ABSTRACT
The purpose of this study was to assess the added diagnostic value of whole genome sequencing (WGS) for patients with inherited retinal diseases (IRDs) who remained undiagnosed after whole exome sequencing (WES). WGS was performed for index patients in 66 families. The datasets were analyzed according to GATK's guidelines. Additionally, DeepVariant was complemented by GATK's workflow, and a novel structural variant pipeline was developed. Overall, a molecular diagnosis was established in 19/66 (28.8%) index patients. Pathogenic deletions and one deep-intronic variant contributed to the diagnostic yield in 4/19 and 1/19 index patients, respectively. The remaining diagnoses (14/19) were attributed to exonic variants that were missed during WES analysis due to bioinformatic limitations, newly described loci, or unclear pathogenicity. The added diagnostic value of WGS equals 5/66 (9.6%) for our cohort, which is comparable to previous studies. This figure would decrease further to 1/66 (1.5%) with a standardized and reliable copy number variant workflow during WES analysis. Given the higher costs and limited added value, the implementation of WGS as a first-tier assay for inherited eye disorders in a diagnostic laboratory remains untimely. Instead, progress in bioinformatic tools and communication between diagnostic and clinical teams have the potential to ameliorate diagnostic yields.
Subject(s)
Genetic Testing , Retinal Diseases , Whole Genome Sequencing , Humans , Retinal Diseases/genetics , Retinal Diseases/diagnosis , Genetic Testing/methods , Whole Genome Sequencing/methods , Male , Female , Switzerland , Cohort Studies , Adult , DNA Copy Number Variations , Exome Sequencing/methods , Computational Biology/methods , Middle Aged , Child , Adolescent , PedigreeABSTRACT
Vision is arguably our most important sense, and its loss brings substantial limitations to daily life for affected individuals. Light is perceived in retinal photoreceptors (PRs), which are highly specialized neurons subdivided into several compartments with distinct functions. The outer segments (OSs) of photoreceptors represent highly specialized primary ciliary compartments hosting the phototransduction cascade, which transforms incoming light into a neuronal signal. Retinal disease can result from various pathomechanisms originating in distinct subcompartments of the PR cell, or in the retinal pigment epithelium which supports the PRs. Dysfunction of primary cilia causes human disorders known as "ciliopathies", in which retinal disease is a common feature. This chapter focuses on PR OSs, discussing the mechanisms controlling their complex structure and composition. A sequence of tightly regulated sorting and trafficking events, both upstream of and within this ciliary compartment, ensures the establishment and maintenance of the adequate proteome and lipidome required for signaling in response to light. We discuss in particular our current understanding of the role of ciliopathy proteins involved in multi-protein complexes at the ciliary transition zone (CC2D2A) or BBSome (BBS1) and how their dysfunction causes retinal disease. While the loss of CC2D2A prevents the fusion of vesicles and delivery of the photopigment rhodopsin to the ciliary base, leading to early OS ultrastructural defects, BBS1 deficiency results in precocious accumulation of cholesterol in mutant OSs and decreased visual function preceding morphological changes. These distinct pathomechanisms underscore the central role of ciliary proteins involved in multiple processes controlling OS protein and lipid composition.
Subject(s)
Cilia , Retinal Diseases , Humans , Cilia/metabolism , Protein Transport , Retinal Diseases/metabolism , Signal Transduction , LipidsABSTRACT
Purpose: De novo variants in CUL3 (Cullin-3 ubiquitin ligase) have been strongly associated with neurodevelopmental disorders (NDDs), but no large case series have been reported so far. Here we aimed to collect sporadic cases carrying rare variants in CUL3, describe the genotype-phenotype correlation, and investigate the underlying pathogenic mechanism. Methods: Genetic data and detailed clinical records were collected via multi-center collaboration. Dysmorphic facial features were analyzed using GestaltMatcher. Variant effects on CUL3 protein stability were assessed using patient-derived T-cells. Results: We assembled a cohort of 35 individuals with heterozygous CUL3 variants presenting a syndromic NDD characterized by intellectual disability with or without autistic features. Of these, 33 have loss-of-function (LoF) and two have missense variants. CUL3 LoF variants in patients may affect protein stability leading to perturbations in protein homeostasis, as evidenced by decreased ubiquitin-protein conjugates in vitro . Specifically, we show that cyclin E1 (CCNE1) and 4E-BP1 (EIF4EBP1), two prominent substrates of CUL3, fail to be targeted for proteasomal degradation in patient-derived cells. Conclusion: Our study further refines the clinical and mutational spectrum of CUL3 -associated NDDs, expands the spectrum of cullin RING E3 ligase-associated neuropsychiatric disorders, and suggests haploinsufficiency via LoF variants is the predominant pathogenic mechanism.
