Piezoelectric mechanosensitive ion channel component 1 (PIEZO1) variants and uses thereof

Abstract

Provided herein are methods of treating a patient suffering from varicose veins, methods of identifying a subject at increased risk of developing varicose veins, and methods of diagnosing varicose veins in a human subject, the methods comprising detecting the presence of a Piezoelectric Mechanosensitive Ion Channel Component 1 (PIEZO1) predicted loss-of-function variant nucleic acid molecule and polypeptide in a biological sample from the patient or subject.

Description

[0001] References to sequence listings

[0002] This application includes a sequence listing submitted electronically as a text file, the text file is named 18923802502SEQ, created on February 5, 2020, and is 132 kilobytes in size. The sequence listing is incorporated herein by reference. Technical Field

[0003] The present disclosure provides methods of treating patients suffering from varicose veins, methods of identifying subjects at increased risk of developing varicose veins, and methods of diagnosing varicose veins in human subjects, the methods comprising detecting the presence of PIEZO1 predicted loss-of-function variant nucleic acid molecules and polypeptides in a biological sample from the patient or subject. Background Art

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

[0005] PIEZO1 is encoded by a 70kb gene located at 16q24.3 and exists in five potential isoforms. The PIEZO1 protein is 2,521 amino acids in length and is a 286kDa transmembrane protein that contains 38 transmembrane domains and functions as a homotetramer. PIEZO1 encodes an evolutionarily conserved endothelial mechanosensitive cation channel that produces a current characterized by a linear current-voltage relationship, the channel being sensitive to ruthenium red and gadolinium. PIEZO1 is ubiquitously expressed and plays a role in epithelial cell adhesion by maintaining integrin activation through the recruitment of R-Ras to the endoplasmic reticulum (most likely 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-related shear stress and promotes endothelial cell organization and alignment along the direction of blood flow, thereby ensuring proper vascularization, remodeling, and maturation. PIEZO1 also appears to be required for lymphatic flap formation. Other reported functions include blood pressure regulation, urine osmolality, red blood cell integrity, pressure sensing, and collecting duct osmotic pressure regulation. Summary of the invention

[0006] The present disclosure provides a method for identifying a human subject with an increased risk of developing varicose veins, wherein the method comprises determining or having determined the presence or absence in a biological sample obtained from the subject: a Piezoelectric mechanosensitive ion channel component 1 (PIEZO1) predicted loss-of-function variant genomic nucleic acid molecule; a PIEZO1 predicted loss-of-function variant mRNA molecule; a PIEZO1 predicted loss-of-function variant cDNA molecule produced from the mRNA molecule; or a PIEZO1 predicted loss-of-function variant polypeptide; wherein: the absence of a PIEZO1 predicted loss-of-function variant genomic nucleic acid molecule, mRNA molecule, cDNA molecule, or polypeptide indicates that the subject does not have an increased risk of developing varicose veins; and the presence of a PIEZO1 predicted loss-of-function variant genomic nucleic acid molecule, mRNA molecule, cDNA molecule, or polypeptide indicates that the subject has an increased risk of developing varicose veins.

[0007] The present disclosure also provides a method for diagnosing varicose veins in a human subject, wherein the method comprises detecting the presence or absence in a sample obtained from the subject: a genomic nucleic acid molecule of a predicted loss-of-function variant of piezoelectric mechanosensitive ion channel component 1 (PIEZO1); a mRNA molecule of a predicted loss-of-function variant of PIEZO1; a cDNA molecule of a predicted loss-of-function variant of PIEZO1 produced by the mRNA molecule; or a polypeptide of a predicted loss-of-function variant of PIEZO1; wherein when the subject has a genomic nucleic acid molecule, mRNA molecule, cDNA molecule or polypeptide of a predicted loss-of-function variant of PIEZO1, and has one or more symptoms of varicose veins, the subject is diagnosed as having varicose veins.

[0008] The present disclosure also provides a method for treating a patient with a therapeutic agent for treating or inhibiting varicose veins, wherein the patient suffers from varicose veins or has an increased risk of developing varicose veins, the method comprising the following steps: determining whether the patient has a PIEZO1 predicted loss-of-function variant nucleic acid molecule encoding a human PIEZO1 polypeptide by: obtaining or having obtained a biological sample from the patient; and performing or having performed a genotyping assay on the biological sample to determine whether the patient has a genotype comprising a PIEZO1 predicted loss-of-function variant nucleic acid molecule; and when the patient is a PIEZO1 reference, then administering or continuing to administer the therapeutic agent for treating or inhibiting varicose veins to the patient in a standard dose amount; and when the patient is heterozygous or homozygous for the PIEZO1 predicted loss-of-function variant nucleic acid molecule, then administering or continuing to administer the therapeutic agent for treating or inhibiting varicose veins to the patient in an amount equal to or greater than the standard dose amount; wherein the presence of a genotype having a PIEZO1 predicted loss-of-function variant nucleic acid molecule encoding a human PIEZO1 polypeptide indicates that the patient has an increased risk of developing varicose veins. BRIEF DESCRIPTION OF THE DRAWINGS

[0009] The accompanying drawings, which are incorporated in and constitute a part of this specification, illustrate several aspects and, together with the description, serve to explain the principles of the disclosure.

[0010] Figure 1 Shown is a representative distribution of IBD sharing for pairs of individuals in the UKB 50k WES; estimated proportion of WES genotypes with no identical by descent (IBD) alleles versus 1 IBD allele in pairs across all UKB 50k exome participants.

[0011] Figure 2 All autosomal variants and the site frequency spectrum (SFS) observed by functional prediction are shown; the UKB50k exome was randomly downsampled to the number of individuals specified on the horizontal axis; the number of genes containing LOF AAF < 1% with at least the count specified in the legend is plotted on the vertical axis; the maximum number of autosomal genes is 18,272.

[0012] Figure 3 Continental ancestry in UK Biobank 500k and 50k is shown; principal components 1 and 2 are available from the UK Biobank data display for n=488,377 individuals; three predefined regions of the graph represent African (blue), East Asian (green) and European (red) ancestry.

[0013] Figure 4 Single point and aggregated results for 65 pLOF variants in PIEZO1 are shown.

[0014] Figure 5 LD assessment of PIEZO1 is shown. DETAILED DESCRIPTION

[0015] Various terms related to various aspects of the present disclosure are used throughout the specification and claims. Unless otherwise indicated, such terms will be given their ordinary meanings in the art. Other specifically defined terms will be interpreted in a manner consistent with the definitions provided herein.

[0016] Unless otherwise expressly stated, it is not intended that any method or aspect described herein be construed as requiring that its steps be performed in a specific order. Therefore, in the claims or specification, when a method claim does not expressly state that the steps are limited to a specific order, no order is intended to be inferred in any respect. This applies to any possible unexpressed principles of interpretation, including matters of logic regarding the arrangement of steps or operational flow, obvious meaning derived from grammatical organization or punctuation, or the number or type of aspects described in the specification.

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

[0018] As used herein, the terms "subject" and "patient" are used interchangeably. The subject can include any animal, including mammals. Mammals include, but are not limited to, farm animals (such as, for example, horses, cows, pigs), companion animals (such as, for example, dogs, cats), laboratory animals (such as, for example, mice, rats, rabbits), and non-human primates. In some embodiments, the subject is a human.

[0019] As used herein, "nucleic acid", "nucleic acid molecule", "nucleic acid sequence", "polynucleotide" or "oligonucleotide" may include a polymeric form of nucleotides of any length, may include DNA and / or RNA, and may be single-stranded, double-stranded or multi-stranded. A strand of a nucleic acid also refers to its complementary sequence.

[0020] As used herein, in certain embodiments, the term "comprising" may be replaced with "consisting of" or "consisting essentially of," as appropriate.

[0021] As used herein, the phrase "corresponding to" or its grammatical variants, when used in the context of the numbering of a particular amino acid or nucleotide sequence or position, refers to the numbering of the reference sequence when the particular amino acid or nucleotide sequence is compared to a reference sequence (e.g., the reference sequence herein is a (wild-type) PIEZO1 nucleic acid molecule or polypeptide). In other words, the residue (e.g., amino acid or nucleotide) number or residue (e.g., amino acid or nucleotide) position of a particular polymer is specified relative to a reference sequence, rather than by the actual numbered position of the residue within the particular amino acid or nucleotide sequence. For example, a particular amino acid sequence can be aligned with a reference sequence by introducing a gap to optimize the residue matching between the two sequences. In these cases, although there is a gap, the numbering of the residues in the particular amino acid or nucleotide sequence is performed relative to the reference sequence to which it is aligned.

[0022] According to the present disclosure, certain variations in PIEZO1 have been observed to be associated with the risk of developing varicose veins. It is believed that variants of the PIEZO1 gene or protein have no known association with varicose veins in humans. Therefore, human subjects with PIEZO1 changes associated with varicose veins can be treated so that varicose veins are inhibited, their symptoms are alleviated, and / or the development of symptoms is inhibited. Therefore, the present disclosure provides methods for identifying or stratifying the risk of developing varicose veins in such subjects using the identification of such variants in subjects, or diagnosing subjects as having varicose veins, so that subjects at risk or subjects with active disease can be treated.

[0023] For purposes of this disclosure, any particular person can be classified as having one of the following three PIEZO1 genotypes: i) PIEZO1 reference; ii) heterozygous for a PIEZO1 predicted loss-of-function variant, and iii) homozygous for a PIEZO1 predicted loss-of-function variant. A person is a PIEZO1 reference when the person has no copies of a PIEZO1 predicted loss-of-function variant nucleic acid molecule. A person is heterozygous for a PIEZO1 predicted loss-of-function variant when the person has a single copy of a PIEZO1 predicted loss-of-function variant nucleic acid molecule. A PIEZO1 predicted loss-of-function variant nucleic acid molecule is any PIEZO1 nucleic acid molecule (such as a genomic nucleic acid molecule, an mRNA molecule, or a cDNA molecule) that encodes a PIEZO1 polypeptide having a partial loss of function, a complete loss of function, a predicted partial loss of function, or a predicted complete loss of function. A person having a PIEZO1 polypeptide with a partial loss of function (or a predicted partial loss of function) is hypomorphic for PIEZO1. The PIEZO1 predicted loss-of-function variant nucleic acid molecule can be any of the variant nucleic acid molecules described herein. A person is homozygous for a PIEZO1 predicted loss-of-function variant when the person has two copies of any of the PIEZO1 predicted loss-of-function variant nucleic acid molecules.

[0024] For human subjects or patients genotyped or determined to be heterozygous or homozygous for a predicted loss-of-function variant nucleic acid molecule of PIEZO1, such human subjects or patients have an increased risk of developing varicose veins. For human subjects or patients genotyped or determined to be heterozygous or homozygous for a predicted loss-of-function variant nucleic acid molecule of PIEZO1, such human subjects or patients can be treated with an agent effective for treating varicose veins.

[0025] The present disclosure provides a method for identifying a human subject with an increased risk of developing varicose veins, wherein the method comprises determining or having determined the presence or absence of a PIEZO1 predicted loss-of-function variant nucleic acid molecule (such as a genomic nucleic acid molecule, mRNA molecule and / or cDNA molecule) or polypeptide in a biological sample obtained from the subject; wherein the absence of a PIEZO1 predicted loss-of-function variant nucleic acid molecule or polypeptide indicates that the subject does not have an increased risk of developing varicose veins; and the presence of a PIEZO1 predicted loss-of-function variant genomic nucleic acid molecule, mRNA molecule, cDNA molecule or polypeptide indicates that the subject has an increased risk of developing varicose veins.

[0026] The present disclosure also provides a method for identifying a human subject with an increased risk of developing varicose veins, wherein the method comprises determining or having determined the presence or absence in a biological sample obtained from the subject: i) a PIEZO1 predicted loss-of-function variant genomic nucleic acid molecule; ii) a PIEZO1 predicted loss-of-function variant mRNA molecule; iii) a PIEZO1 predicted loss-of-function variant cDNA molecule produced by the mRNA molecule; or iv) a PIEZO1 predicted loss-of-function variant polypeptide; wherein: the absence of a PIEZO1 predicted loss-of-function variant genomic nucleic acid molecule, mRNA molecule, cDNA molecule or polypeptide indicates that the subject does not have an increased risk of developing varicose veins; and the presence of a PIEZO1 predicted loss-of-function variant genomic nucleic acid molecule, mRNA molecule, cDNA molecule or polypeptide indicates that the subject has an increased risk of developing varicose veins.

[0027] The present disclosure also provides a method for identifying a human subject with an increased risk of developing varicose veins, wherein the method comprises: determining or having determined the presence or absence of a PIEZO1 predicted loss-of-function variant nucleic acid molecule (such as a genomic nucleic acid molecule, an mRNA molecule and / or a cDNA molecule) or a polypeptide encoding a human PIEZO1 polypeptide in a biological sample obtained from the subject; wherein: i) when the human subject lacks a PIEZO1 predicted loss-of-function variant nucleic acid molecule (i.e., the human subject is genotypically classified as a PIEZO1 reference), then the human subject does not have an increased risk of developing varicose veins; and ii) when the human subject has a PIEZO1 predicted loss-of-function variant nucleic acid molecule (i.e., the human subject is classified as being heterozygous for a PIEZO1 predicted loss-of-function variant or as being homozygous for a PIEZO1 predicted loss-of-function variant), then the human subject has an increased risk of developing varicose veins.

[0028] In any of the embodiments described herein, the PIEZO1 predicted loss-of-function variant nucleic acid molecule can be any PIEZO1 nucleic acid molecule (such as, for example, a genomic nucleic acid molecule, an mRNA molecule, or a cDNA molecule) encoding a PIEZO1 polypeptide having a partial loss of function, a complete loss of function, a predicted partial loss of function, or a predicted complete loss of function. For example, the PIEZO1 predicted loss-of-function variant nucleic acid molecule can be any of the PIEZO1 variant nucleic acid molecules described herein.

[0029] Determining whether a human subject has a PIEZO1 predicted loss-of-function variant nucleic acid molecule in a biological sample can be performed by any of the methods described herein. In some embodiments, these methods can be performed in vitro. In some embodiments, these methods can be performed in situ. In some embodiments, these methods can be performed in vivo. In any of these embodiments, the nucleic acid molecule can be present in a cell obtained from a human subject.

[0030] In any of the embodiments described herein, the varicose veins may be early varicose veins (e.g., C0 according to the CEAP (Clinical, Etiology, Anatomy and Pathophysiology) classification). In some embodiments, the varicose veins may be late varicose veins (e.g., C6 according to the CEAP classification). In some embodiments, the varicose veins may be at any disease stage (e.g., C0-C6 according to the CEAP classification). In some embodiments, the human subject is female.

[0031] In some embodiments, when a human subject is identified as having an increased risk of developing varicose veins, the human subject is further treated with a therapeutic agent for treating or inhibiting varicose veins, as described herein. For example, when a human subject is heterozygous or homozygous for a loss-of-function variant nucleic acid molecule predicted by PIEZO1, and therefore has an increased risk of developing varicose veins, a therapeutic agent for treating or inhibiting varicose veins is administered to the human subject. In some embodiments, when a patient is homozygous for a loss-of-function variant nucleic acid molecule predicted by PIEZO1, a therapeutic agent for treating or inhibiting varicose veins is administered to the patient, and its dosage amount is equal to or greater than the standard dosage amount administered to a patient who is heterozygous for a loss-of-function variant nucleic acid molecule predicted by PIEZO1. In some embodiments, the patient is heterozygous for a loss-of-function variant nucleic acid molecule predicted by PIEZO1. In some embodiments, the patient is homozygous for a loss-of-function variant nucleic acid molecule predicted by PIEZO1.

[0032] The present disclosure provides a method for diagnosing varicose veins in a human subject, wherein the method comprises detecting the presence or absence of a PIEZO1 predicted loss-of-function variant nucleic acid molecule (such as a genomic nucleic acid molecule, mRNA molecule and / or cDNA molecule) or polypeptide in a sample obtained from the subject; wherein when the subject has a PIEZO1 predicted loss-of-function variant genomic nucleic acid molecule, mRNA molecule, cDNA molecule or polypeptide, and has one or more symptoms of varicose veins, the subject is diagnosed as having varicose veins.

[0033] The present disclosure also provides a method for diagnosing varicose veins in a human subject, wherein the method comprises detecting the presence or absence of: i) a PIEZO1 predicted loss-of-function variant genomic nucleic acid molecule; ii) a PIEZO1 predicted loss-of-function variant mRNA molecule; iii) a PIEZO1 predicted loss-of-function variant cDNA molecule produced by the mRNA molecule; or iv) a PIEZO1 predicted loss-of-function variant polypeptide; wherein when the subject has a PIEZO1 predicted loss-of-function variant genomic nucleic acid molecule, mRNA molecule, cDNA molecule or polypeptide, and has one or more symptoms of varicose veins, the subject is diagnosed as having varicose veins.

[0034] The present disclosure also provides a method for diagnosing varicose veins in a human subject, wherein the method comprises detecting the presence or absence of a PIEZO1 predicted loss-of-function variant nucleic acid molecule (such as a genomic nucleic acid molecule, mRNA molecule and / or cDNA molecule) encoding a human PIEZO1 polypeptide in a sample obtained from the subject; wherein when the subject has a PIEZO1 predicted loss-of-function variant genomic nucleic acid molecule, mRNA molecule, cDNA molecule or polypeptide (i.e., the human subject is classified as being heterozygous or homozygous for the PIEZO1 predicted loss-of-function variant nucleic acid molecule) and has one or more symptoms of varicose veins, then the subject is diagnosed as having varicose veins.

[0035] In any of the embodiments described herein, the PIEZO1 predicted loss-of-function variant nucleic acid molecule can be any PIEZO1 nucleic acid molecule (such as, for example, a genomic nucleic acid molecule, an mRNA molecule, or a cDNA molecule) encoding a PIEZO1 polypeptide having a partial loss of function, a complete loss of function, a predicted partial loss of function, or a predicted complete loss of function. For example, the PIEZO1 predicted loss-of-function variant nucleic acid molecule can be any of the PIEZO1 variant nucleic acid molecules described herein.

[0036] Detecting the presence or absence of a PIEZO1 predicted loss-of-function variant nucleic acid molecule in a sample obtained from a subject can be performed by any of the methods described herein. In some embodiments, these methods can be performed in vitro. In some embodiments, these methods can be performed in situ. In some embodiments, these methods can be performed in vivo. In any of these embodiments, the nucleic acid molecule can be present in a cell obtained from a human subject.

[0037] In any of the embodiments described herein, the varicose veins may be early varicose veins (e.g., C0 according to the CEAP (Clinical, Etiology, Anatomy and Pathophysiology) classification). In some embodiments, the varicose veins may be late varicose veins (e.g., C6 according to the CEAP classification). In some embodiments, the varicose veins may be any disease stage (e.g., C0-C6 according to the CEAP classification). In some embodiments, the human subject is female.

[0038] In some embodiments, when a human subject is diagnosed with varicose veins, the human subject is further treated with a therapeutic agent for treating or inhibiting varicose veins, as described herein. For example, when a human subject is determined to be heterozygous or homozygous for a loss-of-function variant nucleic acid molecule predicted for PIEZO1, and has one or more symptoms of varicose veins, a therapeutic agent for treating or inhibiting varicose veins is administered to the human subject. In some embodiments, when a patient is homozygous for a loss-of-function variant nucleic acid molecule predicted for PIEZO1, a therapeutic agent for treating or inhibiting varicose veins is administered to the patient, and the dosage amount is equal to or greater than the standard dosage amount administered to a patient who is heterozygous for a loss-of-function variant nucleic acid molecule predicted for PIEZO1. In some embodiments, the patient is heterozygous for a loss-of-function variant nucleic acid molecule predicted for PIEZO1. In some embodiments, the patient is homozygous for a loss-of-function variant nucleic acid molecule predicted for PIEZO1.

[0039] The present disclosure also provides a method for treating a patient with a therapeutic agent for treating or inhibiting varicose veins, wherein the patient suffers from varicose veins or has an increased risk of developing varicose veins, the method comprising the following steps: determining whether the patient has a PIEZO1 predicted loss-of-function variant nucleic acid molecule encoding a human PIEZO1 polypeptide by: obtaining or having obtained a biological sample from the patient; and performing or having performed a genotyping assay on the biological sample to determine whether the patient has a genotype comprising a PIEZO1 predicted loss-of-function variant nucleic acid molecule; and when the patient is a PIEZO1 reference, then administering or continuing to administer the therapeutic agent for treating or inhibiting varicose veins to the patient in a standard dose amount; and when the patient is heterozygous or homozygous for the PIEZO1 predicted loss-of-function variant nucleic acid molecule, then administering or continuing to administer the therapeutic agent for treating or inhibiting varicose veins to the patient in an amount equal to or greater than the standard dose amount; wherein the presence of a genotype having a PIEZO1 predicted loss-of-function variant nucleic acid molecule encoding a human PIEZO1 polypeptide indicates that the patient has an increased risk of developing varicose veins. In some embodiments, the patient is heterozygous for a PIEZO1 predicted loss-of-function variant nucleic acid molecule. In some embodiments, the patient is homozygous for a PIEZO1 predicted loss-of-function variant nucleic acid molecule.

[0040] In any of the embodiments described herein, the PIEZO1 predicted loss-of-function variant nucleic acid molecule can be any PIEZO1 nucleic acid molecule (such as, for example, a genomic nucleic acid molecule, an mRNA molecule, or a cDNA molecule) encoding a PIEZO1 polypeptide having a partial loss of function, a complete loss of function, a predicted partial loss of function, or a predicted complete loss of function. For example, the PIEZO1 predicted loss-of-function variant nucleic acid molecule can be any of the PIEZO1 variant nucleic acid molecules described herein.

[0041] Genotyping assays to determine whether a patient has a predicted loss-of-function variant nucleic acid molecule of PIEZO1 encoding a human PIEZO1 polypeptide can be performed by any of the methods described herein. In some embodiments, these methods can be performed in vitro. In some embodiments, these methods can be performed in situ. In some embodiments, these methods can be performed in vivo. In any of these embodiments, the nucleic acid molecule can be present in a cell obtained from a human subject.

[0042] In some embodiments, when the patient is homozygous for a predicted loss-of-function variant nucleic acid molecule of PIEZO1, a therapeutic agent for treating or inhibiting varicose veins is administered to the patient in an amount equal to or greater than the standard dosage amount administered to patients who are heterozygous for a predicted loss-of-function variant nucleic acid molecule of PIEZO1.

[0043] The present disclosure also provides a method for treating a patient with a therapeutic agent for treating or inhibiting varicose veins, wherein the patient suffers from varicose veins or has an increased risk of developing varicose veins, the method comprising the following steps: determining whether the patient has a PIEZO1 predicted loss-of-function variant polypeptide by: obtaining or having obtained a biological sample from the patient; and performing or having performed an assay on the biological sample to determine whether the patient has a PIEZO1 predicted loss-of-function variant polypeptide; and when the patient does not have a PIEZO1 predicted loss-of-function variant polypeptide, then administering or continuing to administer a therapeutic agent for treating or inhibiting varicose veins to the patient in a standard dose amount; and when the patient has a PIEZO1 predicted loss-of-function variant polypeptide, then administering or continuing to administer a therapeutic agent for treating or inhibiting varicose veins to the patient in an amount equal to or greater than the standard dose amount; wherein the presence of a PIEZO1 predicted loss-of-function variant polypeptide indicates that the patient has an increased risk of developing varicose veins. In some embodiments, the patient has a PIEZO1 predicted loss-of-function variant polypeptide. In some embodiments, the patient does not have a PIEZO1 predicted loss-of-function variant polypeptide.

[0044] Determination of whether a patient has a predicted loss-of-function variant polypeptide of PIEZO1 can be performed by any of the methods described herein. In some embodiments, these methods can be performed in vitro. In some embodiments, these methods can be performed in situ. In some embodiments, these methods can be performed in vivo. In any of these embodiments, the polypeptide can be present in a cell obtained from a human subject.

[0045] In any of the embodiments described herein, the PIEZO1 predicted loss-of-function variant polypeptide can be any PIEZO1 polypeptide having a partial loss of function, a complete loss of function, a predicted partial loss of function, or a predicted complete loss of function. For example, the PIEZO1 predicted loss-of-function variant polypeptide can be any of the PIEZO1 variant polypeptides described herein.

[0046] In any of the embodiments described herein, the varicose veins may be early varicose veins (e.g., C0 according to the CEAP (Clinical, Etiology, Anatomy and Pathophysiology) classification). In some embodiments, the varicose veins may be late varicose veins (e.g., C6 according to the CEAP classification). In some embodiments, the varicose veins may be any disease stage (e.g., C0-C6 according to the CEAP classification). In some embodiments, the human subject is female.

[0047] Symptoms of varicose veins include, but are not limited to, heaviness in the legs, spider veins in the affected legs (telangiectasia), swelling in the ankles (especially at night), shiny brown-yellow skin discoloration near the affected veins, redness, dryness, and itching of the skin area (called stasis dermatitis or venous eczema), cramping (especially with sudden movements such as standing up), minor injuries to the affected area that bleed more than normal or take a long time to heal, shrinkage of the skin above the ankle (lipodermatosclerosis), restless legs syndrome, white, irregular, scar-like patches on the ankles (atrophic leukoplakia), or any combination thereof.

[0048] 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).

[0049] In some embodiments, the dosage of the therapeutic agent for treating or inhibiting 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., lower than the standard dosage amount) for patients or human subjects who are heterozygous for the predicted loss-of-function variant nucleic acid molecule of PIEZO1, compared to patients or human subjects who are homozygous for the predicted loss-of-function variant nucleic acid molecule of PIEZO1 (who can accept the standard dosage amount). In some embodiments, the dosage of the therapeutic agent for treating or inhibiting varicose veins may be reduced by about 10%, about 20%, about 30%, about 40%, or about 50%. In addition, the dosage of the therapeutic agent for treating or inhibiting varicose veins for patients or human subjects who are heterozygous for the predicted loss-of-function variant nucleic acid molecule of PIEZO1 can be administered less frequently than for patients or human subjects who are homozygous for the predicted loss-of-function variant nucleic acid molecule of PIEZO1.

[0050] The administration of the therapeutic agent for treating or inhibiting varicose veins can be repeated, for example, after one day, two days, three days, five days, one week, two weeks, three weeks, one month, five weeks, six weeks, seven weeks, eight weeks, two months, or three months. Repeated administration can be the same dose or different doses. Administration can be repeated once, twice, three times, four times, five times, six times, seven times, eight times, nine times, ten times or more. For example, according to certain dosage regimens, patients can receive therapy for a longer period of time, such as, for example, 6 months, 1 year, or longer.

[0051] The administration of the therapeutic agent for treating or inhibiting varicose veins can occur 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 and substantially isotonic and manufactured under GMP conditions. The pharmaceutical composition can be provided in unit dosage form (i.e., a single dose). The pharmaceutical composition can be formulated using one or more physiologically and pharmaceutically acceptable carriers, diluents, excipients or adjuvants. The preparation depends on the selected route of administration. The term "pharmaceutically acceptable" means that the carrier, diluent, excipient or adjuvant is compatible with the other ingredients of the preparation and is substantially harmless to its recipient.

[0052] As used herein, the terms "treat," "treating," and "treatment," as well as "prevent," "preventing," and "prevention" refer to inducing a desired biological response, such as a therapeutic and prophylactic effect, respectively. In some embodiments, after administration of an agent or a composition comprising the agent, the therapeutic effect includes one or more of: reduction / reduction of varicose veins, reduction / reduction of severity of varicose veins (e.g., such as, reduction or inhibition of the development of varicose veins), reduction / reduction of 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 recurrence, reducing the number or frequency of recurrent attacks, increasing the latency between symptom attacks, increasing the time to sustained progression, accelerating recovery, and / or increasing the efficacy of or reducing resistance to alternative therapeutic agents. A preventive effect may include complete or partial avoidance / inhibition or development / progression of varicose veins (e.g., such as, complete or partial avoidance / inhibition or delay) after administration of a treatment regimen. Treatment of varicose veins includes treating patients diagnosed with any form of varicose veins at any clinical stage or presentation, delaying the onset or development or aggravation or worsening of symptoms or signs of varicose veins, and / or preventing and / or reducing the severity of varicose veins.

[0053] The present disclosure also provides, in any of the methods described herein, for detecting or determining the presence of a PIEZO1 predicted loss-of-function variant genomic nucleic acid molecule, a PIEZO1 predicted loss-of-function variant mRNA molecule, and / or a PIEZO1 predicted loss-of-function variant cDNA molecule in a biological sample from a human subject. It should be understood that gene sequences within a population and mRNA molecules encoded by such genes may vary due to polymorphisms (such as single nucleotide polymorphisms). The sequences provided herein of the PIEZO1 variant nucleic acid molecules disclosed herein are exemplary sequences only. Other sequences of PIEZO1 variant nucleic acid molecules are also possible.

[0054] The biological sample can be derived from any cell, tissue or biological fluid of the subject. The sample can include any clinically relevant tissue such as a bone marrow sample, a tumor biopsy sample, a fine needle aspirate, or a sample of a body fluid such as blood, gingival crevicular fluid, plasma, serum, lymph, ascites, cystic fluid or urine. In some cases, the sample includes an oral swab. The sample used for the method disclosed herein varies according to the assay format, the nature of the detection method, and the tissue, cell or extract used as the sample. The biological sample can be treated differently according to the assay adopted. For example, when detecting any PIEZO1 variant nucleic acid molecule, a preliminary treatment of a sample designed to separate or enrich genomic DNA can be used. Various known techniques can be used for this purpose. When detecting the level of any PIEZO1 variant mRNA, different techniques can be used to enrich biological samples with mRNA. Various methods for detecting the presence or level of mRNA or the presence of a specific variant genomic DNA locus can be used.

[0055] In some embodiments, detecting a loss-of-function variant nucleic acid molecule of human PIEZO1 in a human subject comprises analyzing or genotyping a biological sample obtained from the human subject to determine whether the PIEZO1 genomic nucleic acid molecule, PIEZO1 mRNA molecule, or PIEZO1 cDNA molecule generated from the mRNA molecule in the biological sample contains one or more variations that result in loss of function (partial or complete) or are predicted to result in loss of function (partial or complete).

[0056] In some embodiments, a method for detecting the presence or absence of a predicted loss-of-function variant nucleic acid molecule of PIEZO1 (e.g., a genomic nucleic acid molecule, an mRNA molecule, and / or a cDNA molecule) in a human subject comprises performing an assay on a biological sample obtained from the human subject, wherein the assay determines whether the nucleic acid molecule in the biological sample contains a specific nucleotide sequence.

[0057] In some embodiments, the biological sample comprises a cell or a cell lysate. Such methods may also include, for example, obtaining a biological sample comprising a PIEZO1 genomic nucleic acid molecule or an mRNA molecule from a subject, and if comprising mRNA, optionally reverse transcribing the mRNA into cDNA. Such assays may include, for example, determining the identity of these positions of a specific PIEZO1 nucleic acid molecule. In some embodiments, the method is an in vitro method.

