HLA and immunoglobulin polymorphisms in idiopathic dilated cardiomyopathy

HLA and Immunoglobulin Polymorphisms in Idiopathic Dilated Cardiomyopathy Miryam Martinetti, Jean Michel Dugoujon, Alida L. P. Caforio, Giselle Schwarz, Antonello Gavazzi, Gabriella Graziano, Eloisa Arbustini, Renata Lorini, William J. McKenna, Gian Franco Bottazzo, and Mariaclara Cuccia ABSTRACT: Dilated cardiomyopathy (DCM) is an idi-

opathic heart muscle disorder. The presence of circulating cardiac antibodies and the association with HLA-DR4 are consistent with autoimmune pathogenesis in a subset of patients. Sixty-eight DCM patients and 277 controls were typed for IgG heavy-chain constant region (Gm) and K light-chain (Km) allotypes. All patients and 210 of the 277 controls were HLA-DR typed. The Gm (I, 3, 17; 23; 5", 21, 28) phenotype was overrepresented in DCM compared with controls (25% vs 13a~, p = 0.0139, Pc = NS, RR = 2.23). The frequency of this phenotype was higher in patients with younger age at onset, shorter symptom duration, and among those who were positive for cardiac as well as for non-organ-specific autoantibodies than in controls. A higher frequency of the Gm (1, -+2, 3, 17; -+23; 5", 21, 28) heterozygous phenotypes was

also found in DCM compared to controls (40.91% vs 26.89%;p = 0.02,pc = 0.04, RR = 1.88). The finding of Gm heterozygosity in DCM was associated with serum positivity for cardiac antibodies. A higher proportion of DCM patients were positive for both the Gm (1, 3, 17; 23; 5", 21, 28) phenotype and HLA-DR4 compared to normals (3/68 vs 0/210;p = 0.04, RR = 22.50). A potentiating interactive effect in susceptibility to DCM was also found between the Gm (1, -+2, 3, 17; -+23; 5", 21, 28) heterozygous phenotypes and HLA-DR4 (7/66 vs 2/207, p = 0.0009, RR = 12.16). These Gm phenotypes may be new markers of susceptibility in DCM, associated with positive autoimmune serology. The potentiating interaction of Gm and HLA-DR4 is in keeping with results reported in other autoimmune diseases. Human Immunology 35, 193-199 (1992)

ABBREVIATIONS

DCM Gm HLA

dilated cardiomyopathy IgG markers human leukocyte antigen

Km NOSA

kappa markers non-organ-specific antibody

INTRODUCTION Individuals with a particular immunogenetic background are predisposed to develop humoral and/or celFrom the lmmunohematology and Transfusion Center, Pavia, Italy; the Centerfor Research into Genetic Polymorphism in Human Populations (CRPG-CNRS) (J.M.D.), Toulouse, France; the Department of Cardiological Sciences, St. George's Hospital Medical School (A.L.P.C., W.J.M.), and the Department of Immunology, London Hospital Medical College (A.L.P.C., G.S., G.F.B.), London, England; the Cardiology Division, IRCCS San Matteo Policlinic (A.G., G.G.), Pavia, Italy; and the Institute of Pathologic Anatomy (E.A.), Pediatric Clinic (R.L.), and Department of Genetics and Microbiology (M.C.), University of Pavia, Pavia, Italy. Address reprint requests to Dr. M. MartinettL Laboratorio HLA, Servizio di lmmunoematologia e Transfusione, IRCCS PoliclinicoSan Matteo, Largo Volontari del Sangue 2, 27100 Pavia, Italy. Received August 1, 1991 ; acceptedAugust 26, 1992. Human Immunology 35, 193-199 (1992) © American Society for Histocompatibility and Immunogenetics, 1992

lular autoimmune responses, which may be triggered by environmental factors, including infectious agents [ 1 6]. A u t o i m m u n e diseases are associated with immunogenetic markers of susceptibility, most frequently human leukocyte antigens (HLAs) [ 7 - 9 ] . I g G heavy-chain allotypic determinants (Gm) or K light-chain determinants (Kin) are additional markers of disease susceptibility either by themselves or possibly, according to some authors, by interaction with genes in the H L A region [ 1 0 - 1 2 ] . This has been shown in insulin-dependent diabetes mellitus [13], coeliac disease [14], Graves' disease [15], chronic autoimmune hepatitis [16], Sjogren's syndrome and uveitis [17, 18], systemic lupus erythematosus [19], and multiple sclerosis [20]. 193 0198-8859/92/$5.00

