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Evidence from family studies for autoimmunity in dilated cardiomyopathy
Evidence from
family studies for autoimmunity in dilated cardiomyopathy
Summary
Introduction
antibodies are found in patients with autoimmune disease and their symptom-free relatives many years before clinical onset. Organ-specific cardiac antibodies can be found in patients with dilated cardiomyopathy (DCM) and their relatives, which supports the idea that DCM is an autoimmune disease. We did non-invasive cardiological assessment and antibody screening in 342 symptom-free relatives (170 male, 172 female, mean [SD] age 31 [16] years). 177 relatives were from 33 families with more than 1 affected individual (familial DCM) and 165 relatives from 31 families with only 1 affected member (non-familial DCM). The frequency of cardiac antibodies was higher among relatives of DCM patients than in controls (20% vs 3·5%, p=0·0001). In 37 (58%) of the families studied, cardiac antibodies were found in the proband and/or in at least 1 family member and were more common in familial than in non-familial DCM (24% vs 15%, p=0·036). Antibody-positive relatives were younger (26 [15] vs 33 [17] years, p=0·01) and had a larger mean echocardiographic left ventricular end-systolic dimension (35 [6] vs 32 [6], p=0·01 mm) and reduced percentage fractional shortening compared with antibody-negative relatives (31 [6] vs 34 [6],
Dilated cardiomyopathy (DCM) is a chronic heart muscle disease of unknown cause, characterised by a poorly contracting and dilated left and/or right ventricle.1 Familial disease has been recognised but non-familial cases predominate.2-s Circulating autoantibodies to distinct cardiac autoantigens, including B1. and 0 myosin heavy chain,6-11 have been described in DCM patients and have led to the suggestion of an autoimmune pathogenesis. We have reported organ-specific and disease-specific cardiac autoantibodies in patients with DCM from Englandl2 and from Italy. 13 Similar observations have been found in DCM patients from the USA.14 The detection of organ-specific cardiac autoantibodies in relatives of familial and non-familial DCM cases would support the autoimmune hypothesis and may identify subjects at risk of developing DCM. In this study we assessed cardiological status and screened for antibodies in relatives of DCM patients.
Organ-specific
p=0 008). Presence of cardiac-specific autoantibodies in symptomfree DCM relatives provides evidence of autoimmunity in a subset of our patients (58%), including familial and nonfamilial forms of DCM. These antibodies are associated with mild left ventricular systolic dysfunction on echocardiography and may be early markers for relatives at risk of DCM.
Patients and methods Patients 33 index patients with familial DCM were identified on the basis of their history and were followed at St Georges’ Hospital or at one of the collaborating institutions. Criteria for the diagnosis of familial DCM were presence of at least 1 first-degree relative with documented DCM during life or at necropsy. For dead relatives with suspected familial DCM, medical records, and necropsy findings were reviewed to confirm or refute the diagnosis. Diagnostic features for DCM at post-mortem examination included dilation and increased heart weight, compensatory myocardial wall thickening, and normal epicardial coronary arteries. We studied 31 cases with non-familial DCM from a consecutive series at St Georges’ Hospital. A three-to-four generation pedigree was constructed for each patient. Families were selected for screening on the basis of a negative family history for DCM and other practicable aspects inherent to family screening. Families with small pedigrees (< 2 available first-degree relatives), uncertain parenthood, or those in which a large number of relatives lived abroad were not offered screening; five pedigrees were excluded for these reasons. Subsequent evaluation of the remaining pedigrees confirmed the absence of familial disease.
Diagnosis Departments of Cardiological Sciences, (A L P Caforio MD, PJ Keeling MD, Prof WJ McKenna MD) and Cardiovascular Pathology (J M Mann MD), St George’s Hospital Medical School, London SW17 0RE, UK; Institute of Clinical Medicine, Department of Cardiology, University of Padua, Padua, Italy (A L P Caforio); Department of Immunology, The London Hospital Medical College, London (Prof G F Bottazzo MD); San Camillo Hospital, Rome, Italy (E Zachara MD); International Centre for Genetic Engineering and Biotechnology (L Mestroni MD); and University Hospital, Trieste, Italy (Prof F Camerini MD)
Correspondence to: Prof William J McKenna
The clinical diagnosis of DCM, with or without familial occurrence, was made according to World Health Organization criteriai and included selective coronary arteriography, left ventriculography, and right or left ventricular endomyocardial biopsy. Exclusion criteria were: coronary artery disease, valvular or congenital heart disease, systemic disease, hypertension, diabetes mellitus, specific heart-muscle disease,l,4 myocarditis, and excessive alcohol ingestion. is All DCM patients had an enlarged left ventricular end-diastolic cavity dimension (LVEDD) on two-dimensional echocardiography (>2-7 cm/m2 body surface area)’ and had impaired left ventricular systolic function (echocardiographic fractional shortening < 26% and angiographic left ventricular ejection fraction < 40%). Endomyocardial biopsies
773
DCM dilated cardiomyopathy; NYHA= New York Heart Association. Table 1: Clinical and diagnostic features of patients with DCM I
