Neut,mu.,cularDuoed~'rs,Vol.I. No, 3. pp. 177-183.1901 Pnnted tn Gntat Bntam

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DYSTROPHIN IMMUNOFLUORESCENCE PATTERN IN MANIFESTING AND ASYMPTOMATIC CARRIERS OF DUCHENNE'S AND BECKER MUSCULAR DYSTROPHIES OF DIFFERENT AGES M. VAINZOF.*'I"R. C. M. PAVANELLO,* I. PAVANELLO,* A. M. TSANACLIS,~ J. A. LEVY,** M. R. PASSOS-BUENO*,D. RAPAPORT* and M. ZATZ* Departamento de Biologia.* lnstituto de Bioci~neias and ~.Faculdade de Medicina, Universidade de $5.o Paulo, S~io Paulo. Brazil

(Received 12 November 1990: accepted 25 February 1991)

Abstract--In order to investigate if the same apparent decrease in dystrophin negative fibers with aging observed in mouse mdx female heterozygotes also occurs in carders of the DMD and BMD gene, we have studied the muscle of 29 DMD carriers (19 adults and 10 young daughters of obligate carriers, including 3 manifesting carriers) and 5 adult asymptomatic heterozygotes for Becket dystrophy (BMD). All young DMD possible carders and ! 1 of 24 adult DMB/BMD heterozygotes had increased serum enzymes activities. A population of dystrophin negative fibers, more evident with the use of the C-terminal antibody, was seen in the three manifesting and in a 9-yr-old possible DMD carrier. In the remaining females, a positive immunohistochemical pattern of dystrophin, which did not differ from normal controls, was observed. Our results suggest that: (I) the increased population of dystrophin negative fibers reported in young mdx female heterozygotes was not seen in young DMD carriers, aged 6-17 yr; and (2) abnormalities in dystrophin immunostaining are not easily observed and are more frequent in manifesting carriers, when the muscle is grossly altered. Key words: Dystrophin, immunohistochemistry, DMD/BMD carriers, muscular dystrophy,

Duchcnne, Becket. This protein has been localized to the sarcolemma of normal muscle fibers by immunofluorescence microscopy [I 3-15]. Dystrophin is also absent in the mdx male mouse, while investigations in heterozygote mdx females showed a mixed population of dystrophin negative, partially positive and totally positive fibers in young animals, but very few negative fibers in older females [I 6]. lmmunohistochemical analysis of dystrophin in muscle biopsies from D M D carriers also showed a population of dystrophin-deficient fibers [17,18]. However, such studies have been carried out predominantly in symptomatic adult heterozygotes and little is known about dystrophin immunohistochemistry in young D M D carriers or adult heterozygotes with no clinical manifestation. In the present investigation we have analysed immunohistochemieally the presence of dystrophin through the use of N-terminal and Cterminal antibodies, in a large sample of obligate carriers of the DMD and BMD genes, with and without increased serum enzyme activities

INTRODUCTION

Duchenne muscular dystrophy (DM D) is an Xlinked recessive progressive, degenerative myopathy, with an incidence of approximately I in 3500 male births [I]. Becker muscular dystrophy (BMD), allelic to DMD [21 is a milder form of X-linked muscular dystrophy, with an incidence of about I in 30,000 male births [3]. D M D / B M D patients may arise from new mutations, or germinal mosaicism [4,5] but the majority are inherited from carrier mothers [6,7]. Serum creatine-kinase (CK) and pyruvate kinase (PK) activities are grossly elevated in all affected patients and slightly increased in about 70% of D M D / B M D carriers [6,8,9]. According to Maunder-Sewry and Dubowitz [10] about 70-75°/, of the carriers have some histopathoiogical muscle abnormality. The absence or a defect of the protein dystrophin is responsible for the severe phenotype of DMD or the milder form of BMD [11,12]. t Author to whom correspondence should be addressed. 177

