CLINICAL STUDIES
Hereditary Complement (C2) Deficiency with Dermatomyositis
JOHN P. LEDDY, M.D. ROBERT C. GRIGGS, M.D. MARTIN R. KLEMPERER, M.D. Rochester, New York MICHAEL M. FRANK, M.D. Bethesda, Maryland
From the Departments of Medicine, Neurology and Pediatrics, University of Rochester School of Medicine and Dentistry, Rochester, New York; and from the National Institute of Allergy and Infectious Diseases, Laboratory of Clinical Investigation, National Institutes of Health, Bethesda, Maryland. This study was supported in part by U.S. Public Health Service Research Grant AM-09810 and by the Pediatric Blood Research Fund of the University of Rochester. Requests for reprints should be addressed to Dr. John P. Leddy, Department of Medicine, University of Rochester Medical Center, 260 Crittenden Boulevard, Rochester, New York 14642. Manuscript accepted April 16. 1974.
A 60 year old white man in previous good health presented with a 6 month history of progressive muscle weakness. Clinical and laboratory findings were typical of dermatomyositis. Muscle biopsy confirmed the presence of inflammatory myopathy; deposits of immunoglobulin G (IgG), immunoglobulin M (IgM) or third component of complement (C3) were not detected by immunoftuorescence. No evidence was found for an associated neoplasm. An unexpected finding was the total absence of serum hemolytic complement activity. Further investigation revealed that the complement defect was attributable to a selective and total absence of the second component of complement (C2), as determined by both functional and immunoprecipitin assays. Family studles indicated that the defect was inherited in an autosomal recessive manner, as has been observed in the previously reported CBdeficient kindreds. This case demonstrates that typical muscle lesions of dermatomyositis can occur in the presence of a complement defect which would preclude activation of the classic (Cl-C4-C2) complement pathway. The case is of further interest as one of a series of recently reported associations of rheumatic diseases with hereditary complement deficiencies. Study of the functional properties of the propositus’ CP-deficient serum demonstrated normal generation of chemotactic activity in the presence of endotoxin or aggregated IgG, and normal or near normal bactericidal activity against Salmonella typhi 0 901 and Hemophilus lnfluenzae, type b. These findings emphasize the importance of the alternate (properdin) pathway of complement activation in these functions. In a growing number of recent reports attention has been called to the occurrence of rheumatic disorders or glomerulonephritis in patients with genetically determined deficiency of serum complement (C) components. Although the propositi of the first four reported kindred with hereditary deficiency of the second C component (C2) were found to be healthy [l-5], the development of systemic lupus erythematosus (SLE) or an SLE-like syndrome has more recently been reported in two other C2-deficient (C2D) subjects [6,7]. Another patient with C2 deficiency has experienced persistent vasculitic purpura [8]. Selective deficiency of the Clr subcomponent of the first C component has been recognized in three persons. One, on whom family studies are not available, had glomerulonephritis
January 1975
The American Journal of Medicine
Volume 58
83
HEREDITARY COMPLEMENT DEFICIENCY WITH DERMATOMYOSITI.S-LEDDY
[9, lo]. The other two are siblings; one has glomerulitis, arthritis, skin rash and inflammatory subcutaneous nodules, and the other has arthralgia, skin rash, recurrent otitis media and bronchitis [ 11,121. Independently of these observations, the presence of immunoglobulin and complement (C3) deposits in intramuscular blood vessels of patients with dermatomyositis or polymyositis has recently been reported, suggesting that immune complex deposition might have a pathogenetic role in such patients [ 131. The discovery of genetically determined absence of C2 in a patient presenting with typical dermatomyositis was, therefore, of considerable interest and is presented herein, together with immunochemical, genetic and biologic studies on the eighth reported human kindred with hereditary C2 deficiency. A preliminary report of this case has been presented [ 141. CASEREPORT A 60 year old white man (J.S.) was admitted to the University of Rochester Medical Center (URMC) because of gradual development of muscle weakness during the preceding 6 months. The patient had been generally well and physically active all his life, and had not experienced previous neuromuscular or rheumatic symptoms. As a child he had an “operation for an abscessed gland” on the right side of his neck and at age 15 or 16 surgical “drainage of pus” from a right inguinal node. He had had several ear infections in childhood, and tonsillectomy and adenoidectomy had been performed at age 10. In other respects his infectious disease history had been unremarkable. At age 30 he underwent an appendectomy at URMC; tissue sections revealed an acute inflammatory reaction with many polymorphonuclear and eosinophilic leukocytes, considered quite typical for appendicitis. The patient’s family history was negative for neuromuscular disease. Seven siblings and four children are in excellent health. His father died of accidental causes at age 59 and his mother of unknown causes at age 90. Physical examination revealed an erythematous rash on the upper eyelids, extensor surfaces of elbows, and over the dorsal surface of the metacarpal-phalangeal joints and fingers; diminished diaphragmatic excursion bilaterally with fine rales at the bases of both lungs; and a slightly enlarged heart with an irregular rhythm consistent with atrial fibrillation. Neurologic testing revealed no pharyngeal weakness or defective cranial nerve functions. There was marked proximal weakness of skeletal muscles including inability to raise his arms above his head, sit up from supine, rise from a chair, squat or lift his legs off the bed. Sensation and muscle stretch reflexes were normal. Laboratory studies included normal blood counts, urinalysis and blood chemistries. Serum glutamic-oxaloacetic transaminase (SGOT) was 83, 89 and 81 Karmen units (normal 10 to 45 Karmen units); creatine phosphokinase (CPK) was 640, 690 and 200 IU (normal 0 to 50 IU), and aldolase was in the upper normal range. Alkaline phosphatase was 24, 19 and 17 Bodansky units (normal 2 to 5
04
January
1975
The American
Journal
of Medicine
Volume
ET AL.
