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PubMed1995

Molecular epidemiology of factor IX germline mutations in Mexican Hispanics: pattern of mutation and potential founder effects.

Germline mutations in patients with hemophilia B generally have arisen within the past 150 years. Evidence suggests that these germline mutations generally result from endogenous processes. However, a unique pattern would be expected if a population were exposed to a physiologically important germline mutagen since mutagens generally produce characteristic patterns, or "fingerprints", of mutation. To determine the pattern of mutation in Mexican Hispanics, the regions of likely functional significance in the factor IX gene were screened by dideoxy fingerprinting (ddF) in 31 families with hemophilia B. Mutations were found in 30 of these families. Haplotype analysis was performed on individuals with identical mutations to help distinguish independent, recurrent mutations from founder effects. Analysis of these 30 mutations, along with 7 mutations reported previously in Mexican Hispanic families, reveals a pattern of independent mutation that is similar to the pattern of mutation observed in 127 U.S. Caucasian families (p = 0.89). These results may reflect either an underlying pattern of germline mutation due to endogenous processes or the presence of an ubiquitous mutagen. Further analyses of the recurrent mutations revealed that two mutations, T296M and R248Q, accounted for 19% of the mutations found in the Mexicans. Haplotype data suggest that the multiple occurrences of T296M and R248Q are associated with founder effects and that screening for these mutations may allow rapid mutation detection and carrier diagnosis in a significant minority of Mexican families with hemophilia B, These two mutations also are associated with founder effects in the U.S, Caucasian population. However, the haplotypes are different in these two populations, indicating independent origins. The occurrence of identical founder mutations in distinct populations provides evidence for the previous hypothesis that the number of different mutations giving rise to mild or borderline mild/moderate hemophilia B is small compared to deleterious mutations causing more severe disease.

Factor IX↗
PubMed1987

Radiation-induced forward and reverse specific locus mutations and dominant cataract mutations in treated strain BALB/c and DBA/2 male mice.

Strain BALB/c and DBA/2 mice were chosen to investigate the effects of genetic background on the radiation-induced mutation rate since they exhibit differences in their radiation sensitivity. Males were exposed to 3 + 3-Gy X-irradiation and mated to untreated specific locus Test-stock females. Offspring resulting from treated spermatogonia were screened for induced specific locus forward and reverse mutations and dominant cataract mutations. Since BALB/c mice are homozygous brown and albino, specific locus forward mutations could be screened at 5 of the 7 specific loci (a, d, se, p, s), while reverse mutations could be screened at the b and c loci. Strain DBA/2 is homozygous non-agouti, brown and dilute. Therefore, specific locus forward mutations could be screened at 4 loci (c, se, p, s) and reverse mutations were screened at the a, b and d loci. Results indicate no effect of genetic background on the sensitivity to mutation induction of specific locus forward mutations, while for the dominant cataract alleles strain DBA/2 exhibited a higher mutation rate than either strain BALB/c or similarly treated (101/El X C3H/El)F1 mice. If, by confirmation, these differences should be demonstrated to be real, it is interesting that strain DBA/2 should exhibit a greater sensitivity to radiation-induced dominant mutations. First, strain DBA/2 was chosen as radiation resistant or repair competent. The observation that DBA/2 exhibited a higher sensitivity to radiation-induced mutation may indicate a role for repair, albeit misrepair, in the mutation process. Second, that the effect of genotype was only observed for the mutation rate to dominant cataract alleles may reflect a difference in the spectrum of DNA alterations which result in dominant or recessive alleles. A dominant allele is more likely misinformation, such that as heterozygote it interferes with the wild-type allele. By comparison, a recessive allele may result from any DNA alteration leading to the loss of a functional gene product. One reverse mutation at each of the a and d loci was recovered in the present experiments. The similarities of the present results for radiation-induced reverse mutations with the extensive data on the spontaneous reverse mutation rates are interesting. Reverse mutations were recovered only at the a and d loci. Further, the reverse mutations recovered at the a locus were to alternate alleles (at, Aw or Asy) while true reverse mutations were apparently recovered at the d locus.

