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Previous Volume 328:476-480 February 18, 1993 Number 7 Next

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Myotonic dystrophy is a multisystem disorder that is transmitted in an autosomal dominant fashion and is characterized by muscular weakness and atrophy, clinical and electromyographic evidence of myotonia, ocular cataract, and various other abnormalities, such as cardiac conduction disturbances, testicular atrophy in males, premature balding, increased risk from anesthesia, and mental retardation in cases with early onset¹. It is the most common inherited muscular dystrophy of adulthood, with an incidence of approximately 1 per 7500 people. The clinical expression of myotonic dystrophy is variable, ranging from neonatal mortality to a complete absence of symptoms. Recently, the disorder has been shown to be caused by an increased number of cytosine-thymidine-guanine (CTG) trinucleotide repeats in the 3' untranslated region of a protein kinase gene located in the q13.3 band of chromosome 19^{2,3,4,5,6,7,8}. The normal gene has between 5 and 40 CTG trinucleotide repeats, whereas myotonic dystrophy alleles have from approximately 50 to several thousand such repeats. The severity of the clinical symptoms of myotonic dystrophy usually increases with transmission to subsequent generations, a phenomenon that has been termed "anticipation"⁹. This is paralleled by an increase in the length of the CTG repeat sequence^2,5,7. It has been suggested that the progressively increasing severity of myotonic dystrophy eventually leads to the extinction of the disease from a given pedigree¹⁰.

Genetic theory assumes that there is equilibrium between the incidence of new deleterious gene mutations and their subsequent loss from the population through the reduced viability and fertility of their carriers. No allowance is made in human genetic epidemiology for mechanisms that change the mutation itself from abnormal to normal. Reverse mutation -- i.e., the spontaneous correction of a deleterious mutation upon transmission to unaffected offspring -- has not been reported in humans, although it has been observed at low frequencies in bacteria and cultured mammalian cells.

We report here on two families with myotonic dystrophy in which a reverse mutation has occurred. In the first family, an expanded CTG trinucleotide repeat found in a clinically affected man decreased in size to a normal allele of 24 CTG trinucleotide repeats in his healthy infant daughter. Similarly, in the second family the expanded allele found in the clinically affected father changed to a normal-sized allele containing 19 CTG trinucleotide repeats in his healthy 25-year-old son. The normalization of the mutated myotonic dystrophy gene in these offspring can be explained by the mitotic and (possibly) meiotic instability of the expanded CTG repeat sequence.

Case Reports

Family 1

In Family 1 (Figure 1), prenatal diagnosis was requested by a 25-year-old woman (Subject II-4) and a 27-year-old man (Subject II-3). Myotonic dystrophy had been diagnosed in the man two years earlier on the basis of mild muscular weakness, clinical and electrical evidence of myotonia, and a family history of the disorder. The family was studied with genetic markers closely linked to the myotonic dystrophy gene. All clinically affected family members carried the same haplotype for the APOC2-VSSM and X75b-VSSM markers, which flank the myotonic dystrophy locus. The proband was heterozygous for these markers. Prenatal diagnosis was therefore considered feasible. The first pregnancy was terminated after DNA-marker analysis performed on a sample of chorionic villus obtained by biopsy indicated that the fetus (Subject III-1) had inherited the myotonic dystrophy mutation. Examination of fetal tissues confirmed the results. The second pregnancy was terminated when intrauterine death was diagnosed by ultrasonography six days after a transcervical chorionic-villus biopsy had been performed. DNA analysis indicated that this fetus (Subject III-2) would have been unaffected. In the third pregnancy, genetic-marker analysis again indicated that the fetus (Subject III-3) had received the abnormal DNA-marker haplotype. In view of the small genetic distances between markers in the region of the myotonic dystrophy gene,^11,12,13 the chance that the fetus carried the myotonic dystrophy mutation was estimated to be greater than 99 percent. Mutation analysis⁷ was subsequently performed to determine the size of the CTG repeat in the myotonic dystrophy gene of this fetus in order to obtain more reliable prognostic information.

