(Investigative Ophthalmology and Visual Science. 2005;46:2264-2270.)
© 2005 by The Association for Research in Vision and Ophthalmology, Inc.
DOI: 10.1167/iovs.04-1280
Autosomal Recessive Retinitis Pigmentosa Is Associated with Mutations in RP1 in Three Consanguineous Pakistani Families
S. Amer Riazuddin,1,2,3
Fareeha Zulfiqar,2,3
Qingjiong Zhang,1
Yuri V. Sergeev,1
Zaheeruddin A. Qazi,2
Tayyab Husnain,2
Rafael Caruso,1
Sheikh Riazuddin,2,3
Paul A. Sieving,1 and
J. Fielding Hejtmancik1,3
1From the Ophthalmic Genetics and Visual Function Branch, National Eye Institute, National Institutes of Health, Bethesda, Maryland; and the
2Centre of Excellence in Molecular Biology, University of the Punjab, Lahore, Pakistan.
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Abstract
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PURPOSE. To localize and identify the gene and mutations causing autosomal recessive retinitis pigmentosa in three consanguineous Pakistani families.
METHODS. Blood samples were collected and DNA was extracted. A genome-wide scan was performed by using 382 polymorphic microsatellite markers on genomic DNA from affected and unaffected family members, and lod scores were calculated.
RESULTS. A genome-wide scan of 25 families gave an hlod = 4.53 with D8S260. Retinitis pigmentosa in all three families mapped to a 14.21-cM (21.19-Mb) region on chromosome 8 at q11, flanked by D8S532 and D8S260. This region harbors RP1, which is known to cause autosomal dominant retinitis pigmentosa. Sequencing of the coding exons of RP1 showed mutations in all three families: two single-base deletions, c.4703delA and c.5400delA, resulting in a frame shift, and a 4-bp insertion, c.1606insTGAA, all causing premature termination of the protein. All affected individuals in these families are homozygous for the mutations. Parents and siblings heterozygous for the mutant allele did not show any signs or symptoms of RP.
CONCLUSIONS. These results provide strong evidence that mutations in RP1 can result in recessive as well as dominant retinitis pigmentosa. The findings suggest that truncation of RP1 before the BIF motif or within the terminal portion results in a simple loss of RP1 function, producing a recessive inheritance pattern. In contrast, disruption of RP1 within or immediately after the BIF domain may result in a protein with a deleterious effect and hence a dominant inheritance pattern.
The term retinitis pigmentosa (RP) refers to bone spicule pigmentation in the midperipheral fundus that simulates inflammation and was first used by the German physician Donders in 1857.1 Patients experience night blindness, followed by loss of peripheral visual field and later loss of central vision, often leading to complete blindness. RP primarily affects the rod photoreceptors whereas the function of the cone receptors is compromised as the disease progresses.2 Ocular findings comprise atrophic changes of the photoreceptors and retinal pigment epithelium (RPE) followed by appearance of melanin-containing structures in the retinal vascular layer. Typical fundus appearance includes attenuated arterioles, bone-spicule pigmentation and waxy pallor of the optic disc. Affected individuals often have severely abnormal or nondetectable rod responses in the electroretinograms (ERGs), even in the early stage of the disease.2
RP is the most common inherited retinal dystrophy, affecting approximately 1 in 5000 individuals worldwide.3 4 It may be inherited as an autosomal recessive (ar), autosomal dominant (ad), or an X-linked recessive trait. To date, 39 loci have been implicated in nonsyndromic RP, of which 30 genes are known.5 These include genes encoding components of the phototransduction cascade, proteins involved in retinoid metabolism, cell–cell interaction proteins, photoreceptor structural proteins, transcription factors, intracellular transport proteins, and splicing factors.5 adRP represents 15% to 20% of all cases; arRP comprises 20% to 25%; X-linked recessive RP 10% to 15%, and the remaining 40% to 55% of cases, in which family history is absent, are called simplex (SRP), but many of these may represent arRP.6 7 8 9 A proportion of patients with hereditary retinal degenerations or malfunctions are considered to have "syndromes," because they have associated extraocular disease. These include Bardet-Biedl syndrome, occurring in up to 5% of patients with RP, in which retinal degeneration is coinherited with polydactyly, short stature, truncal obesity, hypogenitalism, mental retardation, and kidney disease and Usher syndrome, characterized by congenital or early onset of sensorineural deafness and various degrees of vestibular dysfunction, which occurs in approximately 18% of patients with RP.10
