









BRCA1 and BRCA2 Gene Mutations Screening In Sporadic
Breast Cancer Patients In Kazakhstan.


BRCA1 and BRCA2 Gene Mutations Screening In Sporadic Breast
Cancer Patients In Kazakhstan.



Ainur R. Akilzhanova1,2, Bagdat
Nyshanbekkyzy2, Zhannur M.
Nurkina1,2, Ivan I. Shtephanov3,
Abay K. Makishev3, Tasbolat A.
Adylkhanov4,5, Tolebay K. Rakhypbekov4,5,
Erlan M. Ramanculov2, Kuvat
T. Momynaliev2

1Center for Life sciences, Nazarbayev
University, Astana, Kazakhstan;
2National Center for Biotechnology, Astana,
Kazakhstan; 3State Medical
University Astana, Astana Oncological Center, Astana, Kazakhstan;
4Semey Oncological Center, Semey, Kazakhstan;
5Semey State Medical
University, Semey, Kazakhstan




Abstract



Background: A large number of distinct mutations in
the BRCA1 and BRCA2
genes have been reported worldwide, but little is known regarding the
role of these inherited susceptibility genes in breast cancer risk
among Kazakhstan women.




Aim: To evaluate the role of BRCA1/2
mutations in Kazakhstan women
presenting with sporadic breast cancer.




Methods: We investigated the distribution and nature
of polymorphisms
in BRCA1 and BRCA2 entire coding regions in 156 Kazakhstan sporadic
breast cancer cases and 112 age-matched controls using automatic direct
sequencing. 



Results: We identified 22 distinct variants,
including 16 missense
mutations and 6 polymorphisms in BRCA1/2 genes. In BRCA1, 9 missense
mutations and 3 synonymous polymorphisms were observed. In BRCA2, 7
missense mutations and 3 polymorphisms were detected. There was a
higher prevalence of observed mutations in Caucasian breast cancer
cases compared to Asian cases (p<0.05); higher frequencies of
sequence variants were observed in Asian controls. No recurrent or
founder mutations were observed in BRCA1/2 genes. There were no
statistically significant differences in age at diagnosis, tumor
histology, size of tumor, and lymph node involvement between women with
breast cancer with or without the BRCA sequence alterations. 



Conclusions: Considering the majority of breast
cancer cases are
sporadic, the present study will be helpful in the evaluation of the
need for the genetic screening of BRCA1/2 mutations and reliable
genetic counseling for Kazakhstan sporadic breast cancer patients.
Evaluation of common polymorphisms and mutations and breast cancer risk
in families with genetic predisposition to breast cancer is ongoing in
another current investigation.





Introduction



Breast cancer is the most common malignancy in females and one of the
leading causes of death from cancer in women worldwide.1
It accounts
for 23% of all cancers among women and is the second most common cancer
overall when both sexes are considered. Breast cancer showed the
biggest proportional increase in the number of new cases in women from
2006-2011 and remains the leading form of cancer in women in
Kazakhstan.2 



Germline mutations in BRCA1 and BRCA2 genes account for genetic
predisposition and increased risk of breast and ovarian cancers.3
Two
major breast cancer susceptibility genes are BRCA1 (MIM 113705, Genbank
accession no. U14680) and BRCA2 (MIM 600185, Genbank accession no.
U43746), located on the long arms of chromosomes 174
and 13,5
respectively, and both apparently function as tumor suppressor genes.
BRCA1 is a large protein of 1863 amino acids and BRCA2, with 3418 amino
acids, is even larger. Both proteins are involved in the control of
homologous recombination (HR) and double-strand break repair in
response to DNA damage.6-11 BRCA1/2 have been
shown to serve as
important central components in multiple biological pathways that
regulate cell-cycle progression, centrosome duplication, DNA damage
repair, cell growth, and apoptosis.12 Evidence
that the loss of BRCA1
alleles or low expression of BRCA1 in a large proportion of sporadic
breast cancer cases supports the role of BRCA1 in the development of
sporadic breast cancer.13-15 



Genetic linkage analysis4 and refine mapping16,17
provided the evidence
of the location of penetrance as having a germline mutation; it was
found that the most commonly encountered sporadic forms of breast
cancer vary among different populations.18-20
This contributory
variation may be attributed to their different gene pool make and also
due to low penetrance gene involvement. However, some studies have
pointed out that germline mutations in BRCA1/2 contribute little to the
induction of breast cancer in some countries.21,22




Mutations in the BRCA1 and BRCA2 genes were first reported in
conjunction with their identification in 199423
and 1995.24,25 During
last decade BRCA1 and BRCA2 have been extensively screened for
mutations; numerous mutations have been reported to be clearly
associated with cancer susceptibility and have been registered in the
Breast Cancer Information Core Database (BIC).26
Most of these are
frameshift or nonsense mutations leading to truncated and therefore,
inactive BRCA1/2 proteins. These mutations have a pathogenetic role and
are characterized by a high penetrance. Furthermore, they are thought
to increase the lifetime risk of developing breast cancer 50% by age 50
and 85% by age 70.27-29




Hundreds of alterations have been reported for BRCA1/2 genes, but not
all are able to confer a higher risk of developing breast cancer with
age27,28 and, indeed, the pathogenetic effect of
a significant number
of single amino acid changes is still unknown. This is particularly
true for polymorphisms, naturally occurring gene sequence variations,
often affecting only a single nucleotide, that have recently been
associated with altered cancer risk.30-32 There
are also a certain
number of single amino acid changes that have been identified and
classified as non-characterized variants. It is not known whether these
variants may affect BRCA1 function and thus bring about an increased
risk for breast and/or ovarian cancer. 