View larger version (90K): [in this window] [in a new window]   Figure 1. Linkage Analysis and Mutation Analysis in Two Families with Myotonic Dystrophy. The squares denote male family members, and the circles female family members. The slash denotes a deceased family member. Prenatal diagnoses are indicated by diamonds. The shaded symbols indicate clinically affected subjects. Subjects carrying a reverted myotonic dystrophy gene are marked by an asterisk. In Family 1, all the affected subjects carry the 3 allele for the APOC2-VSSM marker and the 1 allele for the X75b-VSSM marker at locus D19S112 (boxed). Subject III-3 has also received these alleles from her father. In Family 2, Subject II-2 has inherited the 1 allele for the pD10 marker at locus D19S63 and the 4 allele for the X75b-VSSM marker at locus D19S112. This haplotype (boxed) carries the myotonic dystrophy mutation in several affected family members, including the father (Subject I-1). Mutation analysis (lower panel) detected both normal alleles (with 5 to 40 CTG trinucleotide repeats) and abnormal alleles (with >50 CTG trinucleotide repeats). The size of the alleles, expressed as the number of CTG trinucleotide repeats, is shown beside the blots. In Family 1, Subject III-3 does not have an expanded allele. However, the marker haplotype suggests that she has inherited the myotonic dystrophy mutation from her father. In Family 2, Subject I-1 has an abnormal expanded allele of 150 to 500 CTG trinucleotide repeats, whereas his son (Subject II-2) does not have an expanded allele, although this would be expected on the basis of the marker haplotype.

 
Family 2

In Family 2 (Figure 1), a 23-year-old man (Subject II-2) and his 24-year-old sister (Subject II-1) were examined for signs of myotonic dystrophy because of a history of the disease in several relatives, including their father (Subject I-1). Clinical examination, including electromyography and slit-lamp examination, was normal in both Subject II-1 and Subject II-2. However, analysis with genetic markers flanking the myotonic dystrophy locus showed that the son (Subject II-2) had inherited the paternal chromosome 19 that carries the myotonic dystrophy gene in this family. Because Subject II-2 was now considered to be a carrier of the myotonic dystrophy gene, the clinical examination, including electromyography of 10 muscles, was repeated when he was 25 years old. This reexamination revealed no signs of myotonic dystrophy. Because of the discrepancy between the clinical findings and the results of the DNA analysis, final diagnosis was deferred until direct detection of the myotonic dystrophy mutation was possible.

Methods

Chromosomal DNA was isolated from peripheral-blood cells, cultured fibroblasts, or chorionic villi¹⁴. Spermatozoa were isolated from semen by single-layer Percoll centrifugation, and DNA was isolated from the sperm pellet¹⁵. The genetic markers flanking the myotonic dystrophy mutation and their respective detection methods have been previously described^11,12,13. All the markers were tested at least twice. Using a recently described polymerase-chain-reaction assay,⁷ we tested the expansion of the CTG trinucleotide repeat in genomic DNA. The CTG trinucleotide repeat was amplified with flanking primers, and the resulting DNA fragments were separated by electrophoresis on 1 percent and 4 percent agarose gels. A Southern blot, made from the 1 percent gel, was probed with a (CTG)₁₀ oligonucleotide end-labeled with phosphorus-32, and the hybridizing fragments were visualized by autoradiography. Normal alleles were identified by the same polymerase-chain-reaction assay, with a ³²P-end-labeled amplification primer. The amplification product was separated by electrophoresis on a 6 percent polyacrylamide-7 M urea sequencing gel and visualized by autoradiography. For the purposes of paternity testing, several highly polymorphic loci were tested on chromosomes 3, 15, 17, and 19^{12,16,17,18,19,20,21,22,23}. The sizes of alleles were determined for each locus by comparison with control samples of known size, as well as by measurement on an automated sequencing system with Gene Scanner software (Applied Biosystems, Foster City, Calif.). An internal-lane size standard (Gene Scan-2500 Rox, Applied Biosystems) was added as a reference control for aligning peak data. The paternity index and the probability of paternity were calculated as previously described²⁴.

The research plan was approved by the medical ethics committee of the Nijmegen University Hospital.

