Genotype frequencies did not deviate from Hardy-Weinberg expectations in both controls and CAD group. The genotype distribution and the allele frequency for LPL gene polymorphisms are summarized in Table 2. H⁺ and H⁻ alleles did not differ significantly between CAD group and controls. For the HindIII genotype, within the CAD group (n = 120), the frequencies of the H⁺H⁺ found in 61 individuals (50.8%), whereas 52 (44.3%) carried the H⁻/H⁺ genotype, and 6 (5%) carried the H⁻/H⁻ genotype. Within the control group (n = 65), the H⁺/H⁺ genotype was found in 29 (44.6%), whereas 23 (35.4%) carried the H⁻/H⁺ genotype, and 13.0 (20%) carried the H⁻H⁻ genotype. the distribution of HindIII demonstrated that CAD patients had lower H⁻H⁻ frequency compared to the control group and higher H⁺H⁻, H⁺H⁺ frequencies compared to the control group Table 3. Odds Ratios were 4.5 and 4.6 respectively when compared with (H⁻H⁻) genotype. Individuals with homozygous (H⁺H⁺) or (H⁺H⁻) genotype were at much higher risk of developing CAD compared to the (H⁻H⁻) genotype (p < 0.005), Table 4. For the PvuII genotype, within the CAD group (n = 120), the frequencies of the P⁺P⁺ found in 50 (41.7%), whereas 52 (43.2%) carried the P⁻P⁺ genotype, and 18 (15%) carried the P⁻P⁻ genotype. Within the control group (n = 65), the P⁺P⁺ was found in 25 (38.5%), 28 (43.0%) carried the P⁻P⁺ genotype, and 12.0 (18.5%) carried the P⁻P⁻ genotype. The distribution of PvuII polymorphism demonstrated that CAD patients had higher P⁺P⁻, P⁻P⁻, P⁺P⁺ frequencies compared with controls, Table 3. The odds ratios of PvuII genotype P⁻P⁻ vs. P⁻P⁺ and P⁺P⁺ genotypes (95% CI) is 1.26 (90.53–2.99) and 1.33 (0.56–3.20) respectively, hence showing no significant association with CAD disease Table 5. In previous studies, the prevalence rate of the LPL-HindIII (H⁺H⁺) genotype was implicated as the most common genotype associated with CAD. Shimo-Nakanishi et al. (2001) [17] demonstrated that HindIII polymorphism is associated with increased risk of atherothrombotic infarction. Likewise, Chamberlain et al. (1989) [30] indicated that there were differences in frequencies of both HindIII and PvuII alleles between Whites and Japanese populations, the statistical analysis for the LPL-HindIII genotypes indicates significant differences in the distribution of these genotypes between the control groups and the CAD groups, which in agreement with previous reported study [20]. Several studies have investigated the possibility that the LPL-HindIII and the LPL-PvuII polymorphisms are associated with the severity of CAD in various populations [1,14,16,19,20,31–36]. However, limited data are available concerning the Saudi population [21], which has not adequately defined the association between LPL polymorphism and CAD. While a large number of studies have found the LPL-HindIII (H⁺H⁺) genotype to be associated with CAD, the finding from the association studies with LPL-PvuII polymorphisms have been inconsistent [14,15,19,33,35]. For example, Thorne et al. (1990) [15] reported an increase in the LPL-HindIII (H⁺H⁺) genotypic frequencies in patients with coronary atherosclerosis in Japanese population whereas Peacok et al. (1992) [33] observed no differences in either LPL-HindIII or LPL-PvuII allelic frequencies in young survivors of myocardial infarction in Spanish population. Ukkola et al. (1995) [35] found reductions in various measures of ischemic heart disease in non-insulin-dependent diabetics who were LPL-PvuII (P⁺P⁺), but noted increases in those who were LPL-HindIII (H⁻H⁻) in Canadian adult population. In contrast, Gerdes et al. (1995) [34] found the LPL- HindIII (H⁺H⁺) genotype to be positively associated with a family history of premature ischemic heart disease in a group of Danish men. Similarly, Mattu et al. (1994) [32] found an association between the LPL-HindIII H⁺H⁺ genotype and CAD, but not between the LPL-PvuII (P⁺P⁺) genotype and CAD in native Brazilian population. On the other hand, Wang et al. (1996) [14] reported an association between the extent, but not occurrence of CAD and the LPL-PvuII (P⁺P⁺) genotype. However, in the same study, Wang et al. (1996) [14] found no association between the LPL-HindIII genotype and CAD. Interestingly, Gambino et al. (1999) [31] found a striking association between the LPL-HindIII (H⁺H⁺) genotype and multi-vessel disease in an Italian group of young survivors of myocardial infarction. Also it has been reported that Anderson et al. (1999) [1] found a moderate association between the LPL-HindIII (H⁺H⁺) genotype and CAD, whereas the LPL-PvuII (P⁻P⁻) genotype was modestly associated with CAD. In summary, the relevance of these genotypes may vary among different populations.

