an mRNA molecule encoding a predicted gain-of-function polypeptide of PIEZO1, or its complement, wherein the mRNA molecule has 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. uracil at a position corresponding to position 6,236 according to SEQ ID NO:18 or its complement; uracil at a position corresponding to position 7,526 according to SEQ ID NO:19 or its complement; uracil at a position corresponding to position 1,336 according to SEQ ID NO:20 or its complement; uracil at a position corresponding to position 361 according to SEQ ID NO:21 or its complement; or uracil at a position corresponding to position 7,776 according to SEQ ID NO:22 or its complement.

[0227] In some embodiments, the subject has a cDNA molecule encoding a predicted gain-of-function polypeptide of PIEZO1, or a complement thereof, the cDNA molecule 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.

[0228] In some embodiments, the subject is identified as having a genomic nucleic acid molecule having a nucleotide sequence encoding a predicted gain-of-function polypeptide of PIEZO1, wherein the nucleotide sequence comprises a thymine at a position corresponding to position 69,579 according to SEQ ID NO:2 or its complement; an mRNA molecule having a nucleotide sequence encoding a predicted gain-of-function polypeptide of PIEZO1, wherein the nucleotide sequence comprises a uracil at a position corresponding to position 7,785 according to SEQ ID NO:13 or its complement; a cDNA molecule having a nucleotide sequence encoding a predicted gain-of-function polypeptide of PIEZO1, wherein the nucleotide sequence comprises a thymine at a position corresponding to position 7,785 according to SEQ ID NO:33 or its complement; or a predicted gain-of-function polypeptide of PIEZO1 comprising a leucine at a position corresponding to position 2,510 according to SEQ ID NO:47.

[0229] In some embodiments, the subject is identified as having a genomic nucleic acid molecule having a nucleotide sequence encoding a predicted gain-of-function polypeptide of PIEZO1, the nucleotide sequence comprising: an mRNA molecule having a nucleotide sequence encoding a predicted gain-of-function polypeptide of PIEZO1, the nucleotide sequence comprising a uracil at a position corresponding to position 1,095 according to SEQ ID NO: 14 or its complement; a cDNA molecule having a nucleotide sequence encoding a predicted gain-of-function polypeptide of PIEZO1, the nucleotide sequence comprising a thymine at a position corresponding to position 1,095 according to SEQ ID NO: 34 or its complement; or a predicted gain-of-function polypeptide of PIEZO1 comprising a leucine at a position corresponding to position 2,024 according to SEQ ID NO: 48.

[0230] In some embodiments, the subject is identified as having a genomic nucleic acid molecule having a nucleotide sequence encoding a predicted gain-of-function polypeptide of PIEZO1, the nucleotide sequence comprising: an mRNA molecule having a nucleotide sequence encoding a predicted gain-of-function polypeptide of PIEZO1, the nucleotide sequence comprising a uracil at a position corresponding to position 6,073 according to SEQ ID NO: 15 or its complement; a cDNA molecule having a nucleotide sequence encoding a predicted gain-of-function polypeptide of PIEZO1, the nucleotide sequence comprising a thymine at a position corresponding to position 6,073 according to SEQ ID NO: 35 or its complement; or a predicted gain-of-function polypeptide of PIEZO1 comprising a leucine at a position corresponding to position 2,079 according to SEQ ID NO: 49.

[0231] In some embodiments, the subject is identified as having a genomic nucleic acid molecule having a nucleotide sequence encoding a predicted gain-of-function polypeptide of PIEZO1, the nucleotide sequence comprising: an mRNA molecule having a nucleotide sequence encoding a predicted gain-of-function polypeptide of PIEZO1, the nucleotide sequence comprising a uracil at a position corresponding to position 1,336 according to SEQ ID NO: 16 or its complement; a cDNA molecule having a nucleotide sequence encoding a predicted gain-of-function polypeptide of PIEZO1, the nucleotide sequence comprising a thymine at a position corresponding to position 1,336 according to SEQ ID NO: 36 or its complement; or a predicted gain-of-function polypeptide of PIEZO1 comprising a leucine at a position corresponding to position 94 according to SEQ ID NO: 50.

