SRSF1 haploinsufficiency is responsible for a syndromic developmental disorder associated with intellectual disability.

SRSF1 (also known as ASF/SF2) is a non-small nuclear ribonucleoprotein (non-snRNP) that belongs to the arginine/serine (R/S) domain family. It recognizes and binds to mRNA, regulating both constitutive and alternative splicing. The complete loss of this proto-oncogene in mice is embryonically lethal. Through international data sharing, we identified 17 individuals (10 females and 7 males) with a neurodevelopmental disorder (NDD) with heterozygous germline SRSF1 variants, mostly de novo, including three frameshift variants, three nonsense variants, seven missense variants, and two microdeletions within region 17q22 encompassing SRSF1. Only in one family, the de novo origin could not be established. All individuals featured a recurrent phenotype including developmental delay and intellectual disability (DD/ID), hypotonia, neurobehavioral problems, with variable skeletal (66.7%) and cardiac (46%) anomalies. To investigate the functional consequences of SRSF1 variants, we performed in silico structural modeling, developed an in vivo splicing assay in Drosophila, and carried out episignature analysis in blood-derived DNA from affected individuals. We found that all loss-of-function and 5 out of 7 missense variants were pathogenic, leading to a loss of SRSF1 splicing activity in Drosophila, correlating with a detectable and specific DNA methylation episignature. In addition, our orthogonal in silico, in vivo, and epigenetics analyses enabled the separation of clearly pathogenic missense variants from those with uncertain significance. Overall, these results indicated that haploinsufficiency of SRSF1 is responsible for a syndromic NDD with ID due to a partial loss of SRSF1-mediated splicing activity.

Bi-allelic variants in DOHH, catalyzing the last step of hypusine biosynthesis, are associated with a neurodevelopmental disorder.

Deoxyhypusine hydroxylase (DOHH) is the enzyme catalyzing the second step in the post-translational synthesis of hypusine [Nε-(4-amino-2-hydroxybutyl)lysine] in the eukaryotic initiation factor 5A (eIF5A). Hypusine is formed exclusively in eIF5A by two sequential enzymatic steps catalyzed by deoxyhypusine synthase (DHPS) and deoxyhypusine hydroxylase (DOHH). Hypusinated eIF5A is essential for translation and cell proliferation in eukaryotes, and all three genes encoding eIF5A, DHPS, and DOHH are highly conserved throughout eukaryotes. Pathogenic variants affecting either DHPS or EIF5A have been previously associated with neurodevelopmental disorders. Using trio exome sequencing, we identified rare bi-allelic pathogenic missense and truncating DOHH variants segregating with disease in five affected individuals from four unrelated families. The DOHH variants are associated with a neurodevelopmental phenotype that is similar to phenotypes caused by DHPS or EIF5A variants and includes global developmental delay, intellectual disability, facial dysmorphism, and microcephaly. A two-dimensional gel analyses revealed the accumulation of deoxyhypusine-containing eIF5A [eIF5A(Dhp)] and a reduction in the hypusinated eIF5A in fibroblasts derived from affected individuals, providing biochemical evidence for deficiency of DOHH activity in cells carrying the bi-allelic DOHH variants. Our data suggest that rare bi-allelic variants in DOHH result in reduced enzyme activity, limit the hypusination of eIF5A, and thereby lead to a neurodevelopmental disorder.

ADGRL1 haploinsufficiency causes a variable spectrum of neurodevelopmental disorders in humans and alters synaptic activity and behavior in a mouse model.

ADGRL1 (latrophilin 1), a well-characterized adhesion G protein-coupled receptor, has been implicated in synaptic development, maturation, and activity. However, the role of ADGRL1 in human disease has been elusive. Here, we describe ten individuals with variable neurodevelopmental features including developmental delay, intellectual disability, attention deficit hyperactivity and autism spectrum disorders, and epilepsy, all heterozygous for variants in ADGRL1. In vitro, human ADGRL1 variants expressed in neuroblastoma cells showed faulty ligand-induced regulation of intracellular Ca2+ influx, consistent with haploinsufficiency. In vivo, Adgrl1 was knocked out in mice and studied on two genetic backgrounds. On a non-permissive background, mice carrying a heterozygous Adgrl1 null allele exhibited neurological and developmental abnormalities, while homozygous mice were non-viable. On a permissive background, knockout animals were also born at sub-Mendelian ratios, but many Adgrl1 null mice survived gestation and reached adulthood. Adgrl1-/- mice demonstrated stereotypic behaviors, sexual dysfunction, bimodal extremes of locomotion, augmented startle reflex, and attenuated pre-pulse inhibition, which responded to risperidone. Ex vivo synaptic preparations displayed increased spontaneous exocytosis of dopamine, acetylcholine, and glutamate, but Adgrl1-/- neurons formed synapses in vitro poorly. Overall, our findings demonstrate that ADGRL1 haploinsufficiency leads to consistent developmental, neurological, and behavioral abnormalities in mice and humans.

