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PubMed2006

BRCA1 and BRCA2 genetic testing in Italian breast and/or ovarian cancer families: mutation spectrum and prevalence and analysis of mutation prediction models.

BACKGROUND: Breast cancer is an extremely complex disease, characterized by a progressive multistep process caused by interactions of both genetic and non-genetic factors. A combination of BRCA1 and BRCA2 gene mutations appears responsible for about 20%-30% of the cases with breast cancer familial history. The prevalence of BRCA1/2 pathogenic mutations largely varies within different populations; in particular, the rate of mutations in Italian breast and/or ovarian cancer families is rather controversial and ranges from 8% to 37%. PATIENTS AND METHODS: Of the 152 breast/ovarian cancer families counseled in our centre, 99 were selected for BRCA1/2 mutation screening according to our minimal criteria. The entire coding sequences and each intron/exon boundary of BRCA1/2 genes were screened by direct sequencing (PTT limited to BRCA1 exon 11). For each proband, the a priori probability of carrying a pathogenic BRCA1/2 germline mutation was calculated by means of different mutation prediction models (BRCApro, IC and Myriad Table) in order to evaluate their performances. RESULTS: Our analysis resulted in the identification of 25 and 52 variants in the BRCA1 and BRCA2 genes, respectively. Seventeen of them represent novel variants, including four deleterious truncating mutations in the BRCA2 gene (472insA, E33X, C1630X and IVS6+1G>C). Twenty-seven of the 99 probands harbored BRCA1 (n = 15) and BRCA2 (n = 12) pathogenic germline mutations, indicating an overall detection rate of 27.3% and increasing by more than 15% the spectrum of mutations in the Italian population. Furthermore, we found the lowest detection rate (19.4%) in pure hereditary breast cancer family subset. All of the prediction models showed praises and faults, with the IC software being extremely sensitive but poorly specific, compared to BRCApro. In particular all models accumulated most false-negative prediction in the HBC subset. Interestingly preliminary results of a study addressing the presence of genomic rearrangements in HBC probands with BRCApro or IC prediction scores >/=95%, provided evidence for additional mutations undetectable with our conventional screening for point mutations. CONCLUSIONS: Altogether our results suggest that HBC families, the largest pool in our series, represent an heterogeneous group where the apparently faulty performances of the prediction models might be at least partially explained by the presence of additional kinds of BRCA1/2 alteration (such as genomic rearrangements) or by mutations on different breast cancer related genes.

Adult↗

PubMed1998

Influence of the UV-activated SOS response on the gamma-radiation-induced mutation spectrum in the lacI gene.

Previous studies of our group have shown that intracellular or extracellular gamma-irradiation of the lacI gene results in different mutational spectra. One cause for these differences might be the error-prone SOS response, which is activated in the intracellular situation by gamma-irradiation but not in the extracellular situation. Since UV-radiation is a well-established strong inducer of the SOS response, we used bacterial host cells, pretreated with UV-light to study the influence of the SOS response on the gamma-radiation-induced mutation spectrum in the lacI gene in the extracellular situation. If the SOS response was activated, mutations on A:T base pairs and frameshift mutations accounted for 16% and 12% of all mutations, respectively, but they were hardly detected in the absence of an induced SOS response. G:C to T:A transversions increased from 14% to 24% in the presence of an activated SOS response. We can therefore conclude from this study, that SOS-induction of host cells by UV-light influences the extracellular mutation spectrum in the lacI gene, with respect to mutations on A:T base pairs, G:C to T:A transversions and frameshift mutations. This conclusion is supported by the fact that the previously obtained intracellular gamma-radiation-induced mutation spectrum in the lacI gene, in which the SOS response is also involved, shows great similarities with the extracellular mutation spectrum in the presence of an activated SOS response in this study.

Bacterial Proteins↗

PubMed2002

Molecular analysis of 30 mucopolysaccharidosis type I patients: evaluation of the mutational spectrum in Italian population and identification of 13 novel mutations.

