709 MIXED CONNECTIVE TISSUE DISEASE IN SIBLINGS JOHN R. HORN, JANET J. KAPUR, and SARA E. WALKER Mixed connective tissue disease (MCTD) was diagnosed in a brother and sister, and 18 additional family members spanning three generations were studied to detect evidence of autoimmune disease. Symptoms or signs of MCTD without complete expression of the disease were found in 8 relatives of the original cases. Antibodies to ribonucleoprotein and high-titer antinuclear antibodies were found only in the affected siblings. Tests for rheumatoid factor were positive in 9 of 17 relatives of the patients; the titers ranged from l :160 to l :2560. The brother and sister with MCTD had an identical HLA genotype11,12/2,12. The same genotype was inherited by 3 of their siblings, who had impressive rheumatic complaints. This report emphasizes the association between inflammatory From the Department of Internal Medicine, College of Human Medicine, Michigan State University, The Veterans Administration Hospital, Ann Arbor, Michigan, and The Rackham Arthritis Research Unit and the Department of Internal Medicine, The University of Michigan Medical Center, Ann Arbor, Michigan. Supported by the Medical Research Service of the Veterans Administration; Grant C A 13297 from the National Cancer Institute, National Institute of Health, United States Public Health Service; and a Research Grant from the Michigan Chapter of the Arthritis Foundation. John R. Horn, M.D.: Associate Clinical Professor in the Department of Medicine, College of Human Medicine; Janet J. Kapur, B.A., M.T. (A.S.C.P.): The Veterans Administration Hospital, Ann Arbor, Michigan; Sara E. Walker, M.D.: Assistant Professor of Internal Medicine, The University of Michigan Medical Center, Ann Arbor, Michigan. Address reprint requests to Sara E. Walker, M.D., R4633 Kresge I, 1405 East Ann Street, Ann Arbor, Michigan 48109. Submitted for publication October 17, 1977; accepted in revised form April 10, 1978. Arthritis and Rheumatism, Vol. 21, No. 6 (July-August 1978) connective tissue disease and a specific HLA type within a single kindred. Mixed connective tissue disease (MCTD) is an inflammatory disease of unknown etiology which may have overlapping characteristics of systemic lupus erythematosus, scleroderma, and polymyositis. Commonly, patients with MCTD have arthritis, swollen hands and fingers, abnormal esophageal motility, Raynaud’s phenomenon, and active myositis. They may also experience lymphadenopathy, fever, hepatomegaly, serositis, and splenomegaly. Renal disease is rare in mixed connective tissue disease (1). Sera from these patients give positive indirect immunofluorescent tests for antinuclear antibodies (ANA) with a characteristic coarse speckled pattern. The diagnosis is confirmed by finding serum antibodies directed against ribonucleoprotein (RNP), a ribonuclease-sensitive component of extractable nuclear antigen (ENA) (2). This report presents the first description of MCTD in 2 members of the same family. In 1975, MCTD was diagnosed in a 20-year-old woman and her 25-year-old brother. Eighteen additional family members were subsequently evaluated for clinical and serologic evidence of autoimmune disease, and serologic HLA typing was performed to determine if occurrence of MCTD was associated with a specific haplotype. Eight relatives of the propositi had histories of arthritis, arthralgia, swollen hands and fingers, Raynaud’s phenomenon, and/or pleurisy. The mother and a maternal HORN ET AL 7 10 aunt had dilated periungual capillaries. The siblings with MCTD and 9 family members, including one 3year-old girl, had positive tests for rheumatoid factor. Anti-RNP and high titer ANA were found only in the patients with MCTD, who had an identical genotype, 11,12/2,12. METHODS The siblings with MCTD were evaluated for connective tissue disease as described in Case Reports. These patients and their I8 relatives gave medical histories and had physical examinations performed by a rheumatologist (JRH or SEW). The integument and musculoskeletal