A Trial of Apolipoprotein E Genotyping in Noninsulin Dependent Diabetes Mellitus by Polymerase Chain Reaction Method

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A Trial of Apolipoprotein E Genotyping in Noninsulin Dependent Diabetes Mellitus by Polymerase Chain Reaction Method

Dept. of Internal Medicine, Endocrine Research Institute, Kyung Hee University Hospital.

Paeng, Jung Ryung. ·Kim, Deog Yoon. • Woo, Jung Taek.

Kim, Sung Woon. • Yang, In Myung. • Kim, Jin Woo.

Kim, Young Seol. • Kim, K wang Won. • Choi, Y ong Kil.

Key words : Genotyping. Apolipoprotein E. isoforms. Polymerase chain reaction.

I . INTRODUCTION

Apo E is one of various factors which regulate the lipid metabolism. It is a constituent of plasma lipoproteins such as chylomicrons, very low density lipoproteins(VLDL) and high density lipoproteins.

The major physiological function of apo E plays a central role in cholesterol transport!), and it serves as a ligand for apo B, E receptor2l. The isoforms, designated E2, E3, and E4, differ by single amino acid residue with substitution of cysteine for argi- nine3·4·5l. In addition to this major isoforms, several other rare apo E isoproteins have recently been de- scribed6·7l. The E4 isoform(112 arg and 158 arg) is associated with increased levels of plasma total cholesterol and betalipoprotein⁸l and increased suscepti- bility to coronary heart disease. Hyperlipidemia, a risk for development of atherosclerosis occurs fre- quently in non-insulin dependent diabetic mellitus (NIDDM). Eto et al. have been reported that apo E polymorphism may be a cause of hyperlipidemia in type

ll

(NIDDM) diabetes. In their study, the E2 isoform is associated with decreased levels of cholesterol and betalipoprotein⁸l. And there are reports concerned with relationship between type

V

hyperlipoproteinemia and apo E4/E4⁹•10l.

A number of isoforms of apo E are separable by one dimentional isoelectric focusing(IEF) method as

well as by two dimentional gel IEF method¹¹^•12·13l,

followed by SDS-polyacrylamide gel electrophoresis (PAGE) 14l.

Because this method is based on charge differenc- es of apo E isoproteins, it is not possible to identify neutral amino acid substitution, nor to locate the site of the mutation.

These methods has been well known for time-consuming as well as there has no clear discrimination of IEF band. Recently, apo E typing at the DNA level was introduced by PCR method15l. In addition, It is now possible to define mutation site of apo E gene by application of DNA typing method16^·17).

However, clinical study from large population is not reported until recently. In this report, we have tried restriction fragment analysis of apo E gene as a simpler and faster method for typing the common apo E isoforms. to elucidate the relationship between diabetes accompanied by hyperlipidemia, apo E genotype and apo E allele frequencies of diabetics were studied.

ll • Materials and Methods 1. Amplification of apo E gene

Blood samples were obtained from the patients with NIDDM. DNA was extracted from the periph-

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eral leucocyte as described previously¹⁸l. And amplified by PCR in a DNA thermal cycler(Hybaid) using oligonecleotide primers F4(5 '-ACAGAATTC GCC CCGGCCTGGT ACAG-3') and F6 ( 5 '-T AAG C TTGGCACGGCTGTCCAAGA-3') as described by Emi et aP³•19l.

Oligonucleotide primers were synthesized by the phosphoramidite method using a DNA synthesizer (Applied Biosystems Inc.). Taq DNA polymerase was obtained from KIST, Korea. Each amplification reagents contained 1 pg of DNA, 100 pmol of each primer, 10% dimethylsulfoxide, and 2.5 unit of Taq polymerase in a final volume of 100 p R and then added few drops of mineral oil to prevent evapora- tion. At first, reaction mixture was heated at 95oC for 5 min for denaturation, and then subjected to 30 cycles of amplification by primer annealing(68°C for 50 sec). Extention(70°C for 50 sec), and denaturation(950C for 50 sec).

2. Electrophoresis and restriction fragments isotyping

After PCR amplification, the amplified DNA was recovered by ethanol precipitation, 5 units of Hha I in recovered DNA were added to each reaction mixture for digestion of PCR products(1.5h at 37°C).

Each reaction mixture was loaded on 8% polyacrylamide gel(l mm thick x 12 em long) and electro-

E4 TAAGCTTG GCA CGG CTG

GCG CAG GCC CGG CTG

E2 T

E3 T 112

E4 G1G CGC GGC CGC CTG GTG CAG GCC ATG CTC CTG CGG GTG CGC CTC CTG CGT AAG CGG CTC

E2 T

E3

c

158

E4 CTG CAG AAG _{CGC CTG} GCGAATTCTGT 244

phoresed for 3h under constant current ( 45 rnA).

