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Molecular analysis for the myotonic dystrophy mutation in neuromuscular disorders
Neuromusc.Disord.,Vol. 2, No, 5/6, pp. 405-41I. 1992 Printed in Great Britain
0960--8966/92$5.00 + 0.00 Pergamon Press Lid
M O L E C U L A R ANALYSIS F O R THE M Y O T O N I C D Y S T R O P H Y M U T A T I O N IN N E U R O M U S C U L A R D I S O R D E R S J. C. MACMILLAN,* J. MYRING, H . G . HARLEY, W . REARDON, P. S. HARPER a n d D. J. SHAW
Institute of Medical Genetics,Universityof WalesCollegeof Medicine,Heath Park, Cardiff, CF4 4XN, Wales (Received 13 August 1992; revised 29 October 1992; accepted 5 November 1992)
Abstract--A variable expansion of an unstable CTG repeat has been identified as the causal mutation for myotonic dystrophy. Standard molecular genetic techniques can now supplement traditional assessment protocols in a variety of clinical neurological situations where diagnostic uncertainty prevailed. Southern analysis using DNA probes which identify the expanded sequence, supplemented by direct PCR analysis for repeat number, provides a specific sensitive diagnostic test for myotonic dystrophy. Key words: Myotonic dystrophy, mutation, unstable DNA, prediction.
INTRODUCTION
Myotonic dystrophy is the commonest muscular dystrophy of adult life and is a disorder of exceptional variability in both severity and range of systems affected. While in the majority of cases a firm diagnosis can be established without the need for detailed investigation, a proportion may give rise to considerable diagnostic difficulty, especially when myotonia is not obvious, or when wasting and weakness are variable. Cases presenting in early childhood may also be confusing, as may those in later life where neuromuscular abnormalities can be minimal. Long established investigations in myotonic dystrophy include electromyography for confirming the presence ofmyotonia, muscle biopsy, and slit-lamp examination of the lenses for the characteristic early opacities, but until recently no specific test has been available. Recent detection, by ourselves and colleagues, of a specific mutation producing the disorder [1-3], an unstable DNA sequence on chromosome 19, has given the possibility of using this in neuromuscular diagnosis [4]. We present here our preliminary experience of the use of this technique in cases referred for molecular diagnosis where myotonic dystrophy had been suspected but not confirmed, as in the case of neuromuscular symptoms.
The specific mutational defect in myotonic dystrophy is now recognized to be the expansion of an unstable CTG sequence [5]. In normal individuals, less than 30 copies of this sequence are present, while in myotonic dystrophy the number varies between 50 and over 2000 [4]. The abnormality can be detected by Southern blot analysis, where appropriate probes detect an EcoRI polymorphism giving 9 and 10 kb bands in the normal population. In myotonic dystrophy the 10 kb band is expanded, giving a variable alteration in band position. In individuals with a minimal expansion, this cada be confirmed by using the enzyme PstI, which gives greater resolution of smaller fragments, or by PCR amplification to determine the precise copy number of the CTG repeats [6]. MATERIALS AND METHODS
The individuals and families reported in this paper were seen by one of the authors or referred for molecular diagnosis by the clinicians acknowledged in the case descriptions. The molecular genetic techniques used have been described previously [6, 7].
CASE REPORTS
Case'l (referred by Professor M. Wiles, Cardiff) *Author to whomall corresponden¢~shouldbe addressed.
A 20 yr old m a n (Ill,Fig. I) presented to the neurology clinic with a 5 yr history of muscle 405
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neurogenetics clinic raised the possibility of myotonia congenita. The parents were asymptomatic, unrelated and both neurologically normal. Slit-lamp examination revealed nonspecific tiny white specks in the crystalline lenses of both parents. DNA analysis showed a small but definite expansion ( + , Fig. 1b) on Southern blotting with enzyme EcoRI. No abnormality was seen in samples from either parent. Using enzyme PstI the abnormality in the proband was confirmed (not seen clearly in Fig. lc but marked + + ) while the father (IJ also showed an abnormal band corresponding to a minimal expansion ( + , Fig. lc) which was confirmed by PCR analysis. The positive molecular analysis for DM in this individual enabled a definitive diagnosis of myotonic dystrophy to be made and in addition revealed the previously unknown risk to other family members. Case 2 (referred by Dr P. Lunt, Bristol)
Molecular analysis was requested on this 62 yr old man (112, Fig. 2a) who, along with his Fig. I b, M 10M6/EeoRI analysis showing expanded sequence brother, suffered a neuromuscular problem from in lane ! (+). childhood. He had frontal balding, a long face with high-arched palate, hypoplastic left chest wall, scoliosis and mild proximal muscle weakII 1 11 12 ness. There was no clinical myotonia and EMG was myopathic with 'bizarre repetitive discharges'. Muscle biopsy (vastus lateralis) - ++ changes were reported as 'typical of myotonic dystrophy' with greatly increased fibre size variation, a marked increase in central nuclei -+ which formed chains in longitudinal section, and a mild focal increase of endomysial collagen. - normal Pyknotic nuclear clumps and occasional fibre splitting were seen but no necrosis, regeneration or group atrophy. Histochemical stains showed 'moth-eaten" fibres, occasional ring fibres but no evidence of mitochondrial accumulation nor Fig. It. M 10M6/PstI analysis showing minimal expansion in nemaline rods. There was no fibre-type disI~( + ) and more marked expansion (+ +, poorly seen) in lit. tinction using any stain which was interpreted as stiffness, particularly involving grip, and inter- suggesting a congenital onset of the disease fering with his work as a mechanic. He had process. noticed occasional stiffness of the jaw while DNA analysis (Fig 2b, lane 5) showed he was chewing, but no weakness. Examination showed heterozygous for the EcoRI polymorphism with marked active and percussion myotonia of the normal 9 and 10 kb fragments, and the PstI digest hands but not definite weakness of the face, neck (not shown) showed no evidence for the enor other muscles. EMG confirmed profuse myo- largement in the 10 kb fragment commenly tonic potentials. Slit-lamp examination of the associated with myotonic dystrophy. This was lenses was normal. interpreted as indicating that this man's signs The initial diagnosis was considered to be were not due to myotonic dystrophy; the precise myotonic dystrophy but reassessment at the nature of his myopathy remains undetermined. I
Myotonic Dystrophy Diagnosis
407
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Case 3 (referred by Dr A. Superti-Furga, Zurich)
Molecular DNA analysis was requested on a fragment of skeletal muscle, stored in cryosectioning medium, from a female neonate who died 12 days postnatally. The patient had been born at 34 weeks gestation by caesarian section with a history of polyhydramnios, breech presentation and pathological cardiotochography. She was floppy on delivery, with no spontaneous movements and Apgars of 1, 2 and 3. Muscle hypotonia persisted after intubation and a muscle biopsy was performed; this was reported as being 'myopathic with a dystrophic component' and compatible with myotonic dystrophy. Both parents were clinically normal but neither EMG nor slit-lamp examination were carried out. The DNA isolation from the muscle biopsy specimen showed a normal heterozygous pattern at the myotonic dystrophy locus using Southern blot mutation screen (not shown). The normal mutation analysis in this case made it most unlikely that the disease in question was congenital myotonic dystrophy and shows the value of stored biopsy specimens in molecular analysis.
Fig. 3. M 10M6/EcoRI analysis of 3 sisters (labelled A) with myotonic dystrophy and gynaecological neoplasia. An abnormal band ( + ) is seen in each with some smearing. A normal control (N) is used in lane 1. Case 5
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D N A analysis was requested on three sisters from a sibship of six (three males and three females). All three had features of myotonic dystrophy with cataracts, ptosis, myopathic
408
J.C. MACMILLAN et al.
fades and clinical myotonia. One had noninsulin dependent diabetes and two had cardiac conduction defects. Two had undergone hysterectomy for adenocareinoma of the uterus, (one had in addition an endometrial carcinoma of one ovary) and the third for uterine leiomyomata and bilateral ovarian cystadeomata. The question had been asked whether there was an unusual mutation or extensive deletion in the myotonic dystrophy gene which had given rise to the additional gynaecological neoplasms in these women. All three sisters showed an abnormal band ( + , Fig. 3) with M10M6/EcoRI for myotonic dystrophy; this being of moderate size, similar in the three and showing some smearing, suggesting some somatic instability. They also all showed a normal 10 kb band. The finding of 'typical' DM molecular mutations in these cases made it unlikely that there was a causal relationship between the disease and the gynaecological abnormalities in these sisters. Case 5
The 23 yr old daughter (II2, Fig. 4) of a woman (now deceased) with clinically definite myotonic dystrophy was assessed in 1983. She was asymptomatic and had a normal clinical examination except for percussion myotonia of the thenar eminence and some polychromatic dots reported on ophthalmic examination. In 1982 she had given birth to an apparently healthy boy (arrowed, III0 who was subsequently reassessed at the age of 2½ yr because of delayed speech. He had no stigmata of either congenital or severe childhood myotonic dystrophy. In view of the clinical findings in his mother it was felt that his mild developmental delay may have been due to myotonie dystrophy. Conventional D N A analysis using linked markers was complicated by the fact that the woman's late father (cause of death at 40 yr was unknown) was a first cousin of her mother and himself at 50% risk of inheriting the gene for myotonic dystrophy (his mother had been affected). Both mother and son were reviewed clinically prior to analysis for the myotonic dystrophy mutation. Neurological examination in the child was normal, as was assessment of muscle strength in his mother. Clinical percussion myotonia was again found but EMG of abductor pollicis brevis was normal. Slit-lamp examination showed pigmented lens dots but was not felt to show polychromatic crystals on this occasion. D N A analysis showed no evidence of
the unstable myotonic dystrophy sequence (not shown). The normal mutation analysis in these cases contributed significantly to the revision of the previous erroneous diagnosis of myotonic dystrophy in the mother and the queried diagnosis in her son. Case 6 (referred by 'Professor V. Dubowitz, London)
Analysis was requested, on D N A from the maternal grandparents (arrowed, 113and 114,Fig. 5a) of a living child (IV2) with congenital myotonic dystrophy to facilitate extended family counselling. Both had been assessed at age 66, at the time of the birth of the proband and had normal clinical and EMG findings. The grandfather, however, had developed non-specific cataracts at age 58 whilst the grandmother had a unilateral cataract at age 74. On Southern blotting (PstI digest, Fig. 5b) the grandfather was found to have a minimally increased band size ( + , lane 1) at the DM locus whilst his wife's were normal (only a constant band is seen with PstI in this situation). The abnormal band was seen to increase in size in subsequent generations ('smear', lane 4: + + , lane 5) consistent with the phenomenon of anticipation. Case 7
