Mutational Analysis Of JAK1 Gene In Human Hepatocellular Carcinoma

To investigate whether genetic mutations of the Janus kinase 1 (JAK1) gene are associated in hepatocellular carcinoma (HCC) progression, we analyzed the normal hepatocyte DNA sample and the tumor hepatocyte DNA sample from the same patient. Of 24 exons of JAK1 gene, 12 exons were previously reported to have mutations, we searched genetic alteration of JAK1 in these exons. First we designed the primers that include the whole lenth of exon.Then we amplified the exons using Polymerase Chain Reaction (PCR). All the products were denatured by heating at 95℃for 5 minutes and chill in ice for 5 minutes, then sample changed into single strand. We did electrophoresis in polyacrylamide gel using the denatured samples for single-strand conformational polymorphism (SSCP) analysing. After radioautography, we extracted DNA samples from the aberrant bands and amplified them, then purified them for sequencing to know the mutational type. The experiment used normal hepatocyte DNA sample as control. The results show that there is no mutation in 12 HCC cell lines, while the sample named as #33 that has aberrant band has missense mutations: 2008G>T,Q646V; 2009G>T,H647F. At the same time we also found the high ratio of single nucleotide polymorphism (SNP) (14%) in exon 14. From our experiment we know that the very low mutational ratio is 1.2% and the high ratio of SNP situation in other 4 exons . Interesting the exon 5 and exon 10 has SNP situation in the same two sample named as #2 and #148. We think the high frequency of the SNP pattern maybe indicate some relationship with HCC.