BRCA1 and BRCA2 germline mutations in Moroccan breast/ovarian cancer families:
Novel mutations and unclassi fi ed variants
Amal Tazzitea,1, Hassan Jouhadib,1, Sellama Nadifia,⁎, Paolo Aretinic, Elisabetta Falaschic, Anita Collavolic, Abdellatif Beniderb, Maria Adelaide Caligoc
aGenetics and Molecular Pathology Laboratory, Medical school of Casablanca, Casablanca, Morocco
bDepartment of Oncology, Ibn Rochd University Hospital, Casablanca, Morocco
cSection of Genetic Oncology, University Hospital and University of Pisa, Pisa, Italy
a b s t r a c t a r t i c l e i n f o
Article history:
Received 17 January 2012 Accepted 6 March 2012 Available online 13 March 2012 Keywords:
Breast cancer BRCA1 BRCA2
Germline mutations Morocco
Objective.Breast cancer is the most common female cancer in Morocco. About 5 to 10% are due to hered- itary predisposition and mutations inBRCA1andBRCA2genes are responsible for an important proportion of high-risk breast/ovarian cancer families. The relevance ofBRCA1/2 mutations in the Moroccan population was not studied. The main objective of this study is to investigate the spectrum ofBRCA1andBRCA2germline mutations in early onset and familial breast/ovarian cancer among Moroccan women.
Methods.We screened the entire coding sequences and intron/exon boundaries ofBRCA1andBRCA2 genes in 40 patients by direct sequencing.
Results.Nine pathogenic mutations were detected in ten unrelated families,five deleterious mutations in BRCA1gene and four mutations inBRCA2gene. Four novel mutations were found: one inBRCA1(c.2805delA/
2924delA) and three inBRCA2(c.3381delT/3609delT; c.7110delA/7338delA and c.7235insG/7463insG). We also identified 51 distinct polymorphisms and unclassified variants (three described for thefirst time).
Conclusions.Our data suggest thatBRCA1andBRCA2mutations are responsible for a significant proportion of familial breast cancer in Moroccan patients. Therefore fullBRCA1/2 screening should be offered to patients with a family history of breast/ovarian cancer.
© 2012 Elsevier Inc. All rights reserved.
Introduction
Most breast malignant tumor cases are sporadic but about 5 to 10% are due to hereditary predisposition. Genetic linkage studies and positional cloning have identified two major genes associated to hereditary breast cancer susceptibility. BRCA1 and BRCA2 are tumor suppressor genes involved in maintaining of genome integrity by engaging in many processes such as repair of DNA double strand breaks, cell cycle control and transcription[1].
TheBRCA1gene, cloned in 1994[2], has been localized to chromo- some region 17q21 comprising 22 coding exons that encode a protein of 1863 amino acids. TheBRCA2gene, cloned in 1995[3,4], has been located on chromosome 13ql2 with 26 coding exons producing a pro- tein of 3418 amino acids. Germline mutations in BRCA1/2 genes confer an increased risk for breast and ovarian cancers; the average cumulative risk of developing breast cancer by age 70 years is about 57% for BRCA1 and 49% for BRCA2 mutation carriers whereas the
ovarian cancer risk forBRCA1 and BRCA2 mutation carriers is 40%
and 18% respectively[5].
More than a thousand different mutations were listed in the Breast Cancer Information Core database (BIC;http://research.nhgri.
nih.gov/bic/). Mutations are distributed throughout the entire coding region andflanking intronic sequences, most of them are frame-shift, nonsense or splice-site alterations that lead to truncated proteins. The majority ofBRCA1/2 germline alterations are unique but some recur- rent mutations with a founder effect have been reported in some ethnic populations[6].
In Morocco, breast cancer seems to be a major public health prob- lem. It is the most common type of cancer among women accounting for 36.5% of all female cancers and with a mortality rate of 19.7%[7].
Cancer registries taking place in the greater area of Casablanca and Rabat have reported a standardized incidence of 35 and 35.8 per 100,000 women respectively[8,9]. Although this incidence appears higher than the other Maghreb countries[10–12], it remains low compared to developed countries[13–15]. On the other side, the mean age at diagnosis of breast cancer in our country is less than in western countries. In the greater area of Casablanca the average age is 48.1 years (±11.3 years)[8]and 7% of cases registered at the city of Rabat are younger than 35 years[9]. The early onset of this cancer may be explained by genetic predisposition.
