Research article
Open Access
Open Peer Review

This article has Open Peer Review reports available.

How does Open Peer Review work?

Next-generation sequencing in familial breast cancer patients from Lebanon

  • Nadine Jalkh1,
  • Eliane Chouery1,
  • Zahraa Haidar1,
  • Christina Khater2,
  • David Atallah3,
  • Hamad Ali4, 5,
  • Makia J. Marafie6,
  • Mohamed R. Al-Mulla7,
  • Fahd Al-Mulla5, 8Email author and
  • Andre Megarbane9Email author
BMC Medical GenomicsBMC series – open, inclusive and trusted201710:8

https://doi.org/10.1186/s12920-017-0244-7

©  The Author(s). 2017

Received: 20 August 2016

Accepted: 2 February 2017

Published: 15 February 2017

Open Peer Review reports

Abstract

Background

Familial breast cancer (BC) represents 5 to 10% of all BC cases. Mutations in two high susceptibility BRCA1 and BRCA2 genes explain 16–40% of familial BC, while other high, moderate and low susceptibility genes explain up to 20% more of BC families. The Lebanese reported prevalence of BRCA1 and BRCA2 deleterious mutations (5.6% and 12.5%) were lower than those reported in the literature.

Methods

In the presented study, 45 Lebanese patients with a reported family history of BC were tested using Whole Exome Sequencing (WES) technique followed by Sanger sequencing validation.

Results

Nineteen pathogenic mutations were identified in this study. These 19 mutations were found in 13 different genes such as: ABCC12, APC, ATM, BRCA1, BRCA2, CDH1, ERCC6, MSH2, POLH, PRF1, SLX4, STK11 and TP53.

Conclusions

In this first application of WES on BC in Lebanon, we detected six BRCA1 and BRCA2 deleterious mutations in seven patients, with a total prevalence of 15.5%, a figure that is lower than those reported in the Western literature. The p.C44F mutation in the BRCA1 gene appeared twice in this study, suggesting a founder effect. Importantly, the overall mutation prevalence was equal to 40%, justifying the urgent need to deploy WES for the identification of genetic variants responsible for familial BC in the Lebanese population.

Keywords

Breast cancer BRCA Next-generation sequencing Exome Familial Lebanon Germline Mutation

Background

Breast cancer (BC) is the most common cancer in women, accounting for around 25% of all new cases of cancer [1]. Most BC cases are sporadic, while 5 to 10% of all BC cases are inherited and cluster in families [1]. While mutations in BRCA1 and BRCA2 genes explain 16–40% of all familial BC cases [1–3], other genes have also been found to increase BC susceptibility, which highlights the polygenic nature of many BC cases [4]. Some of these genes including CDH1, TP53, PTEN and STK11, although less frequently altered compared to the BRCA1/2 genes, they have been linked to high-penetrance autosomal dominant BC [5–7]. Moderate penetrance genes are implicated in around 5% of familial BC. These genes include the Fanconi anemia pathway genes: FANCA, PALB2, BRIP1, RAD51C and XRCC2 [8–10] and non-Fanconi anemia genes: ATM, CHEK2, NBN, RAD50, RAD51B, and RAD51D [11–15].

In Lebanon, BC is the most common cancer type in females and it constitutes one-third of all reported cancer cases. BC incidence rates are expected to reach 137 per 100,000 by 2018 [16]. Yet, to date, only two studies have investigated the role of BRCA1 and BRCA2 mutations in the Lebanese population. These studies reported varied prevalence of pathogenic BRCA mutations ranging between 5.6 to 12.5% in BC cases [17, 18]. The reported prevalences of both BRCA1 and BRCA2 deleterious mutations were lower than those reported for the Western populations, which suggest the involvement of other genes in the pathogenesis of BC cases [19]. The reported low prevalence does not support the hypothesis that BRCA1 and BRCA2 mutations alone are responsible for the majority of the observed Lebanese women with early-onset BC. This finding could well explain the fact that BC is a disease with a high level of genetic heterogeneity and that monogenic and polygenic models of inheritance may exist.

Since the completion of the human genome project, massive leaps have reshaped the field of clinical genomics. The development of Next-generation sequencing (NGS) platforms allowed a more robust, fast and accurate analysis of diseases and syndromes with polygenic nature. NGS platforms including WES are believed to enhance and improve diagnosis and therapy development of many diseases including BC [20–23].

In the presented study, we utilized WES to investigate germline genetic variations in 45 Lebanese cases diagnosed with familial BC and unknown BRCA1 or BRCA2 status. We found several rare variants that can potentially explain BC susceptibility in the analyzed cases.

Methods

Inclusion criteria

From 2012 to 2015, 45 unrelated patients with inherited BC were selected to undergo DNA testing. They were referred from a wide variety of settings from all over the country, ranging from private physicians’ clinics to major academic medical centers because of hereditary BC. The patients fulfilled a personal history of invasive BC and at least one of the following criteria: A) diagnosis at age ≤ 40 years, B) BC at any age at onset with at least 2 first- and/or second-degree relatives, C) BC < 50 years in a first- or second-degree relative, D) ovarian cancer in at least 2 first- and/or second-degree relatives, E) breast and ovarian cancer in at least 2 first- and/or second-degree relatives, F) both breast and ovarian cancer in a single first- or second-degree relative.

Approval to conduct the study was obtained from the Ethics Committee of Saint-Joseph University-Lebanon. After an informed consent was signed and all ethical requirements were fulfilled, a 10 ml of peripheral blood was isolated from each individual enrolled and the DNA was extracted using the salting out methods [24]. All patients signed the informed consent and agreed to share their variant data.

Whole exome sequencing

Exon capture and sequencing: Samples were prepared for whole Exome sequencing and enriched according to the manufacturer’s standard protocol. The concentration of each library was determined using Agilent’s QPCR NGS Library Quantification Kit (G4880A). Samples were pooled prior to sequencing with each sample at a final concentration of 10nM. Sequencing was performed on the Illumina HiSeq2000 platform using TruSeq v3 chemistry.