[0232] In some embodiments, the subject is identified as having a genomic nucleic acid molecule having a nucleotide sequence encoding a predicted gain-of-function polypeptide of PIEZO1, the nucleotide sequence comprising an mRNA molecule having a nucleotide sequence encoding a predicted gain-of-function polypeptide of PIEZO1, the nucleotide sequence comprising a uracil at a position corresponding to position 6,073 according to SEQ ID NO: 17 or its complement; or a cDNA molecule having a nucleotide sequence encoding a predicted gain-of-function polypeptide of PIEZO1, the nucleotide sequence comprising a thymine at a position corresponding to position 6,073 according to SEQ ID NO: 37 or its complement.

[0233] In some embodiments, the subject is identified as having a genomic nucleic acid molecule having a nucleotide sequence encoding a predicted gain-of-function polypeptide of PIEZO1, the nucleotide sequence comprising an mRNA molecule having a nucleotide sequence encoding a predicted gain-of-function polypeptide of PIEZO1, the nucleotide sequence comprising 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 predicted gain-of-function polypeptide of PIEZO1, the nucleotide sequence comprising a thymine at a position corresponding to position 6,236 according to SEQ ID NO: 38 or its complement.

[0234] In some embodiments, the subject is identified as having a genomic nucleic acid molecule having a nucleotide sequence encoding a predicted gain-of-function polypeptide of PIEZO1, the nucleotide sequence comprising an mRNA molecule having a nucleotide sequence encoding a predicted gain-of-function polypeptide of PIEZO1, the nucleotide sequence comprising 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 predicted gain-of-function polypeptide of PIEZO1, the nucleotide sequence comprising a thymine at a position corresponding to position 7,526 according to SEQ ID NO: 39 or its complement.

[0235] In some embodiments, the subject is identified as having a genomic nucleic acid molecule having a nucleotide sequence encoding a predicted gain-of-function polypeptide of PIEZO1, the nucleotide sequence comprising an mRNA molecule having a nucleotide sequence encoding a predicted gain-of-function polypeptide of PIEZO1, the nucleotide sequence comprising 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 predicted gain-of-function polypeptide of PIEZO1, the nucleotide sequence comprising a thymine at a position corresponding to position 1,336 according to SEQ ID NO:40 or its complement.

[0236] In some embodiments, the subject is identified as having a genomic nucleic acid molecule having a nucleotide sequence encoding a predicted gain-of-function polypeptide of PIEZO1, the nucleotide sequence comprising an mRNA molecule having a nucleotide sequence encoding a predicted gain-of-function polypeptide of PIEZO1, the nucleotide sequence comprising a uracil at a position corresponding to position 361 according to SEQ ID NO:21 or its complement; or a cDNA molecule having a nucleotide sequence encoding a predicted gain-of-function polypeptide of PIEZO1, the nucleotide sequence comprising a thymine at a position corresponding to position 361 according to SEQ ID NO:41 or its complement.

[0237] In some embodiments, the subject is identified as having a genomic nucleic acid molecule having a nucleotide sequence encoding a predicted gain-of-function polypeptide of PIEZO1, the nucleotide sequence comprising an mRNA molecule having a nucleotide sequence encoding a predicted gain-of-function polypeptide of PIEZO1, the nucleotide sequence comprising a uracil 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 predicted gain-of-function polypeptide of PIEZO1, the nucleotide sequence comprising a thymine at a position corresponding to position 7,776 according to SEQ ID NO:42 or its complement.

[0238] The present disclosure also provides a PIEZO1 agonist for use in treating or preventing varicose veins in a subject (or for use in preparing a medicament for treating or preventing varicose veins), wherein the subject is heterozygous for any of the PIEZO1 variant genomic nucleic acid molecules, variant mRNA molecules, and / or variant cDNA molecules encoding the predicted gain-of-function polypeptides of PIEZO1 described herein, or the subject is a reference to a PIEZO1 genomic nucleic acid molecule, mRNA molecule, or cDNA molecule. The PIEZO1 agonist may be any of the PIEZO1 agonists described herein. The varicose veins may be any of saphenous varicose veins, reticular varicose veins, spider veins, and pregnancy-related varicose veins.

