Homozygous GRHPR C.494G>A mutation is deleterious that causes early onset of nephrolithiasis in West Bengal, India

Abstract

Pediatric nephrolithiasis (NL) or Kidney stone disease (KSD) is an untethered topic in Asian population. In Western countries, the annual incidence of paediatric NL is around 6-10%. Here, we present data from West Bengal, India, on lower age (LA, 0-20 years) NL and its prevalence for the first time. To discover the mutations associated with KSD, twenty-four (18 + 6) rare LA-NL patients were selected for Whole Exome Sequencing (WES) and Sanger sequencing, respectively. It was found that GRHPR c. 494G>A mutation (MZ826703) is predominant in our study cohort. This specific homozygous mutation is functionally studied for the first time directly from human peripheral mononuclear cell (PBMC) samples. Using expression study with biochemical activity and computational analysis we assumed that the mutation is pathogenic with loss of function. Moreover, three genes, AGXT, HOGA1 and GRHPR with Novel variants known to cause hyperoxaluria were found frequently in the study cohort. Our study analyses the genes and variations that cause LA-NL, as well as the molecular function of the GRHPR mutation, which may serve as a clinical marker in the population of West Bengal, Eastern India.

Keywords: GRHPR; hyperoxaluria; kidney stone disease (KSD); nephrolithiasis (NL); whole exome sequencing (WES).

FIGURE 1

Consort diagram of the Study design.

FIGURE 2

Analysis of Survey; (A) Showing prevalence of KSD in all the age groups (0–20, 21–40, 41–60, 60+) years per year, between 2009–2016 (B) Percentage of affected lower age group (0–20) KSD patients per year, between 2009 and 2016.

FIGURE 3

Chromatogram of rs180177314; (A) showing GG genotype, (B) showing GA genotype, (C) showing AA genotype (D) Chromatogram from CDNA sequence showing no sign of splicing error.

FIGURE 4

Moon diploid: (A) Showing mutation in the region of NAD binding domain; Hope: (B) Mutated residue (ASP) shown in red is large compared to wild residue in green; (C) Phyre: showing relatively high clash (left bar indicates gradient of clash) in the mutated region; and (D) Showing relative high conservation in the mutated region.

FIGURE 5

(A) Showing docking with NDP ligand on NAD binding domain on Wild GRHPR protein in 3D; (B) showing interactions of wild GRHPR protein with NDP ligand in 2D; (C) Enlarged view of the Receptor site of Wild GRHPR protein vs. NDP ligand wrt to H-bond pocket; (D) Showing docking with NDP ligand on NAD binding domain on Mutated GRHPR protein in 2D; (E) 2D image showing unfavorable bump in the mutated residue, here shown in red which missing in wild in NAD site, (F) Enlarged view of the receptor site shows H-bond pocket in mutated GRHPR protein vs. NDP ligand.

FIGURE 6

(A) Relative mRNA expression of GRHPR gene in test (C.494G>A) Vs. Control, (N = 6); (B) Immunoblot showing protein fold change wrt GRHPR gene in Case (N = 6) VS. Control; (C) Immunocytochemistry (ICC); showing monocytes of Case and control. p-value less than 0.05 is represented by * and less than 0.01 **.

FIGURE 7

GRHPR Enzyme activity in the presence of different substrate (Glyoxylate) concentration (µmol/ml) between test (C.494G>A) (N = 6) and control samples (N = 10); and the rate of NADP formation demonstrated as mmol ml⁻¹ min⁻¹.