Bodansky units). Bromsulfalein retention was 16 per cent at 45 minutes. Bilirubin was normal. The electrocardiogram confirmed atrial fibrillation with a ventricular rate of 66, ventricular premature contractions, left axis deviation and an intraventricular conduction defect (QRS 0.10 second). Serum electrophoresis revealed moderately increased gamma globulin and slightly increased alpha, and beta globulin peaks. Serum immunoglobulins, measured by radial immunodiffusion, were IgG 1,900, IgA 96, IgM 392 mg/ 100 ml (normal 600 to 1,700, 50 to 250 and 50 to 200 mg/lOO ml, respectively). lmmunoelectrophoresis revealed that the increased IgG and IgM were heterogeneous (“polyclonal”). Serum hemolytic complement was undetectable (less than 5 CHss units) in three samples of fresh serum. Rheumatoid factor (by latex fixation test) was present at a titer of 1:160 (normal 1:20 or less), and a test for antinuclear antibody (ANA) was positive by immunofluorescence. Tests for lupus erythematosus cells were negative. By radioimmunoassay [ 15,161, binding of native deoxyribonucleic acid (DNA) by the patients’s serum globulins was normal on two occasions, whereas binding of single-strand DNA was 21 and 28 per cent, respectively (normal 5 to 23 per cent). A Venereal Disease Research Laboratory slide test was not reactive. Tests for cryoglobulins in two 10 ml serum samples were negative after the customary 72 to 96 hour incubation at 5%. but minute precipitates (less than 1 per cent cryocrit) did appear after 5 to 7 days. These precipitates were pooled, dissolved in 0.5 ml 37’C buffer, and found to contain IgM and IgG but not C2, C4, IgA or albumin (see “Methods”). A slide latex test (Hyland) for rheumatoid factor activity in the redissolved cryoprecipitate was negative. The patient’s serum was not anticomplementary when incubated with normal serum in various ratios. Studies to demonstrate a possible associated malignancy were negative. These included an intravenous pyelogram, barium enema, roentgenograms of the upper gastrointestinal tract, metastatic survey of skull, spine and long bones, and sigmoidoscopy. A small polyp in the lower sigmoid colon was removed and found to be benign. Electromyography of the triceps at rest disclosed spontaneous, prolonged polyphasic potentials, recurrent fibrillations and doublet and triplet biphasic action potentials. With activity, a full interference pattern of low voltage developed. Individual potentials had an average duration of 4 milliseconds and a markedly decreased amplitude. A muscle biopsy specimen from the left biceps (Figure 1) was consistent with inflammatory myopathy. There were large clusters of mononuclear inflammatory cells consisting of predominantly large and small lymphocytes surrounding vessels and between muscle fibers. A small number of muscle fibers were undergoing phagocytosis, and occasional fibers were regenerating. There was increased variation of muscle fiber size, a moderate increase in endomysial connective tissue and an increase in internal nuclei. The vascular involvement did not extend through the wall of muscular arteries. Neither perifascicular atrophy nor vacuolization of muscle fibers was present. Histochemical stains (myosin adenosine triphosphatase pH 9.4) demonstrated a normal mosaic pattern with no type
59
HEREDITARY
COMPLEMENT
DEFICIENCY
WITH
DERMATOMYOSITIS-LEDDY
ET AL.
Histologic sections of muscle biopsy specimen stained with hematoxylin and eosin. A, low power view showing mononuclear cell infiltrate between muscle fibers and in perivascular distribution. Variation in muscle fiber size and some increase in internal nuclei and endomysial connective tissue are also evident. B, higher power view of an intramuscular blood vessel showing perivascular mononuclear cell infiltrate. Original magnification X 200 (A) and X 430 (B); reduced by 28 per cent.
