Rare de novo gain-of-function missense variants in DOT1L are associated with developmental delay and congenital anomalies.

Misregulation of histone lysine methylation is associated with several human cancers and with human developmental disorders. DOT1L is an evolutionarily conserved gene encoding a lysine methyltransferase (KMT) that methylates histone 3 lysine-79 (H3K79) and was not previously associated with a Mendelian disease in OMIM. We have identified nine unrelated individuals with seven different de novo heterozygous missense variants in DOT1L through the Undiagnosed Disease Network (UDN), the SickKids Complex Care genomics project, and GeneMatcher. All probands had some degree of global developmental delay/intellectual disability, and most had one or more major congenital anomalies. To assess the pathogenicity of the DOT1L variants, functional studies were performed in Drosophila and human cells. The fruit fly DOT1L ortholog, grappa, is expressed in most cells including neurons in the central nervous system. The identified DOT1L variants behave as gain-of-function alleles in flies and lead to increased H3K79 methylation levels in flies and human cells. Our results show that human DOT1L and fly grappa are required for proper development and that de novo heterozygous variants in DOT1L are associated with a Mendelian disease.

An autosomal-dominant childhood-onset disorder associated with pathogenic variants in VCP.

Valosin-containing protein (VCP) is an AAA+ ATPase that plays critical roles in multiple ubiquitin-dependent cellular processes. Dominant pathogenic variants in VCP are associated with adult-onset multisystem proteinopathy (MSP), which manifests as myopathy, bone disease, dementia, and/or motor neuron disease. Through GeneMatcher, we identified 13 unrelated individuals who harbor heterozygous VCP variants (12 de novo and 1 inherited) associated with a childhood-onset disorder characterized by developmental delay, intellectual disability, hypotonia, and macrocephaly. Trio exome sequencing or a multigene panel identified nine missense variants, two in-frame deletions, one frameshift, and one splicing variant. We performed in vitro functional studies and in silico modeling to investigate the impact of these variants on protein function. In contrast to MSP variants, most missense variants had decreased ATPase activity, and one caused hyperactivation. Other variants were predicted to cause haploinsufficiency, suggesting a loss-of-function mechanism. This cohort expands the spectrum of VCP-related disease to include neurodevelopmental disease presenting in childhood.

Bi-allelic variants in INTS11 are associated with a complex neurological disorder.

The Integrator complex is a multi-subunit protein complex that regulates the processing of nascent RNAs transcribed by RNA polymerase II (RNAPII), including small nuclear RNAs, enhancer RNAs, telomeric RNAs, viral RNAs, and protein-coding mRNAs. Integrator subunit 11 (INTS11) is the catalytic subunit that cleaves nascent RNAs, but, to date, mutations in this subunit have not been linked to human disease. Here, we describe 15 individuals from 10 unrelated families with bi-allelic variants in INTS11 who present with global developmental and language delay, intellectual disability, impaired motor development, and brain atrophy. Consistent with human observations, we find that the fly ortholog of INTS11, dIntS11, is essential and expressed in the central nervous systems in a subset of neurons and most glia in larval and adult stages. Using Drosophila as a model, we investigated the effect of seven variants. We found that two (p.Arg17Leu and p.His414Tyr) fail to rescue the lethality of null mutants, indicating that they are strong loss-of-function variants. Furthermore, we found that five variants (p.Gly55Ser, p.Leu138Phe, p.Lys396Glu, p.Val517Met, and p.Ile553Glu) rescue lethality but cause a shortened lifespan and bang sensitivity and affect locomotor activity, indicating that they are partial loss-of-function variants. Altogether, our results provide compelling evidence that integrity of the Integrator RNA endonuclease is critical for brain development.

Recessive PRDM13 mutations cause fatal perinatal brainstem dysfunction with cerebellar hypoplasia and disrupt Purkinje cell differentiation.