[0058] In some embodiments, the determining step, detecting step or genotyping assay comprises sequencing at least a portion of the nucleotide sequence of a PIEZO1 genomic nucleic acid molecule, a PIEZO1 mRNA molecule, or a PIEZO1 cDNA molecule generated from an mRNA molecule in a biological sample, wherein the sequenced portion comprises one or more variations that cause loss of function (partial or complete) or are predicted to cause loss of function (partial or complete).

[0059] In any of the methods described herein, the determining step, the detecting step or the genotyping assay comprises sequencing at least a portion of the nucleotide sequence of the PIEZO1 nucleic acid molecule in the biological sample, wherein the sequenced portion comprises a position corresponding to a predicted loss-of-function variant position, wherein when a variant nucleotide at a predicted loss-of-function variant position is detected, the PIEZO1 nucleic acid molecule in the biological sample is a PIEZO1 predicted loss-of-function variant nucleic acid molecule.

[0060] In some embodiments, the determining step, detecting step or genotyping assay comprises: a) contacting the biological sample with a primer that hybridizes to a portion of the nucleotide sequence of the PIEZO1 nucleic acid molecule adjacent to a predicted loss-of-function variant position; b) extending the primer at least through the predicted loss-of-function variant position; and c) determining whether the extension product of the primer comprises a variant nucleotide at the predicted loss-of-function variant position.

[0061] 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.

[0062] In some embodiments, the determining step, the detecting step or the genotyping assay comprises: a) amplifying at least a portion of a PIEZO1 nucleic acid molecule encoding a human PIEZO1 polypeptide, wherein the portion comprises a predicted loss-of-function variant position; b) labeling the amplified nucleic acid molecule with a detectable label; c) contacting the labeled nucleic acid molecule with a support comprising an alteration-specific probe, wherein the alteration-specific probe comprises a nucleotide sequence that hybridizes to a predicted loss-of-function variant position under stringent conditions; and d) detecting the detectable label.

[0063] In some embodiments, the nucleic acid molecule is mRNA, and the determining step further comprises reverse transcribing the mRNA into cDNA prior to the amplifying step.

[0064] In some embodiments, the determining step, detecting step, or genotyping assay comprises: contacting a nucleic acid molecule in a biological sample with a change-specific probe comprising a detectable label, wherein the change-specific probe comprises a nucleotide sequence that hybridizes to a predicted loss-of-function variant position under stringent conditions; and detecting the detectable label.

[0065] The change specific probe or change specific primer described herein comprises a nucleic acid sequence complementary and / or hybridized or specifically hybridized to a loss-of-function variant nucleic acid molecule predicted by PIEZO1 or its complementary sequence. In some embodiments, the change specific probe or change specific primer comprises at least about 5, at least about 8, at least about 10, at least about 11, at least about 12, at least about 13, at least about 14, at least about 15, at least about 16, at least about 17, at least about 18, at least about 19, at least about 20, at least about 21, at least about 22, at least about 23, at least about 24, at least about 25, at least about 30, at least about 35, at least about 40, at least about 45 or at least about 50 nucleotides or consists of it. In some embodiments, the change specific probe or change specific primer comprises at least 15 nucleotides or consists of it. In some embodiments, the change specific probe or change specific primer comprises at least 15 nucleotides to at least about 35 nucleotides or consists of it. In some embodiments, the alter-specific probe or alter-specific primer hybridizes to a PIEZO1 predicted loss-of-function variant genomic nucleic acid molecule, a PIEZO1 predicted loss-of-function variant mRNA molecule, and / or a PIEZO1 predicted loss-of-function variant cDNA molecule under stringent conditions.

[0066] Change-specific polymerase chain reaction techniques can be used to detect mutations in nucleic acid sequences, such as SNPs. Because DNA polymerase will not extend when there is a mismatch with the template, change-specific primers can be used.

[0067] In some embodiments, the nucleic acid molecule in the sample is mRNA and the mRNA is reverse transcribed into cDNA prior to the amplification step.In some embodiments, the nucleic acid molecule is present within a cell obtained from a human subject.

[0068] In any of the embodiments described herein, the PIEZO1 predicted loss-of-function variant nucleic acid molecule can be any PIEZO1 nucleic acid molecule (such as, for example, a genomic nucleic acid molecule, an mRNA molecule, or a cDNA molecule) encoding a PIEZO1 polypeptide having a partial loss of function, a complete loss of function, a predicted partial loss of function, or a predicted complete loss of function. For example, the PIEZO1 predicted loss-of-function variant nucleic acid molecule can be any of the PIEZO1 variant nucleic acid molecules described herein.

[0069] In some embodiments, the assay comprises contacting the biological sample with a primer or probe, such as an alteration-specific primer or an alteration-specific probe, which specifically hybridizes under stringent conditions to a PIEZO1 variant genomic sequence, a variant mRNA sequence, or a variant cDNA sequence but not to a corresponding PIEZO1 reference sequence, and determining whether hybridization has occurred.

[0070] In some embodiments, the assay comprises RNA sequencing (RNA-Seq). In some embodiments, the assay further comprises reverse transcribing the mRNA into cDNA, such as by reverse transcriptase polymerase chain reaction (RT-PCR).

[0071] In some embodiments, the method utilizes probes and primers with sufficient nucleotide lengths to bind to the target nucleotide sequence and specifically detect and / or identify polynucleotides comprising PIEZO1 variant genomic nucleic acid molecules, variant mRNA molecules, or variant cDNA molecules. Hybridization conditions or reaction conditions can be determined by the operator to achieve this result. The nucleotide length can be any length sufficient for the selected detection method (including any assay described or exemplified herein). Such probes and primers can be specifically hybridized with the target nucleotide sequence under high stringency hybridization conditions. Probes and primers can have complete nucleotide sequence identity of continuous nucleotides within 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. 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 with the nucleotide sequence of the target nucleic acid molecule.

[0072] The illustrative examples of nucleic acid sequencing techniques include but are not limited to chain terminator (Sanger) sequencing and dye terminator sequencing. Other methods include nucleic acid hybridization methods other than sequencing, which include using primers or probes (fluorescence in situ hybridization (FISH)) for the labeling of purified DNA, amplified DNA and fixed cell products. In some methods, the target nucleic acid molecule can be amplified before or simultaneously with detection. The illustrative examples of nucleic acid amplification techniques include but are not limited to polymerase chain reaction (PCR), ligase chain reaction (LCR), strand displacement amplification reaction (SDA) and amplification reaction (NASBA) based on nucleic acid sequence. Other methods include but are not limited to ligase chain reaction, strand displacement amplification reaction and thermophilic SDA (tSDA).

[0073] In hybridization techniques, stringent conditions may be employed so that a probe or primer specifically hybridizes to its target. In some embodiments, the detectable extent of hybridization of a polynucleotide primer or probe to its target sequence under stringent conditions will be higher than the detectable extent of hybridization to other non-target sequences, such as at least 2 times, at least 3 times, at least 4 times or more above background, including more than 10 times above background. Stringent conditions are sequence dependent and will be different in different environments.

[0074] Appropriate stringency conditions to promote DNA hybridization (e.g., 6X sodium chloride / sodium citrate (SSC) at about 45°C, followed by a wash of 2X SSC at 50°C) are known or can be found in Current Protocols in Molecular Biology, John Wiley and Sons, NY (1989), 6.3.1-6.3.6. Typically, stringent conditions for hybridization and detection will be those in which the salt concentration is less than about 1.5 M NaCl at pH 7.0 to 8.3. + ions, usually about 0.01 to 1.0 M Na + ion concentration (or other salts), and the temperature is at least about 30°C for short probes (such as, for example, 10 to 50 nucleotides), and at least about 60°C for longer probes (such as, for example, greater than 50 nucleotides). Stringent conditions can also be achieved by adding destabilizing agents such as formamide. Optionally, the wash buffer can contain about 0.1% to about 1% SDS. The duration of hybridization is generally less than about 24 hours, typically about 4 to about 12 hours. The duration of the wash time will be at least the length of time sufficient to reach equilibrium.

[0075] The present disclosure also provides a molecular complex comprising any one of the PIEZO1 nucleic acid molecules (genomic nucleic acid molecules, mRNA molecules or cDNA molecules) described herein or its complementary sequence and any one 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 complementary sequence in the molecular complex is single-stranded. In some embodiments, the PIEZO1 nucleic acid molecule is any one of the genomic nucleic acid molecules described herein. In some embodiments, the PIEZO1 nucleic acid molecule is any one of the mRNA molecules described herein. In some embodiments, the PIEZO1 nucleic acid molecule is any one of the cDNA molecules described herein. In some embodiments, the molecular complex comprises any one of the PIEZO1 nucleic acid molecules (genomic nucleic acid molecules, mRNA molecules or cDNA molecules) described herein or its complementary sequence and any one of the change-specific primers described herein. In some embodiments, the molecular complex comprises any one of the PIEZO1 nucleic acid molecules (genomic nucleic acid molecules, mRNA molecules or cDNA molecules) described herein or its complementary sequence and any one of the change-specific probes described herein. In some embodiments, the molecular complex comprises a non-human polymerase.

[0076] In some embodiments, detecting the presence of a human PIEZO1 predicted loss-of-function polypeptide comprises assaying a sample obtained from a human subject to determine whether the PIEZO1 polypeptide in the subject contains one or more variations that cause the polypeptide to have a loss of function (partial or complete) or a predicted loss of function (partial or complete). In some embodiments, the assay comprises sequencing at least a portion of the PIEZO1 polypeptide comprising a variant position. In some embodiments, the detection step comprises sequencing the entire polypeptide. Identification of a variant amino acid at a variant position of the PIEZO1 polypeptide indicates that the PIEZO1 polypeptide is a PIEZO1 predicted loss-of-function polypeptide. In some embodiments, the assay comprises an immunoassay for detecting the presence of a polypeptide comprising a variant. Detection of a variant amino acid at a variant position of the PIEZO1 polypeptide indicates that the PIEZO1 polypeptide is a PIEZO1 predicted loss-of-function polypeptide.

[0077] Probes and / or primers described herein (including change-specific probes and change-specific primers) comprise or consist of about 15 to about 100, about 15 to about 35 nucleotides. In some embodiments, change-specific probes and change-specific primers comprise DNA. In some embodiments, change-specific probes and change-specific primers comprise RNA. In some embodiments, probes and primers described herein (including change-specific probes and change-specific primers) have nucleotide sequences that specifically hybridize with any one of the nucleic acid molecules disclosed herein or their complementary sequences. In some embodiments, probes and primers (including change-specific probes and change-specific primers) specifically hybridize with any one of the nucleic acid molecules disclosed herein under stringent conditions. In the context of the present disclosure, "specific hybridization" means that probes or primers (including change-specific probes and change-specific primers) do not hybridize with nucleic acid sequences encoding PIEZO1 reference genome nucleic acid molecules, PIEZO1 reference mRNA molecules and / or PIEZO1 reference cDNA molecules. In some embodiments, probes (such as, change-specific probes) comprise labels. In some embodiments, the label is a fluorescent label, a radioactive label or biotin.

[0078] The nucleotide sequence of the PIEZO1 reference genome nucleic acid molecule is shown in SEQ ID NO: 1, and its length is 69,883 nucleotides. The first nucleotide described in SEQ ID NO: 1 corresponds to the nucleotide at position 88,715,338 of chromosome 16 (see, hg38_knownGene_ENST00000301015.14).

[0079] There are numerous variant genomic nucleic acid molecules of PIEZO1, including, but not limited to (using human genome reference build GRch38): 16:88715629:G:A, 16:88715728:G:T, 16:88715767:G:A, 16:88715802:C:A, 16:88715822:D:4, 16:88715987:I:1, 16:88716359:A:G, 16:88716570:C:T, 16:88716874:G:A, 16:88717213:T:A, 16:88719588:G:A, 16:88719722:C:G, 16:887198 70:G:T、16:88720068:D:2、16:88720229:C:A、16:88720248:D:4、16:8872 0394:C:T、16:88720644:D:1、16:88720698:D:1、16:88720698:I:1、16:88 721165:C:A、16:88721268:D:1、16:88721307:G:A、16:88721586:G:C、16: 88721652:G:C、16:88722217:C:T、16:88722605:I:1、16:88723005:I:7、1 6:88723253:G:A、16:88723311:C:T、16:88725081:C:A、16:88726282:G: A. 16:88726546:C:T. 16:88726619:G:A. 16:88726924:G:A. 16:88727038: C:T、16:88727072:D:1、16:88727163:G:A、16:88731768:D:1、16:8873233 4:C:G、16:88732411:D:1、16:88732720:D:1、16:88733326:G:C、16:88733 337:D:4, 16:88733587:C:A, 16:88733965:D:1, 16:88734017:C:A, 16:887 34042:I:1, 16:88734679:C:T, 16:88734909:I:1, 16:88736167:D:2, 16:8 8736324:G:A, 16:88736391:G:T, 16:88736409:C:T, 16:88736671:G:A, 16 :88737557:A:C、16:88737727:C:G、16:88737815:C:T、16:88738283:G:C、16:88738637:G:A, 16:88738735:D:1, 16:88741477:C:T, 16:88742306:D:1, 16:88749399:G:A and 16:88784929:C:T. Thus, for example, using the SEQ ID NO:1 reference genome nucleotide sequence as a basis (where the first nucleotide listed is designated as position 88,715,338), the first listed variant (16:88715629:G:A) replaces guanine with adenine (referred to as the "variant nucleotide") at position 88,715,629 (referred to as the "variant position"). Those variants designated as "D" followed by a number have the stated number of nucleotide deletions. Those variants designated as "I" followed by a number have the stated number of nucleotide (any nucleotide) insertions. Any of these PIEZO1 predicted loss-of-function variant genomic nucleic acid molecules can be detected in any of the methods described herein.

[0080] The nucleotide sequence of the PIEZO1 reference mRNA molecule is shown in SEQ ID NO: 2 (see NCBI reference sequence: NM_001142864.4), which is 8,089 nucleotides in length. The variant genomic nucleic acid molecules described herein also have corresponding variant nucleotides at their respective variant positions of the variant mRNA molecules based on the PIEZO1 reference mRNA sequence according to SEQ ID NO: 2. Any of these PIEZO1 predicted loss-of-function variant mRNA molecules can be detected in any of the methods described herein.

[0081] The nucleotide sequence of the PIEZO1 reference cDNA molecule is shown in SEQ ID NO: 3 (based on NCBI reference sequence: NM_001142864.4), which is 8,089 nucleotides in length. The variant genomic nucleic acid molecules described herein also have corresponding variant nucleotides at their respective variant positions based on the PIEZO1 reference cDNA sequence according to SEQ ID NO: 3. Any of these PIEZO1 predicted loss-of-function variant cDNA molecules can be detected in any of the methods described herein.

[0082] The amino acid sequence of the PIEZO1 reference polypeptide is shown in SEQ ID NO: 4 (see, UniProt Accession No. Q92508.4 and NCBI RefSeq Accession No. NM_001142864.4), which is 2,521 amino acids in length. Using the translated nucleotide sequence of the PIEZO1 mRNA or cDNA molecule, the PIEZO1 variant polypeptide has the corresponding translated variant amino acid at the variant position (codon). Any of these PIEZO1 predicted loss-of-function variant polypeptides can be detected in any of the methods described herein.

[0083] The nucleotide sequences and amino acid sequences listed in the attached sequence table 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 from the 5' end of the sequence and proceeding (i.e., from left to right in each row) to the 3' end. Only one strand of each nucleotide sequence is shown, but it should be understood that the complementary strand is included by any reference to the strand shown. The amino acid sequences follow the standard convention of starting from the amino terminus of the sequence and proceeding (i.e., from left to right in each row) to the carboxyl terminus.

[0084] As used herein, when used in the context of the numbering of a specific nucleotide or nucleotide sequence or position, the phrase "corresponding to" or its grammatical variants refers to the numbering of a specified reference sequence when a specific nucleotide or nucleotide sequence is compared to a reference sequence. In other words, the residue (e.g., such as, nucleotide or amino acid) number or residue (e.g., such as, nucleotide or amino acid) position of a specific polymer is specified relative to a reference sequence, rather than being specified by the actual numerical position of the residue in a specific nucleotide or nucleotide sequence. For example, a specific nucleotide sequence can be aligned with a reference sequence by introducing a gap to optimize the residue matching between the two sequences. In these cases, although there is a gap, the numbering of the residues in a specific nucleotide or nucleotide sequence is performed relative to the reference sequence to which it is aligned. There are a variety of computational algorithms that can be used to perform sequence alignment to identify the nucleotide or amino acid position in one polymer molecule that corresponds to the nucleotide or amino acid position in another polymer molecule. For example, the 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, the sequences can also be aligned manually.

[0085] All patent documents, websites, other publications, accession numbers, etc. cited above or below are incorporated by reference as a whole for all purposes to the same extent as if each individual project was specifically and individually indicated as being so incorporated by reference. If different versions of the sequence are related to accession numbers at different times, then it means the version related to the accession number at the effective application date of the present application. If applicable, when referring to the accession number, the effective application date means the earlier of the actual application date or the application date of the priority application. Similarly, if different versions of publications, websites, etc. are published at different times, then unless otherwise specified, it means the latest published version at the effective application date of the present application. Unless otherwise specifically specified, any feature, step, element, embodiment or aspect of the present disclosure can be used in combination with any other feature, step, element, embodiment or aspect. Although the present disclosure has been described in detail by illustration and example for the purpose of clarity and understanding, it will be apparent that certain changes and modifications can be made within the scope of the appended claims.

[0086] The following examples are provided to describe the embodiments in more detail. They are intended to illustrate but not limit the claimed embodiments. The following examples provide disclosure and description of how to make and evaluate the compounds, compositions, articles, devices and / or methods described herein for those of ordinary skill in the art, and are intended to be exemplary only and are not intended to limit the scope of any claim. Efforts have been made to ensure accuracy with respect to the numerical values ​​used (e.g., amounts, temperatures, etc.), but some errors and deviations may be considered. Unless otherwise indicated, parts are parts by weight, temperatures are in ° C or at ambient temperature, and pressures are at or near atmospheric pressure.

[0087] Example

[0088] Example 1: Materials and Methods

[0089] WES sample preparation and sequencing

[0090] Genomic DNA samples standardized to about 16 ng / μl were transferred from UK Biobank internally into 0.5 ml 2D matrix tubes (Thermo Fisher Scientific) and stored in an automated sample biobank (LiCONiC Instruments) at -80°C before sample preparation. DNA in one sample was insufficient for sequencing. Exome capture was completed using a high-throughput, fully automated method developed in-house. In brief, DNA libraries were generated by enzymatically shearing 100 ng of genomic DNA into an average fragment size of 200 base pairs using a custom NEBNext Ultra II FS DNA library preparation kit (New England Biolabs), and universal Y-shaped adapters (Integrated DNA Technologies) were connected to all DNA libraries. During library amplification with KAPA HiFi polymerase (KAPA Biosystems), unique asymmetric 10 base pair barcodes were added to DNA fragments to facilitate multiple exome capture and sequencing. Equal amounts of samples were pooled and then subjected to overnight exome capture for approximately 16 hours using a slightly modified IDT xGen probe library format; supplemental probes were added to capture regions of the genome that were adequately covered by the previous capture reagent (NimbleGen VCRome) but inadequately covered by the standard xGen probes. A total of n=38,997,831 bases were included in the targeted regions. The captured fragments were then coupled to streptavidin-coupled (Thermo Fisher Scientific), and nonspecific DNA fragments were removed by a series of stringent washes using the xGen Hybridization and Wash Kit according to the manufacturer's recommended protocol (Integrated DNA Technologies). The captured DNA was PCR amplified using KAPA HiFi and quantified by qPCR using the KAPA Library Quantification Kit (KAPA Biosystems). Multiple samples were pooled and quantified using an Illumina S2 flow cell. Sequencing was performed on the 6000 platform using 75 base pair paired-end reads and two 10 base pair index reads.

[0091] Sequence alignment, variant identification, and genotype assignment

[0092] After sequencing is completed, The raw data of operation is collected in local buffer memory and uploaded to DNAnexus platform for automatic analysis.After uploading, the reading that CBCL file is converted into FASTQ format begins to analyze, and bcl2fastq conversion software (Illumina Inc., San Diego, CA) is used to distribute to sample by specific barcode.Then the sample-specific FASTQ file of all readings produced for the sample represented is aligned with GRCh38 genome reference with BWA-mem.The resulting binary alignment file (BAM) of each sample comprises the genome coordinates, quality information and specific reading of mapping reading and the degree different from the reference at its mapping position.The alignment reading in BAM file is then evaluated to use Picard MarkDuplicates tool (World Wide Web " picard.sourceforge.net ") to identify and mark repeated reading, thereby produce alignment file (duplicatesMarked.BAM), wherein the potential repeated reading of all marks is used to exclude in downstream analysis.

[0093] Then use the WeCall variant calling program from Genomics PLC (world wide web "github.com / Genomicsplc / wecall") to generate a GVCF file including variant calls for each individual sample, thereby identifying both SNVs and INDELs compared to the reference. In addition, each GVCF file carries the zygosity of each variant, the read counts of the reference and alternative alleles, the genotype quality representing the confidence of the genotype call, and the overall quality of the variant call at that position.

[0094] After variant calling was completed, each sample BAM file was converted to a completely lossless CRAM file using samtools. Metrics statistics for each sample were captured to evaluate capture, alignment, insert size, and variant calling quality using Picard (World Wide Web "picard.sourceforge.net"), bcftools (World Wide Web "samtools.github.io / Bcftools"), and FastQC (World Wide Web "bioinformatics.babraham.ac.uk / projects / fastqc").

[0095] After completing sample sequencing, samples showing differences between genetically determined and reported sex (n=15), high heterozygosity / contamination rates (D-stat>0.4) (n=7), low sequence coverage (less than 85% of the targeted bases reach 20X coverage) (n=1) or genetically identified sample duplicates (n=14) and WES variants inconsistent with the genotyping chip (n=9) were excluded. Six samples failed quality control in multiple categories, resulting in 38 individuals being excluded. The remaining 49,960 samples were then used to compile a project-level VCF (PVCF) for downstream analysis. The PVCF was created using the GLnexus joint genotyping tool. Note that all homozygous reference, heterozygous, homozygous alternative, and no-call genotypes were carried into the project-level VCF. An additional filtered PVCF, "Goldilocks," was also generated. In the filtered Goldilocks PVCF, samples carrying SNP variant calls in a single sample pipeline or DP<7 were converted to "no calls." After applying the DP filter, sites where all remaining samples were called heterozygous and all samples had AB < 85% ref / 15% alt were excluded. Samples carrying INDEL variant calls in the single sample pipeline with DP < 10 were converted to "no call". After applying the DP filter, sites where all remaining samples were called heterozygous and all samples had AB < 80% ref / 20% alt were excluded. Multi-allelic variant sites in PVCF files were normalized by left alignment and represented as biallelic.

[0096] Phenotypic definition

[0097] Cases based on ICD10 require one or more of the following: a major or minor diagnosis in the inpatient health event statistics (HES) record. Exclusions based on ICD10 have ≥1 major or minor diagnosis within the code range. Controls based on ICD10 are defined as those non-cases or excluded individuals. Custom phenotype definitions include one or more of the following: ICD-10 diagnoses, self-reported diseases from verbal interviews, and physician-diagnosed diseases from online follow-up and touch screen information. Quantitative traits (such as physical indicators, blood cell counts, cognitive function tests, and imaging-derived phenotypes) were downloaded from the UKBiobank (UKB) library and spanned one or more visits. A total of 1,073 binary traits were tested in the WES association analysis, with case counts ≥50 and 669 quantitative traits.

[0098] Annotation of predicted loss-of-function (LOF) variants

[0099] Variants were annotated using snpEff and gene models from Ensemb version 85. A comprehensive and high-quality transcript set of protein coding regions including all protein coding transcripts with annotated start and stop codons was obtained from the Ensembl gene model. Variants annotated as stop_gained, start_lost, splice_donor, splice_acceptor, stop_lost, and frameshift were considered LOF variants.

[0100] A recent large-scale study of genetic variation in 141,456 individuals provided a catalogue of LOF variants. Direct comparisons with this data are difficult due to a number of factors, such as differences in exome sequencing capture platforms, variant calling algorithms and annotation. Furthermore, the number of individuals and regional distribution of ascertainment (and therefore genetic diversity) in the NFE subset of gnomAD reported in this report are likely greater than those of the UK Biobank using WES. Nevertheless, the gnomAD exome sites labeled “PASS” from gnomAD r2.1 were annotated using the annotation pipeline. Data from gnomAD were lifted to HG38 using PicardLiftoverVcf. Data are restricted to MAF NFE We compared the LOFs in the UKB participants using WES of European ancestry with a subset of non-Finnish Europeans (NFE) (n=56,885 samples, individuals) with <1% of the variant and obtained 261,309 LOFs in any transcript in 17,951 genes. Restricting LOFs to those present in all transcripts, 175,162 LOFs were observed in 16,462 genes. 134,745 LOFs were observed in all gene transcripts in UKB participants using WES of European ancestry.

[0101] LOF burden association analysis method

[0102] Burden association tests were performed for rare LOFs in 49,960 individuals of European descent. For each gene region defined by Ensembl. LOFs with a MAF ≤ 0.01 were collapsed, so that any individual who was heterozygous for at least one LOF in the gene region was considered heterozygous, and only individuals carrying two copies of the same LOF were considered homozygous. Rare variants were not staged, so compound heterozygotes were not considered in this analysis.

[0103] For each gene region, 668 rank-based inverse normal transformation (RINT) quantitative indicators (including all subjects and sex-stratified models) with non-missing phenotypic information for ≥5 individuals were evaluated using additive mixed models implemented in BOLT-LMM v2. Before standardization, the traits were first appropriately transformed (log10, squared) and adjusted for a set of standard covariates, including age, sex, study site, the first four principal components of ancestry, and BMI and / or smoking status in some cases. Before standardization, data points greater than the five median absolute deviations of the median were excluded as outliers. Using the generalized mixed model implemented in SAIGE, 1,073 discrete results (including all subjects and sex-stratified models) for ≥50 cases were evaluated by covariate adjustment for the first four principal components of age, sex, and ancestry. For each quantitative and discrete trait included in the analysis, only gene regions with non-missing phenotypic and covariate information for >3 LOF carriers were evaluated.

[0104] Positive controls were systematically defined using a two-step approach. First, each gene with associated disease, trait, biological or functional evidence was annotated using publicly available resources, including OMIM, NCBIMedGen, and the NHGRI-EBI GWAS catalog. Genes with supporting evidence from at least one source were then hand-picked using NCBI PubMed to validate the relationship between the trait and the LOF variant in the gene of interest. Genes with locus-level support for the trait or associated phenotype in the GWAS catalog but lacking clear supporting evidence for the LOF association are reported as new LOF associations in this article.

[0105] Single variant LOF association analysis method

[0106] Single variant association analysis was performed using the same approach as described in the Methods section for burden association analysis. -7 Single variant association statistics were calculated for gene-trait associations using the phenotype of interest for all LOFs included in the burden test that were observed in 49,960 individuals of European ancestry using WES with a minor allele count ≥ 5. Association statistics for these variants are reported in the extended data (ExtData_SingleVariantLOFs_V1.xlsx).

[0107] The P value leaves one out (LOO) burden is the p value of the absence / presence test, excluding the tested variant. The ΔP value burden is the ratio of the p value in the leave one out analysis compared with the burden test using all 65 variants. The burden summary statistics of all 65 variants using unrelated individuals: B=1.44, SE=0.26, p value=3.12E-08, cMAF=0.00174, cMAC=152, cMAF case=0.0066, cMAC case=17, cMAF control=0.0016, cMAC case=135. Stepwise logistic regression selected 11 / 65 variants (AIC 11451). The burden summary statistics using 11 variants: B = 3.71, SE = 0.437, p value < 2e-16, cMAF = 0.0003, cMAC = 22, cMAF case = 0.005, cMAC case = 12, cMAF control = 0.0001, cMAC control = 10. When the analysis was restricted to variants with MAC> 1, stepwise logistic regression selected 5 / 13 variants (AIC 11484). The burden summary statistics using 5 variants: B = 3.02, SE = 0.53, p value = 8.92e-09.

[0108] The LD (r2) of 11 pLOF variants selected by stepwise regression for PIEZO1 and the positive control (16:88835545_G_A; highlighted in green) was reported in the literature for PIEZO1. None of these 12 variants were in LD, with R2>0.01. When the burden test was adjusted for the previously reported variant rs2911463 (16:88835545_G_A), the burden test p-value remained <2E-16 (AIC 11,444), indicating that the burden did not flag the reported variants.

[0109] Example 2: Demographic and clinical characteristics of sequencing participants

[0110] A total of 50,000 participants were selected, with priority given to individuals with more complete phenotypic data: those with whole-body MRI imaging data from the UKBiobank imaging study, enhanced baseline measurements, hospital event statistics (HES) and / or relevant primary care records (which will be available quickly by approved researchers). During data generation, samples from 40 participants were excluded due to quality control indicator failure or participant withdrawal, resulting in a final collection of 49,960 individuals. In summary, the sequenced samples represent 500,000 UKB participants (Table 1). There were no significant differences in age, sex or ancestry between the sequenced samples and the overall study population. Sequencing participants were more likely to have HES diagnostic codes (84.2% in the sequencing group and 77.3% overall) and enhanced indicators (Table 1).

[0111] Table 1: Clinical characteristics of sequenced whole exome and all UK Biobank participants

[0112]

[0113]

[0114] a Number of samples with at least one non-missing image-derived phenotype value in data downloaded from UK Biobank in November 2018.

[0115] b Number of samples with exome sequencing data and at least one non-missing image-derived phenotype value in data downloaded from UK Biobank in November 2018.

[0116] c The number of samples in the three predefined regions of the first two genetic principal component score plots, where the regions were chosen to represent African, East Asian, and European ancestry (see Figure 3 ).

[0117] WES participants with at least one HES diagnosis code did not differ from unsequenced participants in the median or broad phenotypic distribution of major and minor ICD10 codes, with the exception of asthma (ICD10 J45) and status asthmaticus (ICD10 J46) codes (most abundant in the sequenced sample) and senile cataract codes (ICD10 H25) and unknown and unspecified etiology codes (ICD10 R69) (least abundant). The sequenced subset included 194 parent-child pairs, 613 full sibling pairs, 1 monozygotic twin pair, and 195 secondary relationships. The distribution of relatedness between pairs of individuals in UKB WES included Figure 1 middle.

