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Evidence for the Genetic Heterogeneity of Nephropathic Phenotypes Associated with Denys-Drash and Frasier Syndromes

1999; Elsevier BV; Volume: 64; Issue: 6 Linguagem: Inglês

10.1086/302409

1537-6605

A.B. Koziell, Richard G. Grundy, T M Barratt, Peter Scambler,

Prenatal Screening and Diagnostics

To the Editor: The association of constitutional heterozygous mutations of the Wilms tumor 1 (WT1) gene with the majority of cases of both Denys-Drash syndrome (DDS [MIM 194080]) (Denys et al. Denys et al., 1967Denys P Malvaux P van den Berghe H Tanghe W Proemans W Association d'un syndrome anatomo-pathologique de pseudo-hermaphrodisme masculin, d'un tumeur de Wilms' d'un nephropathie parenchymarteuse et d'un mosaicisme XX/XY.Arch Pediatr. 1967; 24: 729-739Google Scholar; Drash et al. Drash et al., 1970Drash A Sherman F Harmann W Blizzard R A syndrome of pseudohermaphroditism, Wilm's tumour, hypertension and degenerative renal disease.J Pediatr. 1970; 76: 585-593Abstract Full Text PDF PubMed Scopus (328) Google Scholar) and Frasier syndrome (FS [MIM 136680]) (Frasier et al. Frasier et al., 1964Frasier SD Bashore RA Mosier HD Gonadoblastoma associated with pure gonadal dysgenesis in monozygotic twins.J Pediatr. 1964; 64: 740-745Abstract Full Text PDF PubMed Scopus (191) Google Scholar) has been well described; however, the characteristic nephropathies connected with these syndromes—that is, diffuse mesangial sclerosis (DMS [MIM 256370]) and focal segmental glomerulosclerosis (FSGS [MIM 603278])—occur more commonly in early life, as disorders confined to the kidney. It is unclear how frequent WT1 gene mutations are in this population. The WT1 gene encodes a transcription factor critical for the normal development and function of the urogenital tract, reflected by the knockout-mouse homologue of WT1, in which homozygous inactivation of Wt1 causes absent kidneys and malformation of the gonads (Kreidberg et al. Kreidberg et al., 1993Kreidberg JA Sariola H Loring JM Maeda M Pelletier J Houseman DE Jaenisch R WT1 is required for early kidney development.Cell. 1993; 74: 679-691Abstract Full Text PDF PubMed Scopus (1574) Google Scholar). Alternative splicing results in at least 4 different zinc-finger protein isoforms, and the possibility of RNA editing and use of an alternative initiation codon increases this number to 16 (Bruening and Pelletier Bruening and Pelletier, 1996Bruening W Pelletier J A non-AUG translational initiation event generates novel WT1 isoforms.J Biol Chem. 1996; 271: 8646-8654Crossref PubMed Scopus (118) Google Scholar). A correct ratio of isoforms appears crucial for normal gene function. During renal development, maximum WT1 expression occurs in condensing mesenchyme and during the mesenchymal-epithelial switch. In the mature nephron, WT1 expression is confined to the podocytes, a highly specialized layer of epithelial cells in the glomerulus. Here it may have a role in the maintenance of these cells, thus affecting the integrity of the glomerular filter (Pritchard-Jones et al. Pritchard-Jones et al., 1990Pritchard-Jones K Fleming S Davidson D Bickmore W Porteous D Gosden C Bard J et al.The candidate Wilms' tumour gene is involved in genitourinary development.Nature. 