RP1 is a photoreceptor-specific gene with expression that is regulated by oxygen.11 It localizes to the connecting cilia of photoreceptors and may assist in maintenance of cilial structure or transport down the photoreceptor.12 Mutations in RP1 (NM_006269) are a common cause of autosomal dominant RP (adRP). Like many retinal degeneration genes, the mechanism by which mutations in RP1 lead to photoreceptor cell death is not known. This gene encodes the RP1 protein with a molecular weight of 241 kDa. The RP1 protein is required for correct orientation and higher-order stacking of outer segment disks.13 It is part of the photoreceptor axoneme, as recently demonstrated.14 The amino-terminal residues 28-228 of the RP1 protein share sequence homology with the neuronal microtubule-associated doublecortin and stimulate the formation of microtubules in vitro as well as stabilize cytoplasmic microtubules in heterologous cells.
Herein, we report three consanguineous Pakistani families with multiple individuals affected by arRP. Clinical diagnosis of these families indicates a phenotype typical of early onset RP. Linkage analysis provides evidence of linkage to chromosome 8 at q11, a region harboring RP1. Sequencing of RP1 shows that two families have single-base-pair deletions resulting in frame shift mutations, while the third family has a 4-base-pair insertion, all leading to premature termination of the RP1 protein (GenBank accession number NP_006260; http://www.ncbi.nlm.nih.gov/Genbank; provided in the public domain by the National Center for Biotechnology Information, Bethesda, MD). All affected individuals of these families were homozygous for the mutations, whereas heterozygous carriers were unaffected by RP.
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Materials and Methods
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Clinical Ascertainment
Twenty-five consanguineous Pakistani families with nonsyndromic RP were recruited to participate in a collaborative study between the Center of Excellence in Molecular Biology, Lahore, Pakistan, and the National Eye Institute, Bethesda, Maryland, to identify new disease loci causing inherited visual diseases. Institutional review board (IRB) approval was obtained for this study from both institutions. The participating subjects gave informed consent, consistent with the tenets of the Declaration of Helsinki.
The families described in this study are from the Punjab province of Pakistan. A detailed medical history was obtained by interviewing family members. Fundus photographs of affected individuals showed changes typical of RP, including waxy, pale optic discs; attenuation of retinal arteries; and bone-spicule pigment deposits in the midperiphery of the retina. Blood samples were collected from affected and unaffected family members. DNA was extracted by the nonorganic method described by Grimberg et al.15
Genotype Analysis
A genome-wide scan was performed with 382 highly polymorphic fluorescent markers from a linkage mapping set (Prism MD-10; Applied Biosystems, Inc. [ABI], Foster City, CA) having an average spacing of 10 cM. Multiplex polymerase chain reactions (PCR) were performed as previously described.16 Briefly, each reaction was performed in a 5-µL mixture containing 40 ng genomic DNA, various combinations of 10-µM dye-labeled primer pairs, 0.5-µL 10x PCR Buffer II (GeneAmp; ABI), 0.5 µL 10 mM dNTP mix (GeneAmp; ABI), 2.5 mM MgCl2, and 0.2 U Taq DNA polymerase (AmpliTaq Gold Enzyme; ABI). Amplification was performed in a PCR System (GeneAmp 9700; ABI) Initial denaturation was performed for 5 minutes at 95°C, followed by 10 cycles of 15 seconds at 94°C, 15 seconds at 55°C, and 30 seconds at 72°C and then 20 cycles of 15 seconds at 89°C, 15 seconds at 55°C, and 30 seconds at 72°C. The final extension was performed for 10 minutes at 72°C and followed by a final hold at 4°C. PCR products from each DNA sample were pooled and mixed with a loading cocktail containing size standards (HD-400; ABI) and loading dye. The resultant PCR products were separated on a 5% denaturing urea-polyacrylamide gel (Long Ranger; J. T. Baker, Phillipsburg, NJ) in a DNA sequencer (model 377; ABI) and analyzed with accompanying software (GeneScan ver. 3.1; GenoTyper, ver. 2.1; ABI).