Mucopolysaccharidosis type I (MPS-I orMPS1) is an autosomal recessive condition characterized by a broad range of clinical symptoms. Molecular diagnosis of MPS-I is important for analyzing genotype-phenotype correlation and for selecting patients for innovative therapies. In this study we analyzed 30 Italian MPS-I patients with different phenotypes (20 severe, 6 intermediate, 4 mild) in an attempt to recognize the mutational spectrum in our population and to identify major DNA alterations specific to our country. We identified 93% of mutated alleles (56 out of 60) with the reconstruction of the complete genotype in 26 patients out of 30. Twenty-three different mutations were found, 13 of which are novel while the remaining 10 have been already described. Among the novel mutations we found 5 non conservative missense mutations (A160D, E178K, P183R, G197D, D349Y), one nonsense mutation (C53X), 6 deletions (468-470del3, 486-491del6, 755-759del5, 1251delC, 1839-1867del29, 1902-1903del2), and one splice site mutation (IVS11+5G>A). No common mutation for MPS-I is present in our country. Frequently (40% of the alleles), mutations were found in just one or two patients. However, Q70X, P533R, G51D, and W402X mutations were present in several patients (15%, 13.3%, 13.3%, and 11.6% of the alleles respectively) suggesting a Mediterranean origin of the P533R and G51D mutations. In most cases the patients' genotypes were unique combinations of mutations. The great heterogeneity found in our MPS-I population hampers mutation detection and hinders the genotype-phenotype correlation.

Alleles↗

PubMed1997

The mutational spectrum in Treacher Collins syndrome reveals a predominance of mutations that create a premature-termination codon.

Treacher Collins syndrome (TCS) is an autosomal dominant disorder of craniofacial development, the features of which include conductive hearing loss and cleft palate. The TCS locus has been mapped to human chromosome 5q31.3-32 and the mutated gene identified. In the current investigation, 25 previously undescribed mutations, which are spread throughout the gene, are presented. This brings the total reported to date to 35, which represents a detection rate of 60%. Of the mutations that have been reported to date, all but one result in the introduction of a premature-termination codon into the predicted protein, treacle. Moreover, the mutations are largely family specific, although a common 5 bp deletion in exon 24 (seven different families) and a recurrent splicing mutation in intron 3 (two different families) have been identified. This mutational spectrum supports the hypothesis that TCS results from haploinsufficiency.

Chromosomes, Human, Pair 5↗

PubMed2004

In vivo mutagenicity and mutation spectrum in the bone marrow and testes of B6C3F1 lacI transgenic mice following inhalation exposure to ethylene oxide.

The lacI mutant frequency and mutation spectrum were determined in the bone marrow and testes of B6C3F1 lacI transgenic mice exposed by inhalation to ethylene oxide (EO). Groups of male transgenic lacI B6C3F1 mice were exposed to 0, 25, 50, 100 or 200 p.p.m. EO for up to 48 weeks (6 h/day, 5 days/week) and were killed at 12, 24 or 48 weeks of EO exposure for determination of lacI mutant frequency. In the bone marrow, the lacI mutant frequency was significantly increased at the two highest exposure levels (100 and 200 p.p.m.) and at the 48 week exposure time point. The shape of the exposure-response curve for lacI mutant frequency in the bone marrow was non-linear. DNA sequence analysis of the bone marrow mutation spectrum revealed that only AT-->TA transversions occurred at an increased frequency in EO-exposed mice: 25.4% in EO-exposed mice for 48 weeks (200 p.p.m.) compared with 1.4% in air controls. In testes, the lacI mutant frequency was increased at a single exposure level of 200 p.p.m. for 24 weeks. At 48 weeks, the lacI mutant frequency in testes was significantly increased to an equal degree at 25, 50 and 100 p.p.m. EO but not at 200 p.p.m. Analysis of the testes mutation spectrum in air control mice and in mice exposed to 200 p.p.m. EO for 48 weeks revealed that no single mutational type occurred at an increased frequency. In the testes, there was a small increase across all mutational types that was sufficient to increase the overall lacI mutation frequency although not significant individually. The mutation spectrum in testes of EO-exposed mice also revealed that the increased lacI mutant frequency observed at 25 or 50 p.p.m. EO was not due to an increase in mutant siblings (clonality). These data demonstrate that inhalation exposure to EO for up to 48 weeks produces distinct mutagenic responses in bone marrow and testes.

