Repair Loci Research Articles

High resolution deletion mapping reveals frequent allelic losses at the DNA mismatch repair loci hMLH1 and hMSH3 in non‐small cell lung cancer

To study the involvement of DNA mismatch repair genes in non-small cell lung cancer, matched normal and tumoral DNA samples from 31 patients were analyzed for both LOH and microsatellite instability with 34 markers at or linked to hMLH1(3p21),hMSH2(2p16), hMSH3(5q11-q13),hMSH6(2p16), hPMS1(2q32), and hPMS2 (7p22) loci. Chromosomal regions 3p21 and 5q11-q13 were found to be hemizygously deleted in 55% and 42% of the patients, respectively. Sixty five percent of the patients deleted at hMLH1 were also deleted at hMSH3. The shortest regions of overlap for 3p21 and 5q11-q13 deletions delimited by D3S1561/D3S1612 and D5S2107/D5S624, respectively, were restricted to genetic distances of only 1 cM. Currently, the hMLH1 (3p21) and hMSH3 (5q11-q13) genes are the only known candidates located within these regions. The mutational analysis of hMLH1 and hMSH3 in hemizygously deleted patients led to the detection of 2 new polymorphisms in hMSH3. The consequence of these allelic losses remains unclear, but the lack of inactivating mutation might explain that replication error, the hallmark of mismatch repair genes inactivation in cancer cells, was quasi-absent in tumors. We suggest that hMLH1 and hMSH3 genes could be involved in lung tumorigenesis through dosage effect in cellular functions other than replication error correction. Int. J. Cancer 77:173–180, 1998. © 1998 Wiley-Liss, Inc.

Frequency of germline hereditary non-polyposis colorectal cancer gene mutations in patients with multiple or early onset colorectal adenomas

Background—The hereditary non-polyposis colorectal cancer (HNPCC) syndrome is caused by germline mutations in mismatch repair genes and predisposes individuals to cancers of the colon and other specific sites. On theoretical...

Molecular genetics of colon cancer.

Colorectal cancer remains a major health problem. Few therapies are effective apart from surgery, and survival has increased little in recent years. This is despite the fact that screening by colonoscopy can potentially remove nearly all colorectal tumours before they become malignant. Molecular genetics has identified some inherited mutations (such as at APC and the mismatch repair loci) that predispose to colon cancer and some somatic mutations (such as at APC and p53) that cause sporadic colon tumours. We review the likely role of these and other genes in colorectal tumorigenesis. We also highlight areas of relative ignorance in colon cancer and emphasise that many important genes, especially those that cause invasion and metastasis, remain to be identified. Colorectal cancer is, however, a well characterised tumour, as regards both its natural history and its histopathology; there are consequently good prospects for advances in colon cancer genetics, with probable benefits for its treatment. We anticipate: (a) that new genes predisposing to colon tumours, including those conferring relatively minor risks, will be characterised; (b) genes and proteins important in invasion and metastasis will be identified; (c) the network of protein interactions in which molecules such as APC are involved will be elucidated; (d) large-scale studies of somatic mutations in tumours will provide accurate predictions of prognosis and suggest optimal therapeutic regimens; and (e) new potential targets for therapy will be identified. Whilst molecular genetics is by no means sufficient for progress in preventing and treating colon cancer, it is a necessary and central part of such advances.

Allele loss occurs frequently at hMLH1, but rarely at hMSH2, in sporadic colorectal cancers with microsatellite instability.

