De novo missense mutations in neurodevelopmental disorders

Thesis (Ph.D.)--University of Washington, 2019Autism spectrum disorder (ASD) is a pervasive neurodevelopmental disorder (NDD) with a high prevalence in the US (1 in 59 children). It is commonly comorbid with other NDDs such as developmental delay (DD), intellectual disability (ID), and epilepsy (EPI). In this thesis, I examine the role of de novo missense mutations in NDDs with a goal of identifying genes and specific mutations that are candidates for pathogenicity. I characterize the aggregate signal for de novo missense mutations in 8,477 NDD cases, finding both quantitative and qualitative differences between mutations in cases and controls. I also find 40 amino acids that bear de novo substitutions in two or more unrelated individuals and develop a tool to assess the likelihood of these observations in the context of stochastic de novo events. I then use targeted sequencing to further establish the association of these recurrent mutations with disease. Upon finding the same p.Ala646Thr substitution in five cases in glutamate receptor subunit GRIA1, I carry out functional experiments that show alterations in ion flux. I also assessed clustering of de novo missense mutations as this pattern is associated with NDDs, such as Schinzel-Giedion syndrome. I used an unsupervised clustering algorithm, CLUMP, to compare the distribution of de novo missense mutations in NDD cases with private missense events in controls and found 200 genes that were significantly more clustered (p < 0.05). As this set of genes is enriched for neuronal functions, a known association of NDD risk genes, it is likely that clustering is a valid feature for identification of disease genes. With increased exome sequencing on NDD cases, I was able to assess de novo mutation burden in 10,927 cases with ASD, DD, or ID. With two different models, I found 253 total genes with more de novo mutations than expected, 123 of which have a burden of missense mutations. Protein-protein interaction and enrichment analyses of genes with a burden of mutation finds that those with a burden of truncating mutations have roles in transcription regulation while those with missense burden have roles in synaptic signaling. This same neuronal enrichment, including in the amygdala and cortex during fetal development, is seen in genes with clustered de novo missense mutations. Interestingly, the phenotypes of patients with missense mutations in a novel gene, TRRAP, segregate with mutation clustering, suggesting the biological relevance of this pattern of mutation. As burden analysis only identified some of the expected pathogenic NDD genes, I included mutations from patients with EPI to my discovery set. Novel genes identified with this addition are enriched for expression in the striatum. Targeted sequencing of these hotspots of mutation identified additional substitutions at 20 recurrent sites and established 28 new recurrent sites. Eighteen of the sites are known to be pathogenic, and some evidence supports the disease association of the remaining 30 sites. Continued assessment of genes with these patterns of mutation, as well as expansion into gene families, will help to characterize the genetic architecture of NDDs, specifically missense mutations, and provide increased understanding of brain development and pathogenesis

Neurodevelopmental disease genes implicated by de novo mutation and copy number variation morbidity

We combined de novo mutation (DNM) data from 10,927 individuals with developmental delay and autism to identify 253 candidate neurodevelopmental disease genes with an excess of missense and/or likely gene-disruptive (LGD) mutations. Of these genes, 124 reach exome-wide significance (P &lt; 5 × 10-7) for DNM. Intersecting these results with copy number variation (CNV) morbidity data shows an enrichment for genomic disorder regions (30/253, likelihood ratio (LR) +1.85, P = 0.0017). We identify genes with an excess of missense DNMs overlapping deletion syndromes (for example, KIF1A and the 2q37 deletion) as well as duplication syndromes, such as recurrent MAPK3 missense mutations within the chromosome 16p11.2 duplication, recurrent CHD4 missense DNMs in the 12p13 duplication region, and recurrent WDFY4 missense DNMs in the 10q11.23 duplication region. Network analyses of genes showing an excess of DNMs highlights functional networks, including cell-specific enrichments in the D1+ and D2+ spiny neurons of the striatum

Genome Sequencing of Idiopathic Pulmonary Fibrosis in Conjunction with a Medical School Human Anatomy Course

