In silico survey primer design for mutation detection of LDLR and APOB genes caused familial hypercholesterolemia

Gidi thttang SV.NCKH-Eureka ldn thzi- 15 nenn 2013

Ky ye'u khoa hoc

IN SILICO SURVEY — PRIMER DESIGN FOR MUTATION DETECTION OF
LDLR AND APOB GENES CAUSED FAMILIAL HYPERCHOLESTEROLEMIA
Nguyin TN) My, Li Thi Dan Dam, Le Huyb Ai Thar'
Khoa Cling Nghe Sinh Hoc, Ttruong Doi hoc Mu Thenh pho HO Chi Minh

De tai clot gi6i Khuyin khich Giiri Mitring Sinh vie'n nghien eau khoa hoc — Eureka art XV mkt 2013
*Email: lhathuve,gmaiLcom

TOM TAT
Tang cholesterol mau c6 tinh gia dinh (Familial Hypercholesterolemia FH) la r6i loan di truyan phO bien do
gen trOi tren nhiem sic the thuang quy dinh, he luy cua no la cac benh ly ve tim mach, dOt quy,... Muc tieu chinh
cua nghien cfru là tim hien ye ca the phan tin benh cao cholesterol mau co yen to gia dinh tren nguari benh Viet
Nam, tap trung a nhimg Wing ming (hot spot) hay cac clOt bien not trOi tren hai gen c6 vai tra quan tong trong dieu
hem cholesterol, gen LDLR (Low Density Lipoprotein Receptor) va APOB (Apolipoprotein B). Kat qua khai that din
lieu ghi nhan dart bien tren gen LDLR tit da clang va phut tap, trong do exon 4 co ty 14 dOt bien cao nhat; cac dOt
bien tren gen APOB tap trung chit yeu a exon 26 va dOt bien not trOi nhit la R3500Q. Tin do, chiing toi tien hanh
thiet ke va danh gia cac bO moi cho cac phuang phap tiep can trong thirc nghiem: PCR — Giai trinh to (ling vai
exon 4 tren gen LDLR) va PCR dac hiau allele (ling yen do 01 bien R3500Q tren gen APOB). Ch'ing toi da
khuach dai thank ding exon 4 gen LDLR, ghi nhin cac clOt bien nghi ngar va ket lu'in btrac du ve tinh phirc tap
cua dOt bien ten exon 4 nay. Ding thai, chi:mg toi khuech dai thanh ding exon 26 gen APOB, khing dinh dOt
bien R3500Q xuit hien teen ca 20 miu benh phim thuOc benh vin Da khoa Khu Niue Thi, flirt cung cap (yeti cac
chi s6 lam sang cho thy cao cholesterol trong mau). Vai nhimg kat qua buarc diu nay, chfing toi ghi nhan ca hai
loaf dOt bien (tren exon 4 gen LDLR va dot bien tren gen APOB) dau tit co the la mot dau thing phan to tiem
nang cho viac chin doan, tam sok nguy ca din den benh FH a nguiri Viet Nam.
Ter khda: d¢t bien R3500Q, Familial Hypercholesterolemia (FH), gen APOB, gen LDLR, PCR clOc hieu allele,
PCR — Gicii trinh tu.

