Genotype-phenotype correlations in RHOBTB2-associated neurodevelopmental disorders

PurposeMissense variants clustering in the BTB domain region of RHOBTB2 cause a developmental and epileptic encephalopathy with early-onset seizures and severe intellectual disability.MethodsBy international collaboration, we assembled individuals with pathogenic RHOBTB2 variants and a variable spectrum of neurodevelopmental disorders. By western blotting, we investigated the consequences of missense variants in vitro.ResultsIn accordance with previous observations, de novo heterozygous missense variants in the BTB domain region led to a severe developmental and epileptic encephalopathy in 16 individuals. Now, we also identified de novo missense variants in the GTPase domain in 6 individuals with apparently more variable neurodevelopmental phenotypes with or without epilepsy. In contrast to variants in the BTB domain region, variants in the GTPase domain do not impair proteasomal degradation of RHOBTB2 in vitro, indicating different functional consequences. Furthermore, we observed biallelic splice-site and truncating variants in 9 families with variable neurodevelopmental phenotypes, indicating that complete loss of RHOBTB2 is pathogenic as well.ConclusionBy identifying genotype-phenotype correlations regarding location and consequences of de novo missense variants in RHOBTB2 and by identifying biallelic truncating variants, we further delineate and expand the molecular and clinical spectrum of RHOBTB2-related phenotypes, including both autosomal dominant and recessive neurodevelopmental disorders.     

EFNB2 haploinsufficiency causes a syndromic neurodevelopmental disorder

Ephrin B2, one of the ligand of the EphB receptors, is involved in a complex signaling pathway regulating the development of the nervous system, neuronal migration, erythropoiesis and vasculogenesis. We report a patient with a de novo variant in EFNB2 and a family in which segregates a 610‐kb deletion at chromosome 13q33 encompassing only ARGLU1 and EFNB2 genes. The de novo variant was observed in a patient with anal stenosis, hypoplastic left ventricle and mild developmental delay. The deletion was identified in 2 sibs with congenital heart defect and mild developmental delay. One of the affected sibs further had myoclonic epilepsy and bilateral sensorineural hearing loss. The carrier mother was apparently asymptomatic. Because EFNB2 is located in the subtelomeric region of 13q chromosome, we reviewed the previous reports of terminal 13q deletion. We suggest that haploinsufficiency of the EFNB2 could be at the origin of several clinical features reported in 13qter deletions, including intellectual disability, seizures, congenital heart defects, anorectal malformation and hearing loss.     

Further insights into the spectrum phenotype of TRAPPC9 and CDK5RAP2 genes,segregating independently in a large Tunisian family with intellectual disability and microcephaly

Intellectual disability (ID) often co-occurs with other neurologic phenotypes making molecular diagnosis more challenging particularly in consanguineous populations with the co-segregation of more than one ID-related gene in some cases.In this study, we investigated the phenotype of three patients from a large Tunisian family with significant ID phenotypic variability and microcephaly and performed a clinical exome sequencing in two cases. We identified, within the first branch, a homozygous variant in the TRAPPC9 gene (p.Arg472Ter) in two cases presenting severe ID, absent speech, congenital/secondary microcephaly in addition to autistic features, supporting the implication of TRAPPC9 in the “secondary” autism spectrum disorders and congenital microcephaly. In the second branch, we identified a homozygous variant (p.Lys189ArgfsTer15) in the CDK5RAP2 gene associated with an heterozygous TRAPPC9 variant (p.Arg472Ter) in one case harbouring primary hereditary microcephaly (MCPH) associated with an inter-hypothalamic adhesion, mixed hearing loss, selective thinning in the retinal nerve fiber layer and parafoveal ganglion cell complex, and short stature. Our findings expand the spectrum of the recently reported neurosensorial abnormalities and revealed the variable phenotype expressivity of CDK5RAP2 defect. Our study highlights the complexity of the genetic background of microcephaly/ID and the efficiency of the exome sequencing to provide an accurate diagnosis and to improve the management and follow-up of such patients.     

