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Three novel mutations causing a truncated protein within the RP2 gene in Italian families with X-linked retinitis pigmentosa
Mutation Research Genomics 432 (2001) 79–82
Three novel mutations causing a truncated protein within the RP2 gene in Italian families with X-linked retinitis pigmentosa Alessandro De Luca a,c , Isabella Torrente a,c , Massimo Mangino a,c , Rita Danesi b , Bruno Dallapiccola c,d , Giuseppe Novelli a,∗ a
Dipartimento di Biopatologia e Diagnostica per Immagini, Università di Roma Tor Vergata, via di Tor Vergata 135, 00133 Rome, Italy b Ospedale “Infermi”, Rimini, Italy c Istituto CSS-Mendel, Rome, Italy d Dipartimento di Medicina Sperimentale e Patologia, Università di Roma La Sapienza, Rome, Italy Received 31 August 2000; accepted 15 November 2000
Abstract X-linked retinitis pigmentosa (XLRP) results from mutations in a number of loci, including RP2 at Xp11.3, and RP3 at Xp21.1. RP2 and RP3 genes have been identified by positional cloning. RP2 mutations are found in about 10% of XLRP patients. We performed a mutational screening of RP2 gene in patients belonging to seven unrelated families in linkage with the RP2 locus. SSCP analysis detected three conformation variants, within exon 2 and 3. Direct sequencing of exon 2, disclosed a G → A transition at nucleotide 449 (W150X), and a G → T transversion in position 547 (E183X). Sequence analysis of exon 3 variant revealed an insertion (853/854insG), leading to a frameshift. In this patient, we detected an additional sequence alteration (A → G at nucleotide 848, E283G). Each mutation was co-segregating with the disease in the affected family members available for the study. These mutations are expected to introduce a stop codon within the RP2 coding sequence probably resulting in a truncated or unstable protein. © 2001 Elsevier Science B.V. All rights reserved. Keywords: Retinitis pigmentosa; RP2; Mutations
1. Introduction X-linked retinitis pigmentosa (XLRP; OMIM #312600) is a severe form of retinal degeneration which typically shows loss of peripheral visual field and night blindness in the affected males [1]. The disease is progressive and causes complete loss of central vision and a total functional blindness usually in the fourth decade of life. Genetic-mapping studies have identified at least four loci on X chromosome (RP2, RP3, RP15 and RP24) [2–6]. Linkage analysis in the ∗ Corresponding author. Tel.: +39-6-72596078; fax: +39-6-20427313. E-mail address: [email protected] (G. Novelli).
XLRP families have suggested that RP3 and RP2 are the most common types, accounting for 70–75%, and 15–20% of the families, respectively [3,7]. The RP3 gene was positionally cloned in 1996 [8,9] and found to encode for a putative guanine nucleotide exchange factor mutated in about 20% of XLRP patients, a figure significantly lower compared to that estimated by genetic-mapping analysis [7]. However, recently Vervoort et al. [10] described a new 30 terminal exon of the gene in which they have identified the majority of RP3 mutations [10]. The RP2 gene encodes for a protein of 350 amino acids with homology to cofactor-C (30.4%) [11], involved in -tubulin folding which is expressed ubiquitously in human tissues and has a predominantly plasma membrane localization in cul-
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tured cells [12,13]. The function of the gene and his specific role in the retina are still unknown. Present results suggest that RP2 gene is mutated in about 10% of the XLRP patients [14,15]. RP2 mutations include nonsense, missense, frameshifts, insertion and deletion changes, which in the majority of cases results in a severe truncated form of the protein [11,14–18]. We performed a mutational analysis of the RP2 gene in a panel of seven XLRP families linked to the RP2 locus, using a PCR-based single stranded conformation polymorphism (SSCP) analysis. We identified three novel mutations, two nonsense within exon 2 and one frameshift in exon 3.
2. Materials and methods 2.1. Patients The RP families were selected on the basis of X-linked inheritance with absence of male to male transmission. Diagnosis of RP in the affected males was based on ocular examination, electroretinography (ERG) and visual fields [19,20]. Families were first analyzed by linkage analysis using a set of X chromosome markers: DXS1110, OTC, DXS556 as RP3 markers [21], and DXS8083, DXS1055 and DXS6616 as RP2 markers [5]. Linkage analysis was performed using the MLINK program [22]. Two-point linkage analysis was performed between the RP2 locus and each RP2 marker using the MLINK option. Genetic distances between markers were obtained from previous studies [5,21].
firmed in the patient, the corresponding PCR product was sequenced after gel purification with the Thermo Sequenase cycle sequencing Kit (Amersham), using infrared dye IRD41 50 labelled primers (LI-COR). The nucleotide sequence was established using a LI-COR 4000L automated DNA sequencer.
