Miscarriage risk assessment: a bioinformatic approach to identifying candidate lethal genes and variants.

PURPOSE: Miscarriage, often resulting from a variety of genetic factors, is a common pregnancy outcome. Preconception genetic carrier screening (PGCS) identifies at-risk partners for newborn genetic disorders; however, PGCS panels currently lack miscarriage-related genes. In this study, we evaluated the potential impact of both known and candidate genes on prenatal lethality and the effectiveness of PGCS in diverse populations. METHODS: We analyzed 125,748 human exome sequences and mouse and human gene function databases. Our goals were to identify genes crucial for human fetal survival (lethal genes), to find variants not present in a homozygous state in healthy humans, and to estimate carrier rates of known and candidate lethal genes in various populations and ethnic groups. RESULTS: This study identified 138 genes in which heterozygous lethal variants are present in the general population with a frequency of 0.5% or greater. Screening for these 138 genes could identify 4.6% (in the Finnish population) to 39.8% (in the East Asian population) of couples at risk of miscarriage. This explains the cause of pregnancy loss in approximately 1.1-10% of cases affected by biallelic lethal variants. CONCLUSION: This study has identified a set of genes and variants potentially associated with lethality across different ethnic backgrounds. The variation of these genes across ethnic groups underscores the need for a comprehensive, pan-ethnic PGCS panel that includes genes related to miscarriage.

45,X/46,XY mosaicism: Clinical manifestations and long term follow-up.

45,X/46,XY chromosomal mosaicism presents a range of clinical manifestations, including phenotypes from Turner syndrome through genital abnormalities to apparently unaffected phenotypic males; however, the full clinical spectrum has not yet been fully delineated since prior studies on the clinical phenotype and associated risk of gonadal tumors included small cohorts and limited follow-up. To better describe the clinical manifestations and long-term outcome of patients with 45,X/46,XY mosaicism. We conducted a retrospective chart review of patients with 45,X/46,XY from three health centers (Hospital for Sick Children and Mount Sinai Hospital in Canada, and University of Pittsburgh Medical Center in United States). Of 100 patients with 45,X/46,XY karyotype, 47 were raised as females and 53 as males. Females were significantly shorter than males (p = 0.04) and height Z-score was significantly decreased with age for both genders (p = 0.02). Growth hormone (GH) treatment did not result in a significant height increase compared to the untreated group (p = 0.5). All females required puberty induction in contrast to majority of males. Five females were diagnosed with gonadal tumors, while no males were affected. Around 58% of patients exhibited at least one Turner syndrome stigmata. This study expands the clinical spectrum, long-term outcomes, and associated tumor risk in a large cohort of patients with 45,X/46,XY mosaicism. Additionally, it highlights our experience with GH therapy and prophylactic gonadectomy.

Sohlh1 loss of function male and female infertility model impacts overall health beyond gonadal dysfunction in mice†.

Reproductive longevity is associated with health outcomes. Early menopause, loss of ovarian function, and male infertility are linked to shorter lifespan and increased adverse health outcomes. Here we examined the extragonadal effects of whole animal loss of spermatogenesis and oogenesis specific basic helix-loop-helix 1 (Sohlh1) gene in mice, a well-described mouse model of female and male infertility. Sohlh1 encodes a transcription factor that is primarily expressed in the male and female germline and regulates germline differentiation. The Sohlh1 knockout mouse model, just like human individuals with SOHLH1 loss of function, presents with hypergonadotropic hypogonadism and loss of ovarian function in females and impaired spermatogenesis in males, with a seemingly gonad restricted phenotype in both sexes. However, extragonadal phenotyping revealed that Sohlh1 deficiency leads to abnormal immune profiles in the blood and ovarian tissues of female animals, sex-specific alterations of metabolites, and behavior and cognition changes. Altogether, these results show that Sohlh1 deficiency impacts overall health in both male and female mice.

Testis formation in XX individuals resulting from novel pathogenic variants in Wilms' tumor 1 (WT1) gene.

