Apolipoprotein E genotyping and phenotyping in type II diabetes mellitus patients with hypertriglyceridemia

ClinicalBiochemistry,Vol.30, No. 1, pp. 47-52, 1997 Copyright© 1997The CanadianSociet3,of ClinicalChemists Printedin the USA.All rightsreserved 0009-9120/97$17.00+ .00 ELSEVIER

PII S0009-9120(96)00130-0

Apolipoprotein E Genotyping and Phenotyping in Type II Diabetes Mellitus Patients with Hypertriglyceridemia JEONG

H O KIM, 1 E U N J I G L E E , 2 a n d O H H U N K W O N 3

1 D e p a r t m e n t of Clinical P a t h o l o g y a n d 2 D e p a r t m e n t of Internal M e d i c i n e , Y o n g d o n g S e v e r a n c e Hospital, Y o n s e i U n i v e r s i t y C o l l e g e of M e d i c i n e , D o k o k d o n g 1 4 6 - 9 2 , K a n g n a m k u , S e o u l 1 3 5 - 2 7 0 K o r e a , a n d 3 D e p a r t m e n t of Clinical P a t h o l o g y , Y o n s e i U n i v e r s i t y C o l l e g e of M e d i c i n e , 1 3 4 Shinchondong, Sodaemungoo, Seoul 120-752 Korea Objectives: We investigated the possible effect of apolipoprotein E (apo E) polymorphism on hypertriglyceridemia in type II diabetes mellitus (DM) patients by both apo E genotyping and phenotyping methods. Design and Methods: Eighty Korean type II DM patients were evaluated. Restriction isotyping after DNA amplification was used for apo E genotyping. The isoelectric focusing of neuraminidase-treated sera followed by immunoblotting was used for apo E phenotyping. Results: The concordant rate between apo E genotyping and phenotyping was 96.3%. Apo E genotype frequencies for all 77 concordant cases were as follows: 72.7% for e3/3; 16.9% for e3/4; 7.8% for e2/3; 1.3% for e2/4, 1.3% for e4/4; and 0% for e2/2. There were no significant differences in apo E genotype frequencies between hypertriglyceridemic (n = 42) and normotriglyceridemic (n = 35) groups. Conclusions: Our findings could not support the association between apo E polymorphism and hypertriglyceridemia among type II DM patients.

KEY WORDS: apolipoproteins E; polymorphism; type II diabetes mellitus; genotypes; phenotypes; isoelectric focusing; hypertriglyceridemia; gene frequency; restriction fragment length polymorphisms. Introduction

Polipoprotein E (apo E) is not only a major pro-

A tein of very-low-density lipoproteins (VLDL), but

also present in almost all kinds of lipoprotein, including chylomicron, chylomicron remnants, intermediate-density lipoproteins (IDL), and the choles-

This manuscript was presented as an abstract in the 47th Annual National Meeting of the American Association for Clinical Chemistry in 1995. Correspondence: Jeong Ho Kim, M.D., Ph.D. Department of Clinical Pathology, Yongdong Severance Hospital, Yonsei University College of Medicine, Dokokdong 146-92, Kangnamku, Seoul 135-270 Korea (from March 1995 to February 1997, Lipid Research Center, Campus Box 8046, Washington University School of Medicine, 660 S. Euclid, St. Louis, MO 63110 USA). Manuscript received May 7, 1996; accepted September 6, 1996.

