Journal of
J. Neurol. 216, 235--249 (1977)
Neurology © by Springer-Verlag 1977
Quantitative EMG and Histological Carrier Detection of Duchenne Muscular Dystrophy G. Scarlato, G. Valli, G. Meola, and L. Carenini Department of Neurology, University of Milan, Medical School, Milan, Italy
Summary. Seventy-nine women known as, or suspected to be, carriers of the Duchenne type of muscular dystrophy were examined. The 15 known carriers had an estimation of the C P K serum level and a manual quantitative E M G , which gave the high detection rate of 93%. The 64 suspected carriers had CPK determination and quantitative E M G , or CPK and muscle biopsy, and the value of each technic is discussed. The problem of giving a reassuring answer to women considered to be possible carriers on genetic criteria, but who are not really carriers, is solved if the results of all three tests are negative. Key words: Carrier, Duchenne - Muscular dystrophy, Duchenne type E M G quantitative in Duchenne - Muscle histology in Duchenne.
Zusammenfassung. 13 sichere und 56 m6gliche Konduktorinnen einer Duchenne'schen Muskeldystrophie wurden untersucht. Bei sicheren Konduktorinnen wurde mittels CPK-Bestimmung im Serum und manueller quantitativer EMG-Technik eine positive Nachweisrate yon 93% erreicht. Bei 64 m6glichen Konduktorinnen wurde entweder durch CPK-Bestimmung und quantitative EMG-Untersuchung oder durch CPK-Bestimmung und Muskelbiopsie untersucht. Es wird der Wert der einzelnen Untersuchungstechniken besprochen. Es wird auf die Notwendigkeit hingewiesen, die m6glichen Konduktorinnen, die jedoch mittels der durchgefiihrten Untersuchungen nicht als solche gesichert werden konnten, richtig zu orientieren.
Introduction and Subjects The most c o m m o n and most severe form of muscular dystrophy (MD) is the Duchenne type which is generally due to a sex-linked recessive gene. Consequently a pregnant w o m a n carrier of the gene for Duchenne dystrophy will have a 50% probability o f generating an affected son or a carrier daughter.
236
G. Scarlato et al.
Since there is no effective treatment for this tragic disorder, it is extremely important to detect the women who are carriers and several criteria have been evaluated for this purpose. From a clinical point of view both an enlargement of the sural muscles, sometimes asymmetrical (Emery, 1963; Zatz et al., 1973) and a muscular weakness, especially of the proximal limbs (Chung, et al., 1960; Moser et al., 1964) have been noticed. Different authors have also considered a decrease of the total body potassium (Bradley, 1971), a reduction of LDH5 isoenzyme (Emery, 1964; Mannucci et al., 1965; Johnston et al., 1966; Pearson and Kar, 1966), the intensity of the limb blood flow (Emery and Schelling, 1965) an increased amino acid uptake in vitro from the polyrybosomes of the muscle (Ionasescu et al., 1971), high serum levels of piruvate kinase (Alberts and Samaha, 1974), an excess of hemopexin in serum (Askanas, 1966; Danieli and Angelini, 1976), and the red cell cholinesterase and serum pseudocholinesterase activity (Schmitt et al., 1976). However, the most practical and reliable methods seem to be the serum creatine kinase (CPK) estimation (Schapira et al., 1960; Hughes, 1963; Pearce et al., 1964; Stephens and Lewin, 1965; Thompson et al., 1967; Dreyfus et al., 1970; Gardner-Medwin, 1968; Gardner-Medwin et al., 1971; Goto et al., 1967), quantitative electromyography (EMG) (Van den Bosch, 1963; Hausmanova-Petrusewicz et al., 1968; Gardner-Medwin, 1968; Gardner-Medwin et al., 1971; Valli et al., 1976) and muscle biopsy (Emery, 1963, 1965; Pearson et al., 1963; Kowalewski et al., 1966; Pearce et al., 1966; Smith et al., 1966; Roy and Dubowitz, 1970; Morris and Raybould, 1971; Ionescu et al., 1975; etc.). We reexamined the data of CPK estimation, quantitative EMG, and muscle biopsy collected in the Neurological Department, University of Milan Medical School, over the last four years, in order to ascertain if these three combined technics can achieve a detection rate high enough to exclude a carrier condition in a suspected woman. Clinical data were not considered because none of the women examined showed features supposed to be significant by at least three of us. Seventy-nine women known or suspected to be carriers were studied. They were divided into two groups according to genetic criteria (Hausmanova): a) 15 known carriers: mothers of one dystrophic child who had at least another affected male in a female line of inheritance, and mothers of two or more dystrophic sons; b) 64 possible carriers: mothers of only one dystrophic son and women related in a female line to a dystrophic subject. The known carriers were tested by CPK estimation and EMG test except two of them who avoided the CPK level determination. Among the group of the possible carriers 20 were tested by CPK and EMG, 51 by CPK and muscle biopsy (seven women have been considered in both groups).
Serum CPK Estimation
The CPK estimation was first performed by Okinaka et al. (1959). The effectiveness of this test was regarded in different ways by different authors, and the
Quantitative EMG and Histological Carrier Detection
237
Table 1. Results of the serum CPK estimation in known and possible carriers Number Known carriers Possible carriers
mothers sisters
Total possible carriers
High serum % CPK values
13
7
53.8
14 42
6 12
42.9 28.6
56
18
32.1
detection rate varies from 78% (Dreyfus et al., 1970) to 64% (Goto et al., 1967) to 54% (Gardner-Medwin et al., 1971) to 33% (Iwamoto, 1969). Many attempts have been made to improve the sensitivity of the test: some authors have repeated the test three times in each suspected carrier (Wilson et al., 1965), some invited the suspected carrier women to perform a standardized physical exercise just before testing (Wiesmann et al., 1965; Stephens and Lewin, 1965; Emery, 1966); other researchers added the determination of aldolase and piruvatokinase serum levels to the CPK estimation (Schapira et al., 1960; Alberts and Samaha, 1974). On the other hand it is important to note that Perry and Fraser (1973) tested the CPK activity in 10 known carriers, up to 12 times: All 10 carriers had at least one normal value, two had constantly normal CPK levels. On the contrary in a group of 44 controls, five showed significantly high values at least once out of 10 times. Thirteen known and 56 possible carriers were tested1; venous blood samples were taken at rest before breakfast from each woman. The serum CPK activity was determined by Monotest CPK activated (UV metod, Boehringer Mannheim G m b H ) , which normal values are up to 50 U/1 (Szasz et al., 1970). The results were as follows: 7 of the 13 known carriers (53.8%) and 18 of the 56 possible carriers (32.1%) had a significantly increased serum CPK activity. The possible carriers were 14 mothers of a single dystrophic son and 42 women, sisters or other female relatives of affected children. Serum CPK levels were found to be high in 6 mothers (42.9%) and in 12 sisters (28.6%). The results, obtained in the known and possible carriers are shown in Table 1.
Electromyographic Methods The E M G technic was used by several authors to detect the carrier state of Duchenne M D (Smith et al., 1966; Caruso and Buchtal, 1965; HausmanovaPetrusewicz et al., 1968; Jacobs, 1968). In 1963 Van den Bosch first adopted manual quantitative E M G for carrier detection, later Gardner-Medwin improved this technic and in 1968, 1970 and 1971 published extensive studies on this matter. 1 Thesenumbers are different from the ones mentioned in the introduction: in fact we failed to obtain the CPK values in two known carriers tested only by EMG. Eight possible carriers were not considered for a statistical evaluation. They had normal CPK levels obtained in other laboratories, and were sent to us only for EMG or muscle biopsy
G.Scarlato et al.
238 Table 2. Action potential measurements of normal subjects
Deltoid
Biceps
Vastus med.
