Russian Journal of Genetics, Vol. 41, No. 7, 2005, pp. 805–807. From Genetika, Vol. 41, No. 7, 2005, pp. 986–989. Original English Text Copyright © 2005 by Hakki Ta ¸stan, Ozlem Osmanagaoglu, Ayla Tuzun.
HUMAN GENETICS
The Frequencies of the Serum Amyloid A2 Alleles in Healthy (Turkish, Azerbaijani, and Kazakh) Populations* Hakki Tas¸ tan, Ozlem Osmanagaoglu, and Ayla Tuzun Ankara University, Department of Biology, Ankara, Turkey; e-mail:
[email protected] Received August 9, 2004
Abstract—The Amyloid A1 (AA1) and A2 (AA2) proteins, which result from proteolytic cleavage of the Serum Amyloid A1 (SAA1) and A2 (SAA2) proteins, are major protein components of the Amyloid A deposits found in secondary amyloidosis. This study determines frequency of serum amyloid A2 alleles (α, β) in healthy Turkish, Azerbaijani, and Kazakh subjects. Two hundred Turkish, sixty-five Azerbaijani and sixty-five Kazakh healthy individuals were studied by previously described the PCR–RFLP methods. Our data revealed that the frequencies of the α and β alleles at the SAA2 locus in the Turkish healthy population were different when compared to those in Azerbaijani and Kazakh healthy populations (P = 0.014 and 0.02), respectively. In contrast, the difference between α and β alleles at the SAA2 locus was not different in both Kazakh and Azerbaijani healthy populations (P = 0.882).
INTRODUCTION
MATERIALS AND METHODS
The human SAA family consists of SAA1 (α, β, γ, δ), SAA2 (α, β) [1, 2]. They are bound to HDL in circulation. SAA1 and SAA2 are acute phase reactants whose synthesis are regulated by interleukin—1, interleukin—6 and tumor necrosis factor—α [3]. The physiological role of SAA remains unclear, although some have suggested its role in cholesterol metabolism [4]. In addition, others have proposed immuno-suppressive effects expressed via platelets and neutrophils [5].
Material. Blood samples were randomly taken from 200 Turkish (138 male, 62 female; aged 17–22 years), 65 Azerbaijan (50 male, 15 female; aged 18–22 years), and 65 Kazakh (63 male, 2 female; aged 17–22 years) healthy volunteers who are students at the Ankara University. A written consent was obtained from each individual.
Serum amyloid A (SAA) is a serum precursor of amyloid A (AA), the main fibrillar component in reactive amyloid deposits [6]. SAA is a polymorphic protein, for which there are at least for major loci present (SAA1, SAA2, SAA3, SAA4) in humans [7–9]. SAA3 is a pseudogene and has no product [10]. SAA1 and SAA2 are acute phase reactants; their sythesis in the liver is greatly induced by inflammatory cytokines. Both types from amyloid fibrils, AA—derived from SAA1, is predominant in human AA deposits [11]. There are two variants of SAA2 locus (α, β) which differ in a single base pair substitution in exon 4 [12]. Although the frequencies at the SAA2 locus among different populations were previously reported by Yamada and Moriguchi [8, 12], very few data are available from this particular area (middle Asia and Europe). So, we have determined the frequencies of the genotypes and alleles at the SAA2 locus in Turkish, Azerbaijani, and Kazakh healthy populations.
PCR–RFLP for SAA2 alleles. Genomic DNA was isolated as previously described [12, 13]. The polymerase chain reaction (PCR) to amplify a segment of the SAA2 gene including the polymorphic site was performed with 0.5 µM SAA2—forward (5'-AGA GAA TAT CCA GAG ACT CAC AGG C-3') and 0.5 µM SAA2—reverse (5'-CAG GCC AGC AGG TCG GAA GT-3') primers in a PCR mixture (50 µl) containing 0.2 mM dNTP, 10 mM Tris-HCl, pH 8.3, 1.5 mM MgCl2, 50 mM KCl, 2.5 U Taq DNA polymerase and approximately 300 ng genomic DNA. The mixture was heated to 94°C for 5 min and then subjected to 35 cycles of
Coding sequence SAA2 α, β
∆
SAA2 forward primer SAA2 reverse primer
Intron 3 Exon 3
* The text was submitted by the authors in English.
∆
Genomic sequence Exon 4
Fig. 1. Schematic description of the positions of the primers for the PCR amplification.
