Pathological donor splice site mutations beyond the dinucleotide canonical sequence in choroideremia

Abstract

Purpose : Choroideremia (CHM) is an X-linked retinal degeneration caused by mutations in the CHM gene that encodes Rab escort protein-1 (REP1). Here we describe the clinical and molecular findings of two unrelated male patients (P1 & P2), who have an identical c.940+3delA donor splice site (intron 7) CHM mutation but markedly different rates of disease progression. Since this mutation is outside the canonical splice donor site, it raises the possibility that some viable RNA transcripts might be generated in the patient with slower rate of degeneration (P1).

Methods : Clinical assessment, including visual acuity and microperimetry, was performed. Rate of disease progression was estimated based on the areas of remaining fundus autofluorescence (AF) compared to a cohort of 31 choroideremia patients with other mutations. CHM exons ± 50 bp of intronic DNA covering splice donor/acceptor sites were bidirectionally Sanger sequenced for the two index patients and a positive control patient (P3) with a mutation in the consensus splice donor site (c.940+2T>A).

Results : All three patients demonstrated typical choroideremia phenotype. P1 showed an AF area of 168.7 mm² (aged 36 yr), compared with P2 who showed an AF area of 13.6 mm² (aged 43 yr), and P3 who showed an AF area of 38.4 mm² (aged 12 yr). Genotyping and phenotyping of the mother of P1 confirmed carrier status. Analysis of intronic sequence 3' to the +3delA mutation revealed a series of 7 thymine (T) nucleotides in all three patients. Hence the +3delA mutation would cause a 5' shift of the poly-T sequence by one nucleotide, thereby leaving the splice donor site otherwise identical up to the +8 position, with the exception of the A to T transversion at position +3.

Conclusions : Splice site mutations beyond the GT canonical donor sequence in intron 7 can cause choroideremia. A single transversion at position +3 appears sufficient to be pathogenic, although it can be associated with a milder form. This raises the possibility that a low level of splicing might occur despite this mutation, possibly due to alternative splice donor sequence downstream.

This is an abstract that was submitted for the 2018 ARVO Annual Meeting, held in Honolulu, Hawaii, April 29 - May 3, 2018.