Mapping and alignment: Reads files (FASTQ) were generated from the sequencing platform via the manufacturer’s proprietary software. Reads were aligned to the hg19/b37 reference genome using the Burrows-Wheeler Aligner (BWA) package v0.6.1 [25]. Local realignment of the mapped reads around potential insertion/deletion (Indel) sites was carried out with the Genome Analysis Tool Kit (GATK) v1.6 [26]. Duplicate reads were marked using Picard v1.62. Additional BAM file manipulations were performed with Samtools 0.1.18 [27]. Base quality (Phred scale) scores were recalibrated using GATK’s covariance recalibration. SNP and Indel variants called using the GATK Unified Genotyper for each sample [28]. SNP novelty is determined against dbSNP. A list of 134 genes known to be associated with hereditary BC and other cancers were studied (Additional file 1).

Variants evaluation

Variants obtained were reported using five categories according to the Human Genome Mutation Database (HGMD Professional) [29]. These categories are listed in Table 1.
Table 1

Variants reported in five categories according to the HGMD Professional

Category

Category

Variation reported as

DM

Disease-causing mutations

Pathological mutation

DM?

Disease-causing mutations

Likely pathological mutation

DP

Disease-associated polymorphism

Polymorphism in significant association with a disease/phenotype (p < 0.05) that is assumed to be functional

DFP

Disease-associated polymorphism

Polymorphism in significant association with disease (p < 0.05) that has evidence of being of direct functional importance

FP

In vitro/laboratory or in vivo functional polymorphism

Polymorphism that affects the structure, function or expression of the gene (or gene product), but with no disease association reported as yet

The first variant category consists of alleles labeled as disease causing mutations (DM) in HGMD Professional. These alleles must be rare: <1% allele frequency in 6,500 exomes from the National Heart, Lung, and Blood Institute (NHLBI) Exome Sequencing Project (“Exome Variant Server” 2015) and the 1,000 Genomes Project Genomes [30].

The BRCA gene variants identified were checked for pathogenicity in 4 databases: Breast Cancer Information Core (BIC) [31], Leiden Open Variation Database (LOVD) [32], the Catalogue of Somatic Mutations in Cancer database (COSMIC) [33] and BRCA Exchange website (http://brcaexchange.org) providing data from the ENIGMA consortium [34].

Variants confirmation

Sanger’s sequencing was utilized to confirm the relevant variants identified by WES and to study the segregation of these variants with the disease phenotype in members of families included in the study. PCR reactions were run in final volumes of 50 μl containing 100 ng DNA, 0.25 mM dNTPs, 100 ng of each primer and 0.02 unit of Taq polymerase (Invitrogen Life Technologies, Carlsbad, CA, USA). PCR was performed in an ABI9700 thermocycler (Applied Biosystems, Foster City, CA) with initial denaturation at 94 °C for 5 min, followed by 35 cycles of 95 °C for 30s, specific annealing temperature for 30s, 72 °C for 30s. Primer sequences are available on request as well as annealing temperatures of each exon. PCR products were purified using the illustraTM GFX PCR DNA and Gel Band Purification Kit (GE Healthcare, Buckinghamshire, UK). Both strands of the products were sequenced using the BigDyeW Terminator v1.1 Cycle Sequencing Kit (Applied Biosystems, Foster City, CA) under standard conditions. The labeled products were subjected to electrophoresis on an ABI3130 and ABI3500 Genetic Analyzer sequencing system (Applied Biosystems, Foster City, CA, USA). Electropherograms were analyzed using Sequence Analysis Software v5.2 (Applied Biosystems, Foster City, CA, USA) and compared to reference sequences using ChromasPro v1.7.6.1 (Technelysium, Queensland, Australia).

Results

Patient characteristics and sequencing statistics

The mean age at diagnosis of BC for the 45 patients was 44 years (range 29–79). Sixteen patients provided us with their histopathological results. Seven BC were estrogen-receptor (ER) and progesterone-receptor (PR) positive, 5 patients had negative ER and PR disease and 2 patients had negative ER and positive PR disease. Two patients had triple negative disease from which one patient (Family 30) carried p.C44F mutation in BRCA1 (Fig. 1).
Fig. 1

Pedigrees of the families presenting DM mutations. Filled squares (males) and circles (females) indicate the affected individuals. Probands are marked with arrows. DM mutations are bolded

We obtained an average of 44 million reads per sample, with a mean coverage of 94% at a mean X coverage of 20X.

WES analysis

Within this cohort, a total of 126 variants were detected by WES and these are listed in Table 2. In 7 of the 45 patients, not listed in Table 2, no variants in cancer predisposing genes (Additional file 1) were identified.
Table 2

Mutations in BC associated genes detected by NGS in a series of 45 Lebanese patients