Monoallelic loss-of-function variants in GSK3B lead to autism and developmental delay.

De novo variants adjacent to the canonical splicing sites or in the well-defined splicing-related regions are more likely to impair splicing but remain under-investigated in autism spectrum disorder (ASD). By analyzing large, recent ASD genome sequencing cohorts, we find a significant burden of de novo potential splicing-disrupting variants (PSDVs) in 5048 probands compared to 4090 unaffected siblings. We identified 55 genes with recurrent de novo PSDVs that were highly intolerant to variation. Forty-six of these genes have not been strongly implicated in ASD or other neurodevelopmental disorders previously, including GSK3B. Through international, multicenter collaborations, we assembled genotype and phenotype data for 15 individuals with GSK3B variants and identified common phenotypes including developmental delay, ASD, sleeping disturbance, and aggressive behavior. Using available single-cell transcriptomic data, we show that GSK3B is enriched in dorsal progenitors and intermediate forms of excitatory neurons in the developing brain. We showed that Gsk3b knockdown in mouse excitatory neurons interferes with dendrite arborization and spine maturation which could not be rescued by de novo missense variants identified from affected individuals. In summary, our findings suggest that PSDVs may play an important role in the genetic etiology of ASD and allow for the prioritization of new ASD candidate genes. Importantly, we show that genetic variation resulting in GSK3B loss-of-function can lead to a neurodevelopmental disorder with core features of ASD and developmental delay.

Monoallelic de novo variants in DDX17 cause a neurodevelopmental disorder.

DDX17 is an RNA helicase shown to be involved in critical processes during the early phases of neuronal differentiation. Globally, we compiled a case-series of 11 patients with neurodevelopmental phenotypes harbouring de novo monoallelic variants in DDX17. All 11 patients in our case series had a neurodevelopmental phenotype, whereby intellectual disability, delayed speech and language, and motor delay predominated. We performed in utero cortical electroporation in the brain of developing mice, assessing axon complexity and outgrowth of electroporated neurons, comparing wild-type and Ddx17 knockdown. We then undertook ex vivo cortical electroporation on neuronal progenitors to quantitatively assess axonal development at a single cell resolution. Mosaic ddx17 crispants and heterozygous knockouts in Xenopus tropicalis were generated for assessment of morphology, behavioural assays, and neuronal outgrowth measurements. We further undertook transcriptomic analysis of neuroblastoma SH-SY5Y cells, to identify differentially expressed genes in DDX17-KD cells compared to controls. Knockdown of Ddx17 in electroporated mouse neurons in vivo showed delayed neuronal migration as well as decreased cortical axon complexity. Mouse primary cortical neurons revealed reduced axon outgrowth upon knockdown of Ddx17 in vitro. The axon outgrowth phenotype was replicated in crispant ddx17 tadpoles and in heterozygotes. Heterozygous tadpoles had clear neurodevelopmental defects and showed an impaired neurobehavioral phenotype. Transcriptomic analysis identified a statistically significant number of differentially expressed genes involved in neurodevelopmental processes in DDX17-KD cells compared to control cells. We have identified potential neurodevelopment disease-causing variants in a gene not previously associated with genetic disease, DDX17. We provide evidence for the role of the gene in neurodevelopment in both mammalian and non-mammalian species and in controlling the expression of key neurodevelopment genes.

Expanding the genetic and clinical spectrum of Tatton-Brown-Rahman syndrome in a series of 24 French patients.

