Genome wide spontaneous mutation in human cells determined by the spectrum of mutations in hprt cDNA genes

We have studied spontaneous mutagenesis in five hprt cDNA genesintegrated at five different genomic positions in a human lymphoblastoidcell line (TK6). The spectra of 40 mutants from each positionwere combined to obtain a mutation spectrum of the overall genome.This collection of mutants was used to assess the contributionof several mutagenic processes to spontaneous mutagenesis. Deletionsand single base pair changes account for the majority of themutants and arise in approximately equal amounts (43 and 41%,respectively).The majority of the deletions and insertions are >5 bp andare likely to be caused by template-directed misalignment (slippage)during replication. To account for frameshifts at non-iteratedsites we propose a slightly different template-directed replicationerror model. A considerable amount of the observed base pairchanges can also be explained by this last model but severalother processes leading to base pair changes such as depurination,deamination or spontaneously arising DNA damage are likely tocontribute as well. We have compared this spectrum with mutationspectra in the endogenous hprt genes using published mutationdata. It is shown that in the endogenous genes the contributionof base pair substitutions is much larger (71%) than in thehprt cDNA integrates and that deletions are less frequentlyobserved (20%) The mutation rates of the integrated hprt cDNAgenes show a mean increase of 30-foldas compared with the endogenoushprt gene. This results in a 60-fold increase of the absoluterate of deletion in the hprt cDNA genes and in a 15-fold increaseof the base pair substitution rate. Replication errors suchas slippage or the mechanism proposed in this study probablyaccount to a large extent for this increase. ²To whom correspondence should be addressed     

Spontaneous mutation spectrum in the hprt gene in human lymphoblastoid TK6 cells

A spectrum of 100 mutations in the endogenous hprt gene of thehuman lymphoblastoid TK6 cell line is presented. The majorityof the mutations originates in sequences outside the codingregion of the gene. Large deletions are a major cause of inactivationof the hprt gene (57% of the mutants). Mutations in the splicesites that result in several forms of aberrantly spliced mRNAare relatively frequently recovered (16%) compared with mutantscontaining alterations in the coding region of the hprt gene(27%). The majority, but not all, of the splice mutants containan alteration in the consensus sequences of the splice sites.A spectrum of mutations in the coding region of the hprt geneenlarged to a total of 42 mutants shows that basepair substitutionspredominate (71%) and that small deletions and insertions areless frequently recovered. Basepair substitutions arise slightlymore frequently at GC basepairs than at AT basepairs. ³To whom correspondence should be addressed     

Molecular nature of spontaneous mutations at the hypoxanthine-guanine phosphoribosyltransferase (hprt) locus in chinese hamster ovary cells

The hypoxanthine-guanine phosphoribosyltransferase (hprt) locus has been widely used as a selectable genetic marker for studies of mammalian cell mutagenesis. We report here the spontaneous mutation spectrum at the hprt locus in 64 independently isolated mutants of Chinese hamster ovary (CHO) cells. All nine hprt exons were simultaneously analyzed via multiplex polymerase chain reaction (PCR) for rapid detection of gene deletions or insertions. Structural point mutations were identified by direct sequence analysis of the PCR amplified cDNA. The molecular nature of RNA splicing errors and insertions was analyzed by solid-phase direct exon sequencing. Single base substitutions were found in 24 mutants (38%), of which 21 were missense and 3 were nonsense mutations. Transversions were about twice as frequent as transitions. Fifteen mutants (23%) had deletions involving either intragenic small fragments (2), single exons (9), or multiple exons (4). The majority of deletion breakpoints (71%) were located in regions surrounding exons 4, 5, and 6. RNA splicing mutations were observed in 15 mutants (23%) and affected exons 3–8; most (6/15) resulted in the loss of exon 7. Two insertion mutants, one with a 209 bp insert in exon 4 and the other with a 88 bp insert accompanied by a 24 bp deletion in exon 6, represent novel mutations reported for the first time in spontaneous mutants of the mammalian hprt gene. © 1995 Wiley-Liss, Inc.     

