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PubMed2006

NRAS and BRAF mutations in melanoma tumours in relation to clinical characteristics: a study based on mutation screening by pyrosequencing.

We have previously demonstrated the use of pyrosequencing to investigate NRAS [neuroblastoma RAS viral (v-ras) oncogene homolog] mutations in melanoma biopsies. Here, we expanded the analysis to include BRAF (V-raf murine sarcoma viral oncogene homolog B1), another member of the Ras-Raf-mitogen-activated protein kinase (MAPK) signalling pathway, and analysed a total of 294 melanoma tumours from 219 patients. Mutations in BRAF exons 11 and 15 were identified in 156 (53%) tumours and NRAS exon 2 mutations in 86 (29%) tumours. Overall, mutations in NRAS or BRAF were found in 242 of 294 tumours (82%) and were found to be mutually exclusive in all but two cases (0.7%). Multiple metastases were analysed in 57 of the cases and mutations were identical in all except three, indicating that BRAF and NRAS mutations occur before metastasis. Association with preexisting nevi was significantly higher in BRAF mutated tumours (P=0.014). In addition, tumours with BRAF mutations showed a significantly more frequent moderate to pronounced infiltration of lymphocytes (P=0.013). NRAS mutations were associated with a significantly higher Clark level of invasion (P=0.022) than BRAF mutations. Age at diagnosis was significantly higher in tumours with NRAS mutations than in those with BRAF mutations (P=0.019). NRAS and BRAF mutations, however, did not influence the overall survival from time of diagnosis (P=0.7). In conclusion, the separate genotypes were associated with differences in several key clinical and pathological parameters, indicating differences in the biology of melanoma tumours with different proto-oncogene mutations.

Adult↗
PubMed1997

Predominance of the recurrent mutation R635X in the LAMB3 gene in European patients with Herlitz junctional epidermolysis bullosa has implications for mutation detection strategy.

Junctional forms of epidermolysis bullosa (JEB) are characterized by tissue separation at the level of the lamina lucida. We have recently disclosed specific mutations in the LAMA3, LAMB3, and LAMC2 genes encoding the subunit polypeptides of the anchoring filament protein laminin 5 in 66 families with different variants of JEB. Examination of the JEB mutation database revealed recurrence of a particular C-->T substitution at nucleotide position 1903 (exon 14) of LAMB3, resulting in the mutation R635X. The inheritance of this nonsense mutation was noted on different genetic backgrounds, suggesting that R635X is a hotspot mutation. In this study, we have performed mutation evaluation in a European cohort of 14 families with the lethal, Herlitz type of JEB (H-JEB). The families were first screened for the presence of the R635X mutation by restriction enzyme digestion of the PCR product corresponding to exon 14. Four of the probands were found to be homozygous and six were heterozygous for R635X. The remaining alleles were subjected to mutation screening by PCR amplification of individual exons of LAMB3 and LAMC2, followed by heteroduplex analysis and nucleotide sequencing. In three families (six alleles), mutations in LAMC2 were disclosed. In the remaining eight alleles, additional pathogenetic LAMB3 mutations were found. None of the patients had LAMA3 mutation. Thus, LAMB3 mutations accounted for 22 of 28 JEB alleles (79%), and a total of 14 of 22 LAMB3 alleles (64%) harbored the R635X mutation, signifying its prevalence as a predominant genetic lesion underlying H-JEB in this European cohort of patients. This recurrent mutation will facilitate screening of additional JEB patients for the purpose of prenatal testing of fetuses at risk for recurrence.

Antigens↗
PubMed2006

High variability in CYP21A2 mutated alleles in Spanish 21-hydroxylase deficiency patients, six novel mutations and a founder effect.

