CN111172270B - Whole blood transcription gene marker for grading cervical squamous intraepithelial lesions and application thereof - Google Patents

Abstract

The invention discloses a whole blood transcription gene marker for grading cervical squamous intraepithelial lesions, which comprises the following components: the characteristic gene sequences related to cervical intraepithelial neoplasia grading are shown in SEQ ID NO. 1-SEQ ID NO. 10. The invention also discloses application of the gene marker in preparation of products for screening high-grade cervical squamous intraepithelial lesions. The cervical squamous intraepithelial lesion related transcriptional gene marker can be used for screening high-level intraepithelial lesions (HSIL) and low-level intraepithelial lesions (LSIL), effectively finds out the high-level intraepithelial lesions (HSIL) with canceration potential, is beneficial to fundamentally reducing the incidence rate and death rate of cervical cancer through timely intervention, takes whole blood as a detection material, has the advantages of no wound, safe sampling, simple and convenient operation and high accuracy, is beneficial to improving the compliance of the detection of screened crowds, is very suitable for early screening and auxiliary diagnosis of cervical cancer of large-scale crowds, and has wide application prospect.

Description

Whole blood transcription gene marker for grading cervical squamous intraepithelial lesions and application thereof

Technical Field

The invention relates to the technical field of molecular biology, in particular to a whole blood transcriptional gene marker for discriminating and grading low-grade cervical squamous intraepithelial lesion (LSIL) and high-grade cervical squamous intraepithelial lesion (HSIL) and application thereof.

Background

Cervical cancer is the 3 rd most common malignancy among women worldwide after breast cancer and colorectal cancer, the 2 nd most common malignancy in developing countries after breast cancer, and the most common malignancy in female genital tract, and the onset of the malignancy tends to be younger. The pathogenesis of the cervical cancer is not completely clear at present, women who are married early, fertile early, prolific and disordered in sexual life have higher morbidity, and the cervical cytology screening is generally applied in recent decades, so that the cervical cancer and precancerous diseases are discovered and treated in early stages, and the morbidity and mortality of the cervical cancer are obviously reduced.

Cervical Squamous intraepithelial lesions (SIL for short) are a Lesion of epithelial tissue closely related to cervical cancer, and are frequently found in women between 25 and 35 years of age. Cervical Squamous intraepithelial lesions are further classified into Low-grade Squamous intraepithelial lesions (Low-grade Squamous intraepithelial lesions, abbreviated as LSIL) and High-grade Squamous intraepithelial lesions (High-grade Squamous intraepithelial lesions, abbreviated as HSIL) according to the difference in tissue and cellular morphology. Wherein the low grade squamous intraepithelial lesions are naturally regressed, but the high grade squamous intraepithelial lesions have a cancerating potential. In clinical practice, cervical squamous intraepithelial lesions are screened and found by people, high-grade squamous intraepithelial lesions are screened, and the high-grade squamous intraepithelial lesions are treated in time, so that the method is an effective measure for preventing cervical cancer.

A large number of clinical studies prove that the treatment effect of the cervical cancer is closely related to the discovery stage, the earlier the cervical cancer is discovered, the better the treatment effect is, and if the cervical cancer precancerous lesion such as high-grade cervical squamous intraepithelial lesion can be discovered and treated in time, the ideal treatment effect can be obtained. At present, cervical squamous intraepithelial lesions are clinically diagnosed mainly through colposcopic tissue biopsy, the method is a traumatic sampling inspection method, the inspection process is complicated, pain and bleeding risks exist, and the compliance of screening people who accept the invasive inspection is low. Therefore, in the field of cervical cancer screening, a noninvasive, safe and simple method is urgently needed, high-grade cervical squamous intraepithelial lesions can be screened, so that intervention treatment can be timely carried out on the cervical cancer precancerous lesions, and the morbidity and mortality of cervical cancer in China are fundamentally reduced.

Blood is a commonly used test material in clinic, and the blood flowing in circulation can exchange information with almost all tissue cells, wherein abundant disease information is contained. Immune cells (leukocytes) in blood are important components of the immune system, when abnormal tumor cells are generated in vivo, the immune system can perform an immune monitoring function, the immune cells such as leukocytes contained in the blood participate in the interaction between the immune system and the tumor, and the gene expression signals of the immune cells change correspondingly, and the change of the gene expression signals of the immune cells is far earlier than the change of obvious signs of the body due to diseases. Therefore, by closely monitoring the blood cell gene expression signal, micro information of malignant diseases such as in vivo tumor can be sensitively captured, the trace of in vivo tumor can be found earlier, and especially reliable evidence is provided for early screening of cervical squamous intraepithelial lesion and other precancerous lesions.

Disclosure of Invention

The invention aims to solve the problem that the diagnosis of cervical squamous intraepithelial lesions depends on traumatic biopsy sampling examination in the current cervical cancer screening process, so that the compliance of screened people to be examined is low, and provides a whole blood transcription gene marker combination for grading cervical squamous intraepithelial lesions, which is used for screening low-grade intraepithelial lesions and high-grade intraepithelial lesions and has high detection accuracy.

In order to solve the technical problems, the invention is realized by the following technical scheme:

in one aspect of the invention, a whole blood transcription gene marker for grading cervical squamous intraepithelial lesions is provided, which comprises one or any combination of more than two of the following genes: STMN3, TRPC4AP, DYRK2, AGK, KIAA0319L, GRPEL1, ZFC3H1, LYL1, ITGB1, ARHGAP18 genes shown in SEQ ID NO. 1-10.

Preferably, the whole blood transcriptional gene marker comprises the following 10 gene combinations: STMN3, TRPC4AP, DYRK2, AGK, KIAA0319L, GRPEL1, ZFC3H1, LYL1, ITGB1, ARHGAP18 genes shown in SEQ ID NO. 1-10.

In another aspect of the present invention, there is provided a kit for screening and grading cervical squamous intraepithelial lesions, the kit comprising: a reagent for detecting the expression level of any one or any two or more of the following genes: STMN3, TRPC4AP, DYRK2, AGK, KIAA0319L, GRPEL1, ZFC3H1, LYL1, ITGB1, ARHGAP18 genes shown in SEQ ID No. 1-10.

Preferably, the kit comprises: the reagent for detecting the expression level of the following 10 genes: STMN3, TRPC4AP, DYRK2, AGK, KIAA0319L, GRPEL1, ZFC3H1, LYL1, ITGB1, ARHGAP18 genes shown in SEQ ID NO. 1-10.

The reagent is a reagent for detecting the amount of RNA transcribed from the gene, and the reagent is preferably a primer and/or a probe.

The reagent is a reagent for detecting the amount of the gene reverse transcribed into complementary DNA (cDNA), and the reagent is a primer and/or a probe. Because of the strict quantitative correspondence between messenger RNA (mRNA) and its reverse transcribed complementary DNA (cDNA), cervical squamous intraepithelial lesions can be screened by detecting the amount of cDNA.

In another aspect of the present invention, there is also provided a composition comprising primers and/or probes for detecting genes differentially expressed in whole blood of patients with low-grade cervical squamous intraepithelial lesions and high-grade cervical squamous intraepithelial lesions, the genes comprising one or a combination of any two or more of the following genes: STMN3, TRPC4AP, DYRK2, AGK, KIAA0319L, GRPEL1, ZFC3H1, LYL1, ITGB1, ARHGAP18 genes shown in SEQ ID No. 1-10.

Preferably, the genes include the following 10 genes: STMN3, TRPC4AP, DYRK2, AGK, KIAA0319L, GRPEL1, ZFC3H1, LYL1, ITGB1, ARHGAP18 genes shown in SEQ ID No. 1-10.

In another aspect of the present invention, there is also provided a gene chip for screening and grading cervical squamous intraepithelial lesions, the gene chip comprising oligonucleotide probes hybridized with a characteristic gene sequence related to cervical squamous intraepithelial lesions, the gene comprising one or any combination of two or more of the following genes: STMN3, TRPC4AP, DYRK2, AGK, KIAA0319L, GRPEL1, ZFC3H1, LYL1, ITGB1, ARHGAP18 genes shown in SEQ ID NO. 1-10.

The kit or the gene chip can be used for detecting the expression condition of cervical squamous intraepithelial lesion related characteristic genes shown in SEQ ID NO. 1-SEQ ID NO.10 in whole blood of a detected person, and then a prediction model is constructed according to the up-regulation or down-regulation information of the gene expression and a statistical method such as a Logistic regression algorithm (LSIL) and the like to judge whether the detected person is high-level intraepithelial lesion (HSIL) or low-level intraepithelial lesion (LSIL), so that the high-level intraepithelial lesion (HSIL) with canceration potential is screened, the compliance of the screened person for receiving examination is improved, the early discovery of the cervical carcinoma precancerous lesion is realized, timely intervention is carried out, and the morbidity and mortality of the cervical carcinoma are fundamentally reduced.

In another aspect of the invention, the application of the whole blood transcription gene marker in preparing a product for screening high-grade cervical squamous intraepithelial lesions is also provided.

The product for screening high-grade cervical squamous intraepithelial lesions comprises: products for detecting high-grade cervical squamous intraepithelial lesions by using real-time quantitative PCR, RNA sequencing or gene chips.

In the present invention, the term "cervical squamous intraepithelial lesion-related characteristic gene" means a gene that can be used for discriminating between high-grade intraepithelial lesions (HSIL) and low-grade intraepithelial lesions (LSIL), and a significant change in the expression level of the group of genes can be used as an index for discriminating between high-grade intraepithelial lesions (HSIL).

The term "primer" refers to an oligonucleotide that, when paired with a strand of DNA, initiates primer extension synthesis product in the presence of a suitable polymerization reagent. To maximize amplification efficiency, the primer is preferably single-stranded. The length of the primer depends on many factors, including: application area, temperature used, template reaction conditions, other reagents and primer sources. The skilled person can design the primers autonomously according to the characteristic genes of colorectal cancer of the present invention and specific requirements.

The term "probe" refers to a molecule that binds to a specific sequence or subsequence or other portion of another molecule. Unless otherwise indicated, the term "probe" generally refers to a polynucleotide probe that is capable of binding to another polynucleotide (often referred to as a "target polynucleotide") by complementary base pairing. Depending on the stringency of the hybridization conditions, a probe can bind to a target polynucleotide that lacks complete sequence complementarity to the probe. The probe may be directly or indirectly labeled.

Those skilled in the art can design other probes that can be used to implement the present invention according to the characteristic genes related to cervical squamous intraepithelial lesions and specific requirements, and are not limited to the probes specifically used in the embodiments of the present invention.

The term "gene chip" refers to a substrate having oligonucleotide probes with different known sequences deposited at known discrete locations on the surface of the substrate. The substrate may be silicon or glass, have the thickness of a glass slide or cover slip, or may be composed of other synthetic polymers.

The transcription gene marker combination for screening and grading cervical squamous intraepithelial lesions adopts an ROC curve (receiver operating characteristic curve) tool to measure the performance of the gene marker combination. Disease diagnosis is the problem of distinguishing between patients and normal persons, and is mathematically a dichotomous problem, i.e., dividing instances into positive or negative, so that a combination of gene markers essentially behaves as a classifier. Four situations can occur when the classification is correct and incorrect: true Positive (TP) if an instance is Positive and is predicted to be Positive; if the example is negative but predicted to be Positive, it is called False Positive (FP). Accordingly, if an instance is Negative and predicted to be Negative, it is referred to as True Negative (TN), and if the instance is positive but predicted to be Negative, it is False Negative (FN). The classification ability of a classifier is measured in terms of sensitivity (sensitivity) and specificity (specificity). The sensitivity is a True Positive Rate (TPR), and the calculation formula is TPR = TP/(TP + FN), which indicates the proportion of the positive examples identified by the classifier to all the positive examples. The specificity is True Negative Rate (TNR), and the calculation formula is TNR = TN/(FP + TN), which represents the proportion of the Negative examples identified by the classifier to all Negative examples.

The marker genes of the invention can be used to design specific oligonucleotide probes and corresponding primers. Such probes and primers can be of any length that specifically hybridizes to a marker gene sequence and is at least about 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 35, 40, 50, 75, 100, 150, 200, 300, 400, 500 nucleotides in length, and the probes can be up to the full length of the marker gene sequences of the present invention. The probe may also comprise other sequences at its 5 'and/or 3' end so as to be capable of extending beyond the target sequence to which it is hybridised.

When a nucleic acid amplification method is used, these probes and primers enable rapid analysis of peripheral blood samples, i.e., detection or quantitative determination of the transcription products (mainly mRNA) of the marker genes. Such methods include any method or technique known in the art or described herein for replicating or increasing the copy number or content of a target nucleic acid or its complement.

The cervical squamous intraepithelial lesion related transcriptional gene marker can be used for screening high-grade intraepithelial lesions (HSIL) and low-grade intraepithelial lesions (LSIL), effectively finds out the high-grade intraepithelial lesions (HSIL) with canceration potential, and contributes to fundamentally reducing the incidence rate and death rate of cervical cancer through timely intervention. In addition, the invention takes whole blood as a detection material, has the advantages of no wound, safe sampling, simple and convenient operation and high accuracy, is beneficial to improving the compliance of the detection acceptance of screening people, is very suitable for early screening and auxiliary diagnosis of cervical cancer of large-scale people, and has wide application prospect.

Drawings

The present invention will be described in further detail with reference to the accompanying drawings and specific embodiments.

FIG. 1 is a ROC AUC graph for the classification diagnosis of 66 high-grade cervical squamous intraepithelial lesions (HSIL) and 36 low-grade cervical squamous intraepithelial lesions (LSIL) by using the cervical squamous intraepithelial lesion classification diagnosis model constructed by the combination of 10 transcriptional gene markers;

FIG. 2 is a box plot of 66 high-grade cervical squamous intraepithelial lesions (HSIL) and 36 low-grade cervical squamous intraepithelial lesions (LSIL) screened by the 10 combinations of gene markers according to the present invention, wherein the ordinate is the decision score (decision score) of the sample, also called logistic regression log likelihood ratio (log regression), the critical threshold (cutoff value) for determination is set to 0, and if the decision score of the sample is greater than or equal to 0, the high-grade cervical squamous intraepithelial lesions (HSIL) is determined; a low-grade cervical squamous intraepithelial lesion (LSIL) is judged if the sample decision score value is less than 0.

