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Research Article

Characterization of BRCA1/2 mutations in patients with family history of breast cancer in Armenia

[version 1; peer review: 2 approved]
PUBLISHED 10 Jan 2017
Author details Author details
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Abstract

Background. Breast cancer is one of the most common cancers in women worldwide. The germline mutations of the BRCA1 and BRCA2 genes are the most significant and well characterized genetic risk factors for hereditary breast cancer. Intensive research in the last decades has demonstrated that the incidence of mutations varies widely among different populations. In this study we attempted to perform a pilot study for identification and characterization of mutations in BRCA1 and BRCA2 genes among Armenian patients with family history of breast cancer and their healthy relatives. Methods. We performed targeted exome sequencing for BRCA1 and BRCA2 genes in 6 patients and their healthy relatives. After alignment of short reads to the reference genome, germline single nucleotide variation and indel discovery was performed using GATK software. Functional implications of identified variants were assessed using ENSEMBL Variant Effect Predictor tool. Results. In total, 39 single nucleotide variations and 4 indels were identified, from which 15 SNPs and 3 indels were novel. No known pathogenic mutations were identified, but 2 SNPs causing missense amino acid mutations had significantly increased frequencies in the study group compared to the 1000 Genome populations. Conclusions. Our results demonstrate the importance of screening of BRCA1 and BRCA2 gene variants in the Armenian population in order to identity specifics of mutation spectrum and frequencies and enable accurate risk assessment of hereditary breast cancers.

Keywords

breast cancer, BRCA1, BRCA2, mutation screening, targeted exome sequencing

Introduction

Breast cancer (BC) is one of the most common cancers in females worldwide1 and particularly in Armenia2. Despite the high prevalence of this disease in developed countries, it has become highly prevalent in developing countries (50% of all cancer cases) and is characterized by high mortality rate (58% of all breast cancer related deaths)3.

The germline mutations of the BRCA14 and BRCA25 genes are the most significant and well characterized genetic risk factors for hereditary breast cancer, which constitutes about 5–10% of all cases6. Inherited mutations in BRCA1 and BRCA2 genes account for 30–50% of all known mutations associated with this disease7,8. Women who carry BRCA1 mutations are particularly susceptible to the development of breast cancer before the age of 35–40 with a probability rate of 45%–60%, whereas women who inherit a BRCA2 mutation have a 25%–40% risk of developing breast cancer7,8. The association of BRCA1/BRCA2 gene mutations with breast cancer was first well described in Ashkenazi Jews811. Intensive research in the last decades has demonstrated that the incidence of mutations in high-risk families varies widely among different populations6. For example, the mutations in BRCA1 and BRCA2 were each estimated to account for 45–50% of families with multiple cases of breast and ovarian cancer in UK and USA3,12, whereas mutation prevalence among African–Americans with family breast and ovarian cancer history was 16.3% for BRCA1 and 11.3–14.4% for BRCA213,14, which is significantly lower compared to Caucasian populations. Identification of the BRCA1/BRCA2 mutations in different populations and ethnic groups is an important endeavor, which enables geneticists and oncologists to make more specific choices in genetic testing of members of high-risk families1517.

Here we have attempted to perform a pilot study for identification and characterization of mutations in BRCA1 and BRCA2 genes among Armenian patients with family history of breast cancer and their healthy relatives.

Materials and methods

Samples

Six patients with confirmed family history of breast cancer (at least two cases in a family) and their first-degree healthy relatives were recruited in this study (except for the BC10 patient, see Table 1). Patients were admitted to the National Center of Oncology MH RA and ARTMED Medical Rehabilitation CJSC. Written informed consent forms were obtained from all the study participants. This study was approved by the Institutional Review Board (IRB00004079) of the Institute of Molecular Biology NAS RA.

Table 1. Family structure of the studied subjects.

FamilySampleAgec (Age at
Diagnosis)
DiseaseFamily historyHealthy relative
in study
FAM1BC0167BCYes (daughter)BC02
FAM1BC02HC
FAM2BC0341BCYes (sister [BC04],
grandmother)
BC05
FAM2BC0440BCYes (sister [BC03],
grandmother)
BC05
FAM2BC05HC
FAM3BC0642BCYes (mother, aunt)BC07
FAM3BC07HC
FAM4BC0836BCYes (aunt)BC09
FAM4BC09HC
FAM5BC1038BCYes (mother, aunt)NA

Blood samples were collected in EDTA-containing tubes and genomic DNA was extracted according to the protocol described elsewhere18. A260/A280 ratio measured for evaluation of quality and quantity of extracted DNA was in the range of 1.8–2.

