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Corpus callosum defects are frequent congenital cerebral disorders caused by mutations in more than 300 genes. These include genes implicated in corpus callosum development or function, as well as genes essential for mitochondrial physiology. However, in utero corpus callosum anomalies rarely raise a suspicion of mitochondrial disease and are characterized by a very large clinical heterogeneity. Here, we report a detailed pathological and neuro-histopathological investigation of nine foetuses from four unrelated families with prenatal onset of corpus callosum anomalies, sometimes associated with other cerebral or extra-cerebral defects. Next generation sequencing allowed the identification of novel pathogenic variants in three different nuclear genes previously reported in mitochondrial diseases: TIMMDC1, encoding a Complex I assembly factor never involved before in corpus callosum defect; MRPS22, a protein of the small mitoribosomal subunit; and EARS2, the mitochondrial tRNA-glutamyl synthetase. The present report describes the antenatal histopathological findings in mitochondrial diseases and expands the genetic spectrum of antenatal corpus callosum anomalies establishing OXPHOS function as an important factor for corpus callosum biogenesis. We propose that, when observed, antenatal corpus callosum anomalies should raise suspicion of mitochondrial disease and prenatal genetic counselling should be considered.

Purpose: Genome-wide sequencing is increasingly being performed during pregnancy to identify the genetic cause of congenital anomalies. The interpretation of prenatally identified variants can be challenging and is hampered by our often limited knowledge of prenatal phenotypes. To better delineate the prenatal phenotype of Coffin-Siris syndrome (CSS), we collected clinical data from patients with a prenatal phenotype and a pathogenic variant in one of the CSS-associated genes. Methods: Clinical data was collected through an extensive web-based survey. Results: We included 44 patients with a variant in a CSS-associated gene and a prenatal phenotype; 9 of these patients have been reported before. Prenatal anomalies that were frequently observed in our cohort include hydrocephalus, agenesis of the corpus callosum, hypoplastic left heart syndrome, persistent left vena cava, diaphragmatic hernia, renal agenesis, and intrauterine growth restriction. Anal anomalies were frequently identified after birth in patients with ARID1A variants (6/14, 43%). Interestingly, pathogenic ARID1A variants were much more frequently identified in the current prenatal cohort (16/44, 36%) than in postnatal CSS cohorts (5%-9%). Conclusion: Our data shed new light on the prenatal phenotype of patients with pathogenic variants in CSS genes. (C) 2022 American College of Medical Genetics and Genomics. Published by Elsevier Inc. All rights reserved.

Nephronophthisis (NPH) is an autosomal-recessive ciliopathy representing one of the most frequent causes of kidney failure in childhood characterized by a broad clinical and genetic heterogeneity. Applied to one of the worldwide largest cohorts of patients with NPH, genetic analysis encompassing targeted and whole exome sequencing identified disease-causing variants in 600 patients from 496 families with a detection rate of 71%. Of 788 pathogenic variants, 40 known ciliopathy genes were identified. However, the majority of patients (53%) bore biallelic pathogenic variants in NPHP1. NPH-causing gene alterations affected all ciliary modules defined by structural and/or functional subdomains. Seventy six percent of these patients had progressed to kidney failure, of which 18% had an infantile form (under five years) and harbored variants affecting the Inversin compartment or intraflagellar transport complex A. Forty eight percent of patients showed a juvenile (5-15 years) and 34% a late-onset disease (over 15 years), the latter mostly carrying variants belonging to the Transition Zone module. Furthermore, while more than 85% of patients with an infantile form presented with extra-kidney manifestations, it only concerned half of juvenile and late onset cases. Eye involvement represented a predominant feature, followed by cerebellar hypoplasia and other brain abnormalities, liver and skeletal defects. The phenotypic variability was in a large part associated with mutation types, genes and corresponding ciliary modules with hypomorphic variants in ciliary genes playing a role in early steps of ciliogenesis associated with juvenile-to-late onset NPH forms. Thus, our data confirm a considerable proportion of late-onset NPH suggesting an underdiagnosis in adult chronic kidney disease.

The antenatal phenotypic spectrum of Noonan Syndrome (NS) requires better characterization.This multicenter retrospective observational included 16 fetuses with molecularly confirmed NS admitted for fetopathological examination between 2009 and 2016.Among 12 pathogenic variants (PV) in PTPN11 (80%), 5 (42%) fell between position c.179 and c.182. Ultrasound showed increased nuchal translucency (n = 13/16, 93%), increased nuchal fold after 15 weeks of gestation (n = 12/16, 75%), pleural effusions (n = 11/16, 69%), polyhydramnios (n = 9/16, 56%), hydrops (n = 7/16, 44%), cardiovascular (n = 6/16, 38%) and cerebral (n = 4/16, 25%) anomalies. Fetopathological examination found dysmorphic features in all cases, cardiovascular anomalies (n = 12/15, 80%), pulmonary hypoplasia (n = 10/15, 67%), effusions (n = 7/15, 47%) and neuropathological anomalies (n = 5/15, 33%). Hydrops was significantly (p = 0.02) more frequent in the four fetuses with RIT1, NRAS and RAF1 PV versus the 12 fetuses with PTPN11 PV.Increased nuchal translucency and nuchal fold is common in NS. Noonan Syndrome antenatal phenotype showed high in utero fetal death, hydrops, prenatal pleural effusion and pulmonary hypoplasia, although the inclusion of only deceased fetuses will have selected more severe phenotypes. Non-specific cardiovascular and neurological abnormalities should be added to NS antenatal phenotype. Next generation sequencing will help detect more genotypes, clarifying the prenatal phenotype and identifying genotype-phenotype correlations.

