Among Wake Forest participants, nondiabetic nephropathy cases were older, had higher BMI, fewer females, and greater African ancestry, compared to non-nephropathy controls (). In FIND participants, nondiabetic nephropathy cases were slightly older, had fewer females, and lower BMI compared to controls.
Demographic data for the Wake Forest non-diabetic nephropathy samples and the FIND replication samples
Of the 1536 SNPs that were selected for genotyping, 1420 were successful and used in these analyses. The mean SNP call rate was 0.999 (standard deviation 0.001). Of the 1980 DNA samples sent to the Center for Inherited Disease Research at Johns Hopkins University for genotyping, 1846 were retained for analysis after removal of failed or poorly genotyped samples, unexpected duplicates and unexpected relatedness. There was no evidence that individuals removed from the analysis based on quality control analysis differed from those analyzed for BMI, age at hypertension, age at dialysis, age at enrollment, admixture or gender (p>0.20). Phenotype data were missing in the other 87 excluded participants. Genotyping rates by individual ranged from 95.5% to 100%; less than 1% had genotyping rates below 98%.
Association analysis in Wake Forest participants genotyped on the GoldenGate chip resulted in 61 SNPs associated at p-values < 0.01 (). The two most associated SNPs, rs2239785 and rs136148, were in the APOL1
gene, (p = 5.91 × 10−24
[dominant] and p = 1.13 × 10−7
[additive]), consistent with recently published results 7
. Two additional SNPs were associated with p-values < 3.00 × 10−5
(remaining significantly associated after Bonferroni correction), rs1573708 in an intergenic region and rs4820237 in the FOXRED2
gene, both located on chromosome 22. Additional SNPs of interest were rs379489 in the complement factor H gene (CFH
) (p = 2.05 × 10−4
, additive; OR, 0.73) and rs16854341 in the podocin gene (NPHS2;
p = 0.004, dominant; OR, 0.72), as both genes have previously been associated with kidney disease 24;25
. In addition, the APOL1
G1 and G2 variants were strongly associated with nephropathy in these Wake Forest cases (p = 3.27 × 10−32
, recessive; OR, 5.21; 95% CI, 3.96–6.86).
The top 61 associated SNPs from genotyping of Wake Forest non-diabetic nephropathy cases and non-nephropathy controls
To address the issue of multiple comparisons, we estimated the number of independent tests performed. A principal component analysis computed on the 1420 SNPs identified 904 principal components, suggesting 904 independent hypotheses tested. False discovery rate–adjusted p-values suggest that besides the chromosome 22 loci, the CFH rs379489 locus was significant (false discovery rate–adjusted p-value=0.03).
The top two APOL1
SNPs from the association analysis, rs2239785 and rs136148, were tested for replication in 1474 FIND participants (668 nondiabetic nephropathy cases, 804 controls), FIND participants were not included in the initial reports of APOL1
. Both SNPs replicated, with association p-values of 5.03 × 10−21
and 1.92 × 10−5
, respectively (). Adjustment for local ancestry on chromosome 22 did not significantly alter the significance of either APOL1
SNP; rs2239785 and rs136148 remained strongly associated after adjustment for local admixture. Importantly, the MYH9
E1 haplotype SNP rs4821480 (p = 1.12 × 10−10
) remained strongly associated with nondiabetic nephropathy after adjusting for local ancestry and these APOL1
Replication genotyping of selected SNPs in the FIND sample
We attempted to replicate the CFH SNP rs379489 association with nondiabetic nephropathy in the FIND participants. Although the association between this SNP was similar between the FIND and Wake Forest participants, the association was non-significant in FIND alone (p = 0.345; odds ratio, 0.92; additive). When FIND and Wake Forest samples were combined in a meta-analysis, rs379489 remained significantly associated (p = 6.75 × 10−4; odds ratio, 0.81; additive); although association was driven primarily by Wake Forest samples. The heterogeneity p-value in the meta-analysis was 0.0769 (additive).
G1 and G2 variants are known to be strongly associated with nondiabetic nephropathy in AAs, we tested the 1420 SNPs that were genotyped for interaction with the G1 and G2 nephropathy risk variants (). The most significantly associated interactive SNP was rs16854341 on chromosome 1 in the podocin (NPHS2
) gene (p = 0.0001). NPHS2
is independently associated with susceptibility to glomerulosclerosis 13;25–27
. Six other SNPs were associated with p-values < 0.001; however, none were located in known kidney disease candidate genes.
The top 44 SNPs from the APOL1 interaction analysis
In order to increase the statistical power for association, an exploratory analysis was computed using logistic regression models that tested for association with each of the 1420 SNPs adjusting for the APOL1 G1/G2 risk loci (). Most loci provided comparable evidence with and without adjustment for the G1/G2 risk loci. However, five loci showed meaningful improvement in the association: rs1500474 on 2q37 (unadjusted OR, 0.94 [p=0.39]; adjusted OR, 0.61 [p=0.0018]); rs11191727 on 10q24 within the gene neuralized-like protein 1 (NEURL1; unadjusted OR, 0.73 [p=0.012]; adjusted OR, 0.59 [p=0.00033]); rs1355652 on 11q14 (unadjusted OR, 1.36 [p=0.0028]; adjusted OR, 1.58 [p=0.00014]); rs9318258 on 13q22 (unadjusted OR, 0.82 [p=0.0070]; adjusted OR, 0.75 [ p=0.00070]) and rs10483956 on 14q31 (unadjusted OR, 0.76 [p=0.0056]; adjusted OR, 0.68 [p=0.00074]). The novel APOL1 SNP, rs2239785, remained modestly associated (p=0.00363). This SNP also remained associated in the FIND population when adjusted for G1/G2 risk status (p=0.0000518; OR, 0.52) ().
The top 46 SNPs from genotyping of Wake Forest non-diabetic nephropathy cases and non-nephropathy controls, adjusted for APOL1 G1/G2 compound risk