[0239] In some embodiments, the subject is a reference for a PIEZO1 genomic nucleic acid molecule, a PIEZO1 mRNA molecule, or a PIEZO1 cDNA molecule. In some embodiments, the subject is heterozygous for a genomic nucleic acid molecule, or a complement thereof, encoding a predicted gain-of-function polypeptide of PIEZO1, wherein the genomic nucleic acid molecule has a nucleotide sequence that includes a thymine at a position corresponding to position 69,579 according to SEQ ID NO:2 or its complement.

[0240] In some embodiments, the subject has an mRNA molecule encoding a predicted gain-of-function polypeptide of PIEZO1, or its complement, wherein the mRNA molecule has 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. uracil at a position corresponding to position 6,236 according to SEQ ID NO:18 or its complement; uracil at a position corresponding to position 7,526 according to SEQ ID NO:19 or its complement; uracil at a position corresponding to position 1,336 according to SEQ ID NO:20 or its complement; uracil at a position corresponding to position 361 according to SEQ ID NO:21 or its complement; or uracil at a position corresponding to position 7,776 according to SEQ ID NO:22 or its complement.

[0241] In some embodiments, the subject has a cDNA molecule encoding a predicted gain-of-function polypeptide of PIEZO1, or a complement thereof, the cDNA molecule 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 the corresponding position; a thymine at the position corresponding to position 6,236 according to SEQ ID NO:38 or its complement; a thymine at the position corresponding to position 7,526 according to SEQ ID NO:39 or its complement; a thymine at the position corresponding to position 1,336 according to SEQ ID NO:40 or its complement; a thymine at the position corresponding to position 361 according to SEQ ID NO:41 or its complement; or a thymine at the position corresponding to position 7,776 according to SEQ ID NO:42 or its complement, or its complement.

[0242] In some embodiments, the subject is identified as being heterozygous for a genomic nucleic acid molecule having a nucleotide sequence encoding a predicted gain-of-function polypeptide of PIEZO1, the nucleotide sequence comprising a thymine at a position corresponding to position 69,579 according to SEQ ID NO:2 or its complement; an mRNA molecule having a nucleotide sequence encoding a predicted gain-of-function polypeptide of PIEZO1, the nucleotide sequence comprising a uracil at a position corresponding to position 7,785 according to SEQ ID NO:13 or its complement; a cDNA molecule having a nucleotide sequence encoding a predicted gain-of-function polypeptide of PIEZO1, the nucleotide sequence comprising a thymine at a position corresponding to position 7,785 according to SEQ ID NO:33 or its complement; or a predicted gain-of-function polypeptide of PIEZO1 comprising a leucine at a position corresponding to position 2,510 according to SEQ ID NO:47. The PIEZO1 agonist may be any of the PIEZO1 agonists described herein.

[0243] In some embodiments, the subject is identified as being heterozygous for a predicted gain-of-function polypeptide of PIEZO1, comprising a genomic nucleic acid molecule having a nucleotide sequence encoding a predicted gain-of-function polypeptide of PIEZO1, the nucleotide sequence comprising an mRNA molecule having a nucleotide sequence encoding a predicted gain-of-function polypeptide of PIEZO1, the nucleotide sequence comprising a uracil at a position corresponding to position 1,095 according to SEQ ID NO: 14 or its complement; a cDNA molecule having a nucleotide sequence encoding a predicted gain-of-function polypeptide of PIEZO1, the nucleotide sequence comprising a thymine at a position corresponding to position 1,095 according to SEQ ID NO: 34 or its complement; or a predicted gain-of-function polypeptide of PIEZO1 comprising a leucine at a position corresponding to position 2,024 according to SEQ ID NO: 48. The PIEZO1 agonist may be any of the PIEZO1 agonists described herein.