MTSS2-related neurodevelopmental disorder: Further delineation of the phenotype

MTSS2-related neurodevelopmental disorder (MTSS2-related NDD) (MIM 620086) is characterized by intellectual developmental disorder with ocular anomalies and distinctive facial features (IDDOF). The only existing report to date described five individuals who exhibited an identical de novo c.2011C>T (p.Arg671Trp) variant in the MTSS2 gene. Herein, we report a new case of MTSS2-related NND in a male dizygotic twin who presented with IDDOF and severe intellectual disability. This patient also displayed additional clinical features, including low functioning autism, hypothyroidism, duodenal obstruction secondary to Ladd's bands, inguinal hernias, cryptorchidism, transient subperiosteal new bone formation, and short stature with delayed bone age, which had not been previously reported in association with the MTSS2-related NDD. Exome sequencing identified the recurrent c.2011C>T (p.Arg671Trp) variant in the MTSS2 gene. The mother and the other twin tested negative for the pathogenic variant, while the father's participation in the study was unavailable. This case confirms that the MTSS2-related NDD is caused by the recurrent MTSS2 missense variant p.Arg671Trp. The novel findings identified in our patient expand the phenotypic spectrum associated with this new autosomal dominant entity, but further studies on its genetic and clinical manifestations are still needed.     

Copy number variations in neurodevelopmental disorders

Common neurodevelopmental disorders (including autism, speech and language delay, schizophrenia, epilepsy and intellectual disability) have complex aetiology, which is predominantly genomic, but also environmental in origin. They share a paradox, in that high heritability is matched by lowered fecundity, placing them under negative genetic selection. This implicates variants of recent origin, such as de novo mutations or common, very low-risk polymorphisms that escape negative selection. High or moderate risk variants have been discovered by chromosome analysis, genome sequencing and copy number variant (CNV) detection, including a 3Mb deletion causing 22q11.2 deletion syndrome (Velo-Cardio-Facial Syndrome) that has penetrance of up to 50% for schizophrenia. More recently, rare, recurrent and often de novo pathogenic CNVs, including deletions at NRXN1, 1q21.2, 15q11.2 and 15q13.3, 16p11.2 and duplications at VIPR2 and 16p13.11, have also been discovered. These have several unique features that differentiate them from Mendelian disease mutations in that they have incomplete penetrance, with moderate-to-high odds ratios for risk, and show diagnostic pleiotropy, increasing risk across the neurodevelopmental disorder spectrum. Some are also syndromic, with characteristic features such as facial dysmorphology, and other specific risks such as aortic dissection or obesity, implying that they might be better classified as distinct diagnoses. The discovery of pathogenic CNVs provide new opportunities for translation leading to patent benefit, including improvements in clinical genetic diagnosis and genetic counselling, the possibility of clinician decision-making tools for risk prediction, and the identification of drug targets and implementation of personalised medicine using stratification by genotype.     

BAZ2B haploinsufficiency as a cause of developmental delay,intellectual disability,and autism spectrum disorder

The bromodomain adjacent to zinc finger 2B gene (BAZ2B) encodes a protein involved in chromatin remodeling. Loss of BAZ2B function has been postulated to cause neurodevelopmental disorders. To determine whether BAZ2B deficiency is likely to contribute to the pathogenesis of these disorders, we performed bioinformatics analyses that demonstrated a high level of functional convergence during fetal cortical development between BAZ2B and genes known to cause autism spectrum disorder (ASD) and neurodevelopmental disorder. We also found an excess of de novo BAZ2B loss‐of‐function variants in exome sequencing data from previously published cohorts of individuals with neurodevelopmental disorders. We subsequently identified seven additional individuals with heterozygous deletions, stop‐gain, or de novo missense variants affecting BAZ2B. All of these individuals have developmental delay (DD), intellectual disability (ID), and/or ASD. Taken together, our findings suggest that haploinsufficiency of BAZ2B causes a neurodevelopmental disorder, whose cardinal features include DD, ID, and ASD.     

