Angiotensin-converting enzyme insertion/deletion polymorphism genotyping error: the cause and a possible solution to the problem

Rigat and colleagues were the first ones to develop a rapid PCR-based assay for identifying the angiotensin converting enzyme insertion/deletion (I/D) polymorphism. Due to a big difference between the length of the wild-type and mute alleles the PCR method is prone to mistyping because of preferential amplification of the D allele causing depicting I/D heterozygotes as D/D homozygotes. The aim of this study was to investigate whether this preferential amplification can be repressed by amplifying a longer DNA fragment in a so called Long PCR protocol. We also aimed to compare the results of genotyping using five different PCR protocols and to estimate the mistyping rate. The study included 200 samples which were genotyped using standard method used in our laboratory, a stepdown PCR, PCR protocol with the inclusion of 4 % DMSO, PCR with the use of insertion specific primers and new Long PCR method. The results of this study have shown that accurate ACE I/D polymorphism genotyping can be accomplished with the standard and the Long PCR method. Also, as of our results, accurate ACE I/D polymorphism genotyping can be accomplished regardless of the method used. Therefore, if the standard method is optimized more cautiously, accurate results can be obtained by this simple, inexpensive and rapid PCR protocol.     

An alternative method for genotyping of the <Emphasis Type="Italic">ACE</Emphasis> I/D polymorphism

The mistyping of the angiotensin I-converting enzyme insertion/deletion (ACE I/D) has been well documented, and new methods have been suggested here to improve the genotyping efficiency. Buccal cell samples were collected from 157 young Caucasians, and genotyped using previously known and newly developed PCR amplification genotyping techniques, as well as PCR-RFLP tests for three single nucleotide polymorphisms (rs4327, rs4341 and rs4343). Inconsistent genotyping results were found when using only the PCR amplification genotyping techniques across repeated attempts (8% to 45%), however, individual SNP genotyping was highly consistent (100%). Two SNPs (rs4341 and rs4343) were in complete LD and SNP rs4327 was in high LD with the ACE I/D. The ACE I/D was in HW equilibrium in the portion of the population with consistent genotyping results, whereas the three SNPs were not in HW equilibrium. The mistyping of ACE I/D by only PCR amplification can be improved using alternative methods.     

ACE基因多态性与高血压肾脏损害及PAI-1的关系

为探讨血管紧张素转换酶（ACE）基因多态性与高血压肾损害和纤溶酶原激活物抑制物-1（PAI-1）的关系,应用聚合酶链反应（PCR）检测96例正常人、67例高血压无肾脏损害患者和70例高血压伴肾损害患者的ACE基因型,采用ELISA法检测血浆PAI-1。ACE基因I/D多态性与高血压病无明显相关,但高血压肾损害患者DD基因型频率及D等位基因频率显著高于对照组和高血压无肾脏损害组,χ2值分别为6.8589、5.6162 和5.9085、5372。血浆PAI-1在DD型、ID型、II型高血压患者之间亦有显著性差异（P＜0.05）。ACE基因DD型可能是高血压肾损害的危险因素;ACE基因多态性与血浆PAI-1水平相关。 Abstract:The work is to explore the relationship between the polymorphism of angiotensin converting enzyme(ACE) gene and hypertensive kidney lesion/PAI-1 in hypertension patients.ACE genotyping with polymorase chain reaction (PCR) was performed in 96 unrelated healthy controls,67 hypertensives without kidney lesion and 70 hypertensives complicated with kidney lesion.The plasma PAI-1 were determined with ELISA.No significant differences could be detected between ACE gene I/D polymorphism and hypertension.However,the frequencies of DD genotype and deletion allele among the hypertensives complicated with kidney lesion were higher than those among the healthy controls and those among the hypertensives without kidney lesion."χ2" values were 6.8589,5.6162 and 5.9085,5.372 respectively.The plasma PAI-1 level showed significant differences among DD genotype,ID genotype and II genotype(P＜0.05).The DD genotype of ACE gene may be a risk for hypertensive kidney lesion.The plasma PAI-1 level is associated with ACE gene polymorphism.     