1990; 346: 194-197Crossref PubMed Scopus (741) Google Scholar). Accumulating evidence supports a regulatory role for WT1 in kidney development, although little is known about either its targets or which genetic cascades are affected by its abnormal function. A wide variety of WT1 mutations is seen in DDS—a triad of intersex, nephropathy due to DMS, and Wilms tumor—making genotype-phenotype correlation difficult even with the aid of computer programs (Jeanpierre et al. Jeanpierre et al., 1998aJeanpierre C Beroud C Niaudet P Junien C Software and database for the analysis of mutations in the human WT1 gene.Nucleic Acids Res. 1998a; 26: 271-274Crossref PubMed Scopus (37) Google Scholar). In contrast, FS is caused by specific intronic point mutations that disrupt the exon 9 alternative splice-donor site, reversing the normal WT1 +/− KTS isoform ratio (Klamt et al. Klamt et al., 1998Klamt B Koziell AB Poulat F Wieacker P Scambler P Berta P Gessler M Frasier syndrome is caused by defective alternative splicing leading to an altered ratio of WT1 +/− KTS splice isoforms.Hum Mol Genet. 1998; 7: 709-714Crossref PubMed Scopus (270) Google Scholar). It is also associated with intersex, but there is no predisposition to Wilms, and the nephropathy typically results from FSGS. These conditions act as human disease models of the effects of WT1 gene mutations and provide further strong evidence of WT1's crucial role in both renal and gonadal development. How WT1 mutations affect glomerular development remains a matter of debate; some mutations may be mediated during the progression of nephrogenesis, perhaps through aberrant interactions of WT1 with genes and proteins important for this process, thereby causing abnormal glomerular differentiation. It is equally possible is that the problem could lie in the terminally differentiated, nondividing podocytes, particularly if silencing of the normal allele were to occur, as in some tissues (e.g., placenta and brain [Jinno et al. Jinno et al., 1994Jinno Y Yun K Nishiwaki K Kubota T Ogawa O Reeve AE Niikawa N Mosaic and polymorphic imprinting of the WT1 gene in humans.Nat Genet. 1994; 6: 305-309Crossref PubMed Scopus (160) Google Scholar]), and if the mutant WT1 protein were incapable of maintaining normal podocyte function. Further study of animal models with targeted gene mutations may shed light on this increasingly complex picture. Jeanpierre et al. (Jeanpierre et al., 1998bJeanpierre C Denamur E Henry I Cabanis M-O Luce S Cécille A Elion J et al.Identification of constitutional WT1 mutations, in patients with isolated diffuse mesangial sclerosis, and analysis of genotype/phenotype correlations by use of a computerized mutation database.Am J Hum Genet. 1998b; 62: 824-833Abstract Full Text Full Text PDF PubMed Scopus (197) Google Scholar) demonstrated that 4 of their 10 patients with DMS but no other features of DDS had mutations in exons 8 and 9 of WT1; 3 of these 4 patients were female. One mutation, 1147T→C (F383L) in exon 9, was novel. Two other mutations, 1186G→A (D396N) in exon 9 and 1129C→T (H377Y) in exon 8, had been found in previously reported cases of DDS. The fourth mutation, (+4C→T) in intron 9, had been shown to cause FS (Barbaux et al. Barbaux et al., 1997Barbaux S Niaudet P Gubler MC Grunfeld JP Jaubert F Kuttenn F Fekete CN et al.Donor splice site mutations are responsible for Frasier syndrome.Nat Genet. 1997; 17: 467-470Crossref PubMed Scopus (534) Google Scholar; Kikuchi et al. Kikuchi et al., 1998Kikuchi H Takata A Akasaka Y Fukuzawa R Yoneyama H Kurosawa Y Honda M et al.Do intronic mutations affecting splicing of WT1 exon 9 cause Frasier syndrome?.J Med Genet. 