Myotonic dystrophy type 1-associated CTG repeats disturb the expression and subcellular distribution of microtubule-associated proteins MAP1A, MAP2, and MAP6/STOP in PC12 cells

To study the effect of DM1-associated CTG repeats on neuronal function, we developed a PC12 cell-based model that constitutively expresses the DMPK gene 3′-untranslated region with 90 CTG repeats (CTG90 cells). As CTG90 cells exhibit impaired neurite outgrowth and as microtubule-associated proteins (MAPs) are crucial for microtubule stability, we analyzed whether MAPs are a target of CTG repeats. NGF induces mRNA expression of Map2, Map1a and Map6 in control cells (PC12 cells transfected with the empty vector), but this induction is abolished for Map2 and Map1a in CTG90 cells. MAP2 and MAP6/STOP proteins decrease in NGF-treated CTG90 cells, whereas MAP1A increases. Data suggest that CTG repeats might alter somehow the expression of MAPs, which appears to be related with CTG90 cell-deficient neurite outgrowth. Decreased MAP2 levels found in the hippocampus of a DM1 mouse model indicates that targeting of MAPs expression by CTG repeats might be relevant to DM1.     

Influence of sex of the transmitting parent as well as of parental allele site on the CTG expansion in myotonic dystrophy (DM)

In patients with myotonic dystrophy (DM), the severity of clinical signs is correlated with the length of a (CTG)_n trinucleotide repeat sequence. This sequence tends to expand in subsequent generations. In order to examine the kinetics of this process and, in particular, the influence of the mutant-allele size and the sex of the transmitting parent, we have studied (CTG)_n repeat lengths in the offspring of 38 healthy carriers with small mutations (less than 100 CTG trinucleotides, mean length [CTG]₆₇). In these studies, we found a weakly positive correlation between the size of the mutation in the carrier parents and that in their offspring. Furthermore, we observed that, in the offspring of male transmitters, repeat lengths exceeding 100 CTG trinucleotides were much more frequent than in the offspring of carrier females (48 [92%] of 52 vs. 7 [44%] of 16, P = .0002). Similarly, in genealogical studies performed in 38 Dutch DM kindreds, an excess of nonmanifesting male transmitters was noted, which was most conspicuous in the generation immediately preceding that with phenotypic expression of DM. Thus, two separate lines of evidence suggest that the sex of the transmitting parent is an important factor that determines DM allele size in the offspring. On the basis of our data, we estimate that when both parents are asymptomatic, the odds are approximately 2:1 that the father carries the DM mutation. Because expansion of the CTG repeat is more rapid with male transmission, negative selection during spermatogenesis may be required to explain the exclusive maternal inheritance of severe congenital onset DM.     

Myotonic dystrophy: An unstable CTG repeat in a protein kinase gene

Myotonic dystrophy (DM) is caused by the amplification of CTG repeats in the 3′ untranslated region of a gene encoding a protein homologous to serine/threonine protein kinases. In DM patients the CTG repeats are extremely unstable, varying in length from patient to patient and generally increasing in length in successive generations. There is a strong correlation between the size of the repeats and the age of onset and severity of the disease. The molecular basis of the effect of the CTG expansion on the development of the DM phenotype continues to be investigated. The first working hypothesis of the molecular mechanism of DM was a reduction in steady-state myotonin-protein kinase (Mt-PK) mRNA and protein levels. However, although the consensus finding is that the Mt PK mRNA and protein levels are decreased in DM patients, it is still not clear if this reduction leads directly to the DM phenotype. In this short review we discuss the molecular aspects of CTG instability and the expression of the myotonin-protein kinase gene in normal and DM populations.     

Pathogenic RNA repeats: an expanding role in genetic disease

Fragile X mental retardation and Friedreich's ataxia were among the first pathogenic trinucleotide repeat disorders to be described in which noncoding repeat expansions interfere with gene expression and cause a loss of protein production. Invoking a similar loss-of-function hypothesis for the CTG expansion causing myotonic dystrophy type 1 (DM1) located in the 3' noncoding portion of a kinase gene was more difficult because DM is a dominantly inherited multisystemic disorder in which the second copy of the gene is unaffected. However, the discovery that a transcribed but untranslated CCTG expansion causes myotonic dystrophy type 2 (DM2), along with other discoveries on DM1 and DM2 pathogenesis, indicate that the CTG and CCTG expansions are pathogenic at the RNA level. This review will detail recent developments on the molecular mechanisms of RNA pathogenesis in DM, and the growing number of expansion disorders that might involve similar pathogenic RNA mechanisms.     

