Mutation screening of the USH2A gene reveals two novel pathogenic variants in Chinese patients causing simplex usher syndrome 2

Unrelated Chinese patients diagnosed with USH2 were included in this study. Two hundred unrelated normal individuals were recruited in this study as healthy controls. All patients underwent careful clinical examinations in Shanghai Tenth People’s Hospital and Clinical diagnosis of Usher syndrome were based on examination of optical coherence tomography (OCT) and electroretinogram (ERG), the typical RP fundus appearance, intact vestibular function, and sensorineural hearing impairment. The reference sequence from NCBI served as controls. This study was granted approval by the Declaration of Helsinki and approved by the institutional review board (IRB) of Tongji Eye Institute of Tongji University School of Medicine (Shanghai, China). Written informed consent was obtained from all participants.

The severity of hearing impairment can be judged according to the pure tone hearing threshold: mild hearing loss: 26–40 dB HL, moderate hearing loss: 41–80 dB HL, severe hearing loss: > 80 dB HL. Vestibular function tests include position tests and hot and cold water tests. (1) Position test: Dix-Hallpike technique was used to induce dizziness. Keeping the patient horizontal with his head pressed down by 30°. The head and eyes of the patient first turn to the right and then to the left, and repeated it several times to observe the severity and duration of nystagmus and dizziness. (2) Hot and cold water test: otoscopy should be performed before the test, and it can be performed without tympanic membrane perforation. The patient lies on his back and raises his head 30°to keep the lateral semicircular canal becomes upright. Each external ear canal was filled with cold or warm water for 40 s. Discomfort from warm water is usually lighter than cold water. In normal patients, cold water stimulates the nystagmus of the slow-phase stimulus side and the fast phase deviates from the stimulus side; warm water stimulus has the opposite response; in patients with vestibular cochlear nerve and vestibular nucleus disease, irrigation on the lesion side cannot induce nystagmus or nystagmus appears healthy slightly slower or shorter duration.

Peripheral blood samples from all the participants were collected in EDTA tubes. Standard protocols of RelaxGene Blood DNA System (TianGen, Beijing, China) were used to extract Genomic DNA according to the manufacturer’s instructions. DNA samples were stored at − 80 °C environment before used. Using the Primer3 software (http://​primer3.​sourceforge.​net/​) designed specific primers encompassing USH2A exons 2 to 72 (Table S1) (including the intron-exon boundary). The coding region was amplified by polymerase chain reaction (PCR) and using Sanger sequencing which was performed using ABI3730 Automated Sequencer (PE Biosystems, Foster City, CA, USA) study alleles. Nucleotide sequences assayed by Sanger sequencing were compared with the published DNA sequence of the USH2A gene (NCBI Reference Sequence: NM_206933.3(http://​genome.​ucsc.​edu/​cgi-bin/​hgc?​hgsid=​785073911_​5XSAy4TZHazZdzeK​szSK5wYZ4AfE&​g=​htcCdnaAli&​i=​NM_​206933&​c=​chr1&​l=​215796232&​r=​216596790&​o=​215796232&​aliTable=​refSeqAli)). The cDNA numbering + 1 position corresponds to A in the ATG translation initiation codon for USH2A.

We used several different computer algorithms: SIFT and PROVEAN (http://​provean.​jcvi.​org/​genome_​submit_​2.​php), PolyPhen-2 (http://​genetics.​bwh.​harvard.​edu/​pph2/​) and MutationTaster (http://​www.​mutationtaster.​org/​) to predict the pathogenic effect of missense variants. Human Splicing Finder (HSF) (http://​www.​umd.​be/​HSF3/​) was used to predict the pathogenicity of Splicing-site. Evolutionary conservation across species was evaluated through the alignment of orthologous USH2A protein sequences (including Mouse, Troglodyte, Bovine, Chicken, Mulatta and Zebrafish) with the human USH2A protein sequence, using Clustal Omega (https://​www.​ebi.​ac.​uk/​Tools/​msa/​clustalo/​).

According to the data of their families, all the recruited patients followed the pattern of autosomal recessive inheritance. Representative fundus photographs indicated typical RP features (Fig. 1a), and representative OCT imaging suggested significantly diminished retinal thickness in patients (Fig. 1b). Moreover, most patients have moderate-to-severe hearing impairment, and analysis of pure tone audiogram testing demonstrated bilateral decrease of air-conduction and bone-conduction auditory (Fig. 1c). The tympanograms were showed type As which means limited activity of the middle ear transmission system (Fig. 1d). The ERG wave amplitude of patients were undetectable (Fig. 1e). Those features indicate the diagnosis of USH2, and detailed clinical information of the patients is summarized in Table 1.

In this study, we found 19 changes among four USH2 patients by exon sequencing of the USH2A gene. According to the result of computer algorithms, five of them were predicted to be pathogenic variants (Table 2). All the other 14 variants predicted non-pathogenic are listed in the Table S2.

These five heterozygous mutations include a nonsense mutation (c.4217C > A (p.Ser1406X)), two splice site mutations (c.8559-2A > G and c.11389 + 3A > T), and two missense mutations (c.8232G > C (p.Trp2744Cys) and c.11780A > G (p.Asp3927Gly)). All of these can be predicted as harmful by the computer prediction tool.

In the five pathogenic variants, two of them (c.4217C > A (p.Ser1406X) and c.11780A > G (p.Asp3927Gly)) were novel (can not be found in the variants in publicly available human genome aggregation data sets) and three (c.8559-2A > G, c.8232G > C (p.Trp2744Cys) and c.11389 + 3A > T) were reported. All the variants predicted to be pathogenic were absent in 200 Chinese unrelated healthy controls.

Two novel variants (c.4217C > A (p.Ser1406X) (Fig. 2a) in exon 19 and c.11780A > G (p.Asp3927Gly) (Fig. 2e) in exon 61) were found in patient No.002. In the family of patient No.002, c.4217C > A (p.Ser1406X) was found in his mother (Figure S1 B) and c.11780A > G (p.Asp3927Gly) was found in his father (Figure S1 C). Parents of patient No.002 are normal. Reported intron sequence variant c.8559-2A > G (Fig. 2c) and missense variant c.8232G > C (p.Trp2744Cys) (Fig. 2b) in exon 42 were found in patient No.004. Interestingly, intron sequence variant c.8559-2A > G also found in patient No.001 and his unaffected father (Figure S1 A). Finally, an intron sequence variant c.11389 + 3A > T (rs753886165) (Fig. 2d) was found in patient No.003. However, in patient No.001 and No.003, we do not find the allelic variant in the USH2A gene. The pedigrees of the four patients with variants in USH2A are shown in the Fig. 3.

For the exon pathogenic variants identified in this study, we examined the location of them along the usherin. Finally, we identified functional domains the exon variants located within (Fig. 4a). Additionally, we aligned USH2A sequences among different species, including Human, Troglodyte, Mulatta, Bovine, Chicken, Mouse and Zebrafish for each of the two novel missense variants by Clustal Omega. The results of the conservative analysis of amino acid sequences were shown in Fig. 4.