An atypical phenotype of macular and peripapillary retinal atrophy caused by a mutation in the RP2 gene
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《英国眼科学杂志》
1 Moorfields Eye Hospital, London, UK
2 Division of Molecular Genetics, Institute of Ophthalmology, University College London, UK
3 Division of Pathology, Institute of Ophthalmology, University College London, UK
4 Division of Visual Science, Institute of Ophthalmology, University College London, UK
Correspondence to:
Dr A Webster
Division of Molecular Genetics, 11–43 Bath Street, Institute of Ophthalmology, University College London, London EC1V 9EL, UK; Andrew.webster@ucl.ac.uk
Accepted for publication 5 September 2003
ABSTRACT
Aims: To determine the molecular basis and describe the phenotype of an atypical retinal dystrophy in a family presenting with bilateral, progressive central visual loss.
Methods: Family members were examined. Investigations included Goldman perimetry, electrophysiology, and autofluorescence imaging. Candidate gene screening was performed using SSCP and sequence analysis. The proband’s lymphoblastoid cells were examined for protein expression.
Results: Fundal examination of the proband, his mother, and brother revealed peripapillary and macular atrophy. Autosomal dominant retinal dystrophy was suspected, but less severe disease in the mother led to screening for mutations in X linked genes. A 4 bp microdeletion in exon 3 of the RP2 gene, segregating with disease, was identified. No RP2 protein expression was detected.
Conclusion: The distinct phenotype in this family, caused by this frameshifting mutation in RP2, broadens the phenotypic spectrum of X linked retinitis pigmentosa. The absence of RP2 protein suggests that loss of protein function and not novel gain of function could account for the atypical phenotype. A definitive diagnosis of X linked retinitis pigmentosa permits appropriate genetic counselling with important implications for other family members. Clinicians should have a low threshold for screening RP2 in families with retinal dystrophy, including posterior retinal disease, not immediately suggestive of X linked inheritance.
Keywords: RP2; retinitis pigmentosa; phenotype
Abbreviations: ERG, electroretinogram; RP, retinitis pigmentosa; VA, visual acuity; XLRP, X linked retinitis pigmentosa
Retinitis pigmentosa (RP) defines a clinically and genetically diverse group of retinal dystrophies characterised by progressive photoreceptor cell degeneration. X linked retinitis pigmentosa (XLRP) affects about 10–20% of families with RP.1–3 It is a severe form of RP in terms of age of onset and progression, typically presenting with symptoms of night blindness and loss of dark adaptation within the first decade of life and reduction of visual fields in the second decade.1 Disease may progress to complete blindness by the third or fourth decade. Premature cell death occurs initially in the mid-periphery of the retina and primarily affects rod photoreceptors. Clinically this is seen as bone spicules in the periphery caused by pigment epithelial atrophy and pigment migration into the retina. Other signs in the late stages of this degenerative disease include optic atrophy and attenuated blood vessels.1,2
Five loci have been mapped for XLRP, of which RP3 and RP2 account for the majority of disease.4–7 Positional cloning resulted in the identification of the highly complex RPGR (RP guanosine triphosphatase (GTPase) regulator) gene in the RP3 region8–10 and the RP2 gene in the RP2 region.11 Many different mutations have been described in RPGR and RP2 as a cause of XLRP. Mutations in RPGR, including the more recently identified ORF15, are the most common and account for approximately 70–90% of XLRP cases.8–10,12–18RP2, the second most frequent cause of XLRP, accounts for approximately 6–20% of cases in the populations studied.11–13,19–22 The RP2 gene has 5 exons and encodes a 350 amino acid polypeptide (RP2) which is ubiquitously expressed.23,24 RP2 shows homology to cofactor C, which functions in the assembly of native tubulin heterodimers with other cofactors (A–E).25,26 Missense, nonsense, frameshift, insertion, and deletion mutations have been identified in the RP2 gene, many of which occur at residues conserved with cofactor C, suggesting functional homology between these proteins.11,12,19 RP2 has been localised to the plasma membrane of a variety of cultured cells including rod and cone photoreceptors in the human retina.23,27–29 Although the function of RP2 remains unknown, it has been shown to interact with GTP bound ADP ribosylation factor like 3 protein (Arl3).27
We report a family with an atypical phenotype, initially thought to represent an autosomal dominant retinal dystrophy. Screening for mutations in X linked retinal disease genes were undertaken and the proband’s leukocytes were examined for the presence of RP2 protein.
MATERIALS AND METHODS
Clinical examination and investigations
The family pedigree is shown in figure 1. The proband, his mother, and younger brother were examined, and fundus photographs taken (Topcon imagenet digital imaging, Topcon Ltd, Newburg, UK). Investigations including Goldmann perimetry (see fig 3) and electrophysiology under ISCEV conditions were carried out on the proband and on the mother. Autofluorescence images using a confocal laser scanning ophthalmoscope (Zeiss, Oberkochen, Germany) were recorded on the younger brother using published techniques.30
Figure 1 Pedigree structure. Affected individuals are drawn as blackened circles (females) and squares (males).
