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PubMed2000

Update of genetics in colorectal carcinomas: genomic instability and somatic evolution.

INTRODUCTION: Currently, there are two contrasting viewpoints on what drives the process of carcinogenesis. The genomic (DNA or chromosomal) instability model contends that an increased mutation rate early in carcinogenesis is necessary for the multistage process, while the somatic evolution model postulates that normal mutation rate with selective advantage and clonal expansion is sufficient to cause cancer. METHODS: Evidence from colorectal carcinoma (CRC) for and against the two models are compared and contrasted. RESULTS: With the exception of hereditary non-polyposis colorectal carcinoma (HNPCC) where DNA instability attributable to mismatch repair deficiency is clearly demonstrated, the majority of CRC appear to progress through the selection of a series of mutations without the need of first acquiring a mutator phenotype. Aneuploidy or chromosomal instability is more likely to be a consequence of non-random selection of mutations in genes residing on the chromosome rather than the direct cause of cancer. Nevertheless, aneuploidy and/or DNA alterations can lead to secondary instability, hence, contributing to the phenotypes associated with carcinoma. CONCLUSIONS: Present knowledge, thus, points to multiple, mutually non-exclusive pathways for different cancer populations, further emphasising tumour heterogeneity.

Adenomatous Polyposis Coli↗
PubMed1995

Genomic instability of microsatellite repeats in prostate cancer: relationship to clinicopathological variables.

Sixty-six patients with prostatic adenocarcinoma were screened for somatic instability at 8 microsatellite marker loci on 5 chromosomes. Differences in unrelated microsatellites for tumor and normal DNA were detected in 13 (19.7%) patients. Only extraglandular spread (nodal involvement and distant metastasis) was found to show significant association with somatic instability after controlling for other clinicopathological variables (P < 0.05). Microsatellite instability may possibly occur during the early stages of neoplastic transformation in a subset of prostate cancer rather than as a late event. This may be related to a phenotype with growth advantage. The frequency of this mutator phenotype is much higher in the United States than Japan, reflecting racial differences in the molecular tumorigenesis of this malignancy.

Adenocarcinoma↗
PubMed1996

Instability of the expanded (CTG)n repeats in the myotonin protein kinase gene in cultured lymphoblastoid cell lines from patients with myotonic dystrophy.

The mutation associated with myotonic dystrophy (DM) is the expansion of an unstable trinucleotide repeat, (CTG)n, in the 3'-untranslated region of the myotonin protein kinase gene. Although expanded repeats show both germline and somatic instability, the mechanisms of the instability are poorly understood. To establish a model system in which somatic instability of the DM repeat could be studied in more detail, we established lymphoblastoid cell lines (LBCL) from DM patients. Analysis of the DNA from DM LBCL using Southern blotting showed that the (CTG)n repeats were apparently stable up to 29 passages in culture. To study infrequent repeat size mutations that are undetectable due to the size heterogeneity, we established LBCL of single-cell origins by cloning using multiple steps of limiting dilution. After expansion to approximately 10(6) cells (equivalent to approximately 20 cell cycles), the DNAs of these cell lines were analyzed by the small pool PCR technique using primers flanking the (CTG)n repeat region. Two types of mutations of the expanded (CTG)n repeat alleles were detected: (1) frequent mutations that show small changes of the (CTG)n repeat size, resulting in alleles in a normal distribution around the progenitor allele, and (2) relatively rare mutations with large changes of the (CTG)n repeat size, with a bias toward contraction. The former may represent the mechanism responsible for the somatic heterogeneity of the (CTG)n repeat size observed in blood cells of DM patients. This in vitro experimental system will be useful for further studies on mechanisms involved in the regulation of the somatic stability of the (CTG)n repeats in DM.

Alleles↗
PubMed2003

Regional differences of somatic CAG repeat instability do not account for selective neuronal vulnerability in a knock-in mouse model of SCA1.

Expression of unstable translated CAG repeats is the mutational mechanism in nine different neurodegenerative disorders. Although the products of genes harboring these repeats are widely expressed, a subset of neurons is vulnerable in each disease accounting for the different phenotypes. Somatic instability of the expanded CAG repeat has been implicated as a factor mediating the selective striatal neurodegeneration in Huntington disease. It remains unknown, however, whether such a mechanism contributes to the selective neurodegeneration in other polyglutamine diseases or not. To address this question, we investigated the pattern of CAG repeat instability in a knock-in mouse model of spinocerebellar ataxia type 1 (SCA1). Small pool PCR analysis on DNA from various neuronal and non-neuronal tissues revealed that somatic repeat instability was most remarkable in the striatum. In the two vulnerable tissues, cerebellum and spinal cord, there were substantial differences in the profiles of mosaicism. These results suggest that in SCA1 there is no clear causal relationship between the degree of somatic instability and selective neuronal vulnerability. The finding that somatic instability is most pronounced in the striatum of various knock-in models of polyglutamine diseases highlights the role of trans-acting tissue- or cell-specific factors in mediating the instability.