Linkage Analysis
Two-point linkage analyses were performed by using the FASTLINK version of MLINK from the LINKAGE program package.17 18 Maximum lod scores were calculated with ILINK. arRP was analyzed as a fully penetrant trait with an affected allele frequency of 0.001. The marker order and distances between the markers were obtained from the Gènèthon database (http://www.genethon.fr/ provided in the public domain by the French Association against Myopathies, Evry, France) and chromosome 8 sequence maps from the National Center for Biotechnology Information (NCBI, Bethesda, MD; http://www.ncbi.nlm.nih.gov/mapview/). For the initial genome scan, equal allele frequencies were assumed, whereas for fine mapping, allele frequencies were estimated from 125 unrelated and unaffected individuals from the Punjab province of Pakistan. Admixture analysis was performed with the HOMOG1 program19 comparing linkage to D8S260 at 
s of 0.001, 0.01 0.02, 0.03, 0.04, 0.05, 0.06, 0.07, 0.08, 0.09, 0.1, 0.2, 0.3, and 0.4, with absence of linkage.
Mutation Screening
Individual exons of RP1 were amplified as described previously.20 The conditions for PCR reactions and the sequences of primer are available on request. The PCR products were analyzed on 2% agarose gel and were purified by gel extraction (QIAQuick; Qiagen, Valencia, CA) or by vacuum filtration manifold plate (Millipore, Billerica, MA). The PCR primers for each exon were used for bidirectional sequencing with a reaction mix (BigDye Terminator Ready; ABI), according to the manufacturer’s instructions. Sequencing products were resuspended in 10 µL of formamide (ABI) and denatured at 95°C for 5 minutes. Sequencing was performed on an automated sequencer (Prism 3100; ABI). Sequencing results were assembled and analyzed (Seqman program; DNAStar, Inc., Madison, WI).
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Results
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All affected individuals examined in all three families fit the diagnostic criteria for RP, as described herein. Fundus photographs of affected individuals showed changes typical in RP, including a waxy, pale optic disc, attenuation of retinal arteries, and bone-spicule pigment deposits in the midperiphery of the retina (Figs. 1C 1D 1F) . Unaffected parents did not show any funduscopic signs of RP (Figs. 1A 1B 1E) . Neither attenuation of retinal arteries nor presence of bone-spicule pigment deposits in the midperiphery of the retina was observed in unaffected individuals, although some older individuals showed mild atrophic changes in the peripheral retina (Fig. 1A , individual 09 of family 61043, 64 years old). No unaffected individuals in these families reported night blindness. Affected individuals had typical RP changes on ERG, including loss of both the rod and cone responses, whereas the parents showed no changes consistent with RP (Fig. 2) .
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FIGURE 1. Funduscopy photographs of (A) individual 09 of family 61043 (unaffected, 64-year-old obligate carrier), (B) individual 10 of family 61043 (unaffected, 59-year-old obligate carrier), (C) individual 11 of family 61043 (affected, 27 years old), (D) individual 13 of family 61043 (affected, 23 years old), (E) individual 07 of family 61006 (unaffected, 59-year-old obligate carrier) and (F) individual 12 of family 61006 (affected, 19 years old).
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FIGURE 2. ERG recordings for individual 09 of family 61043 (unaffected father): (A) combined rod and cone response, (B) cone response. Individual 10 of family 61043 (unaffected mother): (C) combined response, (D) cone response. Individual 07 of family 61006 (unaffected father): (E) combined response, (F) cone response. Individual 12 of family 61006 (affected): (G) combined response, (H) cone response.
