Vol.25, pp. 471-478. 1992 Printed in the USA.All rightsreserved.
Clin Bib:hem,
0009-9120/92$5.00 + .00 Copyrightc 1992The CanadianSocietyof ClinicalChemists.
Apolipoprotein B Gene Polymorphism and Plasma Lipids and Lipoproteins in a Canadian Caucasian Population GODWIN OGBONNA, 1 RAPHAEL M.C. CHEUNG,2 GEORGE WONG,3 and KHOSROW ADELI 1 1Clinical Chemistry Division, Department of Chemistry and Biochemistry, University of Windsor, Windsor, Ontario, Canada and 2Lipid Research Clinic and 3Cardiovascular-Pulmonary Unit, Windsor Western Hospital Center, Windsor, Ontario, Canada We have investigated the frequency of Hind Ill DNA polymorphism of the human apolipoprotein B gene in a Canadian Caucasian population with coronary artery disease, as documented by angiography, and a healthy control population. Patients had significantly (p < 0.05) higher levels of cholesterol, triglycerides, LDL-cholesterol, apolipoprotein B, and lower level of apoAI compared to the controls. Restriction fragment-length polymorphism analysis detected nine hybridizable fragments denoted as H1 to H9. The H1, H2, H3, and H7 alleles were polymorphic. The [H4-H9] genotype seems to be the normal genotype within the population studied since it was detected in 69% of the control group. The [H1-H9] genotype was most frequently observed in the patients (frequency = 0.68). We were unable to strongly associate any of the alleles or genotypes detected with the changes in lipids. The additional alleles observed in the patient group may indicate possible mutations at the 3' end of the apolipoprotein B gene locus.
Correspondence: Dr. K. Adeli, Clinical C h e m i s t r y Division, D e p a r t m e n t of C h e m i s t r y a n d Biochemistry, 401 S u n s e t Avenue, U n i v e r s i t y of Windsor, Windsor, Ontario, C a n a d a N9B 3P4. M a n u s c r i p t received M a y 4, 1992; r e v i s e d A u g u s t 4, 1992; accepted A u g u s t 25, 1992.
sible for the maintenance of the structural integrity of LDL and mediates uptake of about 70% of plasma cholesterol through the LDL receptor (4). Abnormalities in either production or clearance of apoB have been shown to result in elevated levels of plasma apoB and LDL. Genetic variations at the apoB locus may be an important factor in determining the plasma concentrations of apoB and LDLc. A number of common restriction fragment-length polymorphisms (RFLP) have been detected in the apoB gene. RFLPs in this gene have been shown in some cases to be associated with the concentrations of plasma apoB and LDLc as well as with the incidence of CAD (5). This suggests a substantial genetic component in the etiology of CAD that is not surprising. In a study of the genetic determinants of serum lipids and lipoproteins in French Canadian families, it was observed that genetic components accounted for about 50-60% of the variation of every lipid measurement (6). Genetic variations caused by mutation (insertion and/or deletion) at the 3' end of the apoB gene may lead to nonrecognition of LDL-apoB by the LDL receptor and a subsequent delay in LDL clearance resulting in LDL accumulation. Berg detected a highly significant association between serum levels of apoB and XbaI allele at the apoB gene locus (7). Similar observations were made by Law et al. in a different population (8). ApoB gene alleles associated with increases in apoB and serum lipids may therefore be used to predict an individual's predisposition to CAD. Several reports on XbaI, EcoRI, and MspI polymorphisms of the apoB gene have appeared in the literature, but no study has been reported on Hind III polymorphism of this gene (5,9,10). We have investigated genetic variations of the apoB gene at the Hind HI sites and its relationship to plasma lipid and lipoprotein levels of patients with angiographically documented CAD as well as those of healthy controls selected from the Caucasian population in Southwestern Ontario.
