Heart ~"Vessels

Short Communications

© Springer-Verlag1992

Heart Vessels (1992) 7:155-160

HLA Gene analysis in a Japanese family with hypertrophic cardiomyopathy by restriction fragment length polymorphism T s u g i y a s u K a n d a , N o r i a k i T a k e u c h i , A k i r a H a s e g a w a , T a d a s h i Suzuki, a n d K a z u h i k o M u r a t a The Second Department of Internal Medicine, Gunma University School of Medicine, 3-39-15 Showa-machi, Maebashi, Gunma 371, Japan

Summary. A large Japanese family with hypertrophic cardiomyopathy (HCM) was examined (n = 61). Ten of 14 affected family members who showed H C M on the basis of electrocardiography and two-dimensional echocardiography were subjected to human leukocyte antigen (HLA)-A,B,C typing by the lymphocyte cytotoxicity test and D, D R typing by restriction fragment length polymorphism (RFLP). As control, the H L A genotype of 14 non-affected family members was analyzed. H L A typing of the affected subjects with H C M revealed a very close association (67%) of HLAD R (4w8) among A , B , C , D and DR. However, the LOD scores of H L A - D R 4 and -DR w8 were 0.693 (0 = 0.35) and 0.642 (0 = 0.30) respectively. These data suggest that HLA-loci, analyzed on the basis of RFLP, may not be related with familial H C M with or without obstruction. Key words: Human leukocyte antigen (HLA) Hypertrophic cardiomyopathy (HCM) - Hypertrophic obstructive cardiomyopathy (HOCM) - Restriction fragment length polymorphism (RFLP)

Introduction Hypertrophic cardiomyopathy (HCM) is a myocardial disease of unknown etiology. Most cases of H C M show familial occurrence consistent with autosomal dominant inheritance. The gene locus responsible for familial H C M is believed to be linked to the major histo-compatibility complex [1-3]. Recently, some investigators [4-6] have used D N A probes to examine restriction fragment length polymorphism (RFLP) and

Address correspondence to: T. Kanda Received April 10, 1991; revision received December 24, 1991; accepted December 27, 1991.

have found a different chromosomal location from chromosome 6, whieh bears the genes for histocompatibility leukocyte antigens (HLA). A large Japanese family (61 members), in which H C M with (n = 3) or without (n = 12) obstruction was evaluated clinically, was subjected to analysis of H L A class I and II antigens by the R F L P method, and a plausible genetic explanation for the disease was proposed on the basis of H L A genes.

Methods Clinical Design A large family, comprising 13 members with hypertrophic cardiomyopathy, 3 members with unexplained left ventricular hypertophy (LVH) and 45 members without cardiac abnormalities was examined (Fig. 1). For all affected members, the diagnosis was established by 12-1ead electrocardiography (ECG) and typical 2-dimensional (2-D) echocardiography. The patients with HCM were also examined by cardiac catheterization and angiography, and endomyocardial biopsy. There were three patients with hypertrophic obstructive cardiomyopathy (HOCM), ten with hypertrophic non-obstructive cardiomyopathy (HNCM) and two with LVH according to both ECG criteria [7] and 2-D echo-cardiography. The other patient had LVH only according to 2-D echocardiography. The diagnosis of HCM was based on 2-D echocardiographic demonstration of unexplained left, right or bilateral ventricular hypertrophy. The criteria for HCM were a septal to left ventricular posterior wall ratio of greater than 1.3:1 and a septal thickness of more than 14mm. We excluded members with LVH, in whom the rneasurements did not satisfy the criteria for HCM. Obstruction of the left ventricle was confirmed by both echocardiographic and hemodynamic findings: the presence of mitral systolic anterior motion and a significant peak systolic intracavitary pressure gradient (30 mmHg or more) at rest or after provocation. None of the family members had hypertension.

