Association between angiotensin I-converting enzyme gene insertion/deletion polymorphism and mitral valve prolapse syndrome

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1 Valvular and Congenital Heart Disease Association between angiotensin I-converting enzyme gene insertion/deletion polymorphism and mitral valve prolapse syndrome Hsiang-Tai Chou, MD, PhD, a Yng-Tay Chen, MS, b Yi-Ru Shi, MS, b and Fuu-Jen Tsai, MD, PhD b Taichung, Taiwan Background Some studies have reported that patients with mitral valve prolapse syndrome (MVPS) also have a disorder in the autonomic or neuroendocrine function, which can cause a host of related symptoms. A potential role of the renin-angiotensin system in the pathogenesis of MVPS has been addressed. However, the role of angiotensin I-converting enzyme (ACE) genetic variant in MVPS has not been studied. We therefore performed a case-control study investigating the possible relation between ACE gene polymorphisms and MVPS in Taiwan Chinese. Methods We studied 100 patients with MVPS diagnosed by echocardiography and 100 age- and sex-matched normal control patients. ACE gene insertion/deletion (I/D), A-240T, and G2350A polymorphisms were identified by polymerase chain reaction-based restriction analysis. Results There was a significant difference in the distribution of ACE I/D genotypes (P.003) and allelic frequencies (P.001) between MVPS cases and control patients. An odds ratio for the risk of MVPS associated with the ACE II genotype was 2.14 (95% CI ). An odds ratio for the risk of MVPS associated with ACE I allele was 1.96 (95% CI ). The A-240T and G2350A polymorphisms of the ACE gene showed no association with MVPS (P.20, P.13, respectively). Conclusions This study showed that patients with MVPS have a higher frequency of ACE II genotype, which supports a role of the ACE I/D gene polymorphism in determining the risk of MVPS among the Chinese population in Taiwan. (Am Heart J 2003;145: ) Mitral valve prolapse (MVP) is one of the most common cardiac abnormalities in human beings. It has a prevalence of 2% to 8% among the adult population. 1,2 It is described as excessive mitral valve tissue leading to billowing of the mitral valve leaflet, with or without prolapse and mitral regurgitation. 3-6 Mitral valve prolapse syndrome (MVPS) is a widely used term that has been applied to patients with MVP who have a variety of symptoms, including chest pain, dyspnea, dysrhythmia, anxiety, insomnia, and syncope. Some studies report that patients with MVPS also have a disorder in the autonomic or neuroendocrine function as well as renin-aldosterone regulation, which can lead to various From the Division of Cardiology, Departments of a Medicine, and b Pediatrics, Medical Research and Medical Genetics, China Medical College Hospital, Taichung, Taiwan. Submitted January 31, 2002; accepted May 28, Reprint requests: Fuu-Jen Tsai, MD, PhD, Department of Pediatrics, Medical Research and Medial Genetics, China Medical College Hospital, 2 Yuh Der Road, Taichung, Taiwan. d0704@ Copyright 2003, Mosby, Inc. All rights reserved /2003/$ doi: /mhj other symptoms. 7,8 The renin-angiotensin system may play a role in the pathogenesis of MVPS. Angiotensin I-converting enzyme (ACE), a key component of the circulating and vascular renin-angiotensin system, promotes the synthesis of angiotensin II, which is the principal effector of the renin-angiotensin cascade. This has been shown to modulate central and peripheral autonomic function The evidence that the circulating ACE level is under substantial genetic control was first found in a segregation analysis of ACE levels in 87 healthy nuclear families. 12 The human ACE gene has been cloned and localized to chromosome 17q An insertion/deletion (I/D) polymorphism in intron 16 has been identified for use as a genetic marker. 14 There is evidence to suggest that the I/D polymorphism is in strong linkage disequilibrium with a major gene effect at the ACE gene locus, which controls up to 44% of the variability in ACE levels. 15 Subsequent studies have also suggested the possibility of a second functional site possibly unlinked to the ACE gene. 16 One of the candidate regions for an active site influencing ACE levels is the

