Denys-Drash syndrome: a role for the WT1 tumour suppressor gene in urogenital development

The study of inherited disease provides unique insight into basic developmental and biochemical processes. By linking the pathogenesis of complex malformations to mutations of specific genes, the function of those genes in normal developmental biology can be inferred. One such disorder is the Denys-Drash syndrome, where identification of genetic lesions within the WT1 tumour suppressor gene has provided astonishing insight into events regulating development of the urogenital system.     

Germline missense mutations affecting KRAS Isoform B are associated with a severe Noonan syndrome phenotype

Noonan syndrome (NS) is a developmental disorder characterized by short stature, facial dysmorphia, congenital heart disease, and multiple skeletal and hematologic defects. NS is an autosomal dominant trait and is genetically heterogeneous. Gain of function of SHP-2, a protein tyrosine phosphatase that positively modulates RAS signaling, is observed in nearly 50% of affected individuals. Here, we report the identification of heterozygous KRAS gene mutations in two subjects exhibiting a severe NS phenotype with features overlapping those of cardiofaciocutaneous and Costello syndromes. Both mutations were de novo and affected exon 6, which encodes the C-terminal portion of KRAS isoform B but does not contribute to KRAS isoform A. Structural analysis indicated that both substitutions (Val152Gly and Asp153Val) perturb the conformation of the guanine ring-binding pocket of the protein, predicting an increase in the guanine diphosphate/guanine triphosphate (GTP) dissociation rate that would favor GTP binding to the KRASB isoform and bypass the requirement for a guanine nucleotide exchange factor.     

The Wilms' tumor suppressor Wt1 is associated with the differentiation of retinoblastoma cells.

We have demonstrated recently that Wilms' tumor suppressor 1 (Wt1),in addition to its role in genitourinary formation,is required for the differentiation of ganglion cells in the developing retina. Here we provide further evidence that Wt1 is associated with neuronal differentiation. Thus, the retinoblastoma-derived human cell line, Y-79, contained robust amounts of Wt1 mRNA and protein. Wt1 expression was down-regulated upon laminin-induced differentiation of Y-79 into neuron-like cells. Inhibition of Wt1 with antisense oligonucleotides dramatically reduced the capacity of undifferentiated Y-79 cells to undergo neuronal differentiation, whereas sense and missense oligonucleotides had no effect. Wt1 immunoreactivity was also detected in solid retinoblastomas, in which it resided mainly in areas with moderate proliferative activity. These findings suggest a role for Wt1 in the differentiation of retinoblastoma cells. Furthermore, Wt1 expression in retinoblastoma may reflect the potential of these tumors to initiate the early steps of neuronal differentiation.     

Regulation of ornithine decarboxylase gene expression by the Wilms' tumor suppressor WT1.

The importance of ornithine decarboxylase (ODC) to cell proliferation is underscored by the complex array of cell-specific mechanisms invoked to regulate its synthesis and activity. Misregulation of ODC has severe negative consequences on normal cell function, including the acquisition of tumorigenic growth properties by cells overexpressing ODC. We hypothesize that ODC gene expression is a candidate target for the anti-proliferative function of certain tumor suppressors. Here we show that the Wilms' tumor suppressor WT1 binds to multiple sites within the human ODC promoter, as determined by DNase I protection and methylation interference assays. The expression of WT1 in transfected HCT 116, NIH/3T3 and HepG2 cells represses activity of the ODC promoter controlling expression of a luciferase reporter gene. In contrast WT1 expression enhances ODC promoter activity in SV40-transfected HepG2 cells. Both the extent of modulation of ODC gene expression and the mediating WT1 binding elements are cell specific. Constructs expressing WT1 deletion mutants implicate two regions required for repressor function, as well as an intrinsic activation domain. Understanding the regulation of ODC gene expression by WT1 may provide valuable insights into the roles of both WT1 and ODC in development and tumorigenesis.     