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as compared with young DMD heteroz}gotes (sisters of affected patients) withgrossly elevated serum enzymes. Three females (one young girl and two adults) had muscular weakness. The purpose of this study was: (I) to investigate if there is a decrease in dystrophin negative fibers with aging in carriers of the DMD gene; and (2) to verify if there is a correlation of muscle immunodystrophin pattern with histological findings with serum enzyme levels. PATIENTS AND METHODS

The present investigation included muscle biopsies (biceps) from: 18 DMD obligate carriers (aged 23-43 yr, including I manifesting); il DMD probable carriers (9 asymptomatic and I manifesting young daughters of an obligate carrier aged 6-17 yr; I symptomatic mother of isolated case, aged 39 yr); 5 BMD obligate carriers (aged 32-58 yr) 3 of them related to affected males in which an additional degradation fragment of dystrophin (230-250 kd) was detected through Western blot analysis. Muscles from normal controls were obtained from individuals without any history of neuromuscular disorder who were submitted to orthopaedic surgery. All studies were done after informed written consent. Patients' mothers were classified as obligate carriers if they had at least one child with muscular dystrophy and another affected relative through maternal lines. Sisters of affected patients or mothers of isolated cases, all with elevated serum enzyme activities (1.3-120-fold) were classified as probable carriers. All samples were frozen in liquid nitrogen immediately after removal and stored at - 70"C until use. Dystrophin immunohistochemistry was done as reported previously [19]. The primary antibodies used were the original polyclonal 30 kd + 60 kd cardiac dystrophin raised in sheep [12] and antibody 1461, raised in rabbit, against the 17 last amino acids of dystrophin (by Dr E. E. Zubrzycka-Gaarn). Both antibodies were used concomitantly in all samples. The second antibodies used were: biotinylated anti-sheep and Texas Red (Amersham) and FITC conjugated anti-rabbit (Sigma). The sections were analysed and photographed with a Zeiss photomicroscope with epi-illumination. Additional sections were stained with hematoxylin and eosin (HE) and myofibrillar

ATPases (pH 9.4, 4.6 and 4.3 [20]) to evaluate in a blind test the presence of morphologic and morphometric alterations. For each subject, the smaller diameters of 200 fibres were measured, using a computer morphometric program, and the differences of mean diameter between type I and II fibers were estimated. In addition, the proportion of type I and lI fibers, the total Variation Coefficient (VC) and type I and II fibers diameter VC were calculated. Serum CK was determined with Sigma kits, according to the colorimetric procedure 520-C published by Sigma [21]. Serum PK was measured according to methodology described previously [8]. In all cases, enzyme determinations were repeated three times and the mean of the three results was considered.

Fig. 1. Pedigreesof the manifestingcarriers. (A) Case I, (B) case 2, (C) case 3.

DESCRIPTION OF MANIFESTING CARRIERS

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This female, aged 32 yr, is an obligate carrier of the DMD gene [Fig. I(A)]. On clinical examination she showed hypertrophy of the calves, difficulties for climbing stairs and she performs Gowers manoeuvre to raise up from the floor. Her serum enzymes are increased 13-fold. Histopathology analysis of her muscle biopsy showed an early myopathic process.

Dystrophin in D M D BMD Carners

Patient 2 This 5-yr-old girl was referred to our center with parents' complaint of frequent falling and a positive family history of D M D [Fig. I(B)]. On clinical examination she had calves" hypertrophy but no visible weakness. She was able to jump and run freely. Elcctromyography was apparently normal but her serum enzymes were increased 120-fold while her mother and younger sister have slightly elevated serum activities (!.5 and 3-fold. respectively). Karyotypc analysis showed no chromosomal abnormality and her muscle biopsy showed discrete alterations characteristic of a myopathic process.