sectioned, stained with haematoxylin and eosin, and assessed by light microscopy using the Dallas criteria.16 Our study included 177 living symptom-free relatives (107 first-degree, 70 second-degree) of the 33 index DCM cases with familial disease (a further 87 first-degree relatives had died and 87 were living but were unavailable for study) and 165 relatives (88 first-degree, 77 second-degree) of the 31 non-familial DCM cases (another 36 first-degree relatives had died and 59 were unavailable for assessment). The study was done after we received approval from the ethics committee of participating institutions. Screening consisted of a clinical examination with blood pressure measurement, 12-lead electrocardiogram, M-mode and 2dimensional echocardiography, and assessment of sera for cardiac antibodies. Relatives with a history of mild systemic hypertension were included in the study but those with clinically important or documented hypertension during the physical examination were excluded. Systemic hypertension was defined as a resting blood pressure measurement of above or equal to 150/90 mm Hg on at least two distinct occasions. The electrocardiogram was taken to be abnormal according to conventional criteria.17,18 Echocardiograms were taken by an experienced operator who used high-quality equipment and was unaware of family history. Echocardiographic measurements of chamber and wall thickness were obtained at the level of the papillary muscle from twodimensional guided M-mode recordings, in the short axis view. All echocardiograms, including those done at other institutions, were reviewed at St Georges’ Hospital. Normal values for LVEDD were calculated from a standard formula and corrected for age and body surface area (BSA).19 The regression equation used to calculate the predicted normal LVEDD (LVEDD pred) was: LVEDD pred=45-3 (BSA) -0-03 (age) -7-2 ±12%. Left ventricular enlargement (LVE) was defined as LVEDD > 12% (equivalent to > 25D) of the predicted normal value. %LVE was calculated as %LVE=100 (LVEDD-LVEDD pred). The specificity of these established cut-off criteria was assessed in a separate study of 223 symptom-free normal subjects (114 male, 109 female, mean [SD] age 32 [16] years). Symptom-free relatives were classified as normal, potentially affected (either left ventricular enlargement or depressed % fractional shortening, or suspected of having DCM (left ventricular enlargement and depressed % fractional shortening). Relatives thought to have DCM underwent invasive evaluation with selective coronary arteriography when over 30 years old or when ischaemic heart disease was suspected, for example a history of chest pain or electrocardiographic evidence of cardiac ischaemia on treadmill testing. were
Immunology We used standard indirect immunofluorescence at 1 in 10 dilution on 4 lim unfixed fresh frozen cryostat sections of blood group 0 normal human atrium, ventricle, and skeletal muscle to detect cardiac antibodies.12 We measured cardiac antibody titres by doubling dilutions of sera in phosphate-buffered solution and classified the cardiac antibody patterns.12 Briefly, organ-specific antibodies produced a diffuse cytoplasmic staining of atrial, and to a lesser extent, ventricular myocytes; cross-reactive 1 antibodies gave a fine striational immunofluorescence on cardiac tissue, but
774
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2-D Echo: 2-dimensional echocardiagraphy; ECG: electrocardiogram; % FS: % fractional shortening; LVEDD: left ventricular end-diastolic dimension; LVESD: left ventricular end-systolic dimension;%LVE: 100 (LVEDD-LVEDD predicted) =% left ventricular enlargement; NYHA: New York Heart Association.
Table 2: Clinical and diagnostic features of the asymptomatic relatives of DCM patients
only weakly stained skeletal muscle fibres; cross-reactive
2 antibodies stained with a broad striational pattern both heart and skeletal muscle sections. Absorption studies with relevant tissues had confirmed the organ-specificity and cross-reactivity of the three antibody types." Cross-reactive antibodies can be found in a small proportion of DCM patients, disease controls,12 and normal controls a finding that applied to the patients included in our study. Two sera were used as standard positive (antibody titre 1 in 40) and negative controls and were titrated in every assay. The intensity of immunofluorescence of the positive standard at 1 in 40 dilution was used as the threshold for positivity. All sera tested at 1 in 10 dilution were read blindly against these standards. An additional positive control serum was titrated to assess reproducibilityP End-point titres for this serum were reproducible within one double dilution in all assays. The frequency of organ-specific cardiac antibodies in DCM patients and their relatives was compared with that observed in our previously established normal control population of blood donors (91 male, 109 female, age 35 [11] years)p,20 20 spouses (5 male, 15 female, age 42 [8] years) of patients with familial DCM were also assessed for cardiac antibodies, and represented our control group of genetically unrelated individuals sharing the patients’ environment.
Statistical analysis Student’s t-test, one-way analysis of variance, chi-squared test, or Fisher’s exact test were used as appropriate. All p values were two-tailed; p values below 0-05 indicated statistical significance.
Results Table 1 shows that clinical and diagnostic features did not differ between patients with familial and non-familial DCM. Table 2 shows the baseline characteristics of 177 symptom-free relatives of patients with familial DCM. Physical examination was normal for all subjects. 12-lead electrocardiogram was abnormal in 12 relatives (7%). In 155 relatives (87%) echocardiographic studies were of adequate quality; in the remaining 22 relatives quantitative measurements were not recorded, but left ventricular function was normal. Of the 155 relatives, 109 (70%) were normal and 46 (30%) were potentially affected. All 20 had normal examinations, physical electrocardiograms, and echocardiograms. Clinical and diagnostic features of the 165 symptom-free relatives of non-familial DCM patients are also shown in spouses
was confirmed when relatives from non-familial pedigrees were analysed
p= 000001). This familial
and
separately. Table 3 lists the results of cardiac antibody screening. Figure 1 shows antibody status in two representative pedigrees with familial DCM. The frequency of organspecific cardiac antibodies was higher among patients with familial DCM (48%) and their symptom-free relatives (24%) than in normal blood donors (3-5%, p=00001).
