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.     

Dominantly inherited,non-coding microsatellite expansion disorders

Dominantly inherited diseases are generally caused by mutations resulting in gain of function protein alterations. However, a CTG expansion located in the 3' untranslated portion of a kinase gene was found to cause myotonic dystrophy type 1, a multisystemic dominantly inherited disorder. The recent discovery that an untranslated CCTG expansion causes the same constellation of clinical features in myotonic dystrophy type 2 (DM2), along with other recent discoveries on DM1 pathogenesis, have led to the understanding that both DM1 and DM2 mutations are pathogenic at the RNA level. These findings indicate the existence of a new category of disease wherein repeat expansions in RNA alter cellular function. Pathogenic repeat expansions in RNA may also be involved in spinocerebellar ataxia types 8, 10 and 12, and Huntington's disease-like type 2.     

De novo myotonic dystrophy mutation in a Nigerian kindred.

An expansion of an unstable (CTG)n trinucleotide repeat in the 3' UTR of a gene encoding a putative serine/threonine protein kinase (DMPK) on human chromosome 19q13.3 has been shown to be specific for the myotonic dystrophy (DM) disease phenotype. In addition, a single haplotype composed of nine alleles within and flanking DMPK over a physical distance of 30 kb has been shown to be in complete linkage disequilibrium with DM. This has led to two hypotheses: (1) predisposition for (CTG)n instability results from a founder effect that occurred only once or a few times in human evolution; and (2) elements within the disease haplotype may predispose the (CTG)n repeat to instability. A detailed haplotype analysis of the DM region was conducted on a Nigerian (Yoruba) DM family, the only indigenous sub-Saharan DM case reported to date. Each affected member of this family had an expanded (CTG)n repeat in one of his or her DMPK alleles. However, unlike all other DM populations studied thus far, disassociation of the (CTG)n repeat expansion from other alleles of the putative predisposing haplotype was found. We conclude that the expanded (CTG)n repeat in this family is the result of an independent mutational event. Consequently, the origin of DM is unlikely to be a single mutational event, and the hypothesis that a single ancestral haplotype predisposes to repeat expansion is not compelling.     

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.     

(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.     

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.     

Stability of triplet repeats of myotonic dystrophy and fragile X loci in human mutator mismatch repair cell lines

At least nine human genetic diseases, including myotonic dystrophy (DM) and fragile X syndrome have been associated with the expansion of CTG or CGG trinucleotide repeats within the disease loci. Little is known about the molecular mechanisms or the genetic control of the expansion of triplet repeats. Mutations in human mismatch repair genes are associated with the increased polymorphism of many microsatellites, including dinucleotide repeats. The effect of mutations in two mismatch repair genes on the size of trinucleotide repeats in the DM and FRAXA loci has been analyzed. PCR and Southern analysis of the triplet repeat regions of the DM and fragile X mental retardation (FRAXA) loci in cell lines HTC116 and LoVo, which contain mutations in both alleles of the hMLH1 and hMSH2 genes, respectively, indicated that the size of the endogenous (CTG)_n and (CGG)_n tracts fall within the range observed in the normal population. This suggests that mutations in hMLH1 or hMSH2 do not result in the instability of CTG or CGG tracts to the levels observed in individuals with myotonic dystrophy or fragile X syndrome. Received: 4 December 1995 / Revised: 29 January 1996, 7 March 1996     

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     

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)     

Fen1 does not control somatic hypermutability of the (CTG)(n)*(CAG)(n) repeat in a knock-in mouse model for DM1

The mechanism of trinucleotide repeat expansion, an important cause of neuromuscular and neurodegenerative diseases, is poorly understood. We report here on the study of the role of flap endonuclease 1 (Fen1), a structure-specific nuclease with both 5' flap endonuclease and 5'-3' exonuclease activity, in the somatic hypermutability of the (CTG)(n)*(CAG)(n) repeat of the DMPK gene in a mouse model for myotonic dystrophy type 1 (DM1). By intercrossing mice with Fen1 deficiency with transgenics with a DM1 (CTG)(n)*(CAG)(n) repeat (where 104n110), we demonstrate that Fen1 is not essential for faithful maintenance of this repeat in early embryonic cleavage divisions until the blastocyst stage. Additionally, we found that the frequency of somatic DM1 (CTG)(n)*(CAG)(n) repeat instability was essentially unaltered in mice with Fen1 haploinsufficiency up to 1.5 years of age. Based on these findings, we propose that Fen1, despite its role in DNA repair and replication, is not primarily involved in maintaining stability at the DM1 locus.     

