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Genetic counselling in X-linked muscular dystrophy
579
Journal of the neurological Sciences
Elsevier Publishing Company, Amsterdam - Printed in The Netherlands
Genetic Counselling in X-linked Muscular Dystrophy A. E. H. E M E R Y University Department of Human Genetics, Edinburgh (Great Britain) At its Meeting in Montreal on 21 September, 1967, the Executive Committee of the Research Group on Neuromuscular Diseases of the W.F.N. appointed a Sub-Committee on the Detection of Carriers of the Gene responsible for X-linked Muscular Dystrophy, consisting of Prof. A. E. H. Emery (University Department of Human Genetics, Edinburgh, Great Britain), Prof. P. E. Becker (lnstitut fiir Humangenetik, Gi~ttingen, West-Germany) and Dr. A. T. Milhorat (Institute for Muscle Disease, Inc., New York, N.Y., U.S.A.) to consider this matter. A report was drafted in detail by Professor Emery, approved by the other members of the Sub-Committee and subsequently it was circulated to all members of the Executive Committee and to the corresponding members of the Research Group before being finally approved for publication.
INTRODUCTION The commonest and most severe form of muscular dystrophy is the sex-linked variety of Duchenne muscular dystrophy. Since the mutant gene responsible for this condition is carried on the X chromosome, a male who inherits the gene must be affected because he is hemizygous. Heterozygous females (carriers) are usually perfectly healthy though very occasionally they may have some weakness of the limb girdle musculature and some enlargement of the calf muscles. These manifestations in heterozygous females have been interpreted in terms of the Lyon hypothesis concerning gene action in the X chromosome (EMERY 1963). Because the gene for Duchenne muscular dystrophy is sex-linked (or more precisely "X-linked") the condition is transmitted by female carriers but about one-third of all cases are the result of new mutations occurring in genetically-normal mothers. GENETIC COUNSELLING The first step in giving genetic counselling to a woman who suspects that she may be a carrier of Duchenne muscular dystrophy is to establish the diagnosis in the affected relative by a careful clinical assessment, serum enzyme studies and if possible electromyography and muscle histology. For example, the congenital forms of muscular dystrophy, which are often inherited as autosomal recessive traits, in the early stages may resemble clinically X-linked Duchenne muscular dystrophy but the serum level of creatine kinase is only slightly raised in congenital muscular dystrophy (EMERY AND WALTON 1967). An attempt should also be made, as far as possible, to establish the J. neurol. Sci. (1969) 8:579-587
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precise mode of inheritance and distinguish between the X-linked and autosomal recessive forms of Duchenne muscular dystrophy. X-linked versus autosomal recessive inheritance Though the autosomal r ~,cessive form of Duchenne muscular dystrophy has a slightly later age of onset and somewhat slower progression than the X-linked type (JACKSON AND CAREV 1961), differentiation on clinical grounds may be difficult. The distinction between autosomal and X-linked recessive modes of inheritance is clear if an affected male has an affected sister or if several males are affected in a family and the pattern of inheritance is consistent with that of an X-linked recessive trait. The distinction, however, may be impossible in a sporadic male case. Evidence which would suggest the autosomal recessive form of Duchenne muscular dystrophy in such a case would be parental consanguinity and a normal algebraic sum of the R and S waves in the first precordial lead of the electrocardiogram in the affected male (SKYRING AND McKuSlCK 1961). The X-linked form of Duchenne muscular dystrophy appears to be at least 10 times more common than the autosomal recessive form (EMERY AND WALTON 1967). Heterozygotes for the autosomal recessive form of Duchenne muscular dystrophy do not usually have raised serum levels of creatine kinase (JACKSON AND STREHLER 1968). Differentiation between the two types of muscular dystrophy is most important from the point of view of genetic counselling because in the case of the autosomal recessive form an unaffected sister is unlikely to have an affected child, but in the X-linked form of the disease her chances of having an affected son may be as high as 1 in2. Severe versus benign X-linked muscular dystrophy Apart from the severe Duchenne type of X-linked muscular dystrophy, in recent years a more benign form of X-linked muscular dystrophy has been recognised by BECKER (1955, 1957, 1962). This type of muscular dystrophy is much less common than the severe Duchenne type. Becker type muscular dystrophy is clinically similar to Duchenne muscular dystrophy in that weakness begins in the pelvic girdle musculature, only later affecting the upper limbs, and there is pseudohypertrophy of the calf muscles. However it differs from Duchenne muscular dystrophy because the disease does not manifest itself until the teens or early twenties and affected individuals often survive into middle age. Approximately 50% of carriers of this form of muscular dystrophy have raised serum levels of creatine kinase (EMERY et al. 1967). The following discussion is concerned only with the severe Duchenne type of X-linked muscular dystrophy and not with the benign Becker type of X-linked muscular dystrophy.
DEFINITIONOF CARRIER STATUSIN X-LINKED DUCHENNE MUSCULAR DYSTROPHY The carrier state may be defined in the following terms (THOMPSON et al. 1967): (1) A woman is an obligate or definite carrier if she has at least one affected son or at least one proven carrier daughter and a family history, consistent with the pattern J. neurol. Sci (1969) 8:579-587
GENETIC COUNSELLINGIN X-LINKED MUSCULARDYSTROPHY
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of inheritance of an X-linked recessive trait, of one or more male relatives having been affected. (2) A woman can be referred to as a probable carrier if there is no family history of Duchenne muscular dystrophy but she has more than one affected son. In such instances it is possible that the condition could be the autosomal recessive form of Duchenne muscular dystrophy. However, for several reasons, including the fact that the autosomal recessive form is much less common, in such instances it is much more likely that the mother is a carrier of the X-linked variety of Duchenne muscular dystrophy. For this reason some investigators combine together definite and probable carriers. (3) A woman is a possible carrier if: (i) she is related to a definite carrier and therefore has a known statistical risk of being a carrier; (ii) she is a mother, sister or other female relative of an isolated case. The main problem in genetic counselling is to determine whether or not a "possible" carrier is really a "definite" carrier. Several methods for detecting the carrier state are currently being investigated. METHODSFOR DETECTINGTHE CARRIER STATE (1) Genetic linkage Genetic linkage studies have shown that the gene for Duchenne muscular dystrophy is not sufficiently close to the loci for colour blindness (PHILIP et al. 1956; EMERY 1966) and the Xg blood group system (CLARKe t al. 1963 ; BLYTHet al. 1965) to be of any practical value in the recognition of preclinical cases or heterozygous carriers. No data are yet available for other X-linked loci. (2) Limb blood flow DEMOS et al. (1962) reported a significant reduction in the "peripheral circulation time" in a proportion of carriers. The method, however, was complex and hardly a practical proposition in most laboratories. Using the technique of venous occlusion plethysmography, EMERY AND SCHELLING (1965) found no significant difference in the total limb blood flow in a number of carriers and healthy women of comparable age. (3) Abnormalities of the fl-globulin fraction of serum proteins Using the technique of immunoelectrophoresis, ASKANAS (1967) has recently reported abnormalities of the fl-globulin fraction of serum proteins in some carriers of Duchenne muscular dystrophy. These findings await confirmation in other laboratories. (4) Total body potassium A significant reduction in total body potassium has been demonstrated in some carriers of Duchenne muscular dystrophy (BLAND et al. 1964, 1967). However the technique for determining total body potassium is complex, requires expensive instrumentation and can be performed in only a few special centres.