ABSTRACT
Vision is one of our dominant senses and its loss has a profound impact on the life quality of affected individuals. Highly specialized neurons in the retina called photoreceptors convert photons into neuronal responses. This conversion of photons is mediated by light sensitive opsin proteins, which are found in the outer segments of the photoreceptors. These outer segments are highly specialized primary cilia, explaining why retinal dystrophy is a key feature of ciliopathies, a group of diseases resulting from abnormal and dysfunctional cilia. Therefore, research on ciliopathies often includes the analysis of the retina with special focus on the photoreceptor and its outer segment. In the last decade, the zebrafish has emerged as an excellent model organism to study human diseases, in particular with respect to the retina. The cone-rich retina of zebrafish resembles the fovea of the human macula and thus represents an excellent model to study human retinal diseases. Here we give detailed guidance on how to analyze the morphological and ultra-structural integrity of photoreceptors in the zebrafish using various histological and imaging techniques. We further describe how to conduct functional analysis of the retina by electroretinography and how to prepare isolated outer segment fractions for different -omic approaches. These different methods allow a comprehensive analysis of photoreceptors, helping to enhance our understanding of the molecular and structural basis of ciliary function in health and of the consequences of its dysfunction in disease.
Subject(s)
Ciliopathies , Zebrafish , Animals , Humans , Zebrafish/metabolism , Cilia/metabolism , Retina , Zebrafish Proteins/metabolism , Ciliopathies/metabolism , Retinal Cone Photoreceptor Cells/metabolismABSTRACT
Next generation sequencing (NGS) can detect carrier status for rare recessive disorders, informing couples about their reproductive risk. The recent ACMG recommendations support offering NGS-based carrier screening (NGS-CS) in an ethnic and population-neutral manner for all genes that have a carrier frequency >1/200 (based on GnomAD). To evaluate current challenges for NGS-CS, we focused on the ciliopathies, a well-studied group of rare recessive disorders. We analyzed 118 ciliopathy genes by whole exome sequencing in ~400 healthy local individuals and ~1000 individuals from the UK1958-birth cohort. We found 20% of healthy individuals (1% of couples) to be carriers of reportable variants in a ciliopathy gene, while 50% (4% of couples) carry variants of uncertain significance (VUS). This large proportion of VUS is partly explained by the limited utility of the ACMG/AMP variant-interpretation criteria in healthy individuals, where phenotypic match or segregation criteria cannot be used. Most missense variants are thus classified as VUS and not reported, which reduces the negative predictive value of the screening test. We show how gene-specific variation patterns and structural protein information can help prioritize variants most likely to be disease-causing, for (future) functional assays. Even when considering only strictly pathogenic variants, the observed carrier frequency is substantially higher than expected based on estimated disease prevalence, challenging the 1/200 carrier frequency cut-off proposed for choice of genes to screen. Given the challenges linked to variant interpretation in healthy individuals and the uncertainties about true carrier frequencies, genetic counseling must clearly disclose these limitations of NGS-CS.
