Association Study Of The Polymorphism In DNA Double Strand Break Repair Gene And Gene Rearrangement In Papillary Thyroid Carcinoma

ObjectivesTo investigate the prevalance and clinicopathological significance of RET rearrangement,BRAF mutation,H4-PTEN gene rearrangement in papillary thyroid carcinoma,the association of XRCC3 gene(one of DNA double strand break repair genes) polymorphism and predisposition to papillary thyroid carcinoma or with gene rearrangement.Subjects and MethodsPARTâ… One hundred and fourty three thyroid tumors were enrolled in this study which included 6 nodular goiter,2 folicular tumor,7 medullary thyroid carcinoma and 126 papillary thyroid carcinoma.Total RNA were isolated from the tumor tissues and para-tumor tissues and reversely transcribed to cDNA.RET/PTC-1,RET/PTC-2,RET/PTC-3 and ELKS/RET fusion genes were amplified by PCR and confirmed by sequencing.The clinicopathological significance of RET rearrangement was analyzed.PARTâ…¡Total RNA and DNA of tumor and para-tumor tissues of 125 papillary thyroid carcinoma were isolated.BRAF gene coding sequence were amplified by PCR with DNA as template and sequenced.H4 and PTEN rearrangement,H4/PTEN,was amplified by PCR with cDNA as template and sequenced.According to the sequencing results of H4/PTEN,new primers were designed to amplify PTEN/H4,the reciprocal rearrangement of H4/PTEN,and the PCR products were sequenced to confirm the rearrangement style.The interaction,exclusivity and clinicopathological significance of RET rearrangement,BRAF mutation and H4-PTEN rearrangement were analyzed.PARTâ…¢The genomic DNA of 173 papillary thyroid carcinoma patients and 120 normal controls were isolated and used as the template to amplify the DNA double strand break repair gene XRCC3 exons.The PCR products were electrophoresed on DGGE(Denatured Gradient Gel Electrophoresis) system to detect the mutaions.The mutation positive exons were reamplified and sequenced from both the cases and controls to get the mutation allele. Association study of XRCC3 polymorphism and papillary thyroid carcinoma predisposition were analyzed by case-control study including the patients and normal controls.The XRCC3 polymorphisms were compared between the gene rearrangement positive patients and negative patients to get the relationship of DNA double strand break repair gene polymorphisms and gene rearrangement.ResultsPARTâ… RET rearrangement,which included 12 RET/PTC-1 and 6 RET/PTC-3,was only detected in 14.3%(18 of 126) of PTC and was not observed in non-PTC thyroid tumor and para-tumor normal tissues.No RET/PTC-2 and ELKS/RET were detected in this serial study.The patient group of age＜20 years had the highest frequency(50%) of RET rearrangement among the groups of age＜20 years,20-40 years and≥40 years(P = 0.033).RET/PTC-1 positive patients were more easily to suffer from another benign thyroid disease simultaneously(P=0.021),especially thyroiditis.RET/PTC-3 positive patients had a higher frequency of extrathyroidal extension(P =0.005) and advanced T classification(P = 0.007).RET rearrangement (OR = 8.703,95%CI 1.690-44.818),male(OR = 3.875,95%CI 1.405-10.685), age(OR = 0.964,95%CI 0.933-0.996),multifocality(OR = 3.543,95%CI 1.333-9.416) and advanced T classification(OR = 7.318,95%CI 2.910-18.400) were all identified as risk factors of levelâ…¡-â…¤lymph node involvement in the multivariate analysis.PARTâ…¡Among 125 cases of papillary thyroid carcinoma,58 cases of BRAF mutation(46.4%),12 cases of RET/PTC-1(9.6%),6 cases of RET/PTC-3(4.8%),6 cases of H4/PTEN(4.8%) and 7 cases of PTEN/H4(5.6%) were dectected.Eleven cases(19.0%) of BRAF mutation positive patients were found with another genetic aberration and the frequency of multi-genetic aberrantions of RET rearrangement and H4-PTEN rearrangement were 38.9%(7/18)and 75%(9/12),respectively.H4 gene (CCDC4),the fusion