1998; 35: 45-48Crossref PubMed Scopus (72) Google Scholar; Klamt et al. Klamt et al., 1998Klamt B Koziell AB Poulat F Wieacker P Scambler P Berta P Gessler M Frasier syndrome is caused by defective alternative splicing leading to an altered ratio of WT1 +/− KTS splice isoforms.Hum Mol Genet. 1998; 7: 709-714Crossref PubMed Scopus (270) Google Scholar). Schumacher et al. (Schumacher et al., 1998Schumacher V Scharer K Wuhl E Altrogge H Bonzel KE Guschmann M Neuhaus TJ et al.Spectrum of early onset nephrotic syndrome associated with WT1 missense mutations.Kidney Int. 1998; 53: 1594-1600Crossref PubMed Scopus (119) Google Scholar) studied a broader spectrum of patients with early-onset nephrotic syndrome, to identify possible WT1 gene mutations; two of four patients who had DMS but lacked other features of either DDS or FS had mutations; one of them had a newly discovered mutation, 1135G→T (G379C) in exon 8, and the other had a mutation, 1180C→T (R394W) in exon 9, that previously had been reported described in a case of DDS; both patients were female and were reported to have other unspecified features consistent with this syndrome. We tested the hypothesis that WT1 gene mutations occur in cases of DMS and of congenital/early-onset FSGS occurring in the absence of other features of DDS or FS. Our intent was to identify how common mutations of the WT1 gene were in this population and to begin establishing the boundaries of the DDS/FS spectrum of disease. A series of 30 patients, 22 with DMS and 8 with FSGS, were screened for mutations of WT1 (table 1). The diagnosis of DMS or FSGS was established on the basis of either renal biopsy or postmortem renal histological findings. The majority of surviving patients with DMS were followed-up with yearly renal ultrasounds, to exclude Wilms tumor. Twenty-seven of the 30 patients had documented karyotype analysis. Congenital malformations were investigated by clinical examination and the appropriate investigations. None of the patients with psychomotor abnormalities were given a diagnosis of Galloway-Mowat syndrome. Abnormalities of the internal gonads were examined by pelvic ultrasound, and in some cases, by laparotomy. Patient 14 had an equivocal report with regard to karyotype—and, therefore, possible intersex status—but no other features of DDS. Two patients with DMS (patients 21 and 22) had DDS with known 1180C→T (R394W) mutations in exon 9 and were used as positive controls. Genomic DNA was obtained from blood of 23 patients and from paraffin-section material in 7 patients, by standard phenol-chloroform extraction methods. The two most common DDS mutations located in exon 9 (i.e., 1180C→T [R394W], 60%; and 1186G→A [D396N], 15%) abolish an RsrII site in zinc finger 3 (Little et al. Little et al., 1993Little MH Williamson KA Mannens M Kelsey A Gosden C Hastie ND van Heyningen V Evidence that WT1 mutations in Denys-Drash syndrome patients may act in a dominant-negative fashion.Hum Mol Genet. 1993; 2: 259-264Crossref PubMed Scopus (131) Google Scholar). Heterozygous loss of the site was observed in patients 21 and 22, as expected, whereas patients 1–20 showed normal restriction-enzyme digests. We used SSCP to analyze exons 1–10 after PCR amplification, using oligonucleotides that had also been published by Baird et al. (Baird et al., 1992aBaird PN Groves N Haber DA Housman DE Cowell JK Identification of mutations in the WT1 gene in tumours from patients with the WAGR syndrome.Oncogene. 