Figure 3 Goldmann perimetry of proband. Goldmann perimetry shows preserved inferior islands in both eyes to targets III4e and V4e (age 43) VA 6/60 OU.
Mutation identification
Genomic DNA was extracted from peripheral blood leukocytes using the Nucleon II kit (Scotlab Limited, Strathclyde, UK) according to manufacturer’s instructions. The five exons of the RP2 gene were screened for sequence changes using SSCP as described.19 Exon 3 showed a distinct band shift in the proband compared with wild type control. This same amplimer was bidirectionally sequenced for the proband, brother, and mother using the ABI Prism Ready Reaction Dye Terminator cycle sequencing kit (FS kit, Perkin-Elmer Boston, MA, USA) and fragments separated on an ABI 373A automated sequencer. Primer sequences and reaction conditions have been described previously.19
Cell culture
Human lymphoblastoid cell lines corresponding to the proband, (patient III:2), affected members of a different pedigree at Moorfields Eye Hospital with the Arg120stop nonsense mutation in RP228 and randomly selected control males were obtained from the European Collection of Cell Cultures (ECACC, Salisbury, UK). Cells were maintained in suspension culture in RPMI 1640 Glutamax-I (Life Technologies, Paisley, UK) supplemented with 10% fetal bovine serum (Sigma, Poole, UK) with media changes every 2 to 3 days.
Preparation of cell lysates
Lymphoblastoid cells were Dounce homogenised on ice in 20 mM Tris-HCL, pH 7.5, 500 mM NaCl, 12.5 mM KCl, 1 mM EDTA, 1 mM dithiothreitol containing a protease inhibitor cocktail (Sigma). The concentration of protein in the homogenates was determined using the Bio-Rad DC assay (Bio-Rad, Hemel Hempstead, UK), following the manufacturer’s protocols.
Immunoblotting
The cell lysates were prepared for electrophoresis by the addition of sample buffer and heating to 96°C for 5 minutes as described previously.28 Fifty μg or 100 μg of total protein were loaded on a 12% SDS polyacrylamide gel and, after electrophoresis, were electroblotted onto nitrocellulose membrane (Schleicher and Schuell, Dassel, Germany). Non-specific binding sites on the nitrocellulose were blocked by incubation with 5% marvel, 1 x phosphate buffered saline, 0.05% Tween 20 overnight at 4°C. The blots were hybridised with sheep polyclonal antisera S974, and immunoreactive bands were visualised as described previously.23
RESULTS
Clinical evaluation
Case III-2
The 46 year old proband first developed eye problems in childhood, with the onset of myopia. At age 16 his refraction was -12 dioptres in the right eye, and -13 dioptres in the left eye, and he was noted to have peripheral retinal myopic degeneration treated with cryotherapy. He reported reduced night vision from his teenage years, which was initially thought to be caused by his myopia, and his impaired acuity prevented him from being able to drive. He developed progressive loss of central vision over the following years with relative sparing of the peripheral field. His general health was otherwise good and family history revealed an affected younger brother and mother (figs 1 and 2).
Figure 2 Fundus images for affected males and their carrier mother. (A) and (C) fundus photographs of right eye of proband III:2 (aged 45) visual acuity (VA) 1/60. (B) and (D) fundus photographs of left eye of proband III:2 (aged 45) VA 1/60. (E) and (F) fundus photographs of right and left eye of proband’s mother II:1 (aged 68) VA 6/60 OU. (G) and (H) autofluorescence images of the right and left eyes of the proband’s younger brother III:3 showing macular and peripapillary hypo-autofluorescence indicating atrophy of the retinal pigment epithelium (aged 39) VA 6/36 OU.
When examined aged 43, his Snellen visual acuity was 6/60 in both eyes. He was photophobic. There was no evidence of lens opacity or iris transillumination. Fundal examination revealed marked peripapillary chorioretinal atrophy in both eyes extending to the posterior pole and involving the macula (fig 2A–D). Cryotherapy scars were present peripherally. Field testing to confrontation and Goldmann perimetry demonstrated an inferior island of preserved visual field in both eyes (fig 3). Autofluorescence imaging showed no detectable retinal signal.
ISCEV standard Ganzfeld electroretinogram (ERG) responses were almost extinguished, but there was some very low amplitude, grossly delayed flicker response activity. Vision continued to deteriorate progressively, reducing to 1/60 in both eyes a year later.
Case III-3
The proband’s younger brother lives in North America and had symptoms of night blindness since the age of 24. He required a low myopic prescription (-0.25/-1.25x32 RE,-0.25/-1.25x166 LE) and had Snellen visual acuities recorded as 6/12 and 6/24 at age 32. He went on to develop progressive loss of his central and peripheral vision. Fundal examination revealed pallor of both optic discs, sparse bone spicule pigmentation peripherally and the macular was atrophic in both eyes. Autofluorescence imaging confirmed areas of atrophy (decreased autofluorescence) at the maculae and around the disc in both eyes. (fig 2G and H).