J. neurok ScL (1969) 8:579-587
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(5) Muscle lactate dehydrogenase ( L D H ) isoenzymes A reduction in the proportion of the most slowly migrating isoenzyme of LDH (LDH-5) in muscle biopsy specimens has been reported in some carriers of Duchenne muscular dystrophy (EMERY 1964; MANNUCCIet al. 1965; PEARSONAND KAR 1966; JOHNSTON et al. 1966). (6) Muscle histology A number of investigators have demonstrated abnormalities in the histology of muscle biopsy specimens in a proportion of carriers of Duchenne muscular dystrophy. Abnormalities have been demonstrated both with light microscopy (DuRowITZ 1963; EMERY 1963, 1965a; PEARSON et al. 1963; MACCIOTTAet al. 1964; STEPHENSAND LEWIN 1965; KOWALEWSKIet al. 1966; PEARCEet al. 1966; SMITHet al. 1966; RADU et al. 1968) and electron microscopy (MILHORAT et al. 1966; KOWALEWSKIet al. 1966; SMITHet aL 1966). Some investigators claim that histological abnormalities are present in most carriers. However, though the changes are obvious and diagnostic in some carriers, in others they may be minimal and rather non-specific (EMERY 1965a; STEPHENS AND LEWIN 1965; KOWALEWSKIet al. 1966; MILHORATet al. 1966; PEARCE et al. 1966). (7) Electromyography There is general agreement that qualitative electromyography is of no value in detecting carriers and that precise quantitative methods are required. Quantitative methods which have been used with varying success include frequency analysis of interference patterns (BARWlCK 1963; GERSTEN et al. 1967), the number of cycles/second in polyphasic potentials (VAN DEN BOSCH1963), the absolute refractory period (CARUSO AND BUCHTHAL 1965), amplitude of potentials and proportion of polyphasic potentials (SMITH et al. 1966), continuous analysis of the electromyogram (WILLISON 1968) and age-corrected mean potential duration (WALTON et al. 1968) though without age-correction the mean potential duration appears to be of no value in recognising carriers (EMERYet al. 1966). The ratio of the mean number of phases to the mean potential duration (the "phi" value) appears to be successful in identifying some carriers in whom the serum creatine kinase is consistently normal (GARDNERMEDWIN 1968). (8) Serum level o f creatine kinase The simplest and probably the most reliable means of detecting carriers is the serum level of creatine kinase. Results on over 200 carriers have so far been reported and of these approximately two-thirds have been found to have significantly elevated levels of serum creatine kinase (EMERY 1967). In attempts to increase the success rate of this test, investigators have explored the effects of exercise on enzyme levels in carriers and of using other tests in conjunction with serum creatine kinase determinations. WIESMANNet al. (1965) studied the effect of ischaemic exercise of the forearm and found that without such exercise 7 out of 14 carriers had an elevated serum creatine kinase, but after exercise the proportion was increased to 11 out of 14. EMERY(1967) J. neurol. Sci. (1969) 8:579-587
GENETIC COUNSELLINGIN X-LINKED MUSCULARDYSTROPHY
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reported that strenuous exercise over a period of about 30 min produces a significant increase in enzyme level in some carriers but not in normal women. HUDGSON et al. (1967) have also reported that the serum level of creatine kinase in carriers may rise above the upper limit of normal after exercise and EMERY (1967) has suggested that an exercise test may be valuable in suspected carriers, who, with everyday activity, have borderline levels of serum creatine kinase. Unfortunately exercise appears to have little effect in those carriers who have normal levels of serum creatine kinase. Several investigators have used the serum creatine kinase in conjunction with other tests. Unfortunately in some of the reported studies women have been designated as definite carriers when in fact they were possible carriers (e.g. sisters of sporadic males). The results of those studies in which carrier status was clearly defined suggest that there is some justification for carrying out electromyography and a muscle biopsy for histology and possibly L D H isoenzyme studies in a suspected carrier who is found to have a normal level of serum creatine kinase because abnormalities may be detected by these techniques even when the serum creatine kinase is normal (EMERY 1965b; MILHORAT et al. 1966; WALTON et al. 1968). (9) Statistical methods: the probability that a woman who has a normal serum level o f creatine kinase is a carrier
There remains the problem of counselling a suspected carrier who is found to have a normal serum level of creatine kinase and electromyography and muscle histology have either not been carried out or have given normal results. This problem can be partly resolved by using Bayesian methods in the probability calculations. Details of the method have been published elsewhere (MURPHY 1968; EMERY et al. 1968). Further refinements in the method of analysis have recently been described (EMERYAND MORTON 1968). The suspected carrier who seeks genetic counselling will be referred to as the consultand. In calculating the probability of a consultand being a carrier, Bayesian methods not only take into account information about the consultand's sibs and antecedents, which is the usual procedure, but also information about the consultand's descendants and the results of special tests such as the serum level of creatine kinase and perhaps electromyography. The application of Bayesian methods is illustrated in the following examples. In making these calculations it has been assumed that all cases are due to an X-linked recessive gene. I f it is assumed that the risk of being a carrier may be acceptable if less than 10% (in which case the chance of having an affected son or a carrier daughter would be less than 1 in 20) then if a consultand has an affected brother but no children of her own and if we have no information about her serum creatine kinase, then the probability of her being a carrier may reach acceptable limits if she has three or more normal brothers (Fig. 1). However, if the consultand's serum level of creatine kinase is known this information may alter considerably the probability of her being a carrier. In fact the actual level of serum creatine kinase can be used to work out relative probabilities for genetic counselling (WILSON e t al. 1965). I f serum levels of creatine kinase are determined under identical conditions in a number of healthy controls and known carriers, it is possible to calculate the relative probabilities of normal homozygosity to heterozygosity for various levels of J. neurol. Sci. (1969) 8:579-587
GENETIC COUNSELLING IN X-LINKED
MUSCULAR DYSTROPHY
585
TABLE 1 THE RELATIVE PROBABILITIES OF NORMAL HOMOZYGOSITY TO HETEROZYGOSITY (Y1/~r~) FOR VARIOUS LEVELS OF SERUM CREATINE KINASE*
Serum CK
Controls No. %(Y1)
Carriers No. %(]I2)
Y1/ ]I2
0.0-0.3 0.4-4).7 0.8-1.1 1.2-1.5 1.6-1.9 1>2.0
43 35 19 6 4 0 107
0 3 5 4 4 18 34
-3.72 1.21 0.47 0.31 --
Total
40.2 32.7 17.8 5.6 3.7 0.0 100.0
0.0 8.8 14.7 11.8 11.8 52.9 100.0
* Determination by the method of TANZERAND GILVARG(1959); expressed as/~moles/min/l serum at 25°C.
