Journal o f the Neurological Sciences, 1979, 43: 377-394

377

© Elsevier/North-Holland Biomedical Press

A N E V A L U A T I O N OF SOME C A R R I E R D E T E C T I O N T E C H N I Q U E S IN DUCHENNE MUSCULAR DYSTROPHY

R. J. M. LANE, P. MASKREY, G. A. NICHOLSON, P. Q. R. SIDDIQUI, MEGAN NICHOLSON, PATSY GASCOIGNE, R. J. T. PENN]NGTON, D. GARDNER-MEDWIN and J. N. WALTON Muscular Dystrophy Research Laboratories, Regional Neurological Centre, Newcastle General Hospital and the Department of Neurology, the University of Newcastle upon Tyne, Newcastle upon Tyne (Great Britain)

(Received 29 March, 1979) (Accepted 14 May, 1979)

SUMMARY Some recently described abnormalities in the serum and red cell membranes in Duchenne dystrophy have been examined as methods of carrier detection in a singleblind controlled study. Twelve carriers (4 definite, 3 probable and 5 possible carders previously found to have raised creatine kinase levels) and 12 normal female controls of the same age, were examined on 3 separate occasions at approximately two-weekly intervals. Analysis of age-dependent red cell shape changes, serum haemopexin levels, red cell K + efflux rate, sensitivity of red cell ghost membrane ATPase to ouabain, membrane protein phosphorylation studies and lactate dehydrogenase isoenzyme profiles on agarose gel electrophoresis all failed to distinguish carriers from controls. The carriers suffered muscle cramps more frequently than the controls and all but one carrier and two control subjects were correctly identified by manual muscle strength testing, certain proximal muscles in particular being consistently weaker in carriers than in the control group subjects. Scalar electrocardiography revealed higher values for the R/S ratio in Leads V1 and V2 and the sum (R-S) in V2. INTRODUCTION Duchenne muscular dystrophy is due to an X-linked recessive gene and affects only males, except in very rare circumstances (Gomez et al. 1977). However, some 10 % of female carders are "manifesting" and have abnormalities in muscle bulk or strength detectable on routine clinical examination (Gardner-Medwin et al. 1971) This work was supported by the Muscular Dystrophy Group of Great Britain and the Medical Re;earch Council. Reprint requests to Dr. R. J. T. Pennington.

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0.05). There was no significant difference in the incidence of postural or gait abnormalities between the two groups, confirming the findings of Roses et al. (1977c). The average total analogue scores for the groups were: definite carriers 48.5; probable carriers 31.7; possible carriers 39.3 and controls 14.8. The difference between the different carrier groups was not significant, but a highly significant difference (P < 0.01) was found between the carriers as a group and the control subjects (Fig. 1). Fig. 2 shows that the carriers were consistently weaker in the proximal muscle groups than the control group, but that the difference in the distal muscles was negligible, as found by Roses et al. (1977c). Muscles which were never weak in the controls, like biceps, were occasionally found to be so in the carriers, but no subject in the study scored less than 3 ÷ (Table 2) in any muscle or muscle group. The physiotherapists who performed the testing were correct in their assessments regarding the subjects' status as carriers or normal controls in all but 3 cases. One carrier was found to be strong on the single occasion she was tested (No. 9). She was employed as a domestic help. One control subject (No. 15) who was in the early stages of pregnancy, was identified as a carrier because of weakness, but there was no clear explanation for the weakness found in her or in the other control who was incorrectly diagnosed (No. 21). Figure 3 shows the effect of age. Of the 16 paired manual muscle tests, 10 total analogue scores corresponded quite closely on the two occasions, 5 of these each being conducted by different examiners. Overall, the correlation coefficients for the analogue scores on the two occasions were 0.51 in the carrier group and 0.94 for the controls. One high analogue score was recorded in subject No. 16 on the first visit, when she was suffering from a severe "hangover"; her score was within normal limits on the second visit.

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Fig. 2. Ranked analogue scores in MMT of carriers and controls. Key to Fig. 2: PD, posterior deltoid; MT, middle trapezius; LT, lower trapezius; H, hamstrings; ANF, anterior neck flexors; MD, middle deltoid; Rh, rhomboids; Gas, gastrocnemius; G. Max, gluteus maximus; P. Maj, pectoralis major; SCM, sternocleidomastoid; Tr., triceps; Q, quadriceps femoris; S. Ant, serratus anterior; G. Med, gluteus medius; AD, anterior deltoid; Bi, biceps; F. Ext, finger extensors; Br, brachioradialis; F. Flex, finger flexors; Wr. Ext, wrist extensors; Wr. Flex, wrist flexors; UT, upper trapezius.