Missense variants in RPH3A cause defects in excitatory synaptic function and are associated with a clinically variable neurodevelopmental disorder

PurposeRPH3A encodes a protein involved in the stabilization of GluN2A subunit of N-methyl-D-aspartate (NMDA)-type glutamate receptors at the cell surface, forming a complex essential for synaptic plasticity and cognition. We investigated the effect of variants in RPH3A in patients with neurodevelopmental disorders.MethodsBy using trio-based exome sequencing, GeneMatcher, and screening of 100,000 Genomes Project data, we identified 6 heterozygous variants in RPH3A. In silico and in vitro models, including rat hippocampal neuronal cultures, have been used to characterize the effect of the variants.ResultsFour cases had a neurodevelopmental disorder with untreatable epileptic seizures [p.(Gln73His)dn; p.(Arg209Lys); p.(Thr450Ser)dn; p.(Gln508His)], and 2 cases [p.(Arg235Ser); p.(Asn618Ser)dn] showed high-functioning autism spectrum disorder. Using neuronal cultures, we demonstrated that p.(Thr450Ser) and p.(Asn618Ser) reduce the synaptic localization of GluN2A; p.(Thr450Ser) also increased the surface levels of GluN2A. Electrophysiological recordings showed increased GluN2A-dependent NMDA ionotropic glutamate receptor currents for both variants and alteration of postsynaptic calcium levels. Finally, expression of the Rph3A^Thr450Ser variant in neurons affected dendritic spine morphology.ConclusionOverall, we provide evidence that missense gain-of-function variants in RPH3A increase GluN2A-containing NMDA ionotropic glutamate receptors at extrasynaptic sites, altering synaptic function and leading to a clinically variable neurodevelopmental presentation ranging from untreatable epilepsy to autism spectrum disorder.     

De novo loss-of-function variants of ASH1L are associated with an emergent neurodevelopmental disorder

De novo variants of ASH1L, which encodes a histone methyltransferase, have been reported in a few patients with intellectual disability and autistic features. Here, we identified a novel de novo frame-shift variant, c.2422_2423delAAinsT which predicts p.(Lys808TyrfsTer40), in ASH1L in a patient with multiple congenital anomalies (MCA), fine motor developmental delay, learning difficulties, attention deficit hyperactivity disorder, sleep apnea, and scoliosis. This frame-shift variant is expected to result in loss-of-function. Our report provides further evidence to support loss-of-function alterations of ASH1L as causative for an emergent neurodevelopmental syndrome characterized by MCA, intellectual disability, and behavioral problems, and further delineates this genetic disorder.     

ZNF142 mutation causes neurodevelopmental disorder with speech impairment and seizures: Novel variants and literature review

The ZNF142 gene on chromosome 2q35 contains ten exons and encodes a zinc finger protein 142 with 31 C2H2-type zinc fingers domain. Pathogenic variants in ZNF142 result in an autosomal recessive neurodevelopmental disorder with impaired speech and developmental delay. Here, we report two novel variants (NM_001105537: c.25C > T/c.1741C > T, p.Gln9*/p.Arg581Cys) in ZNF142 in an Iranian family identified by Whole-Exome sequencing and confirmed by Sanger sequencing. These variants are categorized as “pathogenic” and “variant of unknown significance” based on the standards for the interpretation of sequence variations recommended by ACMG, respectively. The proband is a five-year-old male born to consanguineous parents. The compound heterozygous variant (NM_001105537: c.25C > T/c.1741C > T, p.Gln9*/p.Arg581Cys) in ZNF142 was identified in the proband with moderate intellectual disability, global developmental delay, speech impairment, and seizures. This paper reported the sixth family in the world with novel pathogenic variants in the ZNF142 gene as the reason for neurodevelopmental Disorder with Impaired Speech and Hyperkinetic Movements (NEDISHM) and determining the phenotype spectrum of this disease. In this study, we also reviewed the phenotype of the former cases. In contrast to the Malaysian cases, proband in the present paper does not manifest any facial features similar to the patients in the initial study. Further studies on the NEDISHM patients could be valuable to determine the phenotype precisely.     