Returning to the problem of the consultand with an affected brother, the probability of her being a carrier may reach acceptable limits even if there is no information about other brothers provided her serum creatine kinase is sufficiently low (Fig. 1). I f the consultand has an affected son, the probability of her being a carrier does not reach acceptable limits even if her serum creatine kinase is in the range 0.4-0.7 units and she has several normal brothers (Fig. 2). However, if both the consultand and her mother have serum levels of creatine kinase in the range 0.4--0.7 units then the probability of the consultand being a carrier may approach acceptable limits if she has at least one unaffected son and a number of normal brothers (Fig. 3). Clearly the risks of a consultand being a carrier are in general greater if she has an affected son rather than if she has an affected brother. The number of normal brothers a consultand has reduces the probability of her being a carrier more if she has an affected brother rather than if she has an affected son. For reliable genetic counselling information should be obtained on as many first-degree relatives as possible and serum levels of creatine kinase should be determined not only on the consultand herself but also on her mother. ACKNOWLEDGEMENT I am grateful to Dr. M. W. Thompson for reading the manuscript and for helpful suggestions.
REFERENCES
ASKANAS)W. (1967) Immunoelectrophoretic investigations of blood serum proteins in muscular diseases, J. Neurol. Neurosurg. Psychiat., 30: 43--46. BAgWZeK,D. D. (1963) Investigations of the carrier state in the Duchenne type dystrophy. In: Research in Muscular Dystrophy (Proc. 2nd Symposium on Current Research in Muscular Dystrophy), Pitman, London, pp. 10-19. BECKER,P. E. (1955) Zur Genetik der Myopathien, Dtsch. Z. Nervenheilk., 173: 482-497. BECrd/R, P. E. (1957) Neue Ergebnisse der Genetik der Muskeldystrophien, Acta genet. (Basel),
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BECKER, P. E. (1962) Two new families of benign sex-linked recessive muscular dystrophy, Rev. canad. Biol., 21 : 551-566. BLAND, W. H., B. CASSEN AND M. LEOERER (1964) Decreased body potassium in non-dystrophic relatives of patients with muscular dystrophy: a biochemical trait, New Engl. J. Med., 270: 197-198. BLAHD, W. H., M. LEDERER AND B. CASSEN (1967) The significance of decreased body potassium concentrations in patients with muscular dystrophy and non-dystrophic relatives, New Engl. J. Med., 276: 1349-1352. BLYTH, H., C. O. CARTER, V. DUBOWITZ, A. E. H. EMERY, J. GAVIN, H. A. JOHNSTON, V. A. McKusICK, R. R. RACE, R. SANGER AND P. TIPPETT (1965) Duchenne's muscular dystrophy and the Xg blood groups: a search for linkage, J. med. Genet., 2: 157-160. BOSCH, J. VAN DEN (1963) Investigations of the carrier state in the Duchenne type dystrophy. In: Research in Muscular Dystrophy (Proc. 2nd Symposium on Current Research in Muscular Dystrophy), Pitman, London, pp. 23-30. CARUSO, G. AND F. BUCHTHAL (1965) Refractory period of muscle and electromyographic findings in relatives of patients with muscular dystrophy, Brain, 88 : 29-50. CLARK, J. 1., R. H. PUITE, R. MARCZYNSKI AND J. D. MANN (1963) Evidence for the absence of detectable linkage between the genes for Duchenne muscular dystrophy and the Xg blood group, Amer. J. hum. Genet., 15: 292-297. DEMOS, J., J. C. DREYFUS, F. SCHAPIRA AND G. SCHAP1RA (1962) Anomalies biologiques chez les transmetteurs apparemment sains de la myopathie, Rev. canad. Biol., 21 : 587-597. DUBOWITZ, V. (1963) Myopathic changes in muscular dystrophy carriers, Proc. roy. Soc. Med., 56: 810-812. EMERY, A. E. H. (1963) Clinical manifestations in two carriers of Duchenne muscular dystrophy, Lancet, i : 1126-1128. EMERY, A. E. H. (1964) The electrophoretic pattern of lactic dehydrogenase in carriers and patients with Duchenne muscular dystrophy, Nature, 201 : 1044-1045. EMERY, A. E. H. (1965a) Muscle histology in carriers of Duchenne muscular dystrophy, J. med. Genet., 2: 1-7. EMERY, A. E. H. (1965b) Carrier detection in sex-linked muscular dystrophy, J. Gdndt. hum., 14: 318-329. EMERY, A. E. H. (1966) Genetic linkage between the loci for colour blindness and Duchenne type muscular dystrophy, J. reed. Genet., 3: 92-95. EMERY, A. E. H. (1967) The use of serum creatine kinase for detecting carriers of Duchenne muscular dystrophy. In: A. T. M1LHORAT (Ed.), Exploratory Concepts in Muscular Dystrophy and Related Disorders (International Congress Series, No. 147), Excerpta Medica Foundation, Amsterdam, pp. 90-97. EMERY,A. E. H. AND R. MORTON (1968) Genetic counselling in lethal X-linked disorders, Acta genet. (Basel), 20: 534-542. EMERY, A. E. H. AND J. L. SCHELLING (1965) Limb blood flow in patients and carriers of Duchenne muscular dystrophy, Acta genet. (Basel), 15: 337-344. EMERY, A. E. H. AND J. N. WALTON (1967) The genetics of muscular dystrophy. In: A. G. STEINBERG AND A. C. BEARN (Eds.), Progress in Medical Genetics, Vol. 5, Grune and Stratton, New York, pp. 116-145. EMERY, A. E. H., R. D. TEASDALLAND