Breast Cancer Susceptibility Gene (BRCA2) Report

Malignancies affecting humans have a tendency to bear association with genes.

This link is considered responsible for a variety of health consequences which might become undetected and unmanageable. Evolutionary adaptations have favored the success of many genes in contributing to the both positive and negative health outcomes. The mechanisms underlying the genetic predisposition to a particular disease are manifold and this concept is the challenging one to the investigators since the advent of Molecular Biology and database resources. This includes gene localization or identification on a specific chromosome loci and designing a primer to amplify that particular gene in a target organism tissue sample. The present description is concerned with, BRCA2 gene known as Breast Cancer susceptibility gene. This gene is the most studied one in the area of Cancer Research. It was identified through genomic linkage studies and described to be localized on chromosome 13q12-13 (1). BRCA2 gene codes for the BRCA2 protein and represents tumor suppressor gene family.BRCA2 protein involves in correcting the chromosomal damage and especially produces a DNA double strand without any breaks thus ensuring an error free repair (2). However, the role of BRCA2 in the transcriptional regulation has few research limitations. Reports describe that the product of region BRCA2 exon 3 that has approximately 23–105 residues triggers transcription whereas a missense mutation (Tyr42Cys) of BRCA2 minimizes the transactivation potential(3). This concept of mutation and its relationship to cancer biology need additional fundamental insights.

BRCA2 overexpression is connected with basal p53 transcriptional activity leading to its down-regulation (3). On the other hand BRCA2 contributes to the modulation of histone acetylation thus reflecting its active role in transcription (3). Thus,the interaction between BRCA2 interacts and transcriptional co-activator protein P/CAF (p300/CBP-associated factor) and p300/CBP, is considered significant as the two proteins function for histone acetylase activity(3). BRCA 2 gene has several associations with other proteins which strengthen its involvement in the Breast Cancer. EMSY, a novel protein was known to bind to the exon 3 region of BRCA2 (4). This protein was shown to co-localize with H2AX and BRCA2 in various experiments thus supporting its role in the DNA damage response. EMSY is a considered as transcriptional repressor as it suppresses the promoter activity. It also involves in chromatin remodeling and DNA repair. EMSY over expression is connected to high-grade sporadic ovarian carcinomas thus supporting the involvement of the BRCA2 pathway in sporadic breast and ovarian cancers (4). But, there is a need of additional mechanistic information on the association between BRCA2-EMSY which could enable to understand the altered pathways in breast cancer (4). The role of BRCA2 in cell cycle regulation or checkpoint functions although known requires further research support (4). It was described that BRCA2 facilitates G2/M-phase control by associating with BRAF35 a novel protein, BRCA2-associated factor 35 which connects with branched DNA structures (5).

This BRAF35/BRCA2 complex association with condensed chromatin coincides phosphorylation of Histone H3 region as revealed from the nuclear staining experiments (5). The main role of BRCA2 in DNA repair makes its suppressing activity more robust for uncorrected DNA lesions, which contribute to cell cycle arrest by triggering mitotic progression and checkpoint signaling (5). BRCA2 functional impairment results in genetic instability which could activate mutations in checkpoint genes like p53(5).

This defect reflecting the mutation would cause unregulated cell cycle and failure of check points thus enabling invasive growth from uncontrolled proliferation.

Thus, BRCA2 gene has pivotal role to play in cancer research. It has multiple functions with special emphasis on DNA repair, transcriptional regulation, and control of cell cycle (5). There is a need to review the information on the BRCA2 gene based on databases. Firstly, chromosome map position of the gene was accessed by taking the assistance of Ensembl, NCBI website. It was revealed that BRCA 2 gene location as given in the Ensembl website is Chromosome 13: 32,889,611-32,973,805 indicating that the BRCA gene is localized on chromosome 13q12-13(6). This information was obtained by selecting ‘Humans’ in the search bar and entering the BRCA 2 gene as key word in the empty space provided adjacent to the ‘Go’ in the website (6). This would take to the page of ‘Results Summary’ after a click on “Homo sapiens 833” followed by “Gene (4)”. Next page gives the heading “4 Genes match your query (‘BRCA2’) in Homo sapiens”.

BRCA2 was selected and ‘location’ which would give the new page showing the chromosome location and regions in detail (6). BRCA2 gene is also known as BRCC2, FACD, FAD, FAD1, FANCD, FANCD1, GLM3, BROVCA (7, 8). This proceeded from the google search where ‘BRCA2’ was typed as key word and the websites were visited.

