Somatic instability of the myotonic dystrophy (CTG)n repeat during human fetal development
Somatic instability of the myotonic dystrophy (CTG) n repeat during human fetal developmentLoreto Martorell1, Keith Johnson2, Catherine A. Boucher2 and Montserrat Baiget1,*
1Unitat de Genètica Molecular, Hospital Sant Pau, Avda Pare Claret 167, 08025 Barcelona, Spain and 2Division of Molecular Genetics, Institute of Biomedical and Life Sciences, University of Glasgow, Anderson College, 56 Dumbarton Road, Glasgow G12 8QQ, UK
Received October 11, 1996;Revised and Accepted February 26, 1997
Myotonic dystrophy is characterised by the striking level of somatic heterogeneity seen between and within tissues of the same patient, which probably accounts for a significant proportion of the pleiotropy associated with this disorder. The congenital form of the disease is associated with the largest (CTG)n repeat expansions. We have investigated the timing of instability of myotonic dystrophy (CTG)n repeats in a series of congenitally affected fetuses and neonates. We find that during the first trimester the repeat is apparently stable and that instability only becomes detectable during the second and third trimesters. In our series repeat instability is apparent only after 13 weeks gestational age and before 16 weeks. The appearance of heterogeneity shows some tissue specificity, with heart most commonly having the largest expansion. The degree of heterogeneity is not correlated with initial expansion size as gauged by chorionic villus and blood (CTG)n repeat sizes.
Myotonic dystrophy (DM) is an autosomal dominant multisystemic disorder and the most prevalent muscular dystrophy in adults, with an average incidence of 1 in 8000 (1 ). The phenotypic expression of the disease mutation is highly variable and different in affected individuals from the same family. DM shows anticipation (progressively earlier onset and concomitant increased severity of the disease in successive generations) (2 ). The molecular basis is mutational expansion of the unstable (CTG)n repeat in the 3' untranslated region (UTR) of the myotonic dystrophy protein kinase (DMPK) gene (3 -6 ). The size of the expansion progressively increases in successive generations of DM families, providing the molecular mechanism for anticipation (7 ,8 ).
Somatic instability of the (CTG)n repeat at the DM locus has been documented by: (i) the broad smears visualised on genomic Southern blots representing heterogeneous length fragments of the expanded alleles (9 ,10 ); (ii) the different expansion pattern observed in some sets of identical DM twins (11 ,12 ); (iii) the different expansion observed between peripheral blood lymphocytes of DM patients compared to their transformed lymphoblastoid cell lines after many passages (13 ); (iv) the finding that the repeat length in patient's blood cells is continuing to expand with time (14 ,15 ); (v) the expansion patterns seen in DNA samples from a variety of tissues of the same patient (16 -19 ).
These data indicate that, although clearly present in male gametes, instability of the repeat occurs post-zygotically, and there is a continuing instability in mitotically dividing cells. The somatic behaviour of expanded triplet repeats in tissues from a small number of DM fetuses has also been studied (13 ,18 -21 ). However, no pattern for the course of the expansion of the (CTG)n repeat during intrauterine life has been established. To address this question we analysed repeat expansions in a broad range of tissues from five DM fetuses, four having expanded alleles corresponding to those predicted to cause the congenital form (CDM) and one with an allele size predicted to correspond to a mild form. In addition we analysed tissues from three CDM infants who died in the neonatal period. We aimed to determine the timing of somatic instability as represented by tissue variability of the (CTG)n repeat size during human development.
We have investigated the somatic instability of expanded (CTG)n repeats in tissues from DM fetuses and neonates, representing a range from 11 weeks gestational age to birth at the time of post mortem (Table 1 ).
Case 1 inherited the DM allele from an affected mother with the adult onset (classical) form of the disease, showing an expanded DM allele of 13 kb. Fetal DNA, obtained from chorionic villus samples for prenatal diagnosis, showed an expanded DM allele of ~2000 repeats, indicating a high risk of congenital DM. After abortion, at week 11, the following tissues were analysed by EcoRI and BglI digests: chorionic villus, umbilical cord, small bowel, skeletal muscle and stomach. No somatic variation in the expanded allele among these fetal tissues was detected.
Case 2 also inherited the disease from an affected mother with the adult onset form of the disease, with a DM allele showing a repeat length of 12.8 kb (~900 repeats). When a prenatal diagnosis was requested, fetal DNA was obtained from chorionic villus sampling. The pregnancy was terminated at week 12 after the detection of an expanded allele of ~2300 repeats. A wide range of fetal tissues were obtained: chorionic villi, umbilical cord, skeletal muscle, stomach, small bowel, kidney, liver, lung, heart, skin and adrenal gland. Analysis by EcoRI and BglI digests did not detect heterogeneity in the expanded allele among these fetal tissues.
