CN115807080A

CN115807080A - Thyroid cancer diagnostic method

Info

Publication number: CN115807080A
Application number: CN202111078925.4A
Authority: CN
Inventors: 苏志熙; 刘轶颖; 徐敏杰; 刘琪; 刘蕊
Original assignee: Shanghai Fuyuan Biotechnology Co ltd; Jiangsu Fuyuan Biotechnology Co ltd
Current assignee: Shanghai Fuyuan Biotechnology Co ltd; Jiangsu Fuyuan Biotechnology Co ltd
Priority date: 2021-09-15
Filing date: 2021-09-15
Publication date: 2023-03-17

Abstract

The present invention relates to a method for diagnosing thyroid cancer. Specifically, the present invention provides the use of (a) an agent or device for determining the methylation level of a DNA sequence or fragment thereof or one or more CpG dinucleotides therein in a sample from a subject, (b) a treated nucleic acid molecule of said DNA sequence or fragment thereof, said treatment converting unmethylated cytosine to a base having a lower binding capacity for guanine than cytosine, in the manufacture of a kit for diagnosing thyroid cancer in a subject or identifying benign or malignant thyroid nodules in a subject. The method and the application have high specificity and sensitivity, and obviously reduce the target detection area for diagnosing the thyroid cancer.

Description

Thyroid cancer diagnostic method

Technical Field

The invention relates to the field of cancer diagnosis, in particular to a thyroid cancer diagnosis method.

Background

Thyroid cancer is the most common type of endocrine tumor, accounting for 90% of endocrine malignancies. In recent years, the incidence of thyroid cancer has been on the rise due to the widespread use of ultrasound and cytopathology screening techniques.

Ultrasound examination is highly suspected of malignant thyroid nodules and requires further fine needle aspiration cytology (FNA) examination for a definitive diagnosis. Unlike other cancers, malignant and benign nodules are difficult to diagnose in some differentiated thyroid cancers due to their approximate cytological characteristics, and up to 40% of thyroid nodules are difficult to diagnose accurately by cytological characteristics. This can lead to over-diagnosis of over-treatment, which can result in unnecessary thyroidectomy in some patients, be costly, and can present serious post-operative complications; patients who receive the total thyroidectomy must take levothyroxine tablets for a long time after the operation, so that the problems of cardiovascular diseases (atrial fibrillation and arrhythmia), osteoporosis and the like can be caused, and the health is seriously influenced.

Although current molecular diagnostic methods improve the accuracy of identification, the efficacy of these methods remains to be improved. The most widely used thyroid nodule molecular diagnostic technique is

Gene Expression Classifier, however, has a Positive Predictive Value (PPV) of only 47%, and only fresh, punctured tissue can be detected due to RNA degradation. Furthermore, SEQv2 in thyo is a gene mutation-based method for diagnosing thyroid cancer, which detects H/K/NRAS gene mutations and RET/PTC gene rearrangements that are frequently carried by benign nodules, but whose PPV is only 42% to 77%. Hair in 2019A paper on Clinical Cancer Research developed a Diagnostic method called DNA Methylation Signature approach (DDMS) for the identification of benign and malignant tissues of thyroid Cancer. Although the method is high in accuracy, the article mentions that some samples are not suitable for detection by the method for technical reasons.

Disclosure of Invention

<xnotran> , , (1) SEQ ID NO:1 , SEQ ID NO:2 , SEQ ID NO:3 , SEQ ID NO:4 , SEQ ID NO:6 , SEQ ID NO:7 , SEQ ID NO:8 , SEQ ID NO:9 , SEQ ID NO:10 , SEQ ID NO:11 , SEQ ID NO:12 , SEQ ID NO:13 , SEQ ID NO:14 , SEQ ID NO:15 , SEQ ID NO:16 , SEQ ID NO:17 , SEQ ID NO:18 , SEQ ID NO:19 , SEQ ID NO:20 , SEQ ID NO:21 , SEQ ID NO:22 , SEQ ID NO:23 , SEQ ID NO:24 , SEQ ID NO:25 , SEQ ID NO:26 , SEQ ID NO:27 , SEQ ID NO:28 , </xnotran> SEQ ID NO:29 or a complement or variant thereof, SEQ ID NO:30 or a complement or variant thereof, SEQ ID NO 32 or a complement or variant thereof, SEQ ID NO 33 or a complement or variant thereof, SEQ ID NO 34 or a complement or variant thereof, SEQ ID NO 35 or a complement or variant thereof, SEQ ID NO 36 or a complement or variant thereof, SEQ ID NO 37 or a complement or variant thereof, SEQ ID NO 39 or a complement or variant thereof, SEQ ID NO 40 or a complement or variant thereof, SEQ ID NO 41 or a complement or variant thereof, SEQ ID NO 42 or a complement or variant thereof, SEQ ID NO 43 or a complement or variant thereof, SEQ ID NO 44 or a complement or variant thereof, SEQ ID NO 45 or a complement or variant thereof, SEQ ID NO 46 or a complement or variant thereof, SEQ ID NO 47 or a complement or variant thereof, SEQ ID NO 48 or a complement or variant thereof, SEQ ID NO 49 or a complement or a variant thereof, SEQ ID NO 50 or a complement or variant thereof, SEQ ID NO 52 or a complement or variant thereof, SEQ ID NO 53 or a variant thereof, SEQ ID NO 52 or a complement or a variant thereof, SEQ ID NO 52 or a variant thereof, SEQ ID NO 54 or a variant thereof, SEQ ID NO 52 or a variant thereof, SEQ ID NO 55 or a variant thereof, SEQ ID NO: 5or a complementary sequence or variant thereof, 31 or a complementary sequence or variant thereof, 38 or a complementary sequence or variant thereof, said variant being a variant having at least 70% identity to the corresponding sequence and in which the methylation site is not mutated, and (2) a treated sequence of (1) which converts unmethylated cytosine to a base which has less ability to bind guanine than cytosine.

In one or more embodiments, the methylation sites are consecutive cpgs.

In one or more embodiments, the methylation marker can be any one or more CpG sites in the sequence region.

In one or more embodiments, the nucleic acid molecule is used as an internal standard or control for detecting the level of DNA methylation of the corresponding sequence in a sample.

In a second aspect, the present invention provides a reagent for detecting DNA methylation, the reagent comprising a reagent for detecting the methylation level of a DNA sequence or a fragment thereof or one or more CpG dinucleotides in a sample from a subject, the DNA sequence comprising the following gene sequences: ACSL5, ACTR3B, AIM, ASB2, C15orf62, C2CD4B, CCDC65, CCNB2, CD200, CD3G, CEBPD, CIITA, CSK, DLEU7, DYNLT3, ELF4, FABP3, HLA-E, HRH, IL12RB1, IL17C, ITGB2, KRTAP5-9, LAT2, LGALS1, LPIN1, OAS3, KR 3, and so forth PCYT1B, PITX, PTAFR, PTPN7, S100A10, SH3BP4, SIGLEC14, SIGLEC7, SLC29A3, SLC2A10, SLC5A5, SLFN13, SNURF, STAT6, STEAP4, SUMF1, TBC1D10C, TEK, THEMIS2, TIMP1, TMEM119, TNFRSF6B, TUBB, VAMP5, VAV1, 3534 zxft 3511B, optionally further comprises one or more of ATF7IP2, PLVAP, SIRPB2.

In one or more embodiments, the reagents are used to detect benign or malignant thyroid cancer.

In one or more embodiments, the DNA sequence further comprises the following gene sequences: (1) ATF7IP2, PLVAP, or (2) SIRPB2, or (3) ATF7IP2, PLVAP, and SIRPB2.

In one or more embodiments, the DNA sequence comprises at least 3 CpG dinucleotides.

In one or more embodiments, the DNA sequence comprises a sense strand or an antisense strand of DNA.

In one or more embodiments, the fragment is 1-1000bp in length, preferably 1-700bp in length.

In one or more embodiments, the fragment is a promoter region of a gene sequence.

In one or more embodiments, the fragment comprises at least 1, preferably at least 3 CpG dinucleotides.

In one or more embodiments, the DNA sequence comprises the following sequences: <xnotran> SEQ ID NO:1 , SEQ ID NO:2 , SEQ ID NO:3 , SEQ ID NO:4 , SEQ ID NO:6 , SEQ ID NO:7 , SEQ ID NO:8 , SEQ ID NO:9 , SEQ ID NO:10 , SEQ ID NO:11 , SEQ ID NO:12 , SEQ ID NO:13 , SEQ ID NO:14 , SEQ ID NO:15 , SEQ ID NO:16 , SEQ ID NO:17 , SEQ ID NO:18 , SEQ ID NO:19 , SEQ ID NO:20 , SEQ ID NO:21 , SEQ ID NO:22 , SEQ ID NO:23 , SEQ ID NO:24 , SEQ ID NO:25 , SEQ ID NO:26 , SEQ ID NO:27 , SEQ ID NO:28 , SEQ ID NO:29 , SEQ ID NO:30 , SEQ ID NO:32 , SEQ ID NO:33 , </xnotran> 34 or a complement or variant thereof, 35 or a complement or variant thereof, 36 or a complement or variant thereof, 37 or a complement or variant thereof, 39 or a complement or variant thereof, 40 or a complement or variant thereof, 41 or a complement or variant thereof, 42 or a complement or variant thereof, 43 or a complement or variant thereof, 44 or a complement or variant thereof, 45 or a complement or variant thereof, 46 or a complement or variant thereof, 47 or a complement or variant thereof, 48 or a complement or variant thereof, 49 or a complement or variant thereof, 50 or a complement or variant thereof, 51 or a complement or variant thereof, 52 or a complement or variant thereof, 53 or a complement or variant thereof, 54 or a complement or variant thereof, or a variant thereof, optionally further comprises one or more of the following: 5or a complement or variant thereof, 31 or a complement or variant thereof, 38 or a complement or variant thereof, said variant being a variant having at least 70% identity to the corresponding sequence and wherein the methylation site is not mutated.

In one or more embodiments, the DNA sequence further comprises the following sequence:

(1) 5or a complementary sequence or variant thereof, and 31 or a complementary sequence or variant thereof,

(2) 38 or its complement or variant, or

(3) SEQ ID NO. 5or a complement or variant thereof, SEQ ID NO. 31 or a complement or variant thereof, and SEQ ID NO. 38 or a complement or variant thereof.

In one or more embodiments, the agent is a primer molecule that hybridizes to the DNA sequence or fragment thereof or their transformed sequences. Said primer molecules being capable of amplifying said DNA sequences or fragments thereof or transformed variants thereof. In one or more embodiments, the primer sequence is methylation specific or non-specific. The primer molecule is at least 9bp.

In one or more embodiments, the agent is a probe molecule that hybridizes to the DNA sequence or fragment thereof or their transformed sequences. In one or more embodiments, the probe further comprises a detectable substance. In one or more embodiments, the detectable species is a5 'fluorescent reporter and a 3' labeled quencher. In one or more embodiments, the fluorescent reporter gene is selected from Cy5, FAM, and VIC. The probe molecule is at least 12bp.

In one or more embodiments, the agent comprises a nucleic acid molecule as described herein in the first aspect.

In one or more embodiments, the sample is from a mammal, preferably a human.

In a third aspect the invention provides a medium bearing a DNA sequence, or fragment thereof, and/or methylation information thereof, said DNA sequence comprising (i) the following gene sequence: ACSL5, ACTR3B, AIM, ASB2, C15orf62, C2CD4B, CCDC, CCNB2, CD200, CD3G, CEBPD, CIITA, CSK, DLEU7, DYNLT3, ELF4, FABP3, HLA-E, HRH, IL12RB1, IL17C, ITGB2, KRTAP5-9, LAT2, LGALS1, LPIN1, OAS3, PCYT1B, PITX, PTR, PTPTPN 7, S100A10, SH3BP4, SIGLEC14, SIGLEC7, SLC29A3, SLC2A10, SLC5A5, FN13, AFURF, STAT6, STEAP4, SUMF1, PB 1D10C, TEK, THIS 2, THIS 1, SLC 24, VAMP 6, TMFRSF 24, VAMP 34, or a sequence that is less capable of converting to a cytosine 34 when treated with one or more than one of said MTBF 24, VA8, or more bases (or more) that is capable of converting to cytosine 34, optionally treated with a cytosine.

In one or more embodiments, the medium is used to align with gene methylation sequencing data to determine the presence, amount, and/or level of methylation of a nucleic acid molecule comprising the sequence or fragment.

In one or more embodiments, the DNA sequence comprises: <xnotran> SEQ ID NO:1 , SEQ ID NO:2 , SEQ ID NO:3 , SEQ ID NO:4 , SEQ ID NO:6 , SEQ ID NO:7 , SEQ ID NO:8 , SEQ ID NO:9 , SEQ ID NO:10 , SEQ ID NO:11 , SEQ ID NO:12 , SEQ ID NO:13 , SEQ ID NO:14 , SEQ ID NO:15 , SEQ ID NO:16 , SEQ ID NO:17 , SEQ ID NO:18 , SEQ ID NO:19 , SEQ ID NO:20 , SEQ ID NO:21 , SEQ ID NO:22 , SEQ ID NO:23 , SEQ ID NO:24 , SEQ ID NO:25 , SEQ ID NO:26 , SEQ ID NO:27 , SEQ ID NO:28 , SEQ ID NO:29 , SEQ ID NO:30 , SEQ ID NO:32 , SEQ ID NO:33 , </xnotran> 34 or a complement or variant thereof, 35 or a complement or variant thereof, 36 or a complement or variant thereof, 37 or a complement or variant thereof, 39 or a complement or variant thereof, 40 or a complement or variant thereof, 41 or a complement or variant thereof, 42 or a complement or variant thereof, 43 or a complement or variant thereof, 44 or a complement or variant thereof, 45 or a complement or variant thereof, 46 or a complement or variant thereof, 47 or a complement or variant thereof, 48 or a complement or variant thereof, 49 or a complement or variant thereof, 50 or a complement or variant thereof, 51 or a complement or variant thereof, 52 or a complement or variant thereof, 53 or a complement or variant thereof, 54 or a complement or variant thereof, or a variant thereof, optionally further comprises one or more of the following: 5or a complement or variant thereof, 31 or a complement or variant thereof, 38 or a complement or variant thereof, said variant being a variant having at least 70% identity to the corresponding sequence and wherein the methylation site is not mutated.

(1) SEQ ID NO. 5or its complement or variant, and SEQ ID NO. 31 or its complement or variant, or

(2) 38 or its complement or variant, or

(3) 5or a complement or variant thereof, 31 or a complement or variant thereof, and 38 or a complement or variant thereof.

In one or more embodiments, the medium is a support, including cards, such as paper, plastic, metal, glass cards, printed with the DNA sequence or fragment thereof and/or methylation information thereof.

In one or more embodiments, the medium is a computer readable medium having stored thereon the sequence and/or methylation information thereof and a computer program which, when executed by a processor, performs the steps of: comparing the methylation sequencing data of the sample to the sequence or information, thereby obtaining the presence, amount and/or level of methylation of nucleic acid molecules comprising the sequence in the sample. The presence, amount and/or methylation level of nucleic acid molecules comprising said sequences are useful for diagnosing thyroid cancer or identifying thyroid nodule benign or malignant.

The invention also provides the use of (a) and/or (b) in the preparation of a kit for diagnosing thyroid cancer or identifying benign and malignant thyroid nodules in a subject,

(a) Reagents or means for determining the methylation level of a DNA sequence or a fragment thereof or one or more CpG dinucleotides therein in a sample of a subject,

(b) A treated nucleic acid molecule of said DNA sequence or fragment thereof, said treatment converting unmethylated cytosine to a base having a lower binding capacity for guanine than cytosine,

wherein the DNA sequence comprises the following gene sequences: ACSL5, ACTR3B, AIM, ASB2, C15orf62, C2CD4B, CCDC65, CCNB2, CD200, CD3G, CEBPD, CIITA, CSK, DLEU7, DYNLT3, ELF4, FABP3, HLA-E, HRH, IL12RB1, IL17C, ITGB2, KRTAP5-9, LAT2, LGALS1, LPIN1, OAS3, KR 3, and so forth PCYT1B, PITX, PTAFR, PTPN7, S100A10, SH3BP4, SIGLEC14, SIGLEC7, SLC29A3, SLC2A10, SLC5A5, SLFN13, SNURF, STAT6, STEAP4, SUMF1, TBC1D10C, TEK, THEMIS2, TIMP1, TMEM119, TNFRSF6B, TUBB, VAMP5, VAV1, 3534 zxft 3511B, optionally further comprises one or more of ATF7IP2, PLVAP, SIRPB2.

In one or more embodiments, the fragment is 1-1000bp, preferably 1-700bp in length.

(2) 38 or its complement or variant, or

In one or more embodiments, the nucleic acid molecule is a nucleic acid molecule as described in the first aspect herein.

In one or more embodiments, the reagent comprises a primer molecule and/or a probe molecule.

In one or more embodiments, the agent comprises a primer molecule that hybridizes to the DNA sequence or fragment thereof or their transformed sequences. Said primer molecules being capable of amplifying said DNA sequences or fragments thereof or transformed variants thereof. In one or more embodiments, the primer sequence is methylation specific or non-specific. The primer molecule is at least 9bp.

In one or more embodiments, the reagent comprises a medium as described in any embodiment herein.

In one or more embodiments, the kit is a minimally invasive diagnostic kit.

In one or more embodiments, the subject is a mammal, preferably a human.

In one or more embodiments, the sample is from a tissue, cell, e.g., thyroid tissue, of a mammal. In one or more embodiments, the sample is a thyroid nodule fine needle biopsy.

In one or more embodiments, the sample comprises genomic DNA.

In one or more embodiments, the DNA sequence is transformed, wherein unmethylated cytosines are converted to bases that have less ability to bind guanine than cytosines. The conversion is carried out using an enzymatic method, preferably a deaminase treatment, or the conversion is carried out using a non-enzymatic method, preferably a treatment with bisulfite, bisulfite or metabisulfite or a combination thereof.

In one or more embodiments, the DNA sequence is treated with a methylation sensitive restriction endonuclease.

In one or more embodiments, the kit further comprises PCR reaction reagents. Preferably, the PCR reaction reagents include DNA polymerase, PCR buffer, dNTP, mg2+.

In one or more embodiments, the kit further comprises additional reagents for detecting DNA methylation, the additional reagents being reagents used in one or more methods selected from the group consisting of: bisulfite conversion based PCR (e.g., methylation specific PCR), DNA sequencing (e.g., bisulfite sequencing, whole genome methylation sequencing, simplified methylation sequencing), methylation sensitive restriction enzyme analysis, fluorometry, methylation sensitive high resolution melting curve, chip-based methylation profile analysis, mass spectrometry (e.g., flight mass spectrometry). Preferably, the additional agent is selected from one or more of: bisulfite, bisulfite or pyrosulfite or their derivatives, restriction endonucleases sensitive or insensitive to methylation, enzyme digestion buffer, fluorescent dyes, fluorescence quenchers, fluorescence reporters, exonucleases, alkaline phosphatase, internal standards, and controls.

In one or more embodiments, the reaction solution for PCR comprises Taq DNA polymerase, PCR buffer, dNTPs, KCl, mgCl ₂ And (NH) ₄ ) ₂ SO ₄ . Preferably, the Taq DNA polymerase is a hot start Taq DNA polymerase. Preferably, mg ²⁺ The final concentration is 1.0-10.0mM.

In one or more embodiments, the diagnosing comprises: and calculating to obtain a score, and diagnosing the thyroid cancer according to the score. In one or more embodiments, the calculation is calculated by constructing a logistic regression model.

In still another aspect, the present invention provides a method for diagnosing thyroid cancer or thyroid nodules benign and malignant, comprising:

(1) Detecting the methylation level of a DNA sequence or a fragment thereof or one or more CpG dinucleotides therein in a sample of a subject, said DNA sequence comprising the following gene sequence: ACSL5, ACTR3B, AIM, ASB2, C15orf62, C2CD4B, CCDC, CCNB2, CD200, CD3G, CEBPD, CIITA, CSK, DLEU7, DYNLT3, ELF4, FABP3, HLA-E, HRH, IL12RB1, IL17C, ITGB2, KRTAP5-9, LAT2, LGALS1, LPIN1, OAS3, PCYT1B, PITX, PTR, PTPTPN 7, S100A10, SH3BP4, SIGLEC14, SIGLEC7, SLC29A3, SLC2A10, SLC5A5, SLFN13, AFURF, STAT6, STEAP4, SUMF1, PB 1D10C, TEK, THIS 2, THIS 1, VAzmP 24, SIR 24, VAxft 3934, and optionally one or more of TBCF 3, TFL 24, TMOFFT 3934, TMOFFSP 34, TMOFFT 3, and TMPLS 3.

(2) Comparing with a reference, or calculating a score,

(3) And diagnosing thyroid cancer or identifying thyroid nodule benign and malignant according to the score.

In one or more embodiments, the fragment is a promoter region of a gene.

In one or more embodiments, the method further comprises DNA extraction and/or quality control prior to step (1).

In one or more embodiments, the DNA sequence comprises the following sequences: <xnotran> SEQ ID NO:1 , SEQ ID NO:2 , SEQ ID NO:3 , SEQ ID NO:4 , SEQ ID NO:6 , SEQ ID NO:7 , SEQ ID NO:8 , SEQ ID NO:9 , SEQ ID NO:10 , SEQ ID NO:11 , SEQ ID NO:12 , SEQ ID NO:13 , SEQ ID NO:14 , SEQ ID NO:15 , SEQ ID NO:16 , SEQ ID NO:17 , SEQ ID NO:18 , SEQ ID NO:19 , SEQ ID NO:20 , SEQ ID NO:21 , SEQ ID NO:22 , SEQ ID NO:23 , SEQ ID NO:24 , SEQ ID NO:25 , SEQ ID NO:26 , SEQ ID NO:27 , SEQ ID NO:28 , SEQ ID NO:29 , SEQ ID NO:30 , SEQ ID NO:32 , SEQ ID NO:33 , </xnotran> 34 or a complement or variant thereof, 35 or a complement or variant thereof, 36 or a complement or variant thereof, 37 or a complement or variant thereof, 39 or a complement or variant thereof, 40 or a complement or variant thereof, 41 or a complement or variant thereof, 42 or a complement or variant thereof, 43 or a complement or variant thereof, 44 or a complement or variant thereof, 45 or a complement or variant thereof, 46 or a complement or variant thereof, 47 or a complement or variant thereof, 48 or a complement or variant thereof, 49 or a complement or variant thereof, 50 or a complement or variant thereof, 51 or a complement or variant thereof, 52 or a complement or variant thereof, 53 or a complement or variant thereof, 54 or a complement or variant thereof, 56 or a variant thereof, and optionally one or more of the following, 54 or a variant thereof, 56 or a variant thereof, or a complement or variant thereof, or a variant thereof, 52 or a complement or variant thereof, 53 or a complement or a variant thereof, and optionally: 5or its complement or variant, 31 or its complement or variant, 38 or its complement or variant, said variant being a variant having at least 70% identity to the corresponding sequence and wherein the methylation site is not mutated.

(1) SEQ ID NO 5or its complementary sequence or variant, and SEQ ID NO 31 or its complementary sequence or variant, or

(2) 38 or its complement or variant, or

In one or more embodiments, step (1) comprises performing the detecting using a nucleic acid molecule, primer molecule, probe molecule and/or medium as described herein.

In one or more embodiments, the detection includes, but is not limited to: PCR based on bisulfite conversion, DNA sequencing, methylation sensitive restriction enzyme analysis, fluorescence quantification, methylation sensitive high resolution melting curve method, chip-based methylation map analysis, and mass spectrum.

In one or more embodiments, the detecting is DNA sequencing. In one or more embodiments, the DNA sequencing is at least 10X, preferably 20X, more preferably 30X deep.

In one or more embodiments, the sample is from a tissue, cell, e.g., thyroid tissue, of a mammal. The mammal is preferably a human. In one or more embodiments, the sample is a thyroid nodule fine needle biopsy.

In one or more embodiments, the sample comprises genomic DNA.

In one or more embodiments, the DNA sequence is transformed in which unmethylated cytosines are converted to bases that do not bind guanine. The conversion is carried out using an enzymatic method, preferably a deaminase treatment, or the conversion is carried out using a non-enzymatic method, preferably a treatment with bisulfite, bisulfite or metabisulfite or a combination thereof.

In one or more embodiments, the score in step (2) is calculated by a logistic regression model.

In one or more embodiments, step (3) comprises: when the score meets a threshold, the subject is identified as having thyroid cancer or a nodule thereof as malignant.

In another aspect of the present invention, there is provided a kit for identifying thyroid cancer or thyroid nodule benign or malignant, comprising:

(a) An agent or device for determining the methylation level of a DNA sequence or a fragment thereof or one or more CpG dinucleotides therein in a sample from a subject, and

optionally (b) a treated nucleic acid molecule of said DNA sequence or fragment thereof, said treatment converting unmethylated cytosine to a base having less ability to bind guanine than cytosine,

wherein the DNA sequence comprises the following gene sequences: ACSL5, ACTR3B, AIM, ASB2, C15orf62, C2CD4B, CCDC, CCNB2, CD200, CD3G, CEBPD, CIITA, CSK, DLEU7, DYNLT3, ELF4, FABP3, HLA-E, HRH, IL12RB1, IL17C, ITGB2, KRTAP5-9, LAT2, LGALS1, LPIN1, OAS3, PCYT1B, PITX, PTR, PTPTPN 7, S100A10, SH3BP4, SIGLEC14, SIGLEC7, SLC29A3, SLC2A10, SLC5A5, SLFN13, AFURF, STAT6, STEAP4, SUMF1, PB 1D10C, TEK, THIS 2, THIS 1, VAzmP 24, SIR 24, VAxft 3934, and optionally one or more of TBCF 3, TFL 24, TMOFFT 3934, TMOFFSP 34, TMOFFT 3, and TMPLS 3.

In one or more embodiments, the fragment is a promoter region of a gene.

