Ricci et al1 report in this issue that, contrary to prior suggestions,2 FMR1 mutations may not be predictive of BRCA mutations status. Collaborating with Austrian colleagues, we in 2011/2012, almost accidentally, stumbled over unexpected results when looking at CGG repeats (CGGn) on the FMR1 gene in carriers of BRCA mutations (BRCA1 and BRCA2).2 The idea for this study came from reports that selected FMR12, 3 and BRCA14 mutations, independently, appeared associated with premature ovarian senescence. Austrian colleagues, who had access to a range of BRCA1/2 mutation samples (64 different mutations) from a local Austrian population, and ability to utilize a well-established FMR1 assay, graciously offered their cooperation.2
BRCA1/2 and FMR1 results from investigated patients were, independently, transmitted from two University centers in Graz and Vienna to our center in New York, where associations were determined. Mutations of FMR1 were defined based on a newly defined ‘normal’ CGGn of 26–34.3 Based on this range, patients are defined as normal (norm, with both alleles in range), as heterozygous (het, with one allele in and one outside range) and as homozygous (hom, with both alleles outside range). Het and hom patients are then further sub-categorized as low (CGGn<26) or high (CGGn>34).
Utilizing these newly defined mutations, we reported significant associations with ovarian aging patterns,2, 3 ethnicities/races,5, 6 evidence for immune system activation2 and, most recently, methylation patterns.7 Most striking variations were seen among het mutations, with low alleles associated with the most severe adverse outcomes, like lowered pregnancy chances with in vitro fertilization (IVF),2, 6 increased risk toward immune system activation2, 6 and abnormal methylation.7
In different female populations norm represented uniformly approximately 55–60% of women, het 40–45% and hom approximately 6–8%, a distribution pattern also confirmed in Austrian non-BRCA carrier women.2 Austrian BRCA1/2 mutation carriers, however, differed to surprising degrees: they demonstrated almost complete absence of all constitutional FMR1 genotypes except for those with low (CGGn<26) alleles.2
In their here presented report Ricci et al1 repeated this study, though with greatly differing results. Largest differences were seen in norm and het-norm/low women: while Austrian BRCA1/2 mutation carriers, comparatively, demonstrated significantly reduced numbers of norm FMR1 genotypes (6.1 vs 58.0%) and significantly increased numbers of het-norm/low (78.8 vs 20.5%), Italian BRCA1/2 carriers demonstrated practically identical numbers for norm FMR1 genotypes as non-carriers (51.2 vs 51.0%), although still somewhat elevated numbers for het-norm/low (32.6 vs 23.1%). Het-norm/high and hom patients (combined for all sub-genotypes) in both studies represented small numbers, and, therefore, appear insignificant for this discussion.
Observed differences between the data reported by Ricci et al1 and ours,2 therefore, raise three important questions; the first one being why are there such differences between studies performed in neighboring countries?
Explanations can, most likely, be found in two significant differences between the two studies: both studies are small but the Italian study is even smaller (43 vs 99 patients), increasing risks for patient selection biases. At 51.0%, for example, the norm FMR1 population appears significantly underrepresented in comparison to average populations in Austria2 and the United States.2, 8 Likely more important is, however, that none of the 32 BRCA1/2 mutations in the Italian study was also represented among 64 different mutations observed in Austrian patients. In other words, Austrian and Italian women represented completely different BRCA1/2 mutations.
This may surprise, especially in neighboring countries. Austria and Italy, however, are known to demonstrate highly varying BRCA mutations (Table 1),9 and even in very small regions within Europe, and in relatively small populations considerable variations in BRCA mutations have been reported.9, 10
If one assumes that differences in BRCA mutations in both studies are responsible for variations in the distribution of observed FMR1 mutation, the next question to be resolved is why different BRCA mutations may affect FMR1 mutation distribution differently?
Observing in Austrian BRCA mutation carriers, with relatively few exceptions, only low FMR1 alleles, we concluded that BRCA mutations, almost universally, had to be embryo-lethal, unless rescued by a low FMR1 allele.2 Ricci et al1 now, rightly, questions our conclusions, and suggest that low FMR1 alleles, therefore, cannot be used as a surrogate for BRCA1/2 screening, as we had suggested. They may be correct if the small study size of our data set resulted in an unfortunate statistical coincidence. Another possible explanation for observed discrepancy in findings, however, is that not all but only selected BRCA1/2 mutations are embryo-lethal. Interesting to note in such a context is that even Ricci’s Italian BRCA mutation carriers, comparatively, demonstrate nominally more het-low FMR1 alleles than their own non-carriers (32.6 vs 23.1%) and than reported by us in general populations.2, 8
Rodent models of BRCA support potential embryo lethality of the BRCA gene, though degrees of lethality vary between models,2 suggesting that embryo lethality, indeed, may vary between BRCA mutations.