In the human genome, about 750 genes contain one intron excised by the minor spliceosome. This spliceosome comprises its own set of snRNAs, among which U4atac. Its noncoding gene, RNU4ATAC , has been found mutated in Taybi-Linder (TALS/microcephalic osteodysplastic primordial dwarfism type 1), Roifman (RFMN), and Lowry-Wood (LWS) syndromes. These rare developmental disorders, whose physiopathological mechanisms remain unsolved, associate ante- and post-natal growth retardation, microcephaly, skeletal dysplasia, intellectual disability, retinal dystrophy, and immunodeficiency. Here, we report bi-allelic RNU4ATAC mutations in five patients presenting with traits suggestive of the Joubert syndrome (JBTS), a well-characterized ciliopathy. These patients also present with traits typical of TALS/RFMN/LWS, thus widening the clinical spectrum of RNU4ATAC -associated disorders and indicating ciliary dysfunction as a mechanism downstream of minor splicing defects. Intriguingly, all five patients carry the n.16G>A mutation, in the Stem II domain, either at the homozygous or compound heterozygous state. A gene ontology term enrichment analysis on minor intron-containing genes reveals that the cilium assembly process is over-represented, with no less than 86 cilium-related genes containing at least one minor intron, among which there are 23 ciliopathy-related genes. The link between RNU4ATAC mutations and ciliopathy traits is supported by alterations of primary cilium function in TALS and JBTS-like patient fibroblasts, as well as by u4atac zebrafish model, which exhibits ciliopathy-related phenotypes and ciliary defects. These phenotypes could be rescued by WT but not by pathogenic variants-carrying human U4atac. Altogether, our data indicate that alteration of cilium biogenesis is part of the physiopathological mechanisms of TALS/RFMN/LWS, secondarily to defects of minor intron splicing.

Congenital combined vitamin K-dependent clotting factors deficiency (VKCFD) is a rare autosomal recessive disease resulting in hemorrhagic symptoms usually associated with developmental disorders and bone abnormalities. Pathogenic variants in two genes encoding enzymes of the vitamin K cycle, GGCX and VKORC1, can lead to this disorder. We present the case of a male fetus with a brachytelephalangic chondrodysplasia punctata (CDP), absence of nasal bone, growth restriction, and bilateral ventriculomegaly at 18 weeks of gestation. Pathological examination showed a Binder phenotype, hypoplastic distal phalanges, stippled epiphyses, and brain abnormalities suggestive of a brain hemorrhage. Two GGCX pathogenic variants inherited respectively from the mother and the father were identified. To our knowledge, this is the first prenatal description of VKCFD. Even if it remains a rare etiology, which is mostly described in children or adult patients, VKCFD should be considered in fetuses with CDP.

Background Neuronal ceroid lipofuscinoses (NCLs) form a clinically and genetically heterogeneous group of inherited neurodegenerative disorders that share common neuropathological features. Although they are the first cause of neurodegenerative disorders in children, their congenital forms are rarely documented. They are classically due to mutations in the CTSD gene (the CLN10 disease). Affected newborns usually present severe microcephaly, seizures and respiratory failure leading to death within the first postnatal days or weeks. Cases We report on two siblings, in which exome sequencing identified a novel homozygous CTSD variant. The first sib presented at birth with seizures, rapidly progressive postnatal microcephaly and visual deficiency related to retinal dysfunction. Progressive neurological deterioration leads to death at the age of 24 months. Cathepsin D activity was reduced in the cultured fibroblasts of this patient. The second sib, a fetus of 36 weeks of gestation, was delivered after pregnancy termination for brain abnormalities (in accordance with French Legislation) suggesting a recurrence of the disease. Fetal postmortem examination disclosed neuropathological features consistent with NCL. Conclusions Congenital NCL related to CTSD mutations is a neuronal storage disorder that produces in the developing brain diffuse neurodegeneration and white matter atrophy resulting in a progressive and rapidly lethal microcephaly.

Purpose Despite a few recent reports of patients harboring truncating variants in NSD2, a gene considered critical for the Wolf–Hirschhorn syndrome (WHS) phenotype, the clinical spectrum associated with NSD2 pathogenic variants remains poorly understood. Methods We collected a comprehensive series of 18 unpublished patients carrying heterozygous missense, elongating, or truncating NSD2 variants; compared their clinical data to the typical WHS phenotype after pooling them with ten previously described patients; and assessed the underlying molecular mechanism by structural modeling and measuring methylation activity in vitro. Results The core NSD2-associated phenotype includes mostly mild developmental delay, prenatal-onset growth retardation, low body mass index, and characteristic facial features distinct from WHS. Patients carrying missense variants were significantly taller and had more frequent behavioral/psychological issues compared with those harboring truncating variants. Structural in silico modeling suggested interference with NSD2’s folding and function for all missense variants in known structures. In vitro testing showed reduced methylation activity and failure to reconstitute H3K36me2 in NSD2 knockout cells for most missense variants. Conclusion NSD2 loss-of-function variants lead to a distinct, rather mild phenotype partially overlapping with WHS. To avoid confusion for patients, NSD2 deficiency may be named Rauch–Steindl syndrome after the delineators of this phenotype., Genetics in Medicine, 23 (8)

Polydactyly is a hallmark of GLI3 pathogenic variants, with Greig cephalopolysyndactyly syndrome and Pallister-Hall syndrome being the two main associated clinical presentations. Homozygous GLI3 variants are rare instances in the literature, and mendelian dominance is the accepted framework for GLI3-related diseases. Herein, we report three unrelated probands, presenting with polydactyly, and homozygous variants in the GLI3 gene. First, a 10-year-old girl, whose parents were first-degree cousins, presented with bilateral postaxial polydactyly of the hands, developmental delay and multiple malformations. Second, a male newborn, whose parents were first-degree cousins, presented with isolated bilateral postaxial polysyndactyly of the hands and the feet. Third, an adult male, whose parents were first-degree cousins, had bilateral mesoaxial polydactyly of the hands, with severe intellectual disability and multiple malformations. All three probands carried homozygous GLI3 variants. Strikingly, the parents also carried the child's variant, in the heterozygous state, without any clinical sign of GLI3 disease. Given the clinical presentation of our patients, the rarity and predicted high pathogenicity of the variants observed, and the absence of other pathogenic variants, we suggest that these GLI3 homozygous variants are causal. Moreover, the parents were heterozygous for the observed variants, but were clinically unremarkable, suggesting that these variants are hypomorphic alleles.