A.P. duration a (mean + 2 SD)
9.4 ms (+ 1.26)
9.5 ms (+ 2.12)
10.7 ms (+ 2.54)
Per cent differences from Buchthal's norm
- 17.9% (P< 0.01)
+1.02%
+ 1.03%
Mean phases per potential + 2 SD
3.04 (+ 0.69)
"
3.14 (+ 0.88)
2.71 (+ 0.65)
Valuesof a group of 9 normal subjects with ages from 23 to 27 years
However in 1971 he concluded that EMG could provide only a marginal contribution and suggested that more versatile methods were needed. In 1972 Mooser et al. published a new automatic technic for quantitative E M G which integrates the O index of Gardner-Medwin, but this method has not been used again. The manual quantitative E M G method, according to Gardner-Medwin was readopted by Valli et al. (1976); they examined three different muscles, deltoid, biceps brachii and vastus medialis, instead of one. Following the same procedure we tested 15 known carriers and 20 possible carriers (two mothers, each of them with one dystrophic son, and 18 sisters). Three muscles of every woman were examined (deltoid, biceps brachii and vastus medialis). Ten potentials were photographed from every muscle; each potential was photographed several times in order to have at least three pictures of the same potential with almost constant shape and size. Amplitude, duration and number of phases were measured, but only the mean action potential duration and the mean number of phases in each muscle were considered. The duration was measured from the first to the last deflection, the phases were defined as oscillations crossing the baseline, but initial and terminal oscillations of less than 20~tV were excluded. We did not consider the ratio between the mean number of phases and the mean action potential duration, i.e. the ~ index of Van den Bosch (1963) modified by Gardner-Medwin (1968), because it is difficult to find significant normal limits from a statistical point of view. A Medelec M S 6 E M G instrument (4 sweeps, 2 channels) was used; we adopted a 200 ~tV/cm gain, and the speed of the sweep was 20 ms/cm. The needle electrodes were Medelec MC 25. Table 2 shows the data considered normal according to the previous study carried out in our laboratory (Valli et al., 1976). The results from the deltoid muscle are presented in Figure 1: in graph A the mean action potential durations are plotted against age, and graph B shows the mean number of phases. In graph A the broken line shows Buchthal's mean, whereas the continuous lines show the corrected Buchtal mean _+2 SD. Figures 2 and 3 present the results from the biceps brachii and vastus medialis muscles, but in graph A the means were not plotted because the results obtained from these muscles agree with Buchthal's values.
Quantitative E M G and Histological Carrier Detection
deltoid
19
msec
239
muscle
Mean
A.P.
Duration
18 17 16 15
14 13 12 11 10 9
. . . . --
j
ooO= o o . .
-
8
O
7 6 5 4 0
10
20
30
40
0 O
50
60
70
years
A Phases
5 e, =
o" o 4.5
0
•'or~
Becket
n
nnn
carriers
11
e'ooo o o o
o •oafO
nnnnnn •
25 No
Carriers gene
e
3.5
3
carriers
Possible
=
0 00• n
2
0 =
o-O
=°o o
4
Known
r rnals
o o 0 0
•
0
Carriers
Known
carriers:
Total rate
detectlorr =
46,6~/o
B Fig. 1 A and B. Results in deltoid muscle. A Mean A.P. durations of carriers plotted against age. B Mean number of phases of normals and carriers. (For further explanation see text)
240
G. Scarlato et al.
biceps brachii muscle
msec
Mean
19
A.P.
Duration
18 17 16 15 14 13 12
~0
11
0
10
•
e"
e"
U_____~-~-~ e
•
9 oOO
8
_ it
0
.
"
7 6 5 4
A
0
10
20
30
40
50
60
70
years
I•
e" Phases
5 O0
45
ior 0
• =
Known
0 =
Possible
O" =
nnn n n n n
3
n
n
0
10l 000 •00
rl
B
K n o w n
Total
Normals
Becker
gene
carriers
O" 0 0 0 0 00•00 •
25 2
carriers
•0
4
35
carriers
0 Carriers
rate
cal
ri
e rs:
detection
=
46,6o,/°
Fig. 2 A and B. Results in biceps brachii muscle. A Mean A.P. durations of carriers plotted against age. B Mean number of phases of normals and carriers. (For further explanation see text)
Quantitative EMG and Histological Carrier Detection
vastus
msec
19
medialis
Mean
A.P.
241
muscle
Duration
18 17 16 15 14 0
13 12 1 1
oO°O oO0o o L _ ~
10 9 8
,
;
7 6 5 4
A
10
20
30
40
50
60
e= Phases
70
Known
years
carriers
5 0---
Possible
If o r 0"=
4.5
carriers
Becker
gene
carr.lers OO
4
0
o0~ 3.5
nn n
•
0~•
K n o w n
c a r r i e r s :
3 nnn
^ ^ 0 = 0 0 ~ -~
nn
2.5
nnn n
2
v~, n
Normals
0
Total
detection
-
•
rate
=
60,0
~o
Carriers
B Fig. 3 A and B. Results in vastus medialis muscle. A Mean A.P. durations of carriers plotted against age. B Mean number of phases of normals and carriers. (For further explanation see text)
242
G. Scarlato et al.
Fig.4. Known carrier (D.E.): short and polyphasic potentials recorded from the right deltoid muscle
F i g u r e 4 gives a n e x a m p l e o f s h o r t a n d p o l y p h a s i c p o t e n t i a l s f o u n d in a carrier. T a b l e 3 s u m m a r i z e s the E M G d a t a f r o m the k n o w n carriers. Thirteen o f 15 w o m e n t u r n e d o u t to be carriers a n d the detection rate was 86.7%. I f only one muscle h a d been tested the E M G significance w o u l d have been m u c h reduced; the
243
Quantitative EMG and Histological Carrier Detection Table 3. Abnormal values obtained from known carriers Known
Age
CPK
Electromyography
carriers
B.E.
32
C.R.
48
S.C.
+
mean A.P. duration
mean number of phases per potential
deltoid biceps
vastus med.
deltoid biceps
-
-
-
+
-
+
+
+
+
-
+
+
+
+
+
+
+
-
+
-
-
-
+
-
30
+
53
.
D.E. D. A . b
42 35
+ +
S.F.
48
.
P.F.
62
+
-
P.R.
43
+
.
C.L.
33
M.S.
a
.
.
-
-
.
.
+ .
.
.
.
.
.
. -
L.L.
63
.
.
.
52
.
.
.
P.G.
44
+
.
.
. -
.
-
. +
.
B.I.
+
+
-
-
.
+
. .
+
.
P.M.
39
.
.
.
.
36
.
.
.
.
-
-
+
+
-
-
-
+
+ .
.
+
.
-
G.T.
vastus med.
.
.
.
+
CPK detection rate = 54%; quantitative EMG = 8&7%; Total detection rate =93.3% b
Carrier of the gene for the Becker type of muscular dystrophy Carrier affected by diabetes and suffering from mild diabetic polyneuropathy
Note: The first 10 women are definite carriers; the last 5 are probable carriers (according to Pearce et al., 1964)
r a t e o f s i g n i f i c a n c e f r o m t h e d e l t o i d a n d b i c e p s m u s c l e s in p a r t i c u l a r w o u l d h a v e b e e n r e d u c e d t o a b o u t o n e h a l f , w h i c h is s i m i l a r t o t h e v a l u e s o b t a i n e d b y G a r d n e r - M e d w i n (1970). T a b l e 4 g r o u p s t h e 20 p o s s i b l e c a r r i e r s t e s t e d ; o n e m o t h e r a n d 12 s i s t e r s o f d y s t r o p h i c c h i l d r e n w e r e f o u n d t o b e c a r r i e r s b y t h e E M G test. I f w e c o n s i d e r t h e C P K v a l u e s a l s o t h e p o s i t i v e c a s e s a r e 14, t h a t is 7 0 % i n s t e a d o f t h e 5 0 % s t a t i s t i c a l l y e x p e c t e d . T h i s h i g h p e r c e n t a g e o f c a r r i e r s d e t e c t e d is p r o b a b l y d u e t o t h e r a n d o m s a m p l i n g . A t a n y r a t e it is n o t e w o r t h y t h a t t h e c a s e C D (sister), w h o h a d a n e g a t i v e E M G t e s t b u t a h i g h C P K e s t i m a t i o n , s h o w e d t h e h i g h s e r u m level of the enzyme only a few months after she had had a baby, whereas more recent estimations were negative.