Case II-1
The proband’s mother had experienced problems attributable to reduced acuity but, because of a previous stroke and dementia, history and examination were limited. At aged 68, her visual acuity was 6/60 in each eye with a hyperopic correction; right +4.25/-0.50x141 and left +5.00/-1.00x43. Examination of the fundus showed peripapillary atrophy with extension to the posterior pole, similar to that of the proband. (fig 2E and F).
Previous electrophysiology, when the patient was aged 59, had revealed rod ERGs reduced to 20–30% of normal, a maximal (mixed rod-cone) response reduced to 15% of normal, and delayed 30Hz flicker ERG reduced to a comparable level.
Genetic analysis
In view of the family history and fundoscopic appearance of the mother and proband, an initial diagnosis of autosomal dominant retinal dystrophy, primarily affecting central retina, with variable expressivity was suspected. The fact that the mother was less severely affected, and that no male to male transmission had occurred, led to screening for mutations in X linked retinal disease genes. A change was detected by SSCP analysis of the RP2 gene, and following sequence analysis, a frameshifting, 4 bp microdeletion in exon 3 (796–799delCAGA) was found in the coding region of RP2 (fig 4). This segregated with disease in the family, in that this maternal mutation was inherited by both affected sons (fig 4). This microdeletion was not detected in a screen of over 100 control chromosomes. It would be predicted to result in a frameshift at codon 266, causing a premature stop codon, three amino acids further downstream (Gln266fsTer269).
Figure 4 Segregation of a 4 bp deletion in the RP2 gene with disease. Electropherograms depicting a 4 bp deletion in the RP2 gene in affected males III:2 and III:3 (delCAGA). Wild type sequence is shown in the father (II:2), and the carrier mother (II:1) is heterozygote for the mutation. Note: the sequence software attempts to read through the carrier mother’s chromosomes, but inspection of the raw data shown demonstrates the two alleles.
RP2 protein expression
RP2 protein is expressed in lymphoblastoid cells, and cells derived from patients of another family with an Arg120stop mutation, have previously been studied.28 As the phenotype of the family presented here is atypical, we exploited the ubiquitous expression of RP2 and investigated the proband’s (III:2) lymphoblastoid cell RP2 expression to test if the presence of an aberrant protein product could account for the differences in phenotypic expression. A band of the predicted molecular mass of RP2, 39.6 kDa, was detected in the control male cell lines (fig 5). No RP2 protein, truncated or full length, was detected in the two different male patient cell lines (fig 5) by antibody S974, which can recognise the first 15 amino acids of RP2 alone.28 The predicted mass of the truncated RP2 protein products were 14 kDa for the Arg120stop and 31 kDa for the proband (III:2) Gln266fs. An immunoreactive band corresponding to an unknown 68 kDa protein was observed in both patient and control lymphoblastoid cells (fig 5); this band is a not a product of the RP2 gene. However, the crossreactivity provided a useful internal loading and transfer control for the western blot assays.28
Figure 5 RP2 protein expression in patient and control lymphoblast cell lines. The western blot detects a band of the expected size in the control male cell line, but no RP2 protein was detected in the proband (Gln266fs) or a male patient with an Arg120stop mutation. The predicted molecular weight of RP2, 39.6 kDa, is marked on the left. *A 68 kDa band that crossreacts with sera S974 was seen in all cell lines.29 Molecular weight markers are indicated on the right.
DISCUSSION
The clinical phenotype and molecular pathology in a family presenting with bilateral, progressive central visual loss are described. The macular and peripapillary atrophic changes in the family did not immediately suggest a diagnosis of RP. They are unlikely to be solely attributable to myopia as the mother and brother (II:1 and III:3), who showed similar posterior retinal signs, were hyperopic and mildly myopic respectively. Autosomal dominant retinal dystrophy with variable expressivity was initially suspected, but the milder phenotype in the mother and the severe ERG abnormality led to the screening for mutations in X linked retinal disease genes. This led to the detection of the microdeletion mutation in exon 3 of the RP2 gene.
The proband’s mother showed an unusual phenotype for an XLRP carrier. Heterozygotes have been shown to vary from being asymptomatic with a normal fundus appearance (4%), to having characteristic retinal changes (87%).31 Profound visual loss has also been described in later life.32,33 Retinal changes include a tapetal reflex (52%) and peripheral pigmentary pigmentation (43%), especially inferiorly.31 These changes are present in 60% to 100% of carriers, but neither was seen in our case.1,32,33 Electrophysiological testing can be useful in those carriers where the fundus appears normal, and is found to be reduced in amplitude in up to 86% of carriers.31 Peripapillary and macular atrophy however are not typical findings.