Mutations at the hMSH2 and hMLH1 mismatch repair loci have been implicated in the pathogenesis of colorectal cancer. Tumours with two allelic mutations at a mismatch repair locus develop replication errors (RERs). In the hereditary non-polyposis colorectal cancer (HNPCC) syndrome, one mutation is inherited and the other acquired somatically: in RER+ sporadic colorectal cancers, both mutations are somatic. RER+ tumours tend to have a low frequency of allele loss, presumably because they acquire most mutations through RERs. However, before a second mismatch repair mutation has occurred somatically, there is no reason to suppose that allele loss occurs less frequently in tumours that are to become RER+. Indeed, this second mutation might itself occur by allele loss. We have searched for allele loss at the hMSH2 and hMLH1 loci in RER+ and RER- sporadic colorectal cancers. Loss occurred at the hMLH1 locus in 7/17 (41%) RER+ tumours, compared with 6/40 (15%) RER- cancers (chi2=3.82, P approximately 0.05). At hMSH2, 2/22 RER+ sporadic cancers (9%) had lost an allele, compared with 2/40 (5%) RER- cancers (chi2=0.03, P>0.5). Taken together with previous studies which focused on colorectal cancers from HNPCC families, the data suggest that allele loss at hMLH1, but not at hMSH2, contributes to defective mismatch repair in inherited and sporadic colorectal cancer.

Microsatellite instability in oral cancer

Generalized genomic instability, detected as somatic changes in allele sizes at microsatellite loci in tumors compared to peripheral lymphocyte DNA, is a recently recognized mechanism of mutation in cancer. Such instability results from the somatic loss of DNA mismatch repair capability. Germ-line mutations at DNA mismatch repair loci confer susceptibility to colon cancer in hereditary non-polyposis colorectal cancer. Somatic loss of DNA mismatch repair has been reported in a large variety of other tumor types. Our goal was to determine the frequency of microsatellite instability in a large series of oral tumors. Out of 91 tumors analyzed for microsatellite instability, 6 (7%) showed microsatellite instability. Instability was observed at multiple loci with a range of 50-74% of loci affected. Alterations include both increase (74%) and decrease (26%) in allele sizes. The proportion of alleles affected ranged from 30-58% of all alleles. Our data suggest that somatic genomic instability plays a role in the pathogenesis of a small subset of oral tumors.

X-radiation-induced chromosome breakage in retinoblastoma lymphocytes

We have examined the spontaneous and X-radiation-induced chromosomal damage in normal humans and in patients with retinoblastoma using the BudR-Giemsa technique in lymphocytes cultured for 48 h.9 sporadic unilateral non-hereditary cases, 11 hereditary cases (8 bilateral sporadic and 3 unilateral hereditary cases) and 20 healthy individuals were studied simultaneously. No difference in the spontaneous frequency of chromatid and chromosome aberrations was observed between patients and controls. After treatment with 150 rad the frequency of chromosome exchange aberrations was higher in unilateral hereditary cases than the controls (42.0% ± 5.3 and 22.3 ± 2.6 respectively; p = 0.05). In bilateral sporadic retinoblastoma 2 different groups were observed. A hypersensitive group showed a significant increment in radiation-induced chromosomal exchange aberrations over the control group (46.2% ± 5.4 and 24.2% ± 2.1 respectively; p = 0.01). The other group had a chromosomal exchange frequency similar individuals (26/5% ± 2.0 and 24.2% ± 0.4 respectively; p = 0.10). Sporadic unilateral non-hereditary retinoblastoma had an exchange chromosomal aberration frequency similar to control individuals (26.1% ± 2.8 and 24.6% ± 2.7 respectively; p > 0.10). These results suggest that: 1. (a) There is no relationship between spontaneous chromosome fragility and retinoblastoma. 2. (b) Sporadi unilateral non-hereditary retinoblastoma has normal chromosome sensitivity to X-irradiation. 3. (c) Some hereditary cases of retinoblastoma are sensitive to X-rays while other behave like normals. A mutation or a submicroscopic deletion at a DNA repair locus which is independent of the retinoblastoma gene may cause this radiosensitivity.