CLINICAL BIOCHEMISTRY, VOLUME 25, DECEMBER 1992
471
K E Y WORDS: apolipoprotein B; r e s t r i c t i o n f r a g m e n t l e n g t h polymorphism; H i n d III; coronary a r t e r y disease.
Introduction
A
positive correlation has been shown between coronary artery disease (CAD) and plasma total cholesterol, low density lipoprotein (LDL) cholesterol, and apolipoprotein B (apoB). Determination of plasma apoB, an indicator of the number of LDL particles, provides additional information in assessing the risk of developing CAD. Increased levels of apoB have been found in survivors of myocardial infarction with normal LDL-cholesterol (LDLc) (1). ApoB has also been shown to best discriminate between CAD cases and controls (2,3). ApoB is the major protein of the cholesterol-rich LDL. It is respon-
OGBONNA, CHEUNG, WONG, ANDADELI
Materials and methods CHEMICALS
General laboratory chemicals were obtained from Sigma Chemical Co. (St. Louis, MO, USA), Fisher Scientific (Fair Lawn, NJ, USA), and BDH (Toronto, Ont, Canada). Restriction endonuclease and nick translation system were obtained from Bethesda Research Laboratories (BRL, Gaithersburg, MD, USA). S & S nytran nylon membrane was from Mandel Scientific Inc. (Guelph, Ont). Deoxycytidine-5'triphosphate, [a.32p] was from ICN Biomedicals Inc. (Irvine, CA, USA). Autoradiography materials were purchased from Eastman Kodak (Rochester, NY, USA). The probe used was purchased from the American Type Culture Collection (Rockville, MD, USA). REAGENTS
a
Reagents used were prepared as stock solutions and sterilized either by autoclaving or filtration, and working concentrations were prepared from the stock. They included 5 × tris-borate (0.45 mol/L trisborate, pH 8.0, containing 0.01 mol/L EDTA), 20× SSPE pH 7.4 (3.0 mol/L NaC1, containing 0.2 mol/L NaH2PO4" H20 and 0.02 mol/L EDTA), 20x SSC pH 7.0 (3 mol/L NaC1 containing 0.34 mol/L sodium citrate), and hybridization solution (2x SSPE containing 1% (w/v) SDS, 0.5% (w/v) Blotto, 10% (w/v) dextran sulphate, and 0.5 mg/mL salmon sperm DNA). SELECTION OF STUDY S U B J E C T S
The patients were 50 Caucasians (36 males and 14 females) age 33-69 years proven to have at least 80% occlusion in at least one of the coronary arteries as shown by angiography at the Windsor Western Hospital Center. The control subjects (27 males and 31 females) were healthy Caucasians age 25-66 years selected from a population screen at the lipid clinic of the same hospital and at the blood donor clinic of the Red Cross Society of Windsor. In both the patient and the control populations, previous history of diabetes, hypertension, elevated serum cholesterol, high blood pressure, smoking, and family history of coronary artery disease were noted. LIPID AND LIPOPROTEIN ANALYSIS
Fasting blood samples were collected in two 5 mL EDTA-containing vacutainer tubes (Becton Dickin-
son, Rutherford, NJ, USA) from each subject. One tube was centrifuged at 1800 × g in a clinical centrifuge for 5 min and the separated plasma was used for lipid and lipoprotein analysis. The other tube was either used immediately for DNA purification or stored at - 20 °C until use. Total cholesterol (TC), triglycerides (TG), and high-density lipoprotein (HDL) cholesterol were determined using the Kodak 700 Ektachem analyzer. LDLc was derived by the Friedewald formula, LDLc = TC - (HDLc + TG/ 2.2). Apolipoproteins A1 and B were measured by the Cobas Fara II using immunonephelometry assay kits from Atlantic Antibodies, Incstar Corp. (Stillwater, MN, USA). GENOTYPE ANALYSIS
Restriction digestion and southern blotting Genomic DNA was purified from whole blood using a rapid method developed in our laboratory (11). Ten micrograms of pure genomic DNA was digested with 20 units of the restriction endonuclease, Hind III, at 37 °C for 1 h following standard protocol (12). The digested DNA was electrophoresed for 18 h in 0.9% (w/v) agarose gel and stained in 5 ~g/mL of ethidium bromide to assure complete digestion. The restricted DNA was fragmented in 0.25 mol/L HC1 for 10 min, soaked in transfer buffer (0.4 mol/L NaOH/0.6 mol/L NaC1) for 30 min and then transferred for 20 h to an S & S Nytran nylon membrane using the standard Southern blotting technique protocol (12). The membrane was rinsed in 2 x SSC after transfer and air dried. Membranes not used immediately were baked at 80 °C for 2 h and stored under vacuum until use.