Muscle
Biopsy
M a n y h i s t o l o g i c a l s t u d i e s h a v e b e e n p e r f o r m e d d u r i n g t h e l a s t 15 y e a r s in o r d e r t o i d e n t i f y t h e c a r r i e r s t a t e o f D u c h e n n e m u s c u l a r d y s t r o p h y . I n 1971 G a r d n e r -
244
G . S c a r l a t o et al.
Table 4. A b n o r m a l values obtained f r o m possible carriers Possible
Age
CPK
Electromyography
carriers
S.A.m.
45
+
C.G.m.
27
.
F.B.s.
30
.
B.P.s.
21
.
mean A.P. duration
mean n u m b e r of phases per potential
deltoid
biceps
deltoid
+
.
. .
. .
vastus med. .
.
.
.
.
.
.
.
.
-
+
. +
.
vastus med.
.
.
.
biceps
.
B.D.s.
24
+
-
-
-
+
+
-
C.F.s.
28
+
-
-
-
+
-
+
P.A.s.
26
.
C.D.s.
26
+
C.S.s.
22
.
.
.
.
+
-
-
S.M.s. a
27
.
.
.
.
+
-
-
Z.G.s.
20
.
.
.
.
S.E.s.
27
.
.
.
.
P.L.s.
26
-
B.S.s.
32
.
B.E.s.
27
+
S.D.s.
18
.
.
.
.
-7
.
.
.
. .
.
.
.
.
.
+
. .
. .
.
. .
. .
+
. .
.
.
.
+ .
.
. .
+ .
+
-
+
-
-
-
-
-
.
.
.
P.M.s.
18
.
P.R.s.
28
-
L.F.s.
25
.
.
.
.
+
-
-
C.L.s.
22
.
.
.
.
+
-
-
-
-
+
C P K detection rate = 25%; quantitative E M G = 65%; total detection rate = 70% m. = m o t h e r of one dystrophic child only s. = sister o f one dystrophic child a
Carrier of the gene for the Becker type of muscular dystrophy
Medwin
et al. s t a t e d t h a t m u s c l e b i o p s y is o f little v a l u e f o r t h e d i a g n o s i s o f t h e
carrier condition
because the myopathic
c h a n g e s t e n d to o c c u r o n l y in c a r r i e r s
whose serum CPK levels are high; while Roy and Dubowitz (1970), Emery (1965), Pearce
et al. ( 1 9 6 6 ) , S m i t h
et al. ( 1 9 6 6 ) , M o r r i s
significant histological changes However
as Dubowitz
and Raybould
also in subjects with norm a l
reported
i n 1973 " a c o m m o n
(1971) found
CPK
levels.
difficulty in this histo-
l o g i c a l s t u d y is t r y i n g t o d e c i d e t h e s i g n i f i c a n c e o f m i n i m a l o r b o r d e r l i n e c h a n g e s i n t h e b i o p s y a n d f i n d i n g a d i v i d i n g l i n e b e t w e e n t h e m i n o r c h a n g e s w h i c h fall within the range of normal
and those which are just beyond
it".
From our experience we consider the following histopathological findings to be significant: variation necrosis,
phagocytosis
i n f i b e r size, i n t e r n a l and
hyaline
nuclei, degenerative
degeneration),
regenerative
aspects (fiber aspects
(fiber
Quantitative EMG and Histological Carrier Detection
245
Fig. 5 A--D. Transverse cryostat section of left deltoid muscle biopsy. A Haematoxylin and Eosin × 100: two enlarged rounded opaque fibres. B H. &E. × 250: increased variability in the size of fibres with some internal nuclei. C H. & E. × 250: small groups of basophilic fibres. D N A D H - T R x 100: irregularity and increase of the histochemical activity in the subsarcolemmal areas
246
G. Scarlato et al.
splitting, basophilic small fibers with vesicular nucleus and prominent nucleolus), increase of endomysial and perimysial connective tissue; and from a histochemical point of view the following: some irregularity in the distribution of mitochondrial enzyme activity such as central cores, moth-eaten, whorled fibers or an increase of the histochemical activity in the subsarcolemmal areas. Each biopsy was considered to be significant which showed at least three of the elementary histological or histochemical changes mentioned above (Fig. 5). In this study 51 possible carriers were tested, 18 mothers of one dystrophic son, and 33 sisters or relatives of dystrophic patients. A biopsy specimen was obtained from the deltoid muscle of each carrier. The cryostat sections were studied by light microscopy after histological (haematoxylin and eosin, modified Gomori thricrome) and histochemical staining (myofibrillar ATPase at pH 9.4; 4.6; 4.3; N A D H diaphorase; phosphorylase; acid phosphatase; PAS). The biopsies of 26 out of 51 possible carriers, 11 mothers and 15 sisters, were positive. All 5 mothers with increased CPK levels, and 6 of the 13 mothers with normal CPK levels, showed significant histological a n d / o r histochemical changes. In the group of sisters of patients, 10 had elevated CPK activity and 8 of them also had a positive biopsy. Seven of the 23 sisters with normal CPK levels had a pathological muscle biopsy. Two sisters had high levels of CPK and a normal muscle biopsy (Table 5).
Discussion
Quantitative EMG and serum CPK tests enabled us to identify 93% of the known carriers (see Table 3); serum CPK determination was successful in detecting 54% of the carriers, quite close to the data reported by others (Sugita and Tyler, 1963; Emery, 1966; Goto et al., 1967; Thompson et al., 1967; Gardner-Medwin et al., 1971), while the E M G test on three different muscles achieved a detection rate of 86.7% Some of the possible carriers were examined both by quantitative E M G and CPK tests, but not by muscle biopsy (see Table 4), whereas other subjects were tested only for CPK and by histological methods (see Table 5). A scrutiny of these tables discloses that one technic often gives positive results when another is negative, therefore all three tests should be performed on each subject in order to maximise the detection rate. Only seven possible carriers were examined by all three tests. Unfortunately they can not be used for a statistical
Table 5. Comparison between biopsy and CPK of possible carriers
Mothers Sisters
Number
Significant % biopsies
High serum % CPK values
18 33
11 15
5 10
61.1 45.4
27.8 30.3
Quantitative EMG and Histological Carrier Detection
247
comparison, not only due to their small number, but also because of bias in sampling (as mentioned above, for the last two years suspected carriers were sent to us by the regional Committee for MD only if they had normal CPK levels). According to Perry and Fraser (1973) "a negative result from a test with a 20% false negative rate is not likely to reduce the odds of being a carrier to a level where most women would consider having a family without prenatal sex determination and abortion of all males". This means that technics with a very high detection rate are necessary to allow a suspected woman to set up a family without too much worry about the future of her offspring. We think that the CPK estimation, quantitative E M G and muscle biopsy combined can attain almost complete detection of the carrier state. Indeed we believe that with these three technics there is more risk of defining a healthy woman as a carrier than of missing a carrier. The serum CPK estimation is the most common test for carrier detection. However muscle fatigue or sporadic accidents such as muscle contusions, latent flogosis, intramuscular injection and others, sometimes unknown at the moment of the test, cause variations in serum CPK activity and make the test not quite specific. The E M G test is easily borne by adult subjects and it is not really painful; it enables one to test more muscles and different areas of each muscle. Muscular changes in the carriers are usually slight, and a detailed E M G examination, such as manual quantitative EMG, is needed. E M G methods, based only on the visual evaluation of the potentials and of the interference pattern on the screen of the oscilloscope, are often tricky and insufficient. The muscle biopsy supplies a further possible aid in the diagnosis of the carrier state, as is stressed by Dubowitz and Brooke (1973); in fact it shows clear changes e v e n if serum enzyme levels are normal. In conclusion, for the carrier detection of Duchenne muscular dystrophy, we suggest estimating serum CPK activity first; whenever this test gives a normal result, it is possible to confirm the carrier state by quantitative EMG, performed on three muscles. When the E M G test is also normal, we recommend muscle biopsy. We believe that these three technics combined form a rather close sieve which allows genetically suspected women to set up a family, when all three tests are normal.