Figure 1.
atrophy, grouping or predominance. Phosphorylase was present. Direct immunofluorescent studies with specific, fluorescein-conjugated antiserums (see “Methods”) did not demonstrate staining of blood vessels or muscle fibers with antiserums to IgG, IgM, C3, albumin or fibrinogen. During therapy with prednisone, 20 mg every 6 hours, the skin rash and muscle tenderness resolved rapidly and within 3 weeks muscle strength had improved markedly. Serum CPK and SGOT enzyme levels gradually returned to normal. The serum complement (CH,,) titer did not change during this remission. Daily prednisone dosage was gradually reduced and later changed to 60 mg on alternate mornings. There was continued improvement in strength to normal. Positive tests for ANA and rheumatoid factor (1:60 to 1:320) and elevated IgM levels persisted. Serum IgG returned to normal. Alkaline phosphatase levels remained elevated and a repeat bromsulfalein test showed 20 per cent retention at 45 minutes. Antimitochondrial antibody titers (largely IgG class) were high (1:640) on several occasions (normal negative -1: 10). Tests for smooth muscle antibodies were negative. The persistently abnormal liver chemistry studies suggested some form of mild liver disease. In view of the high titer of antimitochondrial antibody and elevated serum IgM level the possibility of a developing primary biliary cirrhosis was considered, but a liver biopsy has not yet been performed. Laboratory screening of the seven siblings and a daughter yielded negative tests for ANA, antimitochondrial and antismooth muscle antibodies, and rheumatoid factor except for sibling II-4 (see Figure 2, Table II), a normal subject, who had a latex fixation titer of 1: 160. MATERIALS
was kindly supplied by Dr. Ulf Nilsson and goat antiserum to human glycine-rich P-glycoprotein (GBG, C3 proactivator, properdin factor B) by Dr. Chester A. Alper. Antiserum to human C2 was obtained by immunizing rabbits with human C2 purified as previously described [ 181. Antiserum to human Clq was prepared in rabbits by immunization with Clq purified by the method of Yonesmasu and Stroud [ 191. Goat antialbumin was purchased from Kallestad Laboratories, Minneapolis, Minn. The specificity of these antiserums was verified by immunoelectrophoresis and/or by immunodiffusion against appropriate reference antigens, i.e., normal serum, known C2-deficient serum, purified C3 and C4 (gift of Dr. H. J. Muller-Eberhard). isolated immunoglobulins and albumin. Crycglobulins. These were measured, isolated and identified as previously described [ 201. Immunofluorescence. A portion of biopsied skeletal muscle was immediately frozen in isopentane chilled to -180°C, and 4 and 8 p cryostat sections were prepared. Fluorescein-conjugated antiserums to IgG, IgM and fibrinogen were prepared in our laboratory and passed through DEAE-cellulose columns to obtain fractions with fluorescein: protein ratios of approximately 1.5:l - 2: 1 [21]. Conjugated antiserums to human C3 and to albumin were
t90
p&&
73
72
70
68
64
AND METHODS
Human Serums. Serum for complement (C) assays was either freshly drawn or stored as small aliquots at -70% Specific Antiserums. Specific antiserums to human IgM, IgG and IgA were prepared in rabbits as previously described [ 171. Rabbit antiserums to human C3 and C4 were the gift of Dr. John T. Boyer. Goat antiserum to human C5
I
.
.
t59
/”
6o I
q
C2 NORMAL
0
C2 PRESENTBUT SUBNORMAL
n q
C2 NOT DETECTABLE
5a
5a 4
2 &b
;8
5o
III
25
18
ia
NOT STUDIED
Figure 2. tive to
January 1975
Family pedigree showing
age and status
rela-
C2 deficiency.
The American Journal of Medicine
Volume 58
85
HEREDITARY COMPLEMENTDEFICIENCY WITH DERMATOMYOSfTlS-LEDDY ET AL.