This 29 yr old man (arrowed, III2, Fig. 6) was referred for assessment as his mother had myotonic dystrophy. He had a normal clinical and E M G assessment but slit-lamp examination showed bilateral polychromatic lens opacities. Conventional linkage analysis was not of help as the family structure revealed a two generation phase unknown situation. He had been counselled that he was a carder of the DM gene on the basis of his eye changes. Direct mutation analysis confirmed the expandexl D N A segment in his affected mother but showed that the consultand had inherited her normal 10 kb allele (not shown). This case shows that polychromatic lens crystals, even at an early age, are not specific for myotonic dystrophy. DISCUSSION
Until the identification of a specific mutation for myotonic dystrophy and the subsequent isolation of the gene early this year, the diagnosis
MyotonicDystrophyDiagnosis
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Fig. 5b. M 10M6/Pstl analysisshowinga minimalexpansion (+, lane 1) in the grandfather, a larger expansion (smear, lane 4) in the mother and further enlargement(+ +, lane 5) in the congenitalcase. of myotonic dystrophy has been largely based on clinical findings, the combination o f m y o t o n i a of grip with a characteristic distribution of muscle weakness and wasting allowing a confident diagnosis in most cases, especially in the presence of a known family history.
Electromyography has been a valuable supportive investigation, partly in confirming the clinical finding of myotonia, showing the typical electrophysiological features, as opposed to a presynaptic or electrically silent reaction; and partly in detecting or excluding myotonic potentials in clinically normal relatives at risk of the disorder. Ophthalmologic assessment for the characteristic multicoloured lens opacities often seen early in the disorder has also been a useful investigation, both in the detection of presymptomatic gene carriers and in the differential diagnosis of other myotonic syndromes. Numerous other tests have been suggested as helpful in the diagnosis or presymptomatic
410
J . C . MACMILLAN et al.
detection of myotonic dystrophy, but none has been confirmed as being specific or reliable [8]. Likewise, while linked genetic markers have been useful in raising or lowering genetic risks for family members [9, 10], they have not been applicable in cases without a clear family history. The cases described here, referred over a relatively short period since recognition of the molecular defect, illustrate the wide range of uses of this test in a number of neurological diagnostic situations. In adult patients, molecular analysis is likely to be of most help in cases of myotonia (e.g. case 1) where lack of weakness or progression raises uncertainty as to whether myotonic dystrophy or one of the non-progressive myotonic disorders is the cause. Molecular diagnosis is also becoming possible for some of these, with identification of mutations for hyperkalaemic (myotonic) periodic paralysis in, and linkage of, paramyotonia to the sodium ion channel on chromosome 17 [11, 12]; and mutations in the chloride channel in recessive generalized myotonia [13]. Detection of the gene in older individuals showing a cataract but normal neurological examination and EMG is also important for genetic counselling of their offspring; since these individuals commonly show only a small expansion of the unstable sequence, analysis using PCR as well as Southern blotting should preferably be undertaken to ensure that the number of copies of the repeat sequence can be accurately determined. So far, normal individuals have shown less than 30 copies of the CTG repeat sequence, while minimally affected individuals have shown 50 or more copies [6]. The use of enzyme PstI to give greater resolution of small expansions on Southern blotting has also been noted. In childhood the diagnosis of congenital myotonic dystrophy is frequently not made initially and may, as in the caseoreported here, not be suspected until after the infant's death. In these cases muscle histology is often of not much benefit and can even be misleading where changes are interpreted (erroneously in two patients in this series) as being 'typical' of DM. In such cases the availability of frozen muscle tissue as a source of DNA may be a crucial factor in reaching a specific molecular diagnosis. Since congenital cases of myotonic dystrophy are associated with large expansions of the unstable sequence [4], a clear-cut diagnosis or exclusion of the disorder should be possible. When molecular analysis shows the presence of the specific expanded sequence in a diagnostically uncertain