Family number/Patient code

Genes

Results

HGMD Professional

ExAC allele frequency

1/B19

XRCC3

c.C722T p.T241M

DFP Association with melanoma

0.3075

XRCC1

c.G839A p.R280H

DFP Association with increased lung cancer

0.08811

CASP8

c.*429A > G

Not found

Not found

BRCA2

c.C65T p.A22V

Not found

Not found

MUTYH

c.C1258G p.L420V

Not found

Not found

SLX4

c.C1837T p.Q613X

Not found

Not found

2/B21

BRCA1

c.A536G p.Y179C

DM Breast and/or ovarian cancer

0.0002718

ATM

c.T2572C p.F858L

DP Association with breast cancer

0.009149

ATM

c.C3161G p.P1054R

DFP Association with breast cancer

0.01692

TP53

c.C215G p.P72R

DFP Association with Lung cancer

0.6600

3/B22

BRCA2

c.C5744T p.T1915M

DP Association with breast cancer risk

0.01790

ARL11

c.G446A p.W149X

DP Association with cancer

0.009898

TP53

c.C215G p.P72R

DFP Association with Lung cancer

0.6600

ERCC3

c.C508T p.R170X

Not found

Not found

VHL

c.A631C p.M211L

Not found

0.00004623

MRE11A

c.C1491T p.I497I

Not found

0.0006514

PTCH1

c.G4054A p.V1352I

Not found

Not found

4/B23

TP53

c.C215G p.P72R

DFP Association with Lung cancer

0.6600

TP53

c.C245T p.P82L

DM Breast cancer

0.00001657

BRCA2

c.A1114C p.N372H

DFP Association with Breast cancer

0.2779

FANCA

c.G1038C p.W346C

Not found

0.00006621

POLE

c.C3890T p.S1297L

Not found

0.00002580

POLD1

c.T2257C p.Y753H

Not found

Not found

GATA2

c.C1040T p.T347I

Not found

Not found

5/B24

BRCA2

c.G8775C p.Q2925H

Not found

0.000008322

APC

c.C6821T p.A2274V

DM Adenomatous polyposis coli

0.0009917

EZH2

c.C349T p.Q117X

Not found

Not found

6/B25

XRCC3

c.C722T p.T241M

DFP Association with melanoma

0.3075

MLH1

c.A655G p.I219V

DP Colorectal cancer, non-polyposis

0.2325

RAD51D

c.G494A p.R165Q

Not found

Not found

ATM

c.496 + 4 T > C

Not found

0.00009891

PPM1D

c.G275C p.C92S

Not found

Not found

STK11

c.375-1C > T

DM Colorectal cancer

Not found

7/B26

XRCC3

c.C722T p.T241M

DFP Association with melanoma

0.3075

MSH2

c.C1045G p.P349A

DM Renal cell carcinoma

0.00009062

MUTYH

c.C1174A p.L392M

Not found

Not found

MUTYH

c.C1258A p.L420M

DM? Colorectal cancer

Not found

ATM

c.496 + 4 T > C

Not found

0.00009891

RB1

c.C1505T p.T502I

Not found

0.00001098

PPM1D

c.G275C p.C92S

Not found

Not found

8/B27

XRCC3

c.C722T p.T241M

DFP Association with melanoma

0.3075

BRCA1

c.A1067G p.Q356R

DP Association with breast and/or ovarian cancer

0.04407

XRCC1

c.C580T p.R194W

DFP Benign breast disease

0.09276

CDH1

c.G1774A p.A592T

DM? Breast cancer

0.003212

35/B28

BARD1

c.1071_1091del p.357_364del

Not found

Not found

ABCC12

c.G490T p.G164X

DM Bladder cancer

0.003185

MCC

c.G152T p.G51V

Not found

0.0001346

9/B31

ATM

c.T2119C p.S707P

DFP Association with Breast cancer

0.007927

FANCA

c.C4232T p.P1411L

Not found

0.0001318

36/B32

ATM

c.C2770T p.R924W

Not found

0.00004942

ALK

c.T4211C p.L1404P

Not found

0.00008370

10/B33

BRIP1

c.A3571G p.I1191V

Not found

0.00004967

NSD1

c.2224_2243del p.P742fs

Not found

Not found

FANCG

c.G1298C p.R433P

Not found

0.00004118

FLCN

c.T1387C p.Y463H

Not found

0.00003298

PTCH1

c.A3749G p.Y1250C

Not found

Not found

11/B34

XRCC4

c.T401C p.I134T

DP Association with Lung cancer

0.02505

RAD51C

c.G376A p.A126T

FP Reduced activity

0.003529

12/B35

ARL11

c.G571A p.G191R

Not found

0.00002188

Rad50

c.A280C p.I94L

DM? Breast and/or ovarian cancer

0.003473

POLE

c.G2276A p.R759H

Not found

0.00001647

13/B36

BRCA1

c.G131T p.C44F

DM Breast and/or ovarian cancer

Not found

SLX4

c.G421T p.G141W

DM Breast and/or ovarian cancer

0.0008237

STK11

c.C1211T p.S404F

DM Peutz-Jeghers syndrome

0.0009281

PALB2

c.G2993A p.G998E

DP Breast cancer, increased risk-

0.01579

BRCA2

c.C1151T p.S384F

DM? Breast cancer

0.0006789

DICER1

c.A5276G p.K1759R

Not found

0.00004942

CEBPA

c.T122C p.I41T

Not found

Not found

RECQL4

c.G3314A p.G1105D

Not found

0.005430

14/B37

RAD50

c.G379A p.V127I

Not found

0.001653

CASP8

c.A1117G p.I373V

Not found

Not found

RECQL4

c.C3184T p.R1062W

Not found

0.0003129

WRN

c.G4129A p.G1377S

Not found

0.00002483

15/B38

BARD1

c.C119T p.A40V

Not found

0.00004775

PTCH1

c.169_170delCT p.57_57del

Not found

0.000008913

PTCH1

c.A3749G p.Y1250C

Not found

Not found

PTCH1

c.C4126T p.R1376W

Not found

Not found

ERCC5

c.A1904G p.H635R

Not found

Not found

DICER1

c.C3811T p.L1271F

Not found

Not found

16/B39

CDKN2A

c.G442A p.A148T

DP Association with melanoma

0.02278

RAD51D

c.A758G p.E253G

Not found

0.01144

ERCC6

c.C2800A p.P934T

DM Cockayne syndrome

Not found

17/B40

MSH2

c.A1787G p.N596S

DM Colorectal cancer, non-polyposis

0.0002558

ATM

c.A1982C p.D661A

Not found

Not found

PMS2

c.G1688T p.R563L

DM? Colorectal cancer, non-polyposis

0.005813

GPC3

c.78_79insCCG p.P27delinsPP

Not found

Not found

18/B41

BRCA2

c.658_659delGT p.V220I*

DM Breast and/or ovarian cancer

0.00006119

SLX4

c.G3337C p.G1113R

Not found

0.000008237

SMARCA4

c.C1098G p.I366M

Not found

0.00002715

EPHX1

c.G1040C p.R347T

Not found

0.00003296

19/B42

wwoxtv2

c.A544G p.K182E

DM? cancer

Not found

ATM

c.A5558T p.D1853V

DP Association with breast cancer, contralateral

0.005186

RET

c.C2508T p.S836S

DP Association with thyroid cancer

0.04666

BRCA1

c.5090_5093delGTTA p.L1697fs

Not found

Not found

20/B43

PALB2

c.G2014C p.E672Q

DM? Breast cancer? (common variant)

0.02239

PALB2

c.G2993A p.G998E

DP Breast cancer (common variant)