Subject(s)
Ciliopathies , High-Throughput Nucleotide Sequencing , Humans , Genetic Counseling , Exome Sequencing , Ciliopathies/diagnosis , Ciliopathies/genetics , Genetic Carrier ScreeningABSTRACT
Neurodevelopmental disorders (NDDs) result from highly penetrant variation in hundreds of different genes, some of which have not yet been identified. Using the MatchMaker Exchange, we assembled a cohort of 27 individuals with rare, protein-altering variation in the transcriptional coregulator ZMYM3, located on the X chromosome. Most (n = 24) individuals were males, 17 of which have a maternally inherited variant; six individuals (4 male, 2 female) harbor de novo variants. Overlapping features included developmental delay, intellectual disability, behavioral abnormalities, and a specific facial gestalt in a subset of males. Variants in almost all individuals (n = 26) are missense, including six that recurrently affect two residues. Four unrelated probands were identified with inherited variation affecting Arg441, a site at which variation has been previously seen in NDD-affected siblings, and two individuals have de novo variation resulting in p.Arg1294Cys (c.3880C>T). All variants affect evolutionarily conserved sites, and most are predicted to damage protein structure or function. ZMYM3 is relatively intolerant to variation in the general population, is widely expressed across human tissues, and encodes a component of the KDM1A-RCOR1 chromatin-modifying complex. ChIP-seq experiments on one variant, p.Arg1274Trp, indicate dramatically reduced genomic occupancy, supporting a hypomorphic effect. While we are unable to perform statistical evaluations to definitively support a causative role for variation in ZMYM3, the totality of the evidence, including 27 affected individuals, recurrent variation at two codons, overlapping phenotypic features, protein-modeling data, evolutionary constraint, and experimentally confirmed functional effects strongly support ZMYM3 as an NDD-associated gene.
Subject(s)
Intellectual Disability , Nervous System Malformations , Neurodevelopmental Disorders , Humans , Male , Female , Neurodevelopmental Disorders/genetics , Intellectual Disability/genetics , Phenotype , Gene Expression Regulation , Face , Nuclear Proteins/genetics , Histone Demethylases/geneticsABSTRACT
Cilia are quasi-ubiquitous microtubule-based sensory organelles, which play vital roles in signal transduction during development and cell homeostasis. Dysfunction of cilia leads to a group of Mendelian disorders called ciliopathies, divided into different diagnoses according to clinical phenotype constellation and genetic causes. Joubert syndrome (JBTS) is a prototypical ciliopathy defined by a diagnostic cerebellar and brain stem malformation termed the "Molar Tooth Sign" (MTS), in addition to which patients display variable combinations of typical ciliopathy phenotypes such as retinal dystrophy, fibrocystic renal disease, polydactyly or skeletal dystrophy. Like most ciliopathies, JBTS is genetically highly heterogeneous with â¼40 associated genes. Zebrafish are widely used to model ciliopathies given the high conservation of ciliary genes and the variety of specialized cilia types similar to humans. In this review, we compare different existing JBTS zebrafish models with each other and describe their contributions to our understanding of JBTS pathomechanism. We find that retinal dystrophy, which is the most investigated ciliopathy phenotype in zebrafish ciliopathy models, is caused by distinct mechanisms according to the affected gene. Beyond this, differences in phenotypes in other organs observed between different JBTS-mutant models suggest tissue-specific roles for proteins implicated in JBTS. Unfortunately, the lack of systematic assessment of ciliopathy phenotypes in the mutants described in the literature currently limits the conclusions that can be drawn from these comparisons. In the future, the numerous existing JBTS zebrafish models represent a valuable resource that can be leveraged in order to gain further insights into ciliary function, pathomechanisms underlying ciliopathy phenotypes and to develop treatment strategies using small molecules.
ABSTRACT
A hallmark of meiosis is chromosomal pairing, which requires telomere tethering and rotation on the nuclear envelope through microtubules, driving chromosome homology searches. Telomere pulling toward the centrosome forms the "zygotene chromosomal bouquet." Here, we identified the "zygotene cilium" in oocytes. This cilium provides a cable system for the bouquet machinery and extends throughout the germline cyst. Using zebrafish mutants and live manipulations, we demonstrate that the cilium anchors the centrosome to counterbalance telomere pulling. The cilium is essential for bouquet and synaptonemal complex formation, oogenesis, ovarian development, and fertility. Thus, a cilium represents a conserved player in zebrafish and mouse meiosis, which sheds light on reproductive aspects in ciliopathies and suggests that cilia can control chromosomal dynamics.