case, there is now little doubt as to its significance; the same is true if the abnormality is excluded in a relative of a patient shown to have the defect. An apparently normal result in an isolated case should not, however, yet be interpreted as excluding myotonic dystrophy with certainty; although no confirmed cases not showing the defect have yet to be recognized, it is possible that different mutations in other parts of the myotonic dystrophy gene may be responsible for some cases; particularly those with unusual phenotypes, though the family reported here (case 3) showed the usual mutation. An unusual feature of the myotonic dystrophy mutation is its instability, explaining the long debated phenomenon of anticipation [14], since the progressively earlier onset and greater severity in successive generations is, with a few exceptions, correlated with progressive expansion of the unstable sequence. We, along with others, have shown a broad correlation of size of the expansion with phenotype [15, 16], congenital cases showing the greatest degree of expansion (3-6 kb of DNA), while minimal cases with insignificant or absent neuromuscular features have shown small expansions (usually around 0.15 kb) [6]. The significant overlap in degree of expansion between cases from groups, other than those with congenital and minimal disease, means however, that these data are not directly applicable to the clinical situation where molecular diagnosis for an individual has been requested. Similarly, it should be remembered that, in general, analyses are carried out on D N A extracted from peripheral blood lymphocytes and this may not be representative of the findings in other tissues. This has theoretical implications for prenatal diagnosis of myotonic dystrophy in that, to date, there are no data correlating size of expansion in chorionic DNA with disease severity (most 'affected' pregnancies bein~ terminated). We do not feel it appropriate to offer a prediction of prognosis in place of a simple diagnosis from a chorionic biopsy. Finally, it may be asked whether molecular analysis will now make studies such as electromyography redundant in the diagnosis of myotonic dystrophy? Our view is that it should not, at least for the present, since such investigations will give a guide to the likely development of the neurological phenotype, while it is only by comparison of the various tests that we will gain an accurate idea as to the proportion of neurologically normal gene carriers that exists. Equally, however, molecular analysis of the unstable
Myotonic Dystrophy Diagnosis
sequence must now be regarded as part of the diagnostic investigation of myotonic dystrophy. In view of the broad correlation between the degree of expansion and clinical severity we would urge caution in interpreting the possible relationship of DNA expansion and prognosis when molecular diagnosis is made at an early or presymptomatic stage of the disease. In particular we would not recommend the testing of children at risk unless there are specific clinical reasons for doing so [17]. The discovery of the molecular basis of myotonic dystrophy is a striking example of how the new techniques of DNA analysis are changing both our understanding of neurological disorders and patterns of clinical practice in neurology. We can now look ahead to further studies of the specific gene product (myotonin) predicted on the basis of its sequence to be a member of the protein kinase family, helping to elucidate the pathogenesis of the neuromuscular and other clinical features of this disease, while in the more distant future these developments may lead to therapy than can ameliorate or prevent some of the numerous clinical problems encountered by patients with myotonic dystrophy.
REFERENCES 1. Harley H G, Brook J D, Rundle S A, et al. Expansion of an unstable DNA region and phenotype variation in myotonic dystrophy. Nature 1992; 355: 545-546. 2. Buxton J, Shelbourne P, Davies J, el al. Detection ofan unstable fragment of DNA specific to individuals with myotonic dystrophy. Nature 1992; 355: 547-548. 3. Aslanidis C, Jansen G, Amemiya C, el al. Cloning ofthe essential myotonic dystrophy region and mapping of the putative defect. Nature 1992; 355: 548-551.
411
4. Harley H G, Rundle S A, Reardon W, et al. Unstable DNA sequence in myotonic dystrophy. Lancet 1992; 339: !125-1128. 5. Brook J D, McCurrach M E, Harley H G, et al. Molecular basis of myotonic dystrophy: expansion of a trinucleotide (CTG) repeat at the 3' end of a transcript encoding a protein kinase family member. Cell 1992; 68: 799-808. 6. Reardon W, Harley H G, Brook J D, el al. Minimal expression of myotonic dystrophy: a clinical and molecular analysis. J M e d Genet (in press). 7. Myring J, Meredith A L, Harley H G, et al. Specific molecular diagnosis for the CTG mutation in myotonic dystrophy. J M e d Genet (in press). 8. Harper P S. Myotonic Dystrophy, 2nd Edn. London: Saunders, 1989. 9. Norman A M, Floyd J L, Meredith A L, Harper P S. Presymptomatic detection and prenatal diagnosis for myotonic dystrophy by means of linked DNA markers. J M e d Genet 1989; 26: 750-754. 10. Reardon W, Floyd J L, Myring J, Lazarou L P, Meredith A L, Harper P S. Five years experience of predictive testing for myotonic dystrophy using linked DNA markers. A m J M e d Genel 1992; 43:1006-1011. II. Ebers G C, George A L, Barchi R L, el al. Paramyotonia congenita and hyperkalemic periodic paralysis are linked to the adult muscle sodium channel gene. Ann Neurol 1991; 30: 810-816. 12. Rojas C V, Wang J, Schwartz L S, Hoffman E, Powell B R, Brown R H Jr. A Met-to-Val mutation in the skeletal muscle Na + channel ¢-subunit in hyperkalaemic periodic paralysis. Nature 1991; 354: 387-389. 13. Koch M C, Steinmeyer K, Lorenz C, el al. The skeletal muscle chloride channel in dominant and recessive human myotonia. Science (in press). 14. Harper P S, Harley H G, Reardon W, Shaw D J. Anticipation in myotonic dystrophy: new light on an old problem. A m J Hum Genet 1992; 51: 10-16. 15. Harley H G, Rundle S A, Sandkuijl L, et al. Variable expansion of an unstable CTG repeat sequence in relation to phenotype in myotonic dystrophy (manuscript submitted for publication). 16. Tsilfidis C, MacKenzie A E, Mettler, Barcel6 J, Korneluk R G. Correlation between CTG trinucleotide repeat length and frequency of severe congenital ~nyotonic dystrophy. Nature Genet 1: 192-195. 17. Harper P S, Harley D J, Shaw D J. Detection of minimum mutation carriers in myotonic dystrophy. Lancet 1992; 340: 238-239.
