Genomic approach to Idiopathic Calcium Nephrolithiasis: searching for susceptibility genes in a three generation family

Idiopathic calcium nephrolithiasis (ICN) is a multifactorial disease with a pathogenesis depending upon the interplay of environmental, metabolic, anatomical and genetic factors. The importance of hereditary factors in ICN has emerged from a number of studies on families and twins affected by ICN. Computer programs predicted the best inheritance fit with a model of single gene co-dominant model/polygenic model. Most candidate gene or association studies have produced, up to date, negative or only marginal results, leaving the genetic basis of ICN still an open question. But, genetic analysis techniques are rapidly evolving and promise to improve our knowledge of the genetic basis of nephrolithiasis and allied disorders. Whole-genome scanning techniques, like Next-Generation Sequencing (NGS) and CGH (Comparative Genomic Hybridization) array, are the most recent technical approaches of molecular genetics that allow to trace out a complete draw of variation present in the human genome, namely SNPs (Single Nucleotide Polymorphisms) and heterogeneous structural variants. In the last years, indeed, several studies have recognized the critical role of structural genetic variants, mostly represented by Copy-Number Variants (CNVs), in modulating gene expression and complex diseases phenotype. Aim of this project was to identify some of the major susceptibility genes involved in idiopathic calcium nephrolithiasis applying the potential of whole-genome scanning technologies to a case study very peculiar, both for clinical manifestations both for its genetic background. The proband, since 30 years, suffers of a recurrent and severe form of ICN, with intermittent hypercalciuria and phosphaturic tubulopathy, expelling, up to date, about 300 calculi both spontaneously and by lithotripsy (33 treatments). Strikingly, he belongs to a family with consanguinity in which nephrolithiasis is present in several members and is transmitted in an apparently dominant fashion, with males being more severely affected than females. CGH arrays has been performed in the proband and in eight members of the family in order to detect the presence of CNVs in the genome. A duplication of 308 kb region of the Xq22.2 chromosome has been detected in the proband. This CNV resulted particularly interesting because it was a novel (not known) variant and it was shared by other three family members (maternal inheritance). Moreover, matching the Xq22.2 duplication over a database of 14375 individuals only 5 overlapping duplications were found, confirming its extreme rarity. Strikingly, all these 5 duplication shared a common region that corresponded to NUP62CL gene, suggesting a likely importance of this genetic trait. Furthermore, performing an expression study, we observed a significant and marked down-regulation of NUP62CL. Thus, even if we could not have a definitive proof of principle, this Xq22.2 CNV seemed to have an important role for ICN pathogenesis in our family. Nonetheless, given its presence in a member of the family that was not evidently affected and given the severity of the phenotype of some members of the paternal branch, we postulated that additional genetic determinants were also responsible of the severe phenotype encountered in the proband. In this light, we performed whole exome sequencing of the proband to identify rare single nucleotide variants that may have a role as susceptibility variants for ICN in the case-study family. By bioinformatic analyses and molecular validation, we could draw a polygenic model of heredity, with the identification of four possible susceptibility genes: XDH, ATP6V1B1, HNF1B, NUP62CL. The XDH gene, normally responsible of xanthinuria type I, was highlighted for the presence of two very rare variants with a damaging predicted effect on protein. Next, we discovered a missense homozygous variant in ATP6V1B1 gene, whose role is already known in distal tubular acidosis, that seemed to account for the ICN intermediate phenotype hypocitraturia observed in the family. In addition, we found an extremely rare heterozygous variant in HNF1B gene, a causative factor in cystic nephropathy, and the proband, indeed, recently showed pre-calyceal cysts. Strikingly, we again evidenced NUP62CL gene for the presence of two homozygous variants that operate activating criptic splicing sites, thus eventually generating an aberrant mRNA. This gene has never been associated to ICN but, having a known role in oxalate transport, could be responsible of some of the biological mechanisms that are on the basis of ICN pathogenesis