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.
Myotonic dystrophy (DM) is an autosomal dominant progressive neuromuscular disorder with a highly variable pleiotropic clinical phenotype usually including myotonia, muscle weakness and cataracts (1). It is the commonest form of adult muscular dystrophy, although age of onset and disease severity are highly variable both within and between families. All DM mutations identified to date appear as unstable amplifications of a CTG trinucleotide repeat in the 34-untranslated region (UTR) of a protein kinase gene (DMPK) on chromosome 19q13.3 (2-7). Expansion of the CTG repeat shows a positive correlation with the severity of the disease (8) and the size of the expansion progressively increases in successive generations of DM families, providing the molecular explanation for anticipation (9). Southern blot analysis of the genomic DNA restriction fragments containing the expanded CTG repeat often gives a diffuse hybridization signal, suggesting somatic mosaicism (2-7). Moreover, different tissues present with different allele sizes, again consistent with somatic mosaicism for repeat length. Most notably, allele sizes in muscle cells are consistently larger than those in circulating lymphocytes (10-12), although how this observation correlates with the muscle-based phenotype has not yet been determined.
This somatic mosaicism means that it is not simple to gain a precise measurement of allele size in a sample. Rather an average allele size, as determined by the midpoint of the smear, is usually reported. Although the measured CTG repeat allele length clearly correlates positively with disease severity and inversely with age of onset, these correlations are not precise and an accurate prediction of the prognosis for an individual patient cannot be made on the basis of their measured allele size. Moreover, measured allele size does not always correlate with the clinically observed anticipation. For instance, in one very large study in which apparent intergenerational repeat contractions were observed in 6.4% of all transmissions, over half were still associated with clinical anticipation (13). Some recent work has started to suggest probable molecular explanations for these results (14-16). Restriction digested genomic DNA Southern blot estimates of the extent of repeat length heterogeneity in individual samples have revealed that the level of mosaicism is greater in older patients, a phenomenon that is more apparent for larger repeat lengths (15). In addition, analysis of a small number of samples has shown that the average repeat length in peripheral blood cells of DM patients can increase over a time span of 5 years (14,15). More detailed analyses of an even smaller number of somatic DNA samples using sensitive single molecule and small pool PCR (SP-PCR)-based techniques have indicated that the smear can be resolved into its component parts, revealing somatic distributions skewed towards further expansion (16). These data have been used to conclude that somatic instability is continuous throughout the adult life of patients. Thus, the measured allele size in any one patient will be highly dependent on the age of the individual at sampling. This phenomenon is probably mediated via the stepwise gain of small numbers of repeats at a tissue-specific rate. It was this theretofore unrealized progressive and continuous somatic expansion that almost certainly underlied the apparent paradox in the lack of association of intergenerational contractions with clinical anticipation (16). Correction for this phenomenon would likely also increase the significance of the correlation between repeat size and age of onset, leading to more accurate predictions of patient prognosis. However, no attempts have yet been made to quantify in detail the lifelong dynamics of somatic repeat size and use the data to model somatic repeat instability for eventual use as a diagnostic tool. Towards this end we have determined the average length of the CTG repeat in two peripheral blood samples from DM individuals taken over a period of time ranging from <1 year to 7 years between samples for 111 DM patients. These data have been analysed to see if it is possible to design a simple model that could predict the behaviour of the CTG repeat expansion throughout life in DM patients.
Southern blot analysis of the DM CTG repeat length in genomic DNA from DM patients frequently shows a smear for the expanded allele reflecting somatic mosaicism of the expanded CTG repeat. To confirm that the amount of heterogeneity increases with age we assessed DM CTG repeat length heterogeneity in the blood DNA of 1005 DM patients, of which 100 were congenital cases, 657 were classical forms of the disease, 135 were mild forms and 113 were asymptomatic carriers. Standard techniques revealed that samples from foetuses, newborns and children had low levels of repeat length heterogeneity, whereas older DM patients showed increasing heterogeneity with increasing age, independent of either the sex of the patient or the transmitting parent (data not shown). In a more detailed analysis, six DNA samples that had expansions with an average size of ~300 repeats, but were derived from different patients with widely differing ages (6 months to 62 years), were analysed by the sensitive SP-PCR technique (Fig. 1). The SP-PCR analysis dramatically illustrates the relative increase in repeat length heterogeneity with age. The sample from the newborn showed very little repeat heterogeneity. The level of heterogeneity in the sample from the 8-year-old child was still quite low, but clearly greater than in the newborn. The levels of heterogeneity in the samples from the older individuals were progressively greater, with almost every allele analysed from the oldest patient showing a different repeat length.
