APOLIPOPROTEIN E SEBAGAI FAKTOR RISIKO TIMBULNYA PENYAKIT

JANTUNG KORONER: Kajian Genetika Populasi dan Genetika Klinis

pada Beberapa Kelompok Etnik di Indonesia

Oleh

Pramudji Hastuti

NIM: 99/850/PS

UNIVERSITAS GADJAH MADA

Yogyakarta

2012

i  

APOLIPOPROTEIN E SEBAGAI FAKTOR RISIKO TIMBULNYA PENYAKIT JANTUNG KORONER:

Kajian Genetika Populasi dan Genetika Klinis pada Beberapa Kelompok Etnik

di Indonesia

Disertasi untuk memperoleh

Derajat Doktor dalam Ilmu-Ilmu Kesehatan

Pada

Universitas Gadjah Mada

Dipertahankan terhadap sanggahan

Tim Penguji Pascasarjana Universitas Gadjah Mada

Pada hari: Senin

Tanggal: 21 Mei 2012

Oleh

Pramudji Hastuti

Lahir

Di Klaten, Jawa Tengah

ii  

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BAB 6

RINGKASAN

Apolipoprotein E (apoE) adalah penyusun protein dari lipoprotein plasma yang

mempunyai beberapa fungsi termasuk perannya dalam metabolisme kolesterol dan

sebagai ligan yang penting dalam klirens lipoprotein. Apolipoprotein E pertama kali

diidentifikasi sebagai penyusun very low density lipoprotein (VLDL) yang berfungsi

sebagai transport trigliserida dari hepar ke jaringan perifer (Mayes & Botham, 2006).

Gena apoE terutama diekspresikan di hepar (90%), namun juga terdapat di jaringan

lain, termasuk otak, limpa, paru, gonad, adrenal, ovarium, ginjal dan otot. Makrofag

masak yang berasal dari monosit hepar juga memproduksi apoE dalam jumlah yang

signifikan, yang memberikan sampai 10% protein sirkulasi. Apolipoprotein E bekerja

sebagai ligan berafinitas tinggi untuk beberapa reseptor lipoprotein hepar: reseptor

LDL, LDL receptor-related protein (LRP), reseptor VLDL, dan scavenger receptor

type 1 class B (SRB-I). Glikoprotein ini memperantarai klirens lipoprotein yang

mengandung apoE yaitu kilomikron, VLDL, IDL, dan HDL. Apolipoprotein E juga

terdapat dalam jumlah sedikit dalam LDL dan larut dalam plasma (Mahley et al.,1999;

Mc Neale et al., 2000, Moghadasian et al., 2001, Zechner et al., 1991). Salah satu

fungsi metabolik apoE dalam mentransport kolesterol dari jaringan perifer ke hepar

untuk didegradasi, disebut transport kolesterol balik. Apolipoprotein E juga

memodulasi aktivitas beberapa enzim termasuk dalam metabolisme lipid (Sima et al.,

2006).

Gena apoE adalah polimorfik dan terdapat 6 bentuk protein berbeda,

dinamakan E2/E2, E2/E3, E2/E4, E3/E3, E3/E4, dan E4/E4 yang merupakan produk

gena dari 3 alel apoE masing-masing ε2, ε3 dan ε4 (Belkovets et al., 2001). Hubungan

terkuat antara kadar apoE dengan mortalitas kardiovaskular tampak pada pasien yang

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mempunyai kadar CRP yang rendah pada umur 85 tahun. Pada sebagian besar pasien

ini, kadar CRP meningkat jika umur meningkat, menunjukkan bahwa tingginya kadar

apoE terjadi sebelum terjadi inflamasi. Aktivitas biologis apoE dapat dipengaruhi oleh

modifikasi struktur dan atau kuantitasnya. Perubahan struktural dapat terjadi pada

polimorfisme apoE, yang mengkode apoE2, apoE3, dan apoE4. Apolipoprotein E2

menunjukkan afinitas yang lebih rendah ke reseptor LDL, menghasilkan klirens apoE

yang lebih lambat dan meningkatkan kadar apoE plasma. Keadaan ini selanjutnya

akan direspon dengan mengatur reseptor LDL di hepar untuk menurunkan kadar

kolesterol. Apolipoprotein E4 sebaliknya diambil lebih efisien, menghasilkan kadar

apoE yang lebih rendah dan meningkatkan kadar kolesterol. Oleh karena itu, variasi

genetik yang mempengaruhi metabolisme lipid akan mengubah risiko penyakit

kardiovaskular dan demensia. Polimorfisme apoE hanya berpengaruh sebagian

terhadap kadar apoE plasma, dan kadar apoE plasma bervariasi di antara individu

dengan genotip apoE yang sama. Kadar apoE plasma juga berhubungan dengan kadar

kolesterol. Apolipoprotein E juga memperantarai presentasi antigen lipid terhadap

sistem imunitas dan jalur ini mempengaruhi proses inflamasi. Baik lipid dan inflamasi

terlibat dalam patogenesis aterosklerosis, namun hubungan kadar apoE plasma dan

risiko penyakit kardiovaskular masih belum jelas (Eichner et al., , 2002, Mahley et

al.,1999, Souza et al., 2007)

Frekuensi apoE berbeda pada kelompok etnis berbeda. Penelitian secara

intensif menunjukkan variasi alel apoE mempunyai efek yang signifikan pada variasi

lipid plasma antar individu dan kadar lipoprotein serta risiko penyakit kardiovaskular

pada populasi umumnya (Everaldo et al., 2004). Di antara varian gena apoE, apo ε3

memiliki frekuensi paling banyak sampai lebih dari 60% pada semua populasi yang

diperiksa (Eicner et al., 2002).

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Apolipoprotein E adalah salah satu dari kelompok famili gena apolipoprotein.

Kelompok gena ini, terdapat multigena yang menghasilkan berbagai jenis apoprotein

termasuk apoA-I, apoA-II, apoA-IV, apoC-I, apoC-II dan apoC-III. Gena apo ε

terletak pada kromosom 19q13.2 dan berhubungan erat dengan kompleks gena apoC-

I/C-II. Gena apoE tersusun dari 4 ekson dan 3 intron sepanjang 3.597 nukleotida dan

menghasilkan 299 asam amino (Eichner et al., 2002).

Apolipoprotein E, sama dengan apolipoprotein lainnya membantu

menstabilkan dan melarutkan lipoprotein sehingga dapat bersirkulasi dalam darah.

Peran umum apolipoprotein dalam metabolisme lipid antara lain termasuk

mempertahankan integritas struktural lipoprotein, berfungsi sebagai kofaktor dalam

reaksi enzimatis, dan bekerja sebagai ligan untuk reseptor lipoprotein. Apolipoprotein

E penting dalam pembentukan VLDL dan kilomikron (Corella et al., 2002; Eichner et

al., 2002; Moghadasian et al., 2001).

Penelitian epidemiologis yang melihat peran langsung apoE pada PJK

menunjukkan bahwa 6% variasi risiko PJK di Amerika Utara diperankan oleh lokus

gena ini. Penelitian pada laki-laki umur tengah baya dari 9 populasi memperkirakan

adanya kenaikan sekitar 40% risiko mortalitas PJK oleh pembawa gena ε4 dibanding

pembawa ε3 dan ε2 (Stengard et al., 1998). Beberapa penelitian lain menunjukkan

bahwa pembawa gena ε4 terutama mempunyai kecenderungan lebih besar untuk

menderita lesi koroner atau mempunyai risiko lebih besar untuk mengalami kematian

akibat PJK (Eichner et al., 1993, Lehtinen et al., 1995, Stengard et al., 1995, Wang

et al., 1995). Mekanisme biokimiawi berhubungan dengan disfungsi isoform apoE4

pada metabolisme protein adalah menaikkan kadar kolesterol dan trigliserida serum.

Penelitian di Finlandia, Scotlandia, dan Irlandia Utara menunjukkan bahwa populasi

dengan kadar kolesterol yang tinggi menaikkan risiko mortalitas PJK dan juga

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mempunyai frekuensi alel ε4 yang lebih tinggi. Penelitian lain juga menunjukkan

adanya hubungan alel ε2 dengan naiknya risiko PJK (Eichner et al., 2000; Mahley et

al., 2006; Zannis et al., 1996).