Disease-associated mutations are distributed over the entire coding
regions of these genes, and these demonstrate considerable
ethnogeographic variation.33 For example, in the
Ashkenazi Jewish34,35
or in the Icelanders36 breast cancer
predisposition has been
demonstrated to be due to recurrent mutations (founder mutations)
originating from a single ancestor. 



Molecular analysis of BRCA1/2 in different populations has demonstrated
a very large mutational spectrum and variable mutation prevalence
related to the different techniques employed, selection criteria, and
ethnic origin of the patients. In families with no prior history of
breast cancer, frequency of BRCA mutation was found significantly low
from 0.02% to 10%.33 In Asia, the prevalence of
BRCA1/2 mutations among
unselected breast cancer cases was reported at 5.1% in Philippines,37
and 2.5%–3.1% in Korea.38,39 The frequency of
BRCA1 mutations in
Chinese women with breast cancer without family history was 5.4%.40
On
the other hand, 2% of breast cancer cases in the largest breast cancer
population-based study in a UK population showed association with BRCA
genes with 0.7% attribution of BRCA1 (Anglian Breast Cancer Study
Group, 2000).41 Several hospital-based series of
unselected breast
cancers implicate BRCA1 and BRCA2 in 2–5% and 0–2% of all cases,
respectively.41 This variation may be attributed
to differences on the
genome level among various ethnic and population heterogeneity. The
reason of marginally low penetrance of BRCA1/2 germline mutations may
be attributed to the polygenic involvement and heterogeneity of samples
origin too. As in Asia, the overall prevalence of germline mutation
varies from 0.8% in Japanese42 to 8.0% in
Singapore region,43
indicating involvement of other genes and population response with
respect to various types and origin of cancers. Moreover,
inter-individual variation does exist among the ethnic groups in
association with various risk factors as reported by Peto et al.44
showing mutation prevalence as 3.5% before age 35 declining to 0.49% in
≥ 50 years.




Studies on BRCA gene mutations have been mainly performed in western
populations and the majority of these have involved hereditary breast
and ovarian cancer families. Thus, studies focusing on sporadic breast
cancer and data collection in Asians, especially in the Kazakh
population, remain relatively sparse. To our knowledge, this is the
first study to evaluate the frequency and type of sequence alterations
of BRCA1/2 genes in Kazakhstan breast cancer patients. In order to
evaluate the role of BRCA1 and BRCA2 germline mutations in the
Kazakhstan population, 156 sporadic breast cancer patients were
analyzed for mutations throughout the entire coding regions of the
BRCA1 and BRCA2 genes by using direct sequencing.





Materials and
Methods



Patients




Prior to this study, ethical approval was obtained from the Ethical
committees of Semey State Medical University and in National Center for
Biotechnology. A total of 156 Kazakhstan women with pathologically
confirmed breast cancer; 83 women operated in Oncological Center in
Semey, Semey, Kazakhstan from 1984-2005 year and 73 women operated in
Astana Oncological Center, Astana, Kazakhstan from 2008-2011; year were
enrolled in this study (breast cancer group). Family histories were
obtained through individual interviews and only cases without family
history of affected first- or second-degree relatives with breast
and/or ovarian cancer were included in this study.



Clinical and
pathological characteristics such as: age at diagnosis (operation),
histological subtype (WHO histological classification)45 histological
grade, T stage (TNM clinical classification, and lymph node involvement
were obtained from medical records. Control subjects, matched to cases
based on age and ethnicity were randomly selected from the community in
Semey and Astana (n=112). None of the controls had a personal history
of malignancy at the time of ascertainment. Before the study, written
informed consent was obtained from all participants. Both cases and
controls were divided in to two groups by race (by first name of study
participants and their parents): Caucasian (n=88 and 40 cases and
controls, respectively) and Asian (n=68 and 72 cases and controls,
respectively). 





Blood sample collection 



Peripheral blood samples (ca.10 ml) were collected into vacutainers
with K2EDTA. 





DNA extraction 



Genomic DNA was extracted from whole blood samples from cases with
breast cancer as well as controls using a Master Pure DNA purification
Kit (EPICENTRE Biotechnologies, USA) and DNA extraction kit (Promega,
USA) in accordance with the manufacturer’s protocols. 