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and Pearson chi-square tests were used for determination of the SAA2 genotype and allele frequencies. RESULTS A segment at the genomic SAA2 gene, including one polymorphic site was determined with both PCR/restriction fragment length polymorphism (PCR–RFLP). Both methods successfully separated the α, β alleles (Fig. 2). The data of the numbers and frequencies of the individuals with various genotypes and alleles at the SAA2 locus in the Turkish, Azerbaijani, and Kazakh healthy populations are shown in table. The frequencies of α/β and β/β genotype were different in Azerbaijani and Kazakh healthy populations when compared to those in the Turkish healthy population (41.5, 43.0 versus 52.5, p < 0.01) (1.5, 1.5 versus 7.5%, P < 0.01). The frequencies of α and β alleles were found to be 77.7, 76.9 and 66.3 and 22.3, 23.1 and 33.7% in Azerbaijani, Kazakh, and Turkish healthy populations, respectively. The frequencies of the α and β alleles at the SAA2 locus in Turkish healthy population when compared to the Azerbaijani and Kazakh healthy populations were (0.014 < 0.05) and (0.02 < 0.05), respectively. In contrast, there was not any difference for the α and β alleles at the SAA2 locus between Kazakh and Azerbaijani healthy populations (0.882 > 0.05).
115 bp 90 bp
M ud
1
2
3
4
5
6
7
8
Fig. 2. PCR–RFLP analysis of the SAA2 gene. The PCR products were digested with the NCO restriction enzyme and subjected to electrophoresis. M, molecular weight markers (QX 174 HaeIII, Boehringer Mannheim); ud, undigested PCR product; lanes 1, 4, 5, α/α; lanes 2, 3, 6, 7, α/β; lane 8, β/β genotypes.
94°C for 1 min, 60°C for 1 min, and 72°C for 30 s. The final extension was carried out for 5 min at 72°C. The target sequence of the PCR amplificatio of the SAA2 gene was a 115 bp segment spanning from 10 to 124 bp downstream of the 5' end of exon 4: this segment includes an important polymorphic site (Fig. 1). The PCR products from SAA2 gene were digested overnight at 37°C with NcoI restriction enyzme (Fermentas Vilnius, Lithvania) and then subjected to 4% agarose gel electrophoresys. The DNA was visualized by using ethidium bromide under UV light (Fig. 2). Statistical analysis. Statistical analysis was performed with the χ2 test. Hardy–Weinberg’s equilibrium
DISCUSSION Although the frequencies of the alleles at the SAA1 and SAA2 locus among Caucasians who have different
Comparison of the frequencies of the genotypes and alleles at the SAA2 locus among Turkish, Azerbaijani and Kazakh healthy populations Azerbaijan N (%)
Kazakh N (%)
Turkish N (%)
P
Genotypes α/α
37(56.9)
36(55.3)
80(40)
α/β
27(41.5)
28(43.0)
105(52.5)
β/β
1(1.5)
1(1.5)
15(7.5)
Total
65
65
(n = 2, P < 0.01)
200
Alleles frequencies α
101(77.7)
100(76.9)
265(66.3) *P (.014 < 0.05) P
(.882 > 0.05)
°P (.02 < 0.05) β
29(22.3)
30(23.1)
135(33.7)
*P: between Turks and Azerbaijani, P: between Azerbaijani and Kazakh, °P: between Turkish and Kazakh populations. RUSSIAN JOURNAL OF GENETICS
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THE FREQUENCIES OF THE SERUM AMYLOID A2 ALLELES IN HEALTHY
disease or healthy groups were previously reported [8, 12–16], very few data are available about frequencies of alleles at the SAA2 locus in middle Asian and European healthy populations. Thus, we have determined the frequencies of the genotypes and alleles at the SAA2 locus in Turkish, Azerbaijani, and Kazakh healthy individuals (table). A previous study reported that the prevalence of SAA2β homozygotes in the control groups and the adult rheumatoid arthritis patients with amyloidosis was zero [8, 12]. Similarly, homozygotes for the SAA2β allele seemed to be rare in all control subjects from Turkey, Azerbaijan and Kazakhstan in that study (15 : 7.5%; 1 : 1.5%; 1 : 1.5%). The other previous report found that the frequency of SAA2α allele was approximately 90% in the healthy Japanese population, similar to that in Dutch subjects [8]. In contrast, the frequency of SAA2α allele was lower approximately 66.3% in Turkish, 77.7% Azerbaijani, and 76.9% Kazakh healthy individuals than that of healthy Japanese and Dutch subjects. In addition, the frequency of SAA2β allele was lower in Japanese healthy subjects, similar to that in Dutch subjects (10.9, 11.2%) [8]. However, the frequency of SAA2β was significantly higher in Turkish healthy individuals (33.7%), similar to that in Azerbaijani (22.3%), and Kazakh healthy subjects (33.1%). The frequency of SAA2α/β genotype was significantly higher in Turkish (105 : 52.5%), Azerbaijani (27 : 41.5%), and Kazakh (28 : 43%) healthy individuals than that of healthy Japanese subjects (25 : 21.7%) [8]. We determined the allele frequencies at the SAA2 locus randomly selected in Turkish, Azerbaijani, and Kazakh healthy subjects which may be important from the clinical and genetic point of view. As β allele of the SAA2 gene is higher among Turkish, Azerbaijani, and Kazakh healthy subjects, this may be an important allele for the occurrence of amyloidosis [16]. This investigation can be improved, in this aspect, in future. ACKNOWLEDGMENTS This study was supported by a grant from Ankara University Research Foundation (2003-07-05-045).
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