Detailed Description

The invention is based on the quantitative analysis of the whole blood whole gene expression profile, the whole blood gene expression difference of patients with higher-level cervical squamous intraepithelial lesions (HSIL) and lower-level cervical squamous intraepithelial lesions (LSIL) is screened out 10 characteristic gene expression signals (transcription gene markers, messenger RNA) of the high-level intraepithelial lesions (HSIL) from the whole blood gene expression profile by a characteristic recursion Elimination (Recurved Feature Elimination): STMN3, TRPC4AP, DYRK2, AGK, KIAA0319L, GRPEL1, ZFC3H1, LYL1, ITGB1, ARHGAP18. Based on the 10 gene markers obtained by screening, a classification model for cervical squamous intraepithelial lesions is constructed by using a Logistic Regression algorithm (Logistic Regression). Quantitatively detecting the relative expression data of the 10 gene markers in the whole blood of a detected person by utilizing a fluorescence quantitative RT-PCR (reverse transcription-polymerase chain reaction), a gene expression profiling chip or an RNA sequencing technology, substituting the relative expression data into the constructed intraepithelial lesion classification diagnosis model, calculating a decision score (decision score) of a sample, also called a logistic regression log likelihood ratio (log likelihood ratio), setting a judgment critical threshold (cutoff value) to be 0, and judging high-grade cervical squamous intraepithelial lesions (HSIL) if the decision score of the sample is greater than or equal to 0; a low-grade cervical squamous intraepithelial lesion (LSIL) is judged if the sample decision score value is less than 0.

Example 1 screening of Gene markers characteristic of cervical squamous intraepithelial lesions

The screening of the cervical squamous intraepithelial lesion peripheral blood characteristic gene comprises the following steps:

1) Peripheral blood samples of 20 patients with high-grade cervical squamous intraepithelial lesions (HSIL) and 20 patients with low-grade cervical squamous intraepithelial lesions (LSIL) confirmed by pathological examination were collected using PAXgeneTMblood RNA Tube blood collection tubes, and 2-3mL of peripheral blood was collected from each sample.

2) Total RNA of the peripheral Blood sample was extracted using PAXgene Blood RNA Kit extraction Kit. The RNA samples were tested for fragment integrity (RIN) using an Agilent Bioanalyzer 2100 and for purity using a Nano1000 micro-UV spectrophotometer. All RNA samples must meet the following quality control conditions: the RNA yield is more than 2 micrograms, the ratio of 28S/18S peak is more than 1, the RIN value is more than 7, and the absorbance ratio of 260nm/280nm is more than 1.8.

3) The Gene expression signal of the RNA sample was detected by Affymetrix Gene Profiling Array cGMP U133P 2 chip (human whole Gene expression Profiling chip).

4) The MAS5 statistical method in Affymetrix Expression vector software is utilized to carry out Normalization processing (Normalization) on the detection result of the gene Expression profile chip of each sample, and genes with proper Expression in all samples are selected for subsequent analysis.

5) Screening characteristic gene expression signals related to cervical squamous intraepithelial lesions by using a characteristic recursive elimination method to obtain 10 genes which can be used for screening high-level intraepithelial lesions (HSIL) and low-level intraepithelial lesions (LSIL) and have the highest prediction accuracy (accuracy) and serve as gene markers: STMN3, TRPC4AP, DYRK2, AGK, KIAA0319L, GRPEL1, ZFC3H1, LYL1, ITGB1, ARHGAP18;

6) And detecting the expression signals of the 10 characteristic genes related to the cervical squamous intraepithelial lesions by using a real-time fluorescent quantitative PCR method, wherein the results show that the expression trends of all the 10 characteristic genes on a PCR platform and an expression profile chip platform are consistent. The sequences of the 10 gene markers are shown in table 1 below.

TABLE 1 Gene sequences of cervical squamous intraepithelial lesion-associated Gene markers

As can be seen from table 1 above:

the correlation between the expression level of STMN3 gene and high-grade cervical squamous intraepithelial lesion (HSIL) is 0.74, and the high expression of the gene can obviously increase the risk of HSIL.

The correlation between the expression level of TRPC4AP gene and high-grade cervical squamous intraepithelial lesion (HSIL) is 0.70, and the high expression of the gene can obviously increase the risk of HSIL.

The correlation between the expression level of DYRK2 gene and high-grade cervical squamous intraepithelial lesion (HSIL) is 0.62, and the high expression of the gene can obviously increase the risk of HSIL.

The correlation between the expression level of the AGK gene and high-grade cervical squamous intraepithelial lesion (HSIL) is 0.65, and the high expression of the AGK gene can obviously increase the risk of suffering from the HSIL.

The correlation between the expression level of the KIAA0319L gene and high-grade cervical squamous intraepithelial lesion (HSIL) is 0.62, and the high expression of the gene can obviously increase the risk of suffering from HSIL.

The correlation between the expression level of GRPEL1 gene and high-grade cervical squamous intraepithelial lesion (HSIL) is 0.66, and the high expression of the gene can obviously increase the risk of HSIL.

The correlation between the expression level of the ZFC3H1 gene and high-grade cervical squamous intraepithelial lesion (HSIL) is 0.65, and the high expression of the gene can obviously increase the risk of suffering from HSIL.

The correlation between the expression level of LYL1 gene and high-grade cervical squamous intraepithelial lesion (HSIL) is 0.61, and the low expression of the gene can obviously increase the risk of HSIL.

The correlation between the expression level of the ITGB1 gene and high-grade cervical squamous intraepithelial lesion (HSIL) is 0.68, and the low expression of the gene can obviously increase the risk of suffering from the HSIL.

The correlation between the expression level of the ARHGAP18 gene and high-grade cervical squamous intraepithelial lesion (HSIL) is 0.75, and the low expression of the gene can obviously increase the risk of suffering from the HSIL.

8) Based on 10 screened cervical squamous intraepithelial lesion related gene markers, analyzing relative expression values of samples by using a Logistic Regression algorithm (Logistic Regression), constructing a cervical squamous intraepithelial lesion classification model, calculating a decision score (decision score) of each sample, also called a Logistic Regression log likelihood ratio (Logistic Regression), setting a critical threshold (cutoff value) of the Logistic Regression score to be 0, taking the critical threshold as a judgment standard, and judging the sample to be positive, namely HSIL if the decision score of the sample is greater than or equal to 0; and if the decision score of the sample is less than 0, judging the sample to be negative, namely LSIL.

Example 2 screening and grading of cervical squamous intraepithelial lesions Using 10 selected Whole blood immunogene markers

1. The method comprises the following steps:

1) Collecting peripheral blood samples of the sample to be detected: collecting peripheral blood of elbow vein of 2ml of the subject by using PAXgeneTMblood RNA Tube;

2) Extracting and purifying total RNA in a peripheral blood sample of a sample to be detected: extracting and purifying total RNA in peripheral Blood by using a PAXgene Blood RNA Kit extraction Kit, identifying the integrity and yield of the extracted total RNA fragment by using an Agilent BioAnalyzer model 2100 micro electrophoresis analyzer, and detecting the purity of an RNA sample by using a Nano1000 micro ultraviolet spectrophotometer;

3) Reverse transcription reaction: performing Reverse Transcription reaction by using a High-Capacity cDNA Reverse Transcription kit of Life Techonlgy company and taking Olig (dT) as a Reverse Transcription primer by using total RNA as a template to synthesize cDNA;

TABLE 2 reaction System for reverse transcription Synthesis of cDNA

4) Fluorescent quantitative RT-PCR detection: according to messenger RNA (mRNA) sequences of 10 cervical squamous intraepithelial lesion related gene markers, specific primers SEQ ID NO. 11-SEQ ID NO.30 are designed (wherein, the primers SEQ ID NO. 11-SEQ ID NO.12 are used for specifically amplifying an STMN3 gene marker shown in SEQ ID NO.1, the primers SEQ ID NO. 13-SEQ ID NO.14 are used for specifically amplifying a TRPC4AP gene marker shown in SEQ ID NO.2, the primers SEQ ID NO. 15-SEQ ID NO.16 are used for specifically amplifying a DYRK2 gene marker shown in SEQ ID NO.3, the primers SEQ ID NO. 17-SEQ ID NO.18 are used for specifically amplifying an AGK gene marker shown in SEQ ID NO.4, the primers SEQ ID NO. 19-SEQ ID NO.20 are used for specifically amplifying a KIAA0319L gene marker shown in SEQ ID NO.5, the primers SEQ ID NO. 21-SEQ ID NO.22 are used for specifically amplifying a GRID NO.1 gene marker shown in SEQ ID NO.6, primers SEQ ID NO. 23-SEQ ID NO.24 are used for specifically amplifying a ZFC3H1 gene marker shown in SEQ ID NO.7, primers SEQ ID NO. 25-SEQ ID NO.26 are used for specifically amplifying a LYL1 gene marker shown in SEQ ID NO.8, primers SEQ ID NO. 27-SEQ ID NO.28 are used for specifically amplifying an ITGB1 gene marker shown in SEQ ID NO.9, and primers SEQ ID NO. 29-SEQ ID NO.30 are used for specifically amplifying an ARHGAP18 gene marker shown in SEQ ID NO. 10), and specific probes SEQ ID NO. 31-SEQ ID NO.40 (wherein SEQ ID NO. 31-SEQ ID NO.40 are probe sequences of characteristic gene sequences of SEQ ID NO. 1-SEQ ID NO.10 respectively) are designed, the SEQ ID NO. 11-SEQ ID NO.30 are used as primers, the SEQ ID NO. 31-SEQ ID NO.40 are used as probes or SYBR Green which can be combined with PCR amplification fragment specific dyes, and (3) carrying out real-time fluorescent quantitative RT-PCR reaction by using cDNA obtained by reverse transcription as an amplification template to obtain the mRNA relative content of the 10 gene markers in the peripheral blood sample.

The sequence of the fluorescent quantitative PCR amplification primer is as follows:

STMN3 gene

A forward primer: 5 'GCTGTCCACTACTGGCTTTTG 3' (SEQ ID NO. 11)

Reverse primer: 5 'GGTGCATATTACAGGAGGCTTTCG 3' (SEQ ID NO. 12)

TRPC4AP Gene

A forward primer: 5'CTCGGGCTGTGGAGAGTTTC 3' (SEQ ID NO. 13)

Reverse primer: 5'GCCTCGCTTCAGCAGGAA' (SEQ ID NO. 14)

DYRK2 gene

A forward primer: 5 'CACGGACACGATCCAGGTTCCA 3' (SEQ ID NO. 15)

Reverse primer: 5'CCGTTTTGCCCACTGTTGTA 3' (SEQ ID NO. 16)

AGK gene

A forward primer: 5 'TCTCTCTTCTTTGACCGTGCTAGGA 3' (SEQ ID NO. 17)

Reverse primer: 5 'TGTGGAGGAGCCTAGACACTAGGA 3' (SEQ ID NO. 18)

KIAA0319L gene

A forward primer: 5 'GGAAGGCTATGTGTGAACGTGACCA 3' (SEQ ID No. 19)

Reverse primer: 5'CTGTAGAAGTGGTTGGCAAAGAGA 3' (SEQ ID NO. 20)

GRPEL1 gene

A forward primer: 5'TTCGGGAGTCTGTTCCCTTT 3' (SEQ ID NO. 21)

Reverse primer: 5 'GGCCCTGTCTGAGCTCTTCT 3' (SEQ ID NO. 22)

ZFC3H1 gene

A forward primer: 5 'CAACCGTGAAACAGCGTTT 3' (SEQ ID NO. 23)

Reverse primer: 5'CTGCCAATACCATCAATTTCCA 3' (SEQ ID NO. 24)

LYL1 gene

A forward primer: 5 'CCCCTCTCTCCTCAACAGTGTCTACAT 3' (SEQ ID NO. 25)

Reverse primer: 5'AGCCAGGTCCAGCTCACAGT 3' (SEQ ID NO. 26)

ITGB1 gene

A forward primer: 5'TCAGAATTGGATTTGGCTCATTT 3' (SEQ ID NO. 27)

Reverse primer: 5'TGGTGCAGTTCTGTTCACTTGTG 3' (SEQ ID NO. 28)

ARHGAP18 gene

A forward primer: 5 'GATGATGCCCACATTACTAGTTTCA 3' (SEQ ID NO. 29)

Reverse primer: 5 'GGTGCGAGGTCACCATCC 3' (SEQ ID NO. 30)

The fluorescent quantitative PCR probe sequence is as follows:

STMN3 gene

The probe sequence is as follows: 5 'ATATTCCTCATGGGCCAGCTCA 3' (SEQ ID NO. 31)

TRPC4AP Gene

The probe sequence is as follows: 5 'CCGAGGGACCTCTCTATGCAGA 3' (SEQ ID NO. 32)

DYRK2 gene

The probe sequence is as follows: 5'AAGCGGACAGTGCTCACGACACAACC 3' (SEQ ID NO. 33)

AGK gene

The probe sequence is as follows: 5 'AAAGTGCATTCTGCCTGGCCCTGGCGCGACC 3' (SEQ ID NO. 34)

KIAA0319L gene

The probe sequence is as follows: 5 'AGAATCGGCCCCCCCATTGCTATTGT 3' (SEQ ID No. 35)

GRPEL1 gene

The probe sequence is as follows: 5 'TGCCACGCGTTGAATAGTTCCACTACTT 3' (SEQ ID NO. 36)

ZFC3H1 gene

The probe sequence is as follows: 5 'AGACAAGTTCATCCCCAGCAAACTCTG 3' (SEQ ID No. 37)

LYL1 gene

The probe sequence is as follows: 5 'CATCTTTCCCTAGCAGCCGGTTGAAGC 3' (SEQ ID NO. 38)

ITGB1 gene

The probe sequence is as follows: 5 'TAGCACACACCAGCTAAGCTCAGGAACCC 3' (SEQ ID No. 39)

ARHGAP18 gene

The probe sequence is as follows: 5'ATTGCCAAAAGACAAAACGGGTACCACA 3' (SEQ ID NO. 40)

TABLE 3 real-time fluorescent quantitative PCR reaction System

5) And (3) diagnosis of the result of the sample to be detected: according to the relative expression values of 10 transcription gene markers such as STMN3, TRPC4AP, DYRK2, AGK, KIAA0319L, GRPEL1, ZFC3H1, LYL1, ITGB1, ARHGAP18 and the like in a whole blood sample obtained by real-time fluorescent quantitative PCR detection, substituting into a constructed cervical squamous intraepithelial lesion classification model, calculating the decision score value of each sample, taking the critical threshold value 0 as a judgment standard, and judging that the sample is positive if the decision score value of the sample is greater than or equal to 0, namely judging that the sample is HSIL; if the result of the decision score value of the sample is less than 0, the sample is determined to be negative, i.e., the sample is determined to be LSIL.