Apolipoprotein E ε2/ε2 tampak berhubungan dengan hiperlipoproteinemia tipe

III (HLIII) dan telah diketahui pada satu dekade ini. Gangguan ini dicirikan oleh

naiknya kadar kolesterol, trigliserida dan β-VLDL (remnan kilomikron usus dan

VLDL hepar kaya kolesterol), xantoma dan penyakit vascular prematur, baik PJK

maupun penyakit arteri perifer (Mahley et al., 1995). Angka kejadian HLIII dengan

frekuensi 1 – 5 per 5000, sedangkan homosigositas E2-2 frekuensinya 0,5 – 1 per 100

pada populasi Kaukasia. Oleh karena itu, genotip ini menyokong fenotip HLIII tanpa

memperhatikan penyebabnya (Eichner et al., 2000; Fullerton et al., 2000)

Indonesia merupakan suatu negara yang terletak antara benua Asia dan

Oceania, terdiri dari 17.508 pulau yang tersebar di sekitar garis equator. Luasnya,

serta iklim tropis yang dimiliki didukung geografi kepulauan di Indonesia,

mendukung biodiversitas besar kedua setelah Brazil, yang mempunyai flora dan fauna

campuran antara spesies Asia dan Australasia. Sumatra, Jawa, Kalimantan dan Bali

mempunyai kesamaan flora dan fauna dengan Asia. Sulawesi, Nusa Tenggara, dan

Maluku, mempunyai flora dan faunanya sendiri yang unik. Papua yang merupakan

bagian dari daratan Australia, mempunyai fauna dan flora yang sangat erat

berhubungan dengan Australia, termasuk lebih dari 600 jenis burung (van Oosterzee,

1997).

Alfred Wallace, membagi Indonesia menjadi 2 kawasan dengan garis yang

memanjang dari utara ke selatan antara Kalimantan dan Sulawesi, dan sepanjang

Pulau Lombok , antara Lombok dan Bali yang dinamakan garis Wallace. Di sebelah

barat garis mempunyai flora dan fauna yang cenderung sama dengan Asia; di sebelah

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timur Lombok, cenderung sama dengan Australia. Perbedaan tersebut juga ditunjang

oleh penanda genetik darah dari kedua populasi tersebut yang berbeda. Penelitian

yang dilakukan oleh Sofro (1982), yang meneliti tentang genetika populasi di

Indonesia menunjukkan bahwa frekuensi fenotip beberapa enzim di kawasan barat

Indonesia tinggi dan semakin ke timur frekuensi tersebut semakin turun. Beberapa

contoh di antaranya adalah fenotip enzim glioksilase-1 (GLO-1), glutamat-piruvat

transferase-1 (GPT-1), dan fosfoglukonat dehidrogenase-C (PGD-C). Demikian juga

frekuensi thalassemia-β di Indonesia, misalnya di Palembang 9% (Sofro et al., 1994),

di Ujung Pandang 8% (Sofro et al., 1994), di Maumere Flores 6% (Sofro et al.,

1993) dan di Ambon Maluku 6% (Sofro et al., 1994). Haptoglobin-1 (Hp-1) dan

defisiensi glukose-6-fosfat dehidrogenase (G6PD) berkebalikan dengan HbE, yang di

kawasan Indonesia barat rendah, semakin ke timur frekuensinya semakin naik.

Fenotip transferin yang dominan di kawasan barat Indonesia adalah tipe D-Chi yang

merupakan petanda gena Mongoloid, sedangkan kawasan timur Indonesia transferin

tipe D-1 yang merupakan petanda gen melanesid. Penelitian yang dilakukan oleh

Lanni (2002) menunjukkan bahwa secara genetik populasi Indonesia dapat dibagi

menjadi 3 klaster; klaster pertama merupakan populasi Indonesia yang sangat kuat

dipengaruhi oleh unggun gena (gene pool) Mongoloid, klaster kedua terdiri dari

populasi yang merupakan campuran antara unggun gena Mongoloid dan

Austromelanesid dan klaster ketiga merupakan klaster yang sangat dipengaruhi oleh

unggun gena Austromelanesid.

Indonesia mempunyai dua unggun gena utama, yaitu Mongoloid di sebelah

barat dan Melanesid di sebelah timur, dan di tengah terdapat campuran kedua gena

tersebut, masing-masing populasi ini mempunyai unggun gena yang berbeda, maka

penelitian ini dilakukan untuk mengkaji genotip dan alel apo E pada beberapa etnik

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populasi kawasan barat, tengah dan timur Indonesia. Selain itu pada penelitian ini juga

mengkaji polimorfisme genotip dan alel apoE pada penderita penyakit jantung

koroner pada kelompok etnik Indonesia barat.

Penelitian ini secara umum bertujuan untuk mengetahui peran apoE sebagai

salah satu petanda genetik terhadap timbulnya penyakit jantung koroner. Hal ini

didasarkan atas kenyataan bahwa apo E sebagai faktor genetik memiliki sebaran

dalam populasi yang terkait dengan unggun gena dari suatu populasi. Bukti

antropologi fisis dan genetik menunjukkan bahwa penduduk Indonesia secara garis

besar memiliki dua unggun gena yang berbeda yaitu kawasan barat Indonesia

umumnya menggambarkan pengaruh unggun gena Mongoloid, kawasan timur

Indonesia menggambarkan pengaruh unggun gena Melanesid dan kawasan tengah

Indonesia merupakan unggun gena campuran.

Subyek pada penelitian ini terdiri atas 2 kelompok. Penelitian pertama

meliputi penelitian potong lintang yaitu penderita penyakit jantung koroner (PJK)

yang berasal dari RSUP Dr. Sardjito Yogyakarta sebagai kasus dibandingkan dengan

kontrol yang berasal dari anggota kelompok senam penduduk di Yogyakarta.

Penelitian kedua dari populasi di Indonesia yang diambil dari 3 daerah untuk mewakili

kawasan barat Indonesia diambil dari Surabaya, kawasan tengah Indonesia dari Palu

dan kawasan timur Indonesia dari Alor. Darah yang berasal dari kasus dan kontrol

ditentukan profil lipidnya menggunakan kit Diasys dan buffy coat digunakan untuk

isolasi DNA. Terhadap DNA dari kasus, kontrol dan dari ketiga populasi dilakukan

pemeriksaan gneotip apoE dengan metode Zivelin et al. (1997). Metoda ini akan

mengamplifikasi ekson 4 dari gena apoE. Hasil PCR kemudian dipotong dengan

enzim restriksi HaeII dan Afl III yang akan membedakan antara gena ε2, ε3 dan ε4 (ε4

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dengan pita masing-masing 195 dan 23 pasangan basa; ε3 dengan pita 23, 50 dan 145

pasangan basa dan ε2 mempunyai 50 dan 168 pasangan basa)

Hasil yang diperoleh dianalisis:

a. Membandingkan profil lipid antara penderita PJK dengan kontrol, kemudian

dianalisis dengan uji-t dan perbedaan dinyatakan bermakna dengan taraf

kemaknaan <0,05; faktor risiko dihitung dengan Odd ratio

b. Menghubungkan polimorfisme apoE dengan profil lipid dengan uji korelasi

Pearson

c. Dengan mengendalikan profil lipid, membandingkan genotip dan alel apoE

sebagai faktor risiko PJK dengan Odd ratio.

d. Frekuensi genotip apoE: dihitung dari persentase genotip apoE terhadap

jumlah subjek

e. Frekuensi alel apoE dihitung dari jumlah seluruh alel apoE dan dihitung

persentasenya

f. Membandingkan frekuensi genotip, dan alel apoE di antara populasi dengan

Chi square dan dinyatakan berbeda bermakna dengan taraf kemaknaan < 0,05

g. Membandingkan genotip dan alel apoE antara penderita PJK dengan populasi

sebagai faktor risiko PJK dengan chi square dan Odd ratio.

Hasil

Pemeriksaan polimorfisme apoE pada penderita PJK dan kontrol diperiksa

masing-masing terhadap 33 subjek yang berasal dari penderita PJK dan 38 subjek

kontrol. Jenis kelamin, berat badan, tinggi badan, IMT, tekanan darah dan kadar gula

darah antara penderita PJK dan kontrol yang diuji dengan t-tes tidak menunjukkan

perbedaan bermakna (p>0,05). Profil lipid antara penderita PJK dan kontrol dengan

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analisis t-tes tampak tidak terdapat perbedaan bermakna walaupun rata-rata kadar

trigliserida, kolesterol, maupun LDL-C pada penderita PJK lebih tinggi dan kadar

HDL-C nya lebih rendah jika dibandingkan dengan kontrol (p>0,05). Dislipidemia

merupakan faktor risiko PJK dengan OR 1,4-3,93 kalinya. Genotip apoE antara

penderita PJK dan kontrol terdapat perbedaan bermakna (p<0,05)

Hubungan polimorfisme apoE dengan profil lipid menunjukkan bahwa

pembawa gena ε2 berhubungan dengan signifikansi tingginya kadar trigliserida dan

berhubungan dengan tingginya kadar kolesterol namun tidak berbeda bermakna dan

gena lainnya tidak berhubungan dengan profil lipid.

