A newly established GDL1 cell line from gpt delta mice well reflects the in vivo mutation spectra induced by mitomycin C

In order to create a novel in vitro test system for detection of large deletions and point mutations, we developed an immortalized cell line. A SV40 large T antigen expression unit was introduced into fibroblasts derived from gpt delta mouse lung tissue and a selected clone was established as the gpt delta L1 (GDL1) cell line. The novel GDL1 cells were examined for mutant frequencies (MFs) and for molecular characterization of mutations induced by mitomycin C (MMC). The GDL1 cells were treated with MMC at doses of 0.025, 0.05, and 0.1 microg/mL for 24h and mutations were detected by Spi- and 6-thioguanine (6-TG) selections. The MFs of the MMC-treated cells increased up to 3.4-fold with Spi- selection and 3.5-fold with 6-TG selection compared to MFs of untreated cells. In the Spi- mutants, the number of large (up to 76 kilo base pair (kbp)) deletion mutations increased. A majority of the large deletion mutations had 1-4 base pairs (bp) of microhomology in the deletion junctions. A number of the rearranged deletion mutations were accompanied with deletions and insertions of up to 1.1 kbp. In the gpt mutants obtained from 6-TG selection, single base substitutions of G:C to T:A, tandem base substitutions occurring at the 5'-GG-3' or 5'-CG-3' sequence, and deletion mutations larger than 2 bp were increased. We compared the spectrum of MMC-induced mutations observed in vitro to that of in vivo using gpt delta mice, which we reported previously. Although a slight difference was observed in MMC-induced mutation spectra between in vitro and in vivo, the mutations detected in vitro included all of the types of mutations observed in vivo. The present study demonstrates that the newly established GDL1 cell line is a useful tool to detect and analyze various mutations including large deletions in mammalian cells.     

Aging and high concentrations of glucose potentiate injury to mitochondrial DNA

Deletions of mitochondrial DNA (mtDNA) are associated with aging and several chronic diseases. We have reported heterogeneous mutations between base pair 8468 and 13446 in mtDNA, the region known as the "common" deletion, in muscle of older humans with impaired glucose tolerance or diabetes mellitus. To further characterize potential effects of age and glycemia on mtDNA integrity, we studied corpulent JCR:LA-cp rats that are characterized by insulin resistance, hyperinsulinemia, and hyperlipidemia, factors strongly associated with both aging and cardiovascular disease. In addition to skeletal muscle, we isolated vascular smooth muscle cells (VSMC) from aortas of 6-, 12-, and 17-month-old rats and exposed them to 5-, 25-, 62-, and 100-mM glucose or a combination of hypoxanthine (100 microM) and xanthine oxidase (0.025 U/ml) to generate reactive oxygen species in separate cultures. Long- and short-fragment and nested polymerase chain reaction was used to detect mutations in the common deletion region. The data demonstrate that aging and the cp genotype confer susceptibility to mtDNA deletions in vivo and that high glucose concentrations can induce mtDNA mutations in vitro. Accordingly, aging and glucose-related oxidative stress and possibly hyperinsulinemia may contribute to alterations in mitochondrial gene integrity and the cp genotype appears to increase the susceptibility of muscle to the age-related accumulation of mtDNA mutations.     

The rates and patterns of deletions in the human factor IX gene.

Deletions are commonly observed in genes with either segments of highly homologous sequences or excessive gene length. However, in the factor IX gene and in most genes, deletions (of > or = 21 bp) are uncommon. We have analyzed DNA from 290 families with hemophilia B (203 independent mutations) and have found 12 deletions > 20 bp. Eleven of these are > 2 kb (range > 3-163 kb), and one is 1.1 kb. The junctions of the four deletions that are completely contained within the factor IX gene have been determined. A novel mutation occurred in patient HB128: the data suggest that a 26.8-kb deletion occurred between two segments of alternating purines and pyrimidines and that a 2.3-kb sense strand segment derived from the deleted region was inserted. For our sample of 203 independent mutations, we estimate the "baseline" rates of deletional mutation per base pair per generation as a function of size. The rate for large (> 2 kb) deletions is exceedingly low. For every mutational event in which a given base is at the junction of a large deletion, there are an estimated 58 microdeletions (< 20 bp) and 985 single-base substitutions at that base. Analysis of the nine reported deletion junctions in the factor IX gene literature reveals that (i) five are associated with inversions, orphan sequences, or sense strand insertions; (ii) four are simple deletions that display an excess of short direct repeats at their junctions; (iii) there is no dramatic clustering of junctions within the gene; and (iv) with the exception of alternating purines and pyrimidines, deletion junctions are not preferentially associated with repetitive DNA.     

