2068 DNA peripheral blood mononuclear cell PBMC DNA 1 2 DNA 1 EDTA DNA 4 6 DNeasy Blood & Tissue Kit QIAGEN DNA - 80 Table 1. 1 Basis clinic data of p

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1 Chinese Journal of Pathophysiology ABCG4 * DNA DNA DNA 627 FZD7 LRP TTBK1 USFP1 SYCE1 NDUSF8 ZNF540 ABCG4 8 PCR 8 ABCG4 ABCG % 32. 4% P < ABCG4 ABCG4 R A doi /j. issn Analysis of abnormal methylation in ABCG4 gene promoter in primary hypertension GUO Jun 1 CAI Jun 2 LI Zi - cheng 1 1 Department of Cardiology The First Affiliated Hospital of Jinan University Guangzhou China 2 Heart Center Beijing Chaoyang Hospital Capital Medical University Beijing China. E - mail zichengli@ 163. net ABSTRACT AIM To analyze the variety of DNA promoter methylation and epigenetic pathogenesis of human primary hypertension. METHODS Genomic DNA was collected from peripheral blood mononuclear cells in normal control group and primary hypertension group. High - throughput screening of 627 diversely - methylated genes was performed by DNA methylation chip. The genes of FZD7 LRP TTBK1 USFP1 SYCE1 NDUSF8 ZNF540 and ABCG4 were considered as candidate hypertension - related genes and they were analyzed with bisulfite sequencing PCR. RESULTS Among 8 candidate genes ABCG4 gene presented the most different DNA methylation. In primary hypertension group the promoter of ABCG4 gene presented hypomethylation and the percentage of methylation was 18. 3% while that in normal control group was 32. 4% P < CONCLUSION ABCG4 gene promoter presents hypomethylation suggesting that it may be related to the pathogenesis of primary hypertension. KEY WORDS Methylation Hypertension Promoter Genes ABCG4 90% DNA X RNA 2 1 PCR * No No No. LYM Tel E - mail zichengli@ 163. net

2 2068 DNA peripheral blood mononuclear cell PBMC DNA 1 2 DNA 1 EDTA DNA 4 6 DNeasy Blood & Tissue Kit QIAGEN DNA - 80 Table 1. 1 Basis clinic data of patients 珋 x ± s Group n Age year Sex M /F SBP mmhg DBP mmhg Body mass index kg /m 2 Duration of hypertension Year Primary hypertension ± / ± ** ± ** ± ± 2. 1 Control ± / ± ± ± ** P < vs control. M male F female. 3 DNA pmd18 - T TaKaRa 2 h 10 EZ DNA Methylation Kit DNA μl DH5α Amp + CG C T DNA NaOH MA2 Multi Sample Amplification 2 Mix MSM Multi Sample Amplification Master Mix h DNA FMS Fragmentation Solution 37 1 h PM1 DNA DNA RA h DNA Gene Primer Product bp h ZNF540 5' - TTGGGTTGTAGTTTGAGAGTTTTG - 3' WB1 PB1 1 min 5' - CACAACCAAACAACTCCTAAAACT - 3' 467 DNA FZD7 5' - GTTGGTGAAGGTGTAGTGTTTTTT - 3' DNA TEM 5' - CCTCTCCTCCTCCTTAAAATACA - 3' 488 SBE biotin - dntp DNP LRP3 5' - GTTTGTTTGGGTGAGTGGGT - 3' 95% /1 mmol /L 5' - ACCTAACCTTTATCCCCAACTC - 3' 354 EDTA DNA ABCG4 5' - GGGTGTGTATAGTTTAGTAGAGTTAGG - 3' LTM ATM 5' - CCACTCCCCATCCCTTAAC - 3' 423 TTBK1 5' - GAGTTTTAGGGTTGGAGGAGAG - 3' PB1 XC4 5' - CCCCTCACCTACTCTATACCC - 3' 362 UFSP1 5' - GTGTTGGTGGGTGTGGTGAA - 3' 5' - ACCTTCTCTCCCTCTACACTACTAAC - 3' 510 SYCE1 5' - GGTTGGGTTTTGTTATAGGAGGT - 3' 4 PCR bisulfite sequencing 5' - CAACTCCTACACCCACTAAAACTC - 3' 476 PCR BSP NDUFS8 5' - TGGAGTTGGGGGGTAAGAAT - 3' DNA 500ng Methyl- 5' - ACCCAACCTTAAACCCTCCA - 3' 394 Code Bisulfite Conversion Kit Invitrogen DNA 5 DNA PCR 2 PCR PCR PCR PCR 4 ml LB 37 Axygen BiQ Analyzer 2 PCR Table 2. Primers for bisulfite sequencing PCR SPSS ± x 珋 ± s

2069 t 2 t 627 132 1 DNA 495 DNA 1 Figure 1. 1 DNA methylation chip results of chromosomes Chr in human primary hypertension. Dense green sites indicate hypermethylation. DNA 2 PCR 18.

