Gonosomal mosaicism in myotonic dystrophy patients: involvement of mitotic events in (CTG)n repeat variation and selection against extreme expansion in sperm.

Myotonic dystrophy (DM) is caused by abnormal expansion of a polymorphic (CTG)n repeat, located in the DM protein kinase gene. We determined the (CTG)n repeat lengths in a broad range of tissue DNAs from patients with mild, classical, or congenital manifestation of DM. Differences in the repeat length were seen in somatic tissues from single DM individuals and twins. Repeats appeared to expand to a similar extent in tissues originating from the same embryonal origin. In most male patients carrying intermediate- or small-sized expansions in blood, the repeat lengths covered a markedly wider range in sperm. In contrast, male patients with large allele expansions in blood (> 700 CTGs) had similar or smaller repeats in sperm, when detectable. Sperm alleles with > 1,000 CTGs were not seen. We conclude that DM patients can be considered gonosomal mosaics, i.e., combined somatic and germ-line tissue mosaics. Most remarkably, we observed multiple cases where the length distributions of intermediate- or small-sized alleles in fathers'' sperm were significantly different from that in their offspring''s blood. Our combined findings indicate that intergenerational length changes in the unstable CTG repeat are most likely to occur during early embryonic mitotic divisions in both somatic and germ-line tissue formation. Both the initial CTG length, the overall number of cell divisions involved in tissue formation, and perhaps a specific selection process in spermatogenesis may influence the dynamics of this process. A model explaining mitotic instability and sex-dependent segregation phenomena in DM manifestation is discussed.     

Chemotherapeutic deletion of CTG repeats in lymphoblast cells from DM1 patients

Myotonic dystrophy type 1 (DM1) is caused by the expansion of a (CTG).(CAG) repeat in the DMPK gene on chromosome 19q13.3. At least 17 neurological diseases have similar genetic mutations, the expansion of DNA repeats. In most of these disorders, the disease severity is related to the length of the repeat expansion, and in DM1 the expanded repeat undergoes further elongation in somatic and germline tissues. At present, in this class of diseases, no therapeutic approach exists to prevent or slow the repeat expansion and thereby reduce disease severity or delay disease onset. We present initial results testing the hypothesis that repeat deletion may be mediated by various chemotherapeutic agents. Three lymphoblast cell lines derived from two DM1 patients treated with either ethylmethanesulfonate (EMS), mitomycin C, mitoxantrone or doxorubicin, at therapeutic concentrations, accumulated deletions following treatment. Treatment with EMS frequently prevented the repeat expansion observed during growth in culture. A significant reduction of CTG repeat length by 100-350 (CTG).(CAG) repeats often occurred in the cell population following treatment with these drugs. Potential mechanisms of drug-induced deletion are presented.     

MSH2-dependent germinal CTG repeat expansions are produced continuously in spermatogonia from DM1 transgenic mice

Myotonic dystrophy type 1 is a neuromuscular affection associated with the expansion of an unstable CTG repeat in the DM protein kinase gene. The disease is characterized by somatic tissue-specific mosaicism and very high intergenerational instability with a strong bias towards expansions. We used transgenic mice carrying more than 300 unstable CTG repeats within their large human genomic environment to investigate the dynamics of CTG repeat germinal mosaicism in males. Germinal mosaicism towards expansions was already present in spermatozoa at 7 weeks of age and continued to increase with age, suggesting that expansions are continuously produced throughout life. To determine the precise stage at which germinal expansions occur during spermatogenesis, we sorted and collected the different germ cell types produced during spermatogenesis from males of different ages and analyzed the CTG repeat mosaicism in each fraction. Strong mosaicisms towards expansions were already observed in spermatogonia before meiosis. In transgenic Msh2-deficient mice, germinal instability of the CTG repeats (only contractions) also occurs premeiotically. No significant difference in mosaicism was detected between spermatogonia and spermatozoa, arguing against continued expansions during postmeiotic stages. This indicates that germinal expansions are produced at the beginning of spermatogenesis, in spermatogonia, by a meiosis-independent mechanism involving MSH2.     

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)     

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%).     

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.     

(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.     