DHPLC analysis of adenomatous polyposis coli (APC) mutations using ready-to-use APC plates: simple detection of multiple base pair deletion mutations

The adenomatous polyposis coli (APC), which is the susceptible gene for familial adenomatous polyposis (FAP) and sporadic colorectal cancer, spans 15 exons. The open reading frame of APC is 8529 bp, which encodes 2843 amino acids. Conventional genetic screening involves extensive time as well as high cost and labor. Thus, we developed a novel APC ready-to-use plate for high-throughput mutational analysis by denaturing high performance liquid chromatography (DHPLC). To prepare the ready-to-use APC plate, all 38 primer pairs and PCR mixtures were aliquoted into individual wells of a 96-well plate, and frozen at -20 degrees C until use. All 38 PCR primers were designed to be amplified at the same temperature (52 degrees C). We examined a total of 27 FAP patient samples with APC germline mutations (17 for multiple bp deletions, 1 for 1 bp deletion, 9 for nonsense mutations) and 50 APC-negative noncarriers. All 17 multiple bp deletion mutations were detected during the initial 50 degrees C running analysis and thus ruled out for further analyses. All other mutations were clearly detected under specific optimized conditions. More than 50% of the APC germline mutations were multiple base pair deletions and efficiently selected by omitting time-consuming partial denaturing conditions.     

Molecular characterisation of camptothecin-induced mutations at the hprt locus in Chinese hamster cells

The capacity of the topoisomerase I inhibitor camptothecin (CPT) to induce single locus mutations at the hypoxanthine-guanine phosphoribosyltransferase (hprt) gene and the DNA changes underlying induced mutations were analysed in Chinese hamster ovary cells. Camptothecin treatments increased hprt mutations up to 50-fold over the spontaneous levels at highly cytotoxic doses. Genomic DNA was isolated from 6-thioguanine resistant clones and subjected to multiplex PCR to screen for gross alterations in the gene structure. The molecular analysis revealed that deletion mutants represented 80% of the analysed clones, including total hprt deletion, multiple and single exon deletions. Furthermore, a fraction of the analysed clones showed deletions of more than one exon that were characterised by the absence of non-contiguous exons. These data show that single locus mutations induced by camptothecin are characterised by large deletions or complex rearrangements rather than single base substitutions and suggest that the recombinational repair of camptothecin-induced strand breaks at replication fork may be involved in the generations of these alterations at the chromatin structure level.     

Use of data from bacteria to interpret data on DNA damage processing in mammalian cells

The most important reason for determining the changes in base sequence in the processing of DNA damage is to determine mechanisms. Currently, much more is known about these mechanisms in prokaryotes, partly because the experiments are easier and quicker to do in bacteria, and partly because of the wealth of well characterized bacterial mutants deficient in various DNA repair pathways. This paper summarizes some information on the mechanisms in bacteria that are involved in the induction by various agents of base change mutations, 1- and 2-base deletions or additions that cause frameshifts, and more complicated insertions and deletions that involve up to tens of base pairs. For gross DNA rearrangements such as large deletions involving hundreds or thousands of base pairs, there is actually more information available in mammalian cells than in bacterial cells. It is suggested that deletions of several kilobases or more in bacteria are not easy to detect because they have a high probability of deleting both the gene under study and an adjacent essential gene, forming a nonviable cell. In mammalian cells, the large size (30-40-kb pairs) of the average gene, including both introns and exons, means that a large deletion is more likely to be confined to a single gene and less likely to lead to a nonviable cell.     