Animals↗
PubMed1994

Genomic instability of microsatellite repeats and its association with the evolution of chronic myelogenous leukemia.

Tumorigenesis has been shown to proceed through a series of genetic alterations involving protooncogenes and tumor-suppressor genes. Investigation of genomic instability of microsatellites has indicated a new mechanism for human carcinogenesis in hereditary nonpolyposis colorectal cancer and sporadic cancer and this instability has been shown to be related to inherited predisposition to cancer. This study was conducted to determine whether such microsatellite instability is associated with the evolution of chronic myelogenous leukemia (CML) to the blast crisis. Nineteen CML patients clinically progressing from the chronic phase to accelerated phase or blast crisis and 20 other patients in the CML chronic phase were studied. By polymerase chain reaction assay, DNAs for genomic instability in five separate microsatellites in chromosome arms 5q (Mfd27), 17p (Mfd41), 18q (DCC), 3p (CI3-9), and 8p (LPL) were examined. Differences in unrelated microsatellites of chronic and blastic phase DNAs in 14 of 19 patients (73.7%) were demonstrated. Somatic instability in five microsatellites, Mfd27, Mfd41, DCC, CI3-9, and LPL, was detected in 2 of 19 (10.5%), 8 of 19 (42.1%), 11 of 19 (57.9%), 4 of 17 (23.5%), and 4 of 17 (23.5%) cases. In 10 of 19 cases (52.6%), genetic instability in at least two of five microsatellites was observed and was categorized as replication error (RER+) phenotype. CML evolution cases with myeloid, lymphoid, and mixed phenotypes and the blast crisis and accelerated phase showed somatic instability in a number of microsatellites. No alterations in leukemic cells at the chronic phase could be detected in any microsatellites. These data indicate instability of microsatellites (RER+) but not familial predisposition to possibly be a late genetic event in the evolution of CML to blast crisis. In the microsatellite of the DCC gene, complicated alterations in band patterns caused by instability as well as loss of heterozygosity (LOH) were observed in 13 of 19 cases (68.4%): instability in 9 cases, instability plus LOH in 2 cases, and only LOH in 2 cases. These highly frequent alterations in microsatellites, including instability and LOH, suggesting that secondary events due possibly to loss of fidelity in replication and repair machinery may be significantly associated with CML evolution.

Adult↗
PubMed2002

Instability of a premutation allele in homozygous patients with myotonic dystrophy type 1.

Myotonic dystrophy type 1 (DM1) is caused by the expansion of an unstable CTG repeat in the DMPK gene on chromosome 19q13.3. We present two siblings with DM1 who each inherited a premutation allele, (CTG)43, stably transmitted from the mother and a full-mutation allele, either (CTG)500 or (CTG)180, derived from a paternal protomutation allele, (CTG)52. Small-pool polymerase chain reaction analysis showed that the (CTG)52 repeat allele was relatively stable in somatic tissues but was highly unstable in the male germline and extremely biased toward further expansion, consistent with the high levels of anticipation observed in DM1 families. The (CTG)43 allele showed subtle somatic instability in the mother, with maximum additions of two repeats and deletions of one repeat. Conversely, in the younger affected siblings the (CTG)43 allele showed a high degree of somatic instability (approximately 70% mutation load), resulting in deletions reverting to the high end of the normal range (down to [CTG]33) and additions up to the proto-mutation range (up to [CTG]64). The difference in the somatic stability of the (CTG)43 allele between the mother and her offspring suggests that interallelic interactions or other mechanisms in trans regulate the stability of the (CTG)43 premutation allele.

Adult↗
PubMed1998

Progression of somatic CTG repeat length heterogeneity in the blood cells of myotonic dystrophy patients.

