| Objective: To establish the general population cohort of Tajik and Kazakh ethnic groups in Xinjiang,describe the baseline lipid levels and dyslipidemia in the cohort,and explore the associated factors of dyslipidemia.To screen,validate,and explore molecular mechanisms of new functional variants of angiopoietin-Like Protein 3(ANGPTL3)gene.To evaluate the efficacy and safety of ANGPTL3 monoclonal antibody evinacumab in the treatment of hyperlipidemia.Methods:(1)From May to October 2021 and from March to June 2022,5635 Tajik and 4835 Kazak residents over 18 years old were enrolled in Tashkurgan Tajik Autonomous County and Fuyun County,Xinjiang,respectively.Questionnaire(general information,smoking and drinking history,disease status,etc.),physical examination(waist circumference,hip circumference,height,weight,blood pressure,heart rate,etc.)and laboratory examination of total cholesterol(TC),low-density lipoprotein cholesterol(LDLC),high-density lipoprotein cholesterol(HDL-C),triglycerides(TG)and lipoprotein(a)[Lp(a)] were conducted to collect patient information,establish natural population cohorts,and analyze baseline prevalence of dyslipidemia and related factors in these cohorts.(2)The extreme phenotypic strategy was adopted to select individuals with very low,very high and normal serum lipids in the Tajik and Kazakh natural population cohorts and the young Uyghur cohort established previously,and the potential functional variants of ANGPTL3 gene was screened by whole exon sequencing and bioinformatics analysis.Plasmids containing wild-type and variant ANGPTL3 coding sequences with myc tags were constructed.Different types of ANGPTL3 were overexpressed in Huh-7 cells and 293 T cells by plasmid transfection,and ANGPTL3 levels in cells and medium were detected by Western blot(WB).Protein stability and degradation pathways of different types of ANGPTL3 were investigated.The ubiquitination levels of different types of ANGPTL3 were detected by co-transfection of different types of ANGPTL3 plasmid and ubiquitin plasmid combined with co-immunoprecipitation technology.Laser confocal microscopy was used to investigate the colocalization of different types of ANGPTL3 with endoplasmic reticulum and Golgi apparatus,and the uptake of Dil LDL by Huh-7 cells.AAV8 containing wild-type and variant ANGPTL3 protein coding sequences with myc tags were constructed,and injected through tail vein to overexpress wild type and L335 S variant ANGPTL3 in mice.The transcription and translation levels of ANGPTL3 in mouse liver were detected by q PCR,WB,ELISA and immunohistochemistry.The serum ANGPTL3 levels were detected by ELISA,and the serum TC,LDL-C,HDL-C and TG levels and TC and TG in liver were determined by the detection kits.HE staining was used to show the tissue structure of mouse liver,and oil red O staining was used to learn about the lipid deposition in mouse liver.Finally,family members of L335 S variant carrier were enrolled.First general sequencing was conducted to investigate the mutation carrying status of family members,ELISA was used to test serum lipids and serum ANGPTL3,and echocardiography and epigastrial CT plain scan were used to understand the lipid deposition status in hearts and livers of family members.(3)The relevant literature on evinacumab published between January 1,2000 and November 30,2023 was searched in Pub Med and Embase databases using subject heading and subheading words.Eligible randomized,controlled clinical studies were included and the results were extracted.Data on the efficacy and safety of evinacumab in the treatment of hyperlipidemia were pooled.Subgroup analysis,meta-regression analysis,and sensitivity analysis were used to evaluate the reliability of the meta-analysis results and to explore potential sources of inter-study heterogeneity.Funnel plots and Egger’s regression tests were used to assess whether there was publication bias in the study.Results:(1)The average age of Tajik and Kazak subjects was 41.9 years and 45.8 years,respectively.The overall standardized prevalence rates(95% confidence interval)of high TC,high LDL-C,high TG,low HDL-C,and dyslipidemia in Tajik were 4.1%(3.6%-4.6%),4.9%(4.3%-5.5%),9.4%(8.6%-10.2%),32.4%(31.2%-33.6%),and 37.0%(35.7%-38.3%),respectively.After adjusting for confounders,male gender,higher education,larger BMI and waist circumference,living in urban areas,and smoking were independently associated with dyslipidemia.The standardized prevalence rates(95% confidence interval)of high TC,high LDL-C,low HDL-C,high TG,and dyslipidemia in Kazak were 24.1%(23.0%-25.2%)、24.8%(23.7%-25.9%)、10.0%(9.2%-10.8%)、7.3%(6.6%-8.0%),and 37.9%(36.6%-39.2%),respectively.After adjusting for confounders,sex,age,marital status,smoking,BMI and waist circumference were independent related factors for dyslipidemia.Lp(a)levels of the two ethnic groups were positively skewed,and the Lp(a)levels ranged from0.40 to 1229.40mg/L and from 0.62 to 2108.58 mg/L,respectively.The Lp(a)level of Tajik was lower than that of Kazak,and the median Lp(a)level was 78.90(38.60,190.20)and103.30(49.57,234.27)mg/L.Lp(a)levels were independently associated with ethnicity and LDL-C levels after adjusting for confounders.(2)A total of 5 potential functional variants located in the exon region of ANGPTL3 gene were screened,and L127 F and L335 S variants were finally selected.The uptake rate of Di LDL in Huh-7 cells overexpressing the two variants of ANGPTL3 were the same as those overexpressing wild-type ANGPTL3.The L127 F mutation did not affect the normal secretion,intracellular levels,and the protein stability of ANGPTL3.The degradation pathway of L127 F ANGPTL3 was consistent with that of wild-type ANGPTL3.ANGPTL3 levels in 293 T cells and mouse liver overexpressing L335 S ANGPTL3 were significantly higher than those in overexpressing wild-type ANGPTL3 group.The levels of ANGPTL3 in cell culture medium and mouse serum,TC and TG levels in mouse serum and liver,and lipid deposition in mouse liver tissue were significantly lower than those in overexpressing wild-type ANGPTL3 group,and family studies also confirmed that the members carrying this variant had lower serum TG levels and no obvious lipid deposition in heart and liver.The ubiquitylation level of L335 S ANGPTL3 was significantly higher than that of wild-type ANGPTL3,leading to the degradation of L335 S mainly through proteasome pathway.(3)Compared with placebo,evinacumab decreased TG,LDL-C,and HDL-C levels by 51.7%,29.2%,and 12.9%,respectively,and increased the risk of treatment-related adverse events by 11.9%.Sensitivity analyses suggested that meta-safety results were unstable.Conclusion:(1)The prevalence of dyslipidemia in Tajik people is mainly characterized by low prevalence of high TC,high LDL-C,and high TG,but high prevalence of low HDL-C.Hypercholesterolemia is the main characteristic of dyslipidemia in Kazak.The difference of BMI and waist circumference may be one of the main reasons for the different characteristics of dyslipidemia between the two ethnic groups.The distribution of Lp(a)in Tajik and Kazak is positively skewed,with obvious variation among individuals.The level of Lp(a)among Tajik is lower than that of Kazak.LDL-C and ethnicity were independent correlation factors of Lp(a)levels.(2)The mutation of L335 S leads to the abnormal secretion of ANGPTL3 protein,which is deposited in hepatocytes and degraded through ubiquitin-proteasome pathway,resulting in the decrease of serum ANGPTL3 concentration,resulting in the decrease of serum TC,TG and LDL-C levels,while not causing damage to liver tissue structure and aggravating liver lipid deposition.(3)Evinacumab has good efficacy in the treatment of hyperlipidemia,and its safety and effectiveness in reducing the risk of cardiovascular adverse events need to be further studied. |
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