MOI DAU
Tang cholesterol mau co tinh gia dinh (Familial Hypercholesterolemia — FH) la tOi loan di fruyan (dOt bien
gen) do gen trOi tren nhiem sic the thtramg quy dinh, ph6 Wan nhit la dOt bien ten gen LDLR (Low Density
Lipoprotein Receptor) va APOB (Apolipoprotein B) voi vai tr6 quan tong trong viac dieu haa cholesterol trong
mau. Cac dOt bien tren hai gen nay lam anh htrang den chic nang cua cac protein ma hai gen ma hoa, lam cho
cholesterol trong mau khong duct hap thy vao to boo, gay tang cholesterol trong mau, din den tang cao nguy ca
mac cac benh tim mach (Fard — Esfahani P et al., 2005; Zakharova FM et al., 2005). Nhiau ding trinh nghien cfru
tren the giai ghi nhan viac tim soot cac dOt bien tren hai gen nay la tien quyat trong nghien dm dich to hoc cua
benh, &Ong that g6p phin nghien ciru sau va rO han va cac phuang phap china benh FH (Al — Allaf FA et aL, 2010;
Al — Khateeb A et al., 2011; Fard — Esfahani P et al., 2005). CS Viet Nam, hien tai van chtra ca, nghien ctiu mang
tinh phan tin nao va benh, ding nhu la cac clich to hoc ye FH. Trong khi do, ty la nguari Viet Nam mac benh tang
cholesterol mau duce phat hien ngay tang nhieu. Vi vay, chi:mg toi xac dinh muc tieu cua nghien cuu buoy diu nay
la: 1) Tim hiau thong tin the giai nghien eau phan to ve benh FH, tap trung tren hai gen LDLR va APOB: cac kieu
dOt bien, Wing tap trung dOt bien, tinh dac trung ve chUng tOc, vung dia ly phan biat... (neu co); 2) Buoy dau xay
dung cac ca sa thuc nghiam nhatn tiep can nghien cuu phan tin va benh FH a Viet Nam.
VAT LIEU VA PHUONG PHAP
Sir dung 20 mau mau cua cac benh nhan duct chin doin lam sang biau hien tang cholesterol trong mau bai
Banh vin Da Khoa Khu vuc Thu Dire cung cap (vari cac chi so lam sang cho thiy cao cholesterol trong mau) de
tach DNA bO gen dua theo phuang phip phenol/chloroform (Chomczynski P, Sacchi N, 1987), thou hien PCR hay
PCR dac hiau allele. San phim PCR duct giri giai tai Cong ty Nam Khoa, TP.HCM.

160


Kj; y'Jzi khoa hoc

Gidi thuong SV.NC KH-Eureka kin ilia. 15 mini 2013
HET QUA VA THAO LUAN
Ket qui khai thac dir lieu

Viec khai that thong tin tren NCBI ( va Pubmed
( dirge thuc hien tir hon 190 ding trinh thuc nghiem (s6 lieu cap nhat den 1/
2013) de cap den dot bien tren hai gen LDLR va APOB cho thay d6t bien LDLR va APOB rat da clang tren the giai
vai Ioai mau sir dung phi bien nhAt IA mau mau.
D6i viii gen LDLR, dot hien trai ring tren man b6 trinh to 18 exon va 17 intron, trong do exon 4 c6 nhieu dot
bien nhat (du lieu khong trinh bay). Exon 4 IA exon dai nhat so vai cac exon khac ten gen LDLR, ma hem cho vimg
chirc nang gin vai ph6i tir dm thu the LDLR vii 7 clop trinh to lap lai giau cysteine, tich dien am de de clang gin
vii cac ph6i tir (tich dien ducmg). Do vay, d6t bien tren exon 4 gen LDLR can thiet phai dtroc kiem tra khi tien hanh
tam
' soat phat hien som b'e'nh FH trong Ong ding. Han ncra, exon 4 co khoang 144 dot bien khac nhau va khong tap
trung thanh vimg ma rai rac nen khip ca exon, da s6 la dot bien diem, lam sai nghia (missense) va dOt bien mat doan
nho. Vi vay, phtrang phap thuc nghiem tiep can de do tim dot bien tren exon nay hieu qua nhit la giai trinh to town
be exon.
D6i vai gen APOB, cac d6t bien hau nhu chi xuat hien tap trung a exon 26 cua gen,. trong d6 dOt bien diem
R3500Q Ia dot bien not tr6i nhat. Day IA ket qua cua qua trinh thay the nucleotide CGG thanh CAG a vi tri 10708
cua gen APOB, clan den su thay the acid amin arginine thanh glutamine o vi tri 3500, lam thay dOi cau vile protein
APOB, pha va hoan town su lien ket giaa thu the LDL vii LDLC, trong khi cac dot bien khac chi lam cho su lien
ket nay yeu di. Nhu vay, chi can co mOt d6t bien R3500Q tren gen APOB xuat hien thi qua trinh thu nhi'n
cholesterol vao to bao se bi can ter, gay tich Iuy va tang cholestrol trong mau. Do vay, chung toi chin thir nghiem
phuong phap PCR dac hieu allele nhim phat hien 'Jet hien dot bien R3500Q, ben canh PCR ket hqp giai trinh ttr
nham khatig dinh tinh dac hieu cua ket qua PCR dac hieu allele a boat dau nay.
Ket qui thitt ke/ danh gisi nuti
Chimg toi tien hanh thiet ke cac be m6i cho phucrng phap PCR — giai trinh tu Wing exon 4 gen LDLR va
phuong phap PCR dac hieu allele phat hien dot bien diem R3500Q gen APOB. Sau do, tien hanh khao sat do dac
hieu, cac th6ng so vat 1j, b6 mLi bet— nhang cong cu tin sinh hoc: chuang trinh Blast
(htta://blast.ncbi.nlm.nih.gov/Blast.cai) IDT analyzer
( Qua qua trinh danh gia, so sanh va phan tich, chimg
Val quyet dinh sir dung cac b6 moi trinh bay a BANG 1.
Bang 1. Bang mOi sir dung trong nghien ciru

