Mutational analysis of patients with the diagnosis of choroideremia

Abstract

All reported mutations in the choroideremia (CHM) gene result in the truncation or complete absence of Rab escort protein 1 (REP1). Molecular analysis was carried out on 57 families diagnosed with CHM. Confirmation of the clinical diagnosis is important as end-stage CHM may be clinically similar to the end stages of other retinal degenerative diseases such as RP. The primary means of confirming the diagnosis of CHM is to sequence all 15 exons. An alternative method involves detection of the REP1 protein, as described in MacDonald et al. [1998]. A monoclonal antibody to REP1 does not detect truncated REP1 by immunoblot analysis, presumably due to instability and subsequent degradation of the truncated protein. This analysis provides relatively fast confirmation of the diagnosis, however, protein samples are not always available and are susceptible to degradation, affecting the accurate interpretation of results. CHM gene mutations were found in 54 of 57 families studied. The majority of mutations (>42%) were transitions and transversions. Complete deletions of the CHM gene and deletion/insertion mutations each accounted for almost 4% of the total, while over 9% had large intragenic and other partial deletions. Almost 28% of the mutations were deletions of fewer than 5 base pairs (bp) and almost 13% were splice site mutations. Despite the fact that mutations are found throughout the gene with no common mutation for the disorder, identical mutations have been characterized in unrelated individuals. The majority of these mutations are C to T transitions, changing an arginine residue (CGA) to a stop codon (TGA). Four of the five CGA codons in the CHM gene are sites of recurring mutations.

Novel types of mutation in the choroideremia (CHM) gene: a full-length L1 insertion and an intronic mutation activating a cryptic exon

“…One of these, R293X, was detected in four patients and has already been reported in two unrelated patients with CHM (MacDonald et al 1998;van Bokhoven et al 1994b). Other recurrent mutations are 116+1G> A (Fujiki et al 1999;McTaggart et al 2002), R239X (McTaggart et al 2002), R253X (Fujiki et al 1999;McTaggart et al 2002), and R270X (Fujiki et al 1999;McTaggart et al 2002). All of these mutations involve C>T or G>A transitions at CpG dinucleotides and may thus reflect the hypermutability of these sites.…”

“…Translocations that disrupt the CHM gene have been found in two female patients with mild clinical symptoms of choroideremia (Cremers et al 1990;van Bokhoven et al 1994a). Among the subtle mutations detected in the CHM gene are nonsense, frameshift, and splice site mutations (Beaufrère et al 1996(Beaufrère et al , 1997(Beaufrère et al , 1998Forsyth et al 1997;Fujiki et al 1999;Hotta et al 1997;McTaggart et al 2002;Nesslinger et al 1996;Trujillo et al 1998;van den Hurk et al 1997). Most, if not all of these mutations give rise to a premature termination codon.…”

“…Deletions vary in size from a few kilobases, removing a single exon, to ~15 Mb, comprising the complete CHM gene and virtually the entire Xq21 band (Fujiki et al 1999;McTaggart et al 2002;van den Hurk et al 1997). Translocations that disrupt the CHM gene have been found in two female patients with mild clinical symptoms of choroideremia (Cremers et al 1990;van Bokhoven et al 1994a).…”

CHM gene molecular analysis and X‐chromosome inactivation pattern determination in two families with choroideremia

“…The majority of CHM gene mutations identified to date in choroideremia patients from different ethnicities are point mutations that directly introduce a premature stop codon [van den Hurk et al, 1997[van den Hurk et al, , 2003Beaufr ere et al, 1999;McTaggart et al, 2002]; Other CHM defects as deletions (partial or total), translocations, insertions, and intronic mutations causing splice defects, have also been recognized in some patients [Sankila et al, 1992;van den Hurk et al, 2003;Yip et al, 2007]. CHM missense mutations have not been yet identified, suggesting that these changes are either embryonic lethal or have no or very mild phenotypic effects.…”

Gene Therapy for Choroideremia Using an Adeno-Associated Viral (AAV) Vector

“…The slow rate of degeneration means that there is a long therapeutic window in which to replace the gene in ocular cells before they are lost. In addition, virtually all cases reported so far are functionally null mutations and are often predicted to result in the severe truncation or absence of endogenous REP1 protein (van den Hurk et al 1997(van den Hurk et al , 2003McTaggart et al 2002;Esposito et al 2011). This is useful because the product of the therapeutic gene will not have to compete with a large pool of mutant protein.…”