BACKGROUND: Tatton-Brown-Rahman syndrome (TBRS; OMIM 615879), also known as DNA methyltransferase 3 alpha (DNMT3A)-overgrowth syndrome (DOS), was first described by Tatton-Brown in 2014. This syndrome is characterised by overgrowth, intellectual disability and distinctive facial features and is the consequence of germline loss-of-function variants in DNMT3A, which encodes a DNA methyltransferase involved in epigenetic regulation. Somatic variants of DNMT3A are frequently observed in haematological malignancies, including acute myeloid leukaemia (AML). To date, 100 individuals with TBRS with de novo germline variants have been described. We aimed to further characterise this disorder clinically and at the molecular level in a nationwide series of 24 French patients and to investigate the correlation between the severity of intellectual disability and the type of variant. METHODS: We collected genetic and medical information from 24 individuals with TBRS using a questionnaire released through the French National AnDDI-Rares Network. RESULTS: Here, we describe the first nationwide French cohort of 24 individuals with germline likely pathogenic/pathogenic variants in DNMT3A, including 17 novel variants. We confirmed that the main phenotypic features were intellectual disability (100% of individuals), distinctive facial features (96%) and overgrowth (87%). We highlighted novel clinical features, such as hypertrichosis, and further described the neurological features and EEG results. CONCLUSION: This study of a nationwide cohort of individuals with TBRS confirms previously published data and provides additional information and clarifies clinical features to facilitate diagnosis and improve care. This study adds value to the growing body of knowledge on TBRS and broadens its clinical and molecular spectrum.

Extending the clinical spectrum of X-linked Tonne-Kalscheuer syndrome (TOKAS): new insights from the fetal perspective.

INTRODUCTION: Tonne-Kalscheuer syndrome (TOKAS) is a recessive X-linked multiple congenital anomaly disorder caused by RLIM variations. Of the 41 patients reported, only 7 antenatal cases were described. METHOD: After the antenatal diagnosis of TOKAS by exome analysis in a family followed for over 35 years because of multiple congenital anomalies in five male fetuses, a call for collaboration was made, resulting in a cohort of 11 previously unpublished cases. RESULTS: We present a TOKAS antenatal cohort, describing 11 new cases in 6 French families. We report a high frequency of diaphragmatic hernia (9 of 11), differences in sex development (10 of 11) and various visceral malformations. We report some recurrent dysmorphic features, but also pontocerebellar hypoplasia, pre-auricular skin tags and olfactory bulb abnormalities previously unreported in the literature. Although no clear genotype-phenotype correlation has yet emerged, we show that a recurrent p.(Arg611Cys) variant accounts for 66% of fetal TOKAS cases. We also report two new likely pathogenic variants in RLIM, outside of the two previously known mutational hotspots. CONCLUSION: Overall, we present the first fetal cohort of TOKAS, describe the clinical features that made it a recognisable syndrome at fetopathological examination, and extend the phenotypical spectrum and the known genotype of this rare disorder.

NR2F1 regulates regional progenitor dynamics in the mouse neocortex and cortical gyrification in BBSOAS patients.

The relationships between impaired cortical development and consequent malformations in neurodevelopmental disorders, as well as the genes implicated in these processes, are not fully elucidated to date. In this study, we report six novel cases of patients affected by BBSOAS (Boonstra-Bosch-Schaff optic atrophy syndrome), a newly emerging rare neurodevelopmental disorder, caused by loss-of-function mutations of the transcriptional regulator NR2F1. Young patients with NR2F1 haploinsufficiency display mild to moderate intellectual disability and show reproducible polymicrogyria-like brain malformations in the parietal and occipital cortex. Using a recently established BBSOAS mouse model, we found that Nr2f1 regionally controls long-term self-renewal of neural progenitor cells via modulation of cell cycle genes and key cortical development master genes, such as Pax6. In the human fetal cortex, distinct NR2F1 expression levels encompass gyri and sulci and correlate with local degrees of neurogenic activity. In addition, reduced NR2F1 levels in cerebral organoids affect neurogenesis and PAX6 expression. We propose NR2F1 as an area-specific regulator of mouse and human brain morphology and a novel causative gene of abnormal gyrification.

Delineation of the adult phenotype of Coffin-Siris syndrome in 35 individuals.

