AIM: To identify the new alternative splicing variants of human CYP2D6 in human extratumoral liver tissue with RT-PCR and sequencing. METHODS: Full length of human CYP2D6 cDNAs was amplificated by reverse transcriptio...AIM: To identify the new alternative splicing variants of human CYP2D6 in human extratumoral liver tissue with RT-PCR and sequencing. METHODS: Full length of human CYP2D6 cDNAs was amplificated by reverse transcription-polymerase chain reaction (RT-PCR) from a human extratumoral liver tissue and cloned into pGEM-T vector. The cDNA was sequenced. Exons from 1 to 4 of human CYP2D6 cDNAs were also amplificated by RT-PCR from extratumoral liver tissues of 17 human hepatocellular carcinomas. Some RT-PCR products were sequenced. Exons 1 to 4 of CYP2D6 gene were amplified by PCR from extratumoral liver tissue DNA. Two PCR products from extratumoral liver tissues expressing skipped mRNA were partially sequenced. RESULTS: One of the CYP2D6 cDNAs had 470 nucleotides from 79 to 548 (3' portion of exons 1 to 5' portion of exon 4), and was skipped. Exons 1 to 4 of CYP2D6 cDNA were assayed with RT-PCR in 17 extratumoral liver tissues. Both wild type and skipped mRNAs were expressed in 4 samples, only wild type mRNA was expressed in 5 samples, and only skipped mRNA was expressed in 8 samples. Two more variants were identified by sequencing the RT-PCR products of exons 1 to 4 of CYP2D6 cDNA. The second variant skipped 411 nucleotides from 175 to 585. This variant was identified in 4 different liver tissues by sequencing the RT-PCR products. We sequenced partially 2 of the PCR products amplified of CYP2D6 exon 1 to exon 4 from extratumoral liver tissue genomic DNA that only expressed skipped mRNA by RT-PCR. No point mutations around exon 1, intron 1, and exon 4, and no deletion in CYP2D6 gene were detected. The third variant was the skipped exon 3, and 153 bp was lost. CONCLUSION: Three new alternative splicing variants of CYP2D6 mRNA have been identified. They may not be caused by gene mutation and may lose CYP2D6 activity and act as a down-regulator of CYP2D6.展开更多
目的:评估细胞色素P450 2D6*10基因多态性,对汉族人群术后吗啡自控镇痛效果的影响。方法:美国麻醉医师协会(American Society of Anesthesiologists,ASA)Ⅰ～Ⅱ级患者共64名被纳入此实验,56名患者完成实验。根据CYP2D6基因型,患者分为3...目的:评估细胞色素P450 2D6*10基因多态性,对汉族人群术后吗啡自控镇痛效果的影响。方法:美国麻醉医师协会(American Society of Anesthesiologists,ASA)Ⅰ～Ⅱ级患者共64名被纳入此实验,56名患者完成实验。根据CYP2D6基因型,患者分为3组:野生型纯合子组(w/w组,n=19),杂合子组(m/w组,n=21)和突变型纯合子组(m/m组,n=16)。在手术结束前10 min给予复合剂量吗啡(静脉注射,0.1 mg/kg)后,患者可通过吗啡(单次给药1 mg)自控输注泵进行自控镇痛(patient-controlled analgesia,PCA)。全身麻醉诱导后采集血样。采用聚合酶链反应-限制性片段长度多态性(PCR-RFLP)进行CYP2D6*10基因分型。记录并比较术后72 h各组间的吗啡用量及视觉模拟评分(visual analogue score,VAS)。结果:与m/m组相比,m/w组吗啡在T0.5～4 h的累积量显著升高(P<0.05)。3组中各时间段的VAS评分对比差异无统计学意义。结论:在全麻术后0.5 h至4 h急性疼痛治疗中,CYP2D6*10 m/w组所需吗啡的量比m/m组高,以此推测m/w组产生的内源性阿片肽比m/m组低。展开更多
基金Supported by the National Key Basic Research and Development Program of China,No.2002CB512901National Natural Science Foundation of China,No.39770868 and Natural Science Foundation of Zhejiang Province,No.397490
文摘AIM: To identify the new alternative splicing variants of human CYP2D6 in human extratumoral liver tissue with RT-PCR and sequencing. METHODS: Full length of human CYP2D6 cDNAs was amplificated by reverse transcription-polymerase chain reaction (RT-PCR) from a human extratumoral liver tissue and cloned into pGEM-T vector. The cDNA was sequenced. Exons from 1 to 4 of human CYP2D6 cDNAs were also amplificated by RT-PCR from extratumoral liver tissues of 17 human hepatocellular carcinomas. Some RT-PCR products were sequenced. Exons 1 to 4 of CYP2D6 gene were amplified by PCR from extratumoral liver tissue DNA. Two PCR products from extratumoral liver tissues expressing skipped mRNA were partially sequenced. RESULTS: One of the CYP2D6 cDNAs had 470 nucleotides from 79 to 548 (3' portion of exons 1 to 5' portion of exon 4), and was skipped. Exons 1 to 4 of CYP2D6 cDNA were assayed with RT-PCR in 17 extratumoral liver tissues. Both wild type and skipped mRNAs were expressed in 4 samples, only wild type mRNA was expressed in 5 samples, and only skipped mRNA was expressed in 8 samples. Two more variants were identified by sequencing the RT-PCR products of exons 1 to 4 of CYP2D6 cDNA. The second variant skipped 411 nucleotides from 175 to 585. This variant was identified in 4 different liver tissues by sequencing the RT-PCR products. We sequenced partially 2 of the PCR products amplified of CYP2D6 exon 1 to exon 4 from extratumoral liver tissue genomic DNA that only expressed skipped mRNA by RT-PCR. No point mutations around exon 1, intron 1, and exon 4, and no deletion in CYP2D6 gene were detected. The third variant was the skipped exon 3, and 153 bp was lost. CONCLUSION: Three new alternative splicing variants of CYP2D6 mRNA have been identified. They may not be caused by gene mutation and may lose CYP2D6 activity and act as a down-regulator of CYP2D6.
