KR101323100B1 - BRCA1 and BRCA2 germline mutations useful for predicting genetic predisposition of breast cancer or ovarian cancer - Google Patents

Abstract

The present invention relates to Korean specific BRCA1 and BRCA2 gene mutations that can be used to determine the predisposition and susceptibility of breast and ovarian cancer.

Description

BRCA1 and BRCA2 germline mutations useful for predicting genetic predisposition of breast cancer or ovarian cancer}

The present invention is derived from a study conducted as part of the industrial source technology development project of the Ministry of Knowledge Economy.

[Project number: 10038662, Assignment name: Development of a low-cost, low-cost mass spectrometer for early diagnosis of breast cancer and genitourinary diseases]

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 BRCA1 and BRCA2 gene mutations that can be used in predicting or diagnosing susceptibility of breast cancer and ovarian cancer, and methods of detecting the same.

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: 6 represent 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 start codon Mark as +1. Mutations within the exons of the BRCA1 and BRCA2 genes are expressed based on SEQ ID NO: 1 and SEQ ID NO: 6, respectively. Insertion mutations are denoted by 'ins', deletion mutations are denoted by 'del', and the letters following 'ins' and 'del' refer to the actual inserted or deleted nucleotides. 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 5 refer to GenBank genome reference sequence NT_010755.15 and SEQ ID NOs 7 to 14 refer to GenBank genome reference sequence NT_024524.13. SEQ ID NOs: 2 to 5 and SEQ ID NOs: 7 to 14 are used to indicate mutations in 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.547 + 14delG' means a mutation in which the nucleotide G located at the 14th position from the 547th 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 the '-' sign and subsequently 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) a polynucleotide consisting of 20-100 contiguous nucleotide sequences comprising a G-to-A substitution of the 28th base (c.28G> A) in the polynucleotide consisting of the sequences of SEQ ID NO: 1;

(2) a polynucleotide consisting of 20-100 contiguous nucleotide sequences comprising the G-to-A substitution of the 824th base in the polynucleotide consisting of the sequence of SEQ ID NO: 1 (c.824G> A);

(3) a polynucleotide consisting of 20-100 contiguous nucleotide sequences comprising a substitution of A to G of the 1067th base (c.1067A> G) in a polynucleotide consisting of the sequence of SEQ ID NO: 1;

(4) a polynucleotide consisting of 20-100 contiguous nucleotide sequences comprising a G-to-A substitution (c.1511G> A) of the 1511th base in the polynucleotide consisting of the sequence of SEQ ID NO: 1;

(5) a polynucleotide consisting of 20-100 contiguous nucleotide sequences comprising a duplication of the 1511th base G (c.1511dupG) in a polynucleotide consisting of the sequence of SEQ ID NO: 1;

(6) a polynucleotide consisting of 20-100 contiguous nucleotide sequences comprising a substitution of G to A of the 2077th base (c.2077G> A) in a polynucleotide consisting of the sequence of SEQ ID NO: 1;

(7) a polynucleotide consisting of 20-100 contiguous nucleotide sequences comprising a duplication of the 2157th base A (c.2157dupA) in a polynucleotide consisting of the sequences of SEQ ID NO: 1;

(8) a polynucleotide consisting of 20-100 contiguous nucleotide sequences comprising a deletion of AG (c.2296_2297delAG) at the 2296 base and the 2297 base in the polynucleotide consisting of the SEQ ID NO: 1;

(9) a polynucleotide consisting of 20-100 contiguous nucleotide sequences comprising a deletion of two bases (c.2856_2857delTT) from bases 2856 to 2857 in the polynucleotide consisting of the sequence of SEQ ID NO: 1;

(10) a polynucleotide consisting of 20-100 contiguous nucleotide sequences comprising the G-to-A substitution of the 2884 th base in the polynucleotide consisting of the sequence of SEQ ID NO: 1 (c.2884G> A);

(11) a polynucleotide consisting of 20-100 contiguous nucleotide sequences comprising the substitution of the A-to-C of the 3213rd base (c.3213A> C) in the polynucleotide consisting of the sequence of SEQ ID NO: 1;

(12) a polynucleotide consisting of 20-100 contiguous nucleotide sequences comprising a C to T substitution (c.3607C> T) of the 3607 base in the polynucleotide consisting of the sequence of SEQ ID NO: 1;

