KR20080112551A - Brca1 and brca2 germline mutations useful for predicting or detecting breast cancer or ovarian cancer - Google Patents

Abstract

A BRCA1 and BRCA2 gene mutation is provided to detect breast cancer or ovarian cancer with low costs and high efficiency by providing the type of the mutation specific in Korean existing in the nucleotide sequential of BRCA1 and BRCA2. A polynucleotide consists of the 20-100 continuous nucleotide sequence. The 20-100 continuous nucleotide sequence includes a BRCA1 gene mutation selected from a group consisting of: the insertion of GGG and the deletion of AT in 38th base and 39th base in the polynucleotide consisting of the sequence of the sequence number 1; the substitution of 154th base from C to T in the polynucleotide consisting of the sequence of the sequence number 1; the substitution of 390th base from C to A in the polynucleotide consisting of the sequence of the sequence number 1; the substitution of 527th base from C to T in the polynucleotide consisting of the sequence of the sequence number 1; and the insertion of A between 720th base and 721th base in the polynucleotide consisting of the sequence of the sequence number.

Description

JRCCA1 and BRCA2 germline mutations useful for predicting or detecting breast cancer or ovarian cancer}

1 schematically illustrates the structure and mutation distribution of the BRCA1 gene. Numbers indicate exons and arrowheads schematically indicate where mutations are present.

2 schematically depicts the structure and mutation distribution of the BRCA2 gene. Numbers indicate exons and arrowheads schematically indicate where mutations are present.

FIG. 3 shows a F-CSGE analysis graph of mutations in which the 5339th nucleotide (located at exon 22) of the BRCA1 gene (SEQ ID NO: 1), one of the BRCA1 gene mutations, is substituted for T to C.

FIG. 4 shows a graph of F-CSGE analysis of a mutation with a G inserted between the 8827th and 8828th nucleotides (located at exon 22) of the BRCA2 gene (SEQ ID NO: 9), one of the BRCA2 gene mutations.

The present invention relates to the BRCA1 and BRCA2 genes known as the onset genes of breast and ovarian cancers. More specifically, the present invention relates to Korean-specific germline mutations that can be used in predicting or diagnosing susceptibility of breast and ovarian cancers and to methods for detecting them from the BRCA1 and BRCA2 genes.

The BRCA1 gene, located on chromosome 17 and consists of 22 exons, was the first gene to be proven to increase the risk of developing breast and ovarian cancer. The BRCA2 gene, located on chromosome 13, consists of 26 exons and has been validated as a gene that increases the risk of developing breast and ovarian cancer following BRCA1. Those who inherited the BRCA1 and BRCA2 gene mutations from one of their parents had a 87% chance of developing breast cancer until they were 70 years old, and a 59% chance of developing breast cancer between 40 and 50 years before menopause.

BRCA1 is involved in the development of ovarian cancer as well as breast cancer, and the incidence of ovarian cancer in people with the BRCA1 gene mutation is 44% before age 70 (Ford, et al., 1994). The mutated BRCA gene can be inherited from not only the mother but also from the father, and if the female and male have a genetic mutation, the incidence of breast cancer is high.

Approximately 7% of breast cancers and 10% of ovarian cancers are known to be associated with mutations in the BRCA1 and BRCA2 genes. Mutations in these two genes increase the incidence of breast cancer by 56-87%, and the incidence of ovarian cancer increases more than 10 times that of normal individuals. It is also known to increase the recurrence rate of breast cancer and ovarian cancer, and to increase the disease sensitivity for prostate cancer, colon cancer and other cancers.

The incidence of mutations in the BRCA1 and BRCA2 genes varies among ethnic groups. To date, more than 3,400 germline BRCA1 and BRCA2 mutations, polymorphisms, and sequence variations have been identified (Breast Cancer Information Core (BIC), National Center for Human Genome Research, National Institute of Health: http://research.nhgri.nih.gov/projects/bic/). Analysis of the BRCA1 and BRCA2 gene mutations has deepened the understanding of the genetics of breast cancer, but little is known about the frequency and specific mutation types of BRCA1 and BRCA2 gene mutations in Koreans. In particular, about 7,600 new cases of breast cancer occur annually in Korea, and women have the second highest incidence after gastric cancer (Korea Central Cancer Registration Annual Report).

Therefore, it is necessary to study nucleotide variation patterns including mutations and monobasic polymorphisms of the BRCA1 and BRCA2 genes in Koreans to identify Korean specific BRCA1 and BRCA2 gene mutations. Analyzing the presence or absence of mutations in the BRCA1 and BRCA2 genes may contribute to minimizing the risk of disease by performing a more proactive prevention program by early diagnosis of the risk of breast and ovarian cancer. Accordingly, the present inventors analyzed a sample obtained from a number of Korean breast cancer patients and normal people, and investigated the type and frequency of mutations present in the BRCA1 and BRCA2 genes to identify gene mutations that can be used to predict and diagnose breast and ovarian cancer. Confirmed.

It is an object of the present invention to provide Korean specific BRCA1 and BRCA2 gene mutations that can be utilized in the diagnosis of breast and ovarian cancer.

It is another object of the present invention to provide a polynucleotide consisting of 20 to 100 contiguous nucleotide sequences comprising Korean specific BRCA1 or BRCA2 gene mutations that can be utilized as primers or probes for the diagnosis of breast or ovarian cancer. will be.

Another object of the present invention is to diagnose a breast cancer and ovarian cancer microarray comprising a polynucleotide consisting of 20 to 100 contiguous nucleotide sequences or complementary sequences thereof comprising a Korean specific BRCA1 or BRCA2 gene mutation as a probe To provide.

Gene mutations used in the present invention are defined as follows:

SEQ ID NO: 1 and SEQ ID NO: 9 indicate the coding sequence (CDS) of BRCA1 (Gen Bank Accession No. NM_007294.1) and BRCA2 (Gen Bank Accession No. NM_000059.1), respectively, and the nucleotide of ATG, the translation initiation codon, is nucleotide. Mark as +1. Mutations within the exons of the BRCA1 and BRCA2 genes are indicated based on SEQ ID NO: 1 and SEQ ID NO: 9, respectively. Insertion mutations are denoted by 'ins', deletion mutations are denoted by 'del', and the letters following 'ins' and 'del' indicate actual nucleotides inserted or deleted. Substitution mutations are described by indicating the symbol '>', where the letters before '>' are normal nucleotides of the BRCA1 and BRCA2 genes, and the letters after '>' refer to nucleotides of the mutant BRCA1 and BRCA2 genes.

SEQ ID NOs 2 to 8 refer to GenBank genome reference sequence NT_010755.15 and SEQ ID NOs 10 to 12 refer to GenBank genome reference sequence NT_024524.13. SEQ ID NOs: 2 to 8 and SEQ ID NOs: 10 to 12 are used to indicate mutations of intron or splicing sites of BRCA1 and BRCA2, respectively, and include sequences of corresponding codons and borders of introns, That is, a sequence including a portion of a codon and an intron. The mutation present at the beginning of the intron describes the number of the last nucleotide of the preceding exon (based on the coding sequence) followed by '+' followed by the number of nucleotides from the nucleotide to the mutation site and then the type of mutation. List it. For example, 'c.548 + 13delG' means a mutation in which the nucleotide G located at the 13th position from the 548th nucleotide on the coding sequence is deleted. On the other hand, the mutations present at the end of the intron are the first nucleotide number of the trailing exon (based on the coding sequence) followed by a '-' sign followed by a nucleotide upstream from the nucleotide on the exon up to the corresponding position in the intron. List the numbers, followed by the type of mutation. For example, 'c.5194-10G> T' refers to a mutation in which the nucleotide G located tenth upwards from the 5194th nucleotide on the coding sequence is replaced with T.

1 and 2 show the structure of the BRCA1 and BRCA2 genes and the schematic distribution of the gene mutations identified in the present invention.

One aspect of the invention provides 20-100 contiguous nucleotide sequences comprising a BRCA1 gene mutation selected from the group consisting of 20-80, 20-60, 20-50, or 20-40 Provided is a polynucleotide consisting of two consecutive nucleotide sequences:

(1) deletion of AT and insertion of GGG in base 38 and 39 in a polynucleotide consisting of the sequence of SEQ ID NO: 1;

(2) substitution of C for T of 154 th base in the polynucleotide consisting of the sequence of SEQ ID NO: 1;

(3) the substitution of C for A of the 390th base in the polynucleotide consisting of the sequence of SEQ ID NO: 1;

(4) substitution of C for T of 527th base in the polynucleotide consisting of the sequence of SEQ ID NO: 1;

(5) insertion of A between 720 and 721 th in the polynucleotide consisting of the sequence of SEQ ID NO: 1;

(6) substitution of G for A of 811 th base in the polynucleotide consisting of the SEQ ID NO: 1;

(7) substitution of the C for T of the 928th base in the polynucleotide consisting of the sequence of SEQ ID NO: 1;

(8) deletion of ACT and insertion of GA from bases 1293 to 1295 in the polynucleotide consisting of the sequences of SEQ ID NO: 1;

(9) a deletion of 5 bases from the 1516th to 1520th base in the polynucleotide consisting of the SEQ ID NO: 1;

(10) substitution of C for T of 1612th base in a polynucleotide consisting of the sequence of SEQ ID NO: 1;

(11) a deletion of the 1716th base A in a polynucleotide consisting of the sequence of SEQ ID NO: 1;

(12) a deletion of 1823 base A in the polynucleotide consisting of the sequence of SEQ ID NO: 1;

(13) a deletion of base 1831 C in a polynucleotide consisting of the sequence of SEQ ID NO: 1;

(14) A to G substitution of the 1840 th base in the polynucleotide consisting of the sequence of SEQ ID NO: 1;

(15) substitution of C for T of 1865 base in a polynucleotide consisting of the sequence of SEQ ID NO: 1;

(16) a deletion of base 1961 base A in a polynucleotide consisting of the sequence of SEQ ID NO: 1;

(17) a deletion of the 2048th base A in a polynucleotide consisting of the sequence of SEQ ID NO: 1;

(18) a deletion of the 2359th base G in a polynucleotide consisting of the sequence of SEQ ID NO: 1;

(19) a deletion of base 2433 base C in a polynucleotide consisting of the sequence of SEQ ID NO: 1;

(20) substitution of the A for C of the 2481 base in a polynucleotide consisting of the sequence of SEQ ID NO: 1;

(21) substitution of the 2726 base A for T in a polynucleotide consisting of the sequence of SEQ ID NO: 1;

(22) a deletion of the 2934th base T in a polynucleotide consisting of the sequence of SEQ ID NO: 1;

(23) G to A of 2968 base in the polynucleotide consisting of the sequence of SEQ ID NO: 1;

(24) a deletion of the 3296th base C in a polynucleotide consisting of the sequence of SEQ ID NO: 1;

(25) a deletion of the 3329th base A in a polynucleotide consisting of the sequence of SEQ ID NO: 1;

(26) G to T of 3340rd base in the polynucleotide consisting of the sequence of SEQ ID NO: 1;

(27) substitution of the 3409 base for A to G in a polynucleotide consisting of the SEQ ID NO: 1;

(28) a deletion of 4 bases from 3436 to 3439 in the polynucleotide consisting of the sequences of SEQ ID NO: 1;

(29) a deletion of the 3442rd base G in a polynucleotide consisting of the sequence of SEQ ID NO: 1;

(30) substitution of C for T of 3448 base in a polynucleotide consisting of the sequence of SEQ ID NO: 1;

(31) the substitution of G for T of the 3472 th base in the polynucleotide consisting of the sequence of SEQ ID NO: 1;

(32) substitution of T for A of 3593 base in the polynucleotide consisting of the sequence of SEQ ID NO: 1;