International audience; Purpose: Abnormality of the corpus callosum (AbnCC) is etiologically a heterogeneous condition and the prognosis in prenatally diagnosed cases is difficult to predict. The purpose of our research was to establish the diagnostic yield using chromosomal microarray (CMA) and exome sequencing (ES) in cases with prenatally diagnosed isolated (iAbnCC) and nonisolated AbnCC (niAbnCC).Methods: CMA and prenatal trio ES (pES) were done on 65 fetuses with iAbnCC and niAbnCC. Only pathogenic gene variants known to be associated with AbnCC and/or intellectual disability were considered.Results: pES results were available within a median of 21.5 days (9-53 days). A pathogenic single-nucleotide variant (SNV) was identified in 12 cases (18%) and a pathogenic CNV was identified in 3 cases (4.5%). Thus, the genetic etiology was determined in 23% of cases. In all diagnosed cases, the results provided sufficient information regarding the neurodevelopmental prognosis and helped the parents to make an informed decision regarding the outcome of the pregnancy.Conclusion: Our results show the significant diagnostic and prognostic contribution of CMA and pES in cases with prenatally diagnosed AbnCC. Further prospective cohort studies with long-term follow-up of the born children will be needed to provide accurate prenatal counseling after a negative pES result.

Megacystis-microcolon-intestinal-hypoperistalsis syndrome (MMIHS) is a severe congenital visceral myopathy characterized by an abdominal distension due to a large non-obstructed urinary bladder, a microcolon and intestinal hypo- or aperistalsis. Most of the patients described to date carry a sporadic heterozygous variant in ACTG2. More recently, recessive forms have been reported and mutations in MYH11, LMOD1, MYLK and MYL9 have been described at the molecular level. In the present report, we describe five patients carrying a recurrent heterozygous variant in ACTG2. Exome sequencing performed in four families allowed us to identify the genetic cause in three. In two families, we identified variants in MMIHS causal genes, respectively a nonsense homozygous variant in MYH11 and a previously described homozygous deletion in MYL9. Finally, we identified compound heterozygous variants in a novel candidate gene, PDCL3, c.[143_144del];[380G>A], p.[(Tyr48Ter)];[(Cys127Tyr)]. After cDNA analysis, a complete absence of PDLC3 expression was observed in affected individuals, indicating that both mutated transcripts were unstable and prone to mediated mRNA decay. PDCL3 encodes a protein involved in the folding of actin, a key step in thin filament formation. Presumably, loss-of-function of this protein affects the contractility of smooth muscle tissues, making PDCL3 an excellent candidate gene for autosomal recessive forms of MMIHS.

The evolutionarily conserved hedgehog (Hh) pathway is essential for organogenesis and plays critical roles in postnatal tissue maintenance and renewal. A unique feature of the vertebrate Hh pathway is that signal transduction requires the primary cilium (PC) where major pathway components are dynamically enriched. These factors include smoothened (SMO) and patched, which constitute the core reception system for sonic hedgehog (SHH) as well as GLI transcription factors, the key mediators of the pathway. Here, we report biallelic loss-of-function variations in SMO in seven individuals from five independent families; these variations cause a wide phenotypic spectrum of developmental anomalies affecting the brain (hypothalamic hamartoma and microcephaly), heart (atrioventricular septal defect), skeleton (postaxial polydactyly, narrow chest, and shortening of long bones), and enteric nervous system (aganglionosis). Cells derived from affected individuals showed normal ciliogenesis but severely altered Hh-signal transduction as a result of either altered PC trafficking or abnormal activation of the pathway downstream of SMO. In addition, Hh-independent GLI2 accumulation at the PC tip in cells from the affected individuals suggests a potential function of SMO in regulating basal ciliary trafficking of GLI2 when the pathway is off. Thus, loss of SMO function results in abnormal PC dynamics of key components of the Hh signaling pathway and leads to a large continuum of malformations in humans.

Pathogenic variants in the core spliceosome U5 small nuclear ribonucleoprotein gene EFTUD2/SNU114 cause the craniofacial disorder mandibulofacial dysostosis Guion-Almeida type (MFDGA). MFDGA-associated variants in EFTUD2 comprise large deletions encompassing EFTUD2, intragenic deletions and single nucleotide truncating or missense variants. These variants are predicted to result in haploinsufficiency by loss-of-function of the variant allele. While the contribution of deletions within EFTUD2 to allele loss-of-function are self-evident, the mechanisms by which missense variants are disease-causing have not been characterized functionally. Combining bioinformatics software prediction, yeast functional growth assays, and a minigene (MG) splicing assay, we have characterized how MFDGA missense variants result in EFTUD2 loss-of-function. Only four of 19 assessed missense variants cause EFTUD2 loss-of-function through altered protein function when modeled in yeast. Of the remaining 15 missense variants, five altered the normal splicing pattern of EFTUD2 pre-messenger RNA predominantly through exon skipping or cryptic splice site activation, leading to the introduction of a premature termination codon. Comparison of bioinformatic predictors for each missense variant revealed a disparity amongst different software packages and, in many cases, an inability to correctly predict changes in splicing subsequently determined by MG interrogation. This study highlights the need for laboratory-based validation of bioinformatic predictions for EFTUD2 missense variants.

