A systematic review and evidence assessment of monogenic gene-disease relationships in human female infertility and differences in sex development.

BACKGROUND: As in other domains of medicine, high-throughput sequencing methods have led to the identification of an ever-increasing number of gene variants in the fields of both male and female infertility. The increasing number of recently identified genes allows an accurate diagnosis for previously idiopathic cases of female infertility and more appropriate patient care. However, robust evidence of the gene-disease relationships (GDR) allowing the proper translation to clinical application is still missing in many cases. OBJECTIVE AND RATIONALE: An evidence-based curation of currently identified genes involved in female infertility and differences in sex development (DSD) would significantly improve both diagnostic performance and genetic research. We therefore performed a systematic review to summarize current knowledge and assess the available GDR. SEARCH METHODS: PRISMA guidelines were applied to curate all available information from PubMed and Web of Science on genetics of human female infertility and DSD leading to infertility, from 1 January 1988 to 1 November 2021. The reviewed pathologies include non-syndromic as well as syndromic female infertility, and endocrine and reproductive system disorders. The evidence that an identified phenotype is caused by pathogenic variants in a specific gene was assessed according to a standardized scoring system. A final score (no evidence, limited, moderate, strong, or definitive) was assigned to every GDR. OUTCOMES: A total of 45 271 publications were identified and screened for inclusion of which 1078 were selected for gene and variant extraction. We have identified 395 genes and validated 466 GDRs covering all reported monogenic causes of female infertility and DSD. Furthermore, we present a genetic diagnostic flowchart including 105 genes with at least moderate evidence for female infertility and suggest recommendations for future research. The study did not take into account associated genetic risk factor(s) or oligogenic/polygenic causes of female infertility. WIDER IMPLICATIONS: We have comprehensively reviewed the existing research on the genetics of female infertility and DSD, which will enable the development of diagnostic panels using validated genes. Whole genome analysis is shifting from predominantly research to clinical application, increasing its diagnostic potential. These new diagnostic possibilities will not only decrease the number of idiopathic cases but will also render genetic counselling more effective for infertile patients and their families.

EDIR: exome database of interspersed repeats.

MOTIVATION: Intragenic exonic deletions are known to contribute to genetic diseases and are often flanked by regions of homology. RESULTS: In order to get a more clear view of these interspersed repeats encompassing a coding sequence, we have developed EDIR (Exome Database of Interspersed Repeats) which contains the positions of these structures within the human exome. EDIR has been calculated by an inductive strategy, rather than by a brute force approach and can be queried through an R/Bioconductor package or a web interface allowing the per-gene rapid extraction of homology-flanked sequences throughout the exome. AVAILABILITY AND IMPLEMENTATION: The code used to compile EDIR can be found at https://github.com/lauravongoc/EDIR. The full dataset of EDIR can be queried via an Rshiny application at http://193.70.34.71:3857/edir/. The R package for querying EDIR is called 'EDIRquery' and is available on Bioconductor. The full EDIR dataset can be downloaded from https://osf.io/m3gvx/ or http://193.70.34.71/EDIR.tar.gz. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

The genetic diagnosis of rare endocrine disorders of sex development and maturation: a survey among Endo-ERN centres.

Differences of sex development and maturation (SDM) represent a heterogeneous puzzle of rare conditions with a large genetic component whose management and treatment could be improved by an accurate classification of underlying molecular conditions, and next-generation sequencing (NGS) should represent the most appropriate approach. Therefore, we conducted a survey dedicated to the use and potential outcomes of NGS for SDM disorders diagnosis among the 53 health care providers (HCP) of the European Reference Network for rare endocrine conditions. The response rate was 49% with a total of 26 HCPs from 13 countries. All HCPs, except 1, performed NGS investigations for SDM disorders on 6720 patients, 3764 (56%) with differences of sex development (DSD), including 811 unexplained primary ovarian insufficiency, and 2956 (44%) with congenital hypogonadotropic hypogonadism (CHH). The approaches varied from targeted analysis of custom gene panels (range: 11-490 genes) in 81.5% of cases or whole exome sequencing with the extraction of a virtual panel in the remaining cases. These analyses were performed for diagnostic purposes in 21 HCPs, supported by the National Health Systems in 16 cases. The likelihood of finding a variant ranged between 7 and 60%, mainly depending upon the number of analysed genes or criteria used for reporting, most HCPs also reporting variants of uncertain significance. These data illustrate the status of genetic diagnosis of DSD and CHH across Europe. In most countries, these analyses are performed for diagnostic purposes, yielding highly variable results, thus suggesting the need for harmonization and general improvements of NGS approaches.