Figure
To determine the rate of progression of somatic mosaicism over time in individual DM patients we measured the average size of the CTG repeat expansion in blood DNA over defined time periods. Two repeat samples were taken from 111 DM patients with time intervals ranging from <1 year up to 7 years. Three of the patients were homozygous for the expansion and five patients were sampled three times, giving a total number of observations of 124. The patient's initial repeat lengths varied from 40 up to 1750 CTG repeats, included all clinical severities of the disease and their ages ranged from 4 to 78 years at the time of their initial sample. Comparison of the measured average allele size between samples from the same individual indicated that in most cases the average allele length increased over time. In most patients this change was clearly detectable on genomic DNA Southern blots after digestion with EcoRI (Fig. 2A), but in a subset of patients a change of CTG expansion was not detectable or was difficult to detect on a standard genomic DNA Southern blot (Fig. 2B). However, in most of these cases it was detectable using SP-PCR as a progression of the heterogeneity from the lower end of the distribution towards the upper end with an increase in mean allele size (Fig. 2C). All patients with time intervals >2 years and with initial expansion sizes of >200 repeats showed a detectable increase in their average repeat length over time (summarized in Fig. 3). Only one out of 34 patients with <200 repeats showed a detectable change in average repeat length during this study, doubling the initial size in 3 years from ~167 to 333 repeats.
Figure
Figure
In an attempt to determine the critical factors regulating the continuing expansion process and quantify these effects, these data were analysed for various correlations. Statistical analysis of the initial and final expansion sizes revealed a very strong positive correlation as expected (r = 0.98, P < 0.0001, n = 124) (Fig. 4A). Considering only time between samples and the increment of expansion, there was also a meaningful correlation, but the magnitude of the coefficient of correlation was lower (r = 0.54, P < 0.0001, n = 124) (Fig. 4B). Not surprisingly the largest initial expansions tended to show the largest increments, but again the correlation was not absolute (r = 0.57, P < 0.0001, n = 124) (Fig. 4C).
Figure
In a further attempt to clarify these interrelationships these data were analysed using multivariate analysis. Using multiple linear regression and the `stepwise' method for the variables: age, clinical form, initial expansion and time, the final model rejected age (P = 0.49) and clinical form (P = 0.08) of patients as factors determining the progression of the repeat expansion. However, time (P < 0.0001) and initial expansion size (P < 0.0001) were confirmed as contributing to determining the size of the increment. From this model the correlation was r = 0.75, accounting for ~56% of the variation in the size of the CTG enlargement over time. The final result was: increment = -90 + (37yT) + (0.146yIE), where T was time in years between samples and IE was initial expansion in number of repeats.
The somatic instability of the expanded CTG repeat allele in DM patients repeats is now well documented, with the expanded allele frequently presenting as a smear on genomic DNA Southern blot analysis and different expansion patterns observed in DNA samples of different tissues from the same patient (12,17-19). Different expansions have been observed between somatic tissues and cultured somatic cells (11,20), and greater levels of repeat heterogeneity have been observed in older patients (15). Data based on a small number of samples have also indicated that the distribution of allele sizes between somatic cells is skewed towards further expansion (16) and that the average repeat length in peripheral blood cells of DM patients can increase over relatively small time periods (1-5 years) (14,15). Our analyses reported here have confirmed that the level of repeat heterogeneity does indeed increase with age, as dramatically exemplified by the SP-PCR analysis of similar sized expansions in patients of differing ages (Fig. 1).
In an attempt to further characterize the dynamics of the process whereby the level of repeat heterogeneity increases with time, we have analysed CTG repeat length in repeat blood samples from 111 DM patients over time periods of <1 year up to 7 years. In all cases (70 patients) where the initial repeat size was >200 repeats and the time interval was >2 years, an increase in average repeat length was detected. These data indicate that the somatic expansion bias suggested by earlier results applies to the vast majority, if not all, DM patients, thus refuting the idea that the CTG repeat size in leukocyte DNA decreases while only the affected tissues show increases in size (13). None of the patients with time intervals of <1 year showed detectable changes in their average repeat length using standard techniques. These data probably reflect the limitations of the detection methods rather than genuine lack of expansion over such short time periods. Of the 31 patients studied with <200 repeats, only in one example was an increase in the average repeat length detected over a time period of <5 years. SP-PCR analysis of the blood DNA of a number of patients with such small expansions has revealed low levels of repeat heterogeneity biased towards expansion (16, and unpublished results) indicating that such alleles are unstable in white blood cells, but that the level of instability is low enough that a shift in the modal allele size is not generally detected in short time periods. In the one case in which a large change was detected in a short time period the average repeat size of ~167 repeats at age 38 had increased to ~333 repeats 3 years later. This male had a far more severe phenotype than would be expected based on his initial repeat size measurement, and a very rapid progression of the symptoms. This case indicates that individual specific factors can influence the somatic dynamics of the repeat and suggest that the process of somatic expansion may be correlated with the clinical progression of the disease.