Cramping occurred in 13 instances in the 19 MMT performed on the carriers and in only one instance in 18 MMT in the controls, confirming the impression that muscle cramping is a prominent feature in carriers (Roses et al., 1977c).

(2) Serological tests Creatine kinase levels. These are shown in Table 5 together with the values obtained when the subjects had been tested previously, at various times during the past few years. LDH isoenzymes. The mean LDH-5 level in carriers and controls was 25.1 (± 5.8) IU/1 and 21.3 ( ~ 5.3) TU/1 respectively, the difference being significant at the 5 % level, but there was a similar relative difference (302 IU/I compared to 264 IU/1) in total LDH levels. The level of LDH-5 as a proportion of total LDH was not significantly different in carriers compared with controls. Findings by other workers have been conflicting. Kowelewski and Rotthawe (1972) found a normal LDH isoenzyme pattern in carriers while Hooshmand et al. (1969) found a relative rise in LDH 1-3 and a fall in LDH 4-5, the total LDH being normal; Hausmanowa-Petrusewicz et al. (1977) also reported a decrease in the proportion of LDH 4 and 5. Conversely, Roses et al. (1977a) found a significantly raised level of LDH-5 in a series of known carriers, using the techniques described here, except that the electrophoretic support medium was epiagar. Comparative studies with polyacrylamide gel electrophoresis (Maskrey and Nicholson 1978) have supported the validity of our technique.

387 TABLE 5 SERUM CREATINE KINASE ACTIVITY IN CARRIERS The genetically "possible" carriers were selected from those which had shown elevated serum CK and could therefore be considered to be "definite" carriers. Upper limit of normal = 60 IU/I. Subject No.

CK(IU/I) present

previous

Definite carriers 1 2 3 4

59.4 36.5 92.3 39.6

93.2 25.0 79.6 27.4

Probable carriers 5 6 7

26.0 31.4 42.2

33.4 33.9 62.8

Possible carriers 8 9 10 11 12

127 133 54.4 576 80.6

153 221 145 218 76.8

Serum haemopexin. The mean haemopexin level in the controls was 96.9 (-419.6) mg/100 ml and in the carriers, 88.3 ( i 9.4). The difference was not significant in a paired t-test. Askanas and Mazurczak (1966) found an abnormal precipitation line when serum from cases of Duchenne dystrophy was run against specific rabbit antiDuchenne serum on immunoelectrophoresis or Ouchterlony immunodiffusion, in 78 of cases tested. This was shown to be due to excessive amounts of the ill-globulin, haemopexin (Askanas 1966). Danieli and Angelini (1976a,b) found significantly higher mean serum haemopexin levels in carriers compared with controls, and stated that a level of more than 96 mg/100 ml suggested carrier status, but as Healey (1976) pointed out, there is considerable overlap of values in the two groups.

(3) Red cell membrane studies Age-dependent red cell shape changes. The percentage of echinocytes recorded in the same person's blood varied widely on different occasions, as found by Matheson et al. (1976), and no overall difference was found in the incidence of echinocytes in the blood from carriers compared with controls. Morse and Howland (1973) found increased numbers of crenated/spiculated red cells ("echinocytes") in the blood of genetic and nutritional dystrophic mice. Matheson and Howland (1974) reported the same phenomenon in cases and carriers of Duchenne dystrophy, compared with controls and other neuromuscular diseases, and support for their findings came from Lumb and Emery (1975) and Howells (1976).

