Investigation And Genetic Analysis Of Chinese Han Graves' Disease Multiplex Pedigrees

Introduction: Graves' disease(GD)is a common endocrine disease,and its morbility is about 1%.At present,both of GD and Hashimoto's thyroiditis(HT),the organ-specific autoimmune disease,were recognized as a kind of autoimmune thyroid disease(AITD).GD is characterized by hyperthyroxinemia,diffuse goiter,and positive thyroid autoimmune antibodies.Although the immune etiopathogenisis of it is the producing of thyroid stimulating antibody(TSAb),which has been known clearly,the precise mechanisms of it in genetics remain unknown.However,there is sufficient evidence showing that genetic factors play an important role in the occurrence and development of GD:(1)The condensation in the homozygotic twins is 20%,which is much higher than the dizygotic twins;(2)There is a familial aggregation phenomenon in GD,the sick risk in compatriots of patient is 15 times higher than the general population.Like other complex polygenetic diseases,GD does not follow the typical Mendel's Law.The genetic susceptibility may be dependent on different penetrance of genes or on environmental factors,which bring great difficulties to study its predisposing genes.Up to now,the research strategy of polygenetic diseases still follow that succeeded in single-gene disease model,such as positional cloning and candidate gene strategies,but it dose not mean that the approach exist in isolation.In 2003,Jin et al.of our group revealed the strongest evidence for linkage in chromosome 5q31(D5S436-D5S434)by linkage analysis in 54 Chinese Han multiplex GD pedigrees.In 2005,Yu et al.of our group finished the systemic association study between UGRP1,IL13,IL4 and IRF1 genes,and more than 1000 bp SNPs in the promoter region and Chinese Han Graves disease in 54 multiplex pedigrees,142 unrelated controls and 146 sporadic Graves patients.The consequences suggested that there was no significant link between the SNPs and GD.But,the onset ages of patients with with TG or GG genotype at IRF1 6477 T/G locus were much later than those who were TT genotype significantly.After Yu examined 74 SNPs,haplotype blocks construction in chromosomal 5q31 region(D5S436-D5S434)was found.This chromosomal region could be separated into 8 haplotype blocks,the length of which were 77 kb,71kb,8kb,18 kb,7kb,29 kb,3kb,and 4kb respectively.Every haplotype block only contained several(1-4 kinds)major haplotypes(the frequency of more than 10%).Moreover,each haplotype could be represented by several tag SNPs.Objective: There were eighty GD multiplex families investigated in the study(469 members,197 GD patients,5.8 members per pedigree).To investigate the inherited situation of thyroid autoimmune antibodies in GD multiplex families,and its effects in the development of GD,we performed a eight-year follow-up study in the pedigrees..Based on the discovery of haplotype in human chromosome,the great prospects on application of tag SNPs,as well as the basic research of our group,the further researches would investigated.(1)The predisposing genes of GD would be mapped finely by more microsatellite makers.(2)GD predisposing genes in the Han nationality in China would be study by tag SNPs in haplotype region,which were constructed by chromosome 5q31.Subjects and Methods: In order to study the developmental pattern of GD,we conducted a eight-yeat follow-up study in the 54 multiplex GD pedigrees.Meanwhile,we collected twenty six multiplex GD pedigrees,which used in the fine mapping and profound study on predisposing genes of GD.We selected 8 microsatellite makers whose heterozygosity more than 70 percent from the chromosome region of D5S436-D5S434 to do the fine mapping study;besides,we also detected the tag SNPs of two blocks from the haplotype blocks construction in chromosomal 5q31 region(D5S436-D5S434)we had founded,which was more likely associated with GD.On that basis,we did Linkage analysis,Transmission Disequlibrium Test(TDT),association study.Results: 1.The prevalence of overt hypothyroidism,subclinical hypothyroidism and subclinical hyperthyroidism in the first and second-degree relatives(FSDR)of GD patients was 0.7%,2.2% and 4.8%,respectively;the positive rate of TPOAb,TgAb,TRAb in euthyroid FSDR of GD patients was 19.3%,16.5% and 17.7% respectively.2.In the multiplex GD pedigrees,the prevalence of GD increased obviously when urinary iodine reached 500~599mg/L.3.In the euthyroid subjects(without GD)at basline,the morbidity of subclinical subclinical hyperthyroidism,overt hypothyrodism and subclinical hypothyrodism was 2.0%,1.0%,6.9% respectively,and the positive incidence of TPOAb and TgAb was 4.2% and 2.8%.4.In the euthyroid subjects(without GD)at basline,the morbidity of subclinical subclinical hyperthyroidism,overt hypothyrodism and subclinical hypothyrodism was 6.7%,16.7%,3.3% respectively in the positve thyroid antibodies group,while 0.0%,2.8%,0.0% respectively in the negative group.5.GD is a kind of complex,of which the inheritance patterns and penetrance is unknown.It has obvious genetic heterogeneity.We did parametric linkage analysis under different inheritance patterns and penetrance.The maximun two-and multiple-points HLOD score were 4.29(?=0.42)and 4.01(?=0.34),that suggested a linkage between GD and 5q31 region.6.Nonparametric linkage analysis showed that the maximun NPL score was 3.12,that also suggested a linkage between GD and 5q31 region.7.No clear difference has been found in alleles and genotypes frequencies of all selected SNPs(SNP1-rs7713010 C/T,SNP2-rs12653715 G/C,SNP3-rs12653081 T/C,SNP4-rs6882292 G/A,SNP5-rs1368408 G/A,SNP6-rs3910207 C/T,SNP7-rs3843496 C/T,SNP8-rs3910183 T/G,SNP9-rs2853697 A/C).8.The GG homozygote at SNP2 was associated with GD(GG vs.GC+CC,OR=1.46,95%CI=1.011~2.108,P=0.043).9.Transmission Disequilibrium Tests showed the G allele at SNP2(P=0.0416),C allele at SNP3(P=0.0005)and the SNP2 G+ SNP3 C hyplotype(P=0.0005)was associated with GD.Those SNPs were in JAKMIP2 gene.10.Transmission Disequilibrium Tests showed that the SNP4,SNP5,SNP6,SNP7,SNP8 in and around SCGB3A2 gene were not related to GD(P>0.05).11.The association study showed that the SNP9 in IL12 B intron was not related to GD.Conclusions: 1.In the first and second-degree relatives(FSDR)of GD patients,the prevalence and morbidity of thyroid dysfunction was not different from general population,while the prevalence of positive thyroid autoimmune antibodies was much higher than that of general population.2.Excessive iodine intake might be a risk factor of GD in subjects with obvious inherited tendency of GD.3.Positive thyroid autoimmune antibodies was a risk factor of GD in FSDR of GD patients.4.Comparison with the results obtained in the original data set showed that the 5q31 region continued to show evidence for linkage in the expanded data set.5.JAKMIP2 gene might be a major predisposing gene of Chinese Graves' disease,and JAKMIP2-1(SNP2 G+SNP3 C)was an risk haplotye for Graves' disease.6.SCGB3A2 gene wasn't associated with Chinese familial Graves' disease.7.The SNP9-rs2853697 A/C locus of IL12 B gene didn't associated with Chinese Graves disease.