Probe preparation The probe was a 1.9 kb apoB gene genomic clone insert and included from part of intron 26 to part of exon 29 which is the last exon of the apoB gene. E. coli containing the recombinant vector, pUC19, was amplified in Luria Bertani medium (LB medium), harvested, lysed in alkali, and the DNA was purified using polyethylene glycol (mol wt 800) according to standard protocol (12). The probe (500 ng) was labeled with 50 ~Ci of deoxycytidine-5'-triphosphate, [a_32p] by nick translation (according to the BRL protocol) to a specific activity of 1 x l0 s cpm/~g DNA.
Hybridization and autoradiography
a SSC, sodium chloride/sodium citrate: a 1× solution contains 0.15 mol/L NaC1, 0.017 mol/L sodium citrate, pH 7.0; SSPE, sodium chloride/sodium phosphate/EDTA: a 1× solution contains 0.15 mol/L NaC1, 0.01 mol/L NaH2PO4 • H20, pH 7.4, 0.001 mol/L EDTA; tris-borate: a 1 × solution contains 0.09 mol/L tris-[hydroxymethyl]aminomethane, pH 8.0, 0.09 mol/L boric acid, and 0.002 mol/L EDTA.
The membranes were prehybridized for 5 h in the hybridization solution after wetting in 2 x SSC and hybridized in the hybridization solution containing 1 }xg of [c~-32p]dCTP labeled probe for 30 h. After hybridization, the membranes were washed under low stringency conditions (2 x SSPE, 1% SDS) for 20 min and medium stringency conditions (0.5 × SSPE, 1% SDS) at 60 °C for 30 min, air dried, and exposed
472
CLINICALBIOCHEMISTRY,VOLUME25, DECEMBER 1992
APOLIPOPROTEIN B GENE POLYMORPHISM
to an X-ray film using double intensifying screen for 3 days at - 80 °C. The film was developed in Kodak's developer and fixer solutions.
GENE ANALYSIS
The v a l u e s a r e m e a n -+ SD. CHL, cholesterol; TG, triglycerides, p v a l u e c a l c u l a t e d from S t u d e n t ' s t-test.
