Original Article
Direct Diagnosis of Myotonic Dystrophy with a Disease-Specific DNA Marker
N Engl J Med 1993; 328:471-475February 18, 1993
- Abstract
Background
Myotonic dystrophy is the most common inherited form of muscular dystrophy affecting adults. Its symptoms are not confined to muscle, and variability in their nature and in the patient's age at their onset can make diagnosis difficult. A specific unstable DNA sequence associated with myotonic dystrophy has recently been identified. We describe the use of a DNA probe (p5B1.4) that can detect this mutation directly, improving the accuracy and speed of diagnosis.
Methods
We analyzed DNA extracted from the peripheral-blood lymphocytes of 112 unrelated patients with myotonic dystrophy and their families, using molecular genetic techniques. Southern blot analysis and amplification with the polymerase chain reaction were used to determine the extent of expansion of the unstable DNA sequence.
Results
Probe p5B1.4 allowed direct identification of the myotonic dystrophy mutation in 108 of the 112 unrelated patients. In three families for whom the clinical and genetic data obtained with linked probes were ambiguous, the probe identified persons at risk for symptoms of this disorder and demonstrated that a possible sporadic case of myotonic dystrophy was familial. In one of these families the size of the unstable myotonic dystrophy-specific fragment decreased on transmission to offspring, who remained asymptomatic.
Conclusions
The diagnosis of myotonic dystrophy is improved by the use of a probe that detects directly the mutation responsible for this disorder.
Media in This Article
Figure 1
Haplotype Analysis and Detection of the Myotonic Dystrophy Mutation in Pedigree 1.Figure 2
Haplotype Analysis and Detection of the Myotonic Dystrophy Mutation in Pedigree 2.Article Activity
- Article
Myotonic dystrophy is an autosomal dominant disorder with an incidence of 1:80001. Its manifestations include muscle wasting, myotonia, cataracts, intellectual impairment, and cardiac conduction defects. The median age of patients at the onset of symptoms is 20 to 25 years, although a potentially fatal maternally transmitted form of the disease can affect neonates. The diagnosis of this disorder can be problematic because of wide variation in the range and severity of symptoms, both within and among families. Although the biochemical defect underlying the disorder is unknown, linkage analysis using nearby DNA markers is available for presymptomatic counseling and prenatal diagnosis. This type of genetic diagnosis is subject to limitations, since the restriction-fragment-length polymorphisms may not be informative and recombination events can confound accurate diagnosis. Such studies also depend on the availability of key family members
We recently described the isolation and initial characterization of a complementary DNA probe, cDNA25, that detected a polymorphism in lymphocyte DNA from the normal population with alleles of 8.6 and 9.8 kb (frequencies, 0.43 and 0.57, respectively)2. Similar studies in patients with myotonic dystrophy revealed that this probe detected one normal allele and a myotonic dystrophy-specific band of variable size in most affected persons. The variable band was always larger than the normal 9.8-kb allele, and its size correlated well with the severity of symptoms in the affected persons. The probe often detected a diffuse smear of DNA fragments corresponding to the myotonic dystrophy allele, rather than a discrete band, indicating that the size of the fragment generated by mitosis in somatic tissues can vary. The patterns of inheritance of the unstable fragment also reflected the phenomenon of anticipation, in that the fragment increases in size as it is passed from one generation to the next, through meiosis in both women and men, with an accompanying increase in disease severity. These findings were independently demonstrated with other probes from the region3-5. DNA sequencing revealed that the variation in the size of the fragment arises from the expansion of a repeat unit containing the three nucleotides, cytosine, thymine, and guanine (CTG), at the 3' end of a gene encoding a member of the protein kinase family5-7. The normal copy number of the repeat unit ranges from 5 to 358. In mildly affected patients with myotonic dystrophy this number is higher than 50, and it may be as high as 2000 in severely affected patients6.
In this paper we describe the use of p5B1.4, a probe derived from cDNA25, in the genetic analysis of affected families. Specific reference is made to patients from three different families who presented particular diagnostic problems that were resolved with the use of this probe. In one of these families the inherited myotonic dystrophy-specific fragment decreased in size on transmission to affected offspring.
