DIS3 Variants are Associated With Primary Ovarian Insufficiency: Importance of Transcription/Translation in Oogenesis.

CONTEXT: A genetic etiology accounts for the majority of unexplained primary ovarian insufficiency (POI). OBJECTIVE: We hypothesized a genetic cause of POI for a sister pair with primary amenorrhea. DESIGN: The study was an observational study. Subjects were recruited at an academic institution. SUBJECTS: Subjects were sisters with primary amenorrhea caused by POI and their parents. Additional subjects included women with POI analyzed previously (n = 291). Controls were recruited for health in old age or were from the 1000 Genomes Project (total n = 233). INTERVENTION: We performed whole exome sequencing, and data were analyzed using the Pedigree Variant Annotation, Analysis and Search Tool, which identifies genes harboring pathogenic variants in families. We performed functional studies in a Drosophila melanogaster model. MAIN OUTCOME: Genes with rare pathogenic variants were identified. RESULTS: The sisters carried compound heterozygous variants in DIS3. The sisters did not carry additional rare variants that were absent in publicly available datasets. DIS3 knockdown in the ovary of D. melanogaster resulted in lack of oocyte production and severe infertility. CONCLUSIONS: Compound heterozygous variants in highly conserved amino acids in DIS3 and failure of oocyte production in a functional model suggest that mutations in DIS3 cause POI. DIS3 is a 3' to 5' exoribonuclease that is the catalytic subunit of the exosome involved in RNA degradation and metabolism in the nucleus. The findings provide further evidence that mutations in genes important for transcription and translation are associated with POI.

Copy Number Loss at Chromosome 14q11.2 Correlates With the Proportion of T Cells in Biopsies and Helps Identify T-Cell Neoplasms.

CONTEXT.­: Evidence of T-cell clonality is often critical in supporting the diagnosis of a T-cell lymphoma. OBJECTIVES.­: To retrospectively explore the significance of copy number losses at the 14q11.2 T-cell receptor α locus in relation to the presence of a T-cell neoplasm and proportion of T cells by targeted next-generation sequencing. DESIGN.­: Targeted next-generation sequencing data from 139 tissue biopsies, including T-cell lymphomas, B-cell lymphomas, classic Hodgkin lymphomas, nonhematopoietic malignancies, and normal samples, were reviewed for copy number losses involving the T-cell receptor α gene segments at chr14q11.2. RESULTS.­: We found that biallelic or homozygous deletion of 14q11.2 was found in most (28 of 33, 84.8%) T-cell lymphomas. The magnitude of 14q11.2 loss showed a statistically significant correlation with the proportion of T cells in lymphoma tissue samples. Copy number losses could also be detected in other lymphomas with high numbers of T cells (8 of 32, 25% of B-cell lymphomas, 4 of 4 classical Hodgkin lymphomas), though biallelic/homozygous deletion of 14q11.2 was not significantly observed outside of T-cell lymphomas. Most nonhematopoietic neoplasms and normal tissues (59 of 64, 92.2%) showed no significant copy number losses involving the T-cell receptor α locus at chr14q11.2. CONCLUSIONS.­: Analysis of copy number losses at the T-cell receptor α locus chr14q11.2 with targeted next-generation sequencing can potentially be used to estimate the proportion of T cells and detect T-cell neoplasms.

Modeling primary ovarian insufficiency-associated loci in C. elegans identifies novel pathogenic allele of MSH5.

