De novo missense variants in FBXO11 alter its protein expression and subcellular localization.

Recently, others and we identified de novo FBXO11 (F-Box only protein 11) variants as causative for a variable neurodevelopmental disorder (NDD). We now assembled clinical and mutational information on 23 additional individuals. The phenotypic spectrum remains highly variable, with developmental delay and/or intellectual disability as the core feature and behavioral anomalies, hypotonia and various facial dysmorphism as frequent aspects. The mutational spectrum includes intragenic deletions, likely gene disrupting and missense variants distributed across the protein. To further characterize the functional consequences of FBXO11 missense variants, we analyzed their effects on protein expression and localization by overexpression of 17 different mutant constructs in HEK293 and HeLa cells. We found that the majority of missense variants resulted in subcellular mislocalization and/or reduced FBXO11 protein expression levels. For instance, variants located in the nuclear localization signal and the N-terminal F-Box domain lead to altered subcellular localization with exclusion from the nucleus or the formation of cytoplasmic aggregates and to reduced protein levels in western blot. In contrast, variants localized in the C-terminal Zn-finger UBR domain lead to an accumulation in the cytoplasm without alteration of protein levels. Together with the mutational data, our functional results suggest that most missense variants likely lead to a loss of the original FBXO11 function and thereby highlight haploinsufficiency as the most likely disease mechanism for FBXO11-associated NDDs.

Detailed Analysis of ITPR1 Missense Variants Guides Diagnostics and Therapeutic Design.

BACKGROUND: The ITPR1 gene encodes the inositol 1,4,5-trisphosphate (IP3 ) receptor type 1 (IP3 R1), a critical player in cerebellar intracellular calcium signaling. Pathogenic missense variants in ITPR1 cause congenital spinocerebellar ataxia type 29 (SCA29), Gillespie syndrome (GLSP), and severe pontine/cerebellar hypoplasia. The pathophysiological basis of the different phenotypes is poorly understood. OBJECTIVES: We aimed to identify novel SCA29 and GLSP cases to define core phenotypes, describe the spectrum of missense variation across ITPR1, standardize the ITPR1 variant nomenclature, and investigate disease progression in relation to cerebellar atrophy. METHODS: Cases were identified using next-generation sequencing through the Deciphering Developmental Disorders study, the 100,000 Genomes project, and clinical collaborations. ITPR1 alternative splicing in the human cerebellum was investigated by quantitative polymerase chain reaction. RESULTS: We report the largest, multinational case series of 46 patients with 28 unique ITPR1 missense variants. Variants clustered in functional domains of the protein, especially in the N-terminal IP3 -binding domain, the carbonic anhydrase 8 (CA8)-binding region, and the C-terminal transmembrane channel domain. Variants outside these domains were of questionable clinical significance. Standardized transcript annotation, based on our ITPR1 transcript expression data, greatly facilitated analysis. Genotype-phenotype associations were highly variable. Importantly, while cerebellar atrophy was common, cerebellar volume loss did not correlate with symptom progression. CONCLUSIONS: This dataset represents the largest cohort of patients with ITPR1 missense variants, expanding the clinical spectrum of SCA29 and GLSP. Standardized transcript annotation is essential for future reporting. Our findings will aid in diagnostic interpretation in the clinic and guide selection of variants for preclinical studies. © 2023 The Authors. Movement Disorders published by Wiley Periodicals LLC on behalf of International Parkinson and Movement Disorder Society.

'Werner Syndrome foot'-A case series of four Irish Traveller siblings with Werner Syndrome, diabetes mellitus and complex foot disease.

