A novel SVA retrotransposon insertion in the CHM gene results in loss of REP-1 causing choroideremia

Abstract

Background

Choroideremia is an X-linked retinal disease characterized by progressive atrophy of the choroid and retinal pigment epithelium caused by mutations in the CHM gene. SVA (SINE-R/VNTR/Alu) elements are a type of non-autonomous retrotransposon that occasionally self-replicate, reinsert randomly into a gene, and cause disease. Intragenic SVA insertions have been reported as the mechanism underlying a number of diseases including a syndromic form of retinal dystrophy, but have never been found in CHM.

Materials and Methods

Here we identified and characterized a novel hemizygous SVA insertion, c.97_98inSVA (p.Arg33insSVA), in exon 2 of CHM in a male choroideremia patient. The SVA insertion’s impact was evaluated by establishing a patient-derived lymphoblastoid cell line as a source of RNA for mRNA analysis of the CHM transcript, and protein for immunoblot analysis of Rab Escort Protein 1 (REP-1).

Results

Immunoblot analysis revealed absence of REP-1 protein, while a smaller than expected PCR product was amplified from cDNA. Sequencing of this PCR product showed skipping of exon 2, denoted r.50_116del. Ophthalmic examination including psychophysical tests, visual electrophysiology, and fundus imaging showed the patient’s phenotype was consistent with severe early manifestations of choroideremia.

Conclusions

This case is the first report of a SVA insertion in the CHM gene causing choroideremia.

Introduction

Choroideremia is an X-linked retinal disease characterized by progressive atrophy of the retinal pigment epithelium and choriocapillaris that affects approximately 1:50,000 males (1). Affected males first experience night blindness early in life and subsequently experience progressive peripheral field loss. Electroretinograms of affected males initially follow a course of rod-cone degeneration with the ERG eventually becoming non-detectable. Central vision is generally preserved until the fourth decade of life (2). Carrier females are typically asymptomatic, but fundus abnormalities including signs of chorioretinal degeneration are occasionally observed, especially at the level of the retinal pigment epithelium.

Choroideremia results from pathogenic mutations in the CHM gene. CHM is located at chromosome Xq21.2, spans over 150 kb, and is comprised of 15 exons. CHM encodes the Rab Escort Protein 1 (REP-1) (3). REP-1 is a key element of the catalytic GTPases prenylation complex necessary for vesicular trafficking. A wide spectrum of CHM mutation types including missense, nonsense, frameshift, and splice site variants along with whole gene rearrangements, have been reported in association with choroideremia (Leiden Open Variation Database; www.lovd.nl). Additionally, rare pathogenic events including large duplications, a substitution mutation within the CHM promoter, a LINE-1 insertion, and an Alu insertion have been reported within CHM (4–7). Recently, a synonymous variant in CHM was reported to disrupt an exonic splicing enhancer leading to exon 11 skipping (8).

SVA (SINE-R/VNTR/Alu) elements are a type of non-autonomous composite retrotransposon comprised of a variable length hexameric repeat (CCCTCT)_n followed by an inverted Alu-like sequence, a variable number of tandem repeats (VNTR), a sequence derived from the 3’ end of a HERV-K10 element (SINE-R), and a poly(A) tail (9–11). Approximately 2,700 copies of SVA elements are estimated to be present in the human genome (12). SVA elements are thought to be transcribed by RNA polymerase II, and the subsequent SVA RNAs sometimes reinsert randomly into a gene by L1-encoded proteins acting in trans (13–15). Intragenic SVA insertions are reported as one mutation type underlying a number of diseases including Bardet-Biedl syndrome, a syndromic form of retinal dystrophy (16), but have never been reported in the CHM gene. Here we describe the phenotype and genotype of a choroideremia patient with a novel SVA insertion, and evaluate the impact of the SVA insertion on the CHM transcript and resultant REP-1 protein.

Methods

All procedures were approved by the Institutional Review Boards of UT Southwestern Medical Center and the University of Alberta, Faculty of Medicine and Dentistry. Informed consent was obtained, and all research was conducted in accordance with the Declaration of Helsinki.

An 11-year-old male with no family history of retinal disease, complaining of reduced acuity and decreased visual field underwent ophthalmic examination including best-corrected visual acuity (BCVA), kinetic fields, full-field electroretinogram (ERG) recordings were obtained using the International Society for Clinical Electrophysiology of Vision (ISCEV) standard protocol. Subsequently, morphologic examinations including fundus photography, fundus auto-fluorescence, and spectral-domain optical coherence tomography (SD-OCT) were performed.