Pontocerebellar hypoplasias (PCHs) are congenital disorders characterized by hypoplasia or early atrophy of the cerebellum and brainstem, leading to a very limited motor and cognitive development. Although over 20 genes have been shown to be mutated in PCHs, a large proportion of affected individuals remains undiagnosed. We describe four families with children presenting with severe neonatal brainstem dysfunction and pronounced deficits in cognitive and motor development associated with four different bi-allelic mutations in PRDM13, including homozygous truncating variants in the most severely affected individuals. Brain MRI and fetopathological examination revealed a PCH-like phenotype, associated with major hypoplasia of inferior olive nuclei and dysplasia of the dentate nucleus. Notably, histopathological examinations highlighted a sparse and disorganized Purkinje cell layer in the cerebellum. PRDM13 encodes a transcriptional repressor known to be critical for neuronal subtypes specification in the mouse retina and spinal cord but had not been implicated, so far, in hindbrain development. snRNA-seq data mining and in situ hybridization in humans show that PRDM13 is expressed at early stages in the progenitors of the cerebellar ventricular zone, which gives rise to cerebellar GABAergic neurons, including Purkinje cells. We also show that loss of function of prdm13 in zebrafish leads to a reduction in Purkinje cells numbers and a complete absence of the inferior olive nuclei. Altogether our data identified bi-allelic mutations in PRDM13 as causing a olivopontocerebellar hypoplasia syndrome and suggest that early deregulations of the transcriptional control of neuronal fate specification could contribute to a significant number of cases.

Biallelic EPB41L3 variants underlie a developmental disorder with seizures and myelination defects.

Erythrocyte Membrane Protein Band 4.1 Like 3 (EPB41L3: NM_012307.5), also known as DAL-1, encodes the ubiquitously expressed, neuronally enriched 4.1B protein, part of the 4.1 superfamily of membrane-cytoskeleton adaptors. 4.1B plays key roles in cell spreading, migration, and cytoskeletal scaffolding that support oligodendrocyte axon adhesions essential for proper myelination. We herein describe six individuals from five unrelated families with global developmental delay, intellectual disability, seizures, hypotonia, neuroregression, and delayed myelination. Exome sequencing identified biallelic variants in EPB41L3 in all affected individuals: two nonsense (c.466C>T, p.(R156*); c.2776C>T, p.(R926*)) and three frameshift (c.666delT, p.(F222Lfs*46); c.2289dupC, p.(V764Rfs*19); c.948_949delTG, p.(A317Kfs*33)). Quantitative-real time PCR and Western blot analysis in human fibroblasts harbouring EPB41L3:c.666delT, p.(F222Lfs*46) indicate ablation of EPB41L3 mRNA and 4.1B protein expression. Inhibition of the nonsense mediated decay (NMD) pathway led to an upregulation of EPB41L3:c.666delT transcripts, supporting NMD as a pathogenic mechanism. Epb41l3-deficient mouse oligodendroglia cells showed significant reduction in mRNA expression of key myelin genes, reduced branching, and increased apoptosis. Our report provides the first clinical description of an autosomal recessive disorder associated with variants in EPB41L3, which we refer to as EPB41L3-associated developmental disorder (EADD). Moreover, our functional studies substantiate the pathogenicity of EPB41L3 hypothesized loss-of-function variants.

The expanding clinical and genetic spectrum of DYNC1H1-related disorders.

