New compound heterozygous USH2A mutations in Usher syndrome

Usher syndrome (USH) is an autosomal recessive disorder with congenital sensorineural deafness and progressive retinitis pigmentosa (RP). The prevalence of USH in different populations ranges from 3.5 to 6.2 per 100 000, and it is the most common cause of deafness and blindness in adult (Yan & Liu 2010; Millan et al. 2011; Yoshimura et al. 2016). Clinically, Usher syndrome is classified into three types: type I (USHl), type II (USH2) and type III (USH3) according to the severity of hearing impairment, vestibular dysfunction and the onset age of retinitis pigmentosa (RP) (Keats & Corey 1999; Pennings et al. 2004). However, according to the category, some cases are not easily classified and can be classified as atypical USH syndrome (Otterstedde et al. 2001). Hearing, vestibular function, vision, visual field, electroretinogram (ERG) and other examinations should be performed when the disease is suspected. Vestibular function test is the most reliable criteria for distinguishing between type I and type II Usher syndrome, and retinitis pigmentosa is cross-typed. To date, 16 loci have been associated with USH, in which 14 genes have been identified, including USHlC, CDH23 (USH1D), PCDH15 (USH1F), USHlG, CIB2 (USH1J), USH2A, GPR98 (USH2C), DFNB31 (USH2D); CLRNl (USH3A), PDZD7, CEP250, HARS, ABHD12 (Eisenberger et al. 2012; Puffenberger et al. 2012; Vache et al. 2012). We present a 22-year-old girl presented to our clinic due to progressive bilateral night blindness for 2 years. She had been wearing hearing aids since 18 year due to a hearing loss but without vertigo. The patient had no other general diseases. No other family members were affected. The uncorrected visual acuity (UCVA) was 0.5 (10/20) in the right eye and 0.9 (nearly 20/20) in the left eye. The intraocular pressure (IOP) was normal in both eyes. With slit lamp examination, the sclera, conjunctiva, cornea and lens were normal in both eyes. The fundus examination showed that retina looks rough and coarse in both eyes. Optical coherence tomography (OCT) showed normal macular thickness (196 μm in the right eye and 205 μm in the left eye) without obvious proliferative pigmentation (Fig. 1). Visual field tests showed that the peripheral parts were mostly affected (Fig. 1). Fundus autofluorescence (FAF) imaging showed a parafoveal ring-shaped area of increased autofluorescence in both eyes (Fig. 1). The electroretinogram (ERG) (RETI-Port/Scan 21, Roland Consult) showed markedly decreased cone and rod responses in both eyes (Fig. 2). The amplitudes of the a- and b-waves were significantly reduced in the photopic (which reflects cone response), scotopic (which reflects rod response) and the maximal scotopic ERG (which reflects cone and rod response) compared to normal values (Fig. 2). After obtaining the approval of the Ethics Committee of Shenzhen Eye Hospital and informed consent from the patient and family members, we extracted genomic DNA from them and a screening of 381 pathogenic genes associated with retinal diseases in the patient was performed using targeted sequence capture array technique. Polymerase chain reaction (PCR) and Sanger sequencing were used to confirm the screening results. We found that the proband (II2, Fig. 1) carries three point mutations in USH2A gene, including c.2187C>A (p.C729X), c.997T>C (p.S333P) and c.538T>C (p.S180P) (Fig. 2). Her father (I1, Fig. 1) carries the c.2187C>A (p.C729X) and c.997T>C (p.S333P) mutations in USH2A gene (Fig. 2), and her mother (I2, Fig. 1) carries the c.538T>C (p.S180P) mutation in USH2A gene (Fig. 2). One of her sisters was not found to carry any mutations in USH2A gene (Fig. 2). The high heterogeneity of clinical characteristics and genetic inheritance of USH makes it difficult to diagnose at molecular level by first-generation DNA sequencing. In this study, we screened the potential pathogenic gene mutations in the patient with USH by Targeted Genes Capture Sequencing. We found three mutations in USH2A gene, including c.2187C>A (p.C729X), c.997T>C (p.S333P) and c.538T>C (p.S180P). The genetic pattern of the family is autosomal recessive inheritance according to genetic analysis. Both the father and the mother have normal phenotypes. That means either c.2187C>A (p.C729X) and c.997T>C (p.S333P) (father) or c.538T>C (p.S180P) (mother) does not cause disease phenotypes. It has been reported that the missense mutation c. 538T>C (p. S180P) is associated with USH2 (Nakanishi et al. 2009). But it is recessive, not dominant. The single point mutation of c. 538T>C (p. S180P) may not be sufficient to cause USH2. Currently, no other evidence shows that the missense mutation c.538T>C (p.S180P) is the cause of the USH. In addition, c.2187C>A (p. C729X) is a potential newly gene mutation due to a premature stop codon formed. The patient carries two point mutations from her father and one mutation from her mother, suggesting that it is a compound heterozygous mutation. Future studies will be performed to elucidate the pathophysiological mechanisms of retinal degeneration caused by the USH2A gene mutations. In this study, we found three new compound heterozygous mutations (c.2187C>A (p.C729X), c.997T>C (p.S333P) and c.538T>C (p.S180P)) in Usher syndrome which were also a new pathogenic cause of USH2A. Our results indicate a distinctive mutation spectrum of USH2A in this population which expand our knowledge of heterogenic phenotypes and genotypes through genetic diagnoses of the patient.