The Egyptian Journal of Medical Human Genetics 18 (2017) 329–334
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The Egyptian Journal of Medical Human Genetics
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Original article
Germline variants in the ATM gene and breast cancer susceptibility in
Moroccan women: A meta-analysis
Chaymaa Marouf a,b,⇑, Omar Hajji c, Amal Tazzite a,b, Hassan Jouhadi d, Abdellatif Benider d,
Sellama Nadifi a,b
a
Laboratory of Genetics and Molecular Pathology–Medical School of Casablanca, Casablanca, Morocco
University Hassan II Ain Chock, Center Of Doctoral Sciences ‘‘In Health Sciences”, Casablanca, Morocco
Department of Oncology, Littoral Clinic, Casablanca, Morocco
d
Mohammed VI Center for Cancer Treatment, Ibn Rochd University Hospital, Casablanca, Morocco
b
c
a r t i c l e
i n f o
Article history:
Received 12 October 2016
Accepted 23 February 2017
Available online 6 March 2017
Keywords:
Breast cancers
ATM gene
Germline mutation
Genetic susceptibility
Moroccan population
a b s t r a c t
Background: The ATM gene encoding a large protein kinase is mutated in ataxia-telangiectasia (AT), an
autosomale recessive disease characterized by neurological and immunological symptoms, and cancer
predisposition. Previous studies suggest that heterozygous carriers of ATM mutations have an increased
risk of breast cancer compared with non carriers, but the contribution of specific variants has been difficult to estimate. However, two functional ATM variants, c.7271T > G and c.1066–6T > G (IVS10–
6T > G), are associated with increased risk for the development of breast cancer.
Methods: To investigate the role of ATM in breast cancer susceptibility, we genotyped 163 case patients
with breast cancer and 150 healthy control individuals for the c.7271T > G and c.1066–6T > G (IVS10–
6T > G) ATM variants using polymerase chain reaction (PCR)-restriction fragment length polymorphism
(RFLP) analysis.
Results: We did not detect the ATM c.7271T > G and c.1066–6T > G (IVS10–6T > G) mutations in any of
150 healthy control individuals and 163 breast cancer patients, including 59 women diagnosed with
breast cancer at an early age (<40 years), 10 women with bilateral breast cancer, and 6 women with ovarian cancer.
Conclusion: These observations suggested that the more common c.1066–6T > G (IVS10–6T > G) mutation and the rare c.7271T > G variant are not a risk factor for developing breast cancer in the Moroccan
population. Larger and/or combined association studies are needed to clarify this issue.
Ó 2017 Ain Shams University. Production and hosting by Elsevier B.V. This is an open access article under
the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).
1. Introduction
Biallelic germline mutations in the ATM gene (MIM# 208900)
are linked to the rare human autosomale recessive disorder called
ataxia-telangiectasia (AT). The syndrome has an estimated frequency of 1in 40.000 to 1in 100.000 live births and is characterized
by various clinical features such as cerebellar neurodegeneration
(ataxia), dilated blood vessels in the eyes and skin (telangiectasia),
immunodeficiency, growth retardation, premature aging, chromosomal instability, increased sensitivity to ionizing radiation and a
highly increased susceptibility to cancer, in particular leukaemia
Peer review under responsibility of Ain Shams University.
⇑ Corresponding author at: University Hassan II Ain Chock, Center Of Doctoral
Sciences ‘‘In Health Sciences”, Casablanca, Morocco.
E-mail address: maroufchaymaa@gmail.com (C. Marouf).
and lymphomas [1–13]. ATM, a member of the phosphotidylinositol 3-kinase-like family, codes for a large serine-threonine kinase
of 3056 amino acids that plays a central role in sensing and signaling the presence of DNA double-strand breaks that may be caused
by exposure to ionizing radiation or other types of DNA damaging
agents [14–16].
