Multiple apical plasma membrane constituents are associated with susceptibility to meconium ileus in individuals with cystic fibrosis

Abstract

Variants associated with meconium ileus in cystic fibrosis were identified in 3,763 affected individuals by genome-wide association study (GWAS). Five SNPs at two loci near SLC6A14 at Xq23-24 (minimum P = 1.28 × 10(-12) at rs3788766) and SLC26A9 at 1q32.1 (minimum P = 9.88 × 10(-9) at rs4077468) accounted for ~5% of phenotypic variability and were replicated in an independent sample of affected individuals (n = 2,372; P = 0.001 and 0.0001, respectively). By incorporating the knowledge that disease-causing mutations in CFTR alter electrolyte and fluid flux across surface epithelium into a hypothesis-driven GWAS (GWAS-HD), we identified associations with the same SNPs in SLC6A14 and SLC26A9 and established evidence for the involvement of SNPs in a third solute carrier gene, SLC9A3. In addition, GWAS-HD provided evidence of association between meconium ileus and multiple genes encoding constituents of the apical plasma membrane where CFTR resides (P = 0.0002; testing of 155 apical membrane genes jointly and in replication, P = 0.022). These findings suggest that modulating activities of apical membrane constituents could complement current therapeutic paradigms for cystic fibrosis.

Figure 1

Meconium Ileus GWAS identifies genome-wide significant SNPs. Association analysis was performed on all SNPs with minor allele frequencies > 2% that passed QC criteria (Online Methods). (a) Genome-wide Manhattan plot for meconium ileus. The black solid line corresponds to the genome-wide significance threshold (P<5×10⁻⁸), and the black dashed line to the suggestive association threshold, expected once per genome scan (P <1/543,927=1.84×10⁻⁶). A total of five SNPs in two regions (SLC6A14 on chromosome X, and SLC26A9 on chromosome 1) have association evidence exceeding the genome-wide threshold. The SNPs, rs4077468 and rs4077469 are in perfect LD and appear as one SNP as they are separated by only 128bp. (b) Regional plot for SLC26A9. LocusZoom viewer was used to display the association evidence around SLC26A9 based on NCBI Build 36/hg18. Symbol coloring reflects HapMap CEU LD r² values with the most significant SNP. The significant SNPs, rs4077468, rs7419153 and rs12047830 (Table 2), occur 2.17 kb, 4.72 kb and 4.12 kb upstream, respectively, of the transcription start site. The significant SNP, rs7512462, occurs in intron 5. A gap occurs in the genomic sequence between the SLC26A9 and FAM72A genes in both NCBI36.3 and GRCh37 primary reference assemblies. (c) Regional plot for SLC6A14. The association evidence around SLC6A14 was displayed as above. Rs3788766 (Table 2) is positioned 0.95 kb upstream of the transcription start site and is within the binding site of the CEBPB transcription factor as annotated by ENCODE (not shown). The mRNA transcript corresponding to CXorf61 is downstream (3’) of SLC6A14.

Figure 2

The apical membrane hypothesis identifies genes associated with meconium ileus. A list of 157 genes was annotated using the AmiGO tool (version 1.7; March 28, 2010) based on the Gene Ontology data (GO:00163245) using the cell location search phrase “apical plasma membrane” with restriction to Homo sapiens. In total, 3,814 GWAS SNPs are within ±10 kb of the boundaries of 155 genes (NCBI36/hg18). Two genes were not tagged by any of the GWAS SNPs; SLC6A14 was not annotated to the apical plasma membrane. (a) QQ-plot of the apical SNPs in the discovery sample. The observed association statistics (red), and the statistics calculated from the 10,000 phenotype-permutated replicates are shown (light gray). (b) Statistical significance of the apical membrane hypothesis in the discovery sample. Statistical significance (permutation P = 0.0002) was established via a sum statistic, summing the association evidence (Wald χ²₁ statistic) over all the 3,814 SNPs with the observed sum statistic displayed as a vertical line (red), and the 10,000 permutation-based sum statistics displayed as a histogram (light gray). (c) QQ-plot of the apical SNPs in the replication sample. The SNPs in the French replication cohort were required to have MAF > 6% because of the reduced sample size (1232 × 6% ≈ 3763 × 2%). In total 3,420 GWAS SNPs are within ±10 kb of the boundaries of 154 apical genes. (d) Statistical significance of the apical membrane hypothesis in the French replication sample (permutation P=22/1,000=0.022).

Figure 3

Assessment of the nuclear envelope null hypothesis. A list of 231 genes was generated from the nuclear envelope as defined by GO annotation (GO:0005635) similarly as for the apical membrane list. In total, 3,537 GWAS SNPs are within ±10 kb of the boundaries of 224 tagged genes (NCBI36/hg18). A priori, the nuclear envelope list should not contain genes associated with meconium ileus (under the null of no association). (a) QQ-plot of the nuclear envelope gene SNPs in the discovery sample. (b) Statistical significance of the nuclear envelope hypothesis in the discovery sample. Statistical evaluation indicates that genes listed in the nuclear envelope are, as expected, not significantly associated with meconium ileus (permutation P=4639/10,000=0.4639).