Familial myopathy associated with thrombocytopenia: A clinical and histomorphometric study

Journal of the Neurological Sciences, 1988, 88:55-67

55

Elsevier JNS 03070

Familial myopathy associated with thrombocytopenia: a clinical and histomorphometric study M. Mahon 1, W.J.K. Cumming 2, F. Kfistmundsdottir 1, D.I.K. Evans a and P.A. Carrington 3 IAnatomy, Department of Cell & Structural Biology, Medical School, University of Manchester, ~Neurology, Withington Hospital, and 3Royal Manchester Children's Hospital, Manchester (U.K.)

(Received 29 March, 1988) (Revised, received 28 June, 1988) (Accepted 28 June, 1988)

SUMMARY We describe an unusual vacuolar myopathy with tubular aggregates in a mother and son from a family presenting with a slowly progressive, predominantly limb girdle, weakness and distal upper limb weakness in association with reduced blood clotting ability. To our knowledge this is the In'st report of a familial clinical defect of both muscle and platelets in the same individuals. The possibility that the primary defect may be due to an abnormality of the tubular intramembranous systems in muscle cells and platelet precursors is discussed.

Key words: Familial myopathy; Muscle biopsy; Histochemistry; Morphometry; Thrombocytopenia

INTRODUCTION Congenital or childhood myopathies involving disorders of cellular and intracellular membranes and their associated enzymes are relatively common (Dubowitz 1978; Fardeau 1982; Morgan-Hughes 1982; Banker 1986; Engel 1986). Some of these Correspondence to: M. Mahon,Anatomy,Departmentof Cell & StructuralBiology,Medical School, University of Manchester, Manchester MI3 9PT, U.K.

0022-510X/88/$03.50 © 1988 Elsevier Science Publishers B.V. (BiomedicalDivision)

56 disorders may be systemic and appear in other organs such as liver, heart, or the nervous system (Morgan-Hughes 1982; Pleasure and Bonilla 1982). Furthermore, patients with neuromuscular diseases may also show biochemical or physiological abnormalities of blood cell membranes, particularly with respect to erythrocytes although platelets have also been implicated (Lucy 1980; Yarom et al. 1983; Engel 1986). Surprisingly, few myopathological studies have concerned platelets (see Nicholson et al. 1984; Bornstein et al. 1988) despite the marked structural and functional similarity of platelet and muscle cell membranes and their role in calcium sequestration (White 1972; Resink et al. 1986). We describe here an unusual familial myopathy associated with thrombocytopenia which in itself is uncommon in familial form. Such a clinical association between muscle and platelets has not hitherto been described. We have therefore analysed and compared muscle biopsy specimens from two members of the same family using histochemical, ultrastrnctural and quantitative morphological techniques. The possibility that the primary defect in both tissues may involve the intracellular tubular membranous systems is discussed. The haematological studies are reported in more detail elsewhere (Carrington et al. 1988).

PATIENTS AND METHODS Case reports

Three generations of this family (maternal grandfather, case 3; mother, case 2; proband, case 1) show evidence of a neuromuscular disorder with each of those affected also having thrombocytopenia. The myopathy has predominantly affected the limb girdles and the distal upper limbs, is asymmetric and slowly progressive. It is not associated with pain or myoglobinuria. The bleeding disorder, which manifested as easy bruising and prolonged bleeding following injury, has been described more fully elsewhere (Carrington et al. 1988) and is summarized briefly below. Case 1

This patient (S.H.), the son of case 2 and grandson of case 3, has followed the family history in all respects. He was born in 1971 following a normal pregnancy and had normal motor milestones. His gait became abnormal by the age of 4 years and when examined at age 8 years 4 months he showed an asymmetric limb girdle syndrome (MRC 4 at worst, MRC 1986) with distal weakness involving the intrinsic hand muscles and the anterior tibial group. At that time he had a positive Gowers' sign and brisk tendon reflexes in the lower limbs with flexor plantar responses although tendon reflexes in the upper limbs were normal. In 1981 (age 9 years 2 months) a quadriceps needle muscle biopsy was obtained as described below and in Results. At that time serum creatine kinase was elevated (800 IU/I) and EMG was normal. Because of a tendency to frequent falls he returned to the clinic in 1987 (aged 15 years 8 months). The limb girdle weakness had progressed moderately as had the distal weakness to the stage of clinically significant foot drop bilaterally. In addition, he