Intracellular trafficking involves an intricate machinery of motor complexes including the dynein complex to shuttle cargo for autophagolysosomal degradation. Deficiency in dynein axonemal chains as well as cytoplasmic light and intermediate chains have been linked with ciliary dyskinesia and skeletal dysplasia. The cytoplasmic dynein 1 heavy chain protein (DYNC1H1) serves as a core complex for retrograde trafficking in neuronal axons. Dominant pathogenic variants in DYNC1H1 have been previously implicated in peripheral neuromuscular disorders (NMD) and neurodevelopmental disorders (NDD). As heavy-chain dynein is ubiquitously expressed, the apparent selectivity of heavy-chain dyneinopathy for motor neuronal phenotypes remains currently unaccounted for. Here, we aimed to evaluate the full DYNC1H1-related clinical, molecular and imaging spectrum, including multisystem features and novel phenotypes presenting throughout life. We identified 47 cases from 43 families with pathogenic heterozygous variants in DYNC1H1 (aged 0-59 years) and collected phenotypic data via a comprehensive standardized survey and clinical follow-up appointments. Most patients presented with divergent and previously unrecognized neurological and multisystem features, leading to significant delays in genetic testing and establishing the correct diagnosis. Neurological phenotypes include novel autonomic features, previously rarely described behavioral disorders, movement disorders, and periventricular lesions. Sensory neuropathy was identified in nine patients (median age of onset 10.6 years), of which five were only diagnosed after the second decade of life, and three had a progressive age-dependent sensory neuropathy. Novel multisystem features included primary immunodeficiency, bilateral sensorineural hearing loss, organ anomalies, and skeletal manifestations, resembling the phenotypic spectrum of other dyneinopathies. We also identified an age-dependent biphasic disease course with developmental regression in the first decade and, following a period of stability, neurodegenerative progression after the second decade of life. Of note, we observed several cases in whom neurodegeneration appeared to be prompted by intercurrent systemic infections with double-stranded DNA viruses (Herpesviridae) or single-stranded RNA viruses (Ross-River fever, SARS-CoV-2). Moreover, the disease course appeared to be exacerbated by viral infections regardless of age and/or severity of NDD manifestations, indicating a role of dynein in anti-viral immunity and neuronal health. In summary, our findings expand the clinical, imaging, and molecular spectrum of pathogenic DYNC1H1 variants beyond motor neuropathy disorders and suggest a life-long continuum and age-related progression due to deficient intracellular trafficking. This study will facilitate early diagnosis and improve counselling and health surveillance of affected patients.

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.

Refining the phenotype of SINO syndrome: A comprehensive cohort report of 14 novel cases.

PURPOSE: Spastic paraplegia, intellectual disability, nystagmus, and obesity syndrome (SINO) is a rare autosomal dominant condition caused by heterozygous variants in KIDINS220. A total of 12 individuals are reported, comprising 8 with SINO and 4 with an autosomal recessive condition attributed to biallelic KIDINS220 variants. METHODS: In our international cohort, we have included 14 individuals, carrying 13 novel pathogenic KIDINS220 variants in heterozygous form. We assessed the clinical and molecular data of our cohort and previously reported individuals and, based on functional experiments, reached a better understanding of the pathogenesis behind the KIDINS220-related disease. RESULTS: Using fetal tissue and in vitro assays, we demonstrate that the variants generate KIDINS220 truncated forms that mislocalize in punctate intracellular structures, with decreased levels of the full-length protein, suggesting a trans-dominant negative effect. A total of 92% had their diagnosis within 3 years, with symptoms of developmental delay, spasticity, hypotonia, lack of eye contact, and nystagmus. We identified a KIDINS220 variant associated with fetal hydrocephalus and show that 58% of examined individuals present brain ventricular dilatation. We extend the phenotypic spectrum of SINO syndrome to behavioral manifestations not previously highlighted. CONCLUSION: Our study provides further insights into the clinical spectrum, etiology, and predicted functional impact of KIDINS220 variants.

Cerebral dural arteriovenous fistulas in patients with PTEN-related hamartoma tumor syndrome.

Central nervous system (CNS) dural arteriovenous fistulas (DAVF) have been reported in PTEN-related hamartoma tumor syndrome (PHTS). However, PHTS-associated DAVF remain an underexplored field of the PHTS clinical landscape. Here, we studied cases with a PTEN pathogenic variant identified between 2007 and 2020 in our laboratory (n = 58), and for whom brain imaging was available. Two patients had DAVF (2/58, 3.4%), both presenting at advanced stages: a 34-year-old man with a left lateral sinus DAVF at immediate risk of hemorrhage, and a 21-year-old woman with acute intracranial hypertension due to a torcular DAVF. Interestingly, not all patients had 3D TOF/MRA, the optimal sequences to detect DAVF. Early diagnosis of DAVF can be lifesaving, and is easier to treat compared to developed, proliferative, or complex lesions. As a result, one should consider brain MRI with 3D TOF/MRA in PHTS patients at genetic diagnosis, with subsequent surveillance on a case-by-case basis.

Growth charts in DYRK1A syndrome.