Haploinsufficiency of yeast FEN1 causes instability of expanded CAG/CTG tracts in a length-dependent manner

Trinucleotide repeat diseases, such as Huntington's disease, are caused by the expansion of trinucleotide repeats above a threshold of about 35 repeats. Once expanded, the repeats are unstable and tend to expand further both in somatic cells and during transmission, resulting in a more severe disease phenotype. Flap endonuclease 1 (Fen1), has an endonuclease activity specific for 5' flap structures and is involved in Okazaki fragment processing and base excision repair. Fen1 also plays an important role in preventing instability of CAG/CTG trinucleotide repeat sequences, as the expansion frequency of CAG/CTG repeats is increased in FEN1 mutants in vitro and in yeast cells defective for the yeast homolog, RAD27. Here we have tested whether one copy of yeast FEN1 is enough to maintain CAG/CTG tract stability in diploid yeast cells. We found that CAG/CTG repeats are stable in RAD27 +/- cells if the tract is 70 repeats long and exhibit a slightly increased expansion frequency if the tract is 85 or 130 repeats long. However for CAG-155 tracts, the repeat expansion frequency in RAD27 +/- cells is significantly higher than in RAD27 +/+ cells. This data indicates that cells containing longer CAG/CTG repeats need more Fen1 protein to maintain tract stability and that maintenance of long CAG/CTG repeats is particularly sensitive to Fen1 levels. Our results may explain the relatively small effects seen in the Huntington's disease (HD) FEN1 +/- heterozygous mice and myotonic dystrophy type 1 (DM1) FEN1 +/- heterozygous mice, and suggest that inefficient flap processing by Fen1 could play a role in the continued expansions seen in humans with trinucleotide repeat expansion diseases.     

Distribution of CTG repeats at the DMPK gene in myotonic distrophy patients and healthy individuals from the Mexican population

Myotonic dystrophy type 1 (DM1), the most common form of adult muscular dystrophy, is caused by anormal expansion of CTG trinucleotide repeats located in the 3′-untranslated region of the DMPK gene. The clinical features of DM1 are multisystemic and highly variable, and the unstable nature of CTG expansion causes wide genotypic and phenotypic presentations. In this study, we described to our knowledge for the first time the molecular diagnosis of myotonic dystrophy type 1 patients in the Mexican population, applying a fluorescent PCR method in combination with capillary electrophoresis analysis of the amplified products. We identified expanded alleles in 45 out of 50 patients (90%) with clinical features of myotonic disease. Furthermore, genotyping of 400 healthy subjects revealed the presence of 25 different alleles, ranging in size from 5 to 34 repeats. The most frequent allele was 13 CTG repeats (38.87%) and the frequency for alleles over 18 CTG repeats was 6.7%. Molecular test is essential for DM1 diagnosis and distribution of the CTG repeat alleles present in the Mexican population are significantly different from those of other populations.     

Characteristics of Intergenerational Contractions of the CTG Repeat in Myotonic Dystrophy

In myotonic dystrophy (DM), the size of a CTG repeat in the DM kinase gene generally increases in successive generations with clinical evidence of anticipation. However, there have also been cases with an intergenerational contraction of the repeat. We examined 1,489 DM parent-offspring pairs, of which 95 (6.4%) showed such contractions in peripheral blood leukocytes (PBL). In 56 of the 95 pairs, clinical data allowed an analysis of their anticipation status. It is surprising that anticipation occurred in 27 (48%) of these 56 pairs, while none clearly showed a later onset of DM in the symptomatic offspring. The contraction occurred in 76 (10%) of 753 paternal transmissions and in 19 (3%) of 736 maternal transmissions. Anticipation was observed more frequently in maternal (85%) than in paternal (37%) transmissions (P < .001). The parental repeat size correlated with the size of intergenerational contraction (r² = .50, P « .001), and the slope of linear regression was steeper in paternal (–.62) than in maternal (–.30) transmissions (P « .001). Sixteen DM parents had multiple DM offspring with the CTG repeat contractions. This frequency was higher than the frequency expected from the probability of the repeat contractions (6.4%) and the size of DM sib population (1.54 DM offspring per DM parent, in 968 DM parents). We conclude that (1) intergenerational contraction of the CTG repeat in leukocyte DNA frequently accompanies apparent anticipation, especially when DM is maternally transmitted, and (2) the paternal origin of the repeat and the presence of the repeat contraction in a sibling increase the probability of the CTG repeat contraction.     