Effect of nucleotide excision repair on hprt gene mutations in rodent cells exposed to DNA ethylating agents

The role of the nucleotide excision repair (NER) pathway inremoval of DNA ethylation damage was investigated by means ofhprt mutational spectra analysis in the NER-deficient Chinesehamster ovary cell line UV5, which lacks ERCC2/XPD, and in itsparental cell line AA8. Both cell lines were exposed to ethylmethanesulfonate (EMS) or N-ethyl-N-nitrosourea (ENU). EMS gavea similar dose-dependent increase in hprt mutants in UV5 comparedwith AA8. In both cell lines EMS-induced mutations in the hprtcoding region consisted almost exclusively of GC     

Mutational spectrum of ICR-191 at the hprt locus in human lymphoblastoid cells

Human TK6 lymphoblasts were treated with the acridine derivative ICR-191, and mutants at the hprt locus were isolated. Mutant hprt cDNA was reverse-transcribed from mRNA, amplified by polymerase chain reaction (PCR), and sequenced. Additions of single G:C base pairs (+1 frameshift mutations) in repetitive G:C sequences were found in 82% (32/39) of the mutants. Sixteen of the +1 frameshifts analyzed were located in a single sequence of six consecutive guanine bases in exon 3. The remaining +1 frameshifts occurred at six different GGG sequences (14 mutants) and a single GGGG sequence (2 mutants) in other hprt exons. The repetitive guanine sequences that underwent frameshift mutagenesis were located in both the transcribed and nontranscribed strands of hprt. No single base deletions (-1 frameshift mutations) were observed. Base substitutions were observed in 13% (5/39) of the clones analyzed and occurred at both G:C and A:T bases. Loss of exon 4 from the cDNA was also observed in 5% (2/39) of the mutants. Hprt mutants containing seven consecutive guanines (produced from a +1 frameshift in a GGGGGG sequence) were treated with ICR-191 and wild-type revertants selected in CHAT medium. Revertants were recovered at a frequency of approximately 10^?7 and contained the wild-type sequence (GGGGGG) in all clones analyzed. The observed frequency of ICR-191-induced -1 frameshift reversion in the GGGGGGG sequence was ～500-fold lower than the estimated frequency of +1 frameshifts observed in the wild-type GGGGGG sequence following the same ICR-191 treatment. These results suggest that ICR-191 produces predominantly +1 frameshift mutations at the hprt locus in human cells. © 1994 Wiley-Liss, Inc.     

Multiplex PCR analysis of in vivo-arising deletion mutations in the hprt gene of human T-lymphocytes

A multiplex polymerase chain reaction (PCR) procedure was adapted for the rapid and efficient evaluation of deletions of the hypoxanthine guanine phosphoribosyltransferase (hprt) gene in human T-lymphocytes. The hprt clonal assay was used to isolate in vivo-arising hprt-deficient T-cells from six healthy males. Mutant frequencies ranged from 9-27 × 10^?6. Simple crude cellular extracts from 223 mutants were analyzed for hprt gene deletion. Sixteen (7.2%) were found to be due to total gene deletion and 22 (9.9%) were due to partial gene deletion. The relatively high frequency of total gene deletions was caused by replicate isolates of a single mutational event as shown by single-strand conformation polymorphism (SSCP) analysis of rearranged T-cell receptor (TCR)-γ genes. Eighteen of the 22 partial hprt gene deletion mutants were determined to be of independent origin based on a unique hprt mutation or SSCP-TCR-γ pattern. One-half (9/18) of the partial deletion mutants involved all or part of exon 4 alone, suggesting that this region of the hprt gene is prone to deletion. The small deletions effecting exon 1 (1 mutant), exon 2 (2 mutants), and exon 4 (6 mutants) would not have been detected by conventional Southern blot analysis and may represent a new, previously unrecognized class of mutations. The ready isolation of such intragenic deletions will allow the characterization of breakpoint junctions and may provide insights into the important processes of DNA breakage and rejoining. © 1994 Wiley-Liss, Inc.     