Genomic DNA from patients and controls were subjected to RFLP analysis. Probing the Hind III digest of the genomic DNA with pB27 clone detected 6 hybridizable fragments in the control group with sizes 5.1 kb, 4.5 kb, 4.1 kb, 3.9 kb, 3.1 kb, and 3.0 kb (Lane C, Figure 1). When the patients were probed, these six fragments were also detected plus three additional fragments (8.1 kb, 7.4 kb, and 6.6 kb), making a total of 9 alleles in most of the patients (Lane P, Figure 1). We designated these alleles based on size as H1 (8.1 kb), H2 (7.4 kb), H3 (6.6 kb), H4 (5.1 kb), H5 (4.5 kb), H6 (4.1 kb), H7 (3.9 kb), H8 (3.1 kb), and H9 (3.0 kb). H1, H2, H3, and H7 alleles were found to be polymorphic. Table 3 shows the allele frequencies and the genotype distributions for the patient and control groups. We did not detect H1 and H2 alleles in any of the control subjects. The frequencies of the H1 and H2 alleles in CAD patients were identical at 0.84. The H3 allele was present in controls and patients with frequencies of 0.31 and 0.96, respectively. The H7 allele was present in all control subjects except one (frequency = 0.98) while it was detected in the patients at a lower frequency of 0.84. Due to the polymorphic nature of some of the alleles, we could designate four genotypes within the whole population; [H1-H9], [H3-H9], [H4-H9], and [H1-H6,H8-H9]. The frequencies of these genotypes within the patients and the controls are given in Table 3. The [H4-H9] genotype seems to be the normal genotype within the population studied since it was detected in 69% of the control group. The [H1H9] genotype was most frequently observed in the patients (0.68) while it was not found in any of the controls. Controls had a higher frequency (0.3) of the [H3-H9] genotype compared to the patients (0.12). The [H1-H6,H8-H9] genotype was detected in a number of patients (0.16) but only in one control (O.O2). The effects of these genotypes on the lipids and lipoprotein parameters within each group were estimated using a single factor factorial ANOVA (Table 4). Between-group differences in lipid values for each specific genotype were also evaluated using the SAS program. Within the patient group, no significant differences in total cholesterol, triglycerides, HDLc, LDLc, apoB, and apoAI were found among the four different genotypes. There were also no significant differences in total cholesterol, triglycerides, LDLc, apoB, and apoAI among the four different genotypes within the control group (Table 4). The SAS analysis for between-group differences showed no differences for total cholesterol. Patients with the [H3-H9] genotype showed significantly higher levels of triglycerides and LDLc and lower levels of apoAI compared to controls with the same genotype. In addition to those with [H1-H6,H8-H9] and [H3-H9] genotypes, patients with the [H4-H9] genotype also had significantly lower levels of apoAI. The [H1-H6,H8-H9] genotype in the patient
CLINICAL BIOCHEMISTRY, VOLUME 25, DECEMBER 1992
473
STATISTICAL ANALYSIS
The Student's two-tailed t-test was used to test for significant differences in the levels of lipids and lipoproteins. Probability values
Clin Bib:hem,
0009-9120/92$5.00 + .00 Copyrightc 1992The CanadianSocietyof ClinicalChemists.
Apolipoprotein B Gene Polymorphism and Plasma Lipids and Lipoproteins in a Canadian Caucasian Population GODWIN OGBONNA, 1 RAPHAEL M.C. CHEUNG,2 GEORGE WONG,3 and KHOSROW ADELI 1 1Clinical Chemistry Division, Department of Chemistry and Biochemistry, University of Windsor, Windsor, Ontario, Canada and 2Lipid Research Clinic and 3Cardiovascular-Pulmonary Unit, Windsor Western Hospital Center, Windsor, Ontario, Canada We have investigated the frequency of Hind Ill DNA polymorphism of the human apolipoprotein B gene in a Canadian Caucasian population with coronary artery disease, as documented by angiography, and a healthy control population. Patients had significantly (p < 0.05) higher levels of cholesterol, triglycerides, LDL-cholesterol, apolipoprotein B, and lower level of apoAI compared to the controls. Restriction fragment-length polymorphism analysis detected nine hybridizable fragments denoted as H1 to H9. The H1, H2, H3, and H7 alleles were polymorphic. The [H4-H9] genotype seems to be the normal genotype within the population studied since it was detected in 69% of the control group. The [H1-H9] genotype was most frequently observed in the patients (frequency = 0.68). We were unable to strongly associate any of the alleles or genotypes detected with the changes in lipids. The additional alleles observed in the patient group may indicate possible mutations at the 3' end of the apolipoprotein B gene locus.
