Abstract
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.
Highlights
Complex genetic diseases are caused by many genetic and environmental factors
Genome-wide association studies (GWAS) have been powerful in identifying novel pathways associated with complex traits, including asthma, the small proportion of genetic risk explained by known associated single nucleotide polymorphisms (SNPs) suggests that other forms of genetic variation may play a substantial role in the development of disease
We sequenced each of these 16 genomes to a coverage of 13-fold using an Illumina paired-end protocol (Table 1) as described previously [48]; all sequencing data are available in dbGaP
Summary
Complex genetic diseases are caused by many genetic and environmental factors. In the case of asthma, it has been estimated that genetic factors comprise 60% of the risk of developing this disease [1,2,3,4]. GWAS have been powerful in identifying novel pathways associated with complex traits, including asthma, the small proportion of genetic risk explained by known associated single nucleotide polymorphisms (SNPs) suggests that other forms of genetic variation may play a substantial role in the development of disease. This conclusion is supported by a recent resequencing study that found evidence for a role of rare variants in the development of asthma [17]. A more comprehensive survey of rare genetic variants in asthma is warranted
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