After electrophoresis, the gel was stained with ethidium bromide for 15 min and DNA fragments were visualized by UV illuminator. The sizes of Hha I fragments were estimated by comparision with DNA molecular-weight marker

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(Boehringer ma- nnheim). Apo E genotyping relies on cleavage at Hha I sites to distinguish E2, E3, and E4 sequences.

PCR product was amplified a specific region of the exon 4(Fig. 1, 2).

plasma apo E amino acids 299

precursor 317

protein amino acids

1163 mRNA

nucleotides exon 1 exon 2 ⁴⁴

• -

⁶⁶ _{3597 bp}exon 3 ¹⁹³ exon 4 ⁸⁶⁰

Fig. 1. Structure of the human apo E gene.

A schematic outline of the human apo E gene is shown in relation to the mRNA which it encodes, the primary translation product of the mRNA, and the mature plasma protein product of the gene.

TCC AAG GAG CTG CAG GCG GGC GCG GAC ATG GAG GAC

GTG CAG TAC CGC GGC GAG GGC CAG AGC ACC GAG GAG GCC TCC CAC CTG CGC AAG CTC CGC GAT GCC GAT GAC

GCA GTG TAC CAG GCC GGG

Rg. 2. DNA sequences of apo E isoforms and location of Hha I cleavage sites.

(substitution site at codons 112 and 158 are boxed)

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The second step is cleavage of amplified products with Hha I for PAGE. Each of the isoforms is dis- tinguished by a unique combination of Hha I fragment sizes. According to Hha I fragment sizes, six apo E genotypes were determined as shown in Fig.

3. The cut sites are shown 6 places in E4, 5 places in E3, 4 places in E2(Fig. 3).

112 158

48 35

£3----~+---~---+----91

91 83

Ez----~r---++--- Fig. 3. Hha I cleavage maps are given for ampli-

fied sequences. The distances(in bp) between polymorphic Hha I sites that distinguish isoforms are shown for each cleavage map.

ill. RESULTS

PCR products(224 bp) electrophoresed on 1.5%

agarose gel(Fig. 4).

244 bp

Fig. 4. Agarose gel electrophoresis after PCR amplification.

lane 1, size marker.

lane 2-8, PCR products ( 244 bp)

PCR product is digested with Hha I restriction enzyme and electrophoresed on polyacrylamide gel.

Each genotype posessed unique combinations of Hha I fragments sizes(Fig. 5). The E3/E3 homozygote contained the 91 bp fragment(112 cys), as well as 48 bp and 35 bp fragments from cleavage at the Hha I site at 158 arg. The E4/E4 homozygote also contained these 48 and 35 bp sizes(l58 arg), as well as a unique 72 bp size from cleavage at 112 arg. E3 /E2 heterozygote contained both sets of fragments from each apo E allele, 91 and 83 bp(Fig. 5).

Fig. 5. 8% polyacrylamide gel electrophoresis of Hha I fragments after PCR amplification.

91 bp 83 72 48 35

lane 1, E4/E4(72 bp). lane 2,4-6, E3/E3(91 bp).

lane 3, E3/E2(91.83 bp). lane 7, size marker.

lane 8, PCR product ( 244 bp)

Apo E genotype frequency in normal subjects was 0% for E2/E2, 9.6% for E3/E2, 72.6% for E3/E3, 0% for E4/E2, 15.1% for E4/E3, 2.7% for E4/E4.

In the patients with NIDDMs, the gene frequency of apo E gene was 0% for E2/E2, 12.7% for E3/E2, 64.6% for E3/E3, 0% for E4/E2, 20.2% for E4/E3, 2.5% for E4/E4.

As shown in table 1, the apo E allele frequencies in normal subjects showed 4.8% for E2, 84.9% for E3, 10.3% for E4. And in NIDDMs showed 6.3%

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for E2, 81.0% for E3, 12.7% for E4. The frequency of apo E3/E3 was most prevalent in both NIDDM and normal groups, whereas apo E2/E2 homozygote was not found in both NIDDM and normal groups.

The distribution of apo E isoforms was no significant difference between normal and NIDDMs.

N. DISCUSSION

The human apo E gone spans 3.59 kb including 4 exons and 3 introns20·21). Genetic variation at the apo E locus is an important determination of lipid levels and regard as a risk for developement of atherosclerosis22>. Until recently most methods for detection of allelic variation have relied on IEF method. It require time-consuming and necessary ultracentrifugation for the separation of lipoproteins.