OBJECTIVE: To detect common as well as rare and novel CYP21A mutations in 21-hydroxylase deficiency patients. To estimate the distribution of mutations and compare them with other European studies. To construct haplotypes linked to a recurrent novel mutation. DESIGN: Genetic analysis by sequencing the entire CYP21A2 gene plus Southern blot. PATIENTS: A total of 138 unrelated Spanish patients: 122 nonclassical forms (NCF) and 16 classical forms (CF) were studied. RESULTS: Among the 266 nonrelated mutated alleles; CYP21A2 deletions/conversions and a spectrum of 27 different mutated alleles were found: 15 different single point mutations, 8 nucleotide deletions in exon 3, 3 mutation clusters in exon 6, 9 alleles with more than one mutation, one 21-nucleotide duplication in exon 10, and one allele with CYP21A2 duplicated and both copies mutated. The most frequent mutation in NCF alleles is V281L (71.8%). Among CFs, the most common is I2 g (20%) and Q318X (16%) and rare alleles (21.9%). Six novel causative mutations were found, four associated with CF: I46+1nt, R444X, P463L and M473_R479dup and two associated with NCF: W302 and D322G. The R444X mutation was found in seven unrelated patients and it appeared only once in an ancestral haplotype. In addition, we found a novel single nucleotide polymorphism with a 31.5% frequency for the rare allele. CONCLUSION: A great diversity of haplotypes with a large spectrum of mutated alleles was found. The frequency of the V281L mutation was the highest reported and the relatively high frequency of R444X was the result of a founder effect.

Adrenal Hyperplasia, Congenital↗
PubMed2006

Male infertility and androgen receptor gene mutations: clinical features and identification of seven novel mutations.

OBJECTIVE: Androgens and a functioning androgen receptor (AR) are essential for development and maintenance of the male phenotype and spermatogenesis. Consistent with this, mutations in the AR gene cause a variety of defects related to androgen insensitivity, ranging from complete feminization to phenotypic males with infertility. The aim of his study was to analyse the prevalence of AR gene mutations in male infertility and to clarify the genotype-phenotype relation. DESIGN: Males with infertility were recruited consecutively at the Centre for Male Gamete Cryopreservation at the University of Padova from January 1996 to January 2005. PATIENTS: One thousand five hundred and seventeen men with < 10 million sperm/ml and 310 age-matched normozoospermic controls. METHODS: Screening for AR gene mutation was done by DHPLC and sequencing, and reproductive hormone concentrations were measured. RESULTS: We found 20 mutations in 26 of 1517 patients (1.7%) and no mutations in controls. A high number of mutations localized in exon 1 of the AR gene coding for the transactivation domain of the protein. Of 20 mutations, 7 represent novel mutations. With respect to men without AR mutations, subjects with AR mutations have lower ejaculate volume, higher testosterone levels, higher oestradiol levels, and higher androgen sensitivity index. However, the ranges for these variables were highly overlapping between men with and without AR gene mutations. Also clinical manifestations of AR mutations are not unique and 22 men had only spermatogenic impairment. CONCLUSIONS: AR gene mutations are quite frequent in unselected infertile men but no specific hormonal or clinical data could be used to preselect patients at risk of mutations.

Adult↗
PubMed1994

Mutations in the muscle sodium channel gene (SCN4A) in 13 French families with hyperkalemic periodic paralysis and paramyotonia congenita: phenotype to genotype correlations and demonstration of the predominance of two mutations.

Hyperkalemic periodic paralysis (hyperPP), paramyotonia congenita (PC) and PC with myotonia permanens are closely related muscle disorders of genetic origin due to allelic mutations in the muscle sodium channel gene, SCN4A. Seven families of French origin with hyperPP were studied. Five of these had the Thr704Met mutation, but 2 families, genetically linked to SCN4A, failed to show any of the known mutations of SCN4A. Correlations between the phenotype and the genotype were made for patients with the Thr704Met mutation. All 12 patients over 30 years old with the Thr704Met mutation presented muscle weakness due to degeneration of muscle fibers in addition to periodic paralysis. Only approximately 12.5% of patients with the Thr704Met mutation presented with clinical myotonia and about 50% with hyperkalemia. One family with PC displayed the Gly1306Val mutation with a phenotype similar to the one already reported for this mutation. Five families with either PC or PC with myotonia permanens had the Thr1313Met mutation indicating that the severity of myotonia and its permanence were variable. Two mutations of SCN4A were found to be predominant in these 13 families: the Thr704Met and the Thr1313Met mutations. Only 2 families with the Thr704Met mutation and 3 families with the Thr1313Met shared the same SCN4A haplotype determined with intragenic dinucleotide repeats. Recurrent mutations of SCN4A may contribute to the predominance of these two mutations in the French population.

