Download FMR1 low sub-genotype does not rescue BRCA1

Human Reproduction, Vol.28, No.8 pp. 2308 –2311, 2013
Advanced Access publication on June 11, 2013 doi:10.1093/humrep/det254
ORIGINAL ARTICLE Reproductive genetics
FMR1 low sub-genotype does not rescue
BRCA1/2-mutated human embryos
and does not explain primary ovarian
insufficiency among BRCA1/2-carriers
R.D. Brandão1,2,*, K. van Roozendaal 1, D. Tserpelis1, and M.J. Blok 1
1
Department of Clinical Genetics, Maastricht University Medical Centre, PO Box 616, 6200 MD Maastricht, The Netherlands 2GROW – School
for Oncology and Developmental Biology, Maastricht University Medical Centre, PO Box 616, 6200 MD Maastricht, The Netherlands
*Correspondence address. E-mail: [email protected]
Submitted on February 13, 2013; resubmitted on April 26, 2013; accepted on May 2, 2013
study question: Can we confirm in our population whether FMRI low sub-genotypes are associated with BRCA1/2 mutations, as recently
proposed?
summary answer: Our results indicate that the distribution of the FMR1 sub-genotypes in female BRCA1/2-mutation carriers is significantly different from what has been reported previously and resembles that of the control population. FMRI low sub-genotypes are not associated
with BRCA1/2 mutations and this association is also absent among male mutation carriers.
what is known already: Recently, BRCA1 mutations were reported to be associated with primary ovarian insufficiency (POI) in
female carriers. In animal models, BRCA2-deficiency also results in impaired oogenesis. A recent study has reported that the POI in BRCA1/
2-mutation carriers is most likely due to low FMR1 sub-genotype (CGG n , 26) and the authors also suggested that low sub-genotypes of
the FMR1 gene might be important to rescue the BRCA1/2 embryos, which would otherwise be embryonically-lethal.
study design, size, duration: This retrospective study was performed in October and November of 2012, using genetic material of
464 patients who underwent genetic screening in our centre in the past.
participants/materials, setting, methods: We tested the FMR1 sub-genotypes in 60 female and 29 males with either
BRCA1 or BRCA2 mutations and 375 controls by PCR amplification and size fragment analysis.
main results: We did not find any evidence for an association of FMR1 low sub-genotypes and BRCA1/2 mutations.
limitations, reasons for caution: This association study assumes that the female BRCA1/2 population tested has POI.
wider implications of the findings: Low FMR1 sub-genotypes are not responsible for the presumed rescue of embryos with
BRCA1/2 mutations. Furthermore, the molecular mechanism of the POI in BRCA1/2-female carriers is not likely to be associated with low FMR1
sub-genotype.
study funding/competing interest(s): The Department of Clinical Genetics of the Maastricht University Medical Centre
supported the study. The authors do not have any competing interests to declare.
Key words: BRCA1/2 / FMR1 / primary ovarian insufficiency / embryo rescue / preimplantation genetic diagnostics
Introduction
BRCA1/2-mutation carriers have increased risk of predominantly breast
and ovarian cancer, but also other cancers. To prevent transmission of
mutations to their offspring, nowadays, BRCA1/2-mutation carriers can
opt for IVF with preimplantation genetic diagnosis (PGD). Oktay et al.
reported that BRCA1-mutation carriers subjected to ovarian stimulation
produce lower number of mature oocytes than non-carriers (Oktay
et al., 2010; Titus et al., 2013). Consequently, the chance of a successful
IVF/PGD procedure is potentially decreased. It is noteworthy that
BRCA2 deficiency in mice and zebrafish was also shown to result in impaired
gametogenesis (Sharan et al., 2004; Rodrı́guez-Marı́ et al., 2011).
Different ranges of trinucleotide (CGG) repeats within the
5′ -untranslated region of the fragile X mental retardation gene (FMR1),
& The Author 2013. Published by Oxford University Press on behalf of the European Society of Human Reproduction and Embryology. All rights reserved.
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2309
No association between FMRI and BRCA1/2 mutations
which codes for FMR1 protein (FMRP), are associated with different phenotypes in women: over 200 repeats (full mutation) are associated with
mental retardation, which is usually mild compared with men with a full
mutation; 55–199 repeats (pre-mutation) are associated with premature
ovarian failure (Sherman, 2000); alleles with repeats ,28 and in the 34–55
range have also been associated with prematurely diminished ovarian
reserve (Gleicher et al., 2009; Gleicher et al., 2010). The phenotypes
observed correlate with the expression levels of FMRP, i.e. FMR1 translation has a peak when CGGn is in the normal range (around 30) and it
decreases when the repeat length is smaller or larger (Chen et al., 2003).