Ten m8i

Trinh tu,

D-i

APOB-F
APOB-R1
APOB-R2
LDLR-F
LDLR-R

GACCACAAGCTTAGCTTGG
GGGTGGCTTTGCTTGTATG
TGCAGCTTCACTGAAGACT
ACTGCGGCAGCGTCCCCGGC
TGGGGGAGCCCAGGGACAGG

19
19
19
20
20

hiet er
chay
53.6
54.3
53.9
70.7

66.7

Kc
phLm
334 by
167 by
527bp

Ktt qui PCR — giii trinh tv exon 4 gen LDLR

Tien hanh phin irng PCR khuech dai vimg trinh tu exon 4 gen LDLR tren cac mau vai cap moi LDLR-F va
LDLR-R vai nhiet do lai 68°C, ket qui cho dung kich thuot 527 bp. Vi vay chimg toi giri giai trinh tu (theo hai
chieu nguoc vi xuoi) vi ghi nh(In dtrcyc met se (let bien kern tinh trrtng cua &A Nen là dOng hey to ho4c di hop to
(dir lieu khong trinh bay). Qua phan tich buoy dau, cac d6t bien tren exon 4 gen LDLR kha da dang, trong do co
nhimg difa bien mai chua timg dugs ding bo tar& day tren the giai (S89P, 1101L, Cl 13L,...) va tong co mOt so
dot bien da co cong bo tren the giai (D147H, C I 07R, C113W). Da so cite dot Wen 6 clang dring hop tir cho nen kha
nang di truyen benh cho the he sau la rat cao.
Ket qui phan sing PCR die hiOu allele phsit hiOn dot bien R3500Q gen APOB
Chang toi thuc hien phan ang PCR dac hieu allele phat hien dot bien R3500Q tren gen APOB vai DNA tach
chief tir 20 mau mau a tren vai be moi APOB (mOt cap mai khuech dal ben ngoai dot bien va met moi phat hien d6t
161


Gidi thuring SV.NCKH-Eureka lan thii. 15 ncim 2013

Ky yeu khoa h9c

bien, BANG 1). Ket qua cho thay 20 miu khao sat deu xuet hien dOt bien R3500Q. Ket qui nay la hoan town bat
nga bai du co. mat' rat nho flitting dOt bien lai xuat hien qua cao, khac vii so lieu da ghi nhan ten the giot
KET LUAN