2. Results

Collecting peripheral blood samples of 66 patients with high-level cervical squamous intraepithelial lesions (HSIL) and 36 patients with low-Level Squamous Intraepithelial Lesions (LSIL), detecting the relative expression quantity of 10 peripheral blood transcription gene markers in the peripheral blood by using fluorescence quantitative RT-PCR (reverse transcription-polymerase chain reaction), inputting the relative expression quantity into a cervical squamous intraepithelial lesion classification model, calculating a decision score of each sample, and judging a positive detection result, namely HSIL, when the sample decision score is not less than 0; otherwise, the result is a negative detection result, namely HSIL. Comparing the sample classification result obtained by model calculation with the sample pathological diagnosis result, the sensitivity (TP, true positive rate) of the classification diagnosis of the cervical squamous intraepithelial lesions based on 10 whole blood transcription gene marker combinations reaches 82%, the specificity (TN, true negative rate) reaches 83%, and the detection accuracy reaches 82%. Meanwhile, an ROC curve is established according to the decision score and the judgment result of the sample, and the area under the ROC curve (ROC AUC) is calculated to be 0.90. The specific test results are shown in Table 4 and FIGS. 1-2.

TABLE 4 sensitivity, specificity and ROC AUC values for colorectal cancer detection

The above-mentioned embodiments only express the implementation manner of the present invention, and the description thereof is specific and detailed, but not to be understood as the limitation of the patent scope of the present invention. It should be noted that, for a person skilled in the art, several variations and modifications can be made without departing from the inventive concept, which falls within the scope of the present invention. Therefore, the protection scope of the present patent shall be subject to the appended claims.

Sequence listing

<110> Huaxia upper garment science and technology Co., ltd

<120> Whole blood transcription gene marker for cervical squamous intraepithelial lesion grading and application thereof

<160> 40

<170> PatentIn version 3.3

<210> 1

<211> 2351

<212> DNA

<213> human (Homo sapiens)

<400> 1

acaccgctgc ggcctcgggg accacccttt gcgacgcccg cgccacctgc tctggccaga 60

aaagccactg ccctttgcgc cgcggggtct ggatggggga ggggcgactg cgatccccaa 120

accctccgga gaccagaggg ctccgcccct cccgcccctc cacctcccgc ggggcggacc 180

caccgccttc gaagcctaca aggagaagat gaaggagctg tcggtgctgt cgctcatctg 240

ctcctgcttc tacacacagc cgcaccccaa taccgtctac cagtacgggg acatggaggt 300

gaagcagctg gacaagcggg cctcaggcca gagcttcgag gtcatcctca agtccccttc 360

tgacctgtcc ccagagagcc ctatgctctc ctccccaccc aagaagaagg acacctccct 420

ggaggagctg caaaagcggc tggaggcagc cgaggagcgg aggaagacgc aggaggcgca 480

ggtgctgaag cagctggcgg agcggcgcga gcacgagcgc gaggtgctgc acaaggcgct 540

ggaggagaat aacaacttca gccgccaggc ggaggagaag ctcaactaca agatggagct 600

cagcaaggag atccgcgagg cacacctggc cgcactgcgc gagcggctgc gcgagaagga 660

gctgcacgcg gccgaggtgc gcaggaacaa ggagcagcga gaagagatgt cgggctaagg 720

gcccgggacg ggcggcgccc atcctgcgac agaacacgtt cgggttttgg ttttgtttcg 780

ttcacctctg tctagatgca acttttgttc ctcctccccc acccccgccc ccagcttcat 840

gcttctcttc cgcactcagc cgccctgccc tgtcctcgtg gtgagtcgct gaccacggct 900

tcccctgcag gagccgccgg gcgtgagacg cggtccctcg gtgcagacac caggccgggc 960

gcggctgggt cccccggggg ccctgtgaga gaggtggcgg tgaccgtggt aaacccaggg 1020

cggtggcgtg ggatcacggg tccttacgct gggctgtctg gtcagcacgt gcaggtcagg 1080

gcaggtcctc tgagccggcg cccctggcca gcaggcgagg ctacagtacc tgctgtcttt 1140

ccagggggaa ggggctcccc atgagggagg ggcgacgggg gaggggggtg atggtgcctg 1200

ggagcctgcg tgtgcagccg gtgcttgttg aactggcagg cgggtgggtg ggggctgcag 1260

ctttccttaa tgtggttgca caggggtcct ctgagaccac ctggcgtgag gtggacaccc 1320

tgggccttcc tggaagcctg cagttggggg cctgccctga gtctgctggg gagtgggcat 1380

tctctgccag ggacccatga gcaggctgca tggtctagag gttgtgggca gcatggacag 1440

tcccccactc agaagtgcaa gagttccaaa gagcctctgg cccaggcccc tccccaccag 1500

ggctttgcag atgtccttga aagacccacc ctagagccct ttggagtgct ggcccctcct 1560

gtgccctctg ccctggtgga agcggcagcc acaagtcctc ctcagggagc cccaaggggg 1620

attttgtggg accgctgccc acagatccag gtgttggaag ggcagcgggt aaggttccca 1680

agccagcccc aacacccttc ccacttggca cccagagggg gctgtgggtg gaggcctgac 1740

tccaggcctc tcctgcccac accctctggg ctgagttcct tctttccctt ggacgcccag 1800

tgctggcctt ggaggacggt cagctggagg atggcggtgg gggaggctgt ctttgtacca 1860

ctgcagcatc ccccacttct ccacggaagc cccatcccaa agctgctgcc tggccccttg 1920

ctgtaaagtg tgaagggggc ggctgagttc tcttaggacc cagagccagg gccctcaact 1980

tccatcctgc gggaggcctt ggccgggcac tgccagtgtc ttccagagtc acacccaggg 2040

accacgggag gatcctgacc cctgcagggc tcaggggtca gcagggaccc actgccccat 2100

ctccctctcc ccaccaagac agccccagaa ggagcagcca gctgggatgg gaacccaagg 2160

ctgtccacat ctggcttttg tgggactcag aaagggaagc agaactgagg gctgggatat 2220

tcctcatggt ggcagcgctc atagcgaaag cctactgtaa tatgcaccca tctcatccac 2280

gtagtaaagt gaacttaaaa attcaatcaa atgaacaatt aaataaacac ctgtgtgttt 2340

aagacatggt t 2351

<210> 2

<211> 3162

<212> DNA

<213> human (Homo sapiens)

<400> 2

gcttcctgtt tgtccgacga ggagacatgg cggcggcgcc ggtagcggct gggtctggag 60

ccggccgagg gagacggtcg gcagccacag tggcggcttg gggcggatgg ggcggccggc 120

cgcggcctgg taacattctg ctgcagctgc ggcagggcca gctgaccggc cggggcctgg 180

tccgggcggt gcagttcact gagacttttt tgacggagag ggacaaacaa tccaagtgga 240

gtggaattcc tcagctgctc ctcaagctgc acaccaccag ccacctccac agtgactttg 300

ttgagtgtca aaacatcctc aaggaaattt ctcctcttct ctccatggag gctatggcat 360

ttgttactga agagaggaaa cttacccaag aaaccactta tccaaatact tacatttttg 420

acttgtttgg aggtgttgat cttcttgtag aaattcttat gaggcctacg atctctatcc 480

ggggacagaa actgaaaata agtgatgaaa tgtccaagga ctgcttgagt atcctgtata 540

atacctgtgt ctgtacagag ggagttacaa agcgtttggc agaaaagaat gactttgtga 600

tcttcctgtt tacattgatg acaagtaaga agacattctt acaaacagca accctcattg 660

aagatatttt gggtgttaaa aaggaaatga tccgactaga tgaagtcccc aatctgagtt 720

ccttagtatc caatttcgat cagcagcagc tcgctaattt ctgccggatt ctggctgtca 780

ccatttcaga gatggataca gggaatgatg acaagcacac gcttcttgcc aaaaatgctc 840

aacagaagaa gagcttgagt ttggggcctt ctgcagctga aatcaatcaa gcggcccttc 900

tcagcattcc tggctttgtt gagcggcttt gcaaactggc gactcgaaag gtgtcagagt 960

caacgggcac agccagcttc cttcaggagt tggaagagtg gtacacatgg ctagacaatg 1020

ctttggtgct agatgccctg atgcgagtgg ccaatgagga gtcagagcac aatcaagcct 1080

ccattgtgtt ccctcctcca ggggcttctg aggagaatgg cctgcctcac acgtcagcca 1140

gaacccagct gccccagtca atgaagatta tgcatgagat catgtacaaa ctggaagtgc 1200

tctatgtcct ctgcgtgctg ctgatggggc gtcagcgaaa ccaggttcac agaatgattg 1260

cagagttcaa gctgatccct ggacttaata atttgtttga caaactgatt tggaggaagc 1320

attcagcatc tgcccttgtc ctccatggtc acaaccagaa ctgtgactgt agcccggaca 1380

tcaccttgaa gatacagttt ttgaggcttc ttcagagctt cagtgaccac cacgagaaca 1440

agtacttgtt actcaacaac caggagctga atgaactcag tgccatctct ctcaaggcca 1500

acatccctga ggtggaagct gtcctcaaca ccgacaggag tttggtgtgt gatgggaaga 1560

ggggcttatt aactcgtctg ctgcaggtca tgaagaagga gccagcagag tcgtctttca 1620

ggttttggca agctcgggct gtggagagtt tcctccgagg gaccacctcc tatgcagacc 1680

agatgttcct gctgaagcga ggcctcttgg agcacatcct ttactgcatt gtggacagcg 1740

agtgtaagtc aagggatgtg ctccagagtt actttgacct cctgggggag ctgatgaagt 1800

tcaacgttga tgcattcaag agattcaata aatatatcaa caccgatgca aagttccagg 1860

tattcctgaa gcagatcaac agctccctgg tggactccaa catgctggtg cgctgtgtca 1920

ctctgtccct ggaccgattt gaaaaccagg tggatatgaa agttgccgag gtactgtctg 1980

aatgccgcct gctcgcctac atatcccagg tgcccacgca gatgtccttc ctcttccgcc 2040

tcatcaacat catccacgtg cagacgctga cccaggagaa cgtcagctgc ctcaacacca 2100

gcctggtgat cctgatgctg gcccgacgga aagagcggct gcccctgtac ctgcggctgc 2160

tgcagcggat ggagcacagc aagaagtacc ccggcttcct gctcaacaac ttccacaacc 2220

tgctgcgctt ctggcagcag cactacctgc acaaggacaa ggacagcacc tgcctagaga 2280

acagctcctg catcagcttc tcatactgga aggagacagt gtccatcctg ttgaacccgg 2340

accggcagtc accctctgct ctcgttagct acattgagga gccctacatg gacatagaca 2400

gggacttcac tgaggagtga ccttgggcca ggcctcggga ggctgctggg ccagtgtggg 2460

tgagcgtggg tacgatgcca cacgccctgc cctgttcccg ttcctccctg ctgctctctg 2520

cctgccccag gtctttgggt acaggcttgg tgggagggaa gtcctagaag cccttggtcc 2580

ccctgggtct gagggcccta ggtcatggag agcctcagtc cccataatga ggacagggta 2640

ccatgcccac ctttccttca gaaccctggg gcccagggcc acccagaggt aagaggacat 2700

ttagcattag ctctgtgtga gctcctgccg gtttcttggc tgtcagtcag tcccagagtg 2760

gggaggaaga tatgggtgac ccccaccccc catctgtgag ccaagcctcc cttgtccctg 2820

gcctttggac ccaggcaaag gcttctgagc cctgggcagg ggtggtgggt accagagaat 2880

gctgccttcc cccaagcctg cccctctgcc tcattttcct gtagctcctc tggttctgtt 2940

tgctcattgg ccgctgtgtt catccaaggg ggttctccca gaagtgaggg gcctttccct 3000

ccatcccttg gggcacgggg cagctgtgcc tgccctgcct ctgcctgagg cagccgctcc 3060

tgcctgagcc tggacatggg gcccttcctt gtgttgccaa tttattaaca gcaaataaac 3120

caattaaatg gagactatta aataacttta ttttaaaaat ga 3162

<210> 3

<211> 8739

<212> DNA

<213> human (Homo sapiens)

