WT1 splice site mutation in a 46,XX female with minimal-change nephrotic syndrome and Wilms' tumour

Chantal Loirat1, Jean Luc André2, Jacqueline Champigneulle3, Cécile Acquaviva4, Dominique Chantereau4, Rosine Bourquard2, Jacques Elion4 and Erick Denamur4,

1 Service de Néphrologie, 4 Laboratoire de Biochimie Génétique and INSERM U458, Hôpital Robert Debré, Paris, 2 Service de Néphrologie Pédiatrique, 3 Service d'Anatomie et de Cytologie Pathologiques, CHU Nancy, Vandoeuvre, France

Keywords: Denys–Drash syndrome; Frasier syndrome; Wilms' tumour; WT1 gene



   Introduction
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 Introduction
 Case
 Discussion
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Denys–Drash syndrome (DDS) and Frasier syndrome (FS) were identified 30 years ago as two rare syndromes leading to end-stage renal failure. DDS includes a specific nephropathy characterized by diffuse mesangial sclerosis, associated to male pseudohermaphroditism, and/or Wilms' tumour [1,2]. FS is defined by the association of focal and segmental glomerulosclerosis, male pseudohermaphroditism and gonadoblastoma [3].

Recently, molecular biology has helped the understanding of the pathophysiology of these syndromes by identifying for both of them heterozygous mutations in the WT1 gene [47]. WT1 encodes a zinc-finger protein involved in kidney and gonadal development [4]. WT1 is composed of 10 exons and multiple proteins are made from this gene by a combination of alternative translation start sites, RNA editing and RNA splicing [4]. A highly conserved feature during species evolution is the use of two splice donor sites in the 5' region of intron 9, resulting in the presence (+) or absence (-) of three amino acids, lysine–threonine–serine (KTS), between zinc fingers 3 and 4. The +KTS and -KTS WT1 isoform proteins seem to have distinct roles within the cell nucleus [4].

Phenotype/genotype correlations, first proposed as missense mutations in the exons coding for zinc-finger domains, lead to DDS by dominant negative effect, whereas splice-site mutations in the alternative splicing site of intron 9 modify the +KTS/-KTS isoform ratio and lead to FS by haploinsufficiency. However, observations mixing clinical and molecular data of each syndrome have been reported. First, DDS (P18 in [8]), index case in [9], patient 2 in [10], or isolated diffuse mesangial sclerosis (P4 in [8]) have been described with intron 9 splice-site mutation. Secondly, intron 9 splice-site mutations have been reported in patients with focal and segmental glomerulosclerosis and either male phenotype with hypospadias and testicular ectopia [11] or Wilms' tumour [12]. In addition, FS are now reported in 46,XX patients [7,9,13,14].

We report here a WT1 splice-site mutation in a 46,XX female child with Wilms' tumour and minimal-change nephrotic syndrome evolving towards end-stage renal failure.



   Case
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 Introduction
 Case
 Discussion
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Our subject, a girl born on 3 November 1992, had Wilms' tumour of the right kidney diagnosed in October 1994. Laboratory investigations indicated proteinuria 8.9 g/l, plasma proteins 46 g/l, serum creatinine 52 µmol/l. External genitalia were normal and karyotype was 46,XX. Chemotherapy with vincristine, actinomycin and epirubicin was started. Right nephrectomy was performed in November 1994. Histology showed stage III nephroblastoma. About 100 glomeruli were available in the non-tumoral renal tissue. Most of them showed minimal-change lesions with some podocyte turgescence and mild segmental mesangial hypercellularity. Chemotherapy was pursued from December 1994 to September 1995. Irradiation of the operated site was performed from December 1994 to January 1995.

From December 1994 to December 1995, proteinuria was 4–10 g/l, plasma proteins 45–56 g/l and serum albumin 22–32 g/l. Serum creatinine increased from 75 µmol/l in December 1994 to 140 µmol/l in December 1995, and to 390 µmol/l in September 1996. Ultrasound examination of the left kidney showed hyperechogenic cortex and loss of corticomedullary differentiation.

Regular haemodialysis was started in September 1996. The child received a cadaver kidney graft in December 1998. At 18 months follow-up, serum creatinine was 62 µmol/l. Analyses of the WT1 gene were performed. In a first step, all the 10 exons and intron/exon boundaries were sequenced from white blood cell genomic DNA as in Jeanpierre et al. [8]. A 1128+5 G->A heterozygous mutation in WT1 intron 9 splice site was identified in March 2000 (Figure 1AGo). The presence of this mutation was confirmed on the other strand (data not shown). In a second step, the sequencing of all the gene exons and intron/exon boundaries was performed on DNA extracted from the tumoral tissue of the right nephrectomy, which had been conserved at -80°C. The same mutation was evidenced at the heterozygous state in the tumour (Figure 1BGo) but no other mutation was found. Interestingly, no loss of heterozygoty was observed in the tumoral tissue as indicated by the presence of two peaks (G and A) at the position 1228+5 (Figure 1Go). This absence of loss of heterozygoty at the WT1 locus has been confirmed by the presence in the tumoral tissue of a neutral polymorphism within the exon 7 at position R301 (CGA->CGG), again with the presence of two alleles (A and G) (data not shown). Left nephrectomy was performed in March 2000 because of the risk of recurrent Wilms' tumour. Histology of the nephrectomy specimen showed unspecific end-stage lesions. The WT1 mutation was absent in both parents and in the healthy sister of the patient.



