Clinical and neurogenetic characterisation of autosomal recessive RBL2-associated progressive neurodevelopmental disorder.

Retinoblastoma (RB) proteins are highly conserved transcriptional regulators that play important roles during development by regulating cell-cycle gene expression. RBL2 dysfunction has been linked to a severe neurodevelopmental disorder. However, to date, clinical features have only been described in six individuals carrying five biallelic predicted loss of function (pLOF) variants. To define the phenotypic effects of RBL2 mutations in detail, we identified and clinically characterized a cohort of 28 patients from 18 families carrying LOF variants in RBL2 , including fourteen new variants that substantially broaden the molecular spectrum. The clinical presentation of affected individuals is characterized by a range of neurological and developmental abnormalities. Global developmental delay and intellectual disability were uniformly observed, ranging from moderate to profound and involving lack of acquisition of key motor and speech milestones in most patients. Frequent features included postnatal microcephaly, infantile hypotonia, aggressive behaviour, stereotypic movements and non-specific dysmorphic features. Common neuroimaging features were cerebral atrophy, white matter volume loss, corpus callosum hypoplasia and cerebellar atrophy. In parallel, we used the fruit fly, Drosophila melanogaster , to investigate how disruption of the conserved RBL2 orthologueue Rbf impacts nervous system function and development. We found that Drosophila Rbf LOF mutants recapitulate several features of patients harboring RBL2 variants, including alterations in the head and brain morphology reminiscent of microcephaly, and perturbed locomotor behaviour. Surprisingly, in addition to its known role in controlling tissue growth during development, we find that continued Rbf expression is also required in fully differentiated post-mitotic neurons for normal locomotion in Drosophila , and that adult-stage neuronal re-expression of Rbf is sufficient to rescue Rbf mutant locomotor defects. Taken together, this study provides a clinical and experimental basis to understand genotype-phenotype correlations in an RBL2 -linked neurodevelopmental disorder and suggests that restoring RBL2 expression through gene therapy approaches may ameliorate aspects of RBL2 LOF patient symptoms.

Biallelic NAA60 variants with impaired n-terminal acetylation capacity cause autosomal recessive primary familial brain calcifications.

Primary familial brain calcification (PFBC) is characterized by calcium deposition in the brain, causing progressive movement disorders, psychiatric symptoms, and cognitive decline. PFBC is a heterogeneous disorder currently linked to variants in six different genes, but most patients remain genetically undiagnosed. Here, we identify biallelic NAA60 variants in ten individuals from seven families with autosomal recessive PFBC. The NAA60 variants lead to loss-of-function with lack of protein N-terminal (Nt)-acetylation activity. We show that the phosphate importer SLC20A2 is a substrate of NAA60 in vitro. In cells, loss of NAA60 caused reduced surface levels of SLC20A2 and a reduction in extracellular phosphate uptake. This study establishes NAA60 as a causal gene for PFBC, provides a possible biochemical explanation of its disease-causing mechanisms and underscores NAA60-mediated Nt-acetylation of transmembrane proteins as a fundamental process for healthy neurobiological functioning.

Zebrafish as a model to investigate a biallelic gain-of-function variant in MSGN1, associated with a novel skeletal dysplasia syndrome.