Several groups have studied the correlation between clinical characteristics and genotype with variable success. Rosenberg and colleagues34 identified three RP2 mutations in families with XLRP in which they revealed striking interfamilial phenotypic differences and correlated disease severity to genotype. Those with a null mutation (Gln26stop) or a missense mutation (Arg118His) were associated with a severe phenotype. This was characterised by reduced visual acuity and subjectively impaired night vision in the first decade and progression to total blindness by the age of 50. Interestingly, the Gln26stop mutation was also associated with fundal lesions affecting the macular and peripapillary retinal pigment epithelium and choriocapillaris. The Arg118His mutation was associated with a more typical phenotype including early onset of night blindness, peripheral visual field constriction, and bone spicule pigmentation. An in-frame deletion, Ser6, was associated with a much milder phenotype.34 Patients with this mutation had a later onset of disease in the second decade with preserved intact central vision, preserved visual fields for large targets, and residual cone ERG activity. It has been shown that this mutation interferes with the normal targeting of the RP2 protein to the plasma membrane.23
One possible explanation for the distinct phenotype seen in this family is a gain of function or novel function of the truncated protein product. However, a lack of RP2 protein in the lymphoblastoid cells of the proband suggests that the mutation entirely abrogates protein expression and so an aberrant protein cannot be the cause of the atypical phenotype. Important modifying factors, genetic and/or environmental, may play a role in phenotypic expression of disease, and these may have been acting independently in each of the three family members. Alternatively, an important retinal modifier may have been co-inherited with the RP2 mutation on a nearby X chromosomal gene.
This family exemplifies the value of genetic testing in retinal disease that may show an atypical phenotype and highlights the spectrum of phenotypes that can occur due to a mutation in a single gene. Mutations in RP3 also appear to cause a broad clinical phenotype; as in a family with macular dystrophy found to be harbouring a mutation in RPGR gene.35 In our family’s case, the discovery of the genetic mutation not only gave a definitive diagnosis but allowed accurate genetic counselling with important implications for other family members, especially the proband’s daughters who would be carriers for the mutation. Hence, the laboratory findings had a direct effect on clinical management.
We encourage clinicians to have a low threshold for screening RP2 and RPGR in families with retinal dystrophy, even when there is predominantly posterior retinal disease both in male and female affected family members, not immediately suggestive of X linked inheritance.
REFERENCES
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Fishman GA. Retinitis pigmentosa. Visual loss. Arch Ophthalmol 1978;96:1185–8.
Jay M. On the heredity of retinitis pigmentosa. Br J Ophthalmol 1982;66:405–16.
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Teague PW, Aldred MA, Jay M, et al. Heterogeneity analysis in 40 X-linked retinitis pigmentosa families. Am J Hum Genet 1994;55:105–11.
Bhattacharya SS, Wright AF, Clayton JF, et al. Close genetic linkage between X-linked retinitis pigmentosa and a restriction fragment length polymorphism identified by recombinant DNA probe L1.28. Nature 1984;309:253–5.
Thiselton DL, Hampson RM, Nayudu M, 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 1996;6:1093–102.
Meindl A, Dry K, Herrmann K, et al. A gene (RPGR) with homology to the RCC1 guanine nucleotide exchange factor is mutated in X-linked retinitis pigmentosa (RP3). Nat Genet 1996;13:35–42.
Roepman R, van Duijnhoven G, Rosenberg T, et al. Positional cloning of the gene for X-linked retinitis pigmentosa 3: homology with the guanine-nucleotide-exchange factor RCC1. Hum Mol Genet 1996;5:1035–41.
Vervoort R, Lennon A, Bird AC, et al. Mutational hot spot within a new RPGR exon in X-linked retinitis pigmentosa. Nat Genet 2000;25:462–6.
Schwahn U, Lenzner S, Dong J, et al. Positional cloning of the gene for X-linked retinitis pigmentosa 2. Nat Genet 1998;19:327–32.
Sharon D, Bruns GAP, McGee TL, et al. X-linked retinitis pigmentosa: Mutation spectrum of the RPGR and RP2 Genes and correlation with visual function. Invest Ophthalmol Vis Sci 2000;41:2712–21.
Breuer DK, Yashar BM, Filippova E, et al. A comprehensive mutation analysis of RP2 and RPGR in a North American cohort of families with X-linked retinitis pigmentosa. Am J Hum Genet 2002;70:1545–54.
Zito I, Thiselton DL, Gorin MB, et al. Identification of novel RPGR (retinitis pigmentosa GTPase regulator) mutations in a subset of X-linked retinitis pigmentosa families segregating with the RP3 locus. Hum Genet 1999;105:57–62.
Buraczynska M, Wu W, Fujita R, 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 1997;61:1287–92.
Vervoort R, Wright AF. Mutations of RPGR in X-linked retinitis pigmentosa (RP3). Hum Mutat 2002;19:486–500.
Demirci FY, Rigatti BW, Wen G, et al. X-linked cone-rod dystrophy (locus COD1): identification of mutations in RPGR exon ORF15. Am J Hum Genet 2002;70:1049–53.
Yang Z, Peachey NS, Moshfeghi DM, et al. Mutations in the RPGR gene cause X-linked cone dystrophy. Hum Mol Genet 2002;11:605–11.