This description was arrived to by initially visiting the website of Genetics Home Reference (GHR) and typing BRCA2 in the search column (7). A new page on BRCA2 provided the complete description. This information was also obtained from the related website of NCBI (8). Few other genes located on this chromosome near by BRCA2 are ATP7B, B3GALTL, GJB2, GJB6, RBI SACS etc (7).

Similarly this was found from the same website and clicking on chromosomes, chromosome 13, what is chromosome 13 and genes on chromosome 13 (7).

The above mentioned genes are then copy pasted into the ENSEMBL website, clicking on BRCA2 which is already given as example by default in the search column (6).

On the results summary page, “By species” column was selected and in the space bar adjacent to “Gene”, the above mentioned genes were copy pasted one after the other. The new page that opened has provided the complete information on the location of these genes on chromosome “13” map (6). This is represented in the appendix 1. BRCA2 gene has a total length of 3,283,984,159 bps. It has known protein coding genes of 318, novel protein coding genes of 20469, pseudogenes 14266, RNA Genes 12499, Gene exons 640,18545, gene transcripts 178,191, Immunoglobulin gene segments 562 etc (6).This information was obtained from the Ensembl website and clicking on the whole genome option on the left side (6).

Next, it was essential to obtain information on the exon / intron structure of the BRCA2 gene. BRCA 2 gene has 27 exons and 26 introns. This was obtained from the website of Leiden Open Variation Database (LOVD) home page and clicking on exon/ intron information table (8).

This was further confirmed in detail from the Ensembl database by initially following the method applied earlier for locating the chromosomal region of the BRCA2 gene. Then the link “Gene:BRCA2” present at the upper side of the page was selected which had given a tabular column consisting of transcripts of the gene with their IDs.

“Transcript ID BRCA2 001 -ENST00000380152” was selected and then “Exons 27” option present under the “Sequence” heading of the “Transcript based displays column” present at the left hand side of the page was selected. This revealed the exon sequence information. For intron sequence information, “configure this page” was selected.

The exon / intron structure of the gene was given in the appendix 2.The method followed here was similar to the method described previously for locating the chromosome region in detail.

Next, BRCA 2 gene mutations are important to consider, as the role of this gene is involved in the susceptibility of other conditions like Fanconi Anemia, Pancreatic Cancer, Wilms tumor included Glioma susceptibility, included Medullablastoma, included pre-B-cell acute lymphoblastic leukemia etc. This is represented in the tabular column of appendix 3.

clicking on “Gene Gateway”, then “Gene and Protein Database Guide”, then on “Gene-Mutation Resources” (9). Next, Online Mendelian Inheritance in Man (OMIM): Allelic Variants was selected and OMIM was selected BRCA2 gene was typed as key word in the column provided and the first option “600185. BRCA2 GENE; BRCA2” was clicked (9). This resulted in a new page with the same title and the text was accessed, scrolled down to select the sub heading Allelic variants and then Table view was clicked (9). The mutations are randomly distributed throughout the BRCA2 gene. Single nucleotide polymorphisms (SNPs) are present in the BRCA2 gene. Strong connection between polymorphisms of the BRCA2 gene and clinical characteristics in breast cancer exists. Investigation on this particular area is very active since many years.

There are 2650 SNP’s in the exons and introns of this gene where the exons are randomly distributed. This information was obtained by visiting the website, typing the key word BRCA2 and then clicking BRCA2 in the results page. This resulted in the detailed description of the gene. The page was scrolled down to look for the tabular column with heading “2650 NCBI SNPs in BRCA2 are shown”.

Finally, PCR primers were generated for the gene using a primer design program (10). This was obtained by following the method applied earlier for determining the exon and intron sequence of the BRCA2 gene. Then on the left hand side of the page, “download view as rtf” was clicked which resulte I nthe full download of the exon/ intron sequence in a MS-Word document. This was saved and used for designing the primers. Two different primer sequences were copy pasted into the sequence input area of the primer 3 website. The primer sequence thus obtained is represented in the appendices 4 and 5. Two primers were generated namely abc and abc2, using the software.

In view of the above, it can be inferred that Breast cancer genetics is the complicated area. Women with increased susceptibility to Breast cancer may either carry a mutation in BRCA2 gene or possess family history of Breast cancer (11). As reported previously, women acquire Breast cancer at a rate between 1% and 2% annually and among gene carriers the risk may be approximately 80% in life time period(11). Magenetic Resonance Imaging (MRI) scan trials are considered to have great sensitivity over conventional mammography although further confirmation is required with regard to their lessening effect breast cancer mortality (11).