0.01579

RAD51C

c.G376A p.A126T

FP Reduced activity

0.003529

Tp53

c.673-36G > C

DFP Breast cancer

Not found

21/B44

SLX4

c.G421T p.G141W

DM Breast and/or ovarian cancer

0.0008237

SLX4

c.C1919A p.T640N

Not found

Not found

FANCM

c.A5224G p.I1742V

Not found

0.008398

POLD1

c.G2793C p.K931N

Not found

Not found

22/B45

ATM

c.A5071C p.S1691R

DM Ataxia telangiectasia

0.002019

BRIP1

c.G2220T p.Q740H

DM? Breast and/or ovarian cancer

0.0004614

RET

c.C2508T p.S836S

DP Association with thyroid cancer

0.04666

FANCA

c.A796G p.T266A

DP Associated with breast cancer

0.5166

23/B46

BARD1

c.1071_1091del p.357_364del

Not found

Not found

FANCA

c.C3412G p.L1138V

Not found

0.001533

MRE11A

c.A1728T p.R576R

Not found

0.000008238

SLX4

c.C1186G p.L396V

Not found

Not found

37/B47

SLX4

c.A5501G p.N1834S

Not found

0.005542

ERCC4

c.G1633C p.G545R

Not found

0.000008243

38/B48

SDHC

c.C31T p.R11C

Not found

0.000008252

FANCD2

c.A1348G p.I450V

Not found

0.0003871

FANCF

c.C959T p.P320L

Not found

0.01264

TSC2

c.A2834G p.K945R

Not found

Not found

DIS3L2

c.1651_1652insGGG p.A551delinsGA

Not found

Not found

GNAS

c.C1046T p.P349L

Not found

Not found

24/B49

BRCA1

c.C4327T p.R1443*

DM Breast cancer

Not found

MSH2

c.C1045G p.P349A

DM Renal cell carcinoma

0.00009062

MSH2

c.G965A p.G322D

DM? Colorectal cancer, non-polyposis

0.01411

BARD1

c.G253T p.V85L

Not found

0.001068

NBN

c.G340T p.V114F

Not found

Not found

RET

c.C2249G p.A750G

Not found

0.000008238

XRCC3

c.C260T p.P87L

Not found

0.00006286

25/B50

POLH

c.A2074G p.T692A

DM Xeroderma pigmentosum

0.0001824

Tp53

c.673-36G > C

DFP Breast cancer

Not found

CTNNB1

c.A2315G p.N772S

Not found

0.00003355

POLD1

c.C519G p.S173R

Not found

0.009212

26/D1

ARL11

c.G446A p.W149X

DP Association with cancer

0.009898

MSH2

c.T1182G p.F394L

Not found

0.00001648

27/D4

CHEK2

c.T470C p.I157T

DFP Li-Fraumeni syndrome

Not found

28/D5

CDH1

c.G2387A p.R796Q

Not found

0.00003300

29/D6

BUB1B

c.A1535G p.E512G

Not found

0.000008239

30/D7

APC

c.C2876T p.S959F

Not found

Not found

BRCA1

c.G131T p.C44F

DM Breast and/or ovarian cancer

Not found

PRF1

c.G3A p.M1I

DM Haemophagocytic lymphohistiocytosis, familial

Not found

31/D8

TP53

c.G469A p.V157I

DM Sarcoma, adult-onset

0.00005776

32/III_4

CDH1

c.G3A p.M1I

DM Gastric cancer

Not found

BRCA2

c.C4061T p.T1354M

DM Breast cancer

0.000008328

BRCA2

c.G4258T p.D1420Y

DM? Breast and/or ovarian cancer

0.006796

33/D12

CDH1

c.A160G p.R54G

Not found

0.00005916

34/D13

BRCA2

c.G223C p.A75P

DM? Breast cancer

0.0001650

DM disease-causing mutation, DM? likely disease-causing mutation, DP disease-associated polymorphism, FP in vitro or in vivo functional polymorphism, DFP disease-associated polymorphism with additional functional evidence

We were able to detect 19 HGMD DM variations of which 9 are specifically associated with breast cancer (Table 2). The distribution of the remaining variants in the HGMD categories was: 11 DM?, 11 DP, 1 FP, and 9 DFP. In addition, 75 novel variations were detected in this study (Table 2).

Six BRCA1 and BRCA2 DM mutations were detected in 5 and 2 patients, respectively in a total prevalence of 15.5% (Table 2).

Nine truncating mutations were detected in 9 different patients (Table 2). Three of these mutations were DM in HGMD: The first woman carried p.R1443* in BRCA1, the second one carried p.V220I* in BRCA2 and the third one carried p.G164X in ABCC12 (Table 2). The six remaining truncating mutations were not found in HGMD: p.Q613X in SLX4, p.R170X in ERCC3, p.Q117X in EZH2, p.P742fs in NSD1, p.357_364del in BARD1 and p.L1697fs in BRCA1 (Table 2).

Three DM mutations were found, each one, in 2 different patients: p.C44F in BRCA1 (Families 13 and 30), p.P349A in MSH2 (Families 7 and 24) and p.G141W in SLX4 (Fig. 1 and Table 2).

In some families where different variants were found, in order to consider, which variant is pathogenic, we analyzed the co-segregation of the variations found with the cancer phenotype within 3 families 12, 13, and 32 (Figs. 1 and 2).
Fig. 2

Pedigree of one family segregated for BRCA2 p.T1354M and CDH1 p.M1I variants. Filled squares (males) and circles (females) indicate the affected individuals. + sign indicates the presence of the variant and – sign the absence of the variant in tested individuals

Two members of family 12 were diagnosed with BC, their mother and maternal uncle were diagnosed with primary lung cancer and bone cancer, respectively. The nonsmoking mother was affected at the age of 63 but the age of the maternal uncle at diagnostic was not accessible. WES, in proband 12/B35 diagnosed with BC at the age of 42, identified 2 variants including one DM? p.I94L in RAD50, according to HGMD Professional database, and one novel variation p.G191R in ARL11 (data not shown). Prediction tool Polyphen2 indicated that both changes are benign and SIFT prediction tool indicated that p.I94L in RAD50 is tolerated and that p.G191R in ARL11 is damaging. Only p.I94L in RAD50 segregated in the affected sisters, diagnosed with BC at the age of 48, but it was also found in their third youngest 51 years old unaffected sister.

