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.     

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.     

Rapid screening of myelin genes in CMT1 patients by SSCP analysis: identification of new mutations and polymorphisms in the P0 gene

Charcot-Marie-Tooth type (CMT1) disease or hereditary motor and sensory neuropathy type I (HMSNI) is an autosomal dominant peripheral neuropathy. In most CMT1 families, the disease cosegregates with a 1.5-Mb duplication on chromosome 17p11.2 (CMT1A). A few patients have been found with mutations in the peripheral myelin protein 22 (PMP-22) gene located in the CMT1A region. In other families mutations have been identified in the major peripheral myelin protein p_o gene localized on chromosome Iq21-q23 (CMT1B). We performed a rapid mutation screening of the PMP-22 and P₀ genes in non-duplicated CMT1 patients by single-strand conformation polymorphism analysis followed by direct polymerase chain reaction sequencing of genomic DNA. Six new single base changes in the P₀ gene were observed: two missense mutations in, respectively, exons 2 and 3, two nonsense mutations in exon 4, and two silent mutations or polymorphisms in, respectively, exons 3 and 6.     

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.     

Papillary renal cell carcinoma: analysis of germline mutations in the MET proto-oncogene in a clinic-based population

Approximately 10% of all renal cell carcinomas (RCCs) present a distinctive papillary histology. Familial papillary RCC (PRCC) has been described, but the majority of cases appear to be sporadic. Recently, germline mutations in the MET proto-oncogene on chromosome 7 have been identified in families with hereditary PRCC. We evaluated 59 patients with PRCC for the frequency of MET germline mutations to determine the value of genetic screening of this patient population. Between 1976 and 1997, 165 patients were identified with PRCC by retrospective chart review. Fifty-nine of 133 surviving patients agreed to provide a family history, a blood specimen, and informed consent for genetic research. DNA was isolated from peripheral blood leukocytes. Denaturing high-performance liquid chromatography (DHPLC) followed by genomic sequencing was performed on eight exons of the MET proto-oncogene, including exons 5-7 of the extracellular domain, exon 14, and exons 16-19 of the tyrosine kinase domain. The 59 patients in this study included 49 men and 10 women with a mean age at diagnosis of 61 years. Bilateral and/or multifocal disease was present in 13 cases (22%). No germline mutations were detected in the studied exons of the MET proto-oncogene (exons previously reported to contain deleterious mutations in familial PRCC). No pathological MET proto-oncogene germline mutations were identified in 59 patients with PRCC. The germline mutation rate in this clinic-based population of individuals with PRCC approaches 0% (CI = 0-6.18). MET proto-oncogene germline mutation screening does not appear to be clinically indicated in patients with PRCC without additional evidence for a genetic predisposition (positive family history, unusual age at onset, bilateral disease).     

Rapid isolation of moderate and highly polymorphic DNA fragments mapping close to WT (Wilms' tumour) and AN2 (aniridia) on chromosome 11

Summary Genes implicated in the development of Wilms' tumour (WT) and aniridia (AN2) have been localised to a subregion of band p13 on chromosome 11 by molecular and cytogenetic characterisation of WAGR syndrome patients carrying variable constitutional deletions. Polymorphic markers for the region would be valuable for linkage analysis in the familial forms of both Wilms' tumour and aniridia, as well as for studying somatic rearrangements of chromosome 11 in a variety of tumour types. Here we describe the isolation and characterisation of three frequently polymorphic arbitrary DNA fragments that map proximal to the AN2 and WT loci.     