Analysis of point mutations in the hprt gene of cancer patients treated with radioimmunoglobulin therapy

The mutagenic impact of various environmental and therapeutic agents can now be directly assayed in humans by the T-lymphocyte cloning assay. We have previously reported that following radioimmu-noglobulin therapy, cancer patients exhibited increased mutant frequency at the hprt locus and an increased yield of large intergenic deletions compared to unexposed controls. Here we report the results of the analysis of 26 independent hprt mutations in nine cancer patients who underwent radioimmunoglobulin therapy. The majority of mutations (52%) had lost exon sequences from the mRNA. The remaining mutations were 20% small deletions and frameshifts and 28% base substitutions. The type of mutations observed were similar to those seen in unexposed controls. The site distribution of the mutations, however, indicates that some sequence contexts may be more sensitive to radiation mutagenesis than others. © 1995 Wiley-Liss, Inc.     

DNA sequence analysis of spontaneous and N-ethyl-N-nitrosourea-induced hprt mutations arising in vivo in cynomolgus monkey T-lymphocytes.

The study of hprt mutations in cynomolgus monkey T-lymphocytes is part of our effort to understand the mechanisms of mutagenesis in vivo. This primate model allows us to study mutations and their kinetics at the molecular level under well-controlled conditions using recently developed techniques for selection of mutant T-cells and polymerase chain reaction (PCR) amplification of hprt cDNA, which is directly sequenced. This is the first report of the sequence of the coding region of the cynomolgus monkey hprt gene and PCR/DNA sequence analysis of seven spontaneous mutant T-cell clones, as well as 23 mutant clones isolated 63 and 601 days after treatment with ethylnitrosourea (ENU, 77 mg/kg, intraperitoneal). cDNA was reverse transcribed from hprt mRNA directly from a lysate of about 2-4 x 10(3) cells, and a 700 bp fragment including the coding region was amplified by PCR and sequenced. Of the seven spontaneous mutants, only one point mutation (GC----AT transition) was detected, and the other six failed to amplify by PCR, possibly due to functional deletions. Of the 14 mutant clones isolated 63 days after ENU treatment, nine base substitutions were detected in ten clones: four transitions (three AT----GC and one GC----AT) and five transversions (four AT----TA and one AT----CG). Of the nine mutants isolated 601 days after ENU treatment, six single base substitutions were detected in six clones (five AT----TA and one AT----CG transversions), and one mutant had a large deletion or insertion. No changes were detected in three clones (one Day 63 and two Day 601 clones). In summary, only one of 15 single base substitutions isolated after ENU treatment was a GC----AT transition mutation and the rest were transitions and transversions at AT sites.     

Germline mutations in the von Hippel–Lindau disease tumor suppressor gene: Correlations with phenotype

Von Hippel-Lindau disease (VHL) is an inherited neoplastic disease characterized by a predisposition to develop retinal angiomas, central nervous system hemangioblastomas, renal cell carcinomas, pancreatie cysts, and pheochromocytomas. The VHL gene was recently isolated by positional cloning. The cDNA encodes 852 nucleotides in 3 exons. The VHL gene is unrelated to any known gene families. We identified germline mutations in 85/114 (75%) of VHL families. Clinical heterogeneity is a well-known feature of VHL. VHL families were classified into 2 types based on the presence or absence of pheochromocytoma. The types of mutations responsible for VHL without pheochromocytoma (VHL type 1) differed from those responsible for VHL with pheochromocytoma (VHL type 2). Fifty-six % of the mutations responsible for VHL type l were microdeletions/insertions, nonsense mutations, or deletions; 96% of the mutations responsible for VHL type 2 were missense mutations. Specific mutations in codon 238 accounted for 43% of the mutations responsible for VHL type 2. The mutations identified in these families will be useful in presymptomatic diagnosis. The identification of mutations associated with phenotypes contributes to the understanding of fundamental genetic mechanisms of VHL disease. © 1995 Wiley-Liss, Inc.     

Correlation between connexin 32 gene mutations and clinical phenotype in X-linked dominant Charcot-Marie-tooth neuropathy

We studied the relationship between the genotype and clinical phenotype in 27 families with dominant X-linked Charcot-Marie-Tooth (CMTX1) neuropathy. Twenty-two families showed mutations in the coding region of the connexin32 (cx32) gene. The mutations include four nonsense mutations, eight missense mutations, two medium size deletions, and one insertion. Most missense mutations showed a mild clinical phenotype (five out of eight), whereas all nonsense mutations, the larger of the two deletions, and the insertion that produced frameshifts showed severe phenotypes. Five CMTX1 families with mild clinical phenotype showed no point mutations of the cx32 gene coding region. Three of these families showed positive genetic linkage with the markers of the Xq13.1 region. The genetic linkage of the remaining two families could not be evaluated because of their small size. © 1996 Wiley-Liss, Inc.     