Frekuensi genotip apoE ε2/ε2, ε2/ε3, ε3/ε3, ε3/ε4 pada penderita PJK masing-

masing 3,1%, 18,1%, 42,4% dan 36,4% sedangkan pada kontrol masing-masing

10,5%; 13,2%; 60,6%, 36,8% dan 10,5%. Oleh karena pada penderita PJK tidak

ditemukan adanya genotip ε2/ε4 dan ε4/ε4 maka tidak dapat dilakukan pembandingan

dengan kontrol. Pembawa genotip apoE antara penderita PJK dan kontrol jika

dibandingkan dengan analisis chi square, terdapat perbedaan bermakna (p<0,05)

hanya pada pembawa genotip apoE ε3/ε4. Pembawa genotip apoE ε3/ε4 ini

mempunyai risiko untuk menjadi PJK 4,86 kali lebih besar dibanding pembawa

genotip lainnya. Pembawa genotip ε2/ε2 mempunyai OR kurang dari 1. Hal ini

menunjukkan bahwa pembawa genotip apoE ε2/ε2 merupakan faktor protektif

terhadap timbulnya PJK.

Frekuensi pembawa alel ε2 pada penderita PJK dan kontrol tidak menunjukkan

perbedaan bermakna dengan OR kurang dari 1. Hal ini berarti bahwa alel ε2

merupakan faktor protektif terhadap terjadinya PJK, sementara pembawa alel ε4

mempunyai risiko 2,05 kali lebih besar untuk menderita PJK. Hal ini menunjukkan

bahwa pembawa alel ε4 mempunyai risiko untuk menderita PJK lebih besar dibanding

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seseorang bukan pembawa alel ε4. Dari hasil ini disimpulkan bahwa pembawa genotip

apoE ε3/ε4 dan pembawa alel ε4 mempunyai risiko lebih besar untuk menderita PJK

dan alel ε2 adalah faktor protektif terhadap PJK.

Dislipidemia sebagai faktor risiko terhadap timbulnya penyakit PJK yang

berhubungan dengan polimorfisme apoE, tampak terjadinya dislipidemia, terutama

naiknya kadar kolesterol dan LDL serta turunnya kadar HDL merupakan faktor risiko

timbulnya PJK untuk semua genotip apoE. Kadar trigliserida yang tinggi ini pada

pembawa gen ε2 bukan merupakan faktor risiko timbulnya PJK. Profil lipidnya jika

dikendalikan dan dihubungkan polimorfisme apoE dengan timbulnya PJK, tampak

pembawa gena ε2 bukan merupakan faktor risiko timbulnya PJK, namun pembawa

gena ε4 merupakan faktor risiko kuat untuk menjadi PJK dengan OR 4 kalinya.

Penelitian tentang polimorfisme apoE pada beberapa populasi di Indonesia,

diperiksa dari ketiga populasi yaitu kawasan barat, tengah dan timur Indonesia.

Sebanyak 195 orang terdiri dari populasi Surabaya mewakili kawasan barat Indonesia

sebanyak 82 orang, Palu mewakili kawasan tengah Indonesia sebanyak 68 orang dan

Alor mewakili kawasan timur Indonesia sebanyak 45 orang. Frekuensi genotipe apoE

ε2/ε2 pada populasi Alor adalah 15,6%, Surabaya 8,5%, dan populasi Palu 7,4%.

Frekuensi genotip apoE ε2/ε3 pada populasi Palu adalah 30,9%, Surabaya 18,3% dan

populasi Alor 15,6%. Frekuensi genotip apoE ε3/ε3 pada populasi Surabaya adalah

54,9%, Palu 36,8% dan populasi Alor 17,8%. Frekuensi genotip apoE ε2/ε4 pada

populasi Surabaya dan Palu masing-masing adalah 6,1% dan 6,7%, sedangkan pada

populasi Alor frekuensinya 2,9%. Genotip apoE ε3/ε4 tampak makin ke timur dari

kawasan barat Indonesia menunjukkan adanya peningkatan frekuensi, yaitu dari

kawasan barat Indonesia sebesar 12,2%, pada populasi kawasan tengah Indonesia

19,1% dan pada populasi kawasan timur Indonesia tampak paling tinggi yaitu 42,1%.

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Genotip apoE ε4/ε4 tidak ditemukan dari 82 subjek populasi Surabaya yang diperiksa,

sedangkan pada populasi Palu dan Alor frekuensinya hampir sama yaitu 2,9% dan

2,2%. Frekuensi alel ε2, ε3, dan ε4 pada populasi Surabaya masing-masing sebesar

20,7%, 70,1% dan 9,2%; populasi Palu masing-masing sebesar 24,3%, 61,7% dan

14%, dan populasi Alor masing-masing sebesar 26,7%, 46,6% dan 26,7%. Frekuensi

alel apoE masing-masing populasi ini setelah dihitung dengan persamaan Hardy-

Weinberg tidak menunjukkan perbedaan bermakna (p>0,05). Frekuensi alel ε2 dan ε4,

menunjukkan kecenderungan ke arah timur Indonesia semakin naik, berkebalikan

dengan frekuensi alel ε3 yang semakin menurun atau rendah ke arah timur Indonesia.

Frekuensi genotip apoE antara populasi Surabaya dan Palu terdapat perbedaan

bermakna hanya pada genotip apoE ε3/ε3 saja (Chi square, p>0,05). Frekuensi

genotip antara populasi Surabaya dan Palu jika dibandingkan dengan populasi Alor

terdapat perbedaan bermakna pada genotip apoE ε3/ε3 dan ε3/ε4 (p<0,05). Frekuensi

genotip ε4/ε4, oleh karena dari populasi Surabaya tidak ditemukan genotip ini,

sehingga tidak dapat dibandingkan dengan populasi lainnya. Frekuensi genotip ε4/ε4

antara populasi Palu dengan Alor tidak menunjukkan perbedaan bermakna (p>0,05).

Pembawa alel apoε pada semua populasi yang diperiksa, tampak tidak terdapat

perbedaan bermakna pada frekuensi alel ε2 untuk ketiga populasi (p>0,05). Frekuensi

alel ε3 dan ε4 antara populasi Surabaya dan Palu jika dibandingkan dengan populasi

Alor menunjukkan perbedaan bermakna (p<0,05).

Polimorfisme apoE yang dihubungkan dengan faktor risiko timbulnya PJK

pada Surabaya dan Palu, tampak pembawa genotip apoE ε3/ε4 dan pembawa alel �4

mempunyai risiko sebagai penyebab terjadinya PJK.

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Pembahasan

Polimorfisme apoE dihubungkan dengan risiko timbulnya penyakit jantung

koroner, dibandingkan frekuensi gena apoE pada penderita PJK dengan kontrol.

Genotip ε2/ε2 dan alel ε2 pada penelitian ini tampak memiliki sifat proteksi terhadap

timbulnya PJK dan genotip ε3/ε4 serta alel ε4 merupakan faktor risiko terjadinya PJK.

Peran polimorfisme apoE terhadap timbulnya PJK di kawasan barat dan tengah

Indonesia dengan populasi lainnya di dunia hampir sama, yaitu pembawa alel ε4

mempunyai risiko lebih besar untuk menderita PJK dibanding pembawa alel ε2 dan ε3

(Eichner et al., 2002; Elousa et al., 2004; Mahley et al., 2006; Mc. Neale et al.,

2000; Pirim et al., 2001). Pembawa alel ε4 ini juga mempunyai risiko lebih besar

untuk menderita infark miokard, aterosklerosis, stroke, neurodegenerative (Elousa et

al., 2004; Frikke-Schmidt et al., 2000 (a); Guera et al., 2003; Leshinsky-Silver et al.,

2006; Mahley et al., 2006; Masemola et al., 2007; Moghadasian et al., 2001; Sheehan

et al., 2000; Sima et al., 2006; Yang et al., 2004). Area dengan prevalensi alel ε4 lebih

tinggi, lebih tinggi pula insidensi penderita penyakit jantung iskemiknya dan

determinan genetik ini dapat dihubungkan dengan mortalitas pada populasi yang

relatif terisolasi (Garces et al., 2004). Beberapa penelitian menunjukkan hasil yang

berbeda yaitu alel ε4 bukan merupakan faktor risiko terjadinya PJK ditemukan pada

populasi China (Liu et al., 2003). Penelitian lain yang membandingkan penderita

penyakit arteria koronaria terhadap kontrol di Yunani, Oman dan Brazilia, genotip

apoE4-4 tidak berperan dalam penyebab timbulnya penyakit tersebut (Al-Yahyaee et

al., 2007; De Franca et al., 2004; Kolovou et al., 2002; Souza et al., 2007)