CLINICAL BIOCHEMISTRY,VOLUME 30, FEBRUARY1997

terol-rich s u b c l a s s of h i g h - d e n s i t y lipoproteins (HDL) (1). Apo E is known to play important roles as a ligand of lipoprotein receptors, namely low-density lipoprotein (LDL) receptor or apo E receptor (or LDL receptor-related protein; LRP) (1,2,3). The h u m a n apo E is a 299-amino-acid protein (4); its gene is located on chromosome 19 (5) and is polymorphic, with three common alleles coding for three isoforms of the apo E protein, E2, E3, and E4. This polymorphism is due to variation of amino acid number 112 and/or 158. Apo E3, the wild type, is most common; In apo E2, Arg is substituted by Cys at 158th amino acid (Arg158~Cys). In apo E4, Cys is substituted by Arg at l l 2 t h amino acid ( C y s l l 2 ~ Arg). Thus, 6 common phenotypes are possible: E2/2, E2/3, E2/4, E3/3, E3/4, or E4/4. Type III and IV hyperlipoproteinemia in noninsulin-dependent diabetes mellitus (type II DM) patients with hypertriglyceridemia were reported to be related to the apo E2 allele, and type IIb and V hyperlipoproteinemia were reported to be related to the apo E4 allele (6). In E2 and E3 allele-bearing h y p e r i n s u l i n e m i c i n s u l i n - r e s i s t a n t diabetic patients, fasting plasma insulin and glucose levels were positively correlated with plasma triglyceride levels, but not in E4 allele-bearing patients (7). This was consistent with the findings that VLDL were metabolized more rapidly in patients with the apo E4 allele than in those with other apo E alleles (8). Triglyceride and VLDL concentration were reported to be significantly increased in diabetic patients with apo E2. Thus, it had been suggested that the apoE2 allele confers a genetic susceptibility for hypertriglyceridemia in type II DM (9,10). But Shriver et al. (11) postulated that apo E phenotyping in diabetes mellitus patients might misclassify apo E3 as apo E2 due to high sialylation in hyperglycemia, and recommended apo E genotyping for those cases. The aim of this study was to evaluate the relation between hypertriglyceridemia in Type II DM and

47

KIM, LEE, AND KWON

bp bp bp bp 48 bp

91 Z8 72 63

100 bp

30 bp

1

2

3

4

5

6

7

8

Figure 1 - - Six apo E genotypes based on apo E gene restriction fragment length polymorphism using HhaI. Lanes 1 and 8; 10 bp DNA ladder size marker (Gibco BRL, Life Technologies, Gaithersburg, MD, USA); lane 2, e3/3; lane 3, e4/4; lane 4, e2/2; lane 5, e2/3; lane 6, e3/4; lane 7, e2/4. Sixty-three bp fragments were seen in every isotyping. The e2 heterozygotes showed the reduced intensities of 48 bp and 30 bp DNA. apo E polymorphism by comparing both apo E genotyping and apo E phenotyping in type II DM patients with and without hypertriglyceridemia.

Methods PATIENTS AND SPECIMEN PROCESSING

Eighty type II DM patients were recruited from inpatients or outpatients of the Department of Internal Medicine of Yongdong Severance Hospital, Yonsei University, Seoul, Korea, from March 1994 to August 1994. They were all Koreans, who reside in Seoul or nearby areas in Korea. After 12 h of overnight fasting, 5 mL of EDTA-anticoagulated blood were drawn from the patients under informed consent, and were centrifuged within 4 h. Plasmas were frozen for apo E phenotyping after determination of total cholesterol and triglycerides. Infranatant RBC and buffy coat layers were refrigerated for DNA extraction with cell lysis buffer method (12) or frozen at - 2 0 °C for either conventional phenol chloroform extraction (13) or commercial DNA extraction (Instagene Purification Matrix, BioRad Laboratories, Hercules, CA, USA). TOTAL CHOLESTEROL AND TRIGLYCERIDE ANALYSIS

The total cholesterol and triglyceride values reported represent the average of the 2 highest levels in the preceding 6 months among those who had no history of taking lipid-lowering drugs (gemfibrozil, lovastatin, pravastatin, etc.). We used the average of the 2 highest values, when available, or the highest single value determined before any lipid-lowering drugs were taken among those who were taking 48