The ATM gene, located on human chromosome 11q22.3-23.1,
extends over 150 kb of genomic DNA composed of 66 exons, giving
rise to a transcript of approximately 13 kb that contains an open
reading frame (ORF). It has been reported to be involved in numerous damage repair signaling pathways and cell-cycle checkpoints
[6,17–20]. Loss of heterozygosity in the region of the ATM gene
has been detected in approximately 40% of human sporadic breast
tumors [21–25]. Breast cancer patients with the combination of
radiation treatment and an ATM missense variant resulted in a
shorter mean interval to develop a second tumor than patients
http://dx.doi.org/10.1016/j.ejmhg.2017.02.002
1110-8630/Ó 2017 Ain Shams University. Production and hosting by Elsevier B.V.
This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).
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C. Marouf et al. / The Egyptian Journal of Medical Human Genetics 18 (2017) 329–334
without radiation treatment and ATM germline variant [26]. Previously, several epidemiological case-control studies based on relatives of AT and breast cancer have estimated that the
heterozygous carriers of ATM mutations are at increased risk of
breast cancer and have a two to thirteen fold risk [4,27–39], with
some evidence of higher relative risk under the age of 50 years.
In spite of that, studies of patients with breast cancer have so far
failed to consistently demonstrate an elevated prevalence of germline ATM gene mutations among breast cancer cases relative to
controls [32,33].
Since the cloning of the ATM gene in 1995 [40], many casecontrol studies have carried out mutation screening and single
nucleotide polymorphism (SNP) genotyping to clarify the role of
ATM genetic variation in breast cancer predisposition [41–47].
Specific functional variants of the ATM gene have been individually
associated with higher breast cancer risk. One particular ATM missense mutation, c.7271T > G, was originally identified in two British
A-T families with atypical clinical presentation and an excess of
breast cancer. That variant was associated with a 12.7-fold (95%
confidence interval [CI] 53.5–45.9; P50.003) increase in breast cancer risk [48].The results have not been replicated in subsequent
studies [29,49,50]. However, Tavtigian and colleagues did show
that missense substitutions in the FAT and Kinase domains, including c.7271T > G, confer greater risk than do truncating variants. The
role of missense substitutions uncovered in this paper also somewhat increases the best estimate for the population carrier frequencies of variants in ATM that are pathogenic for breast cancer [51].
A second, more common, the out-of-frame splicing mutation
c.1066-6T > G (IVS10-6T > G), has also been associated with an
increased risk of breast cancer in some [38,52] but not all [49]
studies. This pathogenic mutation leads to incorrect splicing of
exon 11 and exon skipping, resulting in a frame-shift starting at
codon 355 and subsequently truncation of the protein at codon
371. The loss of exon 11 in the mRNA was the pathogenic consequence of this splicing mutation which produced less than 10% of
full-length ATM mRNA and ATM protein [44]. The frequent occurrence of the IVS10–6T > G and c.7271T > G in breast cancer patients
prompted the question whether this mutation frequently arises de
novo in the population. A complete understanding of this breast
cancer-related ATM variant and its impact on human health
requires, besides a genetic and functional analysis, an insight into
its natural history. Thus, the purpose of the current study was to
investigate the associations between the c.7271T > G and c.1066–
6T > G ATM gene variants and breast cancer risk in case-control series from Moroccan population.
2. Subjects and methods
2.1. Study population
Cases were 163 female patients affected with breast cancer as
the first diagnosed cancer. Among them, 113 were recruited from
Mohammed VI Center for Cancer Treatment of Ibn Rochd University Hospital of Casablanca during 2009–2010 and 50 patients
were recruited from the Department of Oncology of the Littoral
Clinic of Casablanca during 2013.
Clinico-pathological parameters including age at diagnosis,
menopausal status, histology type, tumor size, Scarff-BloomRichardson (SBR) grade, lymph nodes status, and hormone receptors status were obtained from patients’ medical records. The
group of sporadic cases (n = 75) presented a median age at diagnosis of 36.9 years (range 23–59 years). The group of familial breast
and/or ovarian cases (n = 59) presented a median age at diagnosis
of 44.8 years (range of 25–67 years), and included women with
specific family-history criteria (Fig. 1):
– Three or more first or second degree relatives with breast cancer diagnosed in the same familial branch, at any age
– Two first degree relatives with breast cancer, with at least one
early onset breast cancer case (40 years).