(13) a polynucleotide consisting of 20-100 contiguous nucleotide sequences comprising a duplicate of the 3982 base T (c.3982 dupT) in a polynucleotide consisting of the sequence of SEQ ID NO: 1;

(14) a polynucleotide consisting of 20-100 contiguous nucleotide sequences comprising the C-to-T substitution of the 3991th base (c.3991C> T) in the polynucleotide consisting of the sequence of SEQ ID NO: 1;

(15) a polynucleotide consisting of 20-100 contiguous nucleotide sequences comprising a deletion of two bases (c.4092_4093delTT) from base 4092 to 4093 in a polynucleotide consisting of the sequence of SEQ ID NO: 1;

(16) a polynucleotide consisting of 20-100 contiguous nucleotide sequences comprising a C-to-T substitution of the 4327th base (c.4327C> T) in the polynucleotide consisting of the sequence of SEQ ID NO: 1;

(17) a polynucleotide consisting of 20-100 contiguous nucleotide sequences comprising the substitution of the A to G of the 4459th base (c.4459A> G) in the polynucleotide consisting of the sequence of SEQ ID NO: 1;

(18) a polynucleotide consisting of 20-100 contiguous nucleotide sequences comprising a T-to-C substitution (c.4586T> C) of the 4586th base in the polynucleotide consisting of the sequence of SEQ ID NO: 1;

(19) a polynucleotide consisting of 20-100 contiguous nucleotide sequences comprising a T-to-C substitution (c.4985T> C) of the 4985th base in the polynucleotide consisting of the sequence of SEQ ID NO: 1;

(20) a polynucleotide consisting of 20-100 contiguous nucleotide sequences comprising a G to T substitution (c.5080 G> T) of the 5080th base in a polynucleotide consisting of the sequence of SEQ ID NO: 1;

(21) a polynucleotide consisting of 20-100 consecutive nucleotide sequences comprising a deletion of the 5548 base C (c.5548delC) in a polynucleotide consisting of the sequence of SEQ ID NO: 1;

(22) a polynucleotide consisting of 20-100 contiguous nucleotide sequences comprising a G to C substitution for the 279 th base in the polynucleotide consisting of the sequences of SEQ ID NO: 2;

(23) a polynucleotide consisting of 20-100 contiguous nucleotide sequences comprising a C to T substitution of the 200th base in a polynucleotide consisting of the sequence of SEQ ID NO: 3;

(24) a polynucleotide consisting of 20-100 contiguous nucleotide sequences comprising a deletion of 281 base G in a polynucleotide consisting of the SEQ ID NO: 4; And

(25) A polynucleotide consisting of 20-100 contiguous nucleotide sequences comprising a substitution of G to T of the 245 th base in the polynucleotide consisting of the sequences of SEQ ID NO: 5.

In the above, the mutations (1) to (21) are represented on the basis of SEQ ID NO: 1 as a mutation present on the coding sequence of the BRCA1 gene. These each correspond to deletion, duplication, or substitution mutations in the nucleotide at the corresponding position in SEQ ID NO: 1, resulting in an amino acid sequence that is different from the normal BRCA1, which can alter the in vivo function of the BRCA1 protein.

In addition, mutations (22) to (25) above are mutations at the intron or splicing site that are not the coding sequence of the BRCA1 gene:

Mutant (22) is a splicing site wherein the nucleotide G present at the first position (SEQ ID NO: 279) at position 5152 on the coding sequence (SEQ ID NO: 1) is substituted with C. Mutation (c.5152 + 1G> C). 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 (23) is a mutation on an intron wherein nucleotide C present at position 13 (200 of SEQ ID NO: 3) upwards is replaced by T from the nucleotide at position 81 on the coding sequence (SEQ ID NO: 1) (c.81-13C> 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.

Mutation (24) is a mutation on intron (c.547 +) wherein the nucleotide at position 14 (SEQ ID NO: 281) is deleted from the nucleotide at position 547 on the coding sequence (SEQ ID NO: 1). 14delG). 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 (25) is a mutation of a splicing site in which the nucleotide G immediately following (nucleotide number 245 of SEQ ID NO: 5) on the coding sequence (SEQ ID NO: 1) is substituted with T ( c.4986 + 1G> T). 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.

