Genetic Analysis of Steroid Resistant Nephrotic Syndrome (SRNS) in South Indian Children

     Nephrotic syndrome (NS) is a common glomerular disease in children and represents a major cause of morbidity and mortality worldwide. According to the clinical relevant definitions, it requires the presence of edema, massive proteinuria (>40 mg/m2/hr or a urine protein/creatinine ratio >2.0 mg/mg), and hypoalbuminemia (<2.5 g/dl). The progression of NS is a multi-factorial process that involves a number of genetic, environmental and ethnic factors. In recent years of scientific investigations, attention has been drawn to identify the new genes being involved in NS. In this research work has four parts.

       In India, genetic mutation records in SRNS children were too little; our aim was to evaluate the molecular mechanism underlying NS patients in south India. Part I and II focus on mutation analysis in WT1 and NPHS2 genes, and then III and IV deal with polymorphism analysis of HLA- class II and ACE of NS and control subjects. The identification of (nucleus, podocyte) WT1, NPHS2 genetic analysis and HLA, ACE susceptibility/genetic variants requires different methodological approaches. Recent studies suggest, and underline the need to explore the pathogenic, genetic and clinical spectrum of NS.

      The Wilms’ tumor suppressor gene encodes a zinc finger containing nuclear protein. WT1 protein plays crucial role for urogenital development and function. Development of gonad is a complex process, and many genes take part in this function, one of the genes being WT1 and its role is essential. WT1 mutation dominant negative product leads to formulate worse development of gonadal cell types. Mutations in this gene are closely associated with three different syndromes such as Frasier syndrome, Denys-Drash syndrome, WAGAR syndrome. These syndromes are associated with kidney failure, defects in reproductive system and impaired masculinization also. Frasier syndrome (FS) symbolizes a rare and difficult condition to diagnose. FS patients develop nephrotic syndrome in childhood, which is steroid resistant. Molecular diagnosis is the Gold standard to confirm the FS. If there is phenotypic versus genotypic discrepancy, surgical removal of gonads is advisable followed by medical management.  Patients with (FS) are prone to develop Wilms’ tumor, genital anomalies and gonadoblastoma due to a mutation that occurs in the intron region of WT1 gene. Basically, WT1 mutation analysis helps us to understand the management of FS and to offer a reliable prognosis to the family. In this study, WT1 genetic analysis was performed in SRNS children. Genetic analysis confirmed that three cases had WT1 mutation. After confirmation of FS, cytogenetic analysis was carried out for all the three cases. Abdominal scans in the two girls showed normal uterus with streak gonads with a considerable risk of gonadoblastoma. Gonadectomy was done for both and tissue biopsy was reported as dysgerminoma stage I. Two of them were phenotypically females but genetically they were males. The third case was a 9 year old boy with FS. Renal biopsy was done and the histopathology report was MCNS. He is currently on steroid therapy and the renal function is normal.

     In addition, the effect of NPHS2 mutation on NS children in the podocyte function was altered and histopathology results were analyzed and used for the correct diagnosis. The present study puts forward that administration of high dose in podocin mutation patients should consider unnecessary drug toxicity. Once again our study has established that NPHS2 mutation does not frequently (~4-5 %) occur in Asian population. Thus, podocin is turning out to be a major contributor to the genetic burden of NS but in India it is not true. To our knowledge, whether or not NPHS2 is the causative gene in Indian SRNS has not been clearly established (except this single center study). Only a molecular approach is diagnostic in this setting, and it is proposed that resistant to steroids and suggestive of FSGS should undergo the analysis of NPHS2 before planning therapeutic regimens with steroids and other immunosuppressive drugs.

        The diagnosis based analysis for NS cases (total one hundred and eighty three patients) were split into two groups such as Steroid Sensitive NS (SSNS) and SRNS-seventy six patients and one hundred and seven patients respectively, and the data were analyzed by pooled and sex determinations. These experimental groups (SSNS and SRNS) were compared with the control groups.  HLA-Class II- DR and-DQ polymorphism and their association were studied for the above said groups.  The present study observed an elevated frequency of HLA DRB1*07 and HLA DQB1*02 in patients more than controls. In controls, the frequency of HLA-DRB1*15 and HLA-DQB1*10 were observed to be higher. The haplotype analysis revealed that the haplotype HLA- DRB1*07-DQB1*02 was observed in higher frequency in SSNS/SRNS patients. One to four number of predisposing alleles (i.e., DRB1*07/03, DQB1*02/07) co expressed FSGS and leading to early onset of the disease. Haplotypes HLA-DRB1*07-DQB1*02 and HLADRB1*03-DQB1*02 conferred a strong predisposition to SRNS and DRB1*15-DQB1*05 and DRB1*10-DQB1*06 afforded protection. DRB1*07-DQB1*02 was predominantly found among NS cases 38.25%. HLA-A and B typing of these NS cases revealed the presence of an extended haplotype ‘A*03-B*07-DRB1*07-DQB1*02’ NS (19.12%). This extended haplotype was identified in SSNS (14.47%), SRNS (22.42%), MCNS (19.14%) and FSGS (37.93%).Consequently, an earlier age-at-onset of NS was observed from predisposing {DRB1*07- DRB1*11-DQB1*02-DQB1*0301, 0304(DQ7)} allelic combination. Moreover, 62.5% cases possessed the extended haplotype A*03-B*07-DRB1*07-DRB1*11-DQB1*02. Of these 80% of cases were showing an age-at-onset of below 24 months.

       In ACE (I/D), we could not find the significant association for the ungrouped data of NS and control. We segregated  NS  biopsy cases into MCN/FSGS, we observed the ACE 'D' allele was found to be increase in FSGS and 'I' allele was associated with MCN; and finally 'II' genotype is weakly associated with MCN's. Gender specific analysis revealed weak association of 'ID' genotype with female NS. Further, we found the over representation of 'DD' genotype in NS male have the dominance than (II+ID) genotypes.

      The genetic heterogeneity clearly revealed in this work, stresses the role of pathogenesis of NS in different ethnic background. Hence the genetic origins of NS may be diverse from Europe to Asia. The disease may represent mal-adaptation and selection of different genotypes in response to different evolutionary pressures, rather than be derived from a common genotype that is present in most populations. Identifying the genetic components of NS is not only providing insight into the mechanisms, but also allows the identification of susceptible individuals, for whom an early diagnosis can be made and suitable treatment may be given subsequently.