第一部分   家族性高胆固醇血症新突变位点致病性功能鉴定研究

中文摘要

研究背景与目的

家族性高胆固醇血症（Familial Hypercholesterolemia, FH）是一种常染色体遗传脂代谢异常疾病，其病因主要为编码低密度脂蛋白受体（low density lipoprotein cholesterol receptor, LDLR）基因发生致病性突变，肝脏不能有效代谢清除LDL，致使血浆中LDL-C水平显著升高，患者主要表现为早发动脉粥样硬化性心血管疾病。纯合型FH（homozygote FH, HoFH）患病率约为 1/30万，而杂合型FH（heterozygote FH, HeFH）患病率并不少见，约为 1/311。我国在FH诊断及治疗上均存在严重不足，FH基因诊断相关研究尤其缺乏。表型诊断为FH的患者若存在未曾报道的可疑致病突变，则很有可能是 FH 新的致病性突变，需要进一步进行功能鉴定和致病性验证等研究。本研究对1例临床表型确诊的FH病人及其亲属进行基因筛查，发现其LDLR第13号内含子区域存在一尚未报道的可疑致病性点突变，拟运用体外细胞学实验证实该点突变与FH表型之间的关系。

研究方法

对1例符合FH临床表型（DLCN=12分）的患者进行基因检测，未发现明确致病性突变，但其LDLR第13号内含子区域存在可疑致病突变，其一级亲属验证也存在该突变，为验证该突变位点的致病性，首先，我们采用CRISPR-Cas9基因编辑技术对人肝脏HepG2细胞系进行定点突变，获得纯合型LDR c.1988-8 T >A细胞模型，通过western blot测定LDLR受体表达情况，流式细胞术检测细胞膜表面LDLR表达水平，LDL内吞实验测定LDLR的功能。

研究结果

与野生对照组细胞相比，突变组细胞LDLR总体表达水平没有统计学差异，但成熟体LDLR表达量减少、前体LDLR表达水平显著增加，提示LDR c.1988-8 T > A导致LDLR成熟障碍。另外，经流式细胞术证实，突变组细胞的膜LDLR表达水平显著低于野生组细胞。LDL摄取实验显示，LDLR介导的Dil-LDL内吞量在突变组显著低于野生型组，提示突变组细胞LDLR介导的内吞能力下降。

研究结论

LDLR内含子点突变（LDLR c.1988-8 T > A）导致LDLR成熟障碍，细胞膜LDLR表达量下降，进而导致细胞水平上LDLR介导的LDL吞噬能力减弱。本研究证实了LDLR内含子点突变（LDR c.1988-8 T > A）的致病性。另外，本研究也提示对于外显子区正常的FH表型患者，应从常规LDLR外显子测序，扩展到全LDLR基因测序。

关键词  家族性高胆固醇血症，内含子，低密度脂蛋白受体

第二部分 血脂净化治疗家族性高胆固醇血症的阜外单中心研究

中文摘要

目的：探讨对于接受强化降脂药物治疗后低密度脂蛋白胆固醇（LDL-C）不能达标的家族性高胆固醇血症（familial hypercholesterolemia, FH）患者采用脂蛋白分离技术即血脂净化（lipoprotein apheresis, LA）治疗的有效性与安全性。

方法：该研究为回顾性、横断面研究，连续选取2015年2月至2019年11月于阜外医院进行LA治疗的FH患者，所有患者均在强化降脂药物治疗的基础上，采用双重滤过血浆置换（double filtration plasma pheresis, DFPP）法进行LA治疗，收集FH患者的年龄、性别、FH类型等基本临床信息，分析LA治疗前后LDL-C、脂蛋白a [lipoprotein(a), Lp(a)]水平变化及其降幅，同时分析免疫球蛋白（immunoglobulin, Ig）水平变化以及LA治疗相关不良反应的发生情况。

结果：共计纳入115例FH患者，其中杂合型FH患者107例（93.0%）、纯合型8例（7.0%）；年龄（43.9±12.2）岁，男性75例（65.2%），合并冠心病108例（93.8%）。LA治疗前LDL-C和Lp（a）的水平分别为（5.20±2.94）mmol/L和[428.70（177.0，829.50）]mg/L，单次LA治疗后即刻LDL-C和Lp（a）水平均显著降低，分别降至（1.83±1.08）mmol/L和148.90（75.90，317.00）mg/L（P均<0.001），降幅分别达64.2%和59.8%。LA治疗后1 d IgG、IgA均可维持于正常水平（降幅分别为15.1%、25.0%，P均＜0.001），IgM低于正常水平（降幅58.7%，P<0.001）。6例（5.2%）患者出现LA治疗相关不良反应，包括恶心、低血压、气短、心悸等，程度均轻微，经对症处理后症状缓解。

