The patients were examined at Eye Center, Second Affiliated Hospital, School of Medicine of Zhejiang University, China. They underwent detailed ophthalmic evaluation, including best correct visual acuity (BCVA), slit-lamp bio-microscopy, dilated indirect ophthalmoscopy, anterior chamber (ultrasound biomicroscopy, UBM; SUOER UBM scan SW-3200), widefield retinal imaging (Optos 200Tx, Marlborough, MA, USA), optical coherence tomography (OCT) (Heidelberg HRT II, Heidelberg, Germany) and fundus fluorescein angiography (FFA) (Heidelberg HRT II, Heidelberg, Germany) examinations. All research involved in this study adhered to the tenets of the Declaration of Helsinki and was approved by the Institutional Review Board of Second Affiliated Hospital of Zhejiang University. Informed written consents were obtained from all participating individuals for this study.
DNA library preparation and target sequencing
Genomic DNA of the proband and available family members was extracted from peripheral blood. A pre-capture library was prepared by using Kapa LTP library prep kit and then was captured on a custom capture panel (Agilent Sureselect) which containing 256 known retinal disease genes. The enriched DNA library was sequenced on Illumina Xten Analyzers for 150 cycles per read to generate paired-end reads. An average of 172.82X in target region was achieved, and 97.86 % of the target region was covered by 10x.
Bioinformatics analysis and Sanger sequencing
After the sequencing step, Raw reads were aligned to the human genome reference (hg19) using the Burrows-Wheeler Aligner. Single-nucleotide variants (SNVs) and InDels (Insertions and Deletions) were called by Atlas-SNP2 and Atlas-Indel, respectively. SNVs and InDels were filtered against the ExAC, gnomAD, HGVD, CHARGE, 1000 Genome, UK10K databases and the internal database of Clinbytes Inc, with an allele frequency cutoff of 0.5% and 0.1% for recessive and dominant variants, respectively. Variants were annotated using Annotate Variation (ANNOVAR). The variant was validated by PCR and Sanger sequencing. PCR primer sets were designed via Primer3 and products were sequenced on an ABI 3700XL Genetic Analyzer.
UHPLC-MS/MS metabolomics analysis
Patients peripheral blood were collected and plasma were prepared for LC-MS/MS analysis, which were performed using a Vanqiush UHPLC system (Thermo Fisher) coupled with an Orbitrap Q Exactive HF-X mass spectrometer (Thermo Fisher) operating in the data-dependent acquisition mode. The eluents for the positive and negative polarity mode were eluent A (0.1% FA in Water) plus eluent B (Methanol) and eluent A (5 mM ammonium acetate, pH 9.0) plus eluent B (Methanol), respectively. The solvent gradient was set as follows: 2% B, 1.5 min; 2-100% B, 12.0 min; 100% B, 14.0 min；100-2% B, 14.1 min；2% B, 16 min. Q Exactive HF-X mass spectrometer was operated in positive/negative polarity mode with spray voltage of 3.2 kV, capillary temperature of 320°C, sheath gas flow rate of 35 arb and aux gas flow rate of 10 arb. The raw data files were processed using the Compound Discoverer 3.0 (CD 3.0, Thermo Fisher) to perform peak alignment, peak picking, and quantitation for each metabolite. The normalized data was used to predict the molecular formula based on additive ions, molecular ion peaks and fragment ions. And then peaks were matched with the mzCloud (https://www.mzcloud.org/) and ChemSpider (http://www.chemspider.com/) database to obtain the accurate qualitative and relative quantitative results.