(33) Insertion of A between 3627 and 3628 in the polynucleotide consisting of the sequences of SEQ ID NO: 1;

(34) substitution of C for G of base 3722 in a polynucleotide consisting of the sequence of SEQ ID NO: 1;

(35) a deletion of 4 bases from 3756 to 3759 in the polynucleotide consisting of the sequence of SEQ ID NO: 1;

(36) the insertion of a T between the 3812 and 3813 th polynucleotides consisting of the sequence of SEQ ID NO: 1;

(37) substitution of C for T of 3895 base in the polynucleotide consisting of the sequence of SEQ ID NO: 1;

(38) a deletion of two bases from 4041 to 4142 th in the polynucleotide consisting of the sequences of SEQ ID NO: 1;

(39) a deletion of 4 bases from 4065 to 4068 in the polynucleotide consisting of the sequences of SEQ ID NO: 1;

(40) the insertion of four base AGAAs between the 4338th and 4339th bases in the polynucleotide consisting of the sequences of SEQ ID NO: 1;

(41) substitution of the T for C of the 4729 th base in the polynucleotide consisting of the sequence of SEQ ID NO: 1;

(42) substitution of the 4743th base for A for C in a polynucleotide consisting of the sequence of SEQ ID NO: 1;

(43) G to T of 4981th base in the polynucleotide consisting of the sequence of SEQ ID NO: 1;

(44) a deletion of 4 bases 5030-5033 in the polynucleotide consisting of the sequences of SEQ ID NO: 1;

(45) a deletion of two bases from 5102 to 5103 for a polynucleotide consisting of the sequence of SEQ ID NO: 1;

(46) a deletion of 2 bases from 5323 to 5324 in the polynucleotide consisting of the sequences of SEQ ID NO: 1;

(47) T to C substitution of the 5339th base in a polynucleotide consisting of the sequence of SEQ ID NO: 1;

(48) substitution of G for A of 5444th base in a polynucleotide consisting of the sequence of SEQ ID NO: 1;

(49) a deletion of 8 bases from 5470 to 5477 in the polynucleotide consisting of the sequence of SEQ ID NO: 1;

(50) deletion of the 5483th base G in a polynucleotide consisting of the sequence of SEQ ID NO: 1;

(51) a deletion of 10 bases from 5497 to 5506 in the polynucleotide consisting of the sequence of SEQ ID NO: 1; And

(52) an insertion of a G between the 5532 and 5533 th polynucleotides consisting of the sequence of SEQ ID NO: 1; And

(53) an insertion of C between 5553 and 5554 th in the polynucleotide consisting of the sequence of SEQ ID NO: 1;

(54) a mutant c.547 + 13delG in which the G present at 210 is deleted in the polynucleotide having a SEQ ID NO: 2;

(55) the mutation c.4484 + 14A> G wherein the base A present at 218th in the polynucleotide having a sequence of SEQ ID NO: 3 is substituted with G;

(56) the mutant c.5194-10G> T wherein the base G present at 167th of the polynucleotide having a sequence of SEQ ID NO: 4 is substituted with T;

(57) a mutant c.441 + 1G> A wherein the base G present at 171th in the polynucleotide having a sequence of SEQ ID NO: 5 is replaced with A;

(58) the mutant c.302-2A> C wherein the base A present at 149th in the polynucleotide having a sequence of SEQ ID NO: 6 is substituted with C;

(59) the mutation c.671-8A> G wherein the base A present at 124th in the polynucleotide having a sequence of SEQ ID NO: 7 is substituted with G; And

(60) The mutation c.5074 + 1G> T wherein the base G existing at 205 in the polynucleotide having the sequence shown in SEQ ID NO: 8 is substituted with T.

In the above, the mutations (1) to (53) are represented on the basis of SEQ ID NO: 1 as a mutation present on the coding sequence of the BRCA1 gene. These correspond to deletions, insertions or substitution mutations of nucleotides at the corresponding positions in SEQ ID NO: 1, respectively, resulting in amino acid sequences different from normal BRCA1, which can alter the in vivo function of the BRCA1 protein.

In addition, mutations (54) to (60) above are mutations at the intron or splicing site that are not the coding sequence of the BRCA1 gene:

Mutation 54 is a mutation on an intron wherein the nucleotide G present at position 13 (210 of SEQ ID NO: 2) is deleted from the nucleotide at position 547 on the coding sequence (SEQ ID NO: 1). Since introns are not sites encoding amino acids, there is no change in the amino acid sequence, but may result in changes in BRCA1 expression patterns due to differences in RNA production due to changes in splicing.

Mutation 55 is a mutation on intron in which nucleotide A present at position 14 (SEQ ID NO: 218 of SEQ ID NO: 3) is replaced by G from nucleotide present at position 4487 on coding sequence (SEQ ID NO: 1). Since introns are not sites encoding amino acids, there is no change in the amino acid sequence, but may result in changes in BRCA1 expression patterns due to differences in RNA production due to changes in splicing.

Mutation 56 is a mutation on an intron in which nucleotide G present at position 10 (No. 167 of SEQ ID NO: 4) is replaced by T upwards from the nucleotide at position 5194 on the coding sequence (SEQ ID NO: 1). to be. Since introns are not sites encoding amino acids, there is no change in the amino acid sequence, but may result in changes in BRCA1 expression patterns due to differences in RNA production due to changes in splicing.

Mutation 57 is a mutation of a splicing site in which the nucleotide G present immediately after the nucleotide present at position 441 (SEQ ID NO: 1) on the coding sequence (SEQ ID NO: 5) is replaced with A. . Since the splicing region is not a region encoding the amino acid, there is no change in the amino acid sequence, but it may cause a change in the BRCA1 expression pattern due to the difference in RNA production due to the splicing change.

Mutation 58 is a splicing wherein nucleotide A present at the second position (No. 149 of SEQ ID NO: 6) upwards from the nucleotide at position 302 on the coding sequence (SEQ ID NO: 1) is replaced with C. Mutation of the site. Since the splicing region is not a region encoding the amino acid, there is no change in the amino acid sequence, but it may cause a change in the BRCA1 expression pattern due to the difference in RNA production due to the splicing change.

Mutation 59 is a mutation on the intron in which nucleotide A present at position 8 (number 124 of SEQ ID NO: 7) is upwardly replaced by nucleotide present at position 671 on the coding sequence (SEQ ID NO: 1). to be. Since the intron is not an amino acid coding region, there is no change in the amino acid sequence, but it may cause a change in BRCA1 expression pattern due to the difference in RNA production due to a change in splicing.

Mutation 60 is a mutation on an intron in which nucleotide G present immediately following (nucleotide number 205 of SEQ ID NO: 8) located at position 5074 on the coding sequence (SEQ ID NO: 1) is replaced with T. Since introns are not sites encoding amino acids, there is no change in the amino acid sequence, but may result in changes in BRCA1 expression patterns due to differences in RNA production due to changes in splicing.

Polynucleotides of the invention can be used as probes or primers.

Another aspect of the invention also relates to 20-100 contiguous nucleotide sequences comprising a BRCA2 mutation selected from the group consisting of 20-80, 20-60, 20-50, or 20- Polynucleotides consisting of 40 contiguous nucleotide sequences are provided:

(1) G to A substitution of the 53rd base in the polynucleotide consisting of the sequences of SEQ ID NO: 9;

(2) G to T substitution of the 97th base in a polynucleotide consisting of the sequence of SEQ ID NO: 9;

(3) the substitution of the 125th base for A to G in a polynucleotide consisting of the sequence of SEQ ID NO: 9;

(4) the insertion of A between 276 and 277 in the polynucleotide consisting of the sequence of SEQ ID NO: 9;

(5) G to A substitution of the 475th base in the polynucleotide consisting of the sequence of SEQ ID NO: 9;

(6) substitution of the 623th base for T to G in a polynucleotide consisting of the sequence of SEQ ID NO: 9;

(7) a deletion of two bases from the 658 th base to the 659 th base in the polynucleotide having the sequence of SEQ ID NO: 9;

(8) substitution of the 943 base for T for A in a polynucleotide consisting of the sequence of SEQ ID NO: 9;

(9) substitution of the A-C of the 964th base in a polynucleotide consisting of the sequence of SEQ ID NO: 9;

(10) a deletion of the 994th base A in a polynucleotide consisting of the sequence of SEQ ID NO: 9;

(11) a deletion of 4 bases from 1310 base to 1313 base in the polynucleotide having the sequence of SEQ ID NO: 9;

(12) substitution of the 1399 base for A to T in a polynucleotide consisting of the SEQ ID NO: 9;

(13) a deletion of the 1547th base T in a polynucleotide consisting of the sequence of SEQ ID NO: 9;

(14) substitution of the 1744th base for A for C in a polynucleotide consisting of the sequence of SEQ ID NO: 9;

(15) substitution of G for A of base 1847 in a polynucleotide consisting of the sequence of SEQ ID NO: 9;

(16) substitution of the C for T of 1889 bases in a polynucleotide consisting of the sequence of SEQ ID NO: 9;

(17) substitution of G for A of 2465th base in a polynucleotide consisting of the sequence of SEQ ID NO: 9;

(18) a deletion of 2 bases from 2798 bases to 2799 bases in the polynucleotide consisting of the sequences of SEQ ID NO: 9;

(19) a deletion of 4 bases from the 2808 th base to the 2811 th base in the polynucleotide having a sequence of SEQ ID NO: 9;

(20) T to G substitution of the 2912nd base in the polynucleotide consisting of the sequence of SEQ ID NO: 9;

(21) deletion of 15 bases and insertion of T from base 3096 to 3110 in the polynucleotide consisting of the sequences of SEQ ID NO: 9;

(22) a deletion of 4 bases from the 3195 base to the 3198 base in the polynucleotide consisting of the SEQ ID NO: 9;

(23) substitution of A for T of 3220rd base in the polynucleotide consisting of the sequence of SEQ ID NO: 9;

(24) substitution of T for G of base 3341 in a polynucleotide consisting of the sequence of SEQ ID NO: 9;

(25) a deletion of 5 bases from the 3352rd base to the 3356th base in the polynucleotide having the sequence of SEQ ID NO: 9;

(26) a deletion of 4 bases from the 3744 base to the 3747 base in the polynucleotide consisting of the sequences of SEQ ID NO: 9;

(27) substitution of the 3814 base for A to G in a polynucleotide consisting of the SEQ ID NO: 9;

(28) substitution of the 3814th base for A for T in a polynucleotide consisting of the sequence of SEQ ID NO: 9;

(29) substitution of the 4376 base A for G in a polynucleotide consisting of the SEQ ID NO: 9;

(30) a deletion of 4766 base C in a polynucleotide consisting of the sequence of SEQ ID NO: 9;

(31) a deletion of two bases from base 4829 to 4830 in a polynucleotide consisting of the sequence of SEQ ID NO: 9;

(32) substitution of T for A of 4854 base in a polynucleotide consisting of the sequence of SEQ ID NO: 9;

(33) a deletion of two bases from the 5207 base to the 5208 base in the polynucleotide consisting of the sequences of SEQ ID NO: 9;

(34) a deletion of the 5250th base C in a polynucleotide consisting of the sequence of SEQ ID NO: 9;

(35) a deletion of 4 bases from 5576 to 5579 bases in the polynucleotide consisting of the sequences of SEQ ID NO: 9;

(36) substitution of C for T of 5656th base in a polynucleotide consisting of the sequence of SEQ ID NO: 9;

(37) substitution of C for T of 5744th base in a polynucleotide consisting of the sequence of SEQ ID NO: 9;

(38) a deletion of 4 bases from base 5946 to 5949th in the polynucleotide consisting of the sequences of SEQ ID NO: 9;

(39) substitution of the 5969th base from A to C in a polynucleotide consisting of the SEQ ID NO: 9;