BACKGROUND/OBJECTIVES: Rare genetic disorders causing specific congenital developmental abnormalities often manifest in single families. Investigation of disease-causing molecular features are most times lacking, although these investigations may open novel therapeutic options for patients. In this study, we aimed to identify the genetic cause in an Iranian patient with severe skeletal dysplasia and to model its molecular function in zebrafish embryos. RESULTS: The proband displays short stature and multiple skeletal abnormalities, including mesomelic dysplasia of the arms with complete humero-radio-ulna synostosis, arched clavicles, pelvic dysplasia, short and thin fibulae, proportionally short vertebrae, hyperlordosis and mild kyphosis. Exome sequencing of the patient revealed a novel homozygous c.374G > T, p.(Arg125Leu) missense variant in MSGN1 (NM_001105569). MSGN1, a basic-Helix-Loop-Helix transcription factor, plays a crucial role in formation of presomitic mesoderm progenitor cells/mesodermal stem cells during early developmental processes in vertebrates. Initial in vitro experiments show protein stability and correct intracellular localization of the novel variant in the nucleus and imply retained transcription factor function. To test the pathogenicity of the detected variant, we overexpressed wild-type and mutant msgn1 mRNA in zebrafish embryos and analyzed tbxta (T/brachyury/ntl). Overexpression of wild-type or mutant msgn1 mRNA significantly reduces tbxta expression in the tailbud compared to control embryos. Mutant msgn1 mRNA injected embryos depict a more severe effect, implying a gain-of-function mechanism. In vivo analysis on embryonic development was performed by clonal msgn1 overexpression in zebrafish embryos further demonstrated altered cell compartments in the presomitic mesoderm, notochord and pectoral fin buds. Detection of ectopic tbx6 and bmp2 expression in these embryos hint to affected downstream signals due to Msgn1 gain-of-function. CONCLUSION: In contrast to loss-of-function effects described in animal knockdown models, gain-of-function of MSGN1 explains the only mildly affected axial skeleton of the proband and rather normal vertebrae. In this context we observed notochord bending and potentially disruption of pectoral fin buds/upper extremity after overexpression of msgn1 in zebrafish embryos. The latter might result from Msgn1 function on mesenchymal stem cells or on chondrogenesis in these regions. In addition, we detected ectopic tbx6 and bmp2a expression after gain of Msgn1 function in zebrafish, which are interconnected to short stature, congenital scoliosis, limb shortening and prominent skeletal malformations in patients. Our findings highlight a rare, so far undescribed skeletal dysplasia syndrome associated with a gain-of-function mutation in MSGN1 and hint to its molecular downstream effectors.

ZSCAN10 deficiency causes a neurodevelopmental disorder with characteristic oto-facial malformations.

Neurodevelopmental disorders are major indications for genetic referral and have been linked to more than 1,500 loci including genes encoding transcriptional regulators. The dysfunction of transcription factors often results in characteristic syndromic presentations, however, at least half of these patients lack a genetic diagnosis. The implementation of machine learning approaches has the potential to aid in the identification of new disease genes and delineate associated phenotypes. Next generation sequencing was performed in seven affected individuals with neurodevelopmental delay and dysmorphic features. Clinical characterization included reanalysis of available neuroimaging datasets and 2D portrait image analysis with GestaltMatcher. The functional consequences of ZSCAN10 loss were modelled in mouse embryonic stem cells (mESC), including a knock-out and a representative ZSCAN10 protein truncating variant. These models were characterized by gene expression and Western blot analyses, chromatin immunoprecipitation and quantitative PCR (ChIP-qPCR), and immunofluorescence staining. Zscan10 knockout mouse embryos were generated and phenotyped. We prioritized bi-allelic ZSCAN10 loss-of-function variants in seven affected individuals from five unrelated families as the underlying molecular cause. RNA-Seq analyses in Zscan10-/- mESCs indicated dysregulation of genes related to stem cell pluripotency. In addition, we established in mESCs the loss-of-function mechanism for a representative human ZSCAN10 protein truncating variant by showing alteration of its expression levels and subcellular localization, interfering with its binding to DNA enhancer targets. Deep phenotyping revealed global developmental delay, facial asymmetry, and malformations of the outer ear as consistent clinical features. Cerebral MRI showed dysplasia of the semicircular canals as an anatomical correlate of sensorineural hearing loss. Facial asymmetry was confirmed as a clinical feature by GestaltMatcher and was recapitulated in the Zscan10 mouse model along with inner and outer ear malformations. Our findings provide evidence of a novel syndromic neurodevelopmental disorder caused by bi-allelic loss-of-function variants in ZSCAN10.

A syndromic neurodevelopmental disorder caused by rare variants in PPFIA3.