388 However, Miale et al. (1975), Miller et al. (1975) and Soltan (1977) could not confirm these results, and a later blind-control study by one of the original investigators demonstrated that echinocyte counts in the same subject varied considerably at different times using variations of the original technique, which was therefore considered unreliable (Matheson et al. 1976). Miller et al. (1976) examined red cells by scanning electron microscopy without manipulation prior to fixation, and found increased numbers of stomatocytes (cup cells) in cases and carriers of Duchenne dystrophy, although the finding was not specific; but the technique was found useful in carrier detection (Roses et al. 1976). Korczyn et al. (1977), on the other hand, found increased echinocyte counts in cases of Duchenne dystrophy and other neuromuscular diseases using the scanning electron microscope under similar conditions. Sensitivity of red cell ghost ATPase to ouabain. Using the technique of NiebrojDobosz (1976) we obtained figures of 8.4 (-k 1.8) and 7.6 ( ± 1.6) nmol/min/mg protein for ATP hydrolysis rates in carrier and control red cell ghosts, respectively. The percentage inhibition by ouabain was 34.0 ( ~ 6.6) and 35.1 ( ± 6.2) in the two groups and a paired t-test showed no significant difference in either case; neither was there any relationship with carrier status or CK level. Ouabain, a cardiac glycoside, inhibits the rate of ATP hydrolysis by membrane ATPase in normal red cells. However, Brown et al. (1967) and Chattopadhyay and Brown (1972) reported that ouabain stimulated rather than inhibited red cell ghost ATPase in a variety of myopathic and myotonic disorders, including Duchenne dystrophy. This was confirmed by Peter et al. (1969) and by Araki and Mawatari (1971), but Klassen and Blostein (1969) and Souweine et al. (1978) obtained normal degrees of inhibition in Duchenne dystrophy. Siddiqui and Pennington (1977) and Mawatari et al. (1976) found significantly reduced ouabain inhibition in Duchenne dystrophy red cell ghosts. Studies on carriers have also produced conflicting results. Araki and Mawatari (1971) and Mawatari et al. (1976) found normal ouabain sensitivity in the few carriers they tested, but Niebroj-Dobosz (1976) reported reduced ouabain sensitivity, while Pearson (1978) found significant ATPase stimulation by ouabain in all the 5 carriers which he tested. Endogenous membrane protein phosphorylation. The band II phosphorylation (pmol/mg ghost protein) was 47.2 ( ± 11.8) and 44 ( ± 13.3) in the carriers and controls, respectively, and a paired t-test showed no significant difference between the two groups. Similarly no difference was found when phosphorylation of the combined bands I and I[ was considered, nor if the phosphorylation was related to the total spectrin content of the sample. Roses and colleagues (Roses et al. 1975, 1976) reported abnormally high [~-32P]ATP incorporation into band II in red cells from Duchenne patients and furthermore found the same abnormality in all the carriers that they studied (77.0 and 55.8 in the carrier and control groups, respectively). Red cell K + efflux. The mean rate of K + efflux (nmol/min per l0 S cells, measured with a K + electrode) was 8.8 (_+_ 3.6) and 7.8 ( ~ 3.2) for carriers and controls, respectively. A paired t-test showed no significant difference between the two groups.

389 TABLE 6 ECG FINDINGS IN 11 CARRIERS A N D 12 N O R M A L CONTROLS Mean values

Carriers

(R-S)vl R/Svl (R-S)v2 R/Sv2

--3.64 0.43 --3.2 4.697

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2.68 0.139 2.95 0.025

--6.5 0.281 --8.71 0.38

± 5.3 ± 0.108 ± 5.54 5_ 0.191

(4) Electrocardiographic analysis Table 6 shows the analysis of ECG parameters in the carrier and control subjects. No significant differences were found between the groups as regards heart rate, QRS axis or duration, PR or QT interval. A Student' t-test revealed a significantly higher value for the sum (R-S) in leads V1 (P < 0.05) and V2 (P < 0.01) and the ratio R/S in leads V1 and V2 (P < 0.01), R/S V2 showing the greater degree of discrimination. Emery (1969) found a mean (R-S) sum in V1 of--6.8 (4- 2.83) in 107 healthy control women aged 20-56 years and quoted Turner and Price's figure of--6.83 (-43.87) calculated from a series of 158 women aged 20--60. Our mean value of--6.5 (-t5.3) is similar, derived from a far smaller sample. Emery's (1969) mean value for (R-S) in V1 in 25 definite, 18 probable and 7 possible carriers was ---4.23 (4- 3.18) and we found a value of--3.64 (-t- 2.68) in our 11 "known" carriers. While there was quite O

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An evaluation of some carrier detection techniques in Duchenne muscular dystrophy.

Journal o f the Neurological Sciences, 1979, 43: 377-394 377 © Elsevier/North-Holland Biomedical Press A N E V A L U A T I O N OF SOME C A R R I E...
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