Subject(s)
Chromosome Pairing , Cilia , Oocytes , Oogenesis , Ovary , Animals , Centromere/genetics , Centromere/physiology , Chromosome Pairing/genetics , Chromosome Pairing/physiology , Cilia/physiology , Female , Fertility/physiology , Mice , Morphogenesis , Oocytes/growth & development , Oogenesis/genetics , Oogenesis/physiology , Ovary/growth & development , Telomere/genetics , Telomere/physiology , Zebrafish/genetics , Zebrafish/physiologyABSTRACT
Primary cilia are key sensory organelles whose dysfunction leads to ciliopathy disorders such as Bardet-Biedl syndrome (BBS). Retinal degeneration is common in ciliopathies, since the outer segments (OSs) of photoreceptors are highly specialized primary cilia. BBS1, encoded by the most commonly mutated BBS-associated gene, is part of the BBSome protein complex. Using a bbs1 zebrafish mutant, we show that retinal development and photoreceptor differentiation are unaffected by Bbs1-loss, supported by an initially unaffected transcriptome. Quantitative proteomics and lipidomics on samples enriched for isolated OSs show that Bbs1 is required for BBSome-complex stability and that Bbs1-loss leads to accumulation of membrane-associated proteins in OSs, with enrichment in proteins involved in lipid homeostasis. Disruption of the tightly regulated OS lipid composition with increased OS cholesterol content are paralleled by early functional visual deficits, which precede progressive OS morphological anomalies. Our findings identify a role for Bbs1/BBSome in OS lipid homeostasis, suggesting a pathomechanism underlying retinal degeneration in BBS.
Subject(s)
Bardet-Biedl Syndrome , Animals , Bardet-Biedl Syndrome/genetics , Cilia/metabolism , Lipids , Microtubule-Associated Proteins/genetics , Microtubule-Associated Proteins/metabolism , Zebrafish/metabolismABSTRACT
Mutations in CEP290 (also known as NPHP6), a large multidomain coiled coil protein, are associated with multiple cilia-associated syndromes. Over 130 CEP290 mutations have been linked to a wide spectrum of human ciliopathies, raising the question of how mutations in a single gene cause different disease syndromes. In zebrafish, the expressivity of cep290 deficiencies were linked to the type of genetic ablation: acute cep290 morpholino knockdown caused severe cilia-related phenotypes, whereas deficiencies in a CRISPR/Cas9 genetic mutant were restricted to photoreceptor defects. Here, we show that milder phenotypes in genetic mutants were associated with the upregulation of genes encoding the cilia-associated small GTPases arl3, arl13b and unc119b. Upregulation of UNC119b was also observed in urine-derived renal epithelial cells from human Joubert syndrome CEP290 patients. Ectopic expression of arl3, arl13b and unc119b in cep290 morphant zebrafish embryos rescued Kupffer's vesicle cilia and partially rescued photoreceptor outer segment defects. The results suggest that genetic compensation by upregulation of genes involved in a common subcellular process, lipidated protein trafficking to cilia, may be a conserved mechanism contributing to genotype-phenotype variations observed in CEP290 deficiencies. This article has an associated First Person interview with the first author of the paper.
