Association of angiotensin converting enzyme gene insertion/deletion polymorphism and familial hypercholesterolemia in the Saudi population
- Khalid K Alharbi†1,
- Tarek S Kashour2,
- Wejdan Al-Hussaini†1, 3,
- May Salem Al-Nbaheen†3, 4,
- Sarar Mohamed†5,
- Rana MW Hasanato6,
- Waleed Tamimi7,
- Mohammed Yahya Al-Naami8 and
- Imran Ali Khan†1Email author
© Alharbi et al.; licensee BioMed Central Ltd. 2013
Received: 26 October 2013
Accepted: 18 November 2013
Published: 1 December 2013
The study of the association between genotype and phenotype is of great importance for the prediction of multiple diseases and pathophysiological conditions. The relationship between angiotensin converting enzyme (ACE) Insertion/Deletion (I/D) polymorphism and Familial Hypercholesterolemia (FH) has been not fully investigated in all the ethnicities. In this study we sought to determine the frequency of I/D polymorphism genotypes of ACE gene in Saudi patients with FH.
This is a case–control study carried out purely in Saudi population. Genomic DNA was isolated from 128 subjects who have participated in this study. ACE gene I/D polymorphism was analyzed by polymerase chain reaction in 64 FH cases and 64 healthy controls. There was no statistically significant difference between the groups with respect to genotype distribution. Furthermore, we did not find any significant difference in the frequency of ACE I/D polymorphism in FH subjects when stratified by gender (p = 0.43).
Our data suggest that ACE gene I/D polymorphism examined in this study has no role in predicting the occurrence and diagnosis of FH.
Familial hypercholesterolemia (FH, OMIM 143890) is an inherited disorder of lipoprotein metabolism, transmitted in an autosomal dominant manner and clinically characterized by elevated serum levels of total and low-density lipoprotein (LDL) cholesterol, the presence of tendon xanthomas and premature atherosclerosis. The genetic basis of FH is a large array of mutations in the LDL-receptor (LDLR) gene (OMIM 606945), resulting in a lack of functional receptors for LDL on the liver cell surface, giving rise to increased plasma LDL levels. Population frequency of heterozygous familial hypercholesterolemia (hFH) is approximately 1/500. Although genetically the disease is caused by mutations in the LDLR gene, the clinical phenotype of FH can vary regardless of the mutation and this variability is assumed to be due to both environmental and genetic factors. The clinical phenotype caused by mutations in LDLR, APOB or PCSK9 and characterized by elevated levels of plasma LDL-Cholesterol (LDL-C) is currently referred to as Autosomal Dominant Hypercholesterolemia (ADH, OMIM 603776).
Gene polymorphisms are markers of biologic diversity, and some genotypic variations correlate with specific phenotypes relevant to the human diseases. However, it is not clear whether many of these variants are involved in the pathogenesis of diseases or are merely in proximity to other pathogenic genetic factors-a phenomenon known as linkage disequilibrium. Angiotensin-converting enzyme (ACE) gene insertion/deletion (I/D) polymorphism is responsible for inter individual differences in ACE plasma levels. It is known that the frequency of ACE D alleles varies in different ethnic groups. A previous study by O'Malley et al. have shown an association with FH patients who have myocardial infarction (MI) and coronary heart disease (CHD). I/D polymorphism have also been associated with risk of MI. ACE gene is involved in the conversion of Angiotensin I to Angiotensin II by its metalloproteinase enzymatic activity. It plays a major role in the rennin–angiotensin (RAS) system. I/D polymorphism (Consisting of a 287-bp fragment) in intron 16 of the gene has been related to the amount of circulating enzyme. Individuals homozygous for the deletion have an approximately two-fold higher level of circulating enzyme in comparison to individuals homozygous for the insertion. Angiotensin II increases vascular smooth muscle cell proliferation, monocyte adhesion, platelet adhesion and aggregation. ACE genotype acting through the tissue or possible plasma ACE concentrations has been implicated in the aetiology of the disease.
ACE is a zinc metalloprotease that is widely distributed on the surface of epithelial and endothelial cells. Its gene consists of 26 exons and spans 21 kb on the human chromosome 17. The sequence codes a 1306-amino-acid protein, including a signal peptide. It has a common polymorphism that consists of the presence (I allele) or the absence (D allele) of a 287-bp Alu repeat sequence within intron 16. Although the I/D polymorphism is in the intronic region of the ACE gene, studies have shown that the DD genotype is strongly associated with increased plasma or serum ACE levels. ACE activity in individuals with DD genotype is twice that found in those with II genotypes. Subjects with ID genotype exhibit intermediate levels of ACE. This ACE I/D gene polymorphism has been implicated as a risk factor for a number of pathologies, such as MI, stroke, Type 2 Diabetes Mellitus (T2DM), Diabetic Nephropathy (DN), and hypertension in different ethnic groups, but these findings are far from conclusive[4, 10]. The aim of this study was to analyze whether the ACE gene I/D polymorphism is associated with the FH patients in Saudi population.
