Molecular analysis of the sex-determining region from the Y chromosome in two patients with Frasier syndrome.

In the Frasier syndrome there is an association between XY gonadal dysgenesis and chronic renal failure. Owing to an observed sex reversal, the Y chromosomes of two girls with this syndrome have been analyzed. Using molecular-biology techniques, no major alterations of the known sex-determining area of the Y chromosome were found. Furthermore, the sequence did not reveal impairment of the recently described testis-determining factor SRY. These data suggest that in the Frasier syndrome, XY sex reversal and renal failure could be the result of either faulty gene(s) located downstream in the sex differentiation pathway during embryogenesis, or impaired SRY regulation. Preliminary results on the Wilms' tumor suppressor gene WT1, a candidate for acting downstream to SRY, are also provided.     

46,XY phenotypic male with focal segmental glomerulosclerosis caused by the WT1 splice site mutation

OBJECTIVE: Frasier syndrome is characterized by progressive glomerulopathy due to nonspecific focal and segmental glomerulosclerosis (FSGS), 46,XY sex reversal and the development of gonadoblastoma from dysgenetic gonads. Donor splice site heterozygous mutations in intron 9 of the Wilms' tumor gene (WT1) cause this disease. We investigated whether WT1 mutations showed clinical heterogeneity. PATIENTS AND METHODS: A 6-year-old phenotypic boy was diagnosed as having FSGS. His karyotype was 46,XY. Gonadotropin-releasing hormone and human chorionic gonadotropin stimulation tests revealed normal luteinizing hormone, follicle-stimulating hormone and testosterone responses. The other patient was a 7-year-old 46,XY female with FSGS. Prophylactic gonadectomy was performed and gonadoblastoma was found. By polymerase chain reaction and direct sequencing, WT1 was analyzed in these patients. RESULTS AND CONCLUSION: Both patients had IVS9 + 5G-->A in intron 9 of the WT1. Our study indicates a normal 46,XY phenotypic male patient with FSGS. The phenotypic variations of the WT1 splice site mutations are further expanded.     

The human sex-determining gene SRY is a direct target of WT1

The product of the Wilms' tumor gene, WT1, is essential for male sex determination and differentiation in mammals. In addition to causing Wilms' tumor, mutations in WT1 often cause two distinct but overlapping urogenital defects in men, Denys-Drash syndrome and Frasier syndrome. In this study we investigated the regulation of the sex determination gene SRY by WT1. Our results showed that WT1 up-regulates the SRY gene through the proximal early growth response gene-1-like DNA-binding sequences in the core promoter. Mutant WT1 proteins in Denys-Drash syndrome patients were unable to activate this promoter. These mutants did not act in a dominant negative manner, as expected over the wild-type WT1 in this promoter. We also found that WT1 could transactivate the endogenous SRY gene. These observations, together with the overlapping expression patterns of WT1 and SRY in human gonads, led us to propose that WT1 regulates SRY in the initial sex determination process in humans and activates a cascade of genes ultimately leading to the complete organogenesis of the testis.     

A 46,XY female DSD patient with bilateral gonadoblastoma, a novel SRY missense mutation combined with a WT1 KTS splice-site mutation

Patients with Disorders of Sex Development (DSD), especially those with gonadal dysgenesis and hypovirilization are at risk of developing malignant type II germ cell tumors/cancer (GCC) (seminoma/dysgerminoma and nonseminoma), with either carcinoma in situ (CIS) or gonadoblastoma (GB) as precursor lesion. In 10-15% of 46,XY gonadal dysgenesis cases (i.e., Swyer syndrome), SRY mutations, residing in the HMG (High Mobility Group) domain, are found to affect nuclear transport or binding to and bending of DNA. Frasier syndrome (FS) is characterized by gonadal dysgenesis with a high risk for development of GB as well as chronic renal failure in early adulthood, and is known to arise from a splice site mutation in intron 9 of the Wilms' tumor 1 gene (WT1). Mutations in SRY as well as WT1 can lead to diminished expression and function of SRY, resulting in sub-optimal SOX9 expression, Sertoli cell formation and subsequent lack of proper testicular development. Embryonic germ cells residing in this unfavourable micro-environment have an increased risk for malignant transformation. Here a unique case of a phenotypically normal female (age 22 years) is reported, presenting with primary amenorrhoea, later diagnosed as hypergonadotropic hypogonadism on the basis of 46,XY gonadal dygenesis with a novel missense mutation in SRY. Functional in vitro studies showed no convincing protein malfunctioning. Laparoscopic examination revealed streak ovaries and a normal, but small, uterus. Pathological examination demonstrated bilateral GB and dysgerminoma, confirmed by immunohistochemistry. Occurrence of a delayed progressive kidney failure (focal segmental glomerular sclerosis) triggered analysis of WT1, revealing a pathogenic splice-site mutation in intron 9. Analysis of the SRY gene in an additional five FS cases did not reveal any mutations. The case presented shows the importance of multi-gene based diagnosis of DSD patients, allowing early diagnosis and treatment, thus preventing putative development of an invasive cancer.     