Correspondence: Dr. K. Adeli, Clinical C h e m i s t r y Division, D e p a r t m e n t of C h e m i s t r y a n d Biochemistry, 401 S u n s e t Avenue, U n i v e r s i t y of Windsor, Windsor, Ontario, C a n a d a N9B 3P4. M a n u s c r i p t received M a y 4, 1992; r e v i s e d A u g u s t 4, 1992; accepted A u g u s t 25, 1992.
sible for the maintenance of the structural integrity of LDL and mediates uptake of about 70% of plasma cholesterol through the LDL receptor (4). Abnormalities in either production or clearance of apoB have been shown to result in elevated levels of plasma apoB and LDL. Genetic variations at the apoB locus may be an important factor in determining the plasma concentrations of apoB and LDLc. A number of common restriction fragment-length polymorphisms (RFLP) have been detected in the apoB gene. RFLPs in this gene have been shown in some cases to be associated with the concentrations of plasma apoB and LDLc as well as with the incidence of CAD (5). This suggests a substantial genetic component in the etiology of CAD that is not surprising. In a study of the genetic determinants of serum lipids and lipoproteins in French Canadian families, it was observed that genetic components accounted for about 50-60% of the variation of every lipid measurement (6). Genetic variations caused by mutation (insertion and/or deletion) at the 3' end of the apoB gene may lead to nonrecognition of LDL-apoB by the LDL receptor and a subsequent delay in LDL clearance resulting in LDL accumulation. Berg detected a highly significant association between serum levels of apoB and XbaI allele at the apoB gene locus (7). Similar observations were made by Law et al. in a different population (8). ApoB gene alleles associated with increases in apoB and serum lipids may therefore be used to predict an individual's predisposition to CAD. Several reports on XbaI, EcoRI, and MspI polymorphisms of the apoB gene have appeared in the literature, but no study has been reported on Hind III polymorphism of this gene (5,9,10). We have investigated genetic variations of the apoB gene at the Hind HI sites and its relationship to plasma lipid and lipoprotein levels of patients with angiographically documented CAD as well as those of healthy controls selected from the Caucasian population in Southwestern Ontario.
CLINICAL BIOCHEMISTRY, VOLUME 25, DECEMBER 1992
471
K E Y WORDS: apolipoprotein B; r e s t r i c t i o n f r a g m e n t l e n g t h polymorphism; H i n d III; coronary a r t e r y disease.
Introduction
A
positive correlation has been shown between coronary artery disease (CAD) and plasma total cholesterol, low density lipoprotein (LDL) cholesterol, and apolipoprotein B (apoB). Determination of plasma apoB, an indicator of the number of LDL particles, provides additional information in assessing the risk of developing CAD. Increased levels of apoB have been found in survivors of myocardial infarction with normal LDL-cholesterol (LDLc) (1). ApoB has also been shown to best discriminate between CAD cases and controls (2,3). ApoB is the major protein of the cholesterol-rich LDL. It is respon-
OGBONNA, CHEUNG, WONG, ANDADELI
Materials and methods CHEMICALS
General laboratory chemicals were obtained from Sigma Chemical Co. (St. Louis, MO, USA), Fisher Scientific (Fair Lawn, NJ, USA), and BDH (Toronto, Ont, Canada). Restriction endonuclease and nick translation system were obtained from Bethesda Research Laboratories (BRL, Gaithersburg, MD, USA). S & S nytran nylon membrane was from Mandel Scientific Inc. (Guelph, Ont). Deoxycytidine-5'triphosphate, [a.32p] was from ICN Biomedicals Inc. (Irvine, CA, USA). Autoradiography materials were purchased from Eastman Kodak (Rochester, NY, USA). The probe used was purchased from the American Type Culture Collection (Rockville, MD, USA). REAGENTS
a
