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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 CYP2D6cDNA.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.
METHODS: Full length of human CYP2D6 cDNAs was amplified 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 genes were amplified by PCR from extratumoral liver tissue DNA. Two PCR products from extratumoral liver tissues expressing skipped mRNA were 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. TheThis 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. third third was was 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.