Methods for MHC genotyping in non-model vertebrates

Genes of the major histocompatibility complex (MHC) are considered a paradigm of adaptive evolution at the molecular level and as such are frequently investigated by evolutionary biologists and ecologists. Accurate genotyping is essential for understanding of the role that MHC variation plays in natural populations, but may be extremely challenging. Here, I discuss the DNA-based methods currently used for genotyping MHC in non-model vertebrates, as well as techniques likely to find widespread use in the future. I also highlight the aspects of MHC structure that are relevant for genotyping, and detail the challenges posed by the complex genomic organization and high sequence variation of MHC loci. Special emphasis is placed on designing appropriate PCR primers, accounting for artefacts and the problem of genotyping alleles from multiple, co-amplifying loci, a strategy which is frequently necessary due to the structure of the MHC. The suitability of typing techniques is compared in various research situations, strategies for efficient genotyping are discussed and areas of likely progress in future are identified. This review addresses the well established typing methods such as the Single Strand Conformation Polymorphism (SSCP), Denaturing Gradient Gel Electrophoresis (DGGE), Reference Strand Conformational Analysis (RSCA) and cloning of PCR products. In addition, it includes the intriguing possibility of direct amplicon sequencing followed by the computational inference of alleles and also next generation sequencing (NGS) technologies; the latter technique may, in the future, find widespread use in typing complex multilocus MHC systems.     

The frequency of factor V Leiden mutation,ACE gene polymorphism,serum ACE activity and response to ACE inhibitor and angiotensin II receptor antagonist drugs in Iranians type II diabetic patients with microalbuminuria

The aim of present study was to determine if factor V Leiden (FVL) mutation and angiotensin converting enzyme insertion/deletion (ACE I/D) polymorphism are associated with diabetic nephropathy (DN) among Kurdish population from Western Iran. This case–control study comprised 144 unrelated adult type 2 diabetic mellitus patients (T2DM) including 72 patients with microalbuminuria and 72 age and sex matched patients without nephropathy. The ACE I/D polymorphism and FVL mutation were detected by polymerase chain reaction (PCR) and PCR–RFLP, respectively. The frequency of FVL G1691A and ACE D allele in T2DM patients with microalbuminuria were 1.6 and 57%, respectively and in normoalbuminuric T2DM patients were 4.9 and 58.3%, respectively (P > 0.05). ACE genotypes affected on serum ACE activity and a better response to ACE inhibitor therapy (captopril) compared to angiotensin II receptor antagonist (losartan) was obtained with significant reduction of ACE activity in diabetic patients without nephropathy carrying DD genotype. However, the beneficial effect of losartan therapy was observed in microalbuminuric patients with II genotype compared to ID and DD genotypes.     

Homozygosity for multiple contiguous single-nucleotide polymorphisms as an indicator of large heterozygous deletions: identification of a novel heterozygous 8-kb intragenic deletion (IVS7-19 to IVS15-17) in a patient with glycogen storage disease type II

Current methods for detection of mutations by polymerase chain reaction (PCR) and sequence analysis frequently are not able to detect heterozygous large deletions. We report the successful use of a novel approach to identify such deletions, based on detection of apparent homozygosity of contiguous single-nucleotide polymorphisms (SNPs). The sequence analysis of genomic DNA PCR products containing all coding exons and flanking introns identified only a single heterozygous mutation (IVS18+2t-->a) in a patient with classic infantile-onset autosomal recessive glycogen storage disease type II (GSDII). Apparent homozygosity for multiple contiguous SNPs detected by this sequencing suggested presence of a large deletion as the second mutation; primers flanking the region of homozygous SNPs permitted identification and characterization by PCR of a large genomic deletion (8.26 kb) extending from IVS7 to IVS15. The data clearly demonstrate the utility of SNPs as markers for large deletions in autosomal recessive diseases when only a single mutation is found, thus complementing currently standard DNA PCR sequence methods for identifying the molecular basis of disease.     

