Absence of apolipoprotein B-3500 mutation in Turkish patients with coronary and cerebrovascular atherosclerosis/Koroner ve serebrovaskuler aterosklerozlu Turk hastalarda apolipoprotein B-3500 mutasyonu yoklugu.

ABSTRACT

Objective: The arginine-to-glutamine change at codon 3500 of the apolipoprotein B-100 (apo B) is a well-known genetic cause of hypercholesterolemia. Since increased cholesterol levels lead to atherosclerosis, identification of the carries of the apo B-3500 mutation is an important step in the risk stratification of individuals and families with hypercholesterolemia. The prevalence of this mutation in Turkish population is not well known. We aimed to investigate the frequency of apo B-100 mutations (colon 3500) C9774T (Arg 3500 [right arrow] Trp) and G9775A (Arg 3500 [right arrow] Gln) in patients with atherosclerosis in comparison with healthy subjects.

Methods: This cross-sectional study included 442 unrelated subjects living on the West coast of Turkey. Subgroups consisted of 165 patients with coronary artery disease, 163 patients with ischemic stroke, and 114 healthy control subjects.

Results: We did not find any apo B-100 mutation both in the patient and control groups.

Conclusion: As it is hypothesized that this mutation arose within the Central European region from a common ancestor approximately 7000 years ago and spread across Europe, our result of the absence of the R35000 mutation in Turkish patients give an important information about the geographical distribution of the apo B-R35000, that the mutation has not reached to Anatolia.

Key words: Apolipoprotein B-100, hypercholesterolemia, atherosclerosis

OZET

Amac: Apolipoprotein B-100 (apo B)'un 3500. kodonunda arginin yerine glutamin degisimi bilinen bir genetik hiperkolesterolemi nedenidir. Yuksek kolesterol duzeyleri ateroskleroza yol actigindan, apo B-100 mutasyonunu tosiyan bireylerin saptanmasi hiperkolesterolemili aile ve kisilerin risk mucadelesinde onemli bir basamagi teskil etmektedir. Bu mutasyonun Turk toplumundaki prevalansi iyi bilinmemektedir. Bu calismada 2 farkli apo B-100 mutasyonunun (3500 kodondaki C9774T (Arg 3500 [right arrow] Trp) ve G9775A (Arg 3500 [right arrow] Gln)) aterosklerozlu hastalardaki sikligini saglikli bireylerle karsilastirmali olarak arastirmayi amacladik.

Yontemler: Bu kesitsel calismada Turkiye'nin bati kiyisinda yasayan ve kan bagi olmayan 442 kisi incelenmistir. Koroner hastaligi olan 165 kisi, iskemik inme oykusu olan 163 kisi ve 114 saglikh gonullu calismanin alt gruplarini olusturmusur.

Bulgular: Hem hastalarda, hem de kontrol grubunda hic apo B-100 mutasyonu saptamadik.

Sonuc: Bu mutasyonun yaklasik 7 bin yil once orta Avrupa'da ortak bir atadan cikip tum Avrupa'da yayildigi hipotezinden hareketle Turk hastalarda R3500Q apo B mutasyonunu saptamamis olmamiz bu mutasyonun cografik dagilimi hakkinda onemli bir bilgi vermektedir: Mutasyon henuz Anadolu'ya ulasmamistir.

Anahtar kelimeler: Apolipoprotein B-100, hiperkolesterolemi, ateroskleroz

Introduction

Apolipoprotein B-100 (apo B-100) is the major protein component of the circulating atherogenic low-density lipoprotein (LDL) particle and serves as the ligand for the LDL receptor (1). Similar to LDL receptor defects, gene mutations in the receptor-binding zone of apo B-100 can disrupt binding and impair removal of circulating LDL. Several point mutations of the LDL receptor binding domain of apo B-100 leading to familial defective apo B-100 (FDB) disorder have been identified (2, 3). Familial defective apo B-100 disease, a genetic disorder of LDL metabolism characterized by hype rcholesterolemia and premature atherosclerosis (3), is estimated to occur in one of 500 to one in 700 people in several Caucasian populations. In most cases, it results from the mutations (C9774T and G9775A) in the codon for amino acid 3500 leading to the substitution of glutamine for arginine. Identification of the apo B-3500 mutation positive individuals is an important step in risk stratification of patients with hypercholesterolemia. The prevalence of these two mutations in Turkish population is not well known.

In the present study, we aimed to investigate the frequency of apo B-100 mutations (codon 3500) C9774T (Arg 3500 [right arrow] Trp) and G9775A (Arg 3500 [right arrow] Gln) in patients with atherosclerosis in comparison to healthy subjects living in Aegean coast of Turkey.

Methods

Study population and design

The study design was cross-sectional and observational. We enrolled 442 unrelated subjects living on the West coast of Turkey. These patients constituted three study groups.