system were examined carefully for signs of connective tissue disease. Periungual capillaries were evaluated by direct visualization using the naked eye. Sera from each subject were tested for antibodies to ENA by means of a hemagglutination technique (2). The 2 positive serum samples from the propositi were tested further by Ouchterlony immunodiffusion to determine whether the anti-ENA antibodies were directed against RNP, Sm, or both (3). Although ENA is active in the hemagglutination test, it precipitates poorly with anti-ENA antibodies in agarose gel diffusion (4). Therefore, saline-soluble calf thymus nuclear extract (CTE) (5) was used as antigen in the immunodiffusion studies. The patients’ sera were placed in wells on diffusion plates and tested for ability to react with CTE. Standard reference sera previously characterized as containing only antiR N P or anti-Sm antibodies were placed in adjacent wells. Additional immunodiffusion testing employed ribonucleasedigested CTE. The following tests were performed on each patient in this study: hemoglobin, hematocrit, white blood cell count; erythrocyte sedimentation rate (Westergren); indirect immunofluorescent ANA test (6) utilizing sera diluted l:lO, 1:40; 1:160, 1:640, and 1:2560; modified Farr test for anti-doublestranded D N A (7); modified Singer-Plotz agglutination test for rheumatoid factor (8). Peripheral blood lymphocytes were tested for HLA antigens using a standard microcytotoxicity assay (9). There was no clinical evidence of myositis or esophageal dysfunction in members of this family when they were examined. Therefore, muscle enzyme tests, electromyograms, muscle biopsies, and esophageal motility studies were not done routinely. CASE REPORTS Patient I1 5. A 26-year-old man employed as a stock mover in a foundry became ill in January 1975. He developed an urticaria-like skin eruption with raised, pruritic, serpiginous lesions which persisted for 2 days. His joints and muscles were painful, and his oral temperature was 105°F. The fever, arthralgia, and myalgia resolved after 2 weeks. Three months later, fever and urticaria recurred. The elbows, wrists, metacarpophalangeal and proximal interphalangeal finger joints, knees, ankles, and metatarsophalangeal joints became swollen and painful. Morning stiffness appeared lasting 1 to 3 hours. There was no history of skin edema or tightening, dysphagia, or muscle weakness. Initial physical examination was performed in June 1975. Blood pressure was 1l6/76, the pulse 88, and the respirations 16. The patient was afebrile. Oropharyngeal mucous membranes were erythematous. The skin was normal. Periungual capillaries were not dilated. There were no enlarged lymph nodes. N o abnormalities were found in examining the lungs, heart, and abdomen. The spleen was not palpable. Both. elbows had 15” flexion contractures, and the metacarpophalangeal and proximal interphalangeal finger joints were swollen. Muscle bulk and strength were normal. The ANA test was positive in serum diluted 1 :640, and a coarse speckled pattern was observed. Anti-ENA were detected by hemagglutination in a serum titer greater than 1 : 100,000. Ouchterlony immunodiffusion of this patient’s serum and CTE gave a single line which was continuous with a line formed by reacting CTE with anti-RNP in an adjacent well. The serum did not react with anti-Sm. Additional testing showed that ribonuclease-treated CTE did not react with the patient’s serum or with anti-RNP. The erythrocyte sedimentation rate (Westergren) was 28 mm/hr, and the test for rheumatoid factor was present in serum diluted 1:2560. The following laboratory studies were normal or nonreactive: hematocrit, leukocyte count, differential leukocyte count, urinalysis, serum creatinine, creatine phosphokinase, aldolase, serum protein electrophoresis, VDRL, C3, and hemagglutination test for anti-DN A. Electromyogram and muscle biopsy were not performed because there was no clinical evidence of myopathy. Esophageal motility was not tested. His symptoms improved initially with