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In the genome-wide scan, significantly positive lod scores were obtained with D8S260, with families 61006 and 61043 showing lod scores of 3.71 and 3.19 with D8S260 at = 0, and family 61040 showing suggestive linkage, with a lod score of 1.91 at = 0.06 and a maximum lod score of 3.55 with D8S285, an adjacent marker. For fine mapping additional markers, D8S532, D8S1110, and D8S1113 from the Gènèthon database were analyzed, and the results are shown in Table 1 and Figure 3 . Two-point linkage analyses gave evidence of linkage to markers on the long arm of chromosome 8 ( = 0, for all markers in all three families) with maximum lod scores of 3.71 with D8S260 and 3.22 with D8S285 for family 61006; 3.19 with D8S260 and 3.21 with D8S285 for family 61043 and 3.27 with D8S1113 and 3.55 with D8S285 for family 61040. When linkage results from the entire data set are subjected to admixture analysis, heterogeneity is suggested with 
= 0.15 and a maximum ln(likelihood) for linkage with heterogeneity of 10.4, corresponding to an hlod = 4.5. These values give a likelihood ratio R = 34,000:1 favoring linkage, and a 
2 = 20.873, corresponding to a P < 0.0001, if an asymptotic 2 distribution is assumed. The conditional probabilities of linkage of families 61006, 61043, and 61040 are 0.9985, 0.9953, and 0.8942, respectively, while conditional probabilities of linkage for the remaining families are <0.004. Taken together, the three linked families localize the arRP gene to a 14.21-cM (21.19-Mb) region on chromosome 8, region q11, flanked by D8S532 and D8S260.
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FIGURE 3. Pedigree of (A) 61043, (B) 61006, and (C) 61040 showing 8q11 haplotypes. Squares: males; circles: females; filled symbols: affected individuals; double lines between individuals: consanguineous matings; diagonal line through a symbol: a deceased family member. Haplotypes of six adjacent 8q11 microsatellite markers are shown with alleles forming the risk haplotype (black), alleles cosegregating with RP but not showing homozygosity (gray), and alleles not cosegregating with RP (white).
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Visual inspection of the haplotypes in this region support the linkage analysis (Fig. 3) , localizing the disease to the q11 region of chromosome 8 flanked by D8S532 and D8S260. Recombination events at D8S505 in affected individual 9 of family 61006, at D8S1771 in affected individual 12 of family 61006 and individual 16 of family 61043, and at D8S532 in individual 17 of family 61040 identified marker D8S532 as the proximal flanking marker, whereas lack of homozygosity at D8S1110 in affected individuals of family 61040 suggests that the locus may lie distal to that marker as well. Similarly, recombination events at D8S270 in affected individuals 13 of family 61006 and 13 of family 61043 and in unaffected individual 18 of family 61043, and at D8S260 in affected individual 11 of family 61040, as well as lack of homozygosity in affected individuals of family 61040, identify D8S260 as the distal flanking marker. Alleles for D8S285 are homozygous for all affected individuals in families 61006, 61040, and 61043.
The linked region on chromosome 8 harbors RP1 (NM_006269) which has been described as a cause of adRP. RP1 contains 4 exons and encodes a 2156 amino acid protein (NP_006260). Sequencing of the coding exons of RP1 discloses two different single-base-pair deletions and a 4-base-pair insertion (Fig. 4) . In affected individuals of family 61006, RP1 shows a single-base-pair deletion in exon 4, c.4703delA, resulting in a frame shift, p.R1519fsX1521. In family 61043 the RP1 gene shows a single-base-pair deletion in exon 4, c.5400delA, resulting in a frame shift, p.N1751fsX1754. In affected individuals of family 61040, RP1 shows a 4-base-pair insertion in exon 4; c.1606insTGAA, leading to a premature termination of the protein, p.E488X. All these mutations are predicted to result in premature termination of the protein and produce truncated products. None of these mutations was detected in 96 DNA samples of unaffected control individuals from the Punjab province of Pakistan.