3. Results and discussion Two-point linkage analysis showed a positive lod score (Z score ranging from 0.90 to 1.89 at θ = 0.00) to RP2 markers (DXS8083, DXS1055, DXS6616). RP3 locus was excluded on the basis of recombination events occurring between RP2 markers and RP3 markers. SSCP analysis performed on at least one affected of each family detected three conformation variants, two within exon 2, respectively, in SSCP fragments 2B (Fig. 1a) and 2C (Fig. 1b), and one within exon 3 (Fig. 1c). Direct sequencing of exon 2 revealed in patients 4–13, a G → A transition at nucleotide 449, converting a tryptophan in a stop codon at position 150 (W150X) (Fig. 2a), and in patients 10–7, a G → T transversion at position 547, resulting in a substitution of the wild type glutamic acid for a stop codon at amino acid position 183 (E183X) (Fig. 2b). Sequence analysis of exon 3 in patients 9–10 revealed an insertion (853/854insG) (Fig. 2c), causing a frameshift coding for two novel amino acids, followed by premature termination. In this patients, we detected an
2.2. Mutation screening Genomic DNA was isolated from peripheral lymphocytes and amplified by PCR, using eight primer pairs according to Schwahn et al. [11]. As suggested, exon 2 was amplified into four different PCR fragments, named 2A, 2B, 2C, and 2D, respectively. SSCP analysis was performed at 600 V at 10–15◦ C on a GenePhor Electrophoresis Unit (Pharmacia Biotech), using GeneGel Excel 12.5%/24 followed by silver staining. When bandshifts were identified in one of the patients, 30 control DNAs were amplified together with the patient and subjected to electrophoresis under the same conditions. When the SSCP variant was con-
Fig. 1. SSCP analysis showing bandshifts in: (a) exon fragment 2B in patients 4–13 and his heterozygote mother 4–3; (b) exon fragment 2C in patients 10–7 and his heterozygote mother10–1; (c) exon 3 in patients 9–10 and his heterozygote mother 9–4.
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Fig. 2. (a) Sequence analysis illustrating the G449A mutation in patients 4–13; (b) sequence analysis illustrating the G547T mutation in patients 10–7; (c) sequence analysis showing the variations A848G and 853/854insG in patients 9–10.
additional sequence alteration (A → G at nucleotide 848, leading to the E283G change) (Fig. 2c), segregating in all affected patients of the family. Whether this A → G alteration represents a disease-causing substitution was not determined. Since mutation W150X destroys a RsaI restriction site, and 853/854insG creates a new BanI restriction site, restriction analysis was performed in all members of the two families, which included four and three affected individuals, respectively. Mutations were found to segregate with the disease. The segregation of E183X in the third family, comprising two affected subjects, was confirmed by SSCP and direct sequencing (data not shown). In conclusion, we describe three new mutations in a subset of Italian XLRP families in linkage with the RP2 locus. This results confirm a high rate of new mutations in this disease, similarly to the RPGR mutations in RP3 families [23]. Personal and published results [11,14–18] indicate that the spectrum of RP2 mutations has a trend toward severe protein truncation. Mutations in the RP2 gene are rare (approximately 10–20%) [11,14,15]. This contrasts with the high detection rate found in this study (40%). A possible explanation of this discrepancy could be attributable to absence of a preliminary linkage study or to the inclusion in the mutation cohort screening of isolated male patients. However, the failure in detecting RP2 mutations in four of the selected families suggests that mutations could be situated in promoter or intronic regions which were not analyzed, or they could have been missed by the SSCP screening method used.