those drugs at the time of this study. Total cholesterol was determined with a cholesterol oxidase method. Triglycerides were determined with glycerol 3-phosphate oxidase-peroxidase (without glycerol blanks) on the Synchron CX5 (Beckman Instruments, Brea, CA, USA) or Hitachi 747 (Hitachi Co., Hitachi, Japan). Hypertriglyceridemia was defined as/>1.81 mmol/L Hypercholesterolemia was defined as/>6.47 mmol/L. APo E GENOTYPING Apo E genotyping was based on the method of Kontula et al. (14), slightly modified. Two pairs (P1, P4; P2, P3) of oligonucleotide primers as described (14) were synthesized by Korea Biotech. Inc. (Taejon, Korea). For DNA amplification, 0.4 mol/L of oligonucleotides were used as a final concentration in 50 ~L reactions. For DNA extraction by the Instagene Purification Matrix, 20 ~tL of extracted DNA was added as instructed. In the Thermal Reactor (Hybaid, Teddington, Middlesex, UK), DNA was amplified as follows. After initial denaturation of DNA for 5 min at 95 °C, the 1.5 Units of Taq polymerase were added at 72 °C. Cycles consisted of 96 °C for 1 min, 55 °C for 1 min, and 72 °C for 1 min, with a total of 25 cycles. The conditions for the second DNA amplification were the same as noted, except that 1 ~L of first DNA amplification products was used as template DNA, and the other set of primer was used. Products were digested with HhaI (Gibco BRL, Life Techonologies, Inc., Gaithersburg, MD, USA) and s e p a r a t e d in 4% M e t a P h o r T M agarose (FMC BioProducts, ME, USA) gel, prepared according to the instruction manual, electrophoresed at 50 V for 80 min. DNA bands were identified with UV transilluminator. APO E PHENOTYPING Apo E phenotyping was performed by the method of Hill and Pritchard (15). Briefly, 10 ~L of EDTA plasma were treated with 40 m U neuraminidase (EC 3.2.1.18, Clostridium perfringens type V; Sigma Chemical Co., St. Louis, MO, USA) for 30 min at 37 °C, then delipidated with cold ethanol/diethyl ether solution (3:1 v/v) and diethyl ether. The apo E isoforms were separated by polyacrylamide gel isoelectric focusing in mini-vertical lab gel (Miniprotean II system®, BioRad Laboratories, Hercules, CA, USA) with a constant current of 3.4 mA per gel until the voltage were increased to reach 500 to 600 V. The protein bands were transferred from the gel to a nitrocellulose membrane (Gelman, Ann Arbor, MI, USA) in a Mini Trans-Blot cell (BioRad Laboratories, Hercules, CA, USA) at constant voltage of 100 V for I h. The apo E isoforms were detected with a goat a n t i - h u m a n apo-E Ab (Calbiochem, Calbiochem-Novabiochem Co., La Jolla, CA, USA) as a primary antibody (1:1000 dilution) and a rabbit antigoat IgG (Fc) horseradish peroxidase conjugate (Calbiochem) as a secondary antibody (1:1430 dilution). 3 , 3 - d i a m i n o b e n z i d i n e t e t r a h y d r o c h l o r i d e (DAB, Gibco BRL, Life Techonologies, Inc., GaiCLINICAL BIOCHEMISTRY, VOLUME 30, FEBRUARY 1997

apo E GENOTYPING AND PHENOTYPING IN TYPE II DM

(-) 6.5

pl 6 . 0 pl 5 . 9 pl 5 . 8

E4 E3 E2

1

2

3

4

5

p14.0

6

7

8

9

10 11 12

(+)

Figure 2 - - Apo E phenotyping by isoelectric focusing, using a mini-vertical slab gel and goat antihuman apo E antibody. apo E4 was located at pI 6.0; apo E3, at pI 5.9; and apo E2, at pI 5.8. Six apo E phenotypes were identified. Lanes 1 and 2 showed the E3/3; lanes 3 and 4, E4/4; lanes 5 and 6, E2/2; lanes 7 and 8, E3/4; lanes 9 and 10, apo E 2/3; and lanes 11 and 12, apo E2/4. Apo E2/2 was difficult to differentiate from apo E2/3. t h e r s b u r g , MD, USA) a n d COC12.12H20 w e r e u s e d as s u b s t r a t e s . STATISTICAL ANALYSIS G r o u p t-test, X2 t e s t , F i s h e r ' s e x a c t t e s t , a n d ANOVA t e s t w e r e used for analysis. T h e level of significance was defined as p ~< 0.05.