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Fig. 1.  DNA sequence analyses of the exon 9/intron 9 boundary of the WT1 gene in the white blood cell (A) and the tumour (B). KTS correspond to the alternative spliced amino acids. The heterozygous mutation 1228+5 G->A corresponds to a superposition of both G and A peaks, each at the same proportion, and is indicated by the N in the base sequence and the arrow.

 



   Discussion
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 Introduction
 Case
 Discussion
 References
 
The observation presented here adds to the previously reported complexity in defining DDS and FS. In FS, the molecular defects affect the intron 9 splice-site with, in over 24 cases [57,12,1418], a predominance of the 1228+4 C->T mutation followed by the 1228+5 G->A mutation. Until recently, the absence of Wilms' tumour was considered as a characteristic of FS, in contrast to its high incidence in DDS. Nevertheless, the occurrence of Wilms' tumour in our patient and in a 3-year-old 46,XY FS patient with the WT1 intron 9+4 C->T mutation [12] demonstrates that such a criterion cannot be relied on. Our data suggest that the partial loss of one splice form of WT1 is sufficient to induce Wilms' tumour formation, as we did not find other mutations in the WT1 gene or loss of heterozygoty in the tumour. However, we did not exclude the alternative hypothesis of a modification of the methylation pattern of the gene or regulatory regions [19,20], which could have occurred on the other allele. Interestingly, Baudry et al. [21] have shown that splicing alterations are present in 63% of sporadic Wilms' tumours. Alternatively, as Wilms' tumour has only been reported in two patients with FS and splice-site mutation ([12] and this case), it may be hypothesized that its development in these two patients is linked to alterations of other genes involved in Wilms' tumour [22]. From a practical point of view, despite the low incidence of Wilms' tumour in FS, it may be justified to perform regular renal ultrasonography in these patients, just as proposed for patients with DDS.

The occurrence of nephrotic syndrome and Wilms' tumour in a 2-year-old child is more suggestive of DDS than of FS. Renal histology in our patient did not show diffuse mesangial sclerosis characteristic of DDS. It showed minimal-change lesions as observed in FS at the early phase of the glomerulopathy. In our patient, the nephrotic syndrome was discovered when the child was 2 years old. Early onset of proteinuria has also been reported in several FS patients, at the age of 7 months in two cases [7,12], 11 months in another case [3], and between 1 and 6 years of age in 17 cases [3,57,14,15,18]. Evolution towards end-stage renal disease occurred within 2 years in our patient. Only six FS patients have been reported to be in end-stage renal failure before the age of 10 years [3,5,7,15,18], but none earlier than 7.5 years [3]. Most patients only reached that stage during the second or third decade or later. It may be speculated that unilateral nephrectomy in our patient accelerated the deterioration of renal function, possibly because of the podocyte dysfunction induced by the mutation [23].

Another point is that our patient is an additional case of FS in a 46,XX girl. Four similar cases with intronic splice-site mutations have been reported [7,9,13,14]. All had normal female genitalia and sexual development, as in 46,XY patients. In fact, the first 46,XX patient with FS, a 14-year-old girl with primary hypogonadism, amenorrhoea, and absent uterus, fallopian tubes and gonads, was reported in 1992 by Bailey et al. [24]. However, no WT1 molecular data was available in this patient. It must also be stressed that female external genitalia can no longer be diagnostic criteria for FS, as a 46,XY boy with intron 9 mutation, focal and segmental glomerulosclerosis, genital anomalies (hypospadias and testicular ectopia) and diaphragmatic hernia, has been reported [11].

In this context, the question is raised as to which patients with steroid-resistant proteinuria/nephrotic syndrome with minimal-change lesions/focal and segmental glomerulosclerosis should be screened for WT1 intron 9 mutations. We propose that this search should be performed at least in female adolescents with amenorrhoea, in addition to karyotype, and in boys with genital anomalies (testicular ectopia, hypospadias) [11], in boys and girls with diaphragmatic hernia [11], and whenever more than one girl in a family has proteinuria, steroid-resistant nephrotic syndrome, or focal and segmental glomerulosclerosis [7,14].



   Notes
 
Correspondence and offprint requests to: Erick Denamur, Laboratoire de Biochimie Génétique, Hôpital Robert Debré, 48 Boulevard Sérurier, 75935 Paris cedex 19, France. Email: denamur{at}infobiogen.fr Back



   References
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 Introduction
 Case
 Discussion
 References
 

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Received for publication: 27. 7.02
Accepted in revised form: 5.12.02