2 170 Chou et al American Heart Journal January 2003 Table I. Clinical characteristics of patients with MVPS and healthy control subjects Patients with MVPS (n 100) Control subjects (n 100) Age (y) Sex (M/F) 30/70 38/62 SBP (mm Hg) * DBP (mm Hg) * BMI (kg/m 2 ) * Data are presented as the mean value SD. MVPS, Mitral valve prolapse syndrome; SBP, systolic blood pressure; DBP, diastolic blood pressure; BMI, body mass index. *P.01 versus control. ACE gene promoter. The A-240T polymorphism on the promoter region of ACE gene was reported to have the greatest association with ACE levels, being associated with 14% of the variability in levels. 17 The G2350A polymorphism in intron 17 was reported to have the most significant effect, accounting for 19% of the total variance in ACE. 18 We hypothesize that the effects of ACE might be involved in the pathogenesis of symptoms (ie, low blood pressure, chest pain) related to abnormal vasomotion in MVPS. Because there are no data available in the literature regarding the role of the ACE gene polymorphism in MVPS, the current study was designed to determine whether the I/D, A-240T, and G2350A polymorphisms of the ACE gene are associated with MVPS among the Chinese population in Taiwan. Methods Study population A total of 100 patients (30 men and 70 women, ranging in age from 14 to 75 years, mean age years) with echocardiographically diagnosed MVP were enrolled in this study. Patients with Marfan syndrome and congenital heart disease were excluded. All patients had a mid-systolic click and various symptoms including chest pain, palpitation, anxiety, dyspnea, syncope, and insomnia. The control group consisted of 100 age- and sex-matched healthy volunteers (38 men and 62 women ranging in age from 16 to 76 years, mean age years) with normal echocardiography verified before inclusion. All people were of Chinese Han nationality and lived in central Taiwan. The study was approved by the institutional research ethics committee, and informed consent was obtained from all participants. Echocardiography Echocardiographic examination (Hewlett-Packard Sonos 2500, Cupertino, Calif) was performed according to the recommendations of the American Society of Echocardiography. 19 The diagnostic criteria for MVP included displacement of the leaflet edges and increased thickness and redundancy of the mitral valve. 20 The displacement of each leaflet was measured in the parasternal long-axis view above a line connecting the mid-portions of the annular hinge points. The thickness of the mitral valve was measured by M-mode recording. The severity of mitral regurgitation was estimated in the parasternal long-axis view by color Doppler echocardiography. 21 To take into account the great variability in the expression of the disease, patients were classified according to the following criteria: (1) a positive diagnosis for MVP was made when patients had a floppy mitral valve with leaflet thickness of 4 mm and total leaflet displacement of 2 mm; (2) a case was regarded as severe when patients had valve thickness 6 mm or total leaflet displacement 8 mmor significant mitral regurgitation (grade 2 assessed according to the criteria of Miyatake et al 21 ). Genotyping of I/D, A-240T, and G2350A polymorphisms of the ACE gene The genomic DNA was prepared from peripheral blood leukocytes by use of a genomic DNA isolation kit (Blossom, Taipei, Taiwan). The ACE gene I/D polymorphism was determined by polymerase chain reaction, by use of primers flanking the polymorphic region of intron 16 with the following primer sequences: 5 -TGGAGACCACTCCCATCCTTTCT-3 and 5 -CAGGTCTTCATATTTCCGATGTGG-3. Reactions were carried out in 50- L volumes containing genomic DNA, 2 to 6 pmol of each primer, 1X Taq polymerase buffer (1.5 mmol/l MgCl 2 ), and 0.25 units of AmpliTaq DNA polymerase (Perkin Elmer, Foster City, Calif). The cycling conditions for I/D were set as follows: 1 cycle at 94 for 3 minutes, 30 cycles at 94 for 30 seconds, 60 for 30 seconds, 72 for 60 seconds, and 1 final cycle of extension at 72 for 10 minutes. Electrophoresis of the amplified products in 2% agarose gel allowed detection of a 495-base pair fragment (insertion) and of a 208-base pair fragment (deletion). The A-240T and G2350A polymorphisms were typed by the restriction fragment length polymorphism method previously described by Keavney et al. 22 Statistical analysis Differences in genotype distribution between patients with MVPS and control patients were tested by the 2 test with 2 degrees of freedom (df). For statistical analysis of the allelic frequency distribution in the I/D, A-240T, and G2350A polymorphisms, the 2 groups were compared by use of the 2 test with 1 df. Allelic frequencies were calculated from genotype frequencies in patients with MVPS and control patients. Because of the small numbers of the ACE DD homozygotes, the I/D and the DD genotypes were collapsed for the calculation of the odds ratio (I/D DD vs II). The differences among the mild MVP, severe MVP, and control groups were estimated by the 2 test, with 4 df. All statistical analyses were performed with NCSS 2000 Software (Kaysville, Utah). A value of P.05 was considered statistically significant. Results Patient characteristics Table I shows the main characteristics of the case and control groups. Blood pressure and body mass in-