The molecular mechanism that gives rise to the increment in size of the repeat expansion in somatic tissues is currently not known. These data give more credence to the idea that this process is continuous and related to the size of the repeat. This last point is well demonstrated in a follow up of one of the patients homozygous for a repeat expansion (21): she has a 60 CTG repeat allele which has been stable for 2 years, whereas her other allele with an initial expansion of 1000 repeats has shown an increase of 166 CTG repeats during the same time interval.
In this study, analysis of two samples from the same patient at different times always detected an overlap between repeat ranges seen in the samples, although this overlap decreased as the time difference between samples increased. One of our patients who was sampled on three occasions over 7.5 years showed only a small overlap between the first and last sample repeat size ranges (data not shown). Over this time period the average allele size as measured by genomic DNA Southern blot analysis increased by ~400 repeats from ~667 to ~1067 repeats. These data clearly indicate that it would be quite possible over long time periods for repeat samples to not overlap in their range. These results further highlight the fact that the age of the patient at the time of sampling can have dramatic effects on the measured allele size. These results must be considered for genetic counselling in families where measured allele sizes and the intergenerational difference, measured by comparing the size of the expanded allele in the leukocyte DNA between parent and child, are used as a predictors of likely clinical severity and age of onset.
In light of the now firmly established expansion biased and progressive nature of the somatic instability in the blood cells of DM patients, some process whereby this phenomenon could be taken into account in assessing measured allele sizes would clearly be of benefit. It seems likely that the best estimate of clinical severity and age of onset in DM patients would be based on knowledge of the allele size inherited by the zygote. In young patients for which the level of heterogeneity in blood DNA is usually very low, the measured allele size in blood DNA is probably a good estimate for this value. However, in older patients for whom levels of heterogeneity can be quite high, the measured allele size may not be a good indicator of inherited allele size unless some account is taken of the effect of the age of the patient. To this end we have performed statistical analysis of our data in an attempt to derive a mathematical model that could be used to predict the behaviour of the repeat expansion over time. Multivariate analysis revealed that neither age nor clinical form had a significant effect on the size of the increment observed over time, although time and initial expansion size were found to be important factors. However, the overall correlation coefficient for the derived equation was still only 0.75, time and initial expansion size thus accounting for only 56% of the variation in increment over time. Thus our current model is not accurate enough to be used as a good indicator of repeat dynamics and could not be used to reliably estimate inherited allele size from defined measurements of repeat length and age. It is likely that technical difficulties involved in defining the average allele length and measuring small shifts in repeat length over time account for some of this variation. However, the magnitude of the deficit and the example of the individual with the rapid expansion suggest that other individual specific factors also play a role in modifying repeat dynamics. The nature of such modifiers remains speculative at the moment, but could reasonably be envisioned to include trans acting genetic factors such as DNA repair gene variants or environmental exposure to genotoxic agents.
In conclusion, we have confirmed that the expansion of the CTG repeat in the blood cells of DM patients is continuous throughout life and that the major factors affecting the rate of expansion determined by this study are initial size of the expanded allele and time, but that expansion process is also affected by as yet unidentified individual specific factors. To take these studies further will probably require more in-depth studies over time in individuals, with more accurate measurement of repeat length distributions and more elaborate modelling of repeat dynamics at the level of cell populations to generate testable individual specific models that may form the basis of a more widely applicable model.