Peran polimorfisme apoE terhadap timbulnya dislipidemia dibandingkan

antara penderita PJK yang mengalami dislipidemia dibanding kontrol. Pembawa gena

ε2 yang mempunyai kadar trigliserida yang tinggi bukan merupakan faktor risiko

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timbulnya PJK. Pembawa gen ε3 dan ε4 yang mengalami dislipidemia merupakan

faktor risiko timbulnya PJK. Profil lipid darahnya jika dikendalikan, pembawa gen ε4

merupakan faktor risiko timbulnya PJK. Peran polimorfisme apoE jika dibandingkan

pada populasi Indonesia dengan populasi lainnya di dunia, hasilnya hampir sama yaitu

pembawa alel ε2 memiliki faktor proteksi terhadap timbulnya PJK walaupun

mengalami dislipidemia (Chaaba et al., 2009; Chanprasetyothin et al., 2000; Eichner

et al., 2002; Elousa et al., 2004; Mahley et al., 2006; Masemola et al., 2007;

Moghadasian et al., 2001; Rodsari et al., 2002; Sheehan et al., 2000; Yang et al.,

2004; Zannis et al., 1996). Beberapa penelitian lain menunjukkan hasil yang berbeda

yaitu pembawa alel ε2 berhubungan dengan naiknya kadar trigliserida dan berperan

terhadap timbulnya hiperlipoproteinemia tipe III (Batal et al., 2000; Bennet et al.,

2007; Eichner et al., 2002; Letonja et al., 2004; Liberopoulos et al., 2004; Pallaud et

al., 2001). Pembawa alel ε4 pada populasi Indonesia barat dan tengah dibanding

populasi lainnya di dunia hasilnya sebagian besar sama yaitu pembawa alel ε4

mempunyai risiko dislipidemia lebih besar dibanding bukan pembawa alel ε4 (Al-

Yahyaee et al., 2007; Chaaba et al., 2009; Eichner et al., 2002; Elousa et al., 2004;

Frikke-Schmidt et al., 2000 (a) ; Guera et al., 2003; Hanon et al., 2000; Liu et al.,

2003; Mahley et al., 2006; Masemola et al., 2007; Mc Neale et al., 2000;

Moghadasian et al., 2001; Pirim et al., 2001; Rodsari et al., 2002; Saidi et al., 2007;

Sheehan et al., 2000; Sima et al., 2006; Tan et al., 2003; Yang et al., 2004; Zannis et

al., 1996). Penelitian lain juga menunjukkan adanya hubungan polimorfisme apoE

mempunyai variabilitas profil lipid lebih besar maupun penyakit metabolik (Eichner et

al., 2002; Fuzikawa et al., 2008; Pallaud et al., 2001).

Pengaruh polimorfisme apoE terhadap dislipidemia disebabkan di antaranya

plasma dari seseorang dengan apoE3 dapat menerima kolesterol lebih banyak dari

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fibroblas dibanding plasma seseorang dengan homosigot E2 dan E4 (Huang et al.,

2009). Apolipoprotein E3 terikat lebih baik dibanding apoE4 pada HDL3, domain

ujung C pada apoE4 strukturnya lebih tidak teratur dan lebih terekspose ke lingkungan

air dibanding apoE3, domain ujung C tersusun berbeda antara apoE3 dan apoE4;

perbedaan ini menyebabkan rangkaian patologis penyakit kardiovaskular dan

neurodegenerative (Sakamoto et al., 2008). Hasil ini menunjukkan bahwa faktor

genetik dan kadar lipid bervariasi sesuai dengan konteksnya yaitu umur, jenis kelamin

dan adanya perbedaan faktor lingkungan (Pallaud et al. 2001). Penelitian yang

dilakukan Eichner et al. (2002) dalam menurunkan profil lipid menunjukkan

pemberian obat yang menghambat enzim HMG-CoA reduktase maupun obat yang

mengikat asam empedu efektif untuk menurunkan profil lipid pada pembawa gen ε2

dan ε3, sedangkan pembawa gen ε4 tidak mudah dipengaruhi oleh intervensi medis.

Diet rendah lemak dan kolesterol menginduksi penurunan kolesterol dan LDL lebih

besar pada pembawa gen ε4 dibanding pembawa gen ε2 dan ε3. Hal ini menunjukkan

bahwa adanya polimorfisme apoE merespon obat hipolipidemiak berbeda. Pemberian

fenofibrat menunjukkan bahwa alel ε2 mempunyai penurunan lebih kecil kadar

trigliserida dibanding bukan pembawa alel ε2 (Irvin et al., 2010). Alel ε4 karena defek

pada proteinnya, efikasi dalam pengikatan dan transport lipid mengalami penurunan.

Obat statin untuk menurunkan profil lipid tidak memberikan respon pada beberapa

orang karena besarnya variabilitas respon obat penurun lipid termasuk pengaruh

genotip apoE. Oleh karena itu mendeteksi variasi genetik yang mempengaruhi kadar

lipoprotein plasma dapat membantu memprediksi respon terapetik (Morrison, 2007).

Alel ε4, secara konsisten menunjukkan penurunan kadar kolesterol total yang lebih

rendah secara signifikan setelah terapi obat penurun lipid pada penelitian di Portugis

(Withers, 2011). Efek intervensi terapetik yang memodifikasi penyakit yang

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tergantung apoE, pembawa gen ε4 mempunyai efek paling tidak baik (Cacabelos et

al., 2010).

Hasil penelitian tentang polimorfisme apoE pada tiga populasi yaitu populasi

Surabaya, Palu dan Alor, frekuensi alel ε2 antara ketiga populasi tersebut masing-

masing 0,207, 0,243 dan 0,267. Frekuensi alel ε3 di Surabaya, Palu dan Alor masing-

masing 0,701, 0,617 dan 0,466 dan frekuensi alel ε4 populasi Surabaya, Palu dan Alor

masing-masing 0,092, 0,14 dan 0,267. Frekuensi alel apoE baik ε2, ε3 maupun ε4

antara populasi Surabaya dan Palu dengan chi square tidak berbeda bermakna.

Frekuensi alel ε3 dan alel ε4 antara populasi Surabaya dengan Alor dan Palu dengan

Alor berbeda bermakna.

Frekuensi apoE antara populasi Indonesia jika dibandingkan dengan populasi

lain di dunia, frekuensi alel ε3 adalah paling besar dibanding alel ε2 dan ε4, (0,466-

0,701) sama dengan populasi dunia lainnya. Alor yang merupakan daerah yang relatif

terisolir, mempunyai frekuensi alel ε3 paling rendah dibanding dengan populasi

Indonesia lainnya, namun frekuensi alel ε3 nya (0,466) hampir sama dengan populasi

Papua New Guinea (0,49)(Siest et al., 1995). Kedua populasi ini termasuk pada

kelompok populasi Melanesia. Frekuensi alel ε2 di Indonesia (0,207-0,267) lebih

tinggi dibanding populasi lainnya di dunia, apalagi jika dibandingkan dengan daerah

yang mempunyai frekuensi 0 atau tidak ditemukan adanya alel ε2 seperti pada

kelompok orang asli Amerika sampai frekuensi 0,14 di beberapa populasi (Eichner et

al., 2002; Marin et al., 1997; Ricardo et al., 2000). Di negara-negara Eropa frekuensi

alel ε2 juga lebih rendah dibanding populasi Indonesia yaitu antara 0,02 – 0,119

(Becher et al., 2005; Ho et al., 2000; Kumar et al., 2002; Rodriques et al., 2005). Di

pedesaan Afrika, frekuensi alel ε2 ditemukan antara 0,031 sampai 0,19. Frekuensi ini

hampir sama dengan frekuensi di Surabaya sebesar 0,207 (Becher et al., 2005; Chaaba

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et al., 2005; Eichner et al., 2002; Willis et al., 2003). Populasi Asia lainnya, frekuensi

alel ε2 ini juga sangat bervariasi. Genotip apoE ε2/ε2, ε2/ε4 dan ε4/ ε4 tidak

ditemukan pada populasi Mongoloid (Yin et al., 2008), frekuensi alel apo ε2 sebesar

0,051 di Kuwait (Al Bustan et al., 2005), frekuensi alel ε2 0,075 di Taiwan (Lin et al.,

2004), Bangsa Han di China dengan frekuensi alel ε2 sebesar 0,193 (Yang et al.,

2004), India 0,039 - 0,043 (Singh et al., 2001; Thelma et al., 2001), populasi

Thailand sebesar 0,117 (Chanprasertyothin et al., 2000), dan pada populasi Malaysia

ditemukan frekuensi alel ε2 sebesar 0,14 (Gajra et al., 1994) dan 0,051 (Seet et al.,

2004). Frekuensi alel ε2 di Asia secara statistik juga menunjukkan penurunan

signifikan ke arah utara, dan alel ini tidak bertanggung jawab terhadap timbulnya

penyakit PJK (Sing et al., 2006). Frekuensi alel ε2 pada beberapa populasi di China

juga menunjukkan adanya heterogenitas dan hampir sama dengan populasi Jepang

antara 0,03 – 0,04 dan berbeda dengan Eropa dan Amerika (Kao et al., 1995)