References Alberts, M. C., Samaha, F. J.: Serum pyruvate kinase in muscle disease and carrier states. Neurology 24, 462--464 (1974) Askanas, W.: Life Science 5, 1767 (1966) Bradley, W. G., Gardner-Medwin, D.,, Haggith, J., Walton, J. N., Hesp, R.: Duchenne muscular dystrophy. Use of rubidium chloride Rb 86 in the detection of carriers of the gene. Arch. Neurol. 25, 193--197 (1971) Buchthal, F.: An Introduction to Electromyography. Copenhagen: Glyndal 1957 Buchthal, F., Rosenfalk, P.: Action potential parameters in different human muscles. Acta psychiat, neurol, scand. 30, 125--131 (1955) Caruso, G., Buchthal, F.: Refractory period of muscle and electromyographic findings in relatives of patients with muscular dystrophy. Brain 88, 29--50 (1965)
248
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Chung, C. S., Morton, N. E., Peters, H. A.: Serum enzymes and genetic carriers in muscular dystrophy. Amer. J. Hum. Genet. 12, 52--66 (1960) Danieli, G. A., Angelini, C.: Duchenne carrier detection. Lancet 10, 86--89 (July 1976) Dreyfus, J. C., Shapira, G., Shapira, F., Demos, J.: Nature of the carrier state in progressive muscular dystrophy. Muscle diseases, eds. J. N. Walton, N. Canal, G. Scarlato, pp. 417--423. Amsterdam: Excerpta Medica 1970 Dubowitz, V., Brooke, M. H.: Muscle Biopsy: a Modern Approach. Philadelphia: Saunders 1973 Emery, A. E. H.: Clinical manifestations in two carriers of Duchenne muscular dystrophy. Lancet 1963 I, 1126--1128 Emery, A. E. H.: Electrophoretic pattern of lactic dehydrogenase in carriers and patients with Duchenne muscular dystrophy. Nature 201, 1044--1045 (1964) Emery, A. E. H., Schelling, J. L.: Limb blood flow in patients and carriers of Duchenne muscular dystrophy. Acta Genet. 15, 337 (1965) Emery, A. E. H.: Muscle histology in carriers of Duchenne muscular dystrophy. J. Med. Genet. 2, 1 - - 7 (1965) Emery, A. E. H.: The use of serum creatine kinase for detecting carriers of Duchenne muscular dystrophy. A. T. Milhorat, Exploratory Concepts in Muscular Dystrophy and Related Disorders. International Congress Series, No. 147, Excerpta Medica, 90--96 (1966) Emery, A. E. H.: The use of serum creatine kinase for detecting carriers of Duchenne muscular dystrophy. J. Med. Genet. 3, 92 (1967) Gardner-Medwin, D.: Some problems encountered in the use of electromyography in carrier detection. Proc. 4th Symposium on Research in Muscular Dystrophy, pp. 420---432. London: Pitman 1968 Gardner-Medwin, D.: Studies of the carrier state in the Duchenne type of muscular dystrophy. J. Neurol. Neurosurg. Psychiat. 31, 124--134 (1968) Gardner-Meclwin, D.: The use of serum creatine kinase and electromyography in carrier detection. Muscle diseases, eds. J. N. Walton, N. Canal, G. Scarlato, pp. 431--438. Amsterdam: Excerpta Medica 1970 Gardner-Medwin, D., Pennington, R. J., Walton, J. N.: The detection of carriers of X-linked muscular dystrophy genes. J. Neurol. Sci. 13, 449--454 (1971) Goto, I., Peters, H. A., Resee, H. H.: CPK in neuromuscular disease. Patients and families. Arch. Neurol. 16, 529--535 (1967) Hausmanova-Petrusewicz, I., Prot, J., Niebroj-Dobosz, I., Hetnarska, L., Emeryk, B., Wasowicz, B., Askanas, W., Slucka, C.: Studies of healthy relatives of patients with Duchenne muscular dystrophy. J. Neurol. Sci. 7, 465--480 (1968) Hughes, B. P.: Serum enzyme studies special reference to the Duchenne type. Research in Muscular Dystrophy, Proc. 2nd Symp. on Current Research in Muscular Dystrophy, 167 (1963) Ionasescu, V., Zellweger, H., Conway, T. W.: A new approach for carrier detection in Duchenne muscular dystrophy: protein synthesis of muscle polyribosomes in vitro. Neurology 21, 703--709 (1971) Ionescu, V., Radu, H., Nicolescu, P.: Identification of Duchenne muscular dystrophy carriers. Electron microscopical investigation of skeletal muscle. Arch. Pathol. 99,436--441 (1975) Iwamoto, A.: Studies on serum creatine phosphokinase of various diseases in orthopedic surgery. Acta Med. (Fukuoka) 39, 108--121 (1969) Jacobs, K.: Carrier detection in the Duchenne type muscular dystrophy: preliminary observation on the place of electromyography. J. Neurol. Sci. 6, 347--356 (1968) Johnston, H. A., Wilkinson, J. H., Withycome, W. A., Raymond, S.: Alpha-hydroxybutyrate dehydrogenase activity in sex-linked muscular dystrophy. J. Clin. Path. 19, 250 (1966) Kowalewski, S., Rotthauwe, H. W., Molbert, E., Mumenthaler, M.: Female carriers of muscular dystrophy. Lancet 1966 I, 1216--1217 Mannucci, P. M., Ideo, g., Cao, A., Macciotta, A.: Gli isoenzimi della latticodeidrogenasi (L.D.H.) e della transaminasi glutammico (T.G.O.) nel muscolo fetale, adulto e di soggetti affetti da distrofia muscolare progressiva tipo Duchenne. Rass. Med. Sarda 68, 287 (1965) Moosa, A., Brown, B. H., Dubowitz, V.: Quantitative electromyography: carrier detection
Quantitative EMG and Histological Carrier Detection
249
in Duchenne type muscular dystrophy using a new automatic technique. J. Neurol. Neurosurg. Psychiat. 35, 841--844 (1972) Morris, C. J., Raybould, J. A.: Histochemically demonstrable fibre abnormalities in normal skeletal muscle and in muscle from carriers of Duchenne muscular dystrophy. J. Neurol. Neurosurg. Psychiat. 34, 348 (1971) Moser, H. yon, Wiesmann, U., Richterich, R., Rossi, E.: Progressive Muskeldystrophie.VI. H~iufigkeit, Klinik und Genetik der Duchenne-Form. Schweiz. Med. Wschr. 94, 1610-- 1621 (1964) Okinaka, S., Sugita, H., Momoi, H., Toyokura, Y., Kumagai, I., Ebashi, S., Fujie, Y.: Serum creatine phosphokinase and aldolase activity in neuromuscular disorders. 84th Ann. Meet. Amer. Neurol. Assoc. 1959 Pearce, J. M. S., Pennington, R. J., Walton, J. N.: Serum enzymes studies in muscle disease. III. Serum creatine kinase activity in relatives of patients with the Duchenne type of muscular dystrophy. J. Neurol. Neurosurg. Psychiat. 27, 181--185 (1964a) Pearce, G. W., Pearce, J. M. S., Walton, J. N.: The Duchenne type muscular dystrophy: histopathological studies of the carrier state. Brain 89, 109--120 (1966) Pearson, C. M., Fowler, W. M., Wright, S. W.: X-chromosome mosaicism in females with muscular dystrophy. Proc. Nat. Acad. Sci. (Wash.) 511,24--31 (1963) Pearson, C. M., Kar, N. C.: Isoenzymes: general considerations and alterations in human and animal myopathies. Ann. N. Y. Acad. Sci. 138, 293 (1966) Perry, T. B., Fraser, F. C.: Variability of serum creatine phosphokinase activity in normal women and carrier of the gene for Duchenne muscular dystrophy. Neurology 23, 1316-- 1323 (1973) Roy, S., Dubowitz, V.: Carrier detection in Duchenne muscular dystrophy. A comparative study of electron microscopy, light microscopy and serum enzymes. J. Neuroi. Sci. 11, 65--79 (1970) Schapira, F., Dreyfus, J. C., Schapira, G., Demos, J.: Etude de l'aldolase et de la cr6atine kinase du s6rum chez les mores de myopathes. Rev. Franc. Etud. Clin. Biol. 5, 990--994 (1960) Schmitt, J., Royer, R., Schmidt, C., Mucka, R.: Approche nosografique des myopathies h6r6ditaires. Essai de classification et de diagnostic par les cholinest6rases globulaires et les pseudocholinest6rases s6riques. Revue Neurologique 132, 7, 481--487 (1976) Smith, H. L., Amick, L. D., Johnston, W. W.: Detection of subclinical and cartier states in Duchenne muscular dystrophy. J. Pediatrics 69, 67--79 (1966) Stephens, J., Lewin, E.: Serum enzyme variations and histological abnormalities in the carrier state in Duchenne dystrophy. J. Neurol. Neurosurg. Psychiat. 