Abnormalities, Radiation-Induced↗
PubMed1994

X-ray-induced specific-locus mutations in the ad-3 region of two-component heterokaryons of Neurospora crassa. XII. Analysis of multiple-locus ad-3 mutations reveals a nonrandom distribution of the separate sites of recessive lethal damage throughout the genome.

Previous studies on X-ray-induced adenine-3 mutations induced in heterokaryon 12 of Neurospora crassa showed that they consisted of gene/point mutations, multilocus deletion mutations, and 3 different genotypic classes of multiple-locus mutations (designated [-3]IR + RLCL, ad-3R + RLCL, and ad-3R + RL). In the present paper, multiple-locus mutations consisting of gene/point mutations at the ad-3A or the ad-3B locus with sites of recessive lethal damage closely linked to the ad-3 region (designated ad-3R + RLCL) or with sites of recessive lethal damage elsewhere in the genome (designated ad-3R + RL) were analyzed to determine whether they resulted from mutations at the same sites or different sites throughout the genome. It was assumed that if the recessive lethal mutations in individual multiple-locus mutations showed complementation on adenine-supplemented medium, they resulted from mutations at different sites. Multiple-locus mutations from both major genotypic classes were combined, as forced heterokaryons, in all possible pairwise combinations and then were plated out on adenine-supplemented medium. These studies indicated that 89.3% (50/56) of the recessive lethal mutations in these 2 classes of multiple-locus mutations complement one another. Thus, they are presumed to have resulted predominantly from genetic damage at different sites throughout the genome. Within the group of 20 multiple-locus mutations that did not complement in various pairwise combinations, 90% (18/20) appear to map in a region, distal to the ad-3 region, defined by a series of overlapping multilocus deletion mutations in 6 mutations of genotype ad-3R + RLCL. The other 10% (2/20) are located elsewhere on Linkage Group I or elsewhere in the genome. The present data base on multiple-locus mutations is unique; such events either can not be detected, or can only be detected with difficulty, in other eukaryotic specific-locus assay systems such as mammalian cells in culture, Drosophila or mice. Our data on X-ray-induced ad-3 specific-locus mutations from the present and previous studies demonstrate the presence of additional sites of genetic damage, both closely linked with the ad-3 region or elsewhere in the genome, in ad-3 specific-locus mutations. Because the frequencies of each class of multiple-locus mutations is dose-dependent, they must be taken into account in genetic risk assessment exercises. Failure to acknowledge the presence of such additional sites of genetic damage in the utilization of specific-locus data could result in underestimation of the risk of human exposure to environmental mutagens.

Adenine↗
PubMed1996

Characterization of the factor VIII defect in 147 patients with sporadic hemophilia A: family studies indicate a mutation type-dependent sex ratio of mutation frequencies.

The clinical manifestation of hemophilia A is caused by a wide range of different mutations. In this study the factor VIII genes of 147 severe hemophilia A patients--all exclusively from sporadic families--were screened for mutations by use of the complete panel of modern DNA techniques. The pathogenous defect could be characterized in 126 patients (85.7 percent). Fifty-five patients (37.4 percent) showed a F8A-gene inversion, 47 (32.0 percent) a point mutation, 14 (9.5 percent) a small deletion, 8 (5.4 percent) a large deletion, and 2 (1.4 percent) a small insertion. Further, four (2.7 percent) mutations were localized but could not be sequenced yet. No mutation could be identified in 17 patients (11.6 percent). Sixteen (10.9 percent) of the identified mutations occurred in the B domain. Four of these were located in an adenosine nucleotide stretch at codon 1192, indicating a mutation hotspot. Somatic mosaicisms were detected in 3 (3.9 percent) of 76 patients, mothers, comprising 3 of 16 de novo mutations in the patients mothers. Investigation of family relatives allowed detection of a de novo mutation in 16 of 76 two-generation and 28 of 34 three-generation families. On the basis of these data, the male:female ratio of mutation frequencies (k) was estimated as k = 3.6. By use of the quotients of mutation origin in maternal grandfather to patients mother or to maternal grandmother, k was directly estimated as k = 15 and k = 7.5, respectively. Considering each mutation type separately, we revealed a mutation type-specific sex ratio of mutation frequencies. Point mutations showed a 5-to-10-fold-higher and inversions a >10-fold-higher mutation rate in male germ cells, whereas deletions showed a >5-fold-higher mutation rate in female germ cells. Consequently, and in accordance with the data of other diseases like Duchenne muscular dystrophy, our results indicate that at least for X-chromosomal disorders the male:female mutation rate of a disease is determined by its proportion of the different mutation types.

