Cone-rod dystrophy (CRD) is an inherited retinal disorder (IRD) with prevalence 1 in 40,000, characterized by visual loss, color vision defects, decreased sensitivity in the central visual field, and a variable degree of nystagmus and photophobia. It represents an important cause of visual impairment in children and adults. CRD is characterized by progressive loss of cone photoreceptor function followed by progressive loss of rod photoreceptor function and is often accompanied by retinal degeneration [1]. Over time, affected individuals develop night blindness and loss of peripheral field. At end stage, CRD may not differ from the rod-cone dystrophies, also called retinitis pigmentosa (RP, with prevalence of 1 in 4000) [2]. Typically, fundus imaging of CRD patients presents pigmentary deposits resembling bone spicules (often in macular or paramacular area), retinal vessels attenuation, pale optic disc and various degrees of retinal atrophy [2]. Progressive degenerative changes of the macula occur rarely. X-linked cone-rod dystrophy (XLCRD) is a rare progressive retinal degeneration and usually manifests with early visual impairment affecting predominantly male patients (hemizygotes), who are legally blind before the end of their third decade, while carrier heterozygous women present various degrees of visual dysfunction, ranging from asymptomatic to severe phenotype [3]. The retinas of some affected males had a bronze-green tapetal-like sheen. The degree of rod-photoreceptor involvement can be variable, with degeneration increasing as the disease progresses. Although penetrance appears to be nearly 100%, there is variable expressivity with respect to age at onset, severity of symptoms, and findings [4]. Moderate or high myopia is often secondary to XLCRD phenotype both on patients and carrier [5].

RPGR gene is a major cause of X-linked retinitis pigmentosa (XLRP) which is the most severe type of RP (OMIM #300029), and is also responsible for XLCRD (OMIM #304020) and atrophic macular degeneration (OMIM #300834) [6]. The RPGR gene encodes the retinitis pigmentosa GTPase regulator (RPGR) protein and is able to express multiple retinal isoforms through alternative splicing. The two major isoforms include RPGR ^1-19, which spans exons 1 - 19 and encodes an 815-aa polypeptide, and RPGR ^ORF15, which spans exons 1 - 15 plus a part of intron 15 and encodes a 1152-aa polypeptide [7] [8]. It shares exons 1 - 14 with RPGR ^1-19 plus the exon ORF15, encoding 567 amino acids with a repetitive glycine and glutamic acid-rich domain and a conserved basic C-terminal domain. In addition to these two major transcripts of the gene, RPGR encodes complex alternative spliced transcripts and many novel tissue-specific exons have been reported. All of the transcripts encode an amino (N)-terminal RCC1-like domain that is structurally similar to the RCC1 protein, a guanine nucleotide exchange factor for the small GTP-binding protein, Ran [9]. RPGR ^1-19 is widely distributed in ciliated tissues, whereas RPGR ^ORF15 is found primarily in the connecting cilia of photoreceptor cells, predominantly in the outer segment of rod photoreceptors [10]. Due to the presence of highly repetitive purine-rich sequences, the exon ORF15 is a mutational hotspot for XlRP (accounting for 2/3 of all disease-causing mutations) and for most XLCRD cases [11].

Most reported mutations in the first 14 exons are nonsense or frameshift mutations that can lead to nonsense-mediated decay of the mRNA (NMD), and low or absent levels of the transcript. In contrast, nonsense or frameshift mutations in ORF15 are less likely to lead to NMD since this is the last exon of the transcript [12], and a series of truncated proteins of varying length can be found [13].

From a genetic point of view, IRDs displays locus and allelic heterogeneity, with more than 300 causative genes (https://sph.uth.edu/retnet/) that make the genetic characterization very difficult. The advent of next-generation sequencing (NGS) has opened new frontiers in genetic diagnostics of IRDs, exploiting the high-throughput parallel sequencing and the simultaneous analysis of many samples. The overall mutation detection rate for IRDs is variable and ranges from 36% to 60%, leaving many cases still genetically unsolved [14]. Although whole-exome sequencing (WES) is a more appropriate tool for genetic diagnostics of the extreme heterogeneous IRDs than gene panels, commercialized gene panels comprising all known disease-related genes, called “clinical exome” have been successfully applied for genetic testing of retinopathies [14] giving a diagnostic yield of over 80% for IRD cases in Bulgaria (personal unpublished data).

Here, we describe a male patient with clinical suspicion of CRD in whom clinical exome sequencing (CES) found a novel nonsense mutation in the exon ORF15 (c.1905 + 223C > T, p.Q710X) of RPGR gene. The presence of RPGR-c.1905 + 223C > T variant in the myopic female carriers is consistent with X-linked dominant mode of inheritance and milder phenotype in the studied pedigree.

Patient and clinical assessment

The study adhered to the tenets of the Declaration of Helsinki and was approved by the Ethics Committee of Medical University of Sofia (Bulgaria). Written informed consent form was obtained from all participants.

An 11-year-old boy complaining from impaired vision, photophobia, and myopia noticed since about 7 years of age, was referred by the University Hospital “Alexandrovska”, Sofia. The proband underwent a complete ophthalmic examination, including autorefractometry after cycloplegia (Canon autorefractometer RK-F2), determination of best-corrected visual acuity (BCVA), intraocular pressure (IOP), slit lamp examination of the anterior eye segment, automated perimetry (Octopus perimeter), slit lamp examination of the ocular fundus using + 90D lens with dilated pupils, multifocal electroretinogram (mfERG, RETIscan Standard 6.11), fundus photograph (Eidon true color confocal scanner), Ishihara color vision test, fundus autofluorescence (FAF, Eidon true color confocal scanner), optical coherence tomography (OCT, NIDEK, RS 3000).

