A novel mutation in the RPE65 gene causing Leber congenital amaurosis and its transcriptional expression in vitro

Abstract

The retinal pigment epithelium-specific 65 kDa protein is an isomerase encoded by the RPE65 gene (MIM 180069) that is responsible for an essential enzymatic step required for the function of the visual cycle. Mutations in the RPE65 gene cause not only subtype II of Leber congenital amaurosis (LCA) but also early-onset severe retinal dystrophy (EOSRD). This study aims to investigate a Chinese case diagnosed as EOSRD and to characterize the polymorphisms of the RPE65 gene. A seven-year-old girl with clinical symptoms of EOSRD and her parents were recruited into this study. Ophthalmologic examinations, including best-corrected visual acuity, slit-lamp, Optical coherence tomography (OCT), and fundus examination with dilated pupils, were performed to determine the clinical characteristics of the whole family. We amplified and sequenced the entire coding region and adjacent intronic sequences of the coding regions of the RPE65 gene for the whole family to explore the possible mutation. Our results demonstrate that the patient exhibited the typical clinically features of EOSRD. Her bilateral decimal visual acuity was 0.3 and 0.4 in the left and right eyes, respectively. Spectral-domain optical coherence tomography (SD-OCT) was used to assess the retinal stratification for the whole family. All together, we identified four mutations within the RPE65 gene (c.1056G>A, c.1243+2T>A, c.1338+20A>C and c.1590C>A) in the patient. Among the four mutations, c.1056G>A and c.1338+20A>C had been reported previously and another two were found for the first time in this study. Her mother also carried the novel mutation (c.1243+2T>A). Either a single or a compound heterozygous or a homozygous one mutation is expected to cause EOSRD because mutations of RPE65 gene usually cause an autosomal recessive disease. Therefore, we speculate that the c.1590C>A mutation together with the c.1243+2T>A mutation may cause the patient's phenotype.

Figure 1. LCA related mutations in RPE65 gene.

The black arrow indicates the novel mutation discovered in this study.

Figure 2. Fundus photographs of both eyes in the family.

Color fundus photographs of both eyes (a: left eye; b: right eye) show mildly attenuated retinal vessels, some whitish dots, and numerous grayish deposits in the mid-peripheral retina of the patient. The inserted panels (g, h) show a magnification of the indicated areas. Whitish dots are marked by white arrowheads. Fundus photographs of her father (c, d) and her mother (e, f) show no whitish dots or grayish deposits in the mid-peripheral retina.

Figure 3. Foveal spectral-domain optical coherence tomography (SD-OCT) recordings of eyes in the whole family.

The green circle in fundus photographies (a-1, b-3, c-1, d-3, e-1, and f-3) shows the scanning position of the presented SD-OCT recordings of the family using 3D macular protocol. Foveal SD-OCT pictures of the patients (a-2 and b-2) show abnormalities of retinal stratification including extremely thinned ONL (outer nuclear layer), heavily thinned RPE (retinal pigment epithelial) layer, altered photoreceptor layers (ELM, IS, ISe, and OS). The patient’s grayscale SD-OCT recordings (a-2, and b-2) show that the boundaries of ELM, IS, ISe and OS layers are not well demarcated and the ISe layer could hardly be discerned. The white box in grayscale SD-recordings (a-2, b-2, c-2, d-2, e-2, and f-2) indicates the area of enlargement. The enlargement of grayscale SD-OCT recordings at the fovea of both eyes (a-3, and b-1) show that the ELM, IS, ISe and OS layers are not continuous. The abnormalities of retinal stratification are not present on the foveal SD-OCT recordings of her father (c and d) or mother (e and f). We measured the thickness of ISe, RPE, and RPE-choroid of the patient and her parents’s right eyes (Figure 3g, 3h, and 3i). Her father/mother’ results were 15 µm/15 µm (Figure 3h-2 and 3i-2), 16 µm/16 µm (Figure 3h-3 and 3i-3), and 40 µm/35 µm (Figure 3h-4 and 3i-4). Because the boundaries of IS, ISe, OS, and RPE are not clear, we measured the noncontiguous area of ISe layer, RPE-choroid and ISe-choroid,and the results were 239 µm (Figure 3g-2), 24 µm (Figure 3g-3), and ISe-choroid (Figure 3g-4), respectively.

Figure 4. Pedigree of the Chinese family and mutations of RPE65 gene.

In the family structure, male and female are represented by squares and circles, respectively. The filled square symbol represents the ESORD-affected daughter (c). One mutation (c.1338+20A>C) is detected in RPE65 gene of her father (a); two other mutations (c.1243+2T>A and c.1590C>A) are detected in her mother (b); four mutation are found in the daughter (a). The red color highlights the novel mutation.

Figure 5. Structure of RPE65 minigene.

The pCIneo minigenes of RPE65 gene were constructed to contain three exons (exon 11, 12, and 13) and flanking intronic sequences (intron 11 and 12) from wild or mutant type (c.1243+2T>A) of RPE65 gene. EcoRI and SalI represent restriction enzyme sites. Horizontal arrows indicate the positions and the directions of the primers. The red 483 bp indicates the amplified product.

Figure 6. Analysis of pre-mRNA splicing of pCIneo minigenes in the transfected 293T cell line.

A. Graphic representation of pre-mRNA splicing of wild type and mutant (C.1243+2T>A) minigenes of RPE65 gene. B. The isolated RNA of transfected cells was amplified by RT-PCR analysis. The different splicing products for wild type (264 bp) and mutant (358 bp) are shown on a 2% agarose gel. The mutant lane demonstrated that only a 358 bp band was obtained from the mutant-RPE65 minigene (a). The wild-type lane showed two different size DNA bands: one is a 264 bp band, and another is ∼200 bp band. The 264 bp DNA band is the expected size (a). The 200 bp band is the amplification caused by mispriming.