salicylate therapy. In September 1975 he developed Raynaud’s phenomenon with cold-induced finger pain and color change from white to blue. The leukocyte count was 4,300, and LE cell preparations and a Farr anti-DNA test were negative. Two months later, his spleen was palpable 3 cm below the left costal margin and he developed transient alopecia. In February 1976 ulcers were present on the oral mucous membranes for 2 weeks. In March 1976 he described bilateral chest pains that occurred with deep breathing. The pains resolved after 2 days. In May 1976 fever recurred and he complained of pleuritic chest pain on the left. A chest roentgenogram showed blunting of the left costophrenic angle, and pleural fluid was seen layered above the left chest wall in a left lateral decubitus film. Prednisone, 30 mg/day, was added to the therapeutic regime. He improved and returned to work. His dose of prednisone was decreased to 20 mg every other day, and his only complaints at present are arthralgia and Raynaud’s phenomenon. in January I978 physical examination showed normal skin over the face and extremities, with no evidence of scleroderma. Patient I1 9. An 18-year-old machine operator in a shoe factory, a sister of patient I1 5, developed fatigue and enlarged lymph nodes in 1973. The following year, her hands and fingers swelled and her elbows and knees became enlarged and painful. Three months later her hair began to fall out. Anxiety or exposure to cold induced Raynaud’s phenomenon in her hands and feet, with characteristic color changes from white to blue to red. Initial physical examination was performed in July 1975. Blood pressure was 122/82, the pulse 82, and the respirations 16. She was afebrile. Her fingers and hands were diffusely MCTD IN SIBLINGS 71 1 Table I . Clinical Abnormalities and Auroanribodies in 2 Siblings with Mixed Connective Tissue Disease and 18 Family Members ~~ Family Member 11 5t II 9t I I 2 3 4 5 6 Sib, MCTD Sib, MCTD Aunt Mother Aunt Father Aunt Uncle Age 21 21 56 58 55 62 58 53 Symptoms and Signs of MCTD* Fever, Arthritis, PI, Ray Large Spleen, Alopecia, Ulc Large Lymph Nodes, SwH Arthritis, Alopecia, Ray Arthritis, SwH, Dil Cap PI, SwH, Dil Cap ANA ~~~ Anti-ENA (CTE) Rheumatoid Factor 1:640, Coarse > 1: 100,Ooo speckled 1:640,Coarse speckled (RNP+, Sm-) 0 0 0 0 0 0 0 0 0 0 1:320 0 0 1:320 1:2560 1:640 0 0 0 1:lO. Nucleolar 1:40, Coarse I :2560 1 :100.oO0 1:640 ( R N P + , Sm-) speckled 11 1 Cousin 2 3 4 6 Sib Sib Sib Sib 1 Sib Sib 8 111 I 2 3 4 5 Nephew Nephew Nephew Daughter Niece 26 34 32 30 26 24 23 8 12 2 3 8 0 0 0 0 0 0 0 Arthralgia, SwH 0 0 0 0 1:160 ND 1:320 0 1:320 0 I :640 0 0 0 0 Arthralgia 0 0 0 0 0 Arthritis Arthralgia, PI, Ray Arthritis PI * PI = pleurisy, Ray = Raynaud’s phenomenon, Ulc capillaries. t Propositi with MCTD. $ Nonspecific agglutination. = oral ulcers, SwH swollen, but there was no hidebound skin. Periungual capillaries were not dilated. Enlarged anterior and posterior cervical lymph nodes were palpated. The lungs, heart, and abdomen were normal. The spleen was not enlarged. The typical color changes of Raynaud’s phenomenon were observed in the fingers. The muscles and joints were normal. The A N A test was positive in a serum dilution of 1:640, with a coarse speckled pattern. Anti-ENA were detected by hemagglutination testing in a serum titer of 1:100,000. After ribonuclease digestion of ENA-coated sheep erythrocytes, the positive titer fell t o 1:IOO. In the Ouchterlony immunodiffusion test, the patient’s serum was tested with CTE, antiRNP, and anti-Sm. A single precipitin line formed between wells containing the patient’s serum and CTE; this line formed a line of identity with the precipitin line between anti-RNP and CTE. Ribnuclease-digested CTE did not react with the patient’s serum or with anti-RNP. The Westergren erythrocyte sedimentation rate was 29 mm/hr. These laboratory tests were within normal limits or negative: hematocrit, leukocyte count, differential leukocyte count, urinalysis, serum creatinine, serum IgG, IgM, IgA, rheumatoid