Case report: Coexistence of myotonia congenita and Brugada syndrome in one family.

Myotonia congenita is a rare neuromuscular disorder caused by CLCN1 mutations resulting in delayed muscle relaxation. Extramuscular manifestations are not considered to be present in chloride skeletal channelopathies, although recently some cardiac manifestations have been described. We report a family with autosomal dominant myotonia congenita and Brugada syndrome. Bearing in mind the previously reported cases of cardiac arrhythmias in myotonia congenita patients, we discuss the possible involvement of the CLCN1-gene mutations in primary cardiac arrhythmia.

Ascites in infantile onset type II Sialidosis.

Sialidosis is a rare autosomal-recessive lysosomal storage disease due to mutations in the NEU1 gene leading to a deficit of alpha-n-acetyl neuraminidase and causing aberrant accumulation of sialylated glycoproteins/peptides and oligosaccharides in the lysosomes of various organs and tissues. Type II sialidosis (dysmorphic form) is classified into three subgroups based on the age of onset and the clinical severity: Congenital or neonatal, infantile (onset 0-12 months) and juvenile form (onset 13 months-20 years). We report the case of a 3-year-old boy with sialidosis type II infantile form, who developed a voluminous ascites. To the best of our knowledge, ascites is not described in the infantile form but in the congenital form of the disease. Ascites seems to be of a multifactorial origin regarding our investigations: on the one hand, portal hypertension and on the other hypoalbuminemia maintained by proteinuria secondary to nephrosialidosis. Loss of plasma proteins in the gastrointestinal tract (protein-losing enteropathy) should also be considered in the case of portal hypertension and damages of the reticuloendothelial system.

Myotonic dystrophy type 1 embryonic stem cells show decreased myogenic potential, increased CpG methylation at the DMPK locus and RNA mis-splicing.

Skeletal muscle tissue is severely affected in myotonic dystrophy type 1 (DM1) patients, characterised by muscle weakness, myotonia and muscle immaturity in the most severe congenital form of the disease. Previously, it was not known at what stage during myogenesis the DM1 phenotype appears. In this study we differentiated healthy and DM1 human embryonic stem cells to myoblasts and myotubes and compared their differentiation potential using a comprehensive multi-omics approach. We found myogenesis in DM1 cells to be abnormal with altered myotube generation compared to healthy cells. We did not find differentially expressed genes between DM1 and non-DM1 cell lines within the same developmental stage. However, during differentiation we observed an aberrant inflammatory response and increased CpG methylation upstream of the CTG repeat at the myoblast level and RNA mis-splicing at the myotube stage. We show that early myogenesis modelled in hESC reiterates the early developmental manifestation of DM1.

Impaired catabolism of free oligosaccharides due to MAN2C1 variants causes a neurodevelopmental disorder.

Free oligosaccharides (fOSs) are soluble oligosaccharide species generated during N-glycosylation of proteins. Although little is known about fOS metabolism, the recent identification of NGLY1 deficiency, a congenital disorder of deglycosylation (CDDG) caused by loss of function of an enzyme involved in fOS metabolism, has elicited increased interest in fOS processing. The catabolism of fOSs has been linked to the activity of a specific cytosolic mannosidase, MAN2C1, which cleaves α1,2-, α1,3-, and α1,6-mannose residues. In this study, we report the clinical, biochemical, and molecular features of six individuals, including two fetuses, with bi-allelic pathogenic variants in MAN2C1; the individuals are from four different families. These individuals exhibit dysmorphic facial features, congenital anomalies such as tongue hamartoma, variable degrees of intellectual disability, and brain anomalies including polymicrogyria, interhemispheric cysts, hypothalamic hamartoma, callosal anomalies, and hypoplasia of brainstem and cerebellar vermis. Complementation experiments with isogenic MAN2C1-KO HAP1 cells confirm the pathogenicity of three of the identified MAN2C1 variants. We further demonstrate that MAN2C1 variants lead to accumulation and delay in the processing of fOSs in proband-derived cells. These results emphasize the involvement of MAN2C1 in human neurodevelopmental disease and the importance of fOS catabolism.