In one or more embodiments, the DNA sequence comprises the following sequences: <xnotran> SEQ ID NO:1 , SEQ ID NO:2 , SEQ ID NO:3 , SEQ ID NO:4 , SEQ ID NO:6 , SEQ ID NO:7 , SEQ ID NO:8 , SEQ ID NO:9 , SEQ ID NO:10 , SEQ ID NO:11 , SEQ ID NO:12 , SEQ ID NO:13 , SEQ ID NO:14 , SEQ ID NO:15 , SEQ ID NO:16 , SEQ ID NO:17 , SEQ ID NO:18 , SEQ ID NO:19 , SEQ ID NO:20 , SEQ ID NO:21 , SEQ ID NO:22 , SEQ ID NO:23 , SEQ ID NO:24 , SEQ ID NO:25 , SEQ ID NO:26 , SEQ ID NO:27 , SEQ ID NO:28 , SEQ ID NO:29 , SEQ ID NO:30 , SEQ ID NO:32 , SEQ ID NO:33 , </xnotran> 34 or a complement or variant thereof, 35 or a complement or variant thereof, 36 or a complement or variant thereof, 37 or a complement or variant thereof, 39 or a complement or variant thereof, 40 or a complement or variant thereof, 41 or a complement or variant thereof, 42 or a complement or variant thereof, 43 or a complement or variant thereof, 44 or a complement or variant thereof, 45 or a complement or variant thereof, 46 or a complement or variant thereof, 47 or a complement or variant thereof, 48 or a complement or variant thereof, 49 or a complement or variant thereof, 50 or a complement or variant thereof, 51 or a complement or variant thereof, 52 or a complement or variant thereof, 53 or a complement or variant thereof, 54 or a complement or variant thereof, or a variant thereof, optionally further comprises one or more of the following: 5or its complement or variant, 31 or its complement or variant, 38 or its complement or variant, said variant being a variant having at least 70% identity to the corresponding sequence and wherein the methylation site is not mutated.

(2) 38 or a complementary sequence or variant thereof, or

In one or more embodiments, the kit is suitable for use as described in any of the embodiments herein.

In one or more embodiments, the reagents comprise primer molecules and/or probe molecules.

In one or more embodiments, the agent comprises a primer molecule that hybridizes to the DNA sequence or fragment thereof or their transformed sequences. Said primer molecules are capable of amplifying said DNA sequences or fragments thereof or transformed variants thereof. In one or more embodiments, the primer sequence is methylation specific or non-specific. The primer molecule is at least 9bp.

In one or more embodiments, the kit is a minimally invasive diagnostic kit.

In one or more embodiments, the subject is a mammal, preferably a human.

In one or more embodiments, the sample comprises genomic DNA.

In one or more embodiments, the kit further comprises reagents for detecting DNA methylation, the reagents being reagents used in one or more of the following methods selected from: bisulfite conversion based PCR (e.g., methylation specific PCR), DNA sequencing (e.g., bisulfite sequencing, whole genome methylation sequencing, simplified methylation sequencing), methylation sensitive restriction enzyme analysis, fluorometry, methylation sensitive high resolution melting curve, chip-based methylation profile analysis, mass spectrometry (e.g., flight mass spectrometry). Preferably, the agent is selected from one or more of: bisulfite and its derivatives, restriction enzyme sensitive or insensitive to methylation, enzyme digestion buffer, fluorescent dye, fluorescence quencher, fluorescence reporter, exonuclease, alkaline phosphatase, internal standard, and reference substance.

In another aspect, the present invention provides an apparatus for diagnosing thyroid cancer or identifying benign or malignant thyroid nodules, the apparatus comprising a memory, a processor, and a computer program stored on the memory and executable on the processor, wherein the processor when executing the program implements the steps of:

(1) Obtaining the methylation level of a DNA sequence or fragment thereof or the methylation level of one or more cpgs in said DNA sequence or fragment in a sample of a subject, said DNA sequence comprising the following gene sequence: ACSL5, ACTR3B, AIM, ASB2, C15orf62, C2CD4B, CCDC, CCNB2, CD200, CD3G, CEBPD, CIITA, CSK, DLEU7, DYNLT3, ELF4, FABP3, HLA-E, HRH, IL12RB1, IL17C, ITGB2, KRTAP5-9, LAT2, LGALS1, LPIN1, OAS3, PCYT1B, PITX, PTR, PTPTPN 7, S100A10, PB 3BP4, SIGLEC14, SIGLEC7, SLC29A3, SLC2A10, SLC5A5, SLFN13, AFURF, STAT6, STEAP4, SUMF1, SLF 1D10C, TEK, THIS 2, THIS 1, VAzmP 24, SIR 3934, SIR 24, VAxft 3934, and optionally one or more of TBCP 3,

(2) Calculating a score, and

(3) And identifying the benign and malignant thyroid nodules according to the scores.

In one or more embodiments, step (1) is preceded by a step of obtaining DNA, such as DNA extraction and/or quality control.

In one or more embodiments, the fragment is a promoter region of a gene.

(2) 38 or a complementary sequence or variant thereof, or

In one or more embodiments, step (1) comprises detecting the level of methylation of the sequence in the sample using a nucleic acid molecule, primer molecule, probe molecule and/or medium described herein. In one or more embodiments, the detection includes, but is not limited to: bisulfite conversion based PCR, DNA sequencing (e.g., bisulfite sequencing, whole genome methylation sequencing, simplified methylation sequencing), methylation sensitive restriction enzyme analysis, fluorometry, methylation sensitive high resolution melting curve, chip-based methylation profile analysis, mass spectrometry (e.g., flight mass spectrometry). In one or more embodiments, the detecting is DNA sequencing. Preferably, the sequencing depth of the DNA sequencing is at least 10X, preferably 20X, more preferably 30X.

In one or more embodiments, the sample comprises genomic DNA.

In one or more embodiments, the sequence is transformed, wherein unmethylated cytosines are converted to bases that do not bind guanine. The conversion is carried out using an enzymatic method, preferably a deaminase treatment, or the conversion is carried out using a non-enzymatic method, preferably a treatment with bisulfite, bisulfite or metabisulfite or a combination thereof.

In one or more embodiments, the score in step (2) is calculated by constructing a logistic regression model.

In one or more embodiments, step (3) comprises: when the score meets a threshold, then the subject is identified as having or not having thyroid cancer or its thyroid nodule as benign or malignant.

The invention has the advantages that:

the sensitivity of the gene combination for identifying benign and malignant thyroid gland can reach 80% when the specificity is 100%. Compared with the existing molecular diagnosis thyroid benign and malignant technology, the gene combination marker provided by the invention has the highest Positive Predictive Value (PPV) of malignant thyroid nodules, and the Positive Predictive Value (PPV) reaches 100%. Effectively solves the problem of over-diagnosis and over-treatment of the existing thyroid cancer.

The invention can detect benign and malignant genes only by detecting the methylation levels of more than fifty genes, obviously reduces target detection areas, improves the application range of the technology and can contain more samples.

Drawings

FIG. 1 ROC curves for diagnosing benign and malignant thyroid nodules in training and test set samples using a predictive model of the group 1 gene combinations.

FIG. 2 ROC curves for diagnosing benign and malignant thyroid nodules in training and test set samples using a predictive model of the group 2 gene combinations.

FIG. 3 ROC curves for diagnosing benign and malignant thyroid nodules in training and test set samples using a predictive model of the group 3 gene combinations.

Detailed Description

The invention aims to provide a molecular detection method for diagnosing benign and malignant nodules by using gene combination, aiming at the problems that the existing thyroid benign and malignant nodule molecular diagnosis has low accuracy or can not cover all samples due to technical reasons and the like.

As used herein, the term "methylation marker" refers to a nucleic acid or gene region of interest, a methylation site: the methylation level or score of a computational model based on the methylation level is indicative of benign or malignant thyroid nodules. The term "methylation marker" shall be taken to include all transcriptional variants thereof and all promoter and regulatory elements thereof. As understood by those skilled in the art, certain genes are known to exhibit allelic variation or single nucleotide polymorphisms ("SNPs") between individuals. SNPs include insertions and deletions of simple repetitive sequences of different lengths (e.g., di-and trinucleotide repeats). Thus, the present application should be understood to extend to all forms of markers/genes resulting from any other mutation, polymorphism or allelic variation. In addition, it is to be understood that the term "methylation marker" shall include both the sense strand sequence of a marker or gene and the antisense strand sequence of a marker or gene.

The term "methylation marker" as used herein is to be broadly interpreted to include both 1) the original marker (at a specific methylation) found in a biological sample or genomic DNA, and 2) its processed sequence (e.g., the corresponding region after bisulfite conversion or the corresponding region after treatment with the methylation-sensitive restriction enzyme MSRE). The corresponding region after bisulfite conversion differs from the target marker in the genomic sequence in that one or more unmethylated cytosine residues are converted to uracil bases, thymine bases or other bases that differ in hybridization behavior from cytosine. The corresponding region treated with MSRE differs from the target marker in the genomic sequence in that the sequence is cleaved at one or more MSRE cleavage sites.

The inventors found that the nature of thyroid nodules is related to methylation of the following genes (e.g., promoter regions): ACSL5, ACTR3B, AIM, ASB2, C15orf62, C2CD4B, CCDC, CCNB2, CD200, CD3G, CEBPD, CIITA, CSK, DLEU7, DYNLT3, ELF4, FABP3, HLA-E, HRH, IL12RB1, IL17C, ITGB2, KRTAP5-9, LAT2, LGALS1, LPIN1, OAS3, PCYT1B, PITX, PTR, TBPTPN 7, S100A10, SH3BP4, SIGLEC14, SIGLEC7, SLC29A3, SLC2A10, SLC5A5, SLFN13, AFURF, STAT6, STSTEAP 4, SUMF1, STF 1D10C, TEK, THIS 2, THIS 1, VAzmP 3934, SIR 24, ATxPLF 24, ATxP 3934, and optionally further comprising TMxPF 3911. The invention provides methylation detection of the genes of a sample, and a mathematical model is used for distinguishing benign nodules and malignant nodules of thyroid gland based on the methylation level of the genes, so that the aim of accurately diagnosing the thyroid cancer nodules is fulfilled.

Herein, the term "gene" includes both coding and non-coding sequences on the genome of the gene in question. Wherein the non-coding sequence includes introns, promoters and regulatory elements or sequences and the like.

In one or more embodiments, the nature of the thyroid nodule is related to methylation of a fragment of the above-described gene. The length of the fragment is 1bp-1kb, preferably 1bp-700bp; the fragments comprise one or more methylation sites in the chromosomal region of the corresponding gene. Such fragments are, for example, the promoter regions of the above-mentioned genes. Generally, a DNA sequence 1kb upstream and 200bp downstream of a Transcription Start Site (TSS) is defined as a promoter region. If a gene has multiple transcripts (i.e., multiple promoter regions), any of the promoter regions may be selected. In some embodiments, the detected fragment contains at least 3 CpG dinucleotides.

Thus, further, the nature of thyroid nodules correlates with the methylation level of the following sequences: <xnotran> ACSL5 SEQ ID NO:1, ACTR3B SEQ ID NO:2, AIM2 SEQ ID NO:3, ASB2 SEQ ID NO:4, C15orf62 SEQ ID NO:6, C2CD4B SEQ ID NO:7, CCDC65 SEQ ID NO:8, CCNB2 SEQ ID NO:9, CD200 SEQ ID NO:10, CD3G SEQ ID NO:11, CEBPD SEQ ID NO:12, CIITA SEQ ID NO:13, CSK SEQ ID NO:14, DLEU7 SEQ ID NO:15, DYNLT3 SEQ ID NO:16, ELF4 SEQ ID NO:17, FABP3 SEQ ID NO:18, HLA-E SEQ ID NO:19, HRH1 SEQ ID NO:20, IL12RB1 SEQ ID NO:21, IL17C SEQ ID NO:22, ITGB2 SEQ ID NO:23, KRTAP5-9 SEQ ID NO:24, LAT2 SEQ ID NO:25, LGALS1 SEQ ID NO:26, LPIN1 SEQ ID NO:27, OAS3 SEQ ID NO:28, PCYT1B SEQ ID NO:29, PITX1 SEQ ID NO:30, PTAFR SEQ ID NO:32, PTPN7 SEQ ID NO:33, S100A10 SEQ ID NO:34, SH3BP4 SEQ ID NO:35, SIGLEC14 SEQ ID NO:36, SIGLEC7 SEQ ID NO:37, SLC29A3 SEQ ID NO:39, SLC2A10 SEQ ID NO:40, SLC5A5 SEQ ID NO:41, SLFN13 SEQ ID NO:42, SNURF SEQ ID NO:43, STAT6 SEQ ID NO:44, STEAP4 SEQ ID NO:45, SUMF1 SEQ ID NO:46, TBC1D10C SEQ ID NO:47, TEK SEQ ID NO:48, THEMIS2 SEQ ID NO:49, </xnotran> 50 in TIMP1, 51 in TMEM119, 52 in TNFRSF6B, 53 in TUBB6, 54 in VAMP5, 55 in VAV1, 56 in ZYG B, optionally further comprising one or more selected from: 5 in ATF7IP2, 31 in PLVAP, 38 in SIRPB2.

The "thyroid cancer-associated sequence" as used herein includes any of the above 53 genes and optionally 3 genes, a sequence within 20kb upstream or downstream thereof, any combination of the above 56 sequences (SEQ ID NOS: 1-56), or their complements.

The positions of the above sequences in the human chromosome are shown in the following table, in which the base numbers correspond to the reference genome HG19:

TABLE 1

Sequence numbering	Chromosome	Starting position	End position	Sequence numbering	Chromosome	Starting position	End position
								SEQ ID NO:1	chr10	114134957	114136157	SEQ ID NO:29	chrX	24665153	24666353
SEQ ID NO:2	chr7	152455834	152457034	SEQ ID NO:30	chr5	134370124	134371324
								SEQ ID NO:3	chr1	159046491	159047691	SEQ ID NO:31	chr19	17487959	17489159
SEQ ID NO:4	chr14	94442937	94444137	SEQ ID NO:32	chr1	28520247	28521447
								SEQ ID NO:5	chr16	10478912	10480112	SEQ ID NO:33	chr1	202129551	202130751
SEQ ID NO:6	chr15	41061159	41062359	SEQ ID NO:34	chr1	151964854	151966054
								SEQ ID NO:7	chr15	62457282	62458482	SEQ ID NO:35	chr2	235886347	235887547
SEQ ID NO:8	chr12	49296932	49298132	SEQ ID NO:36	chr19	52149854	52151054
								SEQ ID NO:9	chr15	59396277	59397477	SEQ ID NO:37	chr19	51644558	51645758
SEQ ID NO:10	chr3	112051003	112052203	SEQ ID NO:38	chr20	1471842	1473042
								SEQ ID NO:11	chr11	118214071	118215271	SEQ ID NO:39	chr10	73078015	73079215
SEQ ID NO:12	chr8	48651448	48652648	SEQ ID NO:40	chr20	45337126	45338326
								SEQ ID NO:13	chr16	10970055	10971255	SEQ ID NO:41	chr19	17981782	17982982
SEQ ID NO:14	chr15	75084385	75085585	SEQ ID NO:42	chr17	33775656	33776856
								SEQ ID NO:15	chr13	51417632	51418832	SEQ ID NO:43	chr15	25199135	25200335
SEQ ID NO:16	chrX	37706690	37707890	SEQ ID NO:44	chr12	57522683	57523883
								SEQ ID NO:17	chrX	129244491	129245691	SEQ ID NO:45	chr7	87936006	87937206
SEQ ID NO:18	chr1	31845723	31846923	SEQ ID NO:46	chr3	4508765	4509965
								SEQ ID NO:19	chr6	30456244	30457444	SEQ ID NO:47	chr11	67170660	67171860
SEQ ID NO:20	chr3	11177779	11178979	SEQ ID NO:48	chr9	27108147	27109347
								SEQ ID NO:21	chr19	18209554	18210754	SEQ ID NO:49	chr1	28198079	28199279
SEQ ID NO:22	chr16	88704001	88705201	SEQ ID NO:50	chrX	47440712	47441912
								SEQ ID NO:23	chr21	46340765	46341965	SEQ ID NO:51	chr12	108991700	108992900
SEQ ID NO:24	chr11	71258466	71259666	SEQ ID NO:52	chr20	62327021	62328221
								SEQ ID NO:25	chr7	73623336	73624536	SEQ ID NO:53	chr18	12307058	12308258
SEQ ID NO:26	chr22	38070615	38071815	SEQ ID NO:54	chr2	85810531	85811731
								SEQ ID NO:27	chr2	11885714	11886914	SEQ ID NO:55	chr19	6771737	6772937
SEQ ID NO:28	chr12	113375157	113376357	SEQ ID NO:56	chr1	53191181	53192381

Herein, methods of detecting DNA Methylation are well known in the art, such as bisulfite conversion-based PCR (e.g., methylation-specific PCR (MSP), DNA sequencing (e.g., bisulfite sequencing, whole genome Methylation sequencing, simplified Methylation sequencing), whole genome Methylation sequencing, simplified Methylation sequencing, methylation-sensitive restriction endonuclease analysis, fluorometry, methylation-sensitive high resolution melting curve, chip-based Methylation profiling, mass spectrometry (e.g., flight mass spectrometry), in one or more embodiments, detecting comprises detecting any strand at a gene or locus.

Thus, the present invention relates to a reagent for detecting DNA methylation. Reagents used in the above-described methods for detecting DNA methylation are well known in the art. In detection methods involving DNA amplification, the reagents for detecting DNA methylation include primers. As used herein, a "primer" refers to a nucleic acid molecule having a specific nucleotide sequence that directs the synthesis of a nucleic acid molecule at the initiation of nucleotide polymerization. The primers are usually at least 9bp. The primer sequence may be methylation specific or non-specific. The sequence of the primer may include a non-methylation specific blocking sequence (Blocker). Blocking sequences may enhance the specificity of methylation detection. Typically, primers are designed to amplify a product of 1-2000bp, 10-1000bp, 30-900bp, 40-800bp, 50-700bp, or at least 150bp, at least 140bp, at least 130bp, at least 120bp in length.

The reagent for detecting DNA methylation may further comprise a probe that hybridizes to a sequence to be detected. Typically, the sequence of the probe is labeled at the 5 'end with a fluorescent reporter group and at the 3' end with a quencher group. Illustratively, the sequence of the probe comprises MGB (Minor groove binder) or LNA (Locked nucleic acid). MGB and LNA are used to increase Tm, increase specificity of analysis and increase flexibility of probe design. As used herein, "hybridization" refers primarily to the pairing of nucleic acid sequences under stringent conditions. Exemplary stringent conditions are hybridization and washing of the membrane at 65 ℃ in a solution of 0.1 XSSPE (or 0.1 XSSC), 0.1% SDS.

The term "variant" or "mutant" as used herein refers to a polynucleotide that has a nucleic acid sequence altered by insertion, deletion or substitution of one or more nucleotides compared to a reference sequence, while retaining its ability to hybridize to other nucleic acids. A mutant according to any of the embodiments herein comprises a nucleotide sequence that has at least 70%, preferably at least 80%, preferably at least 85%, preferably at least 90%, preferably at least 95%, preferably at least 97% sequence identity to a reference sequence and retains the biological activity of the reference sequence. Sequence identity between two aligned sequences can be calculated using, for example, BLASTn from NCBI. Mutants also include nucleotide sequences that have one or more mutations (insertions, deletions, or substitutions) in the reference sequence and in the nucleotide sequence, while still retaining the biological activity of the reference sequence. The plurality of mutations typically refers to within 1-10, such as 1-8, 1-5, or 1-3. The substitution may be a substitution between purine nucleotides and pyrimidine nucleotides, or a substitution between purine nucleotides or between pyrimidine nucleotides. The substitution is preferably a conservative substitution. For example, conservative substitutions with nucleotides of similar or analogous properties are not typically made in the art to alter the stability and function of the polynucleotide. Conservative substitutions are, for example, exchanges between purine nucleotides (A and G), exchanges between pyrimidine nucleotides (T or U and C). Thus, substitution of one or more sites with residues from the same in the polynucleotides of the invention will not substantially affect their activity. Furthermore, the methylation sites (e.g., contiguous CGs) in the variants of the invention are not mutated. That is, the method of the present invention detects methylation of a methylatable site in the corresponding sequence, and a mutation may occur in the base of a non-methylatable site. Typically, the methylation sites are consecutive CpG dinucleotides.

As described herein, transformation can occur between bases of DNA or RNA. "transformation", "cytosine transformation" or "CT transformation" as used herein is a process of converting an unmodified cytosine base (C) to a base having a lower binding ability to guanine than cytosine, for example, an uracil base (U), by treating DNA using a non-enzymatic or enzymatic method. Non-enzymatic or enzymatic methods of performing cytosine conversion are well known in the art. Exemplary non-enzymatic methods include treatment with a conversion reagent such as bisulfite, or metabisulfite, for example, calcium bisulfite, sodium bisulfite, potassium bisulfite, ammonium bisulfite, and the like. Illustratively, the enzymatic method includes a deaminase treatment. The transformed DNA is optionally purified. DNA purification methods suitable for use herein are well known in the art.

The invention also provides a methylation detection kit for diagnosing thyroid cancer, which comprises the primer and/or the probe, and is used for detecting the methylation level of the thyroid cancer related sequence discovered by the inventor. The kit may further comprise a nucleic acid molecule as described herein, in particular according to the first aspect, as an internal standard or positive control.

In addition to the primers, probes, nucleic acid molecules, the kit contains other reagents required for detecting DNA methylation. Illustratively, other reagents for detecting DNA methylation may comprise one or more of: bisulfite and its derivative, PCR buffer solution, polymerasedNTP, primer, probe, mg ²⁺ Restriction enzymes sensitive or insensitive to methylation, digestion buffer, fluorescent dye, fluorescence quencher, fluorescence reporter, exonuclease, alkaline phosphatase, internal standard and reference substance. The kit can also include a transformed positive standard in which unmethylated cytosines are converted to bases that do not bind guanine. The positive standard may be fully methylated.

Based on the inventors' findings, the present invention provides a method for thyroid cancer screening, comprising: (1) Detecting the methylation level of a thyroid cancer associated sequence described herein in a sample from a subject; (2) calculating to obtain a score; (3) Diagnosing thyroid cancer or identifying benign or malignant thyroid nodules according to the score. Typically, the method further comprises, prior to step (1): extraction of sample DNA, quality inspection, and/or conversion of unmethylated cytosines on DNA to bases that do not bind guanine.

The terms "benign" and "malignant" as used herein refer to the nature of thyroid nodules. Generally, benign is manifested by slow growth of nodules, uniform texture, good mobility, smooth surface, cystic changes, no enlargement of lymph nodes, no calcification, etc. Malignancy is characterized by uncontrolled malignant cell growth, spread and tissue infiltration. Ultrasound signs that suggest that thyroid nodules are malignant include: the height of the nodule is greater than the width, lack of vocal cord, micro-calcification, irregular boundaries, reduced echo, solid nodules, abundant blood flow inside the nodule, etc. In an embodiment of the invention, the malignant thyroid nodule is Papillary Thyroid Carcinoma (PTC).

The "diagnosis" in the present invention includes not only early diagnosis of thyroid malignant tumor but also late diagnosis of thyroid malignant tumor, and also includes screening, risk assessment, prognosis, and disease identification of thyroid malignant tumor. Early diagnosis refers to the possibility of finding cancer before metastasis, preferably before morphological changes in tissues or cells can be observed.

In a specific embodiment, step (1) comprises: treating the genomic DNA with a converting reagent to convert unmethylated cytosine to a base that has less binding capacity to guanine (e.g., uracil); performing PCR amplification using primers suitable for amplifying transformed sequences of thyroid cancer associated sequences as described herein; the methylation level of at least one CpG is determined by the presence or absence of an amplification product, or by sequence identification (e.g., probe-based PCR detection identification or DNA sequencing identification).

Or the step (1) may further include: treating genomic DNA with a methylation sensitive restriction enzyme; performing PCR amplification using primers suitable for amplifying sequences having at least one CpG dinucleotide in the thyroid cancer associated sequences as described herein; the methylation level of at least one CpG is determined by the content of the amplification product.

As used herein, "methylation level" refers to the level of methylation at a CpG site of interest or the average level of methylation at a plurality or all of the CpG sites in a sequence of interest. In an exemplary embodiment of the invention, the methylation level of a site is generally the percentage of methylated C's at that site, and if all C's at that CpG site are unmethylated, the methylation level is zero. The methylation level can also be other types of calculations, which are within the knowledge of a person skilled in the art. Furthermore, an increase or decrease in the methylation level of a sequence does not indicate an increase or decrease in the methylation level of all CpG sites in the region. Procedures are known in the art for converting results from methods for detecting DNA methylation (e.g., simplified methylation sequencing) to methylation levels. For example, the average methylation is calculated based on the detected methylation level of CpG sites in the promoter region of each gene, and is used as the DNA methylation level in the promoter region of the gene.

An exemplary calculation formula is as follows:

wherein y is the DNA methylation level of the promoter region of the gene, x is the methylation level of CpG sites, and n is the number of CpG sites in the promoter region.

As used herein, "methylation information" includes characteristic information associated with cytosines in a sequence that are likely to be methylated. The cytosine that is likely to be methylated is typically a C in CpG. Such features include, but are not limited to: whether any cytosine (C) residue within a sequence is methylated, the position of one or more methylation sites (e.g., cpG dinucleotides) and/or the methylation level thereof, the methylation level of any particular region of a nucleic acid, the frequency or percentage of methylated C, the relative concentration, absolute concentration or pattern of methylated C or unmethylated C, and methylated allelic differences due to, for example, differences in allelic origins. For example, if one or more cytosine (C) residues within a nucleic acid sequence are methylated, it may be referred to as "hypermethylated" or have "increased methylation", while if one or more cytosine (C) residues within a DNA sequence are unmethylated, it may be referred to as "demethylated" or have "reduced methylation".