Coffin-Siris syndrome (CSS) is a rare multisystemic autosomal dominant disorder. Since 2012, alterations in genes of the SWI/SNF complex were identified as the molecular basis of CSS, studying largely pediatric cohorts. Therefore, there is a lack of information on the phenotype in adulthood, particularly on the clinical outcome in adulthood and associated risks. In an international collaborative effort, data from 35 individuals ≥ 18 years with a molecularly ascertained CSS diagnosis (variants in ARID1B, ARID2, SMARCA4, SMARCB1, SMARCC2, SMARCE1, SOX11, BICRA) using a comprehensive questionnaire was collected. Our results indicate that overweight and obesity are frequent in adults with CSS. Visual impairment, scoliosis, and behavioral anomalies are more prevalent than in published pediatric or mixed cohorts. Cognitive outcomes range from profound intellectual disability (ID) to low normal IQ, with most individuals having moderate ID. The present study describes the first exclusively adult cohort of CSS individuals. We were able to delineate some features of CSS that develop over time and have therefore been underrepresented in previously reported largely pediatric cohorts, and provide recommendations for follow-up.

Further description of two individuals with de novo p.(Glu127Lys) missense variant in the ASCL1 gene.

Achaete-Scute Family basic-helix-loop-helix (bHLH) Transcription Factor 1 (ASCL1) is a proneural transcription factor involved in neuron development in the central and peripheral nervous system. While initially suspected to contribute to congenital central hypoventilation syndrome-1 (CCHS) with or without Hirschsprung disease (HSCR) in three individuals, its implication was ruled out by the presence, in one of the individuals, of a Paired-like homeobox 2B (PHOX2B) heterozygous polyalanine expansion variant, known to cause CCHS. We report two additional unrelated individuals sharing the same sporadic ASCL1 p.(Glu127Lys) missense variant in the bHLH domain and a common phenotype with short-segment HSCR, signs of dysautonomia, and developmental delay. One has also mild CCHS without polyalanine expansion in PHOX2B, compatible with the diagnosis of Haddad syndrome. Furthermore, missense variants with homologous position in the same bHLH domain in other genes are known to cause human diseases. The description of additional individuals carrying the same variant and similar phenotype, as well as targeted functional studies, would be interesting to further evaluate the role of ASCL1 in neurocristopathies.

Prenatal exome sequencing, a powerful tool for improving the description of prenatal features associated with genetic disorders.

OBJECTIVE: Prenatal exome sequencing (pES) is now commonly used in clinical practice. It can be used to identifiy an additional diagnosis in around 30% of fetuses with structural defects and normal chromosomal microarray analysis (CMA). However, interpretation remains challenging due to the limited prenatal data for genetic disorders. METHOD: We conducted an ancillary study including fetuses with pathogenic/likely pathogenic variants identified by trio-pES from the "AnDDI-Prenatome" study. The prenatal phenotype of each patient was categorized as typical, uncommon, or unreported based on the comparison of the prenatal findings with documented findings in the literature and public phenotype-genotype databases (ClinVar, HGMD, OMIM, and Decipher). RESULTS: Prenatal phenotypes were typical for 38/56 fetuses (67.9%). For the others, genotype-phenotype associations were challenging due to uncommon prenatal features (absence of recurrent hallmark, rare, or unreported). We report the first prenatal features associated with LINS1 and PGM1 variants. In addition, a double diagnosis was identified in three fetuses. CONCLUSION: Standardizing the description of prenatal features, implementing longitudinal prenatal follow-up, and large-scale collection of prenatal features are essential steps to improving pES data interpretation.

Multiple molecular diagnoses in the field of intellectual disability and congenital anomalies: 3.5% of all positive cases.

PURPOSE: Wide access to clinical exome/genome sequencing (ES/GS) enables the identification of multiple molecular diagnoses (MMDs), being a long-standing but underestimated concept, defined by two or more causal loci implicated in the phenotype of an individual with a rare disease. Only few series report MMDs rates (1.8% to 7.1%). This study highlights the increasing role of MMDs in a large cohort of individuals addressed for congenital anomalies/intellectual disability (CA/ID). METHODS: From 2014 to 2021, our diagnostic laboratory rendered 880/2658 positive ES diagnoses for CA/ID aetiology. Exhaustive search on MMDs from ES data was performed prospectively (January 2019 to December 2021) and retrospectively (March 2014 to December 2018). RESULTS: MMDs were identified in 31/880 individuals (3.5%), responsible for distinct (9/31) or overlapping (22/31) phenotypes, and potential MMDs in 39/880 additional individuals (4.4%). CONCLUSION: MMDs are frequent in CA/ID and remain a strong challenge. Reanalysis of positive ES data appears essential when phenotypes are partially explained by the initial diagnosis or atypically enriched overtime. Up-to-date clinical data, clinical expertise from the referring physician, strong interactions between clinicians and biologists, and increasing gene discoveries and improved ES bioinformatics tools appear all the more fundamental to enhance chances of identifying MMDs. It is essential to provide appropriate patient care and genetic counselling.

