Relative stability of a minimal CTG repeat expansion in a large kindred with myotonic dystrophy

The genetic basis for myotonic dystrophy (DM) is a CTG trinucleotide repeat expansion. The number of CTG repeats commonly increases in affected individuals of successive generations, in association with anticipation. We identified a large DM family in which multiple members had minimal CTG repeat expansions, and in which the number of CTG repeats remained in the minimally expanded range through at least three, and possibly four, generations. This relative stability of minimal CTG repeat expansions may help to maintain the DM mutation in the population.     

Myotonic dystrophy phenotype without expansion of (CTG)n repeat: an entity distinct from proximal myotonic myopathy (PROMM)?

Myotonic dystrophy (DM) is associated with an expansion of an unstable (CTG)n repeat in the 3' untranslated region of the DM protein kinase (DMPK) gene on chromosome 19q13.3. We studied six patients from two families who showed no expansions of the repeat, in spite of their clinical diagnosis of DM. These patients had multi-systemic manifestations that were distinguishable from those seen in other myotonic disorders, including proximal myotonic myopathy (PROMM). In one additional family, two symptomatic members showed no expanded (CTG)n repeats, while their affected relatives had the expanded repeats. DM haplotype analysis failed to exclude the DMPK locus as a possible site of mutation in each family; however, DMPK mRNA levels were normal. We conclude that a mutation(s) other than the expanded (CTG)n repeat can cause the DM phenotype. The mutation(s) in these families remain(s) to be mapped and characterized.     

Human papillomavirus investigation of patients with cervical intraepithelial neoplasia 3, some of whom progressed to invasive cancer

Unstable expansion of the CTG repeats in the 3' untranslated region encoding a member of the protein kinase family in the q13.3 band on chromosome 19 is a mutation specific for myotonic dystrophy. To examine the correlation between clinical expression and CTG trinucleotide repeat length, we carried out Southern blot analysis in a family with myotonic dystrophy. In this pedigree, the expanded CTG repeats were transmitted maternally. The mother had three female children. The mother had about 200 CTG repeats, and the number of repeats for each child was about 800, 1500 and 1600 in birth order. The mother and the patient with 800 repeats were unaware of muscle weakness or myotonia. Symptoms were present from age 3 years in the patient with 1500 repeats and from birth in the one with 1600 repeats. Although the mother menstruated regularly, the patients with 800 and 1500 repeats both menstruated irregularly, and the one with 1600 repeats has never menstruated. The age of onset and severity of the disease were correlated with the size of the expanded repeats. Endocrinological studies revealed that the basal levels of the gonadotropins, PRL and E2 were within normal range, and a pituitary response to LHRH was observed. These data suggest that the amenorrhea and menstrual irregularities were caused by a suprahypophyseal dysfunction. When expanded CTG repeats are transmitted maternally, abnormal products resulting from the metabolic disturbance in the affected mother may harm the fetus in utero. A heterozygous fetus, who has more CTG repeats, may be unable to metabolize the pathologic products sufficiently and therefore may become more severely affected. This may explain the exclusive maternal transmission of congenital myotonic dystrophy.     

Heterogeneity of DM kinase repeat expansion in different fetal tissues and further expansion during cell proliferation in vitro: evidence for a casual involvement of methyl-directed DNA mismatch repair in triplet repeat stability

We have analysed the mitotic behaviour of expanded CTG repeats in somatic tissues and cultured somatic cells from myotonic dystrophy (DM) fetuses using indirect and direct methods. Heterogeneity of expansions between fetal tissues was demonstrated in a 16 week old fetus whereas there was no evidence for such a somatic heterogeneity in a 13 week old fetus. Dilution plating of cultured cells from an adult patient and a fetus resulted in isolation of clones showing single expanded restriction fragments when the donor showed a heterogeneous smear of expansions or a single expanded fragment. During proliferation in vitro to 45 doublings, DM cells experienced highly synchronous further repeat expansion which first became evident at approximately 15 cell generations and reached a plateau of maximum expansion at approximately 200 days. When mathematically expressed as a function of culture time the dynamics of expansion of restriction fragments followed a sigmoid curve. This unstable behaviour of CTG repeat expansions in DM was compared to the mitotically stable patterns of full mutation in fragile X fetal tissues and led to the hypothesis that methylation of CpGs within the repeat sequence is a stabilizing factor of largely expanded CGG and GCC repeats allowing for efficient methyl-directed strand-specific DNA mismatch repair.     