Association of inflammatory chemokine gene CCL2I/D with bladder cancer risk in North Indian population

Chemokine genes have been proposed as good candidate genes for conferring susceptibility to Bladder cancer (BC). We examined the combined effect of multiple alleles of pro inflammatory chemokine genes for determining the risk of BC. We tested association of three gene polymorphisms of CCL2I/D (rs3917887), CCL2A2518G (rs1024611) and CCR2V64I (rs1799864) with BC risk in North Indian population. Genotypes were assessed in hospital-based case-control study comprising of 200?BC patients and 200 healthy controls. Genomic DNA was isolated from blood and genotyping done using PCR-RFLP method. In CCL2I/D polymorphism, the heterozygous genotype (I/D) showed high risk of BC p?相似文献   

Association of angiotensin converting enzyme (ACE) gene I/D polymorphism and rheumatoid arthritis

We sought to determine the frequency of I/D polymorphism genotypes of angiotensin converting enzyme gene in Turkish patients with rheumatoid arthritis. Genomic DNA obtained from 256 individuals (110 patients with rheumatoid arthritis and 146 healthy controls) was used in the study. ACE gene I/D polymorphism genotypes were determined using polymerase chain reaction using I and D allele-specific primers. There was a statistically significant difference between the groups with respect to genotype distribution (p = 0.001). A significant difference was found in frequencies of ACE I/D alleles between patients and controls, with RA patients having a higher representation of D and lower representation of I alleles compared to controls (p < 0.001). As a result of our study, angiotensin converting enzyme gene I/D polymorphism DD genotype could be a genetic marker in rheumatoid arthritis in the Turkish study population.     

飞行员中血管紧张素转换酶基因插入或缺失多态性研究

为了解飞行员血管紧张素转换酶(ACE)基因插入或缺失（I/D）多态性情况,探讨ACE基因多态性与飞行员耐力可能的关系,用聚合酶链反应（PCR）扩增技术检测118例飞行员和96例健康对照者的ACE基因I/D多态性。 结果位于ACE基因内含子16的I/D多态性经PCR扩增后呈三种基因型：纯合子插入型（II）、纯合子缺失型（DD）和杂合子插入或缺失型（I/D）。飞行员组II基因型（44.07%）和I等位基因频率（0.65）显著高于健康对照组(分别为31.25%和0.52)。 结果表明ACE I基因有可能在飞行员的飞行耐力中起重要作用。 Abstract:In order to understand insertion/delation (I/D) polymorphism of the angiotensin-converting enzyme (ACE) gene in pilots,and to explore the relationship between ACE gene I/D polymorphism and the perfomance of the pilots,the polymerase chain reaction (PCR) was used to determine the genotypes for an I/D polymorphism in intron 16 of the ACE gene in 118 pilots and 96 healthy subjects as controls.The result showed that the I/D polymorphism in intron 16 of the ACE gene was categorized into three genotypes: two deletion alleles (genotype DD),heterozygous alleles (genotype ID),and two insertion alleles (genotype II).The genotype II and I allele frequency were significantly higher in pilots (44.07% and 0.65) than that in healthy subjects (31.25% and 0.52).It is suggested that I gene of ACE may play a role in perfomance of the pilots.     

High-throughput detection of multiple genetic polymorphisms influencing drug metabolism with mismatch primers in allele-specific polymerase chain reaction

Because of genetic polymorphisms of drug-metabolizing enzyme genes, the activities of the enzymes in humans vary widely and alter the metabolism of commonly used clinical agents. Severe adverse effects or resistance to therapy may result. We have developed a rapid and high-throughput genotyping method for detecting polymorphisms of the drug-metabolizing enzyme genes CYP2C9*3, CYP2C19*2, *3, CYP2D6*2, *4, *10, *14, *21, NAT2*5, *6, *7, and TPMT*3 using allele-specific polymerase chain reaction (PCR) with mismatch primers (ASPCR-MP) and CYP2D6*5, *36, and CYP2D6xN using stepdown PCR with detection by SYBR Green I. We analyzed genomic DNA from 139 Japanese volunteers. Identical genotyping results were obtained by using ASPCR-MP, stepdown PCR, and conventional PCR. We found that the methods clearly differentiate three specific profiles with no overlap in the signals. Moreover, both ASPCR-MP and stepdown PCR for genotyping took less than 3-4h. To our knowledge, this is the first report of successful simultaneous detection of multiple genetic polymorphisms with point mutations using ASPCR-MP or multiple genetic polymorphisms with large structural alterations using stepdown PCR. In conclusion, ASPCR-MP and stepdown PCR appear to be suitable for large clinical and epidemiological studies as methods that enable highly sensitive genotyping and yield a high-throughput.     