Subject(s)
Antigens, Neoplasm , Cell Cycle Proteins , Cilia , Cytoskeletal Proteins , Monomeric GTP-Binding Proteins , Adaptor Proteins, Signal Transducing , Animals , Antigens, Neoplasm/genetics , Cell Cycle Proteins/genetics , Cilia/genetics , Cilia/metabolism , Cytoskeletal Proteins/genetics , Humans , Microtubule-Associated Proteins , Mutation/genetics , Up-Regulation/genetics , Zebrafish/genetics , Zebrafish/metabolism , Zebrafish Proteins/genetics , Zebrafish Proteins/metabolismABSTRACT
Joubert syndrome (JBTS) is a recessive neurodevelopmental ciliopathy characterized by a pathognomonic hindbrain malformation. All known JBTS genes encode proteins involved in the structure or function of primary cilia, ubiquitous antenna-like organelles essential for cellular signal transduction. Here, we used the recently identified JBTS-associated protein armadillo repeat motif-containing 9 (ARMC9) in tandem-affinity purification and yeast 2-hybrid screens to identify a ciliary module whose dysfunction underlies JBTS. In addition to the known JBTS-associated proteins CEP104 and CSPP1, we identified coiled-coil domain containing 66 (CCDC66) and TOG array regulator of axonemal microtubules 1 (TOGARAM1) as ARMC9 interaction partners. We found that TOGARAM1 variants cause JBTS and disrupt TOGARAM1 interaction with ARMC9. Using a combination of protein interaction analyses, characterization of patient-derived fibroblasts, and analysis of CRISPR/Cas9-engineered zebrafish and hTERT-RPE1 cells, we demonstrated that dysfunction of ARMC9 or TOGARAM1 resulted in short cilia with decreased axonemal acetylation and polyglutamylation, but relatively intact transition zone function. Aberrant serum-induced ciliary resorption and cold-induced depolymerization in ARMC9 and TOGARAM1 patient cell lines suggest a role for this new JBTS-associated protein module in ciliary stability.
Subject(s)
Abnormalities, Multiple , Armadillo Domain Proteins , Cerebellum/abnormalities , Cilia , Eye Abnormalities , Kidney Diseases, Cystic , Retina/abnormalities , Zebrafish Proteins , Zebrafish , Abnormalities, Multiple/genetics , Abnormalities, Multiple/metabolism , Acetylation , Animals , Armadillo Domain Proteins/genetics , Armadillo Domain Proteins/metabolism , CRISPR-Cas Systems , Cerebellum/metabolism , Cilia/genetics , Cilia/metabolism , Disease Models, Animal , Eye Abnormalities/genetics , Eye Abnormalities/metabolism , Humans , Kidney Diseases, Cystic/genetics , Kidney Diseases, Cystic/metabolism , Peptides/genetics , Peptides/metabolism , Retina/metabolism , Zebrafish/genetics , Zebrafish/metabolism , Zebrafish Proteins/genetics , Zebrafish Proteins/metabolismABSTRACT
The molecular cause of the majority of rare autosomal recessive disorders remains unknown. Consanguinity due to extensive homozygosity unravels many recessive phenotypes and facilitates the detection of novel gene-disease links. Here, we report two siblings with phenotypic signs, including intellectual disability (ID), developmental delay and microcephaly from a Pakistani consanguineous family in which we have identified homozygosity for p(Tyr103His) in the PSMB1 gene (Genbank NM_002793) that segregated with the disease phenotype. PSMB1 encodes a ß-type proteasome subunit (i.e. ß6). Modeling of the p(Tyr103His) variant indicates that this variant weakens the interactions between PSMB1/ß6 and PSMA5/α5 proteasome subunits and thus destabilizes the 20S proteasome complex. Biochemical experiments in human SHSY5Y cells revealed that the p(Tyr103His) variant affects both the processing of PSMB1/ß6 and its incorporation into proteasome, thus impairing proteasome activity. CRISPR/Cas9 mutagenesis or morpholino knock-down of the single psmb1 zebrafish orthologue resulted in microcephaly, microphthalmia and reduced brain size. Genetic evidence in the family and functional experiments in human cells and zebrafish indicates that PSMB1/ß6 pathogenic variants are the cause of a recessive disease with ID, microcephaly and developmental delay due to abnormal proteasome assembly.