The methylation level of the gene being tested can be mathematically analyzed to obtain a score. And for the detected sample, when the score is greater than the threshold value, judging that the result is positive, namely the thyroid cancer or the malignant nodule, otherwise, judging that the result is negative. Methods of conventional mathematical analysis and processes of determining thresholds are known in the art, and exemplary methods are mathematical models, including but not limited to regression models, support vector machines, random forests, and the like. For example, logistic Regression (Logistic Regression) is constructed for the methylation level of differential methylation markers, and the accuracy, sensitivity and specificity of the detection results and the area under the predictive value characteristic curve (ROC) (AUC) are used by the model to count the sample prediction scores in the test set.

In an exemplary embodiment, a logistic regression model is constructed for a training set by the following process. First, the input z of the Sigmoid function is calculated as follows:

z＝Σw*y+w ₀

then, σ (z) of the Sigmoid function (i.e., the malignancy prediction probability) is calculated as follows:

σ(z)＝1/(1+e ^-z )

where w is the regression model coefficient for each gene, w ₀ Is the intercept and y is the DNA methylation level of the promoter region of the gene in the sample. At the position ofIn an exemplary embodiment, the logistic regression model coefficients (w) for each gene in the respective mathematical models for the three sets of genes are shown in tables 2-4. Intercept of three mathematical models (w) ₀ ) 1.786,1.770 and 1.742, respectively. The threshold value of the malignancy prediction probability obtained based on the group 1 gene combination model is 0.503, the sample malignancy prediction probability is greater than the threshold value, and the sample is judged to be malignant, otherwise, the sample is judged to be benign; the threshold value of the malignancy prediction probability obtained based on the group 2 gene combination model is 0.508, the sample malignancy prediction probability is greater than the threshold value, and the sample is judged to be malignant, otherwise, the sample is judged to be benign; the threshold value of the malignancy prediction probability obtained based on the group 3 gene combination model is 0.511, and the sample malignancy prediction probability is greater than the threshold value, so that the sample is judged to be malignant, otherwise, the sample is judged to be benign.

Examples

Example 1, procedure of experiment

Analysis was performed on all samples of benign and malignant thyroid nodules, namely 145 samples of fine needle biopsy samples (65 benign nodules and 80 malignant nodules) in the paper (John H Yi, clin Cancer Res.,2019, https:// pubmed. Ncbi. Nlm. Nih. Gov/30093451 /), published data, namely genome simplified methylation sequencing (RRBS) raw sequencing data (stored in GSE107738, see https:// www.ncbi.nlm.nih.gov/geo/query/acc. Cgiac = GSE 107738), to obtain the methylation level of each CpG site; then, based on the CpG sites detected in the promoter region of each gene, the average methylation is calculated and used as the DNA methylation level of the promoter region of the gene. For comparison with John H Yim, the present invention groups the article samples in accordance with the present invention, wherein Developing cost is used as the training set (28 benign nodules and 39 malignant nodules) and Testing cost is used as the Testing set (37 benign nodules and 41 malignant nodules). In the following examples, three sets of mathematical models constructed from training set samples were used, and the AUC (Area Under Curve) Under the receiver operating characteristic Curve (ROC) was predicted using the corresponding models in the test set samples.

1. Level of methylation

And (3) respectively detecting the DNA methylation levels of the three groups of gene promoter regions in the sample by using a genome simplified methylation sequencing (RRBS) technology. The DNA sequence 1kb upstream and 200bp downstream of the Transcription Start Sites (TSS) is defined as a promoter region. If a gene has multiple transcripts (Ensembl 74 is selected as the transcript version), i.e., multiple promoter regions, the longest transcript is selected as the promoter region of the gene. The genome coordinates of the promoter regions of the 3 groups of genes are shown in Table 1. At least 3 CpG sites are located in each gene promoter region, and the average sequencing depth of all CpG sites in the region is not less than 30x. Then, calculating the average methylation according to the methylation level of the CpG sites detected in each gene promoter region, and taking the average methylation as the DNA methylation level of the gene promoter region, wherein the calculation formula is as follows:

y is the DNA methylation level of the promoter region of the gene, and x is the CpG site methylation level n is the number of CpG sites in the promoter region.

2. Model construction

First, a Logistic Regression (Logistic Regression) model is constructed for a training set. Then, the input z of the Sigmoid function is calculated, which is given by the following formula:

z＝Σw*y+w ₀

y is the DNA methylation level of the promoter region of the gene in the sample, w is the regression model coefficient for each gene, w is the ratio of the methylation level of the promoter region of the gene in the sample ₀ Is the intercept, and w0 are calculated by logistic regression models.

σ(z)＝1/(1+e ^-z )

the logistic regression model coefficients (w) for each gene in the respective mathematical models for the three sets of genes are shown in tables 2-4. The intercepts (w 0) of the three mathematical models are 1.786 (set 1), 1.770 (set 2) and 1.742 (set 3), respectively.

TABLE 2 coefficients of the mathematical model of group 1

Gene	Coefficient of logistic regression model	Gene	Coefficient of logistic regression model
				ACSL5	1.364	PCYT1B	-0.855
ACTR3B	1.317	PITX1	-1.58
				AIM2	-0.335	PTAFR	-1.982
ASB2	1.685	PTPN7	-1.244
				C15orf62	-2.332	S100A10	-0.523
C2CD4B	-0.462	SH3BP4	-1.956
				CCDC65	2.32	SIGLEC14	0.613
CCNB2	-1.118	SIGLEC7	1.953
				CD200	1.553	SLC29A3	1.399
CD3G	-0.127	SLC2A10	-1.537
				CEBPD	1.804	SLC5A5	-0.533
CIITA	-0.449	SLFN13	1.263
				CSK	-0.53	SNURF	1.949
DLEU7	-0.842	STAT6	-1.854
				DYNLT3	-0.657	STEAP4	-0.665
ELF4	-1.507	SUMF1	1.36
				FABP3	-0.302	TBC1D10C	1.941
HLA-E	-0.694	TEK	-0.835
				HRH1	-1.654	THEMIS2	1.029
IL12RB1	-0.665	TIMP1	-0.608
				IL17C	-1.418	TMEM119	-0.361
ITGB2	1.43	TNFRSF6B	0.089
				KRTAP5-9	2.28	TUBB6	-1.009
LAT2	-2.332	VAMP5	-0.862
				LGALS1	-1.198	VAV1	-0.413
LPIN1	1.42	ZYG11B	1.033
				OAS3	-1.354

TABLE 3 coefficients of the mathematical model of group 2

TABLE 4 coefficients of the mathematical model of group 3

Example 2 combination of group 1 genes for prediction

The AUC was tested in the test set samples using the model constructed from the group 1 gene combinations. The results showed that the gene combination had an area under the ROC curve of 0.95% and 95% CI of 0.93 to 0.98 (FIG. 1). In the training set, when the specificity is 100% and the sensitivity is 97%, the malignancy prediction threshold is 0.503, that is, the malignancy prediction probability (malignancy probability for short) is greater than 0.503, and it is judged to be malignant, otherwise, it is judged to be benign. The sensitivity of the threshold value to the diagnosis of thyroid malignant nodules in a test set reaches 80%, the specificity reaches 100%, the PPV reaches 100%, and the NPV (negative predictive value) reaches 82%. The test set samples were used to predict the results using the group 1 gene combinations shown in table 5.

TABLE 5 test set samples prediction results with group 1 Gene combinations

Example 3 combination of group 2 genes for prediction

The AUC was tested in the test set samples using the model constructed from the group 2 gene combinations. The results showed that the gene combination had an area under the ROC curve of 0.95% and 95% CI of 0.93 to 0.98 (FIG. 2). When the specificity in the training set is 100% and the sensitivity is 97%, the malignancy prediction threshold is 0.508, that is, the malignancy prediction probability is greater than 0.508, and the result is judged to be malignant, otherwise, the result is judged to be benign. The sensitivity of the threshold value to the diagnosis of thyroid malignant nodules of the test set reaches 80%, the specificity reaches 100%, the PPV reaches 100%, and the NPV reaches 82%. The test set samples were used to predict the results using the group 2 gene combinations as shown in table 6.

TABLE 6 prediction of group 2 gene combinations for test set samples

Example 4 combination of group 3 genes for prediction

The model constructed with the group 3 gene combinations tested AUC in the test set samples. The results showed that the gene combination had an area under the ROC curve of 0.95% and 95% CI of 0.93 to 0.98 (FIG. 3). When the specificity of the training set is 100% and the sensitivity is 97%, the malignancy prediction threshold is 0.511, that is, the malignancy prediction probability is greater than 0.511, and the result is judged to be malignant, otherwise, the result is judged to be benign. The sensitivity of the threshold value to the diagnosis of thyroid malignant nodules of the test set reaches 80%, the specificity reaches 100%, the PPV reaches 100%, and the NPV reaches 82%. The test set samples were predicted using the group 3 gene combinations and the results are shown in table 7.

TABLE 7 prediction of group 3 Gene combinations for test set samples

Comparative example

John (John H Yim, clin Cancer Res.,2019, https:// pubmed. Ncbi. Nlm. Nih. Gov/30093451/, raw data stored in GSE107738, see https:// www.ncbi.nlm.nih.gov/geo/query/acc. Cgiac = GSE 107738) developed a method for diagnosing benign and malignant tissue of thyroid Cancer, known as a Diagnostic method based on DNA Methylation Signature (DDMS). It is mentioned that 24 samples (11 training sets, 13 test sets, all samples from example 1) are not suitable for detection by this method due to the lack of apparent changes in the target area.

These 24 samples (data from GSE 107738) were selected as comparative examples and the results showed that the predicted accuracy for all 24 samples was 100% (24/24) for all three combinations. The results are shown in tables 8 to 10.

TABLE 8, prediction results of group 1 Gene combinations for 24 samples

Sample ID	Group of samples	Type of sample	Probability of malignancy prediction	Predicted results
					117T	Training set	Malignancy	0.646	Malignant disease
134T	Training set	Malignancy	0.836	Malignancy
					109T	Training set	Malignancy	0.930	Malignancy
102T	Training set	Malignancy	0.838	Malignant disease
					125T	Training set	Malignancy	0.694	Malignancy
11T	Training set	Malignancy	0.759	Malignancy
					122T	Training set	Malignancy	0.835	Malignancy
506B	Training set	Benign	0.442	Benign
					502B	Training set	Benign	0.273	Benign
17B	Training set	Benign	0.197	Benign
					7B	Training set	Benign	0.036	Benign
610T	Sequencing set	Malignancy	0.582	Malignancy
					158T	Sequencing set	Malignancy	0.875	Malignancy
154T	Sequencing set	Malignant disease	0.811	Malignant disease
					150T	Sequencing set	Malignancy	0.623	Malignancy
162T	Sequencing set	Malignancy	0.622	Malignant disease
					613T	Sequencing set	Malignancy	0.546	Malignancy
169T	Sequencing set	Malignancy	0.869	Malignancy
					565B	Sequencing set	Benign	0.163	Benign
579B	Sequencing set	Benign	0.122	Benign
					531B	Sequencing set	Benign	0.043	Benign
572B	Sequencing set	Benign of the body	0.270	Benign
					534B	Sequencing set	Benign	0.136	Benign
564B	Sequencing set	Benign	0.268	Benign

TABLE 9 prediction of group 2 Gene combinations for 24 samples

Table 10, prediction results of group 3 Gene combinations of 24 samples

Sequence listing

<110> Jiangsu Kun Yuanzhi Co., ltd

SINGLERA GENOMICS Inc.

