Gene Detection And Protein Expression Of NPHS2 And WT1 In 25 Cases Of Southwest Chinese Children With Refractory Nephrosis Syndrome

Objective:Primary nephrotic syndrome (PNS) is the most common glomerular disease in children. It is characterized by heavy proteinuria, hyp- oalbuminemia, 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 PNS respond to steroid treatment and show favourable outcomes, of which still have 30% to 40% of children with clinical manifestations of the hormone steroid dependence or frequency recurrence(steroid-dependent nephrotic syndrome, SDNS / Frequent relapses nephrotic syndrome, FRNS);however, approxim- ately 20% of patients are resistant to typical steroid treatment and may progress to end-stage renal disease(ESRD). With the progress of molecular biology in recent years, more and more studies confirm that mutations in the NPHS2 gene can cause autosomal recessive SRNS and sporadic SRNS. And WT1 gene mutations cause isolated SRNS, Denys-Drash syndrome (DDS) ,Frasier syndrome (FS) and other kidney diseases. Currently, there are several of international analysis of the studies about the NPHS2 or WT1 gene mutation in children with sporadic SRNS,while the relationship between NPHS2 gene mutations and SDNS/FRNS is still controversial. In our country the research about the NPHS2 or WT1 gene mutation in children with sporadic SRNS more in Beijing, Shanghai,fuzhou and Guangzhou.In order to understand the mutations of NPHS2 and WT1 in southwest Chinese children,in this study the clinical data of 25 cases with refractory nephrosis syndrome were retrospectively analyzed,at the same time we detected the NPHS2 and WT1 gene mutation and protein expression characteristics,aims to to explore the role that the NPHS2 and WT1 mutations in the pathogenesis of refractory NS played.Methods: A retrospective analysis of the clinical manifestations of 25 southwest Chinese patients with refractory NS(including gender, onset age, disease duration,routine urine, blood biochemical, renal ultrasonography, renal pathology, therapy and so on). Peripheral blood samples were collected, genomic DNA was isolated from peripheral blood leucocytes.The mutational analysis of NPHS2 and WT1 was performed by polymerase chain reaction.After direct sequencing, we used DNAman software to compare the results in the U.S. National Center for Biotechnology Information (NCBI) gene database. Also we showed the podocin and wt1 protein expression in the kidney tissue paraffin sections of some patients by immunohistochemical detection.Results:1,1 case detected the 288C>A (S96S) heterozygous synonymous mutations and the 365G >C (W121S) heterozygous missense mutations in NPHS2. Access to relevant literature,presumably, 365G> C might be a new pathogenic mutation, 288C>A might be a new single nucleotide polymorphism (SNP). At the same time the patient's podocin protein in kidney tisuue without expression.2,Two single nucleotide polymorphism (SNP) of 954C> T, 1038A> G (already reported SNP),were identified in 6 cases of steroid-resistant nephrotic syndrome and 2 cases of children with complex frequency.The minimum allele frequency were 0.20 and 0.08 respectively in these two sites.3,There were no significant difference (P> 0.05)between the patients carrying SNP and without carrying SNP.4,No mutations in WT1 gene exon 8 and 9 were detected in the 25 patients with refractory NS,and also the WT1 protein expression in kidney tissue were visible;Conclusion:1,In this study, 1 case with steroid-resistant NS detect a NPHS2 gene mutation of a heterozygous missense mutation (W121S) and a synonymous mutation (S96S), and the renal tissue of this case without the expression of podocin, speculate this patient with poor response to disease and hormone therapy may be associated with NPHS2 mutation.2,There has two reported NPHS2 mutations in southwest China children. Respectively is 954C> T, 1038 A> G, the minimum allele frequency are 0.20 and 0.08 respectively .These two sites don't affect the expression of podocin protein and the clinical performance;3,This study don't detect the WT1 gene mutations and abnormal expression of WT1 protein, so WT1 gene mutation is not considered as a major genetic disease gene in southwest Chinese children with refractory nephrotic syndrome.