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PubMed1993

Microsatellite instability in cancer of the proximal colon.

Colorectal tumor DNA was examined for somatic instability at (CA)n repeats on human chromosomes 5q, 15q, 17p, and 18q. Differences between tumor and normal DNA were detected in 25 of the 90 (28 percent) tumors examined. This instability appeared as either a substantial change in repeat length (often heterogeneous in nature) or a minor change (typically two base pairs). Microsatellite instability was significantly correlated with the tumor's location in the proximal colon (P = 0.003), with increased patient survival (P = 0.02), and, inversely, with loss of heterozygosity for chromosomes 5q, 17p, and 18q. These data suggest that some colorectal cancers may arise through a mechanism that does not necessarily involve loss of heterozygosity.

Adult↗
PubMed2006

Genetic background modifies nuclear mutant huntingtin accumulation and HD CAG repeat instability in Huntington's disease knock-in mice.

Genetically precise models of Huntington's disease (HD), Hdh CAG knock-in mice, are powerful systems in which phenotypes associated with expanded HD CAG repeats are studied. To dissect the genetic pathways that underlie such phenotypes, we have generated Hdh(Q111) knock-in mouse lines that are congenic for C57BL/6, FVB/N and 129Sv inbred genetic backgrounds and investigated four Hdh(Q111) phenotypes in these three genetic backgrounds: the intergenerational instability of the HD CAG repeat and the striatal-specific somatic HD CAG repeat expansion, nuclear mutant huntingtin accumulation and intranuclear inclusion formation. Our results reveal increased intergenerational and somatic instability of the HD CAG repeat in C57BL/6 and FVB/N backgrounds compared with the 129Sv background. The accumulation of nuclear mutant huntingtin and the formation of intranuclear inclusions were fastest in the C57BL/6 background, slowest in the 129Sv background and intermediate in the FVB/N background. Inbred strain-specific differences were independent of constitutive HD CAG repeat size and did not correlate with Hdh mRNA levels. These data provide evidence for genetic modifiers of both intergenerational HD CAG repeat instability and striatal-specific phenotypes. Different relative contributions of C57BL/6 and 129Sv genetic backgrounds to the onset of nuclear mutant huntingtin and somatic HD CAG repeat expansion predict that the initiation of each of these two phenotypes is modified by different genes. Our findings set the stage for defining disease-related genetic pathways that will ultimately provide insight into disease mechanism.

Animals↗
PubMed1993

Heterologous transposon tagging of the DRL1 locus in Arabidopsis.

The development of heterologous transposon tagging systems has been an important objective for many laboratories. Here, we demonstrate the use of a Dissociation (Ds) derivative of the maize transposable element Activator (Ac) to tag the DRL1 locus of Arabidopsis. The drl1 mutant shows highly abnormal development with stunted roots, few root hairs, lanceolate leaves, and a highly enlarged, disorganized shoot apex that does not produce an inflorescence. The mutation was shown to be tightly linked to a transposed Ds, and somatic instability was observed in the presence of the transposase source. Some plants showing somatic reversion flowered and produced large numbers of wild-type progeny. These revertant progeny always inherited a DRL1 allele from which Ds had excised. Analysis of the changes in DNA sequence induced by the insertion and excision of the Ds element showed that they were typical of those induced by Ac and Ds in maize.

Arabidopsis↗
PubMed1999

Length-dependent gametic CAG repeat instability in the Huntington's disease knock-in mouse.

The CAG repeats in the human Huntington's disease (HD) gene exhibit striking length-dependent intergenerational instability, typically small size increases or decreases of one to a few CAGs, but little variation in somatic tissues. In a subset of male transmissions, larger size increases occur to produce extreme HD alleles that display somatic instability and cause juvenile onset of the disorder. Initial efforts to reproduce these features in a mouse model transgenic for HD exon 1 with 48 CAG repeats revealed only mild intergenerational instability ( approximately 2% of meioses). A similar pattern was obtained when this repeat was inserted into exon 1 of the mouse Hdh gene. However, lengthening the repeats in Hdh to 90 and 109 units produced a graded increase in the mutation frequency to >70%, with instability being more evident in female transmissions. No large jumps in CAG length were detected in either male or female transmissions. Instead, size changes were modest increases and decreases, with expansions typically emanating from males and contractions from females. Limited CAG variation in the somatic tissues gave way to marked mosaicism in liver and striatum for the longest repeats in older mice. These results indicate that gametogenesis is the primary source of inherited instability in the Hdh knock-in mouse, as it is in man, but that the underlying repeat length-dependent mechanism, which may or may not be related in the two species, operates at higher CAG numbers. Moreover, the large CAG repeat increases seen in a subset of male HD transmissions are not reproduced in the mouse, suggesting that these arise by a different fundamental mechanism than the small size fluctuations that are frequent during gametogenesis in both species.