E. N. CooMEs (1966) Electromyographic studies in carriers of Duchenne muscular dystrophy, Bull. Johns Hopkins Hosp., 118: 439-443. EMERY, A. E. H., E. WILKINSON AND E. A. MURPHY (1968) Genetic counselling in Duchenne muscular dystrophy, Proc. 2nd International Congress of Neurogenetics and Neuro-ophthalmology, Excerpta Medica Foundation, Amsterdam, In the press. EMERY, A. E. H., E. R. CLACK, S. SIMON AND J. L. TAYLOR (1967) Detection of carriers of benign X-linked muscular dystrophy, Brit. reed. J., iv: 522-523. GARDNER-MEowIN, D. (1968) Studies of the carrier state in the Duchenne type of muscular dystrophy, Part 2 (Quantitative electromyography as a method of carrier detection), J. Neurol. Neurosurg. Psychiat., 31 : 124-134. GERSTEN, J. W., D. M. STILLWELL AND N. A. ROSE (1967) Harmonic analysis in carriers of Duchenne muscular dystrophy, Arch. phys. Med., 48: 164-169. HUDGSON, P., D. GARDNER-MEDWIN, R. J'. T. PENNINGTON AND J. N. WALTON (1967) Studies of the carrier state in the Duchenne type of muscular dystrophy, Part 1 (Effect of exercise on serum creatine kinase activity), J. Neurol. Neurosurg. Psychiat., 30: 416-419. JACKSON, C, E. AND J. H. CAREY (1961) Progressive muscular dystrophy: autosomal recessive type, Pediatrics, 28: 77-84.
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JACKSON,C. E. AND D. A. STREHLER(1968) Limb girdle muscular dystrophy: Clinical manifestations and detection of preclinical disease, Pediatrics, 41 : 495-502. JOHNSTON, H. A., J. H. WILKINSON, W. A. WITHYCOMBEAND S. RAYMOND(1966) Alpha-hydroxybutyrate dehydrogenase activity in sex-linked muscular dystrophy, J. clin. Path., 19: 250-256. KOWALEWSKI,S., H. W. ROTTHAUWE,E. M6LBERT AND M. MUMENTHALER(1966) Female carriers of muscular dystrophy, Lancet, i: 1216-1217. MACCIOTTA,A., V. COSTA, A. CAO, F. SFORZAAND V. SCANO(1964) Studio istologico sul tessuto muscolare dei genitori di un bambino con miodistrofia pelvifemorale tipo Duchenne, Ann. ital. Pediat., 17: 1-11. MANNUCCI,P. M., G. IDEO, A. CAO ANDA. MACCIOTTA(1965) Gli isoenzimi della latticodeidrogenasi (LDH) e della transaminasi glutammico-ossalacetica (TGO) nel muscolo fetale, adulto, e di soggetti affetti da distrofia muscolare progressiva tipo Duchenne, Rass. reed. Sarda, 68: 287-300. MILHORAT,A. T., S. A. SHAFIQAND L. GOLDSTONE(1966) Changes in muscle structure in dystrophic patients, carriers and normal siblings seen by electron microscopy; correlation with levels of serum creatinephospho-kinase (CPK), Ann. N. Y. Acad. Sci., 138: 246-292. MURPHY, E. A. (1968) The rationale of genetic counselling, J. Pediat., 72: 121-130. PEARCE, G. W., J. M. S. PEARCEAND J. N. WALTON(1966) The Duchenne type muscular dystrophy: histopathological studies of the carrier state, Brain, 89: 109-120. PEARSON,C. M. AND N. C. KAR (1966) Isoenzymes: general considerations and alterations in human and animal myopathies, ~Inn. N. Y. Acad. Sci., 138: 293-303. PEARSON, C. M., W. M. FOWLER AND S. W. WRIGHT (1963) X-chromosome mosaicism in females with muscular dystrophy, Proc. nat. Acad. Sci. (Wash.), 50: 24-31. PHILIP, U., J. N. WALTON AND C. A. B. SMIrrt (1956) Colour blindness and the Duchenne type muscular dystrophy, Ann. hum. Genet., 21 : 155-158. RADtr, I-L, S. MIGEA, Z. T6R6K, L. BORD~IANtrAND A. RADU (1968) Carrier detection in X-linked Duchenne type muscular dystrophy, J. neurol. Sci., 6: 289-300. SKYRING, A. AND V. A. MCKUslcK (1961) Clinical, genetic and electrocardiographic studies in childhood muscular dystrophy, Amer. J. med. Sci., 242: 534-547. SMITrL H. L., L. D. AMICK AND W. W. JOHNSON(1966) Detection of subclinical and carrier states in Duchenne muscular dystrophy, J. Pediat., 69: 67-79. STEPHENS, J. AND E. LEWIN (1965) Serum enzyme variations and histological abnormalities in the carrier state in Duchenne dystrophy, J. Neurol. Neurosurg. Psychiat., 28: 104-108. TANZER, M. L. AND C. GILVARG (1959) Creatine and creatine kinase measurement, J. biol. Chem., 234: 3201-3204. THOMPSON, M. W., E. G. MURPHy AND P. J. MCALPINE (1967) An assessment of the creatine kinase test in the detection of carriers of Duchenne muscular dystrophy, J. Pediat., 71 : 82-93. WALTON, J. N., D. GARDtCER-MEOWINAND P. HLrDGSON(1968) Carrier detection in the Duchenne type muscular dystrophy, Proc. 2nd International Congress of Neuro-genetics and Neuro-ophthalmology, Excerpta Medica Foundation, Amsterdam, In the press. WIESMANN, U., H. MOSER, R. RaCHTERICH AND E. Rossl (1965) Progressive Muskeldystrophie, Part 7 (Die Erfassung von Heterozygoten der Duchenne-Muskeldystrophie durch Messung der Serum-Kreatin-Kinase unter lokalisierter Arbeitsbelastung in Anoxie), Klin. V/schr., 43 : 1015-1022. WILLISON, R. G. (1968) Quantitative electromyography: the detection of carriers of Duchenne dystrophy, Proc. 2nd International Congress of Neuro-genetics and Neuro-ophthalmology, Excerpta Medica Foundation, Amsterdam, In the press. WILSON, K. M., K. A. EVANSAND C. O. CARTER(1965) Creatine kinase levels in women who carry genes for three types of muscular dystrophy, Brit. reed. J., 1 : 750-753.