A recent study has reported that the primary ovarian insufficiency
(POI) among BRCA1/2-female mutation carriers is in fact due to a low
FMR1 sub-genotype (CGG n , 26) (Weghofer et al., 2012). The
authors also suggested that low sub-genotypes of the FMR1 gene
might be important to rescue BRCA1/2+/2 embryos, which would
otherwise be embryonically lethal. Biallellic inactivation of BRCA1 is
thought to be embryonically lethal, as observed in mice (Howlett et al.,
2002). Additionally, no individuals with homozygous BRCA1 mutations
were reported. Not even among the Ashkenazi Jewish population,
who have a very high incidence of two specific BRCA1 and one BRCA2 mutation. BRCA2 compound heterozygotes are rare and these individuals
are affected with Fanconi anaemia, type D1 (Hakem et al., 1996). It
was observed that the embryonic lethality of BRCA1 2/2 mice could
be rescued by deletion of components of the DNA damage repair
(DDR) pathway, such as p53, ATM, Chek2 and 53BP1 (Xu et al.,
2001; Cao et al., 2006, 2009). We therefore found the FMR1 rescue hypothesis for the presumed embryonic lethality of human BRCA1/2 +/2
mutation carriers very interesting. Testing additional BRCA1/2 carriers,
including males, who carry only one FMR1 allele, would allow us to
confirm this hypothesis. Therefore, we aimed at testing the FMR1 subgenotypes among BRCA1/2-mutation carriers in our centre.
Materials and Methods
Study population
In total, we tested the FMR1 sub-genotypes in 60 unrelated females (30
BRCA1- and 30 BRCA2-mutation carriers) and 29 unrelated males (14
BRCA1- and 15 BRCA2-mutation carriers) and 375 female controls. The
female controls were relatives of Fragile-X syndrome patients and had
been tested for FMR1 repeat size for diagnostic purposes but were anonymized for this study. After removing samples with at least one FMR1 allele
containing CGGn.55, as these are pre-mutations, we had normal genotypes from 325 female controls.
DNA samples from BRCA1/2 mutation carriers were anonymized after selection and could not be traced back to the patient after genotyping and analysis, in agreement with the informed consent of these patients.
Genotyping
Assessment of the sub-genotypes was performed by PCR amplification using
previously described primers (Monet et al., 1996). Size fragment analysis was
performed by capillary electrophoresis in an ABI PRISM 3730 automatic sequencer (Applied Biosystems). Since this methodology only detects alleles up
to a maximum of 70 repeats, confirmation of FMR1 alleles with equal length
was performed with the AmplideXTM FMR1 PCR Kit (CE IVD), allowing the
exclusion of expansions of the FMR1 repeats. Genotypes and sub-genotypes
are defined as previously reported by Gleicher et al. (2010). Briefly, a normal
genotype refers to both alleles in the normal range (26 ≤ CGGn ≤ 34),
Figure 1 The distribution of both FMR1 alleles of CGGn among
women with BRCA1/2 mutations and controls from our population.
Horizontal and vertical parallel lines in scattergrams define the normal
distribution area (26 ≤ CGGn ≤34).
homozygous refers to low– low, high – high or low– high sub-genotypes
(i.e. both alleles outside the normal range), whereas heterozygous refers
to normal – low or normal – high sub-genotypes (i.e. one allele in the
normal range). Comparison of the sub-genotype distribution was performed
using the x 2 statistical test and 5% significance level.
Results
Our results for the female population are shown in Fig. 1 and the subgenotype distribution from our and previously published female populations (Weghofer et al., 2012) are depicted in Fig. 2. Briefly, 45% of the
female BRCA1/2-mutation carriers had two normal alleles (26 ≤
CGGn ≤34), 50% were heterozygous [20% had a normal and a high
allele (n . 34), 30% had a normal and a low allele (n , 26)] and the
remaining 5% were homozygous (2% had high/low alleles and 3%
were low/low, no high/high genotypes were found). There were no significant differences observed between the BRCA1 and BRCA2 populations (P ¼ 0.333). The distribution of the FMR1 sub-genotypes among
female BRCA1/2-mutation carriers resembles that of the controls in
2310
Brandão et al.