Polymerase chain reaction: 



For PCR amplification of
the 22 coding regions of BRCA1 and the 26 coding regions of BRCA2,
primers using Primer 3 v. 0.4.0 program were designed (available from
authors on request). Since exon 11 of BRCA1 varies by 3426 base pairs,
we amplified 10 overlapping regions of this exon; for exon 10 of BRCA2,
we designed 2 pairs of primers and for exon 11 of BRCA2 - 3 pairs of
primers.



Amplification of DNA fragments was performed in Tetrad BioRad Thermal
Cycler (BIO RAD, USA) in 25μl of solution containing 150 mM Tris–HCl
(pH 8.0), 500 mM KCl, 25 mM MgCl2, 10 mM each dNTP, 10 pmol of primers,
25–125 ng of genomic DNA, and 2 units of AmpliTaq Gold DNA polymerase.
The PCR was performed according to the following conditions: initial
denaturation at 95°C for 10 minutes, followed by 35 cycles of 95°C for
15 seconds, 58°C for 30 seconds, and 72°C for 30 seconds. The quality
of amplification was determined by separation of the PCR products on a
1.5% agarose gel. The PCR products were purified using ExoSAP-IT (USB,
USA) and incubated at 37°C for 40 min, 80°C for 20 min, and stored at
4°C. Purified PCR products were further used in the sequencing reaction
process.






DNA sequencing 



All amplified products were sequenced in forward and reverse directions
using the BigDye Terminator v3.1 Cycle sequencing Kit (Applied
Biosystems, USA) on an ABI 3130xL DNA Analyzer (Applied Biosystems,
Foster city, CA, USA). The PCR products were sequenced using the same
primers as the ones used for PCR amplification. Sequence PCR products
were cleaned using Sefadex 50 and MultiScreen filtration colons
(Millipore Corporation, USA). A chromatographic tracing of each
amplicon was analyzed by proprietary sequence analysis software
(Sequence Analysis 5.3.1, SeqScape v.2.6, Finch TV v1.3.1) followed by
visual inspection and confirmation. The sequence was compared with the
Breast Cancer information Core, BICdatabase;
http://research.nhgri.nih.gov/bic), Human Genome Mutation Database
(http://www.hgmd.cf.ac.uk/ac/index.php)
and the National center for
Biotechnology Information database (http://
www.ncbi.nlm.nih.gov). 





Mutation Nomenclature 



Approved recommendations of nomenclature for the description of
sequence variants were adopted ((http://www.hgvs.org/mutnomen/).
Numbering according to GenBank Accession no. NM_007294.1 for BRCA1 and
NM_000059.1 for BRCA2, the A of the ATG translation initiation codon is
+1, according to approved guidelines were used.



Also, traditional
mutation nomenclature used in BIC database26
where nucleotide numbers
refer to the wild type cDNA sequence of BRCA1 (RefSeq accession number
U14680) with numbering starting at the A of the first ATG at the
position 120 and to the wild type cDNA sequence of BRCA2 (RefSeq
accession number U43746) with numbering starting at the A of the first
ATG at the position 229 were used.



We used the term "sequence
variation" and “sequence alteration” to prevent confusion with the
terms "mutation" and "polymorphism", mutation meaning "change" or
"disease-causing change" and polymorphism meaning "non disease-causing
change" or "change found at a frequency of 1% or higher in the
population".



Single-nucleotide polymorphisms may fall within coding sequences of
genes, non-coding regions of genes, or in the intergenic regions
(regions between genes). SNPs within a coding sequence do not
necessarily change the amino acid sequence of the protein that is
produced, due to degeneracy of the genetic code. A SNP in which both
alleles produce the same polypeptide sequence is called a synonymous
polymorphism (sometimes called a silent mutation). If a different
polypeptide sequence is produced, the polymorphism is a replacement
polymorphism. A replacement polymorphism change may be either missense,
which results in a different amino acid, or nonsense, which results in
a premature stop codon. Over half of all known disease mutations come
from replacement polymorphisms.24 



A variation in a genetic sequence
whose association with disease risk is unknown is also called a variant
of uncertain significance, unclassified variant, and VUS (an alteration
in the normal sequence of a gene, the significance of which is unclear
until further study of the genotype and corresponding phenotype in a
sufficiently large population).






Statistical analysis 



Clinical and pathological characteristics and BRCA mutation results
were analyzed using SPSS 19.0 (SPSS, Tokyo, Japan). Differences in
categorical variables between mutation-positive and mutation-negative
group were compared using chi-square analysis, cross tables or Fisher’s
exact test. A probability value of less than 0.05 was considered to
indicate significance.






Results



Mean age of breast cancer patients and controls was 51.2±9.5 years and
57.8±9.1 years, respectively. Sixty-eight women with breast cancer were
Asian and eighty eight – Caucasian. Among healthy women, 72 were Asian
and 40 - Caucasian. All breast cancer cases were distributed by size of
primary breast carcinoma (TNM clinical classification45): T1, T2, T3,
T4 – 15, 96, 39, 6 cases respectively, 14 cases with N1 lymph node
metastasis, 15 women with N1 lymph node metastasis, and 5 women had N3
lymph node involvement.