[0118] Example 3: Summary and characterization of coding variants from WES

[0119] Targeting protein coding regions and exon-intron splice sites of 19,467 genes. Autosomal variant counts observed in all individuals by type / functional category with all and MAF <1% frequency. All variants passed QC criteria with individual and variant missing <10% and Hardy Weinberg p-value >10 -15. Median and interquartile range (IQR) of variant counts for all variants and variants with MAF<1%. The average proportion of targeted bases (n=38,997,831) reaching at least 20-fold coverage in each sample was 94.6% (standard deviation 2.1%). 10,028,025 single nucleotide and indel variants were observed after quality control, 98.5% with minor allele frequency (MAF)<1% (Table 2). Of the total variants, 3,995,785 were within the targeted region. These variants included 2,431,680 nonsynonymous (98.9% with MAF < 1%), 1,200,882 synonymous (97.8% with MAF < 1%), and 205,867 predicted loss-of-function (pLOF) variants (start codon loss, premature stop codon, splicing, and frameshift indel variants; 99.7% with MAF < 1%) affecting at least one coding transcript ( Figure 2 ). The counts of 9,403 synonymous (IQR 125), 8,369 nonsynonymous (IQR 132), and 161 pLOF variants (IQR 14) per individual (median) are comparable to previous exome sequencing studies. If the analysis was restricted to pLOF variants affecting all transcripts of a gene, the number of pLOF variants dropped to 140,850 overall and 96 per individual (decreases of approximately 31.6% and approximately 40.4%, respectively), which is consistent with previous studies.

[0120] Table 2: Summary statistics of variants in the sequenced exomes of 49,960 UKB participants

[0121]

[0122] Example 4: Phenotypic association with LOF variation

[0123] The combination of WES and rich health information allows extensive studies of the phenotypic consequences of human genetic variation. LOF variants can provide insight into gene function; however, imputed datasets have lost most of these variants. WES is well suited to identifying LOF variants and evaluating their phenotypic associations. In n = 46,979 individuals of primarily European ancestry, 1,741 traits (1,073 discrete traits with at least 50 case counts defined by hospital event statistics and self-report data; 668 quantitative, anthropometric, and blood traits) were used to perform gene burden association tests for rare (AAF < 1%) pLOF variants (pLOF variants identified in all genes in > 3 pLOF variant carriers in WES). For each gene-trait association, the association strength of the pLOF gene burden test was also compared with the association results of each SNV included in the burden test.

[0124] Example 5: LOF association and novel gene discovery

[0125] In the pLOF gene burden association analysis, a novel association between PIEZO1 LOF (cumulative allele frequency = 0.2%) and a substantially increased risk of varicose veins was identified. Table 3 shows the PIEZO1 results for the binary phenotype of asymptomatic varicose veins of the lower extremities within the UKB 50k exome and UKB 150k exome.

[0126] Table 3: PIEZO1 LOF gene burden association

[0127]

[0128] This finding was driven by the burden of rare LOF variants, with the most significant PIEZO1 single variant LOF association in the UKB 50k exome achieving 2.29x10 -3 Leave-one-out analysis of the UKB 50k exon group indicated that no single variant explained the entire signal, and stepwise regression analysis indicated that 11 individual variants (5 of which had MAC>1) contributed to the overall burden signal ( Figure 4 ).

