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Asthma is a complex genetic disease caused by a combination of genetic and environmental risk factors. We sought to test classes of genetic variants largely missed by genome-wide association studies (GWAS), including copy number variants (CNVs) and low-frequency variants, by performing whole-genome sequencing (WGS) on 16 individuals from asthma-enriched and asthma-depleted families. The samples were obtained from an extended 13-generation Hutterite pedigree with reduced genetic heterogeneity due to a small founding gene pool and reduced environmental heterogeneity as a result of a communal lifestyle. We sequenced each individual to an average depth of 13-fold, generated a comprehensive catalog of genetic variants, and tested the most severe mutations for association with asthma. We identified and validated 1960 CNVs, 19 nonsense or splice-site single nucleotide variants (SNVs), and 18 insertions or deletions that were out of frame. As follow-up, we performed targeted sequencing of 16 genes in 837 cases and 540 controls of Puerto Rican ancestry and found that controls carry a significantly higher burden of mutations in IL27RA (2.0% of controls; 0.23% of cases; nominal p = 0.004; Bonferroni p = 0.21). We also genotyped 593 CNVs in 1199 Hutterite individuals. We identified a nominally significant association (p = 0.03; Odds ratio (OR) = 3.13) between a 6 kbp deletion in an intron of NEDD4L and increased risk of asthma. We genotyped this deletion in an additional 4787 non-Hutterite individuals (nominal p = 0.056; OR = 1.69). NEDD4L is expressed in bronchial epithelial cells, and conditional knockout of this gene in the lung in mice leads to severe inflammation and mucus accumulation. Our study represents one of the early instances of applying WGS to complex disease with a large environmental component and demonstrates how WGS can identify risk variants, including CNVs and low-frequency variants, largely untested in GWAS. [ABSTRACT FROM AUTHOR]

It is well established that autism spectrum disorders (ASD) have a strong genetic component; however, for at least 70% of cases, the underlying genetic cause is unknown. Under the hypothesis that de novo mutations underlie a substantial fraction of the risk for developing ASD in families with no previous history of ASD or related phenotypes-so-called sporadic or simplex families-we sequenced all coding regions of the genome (the exome) for parent-child trios exhibiting sporadic ASD, including 189 new trios and 20 that were previously reported. Additionally, we also sequenced the exomes of 50 unaffected siblings corresponding to these new (n = 31) and previously reported trios (n = 19), for a total of 677 individual exomes from 209 families. Here we show that de novo point mutations are overwhelmingly paternal in origin (4:1 bias) and positively correlated with paternal age, consistent with the modest increased risk for children of older fathers to develop ASD. Moreover, 39% (49 of 126) of the most severe or disruptive de novo mutations map to a highly interconnected ?-catenin/chromatin remodelling protein network ranked significantly for autism candidate genes. In proband exomes, recurrent protein-altering mutations were observed in two genes: CHD8 and NTNG1. Mutation screening of six candidate genes in 1,703 ASD probands identified additional de novo, protein-altering mutations in GRIN2B, LAMC3 and SCN1A. Combined with copy number variant (CNV) data, these results indicate extreme locus heterogeneity but also provide a target for future discovery, diagnostics and therapeutics. [ABSTRACT FROM AUTHOR]

Evidence for the etiology of autism spectrum disorders (ASDs) has consistently pointed to a strong genetic component complicated by substantial locus heterogeneity. We sequenced the exomes of 20 individuals with sporadic ASD (cases) and their parents, reasoning that these families would be enriched for de novo mutations of major effect. We identified 21 de novo mutations, 11 of which were protein altering. Protein-altering mutations were significantly enriched for changes at highly conserved residues. We identified potentially causative de novo events in 4 out of 20 probands, particularly among more severely affected individuals, in FOXP1, GRIN2B, SCN1A and LAMC3. In the FOXP1 mutation carrier, we also observed a rare inherited CNTNAP2 missense variant, and we provide functional support for a multi-hit model for disease risk. Our results show that trio-based exome sequencing is a powerful approach for identifying new candidate genes for ASDs and suggest that de novo mutations may contribute substantially to the genetic etiology of ASDs. [ABSTRACT FROM AUTHOR]

We demonstrate the successful application of exome sequencing to discover a gene for an autosomal dominant disorder, Kabuki syndrome (OMIM%147920). We subjected the exomes of ten unrelated probands to massively parallel sequencing. After filtering against existing SNP databases, there was no compelling candidate gene containing previously unknown variants in all affected individuals. Less stringent filtering criteria allowed for the presence of modest genetic heterogeneity or missing data but also identified multiple candidate genes. However, genotypic and phenotypic stratification highlighted MLL2, which encodes a Trithorax-group histone methyltransferase: seven probands had newly identified nonsense or frameshift mutations in this gene. Follow-up Sanger sequencing detected MLL2 mutations in two of the three remaining individuals with Kabuki syndrome (cases) and in 26 of 43 additional cases. In families where parental DNA was available, the mutation was confirmed to be de novo (n = 12) or transmitted (n = 2) in concordance with phenotype. Our results strongly suggest that mutations in MLL2 are a major cause of Kabuki syndrome. [ABSTRACT FROM AUTHOR]

We demonstrate the first successful application of exome sequencing to discover the gene for a rare mendelian disorder of unknown cause, Miller syndrome (MIM%263750). For four affected individuals in three independent kindreds, we captured and sequenced coding regions to a mean coverage of 40× and sufficient depth to call variants at ∼97% of each targeted exome. Filtering against public SNP databases and eight HapMap exomes for genes with two previously unknown variants in each of the four individuals identified a single candidate gene, DHODH, which encodes a key enzyme in the pyrimidine de novo biosynthesis pathway. Sanger sequencing confirmed the presence of DHODH mutations in three additional families with Miller syndrome. Exome sequencing of a small number of unrelated affected individuals is a powerful, efficient strategy for identifying the genes underlying rare mendelian disorders and will likely transform the genetic analysis of monogenic traits. [ABSTRACT FROM AUTHOR]

The extent to which non-coding mutations contribute to Mendelian disease is a major unknown in human genetics. Relatedly, the vast majority of candidate regulatory elements have yet to be functionally validated. Here, we describe a CRISPR-based system that uses pairs of guide RNAs (gRNAs) to program thousands of kilobase-scale deletions that deeply scan across a targeted region in a tiling fashion (“ScanDel”). We applied ScanDel to HPRT1 , the housekeeping gene underlying Lesch-Nyhan syndrome, an X-linked recessive disorder. Altogether, we programmed 4,342 overlapping 1 and 2 kb deletions that tiled 206 kb centered on HPRT1 (including 87 kb upstream and 79 kb downstream) with median 27-fold redundancy per base. We functionally assayed programmed deletions in parallel by selecting for loss of HPRT function with 6-thioguanine. As expected, sequencing gRNA pairs before and after selection confirmed that all HPRT1 exons are needed. However, HPRT1 function was robust to deletion of any intergenic or deeply intronic non-coding region, indicating that proximal regulatory sequences are sufficient for HPRT1 expression. Although our screen did identify the disruption of exon-proximal non-coding sequences (e.g., the promoter) as functionally consequential, long-read sequencing revealed that this signal was driven by rare, imprecise deletions that extended into exons. Our results suggest that no singular distal regulatory element is required for HPRT1 expression and that distal mutations are unlikely to contribute substantially to Lesch-Nyhan syndrome burden. Further application of ScanDel could shed light on the role of regulatory mutations in disease at other loci while also facilitating a deeper understanding of endogenous gene regulation. [ABSTRACT FROM AUTHOR]