Intra-amniotic levothyroxine infusions in a case of fetal goiter due to novel Thyroglobulin gene variants.

Indications and administration of intra-amniotic infusions of L-thyroxine in the context of non-immune fetal hypothyroidism with goiter lack of standardization. Systematic follow-up of clinical features related to thyroid hormonal homeostasis may be useful to evaluate their efficiency and develop standardized management guidelines.

MSH2 knock-down shows CTG repeat stability and concomitant upstream demethylation at the DMPK locus in myotonic dystrophy type 1 human embryonic stem cells.

Myotonic dystrophy type 1 (DM1) is caused by expansion of a CTG repeat in the DMPK gene, where expansion size and somatic mosaicism correlates with disease severity and age of onset. While it is known that the mismatch repair protein MSH2 contributes to the unstable nature of the repeat, its role on other disease-related features, such as CpG methylation upstream of the repeat, is unknown. In this study, we investigated the effect of an MSH2 knock-down (MSH2KD) on both CTG repeat dynamics and CpG methylation pattern in human embryonic stem cells (hESC) carrying the DM1 mutation. Repeat size in MSH2 wild-type (MSH2WT) and MSH2KD DM1 hESC was determined by PacBio sequencing and CpG methylation by bisulfite massive parallel sequencing. We found stabilization of the CTG repeat concurrent with a gradual loss of methylation upstream of the repeat in MSH2KD cells, while the repeat continued to expand and upstream methylation remained unchanged in MSH2WT control lines. Repeat instability was re-established and biased towards expansions upon MSH2 transgenic re-expression in MSH2KD lines while upstream methylation was not consistently re-established. We hypothesize that the hypermethylation at the mutant DM1 locus is promoted by the MMR machinery and sustained by a constant DNA repair response, establishing a potential mechanistic link between CTG repeat instability and upstream CpG methylation. Our work represents a first step towards understanding how epigenetic alterations and repair pathways connect and contribute to the DM1 pathology.

Novel inactivating follicle-stimulating hormone receptor mutations in a patient with premature ovarian insufficiency identified by next-generation sequencing gene panel analysis.

OBJECTIVE: To find the genetic etiology of premature ovarian insufficiency (POI) in a patient with primary amenorrhea and hypergonadotropic hypogonadism. DESIGN: Case report. SETTING: University hospital. PATIENTS: A Belgian woman aged 32 years with POI at the age of 17, her parents, and her sister whose POI was diagnosed at age 29. INTERVENTIONS: Analysis of a panel of 31 genes implicated in POI (POIGP) using next-generation sequencing (NGS), Sanger sequencing, and in vitro functional study. MAIN OUTCOME MEASURES: Gene variants, family mutational segregation, and in vitro functional impact of the mutant proteins. RESULTS: The analysis of the gene panel using NGS identified the presence of two novel follicle-stimulating hormone receptor (FSHR) missense mutations at a compound heterozygous state in the affected patient: c.646 G>A, p.Gly216Arg, and c.1313C>T, p.Thr438Ile. Sanger sequencing showed the presence of each mutation at heterozygous state in the patient's parents and at heterozygous compound state in the affected sister. Both substituted amino acids (Gly216 and Thr438) were conserved in FSHR of several vertebrate species as well as in other glycoproteins receptors (TSHR and LHCGHR), suggesting a potentially important role in glycoprotein receptor function. An in vitro functional study showed similar results for both variants with more than 90% reduction of their cell surface expression and a 55% reduction of their FSH-induced cyclic adenosine 3':5' monophosphate (cAMP) production compared with the wild-type FSHR. CONCLUSIONS: The analysis of a gene panel of 31 genes implicated in POI allowed us to identify two novel partially inactivating mutations of FSHR that are likely responsible for the POI phenotype of the proband and of her affected sister.

