Download Mutation pattern in BRCA1/2 genes in Indian hereditary breast

Mutation pattern in BRCA1/2
genes in Indian hereditary
breast/ovarian cancers
Prof. Dr. Kannan Vaidyanathan, MBBS, MD
Professor & Head, Dept of Biochemistry,
Pushpagiri Institute of Medical Sciences & Research Center,
Tiruvalla, Kerala, INDIA
FAMILIAL BREAST CANCER
Breast cancer is one of the most common malignancies affecting
women all over the world
About 5 - 10% of all breast cancers have a familial basis and are
inherited in autosomal dominant manner.
Familial breast cancer has an earlier onset of occurrence. It is
frequently bilateral and associated with ovarian cancer.
BRCA1 and 2 are the most important genes involved.
Other genes are p53, CHK2, PTEN, ATM, etc.
ROLE OF DIFFERENT GENES IN BREAST CANCER
BRCA1
Inherited
Unknown
BRCA2
Sporadic
CHEK2
TP53
BRCA1 gene structure
BRCA2 gene structure
CHK2
1
113
175
FHA
220
ATP
486
Protein kinase
543
BRCA1 & BRCA2 – MORE ASPECTS…
Penetrance – Lifetime risk of developing cancer – 80% for BRCA1
and 2.
Different factors including ethnic, OCP use affect penetrance
values.
Both important in DNA repair, different types and in other
cellular functions broadly related to cell cycle.
Other cancers related to BRCA1,2 include ovarian, pancreatic,
male breast cancer, Wilm’s tumor, Fanconi’s anemia etc.
Cellular signal mechanisms could be responsible for the
development of one particular tissue cancer and the sparing of
the others.
IMPORTANCE
Mutation carriers have greater chance of developing breast cancer
compared to mutation negative individuals.
Scope for genetic counseling in affected families; early detection and
cure .
Can opt for prophylactic therapy, prophylactic oophorectomy or
mastectomy. Controversial.
Awareness increased; more precaution and can opt for more strict
screening measures, including mammography.
Risk of Breast/Ovarian Cancer in Mutation Carriers
Gene
Breast cancer
Inherited
Risk to
All cases
cases
age 70
NONE
85%
BRCA1
5-10%
BRCA2
Ovarian Cancer
Inherited
Risk to
All cases
cases
age 70
9.50%
85%
1.2%
35%
50-80%
3-5%
75%
20-50%
5-10%
40%
50-80%
3-5%
10%
10-30%
MMR: MLH1,
MSH2, MSH6,
PMS1, PMS2
<1%
<5%
10%
<2%
<10%
<10%
TP53
<1%
<1%
90%
<1%
<1%
<1%
PTEN
<1%
<1%
50%
<1%
<1%
<5%
*Breast cancer risk to age 50 years : 30% for BRCA carriers compared
with 2% for general population!!!!!!
Aims and objectives
• To study 61 familial breast/ovarian cancer patients for
mutations/polymorphisms in BRCA1/BRCA2/CHEK2 genes using
conformation sensitive gel electrophoresis (CSGE) and DNA
sequencing (sense and anti-sense directions).
• To study 110 control subjects for deleterious
mutations/polymorphisms using conformation sensitive gel
electrophoresis (CSGE) and DNA sequencing (sense and anti-sense
directions).