Adolescent↗
PubMed2003

Detection of BCR-ABL mutations in patients with CML treated with imatinib is virtually always accompanied by clinical resistance, and mutations in the ATP phosphate-binding loop (P-loop) are associated with a poor prognosis.

Imatinib-treated chronic myeloid leukemia (CML) patients with acquired resistance commonly have detectable BCR-ABL kinase domain mutations. It is unclear whether patients who remain sensitive to imatinib also have a significant incidence of mutations. We evaluated 144 patients treated with imatinib for BCR-ABL kinase domain mutations by direct sequencing of 40 accelerated phase (AP), 64 late chronic phase (> or = 12 months from diagnosis, late-CP), and 40 early-CP patients. Mutations were detected in 27 patients at 17 different residues, 13 (33%) of 40 in AP, 14 (22%) of 64 in late-CP, and 0 of 40 in early-CP. Acquired resistance was evident in 24 (89%) of 27 patients with mutations. Twelve (92%) of 13 patients with mutations in the adenosine triphosphate (ATP) binding loop (P-loop) died (median survival of 4.5 months after the mutation was detected). In contrast, only 3 (21%) of 14 patients with mutations outside the P-loop died (median follow-up of 11 months). As the detection of mutations was strongly associated with imatinib resistance, we analyzed features that predicted for their detection. Patients who commenced imatinib more than 4 years from diagnosis had a significantly higher incidence of mutations (18 [41%] of 44) compared with those treated within 4 years (9 [9%] of 100), P <.0001. Lack of a major cytogenetic response (MCR) was also associated with a higher likelihood of detecting a mutation; 19 (38%) of 50 patients without a MCR had mutations compared with 8 (8.5%) of 94 with an MCR, P <.0001. In conclusion, the detection of kinase domain mutations using a direct sequencing technique was almost always associated with imatinib resistance, and patients with mutations in the P-loop had a particularly poor prognosis.

Adenosine Triphosphate↗
PubMed1999

Several mutations in the melanocortin-4 receptor gene including a nonsense and a frameshift mutation associated with dominantly inherited obesity in humans.

The melanocortin-4 receptor gene (MC4-R) has been implicated in weight regulation. Recently, two independent groups reported frameshift mutations associated with a dominant form of obesity (1, 2). We screened the coding region of the MC4-R in 306 extremely obese children and adolescents (mean body mass index: BMI 34.4 +/- 6.6 kg/m2), 25 healthy underweight students (mean BMI 17.1 +/- 0.8 kg/m2), 52 normal weight individuals (mean BMI 22.0 +/- 1.0 kg/m2), 51 inpatients with anorexia nervosa (AN, DSM IV criteria, mean BMI 14.3 +/- 1.5 kg/m2) and 27 patients with bulimia nervosa (BN, DSM IV criteria, mean BMI 21.7 +/- 5.8 kg/m2) by single strand conformation polymorphism analysis (SSCP). Several mutations were identified, including the frameshift mutation described (1). The mutations were as follows: a) The deletion of 4 bp (delta of CTCT at codon 211) results in a frameshift, thus rendering a truncated protein. This mutation has been assumed to be associated with dominantly-inherited morbid obesity in humans (1). Both the index patient (BMI 42.06 kg/m2, height 171 cm, age 19.6 years) and her mother (BMI 37.55 kg/m2, height 164 cm, age 42.5 years) were heterozygous for the deletion. b) A nonsense mutation at position 35 of the MC4-R was detected in two obese probands (BMI 31.29 kg/m2 and BMI 45.91 kg/m2). This mutation leads to a truncated protein that encompasses the N-terminal extracellular domain. Both carriers additionally showed (c) a missense mutation (Asp-37-Val). In both of these cases Tyr-35-Stop and Asp-37-Val were maternally transmitted, thus these variations form a haplotype. d) e) A male obese proband harbored two missense mutations (Ser-30-Phe, Gly-252-Ser). f)-i) Four different missense mutations (Pro-78-Leu, Thr-112-Met, Arg-165-Trp, Ile-317-Thr) were detected in four different male probands, respectively. All of these mutations (a to i) were found solely in extremely obese individuals whose BMIs were all above the 99th percentile. j) A silent mutation (C-579-T, Val-193-Val) was detected in a male underweight individual. k) A previously described polymorphism (Val-103-Ile; 3) was detected with similar frequencies in all different study groups. 1) We identified a novel polymorphism (Ile-251-Leu) with similar allele frequencies in all groups under study. In conclusion, our data indicate that mutations in the MC4-R are not uncommon. Whereas our data support the evidence for dominantly inherited obesity as revealed by the three obese probands with haplo-insufficiency, the functional significance of the missense mutations remains to be determined.