J. neurol. ScL (1969) 8:579-587
Journal of the neurological Sciences
Elsevier Publishing Company, Amsterdam - Printed in The Netherlands
Genetic Counselling in X-linked Muscular Dystrophy A. E. H. E M E R Y University Department of Human Genetics, Edinburgh (Great Britain) At its Meeting in Montreal on 21 September, 1967, the Executive Committee of the Research Group on Neuromuscular Diseases of the W.F.N. appointed a Sub-Committee on the Detection of Carriers of the Gene responsible for X-linked Muscular Dystrophy, consisting of Prof. A. E. H. Emery (University Department of Human Genetics, Edinburgh, Great Britain), Prof. P. E. Becker (lnstitut fiir Humangenetik, Gi~ttingen, West-Germany) and Dr. A. T. Milhorat (Institute for Muscle Disease, Inc., New York, N.Y., U.S.A.) to consider this matter. A report was drafted in detail by Professor Emery, approved by the other members of the Sub-Committee and subsequently it was circulated to all members of the Executive Committee and to the corresponding members of the Research Group before being finally approved for publication.
INTRODUCTION The commonest and most severe form of muscular dystrophy is the sex-linked variety of Duchenne muscular dystrophy. Since the mutant gene responsible for this condition is carried on the X chromosome, a male who inherits the gene must be affected because he is hemizygous. Heterozygous females (carriers) are usually perfectly healthy though very occasionally they may have some weakness of the limb girdle musculature and some enlargement of the calf muscles. These manifestations in heterozygous females have been interpreted in terms of the Lyon hypothesis concerning gene action in the X chromosome (EMERY 1963). Because the gene for Duchenne muscular dystrophy is sex-linked (or more precisely "X-linked") the condition is transmitted by female carriers but about one-third of all cases are the result of new mutations occurring in genetically-normal mothers. GENETIC COUNSELLING The first step in giving genetic counselling to a woman who suspects that she may be a carrier of Duchenne muscular dystrophy is to establish the diagnosis in the affected relative by a careful clinical assessment, serum enzyme studies and if possible electromyography and muscle histology. For example, the congenital forms of muscular dystrophy, which are often inherited as autosomal recessive traits, in the early stages may resemble clinically X-linked Duchenne muscular dystrophy but the serum level of creatine kinase is only slightly raised in congenital muscular dystrophy (EMERY AND WALTON 1967). An attempt should also be made, as far as possible, to establish the J. neurol. Sci. (1969) 8:579-587
580
A.E.H. EMERY
precise mode of inheritance and distinguish between the X-linked and autosomal recessive forms of Duchenne muscular dystrophy. X-linked versus autosomal recessive inheritance Though the autosomal r ~,cessive form of Duchenne muscular dystrophy has a slightly later age of onset and somewhat slower progression than the X-linked type (JACKSON AND CAREV 1961), differentiation on clinical grounds may be difficult. The distinction between autosomal and X-linked recessive modes of inheritance is clear if an affected male has an affected sister or if several males are affected in a family and the pattern of inheritance is consistent with that of an X-linked recessive trait. The distinction, however, may be impossible in a sporadic male case. Evidence which would suggest the autosomal recessive form of Duchenne muscular dystrophy in such a case would be parental consanguinity and a normal algebraic sum of the R and S waves in the first precordial lead of the electrocardiogram in the affected male (SKYRING AND McKuSlCK 1961). The X-linked form of Duchenne muscular dystrophy appears to be at least 10 times more common than the autosomal recessive form (EMERY AND WALTON 1967). Heterozygotes for the autosomal recessive form of Duchenne muscular dystrophy do not usually have raised serum levels of creatine kinase (JACKSON AND STREHLER 1968). Differentiation between the two types of muscular dystrophy is most important from the point of view of genetic counselling because in the case of the autosomal recessive form an unaffected sister is unlikely to have an affected child, but in the X-linked form of the disease her chances of having an affected son may be as high as 1 in2. Severe versus benign X-linked muscular dystrophy Apart from the severe Duchenne type of X-linked muscular dystrophy, in recent years a more benign form of X-linked muscular dystrophy has been recognised by BECKER (1955, 1957, 1962). This type of muscular dystrophy is much less common than the severe Duchenne type. Becker type muscular dystrophy is clinically similar to Duchenne muscular dystrophy in that weakness begins in the pelvic girdle musculature, only later affecting the upper limbs, and there is pseudohypertrophy of the calf muscles. However it differs from Duchenne muscular dystrophy because the disease does not manifest itself until the teens or early twenties and affected individuals often survive into middle age. Approximately 50% of carriers of this form of muscular dystrophy have raised serum levels of creatine kinase (EMERY et al. 1967). The following discussion is concerned only with the severe Duchenne type of X-linked muscular dystrophy and not with the benign Becker type of X-linked muscular dystrophy.