Results

In Family 1, genetic markers flanking the myotonic dystrophy mutation indicated that the third fetus (Subject III-3) had received the abnormal chromosome 19 from the father. Analysis of the causative myotonic dystrophy mutation showed an abnormal expanded allele in the father, but not in a sample of chorionic villus from the fetus (Figure 1). The fetus had two normal alleles (Figure 2), the larger of which (with 24 CTG trinucleotide repeats) was apparently inherited from the father. However, this allele was clearly different from the father's normal chromosome 19, which carried an allele of 11 CTG trinucleotide repeats. This suggested that a reverse mutation had occurred, through which the abnormal expanded CTG trinucleotide repeat in the father (approximately 150 to 600 CTG trinucleotide repeats) (Figure 1) had decreased in size to a normal allele of 24 CTG trinucleotide repeats in the fetus. After the birth of a normal girl, identical results were obtained in a sample of cord blood and in fibroblasts from the umbilical cord. Attempts to detect mosaicism for the reverted allele were unsuccessful in other tissues from the father. We did not find additional reverted alleles after analyzing DNA from cultured skin fibroblasts and sperm from Subject II-3 (Figure 3). We used several highly polymorphic systems from chromosomes 3, 15, 17, and 19 to exclude the possibility that Subject II-3 was not the girl's father. The probability that Subject II-3 was the biologic father of Subject III-3 was greater than 0.99998 (Table 1).

View larger version (95K): [in this window] [in a new window]   Figure 2. Precise Sizing of CTG Trinucleotide Repeats in Two Families with Myotonic Dystrophy. Only normal alleles (with 5 to 40 CTG trinucleotide repeats) are visualized on 6 percent polyacrylamide-gel electrophoresis. The abnormal expanded (Exp) alleles fall outside the range of fragments detected in this assay. Affected subjects (Subjects II-2, II-3, and III-1 in Family 1 and Subject I-1 in Family 2) have only a single band, representing their single normal chromosome 19. Normal subjects (Subjects I-2, II-1, II-4, II-5, and III-2 in Family 1 and Subject II-1 in Family 2) have two bands; Subject I-2 in Family 2 has only a single band, since she inherited identical normal alleles of five CTG trinucleotide repeats from both her parents. In Family 1, Subject III-3 inherited a new normal allele of 24 CTG trinucleotide repeats from her affected father. In Family 2, the mutated allele has decreased in size, from approximately 150 to 500 CTG trinucleotide repeats (Fig. 1) in the affected father (Subject I-1) to 19 CTG trinucleotide repeats in his son (Subject II-2). The pedigree symbols are as described in Figure 1.

 

View larger version (23K): [in this window] [in a new window]   Figure 3. Tissue Testing in Two Families with Reversal of the Myotonic Dystrophy Mutation. The pedigree symbols are as described in Figure 1. The reverted (normal) allele is indicated by arrows. In Family 1, the reverted allele was not found in either sperm (S) or cultured fibroblasts (F) from the father (Subject II-3). In both families the reverted allele was present in various tissues from the healthy offspring. These included a chorionic-villus sample (CV), umbilical-cord blood (CB), and umbilical-cord fibroblasts (CF) from Subject III-3 in Family 1 and peripheral-blood cells (B), cultured skin fibroblasts, and sperm from Subject II-2 in Family 2.

 

View this table: [in this window] [in a new window]   Table 1. Paternity Testing in Two Families with a Reversal of the Myotonic Dystrophy Mutation.

 
In Family 2, an asymptomatic 25-year-old man (Subject II-2) had inherited the abnormal chromosome 19 from his father. Expansion of the CTG trinucleotide repeat could not be demonstrated, however (Figure 1). Instead, there were two normal alleles (Figure 2), the larger of which (19 CTG repeat units) was inherited from the affected father. The father's mutated myotonic dystrophy gene, which contained approximately 150 to 500 CTG trinucleotide repeats, had therefore changed to a normal-sized allele of 19 CTG trinucleotide repeats in the son. The reverted allele was also present in skin fibroblasts and semen from the son (Figure 3). There were no abnormal expanded alleles in these tissues. Thus, no evidence for either somatic or germline mosaicism was found in this subject. We used several highly polymorphic DNA markers from chromosomes 3, 15, 17, and 19 to exclude the possibility that Subject I-1 was not the man's father. The probability that Subject I-1 was the biologic father of Subject II-2 was greater than 0.99999 (Table 1).