The genetic basis of myotonic dystrophy (DM) is the expansion of an unstable CTG repeat in the 34 UTR of the DM protein kinase gene on chromosome 19. One of the principal features of the DM mutation is an extraordinarily high level of somatic mosaicism, due to an extremely high degree of somatic instability both within and between different tissues. This instability appears to be biased towards further expansion and continuous throughout the life of an individual, features that could be associated with the progressive nature of the disease. Although increasing measured allele size between patients clearly correlates with an increased severity of symptoms and an earlier age of onset, this correlation is not precise and measured allele length cannot be used as an accurate predictor of age of onset. In order to further characterize the dynamics of DM CTG repeat somatic instability, we have studied repeat length changes over time in 111 myotonic dystrophy patients with varying clinical severity and CTG repeat size over time intervals of 1-7 years. We have found a direct progression of the size heterogeneity over time related to initial CTG repeat size and the time interval and always biased towards further expansion. Attempts to mathematically model the dynamics have proved only partially successful suggesting that individual specific genetic and/or environmental factors also play a role in somatic mosaicism.

Adult↗
PubMed1993

Myotonic dystrophy: size- and sex-dependent dynamics of CTG meiotic instability, and somatic mosaicism.

Myotonic dystrophy (DM) is a progressive neuromuscular disorder which results from elongations of an unstable (CTG)n repeat, located in the 3' untranslated region of the DM gene. A correlation has been demonstrated between the increase in the repeat number of this sequence and the severity of the disease. However, the clinical status of patients cannot be unambiguously ascertained solely on the basis of the number of CTG repeats. Moreover, the exclusive maternal inheritance of the congenital form remains unexplained. Our observation of differently sized repeats in various DM tissues from the same individual may explain why the size of the mutation observed in lymphocytes does not necessarily correlate with the severity and nature of symptoms. Through a molecular and genetic study of 142 families including 418 DM patients, we have investigated the dynamics of the CTG repeat meiotic instability. A positive correlation between the size of the repeat and the intergenerational enlargement was observed similarly through male and female meioses for < or = 0.5-kb CTG sequences. Beyond 0.5 kb, the intergenerational variation was more important through female meioses, whereas a tendency to compression was observed almost exclusively in male meioses, for > or = 1.5-kb fragments. This implies a size- and sex-dependent meiotic instability. Moreover, segregation analysis supports the hypothesis of a maternal as well as a familial predisposition for the occurrence of the congenital form. Finally, this analysis reveals a significant excess of transmitting grandfathers partially accounted for by increased fertility in affected males.

Adult↗
PubMed2004

Mre11 deficiency in Arabidopsis is associated with chromosomal instability in somatic cells and Spo11-dependent genome fragmentation during meiosis.

The Mre11/Rad50/Nbs1 complex is involved in many aspects of chromosome metabolism. Aberrant function of the complex is associated with defects in the DNA checkpoint, double-strand break repair, meiosis, and telomere maintenance. In this article, we report the consequences of Mre11 dysfunction for the stability of mitotic and meiotic chromosomes in Arabidopsis thaliana. Although plants homozygous for a T-DNA insertion in a conserved region of the MRE11 gene are viable, they exhibit growth defects and are infertile. Analysis of mitotic chromosomes prepared from the mutant plants revealed abundant dicentric chromosomes and chromosomal fragments. Fluorescence in situ hybridization showed that anaphase bridges are often formed by homologous chromosome arms. The frequency of chromosome fusions was not reduced in mre11 ku70 double mutants, suggesting that plants possess DNA end-joining activities independent of the Ku70/80 and Mre11 complexes. Cytogenetic examination of pollen mother cells revealed massive chromosome fragmentation and the absence of synapsis in the initial stages of meiosis. The fragmentation was substantially suppressed in mre11 spo11-1 double mutants, indicating that Mre11 is required for repair but not for the induction of Spo11-dependent meiotic DNA breaks in Arabidopsis.

Amino Acid Sequence↗
PubMed2004

Involvement of the Arabidopsis thaliana AtPMS1 gene in somatic repeat instability.

Mismatch repair (MMR) genes participate in the maintenance of genome stability in all organisms. Based on its high degree of sequence conservation, it seems likely that the AtPMS1 gene of Arabidopsis thaliana is part of the MMR system in this model plant. To test this hypothesis, we aimed to disrupt AtPMS1 function by over-expressing mutated alleles expected to result in a dominant negative effect. To create one mutant allele we substituted two amino acids in the MutL-box, and for the other mutant allele we deleted 87 amino acids comprising the whole MutL-box. Contrary to published reports in some eukaryotes, transgenic plants expressing these alleles did not exhibit a decrease in fertility nor any other visible phenotype. To examine the impact of these mutations on microsatellite instability, the phenotype most often observed in organisms defective in MMR, reporter lines containing a uidA (GUS) gene inactivated by the insertion of a synthetic microsatellite (G7 or G16) were used. GUS gene function in these lines can be restored following the loss of one base or the gain of two bases in the repetitive tract. This results in the observation of blue sectors on a white background following histochemical staining. In a subset of the transformants, a significant increase (2- to 28-fold) in microsatellite instability was observed relative to wild-type. This report shows that MMR function can be disrupted via a dominant negative approach, and it is the first report to describe the phenotypic consequence of disrupting the function of a MutL homolog in plants.