Erythropoietin-driven proliferation of cells with mutations in the tumor suppressor gene TSC2

Lymphangioleiomyomatosis (LAM) is characterized by cystic lung destruction, resulting from proliferation of smooth-muscle-like cells, which have mutations in the tumor suppressor genes TSC1 or TSC2. Among 277 LAM patients, severe disease was associated with hypoxia and elevated red blood cell indexes that accompanied reduced pulmonary function. Because high red cell indexes could result from hypoxemia-induced erythropoietin (EPO) production, and EPO is a smooth muscle cell mitogen, we investigated effects of EPO in human cells with genetic loss of tuberin function, and we found that EPO increased proliferation of human TSC2-/-, but not of TSC2+/-, cells. A discrete population of cells grown from explanted lungs was characterized by the presence of EPO receptor and loss of heterozygosity for TSC2, consistent with EPO involvement. In LAM cells from lung nodules, EPO was localized to the extracellular matrix, supporting evidence for activation of an EPO-driven signaling pathway. Although the high red cell mass of LAM patients could be related to advanced disease, we propose that EPO, synthesized in response to episodic hypoxia, may increase disease progression by enhancing the proliferation of LAM cells.     

Inhibition of cellular proliferation by the Wilms' tumor suppressor WT1 is associated with suppression of insulin-like growth factor I receptor gene expression.

We have investigated the regulation of the insulin-like growth factor I receptor (IGF-I-R) gene promoter by the Wilms' tumor suppressor WT1 in intact cells. The levels of endogenous IGF-I-R mRNA and the activity of IGF-I-R gene promoter fragments in luciferase reporter constructs were found to be significantly higher in G401 cells (a Wilms' tumor-derived cell line lacking detectable WT1 mRNA) than in 293 cells (a human embryonic kidney cell line which expresses significant levels of WT1 mRNA). To study whether WT1 could suppress the expression of the endogenous IGF-I-R gene, WT1-negative G401 cells were stably transfected with a WT1 expression vector. Expression of WT1 mRNA in G401 cells resulted in a significant decrease in the rate of cellular proliferation, which was associated with a reduction in the levels of IGF-I-R mRNA, promoter activity, and ligand binding and with a reduction in IGF-I-stimulated cellular proliferation, thymidine incorporation, and anchorage-independent growth. These data suggest that a major aspect of the action of the WT1 tumor suppressor is the repression of IGF-I-R gene expression.     

HRPT2, a tumor suppressor gene for hyperparathyroidism-jaw tumor syndrome.

Hyperparathyroidism-jaw tumor (HPT-JT) syndrome is a familial multi-tumor syndrome resulting from inactivating mutations in the HRPT2 tumor suppressor gene, which encodes a protein product named parafibromin. Here, we will review recent advances in genetic and protein studies on parafibromin, and examine its biological functions.     

TGFBR1 mutations associated with Loeys-Dietz syndrome are inactivating

To assess the effect of Loeys-Dietz syndrome (LDS) mutations affecting TGFΒR1 a selection of seven disease-associated amino acid substitutions were introduced into wild type TGFβR1 and constitutively active TGFβR1^T204D. Receptor function was tested by co-transfection with a luciferase reporter or EGFP-tagged SMAD2 in HEK293 cells. All of the mutations were found to be inactivating for canonical TGF-β signaling. Differences in residual activity were not found to correlate with disease subtype. In co-transfection experiments with equal amounts wild-type receptor, the LDS mutations were found to confer a modest dominant negative effect. These results are discussed in relation to LDS and the related Marfan syndrome.     

