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Contactin-associated protein-like 2 (CNTNAP2) gene encodes for CASPR2, a presynaptic type 1 transmembrane protein, involved in cell–cell adhesion and synaptic interactions. Biallelic CNTNAP2 loss has been associated with “Pitt-Hopkins-like syndrome-1” (MIM#610042), while the pathogenic role of heterozygous variants remains controversial. We report 22 novel patients harboring mono- (n = 2) and bi-allelic (n = 20) CNTNAP2 variants and carried out a literature review to characterize the genotype–phenotype correlation. Patients (M:F 14:8) were aged between 3 and 19 years and affected by global developmental delay (GDD) (n = 21), moderate to profound intellectual disability (n = 17) and epilepsy (n = 21). Seizures mainly started in the first two years of life (median 22.5 months). Antiseizure medications were successful in controlling the seizures in about two-thirds of the patients. Autism spectrum disorder (ASD) and/or other neuropsychiatric comorbidities were present in nine patients (40.9%). Nonspecific midline brain anomalies were noted in most patients while focal signal abnormalities in the temporal lobes were noted in three subjects. Genotype–phenotype correlation was performed by also including 50 previously published patients (15 mono- and 35 bi-allelic variants). Overall, GDD (p p p = 0.012), ASD (p = 0.009), language impairment (p = 0.020) and severe cognitive impairment (p = 0.031) were significantly associated with the presence of biallelic versus monoallelic variants. We have defined the main features associated with biallelic CNTNAP2 variants, as severe cognitive impairment, epilepsy and behavioral abnormalities. We propose CASPR2-deficiency neurodevelopmental disorder as an exclusively recessive disease while the contribution of heterozygous variants is less likely to follow an autosomal dominant inheritance pattern.

In the field of rare diseases, progress in molecular diagnostics led to the recognition that variants linked to autosomal-dominant neurodegenerative diseases of later onset can, in the context of biallelic inheritance, cause devastating neurodevelopmental disorders and infantile or childhood-onset neurodegeneration. TOR1A-associated arthrogryposis multiplex congenita 5 (AMC5) is a rare neurodevelopmental disorder arising from biallelic variants in TOR1A, a gene that in the heterozygous state is associated to torsion dystonia-1 (DYT1 or DYT-TOR1A), an early-onset dystonia with reduced penetrance. While 15 individuals with TOR1A-AMC5 have been reported (less than 10 in detail), a systematic investigation of the full disease-associated spectrum has not been conducted. Here, we assess the clinical, radiological and molecular characteristics of 57 individuals from 40 families with biallelic variants in TOR1A. Median age at last follow-up was 3 years (0-24 years). Most individuals presented with severe congenital flexion contractures (95%) and variable developmental delay (79%). Motor symptoms were reported in 79% and included lower limb spasticity and pyramidal signs, as well as gait disturbances. Facial dysmorphism was an integral part of the phenotype, with key features being a broad/full nasal tip, narrowing of the forehead and full cheeks. Analysis of disease-associated manifestations delineated a phenotypic spectrum ranging from normal cognition and mild gait disturbance to congenital arthrogryposis, global developmental delay, intellectual disability, absent speech and inability to walk. In a subset, the presentation was consistent with fetal akinesia deformation sequence with severe intrauterine abnormalities. Survival was 71% with higher mortality in males. Death occurred at a median age of 1.2 months (1 week - 9 years) due to respiratory failure, cardiac arrest, or sepsis. Analysis of brain MRI studies identified non-specific neuroimaging features, including a hypoplastic corpus callosum (72%), foci of signal abnormality in the subcortical