Three members of Family 13 were diagnosed with BC. WES identified 3 DM according to HGMD Professional database, including p.C44F in BRCA1, p.G141W in SLX4 and p.S404F in STK11 (Table 2). Leiden Open Variation Database indicated that p.C44F in BRCA1 affects protein function and it segregated with the disease (Table 3) (Fig. 1).
Table 3

BRCA variations found and their evaluations in BRCA databases

Gene

Variation

BIC database Clinically Importance/ Clinical Classification

COSMIC

Leiden Open Variation Database (LOVD)

BRCA Exchange

BRCA1

c.G131T p.C44F

unknown/ pending

Not found

Affects function

Not found

c.A536G p.Y179C

unknown/ pending

Not found

Does not affect function

Benign

c.C4327T p.R1443*

yes/ class 5

Neutral

Affects function

Not found

c.A1067G p.Q356R

unknown/ pending

Pathogenic

Does not affect function

Benign

c.5090_5093delGTTA p.L1697fs

Not found

Not found

Not found

Not found

BRCA2

c.C65T p.A22V

unknown/pending

Not found

Effect unknown

Not found

c.G223C p.A75P

unknown/ pending

Not found

Does not affect function

Benign

c.658_659delGT p.V220I*

yes/ class 5

Not found

Affects function

Not found

c.C4061T p.T1354M

unknown/ pending

Neutral

Does not affect function

Benign

c.G4258T p.D1420Y

no/ pending

Neutral

Does not affect function

Benign

c.C5744T p.T1915M

no/ class 1

Neutral

Does not affect function

Not found

c.G8775C p.Q2925H

unknown/ pending

Not found

Effect unknown

Not found

c.A1114C p.N372H

no/ class 1

Neutral

Not found

Benign

c.C1151T p.S384F

no/ pending

Not found

Not found

Benign

Descriptions of the classes in the BIC database:

Class 1: Not pathogenic/low clinical significance: There is significant evidence against this variant being a dominant high-risk pathogenic mutation

Class 5: Pathogenic: There is significant evidence to suggest that this variant is a dominant high-risk pathogenic mutation

Six members of family 32 were diagnosed with BC (Fig. 2). Members III-3, III-4 and III-6 were diagnosed with BC at the age of 56, 48 and 50, respectively. WES in proband III-4 identified 2 relevant variants including p.M1I in CDH1 and p.T1354M in BRCA2. Prediction tool SIFT indicated that both changes are damaging and are DM according to HGMD Professional database (Table 2 and Fig. 2). The analysis of this family showed that these variations were carried by affected and siblings that are not affected to date (Fig. 2). However, they were advised to join our screening program.

We have noted that the most frequently altered genes involved in our familial cases are DNA repair genes (Fig. 3a) and that some variants were recurrent in our cohort: p.W149X in ARL11, p.S836S in RET, p.A126T in RAD51C, p.T241M in XRCC3, p.G998E in PALB2 and c.673-36G > C in TP53 (Table 2 and Fig. 3b). In four cases, like the 4 families shown in Fig. 1, individuals appear to co-inherit multiple cancer causing or predisposing gene mutations. Unlike, the old strategy where one stops the investigation once a pathogenic mutation was identified, NGS gives us the capability of collating all known mutations/variants in a sample, which may permit a more comprehensive understanding of the polygenic landscape model of cancer. An important question to be answered is: Does an individual in Family 13 harboring all three DM mutation have different penetrance, genotype to phenotype correlation, type or age of onset of cancer than a sibling with only one DM variant? This critical question can only be answered when we start to combine all germline variant data of cancer patients and their comprehensive phenotypes from around the world in well-curated databases.
Fig. 3

Frequencies of all variants in breast cancer predisposing genes from our 45 patients’ cohort (a) and the details of the most frequent variants shown in (b)

Discussion

We identified, in 45 patients with familial BC, 19 pathogenic mutations that are DM mutations according to the HGMD Professional database (Table 2). These 19 mutations were found in 13 different genes including ABCC12, APC, ATM, BRCA1, BRCA2, CDH1, ERCC6, MSH2, POLH, PRF1, SLX4, STK11, and TP53. Six mutations were found in BRCA1 and BRCA2 presenting a lower prevalence (15.5%) of deleterious BRCA mutations compared to the published literature [21–23].

In the Lebanese population, p.C44F mutation in the BRCA1 gene was found twice in this study and 5 times in previous studies [17, 18] in a total of 7 from 367 cases studied (1.9%). In fact, 2 of 9 patients carried a deleterious BRCA mutation in a cohort of 72 patients and 3 of 14 patients carried a deleterious BRCA mutation in a cohort of 250 patients. Our findings suggest it is the most recurrent mutation in the Lebanese population.

In families 23 and 35, we identified the truncating mutation p.357_364del in BARD1 (Table 2). A previous study, on this variation, showed the absence of co-segregation with the disease and it was considered as neutral polymorphisms [35]. We have observed this variant in our population and breast cancer patients and it is recommended that a more thorough and functional examination of this variant be conducted in the future.

In families 12, 13 and 32, we identified 7 variants in ARL11, BRCA1, BRCA2, CDH1, RAD50, SLX4, and STK11. The association of which variation towards increasing predisposition to BC remains unknown. Therefore, we analyzed the segregation of these variations and BC within the families. In family 13, only p.C44F in BRCA1 segregated with BC in the family. In family 12, p.I94L in RAD50 (a DM? mutation) was found in affected and healthy sisters and could therefore not lead to a conclusion regarding predisposition to BC. In family 32, p.M1I in CDH1 and p.T1354M in BRCA2 are implicated in gastric cancer and BC respectively and knowing that the family presented with only BC, two hypothesis can be formulated. First, III-6 can be considered as phenocopy and second healthy, till now, sisters III-5, III-7 and III-9 are at high risk (Fig. 2). In fact, in high-risk families, women testing negative for the familial BRCA mutation have an increased risk of BC and should be considered for continued surveillance [36]. Interestingly, two members of this family, III-4 and III-6 presented with invasive lobular breast cancer (Fig. 2). The association between CDH1 gene mutation and lobular cancer has been well established previously [37], and it is not unrealistic to suggest that this CDH1 variant may be the cause of lobular breast cancer in this family.

