Identification of novel USH2A mutations in patients with autosomal recessive retinitis pigmentosa via targeted next‑generation sequencing

Abstract

Retinitis pigmentosa (RP) is a group of inheritable blindness retinal diseases characterized by the death of photoreceptor cells and a gradual loss of peripheral vision. Mutations in Usher syndrome type 2 (USH2A) have been reported in RP with or without hearing loss. The present study aimed to identify causative mutations in a cohort of families with RP from China. A cohort of 62 non‑syndromic families with RP and 30 sporadic cases were enrolled in this study. All affected members underwent a complete ophthalmic examination, including fundus photography, visual‑field test and optical coherence tomography examination. Next‑generation sequencing‑targeted sequencing of 163 genes involved in inheritable retinal disorders was performed on the probands. Stringent bioinformatics data analysis was applied to identify potential candidate variants. In total, 6 novel mutations and 2 known mutations of USH2A were identified in 4 families with RP. A stop‑gain mutation (c.C1731A) and a missense mutation (c.G8254A) were identified in RP family RP‑2148. In another RP family, RP‑2150, a known mutation (c.G802A) and a novel frameshift insertion mutation (c.12086dupA) were discovered. A novel stop‑gain mutation (c.G11754A) and a missense mutation (c.G13465A) were identified in family rpz05. A novel missense mutation (c.C9328G) and a known missense mutation (c.G8232C) were also identified. These mutations were subsequently confirmed by Sanger sequencing. All 6 novel mutations affected highly conserved amino acid residues, and were absent in 1,000 ethnically matched controls. Taken together, the present study has reported on 6 novel USH2A mutations in 4 families with RP, and has expanded the mutation spectrum of USH2A in autosomal recessive RP in the Chinese population, thus providing important information for the molecular diagnosis and screening of RP.

Keywords: targeted sequencing; ene mutation; usher syndrome type 2; USH2A; retinitis pigmentosa.

Figure 1.

Fundus photographs of patients and an unaffected individual in RP-2148. (A) Fundus photographs show typical changes (waxy-pale disc, arteriolar attenuation and bone-spicule pigment) of fundus in the OS and OD. (B) Fundus photographs show waxy-pale disc and bone-spicule pigment in patient II:6. (C) Fundus photographs of the unaffected control III:1 show normal retina. OS, oculus sinister/left eye; OD, oculus dexter/right eye; Ctrl, control.

Figure 2.

Visual field test and OCT pictures of the proband in RP-2150 and a healthy control. Loss of peripheral vision in both eyes was revealed by a visual field test in the (A) proband, as compared with that of the (B) control individual. (C) OCT examination of the proband revealed disorganized photoreceptor layers and thinner retina due to atrophy. (D) OCT examination result of a healthy control. OCT, optical coherence tomography; OS, oculus sinister/left eye; OD, oculus dexter/right eye; Ctrl, control.

Figure 3.

Pedigree of the families and USH2A mutations identified by Sanger sequencing analysis. (A) Diagram shows the segregation of compound heterozygous mutations c.G8254A, p.G2752R (MUT1) and c.C1731A, p.C577X (MUT2). Genotypes are presented as follows: MUT1/MUT2 represents the proband II:1 and affected individual II:6 carrying both mutations as compound heterozygous; MUT1/+ and MUT2/+ indicate heterozygous carriers. The proband's mother carried the heterozygous MUT1 mutation, and his father carried heterozygous MUT2 mutations. (B) Diagram showing a missense mutation MUT3:c.G802A [p.G268R] and a frameshift insertion mutation MUT4:c.12086dupA [p.H4029fs] in the proband of RP-2150 II:1. His unaffected father I:2 carried heterozygous MUT4. Black circles represent affected females. Black squares represent affected males. White circles represent unaffected females. White squares represent unaffected males. Arrows indicate the proband. USH2A, Usher syndrome type 2.

Figure 4.

Protein sequence alignment of USH2A protein across different species. The amino acid residues affected by the identified mutations are conserved in 9 species. The orthologs are Homo sapiens, Pan troglodytes, Macaca mulata, Canis lupus, Bos taurus, Mus musculus, Gallus gallus, Danio rerio and Xenopus tropicalis. Orthologous alignments of the detected mutations in the present study suggest their evolutionarily important functions. USH2A, Usher syndrome type 2.

Figure 5.

Diagram showing the location of the identified USH2A mutations in the current study. USH2A encodes a large transmembrane protein, and all 8 mutations are located in the exocytoplamic region. USH2A, Usher syndrome type 2.