Mutations in the Pre-mRNA Splicing-Factor Genes PRPF3, PRPF8, and PRPF31 in Spanish Families with Autosomal Dominant Retinitis Pigmentosa Marı ´a Martı ´nez-Gimeno, 1,2 Marı ´a Jose ´ Gamundi, 1,2 Imma Hernan, 1 Miquel Maseras, 3 Elena Milla´, 3 Carmen Ayuso, 4 Blanca Garcı ´a-Sandoval, 5 Magdalena Beneyto, 6 Concha Vilela, 7 Montserrat Baiget, 8 Guillermo Antin˜olo, 9 and Miguel Carballo 1 PURPOSE. Mutations in the systemically expressed pre-mRNA splicing-factor genes PRPF3, PRPF8, and PRPF31 have re- cently been associated with autosomal dominant retinitis pig- mentosa (adRP). This study was intended to identify mutations in PRPF3, PRPF8, and PRPF31 in 150 Spanish families affected by adRP, to measure the contribution of mutations in these genes to adRP in that population, and to correlate RP pheno- type expression with mutations in pre-mRNA splicing-factor genes. METHODS. Denaturing gradient gel electrophoresis (DGGE) and direct genomic sequencing were used to evaluate the complete coding region and flanking intronic sequences of the PRPF31 gene, exon 42 of PRPF8, and exon 11 of PRPF3 for mutations in 150 unrelated index patients with adRP. Ophthalmic and electrophysiological examination of patients with RP and their relatives was performed according to preexisting protocols. RESULTS. Three nonsense mutations caused by insertion and deletion sequences and two missense mutations (Arg2310Gly) and within the stop codon of the PRPF8 gene (TGA3 TTG), were detected in five unrelated heterozygous patients. Three patients were heterozygous carriers of different nonsense mu- tations in exon 8 of the PRPF31, gene and one Thr494Met mutation was found in exon 11 of the PRPF3 gene. Cosegre- gation of the mutation in PRPF8 and PRPF3 with adRP was observed. However, two nonsense mutations in PRPF31 caus- ing adRP detected in two families showed asymptomatic car- riers. CONCLUSIONS. Nine mutations, six of which are novel, in the pre-mRNA splicing-factor genes PRPF3, PRPF8, and PRPF31, causing adRP have been identified in the Spanish population. Their contribution to adRP is approximately 5% after correc- tion in relation to mutations found in other genes causing adRP. The patients carrying a mutation in the pre-mRNA splic- ing-factor PRPF8 gene showed a type 1 diffuse RP. The exis- tence of asymptomatic carriers of the nonsense mutation in the PRPF31 gene suggests incomplete penetrance for these muta- tions in the families. (Invest Ophthalmol Vis Sci. 2003;44: 2171–2177) DOI:10.1167/iovs.02-0871 R etinitis pigmentosa (RP) is a group of hereditary retinal degenerations with a worldwide incidence of approxi- mately 1 in 3500 individuals. Clinical characteristics include night blindness, loss of peripheral visual field, characteristic changes in the ocular fundus, and depression of the normal ocular electrophysiological responses. 1 The genetics of RP is complex, and the disorder may be inherited through an auto- somal dominant (adRP), autosomal recessive (arRP), X-linked (XLRP), 2,3 or digenic 4 trait. Mutations within six genes (RHO, peripherin/RDS, RP1, NRL, CRX, and FSCN2) that encode proteins specifically expressed in photoreceptor cells have been reported to cause adRP. 5–9 Such proteins are involved in specific functions in the retina, such as the visual transduction cycle and structural components of the rod and cone photo- receptor cells or transcription factors. Furthermore, genetic linkage studies and mutation detection resulted in the charac- terization of mutation in genes with systemic expression im- plicated in adRP. 10 –12 Three of these genes, PRPF3 (RP18), PRPF8 (RP13), and PRPF31 (RP11), are members of the pre-mRNA splicing-factor components of the U4/U6-U5 tri-snRNP particle 13–15 that is dynamically assembled and dissociated at each round of the splicing cycle. Although little is known about the association and dissociation of the U4/U6 and U5 snRNP, protein–protein interaction and rearrangement are believed to take part in the formation and function of the tri-snRNP particle. 16 It is there- fore plausible that some mutation in one of the protein com- ponents of the tri-snRNP affects its structure and the splicing function. Mutations in the pre-mRNA splicing-factor genes reported so far in humans seem to produce pathologic ef- fects in the retina only. The mutations reported to date in PRPF8 11 and PRPF3 12 are clustered within a 14 (exon 42) and 2 (exon 11)-codon stretch respectively, whereas mutations in PRPF31 10 are more dispersed within the transcript sequence. Mutations in the rhodopsin (RHO) and peripherin/RDS genes are the cause of 20% of the adRP in the Spanish popu- lation. 17 Mutation in other genes associated with adRP such as NRL 18 and RP1 (unpublished results) have also been detected in the Spanish population, contributing to 5% of total cases of adRP in Spain. To determine the contribution of mutation in the pre-mRNA splicing-factor genes PRPF3, PRPF8, and PRPF31 in adRP-carrying Spanish families, screening for muta- tion in the complete coding region of the PRPF31 gene was performed. Because mutations in the PRPF3 and PRPF8 gene causing adRP reported so far are clustered in exons 11 and 42 From the 1 Laboratory of Biology and Molecular Genetics, Labora- tory of Service and the 3 Service of Ophthalmology, Consorci Sanitari de Terrassa, Hospital de Terrassa, Terrassa, Spain; the Services of 4 Genet- ics and the 5 Ophthalmology, Fundacio´n Jime´nez Dı ´az, Madrid, Spain; the Services of 6 Genetics and of 7 Neurophysiology, Hospital La Fe, Valencia, Spain; the 8 Service of Molecular Genetics, Hospital de La Santa Creu i Sant Pau, Barcelona, Spain; and the 9 Service of Genetics and Prenatal Diagnostics, Hospital Virgen del Rocı ´o, Sevilla, Spain. 2 Contributed equally to the work and therefore should be consid- ered as equivalent first authors. Supported in part by grants from Fondo de Investigacio´n Sanitaria (FIS 99/0780, 01/0081-01), ONCE, and Fundacio´n ONCE. Submitted for publication August 26, 2002; revised December 3, 2002; accepted December 14, 2002. Disclosure: M. Martı ´nez-Gimeno, None; M.J. Gamundi, None; I. Hernan, None; M. Maseras, None; E. Milla´ , None; C. Ayuso, None; B. Garcı ´a-Sandoval, None; M. Beneyto, None; C. Vilela, None; M. Baiget, None; G. Antin˜olo, None; M. Carballo, None The publication costs of this article were defrayed in part by page charge payment. This article must therefore be marked “advertise- ment” in accordance with 18 U.S.C. §1734 solely to indicate this fact. Corresponding author: Miguel Carballo, Hospital de Terrassa, Ctra. Torrebonica s/n, 08227 Terrassa, Spain; carballolab@terra.es. Investigative Ophthalmology & Visual Science, May 2003, Vol. 44, No. 5 Copyright © Association for Research in Vision and Ophthalmology 2171