Frekuensi alel ε3 populasi Indonesia yang berasal dari Surabaya, Palu dan

Alor timur masing-masing sebesar 0,701; 0,617; 0,466. Jika frekuensi alel ini

dibandingkan dengan populasi lainnya di dunia, frekuensi yang ditemukan pada

populasi Alor hampir sama dengan yang terdapat di Papua New Guinea sebesar 0,49

(Siest et al., 1995), Oceania sebesar 0,486 dan Afrika sebesar 0,536 (Eichner et al.,

2002). Frekuensi alel ε3 di Afrika pada beberapa penelitian juga terdapat variasi yang

besar dari 0,536-0,850 (Eichner et al., 2002; Chaaba et al., 2009; Becher et al., 2005;

Willis et al., 2003). Frekuensi alel ε3 pada populasi Amerika pada umumnya lebih

besar dibanding populasi dunia lainnya yaitu antara 0,720- 0,911 (Eichner et al., 2002;

Ricardo etal., 2000). Frekuensi alel ε3 pada populasi Eropa antara 0,640 – 0,898

(Becher et al., 2005; Eichner et al., 2002; Frikke-Schmidt et al., 2000 (b); Ho et al.,

2000; Kumar et al., 2002; Luccote et al., 1997; Rodriques et al., 2005; Willis et al.,

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2003). Frekuensi alel ε3 pada populasi Asia bervariasi, antara 0,60 – 0,913 (Bennet et

al., 2007; Bustan et al., 2005; Chanprasertyothin et al., 2000; Eichner et al., 2002;

Liberopoulos et al., 2004; Lin et al., 2004; Rodsari et al., 2002; Seet et al., 2004; Siest

et al., 1995; Sing et al., 2001; Thelma et al., 2001; Willis et al., 2003, Yang et al.,

2004). Frekuensi alel ε3 populasi Indonesia barat dan tengah, jika dibandingkan

dengan populasi Asia lainnya tidak berbeda, namun frekuensi alel ε3 dari kawasan

timur Indonesia, tampak berbeda dengan populasi Asia pada umumnya. Data ini

menunjukkan bahwa kawasan barat dan tengah Indonesia hampir sama dengan

populasi Asia yang termasuk ras Mongoloid, sedangkan kawasan timur Indonesia

lebih cenderung sama dengan populasi Melanesia lainnya seperti populasi New

Guinea yang frekuensi alel ε3 nya hampir sama dengan yang ditemukan pada populasi

Alor.

Frekuensi alel ε4 pada populasi Surabaya sebesar 9,2%, populasi Palu sebesar

14% dan Alor sebesar 26,7%. Frekuensi alel ε4 pada populasi Alor tampak paling

besar dan berbeda bermakna dengan dua populasi dari Surabaya dan Palu. Frekuensi

alel ε4 yang tinggi pada populasi Alor dan berbeda dibanding Surabaya dan Palu ini

karena populasi ini cenderung sama dengan ras Mongoloid, sedangkan Alor

cenderung masuk kelompok ras Melanseid. Populasi Afrika Selatan, Papua New

Guinea, Aborigin Australia dan Kulit hitam Afrika (Nigeria, Sudan) mempunyai

frekuensi tinggi alel ε4. Frekuensi alel ε4 pada populasi Wayampi Perancis sejauh ini

menunjukkan frekuensi paling tinggi (0,423 ) diperkirakan karena terisolasinya

populasi ini dan adanya drift genetik. Alel ε2 yang tidak ada dan dengan tingginya

frekuensi alel ε4 pada populasi Wayampi Perancis dapat berpengaruh pada prevalensi

penyakit PJK pada populasi tersebut, suatu keadaan yang harus diamati pada waktu

mendatang (Marin et al., 1997). Frekuensi alel ε4 di Sardinia mencapai 0,40 (Eichner

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et al., 2002). Frekuensi alel ε4 yang tinggi pada populasi Indonesia timur ini hampir

sama dengan yang ditemukan pada populasi Finlandia dan Swedia sebesar 0,20;

penduduk asli Amerika dengan frekuensi sebesar 0,28 (Becher et al., 2005, Eichner et

al., 2002), di pedesaan Afrika dan Tunisia sebesar 0,293 (Masemola et al., 2007,

Chaaba et al., 2009). Populasi yang mempunyai frekuensi alel ε4 yang rendah di dunia

ditemukan di Taiwan sebesar 0,05 (Corbo & Scacchi, 1999), di Eropa selatan 0,08 –

0,12 (Becher et al., 2005, Rodriques et al., 2005); di Kuwait dengan frekuensi 0,065

(Al-Bustan et al., 2005). Frekuensi alel ε4 yang ditemukan pada populasi Indonesia

barat juga termasuk rendah yaitu sebesar 0,092. Frekuensi alel ε4 pada populasi Asia

yang ditemukan bervariasi, Kuwait dengan frekuensi sebesar 0,065 (Masemola et al.,

2007), Taiwan sebesar 0,05 – 0,79 (Lin et al., 2004, Siest et al., 1995), pada populasi

Uygur dan populasi Han masing-masing sebesar 0,197 dan 0,146 (Yang et al., 2004),

beberapa etnik di Malaysia dengan frekuensi alel ε4 sebesar 0,114 (Seet et al., 2004),

beberapa kelompok etnik di India frekuensinya sebesar 0,043 – 0,071 (Singh et al.,

2001; Thelma et al., 2001), dari populasi Thailand ditemukan frekuensi alel ε4

sebesar 0,09. Frekuensi alel ε4 dari beberapa populasi di Asia ini tampak sangat

bervariasi, populasi Indonesia timur termasuk mempunyai frekuensi tinggi dan

berbeda dengan frekuensi yang ditemukan pada beberapa populasi di Asia yang

menunjukkan bahwa Indonesia timur cenderung termasuk kelompok ras Melanesid.

Hasil dari penelitian ini dapat disimpulkan:

1. Dislipidemia merupakan faktor risiko terjadinya PJK. Pembawa genotip apoE ε3/ε4

dan alel ε4 merupakan faktor risiko terjadinya PJK, sedangkan pembawa genotip

apoE ε2/ε2 dan alel ε2 merupakan faktor protektif terhadap timbulnya PJK.

2. Adanya polimorfisme apoE menyebabkan terjadinya variabilitas kadar trigliserida

namun tidak menyebabkan terjadinya variabilitas profil lipid yang lain.

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3. Terdapat perbedaan distribusi genotip dan alel apoE antara populasi kawasan barat

Indonesia, kawasan tengah Indonesia dan kawasan timur Indonesia.

4. Genotip apoE ε3/ε4 dan alel ε4 merupakan faktor risiko PJK pada populasi

Indonesia barat dan tengah.

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SUMMARY

Apolipoprotein E is a protein constituent of plasma lipoproteins that performs

several functions including a role in cholesterol metabolism and as a ligand important

in lipoprotein clearance. Apolipoprotein E was first identified as a constituent of very

low density lipoprotein (VLDL) which have function to transport triglycerides from

the liver to peripheral tissues (Mayes & Botham, 2006). Apolipoprotein E gene is

mainly expressed in the liver (90%), but also in other tissues, including brain, spleen,

lungs, gonads, adrenal, ovarium, kidney and muscle. Mature macrophages derived

from human monocytes also produce significant amounts of apoE, which could

account for up to 10% of the circulating protein. Apolipoprotein E acts as a high

affinity ligand for several hepatic lipoprotein receptors i.e. LDL receptor, LDL

receptor-related protein (LRP), VLDL receptor, apoE receptor and the scavenger

receptor type 1 class B (SRB-I). This glycoprotein mediates the clearance of apoE-

containing lipoproteins i.e. chylomicrons, VLDL, IDL, and HDL. It is also found at

very low levels in LDL and soluble in the plasma (Mahley et al.,1999; Mc Neale et

al., 2000, Moghadasian et al., 2001, Zechner et al., 1991). One of the metabolic

functions of apoE is to transport cholesterol from peripheral tissue to the liver for

degradation, a process called reverse cholesterol transport. In addition, apoE

modulates the activity of several enzymes involved in the lipid metabolism (Sima et

al., 2006).