Beckwith-Wiedemann syndrome: multiple molecular mechanisms

Beckwith-Wiedemann syndrome (BWS) is a congenital overgrowth condition with an increased risk of developing embryonic tumours, such as Wilms' tumour. The cardinal features are abdominal wall defects, macroglossia and gigantism. BWS is generally sporadic; only 10-15% of cases are familial. A variety of molecular aberrations have been associated with BWS. The only mutations within a gene are loss-of-function mutations in the CDKN1C gene, which codes for an imprinted cell-cycle regulator. CDKN1C mutations appear to be particularly associated with umbilical abnormalities, but not with increased predisposition to Wilms' tumour. In the remaining BWS subgroups, a disturbance of the tight epigenetic regulation of gene expression (patUPD 11p, microdeletions or epimutations) seems to be the cause of the syndrome. Here we describe the clinical presentation of BWS and its dissociation from phenotypically overlapping overgrowth syndromes. We then review the current concepts of causative molecular genetic and epigenetic mechanisms, and discuss future directions of research.     

WT1 mutations in patients with Denys-Drash syndrome: a novel mutation in exon 8 and paternal allele origin

Denys-Drash syndrome (DDS) is characterized by early onset nephropathy, pseudohermaphroditism in males and a high risk for developing Wilms' tumour (WT). The exact cause of DDS is unknown but germline mutations in the Wilms' tumour suppressor gene (WT1) have recently been described in the majority of DDS patients studied. These mutations occur de novo and are clustered around the zinc finger (ZF) coding exons of the WT1 gene. Analysis of exons 2–10 of the WT1 gene in constitutional DNA from five patients with DDS was carried out using the polymerase chain reaction (PCR) and direct DNA sequencing. In four out of the five patients, heterozygous germline mutations were found: a novel point mutation in exon 8 (ZF₂) at codon 377 altering the wild-type histidine to arginine, and three previously described point mutations in exon 9 (ZF₃) in the codons corresponding to amino acids ³⁹⁴Arg and ³⁹⁶Asp. In one patient, no mutations could be demonstrated. In three patients where parental DNA was available, the mutations were shown to have occurred de novo. Furthermore, since tumour DNA in two of these cases had lost the wild-type allele, polymorphic markers from the short arm of chromosome 11 were used to determine the parental origin of the mutant chromosome. In both cases, the mutant chromosome was shown to be of paternal origin. Since the majority of published WT1 mutations in DDS patients alter a RsrII restriction site in exon 9, we were able to perform PCR-based diagnosis in a female patient with early renal insufficiency and normal external genitalia.     

Germline mutations in the Wilms' tumor suppressor gene are associated with abnormal urogenital development in Denys-Drash syndrome.

Denys-Drash syndrome is a rare human condition in which severe urogenital aberrations result in renal failure, pseudohermaphroditism, and Wilms' tumor (nephroblastoma). To investigate its possible role, we have analyzed the coding exons of the Wilms' tumor suppressor gene (WT1) for germline mutations. In ten independent cases of Denys-Drash syndrome, point mutations in the zinc finger domains of one WT1 gene copy were found. Nine of these mutations are found within exon 9 (zinc finger III); the remaining mutation is in exon 8 (zinc finger II). These mutations directly affect DNA sequence recognition. In two families analyzed, the mutations were shown to arise de novo. Wilms' tumors from three individuals and one juvenile granulosa cell tumor demonstrate reduction to homozygosity for the mutated WT1 allele. Our results provide evidence of a direct role for WT1 in Denys-Drash syndrome and thus urogenital system development.     