<120> method for diagnosing thyroid cancer

<130> 214176 1CNCN

<160> 56

<170> PatentIn version 3.5

<210> 1

<211> 1201

<212> DNA

<213> Homo sapiens

<400> 1

tgcctcttgg ggagctgagt tcctgaaact aagcaaatga caagtgctcc tccagccgtg 60

agatgagcca gaggatggaa tgccacaggc cccacttcgt ctcccagccc ttcatctttc 120

ttccatgtcc aaaggcggct ccccagaggc caagggctca gctcttgtgt tctctgagac 180

tgtggcacag ctcaccctgg cttccttagt aggaagcttt taagaactgc tctgtagaaa 240

gaacaaaccc taatctagct ccagcaaaga gccaggatca tgctgaactt cctgtgagcc 300

ttcgtggggg atcgaggctg gagaaagtgc tgcttcattc tgaggtatct gtgaggtggg 360

gaaggggcag gaaaatttgt catttcggct tcctaatctg agaatggtag ctccagggcc 420

cccctgtagc cacagcctta ggcggatcaa aaatgagact aatacaggtc ctggtaatac 480

tgggggtgac caaggcgaaa gaatcgattt ttaagttaag ttttgaggtg tgactgtatc 540

agagggaaag aaaccatgcc aattgaacga ctcacagccc tgtcgttcct gacttgctta 600

ttttcttggc tttgccagaa cttcttcaga gaccaaggca ggcagctctg gcgactggag 660

agaggcgggg tagaaggtgt ttgtgtgggt ctgagggctg aggtaccctc tagccagact 720

ttcctgtcct ccgaatccag cagggaacag cgtcacctct gcctctgtga ggcttctgat 780

ctgagggggc acctcaagtc attttagttt gtttgttttc atcttagttg aaagatgata 840

ggccacagga gggcagggtc aactgtgttg aggcaagctg gcttttgtgt cggcccttag 900

gggctgaggt gaaaggcttt gacacggagc cagggtagct ttggcagctt ctcccctcct 960

ggtcagaaca cttccacttt cagttgtgaa aaaaaaaatt aaaaccagga agtgaagtcc 1020

ccgagcacgt tagaaagcct gacatggcct gactcgggac agctcagagc agggcagaac 1080

tggggacact ctgggccggc cttctgcctg catggacgct ctgaagccac cctgtctctg 1140

gaggaaccac gagcgaggga agaaggacag ggactcgtgt ggcaggaaga actcagagcc 1200

g 1201

<210> 2

<211> 1201

<212> DNA

<213> Homo sapiens

<400> 2

ttttccagga gtgatcttgg tgtggtttac tcagtctaag gccattcatt ccttaaggct 60

ggccaagcat ttgttttgca gatttttctt cttagaaaaa tttaggccgt gttaggtata 120

ataattccaa ctaatttttt ttgttgtttt tttgagacag actctcgccc tgttgtccag 180

gctggagtgc aatggcgcga tcttggctca ctgcaacctc cgcctcccag gttcaagcga 240

ttctcctgcc tcagcctccg gagcagctgg gattacaggc acccgccacc acgcccggct 300

aatttttgta ttttcagtag agaaggggtt tcaccatgtt ggccaggctg gtctggaact 360

cctgaccttg tgatccaccc tcagcctccc aaagtgctgg gattacaggc gtgagccacc 420

gcgcccggcc aattccaact aatttttttt ttttttttta ttatatgggg aaggttactc 480

agtcattcaa caaacatttg ggcacttcca caggtacacc aaatactgtg ctaagcacct 540

gctgctcatc aatagtgatt aagacagact caagccctat cctagaagat ttgatagtcc 600

agtaggggag gcagacaaca aatacataaa tataaaaata taaacacttt gctttaaggg 660

ttaggaatga aaccaacaga ttgttgagac agaggataca gtaccggtag gggacatagt 720

cagggtgatc aggaacaact tccctcgcaa ggtgacattc gagcgaagac ttacaggagg 780

agaaaggcgc caggcctcgg aaatggggtc acgttcatgt aagtcagtta aaaataaacc 840

ccaatctctc cagcagcaag gttggacgta tggggccaag aagcctcgct gccagcacta 900

aagatggcgc gccggagtgg gggtgggggt gttgggggag gcggggcgtg cgcgcgttct 960

gattggccga ccggggagtg acgtcacgtg tcggccgccg agcatccggg ctcccggcag 1020

cggcgctgcg gcggctcgcg ggagacgctg cgcgcggggc tagcgggcgg cggagcggac 1080

ggcgacgggg cgctctcggg ctgccggcgg ggccgagcgc cgcgcgtccc gagcatggca 1140

ggctccctgc ctccctgcgt ggtggactgt ggcaccgggt aagagcagct cggcgcccac 1200

c 1201

<210> 3

<211> 1201

<212> DNA

<213> Homo sapiens

<400> 3

ggactcagga gagcatgccc aaataaccta ggtgactttc aaagctacaa agactctgac 60

acttctggac ggcttcacat gtacccctaa taaacacttc ccttcacccc caggcccagt 120

gtgggtagga gtgacaactg gagctctaat tggctgagct ggtttgggtt ctcactagca 180

gccacagaag aaaatgtcta tagaaacttg tatctgagtc atgccacata ggaactacac 240

tgattggaaa cagaaagtgg cctagaaact gtaaaaaatg accgcagatg acttcttgat 300

acactttgtg actaaggggc ctggtgatga catttttctg gtataaacaa gcacactgat 360

ctcagagggt atctaaaagt gttgccagta gggcagtgca ggttcttgtc ttcaaagcat 420

aagagaatct gaaaatgaga cccaagcgaa agtatagaca ttttattgtg gagcaaaacc 480

aacgacaccc tcaagggagg agtgcaggca ctcaaagatt tgagtcacag gcaatgtggt 540

tcacgtacta tatgaataag cacaatgaag gtatggagta ctctgtaatc aaggggaggg 600

gttttctggg aaatgggcgg gcaattcccg gaatcggggt gccacccttt tcttgactaa 660

agatggttaa tccggaactg tcatggcgtc aggtgcatga tgggagtggg aatttcccca 720

tggaagtttt atgatattaa gggcataatg aagctaaggg tcagcagtca gccaagtttt 780

cgaccatctt ggctttaacc agttgcggcc agtttcttct gtgttacatt ctcatctgtg 840

cctaagtagg aagtttgcaa tctgtcttca ttgtcacagg ctgttgtaac agttcctatc 900

tactagtggg gcaatccctc tgctaacctt tttgaccgca tttttattgc ttgtagccac 960

ctgcaagtaa tcatgcctca ttccctcact gactgcttgc ctcaaaagca tatatacagt 1020

ctttcttcct aacccaggct cctaacccta aattcagagc ttgagggctt gattctagtc 1080

agcaatttac ttgaggtttt cctttcttat tcattgaatt aacaattatg cactgaacat 1140

ctaatatgat gtattagatt ctgagaatat aaaagttagt aagacaaagt tccagatgtt 1200

g 1201

<210> 4

<211> 1201

<212> DNA

<213> Homo sapiens

<400> 4

cagggagttt attttggggc caccgcatga ccaacgttga caagccaaac tataagatgt 60

tatacatctc agatttcctg tttggaggtt gcactgagga agctgcaatc ctgccttccg 120

ttcctccggg tgcatcgaat acagactctc acttcagaga ggatcccgga gctttggccc 180

tcacgtgatg tcatcatttg tgatccttga catggaggcc atatatgggt atgtttctgg 240

aactacccag gggagagcca gacaggagag agatgcagtg ggtcagagac agaggcaggg 300

aacgaggacg aggagacgca tagttgggag acaaagagtg aggagcaagg gaggaggaag 360

aaaggggaag aggagaagta aaggcaacaa aggagggaag gcaagacagc aggaaggaag 420

aggatggagt gaaagagaag ggaagaaggg gagagaggag atgaaggagt tgacgtaaaa 480

gggctttgga gaaatcaagg tttggtgctg agcttgtggc agcatttggg gatagcaggt 540

gtctggctgc tggatactgt ggggtggggc tggggataca aagggccagg gtacaaatga 600

gccaacaaac aaacacagag gcatgcgggc aagccctggg cagagatata caacagaaga 660

gagcccttcc tcctggtcta gggaagaacg tcccatttct ggaataggtc aaatgtcaaa 720

gacaacacat acacagccac atgcactgag catccgggat gtgacaggac atggccaggc 780

acctactctc ccacccgtgt gttcacatct catgcctcag agttaggggt ccagaaacat 840

gacttggtct gttcctctaa ggggagaatc atttttgttt cagtggccca ttccccttgg 900

aggatgtgga tttatgatgg ccacagcatg atttctcact gattcaaact caggaaacct 960

caggatgcct ttacacgcac ttcgaatatt ccacaaggaa atacatctca gtatacaagg 1020

tgccacgggg ttgagtcaag gttctggagg tctgactggt gctaaagatg ggaaggacac 1080

cagtggtccc caggcagtcc cccagcttcc ctggtacctg ccaaattgct catcacccca 1140

gctgggtcag tcttcccagg ccactgtggc tgggtggccc tcaaagttag ctcaagttca 1200

g 1201

<210> 5

<211> 1201

<212> DNA

<213> Homo sapiens

<400> 5

gagcaaaact ctgtcaaata aataaataaa ttaattaatg aaaaagaggg cattatggat 60

ctaatctact gttgtatttc ctgtattttc tacacagtgg gcattcagtg accacctgtt 120

gagtgagttc atggcatcca agtgcacagc cacctaaggc atcgggcctc atgaaatcac 180

aagcagccaa ggctggcagg ttcctgggac aatttagcaa actcaaggct atggtgcggg 240

tgggtgcagt ggctcatgcc tgtaatccca acatatgggg aggcagaggt ggcggatcac 300

ctgaggtcca ggagttcaag accagcctgg ccaacatagt gaaaccccat gtctattaaa 360

agtacaaaaa ttagccgggc atggtggtgc acacctgtgg tcccagctac tcgggaggct 420

aaggcaggag aatcgcttga acctgggagg tgggaaatgg gggtacagtg agctgagatc 480

acgccactga actccagcct gggcgacaag agcgaaactc cgtctcaaaa aaaaaaaaaa 540

aaaaaaaaga ctacggtgct aggaccaatc tcctgagtca caaaatttaa ggagtcgtcc 600

aaaaaagtaa ataagataaa tattttcaaa caatattaga aaattcaaat cggaaactac 660

cgaccgggtg aggagttgtt tttgtaaata aggtcttatt cctaataagt ttcgaaagtc 720

taaatcttca cacgagtcac tcaaaagaaa ggcccagctc aggagcctgc atctgcgcta 780

tggcgggtaa gccccggtgg ctgaccggaa gaagcgccca cggaggcgaa ccccaggcgc 840

ccctcagcct acttccattt cccctcgcga cccttcgccc cgcccactct agctggctgt 900

gcctgactgg ctgggctgcc gcatattcgc gctgccattg gtccataatc agctgggggt 960

gggcgtaggg agacctactt cagcaggtcc tggccggacc agcattgtta acggctgcgg 1020

ttcgaagtgg ctgtgggtat tgctgcggct ggtgcgtcgt cgctctaggt acgttggggc 1080

cgggcgagtg tcctgcgaga gcggcgattg gcggggtggc ggctctagga gcgtcggtcg 1140

cctctctgag ggccggcccc ggaggcctcg gagcgccggg cctaccggac gtttgcggcg 1200

a 1201

<210> 6

<211> 1201

<212> DNA

<213> Homo sapiens

<400> 6

ccccgcatgg ggacgctgtc acagagcaag gcaggaggaa aatgtggtgg tcagtgaccc 60

ggggaatagg aagggagcag ctagggttga gatgccaggc cctggccagc aggtttggag 120

taaggggtgt agagaaacag gaagtgttac tcagcctggg ttcccctgtt cctcccgatg 180

gctgcacccc tcccctctcc ccggctgcag cagtcccagc taggccggac tgctaagcct 240

ggcccagagc acggctgaca attcacggca gcctccctgg cccagcctcc tgcaagagaa 300

ggaggacccc agaggaagcc cggtagccag aggggaaggg ctggagcaca gccaggtagg 360

aggcaacacg gcccggcctt cagcagccgc tctcctggca ggagccctct agggtaaaac 420

aagggaggac aaggaaatag cagccaggct gatggcaggc caagaggagg ccacatctcc 480

catccccagg ttaatgctgc tctgccaaga aaacttggag ccacccttgg cgggccccca 540

gcccaaccct gggcagcaga gcccgcccat cacaggcatg gcgctgctcc ccattaacac 600

accctctgcc ccccttcctg ttcctgctcg tgagggcctg acagcggggc tgagtcaccc 660

ataccagcta caggtctctt ccttccccaa caatcgttcc acacgcctgc ctttcaatta 720

actcccaccc gccccgaact cttcctaccc agacactgtg ggcagggagc caggcgcacc 780

tgctgggggc ccagggtggt ggggctgtga tcagaaagga agccgcgtgg tgacacataa 840

agcagggctg cccccacacg agtccccaga ggaatcttaa gctccccatt gccctggggt 900

gttggtccca gctgccccat ccctgcacgc ggatgctgca attacccaag agcacgaaac 960

ctggcttccc acctgactga gtcagctgag aacaaaaaca catctccacc gccacaaaca 1020

gccaggccac tcatcagagt ccgggagcca ctggcagaga gggcacggcc ccacactgcc 1080

ccagagacat ataggagcca ggcaaacacc agggcgagtg agctcgggcc atctgctgcc 1140

tgcctgtgga aggagcgctc gccagcctca cccagcccga gaaggtccac ctctgcctcc 1200

g 1201

<210> 7

<211> 1201

<212> DNA

<213> Homo sapiens

<400> 7

cggctggatc cctcccccag accctgtaaa cgtcccccac gtgtttaaga ccccagacca 60

gttccagcct tcctctcccc tgcacccaca acaaatcagc cccgcttcag acctttctct 120

cttcttcagt gtctctggat ctggttgcaa gctcagtgcc agtggcctct agcccgctgc 180

cgaggcgcca ccttcagtac tgcggccagg agagtggcgc tcccctctta taccgtaggc 240

tgagtcccgc ccagccgcca gcggcccaac ccggaggtgc tgcacagccc gccttcccgc 300

caccctctcc cacgacgccg ggaggtctgc atgcactggg cgctcaggtc tgcgcgaagc 360

tgtcgcggga gcaggcacct tgtggtcagc tctgggaccc gcatttgcag tgagtgcgtg 420

gagccccagg caaggcctgg gaaaggttgc aggtgaagag agggcaccct gtcgtcctgc 480

caaggaaaaa ttggcggctg aaaatgtttc tggcttcttg gagaaggtga ggctggacga 540

ggaaacagga agttaatttg gctaggagat aaaccgtcac tcaccctgca tgtaacctgg 600

acgtgacctg cacgtcctgg gagcttctgt tttcctgtcc ccacgcactc gcagcgtgca 660

gaccaatatg gtttccagtt atttatatac ttatttactg tctgtctccc tctgctagac 720

tgtgctcttt ttgatagcat aggccctggc tcagtccatg ccctaagtac tggtgaagtg 780

ggtaaattaa tcgttgagaa aatttctgat cgcttcttgg tcttttggct aagatcaagt 840

ggagaaagcc tctgaatggc tgcaggctct gcctgtcatc ttgatagtta caaaacatgg 900

ggcttctggc tcctgcccta aaataacact tgacgttttt aaaactaccc cttttttttt 960

gctctagcac ggaggtatcc agttagaaag caccttcccg aaatatagat tccttctttc 1020

cacccccgac cttcattttc tgagatgtga agactgaaat cccagaggga cctgaggaaa 1080

ttccagaaag gttaaaaaga gccagttaat ggcagaagct ggaactagca atttcctgtt 1140

tctttggctt tgcatgtgat gcttcctgtg atctgaaatg ccttttctct gctttttctt 1200

t 1201

<210> 8

<211> 1201

<212> DNA

<213> Homo sapiens

<400> 8

tgtatacatg ctttcatttc tcttaggcag atccctaaga gtgaaattat tgggtcacat 60

ggtaaattta tgtttaacat tttaaaaaat tgccaaactg ttttccaaag tggctacacc 120

attttacatt tctaccacca atatatgagg gttctcgttt ctccacattc tctccaacac 180

ttatcttttt aaaaaaggtt tttgttgttg ttgttgtttg cagtttttac agttttattt 240

aaacagaaaa catacacatg agctgtctac tcattttctt tgctgcgcag cctgggattg 300

gggttggtga ctctgacggc cagctgggca gctcttttcc acgatggctt tgcggttctt 360

ggaggaaaca ttgtgagcga tctcagcaca gtaagatttg ttgcacaaca gcagcacttc 420

cagctccttg acgtgtggac caggaatttc cggaagccac tgggcagcat gtgctttgtt 480

ttcttgttgc tcccataatc aatgttgggc atcaagatct aacccttgaa ccttccacga 540

accctgttgt caatacctct gggtttccgc cggttacgct taattttgac atatcggtca 600

gactggtgcc ggataaactt cttggttctc tttttgacga tcttgggctt cccaagggtt 660

ctgaggacgg tttataaaag tgcactgagg ggaggctgag acaggagaat tgcttgaacc 720

cgggaggcgg aggttgcagt gagccgagat cgtgcccatc ctaggcgaca gagagagact 780

ctatgtcaaa aataaataaa taaataaata aataaataaa taaataaata aataaaatgc 840

actgaggaga cagatttttg agagttaaaa ggaccctctc aaaaaccgac cgcggtcctc 900

cactgggtag ggggcgctcc aagacaggga ctcttctagc ctgttgcctc tccatgttag 960

cgatatcctc gttgctagga gactagacgc gtcttacagt gggagagctc agcctcactg 1020

atagccgggg atctctcctg ttcgagggct gagatctccg gtcccacaac cgcccacaac 1080

caggggcact tctggaagtc ccctttctag ggacattttt cttctaagct ctgtaacgcg 1140

ggccgaggca gaccgaggct tctttcccgg atccaatccc ctgcccccat gcctaagaaa 1200

g 1201

<210> 9

<211> 1201

<212> DNA

<213> Homo sapiens

<400> 9

gggattacag gtgtgagcca ctgcgcccgg cctttttttc tgacttttta atgatggcca 60

ttcttgcagg agtaaggtgg tatctccttg tggtattaac ttgcatttct tcgatgatca 120

gtgatgttga gcattttttc atgtatttgt tggccatttg aatatctttt gagaaatatc 180

tattcatgtc ctttgcccat tttttgatgg cattattttc tttcttgctg atttgagttc 240

cttctagatt ctggacacta ggcctttttc cgatgcatag tttgcaaata ttttctctta 300

atctgtggtt gtctattatt tcttttgatg tgcagaagct ttttagttta attaggtccc 360

atttatgtat ttttgttttt gttgcatttg cttttggggt cttagacatg aattctttgc 420

ttgggctgat tattagacga acatttattg agcaccaacc atatacgaca aacgatgtac 480

taggtgttgg gaatacaatg gagaacttac atgaatcacc agtcacacaa atctctttta 540

tctttttttt tttttttttg ccttacttga gaatttgggt ttttgagtaa acgccattat 600

tctctctttt tttaagcaca cttttgcatc tcaaccacct aacacactgc atccctgagc 660

aacccggagg ggccggttcc tgcacccagg ccaacacaac ttaaacccca acacaccaga 720

agaggaataa aggccaacca acttccgccc acccactaca aacgtcgaga gaaattcgta 780

gatgaggcct tgtcaaaatt cagaggcgtc ctacgtctgc tttcccaatg agacagttcc 840

tttttgtttg tctcagagac tggggttttt ttttttttaa aagactgtag gcagaaaaaa 900

caccccaacc ggctgttgtg acaatcaaac gtgtctaaga aaattcagcc aatgagagtg 960

cgagagtgca tcttgtgttg gccaatgaga acagcgaccc gtgcgcaggg ccgcccaatg 1020

gggcgcaagc gacgcggtat ttgaatcctg gaacaaggct acagcgtcga agatccccag 1080

cgctgcgggc tcggagagca gtcctaacgg cgcctcgtac gctagtgtcc tcccttttca 1140

gtccgcgtcc ctccctgggc cgggctggca ctcttgcctt ccccgtccct catggcgctg 1200

c 1201

<210> 10

<211> 1201

<212> DNA

<213> Homo sapiens

<400> 10

tttctttgtc cttttttttt tttttttaag agccaagaac tctggaacaa gaagaagaag 60

acgaaggagg aggaggaggg ggagggggag gaggagaagg agaaggagga gaaggagaag 120

gagaaggaga cggagaagga ggaagaggag gaggagggcc gggcgcagtg gctcacgcct 180

gtaatcccag cattttggga ggccgaggcg ggcggatcac gaggtcaaga gaccgagacc 240

atcctggcta acatggtgaa accccgtctc tactaaaaat acaaaaaatt agccgggcat 300

ggtggcgggc gtctgtagtc ccagctactc gggaggctga ggcaggagaa tggcgggaac 360

ccgggaggcg gagcttgtag tgagccaaga tagcgccact gcactccagc ctggaccaca 420

gagcgagact ccgtctcaag aagaagaaga aggagaagaa ggagaaggag aagggaaaaa 480

agaatcctca tcattaatgc aagtggaagg aaactcttca ccaaagaatt gatcacatca 540

tgaaaggtga aatcattacg gaattgctta aatatataat ttgaatctgg atttaaaaat 600

aataaatgta agttttttct ttaaaaaaaa aaaaaagtcc attggttaaa ttcagcttga 660

atcctttgcc actacatcaa gagctaacag gtcaacaact gatctccaca acctcccacc 720

tcatttcctt tctcagtcca ggtagcagga aaatggaaaa aaaaaaatga tttttttttt 780

ccaaacactt tgtcagtttc cccagcggtc acctttgaaa agggaaaaat gtctgaaaat 840

agacaaagct gaatataaac atcatttaat tccccccaca cagacagcct ccgctcctgt 900

gagggcgtgg ggaaaacgga gtgggagaag ggggctagcg aggaggaaga ggcgggaggt 960

gcggcagggg cacaggtgac gctcctcccg cctgcctagc agagctccag gcgcacatcc 1020

gcagtcagcc acctcgcgcg cgcctccagg agcaaggatg gagaggctgg tgagcggggc 1080

cggggcttgg gaaggagggc gcagggcagg cgatgttgag ccggtggccc tggggcgggc 1140

ggcgagtgga aggcgcggag cttcggctct tgccacaatc tggtccgggg actagatcag 1200

c 1201

<210> 11

<211> 1201

<212> DNA

<213> Homo sapiens

<400> 11

ctgcagttag actgtcactt acctggggac agagtcatgc ctgtcttgct cactgctgta 60

tcttgtgcct ggcacataac gggagctctg cacatttttg ttggctcact gactgactgg 120

ctgagggaga taggggcctg agatcctgga cattcagtcc gggctctggc ccctgaaaat 180

gtgctggcct gtcctcggaa ttgttccacc tattgccttc caggcgcctc tttcatgatc 240

tcaaaagaat agtgaaacca ggtgccgtgt ctcacgcctg taatcccaac actttgggag 300

gctgaggcag gtggatcaca aggtcaggag ttcgagacca gcctgaccaa caaggtgaaa 360

ccccgtctct actaaaaata caaaaattag ccgggcttgc tggcacgcac ctgtaatctc 420

agctactcag gaggctgagg caggagaatc gcttgaaccc cggaggtgga ggttgcagtg 480

agctgagata gcgccactgc actccagcct gggcaacaaa gtgagactct gtctcaaaaa 540

agaaaaagaa aaaaaaagtg aaaaaaattc ctgaatgaag gcctggactg aggtggcttt 600

ccatttggag gtccagcccc aagcatctga gagtccctcc taaattcatt acctacagca 660

acaacaacta gcaacaagta acaactggct acgatcctaa caactaatga cagggacatt 720

tatctccctc atgaagaaac ggtcccagga ccatctccca cccagcatcc attgcggttc 780

cctgtgcaag atgagtctct gagtgggaat ccagcactct ctccctcttc ttccccacca 840

ccttcaccct ccttaacgga aaaacaaaag gcatctgcac ctgcagccct gctgaggccc 900

ctgctgctca cacttgcagc agagggtgga ggctctgggt tcttgccttc tctcaaaggc 960

cccagcccca acagtgatgg gtggagccag tctagctgct gcacaggctg gctggctggc 1020

tggctgctaa gggctgctcc acgcttttgc cggaggacag agactgacat ggaacagggg 1080

aagggcctgg ctgtcctcat cctggctatc attcttcttc aaggtaaggg cctactaggg 1140

gtctggaagc ctggggaagg gctcaaggga agagcccatc actagtgaga caggaatatt 1200

g 1201

<210> 12

<211> 1201

<212> DNA

<213> Homo sapiens

<400> 12

ggccgacggg acgctgtcgc caaaagcaga gcagatgggg gagacgcggt cacgacaccg 60

cgtcctccgc cggctcctcg aggtgcgggc tctccggccg gacgctactc cccgaccccg 120

cagacccggg cgtccgccgc cagctcccgc gcccgcgttt cttccaggtc tacggaagca 180

gtgccatcgt gtggcggcat cttgcgttag acctgttttt agttccaggc tctttccctg 240

ttaaatattc caaaaaagtg ccttgaacct aaagtagttg ggtccttttt gggaaacagg 300

taagatctac cagtagctat agcagggtgc agatgggcct cgactcacga tggggttaca 360

tcccggtaga cccatcgcac actgaaaatg tcttaagtaa gaatgggctt tgtcatgtgt 420

cctgcaattt gaggcaggca tttctggtca gacccattta ttattttaaa ttaaaaatat 480

ggtttttttc agccgagcac agtggctcat gccggtaatc ccagcatttt gggaggctga 540

ggtgggcgga tcacttgagg tcaggagttt gaaaccagcc tggtcaacat ggtgaaaccc 600

catttctact aaaaatacaa aaattagcca agcgtagtgg cgtgcgcctg taatcccagc 660

tactcgggag gctgaggcag gagaatcgct tgaacccggg aggcggaggt tgcagtgagc 720

cgagatcgtt ccattgcact ccagcctggg cgacagagcg agactccatc tcaaaaaaaa 780

aattgagatg ggatctcaca atattgtcca ggctggtttc aaactcctgg gcttaagaga 840

ttcgccggcc tcagcctccc aaagtgctgg gattacaggc gtgagccacc atgtcgggcc 900

caaatccatt aaatcactat cgctagtcac acctgctcca tgtcgagtct cttcctgcgc 960

aggcctctca ccagatctgc gtcggaacac caagctctac taactgctgt gcagtttctg 1020

cagtcagttc cagaggtcat ttctaacgtt gcactatggg atatttaata ggtttcctaa 1080

agaacaaaca tatttcttta gagttactca gagggtacac aatgatgatg tcacacaatt 1140

aattacctat taagactgaa atccagcaat gcatagagtg tggacttacg cacatccaga 1200

a 1201

<210> 13

<211> 1201

<212> DNA

<213> Homo sapiens

<400> 13

agacataggg tgtcactatg ttgcccaggc tagcctccaa ctcccggctt caagcaatcc 60

tcctgcttcg gcctcccaaa atgttggaat tacaggcaca agccacctgg cccagccatc 120

tactttatat tcaaataaaa ctttacgtcc cattataaag ggaaaaaatg gcaaaaacag 180

gaggtaacca tttaacaaga aagcagagtg atgttagatt atagcaagat actgttgact 240

gtagaaggct ctgaggctag agagctgctt tctataaaac agagtgatca tatattagaa 300

gaggtgttaa agacatgttc acaccaagct gagacttcct ccttgatacc accaggagga 360

tgggcagaga ctggaaaaga cactaacttt ctccctatgg gagtcagtat tatttagcat 420

cactttggcg ggtcacccca aaccatctga ctacaagggt accatatttg ggttaacact 480

cttttggtat aatttatgtt ttagtccaat gtcttgggat gaaaatgaca ggtgggccac 540

ttatgatctc cagagaaatt cagggcaatt tggtgtggga gtaggcatgg tagaggagag 600

cagcatctaa gaagtcccca gcagaggctc tcagcttgtc ttgaggcatc tgggcggagg 660

gctatgatac tggccccatc ctgcagaagg tggcagatat tggcagctgg caccagtgcg 720

gttccattgt gatcatcatt tctgaacgtc agactgttga aggttccccc aacagacttt 780

ctgtgcaact ttctgtcttc accaaattca gtccacagta aggaagtgaa attaatttca 840

gaggtgtggg gagggcttaa gggagtgtgg taaaattaga gggtgttcag aaacagaaat 900

ctgaccgctt ggggccacct tgcagggaga gtttttttga tgatccctca cttgtttctt 960

tgcatgttgg cttagcttgg cgggctccca actggtgact ggttagtgat gaggctagtg 1020

atgaggctgt gtgcttctga gctgggcatc cgaaggcatc cttggggaag ctgagggcac 1080

gaggaggggc tgccagactc cgggagctgc tgcctggctg ggattcctac acaatgcgtt 1140

gcctggctcc acgccctgct gggtcctacc tgtcagagcc ccaaggtaaa aaggccggga 1200

a 1201

<210> 14

<211> 1201

<212> DNA

<213> Homo sapiens

<400> 14

ctctatcatg gggagtgttc agtatcggca tctagagatc tcccctggcc ccatcacagc 60

tagagctatg ctgtcccctt tcagggacat cttgtaattt atccacccag cccccaactg 120

atggacataa aggctgtctc cccatcatct cctgctacta cagacagcac tgcagggact 180

gtcctgctgt gtttttttaa ggcatgggta ctccagaagc agttgctcag ctgcaccccc 240

aaggttgagt ggaagtccct cggtaaagga ggaggagaga gtgtgaaggg aatggcaagg 300

cggggaggga gacagggcac aggtgtcctg gcaacagcag atgggaaaca ggtctggcta 360