Analysis of CAG/CTG repeat size in Chinese subjects with schizophrenia and bipolar affective disorder using the repeat expansion detection method

BACKGROUND: Family studies of schizophrenia and bipolar affective disorder provide evidence for genetic anticipation, which (in common with a number of mendelian disorders), may be caused by triplet repeat expansion. This hypothesis is strengthened by evidence from repeat expansion detection (RED) analysis revealing association between the psychoses and long CAG/CTG trinucleotide repeats. METHODS: We performed RED on Han Chinese subjects with schizophrenia (82), bipolar affective disorder (43), and normal controls (61), using a CTG10 oligonucleotide. RESULTS: Comparison between cases and controls revealed no significant association between long repeats and affected status. We also found no detectable association with age at onset and repeat length in either bipolar affective disorder or schizophrenia. Overall, the size distribution of CAG/CTG repeats in Chinese subjects was not significantly different from those reported previously for Caucasian subjects. CONCLUSIONS: These findings indicate that CAG/CTG repeat expansion is not likely to be a major etiological factor for psychosis in Chinese populations.     

Diameter of the inferior vena cava as an index of dry weight in patients undergoing CAPD

Trinucleotide microsatellites are widespread in the human and other mammalian genomes. Expansions of unstable trinucleotide repeats have been associated so far with a number of different genetic diseases including fragile X, myotonic dystrophy (DM) and Huntington disease. While ten possible trinucleotides can occur at the DNA level, only CTG and CCG repeats are involved in the disorders described so far. However, the repeat expansion detection (RED) technique has identified additional large repeats of ATG, CCT, CTT, and TGG of potentially pathological significance in the human genome. We now show that conclusive information about the chromosomal localization of long trinucleotide repeats can be achieved in a relatively short time using fluorescence in situ hybridization (FISH) with biotin-labelled trinucleotide polymers. Large CTG expansions (> 1 kb) in DM and an unstable (CTG)306 repeat in a patient with schizophrenia were detected by eye through the microscope without electronic enhancement. Digital imaging was used to analyse the chromosomal distribution of long CCA and AGG repeats. Our results suggest that long trinucleotide repeats occur in the normal human genome and that the size of individual repeat loci may be polymorphic.     

Exclusion of expansion of 50 CAG/CTG trinucleotide repeats in bipolar disorder

OBJECTIVE: The purpose of this study was to identify the specific expanded CAG/CTG trinucleotide repeat associated with bipolar disorder. METHOD: The study employed an efficient multistage approach for using a genomic CAG/CTG screening set. RESULTS: The authors found no evidence of expanded repeats at 43 polymorphic autosomal loci and seven X chromosomal loci. Secondary screening was pursued at the only locus that contained a large allele (37 repeats) in the primary screening. No association was found between allele size and diagnostic status. CONCLUSIONS: It is highly unlikely that expansions in repeat size at any of the 50 candidate trinucleotide repeat loci examined are responsible for the association between expanded CAG/ CTG repeats and bipolar disorder. However, although the authors prioritized the repeats that were a priori most likely to be involved, the study does not reject the more general hypothesis that expanded CAG/CTG repeats are implicated in the pathogenesis of bipolar disorder.     

Nucleosome assembly on CTG triplet repeats

Expansion of CTG repeat sequences is associated with several human genetic diseases. We have examined the consequences of CTG repeat expansion for nucleosome assembly and positioning. Short CTG repeats are found within the most favored DNA sequences yet defined for nucleosome assembly. We find that as few as six CTG repeats will facilitate nucleosome assembly to a similar extent as the 50 or more repeats found in disease genes. Thus an increase in nucleosome stability on expansion of existing triplet repeats is unlikely to explain the acquisition of the disease phenotype. However, the CTG repeat sequence is efficiently wrapped around the histone octamer, preferring to associate with histones at the nucleosomal dyad. Thus short segments CTG repeat sequence will facilitate the assembly of a stable positioned nucleosome which might contribute to the expansion phenomenon and the functional organization of chromatin.     