Wilms' tumor protein Wt1 is an activator of the anti-Müllerian hormone receptor gene Amhr2

The Wilms' tumor protein Wt1 plays an essential role in mammalian urogenital development. WT1 mutations in humans lead to a variety of disorders, including Wilms' tumor, a pediatric kidney cancer, as well as Frasier and Denys-Drash syndromes. Phenotypic anomalies in Denys-Drash syndrome include pseudohermaphroditism and sex reversal in extreme cases. We have used cDNA microarray analyses on Wt1 knockout mice to identify Wt1-dependent genes involved in sexual development. The gene most dramatically affected by Wt1 inactivation was Amhr2, encoding the anti-Müllerian hormone (Amh) receptor 2. Amhr2 is an essential factor for the regression of the Müllerian duct in males, and mutations in AMHR2 lead to the persistent Müllerian duct syndrome, a rare form of male pseudohermaphroditism. Here we show that Wt1 and Amhr2 are coexpressed during urogenital development and that the Wt1 protein binds to the promoter region of the Amhr2 gene. Inactivation and overexpression of Wt1 in cell lines was followed by immediate changes of Amhr2 expression. The identification of Amhr2 as a Wt1 target provides new insights into the role of Wt1 in sexual differentiation and indicates, in addition to its function in early gonad development and sex determination, a novel function for Wt1, namely, in Müllerian duct regression.     

Distinct molecular origins for Denys-Drash and Frasier syndromes

The direct involvment of the Wilm's tumor suppressor gene (WT1) in Denys-Drash syndrome through mutations within exons 8 or 9 has recently been established. The absence of such alterations in three patients with Frasier syndrome provides a molecular basis for distinguishing these two syndromes that are associated with streak gonads, pseudohermaphroditism and renal failure.     

Nephropathy and defective spermatogenesis in mice transgenic for a single isoform of the Wilms' tumour suppressor protein, WT1-KTS, together with one disrupted Wt1 allele

The Wilms' tumour suppressor protein, WT1, is a zinc finger protein essential for the development of several organs, including the kidney and gonads. In each of these tissues WT1 is required at multiple stages of development and its persistent expression in podocytes and Sertoli cells suggests WT1 may also have a role in the maintenance of kidney and testis function throughout adult life. Naturally occurring isoforms of WT1 are generated by alternative mRNA splicing. An altered ratio of the splice isoforms WT1-KTS and WT1 + KTS appears to be sufficient to account for the developmental abnormalities (pseudohermaphroditism and nephropathy) characteristic of Frasier syndrome. We show that mice with a transgene encoding WT1-KTS do not differ from their wild-type littermates unless they are also heterozygous for a null mutation at the endogenous Wt1 locus. Animals with both genetic modifications develop proteinuria, together with multiple glomerular cysts, and male infertility. These pathologic changes may be explained as a consequence of altering the WT1 isoform ratio in tissues that express WT1 during adulthood. The results suggest WT1 misexpression could contribute to human glomerulocystic kidney disease.     

Molecular analysis of aniridia patients for deletions involving the Wilms' tumor gene

A human aniridia candidate (AN) gene on chromosome 11p13 has been cloned and characterized. The AN gene is the second cloned gene of the contiguous genes syndrome WAGR (Wilms' tumor, aniridia, genitourinary malformations, mental retardation) on chromosome 11p13, WT1 being the first gene cloned. Knowledge about the position of the AN and WT1 genes on the map of 11p13 makes the risk assessment for Wilms' tumor development in AN patients possible. In this study, we analyzed familial and sporadic aniridia patients for deletions in 11p13 by cytogenetic analyses, in situ hybridization, and pulsed field gel electrophoresis (PFGE). Cytogenetically visible deletions were found in 3/11 sporadic AN cases and in one AN/WT patient, and submicroscopic deletions were identified in two sporadic AN/WT patients and in 1/9 AN families. The exact extent of the deletions was determined with PFGE and, as a result, we could delineate the risk for Wilms' tumor development. Future analyses of specific deletion endpoints in individual AN cases with the 11p13 deletion should result in a more precise risk assessment for these patients.     