Sex determination in mammals is governed by antagonistic interactions of two genetic pathways, imbalance in which may lead to disorders/differences of sex development (DSD) in human. Among 46,XX individuals with testicular DSD (TDSD) or ovotesticular DSD (OTDSD), testicular tissue is present in the gonad. Although the testis-determining gene SRY is present in many cases, the etiology is unknown in most SRY-negative patients. We performed exome sequencing on 78 individuals with 46,XX TDSD/OTDSD of unknown genetic etiology and identified seven (8.97%) with heterozygous variants affecting the fourth zinc finger (ZF4) of Wilms' tumor 1 (WT1) (p.Ser478Thrfs*17, p.Pro481Leufs*15, p.Lys491Glu, p.Arg495Gln [x3], p.Arg495Gly). The variants were de novo in six families (P = 4.4 × 10-6), and the incidence of WT1 variants in 46,XX DSD is enriched compared to control populations (P < 1.8 × 10-4). The introduction of ZF4 mutants into a human granulosa cell line resulted in up-regulation of endogenous Sertoli cell transcripts and Wt1Arg495Gly/Arg495Gly XX mice display masculinization of the fetal gonads. The phenotype could be explained by the ability of the mutated proteins to physically interact with and sequester a key pro-ovary factor ß-CATENIN, which may lead to up-regulation of testis-specific pathway. Our data show that unlike previous association of WT1 and 46,XY DSD, ZF4 variants of WT1 are a relatively common cause of 46,XX TDSD/OTDSD. This expands the spectrum of phenotypes associated with WT1 variants and shows that the WT1 protein affecting ZF4 can function as a protestis factor in an XX chromosomal context.

Heterozygous TP63 pathogenic variants in isolated primary ovarian insufficiency.

PURPOSE: Our study aimed to identify the genetic causes of non-syndromic primary ovarian insufficiency (POI) in female patients. METHODS: We performed whole exome sequencing in females suffering from isolated POI and in their available family members. Copy number variations were validated by long-range PCR and Sanger sequencing, and conservation analysis was used to evaluate the impact of sequence variants on protein composition. RESULTS: We detected two pathogenic TP63 heterozygous deleterious single nucleotide variants and a novel TP63 intragenic copy number alteration in three unrelated women with isolated POI. Two of these genetic variants are predicted to result in loss of transactivation inhibition of p63, whereas the third one affects the first exon of the ΔNp63 isoforms. CONCLUSION: Our results broaden the spectrum of TP63-related disorders, which now includes sporadic and familial, isolated, and syndromic POI. Genomic variants that impair the transactivation inhibitory domain of the TAp63α isoform are the cause of non-syndromic POI. Additionally, variants affecting only the ΔNp63 isoforms may result in isolated POI. In patients with isolated POI, careful evaluation of genomic variants in pleiotropic genes such as TP63 will be essential to establish a full clinical spectrum and atypical presentation of a disorder.

Motifs of the C-terminal domain of MCM9 direct localization to sites of mitomycin-C damage for RAD51 recruitment.

The MCM8/9 complex is implicated in aiding fork progression and facilitating homologous recombination (HR) in response to several DNA damage agents. MCM9 itself is an outlier within the MCM family containing a long C-terminal extension (CTE) comprising 42% of the total length, but with no known functional components and high predicted disorder. In this report, we identify and characterize two unique motifs within the primarily unstructured CTE that are required for localization of MCM8/9 to sites of mitomycin C (MMC)-induced DNA damage. First, an unconventional "bipartite-like" nuclear localization (NLS) motif consisting of two positively charged amino acid stretches separated by a long intervening sequence is required for the nuclear import of both MCM8 and MCM9. Second, a variant of the BRC motif (BRCv) similar to that found in other HR helicases is necessary for localization to sites of MMC damage. The MCM9-BRCv directly interacts with and recruits RAD51 downstream to MMC-induced damage to aid in DNA repair. Patient lymphocytes devoid of functional MCM9 and discrete MCM9 knockout cells have a significantly impaired ability to form RAD51 foci after MMC treatment. Therefore, the disordered CTE in MCM9 is functionally important in promoting MCM8/9 activity and in recruiting downstream interactors; thus, requiring full-length MCM9 for proper DNA repair.

