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Original article 65

Angiotensin-converting enzyme insertion/deletion polymorphism in an Egyptian cohort of hypertrophic cardiomyopathy Heba Sh. Kassema,b,*, Sherif A. Algendya,*, Remon S. Azera, Gehan Magdya,b, Sarah Moharem-Elgamala,c, Maha S. Ayada,d, Ahmed Elguindya, Besra S. Abdelghanya and Magdi H. Yacouba,e a Magdi Yacoub Heart Foundation, Aswan Heart Center, bAlexandria Faculty of Medicine, Alexandria, c National Heart Institute, Giza, Egypt, dCairo Faculty of Medicine and College of Medicine, University of Sharjah, Sharjah, United Arab Emirates and eNational Heart and Lung Institute, Imperial College, London, UK

Correspondence to Heba Sh. Kassem, MD, PhD, MagdiYacoub Molecular Genetics Laboratory, Bibliotheca Alexandrina, 116 Horreya Avenue, Shallalat, Alexandria 21131, Egypt Tel: + 20 100 175 2274; e-mail: [email protected] *Heba Sh. Kassem and Sherif A. Algendy have contributed equally to the writing of this article. Received 29 November 2015 Accepted 10 February 2016 Middle East Journal of Medical Genetics 2016, 5:65–70

The present study evaluated the controversial role of angiotensin-converting enzyme (ACE) I/D polymorphism in patients with hypertrophic cardiomyopathy (HCM). Two hundred and eleven unrelated HCM patients (138 sporadic and 73 familial) and 203 age and sex-matched healthy volunteers were included. The ACE DD genotype was higher in HCM patients (P = 0.049) and significantly more common in the sporadic HCM group (P = 0.0001). Although the distribution of ACE I/D genotype in the control group was in Hardy–Weinberg equilibrium (P = 0.778), it was not so in HCM patients (P = 0.0010). Among the patients with known sarcomeric mutation, the distribution of D allele was observed to be higher among those having mutations in TNNT2 and MYH7 (P = 0.0476). Similar to other studies undertaken in different populations, there was no significant effect of the ACE I/D genotype observed on HCM phenotypic characterization in the present cohort. ACE I/D allelotyping probably plays a contributing role in initiation of HCM phenotype synergistically with the causative pathogenic sarcomeric mutation, rather than directly influencing variable disease expressivity. Keywords: ACE I/D polymorphism, Egypt, genetics, hypertrophic cardiomyopathy Middle East J Med Genet 5:65–70 & 2016 Middle East Journal of Medical Genetics 2090-8571

Introduction Hypertrophic cardiomyopathy (HCM) characteristically expresses marked heterogeneous clinical and genetic profiles (Marian, 2001; Arad et al., 2002; Maron, 2002; Olivotto et al., 2009). Hundreds of mutations in both sarcomeric, and, more recently, nonsarcomeric genes are being incriminated as the underlying cause (Marian et al., 2001; Alcalai et al., 2008; Konno et al., 2010). Mutations in sarcomeric genes generally have highly variable morphological and clinical expression, and with very few exceptions, no particular phenotype is mutationspecific (Fananapazir and Epstein, 1994). The variable disease expressivity does not only exist among genetically unrelated cases but also within the same family members sharing a common pathogenic mutation. These observations denote that the phenotypic expression of the disease is not solely dependent on the effect of a single gene mutation but probably some other modifying factors exist (Marian, 2002). Potential modifier genes constituting the genetic background and environmental factors through epigenetic mechanisms are anticipated to play a significant role in the pathogenesis of HCM (Kalozoumi et al., 2012). The renin–angiotensin–aldosterone system (RAAS) plays an important role in cardiovascular physiology and disease 2090-8571 & 2016 Middle East Journal of Medical Genetics

(Padmanabhan et al., 2000; Wang and Staessen, 2000;Sayed-Tabatabaei et al., 2006). Polymorphisms in genes encoding the RAAS proteins (angiotensin-converting enzyme, ACE; angiotensinogen gene, AGT; angiotensin II receptor type 1 gene, AGTR1; aldosterone synthase gene, CYP11B2) are plausible HCM disease modifiers. ACE is a major component of the RAAS. The serum level of the ACE enzyme is reported to be genetically determined. In a study by Rigat et al. (1990), ACE I/D polymorphism (due to the presence or absence of a 287 bp Alu repeat in intron 16 of the ACE gene) accounted for about 50% of the variability in its serum level. The ACE DD genotype is reported to be associated with higher plasma levels of the ACE enzyme, being twice as that found in association with II genotype individuals, and individuals with ID genotype have intermediate levels (Rigat et al., 1990). The ACE I/D polymorphism has been suggested to be associated with many cardiovascular diseases, but its role in HCM remains controversial (Lopez-Haldon et al., 1999; Orenes-Pinero et al., 2011). Considering the fact that the D allele frequency is among the highest in Arabs and Egyptian population (0.68) (Salem and Batzer, 2009; Abd El-Aziz et al., 2012), the present study sought to investigate the possible association of ACE gene I/D polymorphism and HCM in an Egyptian case–control cohort. DOI: 10.1097/01.MXE.0000484368.11655.d4

Copyright r 2016 Middle East Journal of Medical Genetics. Unauthorized reproduction of this article is prohibited.