Thus Breast cancer is the malignant disorder targeting women with a high tumor inducing potential. Although BRCA 2 Gene has functional role in repair process mutations and / or SNPs have contributed to the success of Breast cancer from various corners. In association with other genes, BRCA2 gene is co-inheriting and producing the adverse health outcome. There is a need to understand the in depth complexity of BRCA2 genetics through the available online databases. The evolving role of this gene should be understood and further studies need to be carried out at the earliest in an evidence based practice approach.

References

Wooster R, Neuhausen SL, Mangion J, Quirk Y, Ford D, Collins N, Nguyen K, Seal S, Tran T, Averill D, et al. Localization of a breast cancer susceptibility gene, BRCA2, to chromosome 13q12-13.Science 1994 ;265(5181):2088-90.

Duncan JA, Reeves JR, Cooke TG (1998). “BRCA1 and BRCA2 proteins: roles in health and disease”. Molecular pathology.1998; 51 (5): 237–47.

Role of BRCA1 and BRCA2 as regulators of DNA repair, transcription, and cell cycle in response to DNA damage. Cancer Sci 2004;95(1):866-871.

Hughes-Davies L, Huntsman D, Ruas M, Fuks F, Bye J, Chin SF, Milner J,Brown LA, Hsu F, Gilks B, Nielsen T, Schulzer M, Chia S, Ragaz J, Cahn A,Linger L, Ozdag H, Cattaneo E, Jordanova ES, Schuuring E, Yu DS, Venkitaraman A, Ponder B, Doherty A, Aparicio S, Bentley D, Theillet C, Ponting CP, Caldas C, Kouzarides T. EMSY links the BRCA2 pathway to sporadic breast and ovarian cancer. Cell 2003; 115: 523–3.

Marmorstein LY, Kinev AV, Chan GK, Bochar DA, Beniya H, Epstein JA,Yen TJ, Shiekhattar R. A human BRCA2 complex containing a structural DNA binding component influences cell cycle progression. Cell 2001; 104:247–5.

BRCA2. 2011. Web.

Genetics Home Reference: Genes. BRCA2. 2011. Web.

Leiden Open Variation Database: Breast Cancer 2 – literature unclassified variants (BRCA2). 2011. Web.

BRCA2. 2011. Web.

Primer 3. 2011. Web.

Narod SA. Screening of women at high risk for breast cancer. Prev Med 2011 Sep 1;53(3):127-30.

Appendices

Appendix-3

Mutations in the BRCA2 gene.