The controls were 150 female blood donors collected through
the Service of Genetics and Molecular Pathology Laboratory. All
blood donors were healthy and with no personal history of breast
cancer. Median age at blood donation was 36.4 years (range of 20–
77 years). All the individuals included in the present case-control
study signed an informed consent to the use of their biological
material for genetic research purposes approved by the Ethic Committee for Biomedical Research in Casablanca (CERBC) of the Faculty of Medicine and Pharmacy in accordance with The Code of
Ethics of the World Medical Association (declaration of Helsinki)
for experiments in humans.
2.2. DNA isolation and PCR-RFLP amplification
Genomic DNA was extracted from peripheral blood leukocytes
using the salting out procedure [53]. Genomic DNA was dissolved
in TE (10 mM Tris-HCl and 0.1 mM EDTA, pH 8.0), confirmed by
agarose gel electrophoresis. Spectrophotometry was used to quantify DNA using the Nanovue TM Plus spectrophotometer.
Polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) analysis was performed to determine the
T7271G and IVS10–6T > G variants of ATM gene.
2.3. ATM T7271G variant analysis
The ATM c.7271T > G mutation was detected by amplifying
genomic DNA with the forward primer 50 -TGAAAAGAGCCAAAaAG
GAGG-30 and the reverse primer 50 -TAACTGGTGAACATAAAATTGT
CAC-30 using The Veriti Thermal Cycler (Applied Biosystems). The
lowercase ‘‘a” in the forward primer is a mismatch (the nucleotide
is G in the published sequence) that was specifically introduced to
destroy an MnlI site adjacent to and overlapping with the MnlI site
created by the G substitution at nucleotide 7271, thus allowing the
7271 nucleotide substitution to be detected by MnlI digestion [52].
The PCR reaction was carried out to amplify ATM gene in a final
volume of 25 mL containing 10X reaction buffer, 25 mM MgCl2,
5 mM dNTPs, 5 mM primers, 500 U of Taq DNA polymerase, and
150 ng of genomic DNA. The PCR amplification conditions were
as follows: 5 min at 94 °C, followed by 35 cycles of 20 s at 94 °C,
20 s at 55 °C, and 20 s at 72 °C, with a final extension step of
5 min at 72 °C. The PCR products were digested with the MnlI
restriction enzyme.
Thus, 10 mL of PCR product was digested with 3 U of MnlI
restriction enzyme in a 20 mL reaction with 1X reaction buffer
and bovine serum albumin (0.1 mg/mL) at 37 °C overnight. The
digested products were separated by electrophoresis in a 4.5%
agarose gel containing éthidium bromide and visualized under
UV illumination. Wild type allele resulted in 101 bp fragment,
and variant allele resulted in 13 and 88 bp fragments. All fragments were observed for heterozygous genotype.
2.4. ATM IVS10–6T > G variant analysis
A PCR– RFLP assay was used to detect the ATM IVS10–6T > G
variant using The Veriti Thermal Cycler (Applied Biosystems). A
193 bp PCR product was amplified using the following primers:
Forward; 50 -ACAGCGAAACTCTGGCTCAAA-30 , Reverse; 50 -TGATCTT
TTATTACTTCCCAGCCTAGT-30 in a final volume of 25 mL as
described above [52].
Cycling conditions were 94 °C for 4 min, followed by 35 cycles
of 20 s of denaturing at 94 °C, 20 s at 53 °C, and 20 s of annealing
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C. Marouf et al. / The Egyptian Journal of Medical Human Genetics 18 (2017) 329–334
Fig. 1. Pedigree corresponding to one of the families included in the present study of ATM mutation in hereditary breast cancer. The index case is indicated with an arrow.
at 72 °C, with a final extension at 72 °C for 7 min. The PCR products
were digested with the RsaI restriction enzyme.
Digestion was performed in a total volume of 20 ml containing
10 ml PCR product, 1X digestion buffer, 0.1 mg/mL BSA and 3U of
RsaI by overnight incubation at 37 °C. The digested products were
separated by electrophoresis on a 4.5% agarose gel staining with
ethidium bromide, and the genotype was determined by the banding pattern observed. The variant allele was identified by the presence of 58 bp and 135 bp fragments, and the Wild type allele,
which lacks a RsaI restriction site, was identified by a single 193bp fragment. All fragments were observed for heterozygous
genotype.