[0129] This finding was replicated in 2,953 varicose vein cases and 75,694 controls with previous exome sequencing (OR = 2.7, p = 1.86 x 10 -9 This region has been previously associated with common noncoding variants with small effects on disease risk, with rs2911463 and other common variants on nearby chromosome 16 recently associated with varicose veins (frequency = 0.69, OR = 0.996, p-value = 1.0x10 in a GWAS of 408,455 genotyped UKBiobank participants). -27 The rs2911463 variant was not in LD and no key variant was identified ( Figure 5 ), and the burden test remained significant when adjusted for rs2911463. Sequence Listing <110> Regeneron Pharmaceuticals, Inc. <120> Piezoelectric mechanosensitive ion channel component 1 (PIEZO1) variants and their uses <130> 189238.02502 (3172) <150> 62 / 862,847 <151> 2019-06-18 <150> 62 / 806,932 <151> 2019-02-18 <160> 4 <170> PatentIn version 3.5 <210> 1 <211> 69883 <212> DNA <213> Homo sapiens <400> 1 gggagccgcc gtccggccca gctcggcccc agtgagccga gcgctgcgct ccgccgaggg 60 gcagggcggt cgcctgagcg agcgcgggcc cgggacgtcg gcaccggcgg ggcggccgaa 120 ggagaaggag gaagaggaga aggcggcgcg cgggtccccg cgggtcagcc atggcgcgcc 180 ggccccgggg cccccgcacc gccccatagc gccgcggcgt ccgctcggtc tgggccgggc 240 cctgggccct ccagccatgg agccgcacgt gctcggcgcg gtcctgtact ggctgctgct 300 gccctgcgcg ctgctggctg gtgagtgggg ggcgggcgcc tgggggcgac gggagggggc 360 tgcgtctcgg ctccccacgg cctggacacc ggacgacgcc ggccggggcg agggctgcgg 420 gcgagcgggc gcggaaattc ccagggacgc gcgacccggg cgcccgcatt cctgaagcat 480 gagcgcgcca ggcggcggcg gggctcctgt cccagggccg ggctggaagg gcggcggcgg 540 ctgggggaga cggcaccgcg tgcccacggg ggcggtcgag cgagcgccgg ccatagcgcg 600 gctggcgtct ccgccggggc gctgcggaga ggaggccgcc gggcgaggcg gtgtttgccc 660 cggtgggaag ggccgcggcg gtggtgggg gagcacgaat ctctttttct ctttcgggtt 720 taaaaaaaaa agcgcaaagt tgcatcagga cttcctgaca atctgggaga aggcgggctt 780 cctgcctgga gctgtttaat ttggagcttc ccgagcccaa cgaacgtccg tgcccagggc 840 ccagccccgc tcaccgctgc accccccctct gccggactga ggcggtccca cactttggaa 900 aaaaatagtg tgggttcctc cctggtcctc ccttgcccta ctgggctcag tttcgcaggg 960 gccggggccg gcctctgccc tggtctgggg gaggggacac ccccggaggc tgtggcctgg 1020 tgtcagggcg gggcaggggt ccccagtcct ggcatctgtg ttccctgctt gccgggcagt 1080 ggtgcccctt tcgcgaagca cacccgggtg gcttggtgct gacggcctg cacccctac 1140 ccttccccga ccctggccta gccgggaccc agggtccgcg ccctccgcccc gggggctccc 1200 cacgtgtgat tgatctggga agcagtcgga tggaattaac ccacggacaa gtgggacggt 1260 ttgcattggg agtccgccat ggacacggca ggtggggcct tttgattgta aaagcccttt 1320 cggagccctt gcctcgctcc aggtgggagc tcgcccagcg ctagctttgg ggatctagag 1380 ccgcctgcct gaggctccca gacagactgc gttttgatcg gtcgcacaga aaggtggtga 1440 aacttgggga agattttcta gacaggaatc aatgaaaacc attgaggctg gagaggagag 1500 gttttgagca actctcttca gtgcggtcag ccctgtgtgg actgggcagc ctgggacctg 1560 ctcccagtgc agggtcagat gggccgtaaa cagggcccgg ctgtgttcct tcctgtgcct 1620 tgaaaacaag caggacagcc tggcacagag gcagagtcta gagctgacag gccttagaga 1680 gggaaacagg aaagcttctg aaacgtcccg ttcacactga tcgttccatt tcctcttgtg 1740 tctgagtggg agcgggtgtc ctccctgcag ggaatgcccc ccctctcaga tggcagctgc 1800 tccttgggca gagttggcaa tgtttttctt taaatgacca gatggtaaat attttcatgt 1860 cacaaaatct tcttcttctg gtatttttcc agccattaaa tgtaaaagcc ctcctgagtt 1920 catgggctgt acctaaacag gtgttgagcc cgattctgtg gcacatgtgg tttgctgccc 1980 cctgggcatt ggtcaggggg cctgggttct gccttctcga ttgctatccg cgtgggggat 2040 ctgggggagg gatcactgtt cttcttgctt ttggcctcct tggggaggat ggggaggtag 2100 cccaggggtg ctcacccagg ccccgtgtca gtcttctatg aaacttttaa agaatagtga 2160 tgactgactg tctgtctgta tggtactttc cttaaaccta aaactggtcc caaataaagt 2220 ctcttaattt gaaagatgct gaagcccggg ccatacccca cactgattct gtgtctgggg 2280 atggggcgtg gggcctgggc ctcactcagt gttttctctc agtcacctgg gggagatgga 2340 agtggagccg gccaagaacc ctgcctgcct gcctgctggc cgggactcct gagtcaggct 2400 ctctggccct ggggtgtggg cagctccaga tggacccgcg atgtgcaggt tcagctggcc 2460 tggccggagg tgggacactg gctttgctgt ctttggagtg ccccctccct ctctggcgag 2520 ctttggctgg aagcagttct accgtgtttt ggaaatgaat gaggccttca gaaggcatta 2580 gtcagtgtgt gcctgcgctg gctcagacag tgcctggtga gggtttgagt catcctgggg 2640 tgcccctggc ccccacgccc tccctctcca gtgcaggatc attacccaaa aatctggcag 2700 ggagctgccc cacccacagg gagcaggggc ctccttcagc agtctcacct aatgttgctg 2760 gagccttggg ggatcagggc ccatctcttc tagagagatg tcagggcagg gctgggcgcg 2820 atggctcaca cctgtaatcc cagagcttcg ggaggccaag gtgggaggat tgcttgagcg 2880 tagccattcg agagcagcct gggcaacgta gagatcccca tctctatgaa aaatatttaa 2940 aaattagctg ggcatggtgg tagtgcacct gtagtcccag ctactcagga ggccaaggtg 3000 ggatgattgt ttgagtccag gagttggagg ctacagtgag ccatgattgt atgactgcac 3060 tccagcctgg gtgacagacc ctgtctcaaa aaagaaaaag gaaaaaaaag ggcaggacat 3120 ttgtgattct gataatatgg gaccgcacct ccagttctat cagtgggaaa tctagacagg 3180 gctgcggaaa gccagctggt gcgagaggag ccaccgtgtc actgactgtg ggacacccac 3240 gtgggctgac aatatggctt ctgcttttca gggtgcctcg tggcacagcc taggggcaca 3300 cccacggccg gcaagctggg cgtcacctcc ttcaggctga ttgtcactga gaagtgtcac 3360 catttagcat gagggatgct gcctcctttt tcagaacatt gtcaccatca gggtctcacc 3420 actcctggga ggcggccgag aagctgggga acagcaggca ctcggctcac agttgctcag 3480 cagtgcagac cctctgagct gagcatggca gagtcaccct tcggaggcct gtgcccgggt 3540 ctcaggacct gcacagaacc ctggcctgtc ccatccgagg gtgctgggaa gagcatggcc 3600 gtggcagagt agggtgggag ctgctttcct ctgtggcttg ggggcccctt ctgagcatca 3660 gctccctggt gtgacagagg ggcgcactct gtccccatgc tgggcctgga ggctggatga 3720 gtcagcagga gagcctgggg cctgcctcac agcaccaagg gctgcaggtg tgagtgtgca 3780 catgtgtgcg tgtttgggga aggggccagg gactgcccag gagctgagga tgggtcacag 3840 cgggtgctcg tcccgcagcg ggtcactggt gcccaggaca cagggagctc cagccccagc 3900 tgccagggtc ccacagagag gaagtttcct ctgggggtgg gtgggggcgc acagtctctg 3960 atcctggccc caaggcagct tcctgggcgg tgtctctcct gtgctgactc ggcagtgcat 4020 ttgctttcgg tgctcaaaga tgaaggggaa ccaccgtggg ccttgacggc ctcatctgcc 4080 cgctgcagcc cactcctgag atgggaccac cgcagtcgtc agggtccagt gagagccgca 4140 tcttgcagga agccattcct ggcctctctg gcctcagaaa tccccttttc aattcaacaa 4200 aatgttagtc ttctgtttag ctattttaga aatagacagt taggcttttt tctctttttt 4260 cttaaagaca gaggctcact ctgtcgccca ggctggagagtg tagcggcgtg attttggctc 4320 cctacaactt cagcctccag ggctcaagcc atcctcctgc ctcagcctcc tgagtagctg 4380 agattacagg tgtgtagcac catacctaat ttttgtattt tttgtagaga cggggtttca 4440 ccactttggc caggctggcc ttgaactcct gaccttaagc gatctgcccg cctcagcctc 4500 caaaagtgct ggggttacag gcatgagcca ccgcgcccgg ctgacagtta gacttttgct 4560 tctttgttta tataagcttt ttcttctggt tccaaaagca agtttgccct tccttgtggt 4620 agagaatcct agctcacaga gcagtttaga agccagcaca gtatcccaca cacacatctg 4680 gcatggacag acccttcctt gctgggtgtg ggtcctgtgt tcttctgaaa ggcaagctgt 4740 tcccagccaa cccctgcccc tctctgcctt agcctgcctg gaggcctgag tctcctgggt 4800 gactgtgagg tgggacccc ccttccccta cccccaccca catcctctgt atctgccttc 4860 tgtcctgctc ttgacctttg agctcctctg ctggcttcag ggtgcggctg ttgagcctat 4920 tttttggatt aggactctgg ggtgagggaa gttaattcac acacccaaga tcacactggt 4980 gggaagggac aggcccgggg tgaaggcttc tcctctcctt ggcggttgag tcccacacct 5040 gctggccgag gcacctgaag gggacttggg gtccagggtc actgggagga cgggggcagg 5100 cagaggggtg gccgacctgg tggcggctcg tgggcagcag ccgaccctat cttgctctga 5160 acgtgtgggg ccctcacccc cttctctggg tctgggtttc ctcccctgta agtgacaccg 5220 taaaagcttc acagccgctt ccaagtctca ggtctctcgg gctttggatc tcacacccag 5280 gctgggtggg ggtaggggcg ggacagccgt cccccccgga aggctcagaa ttcctcgcac 5340 aatcgtgggg ccaggagacc cgcaacacag gctttcccag ctgcgctgag tgccgcggtg 5400 gccgggggtc cgtcgggcct ccatggagct gaggggaagg ggccactcac cgcctggtcc 5460 cggagcacac agggcagctc ccaggagcac agaggctcct tgggacctgt ggggctgtcg 5520 gcctccctca tgctgcacac acagcgcgtc cccaggggtg tctgcagccc agcccatccc 5580 actgcagatt cccgaccact cagattcatt catgcatcct ctcacgccgg tcctgtggga 5640 cagagctctg ggcagcagcc agaagtccaa gttctggttc agggccagtg gaggtgggtg 5700 ttggggtggg gctggagctc cctgctctcc ctcccaagct agccaggaaa ggaggttggg 5760 ggccccgcac ggtcattgct gtttattcac aaagcgcgat gctgagcaca ggcggggaaa 5820 gaaaagtgcg atcagtgcca ggaaaatggg gctcccccga cgccgtccaa aatgggatcc 5880 cttgccgggc gcggtggctc acacctgtaa tcccagcact ttgggaagcc gaggcaggtg 5940 gatcacctga ggtcaggagt tcgagaccag cctgaccaac atggcaaaac tccatctcag 6000 ctaaaaatac aaaaattagc tgggcatggt ggcgggcacc tgtattccca gctgggaagg 6060 ctgagacagg agaatcgccg gaacccagga ggtagaggtt gcagtgagcc acagtcgtgc 6120 cactgcactc cagcttgggg gcttggggga cagcaagact ccctctcaaa aaaaaaaaaa 6180 aaaagtgaag tcgtaaatca gattaaattc cctttttaac cctttgaacc tctgtcctcc 6240 cctgttccca gcgggaagcc tctggtgaac gcgccatgca ccccacctgc ccccgctctc 6300 tgggtctctc tcccagctgg aacggccgct tccccaggtg ccttccctgg gccacagcct 6360 tgtgcctcgg cggctgctgg atgcctgggt gtgtgggtgg ctcccagtgt gtgggatggc 6420 acacgagcct ctcgcccttc tgtgtggggt cgcacaccca ccgcaggccg tatttttgct 6480 cacgttcatg tttctccacg gtggacgctg ggttgcaggg acccttcctg tgtgcgggtg 6540 aggatctggg cagctgctgg tgccgggccc atggggacgc tgaccgtccc gggtgccggc 6600 tctgaggtgt gcagtggacg gctgtcctgc cgggcgctgc cagggcccct taggccgacg 6660 tgcgtggcca cccgattccc cgccgttgtc tcaggaacct ttgccgagtg gggtggatca 6720 atttttcggg tgtgttttaa tagcataatt acagggagta tttcaggctc cctctgacgg 6780 gccggcaggg tttggctgcc ggctgtttac caggctccaa tctgcacact atttttctgt 6840 gggtatatat agctggggct gcttctcctt cctcaggttc aggctaaaga gggacagcag 6900 ccgcctcagc caccccctgt ggtttccttt gcctgtggat gggcggctaa aatgggccca 6960 ggaagagtca agaacaaggc cggctctcgg tgccacagct ctacccccaa aagcaggaag 7020 ggggctcggg ccatgcccat ctgtgagcta caccggtccg ggagcggcat caggcagggg 7080 agtcctggac ccccgcagtg ctggggtgtg tttgtccgcc ctccctcccg tgtgtctaga 7140 agcctccagc ctcggggaaa acaatgaaac tcaactgtga cttaaacaga ttcccaggcc 7200 cgcaggagct cccggaggct tgtggctgtg gcgagacctg gagggccatg cgggagggac 7260 agacgcaggt ttgcggaggc cgcctgccca ggaggggcgt caaaggaggg gacagatgtg 7320 ggtttaggga ggccacctgc ccgggaggag cctcgaagga aggcacaggc gtgggtttgg 7380 ggctgcctgc cctggagggg cctcaaggac cccaggtctg gtcttggtct cactcacctc 7440 ctggaccccc caaggcctgc agtttgcaat ctgtcgcctg ggacccccac tgtctgcctt 7500 tacgcagctc agccaccacg cggccctcgc tccctcattt acttgatttc tgtttatggt 7560 taaagtaccg tttaaaacga cacatcatta aagcaacatg aaagggagtt ttgaaaaggg 7620 aagccatcgt ccatcccact gcccctgcct cagcggggat ttacttttcc tttcctgtct 7680 gcgggccagt gacaatgagg accccgcaat gtgtctgcgg gccagtgaca atgaggaccc 7740 cgcaatgtgt ctgcgggcca gtgacagtga ggaccccgca atgtgtctgc gggccagtga 7800 cagtgaggac cctgcaatgt gtctgcgggc cagtgacaat gaggaccctg caatgggcgg 7860 cctgtaaggc tctgccctgg cctccgctgc cttcgctttc tccctccttg gtgggtgcac 7920 gcccttgtgc ttttcctaaa agagcaggtc ctccgggcat ggtggctcac gccggtaatc 7980 ccagcacttt gggaggccga gacgggtgga tcccaaggcc aggagttcaa gaccagcctg 8040 gccaacatgg caaaaccctg tccctactga aaatacaaaa attagctgggg tatggtgaca 8100 ggcatctgta atcctagcta ctcgggaggc tgaggcagga gaatcacttg aatccgggag 8160 acggagtttg cagtgagccg agatcacacc attgcactcc agcctgggca acaagagcga 8220 aactccatct caaaaaagaa gaagaaa atcctgacac ttcagcctgg tgcaggccct 8280 tccctcttc tagtccctgc aagaagtga gccgggccca gatctcctgc cgggcgggga 8340 8400 ggacgtacag gctcaggggc cgtgggtggc agagaggcta tcagcgccgg actggcccgc 8460 cccgagccag ggtccagccc cacagtcctg tccccaagcc ctggcccttc tgcggtcact 8520 cccgtctgct gaatccccta ctctgcccct ggtgtgtggc cccccagttc cctcctgtgt 8580 tcattccctg ctaacctccc gtggcttcgc ctccccagat gccctgagca cacagcctct 8640 ccccttcctc ccctcctaga tgtgcacata ggagccgcc aaaggctggg gcagctagtg 8700 gggcccctcc aagggaagct gggccccggg caatgccctg agccaccagg tcctggccct 8760 gcgtctcatc ccttcttttt tttttttgag acagagtctc gctctgtcgc cctggctgga 8820 gtgcagtggc ccagtctggg ctcactgcaa gctctgcctc ccaggttcac gccattctcc 8880. ttcctcagcc tcccgagtag ctgggactac aggcgcccac caccacactc cgctaatttt ttgtattttt acg gggtttcacc gtgttaacca ggatggtctc aatctcctga cctcatgatc cacccgcctc ggcctcccag aatgctggga tgacaggcgt gagccaccgc 9060. gcccggcctc atttggttcc ttctgtgcac cacagtgtgg gatctcgggt cctgggtggc 9120 acgtgtttaa cctgaaagga aactgcccct gcgctcccca gcttgtagcc cgtgggcctg gctctgaggc cccgactgcc ccacggcctg tgctgcgcat gctggagcca ggctccggct 9240 ctggttggcg cctcccgtgg ctttaatctg cagtgacctg ggtgcttaac gagggccttt 9300 gcctctgcgt gtgcactgcc ttcttctgag gagtctcctc aagtctccca gctgtttaaa 9360 caatggggtc ttttgtcttt tgacctgttg gtgccatcag ccagcctcct gcacatactc 9420 tccctaccct tggctgccac tcctgtcctg cccttcgcca tgtctttttt cttttctttt 9480 tttttttttt ttttgacagc gtcttattct gccacccagg ctgtagtgga gtggcacgat 9540 cttggctcac tgcaccctcc gcctccctgg ttcaaccgat tctcctgcct tagcctccca 9600 agtagctagg actacaggtg cccaccacca cacccggcta atttttgtat tgttagtgga 9660 gatggagttt caccattttg gccaggctgg tctcaaactc ctgaccttag gtgatccgcc 9720 tgccttggcc tcccaaagtg ctgggattac aggcgtgagc cactgcgccg ggcccccgcc 9780 atgtgttttt aagagcaggc atttcattca gatgaagccc ggacactctt gggggttctg 9840 ctcaggggct ctacctgcca ctgaacgtcc tctccctgtg tgggtggcca tggcattcag 9900 cctgtgttgg gccttgttct gcctggctac tggccgctag gtctgccccg gcatcctctg 9960 tgtcctgtgt gggcgcctct gcctcctgct ccagcctgtg ccaggcaatc ctgctcacct 10020 tccaggagcc aggcctctcc ccagggcctg cgtcctgtca gggtcaggga cggcccctct 10080 gccatgctcc ggagtccctg gtcccctcac tccgttacgt cctgggtgtc acgggtacgg 10140 ccgggaactc tgtcgtcttc actgtctggg ccggggcctc cctgggtgtc tgctggatgg 10200 agtgggtgcc tttgggtccc tgcaaagtga gcctgcctcc cagcaccgcc ctgtcgttac 10260 atagccacta tctttgcgcc tgtttttcct tcctttgact ggttcctctg gggttaattc 10320 ccaggcctgg tattaccatc tctgaatgcc tgggtggttt cagcgcccag gaggctgtgc 10380 tgaggtatct tagaccatgt gggcaccgtt cgctcctgcg cagccggctc cggggtgccc 10440 tgtttttgcg gaatcctgca gggaacccca tgtaccctaa gagtgctctc cccagccact 10500 gtggcataag acaagcggtc tctttgccct tgggccccat ccttgtctgg tcggcccttc 10560 ttcatgggtc cagcgcggga ttgccggctt ccttttcagg cttcctggga cccccactca 10620 gacctgcagc tgggccagcg atgcccaccc gtttctcctc cacgtggtat acagaggtgc 10680 ccaggctgct gctggggact ctggagccca ggagtgagtc tccttgaccc tgagctgtcc 10740 tggctatcac agctgggtcc tgttttcccc ttcagcaccc acgggtgtct ttctccagtt 10800 tatattgtta tttttattta tttacttatc gagacagagt ctcgctctgt cgcccaagct 10860 ggtgtgcagt ggcacaatct cgactcatcg caaactccgc ctcccgggtt caagcgattc 10920 tcatgcctca gcctcctgag tagctgggat tacaggcatg tatcaccagg ctggacctct 10980 agtttatatt attacagcct ggtcagggag tcactgggcg tcgccatcct gtgggtggga 11040 agggggccca ggcagaggcc aggaggaggt gacagtcatg cgcttagaag ctcaggccac 11100 gcccacccag ccctgcctgg tgaccggttc ctgctgtgtt gggagctgca tcccagacct 11160 tatcgccggg cacagaactt ccaagccagg ggagagggag gcctggaggg gccccaatct 11220 ctgaaggtca gcagtggcgg ggaggaggct tgcggtgctg aagggactcg ggggacctgc 11280 agggaggtgt tggtggtcag ggatggtggc acctgagggg acttttggca gggccagaag 11340 tgcccagagc aagctccggt ggggccctgc actggagggc tggggtcgag tgaccctctt 11400 cctaagacca cccaggagga cactgggtgc agggtggccg gagccctcac ccccagtagg 11460 cagctgctgt ccactccgcc gacctgcctg tcacccagta ggcagctgct gtccactccc 11520 ccgacctgcc tgtcacccag taggcagctg ctgtccactc ccccgacctg cctgtcaccc 11580 aataggcagc tgctgtccag tcccccgacc tgcctgtcag ccttttccct gtcaggccct 11640 gttcctagga gcctggagac ctcaggggtg gccttgagcc cccagggttt ttttgagggg 11700 aagcgccagc tgctgtcttc acccttcccc tagtgaggcc aggctgtgca gggccacgtg 11760 gaggcagtct gtgctgcgcc catcggtcgc ctggcttcct gccgaccctc ggcccccagc 11820 cacctctggt ctcgggcaag gcccctcccc ctgcccacct cctccctggc ccccacgcca 11880 ggtgggcaga cctcctctgc ggtttttatt cagggggtcc ctcgttggct gcccactctt 11940 ggagggctgt cctgactcag gcctcccctc tactcagatc ccctgagcga gggcctgggc 12000 gtgacgccgg gagttactgg ggggccagag ggggaggctc agcctgaatc aatgagaccc 12060 aggaggaagg aggcacgtgg acctgagggc tggctggagc cgctggtgac agatggagga 12120 gtaattgctg cttccagagc acagcgagct cgagctccct ggagtgccag aagcttctgg 12180 gtggacagac aggccgcctc acattccaga ggctgacaca gtcttccagg cacccctggg 12240 gccagctgga agccatgtgc ctccactcac gtgtcccgtg ggtgtttgga ggggaggctg 12300 gccctgccat ggcccacccc agcctgcggc ctcaggagct tgcacatttg caagggggtg 12360 acatgaagac tgagccaggg ctgcggcggg catccccctc agagaacagg gaggaggggg 12420 cacaggcctt tttttttttt tttttgacag agtctcactc tttggccagg ctggaatgca 12480 gtggcacgat ctcagctcac tgccaccttc gcctcccagg ttcaagcgat tctcctgcct 12540 cagcctcccc agtagctgag attacaggca cccaccatca cgcccggctc atttttgtat 12600 ttttagtaga gacggggttt taccatgttg tccaggatgg tcttgatctg ttgacctcgt 12660 gatctgcttg cctcggcctc ccaaagtgct gggattacag gcgtgagcca ctgtgccctg 12720 ccaggcacgg gcctttttat gagggaggaa gccgtgccct ctgccacctg cctgtgggct 12780 ggggcctgca ggctcccagg gccctcagct ccagcgtggc tgttggttat tggcacctgg 12840 gcctcatgtc cacatggaaa gtatattccg agctggtggc taagccacaa gagcctgctt 12900 acctgtccca acccccggct cacttgctgc catgatacgt ctgggattct gggccctcct 12960 tctgagcttg gacacagact tgggacttca gcgctggaca caggtgctgg atctggggtc 13020 tcaatggcac cagggatggt cattctgaca gccatgccca gagtctcagg agggcctggg 13080 gccgcaggtg gacaggcatc ctgaccagag tctcaggagg gcctggggcc gcgggtggac 13140 aggcatagag tctcaggtgg gcctggggcc gcgggtggac gggcatagag tctcaggagg 13200 gcctggggcc gcgggtggat gggcatagag tctcaggagg gcctggggcc gcgggtggac 13260 gggcatagag tctcaggtgg gcctggggcc gcgggtggac gggcatagag tctcaggagg 13320 gcctggggcc gcgggtggac gggcatagag tctcaggagg gcctggggcc gcgggtggac 13380 gggcatagag tctcaggagg gcctggggcc gcgggtggac gggcatagag tctcaggagg 13440 gcctggggcc gcgggtggac gggcatagag tctcaggagg gcctggggcc gcgggtggac 13500 gggcatctgg acgtcggctc cactagtgtc acctctgcaa ccttgtccca cccagcacct 13560 ccctatggcc ctgctctgac tgtgggctga gggggacccc catggctagc ttgggacctc 13620 cgctggacac tgtcataggg cacttgtcga gtggccctgc tgaggttcag gcttgggcgt 13680 gtgtggcccc tgagcccagc agctgcgtga gccgtgggga gccgagcccc cagtcaagaa 13740 agctgtgtgc tccttaccct gcctgctggg cttggggcct gcagggcaag ggaggcggtg 13800 ggatgggagc cccgcctggc ctggtttctt gttgtcgccg cactcctagc tgtatgtagt 13860 cagatgaggc tcccagtcct ggctctgctg ctgagcacca ttgggccggt ggttgggtga 13920 atctctcagc ctctctgagc ctccagcact tcgtccttaa gagtggaact ggtctggacc 13980 tcccaagggg gggtcactgg gtgcagagtt cctgcaaggc tgggcccggt gccagtagcg 14040 gtcgctctgt ggggcctttc gcacggttga tttttatctc cctgtgagat tagcgcctgc 14100 cctgctctgc ctcacggctc tggctctccg ggactgagac cacctgctaa gtgacagggg 14160 cggaaggacc ttgcaacaca gagcaagacg tgatagggtg gagctgctcc gcccaggccc 14220 cgcctgcaga tgagagggcc cagagcctct taatcccctg cgctcagggc atgtgacggc 14280 agggagtctc tccagtgccg cccaccggat tcctatagtt agagctcctc gcccaagccc 14340 ccagggcacc tcactggcct cccaggtgag gacatgaggc ccagcgactc tggtggtggc 14400 tggtgtcacc tatggcaggg ccgcgcgggg cccagtggag gacgcctggc ccggacctga 14460 ctgctccgtg ggaaccgtgg ggcgtgctca gggtgacttg gtgcggctgg gctgccagcc 14520 ggcctggctg agtcactggc agtgaaggca ccaggtggtg gacgtgctcg gcagctggcg 14580 ggagagtacc cccacccagg accgtgttca ggctgagggg gaagggagcg gcccaggcag 14640 gcaccgccca tggcggggcc cccgcactaa cggccacctc tcccgatgga ctgcagttct 14700 ggagccgccc ctgaggagaa cgccctgctc agtgtcctgg cagactctgg actcttcacc 14760 gtgccatcct cgctacagcc tggccaggca gccgctcccg tccccccacc ccacagacag 14820 gaactgcaga ctcctaaagg tcgagccagc tcccaagtcc atagccagaa agtgatgcag 14880 gcggggcccg gctggggggc aggtgtgacc gggtcatggg ggagcgggct gtgcacaggc 14940 atcaggctga gaggtctcgg ggatacgtcc tgggagccgc ctgccaggcg tcctgtgggg 15000 ctgcctgcag gcctcctctg gctttggcag gttctgtccc gacagaccga ggcttgggaa 15060 gaccggattg tgcactctct gcctggagag agggtgggga ggggctgggg cgtgtgggca 15120 gggagaacac cctgggaggc atcagtcctc ttccctgggg acgggccatg cgtcaacagt 15180 ccctcacttc ctctctcagg ggcagaggcg tgggggtccc gagcctccag gttggagtag 15240 gttaggaggg cctcagagcc ccctgctgcc ccgccatgtg gcattggaag gggtgtgacc 15300 ccctagggga tctgttaacc acactccgcc ccatgccggc ctctctgttc cctctaacct 15360 gcgcaggagc agggccagct ctgagctagc agaggtgaca tgaggctcgt cctggccccg 15420 cccaccctgg cccctccctg tttcccacca cgctccgccc accagagcta catgttgtgt 15480 ttgagcctca gccccagacc tggctcaggt cctcaggagg aagcagtgac agccaggccc 15540 ggggtcctct ccctgctctg ctccctgcgt gctgactgga ggctgcaggt ccccaggctt 15600 ggccctgacc ctcaggaatg gagccggccg atgtgaggtg ggggctccgg tcatgtgcag 15660 tggtaggaga ggaggcggga ccggtcccac agctccattg ctgccgaggc gttccgcagg 15720 tctgtcttca tattggtaag gaaaatgcga ggatggtgta gcctgggcat cccagtcccc 15780 aagtccgaaa tctaaatcca agcggaaaat tccaggcctg acttcatacg ccaggtcaca 15840 gtcagaagcc agaattattg aaagtatgct ctgggcgtgg tggcctcaca cctgtaatcc 15900 cagcactttg ggaggccatg gtgggaggat cacttgaggc caggagtttg agagcagcct 15960 gggtaacata gtgagacccc cgtcattatt tacacaaaat tatgtgactg cctttagggt 16020 atgtgtacga ggtatatatt gaaatgtgag tagaattttg tgttgagtcc catccccaag 16080 atacctcatt atgcgtgtgc aaatattcca aaatccagaa aatcctaaac tggaaatact 16140 tctggcccct ttggatgagg ggcccccact ctgcatagca agtgtgggcg cgggtgctgc 16200 tgtgtcctag cgtacttcag tgtgtggcct ttgcagtgaa tagggctggt gtcctcactg 16260 tacagatgag gaaactgagg cccagcttgc tttgccaagg tgcatccggc cccggccatg 16320 gctattctgg ctccagatcc catggtctgc agccacaata ctgctgtgcc ccggactggg 16380 ccctgcagct cgcggtgtcg ctggcctgcc cattgtgggc accgccccca ccccagactg 16440 gccgaggcct agaagggagc agggcctggc tgaggctgca ggggtgggga cggtcagcca 16500 gcccctcact gccaggaagg gcgcatccat cctggcctct ccccagggag aagggaggag 16560 cggctgagag ggaagcgctc ttgccctgtg gacgagctcc tgccccacgg actagggagc 16620 ccccgcccac aacctgcttg tcagggccac ccgggacccc cgggagttcg gctgctcgct 16680 ctgctgttag gaattggatt agttttccat aaaaacagga tgtggtgggt gagagggcag 16740 tgtgtccgtc tttctcactc ccctttttcc aggaactgag cacgcgcata ggttttagcc 16800 agggccgtcc agtcccctcc ccacccccca cagggaacaa tccactctct gctcttaagt 16860 ggccacttaa tcagcttctc ctcctggccc ggggagcttc ttggagccgg cctgccgtgg 16920 tgggaacagc tatggggaca ccctgccata aggtccagca gctaagctgg gatgtggggg 16980 gaggggctgc gaggcccagg cagtgtgcca ggccgcacaa gaggagccca gctcttgccc 17040 caccagctgg cagccctgga ccgaggttgg gcccgtgagg ttggctgggc cctgggccct 17100 gggcccccct ccccaggaca cgactgtggt ggcacatggc tttgggggct cgtgggtccc 17160 actttgcaga cctctgcttt aaggggtctg gtccacgggg tcccctctgg agggcctggg 17220 ggaaatctca gggacctggg gtctggaccc gggggaggga gcgggagaag catgtgcgtg 17280 agtctcgtgc tgtcagggag cccgggaagt ctgcgagggc ttgggggttg tgtcagggag 17340 tgtgggtttt gcccttcagt ggtggaagct ggcttgagtc ccctcgatcc ctcaaggctg 17400 tagtcctgac tcggggctgt agggcagggc agggtggggt caccctgagg cagaaggctc 17460 agcggagatg ttctggcttg gctctgcccc tccctggctg ggtggcctca ccctgtgacc 17520 tttggggacc ctggttcctc tgagccaggg gacagcagtg actccgcctt cctaggtggc 17580 tgaggatgac atgggctccc cctgcaaatg tgggttctgg ctcagtggcc aagtgtatga 17640 tggtatgtgg ctcttggggt cctgagagag atgggagagg agcaggggtt tgtagggaag 17700 ctggcggctt ccaccccagc cagtcacctg cagtggggga gttccaaagc tgactgaagc 17760 ttcgaccttg tggctggtcc ccttccttcc tgcctcagtc attctggtct ctgggggatc 17820 agggctgggg ggctctgggc tgtgggggcc tgtttttgtg acttaaagct ctcccagcac 17880 agccccctga cctccttcct catgggcagg acctggccca ggggtctcag cacagccaca 17940 ggccagggat gcccttgcag atggccctgg atggaattcc agaactcaga aatgtctcct 18000 tcccgtaagg atgtcccgag actcatgaga ccgtttcctt ctgggaaggg ggaggaatgg 18060 ggagatgagt gaagagcgca ctgcagctca atccgggaag aagctaatca atcaatcagg 18120 gaagccaatc gatcggagaa gctgatggga aagctgtgct tggtagaata ggctcgtgcg 18180 ggcagagcag tcgcggcact cacaggctga ctgtgaggac cttgggtgtt actttgtgct 18240 tctctgcatt ttaaaaactt ttgaaagtgg agaagaggaa agtgaatgtt ctctgagatt 18300 tcatggaaag gggaaactga ggcccactct agccagtttg ggcccagggt tccaacctgg 18360 ggtggccccg gccctcgtgg cctgaggtga tcgtccctgt ggctctgaga gcagctgggg 18420 ccgggtcccc gttctggggc tggtgatcct ggggaagagc caggcagtgc cctgcccacc 18480 tagtggttat gagcccagaa tgttgatttt tttcccttgg ttgcttcatg actttgttga 18540 atttccagag tatgtgtggg ggcccccggc gtcccactcg ccccagccct gtggcagcag 18600 agctggctgt caagctcagt cagctgggcc cagggccccg gaggtaggtg ggtgtgtgcc 18660 tgagcttccc ctttgggccc tgccaggtgc tgggagggac cacacaggcg gcaggaactc 18720 ggggtcccca ggcctcggcc acaccagcct ggtgcttgtt atatattgat atgtctctct 18780 acctgtgaaa tgggtattta ttttaaggag ctgacgcacg cgattgtggg gctgtcacgt 18840 ctgaagttcg cagggcgcgc tggctgtcaa gagccgctgt tgcagtctcg agtccaggct 18900 gtgcgctcag gccgggcttc tctgtggcgg tcctgggaat tcctcctccg ggcccttagg 18960 cgacactccc ctgccttcag ctgattggat gaggccctca tgccgtctgc tgatttaaat 19020 caatctcatt ttaaaaacac ctgcacacca aattgcgatt ggcgtttgac taaactgggc 19080 agcgtggccc agcagcgctg acacatgaat caaccctcac accccacccg caggcgtgac 19140 cacggcaccc acgctgccga ctgagaacac agccgtgcgc tgactcgcat gtgatgtctc 19200 tggtcgcacc tcctccatcc tcagcaccct gttcagaatg gagatagcgg ccgggtgcag 19260 tgctcacacc tgtatcccca gcaccttggg aggcctgggt gggcgggtca cctgaggtca 19320 ggagttcgag accagcctgg ccaacatggt gaaaccccat ctctactgaa aatagaaaaa 19380 ttagccgggt gtggtggcag gtgtctgtag tcccagctac tcgggaggct gaggcaggag 19440 aatcgcttga acccaggagg tagaggttgt agtgcgcgag attgtgccac tgcactccag 19500 cctgggcggg cgacagagtg agactctgtc tcaaaaaaaa aaaaaaaaaa aattagagat 19560 aacgacttgg gctctctaga gaccagggag ccaggctcca ggcgctgctg tccagcccct 19620 tgccagctat acgaggtgct cacctggcca cgtgtccggg cagtgcttct ggggtggcat 19680 ccactgggag ggcaagatgg ttctggtaca ggtgcccaat tctgtcccca tttcacttac 19740 tgggaactca aggcacagag aggggagggc tgagctagga ccagacccca gtctcctgca 19800 gaagtacaca gacatgatgt acgaccatcc tggacacctg ccctgagatt cccctccctc 19860 tcctgccctg tcccagtggc ctggggggaa ggggaagcaa ggttctgaag ggagtgtgac 19920 cagacacctg cccgtgacac cccctctcca ggctgcctcc gagtggctgg tgactcccct 19980 cctgcctgcg agggaggtgg ccaggttgca ttcctctctg agtgccgggg aagtccctag 20040 agagcaggcc agcctgtgac tgggccctgg ggcagtctag acaggccaga ctggacaggc 20100 cagggggctg ggtgccgctg ggtaaatcac agggtgaggg ctctgagtca gcaccccatc 20160 ttctgtcctg ggtccagcac cgctgaggac acagtgggca gccgggtctg ccagggccag 20220 gtagctgtgt tgagaaggca gtgctcctga gaggcggcta ccgggaggtt ttcaatggcc 20280 aggcttctta ggaagccctt gttgcctctc tggggtgagt tgctggggcc atggttggag 20340 tggtcgccag tgtctgcccc tggtgccgag ggcggagtcc tcgttttggg aggtcacggc 20400 atgatgctgg gagtcaaagg caggccgtgg caggggcatt ccttttttct ttcttttttt 20460 tttttttttg agacggagtt tcactcttgt cgcccaggct ggagtgcagt ggtgcgatcc 20520 cagctcactg ggatcccagg ctcccaggtt caagcgattc tcctgcctca gcctcccgag 20580 tagctgggat tacaggtacg tgccaccatg cccggctaat ttttgtattt ttagtagcga 20640 cggggtttcg tcatgttggc caggctggtc tcgaactcct gacctcatga tccacccgcc 20700 ttagcttccc aaagtgttgg cattacaggt gtgagccacc gcgcccggcc tggcagggga 20760 atgtcgacgc gtgatctctg cctggagagc acgttcatgt ttcccagagg acactttaga 20820 acatggcgcc tgggtttgga tgaacctcag cctaagaatc tacctgctca ggatccagcg 20880 acgctggtgg tgtggacttc agctctggag aatgggttat atggaacctg ggcgccggga 20940 gggcattgcc acgtgcttgc tgctggggct tcaagaggac cccatctcct gtggccgaga 21000 ccccgtgtct caaggcacat cccctttcgt accccgcccc accctccgca gcttcatgac 21060 ctctgggttt cccccaggac cttcgcatct gatgttccca gatccttcct gccactgggt 21120 cctgctctgg tgccccccgg ggaagccttc cctgaggacc cagctcagca tctggtgcta 21180 actgtggctc atcgtgcact ttggccccca ggaggctgtg ggctcctgaa acccctctga 21240 accacgggct tccaaccaca cctgaccccc tgcgtgccga ccctgtgtgc aggtgtacag 21300 gtgtgcctgg ggcaggatgg ctgtgacggc ctcacagagc ccggagagct gcctcctagc 21360 ttccaaagcc ttcatttcag gaatcttacc ctctcaatta gtctggaatg ctgggggcgg 21420 ggccagctcc aggtcacaga gcgaccttgt ttacccagac cttaacatcg gcccttccat 21480 gctaatcaat gtaatcattg tggtccatgc ggcctctgga atgtgctgtc cactccctgg 21540 gtcagccaga gagtgtcagg gagcacctac cggctgttac ccagtgctgc cctgacctgg 21600 ttcttcactc tgcacatttg tatcacgcca gaccctggct ggcagctcca ggtgacgagg 21660 catgtcagtg ccttcctgtt ctgttctgtt ttgttttgtt ttttaaatcg ggatgaggcc 21720 ttcctgtgtt gcccaggttg gtcttgaact ctcagggtca agcgaacctt ctgccttggc 21780 ctcataaact gctggattac aggcaggagt caccatacct ggcccactgc tactttctag 21840 atgaagagac agaatcccag agaagaagca ggggtttggc tgctggtctg gagccggttc 21900 tgctcacctc cagcttctgc cttgggccgc cctgttcaca caggagctgc tcacaggctg 21960 agacctcgag cagggccctc ctagaggaac tgggcccccg taagtgccct gagccgccag 22020 gagccggccc tgcgtctcat ccctatctcc ggaggacatt ggctgctagc tcaccagctg 22080 gcccctgggc aggcttgaat catgacctgg aacgccaggt gtctctggct ctacccctgg 22140 acctgcaccc tgtccaagtg ccccagggcc agactgtttt ggttgcaccc tctggacggg 22200 ctacccccat gatggctgct catggaaagc tgtggttctt agggagctgc caattcctag 22260 tcctgcagcc tggagctcct gggtataagg tgggggctcg ggcgtattgg ggactggggg 22320 tctcaggaaa gagcctgtgg gacctgtgaa ctcatagctg ctggccgagg gaccaccttg 22380 tggctgtcct tctcagctag gcctggtcag ggcttgtgtg cagggcggct gaagctgtgg 22440 gaggccacac tgtccacaca gtgccctgta agcccaccgt gcctcagttt ccccgtctga 22500 caagtggcac aacagaagcc acttcctggc acacagctca gggtcagggc cgacacagca 22560 cttgtgggct gccgggagac ccgagaggct gccccttcct tgccttggct gccacgggtg 22620 acctggcaaa cccctctggc cgtggcacca ctgggggtct acccttggca gtcagggttg 22680 gccgcttggc tgggagcccc ttctcctccc cagacacatc ctctccttgg ggctggaggg 22740 ggtcctgccg tccccgggat tgtcgagcag caggaatcca ggagggcagt gcctgcagct 22800 cagatggggg ccagtgggca gggccgatcc aagggtggca ggaaagtgcc catcactgac 22860 ctcaggtggg ggaggccatg gtgtgtgaag gaaggagagc tgagtgggag gtcttacttt 22920 gtcaccgccc ctctgagtgc ctgctgtgtg caaaggccag cgggggccct ttcttcagct 22980 gggctctgcc cagacccctg agctctgggt gggccgggag ggagaccttg ctgctcacag 23040 aggtggctct gcctgaccca gctcccttcc cagggcacag tgggggcata gggtcggctc 23100 cgtcagacat tccgggacct gcgtccttcc tggggccaca ccctctaccc actgtccccc 23160 acctatttac ctgtatctgc gccagagatg gctgcccaga taagccctgg tttcctccct 23220 ttctggagag gctgcggggg ctggcgagga acccacctgc gcagagagtc aggggattgc 23280 tctgtgtgga acgcaggcct cacccatgcc ctggaatctg tccccttctc tgttgctgag 23340 ggatgagtcc cagatccctg accctgcagg ggagcccagc acagaaaaac tctgaggcct 23400 ccacaccctg gcagcgctgc tggtcgtctg tggggaagga caggccctgg gagggagggg 23460 gaggtgcgga gggcagtggg