Genome-Wide Association Analysis of Protein-Coding Variants in IgA Nephropathy

Abstract

Significance statement: Genome-wide association studies have identified nearly 20 IgA nephropathy susceptibility loci. However, most nonsynonymous coding variants, particularly ones that occur rarely or at a low frequency, have not been well investigated. The authors performed a chip-based association study of IgA nephropathy in 8529 patients with the disorder and 23,224 controls. They identified a rare variant in the gene encoding vascular endothelial growth factor A (VEGFA) that was significantly associated with a two-fold increased risk of IgA nephropathy, which was further confirmed by sequencing analysis. They also identified a novel common variant in PKD1L3 that was significantly associated with lower haptoglobin protein levels. This study, which was well-powered to detect low-frequency variants with moderate to large effect sizes, helps expand our understanding of the genetic basis of IgA nephropathy susceptibility.

Background: Genome-wide association studies have identified nearly 20 susceptibility loci for IgA nephropathy. However, most nonsynonymous coding variants, particularly those occurring rarely or at a low frequency, have not been well investigated.

Methods: We performed a three-stage exome chip-based association study of coding variants in 8529 patients with IgA nephropathy and 23,224 controls, all of Han Chinese ancestry. Sequencing analysis was conducted to investigate rare coding variants that were not covered by the exome chip. We used molecular dynamic simulation to characterize the effects of mutations of VEGFA on the protein's structure and function. We also explored the relationship between the identified variants and the risk of disease progression.

Results: We discovered a novel rare nonsynonymous risk variant in VEGFA (odds ratio, 1.97; 95% confidence interval [95% CI], 1.61 to 2.41; P = 3.61×10 -11 ). Further sequencing of VEGFA revealed twice as many carriers of other rare variants in 2148 cases compared with 2732 controls. We also identified a common nonsynonymous risk variant in PKD1L3 (odds ratio, 1.16; 95% CI, 1.11 to 1.21; P = 1.43×10 -11 ), which was associated with lower haptoglobin protein levels. The rare VEGFA mutation could cause a conformational change and increase the binding affinity of VEGFA to its receptors. Furthermore, this variant was associated with the increased risk of kidney disease progression in IgA nephropathy (hazard ratio, 2.99; 95% CI, 1.09 to 8.21; P = 0.03).

Conclusions: Our study identified two novel risk variants for IgA nephropathy in VEGFA and PKD1L3 and helps expand our understanding of the genetic basis of IgA nephropathy susceptibility.

Graphical abstract

Figure 1

Manhattan plot showing significance of the association of each SNP allele with IgA nephropathy risk in the discovery cohort. The x axis shows the chromosome positions. The y axis indicates the level of statistical significance expressed as −log10 (P value). The horizontal red and blue lines indicate the genome-wide significant threshold (P = 5×10⁻⁸) and suggestive threshold (P = 1×10⁻⁴), respectively. Black, new genome-wide significant coding variants associated with IgA nephropathy; green, previously reported coding variants in GWASs for IgA nephropathy; the numbers represent variants with suggestive associations but did not exceed genome-wide significance in combined analyses (① SSUH2 p.C372S; ② ADAMTS9 p.R209C; ③ RPGRIP1L p.G1025S; ④ TCF3 p.T531M; ⑤ CD177 p.A3P; ⑥ MCM3AP p.V1363L). GWAS, genome-wide association study.

Figure 2

Regional association plots for two distinct genome-wide significant loci. (A) Regional plot for IgA nephropathy association at the VEGFA locus. (B) Regional plot for IgA nephropathy association at the PKD1L3 locus. The x axis shows the chromosome positions. The y axis indicates the level of statistical significance expressed as −log10 (P value). The gray dashed line corresponds to the genome-wide significance threshold. The blue line corresponds to the suggestive significance threshold (P = 1×10⁻⁴). Dot color indicates LD of each variant with the highlighted lead variant (purple diamond). LocusZoom was used to provide regional visualization of results with the East Asian panel of 1000 Genomes Phase 3 v5 (GRCh37/hg19). V1, validation 1; v2, validation 2; VEGFA, vascular endothelial growth factor A; PKD1L3, polycystin 1 like 3.

Figure 3

Rare coding variants in VEGFA that were identified by sequencing in 2148 IgA nephropathy cases and 2732 controls. The number of heterozygotes in IgA nephropathy and controls are listed. The p.V167I variant is shown in bold font. Variants are annotated on the longest RefSeq isoform NM_001025366.2/NP_001020537.2 but numbered according to NM_001171623.1/NP_001165094.1 (VEGFA-206). VEGFA, vascular endothelial growth factor A.

Figure 4

The effect of V167I mutation on binding affinity of VEGFA to VEGFR1 and VEGFR2. The interaction mode between (A) VEGFA V167, (B) VEGFA V167I, and VEGFR1. VEGFA is colored with yellow; VEGFR1 is colored with marine. The interaction mode between (C) VEGFA V167, (D) VEGFA V167I, and VEGFR2. VEGFA is colored with yellow; VEGFR2 is colored with magenta. The key residues I167/V167 in VEGFA are shown as green sticks. The red dashes represent hydrogen bond interaction. The blue dashes represent salt bridge. VEGFA, vascular endothelial growth factor A.