Hardcastle AJ, Thiselton DL, Van Maldergem L, et al. Mutations in the RP2 gene cause disease in 10% of families with familial X-linked retinitis pigmentosa assessed in this study. Am J Hum Genet 1999;64:1210–15.
Mears AJ, Gieser L, Yan D, 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 1999;64:897–900.
Thiselton DL, Zito I, Plant C, et al. Novel frameshift mutations in the RP2 gene and polymorphic variants. Hum Mutat 2000;15:580.
Miano MG, Testa F, Filippini F, et al. Identification of novel RP2 mutations in a subset of X-linked retinitis pigmentosa families and prediction of new domains. Hum Mutat 2001;18:109–19.
Chapple JP, Hardcastle AJ, Grayson C, et al. 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 2000;9:1919–26.
Schwahn U, Paland N, Techritz S, et al. Mutations in the X-linked RP2 gene cause intracellular misrouting and loss of the protein. Hum Mol Genet 2001;10:1177–83.
Tian G, Lewis SA, Feierbach B, et al. Tubulin subunits exist in an activated conformational state generated and maintained by protein cofactors. J Cell Biol 1997;138:821–32.
Tian G, Bhamidipati A, Cowan NJ, et al. Tubulin folding cofactors as GTPase-activating proteins: GTP hydrolysis and the assembly of the alpha/beta-tubulin heterodimer. J Biol Chem 1999;274:24054–8.
Bartolini F, Bhamidipati A, Thomas S, et al. Functional overlap between retinitis pigmentosa 2 protein and the tubulin-specific chaperone cofactor C. J Biol Chem 2002;277:14629–34.
Chapple JP, Hardcastle AJ, Grayson C, et al. Delineation of the plasma membrane targeting domain of the X-linked retinitis pigmentosa protein, RP2. Invest Ophthalmol Vis Sci 2002;43:2015–20.
Grayson C, Chapple JP, Willison KR, et al. In vitro analysis of aminoglycoside therapy for the Arg120Stop premature stop mutation in RP2 patients. J Med Genet 2002;39:62–7.
Von Ruckmann A, Fitzke FW, Bird AC. Distribution of fundus autofluorescence with a scanning laser ophthalmoscope. Br J Ophthalmol 1995;119:543–62.
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Bird AC, Blach RK. X-linked recessive fundus dystrophies and their carrier states. Trans Ophth Soc UK 1970;90:127–38.
Berson EL, Rosen JB, Simonoff EA. Electroretinographic testing as an aid in detection of carriers of X-chromosome-linked retinitis pigmentosa. Am J Ophthalmol 1979;87:460–8.
Rosenberg T, Schwahn U, Feil S, et al. Genotype-phenotype correlation in X-linked retinitis pigmentosa 2 (RP2). Ophthalmic Genetics 1999;20:161–72.
Ayyagari R, Demirci FY, Liu J, et al. X-linked recessive atrophic macular degeneration from RPGR mutation. Genomics 2002;80:166–71.(S S Dandekar1,2, N D Eben)
2 Division of Molecular Genetics, Institute of Ophthalmology, University College London, UK
3 Division of Pathology, Institute of Ophthalmology, University College London, UK
4 Division of Visual Science, Institute of Ophthalmology, University College London, UK
Correspondence to:
Dr A Webster
Division of Molecular Genetics, 11–43 Bath Street, Institute of Ophthalmology, University College London, London EC1V 9EL, UK; Andrew.webster@ucl.ac.uk
Accepted for publication 5 September 2003
ABSTRACT
Aims: To determine the molecular basis and describe the phenotype of an atypical retinal dystrophy in a family presenting with bilateral, progressive central visual loss.
Methods: Family members were examined. Investigations included Goldman perimetry, electrophysiology, and autofluorescence imaging. Candidate gene screening was performed using SSCP and sequence analysis. The proband’s lymphoblastoid cells were examined for protein expression.
Results: Fundal examination of the proband, his mother, and brother revealed peripapillary and macular atrophy. Autosomal dominant retinal dystrophy was suspected, but less severe disease in the mother led to screening for mutations in X linked genes. A 4 bp microdeletion in exon 3 of the RP2 gene, segregating with disease, was identified. No RP2 protein expression was detected.
Conclusion: The distinct phenotype in this family, caused by this frameshifting mutation in RP2, broadens the phenotypic spectrum of X linked retinitis pigmentosa. The absence of RP2 protein suggests that loss of protein function and not novel gain of function could account for the atypical phenotype. A definitive diagnosis of X linked retinitis pigmentosa permits appropriate genetic counselling with important implications for other family members. Clinicians should have a low threshold for screening RP2 in families with retinal dystrophy, including posterior retinal disease, not immediately suggestive of X linked inheritance.
