Targeted Next-Generation Sequencing Reveals a Novel Frameshift Mutation in the MERTK Gene in a Chinese Family with Retinitis Pigmentosa

Mu Yang,Shujin Li,1–3,* Wenjing Liu,3 Yeming Yang,3 Lin Zhang,3 Shanshan Zhang,3 Zhilin Jiang,3 Zhenglin Yang,1–3,5 and Xianjun Zhu1–5

Abstract

Background: Retinitis pigmentosa (RP) is a group of inherited retinal diseases that result in severe progressive visual impairment.
Aims: The purpose of this article was to apply targeted next-generation sequencing (NGS) to identify the causative mutation in a Chinese RP family.
Methods: Blood samples were collected from a Chinese proband diagnosed with RP and her family members. A total of 163 genes that have been previously found to be involved in inherited retinal diseases were selected for NGS. Rigorous NGS data analysis; Sanger sequencing validation; and segregation analysis were applied to evaluate a novel frameshift mutation.
Results: Sequence analysis revealed that the proband and her affected sister both carried a novel homozygous frameshift mutation in MERTK (p.I103Nfs*4). Other family members carrying a heterozygous mutation were unaffected. This mutation was found to cosegregate with the disease phenotype in this family. This mutation was not found in 1,000 control individuals.
Conclusions: The targeted NGS strategy employed provides an efficient tool for RP pathogenic gene detection. This study identified a new autosomal recessive mutation in the RP-related gene MERTK, which expands the spectrum of RP disease-causing mutations.

Keywords: MERTK, next-generation sequencing, genetics, autosomal recessive retinitis pigmentosa

Introduction

Retinitis pigmentosa (RP; MIM No. 268000) is the inheritance mannersthat account fora smallproportionoftraits, most frequent form of inherited retinopathies with high including mitochondrial inherited and digenic inherited traits, clinical and genetic heterogeneity (Neveling et al., 2012). Between 1 in 2,500 to 1 in 4,000 people worldwide were affected with RP due to abnormalities of photoreceptors (Haim, 2002; Hamel, 2006). Patients share common clinical symptoms, such as progressive impairment of rod photoreceptor cells in the early stages that leads to night blindness and defective dark adaptation, followed by loss of peripheral vision and eventually to blindness due to cone photoreceptor death. However, the time of disease onset and disease progression varies remarkably among patients (Fu et al., 2013; Wang et al., 2014; Fernandez-San Jose et al., 2015).
Genetic heterogeneity of RP includes a monogenic pattern of inheritance, such as autosomal-recessive (50–60%), autosomal- have also been reported (Kajiwara et al., 1994; Mansergh et al., 1999; Katsanis et al., 2001; Hartong et al., 2006). To date, >80 genes with their >3,100 mutations have been identified as associating with RP; however, these known mutations are responsible for only *60% of RP cases, leaving 40% of patients with no molecular diagnosis (Wang et al., 2016; Al-Khersan et al., 2017; Anasagasti et al., 2012).
One of the genes identified with RP, the mer tyrosine kinase (MERTK—OMIM No. 604705), is a member of the Tyro3/Axl/Mer receptor kinase family (Duncan et al., 2003; Prasad et al., 2006; Al-Khersan et al., 2017). MERTK has also been reported to participate in many cellular processes, such as cell migration, adhesion, and proliferation, as well as the regulation of platelet function and inflammation (Chen et al., 2004; Zagorska et al., 2014). As an RP pathogenic gene, MERTK was first implicated in retinal degeneration in Royal College of Surgeons (RCS) rats (D’Cruz et al., 2000). In the same year, Gal et al. (2000) mapped human MERTK and identified the first three mutations in RP patients. Several new RP mutations in MERTK have been reported in recent decades, including five large exonicdeletions,a 91kbdeletionmutation fromexons1to7 (Ostergaard etal., 2011),a*9kbdeletionof exon 8 (Mackay et al., 2010), a 1.73kb deletion of exon 15 (Siemiatkowska et al., 2011), a start codon mutation that leads to the loss of entire protein (Jinda et al., 2016), and a recently described 25kb deletion of exons 6–8 (Evans et al., 2017). However, compared with other genes implicated in RP, the discovery of new MERTK mutations has been slow. In humans, mutations in the MERTK gene account for only *1% of autosomal recessive RP cases (Strick and Vollrath, 2010).
Next-generation sequencing (NGS) has been demonstrated to be an efficient strategy for the identification of diseasecausing genes (Buermans and den Dunnen, 2014). Recently, Wang et al. (2013) developed the targeted NGS method that allows the systematic screening of mutations in retinal dystrophies. The screen panel enriches the exonic DNA of 163 retinal disease genes, among which 48 are RP genes (Wang et al., 2014). In this study, we performed a comprehensive molecular screening of a Chinese proband, her family members, and 1,000 control samples using the targeted NGS strategy. A novel frameshift homozygous mutation, predicted to result in a stop-gain in exon 2 of the RP-related gene MERTK (p.I103Nfs*4), was identified.

