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Novel idiopathic pulmonary fibrosis susceptibility variants revealed by deep sequencing.

ABSTRACT: Background:Specific common and rare single nucleotide variants (SNVs) increase the likelihood of developing sporadic idiopathic pulmonary fibrosis (IPF). We performed target-enriched sequencing on three loci previously identified by a genome-wide association study to gain a deeper understanding of the full spectrum of IPF genetic risk and performed a two-stage case-control association study. Methods:A total of 1.7?Mb of DNA from 181 IPF patients was deep sequenced (>100×) across 11p15.5, 14q21.3 and 17q21.31 loci. Comparisons were performed against 501 unrelated controls and replication studies were assessed in 3968 subjects. Results:36 SNVs were associated with IPF susceptibility in the discovery stage (p<5.0×10-8). After meta-analysis, the strongest association corresponded to rs35705950 (p=9.27×10-57) located upstream from the mucin 5B gene (MUC5B). Additionally, a novel association was found for two co-inherited low-frequency SNVs (<5%) in MUC5AC, predicting a missense amino acid change in mucin 5AC (lowest p=2.27×10-22). Conditional and haplotype analyses in 11p15.5 supported the existence of an additional contribution of MUC5AC variants to IPF risk. Conclusions:This study reinforces the significant IPF associations of these loci and implicates MUC5AC as another key player in IPF susceptibility.

SUBMITTER: Lorenzo-Salazar JM

PROVIDER: S-EPMC6556557 | biostudies-literature | 2019 Apr

REPOSITORIES: biostudies-literature

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Novel idiopathic pulmonary fibrosis susceptibility variants revealed by deep sequencing.

Lorenzo-Salazar Jose M JM Ma Shwu-Fan SF Jou Jonathan J Hou Pei-Chi PC Guillen-Guio Beatriz B Allen Richard J RJ Jenkins R Gisli RG Wain Louise V LV Oldham Justin M JM Noth Imre I Flores Carlos C

ERJ open research 20190401 2

<h4>Background</h4>Specific common and rare single nucleotide variants (SNVs) increase the likelihood of developing sporadic idiopathic pulmonary fibrosis (IPF). We performed target-enriched sequencing on three loci previously identified by a genome-wide association study to gain a deeper understanding of the full spectrum of IPF genetic risk and performed a two-stage case-control association study.<h4>Methods</h4>A total of 1.7 Mb of DNA from 181 IPF patients was deep sequenced (>100×) across 1 ...[more]

PMID: 31205927

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Project description:BackgroundIdiopathic pulmonary fibrosis (IPF) is a devastating disease that probably involves several genetic loci. Several rare genetic variants and one common single nucleotide polymorphism (SNP) of MUC5B have been associated with the disease. Our aim was to identify additional common variants associated with susceptibility and ultimately mortality in IPF.MethodsFirst, we did a three-stage genome-wide association study (GWAS): stage one was a discovery GWAS; and stages two and three were independent case-control studies. DNA samples from European-American patients with IPF meeting standard criteria were obtained from several US centres for each stage. Data for European-American control individuals for stage one were gathered from the database of genotypes and phenotypes; additional control individuals were recruited at the University of Pittsburgh to increase the number. For controls in stages two and three, we gathered data for additional sex-matched European-American control individuals who had been recruited in another study. DNA samples from patients and from control individuals were genotyped to identify SNPs associated with IPF. SNPs identified in stage one were carried forward to stage two, and those that achieved genome-wide significance (p<5 × 10(-8)) in a meta-analysis were carried forward to stage three. Three case series with follow-up data were selected from stages one and two of the GWAS using samples with follow-up data. Mortality analyses were done in these case series to assess the SNPs associated with IPF that had achieved genome-wide significance in the meta-analysis of stages one and two. Finally, we obtained gene-expression profiling data for lungs of patients with IPF from the Lung Genomics Research Consortium and analysed correlation with SNP genotypes.FindingsIn stage one of the GWAS (542 patients with IPF, 542 control individuals matched one-by-one to cases by genetic ancestry estimates), we identified 20 loci. Six SNPs reached genome-wide significance in stage two (544 patients, 687 control individuals): three TOLLIP SNPs (rs111521887, rs5743894, rs5743890) and one MUC5B SNP (rs35705950) at 11p15.5; one MDGA2 SNP (rs7144383) at 14q21.3; and one SPPL2C SNP (rs17690703) at 17q21.31. Stage three (324 patients, 702 control individuals) confirmed the associations for all these SNPs, except for rs7144383. Linkage disequilibrium between the MUC5B SNP (rs35705950) and TOLLIP SNPs (rs111521887 [r(2)=0·07], rs5743894 [r(2)=0·16], and rs5743890 [r(2)=0·01]) was low. 683 patients from the GWAS were included in the mortality analysis. Individuals who developed IPF despite having the protective TOLLIP minor allele of rs5743890 carried an increased mortality risk (meta-analysis with fixed-effect model: hazard ratio 1·72 [95% CI 1·24-2·38]; p=0·0012). TOLLIP expression was decreased by 20% in individuals carrying the minor allele of rs5743890 (p=0·097), 40% in those with the minor allele of rs111521887 (p=3·0 × 10(-4)), and 50% in those with the minor allele of rs5743894 (p=2·93 × 10(-5)) compared with homozygous carriers of common alleles for these SNPs.InterpretationNovel variants in TOLLIP and SPPL2C are associated with IPF susceptibility. One novel variant of TOLLIP, rs5743890, is also associated with mortality. These associations and the reduced expression of TOLLIP in patients with IPF who carry TOLLIP SNPs emphasise the importance of this gene in the disease.FundingNational Institutes of Health; National Heart, Lung, and Blood Institute; Pulmonary Fibrosis Foundation; Coalition for Pulmonary Fibrosis; and Instituto de Salud Carlos III.