International audience; Primary cilia (PC) are non-motile dynamic microtubule-based organelles that protrude from the surface of most mammalian cells. They emerge from the older centriole during the G1/G0 phase of the cell cycle, while they disassemble as the cells re-enter the cell cycle at the G2/M phase boundary. They function as signal hubs, by detecting and transducing extracellular signals crucial for many cell processes. Similar to most cell types, all neocortical neural stem and progenitor cells (NSPCs) have been shown harboring a PC allowing them to sense and transduce specific signals required for the normal cerebral cortical development. Here, we provide detailed protocols to generate and characterize two-dimensional (2D) and three-dimensional (3D) cell-based models from human induced pluripotent stem cells (hIPSCs) to further dissect the involvement of PC during neocortical development. In particular, we present protocols to study the PC biogenesis and function in 2D neural rosette-derived NSPCs including the transduction of the Sonic Hedgehog (SHH) pathway. To take advantage of the three-dimensional (3D) organization of cerebral organoids, we describe a simple method for 3D imaging of in toto immunostained cerebral organoids. After optical clearing, rapid acquisition of entire organoids allows detection of both centrosomes and PC on neocortical progenitors and neurons of the whole organoid. Finally, we detail the procedure for immunostaining and clearing of thick free-floating organoid sections preserving a significant degree of 3D spatial information and allowing for the high-resolution acquisition required for the detailed qualitative and quantitative analysis of PC biogenesis and function.

Congenital anomalies of the kidney and urinary tract (CAKUT) represent the most common cause of chronic kidney failure in children. Despite growing knowledge of the genetic causes of CAKUT, the majority of cases remain etiologically unsolved. Genetic alterations in roundabout guidance receptor 1 (ROBO1) have been associated with neuronal and cardiac developmental defects in living individuals. Although Slit-Robo signaling is pivotal for kidney development, diagnostic ROBO1 variants have not been reported in viable CAKUT to date. By next-generation-sequencing methods, we identified six unrelated individuals and two non-viable fetuses with biallelic truncating or combined missense and truncating variants in ROBO1. Kidney and genitourinary manifestation included unilateral or bilateral kidney agenesis, vesicoureteral junction obstruction, vesicoureteral reflux, posterior urethral valve, genital malformation, and increased kidney echogenicity. Further clinical characteristics were remarkably heterogeneous, including neurodevelopmental defects, intellectual impairment, cerebral malformations, eye anomalies, and cardiac defects. By in silico analysis, we determined the functional significance of identified missense variants and observed absence of kidney ROBO1 expression in both human and murine mutant tissues. While its expression in multiple tissues may explain heterogeneous organ involvement, variability of the kidney disease suggests gene dosage effects due to a combination of null alleles with mild hypomorphic alleles. Thus, comprehensive genetic analysis in CAKUT should include ROBO1 as a new cause of recessively inherited disease. Hence, in patients with already established ROBO1-associated cardiac or neuronal disorders, screening for kidney involvement is indicated.

International audience; Free oligosaccharides (fOSs) are soluble oligosaccharide species generated during N-glycosylation of proteins. Although little is knownabout fOS metabolism, the recent identification of NGLY1 deficiency, a congenital disorder of deglycosylation (CDDG) caused by loss offunction of an enzyme involved in fOS metabolism, has elicited increased interest in fOS processing. The catabolism of fOSs has beenlinked to the activity of a specific cytosolic mannosidase, MAN2C1, which cleaves a1,2-, a1,3-, and a1,6-mannose residues. In this study,we report the clinical, biochemical, and molecular features of six individuals, including two fetuses, with bi-allelic pathogenic variants inMAN2C1; the individuals are from four different families. These individuals exhibit dysmorphic facial features, congenital anomaliessuch as tongue hamartoma, variable degrees of intellectual disability, and brain anomalies including polymicrogyria, interhemisphericcysts, hypothalamic hamartoma, callosal anomalies, and hypoplasia of brainstem and cerebellar vermis. Complementation experimentswith isogenic MAN2C1-KO HAP1 cells confirm the pathogenicity of three of the identified MAN2C1 variants. We further demonstratethat MAN2C1 variants lead to accumulation and delay in the processing of fOSs in proband-derived cells. These results emphasize theinvolvement of MAN2C1 in human neurodevelopmental disease and the importance of fOS catabolism.

BackgroundArthrogryposis multiplex congenita (AMC) is characterised by congenital joint contractures in two or more body areas. AMC exhibits wide phenotypic and genetic heterogeneity. Our goals were to improve the genetic diagnosis rates of AMC, to evaluate the added value of whole exome sequencing (WES) compared with targeted exome sequencing (TES) and to identify new genes in 315 unrelated undiagnosed AMC families.MethodsSeveral genomic approaches were used including genetic mapping of disease loci in multiplex or consanguineous families, TES then WES. Sanger sequencing was performed to identify or validate variants.ResultsWe achieved disease gene identification in 52.7% of AMC index patients including nine recently identified genes (CNTNAP1, MAGEL2, ADGRG6, ADCY6, GLDN, LGI4, LMOD3, UNC50 and SCN1A). Moreover, we identified pathogenic variants in ASXL3 and STAC3 expanding the phenotypes associated with these genes. The most frequent cause of AMC was a primary involvement of skeletal muscle (40%) followed by brain (22%). The most frequent mode of inheritance is autosomal recessive (66.3% of patients). In sporadic patients born to non-consanguineous parents (n=60), de novo dominant autosomal or X linked variants were observed in 30 of them (50%).ConclusionNew genes recently identified in AMC represent 21% of causing genes in our cohort. A high proportion of de novo variants were observed indicating that this mechanism plays a prominent part in this developmental disease. Our data showed the added value of WES when compared with TES due to the larger clinical spectrum of some disease genes than initially described and the identification of novel genes.