DEFINITIONOF CARRIER STATUSIN X-LINKED DUCHENNE MUSCULAR DYSTROPHY The carrier state may be defined in the following terms (THOMPSON et al. 1967): (1) A woman is an obligate or definite carrier if she has at least one affected son or at least one proven carrier daughter and a family history, consistent with the pattern J. neurol. Sci (1969) 8:579-587
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of inheritance of an X-linked recessive trait, of one or more male relatives having been affected. (2) A woman can be referred to as a probable carrier if there is no family history of Duchenne muscular dystrophy but she has more than one affected son. In such instances it is possible that the condition could be the autosomal recessive form of Duchenne muscular dystrophy. However, for several reasons, including the fact that the autosomal recessive form is much less common, in such instances it is much more likely that the mother is a carrier of the X-linked variety of Duchenne muscular dystrophy. For this reason some investigators combine together definite and probable carriers. (3) A woman is a possible carrier if: (i) she is related to a definite carrier and therefore has a known statistical risk of being a carrier; (ii) she is a mother, sister or other female relative of an isolated case. The main problem in genetic counselling is to determine whether or not a "possible" carrier is really a "definite" carrier. Several methods for detecting the carrier state are currently being investigated. METHODSFOR DETECTINGTHE CARRIER STATE (1) Genetic linkage Genetic linkage studies have shown that the gene for Duchenne muscular dystrophy is not sufficiently close to the loci for colour blindness (PHILIP et al. 1956; EMERY 1966) and the Xg blood group system (CLARKe t al. 1963 ; BLYTHet al. 1965) to be of any practical value in the recognition of preclinical cases or heterozygous carriers. No data are yet available for other X-linked loci. (2) Limb blood flow DEMOS et al. (1962) reported a significant reduction in the "peripheral circulation time" in a proportion of carriers. The method, however, was complex and hardly a practical proposition in most laboratories. Using the technique of venous occlusion plethysmography, EMERY AND SCHELLING (1965) found no significant difference in the total limb blood flow in a number of carriers and healthy women of comparable age. (3) Abnormalities of the fl-globulin fraction of serum proteins Using the technique of immunoelectrophoresis, ASKANAS (1967) has recently reported abnormalities of the fl-globulin fraction of serum proteins in some carriers of Duchenne muscular dystrophy. These findings await confirmation in other laboratories. (4) Total body potassium A significant reduction in total body potassium has been demonstrated in some carriers of Duchenne muscular dystrophy (BLAND et al. 1964, 1967). However the technique for determining total body potassium is complex, requires expensive instrumentation and can be performed in only a few special centres.
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(5) Muscle lactate dehydrogenase ( L D H ) isoenzymes A reduction in the proportion of the most slowly migrating isoenzyme of LDH (LDH-5) in muscle biopsy specimens has been reported in some carriers of Duchenne muscular dystrophy (EMERY 1964; MANNUCCIet al. 1965; PEARSONAND KAR 1966; JOHNSTON et al. 1966). (6) Muscle histology A number of investigators have demonstrated abnormalities in the histology of muscle biopsy specimens in a proportion of carriers of Duchenne muscular dystrophy. Abnormalities have been demonstrated both with light microscopy (DuRowITZ 1963; EMERY 1963, 1965a; PEARSON et al. 1963; MACCIOTTAet al. 1964; STEPHENSAND LEWIN 1965; KOWALEWSKIet al. 1966; PEARCEet al. 1966; SMITHet al. 1966; RADU et al. 1968) and electron microscopy (MILHORAT et al. 1966; KOWALEWSKIet al. 1966; SMITHet aL 1966). Some investigators claim that histological abnormalities are present in most carriers. However, though the changes are obvious and diagnostic in some carriers, in others they may be minimal and rather non-specific (EMERY 1965a; STEPHENS AND LEWIN 1965; KOWALEWSKIet al. 1966; MILHORATet al. 1966; PEARCE et al. 1966). (7) Electromyography There is general agreement that qualitative electromyography is of no value in detecting carriers and that precise quantitative methods are required. Quantitative methods which have been used with varying success include frequency analysis of interference patterns (BARWlCK 1963; GERSTEN et al. 1967), the number of cycles/second in polyphasic potentials (VAN DEN BOSCH1963), the absolute refractory period (CARUSO AND BUCHTHAL 1965), amplitude of potentials and proportion of polyphasic potentials (SMITH et al. 1966), continuous analysis of the electromyogram (WILLISON 1968) and age-corrected mean potential duration (WALTON et al. 1968) though without age-correction the mean potential duration appears to be of no value in recognising carriers (EMERYet al. 1966). The ratio of the mean number of phases to the mean potential duration (the "phi" value) appears to be successful in identifying some carriers in whom the serum creatine kinase is consistently normal (GARDNERMEDWIN 1968). (8) Serum level o f creatine kinase The simplest and probably the most reliable means of detecting carriers is the serum level of creatine kinase. Results on over 200 carriers have so far been reported and of these approximately two-thirds have been found to have significantly elevated levels of serum creatine kinase (EMERY 1967). In attempts to increase the success rate of this test, investigators have explored the effects of exercise on enzyme levels in carriers and of using other tests in conjunction with serum creatine kinase determinations. WIESMANNet al. (1965) studied the effect of ischaemic exercise of the forearm and found that without such exercise 7 out of 14 carriers had an elevated serum creatine kinase, but after exercise the proportion was increased to 11 out of 14. EMERY(1967) J. neurol. Sci. (1969) 8:579-587
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reported that strenuous exercise over a period of about 30 min produces a significant increase in enzyme level in some carriers but not in normal women. HUDGSON et al. (1967) have also reported that the serum level of creatine kinase in carriers may rise above the upper limit of normal after exercise and EMERY (1967) has suggested that an exercise test may be valuable in suspected carriers, who, with everyday activity, have borderline levels of serum creatine kinase. Unfortunately exercise appears to have little effect in those carriers who have normal levels of serum creatine kinase. Several investigators have used the serum creatine kinase in conjunction with other tests. Unfortunately in some of the reported studies women have been designated as definite carriers when in fact they were possible carriers (e.g. sisters of sporadic males). The results of those studies in which carrier status was clearly defined suggest that there is some justification for carrying out electromyography and a muscle biopsy for histology and possibly L D H isoenzyme studies in a suspected carrier who is found to have a normal level of serum creatine kinase because abnormalities may be detected by these techniques even when the serum creatine kinase is normal (EMERY 1965b; MILHORAT et al. 1966; WALTON et al. 1968). (9) Statistical methods: the probability that a woman who has a normal serum level o f creatine kinase is a carrier
There remains the problem of counselling a suspected carrier who is found to have a normal serum level of creatine kinase and electromyography and muscle histology have either not been carried out or have given normal results. This problem can be partly resolved by using Bayesian methods in the probability calculations. Details of the method have been published elsewhere (MURPHY 1968; EMERY et al. 1968). Further refinements in the method of analysis have recently been described (EMERYAND MORTON 1968). The suspected carrier who seeks genetic counselling will be referred to as the consultand. In calculating the probability of a consultand being a carrier, Bayesian methods not only take into account information about the consultand's sibs and antecedents, which is the usual procedure, but also information about the consultand's descendants and the results of special tests such as the serum level of creatine kinase and perhaps electromyography. The application of Bayesian methods is illustrated in the following examples. In making these calculations it has been assumed that all cases are due to an X-linked recessive gene. I f it is assumed that the risk of being a carrier may be acceptable if less than 10% (in which case the chance of having an affected son or a carrier daughter would be less than 1 in 20) then if a consultand has an affected brother but no children of her own and if we have no information about her serum creatine kinase, then the probability of her being a carrier may reach acceptable limits if she has three or more normal brothers (Fig. 1). However, if the consultand's serum level of creatine kinase is known this information may alter considerably the probability of her being a carrier. In fact the actual level of serum creatine kinase can be used to work out relative probabilities for genetic counselling (WILSON e t al. 1965). I f serum levels of creatine kinase are determined under identical conditions in a number of healthy controls and known carriers, it is possible to calculate the relative probabilities of normal homozygosity to heterozygosity for various levels of J. neurol. Sci. (1969) 8:579-587
GENETIC COUNSELLING IN X-LINKED
MUSCULAR DYSTROPHY
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TABLE 1 THE RELATIVE PROBABILITIES OF NORMAL HOMOZYGOSITY TO HETEROZYGOSITY (Y1/~r~) FOR VARIOUS LEVELS OF SERUM CREATINE KINASE*
Serum CK
Controls No. %(Y1)
Carriers No. %(]I2)
Y1/ ]I2
0.0-0.3 0.4-4).7 0.8-1.1 1.2-1.5 1.6-1.9 1>2.0
43 35 19 6 4 0 107
0 3 5 4 4 18 34
-3.72 1.21 0.47 0.31 --
Total
40.2 32.7 17.8 5.6 3.7 0.0 100.0
0.0 8.8 14.7 11.8 11.8 52.9 100.0
* Determination by the method of TANZERAND GILVARG(1959); expressed as/~moles/min/l serum at 25°C.