Apolipoprotein E gene is polymorphic and exists in six different isoprotein

forms, designated E2/E2, E2/E3, E2/E4, E3/E3, E3/E4, and E4/E4 which are the gene

products of three ApoE allele ε2, ε3 and ε4 respectively (Belkovets et al., 2001). The

strongest relationship between apoE levels and cardiovascular mortality was seen in

patients who had low CRP levels at age 85. In most of these patients, CRP levels rose

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in the following years, suggesting that high ApoE levels come before an increase in

inflammation. The biological activity of ApoE can be influenced by modification of

its structure and/or quantity. A structural alteration arises from the two common ApoE

polymorphisms, encoding ApoE2, ApoE3, and ApoE4, respectively. Apolipoprotein

E2 exhibits lower affinity for the LDL receptor, resulting in slower clearance of ApoE

and higher plasma apoE levels. In response, the liver up-regulates the LDL receptor,

resulting in lower cholesterol levels. Conversely, ApoE4 is cleared more efficiently,

resulting in lower ApoE levels and higher cholesterol levels. The genetic variations

thus affect lipid metabolism and have been shown to alter risk of cardiovascular

disease and dementia. Plasma apoE levels are only partially explained by the 2/3/4

polymorphism, and plasma apoE levels vary between individuals with the same apoE

genotype. Irrespective of apoE genotype, plasma apoE levels are also associated with

cholesterol levels. Moreover, it was shown recently that apoE mediates the

presentation of lipid antigens to the immune system and in this way influences the

inflammatory process. Both lipids and inflammation are involved in the pathogenesis

of atherosclerosis, but the relation of plasma apoE levels and cardiovascular disease

risk has not yet been reported (Eichner et al., 2002, Mahley et al.,1999, Souza et al.,

2007).

The frequencies of apoE alleles differ significantly among diverse ethnic

groups. Intensive investigation has established that allele variation in the apoE gene

has a significant effect on interindividual variation in plasma lipid and lipoprotein

levels and on risk of cardiovascular disease in the general population (Everaldo et al.,

2004). Among the variants the allele ε3 is the most frequent (>60%) in all population

studied (Eicner et al., 2002).

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Apolipoprotein E is a member of apolipoprotein gene family. Other members

of this multigene family include apoA-I, apoA-II, apoA-IV, apoC-I, apoC-II and

apoC-III. The Apo ε gene is located at chromosome 19q13.2 and is closely linked to

the apoC-I/C-II gene complex. It consists of four exons and three introns spanning

3,597 nucleotides and produces a 299 amino acid polypeptide (Eichner et al., 2002).

Apolipoprotein E, similar to other apolipoproteins, helps to stabilize and

solubilize lipoproteins as they circulate in the blood. In general, the role of

apolipoproteins in lipid merabolism includes maintaining the structural integrity of

lipoproteins, serving as cofactors in enzymatic reactions, and acting as ligands for

lipoprotein receptors. Apolipopfrotein E is critical in the formation of very low

density lipoprotein (VLDL) and chylomicrons (Corella et al., 2002; Eichner et al.,

2002, Moghadasian et al., 2001).

Epidemiologic studies addressing the contribution of apo ε to CHD, reported

that 6 percent of the variation in risk for CHD in North America can be attributed to

this locus. Another study of middle-aged men from nine populations estimated a 40

percent increased risk for CHD mortality for ε4 carriers compared with ε3 carriers or

ε2 carriers (Stengard et al., 1998). Some studies have also suggested that ε4 carriers

are particularly prone to developing disseminated coronary lesions or to have an

increased risk of death from CHD (Eichner et al., 1993, Lehtinen et al., 1995;

Stengard et al., 1995, Wang et al., 1995). Coronary heart disease is related to

dysfunction of the E4 isoform in lipoprotein metabolism and an increased

concentration of serum cholesterol and triglycerides. Studies from Finland, Scotland,

and northern of Ireland have shown that populations with higher cholesterol levels and

higher CHD mortality rates also have a higher frequency of the ε4 allele. Other studies

 

 

 

 

 

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have also associated the ε2 allele with increased CHD risk (Eichner et al., 2000,

Zannis et al., 1996, Mahley et al., 2006).

An association between apoE ε2/ε2 and type III hyperlipoproteinemia has been

known for decades. This disorder is characterized by increased cholesterol and

triglyceride levels, the presence of ß-VLDL (cholesterol-enriched remnants of

intestinal chylomicrons and hepatic VLDL), xanthomas, and premature vascular

disease, both CHD and peripheral artery disease (Mahley et al., 1995). Overt

hyperlipoproteinemia III occurs with a frequency of 1–5 per 5,000, whereas

homozygosity for E2/2 occurs with a frequency of 0.5–1.0 per 100 in Caucasian

populations. Thus, this genotype contributes to the hyperlipoproteinemia III phenotype

without being its sole cause (Eichner et al., 2002, Fullerton et al., 2000).

Indonesia is a country in Southeast Asia and Oceania, comprises 17,508

islands. These are scaterred over both sides of the equator. Indonesia’s size, tropical

climate and archipelagic geography, support the world’s second highest level of

biodiversity (after Brazil), and its flora and fauna is a mixture of Asian and

Australasian species. Once linked to the Asian mainland, the islands of the Sunda

Shelf (Sumatra, Java, Borneo, and Bali) have a wealth of Asian fauna. Sulawesi, Nusa

Tenggara, and Maluku, having been long separated from the continental landmasses-

have developed their own unique flora and fauna. Papua was part of the Australian

landmass, and is home to a unique fauna and flora closely related to that Australia,

including over 600 bird species (van Oosterze, 1997).

The British naturalist, Alfred Wallace, described a dividing line between the

distribution and peace of Indonesia's Asian and Australasian species. Known as the

Wallace Line, it runs roughly north-south along the edge of the Sunda Shelf, between

Kalimantan and Sulawesi, and along the deep Lombok Strait, between Lombok and

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Bali. West of the line the flora and fauna are more Asian; moving east from Lombok,

they are increasingly Australian. This differences is characteristic by different marker

genetic in their populations (van Oosterze, 1997).

Research by Sofro, (1982), on the population genetic of Indonesia showed

clinal pattern in the distribution some enzyme across the Indonesia archipelago. There

were trend of increasing Glutamic-pyruvic Transaminase1 (GPT1) also

Phosphogluconate DehydrogenaseC (PGDc) gene frequencies and of decreasing

Glyoxylase1 (GLO1) gene frequency towards the east. This pattern was found also in

β-Thalassemia frequency in Indonesia i.e. 9% in Palembang (Sofro et al., 1994), 8%

in Makassar (Sofro et al., 1994), 6% in Maumere, Flores (Sofro et al., 1993) and 6%

in Ambon, Maluku (Sofro et al., 1994). Contrary with Hb E, haptoglobin-1 (Hp1) and

glucose-6-phosphate dehydrogenase (G6PD) deficiency, low frequency was observed

in the western part of Indonesia and higher in the eastern part of Indonesia. Phenotype

of Transferrin in the western part of Indonesia was D-chi type as Mongoloid gene

marker and in the eastern part of Indonesia is Transferrin D1, the Melanesid gene

marker. Lanni (2002) showed Indonesia consisted of three clusters; first cluster was

Indonesian population with Mongoloid gene pool, second is mixed Mongoloid and

Australomelanosid and the third was cluster with Australomelanosid gene pool.

Considering the existence of three different gene pools in Indonesia i.e.

Mongoloid, mixed Mongoloid-Australomelanosid and Australomelanosid in the

western, middle and eastern region of Indonesia respectively, this study examines

polymorphism of apoE genotype in three ethnics of Indonesia accordingly. In

addition, it examines the relationship of this genotype in coronary heart disease

(CHD) patients compared to the specified controls.

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Blood samples were collected from three group of Indonesian populations

and from CHD patients in Dr. Sardjito Hospital Yogyakarta compared with controls

collected from an exersice group in Yogyakarta. Plasma from patients and controls

were separated and the lipid levels were examined. Buffy coat from patients, controls

and three population groups were prepared for DNA isolation. Polymerase Chain

Reacrion (PCR) was performed with Zivelin et al., (1997) method to amplify exon 4

and then followed by HaeII dan Afl IIIdigestion to identify the ε2, ε3 and ε4 allele

with 195 and 23 bp for ε4; 23, 50 and 145 bp for ε3 and 50 and 168 bp for ε2

respectively.

The result were analyzed to:

a. Compare lipid profile in CHD patients and controls with t-test. Significant rate

was applied for p <0,05; and Odd Ratio was used to determine the risk factors

b. Compare the frequency of apoE genotype, and allele as risk factor of CHD with

Pearson correlation test.

c. Compare the apoE genotype and allele as risk factor of CHD by Odd Ratio with

controlled lipid profile.

d. Calculate the genotype frequency

e. Calculate the ε allele frequency

f. Compare the frequency of genotype, genotype carrier and the allele of apoE in

population. Chi square was tested with significant different p < 0,05.

g. Compare the frequency of apoE genotype and allele in CHD patients with

populations as risk factor employing chi square and Odd Ratio.