<400> 3

gcgcgcgggc ccgggagagg ctcccgagcc aggcggtctt cggtcctcgc agcgcttcca 60

gctccccgcg cccctatgtg agggagacgg ggaggcccgc ggcgcgcagg ggagggcgag 120

gcatgtgcac gggccggagg gtgctgcagc cgcccgagga agaggaggac ggcggcgagg 180

aggagagcgg ggggctcgcg gcggcgggcc ccggccgagg ggatgcagtg gactgtgtgt 240

gtctggctgt agcagacgcg aggcggcgac gaggcgccgg ggacccgcgc gaggggcggc 300

cgggaggcgg cggcggcggc cgccagaagt agcagcagga ccggcggcgg cgacggcagc 360

cctgaaatgc attttcctct ccagcggcca tgttaaccag gaaaccttcg gccgccgctc 420

ccgccgccta cccgaccgat tggcggcagt aagcacacaa tgaatgatca cctgcatgtc 480

ggcagccacg ctcacggaca gatccaggtt caacagttgt ttgaggataa cagtaacaag 540

cggacagtgc tcacgacaca accaaatggg cttacaacag tgggcaaaac gggcttgcca 600

gtggtgccag agcggcagct ggacagcatt catagacggc aggggagctc cacctctcta 660

aagtccatgg aaggcatggg gaaggtgaaa gccaccccca tgacacctga acaagcaatg 720

aagcaataca tgcaaaaact cacagccttc gaacaccatg agattttcag ctaccctgaa 780

atatatttct tgggtctaaa tgctaagaag cgccagggca tgacaggtgg gcccaacaat 840

ggtggctatg atgatgacca gggatcatat gtgcaggtgc cccacgatca cgtggcttac 900

aggtatgagg tcctcaaggt cattgggaag gggagctttg ggcaggtggt caaggcctac 960

gatcacaaag tccaccagca cgtggcccta aagatggtgc ggaatgagaa gcgcttccac 1020

cggcaagcag cggaggagat ccgaatcctg gaacacctgc ggaagcagga caaggataac 1080

acaatgaatg tcatccatat gctggagaat ttcaccttcc gcaaccacat ctgcatgacg 1140

tttgagctgc tgagcatgaa cctctatgag ctcatcaaga agaataaatt ccagggcttc 1200

agtctgcctt tggttcgcaa gtttgcccac tcgattctgc agtgcttgga tgctttgcac 1260

aaaaacagaa taattcactg tgaccttaag cccgagaaca ttttgttaaa gcagcagggt 1320

agaagcggta ttaaagtaat tgattttggc tccagttgtt acgagcatca gcgtgtctac 1380

acgtacatcc agtcgcgttt ttaccgggct ccagaagtga tccttggggc caggtatggc 1440

atgcccattg atatgtggag cctgggctgc attttagcag agctcctgac gggttacccc 1500

ctcttgcctg gggaagatga aggggaccag ctggcctgta tgattgaact gttgggcatg 1560

ccctcacaga aactgctgga tgcatccaaa cgagccaaaa attttgtgag ctccaagggt 1620

tatccccgtt actgcactgt cacgactctc tcagatggct ctgtggtcct aaacggaggc 1680

cgttcccgga gggggaaact gaggggccca ccggagagca gagagtgggg gaacgcgctg 1740

aaggggtgtg atgatcccct tttccttgac ttcttaaaac agtgtttaga gtgggatcct 1800

gcagtgcgca tgaccccagg ccaggctttg cggcacccct ggctgaggag gcggttgcca 1860

aagcctccca ccggggagaa aacgtcagtg aaaaggataa ctgagagcac cggtgctatc 1920

acatctatat ccaagttacc tccaccttct agctcagctt ccaaactgag gactaatttg 1980

gcgcagatga cagatgccaa tgggaatatt cagcagagga cagtgttgcc aaaacttgtt 2040

agctgagctc acgtcccctg atgctggtaa cctgaaagat acgacattgc tgagccttac 2100

tgggttgaaa aggagtagct cagacctgtt tttatttgct caataactct actcatttgt 2160

atcttttcag cacttaattt taatgtaaga aagttgttca ttttgttttt ataaaataca 2220

tgaggacaat gctttaagtt tttatacttt cagaaacttt ttgtgttcta aaagtacaat 2280

gagccttact gtatttagtg tggcagaata ataacatcag tggcaggcca ctgattactt 2340

catgactgcc acgcatttac agattggtgt caaagacatt cactatgttt ttatggttca 2400

tgttatatcc tccccagggt gacagcccct taaggccctc cttttccctc catgctccag 2460

gtccatgcac aggtgtagca tgtcctgctt ccgtttttca taaattaatc tgggtgttgg 2520

gggtagtggg aggagaacgg tcagaatcaa agtgacattc taagaaaaac tgtaccttag 2580

agattttcct ctagtgctca aacaaataca aaataagatc cccaaggttt aaactgccca 2640

gttagcattc tgacattcta aaagccggca aagcagcttt tagtggataa atgggaatgg 2700

aaacgtgtgt gttcctccaa attttctagt atgatcggtg agctgttttg taaagaagcc 2760

tcatattaca gagttgcttt tgcacctaaa tttagaattg tattccatga actgttcctc 2820

ccttttctct gcttttctcc tctctgttcc tcttttaata ccacacgtct gttgcttgca 2880

tttagtttgt cttcttcctt cagctgtgta tcccagactg ttaatacaga aaagagacat 2940

ttcagctgtg attatgacca ttgtttcata ttccaattaa aaaaagaaca gcagcctagc 3000

tacttaaggt ggggatttcc atagttccaa agaagattta gcagattaga gtgagttcac 3060

acttttcagg tgccactgta aggttctctc agcctgggaa actatcaact ctttctttaa 3120

aaagaaagag ggttgaaaat cctctggacg aacagaagtc actttggctg ttcagtaagg 3180

ccaatgttaa caacacgttt agaggaggaa aagttcaacc tcaagttaaa tggtttgact 3240

tattcttcgt atcattagaa gaaccccaga gatagcattc ctctatttta ttttactttc 3300

ttttggattg cactgattgt ttttgtggga atgacacttt atctggcaaa gtaactgaga 3360

gtttggtaaa agaatatttt cttctctgaa taataattat tttcacagtg aaaatttcag 3420

tattttatca ctaatgtatg agcaatgatc tatatcaatt tcaaggcacg tgaaaaaaat 3480

tttttagtat gtgcaattta atatagaaag atttctgcct gtttggacaa taggttttgg 3540

gtagtacaga ttaggataag taagcttata tatgcacaga gattattgta ttacctgtaa 3600

attgatttac aagtacttaa aagcgtggtc cccagtgagg ccaagaaagt ttccggttaa 3660

gttctttaat aataatccta cagtttatct taagaaaaaa aaaaaggttt gaaaaaaaca 3720

ctttaattta ggcttcgttg gttgatggtg gaaaaaaatg ctcaggaaat atttcagata 3780

tttgccaaaa aaccagtaat aaggttacct tattaaaata gtgatatttg ctttactatt 3840

taaggtgtca ctgataaatt aatttgtact tctgtgttta gaaattatag cttcttttcc 3900

cttagtcaaa ttttttagca tattatacac atttctgtgt aatctgtgga agtgcaataa 3960

tatgttagta agttacattt taaataatgc tcatgtgaca atactcccaa tcaatggctt 4020

atagaattta aagatctgta tattagattt tggcttaaag gcatgagaag tataagactt 4080

ggtttggtgg ctttgtaaga ccaccagcct cttaatgatg gttagcttct ttaggtcatt 4140

aaatcaataa aaacatataa tgctgttttg ctcttctaat gctcctcttc catttccagt 4200

tatcttcaca tttacattta aatatacaaa cctgagcctg ccattattaa tttccctata 4260

aaatgacgat acatgtgaac atttataaat ggactaatac tgcttgtctt tcccccaccg 4320

cacaaaactg gttcttaaga tgccagcaat gaatttgaga ctatctttat ttataaatgg 4380

aaaccggaaa cttttatacc aaactataat aatgtgcagc actgtagggc tttttttttt 4440

ttccctccaa atacagtgaa atttttttat tcacaagagc tgccacatct cagcatttag 4500

taatagagct gctttaataa aattctagtt tgattgtcat gtcaaaaaaa gaaaaatgtt 4560

gcatctttgt gattttaaaa cataaattaa tgaaggctct gataggctat taggagttgg 4620

cttggaaaca gtttttggtc tcacaggtta ccattgtttg gggatgtctg agctgttttc 4680

agatctagga atagcacagt gttgtcttgt ctttggcagt ctcatttggc tctgtttctt 4740

gcaccaccag cgtgttcatt accacttaaa tatattgcta cagcagtgga acaacagagt 4800

ggtgcaagac actgtagatt aacggtagag gagaaattgt gcccttagtg ttaacaatgt 4860

gccttttgtt ctgaatgcca tgttgtaggg catgcatttt ttggcctctt taactcttcg 4920

aattctagtc agtaagaatg gaacccatct ctgcaaagat acatctgtct taaatatcta 4980

gttacaggcc ttaatagaaa ccataaggca tgactcatct tcaggcactg aaaaaagata 5040

accatcaggt agtgttacac aaggacttcc tatatttaag gggttaaaga tggtctttgt 5100

tgtatcttaa catcagactg atttttacat tttttttttg ttatgctaac actagacaaa 5160

aatcaactgt atttgtaaaa atttacctca aaccatttaa ttttatagtg tgattaatcc 5220

cagggcattt ggtatgaacc aaagtgcatt ccttttatat gtgcctggct ctagtaagga 5280

tggccaggga tttttacaat ttgggtgcaa ggcacttaag ccacttttaa acttaatggg 5340

tggtttgggg tcgtgttaaa tgactccatc agaatgttag aaaacacttt aggcatcagt 5400

agcattgggc catattggaa tcctaaagtg tgaattattt taaggagagc attcattttt 5460

gtaatttttt tcatcaaaaa tatttctggt aagcagaaga ctttttaaaa aaactgatct 5520

ggtctcggta aaggttttaa tattgcccaa cataatgctg taatagcatt aaaaaaagta 5580

tttgtgaact ctgtttctta ggggcttgta catctctctg ctatggacat acataaaatt 5640

aattgtaatt atactcagct caactgctac agttctgtct aggcagtggc ttgggttttt 5700

atcgagcaac aacttagaca cgtgactgta atatgctgca actgtgtgta ctgaaaatat 5760

gtgaaaatgg ttgaatgtgg actgtgtata tatgtatgta aaaatttctg tgagatgctg 5820

ctgtcgccac ttaacattaa atatgttcta gtggatttta atcctagtgg ccagttctat 5880

gatactgtat gtattataca gctgatgaca ggagtaagac tgtttagtga atatctgtta 5940

aattttattg ttgtggccag agataatttc agaataaaat tttaatgtcc taccttactt 6000

ctctcccctt tctaactata atttaactcc tgatctattt ctggtattat cctttgtcaa 6060

ttagcctgga gagggtttag aatgcaaaat ggatagctct atatcagggt cttgagaagt 6120

taaaataatt cattctacta gtttgttcat agcaatactt tattagttca atataagtat 6180

gcagattcca agtggaaaaa caaaggtttt agtaagtagt tcttgctttt cccaggatat 6240

tctattcctt cacctgtcag atttttatgt taactttttt atatatagcc tctgctgtgt 6300

tctttattat gctagagctt catatcttct tttattttaa ccttcacaat gatagttaag 6360

ctgggggagc agaacctgtc aattattccc caccagtctt cattcttttc tctgcacaag 6420

ttctggtaaa ttattctaat gtgctctaac cagttccacc caatgttttt taatgcttat 6480

gagtttgcag tttttgtact tggtggtctc tgtggttcac atttttttgt gttgtagtgt 6540

ctcctgcctc ggttggctca gaaaataagg cctaggaact gcaattagct catgatttgt 6600

caagttcagt tagtaccaag ctaagagttt acttacagat gacagcaagc agatgctcta 6660

gtaattcgtc agacattgca gggatattgt gtagtcagat attaccctct tgtggaaaga 6720

actacctcac atcattattt atttcccttc tgttaccaac agccaaggaa ttacttagtg 6780

tggctccctg catcaatact gggatatgct taaacaaggg aatgccataa gagttcccaa 6840

ttgcctcgtc atagcctggg ccatagattt ttgttactgc taatcttgct tcttaaagtt 6900

cacacccagt gcaaaaaacc caatcagcaa actaacccca aaatccaata tatttagaaa 6960

tgtaagtgtt aagagatgtg cattatgtac aaaattgaaa attggtgcta aagtggcaat 7020

gtcaatttaa aatttcttgt ccagatctga ctgactgact caggagatta tagttcctac 7080

tcattctctt tccttttatc agatctttca aaagcgcttt ttaaaattga tcaatgtgca 7140

attctgtttg agtctctaca gtcctatccc ttttggaatg aacagctaca caaataaata 7200

ttcttggaac ttcgaagttg tcttcaaact agctctagaa aatgtcccta gttgacatta 7260

gtgttggtct gttagaaggg gaaattgatt tgactgcttc atgttttgaa aagagcatta 7320

agagggattt gaggaaggta tatgtggctt tctttttttt tcttaagttt gaattacact 7380

tgatttcctt tgggttatta ggcagataaa cccttgtcat tagtaatact tagcactgtc 7440

caaaatgaaa aatgactgcc agtgtgttgc acatacataa gaaaattaca tcttactgcg 7500

aagtctttgg tttgtttcct tatttggttt gactttgact tactattttg ctatggtcat 7560

atccttaata tctgcggtac atttcatata tatatatggg gtgggagcat aattgccatt 7620

cccatgtgtc tcaaggaagc ttaatgagga aactaatgag ttaatattaa ctcttctgaa 7680

gaaagctgac attttaggag tattaagtca tctgtcgtta aggagcagca agaatatatt 7740

atgattgtat cacatgttcc agaagagcat taccccttac atagaaagct ccttggagat 7800

ttcatgatgt tggatgaaca tcagtttatc cttatacatc cctatgagct ataattatcc 7860

tcatttgata cttgaggaaa ctgaagtgac ttgctccaaa gttgactaca aaaacattgc 7920

agtttgaggc ttaggttgta atcaggtaat tttggtcttg gacagtaagg gaaaatccat 7980

aatacataaa acacaaaagt ttagaaaggg ggggaagttg agctagcaat gggcatttct 8040

attatttaag ttacatatca gtttccagga atatttttca aatgggacaa actaaaaagt 8100

tgaggactag agcatcgtag tgtctaagtg cacctaataa cttaatgcat gtgcacacac 8160

cctcagttaa tttgtaagag agtgacttct cattgttatt tgtggagagg ctaattatag 8220

tagcttcatt ctttattttc tgtgttctta aatttttttt tacatttgaa agtatgaaaa 8280

tactattttt gacactttac tgatactcag ggattaaaag ttaaatttta ctgactgtag 8340

cagcaggtaa atctcttaaa tttactcagg tttcctatcc tttgcctcca tatttaagaa 8400

aatatagaat gcctgccagg gaaatatata aaaataaaaa tttcaaatac aattttaaaa 8460

taccaatttg taaataaacc aattttacaa actaaaatat tgtgtctttc cttgcttttt 8520

atcccaattt atctcccctc ctaagtttga ggagttttat atattaaggc tatttcttca 8580

tttgtaaaaa tgaagtatgc tatttgcaaa gcaaaagccc atgaaaagcc ccaaatcatt 8640

taagtaaagc ttactgaatt gttttcaatt gttttgggag aaatgtaact aattataagc 8700

aatttatgtg gaccagcagc aaataaatgt atgagtaca 8739

<210> 4

<211> 3628

<212> DNA

<213> human (Homo sapiens)

