Genetic and phenotypic characteristics of four Chinese families with fundus albipunctatus

Fundus albipunctatus (FA) is a rare autosomal recessive form of stationary night blindness characterized by the presence of white or white-yellow dots in the perimacular area and the periphery of the retina, with or without macular involvement. In this study, we examined four Chinese families with FA. Patients were given complete ophthalmic examinations, and blood samples were collected for DNA extraction. Three genes, RDH5, RLBP1 and RPE65, were screened by direct sequencing. Mutations in RDH5 were identified in three families and mutations in RPE65 were identified in one family. This is the second reported case of FA caused by mutations in RPE65.

no rod responses according to the ERG standards of the International Society for Clinical Electrophysiology of Vision (ISCEV, 2008 Version). After prolonged dark adaption (3 h, 10 h), the rod responses almost recovered to normal levels (Fig. 7, Table 2). Physical examinations excluded systemic disorders in all patients.

Mutation analysis.
Mutations of RDH5 were identified in three families, including a homozygous c.928delCinsGAAG (Leu310 to GluVal) mutation in family 1, heterozygous c.500 G > A (p.Arg167His) and c.719insG (p.Ala240Glyfs17) mutations in family 2 and heterozygous c.928delCinsGAAG (Leu310 to GluVal) and c.500 G > A (p.Arg167His) mutations in family 3. In family 1, mother and father both carried the c.928delCins-GAAG mutation. In family 2, the father carried c.500 G > A mutation and the mother carried the c.719insG mutation respectively. Mutations of RPE65 were identified in family 4, including heterozygous c.639_640insA and L328F mutations (Fig. 8). The c.639_640insA was predicted to lead to premature stop codons (p.A214Sfs20) and cause a loss of function. The p.L328F change is predicted to damage the function of RPE65 when analysed using SIFT (0.998) and Polyphen-2 (0.01) websites.

Discussion
In this study, four patients were ascertained who exhibited typical FA. Four patients in four different families include three children and one middle-aged woman. They all suffered from night blindness from birth. We identified three disease-causing mutations in RDH5 in three unrelated families and two potential disease-causing        11 . The retinoid (visual) cycle is an enzyme pathway that occurs to regenerate the visual chromophore following light exposure. The RDH5 and RPE65 encode two key catalytic enzymes participating in the retinoid (visual)   19 . The accumulation of cis-retinoids may be responsible for the pathology of FA. Other enzymes in the RPE may compensate for the isomerohydrolase activity. A study confirmed that RDH10 can partially compensate for the loss of RDH5 function 20 . This may explain why the ffERG response can recover after dark adaption in patients with FA and why FA has relatively mild symptoms compared with RP.
The white dots in the fundus varied with age and genotype. Fundus autofluorescence (FAF) was performed in three patients. Two patients showed subnormal FAF. Autofluorescence in the retinal pigment epithelium represented the accumulation level of lipofuscin. A2E is a major component of lipofuscin. The mutations in RDH5 and RPE65 affected retinoid metabolism in the visual cycle and the production of A2E in the RPE. Spectral domain-OCT in the patients showed a lot of hyper-reflective lesions corresponding to white-yellow dots on fundus. Hyper-reflective lesions extend from the RPE to the external limiting membrane and distributed in the periphery except for the macular area. After prolonged dark adaption (> 3 h), the rod responses almost recovered to normal levels. The reason may be explained by an alternative compensation mechanism in the visual cycle. Further work is needed to understand the dark adaptation in fundus albipunctatus.
Gene therapy for RPE65-related Leber congenital amaurosis has been shown to be a successful and innovative technology in translational research [21][22][23] . It has become a research hotspot. Mutations of RPE65 can cause different phenotypes, but the mechanisms are still not clear. Our study provides valuable material and clues for further research about the biological functions of RPE65 and the pathogenesis of FA.

Methods
Patients and clinical data. The four families enrolled in this study were from Hebei province, China.
Clinical examination, peripheral blood collection and DNA extraction were performed at the Department of Ophthalmology at Hebei Ophthalmic Hospital. This study followed the tenets of the Declaration of Helsinki, and was approved by the Ethics Committee of Hebei Provincial Ophthalmic Hospital. The methods were carried out in accordance with the approved guidelines. Written informed consent was obtained from all participants. Families 1, 2 and 4 included one confirmed patient each, and Family 3 included two confirmed patients. The parents of family 1 and family 2 also participated in the study. Clinical data for these subjects was ascertained by detailed ocular examinations, including fundus photograph, multicolor, infrared reflectance (IR), fundus autofluorescence (FAF), fundus fluorescein angiography (FFA)/indocyanine green angiography (ICGA) imaging (HRA2 Heidelberg Engineering, Heidelberg, Germany), optical coherence tomography (OCT; Heidelberg Engineering, Heidelberg, Germany), visual field (VF) (Octopus 900, Switzerland), and full field electroretinography (ffERG; RetiPort ERG system, Roland Consult, Wiesbaden, Germany). Proband II-1 in family 4 is a three-year-child, so we only got the ERG after standard dark adaption under anesthesia. In addition, physical examinations were performed to exclude systemic diseases.

Mutation analysis.
Coding exons of RDH5, RLBP1, and RPE65 were amplified by a polymerase chain reaction (PCR) using primers previously described 24 . The PCR products were sequenced on an ABI3730 Automated Sequencer (PE Biosystems, Foster City, CA). SIFT(http://sift.jcvi.org/) 25 and Polyphen(http://genetics.bwh.harvard.edu/pph2/) 26 were used to predict the possible impact of an amino acid substitution on the structure and function of a human protein.