and periventricular white matter (55%), diffuse white matter volume loss (45%), mega cisterna magna (36%) and arachnoid cysts (27%). The molecular spectrum included 22 distinct variants, defining a mutational hotspot in the C-terminal domain of the Torsin-1A protein. Genotype-phenotype analysis revealed an association of missense variants in the 3-helix bundle domain to an attenuated phenotype, while missense variants near the Walker A/B motif as well as biallelic truncating variants were linked to early death. In summary, this systematic cross-sectional analysis of a large cohort of individuals with biallelic TOR1A variants across a wide age-range delineates the clinical and genetic spectrum of TOR1A-related autosomal-recessive disease and highlights potential predictors for disease severity and survival. CC BY 4.0Correspondence to: Reza MaroofianDepartment of Neuromuscular Diseases, Queen Square Institute of Neurology, University College London, London WC1N 3BG, UKE-mail: R.Maroofian@ucl.ac.ukA.S. is funded by the Deutsche Forschungsgemeinschaft (DFG, German Research Foundation) – SA 4171/1-1. H.H. is funded by The MRC (MR/S01165X/1, MR/S005021/1, G0601943), The National Institute for Health Research University College London Hospitals Biomedical Research Centre, Rosetree Trust, Ataxia UK, MSA Trust, Brain Research UK, Sparks GOSH Charity, Muscular Dystrophy UK (MDUK), Muscular Dystrophy Association (MDA USA). S.E. is supported by an MRC strategic award to establish an International Centre for Genomic Medicine in Neuromuscular Diseases (ICGNMD) MR/S005021/1’. This work was supported in part by the US National Institutes of Health R35 NS105078 and MDA#512848 to J.R.L., a jointly funded National Human Genome Research Institute (NHGRI) and National Heart, Lung, and Blood Institute (NHLBI) grant to the Baylor-Hopkins Center for Mendelian Genomics (UM1 HG006542) and NHGRI Genomics Research to Elucidate Genetics of Rare (BCM-GREGoR U01 HG011758). D.P. is supported by Clinical Research Training Scholarship in Neuromuscular Disease partnered by the American Brain Foundation (ABF) and Muscle Study Group (MSG). The research conducted at the Murdoch Children’s Research Institute was supported by the Victorian Government’s Operational Infrastructure Support Program. The Chair in Genomic Medicine awarded to J.C. is generously supported by The Royal Children’s Hospital Foundation. H.T. has been supported by the European Union’s Seventh Framework Program for research, technological development and demonstration under grant agreement no. 608473. H.S.D. is supported by the Cologne Clinician Scientist Program/Faculty of Medicine/University of Cologne and funded by the Deutsche Forschungsgemeinschaft (DFG, German Research Foundation, Project No. 413543196). H.J. has been supported by a grant from the European Union (H2020- MSCA-ITN-2017). A.M.D. is supported by the Eunice Kennedy Shriver National Institute of Child Health and Human Development of the National Institutes of Health (grant 1R01HD104938-01A1). H.L. receives support from the Canadian Institutes of Health Research (Foundation Grant FDN-167281), the Canadian Institutes of Health Research and Muscular Dystrophy Canada (Network Catalyst Grant for NMD4C), the Canada Foundation for Innovation (CFI-JELF 38412), and the Canada Research Chairs program (Canada Research Chair in Neuromuscular Genomics and Health, 950-232279). This research was funded in part by the Wellcome Trust (WT093205MA, WT104033AIA and the Synaptopathies Strategic Award, 165908) as well as the Medical Research Council (MRC) (MR/S01165X/1, MR/ S005021/1, G0601943). Additonally, we are grateful for funding from The MSA Trust, The National Institute for Health Research University College London Hospitals Biomedical Research Centre, The Michael J Fox Foundation (MJFF), BBSRC, The Fidelity Trust, Rosetrees Trust, Ataxia UK, Brain Research UK, Sparks GOSH Charity, Alzheimer’s Research UK (ARUK) and CureDRPLA.

