Frequency Investigation Of IL-1A-889, +4716 And +4845 SNP Loci In Liaoning Han And Their Forensic Medicine Application

Because of its abundance quantity and stable character in human genome, SNP (single nucleotide polymorphism) as the third genetic marker after RFLP and STR, has a promising future in Forensic Medicine.Interleukin-1 is a pre-inflammation cytokine which participates in variety of physiological and pathological process and makes key accommodating roles in inflammation and immune reaction. IL-1 includesαandβforms and IL-1r acceptor. IL-1 cluster is located in 2q1.4, including IL-1A, IL-1B, IL-1RN, about 430kb long. Each of three is ranging in 0 kb～35 kb, IL-1B 70 kb～110 kb, IL-1RN 330 kb～430 kb, separately. IL-1A and IL-1B have both exon 7 and intron 6. IL-1RN has exon 4 and intron 3. There are 91 SNP in IL-1A locus reported by NCBI till now, since Yasuji Furutani reported the complete nucleotide sequence of the gene for human interleukin 1 alpha in 1986. The researches involved only a few SNP in IL-1A locus, which all focused on diseases. No researches on genetic polymorphisms of IL-1A in Liaoning Han and their application to forensic medicine are reported till now in the world.In this study, we selected 3 SNP (-889, +4716 and +4845) in IL-1A locus to investigate their frequency in Liaoning Han population in order to provide data for Human Genetics and Forensic Medicine and to discuss their meaning of Forensic Medicine.Materials and Methods1. SamplesTotal 234 anticoagulated blood samples are collected from non-relative Liaoning Han. Total 68 core families are provided by School of Forensic Medicine China Medical University.2. ReagentsProteinase K(Takara); Taq DNA polymerase; 10×Buffer(Takara); dNTP(Takara); DNA Marker(Beijing Xinyuan Company); Acrylamide, N,N'-Methylenebisacrylamide(MERCK); Agarose, Tri (hydroxymethyl) aminomethane (Huamei Bioengneering Company).Thermal Cycler(UNOⅡ, BIOMETRA); Electrophoresis System(Pharmacia, EPS 500/400); Electrocoat tank (Beijing Liuyi Factory, DYY-Ⅲ33A); Ultraviolet Light (Pharmacia LKB, 2011 Macro VUE); Centrifuge (TGL- 16G).3. MethodsThe genotypes of the three SNPs (-889, +4716 and +4845) in IL-1A cluster are detected by Bi-PASA, PAGE and silver staining. The Hardy-Weinberg equilibrium test was performed. Frequency distributions in different populations were compared by x~2 test using SPSS13.0. Probability of Discrimination Power and Probability of Exclusion were calculated according to Fisher method and Garber method respectively. Heterozygosity was calculated according to Nei and Chakraborty method.ResultsThrough frequency investigation of 234 non-relationship healthy individuals, two alleles and three genotypes of IL-1A -889 SNP are detected in Liaoning Han. Frequency of the two alleles, T and C, are 0.0983 and 0.9017. Frequency of the three genotypes, TT, TC and CC, are 0.0043, 0.1880 and 0.8077 respectively.Two alleles and three genotypes of IL-1A +4716 SNP are detected in Liaoning Han population. Frequency of the two alleles, A and C, are 0.6325 and 0.3675. Frequency of the three genotypes, AA, AC and CC, are 0.4188, 0.4274 and 0.1538 respectively.Two alleles and three genotypes of IL-1A +4845 SNP are detected in Liaoning Han population. Frequency of the two alleles, T and G, are 0.0983 and 0.9017. Frequency of the three genotypes, TT, TG and GG, are 0.0043, 0.1880and 0.8077 respectively.68 core family investigations are performed. Four of them do not hew to the law of biology genetics. Two of the four are denied by all of the three SNP and the other are denied by +4716 only.DiscussionBi-PASA, a method meliorated from PCR-ASA, is proofed to be a method of genotyping, which can save time and money compared to PCR-RFLP, PCR-ASA, Taqman and DNA sequencing. In Bi-PASA there are two pairs of primers are used in one PCR system once. Two inner primers are each specific for different alleles. In heterozygotes, three segments are amplified. In homozygotes, two segments are amplified, but the lengths of the segments in two homozygotes are different. Bi-PASA is a rapid, inexpensive and high specific method, which fits to apply in average lab.According to the result of this study, H, DP, PIC and PE are calculated. Identification power of IL-1A -889 SNP and +4845 SNP are low, which should belong to middle power genetic markers. H, DP, PIC and EPP of +4716 SNP are 0.4274, 0.6182, 0.4649 and 0.1784 respectively, which belong to high power genetic markers. In this study three SNPs were applied to research in 68 core families. The biology genetic relationships were denied in four core families totally. Two of the four are denied by all of the three SNP and the other two are denied by +4716 only, which are the same as the results from other genetic markers testing system. +4716 SNP has high power of identification than -889 and +4845 SNP and fits to individual identification and parentage test.We compare -889 SNP frequencies distribution in Liaoning Han with which in other 14 populations or groups. From the result we can see Liaoning Han has significant difference with European and American White and American black (P＜0.05) except groups of Hubei Han, Guangxi Zhuang, Korea and South Korea in Asia (P＞0.05).Conclusions1. Bi-PASA is a rapid, high specific and high power method, which has high value in Forensic Medicine application.2. IL-1A -889, +4716 and +4845 SNPs have polymorphisms in Liaoning Han and their frequency distributions are all accorded to Hardy-Weinberg equilibrium law.3. IL-1A -889 and +4845 SNPs belong to middle power genetic markers; +4716 SNP belongs to high power genetic markers. They all can be used to Forensic Medicine Testing.4. Frequency distribution of IL-1A-889 SNP in Liaoning Han is different with that in European and American White and American black, but not in Asian.