aggtaccaga agccaacaag tcccagaaag gtcaagtgac tttcccaagg tcacacagca 420

agttgatggc agagctgggt acaggactca gagcatctga cacccaggac gatgttcctg 480

cactgttcca tagagtagcc cgactcttaa gtttaaataa ccccaggaag gaccccctct 540

ctgtgcctga cccccgctgg gttgtagagg cctaagtttg cagtagctcc tcccagccac 600

tctggctgtc agcctctgac ctgcagtggg agggtctcac cttctggaga tgttttaagg 660

aggggacccc ggcctaggca ctagctggga agccactgag ccatcttgtc tgcggccact 720

gacgtcagtg cttgggcaca atgccagcct gttcccggga ccccagcaca agggctactg 780

ggagccgggt ttggggcagg aagtcagcgt gaagcactgg tcctgccaca tcccaagccc 840

cttgtcccct agctctgtgg gggtggggtg acagctgcta tcgctttggg gttaactata 900

gtccctttgg ggttaactat agtcccattg gttatgggac ctcagtgtga tgtagccagg 960

ctggattccc ccttttgttt gttcatttat tcattcattc aggggtctca tgaccccacc 1020

agggaggagc cgtccaagag agttagcagc tcccacttgc aagatgtctg tggcaaggca 1080

ggcactgttc tggtgctgag agctcaactc cagtcctcac agcagccccc agagccaagg 1140

agtatcacca gtccattgca cagatgggga agctgggttc tagagtcaca gccagcagag 1200

t 1201

<210> 15

<211> 1201

<212> DNA

<213> Homo sapiens

<400> 15

ctggagcggt ggacacgtgg tctgggtccc gcgggttccc gggggcgact ggaccgtccc 60

cccagcccca ctcctgctgc agcagctgca aggtctgcag agccaccatt tggtggctga 120

tggaggccac taagggcgca gggctggcca tcgcctccgc tggcggcccg gcgcgctccg 180

cgtgcaggtg gagcagcagc gagggaggcg ggggcgttgg ggtcgccaag gtcacatttt 240

ctaacccgcg gctcctcggc ctctgggtct cacagcgtgt gtggtcggcc ccgcccccgg 300

ctctgaatcg gtgacccgtg ctggcctctg ccttcgcctg aagtccgaat caggcgtgtt 360

ctgacccaaa gctgcctgaa gcgctgctcg gtgtgcactt gaaaatcagc gaaatgctga 420

gcgttgcttc ttccctctct ccagggcttt gatgagaaaa cgtgctgctt tgtgcgggcg 480

catccatcgg cggggatacc ccgcccccac ctccttcgaa ggatgcctgc caatgaccac 540

tgaggtctgg gaggtggggg tgcaaaagac ccactctagg gcaaaagtac gaagggctct 600

agaaaaactt gcaaaaggaa gactgtgcct ttcccaaccc gaggggaatt ttcagaaact 660

tgttgaagca gccttcaagg ccttgcatcc cccaaacaaa agaaggagga gttgtgtatc 720

tggaagttct gctcttctct gctttgctgg gtgccacttg agcaccgaga gcctggtggc 780

tgaactagca tcagcaagcc tcaaggccca ggcctggcac tgcccagaaa agcctaaatg 840

tttttttgtg acactcccaa aagacttagg cagcaaggtt gaagttaatc cttttatgag 900

ccatgaactt agggacagct gtacaaatag ttgctgtttt taattaaacc caacatggct 960

acttccttaa cttagaaaaa agcgactagc aatgttatgc tttgatagat tggaaaccat 1020

gggtgtgtga tcacacttaa gaaaatagaa gatatttgag aggagtggta tggaaaacca 1080

gtccggagca gcattgttcc tgcttgcttt ctgcaaggaa aaccactaga caacccaaca 1140

gcccagtaaa gccagggcaa tggtgcatgc ccacgacaga caggtgggcc ctggcagacc 1200

t 1201

<210> 16

<211> 1201

<212> DNA

<213> Homo sapiens

<400> 16

aaggcgggag tggggggcgc tcccaggtag ccaaggggcg gtacctcgtc gcagtggcga 60

tggtactcct ccatggtagc gccggctctc cctggggcgg agcgacacgc cggtagagca 120

ccgcggccgc gcactggcct ccggggaggc cccacccccg tttatggcct agggcctttc 180

cctacgcaat ccccgccttt gtgtcgcagt ggcgtcacct ggcagcgctg ggcgggcttc 240

gggcaacgcc aggcgtcact tcaggccacg cactttttcc tcgcttcctc tgccctttct 300

ggaaacaagg gatgaatgta aagaactggg aatggcgcca gtgtggatca gatggggtga 360

cctagtggca gttagtctag actccgcact agtggttgag tgcccactgc tgtgattact 420

tgcacgtatt tgttcattca ccatcatgtg gcacataaat gtagttaatt tcgtggtaaa 480

ccagccactt tgatttgtcc acttactagc tatgttactt ggaaaaattc acctcccaca 540

gcttcagttt tctcatatgg aaacttacca tcatacctat ttcactactg gttgtaagga 600

tctgatgaga agctgtatac acggctccta aagtagatgc ttctcaacaa gagaacaatg 660

actacatttt aaaataaatg tagtaatttt aaattagtaa ttttaaaata aatgtagtaa 720

ttttaaatta gtaattttaa aataaattac taatttccag atgatcctag acacttcctc 780

aaaacctcac aataagtcag aggagttagt tcttccatca ctgcttttat caagatgaaa 840

ctgaggtata gagaatttaa tttgcccaag gtcatacagc cagtagggtg tagaaaaggg 900

acttgagctc aagtagtctg atagcagagc ccaagctctt aaccactgta ttactttcct 960

attgctactg taacaaatta ccgtaaatgt agtgccttaa agaaacacag tcctgttctc 1020

ttacagttct gttaccagtg tagggtgtcc aggttcttgg ctctttgaac aaagaattgg 1080

acaaaacgca caaacaaagc aaggaaagaa tgaagcaaca aaagcagaga tttattgaaa 1140

acgaaagtac actctacagg gtgggagcgg gcagagaagc cgctcaaggg ccgggatcca 1200

g 1201

<210> 17

<211> 1201

<212> DNA

<213> Homo sapiens

<400> 17

ccggtcggtc cccgcggccg ccggggccgc cgtcccccgc cctcctcccc gcccgccctt 60

cttccccgcc cgcctgcccc cgccaccccg cgcccgtgac tcacctcggc gccggggcag 120

gctggtccgg gcgggcgagg cgcagggctc tcccacactt cgcgccgcca atggcaagtt 180

ggagggagag atgcaaatac caggtgagac ggcggcgccc ctgataaaac ggggcggggg 240

gcggggtgga gcgcgcgggg agcggggcgg acgccgggga gggccgggcc aagacacggg 300

gcggcagggg cgcccgggga gggggcgcga accggcgcag acatgcaccg aaacgagcga 360

gcgaaagggg aaatgcagta gagacacaca cgtccccgca ggcggcggat ggggcgggcg 420

cgaaggaggc agggcgggag ggagggaaga gccggaggga gggaccgacg ggagggactg 480

cgggcgggcg gggtggggga gggaacgagg gagggacgag gcatctcggt ggaaacagcg 540

cccgtccgcc ccggcccgcc cgccgcggtc gccagcacgg catggggagc cgtgccaggg 600

gcccgcgcgg cctctcattc ccctcgcggc tcgctcgcgc ccgggggacc acgaggcagg 660

gcgcccgcag agcccgcgtc gtgcgccgcg gcccgggcgg gcgctcccca ctctgcttct 720

agccagcgag actccgagct ctctgctcgt tccgcgccct tcccggtgcg aagcccccat 780

acaacgcccc gcagagaaat cccttccccg cggactcggg aaggtttcgt ccagccccct 840

cggcccgtgc aagtggagac ttgcttgacg cctttgggcc aagccctgcg ctccacatta 900

ttgagctcct ggggtgtgca ggccccagcg ggagggcggg ccgtgcggcg ggtgaggccg 960

cgagatgccc agaggataca attttggaac catatgcccg actccgcaga atcctgggcg 1020

taacacctgg ggtgactgga gagtttcggc gacttcttag ggaagtcgag tctaggactc 1080

ctgcgggaag ttggggtggg ggacgctggc aagatatgag tcaggggaag ggctaggggg 1140

agaggggctg gcccagcaag tgcaaagaga gccagagttc gggtgctgga agaaagccca 1200

t 1201

<210> 18

<211> 1201

<212> DNA

<213> Homo sapiens

<400> 18

gcgtggggct aggcacgcca ccagccagtc cccaagccaa catcctgagc cccgcggctt 60

gctcaccgag tgacttcatg tagtcatcga aattcttgct gtccactagc ttccaggtgc 120

ccaggaaagc gtccaccata gtgatgctgg gctaggctga gagaagctac aagagagcag 180

gcgtgcaagg gctccgacgg cggctccctg cccgggctgc cgctttaaat agccctcgca 240

tcacatgagg agacgtggcc cacgcccccg gctcccgaaa taggaagccc caggctaggg 300

ctcacccagg aggggctggt gcgcccatcc cccaacccac cgaggtgggt ggggaccagc 360

cagtggcgcc ctgctagcct gcgtcagcgc cagtgcctgg cccccgagtc cctaccccag 420

gctggtagga ccttctccct ttcctggcct ctaggcactc tggaatctga gaaggaagca 480

gccaacacca gcccgagagc tagggcgcat gccatggtgg atatcttccc gtcccccagt 540

ccctctttga gcttagcgtc agagcagaga tgagactaga aaagcaaaac gcccttgggc 600

tggtgcccac gccggggaaa gctctggttg acttctggca cgggcatctg tcctcgtttt 660

acaagtgagg agtccgaggc tcacagggga actgacaagg tctcaagaaa taggaggggg 720

tgaatggagc tcagattgca agcttagtct agcgggcttc agagctcgcg ctctcccttc 780

actctcactg cctggtggcg gagacagtca ccagcggctc ggtctgcggc tgagctgacc 840

ttgtcctctg cctgttttga gcaaaagcct gacccttcgc gccccgcccg cctcacacgt 900

gcttgcccct ccctgcggaa tgtctcctgc tggccctcct ccgtccccac cccaccccac 960

catctagctc cccacccgcc cctgcaagtg ctctgggctt ttccggtttc ttcgcacagc 1020

tccgagcttt agctcaggtc gttcccagct cccctcctgc agaaattctg ccaattcggt 1080

cagctcttca aggcccatgg ctgccttccc cacctgccgc cctttctttg gcctgtgcac 1140

actagcaagc cgcctgcact tttgggtaat ttgctgcctc ctttcgagag cctgttgtgc 1200

g 1201

<210> 19

<211> 1201

<212> DNA

<213> Homo sapiens

<400> 19

gccaggttag tcttgaactc ctgacctcgt gatctgcctg cctcggccta ccaaaatgct 60

gcgattacag gcgtgagcca ccgttcccgg cctatacgtt gtttattttg gaaaaattaa 120

aaattaagtt ttttttcatt aaagatatgt tatttccgat caagagatca agaccatcct 180

ggccaacatg gtgaaacccc gtctctacta aaaacacaaa aattagctgg gtgtggtggc 240

acacgcctgt agttccagtt actggggagg ctgaggcagg agaatcgctt gaacccggga 300

gaaggaggtt gcagtgagcc gagatcatgc cactgcactc cagcctgggg acagagcaag 360

actctgactc aaaaaaaaaa aaaagttgtt tctattaaca tgtaatgggt tattaatatt 420

ctcttaaatg aattaatatt tttaatattt tgttttaata tcttttaatt tatatatgat 480

aaaaattgat acaatccaca gaaacaaaat ttatttgggt cctcactaat ttcttttttc 540

ttgttgccca ggctggaggg caatggcacg atcttggctc accgcaacct cctcctcctg 600

ggttcaagtg attctcctgc ctcagcctcc caagtagcca ggattacagc catgcgccac 660

cacgccggct aattttttgg acttttagta gagacagggt ttctccatat tggtcgggct 720

ggtctcgaac tcccaacctc aggtgatcag cccgccttgg cctcccaaag tgctgagatt 780

acaggcgtga gccaccgcgc ccagccagga ctaatttcta agagtgtgca gagataccga 840

aacctaaaag tttaagaact gctgattgct gggaaactct gcagtttccc gttcctctcg 900

taacctggtc atgtgtcctt cttcctggat actcatgacg cagactcagt tctcattccc 960

aatgggtgtc gggtttctag agaagccaat cagcgtcgcc acgactcccg actataaagt 1020

ccccatccgg actcaagaag ttctcaggac tcagaggctg ggatcatggt agatggaacc 1080

ctccttttac tcctctcgga ggccctggcc cttacccaga cctgggcggg tgagtgcggg 1140

gtcgggatgg aaacggcctc taccgggagt agagaggggc cggcccggcg ggggcgaagg 1200

a 1201

<210> 20

<211> 1201

<212> DNA

<213> Homo sapiens

<400> 20

tgtgcccggc cctaaaccca gctcatttta aacattggtt tcaattccat tcaccctatg 60

atggagcatc ctgcccctcc ccgacaactc ccatacacca ctgagcctcc cctaccccag 120

gtgcctgcaa gcccctcaga gaccaaggct gtgtgccagg gcttttctct cccagaacat 180

ccttgatagc cacatttaga aaacagtcgc cagaatggcc tctgtattgc cagcagtttg 240

gcagtgggaa agccagcttt tggcttccag acaggaaaag caaggcgtgc tgaccacaca 300

cagcctgcac ctcagtgacc ctctggccag aggctgcctt aggattctgg gaggagagct 360

agacctggtt ccctgcagca acaccttggg aggttaaaaa atacagatga ctgggtctct 420

accccaaccc agcagtagga tccagggatc cttatttcta acaaactccc ccgatgattc 480

caacacacgt caaggctgga agcaaccgat tcgtttcttt gggcctcagt ttccccatcc 540

aacttaaaac tcacacagga gtctagataa tcttctttga gcctcacggc catctaaaaa 600

aatgtcattg ttcccatttt tgagatggga agaatggagg tttatagagg caaagggatg 660

aaggggtggg ccacatggtc attagtcttt ttcggcgctg gggtttgaag atcctaagac 720

gacctaagga aggcagctga atccgccttc caaacccctg gggtagactc aggctctcac 780

acacccagga gagtcatttc taggcacatc ttctcgggtg actcatgagc accaaggcag 840

gcctgtgtgt ccaagcgcct gcacaggcag aagcgaggct ctttcccata accggaaccc 900

ttaccgtgcc cctagcaaac tcccagctcc tgcctgacaa gttactgcct aacccgtgac 960

ttatttcggg aatccctgac gcatttcttt accgtccctc atttcaaagc acaccatgcc 1020

ggcagcactt ggaggtggta tgtgctggga aactgaccaa cagtttagtc agactgagtc 1080

acttcaagga ggggagtgga gacagcagtg ggtgccagat gacacggagc tgtcggcttc 1140

cgtgtgcatt ttcttttgat agtagccgta gagaagttgt ccgccaggca gccctcaatg 1200

g 1201

<210> 21

<211> 1201

<212> DNA

<213> Homo sapiens

<400> 21

tcagcactta cccaacgaac atttatggag cgcctgctgt gtgcctggct ctggagaggc 60

agcggagtgt tctgcaacct ctggagctgc cctcctagaa accccaacct gttgggggag 120

gtagacgcca gagcagcggt gaccccacgg agtgatcagt tgtgataagg gactcacagc 180

aagtgaagga ggcgcctgac ccagtcattg ctgggagccc tggggtagag gaggcagcca 240

agggtggcga ggcattgcag cggggggcgg tatttaggga aatgaaagag gttgacagaa 300

gctggaacct agggaggact gagcaggtgg ggtgggggtc ggggactgag gttagctccg 360

ggcgggttga gggggtgaga ggaatggggc cggcgggtct ccaacctgga gccgcaccgg 420

tagctgcggg actgcgaact cagcaggtga tgggaagcca ggtcgggagt ggtcagggaa 480

ggcacgggct gagctgcaga tcagagacac ccggtctctg gccgcgcggg ggtggggcag 540

aactggaggc gctgaggcca gaggcaagag tgggggactt gggcggaaag gagggggcgg 600

ggcgggattg gcgagcagcg tggtgttggt gggggggcag tcccgcctcc agccccagtt 660

agatgccaac ccgcccgcag cctcttaaaa tagagaaacc gcctccgggt aggcgaatgg 720

gggtgctgct gacgtcaaga agtggggcct gttccatggt gggggggtca aacatttcct 780

gatggtaagt tctatgttac aaaaggggaa actgaggttc agagaagcat agccactgtc 840

cgggtttcac aaagtatagg tgacagagtc agacaggatt tgaacccacg cagttccggc 900

ttcctctcca aaccaaccca gcacaggcgt ccctccacga aggtggaggg atgagcacgg 960

attccccgac tgaatgaggg cattccgggc agaggccaca gcacgtgcaa aggcccagag 1020

gcatgtagcc tttcttacct gtttatatgg ggaaacaaga acttgaacct atttcttagc 1080

actagaggct gatggccagg acttaatgct tccagggcct tcagtgctga ttttgaagag 1140

gttagacttt ccccctaggg tgatggggag ctatggaagg tgctggagca ggagaggaca 1200

t 1201

<210> 22

<211> 1201

<212> DNA

<213> Homo sapiens

<400> 22

tgtagtccca gctacccggc aggctgaggc aggagaatgg cgtgaaccca ggaagctgag 60

cttgtagtga gccgagatcg caccactgta ctccccgcct gggcgacaga gtcagactcc 120

gcctcaaaaa aacaacaaaa aaaaaaacaa cacagaaacc caacagagac ctgaagacgt 180

gcctgcctcc cccatctccg ggacctctcc cacctgggtg aagggggcac gtgagcctca 240

cctgatcttg accatctcag ctaaaacact gaaccaccct tggcacccac cagcagcaga 300

cggagaagaa ctgtataggg tgcaaaagcg ccccgccacc atcaggtcca gatgaaaacc 360

tacaagttga atcagcaagg tgctgacgga tacgcaagca gaaaccctct tataggaggt 420

tcaaggctgg gcgcagtggc tcactcctgt aatcccagca ctttgggagg ccaaggcgag 480

cagatcactt gaggtcagga gcctcagacc agcctggccg atatggcaaa accctgtctg 540

tactaaaaat acaaaaatta gccaggcatg gtggggggtg cctgtaatcc cagctactcg 600

gaagcctgag gcaggagaat cgcttgaacc tgggatgtgg aggttgcagt gagctgggac 660

agcaccattt ccctccagct cgggtgacag caagaccctg tctcaaaaaa aaaaaaaaag 720

tttgagaatg tcagacaagg cagcaccctg tgtaggtgct caggttgggc ggagtctctc 780

tcgggggagg ggggtaacag gacagcgcct cccggggcct ccttgcatgt ctgtgtcctg 840

atgcacagca ggcgggggct gcggggacca gagaaggaag aagcgcccca gctgggcact 900

tcccgaggag gaagtggtgc cggaatttcc cccaggtgtg gcctcaggta taagagcggc 960

tgctgccagg tgcatggcca ggtgcacctg tgggattgcc gccaggtgtg caggccgctc 1020

caagcccagc ctgccccgct gccgccacca tgacggtgag cctctccaca tgccgctcgg 1080

atggatgctg cttggatgtg cagcctggca tggaagggct ggggttctct gagcctggga 1140

ggagggtgtt gggatggggt ctgggaaacc cctcagtctg ggggtagggg aggcagctgc 1200

a 1201

<210> 23

<211> 1201

<212> DNA

<213> Homo sapiens

<400> 23

tcctggctgg gcgtgggggc tttgctacca gtctgccctg ggtcacgtct agaaacctca 60

gctggaggcg cgggtcgtaa agtcctgaca cttcctgttc ctctcagctc ctccttggag 120

gaagtggtgg gttgcaccac cgtggccctt cacccctgcc cctctccgct ctacagcagc 180

tctgatgtca gagagcggcc ccttgagaca ggtgggacat gtcatttctc acatgaggtc 240

ccggcgagaa ggacctgctt ccatcagatt cacacttggg agaggggccc ggcctccagg 300

agcgccaggc agaaagggca cccattagga agaagaacac agaggccatg ggagtgaaaa 360

gtttgatgct ggaaagcaac agtagcacat tctgctacag agcggaatga aaaaccctgg 420

ggagcaaatt cctgagccag agaatggagc ggaggaacca gccaaggagg ataaagaggt 480

gggaacaggc aagaaaacct gggtgcgtgc acgcattgac atccacctgc atgtgagggc 540

gggaggggac agaacaggga cagcgaggag gagacactca ggtcacagga ggaaggctgc 600

ggatctgggc ggggacgtgg attgcgaggc cactgaacac cccagggatg gatggatgag 660

gtcctgttaa ctagggacat taggaggata ccacagaaca cgaaagggaa aatactacag 720

gctcagagag agagagaagg gagaggagga ggaggaggaa gaggagggaa agagattata 780

cctagaaaaa aaacccaaat cagacatctt cagagcaagg actcagacac attactaccc 840

taaactctgt tttatttatt tatttatttt tatgttttca gttttttttg agacagagtt 900

ttgcttttgt cacccaggct ggagtgcagt ggcgtgatct cagctcactg caacctctgc 960

ctcccgggtt caagcaattc tcctgcctca gcctcccaag tagctgggat tacaggcaca 1020

tgtaccacca cgcctgggta atttttgtat tcttagtaga gacagggttt cactatgttg 1080

accaggctgg tcttgaactc ctggcttcaa gtgatccatc cacctctgcc ttccaaagtg 1140

ctgggattac tggcgtgagc catggctccc ggctcctaaa ctctgtttta aagatgtctt 1200

g 1201

<210> 24

<211> 1201

<212> DNA

<213> Homo sapiens

<400> 24

gaccccacaa atcatgcggc ctgtgccacc atccagtgcc ggatctgaac tactgatcca 60

ctgagctttg ggctgattgg ttctgcaacg atgggtggcc aaggccacgg tcaaatccag 120

ctggagaggt ggatggggcc agtgtcaggg gtttggattt caccccaagt cagctgaggc 180

acactggaat gtcgtaagcc tgcacatcca ctcatcacac attttaaccg catctgagtg 240

tggaccgtac cgatgggttc ctgcacctgc agcctccaca ccattgctgg tgccctgcct 300

cctagctgga gtatccttcc tgtctcttgg ctgctctttg tacccatcat ggtcccccta 360

ccaccctcca agttctgctc caagctttgt tttcctccaa gagaagaacc tgtccagaca 420

atagtttcaa agcagcggga agctctgctc acgtgtcccc aaggaccatg ctgtgtgaaa 480

ttcccttctg taatcacaga gcccattgcc ctggcctatt cctggtccag gaatagggag 540

gaggtagaca gaggatgcct ccttccccac tgctgagaac cctgccatcc tcagccacag 600

ttgccacaga gaagatacca catccctggg ggaatcagca ggaatcaggt agagagtggc 660

actgctctgg ggagggaggg cgtctcacag catcaaacgt caaaaaccca caacattgac 720

ccagtcctgc caagacggaa ccctgcatga gcatggggga tggggagttg gggtgttgca 780

aaagacgcaa tacatgaatg atctcaggta attctcaggc aacccccgga ggctggtgtt 840

gctagcaccc ctctgcagga gaagaagctg gggctcggga gctgactgga tctgctcaaa 900

ggcccaggaa gaataagagt taggaactgg gacagacctt gaggaagctg cacttcctcc 960

tgaggtgagc cagcgttgga gctgtttttc ctttcagtat gaattccaca aggaaatcat 1020

ctcaggagga agggctcata cttggatcca gaaaatatca acatagccaa agaaaaacaa 1080

tcaagacata cctccaggag ctgtgtaaca gcaaccggaa agagaaacaa tggtgtgttc 1140

ctatgtggga tataaagagc cggggctcag ggggctccac acctgcacct ccttctcacc 1200

t 1201

<210> 25

<211> 1201

<212> DNA

<213> Homo sapiens

<400> 25

gtcttcttct tttttttttt ttttttgaga tgaagtcttg ctctgtcacc caggctggag 60

tgcagtggcg ctatcttggc tcactgcaac ctccacctcc tgggttcaag caattctcct 120

gcctcagcct cccgagtagc tgggattaca ggcacccgcc accacaccca gataattttt 180

gtatttttgg tagatatggg gtttcaccat gttggtcagg ctggttttga acccctgact 240

tcatgacctg cctgccttgg cctcccaaag tgctgggatt acaggcatga gccaccgcgc 300

ccggcccatg ggtgtgaatt tctgcctgtg tgtgtctttt tagcgtatgt gtgtctccat 360

atctgccatc tgcaggagca cctcagtttc attatcttgc tccctgtcta ttggagtgta 420

tctctcttcc tctctgtgtg actttttgtg tttatgtgtg tgtttagtgt gtgtccatat 480

tttcctgttc tcttgtgtct ctctgctgtg tctctctctt tttctttctg ttttttgaga 540

cggagtctcc ctctgtcacc caggctggag tgcagtggtg cgatctcggc tcactgcaac 600

ctctgcttct gaggttcaaa cgattctcct gcctcagcct cccgagtagc tggaattaca 660

gacacatgcc accatgccca gctaattttg tatttttaat agagacgggg tttcaccatg 720

ttggccaggc tggtcttaaa ctcttgacct tacgtgatcc acccgcctcg gcctcccaaa 780

gtttttggat tacaggtgtg agccacctcg cccggtccta tctctctttc ttctctttct 840

ccctctcact ttgtttctct tctctctctc tctcccgcct ccttcccccg tctcccctcc 900

cttcccccca ccgccctctt catagctgag cctgtccggc agtgcggcgg atgtacggat 960

gattcagtgg ctggcaggaa gcccgccctg cccgcccgcc agtgtcagtg gtgttggcat 1020

cagcttgggc aggtgtgcgg gctcaggatg gggcggccgt ggtgaggaac cctggactct 1080

caggtaagcc tttcccaggg gtgcctcagt cctcaggctg tcccccatcc cccaggagct 1140

cctggcccca ctgggtgggg tgggaagagg cctggatgcc tggcgccccc tcccgcagcc 1200

c 1201

<210> 26

<211> 1201

<212> DNA

<213> Homo sapiens

<400> 26

agaccagcct ggccaacgtg gtgaaacccc gtctctacta aaagtacata aattagccgg 60

gtgtggtggt gggggcctgt agtcccagct actcgggagg ctgaggcagg agaatcagtt 120

gaacctggga ggcagaggtt gcagtgagct gagatcacgc cactgcactc cagcctgggc 180

gacagagcga gactccatct caaaaataaa tatataaata aataaaaata aaaaagagaa 240

aggacagggt gcacagagcc aggaccccac cccctcctgg tctggaccac cctgggccat 300

ctcctaagct tgacactctc aggcctgagg ctgtcaggcc agggcgcttt gactatcccc 360

tgcctgcccc tgaacgcggc caagcccgag gccttagcca agccctgcag cctcaggctt 420

ggcgcccggc ccagcctttc tttagccttc ccgaggagcc agcgagagag cgctgcagca 480

gccgcagtcc ttgcttccgg ggaaggcccg ggtggggcca gggcctgtgg ttaagtcagt 540

ggccctctgc agacgcgcac gcagcaggag ccacgccctg gtccctgccc gggagtggcc 600

tggtctcccc cagctctgat tccctgaagg accctgcacc tcctcagcca tcttctctgg 660

gcaccccctg agcccagcct gctggccaca ggccccttcc cgccaacttg tgggcctagc 720

tcatcatcaa actattttcc actggcttcc cccaatctct gccacagcac aattttggtg 780

gggaggtgga gagggatgaa ggagaatagg aggtggagct tggactctgg ccgggggcgg 840

cggggtgagg gggggcagca gctcgccact ctgattggtc acctctgctc caaaactggc 900

ttcaaaattc cacggactcc gcccccagtg gcccccagcc ctatcctgac ttgcaattgg 960

ctgaactttc agggggcggg gctcacccgg tccggtccag ttaaaagggt gggagcgtcc 1020

gggggcccat ctctctcggg tggagtcttc tgacagctgg tgcgcctgcc cgggaacatc 1080

ctcctggact caatcatggc ttgtgtgagt gtggggaccc ccccccaagg tccaggggat 1140

agggcaggaa ctgatggcca gaggagagct gggcagatcg ggagcagatt ctagccccag 1200

c 1201

<210> 27

<211> 1201

<212> DNA

<213> Homo sapiens

<400> 27

tggtcccagc tacttgagag gctgaggcag gaggattgcc tgaggccaga aggtgaaggc 60

tgcagtgaac cgagattgtg ccactgcact gtagcttggg tgacagagtg ggaccctgtc 120

tcaaaaagaa ttggtttgga ataacattta aataatacct aaactccttg ccttggccta 180

caagacctga atatttggcc cctgccttcc ctccagccca cagcaccggc cccaggacct 240

ttgcacctgc catttctgct ctccagaaag ccctgcctcc tccccatctt tgtagtcata 300

ggtgtttctt gtcatttaga gtttcagttc aaatatgaca ccctcccatt gatggaaatc 360

atttcaccac cagacgttat gtggctcatt gtttatccgc tgcctgcctc ccggctggag 420

aggcgcaggg agctgcctgt ccctcctgtt cagagccacc gttccgcacc tggaccaccg 480

cttggcagag gcgggagtgg aggctgtcac catcaggatg aaggacaggg gtggcccggc 540

gtggagaagc taagaagaga tggcgatagg tcaacgcgat tgcgccttta aggtggaaga 600

aacgaagggc gagggtcagt aaagtcaagg acgggctcag atcccgccac tggtgataag 660

cgggacccat aggaccgacg tgatcgcggc caggccctgg gtggtttacc tggactttta 720

cctggctgcc ttttctctgc acccctccgt cctcactccc ccagtcatcg gcataggtca 780

agtcagagtg ttgaagaaac acacgagttt ggctctggaa aggaaaatca gaaaggcggc 840

ctaaggggcc ccacccccag gaacgctaag ataggcggcc aggggtgagc gggcagggcc 900

