APOLIPOPROTEIN E SEBAGAI FAKTOR

RISIKO TIMBULNYA PENYAKIT

J ANTUNG KORONER:
Kajian Genetika Populasi dan Genetika Klinis
pada Beberapa Kelompok Etnik
di Indonesia








Oleh
Pramudji Hastuti
NIM: 99/850/PS


UNIVERSITAS GADJ AH MADA
Yogyakarta
2012
i

APOLIPOPROTEIN E SEBAGAI FAKTOR RISIKO

TIMBULNYA PENYAKIT J ANTUNG 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, J awa Tengah

ii

157

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 2, 3 dan 4 (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 3
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
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 4 dibanding
pembawa 3 dan 2 (Stengard et al., 1998).

Beberapa penelitian lain menunjukkan
bahwa pembawa gena 4 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 4 yang lebih tinggi. Penelitian lain juga menunjukkan
adanya hubungan alel 2 dengan naiknya risiko PJK (Eichner et al., 2000; Mahley et
al., 2006; Zannis et al., 1996).
Apolipoprotein E 2/2 tampak berhubungan dengan hiperlipoproteinemia tipe
III (HLIII) dan telah diketahui pada satu dekade ini. Gangguan ini dicirikan oleh
naiknya kadar kolesterol, trigliserida dan -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- 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 2, 3 dan 4 (4
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dengan pita masing-masing 195 dan 23 pasangan basa; 3 dengan pita 23, 50 dan 145
pasangan basa dan 2 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 2 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 2/2, 2/3, 3/3, 3/4 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 2/4 dan 4/4 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 3/4. Pembawa genotip apoE 3/4 ini
mempunyai risiko untuk menjadi PJK 4,86 kali lebih besar dibanding pembawa
genotip lainnya. Pembawa genotip 2/2 mempunyai OR kurang dari 1. Hal ini
menunjukkan bahwa pembawa genotip apoE 2/2 merupakan faktor protektif
terhadap timbulnya PJK.
Frekuensi pembawa alel 2 pada penderita PJK dan kontrol tidak menunjukkan
perbedaan bermakna dengan OR kurang dari 1. Hal ini berarti bahwa alel 2
merupakan faktor protektif terhadap terjadinya PJK, sementara pembawa alel 4
mempunyai risiko 2,05 kali lebih besar untuk menderita PJK. Hal ini menunjukkan
bahwa pembawa alel 4 mempunyai risiko untuk menderita PJK lebih besar dibanding
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seseorang bukan pembawa alel 4. Dari hasil ini disimpulkan bahwa pembawa genotip
apoE 3/4 dan pembawa alel 4 mempunyai risiko lebih besar untuk menderita PJK
dan alel 2 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 2 bukan merupakan faktor risiko timbulnya PJK. Profil lipidnya jika
dikendalikan dan dihubungkan polimorfisme apoE dengan timbulnya PJK, tampak
pembawa gena 2 bukan merupakan faktor risiko timbulnya PJK, namun pembawa
gena 4 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
2/2 pada populasi Alor adalah 15,6%, Surabaya 8,5%, dan populasi Palu 7,4%.
Frekuensi genotip apoE 2/3 pada populasi Palu adalah 30,9%, Surabaya 18,3% dan
populasi Alor 15,6%. Frekuensi genotip apoE 3/3 pada populasi Surabaya adalah
54,9%, Palu 36,8% dan populasi Alor 17,8%. Frekuensi genotip apoE 2/4 pada
populasi Surabaya dan Palu masing-masing adalah 6,1% dan 6,7%, sedangkan pada
populasi Alor frekuensinya 2,9%. Genotip apoE 3/4 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 4/4 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 2, 3, dan 4 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 2 dan 4,
menunjukkan kecenderungan ke arah timur Indonesia semakin naik, berkebalikan
dengan frekuensi alel 3 yang semakin menurun atau rendah ke arah timur Indonesia.
Frekuensi genotip apoE antara populasi Surabaya dan Palu terdapat perbedaan
bermakna hanya pada genotip apoE 3/3 saja (Chi square, p>0,05). Frekuensi
genotip antara populasi Surabaya dan Palu jika dibandingkan dengan populasi Alor
terdapat perbedaan bermakna pada genotip apoE 3/3 dan 3/4 (p<0,05). Frekuensi
genotip 4/4, oleh karena dari populasi Surabaya tidak ditemukan genotip ini,
sehingga tidak dapat dibandingkan dengan populasi lainnya. Frekuensi genotip 4/4
antara populasi Palu dengan Alor tidak menunjukkan perbedaan bermakna (p>0,05).
Pembawa alel apo pada semua populasi yang diperiksa, tampak tidak terdapat
perbedaan bermakna pada frekuensi alel 2 untuk ketiga populasi (p>0,05). Frekuensi
alel 3 dan 4 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 3/4 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 2/2 dan alel 2 pada penelitian ini tampak memiliki sifat proteksi terhadap
timbulnya PJK dan genotip 3/4 serta alel 4 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 4
mempunyai risiko lebih besar untuk menderita PJK dibanding pembawa alel 2 dan 3
(Eichner et al., 2002; Elousa et al., 2004; Mahley et al., 2006; Mc. Neale et al.,
2000; Pirim et al., 2001). Pembawa alel 4 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 4 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 4 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
2 yang mempunyai kadar trigliserida yang tinggi bukan merupakan faktor risiko
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timbulnya PJK. Pembawa gen 3 dan 4 yang mengalami dislipidemia merupakan

