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Association of a 3-Amino Acid Deletion Mutation of NPHS2 gene with Congenital Nephrotic Syndrome (CNS) in a consanguineo

Association of a 3-Amino Acid Deletion Mutation of NPHS2 gene with Congenital Nephrotic Syndrome (CNS) in a consanguineous Pakistani family. Muhammad Ajmal, Muhammad Nasir and Abdul Hameed Institute of Biomedical and Genetic Engineering 24-Mauve Area, G-9/1 Islamabad. Introduction.

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Association of a 3-Amino Acid Deletion Mutation of NPHS2 gene with Congenital Nephrotic Syndrome (CNS) in a consanguineo

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  1. Association of a 3-Amino Acid Deletion Mutation of NPHS2 gene with Congenital Nephrotic Syndrome (CNS) in a consanguineous Pakistani family Muhammad Ajmal, Muhammad Nasir and Abdul Hameed Institute of Biomedical and Genetic Engineering 24-Mauve Area, G-9/1 Islamabad

  2. Introduction Autosomal recessive nephrotic syndrome (NS) belongs to the heterogeneous group of familial nephrotic syndrome. The disease is characterized by childhood onset of proteinuria, progression to end-stage renal disease, and minimal change nephrotic syndrome. NS results from alterations of the glomerular capillary wall. Proteinuria and hypoalbuminemia, often associated with edema and generalized hyperlipidemia

  3. Genetic of NPHS • Majority of nephrotic syndrome (NS) occurs as a sporadic form, the incidence of familial cases are from 3 to 5%. • Mutations of seven genes, NPHS1 (19q13.1), NPHS2 (1q25-31), ACTN4 (19q13), CD2AP, WT1, TRPC6 (11q21-22), and LAMB2 have been recognized to date are responsible for various forms of NS are summarised in the table.

  4. NPHS: Known Loci

  5. NPHS Family: Sample collection A three generation consanguineous Pakistani family, in which congenital nephrotic syndrome was segregating as an autosomal recessive trait was ascertained through nephrology clinic in Peshawar, Pakistan Three living members of the family were found to be affected with NPHS Detailed clinical examination was performed on the available family members including 3 affected individuals

  6. A pedigree of Pakistani NPHS Family Legend Male Normal Female Normal Male affected Female affected Marriage Cousin Marriage

  7. Aim of the study The aim of present study was • To locate and study Pakistani families affected with autosomal recessive congenital nephrothic syndrome. • To identify the genes responsible for producing specific phenotypes in Pakistani family with CNS. • To establish the prenatal diagnosis and carrier screening tests of the disease in Pakistan. 4.Genetic Counseling Avoid cousin marriages and /or avoid children

  8. NPHS Family: sample collection & DNA extraction • Blood samples from affected individuals, their parents and clinically normal siblings of the family members were collected with informed consent • Blood samples were also collected from 100 ethnically matched unrelated normal Pakistani individuals and were used as controls for allele frequencies and confirmation of disease associated mutation • Genomic DNA for linkage analysis was extracted from peripheral blood by the standard phenol chloroform extraction procedure

  9. Genotyping and Linkage Analysis • To identify the locus responsible for the disease in this family, genomic DNA from each individual was genotyped using microsatellite marker for the known NS loci • Microsatellite markers in the disease region were amplified by polymerase chain reaction (PCR)

  10. PCR Conditions • PCR reactions were performed in 10 l volume, each containing 1.5mM MgCl2, 0.6M of each primer, 0.2mM dNTPs, 1U TaqDNA polymerase and PCR buffer [16mM (NH4) 2SO4, 67mMTris-HCI (pH 8.8), and 0.01% of the nonionic detergent Tween-20] • Amplification was performed with an initial denaturation for 4 min at 94oC, followed by 35 cycles of denaturation at 94oC for 45sec, annealing at 55oC for 45 sec, extension at 72oC for 45 sec and a final extension at 72oC for 5 min

  11. PAGE Electrophoresis • The PCR products were separated on 10-12% non-denaturing polyacrylamide gels • The gel was stained with ethidium bromide, visualized under UV illumination and photographed. • Alleles were assigned to each individuals and genotyping was carried out.

