Genomic rearrangements of the PRPF31 gene account for 2.5% of autosomal dominant retinitis pigmentosa

Abstract

Purpose: To determine whether genomic rearrangements in the PRPF31 (RP11) gene are a frequent cause of autosomal dominant retinitis pigmentosa (adRP) in a cohort of patients with adRP.

Methods: In a cohort of 200 families with adRP, disease-causing mutations have previously been identified in 107 families. To determine the cause of disease in the remaining families, linkage testing was performed with markers for 13 known adRP loci. In a large American family, evidence was found of linkage to the PRPF31 gene, although DNA sequencing revealed no mutations. SNP testing throughout the genomic region was used to determine whether any part of the gene was deleted. Aberrant segregation of a SNP near exon 1 was observed, leading to the testing of additional SNPs in the region. After identifying an insertion-deletion mutation, the remaining 92 families were screened for genomic rearrangements in PRPF31 with multiplex ligation-dependent probe amplification (MLPA).

Results: Five unique rearrangements were identified in the 93 families tested. In the large family used for linkage exclusion testing, an insertion-deletion was found that disrupts exon 1. The other four mutations identified in the cohort were deletions, ranging from 5 kb to greater than 45 kb. Two of the large deletions encompass all PRPF31 as well as several adjacent genes. The two smaller deletions involve either 5 or 10 completely deleted exons.

Conclusions: In an earlier long-term study of 200 families with adRP, disease-causing mutations were identified in 53% of the families. Mutation-testing by sequencing missed large-scale genomic rearrangements such as insertions or deletions. MLPA was used to identify genomic rearrangements in PRPF31 in five families, suggesting a frequency of approximately 2.5%. Mutations in PRPF31 now account for 8% of this adRP cohort.

Figures

Figure 1

(1) Hybridization of two half-probes to each unique target site in the genome. (2) Ligation of each half-probe pair, unless one or both half-probes did not hybridize correctly due to deletion or base mismatches. (3) Amplification of each probe with fluorescently labeled universal primers. Amplification products from each target are a unique size. (4) Capillary electrophoresis of amplified probes. Areas under each probe peak were calculated and compared to control samples. (5) DQs were calculated relative to control samples. A DQ of 1.0 indicates that two alleles are being amplified. DQs <0.6 or greater than 1.4 were investigated further. In this example, probe 1 is normal, whereas probe 2 appears to be hemizygous.

Figure 2

Abnormal segregation of SNP rs4806711 in BCMAD014. Actual genotypes are shown in parentheses, observed genotypes are shown above. X, the allele carrying the insertion–deletion that no longer contains the SNP. Multiple individuals appeared to be incompatible with their parents but were actually hemizygous for the SNP.

Figure 3

Mapping of deletions in five families by using MLPA. (A) Twenty-one MLPA probes (green stars) within the PRPF31 gene were tested in all families. Four additional internal probes (blue stars) were used to refine the deletion breakpoints in UTAD069 and UTAD082. Red bars: the extent of each deletion. (B) Thirteen flanking probes (orange stars) were tested to determine the extent of the deletions in UTAD119 and UTAD034. Probes are spaced at approximate 5-kb intervals on either side of PRPF31. The deletion in UTAD034 extends beyond the most 5′ probe tested. (C–G) Calculated dosage quotients (DQs) for probe set D and control probes from the probe set (P115 Retina; MRC-Holland, Amsterdam, The Netherlands). Green: PRPF31 probes with normal DQs; red: PRPF31 probes with abnormal DQs. Black: control probes from the set, including probes for rhodopsin, RP1, and RPE65 as well as other controls.