faktor risiko timbulnya PJK. Profil lipid darahnya jika dikendalikan, pembawa gen 4
merupakan faktor risiko timbulnya PJK. Peran polimorfisme apoE jika dibandingkan
pada populasi Indonesia dengan populasi lainnya di dunia, hasilnya hampir sama yaitu
pembawa alel 2 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 2 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 4 pada populasi Indonesia barat dan tengah dibanding
populasi lainnya di dunia hasilnya sebagian besar sama yaitu pembawa alel 4
mempunyai risiko dislipidemia lebih besar dibanding bukan pembawa alel 4 (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
169

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

2009). Apolipoprotein E3 terikat lebih baik dibanding apoE4 pada HDL
3,
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 2
dan 3, sedangkan pembawa gen 4 tidak mudah dipengaruhi oleh intervensi medis.
Diet rendah lemak dan kolesterol menginduksi penurunan kolesterol dan LDL lebih
besar pada pembawa gen 4 dibanding pembawa gen 2 dan 3. Hal ini menunjukkan
bahwa adanya polimorfisme apoE merespon obat hipolipidemiak berbeda. Pemberian
fenofibrat menunjukkan bahwa alel 2 mempunyai penurunan lebih kecil kadar
trigliserida dibanding bukan pembawa alel 2 (Irvin et al., 2010). Alel 4 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 4, 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
170

tergantung apoE, pembawa gen 4 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 2 antara ketiga populasi tersebut masing-
masing 0,207, 0,243 dan 0,267. Frekuensi alel 3 di Surabaya, Palu dan Alor masing-
masing 0,701, 0,617 dan 0,466 dan frekuensi alel 4 populasi Surabaya, Palu dan Alor
masing-masing 0,092, 0,14 dan 0,267. Frekuensi alel apoE baik 2, 3 maupun 4
antara populasi Surabaya dan Palu dengan chi square tidak berbeda bermakna.
Frekuensi alel 3 dan alel 4 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 3 adalah paling besar dibanding alel 2 dan 4, (0,466-
0,701) sama dengan populasi dunia lainnya. Alor yang merupakan daerah yang relatif
terisolir, mempunyai frekuensi alel 3 paling rendah dibanding dengan populasi
Indonesia lainnya, namun frekuensi alel 3 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 2 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 2 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 2 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 2 ditemukan antara 0,031 sampai 0,19. Frekuensi ini
hampir sama dengan frekuensi di Surabaya sebesar 0,207 (Becher et al., 2005; Chaaba
171

et al., 2005; Eichner et al., 2002; Willis et al., 2003). Populasi Asia lainnya, frekuensi
alel 2 ini juga sangat bervariasi. Genotip apoE 2/2, 2/4 dan 4/ 4 tidak
ditemukan pada populasi Mongoloid (Yin et al., 2008), frekuensi alel apo 2 sebesar
0,051 di Kuwait (Al Bustan et al., 2005), frekuensi alel 2 0,075 di Taiwan (Lin et al.,
2004), Bangsa Han di China dengan frekuensi alel 2 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 2 sebesar 0,14 (Gajra et al., 1994) dan 0,051 (Seet et al.,
2004). Frekuensi alel 2 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 2 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 3 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 3 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 3 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 3 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.,
172

