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Study on Abnormal Methylation of Several Tumor Suppressor Genes in Pheochromocytoma and Paraganglioma

Author: FuChunLi
Tutor: ZengZhengPei;XingXiaoPing;SunZuo
School: Beijing Union Medical College
Course: Endocrine and metabolic diseases
Keywords: Pheochromocytoma Paraganglioma Adrenal medulla Methylation specific PCR CpG island methylation phenotype Methylation index Multiple tumor suppressor gene ( MTSI , p16INK4a ) Ras association domain family 1A gene Decoy receptor 2 (DcR2) O ~ 6 - methylguanine - DNA methyltransferase (MGMT ) Semi- nested methylation -specific PCR p16INK4a RASSF1A Messenger ribonucleic acid Real - time quantitative PCR
CLC: R739.4
Type: PhD thesis
Year: 2011
Downloads: 103
Quote: 0
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Abstract


Objective: identification of benign and malignant pheochromocytoma (PHEO) and paraganglioma (PGL), and effective treatment of malignant pheochromocytoma neuroendocrine tumor research unsolved problem. Epigenetics addition to genetic alterations in tumors and diseases is another important area of ??current research. Pheochromocytoma epigenetic research abroad has only just begun, the choice of the study of genes related to cell cycle regulation of p16INK4a DNA repair gene (?) MLH1, MGMT, BRCA1, ras-related signal transduction pathway genes RASSFIA related apoptosis DcR2 gene, the mutation causes pheochromocytoma occurred SDHB and SDHD, VHL gene, and preliminary studies by our group show low expression in pheochromocytoma of LRP1B epigenetic in methylation-specific PCR (MSP) to study the gene methylation status, looking for pheochromocytoma CpG island methylation phenotype; analysis of changes in gene methylation patients with benign and malignant tumors and other clinical features the relationship between the. Methods: 1. Of use EZNATM DNA / RNA Isolation Kit to extract 24 cases PHEO (10 malignant and 14 benign), 29 cases of PGL (14 malignant and 15 benign) and 7 patients with normal adrenal medullary tissue DNA; methylation specific PCR method to detect the different tissues of p16INK4a, RASSF1A, DcR2, MGMT, SDHB, SDHD, VHL, LRP1B, BRCA1, hMLH1 gene methylation status, explore the DNA methylation of these genes in PHEO and PGL ; 3. analysis of the methylation status of gene promoter region in patients with benign and malignant tumors and other clinical data, understanding of the pathophysiological significance PHEO and PGL. Results: 1 of p16INK4a methylation of RASSF1A, MGMT, DcR2 gene PHEO and PGL, change 1) were detected in 53 cases of PHEO / PGL organization of p16INK4a, RASSF1A, MGMT, DcR24 gene methylation, the prosecution a rate of 52.8%, 52.8%, 35.8%, 60.4%. In PHEO 4 gene methylation detection rate were 58.3% (14/24), 58.3% (14/24), 41.7% (10/24), 54.2% (13/24), including malignant respectively 33.3% (8/24), 25.0% (6/24), 20.8% (5/24), 20.8% (5/24); 4 in PGL gene methylation detection rates were 48.3% (14 / 29), 48.3% (14/29), 31.0% (9/29), 65.6% (19/29), malignant were 37.9% (11/29), 34.5% (10/29), 20.7% ( 6/29) and 48.3% (14/29); 2) p16INK4a in benign and malignant PHEO, the the methylation detection rate of benign and malignant PGL were 42.9% (6/14), 80.0% (8/10) 20.0% (3/15) and 78.6% (11/14); 3) RASSF1A gene methylation in benign and malignant PHEO, benign and malignant PGL detection rate was 57.1% (8/14), 60.0% ( 6/10), 26.7% (4/15) and 71.4% (10/14); 4) MGMT gene methylation in benign and malignant PHEO, benign and malignant PGL detection rate was 35.7% (5/14) , 50.0% (5/10), 20.0% (3/15) and 42.9% (6/14); 5) DcR2 gene methylation in benign and malignant PHEO, benign and malignant PGL detection rate of 57.14% ( 8/14), 50.00% (5/10), 33.33% (5/15) and 100% (14/14); 6) in malignant PHEO and PGL at least one gene methylation in benign specimens (1 case PHEO, 7 cases the PGL) none of the four genes methylation occurs. CpG island methylation phenotype (CIMP) positive in 19 cases, including five cases of malignant PHEO, 4 benign PHEO; 9 malignant PGL, 1 benign PGL. 2 of p16INK4a, RASSF1A, MGMT, DcR2 gene methylation clinical significance) in patients with malignant PGL 24h urinary norepinephrine (NE), neuron-specific enolase (NSE) levels than benign patients, statistical difference learn significance (p lt; 0.05) 2) the PHEO p16INK4a gene methylation patients 24h urinary epinephrine (E) is higher than the non-methylated, the difference was statistically significant (p = 0.014). 