Whole-Genome Sequencing of Individuals from a Founder Population Identifies Candidate Genes for Asthma

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.

The Nedd4-2/Ndfip1 axis is a negative regulator of IgE-mediated mast cell activation

“…Human genetic studies have associated variants in NDFIP1 with asthma52 and other inflammatory diseases (that is, rheumatoid arthritis53 and inflammatory bowel disease54). More recently, in a whole-genome sequencing study on individuals from a Hutterite population, a 6 kbp deletion in an intron in NEDD4L has been associated with increased risk of asthma23. Our experimental findings here, raise the possibility that such genetic alterations perturb the ability of Nedd4-2 to negatively regulate mast cell signalling, as well as that of other cell populations expressing this ligase and adaptor50, resulting in sustained inflammatory responses and a contribution to the sequelae of allergic disease in affected individuals.…”

“…To date, Nedd4-2 is best known for its ability to regulate stability and activity of ion channels and transporters, particularly in epithelial cells22, but little is known about the role of this ubiquitin ligase in allergic inflammation. Recently, genetic studies from asthma-enriched families have identified a variant in NEDD4L associated with increased risk of the disease23. We have found that mast cells express Nedd4-2 and importantly, loss of Nedd4-2 in foetal liver-derived mast cells (FLMCs) or bone marrow-derived cultured mast cells (BMCMCs) not only results in heightened and sustained pro-inflammatory mediator release by mast cells in vitro , but also in prolonged IgE-mediated passive cutaneous anaphylaxis reactions in three different types of mast cell-deficient mice engrafted with Nedd4-2 −/− mast cells.…”

Complex genetics of pulmonary diseases: lessons from genome-wide association studies and next-generation sequencing

“…They were unable to show that the asthmatic members have significantly higher burden of rare variants. Recently, Campbell et al 60 conducted a whole genome sequencing (WGS) on 16 individuals (8 asthmatic) from a Hutterite population in order to find novel asthma genetic variants that have not been uncovered in previous GWASs. However, none of their discovered variants met genome-wide significance threshold, specifically mutations within gene NEDD4L, which contributes to the ubiquitination of specific proteins for lysosomal degradation.…”

Systems biology of asthma and allergic diseases: A multiscale approach

“…9 candidate asthma genes in 965 subjects 17 ) or for discovery in limited numbers of individuals (e.g. whole genome sequencing in 16 subjects with well-characterized asthma 18 ).…”