purchased from Hyland Laboratories, Costa Mesa, Calif. Direct immunofluorescence examination of muscle tissue was carried out as previously described [22]. We are indebted to Dr. Mary Lou Callerame for these studies. Aliquots of these same conjugated antiserums to IgG, IgM or C3 have, during this period of study, given positive staining of renal glomeruli and skin biopsy specimens from patients with systemic lupus erythematosus. Complement Reagents and Assays. Hemolytic complement (CH,,) titrations were performed as previously reported [23]. Materials, buffers and methods for the preparation of Cl, C2 and cellular intermediates and for stoichiometric assays of Cl, C4 and C2 have been described [24,25]. Other functionally pure human complement components were obtained from the Cordis Corporation, Miami, Fla. In titrations of C2, the cell intermediate EAC14 hu was prepared.with a T max for C2 binding of less than 5 minutes. Titrations were performed by the standard assay (method I) [24,25] and by a modification of a tenfold more sensitive assay (method II) [ 261. Plates and reference standards for measuring human C3 and C4 by radial immunodiffusion were purchased, respectively, from Hyland Laboratories and from Behring Diagnostics, Somerville, N.J. Normal ranges were established with serum samples from 20 healthy donors. Agar plates for the measurement of C5 and properdin factor B (C3 proactivator) by the same technic were prepared in our own laboratory. The normal range for C5 was established with 30 normal serums with reference to a serum which had been standardized against isolated C5 (reference serum supplied by Dr. Peter Schur). C2 and Clq were estimated by double diffusion (Oucherlony) in 1 per cent agar, employing serial doubling dilutions of test serums against a given antiserum. A pool of 30 normal serums, tested concurrently, served as reference standard. The highest dilution of test serum giving a visible precipitin line was taken as the endpoint in such studies. lmmunoelectrophoretic analysis of C3 and properdin factor B (C3 proactivator) in serum was performed in 1 per cent agarose gel prepared in a pH 6.4 buffer containing 0.02 M Veronale and 0.01 M sodium EDTA. Fresh normal serum and normal serum activated by inulin [44] served as reference serums. Since the serum samples from patient J.S. had been stored for 1 to 2 years at -70°C. normal serums stored for a similar time were studied concurrently. Serum Bactericidal Activity. Serum bactericidal activity against Salmonella typhi 0 901 was assayed by previously described modifications [27] of a published method [26]. Bactericidal assay against Hemophilus influenzae, type b, was performed as follows. Rabbit antiserum to this organism was donated by Dr. Richard Robertson, Department of Microbiology, and was heat inactivated (56%, 30 minutes) before use. An “optimal” dilution (1:200) was selected for subsequent assays by titration with agammaglobulinemic serum, as described [27] for the anti-Salmonella antibody. From an overnight broth culture of H. influenzae, a subculture in Levinthal’s broth was incubated at 37°C until the O.D. at 650 nm reached 0.065. This subculture was then diluted 1:200,000 in phosphate buffered saline
88
January 1975
The American Journal of Medicine
solution, pH 7.1 to 7.3 containing 3 X 10m4 M calcium ion and 2 X 10m3 M magnesium ion. For the actual assays the following chilled reactants were added to sterile, glass tubes in an ice bath: phosphate buffered saline solution 0.2 ml; 1:200 rabbit antibody (in phosphate buffered saline solution) or phosphate buffered saline solution alone, 0.1 ml; 1:200,000 suspension of H. influenzae, stirred by Vortex mixer immediately before each pipetting, 0.1 ml; and test serum (or dilution in phosphate buffered saline solution) as the source of complement, 0.2 ml. Each assay tube was stirred (Vortex), and a 0.1 ml aliquot was inoculated on chocolate agar (zero time). The tubes were then placed in a 37% bath for 60 minutes, followed by Vortex mixing and inoculation of another 0.1 ml aliquot on chocolate agar (60 minute sample). The plates were placed overnight in a 37’C incubator in 5 per cent carbon dioxide. Colonies were counted, and the degree of killing in each 60 minute sample was calculated by reference to the zero-time count on the same tube. Zero-time plates typically showed from 120 to 130 colonies per plate. Control mixtures omitting a source of complement, or substituting heat inactivated serum for fresh serum, showed no killing. Chemotactic Assays. These assays were kindly performed by Dr. John Baum as described elsewhere [27,29]. Results are expressed as a ratio, the chemotactic index, explained in [29]. In all experiments, normal and C2 deficient serums were tested concurrently. Controls included activating agent without complement or with heat inactivated serum, and test serum without activating agent. Antimitochondrial and Antismooth Muscle Antibodies. These antibodies were generously assayed by Dr. Marilyn Brown, Gastroenterology Section, The Genesee Hospital, using immunofluorescence with rabbit stomach, kidney and uterus as substrates. RESULTS
Complement Assays. Table I presents the results of selected complement assays performed on our patient and available family members. Additional data from other assays carried out only on the propositus and his youngest sibling are shown in Table II. Genetic relationships in this family are presented schematically in Figure 2. As shown in Table I, our patient (N-6, Figure 2) and his youngest brother (11-8) lacked detectable C2 by standard hemolytic assay (method I) and by immunoprecipitin (Ouchterlony) analysis with rabbit antiserum to human C2. A second, more sensitive functional C2 assay (method II, [26]) also failed to detect C2 in the serum of the propositus or his brother (not shown). This assay yielded titers of 10,000 units/ml or greater in normal serums tested concurrently. This indicates that in these two siblings (II-6 and 11-8)functional C2 activity is not more than 0.1 per cent of normal. Data on the other complement components in these two serums (Tables I and II) are consistent with a selective deficiency of C2. Clq levels (not
Volume 58
HEREDITARY COMPLEMENT DEFICIENCY WITH DERMATOMYOSITIS-LEDDY
I
TABLE
Assay of Selected
Family Member* II-1 II-2 II-3 II-4 II-5 II-6 (propositus) II-7 I l-8 Ill-2 Normal subjects _____
___
Cbmplement
Hemolytic Complement (CH,,) Titer (units/ml) 120 88 84 145 73
Hereditary Complement (C2) Deficiency with Dermatomyositis
JOHN P. LEDDY, M.D. ROBERT C. GRIGGS, M.D. MARTIN R. KLEMPERER, M.D. Rochester, New York MICHAEL M. FRANK, M.D. Bethesda, Maryland
From the Departments of Medicine, Neurology and Pediatrics, University of Rochester School of Medicine and Dentistry, Rochester, New York; and from the National Institute of Allergy and Infectious Diseases, Laboratory of Clinical Investigation, National Institutes of Health, Bethesda, Maryland. This study was supported in part by U.S. Public Health Service Research Grant AM-09810 and by the Pediatric Blood Research Fund of the University of Rochester. Requests for reprints should be addressed to Dr. John P. Leddy, Department of Medicine, University of Rochester Medical Center, 260 Crittenden Boulevard, Rochester, New York 14642. Manuscript accepted April 16. 1974.
