priya brca1

25
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 3 rd Year Session: 2012-15 Under the Supervision of: Mr. Vaibhav Kumar Sharma

Upload: priya-yadav

Post on 15-Aug-2015

101 views

Category:

Documents


2 download

TRANSCRIPT

Page 1: priya brca1

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

Page 2: priya brca1

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

Page 3: priya brca1

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.

Page 4: priya brca1

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.

Page 5: priya brca1

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

Page 6: priya brca1

Mechanism of the Disease

Page 7: priya brca1

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

Page 8: priya brca1

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.

Page 9: priya brca1

Mutations reported in BRCA1 gene:

Page 10: priya brca1

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.

Page 11: priya brca1

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.

Page 12: priya brca1

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.

Page 13: priya brca1

2) pBLAST

Page 14: priya brca1

3) Primary structure analysis by using ProtParam:

Page 15: priya brca1

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.

Page 16: priya brca1

4) Secondary Structure Analysis using GOR:

Page 17: priya brca1

4)Secondary Structure Analysis using GOR:

• GOR analyzes provided a.a. sequence to predict alpha- helix, beta- sheets, turns/ coils.• After mutation in BRCA1:- Alpha- helix = 24.30 %- Extended strands= 32.24 %- Random coils= 55.29%• Results into the altered protein.• Tumor Suppressing functionality is lost.

Page 18: priya brca1

5) Protein Model Building using Swissmodel- my workspace:

Page 19: priya brca1
Page 20: priya brca1

6) Model Validation by UCLA server ( verify 3D, ERRAT)

Page 21: priya brca1

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).

Page 22: priya brca1

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.

Page 23: priya brca1

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.

Page 24: priya brca1

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.

Page 25: priya brca1