Opposite Modulation of RAC1 by Mutations in TRIO Is Associated with Distinct, Domain-Specific Neurodevelopmental Disorders.

The Rho-guanine nucleotide exchange factor (RhoGEF) TRIO acts as a key regulator of neuronal migration, axonal outgrowth, axon guidance, and synaptogenesis by activating the GTPase RAC1 and modulating actin cytoskeleton remodeling. Pathogenic variants in TRIO are associated with neurodevelopmental diseases, including intellectual disability (ID) and autism spectrum disorders (ASD). Here, we report the largest international cohort of 24 individuals with confirmed pathogenic missense or nonsense variants in TRIO. The nonsense mutations are spread along the TRIO sequence, and affected individuals show variable neurodevelopmental phenotypes. In contrast, missense variants cluster into two mutational hotspots in the TRIO sequence, one in the seventh spectrin repeat and one in the RAC1-activating GEFD1. Although all individuals in this cohort present with developmental delay and a neuro-behavioral phenotype, individuals with a pathogenic variant in the seventh spectrin repeat have a more severe ID associated with macrocephaly than do most individuals with GEFD1 variants, who display milder ID and microcephaly. Functional studies show that the spectrin and GEFD1 variants cause a TRIO-mediated hyper- or hypo-activation of RAC1, respectively, and we observe a striking correlation between RAC1 activation levels and the head size of the affected individuals. In addition, truncations in TRIO GEFD1 in the vertebrate model X. tropicalis induce defects that are concordant with the human phenotype. This work demonstrates distinct clinical and molecular disorders clustering in the GEFD1 and seventh spectrin repeat domains and highlights the importance of tight control of TRIO-RAC1 signaling in neuronal development.

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

PURPOSE: Within the Solve-RD project (https://solve-rd.eu/), the European Reference Network for Intellectual disability, TeleHealth, Autism and Congenital Anomalies aimed to investigate whether a reanalysis of exomes from unsolved cases based on ClinVar annotations could establish additional diagnoses. We present the results of the "ClinVar low-hanging fruit" reanalysis, reasons for the failure of previous analyses, and lessons learned. METHODS: Data from the first 3576 exomes (1522 probands and 2054 relatives) collected from European Reference Network for Intellectual disability, TeleHealth, Autism and Congenital Anomalies was reanalyzed by the Solve-RD consortium by evaluating for the presence of single-nucleotide variant, and small insertions and deletions already reported as (likely) pathogenic in ClinVar. Variants were filtered according to frequency, genotype, and mode of inheritance and reinterpreted. RESULTS: We identified causal variants in 59 cases (3.9%), 50 of them also raised by other approaches and 9 leading to new diagnoses, highlighting interpretation challenges: variants in genes not known to be involved in human disease at the time of the first analysis, misleading genotypes, or variants undetected by local pipelines (variants in off-target regions, low quality filters, low allelic balance, or high frequency). CONCLUSION: The "ClinVar low-hanging fruit" analysis represents an effective, fast, and easy approach to recover causal variants from exome sequencing data, herewith contributing to the reduction of the diagnostic deadlock.

Consolidation of the clinical and genetic definition of a SOX4-related neurodevelopmental syndrome.