⁎Corresponding author at: Genetics and Molecular Pathology Laboratory, Medical School of Casablanca, 19 Rue Tarik Ibnou Ziad, B.P. 9154, Morocco.
E-mail address:labgenmed@yahoo.fr(S. Nadifi).
1The authors contributed equally to this work.
0090-8258/$–see front matter © 2012 Elsevier Inc. All rights reserved.
doi:10.1016/j.ygyno.2012.03.007
Contents lists available atSciVerse ScienceDirect
Gynecologic Oncology
j o u r n a l h o m e p a g e : w w w . e l s e v i e r . c o m / l o c a t e / y g y n o
Moreover, Morocco is a country of northwestern Africa composed predominantly of Berbers and Arabs ethnic groups. The Berbers are an indigenous population with specific language and culture. They are thefirst inhabitants of Morocco and are probably the descendants of Mesolithic Capsian populations and Neolithic people who came from the Near East[16]. In the 7th century, the Arabs conquered Mo- rocco and introduced their civilization. The contribution of these eth- nic groups to the genetic diversity of Moroccan population is evident.
The relevance ofBRCA1/2 mutations in the Moroccan population was not studied. In fact, the main objective of this study is to investi- gate the spectrum ofBRCA1andBRCA2germline mutations in early onset and familial breast/ovarian cancer among Moroccan patients.
Material and methods Patients
The present study included 40 Moroccan early onset and familial breast and/or ovarian cancer cases selected from patients treated in oncology center of Ibn Rochd University Hospital of Casablanca and referred for genetic counseling and consideration ofBRCA1/2 testing to the Genetics and Molecular Pathology Laboratory at the Medical school of Casablanca between 2009 and 2010.
Families were selected according to specific family-history criteria:
a. Three or morefirst or second degree relatives with breast cancer diagnosed at any age in the same familial branch (n = 17);
b. Twofirst degree relatives with breast cancer, with at least one early onset breast cancer case (≤40 years) or male breast cancer case or ovarian cancer case (n = 16).
Single cases were selected from patients diagnosed with breast cancer before age 40 or with breast and ovarian cancer (n = 6).
All patients were asked to provide detailed information regarding personal and family history of cancer by interview. Clinical and path- ological characteristics were collected from medical records and pathology reports. The study was approved by the local ethics com- mittee of our institution and written informed consent was obtained from each subject. Blood samples were taken from at least one affected woman of each family and stored in EDTA tubes.
Molecular analysis
DNA was extracted using the salting out method, confirmed by agarose gel electrophoresis and then quantified using the NANODROP 2000 spectrophotometer (Thermo Scientific).
All exons and exon–intron boundaries ofBRCA1andBRCA2genes were amplified in afinal volume of 25μl containing: 1x reaction buff- er, 1.5 mM MgCl2, 0.2 mM dNTPs, 5μM primers (sequences available on request), 1.25 U Taq polymerase and 50 ng genomic DNA. Exon 11 ofBRCA1andBRCA2genes was analyzed in 8 and 12 overlapping PCR fragments respectively.
Amplification cycles were: 94 °C for 7 min followed by 4 cycles of 94 °C for 0.1 min, 64 °C for 0.1 min, and 72 °C for 1 min, 4 cycles of 94 °C for 0.1 min, 64 °C for 0.1 min, and 72 °C for 1 min, 35 cycles of 94 °C for 0.1 min, 58 °C for 0.1 min, and 72 °C for 1 min and 1 cycle at 72 °C for 7 min, except for exon 15 ofBRCA2for which the amplification conditions were: 94 °C for 7 min followed by 40 cycles of 94 °C for 1 min, 55 °C for 1 min, 72 °C for 2 min and ended with a 7 min incubation at 72 °C. Amplicons were purified by membrane retention (Multiscreen, Millipore).
Purified PCR products were sequenced in both forward and reverse strands, using BigDye Terminator v3.1 cycle sequencing kit (Applied Biosystems), purified over sephadex G50 (GE Healthcare), denatured with deionized formamide and then runned on a ABIPRISM 3130 XL Genetic analyzer (Applied Biosystems). Sequence analyses were performed using SeqScape v2.6 (Applied Biosystems)
software. The samples were not screened for the presence of large deletions and duplications.