Qua trinh thus hien nghi'en eau, chung tai rat ra &or mOt s6 ket lino nhtr sau: (1) DOt bien tren gen LDLR rat
da clang va phirc tap, trong do exon 4 co tY le dot bien cao nhat; dOt Wen not trOi Mat tren gen APOB la dOt bien
R3500Q; (2) Khuech dai thanh cong exon 4 gen LDLR, ghi nhan cac dOt hien va ket luan btrac du ve tinh pink tap
caa dqt bien tren exon 4 nay; (3) Khuech dai thanh cong exon 26 gen APOB, khAng dinh dOt bien R3500Q xuat hien
tren ca 20 mau b'enh pharn. Nhu vay ca hai loaf dOt bien (tren exon 4 gen LDLR va dOt bien R3500Q tren gen
APOB)deu rat co the la mOt dau chirng phan tir tiem nang cho viec char down, tam soat nguy ca dan den benh FH
'Toed Viet Nam. Huong nghien cfru sip tai cua chang t8i la tien hanh tren ca mall Ion xac nhan tan silk xuat
hien dm dot bien R3500Q gen APOB va dOt bien gen LDLR, tren cong ding nguoi Viet Nam.
TAI LIEU THAM KHAO
Al — Allaf FA, Coutelle C, Waddington SN, David AL, Harbottle R, Themis M (2010) Gene therapy for
familial hypercholesterolaemia problems, progress, and perspectives. International Archives of Medicine 3: 36.
Al — Khateeb A, Zahri MK, Mohamed MS, Sasongko TH, Ibrahim S, Yusof Z, Zilfalil BA (2011) Analysis of
sequence variations in low — density lipoprotein receptor gene among Malaysian patients with familial
hypercholesterolemia. BMC Medical Genetics 12: 40.
Chomczynski P, Sacchi N (1987) Single — step method of RNA isolation by acid guanidinium thiocyanate —
phenol — chloroform extraction. Anal Biochem 162: 156 —159.
Fard — Esfahani P, Mohammadi — Torbati P, Khatami S, Zeinali S, Taghikhani M, Allahyari M (2005)
Familial defective Apoliporrotein b 100: frequency of R3500Q mutation of Apoliporotein B gene in Iranian
hypercholesterolemic patients. Acta Medica Iranic 43: 193 —196.
Fard — Esfahani P, Zeinali C, Dehboneh SR, Taghikhani M, and Khatami S (2008) A novel mutation in exon 4
of the Low Density Lipoprotein (LDL) receptor gene in an Iranian familial hypercholesterolemia patient. Iran
Biomed J. 9: 139 —142.
Zakharova FM, Damgaard D, Mandelshtam MY, Golubkov VI, Nissen PH, Nilsen GG, Stenderup A,
Lipovetsky BM, Konstantinov VO, Denisenko AD, Vasilyev VB, Faergeman 0 (2005) Familial
hypercholesterolemia in St. — Petersburg the known and novel mutations found in the low density lipoprotein
receptor gene in Russia. BMC Medical Genetics 6: 6.

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Giai thuong SV.NCKH-Eureka lan th& 15 neim 2013

KY .0u khoa hoc

IN SILICO SURVEY - PRIMER DESIGN FOR MUTATION DETECTION OF
LDLR AND APOB GENES CAUSED FAMILIAL HYPERCHOLESTEROLEMIA
Nguyen Tra My, Le Thi Dan Diem, Le Huyen Ai Thuy*
Faculty of Biotechnology, Ho Chi Minh City Open University
*Email.. lhathuvezmail.com
SUMMARY
Familial Hypercholesterolemia (FH) is the most common genetic disorder, autosomal dominant inheritance
and the consequence of heart diseases, peripheral vascular and ischemic cerebrovascular diseases, etc. The primary
objective of this study was to find out the molecular mechanism of FH in Vietnam patients, to focus on hotspot or
dominant mutations of two important genes which are responsible for the regulation of cholesterol metabolism as
LDLR gene (Low Density Lipoprotein Receptor) and APOB gene (Apolipoprotein B). The results of data mining
reported on the high diverse, complicated mutations of LDLR gene, the highest mutation frequency was in exon 4,
the mutations of APOB gene turned on exon 26 and the dominant mutation of R3500Q. Thereby, we carried out to
design and evaluate the primer sets for the experimental approach including the combination of PCR and
sequencing which corresponded with exon 4 of LDLR gene and Allele Specific PCR method for identification of the
R3500Q mutation of the APOB gene. The results showed the successful amplification in exon 4 of LDLR gene as
confirmed the suspicious mutations and concluded that the complexity of mutations in exon 4. In addition, we
successfully amplified the exon 26 of the APOB gene, that affirmed the occurrence of R3500Q mutation in 20
samples of Thu Duc Hospital which had high levels of blood cholesterol. Our initial results reported the mutations in
exon 4 of LDLR gene and R3500Q of APOB gene can be the potential molecular markers for the diagnosis,
screening the risk of FH diseases in Vietnamese people.
Keywords:Allele Specific PCR, ApoB gene, Familial Hypercholesterolemia (F1V, LDLR gene, PCR and
sequencing, R3500Q mutation.

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