<400> 4

agagccgcga gctggaccag ccgtgcaaat ctctagaaga tgacggtgtt ctttaaaacg 60

cttcgaaatc actggaagaa aactacagct gggctctgcc tgctgacctg gggaggccat 120

tggctctatg gaaaacactg tgataacctc ctaaggagag cagcctgtca agaagctcag 180

gtgtttggca atcaactcat tcctcccaat gcacaagtga agaaggccac tgtttttctc 240

aatcctgcag cttgcaaagg aaaagccagg actctatttg aaaaaaatgc tgccccgatt 300

ttacatttat ctggcatgga tgtgactatt gttaagacag attatgaggg acaagccaag 360

aaactcctgg aactgatgga aaacacggat gtgatcattg ttgcaggagg agatgggaca 420

ctgcaggagg ttgttactgg tgttcttcga cgaacagatg aggctacctt cagtaagatt 480

cccattggat ttatcccact gggagagacc agtagtttga gtcataccct ctttgccgaa 540

agtggaaaca aagtccaaca tattactgat gccacacttg ccattgtgaa aggagagaca 600

gttccacttg atgtcttgca gatcaagggt gaaaaggaac agcctgtatt tgcaatgacc 660

ggccttcgat ggggatcttt cagagatgct ggcgtcaaag ttagcaagta ctggtatctt 720

gggcctctaa aaatcaaagc agcccacttt ttcagcactc ttaaggagtg gcctcagact 780

catcaagcct ctatctcata cacgggacct acagagagac ctcccaatga accagaggag 840

acccctgtac aaaggccttc tttgtacagg agaatattac gaaggcttgc gtcctactgg 900

gcacaaccac aggatgccct ttcccaagag gtgagcccgg aggtctggaa agatgtgcag 960

ctgtccacca ttgaactgtc catcacaaca cggaataatc agcttgaccc gacaagcaaa 1020

gaagattttc tgaatatctg cattgaacct gacaccatca gcaaaggaga ctttataact 1080

ataggaagtc gaaaggtgag aaaccccaag ctgcacgtgg agggcacgga gtgtctccaa 1140

gccagccagt gcactttgct tatcccggag ggagcagggg gctcttttag cattgacagt 1200

gaggagtatg aagcgatgcc tgtggaggtg aaactgctcc ccaggaagct gcagttcttc 1260

tgtgatccta ggaagagaga acagatgctc acaagcccca cccagtgagc agcagaagac 1320

aagcactctg agaccacact ttaggccacc ggtgggacca aaagggaaca ggtgcctcag 1380

ccatcccaac agtgtcgtca gagggtcccc agggcatttt catggcaagt acccctctgc 1440

ccccactcca gcagtgcttc ccaaagtgtg ctctgtcacc tgctttgcaa tcggcttcca 1500

ttagcgcatg ttttattttg gtgtgacggt tggccctcct aaacacggac tttcctcagg 1560

ctggttcaag acggaaaagg actttcttct gttttcttcc aaagtgcaac cacagtggag 1620

agcccacggt gggcttagcc tgcctaggcc cttccatttc tcttctttga ccgtgctagg 1680

aattccagga aagtgcattc ctgccctggt gaccttttcc tatgtctagg ctcctccaca 1740

ggtgctgcta ttttgtgagc tccggctcct gtttagcttt tatttcagtt ctaacctcag 1800

tccagaaaca tatgtgaggt tgtttccctc ttcagccacg gctacaatac cggaaaatgc 1860

tagtttttat ttattttttt aagtagtgct tcctaaatgg tttgcatgag agccacctgg 1920

ggtacatgtt gaaaacttat ttggggtcta ccccaaacct aataacccaa atttggggat 1980

ggggcccagg aatatgcatt tttaaaaagt catctgccct tcccaggtga ttctgtaagt 2040

tgtccctcaa ctgtacttgg agaaatcgtg ttttaaagca gtagtccaca aagtattctg 2100

ctcatgtgcc cccaaaagta ttttgaaaaa tcatgtatac cctcacccat ctaagttgat 2160

atctaaaatt ttatctaagt tggtatctaa aatttttcat gggaagttaa atagttgaca 2220

aagtatgtat ttgctggtgt cgtgtaaata ttggtatttt aaaataaaaa ctgttacatc 2280

actattttaa acatatccag tacaatttaa atatcacaac aatttgacac ccttcattca 2340

tttataaaaa taaatgagct agttctttag tagttaaaca tttcaaattg gcttttctcc 2400

ttctgtattt ccataccact tttcagccaa gaatcctatc ataatgtaat ctattatgcc 2460

cgacatcttt taatcattca ccccattact tcttgtcaac aaaaaatata aatggaaatt 2520

ttttttttag ctcttgcttt aagtgtttgt ttgttatctc agtccagaac caatattatc 2580

gtaattaatt attggtatat aatgaaaacg gtattaattc ttggatgatt aaaagttttt 2640

ttattagaat gttctttatc ctaattagtt catttatcca agaatacatg aatgtgattt 2700

acagctgaga tggggttcaa cctcagctgt attccttgtt tctgtataga tgtaagcaca 2760

taaattcgat ggaatagaat tacgttaaca atgtttttac agttctttgg attcctttgg 2820

cattttgaca aagatcacag tgctctatca tcaagaatta ttaatgatga tctatcaact 2880

aacaaacaac ttgattagat tctcctttag tctgttgaaa gcagagaact gaaatccacc 2940

tgatttacca tggctttgcc agccagtcat tagcaccatt tacttttact atcgctgaca 3000

ttttcctttg ttcagtggcc ctgaggttct tacactctag ggggcagtgc accacaggaa 3060

gatagatcaa tgagggagga ttgcgagggg gaaggggagg aagcagagct ggcaggcctt 3120

agctacaggc tctctctcag gcagatccct tttaagatac atacaccatg cccacacatc 3180

ccatggagag agaccaatgc tttagtagat tacagaacag ctatgaaaag tccatgaatg 3240

aagatcacaa aaaggaaggc tttcttattt catactgtat tcttcagggt ggtaaaattt 3300

ctgcttttgg caaaaacata acagacggtt ccaaacatca gcataaagat cactcatccc 3360

ataccaccca caggtaggga ggaaggatgc tgtagtatat gaaaacaaaa gttttcacct 3420

gagctgagag catttagcat atcgtggttc tgtaacaata tcaaggacca gtgcagaatc 3480

tggctttctt ttctgatagg ctaccagtgt gtgtttatgt gtgctcattt tgtggttcta 3540

atcataatgg tacatataat tagggaagga tatggaagcc actttagaat cttattcatt 3600

tttaaatata aatatgcctt gtttcaaa 3628

<210> 5

<211> 4777

<212> DNA

<213> human (Homo sapiens)

<400> 5

gtttccggcc gccgtcgctg tccagggagg ctgaggcgag aggtagctgt ccgggtgggg 60

agcccgcact accttcttcc tcttcctcct cctcctccgg gtgaggggag cgaaggttgg 120

gggtccccga gcccatggac caggaggagg cggaggccgc cgagagccgg ggccccgcta 180

tgtggccctg agccccgtgt actggttctg cctgtctgga gggccatgga gaagaggctg 240

ggagtcaagc caaatcctgc ttcctggatt ttatcaggat attattggca gacatctgcg 300

aagtggttga gaagcctgta cctgttttat acttgctttt gcttcagcgt tctgtggttg 360

tcaacagatg ccagtgagag caggtgccag caggggaaga cacaatttgg agttggcctg 420

agatctgggg gagaaaatca cctctggctt cttgaaggaa ccccctctct ccagtcatgt 480

tgggctgcct gctgccagga ctctgcctgc catgtctttt ggtggctaga agggatgtgc 540

attcaggcag actgcagcag gccccagagc tgccgggctt ttaggacaca ctcctccaat 600

tccatgctgg tgtttttaaa aaaattccaa actgcagatg atttgggctt tctacctgaa 660

gatgatgtac cacatcttct ggggctaggt tggaactggg catcttggag gcagagccca 720

cccagagctg cactcagacc tgctgtatct tccagtgacc agcagagctt aatcaggaag 780

cttcagaaga gaggtagtcc cagtgacgta gttacaccta tagtgacaca gcattctaaa 840

gtgaatgact ccaacgaatt aggtggtctg actaccagtg gctctgcaga ggtccacaag 900

gcgattacaa tttccagtcc cctaaccaca gacctgactg cagagctgtc tggtgggcca 960

aagaatgtat cagtgcaacc tgaaatatca gagggtcttg ctactacgcc cagcactcaa 1020

caagtaaaaa gttctgagaa aacccagatt gctgtccccc agccagtggc tccctcctac 1080

agttatgcta cccctacccc ccaggcctct ttccagagca cctcagcacc atacccagtt 1140

ataaaggaac tggtggtatc tgctggagag agtgtccaga taaccctgcc taagaatgaa 1200

gttcaattaa atgcatatgt tctccaagaa ccacctaaag gagaaaccta cacctacgac 1260

tggcagctga ttactcatcc tagagactac agtggagaaa tggaagggaa acattcccag 1320

atcctcaaac tatcgaagct cactccaggc ctgtatgaat tcaaagtgat tgtagagggt 1380

caaaatgccc atggggaagg ctatgtgaac gtgacagtca agccagagcc ccgtaagaat 1440

cggcccccca ttgctattgt gtcacctcag ttccaggaga tctctttgcc aaccacttct 1500

acagtcattg atggcagtca aagcactgat gatgataaaa tcgttcagta ccattgggaa 1560

gaacttaagg ggcctctaag agaagagaag atttctgaag atacagccat attaaaacta 1620

agtaaactcg tccctgggaa ctacactttc agcttgactg tagtagactc tgatggagct 1680

accaactcta ctactgcaaa cctgacagtg aacaaagctg tggattaccc ccctgtggcc 1740

aacgcaggcc ccaaccaagt gatcaccctg ccccaaaact ccatcaccct ctttgggaac 1800

cagagcactg atgatcatgg catcaccagc tatgagtggt cactcagccc aagcagcaaa 1860

gggaaagtgg tggagatgca gggtgttaga acaccaacct tacagctctc tgcgatgcaa 1920

gaaggagact acacttacca gctcacagtg actgacacaa taggacagca ggccactgct 1980

caagtgactg ttattgtgca acctgaaaac aataagcctc ctcaggcaga tgcaggccca 2040

gataaagagc tgacccttcc tgtggatagc acaaccctgg atggcagcaa gagctcagat 2100

gatcagaaaa ttatctcata tctctgggaa aaaacacagg gacctgatgg ggtgcagctc 2160

gagaatgcta acagcagtgt tgctactgtg actgggctgc aagtggggac ctatgtgttc 2220

accttgactg tcaaagatga gaggaacctg caaagccaga gctctgtgaa tgtcattgtc 2280

aaagaagaaa taaacaaacc acctatagcc aagataactg ggaatgtggt gattacccta 2340

cccacgagca cagcagagct ggatggctct aagtcctcag atgacaaggg aatagtcagc 2400

tacctctgga ctcgagatga ggggagccca gcagcagggg aggtgttaaa tcactctgac 2460

catcacccta tcctttttct ttcaaacctg gttgagggaa cctacacttt tcacctgaaa 2520

gtgaccgatg caaagggtga gagtgacaca gaccggacca ctgtggaggt gaaacctgat 2580

cccaggaaaa acaacctggt ggagatcatc ttggatatca acgtcagtca gctaactgag 2640

aggctgaagg ggatgttcat ccgccagatt ggggtcctcc tgggggtgct ggattccgac 2700

atcattgtgc aaaagattca gccgtacacg gagcagagca ccaaaatggt attttttgtt 2760

caaaacgagc ctccccacca gatcttcaaa ggccatgagg tggcagcgat gctcaagagt 2820

gagctgcgga agcaaaaggc agactttttg atattcagag ccttggaagt caacactgtc 2880

acatgtcagc tgaactgttc cgaccatggc cactgtgact cgttcaccaa acgctgtatc 2940

tgtgaccctt tttggatgga gaatttcatc aaggtgcagc tgagggatgg agacagcaac 3000

tgtgagtgga gcgtgttata tgttatcatt gctacctttg tcattgttgt tgccttggga 3060

atcctgtctt ggactgtgat ctgttgttgt aagaggcaaa aaggaaaacc caagaggaaa 3120

agcaagtaca agatcctgga tgccacggat caggaaagcc tggagctgaa gccaacctcc 3180

cgagcaggca tcaaacagaa aggccttttg ctaagtagca gcctgatgca ctccgagtca 3240

gagctggaca gcgatgatgc catctttaca tggccagacc gagagaaggg caaactcctg 3300

catggtcaga atggctctgt acccaacggg cagacccctc tgaaggccag gagcccgcgg 3360

gaggagatcc tgtagccacc tggtctgtct cctcagggca gggcccagca cactgcccgg 3420

ccagtcctcc tacctcccga gtctgcgggc agctgctgtc ccagcatctg ctggtcattt 3480

cgccctgaca gtcccaacca gaacccctgg gacttgaatc cagagacgtc ctccaggaac 3540

ccctcaacga agctgtgaat gaagaggttt cctctttaaa cctgtctggt gggcccccag 3600

atatcctcac ctcagggcct cctttttttg caaactcctc ccctcccccg agggcagacc 3660

cagccagctg ctaagctctg cagctcccca gtggacagtg tcattgtgcc cagagtgctg 3720

caaggtgagg cctgctgtgc tgcccgcaca cctgagtgca aaaccaagca ctgtgggcat 3780

ggtgtttccc tctctggggt agagtacgcc ctctcgctgg gcaaagagga agtggcaccc 3840

ctcccctcac cacagatgct gagatggtag catagaaatg atggccgggc gcggtggctc 3900

acgcctgtaa tcccagcact ttgggaggcc gaggcgggcg gatcatgagg tcaggagatc 3960

aagaccaccc tggctaacac ggtgaaaccc catctctact aaaaataaaa aaaaaaatta 4020

gccgggtttg gtggcgtatg cctgtaatcc cagctactcg ggaggctgag gcaggagaat 4080

tgcttaaacc tgggaggtgg aggctgcagt gagccaagat cgtgccactg cactccagcc 4140

tgagtgacag agcaagactc cgtcaaaaaa aaaaaaaaaa aaaaagaaat gatatctggc 4200

ccccccttaa cactggagcc ccactccctt ctcccatccg gcccgagatt agggaggatt 4260

gactgtgtca gggatggcgg gtggcctctc tcgctgccag ggcccttgtc agagcagcca 4320

ggctggacag acggcctccc tcctctccat ctgaccggca cctgctgctt cggggcttag 4380

gccaccgctc cctgtcccca gaggagatag ccccagatgg actggaatgt tgtggcatga 4440

gagcgcatgt gtgcgatggc cccgctgtgg tcccctctct gtccctccat ctgtatgtgt 4500

tctgtgtccc ttgcatgtgt gcgtgttaga gtgagcgcgt atgcatcaac tcattgggct 4560

cttggctgct cacaaggcaa atttgacttg gaaagacttt catctccttg gaaccaagac 4620

ttcctgagtc cccctcaccc tggccctgtt ccaccatggt tatctgggta ttggggaatg 4680

gaaactttgg gggagtgact ttttaaagag acacttataa tttctactac tgcactactg 4740

tccattgtgg gatgattaaa catggtattt aactgtg 4777

<210> 6

<211> 2653

<212> DNA

<213> human (Homo sapiens)