57 showed tightening of the right tendo-AchiUes. A second muscle biopsy was obtained as described below and in Results. His platelet count varied from 20 to 60 x 109/1although platelet morphology was normal. Case 2 J.H., the mother of case 1 and daughter of case 3 was born in 1948 at term following a normal delivery. She had normal motor milestones and walked at the age of 12 months. However, by the age of 4 years she displayed an abnormal gait and was unable to skip, by the age of 12 years she had hand weakness. Her symptoms have been asymmetric from the onset with the left side more affected than the right. In adolescence and her early twenties she was aware of slowly progressive lower limb girdle weakness with wasting of the quadriceps muscles. At the time of the family investigation (aged 32 years) she showed moderately severe limb girdle pattern of weakness with distal involvement only in the upper limbs. At this time she had depressed reflexes in the upper limbs and brisk reflexes in the lower limbs with flexor plantar responses. ECG was normal and the only abnormality in the EMG was a high amplitude motor unit with a slightly diminished interference pattern. Her serum creatine kinase was elevated (600 IU/I). A muscle biopsy was obtained from the vastus medialis and is described in Results. Her platelet count ranged from 25 to 80 x 109/1. Case 3 This patient (D.H.), was born in 1925 and has a history of normal motor milestones, walking at the age of 14 months. His parents were concerned about his walking ability about the age of 4 years but no definite diagnosis was made at that time. He was not athletic at school and by his teens was experiencing difficulty walking. This progressed slowly up to his mid-thirties when he was investigated (1960) by the orthopaedic department. At that time he showed weakness of grip in both hands and asymmetric weakness of the lower limb girdle. Investigations showed a normal CSF, raised CK (524 IU/1) and low platelet count (53 x 109/1). A muscle biopsy was performed. He was subsequently lost to follow-up and next seen when his daughter and grandson were investigated (1981). At that time he gave a history of slowly progressive lower limb weakness, affecting the left more than the right, as a consequence of which he had become wheelchair confined in 1978. Examination showed a severe (MRC 3 at worst) symmetrical limb girdle weakness with moderate distal weakness in the upper and lower limbs. Consent was not obtained for a further muscle biopsy. Muscle biopsies Muscle biopsy samples from case 1 (at age 9 years 2 months and 15 years 8 months) were obtained from the lateral portion of the right quadriceps femoris using a needle biopsy technique as previously described (Young et al. 1978; Mahon et al. 1984). Serial transverse 10-/~m thick cryostat sections were prepared for histological and histochemical staining and morphometric analysis. Fascicular architecture, muscle fibre morphology, storage material and enzyme content were assessed from sections stained with haematoxylin and eosin (H&E), Masson's and modified Gomori trichrome,

58 Sudan black B, periodic acid-Schiff (PAS), von Kossa, nicotinamide adenine dinucleotide tetrazolium reductase (NADH-TR), succinate dehydrogenase, acid phosphatase, phosphorylase and non-specific esterase. Muscle fibre types were characterized as type I, IIA, liB, or IIC using myofibrillar adenosine triphosphatase (ATPase) staining at pH 10.4, 4.6 and 4.35 (see Dubowitz (1985) for staining methods and fibre typing). Histological material from cases 2 and 3, and resin-embedded tissue blocks from case 1 were also made available for investigation. The histological composition (connective tissues, blood vessels, nerves, muscle fibres) of biopsy samples from cases 1 and 2 was estimated by point counting methods using trichrome stained sections (HaUy 1964; Weibel 1969). From representative areas of each biopsy the relative frequency of muscle fibre types and pathological features were enumerated. The cross-sectional area, perimeter length and minimum diameter of 200 fibre profiles from each specimen was measured using a Leitz Video Imagan digitising system. For each fibre profde a form factor was calculated from the formula 4 x n x area/perimeter 2 where a form factor of 1.00 represents a circle. Morphometric data was recorded separately for type I and type II fibres. The spatial distribution of the two major fibre types within each specimen was also estimated using the SPAM technique (Mahon and Cumming 1985; Mahon et al. 1987) to provide data on deviations (either clumping or dispersion) from a random distribution.