In the human genome, about 750 genes contain one intron excised by the minor spliceosome. This spliceosome comprises its own set of snRNAs, among which U4atac. Its non-coding gene,RNU4ATAC, has been found mutated in Taybi-Linder (MOPD1/TALS), Roifman (RFMN) and Lowry-Wood syndromes (LWS). These rare developmental disorders, whose physiopathological mechanisms remain unsolved, associate ante- and post-natal growth retardation, microcephaly, skeletal dysplasia, intellectual disability, retinal dystrophy and immunodeficiency. Here, we report a homozygousRNU4ATACmutation in the Stem II domain, n.16G>A, in two unrelated patients presenting with both typical traits of the Joubert syndrome (JBTS), a well-characterized ciliopathy, and of TALS/RFMN/LWS, thus widening the clinical spectrum ofRNU4ATAC-associated disorders and indicating ciliary dysfunction as a mechanism downstream of minor splicing defects. This finding is supported by alterations of primary cilium function in TALS and JBTS/RFMN fibroblasts, as well as byu4ataczebrafish model, which exhibit ciliopathy-related phenotypes and ciliary defects. Altogether, our data indicate that alteration of cilium biogenesis is part of the physiopathological mechanisms of TALS/RFMN/LWS, secondarily to defects of minor intron splicing.

Cerebro-oculo-facio-skeletal syndrome (COFS) is a rare autosomal recessive neurodegenerative disease belonging to the family of DNA repair disorders, characterized by microcephaly, congenital cataracts, facial dysmorphism and arthrogryposis. Here, we describe the detailed morphological and microscopic phenotype of three fetuses from two families harboring ERCC5/XPG likely pathogenic variants, and review the five previously reported fetal cases. In addition to the classical features of COFS, the fetuses display thymus hyperplasia, splenomegaly and increased hematopoiesis. Microencephaly is present in the three fetuses with delayed development of the gyri, but normal microscopic anatomy at the supratentorial level. Microscopic anomalies reminiscent of pontocerebellar hypoplasia are present at the infratentorial level. In conclusion, COFS syndrome should be considered in fetuses when intrauterine growth retardation is associated with microcephaly, arthrogryposis and ocular anomalies. Further studies are needed to better understand XPG functions during human development.

Objective Neural tube defects (NTDs) are one of the most common congenital anomalies caused by a complex interaction of many genetic and environmental factors. In about 10% of cases, NTDs are associated with genetic syndromes or chromosomal anomalies. Among these, SOX3 duplication has been reported in some isolated cases. The phenotype associated with this microduplication is variable and includes myelomeningocele (MMC) in both sexes as well as hypopituitarism and cognitive impairment in males. In order to determine the prevalence of this anomaly in fetuses with MMC, a retrospective cohort of fetuses with MMC was analyzed by quantitative PCR (qPCR) targeting SOX3 locus. Methods The detection of an SOX3 microduplication by chromosomal microarray analysis (CMA) in two female fetuses with MMC prompted us to analyze retrospectively by qPCR this gene in a cohort of 53 fetuses with MMC. Results In addition to our two initial cases, one fetus harboring an Xq27.1q28 duplication that encompasses the SOX3 gene was detected. Conclusion Our data demonstrate that SOX3 duplication is a genomic imbalance involved in the pathogenesis of NTDs. In addition, our survey highlights the importance of CMA testing in fetuses with NTDs to enable genetic counseling upstream of any considerations of in utero fetal surgery.

Hydrolethalus syndrome (HLS) is a rare lethal fetal malformation disorder related to ciliogenesis disruption. This condition is more frequent in Finland where a founder missense variant in the HYLS1 gene was identified. No other HYLS1 variant has hitherto been implicated in HLS. We report two unrelated French fetuses presenting with a phenotype of HLS with brain abnormalities, limbs malformations with pre and postaxial hexadactyly and abnormal genitalia. These two fetuses have compound heterozygous variants in HYLS1. The first allele carries the same Finnish missense variant (NM_145014.2: c.632A > G, p.[Asp211Gly]) in both fetuses and the second allele carries a new missense variant (c.662G > C, p.[Arg221Pro]) in the first fetus, and a new nonsense variant (c.613C > T, p.[Arg205*]) in the second fetus. This is the first report of HYLS1 mutated cases outside Finland. Both cases presented here are consistent with HLS with additional malformations, allowing expansion of the phenotypic presentation previously described.

TALPID3/KIAA0586 is an evolutionary conserved protein, which plays an essential role in protein trafficking. Its role during gastrointestinal (GI) and enteric nervous system (ENS) development has not been studied previously. Here, we analysed chicken, mouse and human embryonic GI tissues with TALPID3 mutations. The GI tract of TALPID3 chicken embryos was shortened and malformed. Histologically, the gut smooth muscle was mispatterned and enteric neural crest cells were scattered throughout the gut wall. Analysis of the Hedgehog pathway and gut extracellular matrix provided causative reasons for these defects. Interestingly, chicken intra-species grafting experiments and a conditional knockout mouse model showed that ENS formation did not require TALPID3, but was dependent on correct environmental cues. Surprisingly, the lack of TALPID3 in enteric neural crest cells (ENCC) affected smooth muscle and epithelial development in a non cell-autonomous manner. Analysis of human gut fetal tissues with aKIAA0586mutation showed strikingly similar findings compared to the animal models demonstrating conservation of TALPID3 and its necessary role in human GI tract development and patterning

ObjectiveTo describe a real-time decision support system (DSS), named SONIO, to assist ultrasound-based prenatal diagnosis and to assess its performance using a clinical database of precisely phenotyped postmortem examinations.Population and MethodsThis DSS is knowledge-based and comprises a dedicated thesaurus of 294 syndromes and diseases. It operates by suggesting, at each step of the ultrasound examination, the best next symptom to check for in order to optimize the diagnostic pathway to the smallest number of possible diagnoses. This assistant was tested on a single-center database of 251 cases of postmortem phenotypes with a definite diagnosis. Adjudication of discordant diagnoses was made by a panel of external experts. The primary outcome was a target concordance rate >90% between the postmortem diagnosis and the top-7 diagnoses given by SONIO when providing the full phenotype as input. Secondary outcomes included concordance for the top-5 and top-3 diagnoses; We also assessed a “1-by-1” model, providing only the anomalies sequentially prompted by the system, mimicking the use of the software in a real-life clinical setting.ResultsThe validation database covered 96 of the 294 (32.65%) syndromes and 79% of their overall prevalence in the SONIO thesaurus. The adjudicators discarded 42/251 cases as they were not amenable to ultrasound based diagnosis. SONIO failed to make the diagnosis on 7/209 cases. On average, each case displayed 6 anomalies, 3 of which were considered atypical for the condition. Using the ‘full-phenotype’ model, the success rate of the top-7 output of Sonio was 96.7% (202/209). This was 91.9% and 87.1% for the top-5 and top-3 outputs respectively. Using the “1-by-1” model, the correct diagnosis was within the top-7, top-5 and top-3 of SONIO’s output in 72.4%, 69.3% and 63.1%.ConclusionSonio is a robust DSS with a success-rate >95% for top-7 ranking diagnoses when the full phenotype is provided, using a large database of noisy real data. The success rate over 70% using the ‘1-by-1’ model was understandably lower, given that SONIO’s sequential queries may not systematically cover the full phenotype.