194

Dilated cardiomyopathy (DCM) is a chronic heart muscle disorder of unknown cause and of probable heterogeneous etiology, which causes dilatation and failure of the left and/or right ventricle. It predominantly affects young males and its prevalence is around 20 per 100,000 population [21]. It has been suggested that it is an autoimmune disorder, possibly initiated or triggered by viral infections in individuals with predisposing HLA alleles [22-25]. The recent finding of organ-specific circulating cardiac antibodies is consistent with this hypothesis [26]. HLA-DR4 is associated with DCM, but is a marker of susceptibility in only a subset of patients [27, 28]. In this study, we assessed the Gm and Km phenotypic frequencies and HLA-DR in DCM patients from northern Italy and in healthy controls from the same region. Aims of the study were (a) to identify further immunogenetic markers of susceptibility in DCM; (b) to relate the presence of the Gm and Km markers to clinical features, cardiac and non-organspecific autoantibody status; and (c) to assess potential interactive effects of Gm and HLA-DR.

MATERIALS AND METHODS

Study patients. A total of 68 patients with DCM re-

M. Martinetti et aI.

24, and 28), and Km (1) allotypes, using the standard hemagglutination inhibition assay on opaline plates [30, 31]. The allotypes G3m (5, 10, 11, 13, and 14) are designated by 5*. The reagents were all of human origin, except for G2m (23) (murine monoclonal antibody). The antisera were either produced in the C N R S - C R P G or kindly provided by international exchanges.

HLA typing. All 68 DCM patients and 210 of the 277 normals were typed for HLA-DR in the Tissue Typing Laboratory, AVIS Transfusion Center (Pavia, Italy). A standard microlymphocytotoxicity assay was used as previously described [27].

Cardiac

autoantibody screening. Cardiac antibody screening was performed in 64 of the 68 DCM patients. Sera were tested by standard indirect immunofluorescence technique on fresh frozen cryostat sections of blood group O normal human atrium, ventricle and skeletal muscle, and cardiac antibody types were classified as previously described [26, 32]. Briefly, sera that gave diffuse cytoplasmic staining of both atrial and ventricular myocytes and were negative on skeletal muscle were classified as containing organ-specific cardiac antibodies; antibodies that gave fine striational fluorescence on cardiac tissue, but weakly stained skeletal muscle sections were classified as cross-reactive 1; and antibodies that showed a broad striational pattern on longitudinal sections of heart and skeletal muscle were classified as cross-reactive 2.

ferred to the department of Cardiology, IRCCS, San Matteo Hospital (Pavia, Italy) were studied. They were aged 20-57 years (mean, 43 -+ 11); there were 54 men and 14 women. The diagnosis of DCM was based on the demonstration of a dilated and poorly contracting left or right ventricle, or both, according to the World Health Organization criteria [29]. In all patients, a detailed personal history for cardiovascular disease was obtained; age at disease onset and mean duration of cardiac symptoms at the time of diagnosis were clinically ascertained. The duration of cardiac symptoms ranged from 1 to 120 months (median, 24 months); at diagnosis one patient was in New York Heart Association (NYHA) functional class I, 21 were in class II, 39 were in class III, and five were in class IV. All patients underwent right and left heart catheterization, selective coronary angiography, echocardiography, and right and/or left ventricular endomyocardial biopsy. Mean angiographic left ventricular ejection fraction was 28 -+ 14%.

patients were tested by indirect immunofluorescence for NOSAs (antinuclear, antireticulin, antimitochondrial, anti-smooth-muscle, antiribosoma, anti-liver/kidney microsomal) on cryostat sections of rat liver and kidney with the use of standard protocols [33]. Antibodies to thyroglobulin and to thyroid microsomal antigen were detected by passive hemagglutination using Wellcome Thymune kits. Sera from 70 age- and sexmatched healthy volunteers were tested as controls; subjects with a personal or family history of autoimmune disease were excluded.