gagggtcagg aggaagtggg ggaagggcca cccaggcccc 23520 gatgtggggg atgtctcacg cgtggggtgg ggcattctca tctctgcttg gtctcctgcc 23580 atgctggggg tcgttcactg cggaccccaa gtaccatgaa gatggggatg ggatgctgag 23640 cagcatcggg gagaacgcaa aggcacctcc cagctcaccc gcccccaccc cgcaagcaca 23700 accattgcca tggtgtgggg accggaaggc ggcggctttg ggaccagact gcttctgcct 23760 cgggccgtgc cgctgggcct ttggtcagca ccatcgtgcc ctgcaagtca ctcttagcct 23820 ggtgccctcc tgggcagggc agtgccacaa gagctcaggc ccagatgtgc aggtgccact 23880 gttccaccca ccagcaggtc acctggggaa ccctcccctc ctgcagcctc tgtgtgctca 23940 tctgtgaaat gggcgtggtg gagtcacccc tggcttgtgg gagaatccca ggggctgatg 24000 cctgcaggac cctgtgggct ttgccccgct cctgggcag agacagttcc cccagtccca 24060 cccacgtggc tgtgtgcagc aggtgcctgc tgaccctctg ttcctgcaca gtcaccttct 24120 tcacagacgg acccccaccc tgccctgcaa acccctccag gggctgcggg ctgagtgtgt 24180 ccgggagggt gtcctgactc tccacgccag caggtctgag agcagatggc tgtggcaggt 24240 gcggtgggtg cccagcccac agcagccacc aggcctgcag gaccctgcc cgtgtaggtc 24300 agatgagcca taaaactgag tttcctggac actgagctaa ttaaacctgg acaccgagct 24360 aattaaatgg tccagaagct cctcagtgcc ccaggctgct ggccgggctc caagtaggtg 24420 aggaacattc cgtttacctc ctgctgcgtc gaaggcgggt ggctcccctc gggcccctgc 24480 ctgtcccggg ccccctgggt gctgctgcgt caaaggcggg tgtctccgct tgggaccctg 24540 cctgtcctgg gcttcctggg tgctgctgcc tcgaagacgg gtggctcccc tcgggcccct 24600 gcctgtcctg ggctccctgg gtcctgctgc cttaaaggcg ggtcgttccc ctcggatccc 24660 tgcctgtctt gagctccctg ggtgctgcct gccctgtggc cacgtcccac tgttgccttt 24720 ggacaaagct ctcctagggc ctggggttct cccatgagat gacaggggtt ggttcaaagg 24780 tctctgaggt ttttagagct ttgtgaagtg ttctagaatc cattttctct ctcattgctg 24840 gacagagagt atagactggg ctttttcttg agtttctctc aactcttctt tgtccatgag 24900 gcaggaagag gggcttcctc agtcctcagc ttggggtgga ttgggatgga cagagaaccg 24960 tgcaggatcc cagcctgaga cggtccagcg ccccggggga gcggagcccg tggctcagcc 25020 gtctgtagcc acggccgggg tcactgtcac tgagggcacg gaggccccgc cggcgaggtc 25080 cgaggtgggc gtgtggggtg gtgggcgctg gaggcaggac ctctgcttgt ggagggtggg 25140 ccaggcgtgg accaggtgtc acgtccgctg gggcctttga ggggcaggtg gggattggcg 25200 cggagaagga gtgaagcagc tggggttttg ggagcctgtg cggagcaggc ccggatgcca 25260 gggtggctgg tgaaggtggg cctggccggg ctcggcctcc attgggggag ccccccaggt 25320 cccctcccca cacagcttcc ccctctgtct gctgtccaag gctcctgtgc tggcactctg 25380 gggtggtagg ccatgcagcc cgtgtgaacc tccatagacc ttgctgtgaa cgctgcacgg 25440 ggcgtctggg ggcgggctgg cttccccccg tcccctgggc caaccttgcc agccttcttc 25500 ttccataaaa gtgggatccg tctgagccct catggcccct ctgggtctgg gtttgctttt 25560 tgtgctctgc ctggggtcat ggcagggaag gcccagcagg ctcccttgca gagcagggca 25620 gagcagcggc cgccaggagc tgcctgtacc acgtctgctc ttcttcctct cttcctctgc 25680 ccggccccgc ccaccgtcga aagcactagc acgggagtgt ttatcggttt ctcttccaag 25740 ccaaataagg cagagagcgc tcttgcagga gttggagcag gccaggggga gggcagctgg 25800 ggcttggacc aggaccccgg ctccctgtag ctgccctggc ctggcagctg tcatccgcga 25860 actctgatcc tcgcctgccc tcacccggcc cctttcagag cttcccatgc tcagctggta 25920 gcaaaagaag tcaagaccta gcaaggtgcc tgccccggcc aggaggccca ggttctagcc 25980 ctgcagctgc cgctgtgagc tctggggact cagacgactg atgtccttcc cagcttctgt 26040 ttcctttgat gagaggaagg ttggaccaag cgacctgccc tgcttctgtc tgtttcctcc 26100 agtccttttc ctttttgacc agttcacttg ttgataactc cagcaacgca tgaaaacatt 26160 ccccttgtaa aaaagggaaa tgccgtgggt aggggggacc aaggacctct tctgcaggag 26220 cagacgcaac agcttggtgt ggatcttcct cagccttttt gtccatccta gacatgtgga 26280 atttcagttt tacacaaaag gaatggtagg cgtctgactc tctgtggctt gtatcacttg 26340 gcagtgccac agggattggt gcatgtttgc tctctcatgc tatgtgtcag gcactgttgt 26400 ctctgcgaca atcctgtgag gagggagtta tggttcttga tttatagacg aacaaactaa 26460 gacagaggag attgggctca tctgtggtca tctcagcagt tgtgagaggt cagggccaga 26520 gcccagtggt gtcattctgc agggcacata cagacacgac ccacagggtt ctgccgcacc 26580 ccctgccgct tggcctgctc tgcaggtggg tgccgaggct gttcctgcat ctgctgtgtc 26640 ttgtgagtcc gtgggtgcct gtggagtggg ggcagtgggt ttgcatttcc aagcactgcc 26700 tctgtcatag gctcagggag aagcagttac gcatgagctg ctatgagcca cgggtatgac 26760 tggcatcaca gcaggaggag ccctggggat ggctgagtga cgatgggtgg ggacagagct 26820 cacagaagcc agggttgccc ccaggccagg ccactgctgg ggccaaggct gggatgcagc 26880 acgatgttgg tctgtcaggg agtggcagga agcccacagg gtcagctgac acttaaccca 26940 gagtggtggg agccttgaat gccagactga gggccacaca gttggtctcg gtgcacaagg 27000 acttgggctg agagggctgt tgctcctcaa tggccatctc cttgtgggag gagagtccgg 27060 gcagccgctt ccaggaaccc acccatgctt gaaatagtgg gaccacaggg ctgaggaggc 27120 tcctggggat tcctcccagg attcagctgc tctgtcccca accccggagg gcctattcgt 27180 ctagccctat atcctctctg ccagttaggt cagggcagag aagtagaggc acagagggtg 27240 gcggaggggt gtccagccag gcctttctgt ggcaccttct gaggctggct ggagtctgtg 27300 aaatctgtga taactggccc agacccttcc tgcctcctgg ggcatctgct ggagctgagg 27360 ctgctggggg agtctgcctg tgacttgagt ctcttgctgg gccatgctgg gctcagttcg 27420 cctgtctgtg gggtgggatg gtgccaccct taggttgttg ggaggaccca aggagagtga 27480 tgcctccagc catggcagct ggcccagctc cggcccgcag cctggctcct tcagggccag 27540 gaacccccag gtcatggact ccaaccctcg ggctctcctg cttcccaggt gacgaagctt 27600 caacccatgg gctctccttc ctgggtgtgg aagccctgga gaaggtggag atgggatctc 27660 agtccagccc attggctcca cttcctttga gaagctcttt cccccgtctt ggcccccagc 27720 ccttgttcaa gcacctgtcc ctctcctgtc tcctaggccc cgtcagtcct tttggagacc 27780 tgttcccacc cgcccctgct ctcacaggct gccagtgtcc acacactctc aggcactgtc 27840 attggagcct tttaaccaca cgggaaagca ggcaggttag gtaactaccc cccacccccc 27900 cgccagcaac gccccctgca tacccagggc ctgcaccagg tgctgctgcg ctgccagctc 27960 tctcgaggtc cctcctccta acctcaatgc atcgcgtctt ccagccccg gctccgaggg 28020 ctcagcctcc aggtggtcta acctggagtc taaacctgga ttgccatctg cctgcctcag 28080 cccttccctt ccccagggac cgagggccag tgcggctccc cagccaccca tttttgttt 28140 gttttgagac ggagtctcgc tctgtcgccc aggctggagt gcagtgtcct gatctcggct 28200 cactgcaagc tccatctcct gggttcacgc cattctcctg cctcagcccc ccaagtagct 28260 gggactacag gtgcccgcca ccacacccgg ctactttttt gtatttttag tagagacagg 28320 gtttcaccat gttggccagg atggtctcga tctcctgacc ttctgatctg cccgctcgg 28380 cctcccaaag tgctgggatt acaggcatga gccactgcac ctggcttgtt tgtttttatt 28440 tttattgttt tttgagacgg aatcttgctc tattgcccag gctggagtgc agtgacataa 28500 tctcagctca ctgcaacctc cacctcccag gttcaagcga ttctcttgcc tcagcctccc 28560 aagtagctgg gactacaggc acccaccacc acacccagat acattttgta tttttagtag 28620 agatggggtt tcaccatgtt ggccaggctg gtttcgaact cctgaccatg agatggcggg 28680 cttgagccat cttcccgcct cggcctccca aagtgctggg attacaggcg tgagccacgg 28740 tgcccggccc cagccaccag ttttgtacag cactcaggtg ggtgtggttc ctgaggacat 28800 gtcacacgag atccttccgg tccattagcc caccccgccc catcagtggc cagtgcggcc 28860 attgtggggt ccagagtccc gaggcagaac gactgactcc aggttgtgga ggggcctctt 28920 aggatccttc taggcaccca ggttgggcag gtgcctccat gcctaagact gagctcctgg 28980 tggacatcgg gctgggtgtg ggcgtcctgc acaccctccc tgtggcctgg tgcccgtgag 29040 gagtggacag tcaccccgag agcatagacc accctcgagg ctcccagatg gcggccgcag 29100 agccccactt ccctgcctgg tgtgtgatac cttccatggc tgaggacacc tctgactgct 29160 cactcctgtg accctggacc caagtgctgg gccacacctg gactccaggc cccagtcctt 29220 ccttcttctg ctgtcctggc tcctgggtgg cccccgccct ccccgctccg aggctctgcc 29280 ctgtgctgggg aacccaggaa ttgccacccc ctggccccgg tttctcatgt caggatctgc 29340 tggccaggtt gactcctgag cgccactgtc tcctcagact tgctggtccc acccggtccc 29400 cacctgtcca ccgcccgagg ctctcctgtg aggccgctct gcagctcttg gcctggggc 29460 ccccggcctc tcatggtgcc ttctgcactg tcccctttgg agatgggagc tttgagggta 29520 ggcgccaccacg ttcggtgaaa aggcggtata agcaaagcaa cccccagata ctgaaggagc 29580 cgggaataa aaagggcag acagagcaag tgtgttggta cccgcctatt tactggcagg 29640 aacttacaga cggaaacatg gcctcaggca gcctccaggc aggtagagct ctgaacccaa 29700 acgccccgatc tggggctcgt atcgtgccct gggaggaatg tgcaggtggc tgggaacgtg 29760 gcgggtgggg tggccagatt cctgctgcag caccgggttt gtttaggaag aaacgtacga 29820 ggactagaca ctcttttttt tctttcctgc acgtgaatgc cagggagtag gtgctcctgc 29880 aggaagatga tgcatcaacc agccagtctg gaggcattcc gagacccggg gttaatcagc 29940 agattagcat ttaaataaag ttactctgtc cccacagcca gctaggcccg agccacatgg 30000 cgggggcggc gggggcggct gttcgccgca tcccaggctt gctccatctc ctcgtctgca 30060 atgtggaggt gacagtcgtg gtcctcggct agtgccacag gactgtacgg gacagatcac 30120 ccagccccgt gcctgcactg ggggcggcgg tgacgtgatg gccgcggcgg tgacctgatg 30180 gccgcaggtg tgtccaccct ctggttaggc ctctcctctc cctggggcgc aacaggtgtc 30240 ctcgggtggg aggttcagct gctgggcctg gggccccagc tttcagctcc tgtgcaggcg 30300 tggagcggga cggcacgccg gggcgttcgt tcacacgatg cccccagctc gtctctgcct 30360 gctctgtgat gcagcctcag ctcctgtggg tcccgggtca cccacccgtg tgggccgcag 30420 tctttctgca ccacgtcctg ctctcggcca gcgctggcct gcgggccccg gcagtgtggg 30480 gtcacacaca ggccgcaccg gctgtttcac ggagcccccc tgctcaaaat cctggccttg 30540 ggtctctgtg tggctcctgg agggagcatg tatgaggtgt ccaggttgtg aaagccccat 30600 ccggcagccc tccctgtgac agtccttcag gcctcgtcag gcttggttca gcccgtgtta 30660. gaggaggcca gacagctcct cctcgatcac cacagggac gaggggttggg cagtttctcc tctcccaccc gggcgtgggt cagctgcag ggtctcatcc caggaactgg atcctggcag 30780 30840. gggggctcag gcatctgaca tcccctggca ggtcttccta tgtgccagtg ggagtgggtg gtgtccagcc ctctttgtgg aatgttccgg ttgagtggtg tgtggtcctg gttaccacca 30900 tgtggcattt gtggctttcc tggccagcac ccactgaaag cagaggccag cccagccctt 31020. ggacacccag gggcccagca gctaccgtgg gggcagcctc tcccctcctg actgtgctcc tctcagctgt aggatgaagg tctgtggatg ggtgatggct gggctctctg cttcccaagg 31080 agctggatgt gggccatagt caggcgtgtg ctggtgaggc gaggggttg ggggttggga 31140 accacagctc tctctgaccc catcccattg cagctgactc aggaaaaggc attackccccca cggggtcaca ggcccagcct gggcagctcc tgatgggac atgggagcca cagggcaggg 31260. caggctcctg tccccgctcc ttggattgtc tcaggccccg cctccccttt ggctgaattt 31320 cttccttctc tgcctgagcc tgatctgcga agcttccgac ctgccctgag ttggagccag 31380 tcttcagggc caaaagcaat gccgtctcct ccaggcagcc tgctctgaag tctccccctc 31440 tggctttagc ccacaaagtc acagctgagg ccacatcaac acacttagac caagagcact 31500 gccggctgca tagacagcac aggtagatcc aaggagtgtt tcgaaaagcc gtggccctgc 31560 ggccgggatg cccggacctg ggtcctgacc actcttggga gctcctcccc tctcagagcc 31620 catcccatct gaaaatgggg gcattggagc attggagag tgctcctcac tcctgccctg 31680 ctagccggct actccgagga ccccaagacc agatgatggg agaagtggtg ggctggggctg 31740 tgaccctgag cccccttctc atcccacccg tgaggctgcc cctgcttggt gtctgtggca 31800 aggctgacag aagatggcgc cagggaccag ccagggccct cctgtctcag cccgtccttg 31860 ggtgagtggg tgtgctctgg gggtggcagg tgtgctccgg gggcgggagg tgtgctgcgg 31920 ggggccgggt acgctctggg ggggcgggtg agctctgggg ggctgggtgc gctctgggg 31980 ggctggtgtg ctctggggat ggacaggtgt gctctggggg gcggggtgtg ctctggggat 32040 ggataggtgt gctctggggg ggcggttgtg ctctgggggg gcaggggtgc tccgggggtg 32100 agtgtgctct ggggggcggt gggtgtgctc tgggggcggt gggtgggctt tgggctaagt 32160 agccctcttc ctctctgagc cttttccttg tctgtgttgt tggtgagtgg ccgccacctg 32220 ggacaagcct tagctgtctg tccaactgtc caccatgtcc ccggacccta gaccaagccc 32280 gctgcctggt ggggcctgct tggtattgaa tgaatgaatg aatggatgaa tggatgaatg 32340 gatgaatgaa tgagtgaaca aactctgggg ggcgtgttgc tgatcacccc agggcaggcc 32400 cgtggtgggt catggtctca ggcaggggac ttgccctgcc gggtgacgtt ggcctgccgg 32460 ggaacccctg ccaaggtgcc tcccatgccc ccatcgtcct ctgcctttga gtccactctg 32520 cccctcaagg ttcccctctg gctcggccag gggcagggcg gtcagggagg cctccagctc 32580 cagcccctcc ctcatctcca tcctcagccc ctgcacccgc ctcatctcca gccccagccc 32640 cttgcagcct caggctcggg cttctgagaa tcctttgcag atttcagggc tggttatttt 32700 tttattctgg caaaatatac atccataaag ctttgtttcc tgagatgatc tcaccctgtc 32760 gcccaggctg gagtgcggca gcgtgatcat ggctcactgc ggcctcagcc tcctgggctc 32820 aagtgatcct cctgctccag cctcctgagt agctgggacc acaggcatgc accatcatcc 32880 ccggctaatt tttgaatttt tttgtagaga tggggtctct gtgttgcctg gtctggtctc 32940 gaactcctgg gctcaagtga tccgcctgcc ttggcctccc aaagtgctgg gattacaggc 33000 ctcagccacc acacccggcc taaagtttgt cacgttagcc gcattgaagg gcacaggtca 33060 gcagcggtaa gcatgttcgc attgttgtgc agccatcact gacacccatc tctagaactt 33120 cttcgttttc ccaaaggaaa ctgcacgcac ggcaccacct ccctggcctc cccacagcct 33180 cagggtctgg tgaccttcat ccccaggacc tacgcttctg actccaggag ccctgcctgg 33240 tgtccacacc acactaaggt gccctgccac gggcttcgaa cccaaccctg gtgcacttgt 33300 tgagcattgg acgttcctgg caggtcagac cgtgagctcc tctgctctga cggatgcatg 33360 gacggctgcc acctggctgg acaggtgcag aggggaggtg ccaccttctc tggggacagg 33420 actggggcag aactcacccg gctgaaaagt gcagctgtgg agaacttcag aacttcagaa 33480 gcctttgctg tgaatgcatt ttcctcctgc ctcttgggcc agatcttagg gatttttttt 33540 ttttttaatg aaaatgtata atcgtcaaaa aactttagat ttaaaagcat ggagccgatg 33600 gctatgcttg gtgcgttaat gggaggaaga ggggatctta acttcaggga gggctcctgg 33660 gggagctggg ggaggctgct gtgtccagca gggcgggcgg tgccccccgg agcccggcac 33720 tccgcgatgt gtgcgctaag ccgaggccgc cttgagcccg tagcagcgcc gagcgcgatt 33780 ctttcgtgtc tgcttccggg agggtggaag ggtgaagctg ttgagagtgg gaagggaggg 33840 gctgtgcttc ttgagtttgc cgtgtgcccc tgaccctttc aggtgcaagc gtcagctcca 33900 cggtgccaat ggggagatgg gtctggaggc cccaccaggc tgtgaaaggc ccggcctcct 33960 gtgctgtccg tggaggtcgg ggtctccctc tgccctgcgc ctcctctaac ttgccctgcg 34020 ttttccgtgc ccctggtcta gggtgaggct cgggtcttgg ggactgaggg gcaggtgggt 34080 gtcaaggtga gagctgggct gggctgggcc tccctccaga agacttcccg gggctccttt 34140 ggcaccaagg atggggcaga tggtggcacg catggcctcc cctgggcaca gagccagact 34200 atggagggat ctcgttggct acatcctggc tggggctttg gggtcatggt gggagcagca 34260 gccaggggtc tcgcttggac acgggtctga gctcactgtg gcctccttcc tcagcctcct 34320 gaccggggtc ctgaggctgg agggtgggtg gagggtggag ggtgggtgga tagtgggctg 34380 gaggatgggc tgctggcgcc ccttaggcag catgaggcgc ctttccagtc ccagtgtcct 34440 ccctggtagc cccggaaagg ctgagtggtc cctcaggtcc cctgcgagga gacgacatca 34500 ggccccaccc ctcatctcct gacaaacctc acattcctag gaggaagcag gcctggctag 34560 cacgtggcag gcacagcctc cctgactcca gaccttatca gatcagtgca agtgccaggg 34620 agaccccggg tgggagctgt gtggttggga ctgatgcagg cactggccgg actctcagga 34680 tgagggtggg aggccaggcc ccgggacctt ctgggacagg caggagggca gacacgggac 34740 atggtgtggg ggctgtggcg agtgcggcca cgcagagcat ttgggctgaa ggcgcgctgt 34800 gtcccactgc cccatacgtg tgcgggggct cccagacacg tgctgggggc aggaatcata 34860 gcattttttt gtttgattgt tttttgagat ggagtctcgc tctgtcgccc aggctggagt 34920 gcagtggctc aatctcggct cactgcaacc tccacctccc gggttcaagc gattctcctg 34980 cctcagcctc ctgagtagct ggaattacag gtgcccgcca ccacacccag ctaatttttg 35040 tatttttttg tcaagacagg gattcaccat attggccagg ctggtcttga actcctgacc 35100 tcaggtgatc ctcctgctgc agcctcccaa actgctggga ttacaggcgt gagccaccac 35160 acccagccaa gaatagcatt ttctttcttt ctctttttga ggcggagtct tgctctgttg 35220 cccaggctgg agtgcagtgg tgccatctca gtgtaacctc cgcctcccgg tttcaagcaa 35280 ttcttctgcc tcagcctccc gagtagctgg gattacaggt gcccgccacc atgcccggct 35340 aatttttgta ttttcggtgg agacagggtt tcaccacgtt ggccaggctg gtcttgaact 35400 cctgacctca agtgatccgc ccgccttggc ctcccaaagt gctgggatga caggtgtgaa 35460 ccactgcacc cagcccaaga atagcatttt taaaggaaaa aaagcaaggt agaaagatgg 35520 gtcaaaaccc cagcgccccg ctgggaggct ggtgcccgtg tcgtggcgtg tgtgagatat 35580 gtacggaagc gcgaggcggc ttccacggct ctggctcagg gcagcactca cagggcacac 35640 ggcctcggcg gtgtgacgag ctgggccgtg ggtgcgctgt cctctggggt ggggctggcc 35700 atcctctgtt ggcgacctag ttaacgccca tctccccgca gcctgcctgc tccgcttcag 35760 cggactctcg ctggtctacc tgctcttcct gctgctgctg ccctggttcc ccggccccac 35820 ccgatgcggc ctccaaggta aggccagggg accctgccca cgctctcagg gtgggagggg 35880 gtgggcattc gtttggggcc aaaattcgta cagctttcct cacctttaac agcccggggt 35940 ggaagatgag gggtcccgaa ataaaatttt tttttttttt tgagacggag tctcgctcag 36000 tcgcccaggc tggagtgcag tggcgcgatc tctgctcact gcaggcttcg cctcccaggt 36060 tcacgccatt ctcctgcctc agcctcccgg gtagctggga ctacaggcgc ccgccaccac 36120 gcctggctta tttttttttg tatttttagt agagacgggg tttcaccgtg atagccagga 36180 tggtctcgat ctcctgacct tgtgatccgc ccgcctcggc ctcccaaagt gctgggatta 36240 cagacgtgag ccaccgcgcc cggctttttt tttttttttt ttttttttga gaccgagtct 36300 cgctctatct cccaggctgg agtgcagtgg cacaatcttg gctcactgca acctccgcat 36360 cccaggttca agtaatcctc gcatcccagc ctcccaagta gctgcaatta caggtgtgag 36420 ccaccacgcc cagccatcaa agtttcttat tttcattgta gaccccaagc taagctcaga 36480 atcttttctg atccgtgttg caggggctgt gaggggcgag gagggtcatg ggggtggagt 36540 ctctgtgtcc cttgggaggc cacagggagt ggatgggccc caactgccca ctctctgcag 36600 gctccaggca cgtttcctgc ctgctgaggc tctggcagcc tgggagttcc agccgcctgg 36660 accgccctcc cgctgcccgc tgtggggttg gacctcagtt gactttgatc cacttgtgcg 36720 gggggggggg ggagctgaga ccccccttgg gaaccacctt ggggtctgag ctgggccagg 36780 actgtgttgc tctcccgtat cctccaaaca gagggcggtg aggccacgtg ccaagcctgc 36840 agcctgggtg acgagggtag cagcaaaccc tccatcctgt gacatgtcac ctacctgccc 36900 tgtggggcc atgtccattt tgtgcccacg ttttaggaga gcgcgttttg gagcaaacac 36960 tcagagacct gtccccaaag gccatgggcc aaacgtgggt ggggccgggc cctccgctgg 37020 tctttgggtt tggagggtga agagccactg aaaggaag aggtccccag ggagggcaag 37080 tgtaggcagc aagagacctt cccagcaga gggcagagag gaggccaagc aggaccaggg 37140 ggcgtgactg tggagacagg ctcttgggtg ccgcccttct gggaggtgct ggacgcatca 37200 ggctggcagc gtcggggcct gtccatggct acgaggtgtc caccgggctg cgtttttcca 37260 ggggactctg gggaggatct gcttctctctt ccagctgctc caggtgccta ccttcctggg 37320 cttggggccc ctccctccat ctccagagcc ggtggggtgg ggtcaggatc acaccctgtc 37380 tctcctgctc cctctcccct gcggtaaccc tgtggcttcg tcccatctgc ctccccggct 37440 aatccaggcc gacttccctg gtttaaggtc agctgttggg cagcccccat ttcctctgct 37500 gcctggactc gcctctgcat gtgagcttct gcgttcacag attctgggga ctggggtgca 37560 ggcatcattg gggtccatcg ttctgccagc tgcaggtgga aggtcgctct ccggcctcgc 37620 gacatctggc tggaagcatc ccagaggctc ctcctagctc gtggtgctgt ggggtgggca 37680 atggtctttt ttattattt tttttgagat ggagtctcac tctgtcaccc aggctggagt 37740 gcaatggcac gatctgggct cactgcaacc tccgcctcgc gggttcaagc gattctcccg 37800 cctcggcctc ccgagtagct gggattacag gcgcccacca tcatgcctgg ctaatttttg 37860 tgtttttgta gagacagggg ttttaccatg ttggccaggc tggtcttgaa ctcctgacct 37920 caggtgatct gcccacctcg gccgagacta caggcatgag ctaccgtgct tggctttttt 37980 tttttttttc gagtggggt cttgcttggt tgcccaggct ggagagcagt ggcatgatca 38040 tggctcactg cagcctccac ctcctgggct caagcgatcc tcctgcctta gcctaccaaa 38100 gtgctgagat tacaggtgtg agtcactgtg cccagcgcag gcactgatct tggatcatgg 38160 gtagaattca ggggtgcaga gaggcatttt gggaggggcc cagtgcggcc gagtgctggg 38220 agtcgctggg gcccgaggcc caaacccctc caggatgcat ggctggggtg ggtgggcacc 38280 cccactcccg gtccctatcc tgggcctccc ctttgctctg tggagccggt tctgtctgtt 38340 cccaggcccc tggcgtttcg ccgtttgttc cgtaaatatt tcatgcccag ggcgcaattg 38400 gaaacacttt cctttaatca gcagtggggg aaggcaggcg cccagccagg ccaggggagg 38460 agctggggtg ggaagatttg gagaggaccc gggaggactt ccctgcctga gcctcgtcag 38520 aggcccttca gagacggaga tgctgcccag ttttccggga gggagagagg aaagtgtgag 38580 gcttgcccga gccgagtgcc cggggcttta tgatttgtgc agctgctggg cttggcgtgg 38640 ccctggtgga agctctgacg ccatctgagc cttggtcccc ttgtaggcgt cagcctgatc 38700 cttggggggc tggaggttga gctcagactg aacagggagg agaggggtag gggctggggc 38760 tggggcagag ggaacagacc ctggtgctcc aggcagggct gcaggcagag ccacaggggg 38820 tggctcccag agacctgctt gtcatttagg gactcagaag ccccatcctg ccttaggata 38880 aaacccacct ccaagactga ccctgctgtc tggagagaga tcacccccca cctcttacca 38940 tgccccgtgc agggaggccc tgccaccctc tccagcttca ctggtccccc ggctctgcca 39000 gccttgtgta gtctccatcc tctgtctcaa aaggcccctc tcgtggccct accccgcagc 39060 tcagccctag ctgtcgctgg gtgggcctca tggctcagcc tcaaggcacc ctgggtccct 39120 tggctggtcc catgcacacc ccatggatca ggttccaggg ggctcccatg cacaccccac 39180 ggatcagact cagggcaact ccacactccc cacagaacaa tcaggcccag cccttgtccg 39240 gcaggtgtgg ggatggtggc tatggaccac gcatcaggat gctgccgtgg tggggcccct 39300 cagcctttgc aggccgtgcc ggggtccaag ggctccggct ctgggggagg tccatgctct 39360 gtcaaccatc cggtctgcaa taccctgggg cctccccgcc ccttcctgtt tcctcctgag 39420 agccctgcaa gctggagggg gagccccggc cccttttttt tttttttttt ttgagacgga 39480 gtcttgttct gttgcccagg ctggagtgca gtggcgcgat ctcaactcac tgcaacctcc 39540 gcctcctggg tttaagtgaa agtgattctc ctgcctcagc ctcctgagta gctgagatta 39600 caggtgccca tcaccacgcc tggctaattt ttgtattttc agtagaaaca gggtttcacc 39720. atgttggcca ggctggtctc aaactcctga cctgaggtga tccacccacc tcggcctccc 39780. aaaggtgtga accaccacgc caggccccct gcaccttctt ctggggtgggc aagagccatg ctgagcccct cccctgccat ggataggggt ggcaagaata gtccattccc catgggctcc 39840. ctccggcagg cagcggatgg gttggggccc acacagtggt tcaggaaagg ctgaaggggcg 39900 gttgccggcc agagctgggc gggtccctgg cctgagcctc ctctcactcc ctttcccagg 39960 atggaaaggc cactgtgagc tgggtgtgtc ctgccaggag ggagttggag tcgggggcag 40020. gagacccact gggtctcctg ggctcctgga agaggcagca gtgggtccct gcggaggtgg 40080 cctgatcccc ggctcagcct ggttttccca gctttgcatt tgggggtggg acctgcaggg 40140 gggagtgac ggggtggggcg gggacagggg ctgcaggtgg aggcacgaga aagccacccg 40200 accccttgag gtcatgctgt ttgcgtcgga gaagagggcc ggtgtcaggg tgccccctcg 40260 gcctctgtgt gcacctcccc gctccccgcc ccgtgcaggt cagcagagcc tcgatccctc 40320 tgcacgttcc ggccccctcc catctccagc agcgtctctt cccggggtgc ccctcggact 40380 ctcagctccc tagtctgatc cagcccaggc agggagcggg cgtgccacgt ggccaggagt 40440 ggcctccagg cccagctgcg gccccttcga ccctgcggag ggatcatggc ccagctgttc 40500 taacatggcc agaccaggac actctggccg gcccgagaac tgaaccggga aggaggcagg 40560 gaaaggggag ggaggaagac aatggggaag cagtgacatc cgagatgtag ccagagacgg 40620 acatcctgga ctgtcgtgca gggcaaggcg ggtgggcggg agccaggtgg cctgagagcc 40680 cctccctgcc caggggtctc tggtcaggca gttccttccc gggctgctgg atcgtgtgtg 40740 cagggaaccc gtcagcctgg ctgcccaagg gcccagatgt cctctcagtg cccgggggtc 40800 cttgacagcc ccagcaggag ccccacgtgc ctgtggggca ggccccgcag gtccctctct 40860 gtaggactca gaataccttc tccaatgcca cgtgctctcc ctgagtgccc agtgccacag 40920 agggccccgc tggcgaggtt acttcaacgt caggctagag ggtgcagcga gcaggactca 40980 cagcccaggt tcccaggcag gtgtggcagg agcccattcc ctgggtcggt gttctcatgt 41040 caccccacgg tgaccctgct gtacagaggc gggggcgcgg ggcccctggc tggtgccctc 41100 ctgctgccag acttgtgctc tgctgaagag gggctggcgc ggcaggtatg agcccggcac 41160 ggaggtcgtg agcagtgaga agcctggcac ctatgggttc ggggcaggga ggccctggaa 41220 ggtcctgtcc tccgggagcc ctgcacagca ccccctggtg gtggctcctg ttcgtgtcgg 41280 ggctgcagcc tcccttcctg gacggttttc cattcctcct tcccactctc cctgctgctc 41340 acccctccca tcctccacag gtcagaggtc atagctgcag ggtcagcagg tcagggcctg 41400 gggcaaccct aggaaggtgt gagtgtgaga agctggtcca aacctgcctc ggctgcctgg 41460 tggtgcggac agggagtcct gggcatccgt gagggctgct tcacggtcag gcttagggat 41520 gtgcagggtg acttggacgt gggtcatgag tctttgctcc agaaagaggg ggctgagtgt 41580 ccaaggccaa tcccgagtct gtcacctaac accatttgtg ctcaaaaaac tgaacagagt 41640 ggacacaggc cctgagtttg cgccccgagg ccgagagagg gcagatgtgg tggtgacatt 41700 caccaccttg gacccagacc cagatgctac ctgtccctga gctccaggaa gttgtgagaa 41760 gggcctgagc tgttctgcac tttctcgtgg ccgggcgtgg ctggtggtgc aggagttgct 41820 gccccagggt gagggcccgg aagctcctgc cagcacgtgc cggggtggaa agggaagctg 41880 tccacagccc tgtcaggact cagaacccgg tgggtcaagg actttggtcc ggacccctgc 41940 tgaagggtga gctgtccaca tgtgcgccga gagcagaggt gaagccaggg ctcctgagtg 42000 cccccagcca cagggtgcgc ccgcccagcc cctgccctgc agccgaaggc ctccctgctg 42060 gggggctgag tccagtgggg ccacaggcag ctgggagcag gacaaggctg ccaggcaacc 42120 agatggtgct gccgcttcct gccaggtgtg ggtgcacaga gagagagagg atgccggtct 42180 ggggcctgac ctggtgcaca gcaggtgcct gaaatgccag ggtggccatg gggactgggt 42240 accatgcata ggccatgcat cgggatgcaa cttctccttg cagcccctca gccccaggga 42300 ggcagctgcc tgccccactt ttctccagag ccatcatggc cctgctccca ccccagccac 42360 ggctgctcag gggcgctccg catgctctgg tctccatcca cctgcagccc ccactggggt 42420 gaggtggagc ttcttgcctc tccttgtgtc tatttcctct gcttccacaa ctgaacggtg 42480 acaggtattt gctggatgag ggagcacacc ccaggtggtt tcctctgagc ctgggaggcc 42540 ttttcctgcc tgtgggcccc aggcccatcc tgctgccacc cccaggagga tgcccggctc 42600 cttgtgacaa gagtgaccct cgggaggcgt ggggagtggg gctggccggc ctgcctgatg 42660 gggtcctgag tccatggcgg gtttgcatct caggcctctg ggctctggcc gggctggggg 42720 ctattgtccg gctgagcggc ctgggctgcg gcccctcccc gtccccggga ccagcctcac 42780 ccactcgctc tgccgcaggt cacacaggcc gcctcctgcg ggcattgctg ggcctcagcc 42840 tgctcttcct ggtggcccat ctcgccctcc agatctgcct gcatattgtg ccccgcctgg 42900 accagctcct gggacccagc tgtgagtcgc tgagggggcg gggtagggat agccatcctg 42960 ggggtcaggg agagggccct gcagtgaccc cgagtctcct gggggggttg actcagcctg 43020 atttatgtct ggcctggatg gtccaggtga aacgctccag ggatgaccag gccacggtgc 43080 tggctgggca gagcctgacc tgggttcccc cgtctttctc tgcaggcagc cgctgggaga 43140 ccctctcgcg acacataggg gtcacaaggt aagaccattc ctcccacccc caaccagcaa 43200 gcctcccttg gggatttcag gccccaggaa gtggggggac ccaggaggga cagaggggga 43260 cctggagact catccacact cccacccaca cctggagacc catccacgct cccacccaca 43320 cctggagacc catccacact ctcacccgca cctggagacc catccacact cccacccgca 43380 cctggagacc catccacact cccacccgca cctgcagacc catccacact ctcacccgca 43440 cctggagacc catccacact cccacccgca cctggagacc catccacact cccacccgca 43500 cctggagacc catccacact cccacccgca cctggagacc catccacact cacacccaca 43560 cctgtcctct tggtctgacc gcggctgctc cctgctctcc gcaggctgga cctgaaggac 43620 atccccaacg ccatccggct ggtggcccct gacctgggca tcttggtggt ctcctctgtc 43680 tgcctcggca tctgcgggcg ccttgcaagg aacacccggc agagcccaca tccacgggag 43740 ctggtgaggg cagctgcgtc acccgtgtgt cagggaggtc attcgagagc tgtggtctca 43800 gccattttga gggttatttt aatcttttta aaacagatgt agacgttttg gttgtaagtt 43860 ggtgttaaag agaggaggaa gttccaaatc ccaccccggg gcccagcctg cagttccatc 43920 cgttcagacc tgtttctact cgggctctgc ctctagtcag aaacctccac gccccgacat 43980 ggcatctgtg cccttaggaa ctcttcacag gggaattatt tggggccacg cggtggtgga 44040 aacctgcagt gctgggcagt gggtctggct ggagagccac tgcagaaggg ctgagaaggg 44100 gcggccccag caggccccat gcacttacag gcaaacaggc tgtcggccca gagccccagc 44160 aggggcctgg accccaggag cgacgggcct gaagcagggc ctctgtcctc ggagtgggag 44220 gcagagtgaa ctttagctgc tacaagactt ggaggtccgg ccccgggaat ctctgagcta 44280 tggccccctc acacggacag ggcatgagct ggggcctgcg acacccaacg ttgactgctc 44340 agaccctctc gtccctgcct gggcccacac ttgccatccc caggctcagc caggatttat 44400 ggccacctgg gtgtcctggc ccacgaactc cctgccccgg gcacccagcc tcctcccacc 44460 tcaccctccc agtgcccact cgtcagatag ctgatgctcg ccacgcacat gggcctcaac 44520 ttctagagga gtcccgagag ggaagggt tccccaagcc acacggtagg tcgagtacac 44580 ctgctcagcc tgcagaggcg gcctgccctc ctgcctgtcc tgtcgccacc ccatggggca 44640 aggctggtgc ccgcatttca gaagtgggaa cggggacctg gaggtcaagt ggcttttccg 44700 ggcatttgag ggggtccagc gctcatgagg actgtgctgc tgcctgtcct ccaagagacc 44760 cgcccctccc tcgtcagccc ctccatgctg atggggcat ggggcatgag cttcctagag 44820 tttggctgct gggaaggggc tgggtggctg gactctggtc atcttcatct tggtgtgatg 44880 ggagcctgag tgtgcagctg cgccctgggc ggccagcgtg gacagacttc agagtgagct 44940 gggggcacca ggcacctgca ggataccgtt ccgaaagatc tagaagcccc atgacccgg 45000 ctggcagggc ccccgggcga ccctcactcc cacatatggg caacacacac acgtgtttgc 45060 aggcatgcga cggagtgtgc cagccagccc gaggggtgct ggccgctaga ggagggtgag 45120 gggcgtctgc agcatggctg cggcacgtgg ggcccaagtc agggccgggc ctgcccctcg 45180 gtggcctcac accccaagtc agggccgggc ctgcccctcg gtggcctcat accccaagtc 45240 agggccgggc ctgcccctcg gtggcctcac acctgttctc tcacagcagc tgcctctgtt 45300 ctttccactc ccagaaatgc tcatttttca cggctctgct cagaagattt cccagggcca 45360 gggccagggc caggctgtgg gtggggtgag cgatggcgcc ctgcgttctg atccagcctc 45420 gcccccagct gctgtgtggt ctctgagagg ggtgggctca gggcctgggc ctctcctgcc 45480 ctgatctccc tgttaccctg gcctcggagc ctcccagcag cccctgcagc cctggaggcc 45540 tgtgaggaca ggactggtct ggttcatctg tcccgtcaag cccccatccc atttgacaga 45600 aggggaatag gctccgagag gctgtgactt gcctcggcct gcagagcacg gagactgtca 45660 gagctggggt ggggcctatc gtccagcccc acagcctgcg gtctcagccc gggcacctgg 45720 cgcctcagcc accaggagag cccccgagtc acagatgggg acactgggcc ggtggcagcc 45780 aggccagagc cagggcatct gacgacagcc gtgttctttc caccacacct ggctttccct 45840 ggtgtttgg aaatggctgt gttctgggaa atctttagtc aggtccaggg aaaacaggcc 45900 cggcaggtct ctctccccag ccccaggccg gcctgtcact tcttcttaggc agctggccac 45960 ctctgtcccc ccagaggctt ggggagggca ggagtaccac ccctatcttc caggcagagc 46020 cacccagaag gctggcaggg gtacacagga gaccctggag cctggccatg tccctcaggc 46080 ccccttggtc tctagagacc ccccggggta tagacagggc cccgctgctc cctgggtgcg 46140 tggttcgggg agatgaggtg gtataggaca gtctgtggtc tgtctgtacc tggcaaggtc 46200 atcacgtgcc tgggcttggc aggagagacc ctgggtcttc ggccagggtg ggagctgcta 46260 ccaggaaggc ctgcaggaac tgtgagcttg agtgaggaag taggaaggtg tcaggcagac 46320 ctcaggagcg gctggggcct gtgcccgggg aggctgtcct gtggccctca gaggagcagc 46380 tgtggatgtg gccgcctccc acgctcctgg ctgggcaggt gtgggctgga gaggtggcgt 46440 cagtgcgata cacctgacct tgcccctgtc cgtgaccttg gcaggatgat gatgagaggg 46500 atgtggatgc cagcccgacg gcagggctgc aggaagcagc aacgctggcc cctacacgga 46560 ggtcacggct ggccgctcgt ttccgagtca cggcccactg gctgctggtg gcggctgggc 46620 gggtcctggc cgtaacactg cttgcactgg caggtacgca ccgaggcagg gggcactggc 46680 agtcacactg ggaggggtct tgggagttcc ctgatgactg tggagacagc gggacacatg 46740 gcactggcca ggtaccaccc tgtgtgcccc tgccccgcag gcatcgccca cccctcggcc 46800 ctctccagtg tctacctgct gctcttcctg gccctctgca cctggtgggc ctgccacttt 46860 cccatcagca ctcggggctt cagcagactc tgcgtcgcgg tggggtgctt cggcgccggc 46920 catctcatct gcctctactg ctaccagatg cccttggcac aggctctgct cccgcctgcc 46980 ggcatctggg ctaggtaacg gcttgccaca cagccccttt ttcctgccac cctggtcccg 47040 cccacctggc tcgtctagcc cctgtggccc cactgcctct ggggtggtag gctgtgacgg 47100 gtcttctctg gacagggtgc tgggtctcaa ggacttcgtg ggtcccacca actgctccag 47160 cccccacgcg ctggtcctca acaccggcct ggactggcct gtgtatgcca gccccggcgt 47220 cctcctgctg ctgtgctacg ccacggcctc tctgcgcaag ctccgcgcgt accgcccctc 47280 cggccaggtg agcacctgcc acccatggtg ggtgggctga ggccaggcca tggggctggt 47340 ctcaggacct cctgcctctg ggtggggtgt ggagctggtt tgggctcaag acgctggtct 47400 ctgcagagga aggaggcggc aaaggggtat gaggctcggg agctggagct agcagagctg 47460 47520. cccagtggc cccaggacg ggagtctgac gccaggcagg tggagacgcc agcgtggggg gcgcccggcc agcccgtgca tggctcagcg ctgcttgccc