Administration, Inhalation↗

PubMed2003

The amino-acid mutational spectrum of human genetic disease.

BACKGROUND: Nonsynonymous mutations in the coding regions of human genes are responsible for phenotypic differences between humans and for susceptibility to genetic disease. Computational methods were recently used to predict deleterious effects of nonsynonymous human mutations and polymorphisms. Here we focus on understanding the amino-acid mutation spectrum of human genetic disease. We compare the disease spectrum to the spectra of mutual amino-acid mutation frequencies, non-disease polymorphisms in human genes, and substitutions fixed between species. RESULTS: We find that the disease spectrum correlates well with the amino-acid mutation frequencies based on the genetic code. Normalized by the mutation frequencies, the spectrum can be rationalized in terms of chemical similarities between amino acids. The disease spectrum is almost identical for membrane and non-membrane proteins. Mutations at arginine and glycine residues are together responsible for about 30% of genetic diseases, whereas random mutations at tryptophan and cysteine have the highest probability of causing disease. CONCLUSIONS: The overall disease spectrum mainly reflects the mutability of the genetic code. We corroborate earlier results that the probability of a nonsynonymous mutation causing a genetic disease increases monotonically with an increase in the degree of evolutionary conservation of the mutation site and a decrease in the solvent-accessibility of the site; opposite trends are observed for non-disease polymorphisms. We estimate that the rate of nonsynonymous mutations with a negative impact on human health is less than one per diploid genome per generation.

Amino Acid Substitution↗

PubMed2005

P53 gene mutation spectrum and the relationship between gene mutation and protein levels in pterygium.

PURPOSE: To investigate the spectrum of p53 gene mutations and the relationship between gene mutation and p53 protein levels in pterygium. METHODS: Pterygial samples were harvested from 51 patients undergoing pterygium surgery. DNA samples for p53 mutation analyses were extracted from epithelial cells and subjected to DNA sequencing for examination of mutations in exons 4, 5, 6, 7, and 8 of the p53 gene. In situ levels of p53 protein were studied by immunohistochemistry (IHC) and the percentage of positively stained cells quantified. Ten normal conjunctiva samples were included in this study as controls. RESULTS: Mutations within the p53 gene were detected in 8 pterygial samples (15.7%) with only one mutation found in each sample. All the mutations observed were point mutations, with 6 being substitutions and 2 deletions. Three mutations were identified in exon 6, two in exon 7, and a single mutation found in each of exons 4, 5, and 8. P53 protein levels were scored as 0 (negative) in 31 pterygial specimens (60.8%), +1 in 9 samples (17.6%), +2 in 5 samples (9.8%), and +3 in 6 samples (11.8%) by IHC. The 8 samples found to have p53 gene mutations were equally distributed among the different levels of p53 protein observed using IHC, with 2 samples in each group. The two deletion mutations, which caused a frame shift to occur, were found in samples negative for p53 immunostaining (score 0), while substitution mutations were found in samples positively stained (score +1, +2, and +3). CONCLUSIONS: Mutations within p53 gene exons 4-8 were detected in pterygial epithelium and the mutations showed no correlation with p53 protein levels as seen by IHC.

Aged↗

PubMed1993

Mutation enhancing effect of o-vanillin in the lacZ gene of Escherichia coli: characterization of mutational spectrum.

The enhancing effect of o-vanillin (2-hydroxy-3-methoxybenz-aldehyde) on mutations induced by N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) was characterized with mutational specificity. The mutational spectrum of MNNG-induced mutations in the presence of o-vanillin was compared with that in the absence of o-vanillin by means of a series of mutant lacZ genes in E. coli that can detect each of the six types of base substitutions and five kinds of frameshift events. In the absence of o-vanillin, the mutational spectrum induced by MNNG consisted mainly of G.C-->A.T transitions and, to a lesser extent, -1(G.C) frameshift mutations. By adding o-vanillin at 75 micrograms/plate, a marked enhancement was observed in two transitions, G.C-->A.T and A.T-->G.C, and in two frameshift mutations, +1(G.C) and -1(G.C). Only a small change was observed in the -2(C.G-G.C) fraction. Regarding the MNNG-induced frameshifts at the A.T base pair, the +1(A.T) frameshift was much more enhanced by o-vanillin than the -1(A.T) frameshift.

