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, United States) 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.
Genomic DNA was extracted from peripheral blood lymphocytes by standard procedures using Axygen® AxyPrep™-96 Blood Genomic DNA Kit (Axygen,United States). The DNA libraries were constructed and sequenced using the Illumina 2000 platform (Illumina, United States), providing an average sequencing depth of > 100-fold of targeted exons.
Data filtering and bioinformatics analysis
Minor allele frequencies (MAF) of the identified variants in the matched population were compared with 3 major SNP databases: ExAc (http://exac.broadinstitute.org/), 1000 genomes (http://www.1000genomes.org/) and ESP6500 (http://evs.gs.washington.edu/EVS/), as well as in-house database. Known or published genetic determinants for the human inherited disease were reviewed with HGMD (http://www.hgmd.cf.ac.uk/ac/), OMIM (http://www.omim.org/) and ClinVar databases (https://www.ncbi.nlm.nih.gov/clinvar/). The in silico prediction analysis of non-synonymous variants were performed 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/). Sanger sequencing was performed bidirectionally for the verification of AKAP9 c.10714C>G, FLNC c.7778C>G 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)[13]. 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.
Western Blots
HEK293T cells were transfected with either wild type or mutant plasmids using lipofectamine 3000 (Invitrogen, USA). Total cell extracts were lysed by RIPA lysis buffer. Nuclear and cytoplasmic extracts were separated using Nuclear and Cytoplasmic Protein Extraction Kit (Beyotime Biotechnology, China). Protein samples were separated by SDS-PAGE, transferred to PVDF membranes and immunoblotted using the corresponding antibodies.
Confocal Microscopy
Cells seeded on cover slips were fixed with 4% paraformaldehyde (PFA)/PBS, permeabilized in 0.5%(v/v) Triton X-100 (Sigma-Aldrich, USA) and blocked with 5% (w/v) BSA. Then the cells were immunoblotted using the corresponding antibodies, and the cover slips were mounted on microscope slides using mounting medium containing DAPI (Burlingame, CA, USA). Optical sections of cells were obtained using Leica TCS SP8 (Leica Microsystems Inc., Buffalo Grove, IL, USA) with a 63× objective lens.
Statistical analysis
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. P values of less than 0.05 were considered statistically significant. Statistical calculations were carried out using GraphPad Prism 8.0.1.