A systematic review and standardized clinical validity assessment of male infertility genes

Publisher Copyright: © The Author(s) 2019. Published by Oxford University Press on behalf of the European Society of Human Reproduction and Embryology.STUDY QUESTION: Which genes are confidently linked to human monogenic male infertility? SUMMARY ANSWER: Our systematic literature search and clinical validity assessment reveals that a total of 78 genes are currently confidently linked to 92 human male infertility phenotypes. WHAT IS KNOWN ALREADY: The discovery of novel male infertility genes is rapidly accelerating with the availability of next-generating sequencing methods, but the quality of evidence for gene-disease relationships varies greatly. In order to improve genetic research, diagnostics and counseling, there is a need for an evidence-based overview of the currently known genes. STUDY DESIGN, SIZE, DURATION: We performed a systematic literature search and evidence assessment for all publications in Pubmed until December 2018 covering genetic causes of male infertility and/or defective male genitourinary development. PARTICIPANTS/MATERIALS, SETTING, METHODS: Two independent reviewers conducted the literature search and included papers on the monogenic causes of human male infertility and excluded papers on genetic association or risk factors, karyotype anomalies and/or copy number variations affecting multiple genes. Next, the quality and the extent of all evidence supporting selected genes was weighed by a standardized scoring method and used to determine the clinical validity of each gene-disease relationship as expressed by the following six categories: no evidence, limited, moderate, strong, definitive or unable to classify. MAIN RESULTS AND THE ROLE OF CHANCE: From a total of 23 526 records, we included 1337 publications about monogenic causes of male infertility leading to a list of 521 gene-disease relationships. The clinical validity of these gene-disease relationships varied widely and ranged from definitive (n = 38) to strong (n = 22), moderate (n = 32), limited (n = 93) or no evidence (n = 160). A total of 176 gene-disease relationships could not be classified because our scoring method was not suitable. LARGE SCALE DATA: Not applicable. LIMITATIONS, REASONS FOR CAUTION: Our literature search was limited to Pubmed. WIDER IMPLICATIONS OF THE FINDINGS: The comprehensive overview will aid researchers and clinicians in the field to establish gene lists for diagnostic screening using validated gene-disease criteria and help to identify gaps in our knowledge of male infertility. For future studies, the authors discuss the relevant and important international guidelines regarding research related to gene discovery and provide specific recommendations for the field of male infertility. STUDY FUNDING/COMPETING INTEREST(S): This work was supported by a VICI grant from The Netherlands Organization for Scientific Research (918-15-667 to J.A.V.), the Royal Society, and Wolfson Foundation (WM160091 to J.A.V.) as well as an investigator award in science from the Wellcome Trust (209451 to J.A.V.).None.publishersversionPeer reviewe

Rapid whole exome sequencing in pregnancies to identify the underlying genetic cause in fetuses with congenital anomalies detected by ultrasound imaging

Objective: The purpose of this study was to explore the diagnostic yield and clinical utility of trio-based rapid whole exome sequencing (rWES) in pregnancies of fetuses with a wide range of congenital anomalies detected by ultrasound imaging. Methods: In this observational study, we analyzed the first 54 cases referred to our laboratory for prenatal rWES to support clinical decision making, after the sonographic detection of fetal congenital anomalies. The most common identified congenital anomalies were skeletal dysplasia (n = 20), multiple major fetal congenital anomalies (n = 17) and intracerebral structural anomalies (n = 7). Results: A conclusive diagnosis was identified in 18 of the 54 cases (33%). Pathogenic variants were detected most often in fetuses with skeletal dysplasia (n = 11) followed by fetuses with multiple major fetal congenital anomalies (n = 4) and intracerebral structural anomalies (n = 3). A survey, completed by the physicians for 37 of 54 cases, indicated that the rWES results impacted clinical decision making in 68% of cases. Conclusions: These results suggest that rWES improves prenatal diagnosis of fetuses with congenital anomalies, and has an important impact on prenatal and peripartum parental and clinical decision making

Establishing the phenotypic spectrum of ZTTK syndrome by analysis of 52 individuals with variants in SON

Zhu-Tokita-Takenouchi-Kim (ZTTK) syndrome, an intellectual disability syndrome first described in 2016, is caused by heterozygous loss-of-function variants in SON. Its encoded protein promotes pre-mRNA splicing of many genes essential for development. Whereas individual phenotypic traits have previously been linked to erroneous splicing of SON target genes, the phenotypic spectrum and the pathogenicity of missense variants have not been further evaluated. We present the phenotypic abnormalities in 52 individuals, including 17 individuals who have not been reported before. In total, loss-of-function variants were detected in 49 individuals (de novo in 47, inheritance unknown in 2), and in 3, a missense variant was observed (2 de novo, 1 inheritance unknown). Phenotypic abnormalities, systematically collected and analyzed in Human Phenotype Ontology, were found in all organ systems. Significant inter-individual phenotypic variability was observed, even in individuals with the same recurrent variant (n = 13). SON haploinsufficiency was previously shown to lead to downregulation of downstream genes, contributing to specific phenotypic features. Similar functional analysis for one missense variant, however, suggests a different mechanism than for heterozygous loss-of-function. Although small in numbers and while pathogenicity of these variants is not certain, these data allow for speculation whether de novo missense variants cause ZTTK syndrome via another mechanism, or a separate overlapping syndrome. In conclusion, heterozygous loss-of-function variants in SON define a recognizable syndrome, ZTTK, associated with a broad, severe phenotypic spectrum, characterized by a large inter-individual variability. These observations provide essential information for affected individuals, parents, and healthcare professionals to ensure appropriate clinical management

