Progression of somatic CTG repeat length heterogeneity in the blood cells of myotonic dystrophy patients

The genetic basis of myotonic dystrophy (DM) is the expansion of an unstable CTG repeat in the 34 UTR of the DM protein kinase gene on chromosome 19. One of the principal features of the DM mutation is an extraordinarily high level of somatic mosaicism, due to an extremely high degree of somatic instability both within and between different tissues. This instability appears to be biased towards further expansion and continuous throughout the life of an individual, features that could be associated with the progressive nature of the disease. Although increasing measured allele size between patients clearly correlates with an increased severity of symptoms and an earlier age of onset, this correlation is not precise and measured allele length cannot be used as an accurate predictor of age of onset. In order to further characterize the dynamics of DM CTG repeat somatic instability, we have studied repeat length changes over time in 111 myotonic dystrophy patients with varying clinical severity and CTG repeat size over time intervals of 1-7 years. We have found a direct progression of the size heterogeneity over time related to initial CTG repeat size and the time interval and always biased towards further expansion. Attempts to mathematically model the dynamics have proved only partially successful suggesting that individual specific genetic and/or environmental factors also play a role in somatic mosaicism.      

Instability of normal (CTG)n alleles in the DM kinase gene.

We report on a myotonic dystrophy (DM) family exhibiting instability of normal sized (CTG)n alleles in the DM kinase gene on the non-DM chromosome. At least two mutational events involving normal DM alleles must have occurred in this family; one was characterised as a 34-35 (CTG)n repeat mutation. These findings represent a dissociation between (CTG)n repeat instability and myotonic dystrophy. Furthermore, this family highlights genetic counselling issues relating to the pathogenicity of alleles at the upper end of the normal size range and the risk of further expansion into the disease range.     

Somatic cell heterogeneity between DNA extracted fromllymphocytes and skeletal muscle in congenital myotonic dystrophy

Summary Myotonic dystrophy (DM) results from the expansion of an unstable CTG trinucleotide repeat in the 3 untranslated region of mRNA encoding a putative serine/threonine protein kinase. The degreeoof the CTG repeat amplification in genomic DNAs extracted from lymphocytes correlates with disease severity. We have analyzed the amplification of the CTG repeat of DNAs extracted from skeletal muscles and lymphocytes in five congenital DM patients. The amplification from skeletal muscles showed an increase of about 1.5 kb to 3.5 kb larger than that from lymphocytes in all patients. Furthermore, we have investigated the somatic instability of the CTG repeat in various tissues from a severe congenital DM patient.     

Transgenic mice carrying large human genomic sequences with expanded CTG repeat mimic closely the DM CTG repeat intergenerational and somatic instability

Myotonic dystrophy (DM) is caused by a CTG repeat expansion in the 3'UTR of the DM protein kinase (DMPK) gene. A very high level of instability is observed through successive generations and the size of the repeat is generally correlated with the severity of the disease and with age at onset. Furthermore, tissues from DM patients exhibit somatic mosaicism that increases with age. We generated transgenic mice carrying large human genomic sequences with 20, 55 or >300 CTG, cloned from patients from the same affected DM family. Using large human flanking sequences and a large amplification, we demonstrate that the intergenerational CTG repeat instability is reproduced in mice, with a strong bias towards expansions and with the same sex- and size-dependent characteristics as in humans. Moreover, a high level of instability, increasing with age, can be observed in tissues and in sperm. Although we did not observe dramatic expansions (or 'big jumps' over several hundred CTG repeats) as in congenital forms of DM, our model carrying >300 CTG is the first to show instability so close to the human DM situation. Our three models carrying different sizes of CTG repeat provide insight on the different factors modulating the CTG repeat instability.     

Comparison of CTG repeat length expansion and clinical progression of myotonic dystrophy over a five year period.

Myotonic dystrophy (DM) is associated with an underlying CTG trinucleotide repeat expansion at a locus on chromosome 19q13.3. We have determined the repeat length in 23 DM patients with varying clinical severity of symptoms and various sizes of repeat amplification. We confirm that as in previous studies there is no strong correlation between repeat length and clinical symptoms but find that the repeat length in peripheral blood cells of patients increases over a time span of five years indicating continuing mitotic instability of the repeat throughout life. Repeat length progression does not appear to be indicative of clinical progression but age probably is. The degree of expansion correlates with the initial repeat size and 50% of the patients with continuing expansions showed clinical progression of their disease symptoms over the five year study period.     