factor test, VDRL, infectious mononucleosis spot test, cryoproteins, C3, LE cell preparations, and hemagglutination test for anti-DNA. Tests for creatine phosphokinase = 0 0 0 1:640 0 NSAS diffusely swollen hands and fingers, Dil Cap = dilated periungual and aldolase were not performed. A chest roentgenogram was normal. Electromyogram and muscle biopsy were not performed. Esophageal motility was not tested. Treatment with aspirin and phenoxybenzamine was instituted, and the patient received a 3-month course of prednisone therapy, 7.5 mg/day. In September 1976 the rheumatoid factor test reverted from negative to positive in serum titered 1:640, and a modified Farr test for anti-DNA was negative. The patient quit work because activity caused pains in the elbows, wrists, and metacarpophalangeal joints. In 1976, she became pregnant and discontinued all medications. She had a spontaneous delivery in August 1977 without exacerbation of her disease. Six weeks postpartum, she was found to have carcinoma in situ of the cervix. Physical examination in January 1978 showed no scleroderma changes in the skin and no evidence of active disease. FAMILY STUDIES All family members were questioned about exposure to a possible common source of disease. The adults in this study lived in different households, and there was no history of keeping sick pets. In patient I 1 9, M C T D began 2 years before HORN ET A L patient I1 5 became ill. Although the siblings with M C T D lived in the same small town, they did not live in the same house or work in the same factory when their disease began. They denied exposure to vibrating tools, vinyl chloride reactors, and toxic substances. Clinical abnormalities and tests for autoantibodies in 20 family members are listed in Table I. Swollen hands and fingers, dilated periungual capillaries, arthritis, arthralgia, pleurisy, and Raynaud's phenomenon were found in 8 of 18 first- and second-degree relatives of the siblings with MCTD. Nine of these relatives had histories of disabling allergies to dust and pollens. Family member 1 3 had a leukocyte count of 3500/mma. A paternal aunt of the affected siblings (I 5 ) had persistent, unexplained elevations of the sedimentation rate (79-85 mm/hr). Otherwise, blood counts and sedimentation rates were normal. Two asymptomatic relatives in generation I had positive ANA tests, but high-titer ANA and anti-ENA were restricted to the 2 siblings with clearcut MCTD. Farr tests for anti-DNA were negative in all 20 family members. Sera from 9 of 17 relatives of the affected siblings were positive for rheumatoid factor, with titers ranging from 1:160 to 1 :2560. Figure 1 illustrates the family tree and inheritance of HLA types within this kindred. I 2, the mother of the brother and sister with MCTD, had a history of pleurisy and diffusely swollen hands. Physical examination showed prominent dilated periungual capillaries. Her sister, I 1, stated that her metacarpophalangeal and proximal interphalangeal finger joints had been painful and swollen. She also gave a history of intermittent, diffuse swelling of her hands. H e r only abnormal physical finding was striking dilation of the periungual capillaries. I 2 married 1 4 , a man with no evidence of rheumatic disease who has a positive latex agglutination test for rheumatoid factor. Two of their offspring, who inherited the identical genotype 1 I,l2/2,12, developed typical MCTD. This genotype was shared by 2 brothers ( I 1 3 and I1 4 ) and a sister (11 8). Although these H LA identical siblings of the affected individuals were not severely ill, they had abnormalities that are common in patients with MCTD. I1 3, a 32-year-old male factory worker, had pleurisy, painful joints, and Raynaud's phenomenon. I1 4, his brother, had a history of pain and swelling involving the shoulders, elbows, metacarpophalangeal and proximal interphalangeal finger joints, and knees. Their sister, I1 8, complained of arthralgia and recurring swelling of the hands and fingers. Clinical and serologic abnormalities were common in relatives who inherited haplotype 2, I2 without 11,12. Three of 6 individuals with 2,12 had rheumatic complaints. Positive tests for rheumatoid factor were associated with the 2,12 haplotype in 3 family members. 