28, 104--108 (1965) Sugita, H., Tyler, F. H.: Pathogenesis of muscular dystrophy. Trans. Ass. Amer. Phycns. 76, 231--236 (1963) Szasz, G., Busch, E. W., Farohs, H. B.: Serumkreatinkinase. Methodische Erfahrungen und Normalwerte mit einem neuen handelsiiblichen Test. Dtsch. med. Wschr. 95,829 (1970) Thompson, M. W., Murphy, E. G., McAlpine, P. J.: An assessment of the creatine kinase test in the detection of carriers of Duchenne muscular dystrophy. J. Pediat. 71, 82--93 (1967) Valli, G., Scarlato, G., Contartese, M.: Quantitative electromyography in the detection of the carriers in Duchenne type muscular dystrophy. J. Neurol. 212, 139--149 (1976) Van den Bosch, J.: Investigation of the carrier state in the Duchenne type dystrophy. In: 2nd Symposium on Research in Muscular Dystrophy, pp. 23--30. London: Pitman 1963 Wiesmann, V., Moser, H., Richterich, R., Rossi, E.: Progressive Muskeldystrophie. VII. Die Erfassung von Heterozygoten der Duchenne-Muskeldystrophiedutch Messung der Serumkreatinkinase unter iokalisierter Arbeitsbelastung in Anoxie. Klin. Wschr. 43, 1015--1022 (1965) Wilson, K. M., Evans, K. A., Carter, C. O.: Creatine kinase levels in women who carry genes for three types of muscular dystrophy. Brit. Med. J. 1965 I, 750--753 Zatz, M., Levisky, R. B., Levy, J. A., Valente, B. O., Gianotti, M., Frota-Pessoa, O.: Clinical symptoms in a female carrier of Duchenne muscular dystrophy. J. Genet. Human 21, 297--305 (1973) Received March 11, 1977
J. Neurol. 216, 235--249 (1977)
Neurology © by Springer-Verlag 1977
Quantitative EMG and Histological Carrier Detection of Duchenne Muscular Dystrophy G. Scarlato, G. Valli, G. Meola, and L. Carenini Department of Neurology, University of Milan, Medical School, Milan, Italy
Summary. Seventy-nine women known as, or suspected to be, carriers of the Duchenne type of muscular dystrophy were examined. The 15 known carriers had an estimation of the C P K serum level and a manual quantitative E M G , which gave the high detection rate of 93%. The 64 suspected carriers had CPK determination and quantitative E M G , or CPK and muscle biopsy, and the value of each technic is discussed. The problem of giving a reassuring answer to women considered to be possible carriers on genetic criteria, but who are not really carriers, is solved if the results of all three tests are negative. Key words: Carrier, Duchenne - Muscular dystrophy, Duchenne type E M G quantitative in Duchenne - Muscle histology in Duchenne.
Zusammenfassung. 13 sichere und 56 m6gliche Konduktorinnen einer Duchenne'schen Muskeldystrophie wurden untersucht. Bei sicheren Konduktorinnen wurde mittels CPK-Bestimmung im Serum und manueller quantitativer EMG-Technik eine positive Nachweisrate yon 93% erreicht. Bei 64 m6glichen Konduktorinnen wurde entweder durch CPK-Bestimmung und quantitative EMG-Untersuchung oder durch CPK-Bestimmung und Muskelbiopsie untersucht. Es wird der Wert der einzelnen Untersuchungstechniken besprochen. Es wird auf die Notwendigkeit hingewiesen, die m6glichen Konduktorinnen, die jedoch mittels der durchgefiihrten Untersuchungen nicht als solche gesichert werden konnten, richtig zu orientieren.
Introduction and Subjects The most c o m m o n and most severe form of muscular dystrophy (MD) is the Duchenne type which is generally due to a sex-linked recessive gene. Consequently a pregnant w o m a n carrier of the gene for Duchenne dystrophy will have a 50% probability o f generating an affected son or a carrier daughter.
236
G. Scarlato et al.
Since there is no effective treatment for this tragic disorder, it is extremely important to detect the women who are carriers and several criteria have been evaluated for this purpose. From a clinical point of view both an enlargement of the sural muscles, sometimes asymmetrical (Emery, 1963; Zatz et al., 1973) and a muscular weakness, especially of the proximal limbs (Chung, et al., 1960; Moser et al., 1964) have been noticed. Different authors have also considered a decrease of the total body potassium (Bradley, 1971), a reduction of LDH5 isoenzyme (Emery, 1964; Mannucci et al., 1965; Johnston et al., 1966; Pearson and Kar, 1966), the intensity of the limb blood flow (Emery and Schelling, 1965) an increased amino acid uptake in vitro from the polyrybosomes of the muscle (Ionasescu et al., 1971), high serum levels of piruvate kinase (Alberts and Samaha, 1974), an excess of hemopexin in serum (Askanas, 1966; Danieli and Angelini, 1976), and the red cell cholinesterase and serum pseudocholinesterase activity (Schmitt et al., 1976). However, the most practical and reliable methods seem to be the serum creatine kinase (CPK) estimation (Schapira et al., 1960; Hughes, 1963; Pearce et al., 1964; Stephens and Lewin, 1965; Thompson et al., 1967; Dreyfus et al., 1970; Gardner-Medwin, 1968; Gardner-Medwin et al., 1971; Goto et al., 1967), quantitative electromyography (EMG) (Van den Bosch, 1963; Hausmanova-Petrusewicz et al., 1968; Gardner-Medwin, 1968; Gardner-Medwin et al., 1971; Valli et al., 1976) and muscle biopsy (Emery, 1963, 1965; Pearson et al., 1963; Kowalewski et al., 1966; Pearce et al., 1966; Smith et al., 1966; Roy and Dubowitz, 1970; Morris and Raybould, 1971; Ionescu et al., 1975; etc.). We reexamined the data of CPK estimation, quantitative EMG, and muscle biopsy collected in the Neurological Department, University of Milan Medical School, over the last four years, in order to ascertain if these three combined technics can achieve a detection rate high enough to exclude a carrier condition in a suspected woman. Clinical data were not considered because none of the women examined showed features supposed to be significant by at least three of us. Seventy-nine women known or suspected to be carriers were studied. They were divided into two groups according to genetic criteria (Hausmanova): a) 15 known carriers: mothers of one dystrophic child who had at least another affected male in a female line of inheritance, and mothers of two or more dystrophic sons; b) 64 possible carriers: mothers of only one dystrophic son and women related in a female line to a dystrophic subject. The known carriers were tested by CPK estimation and EMG test except two of them who avoided the CPK level determination. Among the group of the possible carriers 20 were tested by CPK and EMG, 51 by CPK and muscle biopsy (seven women have been considered in both groups).