[0244] In some embodiments, the subject is identified as being heterozygous for a predicted gain-of-function polypeptide of PIEZO1, comprising a genomic nucleic acid molecule having a nucleotide sequence encoding a predicted gain-of-function polypeptide of PIEZO1, the nucleotide sequence comprising an mRNA molecule having a nucleotide sequence encoding a predicted gain-of-function polypeptide of PIEZO1, the nucleotide sequence comprising a uracil at a position corresponding to position 6,073 according to SEQ ID NO: 15 or its complement; a cDNA molecule having a nucleotide sequence encoding a predicted gain-of-function polypeptide of PIEZO1, the nucleotide sequence comprising a thymine at a position corresponding to position 6,073 according to SEQ ID NO: 35 or its complement; or a predicted gain-of-function polypeptide of PIEZO1 comprising a leucine at a position corresponding to position 2,079 according to SEQ ID NO: 49. The PIEZO1 agonist may be any of the PIEZO1 agonists described herein.

[0245] In some embodiments, the subject is identified as being heterozygous for a genomic nucleic acid molecule having a nucleotide sequence encoding a predicted gain-of-function polypeptide of PIEZO1, the nucleotide sequence comprising an mRNA molecule having a nucleotide sequence encoding a predicted gain-of-function polypeptide of PIEZO1, the nucleotide sequence comprising a uracil at a position corresponding to position 1,336 according to SEQ ID NO: 16 or its complement; a cDNA molecule having a nucleotide sequence encoding a predicted gain-of-function polypeptide of PIEZO1, the nucleotide sequence comprising a thymine at a position corresponding to position 1,336 according to SEQ ID NO: 36 or its complement; or a predicted gain-of-function polypeptide of PIEZO1 comprising a leucine at a position corresponding to position 94 according to SEQ ID NO: 50. The PIEZO1 agonist may be any of the PIEZO1 agonists described herein.

[0246] In some embodiments, the subject is identified as being heterozygous for a genomic nucleic acid molecule having a nucleotide sequence encoding a predicted gain-of-function polypeptide of PIEZO1, the nucleotide sequence comprising an mRNA molecule having a nucleotide sequence encoding a predicted gain-of-function polypeptide of PIEZO1, the nucleotide sequence comprising a uracil at a position corresponding to position 6,073 of SEQ ID NO: 17 or its complement; or a cDNA molecule having a nucleotide sequence encoding a predicted gain-of-function polypeptide of PIEZO1, the nucleotide sequence comprising a thymine at a position corresponding to position 6,073 of SEQ ID NO: 37 or its complement. The PIEZO1 agonist may be any of the PIEZO1 agonists described herein.

[0247] In some embodiments, the subject is identified as being heterozygous for a genomic nucleic acid molecule having a nucleotide sequence encoding a predicted gain-of-function polypeptide of PIEZO1, the nucleotide sequence comprising an mRNA molecule having a nucleotide sequence encoding a predicted gain-of-function polypeptide of PIEZO1, the nucleotide sequence comprising 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 predicted gain-of-function polypeptide of PIEZO1, the nucleotide sequence comprising a thymine at a position corresponding to position 6,236 according to SEQ ID NO: 38 or its complement. The PIEZO1 agonist may be any of the PIEZO1 agonists described herein.

[0248] In some embodiments, the subject is identified as being heterozygous for a genomic nucleic acid molecule having a nucleotide sequence encoding a predicted gain-of-function polypeptide of PIEZO1, the nucleotide sequence comprising an mRNA molecule having a nucleotide sequence encoding a predicted gain-of-function polypeptide of PIEZO1, the nucleotide sequence comprising a uracil at position corresponding to position 7,526 of SEQ ID NO: 19 or its complement; or a cDNA molecule having a nucleotide sequence encoding a predicted gain-of-function polypeptide of PIEZO1, the nucleotide sequence comprising a thymine at position corresponding to position 7,526 of SEQ ID NO: 39 or its complement. The PIEZO1 agonist may be any of the PIEZO1 agonists described herein.

[0249] In some embodiments, the subject is identified as heterozygous for a genomic nucleic acid molecule having a nucleotide sequence encoding a predicted gain-of-function polypeptide of PIEZO1, the nucleotide sequence comprising an mRNA molecule having a nucleotide sequence encoding a predicted gain-of-function polypeptide of PIEZO1, the nucleotide sequence comprising 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 predicted gain-of-function polypeptide of PIEZO1, the nucleotide sequence comprising a thymine at a position corresponding to position 1,336 according to SEQ ID NO:40 or its complement.