Reagents used were prepared as stock solutions and sterilized either by autoclaving or filtration, and working concentrations were prepared from the stock. They included 5 × tris-borate (0.45 mol/L trisborate, pH 8.0, containing 0.01 mol/L EDTA), 20× SSPE pH 7.4 (3.0 mol/L NaC1, containing 0.2 mol/L NaH2PO4" H20 and 0.02 mol/L EDTA), 20x SSC pH 7.0 (3 mol/L NaC1 containing 0.34 mol/L sodium citrate), and hybridization solution (2x SSPE containing 1% (w/v) SDS, 0.5% (w/v) Blotto, 10% (w/v) dextran sulphate, and 0.5 mg/mL salmon sperm DNA). SELECTION OF STUDY S U B J E C T S
The patients were 50 Caucasians (36 males and 14 females) age 33-69 years proven to have at least 80% occlusion in at least one of the coronary arteries as shown by angiography at the Windsor Western Hospital Center. The control subjects (27 males and 31 females) were healthy Caucasians age 25-66 years selected from a population screen at the lipid clinic of the same hospital and at the blood donor clinic of the Red Cross Society of Windsor. In both the patient and the control populations, previous history of diabetes, hypertension, elevated serum cholesterol, high blood pressure, smoking, and family history of coronary artery disease were noted. LIPID AND LIPOPROTEIN ANALYSIS
Fasting blood samples were collected in two 5 mL EDTA-containing vacutainer tubes (Becton Dickin-
son, Rutherford, NJ, USA) from each subject. One tube was centrifuged at 1800 × g in a clinical centrifuge for 5 min and the separated plasma was used for lipid and lipoprotein analysis. The other tube was either used immediately for DNA purification or stored at - 20 °C until use. Total cholesterol (TC), triglycerides (TG), and high-density lipoprotein (HDL) cholesterol were determined using the Kodak 700 Ektachem analyzer. LDLc was derived by the Friedewald formula, LDLc = TC - (HDLc + TG/ 2.2). Apolipoproteins A1 and B were measured by the Cobas Fara II using immunonephelometry assay kits from Atlantic Antibodies, Incstar Corp. (Stillwater, MN, USA). GENOTYPE ANALYSIS
Restriction digestion and southern blotting Genomic DNA was purified from whole blood using a rapid method developed in our laboratory (11). Ten micrograms of pure genomic DNA was digested with 20 units of the restriction endonuclease, Hind III, at 37 °C for 1 h following standard protocol (12). The digested DNA was electrophoresed for 18 h in 0.9% (w/v) agarose gel and stained in 5 ~g/mL of ethidium bromide to assure complete digestion. The restricted DNA was fragmented in 0.25 mol/L HC1 for 10 min, soaked in transfer buffer (0.4 mol/L NaOH/0.6 mol/L NaC1) for 30 min and then transferred for 20 h to an S & S Nytran nylon membrane using the standard Southern blotting technique protocol (12). The membrane was rinsed in 2 x SSC after transfer and air dried. Membranes not used immediately were baked at 80 °C for 2 h and stored under vacuum until use.
Probe preparation The probe was a 1.9 kb apoB gene genomic clone insert and included from part of intron 26 to part of exon 29 which is the last exon of the apoB gene. E. coli containing the recombinant vector, pUC19, was amplified in Luria Bertani medium (LB medium), harvested, lysed in alkali, and the DNA was purified using polyethylene glycol (mol wt 800) according to standard protocol (12). The probe (500 ng) was labeled with 50 ~Ci of deoxycytidine-5'-triphosphate, [a_32p] by nick translation (according to the BRL protocol) to a specific activity of 1 x l0 s cpm/~g DNA.
Hybridization and autoradiography
a SSC, sodium chloride/sodium citrate: a 1× solution contains 0.15 mol/L NaC1, 0.017 mol/L sodium citrate, pH 7.0; SSPE, sodium chloride/sodium phosphate/EDTA: a 1× solution contains 0.15 mol/L NaC1, 0.01 mol/L NaH2PO4 • H20, pH 7.4, 0.001 mol/L EDTA; tris-borate: a 1 × solution contains 0.09 mol/L tris-[hydroxymethyl]aminomethane, pH 8.0, 0.09 mol/L boric acid, and 0.002 mol/L EDTA.