A de novo paradigm for male infertility

De novo mutations are known to play a prominent role in sporadic disorders with reduced fitness. We hypothesize that de novo mutations play an important role in severe male infertility and explain a portion of the genetic causes of this understudied disorder. To test this hypothesis, we utilize trio-based exome sequencing in a cohort of 185 infertile males and their unaffected parents. Following a systematic analysis, 29 of 145 rare (MAF < 0.1%) protein-altering de novo mutations are classified as possibly causative of the male infertility phenotype. We observed a significant enrichment of loss-of-function de novo mutations in loss-of-function-intolerant genes (p -value = 1.00 × 10 −5) in infertile men compared to controls. Additionally, we detected a significant increase in predicted pathogenic de novo missense mutations affecting missense-intolerant genes (p -value = 5.01 × 10 −4) in contrast to predicted benign de novo mutations. One gene we identify, RBM5, is an essential regulator of male germ cell pre-mRNA splicing and has been previously implicated in male infertility in mice. In a follow-up study, 6 rare pathogenic missense mutations affecting this gene are observed in a cohort of 2,506 infertile patients, whilst we find no such mutations in a cohort of 5,784 fertile men (p -value = 0.03). Our results provide evidence for the role of de novo mutations in severe male infertility and point to new candidate genes affecting fertility. Germline de novo mutations can impact individual fitness, but their role in human male infertility is understudied. Trio-based exome sequencing identifies many new candidate genes affecting male fertility, including an essential regulator of male germ cell pre-mRNA splicing

Evidence for 28 genetic disorders discovered by combining healthcare and research data

De novo mutations in protein-coding genes are a well-established cause of developmental disorders. However, genes known to be associated with developmental disorders account for only a minority of the observed excess of such de novo mutations. Here, to identify previously undescribed genes associated with developmental disorders, we integrate healthcare and research exome-sequence data from 31,058 parent–offspring trios of individuals with developmental disorders, and develop a simulation-based statistical test to identify gene-specific enrichment of de novo mutations. We identified 285 genes that were significantly associated with developmental disorders, including 28 that had not previously been robustly associated with developmental disorders. Although we detected more genes associated with developmental disorders, much of the excess of de novo mutations in protein-coding genes remains unaccounted for. Modelling suggests that more than 1,000 genes associated with developmental disorders have not yet been described, many of which are likely to be less penetrant than the currently known genes. Research access to clinical diagnostic datasets will be critical for completing the map of genes associated with developmental disorders

Heterozygous Mutations of FREM1 Are Associated with an Increased Risk of Isolated Metopic Craniosynostosis in Humans and Mice

The premature fusion of the paired frontal bones results in metopic craniosynostosis (MC) and gives rise to the clinical phenotype of trigonocephaly. Deletions of chromosome 9p22.3 are well described as a cause of MC with variably penetrant midface hypoplasia. In order to identify the gene responsible for the trigonocephaly component of the 9p22.3 syndrome, a cohort of 109 patients were assessed by high-resolution arrays and MLPA for copy number variations (CNVs) involving 9p22. Five CNVs involving FREM1, all of which were de novo variants, were identified by array-based analyses. The remaining 104 patients with MC were then subjected to targeted FREM1 gene re-sequencing, which identified 3 further mutant alleles, one of which was de novo. Consistent with a pathogenic role, mouse Frem1 mRNA and protein expression was demonstrated in the metopic suture as well as in the pericranium and dura mater. Micro-computed tomography based analyses of the mouse posterior frontal (PF) suture, the human metopic suture equivalent, revealed advanced fusion in all mice homozygous for either of two different Frem1 mutant alleles, while heterozygotes exhibited variably penetrant PF suture anomalies. Gene dosage-related penetrance of midfacial hypoplasia was also evident in the Frem1 mutants. These data suggest that CNVs and mutations involving FREM1 can be identified in a significant percentage of people with MC with or without midface hypoplasia. Furthermore, we present Frem1 mutant mice as the first bona fide mouse model of human metopic craniosynostosis and a new model for midfacial hypoplasia

Inherited variants in CHD3 show variable expressivity in Snijders Blok-Campeau syndrome