Results F a i n t DNA p r o d u c t s of 328 bp size could be identified by first amplification only in some samples. B u t t h e DNA p r o d u c t s of the second amplification, 266 bp in size, could be i d e n t i f i e d in a l m o s t all samples. Six g e n o t y p e s of apo E w e r e identified (Figu r e 1). Apo E 4 was located on the c a t h o d a l side; apo E2, on the a n o d a l side; a n d apo E3, b e t w e e n t h e m (Figures 2 and 3). But, for some cases, b a n d intensities should be c o n s i d e r e d for exact d e t e r m i n a t i o n of apo E p h e n o t y p i n g (Figures 2 a n d 3). T h r e e cases w e r e excluded for analysis b e c a u s e d i s c o r d a n t results w e r e o b t a i n e d b e t w e e n apo E g e n o t y p i n g a n d p h e n o t y p i n g , despite r e p e a t e d e x p e r i m e n t s , apo e2/3 vs E3/3, e3/3 vs E2/3, a n d e2/2 vs E2/3, respectively. M e a n ages t i m e d u r a t i o n s from diagnosis of type II DM to t h e d a y of blood collection, w e r e not significantly different between the hypertriglyceridemic group (n = 42) a n d n o r m o t r i g l y c e r i d e m i c groups (n = 35) (Table 1). Total cholesterol v a l u e s w e r e significantly h i g h e r in the H y p e r t r i g l y c e r i d e m i c G r o u p t h a n in the N o r m o t r i g l y c e r i d e m i c G r o u p b y group t-test (Table 1). T h e r e w e r e no significant differences in t h e apo E g e n o t y p e f r e q u e n c i e s b e t w e e n h y p e r t r i g l y c e r i d e m i c s a n d n o r m o t r i g l y c e r i d e m i c s in type II DM subjects (p = 0.387, T a b l e 2). T h e r e w e r e also no significant differences of apo E allele frequencies b e t w e e n t h e 2 G r o u p s b y F i s h e r ' s exact t e s t (e2,p = 0.093; e 3 , p = 0.089; e 4 , p = 0.345, Table 2). CLINICAL BIOCHEMISTRY, VOLUME 30, FEBRUARY 1997

T h e r e w e r e no significant differences in d u r a t i o n of disease (p = 0.988), and total cholesterol (p = 0.269) or triglyceride (p = 0.938) v a l u e s according to apo E g e n o t y p e s by ANOVA t e s t (Table 3). T h e r e w e r e no significant differences of apo E g e n o t y p e s in h y p e r c h o l e s t e r o l e m i c (n = 13) vs normocholesterolemic (n = 64) p a t i e n t s w i t h t y p e II DM (×2 = 2.462, p -- 0.652). F o r apo E allele frequencies b e t w e e n 2 groups, Fisher's exact t e s t s h o w e d no significant differences (e2,p = 0.664; e 3 , p = 0.211; e 4 , p = 0.273).

Discussion I m a r i et al. (9) s u g g e s t e d t h a t the apo E2 allele conferred a genetic susceptibility for h y p e r t r i g l y c e r i pl 6.5

(-)

E4 E3 E2

1

2

3

4

5

6

7

6

g

10 11

12'

(+) p| 4 0

Figure 3 - - Apo E phenotyping by isoelectric focusing, using a mini-vertical slab gel and a goat antihuman apo E antibody. (lanes 1, 4, and 11, E3/4; lane 2, E4/4; lanes 3, 5, 6, 7, 8, 9, 10, and 12, E3/3). The more dense apo E2 bands in lanes 6, 8, and 10 were due to high serum triglyceride levels (lane 6, 6.07 mmol/L; lane 8, 2.81 mmol/L; lane 10, 3.83 mmol/L). 49

KIM, LEE, AND KWON TABLE 1 Comparisons of Normotriglyceridemic a n d Hypertriglyceridemic P a t i e n t s With Type II Diabetes Mellitus (DM) Normotriglyceridemic (n = 35) Ages Duration§ T. cholesterol II Triglyceride II

57.2 9.9 4.75 1.27

± ± ± +

Hypertriglyceridemic* (n = 42)

10.8 0.3 1.16 0.32

56.5 6.0 5.53 3.05

± ± ± ±

Total (n = 77)

11.4 7.1 1.30 1.16

56.8 7.8 5.17 2.24

± ± ± +

p valuet

11.0 8.9 1.29 1.25

>0.05 >0.05 0.007 <0.001

* Hypertriglyceridemia was considered w h e n s e r u m triglyceride levels were at or higher t h a n the u p p e r reference limit, 1.81 mmol/L. t p value by group t test; S years old; § years, the period elapsed from the diagnosis of type II DM to w h e n the apo E genotyping was tested; II mmol/L.