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:

(26) a polynucleotide consisting of 20-100 consecutive nucleotide sequences comprising the G-T substitution of the 880th base in the polynucleotide consisting of the sequence of SEQ ID NO: 6 (c.880G> T);

(27) a polynucleotide consisting of 20-100 contiguous nucleotide sequences comprising a substitution of A to G of the 1046th base (c.1046A> G) in a polynucleotide consisting of the sequence of SEQ ID NO: 6;

(28) a polynucleotide consisting of 20-100 contiguous nucleotide sequences comprising the substitution of the 1115th base A for T (c.1115A> T) in the polynucleotide consisting of SEQ ID NO: 6;

(29) a polynucleotide consisting of 20-100 contiguous nucleotide sequences comprising a deletion of 5 bases (c.1176_1180delCTGTG) from base 1176 to base 1180 in the polynucleotide consisting of the sequence of SEQ ID NO: 6;

(30) a polynucleotide consisting of 20-100 contiguous nucleotide sequences comprising the substitution of the A to G of the 1568th base (c.1568A> G) in the polynucleotide consisting of the sequence of SEQ ID NO: 6;

(31) a polynucleotide consisting of 20-100 consecutive nucleotide sequences comprising the C-to-T substitution of the 1817 th base in the polynucleotide consisting of the sequence of SEQ ID NO: 6 (c.1871C> T);

(32) a polynucleotide consisting of 20-100 contiguous nucleotide sequences comprising the substitution of the A-to-T of the 2271st base (c.2271A> T) in the polynucleotide consisting of the sequence of SEQ ID NO: 6;

(33) a polynucleotide consisting of 20-100 consecutive nucleotide sequences comprising the C-to-A substitution of the 4347th base (c.4347C> T) in the polynucleotide consisting of the sequence of SEQ ID NO: 6;

(34) a polynucleotide consisting of 20-100 contiguous nucleotide sequences comprising a deletion of 4 bases (c.4478_4481delAAAG) from base 4478 to base 4481 in the polynucleotide having the sequence of SEQ ID NO: 6;

(35) a polynucleotide consisting of 20-100 contiguous nucleotide sequences comprising a deletion of the 4593 base A (c.4593delA) in a polynucleotide consisting of the sequence of SEQ ID NO: 6;

(36) a polynucleotide consisting of 20-100 contiguous nucleotide sequences comprising a substitution of A to G (c.4802A> G) of the 4802 base in a polynucleotide consisting of the sequence of SEQ ID NO: 6;

(37) a polynucleotide consisting of 20-100 contiguous nucleotide sequences comprising a deletion of the 5351st base A (c.5351delA) in a polynucleotide consisting of the sequences of SEQ ID NO: 6;

(38) a polynucleotide consisting of 20-100 contiguous nucleotide sequences comprising a deletion of two bases (c.5592_5593delCA) from the 5592 to 5593 base in the polynucleotide consisting of the sequence of SEQ ID NO: 6;

(39) a polynucleotide consisting of 20-100 contiguous nucleotide sequences comprising the C-to-A substitution of the 5645th base in the polynucleotide consisting of the sequence of SEQ ID NO: 6 (c.5645C> A);

(40) a polynucleotide consisting of 20-100 contiguous nucleotide sequences comprising a deletion of two bases (c.5722_5723 delCT) from base 5722 to 5723 in a polynucleotide consisting of the sequence of SEQ ID NO: 6;

(41) a polynucleotide consisting of 20-100 contiguous nucleotide sequences comprising a C-to-T substitution of the 6100th base (c.6100C> T) in the polynucleotide consisting of the sequence of SEQ ID NO: 6;

(42) a polynucleotide consisting of 20-100 contiguous nucleotide sequences comprising a deletion of the 6688th base A (c.6688delA) in a polynucleotide consisting of the sequence of SEQ ID NO: 6;

(43) a polynucleotide consisting of 20-100 contiguous nucleotide sequences comprising the G-to-A substitution of the 7051st base (c.7051 G> A) in the polynucleotide consisting of the sequence of SEQ ID NO: 6;

(44) a polynucleotide consisting of 20-100 contiguous nucleotide sequences comprising the substitution of the A to T of the 7375th base (c.7375A> T) in the polynucleotide consisting of the sequence of SEQ ID NO: 6;

(45) a polynucleotide consisting of 20-100 contiguous nucleotide sequences comprising the substitution from A to G of the 8023th base (c.8023A> G) in the polynucleotide consisting of the sequence of SEQ ID NO: 6;