Objectives: Agnathia-otocephaly complex is a rare condition characterized by mandibular hypoplasia or agnathia, ear anomalies (melotia/synotia) and microstomia with aglossia. This severe anomaly of the first branchial arch is most often lethal. The estimated incidence is less than 1 in 70.000 births, with etiologies linked to both genetic and teratogenic factors. Most of the cases are sporadic. To date, two genes have been described in humans to be involved in this condition: OTX2 and PRRX1. Nevertheless, the overall proportion of mutated cases is unknown and a significant number of patients remain without molecular diagnosis. Thus, the involvement of other genes than OTX2 and PRRX1 in the agnathia-otocephaly complex is not unlikely. Heterozygous mutations in Cnbp in mice are responsible for mandibular and eye defects mimicking the agnathia-otocephaly complex in humans and appear as a good candidate. Therefore, in this study, we aimed (i) to collect patients presenting with agnathia-otocephaly complex for screening CNBP, in parallel with OTX2 and PRRX1, to check its possible implication in the human phenotype and (ii) to compare our results with the literature data to estimate the proportion of mutated cases after genetic testing. Materials and methods: In this work, we describe 10 patients suffering from the agnathia-otocephaly complex. All of them benefited from array-CGH and Sanger sequencing of OTX2, PRRX1 and CNBP. A complete review of the literature was made using the Pubmed database to collect all the patients described with a phenotype of agnathia-otocephaly complex during the 20 last years (1998-2019) in order (i) to study etiology (genetic causes, iatrogenic causes...) and (ii), when genetic testing was performed, to study which genes were tested and by which type of technologies. Results: In our 10 patients’ cohort, no point mutation in the three tested genes was detected by Sanger sequencing, while array-CGH has allowed identifying a 107-kb deletion encompassing OTX2 responsible for the agnathia-otocephaly complex phenotype in 1 of them. In 4 of the 70 cases described in the literature, a toxic cause was identified and 22 out the 66 remaining cases benefited from genetic testing. Among those 22 patients, 6 were carrying mutation or deletion in the OTX2 gene and 4 in the PRRX1 gene. Thus, when compiling results from our cohort and the literature, a total of 32 patients benefited from genetic testing, with only 34% (11/32) of patients having a mutation in one of the two known genes, OTX2 or PRRX1. Conclusions: From our work and the literature review, only mutations in OTX2 and PRRX1 have been found to date in patients, explaining around one third of the etiologies after genetic testing. Thus, agnathia-otocephaly complex remains unexplained in the majority of the patients, which indicates that other factors might be involved. Although involved in first branchial arch defects, no mutation in the CNBP gene was found in this study. This suggests that mutations in CNBP might not be involved in such phenotype in humans or that, unlike in mice, a compensatory effect might exist in humans. Nevertheless, given that agnathia-otocephaly complex is a rare phenotype, more patients have to be screened for CNBP mutations before we definitively conclude about its potential implication. Therefore, this work presents the current state of knowledge on agnathia-otocephaly complex and underlines the need to expand further the understanding of the genetic bases of this disorder, which remains largely unknown. Clinical relevance: We made here an update and focus on the clinical and genetic aspects of agnathia-otocephaly complex as well as a more general review of craniofacial development.

Herein, we report the screening of a large panel of genes in a series of 80 fetuses with congenital heart defects (CHDs) and/or heterotaxy and no cytogenetic anomalies. There were 49 males (61%/39%), with a family history in 28 cases (35%) and no parental consanguinity in 77 cases (96%). All fetuses had complex CHD except one who had heterotaxy and midline anomalies while 52 cases (65%) had heterotaxy in addition to CHD. Altogether, 29 cases (36%) had extracardiac and extra-heterotaxy anomalies. A pathogenic variant was found in 10/80 (12.5%) cases with a higher percentage in the heterotaxy group (8/52 cases, 15%) compared with the non-heterotaxy group (2/28 cases, 7%), and in 3 cases with extracardiac and extra-heterotaxy anomalies (3/29, 10%). The inheritance was recessive in six genes (DNAI1, GDF1, MMP21, MYH6, NEK8, and ZIC3) and dominant in two genes (SHH and TAB2). A homozygous pathogenic variant was found in three cases including only one case with known consanguinity. In conclusion, after removing fetuses with cytogenetic anomalies, next-generation sequencing discovered a causal variant in 12.5% of fetal cases with CHD and/or heterotaxy. Genetic counseling for future pregnancies was greatly improved. Surprisingly, unexpected consanguinity accounts for 20% of cases with identified pathogenic variants.