AIMSWERNER SYNDROME IS A RARE PREMATURE AGEING AUTOSOMAL RECESSIVE DISORDER CAUSED BY PATHOGENIC VARIANTS IN THE WRN GENE. PEOPLE WITH WERNER SYNDROME MAY DEVELOP DIABETES MELLITUS. CHRONIC FOOT ULCERATION IS SEEN, WITH SOME CHARACTERISTICS OVERLAPPING WITH DIABETIC FOOT DISEASE. HOWEVER, THE CLINICAL COURSE OF THE ULCERATION IS ATYPICAL OF DIABETIC FOOT DISEASE. WE PRESENT FOUR SIBLINGS FROM AN IRISH TRAVELLER FAMILY WITH WERNER SYNDROME TO HIGHLIGHT THE COMPLEXITY OF THIS CONDITION. THE IRISH TRAVELLER POPULATION ARE AN INDIGENOUS, ENDOGAMOUS POPULATION IN WHICH CONSANGUINITY IS COMMON. AS A RESULT, RARE AUTOSOMAL RECESSIVE DISORDERS ARE PREVALENT AMONG THIS POPULATION: . METHODS: We describe our experience managing the complex foot disease seen in all four siblings. Foot complications present in the siblings include painful peripheral neuropathy, chronic foor ulceration, underlying osteomyelitis and acral melanoma. RESULTS: The cases are described individually, with a particular focus on the complex foot disease associated with the condition. CONCLUSIONS: Although the siblings attend a diabetic foot clinic, we suggest that the combination of clinical features seen in these cases is unique to Werner syndrome and warrants the title 'Werner Syndrome' (rather than 'Diabetic') foot.

Gain-of-function and loss-of-function variants in GRIA3 lead to distinct neurodevelopmental phenotypes.

AMPA (α-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid) receptors (AMPARs) mediate fast excitatory neurotransmission in the brain. AMPARs form by homo- or heteromeric assembly of subunits encoded by the GRIA1-GRIA4 genes, of which only GRIA3 is X-chromosomal. Increasing numbers of GRIA3 missense variants are reported in patients with neurodevelopmental disorders (NDD), but only a few have been examined functionally. Here, we evaluated the impact on AMPAR function of one frameshift and 43 rare missense GRIA3 variants identified in patients with NDD by electrophysiological assays. Thirty-one variants alter receptor function and show loss-of-function (LoF) or gain-of-function (GoF) properties, whereas 13 appeared neutral. We collected detailed clinical data from 25 patients (from 23 families) harbouring 17 of these variants. All patients had global developmental impairment, mostly moderate (9/25) or severe (12/25). Twelve patients had seizures, including focal motor (6/12), unknown onset motor (4/12), focal impaired awareness (1/12), (atypical) absence (2/12), myoclonic (5/12), and generalized tonic-clonic (1/12) or atonic (1/12) seizures. The epilepsy syndrome was classified as developmental and epileptic encephalopathy in eight patients, developmental encephalopathy without seizures in 13 patients, and intellectual disability with epilepsy in four patients. Limb muscular hypotonia was reported in 13/25, and hypertonia in 10/25. Movement disorders were reported in 14/25, with hyperekplexia or non-epileptic erratic myoclonus being the most prevalent feature (8/25). Correlating receptor functional phenotype with clinical features revealed clinical features for GRIA3-associated NDDs and distinct NDD phenotypes for LoF and GoF variants. GoF variants were associated with more severe outcomes: patients were younger at the time of seizure onset (median age one month), hypertonic, and more often had movement disorders, including hyperekplexia. Patients with LoF variants were older at the time of seizure onset (median age 16 months), hypotonic, and had sleeping disturbances. LoF and GoF variants were disease-causing in both sexes but affected males often carried de novo or hemizygous LoF variants inherited from healthy mothers, whereas all but one affected females had de novo heterozygous GoF variants.

DNA Methylation Signature for EZH2 Functionally Classifies Sequence Variants in Three PRC2 Complex Genes.

Weaver syndrome (WS), an overgrowth/intellectual disability syndrome (OGID), is caused by pathogenic variants in the histone methyltransferase EZH2, which encodes a core component of the Polycomb repressive complex-2 (PRC2). Using genome-wide DNA methylation (DNAm) data for 187 individuals with OGID and 969 control subjects, we show that pathogenic variants in EZH2 generate a highly specific and sensitive DNAm signature reflecting the phenotype of WS. This signature can be used to distinguish loss-of-function from gain-of-function missense variants and to detect somatic mosaicism. We also show that the signature can accurately classify sequence variants in EED and SUZ12, which encode two other core components of PRC2, and predict the presence of pathogenic variants in undiagnosed individuals with OGID. The discovery of a functionally relevant signature with utility for diagnostic classification of sequence variants in EZH2, EED, and SUZ12 supports the emerging paradigm shift for implementation of DNAm signatures into diagnostics and translational research.