1992a; 7: 2141-2149PubMed Google Scholar) and radiolabeling them with [32P]. Products were electrophoresed, at 4°C, on both 5% and 10% nondenaturing polyacrylamide gels. In addition, exons 5, 8, and 9 were directly sequenced on an ABI 377 automated sequencer. PCR products were purified either on spin columns (Promega Biotec) or by gel extraction (Qiagen Gel Extraction Kit). Samples (2–5 μl) of the purified product were sequenced by use of a Thermo-Sequenase dye terminator cycle sequencing kit and 5 pmol of the original primer.Table 1Results and Clinical StatusNephropathyCaseKaryotypeOnset of ProteinuriaESRF/ Dialysis?Nephrectomy?Transplant?Syndromal Tumor?Genital Status (Internal or External)Other Renal or Extrarenal Defects?Family History?WT1 Gene Mutation?Age at Last Follow-upDMS: 146,XXDay 1YesNoYesNoFemalePsychomotorYesNo; exons 1–1010 years 246,XXDay 1YesYes, right and leftYesNoFemaleNoNoNo; exons 1–104 years 346,XXDay 1YesYes, right and leftNoNoFemaleNoYesNo; exons 1–108 years 446,XYDay 1YesNoNoNoMaleNoNoNo; exons 1–10(Died at age 3½ years) 546,XY>1 yearYesYes, right and leftYesNoMaleNoNoNo; exons 1–1013 years 646,XY>1 yearYesYes, right and leftYesNoMaleNoNoNo; exons 1–1011½ years 746,XY>1 yearNoNoNoNoMaleNoNoNo; exons 1–103 years 846,XXDay 1NoNoNoNoFemalePsychomotor, hypothyroidismNoNo; exons 1–10(Died at age 7 mo) 946,XY6 moYesNoYesNoMalePulmonary stenosisNoNo; exons 1–1014½ years 1046,XX>1 yearNoNoNoNoFemalePsychomotorNoNo; exons 1–10(Died at age 3½ years) 1146,XY18 moYesNoNoNoMaleNoNoNo; exons 1–105½ years 1246,XXDay 1YesNoNoNoFemalePsychomotor?No; exons 2–10(Died at age 2 mo) 13UnknownDay1YesNoNoNoFemaleNoYesNo; exons 2–10(Died at age 7 mo) 1446,XYDay1NoNoNoNoFemaleNoNoExon 4 polymorphism(Died at age 3 mo) 1546,XXDay1NoNoNoNoFemalePsychomotorNoNo; exons 2–10(Died at age 10 mo) 1646,XYDay 1NoNoNoNoMaleCryptorchidism?NoAflIII polymorphism(Died at age 2 mo) 17UnknownDay 1NoNoNoNoUnknownNoNoNo; exons 2–10(Died at age 10 wk) 18UnknownDay 1NoNoNoNoUnknownNoNoNo; exons 2–10(Died at age 8 mo) 1946,XX1 yearYesNoNoNoFemaleLeft kidney dysplasticNoNo; exons 5, 8, 92½ years 2046,XY9 moNoNoNoNoMalenoNoNo; exons 5, 8, 914 mo 2146,XY>2 yearsYesYes, right and leftYesWilmsFemaleNoNoExon 9: 1180C→T (R394W)13 years 2246,XY1 yearYesYes, right and leftYesWilmsAmbiguousNoNoExon 9: 1180C→T (R394W)11½ yearsFSGS: 2346,XX4 moNoNoNoFemaleNoNoExons 1–107 years 2446,XX>1 year???NoFemaleNoYesExons 1–10Lost to follow-up 2546,XY>1 year???NoMaleNoYesExons 1–10Lost to follow-up 2646,XXDay 1NoNoNoNoFemaleNoYesExons 1–103½ years 2746,XY18 moYesNoYesNoMaleNoYesExons 1–1013 years 2846,XY>1 yearYes?YesNoMaleNoNoExons 1–107 years 2946,XX1 yearYesYes, right and leftNoNoFemaleSpondyloepiphyseal dysplasiaNoExons 1–10(Died at age 6 years) 3046,XY5 years8 yearsNoYesGonadoblastomaFemaleNo?Intron 9 +5G→A16 years Open table in a new tab No WT1 mutations were detected in either the 20 patients with isolated DMS or the 7 patients with isolated FSGS, although patient 14 demonstrated a newly discovered polymorphism in codon 178 C→T in exon 4, which conserves a threonine, and patient 16 a demonstrated previously reported (Groves et al. Groves et al., 1992Groves N Baird PN Hogg A Cowell JK A single base pair polymorphism in the WT1 gene detected by single-stranded conformational polymorphism analysis.Hum Genet. 1992; 90: 440-442Crossref PubMed Scopus (8) Google Scholar) polymorphism, an A→G transition in codon 313 (arginine) in exon 7, which destroys an AflIII restriction-enzyme recognition site. Both DDS patients showed the expected (as reported by Baird et al. Baird et al., 1992bBaird PN Santos A Groves N Jadresic L Cowell JK Constitutional mutations in the WT1 gene in patients with Denys-Drash syndrome.Hum Mol Genet. 