(40) substitution of T for G of base 6029 in a polynucleotide consisting of the sequence of SEQ ID NO: 9;

(41) substitution of G for T of base 6082 in a polynucleotide consisting of the sequence of SEQ ID NO: 9;

(42) substitution of the 6086 base for A to G in a polynucleotide consisting of the SEQ ID NO: 9;

(43) G-T substitution of the 6131 th base in the polynucleotide consisting of the sequence of SEQ ID NO: 9;

(44) T to G substitution of the 6239rd base in the polynucleotide consisting of the sequence of SEQ ID NO: 9;

(45) substitution of G for A of 6325 base in the polynucleotide consisting of the sequence of SEQ ID NO: 9;

(46) substitution of the 6448 base A for C in a polynucleotide consisting of the SEQ ID NO: 9;

(47) G to T substitution of the 6496th base in a polynucleotide consisting of the sequence of SEQ ID NO: 9;

(48) a deletion of 6553 base G in a polynucleotide consisting of the sequence of SEQ ID NO: 9;

(49) a deletion of two bases from base 6724 to base 6725 in the polynucleotide consisting of the sequences of SEQ ID NO: 9;

(50) T to G substitution of the 6839th base in the polynucleotide consisting of the sequence of SEQ ID NO: 9;

(51) substitution of the 6875 base from A to C in a polynucleotide consisting of the sequence of SEQ ID NO: 9;

(52) C to G substitution of the 7052th base in a polynucleotide consisting of the sequence of SEQ ID NO: 9;

(53) A to G substitution of the 7088th base in a polynucleotide consisting of the sequence of SEQ ID NO: 9;

(54) T to G substitution of the 7218th base in the polynucleotide consisting of the sequence of SEQ ID NO: 9;

(55) substitution of A for G of base 7411 in a polynucleotide consisting of the sequence of SEQ ID NO: 9;

(56) substitution of the T for C of base 7469 in a polynucleotide consisting of the sequence of SEQ ID NO: 9;

(57) C to T substitution of the 7480 th base in the polynucleotide consisting of the sequence of SEQ ID NO: 9;

(58) substitution of G for A of 7522 base in a polynucleotide consisting of the sequence of SEQ ID NO: 9;

(59) substitution of C for A of 7625th base in a polynucleotide consisting of the sequence of SEQ ID NO: 9;

(60) substitution of G for A of base 7814 in a polynucleotide consisting of the sequence of SEQ ID NO: 9;

(61) substitution of the T for C of the 7876 base for the polynucleotide consisting of the sequence of SEQ ID NO: 9;

(62) substitution of T for A of the 8063th base for a polynucleotide consisting of the sequence of SEQ ID NO: 9;

(63) insertion of an AC between the 8299th and 8300th bases in a polynucleotide consisting of the SEQ ID NO: 9;

(64) a deletion of three bases from the 8437th base to the 8439th base in the polynucleotide having the sequence of SEQ ID NO: 9;

(65) a deletion of two bases from the 8717 th base to the 8718 th base in the polynucleotide having the sequence of SEQ ID NO: 9;

(66) the insertion of G between the 8827th base and the 8828th base in the polynucleotide consisting of the sequence of SEQ ID NO: 9;

(67) T to G substitution of the 8932 th base in the polynucleotide consisting of the sequence of SEQ ID NO: 9;

(68) substitution of C for G of the 8951th base in a polynucleotide consisting of the sequence of SEQ ID NO: 9;

(69) T to G substitution of the 8991th base in the polynucleotide consisting of the sequence of SEQ ID NO: 9;

(70) substitution of C for T of 9076 bases in a polynucleotide consisting of the sequence of SEQ ID NO: 9;

(71) an insertion of A between 9253 and 9254 times in the polynucleotide consisting of the sequences of SEQ ID NO: 9;

(72) substitution of the 9309th base for A for C in a polynucleotide having a sequence of SEQ ID NO: 9;

(73) C to T of the 10150th base in a polynucleotide consisting of the sequence of SEQ ID NO: 9.

(74) the mutation c.68-7delT in which the polynucleotide consisting of the sequences of SEQ ID NO: 10 is deleted with the 171th base T;

(75) the mutation c.425 + 13T> C in which the polynucleotide consisting of the sequence of SEQ ID NO: 11 is substituted for C with the 208th base T; And

(76) The mutant c.517-12C> T wherein the 118th base C is substituted with A in the polynucleotide having a sequence of SEQ ID NO: 12.

In the above, the mutations (1) to (73) are represented on the basis of SEQ ID NO: 9 as a mutation present on the coding sequence of the BRCA2 gene. These correspond to deletions, insertions or substitution mutations of nucleotides at the corresponding positions in SEQ ID NO: 9, respectively, resulting in amino acid sequences different from normal BRCA2, which can alter the in vivo function of the BRCA2 protein.

In addition, mutations (74) to (76) above are mutations in introns that are not coding sequences of the BRCA2 gene:

Mutation 74 is a mutation on the intron wherein the nucleotide T present at position 7 (SEQ ID NO: 171) upwards from the nucleotide at position 68 on the coding sequence (SEQ ID NO: 9) is deleted. Since introns are not sites encoding amino acids, there is no change in the amino acid sequence, but may result in changes in BRCA2 expression patterns due to differences in RNA production due to changes in splicing.

Mutation 75 is a mutation on intron wherein the nucleotide T present at position 13 (SEQ ID NO: 208) at position 425 on the coding sequence (SEQ ID NO: 9) is replaced with C. Since introns are not sites encoding amino acids, there is no change in the amino acid sequence, but may result in changes in BRCA2 expression patterns due to differences in RNA production due to changes in splicing.

Mutation 76 is a mutation on the intron wherein nucleotide C present at position 12 (SEQ ID NO: 118) upwards from the nucleotide at position 517 on the coding sequence (SEQ ID NO: 9) is replaced with T. to be. Since introns are not sites encoding amino acids, there is no change in the amino acid sequence, but may result in changes in BRCA1 expression patterns due to differences in RNA production due to changes in splicing.

According to the present invention, a person having such mutations in the BRCA1 and / or BRCA2 genes is more likely to develop breast cancer and / or ovarian cancer than normal people.

Another aspect of the invention provides a primer or probe consisting of a polynucleotide consisting of 20 to 100 contiguous nucleotide sequences comprising a mutation present in such a BRCA1 or BRCA2 gene or a polynucleotide of a sequence complementary thereto. The primer or probe may be used to confirm the predisposition or susceptibility of breast cancer and / or ovarian cancer by confirming the presence or absence of the aforementioned BRCA1 and / or BRCA2 gene mutations. To this end, in addition to techniques for performing F-CSGE described in detail in the following Examples and then confirming through DNA sequencing, various methods may be used as follows:

(A) Do not perform F-CSGE by amplifying the target fragments including the exon and intron boundary regions with a pair of primers that can identify the mutation, respectively, and perform sequencing of the mutations by a dideoxy reaction. You can check the presence. In this case, the presence or absence of a mutation can be directly confirmed without using an oligonucleotide probe composed of a site where the mutation of the present invention occurs and a base sequence including the same. Alternatively, BRCA1 and BRCA2 may be cloned and inserted into a vector, followed by sequencing. In this case, it is possible to confirm the presence or absence of a mutation by comparing the nucleotide sequence of the normal BRCA1, BRCA2 gene and sequencing results.

(B) Presence of gene mutations can also be confirmed by restriction enzyme digestion. In view of the fact that the nucleotide sequences of the mutant and normal genes of the BRCA1 and BRCA2 genes are different from each other, the normal gene is cleaved and the mutant gene is not cleaved using a compound that produces the same result as a suitable restriction enzyme or restriction enzyme. Conversely, the normal gene is not cut, but the mutant gene is cut using the principle. Similar methods include invaders using the principle that an endonuclease enzyme that cuts a primer bound to a mutation site cannot cleave a misalignment site caused by a mutation, and all of them have a presence or absence of a mutation of the present invention. It can be used to confirm.

(C) There is a method of identifying mutation patterns by using primers that bind before and after the base position on the gene where mutation can occur, depending on the base of the mutation position. Reverse dot blot (reverse dot blot) and the like, using a variety of methods using a fluorescent marker, beads, etc. can be used to determine the presence and aspect of the mutation including the mutation of the present invention using the above principle.

(D) Allele-specific PCR performs PCR using primers that bind to base site sites on the genes where mutations can occur, so that a PCR reaction occurs selectively depending on the base of the mutation site. The principle is to identify the mutation pattern. FRET and AlphaScan, etc., all of which can be used to confirm the presence or absence of a mutation of the present invention.

(E) As another method, SSCP (Single Strand Conformation Polymorphism) can be analyzed for the presence of mutations by analyzing the single strand in the fragment of the gene. This can be used for diagnosis using a system capable of producing electrophoresis or similar results, taking note of the fact that the dual structure of the single chain is different depending on the presence or absence of mutation.

(F) As another method, heteroduplex analysis is a method for generating a heterologous double strand and analyzing it using equipment such as a sequencing analyzer (for example, ABI377). Similar methods include Two-Dimensional Gene Scanning (TDGS), D-HPLC, and DGGE, which separate the resulting heterologous double strands into 2-D, which are all used to confirm the presence or absence of a mutation of the present invention. Can be.

(4) As another method, a primer extension assay is to prepare and attach a primer that selectively binds to the 5 'direction of a base position on a gene where mutations can occur when the presence of a mutation is desired. , Using primers to extend the primer to check for the presence of mutations.

(H) As another method, a real time PCR (real time PCR) assay produces a primer that complementarily binds near a base position on a gene where a mutation can occur when the presence of a mutation is desired, and the amount of primer It is a method of identifying a mutation pattern by labeling a fluorescent marker and a quencher at the ends and measuring the amount of the fluorescent marker liberated with the progress of PCR. Taqman et al. And the like, Molecualr beacon. These can all be used to confirm the presence or absence of a mutation of the present invention.

(4) As another method, an oligonucleotide ligation assay (OLA) is used to prepare primers that bind before and after base positions on genes where mutations can occur when the presence of mutations is desired. It is a method of identifying mutation patterns using the principle that only primers complementary to the mutation site are bound by a ligase. OLA, Colorimetric OLA, and OLA-based DNA chips. These can all be used to confirm the presence or absence of a mutation of the present invention.

Polynucleotides of the invention can be used as probes or polynucleotides.

Another aspect of the present invention is for diagnosing breast cancer and / or ovarian cancer comprising a polynucleotide consisting of 20 to 100 contiguous nucleotide sequences comprising a mutation present in a BRCA1 or BRCA2 gene or a polynucleotide of a sequence complementary thereto. Provide a microarray.

In the microarray according to one aspect of the invention, the probe polynucleotide is preferably a polynucleotide consisting of 20 to 80, 20 to 60, or 20 to 50 consecutive nucleotide sequences.

In a microarray according to one aspect of the present invention, the polynucleotide that is a probe may be labeled by radiolabeling, fluorescent labeling, enzyme labeling, sequence tag, or biotin.

In one aspect of the invention, the microarray comprises a polynucleotide comprising a BRCA1 or BRCA2 gene mutation or a polynucleotide having a sequence complementary thereto on a small solid substrate surface made of surface-modified glass, silicone, or nylon. It may be a microchip attached at a high density. By detecting hybridization between the probe on the microarray and the target material in the sample, the presence or absence of mutations in the BRCA1 or BRCA2 genes in the sample can be used to predict the predisposition or susceptibility of the subject's breast and / or ovarian cancer. .

Another aspect of the present invention relates to a kit for diagnosing breast cancer and / or ovarian cancer caused by the BRCA1 and / or BRCA2 gene mutations. The kit comprises one or more container means and carrier means compartmentalized to receive the one or more container means in a confined space, wherein the one or more container means comprises the BRCA1 and / or Or polynucleotides comprising a BRCA2 gene mutation.