BICRA, a SWI/SNF Complex Member, Is Associated with BAF-Disorder Related Phenotypes in Humans and Model Organisms.

SWI/SNF-related intellectual disability disorders (SSRIDDs) are rare neurodevelopmental disorders characterized by developmental disability, coarse facial features, and fifth digit/nail hypoplasia that are caused by pathogenic variants in genes that encode for members of the SWI/SNF (or BAF) family of chromatin remodeling complexes. We have identified 12 individuals with rare variants (10 loss-of-function, 2 missense) in the BICRA (BRD4 interacting chromatin remodeling complex-associated protein) gene, also known as GLTSCR1, which encodes a subunit of the non-canonical BAF (ncBAF) complex. These individuals exhibited neurodevelopmental phenotypes that include developmental delay, intellectual disability, autism spectrum disorder, and behavioral abnormalities as well as dysmorphic features. Notably, the majority of individuals lack the fifth digit/nail hypoplasia phenotype, a hallmark of most SSRIDDs. To confirm the role of BICRA in the development of these phenotypes, we performed functional characterization of the zebrafish and Drosophila orthologs of BICRA. In zebrafish, a mutation of bicra that mimics one of the loss-of-function variants leads to craniofacial defects possibly akin to the dysmorphic facial features seen in individuals harboring putatively pathogenic BICRA variants. We further show that Bicra physically binds to other non-canonical ncBAF complex members, including the BRD9/7 ortholog, CG7154, and is the defining member of the ncBAF complex in flies. Like other SWI/SNF complex members, loss of Bicra function in flies acts as a dominant enhancer of position effect variegation but in a more context-specific manner. We conclude that haploinsufficiency of BICRA leads to a unique SSRIDD in humans whose phenotypes overlap with those previously reported.

ATP2B2 de novo variants as a cause of variable neurodevelopmental disorders that feature dystonia, ataxia, intellectual disability, behavioral symptoms, and seizures.

PURPOSE: ATP2B2 encodes the variant-constrained plasma-membrane calcium-transporting ATPase-2, expressed in sensory ear cells and specialized neurons. ATP2B2/Atp2b2 variants were previously linked to isolated hearing loss in patients and neurodevelopmental deficits with ataxia in mice. We aimed to establish the association between ATP2B2 and human neurological disorders. METHODS: Multinational case recruitment, scrutiny of trio-based genomics data, in silico analyses, and functional variant characterization were performed. RESULTS: We assembled 7 individuals harboring rare, predicted deleterious heterozygous ATP2B2 variants. The alleles comprised 5 missense substitutions that affected evolutionarily conserved sites and 2 frameshift variants in the penultimate exon. For 6 variants, a de novo status was confirmed. Unlike described patients with hearing loss, the individuals displayed a spectrum of neurological abnormalities, ranging from ataxia with dystonic features to complex neurodevelopmental manifestations with intellectual disability, autism, and seizures. Two cases with recurrent amino-acid variation showed distinctive overlap with cerebellar atrophy-associated ataxia and epilepsy. In cell-based studies, all variants caused significant alterations in cytosolic calcium handling with both loss- and gain-of-function effects. CONCLUSION: Presentations in our series recapitulate key phenotypic aspects of Atp2b2-mouse models and underline the importance of precise calcium regulation for neurodevelopment and cerebellar function. Our study documents a role for ATP2B2 variants in causing heterogeneous neurodevelopmental and movement-disorder syndromes.

Biallelic variants in PIGN cause Fryns syndrome, multiple congenital anomalies-hypotonia-seizures syndrome, and neurologic phenotypes: A genotype-phenotype correlation study.

