Study On Graves Susceptible Gene And Haplotype Block Construction Of Chinese Han In Chromosomal 5q31 Regions

Study on Graves Susceptible Gene and Haplotype Block Construction of Chinese Han in Chromosomal 5q31 RegionsObjectivesGraves' disease was a kind of common autoimmune thyroid disease, and genetic factors played a pivotal effect in its etiology. Graves disease had significant familiar history, and an increased relative risk of more than 15 was seen in siblings, compared with the general population (λs). Moreover, an increased concordance rate in monozygotic, compared with dizygotic twin, and the thyroid auto-antibodies, markers for AITD, were positive in 50-70% Graves family members. The research models of complex polygenic disease susceptible genes usually referred those of monogenic diseases, i.e. linkage analysis based on pedigrees and association study based on random populations.Single Nucleotide Polymorphism was the single base mutation in the genome sequence. As the third genetic marker, SNP has important significance in susceptible genes studies, especially those located in the gene regulatory or coding regions, which could result in the quantity or quality alteration of the coding proteins. Recent studies showed that, the SNPs in human genome were not randomly assembled, but in typical haplotype block structures separated by the recombination hot spots. Within each haplotype block, the linkage disequilibrium degree was very high, and only several major haplotypes existed. If the pathogenic polymorphisms located in the haplotype block structures, their frequencies should be accord with the examined polymorphisms in the haplotype blocks, or at least accord with the major haplotypes determined by the examined SNPs.In 2003, Jin Ying et al succeeded in mapping a major susceptible locus for Chinese Graves disease in chromosomal 5q31 regions (D5S436-D5S434), through genome wide scan in 54 multiplex Graves pedigrees. This finding was consistent with the Japanese researchers' results through affecting spring pairs, however, was different from the findings in Caucasian populations. These suggested that, the East Asians might share similar Graves susceptible locus.This study was aimed to research the Graves susceptible loci in the chromosomal 5q31 regions, evaluate the plausible associate gene(s) or SNPs for Graves disease, and study the clinical variables difference of Graves patients with different genotypes at certain SNPs loci.Materials and MethodsIn order to further study the Graves susceptible locus, we selected 14 candidate genes for Graves' disease in the mapping regions, including IL4, IL13, IL12B, IRF1and UGRP1 genes, sequenced all the coding areas and about 1,000bp promoter areas to screen the SNPs, and carried out association study in the plausible significant SNPs through multiplex Graves pedigrees and random population. According to the current knowledge of Graves pathogenesis, we performed systemic and detailed study in IL4, IL13, IL12B, IRF1, UGRP1 and ADRB2 genes from the above 14 candidate genes, including association study in extended samples, constructing haplotype block structure, intra-pedigrees transmission disequilibrium test, and comparisons in Graves' patients different clinical variables at certain polymorphism loci.We further selected 74 SNPs between the two micro-satellite markers D5S436 and D5S434. The enrolled criteria were that minor allelic frequency of the selected polymorphisms was higher than 20% and these SNPs should be located adjacent the gene coding areas or promoter regions. We constructed the haplotype block structures and identified the represent tagSNPs via pyro-sequencing protocols.Results Totally, we sequenced 63,498bp in the 14 candidate genes in chromosomal 5q31 regions, and found 116 SNPs, including 17 coding SNPs and 35 regulatory SNPs. About every 550bp had one SNP, and the SNPs density in regulatory regions or introns is higher than that in coding regions. Of the examined 116 SNPs, the ratio of transition and transversion was about 2:1. Association Study of IL12B and ADRB2 genes in larger samples showed that, the rs2853697 A/C SNP of IL12B gene had significant different distribution between Graves patients and normal controls (P=0.0024, Pc=0.0168), and no difference were found in other SNP loci.We performed systemic association study for IL4, IL13, IRF1, UGRP1 genes and Chinese Graves disease in 54 multiplex Graves pedigrees, 146 sporadic Graves patients and 142 unrelated controls. The results showed that, none of the 22 polymorphisms in these candidate genes was associated with the etiology of Graves' disease in random samples or had dominant transmission from heterozygous parents to affected offspring. The haplotype analysis in IRF1 and UGRP1 genes didn't found statistical difference, either. However, the patients' onset ages who were TT allele at IRF1 6477 T/G locus were 37.73±11.69 years, which were much earlier than those who were TG (42.16±13.75 ) or GG ( 43.24±15.20 ) alleles, and had statistical significance (P=0.005, Pc=0.020).The results of haplotype blocks construction in chromosomal 5q31 regions (D5S436-D5S434) indicated that, this chromosomal region could be separated by 8 haplotype blocks, and the longest haplotype block even reached 77kb. In every haplotype block, the chromosomal recombination events seldom happened, and only contained several (1-4 kinds) major haplotypes. Moreover, each haplotype structures could be represented by several tagSNPs. These results suggested that, in the future studies, only these tagSNPs need to be genotyped, and could represent the whole haplotype block structure of the study samples. Moreover, in the 6th and 8th haplotype blocks, the major haplotype AGCGTC and GGCCT showed different distribution between the probands in Graves' multiplex pedigrees and normal control individuals, which had statistical significance (P=0.0064 and 0.0274, respectively, the former P value became 0.0256 after correction).Conclusions1. In the screened 14 candidate genes, SNPs density was about one per 550bp bases, and the promoter regions and introns had higher SNPs density. The ratio of transition and transversion was about 2:1.2. The rs2853697 locus of IL12B gene might be associated with Chinese Graves disease.3. The ADRB2 gene was not associated with Chinese Graves disease.4. IL4, IL13, IRF1 and UGRP1 genes were not associated with Chinese Graves disease.5. In the IRF1 6477 T/G locus, those Graves patients with TT genotype had earlier onset ages than those with TG or GG alleles.6. The Chinese Han population had typical haplotype block structures in chromosomal 5q31 regions (D5S436-D5S434), and the longest haplotype block was 77kb. In each haplotype block, the chromosomal recombination events were seldom seen, and only several kinds of major haplotypes existed. Moreover, each haplotype block could be represented by few tagSNPs.7. The AGCGTC haplotype and GGCCT haplotype in the sixth and eighth haplotype blocks had statistical different distribution between Graves probands in multiplex pedigrees and normal control individuals.