Hereinafter, the present invention will be described in more detail with reference to Examples. However, these examples are for illustrative purposes only and the scope of the present invention is not limited to these examples.

Example

Example  One. BRCA1  And / or BRCA2  Detection and Identification of Mutations in Genes

1. Isolation of Genomic DNA

Genomic DNA of breast and ovarian cancer patients was isolated from 200 μl EDTA-peripheral blood using QIAmp DNA Mini Kit (Catalog No. 13323, Quiagen, USA).

2. Amplification of BRCA1 and BRCA2 Genes by Polymerase Chain Reaction (PCR)

The polymerase chain reaction was performed by a multiplex method. This is done by putting the same template in each PCR unit tube and adding a pair of primers in a combination of not one, duplex, triple or quadraplex. Multiple polymerase chain reaction reduces the cost of the entire polymerase chain reaction to one third. BRCA1 and BRCA2 are large genes containing 22 and 26 exons, respectively, as shown in FIGS. 1 and 2. In particular, exon 11 of BRCA1 was so large that the polymerase chain reaction could not be performed by a pair of primers, and amplified using ten pairs of primers. The ten segments of this exon 11 are named 11-1, 11-2, 11-3, 11-4, 11-5, 11-6, 11-7, 11-8, 11-9, and 11-10. It was. Since BRCA2 has the larger size of exon 10, exon 11 and exon 27 out of 26 exons, exon 10 is divided into 10-1, 10-2, 10-3, and 10-4, and exon 11 is 11-1, 11 -2, 11-3, 11-4, 11-5, 11-6, 11-7, 11-8, 11-9, 11-10, 11-11, 11-12, 11-13, 11-14 And 11-15, and exon 27 was divided into 27-1 and 27-2 to perform a polymerase chain reaction. As a result, BRCA1 and BRCA2 were amplified into a total of 75 fragments through 25 multiplex polymerase chain reactions as shown in Table 1. The product of each polymerase chain reaction was designed to include 20 bp forward and reverse primer sequences and at least 40 bp 5'- and 3'-flanknig regions.

Table 1A. Amplification of the BRCA1 Gene

Table 1B. Amplification of the BRCA2 Gene

The composition and reaction conditions of the multiple PCR reaction are as shown in Table 2 and Table 3, respectively.

Table 2. PCR reaction solution

ingredient Volume (μl) 10X Taq-buffer without MgCl ₂ One MgCl ₂ (25 mM) One Primer mix 4 dNTPs (25 mM each) 1.4 Taq polymerase 0.14 DNA (genomic DNA) 1.45 5X Band Doctor One Total volume 10

Table 3. PCR reaction conditions

order Temperature time Cycles One 95 ℃ 10 minutes One 2 95 ℃ 30 seconds  35 57 ℃ 1 minute 72 ℃ 45 sec 3 72 ℃ 7 minutes One 4 95 ℃ 5 minutes One 5 68 ℃ 30 minutes One 6 4 ℃ ∞ -

3.F-CSGE (Fluorescence Conformation Senstitive Gel Electrophoresis)

F-CSGE is a method used to search for genetic variations present on gene sequences. In the presence of mutations in the gene, heteroduplexes can be formed between the mutant allele and the normal allele. As a result, the shape of the tertiary structure of the double DNA (double strand) is different, the mutation can be detected by using the difference in the movement speed during electrophoresis.

The polymerase chain reaction product obtained above was subjected to 0.2 mm thick 12% polyacrylamide gel (acrylamide (BioRad): 1,4-bis (acryloyl) piperazine (BAP, Fluka, Deisenhofen, Germany)) = 99 : 1) were electrophoresed with 1 × TBE buffer in 15% formamide (v / v). Electrophoresis was performed at 40 ° C. and 2000 V for 5 hours, and each lane was loaded with one type of multiple polymerase chain reaction product (components of the loading mixture: 0.5 μl of size standard with TAMRA attached, deionized). 0.5 μl formamide). Data analysis was performed using GeneScan and Genotyper (PE Biosystems) software. As shown in FIG. 3 and FIG. 4, since the analysis results of the genes having mutations and the normal genes without mutations are different from those of F-CSGE, DNAs having different analysis results on F-CSGE are sequenced. The final analysis of the variation was performed.

4. DNA sequencing

The polymerase chain reaction product predicted for the presence of mutations and single nucleotide polymorphisms (SNPs) from the F-CSGE analysis was amplified again using the same oligonucleotide primers as those used for the F-CSGE analysis. ) Using a POP6 gel. As a result, specific mutation patterns on DNA fragments identified by the presence of mutations through F-CSGE were analyzed.

3 shows the results of analyzing exon 22 of the BRCA1 gene. According to the F-CSGE, the shape of the graph was significantly different between the normal gene and the mutant gene, and sequencing confirmed that the 5339th nucleotide T of the BRCA1 gene (SEQ ID NO: 1) was substituted with C. 4 shows the results of analyzing exon 22 of the BRCA2 gene. According to the F-CSGE, the shape of the graph was significantly different between the normal gene and the mutant gene, and sequencing confirmed that the G was inserted between the 8827th and 8828th nucleotides of the BRCA2 gene (SEQ ID NO: 9).

The polynucleotide of the present invention provides a type of Korean-specific mutation present on the nucleotide sequences of the breast cancer and / or ovarian cancer inducing genes BRCA1 and BRCA2, so that the predisposition of breast cancer and ovarian cancer can be diagnosed with low cost and high efficiency. do.