(46) a polynucleotide consisting of 20-100 contiguous nucleotide sequences comprising the substitution of the A-to-C of the 8944th base (c.8944A> C) in the polynucleotide consisting of the sequence of SEQ ID NO: 6;

(47) a polynucleotide consisting of 20-100 consecutive nucleotide sequences comprising a duplication of the 9097 base A (c.9097dupA) in a polynucleotide consisting of the sequence of SEQ ID NO: 6;

(48) a polynucleotide consisting of 20-100 contiguous nucleotide sequences comprising a deletion of the 9431 base C (c.9431delC) in a polynucleotide consisting of the sequence of SEQ ID NO: 6;

(49) a polynucleotide consisting of 20-100 contiguous nucleotide sequences comprising a deletion of two bases (c.9435_9436delGT) from the 9435 base to the 9436 base in the polynucleotide consisting of the sequence of SEQ ID NO: 6;

(50) a polynucleotide consisting of 20-100 contiguous nucleotide sequences comprising a substitution of the A-to-C of the 10131th base (c.10131A> C) in the polynucleotide consisting of the sequence of SEQ ID NO: 6;

(51) a polynucleotide consisting of 20-100 contiguous nucleotide sequences comprising a substitution of G for A of the 258th base in the polynucleotide consisting of the sequence of SEQ ID NO: 7;

(52) a polynucleotide consisting of 20-100 contiguous nucleotide sequences comprising a substitution of G for A of a 96th base in a polynucleotide having a sequence of SEQ ID NO: 8;

(53) a polynucleotide consisting of 20-100 contiguous nucleotide sequences comprising a substitution of G for A of the 294 th base in the polynucleotide consisting of the sequence of SEQ ID NO: 9;

(54) a polynucleotide consisting of 20-100 contiguous nucleotide sequences comprising a substitution of the A to G of the 167th base in the polynucleotide consisting of the sequences of SEQ ID NO: 10;

(55) a polynucleotide consisting of 20-100 consecutive nucleotide sequences comprising the substitution of the A to G of the 145th base in the polynucleotide consisting of the sequence of SEQ ID NO: 11;

(56) a polynucleotide consisting of 20-100 contiguous nucleotide sequences comprising a substitution of C for T for the 124th base in the polynucleotide consisting of the sequence of SEQ ID NO: 12;

(57) a polynucleotide consisting of 20-100 contiguous nucleotide sequences comprising a T-to-C substitution of the 270th base in the polynucleotide consisting of the SEQ ID NO: 13; And

(58) A polynucleotide consisting of 20-100 contiguous nucleotide sequences comprising a substitution of G for A of the 124th base in the polynucleotide having the sequence of SEQ ID NO: 14.

In the above, mutations (26) to (50) are represented on the basis of SEQ ID NO: 6 as mutations 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: 6, respectively, resulting in amino acid sequences different from normal BRCA2, which may alter the in vivo function of the BRCA2 protein.

In addition, mutations (51) to (58) above are mutations in introns that are not coding sequences of the BRCA2 gene:

Mutation (51) is a mutation on intron (c) wherein nucleotide G present at position 24 (SEQ ID NO: 258) is replaced with A from the nucleotide at position 7976 of the coding sequence (SEQ ID NO: 6). .7976 + 24G> A). 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 52 is a splicing in which nucleotide T present at position 1 (SEQ ID NO: 96) upwards from the nucleotide at position 8488 on the coding sequence (SEQ ID NO: 6) is replaced with C Mutation of the site (c.8488-1G> A). Since the splicing site is not an amino acid coding site, there is no change in the amino acid sequence, but a change in the expression of BRCA2 may occur due to a difference in RNA production due to a change in splicing.

Mutation 53 is a mutation of a splicing site (c.9117G> A) in which nucleotide G present at position 9117 (SEQ ID NO: 294) on the coding sequence (SEQ ID NO: 6) is substituted with A. to be. Since the splicing site is not an amino acid coding site, there is no change in the amino acid sequence, but a change in the expression of BRCA2 may occur due to a difference in RNA production due to a change in splicing.