Recently much attention has been focused on the relationship between apo E alleles and hyperlipoproteinemia. Utermann et al. found that the apo E2 /E2 homozygote is related to type

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hyperlipopro- teinemia23>. And other apo E2 allele has been reported to be associated with higher levels of plasma tri- glyceride and VLDU'>. PCR technology has allowed genotyping of apo E isof orms by direct detection of nucleotide substitutions in genomic DNA rather than IEF of isoproteins. Our results clearly show that typing of the three major apo E isoforms from minute quantities of DNA can be done using the PCR methodology(Fig. 3).

As shown in Table 1, apo E3/E3 were a prevalent genotype, and a lower frequency of genotype E3/E2 were found in the Korean than those reported for the Black et. al from the caucasian. But apo E4/E3 and E2/E2 showed a similar patterns. Black et al.

suggested that the frequency of apo E3 was signifi- cantly less in diabetic than in normal subjects25>.

Table 2 shows distribution of apo E allele f requen- cies in our study and a patients with NIDDM previously repoted. The frequencies of apo E genotype in our population agree almost with those reported by previous authors although their method based on protein characterization26· 27 · 28>.

The apo E allele frequencies in diabetic subjects (E2, 6.3% ; E3, 81.0% ; E4, 12.7%) are similar to those in Japanese diabetic subjects(E2, 4.3% ; E3, 83.8% ; E4, 11.9%). Besides Eto et al. have been

Table 1. Apo E genotypes and phenotypes in Korean and previous report Apo E

2/2 3/2 3/3 4/2 4/3 4/4 Genotype(%)

NIDDM (n=79) 0.0 12.7 64.6 0.0 20.2 2.5 Normal (n=73) 0.0 9.6 72.6 0.0 15.1 2.7 Black et al. 0.0 26.0 43.0 4.0 19.0 7.0 Phenotype(NIDDM, n=72)

Apo E allele frequencies(%) E2 E3 E4

NIDDM (n=79) 6.3 81.0 12.7

Normal (n = 73) 4.8 84.9 10.3

Table 2. Apo E Allele Frequencies in NIDDM Frequencies ( % ) E2 E3 E4 Korean (n=79) 6.3 81.0 12.7 Japanese (n= 105) 4.3 83.8 11.9 British (n=72) 16.2 67.6 16.2

proposed that the apo E3/E2 and apo E4/E3 diabetic subjects is closely related to hyperlipemia26>. In particular, they suggested that E4 allele is closely related to higher levels of plasma cholesterol in NIDDM, although some investigator did not find any difference29·30·31>.

In our present study it was not clear which type of hyperlipoproteinemia is associated with the apo E allele because detailed lipoprotein analysis was not included. Table 3 shows a comparison between our present study(genotyping) and previously reported (phenotyping) in normal groups. Both Korean and Japanese have also a similar pattern as shown in NIDDMs.

Table 3. Apo E Genotype in Korean and Apo E Phenotype Frequencies in Other Races ApoE

2/2 3/2 3/3 4/2 4/3 4/4 Genotype (% )

NIDDM (n=79) 0.0 12.7 64.6 0.0 20.2 2.5 Normal (n=73) 0.0 9.6 72.6 0.0 15.1 2.7 Apo E Phenotype(%) : Nondiabetic subjects Japanese (n=68) 0.0 10.3 76.4 1.5 10.3 1.5 Finnish (n=490) 1.0 13.1 58.9 0.0 24.3 2.7

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Interestingly, our results showed a great difference in the apo E allele frequencies when compared with those of the Europians(British, Finnish). Fur- ther studies will be required for setting the relationship between the apo E4 allele and hyperlipoproteinemia in NIDDM.

Futhermore, Hha I apo E genotyping could not detect rare variants, unless substituions alter Hha I sites within the amplified region using F4 and F6 primer. 'And a possibility of methylation may have prevent~d Hha I cleavage of PCR products. Howev- er, sequencing or single-strand conformation polymorphism analysis of apo E genes may be useful to detect rare variants. PCR technique could be a useful for diagnosis of genetic desease, or for detection of unknown polymorphisms using survey of many different restriction enzymes.

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CONCLUSION

There was no significant difference of apo E isoforms distribution between diabetic and normal groups in apo E genotypig. The apo E allele frequencies of Korean and Japanese diabetics were similar, but there was a great difference as compared with those of caucasian. In order to prove this difference between isoelectric focusing, we suggest tha:t fur- ther studies should be subdivided by type of lipoproteinemia. Apo E genotyping by PCR method may be identified in conjunction with apo E phenotyping.

ACKNOWLEGEMENTS

This study was supported by Kyung Hee University Medical Center. We also thank Dr, Dae-sil Lee for kindly providing Taq polymerase.

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