111 4, the 3-year-old daughter of the male propositus with MCTD, inherited 2,12 from her father. Although she had no rheumatic complaints, she had a positive latex test i n serum diluted 1:640. DISCUSSION This r e p o r t is t h e first description of MCTD occurring in t w o members of t h e same family. Other investigators h a v e mentioned t h e association of MCTD with o t h e r connective tissue diseases i n close relatives. In one kindred, a p a t i e n t with MCTD had a sister who died with severe systemic lupus erythematosus (10). Two additional families in which MCTD coexisted with systemic lupus erythematosus and/or rhematoid arthritis h a v e been discussed briefly ( 1 1). T h e s e familial clusters of connective tissue diseases may be interpreted as showing t h a t MCTD is a variant of a n o t h e r disorder such as systemic lupus erythematosus. However, in t h e family described in this report, MCTD a p p e a r e d as a distinct II 24,7/2,40 24,7/-,40 11,12/2,12 11,12/2,12 @ ll,l2/-,- 2,7/-,12 2,12/1,8 2,12/3,7 11,12/24,- Y a MIXED CONNECTIVE TISSUE DISEASE Figure 1. This family tree illustrates three generations of a family in which two siblings (I15 and I1 9)had MCTD. HLA types in 19 family members are listed. MCTD IN SIBLINGS clinical entity. The affected brother and sister had typical symptoms, physical signs, and serologic findings of MCTD. ~~~~~~~~~~~i~and the hidebound skin ofscleroderma did not develop during a followup period Of Years. Furthermore, negative tests for anti-DNA, norma1 serum complement levels, and absence of clinical renal disease provided strong evidence that these patients had MCTD and not systemic lupus erythematosus. Although both patients had positive tests for rheumatoid factor, they had no typical deformities of rheumatoid disease. Positive latex agglutination tests are compatible with MCTD; rheumatoid factor is present in sera from 55% of MCTD patients (5). Limitation of overt disease to only 2 members of this family is not explained easily. Eight of 18 relatives of these patients had complaints which may be associated with MCTD-arthritis, arthralgia, diffusely swollen hands, Raynaud’s phenomenon, and pleurisy. Dilated periungual capillaries were present in an aunt and the mother of the propositi. These vascular lesions are an important physical finding in MCTD. In a preliminary study, 13 of 26 patients with MCTD had abnormal nailfold capillaries (12). It may be postulated that the high frequency of rheumatic complaints and positive latex agglutination tests in this kindred represented incomplete forms of disease. Characteristic MCTD with antibodies to R N P in a brother and sister may have been caused by unknown environmental o r genetic factors in susceptible hosts. Histocompatibility typing has been performed in many patients with inflammatory disorders of connective tissue to search for associations between specific HLA types and disease. An early report showed an increased frequency of HLA-BS and B15 in systemic lupus erythematosus, a disease which closely resembles MCTD (13). However, a more recent study showed that systemic lupus erythematosus was associated only with HLA-B5 in black Americans (14). In families with multiple cases of systemic lupus erythematosus, disease inheritance is often associated with the same HLA types within a well-defined kindred ( I 5). The family with MCTD described in this report may represent inheritance of MCTD or “MCTD diathesis” associated with certain histocompatability antigens. Surveys of additional patients will be needed to determine if HLA-A2, All, or B12 are associated consistently with MCTD. ACKNOWLEDGMENTS Mrs. Noel Horn and Eric Hodeen, M.D., assisted the authors by obtaining valuable clinical information from 713 M C T D patients and family members. Sera were tested for E N