3) PGL the, p161NK4a gene methylation more common in malignant patients (vs benign), the difference was statistically significant (p = 0.002) significantly, how seen in the trend of the age of onset, but the difference was not statistically significant (p = 0.085). RASSFIA gene methylation is more common in malignant patients (vs benign), the difference was statistically significant (p = 0.016) significantly. DcR2 gene methylation was more common in malignant patients younger onset patients, the difference was significant statistically significant (p lt; 0.01). 4) methylation index (MI) was significantly higher than that in patients with malignant PGL benign patients, a statistically significant difference (p lt; 0.001). CpG island methylation phenotype (CIMP) positive will rise seen in patients with malignant PGL, compared with benign patients the difference was statistically significant (p = 0.002) significantly. 3 p16INKa, RASSF1A, MGMT, DcR2 gene methylation analysis) PHEO in RASSFIA and DcR2 gene methylation has a correlation (r = 0.410, p lt; 0.05). 2) PGL p16INK4a and DcR2 gene methylation correlation (r = 0.556, p lt; 0.01); RASSF1A and MGMT, DcR2 gene methylation correlation (r = 0.396, p lt; 0.05 and r = 0.411 , p lt; 0.05). PHEO and PGL SDHB, SDHD, VHL, LRP1B, BRCA1, hMLH1 gene methylation changes not detected the six gene DNA methylation changes. Conclusion: 1.84.9% (45/53) PHEO and PGL of p16INK4a, more than one of the four gene RASSF1A, DcR2 and MGMT, promoter methylation, suggesting that the tumor suppressor gene DNA methylation play an important role in the occurrence and development of PHEO and PGL; of p16INK4a, RASSF1A, DcR2 gene methylation and the PGL vicious behavior, prompted of p16INK4a, RASSF1A the DcR2 gene promoter methylation state as a differential diagnosis of benign malignant PGL laboratory based on methylation-positive patients should closely follow-up observation. Methylation index (MI) and CpG island methylation phenotype (CIMP) are associated with the degree of malignancy of the PGL prompt malignant PGL multiple tumor suppressor gene methylation level detection of multiple gene promoter region The methylation status of benign and malignant identify the PGL has some significance. Objective: same patient tumor tissue and plasma DNA methylation of the similarities and differences between the experimental part of the patients in the first part of the plasma of p16INK4a the RASSF1A gene DNA methylation, and analysis with the corresponding tumor gene A -provide the basis for the clinical detection. Method: 1. Extracted using the Qiagen DNA blood mini kit malignant paraganglioma (n = 8), benign paraganglioma (n = 6), malignant pheochromocytoma (n = 7), benign pheochromocytoma ( n = 8) of plasma DNA; semi-nested methylation-specific PCR method to detect the plasma of p16INK4a, RASSF1A gene methylation status; comparison of plasma gene promoter region methylation and organizations in gene methylation status and and the relationship between the clinical characteristics. Results: 1.29 cases plasma corresponding tumor tissue p16INK4a gene methylation detection rate were 44.83% (13/29) and 55.17% (16/29), plasma and tumor tissue methylation detection rate difference was not statistically significance; p16INK4a promoter methylation in the plasma and tumor tissue of PHEO / PGL significant positive correlation (r = 0.813, p lt; 0.001); 2.29 cases of plasma and corresponding tumor tissue RASSF1A gene methylation detection rate were 31.03% (9/29) and 44.83% (13/29), no significant difference between the plasma and tumor tissue methylation detection rate; RASSF1A gene promoter methylation in PHEO / PGL plasma and tumor organization a significant positive correlation (r = 0.694, p lt; 0.001); 3 of p16INK4a gene methylation