Statistical analysis Histograms of fibre size distributions were constructed and analysed for normality using the Filliben-Grubbs test (Wainwright and Gilbert 1981), and for skewness and kurtosis (Snedecor and Cochran 1980) and compared using the unpaired Student's t-test. Differences were considered significant at P < 0.05. Variability was described using the coefficient of variation (CV) calculated from standard deviation of the mean/mean × 100.

RESULTS

Qualitative analysis of the muscle biopsies Case 1 (S.H.) The first biopsy obtained at 9 years 2 months was inadequate for histomorphometry (less than 100 muscle fibres present) and provided only qualitative data. This appeared completely normal except for the presence of a few fibres possessing small granular deposits of reaction product on NADH-TR stained specimens. The second biopsy (at age 15 years 8 months) showed normal fascicular architecture and perimysial and endomysial connective tissue content without inflammatory infiltrate. The majority of muscle fibres presented normal polygonal outlines although scattered atrophic and angular fibres were also present, a few of which were extremely basophilic and esterase positive. About 20~o of fibres possessed internal nuclei (Fig. la). Many of the fibres of normal size and shape contained small 'slit-like' centrally located

Fig. 1. Quadriceps muscle biopsy from case 1 (age 15 years 8 months). Bar line 50 #m. (a) H&E, showing normal muscle architecture, increased fibre size variation and the presonce of vacuoles and internal nuclei. Inset: the presence of slit like vacuoles with basophili¢ staining rims in some fibres is clearly evident. (b) NADH-TR, showing dense internal and subsarcolemmal stain deposits. (c)Non-specific esterase is prominent in association with the vacuolar regions. (d) ATPas¢, pH 10.2, there is a marked predominance of type I fibres with evidence of type II fibre atrophy.

60 vacuoles with granular outlines (Fig. la, inset). These vacuolar regions were unstained with modified Gomori trichrome, Sudan black B, von Kossa, succinate dehydrogenase, phosphorylase and acid phosphatase. The vacuolar rims were, however, positive for non-specific esterase (Fig. lc) and some stained lightly for PAS. In contrast, NADH-TR showed dense granular deposits within many fibres corresponding to the location of the vacuoles (Fig. ld). Otherwise, NADH-TR staining throughout the specimen was fairly homogeneous with poor fibre type differentiation. Myofibrillar ATPase staining showed clear differentiation of fibre types with an apparent excess of type I fibres and type II atrophy (Fig. ld). Vacuoles occurred in both fibre types and were unstained with ATPase. Ultrastructural abnormalities were found within the majority of muscle fibres. These included tubular aggregates of varying mass and location (Fig. 2a-d), dilated sacs of sarcoplasmic reticulum (Fig. 2e), autophagic vacuoles with myelin bodies (Fig. 2f), and a mild increase in interfibfillar glycogen (Fig. 2e).

