Frequency and phenotypic characteristics of RPE65 mutations in the Chinese population

A total of 1434 Chinese patients with IRDs and their available family members (total participants: 3576) were recruited from the eye genetic disease clinic of the Eye and ENT Hospital of Fudan University between January 2017 and June 2019. Of these, 956 patients had been mentioned in our previous report [Gao and others 2019]. Written informed consent in accordance with the tenets of the Declaration of Helsinki was obtained from all participants or their guardians. This study was approved by the Ethics Committee of the Eye and ENT Hospital of Fudan University. DNA was isolated from peripheral blood using the FlexiGene DNA Kit (Qiagen, Venlo, the Netherlands) according to the manufacturer’s protocol. NGS analysis and bioinformatics analysis were performed as previously reported [7]. We designed a high-throughput targeted enrichment approach to exon-capture regions of 586 genes that are involved in common inherited eye diseases. The probe length of the panel is 90 nt, the total target area obtained is 2.3 M. On average, the mean coverage depth was more than 400X, and the coverage of target region was ~ 99.9% using BGISEQ-2000. Then the sequence data obtained were analyzed as previously reported [7]. Previous reported variants were determined using Human Gene Mutation Database (HGMD, professional version 2019.2). For variants that passed the initial filtration, Sanger sequencing was carried out for segregation analysis and variants validation.

All patients with pathogenic mutations in RPE65 underwent a full ophthalmic examination, including best Snellen corrected visual acuity testing (BCVA, they were converted to equivalent value of logarithm of minimal angle of resolution (logMAR) unit), slit lamp biomicroscopy, tonometry, fundus examination, wide-field fundus imaging (Optos PLC, Dunfermline, United Kingdom), swept-domain optical coherence tomography (SD-OCT, Spectralis HRA + OCT, Heidelberg, Engineering Inc., Heidelberg, Germany), visual field (Humphrey Visual Field Analyzer, Carl Zeiss Inc., CA, USA), and full-field electroretinography (ERG, according to the standards of the International Society for Clinical Electrophysiology of Vision; available at www.​iscev.​org).

Of the 1434 patients with IRD, 74.55% of patients (n = 1069) received a genetic diagnosis, and a total of 1516 variants involved 87 genes were identified. Only 41 alleles representing 26 distinct variants in 17 families (20 patients: 10 males, 10 females) were identified in the RPE65 gene (NM_000329.2, Table 1 and Additional file 5: Table S1), accounting for 2.83% of all the variants, and the gene was ranked as the seventh most common gene detected in this cohort of patients with IRD (Additional file 1: Figure S1A and S1B). However, the number of patients with RPE65 mutations only accounted for 1.87% (20/1069) of all patients with genetic diagnoses and was the 14th most common among all the patients (Additional file 1: Fig. 1c, d). Pedigrees and mutations of the 17 families are available in Additional file 2: Figure S2. Of the 26 distinct variants identified in this study, 10 (c.1039C>T p.Arg347Cys, c.1255C>T p.Pro419Ser, c.1444G>A p.Asp482Asn, c.334T>A p.Cys112Ser, c.354-2A>G, c.376del p.Val126*fs1, c.806_809delinsTGGAGCCATGAAG p.SerLeu269MetGluProTer, c.837del p.Phe279Leufs46, c.886del p.Arg296*fs1, c.94+2T>A) were novel (Additional file 3: Figure S3), including seven likely pathogenic variants and three missense variants of uncertain significance (p.Pro419Ser, p.Cys112Ser and p.Arg347Cys), which are localized in highly conserved residues (Additional file 4: Figure S4A). Bioinformatics analysis results of the novel variants are shown in Additional file 6: Table S2. Of the 16 variants reported previously, p.His68Tyr and c.998+1G>A were firstly reported to be associated with LCA.

To date, a total of 39 patients from 27 unrelated Chinese families have been reported with pathogenic mutations in RPE65 (Additional file 5: Table S1). Together with the 20 patients (two have been previously reported: F4-1 and F5-1) in this study [8], 57 patients of Chinese origin were diagnosed. Of the 115 variants identified in these patients, the majority of pathogenic defects (71.3%, n = 82) were missense variants, and 28.7% (n = 33) were nonsense, frameshift, or splice-site mutations that severely affected protein function (Fig. 1a). Variants were distributed from exon 2 to exon 14 (Fig. 1b).