Keywords: RP2; retinitis pigmentosa; phenotype
Abbreviations: ERG, electroretinogram; RP, retinitis pigmentosa; VA, visual acuity; XLRP, X linked retinitis pigmentosa
Retinitis pigmentosa (RP) defines a clinically and genetically diverse group of retinal dystrophies characterised by progressive photoreceptor cell degeneration. X linked retinitis pigmentosa (XLRP) affects about 10–20% of families with RP.1–3 It is a severe form of RP in terms of age of onset and progression, typically presenting with symptoms of night blindness and loss of dark adaptation within the first decade of life and reduction of visual fields in the second decade.1 Disease may progress to complete blindness by the third or fourth decade. Premature cell death occurs initially in the mid-periphery of the retina and primarily affects rod photoreceptors. Clinically this is seen as bone spicules in the periphery caused by pigment epithelial atrophy and pigment migration into the retina. Other signs in the late stages of this degenerative disease include optic atrophy and attenuated blood vessels.1,2
Five loci have been mapped for XLRP, of which RP3 and RP2 account for the majority of disease.4–7 Positional cloning resulted in the identification of the highly complex RPGR (RP guanosine triphosphatase (GTPase) regulator) gene in the RP3 region8–10 and the RP2 gene in the RP2 region.11 Many different mutations have been described in RPGR and RP2 as a cause of XLRP. Mutations in RPGR, including the more recently identified ORF15, are the most common and account for approximately 70–90% of XLRP cases.8–10,12–18RP2, the second most frequent cause of XLRP, accounts for approximately 6–20% of cases in the populations studied.11–13,19–22 The RP2 gene has 5 exons and encodes a 350 amino acid polypeptide (RP2) which is ubiquitously expressed.23,24 RP2 shows homology to cofactor C, which functions in the assembly of native tubulin heterodimers with other cofactors (A–E).25,26 Missense, nonsense, frameshift, insertion, and deletion mutations have been identified in the RP2 gene, many of which occur at residues conserved with cofactor C, suggesting functional homology between these proteins.11,12,19 RP2 has been localised to the plasma membrane of a variety of cultured cells including rod and cone photoreceptors in the human retina.23,27–29 Although the function of RP2 remains unknown, it has been shown to interact with GTP bound ADP ribosylation factor like 3 protein (Arl3).27
We report a family with an atypical phenotype, initially thought to represent an autosomal dominant retinal dystrophy. Screening for mutations in X linked retinal disease genes were undertaken and the proband’s leukocytes were examined for the presence of RP2 protein.
MATERIALS AND METHODS
Clinical examination and investigations
The family pedigree is shown in figure 1. The proband, his mother, and younger brother were examined, and fundus photographs taken (Topcon imagenet digital imaging, Topcon Ltd, Newburg, UK). Investigations including Goldmann perimetry (see fig 3) and electrophysiology under ISCEV conditions were carried out on the proband and on the mother. Autofluorescence images using a confocal laser scanning ophthalmoscope (Zeiss, Oberkochen, Germany) were recorded on the younger brother using published techniques.30
Figure 1 Pedigree structure. Affected individuals are drawn as blackened circles (females) and squares (males).
Figure 3 Goldmann perimetry of proband. Goldmann perimetry shows preserved inferior islands in both eyes to targets III4e and V4e (age 43) VA 6/60 OU.
Mutation identification
Genomic DNA was extracted from peripheral blood leukocytes using the Nucleon II kit (Scotlab Limited, Strathclyde, UK) according to manufacturer’s instructions. The five exons of the RP2 gene were screened for sequence changes using SSCP as described.19 Exon 3 showed a distinct band shift in the proband compared with wild type control. This same amplimer was bidirectionally sequenced for the proband, brother, and mother using the ABI Prism Ready Reaction Dye Terminator cycle sequencing kit (FS kit, Perkin-Elmer Boston, MA, USA) and fragments separated on an ABI 373A automated sequencer. Primer sequences and reaction conditions have been described previously.19
Cell culture
Human lymphoblastoid cell lines corresponding to the proband, (patient III:2), affected members of a different pedigree at Moorfields Eye Hospital with the Arg120stop nonsense mutation in RP228 and randomly selected control males were obtained from the European Collection of Cell Cultures (ECACC, Salisbury, UK). Cells were maintained in suspension culture in RPMI 1640 Glutamax-I (Life Technologies, Paisley, UK) supplemented with 10% fetal bovine serum (Sigma, Poole, UK) with media changes every 2 to 3 days.
Preparation of cell lysates
Lymphoblastoid cells were Dounce homogenised on ice in 20 mM Tris-HCL, pH 7.5, 500 mM NaCl, 12.5 mM KCl, 1 mM EDTA, 1 mM dithiothreitol containing a protease inhibitor cocktail (Sigma). The concentration of protein in the homogenates was determined using the Bio-Rad DC assay (Bio-Rad, Hemel Hempstead, UK), following the manufacturer’s protocols.