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Materials and methods

Figure 1

Study population

The study comprised 211 consecutive unrelated index HCM patients (138 sporadic and 73 familial cases). The current study was undertaken during 2012–2013 and was approved by the Research Ethics Committees of Magdi Yacoub Heart Foundation/Aswan Heart Center and Alexandria Faculty of Medicine. All research participants provided informed written consent for enrollment in the study and donation of blood sample for genetic analysis. The diagnosis of HCM was established by means of 2D and M-mode echocardiographic analysis in satellite HCM clinics in Aswan, Alexandria, and Cairo. The degree of left ventricular hypertrophy (LVH) was determined by assessing the maximum thickness of the interventricular septum (IVS). Age and sex-matched healthy adult controls (203) were recruited to participate in the study to assess the prevalence of the ACE I/D polymorphism as a representative of ethnic-matched normal population and to correlate I/D allele frequencies to that of the HCM cohort. Control subjects were selected based on lack of a self-reported or a family history of cardiovascular disease and with normal electrocardiographic (ECG) screening. Genetic analysis

Blood samples were collected in EDTA tubes and DNA was extracted using Wizard Genomic DNA purification kit (Promega Corporation, Fitchburg, Wisconsin, USA). ACE I/D polymorphism genotyping was performed using sense and antisense primers previously described by Rigat et al. (1992) (F: 50 -CTGGAGACCACTCCCATCCTT TCT-30 and R: 50 -GATGTGGCCATCACATTCGTCAG AT-30 ). The PCR reaction was performed in a final volume of 25 ml and contained 25 ng of genomic DNA and 25 pmol of each primer. Dimethyl sulfoxide 5% was added to enhance the amplification of the insertion allele (Fogarty et al., 1994). DNA was amplified for 30 cycles (denaturation at 941C for 1 min, annealing at 621C for 1 min, extension at 721C for 1 min, and a final extension at 721C for 5 min). PCR products were resolved on 1.8% agarose gel and visualized with ethidium bromide staining. The 190 bp band represents the D allele and the 490 bp band represents the I allele (Fig. 1). To avoid potential misgenotyping of heterozygote ID participants as DD due to preferential amplification of D allele, all samples scored as DD genotype in primary PCR were subjected to a secondary confirmatory PCR reaction using insertion-specific primer set (F: 50 -TGGGACCACA GCGCCCGCCACTAC-30 and R: 50 -TCGCCAGCCCTC CCATGCCCATAA-30 ) using similar PCR program conditions of the primary reaction except that the annealing temperature was 671C and the reaction did not contain dimethyl sulfoxide (Lindpaintner et al., 1995). A band of 335 bp is only evident in the presence of an I allele and was not found in samples homozygous for DD (Fig. 1). All samples that were scored as DD in the first PCR and proven to be ID in second PCR reaction were analyzed twice. One hundred and seventy-two (81.5%) patients

Captured gel image of ACE I/D genotyping. Lane 1: Negative control; second and sixth lanes scored as ID genotype using primary PCR reaction; third and eighth lanes show D allele using primary PCR reaction; fourth, fifth, and seventh lanes show I allele using secondary PCR reaction with insertion-specific primers. ACE, angiotensinconverting enzyme.

included in the present study were also screened for mutations in the three most commonly involved sarcomeric genes (MYBPC, MYH7, and TNNT) (Kassem et al., 2012).

Statistical analysis

Statistical analysis was performed using MedCalc Software for Windows, version 11.6.1.0 (MedCalc, Oostende, Belgium) and the GraphPad prism software for Windows, version 5.0 (GraphPad Software Inc., San Diego, California, USA). The w2-test was used to test qualitative variables. The Mann–Whitney U-test was used to assess quantitative parameters and analysis of variance (Kruskal–Wallis test) was used to compare the difference between the positive three sarcomeric groups. A P-value of less than or equal to 0.05 was considered statistically significant.

