Electrophysiological evaluation in myotonic dystrophy: correlation with CTG length expansion

Avaliação eletrofisiológica na distrofia miotônica: correlação com a expansão de tripletos CTG

ELECTROPHYSIOLOGICAL EVALUATION IN MYOTONIC DYSTROPHY

Correlation with CTG length expansion

Beatriz Helena Miranda Pfeilsticker¹, Carmen Sílvia Bertuzzo², Anamarli Nucci¹

ABSTRACT - In myotonic dystrophy (MD), disease severity has been correlated with expansion of CTG repeats in chromosome 19. The aims of this study were to evaluate efficacy of electromyography in the diagnosis of MD, access the frequency and the characteristics of peripheral involvement in the disease and to verify whether the CTG repeats correlated with the electrophysiological abnormalities. Twenty-five patients and six relatives at risk of carrying the MD gene were examined. Electrical myotonia (EM) was scored. Sensory and motor conduction velocity (CV) were studied in five nerves. Leukocyte DNA analysis was done in 26 subjects. Myopathy and myotonia were found in 27 cases. EM was most frequent in muscles of hand and in tibialis anterior. No significant correlation was found between EM scores and length of CTG expansions. EM scores correlated significantly with the degree of clinical myopathy, expressed by a muscular disability scale. Peripheral neuropathy was found in eight subjects and was not restricted to those who were diabetics.

KEY WORDS: myotonic dystrophy, electromyography, myotonia, myopathy, peripheral neuropathy, CTG repeat.

Avaliação eletrofisiológica na distrofia miotônica: correlação com a expansão de tripletos CTG.

RESUMO - Na distrofia miotônica (DM) a severidade da doença tem sido correlacionada com a expansão de tripletos CTG, no cromossomo 19. Foram objetivos do estudo avaliar a eficácia da eletromiografia no diagnóstico, verificar a freqüência e tipos de neuropatias periféricas e determinar se a expansão CTG correlacionava-se com as anormalidades eletrofisiológicas nesta doença. Examinamos 25 pacientes e seis familiares sob risco de herdar a doença. A miotonia elétrica (ME) foi graduada, cinco velocidades de condução sensitivas e cinco motoras estudadas. O DNA leucocitário foi analisado em 26 indivíduos. Encontrou-se miopatia miotônica em 27 pacientes, sendo a ME mais freqüente nos músculos da mão e no tibial anterior. Não houve correlação significativa entre os graus de ME e o número de repetições CTG. Os graus de ME correlacionaram-se significantemente com a severidade da miopatia clínica, expressa por escala de disfunção muscular. Neuropatia periférica foi encontrada em oito indivíduos, não exclusivamente em diabéticos.

PALAVRAS-CHAVE: distrofia miotônica, eletromiografia, miotonia, miopatia, neuropatia periférica, repetição de tripletos.

Myotonic dystrophy (MD) is a systemic disorder, with a variable phenotype including myopathy and myotonia¹. Disease severity has been correlated with unstable expansion of CTG repeats in the gene encoding myotonin protein kinase in chromosome 19^2,3. Electromyography (EMG) is a master tool for the diagnosis, but the incidence of EMG myotonia has varied in different reports because of selection and number of muscles examined and interpretation of electromyographic findings, as well as diagnostic criteria⁴. Electroneuromyography may also detect peripheral neuropathy (PN) in MD patients^5,6.

The aims of this paper were to study prospectively the efficacy of EMG in MD diagnosis, access the frequency and the characteristics of peripheral neuropathy in the disease and to correlate the CTGn with abnormalities documented in EMG.

METHOD

Thirty one subjects from 20 different families participated, by writing informed consent; 25 with clinical hypothesis of MD and six relatives at risk of carrying the MD gene. Inclusion criteria were presence of clinical myopathy with myotonia and others systemic features of MD and asymptomatic subjects or with minimal MD signs or symptoms belonging to MD families. They were submitted to neurological examination, EMG protocol, routine blood analyses to detect common causes of PN and blood sample for DNA analysis. Clinical and EMG results were established before the genetic study. They were classified according to Mathieu et al.⁷, into a muscular disability rating scale, as follow: Grade 1 – no clinical muscular impairment (diagnosis made by EMG, slit-lamp examination or DNA analysis); Grade 2 - minimal signs (myotonia, jaw and temporal wasting, facial weakness, sternomastoids wasting / weakness, ptosis, nasal speech, no distal weakness except isolated digitis flexor weakness); Grade 3 - distal weakness (no proximal weakness, except isolated triceps brachii weakness); Grade 4 - mild or moderate proximal weakness; Grade 5 - severe proximal weakness (confined to wheelchair for short and long distances).