International audience; Background: Despite undisputable benefits, midtrimester prenatal surgery is not a cure for myelomeningocele (MMC): residual intracranial and motor deficits leading to lifelong handicap question the timing of prenatal surgery. Indeed, the timing and intensity of intrauterine spinal cord injury remains ill defined.Objective: We aimed to describe the natural history of neuronal loss in MMC in utero based on postmortem pathology.Study design: Pathology findings were analyzed in 186 cases of myelomeningocele with lesion level between S1 and T1. Using a case-control, cross-sectional design, we investigated the timewise progression and topographic extension of neuronal loss between 13 and 39 weeks. Motor neurons were counted on histology at several spinal levels in 54 isolated MMC meeting quality criteria for cell counting. These were expressed as observed-to-expected ratios, after matching for gestational age and spinal level with 41 controls.Results: Chiari II malformation increased from 30.7% to 91.6% after 16 weeks. The exposed spinal cord displayed early, severe, and progressive neuronal loss: the observed-to-expected count dropped from 17% to ≤2% after 16 weeks. Neuronal loss extended beyond the lesion to the upper levels: in cases

OBJECTIVE Fetal anomalies of the corpus callosum (CC) have been reported in the prenatal imaging literature since 1985, and, especially when isolated, pose challenges for both the patient and fetal medicine specialist. The purpose of this study was to review systematically the literature on prenatally diagnosed abnormalities of the CC, focusing on the terminology used to describe abnormalities other than complete agenesis of the CC, and to assess the heterogeneity of the nomenclature and definitions used. METHODS This study was conducted in accordance with the PRISMA statement for reporting systematic reviews. A literature search was performed to identify prospective or retrospective case series or cohort studies, published in English, French, Italian, German or Spanish, reporting fetal imaging findings and describing anomalies of the CC. Quality and risk of bias of the studies were evaluated using the Newcastle-Ottawa scale and a modification of the scale developed by Conde-Agudelo et al. for other fetal imaging studies. The data extracted included the number of patients, the number of different anomalies identified, the descriptive names of the anomalies, and, where applicable, the definitions of the anomalies, the number of cases of each type of anomaly and the biometric charts used. Secondary tests used to confirm the diagnosis, as well as the postnatal or post-termination tests used to ascertain the diagnosis, were also recorded. RESULTS The search identified 998 records, and, after review of titles and abstracts and full review of 45 papers, 27 studies were included initially in the review, of which 24 were included in the final analysis. These 24 studies had a broad range of quality and risk of bias and represented 1135 cases of CC anomalies, of which 49% were complete agenesis and the remainder were described using the term partial agenesis or nine other terms, of which five had more than one definition. CONCLUSIONS In comparison to the postnatal literature, in the prenatal literature there is much greater heterogeneity in the nomenclature and definition of CC anomalies other than complete agenesis. This heterogeneity and lack of standard definitions in the prenatal literature make it difficult to develop large multicenter pooled cohorts of patients who can be followed in order to develop a better understanding of the genetic associations and neurodevelopmental and psychological outcomes of patients with CC anomalies. As this information is important to improve counseling of these patients, a good first step towards this goal would be to develop a simpler categorization of prenatal CC anomalies that matches better the postnatal literature. © 2020 International Society of Ultrasound in Obstetrics and Gynecology.

We report two fetal cases carrying a de novo MID1 mutation and presenting with severe hydrothorax, suggesting the expansion of the phenotype of Opitz GBBB syndrome.

PurposeMolecular diagnosis based on singleton exome sequencing (sES) is particularly challenging in fetuses with multiple congenital abnormalities (MCA). Indeed, some studies reveal a diagnostic yield of about 20%, far lower than in live birth individuals showing developmental abnormalities (30%), suggesting that standard analyses, based on the correlation between clinical hallmarks described in postnatal syndromic presentations and genotype, may underestimate the impact of the genetic variants identified in fetal analyses.MethodsWe performed sES in 95 fetuses with MCA. Blind to phenotype, we applied a genotype-first approach consisting of combined analyses based on variants annotation and bioinformatics predictions followed by reverse phenotyping. Initially applied to OMIM-morbid genes, analyses were then extended to all genes. We complemented our approach by using reverse phenotyping, variant segregation analysis, bibliographic search and data sharing in order to establish the clinical significance of the prioritised variants.ResultssES rapidly identified causal variant in 24/95 fetuses (25%), variants of unknown significance in OMIM genes in 8/95 fetuses (8%) and six novel candidate genes in 6/95 fetuses (6%).ConclusionsThis method, based on a genotype-first approach followed by reverse phenotyping, shed light on unexpected fetal phenotype-genotype correlations, emphasising the relevance of prenatal studies to reveal extreme clinical presentations associated with well-known Mendelian disorders.

Bardet-Biedl syndrome (BBS) is an emblematic ciliopathy associated with retinal dystrophy, obesity, postaxial polydactyly, learning disabilities, hypogonadism and renal dysfunction. Before birth, enlarged/cystic kidneys as well as polydactyly are the hallmark signs of BBS to consider in absence of familial history. However, these findings are not specific to BBS, raising the problem of differential diagnoses and prognosis. Molecular diagnosis during pregnancies remains a timely challenge for this heterogeneous disease (22 known genes). We report here the largest cohort of BBS fetuses to better characterize the antenatal presentation. Prenatal ultrasound (US) and/or autopsy data from 74 fetuses with putative BBS diagnosis were collected out of which molecular diagnosis was established in 51 cases, mainly in BBS genes (45 cases) following the classical gene distribution, but also in other ciliopathy genes (6 cases). Based on this, an updated diagnostic decision tree is proposed. No genotype/phenotype correlation could be established but postaxial polydactyly (82%) and renal cysts (78%) were the most prevalent symptoms. However, autopsy revealed polydactyly that was missed by prenatal US in 55% of the cases. Polydactyly must be carefully looked for in pregnancies with apparently isolated renal anomalies in fetuses.