An internal deletion within an 11p13 zinc finger gene contributes to the development of Wilms' tumor

We have recently described the isolation of a candidate for the Wilms' tumor susceptibility gene mapping to band p13 of human chromosome 11. This gene, primarily expressed in fetal kidney, appears to encode a DNA binding protein. We now describe a sporadic, unilateral Wilms' tumor in which one allele of this gene contains a 25 bp deletion spanning an exon-intron junction and leading to aberrant mRNA splicing and loss of one of the four zinc finger consensus domains in the protein. The mutation is absent in the affected individual's germline, consistent with the somatic inactivation of a tumor suppressor gene. In addition to this intragenic deletion affecting one allele, loss of heterozygosity at loci along the entire chromosome 11 points to an earlier chromosomal nondisjunction and reduplication. We conclude that inactivation of this gene, which we call WT1, is part of a series of events leading to the development of Wilms' tumor.     

Heteroduplex analysis detects frameshift and point mutations in patients with acute intermittent porphyria

We used heteroduplex analysis to screen for mutations in the porphobilinogen deaminase gene in 21 patients with acute intermittent porphyria (AIP). Unique banding patterns were investigated by direct sequencing of polymerase chain reaction products and, when indicated, sequencing of cloned DNA containing the exon of interest. Two frameshift mutations were found, a 2-bp deletion in exon 5 and a 1-bp insertion in exon 7. Both mutations generate a premature stop codon. Two point mutations, in exons 10 and 14, were also observed. The CT mutation in exon 10 codes for an Arg¹⁷³ to Trp substitution, while a GA mutation in exon 14 changes Trp²⁸³ into a premature stop codon. This study extends the spectrum of mutations that cause AIP and demonstrates the utility of heteroduplex analysis as a screening technique.     

Two novel mutations in exons 19a and 20 and a BsaI polymorphism in a newly characterized intron of the neurofibromatosis type 1 gene

Neurofibromatosis type 1 (NF1) is a common autosomal dominant disorder. It is caused by mutations in the NF1 gene, which comprises 60 exons and is located on chromosome 17q11.2. A total of 170 unrelated NF1 patients were screened for mutations in four exons by temperature-gradient gel electrophoresis. Preparatory work revealed the presence of a previously uncharacterized intron (19a) in what was previously designated exon 19; this allowed us to develop assays for genomic mutation screening in the newly defined exons 19a and 19b. Two novel NF1 mutations were detected: a single-base insertion in exon 19a creating a frameshift, and a second mutation affecting the splice donor site of intron 20 and leading to skipping of exon 20. A novel BsaBI polymorphism was identified in intron 19a. Received: 11 August 1997 / Accepted: 13 November 1997     

Genomic rearrangements of the APC tumor-suppressor gene in familial adenomatous polyposis

Germline mutations of the adenomatous polyposis coli (APC) tumor-suppressor gene result in the hereditary colorectal cancer syndrome familial adenomatous polyposis (FAP). Almost all APC mutations that have been identified are single-nucleotide alterations, small insertions, or small deletions that would truncate the protein product of the gene. No well-characterized intragenic rearrangement of APC has been described, and the prevalence of this type of mutation in FAP patients is not clear. We screened 49 potential FAP families and identified 26 different germline APC mutations in 30 families. Four of these mutations were genomic rearrangements resulting from homologous and nonhomologous recombinations mediated by Alu elements. Two of these four rearrangements were complex, involving deletion and insertion of nucleotides. Of these four rearrangements, one resulted in the deletion of exons 11 and 12 and two others resulted in either complete or partial deletion of exon 14. The fourth rearrangement grossly altered the sequence within intron 14. Although this rearrangement did not affect any coding sequence of APC at the genomic DNA level, it caused inappropriate splicing of exon 14. These rearrangements were initially revealed by analyzing cDNAs and could not have been identified by using mutation detection methods that screened each exon individually. The identification of a rearrangement that did not alter any coding exons yet affected the splicing further underscores the importance of using cDNA for mutation analysis. The identification of four genomic rearrangements among 30 mutations suggests that genomic rearrangements are frequent germline APC mutations.     