Returning to the problem of the consultand with an affected brother, the probability of her being a carrier may reach acceptable limits even if there is no information about other brothers provided her serum creatine kinase is sufficiently low (Fig. 1). I f the consultand has an affected son, the probability of her being a carrier does not reach acceptable limits even if her serum creatine kinase is in the range 0.4-0.7 units and she has several normal brothers (Fig. 2). However, if both the consultand and her mother have serum levels of creatine kinase in the range 0.4--0.7 units then the probability of the consultand being a carrier may approach acceptable limits if she has at least one unaffected son and a number of normal brothers (Fig. 3). Clearly the risks of a consultand being a carrier are in general greater if she has an affected son rather than if she has an affected brother. The number of normal brothers a consultand has reduces the probability of her being a carrier more if she has an affected brother rather than if she has an affected son. For reliable genetic counselling information should be obtained on as many first-degree relatives as possible and serum levels of creatine kinase should be determined not only on the consultand herself but also on her mother. ACKNOWLEDGEMENT I am grateful to Dr. M. W. Thompson for reading the manuscript and for helpful suggestions.
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BECKER, P. E. (1962) Two new families of benign sex-linked recessive muscular dystrophy, Rev. canad. Biol., 21 : 551-566. BLAND, W. H., B. CASSEN AND M. LEOERER (1964) Decreased body potassium in non-dystrophic relatives of patients with muscular dystrophy: a biochemical trait, New Engl. J. Med., 270: 197-198. BLAHD, W. H., M. LEDERER AND B. CASSEN (1967) The significance of decreased body potassium concentrations in patients with muscular dystrophy and non-dystrophic relatives, New Engl. J. Med., 276: 1349-1352. BLYTH, H., C. O. CARTER, V. DUBOWITZ, A. E. H. EMERY, J. GAVIN, H. A. JOHNSTON, V. A. McKusICK, R. R. RACE, R. SANGER AND P. TIPPETT (1965) Duchenne's muscular dystrophy and the Xg blood groups: a search for linkage, J. med. Genet., 2: 157-160. BOSCH, J. VAN DEN (1963) Investigations of the carrier state in the Duchenne type dystrophy. In: Research in Muscular Dystrophy (Proc. 2nd Symposium on Current Research in Muscular Dystrophy), Pitman, London, pp. 23-30. CARUSO, G. AND F. BUCHTHAL (1965) Refractory period of muscle and electromyographic findings in relatives of patients with muscular dystrophy, Brain, 88 : 29-50. CLARK, J. 1., R. H. PUITE, R. MARCZYNSKI AND J. D. MANN (1963) Evidence for the absence of detectable linkage between the genes for Duchenne muscular dystrophy and the Xg blood group, Amer. J. hum. Genet., 15: 292-297. DEMOS, J., J. C. DREYFUS, F. SCHAPIRA AND G. SCHAP1RA (1962) Anomalies biologiques chez les transmetteurs apparemment sains de la myopathie, Rev. canad. Biol., 21 : 587-597. DUBOWITZ, V. (1963) Myopathic changes in muscular dystrophy carriers, Proc. roy. Soc. Med., 56: 810-812. EMERY, A. E. H. (1963) Clinical manifestations in two carriers of Duchenne muscular dystrophy, Lancet, i : 1126-1128. EMERY, A. E. H. (1964) The electrophoretic pattern of lactic dehydrogenase in carriers and patients with Duchenne muscular dystrophy, Nature, 201 : 1044-1045. EMERY, A. E. H. (1965a) Muscle histology in carriers of Duchenne muscular dystrophy, J. med. Genet., 2: 1-7. EMERY, A. E. H. (1965b) Carrier detection in sex-linked muscular dystrophy, J. Gdndt. hum., 14: 318-329. EMERY, A. E. H. (1966) Genetic linkage between the loci for colour blindness and Duchenne type muscular dystrophy, J. reed. Genet., 3: 92-95. EMERY, A. E. H. (1967) The use of serum creatine kinase for detecting carriers of Duchenne muscular dystrophy. In: A. T. M1LHORAT (Ed.), Exploratory Concepts in Muscular Dystrophy and Related Disorders (International Congress Series, No. 147), Excerpta Medica Foundation, Amsterdam, pp. 90-97. EMERY,A. E. H. AND R. MORTON (1968) Genetic counselling in lethal X-linked disorders, Acta genet. (Basel), 20: 534-542. EMERY, A. E. H. AND J. L. SCHELLING (1965) Limb blood flow in patients and carriers of Duchenne muscular dystrophy, Acta genet. (Basel), 15: 337-344. EMERY, A. E. H. AND J. N. WALTON (1967) The genetics of muscular dystrophy. In: A. G. STEINBERG AND A. C. BEARN (Eds.), Progress in Medical Genetics, Vol. 5, Grune and Stratton, New York, pp. 116-145. EMERY, A. E. H., R. D. TEASDALLAND E. N. CooMEs (1966) Electromyographic studies in carriers of Duchenne muscular dystrophy, Bull. Johns Hopkins Hosp., 118: 439-443. EMERY, A. E. H., E. WILKINSON AND E. A. MURPHY (1968) Genetic counselling in Duchenne muscular dystrophy, Proc. 2nd International Congress of Neurogenetics and Neuro-ophthalmology, Excerpta Medica Foundation, Amsterdam, In the press. EMERY, A. E. H., E. R. CLACK, S. SIMON AND J. L. TAYLOR (1967) Detection of carriers of benign X-linked muscular dystrophy, Brit. reed. J., iv: 522-523. GARDNER-MEowIN, D. (1968) Studies of the carrier state in the Duchenne type of muscular dystrophy, Part 2 (Quantitative electromyography as a method of carrier detection), J. Neurol. Neurosurg. Psychiat., 31 : 124-134. GERSTEN, J. W., D. M. STILLWELL AND N. A. ROSE (1967) Harmonic analysis in carriers of Duchenne muscular dystrophy, Arch. phys. Med., 48: 164-169. HUDGSON, P., D. GARDNER-MEDWIN, R. J'. T. PENNINGTON AND J. N. WALTON (1967) Studies of the carrier state in the Duchenne type of muscular dystrophy, Part 1 (Effect of exercise on serum creatine kinase activity), J. Neurol. Neurosurg. Psychiat., 30: 416-419. JACKSON, C, E. AND J. H. CAREY (1961) Progressive muscular dystrophy: autosomal recessive type, Pediatrics, 28: 77-84.