Base Sequence↗
PubMed1979

Is there a proportionality between the spontaneous and the X-ray-induction rates of mutations? Experiments with mutations at 13 X-chromosome loci in Drosophila melanogaster.

The X-ray induction of recessive visible specific locus mutations at 14 X-chromsome loci was studied in Drosophila melanogaster using the "Maxy" technique. The X-ray exposure was 3000 R to 5-day-old males and the sampling of germ cells was restricted to mature spermatozoa. Presumptive mutant females recovered in the F1 generation were tested for transmission, allelism, fertility and viability in males. A total of 128 mutations (115 completes and 13 mosaics including those that were male viable as well as male-lethal) recovered among 38 898 female progeny were found to be transmitted. On the basis of the above frequency, the average mutation rate can be estimated as 7.8 X 10(-8)/locus/R; for mutations that were viable and fertile in males, the rate is 3.0 X 10(-5)/locus/R (49 mutations among 38 898 progeny). The frequency of mutations at the different loci encompassed a wide range: while no mutations were recovered at the raspberry and carnation loci, at others, the numbers ranged from 1 at echinus to 31 at garnet; in addition, the proportion of mutations that was male-viable was also different, depending on the locus. Schalet's extensive data on spontaneous mutations at 13 (of the 14 loci employed in the present study) loci permit an estimate of the spontaneous rate which is 6.1 X 10(-6)/locus (a total of39 mutations among 490 000 progeny); for mutations that were viable and fertile in males, the rate is 3.0 X 10(-6)/locus (19 mutations among 490 000 progeny). The mutability of the different loci varied over a 9-fold range. When the different loci are ranked depending on their relative mutability (for spontaneous and induced mutations) it is found that in general, loci that mutate spontaneously relatively more frequently are also those at which more mutations have been recovered in the radiation experiments and likewise, those that are less mutable spontaneously are also those that mutate less after irradiation. Since the data are limited, it is concluded that the above finding is not inconsistent with the assumption of proportionality between spontaneous and induction rates of mutations. On the basis of the above results, a doubling dose of 100 R can be calculated for the X-ray induction of specific-locus mutations in Drosophila spermatozoa.

Animals↗
PubMed1997

Development of a rapid assay for detecting gyrA mutations in Escherichia coli and determination of incidence of gyrA mutations in clinical strains isolated from patients with complicated urinary tract infections.