Acknowledgements This work was supported in part by Italian Ministry of Health. References [1] A.C. Bird, X-linked retinitis pigmentosa, Br. J. Ophthalmol. 59 (1975) 177–199. [2] S.S. Bhattacharya, A.F. Wright, J.F. Clayton, W.H. Price, C.I. Phillips, C.M. McKeown, M. Jay, et al., Close genetic linkage between X-linked retinitis pigmentosa and a restriction fragment length polymorphism identified by recombinant DNA probe L1.28, Nature 309 (1984) 253–255. [3] J. Ott, S. Bhattacharya, J.D. Chen, M.J. Denton, J. Donald, C. Dubay, G.J. Farrar, et al., Localizing multiple X chromosome-linked retinitis pigmentosa loci using multilocus homogeneity tests, Proc. Natl. Acad. Sci. U.S.A. 87 (1990) 701–704. [4] R.E. McGuire, L.S. Sullivan, S.H. Blanton, M.W. Church, J.R. Heckenlively, S.P. Daiger, X-linked dominant cone-rod degeneration: linkage mapping of a new locus for retinitis pigmentosa (RP15) to Xp22.13–p22.11, Am. J. Hum. Genet. 57 (1995) 87–94. [5] D.L. Thiselton, R.M. Hampson, M. Nayudu, L. Van Maldergem, M.L. Wolf, B.K. Saha, S.S. Bhattacharya, et al., Mapping the RP2 locus for X-linked retinitis pigmentosa on proximal Xp: a genetically defined 5-cM critical region and exclusion of candidate genes by physical mapping, Genome Res. 6 (1996) 1093–1102. [6] L. Gieser, R. Fujita, H.H.H. Goring, J. Ott, D.R. Hoffman, A.V. Cideciyan, D.G. Birch, et al., A novel locus (RP24) for X-linked retinitis pigmentosa maps to Xq26–27, Am. J. Hum. Genet. 63 (1998) 1439–1447. [7] P.W. Teague, M.A. Aldred, M. Jay, M. Dempster, C. Harrison, A.D. Carothers, L.J. Hardwick, et al., Heterogeneity analysis in 40 X-linked retinitis pigmentosa families, Am. J. Hum. Genet. 55 (1994) 105–111.
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[8] A. Meindl, K. Dry, K. Herrmann, F. Manson, A. Ciccodicola, A. Edgar, M.R.S. Carvalho, et al., A gene (RPGR) with homology to the RCC1 guanine nucleotide exchange factor is mutated in X-linked retinitis pigmentosa (RP3), Nat. Genet. 13 (1996) 35–42. [9] R. Roepman, G. van Duijnhoven, T. Rosenberg, A.J.L.G. Pinckers, L.M. Bleeker-Wagemakers, A.A.B. Bergen, J. Post, et al., Positional cloning of the gene for X-linked retinitis pigmentosa 3: homology with the guanine-nucleotide-exchange factor RCC1, Hum. Mol. Genet. 5 (1996) 1035–1041. [10] R. Vervoort, A. Lennon, A.C. Bird, B. Tulloch, R. Axton, M.G. Miano, A. Meindl, et al., Mutational hot spot within a new RPGR exon in X-linked retinitis pigmentosa, Nat. Genet. 25 (2000) 462–466. [11] U. Schwahn, S. Lenzner, J. Dong, S. Feil, B. Hinzmann, G. van Duijnhoven, R. Kirschner, et al., Positional cloning of the gene for X-linked retinitis pigmentosa 2, Nat. Genet. 19 (1998) 327–332. [12] G. Tian, Y. Huang, H. Rommelaere, J. Vandekerckove, C. Ampe, N.J. Cowan, Pathways leading to correctly folded -tubulin, Cell 86 (1996) 287–296. [13] J.P. Chapple, A.J. Hardcastle, C. Grayson, L.A. Spakman, K.R. Willison, M.E. Cheetham, Mutations in the N-terminus of the X-linked retinitis pigmentosa protein RP2 interfere with the normal targeting of the protein to the plasma membrane, Hum. Mol. Genet. 9 (2000) 1919–1926. [14] A.J. Mears, L. Gieser, D. Yan, C. Chen, S. Fahrner, S. Hiriyanna, R. Fujita, et al., Protein-truncation mutations in the RP2 gene in a North American cohort of families with X-linked retinitis pigmentosa, Am. J. Hum. Genet. 64 (1999) 897–900. [15] A.J. Hardcastle, D.L. Thiselton, L.V. Maldergem, B.K. Saha, M. Jay, C. Plant, R. Taylor, et al., Mutations in the RP2
[16]
[17]
[18]
[19]
[20]
[21]
[22]
[23]