Modification of the coronary artery disease risk associated with the presence of traditional risk factors by insertion/deletion polymorphism of the ACE gene

Cigarette smoking, hypercholesterolemia, and obesity influence the renin-angiotensin system (RAS) functions including an increased synthesis of the angiotensin I converting enzyme (ACE). Thus in the present work we explore the interactions of the ACE gene insertion/deletion (I/D) polymorphism and traditional risk factors. The study cohort included 341 subjects composed of 172 patients with angiographically confirmed CAD and 169 blood donors without a history of cardiovascular diseases. The I/D polymorphism was genotyped using polymerase chain reaction (PCR) methodology. To determine the interactions between the ACE genotypes and traditional risk factors the epidemiologic approach was used (4 x 2 tables and the synergy measures). The frequency of the DD genotype was significantly higher in patients than in controls (33.7% versus 21.3%, odds ratio [OR] = 1.88, 95% CI; 1.13-3.15, p = 0.010), but greater differences were found in males (35.7% versus 20.5%, OR = 2.15, 95% CI: 1.14-4.04, p = 0.010). We found a synergy of the DD genotype with smoking (SI = 1.88, SIM = 1.22), total cholesterol > or =5 mmol/l (SI = 2.12, SIM = 1.31) and elevated low density lipoprotein (LDL) cholesterol level (> or =3 mmol/l) (SI = 1.78, SIM = 1.14). The presence of the D allele (DD + ID subjects) also increased the risk of coronary artery disease (CAD) associated with the presence of elevated total cholesterol and LDL cholesterol (SI = 1.69, SIM = 1.18, in both cases), elevated level (> or =1.7 mmol/l) of triacylglycerols (SI = 1.81, SIM = 1.18) and overweight/obesity (SI = 4.25, SIM = 2.36). In each case the estimated CAD risk was greater than that predicted by assuming the additivity of effects (the risk increased from 69% for the D allele - total cholesterol interaction to 325% for the D allele - overweight/obesity). The statistical significance was also confirmed by a multiplicative model of synergy. The DD genotype/D allele of the ACE gene increases the risk of CAD associated with the presence of traditional risk factors.     

Parentage analysis for three Romanian families by DNA-fingerprinting

In forensic medicine, DNA fingerprinting for human identification and paternity testing is becoming a necessary procedure. The genetic locus D1S80 (MCT118) with Hinf I polymorphism of its 5' flanking sequence, HUMTH01 and D21S11 have been successfully amplified from human genomic DNA isolated from blood (50 ng from each sample) by the polymerase chain reaction (PCR) using oligonucleotide primers complementary to the flanking sequences as primers for amplification. DNA bands were detected by ethidium bromide staining after electrophoresis on agarose gels or high-resolution SDS-PAGE. Analysis of these VNTR loci was thus achieved without the need for Southern blot or radioactive material. The small size of the DNA fragments produced in the PCR amplification permitted good resolution of individual alleles. The precise specification of the number of tandem repeats present in each allelic fragment was reproducible from one analysis to another. The aim of this study includes three paternity testing cases; they are the first three human DNA-fingerprints performed in Romania.     

Association of AGTR1 (A1166C) and ACE (I/D) Polymorphisms with Breast Cancer Risk in North Indian Population

Renin angiotensin system (RAS) comprising Angiotensin converting enzyme (ACE), Angiotensin II (Ang II) and its receptor Angiotensin II receptor type I (AGTR1), plays a critical role in several diseases including cancer. A single nucleotide polymorphism (SNP) A1166C located in 3′ untranslated region (UTR) of AGTR1 and an insertion/deletion (I/D) polymorphism present in intron 16 of ACE gene have been associated with many diseases, but their association with Breast cancer (BCa) is still debatable. Here, we for the first time investigated the association of these polymorphisms in a North Indian BCa cohort including 161 patients and 152 healthy women. The polymorphisms were evaluated by polymerase chain reaction (PCR) and restriction fragment length polymorphism (RFLP) respectively. The association between these polymorphisms and BCa risk was estimated by calculating Odds Ratio (OR) and chi-square (χ²) test. The DD genotype/D allele of ACE (I/D) polymorphism and “AC and CC” genotype/C allele of AGTR1 (A1166C) polymorphism were associated with higher risk of BCa when evaluated independently. Furthermore, interaction analysis of “AC and CC” and DD genotype and combination of “C and D” alleles of both polymorphisms revealed significantly greater BCa risk than that observed independently. Conclusively, women harboring “AC or CC” genotype/C allele for AGTR1 (A1166C) polymorphism and DD genotype/D allele for ACE (I/D) polymorphisms have a predisposition to develop more aggressive disease with advanced staging and larger tumor size. Our study indicates importance of genetic screening based on these polymorphisms for women, who may have higher risk of BCa.     