PPFIA3 encodes the protein-tyrosine phosphatase, receptor-type, F-polypeptide-interacting-protein-alpha-3 (PPFIA3), which is a member of the LAR-protein-tyrosine phosphatase-interacting-protein (liprin) family involved in synapse formation and function, synaptic vesicle transport, and presynaptic active zone assembly. The protein structure and function are evolutionarily well conserved, but human diseases related to PPFIA3 dysfunction are not yet reported in OMIM. Here, we report 20 individuals with rare PPFIA3 variants (19 heterozygous and 1 compound heterozygous) presenting with developmental delay, intellectual disability, hypotonia, dysmorphisms, microcephaly or macrocephaly, autistic features, and epilepsy with reduced penetrance. Seventeen unique PPFIA3 variants were detected in 18 families. To determine the pathogenicity of PPFIA3 variants in vivo, we generated transgenic fruit flies producing either human wild-type (WT) PPFIA3 or five missense variants using GAL4-UAS targeted gene expression systems. In the fly overexpression assays, we found that the PPFIA3 variants in the region encoding the N-terminal coiled-coil domain exhibited stronger phenotypes compared to those affecting the C-terminal region. In the loss-of-function fly assay, we show that the homozygous loss of fly Liprin-α leads to embryonic lethality. This lethality is partially rescued by the expression of human PPFIA3 WT, suggesting human PPFIA3 function is partially conserved in the fly. However, two of the tested variants failed to rescue the lethality at the larval stage and one variant failed to rescue lethality at the adult stage. Altogether, the human and fruit fly data reveal that the rare PPFIA3 variants are dominant-negative loss-of-function alleles that perturb multiple developmental processes and synapse formation.

Bi-allelic truncating variants in CASP2 underlie a neurodevelopmental disorder with lissencephaly.

Lissencephaly (LIS) is a malformation of cortical development due to deficient neuronal migration and abnormal formation of cerebral convolutions or gyri. Thirty-one LIS-associated genes have been previously described. Recently, biallelic pathogenic variants in CRADD and PIDD1, have associated with LIS impacting the previously established role of the PIDDosome in activating caspase-2. In this report, we describe biallelic truncating variants in CASP2, another subunit of PIDDosome complex. Seven patients from five independent families presenting with a neurodevelopmental phenotype were identified through GeneMatcher-facilitated international collaborations. Exome sequencing analysis was carried out and revealed two distinct novel homozygous (NM_032982.4:c.1156delT (p.Tyr386ThrfsTer25), and c.1174 C > T (p.Gln392Ter)) and compound heterozygous variants (c.[130 C > T];[876 + 1 G > T] p.[Arg44Ter];[?]) in CASP2 segregating within the families in a manner compatible with an autosomal recessive pattern. RNA studies of the c.876 + 1 G > T variant indicated usage of two cryptic splice donor sites, each introducing a premature stop codon. All patients from whom brain MRIs were available had a typical fronto-temporal LIS and pachygyria, remarkably resembling the CRADD and PIDD1-related neuroimaging findings. Other findings included developmental delay, attention deficit hyperactivity disorder, hypotonia, seizure, poor social skills, and autistic traits. In summary, we present patients with CASP2-related ID, anterior-predominant LIS, and pachygyria similar to previously reported patients with CRADD and PIDD1-related disorders, expanding the genetic spectrum of LIS and lending support that each component of the PIDDosome complex is critical for normal development of the human cerebral cortex and brain function.

Hemizygous variants in protein phosphatase 1 regulatory subunit 3F (PPP1R3F) are associated with a neurodevelopmental disorder characterized by developmental delay, intellectual disability and autistic features.