The detection of mutations induced in vitro in the human p53 gene by hydrogen peroxide with the restriction site mutation (RSM) assay

We have analysed five mutation hotspots within the p53 gene (codons 175, 213, 248, 249, and 282) for mutations induced by hydrogen peroxide (H₂O₂), employing the restriction site mutation (RSM) assay. In addition, four other restriction sites covering non-hotspot codons of exons 5–9 of the p53 gene (codons 126, 153/54, 189 and the 3′ splice site of exon 9) were analysed by the RSM assay for H₂O₂-induced mutations. Two cell types were concurrently analysed in this study, i.e. primary fibroblast cells and a gastric cancer cell line. Using the RSM assay, H₂O₂-induced mutations were only detected in exon 7 of the p53 gene. This was true for both cell types. These mutations were mainly induced in the Msp I restriction site (codon 247/248) and were predominantly GC to AT transitions (71%). Hence these GC to AT mutations were presumably due to H₂O₂ exposure, possibly implicating the 5OHdC adduct, which is known to induce C to T mutations upon misreplication. Importantly, this study demonstrates that the RSM methodology is capable of detecting rare oxidative mutations within the hotspot codons of the p53 tumour suppressor gene. Hence, this methodology may allow the detection of early p53 mutations in pre-malignant tissues.     

WT1 exon 1 deletion/insertion mutations in Wilms tumor patients, associated with di- and trinucleotide repeats and deletion hotspot consensus sequences.

The WT1 gene is known to play a role in at least some cases of Wilms tumor (WT). The first exon of the gene is highly GC rich and contains many short tandem di- and trinucleotide repeats, interrupted direct repeats, and CCTG (CAGG) motifs that have been identified as hotspots for DNA deletions. We have analyzed 80 WT patient samples for mutations in the first exon of WT1, either by SSCP analysis of the first 131 bp of the coding portion of WT1 exon 1 or by size analysis of a PCR product encompassing the coding region of exon 1 in addition to flanking noncoding regions. We report here the occurrence of somatic and germ-line deletion and insertion mutations in this portion of the gene in four WT patients. The mutations are flanked by short direct repeats, and the breakpoints are within 5 nt of a CCTG (CAGG) sequence. These data suggest that a distinctive mutational mechanism, previously unrecognized for this gene, is important for the generation of DNA mutations at the WT1 locus.     

Parental origin of germ-line and somatic mutations in the retinoblastoma gene

Segregation analysis of polymorphic sites within the retinoblastoma (RB) gene and on chromosome 13, as well as the parental origin of the lost allele in the tumor, were analyzed in 24 families with RB patients. Four mutant alleles transmitted through the germ-line and seven de novo germ-line mutant alleles were identified in 11 patients with hereditary RB. Segregation analysis within the RB gene and on chromosome 13 was useful for DNA diagnosis of susceptibility to RB in relatives of hereditary patients, even if mutations were not identified. All seven de novo germ-line mutant alleles were paternally derived. The bias toward the paternal allele for de novo germ-line mutations of the RB gene was statistically significant. Seven paternal alleles and six maternal alleles were lost in 13 non-hereditary RB tumors with no bias in the parental origin of the somatic allele loss. These results suggest that the physical environment or a deficiency in DNA repair during spermatogenesis may be associated with significant risk factors for de novo germ-line mutations.     

A common region of loss of heterozygosity in Wilms' tumor and embryonal rhabdomyosarcoma distal to the D11S988 locus on chromosome 11p15.5

The development of Wilms' tumor has been associated with two genetic loci on chromosome 11: WTI in 11p13 and WT2 in 11p15.5. Here, we have used loss of heterozygosity (LOH) in Wilms' tumors to narrow the WT2 locus distal to the D11S988 locus. A similar region was apparent for the clinically associated tumor, embryonal rhabdomyosarcoma. We have also demonstrated that a constitutional chromosome translocation breakpoint associated with Beckwith-Wiedemann syndrome and an acquired somatic chromosome translocation breakpoint in a rhabdoid tumor each occur in the same chromosomal interval as the smallest region of LOH in Wilms' tumors and embryonal rhabdomyosarcoma. Finally, we report the first Wilms' tumor without a cytogenetic deletion that shows targeted LOH for 11p15 and 11p13 while maintaining germline status for 11p14.     