<div><p>Even in cases where there is no obvious family history of disease, genome sequencing may contribute to clinical diagnosis and management. Clinical application of the genome has not yet become routine, however, in part because physicians are still learning how best to utilize such information. As an educational research exercise performed in conjunction with our medical school human anatomy course, we explored the potential utility of determining the whole genome sequence of a patient who had died following a clinical diagnosis of idiopathic pulmonary fibrosis (IPF). Medical students performed dissection and whole genome sequencing of the cadaver. Gross and microscopic findings were more consistent with the fibrosing variant of nonspecific interstitial pneumonia (NSIP), as opposed to IPF <i>per se</i>. Variants in genes causing Mendelian disorders predisposing to IPF were not detected. However, whole genome sequencing identified several common variants associated with IPF, including a single nucleotide polymorphism (SNP), rs35705950, located in the promoter region of the gene encoding mucin glycoprotein MUC5B. The <i>MUC5B</i> promoter polymorphism was recently found to markedly elevate risk for IPF, though a particular association with NSIP has not been previously reported, nor has its contribution to disease risk previously been evaluated in the genome-wide context of all genetic variants. We did not identify additional predicted functional variants in a region of linkage disequilibrium (LD) adjacent to <i>MUC5B</i>, nor did we discover other likely risk-contributing variants elsewhere in the genome. Whole genome sequencing thus corroborates the association of rs35705950 with <i>MUC5B</i> dysregulation and interstitial lung disease. This novel exercise additionally served a unique mission in bridging clinical and basic science education.</p></div

Integrated Genomics Viewer (IGV) screenshot of the rs35705950 variant.

<p>Integrated Genomics Viewer (IGV) screenshot of the rs35705950 variant.</p

Genes previously associated with familial IPF.

<p>Genes previously associated with familial IPF.</p

Variants associated with IPF that were also seen in this individual. Allele frequencies accessed 4/16/2014.

A<p>Discovery and ^Breplicate GWAS.</p><p>Variants associated with IPF that were also seen in this individual. Allele frequencies accessed 4/16/2014.</p

Sequencing coverage and variant distribution within the <i>MUC5B</i> locus.

<p>(<b>A</b>) Sequence coverage of the <i>MUC5B</i> gene. (<b>B</b>) Rare variants neighboring the <i>MUC5B</i> promoter variant rs35705950. Variant track is colored by allele frequency (blue: AF<2%, red: AF<5%, black: AF<10%). No other rare variants in this region overlap with putative transcription factor binding sites, consistent with the hypothesis that the rs35705950 is causative of <i>MUC5B</i> dysregulation. Plots were generated using the UCSC genome browser (<a href="http://www.genome.ucsc.edu" target="_blank">http://www.genome.ucsc.edu</a>).</p

Histological demonstration of the NSIP pattern of IPF in the patient's lungs. Microscopic examination of the lungs.

<p>(<b>A</b>) 40×; (<b>B</b>) 400×. Note uniform fibrotic thickening of the alveolar septae and type II pneumocyte hypertrophy. There was no histologic evidence of sarcoidosis, hypersensitivity pneumonitis, organizing pneumonia, or diffuse alveolar damage.</p

Hotspots of missense mutation identify neurodevelopmental disorder genes and functional domains

Although de novo missense mutations have been predicted to account for more cases of autism than gene-truncating mutations, most research has focused on the latter. We identified the properties of de novo missense mutations in patients with neurodevelopmental disorders (NDDs) and highlight 35 genes with excess missense mutations. Additionally, 40 amino acid sites were recurrently mutated in 36 genes, and targeted sequencing of 20 sites in 17,688 patients with NDD identified 21 new patients with identical missense mutations. One recurrent site substitution (p.A636T) occurs in a glutamate receptor subunit, GRIA1. This same amino acid substitution in the homologous but distinct mouse glutamate receptor subunit Grid2 is associated with Lurcher ataxia. Phenotypic follow-up in five individuals with GRIA1 mutations shows evidence of specific learning disabilities and autism. Overall, we find significant clustering of de novo mutations in 200 genes, highlighting specific functional domains and synaptic candidate genes important in NDD pathology.status: publishe