WT1 splice-site mutations are rarely associated with primary steroid-resistant focal and segmental glomerulosclerosis

Background Donor splice-site de novo heterozygous mutations in intron 9 of the Wilms' tumor gene (WT1) have been reported in Frasier syndrome, which i s defined by the association of focal and segmental glomerulosclerosis (FSG S), male pseudohermaphroditism, and gonadoblastoma. These splice-site mutat ions alter the WT1 alternative splicing leading to two WT1 isoforms, with ( +) or without (-) three amino acids, lysine-threonine-serine (KTS), between zinc fingers 3 and 4. The aim of this work was to investigate the possibil ity that some cases of primary steroid-resistant nephrotic syndrome associa ted with FSGS may be caused by WT1 splice-site mutations. Methods. We analyzed WT1 exons 8 and 9 and the surrounding exon/intron boun dary DNA sequences in 37 children with nonfamilial primary steroid-resistan t nephrotic syndrome. Semiquantitative reverse transcription-polymerase cha in reaction (RT-PCR) was used to determine the relative ratio of +KTS/-KTS transcripts from immortalized lymphocyte RNA. Results. One boy with FSGS and associated pathologies (diaphragmatic hernia , proximal hypospadias, and unilateral testicular ectopia) was found to car ry the heterozygous 1228 +4 C-->T splice-site mutation. RT-PCR quantitation of the +KTS/ -KTS transcripts from immortalized lymphocyte RNA of this pat ient showed a diminution of the +KTS/-KTS isoform ratio (0.43), which is id entical to that reported in patients with Frasier syndrome. Using the same approach, healthy control subjects have +KTS/-KTS ratios ranging from 1.50 to 2.00. Conclusions. This study expands the range of the phenotypic presentation of the intron 9 splice-site WT1 mutations and adds to the already reported he terogeneity of primary steroid-resistant nephrotic syndromes. We suggest th at these mutations are not likely to be a common cause of isolated steroid- resistant nephrotic syndrome, and recommend a WT1 exon 9/intron 9 splice-si te study in children with primary steroid-resistant nephrotic syndrome if g enital or diaphragmatic anomalies are associated. The identification of suc h WT1 mutations has practical implications for the management of these pati ents.