PURPOSE: Biallelic PIGN variants have been described in Fryns syndrome, multiple congenital anomalies-hypotonia-seizure syndrome (MCAHS), and neurologic phenotypes. The full spectrum of clinical manifestations in relation to the genotypes is yet to be reported. METHODS: Genotype and phenotype data were collated and analyzed for 61 biallelic PIGN cases: 21 new and 40 previously published cases. Functional analysis was performed for 2 recurrent variants (c.2679C>G p.Ser893Arg and c.932T>G p.Leu311Trp). RESULTS: Biallelic-truncating variants were detected in 16 patients-10 with Fryns syndrome, 1 with MCAHS1, 2 with Fryns syndrome/MCAHS1, and 3 with neurologic phenotype. There was an increased risk of prenatal or neonatal death within this group (6 deaths were in utero or within 2 months of life; 6 pregnancies were terminated). Incidence of polyhydramnios, congenital anomalies (eg, diaphragmatic hernia), and dysmorphism was significantly increased. Biallelic missense or mixed genotype were reported in the remaining 45 cases-32 showed a neurologic phenotype and 12 had MCAHS1. No cases of diaphragmatic hernia or abdominal wall defects were seen in this group except patient 1 in which we found the missense variant p.Ser893Arg to result in functionally null alleles, suggesting the possibility of an undescribed functionally important region in the final exon. For all genotypes, there was complete penetrance for developmental delay and near-complete penetrance for seizures and hypotonia in patients surviving the neonatal period. CONCLUSION: We have expanded the described spectrum of phenotypes and natural history associated with biallelic PIGN variants. Our study shows that biallelic-truncating variants usually result in the more severe Fryns syndrome phenotype, but neurologic problems, such as developmental delay, seizures, and hypotonia, present across all genotypes. Functional analysis should be considered when the genotypes do not correlate with the predicted phenotype because there may be other functionally important regions in PIGN that are yet to be discovered.

A novel report of a fertile female with partial Y chromosome gain completing a healthy pregnancy.

We present a female patient with a complex sex chromosomal rearrangement [GRCh38] Xp22.33(10701_981101)x1,Yq11.221q11.23(13948013_26483746)x1 who conceived spontaneously and carried a healthy pregnancy to term. The patient presented with extreme short stature (more than 4SD below expected) and a bilateral Madelung deformity suggesting a possible SHOX deletion. The patient was otherwise medically well. This patient's short stature was found to be a result of a complex chromosome rearrangement involving a partial X chromosome deletion, which included the SHOX gene and a gain of Y chromosomal material. The Y chromosome material did not contain the SRY gene locus. This is the first recorded case to date of this rearrangement in a female who spontaneously conceived which resulted in a live birth. This patient had normal external and internal anatomy and normal endocrine evaluation with normal puberty. X-inactivation studies revealed no evidence of skewed inactivation.

Triplications of chromosome 1p36.3, including the genes GABRD and SKI, are associated with a developmental disorder and a facial gestalt.

Triplication of chromosomal region 1p36.3 is a rare genomic rearrangement. In this report, we delineate the phenotypic spectrum associated with 1p36.3 triplications. We describe four patients with microtriplications of variable size, but with a strong phenotypic overlap, and compare them to previously described patients with an isolated triplication or duplication of this region. The 1p36.3 triplication syndrome is associated with a distinct phenotype, characterized by global developmental delay, moderate intellectual disability, seizures, behavioral problems, and specific facial dysmorphic features, including ptosis, hypertelorism, and arched eyebrows. The de novo occurrence of these microtriplications demonstrates the reduced reproductive fitness associated with this genotype, in contrast to 1p36.3 duplications which are mostly inherited and can be associated with similar facial features but with a less severe developmental phenotype. The shared triplicated region encompasses four disease-related genes of which GABRD and SKI are most likely to contribute to the phenotype.

Familial Bainbridge-Ropers syndrome: Report of familial ASXL3 inheritance and a milder phenotype.

De novo truncating and splicing pathogenic variants in the Additional Sex Combs-Like 3 (ASXL3) gene are known to cause neurodevelopmental delay, intellectual disability, behavioral difficulties, hypotonia, feeding problems and characteristic facial features. We previously reported 45 patients with ASXL3-related disorder including three individuals with a familial variant. Here we report the detailed clinical and molecular characteristics of these three families with inherited ASXL3-related disorder. First, a father and son with c.2791_2792del p.Gln931fs pathogenic variant. The second, a mother, daughter and son with c.4534C > T, p.Gln1512Ter pathogenic variant. The third, a mother and her daughter with c.4441dup, p.Leu1481fs maternally inherited pathogenic variant. This report demonstrates intrafamilial phenotypic heterogeneity and confirms heritability of ASXL3-related disorder.

Pathogenicity and selective constraint on variation near splice sites.