EMG protocol studied the anatomical distribution of electrical myotonia (EM), in 13 muscles and was scored according to Streib and Sun⁴: Grade 1 - occasional short run of positive waves following needle movement (interpreted as nonspecific); Grade 2 - myotonic discharges of more than 0.5 sec duration in two or more muscle areas; Grade 3 - myotonic discharges in most needle locations; Grade 4 - myotonic discharges in each movement in all examined areas. Score for individual muscle: sum of the grades divided by the number of times the muscle was examined. Total EMG myotonia score for a subject: summed grades of EMG-myotonia, divided by the number of muscles examined.

H reflex (tibial nerve), CV in five motor and in five sensory nerves were evaluated in each patient. CV techniques and normal values were considered according to Kimura⁸. Investigations were performed using a Neuropack equipment (Nihon Khoden Co.), superficial electrodes for stimulation and recording and monopolar needle for EMG. Temperature was controlled by a Cole Palmer Digi Sense Termometer and maintained over 32°C.

Routine blood examination included hemogram, creatine kinase, alanine and aspartate aminotransferase, total calcium, potassium, sodium, lactate dehydrogenase, cholesterol total, HDL and LDL-cholesterol, creatinine, glucose, triglycerides, urea nitrogen, thyroxine free and thyrotropin, serum B12 and folic acid.

Genomic DNA was prepared from peripheral blood leukocytes ^9,10. The size of the CTG-repeat expansion was analysed by PCR-Elongase. PCR analyses were performed in a volume of 30 µml, containing 100 ng template DNA,60 mM Tris-SO4 (ph 9.1), 18 mM (NH4)2SO4, 1.1 mM Mg SO4, 10 mM dNTP mix, 10% Dimethylsulfonamide (DMSO), 1 µml Elongaseââ (mixture of Taq and Pyrococcus species GB-D DNA polymerases from Life Technologies, 10480-010), 0.2 µmM of primers 101 and 102 (101: 5'-CTTCCCAGGCCTGCAGTTTGCCCATC - 3'; 102: 5'-GAACGGGGCTCGAAGGGTCTTGTAGC 3'). PCR involved heating to 94°C for 6 min, 65°C for 1 min, 72°C for 1 min and then 30 cycles of 94°C for 1 min, 65°C for 1 min, 72°C for 1 min. Amplified product (2 µml) was mixed with 2µml of formamide loading buffer, heated for 5 min at 100°C, subjected to electrophoresis in a 2% agarose gel, blotted on to nylon membrane and probed with the 3'-end-labelled 101 primer.

Statistical analyses were based on linear correlation coefficient of Spearman (non-parametric); Kruskal-Wallis test was used to verify homogeneity between myotonic score and the degree of muscular disability.

RESULTS

Myotonia and myopathy were found in 27 subjects, with mean age of 34,4 years. None of them presented congenital MD. The mean grade of muscular disability was 2,77. Total EMG-myotonia score, the number of CTG repeats, grade of muscular disability and age of each case is presented in Table 1.

Six subjects (Cases 1,2,3,4,13,21,Table 1), belonging to three different families with MD, were initially considered relatives at risk to have the abnormal gene. They were clinically normal (force grade five in all muscles, no clinical myotonia, no muscular atrophy, no other clinical signs of MD). In cases 1,2,3 and 4, EMG and muscle enzimes were also normal. They were daughter (Case 1), son (Case 2) and sisters (Cases 3 and 4) of one patient (Case 26). Cases 13 and 21 were also clinically normal, had mild abnormalities in EMG, and DNA confirmed MD. Case 13 was sister of patients (Cases 22 and 25). The deceased mother of case 21 had a definitive diagnosis of MD.

The frequency of EM and composite score for each muscle examined is shown in Table 2.

A significant correlation (0,617), at 0,05 level with Spearman's test, was found between myotonic score and the grade of muscular disability. There was not a significant correlation (0,232), between CTG expansion and the score of myotonia, neither with the degree of muscular disability (0.026).The p-value in Kruskal-Wallis test was 0,012 for the score of myotonia and muscular disability.