Serum CPK Estimation
The CPK estimation was first performed by Okinaka et al. (1959). The effectiveness of this test was regarded in different ways by different authors, and the
Quantitative EMG and Histological Carrier Detection
237
Table 1. Results of the serum CPK estimation in known and possible carriers Number Known carriers Possible carriers
mothers sisters
Total possible carriers
High serum % CPK values
13
7
53.8
14 42
6 12
42.9 28.6
56
18
32.1
detection rate varies from 78% (Dreyfus et al., 1970) to 64% (Goto et al., 1967) to 54% (Gardner-Medwin et al., 1971) to 33% (Iwamoto, 1969). Many attempts have been made to improve the sensitivity of the test: some authors have repeated the test three times in each suspected carrier (Wilson et al., 1965), some invited the suspected carrier women to perform a standardized physical exercise just before testing (Wiesmann et al., 1965; Stephens and Lewin, 1965; Emery, 1966); other researchers added the determination of aldolase and piruvatokinase serum levels to the CPK estimation (Schapira et al., 1960; Alberts and Samaha, 1974). On the other hand it is important to note that Perry and Fraser (1973) tested the CPK activity in 10 known carriers, up to 12 times: All 10 carriers had at least one normal value, two had constantly normal CPK levels. On the contrary in a group of 44 controls, five showed significantly high values at least once out of 10 times. Thirteen known and 56 possible carriers were tested1; venous blood samples were taken at rest before breakfast from each woman. The serum CPK activity was determined by Monotest CPK activated (UV metod, Boehringer Mannheim G m b H ) , which normal values are up to 50 U/1 (Szasz et al., 1970). The results were as follows: 7 of the 13 known carriers (53.8%) and 18 of the 56 possible carriers (32.1%) had a significantly increased serum CPK activity. The possible carriers were 14 mothers of a single dystrophic son and 42 women, sisters or other female relatives of affected children. Serum CPK levels were found to be high in 6 mothers (42.9%) and in 12 sisters (28.6%). The results, obtained in the known and possible carriers are shown in Table 1.
Electromyographic Methods The E M G technic was used by several authors to detect the carrier state of Duchenne M D (Smith et al., 1966; Caruso and Buchtal, 1965; HausmanovaPetrusewicz et al., 1968; Jacobs, 1968). In 1963 Van den Bosch first adopted manual quantitative E M G for carrier detection, later Gardner-Medwin improved this technic and in 1968, 1970 and 1971 published extensive studies on this matter. 1 Thesenumbers are different from the ones mentioned in the introduction: in fact we failed to obtain the CPK values in two known carriers tested only by EMG. Eight possible carriers were not considered for a statistical evaluation. They had normal CPK levels obtained in other laboratories, and were sent to us only for EMG or muscle biopsy
G.Scarlato et al.
238 Table 2. Action potential measurements of normal subjects
Deltoid
Biceps
Vastus med.
A.P. duration a (mean + 2 SD)
9.4 ms (+ 1.26)
9.5 ms (+ 2.12)
10.7 ms (+ 2.54)
Per cent differences from Buchthal's norm
- 17.9% (P< 0.01)
+1.02%
+ 1.03%
Mean phases per potential + 2 SD
3.04 (+ 0.69)
"
3.14 (+ 0.88)
2.71 (+ 0.65)
Valuesof a group of 9 normal subjects with ages from 23 to 27 years
However in 1971 he concluded that EMG could provide only a marginal contribution and suggested that more versatile methods were needed. In 1972 Mooser et al. published a new automatic technic for quantitative E M G which integrates the O index of Gardner-Medwin, but this method has not been used again. The manual quantitative E M G method, according to Gardner-Medwin was readopted by Valli et al. (1976); they examined three different muscles, deltoid, biceps brachii and vastus medialis, instead of one. Following the same procedure we tested 15 known carriers and 20 possible carriers (two mothers, each of them with one dystrophic son, and 18 sisters). Three muscles of every woman were examined (deltoid, biceps brachii and vastus medialis). Ten potentials were photographed from every muscle; each potential was photographed several times in order to have at least three pictures of the same potential with almost constant shape and size. Amplitude, duration and number of phases were measured, but only the mean action potential duration and the mean number of phases in each muscle were considered. The duration was measured from the first to the last deflection, the phases were defined as oscillations crossing the baseline, but initial and terminal oscillations of less than 20~tV were excluded. We did not consider the ratio between the mean number of phases and the mean action potential duration, i.e. the ~ index of Van den Bosch (1963) modified by Gardner-Medwin (1968), because it is difficult to find significant normal limits from a statistical point of view. A Medelec M S 6 E M G instrument (4 sweeps, 2 channels) was used; we adopted a 200 ~tV/cm gain, and the speed of the sweep was 20 ms/cm. The needle electrodes were Medelec MC 25. Table 2 shows the data considered normal according to the previous study carried out in our laboratory (Valli et al., 1976). The results from the deltoid muscle are presented in Figure 1: in graph A the mean action potential durations are plotted against age, and graph B shows the mean number of phases. In graph A the broken line shows Buchthal's mean, whereas the continuous lines show the corrected Buchtal mean _+2 SD. Figures 2 and 3 present the results from the biceps brachii and vastus medialis muscles, but in graph A the means were not plotted because the results obtained from these muscles agree with Buchthal's values.
Quantitative E M G and Histological Carrier Detection
deltoid
19
msec
239
muscle
Mean
A.P.
Duration
18 17 16 15
14 13 12 11 10 9
. . . . --
j
ooO= o o . .
-
8
O
7 6 5 4 0
10
20
30
40
0 O
50
60
70
years
A Phases
5 e, =
o" o 4.5
0
•'or~
Becket
n
nnn
carriers
11
e'ooo o o o
o •oafO
nnnnnn •
25 No
Carriers gene
e
3.5
3
carriers
Possible
=
0 00• n
2
0 =
o-O
=°o o
4
Known
r rnals
o o 0 0
•
0
Carriers
Known
carriers:
Total rate
detectlorr =
46,6~/o
B Fig. 1 A and B. Results in deltoid muscle. A Mean A.P. durations of carriers plotted against age. B Mean number of phases of normals and carriers. (For further explanation see text)
240
G. Scarlato et al.
biceps brachii muscle
msec
Mean
19
A.P.
Duration
18 17 16 15 14 13 12
~0
11
0
10
•
e"
e"
U_____~-~-~ e
•
9 oOO
8
_ it
0
.
"
7 6 5 4
A
0
10
20
30
40
50
60
70
years
I•
e" Phases
5 O0
45
ior 0
• =
Known
0 =
Possible
O" =
nnn n n n n
3
n
n
0
10l 000 •00
rl
B
K n o w n
Total
Normals
Becker
gene
carriers
O" 0 0 0 0 00•00 •
25 2
carriers
•0
4
35
carriers
0 Carriers
rate
cal
ri
e rs:
detection
=
46,6o,/°
Fig. 2 A and B. Results in biceps brachii muscle. A Mean A.P. durations of carriers plotted against age. B Mean number of phases of normals and carriers. (For further explanation see text)
Quantitative EMG and Histological Carrier Detection
vastus
msec
19
medialis
Mean
A.P.
241
muscle
Duration
18 17 16 15 14 0
13 12 1 1
oO°O oO0o o L _ ~
10 9 8
,
;
7 6 5 4
A
10
20
30
40
50
60
e= Phases
70
Known
years
carriers
5 0---
Possible
If o r 0"=
4.5
carriers
Becker
gene
carr.lers OO
4
0
o0~ 3.5
nn n
•
0~•
K n o w n
c a r r i e r s :
3 nnn
^ ^ 0 = 0 0 ~ -~
nn
2.5
nnn n
2
v~, n
Normals
0
Total
detection
-
•
rate
=
60,0
~o
Carriers
B Fig. 3 A and B. Results in vastus medialis muscle. A Mean A.P. durations of carriers plotted against age. B Mean number of phases of normals and carriers. (For further explanation see text)
242
G. Scarlato et al.
Fig.4. Known carrier (D.E.): short and polyphasic potentials recorded from the right deltoid muscle
F i g u r e 4 gives a n e x a m p l e o f s h o r t a n d p o l y p h a s i c p o t e n t i a l s f o u n d in a carrier. T a b l e 3 s u m m a r i z e s the E M G d a t a f r o m the k n o w n carriers. Thirteen o f 15 w o m e n t u r n e d o u t to be carriers a n d the detection rate was 86.7%. I f only one muscle h a d been tested the E M G significance w o u l d have been m u c h reduced; the
243
Quantitative EMG and Histological Carrier Detection Table 3. Abnormal values obtained from known carriers Known
Age
CPK
Electromyography
carriers
B.E.
32
C.R.
48
S.C.
+
mean A.P. duration
mean number of phases per potential
deltoid biceps
vastus med.
deltoid biceps
-
-
-
+
-
+
+
+
+
-
+
+
+
+
+
+
+
-
+
-
-
-
+
-
30
+
53
.