Protein phosphatase 1 regulatory subunit 3F (PPP1R3F) is a member of the glycogen targeting subunits (GTSs), which belong to the large group of regulatory subunits of protein phosphatase 1 (PP1), a major eukaryotic serine/threonine protein phosphatase that regulates diverse cellular processes. Here, we describe the identification of hemizygous variants in PPP1R3F associated with a novel X-linked recessive neurodevelopmental disorder in 13 unrelated individuals. This disorder is characterized by developmental delay, mild intellectual disability, neurobehavioral issues such as autism spectrum disorder, seizures and other neurological findings including tone, gait and cerebellar abnormalities. PPP1R3F variants segregated with disease in affected hemizygous males that inherited the variants from their heterozygous carrier mothers. We show that PPP1R3F is predominantly expressed in brain astrocytes and localizes to the endoplasmic reticulum in cells. Glycogen content in PPP1R3F knockout astrocytoma cells appears to be more sensitive to fluxes in extracellular glucose levels than in wild-type cells, suggesting that PPP1R3F functions in maintaining steady brain glycogen levels under changing glucose conditions. We performed functional studies on nine of the identified variants and observed defects in PP1 binding, protein stability, subcellular localization and regulation of glycogen metabolism in most of them. Collectively, the genetic and molecular data indicate that deleterious variants in PPP1R3F are associated with a new X-linked disorder of glycogen metabolism, highlighting the critical role of GTSs in neurological development. This research expands our understanding of neurodevelopmental disorders and the role of PP1 in brain development and proper function.

Gain and loss of function variants in EZH1 disrupt neurogenesis and cause dominant and recessive neurodevelopmental disorders.

Genetic variants in chromatin regulators are frequently found in neurodevelopmental disorders, but their effect in disease etiology is rarely determined. Here, we uncover and functionally define pathogenic variants in the chromatin modifier EZH1 as the cause of dominant and recessive neurodevelopmental disorders in 19 individuals. EZH1 encodes one of the two alternative histone H3 lysine 27 methyltransferases of the PRC2 complex. Unlike the other PRC2 subunits, which are involved in cancers and developmental syndromes, the implication of EZH1 in human development and disease is largely unknown. Using cellular and biochemical studies, we demonstrate that recessive variants impair EZH1 expression causing loss of function effects, while dominant variants are missense mutations that affect evolutionarily conserved aminoacids, likely impacting EZH1 structure or function. Accordingly, we found increased methyltransferase activity leading to gain of function of two EZH1 missense variants. Furthermore, we show that EZH1 is necessary and sufficient for differentiation of neural progenitor cells in the developing chick embryo neural tube. Finally, using human pluripotent stem cell-derived neural cultures and forebrain organoids, we demonstrate that EZH1 variants perturb cortical neuron differentiation. Overall, our work reveals a critical role of EZH1 in neurogenesis regulation and provides molecular diagnosis for previously undefined neurodevelopmental disorders.

Genotype-phenotype correlations in RHOBTB2-associated neurodevelopmental disorders.

PURPOSE: Missense variants clustering in the BTB domain region of RHOBTB2 cause a developmental and epileptic encephalopathy 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. 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 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. CONCLUSION: By 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.

Broadening the phenotypic and molecular spectrum of FINCA syndrome: Biallelic NHLRC2 variants in 15 novel individuals.

FINCA syndrome [MIM: 618278] is an autosomal recessive multisystem disorder characterized by fibrosis, neurodegeneration and cerebral angiomatosis. To date, 13 patients from nine families with biallelic NHLRC2 variants have been published. In all of them, the recurrent missense variant p.(Asp148Tyr) was detected on at least one allele. Common manifestations included lung or muscle fibrosis, respiratory distress, developmental delay, neuromuscular symptoms and seizures often followed by early death due to rapid disease progression.Here, we present 15 individuals from 12 families with an overlapping phenotype associated with nine novel NHLRC2 variants identified by exome analysis. All patients described here presented with moderate to severe global developmental delay and variable disease progression. Seizures, truncal hypotonia and movement disorders were frequently observed. Notably, we also present the first eight cases in which the recurrent p.(Asp148Tyr) variant was not detected in either homozygous or compound heterozygous state.We cloned and expressed all novel and most previously published non-truncating variants in HEK293-cells. From the results of these functional studies, we propose a potential genotype-phenotype correlation, with a greater reduction in protein expression being associated with a more severe phenotype.Taken together, our findings broaden the known phenotypic and molecular spectrum and emphasize that NHLRC2-related disease should be considered in patients presenting with intellectual disability, movement disorders, neuroregression and epilepsy with or without pulmonary involvement.