For the Ser447Ter genotype, within the CAD group (n = 120), the frequencies of the CC found in 100 (83.3%), whereas 20 (16.7%) carried the CG genotype. Within the control group (n = 65), the CC was found in 57 (87.7%) and 8 (12.3%) carried the CG genotype. We did not get any GG genotypes in the control or any patients for this gene. The homozygote of G allele was incorporated with the heterozygote due to the low frequency of GG, higher frequencies of CC and CG genotypes in the CAD group Table 3. OR of CC vs. CG+GG (95% CI) is 1.43 from 0.59 to 3.44, which is insignificant, Table 4. Interestingly, no GG genotype was observed in the CAD and control groups in the distribution of Ser447Ter polymorphism in the present study. However, we found for the first time in Saudi populations new genotype frequencies of Ser447-Ter mutations of the LPL gene between the CAD patients and the healthy controls. In previous reported studies, the frequency of the Ser447-Ter genotype of GG and CG was significantly lower in a group of Japanese CAD patients than in the control [37], representing a significantly low risk of CAD. Also Shimo-Nakanishi et al. (2001) [17] found that Ser447 Stop mutations of the LPL gene are a novel genetic marker for low risk of atherothrombotic cerebral infarction. Kozaki et al. (1993) [37] originally found an approximately 2-fold increase in the enzymatic activity of LPL resulting from the Ser447-Ter mutations at the carboxy terminal of LPL transfected and expressed in vitro, conflicting results that demonstrated that Ser447-Ter mutations might have either no or an inverse effect on LPL function may have been caused by different methodology.

Haplotype frequencies for the HindIII, PvuII and Ser447Ter alleles were estimated for controls and patients (Table 6). A score for each haplotype (Hap-score) was calculated and the P-value was obtained for the significance of each Hap-score. The haplotypes −/−/C, +/−/C, −/−/G, −/+/C, −/+/G, +/−/G, +/+/C and +/+/G are the most common in the CAD patients group. There were differences between the CAD group and control group with regards to the haplotype distribution. Comparison of the common haplotype frequencies between the CAD patients group and control group revealed a significant increase in the frequency of the most common haplotype in those with CAD. This also suggests an increase in the frequency of less common haplotypes among some cases. Haplotypes −/−/G and +/+/C were associated with a significantly increased risk factor of CAD (p = 0.06, 0.04 respectively). Table 6 Shows that combined genotypes may play an important role in the process of atherogenesis. It has been proposed that the H⁻allele is a marker either for a mutation that alters an amino acid within LPL, causing enzyme activity or lipid binding to be more efficient, or possibly for a sequence change within the promoter, causing higher levels of LPL expression [20]. The frequencies of H⁻/C and P⁻/C were significantly higher in CI patients than in controls, and the H⁻/P⁻/C combined genotype was the most common genotype of HindIII/PvuII/Ser447Ter combined genotypes in the cerebral infarction group. P⁻allele is more frequent and G allele is less frequent in cerebral infarction (CI) patients than in controls. The data also provide strong evidence of an association between polymorphisms in the LPL gene with lipid levels. Moreover, H⁻/C and P⁻/C mutations, especially H⁻/P⁻/C mutation of the LPL gene, are relevant to CI. PvuII polymorphism is a risk factor for CI while Ser447Ter mutation is a protective factor, indicating that the LPL polymorphism remains a useful genetic marker for predicting CI risk in the Chinese [19]. Goodarzi et al. 2003 [38] showed no differences between cases and controls, in comparison of genotype frequencies except for a modestly significant difference for the 8393 (HindIII) variant (p = 0.05). However, comparison of the common haplotype frequencies between the Mexican-Americans with and without coronary artery disease revealed a significant decrease in the frequency of the most common haplotype in those with disease. Goodarzi et al. 2003 found that six markers in the 3′ end of LPL allowed us to distinguish the most common haplotypes occurring in two major U.S. ethnic groups, Hispanic and non-Hispanic Caucasians. The allele and haplotype frequencies were different between the Mexican-Americans and non-Hispanic Caucasians. Furthermore, within the Mexican-Americans, there were positive and negative associations of particular haplotypes with coronary artery disease, suggesting a role of this gene in contributing to CAD in this population. In comparing two different ethnic groups, Goodarzi et al. 2003 [38] found several differences in the allele and haplotype frequencies observed in the 3′LPL markers. Such differences may affect the results of association studies conducted in different populations.

Study patients and control subjects consisted of 120 patients (58 male and 62 female, aged 36–80 years) who were admitted to Department of Cardiology, King Khalid University Hospital, Riyadh, Saudi Arabia, and 65 healthy subjects (29 male and 36 female, aged 32–80) who had no history of CAD as control. Included subjects were of unrestricted age and gender who gave written informed consent to draw blood at the time of angiography or at the time of screening for research deoxyribonucleic acid (DNA) extraction to be used in studies approved by the hospital’s institutional review board and was conducted in accordance with the guidelines set by the ethics committee of College of Medicine and Research Centre (CMRC) of King Saud University, Riyadh, Saudi Arabia. All subjects enrolled in this study were Saudi residents with similar dietary pattern. Key demographic data of subjects were recorded including age and gender lipid profile. Assessment of CAD was made by review of angiograms by the patient’s cardiologist.