One hundred and sixty-five of the patients were classified as having coronary artery disease (CAD group). The diagnosis of CAD was based on the case history of past coronary revascularization procedure or was confirmed by coronary angiography (ie they had stenosis with greater than 50% narrowing in the cross-sectional area of one of the major coronary arteries).

Cerebrovascular disease (CVD) group constituted of 163 patients with a history of ischemic stroke. The CAD and CVD groups were randomly recruited from the Cardiology and Neurology departments of Ege University Medical School. We did not use any exclusion criteria.

One hundred and fourteen healthy volunteers among the medical and paramedical staff (sixty-eight males and forty-six females) served as the control group. None of the control subjects had prior history of CAD or CVD and all had normal resting elctrocardiogram.

Study protocol was in agreement with the guidelines of our Institutional Review Board and written informed consent was obtained from all subjects for the use of their blood samples for the study after the nature of the study had been explained.

Lipid and lipoprotein analysis

Blood lipid parameters were available for only the CAD and control groups. The CVD group characteristics were obtained from hospital charts. Blood samples were collected after an overnight fasting by using antecubital vein. Total cholesterol, high-density lipoprotein (HDL) cholesterol and triglycerides were assessed enzymatically by auto-analyzer (Bayer Diagnostics Dax 48, Toshiba, Japan). The LDL cholesterol was calculated by the Friedewald formula (4).

DNA analysis and mutation detection

Genomic DNA was extracted from peripheral leukocytes of the subjects using the High Pure PCR Template Preparation Kit (Roche Applied Science). All experiments were carried out on the LightCycler[TM} Instrument (Roche Applied Science) according to the protocols provided by the manufacturer. The polymerase chain reaction (PCR) and melting curve determination were performed in 20-[micro]l volumes in glass capillaries (Roche Applied Science) polymorphic, mutated and wild type alleles were identified by the specific melting temperature (Tm) of the resulting amplicons. For the detection of the apo B codon 3500 mutations the LightCycler apo B (codon 3500) Mutation Detection kit was used (Roche Applied Science). The temperature of the wild type allele of apo B was 64.0[degrees]C, C9774T allele--58.4[degrees]C, and G9775A allele--54.6[degrees]C.

Statistical analysis

Statistical analysis was performed by Microsoft Excel programme. The distribution of the variables of age and lipid levels were normal. One-way ANOVA test was used for the comparison of the lipid parameters between the groups.

Results

The gender characteristics and serum lipid levels are presented in Table 1. As expected, total cholesterol, LDL, and triglyceride levels were significantly higher both in CAD and CVD groups than in control subjects (p=0.038, p=0.0001, and p=0.0001, respectively). The HDL cholesterol levels were markedly lower in CAD group than stroke and control groups (p<0.0001).

None of the patients in three groups had C9774T (Arg 3500 [right arrow] Trp) or G9775A (Arg 3500 [right arrow] Gln) mutations for apo B codon 3500.

Discussion

The FOB is a disorder of LDL metabolism characterized by hypercholesterolemia and premature atherosclerosis (2, 3). The FOB phenotype closely resembles the familial hypercholesterolemia phenotype (5, 6). The main genetic cause of FOB is an apo B gene mutation that substitutes a glutamine for an arginine at position 3500 of the apo B protein. This abnormal apo B protein cannot bind well to the LDL receptor leading to the accumulation of LDL in plasma. In addition to the 1335000. mutation, other forms were described (R3531C, R3480W, and R3500W) with low rates of occurrence. Among these, the R3500Q and R3531C mutations are frequent in Caucasians (0.08%), whereas the R3500W mutation is very rare in that population, but more frequent in the South Asian population (7). The frequency of 1335000. mutation in hypercholesterolemic subjects largely differs across Europe: populations with highest frequencies cluster in Central Europe, and the mutation's frequency decreases with increasing distance from the Central Europe (8-13). As almost all subjects with the mutation carry the same haplotype in Europe, it is hypothesized that this mutation arose within the Central European region from a common ancestor approximately 7000 years ago, and spread across Europe (14-15). Recent studies from different European populations also suggest that clear distribution gradients could be tracked from Central Europe in all directions, including southeast (16-20).

We did not detect the apo B-1335000. mutation in any of our patients. This finding is in agreement with the previous observations that the R3500Q. mutation had not been found in hyperlipidemic patients in Turkey (21-22). Tamer et al. (21) failed to identify the mutation in 596 people (272 healthy controls, 145 hypercholesterolemic patients, and 179 patients with atherosclerotic coronary artery disease) living on the east Mediterranean coast of Turkey. Mahley et al. (22) also did not detect the R3500Q. mutation in the survey of 2,450 participants in the Turkish Heart Study. The absence of the R3500Q. mutation also supports the hypothesis of a common origin of the mutation.

