A family presenting with bilateral FEVR was recruited for the present study. A total of five family members underwent complete ophthalmic examinations in the Zhongshan Ophthalmic Center, Sun Yat-sen University (Guangzhou, China). Visual acuity was examined using the early treatment diabetic retinopathy study chart (Precision Vision, Lasalle, IL, USA). Anterior segment photography was performed using the BX 900 Slit Lamp (Haag-Streit AG, Koeniz, Switzerland). Fundus photography and fundus fluorescein angiography (FFA) were performed using the Heidelberg Retina Angiograph (Heidelberg Engineering GmbH, Heidelberg, Germany) and the Ret Cam imaging system (Clarity Medical Systems, Inc., Pleasanton, CA, USA). The amplitudes of the rod and cone responses were assessed using the Espion electrophysiology system (Diagnosys LLC, Lowell, MA, USA) according to the electroretinogram (ERG) standards of the International Society for Clinical Electrophysiology of Vision (ISCEV; 2008 update). Physical examinations and B-scan ultrasonography were performed to confirm the diagnosis and exclude systemic diseases.

A total volume of 15 ml venous blood samples from the patient, the unaffected family members, and 200 unrelated control subjects from the same population were collected. Genomic DNA from peripheral blood leucocytes was extracted using the QIAamp DNABlood Midi kit (cat. no. 51104, Qiagen GmbH, Hilden, Germany) according to the manufacturer's instructions. The genomic DNA was fragmented by the Covaris LE220 Sonicator (Covaris, Woburn, MA, USA) to generate a paired-end library (200–250 bp). The library was enriched by array hybridization as previously described (21), followed by elution and post-capture amplification. The products were then subjected to the Agilent 2100 Bioanalyzer (Agilent Technologies, Inc., Santa Clara, CA, USA) and StepOnePlus Real-Time PCR System (Thermo Fisher Scientific, Inc., Waltham, MA, USA) for estimation of the enrichment magnitude. Subsequent to quality control, captured library sequencing was performed on the Illumina HiSeq2500 Analyzers (Illumina, Inc., San Diego, CA, USA) for 90 cycles per read to generate paired-end reads. Image analysis, error estimation and base calling were performed using the Illumina Pipeline software (version 1.3.4; Illumina, Inc.) to generate raw data (22).

A capture panel of inherited retinal-disease genes was previously designed and assessed in the present study. The capture panel comprised 708,919 bp that covered all exons together with the flanking exon and intron boundaries (±15 bp) of 175 genes, including 138 genes causing common inherited non-syndromic eye diseases and 54 genes causing syndromic eye diseases (22,23).

To detect the potential variants in the family, bioinformatics processing and data analysis were performed after receiving the primary sequencing data. Previously published filtering criteria were used to generate 'clean reads' for further analysis (21). The 'clean reads' (with a length of 90 bp) derived from targeted sequencing and filtering were subsequently aligned to the human genome reference (hg19) using the Burrows Wheeler Aligner (BWA) Multi-Vision software package (version 0.7.12, http://bio-bwa.sourceforge.net) (24). Following alignment, the output files were used to perform sequencing coverage and analysis of the target region, single-nucleotidevariant (SNV), and insertion/deletion (indel) calling. SOAPsnp software (version 1.03, http://soap.genomics.org.cn/soapsnp.html) (24) and Samtools (version 1.6, http://samtools.sourceforge.net) (25) were used to detect SNVs and indels. All SNVs and indels were filtered and estimated via multiple databases, including the National Center for Biotechnology Information (NCBI) dbSNP (https://www.ncbi.nlm.nih.gov/snp/), HapMap1000 human genome dataset (http://www.internationalgenome.org/home), and a database of 200 Chinese healthy adults based on the blood samples of the 200 untreated control patients included in the present study.