<400> 6

gtgcgcggcg actgcgacgg gcagtggcag tcatggcggc tcagtgcgtg aggttggcgc 60

ggcgcagtct tcctgctttg gcgttgtctc tcaggccatc tccccggttg ttgtgcacag 120

ccacgaaaca aaagaacagt ggccagaacc tggaagagga catgggtcag agtgaacaga 180

aggcagatcc tcctgctaca gagaagaccc tcctggaaga gaaggtcaag ttggaggaac 240

agctgaagga gactgtggaa aaatataaac gagctttggc agacactgag aacttacggc 300

agaggagcca gaaattggtg gaggaggcaa aattatacgg cattcaagcc ttctgcaagg 360

acttgttgga ggtggcagac gttctggaga aggcaacaca gtgtgttcca aaagaagaaa 420

ttaaagacga taaccctcac ctgaagaacc tctatgaggg gctggtcatg actgaagtcc 480

agatccagaa ggtgttcaca aagcatggct tgctcaagtt gaaccctgtc ggagccaagt 540

tcgaccctta tgaacatgag gccttgttcc acacaccggt tgaggggaag gagccaggca 600

cagtggccct agttagcaaa gtggggtaca agctgcatgg gcgcactctg agacccgccc 660

tggtgggggt ggtgaaggaa gcttagctgc tgttgatggg gtgggtgttt ttaaactcac 720

ttgatgtaac tctcaaggct ggttcattgt ttctcatcta tgagtacgtg tgaccttttc 780

ccaaacctta ttggaaacct taagtaacca gtggctaaac agaaaagccg gttgcccaac 840

tgcattaatg aactctaatt cgggagtctg ttccctttta gtgccacgcg ttgaatagtt 900

ccacatactt tcagaagagc tcagcagggc cctgcctggt ctcccgagca tcatgagtaa 960

cgtgtctgct cagactctgc tgacaccaaa gtattttaaa caaataaaag gtcttgggga 1020

attctgtttg gctacctggg cacgccagtc tgcaccatgt gtccctgcgg cgcatgagtg 1080

actggcgtat ttagcccgtc acatttcatt cgctgaagga aaggcaagag agttgaaaca 1140

tttttcttac ttaaaaaaaa tgatctttgt gaagaacata gtgagttcgt ttgtcttcag 1200

tcaacagcgg ctgaaactga ccactgagaa atgggtgtgg gcactgacag ttctccccca 1260

ttatttggcc aggaattgag cttggcttgg caaagttcct tttaccctgt tctgttcatc 1320

taaatgcaga catatttaaa tcatattcaa ctagttacta atgacctcaa gttgtattcc 1380

ctggcaaaat ggactttctc aaaataggac tgcacgcttg gtgtacttta aatgttaatg 1440

tttaatttaa aatttttatt taagaggatt aaagccctaa tgtttatttt cctactttct 1500

gaaaacaatt ttgtgtcctc tcatggagat ccttgccgct gaggcaagca tggcttagcc 1560

ttcccattct ctactccagc ccagaagctg tcagcttgac acgtgcgtgc gtgcacattc 1620

acgtacacac atatacaggc ttctacacag aaagggagag ccacactccc ccagtgcagc 1680

tgcagcccgt ccggtaagaa tacgaaacac ccaggagcca cagcaagggc acaggatttg 1740

caaaatcatc ttgccaaagg gctgttcctc atttacacgg tcatccaggg cttagaaaaa 1800

ggctgaatga ttggcgtctg ttacaaaaaa gagaaaagaa cctttcgctg cctttgcttc 1860

tagaatggaa gatacttgtg aaagccattt gctgcacagg aaagatgggg cagtttattt 1920

cacttctaat tttaaaagac gcaagtaatg gtcccataat acttcatctt gggataccat 1980

tcccaaacat gttccacctt taagatgcct taagttaatt tggtcagaaa caactaaagg 2040

cagcatcgtg tattgaagaa aacatgactg ggagctccag cgctgcctgt tttcacgctg 2100

tggggttgtg agtgcatcat cacccctgga gctcccgagt taaatggagg ctaagaaaag 2160

taatttatca actcatttct gtgaatgttt gcagtctcct accgtctcaa ctacagctgc 2220

agttgctaaa aagcacttaa ctcgtaacct agaggcaata tagcttaagt agcaaaatat 2280

ttttttacag taagcttcag tcatccatag tcactataag ttggttatca tctgaaatgc 2340

ttagaaccag aattgtttca aacttggggg gttttcggat tttggaatat agtcaagcat 2400

ggcttaacca cagggatgtg ttctgagaaa tgcgctgtca ggcagttttt cctgtgaaca 2460

ccatgtgtac ctacatggac ctaaaaaaca ggtggtggag cctcctccac acctaggcta 2520

tatggtgtat gtagcctgtt gctctagact ggagggcacg ttactgtact gatactgtag 2580

gcaactgtta acacaatggt atctgtgtat ctgaacataa catagtaaag gtacagtgca 2640

aactgtgttc taa 2653

<210> 7

<211> 7037

<212> DNA

<213> human (Homo sapiens)

