Genetic variation in severe cystic fibrosis liver disease is associated with novel mechanisms for disease pathogenesis

Abstract

Background and aims: It is not known why severe cystic fibrosis (CF) liver disease (CFLD) with portal hypertension occurs in only ~7% of people with CF. We aimed to identify genetic modifiers for severe CFLD to improve understanding of disease mechanisms.

Approach and results: Whole-genome sequencing was available in 4082 people with CF with pancreatic insufficiency (n = 516 with severe CFLD; n = 3566 without CFLD). We tested ~15.9 million single nucleotide polymorphisms (SNPs) for association with severe CFLD versus no-CFLD, using pre-modulator clinical phenotypes including (1) genetic variant ( SERPINA1 ; Z allele) previously associated with severe CFLD; (2) candidate SNPs (n = 205) associated with non-CF liver diseases; (3) genome-wide association study of common/rare SNPs; (4) transcriptome-wide association; and (5) gene-level and pathway analyses. The Z allele was significantly associated with severe CFLD ( p = 1.1 × 10 -4 ). No significant candidate SNPs were identified. A genome-wide association study identified genome-wide significant SNPs in 2 loci and 2 suggestive loci. These 4 loci contained genes [significant, PKD1 ( p = 8.05 × 10 -10 ) and FNBP1 ( p = 4.74 × 10 -9 ); suggestive, DUSP6 ( p = 1.51 × 10 -7 ) and ANKUB1 ( p = 4.69 × 10 -7 )] relevant to severe CFLD pathophysiology. The transcriptome-wide association identified 3 genes [ CXCR1 ( p = 1.01 × 10 -6 ) , AAMP ( p = 1.07 × 10 -6 ), and TRBV24 ( p = 1.23 × 10 -5 )] involved in hepatic inflammation and innate immunity. Gene-ranked analyses identified pathways enriched in genes linked to multiple liver pathologies.

Conclusion: These results identify loci/genes associated with severe CFLD that point to disease mechanisms involving hepatic fibrosis, inflammation, innate immune function, vascular pathology, intracellular signaling, actin cytoskeleton and tight junction integrity and mechanisms of hepatic steatosis and insulin resistance. These discoveries will facilitate mechanistic studies and the development of therapeutics for severe CFLD.

Figure 1.. Genetic loci significantly or suggestively associated with severe cystic fibrosis liver disease (CFLD) phenotype.

Genome-wide Manhattan plot of associations in 516 CF patients with severe CFLD versus 3,566 CF patients with no severe CFLD (CFnoLD). Red line shows genome-wide significance of p < 5×10–8. Blue line shows a suggestive significance of p < 5×10–7. The inset shows plot of the genomic inflation factor (lambda).

Figure 2.. Locus Zoom plots for four loci with genome-wide significant (A and B; p < 5×10–8) or suggestive (C and D; p < 5 × 10–7) associations with severe CFLD.

Dotted line shows genome-wide significance of p < 5×10–8.

Figure 3.. Transcriptome-wide association evidence for expression versus severe CFLD.

CXCR1 / AAMP had increased expression associated with increased CFLD risk (p < 9.2×10⁻⁶) (shown as just 1 dot, since SNPs are close in proximity) and TRBV24–1 had increased expression associated with decreased CFLD risk (p < 1.2×10⁻⁵). Horizontal black line corresponds to a transcriptome-wide false discovery q < 0.15.

Figure 4.. Genes that drive core enrichment–significant results for the phospholipase d signaling pathway (KEGG; hsa04072).

This VEGAS2 analysis Gene Set Enrichment Analysis plot includes 25 (of 36) genes that are linked to pathogenic mechanisms associated with liver disease in non-CF subjects (shown in bold). Seven of these genes (note asterisks *) are also in the KEGG lipolysis pathway (hsa04923; see Supplemental Figure 2a). These pathogenic mechanisms (hepatic fibrosis; inflammation; endothelial/vascular pathology; fatty liver/hepatic steatosis; actin/cytoskeleton function; and autoimmune hepatitis) have relevance to the potential pathogenesis of severe CFLD (see Supplemental Table 8).