2003). Frekuensi alel 3 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 3 populasi Indonesia barat dan tengah, jika dibandingkan
dengan populasi Asia lainnya tidak berbeda, namun frekuensi alel 3 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 3 nya hampir sama dengan yang ditemukan pada populasi
Alor.
Frekuensi alel 4 pada populasi Surabaya sebesar 9,2%, populasi Palu sebesar
14% dan Alor sebesar 26,7%. Frekuensi alel 4 pada populasi Alor tampak paling
besar dan berbeda bermakna dengan dua populasi dari Surabaya dan Palu. Frekuensi
alel 4 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 4. Frekuensi alel 4 pada populasi Wayampi Perancis sejauh ini
menunjukkan frekuensi paling tinggi (0,423 ) diperkirakan karena terisolasinya
populasi ini dan adanya drift genetik. Alel 2 yang tidak ada dan dengan tingginya
frekuensi alel 4 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 4 di Sardinia mencapai 0,40 (Eichner
173

et al., 2002). Frekuensi alel 4 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 4 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 4 yang ditemukan pada populasi Indonesia
barat juga termasuk rendah yaitu sebesar 0,092. Frekuensi alel 4 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 4 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 4
sebesar 0,09. Frekuensi alel 4 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 3/4
dan alel 4 merupakan faktor risiko terjadinya PJK, sedangkan pembawa genotip
apoE 2/2 dan alel 2 merupakan faktor protektif terhadap timbulnya PJK.
2. Adanya polimorfisme apoE menyebabkan terjadinya variabilitas kadar trigliserida
namun tidak menyebabkan terjadinya variabilitas profil lipid yang lain.
174

3. Terdapat perbedaan distribusi genotip dan alel apoE antara populasi kawasan barat
Indonesia, kawasan tengah Indonesia dan kawasan timur Indonesia.
4. Genotip apoE 3/4 dan alel 4 merupakan faktor risiko PJK pada populasi
Indonesia barat dan tengah.





















175

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 2, 3 and 4 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
176

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 3 is the most frequent (>60%) in all population
studied (Eicner et al., 2002).
177

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 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 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 4 carriers compared with 3 carriers or
2 carriers (Stengard et al., 1998).

Some studies have also suggested that 4 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 4 allele. Other studies

178

have also associated the 2 allele with

increased CHD risk (Eichner et al., 2000,
Zannis et al., 1996, Mahley et al., 2006).
An association between apoE 2/2 and type III hyperlipoproteinemia

has been
known for decades. This disorder is characterized

by increased cholesterol and
triglyceride levels, the presence

of -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 15

per 5,000, whereas
homozygosity for E2/2 occurs with a frequency

of 0.51.0 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. Indonesias size, tropical
climate and archipelagic geography, support the worlds 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
179

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 Transaminase
1
(GPT
1
) also
Phosphogluconate Dehydrogenase
C
(PGD
c
) gene frequencies and of decreasing
Glyoxylase
1 (
GLO
1
) gene frequency towards the east. This pattern was found also in
-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 (Hp
1
) 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.
180

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 2, 3 and 4 allele
with 195 and 23 bp for 4; 23, 50 and 145 bp for 3 and 50 and 168 bp for 2
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 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.



181

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 2/2 genotype was related with
higher level of triglyceride and cholesterol. Other genotypes were not related with
lipid profile.
The frequency of apoE 2/2, apoE 2/3, apoE 3/3, and apoE 3/4
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 2/4 and apoE
4/4 genotypes were not found in CHD patients. Comparison was not possible with
controls, since apoE 2/4 and apoE 4/4 genotypes were not found in CHD
patients. Significant difference (p<0.05) was only observed in apoE 3/4 genotype
with OR 4.86 times higher than other genotypes between CHD patients and controls.
Apolipoprotein E 2/2 genotype in CHD patients showed OR < 1, indicating that
apoE 2/2 genotype was protective factor for CHD.
182