DYRK1A Syndrome (OMIM #614104) is caused by pathogenic variations in the DYRK1A gene located on 21q22. Haploinsufficiency of DYRK1A causes a syndrome with global psychomotor delay and intellectual disability. Low birth weight, growth restriction with feeding difficulties, stature insufficiency, and microcephaly are frequently reported. This study aims to create specific growth charts for individuals with DYRK1A Syndrome and identify parameters for size prognosis. Growth parameters were obtained for 92 individuals with DYRK1A Syndrome (49 males vs. 43 females). The data were obtained from pediatric records, parent reporting, and scientific literature. Growth charts for height, weight, body mass index (BMI), and occipitofrontal circumference (OFC) were generated using generalized additive models through R package gamlss. The growth curves include height, weight, and OFC measurements for patients aged 0-5 years. In accordance with the literature, the charts show that individuals are more likely to present intrauterine growth restriction with low birth weight and microcephaly. The growth is then characterized by severe microcephaly, low weight, and short stature. This study proposes growth charts for widespread use in the management of patients with DYRK1A syndrome.

Prenatal diagnosis of pontocerebellar hypoplasia with postnatal follow-up.

OBJECTIVE: To describe the MR features enabling prenatal diagnosis of pontocerebellar hypoplasia (PCH). METHOD: This was a retrospective single monocentre study. The inclusion criteria were decreased cerebellar biometry on dedicated neurosonography and available fetal Magnetic Resonance Imaging (MRI) with PCH diagnosis later confirmed either genetically or clinically on post-natal MRI or by autopsy. The exclusion criteria were non-available MRI and sonographic features suggestive of a known genetic or other pathologic diagnosis. The collected data were biometric or morphological imaging parameters, clinical outcome, termination of pregnancy (TOP), pathological findings and genetic analysis (karyotyping, chromosomal microarray, DNA sequencing targeted or exome). PCH was classified as classic, non-classic, chromosomal, or unknown type. RESULTS: Forty-two fetuses were diagnosed with PCH, of which 27 were referred for decreased transverse cerebellar diameter at screening ultrasound. Neurosonography and fetal MRI were performed at a mean gestational age of 29 + 4 and 31 + 0 weeks, respectively. Termination of pregnancy occurred. Pregnancy was terminated in 24 cases. Neuropathological examination confirmed the diagnosis in 24 cases and genetic testing identified abnormalities in 29 cases (28 families, 14 chromosomal anomaly). Classic PCH is associated with pontine atrophy and small MR measurements decreasing with advancing gestation. CONCLUSION: This is the first large series of prenatally diagnosed PCHs. Our study shows the essential contribution of fetal MRI to the prenatal diagnosis of PCH. Classic PCHs are particularly severe and are associated with certain MR features.

New insights into CC2D2A-related Joubert syndrome.

PURPOSE: In this study, we describe the phenotype and genotype of the largest cohort of patients with Joubert syndrome (JS) carrying pathogenic variants on one of the most frequent causative genes, CC2D2A. METHODS: We selected 53 patients with pathogenic variants on CC2D2A, compiled and analysed their clinical, neuroimaging and genetic information and compared it to previous literature. RESULTS: Developmental delay (motor and language) was nearly constant but patients had normal intellectual efficiency in 74% of cases (20/27 patients) and 68% followed mainstream schooling despite learning difficulties. Epilepsy was found in only 13% of cases. Only three patients had kidney cysts, only three had genuine retinal dystrophy and no subject had liver fibrosis or polydactyly. Brain MRIs showed typical signs of JS with rare additional features. Genotype-phenotype correlation findings demonstrate a homozygous truncating variant p.Arg950* linked to a more severe phenotype. CONCLUSION: This study contradicts previous literature stating an association between CC2D2A-related JS and ventriculomegaly. Our study implies that CC2D2A-related JS is linked to positive neurodevelopmental outcome and low rate of other organ defects except for homozygous pathogenic variant p.Arg950*. This information will help modulate patient follow-up and provide families with accurate genetic counselling.

Evaluation of DNA Methylation Episignatures for Diagnosis and Phenotype Correlations in 42 Mendelian Neurodevelopmental Disorders.

