A brief description of cancer associated genes in breast cancer

1. PREVALENCE OF CANCER ASSOCIATED GENES IN
BREAST CANCER PATIENTS IN THE HOSPITAL
BASED STUDY FROM SOUTHERN ASSAM
Presented By:-
Jagadish Hansa
Ph.D Regn. No.:-805, 17/08/2009
A.U. Regn. No.:-74400130 of 2008-2009
Department of Biotechnology
Assam University, Silchar
Serial No: IRB/CCHRC/01/2010

2. Simply being a woman is the main risk factor for developing breast cancer. Male
can also develop.
The Northeastern region have the highest tobacco related cancer incidences
rate compared to that of rest of the country. (Ihshan et al., 2011)
Prevalence of breast cancer in southern Assam is second highest after head and
neck cancer. (Koushal et al., 2010)
North-East region of India has different customs, food habits, life-style, diverse
ethnic groups and type and pattern of tobacco use as compared to the rest of
the country.
Statistics itself define that in this southern part of Assam their will be more
requirement of research work.
BREAST CANCER IN NORTH EASTBREAST CANCER IN NORTH EAST

3. Objective:-
 Survey and collection of samples from hospitals and medical
institutes of Barak valley.
 Immunohistochemistry analysis of tissues from the breast
cancer patients.
 Design of primer for amplification of most responsible genes
in breast cancer. And detection of hotspot sequence
mutation from Breast Associated gene(s).
 Bioinformatical analysis of breast associated genes and the
global databases in breast cancer.

4. MATERIAL & METHODs
Survey of cancer effected patients
Collection of tissue from patients
Pathological analysis
Extraction or isolation
Design of primer
Hotspot mutation detection
DNA Sequencing
Bioinformatical Analysis

5. Good Primer’s Characteristic
Uniqueness*, Length*, Base Composition*, Melting Temperature*, Annealing
Temperature*, Absence of Dimerization Capability*
We have designed 3 sets of primer for BRCA1 and one set of pimer for p53 by
considering all the properties of a primer
Primer Nucleotide Length A TGC
GC
content
Mol.
Weight Tm
Product
Size
BRCA1-185delAG-F ATT GGA ACA GAA AGA AAT GG 20 10 3 6 1 35% 6247.2 52.3 C 185b
p
BRCA1-185delAG-R CCT AGT ATG TAA GGT CAA TTC T 22 6 8 3 5 36% 6724.5 56.4 C
BRCA1-1014delGT-F ACA GCA TGA GAA CAG CAG 18 7 1 4 6 50% 5550.7 53.8 C 195b
p
BRCA1-1014delGT-R CAC AGG GGA TCA GCA TTC AGA 21 6 3 6 6 52% 6464.3 61.2 C
BRCA1-3889delAG-F TCT ACT AGG CAT AGC ACC GTT 21 5 6 4 6 48% 6381.2 59.5 C 192b
p
BRCA1-3889delAG-R CTT CCA ATT CAC TGC ACT GTG 21 4 7 3 7 48% 6332.2 59.5 C
p53-E8-F GCT TCT CTT TTC CTA TCC TG
20 1
1
0
2 7 45% 5975.9 56.4 C 167b
p
p53-E8-R CTT ACC TCG CTT AGT GCT 18 2 7 3 6 50% 5416.6 53.8 C

6. Agarose gel electrophoresis of Islolated DNA and amplified products
Isolation from Blood Isolation from TISSUE
Amplification of 192bp DNA
amplicon in 2% agarose gel.
Amplification of 168bp DNA
amplicon in 2% agarose gel.
1kb BC1 BC2 BC3 BC4 BC5

7. DNA PURIFICATION
All PCR products were purified using PCR Purification Kit (Qiagen, UK)
DNA SEQUENCING
The purified PCR product was sequenced both from forward and reverse
side using automated DNA sequencer (ABI3700).
PCR PRoduCt sequenCing

9. Some deleterious Mutation
a) Normal sequence, no deletion at 185 AG position from exon 2. b) Mutated sequences, deletion at 185 AG position,
from exon 2. c) Normal sequence, no deletion at 1014 GT position from exon 11a. d) Mutated sequence, deletion at
1014 GT position from exon 11a. e) Normal sequence, no deletion at 3889 AG position from exon 11d. f) Mutated
sequence, deletion at 3889 AG position from exon 11d.

10. DISCUSSION
Three previously reported deleterious frame-shift mutations resulting in a
premature termination codon were identified in BRCA1: 185delAG in exon 2;
1014delGT and 3889delAG in exon 11 in southern part of the Assam.
Surprisingly, we have found 185delAG in a Northeast Indian, Hindu patient
residing in Cachar district who claimed to have no Jewish ancestry.
BRCA1 1014delGT was detected in a Muslim index case of very early onset
disease [age 42] without any family history. Interestingly, the same mutation was
reported in a heterogeneous Italian population intermixed with French, German,
and Slovenian ethnic groups and a large number of Muslim immigrants.
The 3889delAG mutation is located towards the C terminus of BRCA1, within
the transcriptional activation domain, a region also reported to interact with the
BRCA2 protein, which plays an important role in double stranded break (DSB)
repair.
The above mutations have been reported in the Breast Cancer Information
Core (BIC) website in breast cancer cases of diverse ethnic origins.