3 American Heart Journal Volume 145, Number 1 Chou et al 171 dex were significantly lower in patients with MVPS in comparison with control patients. There was no difference in age and sex between patients with MVPS and control patients. Of the 100 patients with MVPS, 30 (30%) were male and 70 (70%) were female. Seventyone (71%) patients were aged 40 years; 29 (29%) were aged 40 years. There was no case of mitral chordal rupture. All patients had mitral regurgitation on color Doppler echocardiography. The mitral regurgitation was mild (1 ) in 72, moderate (2 ) in24, and moderate to severe (3 ) in 4. Among these, 14 male patients and 33 female patients had severe MVP. The remaining patients had mild MVP. Distribution of ACE genotypes and allelic frequencies among cases and control patients For each ACE polymorphism I/D, A-240T, and G2350A, we confirmed that the genotype proportions fit the Hardy-Weinberg equilibrium estimated by 2 test. Examination of this distribution showed a significant difference in the I/D polymorphism ( , P.003) among cases and control patients (Table II). An odds ratio for risk of MVPS associated with ACE II genotype was 2.14 (95% CI ). A significant increase in allelic frequencies of insertion of the ACE gene was observed in patients with MVPS (0.72 vs 0.56, , P.001). An odds ratio for risk of MVPS associated with the I allele was 1.96 (95% CI ). The analysis of genotype distribution did not show a statistically significant difference for the A-240T and G2350A polymorphisms of the ACE gene between patients with MVPS and control patients ( , P.20, , P.13, respectively). Allelic frequencies of the A-240T and G2350A of the ACE gene were not significantly different between cases and control patients ( , P.15, , P.25, respectively). As shown in Table III, no statistically significant differences in the A-240T ( , P.42) and G2350A ( , P.21) polymorphisms among the mild MVP and severe MVP subgroups and the control group were observed. There was a significant difference in the distribution of I/D polymorphism among cases and control patients. No significant difference in the distribution of I/D polymorphism between the mild and severe MVP subgroups was observed. Discussion Since Barlow s description, 6 a variety of names have been given to mitral valve prolapse, including clickmurmur syndrome, floppy mitral valve syndrome, myxomatous mitral valve, and idiopathic mitral valve prolapse. This reflects the uncertainty about its pathophysiology. Although most cases appear to be Table II. Distribution of ACE genotypes in patients with MVPS (n 100) and healthy control subjects (n 100) Gene polymorphism Genotype Patients with MVPS, Controls, 2 P ACE-I/D II 48 (48) 30 (30) ID 47 (47) 52 (52) DD 5 (5) 18 (18) A-240T I/D 0.72/ / AA 41 (41) 29 (29) AT 47 (47) 57 (57) TT 12 (12) 14 (14) G2350A A/T 0.65/ / GG 7 (7) 16 (16) GA 50 (50) 43 (43) AA 43 (43) 41 (41) A/G 0.68/ / Genotype frequencies are indicated in absolute values (percentages). Allelic frequencies are indicated in fractions. ACE, Angiotensin I-converting enzyme. sporadic, echocardiographic screening of families for MVP has suggested autosomal dominant inheritance of the trait, with an age- and sex-dependent expression Defects in fibrillin and collagen genes have been identified in syndromic valvular disease. However, genetic studies have failed to find a link between collagen genes and familial MVP. 26 Identification of the locus for autosomal dominant myomatous MVP to chromosome 16p11.2 p12.1 was reported. 27 In addition, X-linked inheritance has been reported in a special form of MVP, called myxomatous valvular dystrophy, which has been mapped to Xq Single nucleotide polymorphism (SNP) is the most abundant type of DNA sequence variation in the human genome. 29,30 SNP is a single base pair on the DNA that varies from person to person. The SNP marker may provide a new method for identification of complex or multifactorial gene-associated diseases. The current study used a method of polymerase chain reaction-based restriction analysis to approach the SNP. Low blood pressure, orthostatic phenomena, and autonomic dysfunction may be regarded as a phenotypic feature of MVPS. 7,31,32 Orthostatic phenomena may be multifactorial in origin. Decreased intravascular volume, abnormal renin-aldosterone response to volume depletion, baroreflex modulation abnormality, hyperadrenergic state, or parasympathetic response abnormality may partially account for these phenomena. 32 The renin-angiotensin system was hypothesized to be involved in the pathogenesis of MVPS. The renin-angiotensin system has been a model system for the study of the genetics of MVPS. In recent years, association between the A/C 1166 polymorphism of the angiotensin II type 1 receptor (AGTR1) gene and MVP in the white