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Pat&'nt 3 This 39-yr-old woman was referred to our center because she has a D M D son, currently 8yr-old, showing a severe progression o f the dystrophic process who has been confined to a wheelchair since the age of 7 yr. He is the only case in the family [Fig. I(C)]. On clinical examination she showed promincnt calves and a significant muscle weakness. She is unable to stand from a chair, to climb stairs or to perform Gowers manoeuvre. Her serum enzymes activities were increased by 5-1k)ld and her muscle biopsy showed a typical myopathic process, with a predominance of type I tibcrs. RESULTS

ilistopathoh~gy Man(]i'sting carr&rs. Muscle analysis from the three manifesting carriers showed histopathological alterations typical of a myopathic process, which were more pronounced in the one who is clinically more severely affected too (case 3). A significant difference in types I and II fiber diameter was seen in subject i while the other two showed a predominance of type I fibers. A VC of fiber diameter greater than 25% was observed in all three females. Non-man~'sthlg carriers. Ten among the seventeen obligate asymptomatic DM D carriers and two of the five BMD carriers had increased serum CK and/or PK (I.5 5-fold) while the remaining had normal serum enzyme activities. The histopathological analysis revealed discrete alterations typical of a myopathic process in 70% of DMD-obligate carriers with elevated

3

Fig. 2. Dystrophin immunofluorescence pattern in manifesting carriers. (A) Normld muscle, (B) case 2, (C) case I, (D) case 3.

enzymes, 14% of DMD-obligated carriers with normal enzymes, 38% of young D M D carriers and two of five BMD-obligate carriers, both with normal enzymes (Table I). These included:

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Table I, Summar~ of abnormal h,,ps? tindmgs m 34 D M D BMD t~bl,gate carrier~ (OC) and prt~bable carriers (PC) DMD-OC Normal enz. Ele,,. enz. N=7 N = I0

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Fbl~ll Ap Ill VC 20-25%

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4 8 8

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6 7 4

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GHA--Global histopathological alterations. Fb 1=:II--Statistically significant difference (P < 0.05) bctwcen types 1 and II fibcr diameter, Ap l li--Altered proportion of t.~pes I and I1 tibers. VC--Variation coefficient.

Fig. 3. Ninc-yr-old D M D - P C showing a dystrophin mosaic pattern. (A) N-tcrnfinal antibodies, (B) (_'-terminal antibody.

variations in fiber size, presence o f split fibers, increased numbers o f internal nuclei, scattered degenerating and regenerating fibers and increased connective tissue. M o r p h o m e t r i c analysis showed: a significant difference between mean fibers I/Ii in 52% o f the cases; a predominance o f type i fibers in 74%; a 2 0 - 2 5 % VC in 61% and greater than 25% in 10% o f the subjects.

Dystrophin analysis Man(/bsting carriers. In the majority of fibers dystrophin immunohistochemistry showed a positive continuous reaction a r o u n d all sarcolemma (Fig. 2). Some regions with totally (24, 16 and 3%, cases I, 2 and 3, respectively) or partially negative labelling (6.4, 4.9 and 4.2%) were observed in the three females with no apparent correlation with age or clinical weakness.

Non-man(['estin.g carrh'rs. Only one 9-yr-old girl showed an immunolluorescence dystrophin mosaic pattern (with 4.5% o f negative or partially labelled fibers) which was more evident with the C-terminal antibody (Fig. 3). With the exception o f this girl, all other subjects showed a positive i m m u n o h i s t o c h c m ical pattern for dystrophin which did not differ from normal controls. A continuous and positive reaction was seen a r o u n d the sarcolemma in the majority o f fibers with no difference in y o u n g as c o m p a r e d with older carriers (Fig. 4). A partial ktbelling was observed in some scattered fibers but this was seen also in normal muscle suggesting an artefact o f the technique. I)l~ft

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Histopathological studies in muscle from D M D carriers have been widely investigated

Dystrophin in D M D B M D Carriers

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l:ig. 4. D)strophin immunolluorescence pattern in (A) D M D - O C with normal serum enzymes, (B) D M D - O C with elevated serum enzymes. (C) young D M D - P C , (D) B M D - O C with elevated serum enzymes.

