Study On Relationship Between Haplotypes Of TIM-3 Gene And Allergic Asthma In Hubei Han Population

Part One Linkage disequilibrium analysis of -1516,-574,4259 single nucleotide polymorphisms in TIM-3 geneObjective To investigate the frequencies of -1516,-574 and 4259 single nucleotide polymorphisms (SNPs) of T cells immunoglobulin mucin–3( TIM-3) gene in Hubei population and address the question whether they are in linkage disequilibrium(LD) .Methods Genotype and allele frequencies of TIM-3 gene were examined by allelic-specific polymerase chain reaction (AS-PCR) methods in 147 healthy Hubei Han individuals. Hardy-Weinberg equilibrium and Two-point LD analyses and Haplotype frequencies were evaluated with Arlequin v3.1 software。Results The allele frequencies of 3 SNPs are in agreement with Hardy-Weinberg equilibrium. Minor allelic frequencies of TIM-3 -1516G/T,-574T/G and 4259G/T were 8.5%,1.0%,2.0%,respectively. The dominant haplotypes comprising the three loci were G-G-G(2.0%),G-G-T(88.4%), T-G-T(8.5%),G-T-T(1.0%). LD analyses revealed that all of coefficients of linkage disequilibrium (D′) were 1.Conclusion -1516,-574 and 4259 loci in TIM-3 gene are in complete linkage disequilibrium. Our study has provided population genetics data on TIM-3 gene in Chinese Hubei Han population and a basis for searching immune-mediated disease-related TIM-3 haplotype. Part Two The relationship between the haplotype of T cells immunoglobulin mucin -3 gene and allergic asthma in Han population from Hubei Province of ChinaObjective To investigate the single nucleotide polymorphisms(SNPs) of -1516G/T in the promoter region and 4259G/T in the exon-3 region of the T cells immunoglobulin mucin -3(TIM-3 ) ,their linkage disequilibrium and detect their haplotypes relationship with allergic asthma of Han population from Hubei Province of China.Methods The two polymorphisms were detected with allelic specific polymerase chain reaction(AS-PCR) in the 175 asthmatic subjects and in the 202 healthy controls collected from June,2004 to October,2007 in the Han population from Hubei Province.The genotype and allele frequencies, the D′value between the two SNPs sites, the haplotype and their frequencies were calculated and analyzed, respectively.Results The genotype frequencies of GG,GT and TT in -1516G/T polymorphism of TIM-3 gene were respectively 82.7%(167/202),17.3%(35/202),0(0/202)in the 202 controls and respectively 82.9%(145/175),17.1%(30/175),0(0/175)in the 175 asthmatic subjects.The genotype frequencies of GG,GT and TT in 4259G/T polymorphism of TIM-3 gene were respectively 0.5%(1/202),2.5%(5/202),97.0%(196/202)in the 202 controls and respectively 0.6%(1/175),5.7%(10/175),93.7%(164/175)in the 175 asthmatic subjects. control group: D′=1.0, asthma group D′=0.9. Three haplotypes were G-G ,G-T,T-T in the Han population from Hubei Province of China, and their haplotype frequencies were distributed similarly in asthma(3.4%(12/350),88.0%(308/350),8.6%(30/350)) and in the controls (1.7%(7/404),89.6%(362/404),8.7%(35/404)). None of these differences were statistically significant (χ2 =2.15,0.47,0.003 respectively,P >0.05).Conclusion There are strong linkage disequilibrium between the two SNPs sites in TIM-3 gene in Han population from Hubei Province, but the haplotype G-G, G-T and T-T is not associated with asthma susceptibility of the Han population from Hubei Province of China. We cannot exclude the possibility that the haplotypes of TIM-3 may may be associated with asthma susceptibility in other ethnic populations or the susceptibility of other atopic diseases and autoimmunity diseases. Part Three Long-range allele-specific polymerase chain reaction method for direct molecular haplotyping of -574G/T and 4259G/T two SNPs of the TIM-3 geneObjective To set up a new molecular haplotyping method, which is used to determine the phase or haplotype of two or more SNPs that are kilobases apart.Methods One hundred and thirty healthy Chinese Han people from Hubei province were enrolled in this study. Polymorphisms at -574G/T and 4259G/T of T cells immunoglobulin mucin–3(TIM-3) gene were haplotyped with the method of long-range Allele-specific polymerase chain reaction.Results Successfully haplotyping of TIM-3 gene, haplotypes of TIM-3 gene were comprised of T-T,G-T,G-G and was not found T-G haplotype .Conclusion We set up a new method for molecular haplotyping based on long-range allele-specific PCR and have successfully haplotyped TIM-3 gene.