TGFBR1 mutations associated with Loeys-Dietz syndrome are inactivating

To assess the effect of Loeys-Dietz syndrome (LDS) mutations affecting TGFΒR1 a selection of seven disease-associated amino acid substitutions were introduced into wild type TGFβR1 and constitutively active TGFβR1(T204D). Receptor function was tested by co-transfection with a luciferase reporter or EGFP-tagged SMAD2 in HEK293 cells. All of the mutations were found to be inactivating for canonical TGF-β signaling. Differences in residual activity were not found to correlate with disease subtype. In co-transfection experiments with equal amounts wild-type receptor, the LDS mutations were found to confer a modest dominant negative effect. These results are discussed in relation to LDS and the related Marfan syndrome.     

APEX disease gene resequencing: mutations in exon 7 of the p53 tumor suppressor gene.

Detection of mutations in disease genes will be a significant application of genomic research. Methods for detecting mutations at the single nucleotide level are required in highly mutated genes such as the tumor suppressor p53. Resequencing of an individual patient's DNA by conventional Sanger methods is impractical, calling for novel methods for sequence analysis. Toward this end, an arrayed primer extension (APEX) method for identifying sequence alterations in primary DNA structure was developed. A two-dimensional array of immobilized primers (DNA chip) was fabricated to scan p53 exon 7 by single bases. Primers were immobilized with 200 microm spacing on a glass support. Oligonucleotide templates of length 72 were used to study individual APEX resequencing reactions. A template-dependent DNA polymerase extension was performed on the chip using fluorescein-labeled dideoxynucleotides (ddNTPs). Labeled primers were evanescently excited and the induced fluorescence was imaged by CCD. The average signal-to-noise ratio (S/N) observed was 30:1. Software was developed to analyze high-density DNA chips for sequence alterations. Deletion, insertion, and substitution mutations were detected. APEX can be used to scan for any mutation (up to two-base insertions) in a known region of DNA by fabricating a DNA chip comprising complementary primers addressing each nucleotide in the wild-type sequence. Since APEX is a parallel method for determining DNA sequence, the time required to assay a region is independent of its length. APEX has a high level of accuracy, is sequence-based, and can be miniaturized to analyze a large DNA region with minimal reagents.     

Identification of novel mutations in the human EXT1 tumor suppressor gene

Hereditary multiple exostoses (EXT) is a genetically heterogeneous bone disorder caused by genes segregating on human chromosomes 8, 11, and 19 and designated EXT1, EXT2 and EXT3, respectively. Recently, the EXT1 gene has been isolated and partially characterized and appears to encode a tumor suppressor gene. We have identified six mutations in the human EXT1 gene from six unrelated multiple exostoses families segregating for the EXT gene on chromosome 8. One of the mutations we detected is the same 1-bp deletion in exon 6 that was previously reported in two independent EXT families. The other five mutations, in exons 1, 6, 9, and the splice junction at the 3′ end of exon 2, are novel. In each case, the mutation is likely to result in a truncated or nonfunctional EXT1 protein. These results corroborate and extend the previous report of mutations in this gene in two EXT families, and provide additional support for the EXT1 gene as the cause of hereditary multiple exostoses in families showing linkage to chromosome 8. Received: 2 August 1996 / Revised: 18 November 1996     

Missense mutations in the BMP15 gene are associated with ovarian failure

Premature ovarian failure (POF) is an unexplained amenorrhoea (>6 months) with raised levels of gonadotropins (FSH>40 U/L) occurring before the age of 40 years. Recent studies have elucidated the role of oocyte derived growth factors (BMP15 and GDF9) in maintenance of folliculogenesis, granulosa cell (GC) proliferation and overall fertility. Our recently published work showed presence of two rare missense variants in the GDF9 gene associated with ovarian failure (Dixit et al. 2005, Menopause 12:749–754). The present case-control study has been structured to establish the role of BMP15 germline status associated with ovarian failure. Sequence analysis of the coding region of the BMP15 gene was carried out in a cohort of women with POF (n=133), primary amenorrhoea (n=60), and secondary amenorrhoea (n=9) compared with control females (n=197). This study revealed a total of 18 germline variants in the coding region of BMP15 gene, including 16 novel variants. These novel variants include one intronic variant, one 3’ flanking variant, one silent variant, and 13 missense variants. Eleven missense variants were present only in cases with complete absence in the control females. The remaining two missense variants viz. c.308A>G (p.Asn103Ser) and c.788_789insTCT (p.Leu263_Arg264insLeu) were present both in the cases and in the controls. The c.788_789insTCT variant was significantly higher in primary amenorrhoea cases than in the controls (Fisher’s exact test, P=0.034). Three frequent variants c.-9C>G, c.308A>G, and c.852C>T were chosen for haplotyping. The haplotype G-G-C was found to be significantly associated with ovarian failure (P=0.0075). In a nutshell, the BMP15 gene is highly associated with etiology of ovarian failure.     