ACE I/D polymorphism in Indian patients with hypertrophic cardiomyopathy and dilated cardiomyopathy

Aim The study was carried to determine the association of angiotensin converting enzyme (ACE) insertion/deletion (I/D) polymorphism with the risk of hypertrophic cardiomyopathy (HCM), dilated cardiomyopathy (DCM), and restrictive cardiomyopathy (RCM). Methods and results A total of 174 patients diagnosed with cardiomyopathy (118 with HCM, 51 with DCM, and 5 with RCM) and 164 ethnically, age- and gender-matched controls were included in the study. ACE I/D genotyping was performed by PCR. In total, 25.86% of the patients were in New York Heart Association (NYHA) class III and IV at presentation. A total of 67.24% patients had dyspnea, 56.89% had angina pectoris, and 25.28% of the patients had at least one event of syncope. Frequency of occurrence of the disease was more in male patients compared to female patients (P < 0.05). After adjustment for age, sex, body mass index (BMI), and smoking habit, the prevalence of ACE DD genotype, and ACE ‘D’ allele was significantly higher in patients as compared to controls and was associated with increased risk (DD: OR 2.11, 95% CI 1.27–3.52, P < 0.05; ‘D’: OR 1.91, 95% CI 1.08–3.35, P < 0.05). The mean septal thickness was higher for DD and ID genotypes (20.40 ± 3.73 mm and 21.82 ± 5.35 mm, respectively) when compared with II genotype (18.63 ± 6.69 mm) in HCM patients, however, the differences were not significant statistically (P > 0.05). The DCM patients with ID genotype showed significantly decreased left ventricular ejection fraction (LVEF) at enrolment (26.50 ± 8.04%) (P = 0.04). Conclusion Our results suggest that D allele of ACE I/D polymorphism significantly influences the HCM and DCM phenotypes.     

DD genotype of ace gene I/D polymorphism is associated in a turkish study population with osteoarthritis

This study was conducted in Turkish osteoarthritis patients to determine the frequency of I/D polymorphism genotypes of angiotensin converting enzyme gene, and to examine the role of this polymorphism in osteoarthritis development. Genomic DNA obtained from 200 persons (135 patients with osteoarthritis and 65 healthy controls) was used in the study. DNA was multiplied by polymerase chain reaction using I and D allele-specific primers. Polymerase chain reaction products were assessed with CCD camera by being exposed to 2% agarose gel electrophoresis. There was statistically significant difference between the groups with respect to genotype distribution (P < 0.001). The D allele frequency was indicated as 69% and I allele was as 31% in the patients, whereas it was 55–45% in the control group. Consequently, in this study, we may assert that ACE gene I/D polymorphism DD genotype determination is significant criteria for identifying patients who are likely to develop osteoarthritis in east population of Turkey.     

Sequence-based genotyping of the sheep MHC class II DRB1 locus

The immunopolymorphism database (IPD) provides a single nomenclature for alleles at the major histocompatibility complex (MHC) loci for a range of different species. The minimum requirements for inclusion of a sheep class II DRB1 sequence is a submission that includes all polymorphic sites within the second exon from at least two independent polymerase chain reactions (PCR). In order to meet these requirements, we have developed a DNA-based genotyping method for the rapid analysis of allelic diversity at the DRB1 locus in domestic sheep, Ovis aries. Using a series of primers located within introns flanking exon 2 and genomic DNA from a cohort of 214 sheep representing 15 different breeds and crossbreeds, the complete exon 2 sequences of 38 Ovar-DRB1 alleles were obtained. This sequence resource allowed the development of a generic set of locus-specific primers which amplify a fragment that includes all polymorphic sites within the second exon. Bidirectional sequence analysis of the PCR product provides a composite sequence where each polymorphic site is represented by the corresponding International Union of Biochemistry nucleotide code. A Basic Local Alignment Search Tool search of alleles held within the IPD or National Center for Biotechnology Information databases allows individual allele sequences to be identified. Low levels of homozygosity (7.48%) within the cohort and verification of previously genotyped samples confirmed the broad allelic specificity of this method. It improves on currently available methods and is broadly applicable to the analysis of MHC diversity in studies investigating linkages with resistance or susceptibility to disease.     