Of the 20 patients in the 17 families, four were diagnosed with RP, one with FAP, and 15 with LCA (Table 2). The mean age at visit was 16.4 ± 12.59 years (range 3–49 years; median, 10 years). All accepted patients (LCA and RP) experienced poor vision at an early age. The mean BCVA with LCA patients was 0.82 ± 0.92 (range 3.00–0.40) LogMAR, and 90% (27/30) of eyes had a BCVA worse than 0.52 LogMAR. Of patients younger than 15 years, the mean BCVA was 0.68 ± 0.92 (range 1.30–0.40) LogMAR, while for patients older than 20 years, the mean BCVA was worse (p < 0.001, mean 1.30 ± 1.30 (range 3.00–1.00) LogMAR. Of the four patients with RP, the mean BCVA was 0.37 ± 1.05 (range 0.52–0.22) LogMAR, while the FAP patient maintained better vision (0/0.05 LogMAR). Bone spicule-like pigment deposits (BSLPs) were observed in six patients from five families (30%, 6/20, Fig. 2a), and no typical deposits were seen in 70% of patients (n = 14, Fig. 2b). Interestingly, we observed that subjects with BSLP were older than those without BSLP (p < 0.05, 26.83 ± 14.86 vs. 11.93 ± 8.65 years, respectively) (Fig. 2d). Eight patients (40%, mean age 9.0 ± 3.74 years) showed white-yellow dots (WYD) scattered in the periphery of the retina (n = 5) or perimacular area (n = 3, Fig. 2c); they were younger than those (n = 12, mean age 21.33 ± 14.09 years) without WYD (p < 0.01) and younger than patients with BSLP (p < 0.05, Fig. 2d). Cone and rod responses on ERG were nonrecordable in 14 of the 15 LCA patients and notably attenuated in the four RP patients.

All RPE65 variants identified in the Chinese population are shown in Fig. 3 and Additional file 5: Table S1. To date, 39 variants have been reported to be associated with LCA, 21 with RP, and 8 with FAP. As no clinical features of the FAP patient with one frameshift were provided by Guoxing Yang et al. [9], the FAP diagnosis requires further confirmation, and this patient was not included in the statistical analysis. Of all the variants associated with LCA (n = 73), 71.2% (n = 52) were missense variants, and 28.8% (n = 21) were nonsense (n = 5), frameshift (n = 9), or splice-site mutations (n = 7) that severely affected protein function, while in the FAP group (n = 16), 93.75% (n = 15) variants were missense, and only one splice-site mutation was identified (Fig. 1c). In the RP group, 58.3% (n = 14) of variants were missense variants, 20.8% (n = 5) were truncating stops, and 20.8% were frameshift (n = 2) or splicing defects (n = 3). Therefore, of all the variants (n = 97) associated with LCA and RP, which have a severe early-onset clinical presentation, only 68% (n = 66) were missense mutations, and the remaining 32% (n = 31) of variants were truncating mutations. Nevertheless, in the FAP group, which showed relatively mild symptoms, 93.8% (n = 15) of all the variants were missense. Of all the LCA and RP patients, 50% had missense + missense mutations, 35.4% had missense + nonsense/frameshift/splice-site mutations, and 14.6% did not have any missense mutations. However, of the eight FAP patients, 7 were missense + missense, and only one was a missense + splice-site mutation.

Only one variant, c.1399C>G (p. Pro467Ala), was associated with LCA, RP and FAP (Fig. 1d). This mutation was located in a highly evolutionarily conserved region (Additional file 4: Figure S4A) and altered the corresponding amino acid from proline to alanine. The 3D structural model of these amino changes is portrayed in Additional file 4: Figure S4B. Of the eight FAP patients, five had the p. Pro467Ala mutation, and the other three had the p.Arg515Trp mutation. It is likely that the two variants were hotspots of FAP. c.131G>A p.Arg44Gln was associated with both LCA and FAP; when the second mutated allele is a truncating mutation (c.858 + 1del), the patients were likely to present an LCA diagnosis, while if the second mutated allele is a missense mutation (p.Arg515Trp), the patients were likely to present an FAP diagnosis. p.Arg515Trp, p.Ala145Asp and p.Ala214Serfs*20 were associated with both RP and FAP, while p.Ala434Glu, p.Ser238Cys, c.94+2T>A, and p. Leu328Phe were only associated with FAP (Fig. 1d). Further analysis of the relationship between BCVA and fundus changes with specific mutations revealed that there was no correlation between them. Different individuals, even those with the same mutation, were found to show different changes. For example, patient F6-2 showed white-yellow dots scattered in the periphery of the retina, while patient F6-1, who had the same mutations, did not display these dots. However, we can't rule out the possibility that these dots would disappear with age.