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 (2019-133) (May, 2019). Informed written consent was obtained from all participating individuals in this study.
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), wide-field 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.
DNA library preparation and target sequencing
Genomic DNA of the proband and the available family members was extracted from peripheral blood by using a DNA isolation kit (Qiagen, Hilden, Germany). A pre-capture library was prepared by using Kapa LTP library prep kit (Kapa Biosystems, Wilmington, USA) and then was captured on a custom capture panel (Agilent Sureselect, USA) which containing 256 known retinal disease genes (Supplementary Table 1). The enriched DNA library was sequenced on Illumina Xten Analyzers (San Diego, USA) 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) by using the Burrows-Wheeler Aligner (Wellcome Trust Sanger Institute, Cambridge, UK). Single-nucleotide variants (SNVs) and Insertions and Deletions (InDels) were called by Atlas-SNP2 and Atlas-Indel, respectively. SNVs and InDels were filtered against the ExAC, gnomAD, HGVD, CHARGE, 1000 Genome, and 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). Conservation analysis of the related homologous proteins in the mutation site was performed using the UCSC Genome Browser database. In silico gene function prediction software (e.g., SIFT, PolyPhen2, and FATHMM) was used for pathogenicity assessment. The variants were further validated and segregated by Sanger sequencing from all available family members. PCR primer sets were designed via Primer3, and the products were sequenced on an ABI 3700XL Genetic Analyzer (Thermo Fisher, USA).
UHPLC-MS/MS metabolomics analysis
The patients’ peripheral blood was collected from the available family members, and plasma was prepared for LC-MS/MS analysis, which was performed using the Vanqiush UHPLC system (Thermo Fisher) coupled with an Orbitrap Q Exactive HF-X mass spectrometer (Thermo Fisher) operating in the data-dependent acquisition mode by Novogene Co., Ltd. (Beijing, China). The detailed information has been provided previously . The samples were divided into three groups according to the genotype in chr11:61725867 loci. The plasma metabolites were compared between the homozygous variation group (A/A, n = 2) and the wild-type group (G/G, n = 3), as well as between the homozygous variation group (A/A, n = 2) and the heterozygosity group (A/G, n = 2).