Chi-square test on 2 x5 table: p =0 0001; p vs normal donors: *=00 0001; p vs asymptomatic relatives of non-familial cases: A=00 036;t = 26 probands and 24 affected relatives were tested. Antibody test not done in 7 probands and 1 affected relative. ttAntibody test not done in 1 proband. Table 3: Frequency of organ-specific cardiac antibodies In patients and relatives with familial and non-familial dilated
None of the 20 spouses from the kindreds with familial disease was autoantibody positive. These antibodies were also more common among patients with non-familial DCM (30%) and their symptom-free relatives (15%) than in normal controls (3-5%, p=00001). The frequency of the antibody markers was higher in relatives of familial than in non-familial DCM (24% vs 15%, p=0036). Cardiac antibody titres in the patients with DCM and their family members were: 1 in 10 in 53 of the 101 positive sera (52%),1 in 20 in 34 (34%) and 1 in 40 in 14 (14%). All positive sera contained IgG autoantibodies. A high proportion (37 of 64, 58%) of the pedigrees studied had the proband and/or at least one relative who was antibody positive. Cardiac antibodies were more common in relatives of antibody-positive compared with antibody-negative probands (38 of 105, 36% vs 27 of 204, 13%, p=00001). We found cardiac antibodies in at least one first-degree relative in 18 of 19 (95%) families whose proband was antibody positive, in 14 of 37 (38%) families whose proband was antibody negative, and in 4 of 8 (50%) of the pedigrees where antibody status was not known
cardlomyopathy table 2. The non-familial DCM relatives were older than familial DCM patients (p = 0-0002). All 165 relatives were symptom-free with normal exercise tolerance; 12-lead electrocardiogram was normal in all but 11 relatives (7%). In 140 relatives of non-familial DCM (85%) echocardiographic studies were adequate. In the other 25
relatives, quantitative measurements were not recorded, although left ventricular function was normal. Of the 140 relatives with good quality echocardiographic images, 104 (74%) were normal and 36 (26%) were potentially affected. Similar proportions of symptom-free relatives from familial and non-familial pedigrees were classified as potentially affected (30% vs 26% p = 0-45). The frequency of potentially affected subjects was significantly higher in the symptom-free relatives of DCM patients than in our control group of unrelated normal subjects who had for echocardiographic screening hypertrophic and were unaffected cardiomyopathy (28% vs 12%,
(p = 0-0002). The familial and the non-familial pedigrees were analysed separately for antibody status in the proband and
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Figure 1: Antibody status in two representative pedigrees with familial
DCM 0 = male; O = female; open symbols: unaffected subjects; solid symbols: subjects with DCM; shaded symbols: subjects who were considered potentially affected on the basis of an abnormal echocardiogram. Deceased family members are indicated by a slash. The arrow indicates the proband, the minus sign indicates negative cardiac antibody status and plus signs positive cardiac antibody status. *Indicates relatives who were also affected by
insulin-dependent diabetes mellitus. 775
A=Detection of antibody
in at least
B=Detection of antibody in
one
at least one
first
degree relative
with DCM
Table 4: Clinical and diagnostic features of asymptomatic DCM relatives in relation to antibody status
asymptomatic first degree relative
shortening was lower (p 0-03) in relatives with high-titre antibody: 34 (6) in antibody-negative relatives, 31 (7) in relatives with (1 in 10) antibody titre and 32 (5) in those patients with antibody titre (1 in 20 or 1 in 40). =
Figure 2: Familial DCM: cardiac antibody status In the proband and first-degree relatives *Indicates pedigrees with positive autoimmune serology in the proband and/or in at least one first-degree relative (n = 22). A Denotes families with negative autoimmune serology in the proband and/or in at least one first-degree relative (n = 11).
in
first-degree relatives (figures 2 and 3). In 22 pedigrees (67%) with familial and in 15 with non-familial DCM (48 %), cardiac antibodies were found in the proband and/or in at least 1 first-degree relative. In the remaining 11 familial and 16 non-familial pedigrees, cardiac antibodies were not found in the proband or in the family. The proportion of pedigrees with positive autoimmune serology was slightly but not significantly higher in familial than in non-familial DCM (22 of 33, 67% vs 15 of 31, 48%, p=0-14), and was similar in smaller (>5 relatives) and larger (> 5 relatives) families (28 of 46,61 % vs 9 of 18,50%, p = 0-42). Table 4 shows clinical and diagnostic features in relation to cardiac antibody status. When symptom-free relatives from familial and non-familial cases were analysed together, the relatives with antibody were significantly younger than those without (p=001). Mean age did not differ in antibody positive relatives from familial and non-familial pedigrees (26 [ 16] vs 25 [15] years, p=0 62). Similar proportions of relatives with and without cardiac antibody were classified as potentially affected on the basis of the established echocardiographic cutoffs (33% vs 26-5%, p=0-30). Conversely, mean left ventricular endsystolic dimension and % left ventricular enlargement were higher and % fractional shortening was lower in antibody positive relatives compared with antibody negative patients (p=0-01, p = 0004, and p = 0-008). Mean % fractional Probands with
non
familial DCM
(n=31) I
Figure 3: Non-familial DCM: cardiac antibody status In the proband and In first-degree relatives *Indicates positive autoimmune serology in the proband and/or in at least one first-degree relative (n = 15). A Denotes families with negative autoimmune serology in the proband and/or in at least one first-degree relative (n= 16). ).