D.E. D. A . b
42 35
+ +
S.F.
48
.
P.F.
62
+
-
P.R.
43
+
.
C.L.
33
M.S.
a
.
.
-
-
.
.
+ .
.
.
.
.
.
. -
L.L.
63
.
.
.
52
.
.
.
P.G.
44
+
.
.
. -
.
-
. +
.
B.I.
+
+
-
-
.
+
. .
+
.
P.M.
39
.
.
.
.
36
.
.
.
.
-
-
+
+
-
-
-
+
+ .
.
+
.
-
G.T.
vastus med.
.
.
.
+
CPK detection rate = 54%; quantitative EMG = 8&7%; Total detection rate =93.3% b
Carrier of the gene for the Becker type of muscular dystrophy Carrier affected by diabetes and suffering from mild diabetic polyneuropathy
Note: The first 10 women are definite carriers; the last 5 are probable carriers (according to Pearce et al., 1964)
r a t e o f s i g n i f i c a n c e f r o m t h e d e l t o i d a n d b i c e p s m u s c l e s in p a r t i c u l a r w o u l d h a v e b e e n r e d u c e d t o a b o u t o n e h a l f , w h i c h is s i m i l a r t o t h e v a l u e s o b t a i n e d b y G a r d n e r - M e d w i n (1970). T a b l e 4 g r o u p s t h e 20 p o s s i b l e c a r r i e r s t e s t e d ; o n e m o t h e r a n d 12 s i s t e r s o f d y s t r o p h i c c h i l d r e n w e r e f o u n d t o b e c a r r i e r s b y t h e E M G test. I f w e c o n s i d e r t h e C P K v a l u e s a l s o t h e p o s i t i v e c a s e s a r e 14, t h a t is 7 0 % i n s t e a d o f t h e 5 0 % s t a t i s t i c a l l y e x p e c t e d . T h i s h i g h p e r c e n t a g e o f c a r r i e r s d e t e c t e d is p r o b a b l y d u e t o t h e r a n d o m s a m p l i n g . A t a n y r a t e it is n o t e w o r t h y t h a t t h e c a s e C D (sister), w h o h a d a n e g a t i v e E M G t e s t b u t a h i g h C P K e s t i m a t i o n , s h o w e d t h e h i g h s e r u m level of the enzyme only a few months after she had had a baby, whereas more recent estimations were negative.
Muscle
Biopsy
M a n y h i s t o l o g i c a l s t u d i e s h a v e b e e n p e r f o r m e d d u r i n g t h e l a s t 15 y e a r s in o r d e r t o i d e n t i f y t h e c a r r i e r s t a t e o f D u c h e n n e m u s c u l a r d y s t r o p h y . I n 1971 G a r d n e r -
244
G . S c a r l a t o et al.
Table 4. A b n o r m a l values obtained f r o m possible carriers Possible
Age
CPK
Electromyography
carriers
S.A.m.
45
+
C.G.m.
27
.
F.B.s.
30
.
B.P.s.
21
.
mean A.P. duration
mean n u m b e r of phases per potential
deltoid
biceps
deltoid
+
.
. .
. .
vastus med. .
.
.
.
.
.
.
.
.
-
+
. +
.
vastus med.
.
.
.
biceps
.
B.D.s.
24
+
-
-
-
+
+
-
C.F.s.
28
+
-
-
-
+
-
+
P.A.s.
26
.
C.D.s.
26
+
C.S.s.
22
.
.
.
.
+
-
-
S.M.s. a
27
.
.
.
.
+
-
-
Z.G.s.
20
.
.
.
.
S.E.s.
27
.
.
.
.
P.L.s.
26
-
B.S.s.
32
.
B.E.s.
27
+
S.D.s.
18
.
.
.
.
-7
.
.
.
. .
.
.
.
.
.
+
. .
. .
.
. .
. .
+
. .
.
.
.
+ .
.
. .
+ .
+
-
+
-
-
-
-
-
.
.
.
P.M.s.
18
.
P.R.s.
28
-
L.F.s.
25
.
.
.
.
+
-
-
C.L.s.
22
.
.
.
.
+
-
-
-
-
+
C P K detection rate = 25%; quantitative E M G = 65%; total detection rate = 70% m. = m o t h e r of one dystrophic child only s. = sister o f one dystrophic child a
Carrier of the gene for the Becker type of muscular dystrophy
Medwin
et al. s t a t e d t h a t m u s c l e b i o p s y is o f little v a l u e f o r t h e d i a g n o s i s o f t h e
carrier condition
because the myopathic
c h a n g e s t e n d to o c c u r o n l y in c a r r i e r s
whose serum CPK levels are high; while Roy and Dubowitz (1970), Emery (1965), Pearce
et al. ( 1 9 6 6 ) , S m i t h
et al. ( 1 9 6 6 ) , M o r r i s
significant histological changes However
as Dubowitz
and Raybould
also in subjects with norm a l
reported
i n 1973 " a c o m m o n
(1971) found
CPK
levels.
difficulty in this histo-
l o g i c a l s t u d y is t r y i n g t o d e c i d e t h e s i g n i f i c a n c e o f m i n i m a l o r b o r d e r l i n e c h a n g e s i n t h e b i o p s y a n d f i n d i n g a d i v i d i n g l i n e b e t w e e n t h e m i n o r c h a n g e s w h i c h fall within the range of normal
and those which are just beyond
it".
From our experience we consider the following histopathological findings to be significant: variation necrosis,
phagocytosis
i n f i b e r size, i n t e r n a l and
hyaline
nuclei, degenerative
degeneration),
regenerative
aspects (fiber aspects
(fiber
Quantitative EMG and Histological Carrier Detection
245
Fig. 5 A--D. Transverse cryostat section of left deltoid muscle biopsy. A Haematoxylin and Eosin × 100: two enlarged rounded opaque fibres. B H. &E. × 250: increased variability in the size of fibres with some internal nuclei. C H. & E. × 250: small groups of basophilic fibres. D N A D H - T R x 100: irregularity and increase of the histochemical activity in the subsarcolemmal areas
246
G. Scarlato et al.
splitting, basophilic small fibers with vesicular nucleus and prominent nucleolus), increase of endomysial and perimysial connective tissue; and from a histochemical point of view the following: some irregularity in the distribution of mitochondrial enzyme activity such as central cores, moth-eaten, whorled fibers or an increase of the histochemical activity in the subsarcolemmal areas. Each biopsy was considered to be significant which showed at least three of the elementary histological or histochemical changes mentioned above (Fig. 5). In this study 51 possible carriers were tested, 18 mothers of one dystrophic son, and 33 sisters or relatives of dystrophic patients. A biopsy specimen was obtained from the deltoid muscle of each carrier. The cryostat sections were studied by light microscopy after histological (haematoxylin and eosin, modified Gomori thricrome) and histochemical staining (myofibrillar ATPase at pH 9.4; 4.6; 4.3; N A D H diaphorase; phosphorylase; acid phosphatase; PAS). The biopsies of 26 out of 51 possible carriers, 11 mothers and 15 sisters, were positive. All 5 mothers with increased CPK levels, and 6 of the 13 mothers with normal CPK levels, showed significant histological a n d / o r histochemical changes. In the group of sisters of patients, 10 had elevated CPK activity and 8 of them also had a positive biopsy. Seven of the 23 sisters with normal CPK levels had a pathological muscle biopsy. Two sisters had high levels of CPK and a normal muscle biopsy (Table 5).