Case 3 was a fetus terminated at 13 weeks gestation. The father, with a relatively mild DM phenotype, had a DM allele with an expansion of 10.7 kb (~250 repeats). Prenatal diagnosis was requested by the parents and fetal DNA analysis revealed an expansion allele also of ~250 repeats. At termination, small bowel and skeletal muscle samples were obtained and shown to have identical expansion sizes to those obtained from chorionic villi, (Fig. 1 ).
Fifteen different tissues (chorionic villus, skeletal muscle, heart, brain, colon, small bowel, stomach, trachea, kidney, skin, lung, adrenal gland, oesophagus, pancreas, spleen and umbilical cord) were obtained from a 16 week old fetus at termination represented by case 4. In this fetus the DM allele was inherited from her mother, who showed a DM allele with an expansion of 11.5 kb. The chorionic villus sample (CVS) demonstrated an expanded allele of 14.5 kb (~1500 repeats). All tissues, except heart, showed hybridisation to bands corresponding to the normal and expanded alleles in the CVS. In heart an expanded allele corresponding to ~1700 repeats was seen.
Case 5 was a 28 week old CDM premature infant born to an affected mother, with a DM allele of 12 kb. DNA from four different tissues (blood, skeletal muscle, kidney and brain) were analysed. Expansion alleles of 17 kb (~2600 repeats) were seen in skeletal muscle, kidney and brain, compared to an expanded allele of ~2300 repeats seen in blood (Fig. 2 ). The expanded alleles are apparently fainter because of the heterogeneity of fragment sizes they represent, longer exposures (data not shown) reveal a wider smear of hybridisation around the most intense band of the expanded allele.
Cases 6 and 7 were CDM neonates in whom the DM allele was maternally inherited and who both died in their perinatal period. The mother of case 6 had an expanded allele of 11.5 kb. The mother of case 7 had an expanded allele of 12 kb. In both cases the smallest expanded alleles in neonatal tissues were 17 and 17.5 kb (~2300 and 2500 repeats, respectively) and were seen in blood. The largest were seen in skeletal muscle (Fig. 2 ).
The somatic behaviour of the expanded repeat in DM has been documented during fetal development in 10 cases in the literature. We have analysed a range of tissues from seven additional cases which together with these literature reports allow us to track the stability or otherwise of the repeat during intrauterine life. Table 1 summarises previous data and those presented here, and allows some preliminary conclusions to be drawn.
Heterogeneity of repeat expansions between tissues does not appear to be detectable before 13 weeks of development, as documented by seven embryos sampled in the interval between 10 and 13 weeks (three cases from this study). From 16 weeks to just after birth heterogeneity is almost always detectable (nine out of ten cases).
Taken together the data from the seven cases presented here and the 10 previously reported in other studies show: (i) five different tissues have been analysed in at least three independent studies, brain in five out of seven, skeletal muscle in four out of seven and heart, skin and blood in three out of seven. These tissues show a gradation in the frequency at which they show the largest expansion of those sampled from a given fetus. The heart shows the largest expansion in 100% (3/3), skin in 66% (2/3), skeletal muscle 50% (2/4), brain 40% (2/5) and blood 0% (0/3); (ii) nine tissues have been investigated in two independent studies. Colon, small bowel and stomach showed no larger repeat expansions than other tissues, whereas adrenal gland, umbilical cord, kidney, diaphragm, ovary and liver showed largest expansions in one of the two studies. Kidney was largest in 2/2 studies; (iii) the tissues studied in only one case always showed the smallest expansions compared to the more frequently studied tissues above. These differences do not appear to reflect numbers of cell divisions during development or lineages.
Extrapolating from these data we appear to be seeing instability of the repeat occurring after week 13 and before week 16 in heart (3/3) and kidney (2/2). Instability may occur in skeletal muscle, brain, skin, adrenal, liver, umbilical cord, diaphragm and ovary and has not yet been documented in other tissues. This may be a sampling bias and larger data sets will be required to be collated in order to confirm these trends. Alternatively, instability may be continuous and cumulative, only reaching detectable levels after 13 weeks. However, the relatively large increases detectable at 16 weeks gestation (170 and 1000 repeats) suggest that this cannot be the only explanation of our observations.