Case 2 (J.H.) The muscle biopsy from the mother showed similar but more extensive pathological changes to those described from her son (case 1). Interstitial connective tissue was increased in amount without evidence of inflammatory response. The majority of fibres were type I (Fig. 3d) and showed extensive hypertrophy contrasting with the atrophic type II fibres. Fibre splitting and necrosis were also present. Many fibres possessed internal nuclei and large centrally located or multiple vacuoles with angular outlines. The vacuoles were pale staining with granular deposits on H&E (Fig. 3a,b), densely stained on NADH-TR (Fig. 3c), PAS-positive, light red with modified Gomori trichrome, but negative for succinate dehydrogenase, Oil red O, methyl green pyronine, ATPase and Sudan black B. The vacuoles were much larger and more distinct than in case 1 and were present in both fibre types. Occasionally, such material occurred in subsarcolemmal positions. Phosphorylase activity was reported as normal. Electronmicroscopy was reported as confLrming that the granular deposits consisted of proliferated tubules. Case 3 (D.H.) Only paraffin processed histological material was available for examination from his biopsy in 1960. This was reported as being normal except for the presence of a few atrophic fibres. Quantitative analysis of the muscle biopsies The volume density of interstitial connective tissue in the muscle biopsies was 10.8~ + 0.98~ (mean + SE) (case 1 aged 15 years 8 months) and 25.4 + 2.40~o (case 2 aged 32years). This compares to normal data from our laboratory of 10.8 + 0.8~o (unpublished data from the study of Mahon et al. 1984). Muscle fibre size in both specimens showed considerable variation. Mean fibre cross-sectional area and narrow fibre diameter were 3887 pm 2 (CV 50~o) and 52 #m (CV 40Yo) for case 1, and 3262 #m 2 (CV 6 6 ~ ) and 44 #m (CV 49 ~o)'for case 2, respectively. Size variability,

Fig. 2. Electronmicrographs of muscle from case 1 (age 15 years 8 months). Bar line 1 #m. Regular arrangements of tubular aggregates present in the majority of muscle fibres are shown in transverse (a) and longitudinal (b) section. Diameters of the outer and inner cylinders were 65 nm and 25 nm, respectively. In some areas tubular aggregates were associated with glycogen deposits and dilated membranous sacs of varying size but lacking inner tubules (c,d). Large sacs of dilated sarcoplasmic reticulum (e) and myelin-like figures (f) were also observed.

Fig. 3. Quadriceps muscle biopsy of case 2 (aged 32 years). Bar line 50/am. (a) H&E, showing increased connective tissue, fibre size variation and the presence of stellate basophilic areas in the majority of muscle fibres. (b) H&E, the large internal vacuoles are densely basophilic and sometimes associated with internal nuclei. (c) NADH-TR, most fibres contain large dense deposits. (d) ATPase, pH 10.2, most fibres are type I, some are very atrophic and many contain unstained vacuoles with irregular outlines.

63 especially fibre atrophy, was most evident in type II fibres (Fig. 4) which were significantly smaller than type I fibres in both cases. Type II fibres in both specimens were also less rounded than type I fibres as shown by their form factors - type II 0.69 (case 1), 0.75 (case 2); and type I 0.85 (case 1), 0.81 (case 2). The relative proportions of fibre types in the 2 biopsies are given in Table 1 where the marked excess of type I fibres in both cases is evident. In both specimens only a small proportion (less than 2%) of fibres were of the 'immature' or 'transitional' type (IIC). Analysis of the spatial distribution patterns of the 2 major fibre types (SPAM technique) in cases 1 and 2 showed distributions not significantly different from a

50-

Case 1 Type I (n=128)

40-

Type I

401

.

~ 62.2

-~ 30z

Case 2

50,

- ~

cv18%

.

54.5 cv 25 */*

30-

20-

20-

10-

10-

50-

50-

Type ~" ( n : 7 2 )

(n:144)

Type IT (n = 56)

40.

4032.5 cv 61%

~, 3 0 ~2o.

20-

10-

10-

7

15

30 45 60 75 Norrow fibre diometer pm

17.6 cv 85 %

-

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30-

15

90

30

45

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Narrow fibre diameter pm

Fig. 4. Histograms of narrow fibre diameter from case 1 (age 15 years 8 months) and case 2 (age 32 years). ,~, mean; CV, coefficient of variation.