Discussion
Quantitative EMG and serum CPK tests enabled us to identify 93% of the known carriers (see Table 3); serum CPK determination was successful in detecting 54% of the carriers, quite close to the data reported by others (Sugita and Tyler, 1963; Emery, 1966; Goto et al., 1967; Thompson et al., 1967; Gardner-Medwin et al., 1971), while the E M G test on three different muscles achieved a detection rate of 86.7% Some of the possible carriers were examined both by quantitative E M G and CPK tests, but not by muscle biopsy (see Table 4), whereas other subjects were tested only for CPK and by histological methods (see Table 5). A scrutiny of these tables discloses that one technic often gives positive results when another is negative, therefore all three tests should be performed on each subject in order to maximise the detection rate. Only seven possible carriers were examined by all three tests. Unfortunately they can not be used for a statistical
Table 5. Comparison between biopsy and CPK of possible carriers
Mothers Sisters
Number
Significant % biopsies
High serum % CPK values
18 33
11 15
5 10
61.1 45.4
27.8 30.3
Quantitative EMG and Histological Carrier Detection
247
comparison, not only due to their small number, but also because of bias in sampling (as mentioned above, for the last two years suspected carriers were sent to us by the regional Committee for MD only if they had normal CPK levels). According to Perry and Fraser (1973) "a negative result from a test with a 20% false negative rate is not likely to reduce the odds of being a carrier to a level where most women would consider having a family without prenatal sex determination and abortion of all males". This means that technics with a very high detection rate are necessary to allow a suspected woman to set up a family without too much worry about the future of her offspring. We think that the CPK estimation, quantitative E M G and muscle biopsy combined can attain almost complete detection of the carrier state. Indeed we believe that with these three technics there is more risk of defining a healthy woman as a carrier than of missing a carrier. The serum CPK estimation is the most common test for carrier detection. However muscle fatigue or sporadic accidents such as muscle contusions, latent flogosis, intramuscular injection and others, sometimes unknown at the moment of the test, cause variations in serum CPK activity and make the test not quite specific. The E M G test is easily borne by adult subjects and it is not really painful; it enables one to test more muscles and different areas of each muscle. Muscular changes in the carriers are usually slight, and a detailed E M G examination, such as manual quantitative EMG, is needed. E M G methods, based only on the visual evaluation of the potentials and of the interference pattern on the screen of the oscilloscope, are often tricky and insufficient. The muscle biopsy supplies a further possible aid in the diagnosis of the carrier state, as is stressed by Dubowitz and Brooke (1973); in fact it shows clear changes e v e n if serum enzyme levels are normal. In conclusion, for the carrier detection of Duchenne muscular dystrophy, we suggest estimating serum CPK activity first; whenever this test gives a normal result, it is possible to confirm the carrier state by quantitative EMG, performed on three muscles. When the E M G test is also normal, we recommend muscle biopsy. We believe that these three technics combined form a rather close sieve which allows genetically suspected women to set up a family, when all three tests are normal.
References Alberts, M. C., Samaha, F. J.: Serum pyruvate kinase in muscle disease and carrier states. Neurology 24, 462--464 (1974) Askanas, W.: Life Science 5, 1767 (1966) Bradley, W. G., Gardner-Medwin, D.,, Haggith, J., Walton, J. N., Hesp, R.: Duchenne muscular dystrophy. Use of rubidium chloride Rb 86 in the detection of carriers of the gene. Arch. Neurol. 25, 193--197 (1971) Buchthal, F.: An Introduction to Electromyography. Copenhagen: Glyndal 1957 Buchthal, F., Rosenfalk, P.: Action potential parameters in different human muscles. Acta psychiat, neurol, scand. 30, 125--131 (1955) Caruso, G., Buchthal, F.: Refractory period of muscle and electromyographic findings in relatives of patients with muscular dystrophy. Brain 88, 29--50 (1965)
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Chung, C. S., Morton, N. E., Peters, H. A.: Serum enzymes and genetic carriers in muscular dystrophy. Amer. J. Hum. Genet. 12, 52--66 (1960) Danieli, G. A., Angelini, C.: Duchenne carrier detection. Lancet 10, 86--89 (July 1976) Dreyfus, J. C., Shapira, G., Shapira, F., Demos, J.: Nature of the carrier state in progressive muscular dystrophy. Muscle diseases, eds. J. N. Walton, N. Canal, G. Scarlato, pp. 417--423. Amsterdam: Excerpta Medica 1970 Dubowitz, V., Brooke, M. H.: Muscle Biopsy: a Modern Approach. Philadelphia: Saunders 1973 Emery, A. E. H.: Clinical manifestations in two carriers of Duchenne muscular dystrophy. Lancet 1963 I, 1126--1128 Emery, A. E. H.: Electrophoretic pattern of lactic dehydrogenase in carriers and patients with Duchenne muscular dystrophy. Nature 201, 1044--1045 (1964) Emery, A. E. H., Schelling, J. L.: Limb blood flow in patients and carriers of Duchenne muscular dystrophy. Acta Genet. 15, 337 (1965) Emery, A. E. H.: Muscle histology in carriers of Duchenne muscular dystrophy. J. Med. Genet. 2, 1 - - 7 (1965) Emery, A. E. H.: The use of serum creatine kinase for detecting carriers of Duchenne muscular dystrophy. A. T. Milhorat, Exploratory Concepts in Muscular Dystrophy and Related Disorders. International Congress Series, No. 147, Excerpta Medica, 90--96 (1966) Emery, A. E. H.: The use of serum creatine kinase for detecting carriers of Duchenne muscular dystrophy. J. Med. Genet. 3, 92 (1967) Gardner-Medwin, D.: Some problems encountered in the use of electromyography in carrier detection. Proc. 4th Symposium on Research in Muscular Dystrophy, pp. 420---432. London: Pitman 1968 Gardner-Medwin, D.: Studies of the carrier state in the Duchenne type of muscular dystrophy. J. Neurol. Neurosurg. Psychiat. 31, 124--134 (1968) Gardner-Meclwin, D.: The use of serum creatine kinase and electromyography in carrier detection. Muscle diseases, eds. J. N. Walton, N. Canal, G. Scarlato, pp. 431--438. Amsterdam: Excerpta Medica 1970 Gardner-Medwin, D., Pennington, R. J., Walton, J. N.: The detection of carriers of X-linked muscular dystrophy genes. J. Neurol. Sci. 13, 449--454 (1971) Goto, I., Peters, H. A., Resee, H. H.: CPK in neuromuscular disease. Patients and families. Arch. Neurol. 16, 529--535 (1967) Hausmanova-Petrusewicz, I., Prot, J., Niebroj-Dobosz, I., Hetnarska, L., Emeryk, B., Wasowicz, B., Askanas, W., Slucka, C.: Studies of healthy relatives of patients with Duchenne muscular dystrophy. J. Neurol. Sci. 7, 465--480 (1968) Hughes, B. P.: Serum enzyme studies special reference to the Duchenne type. Research in Muscular Dystrophy, Proc. 2nd Symp. on Current Research in Muscular Dystrophy, 167 (1963) Ionasescu, V., Zellweger, H., Conway, T. W.: A new approach for carrier detection in Duchenne muscular dystrophy: protein synthesis of muscle polyribosomes in vitro. Neurology 21, 703--709 (1971) Ionescu, V., Radu, H., Nicolescu, P.: Identification of Duchenne muscular dystrophy carriers. Electron microscopical investigation of skeletal muscle. Arch. Pathol. 99,436--441 (1975) Iwamoto, A.: Studies on serum creatine phosphokinase of various diseases in orthopedic surgery. Acta Med. (Fukuoka) 39, 108--121 (1969) Jacobs, K.: Carrier detection in the Duchenne type muscular dystrophy: preliminary observation on the place of electromyography. J. Neurol. Sci. 6, 347--356 (1968) Johnston, H. A., Wilkinson, J. H., Withycome, W. A., Raymond, S.: Alpha-hydroxybutyrate dehydrogenase activity in sex-linked muscular dystrophy. J. Clin. Path. 19, 250 (1966) Kowalewski, S., Rotthauwe, H. W., Molbert, E., Mumenthaler, M.: Female carriers of muscular dystrophy. Lancet 1966 I, 1216--1217 Mannucci, P. M., Ideo, g., Cao, A., Macciotta, A.: Gli isoenzimi della latticodeidrogenasi (L.D.H.) e della transaminasi glutammico (T.G.O.) nel muscolo fetale, adulto e di soggetti affetti da distrofia muscolare progressiva tipo Duchenne. Rass. Med. Sarda 68, 287 (1965) Moosa, A., Brown, B. H., Dubowitz, V.: Quantitative electromyography: carrier detection
Quantitative EMG and Histological Carrier Detection
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