A summary of patient characteristics can be found in:




Table 1: Characteristics of Study Participants


		Breast
Cancer Cases (n=156)	Controls

(n=112)

Abs(%)	Totally

(n=268)

Abs (%)
	Abs	T1 

Abs 

(%)	T2

Abs

(%)	T3

Abs

(%)	T4 

Abs

(%)	N1 

Abs

(%)	N2

Abs

(%)	N3

Abs

(%)
	Age (years) at operation (patients)/sample collection
(controls),Mean±SD	51.2±9.5*	57.8±9.1
	Asian	68	10 

(15.1)	43

(63.6)	14

(21.2)	1

(1.5)	9

(13.2)	6

(9.0)	-	72

(64.3)	144

(53.8)
	Caucasian	88	5

<(6.0)	53

(60.0)	25

(28)	5

(6.0)	5

(6.0)	9

(10.0)	5

(6.0)	40

(35.7)	124

(46.2)
	T – primary tumor: T1
- ≤2cm, T2 –
>2 to 5cm, T3 - >5cm, T4 – tumor of any size with direct
extension to chest wall or skin, N – regional lymph nodes, M – distant
metastasis *- p<0.05 vs controls





Mutational screening of BRCA1 and BRCA2 coding regions was performed
for 156
sporadic breast cancer cases and 112 controls. Mutation analysis of the
BRCA1 and BRCA2 genes revealed the presence of 22 distinct variants,
including 16 missense mutations and 6 polymorphisms (Tables 2 and 3).




The sequence variants identified in BRCA1 gene 9 missense mutations –
c.95G>T (G32V), c.254A>G (N85S), c.1067A>G
(Q356R), c.2612C>T (P871L), c.3113A>G (E1038G),
c.3348A>G (K1183R), c.4744C>G (S1542C), c.5397A>G
(A1627G) and c.5585T>G (T1862G), three synonymous polymorphisms
- Ser694Ser, Leu771Leu and Ser1436Ser. Most of these polymorphisms were
found in exon 11 of BRCA1, which is 60% of all BRCA1 coding region.




Figure 1: Detection of the sequence variants in 11 exon of
BRCA1.






Table 2: Details and the frequency of the variants detected in
the BRCA1 in breast cancer and control groups.


	Exon	Sequence Variant	Amino Acid Variant	Frequency in the Breast
Cancer Group
(n=156), abs (%)	Frequency in the control
group
(n=112), abs (%) 	BIC Entry
	Total	Caucasian/Asian	Total	Caucasian/Asian
	Missense
Mutations
	2	c.95(G>T) †
(214G>T) ‡
	p.Gly32Val (G32V) 	84(54.1)*	51(60.7)/
33(39.3) #
	49(43.7) 	17(36.7)/
32(65.3) #
	Yes
	3	c.254(A>G) (373A>G) 	p.Asn85Ser
(N85S) 	83 (53.0)*	48(57.8)/
35(42.2) #
	42(37.5)	15(35.7)/
27(64.3) #
	Yes
	11	c.1067A>G (1186A>G) 	p.Gln356Arg (Q356R) 	11 (7.2) 	6(54.5)/
5(45.4) 	10 (8.9) 	6(60.0)/
4(40.0) 	Yes
	11	c.2612(C>T) (2731C>T)
	p.Pro871Leu (P871L)	87(55.8)	53(60.9)/
34(39.1) #
	54(48.2)	20(37.0)/
34(63.0) #
	Yes
	11	c.3113(A>G) (3232A>G)
	p.Glu1038Gly (E1038G)	88(56.6)	55(62.5)/
33(37.5) #
	53(47.3)	18(34.0)/
35(66.0) #
	Yes
	11	c.3348(A>G) (3667A>G)
	p.Lys1183Arg (K1183R)	92(59.0)*	58(63.0)/
34(37.0) #
	51(45.5)	17(33.3)/
34(66.7) #
	Yes
	15	c.4744C>G (4863C>G) 	p.Ser1542Cys (S1542C) 	24(15.5) 	14(58.3)/
10(41.7) 	19 (17.0) 	10(52.6)/
9(47.4) 	Yes
	20	c.5397A>G (5516A>G) 	p.Ala1627Gly (A1627G) 	21(13.6)	9(42.8)/
12(57.1) #
	20 (17.8) 	10(50.0)/
10(50.0) 	Yes
	20	c.5585T>G (5704T>G) 	p.Thr1684Gly
(T1862G) 	23(14.8) 	12(52.2)/
11(47.8) 	17 (15.2) 	9(52.9)/
8(47.1) 	Yes
	Polymorphisms
	11	c.2082(C>T) (2201C>T) 	Synonymous Ser694Ser (S694S) 	45(54.2) 	28(62.2)/
17(37.8) #
	48(42.8) 	17(35.4)/
31(64.6) #
	Yes
	11	c.2311(T>C) (2430T>C)
	Synonymous Leu771Leu (L771L) 	45(54.2)	28(62.2)/
17(37.8) #
	48(42.8)	17(35.4)/
31(64.6) #
	Yes
	13	c.4427(T>C) (4546T>C)
	Synonymous Ser1436Ser (S1436S)
	76(49.0)	35(46.0)/
41(53.9) #
	54(48.2)	25(46.3)/
29(53.7)
	Yes
	† Numbering according
to GenBank Accession no. NM_007294.1, the A of the ATG translation
initiation codon is +1,according to approved guidelines
(http://www.hgvs.org/mutnomen/)
‡The nomenclature as used in the BIC database is given in parentheses.
Mutation nomenclature is according to RefSeq accession number U14680
(BRCA1) with numbering starting at the A of the first ATG at the
position 120 * - p<0.05, ** - p<0.001 between cases and
controls
#- p<0.05, ##-- p<0.001 between Caucasians and Asians in
group