New intermediate phenotype between MED and DD caused by compound heterozygous mutations in the DTDST gene

DTDST mutations cause a spectrum of diastrophic dysplasia disorders characterized by defects of proteoglycans sulfation. Reduction of sulfate/chloride antiporter activity is manifested by lower sulfate uptake and depends on a combination of mutations in DTDST. We analyzed a family with an autosomal recessive form of bone dysplasia. Three affected brothers from this family are compound heterozygotes for C653S/A715V mutations. We classified their phenotype as a new intermediate form between diastrophic dysplasia and multiple epiphyseal dysplasia, manifested by shortening of stature, metatarsus adductus/club foot, mild brachydactyly, proximally placed thumbs and clinodactyly of the fifth fingers. Radiographs document platyspondyly most marked in the lower thoracic and upper lumbar spine, epiphyseal dysplasia affecting predominantly the femoral heads, widening of the metaphyses, narrow growth cartilage and multilayered patellae. Exaggerated lesser trochanters of femur, that is, "monkey wrench" sign, elevated greater trochanters, thin upper pubic rami, grossly normal carpal/tarsal bones and severe, early onset osteoarthritis were other notable features.     

Comparison of co-cultivation of V79 cells with rat hepatocytes and rat H4IIE hepatoma cells for studying nitrosamine-induced hprt gene mutations

The induction of mutations by nitrosamines in the hprt locusof V79 Chinese hamster cells was examined after metabolic activationin a co-cultivation system using either freshly isolated rathepatocytes or H4IIE rat hepatoma cells and the results obtainedwere compared with systems which employ the rat liver microsomalfraction (S9-mix). This study was also designed as a first approachto investigating the induction of point mutations by tobacco-specificnitrosamines in mammalian cells in order to obtain informationabout the significance of these compounds in connection withthe carcinogenicity of tobacco smoke. The mutagenicity of twotobacco-specific nitrosamines, 4-(methylnitroso)-1-(3-pyridol)-1-butanone(NNK) and N'-nitrosonornicotine (NNN), were investigated andcompared to two extensively investigated nitrosamines, i.e.dimethylnitrosamine (DMN) and diethylnitrosamine (DEN). DMNwas activated to mutagenic species by primary hepatocytes atµmolar concentrations, i.e. 1/100 of the concentrationsrequired for mutagenesis by DEN and NNK. NNN was not activatedto mutagenic species by liver S9 or primary hepatocytes. Thefindings shown here on the mutagenicities of NNK and NNN withliver preparations are in agreement with their relative carcinogenicpotencies. When the established liver cell line H4IIE was usedfor metabolic activation, DMN and was found to be mutagenic,whereas the results for NNN were borderline and for DEN andNNK were without effect. The fate of these compounds via differentmetabolic pathways is discussed in terms of systems for detectionof mutagenic metabolites and type of mutation induced.     

Interactions between clock gene mutation, circadian phenotype and tumor growth in mice

The relation between circadian physiology (rest-activity and body temperature) and the growth of a grafted tumor (Glasgow osteosarcoma-GOS) was investigated in the mice with mutation of clock gene (ClockDelta19(-)) or gene controlled by the clock (Vpac(-/-)). Circadian rhythms in temperature and activity were stable, with an approximately 24-h period in all the mice synchronized by the alternation of 12 h of light and 12 h of darkness (LD 12:12). Following exposure to constant darkness (DD), both rhythms persisted in ClockDelta19(-), yet with a lengthening of the period by 4.5 h compared to wild type. In DD, the amplitude increased by 45.9% for the temperature rhythm (p<0.001) and by 17.4% for the activity one (p=0.08) as compared to LD 12:12 in ClockDelta19(-). The improvement of circadian coordination and/or the lengthening of the circadian period observed in ClockDelta19(-) kept in DD was associated with a moderate slowing down of tumor growth. Although the exposure to DD ablated the activity and temperature rhythms in Vpac(-/-), no modification in tumor growth was observed as compared to wide type or Vpac(-/-) in LD 12:12. Major alternations of circadian physiology can result from interactions between photoperiodic environment and mutation of clock gene or gene controlled by the clock. In these conditions, we have shown that the alternation of the circadian phenotype does not seem to constitute an essential determinant of the growth of a grafted tumor.     