Appendix 4

Primer3 Output

Primer abc

PRIMER PICKING RESULTS FOR abc

No mispriming library specified

No hyb oligo mishyb library specified

Using 1-based sequence positions

OLIGO start len tm gc% any 3′ seq

LEFT PRIMER 212 20 59.87 45.00 3.00 0.00 AGCAAACGCTGATGAATGTG

RIGHT PRIMER 393 20 59.97 50.00 4.00 1.00 ACCATTCACAGGCCAAAGAC

HYB OLIGO 324 20 59.98 55.00 4.00 3.00 CAGAAGCCCTTTGAGAGTGG

SEQUENCE SIZE: 1116

INCLUDED REGION SIZE: 1116

PRODUCT SIZE: 182, PAIR ANY COMPL: 7.00, PAIR 3′ COMPL: 1.00

1 GATTTGGAAAAACATCAGGGAATTCATTTAAAGTAAATAGCTGCAAAGACCACATTGGAA

61 AGTCAATGCCAAATGTCCTAGAAGATGAAGTATATGAAACAGTTGTAGATACCTCTGAAG

121 AAGATAGTTTTTCATTATGTTTTTCTAAATGTAGAACAAAAAATCTACAAAAAGTAAGAA

181 CTAGCAAGACTAGGAAAAAAATTTTCCATGAAGCAAACGCTGATGAATGTGAAAAATCTA

241 AAAACCAAGTGAAAGAAAAATACTCATTTGTATCTGAAGTGGAACCAAATGATACTGATC

301 CATTAGATTCAAATGTAGCAAATCAGAAGCCCTTTGAGAGTGGAAGTGACAAAATCTCCA

361 AGGAAGTTGTACCGTCTTTGGCCTGTGAATGGTCTCAACTAACCCTTTCAGGTCTAAATG

421 GAGCCCAGATGGAGAAAATACCCCTATTGCATATTTCTTCATGTGACCAAAATATTTCAG 481 AAAAAGACCTATTAGACACAGAGAACAAAAGAAAGAAAGATTTTCTTACTTCAGAGAATT 541 CTTTGCCACGTATTTCTAGCCTACCAAAATCAGAGAAGCCATTAAATGAGGAAACAGTGG 601 TAAATAAGAGAGATGAAGAGCAGCATCTTGAATCTCATACAGACTGCATTCTTGCAGTAA 661 AGCAGGCAATATCTGGAACTTCTCCAGTGGCTTCTTCATTTCAGGGTATCAAAAAGTCTA 721 TATTCAGAATAAGAGAATCACCTAAAGAGACTTTCAATGCAAGTTTTTCAGGTCATATGA 781 CTGATCCAAACTTTAAAAAAGAAACTGAAGCCTCTGAAAGTGGACTGGAAATACATACTG 841 TTTGCTCACAGAAGGAGGACTCCTTATGTCCAAATTTAATTGATAATGGAAGCTGGCCAG 901 CCACCACCACACAGAATTCTGTAGCTTTGAAGAATGCAGGTTTAATATCCACTTTGAAAA 961 AGAAAACAAATAAGTTTATTTATGCTATACATGATGAAACATCTTATAAAGGAAAAAAAA 1021 TACCGAAAGACCAAAAATCAGAACTAATTAACTGTTCAGCCCAGTTTGAAGCAAATGCTT 1081 TTGAAGCACCACTTACATTTGCAAATGCTGATTCAG KEYS (in order of precedence): left primer

right primer

hyb oligo

ADDITIONAL OLIGOS

start len tm gc% any 3′ seq

1 LEFT PRIMER 212 20 59.87 45.00 3.00 0.00 AGCAAACGCTGATGAATGTG

RIGHT PRIMER 432 20 60.03 50.00 4.00 2.00 CCATCTGGGCTCCATTTAGA

HYB OLIGO 324 20 59.98 55.00 4.00 3.00 CAGAAGCCCTTTGAGAGTGG

PRODUCT SIZE: 221, PAIR ANY COMPL: 4.00, PAIR 3′ COMPL: 1.00

2 LEFT PRIMER 212 20 59.87 45.00 3.00 0.00 AGCAAACGCTGATGAATGTG

RIGHT PRIMER 433 20 60.03 50.00 4.00 2.00 TCCATCTGGGCTCCATTTAG

HYB OLIGO 324 20 59.98 55.00 4.00 3.00 CAGAAGCCCTTTGAGAGTGG

PRODUCT SIZE: 222, PAIR ANY COMPL: 4.00, PAIR 3′ COMPL: 2.00

3 LEFT PRIMER 212 20 59.87 45.00 3.00 0.00 AGCAAACGCTGATGAATGTG

RIGHT PRIMER 434 20 60.03 50.00 4.00 2.00 CTCCATCTGGGCTCCATTTA

HYB OLIGO 324 20 59.98 55.00 4.00 3.00 CAGAAGCCCTTTGAGAGTGG

PRODUCT SIZE: 223, PAIR ANY COMPL: 4.00, PAIR 3′ COMPL: 3.00

4 LEFT PRIMER 42 20 60.09 40.00 4.00 1.00 TGCAAAGACCACATTGGAAA

RIGHT PRIMER 220 20 60.08 40.00 5.00 2.00 GCGTTTGCTTCATGGAAAAT

HYB OLIGO 67 22 59.71 40.91 6.00 2.00 TGCCAAATGTCCTAGAAGATGA

PRODUCT SIZE: 179, PAIR ANY COMPL: 5.00, PAIR 3′ COMPL: 1.00

Statistics

con too in in no tm tm high high high

sid many tar excl bad GC too too any 3′ poly end

ered Ns get reg GC% clamp low high compl compl X stab ok

Left 8874 0 0 0 362 0 4840 1239 0 1 64 55 2312

Right 8704 0 0 0 368 0 4613 1246 0 1 87 59 2330

Hyb 10362 0 0 0 437 0 5411 1618 0 0 89 0 2807

Pair Stats:

considered 1974, unacceptable product size 1963, high end compl 1, ok 10

primer3 release 1.1.4

(primer3_results.cgi release 0.4.0)