Exome sequencing

BRCA1 and BRCA2 exome sequencing was performed by an external service provider (Admera Health LLC, South Plainfield, NJ, USA) using the proprietary breast cancer panel iBRCATM, which detects genetic variations in all exons of BRCA1 and BRCA2. According to the service provider’s description, this panel utilizes the targeted amplicon (166 amplicons) sequencing method, based on Seq-Ready™ TE Panels protocol (WaferGen Biosystems Inc, Freemont, CA, USA). Reagent cocktails and samples were aliquoted into a 384-well sample source plate. The source plate and BRCA1/2 SmartChip™ were pre-dispensed with Seq-Ready™ TE BRCA1/2 Primers and were placed into the SmartChip™ Multisample Nanodispenser. The SmartChip™ was then amplified with Bio-Rad T100 SmartChip™ TE Cycler. PCR product was then purified with Agencourt AMPure XP (Beckman Coulter, Inc.), according to manufacturer’s instructions. Samples were then quantified with Qubit® 2.0 Fluorometer (Thermo Fisher Scientific, Inc.) and quality analyzed with Tapestation (Agilent Technologies). Sequencing was performed with Illumina MySeq platform on a single lane. Raw reads for each sequenced sample were stored in separate fastq files. DNA samples were shipped on ice to avoid degradation and were passed internal quality check before processing.

Short-read alignment

For each sample, raw sequences were aligned to the human reference genome sequence (hg19, see Public genome data section) using Burrows-Wheeler Aligner (BWA) version 0.7.10 with default parameters. The resulting bam files were used in downstream variant discovery analysis.

Variant discovery

Variant discovery was performed using Genome Analysis Tool Kit (GATK) version 3.6 according to recommended workflows for germline single nucleotide variations (SNVs) and indel discovery in whole genome and exome sequencing data19. Base quality score recalibration, indel realignment and mate pair fixing were performed in bam files. Variant calling was performed without duplicate read removal. SNV and indel discovery and genotyping were performed simultaneously across all samples using standard hard filtering parameters19.

Public genome data

For the alignment, we have used the human reference genome sequence (NCBI build 36.1/hg19) from the UCSC (University of California, Santa Cruz) database (http://genome.ucsc.edu). Known SNPs (single nucleotide polymorphisms) were annotated using the UCSC database (single nucleotide polymorphism database, dbSNP version 135). 1000 Genomes phase 1 genotype data was used for human genetic variations filtration (ftp://ftp.1000genomes.ebi.ac.uk/vol1/ftp/). Allelic frequencies of detected variants were compared against 1000 Genomes phase 3 genotypes, as well as with the genome-wide association study (GWAS) data from 54 healthy Armenian females that were genotyped in the framework of population genetics study by Harber et al.20 (ftp://ngs.sanger.ac.uk/scratch/project/team19/Armenian). The Data on clinically significant BRCA1 and BRCA2 variants were obtained from Breast Cancer Core DataBase maintained by National Human Genome Research Institute (https://research.nhgri.nih.gov/bic/).

Statistical analysis and functional annotation

Comparison of allele frequency distributions in the study group with 1000 Genomes and healthy Armenians was performed using Fisher’s exact test available in R 3.3.2 base package. Variant functional annotation was performed using ENSEMBL Variant Effect Predictor tool21.

Results

In this study we have performed exome sequencing of BRCA1 and BRCA2 genes in patients with a positive family history of breast cancer and their healthy relatives of Armenian origin. Patients’ clinical data and family structure of the studied subjects are presented in the Table 1. The aligned sequencing data is available in the NCBI Sequence Read Archive (SRA, https://www.ncbi.nlm.nih.gov/sra/) under accession SRP095082. For each sample, a total of 166 different primer pairs were used to amplify all the coding regions of BRCA1 and BRCA2 (as described in the Methods section). The average sequencing depth per base per sample was 6696±606. Detailed NGS statistics are presented in Table 2 and Supplementary file S1.

Table 2. Overall NGS statistics.

