Genetic association for renal traits among participants of African ancestry reveals new loci for renal function

Abstract

Chronic kidney disease (CKD) is an increasing global public health concern, particularly among populations of African ancestry. We performed an interrogation of known renal loci, genome-wide association (GWA), and IBC candidate-gene SNP association analyses in African Americans from the CARe Renal Consortium. In up to 8,110 participants, we performed meta-analyses of GWA and IBC array data for estimated glomerular filtration rate (eGFR), CKD (eGFR <60 mL/min/1.73 m(2)), urinary albumin-to-creatinine ratio (UACR), and microalbuminuria (UACR >30 mg/g) and interrogated the 250 kb flanking region around 24 SNPs previously identified in European Ancestry renal GWAS analyses. Findings were replicated in up to 4,358 African Americans. To assess function, individually identified genes were knocked down in zebrafish embryos by morpholino antisense oligonucleotides. Expression of kidney-specific genes was assessed by in situ hybridization, and glomerular filtration was evaluated by dextran clearance. Overall, 23 of 24 previously identified SNPs had direction-consistent associations with eGFR in African Americans, 2 of which achieved nominal significance (UMOD, PIP5K1B). Interrogation of the flanking regions uncovered 24 new index SNPs in African Americans, 12 of which were replicated (UMOD, ANXA9, GCKR, TFDP2, DAB2, VEGFA, ATXN2, GATM, SLC22A2, TMEM60, SLC6A13, and BCAS3). In addition, we identified 3 suggestive loci at DOK6 (p-value = 5.3×10(-7)) and FNDC1 (p-value = 3.0×10(-7)) for UACR, and KCNQ1 with eGFR (p = 3.6×10(-6)). Morpholino knockdown of kcnq1 in the zebrafish resulted in abnormal kidney development and filtration capacity. We identified several SNPs in association with eGFR in African Ancestry individuals, as well as 3 suggestive loci for UACR and eGFR. Functional genetic studies support a role for kcnq1 in glomerular development in zebrafish.

Figure 1. Regional association plots for DOK6 and FNDC1.

For A) DOK6 and B) FNDC1, Stage 1 only.

Figure 2. Regional association plots for KCNQ1.

For A) KCNQ1 in participants of African ancestry and B) in participants of European ancestry.

Figure 3. Kcnq1 knockdown in zebrafish embryos causes glomerular abnormalities.

(A–E) Zebrafish embryos injected with control morpholino show normal glomerular and tubular morphology, as shown by in situ hybridization for the global kidney marker pax2a (A, inset showing lower-magnification image, with staining in both glomerulus and tubules), the podocyte markers wt1a (B) and nephrin (C), and the proximal and distal tubular markers slc20a1a (D) and slc12a3 (E). (F–J) Injection of the kcnq1 antisense morpholino, targeting the ATG site of the gene, at the one-cell stage results in significant changes in glomerular gene expression (F–H). No changes were observed in the proximal tubule (I) or the distal tubule (J).

Figure 4. Tg(cdh17:GFP) embryos were injected with 200 uM control or kcnq1 morpholino (MO) at the 1-cell stage.

Embryos were subsequently injected in the sinus venosus with 10,000 MW rhodamine dextran at 48 hpf. Dextran clearance was monitored over the next 48 hours by confocal microscopy. Compared to control MO-injected embryos (A–C), kcnq1 MO-injected embryos exhibit edema (D, E) and increased dextran clearance (F) 48 hours post-injection (hpi), suggestive of a filtration defect. At 5 dpf, 87% of kcnq1 morphant embryos (n = 38) develop edema (G).