Subject(s)
Dwarfism/genetics , Microcephaly/genetics , Proteasome Endopeptidase Complex/genetics , Alleles , Animals , Child , Consanguinity , Developmental Disabilities/complications , Developmental Disabilities/genetics , Developmental Disabilities/pathology , Dwarfism/complications , Female , Homozygote , Humans , Intellectual Disability/complications , Intellectual Disability/genetics , Intellectual Disability/pathology , Male , Microcephaly/complications , Microcephaly/pathology , Models, Molecular , Pedigree , Phenotype , Zebrafish/geneticsABSTRACT
Joubert syndrome (JS) is a recessive neurodevelopmental disorder defined by a characteristic cerebellar and brainstem malformation recognizable on axial brain magnetic resonance imaging as the "Molar Tooth Sign". Although defined by the neurological features, JS is associated with clinical features affecting many other organ systems, particularly progressive involvement of the retina, kidney, and liver. JS is a rare condition; therefore, many affected individuals may not have easy access to subspecialty providers familiar with JS (e.g., geneticists, neurologists, developmental pediatricians, ophthalmologists, nephrologists, hepatologists, psychiatrists, therapists, and educators). Expert recommendations can enable practitioners of all types to provide quality care to individuals with JS and know when to refer for subspecialty care. This need will only increase as precision treatments targeting specific genetic causes of JS emerge. The goal of these recommendations is to provide a resource for general practitioners, subspecialists, and families to maximize the health of individuals with JS throughout the lifespan.
Subject(s)
Abnormalities, Multiple/epidemiology , Cerebellum/abnormalities , Eye Abnormalities/epidemiology , Health Personnel , Kidney Diseases, Cystic/epidemiology , Neurodevelopmental Disorders/epidemiology , Retina/abnormalities , Abnormalities, Multiple/genetics , Abnormalities, Multiple/pathology , Abnormalities, Multiple/therapy , Brain Stem/pathology , Cerebellum/pathology , Eye Abnormalities/genetics , Eye Abnormalities/pathology , Eye Abnormalities/therapy , Health Planning Guidelines , Humans , Kidney/pathology , Kidney Diseases, Cystic/genetics , Kidney Diseases, Cystic/pathology , Kidney Diseases, Cystic/therapy , Liver/pathology , Neurodevelopmental Disorders/genetics , Neurodevelopmental Disorders/pathology , Neurodevelopmental Disorders/therapy , Retina/pathologyABSTRACT
Light sensation occurs in photoreceptor outer segments (OS), which derive from highly specialized primary cilia, based on structural and molecular similarities. Ciliary dysfunction causes ciliopathies, in which retinal degeneration is common. The connecting cilium (CC) is the obligate passage for proteins moving between ciliary and cellular compartment, controlling the correct distribution of proteins on either side of its barrier. While new mechanisms for selective entry of ciliary proteins are being elucidated, active transport out of the OS is increasingly studied. We further discuss other recent advances in the field, such as a role for the CC in docking and fusion of incoming transport vesicles, a newly proposed subcompartmentalization into proximal and distal CC, and mechanisms of OS membrane dynamics paralleling ectosome formation in other cilia.
Subject(s)
Cilia/metabolism , Ciliopathies/metabolism , Photoreceptor Cells, Vertebrate/metabolism , Retinal Degeneration/metabolism , Zebrafish Proteins/metabolism , Zebrafish/metabolism , Animals , Cilia/genetics , Cilia/ultrastructure , Ciliopathies/genetics , Gene Expression Regulation, Developmental , Larva/genetics , Larva/metabolism , Larva/ultrastructure , Microscopy, Electron, Transmission , Photoreceptor Cells, Vertebrate/ultrastructure , Retinal Degeneration/genetics , Zebrafish/genetics , Zebrafish/growth & development , Zebrafish Proteins/geneticsABSTRACT
Recent recognition of the key role of primary cilia in orchestrating human development and of the dire consequences of their dysfunction on human health has placed this small organelle in the spotlight. While the causal link between mutations in ciliary genes and central nervous system malformations and dysfunction is well established, the mechanisms by which primary cilia dysfunction acts on development and function of the CNS remain partly unknown. The recent article by Bashford and Subramanian in The Journal of Pathology describes a new mouse model for the neurodevelopmental ciliopathy Joubert syndrome, supporting a role for ciliary-mediated Hedgehog signaling on proliferation, survival, and differentiation of cerebellar granule cell progenitors. © 2019 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.