acagcacgtg 47580. gtgcccacag cacccgacac cgaggctgat aactgcatcg tgcacgagct gaccggccag agctccgtcc tgcggcggcc tggtgagtac cgcacactgc aaggtatggc tgggtgcggg 47700 gggcggggcg gaggccggtg ctgccccctg gtggccgcct ggcgctctcg catgctcgcg 47760 ccgcacctct gcctgccgcc ccctcgggggg cccaggacat cccgggtcg gtgtcagtga 47820 47880. cccccgagac ccccagggca gccgagtggc catgtcactg accaaccccc aagaccccca gggcagctga gtggccgcgt cgttgatccc caagacccca tggggggcct ccaggtcccc 47940 caacccctcc ccagagaatg tggctatgct gtcttgtgct gttagctctg ggagctgctc 48000 caggtggccc agtggcccca ggaggccgct cgtccagggc aggggctggc ctgggaactc 48060 tgtgttggcc acgtcgcctt gggagggcct gggggctctt tctggctact ttctttcttt 48120 accctaaccc ttgattttcc attttgcaat gtgtttctga atgaagcaaa tgaagccacg 48180 gccctggggt gggggtcctg agagtcttca ggtgcgcaga gctggaaagg gggtcagggc 48240 cacctttccc accctttcaa ggaaagtgag gcccagagaa cggcaggtgc tggcagggcc 48300 atccctgacg ctcagggacg gtgtcagccc aattgccgga gccctcgtgt tctgcccata 48360 gcccaccggg ggcctgtctc tcctgctgtg tgcttgccca gggcccagat tttagggcat 48420 agtcagggtg gggaggcctg cagatcaacc tgccgaagct gaccgctgtc cccacctgca 48480 gtgcggccca agcgggctga gcccagggag gcgtctccgc tccacagcct gggccacctc 48540 atcatggacc agagctatgt gtgcgcgctc attgccatga tggtaggcgg ctgtgggggt 48600 tggggtgggc ggccccctct gccgcgcagg tgtggggcat cgcctgggtg gggtgcgctg 48660 ggcagctgtg cagccccctc tgccgcgcag gtgtggggca tcgcctgggt ggggtgcgct 48720 gggcagctgt gcagccccct ctgccgcgca ggtgtggggc atcgcctggg tggggtgcgc 48780 tgggcagctg tgcagccctc tctgccgcgc aggtatggag catcacctac cacagctggc 48840 tgaccttcgt actgctgctc tgggcctgcc tcatctggac ggtgcgcagc cgccaccaac 48900 tggccatgct gtgctcgccc tgcatcctgc tgtatgggat gacgctgtgc tgcctacgct 48960 acgtgtgggc catggacctg cgccctgagc tgcccaccac cctgggcccc gtcagcctgc 49020 gccagctggg gctggagcac acccgctacc cctgtctgga ccttggtgcc atggtgagtg 49080 tgcaccacca catccggggg tgcctgggtg cgcagaccca tcagggttgt cgtcctgttc 49140 aatgtccact tgcccggggg agtggcagcg ccaagaaggc agatgtgtct gtctgtcccc 49200 ttctgcccac ccagagccag cccagagtag cttctcagtg agcgtttgtt gactgaataa 49260 acagacaacc ttgtgttggc acgggcacca cccctgtgcc ctgacactgt gtgagcgtgg 49320 gctctgttgg cacgggcacc acccctgtgc cccgacactg tgtgtgagca tgggctatgc 49380 ccattggcac aggcaccacc cctgcgcccc tgacagtgtg tgtgagcgtg ggctctgccc 49440 attggcacca tgcacagccc tgggtctcag tgacaagctg tgcaggccat gtgttcacag 49500 ggtgcctgcg tgtccatgtg aaacgggtgc cagcatcgtg tctggacacc tgtttgcagg 49560 ccagtgggtc tcatcttgtg aaacttgtga gcctgtgtgc caacatatgc acctgtgagc 49620 ttgtgtgcat atgtgagcac gcatgtgggc aaacatgcac tccagcatgt ggacatgtgt 49680 gcaggtgcgt gcatggatct gtgcccacgt gaactagtga acccgtgtgt gactctgcgt 49740 gtgagcgcaa gtgaacccat gcactcatcc atggatgtga gagtgtgtgc tcgtgtgcct 49800 ctgagtgggt gtgagcgaga gggtgttcgg tgcctgtggg gaggctgcgg tggatgggct 49860 ggtgccagcc gcctgagagc tcttgccccc tgctatagga gggtgctggg tcccccggct 49920 gtgggagggg tgctgggccc cccggctgac tgtgacaccc tgcgcttgtc acagttgctc 49980 tacaccctga ccttctggct cctgctgcgc cagtttgtga aagagaagct gctgaagtgg 50040 gcagagtctc cagctgcgct gacggaggtc accgtggcag acacaggtga gtggtgggcc 50100 agaggcgggg gttgccctcc tgcctgcccg ccctgatgcc atcgcctgcc cctggcttgg 50160 cccacagagc ccacgcggac gcagacgctg ttgcagagcc tgggggagct ggtgaagggc 50220 gtgtacgcca agtactggat ctatgtgtgt gctggcatgt tcatcgtggt cagcttcgcc 50280 ggccgcctcg tggtctacaa gattgtctac atgttcctct tcctgctctg cctcaccctc 50340 ttccaggtgg ctggggggcc gggatggggg ctggggcacg gaccctcccc gcggtcctca 50400 ccacccccac ctcacccggc aggtctacta cagcctgtgg cggaagctgc tcaaggcctt 50460 ctggtggctc gtggtggcct acaccatgct ggtcctcatc gccgtctaca ccttccagtt 50520 ccaggacttc cctgcctact ggcgcaacct cactggcttc accgacgagc agtgagtcca 50580 ggctggggcg gtggggcagg ggcgccgaaa ccccgtgcac ttccccgggg ctgcagcggc 50640 tctgccgggg gccgggccgg tgctgatgct gcccctccac aggctggggg acctgggcct 50700 ggagcagttc agcgtgtccg agctcttctc cagcatcctg gtgcccggct tcttcctcct 50760 ggcctgcatc ctgcagctgc actacttcca caggcccttc atgcagctca ccgacatgga 50820 gcacgtgtcc ctgcctggca cgcgcctccc gcgctgggct cacaggtgcg gccccgccct 50880 ccctgtccgg ccctggagag gtgtagcctc ctgggccagg gagggagcca ggtgggagtt 50940 ggacaggagc cacatcttcc accttcagat cccaaggggc atttgctcat accaagggga 51000 tggcagtagc gtggaggtca cagggacagt gggcatgagt tgcgacacag ctgtgcacct 51060 gaactggcag ctgcagcaga agcggtgccg acagggcttc ttccagcccc aggaaatgag 51120 gggcaggaac ccagttggga gatgacattt tcggaccctc tcccaggcag gatgcagtga 51180 gtgggacccc actgctgcgg gaggagcagc aggagcatca gcagcagcag caggaggagg 51240 aggaggagga ggaggactcc agggacgagg ggctgggcgt ggccactccc caccaggcca 51300 cgcaggtgcc tgaaggtggg ttgggcgggc agagcacagc tgccacccag tctgctgtgc 51360 catgtcccag ctcggggggc gttggcagag tcccctctgg gctccagagc ctcttcctca 51420 caggggaccc gggaatcccc gtttgtgccc cgcactgacc ctcacaccat cacaggggca 51480 gccaagtggg gcctggtggc tgagcggctg ctggagctgg cagccggctt ctcggacgtc 51540 ctctcacgcg tgcaggtgtt cctgcggcgg ctgctggagc ttcacgtttt caagctggtg 51600 gccctgtaca ccgtctgggt ggccctgaag gaggtgagtg tggcaggcaa ctcagcttcc 51660 catctggggt ggggtcgctc tggcctgccc agctggcctc cccaagccca gccccacgtg 51720 cccactgccc tccccaagcc cagccccacg tgcccactgc cctcaggtgt cggtgatgaa 51780 cctgctgctg gtggtgctgt gggccttcgc cctgccctac ccacgcttcc ggcccatggc 51840 ctcctgcctg tccaccgtgt ggacctgcgt catcatcgtg tgtaagatgc tgtaccagct 51900 caaggttgtc aacccccagg agtattccag caactgcacc gaggtaccgg cccccgaggg 51960 ctgggacggg aggaagctcc aggcaactct gtattcgcag cccgaccctc ctggggcagc 52020 tgcctcagtg cagtggggcc agcaatggag atggaggact ctcccctggg ggcgccaagg 52080 gggcttcctg gaggcagcat ccttcgacct caactgtgga ccaggggcgc actccctgca 52140 cacaagggtg tccagtaggg gcggagtccc agggtctccg gcagtgagga cgggagggcc 52200 ccacccctgg acagggagag acagtcaggc atctctgcct gggaccttct cgcacatccc 52260 tccttctccc tggacctctc ttcactcccc cagcccctgc ccgtggtctc cctgtttctc 52320 aaacacctgg tccccttccc cgtgaaggtg gctccaaggc tggcagcccc cgtgtccctg 52380 gctggggagc agtggacctg ccccagagct gtggctgtgg tgggctccgg gcagggccag 52440 ggggcactgt ggcctgggag ggggcactga tgcctggcct cttgccagcc cttccccaac 52500 agcaccaact tgctgcccac ggagatcagc cagtccctgc tgtaccgggg gcccgtggac 52560 cctgccaact ggtttggggt gcggaaaggg ttccccaacc tgggctacat ccaggtgagt 52620 tgaagggctg gtgggcggct gggcgggcga gtacccggct gccccctgac ccttgccctc 52680 cgcagaacca cctgcaagtg ctgctgctgc tggtattcga ggccatcgtg taccggcgcc 52740 aggagcacta ccgccggcag caccagctgg ccccgctgcc tgcccaggcc gtgtttgcca 52800 gcggcacccg ccagcagctg gaccaggatc tgctcggctg cctcaagtac ttcatcaact 52860 tcttcttcta caaattcggg ctggaggtga ggcaaggaca ttgcctcccc ctggggcagg 52920 gcttggcctt cgggagggag ggacggctgc accgtgcagg caccgcaagc ctggccccac 52980 ctgggtttgc ctgggccaca gagggtgggg gactcagggc caggcacggc ttccctggac 53040 tcctgtggtg tgtcggtgct gacaacaggc agggggccaa gttagatctg ctctactgta 53100 cagcccacct cctggagcct cagtttcccc tgcacgatgg caactgccag ccactcctgc 53160 cctcttgaca gcgccgctgg ccctgtcctt gcttgatgcc cgcagcctcc aggcagggct 53220 gctgcaagcc tgaggcctgc tgggtgggac aagaaagtcc ctccccccag actcagtgca 53280 tccccacacc ccgccctctc ccctccccag atctgcttcc tgatggccgt gaacgtgatc 53340 gggcagcgca tgaactttct ggtgaccctg cacggttgct ggctggtggc catcctcacc 53400 cgcaggcacc gccaggccat tgcccgcctc tggcccaact actgcctctt cctggcgctg 53460 ttcctgctgt accagtacct gctgtgcctg gggatgcccc cggccctgtg cattggtgag 53520 gggcacgtgg cttgggtggg agtgggcttt gtggctttgt ggatgcccgt gggggtgttt 53580 cccgcctgcc ccagactcct gtccaccctc ctagacttag ccttggcctc ctccagtccc 53640 tcctctctgc tccacatcct acccaggcgc catcacctgc atcctgtctc tcgggggcgg 53700 cctggccccc tccaggctct gctgttctct tttccttttt tgcccagtat tctatcttga 53760 aatatttcaa accttcagga aagttgtgag tcacacaagg agcactgcgt cctctccggg 53820 ccccgtgcgg gtcggccgtg atgcctcaca cccaagtgct gcctcgagca tgcgtctcct 53880 gggtgagggt gtctgagccc acagccccac acccgtggtc ccgtctcctg gcagtgccat 53940 cgtcaaacgt gtcctctgca ttcaaacagc cccggcctca gcacccttct ttgtggccat 54000 ttggttttca gacgggatct ggtctgatgt ttgctctagt ctcttgtctt gggtctaagc 54060 cgccccccgcc tctcctgtct cttgggaagt tccggaggga ggccggtagc gttgctgacg 54120 ccgtgagact ggatttgcgt ggctgtcctg gtgctgcagg tctgctcaag gcacacagca 54180 ccctgcggtc tgagatgggg agtcacattt gtgcacgtgg ccggctcagg ggcgtcccac 54240 ctgccccaca gtggcaccca gcctgttggc actggtgggc tgtgtggggt cagccttgtg 54300 gttttacgaa acagactttc tctctgctgt ctctgtgtgt ctgtcagctg ggattcccat 54360 caaggacagc tgccatttgt tactggctgc tttccaagaa ttctgtcatc cgcagaccct 54420 gggcctcccc tctgctgagt gggtcctggc ccctccggcc acacactgtt acatcatctc 54480 cccgtatttg gctgggcatg gtggttcacg cctgtgatgc cagcactttg ggaggctgag 54540 gcaggagcat cccttgaggc caggagtttg aaaccactct ggtcaacata gcaagaaccc 54600 tttttttttt tttttttttt tttgagatgg agtcttgctc tgtcacccag gctggagagtac 54660 actggcacaa tctcggctca ctgcagcctc cgcctccccgg gttcaagcaa ttctcctgtc 54720 tcagcctccc tcgtggctag gattacaggc aagcaccacc aggcccggct aatttttgta 54780 tttttagtag agacggggtt tcaccatctt ggccaggctg gtcttgaact cctgacctca 54840 ggtgatccac ccgcctccca aattacaggc ctccctcctg ggattacagg cgtgagctgc 54900 cacgcccggc cccgtcttgt tttctgctcc caggcgctgc tgcctcatct tctgctaccc 54960 aggcccagcc ttgtgctcac agccattgct ccagggagcc caatcgagtt ctaggagcgt 55020 gaggtttaga gcccggggtc tgggcgctgg gtgtgcctgt tgctacaggg ctgcctcagc 55080 ctctgggccc tccagctctt ccttgttgaa acatctgctt tcgagcatca ccgaggccag 55140 ctccccgtct cctgtccacc tcttccttgt tgaaatacct gctatcaagc gtcacctagg 55200 ccagctcccc ttcttctgcc tccttccacg cggctgcgcc atgcagtcgc catcctgtga 55260 gatcagcatg tcctgggttc cccaacatcg agggtaactt tgtttttgta tcgtgaggtt 55320 ccctctgtgg cagatggggc tgtgggttca gcatgtcctg ggttccccag catcgagggt 55380 cactttgttt ttgggtcgcg aggttccctc tgtggcagat ggggctgtgg gttcagcatg 55440 tcctgggttc cccagcatcg agggtcactt tgtttttgtg tcgcgaggtt ccctctgtgg 55500 cagatggggc tgtgggttca gcatgtcctg ggttccccag catcgagggt cactttgttt 55560 ttgggtcgcg aggttccctc tgtggcagat gggggctgtg ggttcagaat gtcctgggtt 55620 ccccagcatc gagggtcact ttgtttttgg gtcacgaggt tccctctgtg gcagatgggg 55680 ctgtgggttc agcatgtcct gggttcccca gcatcgaggg tcactttgtt tttgtgtcgc 55740 gaggttccct ctgtggcaga tggggctgtg agatcagcat gtcctgggtt ccccaacatc 55800 gagggtcact ttgtttttgg gtcgcgaggt tccctctgtg gcagatgggg ctgtgggttc 55860 agcatgtcct gggttcccca gcatcgaggg tcactttgtt tttgggtcgc gaggttccct 55920 ctgtggcaga tggggctgtg ggttcagcat gtcctgggtt ccccagcatc gagggtcact 55980 ttgtttttgg gtcgcgaggt tccctctgtg gcagatgggg ctgtgggttc agaatgtcct 56040 gggttcccca gcatcgaggg tcactttgtt tttgggtcgc gaggttccct ctgtggcaga 56100 tggggctgtg ggttcagcat gtcctgggtt ccccagcatc gagggtcact ttgtttttgg 56160 gtcacgaggt tccctctgtg gcagatgggg ctgtgggttc agcatgtcct gggttcccca 56220 acatcgaggg tcactttgtt tttgggtcgc gaggttccct ctgtggcaga tggggctgtg 56280 agttcagcat gtcctgggtt ccccagcatg gagggtcact ttgtttttgt gtcgcaaggt 56340 tccctctgtg gcagatgggg ctgtgagttc agcatgtcct gggttcccca gcatggaggg 56400 tcactttgtt tttgtgtcgc gaggttccct ctgtggcaga tggggctgtg agttcagcat 56460 gtcctgggtt ccccagcatg gagggtcact ttgtttttgt gtcgcgaggt tccctctgtg 56520 gcagatgggg ctgtgagttc agaatgtcct gggttcccca gcatcgaggg tcactttgtt 56580 tttgtgtcgc gaggttccct ctgtggcaga tggggctgtg agatcagcat gtcctgggtt 56640 ccccaacatc gagggtcact ttgtttttgg gtcgcgaggt tccctctgtg gcagatgggg 56700 ctgtgggttc agcatgtcct gggttcccca gcatcgaggg tcactttgct tttgggtcgc 56760 gaggttccct ctgtggcaga tggggctgtg agttcagcat gtcctgggtt ccccagcatg 56820 gagggtcact ttgtttttgt gtcgcgaggt tccctctgtg gcagatgggg ctgtgggttt 56880 cgcagatgcg tggagtcaca tccatgccct cagtccttag ggaccgaccc tccctgcctc 56940 acacgcctcc caggaagtgt ggccgggggc cggcagtgcc acggctccct ccccagcagg 57000 ccccggccgc tcccatcccc agcacgtggt cctatcagaa cgccacgtca gcaggactcc 57060 cagcaggtgg cctttaggtc tggcttcttt cacttggcag agcacactga ggtctgtcta 57120 ggctgtcgca tggatcccgg tcccacgtgc tgagcagcgc gttcccagct gtggttgctg 57180 caggttgaac ttttcctggc tgcaggcgtc cgtgcagctt ctggccgttg ttttcagagc 57240 tgtcctatca cacgcactgt cctatcatgg aatatgacgc cgtgtgggcc acaactcagg 57300 cccagcagcc cccacccccg tgctctctgg cctcctgctc agttcctttg cccccagggg 57360 cttggtgcag agttgaagga atctgtgtgt gtgaacacac aggacactag agctgtcagt 57420 tctcgagaca ccaggtgtgc gcgaggtgat tcccatggac cctgagggct ggtgatagac 57480 tcgggtcaac gggtggggac cgggtgtctc aggccccagg caggcccggc ccttcctgac 57540 atgacacccc ttcccccaga ttatccctgg cgctggagcc gggccgtccc catgaactcc 57600 gcactcatca agtggctgta cctgcctgat ttcttccggg cccccaactc caccaacctc 57660 atcagtgagt gccccccacc acccccgcct ctgcagagga ccctcagagt acattcacgc 57720 ccccaaatct gctcacaagt gtgcacacag gcgtgcacgg gcggaggtgt ggtcaggcac 57780 atggcggcct gcaggccctg acctcgcacg cacgcacgca gacctcagcc tgtgtgcacg 57840 gcagcccttg tgcagatgcc ctcacaccgg ggctccccca gggacacccg gccactcacc 57900 caggcagacg tgtgtccgct cccagcggct gcacgccgac aggcctgggg tgggaggtgg 57960 gatttatgcg ccgtgcccac ctcgtgtggg tccccgtgtg gcacagcggc ggctcctgtg 58020 tcctgcaggc gactttctcc tgctgctgtg cgcctcccag cagtggcagg tgttctcagc 58080 tgagcgcaca gaggagtggc agcgcatggc tggcgtcaac accgaccgcc tggagccgct 58140 gcggggggag cccaaccccg tgcccaactt tatccactgc aggtgggttc cacgtcaccc 58200 tccacgggga accttctggg aggggtggcc ggggcgcccg ccctgacgct ccggcctggc 58260 aggtcctacc ttgacatgct gaaggtggcc gtcttccgat acctgttctg gctggtgctg 58320 gtggtggtgt ttgtcacggg ggccacccgc atcagcatct tcgggctggg ctacctgctg 58380 gcctgcttct acctgctgct cttcggcacg gccctgctgc agagggacac acgggcccgc 58440 ctcgtgctgt gggactgcct cattctgtac aacgtcaccg tcatcatctc caagaacatg 58500 ctgtcggtga gcctccggcc cccccgcacc caccgccctg gggccccgct ggccccgctg 58560 accctgctct cccccagctc ctggcctgcg tcttcgtgga gcagatgcag accggcttct 58620 gctgggtcat ccagctcttc agccttgtat gcaccgtcaa gggctactat gaccgtgagt 58680 ggccaggacg gtggcggggg agggcgtggg gaagccccct gctcctgggc cctgggcctg 58740 acccttgccg gtgcctgcct tgcagccaag gagatgatgg acagagacca ggactgcctg 58800 ctgcctgtgg aggaggctgg catcatctgg gacagcgtct gcttcttctt cctgctgctg 58860 cagcgccgcg tcttccttag ccattactac ctgcacgtca gggccgacct ccaggccacc 58920 gccctgctag cctccaggca agcttgggcc cagacacagc ccagagctcc cgtcttgggg 58980 ctgggagggg gcaatgggag gttcctcact gtctcaggcc ccggcccgtg gagggcaggc 59040 tctgccactc tgtgacatgg gcgtgtcatc tagagggaga atgaaggccg gcagatcccc 59100 ggcaccatca cactctgccc cagtgctggg tctgtcagag accacaggct gcagtgctga 59160 cggtggctgg tgtctcaccc ccagccaact ttcccactaa gggctaagtt tctccaccag 59220 cgggagggcc actgtgtggt gtcacgactg ccccagggag gggttctggc ttggggccag 59280 ctttgccttc ttccctgcag ctgtggtggg gtgggtgcca ccagacgccc ctgcatctgt 59340 acggcagaag ggcctgtcct cgccgcagac agcacggagg gtgggggcag cagatgcctc 59400 ccccgtgggt gcctcttgtc cagcgtgggc agagaggagc aggctgagct gtcccgggct 59460 gagcggggag cggcggctgc ccatgttgct ggggtcgagt gcctggtgct cacaccccat 59520 ccccgcctcc ctacaggggc ttcgccctct acaacgctgc caacctcaag agcattgact 59580 ttcaccgcag gatagaggag aagtccctgg cccagctgaa aagacagtag gtgcctctgg ggcggggact ccccggctcc tccccccaat gctcagcata ccccacctttt ccccaccaca 59700. ggatggagcg tatccgtgcc aagcaggaga agcacaggca gggccggggtg gaccgcagtc gcccccagga caccctgggc cccaaggacc ccggcctgga gccaggtgag tgcagctgga 59820 gtcgggcacc cagggccccg tgtccagcat gtctgtgcct gctggcgtgt gctgcgtctg 59880 tgcccatgtg acgtcccaca gggctcccag cccgcctgtc ctgtccgcat gatcaccctc 59940 tgtctggcag gccccatggc cgccctgtga ctgtccgtcc acgcacatgg gctctgagcc60000 ccatggcccc acacggcccc cgtcactgtg ggtgtccgtg tctgtctcca cctatcctgt 60060 ctccaagacg ggagcactca cagccccgac ccctcctggt ggcttgactg ctgcctcatg ctcaccctgc ccctccacag ggcccgacag tccagggggc tcctccccgc cacggaggca 60180. gtggtggcgg ccctggctgg accacgccac aggtacccc aattaggccg cctgtggcca 60240 ccctctcagg ccctctgtgc ccccatctgt cctctgcctg gcctgctatc ttcccctccc 60300 ttcccccgac tcccaggccc tgagcgtcag gacgtgctca ggcctcctgg gtcggggggt 60360 gcctcactgg ctgcagaccc ctgggctgac tatgtcctct cctggctatg ccccagccct 60420 tccaacagtg ggagtctcgg agcttgcccc gatgacacat ggtggtcgag cagcgatctc 60480 acctgggacc cagcagcact gcgttattct gtttttgttt ctttttgaga tggagtctcg 60540 ctctgtcact gcaggctgga gtgcagtggc atgatctcag ctcactgcaa tctctacctc 60600 ccgggttcaa gtgatcctcc tgcctcagcc tcccaagtaa ctgagactac aagcatgtgc 60660 cccactccag gccttttttt tttttttttg gagacggtgt cttgccctgt cgcccaggct 60720 acagtgcaat ggcgtgattg cggctcacta caacccccac ctcccaggtt caaaggattc 60780 tcctgcctca gcctcccaag tagctgggat tacaggtgcc cgccaccacg cccagctaat 60840 tttcgtattt ttagtagaga tggggtttca ccatattggc caggctggtc tcgaacctct 60900 gacttcaggt gatccgcccg cctcagcctc ccacagtgct gggattacag gcgtcagcca 60960 ccgtgcccgg cctgttctgt ttttctaact ctcacacagc ctcctgggtt ttccccggtc 61020 ctctgcagtc ggcccactct gcaccccagc ccgcgctggc tctgctcctc agctgccctg 61080 cccacctctg tcttgtccca ccgcgctggc ctgtgtcttg tgcctgcact gctcccggct 61140 actccgcatg ggaagggtgg ctctcgggcc ttggcccatg caggcggagg gggtctggct 61200 gggagtctcc ctgcatggaa ggctggctct cagtgctgcc tgcccacagt catccactcc 61260 ggggactact tcctgtttga gtccgacagt gaggaagagg aggaggctgt tcctgaagac 61320 ccgaggccgt cggcacagag tgccttccag gtgaggtggg agagccccgt cggccccact 61380 ccaaccacag agcttgtggt cctggaccag ggcagcatag agggtgtcag atgcccccag 61440 ggcctgggag ccgagctcct ccacctccag ttagcccacc ccgccccatc caggcctccc 61500 aagtcccatg ggaaaccagg ctacagggac atgggtcatg tgtagcctgc tgccccacgg 61560 tcttggctct gaccacccag gttctggtgg ctgcccgtgg cctgacctgt gagaccggcc 61620 caacaccttt gtgctggccg cctggctgtc ctgggtccat ctttgggccc ctggctcttg 61680 gtgttagacc agcccaccca actcctgaat gggtgggagt cttcccccac agcccctcag 61740 ggtccccatc cgggaggggc tcagggacac ggaggtccct gggagacaca gagcagggat 61800 ctggatctgg cgcccggctt gcccaacccc agcttcccgc ctgggtctga tggctcggga 61860 ggccgggtcc taacccgggg gctggccgac agctggcgta ccaggcatgg gtgaccaacg 61920 cccaggcggt gctgaggcgg cggcagcagg agcaggagca ggcaaggcag gaacaggcag 61980 gacagctacc cacaggtgag ctggggggcg tggggactct gaggggaagc cgcgggactg 62040 ccagtcactc accagcatcc tgtgcccagg aggtggtccc agccaggagg tggagccagc 62100 agagggcccc gaggaggcag cggcaggtac gtgggcccgg ggctggggag tgggaggtct 62160 ctcttggccc cacaggctgc ccctccagcg ccccctcccg ccctcccgca ggccggagcc 62220 atgtggtgca gagggtgctg agcacggcgc agttcctgtg gatgctgggg caggcgctag 62280 tggatgagct gacacgctgg ctgcaggagt tcacccggca ccacggcacc atgagcgacg 62340 tgctgcgggc agagcgctac ctcctcacac aggagctcct gcaggtgagc ctgcccgtgc 62400 accacgctcg tccctgctct gcctgactac gcccctgcct gcttaacagc ctagtcccgc 62460 gcccactgca cgaaaccccg tgtggggaca agagctggac gcagccctga gccccctgct 62520 gtgccctgca gggcggcgaa gtgcacaggg gcgtgctgga tcagctgtac acaagccagg 62580 ccgaggccac gctgccaggc cccaccgagg cccccaatgc cccaagcacc gtgtccaggt 62640 aggtgcgggg gtgacccgag ccccagctgc tgcccctggt gtgtgggcat cgcctagcca 62700 tccccgaccc tcgccattcc cttgtacccc aaaggaccgt gggcactttc caccctgacc 62760 ctccctgtag cctggggtca ggccatagag caggattctc tgtgactcgg cttccctccc 62820 cagtgggctg ggcgcggagg agccactcag cagcatgaca gacgacatgg gcagccccct 62880 gagcaccggc taccacacgc gcagtggcag tgaggaggca gtcaccgacc ccggggagcg 62940 tgaggctggt gcctctctgt accagggact gatgcggacg gccagcgagc tgctcctgga 63000 caggtggggg cgggacgcgc acaacaccag cctcaccatg gccctcgggg agcagccgaa 63060 caggggcagg agactgactg tgaccggcaa cagatcgggc cgtcatgcct tcgggcagtc 63120 ccagactccc ccaaacacgc gggtctccct gtaggcgcct gcgcatccca gagctggagg 63180 aggcagagct gtttgcggag gggcagggcc gggcgctgcg gctgctgcgg gccgtgtacc 63240 agtgtgtggc cgcccactcg gagctgctct gctacttcat catcatcctc aaccacatgg 63300 tcacggcctc cgccggctcg ctggtgctgc ccgtgctcgt cttcctgtgg gccatgctgt 63360 cgatcccgag gcccagcaag cgcttctgga tgacggccat cgtcttcacc gaggtgggcc 63420 gaggccgcgg gggagggggc gcccggccca ccgcgccgtg accctccccg cgtgctgagc 63480 cccctccccc acagatcgcg gtggtcgtca agtacctgtt ccagtttggg ttcttcccct 63540 ggaacagcca cgtggtgctg cggcgctacg agaacaagcc ctacttcccg ccccgcatcc 63600 tgggcctgga gaagactgac ggctacatca agtacgacct ggtgcagctc atggcccttt 63660 tcttccaccg ctcccagctg ctggtgagtg tgagccttgg ctggcaatgc ggggctgggc 63720 aggccctctg ggcacctgtg ctctccacca gggaggcaag gccccctcac cacaccctcc 63780 cgcccctcag tgctatggcc tctgggacca tgaggagac tcaccatcca aggagcatga 63840 caagagcggc gagggagc agggagccga ggaggggcca ggggtgcctg cggccaccac 63900 cgaagaccac attcaggtgg aagccagggt cggacccacg gacgggaccc cagaacccca 63960 agtggagctc aggccccgtg atacgaggcg catcagtcta cgttttagaa gaaggaagaa 64020 ggagggccca gcacgggaag gagcggcagc catcggtata agcgccctgc ctcacaacct 64080 cctgcctacc cagttttctg agtggggcta ctgcagggag ggtctttctc agatgagacg 64140 gccaagccca gtgcgaggcc cacctggatc ccaggaaggt gccacttctg agccacagct 64200 cccggctctg cctacagagc cgtccctgac tgctgcccccc ggggatgctc cccacgtgta 64260 gggtgactgt tggcctgggc tggcccctca cagttgcccc agagagga cacagcccca 64320 gctgtctcct tgccagtgac actgggagct ttcctgtgct ccgtctgctt gtctgtcaaa 64380 cagggagaat gccagcctct tagggtggtc aggagccatg agccaggccc agtccccagg 64440 gggcccaggc agaagtcagc ttttccctac agaagctggag gacagggagg aagaagagg 64500 ggaggaagag aaagaggccc ccacggggag agaagagg ccaagccgct ctggaggaag 64560 agtaagggcg gccgggcgg ggctgcaggg cttctgcctg tccctgtgag tgatggcgg 64620 cggggcagc tggggagtgg gggtggggag gcgggtactg ggcccaggct gagcgcccc 64680 ttccgcagg cccagggcac atatcggccg ctacggcgct tcttccacga catcctgcac 64740 accaagtacc gcgcagccac cgacgtctat gccctcatgt tcctggctga tgttgtcgac 64800 ttcatcatca tcatttttgg cttctgggcc tttggggtga gccaggcccg ggacccaaac 64860 ccagtgtacg cagagctcag cagccaccca catcccctgg gcttggctcc ccctgacctg 64920 tgctctcctg gccacagaag cactcggcgg ccacagacat cacgtcctcc ctatcagacg 64980 accaggtacc cgaggctttc ctggtcatgc tgctgatcca gttcagtacc atggtggttg 65040 accgcgccct ctacctgcgc aagaccgtgc tgggcaagct ggccttccag gtggcgctgg 65100 tgctggccat ccacctatgg atgttcttca tcctgcccgc cgtcactgag aggtgggccc 65160 acgcgtgggg gcgctcggtc tccaggggcg gggcagtgca ggctggggc cctgcggggc 65220 tgtttctgat ggggtccttg acctggccat cccgccccag gatgttcaac agaatgtgg 65280 tggcccagct ctggtacttc gtgaagtgca tctacttgc cctgtccgcc taccagatcc 65340 gctgcggcta ccccacccgc atcctcggca acttctctcac aagagtac aatcatctca 65400 acctcttcct cttccagggg tgagtgcagg tccgccgggg tggggtcac ggcccgggca 65460 tgagggagcc cacctgacgg gaaccctggc tgtgggcagg ttccggctgg tgccgttcct 65520 ggtggagctg cgggcagtga tggactgggt gtggacggac accacgctgt ccctgtccag 65580 ctggatgtgt gtggagaca tctatgccaa catcttcatc atcaaatgca gccgagagac 65640 agagaaggtg cctgggccca gggcggggc cgggacaagg gccagggata tgccctctcc 65700 ctaagacaga ggcactgctg ccacgagaac ccgtggtgct ggaggccctc ccagggctcg 65760 gagcccatgg ggacatgagg cgagcccacc cactagctga tcacgaggcc agtgatcttg 65820 gcagctgcga gtgagtgctg ggcgcagaag tgggcagcgg agttggtcct gttccaggca 65880 gggctggcagc agagcagggc ctggtgcagg gaggaccgga cagccactgt ttgctgcatt 65940 cttgtttaat ggcctttctc agaagagaatt cgtgcgtcag accactcccc cacgtaaaaa 66000 gtacaactca ggggtttcta gtggattcac agttgagcat ctgccttctc accacttcaa 66060 aaagaaaccc cgggccgggc acagcggctc acccctatca tcccagcact gtaagagacc 66120 caggagggag gtactgctca aggcaggag ttcaagatct gcacggccac aagcgagacc 66180 ctgcctcagc aaatgtaaaa ataaacaat taggctggcaa tggcagctca tgcctatggt 66240 cccagcactt tgggaggcca aaggagggagg atcagttgag gccaggagtt ttaagaccag 66300 cctgggcaac atagtgagtg agactctctc tacaaaaaaa taaacgttag ctgggtgtga 66360 tggtgcacac ttgtgctccc agctactctg gaggctgagg tgggaggatg gcttgaggcc 66420 aggagatcag ggctgcatta agcctgatca caccactgca ctccagcttg ggcaacagag 66480 tgagaccctg tctctaaaaa agtaaaaaga agaaacccag tgcccattgg ctgtcactcg 66540 gtttcccctc ccctggcccc tagtaacccc ttgtctaggt cctggttcta tgagatttgc 66600 ctacagtgga tatttcatga aaacaggctc agacagcgtt tgtccttttg taatcagctt 66660 tccttgctca gcatggtgtc tctgaggtcc acccacgtgg cagtatggcc ttcctgttta 66720 tgggcaaatg atattctgtg gcatggatgg accacaacat gctcatctgt tgatgggctg 66780 ttgcccgtga tgctgccagg cacgccggtg tacacgtctg tgtgcccgtg cccctggtcc 66840 tgtggctgca cgccagggcc ggaagtgctg gtggtggttt ccatcatgag gagctgctgg 66900 ttttccatag cagctccacc atctaaggtt cccaccacca acacgaggtt gcggtttctc 66960 cacatcctca acaacctgtt actatgtctt ttttgttctg gccatgctgc tgaacgggtg 67020 gcttgctgtg ggctcttctc agttccctgt tgaccgacgc tgagcgtctt ttcatgtgct 67080 tggccatttg tgtatcttct ctggggaaat gtctattcaa atcctttctc catagtttag 67140 ttgggctttt gagataggat ctcaggctga agtgcagtgg catgaccttc actcactgta 67200 gcctctgcct cccaggttca agcgattctc ccacctcagc ctcccgagta gctgggacta 67260 caggtgtgca ccaccaggcc tggctagtct tttgttttt tggccaggtt cgtctcaaac 67320 tcctgacctc aaagtgctgg gatgacaagt gtgagccgcc acacccagca gttgggttgt 67380 ttttttag agtttttct gtttttctgc agatacattg gctagaaatg actgaattgg 67440 aagaattctc tgtattctct ggttgctaga accttatcaa ttaaaatttg cagaaaattt 67500 ctccaattct atggactgtc tcttaaattt tcttggtgtc tttggaagca caaagtattt 67560 attttggtaa tatctggttt actttgtttc cttcgccaaa tccaggttca tgaagatttg 67620 cccgttttct tctaaaagtt ctatagtttt agctctgaag tttcgctctt tgatccactt 67680 tgaggtaaat tttggcacat ggtatgaggc aggagttgcg tttcattctc ctgcctgggg 67740 cggtgcctgc accgtgttga aaaaggttgt ccgttcccac tgaacggtca cagctccctt 67800 gtctaagatc aacgacccct gaacatgagg gttccaactg gactcttagt tctactccac 67860 tggcctgtgt ctgcccacca ttactaccgc tgtgccatac tgaggtcagg ctggggcttt 67920 tctgggctgc tgggtgagct ggagaatgcg gttgtgtgac ccgcaggaag ggcagagctg 67980 agcgtatgac ctgtgtcgtt cccctccaga aatacccgca gcccaaaggg cagaagaaga 68040 agaagatcgt caagtacggc atgggtggcc tcatcatcct cttcctcatc gccatcatct 68100 ggttcccact gctcttcatg tcgctggtgc gctccgtggt tggggttgtc aaccagccca 68160 tcgatgtcac cgtcaccctg aagctgggcg gctatgaggt gagcatgtgt gggtccgcct 68220 gtccattccc atcccctggg ggttctggcc aaggtggtgc accaccccca gccgctcctc 68280 cacgctcatc ttcgtggccc cgtgtccccg tgcctgcccc agccgctgtt caccatgagc 68340 gcccagcagc cgtccatcat ccccttcacg gcccaggcct atgaggagct gtcccggcag 68400 tttgaccccc agccggtaag tggcctctgc cctgtgaaag ctggtgtggg gaggcggctg 68460 cagtcactga gggtgtcact tgtacccagc tggccatgca gttcatcagc cagtacagcc 68520 ctgaggacat cgtcacggcg cagattgagg gcagctccgg ggcgctgtgg cgcatcagtc 68580 cccccagccg tgcccagatg aagcgggagc tctacaacgg cacggccgac atcaccctgc 68640 gcttcacctg gaacttccag aggttcgtcc tggacttggg gcagtgcctg ggtgggtgga 68700 cccactacag tgggtcacgc tgtgttccca cccccaggga cctggcgaag ggaggcactg 68760 tggagtatgc caacgagaag cacatgctgg ccctggcccc caacagcact gcacggcggc 68820 agctggccag cctgctcgag ggcacctcgg accagtctgt gtgagtgaag ggcccgggtg 68880 gtgggcagga gggctgtgcc aggttggctg ggccaggcct gacctgccag cacctccctg 68940 cagggtcatc cctaatctct tccccaagta catccgtgcc cccaacgggc ccgaagccaa 69000 ccctgtgaag cagctgcagc ccagtgagta tgggcgtggg ggttggggga ggctagagag 69060 gggtgacctg cggcctcaac gatcttctcc ctccatccca gatgaggagg ccgactacct 69120 cggcgtgcgt atccagctgc ggagggagca gggtgcgggg gccaccggct tcctcgaatg 69180 gtgggtcatc gagctgcagg agtgccggac cgactgcaac ctgctgccca tggtcatttt 69240 cagtgacaag gtcagcccac cgagcctcgg cttcctggct ggctacgggt gagtgagtgg 69300 ctgggggggc accccgcagc tcggggggct ccgggcggcc ccaggactca ccagcttccc 69360 ccgcagcatc atggggctgt acgtgtccat cgtgctggtc atcggcaagt tcgtgcgcgg 69420 attcttcagc gagatctcgc actccattat gttcgaggag ctgccgtgcg tggaccgcat 69480 cctcaagctc tgccaggaca tcttcctggt gcgggagact cgggagctgg agctggagga 69540 ggagttgtac gccaagctca tcttcctcta ccgctcaccg gagaccatga tcaagtggac 69600 tcgtgagaag gagtaggagc tgctgctggc gcccgagagg gaaggagccg gcctgctggg 69660 cagcgtggcc acaaggggcg gcactcctca ggccggggga gccactgccc cgtccaaggc 69720 cgccagctgt gatgcatcct cccggcctgc ctgagccctg atgctgctgt cagagaagga 69780 cactgcgtcc ccacggcctg cgtggcgctg ccgtccccca cgtgtactgt agagtttttt 69840 ttttaattaa aaaatgtttt atttatacaa atggacaatc aga 69883 <210> 2 <211> 8089 <212> DNA <213> Homo sapiens <400> 2 gggagccgcc guccggccca gcucggcccc agugagccga gcgcugcgcu ccgccgaggg 60 gcagggcggu cgccugagcg agcgcgggcc cgggacgucg gcaccggcgg ggcggccgaa 120 ggagaaggag gaagaggaga aggcggcgcg cggguccccg cgggucagcc auggcgcgcc 180 ggccccgggg cccccgcacc gccccauagc gccgcggcgu ccgcucgguc ugggccgggc 240 ccugggcccu ccagccaugg agccgcacgu gcucggcgcg guccuguacu ggcugcugcu 300 gcccugcgcg cugcuggcug ccugccugcu ccgcuucagc ggacucucgc uggucuaccu 360 gcucuuccug cugcugcugc ccugguuccc cggccccacc cgaugcggcc uccaagguca 420 cacaggccgc cuccugcggg cauugcuggg ccucagccug cucuuccugg uggcccaucu 480 cgcccuccag aucugccugc auauugugcc ccgccuggac cagcuccugg gacccagcug 540 cagccgcugg gagacccucu cgcgacacau aggggucaca aggcuggacc ugaaggacau 600 ccccaacgcc auccggcugg uggccccuga ccugggcauc uugguggucu ccucugucug 660 ccucggcauc ugcgggcgcc uugcaaggaa cacccggcag agcccacauc cacgggagcu 720 ggaugaugau gagagggaug uggaugccag cccgacggca gggcugcagg aagcagcaac 780 gcuggccccu acacggaggu cacggcuggc cgcucguuuc cgagucacgg cccacuggcu 840 gcugguggcg gcugggcggg uccuggccgu aacacugcuu gcacuggcag gcaucgccca 900 ccccucggcc cucuccagug ucuaccugcu gcucuuccug gcccucugca ccuggugggc 960 cugccacuuu cccaucagca cucggggcuu cagcagacuc ugcgucgcgg uggggugcuu 1020 cggcgccggc caucucaucu gccucuacug cuaccagaug cccuuggcac aggcucugcu 1080 cccgccugcc ggcaucuggg cuagggugcu gggucucaag gacuucgugg gucccaccaa 1140 cugcuccagc ccccacgcgc ugguccucaa caccggccug gacuggccug uguaugccag 1200 ccccggcguc cuccugcugc ugugcuacgc cacggccucu cugcgcaagc uccgcgcgua 1260 ccgccccucc ggccagagga aggaggcggc aaagggguau gaggcucggg agcuggagcu 1320 agcagagcug gaccaguggc cccaggaacg ggagucugac cagcacgugg ugcccacagc 1380 acccgacacc gaggcugaua acugcaucgu gcacgagcug accggccaga gcuccguccu 1440 gcggcggccu gugcggccca agcgggcuga gcccagggag gcgucuccgc uccacagccu 1500 gggccccuc aucauggacc agagcuaugu gugcgcgcuc auugccauga ugguauggag 1560 caucaccuac cagcuggc ugaccucgu acgcugcuc ugggccgcc ucaucuggac 1620 ggugcgcagc cgccaccaac uggccaugcu gugcucgcccc ugcauccugc uguaugggau 1680 gacgcugugc ugccuacgcu acguggggc cauggaccug cgcccugagc ugcccaccac 1740 ccugggcccc gucagccugc gccagcuggg