Immunoblotting
The cell lysates were prepared for electrophoresis by the addition of sample buffer and heating to 96°C for 5 minutes as described previously.28 Fifty μg or 100 μg of total protein were loaded on a 12% SDS polyacrylamide gel and, after electrophoresis, were electroblotted onto nitrocellulose membrane (Schleicher and Schuell, Dassel, Germany). Non-specific binding sites on the nitrocellulose were blocked by incubation with 5% marvel, 1 x phosphate buffered saline, 0.05% Tween 20 overnight at 4°C. The blots were hybridised with sheep polyclonal antisera S974, and immunoreactive bands were visualised as described previously.23
RESULTS
Clinical evaluation
Case III-2
The 46 year old proband first developed eye problems in childhood, with the onset of myopia. At age 16 his refraction was -12 dioptres in the right eye, and -13 dioptres in the left eye, and he was noted to have peripheral retinal myopic degeneration treated with cryotherapy. He reported reduced night vision from his teenage years, which was initially thought to be caused by his myopia, and his impaired acuity prevented him from being able to drive. He developed progressive loss of central vision over the following years with relative sparing of the peripheral field. His general health was otherwise good and family history revealed an affected younger brother and mother (figs 1 and 2).
Figure 2 Fundus images for affected males and their carrier mother. (A) and (C) fundus photographs of right eye of proband III:2 (aged 45) visual acuity (VA) 1/60. (B) and (D) fundus photographs of left eye of proband III:2 (aged 45) VA 1/60. (E) and (F) fundus photographs of right and left eye of proband’s mother II:1 (aged 68) VA 6/60 OU. (G) and (H) autofluorescence images of the right and left eyes of the proband’s younger brother III:3 showing macular and peripapillary hypo-autofluorescence indicating atrophy of the retinal pigment epithelium (aged 39) VA 6/36 OU.
When examined aged 43, his Snellen visual acuity was 6/60 in both eyes. He was photophobic. There was no evidence of lens opacity or iris transillumination. Fundal examination revealed marked peripapillary chorioretinal atrophy in both eyes extending to the posterior pole and involving the macula (fig 2A–D). Cryotherapy scars were present peripherally. Field testing to confrontation and Goldmann perimetry demonstrated an inferior island of preserved visual field in both eyes (fig 3). Autofluorescence imaging showed no detectable retinal signal.
ISCEV standard Ganzfeld electroretinogram (ERG) responses were almost extinguished, but there was some very low amplitude, grossly delayed flicker response activity. Vision continued to deteriorate progressively, reducing to 1/60 in both eyes a year later.
Case III-3
The proband’s younger brother lives in North America and had symptoms of night blindness since the age of 24. He required a low myopic prescription (-0.25/-1.25x32 RE,-0.25/-1.25x166 LE) and had Snellen visual acuities recorded as 6/12 and 6/24 at age 32. He went on to develop progressive loss of his central and peripheral vision. Fundal examination revealed pallor of both optic discs, sparse bone spicule pigmentation peripherally and the macular was atrophic in both eyes. Autofluorescence imaging confirmed areas of atrophy (decreased autofluorescence) at the maculae and around the disc in both eyes. (fig 2G and H).
Case II-1
The proband’s mother had experienced problems attributable to reduced acuity but, because of a previous stroke and dementia, history and examination were limited. At aged 68, her visual acuity was 6/60 in each eye with a hyperopic correction; right +4.25/-0.50x141 and left +5.00/-1.00x43. Examination of the fundus showed peripapillary atrophy with extension to the posterior pole, similar to that of the proband. (fig 2E and F).
Previous electrophysiology, when the patient was aged 59, had revealed rod ERGs reduced to 20–30% of normal, a maximal (mixed rod-cone) response reduced to 15% of normal, and delayed 30Hz flicker ERG reduced to a comparable level.
Genetic analysis
In view of the family history and fundoscopic appearance of the mother and proband, an initial diagnosis of autosomal dominant retinal dystrophy, primarily affecting central retina, with variable expressivity was suspected. The fact that the mother was less severely affected, and that no male to male transmission had occurred, led to screening for mutations in X linked retinal disease genes. A change was detected by SSCP analysis of the RP2 gene, and following sequence analysis, a frameshifting, 4 bp microdeletion in exon 3 (796–799delCAGA) was found in the coding region of RP2 (fig 4). This segregated with disease in the family, in that this maternal mutation was inherited by both affected sons (fig 4). This microdeletion was not detected in a screen of over 100 control chromosomes. It would be predicted to result in a frameshift at codon 266, causing a premature stop codon, three amino acids further downstream (Gln266fsTer269).
Figure 4 Segregation of a 4 bp deletion in the RP2 gene with disease. Electropherograms depicting a 4 bp deletion in the RP2 gene in affected males III:2 and III:3 (delCAGA). Wild type sequence is shown in the father (II:2), and the carrier mother (II:1) is heterozygote for the mutation. Note: the sequence software attempts to read through the carrier mother’s chromosomes, but inspection of the raw data shown demonstrates the two alleles.