Adolescent↗
PubMed2003

Highly recurrent RET mutations and novel mutations in genes of the receptor tyrosine kinase and endothelin receptor B pathways in Chinese patients with sporadic Hirschsprung disease.

BACKGROUND: Hirschsprung disease (HSCR) is a congenital disorder characterized by an absence of ganglion cells in the nerve plexuses of the lower digestive tract. HSCR has a complex pattern of inheritance and is sometimes associated with mutations in genes of the receptor tyrosine kinase (RET) and endothelin receptor B (EDNRB) signaling pathways, which are crucial for development of the enteric nervous system. METHODS: Using PCR amplification and direct sequencing, we screened for mutations and polymorphisms in the coding regions and intron/exon boundaries of the RET, GDNF, EDNRB, and EDN3 genes of 84 HSCR patients and 96 ethnically matched controls. RESULTS: We identified 10 novel and 2 previously described mutations in RET, and 4 and 2 novel mutations in EDNRB and in EDN3, respectively. Potential disease-causing mutations were detected in 24% of the patients. The overall mutation rate was 41% in females and 19% in males (P = 0.06). RET mutations occurred in 19% of the patients. R114H in RET was the most prevalent mutation, representing 7% of the patients or 37% of the patients with RET mutations. To date, such a high frequency of a single mutation has never been reported in unrelated HSCR patients. Mutations in EDNRB, EDN3, and GDNF were found in four, two, and none of the patients, respectively. Two patients with mutations in genes of the EDNRB pathway also harbored a mutation in RET. Three novel and three reported polymorphisms were found in EDNRB, EDN3, and GDNF. CONCLUSION: This study identifies additional HSCR disease-causing mutations, some peculiar to the Chinese population, and represents the first comprehensive genetic analysis of sporadic HSCR disease in Chinese.

Asian People↗
PubMed2007

[Screening for mutations in the hotspot mutation regions of PIK3CA gene in nasopharyngeal carcinoma].

BACKGROUND & OBJECTIVE: Recent studies showed high frequency of phosphatidylinositol 3-kinase catalytic alpha polypeptide (PIK3CA) mutations in various human cancers; notably, these mutations frequently locate in the hotspot mutation regions of PIK3CA exon 9 and exon 20 with functional significance in tumorigenesis, invasion, and anti-apoptosis. This study was to screen for mutations in the hotspot mutation regions of PIK3CA in nasopharyngeal carcinoma (NPC), and explore the correlation of PIK3CA mutations to tumorigenesis of NPC. METHODS: PIK3CA exon 9 and exon 20 in 46 specimens of sporadic primary NPC tissues were screened by polymerase chain reaction (PCR)-clone sequencing; those in 46 samples of matched NPC peripheral blood and 3 NPC cell lines CNE1, CNE2, and SUNE1 were directly sequenced. RESULTS: Among the 46 specimens of NPC, 2 (4.3%) had point mutation in PIK3CA exon 9 [T1563G (521Asn-->Lys) and A1646G (549Asp-->Gly)], 18 had multiple mutations in PIK3CA exon 9 (A1634C-G1658C-del 1659T), which might be the homologous sequence of Cat Eye Syndrome region on 22q11.2; none had mutation in PIK3CA exon 20. Moreover, no mutation was detected in PIK3CA exon 9 and exon 20 in the 46 matched NPC peripheral blood samples and CNE1, CNE2, and SUNE1 cells. CONCLUSIONS: PIK3CA exon 9 and exon 20 rarely mutate in NPC. Clone sequencing is more sensitive than direct sequencing in screening for somatic mutation. A1634C-G1658C-del 1659T mutations in PIK3CA exon 9, detected by clone sequencing, are supposed to be the homologous sequence of Cat Eye Syndrome region on 22q11.2 instead of mutations in PIK3CA.