Age Factors↗
PubMed1999

Instability of the CAG repeat in immortalized fibroblast cell cultures from Huntington's disease transgenic mice.

Huntington's Disease transgenic mice were used for an exploration into the stability of a trinucleotide repeat. The brain shows heterogeneous somatic instability that increases quantitatively with age. To test somatic CAG-repeat alterations during long-term culture, DNA was extracted from transgenic tissue, primary fibroblasts, and SV40-immortalized fibroblasts at intervals of approximately 100 cell doublings. In fibroblasts derived from an adult mouse, there was an initial short truncation of the repeat, followed by an emerging population of cells showing continuous slow expansion. After 15 months in continuous culture (approximately 600 cell doublings following transformation) the major CAG peak has increased from 155 to approximately 170 triplets. This in vitro system can now be used to assay factors that affect instability.

Animals↗
PubMed2003

Two modes of germline instability at human minisatellite MS1 (locus D1S7): complex rearrangements and paradoxical hyperdeletion.

Minisatellite MS1 (locus D1S7) is one of the most unstable minisatellites identified in humans. It is unusual in having a short repeat unit of 9 bp and in showing somatic instability in colorectal carcinomas, suggesting that mitotic replication or repair errors may contribute to repeat-DNA mutation. We have therefore used single-molecule polymerase chain reaction to characterize mutation events in sperm and somatic DNA. As with other minisatellites, high levels of instability are seen only in the germline and generate two distinct classes of structural change. The first involves large and frequently complex rearrangements that most likely arise by recombinational processes, as is seen at other minisatellites. The second pathway generates primarily, if not exclusively, single-repeat changes restricted to sequence-homogeneous regions of alleles. Their frequency is dependent on the length of uninterrupted repeats, with evidence of a hyperinstability threshold similar in length to that observed at triplet-repeat loci showing expansions driven by dynamic mutation. In contrast to triplet loci, however, the single-repeat changes at MS1 exclusively involve repeat deletion, and can be so frequent--as many as 0.7-1.3 mutation events per sperm cell for the longest homogeneous arrays--that alleles harboring these long arrays must be extremely ephemeral in human populations. The apparently impossible existence of alleles with deletion-prone uninterrupted repeats therefore presents a paradox with no obvious explanation.

Alleles↗
PubMed1983

Monoclonal antibodies reveal the structural basis of antibody diversity.

Hybridoma technology has made it possible to introduce into continuous culture normal antibody-forming cells and to obtain large amounts of the immunoglobulin produced by each of these cells. Examination of the structure of a number of monoclonal antibodies that react with a single antigen has provided new information on the structural basis of the specificity and affinity of antibodies. Comparisons of families of monoclonal antibodies derived from a single germ line gene revealed the importance of somatic mutation in generating antibody diversity. Monoclonal antibodies that react with variable regions of other monoclonals allow the further dissection and modulation of the immune response. Finally, the continued somatic instability of immunoglobulin genes in cultured antibody-forming cells makes it possible to determine the rate of somatic mutation and to generate mutant monoclonal antibodies that may be more effective serological reagents.

Amino Acid Sequence↗
PubMed1999

Human minisatellites, repeat DNA instability and meiotic recombination.

Minisatellites include some of the most variable loci in the human genome and are superb for dissecting processes of tandem repeat DNA instability. Single DNA molecule analysis has revealed different mutation processes operating in the soma and germline. Low-level somatic instability results in simple intra-allelic rearrangements. In contrast, high frequency germline instability involves complex gene conversions and is therefore recombinational in nature, almost certainly occurring at meiosis. To determine whether true meiotic crossovers occur at human minisatellites, we have used polymorphisms near the repeat array to recover recombinant DNA molecules directly from sperm DNA. Analysis of minisatellite MS32 has revealed an intense and highly localised meiotic crossover hotspot centred upstream of the array, the first example of a human hotspot defined at the molecular level. This hotspot extends into the beginning of the repeat array, resulting in unequal and equal crossovers. Array crossovers occur much less frequently than array conversions but appear to arise by a common process, most likely by alternative processing of a recombination initiation complex. The location of MS32 at the boundary of a recombination hotspot suggests that this locus has evolved as a by-product of localised meiotic recombination activity, and that minisatellites might in general mark recombinationally proficient hotspots or hot domains in the genome. Finally, sperm crossover analysis makes it possible to explore the molecular rules that govern human meiotic recombination, and to detect phenomena such as meiotic drive that could provide a possible connection between recombination and DNA sequence diversity itself.