RP2 protein expression
RP2 protein is expressed in lymphoblastoid cells, and cells derived from patients of another family with an Arg120stop mutation, have previously been studied.28 As the phenotype of the family presented here is atypical, we exploited the ubiquitous expression of RP2 and investigated the proband’s (III:2) lymphoblastoid cell RP2 expression to test if the presence of an aberrant protein product could account for the differences in phenotypic expression. A band of the predicted molecular mass of RP2, 39.6 kDa, was detected in the control male cell lines (fig 5). No RP2 protein, truncated or full length, was detected in the two different male patient cell lines (fig 5) by antibody S974, which can recognise the first 15 amino acids of RP2 alone.28 The predicted mass of the truncated RP2 protein products were 14 kDa for the Arg120stop and 31 kDa for the proband (III:2) Gln266fs. An immunoreactive band corresponding to an unknown 68 kDa protein was observed in both patient and control lymphoblastoid cells (fig 5); this band is a not a product of the RP2 gene. However, the crossreactivity provided a useful internal loading and transfer control for the western blot assays.28
Figure 5 RP2 protein expression in patient and control lymphoblast cell lines. The western blot detects a band of the expected size in the control male cell line, but no RP2 protein was detected in the proband (Gln266fs) or a male patient with an Arg120stop mutation. The predicted molecular weight of RP2, 39.6 kDa, is marked on the left. *A 68 kDa band that crossreacts with sera S974 was seen in all cell lines.29 Molecular weight markers are indicated on the right.
DISCUSSION
The clinical phenotype and molecular pathology in a family presenting with bilateral, progressive central visual loss are described. The macular and peripapillary atrophic changes in the family did not immediately suggest a diagnosis of RP. They are unlikely to be solely attributable to myopia as the mother and brother (II:1 and III:3), who showed similar posterior retinal signs, were hyperopic and mildly myopic respectively. Autosomal dominant retinal dystrophy with variable expressivity was initially suspected, but the milder phenotype in the mother and the severe ERG abnormality led to the screening for mutations in X linked retinal disease genes. This led to the detection of the microdeletion mutation in exon 3 of the RP2 gene.
The proband’s mother showed an unusual phenotype for an XLRP carrier. Heterozygotes have been shown to vary from being asymptomatic with a normal fundus appearance (4%), to having characteristic retinal changes (87%).31 Profound visual loss has also been described in later life.32,33 Retinal changes include a tapetal reflex (52%) and peripheral pigmentary pigmentation (43%), especially inferiorly.31 These changes are present in 60% to 100% of carriers, but neither was seen in our case.1,32,33 Electrophysiological testing can be useful in those carriers where the fundus appears normal, and is found to be reduced in amplitude in up to 86% of carriers.31 Peripapillary and macular atrophy however are not typical findings.
Several groups have studied the correlation between clinical characteristics and genotype with variable success. Rosenberg and colleagues34 identified three RP2 mutations in families with XLRP in which they revealed striking interfamilial phenotypic differences and correlated disease severity to genotype. Those with a null mutation (Gln26stop) or a missense mutation (Arg118His) were associated with a severe phenotype. This was characterised by reduced visual acuity and subjectively impaired night vision in the first decade and progression to total blindness by the age of 50. Interestingly, the Gln26stop mutation was also associated with fundal lesions affecting the macular and peripapillary retinal pigment epithelium and choriocapillaris. The Arg118His mutation was associated with a more typical phenotype including early onset of night blindness, peripheral visual field constriction, and bone spicule pigmentation. An in-frame deletion, Ser6, was associated with a much milder phenotype.34 Patients with this mutation had a later onset of disease in the second decade with preserved intact central vision, preserved visual fields for large targets, and residual cone ERG activity. It has been shown that this mutation interferes with the normal targeting of the RP2 protein to the plasma membrane.23
One possible explanation for the distinct phenotype seen in this family is a gain of function or novel function of the truncated protein product. However, a lack of RP2 protein in the lymphoblastoid cells of the proband suggests that the mutation entirely abrogates protein expression and so an aberrant protein cannot be the cause of the atypical phenotype. Important modifying factors, genetic and/or environmental, may play a role in phenotypic expression of disease, and these may have been acting independently in each of the three family members. Alternatively, an important retinal modifier may have been co-inherited with the RP2 mutation on a nearby X chromosomal gene.
This family exemplifies the value of genetic testing in retinal disease that may show an atypical phenotype and highlights the spectrum of phenotypes that can occur due to a mutation in a single gene. Mutations in RP3 also appear to cause a broad clinical phenotype; as in a family with macular dystrophy found to be harbouring a mutation in RPGR gene.35 In our family’s case, the discovery of the genetic mutation not only gave a definitive diagnosis but allowed accurate genetic counselling with important implications for other family members, especially the proband’s daughters who would be carriers for the mutation. Hence, the laboratory findings had a direct effect on clinical management.
We encourage clinicians to have a low threshold for screening RP2 and RPGR in families with retinal dystrophy, even when there is predominantly posterior retinal disease both in male and female affected family members, not immediately suggestive of X linked inheritance.
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