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At least 127 records · Page 7Linked to original sources
PubMed1993

Direct molecular diagnosis of myotonic dystrophy.

Myotonic dystrophy (DM) arises from an unstable trinucleotide (CTGn) repeat sequence within the DM locus at 19q13.3. Twenty-three myotonic dystrophy families containing 205 persons with no symptoms, minimal manifestations, classic DM or congenital DM were investigated to validate the application of the pM10M6 probe to direct molecular diagnosis. Affected family members had been diagnosed clinically and the unaffected family members had been assigned carrier probabilities close to either zero or 100%, using closely linked flanking markers. Southern analysis identified all 89 DM gene carriers as having expansions of the unstable element. PstI detected all small expansions of the repeat sequence as easily seen discrete bands; but large expansions were usually seen as diffuse smears, sometimes difficult to distinguish from lane background. EcoRI concentrated these diffuse smears, associated with somatic instability, into discrete bands which were easy to detect; but it did not resolve the smaller expansions present in 9 (10%) of the DM carriers. It is essential that PstI and EcoRI gels are run in parallel to detect all DM gene carriers. The extent of expansion of CTG correlated with age of onset and disease severity. Biopsies of various fetal tissues from two terminated pregnancies confirmed the diagnosis obtained by CVS and revealed no heterogeneity between tissues at this developmental stage. Further expansion occurred during the culture of CVS cells, indicating that direct prenatal diagnosis needs to be carried out on CVS tissue rather than on cultured cells. The intergenerational change of the repeat sequence from DM parent to DM offspring showed a significant parental sex difference for those parents with large expansions. Contraction of the unstable element was observed in the three males carrying the largest expansions and could explain why congenital DM is exclusively of maternal origin.

Female↗
PubMed1997

Mapping of 34 minisatellite loci resolved by two-dimensional DNA typing.

Two-dimensional (2-D) DNA typing is based on electrophoretic separation of genomic DNA fragments in two dimensions according to independent criteria (size and base-pair sequence), followed by hybridization analysis using multilocus probes. The technique allows simultaneous visualization of several hundred loci as spots in a 2-D pattern. The majority of the loci resolved are polymorphic. Using linkage analysis in a large CEPH family, this study reports the mapping of 34 loci detected by the minisatellite core probe 33.6. By multipoint linkage analysis, regional chromosome positions of the 33.6 loci could be deduced, showing no evidence of clustering. In the analysis of spot patterns, use was made of a computerized image analysis system specifically designed for 2-D DNA typing. Since experimental variations between different separation patterns were automatically corrected for with this program, rapid and reliable scorings could be obtained. The results presented demonstrate the availability of reliable genetic information throughout the 2-D separation pattern. Adding the use of semiautomated computerized pattern analysis, this study further substantiates the applicability of 2-D DNA typing in genome scanning, not only in theoretical but also in practical terms. Moreover, it can be anticipated that this method will have a specific advantage in studies that scan for trinucleotide repeat expansions and somatic instability, where the repeat sequences detected by appropriate core probes are of particular interest.

Chromosome Mapping↗
PubMed2003

Myotonic dystrophy type 2: molecular, diagnostic and clinical spectrum.