All mutations and variants are cited according to Human Genome Variation Sequence systematic nomenclature (HGVS; http://www.
hgvs.org/mutnomen/) using GenBank entries: U14680 for BRCA1 and U43746 forBRCA2and following the rule in which the A of the first ATG translational initiation codon nucleotide is +1. The Breast Cancer Information Core (BIC) nomenclature, based on reference sequences stated above and where A of the ATG translation initiation codon is at positions 120 and 229 ofBRCA1andBRCA2respectively, is also indicated in the tables and text.
In silico prediction
To identify non-synonymous amino acid changes likely to disrupt BRCA1/2 genes function we used three comparative evolutionary bio- informatic programs: Sorting Intolerant From Tolerant (SIFT;http://
blocks.fhcrc.org/sift/SIFT.html), Polymorphism Phenotyping v2 (Poly- Phen-2; http://genetics.bwh.harvard.edu/pph2/) and Align GVGD (http://agvgd.iarc.fr/agvgd_input.php). SIFT is a multiple sequence alignment tool based on the premise that important amino acids will be conserved among species in a protein family, so that changes of amino acids conserved in the family should affect protein function [17]. PolyPhen is an automatic tool for prediction of possible impact of an amino acid substitution on the structure and function of a human protein[18]. Align-GVGD is a program that combines biophysical characteristics of amino acids and protein multiple sequence align- ments to predict the effect of mutations on the encoded proteins[19].
Results
We screenedBRCA1andBRCA2mutations in 40 patients from 39 families with personal and family history of breast and/or ovarian cancers. Six patients were single cases, with no family history, and 34 had a family history of breast and/or ovarian cancers. All women were diagnosed with unilateral breast cancer except two cases with bilateral breast cancer and two patients with breast and ovarian cancer. The median age at diagnosis of breast cancer was 38 years (range 25 to 60).
A total of nineBRCA1 and BRCA2mutations were found in ten unrelated families with a frequency of 25.64% (10/39) (Table 1).
Five deleterious mutations were present in BRCA1 gene and five breast cancer families were carriers ofBRCA2deleterious mutations (12.82%). Additionally, theBRCAmutations are estimated to occur in 60% (3/5) of Berber patients and in 20.6% (7/34) of women of Arab origin (Table 1).
FourBRCA1 mutations were found in families with site-specific breast cancer, and one was found in a breast–ovarian cancer family.
All of these families had one or more cases of breast cancer diagnosed before age 45. On the other hand, families withBRCA2 mutations were breast cancer families. Four had a strong family history of hered- itary breast cancer and the remaining one is a single early onset breast cancer case diagnosed at 33 years old.
Three BRCA1 mutations are small frameshift deletions (c.798_
799delTT/917delTT, c.3279delC/3398delC, c.2805delA/2924delA) leading to premature protein termination at codons 285, 1108 and 999 respectively. They are all located in exon 11. The fourth mutation is a missense mutation c.181T>G (300T>G) located in exon 5 result- ing in the conversion of Cysteine to Glycine at position 61 and occurring in the highly conserved cysteine ligating residues in the RING finger domain of BRCA1. Finally, the c.5062–5064delGTT (5181delGTT) is a single amino acid in frame deletion mutation within exon 17 in BRCA1 COOH terminus (BRCT) domain.
Three distinct frameshift mutations were identified inBRCA2gene, one located in exon 11 (c.3381delT/3609delT) and two in exon 14 (c.7110delA/7338delA, c.7235insG/7463insG). These mutations are
leading to premature truncated proteins at positions 1150, 2376 and 2413 respectively. The c.7110delA (7338delA) was detected in a patient with a strong family history of breast cancer; she has a mother with breast cancer at 40 years of age, a maternal aunt diagnosed at age 45 and two maternal aunts died of breast cancer developed at ages 38 and 42 (Fig. 1). The otherBRCA2mutation was an intron 6 splice site substitution (c.517-1G>A/IVS6-1G>A).
All the mutations found were unique, detected only once, except theBRCA2mutation c.7235insG (7463insG) which was found in two unrelated families thus occurring at a frequency of 5.12% (2/39).
Four novel mutations which have not been reported before in the BIC database were found: one inBRCA1(c.2805delA/2924delA) and three inBRCA2(c.3381delT/3609delT; c.7110delA/7338delA and c.7235insG/7463insG). The c.3279delC (3398delC)BRCA1mutation, even if it has not been cited before in the BIC database or any reports, it was mentioned twice in the Universal Mutation Database (UMD;
http://www.umd.be/)[20,21]without information about their geo- graphic origin. We should note that this last mutation was detected in both the proband and her mother diagnosed with breast cancer at ages 32 and 49 respectively.
