Kumaraswamy Sivakumar, Christina Semino-Mora and Marinos C. Dalakas

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1 braini0310 Brain (1997), 120, An inflammatory, familial, inclusion body myositis with autoimmune features and a phenotype identical to sporadic inclusion body myositis Studies in three families Kumaraswamy Sivakumar, Christina Semino-Mora and Marinos C. Dalakas Neuromuscular Diseases Section, National Institutes for Correspondence to: Dr Marinos Dalakas, Building 10, Neurological Disorders and Stroke, National Institutes of Room 4N248, National Institutes of Health, Bethesda, Health, Bethesda, Maryland, USA MD 20892, USA Summary We describe the occurrence of an inflammatory inclusion the presence of the DR3 allele (DRB1*0301/0302) in all body myositis in siblings of a single generation in three seven patients. The combination of the clinical, histological, separate families. The disease in this total of seven patients immunopathological and immunogenetic features indicate was characterized by selective and early involvement of that these patients have a disease identical to sporadic forearm and finger flexors, confirmed by MRI, and inclusion body myositis (s-ibm). We conclude that the weakness of the quadriceps, triceps and foot extensors. classic, inflammatory, s-ibm can also occur in families Muscle biopsies in at least two members from each family (familial inclusion body myositis), in a pattern analogous showed endomysial inflammation, red-rimmed vacuoles, to the familial occurrence of other autoimmune neuro- intracellular amyloid deposition and nm tubulo- logical diseases such as myasthenia gravis and multiple filaments within the vacuolated muscle fibres. Immunocytochemistry sclerosis. These observations strengthen the view that s-ibm on serial muscle biopsy sections demonstrated behaves like other autoimmune diseases and has disease an abundance of CD8 cells invading non-necrotic, MHC- susceptibility linked to the DR3 allele. I-expressing muscle fibres. Immunogenetic studies showed Keywords: myositis, HLA antigens; autoimmune disease; inclusion body myositis; inherited susceptibility Abbreviations: CK creatine kinase; f-ibm familial inclusion body myositis; h-ibm hereditary inclusion body myopathy; HLA histocompatability leukocyte antigen; MHC major histocompatability complex; s-ibm sporadic inclusion body myositis; TNF-α tumournecrosis factor-α Introduction Sporadic inclusion body myositis (s-ibm) is a chronic, heterogenous group of non-inflammatory, red-rimmed acquired, inflammatory myopathy that (i) usually begins vacuolar myopathies that represent various clinical after the age of 50 years, (ii) affects proximal and distal phenotypes which are always distinct from the typical muscle groups with a characteristic early involvement of clinical picture of s-ibm (Cole et al., 1988; Klingman finger flexors and quadriceps muscles and (iii) has and Gibbs, 1991; Neville et al., 1992; Sadeh et al., 1993; characteristic histological features of red-rimmed vacuoles Sivakumar and Dalakas, 1996). containing nm tubulofilaments, small intracellular We now describe three separate families in which the amyloid deposits, and primary endomysial inflammation classic clinicohistological picture of s-ibm was observed in with CD8 cells and macrophages invading non-necrotic a total of seven siblings. In all families, the disease [familial muscle fibres (Lotz et al., 1989; Dalakas, 1991; Sekul and inclusion body myositis (f-ibm)] affected only one Dalakas, 1993; Griggs et al., 1995). In contrast, hereditary generation. inclusion body myopathies (h-ibms) comprise a Oxford University Press 1997