[0250] In some embodiments, the subject is identified as heterozygous for a genomic nucleic acid molecule having a nucleotide sequence encoding a predicted gain-of-function polypeptide of PIEZO1, the nucleotide sequence comprising an mRNA molecule having a nucleotide sequence encoding a predicted gain-of-function polypeptide of PIEZO1, the nucleotide sequence comprising a uracil at a position corresponding to position 361 according to SEQ ID NO:21 or its complement; or a cDNA molecule having a nucleotide sequence encoding a predicted gain-of-function polypeptide of PIEZO1, the nucleotide sequence comprising a thymine at a position corresponding to position 361 according to SEQ ID NO:41 or its complement.

[0251] In some embodiments, the subject is identified as heterozygous for a genomic nucleic acid molecule having a nucleotide sequence encoding a predicted gain-of-function polypeptide of PIEZO1, the nucleotide sequence comprising an mRNA molecule having a nucleotide sequence encoding a predicted gain-of-function polypeptide of PIEZO1, the nucleotide sequence comprising a uracil 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 predicted gain-of-function polypeptide of PIEZO1, the nucleotide sequence comprising a thymine at a position corresponding to position 7,776 according to SEQ ID NO:42 or its complement.

[0252] 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 of the PIEZO1 agonists described herein.

[0253] All patent documents, websites, other publications, accession numbers, etc. cited above or below are incorporated by reference in their entirety for all purposes to the same extent as if each individual item was specifically and individually indicated to be so incorporated by reference. Where different versions of sequences are associated with accession numbers at different times, the version associated with the accession number at the effective filing date of this application is meant. Effective filing date means the earlier of the actual filing date or the filing date of the priority application that references the accession number, if applicable. Similarly, where different versions of publications, websites, etc. are published at different times, the latest published version at the effective filing date of this application is meant unless otherwise indicated. Any feature, step, element, embodiment, or aspect of the present disclosure may be used in combination with any other feature, step, element, embodiment, or aspect unless otherwise specifically indicated. The present disclosure has been described in some detail by illustration and example for purposes of clarity and understanding, but it will be apparent that certain changes and modifications may be practiced within the scope of the appended claims.

[0254] The following examples are provided to more fully describe the embodiments. They are intended to illustrate, not limit, the claimed embodiments. The following examples provide those skilled in the art with a disclosure and description of how the compounds, compositions, articles, devices and / or methods described herein are made and evaluated, are intended to be purely exemplary, and are not intended to limit the scope of any claims. Attempts have been made to ensure accuracy with respect to numbers (e.g., amounts, temperatures, etc.), but some error and deviation can be accounted for. Unless otherwise indicated, parts are parts by weight, temperature is °C or is ambient temperature, and pressure is at or near atmospheric pressure. EXAMPLES

[0255] Example 1: Association of PIEZO1 GOF variants with varicose veins As previously reported (Szustakowski, Advancing Human Genetics Research and Drug Discovery through Exome Sequencing of the UK Biobank. bioRxiv, 2021; and Van Hout et al., Nature, 2020), the exomes of 454,787 UKB study participants were sequenced, with 95.8% of the target bases covered at a depth of 20x or greater. 12 million variants were identified in 39 million base pairs across the coding regions of 18,659 genes (data not shown). Among the variants identified, there were 3,375,252 (median 10,260 per subject) synonymous variants, 7,689,495 (9,284 per subject) missense variants, and 889,957 (212 per subject) putative gain-of-function (pLOF) variants (data not shown), of which approximately half were observed only once in this dataset (singleton variants; data not shown).