Results Clinical characteristics and genetic status of the study population

We examined 211 patients diagnosed with HCM and 203 healthy adults as a control group for the ACE I/D polymorphism. Patients with HCM were further classified according to the presence of family history of HCM into familial (34.5%) and sporadic (65.5%) cases and according to their myofilament/sarcomeric genotype status (Kassem et al., 2012). The demographic, clinical, and echocardiographic characteristics of the study patient cohort are shown in Table 1. The PCR-based method used to assess the ACE I/D variants allowed the detection of three possible genotypes (DD, ID, and II) (Fig. 1).


ACE I/D polymorphism in HCM Kassem et al. 67

Table 1 Clinical and demographic characteristics of patients with hypertrophic cardiomyopathy Feature Sex Male Female Age at diagnosis (years) Median (range) Family history Positive for HCM Negative for sudden death Syncope Atrial fibrillation Echocardiographic measurements Left atrium (mm) LV outflow gradient (Z30 mmHg) Maximum LV thickness (mm) o30 Z30 LV end-diastolic dimension (mm) LV end-systolic dimension (mm) Mitral regurgitation SAM Major adverse cardiac events Sudden cardiac death Previous cardiac arrest Heart failure Interventions for obstruction/symptoms Alcohol septal ablation Surgical septal myectomy ICD Pacemaker

n (%) or mean ± SD 144/211 (68.2) 67/211 (31.7) 35.9 ± 16.9 37 (1–70) 73/211 42/211 114/211 17/211

(34.5) (19.9) (54) (8)

45.02 ± 9.75 113/197 (57.36) 24.04 ± 6.45 175/211 (82.9) 36/211 (17.06) 44.5 ± 8.38 26.54 ± 6.60 144/197 (73.09) 128/191 (67.01) 2/118 (1.69) 3/118 (2.54) 3/118 (2.54) 3/211 52/211 2/211 4/211

(1.42) (24.64) (0.94) (1.89)

Data on major adverse cardiac events, mitral regurgitation, SAM and LV outflow gradient were not available for all patients (clarified by the denominator). HCM, hypertrophic cardiomyopathy; ICD, implantable cardiac defibrillator; LV, left ventricular; SAM; systolic anterior motion of the mitral valve.

In the studied cohort, 160 samples had an ID genotype, of which 145 samples (90.6%) were initially scored as DD in the first PCR reaction and were shown to have an I allele in the secondary PCR reaction using the insertionspecific primer set. This indicates the necessity for screening with insertion-specific primers. The present study showed similar D allele frequency in HCM patients and healthy controls (68 and 68.7%, respectively) (Table 2). However, there was an observed difference in the distribution of ACE DD genotype, being higher among HCM patients compared with healthy volunteers (P = 0.0493; Fig. 2a). Furthermore, a highly significant difference was observed in the presence of DD genotype between sporadic and familial HCM cases (P = 0.0001; Fig. 2b). Among the 172 patients who were genotyped for the three most common sarcomeric gene mutations, 68 (39.5%) were found to be mutation-positive as follows: MYBPC, 37/68 (54.4%); MYH7, 22/68 (32.3%); and TNNT 9/68, (13.2%). Details of sarcomeric mutations detected have been previously reported (Kassem et al., 2012). DD and ID genotypes of ACE were significantly higher compared with II genotype (P = 0.0476; Fig. 2c) in HCM patients with sarcomeric mutations in TNNT2 and MYH7, respectively. Although the distribution of ACE genotypes in healthy volunteers was in Hardy–Weinberg equilibrium

(w2 = 0.079; P = 0.7780), it was not so in HCM patients (w2 = 10.843; P = 0.0010) (Emigh, 1980; Wigginton et al., 2005) (Table 3). There was no significant correlation observed between ACE genotype distribution and variable clinical features in the present study, including the New York Heart Association class (P = 0.8885), age at diagnosis (P = 0.7937), family history of sudden cardiac death (P = 0.37), occurrence of atrial fibrillation (P = 0.1146), and proportion of those who had undertaken surgical relief for outflow tract obstruction (P = 0.221). In addition, no correlation was observed between ACE I/D polymorphism and echocardiographic parameters, including maximum left ventricle thickness greater than or equal to 30 mm (P = 0.5758), left ventricular enddiastolic diameter (P = 0.95), left ventricular end-systolic diameter (P = 0.89), ejection fraction (P = 0.18), or left ventricular outflow tract obstruction (P = 0.096). Absence of any correlation between ACE allelotype and disease parameters and prognosis suggests lack of a direct role of ACE I/D polymorphism in modifying disease clinicopathologic characterstics, but does not preclude a possible role in disease initiation and pathogenesis.