Mutations that perturb normal pre-mRNA splicing are significant contributors to human disease. We used exome sequencing data from 7833 probands with developmental disorders (DDs) and their unaffected parents, as well as more than 60,000 aggregated exomes from the Exome Aggregation Consortium, to investigate selection around the splice sites and quantify the contribution of splicing mutations to DDs. Patterns of purifying selection, a deficit of variants in highly constrained genes in healthy subjects, and excess de novo mutations in patients highlighted particular positions within and around the consensus splice site of greater functional relevance. By using mutational burden analyses in this large cohort of proband-parent trios, we could estimate in an unbiased manner the relative contributions of mutations at canonical dinucleotides (73%) and flanking noncanonical positions (27%), and calculate the positive predictive value of pathogenicity for different classes of mutations. We identified 18 patients with likely diagnostic de novo mutations in dominant DD-associated genes at noncanonical positions in splice sites. We estimate 35%-40% of pathogenic variants in noncanonical splice site positions are missing from public databases.

De Novo ZMYND8 variants result in an autosomal dominant neurodevelopmental disorder with cardiac malformations.

PURPOSE: ZMYND8 encodes a multidomain protein that serves as a central interactive hub for coordinating critical roles in transcription regulation, chromatin remodeling, regulation of super-enhancers, DNA damage response and tumor suppression. We delineate a novel neurocognitive disorder caused by variants in the ZMYND8 gene. METHODS: An international collaboration, exome sequencing, molecular modeling, yeast two-hybrid assays, analysis of available transcriptomic data and a knockdown Drosophila model were used to characterize the ZMYND8 variants. RESULTS: ZMYND8 variants were identified in 11 unrelated individuals; 10 occurred de novo and one suspected de novo; 2 were truncating, 9 were missense, of which one was recurrent. The disorder is characterized by intellectual disability with variable cardiovascular, ophthalmologic and minor skeletal anomalies. Missense variants in the PWWP domain of ZMYND8 abolish the interaction with Drebrin and missense variants in the MYND domain disrupt the interaction with GATAD2A. ZMYND8 is broadly expressed across cell types in all brain regions and shows highest expression in the early stages of brain development. Neuronal knockdown of the DrosophilaZMYND8 ortholog results in decreased habituation learning, consistent with a role in cognitive function. CONCLUSION: We present genomic and functional evidence for disruption of ZMYND8 as a novel etiology of syndromic intellectual disability.

HK1 haemolytic anaemia in association with a neurological phenotype and co-existing CEP290 Meckel-Gruber in a Romani family.

HK1 deficient Haemolytic Anaemia in association with a Neurological Phenotype & co-existing Meckel-Gruber due to CEP290 in a Romani family.

De Novo Variants in the F-Box Protein FBXO11 in 20 Individuals with a Variable Neurodevelopmental Disorder.

Next-generation sequencing combined with international data sharing has enormously facilitated identification of new disease-associated genes and mutations. This is particularly true for genetically extremely heterogeneous entities such as neurodevelopmental disorders (NDDs). Through exome sequencing and world-wide collaborations, we identified and assembled 20 individuals with de novo variants in FBXO11. They present with mild to severe developmental delay associated with a range of features including short (4/20) or tall (2/20) stature, obesity (5/20), microcephaly (4/19) or macrocephaly (2/19), behavioral problems (17/20), seizures (5/20), cleft lip or palate or bifid uvula (3/20), and minor skeletal anomalies. FBXO11 encodes a member of the F-Box protein family, constituting a subunit of an E3-ubiquitin ligase complex. This complex is involved in ubiquitination and proteasomal degradation and thus in controlling critical biological processes by regulating protein turnover. The identified de novo aberrations comprise two large deletions, ten likely gene disrupting variants, and eight missense variants distributed throughout FBXO11. Structural modeling for missense variants located in the CASH or the Zinc-finger UBR domains suggests destabilization of the protein. This, in combination with the observed spectrum and localization of identified variants and the lack of apparent genotype-phenotype correlations, is compatible with loss of function or haploinsufficiency as an underlying mechanism. We implicate de novo missense and likely gene disrupting variants in FBXO11 in a neurodevelopmental disorder with variable intellectual disability and various other features.

ANKRD11 variants: KBG syndrome and beyond.