Histopathologically, one medullary carcinoma was observed in BRCA1mutation carrier (br59) with a frequency of 20% (1/5) among patients with BRCA1 deleterious mutations. Furthermore, 50%
(5/10) of cases withBRCA1/2mutations had triple negative tumors
(with negative estrogen receptors, negative progesterone receptors and negative Her2/neu status), two carryingBRCA1alterations and three withBRCA2mutations.
The majority of mutated patients in our series are familial cases selected according to two criteria: families with three or morefirst or second degree relatives with breast cancer and families with two first degree relatives with early onset breast cancer, male breast can- cer or ovarian cancer. In thefirst group, 29.4% (5/17) carriedBRCA1/2 mutations whereas 25% (4/16) were mutated in the second group.
Concerning single cases, one out of 6 was found mutated with a fre- quency of 16.6% (1/6). These results show that family history and early onset of breast cancer are two important selection criteria for the identification of patients carryingBRCA1/2 mutations.
In addition to the deleterious mutations, we identifiedfifty one variants (22BRCA1and 29BRCA2) including distinct polymorphisms and unclassified sequence variants (Table 2).
Besides the known variants previously reported three were novel.
The c.594-67G>T (IVS9-67G>T) and c.8488-143G>A (IVS19-143G>A) variants, located in the intron–exon boundaries ofBRCA1andBRCA2 genes respectively, could be benign since the“Human Splicing Finder” software (http://www.umd.be/HSF/) [22] reported no splice site at these positions. The other unknown variant is the c.7462A>G (7690A>G) which produces conversion of Arginine to Glycine at posi- tion 2488 of the BRCA2 protein and listed neither in the BIC database Table 1
BRCA1andBRCA2pathogenic germline mutations in Moroccan families.
Gene Patient code
Exon Systematic nomenclature
BIC nomenclature Described in BIC database
Amino acid change
Mutation type
Manifestation, age at diagnosis
Family history Ethnic origin
BRCA1 br18 5 c.181T>G 300T>G Yes C61G MS BC, 34 y Mother, BC 45 y Arab
br59 11 c.798-799delTT 917delTT Yes Stop285 FS BC, 42 y Mother, BOC 50 y
M cousin, BC 47 y Arab
br24 11 c.2805delA 2924delA No Stop999 FS BC, 41 y M aunt, BC 42 y Berber
br23 11 c.3279delC 3398delC No Stop1108 FS BC, 32 y Mother, BC 49 y Berber
br31 17 c. 5062-5064delGTT 5181delGTT Yes V1688del IFD BC, 25 y M aunt, BC 40 y
M cousin, BC 27 y Arab
BRCA2 br15 Intron 6 c.517-1G>A IVS6-1G>A Yes – IVS BC, 33 y No family history Arab
br3 11 c.3381delT 3609delT No Stop1150 FS BC, 38 y Mother, BC 50 y
M aunt, BC 43 y
Berber
br91 14 c.7110delA 7338delA No Stop2376 FS BC, 38 y Mother, BC 40 y
M aunt, BC 38 y M aunt, BC 42 y M aunt, BC 45 y
Arab
br87 14 c.7235insG 7463insG No Stop2413 FS BC, 40 y Sister, BC 33 y Arab
br51 14 c.7235insG 7463insG No Stop2413 FS BBC, 45 y Sister, BC 41 y
P cousin, BC 40 y P cousin, BC 50 y
Arab
MS: missense, FS: frameshift, IFD: in frame deletion, IVS: intervening sequence, BC: breast cancer, BBC: bilateral breast cancer, BOC: breast and ovarian cancers, M: maternal, P:
paternal.
Fig. 1.Pedigree and sequencing result of a patient carrying c.7110delA/7338delABRCA2deleterious mutation. a. Pedigree of patient with BRCA2 c.7110delA/7338delA mutation.
Shaded circles indicate individuals affected with breast cancer. The index case is indicated with an arrow. Diagonal slash indicates deceased. BC: breast cancer. The numbers follow- ing the abbreviations indicate age of diagnosis. b. Electropherogram for sense and anti-sense strands of exon 14 of BRCA2.
nor in the literature. It was found in a breast cancer case diagnosed at age 36 with a family history of breast cancer in her sister at age 37 and in her maternal aunt at age 50. This variant was predicted to be probably damaging with a score of 1.000 by Polyphen-2 software and to affect protein function with a score of 0.00 by SIFT. Using Align- GVGD algorithm, it was classified as C45 (Table 3). Thereby we cannot exclude the possibility that this mutation could be pathogenic.