Subject(s)
Abnormalities, Multiple , Ciliopathies , Eye Abnormalities , Kidney Diseases, Cystic , Animals , Cell Cycle Proteins , Cerebellum , Cilia , Hedgehog Proteins , Humans , Mice , Retina , United KingdomABSTRACT
PURPOSE: Microcephaly is a sign of many genetic conditions but has been rarely systematically evaluated. We therefore comprehensively studied the clinical and genetic landscape of an unselected cohort of patients with microcephaly. METHODS: We performed clinical assessment, high-resolution chromosomal microarray analysis, exome sequencing, and functional studies in 62 patients (58% with primary microcephaly [PM], 27% with secondary microcephaly [SM], and 15% of unknown onset). RESULTS: We found severity of developmental delay/intellectual disability correlating with severity of microcephaly in PM, but not SM. We detected causative variants in 48.4% of patients and found divergent inheritance and variant pattern for PM (mainly recessive and likely gene-disrupting [LGD]) versus SM (all dominant de novo and evenly LGD or missense). While centrosome-related pathways were solely identified in PM, transcriptional regulation was the most frequently affected pathway in both SM and PM. Unexpectedly, we found causative variants in different mitochondria-related genes accounting for ~5% of patients, which emphasizes their role even in syndromic PM. Additionally, we delineated novel candidate genes involved in centrosome-related pathway (SPAG5, TEDC1), Wnt signaling (VPS26A, ZNRF3), and RNA trafficking (DDX1). CONCLUSION: Our findings enable improved evaluation and genetic counseling of PM and SM patients and further elucidate microcephaly pathways.
Subject(s)
Developmental Disabilities/genetics , Genetic Predisposition to Disease , Intellectual Disability/genetics , Microcephaly/genetics , Adolescent , Cell Cycle Proteins/genetics , Child , Child, Preschool , DEAD-box RNA Helicases/genetics , Developmental Disabilities/pathology , Exome/genetics , Female , Gene Expression Regulation/genetics , Humans , Infant , Intellectual Disability/pathology , Male , Microcephaly/pathology , Mutation , Pedigree , Phenotype , Ubiquitin-Protein Ligases/genetics , Exome Sequencing , Wnt Signaling PathwayABSTRACT
BACKGROUND: Deleterious variants in the voltage-gated sodium channel type 2 (Nav1.2) lead to a broad spectrum of phenotypes ranging from benign familial neonatal-infantile epilepsy (BFNIE), severe developmental and epileptic encephalopathy (DEE) and intellectual disability (ID) to autism spectrum disorders (ASD). Yet, the underlying mechanisms are still incompletely understood. METHODS: To further elucidate the genotype-phenotype correlation of SCN2A variants we investigated the functional effects of six variants representing the phenotypic spectrum by whole-cell patch-clamp studies in transfected HEK293T cells and in-silico structural modeling. RESULTS: The two variants p.L1342P and p.E1803G detected in patients with early onset epileptic encephalopathy (EE) showed profound and complex changes in channel gating, whereas the BFNIE variant p.L1563V exhibited only a small gain of channel function. The three variants identified in ID patients without seizures, p.R937C, p.L611Vfs*35 and p.W1716*, did not produce measurable currents. Homology modeling of the missense variants predicted structural impairments consistent with the electrophysiological findings. CONCLUSIONS: Our findings support the hypothesis that complete loss-of-function variants lead to ID without seizures, small gain-of-function variants cause BFNIE and EE variants exhibit variable but profound Nav1.2 gating changes. Moreover, structural modeling was able to predict the severity of the variant impact, supporting a potential role of structural modeling as a prognostic tool. Our study on the functional consequences of SCN2A variants causing the distinct phenotypes of EE, BFNIE and ID contributes to the elucidation of mechanisms underlying the broad phenotypic variability reported for SCN2A variants.