3. Results
In total, 163 breast cancer cases and 150 controls were successfully genotyped for the T7271G and IVS10–6T > G variants of the
ATM gene using the PCR-RFLP technique. The baseline characteristics of the population sample including distribution of tumor characteristic such as histological grade and location of cancer were
obtained from patients’medical records and listed in Table 1.
None of the 163 Moroccan breast cancer patients carried the
ATM T7271G and IVS10–6T > G mutation. Moreover among the
163 cases, 40 patients had been screened for BRCA1 and BRCA2
mutations [54]. Thus, in the 30 patients who were non-BRCA mutation carriers, the analysis of T7271G and IVS10–6T > G mutation
was also found negative.
4. Discussion
The ATM gene has long been hypothesized to be a breast cancer
susceptibility gene, but the evidence has been contradictory. Most
mutation analyses of ATM in patients with breast cancer and in
control subjects have not found the increased frequency of mutations in case patients that would be expected if these mutations
did predispose to breast cancer [32,33,37,42]. However, these studies have been limited by a lack of statistical power [55] and by the
choice of method for mutation detection, most of which exclusively or preferentially detect protein-truncating mutations [56].
Furthermore, these studies have focused largely on patients with
sporadic breast cancer, and few have tested for ATM mutations in
multiple-case breast cancer families in which segregation between
mutation and cancer can be examined [57].
In the present population-based case-control study, we evaluated for the first time the involvement of two ATM mutations,
T7271G and IVS10–6T ? G, on breast cancer susceptibility in the
Table 1
Characteristics of individuals with breast cancer at time of diagnosis, screened for
ATM c.7271T > G and c.1066–6T > G (IVS10–6T > G) mutations.
Characteristics
Samples
Cases/Controls
Age at diagnosis, mean ± SD (years)
Range (years)
163/150
41 ± 11
23–67
Menopausal Status
Premenopausal
Postmenopausal
Missing
No. (%)
85(52.14)
77(47.23)
1(0.61)
Estrogen receptor
Positive
Negative
Missing
120 (73.61)
33 (20.24)
10 (6.13)
Progesterone receptor
Positive
Negative
Missing
103 (63.19)
50(30.67)
10 (6.13)
Estrogen/Progesterone receptor
ER+/PR+
ER+/PR
ER/PR+
ER/PR-
46
34
20
63
Tumor size
<2 cm
>2 cm
>5 cm
Tumor of any size with extension
25 (15.33)
66 (40.49)
41(25.15)
31 (19.01)
Histological grade
1
2
3
22 (13.49)
95 (58.28)
46 (28.22)
Lymph node status
Negative
Positive
65(39.87)
98 (60.12)
Distant metastases
Negative
Positive
133(81.59)
30 (18.40)
(28.22)
(20.85)
(12.26)
(38.65)
Moroccan population. For this purpose, we performed a screening
of those variants in 163 Moroccan breast cancer patients and 150
healthy controls. We found that none of the 313 analyzed samples
carried the ATM T7271G and IVS10–6T ? G mutations, suggesting
that the frequency of those variants is extremely low (or not present) in the Moroccan population.
Furthermore, the existence of two distinct classes of ATM mutations (truncating and missense) might explain some of the contradictory data on cancer risk. Some missense variants in ATM encode
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C. Marouf et al. / The Egyptian Journal of Medical Human Genetics 18 (2017) 329–334
Table 2
Studies investigating the frequency of T7271 G and IVS10–6T > G ATM gene variants in breast cancer cases and controls by ethnicity.