In order to study the genetic basis of the phenotype segregating in this pedigree, we collected peripheral blood samples from the proband for CES analysis as well as from asymptomatic and myopic family members available for segregation study.

Genetic analysis

Peripheral blood samples of the proband, his parents and the sister of his mother (Figure 1) were collected for a molecular genetic analysis and DNA was extracted from leukocytes using Chemagic DNA blood 10 k kit H1 and Chemagen Magnetic Separation Module (PerkinElmer®, Waltham, MA, USA) according the manufacturer’s protocol. Targeted NGS was performed using TruSight One sequencing panel (Illumina, San Diego, CA, USA), which includes 4813 genes associated with known OMIM diseases. TruSight one sequencing

panel includes all reagents required for amplification, amplicon enrichment, and indexing of samples, and protocol was followed according to the manufacturer’s instructions. After preparation of the sequence libraries, MiSeq next-generation platform (Illumina) was used to sequence 150-bp paired-end reads.

Assessment of the pathogenicity of candidate variants

Filtered variants with coverage < 20× and those with MAF higher than 0.005 (in case of presumed autosomal recessive mode of inheritance) in at least one of the searchable databases (dbSNP or gnomAD) were excluded. To detect known disease-associated mutations, the remaining variants were compared to human mutation databases such as HGMD and ClinVar. The pathogenicity of novel SNVs was predicted by PolyPhen-2 (http://genetics.bwh.harvard.edu/pph2/), SIFT (http://sift.bii.a-star.edu.sg), MutationAssessor (http://mutationassessor.org/r3/), and FATHMM (http://fathmm.biocompute.org.uk/) softwares. Pathogenicity of variants was ascertained according to the criteria of American College of Medical Genetics (ACMG) [15], which classify variants according to 5 categories (benign, likely benign, uncertain significance, likely pathogenic, and pathogenic).

The Identified variant was confirmed by Sanger sequencing on an ABI 3130XL Genetic Analyzer (Applied Biosystems, Foster City, CA, USA). The DNA fragments containing the variants were amplified by PCR with specific primers and were sequenced using the Big Dye 3.1 Terminator Sequencing Kit. Sanger sequencing was also employed for segregation study.

Clinical data

Patient has suffered from symptoms consisting with a mild form of CRD, including decreased sensitivity in the central and peripheral visual field, slightly reduced visual acuity, and myopia with no nyctalopia and color vision defects observed. BCVA was declined to 0.7 (metric) in both eyes (with spherical correction of −1.0 D in the left eye). Automated perimetry showed a bilateral peripheral and central perimetric defect (Figure 2(a) and Figure 2(b)). Ophthalmic examination revealed grayish point-like deposits scattered within the posterior pole of the fundus, mainly in the paramacular area, but no attenuation of the retinal vessels, optic disc pallor and retinal atrophy were observed (Figure 3(a) and Figure 3(b)). Fundus autofluorescence did not reveal a pathologic finding (Figure 4(a) and Figure 4(b)). The optical coherence tomography showed normal retinal thickness and absence of any pathological changes (Figure 5(a) and Figure 5(b)). The Ishihara test revealed no abnormalities in color perception. ERG profile of the proband demonstrated normal morphology and polarity with a reduced amplitude of scotopic and photopic response (20% for the right eye and 25% for the left eye) and prolonged latency (Figure 6).

Patient’s mother (II: 2, 44 y) and her sister (II: 1, 50 y) presented with moderate (−4.0 diopters) and high myopia (−8.0 diopters), respectively, from their

20s. Fundus and electrophysiological examination of the two sisters (II: 1 and II: 2) showed no abnormalities. The ophthalmic diagnostic data are documented in Table 1. There was no older male carrier available for study the progression during follow-up years.

Genetic findings

Family pedigree is shown on Figure 1. Genetic testing of proband’s DNA identified a novel variant c.1905 + 223C > T (p.Q710X) in the RPGR ^ORF15. The mean sequence coverage was over 100× and more than 95% of target bases were covered with at least 20×. A novel variation, c.1905 + 223C > T, in RPGR gene resulting in a stop codon and premature translational termination at position 710 (p.Q710X) of the normal 1152-aa polypeptide was identified as the potential disease-causing nonsense mutation. The novel RPGR variant has not been reported in the context of clinical significance (ClinVar, HGMD) and is not found in the gnomAD population database and dbSNP. According to the ACMG classification, c.1905 + 223C > T (NM_001034853.1) was classified as a pathogenic because of 1) its type of null variant (PVS1), 2) absence in healthy individuals (PM2), 3) co-segregation with the disease in the family (PP1), 4) occurrence in a gene that has a low rate of benign missense variation (PP2), 5) computational tools predict a deleterious effect on the coded gene product (PP3) and 6) phenotype and family history specific for X-linked disease with a monogenic etiology (PP4).

Genomic DNA of collected family members was further analyzed by Sanger sequencing. Novel RPGR variant c.1905 + 223C > T was confirmed for the proband as a hemizygous mutation and for the myopic female members (mother and aunt) who were heterozygous carriers (Figure 1). The change was not found in the unaffected father. Therefore, the novel RPGR change c.1905 + 223C > T was shared by affected patients.