A population genetic approach to mapping neurological disorder genes using deep resequencing

Deep resequencing of functional regions in human genomes is key to identifying potentially causal rare variants for complex disorders. Here, we present the results from a large-sample resequencing (n  =  285 patients) study of candidate genes coupled with population genetics and statistical methods to identify rare variants associated with Autism Spectrum Disorder and Schizophrenia. Three genes, MAP1A, GRIN2B, and CACNA1F, were consistently identified by different methods as having significant excess of rare missense mutations in either one or both disease cohorts. In a broader context, we also found that the overall site frequency spectrum of variation in these cases is best explained by population models of both selection and complex demography rather than neutral models or models accounting for complex demography alone. Mutations in the three disease-associated genes explained much of the difference in the overall site frequency spectrum among the cases versus controls. This study demonstrates that genes associated with complex disorders can be mapped using resequencing and analytical methods with sample sizes far smaller than those required by genome-wide association studies. Additionally, our findings support the hypothesis that rare mutations account for a proportion of the phenotypic variance of these complex disorders

MYT1L mutations cause intellectual disability and variable obesity by dysregulating gene expression and development of the neuroendocrine hypothalamus

Deletions at chromosome 2p25.3 are associated with a syndrome consisting of intellectual disability and obesity. The smallest region of overlap for deletions at 2p25.3 contains PXDN and MYT1L. MYT1L is expressed only within the brain in humans. We hypothesized that single nucleotide variants (SNVs) in MYT1L would cause a phenotype resembling deletion at 2p25.3. To examine this we sought MYT1L SNVs in exome sequencing data from 4, 296 parent-child trios. Further variants were identified through a genematcher-facilitated collaboration. We report 9 patients with MYT1L SNVs (4 loss of function and 5 missense). The phenotype of SNV carriers overlapped with that of 2p25.3 deletion carriers. To identify the transcriptomic consequences of MYT1L loss of function we used CRISPR-Cas9 to create a knockout cell line. Gene Ontology analysis in knockout cells demonstrated altered expression of genes that regulate gene expression and that are localized to the nucleus. These differentially expressed genes were enriched for OMIM disease ontology terms “mental retardation”. To study the developmental effects of MYT1L loss of function we created a zebrafish knockdown using morpholinos. Knockdown zebrafish manifested loss of oxytocin expression in the preoptic neuroendocrine area. This study demonstrates that MYT1L variants are associated with syndromic obesity in humans. The mechanism is related to dysregulated expression of neurodevelopmental genes and altered development of the neuroendocrine hypothalamus

Delineating the GRIN1 phenotypic spectrum: a distinct genetic NMDA receptor encephalopathy

Objective:To determine the phenotypic spectrum caused by mutations in GRIN1 encoding the NMDA receptor subunit GluN1 and to investigate their underlying functional pathophysiology.Methods:We collected molecular and clinical data from several diagnostic and research cohorts. Functional consequences of GRIN1 mutations were investigated in Xenopus laevis oocytes.Results:We identified heterozygous de novo GRIN1 mutations in 14 individuals and reviewed the phenotypes of all 9 previously reported patients. These 23 individuals presented with a distinct phenotype of profound developmental delay, severe intellectual disability with absent speech, muscular hypotonia, hyperkinetic movement disorder, oculogyric crises, cortical blindness, generalized cerebral atrophy, and epilepsy. Mutations cluster within transmembrane segments and result in loss of channel function of varying severity with a dominant-negative effect. In addition, we describe 2 homozygous GRIN1 mutations (1 missense, 1 truncation), each segregating with severe neurodevelopmental phenotypes in consanguineous families.Conclusions:De novo GRIN1 mutations are associated with severe intellectual disability with cortical visual impairment as well as oculomotor and movement disorders being discriminating phenotypic features. Loss of NMDA receptor function appears to be the underlying disease mechanism. The identification of both heterozygous and homozygous mutations blurs the borders of dominant and recessive inheritance of GRIN1-associated disorders.Johannes R. Lemke (32EP30_136042/1) and Peter De Jonghe (G.A.136.11.N and FWO/ESF-ECRP) received financial support within the EuroEPINOMICS-RES network (www.euroepinomics.org) within the Eurocores framework of the European Science Foundation (ESF). Saskia Biskup and Henrike Heyne received financial support from the German Federal Ministry for Education and Research (BMBF IonNeurONet: 01 GM1105A and FKZ: 01EO1501). Katia Hardies is a PhD fellow of the Institute for Science and Technology (IWT) Flanders. Ingo Helbig was supported by intramural funds of the University of Kiel, by a grant from the German Research Foundation (HE5415/3-1) within the EuroEPINOMICS framework of the European Science Foundation, and additional grants of the German Research Foundation (DFG, HE5415/5-1, HE 5415/6-1), German Ministry for Education and Research (01DH12033, MAR 10/012), and grant by the German chapter of the International League against Epilepsy (DGfE). The project also received infrastructural support through the Institute of Clinical Molecular Biology in Kiel, supported in part by DFG Cluster of Excellence "Inflammation at Interfaces" and "Future Ocean." The project was also supported by the popgen 2.0 network (P2N) through a grant from the German Ministry for Education and Research (01EY1103) and by the International Coordination Action (ICA) grant G0E8614N. Christel Depienne, Caroline Nava, and Delphine Heron received financial support for exome analyses by the Centre National de Genotypage (CNG, Evry, France)