gene cause disease in 10% of families with familial X-linked retinitis pigmentosa assessed in this study, Am. J. Hum. Genet. 64 (1999) 1210–1215. T. Rosenberg, U. Schwahn, S. Feil, W. Berger, Genotype–phenotype correlation in X-linked retinitis pigmentosa 2 (RP2), Ophthalmic Genet. 20 (1999) 161– 172. D.L. Thiselton, I. Zito, C. Plant, M. Jay, S.V. Hodgson, A.C. Bird, S.S. Bhattacharya, A.J. Hardcastle, Novel frameshift mutations in the RP2 gene and polymorphic variants, Hum. Mutat. 15 (2000) 580. Y. Wada, M. Nakazawa, T. Abe, M. Tamai, A new Leu253Arg mutation in the RP2 gene in a Japanese family with X-linked retinitis pigmentosa, Invest. Ophthalmol. Vis. Sci. 41 (2000) 290–293. S.O. Andreasson, B. Ehinger, Electroretinographic diagnosis in families with X-linked retinitis pigmentosa, Acta Ophthalmol. (Copenh) 68 (1990) 139–144. S.G. Jacobson, K. Yagasaki, W.J. Feuer, A.J. Roman, Interocular asymmetry of visual function in heterozygotes of X-linked retinitis pigmentosa, Exp. Eye Res. 48 (1989) 679– 691. A.F. Roux, J. Rommens, C. McDowell, L. Anson-Cartwright, S. Bell, K. Shappert, G.A. Fishman, et al., Identification of a gene from Xp21 with similarity to the tctex-1 gene of the murine t complex, Hum. Mol. Genet. 3 (1994) 257–263. G.M. Lathrop, J.M. Lalouel, Easy calculatins of lod scores and genetic risks on small computers, Am. J. Hum. Genet. 36 (1984) 460–465. M. Buraczynska, W. Wu, R. Fujita, K. Buraczynska, E. Phelps, S. Andreasson, J. Bennet, et al., Spectrum of mutations in the RPGR gene that are identified in 20% of families with X-linked retinitis pigmentosa, Am. J. Hum. Genet. 61 (1997) 1287–1292.
Three novel mutations causing a truncated protein within the RP2 gene in Italian families with X-linked retinitis pigmentosa Alessandro De Luca a,c , Isabella Torrente a,c , Massimo Mangino a,c , Rita Danesi b , Bruno Dallapiccola c,d , Giuseppe Novelli a,∗ a
Dipartimento di Biopatologia e Diagnostica per Immagini, Università di Roma Tor Vergata, via di Tor Vergata 135, 00133 Rome, Italy b Ospedale “Infermi”, Rimini, Italy c Istituto CSS-Mendel, Rome, Italy d Dipartimento di Medicina Sperimentale e Patologia, Università di Roma La Sapienza, Rome, Italy Received 31 August 2000; accepted 15 November 2000
Abstract X-linked retinitis pigmentosa (XLRP) results from mutations in a number of loci, including RP2 at Xp11.3, and RP3 at Xp21.1. RP2 and RP3 genes have been identified by positional cloning. RP2 mutations are found in about 10% of XLRP patients. We performed a mutational screening of RP2 gene in patients belonging to seven unrelated families in linkage with the RP2 locus. SSCP analysis detected three conformation variants, within exon 2 and 3. Direct sequencing of exon 2, disclosed a G → A transition at nucleotide 449 (W150X), and a G → T transversion in position 547 (E183X). Sequence analysis of exon 3 variant revealed an insertion (853/854insG), leading to a frameshift. In this patient, we detected an additional sequence alteration (A → G at nucleotide 848, E283G). Each mutation was co-segregating with the disease in the affected family members available for the study. These mutations are expected to introduce a stop codon within the RP2 coding sequence probably resulting in a truncated or unstable protein. © 2001 Elsevier Science B.V. All rights reserved. Keywords: Retinitis pigmentosa; RP2; Mutations
1. Introduction X-linked retinitis pigmentosa (XLRP; OMIM #312600) is a severe form of retinal degeneration which typically shows loss of peripheral visual field and night blindness in the affected males [1]. The disease is progressive and causes complete loss of central vision and a total functional blindness usually in the fourth decade of life. Genetic-mapping studies have identified at least four loci on X chromosome (RP2, RP3, RP15 and RP24) [2–6]. Linkage analysis in the ∗ Corresponding author. Tel.: +39-6-72596078; fax: +39-6-20427313. E-mail address: [email protected] (G. Novelli).
