Genetic Variations Of NPHS2 And WT1 In Southern Chinese Children With Glomerular Diseases

Part one: Genetic variations of NPHS2 and WT1 in twenty children with sporadic steroid-resistant nephrotic syndrome in Southern ChineseObjective Primary nephrotic syndrome (PNS) constitutes one of the most common diagnoses in pediatric nephrology. It is characterized by heavy proteinuria, hypoalbuminemia, edema and hyperlipidemia. On the basis of the patients'responses to standard steroid therapy, PNS has been separated into steroid-sensitive nephrotic syndrome (SSNS) and steroid-resistant nephrotic syndrome (SRNS). Approximately 80% of all children with sporadic NS respond to steroid treatment and show favourable outcomes; however, approximately 20% of patients are resistant to typical steroid treatment and may progress to end-stage renal disease (ESRD). Recently, molecular genetics studies have demonstrated that mutations in podocin (NPHS2) and Wilms'tumor gene 1 (WT1) are responsible for SRNS. The NPHS2 gene is mapped to 1q25–31 and encodes podocin. Podocin is an integral membrane protein and is exclusively expressed in glomerular podocytes. Mutations in the NPHS2 gene cause autosomal recessive SRNS and sporadic SRNS. The WT1 gene, which is located in chromosome 11p13, encodes a transcriptional factor of the zinc finger protein family. It plays a critical role during kidney and gonadal development and, when mutated, in the occurrence of some diseases such as Denys-Drash syndrome, Frasier syndrome and isolated SRNS. To our knowledge, there were several studies regarding the incidence of mutations in the NPHS2 and WT1 genes in childhood SRNS. However, there were only two studies about the mutations in NPHS2 in sporadic SRNS in Chinese, and whether or not the WT1 gene is the causative gene in Chinese children with sporadic SRNS has not been established. This study aims to examine genetic variations of NPHS2 and WT1 in children with sporadic SRNS in Southern Chinese. Methods Peripheral blood samples were collected for genetic analysis from 20 children with sporadic SRNS in Southern Chinese, and 50 unrelated adult volunteers with normal urinalysis who were also studied as controls. Genomic DNA was isolated from peripheral blood leucocytes. The mutational analysis of NPHS2 and WT1 was performed by polymerase chain reaction, DNA sequencing directly and restriction fragment length polymorphism analysis. Results No mutations in either NPHS2 or WT1 in their all exons and exon-intron boundaries were detected in the 20 patients with sporadic SRNS, whereas two polymorphisms of NPHS2, 102G>A and 954T>C, and six variants of WT1, 5'-UTR-7G>T, 126C>T, IVS3+16G>A, IVS5-64 A>G, 903A>G and IVS7-32C>A, were identified in some of the patients and the controls. There was no significant difference in the genotypic and allelic frequencies of 102G>A of NPHS2 between the 20 patients and 50 controls. Six variants of WT1, 5'-UTR-7G>T, 126C>T, IVS3+16G>A, IVS5-64A>G, 903A>G and IVS7-32C>A, were also identified in controls, indicating that these six variants of WT1 are polymorphisms. There was no significant difference in the genotypic and allelic frequencies of 5'-UTR-7G>T, 126C>T, IVS3+16G>A, IVS5-64A>G, 903A>G and IVS7-32C>A of WT1 between the 20 patients and 50 controls, respectively. In addition, IVS5-64A>G is a novel polymorphism of WT1. Conclusion Our results suggest that mutations in both NPHS2 and WT1 are not major causes of the children with sporadic SRNS in Southern Chinese in the study. Part two: Genetic variations of NPHS2 and WT1 in seven children with chronic renal failure in Chinese Han ethnic groupObjective Chronic Renal Failure (CRF) is characterized by progressive scarring that ultimately affects all structures of the kidney. The relentless progression of CRF is postulated to result from a self perpetuating vicious cycle of fibrosis activated after initial injury. CRF is clearly a multifactorial disease. New studies of the molecular biology of the podocyte and identification of genes mutated such as NPHS2 and WT1 have given important new insights into mechanisms of progressive glomerulosclerosis. To our knowledge, there were few studies regarding the incidence of mutations in the NPHS2 and WT1 genes in children with CRF. However, whether or not the NPHS2 and WT1 genes are the causative genes in Chinese children with CRF has not been established. This study aims to examine genetic variations of NPHS2 and WT1 in children with CRF in Chinese Han ethnic group. Methods Peripheral blood samples were collected for genetic analysis from seven children with CRF in Chinese Han ethnic group with glomerular diseases, and 50 unrelated adult volunteers with normal urinalysis who were also studied as controls. Genomic DNA was isolated from peripheral blood leucocytes. Eight exons and exon-intron boundaries of NPHS2, and 10 exons and exon-intron boundaries of WT1 were amplified by polymerase chain reaction (PCR). Mutational analysis was performed by DNA sequencing directly and RFLP/PCR (RFLP, restriction fragment length polymorphism). Results No mutations in either NPHS2 or WT1 were detected in the seven patients with CRF, whereas two already reported polymorphisms of NPHS2, 102G>A and 954T>C, and four variants of WT1, 5'-UTR-7G>T, 126C>T, IVS5-9T>C and 903A>G, were identified in some patients. Three already reported variants of WT1 (5'-UTR-7G>T, 126C>T and 903A>G) were also identified in some controls, indicating that these variants are polymorphisms. There was no significant difference in the genotypic and allelic frequencies of 102G>A of NPHS2, and 5'-UTR-7G>T, 126C>T and 903A>G of WT1 between the seven patients and 50 controls, respectively. In addition, a novel variant, IVS5-9T>C of WT1, was identified in one patient and his father with normal urinalysis, whereas it was not found in the 50 controls. Conclusion Our results suggest that mutations in NPHS2 and WT1 are not major causes of the seven children with CRF in Chinese Han ethnic group in the study.