BACKGROUND: Myotonic dystrophy types 1 (DM1) and 2 (DM2/proximal myotonic myopathy PROMM) are dominantly inherited disorders with unusual multisystemic clinical features. The authors have characterized the clinical and molecular features of DM2/PROMM, which is caused by a CCTG repeat expansion in intron 1 of the zinc finger protein 9 (ZNF9) gene. METHODS: Three-hundred and seventy-nine individuals from 133 DM2/PROMM families were evaluated genetically, and in 234 individuals clinical and molecular features were compared. RESULTS: Among affected individuals 90% had electrical myotonia, 82% weakness, 61% cataracts, 23% diabetes, and 19% cardiac involvement. Because of the repeat tract's unprecedented size (mean approximately 5,000 CCTGs) and somatic instability, expansions were detectable by Southern analysis in only 80% of known carriers. The authors developed a repeat assay that increased the molecular detection rate to 99%. Only 30% of the positive samples had single sizeable expansions by Southern analysis, and 70% showed multiple bands or smears. Among the 101 individuals with single expansions, repeat size did not correlate with age at disease onset. Affected offspring had markedly shorter expansions than their affected parents, with a mean size difference of -17 kb (-4,250 CCTGs). CONCLUSIONS: DM2 is present in a large number of families of northern European ancestry. Clinically, DM2 resembles adult-onset DM1, with myotonia, muscular dystrophy, cataracts, diabetes, testicular failure, hypogammaglobulinemia, and cardiac conduction defects. An important distinction is the lack of a congenital form of DM2. The clinical and molecular parallels between DM1 and DM2 indicate that the multisystemic features common to both diseases are caused by CUG or CCUG expansions expressed at the RNA level.

Adolescent↗
PubMed2004

Mouse models of triplet repeat diseases.

Since their discovery in 1991, triplet repeat mutations have been found to be the cause of genomic fragile sites, two of which are linked to mental retardation, myotonic dystrophy, and several late-onset neurodegenerative diseases. In all cases, these mutations exhibit gametic and/or somatic instability once they have expanded into the mutant range. The mutations are located in coding and noncoding gene regions and have been found to act by dominant and recessive mechanisms. A wide range of mouse models has been generated to understand both of the mechanisms that underlie repeat instability and the molecular pathogenesis of the diseases. Mouse models have proved extremely useful in these goals and are now also being used for the preclinical testing of therapeutic compounds. This chapter reviews the successes and limitations of the approaches that have been developed.

Animals↗
PubMedDate not supplied

Biomolecular identification of (CCTG)n mutation in myotonic dystrophy type 2 (DM2) by FISH on muscle biopsy.

Myotonic dystrophy type 2 (DM2) is a dominantly inherited disorder with multisystemic clinical features, caused by a CCTG repeat expansion in intron 1 of the zinc finger protein 9 (ZNF9) gene. The mutant transcripts are retained in the nucleus forming multiple discrete foci also called ribonuclear inclusions. The size and the somatic instability of DM2 expansion complicate the molecular diagnosis of DM2. In our study fluorescence-labeled CAGG-repeat oligonucleotides were hybridized to muscle biopsies to investigate if fluorescence in situ hybridization (FISH), a relatively quick and simple procedure, could be used as a method to diagnose DM2. When FISH was performed with (CAGG)5 probe, nuclear foci of mutant RNA were present in all genetically confirmed DM2 patients (n=17) and absent in all patients with myotonic dystrophy type 1 (DM1; n=5) or with other muscular disease (n=17) used as controls. In contrast, foci were observed both in DM1 and DM2 myonuclei when muscle tissue were hybridized with (CAG)6CA probe indicating that this probe is not specific for DM2 identification. The consistent detection of ribonuclear inclusions in DM2 muscles and their absence in DM1, in agreement with the clinical diagnosis and with leukocyte (CCTG)n expansion, suggests that fluorescence in situ hybridization using (CAGG)5 probes, may be a specific method to distinguish between DM1 and DM2. Moreover, the procedure is simple, and readily applicable in any pathology laboratory.

Adult↗
PubMed2000

Microsatellite DNA in peach (Prunus persica L. Batsch) and its use in fingerprinting and testing the genetic origin of cultivars.