before the identification of dystrophin as the product of the DM D/BM D gene. In a study of 65 females at risk for DMD, Maunder-Sewry and Dubowitz [10] reported that only I1% of the muscle biopsies were qualitatively abnormal. However, the majority of them showed at least one abnormal finding when compared with normal controls and no correlation was observed between histological abnormalities and serum CK activity suggesting that the analysis of muscle biopsies was useful for increasing the rate of carrier detection. The histopathological findings more frequently found in females from the present study ~ere similar to the one observed by these investigators and ,,vc did not observe either any

correlation between such findings and serum enzyme activities. Several reports have been published recently on dystrophin studies in females at risk for D M D and in mdr female heterozygotes. Bonilla et al. [17], in a study of !1 D M D obligate carriers or probable carriers (5 with clinical signs) observed a proportion of fibers (18.2-32.2%) with partial dystrophin labelling in the five manifesting carriers. Among those with no clinical signs, fibers with patchy immunostaining were absent or infrequent (from 0 to 9.1% ). They suggested that more subjects should be studied before drawing any conclusion about the potential use of such a test for improving the rate of carrier detection.

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Arahata et al. [18] compared immunod~strophin and histopathologic findings in three manifesting carriers and observed a population of dystrophin-ncgative or partial labelling fibers (40.6 73.6'!i,). According to these authors. dystrophin assessment might represent a good test lbr carrier detection. Based on dystrophin analysis in clonal myoblasts derived from one D M D carrier, Hurko el a/. [22] also suggest that somatic cell testing of dystrophin cxpression may offer an alternative to established genetic carrier tests. Rcsuhs from the present investigation do not support these suggestions since abnormal dystrophin findings were observed mainly in the manifesting carriers. In thc two adult females, in opposition to the expected, a smaller proportion of negative fibers was seen in the more severely alrected one, which might be a consequence o f m u s c l e fiber degeneration and clintination dt, ring the dystrophic process. In the yot, nger manifesting carrier, serum CK levels wcrc in the same range as those of I ) M D affected boys of comparable age, so a greater proportion of negative fibers should bc expected, which was not seen. tlowevcr, the severity of her phcnotypc in adulthood cannot bc predicted yet. On the other hand, it should be cons!tiered that muscle biopsy samples may bc representing only focal changes and not the total muscle ntass of the patient. In ,tddition, no consistent alteration was sccn in carriers with elevated serum enzymes, but without clinical signs. When samples from carriers and normal controls were e x a m i n e d in a blind test throt, gh the use of the two antibodies, wc were not able to identify' the hcterozygotcs from the normal controls. In mdx hctcrozygotc mice, Watkins et al. [161 observed a large population of dystrophinnegative fibers which decreased with aging. According to these authors the gradual disappearance of negative libcrs would occur more likely due to an increase in protein synthesis. ttowever, a basic dilrerence between the mouse and human dystrophies is the spontaneot, s liber regeneration which occurs in md.v mice but not in DMD. Our findings suggest that if there is an increased proportion of dystrophin-ncgativc tibers in humans, this might occur in ~cry young heterozygotcs, since, with the exception of one girl who showed minor alterations, no abnormal!tics were found in eight young probable carriers aged 6 17 yr old. all ~ith signilicantly elevated serum enzyme activities.