[0256] A missense variant in PIEZO1 (rs61745086:A, Pro2,510Leu, MAF=0.98%) was found to be associated with a reduced risk of varicose veins (7,455 carriers; OR=0.69, 95% CI 0.61-0.70, P=2.61×10 -8 ). PIEZO1 encodes a mechanosensitive cation channel that plays an important role in the formation of venous and lymphatic valves. It was previously shown in the first 50K exomes from UKB that a rare pLOF in this gene increases the risk of asymptomatic varicose veins of the lower extremities by 4.9-fold (162 carriers; 95% CI 2.8 to 8.6, P = 3.2 × 10 -8 ), this association is now estimated to be 2.04-fold using approximately eight times more data (1,302 carriers; OR = 2.04, 95% CI 1.63 to 2.56, P = 5.3 × 10 -10). The novel protective association with rs61745086:A replicated in the GHS cohort (2,243 carriers; OR=0.66, 95% CI 0.47-0.93, P=0.017) suggests that this missense variant likely has a gain-of-function effect. This is important because activation of PIEZO1 may provide a therapeutic pathway for a common pathology with no available pharmacological interventions.

[0257] In addition to those described herein, various modifications of the described subject matter will be apparent to those skilled in the art from the above description. Such modifications are also intended to fall within the scope of the appended claims. Each reference cited in this application (including but not limited to journal articles, U.S. and non-U.S. patents, patent application publications, international patent application publications, gene bank accession numbers, etc.) is incorporated herein by reference in its entirety for all purposes.

Claims

1. An in vitro method for identifying the susceptibility of a subject to the development of venous aneurysms, said method comprising: determining or having determined the presence or absence of a PIEZO1 variant nucleic acid molecule encoding a predicted gain-of-function polypeptide of piezo-type mechanosensitive ion channel component 1 (PIEZO1) in a biological sample obtained from said subject; indicating that a subject that is a PIEZO1 reference is a subject at high risk of developing a venous aneurysm; A method indicating that a subject that is heterozygous or homozygous for a PIEZO1 variant nucleic acid molecule encoding a predicted gain-of-function polypeptide of said PIEZO1 is a subject at low risk of developing a venous aneurysm.

2. The method according to claim 1, wherein said PIEZO1 variant nucleic acid molecule encodes Pro2,510Leu, Pro2,024Leu, Pro2,079Leu, or Pro92Leu.

3. The PIEZO1 variant nucleic acid molecule is a genomic nucleic acid molecule having a nucleotide sequence containing thymine at a position corresponding to position 69,579 according to SEQ ID NO: 2; or uracil at a position corresponding to position 7,785 according to SEQ ID NO: 13; uracil at a position corresponding to position 1,095 according to SEQ ID NO: 14; uracil at a position corresponding to position 6,073 according to SEQ ID NO: 15; uracil at a position corresponding to position 1,336 according to SEQ ID NO: 16; uracil at a position corresponding to position 6,073 according to SEQ ID NO: 17; uracil at a position corresponding to position 6,236 according to SEQ ID NO: 18; uracil at a position corresponding to position 7,526 according to SEQ ID NO: 19; uracil at a position corresponding to position 1,336 according to SEQ ID NO: 20; uracil at a position corresponding to position 361 according to SEQ ID NO: 21; or a mRNA molecule having a nucleotide sequence containing uracil at a position corresponding to position 7,776 according to SEQ ID NO: 22, the method according to claim 2.

4. Use of a therapeutic agent for treating or preventing venous aneurysms in the manufacture of a medicament for treating or preventing venous aneurysms in a subject, wherein said subject is heterozygous or homozygous for a PIEZO1 variant nucleic acid molecule encoding a predicted gain-of-function polypeptide of piezo-type mechanosensitive ion channel component 1 (PIEZO1). Use of a Piezo type mechanically-gated ion channel component 1 (PIEZO1) agonist in the manufacture of a medicament for the treatment or prevention of venous aneurysms in a subject, wherein the subject is: a) a reference for a PIEZO1 genomic nucleic acid molecule, or a PIEZO1 mRNA molecule; or b) heterozygous for a PIEZO1 variant nucleic acid molecule encoding a predicted gain-of-function polypeptide of piezo type mechanically-gated ion channel component 1 (PIEZO1).

6. The use according to claim 5, wherein the agonist is a small molecule agonist.

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

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

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

10. The method according to any one of claims 1 to 3, wherein the venous aneurysm is a latent venous aneurysm, a reticular venous aneurysm, a spider vein, or a pregnancy-related venous aneurysm.