DNA↗
PubMed1999

Segregation analysis of tetra- and pentanucleotide short tandem repeat polymorphisms: deviation from Mendelian expectations.

Short tandem repeat (STR) polymorphisms are powerful tools for linkage studies, chromosome mapping and population analysis. The instability of these microsatellite regions is a prevailing event in several tumors and human genetic diseases and, despite various reports associating instability-related genes and meiosis control, the dynamics of these STR regions in normal cells/individuals has frequently been disregarded. Having previously assayed somatic instability in gastric cancer for some tetra- and pentanucleotide STRs and given the increased application of this type of marker for routine forensic expertise, we report the results of an extensive analysis of segregation in nuclear families of a normal population for the same loci. No mutations were detected in 2374 parent/offspring allelic transfers at TH01, TPO, VWA31/A, MBPB, and CD4 STR loci. Nonsignificant differences were found between gene frequencies of parental and offspring generations. However, the segregation analysis revealed significant deviation from Mendelian expectations for: VWA31/A locus - alleles 19, 17 and 14 and TH01 locus - allele 6. In particular, parental meiosis strongly favored specific allele transmission, depending upon the sex of the offspring. Specific mating types are apparently responsible for most of these abnormal segregations. These results suggest selective factors working either at the gametic or zygotic levels.

Female↗
PubMed2004

Intergenerational instability of the expanded CTG repeat in the DMPK gene: studies in human gametes and preimplantation embryos.

The CTG repeat at the 3' untranslated region of the dystrophia myotonica protein kinase (DMPK) gene shows marked intergenerational and somatic instability in patients with myotonic dystrophy (DM1), when the repeat is expanded to more than approximately 55 repeats. Intensive research has yielded some insights into the timing and mechanism of these intergenerational changes: (1) increases in expansion sizes occur during gametogenesis but probably not during meiosis, (2) the marked somatic mosaicism becomes apparent from the 2nd trimester of development onward and increases during adult life, and (3) DNA repair mechanisms are involved. We have performed preimplantation genetic diagnosis for DM1 since 1995, which has given us the unique opportunity to study the expanded CTG repeat in affected embryos and in gametes from affected patients. We were able to demonstrate significant increases in the number of repeats in embryos from female patients with DM1 and in their immature and mature oocytes, whereas, in spermatozoa and embryos from male patients with DM1, smaller increases were detected. These data are in concordance with data on other tissues from adults and fetuses and fill a gap in our knowledge of the behavior of CTG triplet expansions in DM1.

Blastocyst↗
PubMed1994

The molecular basis of instability of the singed(very weak) mutation in Drosophila melanogaster.

The singed(very weak) mutation was created by the sequential addition of two P transposable elements to the singed gene. The mutation can be somatically unstable through the action of a dominant maternal effect mutation on the second chromosome. It is also unstable in the germ line in these conditions. Sequencing of the region of the P insertions in the mutation reveals that the two inserted elements have single internal deletions, and the larger of the two is a copy of the KP element. The mutation will generate, at high frequencies, strongly singed and pseudo-wild type products by reversions occurred in the germline. These are the result of the precise excision of the smaller and the larger elements respectively. By PCR amplification of dissected thoraces we show that the somatic instability of the mutation, from a weak to a strong singed phenotype, is also caused by the excision of the smaller of the two elements.

Animals↗
PubMed2006

AIB1 gene amplification and the instability of polyQ encoding sequence in breast cancer cell lines.