H L A Testing The gene loci of the HLA-A,B,C antigens were determined by the micro-droplet lymphocyte cytotoxicity test using 80

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Fig. 1. Inheritance of familial hypertrophic cardiomyopathy in a Japanese family. The subject numbers and disease status of each family member analyzed are indicated. The numbers without circles show affected members and the numbers in circles show non-affected members whose HLA haplotypes

were analyzed. Squares denote male family members, and circles females. Solid symbols denote those with hypertrophic cardiomyopathy, clear symbols those non-affected. Crosses denote deceased family members. An arrow shows the proband

standardized antisera to assess 38 specificities. The HLA-D and -DR antigens were analyzed by RFLP. In brief, DNA was isolated and digested with four different restriction enzymes - EcoRI, PstI, TaqI and MspI. Southern blotting of restriction endonuclease-treated DNA transferred to filters was then performed, followed by hybridization with Tenth International Histocompatibility Workshop (IHW) [8] fulllength DQA or DQB cDNA probes. The probes were radiolabeled with [ct-32p] dCTP using the method of Feinburg and Vogelstein. [9] The filters were washed under conditions of high stringency (low salt, high temperature 65°C, 15 mM sodium chloride, 1.5mM sodium citrate, 0.1% sodium dodecyl sulfate) and subjected to autoradiography for 24-72 h with intensifying screens at -70°C.

pressures and no significant stenosis of coronary arteries. There was no personal history of hypertension. The histological findings from biopsy specimens of the leff ventricle showed prominent disärray of the myocardium and interstitial fibrosis. The patient was ädmitted to Gunma University Hospital because of recurrent palpitation. Her electrocardiogram showed ventricular tachycardia, which was not controlled by the treatment of antiarrythmic drugs. After 7 days of admission, she died due to cardiac arrest. At necropsy, extensive scar tissue was found in the posterior wall of the heart (Fig. 4). F a m i l y Mernbers

Results Proband

The patient was a 53-year-old female with a chief complaint of systemic edema. The results of physical examination were within normal limits, but a fourth heart sound was heard. A chest roentgenogram showed cardiomegaly (cardiothoracic ratio = 59%) (Fig. 2), and an electrocardiogram showed high QRS voltage with deep T-wave inversion in V 4 - 6 (Fig. 3). A 2-D echocardiogram revealed hypertrophy of the interventricular septum (20mm), and left ventricular posterior wall (15mm). Subsequent catheterization showed normal left ventricular and right ventricular

Left ventricular hypertrophy was found in 15 family members. Hypertrophic cardiomyopathy was diagnosed in 12 members. The other three (subjects 4, 7 and 10) members did not sufficiently satisfy the criteria for HCM, but showed unexplained LVH by electrocardiography and marked hypertrophy of IVST. The etectrocardiographic and 2-D echocardiographic findings in the patients with HCM are shown in Table 1. The electrocardiographic and echocardiographic findings of the other 37 family members did not satisfy the criteria for H C M by H L A class I (A,B,C) and II (Dw, DR) genotypes in these family members with HCM are shown in Table 2. The proband carried H L A - A (24, 31), -B (35, w61), -Cw (1, 3), -Dw (8, 1,

157

T. Kanda et al. : HLA analysis in familial HCM by RFLP

Fig. 2. Chest roentgenogram showing cardiomegaly in the proband with hypertrophic cardiomyopathy. Cardiothoracic ratio was 0.59

15) and -DR (4, wS) haplotypes. Ten individuals with HCM carried at least orte HLA-A (24, 32), Cw (1, 3) or -DR (4, w8). However, the HLA-DR (4, wS) haplotype was present in 6/10 (60%), showing the highest incidence rate. HLA-A (24, 31) was present in 2/10 (20%), and HLA-Cw (1, 3) in 3/10 (30%), incidently. Two members with symmetrical LVH had the same HLA-DR (4, w8). The antigen frequency of HLA in the Japanese population was shown as below: HLA-A24 (37.2%),-A31 (8.7%), -Cwl (11.1%), Cw3 (26.3%),-DR4 (14.4%),-DR8 (7.2%) [10]. Three members (subjects 7, 9, and 11) in this family were revealed to have HOCM. Their HLA gene haplotype was not characteristic hut coincident with that of HNCM or with a member with symmetrical LVH (subject 7 = subject 6, subject 9 = subject 10 except for HLA-B, subject 12 = subject 9). Next, we typed the HLA-DR loci in fourteen nonaffected members of the same family (Table 3). The HLA-DR4 was present in 5/14 (36%) and -DR w8 in 5/14 (36%). The findings for the affected members were compared with those from the non-affected members by linkage analyses. The affected individuals are those who are numbered without a circle in Table 2. The non-affected members are numbered with

