Centromere protein I (CENPI) is a candidate gene for X-linked steroid sensitive nephrotic syndrome.

BACKGROUND: Individuals with proteinuria in association with hypoalbuminemia, edema, and hyperlipidemia are considered as having nephrotic syndrome (NS). NS is the most common kidney disease seen in the paediatric age group. NS is usually classified into steroid resistant nephrotic syndrome (SRNS) and steroid sensitive nephrotic syndrome (SSNS). More than 58 genes have been identified as a monogenic cause of SRNS, however, the genetic architecture of childhood SSNS remains poorly understood. METHODS: Here in this study, we performed sequencing of 66 NS candidate genes followed by whole genome SNP genotyping and whole exome sequencing in SSNS families with multiple affected individuals. RESULTS: NS candidate genes sequencing did not identify any pathogenic variant in the known genes. Homozygosity mapping based on an autosomal recessive model failed to detect any shared loss of heterozygosity region in the genome. An unbiased and hypothesis-free exome data analysis identified a missense variant (c.383G>A; p.Arg128Gln) in the CENPI gene. Sanger sequencing of both parents, unaffected and affected individuals confirmed an X-linked inheritance pattern of the variant (c.383G>A) with SSNS phenotype. The variant (c.383G>A) is very rare and is potentially damaging. CONCLUSION: Collectively, these observations suggest that a specific pathogenic link between SSNS development and alteration in CENPI exists. However, human mutations in CENPI causing SSNS have not been reported hitherto. Identification of genetic defects underlying SSNS will help in understanding the precise aetiology of SSNS and improved management of children with NS.

Functional alterations due to amino acid changes and evolutionary comparative analysis of ARPKD and ADPKD genes.

A targeted customized sequencing of genes implicated in autosomal recessive polycystic kidney disease (ARPKD) phenotype was performed to identify candidate variants using the Ion torrent PGM next-generation sequencing. The results identified four potential pathogenic variants in PKHD1 gene [c.4870C > T, p.(Arg1624Trp), c.5725C > T, p.(Arg1909Trp), c.1736C > T, p.(Thr579Met) and c.10628T > G, p.(Leu3543Trp)] among 12 out of 18 samples. However, one variant c.4870C > T, p.(Arg1624Trp) was common among eight patients. Some patient samples also showed few variants in autosomal dominant polycystic kidney disease (ADPKD) disease causing genes PKD1 and PKD2 such as c.12433G > A, p.(Val4145Ile) and c.1445T > G, p.(Phe482Cys), respectively. All causative variants were validated by capillary sequencing and confirmed the presence of a novel homozygous variant c.10628T > G, p.(Leu3543Trp) in a male proband. We have recently published the results of these studies (Edrees et al., 2016). Here we report for the first time the effect of the common mutation p.(Arg1624Trp) found in eight samples on the protein structure and function due to the specific amino acid changes of PKHD1 protein using molecular dynamics simulations. The computational approaches provide tool predict the phenotypic effect of variant on the structure and function of the altered protein. The structural analysis with the common mutation p.(Arg1624Trp) in the native and mutant modeled protein were also studied for solvent accessibility, secondary structure and stabilizing residues to find out the stability of the protein between wild type and mutant forms. Furthermore, comparative genomics and evolutionary analyses of variants observed in PKHD1, PKD1, and PKD2 genes were also performed in some mammalian species including human to understand the complexity of genomes among closely related mammalian species. Taken together, the results revealed that the evolutionary comparative analyses and characterization of PKHD1, PKD1, and PKD2 genes among various related and unrelated mammalian species will provide important insights into their evolutionary process and understanding for further disease characterization and management.

Next-generation sequencing for molecular diagnosis of autosomal recessive polycystic kidney disease.

Autosomal recessive polycystic kidney disease (ARPKD) a rare genetic disorder, described by formation of cysts in the kidney. A targeted customized sequencing of genes implicated in ARPKD phenotype was performed to identify candidate variants using the Ion torrent PGM next-generation sequencing. The results identified likely pathogenic disease causing variants during the validation process. Four potential pathogenic variants [c.4870C>T, p.(Arg1624Trp)], [c.5725C>T, p.(Arg1909Trp)], c.1736C>T, p.(Thr579Met)] and [(c.10628T>G), p.(Leu3543Trp)] were observed in PKHD1 gene among 12 out of 18 samples. The rest of the patient samples also showed few variants in ADPKD (Autosomal Dominant Polycystic Kidney Disease) disease causing genes PKD1 and PKD2 i.e. [c.12433G>A, p.(Val4145Ile)] and [c.1445T>G, p.(Phe482Cys)], respectively. All causative variants were validated by capillary sequencing, confirming the presence of a novel homozygous variants [c.10628T>G, p.(Leu3543Trp)] found in exon 61 of a male proband. All potentially deleterious variants identified in PKHD1, PKD1, and PKD2 gene, also exhibited pathologically or clinically significance based on the computational predictions involved in predicting the impact of non-synonymous SNPs (nsSNPs) on protein function such as Sorting Intolerant From Tolerant (SIFT) and Polymorphism Phenotyping (PolyPhen2). SIFT classified 50% of our nsSNPs as "deleterious", while PolyPhen2 identified 45% of our nsSNPs as "Probably damaged" and the results from both programs were largely complementary. Taken together, these results suggest that the NGS strategies provide a fast, accurate and cost-effective molecular diagnostic tool for identifying mutations in targeted genes sequence analysis.

Type 1/type 2 cytokine serum levels and role of interleukin-18 in children with steroid-sensitive nephrotic syndrome.

INTRODUCTION: In view of the conflicting evidence of helper T cell type 1 (Th1) or type 2 (Th2) pattern of cytokine synthesis in steroid sensitive nephrotic syndrome (SSNS), this study aimed to assess type-1/type-2 cytokines level in different stages of SSNS and to evaluate the role of IL-18. METHODS: We prospectively studied thirty children with SSNS, aged 2-12 years. The children were evaluated in the active stage before treatment initiation and re-evaluated again during remission while still on steroid treatment. A subgroup of children (21/30) was also evaluated during remission after steroid withdrawal. The control group included 30 healthy age- and sex-matched siblings. Serum levels of IL-2, IFN-γ, ΙL-4, IL-13 and IL-18 were measured by ELISA. RESULTS: IL-2 levels were not significantly different between children in different stages of SSNS and controls (p>0.05). Levels of IL-4, IL-13 and IL-18 were significantly higher during the active stage of SSNS compared to remission and controls (p<0.05). Serum IFN-γ was significantly lower in children with active disease compared to remission stages and controls (p<0.05). In children with SSNS, serum levels of IL-18 correlated significantly with both IL-4 and IL-13 during all stages (r=0.72 and p<0.0001, r=0.82 and p<0.0001, respectively). CONCLUSION: Children with active SSNS seem to have a shift to type-2 cytokine production, and IL-18 expression is significantly correlated with this type-2 immune response.