gcuggagcac acccgcuacc ccugucugga 1800 ccuuggugcc auguugcucu acacccugac cuucuggcuc cugcugcgcc aguuuugugaa 1860 agagaagcug cugaaguggg cagagucucc agcugcgcug acggagguca ccguggcaga 1920 cacagagccc acgcggacgc agacgcuguu cgagagccug ggggagcugg ugaagggcgu 1980 2040 ccgccucgug gucuacaaga uugucuacau guuccucuuc cugcucugcc ucacccucuu 2100 ccaggucuac uacagccugu ggcggaagcu gcuaagcc uucugguggc ucgugguggc 2160 cuacaccaug cugguccuca ucgccgucua caccuuccag ucccaggacu ucccugccua 2220 cuggcgcaac cucacuggcu ucaccgacga gcagcugggg gaccuggggcc uggagcaguu 2280 cagcgugucc gagcucuucu ggcuuccu ggugcccggc uucuuccucc uggccugcuu 2340 ccugcagcug cuacuucc accgaccug agcacguguc 2400 ccugccuggc acgcgccucc cgcgcuggg ucaggcag gaugcaguga gugggacccc 2460 acugcugcgg gaggagcagc aggagcauca gcagcagcag caggaggagg aggaggagga 2520 ggaggacucc agggacgagg ggcugggcgu ggccacuccc caccaggcca cgcaggugcc 2580 ugaaggggca gccaagugggg gccugguggc ugagcggcug cuggagcugg cagccggcuu 2640 cucggacguc cucucacgcg ugcagguguu ccugcggcgg cugcuggagc ucacguuuu 2700 caagcuggug gcccuguaca ccgucugggu ggcccugaag gaggugucgg ugaugaaccu 2760 gcugcuggug gugcugugggg squawkcccu gcccuaccca cgcuuccggc squash 2820 cugccugucc accgugugga ccugcgucau caucgugugu aagaugcugu acccuca 2880 gguugucaac cccacaggu auuccagcaa cugcaccgag cccuuccccca acagcaccaa 2940 cuugcugccc acggagauca gccagucccu gcuguaccgg gggcccgugg acccugccaa 3000 cugguuuggg gugcggaaag gguuccccaa ccugggcuac auccagaacc accugcaagu 3060 gcugcugcug cugguauucg aggccaucgu guaccggcgc caggagcacu accgccggca 3120 gcaccagcug gccccgcugc cugcccaggc cguuuugcc agcggcaccc gccagcagcu 3180 ggaccaggau cugcucggcu gccucaagua cuucaucaac uucuucuucu acaaauucgg 3240 3300 gacccugcac gguugcuggc ugguggccau ccucacccgc aggcaccgcc aggccauugc 3360 ccgccucugg cccaacuacu gccucuuccu ggcgcuguuc cugcuguacc aguaccugcu 3420 gugccugggg augcccccgg cccugugcau ugauuaucccc uggcgcugga gccgggccgu 3480 ccccaugaac uccgcacuca ucaaguggcu guaccugcu gauuucucc gggcccccaa 3540 cuccaccaac cucaucagcg acuuucuccu gcugcugugc gccucccagc aguggcaggu 3600 guucucagcu gagcgcacag aggaguggca gcgcauggcu ggcgucaaca ccgaccgccu 3660 ggaggccgcug cggggggagc ggccccgu gccshacuuu auccacugca gguccuaccu 3720 ugacaugcug aagguggccg ucuuccgaua ccuguucugg cuggugcugg uggugguguu 3780 ugucacgggg gccacccgca ucaccaucuu cggcugggc uaccugcugg ccugcuucua 3840 ccugcugcuc uucggcacgg cccugcugca gagggacaca cgggcccgcc ucgugcugug 3900 ggacugccuc auucuguaca acguccuc 3960 ggccugcguc uucguggagc agaugcagac cggcuucugc uggcucucc agcucuucc 4020 ccuuguaugc accgucaagg gcuacuauga cccaaggag augauggaca gagaccagga 4080 cugccugcug ccuguggagg cugcuggcu cucugggac agcgucugcu cucuucuccu 4140 gcugcugcag cgccgcgucu uccuuagcca uuacuaccug cacgucaggg ccgaccucca 4200 ggccaccgcc cugcuagccu ccaggggcuu cgcccucuac accgcugcca accucagag 4260 cauugacuuu caccgcagga uagaggagaa gucccuggcc caccugaaa gagagaugga 4320 gcguauccgu gccaagcagg agaagcacag gcagggccgg guggaccgca gucgccccca 4380 ggacacccug ggccccaagg accccggccu ggagccaggg cccgacaguc caggggccuc 4440 cuccccgcca cggaggcagu gguggcggcc cuggcuggac cacgccacag ucauccacuc 4500 cgggacuac uuccuguuug aguccgacag uggaagaag gagaggcug uuccugaagaa 4560 cccgaggccg ucggcacaga gugccuucca gcuggcguac caggcauggg ugaccaacgc 4620 ccaggcggug cugaggcggc ggcaggcagg gcaggcagg gcaggcagg aacaggcagg 4680 acagcuaccc acaggaggug gucccagcca ggagguggag ccagcagagg gccccgagga 4740 ggcagcggca ggccggagcc auguggugca gagggugcug agcacggcgc aguuccugug 4800 gaugcugggg caggcgcuag uggaugagcu gaccgcugg gacgggagu ucaccccggca 4860 ccacggcacc augagcgacg ugcugcgggc agagcgcuac cuccucacac aggagcuccu 4920 gcagggcggc gaagugcaca ggggcgugcu ggaucagcug uacacaagcc aggccgaggc 4980 cacgcugcca ggccccaccg aggcccccaa ugccccaagc accgugucca gugggcuggg 5040 cgcggaggag ccacucagca gcaugacaga cgacaugggc agcccccuga gcaccggcua 5100 ccacacgcgc aguggcagug aggaggcagu caccgacccc ggggagcgug aggcuggugc 5160 cucucuguac cagggacuga ugcggacggc cagcgagcug cuccuggaca ggcgccugcg 5220 caucccagag cuggaggagg cagagcuguu ugcggagggg cagggccggg cgcugcggcu 5280 gcugcgggcc guguaccagu guguggccgc ccacucggag cugcucugcu acuucaucau 5340 cauccucaac cacaugguca cggccuccgc cggcucgcug gugcugcccg ugcucgucuu 5400 ccugugggcc augcugucga ucccgaggcc cagcaagcgc uucuggauga cggccaucgu 5460 cuucaccgag aucgcggugg ucgucaagua ccuguuccag uuuggguucu uccccuggaa 5520 cagccacgug gugcugcggc gcuacgagaa caagcccuac uucccgcccc gcauccuggg 5580 ccuggagaag acugacggcu acaucaagua cgaccuggug cagcucaugg cccuuuucuu 5640 ccaccgcucc cagcugcugu gcuauggccu cugggaccau gaggaggacu caccauccaa 5700 ggagcaugac aagagcggcg aggaggagca gggagccgag gaggggccag gggugccugc 5760 ggccaccacc gaaccaca uucaggugga agccaggguc ggacccacgg acgggacccc 5820 agaaccccaa guggagcuca ggccccguga uacgaggcgc aucagucuac guuuugaag 5880 aaagaaag gagggcccag cacggaagg agcggcagcc aucgaagcug agcaggga 5940 ggaaagag ggggagaag agaaagaggc ccccacgggg agagaagaa ggccaagccg 6000 cucuggagga agaguaaggg cggccgggcg gcggcugcag ggcuucugcc ugucccuggc 6060 ccagggcaca uaucggccgc uacggcgcuu cuuccacgac auccugcaca ccaaguaccg 6120 6180 cauuuuuggc uucugggccu uugggaagca cucggcggcc acagacauca cguccucccu 6240 6300 ggugguugac cgcgcccucu accugcgcaa gaccgugcug ggcaagcugg ccuuccaggu 6360 ggcgcuggug cuggccaucc accuauggau guuucuuc cugcccgccg ucacugagag 6420 gauguucaac cagaaugugg uggcccagcu cugguacuuc gugaagugca ucuacuucgc 6480 ccuguccgcc uaccagaucc gguccggcua ccccacccgc auccucggca acuucccac 6540 caagaaguac aaucaucuca accucuuccu cuuccagggg uuccggcugg ugccguuccu 6600 gguggagcug cggcaguga uggacugggu guggacggac accacgcu cccuguccag 6660 cuggaugugu guggaggaca ucuaugccaa caucuucauc aucaaaugca gccgagagac 6720 agaagaaa uacccgcagc ccaaagggca gaagaagaag aagaucguca aguacggcau 6780 ggguggccuc aucauccucu uccucaucgc caucaucugg uucccagc ucuucauguc 6840 gcuggugcgc uccgugguug ggguugucaa ccagcccauc gaugucaccg ucaccugaa 6900 gcugggcggc uaugagccgc uguucaccau gagcgcccag cagccgucca ucauccccuu 6960 cacggcccag gccuaugagg agcugucccg gcaguuugac ccccagccgc uggccaugca 7020 7080 7140 cacggccgac aucaccguc gcuucccug gaacuuccag agggaccugg cgaagggagg 7200 cacuguggag uaugccaacg agaagcau gcuggcccug gcccccaaca gcacugcacg 7260 gcggcagcug gccagccugc ucgagggcac cucggaccag ucugugguc ucccuaaucu 7320 cuccccaag uacauccgug cccccaacgg gcccgaagcc accccuguga agcagcugca 7380 gcccaugag gaggccgacu accucggcgu gcguauccag cugcggaggg agcaggggc 7440 gggggccacc ggcuuccucg aauggggu caucgagcug caggagugcc ggaccgacug 7500 7560 ggcuggcuac ggcaucaugg ggcuguacgu guccaucgug cuggucaucg gcaaguucgu 7620 gcgcggauuc ucagcgaga ucucgcacuc cauauguc gaggagcugc cgugcgugga 7680 ccgcauccuc agcucugcc aggacucuu ccuggugcgg gagacucggg agcuggagcu 7740 ggaggagg uuguacgcca agcucaucuu ccucuaccgc ucaccggaga ccaucaucauca 7800 guggacucgu gagaggagu aggagcugcu gcuggcgccc gagagggag gagccggccu 7860 gcugggcagc guggccacaa ggggcggcac uccucaggcc gggggagcca cugccccguc 7920 caaggccgcc agcugugaug cauccucccg gccugccuga gcccugaugc ugcugucaga 7980 gaaggacacu gcguccccac ggccugcgug gcgcugccgu cccccacgug uacuguagag 8040 uuuuuuuuuu aauuaaaaaa uguuuuauuu auacaaaugg acaaucaga 8089 <210> 3 <211> 8089 <212> DNA <213> Homo sapiens <400> 3 gggagccgcc gtccggccca gctcggcccc agtgagccga gcgctgcgct ccgccgaggg 60 gcagggcggt cgcctgagcg agcgcgggcc cgggacgtcg gcaccggcgg ggcggccgaa 120 ggagaaggag gaagaggaga aggcggcgcg cgggtccccg cgggtcagcc atggcgcgcc 180 ggccccgggg cccccgcacc gccccatagc gccgcggcgt ccgctcggtc tgggccgggc 240 cctgggccct ccagccatgg agccgcacgt gctcggcgcg gtcctgtact ggctgctgct 300 gccctgcgcg ctgctggctg cctgcctgct ccgcttcagc ggactctcgc tggtctacct 360 gctcttcctg ctgctgctgc cctggttccc cggccccacc cgatgcggcc tccaaggtca 420 cacaggccgc ctcctgcggg cattgctggg cctcagcctg ctcttcctgg tggcccatct 480 cgccctccag atctgcctgc atattgtgcc ccgcctggac cagctcctgg gacccagctg 540 cagccgctgg gagaccctct cgcgacacat aggggtcaca aggctggacc tgaaggacat 600 ccccaacgcc atccggctgg tggcccctga cctgggcatc ttggtggtct cctctgtctg 660 cctcggcatc tgcgggcgcc ttgcaaggaa cacccggcag agcccacatc cacgggagct 720 ggatgatgat gagagggatg tggatgccag cccgacggca gggctgcagg aagcagcaac 780 gctggcccct acacggaggt cacggctggc cgctcgtttc cgagtcacgg cccactggct 840 gctggtggcg gctgggcggg tcctggccgt aacactgctt gcactggcag gcatcgccca 900 cccctcggcc ctctccagtg tctacctgct gctcttcctg gccctctgca cctggtgggc 960 ctgccacttt cccatcagca ctcggggctt cagcagactc tgcgtcgcgg tggggtgctt 1020 cggcgccggc catctcatct gcctctactg ctaccagatg cccttggcac aggctctgct 1080 cccgcctgcc ggcatctggg ctagggtgct gggtctcaag gacttcgtgg gtcccaccaa 1140 ctgctccagc ccccacgcgc tggtcctcaa caccggcctg gactggcctg tgtatgccag 1200 ccccggcgtc ctcctgctgc tgtgctacgc cacggcctct ctgcgcaagc tccgcgcgta 1260 ccgcccctcc ggccagagga aggaggcggc aaaggggtat gaggctcggg agctggagct 1320 agcagagctg gaccagtggc cccaggaacg ggagtctgac cagcacgtgg tgcccacagc 1380 acccgacacc gaggctgata actgcatcgt gcacgagctg accggccaga gctccgtcct 1440 gcggcggcct gtgcggccca agcgggctga gcccagggag gcgtctccgc tccacagcct 1500 gggccacctc atcatggacc agagctatgt gtgcgcgctc attgccatga tggtatggag 1560 catcacctac cacagctggc tgaccttcgt actgctgctc tgggcctgcc tcatctggac 1620 ggtgcgcagc cgccaccaac tggccatgct gtgctcgccc tgcatcctgc tgtatgggat 1680 gacgctgtgc tgcctacgct acgtgtgggc catggacctg cgccctgagc tgcccaccac 1740 cctgggcccc gtcagcctgc gccagctggg gctggagcac acccgctacc cctgtctgga 1800 ccttggtgcc atgttgctct acaccctgac cttctggctc ctgctgcgcc agtttgtgaa 1860 agagaagctg ctgaagtggg cagagtctcc agctgcgctg acggaggtca ccgtggcaga 1920 cacagagccc acgcggacgc agacgctgtt gcagagcctg ggggagctgg tgaagggcgt 1980 gtacgccaag tactggatct atgtgtgtgc tggcatgttc atcgtggtca gcttcgccgg 2040 ccgcctcgtg gtctacaaga ttgtctacat gttcctcttc ctgctctgcc tcaccctctt 2100 ccaggtctac tacagcctgt ggcggaagct gctcaaggcc ttctggtggc tcgtggtggc 2160 ctacaccatg ctggtcctca tcgccgtcta caccttccag ttccaggact tccctgccta 2220 ctggcgcaac ctcactggct tcaccgacga gcagctgggg gacctgggcc tggagcagtt 2280 cagcgtgtcc gagctcttct ccagcatcct ggtgcccggc ttcttcctcc tggcctgcat 2340 cctgcagctg cactacttcc acaggccctt catgcagctc accgacatgg agcacgtgtc 2400 cctgcctggc acgcgcctcc cgcgctgggc tcacaggcag gatgcagtga gtgggacccc 2460 actgctgcgg gaggagcagc aggagcatca gcagcagcag caggaggagg aggaggagga 2520 ggaggactcc agggacgagg ggctgggcgt ggccactccc caccaggcca cgcaggtgcc 2580 tgaaggggca gccaagtggg gcctggtggc tgagcggctg ctggagctgg cagccggctt 2640 ctcggacgtc ctctcacgcg tgcaggtgtt cctgcggcgg ctgctggagc ttcacgtttt 2700 caagctggtg gccctgtaca ccgtctgggt ggccctgaag gaggtgtcgg tgatgaacct 2760 gctgctggtg gtgctgtggg ccttcgccct gccctaccca cgcttccggc ccatggcctc 2820 ctgcctgtcc accgtgtgga cctgcgtcat catcgtgtgt aagatgctgt accagctcaa 2880 ggttgtcaac ccccaggagt attccagcaa ctgcaccgag cccttcccca acagcaccaa 2940 cttgctgccc acggagatca gccagtccct gctgtaccgg gggcccgtgg accctgccaa 3000 ctggtttggg gtgcggaaag ggttccccaa cctgggctac atccagaacc acctgcaagt 3060 gctgctgctg ctggtattcg aggccatcgt gtaccggcgc caggagcact accgccggca 3120 gcaccagctg gccccgctgc ctgcccaggc cgtgtttgcc agcggcaccc gccagcagct 3180 ggaccaggat ctgctcggct gcctcaagta cttcatcaac ttcttcttct acaaattcgg 3240 gctggagatc tgcttcctga tggccgtgaa cgtgatcggg cagcgcatga actttctggt 3300 gaccctgcac ggttgctggc tggtggccat cctcacccgc aggcaccgcc aggccattgc 3360 ccgcctctgg cccaactact gcctcttcct ggcgctgttc ctgctgtacc agtacctgct 3420 gtgcctgggg atgcccccgg ccctgtgcat tgattatccc tggcgctgga gccgggccgt 3480 ccccatgaac tccgcactca tcaagtggct gtacctgcct gatttcttcc gggcccccaa 3540 ctccaccaac ctcatcagcg actttctcct gctgctgtgc gcctcccagc agtggcaggt 3600 gttctcagct gagcgcacag aggagtggca gcgcatggct ggcgtcaaca ccgaccgcct 3660 ggagccgctg cggggggagc ccaaccccgt gcccaacttt atccactgca ggtcctacct 3720 tgacatgctg aaggtggccg tcttccgata cctgttctgg ctggtgctgg tggtggtgtt 3780 tgtcacgggg gccacccgca tcagcatctt cgggctgggc tacctgctgg cctgcttcta 3840 cctgctgctc ttcggcacgg ccctgctgca gagggacaca cgggcccgcc tcgtgctgtg 3900 ggactgcctc attctgtaca acgtcaccgt catcatctcc aagaacatgc tgtcgctcct 3960 ggcctgcgtc ttcgtggagc agatgcagac cggcttctgc tgggtcatcc agctcttcag 4020 ccttgtatgc accgtcaagg gctactatga ccccaaggag atgatggaca gagaccagga 4080 ctgcctgctg cctgtggagg aggctggcat catctgggac agcgtctgct tcttcttcct 4140 gctgctgcag cgccgcgtct tccttagcca ttactacctg cacgtcaggg ccgacctcca 4200 ggccaccgcc ctgctagcct ccaggggctt cgccctctac aacgctgcca acctcaagag 4260 cattgacttt caccgcagga tagaggagaa gtccctggcc cagctgaaaa gacagatgga 4320 gcgtatccgt gccaagcagg agaagcacag gcagggccgg gtggaccgca gtcgccccca 4380 ggacaccctg ggccccaagg accccggcct ggagccaggg cccgacagtc cagggggctc 4440 ctccccgcca cggaggcagt ggtggcggcc ctggctggac cacgccacag tcatccactc 4500 cggggactac ttcctgtttg agtccgacag tgaggaagag gaggaggctg ttcctgaaga 4560 cccgaggccg tcggcacaga gtgccttcca gctggcgtac caggcatggg tgaccaacgc 4620 ccaggcggtg ctgaggcggc ggcagcagga gcaggagcag gcaaggcagg aacaggcagg 4680 acagctaccc acaggaggtg gtcccagcca ggaggtggag ccagcagagg gccccgagga 4740 ggcagcggca ggccggagcc atgtggtgca gagggtgctg agcacggcgc agttcctgtg 4800 gatgctgggg caggcgctag tggatgagct gacacgctgg ctgcaggagt tcacccggca 4860 ccacggcacc atgagcgacg tgctgcgggc agagcgctac ctcctcacac aggagctcct 4920 gcagggcggc gaagtgcaca ggggcgtgct ggatcagctg tacacaagcc aggccgaggc 5040. cacgctgcca ggccccaccg aggccccca tgccccaagc accgtgtcca gtgggctggg cgcggaggag ccactcagca gcatgacaga cgacatgggc agccccctga gcaccggcta ccacacgcgc agtggcagtg aggaggcagt caccgacccc ggggagcgtg aggctggtgc 5160 ctctctgtac cagggactga tgcggacggc cagcgagctg ctcctggaca ggcgcctgcg 5220 catcccagag ctggaggagg cagagctgtt tgcggagggg cagggccggg cgctgcggct 5280 gctgcgggcc gtgtaccagt gtgtggccgc ccactcggag ctgctctgct acttcatcat 5340 catcctcaac cacatggtca cggcctccgc cggctcgctg gtgctgcccg tgctcgtctt 5400 cctgtgggcc atgctgtcga tcccgaggcc cagcaagcgc ttctggatga cggccatcgt 5460 cttcaccgag atcgcggtgg tcgtcaagta cctgttccag tttgggttct tcccctggaa 5520. cagccacgtg gtgctgcggc gctacgagaa caagccctac ttcccgcccc gcatcctggg 5580. cctggagaag actgacggct acatcaagta cgacctggtg cagctcatgg cccttttctt ccaccgctcc cagctgctgt gctatggcct ctggggaccat gaggaggact caccatccaa ggagcatgac aagagcggcg aggagca gggagccgag gaggggccag gggtgcctgc 5760 ggccaccacc gaagaccaca ttcaggtgga agccagggtc ggacccacgg acgggacccc agaaccccaa gtggagctca ggccccgtga tacgaggcgc atcagtctac gttttagaag aaggaagaag gagggcccag cacggaaagg agcggcagcc atcgaagctg aggacaggga ggggagg ggggaggagg ccccacgggg ggccaagccg ctctggagga agagtaaggg cggccggggcg gcggctgcag ggcttctgcc tgtccctggc 6060 ccagggcaca tatcggccgc tacggcgctt cttccacgac atcctgcaca ccaagtaccg cgcagccacc gacgtctatg ccctcatgtt cctggctgat gttgtcgact tcatcatcat 6180 catttttggc ttctgggcct ttggggagca ctcggcggcc acagacatca cgtcctccct 6240 atcagacgac caggtacccg aggctttcct ggtcatgctg ctgatccagt tcagtacat 6300 ggtggttgac cgcgccctct acctgcgcaa gaccgtgctg ggcaagctgg ccttccaggt 6360 ggcgctggtg ctggccatcc acctatggat gttcttcatc ctgcccgccg tcactgagag 6420 gatgttcaac cagaatgtgg tggcccagct ctggtacttc gtgaagtgca tctacttcgc 6480 cctgtccgcc taccagatcc gctgcggcta ccccacccgc atcctcggca acttctccac 6540 aagagtac aatcatctca acctcttcct cttccagggg ttccggctgg tgccgttcct 6600 ggtggagctg cgggcagtga tggactgggt gtggacggac accacgctgt ccctgtccag 6660 ctggatgtgt gtggagaca tctatgccaa catcttcatc atcaaatgca gccgagagac 6720 agagaagaaa tacccgcagc ccaaagggca gaagagaag aagatcgtca agtacggcat 6780 gggtggcctc atcatcctct tcctcatcgc catcatctgg ttcccactgc tcttcatgtc 6840 gctggtgcgc tccgtggttg gggttgtcaa ccagcccatc gatgtcaccg tcaccctgaa 6900 gctggggcggc tatgagccgc tgttcaccat gagcgcccag cagccgtcca tcatcccctt 6960 cacggcccag gcctatgagg agctgtcccg gcagtttgac ccccagccgc tggccatgca 7020 gttcatcagc cagtacagcc ctgaggacat cgtcacggcg cagattgagg gcagctccgg 7080 ggcgctgtgg cgcatcagtc cccccagccg tgcccagatg aagcgggagc tctacaacgg 7140 cacggccgac atcaccctgc gcttcacctg gaacttccag agggacctgg cgaagggagg 7200 cactgtggag tatgccaacg agaagcacat gctggccctg gcccccaaca gcactgcacg 7260 gcggcagctg gccagcctgc tcgagggcac ctcggaccag tctgtggtca tccctaatct 7320 cttccccaag tacatccgtg cccccaacgg gcccgaagcc aaccctgtga agcagctgca 7380 gcccaatgag gaggccgact acctcggcgt gcgtatccag ctgcggaggg agcagggtgc 7440 gggggccacc ggcttcctcg aatggtgggt catcgagctg caggagtgcc ggaccgactg 7500 caacctgctg cccatggtca ttttcagtga caaggtcagc ccaccgagcc tcggcttcct 7560 ggctggctac ggcatcatgg ggctgtacgt gtccatcgtg ctggtcatcg gcaagttcgt 7620 gcgcggattc ttcagcgaga tctcgcactc cattatgttc gaggagctgc cgtgcgtgga 7680 ccgcatcctc aagctctgcc aggacatctt cctggtgcgg gagactcggg agctggagct 7740 ggaggaggag ttgtacgcca agctcatctt cctctaccgc tcaccggaga ccatgatcaa 7800 gtggactcgt gagaaggagt aggagctgct gctggcgccc gagagggaag gagccggcct 7860 gctgggcagc gtggccacaa ggggcggcac tcctcaggcc gggggagcca ctgccccgtc 7920 caaggccgcc agctgtgatg catcctcccg gcctgcctga gccctgatgc tgctgtcaga 7980 gaaggacact gcgtccccac ggcctgcgtg gcgctgccgt cccccacgtg tactgtagag 8040 tttttttttt aattaaaaaa tgttttattt atacaaatgg acaatcaga 8089 <210> 4 <211> 2521 <212> PRT <213> Homo sapiens <400> 4 Met Glu Pro His Val Leu Gly Ala Val Leu Tyr Trp Leu Leu Leu Pro 1 5 10 15 Cys Ala Leu Leu Ala Ala Cys Leu Leu Arg Phe Ser Gly Leu Ser Leu 20 25 30 Val Tyr Leu Leu Phe Leu Leu Leu Leu Pro Trp Phe Pro Gly Pro Thr 35 40 45 Arg Cys Gly Leu Gln Gly His Thr Gly Arg Leu Leu Arg Ala Leu Leu 50 55 60 Gly Leu Ser Leu Leu Phe Leu Val Ala His Leu Ala Leu Gln Ile Cys 65 70 75 80 Leu His Ile Val Pro Arg Leu Asp Gln Leu Leu Gly Pro Ser Cys Ser 85 90 95 Arg Trp Glu Thr Leu Ser Arg His Ile Gly Val Thr Arg Leu Asp Leu 100 105 110 Lys Asp Ile Pro Asn Ala Ile Arg Leu Val Ala Pro Asp Leu Gly Ile 115 120 125 Leu Val Val Ser Ser Val Cys Leu Gly Ile Cys Gly Arg Leu Ala Arg 130 135 140 Asn Thr Arg Gln Ser Pro His Pro Arg Glu Leu Asp Asp Asp Glu Arg 145 150 155 160 Asp Val Asp Ala Ser Pro Thr Ala Gly Leu Gln Glu Ala Ala Thr Leu 165 170 175 Ala Pro Thr Arg Arg Ser Arg Leu Ala Ala Arg Phe Arg Val Thr Ala 180 185 190 His Trp Leu Leu Val Ala Ala Gly Arg Val Leu Ala Val Thr Leu Leu 195 200 205 Ala Leu Ala Gly Ile Ala His Pro Ser Ala Leu Ser Ser Val Tyr Leu 210 215 220 Leu Leu Phe Leu Ala Leu Cys Thr Trp Trp Ala Cys His Phe Pro Ile 225 230 235 240 Ser Thr Arg Gly Phe Ser Arg Leu Cys Val Ala Val Gly Cys Phe Gly 245 250 255 Ala Gly His Leu Ile Cys Leu Tyr Cys Tyr Gln Met Pro Leu Ala Gln 260 265 270 Ala Leu Leu Pro Pro Ala Gly Ile Trp Ala Arg Val Leu Gly Leu Lys 275 280 285 Asp Phe Val Gly Pro Thr Asn Cys Ser Ser Pro His Ala Leu Val Leu 290 295 300 Asn Thr Gly Leu Asp Trp Pro Val Tyr Ala Ser Pro Gly Val Leu Leu 305 310 315 320 Leu Leu Cys Tyr Ala Thr Ala Ser Leu Arg Lys Leu Arg Ala Tyr Arg 325 330 335 Pro Ser Gly Gln Arg Lys Glu Ala Ala Lys Gly Tyr Glu Ala Arg Glu 340 345 350 Leu Glu Leu Ala Glu Leu Asp Gln Trp Pro Gln Glu Arg Glu Ser Asp 355 360 365 Gln His Val Val Pro Thr Ala Pro Asp Thr Glu Ala Asp Asn Cys Ile 370 375 380 Val His Glu Leu Thr Gly Gln Ser Ser Val Leu Arg Arg Pro Val Arg 385 390 395 400 Pro Lys Arg Ala Glu Pro Arg Glu Ala Ser Pro Leu His Ser Leu Gly 405 410 415 His Leu Ile Met Asp Gln Ser Tyr Val Cys Ala Leu Ile Ala Met Met 420 425 430 Val Trp Ser Ile Thr Tyr His Ser Trp Leu Thr Phe Val Leu Leu Leu 435 440 445 Trp Ala Cys Leu Ile Trp Thr Val Arg Ser Arg His Gln Leu Ala Met 450 455 460 Leu Cys Ser Pro Cys Ile Leu Leu Tyr Gly Met Thr Leu Cys Cys Leu 465 470 475 480 Arg Tyr Val Trp Ala Met Asp Leu Arg Pro Glu Leu Pro Thr Thr Leu 485 490 495 Gly Pro Val Ser Leu Arg Gln Leu Gly Leu Glu His Thr Arg Tyr Pro 500 505 510 Cys Leu Asp Leu Gly Ala Met Leu Leu Tyr Thr Leu Thr Phe Trp Leu 515 520 525 Leu Leu Arg Gln Phe Val Lys Glu Lys Leu Leu Lys Trp Ala Glu Ser 530 535 540 Pro Ala Ala Leu Thr Glu Val Thr Val Ala Asp Thr Glu Pro Thr Rec 545 550 555 560 Thr Gln Thr Leu Leu Gln Ser Leu Gly Glu Leu Val Lys Gly Val Tyr 565 570 575 Ala Lys Tyr Trp Ile Tyr Val Cys Ala Gly Met Phe Ile Val Val Ser 580 585 590 Phe Ala Gly Arg Leu Val Val Tyr Lys Ile Val Tyr Met Phe Leu Phe 595 600 605 Leu Leu Cys Leu Thr Leu Phe Gln Val Tyr Tyr Ser Leu Trp Arg Lys 610 615 620 Leu Leu Lys Ala Phe Trp Trp Leu Val Val Ala Tyr Thr Met Leu Val 625 630 635 640 Leu Ile Ala Val Tyr Thr Phe Gln Phe Gln Asp Phe Pro Ala Tyr Trp 645 650 655 Arg Asn Leu Thr Gly Phe Thr Asp Glu Gln Leu Gly Asp Leu Gly Leu 660 665 670 Glu Gln Phe Ser Val Ser Glu Leu Phe Ser Ser Ile Leu Val Pro Gly 675 680 685 Phe Phe Leu Leu Ala Cys Ile Leu Gln Leu His Tyr Phe His Arg Pro 690 695 700 Phe Met Gln Leu Thr Asp Met Glu His Val Ser Leu Pro Gly Thr Arg 705 710 715 720 Leu Pro Arg Trp Ala His Arg Gln Asp Ala Val Ser Gly Thr Pro Leu 725 730 735 Leu Arg Glu Glu Gln Gln Glu His Gln Gln Gln Gln Gln Glu Glu Glu 740 745 750 Glu Glu Glu Glu Asp Ser Arg Asp Glu Gly Leu Gly Val Ala Thr Pro 755 760 765 His Gln Ala Thr Gln Val Pro Glu Gly Ala Ala Lys Trp Gly Leu Val 770 775 780 Ala Glu Arg Leu Leu Glu Leu Ala Ala Gly Phe Ser Asp Val Leu Ser 785 790 795 800 Arg Val Gln Val Phe Leu Arg Arg Leu Leu Glu Leu His Val Phe Lys 805 810 815 Leu Val Ala Leu Tyr Thr Val Trp Val Ala Leu Lys Glu Val Ser Val 820 825 830 Met Asn Leu Leu Leu Val Val Leu Trp Ala Phe Ala Leu Pro Tyr Pro 835 840 845 Arg Phe Arg Pro Met Ala Ser Cys Leu Ser Thr Val Trp Thr Cys Val 850 855 860 Ile Ile Val Cys Lys Met Leu Tyr Gln Leu Lys Val Val Asn Pro Gln 865 870 875 880 Glu Tyr Ser Ser Asn Cys Thr Glu Pro Phe Pro Asn Ser Thr Asn Leu 885 890 895 Leu Pro Thr Glu Ile Ser Gln Ser Leu Leu Tyr Arg Gly Pro Val Asp 900 905 910 Pro Ala Asn Trp Phe Gly Val Arg Lys Gly Phe Pro Asn Leu Gly Tyr 915 920 925 Ile Gln Asn His Leu Gln Val Leu Leu Leu Leu Val Phe Glu Ala Ile 930 935 940 Val Tyr Arg Arg Gln Glu His Tyr Arg Arg Gln His Gln Leu Ala Pro 945 950 955 960 Leu Pro Ala Gln Ala Val Phe Ala Ser Gly Thr Arg Gln Gln Leu Asp 965 970 975 Gln Asp Leu Leu Gly Cys Leu Lys Tyr Phe Ile Asn Phe Phe Phe Tyr 980 985 990 Lys Phe Gly Leu Glu Ile Cys Phe Leu Met Ala Val Asn Val Ile Gly 995 1000 1005 Gln Arg Met Asn Phe Leu Val Thr Leu His Gly Cys Trp Leu Val 1010 1015 1020 Ala Ile Leu Thr Arg Arg His Arg Gln Ala Ile Ala Arg Leu Trp 1025 1030 1035 Pro Asn Tyr Cys Leu Phe Leu Ala Leu Phe Leu Leu Tyr Gln Tyr 1040 1045 1050 Leu Leu Cys Leu Gly Met Pro Pro Ala Leu Cys Ile Asp Tyr Pro 1055 1060 1065 Trp Arg Trp Ser Arg Ala Val Pro Met Asn Ser Ala Leu Ile Lys 1070 1075 1080 Trp Leu Tyr Leu Pro Asp Phe Phe Arg Ala Pro Asn Ser Thr Asn 1085 1090 1095 Leu Ile Ser Asp Phe Leu Leu Leu Leu Cys Ala Ser Gln Gln Trp 1100 1105 1110 Gln Val Phe Ser Ala Glu Arg Thr Glu Glu Trp Gln Arg Met Ala 1115 1120 1125 Gly Val Asn Thr Asp Arg Leu Glu Pro Leu Arg Gly Glu Pro Asn 1130 1135 1140 Pro Val Pro Asn Phe Ile His Cys Arg Ser Tyr Leu Asp Met Leu 1145 1150 1155 Lys Val Ala Val Phe Arg Tyr Leu Phe Trp Leu Val Leu Val Val 1160 1165 1170 Val Phe Val Thr Gly Ala Thr Arg Ile Ser Ile Phe Gly Leu Gly 1175 1180 1185 Tyr Leu Leu Ala Cys Phe Tyr Leu Leu Leu Phe Gly Thr Ala Leu 1190 1195 1200 Leu Gln Arg Asp Thr Arg Ala Arg Leu Val Leu Trp Asp Cys Leu 1205 1210 1215 Ile Leu Tyr Asn Val Thr Val Ile Ile Ser Lys Asn Met Leu Ser 1220 1225 1230 Leu Leu Ala Cys Val Phe Val Glu Gln Met Gln Thr Gly Phe Cys 1235 1240 1245 Trp Val Ile Gln Leu Phe Ser Leu Val Cys Thr Val Lys Gly Tyr 1250 1255 1260 Tyr Asp Pro Lys Glu Met Met Asp Arg Asp Gln Asp Cys Leu Leu 1265 1270 1275 Pro Val Glu Glu Ala Gly Ile Ile Trp Asp Ser Val Cys Phe Phe 1280 1285 1290 Phe Leu Leu Leu Gln Arg Arg Val Phe Leu Ser His Tyr Tyr Leu 1295 1300 1305 His Val Arg Ala Asp Leu Gln Ala Thr Ala Leu Leu Ala Ser Arg 1310 1315 1320 Gly Phe Ala Leu Tyr Asn Ala Ala Asn Leu Lys Ser Ile Asp Phe 1325 1330 1335 His Arg Arg Ile Glu Glu Lys Ser Leu Ala Gln Leu Lys Arg Gln 1340 1345 1350 Met Glu Arg Ile Arg Ala Lys Gln Glu Lys His Arg Gln Gly Arg 1355 1360 1365 Val Asp Arg Ser Arg Pro Gln Asp Thr Leu Gly Pro Lys Asp Pro 1370 1375 1380 Gly Leu Glu Pro Gly Pro Asp Ser Pro Gly Gly Ser Ser Pro Pro 1385 1390 1395 Arg Arg Gln Trp Trp Arg Pro Trp Leu Asp His Ala Thr Val Ile 1400 1405 1410 His Ser Gly Asp Tyr Phe Leu Phe Glu Ser Asp Ser Glu Glu Glu 1415 1420 1425 Glu Glu Ala Val Pro Glu Asp Pro Arg Pro Ser Ala Gln Ser Ala 1430 1435 1440 Phe Gln Leu Ala Tyr Gln Ala Trp Val Thr Asn Ala Gln Ala Val 1445 1450 1455 Leu Arg Arg Arg Gln Gln Glu Gln Glu Gln Ala Arg Gln Glu Gln 1460 1465 1470 Ala Gly Gln Leu Pro Thr Gly Gly Gly Pro Ser Gln Glu Val Glu 1475 1480 1485 Pro Ala Glu Gly Pro Glu Glu Ala Ala Ala Gly Arg Ser His Val 1490 1495 1500 Val Gln Arg Val Leu Ser Thr Ala Gln Phe Leu Trp Met Leu Gly 1505 1510 1515 Gln Ala Leu Val Asp Glu Leu Thr Arg Trp Leu Gln Glu Phe Thr 1520 1525 1530 Arg His His Gly Thr Met Ser Asp Val Leu Arg Ala Glu Arg Tyr 1535 1540 1545 Leu Leu Thr Gln Glu Leu Leu Gln Gly Gly Glu Val His Arg Gly 1550 1555 1560 Val Leu Asp Gln Leu Tyr Thr Ser Gln Ala Glu Ala Thr Leu Pro 1565 1570 1575 Gly Pro Thr Glu Ala Pro Asn Ala Pro Ser Thr Val Ser Ser Gly 1580 1585 1590 Leu Gly Ala Glu Glu Pro Leu Ser Ser Met Thr Asp Asp Met Gly 1595 1600 1605 Ser Pro Leu Ser Thr Gly Tyr His Thr Arg Ser Gly Ser Glu Glu 1610 1615 1620 Ala Val Thr Asp Pro Gly Glu Arg Glu Ala Gly Ala Ser Leu Tyr 1625 1630 1635 Gln Gly Leu Met Arg Thr Ala Ser Glu Leu Leu Leu Asp Arg Arg 1640 1645 1650 Leu Arg Ile Pro Glu Leu Glu Glu Ala Glu Leu Phe Ala Glu Gly 1655 1660 1665 Gln Gly Arg Ala Leu Arg Leu Leu Arg Ala Val Tyr Gln Cys Val 1670 1675 1680 Ala Ala His Ser Glu Leu Leu Cys Tyr Phe Ile Ile Ile Leu Asn 1685 1690 1695 His Met Val Thr Ala Ser Ala Gly Ser Leu Val Leu Pro Val Leu 1700 1705 1710 Val Phe Leu Trp Ala Met Leu Ser Ile Pro Arg Pro Ser Lys Arg 1715 1720 1725 Phe Trp Met Thr Ala Ile Val Phe Thr Glu Ile Ala Val Val Val 1730 1735 1740 Lys Tyr Leu Phe Gln Phe Gly Phe Phe Pro Trp Asn Ser His Val 1745 1750 1755 Val Leu Arg Arg Tyr Glu Asn Lys Pro Tyr Phe Pro Pro Arg Ile 1760 1765 1770 Leu Gly Leu Glu Lys Thr Asp Gly Tyr Ile Lys Tyr Asp Leu Val 1775 1780 1785 Gln Leu Met Ala Leu Phe Phe His Arg Ser Gln Leu Leu Cys Tyr 1790 1795 1800 Gly Leu Trp Asp His Glu Glu Asp Ser Pro Ser Lys Glu His Asp 1805 1810 1815 Lys Ser Gly Glu Glu Glu Gln Gly Ala Glu Glu Gly Pro Gly Val 1820 1825 1830 Pro Ala Ala Thr Thr Glu Asp His Ile Gln Val Glu Ala Arg Val 1835 1840 1845 Gly Pro Thr Asp Gly Thr Pro Glu Pro Gln Val Glu Leu Arg Pro 1850 1855 1860 Arg Asp Thr Arg Arg Ile Ser Leu Arg Phe Arg Arg Arg Lys Lys 1865 1870 1875 Glu Gly Pro Ala Arg Lys Gly Ala Ala Ala Ile Glu Ala Glu Asp 1880 1885 1890 Arg Glu Glu Glu Glu Gly Glu Glu Glu Lys Glu Ala Pro Thr Gly 1895 1900 1905 Arg Glu Lys Arg Pro Ser Arg Ser Gly Gly Arg Val Arg Ala Ala 1910 1915 1920 Gly Arg Arg Leu Gln Gly Phe Cys Leu Ser Leu Ala Gln Gly Thr 1925 1930 1935 Tyr Arg Pro Leu Arg Arg Phe Phe His Asp Ile Leu His Thr Lys 1940 1945 1950 Tyr Arg Ala Ala Thr Asp Val Tyr Ala Leu Met Phe Leu Ala Asp 1955 1960 1965 Val Val Asp Phe Ile Ile Ile Ile Phe Gly Phe Trp Ala Phe Gly 1970 1975 1980 Lys His Ser Ala Ala Thr Asp Ile Thr Ser Ser Leu Ser Asp Asp 1985 1990 1995 Gln Val Pro Glu Ala Phe Leu Val Met Leu Leu Ile Gln Phe Ser 2000 2005 2010 Thr Met Val Val Asp Arg Ala Leu Tyr Leu Arg Lys Thr Val Leu 2015 2020 2025 Gly Lys Leu Ala Phe Gln Val Ala Leu Val Leu Ala Ile His Leu 2030 2035 2040 Trp Met Phe Phe Ile Leu Pro Ala Val Thr Glu Arg Met Phe Asn 2045 2050 2055 Gln Asn Val Val Ala Gln Leu Trp Tyr Phe Val Lys Ile Tyr 2060 2065 2070 Phe Ala Leu Ser Ala Tyr Gln Ile Arg Cys Gly Tyr Pro Thr Arg 2075 2080 2085 Ile Leu Gly Asn Phe Leu Thr Lys Lys Tyr Asn His Leu Asn Leu 2090 2095 2100 Phe Leu Phe Gln Gly Phe Arg Leu Val Pro Phe Leu Val Glu Leu 2105 2110 2115 Arg Ala Val Met Asp Trp Val Trp Thr Asp Thr Thr Leu Ser Leu 2120 2125 2130 Ser Ser Trp Met Cys Val Glu Asp Ile Tyr Ala Asn Ile Phe Ile 2135 2140 2145 Ile Lys Ser Arg Glu Thr Glu Lys Tyr Pro Gln Pro Lys 2150 2155 2160 Gly Gln Lys Lys Lys Ile Val Lys Tyr Gly Met Gly Gly Leu 2165 2170 2175 Where You Leu Where You Where You Trp Where Pro Leu Leu Where 2180 2185 2190 Met Ser Leu Val Arg Ser Val Val Gly Val Val Asn Gln Pro Ile 2195 2200 2205 Asp Val Thr Val Thr Leu Lys Leu Gly Gly Tyr Glu Pro Leu Phe 2210 2215 2220 Thr Met Ser Ala Gln Gln Pro Ser Ile Ile Pro Phe Thr Ala Gln 2225 2230 2235 Ala Tyr Glu Glu Leu Ser Arg Gln Phe Asp Pro Gln Pro Leu Ala 2240 2245 2250 Met Gln Phe Ile Ser Gln Tyr Ser Pro Glu Asp Ile Val Thr Ala 2255 2260 2265 Gln Ile Glu Gly Ser Ser Gly Ala Leu Trp Arg Ile Ser Pro Pro 2270 2275 2280 Ser Arg Ala Gln Met Lys Arg Glu Leu Tyr Asn Gly Thr Ala Asp 2285 2290 2295 Ile Thr Leu Arg Phe Thr Trp Asn Phe Gln Arg Asp Leu Ala Lys 2300 2305 2310 Gly Gly Thr Val Glu Tyr Ala Asn Glu Lys His Met Leu Ala Leu 2315 2320 2325 Ala Pro Asn Ser Thr Ala Arg Arg Gln Leu Ala Ser Leu Leu Glu 2330 2335 2340 Gly Thr Ser Asp Gln Ser Val Val Ile Pro Asn Leu Phe Pro Lys 2345 2350 2355 Tyr Ile Arg Ala Pro Asn Gly Pro Glu Ala Asn Pro Val Lys Gln 2360 2365 2370 Leu Gln Pro Asn Glu Glu Ala Asp Tyr Leu Gly Val Arg Ile Gln 2375 2380 2385 Leu Arg Arg Glu Gln Gly Ala Gly Ala Thr Gly Phe Leu Glu Trp 2390 2395 2400 Trp Val Ile Glu Leu Gln Glu Cys Arg Thr Asp Cys Asn Leu Leu 2405 2410 2415 Pro Met Val Ile Phe Ser Asp Lys Val Ser Pro Pro Ser Leu Gly 2420 2425 2430 Phe Leu Ala Gly Tyr Gly Ile Met Gly Leu Tyr Val Ser Ile Val 2435 2440 2445 Leu Val Ile Gly Lys Phe Val Arg Gly Phe Phe Ser Glu Ile Ser 2450 2455 2460 His Ser Ile Met Phe Glu Glu Leu Pro Cys Val Asp Arg Ile Leu 2465 2470 2475 Lys Leu Cys Gln Asp Ile Phe Leu Val Arg Glu Thr Arg Glu Leu 2480 2485 2490 Glu Leu Glu Glu Glu Leu Tyr Ala Lys Leu Ile Phe Leu Tyr Arg 2495 2500 2505 Ser Pro Glu Thr Met Ile Lys Trp Thr Arg Glu Lys Glu 2510 2515 2520