Missense mutations c.95G>T (G32V), c.254A>G (N85S),
c.3348A>G (K1183R) were detected with frequency in 54.1%, 53.0%,
59.0% of cases and 43.7%, 37.5%, 45.5% of controls, respectively,
showing significantly higher prevalence in cases (p<0.05). There
was no significant prevalence in frequency of missense mutations
c.1067A>G (Q356R), c.2612C>T (P871L), c.3113A>G
(E1038G), c.4744C>G (S1542C), c.5397A>G (A1627G) in cases
comparing to controls. Synonymous polymorphisms S694S and L771L were
detected in 45 (54.2%) of cases and 48 (42.8%) controls. S1436S was
found in 76 (49.0%) cases and 54 (48.2%) controls. 



In BRCA2, gene missense mutations c.865A>C (N289H),
c.10234A>G (I3412V) were detected with higher frequency in 15.2%
and 30.8% of cases compared to controls (10.1%, 18.7%, respectively,
p<0.05).




Table 3: Details and the frequency of the variants detected in
the BRCA2 in breast cancer and control groups
	Exon	Sequence Variant	Amino Acid Variant	Frequency in the Breast
Cancer Group
(n=156), abs (%)	Frequency in the control
group
(n=112), abs (%) 	BIC Entry
	Total	Caucasian/Asian	Total	Caucasian/Asian

	Missense
Mutations
	10	c.865(A>C)†
(1093A>C) ‡
	p.Asn289His (N289H) 	24(15.2)*	13(54.2)/
11(45.8)
	11(10.1) 	7(63.6)/
4(36.4) 	Yes
	10	c.2127(T>C) (2235T>C) 	p.Asn709Arg
(N709R) 	65(41.6)*	36(55.4)/
29(44.6)#-
	52(46.4)	29(55.8)/
23(44.2)
	Yes
	11	c.2350(A>G) (2578A>G) 	p.Met784Val
(M784V) 	26(17.0)	16(61.5)/
10(38.5)#-
	24(21.4)	13(54.2)/
11(45.8)
	Yes
	11	c.2410(G>A) (2638G>A) 	p.Asp804Asn (D804N) 	54(34.7) 	33(61.1)/
21(38.9)#
	38 (33.9) 	16(42.1)/
22(57.9) 	Yes
	11	c.3422(T>C) (3650T>C) 	p.Ile1141Thr (I1141T) 	39(24.9) 	15(38.5)/
14(35.9) 	26 (23.2) 	16(61.5)/
10(38.5)#
	Yes
	11	c.3572(C>T) (3800C>T) 	p.Ser1191Phe (S1191F) 	34(21.8) 	15(44.1)/
19(55.9)# 	23 (20.5) 	10(43.5)/
13(56.5) 	Yes
	27	c.10234(A>G) (10462A>G) 	p.Ile3412Val (I3412V) 	48 (30.8)* 	29(60.4)/
19(39.6) # 	21 (18.7) 	10(47.6)/
11(52.4) 	Yes
	Polymorphisms
	10	c.1365(A>G) (1593A>G) 	Synonymous Ser455Ser (S455S) 	81(52.2*) 	48(59.2)/
33(40.7)#
	48(42.8) 	19(39.6)/
29(60.4)#
	Yes
	11	c.2229(T>C) (2457T>C)
	Synonymous His473His (H473H) 	81(52.2)*	48(59.2)/
33(40.7)#
	48(42.8) 	19(39.6)/
29(60.4)#
	Yes
	14	c.7242(A>G) (7470A>G)
	Synonymous Ser2414Ser (S2414S)
	61(39.0)*	35(57.3)/
26(42.6)#
	36(32.1)	16(44.4)/
20(55.5)
	Yes
	† Numbering according
to GenBank Accession
no. NM_000059.1, the A of the ATG translation initiation codon is +1,
according to approved guidelines (http://www.hgvs.org/mutnomen/).
‡The nomenclature as used in the BIC database is given in parentheses.
Mutation nomenclature is according to GenBank accession number U43746
(BRCA2) with numbering starting at the A of the first ATG at the
posotion 229. * - p<0.05, ** - p<0.001 between cases and
controls
#- p<0.05, ##-- p<0.001 between Caucasians and Asians in
group






There was no significant difference in frequency of missense mutations
c.2350A>G (M784V), c.2410G>A (D804N), c.3422T>C
(I1141T), c.3572C>T (S1191F) in cases compared to controls.
Synonymous polymorphisms S455S, H473H, were detected in 81 (52.2%)
cases and S2114S was detected in 61 (39.0%) cases, p>0.05 (Table
3). 