A 60 year old white man in previous good health presented with a 6 month history of progressive muscle weakness. Clinical and laboratory findings were typical of dermatomyositis. Muscle biopsy confirmed the presence of inflammatory myopathy; deposits of immunoglobulin G (IgG), immunoglobulin M (IgM) or third component of complement (C3) were not detected by immunoftuorescence. No evidence was found for an associated neoplasm. An unexpected finding was the total absence of serum hemolytic complement activity. Further investigation revealed that the complement defect was attributable to a selective and total absence of the second component of complement (C2), as determined by both functional and immunoprecipitin assays. Family studles indicated that the defect was inherited in an autosomal recessive manner, as has been observed in the previously reported CBdeficient kindreds. This case demonstrates that typical muscle lesions of dermatomyositis can occur in the presence of a complement defect which would preclude activation of the classic (Cl-C4-C2) complement pathway. The case is of further interest as one of a series of recently reported associations of rheumatic diseases with hereditary complement deficiencies. Study of the functional properties of the propositus’ CP-deficient serum demonstrated normal generation of chemotactic activity in the presence of endotoxin or aggregated IgG, and normal or near normal bactericidal activity against Salmonella typhi 0 901 and Hemophilus lnfluenzae, type b. These findings emphasize the importance of the alternate (properdin) pathway of complement activation in these functions. In a growing number of recent reports attention has been called to the occurrence of rheumatic disorders or glomerulonephritis in patients with genetically determined deficiency of serum complement (C) components. Although the propositi of the first four reported kindred with hereditary deficiency of the second C component (C2) were found to be healthy [l-5], the development of systemic lupus erythematosus (SLE) or an SLE-like syndrome has more recently been reported in two other C2-deficient (C2D) subjects [6,7]. Another patient with C2 deficiency has experienced persistent vasculitic purpura [8]. Selective deficiency of the Clr subcomponent of the first C component has been recognized in three persons. One, on whom family studies are not available, had glomerulonephritis
January 1975
The American Journal of Medicine
Volume 58
83
HEREDITARY COMPLEMENT DEFICIENCY WITH DERMATOMYOSITI.S-LEDDY
[9, lo]. The other two are siblings; one has glomerulitis, arthritis, skin rash and inflammatory subcutaneous nodules, and the other has arthralgia, skin rash, recurrent otitis media and bronchitis [ 11,121. Independently of these observations, the presence of immunoglobulin and complement (C3) deposits in intramuscular blood vessels of patients with dermatomyositis or polymyositis has recently been reported, suggesting that immune complex deposition might have a pathogenetic role in such patients [ 131. The discovery of genetically determined absence of C2 in a patient presenting with typical dermatomyositis was, therefore, of considerable interest and is presented herein, together with immunochemical, genetic and biologic studies on the eighth reported human kindred with hereditary C2 deficiency. A preliminary report of this case has been presented [ 141. CASEREPORT A 60 year old white man (J.S.) was admitted to the University of Rochester Medical Center (URMC) because of gradual development of muscle weakness during the preceding 6 months. The patient had been generally well and physically active all his life, and had not experienced previous neuromuscular or rheumatic symptoms. As a child he had an “operation for an abscessed gland” on the right side of his neck and at age 15 or 16 surgical “drainage of pus” from a right inguinal node. He had had several ear infections in childhood, and tonsillectomy and adenoidectomy had been performed at age 10. In other respects his infectious disease history had been unremarkable. At age 30 he underwent an appendectomy at URMC; tissue sections revealed an acute inflammatory reaction with many polymorphonuclear and eosinophilic leukocytes, considered quite typical for appendicitis. The patient’s family history was negative for neuromuscular disease. Seven siblings and four children are in excellent health. His father died of accidental causes at age 59 and his mother of unknown causes at age 90. Physical examination revealed an erythematous rash on the upper eyelids, extensor surfaces of elbows, and over the dorsal surface of the metacarpal-phalangeal joints and fingers; diminished diaphragmatic excursion bilaterally with fine rales at the bases of both lungs; and a slightly enlarged heart with an irregular rhythm consistent with atrial fibrillation. Neurologic testing revealed no pharyngeal weakness or defective cranial nerve functions. There was marked proximal weakness of skeletal muscles including inability to raise his arms above his head, sit up from supine, rise from a chair, squat or lift his legs off the bed. Sensation and muscle stretch reflexes were normal. Laboratory studies included normal blood counts, urinalysis and blood chemistries. Serum glutamic-oxaloacetic transaminase (SGOT) was 83, 89 and 81 Karmen units (normal 10 to 45 Karmen units); creatine phosphokinase (CPK) was 640, 690 and 200 IU (normal 0 to 50 IU), and aldolase was in the upper normal range. Alkaline phosphatase was 24, 19 and 17 Bodansky units (normal 2 to 5
04
January
1975
The American
Journal
of Medicine
Volume
ET AL.