Discussion
The molecular genetics revolution has made an outstanding con- tribution to our understanding of genetic disease etiology. Germline mutations inBRCA1andBRCA2have been shown to play an important role in genetic predisposition to breast/ovarian cancer. To our knowl- edge, the contribution of BRCA1 and BRCA2 mutations to familial breast and/or ovarian cancer in Moroccan women is insufficiently
explored up to date. This is thefirst published report of the spectrum and the frequency ofBRCA1andBRCA2mutations among Moroccan women.
Based on our results, the frequency ofBRCA1andBRCA2mutations among Moroccan women with hereditary breast and/or ovarian can- cer is 25.64%. Some studies conducted in neighboring populations such as Tunisia and Algeria have reported frequencies of 19.4%[23]
and 11.4%[24]respectively. Overall, 3 to 50% of familial breast cancer cases are linked to mutations in theBRCA1/2 genes[25]. These differ- ences are mainly due to inclusion criteria of patients and their ethnic- ity. We would like to point out that, in our series,BRCA1mutations are as common asBRCA2mutations. Thisfinding may be due to an underestimation ofBRCA1/2 mutations because of the small number of families analyzed.
In theBRCA1gene, we detectedfive mutations, most of them were identified in exon 11 (3/5). However, this exon alone accounts for 60%
Table 2
Polymorphisms and unclassified variants inBRCA1andBRCA2genes.
Gene Exon Systematic nomenclature BIC nomenclature Amino acid change Type Identified in patients with BRCA1/2 deleterious mutation
Minor allele frequencya
BRCA1 Intron 7 c.442-34C>T IVS7-34C>T Non coding IVS Yes 0.23
7 c.425C>A 544C>A P142H MS Yes 0.01
Intron 8 c.548-58delT IVS8-58delT Non coding IVS Yes 0.22
Intron 9 c.594-67G>T IVS9-67G>T (N) Non coding IVS No 0.01
11 c.981A>G 1100A/G T327T S Yes 0.01
11 c.1067A>G 1185A>G Q356R MS Yes 0.04
11 c.1396C>G 1515C>G R466G MS No 0.01
11 c.1846delTCT 1965delTCT S616del IFD No 0.01
11 c.2077G>A 2196G>A D693N MS Yes 0.05
11 c.2082C>T 2201C>T S694S S Yes 0.25
11 c.2311T>C 2430T/C L771L S Yes 0.25
11 c.2412G>C 2531G>C Q804H MS Yes 0.02
11 c.2612C>T 2731C>T P871L MS Yes 0.44
11 c.2733A>G 2852A>G G911G S Yes 0.01
11 c.3113A>G 3232A>G E1038G MS Yes 0.22
11 c.3418A>G 3537A>G S1140G MS No 0.01
11 c.3548A>G 3667A>G K1183R MS Yes 0.24
13 c.4308T>C 4427T>C S1436S S Yes 0.22
Intron 14 c.4485-63C>G IVS14-63C>G Non coding IVS Yes 0.13
16 c.4837A>G 4956A>G S1613G MS Yes 0.22
16 c.4956G>A 5075G>A M1652I MS No 0.01
Intron 18 c.5152+66G>A IVS18+66G>A Non coding IVS Yes 0.22
BRCA2 Intron 2 c.-26G>A 5′UTR-26G>A Non coding 5′UTR Yes 0.12
Intron 2 c.67+62T>G IVS2+62T/G Non coding IVS No 0.02
Intron 6 c.516+21A>T IVS6+21A>T Non coding IVS No 0.01
Intron 8 c.681+56C>T IVS8+56C>T Non coding IVS Yes 0.18
10 c.865A>C 1093A>C N289H MS No 0.01
10 c.1012G>A 1240G>A A338T MS No 0.01
10 c.1114A>C 1342A>C H372N MS Yes 0.09
10 c.1365A>G 1593A>G S455S S No 0.01
11 c.2229T>C 2457T>C H743H S No 0.01
11 c.2971A>G 3199A>G N991D MS No 0.02
11 c.3396A>G 3624A>G K1132K S Yes 0.16
11 c.3516G>A 3744G>A S1172S S Yes 0.01
11 c.3807C>T 4035C>T V1269V S Yes 0.22
11 c.4090A>C 4318A>C I1364L MS Yes 0.01
11 c.4563G>A 4791G/A L1521L S No 0.01
11 c.6347A>G 6575A>G H2116R MS No 0.01
11 c.6513C>G 6741C/G V2171V S No 0.01
Intron 11 c.6841+80delTTAA IVS11+80del4 Non coding IVS Yes 0.11
14 c.7242A>G 7470A>G S2414S MS Yes 0.12
14 c.7397C>T 7625C>T A2466V MS No 0.01
15 c.7462A>G 7690A>G (N) R2488G MS No 0.01
Intron 16 c.7806-14T>C IVS16-14T/C Non coding IVS Yes 0.33
Intron 19 c.8488-143G>A IVS19-143G>A (N) Non coding IVS No 0.01
20 c.8503T>C 8731G>C S2835P MS No 0.01
Intron 21 c.8735-66T>C IVS21-66T/C Non coding IVS Yes 0.25