The fact that the mosaic pattern was more evident with the C-terminal antibody (which does not share homology with any other known cytoskeletal protein) suggests that the use of more specific antibodies might increase the sensitivity for discriminating carriers through this test. An interesting f i n d i n g is t h e d i f f e r e n c e in t h e dystrophin expression pattern in skeletal a s compared with cardiac muscle reported in md.v carrier mice [23,24]. The age related conversion of dystrophin-negative to positivc fiber segments in skeletal muscle was not observed in cardiac muscle in canine X-linked M D as well [25]. According to Karpati et al. [24], such a finding may be partially due to a spontaneous fusion of dystrophin-competent satellite cells into the dystrophin-negative fiber segments and partly to an expansion of the cytoplasmic domain of dystrophin expression related to the original myonuclei, observed only in skeletal muscle, since cardiac muscle lacks satellite cells. In conclusion our data suggest that although the elevation in serum enzyme activities is an indication of Icakage from muscle probably due to an abnormality in its membrane, dystrophin immunohistochenaistry studies are not sensitive enough to detect minor alter;ttions and therefore do not represent a reliable test I\~r carrier detection in DM D/BM D carriers. Dystrophin quantitation, through Western blot analysis might be of interest, although it is tmlikely that small differences might be detected through such a test. Wc are extremel,, grateful Io Drs E. P. tloffman. L. M. Kt,nkel and R. Worton for their generous gift of anti-dystrophin antibodies and f~r their critical conunents and suggestions. We wish to thank all the ph}sicians for referring patients; to Mrs Maria Canovas, Rober[o $chreibcr, Martha A. B. C Lima and Angck, Levy, l\~r technical assistance" to Marcia L. Nevcs and Thais Zago lot secretarial assistance and to Dr J. M. Amabis, Dr A. WajntaL Dr C. Koiffmann, l)r Y. Yassuda. Dr ('ht,-Tien tlsi, E. (iorab, M. A. Xavier, l)r L. C. C; Simtcs. Dr P. l_eite and to the Department of tlistology and Emhriology of the University of S;]o Paulo. for lahorat~ry I'acililics. This work was supported b} grant~, from Fundaqfit~ de A m p a r o ~ Pesquisa do Estado de $5o Paulo ~FAPESP}. Consclho National de Desenvolvimcnto CicntilJco e Tcchnol6gico {CNPq}. Financiadora dc Esludos e Projetos (I:INEP}, Secretaria da Ci~nda c Tecnologia de Silo Paulo and Ass~ciagfio Brasileira de Distrofia Muscular {ABDIMI. ..lcknowh,dgemcnt.~

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Dystrophin in DMD/BMD Carriers 2.

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14. Zubrzycka-Gaarn E E° Bulman D E. Karpati G. et al. The Duchenne muscular dystrophy gene product is localized in sarcolemma of human skeletal muscle. Nature 1988: 333: 466--469. 15. Arahata K. Ishiura S, lshiguro T, et al. Immunostaining of skeletal and cardiac muscle surface membrane with antibody against Duchenne muscular dystrophy peptide. Nature 1988: 333: 861-863. 16. Watkins S C, Hoffman E P, Slayter H S, Kunkel L M. Dystrophin distribution in heterozygote MDX mice. Muscle Nerve 1989: 12: 861-868. 17. Bonilla E. Schmidt B. Samitt C E. et al. Normal and dystrophin-deficient muscle fibres in carriers of the gene for Duchenne muscular dystrophy. Am J Pathol 1988: 133: 440-445. 18. Arahata K. lshihara T, Kamakura K, et al. Mosaic. expression of dystrophin in symptomatic carriers of Duchenne's muscular dystrophy. N Engl J Med 1989~ 320: 138-142. 19. Vainzof M, Pavanello R C M. Pavanello I. et al. Dystrophin immunostaining in muscles from patients with different types of muscular dystrophy: a Brazilian study. J Neurol Sci 1990: 98:221-233. 20. Dubowitz V, (ed.). Muscle Biopsy: A Practical Approach. London: Bailli~re Tindall, 1985. 21. Sigma Technical Bulletin. Creatine phosphokinase (CPK) in serum or plasma. St. Louis. Missouri: Sigma Chemical Company 1985; 520:12-76. 22. Hurko O. Hoffman E P. McKee L, Johns D R, Kunkel L M. Dystrophin analysis in clonal myoblasts derived from a Duchenne muscular dystrophy carrier. Ant J Hum Gem't 1989: 44: 820-826. 23. Tanaka H. Ikeya K. Ozawa E. Difference in the expression pattcrn of dystrophin on the surface mcmbranc between the skeletal and cardiac muscles of mdx carrier micc. lli.stochemistry 1990: 93: 447--452. 24. Karpati G, Zubrzycka-Gaarn E E. Carpenter S, Bulman D E. Ray P N. Worton R G. Agc-rclated conversion of dystrophin-negative to -positive fiber scgmcnts of skclctal but not cardiac musclc fibres in hctcrozygote mdx mice. J Neurpath Exp Neurol 1990: 49: 96-105. 25. Cooper B J, Gallaghcr E A. Smith C A. Valentine B A. Winand N J. Mosaic cxprcssion on dystrophin in carriers of canine X-linked muscular dystrophy. Lab Invest [990: 62: 171-178.