A novel WT1 gene mutation in a newborn infant diagnosed with Denys-Drash syndrome

Denys-Drash syndrome (DDS) is a rare disorder characterized by glomerulopathy, genital abnormalities and predisposition to Wilms' tumor. It is associated with constitutional Wilms'tumor suppressor 1 (WT1) gene mutations, in which the majority being missense mutations in the zinc-finger region. Here, we present a newborn with DDS, associated with a novel heterozygous missense mutation, p.Asp396His, on exon 9 of WT1.     

Mice lacking the 68-amino-acid,mammal-specific N-terminal extension of WT1 develop normally and are fertile

Mutations in the Wilms' tumor 1 gene, WT1, cause pediatric nephroblastoma and the severe genitourinary disorders of Frasier and Denys-Drash syndromes. High levels of WT1 expression are found in the developing kidney, uterus, and testis--consistent with this finding, the WT1 knockout mouse demonstrates that WT1 is essential for normal genitourinary development. The WT1 gene encodes multiple isoforms of a zinc finger-containing protein by a combination of alternative splicing and alternative translation initiation. The use of an upstream, alternative CUG translation initiation codon specific to mammals results in the production of WT1 protein isoforms with a 68-amino-acid N-terminal extension. To determine the function in vivo of mammal-specific WT1 isoforms containing this extension, gene targeting was employed to introduce a subtle mutation into the WT1 gene. Homozygous mutant mice show a specific absence of the CUG-initiated WT1 isoforms yet develop normally to adulthood and are fertile. Detailed histological analysis revealed normal development of the genitourinary system.     

Insertional inactivation of the WT1 gene in tumour cells from a patient with WAGR syndrome

The WT1 gene was analysed using DNA from a Wilms' tumour derived from a patient with the WAGR syndrome using single strand conformation polymorphism analysis and polymerase chain reaction sequencing. A 14-bp insertion was found in the intron part of the splice donor site of exon 7 and was a tandem duplication of an upstream exon sequence. This mutation would be expected to disrupt the correct processing of the WT1 mRNA and is predicted to result in a non-functional protein. This observation further supports the role of WT1 in Wilms' tumorigenesis in patients with constitutional 11p13 deletions.     

Wilms' tumour: reconciling genetics and biology.

Wilms' tumour, a paediatric malignancy of the kidney, is a striking example of the relationship between aberrant development and cancer. Several different genetic loci have been implicated in the aetiology of the tumour; genomic imprinting also plays a role. One Wilms' tumour predisposition gene (WT1), encoding a zinc finger protein, is expressed in a limited set of tissues, including developing nephrons and gonads. The biology and genetics of Wilms' tumour underline the developmental relationship between kidneys and gonads.     

Genome-wide loss of maternal alleles in a nephrogenic rest and Wilms' tumour from a BWS patient

A patient with Beckwith-Wiedemann syndrome (BWS) presented with Wilms' tumour. Examination of the nephrectomy specimen showed, in addition to the tumour, the presence of nephrogenic rests. Nephrogenic rests are thought to be precursor lesions from which a Wilms' tumour may develop. A molecular analysis examining the loss of constitutional heterozygosity (LOCH), initially for chromosome 11, was performed on peripheral blood, the normal kidney, nephrogenic rest and tumour material. The study was extended to include markers from all 23 chromosomes. At each informative, locus, LOCH of the maternal allele was shown in the nephrogenic rest and tumour material. In addition, the normal kidney displayed allele imbalance. It would appear from these results that either extensive LOCH across the genome was an early genetic event in the development of malignancy in this patient or that the tumour and rest developed from cells containing no maternal chromosomes. The apparent LOCH seen in the normal kidney sample implies that full reduction to homozygosity is consistent with a histologically normal appearance. Putative mechanisms to explain this phenomenon are discussed.