Msh3 is a limiting factor in the formation of intergenerational CTG expansions in DM1 transgenic mice

The CTG repeat involved in myotonic dystrophy is one of the most unstable trinucleotide repeats. However, the molecular mechanisms underlying this particular form of genetic instability—biased towards expansions—have not yet been completely elucidated. We previously showed, with highly unstable CTG repeat arrays in DM1 transgenic mice, that Msh2 is required for the formation of intergenerational and somatic expansions. To identify the partners of Msh2 in the formation of intergenerational CTG repeat expansions, we investigated the involvement of Msh3 and Msh6, partners of Msh2 in mismatch repair. Transgenic mice with CTG expansions were crossed with Msh3- or Msh6-deficient mice and CTG repeats were analysed after maternal and paternal transmissions. We demonstrated that Msh3 but not Msh6 plays also a key role in the formation of expansions over successive generation. Furthermore, the absence of one Msh3 allele was sufficient to decrease the formation of expansions, indicating that Msh3 is rate-limiting in this process. In the absence of Msh6, the frequency of expansions decreased only in maternal transmissions. However, the significantly lower levels of Msh2 and Msh3 proteins in Msh6 -/- ovaries suggest that the absence of Msh6 may have an indirect effect.     

Small increase in triplet repeat length of cerebellum from patients with myotonic dystrophy

Myotonic dystrophy (DM) is genetically characterized by abnormal expansion of an unstable CTG trinucleotide repeat, located in the 3′-untranslated region of mRNA encoding the family of serine-threonine protein kinases. DNA extracted from various organs of patients with DM was analyzed by the Southern blotting method. We identified differently expanded bands in DNAs from various tissues from patients with DM. In studying the length of the CTG repeat in different regions of the brain, we found a noticeably small increase in repeat length in the cerebellum compared with other tissues. While this phenomenon has been reported in other triplet repeat diseases such as Huntington disease, spinocerebellar ataxia type 1, and dentatorubral-pallidoluysian atrophy, we are the first to describe it in DM. Although the mechanism of expansion of the triplet repeat remains to be defined, the tissue-dependent somatic mosaicism suggests that its occurrence may depend on the differentiated state of each tissue. Received: 18 October 1995 / Revised: 20 March 1996     

Mutagenic stress modulates the dynamics of CTG repeat instability associated with myotonic dystrophy type 1

The molecular basis of the myotonic dystrophy type 1 is the expansion of a CTG repeat at the DMPK locus. The expanded disease-associated repeats are unstable in both somatic and germ lines, with a high tendency towards expansion. The rate of expansion is directly related to the size of the pathogenic allele, increasing the size heterogeneity with age. It has also been suggested that additional factors, including as yet unidentified environmental factors, might affect the instability of the expanded CTG repeats to account for the observed CTG size dynamics over time. To investigate the effect of environmental factors in the CTG repeat instability, three lymphoblastoid cell lines were established from two myotonic dystrophy patients and one healthy individual, and parallel cultures were concurrently expanded in the presence or absence of the mutagenic chemical mitomycin C for a total of 12 population doublings. The new alleles arising along the passages were analysed by radioactive small pool PCR and sequencing gels. An expansion bias of the stepwise mutation was observed in a (CTG)₁₂₄ allele of a cell line harbouring two modal alleles of 28 and 124 CTG repeats. Interestingly, this expansion bias was clearly enhanced in the presence of mitomycin C. The effect of mitomycin C was also evident in the normal size alleles in two cell lines with alleles of 13/13 and 12/69 repeats, where treated cultures showed new longer alleles. In conclusion, our results indicate that mitomycin C modulates the dynamics of myotonic dystrophy-associated CTG repeats in LBCLs, enhancing the expansion bias of long-pathogenic repeats and promoting the expansion of normal length repeats.