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

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 the 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.

Visual Function at the Atrophic Border in Choroideremia Assessed with Adaptive Optics Microperimetry.

PURPOSE: Recent advances in retinal imaging allow visualization of structural abnormalities in retinal disease at the cellular level. This study used adaptive optics (AO) microperimetry to assess visual sensitivity with high spatial precision and to examine how function varies across 2 phenotypic features observed in choroideremia: atrophic lesion borders and outer retinal tubulations (ORTs). DESIGN: Cross-sectional study. PARTICIPANTS: Twelve choroideremia patients. METHODS: A custom AO scanning light ophthalmoscope (AOSLO) equipped with both confocal and nonconfocal split-detection imaging methods was used to image the photoreceptor inner and outer segment mosaics. For AO microperimetry, circular 550-nm stimuli were presented through the AOSLO system; stimuli were either 9.6 or 38.3 arcmin2 (approximately 60 or 15 times smaller than a Goldman III stimulus). Test locations were identified in structural images and stimuli were targeted to these locations using real-time retinal tracking combined with measurements of transverse chromatic aberration. Psychophysical detection thresholds were measured at the atrophic border in 12 patients. Additionally, visual sensitivity was probed along ORTs in 4 patients. MAIN OUTCOME MEASURE: Visual sensitivity thresholds measured with AO microperimetry at retinal locations corresponding to structural phenotypes observed on AOSLO retinal images. RESULTS: In choroideremia, sharp borders between intact central islands of the photoreceptor mosaic and complete atrophy of the outer retina and retinal pigment epithelium were observed in both split-detection and confocal structural images. Adaptive optics microperimetry at locations spanning these borders showed a commensurately sharp decrease in function, with readily measurable visual sensitivity on one side and dense scotoma on the other. These functional transitions often occurred over a distance smaller than the diameter of the Goldman III stimulus. Thresholds measured along ORTs showed dense scotoma over the tubule in all 4 participants, despite the visibility of remnant cone inner segments on the AO images. CONCLUSIONS: Choroideremia patients exhibited sharp functional transitions that collocated with structural transitions from intact to severely degenerated retina. We found no evidence of visual sensitivity over ORTs. Measuring cone function with high resolution offered insight into disease mechanisms and may enable precise assessment of whether experimental therapies, such as gene therapy, provide a functional benefit.

Choroideremia: towards a therapy.

PURPOSE: To review what progress has been made towards the application of ocular gene therapy to prevent progressive vision loss in patients affected by choroideremia. DESIGN: A Perspective based on the collective opinions of researchers and clinicians actively engaged in vision research on choroideremia and a review of current literature. METHODS: Researchers from Europe, Canada, Australia, and the United States were convened to the first International Choroideremia Research Symposium held in Sommières, France in September 2011. Attendees shared their collective understanding of the pathophysiology of choroideremia and current trends in the development of treatments, with an emphasis on the potential of gene therapy as an achievable approach. Supplemental perspectives are provided along with an update of progress made since the meeting. RESULTS: The complexity of treating a retinal disease such as choroideremia that affects multiple tissue layers has been brought to light. The genetic basis of choroideremia must be thoroughly deciphered and appropriate clinical tests selected to follow disease progression and evaluate the efficiency of treatments. CONCLUSIONS: Whereas the time frame for the development of therapies for some retinal dystrophies may be in the years hence, gene therapy trials for choroideremia have started in the United Kingdom and results are pending. These first trials may help resolve the remaining issues associated with the treatment of this disease.

Choroideremia: a review of general findings and pathogenesis.

Choroideremia (CHM) is an X-linked retinal dystrophy belonging to the family of blinding disorders. It is characterized by progressive degeneration of the choriocapillaris, retinal pigment epithelium and photoreceptors. CHM is caused by mutations in the Rab Escort Protein 1 (REP-1) gene, which encodes a protein involved in vesicular trafficking. This paper gives an overview of the clinical features, visual function, biochemistry, histology, molecular genetics, pathogenesis, diagnosis and treatment of CHM.

Multiple factors contribute to inefficient prenylation of Rab27a in Rab prenylation diseases.

Post-translational geranylgeranylation of Rab GT-Pases is essential for their membrane association and function as regulators of intracellular vesicular transport. The reaction is catalyzed by Rab geranylgeranyltransferase (RGGT) and is assisted by the Rab escort proteins (REP), which form stable complexes with newly synthesized GDP-bound Rabs. Two genetic diseases involve the Rab geranylgeranylation machinery: choroideremia, an X-linked retinal degeneration resulting from loss-of-function mutations in REP1, and gunmetal, a mouse model of Hermansky-Pudlak syndrome resulting from mutations in the alpha-subunit of RGGT. A small subset of Rab proteins is selectively under-prenylated in both diseases, most notably Rab27a. Here we analyze why Rab27a is selectively affected in diseases of Rab geranylgeranylation. Semi-quantitative immunoblotting suggests that mass action, i.e. the amount of Rab27a relative to other Rabs, is unlikely to be a factor as the expression level of Rab27a is similar to other Rabs not affected in these diseases. In vitro binding assays and fluorescence resonance energy transfer detected by fluorescence lifetime imaging microscopy in intact cells demonstrate that Rab27a binds equally well to both REP1 and REP2, suggesting differential affinity of Rab27a for REP isoforms is not an important factor. However, steady-state kinetic analysis of the geranylgeranylation reaction indicates that REP2-Rab27a has lower affinity for RGGT compared with REP1-Rab27a. Furthermore, we show that Rab27a has relatively low GTPase activity, presumably decreasing the affinity of the REP interaction in vivo. We suggest that the restricted phenotypes observed in these diseases result from multiple contributing factors.