Objective To investigate the clinical significance and mechanism of WW domain containing oxidoreductase (WWOX) gene and p73 gene abnormal expression in acute lymphocytic leukemia (ALL).Methods Case-control study was used in the research.Forty-eight cases of bone marrows from ALL patients were collected,including 32 cases newly diagnosed,11 cases with complete remission and 5 case with relapse.Thirty-one cases of bone marrows from non-leukemia patients were used as control group.All the samples were collected from First Affiliated Hospital of Guangxi Medical University from July 2010 to July 2011.The doctors punctured patients' bone marrows 3 milliliters from the left of posterior superior iliac spine.Samples were bottled up with EDTA anti-coagulation tube.1 milliliter bone marrow was used to extract genome RNA with purity from 1.8 to 2.0.And then,the level of WWOX and p73 gene transcripts were tested immediately using reverse transcriptase-polymerase chain reaction (RT-PCR).Meanwhile genome DNA was also extracted from the other 2 milliliter bone marrow with purity from 1.7 to 1.9,which was used to detect the promoter methylation of WWOX gene and the first exon methylation of p73 gene by methylation PCR (MS-PCP).x2 test and Fisher's exact test were used to compare tbe methylation status of WWOX and p73 gene.Results In 31 controls,expression of WWOX and p73 gene mRNA was 94.00％.The total expression frequency of WWOX gene mRNA in 48 ALL samples was 48.00％ (23/48),much lower than control (x2 =17.434,P =0.000 ).There was significant difference (x2 =10.471,P =0.001 ) between newly diagnosed cases 34.38％ ( 11/32),complcte remission cases (90.91％,10/11 ) and control.The total expression frequency of p73 gene mRNA in 48 ALL samples was 56.00％ (27/48),much lower than control (x2 =12.697,P =0.000).There was significant difference (P =0.012 ) between newly diagnosed cases 43.75％ (14/32) and complete remission cases 90.91％ (10/11).It was unmethylation in 31 controls.The total methylation frequency of WWOX gene promoter region in 48 ALL samples was 44.00％(21/48),much lower than control (x2 =18.473,P =0.000).There was significant difference (P =0.012) between newly diagnosed cases 56.25％ (18/32),complete remission cases 9.09％ (1/11 ) and control.The total methylation frequency of p73 gene the first exon region in 48 ALL samples was 35.00％(17/48),much lower than control (x2 =13.990,P =0.000).There was significant difference (P =0.033) between newly diagnosed cases 46.88％ (15/32),complete remission cases 9.09％ ( 1/11 ) and control.There was a negative correlation between the expression of WWOX gene mRNA and its methylation status(r =- 0.678,P =0.000),the same as p73 gene ( r =- 0.577,P =0.000).Conclusions The abnormal methylation of WWOX and p73 gene may be the major mechanism of gene silence in ALL,which leads to no expression of WWOX mRNA or p73 mRNA.And the abnormal methylation of WWOX and p73 gene may be relevant with the process of occurrence and development in ALL.It may be an effective and significant to detect methylation status of WWOX gene and p73 gene for the diagnosis and treatment of ALL patients.(Chin J Lab Med,2012,35:820-825)%目的 研究含有氧化还原酶的WW域(WW domain containing oxidoreductase,WWOX)和p73基因在急性淋巴细胞白血病(acute lymphocytic leukemia,ALL)中异常表达的临床意义及其机制.方法 采用病例对照研究,收集2010-2011年广西医科大学第一附属医院收治的ALL患者骨髓样本48份,其中包括初诊患者32例、缓解患者11例、复发患者5例；收集同期非白血病患者骨髓样本31份作为对照组.抽取骨髓3 ml,EDTA抗凝,1 ml骨髓样本采用离心柱法立即提取RNA,取纯度在1.8～2.0范围内的产物逆转录后进行逆转录聚合酶链反应(RT-PCR),检测WWOX和p73基因mRNA的表达情况；2 ml骨髓样本采用离心柱法提取DNA,取纯度在1.7 ～1.9范围内的产物进行甲基化特异性聚合酶链反应(MS-PCR),检测WWOX基因启动子区和p73基因第1外显子的甲基化情况.不同组别间甲基化状态的比较采用x2检验或Fisher确切概率法.结果 31份对照组标本中,WWOX与p73基因mRNA阳性表达率均为94.00％(29/31).48份ALL标本中,WWOX mRNA阳性表达率为48.00％(23/48),低于对照组,差异有统计学意义(x2=17.434,P =0.000)；其中初诊组为34.38％(11/32),低于缓解组的90.91％(10/11),差异有统计学意义(x2=10.471,P=0.001).p73基因mRNA阳性表达率为56.00％( 27/48),低于对照组,差异有统计学意义(x2=12.697,P=0.000)；其中初诊组为43.75％( 14/32),低于缓解组的90.91％(10/11),差异有统计学意义(P=0.012).31份对照组标本中,WWOX与p73基因均未见甲基化现象(0/31).48份ALL标本中,WWOX基因甲基化率为44.00％( 21/48),明显高于对照组,差异有统计学意义(x2=18.473,P=0.000)；其中初诊组为56.25％ (18/32),高于缓解组的9.09％ (1/11),差异有统计学意义(P=0.012);p73基因甲基化率为35.00％(17/48),明显高于对照组,差异有统计学意义(x2=13.990,P=0.000),其中初诊组为46.88％( 15/32),高于缓解组的9.09％ (1/11),差异有统计学意义(P=0.033).WWOX与p73基因mRNA阳性表达率均与各自基因甲基化状态呈负相关(r=-0.678、-0.577,P=0.000).结论 WWOX与p73基因的甲基化可能导致基因的沉默,使其mRNA减少或缺失；WWOX与p73基因甲基化的检测可能对ALL的诊断和疗效评估有一定意义.
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