A Comprehensive Analysis of Choroideremia: From Genetic Characterization to Clinical Practice

Abstract

Choroideremia (CHM) is a rare X-linked disease leading to progressive retinal degeneration resulting in blindness. The disorder is caused by mutations in the CHM gene encoding REP-1 protein, an essential component of the Rab geranylgeranyltransferase (GGTase) complex. In the present study, we evaluated a multi-technique analysis algorithm to describe the mutational spectrum identified in a large cohort of cases and further correlate CHM variants with phenotypic characteristics and biochemical defects of choroideremia patients. Molecular genetic testing led to the characterization of 36 out of 45 unrelated CHM families (80%), allowing the clinical reclassification of four CHM families. Haplotype reconstruction showed independent origins for the recurrent p.Arg293* and p.Lys178Argfs*5 mutations, suggesting the presence of hotspots in CHM, as well as the identification of two different unrelated events involving exon 9 deletion. No certain genotype-phenotype correlation could be established. Furthermore, all the patients´ fibroblasts analyzed presented significantly increased levels of unprenylated Rabs proteins compared to control cells; however, this was not related to the genotype. This research demonstrates the major potential of the algorithm proposed for diagnosis. Our data enhance the importance of establish a differential diagnosis with other retinal dystrophies, supporting the idea of an underestimated prevalence of choroideremia. Moreover, they suggested that the severity of the disorder cannot be exclusively explained by the genotype.

Fig 1. Molecular strategy followed up for the diagnosis of CHM families.

Fig 2. Haplotypes from families presenting the recurrent CHM mutations.

Identified pedigrees carrying the exon 9 deletion (A), the p.Arg293* (B) and the p.Lys178Argfs*5 (C) mutations are shown. For exon 9 deletion, haplotypes analysis demonstrated identity by descent in the Spanish families RP-1310, RP-1560 and RP-2128 but independent origin for the Portuguese family RP-0779, defined by the alleles located along the black bar. For the p.Arg293* and p.Lys178Argfs*5 mutations, haplotypes indicates an independent origin for both variants defined by the alleles located along the black bar.

Fig 3. Prenylation status of different CHM mutations.

A) A representative in vitro prenylation assay using a biotinylated prenyl donor followed by western blot analysis. A weaker signal of incorporated biotin can be seen for the wild-type (WT) control as compared to the patients’ cells. B) Semi-quantification of the pool of biotinylated Rabs (ranging in size from 20 to 29 kDa) after normalization of β-actin loading. The levels in control cells were set to 1. The unprenylated Rab levels were significantly higher than wild-type (black bar) in all the patients’ cells regardless of the type of mutation (p<0.05, asterisks; n = 3). In addition, the Rab levels were significantly different between two brothers carrying the same large deletion (dark grey bars) of the CHM gene (p<0.05, asterisk; n = 3).