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FIGURE 4. The forward and reverse sequence chromatograms of (A) unaffected individual 11 of family 61006; (B) individual 12 of family 61006 showing a single-base-pair deletion in exon 4, c.7470delA, resulting in a frame shift, p.R1519fsX1521; (C) individual 12 of family 61043, heterozygous in contrast to (D) individual 16 of family 61043 who is homozygous for a single-base-pair deletion in exon 4, c.8168delA, resulting in a frame shift, p.N1751fsX1754. (E) Unaffected individual 10 of family 61040 and (F) individual 11 of family 61040, showing a 4-base-pair insertion in exon 4, c4377-4378insTGAA, leading to a premature termination of the protein, p.E488X.
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Discussion
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We report three consanguineous Pakistani families with multiple family members affected with arRP, the localization of arRP in these families to a 14.21-cM (21.19-Mb) region on chromosome 8, region q11, flanked by D8S532 and D8S260, and its association with mutations in the RP1 gene. These include two novel single-base deletions, c.4703delA and c.5400delA, both resulting frame shift mutations with premature termination, and a 4-base-pair insertion, c.1606insTGAA, also resulting in premature termination of the RP1 protein.
The RP1 gene consists of four exons and gives rise to a transcript of approximately 7.1 kb encoding a protein of 2156 amino acids.11 Mutations in RP1 previously have been implicated in adRP.11 21 22 Most mutations in RP1 causing adRP are nonsense codons occurring in the last exon of the RP1 gene between codons 658 and 1053. Nonsense mutations in mammalian genes generally lead to unstable mRNAs that are degraded by nonsense-mediated decay.23 However, nonsense-mediated decay does not occur if the mutation is in the last exon. Hence, all three arRP mutations and most reported adRP mutations would be expected to produce stable transcripts translated into truncated proteins. The adRP mutations are located between residues 499 to 1053 and generally would be predicted to truncate the RP1 protein within or immediately after the BIF and cohesin motifs.24
One arRP mutation described herein would be predicted to truncate the protein immediately before the BIF domain, whereas the remaining two mutations truncate the protein within the C-terminal domain. This suggests that disruption of RP1 within or immediately after the BIF domain may result in a protein with a deleterious effect and hence a dominant inheritance pattern. In contrast, truncation of RP1 before the BIF motif or within the terminal portion of the protein appears to result in a simple loss of RP1 function, producing a recessive inheritance pattern. Further characterization of the functional effects of mutations leading to arRP will greatly enhance our understanding of the structure–function relationship of RP1 protein at a molecular level and the physiology of retinal photoreceptors.
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Acknowledgements
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The authors thank the families for their participation in the study; the staff of Layton Rahmatullah Benevolent Trust (LRBT) hospital, especially Muhammad Amer and Amir Niazi for identification of the families and expert clinical evaluation of affected individuals; and Afshan Yasmeen, Muhammad Farooq Sabar, Muhammad Awais, and Assad Riaz for assistance in the work.
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Footnotes
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3 Contributed equally to the work and therefore should be considered equivalent authors. 
Supported in part by the Higher Education Commission and Ministry of Science and Technology Islamabad, Pakistan.
Submitted for publication November 1, 2004; revised January 12, 2005; accepted February 1, 2005.
Disclosure: S.A. Riazuddin, None; F. Zulfiqar, None; Q. Zhang, None; Y.V. Sergeev, None; Z.A. Qazi, None; T. Husnain, None; R. Caruso, None; S. Riazuddin, None; P.A. Sieving, None; J.F. Hejtmancik, None
The publication costs of this article were defrayed in part by page charge payment. This article must therefore be marked "advertisement" in accordance with 18 U.S.C. 
1734 solely to indicate this fact.
Corresponding author: J. Fielding Hejtmancik, OGVFB/NEI/NIH, Building 10, Room 10B10, 10 Center Drive, MSC 1860, Bethesda, MD 20892-1860; f3h{at}helix.nih.gov.
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Abstract
Materials and Methods