A proteomic investigation of glomerular podocytes from a Denys-Drash syndrome patient with a mutation in the Wilms tumour suppressor gene WT1

Glomerular podocytes are essential for blood filtration in the kidney underpinned by their unique cytoskeletal morphology. An increasing number of kidney diseases are being associated with key podocyte abnormalities. The Wilms tumour suppressor gene (WT1) encodes a zinc finger protein with a crucial role in normal kidney development; and in the adult, WT1 is required for normal podocyte function. Denys-Drash Syndrome (DDS) results from mutations affecting the zinc finger domain of WT1. The aim of this study was to undertake, for the first time, a proteomic analysis of cultured human podocytes; and to analyse the molecular changes in DDS podocytes. The morphology of DDS podocytes was highly irregular, reminiscent of a fibroblastic appearance. A reference 2-D gel was generated, and 75 proteins were identified of which 43% involved in cytoskeletal architecture. The DDS and wild-type proteomes were compared by 2-D DIGE. The level of 95.6% of proteins was unaltered; but 4.4% were altered more than two-fold. A sample of proteins involved in cytoskeletal architecture appeared to be misexpressed in DDS podocytes. Consistent with this finding, overall levels of filamentous actin also appeared reduced in DDS podocytes. We conclude that one of WT1 functions in podocytes is to regulate the expression of key components and regulators of the cytoskeleton.     

Three new BLM gene mutations associated with Bloom syndrome

Bloom's syndrome (BS) is a rare autosomal recessive disease predisposing patients to all types of cancers affecting the general population. BS cells display a high level of genetic instability, including a 10-fold increase in the rate of sister chromatid exchanges, currently the only objective criterion for BS diagnosis. We have developed a method for screening the BLM gene for mutations based on direct genomic DNA sequencing. A questionnaire based on clinical information, cytogenetic features, and family history was addressed to physicians prescribing BS genetic screening, with the aim of confirming or guiding diagnosis. We report here four BLM gene mutations, three of which have not been described before. Three of the mutations are frameshift mutations, and the fourth is a nonsense mutation. All these mutations introduce a stop codon, and may therefore be considered to have deleterious biological effect. This approach should make it possible to identify new mutations and to correlate them with clinical information.     

Germline mutations of the MYH gene in Japanese patients with multiple colorectal adenomas

Germline mutations of the MYH gene have been revealed to associate with the recessive inheritance of multiple colorectal adenomas in Caucasian population. However, MYH mutations in Japanese patients have not yet been clarified. In an assessment of MYH mutations, we examined 35 Japanese patients with multiple colorectal adenomas who had neither dominant inheritance of colorectal tumors, nor germline APC mutations. One patient had a homozygous biallelic MYH mutation, R231C and three independent patients had monoallelic MYH mutations at a splice-site on exon 11 (IVS10-2 A to G). These four patients had 21 to around 100 colorectal adenomas and 1-3 synchronous colorectal carcinomas. The most common mutations in Caucasian patients, Y165C and G382D, were not detected in our Japanese cases. The MYH mutations detected in Japanese patients were novel and different from those detected among Caucasian, Indian and Pakistani patients, which suggests the existence of ethnic differentiation in MYH mutations.