Figure 2 FMR1 genotype and sub-genotype distribution among women with BRCA1/2 mutations and controls from our population and from a previous
study (Weghofer et al., 2012). Normal (norm) refers to alleles between 26 and 34 CGG repeats; low and high sub-genotypes refer to CGGn,26 and
CGGn.34, respectively. The sub-genotypes for homozygous samples from the study of Weghofer et al. were not reported by the authors. MUMC, Maastricht University Medical Centre.
Figure 3 Allelic FMR1 distributions among females and males with BRCA1/2 mutations and controls from our population. Normal (norm) refers to alleles
between 26 and 34 CGG repeats; low and high sub-genotypes refer to CGGn,26 and CGGn.34, respectively.
our study and that from the control population used by Weghofer et al.,
whereas it is significantly different from the previously reported BRCA1/
2-female population used by Weghofer et al. (P-value , 0.0001).
Among male carriers, the majority had the normal genotype (26 ≤
CGGn ≤ 34), only eight had the low genotype (20%) and three male carriers had a high genotype. These allelic distribution frequencies are not
different from those of both BRCA1/2 and control female populations
analysed (Fig. 3).
Discussion
Weghofer et al. recently reported that low FMR1 sub-genotype (CGG
n , 26) explained the POI among BRCA1/2-female mutation carriers.
The association found by the authors was so strong that they have also
suggested that low sub-genotypes of the FMR1 gene might be important
to rescue human BRCA1/2+/2 embryos, in which the mutation would
otherwise be embryonically lethal (Weghofer et al., 2012). The results
2311
No association between FMRI and BRCA1/2 mutations
published by Weghofer et al. would have great implications for women
undergoing an IVF procedure for fertility preservation or subsequent
embryo selection by PGD. We aimed at testing their hypothesis in our
BRCA1/2 population. We have also included males, to confirm the
embryo rescue hypothesis, since these carry only one FMR1 allele on
the X chromosome.
Our findings contrast with those from previously reported by Weghofer et al. An explanation for this discrepancy could be that Weghofer et al.
used a control population from a different geographic region to where
their BRCA1/2-mutation carriers originated (USA versus Austria, respectively). However, to test this hypothesis, Austrian women without
BRCA1/2 mutations would have to be genotyped. Furthermore, Weghofer et al. suggested that in human embryos the BRCA1/2 +/2 mutation
may be embryonically lethal. This was based on their results on the
strong selection of FMR1 genotypes, since almost all subjects in their
BRCA1/2 population had at least one low allele and only few normal/
normal genotypes were observed. However, besides their work there is
no evidence of embryonic lethality of human BRCA1/2+/2 genotypes.
Rescue of embryonic lethality of BRCA12/2 mice embryos was observed
in the presence of a deletion of components of the DDR pathway, such as
p53, ATM, Chek2 and 53BP1 (Xu et al., 2001; Cao et al., 2006, 2009). Although it cannot be excluded, Weghofer et al. did not provide experimental evidence that FMR1 is part of the DDR pathway.
In fact, POI in BRCA1/2-mutation carriers is most likely due to impairment of the BRCA1-related DNA damage repair mechanism itself, which
causes an accumulation of double-strand breaks in oocytes leading to
increased apoptosis (Titus et al., 2013). The results of this recent study
suggest that ovarian reserve is affected in all BRCA1 carriers independently of their FMR1 genotypes. Nevertheless, to improve future association
studies on this subject, parameters reflecting ovarian reserve, such as the
level of anti-Müllerian hormone in the serum of age-matched groups,
should be included. Also, larger populations should be studied to allow
for separate analysis of BRCA1 versus BRCA2 carriers. To date, POI has
not been observed among BRCA2 women, despite the fact that
BRCA2 deficiency leads to impaired gametogenesis in animal models.
In summary, if rescue of human embryos with BRCA1/2 mutations
exists, it does not seem likely that it is due to the presence of low
FMR1 sub-genotypes since we did not observe a selection towards
low alleles in our BRCA1/2-mutation carrier population. Moreover,
low FMR1 sub-genotypes do not appear to be the major mediators in
POI observed in BRCA1-mutation carriers, but rather the effect of
DDR pathway mutations as recently shown.