A Population Genetic Approach to Mapping Neurological Disorder Genes Using Deep Resequencing

Deep resequencing of functional regions in human genomes is key to identifying potentially causal rare variants for complex disorders. Here, we present the results from a large-sample resequencing (n = 285 patients) study of candidate genes coupled with population genetics and statistical methods to identify rare variants associated with Autism Spectrum Disorder and Schizophrenia. Three genes, MAP1A, GRIN2B, and CACNA1F, were consistently identified by different methods as having significant excess of rare missense mutations in either one or both disease cohorts. In a broader context, we also found that the overall site frequency spectrum of variation in these cases is best explained by population models of both selection and complex demography rather than neutral models or models accounting for complex demography alone. Mutations in the three disease-associated genes explained much of the difference in the overall site frequency spectrum among the cases versus controls. This study demonstrates that genes associated with complex disorders can be mapped using resequencing and analytical methods with sample sizes far smaller than those required by genome-wide association studies. Additionally, our findings support the hypothesis that rare mutations account for a proportion of the phenotypic variance of these complex disorders

ANK2 loss-of-function variants are associated with epilepsy, and lead to impaired axon initial segment plasticity and hyperactive network activity in hiPSC-derived neuronal networks

PURPOSE: To characterize a novel neurodevelopmental syndrome due to loss-of-function (LoF) variants in Ankyrin 2 (ANK2), and to explore the effects on neuronal network dynamics and homeostatic plasticity in human-induced pluripotent stem cell-derived neurons. METHODS: We collected clinical and molecular data of 12 individuals with heterozygous de novo LoF variants in ANK2. We generated a heterozygous LoF allele of ANK2 using CRISPR/Cas9 in human-induced pluripotent stem cells (hiPSCs). HiPSCs were differentiated into excitatory neurons, and we measured their spontaneous electrophysiological responses using micro-electrode arrays (MEAs). We also characterized their somatodendritic morphology and axon initial segment (AIS) structure and plasticity. RESULTS: We found a broad neurodevelopmental disorder (NDD), comprising intellectual disability, autism spectrum disorders and early onset epilepsy. Using MEAs, we found that hiPSC-derived neurons with heterozygous LoF of ANK2 show a hyperactive and desynchronized neuronal network. ANK2-deficient neurons also showed increased somatodendritic structures and altered AIS structure of which its plasticity is impaired upon activity-dependent modulation. CONCLUSIONS: Phenotypic characterization of patients with de novo ANK2 LoF variants defines a novel NDD with early onset epilepsy. Our functional in vitro data of ANK2-deficient human neurons show a specific neuronal phenotype in which reduced ANKB expression leads to hyperactive and desynchronized neuronal network activity, increased somatodendritic complexity and AIS structure and impaired activity-dependent plasticity of the AIS

SYN2 is an autism predisposing gene: loss-of-function mutations alter synaptic vesicles cycling and axon outgrowth