The MICs of ofloxacin for 743 strains of Escherichia coli isolated from 1988 to 1994 were determined by testing. The strains were from patients with urinary tract infections complicated by functional or anatomical disorders of the urinary tract. Those determined to be ofloxacin resistant (MIC, > or =12.5 microg/ml) comprised 3 of 395 strains (1.3%) from the 1988 to 1990 group, 2 of 166 strains (1.2%) from the 1991 to 1992 group, and 7 of 182 strains (3.8%) from the 1993 to 1994 group. The incidence of resistant strains increased significantly during this period. The percentage of isolates with moderately decreased susceptibilities to ofloxacin (MIC, 0.39 to 3.13 microg/ml) also rose during the same period. To determine the incidence of gyrA mutations in urinary-tract-derived strains of E. coli, we developed a simple and rapid assay based on PCR amplification of the region of the gyrA gene containing the mutation sites followed by digestion of the PCR product with a restriction enzyme. Using this assay, we examined all 182 strains isolated in 1993 and 1994 for the presence of mutations at Ser-83 and Asp-87 in the gyrA gene. Of these strains, 33 (18.1%) had mutations in the gyrA gene. The incidences of mutations at Ser-83, at Asp-87, and at both codons were 10.4 (19 strains), 4.4 (8 strains), and 3.3% (6 strains), respectively. To determine the correlation of the mutations in the gyrA gene with susceptibilities to quinolones (nalidixic acid, ofloxacin, norfloxacin, and ciprofloxacin), we further examined 116 strains for which the MICs of ofloxacin were > or =0.2 microg/ml that were chosen from the isolates in the 1988 to 1992 group. The MICs of nalidixic acid for the strains without mutations at either Ser-83 or Asp-87 were < or =25 microg/ml, whereas those for the strains with single mutations or double mutations were from 50 to >800 microg/ml. For the fluoroquinolones, significant differences in the distributions of the MICs were observed among the strains without mutations, with single mutations, and with double mutations. The accumulation of mutations in the gyrA gene was associated with an increase in fluoroquinolone resistance. Ofloxacin MICs for the majority of the strains with single and double mutations were 0.39 to 3.13 and 6.25 to 100 microg/ml, respectively. This study demonstrates a chronological increase in the percentage of not only highly fluoroquinolone-resistant strains, corresponding to those with double mutations in the gyrA gene, but also strains with moderately decreased susceptibilities to fluoroquinolones, corresponding to those with single mutations. This increase in the incidence of strains with a single mutation in the gyrA gene portends a further increase in the incidence of strains with clinically significant resistance to fluoroquinolones.

Anti-Infective Agents↗
PubMed1996

Two mutations in exon XII of the protein S alpha gene in four thrombophilic families resulting in premature stop codons and depressed levels of mutated mRNA.

Sixteen Danish unrelated thrombophilic families with plasma protein S deficiency of type 1 (or III) are currently under investigation in our laboratory for defects in the protein S alpha gene. The present paper describes a part of this work, which deals with the identification and phenotypical presentation of two unique mutations in exon XII of the protein S alpha gene in four of these families. The mutations were identified by SSCP screening followed by nucleotide sequence analysis or by direct nucleotide sequence analysis. The mutation found in one family (D) is a novel deletion of an A in either the codon for Gly448 (GGA) or Ile449 (ATI) resulting in a frameshift and a premature stop codon at position 454. The other mutation shared by three families (F, G and J) is a previously reported C-->T transition within a hypermutable CG dinucleotide sequence converting Arg410 (CGA) to Stop (TGA). All affected individuals are heterozygotes for their mutation and in each family the protein S genotype, the plasma protein S phenotype (not shown for Family J) and the clinical phenotype cosegregate. The two mutations can fully explain the abnormal protein S phenotype since premature stop codons are known to disrupt gene function of the mutated allele. Analysis of protein S mRNA from platelets showed that both mutations result in a marked reduction in the amount of protein S mRNA from the mutated alleles indicating that the mutations exert their deleterious effects on gene expression at the transcriptional level. The Arg410-->Stop mutation in Families F, G and J is in all instances linked to a G at the site of a common neutral dimorphism in the codon for Pro626 (CCA/G) in exon XV. This indicates that the mutation in these families could have arisen in a common ancestor. The Arg410 (CGA)-->Stop (TGA) mutation is also seen in exon XII of the normal protein S alpha gene. This gives rise to the speculation as to whether the mutation in the protein S alpha gene is the result of an interaction with the protein S beta gene leading to double homologous unequal crossing-over or gene conversion of a short DNA sequence. However, this is unlikely since none of the 7 other protein S beta-specific nucleotides are present in the mutated exon XII sequence of the protein S alpha gene. The common Arg506-->Gln Leiden mutation in coagulation factor V is not an additional risk factor for thrombosis in any of the four families studied.