Variant
Study
T7271G
Africa
Present study
América
Bernstein and al
Bernstein and al
Szabo and al
Bernstein and al
Australia
Chenevix-Trench and al
Europe
Szabo and al
Szabo and al
Szabo and al
Renwick and al
IVS10-6T > G
Africa
Present study
América
Bernstein and al
Sommer and al
Bernstein and al
Szabo and al
Europe
Broeks and al
Dork and al
Szabo and al
Szabo and al
Szabo and al
Lei and al
Broeks and al
Soukupova and al
Australia
Lindeman and al
Thompson and al
Chenevix-Trench and al
Bernstein and al
Country
Year
Overall
Overall
Carriers n (frequency
of carriers,%)
Carriers n (frequency
of carriers,%)
Case
Control
Case
Control
References
MOROCCO
2016
163
150
0(0)
0(0)
Present
study
USA
CANADA
CANADA
USA
2006
2006
2004
2003
1110
1195
43
511
1268
1268
–
638
2
4
0
0
0 (0)
0 (0)
–
1(0.2)
[47]
[47]
[67]
[70]
AUSTRALIA
2002
525
381
1 (0.1)
0 (0)
[52]
THE NETHERLANDS
AUSTRIA
FRANCE
UNITED KINGDOM
2004
2004
2004
2006
501
87
209
443
184
91
–
521
0 (0)
0 (0)
0 (0)
2(0.4)
0 (0)
0 (0)
–
0 (0)
[67]
[67]
[67]
[31]
MOROCCO
2016
163
150
0(0)
0(0)
Present
study
USA
USA
USA
CANADA
AUSTRALIA
CANADA
2003
2002
2006
511
43
3757
638
43
1268
1 (0.2)
0 (0)
13(0.3)
8 (1.3)
1(2.3)
10(0.8)
[70]
[80]
[47]
2004
44
–
0 (0)
–
[67]
THE NETHERLANDS
GERMANY
THE NETHERLANDS
AUSTRIA
FRANCE
SWEDEN AND CZECH
REPUBLIC
THE NETHERLANDS
CZECH REPUBLIC
2000
2001
2004
2004
2004
2002
82
1000
621
87
209
768
268
500
452
91
–
557
3(3.7)
7(0.7)
6(0.9)
2(2.3)
0 (0)
2 (0.2)
2(0,7)
3(0.6)
3(0,6)
1(1.1)
–
1 (0.2)
[38]
[44]
[67]
[67]
[67]
[81]
2003
2008
233
161
268
183
3(1.3)
1(0.6)
2(0.7)
2(1.1)
[82]
[83]
AUSTRALIA
AUSTRALIA
AUSTRALIA
USA
CANADA
AUSTRALIA
2004
2005
2002
2006
495
302
262
3757
725
707
68
1268
7(1.4)
3(1.0)
0 (0)
13(0.3)
6 (0.8)
7 (1.0)
0 (0)
10(0.8)
[79]
[49]
[52]
[47]
stable, but functionally abnormal proteins that could compete in
complex formation with the normal ATM protein, resulting in a
dominant-negative cellular phenotype and confer a particularly
high risk of breast cancer when heterozygous, while causing a
milder form of AT, when homozygous. In contrast, truncating
mutations produce an unstable ATM protein so that heterozygote
individuals still maintain 50% of wild type ATM activity, resulting
in an almost normal phenotype [56,58]. The initial studies that
examined the role of truncating ATM mutations and breast cancer
risk failed to reveal statistically significant disease associations
[33,59]. This was despite the evidence of excess breast cancer incidence within A-T families [34–37,42,60–64]. This apparent dichotomy between the results obtained with these two different study
designs may simply reflect inadequate power rather than true disagreement [55]. However, an analysis of 20 missense ATM mutations provided little support for an association of ATM missense
mutation and breast cancer [65]. Thompson et al. [63] also found
no evidence for a difference in risk of breast or other cancer according to the type of ATM mutation, while the risk estimate of Renwick
et al. [31] was based mainly on truncating mutations. Haplotype
analysis could also reveal a role for common variants in the ATM
gene in causing breast cancer. Five biallelic haplotype tagging sin-
(0.2)
(0.3)
(0)
(0)
gle nucleotide polymorphisms (SNPs) have been estimated to capture 99% of the haplotype diversity in Caucasian populations. In the
Nurses Health Study, there was no evidence that common haplotypes of ATM are associated with breast cancer risk [66]. When
confirmed, this could suggest that less common variation in ATM
is involved in increasing breast cancer risk, which can only be
addressed in much larger studies. A possible example of such a
variant is the c.7271T > G (V2424G), with an allele frequency of
approximately 0.2% among cases and a substantially elevated
breast cancer risk [47,52,67] (Table 2).