Gm and Km typing. Gm and Km typing on serum sam-

Data analysis. Chi-squared test and, when necessary,

ples from DCM patients and controls was performed in the Center for Research into Genetic Polymorphism in Human Populations (CNRS-CRPG), Purpan Hospital of Toulouse, France. The control group included 277 healthy subjects from northern Italy, living in the area of Pavia. The samples were typed for G l m (1, 2, 3, and 17), G2m (23), G3m (5, 6, 10, 11, 13, 14, 15, 16, 21,

Fisher's exact test were used (a) to determine the fit of allotypic combinations to the Hardy-Weinberg distribution, and to compare Gm and Km phenotypic frequencies in DCM vs controls and (b) to compare the proportions of patients and controls positive for both specific Gm phenotypes or for Gm (1, -+2, 3, 17; -+23; 5", 21, 28) heterozygous phenotypes and HLA-DR4. p

Screening for non-organ-specific antibodies (NOSAs) and antithyroidautoantibodies. Sera from 65 of the 68 DCM

Dilated Cardiomyopathy and Ig Allotypes

195

Values were corrected for multiple comparisons as appropriate. G m haplotype frequencies were derived from phenotypic frequencies by maximum-likelihood methods, using a computer program [34]. Strength of associations was measured using the relative risk (RR) value, according to Woolf's formula. Normal values for G m and Km phenotypic frequencies have been reported elsewhere [35].

RESULTS P r e v a l e n c e o f C i r c u l a t i n g A u t o a n t i b o d i e s in D C M Cardiac antibodies were detected in 41 (64%) DCM patients and were classified as organ specific in 23 cases (35.9%), cross-reactive 1 in 13 (20.3%), and cross-reactive 2 in five (7.8%). The prevalence of organ-specific cardiac antibodies in these patients from Italy was similar to that first reported in a patient population from England; these antibodies are found in only a small proportion (3.5%) of healthy individuals [26]. NOSAs were more commonly found in DCM patients than in controls (22 [33%] of 65 vs nine [12.8%] of 70; p = 0.003). Antithyroid antibodies were found only in one patient. D i s t r i b u t i o n o f G m and K m P h e n o t y p e s in D C M The G m and Km phenotypic frequencies in DCM patients and controls are shown in Table 1. Their distribution in both groups did not deviate significantly from

TABLE 1

G m and Km phenotype frequencies in DCM patients Controls (n = 277)

Patients (n = 68)

n

%

n

%

G m phenotypes 3; 23; 5* 3; ..; 5* 1, 3, 17; 23; 5", 21, 28 1, 3, 17; ..; 5", 21, 28 1, 3, 17; 23; 5* 1, 3, 17; ..; 5", 15, 16 1, 3, 17; ..; 5* 1, 17; ..; 21, 28 1, 2, 3, 17; 23; 5*, 21, 28 1, 2, 3, 17; ..; 5*, 21, 28 1, 2, 17; ..; 21, 28

175 10 36 16 8 4 1 6 16 3 2

63.18 3.61 13.00 5.78 2.89 1.44 0.36 2.17 5.78 1.08 0.72

35 3 17 4 1 0 1 0 5 1 1

51.47 4.41 25.00 * 5.88 1.47 0 1.47 0 7.35 1.47 1.47

K m phenotypes 1 - 1

37 240

13.36 86.64

10 58

14.71 85.29

ax2 VS controls = 6.05; p -- 0.0139, p corrected (Pc) = 0.1529 = NS, and RR = 2.23.

H a r d y - W e i n b e r g equilibrium showing an homogeneous ethnic background (data not shown). The G m (1, 3, 17; 23; 5", 21, 28) phenotype was overrepresented in DCM compared with controls ( 17 [25%] of 68 vs 36 [13%] of 277; p = 0.0139, RR = 2.23), although the p value did not reach statistical significance when corrected for multiple comparisons. The frequency of heterozygous and homozygous phenotypes in patients and controls is shown in Table 2. The unusual G m phenotypes (1, 3, 17; 23; 5"), (1, 3, 17; ..; 5", 15, 16), and (1, 3, 17; ..; 5*) were not included in the analysis because they might represent genetic anomalies or non-Caucasian admixture. A higher frequency of heterozygotes was found in DCM compared with controls (27 [40.91%] of 66 vs. 71 [26.89%] of 264; p = 0.02, Pc = 0.04, RR = 1.88). The Km phenotypic frequencies were similarly distributed in patients and controls.

Disease heterogeneity: Gm phenotype/clinical features. The relation between the G m (1, 3, 17; 23; 5", 21, 28) phenotype and clinical features is shown in Table 3. This phenotype was more common among patients who were younger at onset of disease (-<40 years) than in controls (29% vs 1 3 % ; p -- 0.0165, RR 2.74, pc = 0.033), while its frequency in older patients ( > 4 0 years) was similar to that seen in controls (21.2% vs 13%; p -- NS). In patients with DCM, however, the association of this Gm phenotype with age at onset did not reach statistical significance (29% vs 21.2%, p = NS). The phenotype was also more common among patients with shorter symptom duration (-<41 months) than in controls (32.5% vs 1 3 % ; p = 0.0014, RR = 3.22, pc =0.0028); its frequency in patients with longer symptom duration (>41 months) was similar to that seen in controls (16.7% vs 13%; p = NS). In patients with DCM, no significant association between this G m phenotype and symptom duration was found (32.5% vs 16.7%; p = NS). The relationship of G m phenotype with ejection fraction could not be evaluated, because ejection fraction was very low in the majority of the patients, as a result of their end-stage heart failure.