All identified polymorphisms were previously reported in the BIC
database26 (Table 2,3).




Interestingly, frequency of nearly all sequence variants was
significantly different in Caucasian and Asian groups of study
participants (Table 2,3). There was higher prevalence of observed
mutations in Caucasian cases comparing to Asian breast cancer cases
(p<0.05) and higher frequency of sequence variants was observed
in the control group in Asians.


The clinical and pathological characteristics of women with breast
cancer are summarized in:




Table 4: Clinical-pathological profile of breast cancer cases
with/or
without BRCA1/2 polymorphisms
		Cases (n=156)
	Polymorphism Positive 

(n = 111) 71.2%	Polymorphism Negative 

(n = 45) 28.8%

	1 polymorphism	15(13.6)	
	2-3 polymorphisms	17(15.2)	
	4-6 polymorphisms	79(71.2)	
	Age	50.6±8.8	52.7±10.7
	Caucasian	66(59.3)	28(62.5)
	Asian	45(40.7)	17(37.5)
	Tumor
histology
	 Invasive ductal	68(61.0)	32(70.8)
	 Invasive lobular	34(30.5)	13(29.2)
	 Medullary	8(6.8)	
	 Mucinous	2(1.7)	
	Tumor
size
	 T1	1(1.1)	4(8.4)
	T2	66(59.3)	30(66.7)
		30(27.1)	9(20.8)
	 T4	38(3.4) 	2(4.2)
	Lymph
node
involvement
	 Yes	21(18.6)	4(16.7)
	 No	90(81.4)	20(83.3)
	T – primary tumor: T1 -
≤2cm, T2 – >2 to 5cm, T3 - >5cm, T4 – tumor of any size
with direct extension to chest wall or skin, N – regional lymph nodes,
M – distant metastasis 




There was no significant difference between both groups of cases in
mean age at diagnosis (50.6±8.8 years vs. 52.7±10.7 years,
respectively, p=0.36). Assuming that the absolute numbers of
co-existing BRCA1/2 alterations in each case may also play a relevant
biological role, the cases in the mutation-positive group were divided
in three subgroups: (a) with 1 alteration; (b) with 2-3 alterations;
(c) with 4-6 alterations. We found that 79 of 156 women (71.2%) carried
4-6 alterations. This may be due to polymorphisms located in one
haplogroup and inherited together. Also amongst controls, in 48 (73.8%)
of 65 alteration carriers we detected more than 3 mutations
simultaneously (data not shown).



Invasive ductal carcinoma was the predominant histological subtype in
both groups (61.0% and 70.8%, respectively). Primary tumors larger than
5.0 cm were more frequently found in women carrying sequence
alterations. However, the size of tumor (T stage) and lymph node
involvement did not show a statistically significant difference between
these two groups (p=0.92 and p=1.0, respectively).






Discussion



156 Kazakhstan patients with sporadic breast cancer were analyzed for
mutations throughout the entire coding regions of the BRCA1 and BRCA2
genes, using direct sequencing. Whereas the majority of studies on BRCA
gene mutations have focused on western populations with a family
history of breast or ovarian cancer, only a relatively small number of
investigations on the role of the BRCA genes have been undertaken in
Asian sporadic breast cancer populations. A large number of distinct
mutations in the BRCA1 and BRCA2 genes have been reported worldwide,
but little is known regarding the role of these inherited
susceptibility genes in breast cancer risk among Kazakhstan women. So
far there was no information about the role of the BRCA1/2 gene in
breast cancer risk among Kazakhstan women. 



The
incidence of detectable BRCA1 sequence alterations was estimated for
the first time in females with sporadic breast cancer as well as in
healthy women from Semipalatinsk (East Kazakhstan) region of Kazakhstan
in our studies for the first time beginning from 2006.46
We showed a
higher prevalence of BRCA1 sequence alterations in exon 11 in 59
(71.1%) from 83 women with breast cancer and in 65 (58.0%) from 112
healthy women (controls). 



In the present study, the entire
coding regions of BRCA1 and BRCA2 were analyzed, and patients with
sporadic breast cancer were selected by excluding patients at high risk
of being mutation-carriers. This included those with a family history
of breast or ovarian cancer, those diagnosed at less than 35 years of
age, and those with bilateral or multifocal breast cancer. Given that
the great majority of breast cancer cases are sporadic, further
extensive studies are needed to precisely identify the roles of BRCA
genes in sporadic breast cancer. In our present study we sequenced all
coding regions of both BRCA1 and BRCA2 genes in larger group of cases
including an Astana cohort, where women gathered from different
Kazakhstan regions. 