The identified mutations were validated using conventional polymerase chain reaction (PCR)-based sequencing methods. Exon 40 of the ABCA4 gene (OMIM 601691) and the exon 2 of the LRP5 gene were amplified by PCR using the respective primers (Table I) as previously described (26–28). Briefly, the PCR reactions were conducted in a total volume of 50 µl using the following thermal cycling profile: One cycle at 94°C for 5 min, followed by 40 cycles at 94°C for 45 sec, 60–62°C for 45 sec, 72°C for 45 sec, and a final extension step at 72°C for 10 min. The PCR products were sequenced from each direction with an ABI3730 Automated Sequencer (Thermo Fisher Scientific, Inc.). The sequencing results were analyzed using the SeqManII program of the Lasergene package (DNAStar, Inc., Madison, WI, USA) and compared with the reference sequences in the NCBI database (29,30).

To predict the effect of missense variants, polymorphism phenotyping (PolyPhen) and sorting intolerant from tolerant (SIFT) were used to predict the potential impact of a single amino acid substitution on the protein structure and function using straight forward physical and comparative considerations (22,23,31). Variants were considered pathogenic when at least one of the two programs predicted deleterious effect of the amino acid substitution on the protein structure or function. The Human Gene Mutation Database was used to screen mutations reported in the published studies. In addition, HomoloGene (https://www.ncbi.nlm.nih.gov/homologene) was used to assess whether the mutated amino acid residues were conserved across different species.

All experimental protocols were performed according to the guidelines approved by the ethics committee of Zhongshan Ophthalmic Center. Written informed consent forms were obtained from all family members prior to the study. The tenets of the Declaration of Helsinki were followed throughout the study.

The family in the current study was from the southern area of China. The pedigree indicated that the FEVR was inherited in an AD manner (Fig. 1). The affected father (I:1) exhibited a normal anterior segment with transplant cornea and lens in each eye (Fig. 2A–D). Fundus photography and FFA exhibited peripheral retinal degeneration (Fig. 2E and F; black arrows) and brush-like peripheral vessels (Fig. 2G and H; white arrows), which are considered typical signs of FEVR. The mother (I:2) and the 9-year-old daughter (II:1) presented with normal anterior segments and normal retinal appearance without leakage and degeneration (Figs. 3 and 4). The 7-year-old son (II:2; the proband) presented with local retinal detachment and low vision at birth. He exhibited bilateral nystagmus. Anterior-segment examination demonstrated local lens opacities in the right eye (Fig. 5A and B; white arrows), whereas the left eye was relatively normal (Fig. 5C and D). Fundus examination exhibited knife-like retinal fold (falciform retinal fold), macular dragging and retinal detachment (Fig. 5E and F; black arrows). FFA demonstrated peripheral retinal avascularity with abnormal vessels and leakage (Fig. 5G and H; white arrows). A B-scan indicated retinal detachment (Fig. 5I and J; white arrows). ERG exhibited mild abnormal rod responses and severe abnormal cone responses (Fig. 6). Another affected 6-year-old son (II:3) presented with bilateral nystagmus, but normal anterior segment (Fig. 7A–D). Fundus examination revealed straight retinal blood vessels and the absence of a macular arch ring (Fig. 7E and F). The clinical manifestations of the family members are summarized in Table II.

One recurrent heterozygous ABCA4 mutation c.5693G>A (p.R1898H) in exon 40 was identified in the cases of I:1, II:1, II:2 and II:3 (Fig. 8A and Table II). Multiple sequence alignment indicated that the residue at position 1898 of ABCA4 is not highly conserved (Fig. 8B). Polyphen (Fig. 8C) and SIFT predicted that the amino acid substitution R1898H in the protein ABCA4 is benign.

One novel heterozygous LRP5 mutation c.260T>G (p.I87S) in exon 2 was identified in this family (I:1, II:2 and II:3; Fig. 9 and Table II). A single nucleotide polymorphism c.266A>G (p.Q89R, rs41494349) in exon 2 was identified in the cases of I:2, II:1 and II:3 (Fig. 9A) (13). The residues at position 87 and 89 of LRP5 are highly conserved across species (Fig. 9B). PolyPhen (Fig. 9C) and SIFT predicted that the amino acid substitution I87S in the protein LRP5 is damaging. PolyPhen (Fig. 9C) predicted that the amino acid substitution Q89R in the protein LRP5 is benign.

These two mutations [c.5693G>A (p.R1898H) in ABCA4 and c.260T>G (p.I87S) in LRP5] were not observed in the 200 unrelated subjects from the same population.