Identification and Characterization of Pathogenic Mutations in Neurodevelopmental Disorders Discovered by Next-Generation Sequencing

<p>Neurodevelopmental disorders develop over time and are characterized by a wide variety of mental, behavioral, and physical phenotypes. The categorization of neurodevelopmental disorders encompasses a broad range of conditions including intellectual disability, autism spectrum disorder, attention deficit hyperactivity disorder, cerebral palsy, schizophrenia, bipolar disorder, and epilepsy, among others. Diagnostic classifications of neurodevelopmental disorders are complicated by comorbidities among these neurodevelopmental disorders, unidentified causal genes, and growing evidence of shared genetic risk factors. </p><p>We sought to identify the genetic underpinnings of a variety of neurodevelopmental disorders, with a particular emphasis on the epilepsies, by employing next&ndash;generation sequencing to thoroughly interrogate genetic variation in the human genome/exome. First, we investigated four families presenting with a seemingly identical and previously undescribed neurodevelopmental disorder characterized by congenital microcephaly, intellectual disability, progressive cerebral atrophy, and intractable seizures. These families all exhibited an apparent autosomal recessive pattern of inheritance. Second, we investigated a heterogeneous cohort of &sim;60 undiagnosed patients, the majority of whom suffered from severe neurodevelopmental disorders with a suspected genetic etiology. Third, we investigated 264 patients with epileptic encephalopathies &mdash; severe childhood epilepsy disorders &mdash; looking specifically at infantile spasms and Lennox&ndash;Gastaut syndrome. Finally, we investigated &sim;40 large multiplex epilepsy families with complex phenotypic constellations and unclear modes of inheritance. The studied neurodevelopmental disorders exhibited a range of genetic complexity, from clear Mendelian disorders to common complex disorders, resulting in varying degrees of success in the identification of clearly causal genetic variants. </p><p>In the first project, we successfully identified the disease&ndash;causing gene. We show that recessive mutations in <italic>ASNS </italic> (encoding asparagine synthetase) are responsible for this previously undescribed neurodevelopmental disorder. We also characterized the causal mutations <italic>in vitro</italic> and studied Asns&ndash;deficient mice that mimicked aspects of the patient phenotype. This work describes ASNS deficiency as a novel neurodevelopmental disorder, identifies three distinct causal mutations in the ASNS gene, and indicates that asparagine synthesis is essential for the proper development and function of the brain.</p><p>In the second project, we exome sequenced 62 undiagnosed patients and their unaffected biological parents (trios). By analyzing all identified variants that were annotated as putatively functional and observed as a novel genotype in the probands (not observed in the unaffected parents or controls), we obtained a genetic diagnosis for 32% (20/62) of these patients. Additionally, we identify strong candidate variants in 31% (13/42) of the undiagnosed cases. We also present additional analysis methods for moving beyond traditional screens, e.g., considering only securely implicated genes, or subjecting qualifying variants from any gene to two unique analysis approaches. This work adds to the growing evidence for the utility of diagnostic exome sequencing, increases patient sizes for rare neurodevelopmental disorders (enabling more detailed analyses of the phenotypic spectrum), and proposes novel analysis approaches which will likely become beneficial as the number of sequenced undiagnosed patients grows. </p><p>In the third project, we again employ a trio&ndash;based exome sequencing design to investigate the role of <italic>de novo</italic> mutations in two classical forms of epileptic encephalopathy. We find a significant excess of <italic>de novo</italic> mutations in the &sim;4,000 genes that are the most intolerant to functional genetic variation in the human population (P = 2.9 x 10<super>&ndash;3</super>, likelihood analysis). We provide clear statistical evidence for two novel genes associated with epileptic encephalopathy &mdash; <italic>GABRB3</italic> and <italic>ALG13</italic>. Together with the 15 well&ndash;established epileptic encephalopathy genes, we statistically confirm the association of an additional ten putative epileptic encephalopathy genes. We show that only &sim;12% of epileptic encephalopathy patients in our cohort are explained by <italic>de novo</italic> mutations in one of these 24 genes, highlighting the extreme locus heterogeneity of the epileptic encephalopathies. </p><p>Finally, we investigated multiplex epilepsy families to uncover novel epilepsy susceptibility factors. Candidate variants emerging from sequencing within discovery families were further assessed by cosegregation testing, variant association testing in a case&ndash;control cohort, and gene&ndash;based resequencing in a cohort of additional multiplex epilepsy families. Despite employing multiple approaches, we did not identify any clear genetic associations with epilepsy. This work has, however, identified a set of candidates that may include real risk factors for epilepsy; the most promising of these is the <italic>MYCBP2</italic> gene. This work emphasizes the extremely high locus and allelic heterogeneity of the epilepsies and demonstrates that very large sample sizes are needed to uncover novel genetic risk factors. </p><p>Collectively, this body of work has securely implicated three novel neurodevelopmental disease genes that inform the underlying pathology of these disorders. Furthermore, in the final three studies, this work has highlighted additional candidate variants and genes that may ultimately be validated as disease&ndash;causing as sample sizes increase.</p>Dissertatio

The Characterization of Twenty Sequenced Human Genomes

We present the analysis of twenty human genomes to evaluate the prospects for identifying rare functional variants that contribute to a phenotype of interest. We sequenced at high coverage ten “case” genomes from individuals with severe hemophilia A and ten “control” genomes. We summarize the number of genetic variants emerging from a study of this magnitude, and provide a proof of concept for the identification of rare and highly-penetrant functional variants by confirming that the cause of hemophilia A is easily recognizable in this data set. We also show that the number of novel single nucleotide variants (SNVs) discovered per genome seems to stabilize at about 144,000 new variants per genome, after the first 15 individuals have been sequenced. Finally, we find that, on average, each genome carries 165 homozygous protein-truncating or stop loss variants in genes representing a diverse set of pathways

The aptamer core of SAM-IV riboswitches mimics the ligand-binding site of SAM-I riboswitches

A novel family of riboswitches, called SAM-IV, is the fourth distinct set of mRNA elements to be reported that regulate gene expression via direct sensing of S-adenosylmethionine (SAM or AdoMet). SAM-IV riboswitches share conserved nucleotide positions with the previously described SAM-I riboswitches, despite rearranged structures and nucleotide positions with family-specific nucleotide identities. Sequence analysis and molecular recognition experiments suggest that SAM-I and SAM-IV riboswitches share similar ligand binding sites, but have different scaffolds. Our findings support the view that RNA has considerable structural versatility and reveal that riboswitches exploit this potential to expand the scope of RNA in genetic regulation

Inherited and De Novo Genetic Risk for Autism Impacts Shared Biological Networks

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