Association Of TSHR Intron₁Gene Polymorphisms With Graves Disease

ObjectiveTo investigate the allele and genotype of single nucleotide polymorphisms （SNPs） ofthyroid stimulating hormone receptor gene （TSHR） intron₁region in Shandong LinYiHan population with Graves disease. Then identify the association between these SNPs atTSHR intron₁region and Graves disease. The last to discuss the relationship betweenthese SNPs and many clinical manifestations of GD patients including sex, age, thyroiddiffused goiter, ophthalmic signs, serum concentrations of thyroid hormone （FT₃, FT₄, TSH）and thyroid thyrotropin receptor antibody （TRAb）.MethodsSelecting a total of1759individuals with Graves disease of Endocrinology of LinYiPeople′s Hospital from2007to2009（718ones for GWAS and1041ones for replication）.The diagnosis of Graves disease was established on the basis of China2008Graves diseasediagnostic criteria of Guide to Diagnosis and Treatment of thyroid disease in China.Collect1740subjects from LinYi People′s Hospital over the same period whose sex werecomparable with the patients and have no blood relations as a control group （739ones forGWAS and1001ones for reputation）. All the control samples were larger than40years oldto reject late-onset autoimmune disease and were all measured the serum concentrations ofthyroid hormones and thyroid peroxidase antibody （TPOAb） to get rid of thyroid diseases.Peripheral blood were collected on empty stomach from all the members, detach andextract DNA, conducted the genome-wide association study at TSHR intron₁region,genotyped by Taqman probe technique on Fluidigm Ep1platform and do a series oflinkage analysis, logistic regression analysis, correlation analysis. Meanwhile, the level of serum FT₃, FT₄, TSH and TRAb of the patients were measured, compared the relationshipbetween clinical manifetations of Graves disease and gene polymorphisms.Results1.21SNPs had been discovered at the region TSHR intron₁, and5SNPs wereselected for replication stageGWAS research for718patients and739control subjects revealed that21SNPs hadbeen discovered at the candidate region TSHR intron₁, and11SNPs were significantlyassociated with Graves disease with P﹤0.01. Then,5SNPs, rs179247, rs2284722,rs12101261, rs2300525, rs1711947were selected as representative into replication stagethrough linkage and logistic regression analysis.2. rs12101261was a dominant and independently locus contributing risk toGraves diseaseDuring the replication stage and combine stage, correlation analysis showed thatrs12101261was the strongly significantly associated SNP (Pre=1.76×10^-4, ORre=0.772,Pcom=4.85×10-6, ORcom=0.7906); then stepwise logistic regression and binary logisticregression analysis indentified that rs12101261was the most sufficient to model theassociation of these5SNPs, so we considered rs12101261was a dominant andindependently locus contributing risk to Graves disease.3. The association between polymorphism of rs12101261and the clinicalmanifestations of Graves diseaseThe distribution of three genotypes among rs12101261was not uniform between thetwo groups. The frequency of rs12101261_TT was significantly more higher in GD groupthan in control group (49.8%vs41.95%, P=2.53×10^-3) while the positive rate ofrs12101261_CC between the two groups was adverse （9.15%vs11.21%, P=0.029）. Afterone year treatment, the level of serum TRAb was measured again, and then we divided GDgroup into two subgroups using TRAb1.5U/L. We found three genotypes amongrs12101261show significant difference between the two subgroups （P=0.014）. In patientswith persistent positive TRAb level, rs12101261_T was strongly associated with Gravesdisease, however, the association was obviously weaker in patients turn into negativeserum TRAb level. Furthermore, except TRAb, no other clinical feathers like sex, age, thyroid diffused goiter, ophthalmic signs, serum concentrations of thyroid hormone （FT₃,FT₄, TSH） showed significant difference among three genotypes of rs12101261.Conclusion1. The region TSHR intron₁was associated with Graves disease in ShandongLinYi Han population. A series of SNPs reached significant P value had been found at thisregion, some of them were high related to the development of Graves disease.2. rs12101261at the region TSHR intron₁was relatively an independentsusceptibility locus contributing risk to Graves disease. Furthermore, rs12101261wassignificantly associated with patients who showed persistent TRAb positivity and allelers12101261_T was the main pathogenic risk gene to Graves disease. Except TRAb, noother clinical feathers like sex, age, thyroid diffused goiter, ophthalmic signs, serumconcentrations of thyroid hormone （FT₃, FT₄, TSH） showed remarkable difference betweenpolymorphism of rs12101261and Graves disease.