TABLE 2 Apolipopprotein E Genotypc a n d Allele Frequencies in Normotriglyceridemic a n d Hypertriglyceridemic P a t i e n t s With Type II Diabetes Mellitus (DM) Normotriglyceridemic apo E genotype e2/2 e2/3 e2/4 e3/3 e3/4 e4/4 Total

n

(%)

Hypertriglyceridemic n

(%)

Total n

(%)

0

(0)

0

(0)

0

(0)

1

(2.9)

5

(11.9)

6

(7.8)

0 28 6 0

(0) (80.0) (17.1) (0)

1 28 7 1

(2.4) (66.7) (16.7) (2.4)

1 56 13 1

(1.3) (72.7) (16.9) (1.3)

35

(100.0)

42

(100.0)

77

(100.0)

e2 = 0.014 e3 = 0.900 e4 = 0.086

e2 = 0.071 e3 = 0.810 e4 = 0.119

e2 = 0.045 e3 = 0.851 e4 = 0.104

There were no significant apo E genotype frequency differences between normotriglyceridemic a n d hypertriglyceridemic p a t i e n t s in type II DM (X 2 = 4.14, p = 0.387). There were no significant differences in each apo E allele frequency between the 2 groups by Fisher's exact test (e2, p = 0.093; e3, p = 0.089; e4, p = 0.345).

TABLE 3 D u r a t i o n of Diabetes, Total Cholesterol a n d Triglyceride Levels According to Apolipoprotein E Genotype a n d Allele F r e q u e n c y in Type II Diabetes Mellitus (DM) Patients Apo E genotype*

(n)

Durationt

Total cholesterols

Triglycerides

e2/3 e3/3 e3/4 e2/4 e4/4

(6) (56) (13) (1) (1)

9.1 ± 7.6 7.7 + 8.3 8.0 ± 12.5 7.0 0

4.83 ± 1.41 5.08 ± 1.22 5.79 ± 1.51 4.53 5.25

2.46 ± 0.91 2.20 ± 0.16 2.32 ± 1.38 2.69 1.85

Total

(77)

7.8 ± 8.9

5.17 ± 1.29

2.24 ± 1.25

* There were no significant differences in duration, total cholesterol, a n d triglyceride levels a m o n g the various genotypes (Duration, F = 0.044,p = 0.988; Total cholesterol, F = 1.388, p = 0.269; Triglyceride, F = 0.136, p = 0.938 by ANOVA test). t D u r a t i o n is i n years elapsed from the time w h e n diabetes mellitus was first diagnosed to w h e n apo E genotyping was performed. S Values are m e a n s ± s t a n d a r d deviations. Total cholesterol a n d triglyceride levels r e p r e s e n t averages of the 2 highest values i n the preceding 6 m o n t h s in p a t i e n t s who had never t a k e n lipid-lowering drugs, or the highest levels in the 6 m o n t h s before i n i t i a t i o n of t r e a t m e n t with lipid-lowering drugs. 50