Claims

1. Use of an agent for detecting a predicted loss-of-function variant of PIEZO1 in the preparation of a kit for identifying a human subject having an increased risk of developing varicose veins, wherein the kit is for use in a method comprising: determining or having determined the presence or absence in a biological sample obtained from the subject: Piezoelectric mechanosensitive ion channel component 1 (PIEZO1) predicted loss-of-function variant genomic nucleic acid molecule; a predicted loss-of-function variant mRNA molecule of PIEZO1 produced from said genomic nucleic acid molecule; a predicted loss-of-function variant cDNA molecule of PIEZO1 produced from said mRNA molecule; or a predicted loss-of-function variant polypeptide of PIEZO1 encoded by the genomic nucleic acid molecule; in, Using the SEQ ID NO: 1 reference genomic nucleotide sequence as a basis, the PIEZO1 predicted loss-of-function variant genomic nucleic acid molecules include 16:88716359:A:G, 16:88716874:G:A, 16:88719588:G:A, 16:88721268:D:1, 16:88721586:G:C, 16:88723253:G:A, 16:88726546:C:T, 16:88727072:D:1, 16:88727163:G:A, 16:88736324:G:A or 16:88738735:D:1, in: The absence of the PIEZO1 predicted loss-of-function variant genomic nucleic acid molecule, mRNA molecule, cDNA molecule, or polypeptide indicates that the subject does not have an increased risk of developing varicose veins; and The presence of the PIEZO1 predicted loss-of-function variant genomic nucleic acid molecule, mRNA molecule, cDNA molecule or polypeptide indicates that the subject has an increased risk of developing varicose veins.

2. Use of an agent for detecting a predicted loss-of-function variant of PIEZO1 in the preparation of a kit for diagnosing varicose veins in a human subject, wherein the kit is used in a method comprising: determining the presence or absence in a sample obtained from the subject: Piezoelectric mechanosensitive ion channel component 1 (PIEZO1) predicted loss-of-function variant genomic nucleic acid molecule; a predicted loss-of-function variant mRNA molecule of PIEZO1 produced from said genomic nucleic acid molecule; a predicted loss-of-function variant cDNA molecule of PIEZO1 produced from said mRNA molecule; or a predicted loss-of-function variant polypeptide of PIEZO1 encoded by the genomic nucleic acid molecule; Wherein when the subject has a PIEZO1 predicted loss-of-function variant genomic nucleic acid molecule, mRNA molecule, cDNA molecule or polypeptide, wherein, Using the SEQ ID NO:1 reference genomic nucleotide sequence as a basis, the PIEZO1 predicted loss-of-function variant genomic nucleic acid molecule includes 16:88716359:A:G, 16:88716874:G:A, 16:88719588:G:A, 16:88721268:D:1, 16:88721586:G:C, 16:88723253:G:A, 16:88726546:C:T, 16:88727072:D:1, 16:88727163:G:A, 16:88736324:G:A or 16:88738735:D:1, and the subject has one or more symptoms of varicose veins.

3. The use of claim 1 or claim 2, wherein the method further comprises treating the subject having varicose veins or having an increased risk of developing varicose veins with an agent effective for treating varicose veins.

4. The use according to claim 1 or 2, wherein the determining step, the detecting step or the genotyping assay is performed in vitro.

5. The use according to claim 1 or 2, wherein the determining step, detecting step or genotyping assay comprises sequencing at least a portion of the nucleotide sequence of the PIEZO1 nucleic acid molecule in the biological sample, wherein the sequenced portion comprises a position corresponding to a predicted loss-of-function variant position, wherein when a variant nucleotide at the predicted loss-of-function variant position is detected, the PIEZO1 nucleic acid molecule in the biological sample is a PIEZO1 predicted loss-of-function variant nucleic acid molecule.

6. The method according to claim 1 or 2, wherein the determining step, detecting step or genotyping assay comprises: a) contacting the biological sample with a primer that hybridizes to a portion of the nucleotide sequence of the PIEZO1 nucleic acid molecule adjacent to a predicted loss-of-function variant position; b) extending the primer at least through the predicted loss-of-function variant position; and c) determining whether the extension product of the primer comprises a variant nucleotide at the predicted loss-of-function variant position.

7. The use according to claim 5, wherein the determining step, the detecting step or the genotyping assay comprises sequencing the entire nucleic acid molecule.

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

9. Use according to claim 8, wherein the nucleic acid molecule in the sample is mRNA and the mRNA is reverse transcribed into cDNA before the amplification step.

10. The use according to claim 8, wherein the determining step, detecting step or genotyping assay comprises: contacting the nucleic acid molecules in the biological sample with an alteration-specific probe comprising a detectable label, wherein the alteration-specific probe comprises a nucleotide sequence that hybridizes to a predicted loss-of-function variant position under stringent conditions; and The detectable label is detected.

11. The use according to claim 6, wherein the determining step, the detecting step or the genotyping assay comprises sequencing the entire nucleic acid molecule.

12. The method of claim 9, wherein the determining step, detecting step or genotyping assay comprises: contacting the nucleic acid molecules in the biological sample with an alteration-specific probe comprising a detectable label, wherein the alteration-specific probe comprises a nucleotide sequence that hybridizes to a predicted loss-of-function variant position under stringent conditions; and The detectable label is detected.

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