Subject(s)
Epilepsy, Benign Neonatal/genetics , Epileptic Syndromes/genetics , Intellectual Disability/genetics , NAV1.2 Voltage-Gated Sodium Channel/physiology , Adolescent , Child , Epilepsy, Benign Neonatal/physiopathology , Epileptic Syndromes/physiopathology , Genetic Association Studies , HEK293 Cells , Humans , Intellectual Disability/physiopathology , Phenotype , Young AdultABSTRACT
Developmental delay and intellectual disability (DD and ID) are heterogeneous phenotypes that arise in many rare monogenic disorders. Because of this rarity, developing cohorts with enough individuals to robustly identify disease-associated genes is challenging. Social-media platforms that facilitate data sharing among sequencing labs can help to address this challenge. Through one such tool, GeneMatcher, we identified nine DD- and/or ID-affected probands with a rare, heterozygous variant in the gene encoding the serine/threonine-protein kinase BRSK2. All probands have a speech delay, and most present with intellectual disability, motor delay, behavioral issues, and autism. Six of the nine variants are predicted to result in loss of function, and computational modeling predicts that the remaining three missense variants are damaging to BRSK2 structure and function. All nine variants are absent from large variant databases, and BRSK2 is, in general, relatively intolerant to protein-altering variation among humans. In all six probands for whom parents were available, the mutations were found to have arisen de novo. Five of these de novo variants were from cohorts with at least 400 sequenced probands; collectively, the cohorts span 3,429 probands, and the observed rate of de novo variation in these cohorts is significantly higher than the estimated background-mutation rate (p = 2.46 × 10-6). We also find that exome sequencing provides lower coverage and appears less sensitive to rare variation in BRSK2 than does genome sequencing; this fact most likely reduces BRSK2's visibility in many clinical and research sequencing efforts. Altogether, our results implicate damaging variation in BRSK2 as a source of neurodevelopmental disease.
Subject(s)
Developmental Disabilities/genetics , Gene Deletion , Intellectual Disability/genetics , Neurodevelopmental Disorders/genetics , Protein Serine-Threonine Kinases/genetics , Adolescent , Autistic Disorder/genetics , Child , Child Behavior Disorders/genetics , Child, Preschool , Exome , Female , Genetic Predisposition to Disease , Genetic Variation , Heterozygote , Humans , Male , Motor Skills Disorders/genetics , Mutation , Phenotype , Exome Sequencing , Young AdultABSTRACT
A correction to this article has been published and is linked from the HTML and PDF versions of this paper. The error has been fixed in the paper.
ABSTRACT
Ciliopathies are human disorders caused by dysfunction of primary cilia, ubiquitous microtubule-based organelles involved in signal transduction. Cilia are anchored inside the cell through basal bodies (BBs), modified centrioles also acting as microtubule-organization centers. Photoreceptors (PRs) are sensory neurons, whose primary cilium forms a highly specialized compartment called the outer segment (OS) responsible for sensing incoming light. Thus, ciliopathies often present with retinal degeneration. Mutations in KIAA0586/TALPID3 (TA3) cause Joubert syndrome, in which 30% of affected individuals develop retinal involvement. To elucidate the function of TALPID3 in PRs, we studied talpid3 zebrafish mutants and identified a progressive retinal degeneration phenotype. The majority of PRs lack OS development due to defects in BB positioning and docking at the apical cell surface. Intracellular accumulation of the photopigment opsin leads to PR cell death of moderate severity. Electroretinograms demonstrate severe visual impairement. A small subset of PRs display normally docked BBs and extended OSs through rescue by maternally-deposited Talpid3. While localization of the small GTPase Rab8a, which plays an important role in BB docking, appears unaffected in talpid3-/- PRs, overexpression of constitutively active Rab8a rescues OS formation, indicating that the role of Ta3 in early ciliogenesis lies upstream of Rab8a activation in PRs.