Discussion Angiotensin I-converting enzyme enhances the synthesis of angiotensin II, which promotes cell growth and hypertrophy. Angiotensin II stimulates the synthesis and release of aldosterone, which promotes cardiac fibrosis (Orenes-Pinero et al., 2011). Thus, it is anticipated that overexpression of the enzyme could influence HCM pathogenesis. The gene coding for ACE enzyme shows an insertion/deletion polymorphism due to the presence or absence of a 287 bp Alu repeat in intron 16 of the ACE gene. Although the I/D polymorphism is located in a noncoding region (intron) of the ACE gene, several investigators have reported an association between the presence of D allele and increased activity of the enzyme in serum (Rigat et al., 1990; Sayed-Tabatabaei et al., 2006; Abbas et al., 2012). Individuals with the DD genotype have higher tissue levels of ACE, which may thus act as a potentially pro-LVH milieu. Cardiac hypertrophy and interstitial fibrosis are major determinants of morbidity and mortality in HCM. These pathologic features result in an increased left ventricular end-diastolic pressure and diastolic dysfunction contributing to heart failure (Shirani et al., 2000; Spirito et al., 2000). Increased ACE gene expression and ACE activity may contribute to an abnormal pattern of myocardial growth and modify LVH and myocardial fibrosis in HCM pathogenesis. Prinz et al. (2012a, 2012b) reported that myocardial fibrosis, as detected using two-dimensional speckletracking echocardiography, was associated with both left atrial and biventricular dysfunction. Zachariah et al. (2012) showed that circulating matrix metalloproteinases, which is a marker for fibrosis, are significantly higher in patients with ventricular arrhythmias. Myocardial fibrosis


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Figure 2

Bar charts showing distribution of ACE genotypes in different groups. (a) Distribution of ACE genotypes in HCM cases versus healthy volunteers. (b) Distribution of ACE genotypes in sporadic and familial HCM. (c) Distribution of ACE genotypes among sarcomeric genopositive patients for MYBPC3, MYH7, and TNNT2 genes. ACE, angiotensin-converting enzyme; HCM, hypertrophic cardiomyopathy.

Table 2 Distribution of ACE I/D allele and genotype frequencies in different studied groups

HCM cases (n = 211) Controls (n = 203) Sporadic HCM (n = 138) Familial HCM (n = 73) MyBPC (n = 37) MyH7 (n = 22) TNNT (n = 9)

DD [n (%)]

ID [n (%)]

108 95 82 26 16 12 6

71 89 45 26 12 9 3

(51.18) (46.79) (59.4) (35.6) (43.2) (54.5) (66.6)

II [n (%)]

(33.64) (43.84) (32.6) (35.6) (32.4) (40.9) (33.3)

32 19 11 21 9 1 0

(15.16) (9.35) (7.97) (28.7) (24.3) (4.5) (0.0)

P-value

D allele (%)

I allele (%)

0.0493*

68 68.7 75.7 53.4 59.5 75 83.3

32 31.3 24.3 46.6 40.5 25 16.6

0.0001* 0.1701

ACE, angiotensin-converting enzyme; HCM, hypertrophic cardiomyopathy. *Pr0.05 is considered statistically significant.

Table 3 Distribution of ACE genotypes between healthy volunteers and HCM patients in relation to Hardy–Weinberg equilibrium Control

Genotype frequency (%) D allele frequency (%) I allele frequency (%) Hardy–Weinberg – expected frequency [n (%)] w2 P-value

HCM

DD

ID

II

DD

ID

II

46.80

43.84 68.7 31.3 87.27 (42.99) 0.079 0.7780

9.36

51.18

15.17

19.86 (9.78)

97.59 (46.25)

33.65 68.0 32.0 91.81 (43.51) 10.843 0.0010*

95.86 (47.22)

21.59 (10.23)

ACE, angiotensin-converting enzyme; HCM, hypertrophic cardiomyopathy; HWE, Hardy–Weinberg equilibrium. *Pr0.05 = not consistent with HWE.