Benzaldehydes↗

PubMed2001

The p53 mutational spectrum associated with BRCA1 mutant ovarian cancer.

PURPOSE: Cancer-specific p53 mutational spectra have been identified. Data from murine models and human BRCA1-related hereditary breast cancers suggest that both unique and specific BRCA1-associated p53 mutations may be found in BRCA1-related ovarian cancers. EXPERIMENTAL DESIGN: The p53 mutational spectrum from ovarian cancers containing either somatic or germ-line BRCA1 mutations was compared with that of sporadic ovarian cancers defined as those diagnosed with a negative family history for breast/ovarian cancer in a three-generation pedigree. Tumor DNA was screened over exons 2-11 of the p53 gene by the PCR and single-strand confirmation polymorphism analysis of the amplimers. Cycle-based DNA sequencing from separate reactions was used to confirm p53 mutations. RESULTS: p53 gene mutations were detected in 42 of 86 sporadic ovarian cancers, compared with 13 of 15 cancers with somatic BRCA1 mutations (P = 0.007) and 16 of 20 cancers with germ-line BRCA1 mutations (P = 0.01). p53 null mutations were found in 31.4% of BRCA1 mutant cancers, compared with only 9.3% of the sporadic cancers (P = 0.002). The p53 mutational spectrum of germ-line BRCA1-related cancers was shifted toward transversions, frameshifts, and non-CpG transitions relative to the spectrum of sporadic ovarian cancers. Thirty-three unique ovarian cancer p53 mutations were sequenced. However, the specific p53 mutations in the BRCA1 mutant cancers were no more unique to this cohort than the p53 mutations of the sporadic cancers. CONCLUSIONS: Ovarian cancers containing somatic or germ-line BRCA1 mutations are uniformly accompanied by p53 dysfunction. This finding offers additional support to observations regarding the importance of p53/BRCA1 interactions in ovarian carcinogenesis.

BRCA1 Protein↗

PubMed1994

Mutational spectrum induced in Saccharomyces cerevisiae by the carcinogen N-2-acetylaminofluorene.

The spectrum of mutations induced by the carcinogen N-2-acetylaminofluorene (AAF) was analysed in Saccharomyces cerevisiae using a forward mutation assay, namely the inactivation of the URA3 gene. The URA3 gene, carried on a yeast/bacterial shuttle vector, was randomly modified in vitro using N-acetoxy-N-2-acetylaminofluorene (N-AcO-AAF) as a model reactive metabolite of the carcinogen AAF. The binding spectrum of AAF to the URA3 gene was determined and found to be essentially random, as all guanine residues reacted about equally well with N-AcO-AAF. Independent Ura- mutants were selected in vivo after transformation of the modified plasmid into a ura3 delta yeast strain. Plasmid survival decreased as a function of AAF modification, leading to one lethal hit (37% relative survival) for an average of approximately 50 AAF adducts per plasmid molecule. At this level of modification the mutation frequency was equal to approximately 70 x 10(-4), i.e. approximately 50-fold above the background mutation frequency. UV irradiation of the yeast cells did not further stimulate the mutagenic response, indicating the lack of an SOS-like mutagenic response in yeast. Sequence analysis of the URA3 mutants revealed approximately 48% frameshifts, approximately 44% base substitutions and approximately 8% complex events. While most base substitutions (74%) were found to be targeted at G residues where AAF is known to form covalent C8 adducts, frameshift mutations were observed at GC base pairs in only approximately 24% of cases.(ABSTRACT TRUNCATED AT 250 WORDS)

2-Acetylaminofluorene↗

PubMed1996

Erratum to Mutation spectrum of 2-chloroethyl methanesulfonate in Drosophila melanogaster premeiotic germ cells' [Mutation Res. 331 (1995) 213-224].

