Study participants and blood sample collection

According to the diagnostic criteria of the Dutch Lipid Clinic Network (DLCN), individuals with scores of ≥ 8 points and their families were included in this study. After using DLCN diagnostic criteria to evaluate the participants, a family tree was built. Venous blood was collected from participants for a blood lipid analysis and subsequent whole-exome sequencing. Participants had completed informed consent forms, which were authorized by the First Affiliated Hospital Ethics Committee of Ningbo University.

Whole-exome sequencing

Venous blood samples were forwarded to the Beijing Genomics Institution (BGI, Wuhan, China) for whole-exome sequencing. After low-quality reads, adapters, and a high percentage of N-bases were removed from the raw sequencing data, alignments against the human reference genome hg19 sequence were generated using Burrows Wheeler Aligner [30]. Using the Genome Analysis Toolkit (GATK), duplicate reads were tagged, and base mass value recalibration was performed. GATK4’s HaplotypeCaller was used to find single nucleotide polymorphisms (SNPs) and InDels [31]. Rigorous filtering was applied to extract SNPs and InDels that are both highly dependable and of excellent quality.

Sanger sequencing

Utilizing the E Z. N.P. ® Blood DNA Mini Kit (D3392-02; Omega Bio-Tek, Norcross, GA, USA) for DNA extraction, which was then amplified using polymerase chain reaction (PCR). The total PCR system was 50 µL, which includes 25 µL of 2× ES Taq Master Mix, 2 µL of forward primer (5′-CAGGACGAGTTTCGCTGCCAC-3′), 2 µL of reverse primer (5′-ATCCGAGCCATCTTCGCAGTC-3′), 500 ng of DNA and enzyme-free water. After sending the PCR products to BGI for Sanger sequencing, data analysis was conducted using Chromas software.

In silico analysis

MutationTaster was used to predict the pathogenicity of point variants [32]. DynaMut was utilized to evaluate how point variants affected the stability and flexibility of proteins [33]. A normal mode analysis was used to determine the difference in free energy change (ΔΔG) between the structures of the wild-type (WT) and the variant. ENCoM-based difference in vibrational entropy (ΔΔSVib) was used to predict the difference in flexibility [34]. SnapGene v6.0.2 was employed to determine the conservation of protein sequences among species using the multiple sequence comparison by log-expectation (MUSCLE) algorithm.

Plasmid construction, cell culture, and transfection

Shanghai GeneChem Co. (Shanghai, China) completed the construction of human WT LDLR and LDLR c.415G > A with a FLAG epitope close to the N terminus in the GV208 vector. HEK293T cells were used for the plasmid transfection [35]. The cells were grown in Eagle media that had been modified by Dulbecco (high glucose) (Cytiva, Shanghai, China) containing 10% fetal bovine serum (Vivacell, Shanghai, China). For transfection, the cells were transfected with 2500 ng of plasmid DNA using Lipofectamine™ 3000 Reagent (Invitrogen, Shanghai, China) in a six-well plate.

Quantitative real-time PCR

Following transfection, TRIzol (Omega, Norwalk, CT, USA) was used to extract RNA, and the HiFiScript cDNA Synthesis Kit (CW2569M; CWBIO, Beijing, China) was used for reverse transcription. The Mastercycler® Nexus X2 (Eppendorf, Hamburg, Germany) was used to carry out quantitative real-time PCR (qPCR). TaqMan assays were employed for the detection of fluorescence. The relative amplification efficiency of LDLR was established using the comparative Ct method. The primers used were as follows: LDLR, F-5′-AAGTGCATCTCTCGGCAGTT-3′, LDLR, R-5′-CCACTCATCCGAGCCATCTT-3′; GAPDH, F-5′-GGAAATCGTGCGTGACATTA-3′, R-5′-GGAAGGAAGGCTGGAAGAG-3′.

Western blotting

The cells were lysed using RIPA solution (Solarbio, Beijing, China), which contains inhibitors of phosphatase and protease. The proteins were boiled for 10 min with loading buffer (Solarbio, Beijing, China) in preparation for western blotting. Following 7.5% SDS/PAGE resolution, the samples were blotted onto PVDF membranes (Merck, Darmstadt, Germany). After using 5% skim milk to prevent non-specific binding, monoclonal mouse anti-FLAG (1:3000, F1804; Sigma, Shanghai, China) and monoclonal rabbit anti-β-actin (1:10000, AF7018; Affinity Biosciences, San Francisco, California, USA) primary antibodies were added, and the mixture was incubated for a whole night at 4 °C. Then, the samples were treated with the corresponding horseradish peroxidase-conjugated IgG for 60 min. Lastly, the immunoreactive proteins were identified using enhanced chemiluminescence.

Flow cytometry

Cells were added to a six-well plate with 0.05% trypsin and transferred into a 2 mL EP tube. Diluted rabbit anti-human LDLR monoclonal antibody conjugated with allophycocyanin (1:200, ab275614; Abcam, Cambridge, MA, USA) was added, and the mixture was maintained in the dark for an additional hour after blocking with 10% donkey serum for an hour at room temperature. The mean fluorescence levels from at least three replicate estimates were obtained using a Beckman CytoFlex S flow cytometer (Beckman Coulter, Shanghai, China). Data analysis was done with FlowJo software.

Immunofluorescence

After transfection, cells were fixed using 4% paraformaldehyde (P1110; Solarbio, Beijing, China). Following a wash with 1× PBS, the cells were blocked using a 10% goat serum solution to prevent non-specific binding. Next, mouse anti-flag antibody (1;3000, F1840; Sigma-Aldrich, Saint Louis, USA) was diluted in 1x PBS and incubated at 4 °C for 4 h, along with 20 µg/mL labelled human plasma LDL (Dil-LDL; L3482; Thermo Fisher, Shanghai, China). After incubation, the cells were washed with 1× PBS and then treated with goat anti-mouse IgG conjugated with AlexaFluor488 (1:500, ab150113; Abcam, Cambridge, UK). After completing the staining of nucleus with 4′,6-diamidino-2-phenylindole (DAPI), the cells can be observed under a LEICA TCS SP8 confocal laser scanning microscope.

In order to assess the uptake capacity of LDLR, transfected HEK293T cells were treated with 20 µg/mL Dil-LDL for 4 h at 37 °C. Similarly, confocal microscopy was used for analysis after washing with PBS, fixation with 4% paraformaldehyde, and DAPI labeling of cell nuclei.

Statistical analysis

All data was analyzed using GraphPad Prism (version 9.0.0; La Jolla, CA), and presented as means ± SEM. Normal distribution was evaluated using the D’Agostino–Pearson omnibus normality test. Group differences were evaluated using a one-way ANOVA. P < 0.05 was used as the statistical significance criterion.