Adult↗
PubMed1996

BRCA1 germline mutational spectrum in Italian families from Tuscany: a high frequency of novel mutations.

BRCA1 germline mutations confer susceptibility to familial breast and ovarian cancer. Mutational hot spots have never been detected in BRCA1 cDNA. Some mutations have been reported several times whereas some others appear to be population-related. In this study a group of 36 Italian families were analysed for BRCA1 germline mutations. All of them were screened by allele-specific oligonucleotide hybridization (ASO) for three recurrent mutations (185delAG, 5382insC, nt332-T>G). Twenty families, selected because of their high risk of carrying BRCA1 mutations, were subjected to analysis of the entire coding sequence of the gene. A total of eight mutations were found. ASO screening demonstrated only one known mutation in one patient, whereas cycle sequencing revealed five new mutations. Three of these new mutations were frameshifts: one occurred in exon 11 (1499insA), one in exon 16 (4873delCA) and one in the splice site of exon 3 (252delAAgt). Two were missense mutations (Cys64Arg; Asn158Tyr). The same frameshift mutation, 1499insA, was detected in three unrelated families. Haplotype analysis supported the hypothesis that two of these families may have had common ancestors, whereas in the third family the analysis was uninformative. BRCA1 germline mutations were found in one out of two families with ovarian cancer, in five out of eight families with breast-ovarian cancer, and in two out of 11 families with breast cancer. All three families with 1499insA mutations included at least one case of ovarian cancer. The majority of the ovarian cancers (4/5) associated with detectable BRCA1 germline mutations were of serous histotype.

Adult↗
PubMed1978

Gamma-ray-induced dominant mutations that cause skeletal abnormalities in mice. II. Description of proved mutations.

In a mutation-rate experiment described earlier, 31 dominant skeletal mutations were confirmed by breeding tests. Skeletal abnormalities were detected in the skeletons of some of the sons of irradiated males, and for 31 of these sons the study of skeletons in subsequent generations showed that they transmitted abnormalities. The detailed descriptions of these mutations, together with descriptions of 6 presumed mutations found in a later paper, provide the basis for determining which mutations cause effects that would, if they occurred in humans, cause a serious handicap. Such a determination is necessary before these data can be used to estimate genetic hazard to humans. Furthermore, these descriptions of syndromes caused by individual dominant mutations should be useful to clinicians interested in skeletal defects. The statistical analysis of the frequency of each abnormality in the mutant line versus an approximation of the frequency of the malformation in the absence of new mutations is essential to be sure that a mutation is indeed the cause of each abnormality. These analyses, together with analyses of the correlation of abnormalities caused by individual mutations, clearly demonstrate that dominant mutations exhibit low penetrance for many of their effects. A few of the mutations also cause the death of some heterozygotes. No externally visible effects have been detected in heterozygotes for most of these mutations. Externally visible effects found in some of the heterozygotes for a few of the mutations include hydrocephalus, circling behavior, increased nervous activity, gray coat color, webbing of digits, and small size. Two coat-color mutations were found that caused no detected skeletal abnormalities. The data suggest that a few of the mutations may be reciprocal translocations. In most of the mutant lines tested cytologically, however, there was no indication of chromosomal aberrations.

Animals↗
PubMed1991

X-ray-induced specific-locus mutations in the ad-3 region of two-component heterokaryons of Neurospora crassa, IX. Mutational spectra as a function of X-ray dose.