The effectiveness of topical diclofenac for lateral epicondylitis

Clinical evidence for a dominant mode of inheritance and anticipation in periodic catatonia, a distinct subtype of schizophrenia, suggests that trinucleotide repeat expansions may be involved in the aetiology of this disorder. Since genes with triplet repeats are putative canditates for causing schizophrenia, we have analysed the polymorphic B33 CTG repeat locus on chromosome 3 in 45 patients with periodic catatonia and 43 control subjects. The B33 CTG repeat locus was highly polymorphic, but all alleles in both the patient and control groups had repeat lengths within the normal range. We conclude that susceptibility to periodic catatonia is not influenced by variation at the B33 CTG repeat locus. Nevertheless, that periodic catatonia displays dominant inheritance and anticipation, characteristic of genetic disorders involving trinucleotide repeats, justifies further screening for triplet repeat expansions in this illness.     

Intellectual impairment and brain MRI findings in myotonic dystrophy: with a special reference to hippocampal atrophy and white matter lesions

We performed a correlative study between intellectual impairment, CTG repeat expansion and magnetic resonance imaging (MRI) abnormalities, including hippocampal atrophy, white matter lesions and ventricular dilatation in 15 patients with myotonic dystrophy (MD). They included 4 males and 11 females aged from 20 to 66 years, averaging 43 years of age and 15 years of duration of illness. Nine patients had intellectual impairment (WAIS-R<80). Negative correlations were found between full scale IQ (FSIQ), duration of illness (p<0.05) and CTG repeat expansion (p<0.05). Compared with normal controls, the patients with MD showed a significant reduction in size of the hippocampal head (p<0.01), which was positively correlated to FSIQ, verbal IQ and performance IQ levels (p<0.05). Ten patients had white matter lesions. Severer white matter lesions tended to be recognized in patients with longer duration of illness and with decreased FSIQ level. These results suggest that hippocampal atrophy and white matter lesions are related to intellectual impairment in patients with MD.     

Human MSH2 binds to trinucleotide repeat DNA structures associated with neurodegenerative diseases

The expansion of trinucleotide repeat sequences is associated with several neurodegenerative diseases. The mechanism of this expansion is unknown but may involve slipped-strand structures where adjacent rather than perfect complementary sequences of a trinucleotide repeat become paired. Here, we have studied the interaction of the human mismatch repair protein MSH2 with slipped-strand structures formed from a triplet repeat sequence in order to address the possible role of MSH2 in trinucleotide expansion. Genomic clones of the myotonic dystrophy locus containing disease-relevant lengths of (CTG)n x (CAG)n triplet repeats were examined. We have constructed two types of slipped-strand structures by annealing complementary strands of DNA containing: (i) equal numbers of trinucleotide repeats (homoduplex slipped structures or S-DNA) or (ii) different numbers of repeats (heteroduplex slipped intermediates or SI-DNA). SI-DNAs having an excess of either CTG or CAG repeats were structurally distinct and could be separated electrophoretically and studied individually. Using a band-shift assay, the MSH2 was shown to bind to both S-DNA and SI-DNA in a structure-specific manner. The affinity of MSH2 increased with the length of the repeat sequence. Furthermore, MSH2 bound preferentially to looped-out CAG repeat sequences, implicating a strand asymmetry in MSH2 recognition. Our results are consistent with the idea that MSH2 may participate in trinucleotide repeat expansion via its role in repair and/or recombination.     

Somatic instability of the myotonic dystrophy (CTG)n repeat during human fetal development

Myotonic dystrophy is characterised by the striking level of somatic heterogeneity seen between and within tissues of the same patient, which probably accounts for a significant proportion of the pleiotropy associated with this disorder. The congenital form of the disease is associated with the largest (CTG)n repeat expansions. We have investigated the timing of instability of myotonic dystrophy (CTG)n repeats in a series of congenitally affected fetuses and neonates. We find that during the first trimester the repeat is apparently stable and that instability only becomes detectable during the second and third trimesters. In our series repeat instability is apparent only after 13 weeks gestational age and before 16 weeks. The appearance of heterogeneity shows some tissue specificity, with heart most commonly having the largest expansion. The degree of heterogeneity is not correlated with initial expansion size as gauged by chorionic villus and blood (CTG)n repeat sizes.     