circles in Table 3. The maximum logarithm of the odds (LOD) score of HLA-DR4 frequency was 0.693 at 0 = 0.35 and that of HLA-DRw8 was 0.642 at 0 = 0.30 by Moton's maximum likelihood method [11]. Neitheer HLA-A, -Cw or -Dw were linked to the locus for familial HCM (LOD score < -2.0 at O = 0.5)..A LOD score above +3 indicates that the observed data are 1,000-fold more likely to occur. A LOD score of less than - 2 is generally accepted as evidence of nonlinkage between a pair of loci at a given distance. We could not observe the definite correlation between the locus for familial HCM and HLA genes by the RFLP.

Discussion The present findings suggested the following: (1) There was no significant correlation between HLA gene haplotype and familial HCM, and (2) the difference between HNCM and HOCM could not be explained by HLA haplotype in one Japanese family with HCM. Familial hypertrophic cardiomyopathy is an idiopathic disorder of the myocardium. The disease is

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Fig. 3. Electrocardiogram showing high QRS with deep T-wave inversion in the s a m e patient as that in Fig. 2

Fig. 4. Transverse section of the heart in the proband at necropsy. The extensive scar was prominent in the posterior wall of the left ventricle

characterized clinically by myocardial hypertrophy, a wide spectrum of possible symptoms, and an annual mortality rate of 2 % - 4 % due to sudden death [12]. The anatomical distribution of myocardial hypertrophy and the severity of the symptoms may be quite variable, even within a single family. The cause and molecular pathophysiology have remained obscure. There have been several reports suggesting a significant relationship between HLA-DR and HCM in Europe and Japan, a markedly increased frequency of HLA-DR3 or DR6 being noted in Caucasian patients. However, a high occurrence of DR4 has been described in Japanese patients with HOCM by Matsumori et al. [1]. These variations may be due to racial differences or limitations in the number of patients examined. Several diseases are known to be associated with an increased frequency of specific HLA-DR4 types, including juvenile-onset diabetes mellitus, pemphigus vulgaris and rheumatoid arthritis. Recent studies have demonstrated that dilated cardio-

myopathy shows a significant relationship with HLADR4 antigen [13, 14]. On the other hand, several studies performed in North America have failed to show any linkage of familial HCM to HLA region even by the application of RFLP markers. Jarcho et al. showed evidence of no-linkage between HLA and familial HCH by four different DNA probes of HLA genes [4]. Heitnancik et al. concluded that the gene responsible for HCM was not linked to the HLA region by studies with serum markers and with RFLP markers [15]. However, the criteria for HCM are not exactly the same in these studies. Matsumori et al. [1], Kishimoto et al. [2], and Fiorito et al. [3] excluded patients without asymmetrical hypertrophy. Jascho et al. [4] included patients without asymmetric hypertrophy. Twenty percent of affected members had symmetric hypertrophy. Solomon et al. [16] also included such members. Hejtmancik et al. [17] did not mention asymmetrical hypertrophy, but determined HCM by

T. Kanda et al.: HLA analysis in familial HCM by RFLP

159

Table 1. Clinical characteristics of patients with hypertrophic cardiomyopathy and patients with left ventircular hypertrophy from a Japanese family 2-D echocardiogram

Subject no. electrocardiogram

IVST mm

PWT mm

IVST /PWT

LVDd mm

LVDs mm

LAD mm

RAD mm

18 14 16 14 17 16 18 18 15 16 18 18 15 15 21

10 10 10 11 13 11 10 12 10 11 11 10 12 12 18

1.8 1.4 1.6 1.3 1.3 1.5 1.8 1.5 1.5 1.5 1.6 1.8 1.2 1.2 1.2

49 46 42 40 37 53 38 32 40 32 44 39 50 50 40

32 31 30 21 19 32 27 23 21 25 28 27 35 29 21

50 46 38 30 30 46 31 27 29 26 32 23 46 42 38

22 20 20 27 19 24 25 22 20 21 16 18 23 26 22

1 2 3 4 5 6

Af, LAD, POOR-R LVH LVH CRBBB LVH LVH 7 CRBBB, POOR-R 8 Af, LAD, POOR-R 9 LVH 10 LVH 11 POOR-R 12 POOR-R (~) LVH @ POOR-R (~) LVH