776
Discussion
Organ-specific cardiac antibodies were found in 41 % of DCM patients and in 20% of their symptom-free relatives, but were absent or uncommon in normal subjects and in genetically unrelated symptom-free individuals from the same household, in particular the patients’ spouses. This lends strong evidence for genetic predisposition and involvement of organ-specific autoimmunity in DCM. When looked at in the context of other organ-specific autoimmune diseases, the features seen in DCM resemble those found in insulin-dependent diabetes mellitus (IDDM). Both conditions exhibit a male preponderance, an HLA-DR4 association,13,21 familial aggregation in 15-20% of cases,3,22 and an increased frequency of the corresponding organ-specific antibodies among symptomfree relatives. However, the frequency of antibody-positive patients is markedly different in DCM and IDDM. The majority (80-90%) of IDDM patients are positive at diagnosis for at least one of the islet-cell antibodies. Conversely, autoantibodies to each of the cardiac antigens so far reported,6,11 including the adenine nucleotide translocator,9 the &bgr; receptor,6,7 a and &bgr; myosin heavy chains" and the organ-specific cytoplasmic antigen(s) detected by indirect immunofluorescence12 are found in only 30-40% of DCM patients, when these specificities are tested separately. It remains to be seen whether patients classified as seronegative for one antibody marker are positive for another. Exchange of sera among laboratories currently testing the individual cardiac antibodies should clarify this important point. The absence of cardiac antibodies in the majority of DCM patients could also indicate that they are early markers that are no longer detectable with disease progression. This is a well-documented phenomenon in IDDM where islet cell antibodies are often not detectable in longstanding disease. In support of this concept, we have reported a higher frequency of cardiac antibodies in younger DCM patients with shorter disease duration.12 In this study, cardiac antibodies were associated with younger age when found in symptom-free relatives of DCM patients. DCM might be heterogeneous with a proportion of the antibody-negative cases having a non-autoimmune
pathogenesis. Our study examines the issue of disease heterogeneity and indicates that autoimmunity is involved in most pedigrees with familial and non-familial DCM. Cardiac
antibodies were not found in the proband or in the family members in 27 pedigrees (42%). Although follow-up studies might reveal that some symptom-free relatives from these families will develop cardiac antibodies, our data suggest that in antibody-negative families autoimmunity is unlikely to be involved. Will the cardiac antibody be a marker of disease predisposition in first-degree relatives of patients with DCM? Islet-cell antibodies are useful predictive markers for IDDM in symptom-free individuals from high-risk
populations, particularly subjects who are genetically related to the index patients, such as first-degree relatives and discordant monozygotic twins or triplets.23 These antibodies are sometimes found years before the onset of clinical symptoms or detectable abnormalities of 0 cell function. Follow-up is needed to determine whether antibody status is predictive of disease susceptibility in DCM. Our evidence suggests that the antibody, as in IDDM, is associated with early disease, though accurate identification of such patients remains problematic. Echocardiographic evaluation was used as a practical means to assess minor changes in left ventricular structure and function. The frequency of left ventricular enlargement, was higher (28%) in symptom-free relatives of DCM families than in unrelated normal subjects (12%). This is consistent with previous observationsll and may indicate that the echocardiographic abnormalities represent early disease changes. When established cutoffs for the echocardiographic measurements are used there will be an overlap of measurements in early disease with the gaussian distribution of normality. This may explain the presence of left ventricular enlargement in 12 % of our normal subjects, and the detection of a similar proportion of seropositivity in normal and potentially affected relatives. The finding, however, that left ventricular end-systolic dimension was higher, fractional shortening was lower, and left ventricular enlargement was greater in symptom-free relatives who were antibody positive suggests that this antibody is associated with early disease. Organ-specific cardiac antibodies present in relatives of patients with and without familial DCM provides evidence33 for autoimmunity in approximately 60% of both familial and non-familial forms of the disease. However, whether these antibodies have a direct pathogenic role remains to be established. These antibodies were not detected in the remaining 40% of the pedigrees and there may be a DCM subset where autoimmunity is not involved. If the antibody is shown to identify family members at risk, the potential role of pre-clinical introduction of ACE inhibition and immunosuppressive therapy should also be tested
prospectively with an appropriate study design. We thank Prof Sergio Dalla Volta, Prof Stefano Schiaffino, and Prof Gaetano Thiene (University of Padua, Italy) for support and advice, and Prof Michael J Davies (St George’s Hospital, London) for providing laboratory facilities. We are grateful to Dr Daniela Miani (Cardiology, Trieste, Italy), Dr Mauro Giacca, and Prof Arturo Falaschi (ICGEBT, Trieste, Italy) for technical support. We also thank Dr Ludwig Thierfelder (Dept of Cardiology, University of Freiburg, Germany) for allowing us to study his patients, our research sister Miss Sonia E Bent, our echocardiographers Mrs Gill Smith and Dr Bruno Pinamonti, and our immunology technician Mr Les H Haven. This work was supported by a project grant from the British Heart Foundation. Dr A L P Caforio is
supported by the Veneto Region Target Project on Cardiomyopathies (Venice, Italy) and by the National Research Council target project "FAT.MA" (Rome, Italy). References 1
Brandeburg RO, Chazov E, Cherian G, et al. Report of the WHO/
2
cardiomyopathies. Circulation 1981; 64: 437A-38A, Mestroni L, Miani D, De Lenarda A, et al. Clinical and pathological study of familial dilated cardiomyopathy. Am J Cardiol 1990; 65:
ISFC task force on definition and classification of the
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15
16