Conformation sensitive gel electrophoresis (CSGE)
Mutant BRCA2 gene
Normal BRCA2 gene
G
C
A
T
PCR
G
A
C
T
G
A
C
T
CSGE
Heteroduplexing
G
A
T
C
Heteroduplexes
Homoduplexes
BRCA1 mutations identified
Exon
18
2
2
2
2
2
2
2
2
5
11.1
11.9
11.13
9
9
Codon
1716
22/23
22/23
22/23
22/23
22/23
22/23
22/23
22/23
59/60
1111/1112
955
1365
ND
ND
Nucleotide
5271
185/187
185/187
185/187
185/187
185/187
185/187
185/187
185/187
295/297
3450
2883
4213
ND
ND
Type
NS
FS
FS
FS
FS
FS
FS
FS
FS
FS
FS
NS
NS
ND
ND
FS-frame shift mutation; NS-nonsense mutation
Mutation
Y1716X
delAGter39
delAGter39
delAGter39
delAGter39
delAGter39
delAGter39
delAGter39
delAGter39
delCAter64
3450del4
S955X
L1365X
ND
ND
Medical History
Breast/Ovarian
Breast/Ovarian
Ovarian Cancer
Ovarian Cancer
Breast/Ovarian
Breast Cancer
Breast Cancer
Breast Cancer
Breast Cancer
Breast/Ovarian
Ovarian Cancer
Breast Cancer
Breast Cancer
Breast Cancer
Germ Cell Tumor
Remarks
Novel
Reported
Reported
Reported
Reported
Reported
Reported
Reported
Reported
Novel
Reported
Reported
Novel
Ovary
BRCA2 MUTATIONS IDENTIFIED
EXON
CODON
NUCLEOTIDE
TYPE OF
MUTATION
MUTATION
REMARKS
11J
1547
4866InsT
FS
Asp1547Stop
Novel
11N
1951&195
2
6079delAGTT
FS
delAGTTter1961
Reported
4
NA
IVS4+67A>C
Polymorphism
NA
Novel
8
NA
IVS8+56C>T
Polymorphism
NA
Reported
11F
1132
3625A >G
Neutral Change
Lys1132Lys
Reported
25
NA
IVS25-16T>C
Polymorphism
NA
Reported
10C
NA
1593
Neutral (SNP)
Ser 455 Ser
Reported
11S
NA
IVS11+77delTTAA
Polymorphism
NA
Reported
13
NA
IVS13+136dup8nt
Polymorphism
NA
Novel
11d
991
3199 A>G
Polymorphism
Asn3199Asp
Reported
Control
KP12
KP13
KP14
KP6
KP7
KP8
KP9
KP10
KP11
Marker
Control
KP2
K33
KP4
KP5
Analysis of exon 2 of patient KP2,14 showing 185delAG mutation
500
400
300
258
KP 14
M
WT
185delAG
Codon
Control
BRCA1/EXON 2
20
21 22 23
24 25 26
AAA ATC TTA GAG TGT CCC ATC TG
K
I
L
E
C
P
I
Nucleotidede: 185delAG
Codon:22 & 23 to ter 39
Patient
AAA ATC TTA GTG TCC CAT CTG
K
I
L
V S
H
L
DNA sequencing showing 4866insT
pBR322/
HinfI
KP7
185del
AG
Y1716X
CSGE showing 4866insT(11J) in KP-7
T G A A A A A C C T T T T T G A T G A A A A A G A G C A A GG
Control
517
506
4866insT
396
403
G A T G A A A A A G A GC A AGG
T G A A A A A C C T T T T T T G A T G A A AA A G A G C AAG
344
KP 7
298
Codon
Wild type
1543 1544 1545 1546 1547 1548 1549 1550 1551 1552
AAA AAC CTT TTT GAT GAA AAA GAG CAA GGT
K
N
L
F
D
E
K
E
Q
G
Nucleotide: 4866insT
Codon: 1547 (GAT) to ter1547 (TGA)
Patient
AAA AAC CTT TTT TGA TGA AAA AGA GCA AGG
K
N
L
F Stop
CSGE showing heteroduplex in KP 8 – Exon 11N
Sequencing (KP 8) showing 6079delAGTT
KP1
KP2
KP3
KP4
KP5
KP6
KP7
KP8
KP9
KP10
KP11
KP12
KP13
Control
Control
Y1716X
185del
Marker
AG
Control
6079delAGTT
KP8
517
506
423
396
344
298
Codon
Wild Type
1948 1949 1950 1951 1952 1953
1954 1955 1956 1957 1958 1959 1960 1961
T TGT GAT GTT AGT TTG GAA ACT TCA GAT ATA TGT AAA TGT AGT ATA GGG
C
D
V
S
L
E
T
S
D
I
C
K
C
S
I
G
Nucleotide: 6079delAGTT
Codon: 1951/1952 to ter1961
Patient
T TGT GAT GTT TGG AAA CTT CAG ATA TAT GTA AAT GTA GTA TAG GG
C
D
V
S
K
L Q
I
Y
V
N
V
V
STOP
429 bp
13
17
20
26
Sequencing showing IVS11+80delTTAA
pBR322 /HinfI
9
185del AG
8
Y1716X
Control
CSGE in intron 11 showing IVS11+80delTTAA
G G T A T G C T A A C A A
T T
A A G A G T G T T
A T
Control
517
506
Patient
396
G G T A T G C
IVS 11+80delTTAA
T A A C A A
344
298
Wild type: GGTATGCTAACAATTAAGAGTGTTATA
IVS 11+80delTTAA