250 CTG repeats in DMPK is a threshold for correlation of expansion size and age at onset of juvenile–adult DM1

Myotonic dystrophy type 1 (DM1) is associated with an expansion of CTG repeats in the 3′UTR of the DMPK gene. It is accepted, as in other trinucleotide diseases, that the number of the repeats is correlated with age at onset and severity of the disease. However, assessment of genotype–phenotype correlation in DM1 is complicated with the expansion‐biased somatic instability of mutant alleles over time and difficulties in precise assessment of the number of repeats by standard Southern blot hybridization. In order to clarify this issue we defined DM1 expansion size in lymphocytes by three parameters: size of progenitor, average, and largest allele, using a more precise small‐pool/long‐range PCR technique. We found a negative linear correlation of age at onset and average expansion size in juvenile‐adult DM1 patients (35 out of 46) whose progenitor allele is less than 245 repeats long. Our result favors the hypothesis of the existence of a threshold in the progenitor allele size beyond which number of CTG repeats does not influence age at onset. Potential clinical significance is that the average allele size could be a useful indicator for the age at onset in juvenile–adult DM1 patients with relatively short progenitor allele. To test whether somatic instability of mutant alleles influences the progression of DM1, patients were divided in three phenotypic classes according to the severity of neuromuscular symptoms. We showed that the largest expansion in each DM1 phenotypic class reflects somatic instability of mutant allele over time independently of progenitor allele size and patient’s age at sampling. The mean of the largest expansion was significantly different between phenotypic classes, implying the possible association between expansion‐biased somatic instability of mutant alleles over time and progression of neuromuscular symptoms. Hum Mutat 19:131–139, 2002. © 2002 Wiley‐Liss, Inc.     

"Mitotic drive" of expanded CTG repeats in myotonic dystrophy type 1 (DM1)

In myotonic dystrophy type 1 (DM1), an expanded CTG repeat shows repeat size instability in somatic and germ line tissues with a strong bias toward further expansion. To investigate the mechanism of this expansion bias, 29 DM1 and six normal lymphoblastoid cell lines (LBCLs) were single-cell cloned from blood cells of 18 DM1 patients and six normal subjects. In all 29 cell lines, the expanded CTG repeat alleles gradually shifted toward further expansion by "step-wise" mutations. Of these 29 cell lines, eight yielded a rapidly proliferating mutant with a gain of large repeat size that became the major allele population, eventually replacing the progenitor allele population. By mixing cell lines with different repeat expansions, we found that cells with larger CTG repeat expansion had a growth advantage over those with smaller expansions in culture. This growth advantage was attributable to increased cell proliferation mediated by Erk1,2 activation, which is negatively regulated by p21(WAF1). This phenomenon, which we designated "mitotic drive" , is a novel mechanism which can explain the expansion bias of DM1 CTG repeat instability at the tissue level, on a basis independent of the DNA-based expansion models. The lifespans of the DM1 LBCLs were significantly shorter than normal cell lines. Thus, we propose a hypothesis that DM1 LBCLs drive themselves to extinction through a process related to increased proliferation.     

Detection of the CTG repeat expansion in congenital myotonic dystrophy

Summary Myotonic dystrophy (DM) is caused by an abnormal expansion of an unstable CTG trinucleotide repeat in the 3′ untranslated region of mRNA encoding a putative serine/threonine protein kinase. We analyzed 59 patients with DM (28 congenital DM families: 27 families with maternal transmission and 1 paternal transmission) and 27 normal control subjects to evaluate their CTG repeat size between DM patients and the normal controls, and to search for a correlation between the clinical characteristics of congenital DM (CDM) and CTG repeat expansions. Analysis was on the basis of the Southern blot and polymerase chain reaction (PCR) methods, and by direct sequencing of PCR amplified CTG repeats. Analysis of intergenerational differences in the CTG repeat size for mother-child pairs showed a positive correlation (y=1.0384x+1265.2,r ²=0.311). In addition to the strong parental bias, this group showed genetic anticipation. There was a significant correlation of the CTG repeat expansion with disease severity. The largest CTG repeat expansion (2,293 CTG repeats) on average belonged to the severe CDM group, and the smallest (129 CTG repeats) to the subclinical DM group. The mutant allele of an asymptomatic father in the paternally transmitted pedigree revealed 75 CTG repeats, demonstrating that he was a DM protomutation carrier.     

