Autosomal recessive retinitis pigmentosa with early macular affectation caused by premature truncation in PROM1

Abstract

Purpose: To identify the genetic basis of a large consanguineous Spanish pedigree affected with autosomal recessive retinitis pigmentosa (arRP) with premature macular atrophy and myopia.

Methods: After a high-throughput cosegregation gene chip was used to exclude all known RP and Leber congenital amaurosis (LCA) candidates, genome-wide screening and linkage analysis were performed. Direct mutational screening identified the pathogenic mutation, and primers were designed to obtain the RT-PCR products for isoform characterization.

Results: Mutational analysis detected a novel homozygous PROM1 mutation, c.869delG in exon 8 cosegregating with the disease. This variant causes a frameshift that introduces a premature stop codon, producing truncation of approximately two-thirds of the protein. Analysis of PROM1 expression in the lymphocytes of patients, carriers, and control subjects revealed an aberrant transcript that is degraded by the nonsense-mediated decay pathway, suggesting that the disease is caused by the absence of the PROM1 protein. Three (s2, s11 and s12) of the seven alternatively spliced isoforms reported in humans, accounted for 98% of the transcripts in the retina. Given that these three contained exon 8, no PROM1 isoform is expected in the affected retinas.

Conclusions: A remarkable clinical finding in the affected family is early macular atrophy with concentric spared areas. The authors propose that the hallmark of PROM1 truncating mutations is early and severe progressive degeneration of both rods and cones and highlight this gene as a candidate of choice to prioritize in the molecular genetic study of patients with noncanonical clinical peripheral and macular affectation.

Figure 1.

(A) Pedigree and SNP haplotypes on chromosome 4p16.1-p15.31 surrounding the PROM1 locus. Black bars indicate the disease haplotype while open bars represent nondisease haplotypes. (B) Chromatograms identifying the mutation c.869delG, showing the wild-type exon 8 sequence (top), the heterozygous carrier (middle), and the homozygous patient (bottom). (C) PROM1 exon structure showing the reported seven human isoforms (Fargeas et al.). They differ on the inclusion/exclusion of exons 3, 25, 26b, and 27, corresponding either to the N (3)- or C (25, 26b, and 27)-terminal tails. Arrows: specific primers for every isoform are indicated over the exons. The reported mutations are shown as colored stars. (D) RT-PCR analysis of PROM1 isoforms in wild-type human retina; s11 and s12 are the most prominent isoforms; isoform s2 is expressed more faintly, whereas isoforms s1, s7, and s10 are barely detectable. (E) PROM1 topology. Left: wild-type PROM1 is predicted to consist of an extracellular N-terminal domain, five transmembrane domains (TM1–TM5) that define two small intracellular and two large extracellular loops and a C-terminal cytoplasmic tail. The location of the novel c.869delG mutation and the previously described c.1117 C>T, c.1349insT, c.1726 C>T, and c.1876delG mutations are also shown. Right: the assumed PROM1 topologic representation of the truncated protein encoded by the mutant allele.

Figure 2.

Fundus eye photographs, autofluorescence images, and optical coherence tomography (OCT) from affected and unaffected family members. Images correspond to nonaffected heterozygous carrier IV1 (A, B), patient IV2 (C), patient IV3 (D, E), and patient IV4 (F–H). The OCT macular scans from patient IV4 show bilaterally neurosensorial atrophy in the macular area.

Figure 3.

Humphrey's visual field test from affected and unaffected family members, carrier IV1 (A), patient IV3 (B), and patient IV4 (C). Note the correspondence of the preserved central area around the macula in this test with the autofluorescence images of the posterior pole in Figure 2.

Figure 4.

RT-PCR analysis of PROM1 mRNAs from blood of patients IV3 and IV4, the heterozygous carrier and a control individual. (A) Patients IV3 and IV4 showed lower, although detectable levels of PROM1 transcripts compared with carrier IV1 or the control (WT). GAPDH was used as control for normalization. (B) Semiquantitative analysis of PROM1 levels, with GAPDH expression used for normalization and the PROM1 levels of the wild-type control set at 100%.