Patient: GGTATGCTAACAAGAGTGTTATAAACT
T T A A G A G
T G T T A T
G A G T G T T
A T A A A C
Sequencing showing 9 nt duplication
CSGE in intron 13 showing 9 nt duplication in KP - 29
B
29 30 31 32 C
185delAG
22 23 24 25 26 27 28
Y1716X
A
pBR322/Hinf1
AA T T T T A T A A A A C G G G A A G TG T T A A C T T C
Control
517
506
396
470
bp
KP 29
T T T T A T A A A A C GG G A A G T
AA T T T T A T A AA AC G G G A AG T G T T A A C T T C
Duplication 9 nt
344
298
C
Wild Type:- A A T T T T A T A A A A C G G G A A G T G T T A A C T T C
Reverse
complement
INTRON 13 Duplication 9 nt
Patient:- A A T T T T A T A A A A C G G G A A G T G T T A A C T T C
T T T T A T AAAA C G G G AA G T
Reverse complement
Results (Contd.)
• No mutations were identified in CHEK2 gene, including CHK2
1100delC mutation.
• Haplotype analysis was carried out, and it was found to be different
from Ashkenazi Jewish population.
• Population screening was done on 110 control subjects.
• Many of the polymorphisms found in BRCA2 were found in the
control subjects in similar or higher frequencies.
• This suggested normal population variants.
Discussion
• 185delAG mutation has been reported to occur at varying frequencies
among families with breast/ovarian cancer in different populations.
• Some Indian Studies (Kumar et al. 2002; Saxena et al. 2002; Rajkumar
et al. 2003; Valarmathi et al. 2003, 2004; Hedau et al. 2004; Saxena et
al. 2006, Juwle and Saranth. 2012) have identified BRCA1/2 mutations
including 185 delAG mutation.
• Chakraborty et al, 2015, Sharma et al. 2014 did not find 185delAG
(and 6174delT) mutations from Eastern India and in a pilot study from
New Delhi respectively.
• Kim and Choi. 2013 report that BRCA2 mutations are more common
from Asian population except India and Pakistan.
• Not found among Chinese and Japanese families with breast cancer
(Ikeda et al. 2001; Zhi et al. 2002).
• A very high frequency of 31.6% has been reported among non-Jewish
Americans of Spanish ancestry from the San Luis Valley, Colorado
(Mullineaux et al. 2003).
• However, this mutation has been found to occur at a varying low
frequency (1.13–5.9%) among white Americans, the Spanish from
Spain, Polish, Iranian, Pakistani and Turkish women (Grzybowska et al.
2002; Shih et al. 2002; Guran et al. 2005; Weitzel et al. 2005;
Mehdipour et al. 2006; Rashid et al. 2006).
Conclusions
• In this study, 61 breast and/or ovarian cancer patients with a positive
family history of breast and/or ovarian cancer were screened for
BRCA1/2 mutations.
• In the BRCA1 gene, 15 mutations were identified; (mutation
frequency, 24.6%) and in the BRCA2 gene, two mutations were
detected (mutation frequency, 3.28%).
• Of the BRCA1 mutations identified, 3 were novel mutations and 3
more were previously reported mutations.
• The mutation, 185delAG was found in 10 patients at a very high
frequency of 16.4%.
• This mutation is detected in high proportion in Ashkenazi Jewish
population (18% in breast/ovarian cancers and 1% in general
population).
• A large number of polymorphisms were also detected in BRCA2 gene,
which included normal population variants.
• The mutation spectrum of BRCA1/2 in other Indian studies also
indicate a higher incidence of 185delAG mutation.
• The possibility of founder mutation status need to be considered for
BRCA1 185delAG mutation.
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