Epithelial to Mesenchymal transitions (EMT) drive cell plasticity and are associated with cell features such as invasiveness, migration and stemness. They are orchestrated by select families of EMT-associated transcription factors, which exhibit pleiotropic roles in the malignant progression of various cancer types, such as breast and colorectal cancer (CRC). This has spurred interest in EMT as a promising target for the development of novel therapeutic strategies. In this study, we developed a phenotypic dual EMT Sensor screening assay, amendable to efficient high-throughput identification of small molecules interfering with EMT. In a proof-of-concept screening we identified anti-EMT repurposing drugs. From these, we validated RepSox, a selective inhibitor of the TGF-β type I receptor ALK5, and demonstrated that it is potently blocking EMT in both breast and colorectal cancer cell lines in vitro. In addition, utilizing a Drosophila melanogaster metastatic CRC model we confirmed the ability of the identified anti-EMT hits to suppress metastatic behavior in vivo.

Primary ovarian insufficiency (POI) is an important cause of female infertility, stemming from follicle dysfunction or premature oocyte depletion. Pathogenic variants in genes such as NOBOX, GDF9, BMP15, and FSHR have been linked to POI. NOBOX, a transcription factor expressed in oocytes and granulosa cells, plays a pivotal role in folliculogenesis. Loss-of-function variants in NOBOX are reported in 1–2

Summary Delayed puberty in girls is often related to late maturation but is occasionally the first sign of premature ovarian insufficiency (POI). POI is a condition that affects ovarian function and fertility, and its etiology is unknown in most cases. Genetic factors have recently been identified in 20–25% of women with POI, involving genes that regulate various aspects of ovarian development and maintenance. We report a case of delayed puberty due to POI in an adolescent from a non-consanguineous family who carried two variants in the MCM9 gene. MCM9 is essential for DNA replication and repair, and its dysfunction can lead to chromosomal instability and ovarian failure. Our case highlights the importance of targeted gene panel analysis, particularly in POI patients with negative autoimmunity screening, and evidence of ovarian or uterine dysgenesis on pelvic imaging. Learning points Delayed puberty in girls is often self-limiting, but it can also indicate underlying conditions with lifelong implications, such as premature ovarian insufficiency (POI). Patients with POI, negative autoimmune screening, a normal karyotype, and no FMR premutation should undergo further genetic testing, preferably through targeted gene panels. Compound heterozygous variants in MCM9 can cause POI, presenting with delayed puberty and primary amenorrhea in girls without a consanguineous family.

BACKGROUND:Congenital myasthenic syndromes (CMS) are a group of genetic disorders characterized by impaired neuromuscular transmission. CMS typically present at a young age with fatigable muscle weakness, often with an abnormal response after repetitive nerve stimulation (RNS). Pharmacologic treatment can improve symptoms, depending on the underlying defect. Prevalence is likely underestimated. This study reports on patients with CMS followed in Belgium in 2022. METHODS:Data were gathered retrospectively from the medical charts. Only likely pathogenic and pathogenic variants were included in the analysis. RESULTS:We identified 37 patients, resulting in an estimated prevalence of 3.19 per 1,000,000. The patients harbored pathogenic variants in CHRNE, RAPSN, DOK7, PREPL, CHRNB1, CHRNG, COLQ, MUSK, CHRND, GFPT1, and GMPPB. CHRNE was the most commonly affected gene. Most patients showed disease onset at birth, during infancy, or during childhood. Symptom onset was at adult age in seven patients, caused by variants in CHRNE, DOK7, MUSK, CHRND, and GMPPB. Severity and distribution of weakness varied, as did the presence of respiratory involvement, feeding problems, and extraneuromuscular manifestations. RNS was performed in 23 patients of whom 18 demonstrated a pathologic decrement. Most treatment responses were predictable based on the genotype. CONCLUSIONS:This is the first pooled characterization of patients with CMS in Belgium. We broaden the phenotypical spectrum of pathogenic variants in CHRNE with adult-onset CMS. Systematically documenting larger cohorts of patients with CMS can aid in better clinical characterization and earlier recognition of this rare disease. We emphasize the importance of establishing a molecular genetic diagnosis to tailor treatment choices.

