Identification of a novel RHO heterozygous nonsense mutation in a Chinese family with autosomal dominant retinitis pigmentosa

This study was approved by the medical ethics committee of Tianjin Medical University Eye Hospital. A four-generation Chinese family suffered from RP was recruited and written informed consent was obtained. A 5 ml venous blood sample of each enrolled participant was collected from antecubital vein puncture, drawn into an ethylenediamine tetraacetic acid (EDTA) sample tube and stored in − 20 °C refrigerator for further analysis.

The target region capture and next-generation sequencing (NGS) procedure was described previously [9]. Briefly, genomic DNA was firstly extracted according to the manufacturer’s standard procedure (MagPure Buffy Coat DNA Midi KF Kit, Magen, China). After that, the genomic DNA of the proband was sequenced with PE100 + 100 on MGISEQ-2000. Finally, the targeted sequences were captured using the NimbleGen SeqCap EZ Choice XL Library 24 Reaction 150217_HG19_CllnE_EZ_HX1 chip (Roche, Madison, USA) containing 156 genes related to retinal diseases (Table 1).

To detect the potential variants in the family, we performed bioinformatics processing and data analysis after receiving the primary sequencing data. We used previously published filtering criteria to generate “clean reads” for further analysis [10]. The “clean reads” (with a length of 90 bp) derived from targeted sequencing and filtering were then aligned to the human genome reference (hg19) using the BWA (Burrows Wheeler Aligner) Multi-Vision software package [11]. After alignment, the output files were used to perform sequencing coverage and depth analysis of the target region, single-nucleotide variants (SNVs) and INDEL calling. We used GATK software [12] to detect SNVs and indels. All SNVs and indels were filtered and estimated via multiple databases, including NCBI dbSNP, HapMap, 1000 human genome dataset and database of 100 Chinese healthy adults. The Human Gene Mutation Database (HGMD, http://​www.​hgmd.​cf.​ac.​uk/​ac/​index.​php) was used to screen mutations reported in published studies.

All mutations and potential pathogenic variants were validated using conventional Sanger sequencing methods. Segregation analysis was performed in all available family members.

Multiple sequence alignment from different species was performed by CLUSTALW (https://​www.​genome.​jp/​tools-bin/​clustalw). In addition, the structures of homomeric wild-type and the mutant RHO were modeled by Swiss-Model Server (https://​swissmodel.​expasy.​org) and shown using a PyMOL Molecular Graphic system, using the solved structure of rhodopsin coded by RHO gene as template (Protein Data Bank No. 5W0P).

Two patients (III:2, III:3) and three normal individuals (III:4, III:5, IV:2) from this family were enrolled in this study (Fig. 1). The proband was a sixty-year-old man (III:2) and was diagnosed with RP when he was 17 years old. The patient suffered from night blindness when he was young and accepted cataract surgery 20 years ago. On presentation, the visual acuity of both eyes was light perception. The axial length was 25.99 mm and 25.56 mm, the central corneal thickness was 586 μm and 595 μm, the flat K was 42.56D and 43.27 D, the steep K was 43.80 D and 43.79 D, the intraocular pressure was 20.1 mmHg and 18.6 mmHg for the right eye and left eye, respectively. Under slit-lamp microscopy, the cornea was clear, the anterior chamber was deep and quiet, the pupil was sluggish and the intraocular lens was central and clear. Fundoscopic examination revealed attenuated arterioles, waxy pallor of the optic disc, atrophy of the choroid and diffuse bone-spicule pigmentation in both eyes (Fig. 2). The sister of the proband (III:3) also suffered from night blindness and was diagnosed with RP when she was 15 years old and she had similar fundus changes with the proband. There was no consanguineous marriage in this family.

Targeted region sequencing containing 156 retinal diseases-related genes of the proband (III:2) revealed a transversion in exon 5 (c.1015A > T) of RHO, which was not reported previously. The variant causes the replacement of a wild-type Lysine with stop codon at codon 339 (p.Lys339Ter; Fig. 3A). The variant can be detected in all affected patients enrolled in this study (III:2, III:3) and was not found in the unaffected family member (III:4, III:5, IV:2) by further Sanger sequencing. The variant co-segregated with the disease in family members and was not found in NCBI dbSNP, HapMap, 1000 human genome dataset and database of 100 Chinese healthy adults, suggesting the variant may be the pathogenic mutation in this family.

Multiple-sequence alignments of RHO in various species showed codon 339 was located within a highly conserved region (Fig. 3B). Structure model of rhodopsin-arrestin complex showed normally, arrestin ‘reads’ phosphorylation codes of rhodopsin through code-sensing pockets in the C-terminus, while p.K339X mutation of RHO is predicted to produce a truncated protein and interfere with arrestin recruitment (Fig. 4).