Alterations Of Mitochondrial DNA And Epigenetics In Sporadic Papillary Thyroid Carcinoma

Alterations of mitochondrial DNA and epigenetics in sporadic papillary thyroid carcinomaSun Tuan-qi Supervisor: Professor Wu YiObjective: (1) To analyze the somatic mutations in the D-loop of mtDNA and further evaluate the possibility of mitochondrial genetic instability in papillary thyroid carcinoma. (2) To elucidate the relationship between thyroid tumorigenesis and the mtDNA content alterations, and further to explore the possible correlations of the mtDNA content in papillary thyroid carcinoma with traditional clinicopathologic parameters. (3) To investigate promoter methylation status of RASSF1A, P16^INK4a, and ATM genes in papillary thyroid carcinoma, and to explore its correlation with major clinical pathological features.Methods: (1) Hypervariable regions (HVR-Ⅰand HVR-Ⅱ) in the D-loop of mtDNA from the specimen of 35 papillary thyroid carcinomas and matched lymphocytes were amplified by PCR, and then were sequenced. (2) The mtDNA content in 54 pairs of normal and thyroid neoplasm samples were examined by TaqMan quantitative real-time PCR analysis, including 29 papillary thyroid carcinomas, 14 thyroid adenomas and 11 nodular goiters. The relationship between mtDNA content and thyroid aggressiveness is further discussed. (3) Methylation-specific PCR (MSP) was performed to detect the methylation status in the promoter region of RASSF1A, P16^INK4a, and ATM genes in 77 pairs of normal and thyroid neoplasm samples, including 52 papillary thyroid carcinomas, 14 thyroid adenomas and 11 nodular goiters.Results: (1) Comparing the sequences of tumors to those of matched lymphocytes, 5 somatic mutations in 2 patients (5．7%) were found. Two mutations were insertions of C in a poly-cytidine (nt303) microsatellite, and 3 at positions 73, 152 and 194 in HVR-Ⅱ. In addition, of the 294 genetic variants detected, 292 were previously recorded polymorphisms, whereas 2 were new polymorphisms (nt324: C→G; nt16092: T→A). (2) The data showed that the relative mtDNA contents were increased in the PTC samples compared to their normal thyroid tissues taken from the same individual (224．13±217．24 vs. 88．69±44．06; P<0．01) , also, mtDNA contents in PTC samples were higher than that in the thyroid benign lesions (224．13±217．24 vs. 129．48±105．27; P<0．05) . However, no significant difference was observed in mtDNA content between the thyroid benign lesions and matched normal tissues, nor between the thyroid adenoma and nodular goiter (all P>0．05) . We further analyzed the mtDNA content in 29 paired tumor and normal thyroid tissue DNA from patients with PTC. Our results showed that of the 29 pairs, 21of 29 (72．41%) had increased mtDNA content in the tumor samples, 6 of 29 (20．69%) harbored less mtDNA content in the tumor and 2 of 29 (6．90%) did not show any difference between the normal and the tumor DNA. Additionally, mtDNA content changes in PTC samples did not correlate with any clinical pathological features such as age, tumor size, lymph node metastasis, nor conclusive stage (P>0．05) . (3) The frequency of promoter methylation of RASSF1A showed 65．4% and 42．3% in PTC and corresponding normal thyroid tissues respectively, The methylation frequency of PTC tissues was higher than that of corresponding normal tissues and thyroid benign lesions (P＜0．05) . The frequency of promoter methylation of p16^INK4a showed 42．3% and 15．4% in PTC and corresponding normal thyroid tissues respectively, the methylation frequency of PTC tissues was higher than that of corresponding normal tissues and thyroid benign lesions (P＜0．01) . Methylation in the promoter region of ATM gene, however, was not found in all thyroid neoplasm samples. In addition, the methylation status of RASSF1A in PTC samples did not correlate with major clinicopathologic parameters such as age, tumor size, lymph node metastasis, nor conclusive stage (P>0．05) . But, lymph node metastasis status showed a statistically significant prevalence for the p16^INK4a promotor methylation (P=0．035) .Conclusions:1．Mutations in the D-loop of mtDNA are found in PTC, this mutation rate is lower than the reported rate of alteration in tumors of other epithelial origin, and further work is required to elucidate the relationship between this mutations and thyroid tumorigenesis.2．The findings indicate that mtDNA content increased during thyroid tumorigenesis, increased mtDNA may cause increased mitochondrial biogenesis attempting to compensate for the loss of oxidative phosphorylation function.3．Changes in mtDNA content did not correlate with tumor grade and metastasis, suggesting that these alterations may occur in the early stages of thyroid tumorigenesis.4．High frequency of aberrant promoter methylation of RASSF1A and p16^INK4a genes exists in PTC tissues. As tumor suppressor gene, their suppressor activity may besilencing by an epigenetic mechanisim.5．Promoter methylation of RASSF1A gene may be involved in the early-stage during thyroid tumorigenesis, and the similar change in P16^INK4a gene is related to lymphatic metastasis of PTC.