The first variant studied, T7271G, was the only ATM mutation
found in the A-T family reported by Stankovic et al. and is, therefore, likely to be pathogenic, although weakly so, because the three
A-T case patients in this family had an atypically mild clinical phenotype. In the first of the families, two individuals homozygous for
T7271G developed breast cancer at 44 and 50 years and their
mother, an obligate carrier of the mutation, developed breast cancer at 82 years. In the second family, two brothers with ataxiatelangiectasia were compound heterozygotes for T7271G and a
truncating mutation. Three paternal aunts of the brothers developed breast cancer in their 50s one of whom was confirmed to
be a heterozygous T7271G carrier [48]. Subsequently, an Australian
C. Marouf et al. / The Egyptian Journal of Medical Human Genetics 18 (2017) 329–334
family was reported in which five women with breast cancer were
heterozygous T7271G carriers. Expression and activity analyses of
ATM in cell lines from the carriers suggested that the mutation acts
in a dominant negative activity [52]. This mutation has been
shown to be very rare in several other studies [31,52,67,70]. The
T7271G variant is predicted to result in a valine to glycine substitution at position 2424 of the ATM protein (p.Val2424Gly). While
this substitution does not affect any readily-recognizable functional domain, programs such as SIFT [68] and PolyPhen [69],
which attempt to classify mutations based on sequence conservation and structural prediction, suggest that this substitution is
deleterious.
On the other hand, IVS10–6T ? G ATM mutation has been
detected in the homozygous state in one German patient with
fullblown A-T [38,71]. This leaky splicing mutation appears to
be the most common pathogenic ATM gene mutation at the
population level, although it is infrequently compared with other
ATM mutations in ataxia-telangiectasia patients [72,73] suggesting an incomplete penetrance with regard to classical A-T symptoms. In view of its population-wide frequency and its ancient
origin, together with its impairment of ATM function and its linkage with breast cancer, the IVS10–6T > G mutation may constitute
one of the most common inherited susceptibility loci. This variant
had been demonstrated to affect normal splicing of exon 11 and
exon skipping, resulting in a frame shift starting at codon 355
and subsequent truncation of the protein at amino acid 419
(Table 2).
Our results thus refute those of Chenevix-Trench et al. [52] who
proposed that the ATM IVS10–6T > G and 7271T > G mutations are
high-risk breast cancer-susceptibility alleles. They based their estimates of the breast cancer risks conferred by these two mutations
on only 2 and 1 single family, respectively, together including 14
cases with breast cancer. The total likelihood of disequilibrium
score for linkage of breast cancer to the ATM locus from these three
families was 1.18 (odds of 15:1 in favor of linkage), which does not
meet conventional criteria for significant linkage. Given the relatively little linkage information/family (likelihood of disequilibrium scores of 0.14, 0.64, and 0.40), precise estimates of the
breast cancer risks conferred by the two mutations could not be
derived from their dataset, and hence, their Bayes factors should
be viewed with caution. Combining the Bayes factors reported
for the two Australian ATM IVS10–6T > G-positive families [52]
with those of the 5 families in this study gives total Bayes factors
of 0.04 (LCIS case classified as unaffected) and 0.0025 (LCIS case
classified as affected). These results imply overall odds of 25:1
and 400:1 against causality, respectively. The expectation that
many of the breast cancer-susceptibility alleles yet to be identified
will confer low breast cancer risks [74] underlines the need for
stringent thresholds of statistical significance, large sample sizes,
and independent replication before results should be considered
convincing [75,76].
5. Conclusion
Our data do not support an increased breast cancer risk for the
ATM IVS10–6T > G mutation, although a slightly increased risk cannot be formally excluded. Neither the ATM IVS10–6T > G mutation
nor the ATM 7271T > G mutation is likely to have a substantial contribution to familial breast cancer. No evidence currently exists
that any mutation of the ATM gene confers a high risk of breast
cancer [35,33,38,44,48,52,62]. In contrast to others [77,78], we
believe that carrier screening in clinical settings for the purpose
of breast cancer risk assessment is as yet not indicated for any
ATM allele. However, a role for the ATM gene in breast cancer susceptibility is plausible but the exact association remains unclear,
333
and most probably comprises only a modest role in familial breast
cancer susceptibility.
Conflict of interest
We have no conflict of interest to declare.
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