Intron 24 c.9257-83G>A IVS24-83G>A Non coding IVS No 0.11
25 c.9364G>A 9592G>A A3122T MS Yes 0.02
Intron 26 c.9648+106delT IVS26+106delT Non coding IVS No 0.01
27 c.10234A>G 10462A>G I3412V MS No 0.09
N: not reported previously, IVS: intervening sequence, MS: missense, IFD: in frame deletion, S: synonymous.
aEstimated minor allele frequency in our investigated patients (n = 39).
of the coding sequence. The deleterious mutation c.798_799delTT (917delTT) was reported in four Algerian families [24,26] and in three unrelated Tunisian families [27] suggesting a North African founder mutation. Moreover, analysis offive microsatellite markers has shown a common haplotype associated with this mutation in all carriers of both Algerian families[24]. In the present report, this muta- tion was observed in a patient diagnosed with breast medullary carci- noma at age 42. This patient reported to have a mother with breast and ovarian cancer at age 50 and a maternal cousin with breast cancer at 47 years of age. This mutation was also observed in some close Mediterranean countries in particular Spain[28]and Italy (Calabrian) [29]. Thisfinding could be explained by geographical proximity and migrationflow history.
The c.181T>G (300T>G) mutation occurs in the amino-terminal zincfinger motif, an important functional region of BRCA1 protein [30,31]. It has been previously reported as founder mutation in Central European populations[32]and also observed in an Algerian study[24]. The presence of this mutation in Morocco and Algeria too suggests the possibility that this mutation represents a mutation- al hotspot but only the haplotype analysis can establish whether or not all these families had a common ancestor.
The c.5062–5064delGTT (p.V1688del), located in the BRCA1 COOH terminus (BRCT) domain, is a common mutation among Ital- ian families reported in BIC database as variant of uncertain signifi- cance. Several studies suggested its disease causality[33–35]and a recent multimodal analysis confirms the pathogenicity of this muta- tion by destabilizing the BRCT hydrophobic core and altering the protein stability and function[36]. In our study, this mutation was found in a patient with early onset breast cancer (25 years old) with a positive family history. Both her maternal aunt and maternal cousin had been diagnosed with breast cancer at ages 40 and 27 respectively.
BRCA2mutations were found infive families. Three of them are new and only one mutation has been previously described; it's a rare pathogenic splice site mutation IVS6-1G>A which has been cited one time in BIC database in Western Europe and found in our series in an early onset case. Interestingly, the novelBRCA2frameshift mutation c.7235insG (7463insG) was detected in two unrelated families. Therefore, this alteration could be a recurrent mutation in our population but the number of patients enrolled in the study is too small to confirm this conclusion.
The c.3381delT/3609delT (BRCA2) and c.2805delA/2924delA (BRCA1) described for the first time in this paper and the c.3279delC/3398delC (BRCA1) which was found only twice in the UMD database without ethnic origin or nationality, were present in women of Berber origin, an indigenous community of Morocco with
peculiar genetic characteristics suggesting that these mutations may be specific of Morocco.
As previously reported, we have identified variants that have already been elucidated in the BIC database and other publications as neutral polymorphisms or unclassified variants. Further, three novel non- truncating variants have been detected in the coding and intronic se- quences ofBRCA1(IVS9-67G>T) andBRCA2(IVS19-143G>A, R2488G).