Number of samples10
Total aligned reads
(percent aligned reads)
1106492 (90%)
Target aligned reads
(percent aligned reads)
1041136 (94%)
Mean coverage depth 6696.9
Target coverage at 1×100%
Target coverage at 10×99.99%
Target coverage at 50×99.95%

In total, variant calling resulted in detection of 232 sequence variations (200 SNVs and 32 indels, Supplementary datasets S2 and S3). Thirty-nine SNVs and 4 indels passed the thresholds after applying hard filters (Table 3).

Table 3. Polymorphic variants in BRCA1 and BRCA2 genes in patients and their healthy relatives.

This table provides functional annotation of mutations in BRCA1 and BRCA2 genes that passed filters during variant calling with GATK.

HGVSgConsequenceImpactGeneHGVSpSIFTPolyPhenClinical significance
13:g.32889775 G>T5' UTRMD BRCA2 ----
13:g.32889792 A>G5' UTRMD BRCA2 ----
13:g.32890572 G>A5' UTRMD BRCA2 ----
13:g.32899159 C>TintronicMD BRCA2 ---uncertain significance,
not provided
13:g.32900933 T>AintronicMD BRCA2 ----
13:g.32906729 A>CmissenseMO BRCA2 p.Asn372Histoleratedbenignnot provided, benign
13:g.32910561 A>GmissenseMO BRCA2 p.Glu690Glydeleteriouspossibly
damaging
-
13:g.32910594 T>GmissenseMOBRCA2p.Phe701Cystoleratedbenign-
13:g.32911888 A>GsynonymousL BRCA2 p.Lys1132Lys--benign
13:g.32913055 A>GsynonymousL BRCA2 p.Leu1521Leu--benign, likely benign
13:g.32913081 A>GmissenseMO BRCA2 p.Lys1530Argdeleteriousprobably
damaging
not provided
13:g.32913609 A>GmissenseMO BRCA2 p.Asn1706Sertoleratedbenignuncertain significance
13:g.32914236 C>TmissenseMO BRCA2 p.Thr1915Mettoleratedbenignbenign
13:g.32918825 T>CintronicMD BRCA2 ----
13:g.32920905 T>CintronicMD BRCA2 ----
13:g.32929232 A>GsynonymousL BRCA2 p.Ser2414Ser--benign
13:g.32929387 T>CmissenseMO BRCA2 p.Val2466Alatoleratedbenignuncertain significance,
benign
13:g.32929451 A>GintronicMD BRCA2 ----
13:g.32936646 T>CintronicMD BRCA2 ---benign
13:g.32953388 T>CintronicMD BRCA2 ---not provided
13:g.32973012 A>C3' UTRMD BRCA2 ---uncertain_significance
17:g.41216021 G>AsynonymousMD BRCA1 ---uncertain significance
17:g.41223094 T>CmissenseMO BRCA1 p.Ser1634Glytoleratedbenignnot provided, benign,
likely benign
17:g.41226601 G>CintronicMD BRCA1 ----
17:g.41231516 C>TintronicMD BRCA1 ----
17:g.41234470 A>GsynonymousL BRCA1 p.Ser1436Ser---
17:g.41244000 T>CmissenseMO BRCA1 p.Lys1183Argtoleratedbenignbenign
17:g.41244429 C>TmissenseMO BRCA1 p.Ser1040Asntoleratedbenignbenign
17:g.41244434 T>CsynonymousL BRCA1 p.Glu1038Glu---
17:g.41244644 T>AsynonymousL BRCA1 p.Pro968Pro---
17:g.41244936 G>AmissenseMO BRCA1 p.Pro871Leutoleratedbenignnot provided
17:g.41245237 A>GsynonymousL BRCA1 p.Leu771Leu--benign
17:g.41245466 G>AsynonymousL BRCA1 p.Ser694Ser--benign
17:g.41245471 C>TmissenseMO BRCA1 p.Asp693Asndeleteriousbenignbenign
17:g.41249324 A>GintronicMD BRCA1 ---uncertain significance,
likely benign
17:g.41251906 T>AintronicMD BRCA1 ----
17:g.41251931 G>AintronicMD BRCA1 ---not provided
17:g.41256032 G>CintronicMD BRCA1 ----
17:g.41256037 C>TintronicMD BRCA1 ----
13:g.32913172 delCframeshiftH BRCA2 ----
17:g.41249364 delAintronicMD BRCA1 ----
17:g.41256076 delAintronicMD BRCA1 ----
17:g.41256087_41256101
delGAAAAAAAAAAGAAA
intronicMD BRCA1 ----

HGVSg – genomic position of mutation notation by Human Genome Variation Society; Consequence – consequence of mutation; Impact – functional impact of mutation (MD – modifier, MO – moderate, L – low, H – high); HGVSp - protein sequence name notation by Human Genome Variation Society; SIFT - prediction of protein function change depending on amino acid substitution using SIFT software (http://sift.jcvi.org/); PolyPhen - prediction of protein function change depending on amino acid substitution using PolyPhen software (genetics.bwh.harvard.edu/pph2/).