Mutations affecting the transcriptional regulator Ankyrin Repeat Domain 11 (ANKRD11) are mainly associated with the multisystem developmental disorder known as KBG syndrome, but have also been identified in individuals with Cornelia de Lange syndrome (CdLS) and other developmental disorders caused by variants affecting different chromatin regulators. The extensive functional overlap of these proteins results in shared phenotypical features, which complicate the assessment of the clinical diagnosis. Additionally, re-evaluation of individuals at a later age occasionally reveals that the initial phenotype has evolved toward clinical features more reminiscent of a developmental disorder different from the one that was initially diagnosed. For this reason, variants in ANKRD11 can be ascribed to a broader class of disorders that fall within the category of the so-called chromatinopathies. In this work, we report on the clinical characterization of 23 individuals with variants in ANKRD11. The subjects present primarily with developmental delay, intellectual disability and dysmorphic features, and all but two received an initial clinical diagnosis of either KBG syndrome or CdLS. The number and the severity of the clinical signs are overlapping but variable and result in a broad spectrum of phenotypes, which could be partially accounted for by the presence of additional molecular diagnoses and distinct pathogenic mechanisms.

Delineating the Smith-Kingsmore syndrome phenotype: Investigation of 16 patients with the MTOR c.5395G > A p.(Glu1799Lys) missense variant.

Smith-Kingsmore Syndrome (SKS) is a rare genetic syndrome associated with megalencephaly, a variable intellectual disability, autism spectrum disorder, and MTOR gain of function variants. Only 30 patients with MTOR missense variants are published, including 14 (47%) with the MTOR c.5395G>A p.(Glu1799Lys) variant. Limited phenotypic data impacts the quality of information delivered to families and the robustness of interpretation of novel MTOR missense variation. This study aims to improve our understanding of the SKS phenotype through the investigation of 16 further patients with the MTOR c.5395G>A p.(Glu1799Lys) variant. Through the careful phenotypic evaluation of these 16 patients and integration with data from 14 previously reported patients, we have defined major (100% patients) and frequent (>15%) SKS clinical characteristics and, using these data, proposed guidance for evidence-based management. In addition, in the absence of functional studies, we suggest that the combination of the SKS major clinical features of megalencephaly (where the head circumference is at least 3SD) and an intellectual disability with a de novo MTOR missense variant (absent from population databases) should be considered diagnostic for SKS.

De Novo Truncating Mutations in the Last and Penultimate Exons of PPM1D Cause an Intellectual Disability Syndrome.

Intellectual disability (ID) is a highly heterogeneous disorder involving at least 600 genes, yet a genetic diagnosis remains elusive in ∼35%-40% of individuals with moderate to severe ID. Recent meta-analyses statistically analyzing de novo mutations in >7,000 individuals with neurodevelopmental disorders highlighted mutations in PPM1D as a possible cause of ID. PPM1D is a type 2C phosphatase that functions as a negative regulator of cellular stress-response pathways by mediating a feedback loop of p38-p53 signaling, thereby contributing to growth inhibition and suppression of stress-induced apoptosis. We identified 14 individuals with mild to severe ID and/or developmental delay and de novo truncating PPM1D mutations. Additionally, deep phenotyping revealed overlapping behavioral problems (ASD, ADHD, and anxiety disorders), hypotonia, broad-based gait, facial dysmorphisms, and periods of fever and vomiting. PPM1D is expressed during fetal brain development and in the adult brain. All mutations were located in the last or penultimate exon, suggesting escape from nonsense-mediated mRNA decay. Both PPM1D expression analysis and cDNA sequencing in EBV LCLs of individuals support the presence of a stable truncated transcript, consistent with this hypothesis. Exposure of cells derived from individuals with PPM1D truncating mutations to ionizing radiation resulted in normal p53 activation, suggesting that p53 signaling is unaffected. However, a cell-growth disadvantage was observed, suggesting a possible effect on the stress-response pathway. Thus, we show that de novo truncating PPM1D mutations in the last and penultimate exons cause syndromic ID, which provides additional insight into the role of cell-cycle checkpoint genes in neurodevelopmental disorders.

Correction: A restricted spectrum of missense KMT2D variants cause a multiple malformations disorder distinct from Kabuki syndrome.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.