The study protocol conforms to the ethical guidelines of the 1975 Declaration of Helsinki and was approved by Institutional Review Board (IRB) at the Second Affiliated Hospital, Zhejiang University School of Medicine (2016-087). Written informed consent was obtained from all participants. 10 out of total 31 family members in a 4-generation SCD family were recruited in the current study. A complete clinical information including family history, medical history, physical examination, lab test, 12-lead echocardiogram (ECG), 24-hour Holter monitoring, transthoracic echocardiography and CMR were collected.
DNA extraction, target region capture and next-generation sequencing
The proband was selected for next generation sequencing using a commercial capture array (Roche NimbleGen, WI, USA) covering the exons and 50 base pairs of adjacent introns of 1876 cardiovascular diseases associated genes, including inherited cardiomyopathy, arrhythmogenic diseases, congenital heart diseases, mitochondrial diseases, etc (supplementary Table 1).
Genomic DNA was extracted from peripheral blood lymphocytes by standard procedures using Axygen® AxyPrep™-96 Blood Genomic DNA Kit (Axygen, NY, United States). The DNA libraries were constructed and sequenced using the Illumina 2000 platform (Illumina, CA, United States), providing an average sequencing depth of > 100-fold of targeted exons.
Data filtering and bioinformatics analysis
The screening algorithms for potential disease-causing variants were as follows. Initially, intronic and synonymous exonic variants were excluded. Secondly, matched population and in-house database minor allele frequencies (MAF) were used to rule out common variants, defined by MAF>0.01. MAF of 3 major SNP databases were compared: ExAc (http://exac.broadinstitute.org/), 1000 genomes (http://www.1000genomes.org/) and ESP6500 (http://evs.gs.washington.edu/EVS/). Thirdly, rare non- synonymous variants were examined with HGMD (http://www.hgmd.cf.ac.uk/ac/), OMIM (http://www.omim.org/) and ClinVar databases (https://www.ncbi.nlm.nih.gov/clinvar/) and finally analyzed using 3 known prediction tools, namely PolyPhen-2 (http://genetics.bwh.harvard.edu/pph2/), SIFT (http://sift.jcvi.org/) and MutationTaster (http://www.mutationtaster.org/), and categorized according to the recommended guidelines of the American College of Medical Genetics and Genomics (ACMG) and the Association for Molecular Pathology. Sanger sequencing was performed bidirectionally for the verification of AKAP9 c.10714C>G, FLNC c.7778C>G, SYNE1 c.25954C>T and DSP c.832delG in all participants.
Plasmids construction and site-directed mutagenesis
AICSDP-9:DSP-mEGFP was a gift from the Allen Institute for Cell Science (Addgene plasmid # 87424 ; http://n2t.net/addgene:87424 ; RRID:Addgene_87424). In order to facilitate the observation following transfection of mutant plasmid, GFP were cleaved and inserted in between the promoter and DSP gene. The frame-shift mutation was introduced into a wild-type DSP clone using a QuikChange II XL Site-Directed Mutagenesis Kit (Stratagene, La Jolla, CA, USA). The clones were sequenced to confirm the desired mutation and to exclude any other sequence variations.
RT-PCR and real-time PCR
HEK293T cells were transfected with either blank, wild type or mutant plasmids using lipofectamine 3000 (Invitrogen, MA, USA) according to the manufacturer’s instructions. Total RNA was extracted from transfected cells using the Trizol reagent (Invitrogen, MA, USA). cDNA was synthesized using PrimeScript RT reagent Kit (Takara, Shiga, Japan). The resulting cDNA was subjected to real-time PCR using TB Green Premix Ex Taq kits (Takara, Shiga, Japan) on an Applied Biosystems 7500 Fast Real-Time PCR System (ABI, CA, USA). The primers named “N-terminal” detected the mRNA levels in the N-terminal side of the DSP mutation site, and the primers named “C-terminal” detected the mRNA levels in the C-terminal side of the DSP mutation site. GAPDH was used as an endogenous control.
The sequences of primers were listed as follows:
C-terminal-F : 5’-ACATCATTCAGGCCACGT-3’;
C-terminal-R: 5’- CCAGTTGACTCATGCGTA-3’;
24 hours after transfection, total cell extracts were lysed by RIPA lysis buffer. Nuclear and cytoplasmic extracts were separated using Nuclear and Cytoplasmic Protein Extraction Kit (Beyotime Biotechnology, Shanghai, China). Next, proteins were separated by sodium dodecyl sulfate poly-acrylamide gel electrophoresis (SDS-PAGE) and transferred to polyvinylidene fuoride (PVDF) membranes. The membranes were blocked for 1 hour in a blocking solution of 5% (w/v) non-fat milk in PBS containing 0.1% (v/v) Tween-20 and incubated at 4°C overnight with indicated primary antibodies. Primary antibodies included antibodies against JUP (1:1000, sc-8415, Santa Cruz Biotechnology, CA, USA), β-catenin (1:1000, ab6302, Abcam, Cambridge, UK), GFP (1:1000, AF1483, Beyotime Biotechnology), GAPDH (1:5000, 3683S, Cell Signaling Technology, MA, USA), Lamin B1 (1:1000, ET1606-27, HuaBio antibodies, China). Excess primary antibodies were washed off, and then the membranes were incubated with secondary antibodies conjugated with horseradish peroxidase for 1 hour at room temperature. The western blot bands were visualized were visualized using the enhanced chemiluminescence western blotting detection system (Bio-Rad, CA, USA ).
Cells seeded on cover slips were fixed with 4% paraformaldehyde (PFA)/PBS, permeabilized in 0.5%(v/v) Triton X-100 (Sigma-Aldrich, MO, USA) and blocked with 5% (w/v) BSA. Then the cells were incubated using the antibody mouse-anti-JUP (1:1000, sc-8415, Santa Cruz Biotechnology) overnight at 4 °C, followed by secondary antibodies anti-mouse Alexa Fluor 594 (1:200, Thermo Fisher, A-21203, CA, USA) incubation in 5% BSA in PBS for 1h at room temperature. Finally, coverslips were mounted on microscope slides using mounting medium contained with DAPI (H-1200, Vector, CA, USA). Images were acquired using a ﬂuorescence microscope (Leica, IL, USA). Colocalization analysis between JUP and nuclear was performed by Coloc 2 ImageJ in random high-power fields. Pearson's correlation coefficient was used to represent the colocalization quantification, +1 for perfect correlation, 0 for no correlation, and -1 for perfect anti-correlation. Optical confocal microscopies of cells were obtained using Leica TCS SP8 (Leica Microsystems Inc).
Data were presented as the means ± SEM of at least three independent experiments. Student T test was performed to evaluate differences of continuous variables between two groups. One-way ANOVA was used for comparison among three groups. P values of less than 0.05 were considered statistically significant. Statistical calculations were carried out using GraphPad Prism 8.0.1.