A in the laboratory of Gordon C . Sharp, M.D., at the University of Missouri Medical Center. H L A typing was performed in the Veterans Administration laboratory of Jane S. Schultz, Ph.D., Assistant Professor of Human Genetics, University of Michigan Medical School. Barbara Boddy contributed expert technical assistance. REFERENCES I . Sharp G C , lrvin WS, Tan EM, Gould RG, Holman HR: Mixed connective tissue disease-an apparently distinct rheumatic disease syndrome associated with a specific antibody to a n extractable nuclear antigen (ENA). Am J Med 52:148-159, 1972 2. Sharp G C , Irvin WS, LaRoque, RL, Velez C, Daly V, Kaiser AD, Holman HR: Association of autoantibodies t o different nuclear antigens with clinical patterns of rheumatic disease and responsiveness to therapy. J Clin Invest 50350-359, 1971 3. Tan EM, Kunkel H G : Characteristics of a soluble nuclear antigen precipitating with sera of patients with systemic lupus erythematosus. J Immunol 96:464-47 1, 1966 4. Northway J D , Tan EM: Differentiation of antinuclear antibodies giving speckled staining patterns in immunofluorescence. Clin lmmunol lmmunopathol 1 : 140-1 54, I972 5 . Sharp G C , lrvin WS, May C M , Holman H R , McDuffie FC, Hess EV, Schmid FR: Association of antibodies to ribonucleoprotein and Sm antigens with mixed connective-tissue disease, systemic lupus erythematosus and other rheumatic diseases. N Engl J Med 295:1149-1154, 1976 6. Kay D R , Bole GG Jr, Ledger WJ: Antinuclear antibodies, rheumatoid factor and C-reactive protein in serum of normal women using oral contraceptives. Arthritis Rheum 14:239-248, 1971 7. Walker SE, Bole GG Jr: Selective suppression of autoantibody responses in NZB/NZW mice treated with longterm cyclophosphamide. Arthritis Rheum I8:265-272, I975 8. Singer JM: Standardization of the latex test for rheumatoid arthritis serology. Bull Rheum Dis 24:762-769, 19734 9. Amos DB: Cytotoxicity testing, N I A I D Manual of Tissue Typing Techniques (DHEW Publication N o ( N I H ) 76545). Edited by J G Ray Jr, D B Hare, PD Pedersen, DI Mullally. Bethesda, Research Resources Branch (NIAID), 1976, pp 25-28 10. Sharp GC: Autoantibodies and complement in SLE: a reexamination. Hospital Practice 6:109-120, 1971 I I . Sharp GC: Mixed connective tissue disease. Current concepts (Audience discussion). Arthritis Rheum (supplement) 2O:S181-S186, 1977 12. Sharp GC: Personal communication HORN ET AL 7 14 13. Grument FC, Coukell A, Bodmer JG, Bodmer WF, McDevitt H O Histocompatibility (HL-A) antigens associated with systemic lupus erythematosus. A possible genetic predisposition to disease. N Engl J Med 285:193196, 1971 14. Nies K M , Brown JC, Dubois EL, Quismorio FP, Friou G J , Terasaki PI: Histocompatibility (HL-A) antigens and lymphocytotoxic antibodies in systemic lupus erythematosus (SLE). Arthritis Rheum I7:397-402, 1974 15. Cleland LG, Bell DA: HLA-SD haplotypes and immunological findings in SLE families. XIV International Congress of Rheumatology Abstracts, p 236, 1977 Errata The title of the article by Bruce C. Gilliland er al. (April 1978, pages 330-336) should have been: “Synthesis of Rheumatoid Factor by an Established Cell Line from a Rheumatoid Synovium.” The second paragraph on page 529 (June 1978) of the article by Muhammad A. Khan er al.. “A Subgroup of Ankylosing Spondylitis Associated with HLA-B7 in American Blacks,” should have read: Although our earlier study (7) showed no significant difference in age at onset of AS between B27-negative and B27positive patients, selection of the B7-positive subgroup from the larger group of B27-negative patients revealed later onset of disease in these B7-positive American black patients. In a population of European and North-African patients, Feldman el al. (20) noted onset of AS after the age of 40 years in 2 of 42 B27-positive and 3 of 8 B27-negative patients. It is of interest that in psoriasis vulgaris, frequency of Bw17 has been reported to be significantly higher in patients with onset before 35 years of age than in those with later onset (21,22).