Heterogeneous clinical phenotypes and cerebral malformations reflected by rotatin cellular dynamics.

Recessive mutations in RTTN, encoding the protein rotatin, were originally identified as cause of polymicrogyria, a cortical malformation. With time, a wide variety of other brain malformations has been ascribed to RTTN mutations, including primary microcephaly. Rotatin is a centrosomal protein possibly involved in centriolar elongation and ciliogenesis. However, the function of rotatin in brain development is largely unknown and the molecular disease mechanism underlying cortical malformations has not yet been elucidated. We performed both clinical and cell biological studies, aimed at clarifying rotatin function and pathogenesis. Review of the 23 published and five unpublished clinical cases and genomic mutations, including the effect of novel deep intronic pathogenic mutations on RTTN transcripts, allowed us to extrapolate the core phenotype, consisting of intellectual disability, short stature, microcephaly, lissencephaly, periventricular heterotopia, polymicrogyria and other malformations. We show that the severity of the phenotype is related to residual function of the protein, not only the level of mRNA expression. Skin fibroblasts from eight affected individuals were studied by high resolution immunomicroscopy and flow cytometry, in parallel with in vitro expression of RTTN in HEK293T cells. We demonstrate that rotatin regulates different phases of the cell cycle and is mislocalized in affected individuals. Mutant cells showed consistent and severe mitotic failure with centrosome amplification and multipolar spindle formation, leading to aneuploidy and apoptosis, which could relate to depletion of neuronal progenitors often observed in microcephaly. We confirmed the role of rotatin in functional and structural maintenance of primary cilia and determined that the protein localized not only to the basal body, but also to the axoneme, proving the functional interconnectivity between ciliogenesis and cell cycle progression. Proteomics analysis of both native and exogenous rotatin uncovered that rotatin interacts with the neuronal (non-muscle) myosin heavy chain subunits, motors of nucleokinesis during neuronal migration, and in human induced pluripotent stem cell-derived bipolar mature neurons rotatin localizes at the centrosome in the leading edge. This illustrates the role of rotatin in neuronal migration. These different functions of rotatin explain why RTTN mutations can lead to heterogeneous cerebral malformations, both related to proliferation and migration defects.

Genetic diagnosis of subfertility: the impact of meiosis and maternal effects.

During reproductive age, approximately one in seven couples are confronted with fertility problems. While the aetiology is diverse, including infections, metabolic diseases, hormonal imbalances and iatrogenic effects, it is becoming increasingly clear that genetic factors have a significant contribution. Due to the complex nature of infertility that often hints at a multifactorial cause, the search for potentially causal gene mutations in idiopathic infertile couples has remained difficult. Idiopathic infertility patients with a suspicion of an underlying genetic cause can be expected to have mutations in genes that do not readily affect general health but are only essential in certain processes connected to fertility. In this review, we specifically focus on genes involved in meiosis and maternal-effect processes, which are of critical importance for reproduction and initial embryonic development. We give an overview of genes that have already been linked to infertility in human, as well as good candidates which have been described in other organisms. Finally, we propose a phenotypic range in which we expect an optimal diagnostic yield of a meiotic/maternal-effect gene panel.

Rare genetic variants potentially involved in ovarian hyperstimulation syndrome.