  12. NPHS2 Gene: Mutation Screening • NPHS family was mapped to NPHS2 locus on chromosome 1q25.2 • NPHS2 gene was the candidate gene in this region, which comprises of 8 exons. • Polymerase chain reaction (PCR) amplification of the NPHS2 gene was performed with eight pairs of primers spanning all 8 exons by using intronic forward and reverse primers

  13. NPHS2 Gene: PCR Amplification Conditions • PCR amplification was performed in 50 µl reaction volume containing 250 ng of genomic DNA, amplification buffer containing 600 nM of each primer, 1.5 mM MgCl2, 200 mM of dNTPs and 2.5 U Taq polymerase in an PxE thermal cycler. • The amplification conditions were 95oC for 5 min, followed by 35 cycles of 95oC for 45 s, primer specific annealing temperature for 45 s, extension on 72oC for 45 s and final extension at 72oC for 7 min.

  14. NPHS2 Gene: Sequencing • Aliquots (5 µl) of the PCR products were analyzed by 2.5% agarose gel electrophoresis. • PCR products were then purified using QIAquick PCR Purification Kit. • Sequenced directly using Big Dye® Terminator v3.1 cycle sequencing kit in an ABI 3130 genetic analyzer. • Potential mutations were confirmed by bi-directional sequencing and assessing 100 control samples having ethnic backgrounds matching to patients.

  15. NPHS2: Mutation Identified in Pakistani family Patient Patient’s electrophergram showing homozygous 9bp deletion (arrow) in exon 5 of NPHS2 gene. Carrier An electrophergram of a normal carrier showing double peaks at the point of deletion (arrow) in exon 5 of NPHS2 gene.

  16. PCR-RFLP Analysis to Confirm Mutation • Restriction fragment length polymorphism (RFLP) analysis and confirmation of the mutation identified by sequencing within the exon 5 of the NPHS2 gene from the patient and the parents • The sequencing indicated the 9-bp homozygous deletion (704del9) in exon 5 of the NPHS2 in the patient. • The restriction enzyme map of this gene region was analyzed by NEBcutter, version 2.0, software.

  17. PCR-RFLP • To determine the presence of the mutation identified in NPHS2 and confirm its disease-association, we amplified exon 5 of this gene in all the CNS family members and a panel of 100 Pakistani control individuals. • The PCR derived 293 bp fragment was then digested with the restriction enzyme XbaI, and resolved on an agarose (2.0%) gel and visualized on a UV transilluminator.

  18. PCR-RFLP for mutation confirmation • Pedigree of NPHS family shows consanguineous family with three male patients (filled boxes). • (B) Agarose gel showing RFLP analysis .

  19. Results • While testing known loci for NPHS, strong linkage was obtained between the phenotype of NPHS2 family and the markers D1S1677, D1S1589 and D1S518 at chromosome 1q25-31 • This chromosomal region harbors a gene podocin that has previously been reported to be associated withNPHS2 • On mutation screening of NPHS2 gene, a homozygous 9bp deletion (704del9) in exon 5 of NPHS2 gene (Figure 1) was found to be segregating with CNS phenotype in Pakistani family

  20. Results- Continued • The 704del9 deletion result in a 3-amino acid deletion (residues 235-237) of podocin protein product. • In exon 5, 704del9 abolishes the digestion for XbaI restriction enzyme. • To confirm mutation and screening of 100 controls, exon 5 PCR products of NPHS2 gene were digested with XbaI. It confirmed the presence of homozygous deletion in only the affected individuals (Figure 2B).

  21. Discussion • Mutations in the podocin gene, NPHS2, have been shown in familial and sporadic forms of steroid-resistant nephrotic syndrome (SRNS). • Podocin is an integral membrane protein located at the slit diaphragm of the glomerular permeability barrier • The genetic and clinical features of familial SRNS have not been fully studied in Asian patients

  22. Discussion- continued • More than 60 unique NPHS1 mutations have been reported in Caucasian countries and NPHS2 is mutated in 26–38% of the familial and 10.5–23% of the sporadic SRNS in a large European survey • In a study on 36 Japanese sporadic SRNS cases, no NPHS2 mutation were identified, suggesting a significant ethnic difference in disease genes • These observations point to a need for worldwide study to investigate the nature of disease genes in SRNS families outside Caucasian countries.

  23. Identification of 9bp deletion and its association with CNS in Pakistani family is the first of NPHS2 gene involvement in any country of Asian origin • In Pakistan, consanguineous marriages for hundreds of years have resulted into the segregation of recessively inherited disease in large inbred families • The identification of pathogenic NPHS2 gene mutation in Pakistani family may help us in further understanding the functional role of NPHS2 gene, establishing genotype-phenotype correlation, more accurate disease diagnosis, improved genetic counseling and carrier screening.

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