Gm phenotype/autoantibody status. The relation of the G m (1, 3, 17; 23; 5", 21, 28) phenotype to autoantibody status is given in Table 4. This phenotype was more common among DCM patients with cardiac antibodies compared with normal controls (29.27% vs 13%; p = 0.007, RR = 2.77, Pc = 0.014), while its frequency in patients who were cardiac antibody negative did not differ from that seen in normals (17.39% vs 13%; p = NS). In patients with DCM, however, the association of this G m phenotype with cardiac antibody status did not reach statistical significance (29.27% in patients with

196

M. Martinetti et al.

TABLE 2

Frequency of homozygous and heterozygous G m phenotypes in DCM and relation to cardiac autoantibody status Controls (n = 2 6 4 )

Patients (n = 6 6 )

Cardiac Ab negative (n = 23)

Cardiac Ab positive (n = 39)

Gm phenotypes

n

%

n

%

n

%

n

%

Homozygous a Heterozygous b

193 71

73.11 26.89

39 27'

59.01 40.91

17 6

73.91 26.09

19 2 0 d'

48.72 51.28

a Homozygous = G m (3; 23; 5*); G m (3; ..; 5*); G m (1, I7; ..; 21, 28); and G m (1, 2, 17; ..; 21, 28). b Heterozygous = G m (1, 3, 17; 23; 5", 21, 2 8 ) ; G i n ( I , 3, 17; _; 5*, 21, 28); and G m ( 1 , 2, 3, I7; 23; 5", 2I, 28); G m (1, 2, 3, 17; ..; 5", 21, 28). cX2 vs controls = 4.97; p = 0.020, p~ = 0.04, and RR = 1.88. ~X 2 vs controls = 9.62; p = 0.002, p, = 0.004, and RR = 2.86. 'X 2 cardiac Ab positive vs cardiac ab negative = 3.77; p = 0.046, p~ = 0.09, and RR - 2.98.

cardiac antibodies vs 17.39% in patients without; p = NS). The G m (1, 3, 17; 23; 5*, 21, 28) phenotype was also more common among DCM patients with NOSAs compared with normal controls (31.82% vs 13%; p -0.015, RR = 3.12, Pc = 0.030), while its frequency in patients who were N O S A negative was similar to that seen in controls (20.93% vs 13%; p = NS). The frequency o f G m heterozygotes was higher among DCM patients with cardiac antibodies than in those without (51.28% vs 26.09%, respectively; p = 0.04, Pc -- NS, RR -- 2.98) or in controls (51.28% vs 2 6 . 8 9 % ; p = 0.002, Pc = 0.004, RR = 2.86)(Table 2).

HLA and Gm interaction in D C M A significantly higher proportion of DCM patients was positive for both the G m (1, 3, 17; 23; 5", 21, 28) phenotype and H L A - D R 4 compared with controls (3/68 vs 0/210; p = 0.04, RR = 22.50). A potentiating interactive effect was also found between G m heterozygotes and HLA-DR4 (7/66 in DCM vs 2/207 in controls; p = 0.0009, RR -- 12.16).

DISCUSSION This study reports the distribution of G m and Km phenotypic frequencies in a Caucasian patient population with idiopathic DCM from northern Italy. In these patients, the G m (1, 3, 17; 23; 5", 21, 28) phenotype was overrepresented in DCM patients compared with normal controls. Although independent confirmation is

G m and K m H a p l o t y p e s in D C M The distribution of the estimated frequencies for the G m and Km haplotypes was similar among patients and controls (results not shown).

TABLE 3

Relation between G m (1, 3 17; 23; 5*, 21, 28) phenotype and clinical features P a t i e n t s (n = 6 4 y Age of disease onset a Controls (n = 2 7 7 )

Gm phenotypes Gm(1,3, Others a

17; 2 3 ; 5 " , 2 1 , 2 8 )

<40 yr

>40 yr

Disease duration a ---41 months

>41 months

n

%

n

%

n

%

n

%

n

36 241

13.0 87.0

9 22

29.0 b 71.0

7 26

21.2 78.8

13 27

32.5' 67.5

4 20

Data not available in the remaining four patients.

bX2 vs controls = 5.75; p = 0.0165, RR = 2.74, and Pc =0.033. cX2 vs controls = 10.17; p = 0.0014, RR = 3.22, and Pc = 0.0028.