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JACKSON,C. E. AND D. A. STREHLER(1968) Limb girdle muscular dystrophy: Clinical manifestations and detection of preclinical disease, Pediatrics, 41 : 495-502. JOHNSTON, H. A., J. H. WILKINSON, W. A. WITHYCOMBEAND S. RAYMOND(1966) Alpha-hydroxybutyrate dehydrogenase activity in sex-linked muscular dystrophy, J. clin. Path., 19: 250-256. KOWALEWSKI,S., H. W. ROTTHAUWE,E. M6LBERT AND M. MUMENTHALER(1966) Female carriers of muscular dystrophy, Lancet, i: 1216-1217. MACCIOTTA,A., V. COSTA, A. CAO, F. SFORZAAND V. SCANO(1964) Studio istologico sul tessuto muscolare dei genitori di un bambino con miodistrofia pelvifemorale tipo Duchenne, Ann. ital. Pediat., 17: 1-11. MANNUCCI,P. M., G. IDEO, A. CAO ANDA. MACCIOTTA(1965) Gli isoenzimi della latticodeidrogenasi (LDH) e della transaminasi glutammico-ossalacetica (TGO) nel muscolo fetale, adulto, e di soggetti affetti da distrofia muscolare progressiva tipo Duchenne, Rass. reed. Sarda, 68: 287-300. MILHORAT,A. T., S. A. SHAFIQAND L. GOLDSTONE(1966) Changes in muscle structure in dystrophic patients, carriers and normal siblings seen by electron microscopy; correlation with levels of serum creatinephospho-kinase (CPK), Ann. N. Y. Acad. Sci., 138: 246-292. MURPHY, E. A. (1968) The rationale of genetic counselling, J. Pediat., 72: 121-130. PEARCE, G. W., J. M. S. PEARCEAND J. N. WALTON(1966) The Duchenne type muscular dystrophy: histopathological studies of the carrier state, Brain, 89: 109-120. PEARSON,C. M. AND N. C. KAR (1966) Isoenzymes: general considerations and alterations in human and animal myopathies, ~Inn. N. Y. Acad. Sci., 138: 293-303. PEARSON, C. M., W. M. FOWLER AND S. W. WRIGHT (1963) X-chromosome mosaicism in females with muscular dystrophy, Proc. nat. Acad. Sci. (Wash.), 50: 24-31. PHILIP, U., J. N. WALTON AND C. A. B. SMIrrt (1956) Colour blindness and the Duchenne type muscular dystrophy, Ann. hum. Genet., 21 : 155-158. RADtr, I-L, S. MIGEA, Z. T6R6K, L. BORD~IANtrAND A. RADU (1968) Carrier detection in X-linked Duchenne type muscular dystrophy, J. neurol. Sci., 6: 289-300. SKYRING, A. AND V. A. MCKUslcK (1961) Clinical, genetic and electrocardiographic studies in childhood muscular dystrophy, Amer. J. med. Sci., 242: 534-547. SMITrL H. L., L. D. AMICK AND W. W. JOHNSON(1966) Detection of subclinical and carrier states in Duchenne muscular dystrophy, J. Pediat., 69: 67-79. STEPHENS, J. AND E. LEWIN (1965) Serum enzyme variations and histological abnormalities in the carrier state in Duchenne dystrophy, J. Neurol. Neurosurg. Psychiat., 28: 104-108. TANZER, M. L. AND C. GILVARG (1959) Creatine and creatine kinase measurement, J. biol. Chem., 234: 3201-3204. THOMPSON, M. W., E. G. MURPHy AND P. J. MCALPINE (1967) An assessment of the creatine kinase test in the detection of carriers of Duchenne muscular dystrophy, J. Pediat., 71 : 82-93. WALTON, J. N., D. GARDtCER-MEOWINAND P. HLrDGSON(1968) Carrier detection in the Duchenne type muscular dystrophy, Proc. 2nd International Congress of Neuro-genetics and Neuro-ophthalmology, Excerpta Medica Foundation, Amsterdam, In the press. WIESMANN, U., H. MOSER, R. RaCHTERICH AND E. Rossl (1965) Progressive Muskeldystrophie, Part 7 (Die Erfassung von Heterozygoten der Duchenne-Muskeldystrophie durch Messung der Serum-Kreatin-Kinase unter lokalisierter Arbeitsbelastung in Anoxie), Klin. V/schr., 43 : 1015-1022. WILLISON, R. G. (1968) Quantitative electromyography: the detection of carriers of Duchenne dystrophy, Proc. 2nd International Congress of Neuro-genetics and Neuro-ophthalmology, Excerpta Medica Foundation, Amsterdam, In the press. WILSON, K. M., K. A. EVANSAND C. O. CARTER(1965) Creatine kinase levels in women who carry genes for three types of muscular dystrophy, Brit. reed. J., 1 : 750-753.
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