Maternal germline-specific effect of DNA ligase I on CTG/CAG instability

The instability of (CTG)?(CAG) repeats can cause >15 diseases including myotonic dystrophy, DM1. Instability can arise during DNA replication, repair or recombination, where sealing of nicks by DNA ligase I (LIGI) is a final step. The role of LIGI in CTG/CAG instability was determined using in vitro and in vivo approaches. Cell extracts from a human (46BR) harbouring a deficient LIGI (～3% normal activity) were used to replicate CTG/CAG repeats; and DM1 mice with >300 CTG repeats were crossed with mice harbouring the 46BR LigI. In mice, the defective LigI reduced the frequency of CTG expansions and increased CTG contraction frequencies on female transmissions. Neither male transmissions nor somatic CTG instability was affected by the 46BR LigI - indicating a post-female germline segregation event. Replication-mediated instability was affected by the 46BR LIGI in a manner that depended upon the location of Okazaki fragment initiation relative to the repeat tract; on certain templates, the expansion bias was unaltered by the mutant LIGI, similar to paternal transmissions and somatic tissues; however, a replication fork-shift reduced expansions and increased contractions, similar to maternal transmissions. The presence of contractions in oocytes suggests that the DM1 replication profile specific to pre-meiotic oogenesis replication of maternal alleles is distinct from that occurring in other tissues and, when mediated by the mutant LigI, is predisposed to CTG contractions. Thus, unlike other DNA metabolizing enzymes studied to date, LigI has a highly specific role in CTG repeat maintenance in the maternal germline, involved in mediating CTG expansions and in the avoidance of maternal CTG contractions.     

Genome-wide demethylation destabilizes CTG.CAG trinucleotide repeats in mammalian cells

Many neurological diseases, including myotonic dystrophy, Huntington's disease and several spinocerebellar ataxias, result from intergenerational increases in the length of a CTG.CAG repeat tract. Although the basis for intergenerational repeat expansion is unclear, repeat tracts are especially unstable during germline development and production of gametes. Mammalian development is characterized by waves of genome-wide demethylation and remethylation. To test whether changes in methylation status might contribute to trinucleotide repeat instability, we examined the effects of DNA methyltransferase inhibitors on trinucleotide repeat stability in mammalian cells. Using a selectable genetic system for detection of repeat contractions in CHO cells, we showed that the rate of contractions increased >1000-fold upon treatment with the DNA methyltransferase inhibitor 5-aza-deoxycytidine (5-aza-CdR). The link between DNA demethylation and repeat instability was strengthened by similar results obtained with hydralazine treatment, which inhibits expression of DNA methyltransferase. In human cells from myotonic dystrophy patients, treatment with 5-aza-CdR strongly destabilized repeat tracts in the DMPK gene, with a clear bias toward expansion. The bias toward expansion events and changes in repeat length that occur in jumps, rather than by accumulation of small changes, are reminiscent of the intergenerational repeat instability observed in human patients. The dramatic destabilizing effect of DNA methyltransferase inhibitors supports the hypothesis that changes in methylation patterns during epigenetic reprogramming may trigger the intergenerational repeat expansions that lead to disease.     