Base Sequence↗
PubMed1999

The type of somatic mutation at APC in familial adenomatous polyposis is determined by the site of the germline mutation: a new facet to Knudson's 'two-hit' hypothesis.

APC is often cited as a prime example of a tumor suppressor gene. Truncating germline and somatic mutations (or, infrequently, allelic loss) occur in tumors in FAP (familial adenomatous polyposis). Most sporadic colorectal cancers also have two APC mutations. Clues from attenuated polyposis, missense germline variants with mild disease and the somatic mutation cluster region (codons 1,250-1,450) indicate, however, that APC mutations might not result in simple loss of protein function. We have found that FAP patients with germline APC mutations within a small region (codons 1,194-1,392 at most) mainly show allelic loss in their colorectal adenomas, in contrast to other FAP patients, whose 'second hits' tend to occur by truncating mutations in the mutation cluster region. Our results indicate that different APC mutations provide cells with different selective advantages, with mutations close to codon 1,300 providing the greatest advantage. Allelic loss is selected strongly in cells with one mutation near codon 1,300. A different germline-somatic APC mutation association exists in FAP desmoids. APC is not, therefore, a classical tumor suppressor. Our findings also indicate a new mechanism for disease severity: if a broader spectrum of mutations is selected in tumors, the somatic mutation rate is effectively higher and more tumors grow.

Adenoma↗
PubMed2000

Founder BRCA1 mutations and two novel germline BRCA2 mutations in breast and/or ovarian cancer families from North-Eastern Poland.

Germline mutations in the BRCA1 and BRCA2 genes account for the majority of high-risk breast/ovarian cancer families, depending on the population studied. Previously, BRCA1 mutations were described in women from Western Poland. To further characterize the spectrum of BRCA1 mutations and the impact of BRCA2 mutations in Poland, we have analyzed 25 high-risk breast and/or ovarian cancer families from North-Eastern Poland for mutations in all coding exons of the BRCA1 and BRCA2 genes, using combined heteroduplex analysis/SSCP followed by direct DNA sequence analysis. Out of 25 probands a total of five (20%) carried three recurrent BRCA1 mutations (300T>G, 3819del5, 5382insC). The 300T>G mutation accounted for 60% (3/5) of BRCA1 mutations and allelotyping suggested a common founder of this mutation. No unique mutations were found. In addition, we identified three BRCA2 (12%) mutations, one recurrent 4075delGT, and two novel frameshift mutations, 7327ins/dupl19 and 9068delA. We conclude that 30% of high-risk families from North-Eastern Poland may be due to recurrent BRCA1 and unique BRCA2 mutations. Intriguingly, the BRCA1 mutation spectrum seems to be different within subregions of Poland.

Adult↗
PubMed2007

Mutation spectrum in UVB-exposed skin epidermis of Xpa-knockout mice: frequent recovery of triplet mutations.

