CARRIER DETECTION OF DUCHENNE AND BECKER
MUSCULAR DYSTROPHY USING MUSCLE DYSTROPHIN
IMMUNOHISTOCHEMISTRY
ACARY S. BULLE OLIVEIRA * — ALBERTO A. GABBAI * — BENY SCHMIDT ** BEATRIZ HITOMI KIYOMOTO * — J. G. CAMARGO LIMA *
CARLO MINETTI *** — EDUARDO BONILLA ***
S U M M A R Y — T o a s c e r t a i n w h e t h e r d y s t r o p h i n i m m u n o h i s t o c h e m i s t r y c o u l d i m p r o v e D M D / B M D c a r r i e r d e t e c t i o n , w e a n a l y z e d 1 4 m u s c l e b i o p s i e s f r o m 1 3 D M D a n d o n e B M D p r o b a b l e a n d p o s s i b l e c a r r i e r s . A l l w o m e n w e r e a l s o e v a l u a t e d u s i n g c o n v e n t i o n a l m e t h o d s , i n c l u d i n g g e n e t i c a n a l y s i s , c l i n i c a l a n d n e u r o l o g i c a l e v a l u a t i o n , s e r u m C K l e v e l s , K M G , a n d m u s c l e b i o p s y . I n 6 c a s e s , t h e r e wias a m o s a i c o f d y s t r o p h i n - p o s i t i v e a n d d y s t r o p h i n - d e f i c i e n t f i b e r s t h a t a l l o w e d t o m a k e t h e d i a g n o s i s o f a c a r r i e r s t a t e . C o m p a r i n g d y s t r o p h i n i m m u n o h i s t o c h e m i s t r y t o t h e t r a d i t i o n a l m e t h o d s , i t w a s n o t e d t h a t t h i s m e t h o d i s l e s s s e n s i t i v e t h a n s e r u m C K m e a s u r e m e n s , b u t i s m o r e s e n s i t i v e t h a n E M G a n d m u s c l e b i o p s y . T h e u s e o f d y s t r o p h i n i m m u n o -h i s t o c -h e m i s t r y i n a d d i t i o n t o C K , E 1 M G a n d m u s c l e b i o p s y i m p r o v e d t -h e a c c u r a c y o f c a r r i e r d e t e c t i o n . T h i s m e t h o d i s ( a l s o h e l p f u l t o d i s t i n g u i s h m a n i f e s t i n g D M D c a r r i e r s f r o m p a t i e n t s w i t h o t h e r n e u r o m u s c u l a r d i s e a s e s l i k e l i m b - g i r d l e m u s c u l a r d y s t r o p h y a n d s p i n a l m u s c u l a r a t r o p h y .
K E Y W O R D S : m u s c u l a r d y s t r o p h y ( D u c h e n n e a n d B e c k e r ) , c a r r i e r d e t e c t i o n , d y s t r o p h i n .
Detecção de portadora» de distrofia muscular de Duchenne e de Becker utilizando imuno-hi*-toquímica com distrofina
R E S U M O — P a r a d e t e r m i n a r s e i m u n o h i s t o q u í m i c i a c o m d i s t r o f i n a p o d e r i a m e l h o r a r a d e t e c -ç ã o d e p o r t a d o r a s d e d i s t r o f i a m u s c u l a r d e D u c h e n n e ( D M D ) e d e B e c k e r ( D M B ) , a n a l i s a m o s 1 4 b i ó p s i a s m u s c u l a r e s d e 1 3 p o r t a d o r a s p r o v á v e i s o u p o s s í v e i s d e D M D e d e u r n a p o r t a d o r a p r o v á v e l d e D M B . T o d a s a s m u l h e r e s f o r a m t a m b é m a v a l i a d a s u s a n d o m é t o d o s c o n v e n c i o n a i s , i n c l u i n d o a n á l i s e g e n é t i c a , a v a l i a ç ã o c l í n i c a e n e u r o l ó g i c a , n í v e i s s é r i c o s d e C K , E M G e b i ó p s i a m u s c u l a r c o m e s t u d o h i s t o q u í m i c o . E m 6 c a s o s h a v i a u m m o s a i c o d e f i b r a s m u s c u l a r e s d i s t r o f i n a p o s i t i v a s e d i s t r o f i n a n e g a t i v a s , q u e p e r m i t i a c a r a c t e r i z a i * u m e s t a d o d e p o r -t a d o r a . C o m p a r a n d o i m u n o - h i s -t o q u í m i c a c o m d i s -t r o f i n a e m é -t o d o s -t r a d i c i o n a i s , n o -t o u - s e q u e e s t e m é t o d o é m e n o s s e n s í v e l q u e m e d i d a s d e C K , m a s é m a i s s e n s í v e l q u e E M G e b i ó p s i a m u s c u l a r . O u s o d e s t e m é t o d o a s s o c i a d o a C K , E M G e b i ó p s i a m u s c u l a r a u m e n t o u t a p o s s i -b i l i d a d e d e d e t e c ç ã o d e p o r t a d o r a s . E s t e m é t o d o é t a m -b é m ú t i l p a r a d i s t i n g u i r p o r t a d o r a s m a n i f e s t a n t e s d e D M D d e p a c i e n t e s c o m o u t r a s d o e n ç a s n e u r o m u s e u l a r e s c o m o a d i s t r o f i a c i n t u r a - m e m b r o s e a m i o t r o f i a e s p i n h a l p r o g r e s s i v a ,
P A L A V R A S - C H A V E : d i s t r o f i a m u s c u l a r ( D u c h e n n e e B e c k e r ) , d e t e c ç ã o d e p o r t a d o r a s , d i s t r o f i n a .