Copy number variations due to large genomic deletion in X-linked chronic granulomatous disease

Mutations in genes for any of the six subunits of NADPH oxidase cause chronic granulomatous disease (CGD), but almost 2/3 of CGD cases are caused by mutations in the X-linked CYBB gene, also known as NAD (P) H oxidase 2. Approximately 260 patients with CGD have been reported in Japan, of whom 92 were shown to have mutations of the CYBB gene and 16 to have chromosomal deletions. However, there has been very little detailed analysis of the range of the deletion or close understanding of the disease based on this. We therefore analyzed genomic rearrangements in X-linked CGD using array comparative genomic hybridization analysis, revealing the extent and the types of the deletion genes. The subjects were five Japanese X-linked CGD patients estimated to have large base deletions of 1 kb or more in the CYBB gene (four male patients, one female patient) and the mothers of four of those patients. The five Japanese patients were found to range from a patient exhibiting deletions only of the CYBB gene to a female patient exhibiting an extensive DNA deletion and the DMD and CGD phenotype manifested. Of the other three patients, two exhibited CYBB, XK, and DYNLT3 gene deletions. The remaining patient exhibited both a deletion encompassing DNA subsequent to the CYBB region following intron 2 and the DYNLT3 gene and a complex copy number variation involving the insertion of an inverted duplication of a region from the centromere side of DYNLT3 into the deleted region.     

The molecular basis of X-linked spondyloepiphyseal dysplasia tarda

The X-linked form of spondyloepiphyseal dysplasia tarda (SEDL), a radiologically distinct skeletal dysplasia affecting the vertebrae and epiphyses, is caused by mutations in the SEDL gene. To characterize the molecular basis for SEDL, we have identified the spectrum of SEDL mutations in 30 of 36 unrelated cases of X-linked SEDL ascertained from different ethnic populations. Twenty-one different disease-associated mutations now have been identified throughout the SEDL gene. These include nonsense mutations in exons 4 and 5, missense mutations in exons 4 and 6, small (2-7 bp) and large (>1 kb) deletions, insertions, and putative splicing errors, with one splicing error due to a complex deletion/insertion mutation. Eight different frameshift mutations lead to a premature termination of translation and account for >43% (13/30) of SEDL cases, with half of these (7/13) being due to dinucleotide deletions. Altogether, deletions account for 57% (17/30) of all known SEDL mutations. Four recurrent mutations (IVS3+5G-->A, 157-158delAT, 191-192delTG, and 271-275delCAAGA) account for 43% (13/30) of confirmed SEDL cases. The results of haplotype analyses and the diverse ethnic origins of patients support recurrent mutations. Two patients with large deletions of SEDL exons were found, one with childhood onset of painful complications, the other relatively free of additional symptoms. However, we could not establish a clear genotype/phenotype correlation and therefore conclude that the complete unaltered SEDL-gene product is essential for normal bone growth. Molecular diagnosis can now be offered for presymptomatic testing of this disorder. Appropriate lifestyle decisions and, eventually, perhaps, specific SEDL therapies may ameliorate the prognosis of premature osteoarthritis and the need for hip arthroplasty.     

Partial aldolase B gene deletions in hereditary fructose intolerance.

Hereditary fructose intolerance (HFI) is an autosomal recessive condition caused by a deficiency of aldolase B. We have recently shown that three point mutations in this gene account for approximately 85% of HFI alleles in Europe and the United States and are thus of diagnostic importance. In this paper we define three new lesions in the aldolase B gene: two are large deletions, one of 1.65 kb and one of 1.4 kb; the third is a small deletion of 4 bp. We have determined the breakpoints of these deletions and have demonstrated that the presence of such lesions may complicate the genotyping of individuals for diagnosis of HFI.     