The 2-chloroethyl methanesulfonate (2CIEMS)-induced alcohol dehydrogenase (Adh) null germline mutation frequency in treated Drosophila melanogaster second instar larval gonia was two orders of magnitude greater than the spontaneous mutation frequency. DNA sequence analysis of 83 Adh null mutations showed that 40 mutations of independent origin were at 23 sites in the Adh gene. The mutation spectrum contained only GC --> AT transitions with 35 mutations (87.5%) at the middle or 3' guanine. In addition, characteristics of glutathione (GSH)-mediated bioactivation were determined for 2CIEMS in vitro. Rates of GSH-mediated conjugation, catalyzed by purified rat liver glutathione-S-transferase (GST), and binding of [35S]GSH-mediated conjugation products to calf thymus DNA were determined for 2CIEMS, 1,2-dichloroethane (EDC) and 1,2-dibromoethane (EDB). The relative rates of GSH-mediated conjugation were the following: 5 mM EDB > 40 mM 2CIEMS > 40 mM EDC. A similar trend was observed for DNA binding of the [35S]GSH-mediated conjugation products when differences in mutagen concentration were considered: EDB > 2CIEMS > EDC. The ratios of DNA binding to GSH conjugation calculated for EDB, EDC and 2CIEMS were 6.8 x 10(-5), 9.3 x 10(-5) and 19.1 x 10(-5), respectively. A narrow range, less than a 3-fold difference, in the ratios of DNA binding to GSH conjugation indicates that the bioactivation of 2CIEMS is mediated by the same mechanism as EDB and EDC. Consequently, 2CIEMS, EDC and EDB may induce a specific mutation in premeiotic germ cells.

Animals↗

PubMed1995

Mutation spectrum of cigarette smoke condensate in Salmonella: comparison to mutations in smoking-associated tumors.

We used colony probe hybridization and polymerase chain reaction/DNA sequence analysis to determine the mutations in approximately 1600 revertants of Salmonella induced by cigarette smoke condensate (CSC) in the presence of S9. CSC induced approximately 80% GC-->TA transversions and approximately 20% GC-->AT transitions at the base-substitution allele (hisG46) in strain TA100. This spectrum was similar to those of the polycyclic aromatic hydrocarbon (PAH) benzo[alpha]pyrene and various aromatic amines such as 4-aminobiphenyl and Glu-P-1, all of which are present in CSC. This spectrum was also similar to that produced by PAHs in other bacteria, mammalian cells, and rodents as well as to that of the p53 gene in lung tumors from smokers. The results in Salmonella are consistent with a role for the PAH component of cigarette smoke in the base-substitution specificity found in the p53 gene of smoking-associated lung tumors. At the frameshift allele in strains TA1538 and TA98, CSC induced only a hotspot 2-base deletion, which is a mutation spectrum that is identical to that induced by the heterocyclic amine pyrolysate products of amino acids, such as Glu-P-1. This is consistent with bioassay-directed fractionation studies showing that aromatic amines account for most of the frameshift specificity of CSC in Salmonella. Rodent and human studies indicate that aromatic amines are responsible for smoking-associated bladder cancer. Repeated freezing and thawing of the CSC samples changed the chemical composition of the mixtures as evidenced by the production of an altered mutation spectrum. This emphasizes the necessity of proper storage and handling of labile complex mixtures. This study (i) confirms our previous studies showing that the mutation spectrum of a complex mixture reflects the dominance of one or a few classes of chemical mutagens within the mixture, and (ii) illustrates the potential of bioassay-directed molecular analysis for identifying the chemical classes in a complex mixture that are responsible for specific classes of mutation and tumor types produced by the mixture.

Alleles↗

PubMed2004

Extension of the mutation spectrum in Friedreich's ataxia: detection of an exon deletion and novel missense mutations.