Methods
Patients
We selected 112 patients with myotonic dystrophy from our data base for this study. The diagnosis in these patients was based either on linkage-analysis studies using nearby DNA markers or on clinical examination that conformed to the guidelines of the Muscular Dystrophy Association of America/Piton Foundation Working Group for myotonic dystrophy9. The ages of the patients ranged from 3 to 72 years; patients with congenital myotonic dystrophy were excluded. All the studies were approved by the local medical ethics committee, and each subject gave informed consent. The pedigrees of the three families described here are shown in Figure 1Figure 1
Haplotype Analysis and Detection of the Myotonic Dystrophy Mutation in Pedigree 1., Figure 2Figure 2Haplotype Analysis and Detection of the Myotonic Dystrophy Mutation in Pedigree 2., and Figure 3Figure 3
Detection of the Myotonic Dystrophy Mutation in Pedigree 3..Pedigree 1
The 54-year-old proband in Pedigree 1 (Subject II-1) began to have muscle weakness in his hands at the age of 33 years. A cataract was extracted in 1982. In 1992, he had the characteristic facies of myotonic dystrophy, muscle weakness in his neck and arms, and difficulty walking and standing on his heels. His deceased father had been wheelchair-bound in the latter stages of his life, but no other clinical details were available. In 1992, the proband's three sons (Subjects III-1, III-2, and III-3), who were 21, 26, and 29 years old, had no symptoms of myotonic dystrophy. The two older sons had normal neurologic examinations and normal results on electromyography and slit-lamp examination; no data were available for the youngest son.
Pedigree 2
The 44-year-old proband in Pedigree 2 (Subject II-1) first noted myotonia of grip at the age of 21 years, and ultimately had to give up his job because of difficulty with mobility. His father (Subject I-1) had frontal balding and bilateral early cataracts, but a normal electromyogram. His mother (Subject I-2) had weakness and stiffness, particularly in her hands. Electromyography revealed no myotonic discharges but some spontaneous activity, reduction in interference pattern, and polyphasic units; her ophthalmologic examination was normal. Detailed neurologic examinations of the proband's two children (Subjects III-1 and III-2), who were 15 and 17 years of age, respectively, were normal. These children were tested at their request so that they could make career decisions.
Pedigree 3
Subject III-1 in Pedigree 3 was born in 1975 after a normal pregnancy. His psychomotor development was normal, but he had some delay in speech at the age of three years. He was given a diagnosis of myotonic dystrophy at the age of 14 years, after examination revealed a myopathic face, an open mouth, and myotonic contractions of the hands. The diagnosis was confirmed by electromyography. In the next two years, myotonic features and coordination problems became more obvious. His parents (Subjects II-1 and II-2) and his maternal grandparents (Subjects I-1 and I-2) were examined during the period from 1989 to 1991; each had normal physical and slit-lamp examinations and electromyograms. In the light of this information, it was thought that Subject III-1 might have a rare sporadic case of myotonic dystrophy.
Southern Blot Analysis
DNA was prepared from peripheral-blood lymphocytes by standard procedures. Three micrograms of DNA were digested with the appropriate restriction enzyme, after which the fragments were separated by gel electrophoresis, transferred to Hybond N membranes (Amersham International), and hybridized overnight with radiolabeled probes. After being washed, the membranes were subjected to autoradiography for one to three days so that the results could be visualized.
Probe p5B1.4 was a 1.4-kb BamH1 fragment of cDNA252 that spans the variable polymorphic CTG trinucleotide repeat. Other probes used in the haplotype analysis (with the name of the corresponding restriction enzyme or enzymes following in parentheses) were D19S19 (PstI),10 CKM (NcoI11 and TaqI12), D19S63 (PvuII),13 D19S51 (PstI),14 and D19S22 (PstI)15.
Primers and PCR
The region containing the polymorphic NcoI and TaqI sites at the CKM locus was analyzed as described elsewhere16. The region containing the CTG trinucleotide repeat at the myotonic dystrophy locus was amplified as described elsewhere,6 except that Tth polymerase (Hybaid) and 50 pmol of the primers 101 and 102 were used. The reaction mixtures were cycled once for 5 minutes at 94 °C, 35 times for 1.5 minutes at 94 °C, for 1 minute at 62 °C, and for 2 minutes at 72 °C. The products were denatured and separated by polyacrylamide-gel electrophoresis.