0960--8966/92$5.00 + 0.00 Pergamon Press Lid
M O L E C U L A R ANALYSIS F O R THE M Y O T O N I C D Y S T R O P H Y M U T A T I O N IN N E U R O M U S C U L A R D I S O R D E R S J. C. MACMILLAN,* J. MYRING, H . G . HARLEY, W . REARDON, P. S. HARPER a n d D. J. SHAW
Institute of Medical Genetics,Universityof WalesCollegeof Medicine,Heath Park, Cardiff, CF4 4XN, Wales (Received 13 August 1992; revised 29 October 1992; accepted 5 November 1992)
Abstract--A variable expansion of an unstable CTG repeat has been identified as the causal mutation for myotonic dystrophy. Standard molecular genetic techniques can now supplement traditional assessment protocols in a variety of clinical neurological situations where diagnostic uncertainty prevailed. Southern analysis using DNA probes which identify the expanded sequence, supplemented by direct PCR analysis for repeat number, provides a specific sensitive diagnostic test for myotonic dystrophy. Key words: Myotonic dystrophy, mutation, unstable DNA, prediction.
INTRODUCTION
Myotonic dystrophy is the commonest muscular dystrophy of adult life and is a disorder of exceptional variability in both severity and range of systems affected. While in the majority of cases a firm diagnosis can be established without the need for detailed investigation, a proportion may give rise to considerable diagnostic difficulty, especially when myotonia is not obvious, or when wasting and weakness are variable. Cases presenting in early childhood may also be confusing, as may those in later life where neuromuscular abnormalities can be minimal. Long established investigations in myotonic dystrophy include electromyography for confirming the presence ofmyotonia, muscle biopsy, and slit-lamp examination of the lenses for the characteristic early opacities, but until recently no specific test has been available. Recent detection, by ourselves and colleagues, of a specific mutation producing the disorder [1-3], an unstable DNA sequence on chromosome 19, has given the possibility of using this in neuromuscular diagnosis [4]. We present here our preliminary experience of the use of this technique in cases referred for molecular diagnosis where myotonic dystrophy had been suspected but not confirmed, as in the case of neuromuscular symptoms.
The specific mutational defect in myotonic dystrophy is now recognized to be the expansion of an unstable CTG sequence [5]. In normal individuals, less than 30 copies of this sequence are present, while in myotonic dystrophy the number varies between 50 and over 2000 [4]. The abnormality can be detected by Southern blot analysis, where appropriate probes detect an EcoRI polymorphism giving 9 and 10 kb bands in the normal population. In myotonic dystrophy the 10 kb band is expanded, giving a variable alteration in band position. In individuals with a minimal expansion, this cada be confirmed by using the enzyme PstI, which gives greater resolution of smaller fragments, or by PCR amplification to determine the precise copy number of the CTG repeats [6]. MATERIALS AND METHODS
The individuals and families reported in this paper were seen by one of the authors or referred for molecular diagnosis by the clinicians acknowledged in the case descriptions. The molecular genetic techniques used have been described previously [6, 7].
CASE REPORTS
Case'l (referred by Professor M. Wiles, Cardiff) *Author to whomall corresponden¢~shouldbe addressed.
A 20 yr old m a n (Ill,Fig. I) presented to the neurology clinic with a 5 yr history of muscle 405
406
J. C, MACMILLANet al.
(o) Case 1
D
Q
1
2
I
2
B
11"
/
Fig. la. Family pedigree for ease I.