Discussion

A new class of genetic disease mutations has recently been described that is characterized by the amplification of preexisting trinucleotide-repeat units²⁵. Apart from myotonic dystrophy,^{2,3,4,5,6,7,8} the fragile X syndrome^26,27 and X-linked spinal and bulbar muscular atrophy²⁸ have been shown to be associated with this type of mutation. In both the fragile X syndrome and myotonic dystrophy, the length of the respective trinucleotide repeats tends to increase in subsequent generations, and there is a positive correlation between the length of a repeat and the severity of the disease. Although transmission of the fragile X mutation is occasionally accompanied by a reduction in repeat length,²⁷ this has never resulted in an allele of normal size. In our analysis of more than 100 carriers of the myotonic dystrophy mutation, a decreasing repeat length has been found in only one other family. A complete reversal of the mutated allele that yields a CTG trinucleotide repeat of normal size must therefore be an exceptional event.

The mechanism that causes the change from an abnormal expanded allele to a normal-sized allele is unknown. The possibility of single genetic recombination is excluded in these cases, since it would change the DNA-marker haplotype surrounding the mutation. Double recombinants are also highly unlikely, given the small genetic distances in this segment of chromosome 19. Either gene conversion (the substitution of one parental allele for the other²⁹) or direct deletion of the expanded repeat could explain the reversal of the mutation that we found in these two families.

We considered the possibility that the instability of the expanded CTG trinucleotide sequence in somatic tissues was also present in the fathers' germlines in the form of a large array of different-sized alleles that included some normal-sized alleles. However, DNA analysis of cultured skin fibroblasts and a sperm sample from the father in Family 1 (Subject II-3) showed a distribution of expanded repeats that was similar (although not identical) to that found in his peripheral blood. Moreover, this analysis failed to detect the presence of reverted alleles (Figure 3), indicating that if germline mosaicism is present in Subject II-3, the frequency of reverted myotonic dystrophy mutations is very low. Alternatively, the reversal of the mutation in his daughter (Subject III-3) may have occurred in the early embryo.

The fact that the allele lengths in the two subjects described here are entirely within the range found in normal subjects suggests that they should not have myotonic dystrophy in the future. They thus represent true reverse mutations, rather than the nonpenetrance of an abnormal gene. Yet it remains to be established whether their chromosomes have regained the normal stable state, since we do not know whether the expanded trinucleotide-repeat sequence is the only cause of the DNA instability in myotonic dystrophy. Analysis of cultured skin fibroblasts and a sperm sample from Subject II-2 in Family 2 showed 5 and 19 CTG trinucleotide repeats, identical to those detected in his peripheral-blood cells (Figure 3). This suggests that the reverted allele containing 19 CTG trinucleotide repeats is stable in this subject's somatic tissues as well as in his germline.

In conclusion, the two subjects described here are examples of complete spontaneous corrections of myotonic dystrophy mutations. These results should be taken into account when one uses flanking DNA markers for genetic diagnosis,³⁰ since it is possible that other genetic conditions in which the phenotype is highly variable will also prove to be associated with the inheritance of unstable DNA sequences³¹.

Supported by grants from the American Muscular Dystrophy Association, the Prinses Beatrixfonds (87-2694), and the Deutsche Forschungsgemeinschaft (Ro 389/15-4).

We are indebted to Dr. R.J. van Kooy and Dr. G.C.M.L. Christiaens, Department of Obstetrics and Gynecology, University Hospital, Utrecht, the Netherlands, for obtaining sperm and chorionic-villus samples; and to Dr. F. Spaans, Department of Clinical Neurophysiology, University Hospital, Maastricht, for performing additional neurophysiologic examinations.

Source Information

From the Departments of Human Genetics (H.G.B., W.N., M.R.N., B.A.O., H.-H.R., H.J.M.S.) and Cell Biology and Histology (G.J., B.W.), University Hospital and Medical Faculty, Nijmegen; and the Departments of Human Genetics (C.E.M.D.) and Neurology (C.J.H.), University Hospital, Maastricht -- both in the Netherlands.

Address reprint requests to Dr. Brunner at the Department of Human Genetics, University Hospital Nijmegen, P.O. Box 9101, 6500HB Nijmegen, the Netherlands.

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