GENETIC POLYMORPHISM OF PARAOXONASE 3 GENE ALA99ALA IN ADJAANDMAHIETHNIC GROUPS IN BENIN

Julien A. G. Segbo 1* , Chantale Houngnonvi 2 , Antoine Abel Missihoun 2 , Paulin Sedah 2 and Clement Agbangla 2 . 1. Department of Human Biology Engineering, Research Laboratory of Applied Biology, Abomey-Calavi Polytechnic School, Abomey-CalaviUniversity , Republic of Benin. 2. Department of Genetics and Biotechnologies, Faculty of Sciences and Techniques, Abomey-CalaviUniversity, Republic of Benin. ...................................................................................................................... Manuscript Info Abstract ......................... ........................................................................ Manuscript History


2003
). Many studies have been conducted on the relation between PON polymorphisms and genetic susceptibility to coronary heart disease (Erlich et al, 2006 andZhang et al, 2013). The paraoxonase 3 (PON3) have a similar role to PON1 (Sanghera et al, 2008). The expression of PON3 gene occurs mainly in the liver and bind to HDL in blood stream (Reddy et al, 2001). The PON3 gene has five commons Single Nucleotide Polymorphisms (SNPs) Ala99Ala, Asp107Asn, Glu146Lys, Ala179Asp and Tyr233Cys (Robertson et al, 2003;Li et al, 2004;Pasdar et al, 2006 andSanghera et al, 2008). Despite the importance of the paraoxonase gene and its implications on the genetic susceptibility to coronary heart disease, no studies on African black populations have been reported. The purpose of this study was to assess the distribution of PON3 gene Ala99Ala polymorphism in Beninese ethnic groups,Adja and Mahiin order to investigate the genetic basis of PON3 gene related diseases in Beninese population.

Subjects:-
This study has been conducted on two Beninese ethnic groups Adja and Mahi. In June 2015, fifty (50) unrelated volunteers 13 men and 37 women, aged between 17 and 72 years old; were selected randomly from Adjaethnic group from Lokossa, a south-west city in Benin; and in November 2015, ninety-four (94) volunteers,33 men and 61 women, aged between 6 and 70 years old; were selected randomly from Mahiethnic group from Savalou, a central cityin Benin.
DNA extraction and genotyping:-Venous blood samples were collected from each volunteer after written consent. Genomic DNA was extracted by phenol-chloroform method at Genetics and Biotechnologies Laboratory (GBL) at Abomey-Calavi University. The Polymerase Chain Reaction (PCR) primers sequences for the PON3 Ala99Ala SNP (rs1053275) were designed as previously described by Wu et al(2010) and synthesized by SANGON Biotech (Shanghai, China). The sequences were: forward 5'-TCCAGGCATGCCAAACTTT-3' and reverse 5'-TTTCCCTCATTTCCCCCTT-3' were used to amplify 197 bpfragment containing the polymorphism site Ala99Ala on PON3 gene. PCR was performed using thermocycler PTC 100 ™ (Programmable Thermal Controller; Perkin Elmer) in a final volume of 25 μL as follows: 3 μL (3 ng) DNA sample was added to a reaction mixture containing 2.5 μL of 10×PCR buffer, 1.5 μL of 25 mmol/L MgCl 2 , 1μL of 200 μmol/L dNTPs, 1U Taq DNA polymerase (1 μL) (Fermentas) and 2 μL of 0.2 μmol/L of each primer, dimethyl sulfoxide (DMSO) was added to a final concentration of 5% and ultra-pure water (Merck) to a final volume of 25 μL. The fragment amplification was performed under the following conditions: 10 min predenaturation at 94 °C followed by 30 cycles of 45 sec denaturation at 94 °C, 45 sec hybridization at 50 °C and 45 sec extension at 72 °C; and finished by 7 min extension at 72 °C. Then 10 μL of PCR products was digested with 2 μL of 10xNEB buffer, 2 μL of HhaI endonuclease (Promega) and ultra-pure water to a final volume of 20 μL. Tubes were incubated at 37°C for 4h before separation on a 2percentagarose gel and visualization by staining in ethidium bromide under UV trans-illumination.Digestion recognition sequences were GCGC, thus the G allele version would be digested by the enzyme HhaI. The expected results for this polymorphism were the electrophoretic profile with 112 bp, 63 bp and 22 bp bands corresponded to the homozygote genotype G/G; 175 bp and 22 bp bands to the homozygote genotype A/A; and 175 bp, 112 bp, 63 bp and 22 bp bands to the heterozygote genotype G/A. The 63 bpand 22 bp bands were not visible on the agarose gel.
Statistical analysis:-Alleles and genotypes frequencies were calculated by gene counting. The chi-square test on sphinx V 11 software was used both to estimate the Hardy-Weinberg equilibrium and to compare the allelic frequencies observed in Benin population with those reported in other world populations.
Value of p < 0.05 was considered statistically significant.  Table 1.Two alleles and three genotypes were observed in Beninese Adja and Mahi ethnic groups. The homozygote individual G/Gwasfound in these two ethnic groups. The observed and expected frequencies for the polymorphisms were at Hardy-Weinberg equilibrium (p >0.05).
The comparison of PON3 A99A allelic frequencies within Beninese different ethnic groups was shown in Table 2. There were no significant ethnic differences between Adja and Mahifor PON3 gene Ala99Ala polymorphism allelic frequencies(p>0.05).But significant differences were observed for PON3 gene Ala99Ala polymorphism allelic frequencies in these towethnic groups compared to Beninese Abomey-Calavi population respectively (p<0.05).
The comparison of PON3 gene Ala99Ala allelic frequencies in Beninese Adja and Mahiethnic groups with those in other word populations was shown on Table 3. There were significant differences in PON3 Ala99Ala polymorphism allelic frequencies in Beninese AdjaandMahiethnic groups compared to Chinese Li minority and BritishCaucasians.