<110> Lab Genomics <120> BRCA1 and BRCA2 Germline Mutations useful for predicting or          detecting Breast and / or Ovarian Cancer <160> 12 <170> KopatentIn 1.71 <210> 1 <211> 5589 <212> DNA <213> Homo sapiens <400> 1 atggatttat ctgctcttcg cgttgaagaa gtacaaaatg tcattaatgc tatgcagaaa 60 atcttagagt gtcccatctg tctggagttg atcaaggaac ctgtctccac aaagtgtgac 120 cacatatttt gcaaattttg catgctgaaa cttctcaacc agaagaaagg gccttcacag 180 tgtcctttat gtaagaatga tataaccaaa aggagcctac aagaaagtac gagatttagt 240 caacttgttg aagagctatt gaaaatcatt tgtgcttttc agcttgacac aggtttggag 300 tatgcaaaca gctataattt tgcaaaaaag gaaaataact ctcctgaaca tctaaaagat 360 gaagtttcta tcatccaaag tatgggctac agaaaccgtg ccaaaagact tctacagagt 420 gaacccgaaa atccttcctt gcaggaaacc agtctcagtg tccaactctc taaccttgga 480 actgtgagaa ctctgaggac aaagcagcgg atacaacctc aaaagacgtc tgtctacatt 540 gaattgggat ctgattcttc tgaagatacc gttaataagg caacttattg cagtgtggga 600 gatcaagaat tgttacaaat cacccctcaa ggaaccaggg atgaaatcag tttggattct 660 gcaaaaaagg ctgcttgtga attttctgag acggatgtaa caaatactga acatcatcaa 720 cccagtaata atgatttgaa caccactgag aagcgtgcag ctgagaggca tccagaaaag 780 tatcagggta gttctgtttc aaacttgcat gtggagccat gtggcacaaa tactcatgcc 840 agctcattac agcatgagaa cagcagttta ttactcacta aagacagaat gaatgtagaa 900 aaggctgaat tctgtaataa aagcaaacag cctggcttag caaggagcca acataacaga 960 tgggctggaa gtaaggaaac atgtaatgat aggcggactc ccagcacaga aaaaaaggta 1020 gatctgaatg ctgatcccct gtgtgagaga aaagaatgga ataagcagaa actgccatgc 1080 tcagagaatc ctagagatac tgaagatgtt ccttggataa cactaaatag cagcattcag 1140 aaagttaatg agtggttttc cagaagtgat gaactgttag gttctgatga ctcacatgat 1200 ggggagtctg aatcaaatgc caaagtagct gatgtattgg acgttctaaa tgaggtagat 1260 gaatattctg gttcttcaga gaaaatagac ttactggcca gtgatcctca tgaggcttta 1320 atatgtaaaa gtgaaagagt tcactccaaa tcagtagaga gtaatattga agacaaaata 1380 tttgggaaaa cctatcggaa gaaggcaagc ctccccaact taagccatgt aactgaaaat 1440 ctaattatag gagcatttgt tactgagcca cagataatac aagagcgtcc cctcacaaat 1500 aaattaaagc gtaaaaggag acctacatca ggccttcatc ctgaggattt tatcaagaaa 1560 gcagatttgg cagttcaaaa gactcctgaa atgataaatc agggaactaa ccaaacggag 1620 cagaatggtc aagtgatgaa tattactaat agtggtcatg agaataaaac aaaaggtgat 1680 tctattcaga atgagaaaaa tcctaaccca atagaatcac tcgaaaaaga atctgctttc 1740 aaaacgaaag ctgaacctat aagcagcagt ataagcaata tggaactcga attaaatatc 1800 cacaattcaa aagcacctaa aaagaatagg ctgaggagga agtcttctac caggcatatt 1860 catgcgcttg aactagtagt cagtagaaat ctaagcccac ctaattgtac tgaattgcaa 1920 attgatagtt gttctagcag tgaagagata aagaaaaaaa agtacaacca aatgccagtc 1980 aggcacagca gaaacctaca actcatggaa ggtaaagaac ctgcaactgg agccaagaag 2040 agtaacaagc caaatgaaca gacaagtaaa agacatgaca gcgatacttt cccagagctg 2100 aagttaacaa atgcacctgg ttcttttact aagtgttcaa ataccagtga acttaaagaa 2160 tttgtcaatc ctagccttcc aagagaagaa aaagaagaga aactagaaac agttaaagtg 2220 tctaataatg ctgaagaccc caaagatctc atgttaagtg gagaaagggt tttgcaaact 2280 gaaagatctg tagagagtag cagtatttca ttggtacctg gtactgatta tggcactcag 2340 gaaagtatct cgttactgga agttagcact ctagggaagg caaaaacaga accaaataaa 2400 tgtgtgagtc agtgtgcagc atttgaaaac cccaagggac taattcatgg ttgttccaaa 2460 gataatagaa atgacacaga aggctttaag tatccattgg gacatgaagt taaccacagt 2520 cgggaaacaa gcatagaaat ggaagaaagt gaacttgatg ctcagtattt gcagaataca 2580 ttcaaggttt caaagcgcca gtcatttgct ccgttttcaa atccaggaaa tgcagaagag 2640 gaatgtgcaa cattctctgc ccactctggg tccttaaaga aacaaagtcc aaaagtcact 2700 tttgaatgtg aacaaaagga agaaaatcaa ggaaagaatg agtctaatat caagcctgta 2760 cagacagtta atatcactgc aggctttcct gtggttggtc agaaagataa gccagttgat 2820 aatgccaaat gtagtatcaa aggaggctct aggttttgtc tatcatctca gttcagaggc 2880 aacgaaactg gactcattac tccaaataaa catggacttt tacaaaaccc atatcgtata 2940 ccaccacttt ttcccatcaa gtcatttgtt aaaactaaat gtaagaaaaa tctgctagag 3000 gaaaactttg aggaacattc aatgtcacct gaaagagaaa tgggaaatga gaacattcca 3060 agtacagtga gcacaattag ccgtaataac attagagaaa atgtttttaa agaagccagc 3120 tcaagcaata ttaatgaagt aggttccagt actaatgaag tgggctccag tattaatgaa 3180 ataggttcca gtgatgaaaa cattcaagca gaactaggta gaaacagagg gccaaaattg 3240 aatgctatgc ttagattagg ggttttgcaa cctgaggtct ataaacaaag tcttcctgga 3300 agtaattgta agcatcctga aataaaaaag caagaatatg aagaagtagt tcagactgtt 3360 aatacagatt tctctccata tctgatttca gataacttag aacagcctat gggaagtagt 3420 catgcatctc aggtttgttc tgagacacct gatgacctgt tagatgatgg tgaaataaag 3480 gaagatacta gttttgctga aaatgacatt aaggaaagtt ctgctgtttt tagcaaaagc 3540 gtccagaaag gagagcttag caggagtcct agccctttca cccatacaca tttggctcag 3600 ggttaccgaa gaggggccaa gaaattagag tcctcagaag agaacttatc tagtgaggat 3660 gaagagcttc cctgcttcca acacttgtta tttggtaaag taaacaatat accttctcag 3720 tctactaggc atagcaccgt tgctaccgag tgtctgtcta agaacacaga ggagaattta 3780 ttatcattga agaatagctt aaatgactgc agtaaccagg taatattggc aaaggcatct 3840 caggaacatc accttagtga ggaaacaaaa tgttctgcta gcttgttttc ttcacagtgc 3900 agtgaattgg aagacttgac tgcaaataca aacacccagg atcctttctt gattggttct 3960 tccaaacaaa tgaggcatca gtctgaaagc cagggagttg gtctgagtga caaggaattg 4020 gtttcagatg atgaagaaag aggaacgggc ttggaagaaa ataatcaaga agagcaaagc 4080 atggattcaa acttaggtga agcagcatct gggtgtgaga gtgaaacaag cgtctctgaa 4140 gactgctcag ggctatcctc tcagagtgac attttaacca ctcagcagag ggataccatg 4200 caacataacc tgataaagct ccagcaggaa atggctgaac tagaagctgt gttagaacag 4260 catgggagcc agccttctaa cagctaccct tccatcataa gtgactcttc tgcccttgag 4320 gacctgcgaa atccagaaca aagcacatca gaaaaagcag tattaacttc acagaaaagt 4380 agtgaatacc ctataagcca gaatccagaa ggcctttctg ctgacaagtt tgaggtgtct 4440 gcagatagtt ctaccagtaa aaataaagaa ccaggagtgg aaaggtcatc cccttctaaa 4500 tgcccatcat tagatgatag gtggtacatg cacagttgct ctgggagtct tcagaataga 4560 aactacccat ctcaagagga gctcattaag gttgttgatg tggaggagca acagctggaa 4620 gagtctgggc cacacgattt gacggaaaca tcttacttgc caaggcaaga tctagaggga 4680 accccttacc tggaatctgg aatcagcctc ttctctgatg accctgaatc tgatccttct 4740 gaagacagag ccccagagtc agctcgtgtt ggcaacatac catcttcaac ctctgcattg 4800 aaagttcccc aattgaaagt tgcagaatct gcccagagtc cagctgctgc tcatactact 4860 gatactgctg ggtataatgc aatggaagaa agtgtgagca gggagaagcc agaattgaca 4920 gcttcaacag aaagggtcaa caaaagaatg tccatggtgg tgtctggcct gaccccagaa 4980 gaatttatgc tcgtgtacaa gtttgccaga aaacaccaca tcactttaac taatctaatt 5040 actgaagaga ctactcatgt tgttatgaaa acagatgctg agtttgtgtg tgaacggaca 5100 ctgaaatatt ttctaggaat tgcgggagga aaatgggtag ttagctattt ctgggtgacc 5160 cagtctatta aagaaagaaa aatgctgaat gagcatgatt ttgaagtcag aggagatgtg 5220 gtcaatggaa gaaaccacca aggtccaaag cgagcaagag aatcccagga cagaaagatc 5280 ttcagggggc tagaaatctg ttgctatggg cccttcacca acatgcccac agatcaactg 5340 gaatggatgg tacagctgtg tggtgcttct gtggtgaagg agctttcatc attcaccctt 5400 ggcacaggtg tccacccaat tgtggttgtg cagccagatg cctggacaga ggacaatggc 5460 ttccatgcaa ttgggcagat gtgtgaggca cctgtggtga cccgagagtg ggtgttggac 5520 agtgtagcac tctaccagtg ccaggagctg gacacctacc tgatacccca gatcccccac 5580 agccactac 5589 <210> 2 <211> 360 <212> DNA <213> Homo sapiens <400> 2 ttattttgtc catggtgtca agtttctctt caggaggaaa agcacagaac tggccaacaa 60 ttgcttgact gttctttacc atactgttta gcaggaaacc agtctcagtg tccaactctc 120 taaccttgga actgtgagaa ctctgaggac aaagcagcgg atacaacctc aaaagacgtc 180 tgtctacatt gaattgggta agggtctcag gttttttaag tatttaataa taattgctgg 240 attccttatc ttatagtttt gccaaaaatc ttggtcataa tttgtatttg tggtaggcag 300 ctttgggaag tgaattttat gagccctatg gtgagttata aaaaatgtaa aagacgcagt 360                                                                          360 <210> 3 <211> 360 <212> DNA <213> Homo sapiens <400> 3 aaatcagtat tctaacctga attatcacta tcagaacaaa gcagtaaagt agatttgttt 60 tctcattcca tttaaagcag tattaacttc acagaaaagt agtgaatacc ctataagcca 120 gaatccagaa ggcctttctg ctgacaagtt tgaggtgtct gcagatagtt ctaccagtaa 180 aaataaagaa ccaggagtgg aaaggtaaga aacatcaatg taaagatgct gtggtatctg 240 acatctttat ttatattgaa ctctgattgt taattttttt caccatactt tctccagttt 300 tttgcataca ggcatttata cacttttatt gctctaggat acttcttttg tttaatccta 360                                                                          360 <210> 4 <211> 360 <212> DNA <213> Homo sapiens <400> 4 tgtcgaactc ctgacctcaa gtgatctgcc tgcctcagtc tcccaaagtg ctaggattac 60 aggggtgagc cactgcgcct ggcctgaatg ccttaaatat gacgtgtctg ctccacttcc 120 attgaaggaa gcttctcttt ctcttatcct gatgggttgt gtttgggttc