Simultaneous discovery and testing of deletions for disease association in SNP genotyping studies

Copy-number variation (CNV), and deletions in particular, can play a crucial, causative role in rare disorders. The extent to which CNV contributes to common, complex disease etiology, however, is largely unknown. Current techniques to detect CNV are relatively expensive and time consuming, making it difficult to conduct the necessary large-scale genetic studies. SNP genotyping technologies, on the other hand, are relatively cheap, thereby facilitating large study designs. We have developed a computational tool capable of harnessing the information in SNP genotype data to detect deletions. Our approach not only detects deletions with high power but also returns accurate estimates of both the population frequency and the transmission frequency. This tool, therefore, lends itself to the discovery of deletions in large familial SNP genotype data sets and to simultaneous testing of the discovered deletion for association, with the use of both frequency-based and transmission/disequilibrium test-based designs. We demonstrate the effectiveness of our computer program (microdel), available for download at no cost, with both simulated and real data. Here, we report 693 deletions in the HapMap 16c collection, with each deletion assigned a population frequency.     

Deletions in CCM2 are a common cause of cerebral cavernous malformations

Cerebral cavernous malformations (CCMs) are vascular abnormalities of the brain that can result in a variety of neurological disabilities, including hemorrhagic stroke and seizures. Mutations in the gene KRIT1 are responsible for CCM1, mutations in the gene MGC4607 are responsible for CCM2, and mutations in the gene PDCD10 are responsible for CCM3. DNA sequence analysis of the known CCM genes in a cohort of 63 CCM-affected families showed that a high proportion (40%) of these lacked any identifiable mutation. We used multiplex ligation-dependent probe analysis to screen 25 CCM1, -2, and -3 mutation-negative probands for potential deletions or duplications within all three CCM genes. We identified a total of 15 deletions: 1 in the CCM1 gene, 0 in the CCM3 gene, and 14 in the CCM2 gene. In our cohort, mutation screening that included sequence and deletion analyses gave disease-gene frequencies of 40% for CCM1, 38% for CCM2, 6% for CCM3, and 16% with no mutation detected. These data indicate that the prevalence of CCM2 is much higher than previously predicted, nearly equal to CCM1, and that large genomic deletions in the CCM2 gene represent a major component of this disease. A common 77.6-kb deletion spanning CCM2 exons 2-10 was identified, which is present in 13% of our entire CCM cohort. Eight probands exhibit an apparently identical recombination event in the CCM2 gene, involving an AluSx in intron 1 and an AluSg distal to exon 10. Haplotype analysis revealed that this CCM2 deletion occurred independently at least twice in our families. We hypothesize that these deletions occur in a hypermutable region because of surrounding repetitive sequence elements that may catalyze the formation of intragenic deletions.     

Involvement of RecB-mediated (but not RecF-mediated) repair of DNA double-strand breaks in the gamma-radiation production of long deletions in Escherichia coli.

Experiments were designed to determine the association between the repair of gamma-radiation-induced DNA double-strand breaks (DSB) and the induction of 700-1000 bp long deletions (Lac(-)----Lac+), base substitutions (leuB19----Leu+), and frameshifts (trpE9777----Trp+) in Escherichia coli K-12. Over the range of 2.5-20 krad, deletions were induced with linear kinetics, as has been shown for the induction of DSB, while the induction kinetics of base substitutions and frameshifts were curvilinear. Like the repair of DSB, deletion induction showed an absolute requirement for an intact recB gene as well as a dependency on the type of preirradiation growth medium; these requirements were not seen for base substitutions or frameshifts. In addition, about 80% of the spontaneous deletions were absent in the recB21 strain. A recC1001 mutation, which confers a 'hyper-Rec' phenotype, increased the rate of gamma-radiation-induced deletions as well as the low-dose production of base substitutions and frameshifts. A recF143 mutation increased the yield of gamma-radiation-induced deletions without increasing base substitutions or frameshifts. A mutS mutation markedly enhanced the gamma-radiation induction of frameshifts, and had a slight effect on base substitutions, but did not affect the induction of deletions. Resistance to gamma-irradiation and the capacity to repair DSB (albeit at about half the normal rate) were restored to the radiosensitive recB21 strain by the addition of the sbcB21 and sbcC201 mutations. However, the radioresistant recB sbcBC strain, which is recombination proficient via the RecF pathway, was still grossly deficient in the ability to produce deletions. A model for deletion induction as a by-product of the recB-dependent (Chi-dependent) repair of gamma-radiation-induced DSB is discussed, as is the inability to detect deletions in cells that use only the recF-dependent (Chi-independent) mechanism to repair DSB.     