3 2069 t 2 t DNA 495 DNA 1 Figure 1. 1 DNA methylation chip results of chromosomes Chr in human primary hypertension. Dense green sites indicate hypermethylation. DNA 2 PCR 18. 3% FZD7 LRP TTBK1 n = % n USFP1 SYCE1 NDUSF8 ZNF540 ABCG4 8 = 4 BSP 14. 1% P < Table 3. 3 The gene methylation status of primary hypertension group and control group % Group n FZD7 LRP TTBK1 USFP1 SYCE1 NDUSF8 ZNF540 ABCG4 Primary hypertension ** Control ** P < vs control.

2070 Figure 2 The methylation status of ABCG4 gene promoter Open and closed circles indicate unmethylated and methylated CpGs respectively Fork site is undetermined A ABCG4 gene promoter methylation

4 2070 Figure 2 The methylation status of ABCG4 gene promoter Open and closed circles indicate unmethylated and methylated CpGs respectively Fork site is undetermined A ABCG4 gene promoter methylation status in primary hypertension group B ABCG4 gene promoter methylation status in control group 图2 ABCG4 基因启动子区甲基化位点发生甲基化情况讨现高血压大鼠的血管紧张素 II 受体 AT1b 的基因及蛋白表达均显著上调 AT1b 基因近端的启动子高度论原发性高血压的发生机制非常复杂研究者们试图从遗传学改变如单核苷酸多态性的角度寻找原发性高血压的发病机制虽然已经有所发现但是仅能解释少数高血压的发病机制而不能揭示大部分的原发性高血压患者的发病机制近年来一系列的研究报道揭开了高血压表观 3 遗传学机制的冰山一角2007 年 Bogdarina 等发去甲基化新近的临床及基础研究发现高血压患者 2 型 11 β 羟类固醇脱氢酶 11 beta hydroxysteroid dehydrogenase 2 11 β HSD2 基因的启动子区域和 4 5 另外内皮第 1 外显子的 CpG 岛高度甲基化素转换酶 endothelin converting enzyme ECE 1c 的启动子区域的甲基化也被认为和高血压发生密切 6 相关

5 2071 DNA 1 Handy DE Castro R Loscalzo J. Epigenetic modifications basic mechanisms and role in cardiovascular disease J. Circulation ACE DNA I /D J DNA 3 Bogdarina I Welham S King PJ et al. Epigenetic modification of the renin - angiotensin system in the fetal pro- gramming of hypertension J. Circ Res PCR 4 Alikhani - Koopaei R Fouladkou F Frey FJ et al. Epigenetic regulation of 11 β - hydroxysteroid dehydrogenase 627 FZD7 type 2 expression J. J Clin Invest LRP TTBK1 USFP1 SYCE1 NDUSF8 ZNF Friso S Pizzolo F Choi SW et al. Epigenetic control of ABCG4 8 BSP 11 beta - hydroxysteroid dehydrogenase 2 gene promoter is 8 related to human hypertension J. Atherosclerosis % AB- 6 Funke - Kaiser H Thomas A Bremer J et al. Regulation of the major isoform of human endothelin - converting CG4 ATP - ATP - binding cassette ABC G 4 enzyme - 1 by a strong housekeeping promoter modulated 11q23. 3 X by polymorphic microsatellites J. J Hypertens 2003 X ABC 7 Huang Y Sadee W. Membrane transporters and channels in chemoresistance and - sensitivity of tumor cells J. 7 8 GenBank Cancer Lett Oldfield S Lowry C Ruddick J et al. ABCG4 a novel ABCG4 CpG human white family ABC - transporter expressed in the ABDG4 brain and eye J. Biochim Biophys Acta

211 better than those in the control group, with significant difference between two groups (P < 0.05). The ocular hypertension of patients in the cont

211 better than those in the control group, with significant difference between two groups (P < 0.05). The ocular hypertension of patients in the cont 210 毛果芸香碱联合拉坦前列素治疗原发性急性闭角型青光眼的疗效观察符元党 1, 郑振优 2, 林再雄 2, 周吉良 1. 昌江黎族自治县中西医结合医院, 海南昌江 572700 2. 海南医学院附属医院眼科, 海南海口 570100 1 摘要 : 目的探讨