1449-53. Michels VV, Moll PP, Miller FA, et al. The frequency of familial dilated cardiomyopathy in a series of patients with idiopathic dilated cardiomyopathy. N Engl J Med 1992; 326: 77-82. Manolio TA, Baughman KL, Rodeheffer R, et al. Prevalence and etiology of idiopathic dilated cardiomyopathy (Summary of a National Heart, Lung, and Blood Institute workshop). Am J Cardiol 1992; 69: 1458-66. Zachara E, Caforio ALP, Carboni GP, et al. Familial aggregation of idiopathic dilated cardiomyopathy: clinical features and pedigree analysis in 14 families. Br Heart J 1993; 69: 129-35. Limas CJ, Goldenberg IF, Limas C. Autoantibodies against &bgr;-adrenoceptors in human idiopathic dilated cardiomyopathy. Circ Res 1989; 64: 97-103. Magnusson Y, Marullo S, Hoyer S, et al. Mapping of a functional autoimmune epitope on the &bgr;1 adrenergic receptor in patients with idiopathic dilated cardiomyopathy. J Clin Invest 1990; 86: 1658-63. Klein R, Maisch B, Kochsiek K, Berg PA. Demonstration of organspecific antibodies against heart mitochondria (anti-M7) in sera from patients with some forms of heart diseases. Clin Exp Immunol 1984; 58: 283-92. Schulteiss HP, Bolte HD. Immunological analysis of auto-antibodies against the adenine nucleotide translocator in dilated cardiomyopathy. J Mol Cell Cardiol 1985; 17: 603-17. Maisch B, Deeg P, Liebau G, Kochsiek K. Diagnostic relevance of humoral and cytotoxic immune reactions in primary and secondary dilated cardiomyopathy. Am J Cardiol 1983; 52: 1072-78. Caforio ALP, Grazzini M, Mann JM, et al. Identification of the &agr; and &bgr; myosin heavy chain isoforms as major autoantigens in dilated cardiomyopathy. Circulation 1992; 85: 1734-42. Caforio ALP, Bonifacio E, Stewart JT, et al. Novel organ-specific cardiac autoantibodies in dilated cardiomyopathy. J Am Coll Cardiol 1990; 15: 1527-34. Caforio ALP, Martinetti M, Bonifacio E, et al. Idiopathic dilated cardiomyopathy: lack of association between organ-specific cardiac antibodies and HLA-DR antigens. Tissue Antigens 1992; 39: 236-40. Neumann DA, Burek CL, Baughman KL, Rose NR, Herskowitz A. Circulating heart-reactive antibodies in patients with myocarditis or cardiomyopathy. J Am Coil Cardiol 1990; 16: 839-46. Fuster V, Gersh BJ, Giuliani ER, Tajik AJ, Brandenburg AO, Frye RL. The natural history of idiopathic dilated cardiomyopathy. Am J Cardiol 1981; 47: 525-31. Aretz HT, Billingham ME, Edwards WE, et al. Myocarditis: a histopathological definition and classification. Am J Cardiol Pathol
1985; 1: 1-10. 17 Bethesda Conference on Optimal Electrocardiography. Task Force 1: standardization of terminology and interpretation. Am J Cardiol 1978; 41: 130-45. 18 Sokolow M, Lyon TP. The ventricular complex in left ventricular hypertrophy as obtained by unipolar precordial and limb leads. Am Heart J 1949; 37: 161-86. 19 Henry WL, Gardin JM, Ware JH. Echocardiographic measurements in normal subjects from infancy to old age. Circulation 1980; 62: 1054-61. 20 Caforio ALP, Wagner R, Gill JS, et al. Organ-specific cardiac antibodies: serological markers for systemic hypertension in autoimmune polyendocrinopathy. Lancet 1991; 337: 1111-15. 21 Carlquist JF, Menlove RL, Murray MB, O’Connell JB, Anderson JF. HLA class II (DR and DQ) antigen associations in idiopathic dilated cardiomyopathy. Validation study and meta-analysis of published HLA association studies. Circulation 1991; 83: 515-22. 22 Karjalainen J, Salmena P, Ilonen J, Surcel HM, Knip M. A comparison of childhood and adult type I diabetes mellitus. N Engl J Med 1989; 320: 881-86. 23 Srikanta S, Ganda OP, Eisenbarth GS, Soldner JS. Islet cell antibodies in and beta cell function in monozygotic triplets and twins initially discordant for type 1 diabetes mellitus. N Engl J Med 1983; 308: 322-25.
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family studies for autoimmunity in dilated cardiomyopathy
Summary
Introduction
antibodies are found in patients with autoimmune disease and their symptom-free relatives many years before clinical onset. Organ-specific cardiac antibodies can be found in patients with dilated cardiomyopathy (DCM) and their relatives, which supports the idea that DCM is an autoimmune disease. We did non-invasive cardiological assessment and antibody screening in 342 symptom-free relatives (170 male, 172 female, mean [SD] age 31 [16] years). 177 relatives were from 33 families with more than 1 affected individual (familial DCM) and 165 relatives from 31 families with only 1 affected member (non-familial DCM). The frequency of cardiac antibodies was higher among relatives of DCM patients than in controls (20% vs 3·5%, p=0·0001). In 37 (58%) of the families studied, cardiac antibodies were found in the proband and/or in at least 1 family member and were more common in familial than in non-familial DCM (24% vs 15%, p=0·036). Antibody-positive relatives were younger (26 [15] vs 33 [17] years, p=0·01) and had a larger mean echocardiographic left ventricular end-systolic dimension (35 [6] vs 32 [6], p=0·01 mm) and reduced percentage fractional shortening compared with antibody-negative relatives (31 [6] vs 34 [6],
Dilated cardiomyopathy (DCM) is a chronic heart muscle disease of unknown cause, characterised by a poorly contracting and dilated left and/or right ventricle.1 Familial disease has been recognised but non-familial cases predominate.2-s Circulating autoantibodies to distinct cardiac autoantigens, including B1. and 0 myosin heavy chain,6-11 have been described in DCM patients and have led to the suggestion of an autoimmune pathogenesis. We have reported organ-specific and disease-specific cardiac autoantibodies in patients with DCM from Englandl2 and from Italy. 13 Similar observations have been found in DCM patients from the USA.14 The detection of organ-specific cardiac autoantibodies in relatives of familial and non-familial DCM cases would support the autoimmune hypothesis and may identify subjects at risk of developing DCM. In this study we assessed cardiological status and screened for antibodies in relatives of DCM patients.
Organ-specific
p=0 008). Presence of cardiac-specific autoantibodies in symptomfree DCM relatives provides evidence of autoimmunity in a subset of our patients (58%), including familial and nonfamilial forms of DCM. These antibodies are associated with mild left ventricular systolic dysfunction on echocardiography and may be early markers for relatives at risk of DCM.
Patients and methods Patients 33 index patients with familial DCM were identified on the basis of their history and were followed at St Georges’ Hospital or at one of the collaborating institutions. Criteria for the diagnosis of familial DCM were presence of at least 1 first-degree relative with documented DCM during life or at necropsy. For dead relatives with suspected familial DCM, medical records, and necropsy findings were reviewed to confirm or refute the diagnosis. Diagnostic features for DCM at post-mortem examination included dilation and increased heart weight, compensatory myocardial wall thickening, and normal epicardial coronary arteries. We studied 31 cases with non-familial DCM from a consecutive series at St Georges’ Hospital. A three-to-four generation pedigree was constructed for each patient. Families were selected for screening on the basis of a negative family history for DCM and other practicable aspects inherent to family screening. Families with small pedigrees (< 2 available first-degree relatives), uncertain parenthood, or those in which a large number of relatives lived abroad were not offered screening; five pedigrees were excluded for these reasons. Subsequent evaluation of the remaining pedigrees confirmed the absence of familial disease.