BACKGROUND: A neurodevelopmental syndrome was recently reported in four patients with SOX4 heterozygous missense variants in the high-mobility-group (HMG) DNA-binding domain. The present study aimed to consolidate clinical and genetic knowledge of this syndrome. METHODS: We newly identified 17 patients with SOX4 variants, predicted variant pathogenicity using in silico tests and in vitro functional assays and analysed the patients' phenotypes. RESULTS: All variants were novel, distinct and heterozygous. Seven HMG-domain missense and five stop-gain variants were classified as pathogenic or likely pathogenic variant (L/PV) as they precluded SOX4 transcriptional activity in vitro. Five HMG-domain and non-HMG-domain missense variants were classified as of uncertain significance (VUS) due to negative results from functional tests. When known, inheritance was de novo or from a mosaic unaffected or non-mosaic affected parent for patients with L/PV, and from a non-mosaic asymptomatic or affected parent for patients with VUS. All patients had neurodevelopmental, neurological and dysmorphic features, and at least one cardiovascular, ophthalmological, musculoskeletal or other somatic anomaly. Patients with L/PV were overall more affected than patients with VUS. They resembled patients with other neurodevelopmental diseases, including the SOX11-related and Coffin-Siris (CSS) syndromes, but lacked the most specific features of CSS. CONCLUSION: These findings consolidate evidence of a fairly non-specific neurodevelopmental syndrome due to SOX4 haploinsufficiency in neurogenesis and multiple other developmental processes.

High efficiency and clinical relevance of exome sequencing in the daily practice of neurogenetics.

OBJECTIVE: To assess the efficiency and relevance of clinical exome sequencing (cES) as a first-tier or second-tier test for the diagnosis of progressive neurological disorders in the daily practice of Neurology and Genetic Departments. METHODS: Sixty-seven probands with various progressive neurological disorders (cerebellar ataxias, neuromuscular disorders, spastic paraplegias, movement disorders and individuals with complex phenotypes labelled 'other') were recruited over a 4-year period regardless of their age, gender, familial history and clinical framework. Individuals could have had prior genetic tests as long as it was not cES. cES was performed in a proband-only (60/67) or trio (7/67) strategy depending on available samples and was analysed with an in-house pipeline including software for CNV and mitochondrial-DNA variant detection. RESULTS: In 29/67 individuals, cES identified clearly pathogenic variants leading to a 43% positive yield. When performed as a first-tier test, cES identified pathogenic variants for 53% of individuals (10/19). Difficult cases were solved including double diagnoses within a kindred or identification of a neurodegeneration with brain iron accumulation in a patient with encephalopathy of suspected mitochondrial origin. CONCLUSION: This study shows that cES is a powerful tool for the daily practice of neurogenetics offering an efficient (43%) and appropriate approach for clinically and genetically complex and heterogeneous disorders.

O'Donnell-Luria-Rodan syndrome: description of a second multinational cohort and refinement of the phenotypic spectrum.

BACKGROUND: O'Donnell-Luria-Rodan syndrome (ODLURO) is an autosomal-dominant neurodevelopmental disorder caused by pathogenic, mostly truncating variants in KMT2E. It was first described by O'Donnell-Luria et al in 2019 in a cohort of 38 patients. Clinical features encompass macrocephaly, mild intellectual disability (ID), autism spectrum disorder (ASD) susceptibility and seizure susceptibility. METHODS: Affected individuals were ascertained at paediatric and genetic centres in various countries by diagnostic chromosome microarray or exome/genome sequencing. Patients were collected into a case cohort and were systematically phenotyped where possible. RESULTS: We report 18 additional patients from 17 families with genetically confirmed ODLURO. We identified 15 different heterozygous likely pathogenic or pathogenic sequence variants (14 novel) and two partial microdeletions of KMT2E. We confirm and refine the phenotypic spectrum of the KMT2E-related neurodevelopmental disorder, especially concerning cognitive development, with rather mild ID and macrocephaly with subtle facial features in most patients. We observe a high prevalence of ASD in our cohort (41%), while seizures are present in only two patients. We extend the phenotypic spectrum by sleep disturbances. CONCLUSION: Our study, bringing the total of known patients with ODLURO to more than 60 within 2 years of the first publication, suggests an unexpectedly high relative frequency of this syndrome worldwide. It seems likely that ODLURO, although just recently described, is among the more common single-gene aetiologies of neurodevelopmental delay and ASD. We present the second systematic case series of patients with ODLURO, further refining the mutational and phenotypic spectrum of this not-so-rare syndrome.

Copy number variants calling from WES data through eXome hidden Markov model (XHMM) identifies additional 2.5% pathogenic genomic imbalances smaller than 30 kb undetected by array-CGH.

