ABSTRACT Point mutations in the receptor binding domain of low density lipoprotein may increase cholesterol levels in blood. Three mutations of Apo B-100 protein result in defective binding (Arg 3500®Gln, Arg 3500®Trp and Arg 3531®Cys). We estimated the frequency of Apo B point mutations (codon 3500) C9774T (Arg 3500®Trp) and G9775A (Arg 3500®Gln) in 179 atherosclerotic, 145 hyperlipidaemic individuals and 272 healthy individuals in the east Mediterranean region of Turkey. Lipid and lipoprotein levels were measured with routine biochemical analyser and Apo B mutation was detected using real-time PCR. Neither mutation was found. In this region, Apo B-100 protein mutations are rare and causes of hyperlipidaemia and atherosclerosis may therefore be unrelated to them.

RESUME Les mutations ponctuelles sur le site de liaison du récepteur des lipoprotéines de basse densité peuvent faire augmenter le taux de cholestérol sanguin. Trois mutations de la protéine apo B-100 entraînent une liaison défectueuse (Arg 3500 ® Gln, Arg 3500 ® Trp and Arg 3531 ® Cys). Nous avons estimé la fréquence des mutations ponctuelles de l’apo B (codon 3500) C9774T (Arg 3500 ® Trp) et G9775A (Arg 3500 ® Gln) chez 179 patients athérosclérotiques, 145 sujets hyperlipidémiques et 272 sujets en bonne santé de la région est-méditerranéenne en Turquie. Le taux de lipides et de lipoprotéines a été mesuré à l’aide d’un analyseur biochimique et la mutation de l’apo B a été recherchée en utilisant la PCR en temps réel. Aucune mutation n’a été trouvée. Dans cette région, les mutations de la protéine apo B-100 sont rares et les causes de l’hyperlipidémie et de l’athérosclérose peuvent donc ne pas être liées à ces mutations.

Nearly two-thirds of all circulating cholesterol is transported by low density lipo- protein (LDL) particles. The plasma concentration of cholesterol is regulated by which circulating LDL is taken primarily into the hepatocytes. Apolipoprotein B-100 (Apo B-100) is the major protein associated with the LDL particle and contains the ligand that binds LDL to its receptor [1]. Human Apo B-100 is a large, hydrophobic protein of 4536 amino acids and a molecular weight of approximately 540.000 Da. It is synthesized in the liver [2]. Gene mutations in the LDL receptor or in the receptor-binding zone of the Apo B-100 can disrupt binding and impair removal of circulating LDL. More than 150 mutations have been identified in the LDL receptor gene, associated with familial hypercholesterolaemia (FH), an autosomal dominant inherited disorder characterized by severe hypercholesterolaemia, frequent presence of tendon xanthomas, and premature coronary heart disease [3].

To date, several point mutations of the putative receptor binding domain of Apo B-100 have been identified [4,5]. Only 3 of these mutations have been shown to produce binding-defective Apo B-100 by appropriate genetic and functional investigations [6,7]. The first substitution to be discovered, and apparently the most frequent one, is Apo B-100 (Arg 3500®Gln). The other 2 substitutions are Apo B-100 (Arg 3500®Trp) and Apo B-100 (Arg 3531®Cys) and occur less frequently [1,5,6]. Compared with Apo B-100 (Arg 3500®Gln), B-100 (Arg 3531®Cys) is associated with a smaller increase in LDL. Consistently, LDL that contained Apo B-100 (Arg 3531®Cys) exhibits less reduction of LDL receptor binding in vitro than did LDL endowed with Apo B-100 (Arg 3500®Gln) [6,8].

In the present study, we aimed to esti- mate the frequency of Apo B point mutations (codon 3500) C9774T (Arg 3500®Trp) and G9775A (Arg 3500®Gln) in 179 patients with atherosclerosis and compare this with 272 healthy subjects and 185 hypercholesterolaemic patients in the people living on the east Mediterranean coast of Turkey.

DNA samples were isolated from blood samples, collected by venepuncture in sterile siliconized EDTA 2-mL Vacutainer tubes, using High Pure PCR Template Preparation Kit and MagNA Pure LC DNA Isolation Kit I by MagNa Pure LC automated DNA isolation instrument (Roche Molecular Biochemicals, Mannheim, Germany).

The determination of Apo B mutation was done using Real-time PCR and the Apo B mutation detection kit (Roche Diagnostics, GmbH, Mannheim, Germany).