SCA8 CTG repeat: en masse contractions in sperm and intergenerational sequence changes may play a role in reduced penetrance

We recently described an untranslated CTG expansion that causes a previously undescribed form of spinocerebellar ataxia (SCA8). The SCA8 CTG repeat is preceded by a polymorphic but stable CTA tract, with the configuration (CTA)(1-21)(CTG)(n). The CTG portion of the repeat is elongated on pathogenic alleles, which nearly always change in size when transmitted from generation to generation. To better understand the reduced penetrance and maternal penetrance bias associated with SCA8 we analyzed the sequence configurations and instability patterns of the CTG repeat in affected and unaffected family members. In contrast to other triplet repeat diseases, expanded alleles found in affected SCA8 individuals can have either a pure uninterrupted CTG repeat tract or an allele with one or more CCG, CTA, CTC, CCA or CTT interruptions. Surprisingly, we found six different sequence configurations of the CTG repeat on expanded alleles in a seven generation family. In two instances duplication of CCG interruptions occurred over a single generation and in other instances duplications that had occurred in different branches of the family could be inferred. We also evaluated SCA8 instability in sperm samples from individuals with expansions ranging in size from 80 to 800 repeats in blood. Surprisingly the SCA8 repeat tract in sperm underwent contractions, with nearly all of the resulting expanded alleles having repeat lengths of <100 CTGs, a size that is not often associated with disease. These en masse repeat contractions in sperm likely underlie the reduced penetrance associated with paternal transmission.     

Unusual association of a unique CAG interruption in 5′ of DM1 CTG repeats with intergenerational contractions and low somatic mosaicism

Myotonic dystrophy type 1 (DM1) is a dominant multisystemic disorder associated with high variability of symptoms and anticipation. DM1 is caused by an unstable CTG repeat expansion that usually increases in successive generations and tissues. DM1 family pedigrees have shown that ～90% and 10% of transmissions result in expansions and contractions of the CTG repeat, respectively. To date, the mechanisms of CTG repeat contraction remain poorly documented in DM1. In this report, we identified two new DM1 families with apparent contractions and no worsening of DM1 symptoms in two and three successive maternal transmissions. A new and unique CAG interruption was found in 5′ of the CTG expansion in one family, whereas multiple 5′ CCG interruptions were detected in the second family. We showed that these interruptions are associated with maternal intergenerational contractions and low somatic mosaicism in blood. By specific triplet‐prime PCR, we observed that CTG repeat changes (contractions/expansions) occur preferentially in 3′ of the interruptions for both families.     

Cis-acting modifiers of expanded CAG/CTG triplet repeat expandability: associations with flanking GC content and proximity to CpG islands.

An increasing number of human genetic disorders are associated with the expansion of trinucleotide repeats. The majority of these diseases are associated with CAG/CTG expansions, including Huntington's disease, myotonic dystrophy and many of the spinocerebellar ataxias. Recently, two new expanded CAG/CTG repeats have been identified that are not associated with a phenotype. Expanded alleles at all of these loci are unstable, with frequent length changes during intergenerational transmission. However, variation in the relative levels of instability, and the size and direction of the length change mutations observed, between the CAG/CTG loci is apparent. We have quantified these differences, taking into account effects of progenitor allele length, by calculating the relative expandability of each repeat. Since the repeat motifs are the same, these differences must be a result of flanking sequence modifiers. We present data that indicate a strong correlation between the relative expandability of these repeats and the flanking GC content. Moreover, we demonstrate that the most expandable loci are all located within CpG islands. These data provide the first insights into the molecular bases of cis -acting flanking sequences modifying the relative mutability of dispersed expanded human triplet repeats.     

Parental age effects,but no evidence for an intrauterine effect in the transmission of myotonic dystrophy type 1

Myotonic dystrophy type 1 (DM1) is caused by the expansion of an unstable CTG repeat (g.17294_17296(45_1000)) with more repeats associated with increased disease severity and reduced age at onset. Expanded disease-associated alleles are highly unstable in both the germline and soma. Germline instability is expansion biased, providing a molecular explanation for anticipation. Somatic instability is expansion biased, size- and age-dependent, features that have compromised genotype–phenotype correlations and intergenerational studies. We corrected these confounding factors by estimating the progenitor allele length in 54 father–offspring and 52 mother–offspring pairs in Costa Rican DM1 families. Not surprisingly, we found major parental allele length effects on the size of the allele transmitted, the magnitude of the intergenerational length change, the age at onset in the next generation and the degree of anticipation in both male and female transmissions. We also detected, for the first time, an age-of-parent effect for both male and female transmission. Interestingly, we found no evidence for an intrauterine effect in the transmission of congenital DM1, suggesting previous reports may have been an artefact of age-dependent somatic instability and sampling bias. These data provide new insights into the germline dynamics of the CTG repeat and opportunities for providing additional advice and more accurate risk assessments to prospective parents in DM1 families.     