Carrier frequency of autosomal recessive genetic conditions in diverse populations: Lessons learned from the genome aggregation database.

An equitable approach by the American College of Medical Genetics and Genomics (ACMG) has recently recommended carrier screening for genes associated with moderate to severe autosomal recessive conditions with a carrier frequency of ≥1/200 in the Genome Aggregation Database exomes (gnomADv2.0.2). We analyzed carrier frequencies in gnomADv3.1.1 genomes representing diverse populations. ClinVar data on 35 996 pathogenic/likely pathogenic variants in 419 genes were used to estimate the gnomAD frequency of heterozygous carriers. We found that ninety-two genes had a carrier frequency of ≥1/200, of which 63 were shared between v3.1.1 and v2.0.2 and 29 were new in v3.1.1. Addition of new populations (Amish, Finnish and Middle Eastern) increased the number of new genes with a carrier frequency of ≥1/200 to 71. Changes in carrier frequencies were attributed to new gnomAD populations, different sample sizes, new ClinVar data, and technical differences between exomes and genomes. This study highlights the dynamic changes in carrier frequencies due to new datasets from diverse populations and provides updated carrier frequencies based on the combined data from 184 352 genomes and exomes in gnomAD. We recommend a periodic review for inclusion of new population data to update carrier screening panels in the future.

Single-cell sequencing reveals novel cellular heterogeneity in uterine leiomyomas.

STUDY QUESTION: What are the cellular composition and single-cell transcriptomic differences between myometrium and leiomyomas as defined by single-cell RNA sequencing? SUMMARY ANSWER: We discovered cellular heterogeneity in smooth muscle cells (SMCs), fibroblast and endothelial cell populations in both myometrium and leiomyoma tissues. WHAT IS KNOWN ALREADY: Previous studies have shown the presence of SMCs, fibroblasts, endothelial cells and immune cells in myometrium and leiomyomas. However, there is no information on the cellular heterogeneity in these tissues and the transcriptomic differences at the single-cell level between these tissues. STUDY DESIGN, SIZE, DURATION: We collected five leiomyoma and five myometrium samples from a total of eight patients undergoing hysterectomy. We then performed single-cell RNA sequencing to generate a cell atlas for both tissues. We utilized our single-cell sequencing data to define cell types, compare cell types by tissue type (leiomyoma versus myometrium) and determine the transcriptional changes at a single-cell resolution between leiomyomas and myometrium. Additionally, we performed MED12-variant analysis at the single-cell level to determine the genotype heterogeneity within leiomyomas. PARTICIPANTS/MATERIALS, SETTING, METHODS: We collected five MED12-variant positive leiomyomas and five myometrium samples from a total of eight patients. We then performed single-cell RNA sequencing on freshly isolated single-cell preparations. Histopathological assessment confirmed the identity of the samples. Sanger sequencing was performed to confirm the presence of the MED12 variant in leiomyomas. MAIN RESULTS AND ROLE OF CHANCE: Our data revealed previously unknown heterogeneity in the SMC, fibroblast cell and endothelial cell populations of myometrium and leiomyomas. We discovered the presence of two different lymphatic endothelial cell populations specific to uterine leiomyomas. We showed that both myometrium and MED12-variant leiomyomas are relatively similar in cellular composition but differ in cellular transcriptomic profiles. We found that fibroblasts influence the leiomyoma microenvironment through their interactions with endothelial cells, immune cells and SMCs. Variant analysis at the single-cell level revealed the presence of both MED12 variants as well as the wild-type MED12 allele in SMCs of leiomyomatous tissue. These results indicate genotype heterogeneity of cellular composition within leiomyomas. LARGE SCALE DATA: The datasets are available in the NCBI Gene Expression Omnibus (GEO) using GSE162122. LIMITATIONS, REASONS FOR CAUTION: Our study focused on MED12-variant positive leiomyomas for single-cell RNA sequencing analyses. Leiomyomas carrying other genetic rearrangements may differ in their cellular composition and transcriptomic profiles. WIDER IMPLICATIONS FOR THE FINDINGS: Our study provides a cellular atlas for myometrium and MED12-variant positive leiomyomas as defined by single-cell RNA sequencing. Our analysis provides significant insight into the differences between myometrium and leiomyomas at the single-cell level and reveals hitherto unknown genetic heterogeneity in multiple cell types within human leiomyomas. Our results will be important for future studies into the origin and growth of human leiomyomas. STUDY FUNDING/COMPETING INTEREST(S): This work was supported by funding from the National Institute of Child Health and Human Development (HD098580 and HD088629). The authors declare no competing interests.