We hypothesized, based on noted Austrian patient data, that rescue of BRCA mutation-carrying embryos by a low FMR1 allele may explain the so-called ‘BRCA paradox,’ defined by antiproliferative effects of BRCA in embryonic tissue (ie, potential embryo lethality) and proliferative activity in malignancies.2 We furthermore hypothesized that low FMR1 alleles, in rescuing BRCA mutation carrying embryos, block antiproliferative effects of BRCA1/2. Surviving embryos, therefore, lack these antiproliferative characteristics of their BRCA1/2 genes, resulting in surviving adults being exposed to proliferative effects and BRCA1/2-associated cancer risks.
The study of Ricci et al1 allows for a mild adjustment of this hypothesis: since now not all BRCA1/2 mutations appear equally embryo-lethal, not all BRCA1/2 mutations require rescue from embryo lethality by a low FMR1 allele. Only embryo-lethal BRCA1/2 mutations will require such rescue. Only adults with potentially embryo-lethal BRCA1/2 mutations, therefore, will experience the low FMR1 allele-induced effects of blocked antiproliferative BRCA1/2 mutations. A so expanded BRCA/FMR1 hypothesis, therefore, suggests that primarily embryo-lethal BRCA1/2 mutations are also oncogenic.
This modified hypothesis of potential BRCA/FMR1 interactions, paradoxically, is actually easier to confirm (or refute) than the original hypothesis: if correct, low FMR1 alleles can be expected to be disproportionally more frequent in breast and ovarian cancer-stricken BRCA1/2 mutation carriers in comparison to patients with the same cancers lacking BRCA mutations. We are, indeed, currently investigating this question in a North American patient population, and are looking forward to similar investigations from Ricci et al1 and colleagues in other countries.
As proposed in our initial publication,2 the interplay between BRCA and FMR1 genes, as of this moment, remains a hypothesis in need of confirmation. Sometime in the future the paper by Ricci et al1 may be viewed as a first step in its confirmation or repudiation. Either way, their work deserves appreciation
Ricci MT, Pennese L, Gismondi V et al: The FMR1 CGG repeat test is not a candidate pre-screening tool for identifying women with a high probability of being carriers of BRCA mutations. Europ J Hum Genet 2013, (accepted).
Weghofer A, Muy-Kheng T, Barad DH et al: BRCA1/2 mutations appear embryo-lethal unless rescued by low (CGGn<26) FMR1 sub-genotypes: Explanation for the ‘BRCA paradox’? PLoS One 2012; 5: e44753.
Gleicher N, Weghofer A, Barad DH : Ovarian reserve determinations suggest new function of FMR1 (fragie X gene) in regulating ovarian aging. Reprod Biomed Online 2010; 20: 768–775.
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.
Gleicher N, Weghofer A, Barad DH : Effects of race/ethnicity on triple CGG counts in the FMR1 gene in infertile women and egg donors. Reprod Biomed Online 2010; 20: 485–491.
Gleicher N, Weghofer A, Lee IH, Barad DH : Association of FMR1 genotypes with in vitro fertilization outcomes based on ethnicity/race. PLoS One 2011; 6: e18781.
Barad DH, Latham GJ, Filipovic-Sadic S, Kushnir VK, Shohat-Tal A, Gleicher N : Following previously described phenotypes of the FMR1 gene, methylation fractions were significantly skewed between FMR1 genotypes and sub-genotypes. Fertil Steril 2013, (Suppl; ASRM Abstracts; in press).
Gleicher N, Kim A, Weghofer A, Barad DH : Differences in ovarian aging patterns between races are associated with ovarian genotypes and sub-genotypes of the FMR1 gene. Reprod Biol Endocrinol 2012; 10: 77.
Janavičius R : Founder BRCA1/2 mutations in Europe: implications for hereditary-breast-ovarian cancer prevention and control. EPMA J 2010; 1: 397–412.
Verhoog LC, van den Ouweland AM, Berns E et al: Large regional differences in the frequency of distinct BRCA1/BRCA2 mutations in 517 Dutch breast and/or ovarian cancer families. Eur J cancer 2001; 37: 2082–2090.
AW has no potential conflicts to report. NG and DHB are listed as co-owners of a number of already awarded US patents relating to beneficial therapeutic effects of androgen supplementation on female infertility. Pending patents relate to diagnostic claims that the FMR1 gene can be utilized to assess ovarian aging, and to predict fertility treatment chances in association with IVF. NG is a shareholder in Fertility Nutraceuticals, LLC, and owner of the Center for Human Reproduction (CHR). The CHR supported this study through salary support to NG and DHB. NG and DHB receive patent royalties from Fertility Nutraceuticals, LLC.
NG was invited to present this commentary and developed the concept for the manuscript. AW and DHB contributed to the analysis of the manuscript by Ricci et al,1 writing and editing of the manuscript. All authors approved the final manuscript before submission.
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Gleicher, N., Weghofer, A. & Barad, D. Do BRCA1/2 mutations and low FMR1 alleles interact or not?. Eur J Hum Genet 22, 155–156 (2014). https://doi.org/10.1038/ejhg.2013.213
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