Neuropathology does not Correlate with Regional Differences in the Extent of Expansion of CTG Repeats in the Brain with Myotonic Dystrophy Type 1

Myotonic dystrophy (DM1) is known to be an adult-onset muscular dystrophy caused by the expansion of CTG repeats within the 3' untranslated region of the dystrophin myotonin protein kinase (DMPK) gene. The clinical features of DM1 include CNS symptoms, such as cognitive impairment and personality changes, the pathogenesis of which remains to be elucidated. We hypothesized that the distribution of neuropathological changes might be correlated with the extent of the length of the CTG repeats in the DMPK genes in DM1 patients. We studied the neuropathological changes in the brains of subjects with DM1 and investigated the extent of somatic instability in terms of CTG repeat expansion in the different brain regions of the same individuals by Southern blot analysis. The neuropathological changes included état criblé in the cerebral deep white matter and neurofibrillary tangles immunoreactive for phosphorylated tau in the hippocampus and entorhinal cortex, both of which were compatible with the subcortical dementia in DM1 patients. However, the length of the CTG repeats did not correlate with the regional differences in the extent of neuropathological changes. Our data suggested that pathomechanisms of dementia in DM1 might be more multifactorial rather than a toxic gain-of-function due to mutant RNA.     

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.      

Prenatal diagnosis of congenital myotonic dystrophy and counseling of the pregnant mother: Case report and literature review

The molecular basis of the myotonic dystrophy (MD) kinase gene is expansion of the CTG repeat at the 3′-untranslated region of the MD gene. Variability of the CTG repeat size in different tissues of affected individuals has been demonstrated. The objective of this report was to examine and review the feasibility of prenatal diagnosis of congenital myotonic dystrophy (CMD) in pregnant women with MD using CTG repeat sizes in amniocytes or villi. We present a case of a pregnant woman with MD who underwent prenatal diagnosis of MD using amniocytes. The repeat size in the amniocytes was smaller than the repeat size in the maternal leukocytes and smaller than the repeat size in the infant blood. The infant had CMD. We also reviewed the literature for reports on MD cases that were prenatally tested for CTG repeat size using amniocytes or chorionic villi. Data were tabulated based on the number of maternal CTG repeats, prenatal procedure [amniocentesis or chorionic villus sampling (CVS)], CTG repeat size in fetal tissue, fetal/infant blood, and pregnancy outcome. Twenty-seven pregnancies at risk for MD that underwent prenatal diagnosis were reported. Eleven (40.7%) of the 27 pregnancies underwent amniocentesis, and 16 (59.3%) underwent CVS. Fourteen patients (61%) demonstrated an increase in CTG repeat size in the amniocytes or villi compared with the maternal repeat size. Nine (33%) of the 27 pregnancies were terminated because of CMD risk. The outcomes of 11 (40.7%) pregnancies were consistent with diagnosis of CMD. CMD was diagnosed in fetuses demonstrating expansion or contraction of the CTG mutation in the amniocytes. Prenatal diagnosis of MD is possible by using mutation analysis on maternal and fetal DNA and detection of the CTG repeat expansion. Prenatal diagnosis of CMD is more complex. The possible lack of correlation between CTG repeat size in amniocytes, villi, and other fetal tissues is a potential limitation in prenatal diagnosis and counseling of CMD using CTG repeat size. Thus, prenatal diagnosis of CMD should be based on a combination of factors, including maternal pregnancy history, clinical findings, and cautious interpretation of maternal and fetal DNA analysis. Am. J. Med. Genet. 78:250–253, 1998. © 1998 Wiley-Liss, Inc.     

Direct molecular diagnosis of myotonic dystrophy

Hecht BK, Donnelly A, Gedeon AK, Byard RW, Haan EA, Mulley JC. Direct molecular diagnosis of myotonic dystrophy. Clin Genet 1993: 43: 276–285. © Munksgaard, 1993 Myotonic dystrophy (DM) arises from an unstable trinucleotide (CTG_n) 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.