Background Among the 10% of pancreatic cancers that occur in a familial context, around a third carry a pathogenic variant in a cancer predisposition gene. Genetic studies of pancreatic cancer predisposition are limited by high mortality rates amongst index patients and other affected family members. The genetic risk for pancreatic cancer is often shared with breast cancer susceptibility genes, most notably BRCA2, PALB2, ATM and BRCA1. Therefore, we hypothesized that additional shared genetic etiologies might be uncovered by studying families presenting with both breast and pancreatic cancer. Methods Focusing on a multigene panel of 276 DNA Damage Repair (DDR) genes, we performed next-generation sequencing in a cohort of 41 families with at least three breast cancer cases and one pancreatic cancer. When the index patient with pancreatic cancer was deceased, close relatives (first or second-degree) affected with breast cancer were tested (39 families). Results We identified 27 variants of uncertain significance in DDR genes. A splice site variant (c.1605 + 2T > A) in the RAD17 gene stood out, as a likely loss of function variant. RAD17 is a checkpoint protein that recruits the MRN (MRE11-RAD50-NBS1) complex to initiate DNA signaling, leading to DNA double-strand break repair. Conclusion Within families with breast and pancreatic cancer, we identified RAD17 as a novel candidate predisposition gene. Further genetic studies are warranted to better understand the potential pathogenic effect of RAD17 variants and in other DDR genes.

Abstract Study question Are pathogenic NOBOX variants causal for oocyte/zygote/embryo maturation arrest (OZEMA) and what is their molecular mechanism? Summary answer Expression analysis of NOBOX target genes indicates that the examined variants do not influence early folliculogenesis, suggesting a potential involvement in later developmental stages. What is known already Primary ovarian insufficiency (POI) affects around 1% of women under 40 with pathogenic variants in the transcription factor NOBOX being causal in 5-7% of cases. Historically, NOBOX has been linked to premature oocyte depletion. Functional studies in female Nobox knockout (-/-) mice reveal a substantial disruption in germ cell cyst breakdown. This disruption impairs the formation of primordial follicles and the subsequent transition from primordial to primary stages, leading to the early loss of all oocytes. Study design, size, duration Three OZEMA families are presented with two distinct NOBOX variants. A functional study is conducted to explore alterations in mRNA expression associated with the investigated NOBOX variants. Participants/materials, setting, methods Three infertile women with OZEMA underwent multiple in vitro fertilization cycles. Subsequent, exome analysis targeting fertility-related genes revealed the presence of distinct NOBOX variants. To assess their impact on NOBOX target gene expression, we generated mutant NOBOX expressing plasmids via site-directed mutagenesis and transfected them into HEK293T cells. The expression of known NOBOX target genes was monitored by RT-qPCR. Main results and the role of chance We examined three families affected by OZEMA, in which the affected women were heterozygous for NOBOX variants. In one family, we found a heterozygous paternally inherited NM_001080413.3(NOBOX): c.1797_1798delCT, p.(Arg600Aspfs*6) variant in a female with oocyte maturation arrest. In two other families, a heterozygous probably pathogenic paternally inherited NM_001080413.3(NOBOX): c.1849C>T, p.(His617Tyr) variant was detected in females experiencing embryo maturation arrest. Analysis of NOBOX target genes in HEK293T cells revealed that both variants had no impact on the mRNA expression of MOS and OCT4, both of which are implicated in early folliculogenisis. Since both MOS and OCT4 are implicated in earlier folliculogenesis stages, this supports that NOBOX might also be implicated in later stages. Contrastingly, a known pathogenic NOBOX variant, used as a positive control, showed decreased MOS and OCT4 expression. Limitations, reasons for caution Verification of our findings is necessary, given that this is the first report of NOBOX variants in individuals with OZEMA. The precise molecular mechanism underlying OZEMA remains undisclosed at this stage. These results warrant the need of genome-wide mRNASeq to confirm and expand our findings. Wider implications of the findings Since NOBOX pathogenic variants are typically linked to POI, our findings suggest a broader clinical impact, where NOBOX plays a role in oocyte maturation and early embryo development. Hence, the investigation of NOBOX variants is advisable for individuals diagnosed with OZEMA. Trial registration number Not applicable