The pathogenic status of the majority of novel substitutions found and the 6 variations considered as DM? according to HGMD professional, remains problematic (Table 2). In fact, HGMD professional reports DM? as likely pathological mutation reported to be disease causing in the corresponding report, but the author has indicated that there may be some degree of doubt, or subsequent evidence has come to light in the literature, calling the deleterious nature of the variant into question [29]. Further studies are needed to define the pathogenic status of the novel substitutions and the DM? variations that have been found in our cohort of patients with BC. These future studies have to be analyzed in a larger number of affected families and control population samples.

NGS and traditional sequencing methods are not proficient in detecting BRCA genomic rearrangements including large deletions or duplications. Deletion and duplication genomic rearrangements vary significantly among countries and within ethnic groups [38]. We admit, therefore, that our reported BRCA mutation prevalence is underestimated.

Among the DM mutations found, several were associated with syndromes (Peutz-Jeghers), different cancer types (renal cell carcinoma, gastric cancer) and with diseases (Xeroderma pigmentosa, ataxia telangiectasia) (Table 2). Clinically, none of the symptoms found in these diseases were manifested in the different studied families except for family 24. In this family, proband 24/B49 carried the mutation p.R1443* in BRCA1 and two MSH2 variants (Fig. 1). Her mother had ovarian cancer and her sister uterine cancer, both are deceased and could not consequently be tested for these variants. MSH2 mutation is reported in families with endometrial cancer (Lynch syndrome) and breast cancer from Kuwait [39].

This is the first application of NGS on BC in Lebanon. In this study, we showed that the prevalence of deleterious BRCA mutations (15.5%) is lower than expected [17, 18] and that the overall mutation prevalence is equal to 40%, justifying the urgent need for the adoption of high-throughput NGS technologies to identify genes responsible for familial BC in the Lebanese population. Indeed, additional to BRCA mutations, highly penetrant mutations in genes associated with various hereditary cancer syndromes, such as CDH1, TP53, MSH2, ATM and POLH were found in the Lebanese population. Finally, we cannot rule out that some of these families shift a putative explanation towards a polygenic model where moderate and low penetrance alleles, acting together, may play a predominant role [20, 40, 41]. Our findings support the eligibility of performing genetic testing by massively parallel sequencing on Lebanese familial BC cases. Moreover, we would like to use this technology for tumor genome sequencing, in order to identify somatic alterations, which would be a valuable guidance towards individualized cancer therapy of Lebanese patients with BC. However, it is worthy of note that our study reports a small number of variants that are clinically actionable. Given the high rate of novel variants identified in BRCA1/2 and other breast cancer-associated genes, the clinical usefulness of the data is currently limited. Unless larger and rigorous studies are committed in this area of the world to correctly classify variants identified here or in other studies, the diagnosis and treatment of breast cancer will remain suboptimal.

Conclusion

This is the first study that utilized NGS technology to study genetic variants in 45 patients with familial breast cancer from Lebanon. Our deleterious mutation prevalence was 40% with only 15.5% accounted for by the BRCA1 and BRCA2 genes. This data should encourage a different strategy for familial breast cancer genetic screening in Lebanon, one that is based on WES rather than the initial screening of BRCA1/2 genes. We report here novel and rare variants in breast cancer predisposing genes, which will be valuable to researchers and clinicians around the world for variants’ classification and patients’ care in general.

Abbreviations

BC: 

Breast cancer

BIC: 

Breast cancer information core

BWA: 

Burrows-wheeler aligner

COSMIC: 

Catalog of somatic mutations in cancer database

DFP: 

Disease-associated polymorphism

DM: 

Disease-causing mutations

DM?: 

Disease-causing mutations

DP: 

Disease-associated polymorphism

FP: 

In vitro/laboratory or in vivo functional polymorphism

GATK: 

Genome analysis tool kit

HGMD Professional: 

Human genome mutation database

LOVD: 

Leiden open variation database

NGS: 

Next generation sequencing

NHLBI: 

National heart, lung, and blood institute

WES: 

Whole exome sequencing.

Declarations

Acknowledgements

We would like to thank all the patients and their family members who contributed their samples and information for this study.

Funding

This work was supported by a grant from the Kuwait Foundation for the Advancement of Sciences (KFAS No. 2011-1302-06) given to Prof. Fahd Al-Mulla and by grants from the Saint Joseph University and the Mohamad Cheaib Foundation.

Availability of data and materials

Data generated or analyzed during this study are included in this published article and its Additional file. The link to the compiled data will be shared on our website http://www.al-mulla.org, which will be made available once it is connected to international databases in the near future. Meanwhile, data supporting the manuscript can be requested from the any of the two corresponding authors.

Authors' contributions

NJ, FM, AM: drafted the manuscript. NJ, EC, ZH, HA, FM, MRM: carried out the molecular genetic studies and participated in the sequence alignment. CK, DA, MJM: acquisition of data. FM, HA, MRM, NJ: performed statistical analysis and interpretation of data. FM, AM conceived of the study, participated in the design of the study, and in its design and coordination. All authors read and approved the final manuscript.

Competing interests

The authors report no conflicts of interest. The authors alone are responsible for the content and writing of the paper.

Consent for publication

All patients signed the informed consent during the counseling sessions and agreed to share their variant data in a reputable scientific journal.

Ethics approval and consent to participate

Approval to conduct the study was obtained from the Ethics Committee of Saint-Joseph University-Lebanon. Written informed consent was obtained at the time of enrollment for genetic sample collection.

Open AccessThis article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.