Discussion:-
The present study has determined the genotypes distribution and the allelic frequencies of PON3 gene Ala99Ala polymorphism in Beninese AdjaandMahiethnic groups. Two alleles and three genotypes were observed. These genotypic and allelic frequencies of PON3 gene Ala99Ala polymorphism in the Beninese AdjaandMahiethnic groups were compared with previously described frequencies in Beninese Abomey-Calavi population and others world populations (Chineses and Britishs).
No significant ethnic differences in PON3 gene Ala99Ala polymorphism genotypes distribution between Adja and Mahi ethnic groups were observed. These two ethnic groups were referred as homogenous population in Benin, so may have the same characteristics in genetic information's transmission. However, there were significant differences in PON3 gene Ala99Ala polymorphism genotype distribution and alleles frequencies in Beninese AdjaandMahiethnic groups compared to Beninese Abomey-Calavi population respectively (Segbo et al, 2014). In contrast with this previous study, the homozygote individual G/G was found in AdjaandMahi ethnic groups. Abomey-Calavi population, the major ethnic group is among the heterogeneous ethnic group; living in the southcentral area (Abomey-Calavi city), the denser area in the country. While AdjaandMahiethnic groups were considered asmuch homogenouspopulations within which the genetic traits transmission were much conservative. So the transmission of PON3 gene Ala99Ala polymorphism genotypes distribution in Beninese AdjaandMahiethnic groups were much conservative and this polymorphism site may be used as an excellentgenetic marker for DNA analysis in Benin.
There were significant differences in PON3 Ala99Ala polymorphism genotype distribution and allelic frequencies in Beninese AdjaandMahiethnic groupscompared to Chinese Li minority respectively (Wu et al, 2010). The G/G genotype frequency in Adjaethnic group (10.0 %) was similar to that observed in Chinese Li minority (8.0 %), but was significant different from that observed inAdjaethnic group (19.0 %).There were also significant differences in PON3 Ala99Ala polymorphism distribution in Beninese AdjaandMahiethnic groups compared to British Caucasians (p<0.05) (Robertson, 2003;Pasdar, 2006).
Additional studies on large cohorts from different ethnic groups in Benin may be needed to determine the real genotypes distribution of PON3 Ala99Ala polymorphism in Beninese population

Conclusion:-
In this study, the genotypes distribution and allelic frequencies of the PON3 gene Ala99Ala polymorphism in Beninese AdjaandMahiethnic groups were described and compared with other world populations.The PON3 gene Ala99Ala polymorphism distribution in Beninese AdjaandMahiethnic groupswere significant different from other populations. These ethnic variations in PON3 gene polymorphisms can be used as basis for further investigation on the association of this polymorphism with the risk of cardiovascular diseases and other inflammatory diseases. 470