Appendix 5

Primer abc2

PRIMER PICKING RESULTS FOR abc 2

No mispriming library specified

No hyb oligo mishyb library specified

Using 1-based sequence positions

OLIGO start len tm gc% any 3′ seq

LEFT PRIMER 35 20 60.37 45.00 5.00 2.00 AATTTTACCGCACCTGGTCA

RIGHT PRIMER 207 20 59.62 50.00 2.00 0.00 ACTTTGGTTGGTCTGCCTGT

HYB OLIGO 119 21 59.04 42.86 4.00 2.00 GCAGTTTCAGGACATCCATTT

SEQUENCE SIZE: 428

INCLUDED REGION SIZE: 428

PRODUCT SIZE: 173, PAIR ANY COMPL: 3.00, PAIR 3′ COMPL: 1.00

1 CACAACTAAGGAACGTCAAGAGATACAGAATCCAAATTTTACCGCACCTGGTCAAGAATT

61 TCTGTCTAAATCTCATTTGTATGAACATCTGACTTTGGAAAAATCTTCAAGCAATTTAGC

121 AGTTTCAGGACATCCATTTTATCAAGTTTCTGCTACAAGAAATGAAAAAATGAGACACTT

181 GATTACTACAGGCAGACCAACCAAAGTCTTTGTTCCACCTTTTAAAACTAAATCACATTT

241 TCACAGAGTTGAACAGTGTGTTAGGAATATTAACTTGGAGGAAAACAGACAAAAGCAAAA 301 CATTGATGGACATGGCTCTGATGATAGTAAAAATAAGATTAATGACAATGAGATTCATCA 361 GTTTAACAAAAACAACTCCAATCAAGCAGTAGCTGTAACTTTCACAAAGTGTGAAGAAGA 421 ACCTTTAG KEYS (in order of precedence): >>>>>> left primer

right primer

hyb oligo

ADDITIONAL OLIGOS

start len tm gc% any 3′ seq

1 LEFT PRIMER 36 20 60.37 45.00 5.00 3.00 ATTTTACCGCACCTGGTCAA

RIGHT PRIMER 207 20 59.62 50.00 2.00 0.00 ACTTTGGTTGGTCTGCCTGT

HYB OLIGO 119 21 59.04 42.86 4.00 2.00 GCAGTTTCAGGACATCCATTT

PRODUCT SIZE: 172, PAIR ANY COMPL: 3.00, PAIR 3′ COMPL: 1.00

2 LEFT PRIMER 40 20 60.52 50.00 5.00 2.00 TACCGCACCTGGTCAAGAAT

RIGHT PRIMER 207 20 59.62 50.00 2.00 0.00 ACTTTGGTTGGTCTGCCTGT

HYB OLIGO 119 21 59.04 42.86 4.00 2.00 GCAGTTTCAGGACATCCATTT

PRODUCT SIZE: 168, PAIR ANY COMPL: 3.00, PAIR 3′ COMPL: 0.00

3 LEFT PRIMER 31 20 60.86 45.00 6.00 2.00 TCCAAATTTTACCGCACCTG

RIGHT PRIMER 207 20 59.62 50.00 2.00 0.00 ACTTTGGTTGGTCTGCCTGT

HYB OLIGO 119 21 59.04 42.86 4.00 2.00 GCAGTTTCAGGACATCCATTT

PRODUCT SIZE: 177, PAIR ANY COMPL: 5.00, PAIR 3′ COMPL: 1.00

4 LEFT PRIMER 34 20 58.95 45.00 6.00 3.00 AAATTTTACCGCACCTGGTC

RIGHT PRIMER 207 20 59.62 50.00 2.00 0.00 ACTTTGGTTGGTCTGCCTGT

HYB OLIGO 119 21 59.04 42.86 4.00 2.00 GCAGTTTCAGGACATCCATTT

PRODUCT SIZE: 174, PAIR ANY COMPL: 3.00, PAIR 3′ COMPL: 0.00

Statistics

con too in in no tm tm high high high

sid many tar excl bad GC too too any 3′ poly end

ered Ns get reg GC% clamp low high compl compl X stab ok

Left 2585 0 0 0 27 0 1472 266 0 2 22 10 786

Right 2604 0 0 0 130 0 1480 209 1 1 22 8 753

Hyb 3913 0 0 0 130 0 2163 396 0 0 22 0 1202

Pair Stats:

considered 63, unacceptable product size 58, ok 5

primer3 release 1.1.4

(primer3_results.cgi release 0.4.0)