II 1
11
12
,
.~
-+ - lOkb
neurogenetics clinic raised the possibility of myotonia congenita. The parents were asymptomatic, unrelated and both neurologically normal. Slit-lamp examination revealed nonspecific tiny white specks in the crystalline lenses of both parents. DNA analysis showed a small but definite expansion ( + , Fig. 1b) on Southern blotting with enzyme EcoRI. No abnormality was seen in samples from either parent. Using enzyme PstI the abnormality in the proband was confirmed (not seen clearly in Fig. lc but marked + + ) while the father (IJ also showed an abnormal band corresponding to a minimal expansion ( + , Fig. lc) which was confirmed by PCR analysis. The positive molecular analysis for DM in this individual enabled a definitive diagnosis of myotonic dystrophy to be made and in addition revealed the previously unknown risk to other family members. Case 2 (referred by Dr P. Lunt, Bristol)
Molecular analysis was requested on this 62 yr old man (112, Fig. 2a) who, along with his Fig. I b, M 10M6/EeoRI analysis showing expanded sequence brother, suffered a neuromuscular problem from in lane ! (+). childhood. He had frontal balding, a long face with high-arched palate, hypoplastic left chest wall, scoliosis and mild proximal muscle weakII 1 11 12 ness. There was no clinical myotonia and EMG was myopathic with 'bizarre repetitive discharges'. Muscle biopsy (vastus lateralis) - ++ changes were reported as 'typical of myotonic dystrophy' with greatly increased fibre size variation, a marked increase in central nuclei -+ which formed chains in longitudinal section, and a mild focal increase of endomysial collagen. - normal Pyknotic nuclear clumps and occasional fibre splitting were seen but no necrosis, regeneration or group atrophy. Histochemical stains showed 'moth-eaten" fibres, occasional ring fibres but no evidence of mitochondrial accumulation nor Fig. It. M 10M6/PstI analysis showing minimal expansion in nemaline rods. There was no fibre-type disI~( + ) and more marked expansion (+ +, poorly seen) in lit. tinction using any stain which was interpreted as stiffness, particularly involving grip, and inter- suggesting a congenital onset of the disease fering with his work as a mechanic. He had process. noticed occasional stiffness of the jaw while DNA analysis (Fig 2b, lane 5) showed he was chewing, but no weakness. Examination showed heterozygous for the EcoRI polymorphism with marked active and percussion myotonia of the normal 9 and 10 kb fragments, and the PstI digest hands but not definite weakness of the face, neck (not shown) showed no evidence for the enor other muscles. EMG confirmed profuse myo- largement in the 10 kb fragment commenly tonic potentials. Slit-lamp examination of the associated with myotonic dystrophy. This was lenses was normal. interpreted as indicating that this man's signs The initial diagnosis was considered to be were not due to myotonic dystrophy; the precise myotonic dystrophy but reassessment at the nature of his myopathy remains undetermined. I
Myotonic Dystrophy Diagnosis
407
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Case 3 (referred by Dr A. Superti-Furga, Zurich)
Molecular DNA analysis was requested on a fragment of skeletal muscle, stored in cryosectioning medium, from a female neonate who died 12 days postnatally. The patient had been born at 34 weeks gestation by caesarian section with a history of polyhydramnios, breech presentation and pathological cardiotochography. She was floppy on delivery, with no spontaneous movements and Apgars of 1, 2 and 3. Muscle hypotonia persisted after intubation and a muscle biopsy was performed; this was reported as being 'myopathic with a dystrophic component' and compatible with myotonic dystrophy. Both parents were clinically normal but neither EMG nor slit-lamp examination were carried out. The DNA isolation from the muscle biopsy specimen showed a normal heterozygous pattern at the myotonic dystrophy locus using Southern blot mutation screen (not shown). The normal mutation analysis in this case made it most unlikely that the disease in question was congenital myotonic dystrophy and shows the value of stored biopsy specimens in molecular analysis.
Fig. 3. M 10M6/EcoRI analysis of 3 sisters (labelled A) with myotonic dystrophy and gynaecological neoplasia. An abnormal band ( + ) is seen in each with some smearing. A normal control (N) is used in lane 1. Case 5
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Fig.4.Familypedigreeforcase5 Case 4 (referredby Professor N. Nevin, Belfast)
D N A analysis was requested on three sisters from a sibship of six (three males and three females). All three had features of myotonic dystrophy with cataracts, ptosis, myopathic
408
J.C. MACMILLAN et al.
fades and clinical myotonia. One had noninsulin dependent diabetes and two had cardiac conduction defects. Two had undergone hysterectomy for adenocareinoma of the uterus, (one had in addition an endometrial carcinoma of one ovary) and the third for uterine leiomyomata and bilateral ovarian cystadeomata. The question had been asked whether there was an unusual mutation or extensive deletion in the myotonic dystrophy gene which had given rise to the additional gynaecological neoplasms in these women. All three sisters showed an abnormal band ( + , Fig. 3) with M10M6/EcoRI for myotonic dystrophy; this being of moderate size, similar in the three and showing some smearing, suggesting some somatic instability. They also all showed a normal 10 kb band. The finding of 'typical' DM molecular mutations in these cases made it unlikely that there was a causal relationship between the disease and the gynaecological abnormalities in these sisters. Case 5