Results
Patterns of Hybridization with the Probe p5B1.4
Southern blot analysis showed that p5B1.4 and cDNA25 detect identical fragments in EcoRI-digested DNA from normal subjects and patients with myotonic dystrophy. The use of a second enzyme, BglI, in the analysis increased the sensitivity of the test by providing a smaller (3.4-kb) target fragment for the probe17. When a combination of both enzymes was used, a single normal allele accompanied by a larger myotonic dystrophy-specific fragment was detected in 108 of the 112 patients tested. The four patients who did not have myotonic dystrophy-specific bands after DNA digestion with either EcoRI or BglI were heterozygous for alleles, with copy numbers of the CTG repeat that fell within the normal range. These results may be explained either by misdiagnosis or by the occurrence of a mutation (other than the expansion of the CTG repeat) in the myotonic dystrophy gene.
Problematic Cases
Figure 1, Figure 2, and Figure 3 show the inheritance of the myotonic dystrophy-specific fragment detected by p5B1.4 in the three pedigrees described above. Where available, haplotypes constructed from the linkage analysis of informative DNA markers are included in the figures.
Pedigree 1 was investigated by linkage analysis to establish the status of the children in generation III. The father (Subject II-1) was heterozygous for 5 of the 11 markers tested. With the exception of D19S63, however, they were not helpful, because the haplotype of the chromosome carrying the myotonic dystrophy mutation could not be established in the absence of information from the deceased father of Subject II-1. The 3 allele of D19S63 was carried on the affected chromosome of Subject II-1. If it is assumed that there was no recombination between this DNA marker and the myotonic dystrophy locus, all three of his children (Subjects III-1, III-2, and III-3) inherited this allele and therefore the myotonic dystrophy mutation. This prediction was confirmed with probe p5B1.4 (Figure 1). The affected father (Subject II-1) and his sons (Subjects III-1, III-2, and III-3) had one normal (8.6-kb) allele and a larger myotonic dystrophy-specific fragment. Although there was heterogeneity in the size of the fragment in the father, the modal size was approximately 15 kb. The fragment was about 3 kb smaller in the sons, and the bands in the sons were more discrete than that in the father.
In Pedigree 2, clinical examination and segregation analysis with linked markers failed to determine the parental origin of the myotonic dystrophy mutation carried by Subject II-1. Linkage analysis indicated that one of his two sons (Subject III-2) had a risk of more than 99 percent of inheriting myotonic dystrophy. Figure 2 shows that probe p5B1.4 detected a 10.4-kb myotonic dystrophy-specific fragment in the father (Subject II-1) and a 10.8-kb fragment in one of his sons (Subject III-2). As predicted by the linkage analysis, the other son (Subject III-1) had a normal heterozygous pattern. The disease is segregating with the haplotype 212 in this family, suggesting that the grandfather (Subject I-1) was affected. This was confirmed when BglI-digested DNA from this man, probed with p5B1.4, showed the normal 3.4-kb band and an additional 3.6-kb band.
There have been unsubstantiated reports of sporadic cases of myotonic dystrophy, and Subject III-1, the proband in Pedigree 3, was thought to have such a case at presentation (Figure 3). His mother (Subject II-1), however, had one normal (8.6-kb) allele and a 10.6-kb band. Subject III-1 inherited the latter fragment, which increased in size to 10.9 kb. In view of these findings, Subject II-1 was reexamined in the clinic. At the age of 34 years, her neurologic examination was normal, but she had some atrophy of the temporalis muscles. Electromyography showed myotonic discharges in four of the five muscle groups investigated; these muscles had been normal two years earlier. The results of probing with p5B1.4 show clearly that Subject III-1 does not have a sporadic case of myotonic dystrophy, and other family members are now being studied. Although the bands in the grandparents' DNA appeared to be of normal size, polymerase-chain-reaction analysis revealed that the grandfather (Subject I-2) had an allele with 72 copies of the CTG repeat -- well beyond the normal range, and establishing him as the transmitting family member.