AMFR dysfunction causes autosomal recessive spastic paraplegia in human that is amenable to statin treatment in a preclinical model.

Hereditary spastic paraplegias (HSP) are rare, inherited neurodegenerative or neurodevelopmental disorders that mainly present with lower limb spasticity and muscle weakness due to motor neuron dysfunction. Whole genome sequencing identified bi-allelic truncating variants in AMFR, encoding a RING-H2 finger E3 ubiquitin ligase anchored at the membrane of the endoplasmic reticulum (ER), in two previously genetically unexplained HSP-affected siblings. Subsequently, international collaboration recognized additional HSP-affected individuals with similar bi-allelic truncating AMFR variants, resulting in a cohort of 20 individuals from 8 unrelated, consanguineous families. Variants segregated with a phenotype of mainly pure but also complex HSP consisting of global developmental delay, mild intellectual disability, motor dysfunction, and progressive spasticity. Patient-derived fibroblasts, neural stem cells (NSCs), and in vivo zebrafish modeling were used to investigate pathomechanisms, including initial preclinical therapy assessment. The absence of AMFR disturbs lipid homeostasis, causing lipid droplet accumulation in NSCs and patient-derived fibroblasts which is rescued upon AMFR re-expression. Electron microscopy indicates ER morphology alterations in the absence of AMFR. Similar findings are seen in amfra-/- zebrafish larvae, in addition to altered touch-evoked escape response and defects in motor neuron branching, phenocopying the HSP observed in patients. Interestingly, administration of FDA-approved statins improves touch-evoked escape response and motor neuron branching defects in amfra-/- zebrafish larvae, suggesting potential therapeutic implications. Our genetic and functional studies identify bi-allelic truncating variants in AMFR as a cause of a novel autosomal recessive HSP by altering lipid metabolism, which may potentially be therapeutically modulated using precision medicine with statins.

The prevalence and phenotypic range associated with biallelic PKDCC variants.

PKDCC encodes a component of Hedgehog signalling required for normal chondrogenesis and skeletal development. Although biallelic PKDCC variants have been implicated in rhizomelic shortening of limbs with variable dysmorphic features, this association was based on just two patients. In this study, data from the 100 000 Genomes Project was used in conjunction with exome sequencing and panel-testing results accessed via international collaboration to assemble a cohort of eight individuals from seven independent families with biallelic PKDCC variants. The allelic series included six frameshifts, a previously described splice-donor site variant and a likely pathogenic missense variant observed in two families that was supported by in silico structural modelling. Database queries suggested that the prevalence of this condition is between 1 of 127 and 1 of 721 in clinical cohorts with skeletal dysplasia of unknown aetiology. Clinical assessments, combined with data from previously published cases, indicate a predominantly upper limb involvement. Micrognathia, hypertelorism and hearing loss appear to be commonly co-occurring features. In conclusion, this study strengthens the link between biallelic inactivation of PKDCC and rhizomelic limb-shortening and will enable clinical testing laboratories to better interpret variants in this gene.

Biallelic Variants in the Ectonucleotidase ENTPD1 Cause a Complex Neurodevelopmental Disorder with Intellectual Disability, Distinct White Matter Abnormalities, and Spastic Paraplegia.