2 654 K. Sivakumar et al. Fig. 1 Pedigrees of Families A C. In each family there are two (Families A and B) or three (Family C) siblings in only one generation affected with inflammatory IBM that presented with a phenotype identical to s-ibm. Patients and methods level of 608 (normal: U/l) was documented prior to Family A the NIH admission in the older brother. Repeated muscle Two brothers, aged 78 and 73 years and born in the Midwest biopsies in both patients revealed histological, of the USA, developed symptoms of proximal and distal immunopathological and electron microscopic features muscle weakness in the lower extremities and frequent falls typical of s-ibm, as described below. due to knee-buckling, at the age of 73 and 69 years, respectively. The older brother also developed weakness of the forearm flexors and swallowing difficulties. A muscle Family B biopsy was diagnostic of IBM (Lotz et al., 1989) and both Two brothers (Fig. 1), aged 69 and 63 years, developed patients were referred to the NIH (National Institutes of weakness and wasting of the quadriceps muscle and the Health). None of the other family members (Fig. 1) had flexor compartment of the forearm in their mid-fifties. In symptoms of a myopathy. On admission, neurological the older brother, the symptoms began in the lower examination revealed, in both brothers, wasting and weakness extremities with frequent falls and weakness in ambulation; of the quadriceps muscles (Fig. 2), atrophy and weakness of in the younger brother the symptoms began in the upper the finger flexors and weakness of foot extensors, as extremities with weakness of the grip. Subsequently, both classically seen in s-ibm (Dalakas, 1991; Sekul et al., 1994). brothers developed the classic features of s-ibm with severe There was no history of myotoxic drug use, infections or involvement of the quadriceps, forearm flexors, triceps and any systemic disease. The serum creatine kinase (CK) level foot extensors. No bulbar or respiratory symptoms were was 167 and 426 U/l (normal; U/l). An elevated CK noted. By the time the patients were seen at the NIH,

3 Familial inflammatory inclusion body myositis 655 Fig. 2 Photographs of anterior thighs in siblings with inflammatory IBM. Two brothers from Family A demonstrate the selective atrophy of the quadriceps muscle, as typically seen in s-ibm. the older brother was wheelchair-bound but the younger brother was still ambulatory. Their CK levels were 522 and 980 U/l, respectively. Muscle biopsy in both patients had all the histological and immunopathological features of s-ibm, as described below. Family C Three siblings of an African-American family (Fig. 1), two men aged 73 and 69 years and a woman aged 77 years, developed a myopathy with an identical pattern of muscle involvement. The two men developed proximal lower extremity weakness, quadriceps wasting and frequent falls in their early sixties, followed, a few years later, by weakness in the distal muscles of the upper extremities. Muscle biopsy in both male patients showed the typical histological features of s-ibm. Their sister (CI) developed weakness in the lower extremities at the age of 48 years, and developed slowly thereafter weakness and wasting in the proximal and distal muscles of the upper and lower extremities. None of the other members of the family had symptoms of muscle disease. On admission and examination at the NIH, all three patients had wasting and weakness of the quadriceps and foot extensors. A varying degree of weakness and wasting in the finger flexors (forearm flexor compartment) was present in all three siblings but was more prominent in the older brother and sister who both had difficulty making a fist (Fig. 3). Overall, in the upper extremities, the weakness was predominantly distal but both the proximal and the distal muscles were equally affected in the lower extremities. Mild facial- and neck-flexor weakness were present in all three patients. When seen at the NIH, the older brother (CII) had also developed dysphagia. CK levels were 359 and 434 in Fig. 3 Photographs of forearms and hands in siblings with inflammatory IBM. A woman (CI) and two of her brothers (CII and CIII) from Family C demonstrate the selective atrophy of the flexors of the forearm and the weakness in their finger flexors, prominent in CI and CII. Although Patient CIII could make a fist, he demonstrated clinical weakness in the long flexors of digits I, IV and V. Special studies (i) T 1 -weighted MRIs of the thigh muscles were performed in Family C, to confirm the selective and prominent involvement of the knee extensor muscles. The MRIs of the quadriceps were compared with the studies previously performed in patients with s-ibm and h-ibm (Sivakumar the men and 124 U/l in the woman. As described below, the and Dalakas, 1996). previous and repeated biopsies in the two brothers revealed (ii) Open muscle biopsies were performed and processed features identical to those seen in patients with s-ibm. for muscle-enzyme histochemistry and Congo-red staining.

