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Trinucleotide repeat contraction: a pitfall in prenatal diagnosis of myotonic dystrophy

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Editor—Myotonic dystrophy (DM) is a common autosomal dominant disorder characterised by myotonia, muscle weakness, ECG abnormalities, cataracts, hypogonadism, and frontal balding in the typical adult form (MIM 160900). The genetic defect consists of the amplification of an unstable CTG trinucleotide repeat in the 3′ untranslated region of the dystrophia myotonica protein kinase gene (DMPK), which maps to 19q13.3.1 2 Normal subjects have five to 37 repeat copies while affected subjects have over 50 repeats.1 There is some correlation between repeat length and clinical symptoms, especially with respect to the age at onset.3-6 In the vast majority of cases, the number of repeats increases during parent-offspring transmission of the mutant allele, thus providing some molecular basis to the observation of anticipation (increased severity of the disease in successive generations).7 8 However, a decrease in repeat size is occasionally observed in the offspring, mostly in the case of paternal transmission of an expansion of over 600 trinucleotide repeats,9 but contraction of a parental expanded repeat back to the normal range when transmitted to offspring seems to be an extremely rare phenomenon.10-12 Here we report such a case and emphasise the direct impact of this situation on prenatal diagnosis (PND) of DM.

Material and methods


DM was diagnosed in II.2 (fig 1), who presented with mild atrophy of the head and neck muscles, myotonia of the hands, and frontal balding at the age of 37 years, while his first wife (II.1) was pregnant. The sister of patient II.2 (II.4) was more severely affected, with marked impairment of walking from the age of 43 years. Their father, I.2, had no symptoms of DM at the age of 61 years. III.1, the first offspring of II.2, was severely affected with muscular weakness and mental retardation. II.2 was first referred to our unit during the pregnancy of his second wife (II.3) for first trimester PND.

Figure 1

Linkage analysis at the DMPK locus in I.2, II.2, II.3, and III.3. The poly (CA) microsatellite markers used and genetic distances are as follows: cen - D19S223 - (4.2 cM) -D19S217 - (2.1 cM) - D19S412 - (6.3 cM) - D19S606 - (1.4 cM) - D19S596 - (1.3 cM) -D19S879 - tel. The DMPK gene lies between D19S217 and D19S412. Note that III.3 inherited the paternal DM allele. The number of CTG repeat evaluated by PCR amplification is given in parentheses.


DNA was extracted from leucocytes of II.2 and II.3 and from CVS performed at 11 menstrual weeks. Study of the CTG repeat size was carried out by Southern blotting (EcoRI/PM10M6 andPstI/PM10M6)13 and PCR amplification with primers flanking the CTG repeat in parental and fetal DNA.1 Poly (CA) microsatellite markers were used both for linkage analysis at the DM locus and to rule out false paternity (D19S223, D19S412, D19S606, D19S596, D19S879, D20S194, D12S78, with heterozygosity of 81, 80, 81, 53, 76, 91, and 91% respectively, data available through Genebank).


The healthy mother (II.3) had two alleles of 10 CTG repeats, while the father (II.2) displayed a wild type allele of 13 CTG repeats and a mutated allele of approximately 200 CTG repeats (figs 2 and 3). The DM allele in I.2, II.4, and III.1 had previously been estimated as approximately 60, 400, and 600 CTG repeats in size, respectively (data not shown for II.4 and III.1). PCR amplification of the CTG repeat region from the CVS DNA showed two normal alleles, a 10 trinucleotide repeat allele inherited from the mother and a 30 trinucleotide repeat allele not found in the father. Fetal DNA testing by Southern blotting failed to detect any expanded allele (fig 3). Results of the CVS DNA analysis were confirmed both by PCR amplification of the CTG repeat region and Southern blotting of DNA extracted from cultured amniocytes. Linkage analysis in I.2, II.2, II.3, and III.3, using poly (CA) microsatellite markers flanking the DMPKgene, showed that the fetus had inherited the paternal DM allele (fig1). The probability of false paternity was assessed to be less than 10-3. The parents were informed about this unusual situation implying some uncertainty regarding the fetal status and subsequently decided to continue the pregnancy.