XLRP families have suggested that RP3 and RP2 are the most common types, accounting for 70–75%, and 15–20% of the families, respectively [3,7]. The RP3 gene was positionally cloned in 1996 [8,9] and found to encode for a putative guanine nucleotide exchange factor mutated in about 20% of XLRP patients, a figure significantly lower compared to that estimated by genetic-mapping analysis [7]. However, recently Vervoort et al. [10] described a new 30 terminal exon of the gene in which they have identified the majority of RP3 mutations [10]. The RP2 gene encodes for a protein of 350 amino acids with homology to cofactor-C (30.4%) [11], involved in -tubulin folding which is expressed ubiquitously in human tissues and has a predominantly plasma membrane localization in cul-
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tured cells [12,13]. The function of the gene and his specific role in the retina are still unknown. Present results suggest that RP2 gene is mutated in about 10% of the XLRP patients [14,15]. RP2 mutations include nonsense, missense, frameshifts, insertion and deletion changes, which in the majority of cases results in a severe truncated form of the protein [11,14–18]. We performed a mutational analysis of the RP2 gene in a panel of seven XLRP families linked to the RP2 locus, using a PCR-based single stranded conformation polymorphism (SSCP) analysis. We identified three novel mutations, two nonsense within exon 2 and one frameshift in exon 3.
2. Materials and methods 2.1. Patients The RP families were selected on the basis of X-linked inheritance with absence of male to male transmission. Diagnosis of RP in the affected males was based on ocular examination, electroretinography (ERG) and visual fields [19,20]. Families were first analyzed by linkage analysis using a set of X chromosome markers: DXS1110, OTC, DXS556 as RP3 markers [21], and DXS8083, DXS1055 and DXS6616 as RP2 markers [5]. Linkage analysis was performed using the MLINK program [22]. Two-point linkage analysis was performed between the RP2 locus and each RP2 marker using the MLINK option. Genetic distances between markers were obtained from previous studies [5,21].
firmed in the patient, the corresponding PCR product was sequenced after gel purification with the Thermo Sequenase cycle sequencing Kit (Amersham), using infrared dye IRD41 50 labelled primers (LI-COR). The nucleotide sequence was established using a LI-COR 4000L automated DNA sequencer.
3. Results and discussion Two-point linkage analysis showed a positive lod score (Z score ranging from 0.90 to 1.89 at θ = 0.00) to RP2 markers (DXS8083, DXS1055, DXS6616). RP3 locus was excluded on the basis of recombination events occurring between RP2 markers and RP3 markers. SSCP analysis performed on at least one affected of each family detected three conformation variants, two within exon 2, respectively, in SSCP fragments 2B (Fig. 1a) and 2C (Fig. 1b), and one within exon 3 (Fig. 1c). Direct sequencing of exon 2 revealed in patients 4–13, a G → A transition at nucleotide 449, converting a tryptophan in a stop codon at position 150 (W150X) (Fig. 2a), and in patients 10–7, a G → T transversion at position 547, resulting in a substitution of the wild type glutamic acid for a stop codon at amino acid position 183 (E183X) (Fig. 2b). Sequence analysis of exon 3 in patients 9–10 revealed an insertion (853/854insG) (Fig. 2c), causing a frameshift coding for two novel amino acids, followed by premature termination. In this patients, we detected an
2.2. Mutation screening Genomic DNA was isolated from peripheral lymphocytes and amplified by PCR, using eight primer pairs according to Schwahn et al. [11]. As suggested, exon 2 was amplified into four different PCR fragments, named 2A, 2B, 2C, and 2D, respectively. SSCP analysis was performed at 600 V at 10–15◦ C on a GenePhor Electrophoresis Unit (Pharmacia Biotech), using GeneGel Excel 12.5%/24 followed by silver staining. When bandshifts were identified in one of the patients, 30 control DNAs were amplified together with the patient and subjected to electrophoresis under the same conditions. When the SSCP variant was con-
Fig. 1. SSCP analysis showing bandshifts in: (a) exon fragment 2B in patients 4–13 and his heterozygote mother 4–3; (b) exon fragment 2C in patients 10–7 and his heterozygote mother10–1; (c) exon 3 in patients 9–10 and his heterozygote mother 9–4.