Biallelic variants in NSUN6 cause an autosomal recessive neurodevelopmental disorder

Purpose5-methylcytosine RNA modifications are driven by NSUN methyltransferases. Although variants in NSUN2 and NSUN3 were associated with neurodevelopmental diseases, the physiological role of NSUN6 modifications on transfer RNAs and messenger RNAs remained elusive.MethodsWe combined exome sequencing of consanguineous families with functional characterization to identify a new neurodevelopmental disorder gene.ResultsWe identified 3 unrelated consanguineous families with deleterious homozygous variants in NSUN6. Two of these variants are predicted to be loss-of-function. One maps to the first exon and is predicted to lead to the absence of NSUN6 via nonsense-mediated decay, whereas we showed that the other maps to the last exon and encodes a protein that does not fold correctly. Likewise, we demonstrated that the missense variant identified in the third family has lost its enzymatic activity and is unable to bind the methyl donor S-adenosyl-L-methionine. The affected individuals present with developmental delay, intellectual disability, motor delay, and behavioral anomalies. Homozygous ablation of the NSUN6 ortholog in Drosophila led to locomotion and learning impairment.ConclusionOur data provide evidence that biallelic pathogenic variants in NSUN6 cause one form of autosomal recessive intellectual disability, establishing another link between RNA modification and cognition.     

DNA methylation episignature and comparative epigenomic profiling of HNRNPU-related neurodevelopmental disorder

PurposeHNRNPU haploinsufficiency is associated with developmental and epileptic encephalopathy 54. This neurodevelopmental disorder is characterized by developmental delay, intellectual disability, speech impairment, and early-onset epilepsy. We performed genome-wide DNA methylation (DNAm) analysis in a cohort of individuals to develop a diagnostic biomarker and gain functional insights into the molecular pathophysiology of HNRNPU-related disorder.MethodsDNAm profiles of individuals carrying pathogenic HNRNPU variants, identified through an international multicenter collaboration, were assessed using Infinium Methylation EPIC arrays. Statistical and functional correlation analyses were performed comparing the HNRNPU cohort with 56 previously reported DNAm episignatures.ResultsA robust and reproducible DNAm episignature and global DNAm profile were identified. Correlation analysis identified partial overlap and similarity of the global HNRNPU DNAm profile to several other rare disorders.ConclusionThis study demonstrates new evidence of a specific and sensitive DNAm episignature associated with pathogenic heterozygous HNRNPU variants, establishing its utility as a clinical biomarker for the expansion of the EpiSign diagnostic test.     

Delineation of a KDM2B-related neurodevelopmental disorder and its associated DNA methylation signature

PurposePathogenic variants in genes involved in the epigenetic machinery are an emerging cause of neurodevelopment disorders (NDDs). Lysine-demethylase 2B (KDM2B) encodes an epigenetic regulator and mouse models suggest an important role during development. We set out to determine whether KDM2B variants are associated with NDD.MethodsThrough international collaborations, we collected data on individuals with heterozygous KDM2B variants. We applied methylation arrays on peripheral blood DNA samples to determine a KDM2B associated epigenetic signature.ResultsWe recruited a total of 27 individuals with heterozygous variants in KDM2B. We present evidence, including a shared epigenetic signature, to support a pathogenic classification of 15 KDM2B variants and identify the CxxC domain as a mutational hotspot. Both loss-of-function and CxxC-domain missense variants present with a specific subepisignature. Moreover, the KDM2B episignature was identified in the context of a dual molecular diagnosis in multiple individuals. Our efforts resulted in a cohort of 21 individuals with heterozygous (likely) pathogenic variants. Individuals in this cohort present with developmental delay and/or intellectual disability; autism; attention deficit disorder/attention deficit hyperactivity disorder; congenital organ anomalies mainly of the heart, eyes, and urogenital system; and subtle facial dysmorphism.ConclusionPathogenic heterozygous variants in KDM2B are associated with NDD and a specific epigenetic signature detectable in peripheral blood.     