The carriers of these novel variants of unknown significance did not present pathogenic mutations in theBRCAgenes. The missense modi- fication R2488G, located in the BRCA2 C-terminal DNA binding do- main (residues 2478–3185) which is a highly conserved region[37], was predicted by Polyphen-2 to be probably damaging. Otherwise these new alterations need to be explored in an ethnically matched control group to clarify better their implication in breast cancer risk assessment.
Our study had several limitations; the major one is the small sam- ple size. The potential limitation of interview bias also needs to be considered. Family history was ascertained only by an interview of the proband and the majority of cancers in relatives were not histo- pathologically confirmed. Though patients were asked specifically about their family history, there were probably some omissions and confusions. Moreover, the study was limited to breast cancer patients and no patients with only ovarian cancer were considered for the study whereas the mean cumulative cancer risks of ovarian cancer at age 70 years have been estimated at 40% (95% CI, 35% to 46%) for BRCA1and 18% (95% CI, 13% to 23%) forBRCA2mutation carriers[5].
Finally, no evaluation of large rearrangements inBRCA1/2 was ex- plored in this report. The investigation of large genomic deletion or duplication using MLPA method (Multiplex Ligation-dependent Probe Amplification) may resolve more families. Indeed, the standard screening techniques based on PCR amplification of genomic DNA allows detecting only point mutations (single base substitution, small deletions or insertions). However, many studies in Caucasian populations have shown that 10–15% of mutations in the BRCA genes could be due to large genomic rearrangements[38].
In conclusion,BRCA1andBRCA2are responsible for a significant proportion of familial breast cancer in Moroccan patients. It is impor- tant to note that nearly 50% of the mutations found are unique to this country. Furthermore, the mutation detection rate seems to be influ- enced by the presence of breast cancer history and the early age at diagnosis. However, further large studies using the MLPA technique, to screen large BRCA1 and BRCA2rearrangements, are required to better define the full spectrum of mutations in our population.
Conflict of interest statement
The authors declare that they have no competing interests.
Table 3
Predicted effect of unclassified missense variants ofBRCA1andBRCA2genes.
Gene Exon Variant Family HZ/
HM
BIC LOVD IARC In silico prediction
PolyPhen2 Sift Align-GVGD
BRCA1 11 R466G br5 HZ 1 2 (deleterious) NR 0.986 (PrD) 0.02 (APF) C15
BRCA2 10 A338T br82 HZ 2 NR NR 0.000 (B) 1.00 (Tol) C0
BRCA2 14 A2466V br15 HZ 51 2 (neutral) NR 0.664 (PsD) 0.02 (APF) C0
BRCA2 11 I1364L br82 HZ 50 NR NR 0.947 (PsD) 0.32 (Tol) C0
BRCA2 11 H2116R br52 HZ 88 NR NR 0.992 (PrD) 0.15 (Tol) C0
BRCA2 20 S2835P br90 HZ 34 2 (neutral) NR 0.002 (B) 0.28 (Tol) C0
BRCA2 25 A3122T br87; br51 HZ NR NR NR 1.000 (PrD) 0.00 (APF) C0
BRCA2 15 R2488G br82 HZ NR NR NR 1.000 (PrD) 0.00 (APF) C45
HZ: heterozygous, HM: homozygous, NR: not reported; B: benign, PsD: possibly damaging, PrD: probably damaging, Tol: tolerated, APF: affect protein function.
BIC: Breast Information Core Database (http://research.nhgri.nih.gov/bic/).
LOVD: Leiden Open Variation Database (http://chromium.liacs.nl/LOVD2/cancer/home.php).
IARC: International Agency for Cancer Research-Breast Cancer Genes IARC Database (http://brca.iarc.fr/PRIORS/).
PolyPhen2: Polymorphism Phenotyping v2 (http://genetics.bwh.harvard.edu/pph2/).
SIFT: Sorting Intolerant From Tolerant (http://blocks.fhcrc.org/sift/SIFT.html).
Align-GVGD: Align Grantham Variance/Grantham Difference (http://agvgd.iarc.fr/agvgd_input.php).
Acknowledgments
This work was supported by a grant from the Istituto Toscano Tumori 2010–2012. We thank the patients and their families for their participation. We would like to express our great appreciation to ProfessorGeneroso Bevilacquaand staff of Divisione di Anatomia Patologica e Diagnostica Molecolare ed Ultrastrutturale for their sup- port and hospitality. We also thank the clinicians and all the staff of Oncology department for their assistance in data and sample collection.
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