| Objective: This study aimed to search for novel functional variants of the LPA gene involved in the regulation of lipoprotein(a)(Lp(a))levels.In the Xinjiang Uyghur population,we used extreme phenotypic study strategies to perform whole genome sequencing,LPA gene copy number determination,and multiplex PCR enrichment sequencing of target regions to explore the variation pattern of LPA gene KIV-2 copy number variation and its plasma Lp(a)concentration,to screen for new mutant loci affecting Lp(a)levels,to study them at multiple levels such as gene/cell/protein,and to To verify the effect of new functional mutation loci on plasma Lp(a)levels and to provide new theoretical basis and therapeutic strategies for the prevention and treatment of dyslipidemia in the future.Materials and methods: A total of 300 subjects were selected from Uygur patients aged between 18 and 79 with or without coronary heart disease in the first affiliated Hospital of Xinjiang Medical University.First of all,the baseline values were divided into the normal group and extremely high group according to the measured Lp(a)level,(Uyghur: 150 cases in the normal group: Lp(a)≤200 mg/L;150 cases in the very high group: Lp(a)≥ 600 mg/L),the copy number of KIV-2 region of LPA gene was detected,and the study population with KIV-2 copy number ≥ 23,Lp(a)≤ 200 mg/L;KIV-2 copy number < 23,Lp(a)≥ 600 mg/L was defined as the group conforming to the copy number variation law,and KIV-2 copy number ≥ 23,Lp(a)≥ 600 mg/L The population with KIV-2 copy number <23 and Lp(a)≤200 mg/L was defined as the group that did not conform to the copy number variation pattern,and the subjects that did not conform to the copy number variation pattern were considered as the extreme phenotype group,and the population was further screened for new variants of the LPA gene by multiplex PCR sequencing of the target region.The eukaryotic overexpression plasmids of wild-type and mutant LPA genes were constructed,and the functional assay and validation were performed at the cellular level using protein immunoblotting(Western blot),ultrafiltration centrifugation,and confocal microscopy techniques.Results: A total of 300 Uyghur subjects were included in this study,and the extreme phenotype study strategy was used to divide the population into normal and very high groups according to Lp(a)levels.285 samples were included in the final analysis,of which 174 were in the group conforming to the copy number variation pattern and 111 were in the group not conforming to the copy number variation pattern.For the population not conforming to the copy number variation pattern group,seven new mutant loci were screened by multiplex PCR enrichment sequencing of the target region of the LPA gene,among which three new LPA mutants(R2016C,L1372 V,L1358V)were located in the exonic region,and we selected the new variant R2016 C for an in vitro study at the cellular level with a high carrier Lp(a)level.LPA wild-type and LPA-R2016 C mutant overexpression plasmids were constructed for in vitro experiments,and the results of cellular level studies showed that the LPA-R2016 C mutant increased protein expression levels in intracellular and cell culture media compared to wild-type LPA,further verifying that the LPA-R2016 C mutant caused increased Lp(a)secretion and increased plasma Lp(a)levels.Conclusions: In this study,the LPA gene copy number was determined by fluorescence quantitative PCR,and using the extreme value phenotype study strategy,the study subjects were screened out of the group that did not conform to the copy number variation pattern,and a new LPA mutant(R2016C)was screened in this population by multiplex PCR enrichment sequencing of the target region of the LPA gene,which was further validated at the cellular level and found that the LPA-R2016 C mutant may increase plasma Lp(a)levels by increasing the secretion of Lp(a).This provides a theoretical basis for future gene editing therapies as well as lipid-lowering drug development. |
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