Genetic syndromes frequently present with overlapping clinical features and inconclusive or ambiguous genetic findings which can confound accurate diagnosis and clinical management. An expanding number of genetic syndromes have been shown to have unique genomic DNA methylation patterns (called "episignatures"). Peripheral blood episignatures can be used for diagnostic testing as well as for the interpretation of ambiguous genetic test results. We present here an approach to episignature mapping in 42 genetic syndromes, which has allowed the identification of 34 robust disease-specific episignatures. We examine emerging patterns of overlap, as well as similarities and hierarchical relationships across these episignatures, to highlight their key features as they are related to genetic heterogeneity, dosage effect, unaffected carrier status, and incomplete penetrance. We demonstrate the necessity of multiclass modeling for accurate genetic variant classification and show how disease classification using a single episignature at a time can sometimes lead to classification errors in closely related episignatures. We demonstrate the utility of this tool in resolving ambiguous clinical cases and identification of previously undiagnosed cases through mass screening of a large cohort of subjects with developmental delays and congenital anomalies. This study more than doubles the number of published syndromes with DNA methylation episignatures and, most significantly, opens new avenues for accurate diagnosis and clinical assessment in individuals affected by these disorders.

De Novo Variants in CNOT1, a Central Component of the CCR4-NOT Complex Involved in Gene Expression and RNA and Protein Stability, Cause Neurodevelopmental Delay.

CNOT1 is a member of the CCR4-NOT complex, which is a master regulator, orchestrating gene expression, RNA deadenylation, and protein ubiquitination. We report on 39 individuals with heterozygous de novo CNOT1 variants, including missense, splice site, and nonsense variants, who present with a clinical spectrum of intellectual disability, motor delay, speech delay, seizures, hypotonia, and behavioral problems. To link CNOT1 dysfunction to the neurodevelopmental phenotype observed, we generated variant-specific Drosophila models, which showed learning and memory defects upon CNOT1 knockdown. Introduction of human wild-type CNOT1 was able to rescue this phenotype, whereas mutants could not or only partially, supporting our hypothesis that CNOT1 impairment results in neurodevelopmental delay. Furthermore, the genetic interaction with autism-spectrum genes, such as ASH1L, DYRK1A, MED13, and SHANK3, was impaired in our Drosophila models. Molecular characterization of CNOT1 variants revealed normal CNOT1 expression levels, with both mutant and wild-type alleles expressed at similar levels. Analysis of protein-protein interactions with other members indicated that the CCR4-NOT complex remained intact. An integrated omics approach of patient-derived genomics and transcriptomics data suggested only minimal effects on endonucleolytic nonsense-mediated mRNA decay components, suggesting that de novo CNOT1 variants are likely haploinsufficient hypomorph or neomorph, rather than dominant negative. In summary, we provide strong evidence that de novo CNOT1 variants cause neurodevelopmental delay with a wide range of additional co-morbidities. Whereas the underlying pathophysiological mechanism warrants further analysis, our data demonstrate an essential and central role of the CCR4-NOT complex in human brain development.

Characterization of novel CACNA1A splice variants by RNA-sequencing in patients with episodic or congenital ataxia.

Loss of function variants in CACNA1A cause a broad spectrum of neurological disorders, including episodic ataxia, congenital or progressive ataxias, epileptic manifestations or developmental delay. Variants located on the AG/GT consensus splice sites are usually considered as responsible of splicing defects, but exonic or intronic variants located outside of the consensus splice site can also lead to abnormal splicing. We investigated the putative consequences on splicing of 11 CACNA1A variants of unknown significance (VUS) identified in patients with episodic ataxia or congenital ataxia. In silico splice predictions were performed and RNA obtained from fibroblasts was analyzed by Sanger sequencing. The presence of abnormal transcripts was confirmed in 10/11 patients, nine of them were considered as deleterious and one remained of unknown significance. Targeted next-generation RNA sequencing was done in a second step to compare the two methods. This method was successful to obtain the full cDNA sequence of CACNA1A. Despite the presence of several isoforms in the fibroblastic cells, it detected most of the abnormally spliced transcripts. In conclusion, RNA sequencing was efficient to confirm the pathogenicity of nine novel CACNA1A variants. Sanger or Next generation methods can be used depending on the facilities and organization of the laboratories.

1p36 deletion syndrome: Review and mapping with further characterization of the phenotype, a new cohort of 86 patients.