Succinic semialdehyde dehydrogenase (SSADH) deficiency is a rare neurometabolic disorder characterized by defective degradation of gamma-aminobutyric acid (GABA), a major inhibitory neurotransmitter of the brain. Children with SSADH deficiency present with motor mental delay, intractable seizures, infantile onset hypotonia, speech disturbances, extrapyramidal symptoms and ataxia. This wide spectrum results from increased accumulation of 4hydroxy butyric acid (4HBA) leading to down regulation of GABA receptors, which likely explain epileptogenesis but the pathophysiology of stroke in SSADH deficiency is not much elucidated. Here, we report an infant aged 11 months, product of consanguineous marriage with significant family history of motor delay and intellectual disability, presented with sudden onset focal neurological deficit preceded by diarrheal illness. Examination revealed an infant with age-appropriate milestones having left uncrossed hemiplegia along with neuroradiological evidence of right globus pallidus ischemic infarct. Urinary organic acid profile by chromatography was suggestive of 4 hydroxybutyric aciduria.

Objective: To determine the variations in clinical presentation, neuroimaging and electroencephalography patterns of subacute sclerosing panencephalitis. Study Design: Cross-sectional study. Place and Duration of Study: Children’s Hospital & Institute of Child Health, Lahore, Pakistan, from Jul to Dec 2020. Methodology: We recruited children presented with clinical features suggestive of subacute sclerosing panencephalitis, along with positive anti-measles antibodies on cerebrospinal fluid. Association between variables was determined to formulate an early diagnosis of subacute sclerosing panencephalitis. Results: Out of 47 children, 29 were males with a mean age of 6.54 ± 2.9 years. Only 23% were fully immunized against measles, 36.2% were unvaccinated and 40.4% received partial immunization. The mean age of measles infection was 1.49 ± 1.2 years; the mean interval between measles and onset of SSPE was 4.13 ± 3 years. Atypical clinical presentation was seen in 38.3% with intractable epilepsy (8.5%), focal deficit (8.5%) and extrapyramidal symptoms (8.5%) being commonest followed by coma (6.4%), visual loss (4.3%) and psychosis (2.1%). Neuroimaging was suggestive of cortical hyperintensities in 46.8% and was normal in 46.8%. Electroencephalography showed burst suppression in 55.3% and atypical findings in 19.1%. Younger age (1-1.5 years) of measles and unimmunized status were associated with early onset of SSPE with a p-value of 0.001 and 0.05 respectively. Non-immunized status was associated with atypical presentation of SSPE (p-value

Purpose Dioxygenases are oxidoreductase enzymes with roles in metabolic pathways necessary for aerobic life. 4-hydroxyphenylpyruvate dioxygenase-like protein (HPDL), encoded by HPDL, is an orphan paralogue of 4-hydroxyphenylpyruvate dioxygenase (HPD), an iron-dependent dioxygenase involved in tyrosine catabolism. The function and association of HPDL with human diseases remain unknown. Methods We applied exome sequencing in a cohort of over 10,000 individuals with neurodevelopmental diseases. Effects of HPDL loss were investigated in vitro and in vivo, and through mass spectrometry analysis. Evolutionary analysis was performed to investigate the potential functional separation of HPDL from HPD. Results We identified biallelic variants in HPDL in eight families displaying recessive inheritance. Knockout mice closely phenocopied humans and showed evidence of apoptosis in multiple cellular lineages within the cerebral cortex. HPDL is a single-exonic gene that likely arose from a retrotransposition event at the base of the tetrapod lineage, and unlike HPD, HPDL is mitochondria-localized. Metabolic profiling of HPDL mutant cells and mice showed no evidence of altered tyrosine metabolites, but rather notable accumulations in other metabolic pathways. Conclusion The mitochondrial localization, along with its disrupted metabolic profile, suggests HPDL loss in humans links to a unique neurometabolic mitochondrial infantile neurodegenerative condition.

PURPOSE Missense variants clustering in the BTB domain region of RHOBTB2 cause a developmental and epileptic encephalopathy (DEE) with early-onset seizures and severe intellectual disability. METHODS By international collaboration we assembled individuals with pathogenic RHOBTB2 variants and a variable spectrum of neurodevelopmental disorders (NDDs). By western blotting we investigated the consequences of missense variants in vitro. RESULTS In accordance with previous observations, de novo heterozygous missense variants in the BTB domain region led to a severe DEE in 16 individuals. We now identified also de novo missense variants in the GTPase domain in six 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 bi-allelic splice-site and truncating variants in nine families with variable neurodevelopmental phenotypes, indicating that complete loss of RHOBTB2 is pathogenic as well. CONCLUSION By identifying phenotype-genotype correlations regarding location and consequences of de novo missense variants in RHOBTB2 and by identifying bi-allelic truncating variants, we further delineate and expand the molecular and clinical spectrum of RHOBTB2 related disorders including both autosomal dominant and recessive NDDs.

The 2-oxoglutarate dehydrogenase-like (OGDHL) protein is a rate-limiting enzyme in the Krebs cycle that plays a pivotal role in mitochondrial metabolism. OGDHL expression is restricted mainly to the brain in humans. Here, we report nine individuals from eight unrelated families carrying bi-allelic variants in OGDHL with a range of neurological and neurodevelopmental phenotypes including epilepsy, hearing loss, visual impairment, gait ataxia, microcephaly, and hypoplastic corpus callosum. The variants include three homozygous missense variants (p.Pro852Ala, p.Arg244Trp, and p.Arg299Gly), three compound heterozygous single-nucleotide variants (p.Arg673Gln/p.Val488Val, p.Phe734Ser/p.Ala327Val, and p.Trp220Cys/p.Asp491Val), one homozygous frameshift variant (p.Cys553Leufs∗16), and one homozygous stop-gain variant (p.Arg440Ter). To support the pathogenicity of the variants, we developed a novel CRISPR-Cas9-mediated tissue-specific knockout with cDNA rescue system for dOgdh, the Drosophila ortholog of human OGDHL. Pan-neuronal knockout of dOgdh led to developmental lethality as well as defects in Krebs cycle metabolism, which was fully rescued by expression of wild-type dOgdh. Studies using the Drosophila system indicate that p.Arg673Gln, p.Phe734Ser, and p.Arg299Gly are severe loss-of-function alleles, leading to developmental lethality, whereas p.Pro852Ala, p.Ala327Val, p.Trp220Cys, p.Asp491Val, and p.Arg244Trp are hypomorphic alleles, causing behavioral defects. Transcript analysis from fibroblasts obtained from the individual carrying the synonymous variant (c.1464T>C [p.Val488Val]) in family 2 showed that the synonymous variant affects splicing of exon 11 in OGDHL. Human neuronal cells with OGDHL knockout exhibited defects in mitochondrial respiration, indicating the essential role of OGDHL in mitochondrial metabolism in humans. Together, our data establish that the bi-allelic variants in OGDHL are pathogenic, leading to a Mendelian neurodevelopmental disease in humans.