Conclusion

Our results, being in agreement with previous studies (21, 22) give an important information about the geographical distribution of the apo B-1135000 mutation where by the mutation has reached to Balkans but not to Anatolia, provide further evidence to Rosser s (23) suggestion: "populations such as the Hungarians and Turks are unlikely to be separated from surrounding populations by genetic barriers".

References

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(16.) Kalina A, Csaszar A, Czeizel AE, Romics L, Szaboki F, Szalai C, et al. Frequency of the 1335000. mutation of the apolipoprotein B-100 gene in a sample screened clinically for familial hypercholesterolemia in Hungary. Atherosclerosis 2001;154: 247-51.

(17.) Hamalainen T, Palotie A, Aalto-Setala K, Kontula K, Tikkanen MJ. Absence of familial defective apolipoprotein B-100 in Finnish patients with elevated serum cholesterol. Atherosclerosis 1990; 82: 177-83.

(18.) Seripa D, Gravina C, Volpe R, Margaglione M, Papa S, Merla G, et al. Absence of apolipoprotein 133500 mutation in type 2a hyperlipoproteinemia patients and in the general population from southern Italy. J Inherit Metab Dis 1999; 22: 670-1.

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Zuhal Eroglu, Nur Selvi, Buket Kosova, Cigir Biray, Emre Kumral *, Nejat Topcuoglu, Meral Kayikcioglu **

From Departments of Medical Biology, Neurology * and Cardiology **, Faculty of Medicine, Ege University, Izmir, Turkey

Address for Correspondence/Yazisma Adresi: Dr. Meral Kayikcioglu, Ege Universitesi Tip Fakultesi, Kardiyoloji Anabilim Dali, Izmir, Turkey E-mail: [e-mail]

Table 1. Clinical characteristics of the study groups

                              Control               Cardiovascular
Variables                      group                diseases group

Gender, n
Female                          46                        68
Male                            68                        97
Total                           114                      165

Age, years
Female                 27.83 [+ or -] 7.63       41.13 [+ or -] 8.07
Male                   30.33 [+ or -] 10.99      38.76 [+ or -] 5.98
Total                  30.06 [+ or -] 9.84       39.18 [+ or -] 6.34

Cholesterol, mg/dl
Female                151.00 [+ or -] 13.45     191.64 [+ or -] 63.25
Male                  164.14 [+ or -] 40.02     204.19 [+ or -] 56.16
Total                 160.20 [+ or -] 33.88     201.81 [+ or -] 56.54

Triglyceride, mg/dl
Female                 70.00 [+ or -] 14.73     153.07 [+ or -] 99.00
Male                  122.43 [+ or -] 63.83     205.96 [+ or -] 126.28
Total                 106.70 [+ or -] 58.36     201.08 [+ or -] 124.86

HDL, mg/dl
Female                 50.33 [+ or -] 11.85      47.71 [+ or -] 18.28
Male                   45.17 [+ or -] 7.28       39.70 [+ or -] 7.37
Total                  46.89 [+ or -]  8.65      40.99 [+ or -] 10.45

LDL, mg/dl
Female                 86.33 [+ or -] 12.74     106.46 [+ or -] 47.19
Male                   93.50 [+ or -] 30.45     128.24 [+ or -] 55.35
Total                  91.11 [+ or -] 25.16     123.19 [+ or -] 54.01

                          Cerebrovascular
Variables                 diseases group          F *      p *

Gender, n
Female                          34                --        --
Male                            129               --        --
Total                           163               --        --

Age, years
Female                  61.06 [+ or -] 15.176    16.051   0.0001
Male                   62.596 [+ or -] 11.386   139.091   0.0001
Total                   61.96 [+ or -] 13.00     33.992   0.0001

Cholesterol, mg/dl
Female                186.936 [+ or -] 42.02      0.958   0.392
Male                  196.416 [+ or -] 41.08      2.245   0.111
Total                  192.37 [+ or -] 41.44      3.350   0.038

Triglyceride, mg/dl
Female                 139.79 [+ or -] 70.26      5.090   0.010
Male                   136.77 [+ or -] 74.66      8.967   0.0001
Total                  138.06 [+ or -] 72.30     15.563   0.0001

HDL, mg/dl
Female                  47.43 [+ or -] 11.07     15.228   0.0001
Male                    45.38 [+ or -] 10.11     56.124   0.0001
Total                   46.26 [+ or -] 10.50     76.926   0.0001

LDL, mg/dl
Female                 113.32 [+ or -] 32.15     20.658   0.0001
Male                   123.19 [+ or -] 35.81     40.910   0.0001
Total                  118.94 [+ or -] 34.38     66.019   0.0001

Data are represented as numbers and Mean [+ or -] SD

*--Comparisons are made by one-way ANOVA test

HDL--high density lipoprotein, LDL--low density lipoprotein