4 656 K. Sivakumar et al. Glutaradehyde-fixed and Epon-embedded tissue as well as frozen sections (Askanas et al., 1991) were used for electron microscopy. In addition, immunocytochemistry was performed on serial, 5-µm sections, of the muscle biopsy specimens, utilizing monoclonal antibodies directed against major histocompatability complex (MHC) class I antigen, CD8 cells, macrophages, CD4 cells and Natural Killer cells (Leu 19) in an immunoperoxidase technique, as described by Illa et al. (1992). Disease control biopsies, run concurrently with the above sections, included muscle specimens from a patient with typical histological features of s-ibm and a patient with the autosomal dominant form of h-ibm (Sivakumar and Dalakas, 1996). (iii) Histocompatability leukocyte antigen (HLA) Class II genotyping was performed using PCR (polymerase chain reaction) amplification with sequence-specific primers (Park and Tonai, 1992) to determine if the predominance of DR3 haplotype, seen in patients with s-ibm (Garlepp et al., 1994), was also present in the studied patients. (iv) Complete haematological, biochemical and serological studies were performed including a search for anti-nuclear antibodies, and antibodies against double-stranded DNA, ribonucleoprotein, Jo-1, SS-A and SS-B. Results MRI findings MRI performed in one family showed that the extensor compartment of the thighs was more significantly involved than the flexor compartment (Fig. 4), as typically seen in s-ibm (Sekul and Dalakas, 1993). The same pattern was seen clinically as marked wasting of the quadriceps muscle in the other two families (Fig. 2). In Patient II of Family C, who had advanced disease, the flexor compartment was also involved (Fig. 4). Histology The muscle biopsies on all patients showed a myopathy with variation of fibre size, hypertrophic and small fibres, and increased connective tissue. Red-rimmed vacuoles, intramyofibre amyloid deposits, ragged-red fibres and primary inflammation with lymphocytic infiltrates invading non- necrotic muscle fibres were seen in all affected members of Families A and B, and in the two siblings (CII and CIII) of Family C (Fig. 5). Patient CII had two muscle biopsies, one from the least affected deltoid muscle that showed non- specific myopathic features and another from the more affected quadriceps muscle that showed all the diagnostic features of an inflammatory inclusion body myositis, as noted above. In Patient CI, who had the same typical phenotype of s-ibm as her two brothers (Figs 3 and 4), muscle biopsy performed from the least affected deltoid muscle revealed non-specific myopathic features, similar to the deltoid muscle biopsy of her brother (CII) (probable IBM). However, she did not receive any further biopsies. By electron microscopy, Fig. 4 T 1 -weighted MRIs of the thighs in siblings with inflammatory IBM. In two members from Family C (Patients CI and CIII) there is selective involvement of the quadriceps muscle, compared with the hamstrings, seen as increased signal intensity due to fatty replacement. The third member of this family (Patient CII), with advanced, end-stage disease, has involvement of both the quadriceps and the hamstring muscle groups nm diameter tubulofilaments were observed in the muscle biopsy of at least one representative member (AI, AII, BII and CIII) from each family (Fig. 5). Immunocytochemistry The predominant endomysial cells were CD8 cells which surrounded or invaded non-necrotic and MHC-I antigen-

5 Familial inflammatory inclusion body myositis 657 Fig. 5 Cross-section of a trichromatically stained muscle biopsy from Patient AI (A) shows primary endomysial inflammation and vacuolated fibres ( 425). Electron microscopy, performed of a redrimmed-vacuolated muscle fibre of the same patient, identified by the Gomori trichrome stain (B inset, 400), reveals the characteristic tubulofilaments of nm diameter ( , bar represents 160 nm). expressing muscle fibres (Fig. 6). The other cells were (Fig. 7A and B), as previously described (Sivakumar and macrophages and CD4 cells. Natural killer cells were not Dalakas, 1996). detected. The MHC class I antigen was expressed in most fibres, even in areas without inflammation (Fig. 6B), in a pattern identical to the one seen in s-ibm (Engel and Arahata, Immunogenetic studies. 1984). In contrast, in the concurrently stained sections of a The DRB1*0301/0302 allele corresponding to the DR3 patient with h-ibm, MHC class I antigen was not expressed haplotype was seen in all affected members (Table 1). No

6 658 K. Sivakumar et al. Fig. 6 Endomysial CD8 cells in the MHC class I antigen-expressing muscle fibres in f-ibm. Serial sections of a muscle biopsy from Patient BI of Family B immunostained with antibodies to CD8 cells (A) and MHC class I antigen (B) ( 425). CD8 cells invade MHC-I-expressing muscle fibres. In addition, MHC-I is present in fibres not surrounded by cells. autoantibodies were detected, except for a positive antinuclear antibody titre (1:160), with a speckled pattern, observed in the older brother of Family A (Patient A1). Discussion At least two siblings in three different families had the typical clinical phenotype and the radiological, histological, ultrastructural and inflammatory features of s-ibm with prominent endomysial CD8 cytotoxic T cells invading MHC class I antigen-expressing non-necrotic muscle fibres. These observations indicate that the typical s-ibm, with immunopathological features of an autoimmune process, can be also familial, affecting more than one sibling of a generation. The observed familial occurrence of an inflammatory, and probably autoimmune, inclusion body myositis (f-ibm) needs to be distinguished from the hereditary inclusion body myopathies (h-ibm), a heterogenous group of vacuolar myopathies without inflammatory or immunopathological features that often occur in more than one generation of a family, or have predilection for certain ethnic groups (Askanas