Diagnosis Departments of Cardiological Sciences, (A L P Caforio MD, PJ Keeling MD, Prof WJ McKenna MD) and Cardiovascular Pathology (J M Mann MD), St George’s Hospital Medical School, London SW17 0RE, UK; Institute of Clinical Medicine, Department of Cardiology, University of Padua, Padua, Italy (A L P Caforio); Department of Immunology, The London Hospital Medical College, London (Prof G F Bottazzo MD); San Camillo Hospital, Rome, Italy (E Zachara MD); International Centre for Genetic Engineering and Biotechnology (L Mestroni MD); and University Hospital, Trieste, Italy (Prof F Camerini MD)
Correspondence to: Prof William J McKenna
The clinical diagnosis of DCM, with or without familial occurrence, was made according to World Health Organization criteriai and included selective coronary arteriography, left ventriculography, and right or left ventricular endomyocardial biopsy. Exclusion criteria were: coronary artery disease, valvular or congenital heart disease, systemic disease, hypertension, diabetes mellitus, specific heart-muscle disease,l,4 myocarditis, and excessive alcohol ingestion. is All DCM patients had an enlarged left ventricular end-diastolic cavity dimension (LVEDD) on two-dimensional echocardiography (>2-7 cm/m2 body surface area)’ and had impaired left ventricular systolic function (echocardiographic fractional shortening < 26% and angiographic left ventricular ejection fraction < 40%). Endomyocardial biopsies
773
DCM dilated cardiomyopathy; NYHA= New York Heart Association. Table 1: Clinical and diagnostic features of patients with DCM I
sectioned, stained with haematoxylin and eosin, and assessed by light microscopy using the Dallas criteria.16 Our study included 177 living symptom-free relatives (107 first-degree, 70 second-degree) of the 33 index DCM cases with familial disease (a further 87 first-degree relatives had died and 87 were living but were unavailable for study) and 165 relatives (88 first-degree, 77 second-degree) of the 31 non-familial DCM cases (another 36 first-degree relatives had died and 59 were unavailable for assessment). The study was done after we received approval from the ethics committee of participating institutions. Screening consisted of a clinical examination with blood pressure measurement, 12-lead electrocardiogram, M-mode and 2dimensional echocardiography, and assessment of sera for cardiac antibodies. Relatives with a history of mild systemic hypertension were included in the study but those with clinically important or documented hypertension during the physical examination were excluded. Systemic hypertension was defined as a resting blood pressure measurement of above or equal to 150/90 mm Hg on at least two distinct occasions. The electrocardiogram was taken to be abnormal according to conventional criteria.17,18 Echocardiograms were taken by an experienced operator who used high-quality equipment and was unaware of family history. Echocardiographic measurements of chamber and wall thickness were obtained at the level of the papillary muscle from twodimensional guided M-mode recordings, in the short axis view. All echocardiograms, including those done at other institutions, were reviewed at St Georges’ Hospital. Normal values for LVEDD were calculated from a standard formula and corrected for age and body surface area (BSA).19 The regression equation used to calculate the predicted normal LVEDD (LVEDD pred) was: LVEDD pred=45-3 (BSA) -0-03 (age) -7-2 ±12%. Left ventricular enlargement (LVE) was defined as LVEDD > 12% (equivalent to > 25D) of the predicted normal value. %LVE was calculated as %LVE=100 (LVEDD-LVEDD pred). The specificity of these established cut-off criteria was assessed in a separate study of 223 symptom-free normal subjects (114 male, 109 female, mean [SD] age 32 [16] years). Symptom-free relatives were classified as normal, potentially affected (either left ventricular enlargement or depressed % fractional shortening, or suspected of having DCM (left ventricular enlargement and depressed % fractional shortening). Relatives thought to have DCM underwent invasive evaluation with selective coronary arteriography when over 30 years old or when ischaemic heart disease was suspected, for example a history of chest pain or electrocardiographic evidence of cardiac ischaemia on treadmill testing. were
Immunology We used standard indirect immunofluorescence at 1 in 10 dilution on 4 lim unfixed fresh frozen cryostat sections of blood group 0 normal human atrium, ventricle, and skeletal muscle to detect cardiac antibodies.12 We measured cardiac antibody titres by doubling dilutions of sera in phosphate-buffered solution and classified the cardiac antibody patterns.12 Briefly, organ-specific antibodies produced a diffuse cytoplasmic staining of atrial, and to a lesser extent, ventricular myocytes; cross-reactive 1 antibodies gave a fine striational immunofluorescence on cardiac tissue, but
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2-D Echo: 2-dimensional echocardiagraphy; ECG: electrocardiogram; % FS: % fractional shortening; LVEDD: left ventricular end-diastolic dimension; LVESD: left ventricular end-systolic dimension;%LVE: 100 (LVEDD-LVEDD predicted) =% left ventricular enlargement; NYHA: New York Heart Association.
Table 2: Clinical and diagnostic features of the asymptomatic relatives of DCM patients
only weakly stained skeletal muscle fibres; cross-reactive
2 antibodies stained with a broad striational pattern both heart and skeletal muscle sections. Absorption studies with relevant tissues had confirmed the organ-specificity and cross-reactivity of the three antibody types." Cross-reactive antibodies can be found in a small proportion of DCM patients, disease controls,12 and normal controls a finding that applied to the patients included in our study. Two sera were used as standard positive (antibody titre 1 in 40) and negative controls and were titrated in every assay. The intensity of immunofluorescence of the positive standard at 1 in 40 dilution was used as the threshold for positivity. All sera tested at 1 in 10 dilution were read blindly against these standards. An additional positive control serum was titrated to assess reproducibilityP End-point titres for this serum were reproducible within one double dilution in all assays. The frequency of organ-specific cardiac antibodies in DCM patients and their relatives was compared with that observed in our previously established normal control population of blood donors (91 male, 109 female, age 35 [11] years)p,20 20 spouses (5 male, 15 female, age 42 [8] years) of patients with familial DCM were also assessed for cardiac antibodies, and represented our control group of genetically unrelated individuals sharing the patients’ environment.
