Clinical And Functional Characteristics Of APOB Gene Mutation (c.676G>A) In Familial Hypercholesterolemia

Background:Familial hypercholesterolemia(FH)is a common genetic disease characterized by elevated levels of low-density lipoprotein cholesterol(LDL-C),accompanied by skin/tendon xanthoma,fatty cornea,and early atherosclerotic cardiovascular disease.At present,the incidence of FH is increasing year by year,but its visiting rate and social awareness are still low.Early identification,diagnosis and intervention of FH can effectively prevent the occurrence of cardiovascular events.So far,the APOB gene mutation(c.676G>A)has not been reported.Combined with the clinical data and gene sequencing results of the proband and her family members,this study carried out clinical feature analysis and functional verification of the APOB gene mutation(c.676G>A)and explored the pathogenicity and possible pathogenesis of the mutation site,so as to provide genetic basis for finding new prevention and treatment measures for FH.Methods:1.Data collection:Family information of patients was collected according to diagnostic criteria of FH.Fasting peripheral blood of proband and related family members was collected and blood lipid and blood glucose levels were detected,and then we drew the family map.Whole-exome gene sequencing was conducted on the proband and her mother to find the mutation sites related to clinical phenotype,and performed mutation site verification on other related family members.Then,we analyzed the possible pathogenic mutation sites through sequencing and protein structure prediction.2.Plasmid construction:We constructed wild-type and mutant plasmid expressing APOB48-Flag gene,and wild-type and mutant plasmid expressing enhanced green fluorescent protein EGFP and APOB48-Flag gene simultaneously.3.Plasmid transfection:Human hepatoma cells(HepG2)and human embryonic kidney cells(HEK 293T)were cultured in vitro,and co-transfected with Con group(without plasmid transfection),WT group(wild-type plasmid expressing APOB48-Flag gene),Mut group(mutant plasmid expressing APOB48-Flag gene),WT-EGFP group(wild-type plasmid expressing EGFP and APOB48-Flag gene simultaneously)and MUT-EGFP group(mutant plasmid expressing EGFP and APOB48-Flag gene simultaneously).The results of plasmid transfection were observed by fluorescence microscope.Intracellular immunofluorescence expression of wild-type APOB and mutant-type APOB were observed by DAPI staining and fluorescence microscopy.4.Functional verification:The transcription level and protein expression of the mutation site were evaluated by q-PCR and western blot.In addition,the effect of APOB c.676G>A mutation on other blood-lipid regulation genes was studied by q-PCR experiment.5.Statistical analysis:Image J and GraphPad Prism 9 were used for statistical analysis,and P<0.05 was considered statistically significant.Results:1.Through the collection of clinical history data and analysis of lipid test results from three generations of the proband’s family,several patients with a possible diagnosis of FH were screened.The total cholesterol(16.5 mmol/L)and LDL-C(10.66 mmol/L)of the proband were significantly increased,which was consistent with the characteristics of familial hypercholesterolemia.In addition,the mother,uncle and grandmother of the proband can also be diagnosed as hypercholesterolemia.2.Whole-exome gene sequencing of the proband and her mother identified three mutation sites that were highly correlated with clinical phenotypes:APOB c.676G>A,INSR c.4028G>A and AKT2 c.433G>C.By performing mutation site validation on members of related families,it was determined that mutation in the APOB gene(c.676G>A,p.A226T)may be associated with hyperlipidemia in this family.The mutation site has not been reported domestically or internationally,and its clinical significance is unknown.3.The amino acid mutation site of APOB is conserved in various species.APOB(c.676G>A,p.A226T)is a missense heterozygous mutation.The 676th nucleotide in the coding region changes from guanine to adenine,causing the 226th amino acid to change from alanine to threonine.The mutation at this site added a hydrogen bond between amino acid positions 226 and 684,which affected the spatial structural domain of the protein and led to changes in the biological function of the gene.4.After stable transfection of plasmid,wild-type APOB and mutant-type APOB(c.676G>A)were mainly expressed in the cytoplasm under fluorescence microscopy.q-PCR and western blotting showed that the mutation of APOB gene(c.676G>A)did not affect transcription and protein expression levels.The results of q-PCR showed that the mutation of APOB gene(c.676G>A)led to increased expression of triacylglycerol(TAG)synthesis genes(LPIN1,LPIN2,DGAT2),adipogenesis genes(FASN,ACACA,SREBF1,PPARGC1B),and fatty acid oxidation genes(CPT1 A).Conclusions:1.In this study,through the collection of clinical history data and gene sequencing analysis of an FH family,and identified the APOB mutation site(c.676G>A,p.A226T),which is highly correlated with the clinical phenotype of FH.2.The mutation of APOB gene(c.676G>A,p.A226T)led to the increase of the expression levels of TAG synthesis genes(LPIN1,LPIN2,DGAT2)and adipogenesis genes(FASN,AC AC A,SREBF1,PPARGC1B),and caused the increase of cholesterol and triglycerides in the blood circulation,which is related to the proband’s FH history and recurrence of acute pancreatitis.3.For the FH proband in this case,the combination of lipid-lowering drugs,accompanied by a low-fat diet and appropriate exercise,can improve blood lipid control,indicating that early diagnosis can enable the patient to receive early treatment and effectively prevent the occurrence of cardiovascular events.