It is provocative to speculate that the instability appears to occur at a time consistent with the start of the second trimester. This would correlate with the period of onset of rapid growth in the fetus and implies that during the differentiation stage of development, in the first trimester, the repeats are somehow stabilised. This might be due to a greater fidelity of DNA repair mechanisms during the first trimester that cannot be sustained during the rapid growth phase. Alternatively, suppression of repeat expansion during the differentiation phase may occur through methylation of the DM repeat region. Both these mechanisms were proposed by Wohrle et al. (21 ).
Different methods of analysis of the repeat expansions between the studies may explain some of the variation in detection of instability. The analysis of the DM repeat continues to present a technical challenge, in that the largest repeats can only be visualised by Southern analysis. This coupled with the minute quantities of identifiable tissue that are obtained from the lowest gestational age embryos makes it impossible to perform multiple enzyme digests, which would allow the range of heterogeneity to be more accurately assessed. Despite these drawbacks, the data we have presented support our conclusions that there is a marked difference in the detectable degree of heterogeneity between first trimester samples and those of greater gestational age.
Several investigations have approached the question of DNA repair mechanisms in DM based on the evidence of microsatellite instability akin to those seen in inherited cancers. [Interestingly instability of the DM (CTG)n repeat in breast cancers has been reported (22 ).] These findings suggest that alterations of the human mutator mismatch repair system (hMLH1 and hMSH2 genes) could be part of the underlying mechanism by which repeat expansions are propagated. However, cell lines with homozygous hMLH1 or hMSH2 mutations do not show instability at the DM repeat (23 ). These findings could have a trivial explanation, such as the genotypes of these cell lines containing alleles that are inherently stable, as in the proposed population genetic model of Imbert et al. (24 ), the unstable alleles in DM have to have >20 repeats. The (CTG)n repeat genotypes of the cell lines used (22 ) were 10,15 (hMSH2-) and 13,16 (hMLH1-). Alternatively, the mismatch repair loci encode only part of the repair machinery and other elements may be more critical in determining repeat stability. Taking the two sets of findings at face value however, suggests that the mechanism underlying the switch from stability in the first trimester to instability in the second trimester is unlikely to be simply attributable to DNA mismatch repair mutations. This is further supported by the fact that DM patients do not exhibit the spectrum of cancers associated with repair deficiencies. However, the potential saturation of the mismatch repair system by the requirements for rapid cell division in the second trimester remains an attractive hypothesis, which at this time cannot be rigorously tested.
Freshly dissected tissues obtained from five DM fetuses and two CDM who died during the neonatal period, were prepared and stored at -80oC for DNA analysis. DNA was isolated using standard procedures (25 ). Southern blots and PCR analysis were performed to determine the size of the DM (CTG)n alleles. The PCR was used to determine the number of (CTG)n repeats in the normal range and small expansions, reactions were carried out in a total volume of 50 [mu]l with 700 ng of DNA. The conditions used were as described previously (26 ); PCR products were resolved by electrophoresis on 3.5% NuSieve agarose gels.
The Southern blot analysis was used to detect large (CTG)n expansions in tissues from all cases and to determine the degree of heterogeneity in repeat length. DNA (5 [mu]g) was digested with EcoRI and BglI and electrophoresed on 0.6% agarose gels, denatured in 0.5 M NaOH/1.5 M NaCl, neutralised in 0.5 M Tris (pH 7.0)/1.5 M NaCl and transferred onto a Hybond-N membrane in 10* SSC. Filters were probed with cDNA25 (5 ) and labelled with 32P using the random priming method. Blots were washed at 0.2* SSC, 0.2% SDS final stringency at 65oC, and were exposed for 5-10 days at -80oC.
In cases 1-4, the diagnosis was first made on DNA isolated from chorionic villi and pregnancies were terminated after detection of the mutation. In cases 5, 6 and 7, no prenatal diagnosis was requested.
All tissues of newborn and individuals who died during the neonatal period were collected after the autopsy, with the consent of parents.
This work was supported by a grant (94/0350) from the Fondo de Investigaciones de la Seguridad Social, Ministerio de Sanidad y Consumo, Spain. KJJ and CAB acknowledge the support of the MDG of Great Britain. We thank Darren Monckton for critical reading of the manuscript. We are also grateful to Roser González-Duarte for helpful discussions.
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*To whom correspondence should be addressed. Tel: +34 3 291 9361; Fax: +34 3 291 9192; Email: m.baiget@bcn.servicom.es
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