OBJECTIVE: Human genomics established that pathogenic variation in diverse genes can underlie a single disorder. For example, hereditary spastic paraplegia is associated with >80 genes, with frequently only few affected individuals described for each gene. Herein, we characterize a large cohort of individuals with biallelic variation in ENTPD1, a gene previously linked to spastic paraplegia 64 (Mendelian Inheritance in Man # 615683). METHODS: Individuals with biallelic ENTPD1 variants were recruited worldwide. Deep phenotyping and molecular characterization were performed. RESULTS: A total of 27 individuals from 17 unrelated families were studied; additional phenotypic information was collected from published cases. Twelve novel pathogenic ENTPD1 variants are described (NM 001776.6): c.398_399delinsAA; p.(Gly133Glu), c.540del; p.(Thr181Leufs*18), c.640del; p.(Gly216Glufs*75), c.185 T > G; p.(Leu62*), c.1531 T > C; p.(*511Glnext*100), c.967C > T; p.(Gln323*), c.414-2_414-1del, and c.146 A > G; p.(Tyr49Cys) including 4 recurrent variants c.1109 T > A; p.(Leu370*), c.574-6_574-3del, c.770_771del; p.(Gly257Glufs*18), and c.1041del; p.(Ile348Phefs*19). Shared disease traits include childhood onset, progressive spastic paraplegia, intellectual disability (ID), dysarthria, and white matter abnormalities. In vitro assays demonstrate that ENTPD1 expression and function are impaired and that c.574-6_574-3del causes exon skipping. Global metabolomics demonstrate ENTPD1 deficiency leads to impaired nucleotide, lipid, and energy metabolism. INTERPRETATION: The ENTPD1 locus trait consists of childhood disease onset, ID, progressive spastic paraparesis, dysarthria, dysmorphisms, and white matter abnormalities, with some individuals showing neurocognitive regression. Investigation of an allelic series of ENTPD1 (1) expands previously described features of ENTPD1-related neurological disease, (2) highlights the importance of genotype-driven deep phenotyping, (3) documents the need for global collaborative efforts to characterize rare autosomal recessive disease traits, and (4) provides insights into disease trait neurobiology. ANN NEUROL 2022;92:304-321.

Biallelic ADAM22 pathogenic variants cause progressive encephalopathy and infantile-onset refractory epilepsy.

Pathogenic variants in A Disintegrin And Metalloproteinase (ADAM) 22, the postsynaptic cell membrane receptor for the glycoprotein leucine-rich repeat glioma-inactivated protein 1 (LGI1), have been recently associated with recessive developmental and epileptic encephalopathy. However, so far, only two affected individuals have been described and many features of this disorder are unknown. We refine the phenotype and report 19 additional individuals harbouring compound heterozygous or homozygous inactivating ADAM22 variants, of whom 18 had clinical data available. Additionally, we provide follow-up data from two previously reported cases. All affected individuals exhibited infantile-onset, treatment-resistant epilepsy. Additional clinical features included moderate to profound global developmental delay/intellectual disability (20/20), hypotonia (12/20) and delayed motor development (19/20). Brain MRI findings included cerebral atrophy (13/20), supported by post-mortem histological examination in patient-derived brain tissue, cerebellar vermis atrophy (5/20), and callosal hypoplasia (4/20). Functional studies in transfected cell lines confirmed the deleteriousness of all identified variants and indicated at least three distinct pathological mechanisms: (i) defective cell membrane expression; (ii) impaired LGI1-binding; and/or (iii) impaired interaction with the postsynaptic density protein PSD-95. We reveal novel clinical and molecular hallmarks of ADAM22 deficiency and provide knowledge that might inform clinical management and early diagnostics.

Bi-allelic loss-of-function variants in BCAS3 cause a syndromic neurodevelopmental disorder.

BCAS3 microtubule-associated cell migration factor (BCAS3) is a large, highly conserved cytoskeletal protein previously proposed to be critical in angiogenesis and implicated in human embryogenesis and tumorigenesis. Here, we established BCAS3 loss-of-function variants as causative for a neurodevelopmental disorder. We report 15 individuals from eight unrelated families with germline bi-allelic loss-of-function variants in BCAS3. All probands share a global developmental delay accompanied by pyramidal tract involvement, microcephaly, short stature, strabismus, dysmorphic facial features, and seizures. The human phenotype is less severe compared with the Bcas3 knockout mouse model and cannot be explained by angiogenic defects alone. Consistent with being loss-of-function alleles, we observed absence of BCAS3 in probands' primary fibroblasts. By comparing the transcriptomic and proteomic data based on probands' fibroblasts with those of the knockout mouse model, we identified similar dysregulated pathways resulting from over-representation analysis, while the dysregulation of some proposed key interactors could not be confirmed. Together with the results from a tissue-specific Drosophila loss-of-function model, we demonstrate a vital role for BCAS3 in neural tissue development.