<400> 7

agaaccccga tcgctgagga gcaagggggc gctaggaaag ggaactgggt tgcgacggtc 60

cggcgagaga gagctggggt gctggggtgc ggggaagttg gggagcagag gccgcttggt 120

gtccgagtag ggtaagaccg caccgaccca gtccgttagg aaagaaggga aacgaggcaa 180

ttgtcgggcg gatccccgga cggagggcta aggttgtgtg gaaggcgctg ctccccggat 240

ggcgaccgca gatactccgg ccccggcctc cagtggcctc tcgccgaagg aagaagggga 300

gcttgaagat ggggaaatca gtgacgacga taataacagc cagatacgga gtcggagcag 360

cagcagcagc agcggcggcg ggctgttacc ctatccgcgg cgaaggcctc ctcactcggc 420

ccggggcggt ggatctggcg gaggcggtgg ctcttcctcg tcatcgtcct cttctcagca 480

gcagctgagg aatttctcac gctcgcggca cgcgtctgag cggggccacc tcaggggacc 540

cagcagctac cgacccaaag aaccgttccg gtctcatccg ccttctgtac ggatgccttc 600

gagctcactg tccgaaagca gtccccggcc gtctttctgg gagcggagcc acctcgcctt 660

ggaccgtttc cgctttcgag gcaggcctta ccggggtggg agtcgctgga gtcgggggcg 720

aggagtgggt gagcgaggag gcaagccggg gtgcagacct cctctgggag gaggagcagg 780

atccgggttc agcagcagtc agagctggcg agagccctct ccacctcgga agagctccaa 840

aagttttgga aggtctccat caagaaaaca aaattattca tcaaaaaatg aaaactgtgt 900

ggaagaaact tttgaagatt tgcttttaaa gtataaacaa atacagttgg aactagaatg 960

catcaataag gatgaaaaac tagcattgag tagcaaagaa gagaatgtgc aggaagatcc 1020

taaaacattg aacttcgagg accaaactag cactgataat gtcagtatta caaaggattc 1080

aagtaaagaa gtagctcctg aggagaaaac acaagtcaaa acttttcagg catttgaatt 1140

aaaaccactc aggcaaaaat tgactttacc aggagataag aaccgtttga aaaaagttaa 1200

agatggagca aaaccacttt ccctgaaatc cgacactact gattctagtc aaggattaca 1260

agataaagaa caaaatttaa caagaagaat tagtacctca gatattctgt ctgaaaagaa 1320

acttggtgaa gatgaagagg aactatctga attacagctt cgccttttgg ctcttcagtc 1380

agccagtaaa aaatggcaac aaaaagaaca gcaggtgatg aaagaaagca aagaaaagtt 1440

gactaagacg aaaactgtac agcaaaaagt taaaacaagt acaaaaacac attcggccaa 1500

aaaagttagc actacagcta aacaagcatt gaggaagcag caaacaaagg catggaagaa 1560

actacaacaa caaaaagagc aggaaagaca gaaagaagag gatcagcgga aacaagctga 1620

agaagaagag agaaggaaaa gagaggaaga aatcagaaaa attcgagatc tctcaaatca 1680

ggaagaacag tacaatcgat tcatgaaatt ggttggtggc aagaggagat caagaagtaa 1740

atcttcagat cctgacctga ggcgatcctt agataagcaa cctactgata gtggaggagg 1800

catttatcag tatgataact atgaagaagt tgctatggat acagatagtg aaaccagttc 1860

tccagctcct tcaccagtgc aaccgccatt tttctctgaa tgttcattgg ggtatttttc 1920

tccagcacca tctctttctt tgcctccacc acctcaggtt tcttctctgc cacctttgag 1980

ccagccttat gtggaaggct tgtgtgtttc tcttgaacct ctacctcctc taccaccatt 2040

accacctctc ccacctgaag atccagaaca gcctccaaaa ccaccttttg cagatgagga 2100

agaggaggaa gaaatgctgc ttcgagaaga actacttaaa tctctagcaa ataaaagagc 2160

ttttaagcca gaggaaacat ccagtaatag tgacccacct tcacctccag ttctgaacaa 2220

ttcacatcct gtgccaagaa gcaatctatc aatagtcagt attaacacag tgtctcagcc 2280

taggatacag aatccaaagt ttcacagagg accccgtctt ccacgaactg tgatctcgct 2340

tccaaagcat aaatcagtgg ttgtaacact aaatgattca gatgatagtg aatctgatgg 2400

agaggcttcc aagtcaacaa atagtgtttt tggtggatta gagtccatga ttaaagaagc 2460

aagacgaact gctgagcaag cttcaaaacc gaaagtacct ccaaaatctg aaaaagaaaa 2520

tgatcctctg cgaacaccgg aggctttgcc tgaagaaaag aagattgaat atagattgtt 2580

aaaggaagag attgccaacc gtgagaaaca gcgtttgatt aaatcagatc agctgaagac 2640

aagttcatca tccccagcaa actctgatgt ggaaattgat ggtattggca ggatagcaat 2700

ggttactaag caggttacag atgcagaatc aaaactgaaa aaacatagga ttctcttgat 2760

gaaagatgaa tctgttttaa agaatttagt gcaacaagaa gctaagaaga aagaatctgt 2820

tagaaatgct gaagcaaaga ttacaaaact tacagaacag cttcaagcaa ctgaaaaaat 2880

tcttaatgtt aacagaatgt ttttgaagaa gcttcaggaa caaattcaca gagttcaaca 2940

gcgtgttaca attaagaaag ctttgactct aaaatatgga gaagagcttg ctcgggcaaa 3000

ggcagtggcc agtaaagaaa taggaaaacg taaactggaa caagatcgct ttgggccaaa 3060

caaaatgatg agactggaca gttctccagt atcaagtcca agaaagcatt cagcagaact 3120

aattgctatg gagaaaagac ggttacaaaa gctagaatat gaatatgccc tgaaaattca 3180

aaaattaaaa gaagcccgtg cccttaaagc aaaggaacaa caaaatatct ctccagttgt 3240

ggaagaggaa cccgaatttt ctttacctca accctcactt catgatctga cacaagataa 3300

attaaccctg gacactgaag aaaatgatgt tgatgatgaa attttgtctg gttcaagcag 3360

agagcgaaga agatcttttt tagaatccaa ttattttact aaacctaacc ttaagcacac 3420

tgatactgct aacaaagaat gcataaacaa acttaataaa aatactgtag aaaaaccaga 3480

actttttcta gggttaaaaa ttggtgaatt gcaaaaattg tattcaaaag ctgacagcct 3540

aaaacagctg attttaaaaa ccaccacagg cattacagag aaggttttgc atggtcagga 3600

gatttctgta gatgtggatt ttgtcacagc acaaagtaaa acaatggaag tgaagccatg 3660

tccttttaga ccctaccata gtcctcttct agtttttaag tcctacagat ttagtccata 3720

ttatcgaacc aaggaaaaac ttcccctgag ctcagtatca tacagtaata tgattgaacc 3780

ggatcagtgt ttctgccgtt ttgatttaac aggaacatgt aatgatgatg attgtcaatg 3840

gcagcatata caagactata cacttagccg aaaacagtta ttccaggaca ttctgtcata 3900

taatctgtct ttgattggtt gtgcagagac aagtactaat gaagaaatta ctgcttcagc 3960

agaaaaatat gttgagaaac tttttggagt aaacaaagat cgaatgtcaa tggaccagat 4020

ggctgttctc cttgttagca atatcaatga aagtaaaggt catactcctc catttacaac 4080

ctacaaagat aaaagaaagt ggaagccaaa gttttggaga aaacctattt cagataatag 4140

cttcagtagt gatgaggaac agtctacagg accaattaag tatgctttcc agccagagaa 4200

ccaaataaat gttccagctc tggatacagt tgtcactcca gatgatgtca gatactttac 4260

aaatgagact gatgacatcg ctaatttaga agcaagtgtg cttgaaaatc cttctcatgt 4320

acaactttgg ctcaagcttg cgtacaagta cttgaatcaa aatgaggggg agtgctcaga 4380

atccttggat tctgctttaa atgttctggc gcgagcattg gaaaataaca aagacaatcc 4440

agaaatttgg tgccattacc tcagattgtt ctcaaaaaga ggaaccaagg acgaggtgca 4500

ggaaatgtgt gaaacagctg ttgaatatgc tccagattat caaagctttt ggacttttct 4560

acacctagaa agtacctttg aagaaaagga ttacgtatgt gagagaatgt tggagtttct 4620

gatgggagca gccaagcagg aaacatccaa tattttgtcc tttcagcttt tagaggctct 4680

tttgtttaga gttcagctgc acatatttac tggaagatgc caaagtgcac tggcaatttt 4740

acagaatgca ttgaaatctg ctaatgatgg aatagtagct gaatacctta aaaccagtga 4800

tcgatgtttg gcatggttgg cctacataca tcttattgaa ttcaacattc tcccttcaaa 4860

attttatgat ccatctaatg ataatccttc aagaattgtt aacactgaat catttgtaat 4920

gccatggcaa gctgttcaag atgtaaagac taatcctgac atgttgttag cagtttttga 4980

agatgcagtg aaagcttgca cagatgagag ccttgctgtt gaggaaagaa tagaggcctg 5040

ccttccactt tacacaaaca tgattgctct gcaccaactc ctggagaggt atgaggctgc 5100

aatggagctt tgtaaatctt tattggaatc atgtcctatt aactgccagt tgctggaagc 5160

tcttgttgca ttatatttgc aaacaaatca gcatgacaaa gccagagcag tgtggcttac 5220

tgcatttgaa aaaaatcctc agaatgcaga ggttttttat catatgtgca aattcttcat 5280

cttacagaat cgaggcgata atcttcttcc atttttgcgg aaatttattg catccttctt 5340

taaaccgggg tttgagaagt ataataactt ggatctgttt cggtatctct taaatattcc 5400

aggaccaatt gacattccat ctcgtttatg taaagggaat tttgatgatg atatgtttaa 5460

ccaccaagtt ccttatttgt ggctgattta ctgcctttgt catcctcttc aatcaagtat 5520

taaagaaaca gtggaggcat atgaggcagc attaggggtg gctatgagat gtgatatagt 5580

acagaagata tggatggatt atcttgtctt tgcaaataat agagctgctg gatccagaaa 5640

caaagttcaa gaattcaaat tttttactga tttagtgaat agatgtttgg ttacagtccc 5700

tgcccgatac cccattcctt ttagcagtgc tgattactgg tccaactatg aatttcataa 5760

tagggttatt ttcttttatt tgagctgtgt tccaaagacc cagcattcca aaaccttgga 5820

acggttttgt tcagttatgc cagctaattc tggacttgca ttgaggttac ttcaacatga 5880

atgggaagaa agcaatgttc agattctgaa acttcaagcc aagatgttta catataatat 5940

cccaacatgc ctggccacct ggaaaatagc cattgctgct gagattgttc taaagggaca 6000

aagagaggtc caccgtttat atcagagagc cttacagaag ttacctcttt gtgcatcact 6060

gtggaaagat caactcttgt ttgaagcatc agaaggaggt aaaactgata acctgagaaa 6120

actagtttcc aagtgccaag agattggagt cagcctaaat gagctcttaa atttaaacag 6180

taacaaaaca gaaagcaaga atcactgaac actgggtgca gtcagttcta agtccttata 6240

ataattgcca aaattatttg aatgattctt caagattagg ctgatccctg gctaaggtct 6300

gtgtaaggca gacaagcgtt attgatcata tcaagttccc tacaatatcc tgtcctcaaa 6360

accggaagca atgaacatga tcctcttcgg ttggataaat gaacttcctg tttggcctgc 6420

ttctaggccc tgccagattc tcataacatc atatacgtaa gtatagttcc tcaaagtgac 6480

tgacatttat tttaattttg ctttgttttt ttttattttc tcccccattc ctttattttg 6540

tgttattcct gactcacttg acactctctg atgcctgaga gattcctgtt tgggatttaa 6600

tatccagggc tgtgtttaca gtaaaaaaag caggcagtcc cttttagttt ttccttttta 6660

aatttttttg agattcttca tttcaggatt taaaactata gcagtccatc ttaaggaaag 6720

tgtaactgcc atggccacaa gtctgctagt tgcacttgaa tgctctatca gggttgttta 6780

ttaccctttc tacgttctgg actccttgcc gagactgttt aacttgaaga ttaaagaaac 6840

tattgcaaat gccagtgcat cagaacctaa gagtggtcaa atattatgtg caattttttt 6900

gtaaagaaat tttaatttat aataaagttt aacagtttaa agaacagtta atatttgaac 6960

tgctttgtat tgaaatacta ctttgtagta ttgaaattgt ttatatactt ttttaatata 7020

agttaacttt ctaaaaa 7037

<210> 8

<211> 1481

<212> DNA

<213> human (Homo sapiens)

<400> 8

ccttatctgc actgggccag catcctccgg ccgctgcgcc gccaggggtg agagggagga 60

aaccgggccg ccgggggcgg ggagaaggcg ggccggcccg ggagccgctc actttccctg 120

ggggggacct acgcggagac ctcggctatc ctggccttcc gaggcccacg aggaggcgcg 180

gcccaacgcc ggggcctgga gcattgaggc cggaccctcg cgagacagca gagcctggcc 240

tgacgctgga aaccacaccc tggcccagac tgccagccct gacgggacag agccagggca 300

ctcaccaggc tgcaagaaca gtgctggggt gagtaccccc acgtcggggt ccatgtgccc 360

gcctcaggca caggcagagg tgggccccac catgactgag aaggcagaga tggtgtgtgc 420

ccccagccca gcgcctgccc caccccctaa gcctgcctcg cctgggcccc cgcaggtgga 480

ggaggtgggc caccgaggag gctcctcgcc ccccaggctg ccacctggtg taccagtgat 540

cagcctgggc cacagcaggc ccccaggggt agccatgccc accacagagc tgggcactct 600

gcggcccccg ctgctgcaac tctccaccct gggaactgcc ccgcccactt tggccctgca 660

ctaccaccct caccccttcc tcaacagtgt ctacattggg ccagcaggac cttttagcat 720

cttccctagc agccggttga agcggagacc aagccactgt gagctggacc tggctgaggg 780

gcaccagccc cagaaggtgg cccggcgcgt gttcaccaac agccgggagc gctggcggca 840

gcagaacgtt aacggcgcct tcgccgagct gaggaagctg ctgccgacgc acccgcccga 900

ccggaagctg agcaagaacg aggtgctccg cctagccatg aagtacatcg gcttcctggt 960

gcggctgctg cgcgaccaag ccgcagctct ggccgcaggc cccacccctc ccgggcctcg 1020

caaacggccg gtgcaccggg tcccagacga cggcgcccgc cggggatccg gacgcagggc 1080

cgaggcggca gcgcgctcgc agcccgcgcc cccggccgac cccgacggca gccccggtgg 1140

agcggcccgg cccatcaaga tggagcaaac cgctttgagc ccagaggtgc ggtgaccgca 1200

cgcggcagca cctctgagcc ggagggcacc agggactcgg cccagggccg tcaaggaaag 1260

ggcagtggac gtgctgcgca tgttcgggag cgaactcccc cgaagaagga ccagtgaaga 1320

cgtcaggggc aaggtctcgg gggtccggaa gggtgatcat cgacccccaa gggacccgca 1380

gacccttaaa aaaatcaccc acaaccctct ggaagtggcc ttgcccggtc cccttcccag 1440

gggcgaggtc ggcaaagcaa catggcagag cagtcatagg a 1481

<210> 9

<211> 3735

<212> DNA

<213> human (Homo sapiens)

<400> 9

gccagcccgc gggagaggcc cagcgggagt cgcggaacag caggcccgag cccaccgcgc 60

cgggccccgg acgccgcgcg gaaaagatga atttacaacc aattttctgg attggactga 120

tcagttcagt ttgctgtgtg tttgctcaaa cagatgaaaa tagatgttta aaagcaaatg 180

ccaaatcatg tggagaatgt atacaagcag ggccaaattg tgggtggtgc acaaattcaa 240

catttttaca ggaaggaatg cctacttctg cacgatgtga tgatttagaa gccttaaaaa 300

agaagggttg ccctccagat gacatagaaa atcccagagg ctccaaagat ataaagaaaa 360

ataaaaatgt aaccaaccgt agcaaaggaa cagcagagaa gctcaagcca gaggatatta 420

ctcagatcca accacagcag ttggttttgc gattaagatc aggggagcca cagacattta 480

cattaaaatt caagagagct gaagactatc ccattgacct ctactacctt atggacctgt 540

cttactcaat gaaagacgat ttggagaatg taaaaagtct tggaacagat ctgatgaatg 600

aaatgaggag gattacttcg gacttcagaa ttggatttgg ctcatttgtg gaaaagactg 660

tgatgcctta cattagcaca acaccagcta agctcaggaa cccttgcaca agtgaacaga 720

actgcaccag cccatttagc tacaaaaatg tgctcagtct tactaataaa ggagaagtat 780

ttaatgaact tgttggaaaa cagcgcatat ctggaaattt ggattctcca gaaggtggtt 840

tcgatgccat catgcaagtt gcagtttgtg gatcactgat tggctggagg aatgttacac 900

ggctgctggt gttttccaca gatgccgggt ttcactttgc tggagatggg aaacttggtg 960

gcattgtttt accaaatgat ggacaatgtc acctggaaaa taatatgtac acaatgagcc 1020

attattatga ttatccttct attgctcacc ttgtccagaa actgagtgaa aataatattc 1080

agacaatttt tgcagttact gaagaatttc agcctgttta caaggagctg aaaaacttga 1140

tccctaagtc agcagtagga acattatctg caaattctag caatgtaatt cagttgatca 1200

ttgatgcata caattccctt tcctcagaag tcattttgga aaacggcaaa ttgtcagaag 1260

gcgtaacaat aagttacaaa tcttactgca agaacggggt gaatggaaca ggggaaaatg 1320

gaagaaaatg ttccaatatt tccattggag atgaggttca atttgaaatt agcataactt 1380

caaataagtg tccaaaaaag gattctgaca gctttaaaat taggcctctg ggctttacgg 1440

aggaagtaga ggttattctt cagtacatct gtgaatgtga atgccaaagc gaaggcatcc 1500

ctgaaagtcc caagtgtcat gaaggaaatg ggacatttga gtgtggcgcg tgcaggtgca 1560

atgaagggcg tgttggtaga cattgtgaat gcagcacaga tgaagttaac agtgaagaca 1620

tggatgctta ctgcaggaaa gaaaacagtt cagaaatctg cagtaacaat ggagagtgcg 1680

tctgcggaca gtgtgtttgt aggaagaggg ataatacaaa tgaaatttat tctggcaaat 1740

tctgcgagtg tgataatttc aactgtgata gatccaatgg cttaatttgt ggaggaaatg 1800

gtgtttgcaa gtgtcgtgtg tgtgagtgca accccaacta cactggcagt gcatgtgact 1860

gttctttgga tactagtact tgtgaagcca gcaacggaca gatctgcaat ggccggggca 1920

tctgcgagtg tggtgtctgt aagtgtacag atccgaagtt tcaagggcaa acgtgtgaga 1980

tgtgtcagac ctgccttggt gtctgtgctg agcataaaga atgtgttcag tgcagagcct 2040

tcaataaagg agaaaagaaa gacacatgca cacaggaatg ttcctatttt aacattacca 2100

aggtagaaag tcgggacaaa ttaccccagc cggtccaacc tgatcctgtg tcccattgta 2160

aggagaagga tgttgacgac tgttggttct attttacgta ttcagtgaat gggaacaacg 2220

aggtcatggt tcatgttgtg gagaatccag agtgtcccac tggtccagac atcattccaa 2280

ttgtagctgg tgtggttgct ggaattgttc ttattggcct tgcattactg ctgatatgga 2340

agcttttaat gataattcat gacagaaggg agtttgctaa atttgaaaag gagaaaatga 2400

atgccaaatg ggacacgggt gaaaatccta tttataagag tgccgtaaca actgtggtca 2460

atccgaagta tgagggaaaa tgagtactgc ccgtgcaaat cccacaacac tgaatgcaaa 2520

gtagcaattt ccatagtcac agttaggtag ctttagggca atattgccat ggttttactc 2580

atgtgcaggt tttgaaaatg tacaatatgt ataattttta aaatgtttta ttattttgaa 2640

aataatgttg taattcatgc cagggactga caaaagactt gagacaggat ggttactctt 2700

gtcagctaag gtcacattgt gcctttttga ccttttcttc ctggactatt gaaatcaagc 2760

ttattggatt aagtgatatt tctatagcga ttgaaagggc aatagttaaa gtaatgagca 2820

tgatgagagt ttctgttaat catgtattaa aactgatttt tagctttaca aatatgtcag 2880

tttgcagtta tgcagaatcc aaagtaaatg tcctgctagc tagttaagga ttgttttaaa 2940

tctgttattt tgctatttgc ctgttagaca tgactgatga catatctgaa agacaagtat 3000

gttgagagtt gctggtgtaa aatacgtttg aaatagttga tctacaaagg ccatgggaaa 3060

aattcagaga gttaggaagg aaaaaccaat agctttaaaa cctgtgtgcc attttaagag 3120

ttacttaatg tttggtaact tttatgcctt cactttacaa attcaagcct tagataaaag 3180

aaccgagcaa ttttctgcta aaaagtcctt gatttagcac tatttacata caggccatac 3240

tttacaaagt atttgctgaa tggggacctt ttgagttgaa tttattttat tatttttatt 3300

ttgtttaatg tctggtgctt tctgtcacct cttctaatct tttaatgtat ttgtttgcaa 3360

ttttggggta agactttttt tatgagtact ttttctttga agttttagcg gtcaatttgc 3420

ctttttaatg aacatgtgaa gttatactgt ggctatgcaa cagctctcac ctacgcgagt 3480

cttactttga gttagtgcca taacagacca ctgtatgttt acttctcacc atttgagttg 3540

cccatcttgt ttcacactag tcacattctt gttttaagtg cctttagttt taacagttca 3600

ctttttacag tgctatttac tgaagttatt tattaaatat gcctaaaata cttaaatcgg 3660

atgtcttgac tctgatgtat tttatcaggt tgtgtgcatg aaatttttat agattaaaga 3720

agttgaggaa aagca 3735

<210> 10

<211> 4414

<212> DNA

<213> human (Homo sapiens)