11. Work to be done…
Immunohistochemistry analysis of tissues from the breast cancer patients.
Types of antibodies to be used:
a) Monoclonal Anti-Rabbit IgG (Chain specific)−Alkaline Phosphatase
antibody produced in mouse , A2556 SIGMA-ALDRICH
b) Anti-BRCA1 (Ab-1423) antibody, SAB4300490 SIGMA
c) Anti-p53 (SC-101764)
d)Anti-actin (SC-8432)
e)Secondary antibody- Goat Anti-rabbit IgG-Alkaline phosphatase, Merck
Methods of IHC-ALP Method
Deparaffinization
Add Blocking
solution and
secondary antibody
Add Primary
antibody
Blocking SolutionDrying and
Rehydration
Wash with
TTBS
Develop the staining
through BCIP/NBT
solution
Observe the
slides and
analyze the
staining
Required of samples: FFPE samples of the studied
patients…

12. ABSTRACT, ORAL/POSTER PRESENTATION AT DIFFERENT CONFE.
Jagadish Hansa and Sankar Kr Ghosh (2012), “Mutation detection of BRCA1 gene in
breast cancer patients from Southern Assam, India”, 2nd
International Conference on
Perspective of cell signaling and Molecular Medicine, during 8-11 January 2012, Bose
Institute.
Hansa Jagadish, Mondal Rosy, Kannan Ravi and Ghosh K. Sankar (2011), Mitochondrial
DNA Detection at the D310 Region by COLD-PCR in Cancerous Tissue, 98th
ISC, 2011,
Chennai.
Hansa Jagadish, Mondal Rosy, Biswas R. and Ghosh K. Sankar (2010), PCR Based Disgnosis
of HPV from Cancer Patients in the Hospital Based Study from Southern Assam, 97th
ISC,
2010, Tiruvanantapuram.
PAPER PREPARED FOR THE COMMUNICATION
Screening of 185DelAG, 1014DelGT and 3889DelAG Mutations of BRCA1 in Breast
Cancer patients from North-East India: a hospital based study

13. PUUBLISHED SEQUENCE IN NCBI
BankIt1473753 SGSC JN603607; BankIt1473753 SGIS JN603608
BankIt1473753 SGBS JN603609; BankIt1473753 SGSSD JN603610
BankIt1473753 SGSB JN603611; BankIt1473753 SGPG JN603612
BankIt1473753 SGAB JN603613; BankIt1473753 SGARS JN603614
BankIt1473753 SGNSK JN603615; BankIt1473753 SGAMAA JN603616
BankIt1473753 SGBAB JN603617; BankIt1473753 SGBC JN603618
BankIt1473753 SGMKP JN603619; BankIt1473753 SGMKB JN603620
BankIt1473753 SGSDG JN603621; BankIt1473753 SGMS JN603622
BankIt1473753 SGJS JN603623; BankIt1473753 SGSSM JN603624
BankIt1473753 SGBKT JN603625; BankIt1473753 SGAH JN603626
BankIt1473753 SGMMD JN603627;BankIt1473753 SGRKM JN603628
BankIt1473753 SGPRG JN603629

14. References
1) Durant, S.T. and Nickoloff, J.A. Good timing in the cell cycle for precise DNA
repair by BRCA1. Cell Cycle 2005, 4:1216–1222.
2) Honrado, E., Benítez, J., Palacios, J. The molecular pathology of hereditary
breast cancer: Genetic testing and therapeutic implications. Mod. Pathol. 2005,
18(10): 1305-1320.
3) Rennert, G., Bisland-Naggan, S., Barnett-Griness, O., Bar-Joseph, N., Zhang,
S., Rennert, H.S., Narod, S.A. Clinical outcomes of breast cancer in carriers of
BRCA1 and BRCA2 mutations. N. Engl. J. Med.2009, 357: 115-123.
4) www.cancer-genetics.com (BIC)
5) Ihsan, R., T. R. Devi, D. S. Yadav, A. K. Mishra, J. Sharma, E. Zomawia, Y.
Verma, R. Phukan, J. Mahanta, A. C. Kataki, S. Kapur, and S. Saxena. 2011.
Investigation on the role of p53 codon 72 polymorphism and interactions with
tobacco, betel quid, and alcohol in susceptibility to cancers in a high-risk
population from North East India. DNA Cell Biol 30 (3):163-171

15. Acknowledgement
I am sincerely thankful to all the teachers and doctors.
I would also thankful to the Assam University and Cachar
Cancer hospital and research centre institute.
 The moral support given by my friends, family members and
almighty
Thank You all for your
kind
attention