Biallelic loss-of-function variants in NEMF cause central nervous system impairment and axonal polyneuropathy.

We aimed to detect the causative gene in five unrelated families with recessive inheritance pattern neurological disorders involving the central nervous system, and the potential function of the NEMF gene in the central nervous system. Exome sequencing (ES) was applied to all families and linkage analysis was performed on family 1. A minigene assay was used to validate the splicing effect of the relevant discovered variants. Immunofluorescence (IF) experiment was performed to investigate the role of the causative gene in neuron development. The large consanguineous family confirms the phenotype-causative relationship with homozygous frameshift variant (NM_004713.6:c.2618del) as revealed by ES. Linkage analysis of the family showed a significant single-point LOD of 4.5 locus. Through collaboration in GeneMatcher, four additional unrelated families' likely pathogenic NEMF variants for a spectrum of central neurological disorders, two homozygous splice-site variants (NM_004713.6:c.574+1G>T and NM_004713.6:c.807-2A>C) and a homozygous frameshift variant (NM_004713.6: c.1234_1235insC) were subsequently identified and segregated with all affected individuals. We further revealed that knockdown (KD) of Nemf leads to impairment of axonal outgrowth and synapse development in cultured mouse primary cortical neurons. Our study demonstrates that disease-causing biallelic NEMF variants result in central nervous system impairment and other variable features. NEMF is an important player in mammalian neuron development.

Leukoencephalopathy with calcifications and cysts: Genetic and phenotypic spectrum.

Biallelic mutations in SNORD118, encoding the small nucleolar RNA U8, cause leukoencephalopathy with calcifications and cysts (LCC). Given the difficulty in interpreting the functional consequences of variants in nonprotein encoding genes, and the high allelic polymorphism across SNORD118 in controls, we set out to provide a description of the molecular pathology and clinical spectrum observed in a cohort of patients with LCC. We identified 64 affected individuals from 56 families. Age at presentation varied from 3 weeks to 67 years, with disease onset after age 40 years in eight patients. Ten patients had died. We recorded 44 distinct, likely pathogenic, variants in SNORD118. Fifty two of 56 probands were compound heterozygotes, with parental consanguinity reported in only three families. Forty nine of 56 probands were either heterozygous (46) or homozygous (three) for a mutation involving one of seven nucleotides that facilitate a novel intramolecular interaction between the 5' end and 3' extension of precursor-U8. There was no obvious genotype-phenotype correlation to explain the marked variability in age at onset. Complementing recently published functional analyses in a zebrafish model, these data suggest that LCC most often occurs due to combinatorial severe and milder mutations, with the latter mostly affecting 3' end processing of precursor-U8.

Biallelic variants in ADARB1, encoding a dsRNA-specific adenosine deaminase, cause a severe developmental and epileptic encephalopathy.