Deletion and insertion in vivo somatic mutations in the hypoxanthine phosphoribosyltransferase (hprt) gene of human T-lymphocytes

Deletion and insertion mutations have been found to be a major component of the in vivo somatic mutation spectrum in the hypoxanthine phosphoribosyltransferase (hprt) gene of T-lymphocytes. In apopulation of 172 healthy people (average age, 34; mutant frequency, 10.3 × 10⁻⁶), deletion/in sertion mutations constituted 41% (89) of the 217independent mutations, the remainder being base substitutions. Mutations were identified by multiplex PCR assay of genomic DNA for exon regions, by sequencing cDNA, or sequencing genomic DNA. The deletion and insertion mutations were divided among ±1 to 2 basepair (bp) frameshifts (14%, 30), small deletions and insertions of 3–200 bps (13%, 28), large deletions of one or more exons (12%, 27), and complex events (2%, 4). Frameshift mutations were dominated by −1 bp deletions (21 of 30). Exon 3 contained five frameshift mutations in the run of 6 Gs, the only site in the coding region with multiple frameshift mutations, possibly caused by strand dislocation during replication. Both end points were sequenced for 23 of the 28 small deletions/insertions including two tandem duplication events in exon 6. More small deletions (8/28), pos sibly mediated by trinucleotide repeats, occurred in exon 2 than in the other exons. Large deletions included total gene deletions (6), exon 2 + 3 dele tions (4), and loss of multiple (9) and single exons (8) in genomic DNA. The diverse mutation spectrum indicates that multiple mechanisms operated at many different sequences and provides a resource for examination of deletion mutation. Environ. Mol. Mutagen. 30:371–384, 1997 © 1997 Wiley-Liss, Inc.     

The tumor phenotype and the human gene map

The tumor phenotype is associated with the rearrangement of genetic information and the altered expression of many gene products. In this review, genes associated with the tumor phenotype have been arranged on the human gene map and indicate the extent to which the tumor phenotype involves the human genome. Nonrandom chromosomal aberrations that are frequently observed in tumors are presented. Altered metabolic demands of the tumor cell are reflected in altered gene expressions of a wide range of enzymes and other proteins, and these changed enzyme patterns are described. The study of oncogenes increasingly suggests that they may be significant in certain cancers, and the assignment of these genes has been tabulated. The biochemical and metabolic changes observed in tumors are complex; studying the patterns and interactions of these changes will aid our genetic understanding of the origins and development of tumors.     

Somatic mutations in mismatch repair genes in sporadic gastric carcinomas are not a cause but a consequence of the mutator phenotype

In hereditary nonpolyposis colorectal cancer (HNPCC), patients' mismatch repair (MMR) gene mutations cause MMR deficiency, leading to microsatellite instability (MSI-H). MSI-H is also found in a substantial fraction of sporadic gastric carcinomas (SGC), mainly due to MLH1 promoter hypermethylation, although somatic mutations in MMR genes have been described. We aimed to investigate which MMR defects are present in SGC. Twenty-nine MSI-H SGC investigated previously for MLH1 promoter hypermethylation were screened for somatic mutations in MLH1, MSH2, MSH6, MLH3, and MBD4 by denaturing gradient gel electrophoresis and sequencing. Five truncating mutations (three in MSH6, one in MLH3, and one in MBD4) and one missense mutation (MLH1) were identified. Of these, three truncating mutations were in MSI-H cases that lack MLH1 hypermethylation. As all truncating mutations were found in the coding poly-A tracts, it seems likely that they result from the MSI phenotype rather than cause it. In summary, somatic mutations in MMR genes are rare in SGC and do not explain the development of these tumors reflecting, rather than causing, the mutator phenotype. Other MMR genes are probably involved in MSI-H gastric cancer without MLH1 hypermethylation.