Analysis of TGF-beta type I receptor for mutations and polymorphisms in head and neck cancers

Transforming growth factor-beta receptor (TbetaR)-dependent signals are critical for cell growth and differentiation and are often disrupted during tumorigenesis. The entire coding region of TbetaR-I and flanking intron sequences from 30 head and neck carcinomas were examined for alterations using "Cold" SSCP and direct sequencing. No somatic point mutations were found in the TbetaR-I gene. In contrast, 14 polymorphic sequence changes were detected in TbetaR-I in 13 (43%) of the samples, including eight (27%) nucleotide alterations identified as polymorphisms in an exon-1 (GCG)(9) microsatellite repeat, a previously reported tumor susceptibility allele. A nine base pair deletion was found in 23% of the samples including five heterozygous and two homozygous deletions as well as single homozygous 12bp deletion. Additionally, six heterozygous polymorphisms in intronic sequences were determined, including one heterozygous C/A genotype at the +82 nucleotide position of the intron-5 intervening sequence (IVS), and five heterozygous G/A genotypes within intron-7 at the +24 nucleotide position. Exon-1 polymorphisms in the (GCG)(9) microsatellite region of the TbetaR-I gene and their association with head/neck cancers, suggest that development of these cancers may be a direct consequence of loss of responsiveness to TGF-beta mediated growth inhibition.     

Rapid detection of single nucleotide deletions: application to the β 6 (-A) mutation of the β-globin gene and to cystic fibrosis

The formation of heteroduplexes from the amplified products of homologous alleles has been shown to be useful in the identification of heterozygotes carrying deletion or insertion mutations. Here, we describe an improved procedure that allows the detection of single base pair (bp) deletions on nondenaturing polyacrylamide gels. Carriers for a common Mediterranean -thalassemic mutation, 6 (-A), could be easily detected by use of this method, as could carriers of a 1-bp deletion in the cystic fibrosis gene.     

Genome-wide mutagenesis resulting from topoisomerase 1-processing of unrepaired ribonucleotides in DNA

Ribonucleotides are the most common non-canonical nucleotides incorporated into DNA during replication, and their processing leads to mutations and genome instability. Yeast mutation reporter systems demonstrate that 2–5 base pair deletions (Δ2–5bp) in repetitive DNA are a signature of unrepaired ribonucleotides, and that these events are initiated by topoisomerase 1 (Top1) cleavage. However, a detailed understanding of the frequency and locations of ribonucleotide-dependent mutational events across the genome has been lacking. Here we present the results of genome-wide mutational analysis of yeast strains deficient in Ribonucleotide Excision Repair (RER). We identified mutations that accumulated over thousands of generations in strains expressing either wild-type or variant replicase alleles (M644G Pol ε, L612M Pol δ, L868M Pol α) that confer increased ribonucleotide incorporation into DNA. Using a custom-designed mutation-calling pipeline called muver (for mutationes verificatae), we observe a number of surprising mutagenic features. This includes a 24-fold preferential elevation of AG and AC relative to AT dinucleotide deletions in the absence of RER, suggesting specificity for Top1-initiated deletion mutagenesis. Moreover, deletion rates in di- and trinucleotide repeat tracts increase exponentially with tract length. Consistent with biochemical and reporter gene mutational analysis, these deletions are no longer observed upon deletion of TOP1. Taken together, results from these analyses demonstrate the global impact of genomic ribonucleotide processing by Top1 on genome integrity.     

Induction of deletion and insertion mutations by 9-aminoacridine. An in vitro model

The ability of 9-aminoacridine to induce mutagenic lesions during DNA replication in vitro was investigated. The ampicillinase gene of pBR322 was replicated in vitro in the presence of 9-aminoacridine. Transfection of the replicated DNA into Escherichia coli gave Amps mutants. Determination of the base changes in 76 of these mutants indicated that the spectrum of mutations induced by 9-aminoacridine was consistent with its action in vivo. Both large (407-base) and small (1- and 2-base) deletions were induced at repetitive sequences. The frequency of deletion mutations depended on the identity of the base deleted and sequences surrounding the deletions. The characteristics of the frameshift mutations induced were consistent with the interactions of 9-aminoacridine with DNA. These results establish that 9-aminoacridine can induce frameshift mutations during the replication process and provide an in vitro model of frameshift induction for mechanistic studies.     