The electroneurography detected PN in eight subjects (seven patients and one relative at risk). Their data are listed in Table 3. Diabetes mellitus was diagnosed in four patients and ,in the other four individuals, no etiology for PN was defined. Six patients presented mild to moderate sensory-motor axonopathy and one of them had also left carpal tunnel syndrome; one had mild motor axonopathy. A relative at risk had a mild sensitive axonopathy with symptomatic right carpal tunnel syndrome.

DISCUSSION

Despite the discovery of the gene defect in DM the molecular pathological mechanism in the disease is still not completely known^10-13. Currently explanations include: deficiency of the DM protein kinase¹⁴; effect of expanded CTG repeats on neighboring genes^15-18; interference of the mutant DM protein kinase transcripts with others transcripts (trans RNA interference)¹⁹. Studies that are in progress look for new transgenic models that may more closely resemble clinical DM and that provide also a model for anticipation^20,21.

The role of DNA analysis to the diagnosis of DM is well establish. In one of the studies that described the genetic mutation in MD¹³ it was showed that over 98% of MD patients had abnormal CTG repeats in the DMPK gene in chromosome 19. A relationship between the size of the CTG repeat in leukocyte DNA and the disease severity, in its multisystemic aspects, has been reported in several studies^2,3,22. However, the disease is heterogeneous, with clinical variability in all ages considered⁷ and a given triplet size². The somatic instability of the CTG repeats has also been reported and raises the question whether expansions in different tissues might account for the lack of precise correlation between the number of CTG repeats in leukocyte DNA and clinical findings²³. The CTG expansions are larger in skeletal muscle than in leukocytes^24,25, but the pattern of weakness and atrophy in MD do not seem to be related to the differences in CTG-repeat length between different muscles²⁵.

In MD it may be difficult to access the degree of muscular disability using a functional scale, because it may be influenced by multiple and complex factors⁷. We used the muscular disability rating scale constructed by Mathieu et al⁷, based on the clinical muscular involvement and concordant with the usual distal to proximal progression of the muscular involvement in MD. In accordance with this author's observations, we found that the scale allowed registration of significant changes in the degree of muscular deficit. We did not demonstrated a significant correlation between CTG expansion in leukocytes and the EM score, neither with muscular disability. However, patients with CTG equal or over 200 had a tendency to have higher scores of EM. The number of patients examined and clinical variability in all ages are possible explanations for our results.

The pathophysiology responsible for the muscle wasting, weakness and for myotonia is not yet completely defined^26,27. Deficiency in muscle anabolism due to multiple causes¹, deficiency of MD protein kinase leading to dysregulation of the calcium metabolism in muscle fiber²⁶ and reduction of type 2 muscle fiber numbers²⁷ are proposed to explain weakness and wasting. Alteration in the function of slow potassium channels can produce myotonia²⁶. Recently, evidences based on a transgenic mice, support a possible mechanism of RNA gain of function in the pathogenesis of MD¹⁵.

In classical cases, myotonia is often widespread and easy to elicit, but in some patients it may be subtle, limited and difficult to be recognized in clinical level ¹. Electromyography is important specially in these cases and in the evaluation of subjects at risk from MD families⁴. Detailed EMG was diagnostic in two asymptomatic relatives in our data, in whom DNA analysis was confirmatory. Twenty-five cases of MD diagnosed clinically had positive EMG for myopathy and myotonia. DNA study was done in twenty-four of them and CTG expansion was documented in all. Concordance between presence of myotonia on clinical examination and the EMG results was similarly referred by others authors^4,28. The selection and number of muscles examined are fundamental for unequivocal EMG diagnosis, as the sensitivity of diagnosis by EMG exam may vary according to this parameters⁴. However, in patients with minimal MD, molecular analysis is the gold standard for diagnosis.

EM was encountered more frequently in hand muscles (88 to 100%) and tibialis anterior (88%) and less in deltoid (46%). The composite score, showing intensity and frequency of EM, was also greater in hand muscles and tibialis anterior. These data are in agreement with the observations of Streib and Sun⁴. In our series, EM correlated significantly with the degree of myopathy, in accordance with Streib and Sun⁴, although we used a different muscular disability scale⁷. Clinical myotonia may be difficult to elicit in patients with severe wasting and in congenital cases, in the first two years of life²⁹. It should be noticed that no patient in our study had score five for muscular disability and we did not included congenital cases.