Mutation 54 is a mutation on intron wherein nucleotide A present at position 10 (No. 167 of SEQ ID NO: 10) is upwardly replaced by C from the nucleotide at position 317 on the coding sequence (SEQ ID NO: 6). (c.317-10A> G). 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 55 is a mutation on intron wherein nucleotide A present at position 26 (number 145 of SEQ ID NO: 11) is replaced by G upwards from the nucleotide at position 8633 on the coding sequence (SEQ ID NO: 6). (c.8633-26A> G). 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 56 is 5'- wherein nucleotide C present at position 37 (number 124 of SEQ ID NO: 12) is upwardly replaced by nucleotide present at position 1 on the coding sequence (SEQ ID NO: 6). Mutation on UTR (c.1-37C> T). Since 5′-UTR is not an amino acid coding region, there is no change in the amino acid sequence, but it may cause a change in BRCA2 expression pattern due to a difference in RNA production due to a change in splicing.

Mutation 57 is a splicing site wherein the nucleotide T present at the second position (SEQ ID NO: 270) at position 67 on the coding sequence (SEQ ID NO: 6) is substituted with C. Mutation (c.67 + 2T> C). Since the splicing site is not an amino acid coding site, there is no change in the amino acid sequence, but a change in the expression of BRCA2 may occur due to a difference in RNA production due to a change in splicing.

Mutation 58 is a splicing site wherein the nucleotide G present at the first position (No. 124 of SEQ ID NO: 14) is replaced with A from the nucleotide at position 516 on the coding sequence (SEQ ID NO: 6). Mutation (c.516 + 1G> A). Since the splicing site is not an amino acid coding site, there is no change in the amino acid sequence, but a change in the expression of BRCA2 may occur due to a difference in RNA production due to a change 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 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.

According to one embodiment of the invention, a probe set comprising two or more combinations selected from polynucleotides (1) to (25) comprising mutations on the BRCA1 gene to identify predisposition or susceptibility to breast cancer and / or ovarian cancer Can be used.

According to one embodiment of the invention, a probe set consisting of polynucleotides (1) to (25) comprising mutations on the BRCA1 gene can be used to confirm predisposition or susceptibility to breast cancer and / or ovarian cancer.

According to one embodiment of the invention, a probe set comprising two or more combinations selected from polynucleotides (26) to (58) comprising mutations on the BRCA2 gene to identify predisposition or susceptibility to breast cancer and / or ovarian cancer Can be used.

According to one embodiment of the invention, a probe set consisting of polynucleotides (26) to (58) comprising mutations on the BRCA2 gene can be used to confirm predisposition or susceptibility to breast cancer and / or ovarian cancer.

According to one embodiment of the invention, two or more combinations selected from polynucleotides (1) to (58) comprising mutations on the BRCA1 and / or BRCA2 genes to identify predisposition or susceptibility to breast cancer and / or ovarian cancer Including probe sets can be used.

According to one embodiment of the invention, a probe set consisting of polynucleotides (1) to (58) comprising mutations on the BRCA1 and / or BRCA2 genes is used to confirm predisposition or susceptibility to breast cancer and / or ovarian cancer. Can be.

To this end, in addition to the technique of performing DNA sequencing after performing F-CSGE described in detail in the following examples, various methods may be used as follows:

(A) Do not perform F-CSCE by amplifying the target fragments containing the exon and intron boundary portions with a pair of primers each capable of confirming the mutation, and performing sequencing of the mutation through a dideoxy reaction. You can check the presence. In this case, the presence or absence of a mutation can be immediately determined without using an oligonucleotide probe composed of a site where the mutation of the present invention and a base sequence including the same occur. 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 is a principle in which a PCR reaction occurs selectively according to a base of a mutation site by performing PCR using a primer that binds to a base site site on a gene where a mutation can occur. It is a method to identify the mutation pattern using. Fluorescence Resonance Energy Transfer (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 to 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 that are exactly 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.

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 contiguous 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.

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.

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.
Figure 3 shows an F-CSCE analysis graph of a mutation (c.1511dupG) with G inserted at the 1511th nucleotide (located at exon 11) of the BRCA1 gene (SEQ ID NO: 1), one of the BRCA1 gene mutations.
FIG. 4 is a graph of F-CSCE analysis of a mutation (c.9117G> A) in which G is substituted for A in the 9117th nucleotide (located at exon 23) of the BRCA2 gene (SEQ ID NO: 6), which is one of the BRCA2 gene mutations. Illustrated.