Knockout mutations in both alleles of the Xpa gene give rise to a complete deficiency in nucleotide excision repair (NER) in mammalian cells. We used transgenic mice harboring the lambda-phage-based lacZ mutational reporter gene to study the effect of Xpa null mutation (Xpa(-/-)) on damage induction, repair, and mutagenesis in mouse skin epidermis after UVB irradiation. UVB induced equal amounts of cyclobutane pyrimidine dimers (CPDs) and pyrimidine(6-4)pyrimidone photoproducts (64PPs) in mouse skin epidermis of Xpa(-/-) and wild-type mice. Neither photolesion was removed in the Xpa(-/-) epidermis by 12 hr after irradiation whereas removal of 64PPs was observed in the epidermis of wild-type mice. Irradiation with 200 and 300 J/m(2) UVB increased the lacZ mutant frequency in the epidermis of Xpa(-/-) mice, but the induced mutant frequencies were not significantly different from those previously determined for wild-type mice. One-hundred lacZ mutants isolated from the UVB-exposed epidermis of Xpa(-/-) mice were analyzed and compared with mutant sequences previously determined for irradiated wild-type mice. The distribution of the mutations along the lacZ transgene and the preferred dipyrimidine context of the UV-specific mutations were similar in mutants from the Xpa(-/-) and wild-type mice. The spectra of the mutations in the two genotypes were both highly UV-specific and similar in a dominance of C --> T transitions at dipyrimidine sites; however, Xpa(-/-) mice had a higher frequency than wild-type mice of two-base tandem substitutions, including CC --> TT mutations, three-base tandem mutations and double base substitutions that were separated by one unchanged base in a three-base sequence (alternating mutations). These tandem/alternating mutations included a remarkably large number of triplet mutations, a recently reported, novel type of UV-specific mutation, characterized by multiple base substitutions or frameshifts within a three-nucleotide sequence containing a dipyrimidine. We conclude that the triplet mutation is a UV-specific mutation that preferably occurs in NER-deficient genetic backgrounds.

Animals↗
PubMed2002

Mutations in the factor IX gene (F9) during the past 150 years have relative rates similar to ancient mutations.

Pollutants and dietary mutagens have been associated with somatic mutation and cancer, but the extent of their influence on germline mutation is not clear. Since deleterious germline mutations can be transmitted for thousands of years, any influence on germline mutation from the vast increase in man-made chemicals of the past 150 years would be an important public health issue. Observed disease causing mutations in the X-linked factor IX gene (F9) of hemophilia B patients originated predominantly in the past 150 years, since the half-life of these mutations in human populations had been about two generations before effective treatment became available about a generation ago. Recent changes in germline mutational processes may be detected by comparison of the observed hemophilia B causing mutation pattern in F9 with the pattern of neutral polymorphisms which occurred over a much longer period of time. By scanning a total of 1.5 megabases of deep intronic regions of F9 in the genomic DNA from 84 individuals, 42 neutral polymorphisms were found in 23 haplotypes that differed by at least 11 mutations from the ancestral primate haplotype. By sequencing F9 in seven non-human primates, 39 of these polymorphisms were characterized as ancient mutations relative to a unanimous ancestral primate allele. This ancient mutation pattern was compared to the recent pattern of hemophilia B causing mutations. Remarkably, no significant difference was found (P=0.5), suggesting that the vast increase in man-made chemicals during the past 150 years has not had a major impact on the pattern of human germline mutation. This result is consistent with the hypothesis that endogenous processes dominate germline mutation.

Animals↗
PubMed1984

Carcinogen-induced mutation spectrum in wild-type, uvrA and umuC strains of Escherichia coli. Strain specificity and mutation-prone sequences.

Forward mutations induced by the ultimate carcinogen N-acetoxy-N-2-acetylaminofluorene (N-Aco-AAF) in the tetracycline resistance gene carried on plasmid pBR322 are shown to be dependent upon the induction of the host SOS functions in wild-type and umuC Escherichia coli cells. The mutation frequency in the umuC strain is equal to about 40% of the mutation frequency observed in the umu+ background. In the excision-repair-deficient uvrA mutant strain the mutagenic response is the same as in SOS-induced wild-type cells whether or not the uvrA bacteria are SOS-induced. Equal mutation frequencies are obtained in both the wild-type and the uvrA strains for equal modification levels although the survival of AAF-modified plasmid DNA is greatly reduced in the uvrA strain as compared to the wild-type strain. Sequence analysis of the mutations reveals that more than 90% of the N-Aco-AAF-induced mutations are frameshift mutations. Two types of mutational hotspots are observed occurring either at repetitive sequences or at non-repetitive sequences. Both types of mutants appear at similar locations and frequencies in both the wild-type and the uvrA strains. On the other hand, only the non-repetitive sequence mutants are obtained in the umuC background. These non-repetitive sequence mutants preferentially occur within the sequence 5' G-G-C-G-C-C 3' (the NarI restriction enzyme recognition sequence). The analysis of the -AAF binding spectrum to the same DNA fragment shows that there is no direct correlation between the modification spectrum and the mutation spectrum. We suggest that certain sequences are "mutation-prone" in the sense that only these sequences can be efficiently mutated as the result of an active processing mediated by specific proteins. When a sequence is said to be mutation-prone it probably corresponds to a particular structure that is induced within this sequence as a result of the binding to the DNA of the mutagen. This sequence-specific conformational change is the substrate for the protein(s) that fixes the mutation. The mutagenic processing pathway(s) is part of the cellular response to DNA-damaging agents (the so-called SOS response). Two pathways for frameshift mutagenesis are suggested by the data: an umuC-dependent pathway, which is involved in the mutagenic processing of lesions within repetitive sequences; an umuC-independent pathway responsible for the fixation of mutations within specific non-repetitive sequences.