From these variants, 18 were novel (15 SNV and 3 indels), and the rest have already been described in 1000 Genomes populations (Table 4). The novel variants were detected only in one or two subjects (8 in healthy relatives and 7 in patients). We identified 12 missense variants (5 in BRCA1 and 7 in BRCA2), 8 synonymous variants (5 in BRCA1 and 3 in BRCA2), 15 intronic variants (8 in BRCA1 and 7 in BRCA2) and 4 in untranslated regions of BRCA2. The frequency distributions of known BRCA1/2 variants were similar to those in 1000 Genomes populations and/or GWAS of healthy Armenians, except for the g.32914236 C>T (pFisher=8.35E-24 vs Armenians, pFisher=0.013 vs 1000 Genomes) and g.41245471 C>T (pFisher=0.013 vs Armenians, pFisher=4.7-E05). No known clinically significant variants were detected in breast cancer patients and their healthy relatives.

Table 4. Distribution of identified variants in healthy Armenians and in 1000 genomes populations.

The frequency distributions of identified mutations in the study group were compared with data from 1000 Genomes population, as well as the genome-wide association study from 54 healthy Armenian females20.

HGVSgRAFMAFRAF 1000
Genomes
MAF 1000
Genomes
RAF
Armenians
MAF
Armenians
13:g.32889775 G>T0,900,10----
13:g.32889792 A>G0,750,250,850,15--
13:g.32890572 G>A0,750,250,790,210,730,27
13:g.32899159 C>T0,950,05----
13:g.32900933 T>A0,700,300,650,35--
13:g.32906729 A>C0,700,300,750,250,720,28
13:g.32910561 A>G0,950,05----
13:g.32910594 T>G0,950,05----
13:g.32911888 A>G0,700,300,730,270,710,29
13:g.32913055 A>G010,030,97--
13:g.32913081 A>G0,950,05----
13:g.32913609 A>G0,950,05----
13:g.32914236 C>T0,900,100,990,010,980,02
13:g.32918825 T>C0,950,05----
13:g.32920905 T>C0,950,05----
13:g.32929232 A>G0,700,300,770,23--
13:g.32929387 T>C010,020,9801
13:g.32929451 A>G0,950,05----
13:g.32936646 T>C0,600,400,470,530,640,36
13:g.32953388 T>C0,600,400,490,510,630,37
13:g.32973012 A>C0,600,400,840,160,700,30
17:g.41216021 G>A0,950,050,990,01--
17:g.41223094 T>C0,500,500,640,36--
17:g.41226601 G>C0,500,500,650,35--
17:g.41231516 C>T0,500,500,650,35--
17:g.41234470 A>G0,500,500,660,340,550,45
17:g.41244000 T>C0,500,500,650,350,550,45
17:g.41244429 C>T0,950,050,990,010,980,02
17:g.41244434 T>C0,900,10----
17:g.41244644 T>A0,950,05----
17:g.41244936 G>A0,500,500,460,540,530,47
17:g.41245237 A>G0,500,500,660,340,550,45
17:g.41245466 G>A0,500,500,660,340,610,39
17:g.41245471 C>T0,700,300,970,030,920,08
17:g.41249324 A>G0,950,05----
17:g.41251906 T>A0,950,05----
17:g.41251931 G>A0,800,200,900,100,780,22
17:g.41256032 G>C0,950,05----
17:g.41256037 C>T0,950,05----
13:g.32913172 delC0,950,05----
17:g.41249364 delA0,500,500,670,33--
17:g.41256076 delA0,250,50----
17:g.41256087_41256101
delGAAAAAAAAAAGAAA
0,500,18----

MAF – minor allele frequency; RAF – reference allele frequency.