Friedreich's ataxia (FRDA), the most common autosomal recessively inherited ataxia, is due to a homozygous GAA triplet repeat expansion in the first intron of the FRDA gene in about 96% of patients. Approximately 4% of FRDA patients are compound heterozygotes with a GAA repeat expansion in one allele and a point mutation in the coding region of the second allele. To reinvestigate the mutation spectrum, we searched for mutations including exon deletions in six patients heterozygous for the GAA repeat expansion and found two unknown missense mutations, p.Asn146Lys and p.Leu186Arg, in trans to the expanded FRDA allele. Interestingly, we detected a heterozygous 2776 bp deletion including exon 5a in one of our patients. This deletion removes 50 of the 210 residues of the frataxin. Furthermore, since no FRDA case with two-point mutations is known, we screened eight patients with FRDA phenotype but GAA alleles within the normal range but did not reveal a mutation within the FRDA gene. In addition, DNA polymorphisms have been found in four out of 100 control individuals in this study.

Base Sequence↗

PubMed1998

Chemically induced mutations in mitochondrial DNA of human cells: mutational spectrum of N-methyl-N'-nitro-N-nitrosoguanidine.

We have observed a reproducible mitochondrial mutational spectrum in the MT1 human lymphoblastoid line treated with N-methyl-N'-nitro-N-nitrosoguanidine (MNNG). The MNNG spectrum was distinct from the spontaneous mutational spectrum. However, our ability to observe MNNG-induced mitochondrial mutations above the high level of accumulated spontaneous mutations was dependent on the MT1 phenotype. MT1 cells are markedly resistant to the cytotoxicity but not the mutagenicity of MNNG, presumably as a result of inactivation of both copies of the hMSH6 (GTBP) mismatch repair gene. Thus, we were able to use conditions of treatment that yielded induced mitochondrial mutant fractions beyond the practical limits for human cell experiments in mismatch-proficient human cell lines. In contradistinction, when MT1 cells were treated repeatedly with maximum tolerated concentrations of (+/-) anti-benzo(a)pyrene diol-epoxide, no induced mitochondrial mutations above the spontaneous background were observed. A single dose of 4 microM MNNG (survival, 0.85) induced a mutant fraction of 8 x 10(-3) in the nuclear hypoxanthine-guanine phosphoribosyltrans. ferase gene, and a clear and reproducible pattern of seven MNNG-induced hotspot mutations was observed within the mitochondrial DNA target sequence studied (mitochondrial bp 10,030-10,130). All of the MNNG-induced hotspot mutations were G:C to A:T transitions present at frequencies between 6 x 10(-5) and 30 x 10(-5). Additional experiments supported the conclusion that MNNG-induced hotspot mutations observed were generated in living cells as a result of MNNG treatment and not from mismatch intermediates or DNA adducts converted into mutations during the PCR process.

7,8-Dihydro-7,8-dihydroxybenzo(a)pyrene 9,10-oxide↗

PubMed1980

gamma-Ray induced mutational spectrum in the lacI gene of Escherichia coli: comparison of induced and spontaneous spectra at the molecular level.

The spectrum of mutations induced by ionizing radiation (gamma-rays) was determined in the lacI gene of E. coli. Base substitution was the principal type of mutational event following ionizing radiation. Both transitions and transversions were produced, and no strong specificity for a particular base pair was observed. The spectra of spontaneous and of ionizing-radiation-induced base-pair changes differed significantly at several locations within the lacI gene. The location of 3 of these differences corresponded to sites of spontaneous deamination "hot spots" from which we conclude that gamma-rays do not cause extensive deamination. The specific locus rate was calculated as 4.5 X 10(-10) mutations per rad per gene copy per cell, and the nucleotide substitution rate was 2.2 X 10(-12) per rad. The frameshift mutation, trpE997, was not reverted by gamma-rays.

Base Composition↗

PubMed1993

N-acetoxy-N-acetyl-2-aminofluorene-induced mutation spectrum in a human hprt cDNA shuttle vector integrated into mammalian cells.