TABLE 1 RELATIVE FREQUENCY OF MUSCLE FIBRE TYPES Case 1 data at 15 years 8 months. Note: normal range for percent type I fibres in young adult males for our laboratory is 40.1 _+ 12.3 (SD) (from Mahon et al. 1984), and for adult females 47.1 _+ 6.4 (SD) (unpubfished data). Variable

Case 1

Case 2

Fibres counted (n) % type I % type II

457 73.3 26.7

360 71.7 28.3

17.5 7.7 1.5

26.4 1.4 0.5

% type IIA % type IIB % type IIC

64 random pattern composed of the same percentage fibre types. Contiguity indices for type II fibres were low (case 1, 15~o ; case 2, 4~o). The proportion of muscle fibre profiles showing vacuolar appearances was 37~o for case 1 and 65~ for case 2 (250 fibres counted in each case).

DISCUSSION The family shows autosomal dominant inheritance of both myopathy and thrombocytopenia. The myopathy in each affected individual was present from early childhood, initially showing an asymmetric limb girdle involvement. By puberty, distal upper limb weakness had developed. The myopathy was slowly progressive as evidenced by the increasing disability in the mother and her father's deterioration to being wheelchair confined. The asymmetry was lost with increased duration of disease. There does not appear to be any progression of the platelet disorder which was fully expressed in the proband at first presentation. Muscle biopsies from both mother and son showed type I fibre preponderance and hypertrophy, type II fibre atrophy and the presence of rimmed vacuoles richly stained for NADH-TR but negative for succinate dehydrogenase. Electron microscopy revealed the presence of tubular aggregates in both cases although abnormalities were more extensive in the mother. The staining characteristics and ultrastructural dimensions of the tubules corresponded to those described in numerous neuromuscular disorders, especially the periodic paralyses. Tubular aggregates are generally thought to be derived from proliferation of tubular elements of the sarcoplasmic reticulum and are often considered non-specific features of muscle pathology (Dubowitz 1978; Mastaglia and Hudgson 1981; Cullen and Mastagiia 1982; Tome 1982; Carpenter and Karpati 1984; Sewry 1985; Banker 1986; Engel and Banker 1986). Indeed, Chou and colleagues (1980) have implicated viral factors as a cause of tubular proliferation and production of rimmed vacuoles in inclusion body myositis. Although some of the vacuoles observed in the present family resemble those in inclusion body myositis it is more likely that the membrane defects are a primary abnormality since they were present from childhood, occurred in both fibre types, and were found in muscle with no evidence of inflammation or degeneration. It would seem probable, from previous reports of a predilection of tubular aggregates for type II fibres (Engel 1970; Banker 1986), that this fibre type may be particularly susceptible to disruption of the sarcoplasmic reticulum thus accounting for their atrophy, hypoplasia, and subsequent muscle weakness in our family. Specific "myopathies with tubular aggregates" have also been described and Banker (1986) has ascribed these to four clinical categories (myasthenic, exercise induced, autosomal dominant, autosomal recessive). Our family differed significantly from patients in the first two groups. In the majority of these cases, and in the periodic paralyses, tubular aggregates are confined to type II fibres (especially IIB) and are located adjacent to the subsarcolemma, whereas in our family they occurred in both major fibre types and were mainly located centrally in association with slit-like vacuoles. The autosomal dominant group is limited to one family in which three generations