16.7 83.3

Dilated Cardiomyopathy and Ig Allotypes

TABLE 4

197

Relation between Gm (1, 3, 17; 23; 5*, 21, 28) phenotype and cardiac autoantibody status Patients

(n =

64)

Cardiac autoantibody Controls (n = 277)

Positive

Negative

Gm phenotype

n

%

n

%

n

%

G m ( 1 , 3 , 17; 23; 5 " , 2 1 , 2 8 ) Others

36 241

13.0 87.0

12 29

29.27 ~ 71.73

4 19

17.39 82.61

~X 2 vs controls = 7.38; p = 0.007, RR = 2.77, and p~ = 0.014.

needed, because the p value did not reach statistical significance after correction for multiple comparisons, this finding may indicate that not only HLA genes, encoded on chromosome 6, but also genes coding for Gm allotypes, localized on chromosome 14, are genetic markers of susceptibility to idiopathic DCM [22, 28, 36]. The distribution of Gm haplotypes did not show any trends toward an association. These results could be explained either by the greater number of comparisons when comparing Gm phenotype frequencies instead of Gm haplotype frequencies or by an heterozygous effect. The increased frequency of Gm heterozygotes in DCM compared with normals, which is another finding in our study, is consistent with heterozygous effect, and has also been described in systemic lupus erythematosus [19]. In addition, in the present study a potentiating interactive effect was found between the Gm (1, 3, 17; 23; 5", 21, 28) phenotype as well as Gm heterozygosity and the previously reported HLA marker of disease susceptibility HLA-DR4. This is consistent with similar findings reported for other diseases of autoimmune origin [10, 13, 15] and lends indirect support to the theory that autoimmunity is involved in at least a subset of patients with DCM [25, 26]. In the present study the association of the Gm (1, 3, 17; 23; 5", 21, 28) phenotype with DCM was rather weak (RR = 2.23). Possible explanations for this include (a) disease heterogeneity, (b) multiple diseasepredisposing factors, and (c) current limitations of the serotyping methodology in revealing sufficiently large variations in the population. In relation to the last issue, DNA analysis is likely to provide greater understanding of the genes specifically involved in disease predisposition. Interestingly, the Gm (1, 3, 17; 23; 5", 21, 28) phenotype was more common among patients with younger age at disease onset and with shorter symptom duration at the time of diagnosis compared with normals. A simi-

lar association between a subset of patients with distinctive clinical features and an allotype marker has been reported for alopecia areata [ 12]. Our findings may indicate that a Gm phenotype is a marker for a severe form of DCM characterized by earlier onset and accelerated progression of heart dysfunction, but the cross-sectional design of the present investigation does not allow us to draw final conclusions. Prospective studies in newly diagnosed DCM patients should test the hypothesis that the Gm (1, 3, 17; 23; 5", 21, 28) phenotype has a negative prognostic value. In our study, the frequency of this Gm phenotype was significantly higher in the subgroup of DCM patients with positive autoimmune serology (e.g., cardiac and in addition non-organ-specific autoantibodies) compared with controls. Furthermore, the frequency of all Gm heterozygotes was significantly higher among DCM patients with cardiac antibodies than in patients without, or in normals. This result is in agreement with the higher frequency of Gm heterozygotes among healthy subjects who produced high titer IgG in response to bacterial antigens [37], as well as with the excess of Gm heterozygotes in families with atopic diseases, where levels of specific IgE were also associated with a particular Gm type [38]. The association between Gm and cardiac antibodies may be spurious, due to reduction of cardiac antibody levels with disease progression, which may occur similarly to type-1 insulindependent diabetes mellitus [39, 40]. Again, the crosssectional design of the present investigation does not allow us to draw final conclusions; prospective studies are warranted. An alternative explanation for this association would include the presence of an "autoimmune" variant of idiopathic DCM in which Gm heterozygosity is associated with cardiac autoantibody production, as opposed to other forms of the disease with different etiologies [26, 39]. If this would turn out to be the case, linkage disequilibrium between specific Gm genes and V-region genes determining antibody production in response to specific autoantigens (e.g., cardiac specific) might be postulated [41].

ACKNOWLEDGMENTS

We thank M. T. Senegas, C. Burali, E. Guitard, A. Sevin, C. Pizzochero, and D. Zanaboni for their excellent technical assistance.

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