结论：LA治疗后可显著降低FH患者LDL-C和Lp(a)水平，总体耐受性良好。对于接受强化降脂药物治疗后LDL-C不达标的FH患者，尤其是伴Lp（a）升高者，DFPP法LA治疗可进一步大幅度降低LDL-C和Lp（a），是一种高效且安全的降脂治疗措施。

关键词：动脉粥样硬化，家族性高胆固醇血症，血脂净化

第一部分 英文摘要

Function identification of a novel pathogenic mutation in familial hypercholesterolemia

Background and objective

Familial Hypercholesterolemia (FH) is an autosomal-dominant genetic lipid disorder caused mainly by abnormalities in the gene coding the low density lipoprotein receptor (LDLR), resulting in the inability of the liver to sufficiently remove LDL particles from the circulation. Elevated LDL-C concentration leads to premature atherosclerotic cardiovascular disease (ASCVD). The prevalence of the homozygous FH (HoFH) has been estimated at 1 in 160,000 to 1 in 300, 000 and of the heterozygous state from 1 in 311. FH remains underdiagnosed and undertreated in China, especially in the research related to gene diagnosis. When patients with FH phenotype have unreported pathogenic mutations, they are likely to carry new pathogenic mutations, and further functional identification and pathogenicity verification are needed. In this study, genetic cascade screening was performed on a FH patient with clinical phenotype and his relatives. It was found that there was an unreported suspected pathogenic point mutation in intron 13 of LDLR, we evaluate the functional impact of a novel LDLR intronic variant in our patient with FH phenotype by sequencing and in vitro functionality assays.

Methods

Whole genome sequencing of LDLR, apoB and pcsk9 was performed in our patient with the phenotype of FH and his relatives, and a novel intronic variant in LDLR was found. The point mutation was constructed in the HepG2 cell line via the CREISPR-Cas9 technology. Western blot and cytometry were performed to detect the expression of LDLR between the mutant and wild type cells. Moreover, LDL uptake assay were performed to explore whether this mutation altered the ability to uptake which detected by cytometry and Fluorescence microscope.

Results

There was no statistically different expression level of total LDLR between mutant and wild type groups, but the mutant group had less mature LDLR. Moreover, there were less LDLR expressed on the membrane of the mutant group. For LDL uptake assay, we found mutant LDLR had a poor LDL uptake ability.

Conclusions

A novel intronic variant in LDLR ( c.1988-8 T > A) was identified to be responsible for FH. The HepG2 carrying this type of intronic variant had lower levels of mature LDLR, the reduced membrane and the impaired ability to take up LDL. Moreover, our study also suggests the need to widen the scope from LDLR exome sequencing toward whole LDLR gene sequencing in patient with an undetected gene mutation within the LDLR coding sequence.

Key words Familial hypercholesterolemia, intron, Low density lipoprotein receptor

第二部分英文摘要

Objective: We evaluated the safety and efficacy of lipoprotein apheresis (LA) in patients with familial hypercholesterolemia (FH) who can’t reach low-density lipoprotein cholesterol (LDL-C) target goals with the maximal tolerated dose of lipid-lowering agents.

Methods: This was a retrospective cross‑sectional study. Between February 2015 and November 2019, patients with FH who were admitted in Fuwai hospital and treated with LA were consecutively enrolled. Based on intensive lipid-lowering agents, these patients received LA by double filtration plasma pheresis (DFPP) method. The changes of lipid levels such as LDL-C and lipoprotein(a) [Lp(a)] were compared before and after LA treatment, the changes of immunoglobulin (Ig) concentration and LA-related adverse effects were also discussed.

Results: A total of 115 patients with FH were enrolled in this study, of which 8 cases were homozygous and 107 cases were heterozygous. The age was (43.9±12.2) years and there were 75 (65.2%) males, and 108 (93.8%) with coronary artery disease. For pre-and immediately after LA treatment, the LDL-C was (5.20±2.94) mmol/L vs. (1.83±1.08) mmol/L, Lp(a) concentration was 428.70（177.0, 829.50）mg/L vs. 148.90(75.90, 317.00) mg/L (P<0.001), with a decrease of 64.2% and 59.8% respectively. The levels of IgG and IgA measured 1 day after LA treatment were both in the normal range and IgM concentration was below the reference value, the reductions of which were 15.1%、25.0% and 58.7% respectively (P<0.001). Six patients had mild symptoms of nausea, hypotension dyspnea and palpitation, the symptoms were relieved by symptomatic treatment.

Conclusions: Lipoprotein apheresis therapy can significantly decrease blood levels of LDL-C and Lp(a) at short term in FH patients with good tolerance. For patients with FH who do not achieve LDL-C target goal with the maximal tolerated lipid-lowering agents, especially those with elevated Lp(a) levels, LA, which can significantly further reduce LDL-C and Lp(a) levels, is an effective and safe option.

Key words: Atherosclerosis, Familial hypercholesterolemia, Lipoprotein apheresis.