ggccagcagg ccctgaaagc agccgggcca ccagcgcgtc ccgagtcccc ctcgcaccgc 960

cccgcttgcc ccgcccctgc cctctgcccc cgcccctgcc ccgcccccga aggcgagctg 1020

cgctgacagc cggcggcggg ctgggtgttt gcaatacaaa ggcggccacg cgcggcgccg 1080

ctcggtgagt agccgccgcc tccagcctcc cgctgtggag caggggccgc agcctcgggt 1140

taggcggggc ggggcgcggc gccggggcgc tgaggacgcg tggcgaggcg ggggctcggc 1200

c 1201

<210> 28

<211> 1201

<212> DNA

<213> Homo sapiens

<400> 28

aggcagagta aatgctgggt cccggagcag acaggagaga ggacctggga gttttttagt 60

aagatgggga ggaggagata ggctatggct tggaccaggc ctggaagaga gctcaggtgt 120

gcagtctcta ggaacccggg tggagaagca gcaggaaata agcagaaaaa ggagacaggc 180

catgaagata gaagcgcaat ggtcctggat tcaaatctcc actctgcagc ttatagctta 240

cagtccgctt agctttgtgc ccattccaag aggatttccc tattgtagct ttacaatgtt 300

ttaatatgta gtattgttgt gcaaggtacc atcctaaact gactgacttt ccttcctcct 360

tctcctccaa aataattttt aattcttgca gacagacttt agatcgtgtg tttgaagttt 420

ttaaagatgg aatttttatt ggaactgcat tacacgtata aattatttag gaagagttgg 480

cttctgaaga gggcagctct gtgggcttgg gctgggtttg aatccagcca gacaactttc 540

cagctgtgtt accttggaca gttacctagt tcctctgtac cttgacttcc tcatctgtca 600

aatgggtgat gataatagca cctacaccat ggtcgttggg aggagtcagt gagagtctcc 660

aagtgtgctt tattatcgtt ggtggtggta gtggtgttta gaacgcatcc ctcagtcaga 720

cagcgaggta ggacttctcc gtttattaaa tcctgacctt ttttttccat gtaaaacctg 780

cacgtttctg aaatgctcag agtacgttac tcagtatgta cccatatgtt ctgtgggtat 840

actttgttag gttgtgatta gttcgttgga gctggtggtt gcagggacgg ctggaagcaa 900

ggagatgaag gagaggaagt cgtagctggg aaggggacca ggaagtgggt gtcaggtcca 960

agagctgcta gaaagaaacg aaactgaaag cagggaattt cccaagtttg gggaagacag 1020

gaactgcagc gcccctcccc gtttcacgcc acgcgcggga ccgaggacct aggacctggc 1080

cagctgggcg tggttcggag agccgggcgg gaaaacgaaa ccagaaatcc gaaggccgcg 1140

ccagagccct gcttcccctt gcacctgcgc cgggcggcca tggacttgta cagcaccccg 1200

g 1201

<210> 29

<211> 1201

<212> DNA

<213> Homo sapiens

<400> 29

agggaggctc attggaaagg gattttggga tacctgtttc tgactcagca tcagtggtaa 60

ctactggcat ggccagtgaa tgctccctct agctctacac cctcagagag tctcctccca 120

ggcagagaat gttttctttg tcacgtattt ttctcccctc taccctccac ccatcccccc 180

gcttcttctc cccttctttc tgtcttccct aggggaagta aagaggttac cccccgcccc 240

tctctctctc tctctctccc agaagccaag aggcaaggcc tcagtttatc actataacaa 300

ccagacgaca ctgaagcggc tggtgcggga tacagtatct catttgcata gagcgcgctg 360

gttggctact gtcatcatag taccactccg ccgggaagcg gccgggcccc attggctagc 420

cctccgtacc cacgggcccg aacttgctgc ttttgtttct tctgtttaat tcagttgcaa 480

aggtctccgt cctctctggg cacgggaccc gggctgcgcc acccagataa cataaaaccg 540

cttcctgagg ggcctaggac gccttgagaa acaacatagc agacctccac taataaagga 600

ccatgtccct gggatgagct agtgcatgat gtgctgtagt caggatattt gccaaatagg 660

agtcaccctg aagacaaagc cacttttgag cccaaattca ttgtgctaat tgccagtgga 720

gggcccctcc gggcattcac tatgggcacc tgagtgttcg acccctggac ttcatgcaac 780

gtgaaccctg gcagatcccc tcgtggcctg gtgtagccct ggagccctcc cctggggctg 840

ccctaggggg aactttgaag gaggtagatt gtctttaaca gcctgcctcc tactcctggc 900

tgccctttcg cagttgcttc ctctgtcagc cactcttccc cttccccagc tattcaaagt 960

ttcctcaaga agtcttgctg ttgtgtgcct ttcaatacac tgttccatgc actgctctat 1020

tttctcttgg ttcctacttt accagtcgtg ggtcccattt ccgaagtacc ctgctctcag 1080

ccaaatgctc atggcaatca gagatggaat gttaacgatc aggtgcttta atgtctacat 1140

tatctaggga cattcacatt ttaagaggag cattctgaag acacacatcc aaagaactta 1200

c 1201

<210> 30

<211> 1201

<212> DNA

<213> Homo sapiens

<400> 30

caccttcccg ccgcctcccc gccccccgcg gcctcgccgc gcctctccct ccttccccgg 60

tgacacaccg agttctccct cggcctcaca aaagaaacgc atctggcctg cgagtcccgg 120

gcggctcgtg cacacctcag gcctcaaatg acttgtttcg cctagaatca actcccgtct 180

cggccgcctc gaagtcccca gccctggcga gaacagccca ggatgagtga atgtttgccg 240

aacaggatcg caccgagcgg gcgccttcga gccagcgccg gggcgggggc agagcccggg 300

gcccgttccg gcagctccca gcggtgtcct cgtcgagggg ccgcctgccc ctccttctcc 360

agcctcttct cccgggaact cagcccggct gagtgacagc agcctgcgct ctaatgggcc 420

cccggatgct gcctcctaat tagcttggac cttctcccct acggctgctt ctcaggcctc 480

cctctctgca ccgcagtcaa ttttccggga ttgggataga aatgatggct tctaatcaga 540

cgaaacgttt ctgctggctc taggcgtttg gagaatgcag aaggaagacc ccaaggatcc 600

cagccagacc ccacagcctc ttgtacctct ccccttgttt tacaccttag gccttggcca 660

aatgggccag gcctggcctt ggcaggcatg gggttggcct tggcagcctt tgccctcctt 720

cagggtagct ttgtcccagc ctggggcctg cgtcctggct tcccagggct tcaggcatag 780

gcctggtaag ccaggtgact gctgtctccc aaccctgggc cagcagccca ggaagccctc 840

agtcttccag cttctctccc acacagtggt agctgagctg cagctccaat gggccttcac 900

tgggcacagg caaagacatt actctcccac tcctaagagt gattccctcc tcgagtcctt 960

gcacgtaggt gaagtttgtc gaaggatttt gctcaacaat tgttagctgg ccccgagacc 1020

tcagcgtctt gagcaaacag gccacagctg gggcggcaag gcgccgccct gacctgctcc 1080

tctggccata ggcggacccc agactgccct ctgcccctca cctgtgatca gctccaccag 1140

acacagcaac ccaagggagc tgcgccagga ttgtctgcct gggaaagaag cagggaggtg 1200

t 1201

<210> 31

<211> 1201

<212> DNA

<213> Homo sapiens

<400> 31

tgaagagcac gagccccagg atgatgagga attggatgag ggagacgaag aggaagaagt 60

agcgcaggta ataccagcag ccccgagagc tgccccccgc ccgagcgtag gaccctccgt 120

gctccatggc cagacccatt tgctcgatcc cgccgtccgg tgcaccgtcc ctgctcacca 180

ccaggcctgc tctggccccc gcctcgcagg ggggagtgct tatattactc ctcttaatac 240

ttcctctgcc tggctccttc ctggcccgga ggaaacgggg gctgcgggtt atcacaacac 300

tcccactctg gctgtgagaa ggcgggggtg gggctggggg cccagagcca gggcaccggc 360

tggacccctc tgattaatgc tgggtggggc gctggctggc tggtcaggca ttcctggcct 420

cttcgagcct cgcgttcgcc tttacttgct ggcgggtgct gttctcagta cctgctgtga 480

ttaggtcgct gaatcatgcc aggtctgttg ctgtctccct tgtgtggcgg aggaaactga 540

ggcacaggga ggagaagggg ttgcctcggg gtctcacgct gttagttcag cgaacttgac 600

ttacctcaag acaggctggc tacagacgtg cacccacttc accagggcgg tgctgattaa 660

gtgccacaaa acgtgggaaa tgtgccaggc acggtggctc acgcctataa tcccagcact 720

ttgggaggct gaggcggacg gatcacctga ggtcagaagt ttgagaccaa cccggtcacc 780

gtggtgaaac cccccgtctc tactaaaaat acaaaactta gccaggtgtg gtggtgctcg 840

cctgtaattc cagctaccca ggaggctgag gcaggagaat cgcttgaacc caggagtcgg 900

aggttgcggt gagcagagat cgtgccattg cactccagcc tggggtatag agcaagactc 960

tgtttcaaaa aaaaaaaaaa acagaaaaag tgtgaaactc ttagctagcc tagggatgga 1020

gaagcagtag gtgattagta attgcatagg agggcgtcac ggagggctga gccatggatg 1080

gcggtggcgc tggctcccca agctctgtca ttgcccccag tgccctgttc acttagatgc 1140

tcagtaaatg ctccaggaaa ctgcagcaca aggaataatg aacttggagc ggggaagagc 1200

t 1201

<210> 32

<211> 1201

<212> DNA

<213> Homo sapiens

<400> 32

ggacaatggt ccaggcagtg acaaagaggc tgaggcagat gggggtcccc tgagagcatg 60

tttcctggac ggcttctcgg ggaggtcctg gatgacttct cggggagctc aggaagcagg 120

taatgagtgc acaggaggca gaggacgggc ccaggcacag gtgtgtgact cactctgctc 180

taccctccaa tgagcagcag caggatgaag gattaggaaa ggcctgcccc cgggctctgc 240

tgggtgggca gggcagctga ggccaggaag ccagctcctg gggcggggcc ccaacaccaa 300

ggtcaccggg aatacccaca tccagccagg cacaccagct catcccacaa tactgggctg 360

ggcccctctg atgctgcgcc cctctgggag gaagggctgg gcttgggctg gcaggggaga 420

gaggaactgg gtcctttgac tgagggggag atgagccctg acctagagaa ggttttagcc 480

ctgtcctcag gagcccccag cctgggggag atggggacac agccctgtcc tcaggagccc 540

caagcctggg ggagatgggg acacagccct gtcctcagga gtccccagcc tgggggagat 600

ggggacacag ccctgtcctc gggagcccgg gcttcaattc aatgggtaca gccccaaatc 660

cagtgagccc caagttgatg aaaaagaccc aaagctgctt tcagaaggca ccctgtctga 720

ggggacacac agccagtttg cccacaggag gccagtacaa tgggtgtgac aaagccctgc 780

ccacaggtgc cctcaacaga aggggagaca gacctgcctt cagaaatgcc cagcctctgg 840

gaaagagaag tttctgcctt ctgaagccat atccggctga gagggagaca aagccatccc 900

tgtccccata gccatcaggc tgagggagaa gacaaaccct tgcacctcag agttctggca 960

aaagcctagg actgcaccca gtgctccagc tttgcacgtt ctgcactgaa agttactcta 1020

tttctattaa tacctctccc caccgccccc taccacaacc aaaatattgg gcttagcagg 1080

gagcagaggt gtcaggccac agaggaaagc aaactgggcc atggcatagg tgaacattct 1140

caaatgccag ctctaagaat ctgccagatg agggcttaac cgccgcctct ggtcctcagc 1200

t 1201

<210> 33

<211> 1201

<212> DNA

<213> Homo sapiens

<400> 33

agccaggcac tgtggcgccc cactcgccct cccgcactcc ctcctagaga tgccctctta 60

tatccccgga gttcgcaccc cccggggcca caggactccc agtcccccct tcagatactt 120

actgaagcag ctgtggcccc caggctgcct cttgccagct gtctgtctgt ctgtcggtct 180

gtctttgagg gctgagaagg ctccaggaag ccagcttcct ccctccgccc ctccttgctg 240

ccacccacgc acaccccagc tgcattctgc cctcctgtgc ctgctgccgc tgccaccagg 300

ggtcggctgc ctcccgcctg tgcccttctg gggcccaagg ccccgttccc tgggaatggg 360

tgaggggcca ggccttcccg accatcctgc tttccccagc ttccagaccc tctcccctct 420

tctgtgtttc tcctctgggt ctttgtcaca tccagccgct gcctacttgc tgggcacagc 480

tcgcctgacc cccagctctg tgctgtccca caaagaaccc aaggctctcc tgctcaacct 540

gaccttggcc ggaggcttat ttctccttct ctgcttctgc cccacagccc tctccctcaa 600

ggccctctcc ctcaaggccc tctccctcaa agcccttgct gcttcaagcc ttgggaattc 660

atggccaagc acagaccaca tcttcccaga gcagatgtgg cacagactcg gggtggggaa 720

aaagaagaag caagaatcca tgcctcacaa tgtgaaggaa agggccgggg ccaggccaaa 780

aaggtcgtct ccacgcctgt gccacggccc agctctccac agcccacggc ctgagtgtag 840

gccagcccag caggaagcag atagcagagt ccacttgtgg gagggagtcc gaggggcttc 900

ccctgggccc tcagccccct gcaggcctcc tcccagaacc ctgacatcta cttagaagac 960

ttacagacag tggacagtct ctggccctca caccagggga ctcccatgtc ctcttcgctg 1020

ctggggtgcc tggatgcctg ggacctgaag ggtggccccc aagtccaaga ctcctctgcc 1080

cactgcccct gcctgacctt ggcaggctcc ttgcgggcca gcagcccagc cggttcatgc 1140

tcgctgcacg cctcctttta ccttgtaaca tttcaaacgc ccttcccttc ccggccccca 1200

a 1201

<210> 34

<211> 1201

<212> DNA

<213> Homo sapiens

<400> 34

aggctgaggc aggctgccag gcgcatactg gccgataacg aaaagcggtc tcgccaaggc 60

cctgcagatc taattactgc cctcaccggc agttcagtcg agaaacctct cctgcagact 120

ggcagccaga ccaggtgtga ctcacttcct cttggaaaat gccctctgag atggcacagt 180

tctccttttc cttcagggag tgttctgtcc aaaccatcca ccgagagtat ctgcaaccgg 240

ccacacccac atggtctcac agctgagtca caggcctcag accactggga gaatcctgga 300

gccctttgtg ctaaccacgc cctgcagaag ctcccgcaga gacggaactg ggtgggggca 360

gaaaacgggg tgggcttaat gcatcccaga atctggctta gggctcccct gtgctggcct 420

ggcttggagc gctctgcatt ccaccccagc caacaaggca gagctcctgg tttcacttag 480

gcagagctgg actgaggctg gacaggcagc agtgtccacg gctaggggca gcgccattcc 540

ttcggccaaa gcacaggtgg tgaaccaagt accaaaggca cgtctgtctg tttcactgca 600

ccgggccaaa ttaatctatg attggaagat ccttcatcaa gcctgctctt cctccatctc 660

tttctggttc cacttattaa gacagcaagg gaaccctttc agtagagaaa ccacgtcaca 720

cagggccctc tcccacaaac cccagccaaa ctgctgtaga aagaaaacaa gtggacctcc 780

ctccctcagc aggaagaccc tttccgtcga gcagacaaca ggaagccccg accagaaacg 840

cctgtttggc cgacggacta agggttacgg aagggacgcc ccagctgcct actcgtccta 900

cccgcgccta cttgggcggt acagccggga gcaggctacg aacatgcaga cccggcctgc 960

gagcggaacc caccaagaaa cacgccagcc agggcagggg cgtgtaggag ggattcggta 1020

acccgagaga cgagtgggaa agtaggaaag gtgggagtaa agcaacgcaa acataaaggg 1080

aggagggaag aggttcaggc gcaccaatac cacggtcccc tcctaaagaa caagtgcccc 1140

ctcctctggt ccgcgtgggt ctgggggcgg ccgcgcccgg gccggggagg ggcgctgctg 1200

g 1201

<210> 35

<211> 1201

<212> DNA

<213> Homo sapiens

<400> 35

gaggcacctg ctccgtagat gatttttttt tgcttatgca gttagtgcac agagtgagcc 60

ctgcgctggg aaagacacgc acacgcatat gcacacacat gggcacacac acacatgcgt 120

gcacacacac gcagaccctg agaccctgca cttgagtttc tagtcttaag caagaagggt 180

gtcaccgagg ttgtgtcctg cagctgctag tggccagtct gtcccacttg gatggtctgt 240

ttctatttta ttttattttt tacttttttt gagacagagt ttggctcttg ttggccaggc 300

tggagtgcag tggcgccatc tctgctcact gcaacctctg cctcctgggt ttaagcaatt 360

ctcctgcctc agcctcccga gtagctggga ttacaggcat gcgccaccat gccggctaat 420

ttttgtattt tttagtagag aaggggtttc accatgttgg ccaggctgga atcgaactcc 480

cgacctcagg tgatccgcct gcctcagcct cccaaagtgc tgggattaca ggcgtgagcc 540

accatgctgg cagtctgttt ctattttggc atttcctcac tgcacctgga gaagagcttc 600

attcattaat tcatccattc attcattcat tcaacaagta cctgacagcc tgtacagcag 660

tggtgccagg tgaggagggt gggtaaaaat gcagggtcct cctcactgca gtcactgcgg 720

gaccaggtac tcttttcctg cgccccacac tgccccaagg ccccactggt catccgtcca 780

gcactctgtg ctcgctgact ggtgcggtga actctccggc tgggctcctt tcctgctgca 840

ggtgccgcgt ctctttctta agcctgtgta gaaagttctt ttgtccgggt gacattggca 900

tgattgaatc agtgccagag ccaaacagga tgtggttctg ggcgactccc aacttccagc 960

aaatccgtgg ttgttttcag ttaccttaag agggacaact gaaacagaga caagcacccc 1020

aacagtaggg tgcttcgagg tcctggaggg gagccctggc cctgtggaga gagaccgagt 1080

cgtgcagcgt ccgtggcctg catttgaaac tgaagggcaa gaggctggtg agatccgaga 1140

gtcatgccgc tgcgtggagg aacatgctga acctctggag gaacggctct acctccaggt 1200

c 1201

<210> 36

<211> 1201

<212> DNA

<213> Homo sapiens

<400> 36

tgcacttgca gctcgtacac tggcttctcc tgcagggacc ctggggggac acagaagctc 60

agctgcagct ccagcccccc ttctcacccc tgtgcctgtc cctcctccct cagctcactc 120

acccccccac agcaggggca gcagcagcag gggcagcatg tctccatccg ccagggcccc 180

agcccagtcc caggtccggc tgtcagggaa ggaaatgctc caaatgtcca aacgtggggt 240

ggggctgaga aagccccgac aggaagccgg agggtgagtg agagctgtgg acgcgcacag 300

aaggggaact tgggcatcac gtgctgttgg ggtgaggctg gggctgggaa gccattctgc 360

tcccaccccc actggacgcc gatggtgaag atactgaggc ccccagaggg gcttgtccag 420

acttgtaagt ctagactcct gaggtcacca ggtcccatag ctgcagcact ttccttatgc 480

acctttgtct acataggaag gagaacacca cttggccagc tccctgggag tcagggcctc 540

gggaccctca gcgagatggg agaataaatc ctttatgaca tgataggagc tgtggtgagg 600

gaagcacacg ctgggggttt caggtgggga agactttcct ctttccagag ggaaggcaag 660

ttgtcaggga caaacacatc aggtggacat gaccgggtcc ttagcagtca agagagatgt 720

ggtccccacc accagttgga tttgggaacc tcatctcatc cttgtccttg tcccaggcag 780

cgtcagaccc catccctgat gttgacagcc agatgaggct tgagagccgg gcatttgaaa 840

tgtgaacgtt gaggatatgg aataccaagt gtgaacccag acccctcacc taccggctgt 900

tgaactttac aaaagttacc taccctctct gtgcctcagt ttgctcagct gaaaaatgca 960

gacaataata gaattgctgt gtggggattc agtgaggtct catgtgtaaa gtgcttagga 1020

cagtgtctgg ccccgagaat tcactcattc aactgatgct tcatgggcgc ctatggtgca 1080

ccaggccccc tgttttagga gctggagata cagcaaagga taaactctta ggatgagaag 1140

gtaagtcaaa aatcctagtg ggggctcctc tgggcacact gcttatgcat acccctgctc 1200

c 1201

<210> 37

<211> 1201

<212> DNA

<213> Homo sapiens

<400> 37

tttcttctct cagaaaatcg cgcccatcac tgagggtgga cgccccgggg ctggtgtctt 60

tgctccacgt ctcctgttca cctgaacccc taggagcagt tcctgcagga gcaaagcccc 120

agggccagca gacccctcac actctgcact aaccaggcct catgagatag cagtggtgaa 180

gggaaatgtc cttgtgctca gagagctgtg aggatgggaa tagtgtctgc tcttttcata 240

ttgatccttt cccttatgac tttcctgtct accaagcccc acaatacaac aatcacagcc 300

tcagcttccc cggcctcctg caggctgggc tcccctccgt gtccttggcg tgtatcaaca 360

ggcgcaatcc tcaccttcac agccccatgt ccgtctgctc ggtcctcctg agactgaacc 420

cccgacctag gggagagtga ccccaccctg cctgtgcccc ccactgaagc tcctgtcatg 480

gggagctcaa acgtcaggag actcagtgtc tccccaccac acagtgactc cttggggaca 540

gtgtccggct ctgctgtgtt gacctcattg tgcagaaagg gggtgcccac ctcgtggctg 600

ctgagggagg gatgagaggc tgcctgttca ccccacctgc acccctgtct ctcatggccc 660

cgagtcccac ttctgagcta cgcagacatc cacagcctgt gactcagggg tctgggcaga 720

tgggagttat tttcaccctg cagaaaatat cacctaaagg gaaaggcatt gagagggagg 780

caggaggtgg ggtgggccct gcatggggtg ggaatctggg tgagtctgtc tcccgctctg 840

gcctcaggga cccaggagtg aatgtcgggt ggtggacggt ggatctccca gggctgaccc 900

agccctgaca gtgtctgtgt gtgaagttct tcttctaagg aggtcactgt tttgacctca 960

ccaccgactt cctgtagggc ctcttctaag tcttgagccc gcagttcctg agagaagaac 1020

cctgaggaac agacgttccc tcgcggccct ggcacctcca accccagata tgctgctgct 1080

gctgctgctg cccctgctct gggggaggga gagggtggaa ggacagaaga gtaaccggaa 1140

ggattactcg ctgacgatgc agagttccgt gaccgtgcaa gagggcatgt gtgtccatgt 1200

g 1201

<210> 38

<211> 1201

<212> DNA

<213> Homo sapiens

<400> 38

ggcatggcag agcacttagt gcccctctag ggactgacca gccgggggtg gaccctgttc 60

tatcctagac cccacttacc tgaggggaca aggaccagtg ccagcagcag gaagcaggga 120

ggcaagtggg ccaggcaggt gggggccgac atcgtggagc acatggcatc ttctgtggtc 180

cccaagactt ggggctcctc tgctctcttg tgagtgttgg agtctgaggc ggggcccgtg 240

gacaaggcag tttctgcgtc tcttggccac agctgcttgg ggaagtgttt tcattgcagc 300

aacacaaggc tgttggtgca ataggaagga gcccggtcaa agggtttcct gcttttgtgt 360

ctgggctgtg attcctgtag tcactgtgct aaaaccttgc cataggctcc atcctaggtg 420

cccgggaggg aggtgggcac tgaggtcata ggacgcactc cagaatcggg tggaatttgg 480

agcaaacatt ggatcttcct cttcttggct gtgcaagatt tcaaagtcac ttcccatttc 540

tgagcctttt gtttcttcat ctgcagcgta ggaacaacgg cagttccctc ctcagaacag 600

ttttggtaag attatgagaa ataatgtgag ggcacagaac aatgtaaatg ctaaacatat 660

taaccattat tattcatgat gtgcagaaag tagaggaggt gacattgctt aagaagaagg 720

atttattata aagcaagccc catgggggcc tccttttagc actgcaggga gggtacaggc 780

ctaattccta aaagtataat catgtgtaac tctctcagcc tctgtttcct ggggtccaag 840

tgaaacatac tcagattgtg caaacatttg cgtttgtggc atctttgttt cctgtaagtg 900

atctgaactt cctcaggagc ctcctggtgc tggctatcaa gaaggaaaag cttaatcttg 960

ccaattcatg caatccaggc aatctcttta gccacactgt ctgggttcaa atcttggctt 1020

tactcagtct ggctgtgtga ccttaggaca atcatttgac cactttatgc ctcggtttcc 1080

tcatctgtaa aacaggaata aaaatgtggt agcatgtgtg gggtggagta agcactctat 1140

aggctttcac cattgtcttt taggttaact cagtctttta attaactcag ggaaaagtac 1200

a 1201

<210> 39

<211> 1201

<212> DNA

<213> Homo sapiens

<400> 39

caactgcaag aatttactat ccatggtcac agatcttcag gccctgtgaa gcatccagat 60

ccccgctctg aataggccaa ctgggccagg accgggagtc aggaaggttt ccgccttttt 120

tttttttttt ttttttttga gatggagtct tgctctgtcg cccaggctcc aggctggagt 180

gcagtggcac catctcggtt cactgaaatc tccgcctccc gggttcaagc gattctcctg 240

cctcagcctc ccacatagct gggatcactg gcacgcacca ccatgcccgg ctacttttgt 300

attttcagta gagacgaggt ttcgccatgt tggccaggct ggtctcaaac tcccgacctc 360

aggtgatccg cctgcttcgg cctcccaaag tgctgggatt acaggcgtga gccaccgcgc 420

ccggcccctg ctttcttaaa gaacacgcgg cagctgtgtg ttacacgcta tggctgaggt 480

caagtgtgag ggctggaatt gctgactgcc atgttctggg tctactgaaa atagctgcct 540

ggcctgggag gcccagttca gtccgggtgt gggtcccgct tcacctccag agcagcggca 600

gccggtaagg gtagaagcgg cctttggaga ggggtctctg cagcggcctg gggacaagat 660

gagggctcct ttgagtcctg gggtgcaggt tatagtagct aaaggggagg tctgagacag 720

tggggtctgg ctccaaggta aagaaggagc agcaaccaaa aggcctgatg cgcagcgcac 780

ccatcgcaag catgggcagt gcgtttaaga ctgggtgact cactttttct cctataaaat 840

gggcgcacgc aggccccagc tcagcgccag aagggggagc cccggcgcgc cggcagaaga 900

caaaggaaaa aagcgtgttt gcaaacgggg aagcggcggc gtgctgccgc gaggggcggg 960

gcggggccgg gggaggagcc cgcctgccgc ctgccaagcc cagtggtcct ggccgtgcgc 1020

cggaggcagc ggcggcgtgg cgcagcggcg acagtaagtg cgggccggct cgggctcttc 1080

cggctacggt cccggccgcc cccagactcg cgctcagcga cctccctccc gggccctggg 1140

ggcggctgcg ggctgccagg ggagccgcag gaccctaccc catccgtggt cccttcccca 1200

c 1201

<210> 40

<211> 1201

<212> DNA

<213> Homo sapiens

<400> 40

atgtcacaca gccaagacag aactctggat ctcctttccc agccacaagc tgcccctctt 60

ccagtctgta agttcttacg gagcatatat atgtgatctg cctacttttc tccaacctca 120

ccacagtgac atgagcccaa accaacttct caccttgcaa cagcctccca ggtgggaagg 180

ctgagtattc tggcccttaa ccagttagaa ctccccagtt atctgtcctg ctgatggggt 240

tgaaatctac attcctgacc ctggcccacc aaagcccctc ccttagctcc catctccctc 300

ctctctccct gtcttctcct ctgctccaga cactctggct tcatttctgc gttttttgta 360

ccccataagc tccttcccac cccggggcct ttgcctttgc tgttccccct gcggggaatg 420

ccggatctct gctcagatat cctcttctca gatcagcaag ctaaagcagc cacctgtgtc 480

tgcctaaccc accaccgtag tttaactttc tgcctagtct ttatcactag ctgatatttc 540

tcaggatcct ttagttactt ctttttcgtc ttcccctcct agaatgtaaa ctcttcccct 600

cctagaaggt aaacaaaaga cctgttctgt tttgttcttc ggcccatccc aagcctagcg 660

tagtgcctgg tatgtggtgg tgtccaaacc caagcgtgga gtgaatgagg gatgaatcca 720

tgagagagtg agcggctcca gtgggtatgc gcgagtgtct cactcggtgt agatgtgtgt 780

gttgtgtgtg ttgtgtgtgt gcgcacgctg gggaggccag acaagtgtgg accagtgatt 840

ggggcacctc ttccctgcaa agaggccagg ggaagacagt gcgtgtgggg tcttctacca 900

gggaggatgg cttgctggtg tgtccccccc aggggaggac taccaacgaa ggggacccgg 960

gagatggcgg gtgggggccc ccgggaggac agtgggcgag ggagggggtc cttgccaggc 1020

ctggggcggc cgggggcggt cctgggctcc cctccgtccc gcctccaggc ctcggggcct 1080

ggctggccga cgtggcgttg gcggcgctgc gcgcgggagg gcagggcagg agggacagag 1140

gcgggggcgg gccggaaagt ttgtccggcg gcagcggcgt tggggactcc ggcgggggat 1200

g 1201

<210> 41

<211> 1201

<212> DNA

<213> Homo sapiens

<400> 41

gaagggccct gagatgacag ctcgttggtc ctcatggaag cgtgaccccc ccagtagact 60

ttctcccaca cccaaccttg gtttcctcat ctatatgata gggacaagcc agactctacc 120