It has been estimated that Copy Number Variants (CNVs) account for 10%-20% of patients affected by Developmental Disorder (DD)/Intellectual Disability (ID). Although array comparative genomic hybridization (array-CGH) represents the gold-standard for the detection of genomic imbalances, common Agilent array-CGH 4 × 180 kb arrays fail to detect CNVs smaller than 30 kb. Whole Exome sequencing (WES) is becoming the reference application for the detection of gene variants and makes it possible also to infer genomic imbalances at single exon resolution. However, the contribution of small CNVs in DD/ID is still underinvestigated. We made use of the eXome Hidden Markov Model (XHMM) software, a tool utilized by the ExAC consortium, to detect CNVs from whole exome sequencing data, in a cohort of 200 unsolved DD/DI patients after array-CGH and WES-based single nucleotide/indel variant analyses. In five out of 200 patients (2.5%), we identified pathogenic CNV(s) smaller than 30 kb, ranging from one to six exons. They included two heterozygous deletions in TCF4 and STXBP1 and three homozygous deletions in PPT1, CLCN2, and PIGN. After reverse phenotyping, all variants were reported as causative. This study shows the interest in applying sequencing-based CNV detection, from available WES data, to reduce the diagnostic odyssey of additional patients unsolved DD/DI patients and compare the CNV-detection yield of Agilent array-CGH 4 × 180kb versus whole exome sequencing.

MYT1L-associated neurodevelopmental disorder: description of 40 new cases and literature review of clinical and molecular aspects.

Pathogenic variants of the myelin transcription factor-1 like (MYT1L) gene include heterozygous missense, truncating variants and 2p25.3 microdeletions and cause a syndromic neurodevelopmental disorder (OMIM#616,521). Despite enrichment in de novo mutations in several developmental disorders and autism studies, the data on clinical characteristics and genotype-phenotype correlations are scarce, with only 22 patients with single nucleotide pathogenic variants reported. We aimed to further characterize this disorder at both the clinical and molecular levels by gathering a large series of patients with MYT1L-associated neurodevelopmental disorder. We collected genetic information on 40 unreported patients with likely pathogenic/pathogenic MYT1L variants and performed a comprehensive review of published data (total = 62 patients). We confirm that the main phenotypic features of the MYT1L-related disorder are developmental delay with language delay (95%), intellectual disability (ID, 70%), overweight or obesity (58%), behavioral disorders (98%) and epilepsy (23%). We highlight novel clinical characteristics, such as learning disabilities without ID (30%) and feeding difficulties during infancy (18%). We further describe the varied dysmorphic features (67%) and present the changes in weight over time of 27 patients. We show that patients harboring highly clustered missense variants in the 2-3-ZNF domains are not clinically distinguishable from patients with truncating variants. We provide an updated overview of clinical and genetic data of the MYT1L-associated neurodevelopmental disorder, hence improving diagnosis and clinical management of these patients.

HNRNPR Variants that Impair Homeobox Gene Expression Drive Developmental Disorders in Humans.

The heterogeneous nuclear ribonucleoprotein (HNRNP) genes code for a set of RNA-binding proteins that function primarily in the spliceosome C complex. Pathogenic variants in these genes can drive neurodegeneration, through a mechanism involving excessive stress-granule formation, or developmental defects, through mechanisms that are not known. Here, we report four unrelated individuals who have truncating or missense variants in the same C-terminal region of hnRNPR and who have multisystem developmental defects including abnormalities of the brain and skeleton, dysmorphic facies, brachydactyly, seizures, and hypoplastic external genitalia. We further identified in the literature a fifth individual with a truncating variant. RNA sequencing of primary fibroblasts reveals that these HNRNPR variants drive significant changes in the expression of several homeobox genes, as well as other transcription factors, such as LHX9, TBX1, and multiple HOX genes, that are considered fundamental regulators of embryonic and gonad development. Higher levels of retained intronic HOX sequences and lost splicing events in the HOX cluster are observed in cells carrying HNRNPR variants, suggesting that impaired splicing is at least partially driving HOX deregulation. At basal levels, stress-granule formation appears normal in primary and transfected cells expressing HNRNPR variants. However, these cells reveal profound recovery defects, where stress granules fail to disassemble properly, after exposure to oxidative stress. This study establishes an essential role for HNRNPR in human development and points to a mechanism that may unify other "spliceosomopathies" linked to variants that drive multi-system congenital defects and are found in hnRNPs.