The relationship between reaction kinetics and mutagenic action of monofunctional alkylating agents in higher eukaryotic systems: IV. The effects of the excision-defective mei-9L1 and mus(2)201D1 mutants on alkylation-induced genetic damage in Drosophila

Repair-defective mutants of Drosophila melanogaster which identify two major DNA excision repair loci have been examined for their effects on alkylation-induced mutagenesis using the sex-linked recessive lethal assay as a measure of genotoxic endpoint. The alkylating agents (AAs) chosen for comparative analysis were selected on the basis of their reaction kinetics with DNA and included MMS, EMS, MNU, DMN, ENU, DEN and ENNG. Repair-proficient males were treated with the AAs and mated with either excision-defective mei-9 L1 or mus(2)201 D1 females or appropriate excision-proficient control females. The results of the present work suggest that a qualitative and quantitative relationship exists between the nature and the extent of chemical modification of DNA and the induction of genetic alterations. The presence of either excision-defective mutant can enhance the frequency of mutation (hypermutability) and this hypermutability can be correlated with the Swain-Scott constant S of specific AAs such that as the S N1 character of the DNA alkylation reaction increases, the difference in response between repair-deficient and repair-proficient females decreases. The order of hypermutability of AAs with relative to mei-9 L1 is MMS > MNU > DMN = EMS > iPMS = ENU = DEN = ENNG. When the percentage of lethal mutations induced in mei-9 L1 females are plotted against those determined for control females, straight lines of different slopes are obtained. These mei-9 L1/ mei-9 + indices are: MMS = 7.6, MNU = 5.4, DMN = 2.4, EMS = 2.4 and iPMS = ENU = DEN = ENNG = 1. An identical order of hypermutability with similar indices is obtained for the mus(2)201 mutants: MMS(7.3) > MNU(5.4) > EMS(2.0) > ENU(1.1). Thus, absence of excision repair function has a significant effect on mutation production by AAs efficient in alkylating N-atoms in DNA but no measurable influence on mutation production by AAs most efficient in alkylating O-atoms in DNA. The possible nature of these DNA adducts has been discussed in relation to repair of alkylated DNA. In another series of experiments, the effect of alkylation mutagenesis of was studied in males, by comparing mutation induction in males vs. activity in Berlin K (control). Although these experiments suggested the existence of DNA repair in postmeiotic cells during spermatogenesis, no quantitative comparisons could be made. The inherent problem with this approach, mutagen treatment of two different genotypes, is that factors entirely unrelated to DNA repair, e.g., differences in metabolism, may influence the outcome of the comparison. Utilization of the mutant in low dosage experiments did out measurably improve the sensitivity of the assay for sex-linked recessive lethals. Because the mei-9 L1 allele has not been characterized biochemically, we cannot determine at present whether excision deficiency, per se, will not lead to notable shifts in lowest effective concentrations (LECs) or whether the mei-9 L1 allele is ‘leaky’ and providing some wild-type function at low mutagen doses. Nevertheless, ‘high-exposure-level’ experiments with strong reference mutagens may not be indicative of the situation relevant at low genotoxic activities and this aspect should be given special attention in considerations dealing with the possible use of the specific repair-defective alleles in genetic toxicology testing.

Radiosensitivity of fibroblasts from patients with retinoblastoma and chromosome-13 anomalies

Diploid fibroblast cell strains derived from 14 patients with various forms of retinoblastoma (RB) and 5 non-RB patients with constitutional chromosome anomalies involving chromosome 13 were assayed for their clonogenic survival after X-irradiation. Cells from a patient with ataxia telangiectasia (AT) was used as a radiosensitive reference strain. When compared with cell strains from 7 healthy persons as normal controls, a marked radiosensitivity was observed in a strain from an AT patient. However, none of the cell strains derived from RB patients or patients with inborn anomalies in chromosome 13 showed pronounced deviation from the normal range of radiosensitivity. The findings thus did not warrant either the RB as radiosensitive genetic disease or the presence of repair locus on chromosome 13, deletion or triplication of which was previously suggested to link to radiosensitivity.