Çocukluk Dönemi Steroide Dirençli Nefrotik Sendroma Sebep Olan Yeni Genlerin Bulunması

Abstract

Steroid resistant nephrotic syndrome (SRNS), despite being a rare disease, is the second most common cause of end stage kidney failure in childhood and early adulthood. However the heterogeneity and rarity (<1%) of disease causing genes cause significant limitations in identification of a new gene. In this thesis work, identification of a novel gene is aimed via a new approach which combines total genome homozygosity mapping technique with whole human exome capture and massively parallel re-sequencing techniques. 2056 families consisting the cohort were screened via direct DNA sequencing for mutations in NPHS1, NPHS2, LAMB2, PLCE1 and WT1 genes that are known to cause SRNS at a high proportion and the cause of SRNS is identified in 310 families. Out of the remaining 1746, 85 families with known parental consanguinity were chosen to be analyzed by total genome homozygosity mapping technique via Affymetrix 250K (StyI) SNP chip. In homozygosity analysis, the presence of two or more siblings affected by the disease aids in the narrowing of the homozygous segments that are believed to harbor the disease causing gene, therefore 19 families that have multiple affected siblings were chosen among 85 families. One family (A2410) drew attention based on ethnic roots, clinical properties and response to treatment properties amongst 19 families and analyzed with whole human exome capture and massively paralel re-sequencing. Non parametric LOD scores were calculated together for siblings A2410-21 and A2410-22 and cZLR peaks pointing homozygosity by descent were found on chromosomes 3, 10, 14, 17, 21 and 22. Whole human exome capture and massively paralel re-sequencing showed 1968 variants from reference sequence and 48 of them were found not to be SNP?s by comparing to the databases. 11 out of these 48 were found to be non-synonymous changes and harboring in the homozygous segments. Only one of these variants was an insertion/deletion variation. Therefore one basepair homozygous deletion in CUBN (Cubilin) gene exon 53 (c.8355delA) was found that leads to early termination of protein (p.S2785fsX19) via causing a frameshift mutation. The presence of the mutation in homozygous state in affected siblings and in heterozygous state in the parents were shown by direct DNA sequencing. The absence of the mutation in healthy controls was verified by comparison with 1000 genome database. Also the mutation was not found when 92 healthy Turkish control individuals that are ethnic matching to Egyptian population were analyzed by DNA sequencing.