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Fig. 2. (a) Sequence analysis illustrating the G449A mutation in patients 4–13; (b) sequence analysis illustrating the G547T mutation in patients 10–7; (c) sequence analysis showing the variations A848G and 853/854insG in patients 9–10.
additional sequence alteration (A → G at nucleotide 848, leading to the E283G change) (Fig. 2c), segregating in all affected patients of the family. Whether this A → G alteration represents a disease-causing substitution was not determined. Since mutation W150X destroys a RsaI restriction site, and 853/854insG creates a new BanI restriction site, restriction analysis was performed in all members of the two families, which included four and three affected individuals, respectively. Mutations were found to segregate with the disease. The segregation of E183X in the third family, comprising two affected subjects, was confirmed by SSCP and direct sequencing (data not shown). In conclusion, we describe three new mutations in a subset of Italian XLRP families in linkage with the RP2 locus. This results confirm a high rate of new mutations in this disease, similarly to the RPGR mutations in RP3 families [23]. Personal and published results [11,14–18] indicate that the spectrum of RP2 mutations has a trend toward severe protein truncation. Mutations in the RP2 gene are rare (approximately 10–20%) [11,14,15]. This contrasts with the high detection rate found in this study (40%). A possible explanation of this discrepancy could be attributable to absence of a preliminary linkage study or to the inclusion in the mutation cohort screening of isolated male patients. However, the failure in detecting RP2 mutations in four of the selected families suggests that mutations could be situated in promoter or intronic regions which were not analyzed, or they could have been missed by the SSCP screening method used.
Acknowledgements This work was supported in part by Italian Ministry of Health. References [1] A.C. Bird, X-linked retinitis pigmentosa, Br. J. Ophthalmol. 59 (1975) 177–199. [2] S.S. Bhattacharya, A.F. Wright, J.F. Clayton, W.H. Price, C.I. Phillips, C.M. McKeown, M. Jay, et al., Close genetic linkage between X-linked retinitis pigmentosa and a restriction fragment length polymorphism identified by recombinant DNA probe L1.28, Nature 309 (1984) 253–255. [3] J. Ott, S. Bhattacharya, J.D. Chen, M.J. Denton, J. Donald, C. Dubay, G.J. Farrar, et al., Localizing multiple X chromosome-linked retinitis pigmentosa loci using multilocus homogeneity tests, Proc. Natl. Acad. Sci. U.S.A. 87 (1990) 701–704. [4] R.E. McGuire, L.S. Sullivan, S.H. Blanton, M.W. Church, J.R. Heckenlively, S.P. Daiger, X-linked dominant cone-rod degeneration: linkage mapping of a new locus for retinitis pigmentosa (RP15) to Xp22.13–p22.11, Am. J. Hum. Genet. 57 (1995) 87–94. [5] D.L. Thiselton, R.M. Hampson, M. Nayudu, L. Van Maldergem, M.L. Wolf, B.K. Saha, S.S. Bhattacharya, et al., Mapping the RP2 locus for X-linked retinitis pigmentosa on proximal Xp: a genetically defined 5-cM critical region and exclusion of candidate genes by physical mapping, Genome Res. 6 (1996) 1093–1102. [6] L. Gieser, R. Fujita, H.H.H. Goring, J. Ott, D.R. Hoffman, A.V. Cideciyan, D.G. Birch, et al., A novel locus (RP24) for X-linked retinitis pigmentosa maps to Xq26–27, Am. J. Hum. Genet. 63 (1998) 1439–1447. [7] P.W. Teague, M.A. Aldred, M. Jay, M. Dempster, C. Harrison, A.D. Carothers, L.J. Hardwick, et al., Heterogeneity analysis in 40 X-linked retinitis pigmentosa families, Am. J. Hum. Genet. 55 (1994) 105–111.