PURPOSE: We aim to investigate whether there is a genetic predisposition in women who developed ovarian hyperstimulation syndrome (OHSS) after GnRH antagonist protocol with GnRH agonist trigger and freeze-all approach. METHODS: Four patients with OHSS after GnRH agonist trigger and freeze-all approach were gathered from the worldwide patient population. These patients were analyzed through Whole Exome Sequencing. In this study known causes of OHSS were investigated and new causes present in at least two individuals were searched for. RESULTS: In the first part of the study, we evaluated the presence of mutations in genes already known to be involved in OHSS. In PGR and TP53, heterozygous alterations were detected. PGR is predicted to be involved in progesterone resistance with a recessive inheritance pattern and is, therefore, not considered as being causal. The consequences of the variant detected in TP53 currently remain unknown. In part 2 of the study, we assessed the clinical significance of variants in genes previously not linked to OHSS. We especially focused on genes with variants present in ≥ 2 patients. Two patients have variants in the FLT4 gene. Mutations in this gene are linked to hereditary lymphedema, but no link to OHSS has been described. CONCLUSIONS: Defining a genetic predisposition for OHSS is essential in view of prevention. In this study, a potential link between the FLT4 gene and OHSS has been suggested. Future functional studies are essential to define a more precise involvement of the detected variants in the development of OHSS.

Clinical implementation of gene panel testing for lysosomal storage diseases.

BACKGROUND: The diagnostic workup in patients with a clinical suspicion of lysosomal storage diseases (LSD) is often difficult due to the variability in the clinical phenotype. The gold standard for diagnosis of LSDs consists of enzymatic testing. However, due to the sequential nature of this methodology and inconsistent genotype-phenotype correlations of certain LSDs, finding a diagnosis can be challenging. METHOD: We developed and clinically implemented a gene panel covering 50 genes known to cause LSDs when mutated. Over a period of 18 months, we analyzed 150 patients who were referred for LSD testing and compared these results with the data of patients who were previously enrolled in a scheme of classical biochemical testing. RESULTS: Our panel was able to determine the molecular cause of the disease in 22 cases (15%), representing an increase in diagnostic yield compared to biochemical tests developed for 21 LSDs (4.6%). We were furthermore able to redirect the diagnosis of a mucolipidosis patient who was initially suspected to be affected with galactosialidosis. Several patients were identified as being affected with neuronal ceroid lipofuscinosis, which cannot readily be detected by enzyme testing. Finally, several carriers of pathogenic mutations in LSD genes related to the disease phenotype were identified as well, thus potentially increasing the diagnostic yield of the panel as heterozygous deletions cannot be detected. CONCLUSION: We show that the implementation of a gene panel for LSD diagnostics results in an increased yield in comparison to classical biochemical testing. As the panel is able to cover a wider range of diseases, we propose to implement this methodology as a first-tier test in cases of an aspecific LSD presentation, while enzymatic testing remains the first choice in patients with a more distinctive clinical presentation. Positive panel results should however still be enzymatically confirmed whenever possible.

Biallelic mutations in RTTN are associated with microcephaly, short stature and a wide range of brain malformations.

Biallelic mutations in the RTTN gene have been reported in association with microcephaly, short stature, developmental delay and malformations of cortical development. RTTN mutations have previously shown to link aberrant ciliary function with abnormal development and organization of the human cerebral cortex. We here report three individuals from two unrelated families with novel mutations in the RTTN gene. The phenotype consisted of microcephaly, short stature, pachygyria or polymicrogyria, colpocephaly, hypoplasia of the corpus callosum and superior vermis. These findings provide further confirmation of the phenotype related to pathogenic variants in RTTN.

Bi-allelic variants in COL3A1 encoding the ligand to GPR56 are associated with cobblestone-like cortical malformation, white matter changes and cerebellar cysts.

