1. GENE IDENTIFICATION AND TARGET VALIDATION OF GENE (BRCA1)
WITH THE COMPARATIVE STUDY THROUGH GENOMIC AND
PORTEOMIC ANALYSIS USING BIOINFORMATICS TECHNIQUES.
Submitted By: Priya Yadav
B.Sc. (H) Biotechnology
3rd Year
Session: 2012-15
Under the Supervision of: Mr. Vaibhav Kumar Sharma
2. Contents
• Objectives of the study
• Brief introduction to Cancer
• Introduction to the Breast Cancer
• Introduction to BRCA1 gene
• Brief introduction to the Bioinformatics
• Bioinformatics tools & Software which were used during the study
• Results
• Conclusion
• Discussion
• References
3. Objectives of the study
• Comparative Analysis of the genetic information of the gene BRCA1 using Bioinformatics tools and software.
• Study of the application of bioinformatics tools in order to determine whether the DNA contain mutations or not
responsible for the breast cancer.
• To navigate NCBI in order to align sequences using different tools and software and finally to validate the model.
• To analyze the reference sequence, known to be free of cancer causing mutations are used to determine whether the
DNA or protein sequence carry mutations or not.
4. A brief introduction to Cancer
• Medically known as malignant neoplasm.
• Cancer strikes one in three people in the United States; kills 1 in 4.
• As genes switch on and off, they determine when and how fast the cell will grow and divide, when it will stop
dividing.
• Cancer can result when controls over cell division are lost.
5. Abnormal structure of a Cancer Cell
• Larger cell nucleus and less cytoplasm
• Loss of structural specialization
• Cytoskeleton shrinks
• Plasma membrane proteins could be lost or altered
• New plasma membrane proteins may appear
• Changes passed on to cell’s descendants
7. Introduction to the Breast Cancer
• Breast cancer is second only to lung cancer as a cause of cancer deaths in American women.
• Variations of the following genes increases the risk of developing breast cancer:
• BRCA1
• BRCA2
• CHD1
• SET11
• TP53
8. BRCA1 Gene
- Belongs to a class of the genes known to have tumor suppressor function.
- The protein production of BRCA1 in normal conditions help prevent cells from growing and
dividing rapidly and uncontrollably.
- Multi- domain protein; mutated in a great percentile of hereditary breast & ovarian cancers.
- Mutated in the 3 domains basically;
• N- terminus of the RING domain
• Exon- 11, 13
• BRCT domain.
Data unavailable till now for the a.a. encoded by E.- 11, 13 and hence have deleterious effect
on the functions of these domains.
10. What is Bioinformatics?
• Field involves the technology using computers for storage, retrieval, manipulation and
distribution of the biological data.
• Conceptualizing biology in terms of molecules and applying informatics techniques.
• Management information system for molecular biology and has many practical applications.
• Term was coined by Paulin Hogweg (1970).
• First bioinformatics project was carried out by Margret Dayhoff (1965).
• Field carry out prominent roles like:
- Predicting 3D structures
- Gene expression Analysis/ Profiling
- Protein identification by genomic sequence
- Molecular Modelling and Homology Modelling, etc.
11. Bioinformatics tools & Software which were used during the study:
• NCBI Website
• Basic Local Alignment Search Tool (BLAST): nBLAST, pBLAST
• Comparative Proteomics Tools:
- Primary Structure Analysis (ProtParam)
- Secondary Structure Analysis (GOR Method)
- Protein Model Building (Swissmodel- my workspace)
- Model Validation by UCLA server ( verify 3D, ERRAT)
• Phylogenetic relationship study using Clustal W alignment, t-coffee alignment, phylogeny.fr
alignment.
12. Results:
1) nBLAST
- DNA-DNA sequences of the top 5 similar
taking H. sapiens as query species .
- Red Bars indicate most related sequences
matched with the query sequence.
- Pink Bars represent probable homologs.
15. 3) Primary structure analysis by using ProtParam:
• Usually, BRCA1 protein carries substitution and deletion mutations.
