Prophase Study Of Gene Mutation And Mutant Compensation On Familial Hypercholesterolemia Proband

[Obejective]Familial hypercholesterolemia (FH) is one kind of monogenic autosome dominant inherited disease which always lead to atherosclerosis (As) and premature coronary heart disease (CAD) . The objective of this study is to use flow cytometer analysis the binding and internalization activity of the LDL-R protein on the lymphocytes of the FH proband,then amplify the LDL-R cDNA sequence to establish the basis to mutant compensation.[Methods]Lymphocytes were isolated from the whole blood of the health adult and the FH proband. The lymphocytes were cultured in 1640 medium containing 10% human lipoprotein-deficient serum ( LPDS ) for 72 hours to induce LDL receptor's activity. Then the lymphocytes were incubated with fluorescein isothiocyanate (FITC) labeled LDL and LDLR antibody at 4℃ and 37℃ for 1.5 hours respectively, and then the lymphocytes were incubated with PE-CD3 labeled T lymphocytes at 4℃ and 37℃ for 45 min respectively. The binding and internalization of LDL to LDL receptor were detected by flow cytometer (FCM) .Also we use SMART RACE cDNA synthesis and amplify LDL-R cDNA ,and then sequencing the amplified LDL-R cDNA.[Result]The proband exists G→A splice mutation at the third intron. The LDL-R binding and intemalization activity on the lymphocytes of the FH proband is lower than the normal significantly. We get the sequence of LDL-R cDNA from the first exon to the tenth exon by SMART RACE cDNA kits.