2-Acetylaminofluorene↗
PubMed1987

Mechanisms of ultraviolet-induced mutation. Mutational spectra in the Escherichia coli lacI gene for a wild-type and an excision-repair-deficient strain.

We have analyzed the DNA sequence changes in a total of 409 ultraviolet light-induced mutations in the lacI gene of Escherichia coli: 227 in a Uvr+ and 182 in a UvrB- strain. Both differences and similarities were observed. In both strains the mutations were predominantly (60 to 75%) base substitutions, followed by smaller contributions of single-base frameshifts, deletions and frameshift hotspot mutations. The base substitutions proved largely similar in the two strains but differences were observed among the single-base frameshifts, the deletions and the hotspot mutations. Among the base substitutions, both transitions (72.5%) and transversions (27.5%) were observed. The largest single group was G.C----A.T (60% of all base substitutions). The sites where G.C----A.T changes occurred were strongly correlated (97.5%) with sequences of adjacent pyrimidines, indicating mutation targeted ultraviolet photoproducts. Comparable amounts of mutation occurred at cytosine/cytosine and (mixed) cytosine/thymine sites. From an analysis of the prevalence of mutation at either the 5' or 3' side of a dipyrimidine, we conclude that both cyclobutane dimers and (6-4) lesions may contribute to mutation. Despite the general similarity of the base-substitution spectra between the wild-type and excision-defective strains, a number of sites were uniquely mutable in the UvrB- strain. Analysis of their surrounding DNA sequences suggested that, in addition to damage directly at the site of mutation, the potential for nearby opposite-strand damage may be important in determining the mutability of a site. The ultraviolet light-induced frameshift mutations were largely single-base losses. Inspection of the DNA sequences at which the frameshifts occurred suggested that they resulted from targeted mutagenesis, probably at cyclobutane pyrimidine dimers. The prevalence of frameshift mutations at homodimers (TT or CC) suggests that their formation involves local misalignment (slippage) and that base-pairing properties are partially retained in cyclobutane dimers. While the frameshift mutations in the Uvr+ strain were distributed over many different sites, more than half in the UvrB- strain were concentrated at a single site. Ultraviolet light-induced deletions as well as frameshift hotspot mutations (+/- TGGC at positions 620 to 632) are considered to be examples of untargeted or semitargeted mutagenesis. Hotspot mutations in the Uvr+ strain showed an increased contribution by (-)TGGC relative to (+)TGGC, indicating that ultraviolet light may specifically promote the loss of the four bases.(ABSTRACT TRUNCATED AT 400 WORDS)

Base Sequence↗
PubMed1992

Mutational spectra in human B-cells. Spontaneous, oxygen and hydrogen peroxide-induced mutations at the hprt gene.