4 172 Chou et al American Heart Journal January 2003 Table III. Distribution of ACE genotypes in patients with mild (n 53), severe MVP (n 47), and healthy control subjects (n 100) Gene polymorphism Genotype Mild MVP, Severe MVP, Controls, 2 P ACE-I/D II 29 (55) 19 (40) 30 (30) ID 20 (38) 27 (57) 52 (52) DD 4 (7) 1 (3) 18 (18) Significance* Significance A-240T AA 23 (43) 18 (38) 29 (29) AT 25 (47) 22 (47) 57 (57) TT 5 (10) 7 (15) 14 (14) Significance* G2350A GG 3 (6) 4 (9) 16 (16) GA 24 (45) 26 (55) 43 (43) AA 26 (49) 17 (36) 41 (41) Significance* MVP, Mitral valve prolapse. *Comparison among mild MVP, severe MVP, and controls groups. Comparison of mild MVP versus severe MVP groups. population has been reported. 33 However, we did not find an association between A/C 1166 polymorphism of the AGTR1 gene and MVPS in our population. 34 In this case-control study, we found an association between the II genotype of the ACE gene polymorphism and MVPS. The increased risk of MVPS associated with the I allele of the ACE gene was observed. Because the patients with asymptomatic MVP were not enrolled in this study, this fact may represent a selection bias with regard to the question of whether MVP is associated with ACE gene polymorphism. The true incidence of symptoms in patients with MVP is not known; the incidence of symptoms may be exaggerated because most of the studies have been performed in academic institutions and thus may reflect a selection bias. 32 A population-based analysis of unrelated individuals with I/D polymorphism showed an association between this polymorphism and plasma ACE levels. This association followed an additive pattern, with DD and II genotypes being associated with high and low ACE levels and I/D heterozygotes being associated with an intermediate level. 14 These associations have been confirmed in family studies of white European 15 and Afro- Caribbean 16 populations and in Hong Kong Chinese patients. 35 Although the relation of I/D polymorphism and blood pressure was controversial, several studies found a significant association between I/D polymorphism and blood pressure. 36,37 These findings may explain why our patients with MVPS had low blood pressure. The frequency of the ACE DD genotype in our control Taiwan Chinese population was 18%, which is higher than that reported in Hong Kong Chinese (13%) 35 but lower than in a white population (24.8%). 38 The frequencies of the D allele in our control Taiwan Chinese population was 44%, which is higher than that reported in Hong Kong Chinese (37.2%) 35 but lower than in a white population (54%). 38 The discrepancy in the distribution of ACE gene polymorphisms between these studies may be due to ethnic differences. Two recent studies reported that the T allele of A-240T polymorphism was associated with an increase in ACE concentration, as is the G allele of the G2350A polymorphism. 17,18 When both the T allele at A-240T and G allele at G2350A were present, systolic blood pressure and diastolic blood pressure increased, on average, by 2 and 5 mm Hg, respectively. 18 Consistent with previously reported data, we found a trend toward an increased risk of MVPS associated with the A allele of the A-240T or A allele of G2350A polymorphisms of the ACE gene, although these did not reach statistical difference. This also may explain why our patients with MVPS had low blood pressure. Since the ACE gene polymorphisms were hypothesized to be associated with the severity of MVP, we studied the distribution of the I/D, A-240T, and G2350A polymorphisms in the mild and severe MVP subgroups. Our results show no evidence of an association of ACE gene polymorphisms with the severity of MVP. Therefore, other factors should be considered to be involved in the pathogenesis of MVP. Conclusions This study shows that patients with MVPS have a higher frequency of the ACE II genotype, which supports a role of the ACE gene I/D polymorphism in determining the risk of MVPS among the Chinese population in Taiwan. However, this study indicates that the pathogenesis of MVP is complex and requires further investigation.

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