Statistical analysis Student’s t-test, one-way analysis of variance, chi-squared test, or Fisher’s exact test were used as appropriate. All p values were two-tailed; p values below 0-05 indicated statistical significance.
Results Table 1 shows that clinical and diagnostic features did not differ between patients with familial and non-familial DCM. Table 2 shows the baseline characteristics of 177 symptom-free relatives of patients with familial DCM. Physical examination was normal for all subjects. 12-lead electrocardiogram was abnormal in 12 relatives (7%). In 155 relatives (87%) echocardiographic studies were of adequate quality; in the remaining 22 relatives quantitative measurements were not recorded, but left ventricular function was normal. Of the 155 relatives, 109 (70%) were normal and 46 (30%) were potentially affected. All 20 had normal examinations, physical electrocardiograms, and echocardiograms. Clinical and diagnostic features of the 165 symptom-free relatives of non-familial DCM patients are also shown in spouses
was confirmed when relatives from non-familial pedigrees were analysed
p= 000001). This familial
and
separately. Table 3 lists the results of cardiac antibody screening. Figure 1 shows antibody status in two representative pedigrees with familial DCM. The frequency of organspecific cardiac antibodies was higher among patients with familial DCM (48%) and their symptom-free relatives (24%) than in normal blood donors (3-5%, p=00001).
Chi-square test on 2 x5 table: p =0 0001; p vs normal donors: *=00 0001; p vs asymptomatic relatives of non-familial cases: A=00 036;t = 26 probands and 24 affected relatives were tested. Antibody test not done in 7 probands and 1 affected relative. ttAntibody test not done in 1 proband. Table 3: Frequency of organ-specific cardiac antibodies In patients and relatives with familial and non-familial dilated
None of the 20 spouses from the kindreds with familial disease was autoantibody positive. These antibodies were also more common among patients with non-familial DCM (30%) and their symptom-free relatives (15%) than in normal controls (3-5%, p=00001). The frequency of the antibody markers was higher in relatives of familial than in non-familial DCM (24% vs 15%, p=0036). Cardiac antibody titres in the patients with DCM and their family members were: 1 in 10 in 53 of the 101 positive sera (52%),1 in 20 in 34 (34%) and 1 in 40 in 14 (14%). All positive sera contained IgG autoantibodies. A high proportion (37 of 64, 58%) of the pedigrees studied had the proband and/or at least one relative who was antibody positive. Cardiac antibodies were more common in relatives of antibody-positive compared with antibody-negative probands (38 of 105, 36% vs 27 of 204, 13%, p=00001). We found cardiac antibodies in at least one first-degree relative in 18 of 19 (95%) families whose proband was antibody positive, in 14 of 37 (38%) families whose proband was antibody negative, and in 4 of 8 (50%) of the pedigrees where antibody status was not known
cardlomyopathy table 2. The non-familial DCM relatives were older than familial DCM patients (p = 0-0002). All 165 relatives were symptom-free with normal exercise tolerance; 12-lead electrocardiogram was normal in all but 11 relatives (7%). In 140 relatives of non-familial DCM (85%) echocardiographic studies were adequate. In the other 25
relatives, quantitative measurements were not recorded, although left ventricular function was normal. Of the 140 relatives with good quality echocardiographic images, 104 (74%) were normal and 36 (26%) were potentially affected. Similar proportions of symptom-free relatives from familial and non-familial pedigrees were classified as potentially affected (30% vs 26% p = 0-45). The frequency of potentially affected subjects was significantly higher in the symptom-free relatives of DCM patients than in our control group of unrelated normal subjects who had for echocardiographic screening hypertrophic and were unaffected cardiomyopathy (28% vs 12%,
(p = 0-0002). The familial and the non-familial pedigrees were analysed separately for antibody status in the proband and
-
-
+
-
_
-
-
-
-
-
-
_
_
--
Figure 1: Antibody status in two representative pedigrees with familial
DCM 0 = male; O = female; open symbols: unaffected subjects; solid symbols: subjects with DCM; shaded symbols: subjects who were considered potentially affected on the basis of an abnormal echocardiogram. Deceased family members are indicated by a slash. The arrow indicates the proband, the minus sign indicates negative cardiac antibody status and plus signs positive cardiac antibody status. *Indicates relatives who were also affected by
insulin-dependent diabetes mellitus. 775
A=Detection of antibody
in at least
B=Detection of antibody in
one
at least one
first
degree relative
with DCM
Table 4: Clinical and diagnostic features of asymptomatic DCM relatives in relation to antibody status
asymptomatic first degree relative
shortening was lower (p 0-03) in relatives with high-titre antibody: 34 (6) in antibody-negative relatives, 31 (7) in relatives with (1 in 10) antibody titre and 32 (5) in those patients with antibody titre (1 in 20 or 1 in 40). =
Figure 2: Familial DCM: cardiac antibody status In the proband and first-degree relatives *Indicates pedigrees with positive autoimmune serology in the proband and/or in at least one first-degree relative (n = 22). A Denotes families with negative autoimmune serology in the proband and/or in at least one first-degree relative (n = 11).
in
first-degree relatives (figures 2 and 3). In 22 pedigrees (67%) with familial and in 15 with non-familial DCM (48 %), cardiac antibodies were found in the proband and/or in at least 1 first-degree relative. In the remaining 11 familial and 16 non-familial pedigrees, cardiac antibodies were not found in the proband or in the family. The proportion of pedigrees with positive autoimmune serology was slightly but not significantly higher in familial than in non-familial DCM (22 of 33, 67% vs 15 of 31, 48%, p=0-14), and was similar in smaller (>5 relatives) and larger (> 5 relatives) families (28 of 46,61 % vs 9 of 18,50%, p = 0-42). Table 4 shows clinical and diagnostic features in relation to cardiac antibody status. When symptom-free relatives from familial and non-familial cases were analysed together, the relatives with antibody were significantly younger than those without (p=001). Mean age did not differ in antibody positive relatives from familial and non-familial pedigrees (26 [ 16] vs 25 [15] years, p=0 62). Similar proportions of relatives with and without cardiac antibody were classified as potentially affected on the basis of the established echocardiographic cutoffs (33% vs 26-5%, p=0-30). Conversely, mean left ventricular endsystolic dimension and % left ventricular enlargement were higher and % fractional shortening was lower in antibody positive relatives compared with antibody negative patients (p=0-01, p = 0004, and p = 0-008). Mean % fractional Probands with
non
familial DCM
(n=31) I
Figure 3: Non-familial DCM: cardiac antibody status In the proband and In first-degree relatives *Indicates positive autoimmune serology in the proband and/or in at least one first-degree relative (n = 15). A Denotes families with negative autoimmune serology in the proband and/or in at least one first-degree relative (n= 16). ).