CTG repeats distribution and Alu insertion polymorphism at myotonic dystrophy (DM) gene in Amhara and Oromo populations of Ethiopia

Myotonic dystrophy (DM) is a dominantly inherited neuromuscular disease, highly variable and multisystemic, which is caused by the expansion of a CTG repeat located in the 3′ untranslated region of the DMPK gene. Normal alleles show a copy number of 5–37 repeats on normal chromosomes, amplified to 50–3000 copies on DM chromosomes. The trinucleotide repeat shows a trimodal allele distribution in the majority of the examined population. The first class includes alleles carrying (CTG)₅, the second class, alleles in the range 7–18 repeats, and the third class, alleles (CTG)_{≥ 19}. The frequency of this third class is directly related to the prevalence of DM in different populations, suggesting that normal large-sized alleles predispose toward DM. We studied CTG repeat allele distribution and Alu insertion and/or deletion polymorphism at the myotonic dystrophy locus in two major Ethiopian populations, the Amhara and Oromo. CTG allele distribution and haplotype analysis on a total of 224 normal chromosomes showed significant differences between the two ethnic groups. These differences have a bearing on the out-of-Africa hypothesis for the origin of the DM mutation. In addition, (CTG)_{≥ 19} alleles were exclusively detected in the Amhara population, confirming the predisposing role of these alleles compared with the DM expansion-mutation. Electronic Publication     

New founder haplotypes at the myotonic dystrophy locus in southern Africa.

The association between normal alleles at the CTG repeat and two nearby polymorphisms in the myotonin protein kinase gene, the Alu insertion/deletion polymorphism and the myotonic dystrophy kinase (DMK)(G/T) intron 9/HinfI polymorphism, has been analyzed in South African Negroids, a population in which myotonic dystrophy (DM) has not been described. South African Negroids have a CTG allelic distribution that is significantly different from that in Caucasoids and Japanese: the CTG repeat lengths of > or = 19 are very rare. The striking linkage disequilibrium between specific alleles at the Alu polymorphism (Alu(ins) and Alu(del)), the HinfI polymorphism (HinfI-1 and HinfI-2), and the CTG repeat polymorphism seen in Caucasoid (Europeans and Canadians) populations was also found in the South African Negroid population. Numerous haplotypes, not previously described in Europeans, were, however, found. It thus seems likely that only a small number of these "African" chromosomes were present in the progenitors of all non-African peoples. These data provide support for the "out of Africa" model for the origin of modern humans and suggest that the rare ancestral DM mutation event may have occurred after the migration from Africa, hence the absence of DM in sub-Saharan Negroid peoples.     

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.     

Delineation of CTG repeats and clinical features in a Taiwanese myotonic dystrophy family

Myotonic dystrophy (DM) is an inherited, autosomal dominant muscular disease which is primarily caused by a CTG trinucleotide expansion mutation on chromosome 19q13.3. The size of this trinucleotide repeat is related both to the age of onset and to the severity of the clinical manifestation. This disease is very rare in Taiwan, and clinical and genetic study on DM has not yet been documented in this area. Here, we present both clinical features and degrees of CTG expansion for a Taiwanese DM family. All of the DM patients examined in this family showed obvious clinical manifestations by age 30, which included facial and limb muscle weakness with atrophy, myotonia, and ptosis. In addition, individual DM members also exhibited variable phenotypes, which may reflect the complexity of the pathogenic mechanism. Because the collection of blood specimens was considered to be an invasive procedure, a genetic study on this DM family was performed using buccal cells. Our results confirmed that four members showing classic symptoms of DM had CTG repeat expansion in the DMI locus, and that one member with ptosis and minor muscle weakness in the right foot was a normal homozygote for CTG repeat. These data demonstrate that buccal cells can provide clear and reliable results, and thus, are suitable for a family study of DM.     

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.