Af, Atrial fibrillation; LAD, left axial deviation; POOR-R, poor progression of R waves; CRBBB, complete right bundle branch block; AbQ, abnormal Q-wave; LVH, left ventricular hypertrophy; 2-D, two-dimensional; IVST, intraventricular septum thickness; PWT, posterior wall thickness; LVDd, left ventricular diastolic diameter; LVDs, left ventricular systolic diameters; LAD, left atrial diameter

Table 2. HLA Specificities in ten affected patients and two non-affected subjects or family members with LVH

Table 3. HLA-DR genotype in 14 non-affected subjects or family members

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Proband 3 4 5 6 7 9 10 11 12 @ (~)

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B

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Type Dw

DR

24, 31 35, w61 1,3 8.1, 15 4, w8 24, 26 39, w61 1,7 4, 23 4, 9 2, 11 w54, w61 1,3 1, 15 1, 4 2, 20 51, 3, 4, 8.3 4, w8 24, 31 39, w51 1,7 7, 15 4, w8 24, 31 39, w51 1,7 7, 15 4, w8 2, 24 w6l, 3, 1,23 1, 9 2, 24 40, 3, 1, 23 1, 9 2, 24 51, 1,3 8.1, 15 4, w8 26, 31 35, 3, 4, 8.3 4, w8 24, 31 35, 39 3, 7 4, 7 4, w8 26, 31 35, 3, 4, 8.3 4, w8

HNCM HNCM HNCM HNCM HNCM HOCM HOCM HNCM HNCM HOCM LVH LVH

HLA, Human leukocyte antigen; HNCM, hypertrophic nonobstructive cardiomyopathy; HOCM, hypertrophic obstructive cardiomyopathy; LVH, left ventricular hypertrophy wall thickness o f 1 3 m m or more. This point may influence the linkage analysis. Actually, we obtained an elevated (or increased) m a x i m u m L O D score for H L A - D R 4 , w8 after we included three m e m b e r s with symmetric hypertrophy ( D R 4 : 1 . 6 9 4 at 0 = 0.20vs 0.693 at 0 = 0.35, D R w 8 : 2 . 2 9 6 at 0 = 0.20vs 0.642 at

=0.30). Summarizing recent results, Jarcho et al. [4] have reported that the gene responsible for familial H C M is located on c h r o m o s o m e 14 (band q l ) , on the basis of a study of 78 m e m b e r s of a large Canadian family. Moreover, they have identified a mutation in the a and ]3 cardiac myosin heavy chain ( M H C ) gene in this

HLA-DR Type

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family group [15]. The c h r o m o s o m e 14 band ql is closely linked to the M H C gene. Investigators at the National Institutes of Health studied a large n u m b e r of individuals from several families in the United States in w h o m the H C M gene appeared to m a p to chromosome 2ql [18]. Nishi et al. [5] have reported the localization of the H C M gene on c h r o m o s o m e 18q in a Japanese population. However, they did not show the specificity of familial H C M . Recently, Solomon [16] mentioned that familial hypertrophic cardiomyopathy can be caused by defects in at least two loci and that H C M is a genetically heterogenous disorder. They expected other specific loci elsewhere in the genome,

160 which are not related with chromosome 14q 11-12. Our data suggest that H L A - D R on chromosome 6 may not be responsible for familial hypertrophic cardiomyopathy in this family, and that H L A typing may not explain the difference between H O C M and H N C M in familial hypertrophic cardiomyopathy when applying strict criteria for HCM. We further anticipate that a long-term follow-up of individuals in this family and RFLP analysis of the other family with H C M will further our understanding of the H L A genes in cardiac hypertrophy or hypertrophic cardiomyopathy of the Japanese family.