Bodansky units). Bromsulfalein retention was 16 per cent at 45 minutes. Bilirubin was normal. The electrocardiogram confirmed atrial fibrillation with a ventricular rate of 66, ventricular premature contractions, left axis deviation and an intraventricular conduction defect (QRS 0.10 second). Serum electrophoresis revealed moderately increased gamma globulin and slightly increased alpha, and beta globulin peaks. Serum immunoglobulins, measured by radial immunodiffusion, were IgG 1,900, IgA 96, IgM 392 mg/ 100 ml (normal 600 to 1,700, 50 to 250 and 50 to 200 mg/lOO ml, respectively). lmmunoelectrophoresis revealed that the increased IgG and IgM were heterogeneous (“polyclonal”). Serum hemolytic complement was undetectable (less than 5 CHss units) in three samples of fresh serum. Rheumatoid factor (by latex fixation test) was present at a titer of 1:160 (normal 1:20 or less), and a test for antinuclear antibody (ANA) was positive by immunofluorescence. Tests for lupus erythematosus cells were negative. By radioimmunoassay [ 15,161, binding of native deoxyribonucleic acid (DNA) by the patients’s serum globulins was normal on two occasions, whereas binding of single-strand DNA was 21 and 28 per cent, respectively (normal 5 to 23 per cent). A Venereal Disease Research Laboratory slide test was not reactive. Tests for cryoglobulins in two 10 ml serum samples were negative after the customary 72 to 96 hour incubation at 5%. but minute precipitates (less than 1 per cent cryocrit) did appear after 5 to 7 days. These precipitates were pooled, dissolved in 0.5 ml 37’C buffer, and found to contain IgM and IgG but not C2, C4, IgA or albumin (see “Methods”). A slide latex test (Hyland) for rheumatoid factor activity in the redissolved cryoprecipitate was negative. The patient’s serum was not anticomplementary when incubated with normal serum in various ratios. Studies to demonstrate a possible associated malignancy were negative. These included an intravenous pyelogram, barium enema, roentgenograms of the upper gastrointestinal tract, metastatic survey of skull, spine and long bones, and sigmoidoscopy. A small polyp in the lower sigmoid colon was removed and found to be benign. Electromyography of the triceps at rest disclosed spontaneous, prolonged polyphasic potentials, recurrent fibrillations and doublet and triplet biphasic action potentials. With activity, a full interference pattern of low voltage developed. Individual potentials had an average duration of 4 milliseconds and a markedly decreased amplitude. A muscle biopsy specimen from the left biceps (Figure 1) was consistent with inflammatory myopathy. There were large clusters of mononuclear inflammatory cells consisting of predominantly large and small lymphocytes surrounding vessels and between muscle fibers. A small number of muscle fibers were undergoing phagocytosis, and occasional fibers were regenerating. There was increased variation of muscle fiber size, a moderate increase in endomysial connective tissue and an increase in internal nuclei. The vascular involvement did not extend through the wall of muscular arteries. Neither perifascicular atrophy nor vacuolization of muscle fibers was present. Histochemical stains (myosin adenosine triphosphatase pH 9.4) demonstrated a normal mosaic pattern with no type
59
HEREDITARY
COMPLEMENT
DEFICIENCY
WITH
DERMATOMYOSITIS-LEDDY
ET AL.
Histologic sections of muscle biopsy specimen stained with hematoxylin and eosin. A, low power view showing mononuclear cell infiltrate between muscle fibers and in perivascular distribution. Variation in muscle fiber size and some increase in internal nuclei and endomysial connective tissue are also evident. B, higher power view of an intramuscular blood vessel showing perivascular mononuclear cell infiltrate. Original magnification X 200 (A) and X 430 (B); reduced by 28 per cent.