Materials and Methods

Subjects and clinical assessments

All study protocols were approved by the institutional ethics committee of the Sichuan Provincial Peoples’ Hospital. The proband and the family members were recruited from the Sichuan Provincial People’s Hospital. In addition, 1,000 healthy unrelated Chinese individuals without familial history of retinal dystrophy were screened as controls. Written informed consent was obtained from all participants or from their statutory guardians. All participants were diagnosed by a clinical ophthalmologist and geneticist based mainly on fundus photographic and angiographic changes.
Peripheral blood samples were collected using 5mL tubes pretreated with ethylene diamine tetra acetic acid. Genomic DNA was extracted using a Qiagen Blood DNA Kit (Qiagen, Shanghai, China) according to the manufacturer’s protocols. The quantity and quality of the extracted DNA were verified using NanoDrop. DNA was stored at -20C for use in subsequent analysis.

Sequence capture and targeted NGS
The capture panel and targeted NGS were applied as previously described (Fu et al., 2013). The panel covers 2,560 exons and splice junctions of 163 known retinopathy genes. Among them, 48 RP genes were targeted, including 30 autosomalrecessive RP (arRP) genes that had been reported (Wang et al., 2014). In brief, *1mg of genomic DNA samples was sheared into approximately 300–500bp fragments. The sheared fragments were end-repaired by polynucleotide kinase and Klenow fragment. The Klenow exonuclease was applied to add a single adenine base to the 3¢ ends. Then Illumina Y-shape index adapters were applied to generate DNA paired-end libraries. After this, each captured library was loaded on Illumina HiSeq 2000 for quantification and sequencing (Fu et al., 2013; Wang et al., 2013).

Bioinformatics analysis

By using Burrows–Wheeler Aligner, the sequencing reads were aligned to the reference human genome (hg19 UCSC assembly, http://genome.ucsc.edu) (Li and Durbin, 2009). The Genome Analysis Toolkit was applied to the base quality recalibration and local realignment (Challis et al., 2012). SAMtools was applied to identify single nucleotide variants and insertions/deletions. Filtration and annotation of the variants were conducted according to a previously described protocol on the basis of the autosomal recessive inheritance pattern using the following four databases as described previously (Di et al., 2016):

(1) NCBI CCDS (www.ncbi.nlm.nih.gov/CCDS/ccdsBrowse.cgi).
(2) RefSeq (www.ncbi.nlm.nih.gov/RefSeq).
(3) Ensembl (www.ensembl.org).
(4) Encode (http://genome.ucsc.edu/ENCODE).

Variants within intergenic, intronic, and synonymous mutations, as well as mutations in any of the following five databases, were excluded:

(1) dbSNP138 (www.ncbi.nlm.nih.gov/projects/SNP).
(2) 1000 Genome Project (ftp://ftp.1000genomes.ebi.ac.uk/vol1/ftp).
(3) YH database (http://yh.genomics.org.cn).
(4) HapMap Project (ftp://ftp.ncbi.nlm.nih.gov/hapmap).
(5) An ‘‘in house’’ database generated from 1,600 samples sequenced by whole exome sequencing (WES).
According to the bioinformatics pipeline, low-quality variants and variants predicted to be benign by online tools (SIFT/PROVEAN and Variant Effect Predictor) were excluded (Zhang et al., 2016).

Variants validation

To verify whether the remaining variants cosegregated with the phenotype of RP in this family, Sanger sequencing was conducted within all attainable family members and 1,000 healthy controls. Polymerase chain reaction (PCR) primers were designed using the Primer3 Online tool (SourceForge.Net) and synthesized by Sangon Biotech (Shanghai, China) to amplify genomic DNA fragments. The primer sequences were as follows: F, 5¢-CACTGAGGCAAGGG AAGAAG-3¢ and R, 5¢-TTGCTGTAACTTCATCATCTG GA-3¢. The PCR products were purified using FastAP thermosensitive alkaline phosphatase (Thermo Scientific Fermentas) and sequenced by BigDye version 3.1 and an ABI 3730 automated sequencer (Applied Biosystems, Foster City, CA) (Zhang et al., 2016).