Authors’ Affiliations

(1)
Unité de Génétique Médicale, Pôle Technologie Santé, Faculty of Medicine, Saint Joseph University
(2)
Trad Hospital
(3)
Department of Gynecology and Obstetrics, Hôtel-Dieu de France University Hospital, Saint Joseph University
(4)
Department of Medical Laboratory Sciences (MLS), Faculty of Allied Health Sciences, Health Sciences Center (HSC), Kuwait University
(5)
Dasman Diabetes Institute (DDI)
(6)
Kuwait Medical Genetics Center, Maternity Hospital
(7)
Department of Computing Sciences and Engineering, Kuwait University
(8)
Health Sciences Center, Faculty of Medicine, Department of Pathology, Kuwait University
(9)
Institut Jerome Lejeune

References

  1. Melchor L, Benitez J. The complex genetic landscape of familial breast cancer. Hum Genet. 2013;132(8):845–63.View ArticlePubMedGoogle Scholar
  2. Miki Y, Swensen J, Shattuck-Eidens D, Futreal PA, Harshman K, Tavtigian S, et al. A strong candidate for the breast and ovarian cancer susceptibility gene BRCA1. Science. 1994;266(5182):66–71.View ArticlePubMedGoogle Scholar
  3. Wooster R, Bignell G, Lancaster J, Swift S, Seal S, Mangion J, et al. Identification of the breast cancer susceptibility gene BRCA2. Nature. 1995;378(6559):789–92.View ArticlePubMedGoogle Scholar
  4. Scalia-Wilbur J, Colins BL, Penson RT, Dizon DS. Breast Cancer Risk Assessment: Moving Beyond BRCA 1 and 2. Semin Radiat Oncol. 2016;26(1):3–8.View ArticlePubMedGoogle Scholar
  5. Borresen AL, Andersen TI, Garber J, Barbier-Piraux N, Thorlacius S, Eyfjord J, et al. Screening for germ line TP53 mutations in breast cancer patients. Cancer Res. 1992;52(11):3234–6.PubMedGoogle Scholar
  6. Giardiello FM, Brensinger JD, Tersmette AC, Goodman SN, Petersen GM, Booker SV, et al. Very high risk of cancer in familial Peutz-Jeghers syndrome. Gastroenterology. 2000;119(6):1447–53.View ArticlePubMedGoogle Scholar
  7. Lynch ED, Ostermeyer EA, Lee MK, Arena JF, Ji H, Dann J, et al. Inherited mutations in PTEN that are associated with breast cancer, cowden disease, and juvenile polyposis. Am J Hum Genet. 1997;61(6):1254–60.View ArticlePubMedPubMed CentralGoogle Scholar
  8. Rahman N, Seal S, Thompson D, Kelly P, Renwick A, Elliott A, et al. PALB2, which encodes a BRCA2-interacting protein, is a breast cancer susceptibility gene. Nat Genet. 2007;39(2):165–7.View ArticlePubMedGoogle Scholar
  9. Seal S, Thompson D, Renwick A, Elliott A, Kelly P, Barfoot R, et al. Truncating mutations in the Fanconi anemia J gene BRIP1 are low-penetrance breast cancer susceptibility alleles. Nat Genet. 2006;38(11):1239–41.View ArticlePubMedGoogle Scholar
  10. Shamseldin HE, Elfaki M, Alkuraya FS. Exome sequencing reveals a novel Fanconi group defined by XRCC2 mutation. J Med Genet. 2012;49(3):184–6.View ArticlePubMedGoogle Scholar
  11. Loveday C, Turnbull C, Ramsay E, Hughes D, Ruark E, Frankum JR, et al. Germline mutations in RAD51D confer susceptibility to ovarian cancer. Nat Genet. 2011;43(9):879–82.View ArticlePubMedPubMed CentralGoogle Scholar
  12. Meijers-Heijboer H, van den Ouweland A, Klijn J, Wasielewski M, de Snoo A, Oldenburg R, et al. Low-penetrance susceptibility to breast cancer due to CHEK2(*)1100delC in noncarriers of BRCA1 or BRCA2 mutations. Nat Genet. 2002;31(1):55–9.View ArticlePubMedGoogle Scholar
  13. Orr N, Lemnrau A, Cooke R, Fletcher O, Tomczyk K, Jones M, et al. Genome-wide association study identifies a common variant in RAD51B associated with male breast cancer risk. Nat Genet. 2012;44(11):1182–4.View ArticlePubMedPubMed CentralGoogle Scholar
  14. Porhanova NV, Sokolenko AP, Sherina NY, Ponomariova DN, Tkachenko NN, Matsko DE, et al. Ovarian cancer patient with germline mutations in both BRCA1 and NBN genes. Cancer Genet Cytogenet. 2008;186(2):122–4.View ArticlePubMedGoogle Scholar
  15. Renwick A, Thompson D, Seal S, Kelly P, Chagtai T, Ahmed M, et al. ATM mutations that cause ataxia-telangiectasia are breast cancer susceptibility alleles. Nat Genet. 2006;38(8):873–5.View ArticlePubMedGoogle Scholar
  16. Shamseddine A, Saleh A, Charafeddine M, Seoud M, Mukherji D, Temraz S, et al. Cancer trends in Lebanon: a review of incidence rates for the period of 2003–2008 and projections until 2018. Popul Health Metr. 2014;12(1):4.View ArticlePubMedPubMed CentralGoogle Scholar
  17. El Saghir NS, Zgheib NK, Assi HA, Khoury KE, Bidet Y, Jaber SM, et al. BRCA1 and BRCA2 mutations in ethnic Lebanese Arab women with high hereditary risk breast cancer. Oncologist. 2015;20(4):357–64.View ArticlePubMedPubMed CentralGoogle Scholar
  18. Jalkh N, Nassar-Slaba J, Chouery E, Salem N, Uhrchammer N, Golmard L, et al. Prevalance of BRCA1 and BRCA2 mutations in familial breast cancer patients in Lebanon. Hered Cancer Clin Pract. 2012;10(1):7.View ArticlePubMedPubMed CentralGoogle Scholar
  19. Sundquist M, Thorstenson S, Brudin L, Wingren S, Nordenskjold B. Incidence and prognosis in early onset breast cancer. Breast. 2002;11(1):30–5.View ArticlePubMedGoogle Scholar
  20. Gracia-Aznarez FJ, Fernandez V, Pita G, Peterlongo P, Dominguez O, de la Hoya M, et al. Whole exome sequencing suggests much of non-BRCA1/BRCA2 familial breast cancer is due to moderate and low penetrance susceptibility alleles. PLoS One. 2013;8(2):e55681.View ArticlePubMedPubMed CentralGoogle Scholar