tccctggtgg tcatggtctc cgcttattcg ggttcataac cttaaaggcc cctcgcacca 180

cctcagtgag ccatttatgc ctggcacagg gccaactctc agtgcatatc tgcaaaggaa 240

ccaatgaatg aatgaatgaa gtgacaaatg aataaaggaa taaatgaatg aggcacttat 300

catgtaccag gctttcgtta ccacgtccca tttattcctc tgaggcaggg tctattttat 360

ccttgttaca gatggggaaa ctaaggccca gggaggagca aagtcttccc caagtatgta 420

cccactcaga acttgagctc tgaatgtctc ccacccagct tagcccaaga gcggggttca 480

gtgatgccca ccccctaagg ctctagagaa agggggtagg cccacatgcc agtttggggg 540

tggtaaagcc aggtaagttt tctttatggg tcccctgaaa ccctgaaagt gaaccccagt 600

cctgcatgaa agtgagctcc ccatagctca aggtattcaa gcacaatacg gctttgagtg 660

ctgaagcagg ctgtgcaggc ttggatagtg acatgccctc tctgagcctc aatttcccca 720

cctgtcaaca gcagacagtg acagctgtga tcaggggatc acagtgcatg gggatgggtg 780

ggtgcatggg gatggagggg catttgggag ccctccccga taccaccccc tgcagccacc 840

cagatagcct gtcctggcct gtctgtccca gtccagggct gaaagggtgc gggtcctgcc 900

cgcccctagg tctggaggcg gagtcgcggt gacccgggag cccaataaat ctgcaaccca 960

caatcacgag ctgctcccgt aagccccaag gcgacctcca gctgtcagcg ctgagcacag 1020

cgcccaggga gagggacaga cagccggctg catgggacag cggaacccag agtgagaggg 1080

gaggtggcag gacagacaga cagcaggggc ggacgcagag acagacagcg gggacaggga 1140

ggccgacacg gacatcgaca gcccatagat tcctaaccca gggagccccg gcccctctcg 1200

c 1201

<210> 42

<211> 1201

<212> DNA

<213> Homo sapiens

<400> 42

tccagggtcg tgccggacta gaccctaggg cgtgggtaca aacgggagct ccgcagtccc 60

cgtaggagca gggaccctct cttacctctc aagctccagc gcgtcaagct ccaggacact 120

ccgcgtttcc ctggaggcgc caaggtcagg ttcttataac caatggttgg cgcctacctg 180

ggcaggtccc acacagggga tgaacgaacc aatgggtccc aggcagggca tggacgcgcc 240

aatgggagtg tctacaaagc ccgggaacac tttgccacgc tgtctagaag gcctggcggg 300

aggctcgcgc ctatgaaatc ccagcacttt gggaggccta agcgggttca tcgcttgagc 360

caagaagttc gagaccagcc tggacagcac agtgggaccc ccgtctgtac aacaacaaca 420

acaacaacaa caacaacaaa attactcagg cttagtggcg cgcgcctgta gactcaaatg 480

tagtctcaga tgcccgggag gctgaggctg gaggatcact tgagcccgag gaagtccagg 540

gtaaagtgag tcgagatcgc accactgcac tccagcttgg gcgacagagt gagagcctgt 600

tccccctaca aaaaaaaaaa aaaatgtctg gaaacgccaa gttttctgat tgctttcgag 660

tggttcttag ttctttctta ctccccctcc gcccctcctc cgtccctatc tctctctcct 720

ttcccggagc ctgcggtccc gccttccccg gacccctcct ctgagggtcc agccaagctc 780

tcgcccgagc tttccccttc caccgcactg caactgccca taccccagct tgggcttctc 840

tccccactgc ctgccccaga aagtgggcct tgagacccgg gaaccccagg tcaaaatgcg 900

acggatgctc ggatcgggcg gcactcggtt cctggctgga tttgtggctc cgcagacccc 960

gcgccagagt gggatgtggc ggcggccggt gctctgtgct cagtcaacgc cttggagagg 1020

aggactacga aacccataca ctcggacaga accctggcag gtgttaaagg ctgcggcaga 1080

gcagaaaagc agaaccctgg ctggttttct tgtaacacga gcagtgtgac ggttaccggg 1140

gttgaccaag caccgcgcat cggccaagtt ctcactgacg cggactggcc caagggtggg 1200

a 1201

<210> 43

<211> 1201

<212> DNA

<213> Homo sapiens

<400> 43

aacctccact cacatcaccc tcccttcttc gataatgggc aattgagaaa gggcaaatgt 60

gtgtaaaaac tatgcagatg cagtttcagg attattttct tcttttctgc agtgtctttt 120

cccaagctac atcagcgtct aaattttgga gttactgatg cttccgttat ttctgcagat 180

agaatattat gtcgtaaata atgtttactt gtccattagt attatataaa agtgcctgtt 240

tctccccact ccagctatca ttgattattg gtaaaaattt taatgtatcc aattctaata 300

ggcaaagaaa ggctctccta cttagatctt aggtgggggt ctaagttcat atttttgttt 360

tacaaaaatt gaatagccag tgtttttgag ttgaatgatc tatccattcc gtataggaga 420

gtttaattat attgtgattt tgggggagat aattttcaca atttaccccc tcaaaatgta 480

gaaagtagaa aaagggtaga aaaatcgcac caaatttgaa caatgtagca tgcttttaga 540

gttagggata ttttcttttg gaaggatttc aagtctggcg tatttgcatt gattgtggtt 600

atggcgcatt taacttttcc aaaccagctt ttttgtaccg cacataggaa gacctgaggg 660

tgagtgtaaa ttaggattgt ttggccagtg gtgtgtgcaa aaatttattg tagaaccaag 720

aattatttct ttagtatttt acacatttta aaaaacaggt agacatgtcc attgatccca 780

ggttgcttat ggtttctaga ggccccctct cattgcaaca gtgctgtggg gccctagggg 840

tccagtagcc ccctcccccc aggtcattcc ggtgagggag ggagctggga cccctgcact 900

gcggcaaaca agcacgcctg cgcggccgca gaggcaggct ggcgcgcatg ctcaggcggg 960

gatgtgtgcg aagcctgccg ctgctgcagc gagtctggcg cagagtggag cggccgccgg 1020

agatgcctga cgcatctgtc tgaggagcgg tcagtgacgc gatggagcgg gcaaggtcag 1080

ctgtgccggt ggcttctctc aagagacagc ctggggagcg gccactttta ttcatcagat 1140

attccaagtt tttaggactt ggagtactga ataaacggaa tttgggccct aaagtccttt 1200

g 1201

<210> 44

<211> 1201

<212> DNA

<213> Homo sapiens

<400> 44

gggggtgaag gagagaagta gcaggaccag aggggaaggg gctgctgctt gcatcagccc 60

acaccatgct gaccccgccg ttgctcctgc tgctgcccct gctctcagct ctggtcgcgg 120

cggctatcga cggtgagtga gattccgcgt cccccttgga cccctggggg caccctctcc 180

ccagccccca ctcctgcata cggatgggga agggagacgc gggagggggt gccttttgtt 240

atcccagtcc agctgacaca gcagcggccc gactgggggc ggggatgggg tccgatttgg 300

gggatggggg ccctgggcaa atgatgcttc cggggccccc cagcacaaac aaagaccaga 360

ggcctgggta gagagaagag ggctccccat ttttctgatc ctggggagga gaggccttct 420

tttcctatct ctgttcgggg agggcctccg cctcccctac atcctcacaa ccccccaccc 480

cccatctgaa ttgtgaagga atccggattt gcaatgttcg gctgcaaaag ggggtggggg 540

tggggggggt ggcttttgca ctggcctctg tagctcgaaa ggggagccag gccaggggta 600

ccctgccggg cagagggcag gcaggcccgg gaaggctgtg ggtggggaga gggagggccc 660

cctgcttggg cgtggaatcg accccagagc cccggctgga ggcctgggtg ctgaagtagg 720

ctcttccccc tctcagggcc tctgtcagta gcctggggta gggctgtgtt ggggggcaga 780

gagggggagc tcgccatact atgtgacttt gaattttcct tcttaattcc tgggagcttt 840

gagatgaaaa aacgtcagat gtcatcactg gggaagggga ctggagaatg gcctggaatg 900

ctggggtagg agggtggggg aggctgcctt cttcctgact tctggcttca tcacagtttg 960

gattggaagg agagttggag gttggatggg gattatggct gtgcccccac actcctttag 1020

taaatctgtt tattccctgc acccctccaa gcccaaaacc ccatacacac acactttgta 1080

tattgcagag ttaggcttag gctagaactg catccttttg gggaaatggg gtgcctctct 1140

tccctttttt tgtcagctgt ttttttctcc tacttgtgag tcactggttc aagtcttgcc 1200

c 1201

<210> 45

<211> 1201

<212> DNA

<213> Homo sapiens

<400> 45

tcttcagtgc gatgggctgg ggagtggggt cctagcagca gggactggag gtgagctgaa 60

gcagataagg gacgggagcc ttgtggcgaa aagtgaccca cctgagggag aggcgcttcc 120

aaggaggaag cgtccagcgc caacggtgcg cgccgcgggc cgggcgggta gagggaagtt 180

tcgccgcgcc tgcagctcag cgctgtggct tgtggctctc gctgctataa gaagccagag 240

aagggcggga cggtctgctg gatttcgcaa gagaagcgga aaagaacttg tttcctggaa 300

gagttcaagc tgagaatccc agcagctgga aggaccggaa cttctatact gcggtgctgc 360

tgatggtgcc gctgaccccc gggaagcggg gattttaagg gttacacccg agcccttggc 420

atctgtgcat cccagggtga ttgaggtttg gagaggtcta ggaatccccc tttcctaaag 480

gagcaagaag gatctctttg agtagctgag aaaacagcac ctggactagg cttctaggca 540

attcttttgc catcttgtct cagtttatag aaaaggtggt atctttcccc tccgcctgga 600

agccatgggt tcttatgggg ctcataactt ggaagatctt gagttttcag gtcttaattt 660

aggtagatta aagaaataaa acaaatgaaa gagggatcgg gataccttat gatatacatg 720

caaatgtgtc tttctacaga gaaggtcaaa aaccacagcc ctggttgttt aagattgaaa 780

taatcctgat aaactatggg gcaaagcatc catacactcc caaactctgt aatctaaatc 840

ccagagtctt cccaatgatc tccactcagc catgggtctt aaattccaca gcatacgctt 900

gccaagctcc agaggcccgg ccacatggga ggcctgttcc tcccatggaa aggggttgtt 960

gagtcaacct tccctttgtc tggattgcaa ctctcctcat agcttcttgt ggggttggtc 1020

agaccccatg tctttcaaga ggtcttttct tagccattag ttgcaattaa tattttcttt 1080

ctctatacag tagtttatac ttttagcaca gcaattaaga caatttgcct ttcaaaagag 1140

ttattatcat gtgtgtgtgt cttgtcctgg actgcagtct tcgagggatg acaccctagt 1200

t 1201

<210> 46

<211> 1201

<212> DNA

<213> Homo sapiens

<400> 46

gggcgtgccg cagccgcaag aacccgcaag ggaccccgcg cccgcaccgg tcccggcctc 60

ctggctccct gccgctccac acagcagcga gagcagcagc agcaagagga cgagacccag 120

ctcagggcaa cgtccacaca ccagccctag tgcgggcgca gccatgttgt cccgcgggcc 180

atgtgacccg gttggtcacg tggctgagcc ctccttaaag gggcagaatt ttgagctgac 240

ttacgaagtg accaggagag ttgctcttca cctttcttca ggtacttctt tggcgctgct 300

gcttgcctgt gctaacgtca aaacgcagca aaacacaaaa ctccacaaaa ctcaaggcct 360

tccacagacc gtgcacccct ttatgatttc ttggttgaaa aataacagtc aaatttgctg 420

ggcatttggt aagtgccaga gtactatgct aagcacttca ctttttaaaa tctcatttaa 480

tcctcacaaa gacccagtaa tggcgcgctt gtagtcccag ctagtcggga ggccaaggca 540

ggagaatcgt ttgaacctgg gaggcggagg tttcagtgag cccagatccc gccactgcac 600

tccagcctcg gcgacagagc aagactccgt ctcaaaaaaa aaagagaaat acaatacgga 660

atcagaatcc gtggggactg gacacagcaa tccagggttt cacaagctct ccagatgatt 720

ctaatccaca cttaagttta agaaccactg ttctacagtt ccgtaatcga acaaaatgca 780

aaaccaaaac aaaaagaaga tgaaggagaa acttacagat taaaaaacaa catagcaggc 840

cgggcgcggt ggctcaagcc tgtaatccca gcactttggg aggccgaggc gggtggatca 900

cgaggtcagg agatcgagac catcctggct aacacggtga aaccccatct ctactaaaaa 960

atacaaaaaa ttagcccggc gtggtggcac gcgcctgtag tcccagctac tcgggaggct 1020

gaggcaggag aatggcatga atccgggagg tggagcttgc agtgagccga gatggcgcca 1080

ccgcactcca gcctggggac agagcaagac tccatctgaa aaaaaaaaaa aaaaaaaaaa 1140

catagcagcc agctgcaata tgtgaacctt atctaaatac taattcaaac ctactgagtt 1200

c 1201

<210> 47

<211> 1201

<212> DNA

<213> Homo sapiens

<400> 47

tggtctcttg cgtccagcct gggcaacaca gcgaaaccct gtctcaacaa aaaatacaaa 60

aattagctgg gcatggtggt gcgcgcctgc agtcccagct actgtggagg ctgacgcagg 120

aggatctctt gagaccagga ggtggaggtt gcagtgagcc cagatcgtgc cattacactc 180

cagcctgggt gacagagcaa gaccctgtct caagaaacaa acatgaagtc caggcgcagt 240

ggctcatgcc tgcaatccca gcactttggg aggctgaggt ggatggatca cctgaggtca 300

agagtttgac caatgtggca aaaaccctgt ctctactaaa aatacaaaaa ttagccgggc 360

atggtggtgc ttgtctgtag tccctgctac ttggggggct gaggcaggag aatcacttga 420

atccgggagg tggaggttgc agtgagctga gaccgcgcca ctgcactcca gcctgggcaa 480

cagagcgaga ctctgtctca aaagaaacaa acaaaagaac tggaggtggc ccggcccctg 540

cgtctaacag tcctgggccc tccacaccag cacaggaact caagcctgca gtcactgggg 600

ccatggggaa ggtgggagga gcccacagct caggggcact ggggcaggca ggaagagcct 660

cagagaggaa ggggtgacac cccccaccct tgggaggggc ccccagcggt tgccaaagct 720

gtgtgaggag gaggaggaag tgagaggagg aggtgaggtg ctgcgggagg tgagctgggc 780

tggtggggac aggggcaggg cttggggctg ggtctccgga cagaggcctg gcttttctgt 840

cagggcaggg cctagcccct gcccccataa aagaggagac atagggggct tggtgagata 900

ccctgaaacc tcccccctct gaccccgcag ccaggcccca ggctggccgg gagtggcccc 960

tcacactggt tctccccact ttctctgcct gtggcatcga aggccccggg caccatggcc 1020

caggccctgg gggaggacct ggtgcagcct cccgagctgc aggatgactc cagctccttg 1080

gggtccgact cagagctcag cgggcctggc ccatatcgcc aggccgaccg ctatggattc 1140

attgggggca gctcagcaga gccagggtaa gggggcaggg tgagggctgg cggaatgctg 1200

g 1201

<210> 48

<211> 1201

<212> DNA

<213> Homo sapiens

<400> 48

cacctgggaa ggagatattt ttgccatttc acagctgttg aaactgaggc tcaaaaagac 60

taagtaactt ttctcagcta cacatgtggc tgagccagta tttgaaccca gttctgtttg 120

cagacagaac ctgggctttt tcacacctgc aaactggaaa cattaattgg ttcttaagat 180

catcatcgat gtgataaaac ctgggacaga aattagtcaa gactagctgc atctgccttt 240

tcctctggtg ggtaggaaaa ggaggagtat aatgatttcc tcaggcatga aggtcgatga 300

tgagcaaagt gtatactctc taatctaatg tcataattca tattgtggag taattatctg 360

gataagtgta gggtctctga cctcattcta gatattgtac attccatggc tattttcatt 420

ttggtccatg aactctcttt gctctcatga gcaccatttt tatcccaatc taatcctgta 480

tgtttgtgtt tttacacaga ttagttttta aatgttatat ataatttgct tctgaaacac 540

cattgctcaa tgactaccaa atctttctca ttaccaaaat ccttctatgc caacttcttc 600

aagaaatttg atcaccttta gatgaattgt taatgaaaat taaagctata gccggcaaca 660

tgggtatctt tgggctaatg gccaaccaac aggccatctg tgtgaaagaa aacaggctaa 720

caattttgga ctctggtctc ttggggctac attgagcatt gacctcaccg gtgctcactg 780

aaattaattg cttttcaggt tgtattttct catcacggaa accttcttct cccaattcaa 840

accatgtggg ttaaaatgag aaaacaaaag ccaaaacggc ttcccacacc caaaagctcc 900

ttctgtcaga gatcccagta gccccgggag agctgttaga agtctgagaa ggattggtca 960

tcatcgcata ccatacatag gtggagggct tgttattctc agtttcccgc ctatgagagg 1020

atacccctat tgtttctgaa aatgctgacc gggacccaca cttccaacaa aaattcctct 1080

gcccctacag cagcagcaaa agcagcagca gaagcaacag caacagataa gtgttttgat 1140

gaattgcgag atggataggg cttgagtgcc cccagccctg ctgataccaa atgcctttaa 1200

g 1201

<210> 49

<211> 1201

<212> DNA

<213> Homo sapiens

<400> 49

tgcaacctct gcctcccagg ttcaagcgat tctcctgcct cagcctcccg agtagctggg 60

attagaggtg tgtgccacca cgtctggcta ggtttttttg tatttttagt agaaacgggt 120

tttcaccatg ttagtctggt ctcaaactcc tgacctcagg tgatccaccc gcctcggcct 180

cccaaagtgc tgggattata ggcgtgagcc attgcgcccg gccctcttct ccctgtttta 240

gagagtaaga aactgaggtt cacacaatca aagtgatttg cctaacgtca tacaacgagc 300

aagaggagac ctgggatttg aacctatgtt ggtctgactc ctgagggaca gggaaatgga 360

aaaggcttat cccgagcctg tggggtcagg ataagtggtt ggtccggctt ctaagcagat 420

ttctctttag ccttaggaag aatttttaac tagaattggt gaggcccggc gtggtggctc 480

acaggtggat cacacctgtg atcccagcac tttgggaggc cgaggtggga ggatcacttg 540

agtccaggag ttcgaggcca acgtagacaa taattatcga aagagtatgg gagttctgga 600

gaggtaatga gatccccttc accaggaggt ttccgataga ggaccatgga gtgtcctgga 660

gggacattcg ggagccctga cactacatct ccctcctcaa acccaaaggg cagaggcatg 720

tccctcctat gagacgcaga acaagtcggg ccgcgtcccc tttttctaac tgggagcact 780

gctgaggggg cgtctgggac ttcttcatct tcatcatcgc cccttcccca tagggtactg 840

cagggggcgg ggggcggggc ttcggcgggg cggggctgca gggctcccgc gggctggcgg 900

ggcggcttcg gcgtgcgggg cggggcatcg gcggggcggg gctgcagggc tcccggggac 960

cgcccgcccg cccctcagtc tgagcccaga gagccgcggg gaccatggag ccggtgccgc 1020

tgcaggactt cgtgcgcgcc ttggaccccg cctccctccc gcgcgtgctg cgggtctgct 1080

cgggggtcta cttcgagggt gagcgggggc tggaacccct ccgagcactc ccttggtgtg 1140

gggcgggggc agagacccgg agaagacgtt agcaatccgg ggaaactgcc cctgggttca 1200

a 1201

<210> 50

<211> 1201

<212> DNA

<213> Homo sapiens

<400> 50

cgaattaagg atgttaatca gaaccccagg gaaggttcct gcattgtcag aaagctggtg 60

ggcaaggatt gccccacccc accccccacc aggggtacag gtccatgggg agggggcagg 120

gcaggatggg gtggggatta gttttctact gacccactca cttgcttctt tcaccatcat 180

tcatggccac ccccttccca atgctcattc attcacccgc accattccct gcttgctagg 240

acggtgccac tcctgagtta caaccctggt gccaggtagc ctctgtttga atcccggcca 300

tgccctgtga tcttaggaaa attctacagt tcctctacgc cccatatttc ctcatccgta 360

aaacgggaat aagaaccggt acccatctca gagatttgtt gtgagctttg agtgagataa 420

actatgctga gtgcctggta tacagtaggt gctgtataaa tgccggctat ttgcctgtgt 480

tatttgagac cctggctttg gctcctggcc acctgagttc cagtctcagt tctgccatgt 540

attgactctg tgatcctggg taagtcactt aaccactccg tgcctcagtt tccccatttt 600

gtattcctcc cctttcacct gccttatctc cctccactgc tgctacttaa tttgtttcct 660

ctctgccacc cctcaccagc atgtcagaca tacaaaacaa gggatttttg tgtgcttggc 720

acacagtaga tgcacaataa atgttgaagg gctgaactaa tttgggtttg agtcataggg 780

agcttggggg atgtgggtga ttggatagat tctggagact ttaggggact gggccggggg 840

aaatgcggcc tctaagctct ccgctgaggc ggcttggaag gaatagtgac tgacgtggag 900

gtgggggagg tggctggccc gggcgaggcc cagggagagg gagaggaggc gggtgggaga 960

ggaggagggt gtatctcctt tcgtcggccc gccccttggc ttctgcactg atggtgggtg 1020

gatgagtaat gcatccagga agcctggagg cctgtggttt ccgcacccgc tgccaccccc 1080

gcccctagcg tggacattta tcctctagcg ctcaggccct gccgccatcg ccgcagatcc 1140

agcgcccaga gagacaccag aggtaagcag ggcccggggt ggcccagcag ggaccggagc 1200

t 1201

<210> 51

<211> 1201

<212> DNA

<213> Homo sapiens

<400> 51

gatagctcca ggctctcagg acagaggagg ggaccccagg actcaccagt tccttggcgt 60

acaggactgg gtgctcccgc cgaatggagc gagagtgggg ggcacctggg ctggaagaga 120

gagtccgtgc tggtgcccgg ggctgctctg ggctggcgcc tgcccgccct ccctcctccc 180

gtccctcctc ccgtccctcc tccttcctcc ctccccggct gggccgcagg gccagcttga 240

tttcattaag agggtttggg gtggggacag ggggagggga ggggctactc tcgggccaca 300

cgcaatccca cccttgctca cactctcgct ctggggaaga ccagatgcgg cacttcctcc 360

ctgacctcgg gtccccaggg caggctgaga cagacagaca aggctgtcct gcagcgctcc 420

cccagacagt gagctggggt tctgcccacg ctgctgccca ataccctcca aaggggacaa 480

gctcagggag gttcagaggc ctctggaagt gggaaggaaa attccccgtc cttgtgtggc 540

tcaggcttcc aggctcaggg tgcagcgtgg gagctgggat tcgttttaga gacaggcctg 600

atggtgggtc cgagcctgag acagggagcc caactctgcc cttccctctc cagacctcag 660

tttcccattt gtagggatgg gtgggaagct ttctgggcta cctctgactc tgatgttctg 720

ggatctgtgg cctcacaatg cccccaaatg atagtagctt gaataggagt tggagcccca 780

tttcacagag ggggaaagtg aggcagggtg agttctaatg gttcaagcag ggtcctgagg 840

tgagtcagtg gcaggccttg gaaccgggac gtctggctga cctgaggagt cctgtggatt 900

agcagcccct tgcattcatt cattcattca ttcattcatg cattcattcg gcaagtgctc 960

aatgaacacc tgcagcaggc cacgcactgt gctaagcaag ttccagaggt gaatcaggca 1020

cagcccagcc ttggggagct cacagactac cagggagaag gacaccgagg taaattactt 1080

ttttgccaaa cagaaagggg tcaggtcaag ggctccgaga accccaagac agagagatca 1140

atcccccaag ggaaagaggt aggaagccat cctggagggc atgcctgtgt atgagcagaa 1200

g 1201

<210> 52

<211> 1201

<212> DNA

<213> Homo sapiens

<400> 52

acagttcctg ctgggtgtag ccccaggtga tgggctgagg gggaaagggc aggcccttgt 60

cctggtggca acgcctggca gacgtgtgca gtgggccggt tgtctcacag gcctctagga 120

tgtgcccagc ctgccacacc gcctccagga agcagagcgt catgcaggtc ttctggccag 180

agccccagtg agtgcccacg gaggccccca gcacacccaa cgtggcttga tcacctgcct 240

gtccagctct ggtgggccaa gaacccaccc aacagaatag gccagcccat gccagccggc 300

ttggcccgct gcaggcctca ggcaggcggg gcccatggtt ggtccctgcg gtgggaccgg 360

atctgggcct gcctctgaga agccctgagc taccttgggg tctggggtgg gtttctggga 420

aagtgcttcc ccagaacttc cctggctcct ggcctgtgag tggtgccaca ggggcacccc 480

agctgagccc ctcaccggga aggaggagac ccccgtgggc acgtgtccac ttttaatcag 540

gggacagggc tctctaataa agctgctggc agtgcccagg acggtgtctt cgtggcctgg 600

gcttggtggt gggagttgag ggacagggag ttggcagagg cccctcccag cctgccatgt 660

gacactgtac ttcctccacg gtgggctcag ccctgccctc atcctcacag ccgcagccaa 720

gctgcagttg gtaggggatc caccgacaca ccaggctgcc tgggctggtc tctgggttgg 780

gagctgcccc aggtgctgag gagggcagct ccctggctgg tgaggcccct cccagaacca 840

cccttggact gagctctggg gagggatggt accaggtggg tgaggggggc tgcctgggga 900

gggaggggtt cctatggggc gtggcgaggc tggcccagcc ctctccccgc ccatatatgt 960

agggcagcag caggatgggc ttctggactt gggcggcccc tccgcaggcg gaccgggggc 1020

aaaggaggtg gcatgtcggt caggcacagc agggtcctgt gtccgcgctg agccgcgctc 1080

tccctgctcc agcaaggacc atgagggcgc tggaggggcc aggcctgtcg ctgctgtgcc 1140

tggtgttggc gctgcctgcc ctgctgccgg tgccggctgt acgcggagtg gcagaaacac 1200

c 1201

<210> 53

<211> 1201

<212> DNA

<213> Homo sapiens

<400> 53

ggaggcgggg acccaagggt taaaaaaaaa aattcctcaa tttggaaaca tttgcaaaac 60

tccagaagaa aaacttttaa aagttctcca ctcccttcca aacgtacaaa catgcacaca 120

cacaattagg aatttcccaa agttttcatc tgtctaaaaa gaaagtgcca gtgcaaatta 180

cagaacatcg tcgcttcctc atgtgcggcg tttctacggg cctgggaggg gacgcagagg 240

ctaccccggt ggtcccagcc tgcagcgttc acgtgaggaa acttccgcaa aagccctgat 300

gcatgcacat tctgactgca gaggccagag catcccgcat tttggaggct gaaaggctcc 360

cctaggataa ccttttccgt taacagagga gtccgctgaa gcgcagagct aactgaaaac 420

agctagaatt aaaatccaag tcacccggcc ggcatcagca gtcattcctt tccaccacac 480

aaaactggcc aggaagacgg actctggcgc gccctcccag gccgaagcgc atttcttcgc 540

taacgcgcga ggagtaggct gcatagaccc ggcgcaggag gtgttcattt tcggatccag 600

ccctgtagtc actttagcta aacccggttc caaaggggtg ggagtcgtcg gagaggaatg 660

gcctctgtat ctccggagac ggaaacacca ggacgaagag gccctaagaa cgtgtgcccg 720

gggtgcgacc ggggggtggg gggcaggggg cagagaccct gaaaagctcc accgccgaac 780

cccagggcag tgtcccggct tggctggact gggcgcgggc ggctgcaatg ctcccgggcg 840

cggcgctggg accccgcggg ccacgtcggc tccgctgccc cgacgccagg gcccgactcg 900

cgtctccctc tggcggggca ccacaagggg cagcgaaacg gtggtggggg cgggcgccgc 960

gctccttccc gtctcctccc cgccagctcc gggcccgtcc ttttccgccc ggctcccggc 1020

aagggtcccc cgactggcgc ccgcgcgtcc tccctcggct gctgcaggcc gggccgcggc 1080

gtcgagcggg ggcggcgggg cggggcccgc agccattggc gagcggcggg gcgggggcgg 1140

gggcgcggag ggtcggcccc gggacgcgcg cagccggccc gcagttgccg ctgtcgtccg 1200

c 1201

<210> 54

<211> 1201

<212> DNA

<213> Homo sapiens

<400> 54

gctcagcccc agagagggaa actttcaatt ctgttggtca agagaggcct cacttgaggg 60

accaggccca gtggctcaca cctgtaatcc caacactttg gaaacctgag gcagaagaac 120

tgcttgaggc caggagttcg agaccagcct ggtcaacata gtgagaccct gtctctctca 180

aaaaaaaaaa acaaacaaac aaaaaaaaaa aaacgaggga ccttatttta agatgtccga 240

ggcagcttgg agtagtggag aacaggtagc tggtggtttc tctgtagcag tgagaggaca 300

ggatgggtag attcggctca ggaaggctct gaagtggtag tagatacacc agcctccccc 360

atcctctgac tgaggagcaa gtgcatggcg ggtgcatgac agcacagagc tgcacttgcc 420

ctcatggtga ggaaagacat acagaaaggc tagagctagt actttgccca tgtggggctg 480

ctttcttttt gaatagaggt gaaagtcaca caatgtacag tttgttattt taaagtgtac 540

agatcagtga catgtattca gaatgttgtg caaccaccac ttgtctttag tttcaaaact 600