This study was conducted after review and approval of the Institutional Review Board of the Ethics Committee at KKUH (King Khalid University Hospital), and all subjects gave written informed consent prior to participation.

Blood samples for the glucose and lipid measurements were withdrawn from the patients and the control subjects after an overnight fast. The plasma glucose concentration was measured by the glucose oxidase method using a Biotrol Kit on a Bayer opera analyzer. Serum total cholesterol was measured using the Biotrol commercial Kit, HDL cholesterol was determined with a commercial Randox Kit, LDL cholesterol was calculated by the formula of Friedwald, and triglyceride determination was made by the method of Lipase/Glycerol Kinase UV endpoint on the opera analyzer.

Genomic DNA was extracted from peripheral blood specimens, which were in tubes containing EDTA, which performed as an anticoagulant, using QIAamp DNA isolation Kit from QIAGEN (Germany).

Genotyping was performed by polymerase chain reaction (PCR). The primer sets were selected on the basis of previously published information (18): HindIII, forward primer (H1), 5′-TGA AGC TCA AAT GGA AGA GT-3′, reverse primer (H2): 5′-TAC AAG CAA ATG ACT AAA-3′; PvuII, forward primer (P1): 5′-ATG GCA CCC ATG TGT AAG GTG-3′; reverse primer (P2): 5′-GTG AAC TTC TGA TAA CAA TCT C-3′; Ser447Ter, forward primer: 5′-TAC ACT AGC AAT GTC TAG GTG A-3′, reverse primer: 5′-TCA GCT TTA GCC CAG AAT GC-3′. Genomic DNA template 3 μL (150 ng) was added to PCR reaction mixture composed of 12.5 μL 2× Promega master mix, 2 μL each primer and distilled water to a final volume 25 μL. The PCR condition were as follows: initial denaturation at 94 °C for 2 min were followed by 40 cycles of denaturation at 94 °C for 15 s, annealing at 50 °C for 30 s, and extension at 72 °C for 1 min, with final extension at 72 °C for 2 min in My Cycler (Bio-Rad).

Digestion of PCR products was performed by addition of 1 μL of the respective restriction enzyme (HindIII: Promega, Madison WI, USA; PvuII: Promega, Madison WI, USA, MnlI: England Biolabs, Inc.) to 10 μL of PCR products in 2 μL 10× buffer solution (final reaction volume = 20 μL), and centrifuged for 2 min at the speed of 5000 rounds/min, and aqueous bathed at 37 °C for 4 h. The resulting fragments were resolved by electrophoresis (80 V, 60 min) on 2.5% agrose gel, and visualized on UV light.

The HindIII site (intron 8) produces a 600-bp fragment after digestion (Figure 1). The PvuII restriction site (intron 6) yields 330-bp and 110-bp fragments (Figure 2). Genotypes were scored by an experienced reader blinded to clinical and angiographic results. Our convention was to refer to the polymorphic allele displaying the restriction site as (+) and the allele without the site as (−). For both PvuII and HindIII, the (−) allele was less common R [1].

The PCR product of 488 bp contains 2 MnlI restriction sites, of which one is a polymorphic site indicating the Ser447Ter mutation. Digestion of the PCR product with MnlI resulted in 3 fragments of 290, 250, and 200 bp. [17,18]. The identified genotypes were named according to the presence or absence of the enzyme restriction sites, so Ser447Ter GG, CG, and CC are homozygote for the presence of the site, heterozygote for the presence and absence of the site, and homozygote for the absence of the site, respectively (Figure 3).

Measurement data were summarized by mean ± standard deviation (S.D.), and compared with two-sample t-test. Enumeration count data were summarized as number (%) and compared with chi-square test (χ² test). To analysis was used to evaluate the allelic and genotypic frequencies that were calculated from the observed genotypic counts and to assess the Hardy-Weinberg equilibrium expectations. The same methodology was applied to comparisons between allelic and genotypic frequencies. Associations were determined as odds ratios (ORs) and 95% confidence intervals (CIs). (The odds of carrying a specific allele are defined as the frequency of subjects in whom it occurs divided by the frequency of subjects in whom it does not occur. The odds ratio for the LPL genotype distribution χ² analysis was performed. A CAD is the odds of allelic carriage in the diseased [CAD] group divided by the odds in the undiseased [control] group.) Univariate logistic regression was used to determine adjusted ORs for the genetic markers, conditioned on the major CAD risk factors. (p value < 0.05 was considered statistically significant). Statistical analyses were performed with the Statistical Package for Social Sciences for Windows, version 11.5 (SPSS, Inc, Chicago, IL, USA).