The 23 yr old daughter (II2, Fig. 4) of a woman (now deceased) with clinically definite myotonic dystrophy was assessed in 1983. She was asymptomatic and had a normal clinical examination except for percussion myotonia of the thenar eminence and some polychromatic dots reported on ophthalmic examination. In 1982 she had given birth to an apparently healthy boy (arrowed, III0 who was subsequently reassessed at the age of 2½ yr because of delayed speech. He had no stigmata of either congenital or severe childhood myotonic dystrophy. In view of the clinical findings in his mother it was felt that his mild developmental delay may have been due to myotonie dystrophy. Conventional D N A analysis using linked markers was complicated by the fact that the woman's late father (cause of death at 40 yr was unknown) was a first cousin of her mother and himself at 50% risk of inheriting the gene for myotonic dystrophy (his mother had been affected). Both mother and son were reviewed clinically prior to analysis for the myotonic dystrophy mutation. Neurological examination in the child was normal, as was assessment of muscle strength in his mother. Clinical percussion myotonia was again found but EMG of abductor pollicis brevis was normal. Slit-lamp examination showed pigmented lens dots but was not felt to show polychromatic crystals on this occasion. D N A analysis showed no evidence of
the unstable myotonic dystrophy sequence (not shown). The normal mutation analysis in these cases contributed significantly to the revision of the previous erroneous diagnosis of myotonic dystrophy in the mother and the queried diagnosis in her son. Case 6 (referred by 'Professor V. Dubowitz, London)
Analysis was requested, on D N A from the maternal grandparents (arrowed, 113and 114,Fig. 5a) of a living child (IV2) with congenital myotonic dystrophy to facilitate extended family counselling. Both had been assessed at age 66, at the time of the birth of the proband and had normal clinical and EMG findings. The grandfather, however, had developed non-specific cataracts at age 58 whilst the grandmother had a unilateral cataract at age 74. On Southern blotting (PstI digest, Fig. 5b) the grandfather was found to have a minimally increased band size ( + , lane 1) at the DM locus whilst his wife's were normal (only a constant band is seen with PstI in this situation). The abnormal band was seen to increase in size in subsequent generations ('smear', lane 4: + + , lane 5) consistent with the phenomenon of anticipation. Case 7
This 29 yr old man (arrowed, III2, Fig. 6) was referred for assessment as his mother had myotonic dystrophy. He had a normal clinical and E M G assessment but slit-lamp examination showed bilateral polychromatic lens opacities. Conventional linkage analysis was not of help as the family structure revealed a two generation phase unknown situation. He had been counselled that he was a carder of the DM gene on the basis of his eye changes. Direct mutation analysis confirmed the expandexl D N A segment in his affected mother but showed that the consultand had inherited her normal 10 kb allele (not shown). This case shows that polychromatic lens crystals, even at an early age, are not specific for myotonic dystrophy. DISCUSSION
Until the identification of a specific mutation for myotonic dystrophy and the subsequent isolation of the gene early this year, the diagnosis
MyotonicDystrophyDiagnosis
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Fig. 5a. Familypedigree for case 6.
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Fig. 5b. M 10M6/Pstl analysisshowinga minimalexpansion (+, lane 1) in the grandfather, a larger expansion (smear, lane 4) in the mother and further enlargement(+ +, lane 5) in the congenitalcase. of myotonic dystrophy has been largely based on clinical findings, the combination o f m y o t o n i a of grip with a characteristic distribution of muscle weakness and wasting allowing a confident diagnosis in most cases, especially in the presence of a known family history.
Electromyography has been a valuable supportive investigation, partly in confirming the clinical finding of myotonia, showing the typical electrophysiological features, as opposed to a presynaptic or electrically silent reaction; and partly in detecting or excluding myotonic potentials in clinically normal relatives at risk of the disorder. Ophthalmologic assessment for the characteristic multicoloured lens opacities often seen early in the disorder has also been a useful investigation, both in the detection of presymptomatic gene carriers and in the differential diagnosis of other myotonic syndromes. Numerous other tests have been suggested as helpful in the diagnosis or presymptomatic
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detection of myotonic dystrophy, but none has been confirmed as being specific or reliable [8]. Likewise, while linked genetic markers have been useful in raising or lowering genetic risks for family members [9, 10], they have not been applicable in cases without a clear family history. The cases described here, referred over a relatively short period since recognition of the molecular defect, illustrate the wide range of uses of this test in a number of neurological diagnostic situations. In adult patients, molecular analysis is likely to be of most help in cases of myotonia (e.g. case 1) where lack of weakness or progression raises uncertainty as to whether myotonic dystrophy or one of the non-progressive myotonic disorders is the cause. Molecular diagnosis is also becoming possible for some of these, with identification of mutations for hyperkalaemic (myotonic) periodic paralysis in, and linkage of, paramyotonia to the sodium ion channel on chromosome 17 [11, 12]; and mutations in the chloride channel in recessive generalized myotonia [13]. Detection of the gene in older individuals showing a cataract but normal neurological examination and EMG is also important for genetic counselling of their offspring; since these individuals commonly show only a small expansion of the unstable sequence, analysis using PCR as well as Southern blotting should preferably be undertaken to ensure that the number of copies of the repeat sequence can be accurately determined. So far, normal individuals have shown less than 30 copies of the CTG repeat sequence, while minimally affected individuals have shown 50 or more copies [6]. The use of enzyme PstI to give greater resolution of small expansions on Southern blotting has also been noted. In childhood the diagnosis of congenital myotonic dystrophy is frequently not made initially and may, as in the caseoreported here, not be suspected until after the infant's death. In these cases muscle histology is often of not much benefit and can even be misleading where changes are interpreted (erroneously in two patients in this series) as being 'typical' of DM. In such cases the availability of frozen muscle tissue as a source of DNA may be a crucial factor in reaching a specific molecular diagnosis. Since congenital cases of myotonic dystrophy are associated with large expansions of the unstable sequence [4], a clear-cut diagnosis or exclusion of the disorder should be possible. When molecular analysis shows the presence of the specific expanded sequence in a diagnostically uncertain