Discussion

This study provides preliminary characterization of variations in BRCA1 and BRCA2 genes in Armenian patients with family history of breast cancer. Our data suggest that no known clinically significant variants22 contribute to the disease development in these patients. Meanwhile, two other frequent mutations were identified that cause missense substitutions in coding regions of BRCA1 and BRCA2 and were predicted as having pathogenic consequence. The results of this study are in agreement with a a previous report, which also failed to identify known high risk mutations of BRCA1 and BRCA2 genes in Armenian patients using high-resolution melting PCR approach23,24.

Mutations in BRCA1 and BRCA2 genes are known markers for hereditary breast/ovarian cancer25. Currently more than 100 clinically important mutations and polymorphisms have been described. Genetic testing of these mutations was among the first included in the guidelines for cancer prognostics3,4. Nowadays, in many countries genetic testing is routinely prescribed to patients in high-risk groups for hereditary breast and ovarian cancer2628. However, it has also become apparent that the distribution and appearance of particular risk alleles in BRCA1 and BRCA2 genes is population dependent, and in many cases population specific mutations are being identified811. This is especially relevant to populations that have for a long time remained culturally and genetically isolated811, as in the case of Armenians. Recent research has demonstrated that the genetic structure of Armenians “stabilized” about 4000 years ago and has remained almost unchanged since that time20. Furthermore, our own data indicate that the frequencies of genetic variations associated with various complex human diseases share similarities both with European and Asian populations2931. From the other side, Armenian genomes are highly underrepresented in the current human genome sequencing initiatives and little is known about genetic predisposition to complex diseases in this particular population.

In conclusion, despite the small sample size limitation, our results demonstrate the importance of screening of BRCA1 and BRCA2 gene variants in the Armenian population in order to identity specifics of mutation spectra and frequencies and enable accurate assessment of the risk of hereditary breast cancers.

Data availability

The aligned sequencing data is available in the NCBI Sequence Read Archive (SRA) under accession number SRP095082 (https://www.ncbi.nlm.nih.gov/sra/?term=SRP095082). Scripts and vcf files with called and filtered genotypes are available: DOI, 10.5281/zenodo.21561532.

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Atshemyan S, Chavushyan A, Berberian N et al. Characterization of BRCA1/2 mutations in patients with family history of breast cancer in Armenia [version 1; peer review: 2 approved]. F1000Research 2017, 6:29 (https://doi.org/10.12688/f1000research.10434.1)
NOTE: If applicable, it is important to ensure the information in square brackets after the title is included in all citations of this article.
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ApprovedThe paper is scientifically sound in its current form and only minor, if any, improvements are suggested
Approved with reservations A number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.
Not approvedFundamental flaws in the paper seriously undermine the findings and conclusions
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PUBLISHED 10 Jan 2017
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Reviewer Report 17 Mar 2017
Lusine Nazaryan-Petersen, Department of Cellular and Molecular Medicine (ICMM), Faculty of Health Science, University of Copenhagen, Copenhagen, Denmark 
Approved
VIEWS 7
This is a very important pilot study characterizing variations in BRCA1 and BRCA2 genes in Armenian patients with family history of breast cancer. It provides a good background for further large-scale study in Armenia.

I have few notes to consider: ... Continue reading
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Nazaryan-Petersen L. Reviewer Report For: Characterization of BRCA1/2 mutations in patients with family history of breast cancer in Armenia [version 1; peer review: 2 approved]. F1000Research 2017, 6:29 (https://doi.org/10.5256/f1000research.11244.r21044)
NOTE: it is important to ensure the information in square brackets after the title is included in all citations of this article.
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Reviewer Report 01 Mar 2017
David A. Goukassian, PhD Center for Translational Medicine, Temple University School of Medicine, Philadelphia, PA, USA 
Approved
VIEWS 9
The breast cancer is an important health problem in Armenia and identifying specific genetic factors that may predispose to breast cancer development, especially in the families of patient that were already diagnosed with this condition may improve significantly the dire ... Continue reading
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CITE
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Goukassian DA. Reviewer Report For: Characterization of BRCA1/2 mutations in patients with family history of breast cancer in Armenia [version 1; peer review: 2 approved]. F1000Research 2017, 6:29 (https://doi.org/10.5256/f1000research.11244.r20365)
NOTE: it is important to ensure the information in square brackets after the title is included in all citations of this article.

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Alongside their report, reviewers assign a status to the article:
Approved - the paper is scientifically sound in its current form and only minor, if any, improvements are suggested
Approved with reservations - A number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.
Not approved - fundamental flaws in the paper seriously undermine the findings and conclusions
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