BACKGROUND: Collagens are one of the major constituents of the pial membrane, which plays a crucial role in neuronal migration and cortical lamination during brain development. Type III procollagen, the chains of which are encoded by COL3A1, is the ligand of the G protein-coupled receptor 56 (GPR56), also known as adhesion G protein-coupled receptor G1. Bi-allelic mutations in GPR56 give rise to cobblestone-like malformation, white matter changes and cerebellar dysplasia. This report shows that bi-allelic mutations in COL3A1 are associated with a similar phenotype. METHODS: Exome analysis was performed in a family consisting of two affected and two non-affected siblings. Brain imaging studies of this family and of two previously reported individuals with bi-allelic mutations in COL3A1 were reviewed. Functional assays were performed on dermal fibroblasts. RESULTS: Exome analysis revealed a novel homozygous variant c.145C>G (p.Pro49Ala) in exon 2 of COL3A1. Brain MRI in the affected siblings as well as in the two previously reported individuals with bi-allelic COL3A1 mutations showed a brain phenotype similar to that associated with mutations in GPR56. CONCLUSION: Homozygous or compound heterozygous mutations in COL3A1 are associated with cobblestone-like malformation in all three families reported to date. The variability of the phenotype across patients suggests that genetic alterations in distinct domains of type III procollagen can lead to different outcomes. The presence of cobblestone-like malformation in patients with bi-allelic COL3A1 mutations emphasises the critical role of the type III collagen-GPR56 axis and the pial membrane in the regulation of brain development and cortical lamination.

Convert your favorite protein modeling program into a mutation predictor: "MODICT".

BACKGROUND: Predict whether a mutation is deleterious based on the custom 3D model of a protein. RESULTS: We have developed MODICT, a mutation prediction tool which is based on per residue RMSD (root mean square deviation) values of superimposed 3D protein models. Our mathematical algorithm was tested for 42 described mutations in multiple genes including renin (REN), beta-tubulin (TUBB2B), biotinidase (BTD), sphingomyelin phosphodiesterase-1 (SMPD1), phenylalanine hydroxylase (PAH) and medium chain Acyl-Coa dehydrogenase (ACADM). Moreover, MODICT scores corresponded to experimentally verified residual enzyme activities in mutated biotinidase, phenylalanine hydroxylase and medium chain Acyl-CoA dehydrogenase. Several commercially available prediction algorithms were tested and results were compared. The MODICT PERL package and the manual can be downloaded from https://github.com/IbrahimTanyalcin/MODICT . CONCLUSIONS: We show here that MODICT is capable tool for mutation effect prediction at the protein level, using superimposed 3D protein models instead of sequence based algorithms used by POLYPHEN and SIFT.

Polyneuropathy in a young Belgian patient: A novel heterozygous mutation in the WNK1/HSN2 gene.

Hereditary sensory autonomic neuropathy (HSAN) is a rare condition, predominantly affecting the peripheral sensory nervous system, although variable motor and dysautonomic symptoms can be present. At least 7 clinical types of HSAN have been described, and different genetic mutations have been identified for each of these. HSAN IIA (OMIM #201300) is characterized by loss of pain and loss of temperature and touch sensation, with onset usually before the first decade. The mode of inheritance is autosomal recessive.(1) The causative gene, WNK1/HSN2, is located on locus 12p13.33 and is an isoform of the WNK1 (lysine deficient protein kinase 1) gene, which contains the HSN2 exon.(2,3) We describe 2 new heterozygous mutations in the WNK1/HSN2 gene in a Belgian patient with early-onset sensory polyneuropathy.

Sertoli Cell-Only Syndrome: Behind the Genetic Scenes.

Sertoli cell-only syndrome is defined by the complete absence of germ cells in testicular tissues and always results in male infertility. The aetiology often remains unknown. In this paper, we have investigated possible causes of Sertoli cell-only syndrome with a special focus on genetic causes. Our results show that, for a large part of the patients (>23% in an unselected group), the sex chromosomes are involved. The majority of patients had a Klinefelter syndrome, followed by patients with Yq microdeletions. Array comparative genomic hybridization in a selected group of "idiopathic patients" showed no known infertility related copy number variations.

I-PV: a CIRCOS module for interactive protein sequence visualization.

SUMMARY: Today's genome browsers and protein databanks supply vast amounts of information about proteins. The challenge is to concisely bring together this information in an interactive and easy to generate format. AVAILABILITY AND IMPLEMENTATION: We have developed an interactive CIRCOS module called i-PV to visualize user supplied protein sequence, conservation and SNV data in a live presentable format. I-PV can be downloaded from http://www.i-pv.org. CONTACT: ibrahim.tanyalcin@i-pv.org, itanyalc@vub.ac.be or support@i-pv.org SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.