HIV-1: a case of RT67 deletion in a multi-treated non responder patient

The early detection of mutations in the HIV-1 polymerase is a key point in the management of anti-retroviral therapy. While nucleotide substitutions and insertions have been well and frequently desribed in literature as linked to drug resistance, deletions have been rarely observed and desribed (ART67, Imamichi et al.). The aim of this study is to describe a case of deletion of three nucleotides in the RT gene (ART67) of a multi-treated HIV-1 infected patient. As this deletion has not been detected by the oligoprobe assay, the phenotyping test was used to support therapy but without an appreciable success in terms of viral load. Then a sequencing based genotyping system was used to analyse the viral polymerase and a novel deletion was found at codon 67 of RT gene.     

A 3′ splice site consensus sequence mutation in the cystic fibrosis gene

Summary In the cystic fibrosis (CF) gene, recently cloned, a three base pair deletion (ΔF508) has been identified in a majority of CF patients. This deletion has been found in 80% of CF chromosomes in families from north west Brittany. In order to identify new mutations we have selected 43 chromosomes negative for the three base pair deletion from these families and directly sequenced exon 11 after DNA amplification by the polymerase chain reaction. We have detected a base change (G→A) at the 3′ end of the consensus sequence of intron ten (namely 1717-1). This mutation destroys a splice site in the cystic fibrosis gene which probably produces a mutant allele. This single nucleotide mutation has been reported on two other CF chromosomes.     

New mutations in the ataxia telangiectasia gene

We report the detection of four new mutations in the ataxia telangiectasia gene (ATM). Reverse-transcribed RNA extracted from cultured cells was analysed for mutations by polymerase chain reaction amplifications and restriction endonuclease fingerprinting. Three deletions and a base substitution are described. The deletions reported here would result in severe disruptions of the ATM gene product by leading either to a protein truncation (a 4-bp deletion) or the loss of stretches of 53 and 58 amino acids (a 159-bp deletion and a 174-bp deletion, respectively); whereas the base substitution would lead to an amino acid change from a highly conserved glycine to an arginine residue. Received: 15 April 1996 / Revised: 24 April 1996     

Characterization of carbadox-induced mutagenesis using a shuttle vector pSP189 in mammalian cells

Carbadox, a quinoxaline 1,4-dioxide derivative, is a known mutagen with its functional mechanism yet to be well defined. In the present study we used a shuttle vector assay in vitro to uncover the functional details of carbadox-induced mutagenesis in mammalian cells. The plasmid DNA of a shuttle vector pSP189 was treated with different doses of carbadox at 37 degrees C for 1 or 2h with or without the presence of S9. The target gene SupF in the plasmid was sequenced after replication in Vero cells followed by amplification in Escherichia coli MBM7070 to evaluate mutation frequency. DNA sequencing analysis of recovered carbadox-induced mutations revealed 76.3% single base substitution, 7.9% single base insertion, 10.5% single base deletion and 5.3% large fragments deletion. All single base substitutions occurred at G:C base pairs, among which transversion and transition occurred at a 2:1 ratio. The mutations did not occur randomly in the supF gene, but had sequence specificity and hotspots instead: most substitutions were detected at the nucleotide N in a 5'-NNTTNN-3' sequence; 75% of base insertions were seen in the 5'-TCC-3' sequence; whereas all large fragments deletions occurred in the 5'-ANGGCCNAAA-3' sequence. Nucleotide 129, 141 and 155 in the supF gene of plasmid pSP189 were identified as the hotspots for carbadox-induced mutations that accounted for 65% of all single base substitutions. We conclude that carbadox and its metabolites induce sequence-specific DNA mutations at high frequencies, therefore its safe usage in animal husbandry should be seriously considered.