In previous studies, X-ray-induced specific-locus mutations in the adenine-3 (ad-3) region of a two-component heterokaryon (H-12) of Neurospora crassa were combined with a series of tester strains carrying markers in the ad-3 and immediately adjacent regions to map mutants that were presumed multilocus deletions (de Serres, 1989c, 1990a). Two new classes of X-ray-induced mutations were recovered: multiple-locus mutations consisting of gene/point mutations at the ad-3A or ad-3B locus with a closely linked recessive lethal mutation, or multilocus deletions covering the ad-3A, ad-3B and/or nic-2 loci with a closely linked recessive lethal mutation (designated ad-3R + RLCL and [ad-3]IR + RLCL, respectively). Thus, the ad-3 specific-locus assay can detect damage occurring at the ad-3A and the ad-3B loci, as well as at a minimum of 19 other loci in the immediately adjacent regions. The original overall spectrum of ad-3 mutations can be resolved, by genetic analysis, into a series of 30 subclasses. In the present paper, the data from the genetic analysis of 832 X-ray-induced mutants recovered from a series of 4 experiments (Webber and de Serres, 1965) have been presented in terms of Mutational Spectra organized as a function of X-ray dose. Comparison of these Spectra demonstrates the shift from high percentages of gene/point mutations (with a high percentage of mutants at the ad-3B locus showing allelic complementation) at low doses, to low percentages of gene/point mutations (with a low percentage of ad-3B mutants showing allelic complementation) and high percentages of multilocus deletion mutations and multiple-locus mutations (of genotype ad-3R + RLCL or [ad-3]IR + RLCL) at high doses. These Mutational Spectra demonstrate the marked dose-dependence of X-ray-induced specific-locus mutations in a eukaryotic organism.

Alleles↗
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↗
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↗
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↗
PubMed1995

Mutations in the p53 gene in schistosomal bladder cancer: a study of 92 tumours from Egyptian patients and a comparison between mutational spectra from schistosomal and non-schistosomal urothelial tumours.

Much of bladder cancer in East Africa and the Middle East is attributed to chronic urinary infection with Schistosoma haematobium ('schistosomiasis'). Most schistosomal bladder cancer (SBC) is squamous cell carcinoma (SCC) and occurs in the fifth decade of life. In contrast, nonschistosomal bladder cancer (NSBC) in Western countries usually occurs in the seventh decade of life and is largely transitional cell carcinoma (TCC). To shed light on the mechanisms underlying these different patterns of bladder cancer we looked for mutations in the p53 gene in SBC from 92 patients in Egypt, where schistosomiasis is hyperendemic. Patients' mean age at presentation of bladder cancer was 49.4 +/- 9.9 years and 90% had a clinical history of schistosomiasis and/or histological evidence of schistosomal eggs adjacent to the carcinoma. There were 53 SCC, 23 TCC, 13 adenocarcinomas and three other carcinomas. Thirty patients had tumours with mutations in exons 5-8 of the p53 gene: 17/53 SCC, 8/23 TCC, 4/13 adenocarcinomas and 1/3 other tumours. Of 19 mutations in SCC, 16 were base pair substitutions (BPS), two were deletions and one an insertion. Two tumours each contained two mutations. Of the BPS, nine were transitions at CpG dinucleotides and two were G-->T transversions. All the mutations in TCC were BPS: four were transitions at CpG dinucleotides and three were G-->C transversions. One TCC had two mutations. Of four adenocarcinomas with mutations, two had transitions at CpG dinucleotides. Of the 30 BPS mutations, 16 were transitions at CpG dinucleotides, of which 12 were C-->T. We combined these 33 mutations with six obtained from Egyptian SCC reported by Habuchi et al. (Cancer Res., 53, 3795-3799, 1993) to compile a mutational spectrum. This was compared with a NSBC spectrum assembled from 118 mutations reported in the literature. The proportion of BPS at CpG dinucleotides was significantly higher in SBC than in NSBC (18/34 versus 25/103, P = 0.003). There was also a bias away from mutations in exons 7 and 8 towards mutations in exons 5 and 6. We suggest that the excess of transitions at CpG dinucleotides in SBC results from nitric oxide (NO) produced by the inflammatory response provoked by schistosomal eggs. NO could produce such mutations directly, by deamination of 5-methylcytosine, and indirectly, following conversion to nitrate, bacterial reduction to nitrite and endogenous formation of urinary N-nitroso compounds. These produce O6-alkylguanines in DNA, leading to very high rates of G:C-->A:T transitions, a process possibly augmented by inefficient repair of alkylated bases at CpG dinucleotides.

Adenocarcinoma↗