PN have been reported as part of the clinical picture of MD, like polyneuropathy^1,29. In electroneurography, slowing of motor and sensory CV, increase of minimal latency of F wave and delay or absence of the H reflex, reduction of sensory and motor amplitudes of recorded potentials have been described^6,30. Pathological studies of peripheral nerve showed sensory involvement^5,31,32. The abnormalities are probably primary axonal and they do not parallel the severity of muscular involvement⁶. Although the cause of the polyneuropathy remains uncertain, it seems to be one of the manifestations of the disease⁶, but in some patients diabetes mellitus can be associated. Our findings included seven cases of PN out of 27 patients with MD and one case in a relative at risk, in whom the EMG did not diagnosed MD. Six patients presented a mild to moderate sensory-motor axonal polyneuropathy; four were diabetics, and in two, no other evident etiology was found. Two electrophysiological exams showed a carpal tunnel syndrome, one asymptomatic and associated with axonal sensory-motor neuropathy. The other, in a relative at risk, the carpal tunnel syndrome was symptomatic and associated with a mild axonal sensory neuropathy in lower limbs. Mondelli et al.⁶ concluded that PN is not rare in MD, involving about 46% of their patient. In our casuistic, 25,9% had PN, with type and intensity similar the encountered in the literature^5,6,31,32.

Received 20 October 2000, received in final form 23 November 2000. Accepted 27 November 2000.

Dra. Beatriz Helena Miranda Pfeilsticker - Departamento de Neurologia FCM UNICAMP - Caixa Postal 6111 – 13081-970 Campinas SP- Brasil. Email:[e-mail]