ACE I/D polymorphism in HCM Kassem et al. 69

is a hallmark of HCM and is a risk factor for impaired cardiac contractility and ventricular arrhythmia. Previous studies showed that angiotensin II enhances the macrophage-stimulated cardiac fibroblast production of interleukin-6 that leads to cardiac fibrosis (Ma et al., 2012). The D allele frequency is reported to be variable among different populations. It was reported to range from 0.32 to 0.39 among Japanese and from 0.54 to 0.58 in Europeans (Cambien et al., 1992; Yoneya et al., 1995). In the present study, the D allele frequency was found to be among the highest reported (0.68) and is consistent with previous studies in Arab and Egyptian populations (Salem and Batzer, 2009; Abd El-Aziz et al., 2012). ACE I/D polymorphism has been studied in relevance to several cardiovascular disorders. Associations between the DD genotype and premature coronary artery disease (Abd El-Aziz et al., 2012), Hypertension (Zarouk et al., 2012), and development of rheumatic heart disease (Morsy et al., 2011) has been previously reported, suggesting a potential role of ACE in such disorders. The burden of different cardiovascular diseases is variable among different populations and this may be partly explained by difference in underlying genetic background. The relationship between ACE I/D polymorphism and HCM phenotype has been investigated in several earlier studies with controversial results being reported. The fact that D allele is highest among the Egyptian population prompted us to study its possible role in our HCM cohort. The present study demonstrated a slightly higher frequency of DD genotype in the HCM cohort compared with the control population (P = 0.049) and was significantly higher among the sporadic HCM cases compared with familial cases (P = 0.0001). However, this skewed distribution of the ACE polymorphism in our cohort was not found to reflect a significant association with the disease phenotype or outcome. In agreement with our findings, several studies reported an overrepresentation of the ACE DD genotype in HCM cases compared with controls, with no correlation between ACE I/D genotype distribution and disease expression profiles (including outflow tract obstruction and/or the degree of cardiac hypertrophy) (Yoneya et al., 1995Pfeufer et al., 1996; Yamada et al., 1997; Rai et al., 2008). It is also worth mentioning that Buck et al. (2009) previously reported that cardiac hypertrophy was associated with elevated serum ACE activity but not with different ACE genotypes. A recent meta-analysis performed by Luo et al. (2013) on 11 studies suggested a role of ACE I/D polymorphism in HCM, with no significant effect on phenotypic expression of the disease ‘as assessed by maximum IVS thickness’. On the other hand, some studies reported an association between ACE DD genotype (Lechin et al., 1995; Lopez-Haldon et al., 1999; Kolder et al., 2012) and D allele frequency (Lopez-Haldon et al., 1999) with increased LVH and LVH progression (Doolan et al., 2004). Moreover, the ACE gene polymorphism was reported to modify disease expression in specific patient groups according to their sarcomeric gene mutation status

(Tesson et al., 1997). Tesson et al. (1997) showed a significant association between ACE D allele and LVH only in a subgroup of patients who were carriers of Arg403 codon mutation in MYH7 gene. Moreover, Perkins et al. (2005) reported that ACE DD genotype was associated with increased hypertrophy in patients with mutation in MYBPC gene but not with other sarcomeric genes. In the present study, the DD and ID genotypes were significantly higher in patients with mutations in TNNT2 and MYH7 sarcomeric genes. However, it was not possible to detect a correlation between ACE genotypes and mutation-associated disease expression, possibly due to small subgroup sample size. The pro-LVH effect of polymorphisms of RAAS has been addressed by multiple investigators. Previous reports showed that the pooled effect of the pro-LVH RAAS polymorphisms was associated with progression of the disease in terms of development of systolic dysfunction and dilatation (Funada et al., 2010), increased left ventricular mass index, progressive septal hypertrophy, and left ventricular outflow tract obstruction (Kaufman et al., 2007). However, this association was not evident when the IVS thickness and Wigle score were studied (Kolder et al., 2012), further adding to the controversy of the influence of the association. There are some limitations to the present study. The controls were screened only with ECG and not using echocardiography or magnetic resonance imaging. It is a cross-sectional study and accordingly some control participants might develop important cardiac events in the future. However, in the absence of symptoms of cardiac disease and normal finding of ECG screening, there is a very low possibility that the control group included HCM patients that would influence the results significantly. It would also be useful in the future studies to assess the independent effect of ACE gene I/D polymorphism within a genetically dependent cohort – that is, within the same affected family members in which they are sharing most of their genomic constitution, particularly so when they are also having a known sarcomeric mutation. Vertical family members’ genetic and phenotype characterization is still ongoing in the BA HCM program.

Conclusion The DD genotype was slightly over-represented in the HCM cohort, particularly so in the sporadic cases, suggesting that HCM pathogenesis may be influenced by genetically predisposed milieu related to homozygous status of ACE Deletion allele. Further studies of potential disease modifiers and inclusion of further RAAS polymorphisms are warranted to elaborate on HCM phenotype heterogeneity.

Acknowledgements The authors sincerely acknowledge the support of the HCM patients and the healthy volunteers and kind contribution of the referring


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cardiologists to the BA HCM National Program. We also appreciate the support of the Bibliotheca Alexandrina and Magdi Yacoub Heart Foundation.

Conflicts of interest There are no conflicts of interest.

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