7 Familial inflammatory inclusion body myositis 659 Fig. 7 Expression of MHC class I antigen in low-magnification field in the muscle biopsies of patients with f-ibm (A) compared with h-ibm (B). Muscle biopsy of Patient CIII immunostained for MHC-I (A) demonstrates the ubiquitous expression of the MHC-I antigen in many fibres. Several foci of inflammatory cells invading these fibres are also noted ( 270). In a concurrently immunostained section from a control patient with h-ibm (B), no MHC-I-stained fibres are seen ( 350). and Engel, 1995; Sivakumar and Dalakas, 1996). Although pernicious anaemia and common variable immunodeficiency, the phenotype of patients with h-ibm is variable (Sivakumar while other autoimmune diseases including autoimmune and Dalakas, 1996), these patients do not have the selective thyroiditis, myasthenia gravis and pernicious anaemia can pattern of involvement of the quadriceps and the long finger occur in their relatives (Rugero et al., 1995). Some siblings flexor muscles, as typically seen in s-ibm. or relatives of patients with neurological diseases of putative The immunopathological features observed in the present or definite autoimmune origin, e.g. multiple sclerosis, familial cases are identical to those seen in s-ibm and in myasthenia gravis and polymyositis (Cook et al., 1963; polymyositis where an autoimmune pathogenesis has been Leukonia and Buxton, 1973), also demonstrate a variety strongly considered (Engel and Arahata, 1984; Engel et al., of autoantibodies and develop an unrelated (or the same) 1994). s-ibm has been associated with other autoimmune autoimmune disease. The present observations that IBM with conditions, e.g. Sjogrens disease, dermatitis herpetiformis, prominent inflammatory features can also occur in siblings,

8 660 K. Sivakumar et al. Table 1 Alleles in HLA II antigens seen in affected The observed similarities between inflammatory f-ibm siblings of inflammatory f-ibm and s-ibm, prompted us to treat the f-ibm with the same Patient DRB1 DQB1 DRB3 therapeutic strategies used in s-ibm. One patient, from Family A, reported a subjective, transient benefit to steroids. AI 0101, Another patient, from Family B, noted a transient and AII 0101, objective, albeit mild, improvement in his arms after high- BI dose intravenous Ig, as reported for some patients with s- BII 0301, , CI 0301, , IBM (Dalakas et al., 1995). CI 0101, , CIII 0101, , References The DRB1*0301 and 0302 alleles correspond serologically to Askanas V, Engel WK. New advances in the understanding of DR3 haplotypes. sporadic inclusion-body myositis and hereditary inclusion-body myopathies. [Review}. Curr Opin Rheumatol 1995; 7: are consistent with the pattern of familial occurrence noted Askanas V, Serdaroglu P, Engel WK, Alvarez RB. Immunoin the other autoimmune neurological diseases, and suggest localization of ubiquitin in muscle biopsies of patients with inclusion genetic susceptibility to the autoimmune process. body myositis and oculopharyngeal muscular dystrophy. Neurosci Many autoimmune diseases have been associated with a Lett 1991; 130: particular HLA antigen and the susceptibility to autoimmunity Cole AJ, Kuzniecky R, Karpati G, Carpenter S, Andermann E, has been linked to various MHC-associated genes. In Andermann F. Familial myopathy with changes resembling inclusion polymyositis and dermatomyositis, there is a high incidence body myositis and periventricular leucoencephalopathy. Brain 1988; (42%) of the DR3 phenotype (Love, 1989). A very significant 111: (92%) incidence of the HLA DR3 phenotype, com- Cook CD, Rosen FS, Banker BQ. Dermatomyositis and focal pared with 25% in the control population, has been found in scleroderma. Pediatr Clin North Am 1963; 10: s-ibm (Garlepp et al., 1994). 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