A Chinese family was recruited from the First Hospital of Jilin University (Changchun, China), including a total of seven members, with one affected patient and six unaffected individuals (Fig. 1). The control subjects were recruited from Sichuan Provincial People's Hospital (Chengdu, China). All control subjects underwent ophthalmic examination and exhibited no symptoms of RP. The recruitment of the control subjects was previously described (13). The study was implemented in conformity to the tenets of the Declaration of Helsinki and approved by the Institutional Review Boards of Sichuan Provincial People's Hospital (Chengdu, China) and the First Hospital of Jilin University (Changchun, China). Written informed consent was obtained from all participants or parents of children prior to their inclusion in the present study.

Comprehensive ophthalmic examinations were performed on the proband (age, 47 years), the proband's only daughter (age, 23 years), the proband's three brothers (age, 54, 51 and 44 years, respectively), and the proband's only sister (age, 57 years) in the present study. The examinations included visual acuity testing, computerized visual field measurement, tests of dark adaptation and color vision, optical coherence tomography (OCT) and fundus imaging.

Peripheral blood samples were collected in EDTA tubes from the proband (II:4), the proband's daughter (III:1) and the proband's brothers (II:2, II:3 and II:5). Genomic DNA was extracted using the salt precipitation method, involving lysis, precipitation, washing and resuspension (14). DNA samples were stored at −20°C until use.

The DNA of case no. II:4 (the proband) was analyzed by WES at Novogene Biotechnology Inc., with a mean read depth of target regions of 100X. The sequenced samples were prepared according to the Illumina standard protocol (Illumina, Inc.). First, genomic DNA was randomly fragmented to an average size of 180–280 bp by a Covaris S220 sonicator (Covaris, Inc.). The remaining overhangs were converted into blunt ends via exonuclease polymerase activities. Subsequently, DNA fragments were end-repaired and phosphorylated, followed by A-tailing and ligation at the 3′ends with paired-end adaptors (Illumina, Inc.). DNA fragments with ligated adapter molecules on both ends were selectively enriched by PCR. After PCR amplification, libraries were hybridized with a liquid phase containing a biotin-labeled probe and magnetic beads with streptomycin were used to capture the exons of genes. Captured libraries were enriched by PCR to add index tags to prepare for sequencing. Products were purified using an AMPure XP system (Beckman Coulter, Inc.) and quantified using the Agilent high-sensitivity DNA assay on the Agilent Bioanalyzer 2100 system (Agilent Technologies, Inc.). Finally, the DNA library was sequenced on Illumina HiSeq4000 for paired-end 150 bp reads.

Sanger sequencing was used to confirm the identified mutation in CNGA1. All exons of CNGA1 were amplified from the genomic DNAs from subjects II:2-5 and III:1 in the recruited family by PCR using standard conditions (15). Amplified products were purified and sequenced with forward and reverse primers (15) by using the BigDye^® Terminator v3.1 Cycle Sequencing kit (Applied Biosystems; Thermo Fisher Scientific, Inc.) on a 3730 ABI DNA sequencer (Thermo Fisher Scientific, Inc.).

A Chinese family with RP, without any history of consanguineous marriage, was recruited for the present study. The pedigree chart is provided in Fig. 1. Examination of the fundus revealed a normal fundus in subject no. III:1, the proband's daughter (Fig. 2A). By contrast, the proband (II:4) exhibited several classic characteristics of RP, including attenuated blood vessels, pale optic discs and paravascular bone spicule pigmentation (Fig. 2B). OCT revealed thinning of the outer nuclear layer and thinning of the reflex of the retinal pigment epithelium-Bruch's membrane complex (Fig. 2C). The visual field test indicated a significant loss of peripheral vision in the proband, while the vision was normal in the unaffected individuals (II:2, 3, 5 and III:1; Fig. 3A). Electroretinography (ERG) recordings were barely detectable in the proband under either the scotopic or photopic conditions, indicating an almost complete loss of the function of both rods and cones (Fig. 3B-D). The proband's daughter (III:1) had normal visual responses in the ERG tests. In summary, while all of the healthy unaffected family members had normal vision, the affected individual exhibited the typical clinical symptoms of late-stage RP.

To identify the genetic variant causing the RP condition in the family, WES was performed for the proband (subject no. II:4). In total, 24,702 SNPs in coding regions or splicing junctions (2,797 nonsynonymous SNPs, 3,349 synonymous SNPs and 18,556 other types of SNP) and 659 indels were identified. Since neither the proband's parents nor her siblings were affected, the pathogenic variant was more likely to be recessive. Thus, the recessive genetic mode was applied to analyze those SNPs. In order to define the pathogenic variant, the homozygous and compound heterozygous variants were closely followed, particularly frameshift insertions or deletions in the coding region, splicing acceptor and donor site variants, as well as non-synonymous variants that were more likely to be pathogenic. Of note, a homozygous single nucleotide deletion variant in exon 6 of CNGA1 (c.265delC; Chr4:47951883-47951884) was identified in subject no. II:4. This variant causes a frameshift of the open reading frame and loss of function of the protein. CNGA1 is an essential protein involved in the phototransduction cascade in rod photoreceptors. Without CNGA1, the phototransduction cascade would fail in the rods (16). Therefore, this variant was a top candidate subjected to further validation.

The homozygous variant, c.265delC (p.L89Ffs*3) in CNGA1, was confirmed using direct Sanger sequencing in the affected individual (II:4; Fig. 4). It was also determined that, while the proband's daughter (III:1) and one of the proband's brothers (II:2) were heterozygous carriers of c.265delC (p.L89Ffs*3), the other two brothers of the proband (II:3 and II:5) had no variant at this position. The homozygous recessive variant matched the genotype/phenotype in this family. Furthermore, this variant was absent from 600 ethnicity-matched normal control samples (median age, 61; age range, 40–78 years) that underwent Sanger sequencing. Therefore, it was concluded that the homozygous CNGA1 c.265delC variant (p.L89Ffs*3) is the variant responsible for the RP condition in the proband.