The spectrum of mutations induced by N-acetoxy-N-acetyl-2-aminofluorene (N-AcO-AAF) was examined by the pZipHprtNeo shuttle vector in mammalian cells. The vector carries a cDNA of the human hypoxanthine phosphoribosyl transferase (hprt) gene, which is stably integrated into chromosomal DNA of a mouse cell line, VH12. After treatment of the cell with N-AcO-AAF, 48 independent 6-thioguanine-resistant clones were obtained and altered sequences of the mutated cDNA hprt genes were determined. Frameshifts and deletions were the predominant mutational events (68%) induced by N-AcO-AAF and the remainder were base substitutions (32%) of various types. Analysis of sequence alterations at all the sites of mutation revealed that: (i) > 65% of mutations occurred at G:C sites, suggesting C8G adducts are responsible premutagenic lesions for these mutations; and (ii) short sequence repeats were frequently found at the sites of frameshift and deletion, and slippage--misalignment is the suggested mechanism for the induction of mutations at these sites. Implied significance of slippage--misalignment as a fundamental mechanism for mutagenesis is discussed.

3T3 Cells↗

PubMed1992

Mutation spectrum of the p53 gene in bone and soft tissue sarcomas.

We present here an analysis of the spectrum of mutations of the p53 gene seen in 127 bone and soft tissue sarcomas of various histological classifications. Gross rearrangements were analyzed by Southern blotting using a complementary DNA probe from the p53 gene, and subtle alterations in the entire coding sequence (exons 2 through 11) were identified by a combination of single-strand conformation polymorphism analysis and direct genomic sequencing. A total of 42 somatic alterations of the p53 gene were found, of which 21 were gross rearrangements and 21 were subtle alterations. These included 17 cases of a single base substitution, 3 small deletions, and one single base insertion. In contrast to reported findings for other types of cancer, we found that mutations of the p53 gene in sarcomas are quite heterogeneous both in their distribution throughout the gene and in the type of genetic alterations that result. All 13 missense mutations we found occurred at highly conserved residues, whereas 8 nonsense mutations occurred at sites that spanned the gene from codons 46 to 316. Surprisingly, approximately one-half of the osteosarcomas with allelic deletions on 17p did not have detectable alterations in the coding sequence of the p53 gene.

Alleles↗

PubMed2003

Mutational spectrum of congenital adrenal hyperplasia in Slovenian patients: a novel Ala15Thr mutation and Pro30Leu within a larger gene conversion associated with a severe form of the disease.

OBJECTIVE: To analyse the mutational spectrum, the associated haplotypes and the genotype-phenotype correlation, and to design a reliable and rational approach for CYP21 mutation detection in Slovenian congenital adrenal hyperplasia (CAH) patients. DESIGN: Molecular analysis of the CYP21 gene was performed in 36 CAH patients and 79 family members. METHODS: Southern blotting, sequence-specific PCR amplification (PCR-SSP), sequence-specific oligonucleotide hybridisation (PCR-SSO) and sequencing were used to detect CYP21 gene deletions, conversions and point mutations. RESULTS: CYP21 gene deletion was the most frequent mutation (36.4%). Large gene conversions detectable only by Southern blotting represented 12.1%, and gene conversions involving the promoter region represented 7.6% of the mutated alleles. The most frequent point mutations were: intron 2 splice mutation 16.7%, Ile172Asn mutation 7.6%, Gln318Stop 7.5% and Pro30Leu 12.2% of alleles. A correlation between the genotype and the clinical phenotype similar to those described for large populations was observed. The finding of Pro30Leu mutation linked to a gene conversion could explain the simple virilising (SV) phenotype in compound heterozygotes for the Pro30Leu and a severe mutation. In two siblings with a salt wasting form of CAH (SW-CAH), a novel mutation Ala15Thr was found on the allele characterised by Pro30Leu mutation and gene conversion involving the promoter region. CONCLUSIONS: Our genotyping approach allowed reliable diagnosis of CAH in the Slovenian population. The high frequency of CYP21 gene aberrations on Pro30Leu positive alleles justified systematic searching for a gene conversion in the promoter region using the PCR-SSP reaction.

Adrenal Hyperplasia, Congenital↗