Figure 1.
atrophy, grouping or predominance. Phosphorylase was present. Direct immunofluorescent studies with specific, fluorescein-conjugated antiserums (see “Methods”) did not demonstrate staining of blood vessels or muscle fibers with antiserums to IgG, IgM, C3, albumin or fibrinogen. During therapy with prednisone, 20 mg every 6 hours, the skin rash and muscle tenderness resolved rapidly and within 3 weeks muscle strength had improved markedly. Serum CPK and SGOT enzyme levels gradually returned to normal. The serum complement (CH,,) titer did not change during this remission. Daily prednisone dosage was gradually reduced and later changed to 60 mg on alternate mornings. There was continued improvement in strength to normal. Positive tests for ANA and rheumatoid factor (1:60 to 1:320) and elevated IgM levels persisted. Serum IgG returned to normal. Alkaline phosphatase levels remained elevated and a repeat bromsulfalein test showed 20 per cent retention at 45 minutes. Antimitochondrial antibody titers (largely IgG class) were high (1:640) on several occasions (normal negative -1: 10). Tests for smooth muscle antibodies were negative. The persistently abnormal liver chemistry studies suggested some form of mild liver disease. In view of the high titer of antimitochondrial antibody and elevated serum IgM level the possibility of a developing primary biliary cirrhosis was considered, but a liver biopsy has not yet been performed. Laboratory screening of the seven siblings and a daughter yielded negative tests for ANA, antimitochondrial and antismooth muscle antibodies, and rheumatoid factor except for sibling II-4 (see Figure 2, Table II), a normal subject, who had a latex fixation titer of 1: 160. MATERIALS
was kindly supplied by Dr. Ulf Nilsson and goat antiserum to human glycine-rich P-glycoprotein (GBG, C3 proactivator, properdin factor B) by Dr. Chester A. Alper. Antiserum to human C2 was obtained by immunizing rabbits with human C2 purified as previously described [ 181. Antiserum to human Clq was prepared in rabbits by immunization with Clq purified by the method of Yonesmasu and Stroud [ 191. Goat antialbumin was purchased from Kallestad Laboratories, Minneapolis, Minn. The specificity of these antiserums was verified by immunoelectrophoresis and/or by immunodiffusion against appropriate reference antigens, i.e., normal serum, known C2-deficient serum, purified C3 and C4 (gift of Dr. H. J. Muller-Eberhard). isolated immunoglobulins and albumin. Crycglobulins. These were measured, isolated and identified as previously described [ 201. Immunofluorescence. A portion of biopsied skeletal muscle was immediately frozen in isopentane chilled to -180°C, and 4 and 8 p cryostat sections were prepared. Fluorescein-conjugated antiserums to IgG, IgM and fibrinogen were prepared in our laboratory and passed through DEAE-cellulose columns to obtain fractions with fluorescein: protein ratios of approximately 1.5:l - 2: 1 [21]. Conjugated antiserums to human C3 and to albumin were
t90
p&&
73
72
70
68
64
AND METHODS
Human Serums. Serum for complement (C) assays was either freshly drawn or stored as small aliquots at -70% Specific Antiserums. Specific antiserums to human IgM, IgG and IgA were prepared in rabbits as previously described [ 171. Rabbit antiserums to human C3 and C4 were the gift of Dr. John T. Boyer. Goat antiserum to human C5
I
.
.
t59
/”
6o I
q
C2 NORMAL
0
C2 PRESENTBUT SUBNORMAL
n q
C2 NOT DETECTABLE
5a
5a 4
2 &b
;8
5o
III
25
18
ia
NOT STUDIED
Figure 2. tive to
January 1975
Family pedigree showing
age and status
rela-
C2 deficiency.