in sex, age, genetic, tumor location, number, size, Ki-67%, there is no statistical learning differences (p gt; 0.05). p16INK4a gene methylation is more common in malignant patients, the difference was statistically significant (p lt; 0.05); plasma RASSF1A gene promoter region methylation gender, age, type of tumor, tumor number, size, 24h urinary catecholamine levels, Ki-67% and so no significant correlation (p gt; 0.05). Conclusion: The application of the semi-nested MSP detected in plasma the p16INK4a and RASSFIA gene DNA methylation status associated with the methylation status of the corresponding tumor tissue; clinical early detection to identify benign and malignant tumors. Objective: To study the first and second part of the detection of p16INK4a in PHEO and PGL tumor tissue and plasma RASSF1A gene methylation changes in the study was to investigate of p16INK4a, RASSF1A mRNA in normal adrenal pheochromocytoma and vice ganglion tumor expression in the medulla, to explore its relationship with clinical characteristics and gene methylation PHEO and PGL. Methods: 1. Of use EZNATM DNA / RNA Isolation Kit to extract 24 cases PHEO (10 malignant and 14 benign), 29 cases of PGL (14 malignant and 15 benign) and 7 patients with normal adrenal medullary tissue DNA; real-time fluorescence quantitative PCR method to detect the different organizations of p16INK4a, RASSFIA genes and an internal reference β-actin mRNA expression; 3. analysis the p16INK4a, RASSFIA mRNA expression with the clinical characteristics of patients with PHEO and PGL and gene promoter methylation relationship. Results: 1. P16INK4a mRNA expression in PHEO and PGL 1) p16INK4a mRNA expression in benign and malignant PHEO and benign and malignant PGL were significantly lower than the normal adrenal medulla, the difference was significant statistically significant (p lt; 0.01) ; 2) in benign and malignant PHEO between four groups of benign and malignant PGL p16INK4a mRNA expression was no statistically significant difference (vs malignant PGL, p = 0.088,0.213,0.367) 2. RASSF1A mRNA expression in PHEO and PGL 1) RASSF1A mRNA expression in benign and malignant PHEO and benign and malignant PGL were significantly lower than the normal adrenal medulla, the difference was statistically significant (p lt; 0.05); 2) of benign and malignant PHEO between four groups of benign and malignant PGL RASSFIA mRNA expression the difference was not statistically significant (vs malignant PGL, p = 0.314,0.732,0.876) 3. of p16INK4a and RASSF1A mRNA expression with PHEO and PGL patients with clinical features of the relationship between 1) PHEO and PGL mRNA expression in different gender, age, tumor location no significant differences between the clinical features, the number, size, Ki-67-positive rate (p gt; 0.05). 2) PHEO and PGL RASSF1A mRNA expression between patients with different clinical characteristics also no significant difference (p gt; 0.05). 4.p16INK4a and RASSF1A mRNA expression and the relationship between gene promoter methylation 1) p16INK4a mRNA expression detected between p161NK4a gene methylation and non-detected methylation PHEO / PGL difference was not statistically significant (p are gt; 0.05). p16INK4a mRNA expression level of gene methylation status was no significant correlation (p gt; 0.05). 2) RASSF1A mRNA expression detected difference between RASSF1A methylation and non-detected methylation PHEO / PGL was not statistically significant (p gt; 0.05). RASSF1A mRNA expression level of gene methylation status was no significant correlation (p gt; 0.05). Conclusion: p16INK4a] RASSF1A gene mRNA expression decreased in PHEO and PGL p16INK4a. RASSF1A mRNA expression in gene methylated and unmethylated patients the difference was not statistically significant, suggesting that gene promoter methylation of p16INK4a and one of the main mechanism of RASSF1A gene inactivation, at the same time there may be other mechanisms involved in gene inactivation, such as heterozygous deletion, and other abnormal epigenetic (histone acetylation, miRNA interference).

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