Molecular Mechanism Of Familial Hyperchylomicronemia Caused By Lipoprotein Lipase Gene Deficiency (C310R/E396V)

Objective: We first discovered C310R(c.T928C),E396V(c.A1187T)mutations of lipoprotein lipase gene in the world.The purpose of this study is to investigate functional significance and molecular mechanisms of lipoprotein lipase gene in vivo and in vitro,which can help construct the relationship spectrum of LPL gene mutation and phenotype in familial hyperchylomicronemia patients.Methods: Genomic DNA was extracted from proband's family members' peripheral blood cells and screened by whole-exome sequencing to verify candidate gene variations.Polymerase chain reaction(PCR)products were afterwards directly sequenced again to confirm corresponding LPL variants.Fasting plasma of proband MJH,CPH and controls were obtained after intravenous heparin injection(60 IU/kg).The postheparin LPL activity,LPL mass and hepatic lipase(HL)activity were measured by enzyme linked immunosorbent assay(ELISA)and enzyme-fluorescent method.Native gel western blot was conducted to confirm dimer depolymerization of LPL caused by LPL gene mutants.At the cellular level,lentiviruses containing site-directed mutagenized LPL c DNA were constructed and then transfected into COS-1 cells.Functional significance of the mutants was corroborated by analysing LPL activity and mass in the cell medium and lysates.m RNA was assayed by fluorescent real-time polymerase chain reaction(RT-PCR)to confirm the effect on gene transcription.Results: DNA sequence analysis revealed that the proband MJH(I-2)was a heterozygote for a novel T>C substitution(c.T928C)in exon 6 of LPL gene,which resulted in conversion of cysteine to arginine(p.C310R),while his niece MC(II-1)was a compound heterozygote for the mutation(c.T928C)in exon 6 and a novel missense mutation in exon 8(c.A1187T)which induced replacement of glutamic acid with valine(p.E396V).In contrast to normolipidemic controls,the level of postheparin LPL mass as well as LPL activity in the proband MJH and I-1 was dramatically declined.Besides,both of the mutations did not affect LPL dimerization.In vitro studies,these two mutations can cause decreased activity and mass of extracellular lipoprotein lipase.Moreover,further investigation indicated that LPL C310 R mutation tremendously effect post-transcriptional modification of LPL gene,leading to decreased LPL synthesized in COS-1 cells,whereas LPL E396 V mutation dampened intracellular LPL trafficking.Conclusion: Both the mutations are pathogenic by reducing the activity and mass of LPL in the plasma,which affected normal metabolism of triglycerides.