We isolated and sequenced 26 microsatellites from two genomic libraries of peach cultivar 'Redhaven', enriched for AC/GT and AG/CT repeats, respectively. For 17 of these microsatellites, it was possible to demonstrate Mendelian inheritance. Microsatellite polymorphism was assayed in 50 peach and nectarine cultivars. Of the 1300 PCRs carried out, all but two produced amplified products of the expected size. All microsatellites were polymorphic, showing 2-8 alleles per locus. Heterozygosity ranged from 0.04-0.74 (mean 0.47); the discrimination power (PD) ranged from 0.04-0.84 (mean 0.60). Cultivar heterozygosity varied greatly, with one cultivar ('Independence') being homozygous at all loci. The set of microsatellites discriminated all cultivars investigated, except several sport mutations, i.e., 'Dixitime' vs. 'Springcrest', 'Compact Redhaven' vs. 'Redhaven', and two pairs of cultivars, 'Venus' vs. 'Orion' and 'Elegant Lady' vs. 'Rome Star', whose pedigrees are controversial. We were able to analyze the paternity of several cultivars. In most cases, the parenthood was confirmed. The comparison of three long-living 'Redhaven' accessions supplied by different repositories did not provide any evidence of somatic instability of microsatellites. Hence, microsatellites, ranked according to their information content, are recommended as markers of choice for peach fingerprinting and suggestions are provided for interpreting band profiles and the correct sizing of alleles.

Crosses, Genetic↗
PubMed1992

Cytological aberrations in maize populations exhibiting unusual recombinational behaviour.

Earlier studies identified higher recombinational activity and increased somatic instability at various loci in the F1 and F2 progeny of particular stocks of a maize inbred line, A188. Hypothesizing that this may reflect a genome-wide perturbation of chromosome structure which would be detectable cytologically, aberrant meiotic behaviour in these stocks was investigated. Microsporocytes from a population of F2 plants derived from these stocks displayed the following aberrations: varying frequencies of metaphase and anaphase laggards, 'stickiness' at anaphase I resulting in chromosome bridges from pole to pole, acentric fragments and a spontaneous translocation of the NOR on chromosome 6. The frequencies of metaphase I and anaphase I abnormalities in these stocks were higher to a statistically significant extent than in control F2 populations, suggesting that these aberrations may be related to the unusual genetic behaviour of these stocks.

Anaphase↗
PubMed2004

A long PCR-based molecular protocol for detecting normal and expanded ZNF9 alleles in myotonic dystrophy type 2.

Myotonic dystrophy type 2 (DM2) lacks the expansion on chromosome 19q13 present in DM1 and is characterized by a mutation on 3q21. It has been shown that the DM2 mutation is a huge [CCTG]n repeat expansion in intron 1 of the zinc finger protein 9 (ZNF9) gene. The longest normal allele observed has a approximately 30 CCTG repeat, whereas the range of expansion is extremely variable, starting from 75 up to 11,000 CCTGs. Direct analysis by Southern blot, after restriction enzyme digestion of genomic DNA, was the first method chosen for studying the DM2 mutation. However, the expansion size and the elevated grade of somatic instability have limited the sensitivity of the test to approximately 80% of known carriers. We developed a long PCR-formatted protocol, which involves a single genomic in vitro amplification, followed by agarose gel electrophoresis and oligospecific hybridization. We were able to detect normal alleles and expanded ZNF9 alleles, starting from low amounts of genomic DNA (>/= 1 ng) in virtually all the DM2 patients analyzed, obtaining a molecular detection rate of 100%. This method is quick, sensitive, and reproducible, and it reduces the cost of diagnostic laboratory processing for DM2 diagnosis.

Alleles↗
PubMed2004

(CTG)n expansion at DMPK locus seen only in muscle tissue: a novel case.

Triplet repeat expansion in 3 untranslated region of myotonic dystrophy protein kinase (DMPK) gene has been implicated as causative in myotonic dystrophy (DM). In cases of DM, high levels of somatic instability have been reported, in which inter-tissue repeat length differences as large as 3000 repeats have been observed. This study highlights the inter-tissue (CTG)n expansion variability at the DMPK locus. Molecular analysis of DMPK gene, encompassing the triplet repeat expansion, was carried out in 31 individuals (11 clinically identified DM patients, 20 controls). All controls showed a 2.1kb band (upto 35 CTG repeats), while four cases exhibited an expansion (>50 repeats). A novel observation was made in one case, wherein the DNA from lymphocytes showed a normal 2.1kb band while the muscle tissue DNA from the same patient was heterozygous for normal and 4.3 kb band (>700 repeats). Our results suggested that because inter-tissue variability existed in the (CTG)n repeat number at DMPK locus, an attempt should be made to evaluate affected tissue along with blood wherever possible prior to making a final diagnosis. This is important not only for diagnosis and prenatal analysis, but also while providing genetic counseling to families.

