Patients and clinical assessment
The study was adhered to the tenets of the Declaration of Helsinki and approved by the medical ethics committee of Shanghai General Hospital (Shanghai First People’s Hospital). All the members were informed the research and consented to it. And they were all enquired about the family and medical history before taking ophthalmological examinations. Then each member underwent detailed ophthalmic examinations, including best-corrected visual acuity (BCVA)[RT-5100,NIDEX], intraocular pressure (IOP)[TX-20,Canon], optical coherence tomography (OCT) scans [SpectralisOCT, Heidelberg], widefield color fundus imaging and widefield fundus autofluorescence (FAF) [200TX, OPTOS]. In addition, visual field [Carl Zeiss Meditec] and full-field electroretinogram (ERG) [RETI-Port/scan 21] were performed to estimate visual function.
Whole-exome sequencing (WES)
The genomic DNA extracted from peripheral blood samples of the members were collected for whole-exome sequencing (WES). Illumina paired-end libraries were generated according to the Kapa LTP library prep kit protocol (Roche, Basel, Switzerland). Agilent SSELXT Human All Exon V6 was used for whole exome sequencing. The enriched DNA library was sequenced on Illumina Xten Analyzers for 150 cycles per read to generate paired-end reads (following the manufacturer’s standard sequencing protocols). Raw reads were aligned to the human genome reference (hg19) using the BWA (Burrows Wheeler Aligner). Single-nucleotide variants (SNVs) and InDels (Insertions and Deletions) were called by Atlas-SNP2 and Atlas-Indel, respectively. The frequency of all SNVs and InDels were annotated using the ExAC, gnomAD, HGVD, CHARGE, 1000 Genome, UK10K databases and the internal database of Clinbytes Inc. to filter the common variants, with a allele frequency cutoff of 0.5 and 0.1% for recessive and dominant variants, respectively. The WES analysis was provided by Clinbytes.
Assessment of the pathogenicity of candidate variants
PCR amplification and Sanger sequencing were used to further validate candidate variations. Each genomic sequence was obtained from the UCSC genome browser (hg19). The repetitive sequences was masked using RepeatMasker in the huamn genome (available at http://www.repeatmasker.org/cgi-bin/WEBRepeatMasker/). Specific forward and reverse primers were designed at least 100 bp from the variants to amplify 300–500 bp fragments. The amplicons were analyzed by Sanger sequencing.
Prediction of protein structure
The amino acid sequences of both wild type and mutant RPGRORF15 have been processed with the I-TASSER web server. Models were visualized and then exported by PyMol (The PyMOL Molecular Graphics System, Version 1.8 Schrödinger, LLC).
Cloning and plasmid construction
The wild type RPGRORF15(RefSeq NM_001034853.2) was amplified by PCR with primers 5′-agagagcccgaggagctg-3′ and 5′-atgactcgagtcacttcagctccaggtag-3′. The c.2383G > T nonsense mutation was introduced by PCR with primers 5′- agagagcccgaggagctg-3′ and 5′-atgactcgagtcagccctgatcgccttcctc-3′. The plasmid which an HA tag was added in constructs (AAV-MCS) were verified by sanger sequencing. 293 T cells were employed to test the expression of RPGR expressing constructs.
Cell culture and transfection
293 T cells were obtained from the American Type Culture 141 Collection (CRL-2302, Manassas, VA, USA) and cultured in DMEM/ High glucose with 10% FBS. The above plasmids’ transfection was executed by Lipofectamine 3000 (Invitrogen, Carlsbad, CA, USA) according to the manufacturer’s protocol. Samples would be harvested 48 h later .
Transfected Cells were lysed with RIPA buffer containing protease and phosphatase inhibitors. Samples (10 μg total protein) were seperated by 10% SDS-PAGE gels and transferred to electroblotted to polyvinylidene difluoride (PVDF) membranes (IPVH00011, Solarbio). The membranes were blocked by 5% nonfat dry milk in Tris-buffered saline (TBS), containing Tween-20 (TBST) for 1 h at room temperature then incubated with primary antibodies against HA (AF0039 Beyotime) at 4 °C overnight. The membranes were washed with TBST three times for 10 min then incubated with the corresponding secondary antibodies (Rabbit, Proteintech) at room temperature for 1 h. After washing with TBST three times for 10 min, the membranes were visualized by the molecular imaging system (Amersham Imager 600, GE Healthcare, Buckinghamshire, UK).
Cells were fixed with 4% paraformaldehyde for 30 min and blocked with PBS containing 5% goat serum albumin (Beyotime) and 0.05% Triton for 1 h. Then, the cells were incubated at 4 °C overnight with antibodies against HA(1:1000, 11,867,431,001, Roche). After being washed with PBS 3 times for 5 min, the cells were incubated with secondary antibody (Rat, Alexa Fluor 488, Invitrogen, USA) for 1 h. Finally, the cells were visualized with a Leica TCS SP8 confocal laser scanning microscope (Leica TCS NT, Wetzlar, Germany) .