Spinocerebellar ataxia type 6. Molecular and clinical features of 35 Japanese patients including one homozygous for the CAG repeat expansion

Spinocerebellar ataxia type 6 (SCA6) is a newly classified autosomal-dominant cerebellar ataxia (ADCA) associated with CAG repeat expansion. We screened 111 patients with cerebellar ataxia for the SCA6 mutation. Of these, 35 patients were found to have expanded CAG repeats in the SCA6 gene, indicating that second to SCA3, SCA6 is the most common ADCA in Japan. Expanded alleles ranged from 21 to 29 repeats, whereas normal alleles had seven to 17 repeats. There was no change in the CAG repeat length during meiosis. The age at onset was inversely correlated with the repeat length. The main clinical feature of the 35 patients with SCA6 was slowly progressive cerebellar ataxia; multisystem involvement was not common. The 35 patients included nine cases without apparent family history of cerebellar ataxia. The sporadic cases had smaller CAG repeats (21 or 22 repeats) and a later age at onset (64.9 +/- 4.9 years) than the other cases with established family history. We also identified one patient who was homozygous for the SCA6 repeat expansion. The homozygote showed an earlier age of onset and more severe clinical manifestations than her sister, a heterozygote carrying an expanded allele with the same repeat length as the homozygote. This finding suggests that the dosage of the CAG repeat expansion plays an important role in phenotypic expression in SCA6.     

Expansion and deletion of triplet repeat sequences in Escherichia coli occur on the leading strand of DNA replication

Expansions and deletions of triplet repeat sequences that cause human hereditary neurological diseases were previously suggested to be mediated by the formation of DNA hairpins on the lagging strand during replication. The replication properties of CTG.CAG, CGG.CCG, and TTC.GAA repeats were studied in Escherichia coli using an in vivo phagemid system as a model for continuous leading strand synthesis. The repeats were substantially deleted when the CTG, CGG, and GAA repeats were the templates for rolling circle replication from the f1 phage origin. The deletions may be mediated by hairpins formed by these repeat tracts. The distributions of the deletion products of the CTG.CAG and CGG.CCG tracts indicated that hairpins of discrete sizes mediate deletions during complementary strand synthesis. Deletions during rolling circle synthesis are caused by larger hairpins of specific sizes. Thus, most deletion products were of defined lengths, suggesting a preference for specific hairpin intermediates. Small expansions of the CTG.CAG and CGG.CCG repeats were also observed, presumably due to the formation of CTG and CGG hairpins on the nascent complementary strand. Since rolling circle replication has been established in vitro as a model for leading strand synthesis, we conclude that triplet repeat instability can also occur on the leading strand of DNA replication.     

Pathogenesis of sodium and water retention in cardiac failure

A common mechanism for chromosomal fragile site genesis is not yet apparent. Folate-sensitive fragile sites are expanded p(CCG)n repeats that arise from longer normal alleles. Distamycin A or bromodeoxyuridine-inducible fragile site FRA16B is an expanded AT-rich approximately 33 bp repeat; however, the relationship between normal and fragile site alleles is not known. Here, we report that bromodeoxyuridine-inducible, distamycin A-insensitive fragile site FRA10B is composed of expanded approximately 42 bp repeats. Differences in repeat motif length or composition between different FRA10B families indicate multiple independent expansion events. Some FRA10B alleles comprise a mixture of different expanded repeat motifs. FRA10B fragile site and long normal alleles share flanking polymorphisms. Somatic and intergenerational FRA10B repeat instability analogous to that found in expanded trinucleotide repeats supports dynamic mutation as a common mechanism for repeat expansion.     

The relationship between muscle pathology and DM kinase abnormalities in patients with dystrophia myotonica

We studied the relationship between muscle pathology and the number of trinucleotide (CTG) repeats observed in lymphocytes and biopsied muscle tissues from patients with dystrophia myotonica (DM). The diameter of type 1 muscle fibers was smaller than that of type 2 fibers in all patients. The diameter and proportion of each muscle fiber type were related to the patient's age, but not to the number of trinucleotide (CTG) repeats of DM kinase in biopsied muscles. On the other hand, the proportion of type 1 fibers with central nuclei was closely related to the number of trinucleotide (CTG) repeats in muscles. These findings suggest that DM kinase abnormalities influence the muscle nuclei, and increase the number of central nuclei.     