BACKGROUND: Adenosine-to-inosine RNA editing is a co-transcriptional/post-transcriptional modification of double-stranded RNA, catalysed by one of two active adenosine deaminases acting on RNA (ADARs), ADAR1 and ADAR2. ADARB1 encodes the enzyme ADAR2 that is highly expressed in the brain and essential to modulate the function of glutamate and serotonin receptors. Impaired ADAR2 editing causes early onset progressive epilepsy and premature death in mice. In humans, ADAR2 dysfunction has been very recently linked to a neurodevelopmental disorder with microcephaly and epilepsy in four unrelated subjects. METHODS: We studied three children from two consanguineous families with severe developmental and epileptic encephalopathy (DEE) through detailed physical and instrumental examinations. Exome sequencing (ES) was used to identify ADARB1 mutations as the underlying genetic cause and in vitro assays with transiently transfected cells were performed to ascertain the impact on ADAR2 enzymatic activity and splicing. RESULTS: All patients showed global developmental delay, intractable early infantile-onset seizures, microcephaly, severe-to-profound intellectual disability, axial hypotonia and progressive appendicular spasticity. ES revealed the novel missense c.1889G>A, p.(Arg630Gln) and deletion c.1245_1247+1 del, p.(Leu415PhefsTer14) variants in ADARB1 (NM_015833.4). The p.(Leu415PhefsTer14) variant leads to incorrect splicing resulting in frameshift with a premature stop codon and loss of enzyme function. In vitro RNA editing assays showed that the p.(Arg630Gln) variant resulted in a severe impairment of ADAR2 enzymatic activity. CONCLUSION: In conclusion, these data support the pathogenic role of biallelic ADARB1 variants as the cause of a distinctive form of DEE, reinforcing the importance of RNA editing in brain function and development.

Pontocerebellar hypoplasia due to bi-allelic variants in MINPP1.

Pontocerebellar hypoplasia (PCH) describes a group of rare heterogeneous neurodegenerative diseases with prenatal onset. Here we describe eight children with PCH from four unrelated families harboring the homozygous MINPP1 (NM_004897.4) variants; c.75_94del, p.(Leu27Argfs*39), c.851 C > A, p.(Ala284Asp), c.1210 C > T, p.(Arg404*), and c.992 T > G, p.(Ile331Ser). The homozygous p.(Leu27Argfs*39) change is predicted to result in a complete absence of MINPP1. The p.(Arg404*) would likely lead to a nonsense mediated decay, or alternatively, a loss of several secondary structure elements impairing protein folding. The missense p.(Ala284Asp) affects a buried, hydrophobic residue within the globular domain. The introduction of aspartic acid is energetically highly unfavorable and therefore predicted to cause a significant reduction in protein stability. The missense p.(Ile331Ser) affects the tight hydrophobic interactions of the isoleucine by the disruption of the polar side chain of serine, destabilizing the structure of MINPP1. The overlap of the above-mentioned genotypes and phenotypes is highly improbable by chance. MINPP1 is the only enzyme that hydrolyses inositol phosphates in the endoplasmic reticulum lumen and several studies support its role in stress induced apoptosis. The pathomechanism explaining the disease mechanism remains unknown, however several others genes of the inositol phosphatase metabolism (e.g., INPP5K, FIG4, INPP5E, ITPR1) are correlated with phenotypes of neurodevelopmental disorders. Taken together, we present MINPP1 as a novel autosomal recessive pontocerebellar hypoplasia gene.

SCUBE3 loss-of-function causes a recognizable recessive developmental disorder due to defective bone morphogenetic protein signaling.

Signal peptide-CUB-EGF domain-containing protein 3 (SCUBE3) is a member of a small family of multifunctional cell surface-anchored glycoproteins functioning as co-receptors for a variety of growth factors. Here we report that bi-allelic inactivating variants in SCUBE3 have pleiotropic consequences on development and cause a previously unrecognized syndromic disorder. Eighteen affected individuals from nine unrelated families showed a consistent phenotype characterized by reduced growth, skeletal features, distinctive craniofacial appearance, and dental anomalies. In vitro functional validation studies demonstrated a variable impact of disease-causing variants on transcript processing, protein secretion and function, and their dysregulating effect on bone morphogenetic protein (BMP) signaling. We show that SCUBE3 acts as a BMP2/BMP4 co-receptor, recruits the BMP receptor complexes into raft microdomains, and positively modulates signaling possibly by augmenting the specific interactions between BMPs and BMP type I receptors. Scube3-/- mice showed craniofacial and dental defects, reduced body size, and defective endochondral bone growth due to impaired BMP-mediated chondrogenesis and osteogenesis, recapitulating the human disorder. Our findings identify a human disease caused by defective function of a member of the SCUBE family, and link SCUBE3 to processes controlling growth, morphogenesis, and bone and teeth development through modulation of BMP signaling.