Deletion of Gremlin-2 alters estrous cyclicity and disrupts female fertility in mice†.

Members of the differential screening-selected gene aberrative in neuroblastoma (DAN) protein family are developmentally conserved extracellular binding proteins that antagonize bone morphogenetic protein (BMP) signaling. This protein family includes the Gremlin proteins, GREM1 and GREM2, which have key functions during embryogenesis and adult physiology. While BMPs play essential roles in ovarian follicle development, the role of the DAN family in female reproductive physiology is less understood. We generated mice null for Grem2 to determine its role in female reproduction in addition to screening patients with primary ovarian insufficiency (POI) for variants in GREM2. Grem2-/- mice are viable, but female Grem2-/- mice have diminished fecundity and irregular estrous cycles. This is accompanied by significantly reduced production of ovarian anti-Müllerian hormone (AMH) from small growing follicles, leading to a significant decrease in serum AMH. Surprisingly, as AMH is a well-established marker of the ovarian reserve, morphometric analysis of ovarian follicles showed maintenance of primordial follicles in Grem2-/- mice like wild-type (WT) littermates. While Grem2 mRNA transcripts were not detected in the pituitary, Grem2 is expressed in hypothalami of WT female mice, suggesting the potential for dysfunction in multiple tissues composing the hypothalamic-pituitary-ovarian axis that contribute to the subfertility phenotype. Additionally, screening 106 women with POI identified one individual with a heterozygous variant in GREM2 that lies within the predicted BMP-GREM2 interface. In total, these data suggest that Grem2 is necessary for female fecundity by playing a novel role in regulating the HPO axis and contributing to female reproductive disease.

Screening for autosomal recessive and X-linked conditions during pregnancy and preconception: a practice resource of the American College of Medical Genetics and Genomics (ACMG).

Carrier screening began 50 years ago with screening for conditions that have a high prevalence in defined racial/ethnic groups (e.g., Tay-Sachs disease in the Ashkenazi Jewish population; sickle cell disease in Black individuals). Cystic fibrosis was the first medical condition for which panethnic screening was recommended, followed by spinal muscular atrophy. Next-generation sequencing allows low cost and high throughput identification of sequence variants across many genes simultaneously. Since the phrase "expanded carrier screening" is nonspecific, there is a need to define carrier screening processes in a way that will allow equitable opportunity for patients to learn their reproductive risks using next-generation sequencing technology. An improved understanding of this risk allows patients to make informed reproductive decisions. Reproductive decision making is the established metric for clinical utility of population-based carrier screening. Furthermore, standardization of the screening approach will facilitate testing consistency. This practice resource reviews the current status of carrier screening, provides answers to some of the emerging questions, and recommends a consistent and equitable approach for offering carrier screening to all individuals during pregnancy or preconception.

Reproductive outcomes in individuals with chromosomal reciprocal translocations.

PURPOSE: Patients with reciprocal balanced translocations (RBT) have a risk for recurrent pregnancy losses (RPL), affected child, and infertility. Currently, genetic counseling is based on karyotypes found among the products of conception (POC), although factors influencing the success of assisted reproductive technologies (ART) in RBT couples are not established. METHODS: Cytogenetic results from 261 POC and offspring of the parents (113 women and 90 men) with RBT were evaluated. Chromosome segregation modes and number of euploid embryos were assessed in couples undergoing in vitro fertilization. RESULTS: Patients with translocations involving an acrocentric chromosome have a higher risk of unbalanced gametes caused by a 3:1 segregation. Female RBT patients have a statistically higher risk of aneuploidy due to an interchromosomal effect. The rate of euploid embryos is low due to meiosis I malsegregation of RBT, meiosis II nondisjunction, additional whole chromosome or segmental aneusomies. RBT patients with RPL have a higher rate of miscarriage of euploid fetuses with RBT. CONCLUSION: Chromosome-specific factors, female gender, age, and history of RPL are the risk elements influencing pregnancy and in vitro fertilization success in RBT patients. Chromosomal microarray analysis of POC is necessary to provide an accurate and timely diagnosis for patients with adverse reproductive outcomes.