Real Time PCR Principle: a 207 bp fragment of the apolipoprotein B gene was amplified with specific primers from human genomic DNA. The amplicon was detected by fluorescence using a specific pair of hybridization probes. The hybridization probes consist of 2 different short oligo-nucleotides that hybridize to an internal sequence of the amplified fragment during the annealing phase of the PCR cycle. If a mutation is present, the mismatches of the mutation probe with the target destabilize the hybrid. With a wild type genotype, mismatches do not occur, and the hybrid has a higher TM. The temperature is slowly increased and when the mutation probe melts off and the 2 fluorescent dyes are no longer in close proximity, the fluorescence decreases. For mutated genotypes, this occurs at lower temperatures than for the wild type genotype.

In addition, we performed DNA sequencing of 5 randomly selected patients, 5 controls and 5 individuals with hypercholesterolaemia.

Apolipoprotein A (Apo A) and Apolipoprotein B (Apo B) were determined by immunoturbidometric methods. Triglyceride (TG), total cholesterol (TC) and high density lipoprotein (HDL) were analysed by GPO/PAP enzymatic colorimetric, CHOD/PAP enzymatic colorimetric and direct COHD/PAP enzymatic colorimetric me-thods, respectively. LDL content was calculated from the primary measurements using the empirical equation of Friedewald et al. [10]. All these parameters were determined by Cobas Integra 700 biochemical analyser (Roche Diagnostics, GmbH, Mannheim, Germany). The results were expressed in terms of arithmetic means ± standard deviation.

The Apo B arginine-to-glutamine change at codon 3500 by been established as a cause of the failure of the LDL particle to bind to its receptor and the consequent hypercholesterolaemia of familial defective Apo B- 100 [5]. The Apo B (Arg 3500®Gln) mutation has been identified in the USA, Denmark, Germany, Italy, France, Austria, Australia, the Netherlands, South Africa, Switzerland and indirectly in Norway and Sweden since some of the probands from the Oregan study were of Norwegian and Swedish descent [2]. However, the mutation has not been detected in either Finland or former Soviet Union republics. The Apo B-100 (Arg 3500®Gln) mutation has been observed with an approximate frequency of 1 in 500 to 1 in 700 in populations of European (Caucasian) origin [2,8].

Several point mutations of the putative receptor binding domain of Apo B-100 have been identified. Only 3 of these mutations have been shown to produce defective binding Apo B-100 by appropriate genetic and functional investigations [3–6,10,12]. The first substitution to be discovered, and apparently the most frequent one, was Apo B-100 (Arg 3500®Gln). The other 2 substitutions, Apo B-100 (Arg 3500®Trp) and Apo B-100 (Arg 3531®Cys), occur less frequently [8,13].

Teng et al. showed that the prevalence of heterozygote in 373 Chinese individuals with hyperlipidaemia was 0.3% for the Arg 3500®Gln mutation and 2.4 % for the Arg 3500®Trp mutation [13]. When 373 unrelated individuals with hyperlipidaemia were screened for the presence of Apo B-100 mutations, Tai et al. found 9 Arg 3500®Trp index cases, 7 classified as having type IIa and 2 as having type IIb hyperlipidaemia [14]. The authors reported that one of them was of Scottish descent and the others were of Asian descent. They suggested that Arg 3500®Trp alleles were inherited from a common ancestor in Asian populations.

Fisher et al. investigated 297 consecutive individuals with LDL concentrations > 155 mg/dL and triglycerides < 200 mg/dL for Apo B mutation. Apo B-100 (Arg 3500®Trp) was described in just 1 family of European origin, in 2 families with a mixed Chinese and Malayan descent, in 1 family of Asian descent living in the Glasgow region and in another 9 unrelated individuals from Taiwan [15].

In our study we investigated the frequency of Apo B point mutations (codon 3500) C9774T (Arg 3500®Trp) and G9775A (Arg 3500®Gln) in 596 individuals (272 healthy controls, 185 controls with hyphercholesterolaemia and 179 patients with atherosclerosis). Neither C9774T (Arg 3500®Trp) nor G9775A (Arg 3500®Gln) mutation was found in the people living on the East Mediterranean coast of Turkey. As can be seen in our study, the mutations of Apo B-100 protein are very rare. As regards this population, they were not observed in our sample.

15. Tai DY, Pan JP, Lee-Chen GJ. Identification and haplotype analysis of Apo-lipoprotein B-100 Arg 3500 – Trp mutation in hyperlipidemic Chinese. Clinical che-mistry, 1998, 44:1659–65.