Figure 2

PCR amplification of the CTG repeat region. DNA analysis from three controls (C) and patients I.2, II.2, II.3 (leucocytes) and III.3 (CVS) are shown. The PCR products were loaded onto a 2% agarose gel. The estimated number of CTG repeats for both alleles for each subject is as follows: C1: 5/42, C2: 5/20, C3: 5/90, I.2: 20/60, II.2: 13/-, II.3: 10/10, III.3: 10/30. The minor PCR product, larger than the predominant product, seen in the controls, II.2, and III.3, is likely to be a non-specific PCR product.

Figure 3

Southern blotting (EcoRI/ PM10M6) in patients III.3, II.3, I.2, and II.2. The expanded allele in II.2 is not found in his offspring III.3.


Here we report on the contraction of a large expanded DM allele to the normal range during a father-offspring transmission. To our knowledge, this is the first case where contraction of a parental expanded allele back to the normal range has been detected during the prenatal period. No somatic mosaicism could be identified in either choriocytes or amniocytes. A recombination event at the DM locus appears unlikely, based on both haplotype analysis and PCR amplification of the CTG repeat, indicating that the fetus did not inherit the paternal wild type DM allele (fig 1). Such a contraction of a DM allele back to the normal range has seldom been reported.10-12 In one case, a discontinuous gene conversion between the wild type and the DM allele was shown.11 In the other three cases, as in the case reported here, this mechanism appears unlikely, because of the different numbers of CTG repeats in the father/offspring wild type alleles.10 12 A contraction of the DM allele or a double recombination disrupting the CTG repeats is a possibility. These events could be either meiotic or early mitotic. It is worth mentioning that in all cases reported to date, the contraction event was paternal in origin, making the hypothesis of a meiotic event more likely. In one case, long term follow up established that the offspring remained asymptomatic at or beyond the age of disease onset in the transmitting parent, also favouring the hypothesis of a meiotic event.9Understanding when and how the contraction event occurs would be of importance to appreciate both the risk for the children of developing the disease and their own risk of transmission of DM to their offspring.

A partial reduction in size of a trinucleotide repeat above the normal range during parent-offspring transmission seems to be far more frequent than a reduction back to the normal range. In a large series of 1489 DM parent-offspring pairs reported by Ashizawaet al,9 a partial reduction was noted in 6.4% of cases. Such a contraction was more frequently observed in paternal than in maternal transmissions (10% versus 3%, respectively).9 In these cases, the size of the parental DM allele varied from approximately 500 to 1000-1500 CTG repeats. Interestingly, the observed number of sibships whose members had inherited a contracted CTG repeat was greater than expected. This could argue either for a predisposition to reduction during transmission of an expanded allele in some subjects or for negative selection against sperm carrying the largest CTG expansions. This last hypothesis has been raised in FRAXA, where males carrying a full mutation in their somatic cells transmit only premutated alleles to their daughters (MIM 309550).14

While expansion of a mutated DM allele during parent-offspring transmission is almost invariably associated with clinical anticipation, the rare events of contraction raise difficult issues with respect to PND and genetic counselling in DM. In the large series of Ashizawa et al,9approximately half of the offspring who inherited a contracted allele showed clinical anticipation despite the reduced CTG repeat size. In all these cases, however, the size of the contracted allele remained above the normal range. Moreover, Southern blot analysis showed some overlap between the boundaries of the “smear” in some parent-offspring pairs. Conversely, in the four cases where the transmitted DM allele reverted to the normal range, the clinical phenotype seemed to be normal, taking into account the absence of data regarding long term follow up in these cases.9-12

Taken together, these data strengthen the well known fact that direct analysis of fetal DNA should be used as the primary approach in PND, since a reliable prediction of the seriousness of the phenotype cannot be based upon haplotyping using polymorphic markers linked to theDMPK locus. Moreover, the detection of a contraction event in a fetus by Southern blotting warrants further molecular investigations in order to assess the size of the CTG repeat accurately. Indeed, while detection of a DM allele remaining above the normal range does not preclude clinical anticipation, the observation of a contracted allele back to normality should allow reassurance of couples at risk for transmitting DM.