Results

Clinical findings

This 30-year-old female proband of Chinese origin was clinically diagnosed with RP in the Sichuan Provincial People’s Hospital. The patient self-reported difficulty seeing at night since the age of 12 and the onset of progressive periphery visual loss since the age of 23. Fundus examination revealed mild RP phenotype with bits of bone-shaped pigment accumulation in the macula, constricted retinal vessels, and pale optic disk (Fig. 1). However, full-field flash electroretinography response was nonrecordable in both eyes. There was no RP history in this family. However, the proband’s younger sister, a 27-year-old female, also carried the diagnosis of RP with similar symptoms. An unaffected brother did not have any relative phenotype on examination.

Gene findings

Based on pedigree analysis, this RP family was determined to be suitable for the autosomal recessive mode of inheritance. The targeted NGS was developed on genomic DNA extracted from this family. After several filtering steps, a novel deletion mutation, c.303_304delAC, located in the second exon of the MERTK gene, was identified. This frameshift mutation results in a predicted premature termination (p.I103Nfs*4), which may trigger nonsense-mediated messenger RNA (mRNA) degradation. This variant was not observed in 277,202 alleles from gnomAD database. In addition, it was also absent in our in-house database of 1,600 geographically matched individuals analyzed by exome sequencing and was not observed in 1,000 ethnic-matched control individuals. Results showed that the proband (II-2) and her sister (II-3) both carried this homozygous mutation, whereas her parents (I-1 and I-2) and brother (II-1) carrying heterozygous mutation were unaffected (Fig. 2). Taken together, a novel frameshift mutation in the RP gene MERTK was identified, and this mutation cosegregated with the disease phenotype in the family.

Discussion

The targeted NGS allows for a comprehensive molecular diagnosis of genetic diseases with heterogeneity-like retinopathy. It was claimed to be cheaper and more rapid than wholeexome sequencing (Weisschuh et al., 2016). Using this strategy, 163 known retinal disease genes were screened with a parallel sequencing approach, followed by systematic analysis and annotation of all detected genetic mutations (Neveling et al., 2012). In this study, the targeted NGS strategy was applied toanalyze the genetic causesof RPin a Chinese family with an apparent autosomal recessive mode of inheritance. A novel RP mutation located in exon 2 of the MERTK gene was identified (c.303_304delAC). This frameshift mutation was predicted to undergo degradation of the entire mRNA.
Clinical observations of the phenotypes in our patients, who experienced decreased night vision early in life and subsequently suffered a progressive peripheral visual loss, are consistent with previous studies (Al-Khersan et al., 2017). The fundus imaging of both eyes of the proband (Fig. 1) also demonstrated a mild RP phenotype. With regard to the pedigree and Sanger sequencing analysis (Fig. 2), the affected patients were identified to be homozygous carriers of this mutation. The other family members who carried a heterozygous mutation were unaffected, indicating that this MERTK mutation cosegregated with the disease phenotype in the family.
Since the first 3 human MERTK variants were reported to be related to RP in 2000, only 27 different mutations have been identified. These mutations were reported to account for <1% of the autosomal recessive retinal dystrophy diseases, suggesting that more MERTK mutations remain to be discovered by better molecular diagnosis (Gal et al., 2000; Ostergaard et al., 2011; Jinda et al., 2016). Our findings expand the spectrum of RP mutations.
During recent decades, recombinant adeno-associated virus (AAV) has gained prominence in gene therapy for inherited retinal dystrophy. La Vail et al. (2016) and Conlon et al. (2013) reported that subretinal injection of a retinal pigment epithelium (RPE)-specific AAV2-VMD2-hMERTK vector can protect photoreceptors from degeneration in both RCS rats and Mertk knockout mice. The safety of this vector was also proved in RCS rats (Conlon et al., 2013; LaVail et al., 2016). Very recently, the same research group applied this AAV-mediated gene therapy approach to a cohort of MERTK-related RP patients and reported the safe and partial rescue ability of this hMERTK therapy (Ghazi et al., 2016).
Different retinal diseases may share many overlapping clinical phenotypes, leading to a challenge for accurate diagnosis. A comprehensive molecular diagnosis can facilitate more accurate disease management. In our study, we successfully identified a novel pathogenic mutation in the MERTK gene for arRP, which expanded the spectrum of MERTK gene mutations for RP. Furthermore, since the AAV2-based gene therapy for MERTK-related RP has proved its efficiency and safety, the combination of molecular diagnosis, clinic diagnosis, and gene therapy will ultimately help to halt the progression of this disabling disease.

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