Chromosome 1p36 deletion syndrome (1p36DS) is one of the most common terminal deletion syndromes (incidence between 1/5000 and 1/10,000 live births in the American population), due to a heterozygous deletion of part of the short arm of chromosome 1. The 1p36DS is characterized by typical craniofacial features, developmental delay/intellectual disability, hypotonia, epilepsy, cardiomyopathy/congenital heart defect, brain abnormalities, hearing loss, eyes/vision problem, and short stature. The aim of our study was to (1) evaluate the incidence of the 1p36DS in the French population compared to 22q11.2 deletion syndrome and trisomy 21; (2) review the postnatal phenotype related to microarray data, compared to previously publish prenatal data. Thanks to a collaboration with the ACLF (Association des Cytogénéticiens de Langue Française), we have collected data of 86 patients constituting, to the best of our knowledge, the second-largest cohort of 1p36DS patients in the literature. We estimated an average of at least 10 cases per year in France. 1p36DS seems to be much less frequent than 22q11.2 deletion syndrome and trisomy 21. Patients presented mainly dysmorphism, microcephaly, developmental delay/intellectual disability, hypotonia, epilepsy, brain malformations, behavioral disorders, cardiomyopathy, or cardiovascular malformations and, pre and/or postnatal growth retardation. Cardiac abnormalities, brain malformations, and epilepsy were more frequent in distal deletions, whereas microcephaly was more common in proximal deletions. Mapping and genotype-phenotype correlation allowed us to identify four critical regions responsible for intellectual disability. This study highlights some phenotypic variability, according to the deletion position, and helps to refine the phenotype of 1p36DS, allowing improved management and follow-up of patients.

Biallelic variants in ZNF526 cause a severe neurodevelopmental disorder with microcephaly, bilateral cataract, epilepsy and simplified gyration.

BACKGROUND: Next-generation sequencing, combined with international pooling of cases, has impressively enhanced the discovery of genes responsible for Mendelian neurodevelopmental disorders, particularly in individuals affected by clinically undiagnosed diseases. To date, biallelic missense variants in ZNF526 gene, encoding a Krüppel-type zinc-finger protein, have been reported in three families with non-syndromic intellectual disability. METHODS: Here, we describe five individuals from four unrelated families with an undiagnosed neurodevelopmental disorder in which we performed exome sequencing, on a combination of trio-based (4 subjects) or single probands (1 subject). RESULTS: We identified five patients from four unrelated families with homozygous ZNF526 variants by whole exome sequencing. Four had variants resulting in truncation of ZNF526; they were affected by severe prenatal and postnatal microcephaly (ranging from -4 SD to -8 SD), profound psychomotor delay, hypertonic-dystonic movements, epilepsy and simplified gyral pattern on MRI. All of them also displayed bilateral progressive cataracts. A fifth patient had a homozygous missense variant and a slightly less severe disorder, with postnatal microcephaly (-2 SD), progressive bilateral cataracts, severe intellectual disability and unremarkable brain MRI.Mutant znf526 zebrafish larvae had notable malformations of the eye and central nervous system, resembling findings seen in the human holoprosencephaly spectrum. CONCLUSION: Our findings support the role of ZNF526 biallelic variants in a complex neurodevelopmental disorder, primarily affecting brain and eyes, resulting in severe microcephaly, simplified gyral pattern, epileptic encephalopathy and bilateral cataracts.

Delineating the genotypic and phenotypic spectrum of HECW2-related neurodevelopmental disorders.