Association of angiotensin converting enzyme (ACE) gene I/D polymorphism and polycystic ovary syndrome (PCOS)

This study was conducted in Turkish patients with polycystic ovary syndrome to determine the frequency of I/D polymorphism genotypes of angiotensin converting enzyme gene, and to examine the role of this polymorphism in polycystic ovary syndrome development. Genomic DNA obtained from 200 persons (100 patients with polycystic ovary syndrome and 100 healthy controls) was used in the study. DNA was multiplied by polymerase chain reaction using I and D allele-specific primers. Polymerase chain reaction products were assessed with a charge coupled device (CCD) camera by being exposed to 2% agarose gel electrophoresis. There was statistically significant difference between the groups with respect to genotype distribution (p < 0.001). The D allele frequency was indicated as 68% and I allele was as 32% in the patients, whereas it was 51.5-48.5% respectively in the control group. As a result of our study we may assert that angiotensin converting enzyme gene I/D polymorphism DD genotype should be considered as a genetic marker in polycystic ovary syndrome development in this Turkish study population.     

Comparative study of multipoint methods for genotype error detection

Several programs are currently available for the detection of genotyping error that may or may not be Mendelianly inconsistent. However, no systematic study exists that evaluates their performance under varying pedigree structures and sizes, marker spacing, and allele frequencies. Our simulation study compares four multipoint methods: Merlin, Mendel4, SimWalk2, and Sibmed. We look at empirical thresholds, power, and false-positive rates on 7 small pedigree structures that included sibships with and without genotyped parents, and a three-generation pedigree, using 11 microsatellite markers with 3 different map spacings. Simulated data includes 5,000 replicates of each pedigree structure and marker map, with random genotyping errors in about 4% of the middle marker's genotypes. We found that the default thresholds used by these programs provide low power (47-72%). Power is improved more by adding genotyped siblings than by using more closely spaced markers. Some mistyping methods are sensitive to the frequencies of the observed alleles. Siblings of mistyped individuals have elevated false-positive rates, as do markers close to the mistyped marker. We conclude that thresholds should be decided based on the pedigree and marker data and that greater focus should be placed on modeling genotyping error when computing likelihoods, rather than on detecting and eliminating genotyping errors.     

飞行(学)员ACE基因的多态性

血管紧张素转化酶 (ACE)第 16内含子的插入 缺失多态性与运动员耐力水平有关 .为了解这一多态性与飞行员飞行耐力的关系 ,对不同阶段飞行人员ACE第 16内含子基因型进行了分析和比较 .结果显示 ,ACEDD基因型百分率在招飞体检应征人员为 12 5 %、基础飞行学院学员 (未飞 )为 11 5 %、飞行学院初教机飞行学员为 10 0 %、歼击机飞行员为 3 0 % .歼击机飞行员组D等位基因频率及DD基因型明显低于其他 3组 (P <0 0 1) ,而后 3组之间无明显差异 (P >0 0 5 ) .进而观察到 ,飞行员体能测试成绩优者 ,无DD基因型 .提示 ,飞行员体能表现与ACE第 16内含子的插入 缺失多态性有关 ,具有I等位基因者 ,体能较好 ,飞行耐力也较好 .     

Insertion/deletion polymorphism of angiotensin-converting enzyme gene--risk factor for coronary artery disease in the Tuzla region population (Bosnia and Herzegovina)

Angiotensin II is the major effector molecule of renin-angiotensin system; its production can be conveniently interrupted by angiotensin-converting enzyme (ACE). Typical plasma levels of ACE accompany the I/D polymorphism; however, a controversy exists as to whether the DD genotype of the ACE polymorphism affects the risk for the development of coronary artery disease (CAD) and to what extent the ACE polymorphism is associated with CAD in different populations. We compared the I/D polymorphism in 212 CAD patients younger than 50 years with 165 healthy control individuals. They were all from the Tuzla region in Bosnia and Herzegovina. Patients with CAD had a higher prevalence of the DD genotype (36.3%) than controls (25.6%). The odds ratio for the ACE DD genotype in CAD patients was 1.7 (95% confidence interval 1.0-2.7; p < 0.05). We may conclude that the D/D genotype of the ACE gene polymorphism is associated with an increased risk for CAD in the Bosnian population.