Cytogenetic signatures of recurrent pregnancy losses.

OBJECTIVES: To investigate the incidence of chromosomal abnormalities in the products of conception (POC) of patients with spontaneous miscarriages (SM) and with recurrent pregnancy losses (RPL) and to determine biological mechanisms contributing to RPL. METHODS: During a 20-year period, 12 096 POC samples underwent classical chromosome analysis. Cytogenetic findings were compared between the SM and RPL cohorts. RESULTS: Analysis of RPL cohort has identified an increased incidence of inherited and de novo structural chromosome abnormalities, recurrent polyploid conceptions, and complex mosaic alterations. These abnormalities are the signature of genomic instability, posing a high risk of genetic abnormalities to offspring independent of maternal age. Predominance of male conceptions in the RPL cohort points toward an X-linked etiology and gender-specific intolerance for certain genetic abnormalities. CONCLUSIONS: Our study showed several possible genetic etiologies of RPL, including parental structural chromosome rearrangements, predisposition to meiotic nondisjunction, and genomic instability. Loss of karyotypically normal fetuses might be attributed to defects in genes essential for fetal development, as well as aberrations affecting the X chromosome. Molecular studies of parental and POC genomes will help to identify inherited defects in genes involved in meiotic divisions and DNA repair to confirm our hypotheses, and to discover novel fetal-essential genes.

Mutations involving the SRY-related gene SOX8 are associated with a spectrum of human reproductive anomalies.

SOX8 is an HMG-box transcription factor closely related to SRY and SOX9. Deletion of the gene encoding Sox8 in mice causes reproductive dysfunction but the role of SOX8 in humans is unknown. Here, we show that SOX8 is expressed in the somatic cells of the early developing gonad in the human and influences human sex determination. We identified two individuals with 46, XY disorders/differences in sex development (DSD) and chromosomal rearrangements encompassing the SOX8 locus and a third individual with 46, XY DSD and a missense mutation in the HMG-box of SOX8. In vitro functional assays indicate that this mutation alters the biological activity of the protein. As an emerging body of evidence suggests that DSDs and infertility can have common etiologies, we also analysed SOX8 in a cohort of infertile men (n = 274) and two independent cohorts of women with primary ovarian insufficiency (POI; n = 153 and n = 104). SOX8 mutations were found at increased frequency in oligozoospermic men (3.5%; P < 0.05) and POI (5.06%; P = 4.5 × 10-5) as compared with fertile/normospermic control populations (0.74%). The mutant proteins identified altered SOX8 biological activity as compared with the wild-type protein. These data demonstrate that SOX8 plays an important role in human reproduction and SOX8 mutations contribute to a spectrum of phenotypes including 46, XY DSD, male infertility and 46, XX POI.

Copy number alterations involving 59 ACMG-recommended secondary findings genes.

In clinical exome/genome sequencing, the American College of Medical Genetics and Genomics (ACMG) recommends reporting of secondary findings unrelated to a patient's phenotype when pathogenic single-nucleotide variants (SNVs) are observed in one of 59 genes associated with a life-threatening, medically actionable condition. Little is known about the incidence and sensitivity of chromosomal microarray analysis (CMA) for detection of pathogenic copy number variants (CNVs) comprising medically-actionable genes. Clinical CMA has been performed on 8865 individuals referred for molecular cytogenetic testing. We retrospectively reviewed the CMA results to identify patients with CNVs comprising genes included in the 59-ACMG list of secondary findings. We evaluated the clinical significance of these CNVs in respect to pathogenicity, phenotypic manifestations, and heritability. We identified 23 patients (0.26%) with relevant CNV either deletions comprising the entire gene or intragenic alterations involving one or more secondary findings genes. A number of patients and/or their family members with pathogenic CNVs manifest or expected to develop an anticipated clinical phenotype and would benefit from preventive management similar to the patients with pathogenic SNVs. To improve patients' care standardization should apply to reporting of both sequencing and CNVs obtained via clinical genome-wide analysis, including chromosomal microarray and exome/genome sequencing.