3' Untranslated Regions↗
PubMed2006

Italian guidelines for molecular analysis in myotonic dystrophies.

Myotonic dystrophies, the most common form of adult muscular dystrophy, comprise at least two forms, clinically and genetically heterogeneous. Myotonic dystrophy type 1 and type 2 are both caused by unstable repetitions in untranslated gene regions: a [CTG]n expansion in the 3' region of the DMPK gene on chromosome 19q13 (DM1) and [CCTG]n tetranucleotide repeat located in the first intron of the ZNF9 gene on chromosome 3q21 (DM2). DM clinical features are caused by a gain of functions RNA mechanism in which the CUG and CCUG repeats alter nuclear functions, including alternative splicing of shared genes. Southern blot and/or polymerase chain reaction PCR-based approaches allow the detection of DM mutations in almost 100% of cases, however, the expansion size and the elevated grade of somatic instability make molecular testing for DM a diagnostic challenge. The increased use of DNA testing for DM generates many questions regarding the indications and interpretations of the test which require standardized methods, routinely available in molecular genetic laboratories. Here, we propose Guidelines for the molecular diagnosis of DM1 and DM2 approved by the Italian Ministry of Health in 2005 (Piano Nazionale Linee Guida, PNLG). Best practice for DM molecular analysis in diagnostic application, presymptomatic and prenatal testing, using direct and indirect approaches are described, with particular attention focused on ethical, legal and social issues. Overviews of materials used in the molecular diagnosis, as well as internet resources, are also included.

Genetic Testing↗
PubMed1981

Mutational events in mouse myeloma cells.

Cultured mouse myeloma cells frequently generate variants in immunoglobulin expression and structure. In this paper the authors will review the phenotypes of the variants which have been identified and discuss the mechanisms which may be responsible for the somatic instability of the immunoglobulin genes in mouse myeloma cells.

Amino Acids↗
PubMed1995

Somatic heterogeneity of the CTG repeat in myotonic dystrophy is age and size dependent.

The most common form of adult muscular dystrophy, myotonic dystrophy (DM), is caused by the abnormal expansion of the CTG repeat, located in the 3' UTR of the DM gene. The expanded-CTG allele often presents as a diffused band on Southern blot analysis, suggesting somatic mosaicism. In order to study the somatic instability of the CTG repeat, we have investigated the dynamics of the size heterogeneity of the CTG expansion. Size heterogeneity is shown as a smear on Southern blot and is measured by the midpeak-width ratio of the expanded allele to the normal sized allele. The ratio is also corrected for compression in the higher-molecular-weight region. It is found that the size heterogeneity of the expanded-CTG repeats, of 173 DM patients, correlates well with the age of the patient (r = .81, P << .001). The older patients show larger size variation. This correlation is independent of the sex of either the patient or the transmitting parent. The size heterogeneity of the expansion, based on age groups, is also dependent on the size of the expanded trinucleotide repeat. However, obvious size heterogeneity is not observed in congenital cases, regardless of the size of expansion. Comparison of individual patient samples collected at two different times has confirmed that the degree of size heterogeneity increases with age and has revealed a subtle but definite upward shift in the size of the expanded-CTG allele. The progression of the CTG repeat toward larger expansion with age is further confirmed by small-pool PCR assay that resolved the heterogeneous fragments into discrete bands.(ABSTRACT TRUNCATED AT 250 WORDS)

Adolescent↗
PubMed1995

X-linked recessive bulbospinal neuronopathy (SBMA).

