Screening And Functional Mechanism Of New Cholesterol Metabolism Genes And New Mutants

Objective:This study employed extreme phenotype study and family study strategies.A screening study of new genes affecting dyslipidemia was conducted in Xinjiang Uygur and Han university students(18-24 years old).The gene mutation was screened by whole exome sequencing technology.Functional mechanism was carried out at the cellular and animal levels.In this study,through the closed-loop research system of "epidemic survey and sequencing screening-functional verification of candidate new genes and new mutations-family verification-population verification",new genes and new mutation sites affecting dyslipidemia were discovered.This study provides new therapeutic ideas for the prevention and treatment of dyslipidemia and cardiovascular diseases in the future.(1)We used the extreme phenotype strategy to sequence the whole exome of Han and Uygur college students to explore new genes and mutation sites that affect blood lipid metabolism.(2)By using si RNA technology and AAV8-Cas9-g RNA technology to construct cell and animal models,we will conduct functional verification at the cell and animal levels of ten candidate genes detected from Uighur university students.(3)The function of PCSK9 M126 I mutation screened from Han university students was studied.The M126 I mutation of PCSK9 was tested from the cell level to the animal level in order to explore its conservation,stability,catalytic cleavage,cell localization and impact on LDLR affinity.Methods:(1)Han and Uygur university students(18-24 years old)in Xinjiang were selected as the study subjects,and two populations with extremely low LDL-C and normal LDL-C were selected for whole exome sequencing.Enrichment of low-frequency variant genes and mutation sites by bioinformatics.The missense mutation,frameshift mutation and termination mutation affecting the amino acid sequence were analyzed.By comparing the frequency of low-frequency mutations between the extremely low LDL-C group and the control group,the target gene was located,and new genes and mutation sites affecting cholesterol metabolism were found.(2)By using si RNA technology and AAV8-Cas9-g RNA technology to construct cell and animal models,the functional verification of ten candidate genes screened from Uyghur college students were carried out.(3)Functional experiments were carried out on the M126 I mutation of PCSK9 by using Western blot technology,confocal microscope technology,PCR,AAV animal model and other experimental methods to explore its conservation,stability,catalytic cleavage,cell localization and the impact on LDLR affinity.Results:(1)We found that S100A6,CHAT,ALOX15 E,IFNA4,TNIP1,TAF1 A,ATRNL1,AVEN,KRTAP5-4,SEC14L6 genes were highly correlated with LDL-C in the Han college students.We found that ten genes of PCSK9,ACLY,BTN2A1,TRARG1,CARM1,PIGS,SPIRE2,YME1L1,PCSK6 and WASHC5 were highly correlated with LDL-C in the Uighur college students.A functional mutation of protein site 126 in the exon 2 region of PCSK9 was identified in college students of,and the methionine at this position was mutated to isoleucine(M126I).(2)Targeted knockdown of candidate genes in Huh7 cell by small interfering si RNA,detection the LDLR expression level in cells,and four genes TRARG1,CARM1,PIGS,WASHC5 were screened out.Using the AAV8-Cas9-g RNA system,an animal screening model was constructed,and an AAV8-Cas9-PCSK9-g RNA positive reference system was constructed.For the four genes screened in the previous step,the AAV8-Cas9-g RNA system was used to knock down the target gene in mice stably expressing Cas9 protein,and the levels of TC and TG in the mice were detected,and two genes CARM1 and WASHC5 were screened out.The two genes screened in the previous step were overexpressed and verified at the cellular level,and finally WASHC5 was found to be the target gene affecting LDLR.The increase of WASHC5 expression can reduce the expression level of LDLR.(3)The M126 I mutation of PCSK9 at the cellular level makes the PCSK9 protein unable to catalyze the normal cleavage,unable to transport to the Golgi apparatus and then stay in the endoplasmic reticulum,thus blocking the LDLR degradation pathway mediated by the PCSK9 protein,resulting in an increase in the level of LDLR.At the animal level,the new M126 I mutation of PCSK9 prevents PCSK9 protein from being spliced and matured normally in the liver tissue of mice,and thus cannot be secreted into the blood.The lack of PCSK9 protein in the blood led to an increase in the level of LDLR in liver tissue,which in turn enhanced the ability to clear LDL-C in the blood,resulting in a decrease in the level of LDL-C in mice.Conclusion:We conducted a population study among young college students,adopted a combination of extreme phenotypes and family studies,screened new genes and new mutations by using whole exome sequencing technology,and performed functional verification at the cell and animal levels.We found that WASHC5 may be a novel gene affecting cholesterol metabolism.At the same time,a new mutant M126 I was discovered for PCSK9,which is known to affect cholesterol metabolism.The functional mechanism of new genes and new mutants has been proved at the three levels of population,animal and cells.