On the other hand 4 allele although p>0.05 but have OR 2.05 as risk factor for
CHD. It can be summarized that apoE 3/4 genotype and 4 allele were risk factor
for CHD, while apoE 2/2 genotype and 2 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 2
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 2 carrier gene was not
the risk factor for CHD, but 4 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 2/2 genotypes
were 15.6%, 8.5% and 7.4% in Alor, Surabaya and Palu respectively. Frequency of
apoE 2/3 genotype were 30.9%, 18.3% and 15.6% in Palu, Surabaya and Alor
respectively. Frequency of apoE 3/3 genotype were 54.9%, 36.8% and 17.8% in
Surabaya, Palu and Alor respectively. Frequency of apoE 2/4 genotype were 6.1%,
6.7% and 2.9% in Surabaya, Palu and Alor respectively. The frequency of apoE 3/4
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 4/4 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 2, 3, and 4 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
183

different with Hardy-Weinberg equilibrium (p>0.05). The frequency 2 and 4 alleles

tend to increase also from the western part of Indonesia towards the east, contrary to
the 3 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 3/3 genotype (p<0.05). The frequency of apoE 3/3 and
apoE 3/4 genotypes were significantly different for Surabaya and Alor populations
as well as for Palu and Alor populations (p<0.05). The apoE 4/4 genotype was not
found in Surabaya populations, so it was not possible to compare with other
populations. However, apoE 4/4 genotype was not significantly different (p>0.05)
between Palu and Alor populations.
When apo alleles for all Indonesian populations were compared, there were
no significant difference for 2 allele (p>0.05). Significant difference in 3 and 4
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 3/4 genotype and 4 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 2 allele was a protective factor for CHD and 4
allele was a risk factor for CHD. This risk factor was not different in other world
populations, in which 4 gene carrier was the risk factor for CHD than those 2 and
3 gene carriers (Eichner et al., 2002; Elousa et al., 2004; Mahley et al., 2006; Mc.
184

Neale et al., 2000; Pirim et al., 2001). This 4 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 4
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 4 gene carriers was not risk
factor for CHD (Liu et al., 2003), as well as in coronary artery disease, this 4 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 2 allele has protective effect for CHD,
but 3 and 4 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 worlds populations in
which the 2 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 2 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 worlds populations was also found in western and
the middle part of Indonesia, in which 4 allele was a risk factor for dyslipidemia than
185

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 HDL
3
, 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
2 and apoE 3 gene carriers, but apoE 4 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 4 gene carrier than apoE 2 and apoE 3
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
186

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 4 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
4 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 2
allele for all these populations were 0.207, 0.243 and 0.267 respectively. The
frequency of 3 allele in Surabaya, Palu and Alor populations were 0.701, 0.617 and
0.446 respectively and the frequency of 4 allele for all of these population were
0.092, 0.14 and 0.267 respectively. The frequency of 2, 3 and 4 in Surabaya was
not different with Palu population. The frequency of 3 and 4 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 3 allele was the highest than 2 and 4 allele
frequencies (0.466-0.701). Alor which is relatively isolated populations of Indonesia
has 3 allele lowest compared with other populations of Indonesia (0.466), but its 3
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 2 allele in Indonesian populations (0.207-0.267) were higher than other
populations in the World, especially when compared with areas where 2 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, 2 allele
187

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, 2 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 2 allele was varies.
Genotypes of apoE 2/2, apoE 2/4 and apoE 4/4 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 2 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 2 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 3 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 3
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 3 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 3 allele in
188

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 3 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 3 allele
frequency in Surabaya and Palu populations were not different with other Asian
populations, but the frequency of 3 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 4 allele were 9.2%, 14% and 26.7% in Surabaya, Palu and Alor
populations respectively. The frequency of 4 allele in Alor was the highest and
showed significant difference in two populations of Surabaya and Palu. The high
frequency of 4 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
4 allele frequency. Frequency of 4 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 2 allele was not found and a high frequency of 4 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 4 allele frequency was 0.40 in Sardinia
(Eichner et al., 2002). The 4 allele frequency in the eastern part of Indonesia was
189

high and almost identical to those found 0.20 in populations at in Finland and
Swedden ; 0.28 in Native Americas (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 4 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 4 allele in Surabaya was 0.092. Frequency of 4 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 4 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 3/4 genotype and
4 allele were the risk factor for CHD where as apoE 2/2 genotype and 2 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 t of Indonesia.
4. Apolipoprotein E 3/4 genotype and 4 allele were risk factor for CHD in the
western and middle populations of Indonesia.

190

BAB 7

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