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.

Rare variants in PPFIA3 cause delayed development, intellectual disability, autism, and epilepsy.

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 synaptic vesicle transport and presynaptic active zone assembly. The protein structure and function are well conserved in both invertebrates and vertebrates, but human diseases related to PPFIA3 dysfunction are not yet known. Here, we report 14 individuals with rare mono-allelic PPFIA3 variants presenting with features including developmental delay, intellectual disability, hypotonia, autism, and epilepsy. To determine the pathogenicity of PPFIA3 variants in vivo , we generated transgenic fruit flies expressing either human PPFIA3 wildtype (WT) or variant protein using GAL4-UAS targeted gene expression systems. Ubiquitous expression with Actin-GAL4 showed that the PPFIA3 variants had variable penetrance of pupal lethality, eclosion defects, and anatomical leg defects. Neuronal expression with elav-GAL4 showed that the PPFIA3 variants had seizure-like behaviors, motor defects, and bouton loss at the 3 rd instar larval neuromuscular junction (NMJ). Altogether, in the fly overexpression assays, we found that the PPFIA3 variants in the N-terminal coiled coil domain exhibited stronger phenotypes compared to those in 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 protein function is partially conserved in the fly. However, the PPFIA3 variants failed to rescue lethality. 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.

Germline variants in tumor suppressor FBXW7 lead to impaired ubiquitination and a neurodevelopmental syndrome.

Neurodevelopmental disorders are highly heterogenous conditions resulting from abnormalities of brain architecture and/or function. FBXW7 (F-box and WD-repeat-domain-containing 7), a recognized developmental regulator and tumor suppressor, has been shown to regulate cell-cycle progression and cell growth and survival by targeting substrates including CYCLIN E1/2 and NOTCH for degradation via the ubiquitin proteasome system. We used a genotype-first approach and global data-sharing platforms to identify 35 individuals harboring de novo and inherited FBXW7 germline monoallelic chromosomal deletions and nonsense, frameshift, splice-site, and missense variants associated with a neurodevelopmental syndrome. The FBXW7 neurodevelopmental syndrome is distinguished by global developmental delay, borderline to severe intellectual disability, hypotonia, and gastrointestinal issues. Brain imaging detailed variable underlying structural abnormalities affecting the cerebellum, corpus collosum, and white matter. A crystal-structure model of FBXW7 predicted that missense variants were clustered at the substrate-binding surface of the WD40 domain and that these might reduce FBXW7 substrate binding affinity. Expression of recombinant FBXW7 missense variants in cultured cells demonstrated impaired CYCLIN E1 and CYCLIN E2 turnover. Pan-neuronal knockdown of the Drosophila ortholog, archipelago, impaired learning and neuronal function. Collectively, the data presented herein provide compelling evidence of an F-Box protein-related, phenotypically variable neurodevelopmental disorder associated with monoallelic variants in FBXW7.

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.

Clinical and Molecular Spectrum of Nonsyndromic Early-Onset Osteoarthritis.

OBJECTIVE: Osteoarthritis (OA) is the most common joint disease worldwide. The etiology of OA is varied, ranging from multifactorial to environmental to monogenic. In a condition called early-onset OA, OA occurs at an earlier age than is typical in the general population. To our knowledge, there have been no large-scale genetic studies of individuals with early-onset OA. The present study was undertaken to investigate causes of monogenic OA in individuals with nonsyndromic early-onset OA. METHODS: The study probands were 45 patients with nonsyndromic early-onset OA who were referred to our skeletal disease center by skeletal dysplasia experts between 2013 and 2019. Criteria for early-onset OA included radiographic evidence, body mass index ≤30 kg/m2 , age at onset ≤50 years, and involvement of ≥1 joint site. Molecular analysis was performed with a next-generation sequencing panel. RESULTS: We identified a genetic variant in 13 probands (29%); the affected gene was COL2A1 in 11, ACAN in 1, and SLC26A2 in 1. After familial segregation analysis, 20 additional individuals were identified. The mean ± SD age at onset of joint pain was 19.5 ± 3.9 years (95% confidence interval 3-47). Eighteen of 33 subjects (55%) with nonsyndromic early-onset OA and a genetic variant had had at least 1 joint replacement (mean ± SD age at first joint replacement 41 ± 4.2 years; mean number of joint replacements 2.6 per individual), and 21 (45%) of the joint replacement surgeries were performed when the patient was <45 years old. Of the 20 patients age >40 years, 17 (85%) had had at least 1 joint replacement. CONCLUSION: We confirmed that COL2A1 is the main monogenic cause of nonsyndromic early-onset OA. However, on the basis of genetic heterogeneity of early-onset OA, we recommend next-generation sequencing for all individuals who undergo joint replacement prior to the age of 45 years. Lifestyle recommendations for prevention should be implemented.