Alleles↗
PubMed2000

Complex patterns of male germline instability and somatic mosaicism in myotonic dystrophy type 1.

The genetic basis of myotonic dystrophy type 1 (DM1) is the expansion of a CTG repeat in the 3' untranslated region of DM1PK. Once into the disease range, the repeat becomes highly unstable and is biased toward expansion in both somatic and germline tissues. Intergenerational differences usually reveal an increase in allele length, concordant with the clinical anticipation characteristic of DM1, but there have also been cases with intergenerational contractions of the repeat length, accompanied by apparent anticipation. In order to gain a better understanding of this intergenerational behaviour, we have obtained semen samples from six DM males and used single molecule analyses to compare the allele distributions present in their sperm and blood with those of their offspring. We have confirmed that the male germline mutational pathway is distinct from that of the soma, but the extent of variation is highly variable from one individual to another and not obviously correlated with progenitor allele length. Nonetheless, in all cases the alleles present in the father's sperm overlap with those observed in their offspring. These data also provide further indications that the interpretation of intergenerational transmissions by standard analyses is frequently compromised by the masking of germline differences by age-dependent somatic expansion in the parent.

Adolescent↗
PubMed2000

Differential somatic CAG repeat instability in variable brain cell lineage in dentatorubral pallidoluysian atrophy (DRPLA): a laser-captured microdissection (LCM)-based analysis.

Employing a laser-captured microdissection (LCM), we have investigated the somatic instability of CAG repeats in the variable brain cell lineage in three patients with dentatorubral pallidoluysian atrophy (DRPLA). LCM enables the isolation of single lineage brain cells for subsequent molecular analysis. We have found that CAG repeat size and the range of CAG repeats in the cerebellar granular cells is smaller than those in cerebellar glial cells. Similarly, those in the cerebral neuronal cells are significantly shorter than those in cerebral glial cells. These data directly indicate that the CAG repeat is relatively more stable in neuronal cells than in glial cells. Furthermore, cerebellar granular cells show significantly smaller main CAG repeat size and CAG repeat range than either Purkinje cells or cerebral neuronal cells, suggesting that somatic instability in the CAG repeat is markedly variable even among the different types of neuronal populations. The cell-specific CAG repeat instability may thus be more complex than has previously been considered. LCM is a powerful tool for elucidating the mechanism of the triplet repeat instability of each cell type.

Adult↗
PubMed2004

GAA repeat instability in Friedreich ataxia YAC transgenic mice.

Friedreich ataxia (FRDA) is primarily caused by an unstable GAA repeat-expansion mutation within intron 1 of the FRDA gene. However, the exact mechanisms leading to this expansion and its consequences are not fully understood. To study the dynamics of this mutation, we have generated two lines of human FRDA YAC transgenic mice that contain GAA repeat expansions within the appropriate genomic context. We have detected intergenerational instability and age-related somatic instability in both lines, with pronounced expansions found in the cerebellum. The dynamic nature of our transgenic GAA repeats is comparable with previous FRDA patient somatic tissue data. However, there is a difference between our FRDA YAC transgenic mice and other trinucleotide-repeat mouse models, which do not show pronounced repeat instability in the cerebellum. This represents the first mouse model of FRDA GAA repeat instability that will help to dissect the mechanism of this repeat.

Aging↗
PubMed1994

Defective mismatch repair in extracts of colorectal and endometrial cancer cell lines exhibiting microsatellite instability.

