A hypomorphic variant of choroideremia is associated with a novel intronic mutation that leads to exon skipping

Figures & data

Figure 1. (a) Ultra-widefield optomap images of both eyes showing extensive retinal degeneration with choroidal atrophy consistent with choroideremia. (b) Short-wavelength autofluorescence imaging showing typical patterns of sharply demarcated areas of remaining hyperfluorescent tissue against atrophic retina giving an absent autofluorescent signal, with the following FAF areas on 55 degree images: right 7.65 mm² ((OD) RE FAF) and left 13.90 mm² ((OS) LE FAF). (c) Spectral-domain OCT scans showing ellipsoid zone shortening, intraretinal cysts and tubulations. Choroidal hypertransmission reveals the extent of RPE loss. (d) Right and left eye microperimetry sensitivity maps showing retinal sensitivities within the residual island of tissue.

Figure 2. Adapted figure from an article by Aylward et al. (17) showing a correlation between residual FAF area (mm², shown on a log scale) and age in a cohort of 56 eyes (28 patients) for the right (hollow circles) and left (solid triangles) eyes. The green line illustrates that the patient, as a 42-year-old, is an outlier in terms of baseline FAF area for his age, within the orange 95% confidence interval lines.

Figure 3. Agarose gel electrophoresis of PCR products. The wild-type transcript is only detected in the two control samples. Lower molecular weight products are detected in the patient’s sample. These bands correspond to a transcript with skipping of exon 11 (ca. 350 base pairs (bp) band) which results from the mutation c.1350-3C>G, and a transcript with skipping of exons 10 and 11 (ca. 245 bp band) that has also been observed at low levels in healthy subjects (14). Indeed, this band is also visible in the control samples, although with less intensity than that observed in the patient. Please see supplementary Figure S1 for raw Sanger sequencing data. NTC: no template control.

Figure 4. Analysis of REP1 protein expression and function in the CHM patient. Four different samples from healthy donors (no CHM gene mutation) were included in this assay. While REP1 expression was not detected in two of the controls, the four samples show a high concentration of biotinylated RAB6A compared with the patient, which indicates a high prenylation activity in controls. Three technical replicates were conducted with patient’s samples. REP1 protein expression is not detected in the patient carrying the c.1350-3C>G mutation. However, a 24 kDa band corresponding with biotinylated RAB6A is detected in the patient’s samples, which suggests that some residual prenylation activity is present. The 42 kDa band corresponding to the βActin, used as the loading control, is detected in all the samples.

Figure 5. The location of the genetic variant and the impact on pre-mRNA splicing, detailing the two out-of-frame transcripts and lack of REP1 protein. Figure created by BioRender.com; published under MacLaren lab BioRender license.

Table 1. Evidence in this case to satisfy the ACMG criteria for classifying pathogenic variants.

The American College of Medical Genetics and Genomics (ACMG) Criteria for Classifying Pathogenic Variants (18)			Evidence in this case to satisfy requirements
Strong evidence of pathogenicity	PS3	Well-established in vitro or in vivo functional studies supportive of a damaging effect on the gene or gene product	RNA splicing analysis demonstrated splicing abnormalities in a blood sample. Figure 4 confirms the absence of REP1 protein.
Moderate evidence of pathogenicity	PM2	Absent from controls in Exome Sequencing, 100 Genomes, or ExAC	This variant has not been reported before in unaffected human populations. The variant is not listed in the databases of human variation curated by the Genome Aggregation Database (gnomAD) (more than 170,000 alleles screened at this locus). GnomAD V.2.1.1(exomes), accessed December 2022 (12).
Supporting evidence of pathogenicity	PP3	Multiple lines of computational evidence support a deleterious effect on the gene or gene products	In silico prediction tools strongly suggest this variant leads to a dysfunctional splice acceptor site.
	PP4	Patient’s phenotype or family history is highly specific for a disease with a single genetic etiology	Figure 1 details the clinical presentation characteristic of a patient with choroideremia. The patient’s great-grandfather had been diagnosed with retinitis pigmentosa, although this could have been choroideremia incorrectly diagnosed (16).

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