De Novo Frameshift Variants in the Neuronal Splicing Factor NOVA2 Result in a Common C-Terminal Extension and Cause a Severe Form of Neurodevelopmental Disorder.

The neuro-oncological ventral antigen 2 (NOVA2) protein is a major factor regulating neuron-specific alternative splicing (AS), previously associated with an acquired neurologic condition, the paraneoplastic opsoclonus-myoclonus ataxia (POMA). We report here six individuals with de novo frameshift variants in NOVA2 affected with a severe neurodevelopmental disorder characterized by intellectual disability (ID), motor and speech delay, autistic features, hypotonia, feeding difficulties, spasticity or ataxic gait, and abnormal brain MRI. The six variants lead to the same reading frame, adding a common proline rich C-terminal part instead of the last KH RNA binding domain. We detected 41 genes differentially spliced after NOVA2 downregulation in human neural cells. The NOVA2 variant protein shows decreased ability to bind target RNA sequences and to regulate target AS events. It also fails to complement the effect on neurite outgrowth induced by NOVA2 downregulation in vitro and to rescue alterations of retinotectal axonal pathfinding induced by loss of NOVA2 ortholog in zebrafish. Our results suggest a partial loss-of-function mechanism rather than a full heterozygous loss-of-function, although a specific contribution of the novel C-terminal extension cannot be excluded.

Growth charts in Kabuki syndrome 1.

Kabuki syndrome (KS, KS1: OMIM 147920 and KS2: OMIM 300867) is caused by pathogenic variations in KMT2D or KDM6A. KS is characterized by multiple congenital anomalies and neurodevelopmental disorders. Growth restriction is frequently reported. Here we aimed to create specific growth charts for individuals with KS1, identify parameters used for size prognosis and investigate the impact of growth hormone therapy on adult height. Growth parameters and parental size were obtained for 95 KS1 individuals (41 females). Growth charts for height, weight, body mass index (BMI) and occipitofrontal circumference were generated in standard deviation values for the first time in KS1. Statural growth of KS1 individuals was compared to parental target size. According to the charts, height, weight, BMI, and occipitofrontal circumference were lower for KS1 individuals than the normative French population. For males and females, the mean growth of KS1 individuals was -2 and -1.8 SD of their parental target size, respectively. Growth hormone therapy did not increase size beyond the predicted size. This study, from the largest cohort available, proposes growth charts for widespread use in the management of KS1, especially for size prognosis and screening of other diseases responsible for growth impairment beyond a calculated specific target size.

De novo loss-of-function KCNMA1 variants are associated with a new multiple malformation syndrome and a broad spectrum of developmental and neurological phenotypes.

KCNMA1 encodes the large-conductance Ca2+- and voltage-activated K+ (BK) potassium channel α-subunit, and pathogenic gain-of-function variants in this gene have been associated with a dominant form of generalized epilepsy and paroxysmal dyskinesia. Here, we genetically and functionally characterize eight novel loss-of-function (LoF) variants of KCNMA1. Genome or exome sequencing and the participation in the international Matchmaker Exchange effort allowed for the identification of novel KCNMA1 variants. Patch clamping was used to assess functionality of mutant BK channels. The KCNMA1 variants p.(Ser351Tyr), p.(Gly356Arg), p.(Gly375Arg), p.(Asn449fs) and p.(Ile663Val) abolished the BK current, whereas p.(Cys413Tyr) and p.(Pro805Leu) reduced the BK current amplitude and shifted the activation curves toward positive potentials. The p.(Asp984Asn) variant reduced the current amplitude without affecting kinetics. A phenotypic analysis of the patients carrying the recurrent p.(Gly375Arg) de novo missense LoF variant revealed a novel syndromic neurodevelopmental disorder associated with severe developmental delay, visceral and cardiac malformations, connective tissue presentations with arterial involvement, bone dysplasia and characteristic dysmorphic features. Patients with other LoF variants presented with neurological and developmental symptoms including developmental delay, intellectual disability, ataxia, axial hypotonia, cerebral atrophy and speech delay/apraxia/dysarthria. Therefore, LoF KCNMA1 variants are associated with a new syndrome characterized by a broad spectrum of neurological phenotypes and developmental disorders. LoF variants of KCNMA1 cause a new syndrome distinctly different from gain-of-function variants in the same gene.

Morphology and genomic hallmarks of breast tumours developed by ATM deleterious variant carriers.