The patient was enrolled in the Foundation Fighting Blindness My Retina Tracker® Genetic Testing Study. DNA sequence analysis and copy number variation analysis of 266-gene retinal dystrophy panel was performed (Retinal Dystrophy Panel Plus, Blueprint Genetics, Helsinki, FIN). A lymphoblastoid cell line was established and RNA and protein were extracted for mRNA of the CHM transcript and immunoblot analysis for REP-1 as previously described (17).

Results

The proband reported onset of night blindness and loss of side vision at age 5 years. Careful family history revealed that no other family members were symptomatic (Figure 1a). The patient’s mother reported no vision disturbances, but opted to decline vision and genetic testing. The clinical diagnosis of choroideremia was established at age 11. Testing at that time revealed that BCVA was 20/40 in the right eye and slightly worse, 20/80, in the left eye. Visual fields included a central region constricted to 20°, extensive loss of superior field, and infero-temporal peripheral islands. The ERG showed severe reduction in photoreceptor function. ERG rod responses were non-detectable, and 30Hz cone flicker responses were reduced in amplitude (17.7 μV; mean normal 75 μV) and significantly delayed in implicit time (42 msec; mean normal 29 msec). SD-OCT imaging showed extensive loss of ellipsoid zone and outer nuclear layer. A central area of hyper-fluorescence in the macula, along with a scalloped region of atrophy, was observed consistent with a diagnosis of choroideremia. (Figure 1b–d).

Figure 1.

(a) Pedigree of the affected family. (b) Full-field electroretinogram (ffERG) showing loss of rod photoreceptor function below the lower limit of detection, and significantly reduced and delayed cone photoreceptor responses. (c) A central area of fluorescence in the macula, along with a scalloped region of atrophy consistent with a diagnosis of choroideremia was observed with fundus auto-fluorescence imaging. (d) SD-OCT imaging showed extensive loss of ellipsoid zone and outer nuclear layer.

Genetic analysis identified a novel insertion of a retrotransposable element in exon 2 of the CHM gene (Figure 3a). The identified c.97_98insSVA, p.(Arg33insSVA) (NM_000390.2) retrotransposon was initially identified by a drop in coverage of NGS data within exon 2 of CHM. Additional bioinformatic and Sanger sequencing analysis through Blueprint Genetics confirmed the presence of the insertion.

The SVA insertion was estimated to be larger than 265 bp, but the exact size of the insertion could not be determined through NGS methodology. SVA insertions are typically approximately 1000 bp in size, and presence within the exon was predicted to disrupt normal REP-1 production and function. RNA and immunoblot analysis were necessary to evaluate the effect of the SVA insertion at the transcript and protein level. Immunoblot analysis showed absence of REP-1 protein. Amplification from cDNA of a fragment spanning exon 2 showed a smaller than expected product. Sequencing of the PCR product generated from cDNA showed skipping of all of exon 2, denoted r.50_116del (Figure 3b-d).

Discussion

This report demonstrates the pathogenicity of a novel SVA insertion in the CHM gene leading to choroideremia. mRNA analysis and protein expression studies demonstrate skipping of exon 2 leading to absence of detectable REP-1 expression. This type of loss-of-function mutation is likely a rare event, although pathogenic retrotransposon insertions have been recently reported more frequently (18).

This patient presented at an early age and manifested a severe disease phenotype. Extensive chorioretinal atrophy encroaching the macula, extensive loss of ellipsoid zone and outer nuclear layer was observed in this patient by 11 years of age.

Clinical trials for gene therapy in choroideremia are ongoing. The importance of careful molecular screening and diagnosis of young patients will become more critical as gene replacement therapy clinical trials advance. In conclusion, this report extends the diverse mutational spectrum of choroideremia and broadens our understanding of these types of mutations in inherited retinal dystrophies.

Figure 2.

(a) Schematic representation of the inserted SVA element in exon 2 at c.97. (b) cDNA amplification analysis shows a smaller sized product using primers spanning from 5’UTR to exon 3 for the patient (#14594). (c) Sequencing of product generated from cDNA shows skipping of all of exon 2. (d) Western blot analysis shows an absence of REP-1 expression in patient #14594 compared to Wild Type controls.