Novel compound heterozygous nonsense variants, p.L150* and p.Y3565*, of the USH2A gene in a Chinese pedigree are associated with Usher syndrome type IIA

Abstract

Usher syndrome refers to a group of genetically and clinically heterogeneous autosomal recessive diseases with retinitis pigmentosa (RP) and hearing deficiencies. The association between Usher syndrome‑causative genes and resultant Usher syndrome phenotypes in patients are highly variable. In the present study, a Chinese family with Usher syndrome was recruited, and targeted next‑generation sequencing, Sanger sequencing and segregation analysis were performed. The expression profiles and functional effects of the pathogenic variants of USH2A identified were analyzed. Novel nonsense compound heterozygous variants, c.T449G (p.L150*) and c.T10695A (p.Y3565*), were identified in the USH2A gene, which showed co‑segregation with the disease phenotype causing Usher syndrome type IIA in the recruited Chinese pedigree. The p.L150* variant was predicted to produce a truncated protein which lacked almost all the functional domains of USH2A, whereas the p.Y3565* variant is located in one of the fibronectin type 3 domains, resulting in the loss of several fibronectin type 3 domains at the C‑terminus of USH2A by producing the truncated protein. It was shown that Ush2a mRNA expression levels were higher in the retina compared with those in the eye tissues (lens, sclera and cornea), uterus, ovary, breast, testis, spleen, kidney, liver, intestine, brain, skeletal muscle and blood. Additionally, the protein structure was shown to be highly conserved by comparing Homo sapiens USH2A to eight other species. To the best of our knowledge, the present study is the first to identify two novel pathogenic variants, c.T449G (p.L150*) and c.T10695A (p.Y3565*), in the USH2A gene in a patient with Usher syndrome type IIA, thereby expanding the known spectrums of USH2A causative mutations. The present discovery may assist in understanding the molecular pathogenesis underlying the development of RP and Usher syndrome type IIA, and in the development of diagnostic, therapeutic and genetic counseling strategies in patients with Usher syndrome type IIA disease.

Figure 1.

An M147 pedigree with Usher syndrome type IIA. Normal individuals are shown as a clear circle (female) or squares (males). The filled circle indicates the proband (II: 1, arrow) with the compound heterozygous mutation of the USH2A gene: NM_206933.3: c.T449G, c.T10695A. USH2A, usherin.

Figure 2.

Representative retinal phenotypes and ear audiograms of proband II: 1. (A-D) Representative fundus photographs and fundus fluorescent photographs in patient II: 1 of both eyes. (E and F) Physiological audiograms of both ears of proband II:1.

Figure 3.

Pyrogram profiles for variant verification using Sanger sequencing. (A-E) Sequencing results pf I: 1 (heterozygous mutant type), I: 2 (WT), II: 1 (heterozygous mutant type), III: 1 (WT), and II: 2 (WT) of variant c.T449G. (F-J) Sequencing results of I: 1 (WT), I: 2 (heterozygous mutant type), II: 1 (heterozygous mutant type), III: 1 (heterozygous), and II: 2 (WT) of variant c.T10695A. The arrows indicate the mutation position of NM_206933.3: c.T449G or c.T10695A in the USH2A gene. USH2A, usherin; WT, wild-type.

Figure 4.

USH2A comparison, structure and mutant locations between different species. (A) Conservation analysis of USH2A in the indicated species. (B) USH2A domains and mutant positions. Variants p.L150* and p.Y3565* of USH2A are indicated panel B. USH2A, usherin; aa, amino acids. EGF, epidermal growth factor; FN, fibronectin.

Figure 5.

mRNA expression of USH2A in human tissues and Ush2a in mouse tissues. (A) USH2A mRNA expression in human tissues. The source of the data was derived from the link: https://www.ncbi.nlm.nih.gov/gene/7399/?report=expression. (B) Ush2a mRNA levels in the indicated mouse tissues. (C) Ush2a mRNA levels at the indicated developmental time periods in retinal tissue in mice. Whole embryo eyeballs were obtained at 12.5 days (12d) and 20.5 days (20d). d, days; w, weeks; m, months; nc, no DNA template; muscle, skeletal muscle; RPKM, reads per kilobase of transcript per million mapped reads.