BACKGROUND: Variants in HECW2 have recently been reported to cause a neurodevelopmental disorder with hypotonia, seizures and impaired language; however, only six variants have been reported and the clinical characteristics have only broadly been defined. METHODS: Molecular and clinical data were collected from clinical and research cohorts. Massive parallel sequencing was performed and identified individuals with a HECW2-related neurodevelopmental disorder. RESULTS: We identified 13 novel missense variants in HECW2 in 22 unpublished cases, of which 18 were confirmed to have a de novo variant. In addition, we reviewed the genotypes and phenotypes of previously reported and new cases with HECW2 variants (n=35 cases). All variants identified are missense, and the majority of likely pathogenic and pathogenic variants are located in or near the C-terminal HECT domain (88.2%). We identified several clustered variants and four recurrent variants (p.(Arg1191Gln);p.(Asn1199Lys);p.(Phe1327Ser);p.(Arg1330Trp)). Two variants, (p.(Arg1191Gln);p.(Arg1330Trp)), accounted for 22.9% and 20% of cases, respectively. Clinical characterisation suggests complete penetrance for hypotonia with or without spasticity (100%), developmental delay/intellectual disability (100%) and developmental language disorder (100%). Other common features are behavioural problems (88.9%), vision problems (83.9%), motor coordination/movement (75%) and gastrointestinal issues (70%). Seizures were present in 61.3% of individuals. Genotype-phenotype analysis shows that HECT domain variants are more frequently associated with cortical visual impairment and gastrointestinal issues. Seizures were only observed in individuals with variants in or near the HECT domain. CONCLUSION: We provide a comprehensive review and expansion of the genotypic and phenotypic spectrum of HECW2 disorders, aiding future molecular and clinical diagnosis and management.

Loss of Oxidation Resistance 1, OXR1, Is Associated with an Autosomal-Recessive Neurological Disease with Cerebellar Atrophy and Lysosomal Dysfunction.

We report an early-onset autosomal-recessive neurological disease with cerebellar atrophy and lysosomal dysfunction. We identified bi-allelic loss-of-function (LoF) variants in Oxidative Resistance 1 (OXR1) in five individuals from three families; these individuals presented with a history of severe global developmental delay, current intellectual disability, language delay, cerebellar atrophy, and seizures. While OXR1 is known to play a role in oxidative stress resistance, its molecular functions are not well established. OXR1 contains three conserved domains: LysM, GRAM, and TLDc. The gene encodes at least six transcripts, including some that only consist of the C-terminal TLDc domain. We utilized Drosophila to assess the phenotypes associated with loss of mustard (mtd), the fly homolog of OXR1. Strong LoF mutants exhibit late pupal lethality or pupal eclosion defects. Interestingly, although mtd encodes 26 transcripts, severe LoF and null mutations can be rescued by a single short human OXR1 cDNA that only contains the TLDc domain. Similar rescue is observed with the TLDc domain of NCOA7, another human homolog of mtd. Loss of mtd in neurons leads to massive cell loss, early death, and an accumulation of aberrant lysosomal structures, similar to what we observe in fibroblasts of affected individuals. Our data indicate that mtd and OXR1 are required for proper lysosomal function; this is consistent with observations that NCOA7 is required for lysosomal acidification.

TMX2 Is a Crucial Regulator of Cellular Redox State, and Its Dysfunction Causes Severe Brain Developmental Abnormalities.

The redox state of the neural progenitors regulates physiological processes such as neuronal differentiation and dendritic and axonal growth. The relevance of endoplasmic reticulum (ER)-associated oxidoreductases in these processes is largely unexplored. We describe a severe neurological disorder caused by bi-allelic loss-of-function variants in thioredoxin (TRX)-related transmembrane-2 (TMX2); these variants were detected by exome sequencing in 14 affected individuals from ten unrelated families presenting with congenital microcephaly, cortical polymicrogyria, and other migration disorders. TMX2 encodes one of the five TMX proteins of the protein disulfide isomerase family, hitherto not linked to human developmental brain disease. Our mechanistic studies on protein function show that TMX2 localizes to the ER mitochondria-associated membranes (MAMs), is involved in posttranslational modification and protein folding, and undergoes physical interaction with the MAM-associated and ER folding chaperone calnexin and ER calcium pump SERCA2. These interactions are functionally relevant because TMX2-deficient fibroblasts show decreased mitochondrial respiratory reserve capacity and compensatory increased glycolytic activity. Intriguingly, under basal conditions TMX2 occurs in both reduced and oxidized monomeric form, while it forms a stable dimer under treatment with hydrogen peroxide, recently recognized as a signaling molecule in neural morphogenesis and axonal pathfinding. Exogenous expression of the pathogenic TMX2 variants or of variants with an in vitro mutagenized TRX domain induces a constitutive TMX2 polymerization, mimicking an increased oxidative state. Altogether these data uncover TMX2 as a sensor in the MAM-regulated redox signaling pathway and identify it as a key adaptive regulator of neuronal proliferation, migration, and organization in the developing brain.