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. 2023 Jun 20;22(1):77.
doi: 10.1186/s12944-023-01848-6.

In vitro assessment of the pathogenicity of the LDLR c.2160delC variant in familial hypercholesterolemia

Shaoyi Lin #  1 , Tingting Hu #  1 , Kaihan Wang #  1 , Jiaqi Wang  1   2 , Yunyun Zhu  3 , Xiaomin Chen  4   5
Affiliations

In vitro assessment of the pathogenicity of the LDLR c.2160delC variant in familial hypercholesterolemia

Shaoyi Lin et al. Lipids Health Dis. .

Abstract

Background: Familial hypercholesterolemia (FH) is an inherited disorder with markedly elevated low-density lipoprotein cholesterol (LDL-C) and premature atherosclerotic cardiovascular disease. Although many mutations have been reported in FH, only a few have been identified as pathogenic mutations. This study aimed to confirm the pathogenicity of the LDL receptor (LDLR) c.2160delC variant in FH.

Methods: In this study, the proband and her family members were systematically investigated, and a pedigree map was drawn. High-throughput whole-exome sequencing was used to explore the variants in this family. Next, quantitative polymerase chain reaction (qPCR), western blot (WB) assays, and flow cytometry were conducted to detect the effect of the LDLR c.2160delC variant on its expression. The LDL uptake capacity and cell localization of LDLR variants were analyzed by confocal microscopy.

Results: According to Dutch Lipid Clinic Network (DLCN) diagnostic criteria, three FH patients were identified with the LDLR c.2160delC variant in this family. An in-silico analysis suggested that the deletion mutation at the 2160 site of LDLR causes a termination mutation. The results of qPCR and WB verified that the LDLR c.2160delC variant led to early termination of LDLR gene transcription. Furthermore, the LDLR c.2160delC variant caused LDLR to accumulate in the endoplasmic reticulum, preventing it from reaching the cell surface and internalizing LDL.

Conclusions: The LDLR c.2160delC variant is a terminating mutation that plays a pathogenic role in FH.

Keywords: Endoplasmic reticulum; Familial hypercholesterolemia; Low-density lipoprotein cholesterol; Low-density lipoprotein receptor; Whole-exome sequencing.

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Conflict of interest statement

The authors declare no conflicts of interest.

Figures

Fig. 1
Fig. 1
Family tree of the proband. The arrow indicates the proband
Fig. 2
Fig. 2
Pathogenic variants and in silico analysis. (A) The results of whole-exome sequencing. (B) Sanger sequencing confirmed the existence of LDLR c.2160delC. The reverse primer was used for Sanger sequencing. The shaded area represents the actual base. The contents of the box are the stop codon induced by the deletion mutation of the 2160 locus
Fig. 3
Fig. 3
LDLR c.2160delC variant affected the expression of LDLR. (A) The mRNA expression level of LDLR c.2160delC in HEK293T cells. (B) The protein expression of LDLR c.2160delC in HEK293T cells. (C) LDLR expression on the HEK293T cell surface. **** represents P < 0.01
Fig. 4
Fig. 4
Representative confocal microscopy images of the localization of LDLR in the endoplasmic reticulum. Blue fluorescence represents the endoplasmic reticulum, while red fluorescence represents LDLR
Fig. 5
Fig. 5
Representative confocal microscopy images. Blue fluorescence represents DAPI, while red fluorescence represents Dil-LDL
See this image and copyright information in PMC

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