Discussion
These studies illustrate the usefulness of p5B1.4 as a specific probe for the detection of the myotonic dystrophy mutation in over 96 percent of patients. Of the 112 patients with myotonic dystrophy we have tested so far, 4 did not have a myotonic dystrophy-associated expansion of the CTG repeat. Since these 4 patients had two normal alleles (as demonstrated by PCR analysis), it was unclear whether the diagnosis of myotonic dystrophy was incorrect or whether they had another mutation that was responsible for the phenotype.
The use of the probe p5B1.4 improves the accuracy and reliability of diagnosis, particularly when the results of linkage analysis are ambiguous. For example, in Pedigree 2, the equivocal results obtained on electromyography for the grandparents prevented the identification of the affected grandparent. Thus, the haplotype of the proband's affected chromosome could not be deduced, and linked markers could not be used for the presymptomatic diagnosis of his sons. However, p5B1.4 identified the gene carrier directly. In Pedigree 3, direct analysis demonstrated familial myotonic dystrophy, rather than a new mutation, in Subject III-1 and allowed us to identify the transmitting family members.
Pedigree 1 is a rare example of a family in which the reverse of anticipation is occurring at the molecular level. Three asymptomatic sons have inherited a fragment associated with myotonic dystrophy that is smaller than that of their affected father. It will be interesting to see whether the genotype-phenotype correlation is maintained and whether the sons are less severely affected than their father when they reach the age at which the father's symptoms first developed.
Some caution should be exercised in interpreting the test with respect to its predictive power. Within families, the correlation between the size of the myotonic dystrophy-specific variable fragment and the phenotype expressed is good2-4. However, when fragment sizes are compared in unrelated affected patients, the correlation is weaker. For example, Subject III-2 in Pedigree 1, who had an expansion in band size of 2.2 kb above normal, remained asymptomatic at the age of 26, whereas Subject III-1 in Pedigree 3, who had a 1.1-kb expansion, had sufficiently severe symptoms for the diagnosis of myotonic dystrophy to be made at the age of 14. Furthermore, there is uncertainty both about the clinical importance of the somatic instability of the DNA (detected as a smear of bands by the probe) and about the correlation between this heterogeneity of size in lymphocyte DNA and that in other tissues. Differences between tissues may contribute to the variability in clinical presentation.
PCR amplification of the region containing the CTG repeat has demonstrated that a product containing 50 or more copies of the repeat is indicative of myotonic dystrophy6. In the vast majority of patients with this disease, however, the CTG-repeat region has expanded to such an extent (to hundreds or even thousands of copies) that PCR cannot generate a product efficiently. Because for such patients PCR yields a product only for the unaffected chromosome and therefore the patients cannot be distinguished from persons homozygous for a normal allele, the usefulness of PCR in diagnosis is limited.
The principal mutation causing myotonic dystrophy can be detected directly by Southern blot analysis with the probe p5B1.4. This easy and reliable test is superior to current PCR-based methods of detection, and it obviates the need for complex and time-consuming segregation analysis with closely linked DNA markers.
Supported by a grant (RA3/257/1) from the Muscular Dystrophy Group of Great Britain, by a grant from the Task Force in Genetics of the Muscular Dystrophy Association of America/Piton Foundation, and by the Central Research Fund of the University of London. A studentship was provided by the Science and Engineering Research Council.
We are indebted to all our colleagues in the Muscular Dystrophy Association of America/Piton Working Group for myotonic dystrophy for their contributions. This article is dedicated to the memory of our colleague Dr. Richard Lindenbaum, who died during the period between its submission and publication.
Source Information
From the Department of Anatomy, Charing Cross and Westminster Medical School (P.S., J.D., J.B., K.J.); the Department of Neurology, Charing Cross Hospital (Fulham) (R. Lane); and the Department of Biochemistry and Molecular Genetics, St. Mary's Hospital Medical School, Imperial College (R.W.) -- all in London; the Department of Clinical Genetics, Karolinska Hospital, Stockholm, Sweden (M.A., E.B.); the Department of Medical Genetics, Vrije Universiteit Brussel, Brussels, Belgium (M.B., E.S.); and the Department of Medical Genetics, Churchill Hospital, Oxford, United Kingdom (I.G., R. Lindenbaum).
Address reprint requests to Dr. Johnson at the Department of Anatomy, Charing Cross and Westminster Medical School, Fulham Palace Rd., London W6 8RF, United Kingdom.
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