Exome-wide Association Study Identifies GREB1L Mutations in Congenital Kidney Malformations

Abstract

Renal agenesis and hypodysplasia (RHD) are major causes of pediatric chronic kidney disease and are highly genetically heterogeneous. We conducted whole-exome sequencing in 202 case subjects with RHD and identified diagnostic mutations in genes known to be associated with RHD in 7/202 case subjects. In an additional affected individual with RHD and a congenital heart defect, we found a homozygous loss-of-function (LOF) variant in SLIT3, recapitulating phenotypes reported with Slit3 inactivation in the mouse. To identify genes associated with RHD, we performed an exome-wide association study with 195 unresolved case subjects and 6,905 control subjects. The top signal resided in GREB1L, a gene implicated previously in Hoxb1 and Shha signaling in zebrafish. The significance of the association, which was p = 2.0 × 10^-5 for novel LOF, increased to p = 4.1 × 10^-6 for LOF and deleterious missense variants combined, and augmented further after accounting for segregation and de novo inheritance of rare variants (joint p = 2.3 × 10^-7). Finally, CRISPR/Cas9 disruption or knockdown of greb1l in zebrafish caused specific pronephric defects, which were rescued by wild-type human GREB1L mRNA, but not mRNA containing alleles identified in case subjects. Together, our study provides insight into the genetic landscape of kidney malformations in humans, presents multiple candidates, and identifies SLIT3 and GREB1L as genes implicated in the pathogenesis of RHD.

Keywords: CAKUT; EYA1; GATA3; HNF1B; HSPA4L; PAX2; SETBP1; SIX5; T; WNT5A.

Figure 1

Quantile-Quantile (QQ) Plot Expected versus observed p values of the rare loss-of-function model for whole-exome sequencing collapsing analysis in 195 RHD-affected case subjects and 6,905 control subjects. The signal for GREB1L is indicated.

Figure 2

Spatial Overview of the Qualifying GREB1L Heterozygous Variants Discovered in Our RHD Cohort The lollipops in black and gray indicate loss-of-function mutations and non-synonymous mutations, respectively. Numbers on the mRNA schematic indicate the coding exons in GREB1L; numbers on the protein schematic indicate amino acid numbers. The abbreviated variant names correspond to the following amino acidic substitutions: p.Arg13^∗, p.Arg128His, p.Glu761Gln, p.Arg1066Pro, p.Leu1334Profs^∗18, p.Tyr1560^∗, p.Leu1567Pro, p.Tyr1664Cys, p.Val1690Met, p.Gly273Val, p.Ala497Gly, p.Glu761Gln, p.Gln1099^∗, p.Val1549Ala, p.Val1615Ile, p.Ile1655Thr, p.Arg1884His.

Figure 3

Suppression or Mutation of greb1l in Zebrafish Results in Renal Hypoplasia (A) Schematic of a 4-day post fertilization zebrafish larva (dpf; lateral view); renal tubule is outlined in blue with the proximal convolution shown in red (arrow). (B) Representative fluorescent images of renal proximal convoluted tubules immunostained with Na⁺-K⁺-ATPase antibody (a6F) in 4 dpf larvae. Red dashed line in the upper left panel indicates the region measured. (C–E) Quantification of proximal renal tubule size as indicated by measurement of lateral images (see B). Statistical differences were calculated with a Student’s t test and are indicated with ns (not significant), ^∗∗p < 0.01, ^∗∗∗p < 0.001, ^∗∗∗∗p < 0.0001. Abbreviations are as follows: MO, morpholino; WT, wild-type; sgRNA, single guide RNA; F1’, progeny of two sgRNA1 F0 mutants. p.R241Q (p.Arg241Gln; rs147048716) and p.A926V (p.Ala926Val; rs569900756) present in controls are benign (minor allele frequency 0.0001053 and 0.001361 in gnomAD, respectively). p.R1066P (p.Arg1066Pro; hypomorph), p.L1567P (p.Leu1567Pro; null), p.I1655T (p.Ile1655Thr; null), and p.V1690M (p.Val1690Met; hypomorph) are exclusive to RHD-affected case subjects. Error bars represent standard error of the mean (SEM); n = 31–55 larvae/batch, repeated; see Table S7 for details.