tttcagcatg 180 attttgaagt cagaggagat gtggtcaatg gaagaaacca ccaaggtcca aagcgagcaa 240 gagaatccca ggacagaaag gtaaagctcc ctccctcaag ttgacaaaaa tctcacccca 300 ccactctgta ttccactccc ctttgcagag atgggccgct tcattttgta agacttatta 360                                                                          360 <210> 5 <211> 360 <212> DNA <213> Homo sapiens <400> 5 aaacataatg ttttcccttg tattttacag atgcaaacag ctataatttt gcaaaaaagg 60 aaaataactc tcctgaacat ctaaaagatg aagtttctat catccaaagt atgggctaca 120 gaaaccgtgc caaaagactt ctacagagtg aacccgaaaa tccttccttg gtaaaaccat 180 ttgttttctt cttcttcttc ttcttctttt cttttttttt tctttttttt ttttgagatg 240 gagtcttgct ctgtggccca ggctagaagc agtcctcctg ccttagcccc cttagtagct 300 gggattacag gcacgcgcca ccatgccagg ctaatttttg tatttttagt agagacgggg 360                                                                          360 <210> 6 <211> 360 <212> DNA <213> Homo sapiens <400> 6 aatttgattt ttaaaaaaaa tcacaggtaa ccttaatgca ttgtcttaac acaacaaaga 60 gcatacatag ggtttctctt ggtttctttg attataattc atacattttt ctctaactgc 120 aaacataatg ttttcccttg tattttacag atgcaaacag ctataatttt gcaaaaaagg 180 aaaataactc tcctgaacat ctaaaagatg aagtttctat catccaaagt atgggctaca 240 gaaaccgtgc caaaagactt ctacagagtg aacccgaaaa tccttccttg gtaaaaccat 300 ttgttttctt cttcttcttc ttcttctttt cttttttttt tctttttttt ttttgagatg 360                                                                          360 <210> 7 <211> 360 <212> DNA <213> Homo sapiens <400> 7 ttggtagttt atgaggttag tttctctaat atagccagtt ggttgatttc cacctccaag 60 gtgtatgaag tatgtatttt tttaatgaca attcagtttt tgagtacctt gttatttttg 120 tatattttca gctgcttgtg aattttctga gacggatgta acaaatactg aacatcatca 180 acccagtaat aatgatttga acaccactga gaagcgtgca gctgagaggc atccagaaaa 240 gtatcagggt agttctgttt caaacttgca tgtggagcca tgtggcacaa atactcatgc 300 cagctcatta cagcatgaga acagcagttt attactcact aaagacagaa tgaatgtaga 360                                                                          360 <210> 8 <211> 360 <212> DNA <213> Homo sapiens <400> 8 gtgtagaacg tgcaggattg ctacataggt aaacatatgc catggtggaa taactagtat 60 tctgagctgt gtgctagagg taactcatga taatggaata tttgatttaa tttcagatgc 120 tcgtgtacaa gtttgccaga aaacaccaca tcactttaac taatctaatt actgaagaga 180 ctactcatgt tgttatgaaa acaggtatac caagaacctt tacagaatac cttgcatctg 240 ctgcataaaa ccacatgagg cgaggcacgg tggcgcatgc ctgtaatcgc agcactttgg 300 gaggccgagg cgggcagatc acgagattag gagatcgaga ccatcctggc cagcatggtg 360                                                                          360 <210> 9 <211> 10254 <212> DNA <213> Homo sapiens <400> 9 atgcctattg gatccaaaga gaggccaaca ttttttgaaa tttttaagac acgctgcaac 60 aaagcagatt taggaccaat aagtcttaat tggtttgaag aactttcttc agaagctcca 120 ccctataatt ctgaacctgc agaagaatct gaacataaaa acaacaatta cgaaccaaac 180 ctatttaaaa ctccacaaag gaaaccatct tataatcagc tggcttcaac tccaataata 240 ttcaaagagc aagggctgac tctgccgctg taccaatctc ctgtaaaaga attagataaa 300 ttcaaattag acttaggaag gaatgttccc aatagtagac ataaaagtct tcgcacagtg 360 aaaactaaaa tggatcaagc agatgatgtt tcctgtccac ttctaaattc ttgtcttagt 420 gaaagtcctg ttgttctaca atgtacacat gtaacaccac aaagagataa gtcagtggta 480 tgtgggagtt tgtttcatac accaaagttt gtgaagggtc gtcagacacc aaaacatatt 540 tctgaaagtc taggagctga ggtggatcct gatatgtctt ggtcaagttc tttagctaca 600 ccacccaccc ttagttctac tgtgctcata gtcagaaatg aagaagcatc tgaaactgta 660 tttcctcatg atactactgc taatgtgaaa agctattttt ccaatcatga tgaaagtctg 720 aagaaaaatg atagatttat cgcttctgtg acagacagtg aaaacacaaa tcaaagagaa 780 gctgcaagtc atggatttgg aaaaacatca gggaattcat ttaaagtaaa tagctgcaaa 840 gaccacattg gaaagtcaat gccaaatgtc ctagaagatg aagtatatga aacagttgta 900 gatacctctg aagaagatag tttttcatta tgtttttcta aatgtagaac aaaaaatcta 960 caaaaagtaa gaactagcaa gactaggaaa aaaattttcc atgaagcaaa cgctgatgaa 1020 tgtgaaaaat ctaaaaacca agtgaaagaa aaatactcat ttgtatctga agtggaacca 1080 aatgatactg atccattaga ttcaaatgta gcacatcaga agccctttga gagtggaagt 1140 gacaaaatct ccaaggaagt tgtaccgtct ttggcctgtg aatggtctca actaaccctt 1200 tcaggtctaa atggagccca gatggagaaa atacccctat tgcatatttc ttcatgtgac 1260 caaaatattt cagaaaaaga cctattagac acagagaaca aaagaaagaa agattttctt 1320 acttcagaga attctttgcc acgtatttct agcctaccaa aatcagagaa gccattaaat 1380 gaggaaacag tggtaaataa gagagatgaa gagcagcatc ttgaatctca tacagactgc 1440 attcttgcag taaagcaggc aatatctgga acttctccag tggcttcttc atttcagggt 1500 atcaaaaagt ctatattcag aataagagaa tcacctaaag agactttcaa tgcaagtttt 1560 tcaggtcata tgactgatcc aaactttaaa aaagaaactg aagcctctga aagtggactg 1620 gaaatacata ctgtttgctc acagaaggag gactccttat gtccaaattt aattgataat 1680 ggaagctggc cagccaccac cacacagaat tctgtagctt tgaagaatgc aggtttaata 1740 tccactttga aaaagaaaac aaataagttt atttatgcta tacatgatga aacattttat 1800 aaaggaaaaa aaataccgaa agaccaaaaa tcagaactaa ttaactgttc agcccagttt 1860 gaagcaaatg cttttgaagc accacttaca tttgcaaatg ctgattcagg tttattgcat 1920 tcttctgtga aaagaagctg ttcacagaat gattctgaag aaccaacttt gtccttaact 1980 agctcttttg ggacaattct gaggaaatgt tctagaaatg aaacatgttc taataataca 2040 gtaatctctc aggatcttga ttataaagaa gcaaaatgta ataaggaaaa actacagtta 2100 tttattaccc cagaagctga ttctctgtca tgcctgcagg aaggacagtg tgaaaatgat 2160 ccaaaaagca aaaaagtttc agatataaaa gaagaggtct tggctgcagc atgtcaccca 2220 gtacaacatt caaaagtgga atacagtgat actgactttc aatcccagaa aagtctttta 2280 tatgatcatg aaaatgccag cactcttatt ttaactccta cttccaagga tgttctgtca 2340 aacctagtca tgatttctag aggcaaagaa tcatacaaaa tgtcagacaa gctcaaaggt 2400 aacaattatg aatctgatgt tgaattaacc aaaaatattc ccatggaaaa gaatcaagat 2460 gtatgtgctt taaatgaaaa ttataaaaac gttgagctgt tgccacctga aaaatacatg 2520 agagtagcat caccttcaag aaaggtacaa ttcaaccaaa acacaaatct aagagtaatc 2580 caaaaaaatc aagaagaaac tacttcaatt tcaaaaataa ctgtcaatcc agactctgaa 2640 gaacttttct cagacaatga gaataatttt gtcttccaag tagctaatga aaggaataat 2700 cttgctttag gaaatactaa ggaacttcat gaaacagact tgacttgtgt aaacgaaccc 2760 attttcaaga actctaccat ggttttatat ggagacacag gtgataaaca agcaacccaa 2820 gtgtcaatta aaaaagattt ggtttatgtt cttgcagagg agaacaaaaa tagtgtaaag 2880 cagcatataa aaatgactct aggtcaagat ttaaaatcgg acatctcctt gaatatagat 2940 aaaataccag aaaaaaataa tgattacatg aacaaatggg caggactctt aggtccaatt 3000 tcaaatcaca gttttggagg tagcttcaga acagcttcaa ataaggaaat caagctctct 3060 gaacataaca ttaagaagag caaaatgttc ttcaaagata ttgaagaaca atatcctact 3120 agtttagctt gtgttgaaat tgtaaatacc ttggcattag ataatcaaaa gaaactgagc 3180 aagcctcagt caattaatac tgtatctgca catttacaga gtagtgtagt tgtttctgat 3240 tgtaaaaata gtcatataac ccctcagatg ttattttcca agcaggattt taattcaaac 3300 cataatttaa cacctagcca aaaggcagaa attacagaac tttctactat attagaagaa 3360 tcaggaagtc agtttgaatt tactcagttt agaaaaccaa gctacatatt gcagaagagt 3420 acatttgaag tgcctgaaaa ccagatgact atcttaaaga ccacttctga ggaatgcaga 3480 gatgctgatc ttcatgtcat aatgaatgcc ccatcgattg gtcaggtaga cagcagcaag 3540 caatttgaag gtacagttga aattaaacgg aagtttgctg gcctgttgaa aaatgactgt 3600 aacaaaagtg cttctggtta tttaacagat gaaaatgaag tggggtttag gggcttttat 3660 tctgctcatg gcacaaaact gaatgtttct actgaagctc tgcaaaaagc tgtgaaactg 3720 tttagtgata ttgagaatat tagtgaggaa acttctgcag aggtacatcc aataagttta 3780 tcttcaagta aatgtcatga ttctgttgtt tcaatgttta agatagaaaa tcataatgat 3840 aaaactgtaa gtgaaaaaaa taataaatgc caactgatat tacaaaataa tattgaaatg 3900 actactggca cttttgttga agaaattact gaaaattaca agagaaatac tgaaaatgaa 3960 gataacaaat atactgctgc cagtagaaat tctcataact tagaatttga tggcagtgat 4020 tcaagtaaaa atgatactgt ttgtattcat aaagatgaaa cggacttgct atttactgat 4080 cagcacaaca tatgtcttaa attatctggc cagtttatga aggagggaaa cactcagatt 4140 aaagaagatt tgtcagattt aacttttttg gaagttgcga aagctcaaga agcatgtcat 4200 ggtaatactt caaataaaga acagttaact gctactaaaa cggagcaaaa tataaaagat 4260 tttgagactt ctgatacatt ttttcagact gcaagtggga aaaatattag tgtcgccaaa 4320 gagtcattta ataaaattgt aaatttcttt gatcagaaac cagaagaatt gcataacttt 4380 tccttaaatt ctgaattaca ttctgacata agaaagaaca aaatggacat tctaagttat 4440 gaggaaacag acatagttaa acacaaaata ctgaaagaaa gtgtcccagt tggtactgga 4500 aatcaactag tgaccttcca gggacaaccc gaacgtgatg aaaagatcaa agaacctact 4560 ctgttgggtt ttcatacagc tagcgggaaa aaagttaaaa ttgcaaagga atctttggac 4620 aaagtgaaaa acctttttga tgaaaaagag caaggtacta gtgaaatcac cagttttagc 4680 catcaatggg caaagaccct aaagtacaga gaggcctgta aagaccttga attagcatgt 4740 gagaccattg agatcacagc