65 presented with limb girdle dystrophy and tubular aggregates (Rohkamm et al. 1983). Although the histopathological changes were remarkably similar to those described here, their patients differed from ours in having normal CK levels and a much less severe progression of muscle weakness. The autosomal recessive patients were also limited to one family in which a slowly progressive limb girdle weakness was described in 2 siblings who had tubular aggregates in both fibre types and a raised CK (de Groot and Arts 1982). Haematological abnormalities were not specifically mentioned in these reports nor were problems with regard to excessive bleeding as in our family. In the present study dilated sarcoplasmic sacs and autophagic vacuoles with myelin bodies were also encountered and provide further evidence of disruption of the membranous systems within muscle fibres in this myopathy. Additional support for the primary membrane hypothesis is the associated thrombocytopenia in all affected individuals. Familial thrombocytopenia with normal platelet morphology, as in the present study, is rare although minor impairment of platelet function cannot be discounted (Aster 1972; Carrington et al. 1988). Decreased platelet numbers may result from diminished production from megakaryocytes or enhanced destruction and sequestration although the latter often involves infection or immunologic reaction. In mature megakaryocytes demarcation membranes form by invagination of the plasmalemma producing tubular cisterns which eventually give rise to the platelet surface (Fawcett 1986). Any inherent defect in megakaryocyte maturation or membrane integrity could therefore lead to abnormal platelet production as in this family. Furthermore, normal platelet ultrastructure bears many resemblances to that of skeletal muscle fibres since both cell types contain tubular plasmalemmal invaginations, sac-like smooth intracytoplasmic membranes which sequester calcium and a filamentous contractile system (White 1972). Thus, the decreased blood clotting ability of the present family could result from an as yet undetected abnormality of platelet membranes as well as the thrombocytopenia. There have been a few reports in the literature of patients having thrombocytopenia in association with a neuromuscular disorder (Veenhoven et al. 1979; Riggs et al. 1984; Sawdyk and Jundt 1985; Cooper et al. 1986). However, these differed markedly from our cases, involving chronic immune thrombocytopenia and myasthenia gravis, inclusion body myositis or dermatomyositis, all of relatively sudden onset. Although autophagic vacuoles were reported on electron microscopy in some cases (Riggs et al. 1984) tubular aggregates were not encountered and an autoimmune or viral aetiology is the most likely cause of the associated disorders. In the present study the mode of inheritance of both the myopathy and bleeding disorder and the myopathological features suggest a common genetic rather than viral cause of the membrane disturbances. In conclusion it would appear that this family has a rare inherited myopathy involving tubular aggregates which presented initially as a slowly progressive limb girdle myopathy. This lends further support to the view that patients with limb girdle dystrophy should not be seen as a single clinical entity (Munsat 1977). Furthermore, the associated thrombocytopenia in this familial myopathy suggests the possible value of studying both megakaryocytes and platelets in other neuromuscular disorders which are thought to involve defective cellular membranes. Unfortunately, however, biochemical studies of

66 circulating platelets in muscular d y s t r o p h y ( Y a r o m et al. 1983; N i c h o l s o n et al. 1984) and myotonic d y s t r o p h y ( L a n z a et al. 1987; Bornstein et al. 1988) have so far proved equivocal.

NOTES ADDED IN PROOF (Received 6 October, 1988) S t o r m o k e n et al. (Clin. Genet., 28 (1985) 3 6 7 - 3 7 4 ) have described a family with t h r o m b o c y t o p e n i a , muscle fatigue, asplenia, miosis, migraine, dyslexia and ichthyosis. However, only a mild t h r o m b o c y t o p e n i a in one family m e m b e r was reported and muscle biopsy was not carried out. W e are grateful to Professor John H. E d w a r d s (Medical Genetics, Oxford) for bringing this report to our attention. F o r a recent detailed histochemical study o f tubular aggregates in h u m a n muscle see the p a p e r by Meijer et al. (J. Neurol. Sci., 86 (1988) 73-82).

ACKNOWLEDGEMENTS W e are grateful to MiUy Taylor and Julie W a r d for technical assistance with the preparation o f specimens, and are indebted to the N o r t h W e s t Regional N e u r o m u s c u l a r F u n d for the d o n a t i o n o f digitising equipment used in this study. W e t h a n k Dr. A n n a Kelsey for the use o f r e s i n - e m b e d d e d material from case 1 and Dr. Dennis H a r f i m a n for access to histochemical p r e p a r a t i o n s and E M reports for case 2. The support o f the M u s c u l a r D y s t r o p h y G r o u p o f G r e a t Britain is gratefully acknowledged.

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