CLINICAL BIOCHEMISTRY, VOLUME 30, FEBRUARY 1997

apo E GENOTYPING AND PHENOTYPING IN TYPE II DM

demia in Type II DM by apo E phenotyping method. Parhofer et al. (10) found a strong association between apo E2 and hypertriglyceridemia in type II diabetic patients, whereas Shriver et al. (11) could not confirm this hypothesis using apo E genotyping, and they postulated that some apo E3 might be disguised as apo E2 by apo E phenotyping, due to high sialylation in hyperglycemic plasma (9). Shriver et al,(ll) suggested that apo E genotyping would be more suitable than apo E phenotyping. Snowden et al. (16) found that the concordance rate between apo E genotyping and phenotyping in type II DM was only 84%. They thought that the incidence of apo E2 might be increased in type II DM patients due to posttranslational modification detected by apo E phenotyping, and apo E genotyping would avoid that pitfall. We used apo E phenotyping of neuraminidasetreated serum, hoping to avoid the confounding effects of posttranslational modification in diabetes mellitus. The concordance rate, 96.3% (n = 80) in this study, was better than the 84% of Snowden et al. (16) and 76% to 84% of Stavljenic-Rukavina et al. (17), t h o u g h t h e l a t t e r w a s a m o n g insulindependent diabetes mellitus patients, and nearly comparable to the 98% of Kataoka et al. (18). Our high concordance rate seemed to be due to the treatm e n t of s e r a w i t h n e u r a m i n i d a s e . S t a v l j e n i c Rukavina et al. (17) used 0.5 U of neuraminidase, and we used 4 U per mL of serum. Nevertheless, we excluded 3 cases that were discordant between apo E genotyping and phenotyping in repeated experiments. These discrepancies might have been due to other apo E variants (19,20,21). Hansen et al. (22) advocated apo E genotyping as preferable to phenotyping because the latter is affected by posttranslational modification, such as glycosylation, and is more laborious. But, using the mini-vertical slab gel isoelectric focusing system with neuraminidase pretreated whole serum precludes the need for ultracentrifugation or high voltage, and renders the apo E phenotyping method simple and fairly reliable. Thus, the phenotyping method m a y be used for retrospective study, when DNA samples are not available. Though apo E genotyping by Hixon and Vernier (23) has been used conveniently in many institutions, in this study, the double amplification method of apo E gene (14) was selected for possible maximum specificity. We used 4% MetaPhor T M agarose in horizontal submarine minigel electrophoresis cell for separation of small-sized digested DNA products. The advantages of the MetaPhor T M agarose separation were the conveniences of gel casting, the reduction of running time to less than 80 min from 3 to 18 h by polyacrylamide gel electrophoresis, and the avoidance of exposure to toxic acrylamide gel solutions. There are other reports that support speculations that apo E polymorphism might contribute hypertriglyceridemia in type II DM. Lipoproteins with apo E2/2 have decreased bindings affinity for their receptors (24), and most apo E2/2 individuals have inCLINICAL BIOCHEMISTRY, VOLUME 30, FEBRUARY 1997

creased VLDL1, VLDL2, and IDL levels and decreased LDL cholesterol (25). In addition, the met a b o l i s m of i n t e s t i n a l l y d e r i v e d l i p o p r o t e i n is reported to be modulated by apo E polymorphism in normotriglyceridemic type II DM patients (26). Our findings in Korean type II DM subjects did not show any statistical association between apo e2 genotype and hypertriglyceridemia (Tables 2 and 3), although there are limitations of the small sample size. Hypertriglyceridemia in type II DM could be explained by increased VLDL production rate (27,28), although the m e c h a n i s m is not clear. Hyperinsulinemia in insulin-resistant type II DM patients is significantly related to triglyceride levels (29), but the contribution of insulin p e r se to the hypertriglyceridemia still remains an open question (30). Decreased activity of lipoprotein lipase (31,32,33) may contribute to delayed triglyceride clearance. There is a recent finding of relationship between lipoprotein lipase gene mutation and hypertriglyceridemia in type II DM patients (34). Other genes (30,35,36) m a y play a role in hypertriglyceridemia in type II DM, as well. In conclusion, our findings could not support the association between apo E polymorphism and hypertriglyceridemia in type II DM.

Acknowledgements This work was partially supported by the research fund (No. 1993-48) for professors of Yonsei University College of Medicine. We are grateful to Dr. P. Haydn Pritchard and Dr. Jean-Francois Bowden in St. Paul's Hospital Lipid Clinic, University of British Columbia, Vancouver, Canada for their kind demonstration of apo E phenotyping. We also thank Dr. Kyung Soon Song in the Department of Clinical Pathology, Dr. Kyung-Sup Kim in the Department of Biochemistry and Molecular Biology, and Dr. Hyun Chul Lee in the Department of Internal Medicine for advice, and Jong Shin Chung and Dr. Young Sook Park for technical assistances in the Department of Clinical Pathology, Yonsei University College of Medicine, Seoul, Korea. We also thank Dr. Gustav Schonfeld, Kountz Professor of Medicine, Washington University School of Medicine in St. Louis, MO, USA for critiquing this manuscript.

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CLINICAL BIOCHEMISTRY,VOLUME 30, FEBRUARY 1997