11. The PIEZO1 variant nucleic acid molecule encoding the predicted gain-of-function polypeptide of PIEZO1 is: a genomic nucleic acid molecule having a nucleotide sequence containing thymine at a position corresponding to position 69,579 according to SEQ ID NO: 2 or its complement; or, a uracil at a position corresponding to position 7,785 according to SEQ ID NO: 13; a uracil at a position corresponding to position 1,095 according to SEQ ID NO: 14; a uracil at a position corresponding to position 6,073 according to SEQ ID NO: 15; a uracil at a position corresponding to position 1,336 according to SEQ ID NO: 16; a uracil at a position corresponding to position 6,073 according to SEQ ID NO: 17; a uracil at a position corresponding to position 6,236 according to SEQ ID NO: 18; a uracil at a position corresponding to position 7,526 according to SEQ ID NO: 19; a uracil at a position corresponding to position 1,336 according to SEQ ID NO: 20; a uracil at a position corresponding to position 361 according to SEQ ID NO: 21; or a uracil at a position corresponding to position 7,776 according to SEQ ID NO: 22, an mRNA molecule having a nucleotide sequence containing the same. The use according to claim 4, comprising the above.

12. The use according to claim 4 or claim 11, wherein the venous aneurysm is a latent venous aneurysm, a reticular venous aneurysm, a spider vein, or a pregnancy-related venous aneurysm.

13. The use according to claims 4 and 11, wherein the therapeutic agent comprises a flavonoid or an anti-inflammatory agent.

14. The use according to claim 13, wherein the flavonoid comprises diosmin or hesperidin, and the anti-inflammatory agent comprises ibuprofen or aspirin.

15. The PIEZO1 variant nucleic acid molecule encoding the predicted gain-of-function polypeptide of PIEZO1 is: A genomic nucleic acid molecule having a nucleotide sequence containing thymine at a position corresponding to position 69,579 according to SEQ ID NO: 2, or its complement; or Uracil at a position corresponding to position 7,785 according to SEQ ID NO: 13; uracil at a position corresponding to position 1,095 according to SEQ ID NO: 14; uracil at a position corresponding to position 6,073 according to SEQ ID NO: 15; uracil at a position corresponding to position 1,336 according to SEQ ID NO: 16; uracil at a position corresponding to position 6,073 according to SEQ ID NO: 17; uracil at a position corresponding to position 6,236 according to SEQ ID NO: 18; uracil at a position corresponding to position 7,526 according to SEQ ID NO: 19; uracil at a position corresponding to position 1,336 according to SEQ ID NO: 20; uracil at a position corresponding to position 361 according to SEQ ID NO: 21; or a mRNA molecule having a nucleotide sequence containing uracil at a position corresponding to position 7,776 according to SEQ ID NO: 22, The use according to any one of claims 5 to 9, comprising.

16. The use according to any one of claims 5 to 9, wherein the varix is a latent varix, a reticular varix, a spider vein, or a pregnancy-related varix.

17. A pharmaceutical composition for treating a varix in a patient who is heterozygous or homozygous for a PIEZO1 variant nucleic acid molecule encoding the predicted gain-of-function polypeptide of piezo-type mechanosensitive ion channel component 1 (PIEZO1), wherein the pharmaceutical composition comprises, as an active ingredient, a therapeutic agent for treating or preventing a varix. A pharmaceutical composition for treating varicose veins in a patient, which is heterozygous for a) a genomic nucleic acid molecule of the piezo-type mechanically gated ion channel component 1 (PIEZO1) or a PIEZO1 mRNA molecule, or b) a PIEZO1 variant nucleic acid molecule encoding a predicted gain-of-function polypeptide of the piezo-type mechanically gated ion channel component 1 (PIEZO1), wherein the pharmaceutical composition comprises a PIEZO1 agonist as an active ingredient. The pharmaceutical composition according to claim 17 or claim 18, wherein the varicose veins are latent varicose veins, reticular varicose veins, spider veins, or pregnancy-related varicose veins.