* D e p a r t e m e n t o f N e u r o l o g y , N e u r o s u r g e r y a n d E x p e r i m e n t a l N e u r o l o g y o f E s c o l a F t a u -l i s t a d e M e d i c i n a ( E P M ) , S ã o P a u -l o ; * * A d j u n c t P r o f e s s o r , D e p a r t m e n t o f P a t h o -l o g y , E P M ; * * * D e p a r t m e n t o f N e u r o l o g y , C o l l e g e o f P h y s i c i a n s a n d S u r g e o n s o f C o l u m b i a U n i v e r s i t y , N e w Y o r k . S u p p o r t e d p a r t i a l l y b y t h e B r a z i l i a n R e s e a r c h C o u n c i l ( C N P q ) .
The detection of Duchenne (DMD) and Becker (BMD) muscular dystrophy
carriers is one of the major goals in the prevention of these diseases. Traditional
methods of carrier detection have a low sensitivity and are not specific
Ana-lysis of family pedigrees, neurological evaluation, serum creatine kinase (CK)
levels, eleetromyogram (EMG), and muscle biopsy, alone or in combination, lead
to a maximum 80% accuracy
2. Although highly specific, DNA analysis to detect
deletions in the dystrophin gene has not 100% sensitivity
To evaluate the sensitivity of dystrophin immunohistochemistry, we studied
14 possible and probable carriers of DMD and BMD in combination with the
other traditional methods.
PATIENTS A N D METHODS
We studied 14 women, 3 probable and 11 possible DMD (only one a BMD) carriers, aged 6 to 37 years. Thei diagnosis of D M D and BMD in the affected members of tho families, was made according to established criteria 4.
Probable carrier was. defined as a woman with two or more affected sons. Possible carrier was defined as a woman with only one affected son and no other male affected in her fiamily; or a woman without affected son but with affected male(s) in her family 9. Manifesting carrier (or symptomatic carrier) was defined as a carrier with proximal muscle weakness.
All carriers were examined neurologically including a manual muscle test 7, most of them on several occasions. Laboratory tests included electrocardiogram (ECG), serum CK levels, EMG, and muscle biopsy with histochemistry and dystrophin immunostain.
Muscle biopsies were frozen in liquid nitrogen and processed according to standard criteria 3. For immunohistochemistry, the muscle samples were processed by one of us (EB), in New York, following a previously published technique
RESULTS
The clinical history, genetic aspects, clinical manifestations and neurological examina-tion of the 14 patients are summarized in Table 1. Cases 3 and 5 with proximal limb weak-ness were manifesting' carriers of DMD. Case 14 was a B M D carrier.
ECG, EMG, muscle biopsy results and serum CK levels are summarized in Table 2.
Immunohistochemistry with dystrophin was performed in all 14 women.
Normal immunostaining of the muscle fibers (a thin and continuous layer of immuno-fluorescence at the sarcolemma of the fibers) was seen in 8 (cases 6, 7, 8, 9, 10, 11, 12 and 13) (Fig. 1).
In 8 muscle, biopsies (cases. 1, 2, 3, 4, 5 and 14) the dystrophin immunostaining pattern was altered evidencing a deficiency of dystrophin at the sarcolemma either in groups cr in isolated muscle fibers. In these same patients there remained, nonetheless, a population of fibers that showed a normal immunostaining. Comparing serial sections stained with ATPase 9.4, it was seen that the immunostaining abnormalities were randomly distributed not par-ticularly associated to either type I or II fibers.