The membranes were prehybridized for 5 h in the hybridization solution after wetting in 2 x SSC and hybridized in the hybridization solution containing 1 }xg of [c~-32p]dCTP labeled probe for 30 h. After hybridization, the membranes were washed under low stringency conditions (2 x SSPE, 1% SDS) for 20 min and medium stringency conditions (0.5 × SSPE, 1% SDS) at 60 °C for 30 min, air dried, and exposed
472
CLINICALBIOCHEMISTRY,VOLUME25, DECEMBER 1992
APOLIPOPROTEIN B GENE POLYMORPHISM
to an X-ray film using double intensifying screen for 3 days at - 80 °C. The film was developed in Kodak's developer and fixer solutions.
GENE ANALYSIS
The v a l u e s a r e m e a n -+ SD. CHL, cholesterol; TG, triglycerides, p v a l u e c a l c u l a t e d from S t u d e n t ' s t-test.
Genomic DNA from patients and controls were subjected to RFLP analysis. Probing the Hind III digest of the genomic DNA with pB27 clone detected 6 hybridizable fragments in the control group with sizes 5.1 kb, 4.5 kb, 4.1 kb, 3.9 kb, 3.1 kb, and 3.0 kb (Lane C, Figure 1). When the patients were probed, these six fragments were also detected plus three additional fragments (8.1 kb, 7.4 kb, and 6.6 kb), making a total of 9 alleles in most of the patients (Lane P, Figure 1). We designated these alleles based on size as H1 (8.1 kb), H2 (7.4 kb), H3 (6.6 kb), H4 (5.1 kb), H5 (4.5 kb), H6 (4.1 kb), H7 (3.9 kb), H8 (3.1 kb), and H9 (3.0 kb). H1, H2, H3, and H7 alleles were found to be polymorphic. Table 3 shows the allele frequencies and the genotype distributions for the patient and control groups. We did not detect H1 and H2 alleles in any of the control subjects. The frequencies of the H1 and H2 alleles in CAD patients were identical at 0.84. The H3 allele was present in controls and patients with frequencies of 0.31 and 0.96, respectively. The H7 allele was present in all control subjects except one (frequency = 0.98) while it was detected in the patients at a lower frequency of 0.84. Due to the polymorphic nature of some of the alleles, we could designate four genotypes within the whole population; [H1-H9], [H3-H9], [H4-H9], and [H1-H6,H8-H9]. The frequencies of these genotypes within the patients and the controls are given in Table 3. The [H4-H9] genotype seems to be the normal genotype within the population studied since it was detected in 69% of the control group. The [H1H9] genotype was most frequently observed in the patients (0.68) while it was not found in any of the controls. Controls had a higher frequency (0.3) of the [H3-H9] genotype compared to the patients (0.12). The [H1-H6,H8-H9] genotype was detected in a number of patients (0.16) but only in one control (O.O2). The effects of these genotypes on the lipids and lipoprotein parameters within each group were estimated using a single factor factorial ANOVA (Table 4). Between-group differences in lipid values for each specific genotype were also evaluated using the SAS program. Within the patient group, no significant differences in total cholesterol, triglycerides, HDLc, LDLc, apoB, and apoAI were found among the four different genotypes. There were also no significant differences in total cholesterol, triglycerides, LDLc, apoB, and apoAI among the four different genotypes within the control group (Table 4). The SAS analysis for between-group differences showed no differences for total cholesterol. Patients with the [H3-H9] genotype showed significantly higher levels of triglycerides and LDLc and lower levels of apoAI compared to controls with the same genotype. In addition to those with [H1-H6,H8-H9] and [H3-H9] genotypes, patients with the [H4-H9] genotype also had significantly lower levels of apoAI. The [H1-H6,H8-H9] genotype in the patient
CLINICAL BIOCHEMISTRY, VOLUME 25, DECEMBER 1992
473
STATISTICAL ANALYSIS
The Student's two-tailed t-test was used to test for significant differences in the levels of lipids and lipoproteins. Probability values