Genetics of human female infertility†.

About 10% of women of reproductive age are unable to conceive or carry a pregnancy to term. Female factors alone account for at least 35% of all infertility cases and comprise a wide range of causes affecting ovarian development, maturation of oocytes, and fertilization competence, as well as the potential of a fertilized egg for preimplantation development, implantation, and fetal growth. Genetic abnormalities leading to infertility in females comprise large chromosome abnormalities, submicroscopic chromosome deletion and duplications, and DNA sequence variations in the genes that control numerous biological processes implicated in oogenesis, maintenance of ovarian reserve, hormonal signaling, and anatomical and functional development of female reproductive organs. Despite the great number of genes implicated in reproductive physiology by the study of animal models, only a subset of these genes is associated with human infertility. In this review, we mainly focus on genetic alterations identified in humans and summarize recent knowledge on the molecular pathways of oocyte development and maturation, the crucial role of maternal-effect factors during embryogenesis, and genetic conditions associated with ovarian dysgenesis, primary ovarian insufficiency, early embryonic lethality, and infertility.

A high-resolution X chromosome copy-number variation map in fertile females and women with primary ovarian insufficiency.

PURPOSE: Sex-biased expression of genes on the X chromosome is accomplished by a complex mechanism of dosage regulation that leads to anatomical and physiological differences between males and females. Copy-number variations (CNVs) may impact the human genome by either affecting gene dosage or disturbing a chromosome structural and/or functional integrity. METHODS: We performed a high-resolution CNV profiling to investigate the X chromosome integrity in cohorts of 269 fertile females and 111 women affected with primary ovarian insufficiency (POI) and assessed CNVs impact into functional and nonfunctional genomic elements. RESULTS: In POI patients, we observed a 2.5-fold enrichment for rare CNVs comprising ovary-expressed genes, and genes implicated in autoimmune response and apoptotic signaling. Moreover, there was a higher prevalence of deletions encompassing genes that escape X inactivation, noncoding RNAs, and intergenic DNA sequences among POI females, highlighting structural differences between X chromosomes of fertile and POI females. Furthermore, we discovered a ~4% carrier incidence for X-linked disorders among fertile women. CONCLUSION: We constructed a high-resolution map of female-specific CNVs that provides critical insights into the spectrum of human genetic variation, sex-specific disease risk factors, and reproductive potential. We discovered novel CNVs associated with ovarian dysfunction and support polygenic models for POI.

TAF4b Regulates Oocyte-Specific Genes Essential for Meiosis.

TAF4b is a gonadal-enriched subunit of the general transcription factor TFIID that is implicated in promoting healthy ovarian aging and female fertility in mice and humans. To further explore the potential mechanism of TAF4b in promoting ovarian follicle development, we analyzed global gene expression at multiple time points in the human fetal ovary. This computational analysis revealed coordinate expression of human TAF4B and critical regulators and effectors of meiosis I including SYCP3, YBX2, STAG3, and DAZL. To address the functional relevance of this analysis, we turned to the embryonic Taf4b-deficient mouse ovary where, for the first time, we demonstrate, severe deficits in prophase I progression as well as asynapsis in Taf4b-deficient oocytes. Accordingly, TAF4b occupies the proximal promoters of many essential meiosis and oogenesis regulators, including Stra8, Dazl, Figla, and Nobox, and is required for their proper expression. These data reveal a novel TAF4b function in regulating a meiotic gene expression program in early mouse oogenesis, and support the existence of a highly conserved TAF4b-dependent gene regulatory network promoting early oocyte development in both mice and women.

