Novel frame shift mutations (‘A’ deletion) observed in exon 9 of Wilms' tumor (WT1) gene in a patient reported with glomerulosclerosis Santhosh Kumar Pasupuleti a , Venkatesh Katari a , Srikanth Lokanathan a , Venkateswara Prasad Uppu a , Syama Sundar Thummaginjala b , Ram Prasad Reddy Akkamgari b , Tyagi Ayapati b , Radhika Kottu c , Venkata Gurunadha Krishna Sarma Potukuchi a, ⁎ a Department of Biotechnology, Sri Venkateswara Institute of Medical Sciences, Tirupati, AP 517507, India b Department of Urology, Sri Venkateswara Institute of Medical Sciences, Tirupati, AP 517507, India c Department of Pathology, Sri Venkateswara Institute of Medical Sciences, Tirupati, AP 517507, India abstract article info Article history: Received 26 October 2013 Received in revised form 25 April 2014 Accepted 16 May 2014 Available online 20 May 2014 Keywords: Frame shift mutation Immunohistochemistry RMSD Wilms' tumor Wilms' tumor-suppressor gene-1 (WT1) is a transcription factor that contains four zinc-finger motifs at the C-terminus and plays a crucial role in kidney and gonad development. We have identified primitive glomeruloid formation using immunohistochemistry in a patient who was clinically diagnosed with a Wilms' tumor. In order to understand the involvement of mutations in the WT1 gene, the genomic DNA was isolated from peripheral blood of the patient (18/F). Exon 9 of the WT1 gene was amplified and sequenced. The obtained sequence was BLAST searched against the transcript variants (TV) of the WT1 gene. An amplified exon 9 sequence of the WT1 gene showing similarity with exon 9 of TV-A, F and exon 10 of TV-B, D and E with a deletion of single nucleotide ‘A’ causing frame shift in the 4th zinc finger domain of the WT1 protein resulted in Wilms' tumor condition. The deletion position is variable with different transcript variants and they are present at: for TV-A c.1592delA, p.468, for TV-F c.1053delA, p.259, for TV-B c.1643delA, p.485, for TV-D c.1652 delA, p.488, and for TV-E c.1095delA, p.273; all these variations resulted in frame shift mutation. In order to substantiate these results in silico analysis was carried out; the structural superimposition of wild type and mutant WT1 structures showed that the mutated region exhibited a different confirmation with RMSD of 1.759 Å. Therefore, these results conclusively explain the mutation in the WT1 gene that leads to structural changes contributing to glomerulosclerosis. © 2014 Elsevier B.V. All rights reserved. 1. Introduction WT1 is a transcription factor regarded as a tumor suppressor gene that plays a key role in the development and maintenance of glomerular network in the kidney (Wagner et al., 2003). Mutations in the WT1 gene may cause Denys–Drash syndrome (Pelletier et al., 1991) or Frasier syn- drome (Barbaux et al., 1997), and WAGR (Wilms' tumor, aniridia, geni- tourinary malformations, mental retardation) syndrome (Drechsler et al., 1994). Primary (idiopathic) focal and segmental glomerular scle- rosis (FSGS) is a frequent glomerular disease (Huff, 1998) and it is iden- tified in 15 to 20% of adults and 7 to 15% of children presenting with idiopathic nephrotic syndrome and often leads to end-stage renal fail- ure (Denamur et al., 1999). The WT1 gene located at chromosome 11p13 contains 10 exons, en- codes 449 amino acids and has four zinc-finger motifs at the C-terminus and a proline/glutamine-rich DNA-binding domain at the N-terminus. Every zinc finger (Zf) consists of ‘Cys’ and ‘His’ residues linked to a zinc atom and ‘Arg’ is located at the top of the finger (Benetti et al., 2010). The exact ratio of the resulting four isoforms is a prerequisite for normal gene function required for the development of both nephrogenesis and urogenital system (Niaudet and Gubler, 2006). The 9th exon of WT1 majorly contributes in the active site of WT1 structure and moreover very high mutation frequency is observed compared to other exons; therefore, we have chosen this exon for our genetic analy- sis (Adzhubei et al., 2010; Call et al., 1990). In the present study WT1 genetic analysis in exon 9 was carried out in a patient admitted in the Department of Urology who was clinically diagnosed with Wilms' tumor, having prominent glomerulosclerosis. The X-ray crystal structure of WT1 (PDB ID: 2JP9) available in the Protein Data Bank (Berman et al., 2000) with defined active site was used as wild type WT1 conformation in the present study and the mutated Gene 546 (2014) 63–67 Abbreviations: WT1, Wilms' tumor-suppressor gene-1; IHC, immunohistochemistry; TV, transcript variants; BLAST, Basic Local Alignment Search Tool; PDB, Protein Data Bank; SSCP, Single Strand Conformation Polymorphism; TERT, telomerase reverse tran- scriptase; FHL2, factor H-like protein 2; TP53, Tumor Protein 53; WTAP, Wilms' tumor 1 associated protein. ⁎ Corresponding author at: Department of Biotechnology, Sri Venkateswara Institute of Medical Sciences, Tirupati 517 507, India. E-mail address: sarmasvims@gmail.com (V.G.K.S. Potukuchi). http://dx.doi.org/10.1016/j.gene.2014.05.037 0378-1119/© 2014 Elsevier B.V. All rights reserved. Contents lists available at ScienceDirect Gene journal homepage: www.elsevier.com/locate/gene