Acrocallosal syndrome (ACLS) is a rare genetic disorder characterized by agenesis or hypoplasia of corpus callosum (CC), polydactyly, craniofacial dysmorphism and severe intellectual deficiency. We previously identified KIF7, a key ciliary component of the Sonic hedgehog (SHH) pathway, as being a causative gene for this syndrome, thus including ACLS in the group of ciliopathies. In both humans and mice, KIF7 depletion leads to abnormal GLI3 processing and over-activation of SHH target genes. To understand the pathological mechanisms involved in CC defects in this syndrome, we took advantage of a previously described Kif7-/- mouse model to demonstrate that in addition to polydactyly and neural tube closure defects, these mice present CC agenesis with characteristic Probst bundles, thus recapitulating major ACLS features. We show that CC agenesis in these mice is associated with specific patterning defects of the cortical septum boundary leading to altered distribution of guidepost cells required to guide the callosal axons through the midline. Furthermore, by crossing Kif7-/- mice with Gli3Δ699 mice exclusively producing the repressive isoform of GLI3 (GLI3R), we demonstrate that decreased GLI3R signaling is fully responsible for the ACLS features in these mice, as all phenotypes are rescued by increasing GLI3R activity. Moreover, we show that increased FGF8 signaling is responsible in part for CC defects associated to KIF7 depletion, as modulating FGF8 signaling rescued CC formation anteriorly in Kif7-/- mice. Taken together our data demonstrate that ACLS features rely on defective GLI3R and FGF8 signaling.

Beckwith-Wiedemann syndrome (BWS) is the most common overgrowth syndrome. Clinical features are highly variable, including occasional posterior fossa malformations but no femoral shortening.We report two fetuses with BWS associated with short femurs and corpus callosum hypoplasia. Case 2 was growth restricted. BWS was confirmed by molecular studies showing a loss of methylation at ICR2 at 11p15 chromosomic region in case 1 and a gain of methylation at ICR1 and a loss of methylation at ICR2 locus in case 2.Although the phenotype and the genotype of BWS is now well-known, the presence of corpus callosum abnormalities and short femurs expand the phenotypic spectrum of the disorder.

International audience; Background: The recent outbreak of Zika virus (ZIKV) infection and the associated increased prevalence of microcephaly in Brazil underline the impact of viral infections on embryo-fetal development. The aim of the present study is to provide a detailed clinical and histopathological study of the fetal disruption caused by the ZIKV, with a special focus on the associated neuropathological findings. Methods: A detailed feto-placental examination, as well as neuropathological and neurobiological studies were performed on three fetuses collected after pregnancy termination between 22 and 25 weeks of gestation (WG), because brain malforma-tions associated with a maternal and fetal ZIKV infection was diagnosed. Results: In all three cases, the maternal infection occurred during the first trimester of pregnancy. A small head was observed on the ultrasound examination of the second trimester of pregnancy and led to the diagnosis of ZIKV fetopathy and pregnancy termination. The fetal histopathological examination was unremarkable on the viscera but showed on the testis an interstitial lymphocytic infiltrate. The placenta contained a Hofbauer cells hyperplasia with signs of inflammation. Neu-ropathological findings included a meningoencephalitis and an ex vacuo hydrocepha-lus. Immunohistochemical studies showed the presence of T lymphocytic and histiocytic meningitis associated with an abundant cerebral astroglial and macrophagic reaction. In situ hybridization demonstrated, abundant ZIKV particles within the cerebral parenchyma mainly in the ventricular/subventricular zone and in the cortical plate. In addition massive cells death and endoplasmic reticulum damage were present. Conclusion: The present study reports on the clinical and histopathological findings observed in three fetuses infected by the ZIKV. It emphasizes the severity of brain damages and the minimal visceral and placental changes observed upon ZIKV infection. This confirms the selective neurotropism of ZIKV. Finally, it allows us to describe the cascade of multifactorial developmental defects leading to microcephaly.

International audience; CEP290 mutations cause a spectrum of ciliopathies from Leber congenital amaurosis type 10 (LCA10) to embryo-lethal Meckel syndrome (MKS). Using panel-based molecular diagnosis testing for inherited retinal diseases, we identified two individuals with some preserved vision despite biallelism for presumably truncating CEP290 mutations. The first one carried a homozygous 1 base-pair deletion in exon 17, introducing a premature termination codon (PTC) in exon 18 (c.1666del; p.Ile556Phefs*17). mRNA analysis revealed a basal exon skipping (BES) of exon 18, providing mutant cells with the ability to escape protein truncation, while disrupting the reading frame in controls. The second individual harbored compound heterozygous nonsense mutations in exon 8 (c.508A\textgreaterT, p.Lys170*) and exon 32 (c.4090G\textgreaterT, p.Glu1364*), respectively. Some CEP290 lacking exon 8 were detected in mutant fibroblasts but not in controls whereas some skipping of exon 32 occurred in both lines, but with higher amplitude in the mutant. Considering that the deletion of either exon maintains the reading frame in either line, skipping in mutant cells likely involves nonsense-associated altered splicing (NAS) alone (exon 8), or with BES (exon 32). Skipping of PTC-containing exons in mutant cells allowed production of CEP290 isoforms with preserved ability to assemble into a high molecular weight complex and to interact efficiently with proteins important for cilia formation and intraflagellar trafficking. In contrast, studying LCA10 and MKS fibroblasts we show moderate to severe cilia alterations, providing support for a correlation between disease severity and the ability of cells to express shortened, yet functional, CEP290 isoforms.

Ciliopathies comprise a group of clinically heterogeneous and overlapping disorders with a wide spectrum of phenotypes ranging from prenatal lethality to adult-onset disorders. Pathogenic variants in more than 100 ciliary protein-encoding genes have been described, most notably those involved in intraflagellar transport (IFT) which comprises two protein complexes, responsible for retrograde (IFT-A) and anterograde transport (IFT-B). Here we describe a fetus with an unclassified severe ciliopathy phenotype including short ribs, polydactyly, bilateral renal agenesis, and imperforate anus, with compound heterozygosity for c.118_125del, p.(Thr40Glyfs*11) and a c.352 +1G > T in IFT27, which encodes a small GTPase component of the IFT-B complex. We conclude that bilateral renal agenesis is a rare feature of this severe ciliopathy and this report highlights the phenotypic overlap of Pallister-Hall syndrome and ciliopathies. The phenotype in patients with IFT27 gene variants is wide ranging from Bardet-Biedl syndrome to a lethal phenotype.

International audience