Materials and methodology
In this study 64 subjects diagnosed with FH, based on the Deutch working group classification criteria. Patients were recruited from outpatient clinic at King Khalid University Hospital (KKUH), Riyadh. Healthy volunteers (n = 64) were recruited from Medical and laboratory staff at KKUH, and individuals attend KKUH for routine checkup and who were not having chronic metabolic/ medical disease. The research protocol was approved by the Institutional Review Board at KKUH (E-12-829). All participants provided written informed consent prior to enrollment into the study.
Participants were advised to fast overnight for at least 10 hours prior to sample collection. Five mL of blood sample was collected from every participant; 3 mL of the serum sample was used for biochemical parameters like Total Cholesterol (TC), Triglycerides (TG), HDL-C and LDLC to confirm the FH and 2 mL of EDTA blood sample was used for molecular investigation.
Serum samples of TC and TG were measured by the standard enzymatic method (Pars Azmon kit, Iran) using an automated RA-1000 (Technicon, USA). Serum LDL-C and HDL-C levels were measured using commercially available enzyme assay kits (Pars Azmon kit, Iran).
Genomic DNA isolation and genotyping of ACE I/D polymorphism
All the statistical analyses were carried out using SPSS (Chicago, IL, USA) software version 19.0 for Microsoft Windows®. Clinical characteristics of all subjects were expressed as mean ± SD. Continuous variables were compared between the groups using two-tailed student’s t test. Significant cutoff was set at 0.05. Crude ORs with 95% CIs were used to evaluate the strength of the association between ACE I/D polymorphism and FH. Pooled ORs were calculated for allelic contrast (D vs. I), dominant (DD + DI vs. II) and recessive (DD vs. ID + II) genetic model. Z-test was used to determine the significance of the pooled ORs, and p value < 0.05 was considered significant. The effect of I/D genotypes of ACE gene was analyzed using general linear model ANOVA for clinical characteristics. A level of p <0.05 was considered statistically significant.
Demographic characteristics and biochemical profile of FH patients and healthy control
(n = 64)
(n = 64)
52.06 ± 10.27
44.8 ± 6.87
p = 0.0001
p = 0.7
1.3 ± 0.50
1.1 ± 0.36
p = 0.01
6.4 ± 0.68
4.6 ± 0.39
p = 0.04
1.4 ± 0.24
0.9 ± 0.21
p = 0.55
4.8 ± 0.55
3.1 ± 0.32
p = 0.01
Screening of I/D polymorphism in the ACE gene
Genotype and allele frequency of ACE gene
Genotypes and Alleles
FH cases (n = 64)
Healthy controls (n = 64)
Statistical analysis for FH individuals
DD Vs ID + II
OR-1.465;95% CI = 0.7265–2.956; p = 0.28
ID + DD Vs II
OR-1.696;95% CI = 0.7385–3.893; p = 0.21
ID Vs II + DD
OR-1.00;95% CI = 0.4781–2.091; p = 0.85
D Vs I
OR-1.481;95% CI = 0.8953–2.449; p = 0.12
Genotype and allele frequency of ACE gene between males and females
Genotypes and alleles
Males (n = 20)
Females (n = 44)
OR (95% CI)
p = 0.36
p = 0.40
p = 0.86
p = 0.43
To the best of our knowledge this is the first study to investigate the association of ACE gene I/D polymorphism and FH in Saudi population. In our study FH individuals tended to have higher DD (48.4%) genotypes and more ID (32.9%) genotypes than controls. However, there was no significant difference in the allelic frequency between the patients and controls.
Previous studies addressing the association between FH and ACE gene polymorphism focused on the potential risk of CHD coronary heart disease associated with ACE gene polymorphism in FH patients. For example, O’Malley et al. showed that in heterozygote FH patients with ACE-DD genotype, the incidence of MI is 2.5 times higher and CHD is 2.2 higher than in those with DI or II genotypes. On the other hand, another study, did not find any association between ACE gene deletion/insertion polymorphism and increased risk of CHD in patients with FH.
A few studies have been conducted in Saudi Arabia and Middle East looking at the association between ACE gene polymorphism and certain diseases. We have reported previously an association between ACE gene polymorphism and G6PD deficiency. El-Hazmi et al. also showed a significant association between ACE DD genotype and obesity among Saudi patients.
In contrary to that, Dzimiri et al. showed no association between ACE genotype and risk of coronary artery disease in Saudi patients. Another study reported by Chmaisse et al. showed no association between T2DM and ACE gene polymorphism in s mall Lebanese cohort. Our study is limited by small sample size and the fact that our patients have no documented coronary heart disease which may explain our inability to show an association between ACE gene polymorphism and FH.
In conclusion, our study has not shown an association between I/D polymorphism of ACE gene and FH in the Saudi populations. However, considering the relatively small sample sizes of our study, a firm conclusion cannot be made and larger studies in more well-characterized subjects should be conducted in the future.
The authors would like to extend their sincere appreciation to the Deanship of Scientific Research at King Saud University for its funding of this research through the Research Group Project no RGP-VPP-244.