1992b; 1: 301-305Crossref PubMed Scopus (109) Google Scholar) 1180C→T mutations in exon 9; in addition, a +5G→A mutation in intron 9 was detected during this study in patient 30, who had classic features of FS (previously reported by Klamt et al. Klamt et al., 1998Klamt B Koziell AB Poulat F Wieacker P Scambler P Berta P Gessler M Frasier syndrome is caused by defective alternative splicing leading to an altered ratio of WT1 +/− KTS splice isoforms.Hum Mol Genet. 1998; 7: 709-714Crossref PubMed Scopus (270) Google Scholar). In conclusion, when a larger, more generalized population of cases with DMS is examined, mutations of WT1 are both less frequent than initial data have suggested and absent in isolated FSGS. This confirms the genetic heterogeneity of DMS and FSGS, despite the uniform renal histological findings seen throughout this group of conditions. Supportive evidence comes from linkage studies of familial FSGS, which show that the candidate gene for this condition lies on chromosome 1q25-q31 (Fuchshuber et al. Fuchshuber et al., 1995Fuchshuber A Jean G Gribouval O Gubler MC Broyer M Beckmann JS Niaudet P et al.Mapping a gene (SRN1) to chromosome 1q25-q31 in idiopathic nephrotic syndrome confirms a distinct entity of autosomal recessive nephrosis.Hum Mol Genet. 1995; 4: 2155-2158Crossref PubMed Scopus (120) Google Scholar). Isolated DMS and FSGS may therefore also result from abnormalities of other glomerular genes, perhaps downstream of WT1 and mimicking the effects of WT1 mutations seen in DDS and FS. We propose that, if WT1 gene mutations are present in isolated renal DMS or FSGS, this lends strong support to a diagnosis of DDS or FS, and the spectrum of DDS should be broadened to include occasional cases in which the characteristic nephropathy does occur alone. In this situation, WT1 mutations appear more common in phenotypic females, which affirms the less critical role of WT1 in female gonadal development, as has been suggested by experimental data (Nachtigal et al. Nachtigal et al., 1998Nachtigal MW Hirokawa Y Enyeart-VanHouten DL Flanagan JN Hammer GD Ingraham HA Wilms' tumor 1 and Dax-1 modulate the orphan nuclear receptor SF-1 in sex-specific gene expression.Cell. 1998; 93: 445-454Abstract Full Text Full Text PDF PubMed Scopus (476) Google Scholar). This finding also correlates with cases of FS in which individuals with characteristic WT1 mutations in intron 9 and with a 46,XX karyotype develop nephropathy but have no obvious gonadal abnormality (Klamt et al. Klamt et al., 1998Klamt B Koziell AB Poulat F Wieacker P Scambler P Berta P Gessler M Frasier syndrome is caused by defective alternative splicing leading to an altered ratio of WT1 +/− KTS splice isoforms.Hum Mol Genet. 1998; 7: 709-714Crossref PubMed Scopus (270) Google Scholar). Karyotype analysis remains the most important first-line investigation in phenotypic females with isolated renal DMS or FSGS; however, WT1 mutation analysis should also be considered in isolated DMS and FSGS, since the presence of characteristic WT1 gene mutations may be important in the determination of Wilms tumor risk, especially in 46,XX individuals who may not demonstrate any other clues for an underlying diagnosis of DDS. A.B.K. is funded by a National Kidney Research Fund Twistington Higgins Fellowship. We would like to thank the renal units throughout the United Kingdom, which kindly provided patients and clinical information for this study.