BACKGROUND: The poly Q polymorphism in AIB1 (amplified in breast cancer) gene is usually assessed by fragment length analysis which does not reveal the actual sequence variation. The purpose of this study is to investigate the sequence variation of poly Q encoding region in breast cancer cell lines at single molecule level, and to determine if the sequence variation is related to AIB1 gene amplification. METHODS: The polymorphic poly Q encoding region of AIB1 gene was investigated at the single molecule level by PCR cloning/sequencing. The amplification of AIB1 gene in various breast cancer cell lines were studied by real-time quantitative PCR. RESULTS: Significant amplifications (5-23 folds) of AIB1 gene were found in 2 out of 9 (22%) ER positive cell lines (in BT-474 and MCF-7 but not in BT-20, ZR-75-1, T47D, BT483, MDA-MB-361, MDA-MB-468 and MDA-MB-330). The AIB1 gene was not amplified in any of the ER negative cell lines. Different passages of MCF-7 cell lines and their derivatives maintained the feature of AIB1 amplification. When the cells were selected for hormone independence (LCC1) and resistance to 4-hydroxy tamoxifen (4-OH TAM) (LCC2 and R27), ICI 182,780 (LCC9) or 4-OH TAM, KEO and LY 117018 (LY-2), AIB1 copy number decreased but still remained highly amplified. Sequencing analysis of poly Q encoding region of AIB1 gene did not reveal specific patterns that could be correlated with AIB1 gene amplification. However, about 72% of the breast cancer cell lines had at least one under represented (<20%) extra poly Q encoding sequence patterns that were derived from the original allele, presumably due to somatic instability. Although all MCF-7 cells and their variants had the same predominant poly Q encoding sequence pattern of (CAG)3CAA(CAG)9(CAACAG)3(CAACAGCAG)2CAA of the original cell line, a number of altered poly Q encoding sequences were found in the derivatives of MCF-7 cell lines. CONCLUSION: These data suggest that poly Q encoding region of AIB1 gene is somatic unstable in breast cancer cell lines. The instability and the sequence characteristics, however, do not appear to be associated with the level of the gene amplification.

Acetyltransferases↗
PubMed2002

Instability of a premutation-sized CGG repeat in FMR1 YAC transgenic mice.

Fragile X syndrome results from the massive expansion of a CGG repeat in the 5' untranslated region of the gene FMR1. Data suggest that the hyperexpansion properties of FMR1 CGG repeats may depend on flanking cis-acting elements. We have therefore used homologous recombination in yeast to introduce an in situ CGG expansion corresponding to a premutation-sized allele into a human YAC carrying the FMR1 locus. Several transgenic lines were generated that carried repeats of varying lengths and amounts of flanking sequence. Length-dependent instability in the form of small expansions and contractions was observed in both male and female transmissions over five generations. No parent-of-origin effect or somatic instability was observed. Alterations in tract length were found to occur exclusively in the 3' uninterrupted CGG tract. Large expansion events indicative of a transition from a premutation to a full mutation were not observed. Overall, our results indicate both similarities and differences between the behavior of a premutation-sized repeat in mouse and that in human.

Animals↗
PubMed2004

[Instability of repetitive units of foreign centromeric satellite DNA in transgenic mice and transfected cells].

Cytologically detectable instability of centromeric satellite DNA may cause hereditary disorders in human. To study the mechanisms of such instability, two transgenic mouse lines and 11 clones of transfected F9 mouse embryonic teratocarcinoma cells were obtained with the 3.8-kb repetitive unit (Sat) of Bos taurus satellite DNA IV. Intergeneration and somatic instability of exogenous satellite DNA (satDNA) was observed in transgenic mice and transfected cells as a change in nucleotide sequence of an internal Sat region approximately 1000 bp in size. Since Sat was in the hemizygous state in both cases by the experimental protocol, the instability was attributed to intra-allelic processes. Intergeneration instability probably took place in the premeiotic period of gametogenesis or in early embryo development and led to prenatal death of transgenic embryos after at least one generation. No direct or inverse correlation was observed between methylation and instability of Sat. The results testify that submicroscopic changes in highly repetitive noncoding DNA sequences may already affect the genome function in higher eukaryotes.

Animals↗
PubMed2004

Pms2 is a genetic enhancer of trinucleotide CAG.CTG repeat somatic mosaicism: implications for the mechanism of triplet repeat expansion.

The expansion of CAG.CTG repeat sequences is the cause of several inherited human disorders. Longer alleles are associated with an earlier age of onset and more severe symptoms, and are highly unstable in the germline and soma with a marked tendency towards repeat length gains. Germinal expansions underlie anticipation; whereas age-dependent, tissue-specific, expansion-biased somatic instability probably contributes toward the progressive nature and tissue-specificity of the symptoms. The mechanism(s) of repeat instability is not known, but recent data have implicated mismatch-repair (MMR) gene mutS homologues in driving expansion. To gain further insight into the expansion mechanism, we have determined the levels of somatic mosaicism of a transgenic expanded CAG.CTG repeat in mice deficient for the Pms2 MMR gene. Pms2 is a MutL homologue that plays a critical role in the downstream processing of DNA mismatches. The rate of somatic expansion was reduced by approximately 50% in Pms2-null mice. A higher frequency of rare, but very large, deletions was also detected in these animals. No significant differences were observed between Pms2(+/+) and Pms2(+/-) mice, indicating that a single functional Pms2 allele is sufficient to generate normal levels of somatic mosaicism. These findings reveal that as well as MMR enzymes that directly bind mismatched DNA, proteins that are subsequently recruited to the complex also play a central role in the accumulation of repeat length changes. These data suggest that somatic expansion results not by replication slippage, single stranded annealing or simple MutS-mediated stabilization of secondary structures, but by inappropriate DNA MMR.