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Results

Apolipoprotein E polymorphism was examined in 33 CHD patients and 38

controls. There were no significant difference between patients and controls in the

body weight, height, BMI, blood pressure and blood glucose with t-test p>0.05. There

were no significant difference between CHD patients and controls in their lipid profile

(p>0.05) despite higher levels of triglyceride, cholesterol, and LDL-C higher and

lower level of HDL-C in CHD patients. When Chi square analysis was employed,

higher frequency of dyslipidemia in CHD patients was observed with significant

difference (p<0.05) in cholesterol concentration. Odd Ratio statistic showed that

dyslipidemia condition was a strong risk factor for CHD with 1.4 – 3.93 times higher.

From the view point of apolipoprotein E polymorphism, despite no significant

difference in lipid profile it was shown that apoE ε2/ε2 genotype was related with

higher level of triglyceride and cholesterol. Other genotypes were not related with

lipid profile.

The frequency of apoE ε2/ε2, apoE ε2/ε3, apoE ε3/ε3, and apoE ε3/ε4

genotypes in CHD patients were 3.1%, 18.1%, 42.4% and 36.4%, respectively and in

controls were 10.5%, 13.2%, 60.6%, and 15.7% respectively. ApoE ε2/ε4 and apoE

ε4/ε4 genotypes were not found in CHD patients. Comparison was not possible with

controls, since apoE ε2/ε4 and apoE ε4/ε4 genotypes were not found in CHD

patients. Significant difference (p<0.05) was only observed in apoE ε3/ε4 genotype

with OR 4.86 times higher than other genotypes between CHD patients and controls.

Apolipoprotein E ε2/ε2 genotype in CHD patients showed OR < 1, indicating that

apoE ε2/ε2 genotype was protective factor for CHD.

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On the other hand ε4 allele although p>0.05 but have OR 2.05 as risk factor for

CHD. It can be summarized that apoE ε3/ε4 genotype and ε4 allele were risk factor

for CHD, while apoE ε2/ε2 genotype and ε2 allele were protective factors for CHD.

Being as risk factor for CHD, dyslipidemic condition especially the increase of

cholesterol and LDL-C as well as the decrease of HDL-C levels were risk factors for

CHD for all of apoE genotypes. In contrast, the increase of triglyceride level in ε2

carrier gene was not the risk factor for CHD. Apolipoprotein E polymorphism plays a

role as risk factor for CHD by controlling lipid profiles. The ε2 carrier gene was not

the risk factor for CHD, but ε4 carrier gene was the risk factor for CHD with OR 3.94

times higher.

Regarding apoE polymorphism in Indonesian populations, 195 blood samples

were collected from three populations consisting of 85 samples from Surabaya

(western part of Indonesia), 68 samples from Palu (middle part of Indonesia) and 45

samples from Alor (eastern part of Indonesia). The frequency of apoE ε2/ε2 genotypes

were 15.6%, 8.5% and 7.4% in Alor, Surabaya and Palu respectively. Frequency of

apoE ε2/ε3 genotype were 30.9%, 18.3% and 15.6% in Palu, Surabaya and Alor

respectively. Frequency of apoE ε3/ε3 genotype were 54.9%, 36.8% and 17.8% in

Surabaya, Palu and Alor respectively. Frequency of apoE ε2/ε4 genotype were 6.1%,

6.7% and 2.9% in Surabaya, Palu and Alor respectively. The frequency of apoE ε3/ε4

genotype in Surabaya population was the lowest and tend to increase toward the east

i.e. 12.2% in Surabaya, 19.1% in Palu and 42.1% in Alor. The apoE ε4/ε4 genotype

was peculiar, it was not found in Surabaya but it was found 2.9% and 2.2% in Palu

and Alor populations respectively. The frequency of ε2, ε3, and ε4 alleles in Surabaya

were 20.7%, 70.1% and 9.2% respectively; 24.3%, 61.7% and 14% respectively in

Palu and 26.7%, 46.6% and 26.7% respectively in Alor. These result were not

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different with Hardy-Weinberg equilibrium (p>0.05). The frequency ε2 and ε4 alleles

tend to increase also from the western part of Indonesia towards the east, contrary to

the ε3 allele which tend to decrease from the west towards the east. Chi square

analysis of apoE genotype, for Surabaya and Palu populations, showed significant

difference only for apoE ε3/ε3 genotype (p<0.05). The frequency of apoE ε3/ε3 and

apoE ε3/ε4 genotypes were significantly different for Surabaya and Alor populations

as well as for Palu and Alor populations (p<0.05). The apoE ε4/ε4 genotype was not

found in Surabaya populations, so it was not possible to compare with other

populations. However, apoE ε4/ε4 genotype was not significantly different (p>0.05)

between Palu and Alor populations.

When apoε alleles for all Indonesian populations were compared, there were

no significant difference for ε2 allele (p>0.05). Significant difference in ε3 and ε4

alleles were found between Surabaya and Alor populations as well as between Palu

and Alor populations (p<0.05).

Regarding apolipoprotein E polymorphism as risk factor for CHD it was found

that apoE ε3/ε4 genotype and ε4 allele were the risk factor for CHD in Surabaya and

Palu populations.

Discussion

To demonstrate the role of ApoE polymorphism as the risk factor for CHD,

the frequency of ApoE gene in CHD patients was compared to its frequency in various

ethnic groups. It was shown that ε2 allele was a protective factor for CHD and ε4

allele was a risk factor for CHD. This risk factor was not different in other world

populations, in which ε4 gene carrier was the risk factor for CHD than those ε2 and

ε3 gene carriers (Eichner et al., 2002; Elousa et al., 2004; Mahley et al., 2006; Mc.

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Neale et al., 2000; Pirim et al., 2001). This ε4 gene carriers was also the risk factor for

infark myocard, atherosclerosis, stroke, and neurodegenerative (Elousa et al., 2004;

Frikke-Schmidt et al., 2000 (a); Guera et al., 2003 ; Leshinsky-Silver et al., 2006;

Mahley et al., 2006; Masemola et al., 2007; Moghadasian et al., 2001; Sheehan et al.,

2000; Sima et al., 2006; Yang et al., 2004). Population with high frequency of ε4

allele have high incidence of ischemic heart disease and this determinant genetic was

related with mortality in isolated populations (Garces et al., 2004). Different result

were found in China populations, polymorphism of ε4 gene carriers was not risk

factor for CHD (Liu et al., 2003), as well as in coronary artery disease, this ε4 gene

carriers was not risk factor in Oman, Greek and Brazalian populations (Al-Yahyaee et

al., 2007; De Franca et al., 2004; Kolovou et al., 2002; Souza et al., 2007)

The role of apoE polymorphism in causing dyslipidemic condition, was studied in

CHD patients and controls. Apolipoprotein ε2 allele has protective effect for CHD,

but ε3 and ε4 alleles were the risk factor for CHD especially if someone has

dyslipidemic conditions. The role of apoE polymorphism on dyslipidemia in

Indonesia population seems to be almost similar to that in the world’s populations in

which the ε2 allele was a protective factor for CHD despite suffering from

dyslipidemic (Chaaba et al., 2009; Chanprasetyothin et al., 2000; Eichner et al., 2002;

Elousa et al., 2004; Mahley et al., 2006; Masemola et al., 2007; Moghadasian et al.,

2001; Rodsariet al., 2002; Sheehan et al., 2000; Yang et al., 2004; Zannis et al.,

1996). Other studies reported that ε2 allele was related with high triglyceride level and

the incidence of type III hyperlipoproteinemia (Batal et al., 2000; Bennet et al., 2007;

Eichner et al., 2002; Letonja et al., 2004; Liberopoulos et al., 2004; Pallaud et al.,

2001). Similar result with other world’s populations was also found in western and

the middle part of Indonesia, in which ε4 allele was a risk factor for dyslipidemia than

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other alleles (Al-Yahyaee et al., 2007; Chaaba et al., 2009; Eichner et al., 2002;

Elousa et al., 2004; Frikke-Schmidt et al., 2000 (a) ; Guera et al., 2003; Hanon et al.,

2000; Liu et al., 2003; Mahley et al., 2006; Masemola et al., 2007; Mc Neale et al.,

2000; Moghadasian et al., 2001; Pirim et al., 2001; Rodsari et al., 2002; Saidi et al.,

2007; Sheehan et al., 2000; Sima et al., 2006; Tan et al., 2003; Yang et al., 2004;

Zannis et al., 1996). Other studies also found the relation of apoE polymorphism with

variability of lipid profile and metabolic diseases (Eichner et al., 2002; Fuzikawa et

al., 2008; Pallaud et al., 2001).