case, there is now little doubt as to its significance; the same is true if the abnormality is excluded in a relative of a patient shown to have the defect. An apparently normal result in an isolated case should not, however, yet be interpreted as excluding myotonic dystrophy with certainty; although no confirmed cases not showing the defect have yet to be recognized, it is possible that different mutations in other parts of the myotonic dystrophy gene may be responsible for some cases; particularly those with unusual phenotypes, though the family reported here (case 3) showed the usual mutation. An unusual feature of the myotonic dystrophy mutation is its instability, explaining the long debated phenomenon of anticipation [14], since the progressively earlier onset and greater severity in successive generations is, with a few exceptions, correlated with progressive expansion of the unstable sequence. We, along with others, have shown a broad correlation of size of the expansion with phenotype [15, 16], congenital cases showing the greatest degree of expansion (3-6 kb of DNA), while minimal cases with insignificant or absent neuromuscular features have shown small expansions (usually around 0.15 kb) [6]. The significant overlap in degree of expansion between cases from groups, other than those with congenital and minimal disease, means however, that these data are not directly applicable to the clinical situation where molecular diagnosis for an individual has been requested. Similarly, it should be remembered that, in general, analyses are carried out on D N A extracted from peripheral blood lymphocytes and this may not be representative of the findings in other tissues. This has theoretical implications for prenatal diagnosis of myotonic dystrophy in that, to date, there are no data correlating size of expansion in chorionic DNA with disease severity (most 'affected' pregnancies bein~ terminated). We do not feel it appropriate to offer a prediction of prognosis in place of a simple diagnosis from a chorionic biopsy. Finally, it may be asked whether molecular analysis will now make studies such as electromyography redundant in the diagnosis of myotonic dystrophy? Our view is that it should not, at least for the present, since such investigations will give a guide to the likely development of the neurological phenotype, while it is only by comparison of the various tests that we will gain an accurate idea as to the proportion of neurologically normal gene carriers that exists. Equally, however, molecular analysis of the unstable
Myotonic Dystrophy Diagnosis
sequence must now be regarded as part of the diagnostic investigation of myotonic dystrophy. In view of the broad correlation between the degree of expansion and clinical severity we would urge caution in interpreting the possible relationship of DNA expansion and prognosis when molecular diagnosis is made at an early or presymptomatic stage of the disease. In particular we would not recommend the testing of children at risk unless there are specific clinical reasons for doing so [17]. The discovery of the molecular basis of myotonic dystrophy is a striking example of how the new techniques of DNA analysis are changing both our understanding of neurological disorders and patterns of clinical practice in neurology. We can now look ahead to further studies of the specific gene product (myotonin) predicted on the basis of its sequence to be a member of the protein kinase family, helping to elucidate the pathogenesis of the neuromuscular and other clinical features of this disease, while in the more distant future these developments may lead to therapy than can ameliorate or prevent some of the numerous clinical problems encountered by patients with myotonic dystrophy.
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4. Harley H G, Rundle S A, Reardon W, et al. Unstable DNA sequence in myotonic dystrophy. Lancet 1992; 339: !125-1128. 5. Brook J D, McCurrach M E, Harley H G, et al. Molecular basis of myotonic dystrophy: expansion of a trinucleotide (CTG) repeat at the 3' end of a transcript encoding a protein kinase family member. Cell 1992; 68: 799-808. 6. Reardon W, Harley H G, Brook J D, el al. Minimal expression of myotonic dystrophy: a clinical and molecular analysis. J M e d Genet (in press). 7. Myring J, Meredith A L, Harley H G, et al. Specific molecular diagnosis for the CTG mutation in myotonic dystrophy. J M e d Genet (in press). 8. Harper P S. Myotonic Dystrophy, 2nd Edn. London: Saunders, 1989. 9. Norman A M, Floyd J L, Meredith A L, Harper P S. Presymptomatic detection and prenatal diagnosis for myotonic dystrophy by means of linked DNA markers. J M e d Genet 1989; 26: 750-754. 10. Reardon W, Floyd J L, Myring J, Lazarou L P, Meredith A L, Harper P S. Five years experience of predictive testing for myotonic dystrophy using linked DNA markers. A m J M e d Genel 1992; 43:1006-1011. II. Ebers G C, George A L, Barchi R L, el al. Paramyotonia congenita and hyperkalemic periodic paralysis are linked to the adult muscle sodium channel gene. Ann Neurol 1991; 30: 810-816. 12. Rojas C V, Wang J, Schwartz L S, Hoffman E, Powell B R, Brown R H Jr. A Met-to-Val mutation in the skeletal muscle Na + channel ¢-subunit in hyperkalaemic periodic paralysis. Nature 1991; 354: 387-389. 13. Koch M C, Steinmeyer K, Lorenz C, el al. The skeletal muscle chloride channel in dominant and recessive human myotonia. Science (in press). 14. Harper P S, Harley H G, Reardon W, Shaw D J. Anticipation in myotonic dystrophy: new light on an old problem. A m J Hum Genet 1992; 51: 10-16. 15. Harley H G, Rundle S A, Sandkuijl L, et al. Variable expansion of an unstable CTG repeat sequence in relation to phenotype in myotonic dystrophy (manuscript submitted for publication). 16. Tsilfidis C, MacKenzie A E, Mettler, Barcel6 J, Korneluk R G. Correlation between CTG trinucleotide repeat length and frequency of severe congenital ~nyotonic dystrophy. Nature Genet 1: 192-195. 17. Harper P S, Harley D J, Shaw D J. Detection of minimum mutation carriers in myotonic dystrophy. Lancet 1992; 340: 238-239.