- Alonso R, Defesche JC, Tejedor D, Castillo S, Stef M, Mata N, Gomez-Enterria P, Martinez-Faedo C, Forga L, Mata P: Genetic diagnosis of familial hypercholesterolemia using a DNA-array based platform. Clin Biochem. 2009, 42: 899-903. 10.1016/j.clinbiochem.2009.01.017View ArticlePubMedGoogle Scholar
- Goldstein JL, Brown MS: Molecular medicine: the cholesterol quartet. Science. 2001, 292: 1310-1312. 10.1126/science.1061815View ArticlePubMedGoogle Scholar
- Aledo R, Alonso R, Mata P, Llorente-Cortes V, Padro T, Badimon L: LRP1 gene polymorphisms are associated with premature risk of cardiovascular disease in patients with familial hypercholesterolemia. Rev Esp Cardiol. 2012, 65: 807-812. 10.1016/j.recesp.2012.03.013View ArticlePubMedGoogle Scholar
- Movva S, Alluri RV, Komandur S, Vattam K, Eppa K, Mukkavali KK, Mubigonda S, Saharia S, Shastry JC, Hasan Q: Relationship between angiotensin-converting enzyme gene polymorphism with nephropathy associated with Type 2 Diabetes Mellitus in Asian Indians. J Diabetes Complications. 2007, 21: 237-241. 10.1016/j.jdiacomp.2006.07.001View ArticlePubMedGoogle Scholar
- O’Malley JP, Maslen CL, Illingworth R: Angiotensin-converting enzyme DD genotype and cardiovascular risk in heterozygous familial hypercholesterolemia. Circulation. 1998, 97: 1780-1783. 10.1161/01.CIR.97.18.1780View ArticlePubMedGoogle Scholar
- Munshi A, Sultana S, Kaul S, Reddy BP, Alladi S, Jyothy A: Angiotensin-converting enzyme insertion/deletion polymorphism and the risk of ischemic stroke in a South Indian population. J Neurol Sci. 2008, 272: 132-135. 10.1016/j.jns.2008.05.017View ArticlePubMedGoogle Scholar
- Rigat B, Hubert C, Alhenc-Gelas F, Cambien F, Corvol P, Soubrier F: An insertion/deletion polymorphism in the angiotensin I-converting enzyme gene accounting for half the variance of serum enzyme levels. J Clin Invest. 1990, 86: 1343-1346. 10.1172/JCI114844PubMed CentralView ArticlePubMedGoogle Scholar
- Humphries SE, Morgan L: Genetic risk factors for stroke: insights into pathophysiology from candidate gene approaches. Int Congr Ser. 2004, 1262: 482-485.View ArticleGoogle Scholar
- Pasha MA, Khan AP, Kumar R, Ram RB, Grover SK, Srivastava KK, Selvamurthy W, Brahmachari SK: Variations in angiotensin-converting enzyme gene insertion/deletion polymorphism in Indian populations of different ethnic origins. J Biosci. 2002, 27: 67-70. 10.1007/BF02703684View ArticlePubMedGoogle Scholar
- Kunz R, Bork JP, Fritsche L, Ringel J, Sharma AM: Association between the angiotensin-converting enzyme-insertion/deletion polymorphism and diabetic nephropathy: a methodologic appraisal and systematic review. J Am Soc Nephrol. 1998, 9: 1653-1663.PubMedGoogle Scholar
- Benn M, Watts GF, Tybjaerg-Hansen A, Nordestgaard BG: Familial hypercholesterolemia in the Danish general population: prevalence, coronary artery disease, and cholesterol-lowering medication. J Clin Endocrinol Metab. 2012, 97: 3956-3964. 10.1210/jc.2012-1563View ArticlePubMedGoogle Scholar
- Alharbi KK, Khan IA, Abed AS, Syed R: Insertion/Deletion polymorphisms do play any role in G6PD deficiency individuals in the Kingdom of the Saudi Arabia. Bioinformation. 2013, 9: 49-53. 10.6026/97320630009049PubMed CentralView ArticlePubMedGoogle Scholar
- Wierzbicki AS, Lambert-Hammill M, Lumb PJ, Crook MA: Renin-angiotensin system polymorphisms and coronary events in familial hypercholesterolemia. Hypertension. 2000, 36: 808-812. 10.1161/01.HYP.36.5.808View ArticlePubMedGoogle Scholar
- El-Hazmi MA, Warsy AS: Increased frequency of angiotensin-converting enzyme DD genotype in Saudi overweight and obese patients. Ann Saudi Med. 2003, 23: 24-27.PubMedGoogle Scholar
- Dzimiri N, Basco C, Moorji A, Meyer BF: Angiotensin-converting enzyme polymorphism and the risk of coronary heart disease in the Saudi male population. Arch Pathol Lab Med. 2000, 124: 531-534.PubMedGoogle Scholar
- Chmaisse HN, Jammal M, Fakhoury H, Fakhoury R: A study on the association between angiotensin-I converting enzyme I/D dimorphism and type-2 diabetes mellitus. Saudi J Kidney Dis Transpl. 2009, 20: 1038-1046.PubMedGoogle Scholar
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