The role of apoE polymorphism in causing dyslipidemia is due to the ability of

apoE3 to accept more cholesterol from fibroblast than apoE2 and apoE4 (Huang et al.,

2009). In HDL3, apoE3 binds cholesterol better than apoE4, because the structure of

carbon end domain of apoE4 was irregular and more exposed to the water; these

differences causes the pathology of cardiovascular and neurodegenerative disease

(Sakamoto et al., 2008). These condition showed that genetic factors and lipid profile

varies with age, sex, and the diffferences of environmental factors (Pallaud et al.,

2001). Studies by Eichner et al., (2002), giving hypolipidemic drugs to block HMG-

CoA reductase or drugs to bind viles to reduce lipid profiles, was effective for apoE

ε2 and apoE ε3 gene carriers, but apoE ε4 gene carrier was difficult to be influenced

by medical intervention. Low lipid and cholesterol diets induce the decrease of

cholesterol and LDL levels higher in apoE ε4 gene carrier than apoE ε2 and apoE ε3

gene carriers. It was shown that response to hypolidemic drugs was different in apoE

polymorphism. Treatment with phenofibrate showed that �2 allele reduce lower in

triglyceride level than others (Irvin et al., 2010). The defect in E4 protein, causes

efficacy to bind and tranport of lipid was decrease. Statin drug to decrease lipid

profiles was not responded by some individual because of high response variability of

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hypolipidemic drugs. It can be summarized that the detection of genetic variability

that influence lipoprotein levels in the plasma support to predict therapeutic response

(Morrison, 2007). The ε4 allele is consistenly lower in reduced cholesterol level after

hypolipidemic therapeutic in Portugese (Winters, 2011). Regarding the effect of

therapeutic intervention to modify the disease related with apoE polymorphism, apoE

ε4 gene carrier had the worst effect (Cacabelos et al., 2010)

Research with Indonesian population that were from Surabaya, Palu and Alor

represent the western, middle and the eastern part of Indonesia, the frequency of ε2

allele for all these populations were 0.207, 0.243 and 0.267 respectively. The

frequency of ε3 allele in Surabaya, Palu and Alor populations were 0.701, 0.617 and

0.446 respectively and the frequency of ε4 allele for all of these population were

0.092, 0.14 and 0.267 respectively. The frequency of ε2, ε3 and ε4 in Surabaya was

not different with Palu population. The frequency of ε3 and ε4 alleles in Surabaya and

Alor populations were significant different as well as Palu and Alor populations

(p<0.05).

Compared with other population in the world, there were similarity with

Indonesian populations, the frequency of ε3 allele was the highest than ε2 and ε4 allele

frequencies (0.466-0.701). Alor which is relatively isolated populations of Indonesia

has ε3 allele lowest compared with other populations of Indonesia (0.466), but its ε3

allele frequency is almost equal to the Papua New Guinea populations (0.49)(Siest et

al., 1995). Both populations were included in the Austromelanesid population. The

frequency of ε2 allele in Indonesian populations (0.207-0.267) were higher than other

populations in the World, especially when compared with areas where ε2 allele was

not found as in American Native to 0.14 in other populations of America (Eichner et

al., 2002; Marin et al., 1997; Ricardo et al., 2000). In European population, ε2 allele

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frequency was lower than Indonesian population was between 0.02 – 0.119 (Becher et

al., 2005; Ho et al., 2000; Kumar et al.¸2002; Rodriques et al., 2005). In African

villages, ε2 allele frequency were 0.031 – 0.19. This frequency is nearly equal with

Surabaya populations at 0.207 (Becher et al., 2005; Chaaba et al., 2005; Eichner et al.,

2002; Willis et al., 2003). Other Asian populations, apoE ε2 allele was varies.

Genotypes of apoE ε2/ε2, apoE ε2/ε4 and apoE ε4/ε4 were not found in Mongoloid

populations (Yin et al. , 2008). The frequency of �2 allele was 0.051 in Kuwaiti

populations (Al Bustan et al., 2005), 0.075 in Taiwanese population (Lin et al.,

2004), 0.193 in Chinese Han populations (Yang et al., 2004), 0.039 – 0.043 in Indias

populations (Singh et al., 2001; Thelma et al., 2001), 0.117 in Thailand populations

(Chanprasertyothin et al., 2000), 0.14 and 0.051 in Malaysia populations (Gajra et al.

1994, Seet et al., 2004). The ε2 frequency in Asia also showed a statistically decrease

to the north and this allele was not a risk factor for CHD (Sing et al., 2006). There

were heterogeinity the ε2 allele in some populations in China and was similar to the

Japanese populations between 0.03 to 0.04 and different with European and American

populations (Kao et al., 1995)

Frequency of ε3 allele in some populations of Indonesian i.e. 0.701, 0.617,

and 0.466 in Surabaya, Palu and Alor respectively. This frequency if compared with

other populations in the world, the frequency found in Alor population is almost same

as those in Papua New Guinea populations at 0.49 (Siest et al., 1995), 0.486 in

Oceania populations and 0.536 in African population (Eichner et al., 2002). The ε3

allele frequencies in Africa varies from 0.536 to 0.850 (Becher et al., 2005; Chaaba et

al., 2009; Eichner, et al., 2002; Willis et al., 2003). The ε3 allele frequency in

American populations is higher than in other populations of the world i. e. between

0.720 to 0.911 (Eichner et al., 2002; Ricardo etal., 2000). The frequency of ε3 allele in

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European population was between 0.640 to 0.898 (Becher et al., 2005; Eichner et al.,

2002; Frikke-Schmidt et al., 2000 (b); Ho et al., 2000; Kumar et al., 2002; Luccote et

al., 1997; Rodriques et al., 2005, Willis et al., 2003). The frequency of ε3 allele in

Asian populations varied between 0.60 to 0.913 (Bennet et al., 2007; Bustan et al.,

2005; Chanprasertyothin et al., 2000; Eichner et al., 2002; Liberopoulos et al., 2004;

Lin et al. 2004; Rodsari et al., 2002; Seet et al., 2004; Sing et al., 2001; Siest et al.,

1995; Thelma et al., 2001; Willis et al., 2003, Yang et al., 2004). These ε3 allele

frequency in Surabaya and Palu populations were not different with other Asian

populations, but the frequency of ε3 allele from Alor population was different with

other Asian population. This result showed that Surabaya and Palu populations were

Mongoloid gene pool and Alor was Autromelanesid gene pool such as Papua New

Guinea populations.

Frequency of ε4 allele were 9.2%, 14% and 26.7% in Surabaya, Palu and Alor

populations respectively. The frequency of ε4 allele in Alor was the highest and

showed significant difference in two populations of Surabaya and Palu. The high

frequency of ε4 allele in Alor and different than Surabaya and Palu was because these

populations tend to be similar to the Mongoloid populations, while Alor tend to be

similar to the Austromelanesid populations. The populations of South Africa, Papua

New Guinea, Aborigin Australian and Negrito African (Nigeria, Sudan) showed high

ε4 allele frequency. Frequency of ε4 allele in Wayampi, France so far showed the

highest (0.423) estimated for the isolation of this populations and the presence of

genetic drift. The ε2 allele was not found and a high frequency of ε4 allele was shown

in French Wayampi populations, it was carefully noticed as a CHD risk factor in the

next decade (Marin et al., 1997). The ε4 allele frequency was 0.40 in Sardinia

(Eichner et al., 2002). The ε4 allele frequency in the eastern part of Indonesia was

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high and almost identical to those found 0.20 in populations at in Finland and

Swedden ; 0.28 in Native America’s (Becher et al., 2005, Eichner et al., 2002), 0.293

in rural of African and Tunisiana 0.293 (Chaaba et al., 2009; Masemola et al., 2007).

Low frequency of ε4 allele was found in Taiwan at 0.05 (Corbo & Scacchi, 1999), and

0.08 to 0.12 in southern Europe from 0.08 to 0.12 (Becher et al., 2005, Rodriques et

al., 2005). The frequency of ε4 allele in Surabaya was 0.092. Frequency of ε4 allele

varies, 0.065 in Kuwait (Al-Bustan et al., 2005), 0.05 to 0.079 in Taiwan (Lin et al.,

2004; Siest et al., 1995), 0.197 and 0.146 in Uygur and Han populations respectively

(Yang et al., 2004), 0.114 in soem ethnic of Malaysia (Seet et al., 2004), 0.043 to

0.071 in some ethnic of India (Singh et al., 2001; Thelma et al., 2001), 0.09 in

Thailand populations. The frequency of ε4 allele of some populations in Asia was

varied, including the eastern part of Indonesia was high and different with found in

some populations in Asian showed that eastern part of Indonesia was Melanesid

populations.

Conclusion :

1. Dyslipidemia was the risk factor for CHD. Apolipoprotein E ε3/ε4 genotype and

ε4 allele were the risk factor for CHD where as apoE ε2/ε2 genotype and ε2 allele

were protective factor for CHD.

2. Polymorphism of apoE lead to variability of triglyceride level but not to other

lipid profile.

3. There were differences in frequency of apoE genotypes and alleles in the western,

middle and eastern par ε t of Indonesia.

4. Apolipoprotein E ε3/ε4 genotype and ε4 allele were risk factor for CHD in the

western and middle populations of Indonesia.

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