Whole exome sequencing reveals heparan sulfate proteoglycan 2 (HSPG2) as a potential causative gene for kidney stone disease in a Thai family

Abstract

Kidney stone disease (KSD) is a prevalent and complex condition, with an incidence of 85 cases per 100,000 individuals in Thailand. Notably, over 40% of cases are concentrated in the northeastern region, indicating a potential genetic influence, which is supported by genetic mutations reported in several families by our research group. Despite this, the genetic basis of KSD remains largely unknown for many Thai families. This study aimed to identify the genetic mutation responsible for KSD in a specific Thai family, the UBRS131 family, which includes four affected individuals. Whole exome sequencing was performed, and variant filtering using the VarCards2 program identified 10 potentially causative mutations across 9 genes. These mutations were subjected to segregation analysis among family members and screened in 180 control and 179 case samples using real-time PCR-HRM or PCR-RFLP techniques. Prioritization of these variants using GeneDistiller identified the p.Asp775Glu mutation in the heparan sulfate proteoglycan 2 (HSPG2) gene as the likely causative mutation for KSD in this family. The Asp775 residue is highly conserved across vertebrates, and structural analysis suggests that the Glu775 substitution may disrupt the formation of two crucial hydrogen bonds, potentially altering the mutant protein's configuration. Immunohistochemistry confirmed the presence of perlecan (HSPG2 protein) in the proximal tubules in nephrons. These findings highlight the significant role of the HSPG2 gene in familial KSD within this study family.

Keywords: HSPG2; Heparan sulfate proteoglycan 2; Kidney stone disease; Whole exome sequencing.

Fig. 1

Pedigree of the UBRS131 family affected by KSD, segregation analysis of HSPG2, variant validation, and multiple amino-acid sequence alignment. A The pedigree of the UBRS131 family affected by KSD is presented alongside the segregation analysis of HSPG2 c.2325C > G in eight family members using PCR-RFLP and polyacrylamide gel electrophoresis stained with silver. Black circles or squares represent affected individuals with KSD, and the genotypes of c.2325C > G is indicated below each symbol. B Sanger sequencing results confirm the present of the HSPG2 c.2325C > G (p.Asp775Glu) variant in the affected patients. C Multiple amino acid sequence alignment of HSPG2 from eight vertebrate species is shown for the region encompassing the p.Asp775Glu variation

Fig. 2

Schematic diagram and three-dimensional (3D) structure of perlecan. A The schematic diagram illustrates the structure of HSPG2 (perlecan), highlighting the c.2325C > G (p.Asp775Glu) variation in exon 17 (red text) within domain III, along with other variations reported in previous studies. B Three-dimensional and superimposed structures of wild-type (green) and p.D775E (grey) perlecan. The square box indicates the protein region where the mutation is located. The structures of wild-type D775 (blue) and E775 variant (red), as predicted by SWISS-MODEL homology modeling server, are displayed along with their superimposed forms (blue and red). There are four putative H-bonds connecting to D775 in the wild-type structure (bottom-left, blue and green). In contrast, only two putative H-bonds connect to Glu775 (bottom-right, red and grey)

Fig. 3

Expression of HSPG2 mRNA in kidney cell lines and human kidney tissues. A Primer map indicates the relative locations of primers used to amplify HSPG2 mRNA. B HSPG2 mRNA expression in A549 cells, kidney cDNA library, HepG2 cells, HK2 cells, HEK293 cells, HEK293T cells, and human kidney tissues detected by RT-PCR method. Three regions of HSPG2 mRNA, spanning exons 6–7, 32–33, and 56–57, were analyzed. The mRNA of the housekeeping gene, GAPDH, served as an internal control. NTC no template control

Fig. 4

Expression of perlecan protein in human kidney tissues. Perlecan protein expression in the glomerulus and human kidney tubules was assessed by immunohistochemistry (IHC) using perlecan-specific antibody, with an isotype control antibody for comparison. AQP1 was used as a protein marker for the proximal tubule, V-ATPase as a protein marker for the distal tubule and collecting duct, and AQP2 as a protein marker for the collecting duct. The original magnification was 40x. The abbreviations are as follows: Ms mouse; Rb rabbit; GM glomerulus; PT proximal tubule; DT distal tubule; CD collecting duct; H&E hematoxylin and eosin staining. Scale bar: 50 μm