To test the hypothesis that reactive species in the oxygen cascade are responsible for spontaneous mutation, we examined the spectra of oxygen and hydrogen peroxide-induced mutations at the hprt locus in a human B-lymphoblastoid cell line. We compared these spectra with the spontaneous mutational spectrum. Large gene alterations were studied by Southern analysis of individual TGR clones. A combination of high fidelity polymerase chain reaction, denaturing gradient gel electrophoresis and direct DNA sequencing were used to detect and identify point mutations in exon 3 of hprt. With regard to spontaneous mutations, a previous study showed that 39% of the spontaneous TGR clones had large gene alterations. In the present study, the analysis of spontaneous point mutations within exon 3 revealed two hotspots. A one base-pair deletion (-A) at base-pair 256 or 257 and a two base-pair deletion (-GG) at base-pair 237 and 238, were detected in triplicate cultures. Each of the hotspots comprised about 1% of the TGR mutants. The analysis of individual oxygen-induced TGR clones (48 h, 910 microM-O2) showed 43% had large gene alterations similar to the spontaneous TGR clones. However, none of the spontaneous point mutation hotspots was found among triplicate oxygen-treated cultures. Two point mutations in common with H2O2-treated cultures were found in one of the three oxygen-treated cultures. Hydrogen peroxide-induced mutations (1 h, 20 microM) also differed from spontaneous mutations. Only 24% of the hydrogen peroxide-induced TGR clones had large gene alterations. The analysis of point mutations showed three hotspots within exon 3 of hprt. An AT to TA transversion at base-pair 259 had an average frequency of 3% of all TGR mutants (present in all of 3 H2O2-treated cultures). Two GC to CG transversions at base-pairs 243 and 202 were present at a frequency of 0.6% and 0.4%, respectively. A five base-pair deletion (base-pair 274 to 278) was present at an average frequency of 0.3%. The latter three mutations were detected in two of three H2O2-treated cultures. Thus, the point mutation spectra of both oxygen and hydrogen peroxide were significantly different from the spontaneous spectrum. The oxygen and hydrogen peroxide-induced spectra shared some features, suggesting that oxygen and hydrogen peroxide share some but not all pathways for induction of mutations within the DNA sequence studied here.(ABSTRACT TRUNCATED AT 400 WORDS)

B-Lymphocytes↗
PubMed1987

Hyper-mutation caused by the reml mutation in yeast is not dependent on error-prone or excision repair.

The reml mutations of Saccharomyces cerevisiae confer a semi-dominant hyper-recombination/hyper-mutation phenotype. Neither reml mutant allele has any apparent meiotic affect. We have examined spontaneous mutation in reml-2 strains and demonstrate that the reml-2 mutation, like reml-1, confers an average 10-fold increase in reversion and forward mutation rates. Unlike certain yeast rad mutations with phenotypes similar to reml, strains containing reml are resistant to MMS and only slightly UV sensitive at very high doses. To understand the mutator phenotype of reml, we have used a double-mutant approach, combining the reml mutation with radiation-sensitive mutations affecting DNA repair. Double mutants of reml-2 and a mutation in the yeast error-prone repair group (rad6-1) or a mutation in excision repair (rad1-2 or rad4) maintain the hyper-mutation phenotype. Since mutation rates remain elevated in these double-mutant strains, it appears as if the mutations which occur in the presence of reml resemble spontaneous mutation since they do not require the action of a repair system.

Alleles↗
PubMed1996

Differences in the spectrum of spontaneous mutations in the hprt gene between tumor cells of the microsatellite mutator phenotype.

We have determined the frequency and spectrum of spontaneous mutations at the hprt locus in LoVo, HCT116, LS180 and DLD-1 colon carcinoma cell lines exhibiting microsatellite genetic instability. Each cell line has a different mutator gene. LoVo and HCT116 cells have mutated hMSH2 and hMLH1 genes, respectively, which account for the majority of hereditary non-polyposis colorectal cancer (HNPCC). LS180 cells are wild type for these genes and also for hPMS1 and hPMS2 mismatch repair genes. DLD-1 cells harbor a mutated GTBP mismatch binding factor and a mutated DNA Polymerase delta. The mutation rate at the hprt locus was several hundred fold higher in these cell lines relative to control cell lines without microsatellite instability. The mutations were frameshifts (deletions and insertions of a single nucleotide in short repeats) and single base substitutions (transversions and transitions). Some mutations were shared by these four cell lines. However, every cell line also exhibited a distinctive spectrum of mutations suggesting that each mutator gene induces a particular mutator phenotype. These results also suggest that the frequency and spectrum of somatic mutations in tumor cells of the microsatellite mutator phenotype may have diagnostic applications to discriminate among the diverse underlying mutator genes.

Base Sequence↗