Replication inhibitors modulate instability of an expanded trinucleotide repeat at the myotonic dystrophy type 1 disease locus in human cells

Gene-specific CTG/CAG repeat expansion is associated with at least 14 human diseases, including myotonic dystrophy type 1 (DM1). Most of our understanding of trinucleotide instability is from nonhuman models, which have presented mixed results, supporting replication errors or processes independent of cell division as causes. Nevertheless, the mechanism occurring at the disease loci in patient cells is poorly understood. Using primary fibroblasts derived from a fetus with DM1, we have shown that spontaneous expansion of the diseased (CTG)(216) allele occurred in proliferating cells but not in quiescent cells. Expansions were "synchronous," with mutation frequencies approaching 100%. Furthermore, cells were treated with agents known to alter DNA synthesis but not to directly damage DNA. Inhibiting replication initiation with mimosine had no effect upon instability. Inhibiting both leading- and lagging-strand synthesis with aphidicolin or blocking only lagging strand synthesis with emetine significantly enhanced CTG expansions. It was striking that only the expanded DM1 allele was altered, leaving the normal allele, (CTG)(12), and other repeat loci unaffected. Standard and small-pool polymerase chain reaction revealed that inhibitors enhanced the magnitude of short expansions in most cells threefold, whereas 11%-25% of cells experienced gains of 122-170 repeats, to sizes of (CTG)(338)-(CTG)(386). Similar results were observed for an adult DM1 cell line. Our results support a role for the perturbation of replication fork dynamics in DM1 CTG expansions within patient fibroblasts. This is the first report that repeat-length alterations specific to a disease allele can be modulated by exogenously added compounds.     

Risk prediction for clinical phenotype in myotonic dystrophy type 1: data from 2,650 patients

Myotonic dystrophy type 1 (DM1) is a multisystem disorder that affects skeletal and smooth muscle as well as the eye, heart, endocrine system, and central nervous system. DM1 is caused by expansion of a CTG trinucleotidedaggerrepeat in the gene DMPK. Clinical findings in DM1 span a continuum from mild to severe. Although the CTG repeat correlates with the disease phenotype, caution is used in predicting disease severity on the basis of CTG repeat number. This study reports an extensive genotype-phenotype study to evaluate the clinical validity and clinical utility of the molecular genetic test. Data were analyzed by multiple logistic regression, used to estimate the odds ratio (OR) and correlation coefficients for patients phenotype in respect to the categorical variables expansion class, gender, familiarity, and the continuous variables age and disease duration. We assessed disease expression by clinical evaluation and the molecular genetic test in 2,650 patients identified by accurate clinical diagnosis and family segregation. We were able to estimate OR and correlation coefficients for patients phenotype according to CTG number. A genotype-phenotype correlation was established to derivate a clinical predictive risk on the basis of molecular data. This study demonstrates that measurement of triplet expansions in patients' DNA can be considered as a useful tool for DM1 phenotype assessment and presymptomatic testing.     

CTG instability in myotonic dystrophy: molecular genetic analysis of families from south-eastern France with characteristics of intergenerational variation in CGT repeat numbers.

We report clinical, genetical and genealogical findings in 149 French families from the Rh?ne-Alpes area studied over a 5-year period. There was a significant excess of DM females compared to DM males with (CTG) repeat sizes between 1-2 kb. The mean maternal (CTG) repeat size was higher than paternal repeat size. Anticipation phenomenom was significantly higher after maternal than after paternal transmission. A significant correlation between parental (CTG) repeat size and intergenerational variation both in paternal and maternal transmissions was observed. The anticipation phenomenom was more important for sons than daughters particularly after maternal transmission. The mean (CTG) repeat size in mothers of CDM cases was about twice that of mothers of NCDM children. The risk of giving birth to a CDM child increased considerably when the number of maternal (CTG) repeats was over 300 (CTG). A significant excess of DM females was observed. They had on average 24% fewer children than male patients. Paternal transmission (63.6%) of DM occurred more frequently than maternal transmission (52.7%).     

Altered replication in human cells promotes DMPK (CTG)(n) · (CAG)(n) repeat instability

Myotonic dystrophy type 1 (DM1) is associated with expansion of (CTG)(n) · (CAG)(n) trinucleotide repeats (TNRs) in the 3' untranslated region (UTR) of the DMPK gene. Replication origins are cis-acting elements that potentiate TNR instability; therefore, we mapped replication initiation sites and prereplication complex protein binding within the ~10-kb DMPK/SIX5 locus in non-DM1 and DM1 cells. Two origins, IS(DMPK) and IS(SIX5), flanked the (CTG)(n) · (CAG)(n) TNRs in control cells and in DM1 cells. Orc2 and Mcm4 bound near each of the replication initiation sites, but a dramatic change in (CTG)(n) · (CAG)(n) replication polarity was not correlated with TNR expansion. To test whether (CTG)(n) · (CAG)(n) TNRs are cis-acting elements of instability in human cells, model cell lines were created by integration of cassettes containing the c-myc replication origin and (CTG)(n) · (CAG)(n) TNRs in HeLa cells. Replication forks were slowed by (CTG)(n) · (CAG)(n) TNRs in a length-dependent manner independent of replication polarity, implying that expanded (CTG)(n) · (CAG)(n) TNRs lead to replication stress. Consistent with this prediction, TNR instability increased in the HeLa model cells and DM1 cells upon small interfering RNA (siRNA) knockdown of the fork stabilization protein Claspin, Timeless, or Tipin. These results suggest that aberrant DNA replication and TNR instability are linked in DM1 cells.