tgccccaaag tgtaaagaaa tgcagaattc tctcaataat 4800 gataaaaacc ttgtttctat tgagactgtg gtgccaccta agctcttaag tgataattta 4860 tgtagacaaa ctgaaaatct caaaacatca aaaagtatct ttttgaaagt taaagtacat 4920 gaaaatgtag aaaaagaaac agcaaaaagt cctgcaactt gttacacaaa tcagtcccct 4980 tattcagtca ttgaaaattc agccttagct ttttacacaa gttgtagtag aaaaacttct 5040 gtgagtcaga cttcattact tgaagcaaaa aaatggctta gagaaggaat atttgatggt 5100 caaccagaaa gaataaatac tgcagattat gtaggaaatt atttgtatga aaataattca 5160 aacagtacta tagctgaaaa tgacaaaaat catctctccg aaaaacaaga tacttattta 5220 agtaacagta gcatgtctaa cagctattcc taccattctg atgaggtata taatgattca 5280 ggatatctct caaaaaataa acttgattct ggtattgagc cagtattgaa gaatgttgaa 5340 gatcaaaaaa acactagttt ttccaaagta atatccaatg taaaagatgc aaatgcatac 5400 ccacaaactg taaatgaaga tatttgcgtt gaggaacttg tgactagctc ttcaccctgc 5460 aaaaataaaa atgcagccat taaattgtcc atatctaata gtaataattt tgaggtaggg 5520 ccacctgcat ttaggatagc cagtggtaaa atcgtttgtg tttcacatga aacaattaaa 5580 aaagtgaaag acatatttac agacagtttc agtaaagtaa ttaaggaaaa caacgagaat 5640 aaatcaaaaa tttgccaaac gaaaattatg gcaggttgtt acgaggcatt ggatgattca 5700 gaggatattc ttcataactc tctagataat gatgaatgta gcacgcattc acataaggtt 5760 tttgctgaca ttcagagtga agaaatttta caacataacc aaaatatgtc tggattggag 5820 aaagtttcta aaatatcacc ttgtgatgtt agtttggaaa cttcagatat atgtaaatgt 5880 agtataggga agcttcataa gtcagtctca tctgcaaata cttgtgggat ttttagcaca 5940 gcaagtggaa aatctgtcca ggtatcagat gcttcattac aaaacgcaag acaagtgttt 6000 tctgaaatag aagatagtac caagcaagtc ttttccaaag tattgtttaa aagtaacgaa 6060 cattcagacc agctcacaag agaagaaaat actgctatac gtactccaga acatttaata 6120 tcccaaaaag gcttttcata taatgtggta aattcatctg ctttctctgg atttagtaca 6180 gcaagtggaa agcaagtttc cattttagaa agttccttac acaaagttaa gggagtgtta 6240 gaggaatttg atttaatcag aactgagcat agtcttcact attcacctac gtctagacaa 6300 aatgtatcaa aaatacttcc tcgtgttgat aagagaaacc cagagcactg tgtaaactca 6360 gaaatggaaa aaacctgcag taaagaattt aaattatcaa ataacttaaa tgttgaaggt 6420 ggttcttcag aaaataatca ctctattaaa gtttctccat atctctctca atttcaacaa 6480 gacaaacaac agttggtatt aggaaccaaa gtctcacttg ttgagaacat tcatgttttg 6540 ggaaaagaac aggcttcacc taaaaacgta aaaatggaaa ttggtaaaac tgaaactttt 6600 tctgatgttc ctgtgaaaac aaatatagaa gtttgttcta cttactccaa agattcagaa 6660 aactactttg aaacagaagc agtagaaatt gctaaagctt ttatggaaga tgatgaactg 6720 acagattcta aactgccaag tcatgccaca cattctcttt ttacatgtcc cgaaaatgag 6780 gaaatggttt tgtcaaattc aagaattgga aaaagaagag gagagcccct tatcttagtg 6840 ggagaaccct caatcaaaag aaacttatta aatgaatttg acaggataat agaaaatcaa 6900 gaaaaatcct taaaggcttc aaaaagcact ccagatggca caataaaaga tcgaagattg 6960 tttatgcatc atgtttcttt agagccgatt acctgtgtac cctttcgcac aactaaggaa 7020 cgtcaagaga tacagaatcc aaattttacc gcacctggtc aagaatttct gtctaaatct 7080 catttgtatg aacatctgac tttggaaaaa tcttcaagca atttagcagt ttcaggacat 7140 ccattttatc aagtttctgc tacaagaaat gaaaaaatga gacacttgat tactacaggc 7200 agaccaacca aagtctttgt tccacctttt aaaactaaat cacattttca cagagttgaa 7260 cagtgtgtta ggaatattaa cttggaggaa aacagacaaa agcaaaacat tgatggacat 7320 ggctctgatg atagtaaaaa taagattaat gacaatgaga ttcatcagtt taacaaaaac 7380 aactccaatc aagcagcagc tgtaactttc acaaagtgtg aagaagaacc tttagattta 7440 attacaagtc ttcagaatgc cagagatata caggatatgc gaattaagaa gaaacaaagg 7500 caacgcgtct ttccacagcc aggcagtctg tatcttgcaa aaacatccac tctgcctcga 7560 atctctctga aagcagcagt aggaggccaa gttccctctg cgtgttctca taaacagctg 7620 tatacgtatg gcgtttctaa acattgcata aaaattaaca gcaaaaatgc agagtctttt 7680 cagtttcaca ctgaagatta ttttggtaag gaaagtttat ggactggaaa aggaatacag 7740 ttggctgatg gtggatggct cataccctcc aatgatggaa aggctggaaa agaagaattt 7800 tatagggctc tgtgtgacac tccaggtgtg gatccaaagc ttatttctag aatttgggtt 7860 tataatcact atagatggat catatggaaa ctggcagcta tggaatgtgc ctttcctaag 7920 gaatttgcta atagatgcct aagcccagaa agggtgcttc ttcaactaaa atacagatat 7980 gatacggaaa ttgatagaag cagaagatcg gctataaaaa agataatgga aagggatgac 8040 acagctgcaa aaacacttgt tctctgtgtt tctgacataa tttcattgag cgcaaatata 8100 tctgaaactt ctagcaataa aactagtagt gcagataccc aaaaagtggc cattattgaa 8160 cttacagatg ggtggtatgc tgttaaggcc cagttagatc ctcccctctt agctgtctta 8220 aagaatggca gactgacagt tggtcagaag attattcttc atggagcaga actggtgggc 8280 tctcctgatg cctgtacacc tcttgaagcc ccagaatctc ttatgttaaa gatttctgct 8340 aacagtactc ggcctgctcg ctggtatacc aaacttggat tctttcctga ccctagacct 8400 tttcctctgc ccttatcatc gcttttcagt gatggaggaa atgttggttg tgttgatgta 8460 attattcaaa gagcataccc tatacagtgg atggagaaga catcatctgg attatacata 8520 tttcgcaatg aaagagagga agaaaaggaa gcagcaaaat atgtggaggc ccaacaaaag 8580 agactagaag ccttattcac taaaattcag gaggaatttg aagaacatga agaaaacaca 8640 acaaaaccat atttaccatc acgtgcacta acaagacagc aagttcgtgc tttgcaagat 8700 ggtgcagagc tttatgaagc agtgaagaat gcagcagacc cagcttacct tgagggttat 8760 ttcagtgaag agcagttaag agccttgaat aatcacaggc aaatgttgaa tgataagaaa 8820 caagctcaga tccagttgga aattaggaag gccatggaat ctgctgaaca aaaggaacaa 8880 ggtttatcaa gggatgtcac aaccgtgtgg aagttgcgta ttgtaagcta ttcaaaaaaa 8940 gaaaaagatt cagttatact gagtatttgg cgtccatcat cagatttata ttctctgtta 9000 acagaaggaa agagatacag aatttatcat cttgcaactt caaaatctaa aagtaaatct 9060 gaaagagcta acatacagtt agcagcgaca aaaaaaactc agtatcaaca actaccggtt 9120 tcagatgaaa ttttatttca gatttaccag ccacgggagc cccttcactt cagcaaattt 9180 ttagatccag actttcagcc atcttgttct gaggtggacc taataggatt tgtcgtttct 9240 gttgtgaaaa aaacaggact tgcccctttc gtctatttgt cagacgaatg ttacaattta 9300 ctggcaataa agttttggat agaccttaat gaggacatta ttaagcctca tatgttaatt 9360 gctgcaagca acctccagtg gcgaccagaa tccaaatcag gccttcttac tttatttgct 9420 ggagattttt ctgtgttttc tgctagtcca aaagagggcc actttcaaga gacattcaac 9480 aaaatgaaaa atactgttga gaatattgac atactttgca atgaagcaga aaacaagctt 9540 atgcatatac tgcatgcaaa tgatcccaag tggtccaccc caactaaaga ctgtacttca 9600 gggccgtaca ctgctcaaat cattcctggt acaggaaaca agcttctgat gtcttctcct 9660 aattgtgaga tatattatca aagtccttta tcactttgta tggccaaaag gaagtctgtt 9720 tccacacctg tctcagccca gatgacttca aagtcttgta aaggggagaa agagattgat 9780 gaccaaaaga actgcaaaaa gagaagagcc ttggatttct tgagtagact gcctttacct 9840 ccacctgtta gtcccatttg tacatttgtt tctccggctg cacagaaggc atttcagcca 9900 ccaaggagtt gtggcaccaa atacgaaaca cccataaaga aaaaagaact gaattctcct 9960 cagatgactc catttaaaaa attcaatgaa atttctcttt tggaaagtaa ttcaatagct 10020 gacgaagaac ttgcattgat aaatacccaa gctcttttgt ctggttcaac aggagaaaaa 10080 caatttatat ctgtcagtga atccactagg actgctccca ccagttcaga agattatctc 10140 agactgaaac gacgttgtac tacatctctg atcaaagaac aggagagttc ccaggccagt 10200 acggaagaat gtgagaaaaa taagcaggac acaattacaa ctaaaaaata tatc 10254 <210> 10 <211> 360 <212> DNA <213> Homo sapiens <400> 10 cctttctata gattcgcaag agaatggatt aatgatcttg tttaattaat atgccttaac 60 aaaagtaatc catagtcaag atcttaagca tttttttcct tatgatcttt aactgttctg 120 ggtcacaaat ttgtctgtca ctggttaaaa ctaaggtggg attttttttt taaatagatt 180 taggaccaat aagtcttaat tggtttgaag aactttcttc agaagctcca ccctataatt 240 ctgaacctgc agaagaatct gaacataaaa acaacaatta cgaaccaaac ctatttaaaa 300 ctccacaaag gaaaccatct tataatcagc tggcttcaac tccaataata ttcaaagagc 360                                                                          360 <210> 11 <211> 360 <212> DNA <213> Homo sapiens <400> 11 gactttttgt ttttaaacac ttccaaagaa tgcaaattta taatccagag tatatacatt 60 ctcactgaat tattgtactg tttcaggaag gaatgttccc aatagtagac ataaaagtct 120 tcgcacagtg aaaactaaaa tggatcaagc agatgatgtt tcctgtccac ttctaaattc 180 ttgtcttagt gaaaggtatg atgaagctat tatattaaaa tatttaaatg aaacattttc 240 ctacatatat ttgttctata aagatgaatc tgatttttat gctaatattt tggctaagag 300 cctggtagaa gatcttacat ttttaaataa tcttttaggt tgagtccttt aatagaatag 360                                                                          360 <210> 12 <211> 360 <212> DNA <213> Homo sapiens <400> 12 tgtacctagc attctgcctc atacaggcaa ttcagtaaac gttaagtgaa ataaagagtg 60 aatgaaaaaa taatatcctt aatgatcagg gcatttctat aaaaaataaa ctattttctt 120 tcctcccagg gtcgtcagac accaaaacat atttctgaaa gtctaggagc tgaggtggat 180 cctgatatgt cttggtcaag ttctttagct acaccaccca cccttagttc tactgtgctc 240 ataggtaata atagcaaatg tgtatttaca agaaagagca gatgaggttg ataattgtca 300 tctctaatac ttctgttaaa aggaaatatg aaaagaaaat attagataat gtctttgata 360                                                                          360  