  21. Hall MJ, Reid JE, Burbidge LA, Pruss D, Deffenbaugh AM, Frye C, et al. BRCA1 and BRCA2 mutations in women of different ethnicities undergoing testing for hereditary breast-ovarian cancer. Cancer. 2009;115(10):2222–33.View ArticlePubMedPubMed CentralGoogle Scholar
  22. Malone KE, Daling JR, Neal C, Suter NM, O'Brien C, et al. Frequency of BRCA1/BRCA2 mutations in a population-based sample of young breast carcinoma cases. Cancer. 2000;88(6):1393–402.View ArticlePubMedGoogle Scholar
  23. Uhrhammer N, Abdelouahab A, Lafarge L, Feillel V, Ben Dib A, Bignon YJ. BRCA1 mutations in Algerian breast cancer patients: high frequency in young, sporadic cases. Int J Med Sci. 2008;5(4):197–202.View ArticlePubMedPubMed CentralGoogle Scholar
  24. Miller SA, Dykes DD, Polesky HF. A simple salting out procedure for extracting DNA from human nucleated cells. Nucleic Acids Res. 1988;16(3):1215.View ArticlePubMedPubMed CentralGoogle Scholar
  25. Li H, Durbin R. Fast and accurate short read alignment with Burrows-Wheeler transform. Bioinformatics. 2009;25(14):1754–60.View ArticlePubMedPubMed CentralGoogle Scholar
  26. McKenna A, Hanna M, Banks E, Sivachenko A, Cibulskis K, Kernytsky A, et al. The Genome Analysis Toolkit: a MapReduce framework for analyzing next-generation DNA sequencing data. Genome Res. 2010;20(9):1297–303.View ArticlePubMedPubMed CentralGoogle Scholar
  27. Li B, Krishnan VG, Mort ME, Xin F, Kamati KK, Cooper DN, et al. Automated inference of molecular mechanisms of disease from amino acid substitutions. Bioinformatics. 2009;25(21):2744–50.View ArticlePubMedPubMed CentralGoogle Scholar
  28. DePristo MA, Banks E, Poplin R, Garimella KV, Maguire JR, Hartl C, et al. A framework for variation discovery and genotyping using next-generation DNA sequencing data. Nat Genet. 2011;43(5):491–8.View ArticlePubMedPubMed CentralGoogle Scholar
  29. Stenson PD, Mort M, Ball EV, Shaw K, Phillips A, Cooper DN. The Human Gene Mutation Database: building a comprehensive mutation repository for clinical and molecular genetics, diagnostic testing and personalized genomic medicine. Hum Genet. 2014;133(1):1–9.View ArticlePubMedGoogle Scholar
  30. Clarke L, Zheng-Bradley X, Smith R, Kulesha E, Xiao C, Toneva I, et al. The 1000 Genomes Project: data management and community access. Nat Methods. 2012;9(5):459–62.View ArticlePubMedPubMed CentralGoogle Scholar
  31. Szabo C, Masiello A, Ryan JF, Brody LC. The breast cancer information core: database design, structure, and scope. Hum Mutat. 2000;16(2):123–31.View ArticlePubMedGoogle Scholar
  32. Fokkema IF, den Dunnen JT, Taschner PE. LOVD: easy creation of a locus-specific sequence variation database using an "LSDB-in-a-box" approach. Hum Mutat. 2005;26(2):63–8.View ArticlePubMedGoogle Scholar
  33. Forbes SA, Beare D, Gunasekaran P, Leung K, Bindal N, Boutselakis H, et al. COSMIC: exploring the world's knowledge of somatic mutations in human cancer. Nucleic Acids Res. 2015;43(Database issue):D805–811.View ArticlePubMedGoogle Scholar
  34. Spurdle AB, Healey S, Devereau A, Hogervorst FB, Monteiro AN, Nathanson KL, et al. ENIGMA-evidence-based network for the interpretation of germline mutant alleles: an international initiative to evaluate risk and clinical significance associated with sequence variation in BRCA1 and BRCA2 genes. Hum Mutat. 2012;33(1):2–7.View ArticlePubMedGoogle Scholar
  35. De Brakeleer S, De Greve J, Loris R, Janin N, Lissens W, Sermijn E, et al. Cancer predisposing missense and protein truncating BARD1 mutations in non-BRCA1 or BRCA2 breast cancer families. Hum Mutat. 2010;31(3):E1175–1185.View ArticlePubMedGoogle Scholar
  36. Smith A, Moran A, Boyd MC, Bulman M, Shenton A, Smith L, et al. Phenocopies in BRCA1 and BRCA2 families: evidence for modifier genes and implications for screening. J Med Genet. 2007;44(1):10–5.View ArticlePubMedGoogle Scholar
  37. Masciari S, Larsson N, Senz J, Boyd N, Kaurah P, Kandel MJ, et al. Germline E-cadherin mutations in familial lobular breast cancer. J Med Genet. 2007;44(11):726–31.View ArticlePubMedPubMed CentralGoogle Scholar
  38. Ewald IP, Ribeiro PL, Palmero EI, Cossio SL, Giugliani R, Ashton-Prolla P. Genomic rearrangements in BRCA1 and BRCA2: A literature review. Genet Mol Biol. 2009;32(3):437–46.View ArticlePubMedPubMed CentralGoogle Scholar
  39. Marafie MJ, Al-Awadi S, Al-Mosawi F, Elshafey A, Al-Ali W, Al-Mulla F. Impact of 226C > T MSH2 gene mutation on cancer phenotypes in two HNPCC-associated highly-consanguineous families from Kuwait: emphasis on premarital genetic testing. Fam Cancer. 2009;8(4):289–98.View ArticlePubMedGoogle Scholar
  40. Johnson N, Fletcher O, Palles C, Rudd M, Webb E, Sellick G, et al. Counting potentially functional variants in BRCA1, BRCA2 and ATM predicts breast cancer susceptibility. Hum Mol Genet. 2007;16(9):1051–7.View ArticlePubMedGoogle Scholar
  41. Sehl ME, Langer LR, Papp JC, Kwan L, Seldon JL, Arellano G, et al. Associations between single nucleotide polymorphisms in double-stranded DNA repair pathway genes and familial breast cancer. Clin Cancer Res. 2009;15(6):2192–203.View ArticlePubMedPubMed CentralGoogle Scholar

Copyright

© The Author(s). 2017

Advertisement