Authors’ roles
R.D.B. conceived the study, analysed and interpreted the data and drafted
the manuscript. K.R. participated in the collection of data and revised the
manuscript. D.T. collected data and revised the manuscript. M.J.B. participated in study conception and design of the study and revised the manuscript. All the authors approved the version to be published.
Funding
The Department of Clinical Genetics of the Maastricht University
Medical Centre supported the study.
Conflict of interest
None declared.
References
Cao L, Kim S, Xiao C, Wang R, Coumoul X, Wang X, Li W, Xu X, De Soto J,
Takai H et al. ATM-Chk2-p53 activation prevents tumorigenesis at an
expense of organ homeostasis upon Brca1 deficiency. EMBO J 2006;
25:2167– 2177.
Cao L, Xu X, Bunting SF, Liu J, Wang R-H, Cao LL, Wu JJ, Peng T-N, Chen J,
Nussenzweig A et al. A selective requirement for 53BP1 in the biological
response to genomic instability induced by Brca1 deficiency. Mol Cell
2009;35:534 – 541.
Chen L-S, Tassone F, Sahota P, Hagerman PJ. The (CGG)n repeat element
within the 5’ untranslated region of the FMR1 message provides both
positive and negative cis effects on in vivo translation of a downstream
reporter. Hum Mol Genet 2003;12:3067– 3074.
Gleicher N, Weghofer A, Oktay K, Barad D. Relevance of triple CGG repeats
in the FMR1 gene to ovarian reserve. Reprod Biomed Online 2009;
19:385– 390.
Gleicher N, Weghofer A, Barad DH. Ovarian reserve determinations suggest
new function of FMR1 (fragile X gene) in regulating ovarian ageing. Reprod
Biomed Online 2010;20:768 – 775.
Hakem R, de la Pompa JL, Sirard C, Mo R, Woo M, Hakem A, Wakeham A,
Potter J, Reitmair A, Billia F et al. The tumor suppressor gene Brca1 is
required for embryonic cellular proliferation in the mouse. Cell 1996;
85:1009– 1023.
Howlett NG, Taniguchi T, Olson S, Cox B, Waisfisz Q, de Die-Smulders C,
Persky N, Grompe M, Joenje H, Pals G et al. Biallelic inactivation of BRCA2
in Fanconi anemia. Science 2002;297:606 – 609.
Monet E, Chateau C, Hirst MC, Thepot F, Taillandier A, Cibois O, Serre J-L.
Analysis of germline variation at the FMR1 CGG repeat shows variation in
the normal-premutated borderline range. Hum Mol Genet 1996;
5:821 – 825.
Oktay K, Kim JY, Barad D, Babayev SN. Association of BRCA1 mutations with
occult primary ovarian insufficiency: a possible explanation for the link
between infertility and breast/ovarian cancer risks. J Clin Oncol 2010;
28:240– 244.
Rodrı́guez-Marı́ A, Wilson C, Titus TA, Cañestro C, BreMiller RA, Yan Y-L,
Nanda I, Johnston A, Kanki JP, Gray EM et al.. Roles of brca2 ( fancd1) in
oocyte nuclear architecture, gametogenesis, gonad tumors, and genome
stability in zebrafish. PLoS Genet 2011;7:e1001357.
Sharan SK, Pyle A, Coppola V, Babus J, Swaminathan S, Benedict J, Swing D,
Martin BK, Tessarollo L, Evans JP et al. BRCA2 deficiency in mice leads to
meiotic impairment and infertility. Development 2004;131:131 – 142.
Sherman S. Premature ovarian failure in the fragile X syndrome. Am J Med
Genet 2000;97:189– 194.
Titus S, Li F, Stobezki R, Akula K, Unsal E, Jeong K, Dickler M, Robson M,
Moy F, Goswami S et al.. Impairment of BRCA1-related DNA
double-strand break repair leads to ovarian aging in mice and humans.
Sci Transl Med 2013;5:172ra121.
Weghofer A, Tea M-K, Barad DH, Kim A, Singer CF, Wagner K, Gleicher N.
BRCA1/2 mutations appear embryo-lethal unless rescued by low (CGG
< 26) FMR1 sub-genotypes: explanation for the ‘BRCA paradox’? PLoS
ONE 2012;7:e44753.
Xu B, Kim S-T, Kastan MB. Involvement of Brca1 in S-Phase and G2-Phase
Checkpoints after Ionizing Irradiation. Mol Cell Biol 2001;21:
3445 – 3450.