The three cases of asymptomatic carriers (cases 1, 2 and 4) showed lack of immunos-taining in muscle fibers isolatedly disposed (Fig. 2). The two cases of manifesting carriers of D M D (cases 3 and 5) and the asymptomatic B M D carrier showed a marked reduction in the immunostaining at the sarcolemma of fibers, with immunostaining reduced in groups of muscle fibers (Fig. 3).
Two (cases 5 and 14) of the 6 cases with abnormal immunostaining were probable car-riers and 4 (cases 1, 2, 3 and 4) were possible carcar-riers. The immunostaining aspect in case 3, a possible carrier, was indistinguishable from that seen in the two probable carriers (ca-ses 5 and 14). These ca(ca-ses showed lack of immunostaining in groups of muscle fibers. The other three cases (cases 1, 2 and 4) possible carriers showed lack of immunostaining in a few isolated muscle fibers.
COMMENTS
Immunohistochemistry with dystrophin allowed us to make the diagnosis
of definite DMD and BMD carrier status in women with a familial history of
DMD or BMD.
The findings consisted of decreased in the immunostaining at the
sarco-lemma of the muscle fibers isolatedly disposed in 3 patients, and a marked
reduction in the immunostaining in groups of muscle fibers in other 3. The
immunostaining with dystrophin in our cases did not show differences between
type I and II fibers, suggesting that pattern expression of dystrophin is similar
in the two types of muscle fibers.
We found two populations of muscle fibers in the carriers, one containing
normal dystrophin and another with dystrophin deficiency. The proportion of
deficiency of dystrophin was higher in two manifesting carriers of DMD than
in other three asymptomatic carriers. Various clinical, histological and
bioche-mical manifestations in carriers have been explained in terms of the Lyon
hypo-thesis
8. Our findings do not contradict this assumption.
If a DMD carrier has few dystrophin-deficient fibers, she may have only
subclinical muscular dystrophy, whereas if she has numerous defective fibers,
she may be a manifesting carrier.
The immunohistochemmical pattern in the asymptomatic BMD carrier, with
numerous defective fibers, was indistinguishable from that seen in two
mani-festing carriers of DMD. It is possible that in this patient the structural
dys-trophin alteration was not severe enough to allow the disruption of the fibers
and consequent weakness, but was enough to be detected by the anti-dystrophin
antibody we used. A similar phenomenon has recently been reported in a family 5.
The comparison of the results between dystrophin immunohistochemistry
with other traditional methods for the detection of DMD and BMD carriers has
shown:
(1) Calf enlargement was not a good sign to make the diagnosis of definite
carrier. Three cases with dystrophin-deficient fibers did not have calf
enlarge-ment, and two cases with calf enlargement had normal dystrophin immunostain
in muscle fibers (Table 6a).
(2) The serum CK level was more sensitive for detecting the carrier state than
dystrophin immunohistochemistry (Table 6b).
(3) Dystrophin immunohistochemistry was more sensitive than EMG and muscle
biopsy with histochemistry. All cases that had myopathic changes in these exams
showed dystrophin-deficient fibers, and two cases with dystrophin-deficient fibers
had normal EMG and muscle biopsy (Tables 6c and 6d).
(4) The arrangement of CK, EMG, and muscle biopsy allowed us to detect 8
carriers of DMD and BMD out of 14. The use of dystrophin
immunohistoche-mistry improved the accuracy of the genetic counseling. One case, that was not
discriminated as a carrier using traditional methods, showed dystrophin-deficient
fibers, allowing us to identify the carrier state in this case (Tables 5 and 6e).
(5) One woman, case 10, with three sons with DMD, that could be classified
as obligatory carrier did not show any change in the immunostaining with
dystrophin. This result can be interpreted as a false negative. In our previous
publication!, one obligatory carrier also did not show any dystrophin deficiency.
They may be gonadal DMD carriers rather than somatic.
Two of our patients were found to be DMD carriers only after the
demons-tration of dystrophin-deficient fibers in their muscle biopsies. They were referred
to us with the clinical diagnosis of limb-girdle muscular dystrophy (LGD) and
spinal muscular atrophy (SMA). The differential diagnosis between a
mani-festing heterozygote for DMD or BMD and a female case of LGD, both with
the same prevalence in the general population *<> is of great importance, because
of different genetic counseling in these conditions
1 2REFERENCES
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10. Yates JRW, Emery AEH. A population study of adult onset limb-girdle muscular dys-trophy. J Med Genet 1985, 22:250-257.
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