家族性高胆固醇血症(FH)是一种常染色体显性疾病,其特征是LDL-胆固醇水平升高。大约85%的FH病例是由编码低密度脂蛋白受体(LDLR)的LDLR突变引起的。LDLR在内质网(ER)中合成,在此经历翻译后修饰,然后通过高尔基体转运到质膜。已经在FH患者中报道了超过2900种LDLR变体,其中许多的致病性和功能信息有限。这项研究旨在阐明使用生化和功能方法在疑似FH患者中鉴定的LDLR错义变体的细胞运输和功能意义。
■我们使用了HeLa,HEK293T,和LDLR缺陷型CHO-ldlA7细胞,以评估十个LDLR错义变体的亚细胞定位和LDL内化(p。C167F,p.D178N,p.C243Y,p.E277K,p.G314R,p.H327Y,p.D477N,p.D622G,p.R744Q,和p.R814Q)在多种族疑似FH患者中报告。我们还分析了三个变体的功能影响(p。D445E,p.D482H,andp.C677F),其中两个先前显示保留在急诊室。
■我们证明p.D622G,p.D482H,和p.C667F大部分保留在ER中,而p.R744Q部分保留。其他变体主要定位于质膜。CHO-ldlA7细胞中的LDL内化测定表明p.D482H,p.C243Y,p.D622G,和p.C667F在数量上失去了与其他Dil-LDL内化的能力(p。C167F,p.D178N,p.G314R,p.H327Y,p.D445E,p.D477N,p.R744Q和p.R814Q)显示出显著的损失,除了p.E277K保留了全部活性。然而,LDL内化测定仅能够评估变体对LDL内化的影响,而不是确切的功能缺陷,例如未能结合LDL。所表示的数据说明了引起FH的变体的同态性质,这可以解释FH的一些可变表达性。
■我们的组合方法,细胞,功能分析是确定致病性和FH疾病机制的有力策略,可能为新的治疗策略提供机会。
UNASSIGNED: Familial hypercholesterolemia (FH) is an autosomal dominant disorder characterized by increased LDL-cholesterol levels. About 85% of FH cases are caused by LDLR mutations encoding the low-density lipoprotein receptor (LDLR). LDLR is synthesized in the endoplasmic reticulum (ER) where it undergoes post-translational modifications and then transported through Golgi apparatus to the plasma membrane. Over 2900 LDLR variants have been reported in FH patients with limited information on the pathogenicity and functionality of many of them. This study aims to elucidate the cellular trafficking and functional implications of LDLR missense variants identified in suspected FH patients using biochemical and functional methods.
UNASSIGNED: We used HeLa, HEK293T, and LDLR-deficient-CHO-ldlA7 cells to evaluate the subcellular localization and LDL internalization of ten LDLR missense variants (p.C167F, p.D178N, p.C243Y, p.E277K, p.G314R, p.H327Y, p.D477N, p.D622G, p.R744Q, and p.R814Q) reported in multiethnic suspected FH patients. We also analyzed the functional impact of three variants (p.D445E, p.D482H, and p.C677F), two of which previously shown to be retained in the ER.
UNASSIGNED: We show that p.D622G, p.D482H, and p.C667F are largely retained in the ER whereas p.R744Q is partially retained. The other variants were predominantly localized to the plasma membrane. LDL internalization assays in CHO-ldlA7 cells indicate that p.D482H, p.C243Y, p.D622G, and p.C667F have quantitatively lost their ability to internalize Dil-LDL with the others (p.C167F, p.D178N, p.G314R, p.H327Y, p.D445E, p.D477N, p.R744Q and p.R814Q) showing significant losses except for p.E277K which retained full activity. However, the LDL internalization assay is only to able evaluate the impact of the variants on LDL internalization and not the exact functional defects such as failure to bind LDL. The data represented illustrate the hypomorphism nature of variants causing FH which may explain some of the variable expressivity of FH.
UNASSIGNED: Our combinatorial approach of in silico, cellular, and functional analysis is a powerful strategy to determine pathogenicity and FH disease mechanisms which may provide opportunitites for novel therapeutic strategies.