• 1. Harper PS, Rüdel R. Myotonic dystrophy. In Engel AG and Franzini-Armstrong C. Myology. 2Ed. New York: McGraw-Hill, 1994;2:1192-1219.
• 2. Jasper A, Fahsold R, Grehl H, Claus D. Myotonic dystrophy: correlation of clinical symptons with the size of the CTG trinucleotide repeat. J Neurol 1995;242:99-104
• 3. assos-Bueno MR, Cerqueira A, Vainzof M, Marie SK, Zatz M. Myotonic dystrophy: genetic, clinical and molecular analysis of patients from 41 Brazilian families. J Med Genet 1995;32:14-18
• 4. Streib EW, Sun SF. Distribution of electrical myotonia in myotonic muscular dystrophy. Ann Neurol 1983;14:80-82
• 5. Cros D, Harnden P, Pouget J, Pellissier JF, Gastaut JL, Serratrice G. Peripheral neuropathy in myotonic dystrophy: a nerve biopsy study. Ann Neurol 1988;23:470-476
• 6. Mondelli M, Rossi A, Malandrini A, Della Porta P, Guazzi GC. Axonal motor and sensory neuropathy in myotonic dystrophy. Acta Neurol Scand 1993;88:141-148
• 7. Mathieu J, De Braekeleer M, Prevost C, Boily C. Myotonic dystrophy: clinical assessment of muscular dystrophy of muscular disability in an isolated population with presumed homogenous mutation. Neurology 1992;42:203-208
• 8. Kimura J. Electrodiagnosis in diseases of nerve and muscle: principles and practice. Philadelphia: FA Davis, 1989.
• 9. Woodhead JL, Fallon R, Figuered H, Longdale J. Malcom AD. Alternative methodology of gene diagnosis. In Davies K.E. Human genetic diseases: a practical approach. Oxford: IRL Press, 1986:51-64.
• 10. Brook JD, McCurrach ME, Harley HG, et al. Molecular basis of myotonic dystrophy: expansion of trinucleotide (CTG) repeat at the 3' end of a transcript encoding a protein kinase family member. Cell 1992;68:799-808
• 11. Brook JD. Detection of an ustable fragment of DNA specific to individuals with myotonic dystrophy. Nature 1992;355:547-548
• 12. Fu YH, Pizzutti A, Fenwick Jr RG, et al. An unstable triplet repeat in a gene related to myotonic muscular dystrophy. Science 1992;255:1256-1258
• 13. Mahadevan M,Tsilfidis C, Sabourin L, et al. Myotonic dystrophy mutation: an stable CTG repeat in the 3' untranslated region of the gene. Science 1992;255:1253-1255
• 14. Timchenko L, Monckton DG, Caskey CT. Myotonic distrophy: an unstable CTG repeat in a protein kinase gene. Semin Cell Biol 1995;6:13-19.
• 15. Alwazzan M, Newman E, Hamshere MG, Brook JD. Myotonic dystrophy is associated with a reduced level of RNA from the DMWD allele adjacent to the expanded repeat. Hum Mol Genet 1999;8:1491-1497
• 16. Eriksson M, Ansved T, Edstrom L, Anvret M, Carey N. Simultaneous analysis of expression of the three myotonic locus genes in adult skeletal muscle samples:the CTG expansion correlates inversely with DMPK and 59 expression leves but not DMAHP levels. Hum Mol Genet 1999;8:1053-1060
• 17. Korade-Mirnics Z, Tarleton J, Servidei S, et al. Myotonic dystrophy: tissue-specific effect of somatic CTG expansions on allele-specific DMAHP/SIX5 expression. Hum Mol Genet 1999;8:1017-1023
• 18. Timchenko LT. Myotonic dystrophy:the role of RNA CUG repeats. Am J Hum Genet 1999;64:360-364
• 19. Sasagawa N, Takahashi N, Suzuki K, Ishiura S. An expanded CTG trinucleotide repeat causes trans RNA interference: a new hypothesis for the pathogenesis of myotonic dystrophy. Biochem Biophys Res Commun 1999;14:264:76-80.
• 20. Skuk D, Furling D, Bouchard JP, et al. Transplantation of human myoblasts in SCID mice as a potential muscular model for myotonic dystrophy. J Neuropathol Exp Neurol 1999;58:921-931
• 21. Mankodi AK, Logigian EL, Orimo S, Callahan, Thornton C. Transgenic model of myotonic dystrophy. Neurology 2000; 54(Suppl 3):A458
• 22. Genarelli M, Novelli G, Andreasi BF, et al. Prediction of myotonic dystrophy clinical severity based on the number of intragenic [[CTG]] n trinucleotide repeats. Am J Med Genet 1996;65:342-347
• 23. Lavedan C, Hoffmann-Radvanyi H, Shelbourne P, et al. Myotonic dystrophy: size and sex dependent dynamics of CTG meiotic instability and somatic mosaicism. Am J Hum Genet 1993;52:875-883
• 24. Thornton CA, Johnson K, Moxley R III. Myotonic dystrophy patients have larger CTG expansions in skeletal muscle than in leukocytes. Ann Neurol 1994;35:104-107
• 25. Hedberg B, Anvret M, Ansved T. CTG-repeat lenght in distal and proximal leg muscles of symptomatic and non symptomatic patients with myotonic dystrophy: relation to muscle strength and degree of hystopatholigical abnormalities. Eur J Neurol 1999;6:341-346.
• 26. Behrens MI, Jalil P, Serani A, Vergara F, Alvarez O. Possible role of apamin-sensitive K⁺ channels in myotonic dystrophy. Muscle Nerve 1994;17:1264-1270.
• 27. Tohgi H, Kawamorita A, Utsugisawa K, Yamagata M, Sano M. Muscle histopathology in myotonic dystrophy in relation to age and muscular weakeness. Muscle Nerve 1994;17:1037-1043.
• 28. Pryse-Phillips W, Johnson GJ, Larsen B. Incomplete manifestation of myotonic dystrophy in a large kinship in Labrador. Arch Neurol 1982;11:582-591
• 29. Harper PS. Myotonic dystrophy, the clinical picture. In Myotonic dystrophy. Philadelphia: Saunders, 1979:14-36.
• 30. Logullo F, Censori B, Danni M, Del Pesce M, Di Bella P, Provinciali L. Peripheral neuropathy in myotonic dystrophy: electrophysiologycal and clinical features. Electromyogr Clin Neurophysiol 1992;32:515-520
• 31. Pollock M, Dyck PJ. Peripheral nerve morphometry in myotonic dystrophy. Arch Neurol 1976;33:33-40
• 32. Freitas GR, Freitas MRG, Nascimento OJM. Biópsia do nervo sural na distrofia muscular miotônica. Arq Neuropsiquiatr 1996;54:19-24.

¹Departments of Neurology and

Departments of Neurology and ²Medical Genetics, Faculty of Medicine, University of Campinas (UNICAMP) SP, Brazil. Presented partially in poster format at the 10th European Congress of Clinical Neurophysiology, Lyon, France, August 2000.

Publication Dates

• Publication in this collection
  07 June 2001
• Date of issue
  June 2001

History

• Accepted
  27 Nov 2000
• Reviewed
  23 Nov 2000
• Received
  20 Oct 2000