PURPOSE: In women under the age of 40, primary ovarian insufficiency (POI) is a devastating diagnosis with significant prevalence of 1-4% (Rajkovic and Pangas, Semin Reprod Med. 35(3):231-40, 2017). POI is characterized by amenorrhea with elevated levels of follicle stimulating hormone (FSH) and reduced estrogen levels, mimicking the menopausal state. Genetic determinants account for just over 10% of POI cases, yet determining whether particular single nucleotide polymorphisms (SNPs) are pathogenic is challenging. METHODS: We performed exome sequencing on a cohort of women with POI. CRISPR mutagenesis was employed to create a mutation in a conserved amino acid in the nematode protein. Functional relevance was assessed by analysis of bivalents and aberrant DNA morphologies in diakinesis nuclei. RESULTS: We identified a nonsynonymous c.C1051G; p.R351G variant, in a conserved region of the MSH5 protein. Mutation of this conserved amino acid in the C. elegans homolog, msh-5, revealed defective crossover outcomes in the homozygous and hemizygous states. CONCLUSIONS: These studies further implicate MSH5 as a POI gene and c.C1051G; p.R351G variant as likely playing a functional role in mammalian meiosis. This approach also highlights the ability of model organisms, such as C. elegans, to rapidly and inexpensively identify alleles of interest for further studies in mammalian models.

Causal and Candidate Gene Variants in a Large Cohort of Women With Primary Ovarian Insufficiency.

CONTEXT: A genetic etiology likely accounts for the majority of unexplained primary ovarian insufficiency (POI). OBJECTIVE: We hypothesized that heterozygous rare variants and variants in enhanced categories are associated with POI. DESIGN: The study was an observational study. SETTING: Subjects were recruited at academic institutions. PATIENTS: Subjects from Boston (n = 98), the National Institutes of Health and Washington University (n = 98), Pittsburgh (n = 20), Italy (n = 43), and France (n = 32) were diagnosed with POI (amenorrhea with an elevated follicle-stimulating hormone level). Controls were recruited for health in old age or were from the 1000 Genomes Project (total n = 233). INTERVENTION: We performed whole exome sequencing (WES), and data were analyzed using a rare variant scoring method and a Bayes factor-based framework for identifying genes harboring pathogenic variants. We performed functional studies on identified genes that were not previously implicated in POI in a D. melanogaster model. MAIN OUTCOME: Genes with rare pathogenic variants and gene sets with increased burden of deleterious variants were identified. RESULTS: Candidate heterozygous variants were identified in known genes and genes with functional evidence. Gene sets with increased burden of deleterious alleles included the categories transcription and translation, DNA damage and repair, meiosis and cell division. Variants were found in novel genes from the enhanced categories. Functional evidence supported 7 new risk genes for POI (USP36, VCP, WDR33, PIWIL3, NPM2, LLGL1, and BOD1L1). CONCLUSIONS: Candidate causative variants were identified through WES in women with POI. Aggregating clinical data and genetic risk with a categorical approach may expand the genetic architecture of heterozygous rare gene variants causing risk for POI.

Knowledge and opinions regarding BRCA1 and BRCA2 genetic testing among primary care physicians.

BRCA1 and BRCA2 (BRCA1/2) testing is standard for individuals with personal and/or family history suggestive of hereditary breast and ovarian cancer syndrome. The indications for testing have been expanding. To accommodate the need, incorporation of cancer genetic services into the practice of non-genetic healthcare providers should be considered. We carried out a survey to evaluate the knowledge and opinions regarding BRCA1/2 testing among primary care providers. The survey was sent to 245 Obstetrics/Gynecology and 97 Family Medicine physicians in the UPMC network. Eighty-six completed the survey between July 2015 and September 2015. The average correct responses to knowledge questions was 73%. A few respondents reported being completely confident, and ~50% reported being somewhat confident, in providing BRCA1/2-related information. Respondents selected genetic specialists and oncologists as the most qualified to provide cancer genetic services. Several perceived barriers and motivating factors to the implementation of BRCA1/2 testing in primary care were identified. The findings from this study suggested that primary care providers were not uniformly ready to provide BRCA1/2 genetic testing. Availability of professional society guidelines and evidence of testing's usefulness might motivate the incorporation of BRCA1/2 genetic testing into primary care practices. These findings would help guide future educational efforts to promote provision of cancer genetic services by non-genetic professionals.