BACKGROUND: The ataxia telangiectasia mutated (ATM) gene is a moderate-risk breast cancer susceptibility gene; germline loss-of-function variants are found in up to 3% of hereditary breast and ovarian cancer (HBOC) families who undergo genetic testing. So far, no clear histopathological and molecular features of breast tumours occurring in ATM deleterious variant carriers have been described, but identification of an ATM-associated tumour signature may help in patient management. METHODS: To characterise hallmarks of ATM-associated tumours, we performed systematic pathology review of tumours from 21 participants from ataxia-telangiectasia families and 18 participants from HBOC families, as well as copy number profiling on a subset of 23 tumours. Morphology of ATM-associated tumours was compared with that of 599 patients with no BRCA1 and BRCA2 mutations from a hospital-based series, as well as with data from The Cancer Genome Atlas. Absolute copy number and loss of heterozygosity (LOH) profiles were obtained from the OncoScan SNP array. In addition, we performed whole-genome sequencing on four tumours from ATM loss-of-function variant carriers with available frozen material. RESULTS: We found that ATM-associated tumours belong mostly to the luminal B subtype, are tetraploid and show LOH at the ATM locus at 11q22-23. Unlike tumours in which BRCA1 or BRCA2 is inactivated, tumours arising in ATM deleterious variant carriers are not associated with increased large-scale genomic instability as measured by the large-scale state transitions signature. Losses at 13q14.11-q14.3, 17p13.2-p12, 21p11.2-p11.1 and 22q11.23 were observed. Somatic alterations at these loci may therefore represent biomarkers for ATM testing and harbour driver mutations in potentially 'druggable' genes that would allow patients to be directed towards tailored therapeutic strategies. CONCLUSIONS: Although ATM is involved in the DNA damage response, ATM-associated tumours are distinct from BRCA1-associated tumours in terms of morphological characteristics and genomic alterations, and they are also distinguishable from sporadic breast tumours, thus opening up the possibility to identify ATM variant carriers outside the ataxia-telangiectasia disorder and direct them towards effective cancer risk management and therapeutic strategies.

Complete loss of function of the ubiquitin ligase HERC2 causes a severe neurodevelopmental phenotype.

The ubiquitin-proteasome pathway is involved in the pathogenesis of several neurogenetic diseases. We describe a Mauritanian patient harboring a homozygous deletion restricted to two contiguous genes HERC2 and OCA2 and presenting with severe developmental abnormalities. The deletion causes the complete loss of HERC2 protein function, an E3-ubiquitin ligase. HERC2 is known to target XPA and BRCA1 for degradation and a mechanism whereby it is involved in DNA repair and cell cycle regulation. We showed that loss of HERC2 function leads to the accumulation of XPA and BRCA1 in the patient's fibroblasts and generates decreased sensitivity to apoptosis and increased level of DNA repair. Our data describe for the first time the phenotypic consequences, both at the clinical and cellular levels, of a complete loss of HERC2 function in a patient. They strongly suggest that profound ubiquitin ligase - associated dysfunction is responsible for the severe phenotype in this patient, and that dysfunction of this pathway may be involved in other patients with similar neurodevelopmental diseases.

Otopalatodigital spectrum disorders: refinement of the phenotypic and mutational spectrum.

Otopalatodigital spectrum disorders (OPDSD) constitute a group of dominant X-linked osteochondrodysplasias including four syndromes: otopalatodigital syndromes type 1 and type 2 (OPD1 and OPD2), frontometaphyseal dysplasia, and Melnick-Needles syndrome. These syndromes variably associate specific facial and extremities features, hearing loss, cleft palate, skeletal dysplasia and several malformations, and show important clinical overlap over the different entities. FLNA gain-of-function mutations were identified in these conditions. FLNA encodes filamin A, a scaffolding actin-binding protein. Here, we report phenotypic descriptions and molecular results of FLNA analysis in a large series of 27 probands hypothesized to be affected by OPDSD. We identified 11 different missense mutations in 15 unrelated probands (n=15/27, 56%), of which seven were novel, including one of unknown significance. Segregation analyses within families made possible investigating 20 additional relatives carrying a mutation. This series allows refining the phenotypic and mutational spectrum of FLNA mutations causing OPDSD, and providing suggestions to avoid the overdiagnosis of OPD1.

Mutation in TTI2 reveals a role for triple T complex in human brain development.

Tel2-interacting proteins 1 and 2 (TTI1 and TTI2) physically interact with telomere maintenance 2 (TEL2) to form a conserved trimeric complex called the Triple T complex. This complex is a master regulator of phosphoinositide-3-kinase-related protein kinase (PIKKs) abundance and DNA damage response signaling. Using a combination of autozygosity mapping and high-throughput sequencing in a large consanguineous multiplex family, we found that a missense c.1307T>A/p.I436N mutation in TTI2 causes a human autosomal recessive condition characterized by severe cognitive impairment, microcephaly, behavioral troubles, short stature, skeletal anomalies, and facial dysmorphic features. Immunoblotting experiment showed decreased amount of all Triple T complex components in the patient skin fibroblasts. Consistently, a drastically reduced steady-state level of all PIKKs tested was also observed in the patient cells. Combined with previous observations, these findings emphasises the role of the TTI2 gene in the etiology of intellectual disability and further support the role of PIKK signaling in brain development and functioning.