Abstract Study question What is the diagnostic yield of custom designed gene panel for patients with premature ovarian insufficiency (POI)? Summary answer The diagnostic yield of our POI gene panel (POIGP) is 7.3% What is known already POI is a specific female syndrome with a high clinical and genetic heterogeneity. It is characterized by a premature exhaustion of the ovarian function and infertility and affects approximately 1% of women. POI can be related to genetic factors which include chromosomal abnormalities, FMR1 premutation and rare variants in numerous genes. The advent of high throughput sequencing methods has led to the identification of an increasing number of variants implicated in the development of POI over the last decades. However, POI etiologies still remain undetermined in the majority of cases. Study design, size, duration An observational analytic cohort study of 150 patients presenting idiopathic POI (normal karyotype, absence of FMR1 premutation, absence of adrenal and/or ovarian antibodies) recruited prospectively at three Belgian academic and university hospitals between 2016 and 2021. Participants/materials, setting, methods Patients were included if they experienced POI, as defined by ESHRE guidelines on POI (2016). POI genes included in the panel were selected from PubMed using different key words mainly premature ovarian insufficiency, gonadal dysgenesis, hypergonadotropic hypogonadism, ovarian failure and genetics. The panel included 156 genes, variants were filtered based on allele frequency (≤1%) in latest available population databases and classified according to ACMG/AMP (American College of Medical Genetics/Association for Molecular Pathology) guidelines 2015. Main results and the role of chance Our analysis revealed a potential causative variant for 11 patients in the following genes: MEIOB, BMP4, CFTR, FANCA, FSHR, FANCG, MLH1, MRPS22 and STARD9. This means that the diagnostic yield of our POI gene panel (POIGP) is 7.3%. Patients were mainly Caucasian (63%), North African (17%) and sub-Saharan African (13%). They presented primary amenorrhea in 14.7% of cases. Consanguinity and/or a family history of POI or early menopause in 28% of cases. Mean patient’s age (years) at POI diagnosis was 28.9± 8.5 (mean ± SD). The overall mean coverage was 229X, and more than 95% of the target exome was represented with more than 30-fold coverage. Limitations, reasons for caution The present study was limited to monogenic etiologies of POI, potential oligogenic causes have not been searched. Functional studies and/or family segregation were not performed for the identified variants. Wider implications of the findings Our findings show the importance of targeted next generation sequencing in clinical practice and highlight the limit of our current genetic knowledge in the field of POI. A regular update of genes included in POIGP will improve its diagnostic yield. Trial registration number P2016/196/CCB B406201628264

Fabry Disease (FD) is a rare lysosomal storage disorder characterized by α-galactosidase A (α-Gal A) enzyme deficiency, resulting in glycosphingolipid accumulation.Its clinical spectrum ranges from severe classical to milder nonclassical or late-onset phenotypes.Renal involvement, termed Fabry Nephropathy (FN), can vary from mild proteinuria to kidney failure.FN diagnosis, especially in nonclassical cases with a genetic Variant of Unknown Significance (VUS) in the GLA gene, poses challenges.Measurement of plasma ly-

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.

Abstract 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.

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.

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.

Asma Sassi1, Julie Désir2, Alexander Gheldof3, Sonia Van Dooren4, Xavier Peyrassol5, Marc Abramowicz5, Anne Delbaere1* 1Fertility Clinic, Department of Obstetrics and Gynecology, Erasme Hospital, Université Libre de Bruxelles, Brussels, Belgium 2Institut de Pathologie et de Génétique (IPG), Gosselies, Belgium 3Centre for Medical Genetics, Reproduction and Genetics and Regenerative Medicine research cluster, Reproduction and Genetics research group, Vrije Universiteit Brussel-UZ Brussel, Brussels, Belgium 4Brussels Interuniversity Genomics High Throughput core (Bright Core), Brussels, Belgium 5Department of Genetics, Erasme Hospital, Université Libre de Bruxelles, Brussels, Belgium 6Department of Genetic Medicine and Development, Faculty of Medicine, University of Geneva, 1211 Geneva, Switzerland *Correspondence should be addressed to Anne Delbaere, anne.delbaere@erasme.ulb.ac.be