A replication error (RER+) phenotype, characterized by somatic instability in simple repeated sequences, is associated with several types of cancer. To determine if a defect in DNA replication fidelity or repair of replication errors might explain this instability, we compared both processes in cell-free extracts from RER+ endometrial and colorectal cancer cell lines to RER- cell lines. SV40 origin-dependent replication of a microsatellite sequence is highly accurate in cell extracts regardless of their RER phenotype. However, extracts from RER+ cell lines are defective in mismatch repair, while extracts of RER- cell lines are not. Lack of repair was observed when the signal (a nick) for strand-specific repair was either 3' or 5' to the mispair. One colorectal cancer cell line contained deletions in both alleles of the putative mismatch repair gene hMSH2, and one endometrial cancer cell line contained a 4-base pair duplication in one hMSH2 allele. No hMSH2 mutation was detected in the other allele or in the other five RER+ cell lines. Repair was readily detected when each of the defective extracts was mixed with a repair-proficient extract, demonstrating that no trans-acting inhibitor is present. Attempts to complement the repair deficiencies by mixing two different defective extracts identified three combinations that restored repair. The data suggest that: (i) defective repair is associated with colorectal and endometrial cancer and, by extrapolation, with other types of cancer; (ii) mutations in the hMSH2 gene, and possibly other genes, result in defective mismatch repair; (iii) the defect(s) in these lines likely involves pre-incision events or the excision step, but not the incision, polymerization, or ligation steps; and (iv) at least four functional complementation groups for mismatch repair may be involved in human cancer.

Cell Line↗
PubMed1996

CTG repeat analysis in lymphocytes, muscles and fibroblasts in patients with myotonic dystrophy.

The mutation responsible for DM has been identified as the amplification of a polymorphic (CTG)n repeat in the 3' untranslated region of the myotonin proteinase gene. To examine somatic instability of the repeat, we studied tissue variability of the CTG expansion of three mesodermally derived tissues: lymphocytes, cultured fibroblasts and muscle cells. In six patients with adult onset DM, the repeat region was larger in skeletal muscles and fibrolasts as compared to lymphocytes. Our findings indicate that somatic CTG instability between examined tissues might take place postnatally by a selection mechanism in lymphocytes.

Adult↗
PubMed2004

Replication-mediated instability of the GAA triplet repeat mutation in Friedreich ataxia.

Friedreich ataxia is caused by the expansion of a polymorphic and unstable GAA triplet repeat in the FRDA gene, but the mechanisms for its instability are poorly understood. Replication of (GAA*TTC)n sequences (9-105 triplets) in plasmids propagated in Escherichia coli displayed length- and orientation-dependent instability. There were small length variations upon replication in both orientations, but large contractions were frequently observed when GAA was the lagging strand template. DNA replication was also significantly slower in this orientation. To evaluate the physiological relevance of our findings, we analyzed peripheral leukocytes from human subjects carrying repeats of similar length (8-107 triplets). Analysis of 9400 somatic FRDA molecules using small-pool PCR revealed a similar mutational spectrum, including large contractions. The threshold length for the initiation of somatic instability in vivo was between 40 and 44 triplets, corresponding to the length of a eukaryotic Okazaki fragment. Consistent with the stabilization of premutation alleles during germline transmission, we also found that instability of somatic cells in vivo and repeats propagated in E.coli were abrogated by (GAGGAA)n hexanucleotide interruptions. Our data demonstrate that the GAA triplet repeat mutation in Friedreich ataxia is destabilized, frequently undergoing large contractions, during DNA replication.

DNA↗
PubMed2003

Mismatch repair gene Msh2 modifies the timing of early disease in Hdh(Q111) striatum.

Somatic instability of expanded HD CAG repeats that encode the polyglutamine tract in mutant huntingtin has been implicated in the striatal selectivity of Huntington's disease (HD) pathology. Here in Hdh(Q111) mice, we have tested whether a genetic background deficient in Msh2, expected to eliminate the unstable behavior of the 109 CAG array inserted into the murine HD gene, would alter the timing or striatal specificity of a dominant disease phenotype that predicts late-onset neurodegeneration. Our analyses of Hdh(Q111/+):Msh2(+/+) and Hdh(Q111/+): Msh2(-/-) progeny revealed that, while inherited instability involved Msh2-dependent and -independent mechanisms, lack of Msh2 was sufficient to abrogate progressive HD CAG repeat expansion in striatum. The absence of Msh2 also eliminated striatal mutant huntingtin with somatically expanded glutamine tracts and caused an approximately 5 month delay in nuclear mutant protein accumulation, but did not alter the striatal specificity of this early phenotype. Thus, somatic HD CAG instability appears to be a consequence of a striatal-selective disease process that accelerates the timing of an early disease phenotype, via expansion of the glutamine tract in mutant huntingtin. Therefore Msh2, as a striking modifier of early disease onset in a precise genetic HD mouse model, provides a novel target for the development of pharmacological agents that aim to slow pathogenesis in man.

Animals↗