RESUMO
Genetic studies of autism spectrum disorder (ASD) have established that de novo duplications and deletions contribute to risk. However, ascertainment of structural variants (SVs) has been restricted by the coarse resolution of current approaches. By applying a custom pipeline for SV discovery, genotyping, and de novo assembly to genome sequencing of 235 subjects (71 affected individuals, 26 healthy siblings, and their parents), we compiled an atlas of 29,719 SV loci (5,213/genome), comprising 11 different classes. We found a high diversity of de novo mutations, the majority of which were undetectable by previous methods. In addition, we observed complex mutation clusters where combinations of de novo SVs, nucleotide substitutions, and indels occurred as a single event. We estimate a high rate of structural mutation in humans (20%) and propose that genetic risk for ASD is attributable to an elevated frequency of gene-disrupting de novo SVs, but not an elevated rate of genome rearrangement.
Assuntos
Transtorno do Espectro Autista/genética , Deleção de Genes , Duplicação Gênica , Alelos , Sequência de Aminoácidos , Sequência de Bases , Estudos de Casos e Controles , Criança , Variações do Número de Cópias de DNA , Feminino , Frequência do Gene , Rearranjo Gênico , Loci Gênicos , Genoma Humano , Técnicas de Genotipagem , Humanos , Mutação INDEL , Masculino , Análise em Microsséries , Dados de Sequência Molecular , Linhagem , Reprodutibilidade dos Testes , Sensibilidade e EspecificidadeRESUMO
NMDA receptors (NMDARs), ligand-gated ion channels, play important roles in various neurological disorders, including epilepsy. Here we show the functional analysis of a de novo missense mutation (L812M) in a gene encoding NMDAR subunit GluN2A (GRIN2A). The mutation, identified in a patient with early-onset epileptic encephalopathy and profound developmental delay, is located in the linker region between the ligand-binding and transmembrane domains. Electrophysiological recordings revealed that the mutation enhances agonist potency, decreases sensitivity to negative modulators including magnesium, protons and zinc, prolongs the synaptic response time course and increases single-channel open probability. The functional changes of this amino acid apply to all other NMDAR subunits, suggesting an important role of this residue on the function of NMDARs. Taken together, these data suggest that the L812M mutation causes overactivation of NMDARs and drives neuronal hyperexcitability. We hypothesize that this mechanism underlies the patient's epileptic phenotype as well as cerebral atrophy.
Assuntos
Receptores de N-Metil-D-Aspartato/genética , Espasmos Infantis/genética , Regulação Alostérica , Sequência de Aminoácidos , Substituição de Aminoácidos , Criança , Análise Mutacional de DNA , Humanos , Lactente , Magnésio/metabolismo , Masculino , Dados de Sequência Molecular , Mutação de Sentido Incorreto , Receptores de N-Metil-D-Aspartato/agonistas , Receptores de N-Metil-D-Aspartato/metabolismo , Relação Estrutura-AtividadeRESUMO
As whole-genome sequencing becomes commoditized and we begin to sequence and analyze personal genomes for clinical and diagnostic purposes, it is necessary to understand what constitutes a complete sequencing experiment for determining genotypes and detecting single-nucleotide variants. Here, we show that the current recommendation of â¼30× coverage is not adequate to produce genotype calls across a large fraction of the genome with acceptably low error rates. Our results are based on analyses of a clinical sample sequenced on two related Illumina platforms, GAII(x) and HiSeq 2000, to a very high depth (126×). We used these data to establish genotype-calling filters that dramatically increase accuracy. We also empirically determined how the callable portion of the genome varies as a function of the amount of sequence data used. These results help provide a "sequencing guide" for future whole-genome sequencing decisions and metrics by which coverage statistics should be reported.