Project description:Rationale: To identify functionally relevant common genetic risk variants associated with idiopathic pulmonary fibrosis (IPF), we performed expression quantitative trait loci (eQTL) and methylation quantitative trait loci (mQTL) mapping, followed by co-localization of eQTL and mQTL with genetic association signals as well as mediation analysis. Methods: Illumina MEGA genotyping arrays, mRNA sequencing, and Illumina 850k methylation arrays were performed on lung tissue of participants with IPF (234 RNA and 345 DNA samples) and non-diseased control (188 RNA and 202 DNA samples). Transcriptome and methylome datasets were normalized for unknown batch effects using Probabilistic Estimation of Expression Residuals (PEER). eQTL, cell type-interaction eQTL, and mQTL analyses were performed in FastQTL and co-localization analysis in eCAVIAR separately for cases and controls. Benjamini-Hochberg false discovery rate was used for adjustment for multiple comparisons. Results: After appropriate adjustment, we identified 4,745 genes with significant eQTLs in controls and 6,047 in cases (FDR-adjusted p<0.05). Focusing on genetic variants within 10 primary IPF-associated genetic loci, we identified 27 eQTLs in controls and 24 eQTLs in cases (FDR-adjusted p<0.05). Among these signals, we identified association of rs35705950 with expression of MUC5B (Chr11 locus) and rs2076295 with expression of DSP (Chr6 locus) in both cases and controls. To address cell specificity, we performed cell type-interaction eQTL analysis and identified an association of rs2076295 with expression of DSP in smooth muscle cells. mQTL analysis identified CpGs in gene bodies of MUC5B (cg17589883) and DSP (cg08964675) associated with the lead variants in these two loci. eCAVIAR demonstrated strong co-localization of eQTL/mQTL and genetic signal in MUC5B (rs35705950) and DSP (rs2076295) in both cases and controls. Mediation analysis demonstrated partial mediation of the effect of common variants in MUC5B and DSP loci on disease risk though MUC5B and DSP gene expression. Functional validation of the mQTL in MUC5B demonstrates that the CpG is within a putative internal repressor element that interacts through a 3D loop with the enhancer containing the rs35705950 genetic variant. Conclusions: Using lung eQTL/mQTL, co-localization, and mediation analyses, we have established the functional validation of the common IPF genetic risk variants for MUC5B (rs35705950) and DSP (rs2076295). These results provide additional evidence that both MUC5B and DSP are involved in the etiology of IPF.

Project description:BackgroundIdiopathic pulmonary fibrosis (IPF) is a chronic progressive lung disease with high mortality, uncertain cause, and few treatment options. Studies have identified a significant genetic risk associated with the development of IPF; however, mechanisms by which genetic risk factors promote IPF remain unclear. We aimed to identify genetic variants associated with IPF susceptibility and provide mechanistic insight using gene and protein expression analyses.MethodsWe used a two-stage approach: a genome-wide association study in patients with IPF of European ancestry recruited from nine different centres in the UK and controls selected from UK Biobank (stage 1) matched for age, sex, and smoking status; and a follow-up of associated genetic variants in independent datasets of patients with IPF and controls from two independent US samples from the Chicago consortium and the Colorado consortium (stage 2). We investigated the effect of novel signals on gene expression in large transcriptomic and genomic data resources, and examined expression using lung tissue samples from patients with IPF and controls.Findings602 patients with IPF and 3366 controls were selected for stage 1. For stage 2, 2158 patients with IPF and 5195 controls were selected. We identified a novel genome-wide significant signal of association with IPF susceptibility near A-kinase anchoring protein 13 (AKAP13; rs62025270, odds ratio [OR] 1·27 [95% CI 1·18-1·37], p=1·32 × 10-9) and confirmed previously reported signals, including in mucin 5B (MUC5B; rs35705950, OR 2·89 [2·56-3·26], p=1·12 × 10-66) and desmoplakin (DSP; rs2076295, OR 1·44 [1·35-1·54], p=7·81 × 10-28). For rs62025270, the allele A associated with increased susceptibility to IPF was also associated with increased expression of AKAP13 mRNA in lung tissue from patients who had lung resection procedures (n=1111). We showed that AKAP13 is expressed in the alveolar epithelium and lymphoid follicles from patients with IPF, and AKAP13 mRNA expression was 1·42-times higher in lung tissue from patients with IPF (n=46) than that in lung tissue from controls (n=51).InterpretationAKAP13 is a Rho guanine nucleotide exchange factor regulating activation of RhoA, which is known to be involved in profibrotic signalling pathways. The identification of AKAP13 as a susceptibility gene for IPF increases the prospect of successfully targeting RhoA pathway inhibitors in patients with IPF.FundingUK Medical Research Council, National Heart, Lung, and Blood Institute of the US National Institutes of Health, Agencia Canaria de Investigación, Innovación y Sociedad de la Información, Spain, UK National Institute for Health Research, and the British Lung Foundation.

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Novel idiopathic pulmonary fibrosis susceptibility variants revealed by deep sequencing.

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Novel idiopathic pulmonary fibrosis susceptibility variants revealed by deep sequencing.

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