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Discussion
Organ-specific cardiac antibodies were found in 41 % of DCM patients and in 20% of their symptom-free relatives, but were absent or uncommon in normal subjects and in genetically unrelated symptom-free individuals from the same household, in particular the patients’ spouses. This lends strong evidence for genetic predisposition and involvement of organ-specific autoimmunity in DCM. When looked at in the context of other organ-specific autoimmune diseases, the features seen in DCM resemble those found in insulin-dependent diabetes mellitus (IDDM). Both conditions exhibit a male preponderance, an HLA-DR4 association,13,21 familial aggregation in 15-20% of cases,3,22 and an increased frequency of the corresponding organ-specific antibodies among symptomfree relatives. However, the frequency of antibody-positive patients is markedly different in DCM and IDDM. The majority (80-90%) of IDDM patients are positive at diagnosis for at least one of the islet-cell antibodies. Conversely, autoantibodies to each of the cardiac antigens so far reported,6,11 including the adenine nucleotide translocator,9 the &bgr; receptor,6,7 a and &bgr; myosin heavy chains" and the organ-specific cytoplasmic antigen(s) detected by indirect immunofluorescence12 are found in only 30-40% of DCM patients, when these specificities are tested separately. It remains to be seen whether patients classified as seronegative for one antibody marker are positive for another. Exchange of sera among laboratories currently testing the individual cardiac antibodies should clarify this important point. The absence of cardiac antibodies in the majority of DCM patients could also indicate that they are early markers that are no longer detectable with disease progression. This is a well-documented phenomenon in IDDM where islet cell antibodies are often not detectable in longstanding disease. In support of this concept, we have reported a higher frequency of cardiac antibodies in younger DCM patients with shorter disease duration.12 In this study, cardiac antibodies were associated with younger age when found in symptom-free relatives of DCM patients. DCM might be heterogeneous with a proportion of the antibody-negative cases having a non-autoimmune
pathogenesis. Our study examines the issue of disease heterogeneity and indicates that autoimmunity is involved in most pedigrees with familial and non-familial DCM. Cardiac
antibodies were not found in the proband or in the family members in 27 pedigrees (42%). Although follow-up studies might reveal that some symptom-free relatives from these families will develop cardiac antibodies, our data suggest that in antibody-negative families autoimmunity is unlikely to be involved. Will the cardiac antibody be a marker of disease predisposition in first-degree relatives of patients with DCM? Islet-cell antibodies are useful predictive markers for IDDM in symptom-free individuals from high-risk
populations, particularly subjects who are genetically related to the index patients, such as first-degree relatives and discordant monozygotic twins or triplets.23 These antibodies are sometimes found years before the onset of clinical symptoms or detectable abnormalities of 0 cell function. Follow-up is needed to determine whether antibody status is predictive of disease susceptibility in DCM. Our evidence suggests that the antibody, as in IDDM, is associated with early disease, though accurate identification of such patients remains problematic. Echocardiographic evaluation was used as a practical means to assess minor changes in left ventricular structure and function. The frequency of left ventricular enlargement, was higher (28%) in symptom-free relatives of DCM families than in unrelated normal subjects (12%). This is consistent with previous observationsll and may indicate that the echocardiographic abnormalities represent early disease changes. When established cutoffs for the echocardiographic measurements are used there will be an overlap of measurements in early disease with the gaussian distribution of normality. This may explain the presence of left ventricular enlargement in 12 % of our normal subjects, and the detection of a similar proportion of seropositivity in normal and potentially affected relatives. The finding, however, that left ventricular end-systolic dimension was higher, fractional shortening was lower, and left ventricular enlargement was greater in symptom-free relatives who were antibody positive suggests that this antibody is associated with early disease. Organ-specific cardiac antibodies present in relatives of patients with and without familial DCM provides evidence33 for autoimmunity in approximately 60% of both familial and non-familial forms of the disease. However, whether these antibodies have a direct pathogenic role remains to be established. These antibodies were not detected in the remaining 40% of the pedigrees and there may be a DCM subset where autoimmunity is not involved. If the antibody is shown to identify family members at risk, the potential role of pre-clinical introduction of ACE inhibition and immunosuppressive therapy should also be tested
prospectively with an appropriate study design. We thank Prof Sergio Dalla Volta, Prof Stefano Schiaffino, and Prof Gaetano Thiene (University of Padua, Italy) for support and advice, and Prof Michael J Davies (St George’s Hospital, London) for providing laboratory facilities. We are grateful to Dr Daniela Miani (Cardiology, Trieste, Italy), Dr Mauro Giacca, and Prof Arturo Falaschi (ICGEBT, Trieste, Italy) for technical support. We also thank Dr Ludwig Thierfelder (Dept of Cardiology, University of Freiburg, Germany) for allowing us to study his patients, our research sister Miss Sonia E Bent, our echocardiographers Mrs Gill Smith and Dr Bruno Pinamonti, and our immunology technician Mr Les H Haven. This work was supported by a project grant from the British Heart Foundation. Dr A L P Caforio is
supported by the Veneto Region Target Project on Cardiomyopathies (Venice, Italy) and by the National Research Council target project "FAT.MA" (Rome, Italy). References 1
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