X-linked recessive bulbospinal neuronopathy (SBMA) is an adult onset motor neuronopathy with androgen receptor (AR) gene mutation of expanded CAG repeat size in the first exon. The size of CAG repeats in the AR gene is one of the determinant factors of the severity and progression rate of SBMA phenotypes, but the meiotic and somatic instability of CAG repeats is far more stable as compared with other diseases caused by trinucleotide repeat expansions such as HD, DRPLA, MJD and SCA1. Several evidences suggest that aberrant transcriptional activity of androgen through mutant AR is related to the pathogenic mechanism of this disease.

Adult↗
PubMed2004

Regulation of telomere length and suppression of genomic instability in human somatic cells by Ku86.

Ku86 plays a key role in nonhomologous end joining in organisms as evolutionarily disparate as bacteria and humans. In eukaryotic cells, Ku86 has also been implicated in the regulation of telomere length although the effect of Ku86 mutations varies considerably between species. Indeed, telomeres either shorten significantly, shorten slightly, remain unchanged, or lengthen significantly in budding yeast, fission yeast, chicken cells, or plants, respectively, that are null for Ku86 expression. Thus, it has been unclear which model system is most relevant for humans. We demonstrate here that the functional inactivation of even a single allele of Ku86 in human somatic cells results in profound telomere loss, which is accompanied by an increase in chromosomal fusions, translocations, and genomic instability. Together, these experiments demonstrate that Ku86, separate from its role in nonhomologous end joining, performs the additional function in human somatic cells of suppressing genomic instability through the regulation of telomere length.

Antigens, Nuclear↗
PubMed1997

Human minisatellite MS32 (D1S8) displays somatic but not germline instability in transgenic mice.

Human minisatellite MS32 (D1S8) shows instability both in the germline and, at much lower levels, in somatic DNA. To investigate factors that influence somatic and germline mutation, large cosmid-based constructs containing MS32 were introduced into mice, bred to homozygosity and tested for instability in blood and sperm. Analysis of single copy and multicopy transgenic lines revealed somatic mutants occurring at a frequency comparable with that seen in man. As in humans, these mutants arose mainly by simple intra-allelic duplications and deletions. In contrast, analysis of sperm DNA from four different transgenic lines showed no trace of the complex recombination-based germline instability seen in man, even using PCR-based approaches capable of detecting very rare mutants. These data provide further evidence that germline and somatic mutation at human minisatellite MS32 occur via distinct pathways, that a major barrier exists to the transfer of germline instability from humans to mice and that the mouse germline appears to be protected from mitotic instability of the type seen in blood.

Alleles↗
PubMed2003

There is no increase in frequency of somatic mutations in metastases compared with primary colorectal carcinomas with microsatellite instability.

This study investigates the molecular features of metastasis in sporadic colon carcinomas with high-level microsatellite instability (MSI-H). DNA from 51 regions from 10 MSI-H metastatic carcinomas and 26 corresponding metastases was analyzed for mutations in TGFBRII, IGFIIR, BAX, MSH3, MSH6, and TCF4, which are associated with MSI-H carcinomas. In addition, 10 metastatic and 10 non-metastatic MSI-H carcinomas and 10 metastatic microsatellite-stable (MSS) carcinomas were examined for expression of vascular endothelial growth factor (VEGF) and mutant TP53. The frequency of microsatellite instability and somatic mutations was not significantly increased in the metastases compared with the that of primary carcinomas. Although significantly fewer MSI-H carcinomas expressed VEGF (P < 0.01) and mutant TP53 (P < 0.005) than MSS carcinomas, there was no difference in VEGF and mutant TP53 expression in metastatic and non-metastatic MSI-H carcinomas. In conclusion, metastasis does not appear to be associated with an increase in somatic mutation rate in any of the genes examined in MSI-H colon carcinomas. Furthermore, VEGF and TP53 expression did not appear to be involved in metastasis in MSI-H colon carcinomas.

Colorectal Neoplasms↗