Loss of the E2 SUMO-conjugating enzyme Ube2i in oocytes during ovarian folliculogenesis causes infertility in mice.

The number and quality of oocytes within the ovarian reserve largely determines fertility and reproductive lifespan in mammals. An oocyte-specific transcription factor cascade controls oocyte development, and some of these transcription factors, such as newborn ovary homeobox gene (NOBOX), are candidate genes for primary ovarian insufficiency in women. Transcription factors are frequently modified by the post-translational modification SUMOylation, but it is not known whether SUMOylation is required for function of the oocyte-specific transcription factors or if SUMOylation is required in oocytes during their development within the ovarian follicle. To test this, the sole E2 SUMO-conjugating enzyme, Ube2i, was ablated in mouse oocytes beginning in primordial follicles. Loss of oocyte Ube2i resulted in female infertility with major defects in stability of the primordial follicle pool, ovarian folliculogenesis, ovulation and meiosis. Transcriptomic profiling of ovaries suggests that loss of oocyte Ube2i caused defects in both oocyte- and granulosa cell-expressed genes, including NOBOX and some of its known target genes. Together, these studies show that SUMOylation is required in the mammalian oocyte during folliculogenesis for both oocyte development and communication with ovarian somatic cells.

Comparative evaluation of the Minimally-Invasive Karyotyping (MINK) algorithm for non-invasive prenatal testing.

Minimally Invasive Karyotyping (MINK) was communicated in 2009 as a novel method for the non-invasive detection of fetal copy number anomalies in maternal plasma DNA. The original manuscript illustrated the potential of MINK using a model system in which fragmented genomic DNA obtained from a trisomy 21 male individual was mixed with that of his karyotypically normal mother at dilutions representing fetal fractions found in maternal plasma. Although it has been previously shown that MINK is able to non-invasively detect fetal microdeletions, its utility for aneuploidy detection in maternal plasma has not previously been demonstrated. The current study illustrates the ability of MINK to detect common aneuploidy in early gestation, compares its performance to other published third party methods (and related software packages) for prenatal aneuploidy detection and evaluates the performance of these methods across a range of sequencing read inputs. Plasma samples were obtained from 416 pregnant women between gestational weeks 8.1 and 34.4. Shotgun DNA sequencing was performed and data analyzed using MINK RAPIDR and WISECONDOR. MINK performed with greater accuracy than RAPIDR and WISECONDOR, correctly identifying 60 out of 61 true trisomy cases, and reporting only one false positive in 355 normal pregnancies. Significantly, MINK achieved accurate detection of trisomy 21 using just 2 million aligned input reads, whereas WISECONDOR required 6 million reads and RAPIDR did not achieve complete accuracy at any read input tested. In conclusion, we demonstrate that MINK provides an analysis pipeline for the detection of fetal aneuploidy in samples of maternal plasma DNA.

Direct differentiation of human pluripotent stem cells into haploid spermatogenic cells.

Human embryonic stem cells (hESCs) and induced pluripotent stem cells (hiPSCs) have been shown to differentiate into primordial germ cells (PGCs) but not into spermatogonia, haploid spermatocytes, or spermatids. Here, we show that hESCs and hiPSCs differentiate directly into advanced male germ cell lineages, including postmeiotic, spermatid-like cells, in vitro without genetic manipulation. Furthermore, our procedure mirrors spermatogenesis in vivo by differentiating PSCs into UTF1-, PLZF-, and CDH1-positive spermatogonia-like cells; HIWI- and HILI-positive spermatocyte-like cells; and haploid cells expressing acrosin, transition protein 1, and protamine 1 (proteins that are uniquely found in spermatids and/or sperm). These spermatids show uniparental genomic imprints similar to those of human sperm on two loci: H19 and IGF2. These results demonstrate that male PSCs have the ability to differentiate directly into advanced germ cell lineages and may represent a novel strategy for studying spermatogenesis in vitro.