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 G-DEX kit (Catalog No. PK1104, Intron, KOREA). Genomic DNA was isolated from the normal 50 peripheral blood as a control in the same manner as the patient group.

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 24 and 27 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 24 multiplex polymerase chain reactions as shown in Tables 1a and 1b. 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.

ingredient Volume ([mu] l) 10X Taq-buffer without MgCl ₂ One MgCl ₂ (25 mM) 0.6 Primer mix 1 (0.5 forward + 0.5 reverse) dNTPs (10 mM each) 0.125 Taq polymerase 0.1 DNA (genomic DNA) One 5X Band Doctor 0.4 Distilled water 5.78 Total volume 10

order Temperature time Number of cycles One 95 ℃ 5 minutes One 2

95 ℃ 30 seconds
35
57 ℃ 40 seconds 72 ℃ 45 seconds 3 72 ℃ 7 minutes One 4 4 ℃ ∞ -

3.F-CSCE (Fluorescence Conformation Senstitive Capillary Electrophoresis)

F-CSCE 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, mutations can be detected by using the fact that the tertiary structure of the DNA double strand is different and a difference in the moving speed occurs during capillary electrophoresis.

The polymerase chain reaction product obtained above was diluted at a ratio of 1/5 to 1/10. Each well of a 96 well plate was loaded with one kind of multiple polymerase chain reaction product (components of the loading mix: 0.5 μl of diluted product, 0.5 μl of GeneScan-ROX 500 DNA size standard, 8.5 μl of distilled water). Analysis was performed using a conformation analysis polymer in an ABI 3130 sequencer. Data was analyzed using the Genemapper 4.0 program. As shown in FIG. 3 and FIG. 4, the genes with mutations and the normal genes without the mutations (control) show different results from the analysis results through F-CSCE. DNA was finally analyzed for mutants through sequencing.

4. DNA sequencing

The polymerase chain reaction product predicted for the presence of mutations and single nucleotide polymorphisms (SNPs) from the F-CSCE analysis was amplified again using the same oligonucleotide primers as those used for the F-CSCE analysis. ) Using a POP7 polymer. As a result, specific mutation patterns on DNA fragments identified by the presence of mutations through F-CSCE were analyzed.

In order to confirm that the mutations on the BRCA1 and BRCA2 genes identified in the patient group were pathogenic mutations, 50 normal subjects were examined for the presence of the same mutation. Unlike the patient group, no mutations determined to be pathogenic mutations were detected in normal subjects.

Attach an electronic file to a sequence list

Claims (6)

A set of polynucleotides for the diagnosis of breast or ovarian cancer, comprising a polynucleotide comprising the following BRCA1 gene mutation:
(7) a polynucleotide consisting of 20-100 consecutive nucleotide sequences comprising a duplication of the 2157th base A in a polynucleotide consisting of the sequences of SEQ ID NO: 1;
(9) a polynucleotide consisting of 20-100 contiguous nucleotide sequences comprising a deletion of two bases from the 2856nd base to the 2857th base in the polynucleotide consisting of the sequence of SEQ ID NO: 1;
(13) a polynucleotide consisting of 20-100 contiguous nucleotide sequences comprising a duplicate of the 3982th base T in a polynucleotide consisting of the sequences of SEQ ID NO: 1;
(14) a polynucleotide consisting of 20-100 contiguous nucleotide sequences comprising a C to T substitution of the 3991th base in a polynucleotide consisting of the sequence of SEQ ID NO: 1;
(15) a polynucleotide consisting of 20-100 contiguous nucleotide sequences comprising a deletion of two bases from the 4092 base to the 4093 base in the polynucleotide consisting of the sequence of SEQ ID NO: 1;
(21) a polynucleotide consisting of 20-100 contiguous nucleotide sequences comprising a deletion of 5548 base C in a polynucleotide consisting of the sequences of SEQ ID NO: 1; And
(25) A polynucleotide consisting of 20-100 contiguous nucleotide sequences comprising a substitution of G to T of the 245 th base in the polynucleotide consisting of the sequences of SEQ ID NO: 5. A microarray for detecting a BRCA1 gene mutation comprising a polynucleotide set of claim 1 or a polynucleotide set complementary thereto. The microarray of claim 2, wherein the polynucleotide set is labeled with a radiolabel, fluorescent label, enzyme label, sequence tag, or biotin. delete delete delete

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