Adenosine Triphosphatases↗
PubMed2003

Instability of (GATA)n microsatellite loci in the parthenogenetic Caucasian rock lizard Darevskia unisexualis (Lacertidae).

Mini- and microsatellites, comprising tandemly repeated short nucleotide sequences, are abundant dispersed repetitive elements that are ubiquitous in eukaryotic genomes. In humans and other bisexual species hypervariable mini- and microsatellite loci provide highly informative systems for monitoring of germline and somatic instability. However, little is known about the mechanisms by which these loci mutate in species that lack effective genetic recombination. Here, multilocus DNA fingerprinting was used to study M13 minisatellite and (GATA)n microsatellite instability in the parthenogenetic Caucasian rock lizard Darevskia unisexualis (Lacertidae). DNA fingerprinting of 25 parthenogenetic families, from six isolated populations in Armenia (comprising a total of 84 siblings), using the oligonucleotide (GATA)4 as a hybridization probe, revealed mutant fingerprinting phenotypes in 13 siblings that differed from their mothers in several restriction DNA fragments. In three families (8 siblings), the mutations were present in the germline. Moreover, the mutant fingerprint phenotypes detected in siblings were also present in population DNA samples. No intrafamily variations in DNA fingerprint patterns were observed with the M13 minisatellite probe. Estimates of the mutation rate for (GATA)n microsatellite loci in D. unisexualis showed that it was as high as that seen in some bisexual species, reaching 15% per sibling or 0.95% per microsatellite band. Furthermore, in one case, a somatic (GATA)n microsatellite mutation was observed in an adult lizard. These findings directly demonstrate that mutations in (GATA)n microsatellite loci comprise an important source of genetic variation in parthenogenetic populations of D. unisexualis.

Animals↗
PubMed1994

Replication error-type genetic instability at 1q42-43 in human male germ cell tumors.

The replication error phenotype, recognized as microsatellite sequence alterations, has recently been suggested to be associated with hereditary nonpolyposis colorectal cancer and other types of sporadic tumors. We examined paired tumor-normal DNAs from 69 human male germ cell tumors for somatic instability at the 1q42-43 region. Analysis of a variable number of tandem repeats marker (D1S74) and 3 (CA)n type microsatellite loci (D1S235, D1S180, and angiotensinogen) revealed genetic alterations in tumor DNAs of 26 (38.2%) cases. The changes observed comprised rearrangements with D1S74 detected by Southern blot analysis in 4 of 55 (7%) cases; replication error-type alterations with D1S235, D1S180, and angiotensinogen in 12 of 66 (18.2%) cases; and loss of heterozygosity in 12 of 67 (17.9%) cases with the same probes. The microsatellite sequence alterations were more common in histological subsets other than teratomas, while the loss of heterozygosity was significantly more frequent in teratomas compared to other histologies. These results suggest that microsatellite instability and loss of heterozygosity at 1q42-43 may be unrelated genetic events which may play a role in germ cell tumor development.

Chromosomes, Human, Pair 1↗
PubMed2005

High-level microsatellite instability is not involved in gallbladder carcinogenesis.

The molecular alterations involved in the pathogenesis of gallbladder cancer are not yet well defined. Our aim was to determine the microsatellite status of gallbladder carcinomas and its possible correlation with alterations in K-ras and p53 genes as well as the clinicopathological characteristics of these tumors. A group of 37 gallbladder carcinomas was analyzed for alterations in a proposed panel of mononucleotide and dinucleotide markers of microsatellite instability. Somatic frameshift mutations at repeated sequences in the coding regions of TGF-betaRII, Bax, hMSH3, hMSH6 were also examined. The findings were correlated with the presence of K-ras and p53 alterations, and tumors' clinicopathological features. Microsatellite instability and/or LOH was observed in 9 gallbladder carcinomas. Cases showing microsatellite instability displayed alterations only in dinucleotide markers and were classified as MSI-L carcinomas. A subset of gallbladder carcinomas is characterized by low-level instability, based on the analysis of the above mentioned panel of markers. The pathway of microsatellite instability seems to play a minor role in the pathogenesis of gallbladder cancer.

Adult↗