The American Journal of Medicine
Volume 58
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purchased from Hyland Laboratories, Costa Mesa, Calif. Direct immunofluorescence examination of muscle tissue was carried out as previously described [22]. We are indebted to Dr. Mary Lou Callerame for these studies. Aliquots of these same conjugated antiserums to IgG, IgM or C3 have, during this period of study, given positive staining of renal glomeruli and skin biopsy specimens from patients with systemic lupus erythematosus. Complement Reagents and Assays. Hemolytic complement (CH,,) titrations were performed as previously reported [23]. Materials, buffers and methods for the preparation of Cl, C2 and cellular intermediates and for stoichiometric assays of Cl, C4 and C2 have been described [24,25]. Other functionally pure human complement components were obtained from the Cordis Corporation, Miami, Fla. In titrations of C2, the cell intermediate EAC14 hu was prepared.with a T max for C2 binding of less than 5 minutes. Titrations were performed by the standard assay (method I) [24,25] and by a modification of a tenfold more sensitive assay (method II) [ 261. Plates and reference standards for measuring human C3 and C4 by radial immunodiffusion were purchased, respectively, from Hyland Laboratories and from Behring Diagnostics, Somerville, N.J. Normal ranges were established with serum samples from 20 healthy donors. Agar plates for the measurement of C5 and properdin factor B (C3 proactivator) by the same technic were prepared in our own laboratory. The normal range for C5 was established with 30 normal serums with reference to a serum which had been standardized against isolated C5 (reference serum supplied by Dr. Peter Schur). C2 and Clq were estimated by double diffusion (Oucherlony) in 1 per cent agar, employing serial doubling dilutions of test serums against a given antiserum. A pool of 30 normal serums, tested concurrently, served as reference standard. The highest dilution of test serum giving a visible precipitin line was taken as the endpoint in such studies. lmmunoelectrophoretic analysis of C3 and properdin factor B (C3 proactivator) in serum was performed in 1 per cent agarose gel prepared in a pH 6.4 buffer containing 0.02 M Veronale and 0.01 M sodium EDTA. Fresh normal serum and normal serum activated by inulin [44] served as reference serums. Since the serum samples from patient J.S. had been stored for 1 to 2 years at -70°C. normal serums stored for a similar time were studied concurrently. Serum Bactericidal Activity. Serum bactericidal activity against Salmonella typhi 0 901 was assayed by previously described modifications [27] of a published method [26]. Bactericidal assay against Hemophilus influenzae, type b, was performed as follows. Rabbit antiserum to this organism was donated by Dr. Richard Robertson, Department of Microbiology, and was heat inactivated (56%, 30 minutes) before use. An “optimal” dilution (1:200) was selected for subsequent assays by titration with agammaglobulinemic serum, as described [27] for the anti-Salmonella antibody. From an overnight broth culture of H. influenzae, a subculture in Levinthal’s broth was incubated at 37°C until the O.D. at 650 nm reached 0.065. This subculture was then diluted 1:200,000 in phosphate buffered saline
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The American Journal of Medicine
solution, pH 7.1 to 7.3 containing 3 X 10m4 M calcium ion and 2 X 10m3 M magnesium ion. For the actual assays the following chilled reactants were added to sterile, glass tubes in an ice bath: phosphate buffered saline solution 0.2 ml; 1:200 rabbit antibody (in phosphate buffered saline solution) or phosphate buffered saline solution alone, 0.1 ml; 1:200,000 suspension of H. influenzae, stirred by Vortex mixer immediately before each pipetting, 0.1 ml; and test serum (or dilution in phosphate buffered saline solution) as the source of complement, 0.2 ml. Each assay tube was stirred (Vortex), and a 0.1 ml aliquot was inoculated on chocolate agar (zero time). The tubes were then placed in a 37% bath for 60 minutes, followed by Vortex mixing and inoculation of another 0.1 ml aliquot on chocolate agar (60 minute sample). The plates were placed overnight in a 37’C incubator in 5 per cent carbon dioxide. Colonies were counted, and the degree of killing in each 60 minute sample was calculated by reference to the zero-time count on the same tube. Zero-time plates typically showed from 120 to 130 colonies per plate. Control mixtures omitting a source of complement, or substituting heat inactivated serum for fresh serum, showed no killing. Chemotactic Assays. These assays were kindly performed by Dr. John Baum as described elsewhere [27,29]. Results are expressed as a ratio, the chemotactic index, explained in [29]. In all experiments, normal and C2 deficient serums were tested concurrently. Controls included activating agent without complement or with heat inactivated serum, and test serum without activating agent. Antimitochondrial and Antismooth Muscle Antibodies. These antibodies were generously assayed by Dr. Marilyn Brown, Gastroenterology Section, The Genesee Hospital, using immunofluorescence with rabbit stomach, kidney and uterus as substrates. RESULTS
Complement Assays. Table I presents the results of selected complement assays performed on our patient and available family members. Additional data from other assays carried out only on the propositus and his youngest sibling are shown in Table II. Genetic relationships in this family are presented schematically in Figure 2. As shown in Table I, our patient (N-6, Figure 2) and his youngest brother (11-8) lacked detectable C2 by standard hemolytic assay (method I) and by immunoprecipitin (Ouchterlony) analysis with rabbit antiserum to human C2. A second, more sensitive functional C2 assay (method II, [26]) also failed to detect C2 in the serum of the propositus or his brother (not shown). This assay yielded titers of 10,000 units/ml or greater in normal serums tested concurrently. This indicates that in these two siblings (II-6 and 11-8)functional C2 activity is not more than 0.1 per cent of normal. Data on the other complement components in these two serums (Tables I and II) are consistent with a selective deficiency of C2. Clq levels (not
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TABLE
Assay of Selected
Family Member* II-1 II-2 II-3 II-4 II-5 II-6 (propositus) II-7 I l-8 Ill-2 Normal subjects _____
___
Cbmplement
Hemolytic Complement (CH,,) Titer (units/ml) 120 88 84 145 73