An increasing number of genes predisposing to autism spectrum disorders (ASDs) has been identified, many of which are implicated in synaptic function. This 'synaptic autism pathway' notably includes disruption of SYN1 that is associated with epilepsy, autism and abnormal behavior in both human and mice models. Synapsins constitute a multigene family of neuron-specific phosphoproteins (SYN1-3) present in the majority of synapses where they are implicated in the regulation of neurotransmitter release and synaptogenesis. Synapsins I and II, the major Syn isoforms in the adult brain, display partially overlapping functions and defects in both isoforms are associated with epilepsy and autistic-like behavior in mice. In this study, we show that nonsense (A94fs199X) and missense (Y236S and G464R) mutations in SYN2 are associated with ASD in humans. The phenotype is apparent in males. Female carriers of SYN2 mutations are unaffected, suggesting that SYN2 is another example of autosomal sex-limited expression in ASD. When expressed in SYN2 \u200aknockout neurons, wild-type human Syn II fully rescues the SYN2 knockout phenotype, whereas the nonsense mutant is not expressed and the missense mutants are virtually unable to modify the SYN2 knockout phenotype. These results identify for the first time SYN2 \u200aas a novel predisposing gene for ASD and strengthen the hypothesis that a disturbance of synaptic homeostasis underlies ASD

Patients with KCNH1-related intellectual disability without distinctive features of Zimmermann-Laband/Temple-Baraitser syndrome

International audienceDe novo missense variants in KCNH1 encoding Kv10.1 are responsible for two clinically recognisable phenotypes: Temple-Baraitser syndrome (TBS) and Zimmermann-Laband syndrome (ZLS). The clinical overlap between these two syndromes suggests that they belong to a spectrum of KCNH1 -related encephalopathies. Affected patients have severe intellectual disability (ID) with or without epilepsy, hypertrichosis and distinctive features such as gingival hyperplasia and nail hypoplasia/aplasia (present in 20/23 reported cases). We report a series of seven patients with ID and de novo pathogenic KCNH1 variants identified by whole-exome sequencing or an epilepsy gene panel in whom the diagnosis of TBS/ZLS had not been first considered. Four of these variants, p.(Thr294Met), p.(Ala492Asp), p.(Thr493Asn) and p.(Gly496Arg), were located in the transmembrane domains S3 and S6 of Kv10.1 and one, p.(Arg693Gln), in its C-terminal cyclic nucleotide-binding homology domain (CNBHD). Clinical reappraisal by the referring clinical geneticists confirmed the absence of the distinctive gingival and nail features of TBS/ZLS. Our study expands the phenotypical spectrum of KCNH1 -related encephalopathies to individuals with an attenuated extraneurological phenotype preventing a clinical diagnosis of TBS or ZLS. This subtype may be related to recurrent substitutions of the Gly496, suggesting a genotype–phenotype correlation and, possibly, to variants in the CNBHD domain

Clinical characterization of <i>MYT1L</i> variant carriers.

<p>The heatmap depicts the frequency of various clinical features in <i>MYT1L</i> variant carriers.</p

Summary of demographic features, developmental milestones, medical complications and exome sequencing of cohort.

<p>Summary of demographic features, developmental milestones, medical complications and exome sequencing of cohort.</p

Hypothalamic peptide expression in zebrafish knockdown for <i>MYT1L</i> orthologues.

<p>(A)Whole mount in situ hybridization demonstrating that <i>MYT1L</i> orthologs <i>myt1la</i> and <i>myt1lb</i> are expressed throughout the zebrafish central nervous system. T = telencephalon, te = tectum, hy = hypothalamus, h = hindbrain. (B)Whole mount in situ hybridization demonstrating loss of <i>myt1la</i> expression in <i>arnt2</i> mutant zebrafish, top panel shows control fish and bottom panel <i>arnt2</i> mutant fish. The embryos are heavily over-stained to show the low-level expression of <i>myt1la</i> in the ventral diencephalon. The arrow indicates the region of the neuroendocrine preoptic area where oxytocin expressing neurons are located. (C)Whole mount in situ hybridization demonstrating that knockdown of <i>myt1la</i> and <i>myt1lb</i>, alone or in combination, results in loss of oxytocin expression in the neuroendocrine preoptic area. Arrows indicate neuroendocrine preoptic area. (D)Bar chart quantifying loss of oxytocin expression in neuroendocrine preoptic area. WISH for oxytocin was quantified as follows: ~30 cells = wild type expression, 5–15 cells = reduced, 1–4 = highly reduced, 0 = no expression (E)Whole mount in situ hybridization demonstrating that knockdown of <i>myt1la/b</i> results in loss of arginine vasopressin expression in the neuroendocrine preoptic region (indicated by arrow) but not the ventral hypothalamus (indicated by arrowhead). (F) Bar chart quantifying loss of arginine vasopression expression in <i>myt1la/b</i> morphants. WISH for arginine vasopressin was quantified as follows: ~30 cells = wild type expression, 5–15 cells = reduced, 1–4 = highly reduced, 0 = no expression.</p