Diminished ovarian reserve is not observed in infertility patients with high normal CGG repeats on the fragile X mental retardation 1 (FMR1) gene.

STUDY QUESTION: Does an association exist between high normal numbers of CGG trinucleotide repeats on the fragile X mental retardation 1 (FMR1) gene and diminished ovarian reserve (DOR)? SUMMARY ANSWER: This large data set demonstrated that a high normal number of CGG repeats (35-54 repeats) on the FMR1 gene was not significantly correlated with DOR. WHAT IS KNOWN ALREADY: The FMR1 premutation (55-200 repeats) is a known cause of primary ovarian insufficiency. However, the relationship between high normal CGG repeat numbers (35-54 repeats) and ovarian reserve has yet to be conclusively demonstrated. STUDY DESIGN, SIZE, DURATION: This is a retrospective data analysis conducted between January 2012 and February 2014 that included 1287 women. Over 1140 women had complete data. PARTICIPANTS/MATERIALS, SETTING, METHODS: All women, excluding oocyte donors, who presented to a large private practice specializing in reproductive endocrinology and infertility for treatment and who underwent both fragile X and ovarian reserve testing were included. All fragile X testing was performed using triplet repeat PCR, with confirmation of positives by Southern blot. CGG repeat numbers from both alleles were recorded, and the allele with the higher number of repeats was used for statistical calculations. We did not differentiate between patients with one or two high normal alleles. Women with >54 CGG repeats were excluded from the analysis. For our analysis, we considered both a 'high normal' number of CGG repeats (35-44) and an intermediate number of GCC repeats (45-54) as 'high normal'. Ovarian reserve testing was carried out on Cycle Day 2 or 3 and included measurements of FSH, anti-Müllerian hormone (AMH) and antral follicle count (AFC). A generalized linear regression model assuming gamma distribution and log link function that controlled for age was used to assess correlation between CGG repeat number and FSH, AMH and AFC. MAIN RESULTS AND THE ROLE OF CHANCE: As expected, there was a significant correlation between increasing age and increasing FSH and decreasing AFC and AMH for the patients in this study. For every 1-year increase in age, FSH increased by a factor of 1.04, AFC decreased by a factor of 0.93 and AMH decreased by a factor of 0.89. After controlling for age, there was no significant correlation between FMR1 CGG trinucleotide repeat number and FSH (P = 0.23), AFC (P = 0.14) or AMH (P = 0.53). Three subgroup analyses were also performed. We found a significant relationship between increasing CGG repeat number and decreasing AMH levels (P = 0.01) in women >44 years old. The second subgroup analysis included only Caucasian patients and found no significant correlation between CGG repeat number and DOR. In a subgroup analysis comparing women with at least one allele <26 repeats, at least one allele >35 and women with both alleles between 29 and 32, there were no significant associations regarding ovarian reserve in any of these groups. LIMITATIONS, REASONS FOR CAUTION: One limitation of this study is that it involved a heterogeneous population of infertile women with mixed diagnoses. Factors that could affect ovarian reserve, such as medical comorbidities, prior surgeries, family history and endometriosis, were not accounted for. Finally, there was a lack of racial diversity, with Caucasians representing 67.8% of the total population. WIDER IMPLICATIONS OF THE FINDINGS: The findings of this study are generalizable to an infertility population and are in line with several previously published studies. Women who are found to have high normal CGG repeat numbers can be counseled that this is not causative for DOR. Further studies are needed to investigate whether increasing CGG repeat numbers are associated with ovarian responsiveness to gonadotrophin stimulation or IVF outcome.

A rigorous approach for selection of optimal variant sets for carrier screening with demonstration of clinical utility.

Carrier screening for certain diseases is recommended by major medical and Ashkenazi Jewish (AJ) societies. Most carrier screening panels test only for common, ethnic-specific variants. However, with formerly isolated ethnic groups becoming increasingly intermixed, this approach is becoming inadequate. Our objective was to develop a rigorous process to curate all variants, for relevant genes, into a database and then apply stringent clinical validity classification criteria to each in order to retain only those with clear evidence for pathogenicity. The resulting variant set, in conjunction with next-generation DNA sequencing (NGS), then affords the capability for an ethnically diverse, comprehensive, highly specific carrier-screening assay. The clinical utility of our approach was demonstrated by screening a pan-ethnic population of 22,864 individuals for Bloom syndrome carrier status using a BLM variant panel comprised of 50 pathogenic variants. In addition to carriers of the common AJ founder variant, we identified 57 carriers of other pathogenic BLM variants. All variants reported had previously been curated and their clinical validity documented, or were of a type that met our stringent, preassigned validity criteria. Thus, it was possible to confidently report an increased number of Bloom's syndrome carriers compared to traditional, ethnicity-based screening, while not reducing the specificity of the screening due to reporting variants of unknown clinical significance.

Validation for clinical use of, and initial clinical experience with, a novel approach to population-based carrier screening using high-throughput, next-generation DNA sequencing.

Traditional carrier screening assays are designed to look for only the most common mutations within a gene owing to cost considerations. Although this can yield high detection rates in specific populations for specific genes (such as cystic fibrosis in Caucasians), they are suboptimal for other ethnicities or for patients of mixed or unknown ethnic background. Next-generation DNA sequencing provides an opportunity to provide carrier screening using more comprehensive mutation panels that are limited primarily by information about the clinical impact of detected sequence changes. We describe a next-generation DNA sequencing-based assay capable of reliably screening patient samples in a timely and comprehensive manner. The analytic accuracy in a research setting has been documented. Here, we describe the additional studies performed to ensure the accuracy (analytic validity) and robustness of our assay for use in clinical practice and provide data from our experience offering this testing. Our clinical experience using this approach to screen 11,691 in vitro fertilization patients has identified 449 mutant alleles: 447 in carriers and 2 in an affected individual. In total, we found 87 distinct mutations in 14 different genes. Approximately one quarter of the mutations found are not included in traditional, limited, mutation panels, including 16 known mutations unique to our panel, and novel truncating mutations in several genes.

Next-generation DNA sequencing of HEXA: a step in the right direction for carrier screening.

Tay-Sachs disease (TSD) is the prototype for ethnic-based carrier screening, with a carrier rate of ∼1/27 in Ashkenazi Jews and French Canadians. HexA enzyme analysis is the current gold standard for TSD carrier screening (detection rate ∼98%), but has technical limitations. We compared DNA analysis by next-generation DNA sequencing (NGS) plus an assay for the 7.6 kb deletion to enzyme analysis for TSD carrier screening using 74 samples collected from participants at a TSD family conference. Fifty-one of 74 participants had positive enzyme results (46 carriers, five late-onset Tay-Sachs [LOTS]), 16 had negative, and seven had inconclusive results. NGS + 7.6 kb del screening of HEXA found a pathogenic mutation, pseudoallele, or variant of unknown significance (VUS) in 100% of the enzyme-positive or obligate carrier/enzyme-inconclusive samples. NGS detected the B1 allele in two enzyme-negative obligate carriers. Our data indicate that NGS can be used as a TSD clinical carrier screening tool. We demonstrate that NGS can be superior in detecting TSD carriers compared to traditional enzyme and genotyping methodologies, which are limited by false-positive and false-negative results and ethnically focused, limited mutation panels, respectively, but is not ready for sole use due to lack of information regarding some VUS.