Screening Of Novel Pathogenic Genes Of Familial Hypercholesterolemia And Effect Of Soluble Epoxide Hydrolase Gene Mutant On The Function Of LDLR

Objective : Screening of novel pathogenic genes and investigate the relationship between genotype and clinical phenotype in patients with familial hyperlipidemia(FH)in China.Methods:(1) According to the FH clinical diagnostic criteria in China, medical history inquiry, questionnaire survey, biochemical examination and collection of peripheral venous blood were performed on 7 probands and their nuclear families;(2)Gene analysis was finished on 7 probands who were to carry out the target-exon chip combining the two generation sequencing;(3) Analyzing patients whether there are low density lipoprotein receptor(LDLR), apolipoprotein B100(apo B-100) or protein convertase subtilisin / Kexin 9(PCSK9) three known pathogenic gene mutation;(4)Analyzing the clinical characteristics of the mutant negative proband and their core family;(5)Sanger sequencing was used to verify the genotype of mutant negative proband and its core family;Results:(1)7 probands have multiple xanthomas on the skin, tendons and other parts of their body, the plasma cholesterol(TC) significantly increased from 10.98 to 22.62mmol/L;(2) We found a homozygous LDLR deletion mutation of c.632 to634 del ACT. Four LDLR heterozygous missense mutations were for H562 Y, P664 L,C711Y, 818-2A/G. A LDLR stop mutation was W462 X and known pathogenic gene mutation was not screened on another case, but carried soluble epoxide hydrolase(EPHX2) gene mutant loci R287Q;(3) The TC of mutation negative proband was17.06 mmol/L, his parents' TC is lower than the proband but above than normal people and were 10.62 mmol/L,5.23mmol/L respectively;(4) Sanger Sequencing results showed that the mutation negative proband and his core family carried the same soluble epoxide hydrolase gene mutant loci R287 Q.Conclusion: The onset age of FH is uasually earlier, with significantly increased plasma cholesterol, multiple xanthomas on the skin, wrist and other parts of their bodies; most of the probands have gene mutations, the highest frequency of which is LDLR gene mutation; We found that EPHX2 gene mutation in one FH patient might be a new pathogenic gene.Objective:To investigate the effects of the possible novel pathogenic gene soluble epoxide hydrolase gene mutant R287 Q on the function of LDLR.Methods:(1)Mutant plasmid R287 Q was constructed and sequencing was verified according to the wild-type plasmid WT in vitro, then they were transfected into 293 T cell line;(2)Western blot was used for the EPHX2 protein expression;(3)The soluble epoxide hydrolase activity kit for detection of EPHX2 activity;(4) EPHX2 mutant R287 Q on the effect of LDLR binding ability was detected by flow cytometry;(5)Flow cytometry was used for the detection of EPHX2 mutant R287 Q on the effect of LDLR internalization capacity.Results:(1) Sequencing results showed that the wild type WT and mutant R287 Q plasmid were successfully constructed;(2)We observed the expression of protein in293 T cells transfected with wild-type WT and mutant plasmid R287 Q by Western blot;(3)We found that the soluble epoxide hydrolase activity transfected with plasmid R287 Q was decreased significantly than transfected with plasmid WT by using soluble epoxide hydrolase activity kits;(4) Compared to WT, the EPHX2 mutant R287 Q significantly reduced the effect on the LDLR binding ability;(5)Compared to WT, the EPHX2 mutant R287 Q significantly reduced the effect on the internalization ability of LDLR.Conclusion: The mutant EPHX2 gene R287 Q decreased soluble epoxide hydrolase activity, and reduced the cells LDLR binding and endocytosis function, EPHX2 gene may be a susceptibility gene of function of LDLR in FH patients but not a pathogenic gene.