ttttgggtgc ctgtttttgg ttctgggtga gatgcactca ccagggtgat aaagaccctg 660

cctggcttgt tcatggcgtc tccaggacct agtatggtgc ctgaccgtgg cactcatagg 720

gtgtttgttg agtgaatgaa tgatgccttt tcagctgaac tcgagctaga agactccagg 780

gctcactcca ctgtgcccat tccacagatc cggccccgag gtgtgcccgt tcaccaggca 840

gaggtgtcat ccgaattcag gctcctgggg cccgggaggg tccgactcta cggacccagg 900

tcgctgtggc ccatcgcttt cgatttgact tggtttctgt cgccactcgc ggaaggcgcg 960

ccccccgccc tcgctcggcg gcccgccccg ccccgcccct gctcttcctc cggggccgct 1020

ggcactgcgg ccgctccgca ggcagagaag ccgggagcgg gcgaggcggc ggcggcagca 1080

gcgatggtga gggcccaggc ggggccggcc agccctgcga cgggcagagg gcgagtggcg 1140

agggtgggag agaggagtcc aaagtccgcg ggctggggcc tcccctgggg cccacgaggg 1200

c 1201

<210> 55

<211> 1201

<212> DNA

<213> Homo sapiens

<400> 55

agtgagactc cgtctcaaaa aaaaaaaaag aagaaagaaa caaagagaga aagaaaggaa 60

agaaagaaag gaaggaagga aggaaggaag gaaggaagga agaaaggaag ggagggaaag 120

agggagagaa aggaaggaag gaaaaaaata acttaaaaaa tcagatttgt tggacaaaga 180

tcagggctta acctagggag gtgggtagag ctaatggaat gcggaaaagg ctgtgatttg 240

aaatgagggg atttaggaag acctcatgag aaggtagcat ttgagcaaag acatgtaggg 300

gtgagggagc tagccatgaa gttgcttaag gtggaggaca cagcccgtgc aaaggccctg 360

gggcagggcc gtatgttcct ggcatgttgg aggaagagcg aagaggcccg tgtggctgga 420

gcacagtgaa gagggggaga gagggagtgg ggagggcagg gagggaactg ggcaattcaa 480

gcagggtttt gtgggccttg gggaggactt gggcttgtcc ctggaggaaa gtgggagcca 540

tagagagttg tgggcagaag aagggtgtgc cctgactcag atgctcacag gcaacctctg 600

gtggtggctg cagggaggac agactgtggg gtacgggggc tggagtcaga agaccagctg 660

agtgatgacg gggctggacc agacagagga gggggtgaga agtgggtgaa ttctgggtat 720

atttcagagt gtcactgccg ccgtctgcat atggaggaag ctcacccatc tcatagtcta 780

gctggcctga ctcccccagc cccccaactc cccatgccca ggcctgtgtc gagtgggcgg 840

aagaaagaga tgtcagattc tgcatggaag gcgtggggtg gggctgggct gcaggtgctc 900

ccccagctcc cccccgcccc atggctcctc ctcctccacc ccctctcagg gcgacagtta 960

caggcaaaga agaggaagtg gtagcactag ctgtcgctcc acaggcgagc agggcaggcg 1020

tgcgggcggg tgggtggtgg aggctgcgag ggtgcacggc cggccctggg caggcggtag 1080

ccatggagct gtggcgccaa tgcacccact ggctcatcca gtgccgggtg ctgccgccca 1140

gccaccgcgt gacctgggat ggggctcagg tgtgtgaact ggcccaggcc ctccgggatg 1200

g 1201

<210> 56

<211> 1201

<212> DNA

<213> Homo sapiens

<400> 56

aaagtgagta cataaggcca gcgtggggag gaatagcgaa aaggtagttg gatcaatgga 60

tttggggtct tggagttgaa gtttttggag ttggagtatt atcagacaga gtgagctaga 120

aaggaagaag gttgtggccg ggcgcagcgg ctcacgcctg taatcccagc actttggaag 180

gccaaggcaa gcggatcacc tgaggtcagg agttcgagac cagcctggcc aacatggtga 240

agccacttct ctactaaaaa tacaaaaatt agccgggtgt ggtgccgggt gtttgtaatc 300

ccagctactc gggaggctaa ggcaggagaa tcacttgaac ccgggaggtg gaggttgcag 360

tgagccaaga tcgcaccact gcactccagc ctgggcaaca agagcgaaac tccatctcaa 420

aaaaaaaaaa aaaaaagaaa gaaagaaaga aagaaagaaa ggaagaaggt tgcaatggga 480

gttagaggat taaaattgag attgtagaga agttacaatt attcgtatga gcatgagagt 540

gagaggctga gccaagaatg atccctagag aagaatctga gaggccagag gattggaaga 600

attaagcgaa ttttgaaata accaagagtt atgacaatag tagtaatgaa tgacagtgaa 660

ccagaagccc aaatcttaga gaatgatggt gaatgacaga ggagtctata ggtgaccgaa 720

gtgagcgatg ctctaatctc tcgtcatgat gtaatattta aagcttttca tattttgaag 780

ggatgctagg atttgggggt gtgtggctcc tttaagggcc tgggaggggg agaagctgga 840

ggtcgggctg gggggggcgg gaccccctcg tctgagtctg cgcattgaga gcggaggcgg 900

gtccaggcgc gggctcgcgc gcccgggcgg gtcctcgcgg gggcggagtc tgcgctctgg 960

ttcgggctgc ggctgcggct gcggctgcgg ctgctactgc tacgctccta gcttgaggga 1020

aagaggccga ggcctgggcc aagcccggag ccgccgctcg ccggagcctc ctggagcctc 1080

cgcgccggct cagcctgggg gcgggctccg gtccggcccg ccgccgcacc caggacggag 1140

gctgcatgcc cgaggaccag gccggcgcag ccatggtgag ggagcaaggc ctgccctagc 1200

c 1201

Claims

1. Use of (a) and/or (b) in the manufacture of a kit for diagnosing thyroid cancer in a subject or identifying benign or malignant thyroid nodules in a subject,

wherein the DNA sequence comprises the following gene sequences: ACSL5, ACTR3B, AIM, ASB2, C15orf62, C2CD4B, CCDC, CCNB2, CD200, CD3G, CEBPD, CIITA, CSK, DLEU7, DYNLT3, ELF4, FABP3, HLA-E, HRH, IL12RB1, IL17C, ITGB2, KRTAP5-9, LAT2, LGALS1, LPIN1, OAS3, PCYT1B, PITX, PTR, PTPTPN 7, S100A10, PB 3BP4, SIGLEC14, SIGLEC7, SLC29A3, SLC2A10, SLC5A5, SLFN13, AFURF, STAT6, STEAP4, SUMF1, STF 1D10C, TEK, THIS 2, THIS 1, VAzmP 24, SIR 3934, SIR 24, ATxPLP 34, and optionally one or more of TBCP 3,

preferably, the first and second electrodes are formed of a metal,

the DNA sequence also includes the following gene sequences: (1) ATF7IP2, PLVAP, or (2) SIRPB2, or (3) ATF7IP2, PLVAP and SIRPB2, and/or

The fragment is a promoter region of a gene sequence, and/or

The fragment comprises at least 1, preferably at least 3 CpG dinucleotides.

2. The use according to claim 1, wherein the DNA sequence comprises the sequence: <xnotran> SEQ ID NO:1 , SEQ ID NO:2 , SEQ ID NO:3 , SEQ ID NO:4 , SEQ ID NO:6 , SEQ ID NO:7 , SEQ ID NO:8 , SEQ ID NO:9 , SEQ ID NO:10 , SEQ ID NO:11 , SEQ ID NO:12 , SEQ ID NO:13 , SEQ ID NO:14 , SEQ ID NO:15 , SEQ ID NO:16 , SEQ ID NO:17 , SEQ ID NO:18 , SEQ ID NO:19 , SEQ ID NO:20 , SEQ ID NO:21 , SEQ ID NO:22 , SEQ ID NO:23 , SEQ ID NO:24 , SEQ ID NO:25 , SEQ ID NO:26 , SEQ ID NO:27 , SEQ ID NO:28 , SEQ ID NO:29 , SEQ ID NO:30 , SEQ ID NO:32 , SEQ ID NO:33 , </xnotran> 34 or a complement or variant thereof, 35 or a complement or variant thereof, 36 or a complement or variant thereof, 37 or a complement or variant thereof, 39 or a complement or variant thereof, 40 or a complement or variant thereof, 41 or a complement or variant thereof, 42 or a complement or variant thereof, 43 or a complement or variant thereof, 44 or a complement or variant thereof, 45 or a complement or variant thereof, 46 or a complement or variant thereof, 47 or a complement or variant thereof, 48 or a complement or variant thereof, 49 or a complement or variant thereof, 50 or a complement or variant thereof, 51 or a complement or variant thereof, 52 or a complement or variant thereof, 53 or a complement or variant thereof, 54 or a complement or variant thereof, 55 or a complement or variant thereof; the DNA sequence optionally further comprises one or more selected from the group consisting of: 5or a complement or variant thereof, 31 or a complement or variant thereof, 38 or a complement or variant thereof, said variant being a variant having at least 70% identity to the corresponding sequence and wherein the methylation site is not mutated,

preferably, the DNA sequence further comprises the following sequence:

(2) 38 or a complementary sequence or variant thereof, or

3. The use according to claim 1 or 2,

said reagent comprising a primer molecule which hybridizes to said DNA sequence or fragment thereof or transformed variant thereof and which is capable of amplifying said DNA sequence or fragment thereof or transformed variant thereof and/or

Said agent comprising a probe molecule which hybridizes to said DNA sequence or fragment thereof or transformed variant thereof, and/or

The sample is derived from mammalian tissue, cells, e.g., thyroid tissue, and/or

The DNA sequence is transformed in that unmethylated cytosine is converted into a base having a lower binding capacity for guanine than cytosine, and/or the DNA sequence is treated with a methylation-sensitive restriction endonuclease, and/or

The diagnosis includes: and calculating to obtain a score, and diagnosing the thyroid cancer according to the score.

4. <xnotran> , , (1) SEQ ID NO:1 , SEQ ID NO:2 , SEQ ID NO:3 , SEQ ID NO:4 , SEQ ID NO:6 , SEQ ID NO:7 , SEQ ID NO:8 , SEQ ID NO:9 , SEQ ID NO:10 , SEQ ID NO:11 , SEQ ID NO:12 , SEQ ID NO:13 , SEQ ID NO:14 , SEQ ID NO:15 , SEQ ID NO:16 , SEQ ID NO:17 , SEQ ID NO:18 , SEQ ID NO:19 , SEQ ID NO:20 , SEQ ID NO:21 , SEQ ID NO:22 , SEQ ID NO:23 , SEQ ID NO:24 , SEQ ID NO:25 , SEQ ID NO:26 , SEQ ID NO:27 , SEQ ID NO:28 , </xnotran> SEQ ID NO:29 or a complement or variant thereof, SEQ ID NO:30 or a complement or variant thereof, SEQ ID NO 32 or a complement or variant thereof, SEQ ID NO 33 or a complement or variant thereof, SEQ ID NO 34 or a complement or variant thereof, SEQ ID NO 35 or a complement or variant thereof, SEQ ID NO 36 or a complement or variant thereof, SEQ ID NO 37 or a complement or variant thereof, SEQ ID NO 39 or a complement or variant thereof, SEQ ID NO 40 or a complement or variant thereof, SEQ ID NO 41 or a complement or variant thereof, SEQ ID NO 42 or a complement or variant thereof, SEQ ID NO 43 or a complement or variant thereof, SEQ ID NO 44 or a complement or variant thereof, SEQ ID NO 45 or a complement or variant thereof, SEQ ID NO 46 or a complement or variant thereof, SEQ ID NO 47 or a complement or variant thereof, SEQ ID NO 48 or a complement or variant thereof, SEQ ID NO 49 or a complement or a variant thereof, SEQ ID NO 50 or a complement or variant thereof, SEQ ID NO 52 or a complement or variant thereof, SEQ ID NO 53 or a variant thereof, SEQ ID NO 52 or a complement or a variant thereof, SEQ ID NO 52 or a variant thereof, SEQ ID NO 54 or a variant thereof, SEQ ID NO 52 or a variant thereof, SEQ ID NO 55 or a variant thereof, SEQ ID NO: 5or a complementary sequence or variant thereof, 31 or a complementary sequence or variant thereof, 38 or a complementary sequence or variant thereof, said variant being a variant which is at least 70% identical to the corresponding sequence and in which the methylation site is not mutated, and (2) a treated sequence of (1), said treatment converting unmethylated cytosine to a base which has less binding capacity to guanine than cytosine.

5. An agent for detecting methylation of DNA, said agent comprising an agent for detecting the level of methylation of a DNA sequence or fragment thereof or one or more CpG dinucleotides therein in a sample from a subject, said DNA sequence comprising the following gene sequences: ACSL5, ACTR3B, AIM, ASB2, C15orf62, C2CD4B, CCDC, CCNB2, CD200, CD3G, CEBPD, CIITA, CSK, DLEU7, DYNLT3, ELF4, FABP3, HLA-E, HRH, IL12RB1, IL17C, ITGB2, KRTAP5-9, LAT2, LGALS1, LPIN1, OAS3, PCYT1B, PITX, PTR, PTPTPN 7, S100A10, PB 3BP4, SIGLEC14, SIGLEC7, SLC29A3, SLC2A10, SLC5A5, SLFN13, AFURF, STAT6, STEAP4, SUMF1, STF 1D10C, TEK, THIS 2, THIS 1, VAzmP 24, SIR 3934, SIR 24, ATxPLP 34, and optionally one or more of TBCP 3,

preferably, the first and second electrodes are formed of a metal,

the fragment is a promoter region of a gene sequence, and/or

The fragment comprises at least 1, preferably at least 3 CpG dinucleotides, and/or

The DNA sequence comprises the following sequences: <xnotran> SEQ ID NO:1 , SEQ ID NO:2 , SEQ ID NO:3 , SEQ ID NO:4 , SEQ ID NO:6 , SEQ ID NO:7 , SEQ ID NO:8 , SEQ ID NO:9 , SEQ ID NO:10 , SEQ ID NO:11 , SEQ ID NO:12 , SEQ ID NO:13 , SEQ ID NO:14 , SEQ ID NO:15 , SEQ ID NO:16 , SEQ ID NO:17 , SEQ ID NO:18 , SEQ ID NO:19 , SEQ ID NO:20 , SEQ ID NO:21 , SEQ ID NO:22 , SEQ ID NO:23 , SEQ ID NO:24 , SEQ ID NO:25 , SEQ ID NO:26 , SEQ ID NO:27 , SEQ ID NO:28 , SEQ ID NO:29 , SEQ ID NO:30 , SEQ ID NO:32 , SEQ ID NO:33 , </xnotran> 34 or a complement or variant thereof, 35 or a complement or variant thereof, 36 or a complement or variant thereof, 37 or a complement or variant thereof, 39 or a complement or variant thereof, 40 or a complement or variant thereof, 41 or a complement or variant thereof, 42 or a complement or variant thereof, 43 or a complement or variant thereof, 44 or a complement or variant thereof, 45 or a complement or variant thereof, 46 or a complement or variant thereof, 47 or a complement or variant thereof, 48 or a complement or variant thereof, 49 or a complement or variant thereof, 50 or a complement or variant thereof, 51 or a complement or variant thereof, 52 or a complement or variant thereof, 53 or a complement or variant thereof, 54 or a complement or variant thereof, 56 or a variant thereof, and optionally one or more of the following, 54 or a variant thereof, 56 or a variant thereof, or a complement or variant thereof, or a variant thereof, 52 or a complement or variant thereof, 53 or a complement or a variant thereof, and optionally: 5or its complement or variant, 31 or its complement or variant, 38 or its complement or variant, said variant being a variant having at least 70% identity to the corresponding sequence and wherein the methylation site is not mutated.

6. The reagent according to claim 5,

said agent is a primer molecule which hybridizes to said DNA sequence or fragment thereof or transformed variant thereof and which is capable of amplifying said DNA sequence or fragment thereof or transformed variant thereof, or

The agent is a probe molecule that hybridizes to the DNA sequence or fragment thereof or the transformed variant thereof.

7. A medium bearing a DNA sequence or fragment thereof and/or methylation information thereof, said DNA sequence comprising (i) the following gene sequence: ACSL5, ACTR3B, AIM, ASB2, C15orf62, C2CD4B, CCDC, CCNB2, CD200, CD3G, CEBPD, CIITA, CSK, DLEU7, DYNLT3, ELF4, FABP3, HLA-E, HRH, IL12RB1, IL17C, ITGB2, KRTAP5-9, LAT2, LGALS1, LPIN1, OAS3, PCYT1B, PITX, PTR, PTPTPN 7, S100A10, SH3BP4, SIGLEC14, SIGLEC7, SLC29A3, SLC2A10, SLC5A5, FN13, AFURF, SLSTAT 6, STEAP4, SUMF1, PB 1D10C, TEK, THIS 2, THIS 1, SLC 24, VAMP 6, TMR 24, VAMP 6, TMSFF 24, VAzF 34, or a sequence that is less capable of converting to a cytosine 34 when treated with one or more than one of said sequence (S3, TMSFF 24, VAxIFF 34),

preferably, the first and second electrodes are formed of a metal,

the DNA sequence comprises a sense strand or an antisense strand of DNA, and/or

The fragment is a promoter region of a gene sequence, and/or

The DNA sequence comprises: <xnotran> SEQ ID NO:1 , SEQ ID NO:2 , SEQ ID NO:3 , SEQ ID NO:4 , SEQ ID NO:6 , SEQ ID NO:7 , SEQ ID NO:8 , SEQ ID NO:9 , SEQ ID NO:10 , SEQ ID NO:11 , SEQ ID NO:12 , SEQ ID NO:13 , SEQ ID NO:14 , SEQ ID NO:15 , SEQ ID NO:16 , SEQ ID NO:17 , SEQ ID NO:18 , SEQ ID NO:19 , SEQ ID NO:20 , SEQ ID NO:21 , SEQ ID NO:22 , SEQ ID NO:23 , SEQ ID NO:24 , SEQ ID NO:25 , SEQ ID NO:26 , SEQ ID NO:27 , SEQ ID NO:28 , SEQ ID NO:29 , SEQ ID NO:30 , SEQ ID NO:32 , SEQ ID NO:33 , </xnotran> 34 or a complement or variant thereof, 35 or a complement or variant thereof, 36 or a complement or variant thereof, 37 or a complement or variant thereof, 39 or a complement or variant thereof, 40 or a complement or variant thereof, 41 or a complement or variant thereof, 42 or a complement or variant thereof, 43 or a complement or variant thereof, 44 or a complement or variant thereof, 45 or a complement or variant thereof, 46 or a complement or variant thereof, 47 or a complement or variant thereof, 48 or a complement or variant thereof, 49 or a complement or variant thereof, 50 or a complement or variant thereof, 51 or a complement or variant thereof, 52 or a complement or variant thereof, 53 or a complement or variant thereof, 54 or a complement or variant thereof, or a variant thereof, optionally further comprises one or more of the following: 5or its complement or variant, 31 or its complement or variant, 38 or its complement or variant, said variant being a variant having at least 70% identity to the corresponding sequence and wherein the methylation site is not mutated.

8. The medium of claim 7,

the medium is a support on which the DNA sequence or a fragment thereof and/or methylation information thereof is printed, e.g., paper, plastic, metal, glass card, and/or

The medium is a computer-readable medium storing the sequence and/or its methylation information and a computer program which, when executed by a processor, performs the steps of: comparing the methylation sequencing data of the sample to the sequence or information, thereby obtaining the presence, amount and/or level of methylation of nucleic acid molecules comprising the sequence in the sample.

9. A kit for identifying thyroid cancer or thyroid nodule benign and malignant comprising:

(a) Reagents or devices for determining the methylation level of a DNA sequence or a fragment thereof or one or more CpG dinucleotides therein in a sample of a subject, and

wherein the DNA sequence comprises the following gene sequences: ACSL5, ACTR3B, AIM, ASB2, C15orf62, C2CD4B, CCDC, CCNB2, CD200, CD3G, CEBPD, CIITA, CSK, DLEU7, DYNLT3, ELF4, FABP3, HLA-E, HRH, IL12RB1, IL17C, ITGB2, KRTAP5-9, LAT2, LGALS1, LPIN1, OAS3, PCYT1B, PITX, PTR, PTPTPN 7, S100A10, PB 3BP4, SIGLEC14, SIGLEC7, SLC29A3, SLC2A10, SLC5A5, SLFN13, AFURF, STAT6, STEAP4, SUMF1, SLF 1D10C, TEK, THIS 2, THIS 1, VAzmP 24, SIR 3934, SIR 24, VAxft 3934, and optionally one or more of TBCP 3,

preferably, the first and second electrodes are formed of a metal,

the fragment is a promoter region of a gene,

the DNA sequence comprises the following sequences: <xnotran> SEQ ID NO:1 , SEQ ID NO:2 , SEQ ID NO:3 , SEQ ID NO:4 , SEQ ID NO:6 , SEQ ID NO:7 , SEQ ID NO:8 , SEQ ID NO:9 , SEQ ID NO:10 , SEQ ID NO:11 , SEQ ID NO:12 , SEQ ID NO:13 , SEQ ID NO:14 , SEQ ID NO:15 , SEQ ID NO:16 , SEQ ID NO:17 , SEQ ID NO:18 , SEQ ID NO:19 , SEQ ID NO:20 , SEQ ID NO:21 , SEQ ID NO:22 , SEQ ID NO:23 , SEQ ID NO:24 , SEQ ID NO:25 , SEQ ID NO:26 , SEQ ID NO:27 , SEQ ID NO:28 , SEQ ID NO:29 , SEQ ID NO:30 , SEQ ID NO:32 , SEQ ID NO:33 , </xnotran> 34 or a complement or variant thereof, 35 or a complement or variant thereof, 36 or a complement or variant thereof, 37 or a complement or variant thereof, 39 or a complement or variant thereof, 40 or a complement or variant thereof, 41 or a complement or variant thereof, 42 or a complement or variant thereof, 43 or a complement or variant thereof, 44 or a complement or variant thereof, 45 or a complement or variant thereof, 46 or a complement or variant thereof, 47 or a complement or variant thereof, 48 or a complement or variant thereof, 49 or a complement or variant thereof, 50 or a complement or variant thereof, 51 or a complement or variant thereof, 52 or a complement or variant thereof, 53 or a complement or variant thereof, 54 or a complement or variant thereof, or a variant thereof, optionally further comprises one or more of the following: 5or a complement or variant thereof, 31 or a complement or variant thereof, 38 or a complement or variant thereof, said variant being a variant having at least 70% identity to the corresponding sequence and wherein the methylation site is not mutated, and/or

The agent comprising the agent of claim 5or 6 and/or the medium of claim 7,

The kit further comprises a reagent for converting the DNA sequence, said conversion converting unmethylated cytosine to a base having a lower binding capacity for guanine than cytosine, preferably said reagent comprises bisulfite, bisulfite or metabisulfite or a combination thereof, and/or

The kit further comprises a methylation sensitive restriction enzyme, and/or

The kit also comprises PCR reaction reagents.

10. An apparatus for diagnosing thyroid cancer or identifying thyroid nodules as benign or malignant, the apparatus comprising a memory, a processor, and a computer program stored on the memory and executable on the processor, wherein the processor when executing the program performs the steps of:

(2) Calculating a score, and

(3) Diagnosing thyroid cancer or identifying benign or malignant thyroid nodules according to the scores,

step (1) comprises detecting the methylation level of the sequence in a sample, including but not limited to: PCR based on bisulfite conversion, DNA sequencing, methylation sensitive restriction enzyme analysis, fluorescence quantification, methylation sensitive high resolution melting curve method, chip-based methylation map analysis, mass spectrometry,

step (1) comprises detecting the methylation level of said sequence in a sample using a reagent according to claim 5or 6, a medium according to claim 7, or a kit according to claim 8 or 9, and/or

The sequence is transformed in that unmethylated cytosine is converted to a base that does not bind guanine, or the DNA sequence is treated with a methylation sensitive restriction enzyme.