Molecular genetics of myotonic dystrophy--population genetics of the CTG repeat expansion of the MTPK gene

The CTG repeat number in the 3'-untranslated region of the myotonin protein kinase (MTPK) gene varies between 5 and 37 in normal individuals, whereas myotonic dystrophy (DM) patients have expansions from 50 to 3000 copies. However little is known about the molecular mechanisms or the genetic control of the expansion of triplet repeats. To explain the dynamic mutation mechanism and high prevalence in the population, slippage theory, multistep model and meiotic drive hypothesis have been proposed. Recent studies have shown that repeat expansion may affect neighboring genes (59 gene and DMAHP gene), or exert its effect at the RNA level by modulating the binding of (CUG)n-RNA binding proteins which are required for the maturation, stability and translation of specific mRNAs.     

Determination of the epitope of an inhibitory antibody to proliferating cell nuclear antigen

We identified an expansion of the CAG trinucleotide repeat in the coding region of the Machado-Joseph disease gene in 7 of 24 American families diagnosed with autosomal dominant ataxia. All affected individuals were heterozygous for an expanded allele that ranged from 67 to more than 200 CAG repeats, whereas the normal allele had 14 to 33 repeats. In contrast to the Azorean-Portuguese origins of Machado-Joseph disease, the two largest American families were of German and Dutch-African descent. Clinical, pathologic, and genetic evaluations suggest that American families with spinocerebellar ataxia type 3 differ from those with Machado-Joseph disease by their ethnic origins, predominant spinopontine atrophy, lack of dystonic features, and larger CAG repeat expansion.     

CAG repeat expansion in autosomal dominant familial spastic paraparesis: novel expansion in a subset of patients

Autosomal dominant familial spastic paraplegia (FSP) is a genetically heterogeneous neurodegenerative disorder displaying anticipation for which three loci have been mapped to the chromosomal positions 14q11.2-q24.3 (SPG3), 2p21-p24 (SPG4) and 15q11.1 (SPG6). The repeat expansion detection (RED) method has been used to demonstrate expanded CAG repeats in some FSP families that map to SPG4. We analyzed 20 FSP families, including four for which there is evidence for linkage to SPG4, and found that in most cases the repeat expansion detected by RED is due to non-pathogenic expansions of the chromosome 18q21.1 SEF2-1 or 17q21.3 ERDA1 locus. Polymorphic expansions at SEF2-1 and ERDA1 appear frequent and may confound RED studies in the search for genes causing disorders demonstrating anticipation. In six FSP families, however, CAG repeat expansion was detected in a subset of affected and at-risk individuals that did not result from expansion of the SEF2-1 and ERDA1 loci. Overall, 11 of 37 (30%) of the FSP patients with a CAG/CTG repeat expansion are unaccounted for by the SEF2-1 and ERDA1 loci, compared with two of 23 (9%) of the unaffected at-risk individuals and none of 19 controls. In the majority of cases these novel expansions were shorter than those previously reported.     

Expanding complexity in myotonic dystrophy

Myotonic dystrophy (DM) is a highly variable multisystemic disease belonging to the rather special class of trinucleotide expansion disorders. DM results from dynamic expansion of a perfect (CTG)n repeat situated in a gene-dense region on chromosome 19q. Based on findings in patient materials or cellular and animal models, many mechanisms for the causes and consequences of repeat expansion have been proposed; however, none of them has enjoyed prolonged support. There is now circumstantial evidence that long (CTG)n repeats may affect the expression of any of at least three genes, myotonic dystrophy protein kinase (DMPK), DMR-N9 (gene 59), and a DM-associated homeodomain protein (DMAHP). Furthermore, the new findings suggest that DM is not a simple gene-dosage or gain-or-loss-of-function disorder but that entirely new pathological pathways at the DNA, RNA, or protein level may play a role in its manifestation.