Germline mutations of the RET proto-oncogene in eight Japanese patients with multiple endocrine neoplasia type 2A (MEN2A)

Multiple endocrine neoplasia type 2A (MEN2A) is a dominantly inherited cancer syndrome characterized by medullary thyroid carcinoma, pheochromocytoma, and parathyroid hyperplasia. The gene responsible for MEN2A was localized by linkage analysis to chromosome 10q11.2 in 1987, and recently mutations in RET, a proto-oncogene in the candidate region, were discovered in patients with MEN. The majority of mutations found so far in MEN2A patients have been located in nucleotide sequences encoding cysteine residues in the extracellular domain of RET. To characterize MEN2A germline alterations in the Japanese population, we screened DNA from eight unrelated patients for mutations in exons 10 and 11 of the RET proto-oncogene and found mutations in all eight patients, at codons 618, 620, or 634; each of these sites encodes a cysteine residue in the extracellular domain of RET. The mutations were confirmed in other affected individuals in the respective families by digestion of polymerase chain reaction (PCR) products containing the mutated codons with restriction enzymes (RsaI, CfoI, or AluI) for which cleavage sites had been generated by the specific genetic alteration. These PCR-restriction enzyme systems will be useful for genetic diagnosis in members of families carrying these mutations.     

Identification of two new nucleotide mutations (HPRTUtrecht and HPRTMadrid) in exon 3 of the human hypoxanthine-guanine phosphoribosyltransferase (HPRT) gene

Mutations in the X-linked hypoxanthine-guanine phosphoribosyl transferase gene (HPRT) result in deficiencies of HPRT enzyme activity, which may cause either a severe form of gout or Lesch-Nyhan syndrome depending on the residual enzyme activity. Mutations leading to these diseases are heterogeneous and include DNA base substitutions, DNA deletions, DNA base insertions and errors in RNA splicing. Identification of mutations has been performed at the RNA and DNA level. Sequencing genomic DNA of the HPRT gene offers the possibility of direct diagnostic analysis independent on the expression of the mature HPRT mRNA. We describe a Dutch and a Spanish family, in which the Lesch-Nyhan syndrome and a severe partial HPRT-deficient phenotype, respectively, were diagnosed. Direct sequencing of the exons coding for the HPRT gene was performed in both families. Two new exon 3 mutations have been identified. At position 16676, the normally present G was substituted by an A in the Dutch kindred (HPRT_Utrecht), and led to an arginine for glycine change at residue 70. At position 16680, the G was substituted by a T in the Spanish family (HPRT_Madrid); this substitutes a valine for glycine at residue 71. These new mutations are located within one of the clusters of hotspots in exon 3 of the HPRT gene in which HPRT_Yale and HPRT_{New Haven} have previously been identified.     

Four new mutations and polymorphic variants of the low density lipoprotein receptor in patients with familial hypercholesterolemia in Saint Petersburg

In a collection of DNA samples from 100 unrelated patients with clinical features of familial hypercholesterolemia (FH), a search for mutations of exons 4 and 10 of the low-density lipoprotein (LDL) receptor gene was performed using heteroduplex and single-strand conformational polymorphism (SSCP) analyses followed by sequencing of amplified DNA fragments. Four new mutations of the LDL receptor gene were identified: C146R (c.499 T > C), A130P (c.451 G > C), G128G (c.477 T > C), and C188Y (c.626 G > A). Mutation A130P was assigned to the same chromosome with allele variant 447C. Two polymorphic sites in exon 10 of the LDL receptor gene (1413G/A and 1545C/T) were found in the Russian population for the first time. Based on the data obtained, familial hypercholesterolemia was confirmed in seven patients.