Somatic instability of the expanded CTG triplet repeat in myotonic dystrophy type 1 is a heritable quantitative trait and modifier of disease severity (original) (raw)
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Human Molecular Genetics, 2012
Several human genetic diseases are associated with inheriting an abnormally large unstable DNA simple sequence repeat. These sequences mutate, by changing the number of repeats, many times during the lifetime of those affected, with a bias towards expansion. These somatic changes lead not only to the presence of cells with different numbers of repeats in the same tissue, but also produce increasingly longer repeats, contributing towards the progressive nature of the symptoms. Modelling the progression of repeat length throughout the lifetime of individuals has potential for improving prognostic information as well as providing a deeper understanding of the underlying biological process. A large data set comprising blood DNA samples from individuals with one such disease, myotonic dystrophy type 1, provides an opportunity to parameterize a mathematical model for repeat length evolution that we can use to infer biological parameters of interest. We developed new mathematical models by modifying a proposed stochastic birth process to incorporate possible contraction. A hierarchical Bayesian approach was used as the basis for inference, and we estimated the distribution of mutation rates in the population. We used model comparison analysis to reveal, for the first time, that the expansion bias observed in the distributions of repeat lengths is likely to be the cumulative effect of many expansion and contraction events. We predict that mutation events can occur as frequently as every other day, which matches the timing of regular cell activities such as DNA repair and transcription but not DNA replication.
Human Molecular Genetics, 2000
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.
Journal of The Royal Society Interface, 2013
More than 20 human genetic diseases are associated with inheriting an unstable expanded DNA simple sequence tandem repeat, for example, CTG (cytosinethymine-guanine) repeats in myotonic dystrophy type 1 (DM1) and CAG (cytosine-adenine-guanine) repeats in Huntington disease (HD). These sequences mutate by changing the number of repeats not just between generations, but also during the lifetime of affected individuals. Levels of somatic instability contribute to disease onset and progression but as changes are tissue-specific, age-and repeat length-dependent, interpretation of the level of somatic instability in an individual is confounded by these considerations. Mathematical models, fitted to CTG repeat length distributions derived from blood DNA, from a large cohort of DM1-affected or at risk individuals, have recently been used to quantify inherited repeat lengths and mutation rates. Taking into account age, the estimated mutation rates are lower than predicted among individuals with small alleles (inherited repeat lengths less than 100 CTGs), suggesting that these rates may be suppressed at the lower end of the disease-causing range. In this study, we propose that a length-specific effect operates within this range and tested this hypothesis using a model comparison approach. To calibrate the extended model, we used data derived from blood DNA from DM1 individuals and, for the first time, buccal DNA from HD individuals. In a novel application of this extended model, we identified individuals whose effective repeat length, with regards to somatic instability, is less than their actual repeat length. A plausible explanation for this distinction is that the expanded repeat tract is compromised by interruptions or other unusual features. We quantified effective length for a large cohort of DM1 individuals and showed that effective length better predicts age of onset than inherited repeat length, thus improving the genotype-phenotype correlation. Under the extended model, we removed some of the bias in mutation rates making them less length-dependent. Consequently, rates adjusted in this way will be better suited as quantitative traits to investigate cis-or transacting modifiers of somatic mosaicism, disease onset and progression.
Human Molecular Genetics, 1998
There is a good correlation between repeat size (at least in leucocytes), clinical severity and age of onset. The trinucleotide repeat instability mechanisms involved in DM and other human genetic diseases are unknown. We studied somatic instability by measuring the CTG repeat length at several ages in various tissues of transgenic mice carrying a (CTG) 55 expansion surrounded by 45 kb of the human DM region, using small-pool PCR. These mice have been shown to reproduce the intergenerational and somatic instability of the 55 CTG repeat suggesting that surrounding sequences and the chromatin environment are involved in instability mechanisms. As observed in some of the tissues of DM patients, there is a tendency for repeat length and somatic mosaicism to increase with the age of the mouse. Furthermore, we observed no correlation between the somatic mutation rate and tissue proliferation capacity. The somatic mutation rates in different tissues were also not correlated to the relative inter-tissue difference in transcriptional levels of the three genes (DMAHP, DMPK and 59) surrounding the repeat.
Human Molecular Genetics, 2000
Myotonic dystrophy type 1 (DM1) is one of a growing number of inherited human diseases whose molecular basis has been implicated as the expansion of a trinucleotide DNA repeat. Expanded disease-associated alleles of >50 CTG repeats are unstable in both the germline and soma. Expansion of the unstable alleles over time and variation of the level of mutation between the somatic tissues of an individual are thought to account at least partially for the tissue specificity and progressive nature of the symptoms. We previously generated a number of transgenic mouse lines containing a large expanded CTG repeat tract that replicated a number of the features of unstable DNA in humans, including frequent sex-specific changes in allele length during intergenerational transmission. Small length change mutations were apparent in the somatic tissues of young mice in all of the lines generated, but the gross instability observed in human DM1 patients was not replicated. We now show that in one of the lines, Dmt-D, spectacular, expansion-biased, tissue-specific instability is observed in older mice. The highest levels of instability were detected in kidney with gains of >500 repeats, representing a tripling of allele length, in some cells. Mosaicism accumulated in an age-dependent manner, but the tissue specificity did not obviously correlate with cell turnover. Such gross somatic mosaicism was not observed in three other lines examined, further emphasizing a role for flanking DNA in modulating repeat stability.
Human Molecular Genetics, 2020
In myotonic dystrophy type 1 (DM1), somatic mosaicism of the (CTG)n repeat expansion is age-dependent, tissue-specific and expansion-biased. These features contribute toward variation in disease severity and confound genotype-to-phenotype analyses. To investigate how the (CTG)n repeat expansion changes over time, we collected three longitudinal blood DNA samples separated by 8–15 years and used small pool and single-molecule PCR in 43 DM1 patients. We used the lower boundary of the allele length distribution as the best estimate for the inherited progenitor allele length (ePAL), which is itself the best predictor of disease severity. Although in most patients the lower boundary of the allele length distribution was conserved over time, in many this estimate also increased with age, suggesting samples for research studies and clinical trials should be obtained as early as possible. As expected, the modal allele length increased over time, driven primarily by ePAL, age-at-sampling and...
Mitotic drive' of expanded CTG repeats in myotonic dystrophy type 1 (DM1)
Human Molecular Genetics, 2001
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.
The American Journal of Human Genetics, 2003
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.
Nucleic acids research, 2003
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 sm...