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[8] A. Meindl, K. Dry, K. Herrmann, F. Manson, A. Ciccodicola, A. Edgar, M.R.S. Carvalho, et al., A gene (RPGR) with homology to the RCC1 guanine nucleotide exchange factor is mutated in X-linked retinitis pigmentosa (RP3), Nat. Genet. 13 (1996) 35–42. [9] R. Roepman, G. van Duijnhoven, T. Rosenberg, A.J.L.G. Pinckers, L.M. Bleeker-Wagemakers, A.A.B. Bergen, J. Post, et al., Positional cloning of the gene for X-linked retinitis pigmentosa 3: homology with the guanine-nucleotide-exchange factor RCC1, Hum. Mol. Genet. 5 (1996) 1035–1041. [10] R. Vervoort, A. Lennon, A.C. Bird, B. Tulloch, R. Axton, M.G. Miano, A. Meindl, et al., Mutational hot spot within a new RPGR exon in X-linked retinitis pigmentosa, Nat. Genet. 25 (2000) 462–466. [11] U. Schwahn, S. Lenzner, J. Dong, S. Feil, B. Hinzmann, G. van Duijnhoven, R. Kirschner, et al., Positional cloning of the gene for X-linked retinitis pigmentosa 2, Nat. Genet. 19 (1998) 327–332. [12] G. Tian, Y. Huang, H. Rommelaere, J. Vandekerckove, C. Ampe, N.J. Cowan, Pathways leading to correctly folded -tubulin, Cell 86 (1996) 287–296. [13] J.P. Chapple, A.J. Hardcastle, C. Grayson, L.A. Spakman, K.R. Willison, M.E. Cheetham, Mutations in the N-terminus of the X-linked retinitis pigmentosa protein RP2 interfere with the normal targeting of the protein to the plasma membrane, Hum. Mol. Genet. 9 (2000) 1919–1926. [14] A.J. Mears, L. Gieser, D. Yan, C. Chen, S. Fahrner, S. Hiriyanna, R. Fujita, et al., Protein-truncation mutations in the RP2 gene in a North American cohort of families with X-linked retinitis pigmentosa, Am. J. Hum. Genet. 64 (1999) 897–900. [15] A.J. Hardcastle, D.L. Thiselton, L.V. Maldergem, B.K. Saha, M. Jay, C. Plant, R. Taylor, et al., Mutations in the RP2
[16]
[17]
[18]
[19]
[20]
[21]
[22]
[23]
gene cause disease in 10% of families with familial X-linked retinitis pigmentosa assessed in this study, Am. J. Hum. Genet. 64 (1999) 1210–1215. T. Rosenberg, U. Schwahn, S. Feil, W. Berger, Genotype–phenotype correlation in X-linked retinitis pigmentosa 2 (RP2), Ophthalmic Genet. 20 (1999) 161– 172. D.L. Thiselton, I. Zito, C. Plant, M. Jay, S.V. Hodgson, A.C. Bird, S.S. Bhattacharya, A.J. Hardcastle, Novel frameshift mutations in the RP2 gene and polymorphic variants, Hum. Mutat. 15 (2000) 580. Y. Wada, M. Nakazawa, T. Abe, M. Tamai, A new Leu253Arg mutation in the RP2 gene in a Japanese family with X-linked retinitis pigmentosa, Invest. Ophthalmol. Vis. Sci. 41 (2000) 290–293. S.O. Andreasson, B. Ehinger, Electroretinographic diagnosis in families with X-linked retinitis pigmentosa, Acta Ophthalmol. (Copenh) 68 (1990) 139–144. S.G. Jacobson, K. Yagasaki, W.J. Feuer, A.J. Roman, Interocular asymmetry of visual function in heterozygotes of X-linked retinitis pigmentosa, Exp. Eye Res. 48 (1989) 679– 691. A.F. Roux, J. Rommens, C. McDowell, L. Anson-Cartwright, S. Bell, K. Shappert, G.A. Fishman, et al., Identification of a gene from Xp21 with similarity to the tctex-1 gene of the murine t complex, Hum. Mol. Genet. 3 (1994) 257–263. G.M. Lathrop, J.M. Lalouel, Easy calculatins of lod scores and genetic risks on small computers, Am. J. Hum. Genet. 36 (1984) 460–465. M. Buraczynska, W. Wu, R. Fujita, K. Buraczynska, E. Phelps, S. Andreasson, J. Bennet, et al., Spectrum of mutations in the RPGR gene that are identified in 20% of families with X-linked retinitis pigmentosa, Am. J. Hum. Genet. 61 (1997) 1287–1292.