The sequence variants identified in the
BRCA1/2 genes include 16 missense mutations of unknown clinical
significance and 6 synonymous polymorphisms by mutation type (Table 2,
3 and Figure 1). All cases of the single nucleotide changes in BRCA1
and BRCA2 detected in the study were recorded according to the Breast
Cancer Information Core.26




It seems likely that five mutations in BRCA1 (c.95G>T,
c.254 A>G, c.2612C>T, c.3113A>G, c.3348A>G)
and three
mutations in BRCA2 (c.2127T>C, c.2410G>A,
c.10234A>G) are
neutral polymorphisms, in view of the relatively high allele
frequencies (>30%) of these variants. Because of the
unavailability
of a functional BRCA protein assay system, the disease associations of
other mutations remain uncertain. However, the possibility cannot be
ruled out that some of these unverified variants are pathogenically
relevant.




No deleterious mutations were detected in either gene among
studied groups. The lack in the germline of clearly deleterious
alterations might be unsurprising. In fact, these are extremely rare in
patients not selected due to a family history and/or early disease
onset.41 However, the true contribution made by
the BRCA genes to
sporadic breast cancer remains controversial for a number of reasons.
First, missense mutations with an unknown significance could have a
pathogenic effect. Secondly, in addition to missense mutations, silent
polymorphisms may affect the splicing mechanism. However, these
variants cannot be classified as disease associated in the absence of a
good functional assay system for BRCA1 and BRCA2. When a functional
assay becomes available, it will be important to elucidate the
relevance of such variations with unknown clinical significances.




To our knowledge, this report is the first to include
information on the prevalence of missense mutations of unknown
significance, and to provide information on polymorphisms in the
Kazakhstan population in both BRCA1 and BRCA2 genes. Moreover, these
Kazakhstan population-based polymorphisms could be used as potential
markers.




Missense mutation Q356R in BRCA1 was detected in 11 (7.2%)
breast cancer cases and 10 (8.9%) control subjects. A study on the
BRCA1 polymorphisms reported that the Arg356 allele had a higher
genotype distribution in healthy controls than in breast cancer
patients30 and may thus play a protective role
against breast cancer.
In this study, the polymorphism at codon 356 in the BRCA1 gene had
previously been described as being inversely associated with breast
cancer risk (Gln356→ Arg, OR 0.88, 95% confidence interval [CI]
0.63–1.23; Arg356→ Arg, OR 0.00, 95% CI 0.00–0.56).30
Another study
showed that Q356R polymorphism was significantly associated with family
history of ovarian cancer, suggesting that this sequence variant may
increase ovarian cancer risk.31




In contrast, Tommasi et al.47 analyzed BRCA1
mutational risk
using Myriad II software and showed that K1183R, the polymorphism in
exon 11 as reported in BIC,26 resulted inversely
related with BRCA1
mutation carrier status. Also they showed that BRCA1 sequence
alterations such as P871L and E1038G were not significantly related
with higher BRCA1 mutational risk.47 This data
leads us to suggest
further investigation of the effects of these sequence variants on
BRCA1 activity to understand whether these variations have any
pathological role. 



We found that 79 of 156 women (71.2%)
carried 4-6 alterations. This may be due to polymorphisms located in
one haplogroup that are inherited together. Dunning et al examined the
frequency of four polymorphisms: Gln356Arg, Pro871Leu, Glu1038Gly and
Ser1613Gly in large series of breast and ovarian cancer cases and
matched controls.30. Due to strong linkage
disequilibrium, these four
sites generate only three haplotypes with a frequency >1.3%. The
two
most common haplotypes, defined by the alleles
Gln356Pro871Glu1038Ser1613 and Gln356Leu871Gly1038Gly1613, have
frequencies of 0.57 and 0.32, respectively, and these frequencies do
not differ significantly between patient and control groups, indicating
that the most common polymorphisms of the BRCA1 gene do not make a
significant contribution to breast or ovarian cancer risk.30




Common polymorphisms in BRCA1/2 genes appear to be highly
prevalent in Kazakhstan breast cancer cases and in healthy controls
which is in concordance with previously reported findings in some Asian
and European populations.37-43, 47-49
Frequencies of these
polymorphisms were higher in breast cancer cases vs controls,
particularly in the BRCA2 gene, p<0.05. To date, there is little
evidence that highly penetrant, germline mutations in BRCA1/2 are
observed in sporadic cases, but whether common polymorphisms play a
role in disease risk is still controversial. 



The roles of
common missense SNPs, as well as variation in noncoding regions (that
may influence risk through expression levels and alternative splicing),
have yet to be thoroughly explored (at single loci as well as
throughout the genome) as markers of breast cancer susceptibility.
Ongoing efforts to systematically characterize genetic polymorphisms,
such as the International HapMap Project,50
provide the foundation for
conducting comprehensive association studies of common variation.