parter of RET/PTC-1 and H4-PTEN,is the most frequently involved gene in rearrangement(17.6%,22/125).The age of gene rearrangement positive patients was younger than that of the negative patients(36.81±12.416 VS 43.53±13.890,P= 0.025),and the highest frequency of gene rearrangement were detected in patients with age＜20 years(50%).BRAF was the only genetic aberration in patients of age≥60 years(81.8%).The frequency of gene rearrangement was decreasing with the age while the trendency of BRAF mutation was reverse.Rearrangment positive patients had a higher lymph node involvement than negative patients(92.6%VS 70.4%,P=0.018).PARTâ…¢According to the sequence of XRCC3(AF037222),6 SNPs were detected,which included 6390G＞T(located at 5' -UTR),6410A＞G(dSNP number:rs56377012),14242G＞A(Val165Ile),14304C＞T,14362T＞C and 18440C＞T.The heterozygosity of both 6410A＞G and 14304C＞T of normal controls were 2.5%,and their wild type allele frequcncy were 98.8%, while other four polymorphisms were incidental mutation with lower heterozygosity.The allelic and genotypic frequency of 6410A＞G and 14304C＞T were tested with Hardy-Weinburg Equilibrium(P＞0.05).6410A＞G allele G was associated with papillary thyroid carcinoma(the allele frequency of case and control were 4.3%and 1.3%,respectively),P =0.033(OR 3.580,95%CI 1.025-12.505).Haplotylpe analysis of 6410A＞G and 14304C＞T showed that the frequency of haplotype A⁶⁴¹⁰C¹⁴³⁰⁴ at case and control were 97.5%and 93.1%,and haplotype A⁶⁴¹⁰C¹⁴³⁰⁴ had a protective effect on papillary thyroid carcinoma genetic predisposition(P=0.017,OR 2.907,95%CI 1.170-7.224) when compared with the less frequent haplotypes (A⁶⁴¹⁰T¹⁴³⁰⁴,G⁶⁴¹⁰C¹⁴³⁰⁴,G⁶⁴¹⁰T¹⁴³⁰⁴).The frequency of RET rearrangement of tumor samples of XRCC3 6410A＞G A/G and GG cases were higer than the XRCC3 6410A＞G AA cases(25%V.S.14%,P=0.335).ConclusionPARTâ… Only two RET rearrangements,RET/PTC-1 and RET/PTC-3,were detected in Chinese patients with papillary thyroid carcinoma in the present study.The patients with age＜20 years have the highest frequency of RET rearrangement among the four age groups(50%).RET rearrangement is associated with Levelâ…¡-â…¤lymph node metastasis.Male,young age, multifocality,advanced T classification and RET rearrangement were indentified as the risk factors of lateral neck lymph node metastasis. Serious pre-operative scanning and intensive post-operative followup should be performed to the patients with the risk factors.RET/PTC-1 and RET/PTC-3 have different role in the progression of throid cancer,and RET/PTC-1 may be associated with thyroiditis.PARTâ…¡The frequency of gene rearrangement was decreasing with the increasing age.While BRAF mutation was the only genetic aberration of papillary thyroid carcinoma in patients of age≥60 years(81.8%) and have a reverse trendency.RET rearrangement,BRAF mutation and H4-PTEN rearrangement can be detected in one tumor sample simultaneously, especially in patients with young age.The predictive value of molecular changes as a diagnostic and target therapeutic markers will be improved by screening RET rearrangement,BRAF mutation,H4-PTEN rearrangement and other possibly involved genes together.PARTâ…¢XRCC3 gene 6410A＞G(rs56377012)G allele is associated with papillary thyroid carcinoma risk.Haplotylpe analysis of 6410A＞G and 14304C＞T shows that haplotype A⁶⁴¹⁰C¹⁴³⁰⁴ has a protective effect on papillary thyroid carcinoma genetic predisposition compared with the less frequent haplotype(A⁶⁴¹⁰T¹⁴³⁰⁴,G⁶⁴¹⁰C¹⁴³⁰⁴,G⁶⁴¹⁰T¹⁴³⁰⁴).The frequency of RET rearrangement of tumor samples of XRCC3 6410A＞G A/G and GG cases is higer than that of XRCC3 6410A＞G AA cases.These SNPs may affect the papillary thyroid carcinoma risk as a potential etiological factor or just becase of linkage disequilibrium with another etiological SNP.Futher experiments need to be carried out to confirm these associations.