Gene variants identified by whole-exome sequencing in 33 French women with premature ovarian insufficiency.

PURPOSE: To investigate the potential genetic etiology of premature ovarian insufficiency (POI). METHODS: Whole-exome sequencing (WES) was done on DNA samples from women diagnosed with POI. Mutations identified were analyzed by in silico tools and were annotated according to the guidelines of the American College of Medical Genetics and Genomics. Plausible variants were confirmed by Sanger sequencing. RESULTS: Four of the 33 individuals (12%) carried pathogenic or likely pathogenic variants, and 6 individuals carried variants of unknown significance. The genes identified with pathogenic or likely pathogenic variants included PMM2, MCM9, and PSMC3IP. CONCLUSIONS: WES is an efficient tool for identifying gene variants in POI women; however, interpretation of variants is hampered by few exome studies involving ovarian disorders and the need for trio sequencing to determine inheritance and to detect de novo variants.

Importance of complete phenotyping in prenatal whole exome sequencing.

Whole exome sequencing (WES) is an emerging technique in prenatal diagnosis. In this retrospective study, we examined diagnostic utility and limitations of WES in prenatal cases with structural birth defects. DNA from 20 trios (fetal and parental), with normal karyotype and microarray findings, underwent WES and variant interpretation at a reference laboratory. The WES results were later re-evaluated in our academic center utilizing prenatal and postnatal phenotyping. Initial analysis using only prenatal ultrasound findings revealed no pathogenic or likely pathogenic variants in 20 pregnancies with structural birth defects. Re-analysis of WES variants and combination of prenatal and postnatal phenotyping yielded pathogenic variants in at least 20% of cases including PORCN gene in a fetus with split-hand/foot malformation, as well as variants of uncertain significance in NEB and NOTCH1 in fetuses with postnatal muscle weakness and Adams-Oliver syndrome, respectively. Furthermore, Sanger sequencing in a patient with holoprosencephaly, elucidated by postnatal MRI, revealed a pathogenic 47-base pairs deletion in ZIC2 which was missed by prenatal WES. This study suggests that incomplete prenatal phenotyping and lack of prenatal ultrasound-genotype databases are the limiting factors for current interpretation of WES data in prenatal diagnosis. Development of prenatal phenotype-genotype databases would significantly help WES interpretation in this setting. Patients who underwent prenatal clinical WES may benefit from the re-analysis based on detailed postnatal findings.

X-linked TEX11 mutations, meiotic arrest, and azoospermia in infertile men.

BACKGROUND: The genetic basis of nonobstructive azoospermia is unknown in the majority of infertile men. METHODS: We performed array comparative genomic hybridization testing in blood samples obtained from 15 patients with azoospermia, and we performed mutation screening by means of direct Sanger sequencing of the testis-expressed 11 gene (TEX11) open reading frame in blood and semen samples obtained from 289 patients with azoospermia and 384 controls. RESULTS: We identified a 99-kb hemizygous loss on chromosome Xq13.2 that involved three TEX11 exons. This loss, which was identical in 2 patients with azoospermia, predicts a deletion of 79 amino acids within the meiosis-specific sporulation domain SPO22. Our subsequent mutation screening showed five novel TEX11 mutations: three splicing mutations and two missense mutations. These mutations, which occurred in 7 of 289 men with azoospermia (2.4%), were absent in 384 controls with normal sperm concentrations (P=0.003). Notably, five of those TEX11 mutations were detected in 33 patients (15%) with azoospermia who received a diagnosis of azoospermia with meiotic arrest. Meiotic arrest in these patients resembled the phenotype of Tex11-deficient male mice. Immunohistochemical analysis showed specific cytoplasmic TEX11 expression in late spermatocytes, as well as in round and elongated spermatids, in normal human testes. In contrast, testes of patients who had azoospermia with TEX11 mutations had meiotic arrest and lacked TEX11 expression. CONCLUSIONS: In our study, hemizygous TEX11 mutations were a common cause of meiotic arrest and azoospermia in infertile men. (Funded by the National Institutes of Health and others.).