In a large study, Freedman ML et al tested common variation
across the BRCA1 locus in African American, Native Hawaiian, Japanese,
Latino, and White women in the Multiethnic Cohort Study.51
28 single
nucleotide polymorphisms (SNPs) spanning the BRCA1 gene were used to
define patterns of common variation in these populations. The majority
of SNPs were in strong linkage disequilibrium with one another. Nine
tagging SNPs, including five missense SNPs, were selected to predict
the common BRCA1 variants and haplotypes among the non–African American
groups (five additional SNPs were required for African Americans) and
genotyped in a breast cancer case-control study nested in the
Multiethnic Cohort Study (cases, n = 1,715; controls, n = 2,502). In
their another study52 Freedman ML et al observed
most of the common
BRCA2 haplotypes to be shared among Native Hawaiians, Japanese,
Latinos, and Whites; four of the eight common haplotypes were found in
at least three ethnic populations and six of the eight were found in at
least two groups. Authors found no evidence for significant
associations between common variation in BRCA1/2 and risk of breast
cancer.51,52 



In another study, Cox DG et al reported a
modestly positive association between a BRCA1 haplotype and breast
cancer among White women in the Nurses’ Health Study (OR, 1.18, 95% CI,
1.02-1.37).53 



Interestingly, the frequencies of nearly all
sequence variants were significantly different in Caucasian and Asian
groups among study participants (Table 2, 3). There was higher
prevalence of observed mutations in Caucasian cases comparing to Asian
breast cancer cases, (p<0.05) and higher frequency of sequence
variants was observed in control group in Asians. Further studies of
large numbers of cases may give a more accurate estimation of
prevalence and variations between Asian and Caucasian populations in
Kazakhstan. The vast majority of common variation is shared between
populations; however, allele frequencies are known to vary across
populations54 and studies conducted in a
multiethnic
population may
lend insight into better understanding ethnic differences in breast
cancer risk.55




Although the role of common variation in BRCA1 and sporadic
breast cancer risk has been thoroughly addressed, it remains a
possibility that this locus may still prove to be involved in breast
cancer risk. Specifically, rare (<5%) variants may contribute to
disease; to address this hypothesis, however, large-scale resequencing
efforts (to discover the rare variants) and testing of these variants
in larger cohorts, such as the National Cancer Institute Consortium of
Cohorts,56 will be required. Another possibility
is that a sporadic
breast cancer is actually a collection of genetically distinct
subclasses of breast cancer. In this scenario, it would be unlikely
that the same set of underlying susceptibility alleles occur in all
breast cancer cases (i.e., the genetic architecture of disease is not
genetically homogeneous). If these subgroups are not recognized and
analyzed separately, then the power to detect them will be diminished.
At the histologic and molecular levels, breast tumors have different
characteristics; subsets of breast tumors as defined by
immunohistochemistry [e.g., estrogen-receptor (+/-) and HER2/neu(+/-)]
often display different biological behaviors, such as time to disease
progression and response to therapy, which may reflect different
genetic origins. Studies have shown that breast tumors of women with
hereditary breast cancer with mutations in BRCA1 and a subset of women
with sporadic disease (f25%) share similar traits, including a basal
cell histology, higher grade tumors, cytokeratins 5/6, and estrogen
receptor negativity, suggesting they may have a similar etiology.57-59
The ability to stratify breast cancer cases by expression profiling,
immunohistochemistry, methylation patterns, and/or clinical variables
may facilitate the identification of more genetically homogeneous
subsets of cancer cases, and therefore may help to identify causal
variants underlying specific breast cancer phenotypes.




There are several limitations to this study. We did not
include results of screening entire exons of BRCA1 and BRCA2 genes in
families with breast/ovarian cancer members. This study is still
ongoing because family BC cases in Kazakhstan are sparse. Further
evaluation is needed to clarify the relationship between frequency of
sequence alterations in BRCA1/2 genes and breast cancer risks in
Kazakhstan women in family-based and genetically homogeneous cases. 



In
conclusion, 156 Kazakhstan patients with sporadic breast cancer were
analyzed for mutations throughout the entire coding regions of the
BRCA1 and BRCA2 genes, using direct sequencing. The present
investigation revealed 22 different sequence variants. Although we
found none of pathological deleterious mutations in BRCA1/2 genes, we
believe that the present study allows a better evaluation of the need
for the genetic screening of BRCA mutations in sporadic breast cancer
patients in Kazakhstan. However, large population-based screening
studies are needed to establish the frequency, penetrance, and
significance of the broad spectrum of variations in the sequence of
BRCA1/2 genes in Kazakhstan population. It is hoped that similar
mutation surveys in other Central Asian countries will be completed so
that information can be compared and the most common mutations
identified.






Acknowledgements



We are thankful to all patients and healthy women who took part in this
study and to the staff of Semey and Astana Oncological centers who
helped to collect samples. This work was supported by grant-in-aid from
the Kazakhstan Ministry of Education and Science.





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