Claims (6)

A polynucleotide consisting of 20-100 contiguous nucleotide sequences comprising a BRCA1 gene mutation selected from the group consisting of: (1) deletion of AT and insertion of GGG in base 38 and 39 in a polynucleotide consisting of the sequence of SEQ ID NO: 1; (2) substitution of C for T of 154 th base in the polynucleotide consisting of the sequence of SEQ ID NO: 1; (3) the substitution of C for A of the 390th base in the polynucleotide consisting of the sequence of SEQ ID NO: 1; (4) substitution of C for T of 527th base in the polynucleotide consisting of the sequence of SEQ ID NO: 1; (5) insertion of A between 720 and 721 th in the polynucleotide consisting of the sequence of SEQ ID NO: 1; (6) substitution of G for A of 811 th base in the polynucleotide consisting of the SEQ ID NO: 1; (7) substitution of the C for T of the 928th base in the polynucleotide consisting of the sequence of SEQ ID NO: 1; (8) deletion of ACT and insertion of GA from bases 1293 to 1295 in the polynucleotide consisting of the sequences of SEQ ID NO: 1; (9) a deletion of 5 bases from the 1516th to 1520th base in the polynucleotide consisting of the SEQ ID NO: 1; (10) substitution of C for T of 1612th base in a polynucleotide consisting of the sequence of SEQ ID NO: 1; (11) a deletion of the 1716th base A in a polynucleotide consisting of the sequence of SEQ ID NO: 1; (12) a deletion of 1823 base A in the polynucleotide consisting of the sequence of SEQ ID NO: 1; (13) a deletion of base 1831 C in a polynucleotide consisting of the sequence of SEQ ID NO: 1; (14) A to G substitution of the 1840 th base in the polynucleotide consisting of the sequence of SEQ ID NO: 1; (15) substitution of C for T of 1865 base in a polynucleotide consisting of the sequence of SEQ ID NO: 1; (16) a deletion of base 1961 base A in a polynucleotide consisting of the sequence of SEQ ID NO: 1; (17) a deletion of the 2048th base A in a polynucleotide consisting of the sequence of SEQ ID NO: 1; (18) a deletion of the 2359th base G in a polynucleotide consisting of the sequence of SEQ ID NO: 1; (19) a deletion of base 2433 base C in a polynucleotide consisting of the sequence of SEQ ID NO: 1; (20) substitution of the A for C of the 2481 base in a polynucleotide consisting of the sequence of SEQ ID NO: 1; (21) substitution of the 2726 base A for T in a polynucleotide consisting of the sequence of SEQ ID NO: 1; (22) a deletion of the 2934th base T in a polynucleotide consisting of the sequence of SEQ ID NO: 1; (23) G to A of 2968 base in the polynucleotide consisting of the sequence of SEQ ID NO: 1; (24) a deletion of the 3296th base C in a polynucleotide consisting of the sequence of SEQ ID NO: 1; (25) a deletion of the 3329th base A in a polynucleotide consisting of the sequence of SEQ ID NO: 1; (26) G to T of 3340rd base in the polynucleotide consisting of the sequence of SEQ ID NO: 1; (27) substitution of the 3409 base for A to G in a polynucleotide consisting of the SEQ ID NO: 1; (28) a deletion of four bases from 3436 to 3439 in the polynucleotide consisting of the sequences of SEQ ID NO: 1; (29) a deletion of the 3442rd base G in a polynucleotide consisting of the sequence of SEQ ID NO: 1; (30) substitution of C for T of 3448 base in a polynucleotide consisting of the sequence of SEQ ID NO: 1; (31) the substitution of G for T of the 3472 th base in the polynucleotide consisting of the sequence of SEQ ID NO: 1; (32) substitution of T for A of 3593 base in the polynucleotide consisting of the sequence of SEQ ID NO: 1; (33) Insertion of A between 3627 and 3628 in the polynucleotide consisting of the sequences of SEQ ID NO: 1; (34) substitution of C for G of base 3722 in a polynucleotide consisting of the sequence of SEQ ID NO: 1; (35) a deletion of 4 bases from 3756 to 3759 in the polynucleotide consisting of the sequence of SEQ ID NO: 1; (36) the insertion of a T between the 3812 and 3813 th polynucleotides consisting of the sequence of SEQ ID NO: 1; (37) substitution of C for T of 3895 base in the polynucleotide consisting of the sequence of SEQ ID NO: 1; (38) a deletion of two bases from 4041 to 4142 in the polynucleotide consisting of the sequences of SEQ ID NO: 1; (39) a deletion of 4 bases from 4065 to 4068 in the polynucleotide consisting of the sequences of SEQ ID NO: 1; (40) the insertion of four base AGAAs between the 4338th and 4339th bases in the polynucleotide consisting of the sequences of SEQ ID NO: 1; (41) substitution of the T for C of the 4729 th base in the polynucleotide consisting of the sequence of SEQ ID NO: 1; (42) substitution of the 4743th base for A for C in a polynucleotide consisting of the sequence of SEQ ID NO: 1; (43) G to T of 4981th base in the polynucleotide consisting of the sequence of SEQ ID NO: 1; (44) a deletion of 4 bases 5030-5033 in the polynucleotide consisting of the sequences of SEQ ID NO: 1; (45) a deletion of two bases from 5102 to 5103 for a polynucleotide consisting of the sequence of SEQ ID NO: 1; (46) a deletion of 2 bases from 5323 to 5324 in the polynucleotide consisting of the sequences of SEQ ID NO: 1; (47) T to C substitution of the 5339th base in a polynucleotide consisting of the sequence of SEQ ID NO: 1; (48) substitution of G for A of 5444 th base in a polynucleotide consisting of the sequence of SEQ ID NO: 1; (49) a deletion of 8 bases from 5470 to 5477 in the polynucleotide consisting of the sequence of SEQ ID NO: 1; (50) deletion of the 5483th base G in a polynucleotide consisting of the sequence of SEQ ID NO: 1; (51) a deletion of 10 bases from 5497 to 5506 in the polynucleotide consisting of the sequence of SEQ ID NO: 1; And (52) an insertion of a G between the 5532 and 5533 th polynucleotides consisting of the sequence of SEQ ID NO: 1; And (53) an insertion of C between 5553 and 5554 th in the polynucleotide consisting of the sequence of SEQ ID NO: 1; (54) a mutant c.547 + 13delG in which the G present at 210 is deleted in the polynucleotide having a SEQ ID NO: 2; (55) the mutation c.4484 + 14A> G wherein the base A present at 218th in the polynucleotide having a sequence of SEQ ID NO: 3 is substituted with G; (56) the mutant c.5194-10G> T wherein the base G present at 167th of the polynucleotide having a sequence of SEQ ID NO: 4 is substituted with T; (57) a mutant c.441 + 1G> A wherein the base G present at 171th in the polynucleotide having a sequence of SEQ ID NO: 5 is replaced with A; (58) the mutant c.302-2A> C wherein the base A present at 149th in the polynucleotide having a sequence of SEQ ID NO: 6 is substituted with C; (59) the mutation c.671-8A> G wherein the base A present at 124th in the polynucleotide having a sequence of SEQ ID NO: 7 is substituted with G; And (60) The mutation c.5074 + 1G> T wherein the base G existing at 205 in the polynucleotide having the sequence shown in SEQ ID NO: 8 is substituted with T. Microarray for detecting a mutation of the BRCA1 gene comprising the polynucleotide of claim 1 or a polynucleotide complementary thereto. The microarray of claim 2, wherein the polynucleotide is labeled by radiolabeling, fluorescent labeling, enzyme labeling, sequence tag, or biotin. A polynucleotide consisting of 20-100 contiguous nucleotide sequences comprising a BRCA2 mutation selected from the group consisting of: (1) G to A substitution of the 53rd base in the polynucleotide consisting of the sequences of SEQ ID NO: 9; (2) G to T substitution of the 97th base in a polynucleotide consisting of the sequence of SEQ ID NO: 9; (3) the substitution of the 125th base for A to G in a polynucleotide consisting of the sequence of SEQ ID NO: 9; (4) the insertion of A between 276 and 277 in the polynucleotide consisting of the sequence of SEQ ID NO: 9; (5) G to A substitution of the 475th base in the polynucleotide consisting of the sequence of SEQ ID NO: 9; (6) substitution of the 623th base for T to G in a polynucleotide consisting of the sequence of SEQ ID NO: 9; (7) a deletion of two bases from the 658 th base to the 659 th base in the polynucleotide having the sequence of SEQ ID NO: 9; (8) substitution of the 943 base for T for A in a polynucleotide consisting of the sequence of SEQ ID NO: 9; (9) substitution of the A-C of the 964th base in a polynucleotide consisting of the sequence of SEQ ID NO: 9; (10) a deletion of the 994th base A in a polynucleotide consisting of the sequence of SEQ ID NO: 9; (11) a deletion of 4 bases from 1310 base to 1313 base in the polynucleotide having the sequence of SEQ ID NO: 9; (12) substitution of the 1399 base for A to T in a polynucleotide consisting of the SEQ ID NO: 9; (13) a deletion of the 1547th base T in a polynucleotide consisting of the sequence of SEQ ID NO: 9; (14) substitution of the 1744th base for A for C in a polynucleotide consisting of the sequence of SEQ ID NO: 9; (15) substitution of G for A of base 1847 in a polynucleotide consisting of the sequence of SEQ ID NO: 9; (16) substitution of the C for T of 1889 bases in a polynucleotide consisting of the sequence of SEQ ID NO: 9; (17) substitution of G for A of 2465th base in a polynucleotide consisting of the sequence of SEQ ID NO: 9; (18) a deletion of 2 bases from 2798 bases to 2799 bases in the polynucleotide consisting of the sequences of SEQ ID NO: 9; (19) a deletion of 4 bases from the 2808 th base to the 2811 th base in the polynucleotide having a sequence of SEQ ID NO: 9; (20) T to G substitution of the 2912nd base in the polynucleotide consisting of the sequence of SEQ ID NO: 9; (21) deletion of 15 bases and insertion of T from base 3096 to 3110 in the polynucleotide consisting of the sequences of SEQ ID NO: 9; (22) a deletion of 4 bases from the 3195 base to the 3198 base in the polynucleotide consisting of the SEQ ID NO: 9; (23) substitution of A for T of 3220rd base in the polynucleotide consisting of the sequence of SEQ ID NO: 9; (24) substitution of T for G of base 3341 in a polynucleotide consisting of the sequence of SEQ ID NO: 9; (25) a deletion of 5 bases from the 3352rd base to the 3356th base in the polynucleotide having the sequence of SEQ ID NO: 9; (26) a deletion of 4 bases from the 3744 base to the 3747 base in the polynucleotide consisting of the sequences of SEQ ID NO: 9; (27) substitution of the 3814 base for A to G in a polynucleotide consisting of the SEQ ID NO: 9; (28) substitution of the 3814th base for A for T in a polynucleotide consisting of the sequence of SEQ ID NO: 9; (29) substitution of the 4376 base A for G in a polynucleotide consisting of the SEQ ID NO: 9; (30) a deletion of 4766 base C in a polynucleotide consisting of the sequence of SEQ ID NO: 9; (31) a deletion of two bases from base 4829 to 4830 in a polynucleotide consisting of the sequence of SEQ ID NO: 9; (32) substitution of T for A of 4854 base in a polynucleotide consisting of the sequence of SEQ ID NO: 9; (33) a deletion of two bases from the 5207 base to the 5208 base in the polynucleotide consisting of the sequences of SEQ ID NO: 9; (34) a deletion of the 5250th base C in a polynucleotide consisting of the sequence of SEQ ID NO: 9; (35) a deletion of 4 bases from 5576 to 5579 bases in the polynucleotide consisting of the sequences of SEQ ID NO: 9; (36) substitution of C for T of 5656th base in a polynucleotide consisting of the sequence of SEQ ID NO: 9; (37) substitution of C for T of 5744th base in a polynucleotide consisting of the sequence of SEQ ID NO: 9; (38) a deletion of 4 bases from base 5946 to 5949th in the polynucleotide consisting of the sequences of SEQ ID NO: 9; (39) substitution of the 5969th base from A to C in a polynucleotide consisting of the SEQ ID NO: 9; (40) substitution of T for G of base 6029 in a polynucleotide consisting of the sequence of SEQ ID NO: 9; (41) substitution of G for T of base 6082 in a polynucleotide consisting of the sequence of SEQ ID NO: 9; (42) substitution of the 6086 base for A to G in a polynucleotide consisting of the SEQ ID NO: 9; (43) G-T substitution of the 6131 th base in the polynucleotide consisting of the sequence of SEQ ID NO: 9; (44) T to G substitution of the 6239rd base in the polynucleotide consisting of the sequence of SEQ ID NO: 9; (45) substitution of G for A of 6325 base in the polynucleotide consisting of the sequence of SEQ ID NO: 9; (46) substitution of the 6448 base A for C in a polynucleotide consisting of the SEQ ID NO: 9; (47) G to T substitution of the 6496th base in a polynucleotide consisting of the sequence of SEQ ID NO: 9; (48) a deletion of 6553 base G in a polynucleotide consisting of the sequence of SEQ ID NO: 9; (49) a deletion of two bases from base 6724 to base 6725 in the polynucleotide consisting of the sequences of SEQ ID NO: 9; (50) T to G substitution of the 6839th base in the polynucleotide consisting of the sequence of SEQ ID NO: 9; (51) substitution of the 6875 base from A to C in a polynucleotide consisting of the sequence of SEQ ID NO: 9; (52) C to G substitution of the 7052th base in a polynucleotide consisting of the sequence of SEQ ID NO: 9; (53) A to G substitution of the 7088th base in a polynucleotide consisting of the sequence of SEQ ID NO: 9; (54) T to G substitution of the 7218th base in the polynucleotide consisting of the sequence of SEQ ID NO: 9; (55) substitution of A for G of base 7411 in a polynucleotide consisting of the sequence of SEQ ID NO: 9; (56) substitution of the T for C of base 7469 in a polynucleotide consisting of the sequence of SEQ ID NO: 9; (57) C to T substitution of the 7480 th base in the polynucleotide consisting of the sequence of SEQ ID NO: 9; (58) substitution of G for A of 7522 base in a polynucleotide consisting of the sequence of SEQ ID NO: 9; (59) substitution of C for A of 7625th base in a polynucleotide consisting of the sequence of SEQ ID NO: 9; (60) substitution of G for A of base 7814 in a polynucleotide consisting of the sequence of SEQ ID NO: 9; (61) substitution of the T for C of the 7876 base for the polynucleotide consisting of the sequence of SEQ ID NO: 9; (62) substitution of T for A of the 8063th base for a polynucleotide consisting of the sequence of SEQ ID NO: 9; (63) insertion of an AC between the 8299th and 8300th bases in a polynucleotide consisting of the SEQ ID NO: 9; (64) a deletion of three bases from the 8437th base to the 8439th base in the polynucleotide having the sequence of SEQ ID NO: 9; (65) a deletion of two bases from the 8717 th base to the 8718 th base in the polynucleotide having the sequence of SEQ ID NO: 9; (66) the insertion of G between the 8827th base and the 8828th base in the polynucleotide consisting of the sequence of SEQ ID NO: 9; (67) T to G substitution of the 8932 th base in the polynucleotide consisting of the sequence of SEQ ID NO: 9; (68) substitution of C for G of the 8951th base in a polynucleotide consisting of the sequence of SEQ ID NO: 9; (69) T to G substitution of the 8991th base in the polynucleotide consisting of the sequence of SEQ ID NO: 9; (70) substitution of C for T of 9076 bases in a polynucleotide consisting of the sequence of SEQ ID NO: 9; (71) an insertion of A between 9253 and 9254 times in the polynucleotide consisting of the sequences of SEQ ID NO: 9; (72) substitution of the 9309th base for A for C in a polynucleotide having a sequence of SEQ ID NO: 9; (73) C to T of the 10150th base in a polynucleotide consisting of the sequence of SEQ ID NO: 9. (74) the mutation c.68-7delT in which the polynucleotide consisting of the sequences of SEQ ID NO: 10 is deleted with the 171th base T; (75) the mutation c.425 + 13T> C in which the polynucleotide consisting of the sequence of SEQ ID NO: 11 is substituted for C with the 208th base T; And (76) The mutant c.517-12C> T wherein the 118th base C is substituted with A in the polynucleotide having a sequence of SEQ ID NO: 12. Microarray for detecting a mutation of the BRCA2 gene comprising the polynucleotide of claim 4 or a polynucleotide complementary thereto. The microarray of claim 5, wherein the polynucleotide is labeled with a radiolabel, a fluorescent label, an enzyme label, a sequence tag, or a biotin.

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