A new neurodevelopmental disorder linked to heterozygous variants in UNC79

PurposeThe “NALCN channelosome” is an ion channel complex that consists of multiple proteins, including NALCN, UNC79, UNC80, and FAM155A. Only a small number of individuals with a neurodevelopmental syndrome have been reported with disease causing variants in NALCN and UNC80. However, no pathogenic UNC79 variants have been reported, and in vivo function of UNC79 in humans is largely unknown.MethodsWe used international gene-matching efforts to identify patients harboring ultrarare heterozygous loss-of-function UNC79 variants and no other putative responsible genes. We used genetic manipulations in Drosophila and mice to test potential causal relationships between UNC79 variants and the pathology.ResultsWe found 6 unrelated and affected patients with UNC79 variants. Five patients presented with overlapping neurodevelopmental features, including mild to moderate intellectual disability and a mild developmental delay, whereas a single patient reportedly had normal cognitive and motor development but was diagnosed with epilepsy and autistic features. All displayed behavioral issues and 4 patients had epilepsy. Drosophila with UNC79 knocked down displayed induced seizure-like phenotype. Mice with a heterozygous loss-of-function variant have a developmental delay in body weight compared with wild type. In addition, they have impaired ability in learning and memory.ConclusionOur results demonstrate that heterozygous loss-of-function UNC79 variants are associated with neurologic pathologies.     

Expanding the genotype and phenotype spectrum of SYT1-associated neurodevelopmental disorder

PurposeSynaptotagmin-1 (SYT1) is a critical mediator of neurotransmitter release in the central nervous system. Previously reported missense SYT1 variants in the C2B domain are associated with severe intellectual disability, movement disorders, behavioral disturbances, and electroencephalogram abnormalities. In this study, we expand the genotypes and phenotypes and identify discriminating features of this disorder.MethodsWe describe 22 individuals with 15 de novo missense SYT1 variants. The evidence for pathogenicity is discussed, including the American College of Medical Genetics and Genomics/Association for Molecular Pathology criteria, known structure–function relationships, and molecular dynamics simulations. Quantitative behavioral data for 14 cases were compared with other monogenic neurodevelopmental disorders.ResultsFour variants were located in the C2A domain with the remainder in the C2B domain. We classified 6 variants as pathogenic, 4 as likely pathogenic, and 5 as variants of uncertain significance. Prevalent clinical phenotypes included delayed developmental milestones, abnormal eye physiology, movement disorders, and sleep disturbances. Discriminating behavioral characteristics were severity of motor and communication impairment, presence of motor stereotypies, and mood instability.ConclusionNeurodevelopmental disorder–associated SYT1 variants extend beyond previously reported regions, and the phenotypic spectrum encompasses a broader range of severities than initially reported. This study guides the diagnosis and molecular understanding of this rare neurodevelopmental disorder and highlights a key role for SYT1 function in emotional regulation, motor control, and emergent cognitive function.     

Severe neurodevelopmental disorder with intractable seizures due to a novel SLC1A4 homozygous variant

IntroductionBiallelic variants in the SLC1A4 gene have been so far identified as a very rare cause of neurodevelopmental disorders with or without epilepsy and almost exclusively described in the Ashkenazi-Jewish population.Patients and methodsHere we present Czech patient with microcephaly, severe global developmental delay and intractable seizures whose condition remained undiagnosed despite access to clinical experience and standard diagnostic methods including examination with an epilepsy targeted NGS gene panel.ResultsWhole exome sequencing revealed a novel variant NM_003038.4:c.1370G > A p.(Arg457Gln) of the SLC1A4 gene in a homozygous state in the patient, and afterwards Sanger sequencing in both parents confirmed the biallelic origin of the variant. A variant in the same codon, but with a different amino acid exchange, was described previously in a patient that had a very similar phenotype, however, without epilepsy.ConclusionOur data suggest that the SLC1A4 gene should be considered in the diagnosis of patients with severe, early onset neurodevelopmental impairment with epilepsy and encourage the analysis of SLC1A4 gene variants via targeted NGS gene panel or whole exome sequencing.