engleski

Analysis of the hepatocyte growth factor exon 4 polymorphism

Hepatocyte growth factor (HGF) is multifunctional cytokine and the most potent hepatocyte mitogen. Its gene is localized on chromosome 7. It is composed of 18 exons and 17 introns. The HGF structure consists of four kringle domains. The first and second kringle play the crucial role in the HGF binding to HGF-receptor, thus enabling its biologic function. Exons 4 and 5 code the first kringle domain. The aim of this study was to investigate the HGF-exon 4 polymorphism in healthy and liver cirrhotic subjects by two different methods of detection; RFLP and SSCP. The HGF-exon 4 polymorphism was investigated by PCR-RFLP and PCR-SSCP in 30 healthy and 20 cirrhotic subjects. Genomic DNA was isolated from blood by standard procedure. HGF exon 4 fragment (214 bp) was amplified in a total PCR reaction volume of 100 ml including 0.3 mg DNA, 200 mM dNTP-s, 0.1 mM of each primers and 2.5 U Taq polymerase. Each sample was subjected to 35 cycles of 95°C/20 s, 53°C/20 s , and 72 °C/ 30 s on thermal cycler. Initial denaturation was at 95°C/2 min and final extension at 72°C/5 min. For PCR-RFLP the amplicon was cut using the restriction enzymes Mva I, Alu I and Dde I (Boehringer Mannheim) at 37°C/3 hours. For PCR-SSCP the amplicon was denatured by 95% formamide loading buffer at 95°C/5 min. Restriction fragments were detected by 15% PAGE and ssDNA by 10% PAGE, and silver-stained. Results showed neither Mva I, Alu I or Dde I restriction fragment nor ssDNA polymorphism on exon 4. The preliminary results, obtained by the two different methods of polymorphism detection, suggest that there is no mutation on HGF exon 4. Further polymorphism studies are needed to investigate the role of HGF in liver disease patients.

hepatocyte growth factor; HGF; PCR-RFLP; PCR-SSCP; polymorphism; exon 4

nije evidentirano