<400> 10

ctctgtgtca ggatcgcaga aagtatgtcc cttctctcac catgagctgg ctctccagtt 60

cccagggagt ggtactaaca gcctaccacc ccagcggcaa ggaccagacc gtcgggaaca 120

gccatgcaaa ggcaggggag gaagccacct cgagtcgcag atatggccag tacactatga 180

accaggaaag caccaccatc aaagttatgg agaagcctcc atttgatcga tcaatttccc 240

aggattcttt ggatgaacta tctatggaag actattggat agaactagaa aacatcaaga 300

aatctagtga aaacagccaa gaagatcaag aggtggttgt tgtcaaagag cctgatgagg 360

gagaattgga agaagagtgg cttaaagagg ccggtttatc caatctcttc ggagagtctg 420

ctggagatcc acaggaaagc attgtgtttt tatcaacatt gacgcggacc caggcagcag 480

cagttcagaa gcgagtagag acggtctccc agaccttgag gaaaaaaaac aaacagtacc 540

agattcctga cgtcagagac atatttgctc aacagagaga atcaaaagaa acagctccag 600

gtggcactga atcgcagtca cttagaacaa atgaaaacaa ataccaagga agagatgacg 660

aggcatctaa ccttgttggt gaagagaagc tgatcccacc tgaggagacg cctgcccctg 720

aaacagacat caacctggag gtatcatttg ccgagcaagc actcaatcag aaagagagct 780

ccaaggagaa aatccagaag agcaaaggcg atgatgccac attacctagt ttcagattgc 840

caaaagacaa aacgggtacc acaaggattg gtgacctcgc accccaggac atgaagaaag 900

tttgccattt agccctaatt gagctgactg ccctctatga tgtattgggt attgagctga 960

aacaacaaaa agctgtgaaa atcaaaacaa aagattctgg tcttttttgc gttccattga 1020

cagcgctatt agaacaagat cagaggaaag taccaggaat gcgaataccc ttgatctttc 1080

aaaaactgat ttctcgaatt gaagagagag gtttggaaac agaaggcctc ttacggatcc 1140

ctggagctgc cattagaatc aagaatcttt gccaagaact agaagcaaag ttttatgaag 1200

ggacttttaa ttgggaaagt gtcaaacagc atgatgccgc cagcctgctg aagctcttca 1260

ttcgggagtt gccccagcca ctgctcagtg tggagtatct caaagccttt caggctgtcc 1320

agaatcttcc aaccaagaag cagcaactac aggctttgaa ccttcttgtc atcctcctac 1380

ctgatgcaaa cagggacaca ctgaaggccc ttcttgaatt tctccaaaga gtaatagata 1440

ataaagaaaa aaataaaatg acagtcatga atgtagcaat ggtcatggcc ccgaatctct 1500

ttatgtgtca tgcattggga ttgaagtcca gtgaacagcg agaatttgta atggcagctg 1560

ggacagcaaa taccatgcac ttattgatta agtaccaaaa acttctgtgg acaattccca 1620

agtttattgt aaaccaagtg aggaagcaaa acacggaaaa tcataaaaag gataaaagag 1680

ccatgaagaa attgctgaag aaaatggctt atgaccgaga aaaatatgaa aagcaagata 1740

agagtacaaa tgatgctgac gttcctcagg gagtgattcg agtgcaagct ccccatcttt 1800

cgaaagtttc catggcaata cagctaactg aagaactaaa agccagtgat gtacttgcca 1860

ggtttctcag ccaagaaagt ggggttgccc agactctcaa gaaaggagaa gtttttttgt 1920

atgaaattgg aggaaatatt ggggaacgct gccttgatga tgacacttac atgaaggatt 1980

tatatcagct taacccaaat gctgagtggg ttataaagtc aaagccattg tagaagactt 2040

aacaagctgc agataaccat gtggacttct gtcataattc ttgctgagtc aagagtgtaa 2100

ataaaagaaa tggcaggact catattattc agttgtaccc aagtatttaa aaatgactct 2160

cttaagcctt aaaaagtcat agatttgtgc tgctgccaga attatattaa ttattattaa 2220

tgttattatt agaaaaaaaa tttctggagt gagagtaaag aggcttaatt agtttgtggg 2280

cagttttcat atgctctgtg aaatgtgtcc agatgtgaca tagttttttt ttttaatatg 2340

tggaaatgtc ttctcttccc attcttttct cctaaaatca tatatactgt aatatatgct 2400

ctctcacctc tattacctcc tcacatctac cctttcccag ttaggtttgc tttttgacca 2460

aaaagataac aaataccagg tatggcaagt tgtgaagaca gcacattaaa acatacctaa 2520

tttcacagta ttcctgtcac gacagaatgt tagtattcat ctctttgaat catttgctca 2580

aataataaca ttccaccttt tcctgctgta tcacaggaag tgatttgcat tttttttcag 2640

ttcatctgac ttatgttcac agaaccgtat cagcgaccaa gaaaatagga ctgtcagaag 2700

ctgccagtta ttactgaacc attaaatact tatatactaa gaataaataa aatataccca 2760

tgtgaaataa taattggatt atggataaca agagagtgaa agccaaagca ctttctgtct 2820

actgtactct tctaaatgga attttaaaag tcatagctgg ctttacgtgt tgtcattatt 2880

agcattataa atatgcatga tagtataatc cagtaatggt tgaagaatgt attttactta 2940

aagagggatt ttttttttta agtcctgaat aagtctactg gaagaattat tcttctgggt 3000

gaaaaagctt ttgtttgtgt tcttatttta aataatcgga gtcaatttat taaaatgttc 3060

ttgaaagtac tattcccagg gattttaatg cacaaaccat attgtgacaa gagatgagcc 3120

tctgtactgt aaataagaaa tgaagtagag aaatgttaaa tattttatga gtttagaata 3180

tagtaaataa aaggtgatgt aaatgaatgc tgcacaaacg gtgttcatga tacttttagt 3240

agtactttag gaaaaactac acattctcag aagctcttga tgtctctaat gaaggggggg 3300

aatgctgtta atgagaacag tcataaattt ttagcatata attacaagaa cagcctgtgg 3360

atatgatcac ttaaatgatt ttgtggtgat tcgtgccatt gcttttttat ttaaaagaaa 3420

attttgtaat taaatgcctt tttctaaatt atcttctctt ggaatcatta cttttaatcc 3480

tatgtgttta tgagtatttt tgcttttttt ttattaatat tgagaaatgg acttttttgt 3540

tattaaaagt cacctctatt ttctattttc tttgtaattt ttaaagtagg aagatgtcag 3600

agatgtaaat atgttttcgt ctttagtttt tttcctttta caaattttta ttcttcagga 3660

ttttcaaaat acagtttagt ctgtttcttt gacaatatgt attaatttcc caattagcaa 3720

aatggtactt attagtgggt tgaaaacaat taataatata aaagaaaaat taagtgctta 3780

aaacatttta ggagtataca acttcaaaaa aaaagatagc agtgaggata atgatttaag 3840

taaaaggttg tctgaagcat atgccaacta aatttcgcaa cgtttgctac ctacctgaaa 3900

aggagggtca ggagggagac acaacatatt tttgatcatg aaaaagtatc ttaattttaa 3960

aaaagtgaaa atgccatttt attttgaatc ccttttagaa ctcacgaccc aagttcatca 4020

atgttgaata atatcacgtt taaataacaa aaaaatatgg actttaaaaa atctcaaatt 4080

ttttagagac agggtcttgc tctgttttcc agattggagt acagcagtgc attcgcggct 4140

aactgcagcc tcaagcactg gggctcaagc aaacctcctg cctcagcctc gtgagtagct 4200

aggaccacag gtgcatgcca ccatgcctgg ctctaaagag aaaaaaaact tgataccata 4260

gagccttgaa tataaatatc ctgatgttaa cctactgctt ttgctgtgat tttttttcct 4320

tagtgagttt taaatctcag gctagatttt tatttgtttt tctgtgtgtg tatgagacaa 4380

aataaaaata aatatatttg ccttgagttt aaaa 4414

<210> 11

<211> 21

<212> DNA

<213> Artificial sequence (Artificial)

<400> 11

gctgtccaca tctggctttt g 21

<210> 12

<211> 24

<212> DNA

<213> Artificial sequence (Artificial)

<400> 12

ggtgcatatt acagtaggct ttcg 24

<210> 13

<211> 20

<212> DNA

<213> Artificial sequence (Artificial)

<400> 13

ctcgggctgt ggagagtttc 20

<210> 14

<211> 18

<212> DNA

<213> Artificial sequence (Artificial)

<400> 14

gcctcgcttc agcaggaa 18

<210> 15

<211> 20

<212> DNA

<213> Artificial sequence (Artificial)

<400> 15

cacggacaga tccaggttca 20

<210> 16

<211> 20

<212> DNA

<213> Artificial sequence (Artificial)

<400> 16

ccgttttgcc cactgttgta 20

<210> 17

<211> 23

<212> DNA

<213> Artificial sequence (Artificial)

<400> 17

tctcttcttt gaccgtgcta gga 23

<210> 18

<211> 23

<212> DNA

<213> Artificial sequence (Artificial)

<400> 18

tgtggaggag cctagacata gga 23

<210> 19

<211> 22

<212> DNA

<213> Artificial sequence (Artificial)

<400> 19

ggaaggctat gtgaacgtga ca 22

<210> 20

<211> 24

<212> DNA

<213> Artificial sequence (Artificial)

<400> 20

ctgtagaagt ggttggcaaa gaga 24

<210> 21

<211> 21

<212> DNA

<213> Artificial sequence (Artificial)

<400> 21

ttcgggagtc tgttcccttt t 21

<210> 22

<211> 19

<212> DNA

<213> Artificial sequence (Artificial)

<400> 22

ggccctgctg agctcttct 19

<210> 23

<211> 21

<212> DNA

<213> Artificial sequence (Artificial)

<400> 23

caaccgtgag aaacagcgtt t 21

<210> 24

<211> 22

<212> DNA

<213> Artificial sequence (Artificial)

<400> 24

ctgccaatac catcaatttc ca 22

<210> 25

<211> 24

<212> DNA

<213> Artificial sequence (Artificial)

<400> 25

ccccttcctc aacagtgtct acat 24

<210> 26

<211> 20

<212> DNA

<213> Artificial sequence (Artificial)

<400> 26

agccaggtcc agctcacagt 20

<210> 27

<211> 23

<212> DNA

<213> Artificial sequence (Artificial)

<400> 27

tcagaattgg atttggctca ttt 23

<210> 28

<211> 23

<212> DNA

<213> Artificial sequence (Artificial)

<400> 28

tggtgcagtt ctgttcactt gtg 23

<210> 29

<211> 25

<212> DNA

<213> Artificial sequence (Artificial)

<400> 29

gatgatgcca cattacctag tttca 25

<210> 30

<211> 19

<212> DNA

<213> Artificial sequence (Artificial)

<400> 30

ggtgcgaggt caccaatcc 19

<210> 31

<211> 25

<212> DNA

<213> Artificial sequence (Artificial)

<400> 31

atattcctca tggtggcagc gctca 25

<210> 32

<211> 24

<212> DNA

<213> Artificial sequence (Artificial)

<400> 32

ccgagggacc acctcctatg caga 24

<210> 33

<211> 26

<212> DNA

<213> Artificial sequence (Artificial)

<400> 33

aagcggacag tgctcacgac acaacc 26

<210> 34

<211> 25

<212> DNA

<213> Artificial sequence (Artificial)

<400> 34

aaagtgcatt cctgccctgg tgacc 25

<210> 35

<211> 25

<212> DNA

<213> Artificial sequence (Artificial)

<400> 35

agaatcggcc ccccattgct attgt 25

<210> 36

<211> 29

<212> DNA

<213> Artificial sequence (Artificial)

<400> 36

tgccacgcgt tgaatagttc cacatactt 29

<210> 37

<211> 30

<212> DNA

<213> Artificial sequence (Artificial)

<400> 37

agacaagttc atcatcccca gcaaactctg 30

<210> 38

<211> 26

<212> DNA

<213> Artificial sequence (Artificial)

<400> 38

catcttccct agcagccggt tgaagc 26

<210> 39

<211> 30

<212> DNA

<213> Artificial sequence (Artificial)

<400> 39

tagcacaaca ccagctaagc tcaggaaccc 30

<210> 40

<211> 28

<212> DNA

<213> Artificial sequence (Artificial)

<400> 40

attgccaaaa gacaaaacgg gtaccaca 28

Claims (6)

1. A whole blood transcription gene marker for grading cervical squamous intraepithelial lesions, which is applied to the preparation of products for screening high-grade cervical squamous intraepithelial lesions, is disclosed, wherein the genes of the gene marker are STMN3, TRPC4AP, DYRK2, AGK, KIAAO319L, GRPEL1, ZFC3HL, LYL1, ITGB1 and ARHGAP18 genes shown in SEQ ID No. 1-SEQ ID No. 10.

2. The kit for screening and grading the cervical squamous intraepithelial lesions is applied to preparation of products for screening the high-grade cervical squamous intraepithelial lesions, and is characterized in that genes detected by a reagent in the kit are STMN3, TRPC4AP, DYRK2, AGK, KIAAO319L, GRPEL1, ZFC3H1, LYL1, ITGB1 and ARHGAP18 genes shown in SEQ ID No. 1-SEQ ID No.10, and the reagent is a primer and/or a probe.

3. The kit for screening and grading cervical squamous intraepithelial lesions, for use in the preparation of a product for screening high-grade cervical squamous intraepithelial lesions, according to claim 2, characterized in that said reagent is a reagent for detecting the amount of RNA transcribed from said gene, said reagent being a primer and/or a probe.

4. The kit for screening and grading cervical squamous intraepithelial lesions, for use in the preparation of a product for screening high-grade cervical squamous intraepithelial lesions, according to claim 3, wherein the reagent is a reagent for detecting the amount of reverse transcription of the gene into complementary DNA.

5. A composition for use in preparing a product for screening high-grade cervical squamous intraepithelial lesions, comprising primers and/or probes for detecting genes differentially expressed in whole blood of patients with low-grade cervical squamous intraepithelial lesions and high-grade cervical squamous intraepithelial lesions, the genes being STMN3, TRPC4AP, DYRK2, AGK, KIAAO319L, GRPEL1, ZFC3H1, LYL1, ITGB1 and ARHGAP18 genes represented by SEQ ID N0.1 to SEQ ID N0.10.

6. A gene chip for screening and grading cervical squamous intraepithelial lesions is characterized by comprising oligonucleotide probes hybridized with cervical squamous intraepithelial lesion related characteristic gene sequences, wherein the genes are STMN3, TRPC4AP, DYRK2, AGK, KIAAO319L, GRPEL1, ZFC3H1, LYL1, ITGB1 and ARHGAP18 genes shown as SEQ ID N0.1-SEQ ID N0.10.

Images