Purpose: Common diagnostic next-generation sequencing strategies are not optimized to identify inherited variants in genes associated with dominant neurodevelopmental disorders as causal when the transmitting parent is clinically unaffected, leaving a significant number of cases with neurodevelopmental disorders undiagnosed. Methods: We characterized 21 families with inherited heterozygous missense or protein-truncating variants in CHD3, a gene in which de novo variants cause Snijders Blok-Campeau syndrome. Results: Computational facial and Human Phenotype Ontology–based comparisons showed that the phenotype of probands with inherited CHD3 variants overlaps with the phenotype previously associated with de novo CHD3 variants, whereas heterozygote parents are mildly or not affected, suggesting variable expressivity. In addition, similarly reduced expression levels of CHD3 protein in cells of an affected proband and of healthy family members with a CHD3 protein-truncating variant suggested that compensation of expression from the wild-type allele is unlikely to be an underlying mechanism. Notably, most inherited CHD3 variants were maternally transmitted. Conclusion: Our results point to a significant role of inherited variation in Snijders Blok-Campeau syndrome, a finding that is critical for correct variant interpretation and genetic counseling and warrants further investigation toward understanding the broader contributions of such variation to the landscape of human disease

A Solve-RD ClinVar-based reanalysis of 1522 index cases from ERN-ITHACA reveals common pitfalls and misinterpretations in exome sequencing

Purpose: Within the Solve-RD project (https://solve-rd.eu/), the European Reference Network for Intellectual disability, TeleHealth, Autism and Congenital Anomalies aimed to investigate whether a reanalysis of exomes from unsolved cases based on ClinVar annotations could establish additional diagnoses. We present the results of the "ClinVar low-hanging fruit" reanalysis, reasons for the failure of previous analyses, and lessons learned. Methods: Data from the first 3576 exomes (1522 probands and 2054 relatives) collected from European Reference Network for Intellectual disability, TeleHealth, Autism and Congenital Anomalies was reanalyzed by the Solve-RD consortium by evaluating for the presence of single-nucleotide variant, and small insertions and deletions already reported as (likely) pathogenic in ClinVar. Variants were filtered according to frequency, genotype, and mode of inheritance and reinterpreted. Results: We identified causal variants in 59 cases (3.9%), 50 of them also raised by other approaches and 9 leading to new diagnoses, highlighting interpretation challenges: variants in genes not known to be involved in human disease at the time of the first analysis, misleading genotypes, or variants undetected by local pipelines (variants in off-target regions, low quality filters, low allelic balance, or high frequency). Conclusion: The "ClinVar low-hanging fruit" analysis represents an effective, fast, and easy approach to recover causal variants from exome sequencing data, herewith contributing to the reduction of the diagnostic deadlock.The Solve-RD project has received funding from the European Union’s Horizon 2020 research and innovation program under grant agreement number 779257. Data were analyzed using the RD-Connect Genome-Phenome Analysis Platform, which received funding from the EU projects RD-Connect, Solve-RD, and European Joint Programme on Rare Diseases (grant numbers FP7 305444, H2020 779257, H2020 825575), Instituto de Salud Carlos III (grant numbers PT13/0001/0044, PT17/0009/0019; Instituto Nacional de Bioinformática), and ELIXIR Implementation Studies. The collaborations in this study were facilitated by the European Reference Network for Intellectual disability, TeleHealth, Autism and Congenital Anomalies, one of the 24 European Reference Networks approved by the European Reference Network Board of Member States, cofunded by the European Commission. This project was supported by the Czech Ministry of Health (number 00064203) and by the Czech Ministry of Education, Youth and Sports (number - LM2018132) to M.M.S

A MT-TL1 variant identified by whole exome sequencing in an individual with intellectual disability, epilepsy, and spastic tetraparesis

Funder: The Solve-RD project has received funding from the European Union’s Horizon 2020 research and innovation programme under grant agreement No 779257Abstract: The genetic etiology of intellectual disability remains elusive in almost half of all affected individuals. Within the Solve-RD consortium, systematic re-analysis of whole exome sequencing (WES) data from unresolved cases with (syndromic) intellectual disability (n = 1,472 probands) was performed. This re-analysis included variant calling of mitochondrial DNA (mtDNA) variants, although mtDNA is not specifically targeted in WES. We identified a functionally relevant mtDNA variant in MT-TL1 (NC_012920.1:m.3291T > C; NC_012920.1:n.62T > C), at a heteroplasmy level of 22% in whole blood, in a 23-year-old male with severe intellectual disability, epilepsy, episodic headaches with emesis, spastic tetraparesis, brain abnormalities, and feeding difficulties. Targeted validation in blood and urine supported pathogenicity, with heteroplasmy levels of 23% and 58% in index, and 4% and 17% in mother, respectively. Interestingly, not all phenotypic features observed in the index have been previously linked to this MT-TL1 variant, suggesting either broadening of the m.3291T > C-associated phenotype, or presence of a co-occurring disorder. Hence, our case highlights the importance of underappreciated mtDNA variants identifiable from WES data, especially for cases with atypical mitochondrial phenotypes and their relatives in the maternal line