Blood samples were obtained from Spanish DM patients who were referred to the Genetics Unit at the Hospital de Sant Pau, Barcelona, for diagnostic testing. Repeat length was assessed in 1005 DM patients. From these patients, 111 were recontacted and a second blood sample obtained with an intervening time period of from <1 year up to 7.5 years (0 to <1 year, 10 individuals; 1 to <2 years, 17 individuals; 2 to <3 years, 22 individuals; 3 to <4 years, 12 individuals; 4 to <5 years, 27 individuals; 5 to <6 years, 17 individuals; 6 to <7 years, 5 individuals; 7 to <8 years, 1 individual) and from all four severity classes (early onset and congenitally affected, 9 individuals; classic adult onset, 72 individuals; late onset, 10 individuals; asymptomatic, 20 individuals). Five patients were sampled three times and three patients were homozygous for expanded DM alleles. Genomic DNA was prepared from peripheral blood leukocytes using standard procedures (22).
The size of the CTG repeat expansion was analysed by PCR and Southern blotting. Genomic DNA was digested with EcoRI or BglI for Southern blot analysis to detect large expansions and to determine the degree of heterogeneity in repeat length. DNA (5 mg) was digested and electrophoresed on 0.6-0.8% agarose gels, denatured in 0.5 M NaOH/1.5 M NaCl, neutralized in 0.5 M Tris-HCl (pH 7.0)/1.5 M NaCl and transferred onto Hybond-N membrane in 10y SSC. Filters were probed with cDNA25 (4), labelled with 32P using the random priming method (23). Blots were washed at 0.2y SSC, 0.2% SDS final stringency at 65_C, and were exposed for 5-10 days at -80_C. The two or three samples from each patient were run on the same gel. The modal size of the expansion was determined at the point of highest band intensity on the autoradiograph or at the centre of the smear for very diffuse bands.
Polymerase chain reaction (PCR) was used to determine the number of CTG repeats in normal alleles and the small repeat amplifications (<200 repeats) in DM patients. PCR reactions were carried out in a total volume of 50 ml using 700 ng of genomic DNA. Radiolabel was added to the PCR samples for visualization of products on polyacrylamide gels using the previously described conditions (24). The allele lengths were assessed by comparison of the electrophoretic migration pattern of the PCR product in the gel with an M-13 sequence used as a size marker.
Small pool PCR (SP-PCR) (25) analysis was performed to resolve heterogeneous smears of DM repeat length distributions into their component parts essentially as previously described (16) with slight modifications: genomic DNA samples were digested with EcoRI and diluted in 10 mM Tris-HCl (pH 7.5), 1 mM EDTA and 0.1 mM carrier primer DM-A. Then 3-300 pg pools of EcoRI digested DNA were amplified in 7 ml reactions with 0.2 mM primer DM-A, 0.2 mM primer DM-BR and 0.05 U/ml of Taq DNA polymerase (Boehringer Mannheim). Five ml of the reaction product was loaded on 1.5-2% agarose gels and electrophoresis was performed at 90 V for 16 h. The products were analysed by Southern blotting with subsequent hybridization to a radiolabelled (CTG)10 oligonucleotide probe. For each set of PCR amplifications we also set up multiple zero DNA control reactions using equivalent dilutions of EcoRI digest with no DNA added. SP-PCR analysis with ~10 amplifiable expanded molecules per reaction provides both a qualitative and quantitative estimate of repeat length heterogeneity. As a quantitative tool the average allele size in the sample was calculated as a meanof the individual expanded alleles identifiable and sized by comparison to the known molecular weight size markers.
To analyse the possible relationships between the increment of the repeat expansion and the other variables (initial expansion, time between samples, age and clinical form of individuals), we used a linear regression and a multiple linear regression software package (SPSS/WIN 6.0).
This work was supported by a grant (98/1299) from the Fondo de Investigaciones de la Seguridad Social, Ministerio de Sanidad y Consumo, Spain. We are indebted to the patients for their cooperation in performing this study.
Human Molecular Genetics Pages
Progression of somatic CTG repeat length heterogeneity in the blood cells of myotonic dystrophy patients
Introduction
Results
Repeat length heterogeneity increases with age
Expansion size increases over time
Discussion
Subjects And Methods
Subjects
Determination of the CTG repeat size
Small pool PCR
Statistical analysis
Acknowledgements
References
Progression of somatic CTG repeat length heterogeneity in the blood cells of myotonic dystrophy patients
INTRODUCTION
RESULTS
Repeat length heterogeneity increases with age
Expansion size increases over time
DISCUSSION
SUBJECTS AND METHODS
Subjects
Determination of the CTG repeat size
Small pool PCR
Statistical analysis
ACKNOWLEDGEMENTS
REFERENCES
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