• Amino acid composition of the mutated gene is only 214 a.a. instead of 1863 a.a.
• Molecular weight is only 24560.2 daltons.
• pI remains 5.82.
• Half life is only 100 hours (in mammals).
• Instability index is 27.74; protein becomes quite stable.
• Affects the secondary structure formation greatly.
21. 7) Phylogenetic relationship study:
• Complete BRCA1 gene sequences from the 7 primate species was analyzed.
• The specific amino acid sites have been experiencing a repeated selection for the
amino acid replacement over the primate evolution.
• Selection has been focused specifically on Homo sapiens and the closest living
relatives, i.e; Chimpanzees (Pan troglodytes) and Bonobos (Pan paniscus).
22. Conclusion:
• In normal conditions, the BRCA1 gene act as tumor suppressor, producing a
protein that prevents cells from multiplying too quickly.
• Abnormalities in the protective BRCA1 gene are inherited or acquired, cells can
grow and divide uncontrollably and tumors may form.
• Abnormalities translate to a higher-than-average cancer risk and can be inherited
by men or women from either parent.
• BRCA1 gene involved in DNA repair, and is a tumor suppressor gene, is reported
to be evolving rapidly.
• Some more phylogenetic studies and the molecular characterization of the above
mentioned gene- BRCA1, may prove helpful in the process for better drug
development solutions.
23. Discussion:
• BRCA1 are evolving in a similar manner to the highly adaptive immunity genes.
• Additional to BRCA1, other DNA repair genes have also been shown to evolve
under positive selection, but the driver behind this unusual finding remains to be
identified.
• The forces driving the diversifying selection of the gene is still unknown.
• It still remains to be analyzed that whether this is an instance of antagonistic
pleiotropy; where positive selection driven by one force causes functional
consequences in another context, potentially leading to the formation of cancers.
24. References:
• Antoniou A, Pharoah PD, Narod S, et al. Average risks of breast and ovarian cancer associated with BRCA1 or BRCA2 mutations detected in case series unselected for family history: A
combined analysis of 22 studies. American Journal of Human Genetics 2003; 72(5):1117–1130.
• Brose MS, Rebbeck TR, Calzone KA, et al. Cancer risk estimates for BRCA1 mutation carriers identified in a risk evaluation program. Journal of the National Cancer Institute 2002; 94(18):1365–
1372.
• Burk-Herrick A, Scally M, Amrine-Madsen H, Stanhope MJ, Springer MS: Natural selection and mammalian BRCA1 sequences: elucidating functionally important sites relevant to breast
cancer susceptibility in humans. Mamm Genome 2006, 17:257-270.
• Campeau PM, Foulkes WD, Tischkowitz MD. Hereditary breast cancer: New genetic developments, new therapeutic avenues. Human Genetics 2008; 124(1):31–42.
• Chen S, Parmigiani G. Meta-analysis of BRCA1 and BRCA2 penetrance. Journal of * Clinical Oncology 2007; 25(11):1329–1333.
• Easton DF. How many more breast cancer predisposition genes are there? Breast Cancer Research 1999; 1(1):14–17.
• Finch A, Beiner M, Lubinski J, et al. Salpingo-oophorectomy and the risk of ovarian, fallopian tube, and peritoneal cancers in women with a BRCA1 or BRCA2 mutation. JAMA 2006;
296(2):185–192.
• Fleming MA, Potter JD, Ramirez CJ, Ostrander GK, Ostrander EA: Understanding missense mutations in the BRCA1 gene: an evolutionary approach. Proc Natl Acad Sci U S A 2003, 100:1151-
1156.
• Hemel D, Domchek SM: Breast Cancer Predisposition Syndromes. Hematol Oncol Clin North Am 2010, 24:799-814.
• Howlader N, Noone AM, Krapcho M, et al. (eds). SEER Cancer Statistics Review, 1975-2011, National Cancer Institute. Bethesda, MD, http://seer.cancer.gov/csr/1975_2011/ , based on
November 2013 SEER data submission, posted to the SEER web site, April 2014.