PURPOSE: We aimed to define a novel autosomal recessive neurodevelopmental disorder, characterize its clinical features, and identify the underlying genetic cause for this condition.METHODS: We performed a detailed clinical characterization of 19 individuals from nine unrelated, consanguineous families with a neurodevelopmental disorder. We used genome/exome sequencing approaches, linkage and cosegregation analyses to identify disease-causing variants, and we performed three-dimensional molecular in silico analysis to predict causality of variants where applicable.RESULTS: In all affected individuals who presented with a neurodevelopmental syndrome with progressive microcephaly, seizures, and intellectual disability we identified biallelic disease-causing variants in Protocadherin-gamma-C4 (PCDHGC4). Five variants were predicted to induce premature protein truncation leading to a loss of PCDHGC4 function. The three detected missense variants were located in extracellular cadherin (EC) domains EC5 and EC6 of PCDHGC4, and in silico analysis of the affected residues showed that two of these substitutions were predicted to influence the Ca2+-binding affinity, which is essential for multimerization of the protein, whereas the third missense variant directly influenced the cis-dimerization interface of PCDHGC4.CONCLUSION: We show that biallelic variants in PCDHGC4 are causing a novel autosomal recessive neurodevelopmental disorder and link PCDHGC4 as a member of the clustered PCDH family to a Mendelian disorder in humans.

Dioxygenases are oxidoreductase enzymes with roles in metabolic pathways necessary for aerobic life. 4-hydroxyphenylpyruvate dioxygenase-like protein (HPDL), encoded by HPDL, is an orphan paralogue of 4-hydroxyphenylpyruvate dioxygenase (HPD), an iron-dependent dioxygenase involved in tyrosine catabolism. The function and association of HPDL with human diseases remain unknown.We applied exome sequencing in a cohort of over 10,000 individuals with neurodevelopmental diseases. Effects of HPDL loss were investigated in vitro and in vivo, and through mass spectrometry analysis. Evolutionary analysis was performed to investigate the potential functional separation of HPDL from HPD.We identified biallelic variants in HPDL in eight families displaying recessive inheritance. Knockout mice closely phenocopied humans and showed evidence of apoptosis in multiple cellular lineages within the cerebral cortex. HPDL is a single-exonic gene that likely arose from a retrotransposition event at the base of the tetrapod lineage, and unlike HPD, HPDL is mitochondria-localized. Metabolic profiling of HPDL mutant cells and mice showed no evidence of altered tyrosine metabolites, but rather notable accumulations in other metabolic pathways.The mitochondrial localization, along with its disrupted metabolic profile, suggests HPDL loss in humans links to a unique neurometabolic mitochondrial infantile neurodegenerative condition.

Background Neuronal ceroid lipofuscinosis (NCL) is a hereditary lysosomal storage disease with progressive brain neurodegeneration. Mutations in ceroid lipofuscinosis neuronal protein 5 (CLN5) cause CLN5 disease, a severe condition characterized by seizures, visual failure, motor decline, and progressive cognitive deterioration. This study aimed to identify causative gene variants in Pakistani consanguineous families diagnosed with NCL. Methods After a thorough clinical and neuroradiological characterization, whole exome sequencing (WES) was performed in 3 patients from 2 unrelated families. Segregation analysis was subsequently performed through Sanger sequencing Analysis WES led to the identification of the 2 novel homozygous variants c.925_926del, (p.Leu309AlafsTer4) and c.477 T > C, (p.Cys159Arg). Conclusion In this study, we report two novel CLN5 cases in the Punjab region of Pakistan. Our observations will help clinicians observe and compare common and unique clinical features of NCL patients, further improving our current understanding of NCL., Highlights • Neuronal ceroid lipofuscinosis is a hereditary lysosomal storage disease. • It is characterized by progressive brain neurodegeneration. • WES was performed in 3 patients from 2 unrelated Pakistani families. • WES led to the identification of the 2 novel homozygous variants. • We report a novel CLN5 case in the Punjab region of Pakistan.