References 1. Matsumori A, Kawai C, Wakabayashi A, Terasaki PI, Park MS, Sakurami T, Ueno Y (1981) HLA-DR4 antigen linkage in patients with hypertrophic obstructive cardiomyopathy. Am Heart J 14:101-106 2. Kishimioto C, Kaburagi T, Takayama S, Kuwai C (1983) Two forms of hypertrophic cardiomyopathy distinguished by inheritance of HLA haplotypes and leit ventricular outflow tract obstruction. Am Heart J 105: 988-994 3. Fiorito S, Autore C, Fragola PV, Purpura M, Cannata, Sangiorgi M (1986) HLA-DR3 antigen linkage in patients with hypertrophic obstructive cardiomyopathy. Am Heart J 91(111):91-94 4. Jarcho JA, McKenna W, Peter Pare JA, Solomon SD, Holcombe' RF, Dickie S, Levi T, Donis-Keller H, Seidman JG, Seidman CE (1989) Mapping a gene for familial hypertrophic cardiomyopathy to chromosome 14ql. N Engl J Med 16:1372-137 5. Nishi H, Kimura A, Susaki M, Wakisaka A, Matsuyama K, Koga Y, Toshima H, Susazuki T (1989) Localization of the gene for hypertrophic cardiomyopathy of chomosome 18q. Circulation 80(4):II-457 6. Ambrosimi M, Ferraro M, Peale A (1989) Cytogenetic study of familial hypertrophic cardiomyopathy. Identification of a new ffagile site on human chromosome 16 Circulation 80(4):II-458

T. Kanda et al.: HLA analysis in familial HCM by RFLP 7. Surawicz B, Uhley H, Brun R (1978): Task force: Standardization of terminolongy and interpretation. Am J Cardiol 41:130-145 8. Simons MJ, Wheeler R, Lalouel JM, Dupont B (1989) Restriction fragment length polymorphism of HLA genes: Summary of the 10th International Workshop on Southern Blot Analysis. In: Dupont B (ed) Immunobiology of HLA. Springer-Verlag, New York, pp 959-1026 9. Fleinburg AP, Vogelstein B (1983) A technique for radiolabelling DNA restriction endonuclease fragments to high specific activity. Anal Biochem 132:6-10 10. Bodmer WF, Batchelor JR, Bodmer JG, Festenstein H, Morris PJ (1978) Histocompatibility testing 1977. Munksgaard, Copenhagen, pp 259-278 11. Moton NE (1959) Genetic tests under in comlete as certainment; Am J Hum Genet 11:1-16 12. Wigle ED, Sasson Z, Heuderson MA (1985) Hypertrophic cardiomyopathy: The importance of the site and the extent of hypertrophy. A review. Prog Cardiovasc Dis 28:1-8 13. Limas C, Limas C (1989) HLA antigens in idiopathic dilated cardiomyopathy. Br Heart J 62:379-38 14. Anderson JL, Carlquist JF, Murray MB, O'Connell JB (1990) Increased HLA DR4 and DQw4 frequencies in idiopathic dilated cardiomyopathy: Results of validation and meta-analysis species. Circulation 182(4):III-388 15. Tanigawa G, Jarcho JA, Kass S, Solomon SD, Vosberg H-P, Seidman JG, Seidman CE (1990) A molecular basis for familial hypertrophic cardiomyopathy: An a/[3 cardiac myosin heavy chain hybrid gene. Cell 62: 991-998 16. Solomon SD, Jarcho JA, Mckenna W, CeisterferLowrance A, Germain R, Salerni R, Seidman JG, Seidman CE (1990) Familial hypertrophic cardiomyopathy is a genetically heterogenous disease. J Clin Invest 86:993-999 17. Hejtmancik JF, Brink PA, Towbin J, Hill R, Brink L, Raberts R (1991) Localization of gene for familial hypertrophic cardiomyopathy to chromosome 14 q 1 in a diverse US population. Circulation 83 (5): 1592-1597 18. Epstein N, Fananapazir L, Lin H, Maron BJ, Mulvihill J, White R, Lalouel JM, Nianhuis A, Leppert M (1990) Genetic heterogeneity in hypertrophic cardiomyopathy: Evidence that HCM maps to chromosome 2q. Circulation 82 (Suppl III):III-399