Hum Genet (2005) 118: 501–503 DOI 10.1007/s00439-005-0063-3
SHO RT REPOR T
Myrto Papaioannou Æ Christina F. Chakarova De Quincy C. Prescott Æ Naushin Waseem Thorsten Theis Æ Irma Lopez Æ Bhavdip Gill Robert K. Koenekoop Æ Shomi S. Bhattacharya
A new locus (RP31 ) for autosomal dominant retinitis pigmentosa maps to chromosome 9p Received: 14 July 2005 / Accepted: 24 August 2005 / Published online: 28 September 2005 Springer-Verlag 2005
Abstract Retinitis pigmentosa (RP) is a debilitating disease of the retina affecting 1.5 million people worldwide. RP shows remarkable heterogeneity both clinically and genetically, with more than 40 genetic loci implicated, 12 of which account for the autosomal dominant form (adRP) of inheritance. We have recently identified a French Canadian family that presents with early onset adRP. After exclusion of all known loci for adRP, a genome-wide search established firm linkage with a marker from the short arm of chromosome 9 (LOD score of 6.3 at recombination fraction h=0). The linked region is flanked by markers D9S285 and D9S1874, corresponding to a genetic distance of 31 cM, in the region 9p22-p13. Keywords Linkage analysis Æ New adRP locus Æ Chromosome 9p22-p13
Introduction Retinitis pigmentosa (RP) constitutes a clinically and genetically heterogeneous group of severe disorders of the retina, with an estimated prevalence of 1 in 3,000 (Bundey et al. 1984). Rod photoreceptor cells are primarily affected with patients suffering from progressive loss of vision, initially manifesting as night blindness and constriction of the visual fields (rodmediated functions) often progressing to loss of
M. Papaioannou Æ C. F. Chakarova Æ D. Q. C. Prescott N. Waseem Æ T. Theis Æ B. Gill Æ S. S. Bhattacharya (&) Department of Molecular Genetics, Institute of Ophthalmology, UCL, 11-43 Bath Street, EC1V 9EL London, UK E-mail:
[email protected] Tel.: +44-207-6086826 Fax: +44-207-6086863 I. Lopez Æ R. K. Koenekoop McGill Ocular Genetics Laboratory, Montreal Children’s Hospital Research Institute, Montreal, QC, Canada
central vision (largely cone mediated) (Berson 1993). Mode of inheritance can be autosomal dominant (adRP), autosomal recessive (arRP), digenic or Xlinked (Dryja et al. 1995). To date, at least 12 causative genes have been identified for adRP, namely the genes for RHO (3q), RDS (6p), PIM1K (7p), IMPDH1 (7q), RP1 (8q), ROM1 (11q), NRL (14q), CA4 (17q23), FSCN2 (17q25), and the human homologues of yeast pre-mRNA splicing factors PRPF3 (1q), PRPF8 (17p) and PRPF31 (19q) (RetNet, http:// www.sph.uth.tmc.edu/Retnet/). The products of these genes are associated with either photoreceptor structure, cellular function including the phototransduction cascade, or gene expression, e.g. transcription and mRNA-splicing (Himms et al. 2003). As part of our ongoing research to identify new loci for adRP, we have ascertained a three-generation family based in the region of Quebec, Canada. After exclusion of all known loci for adRP, we undertook total genome linkage analysis as described in this paper.
Methods Ascertainment of patients Twenty-six individuals (14 affected, nine unaffected members and three spouses) from the family (Fig. 1) underwent standard ophthalmological evaluations, including dilated retinal examinations, slitlamp biomicroscopy, Snellen visual acuities, Goldmann visual fields and ISCEV standardized electrophysiological assessments at Montreal Children’s Hospital. The study had the approval of the hospital’s ethics committee and conformed to the tenets of the Declaration of Helsinki. Onset of nyctalopia (night blindness) and peripheral visual field loss was highly variable and differed between generations. Informed consent was obtained from all subjects for genetic studies and genomic DNA was isolated from peripheral blood leukocytes by standard techniques.
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Fig. 1 Pedigree structure of the Canadian adRP family that participated in the linkage analysis. DNA was made available from all living individuals. Apparently normal individuals from the bottom generation were not invited to take part in the linkage study because of the difficulty in accurately establishing their clinical status
Genotyping and linkage analysis Fluorescently labelled microsatellite markers from the ABI Prism Linkage Mapping Set v 2.5 (Applied Biosystems, Foster City, CA, USA) were used for genotyping with the polymerase chain reaction (PCR) according to manufacturers‘ instructions. Amplified products were then analysed on an ABI Prism 3100 Genetic Analyser (Applied Biosystems), and the Genotyper v 2.0 software was used to bin alleles. Figure 1 shows the three-generation pedigree of the adRP family. The inheritance pattern is autosomal dominant (male-to-male transmission is seen) with no evidence of incomplete penetrance. The disease gene frequency is estimated at 0.0001. Twenty-two potentially informative meioses were identified. For the purpose of linkage study, subjects were classified as unaffected if they were symptom free, and had a normal ophthalmological examination and a normal ERG at the age of 45 years, because we found one affected female who became symptomatic at around age 40 years. Two-point
LOD scores were calculated using the MLINK option of the Cyrillic pedigree information package version 2.1.3 (Cherwell Scientific Publishing, Reading, UK).
Results and discussion For the 12 known adRP loci, two microsatellite markers flanking the disease gene (marker information available on request) at each locus were used. The markers were D3S3606- RHO-D3S1292, D6S1549- RDS-D6S1650, D7S2252-PIM1K-D7S484, D7S486- IMPDH1-D7S530, D8S532-RP1-D8S285, D11S4191-ROM1-D11S987, D14S972-NRL-D14S275, D17S957-CA4-D17S944, D17S784-FSCN2-D17S928, D1S1653-PRPF3-D1S498, D17S849-PRPF8-D17S831 and D19S572-PRPF31D19S418. LOD scores < 2.0 were observed at recombination fractions ranging between 0.01 and 0.05 for all the markers indicating exclusion of these loci. Consequently, a genome-wide search was undertaken using more than 400 markers from the ABI Mapping Set, with
Table 1 Two-point LOD scores of chromosome 9 markers used in the adRP family DNA marker
Position (cM)a
Recombination fraction (h) 0.00
0.05
0.10
0.20
0.30
¥ 2.40 3.49 6.32 6.32 5.71 ¥
3.13 2.18 3.17 5.80 5.80 5.25 3.07
3.03 1.95 2.84 5.26 5.26 4.75 2.99
2.47 1.46 2.14 4.09 4.09 3.68 2.45
1.67 0.94 1.39 2.76 2.76 2.46 1.71
Zmax
h (Zmax)
3.13 2.40 3.49 6.32 6.32 5.71 3.07
0.05 0.00 0.00 0.00 0.00 0.00 0.05
Telomere D9S285 D9S171 D9S259 D9S161 D9S746b D9S248b D9S1874 Centromere a
30.5 43.8 48 52 53 57.3 62
Marker position on the ABI Prism Linkage Mapping Set v2.5 Marker not present in the ABI Mapping Set v2.5; distance calculated from relative position on ENSEMBL Human Genome Browser (http://www.ensembl.org/) b
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an average spacing of 5–10 cM. The criterion of a LOD score < 2.0 was again used in order to exclude approximately 90% of the genome, before a significant two-point LOD score of 6.3 at h=0 was detected with marker D9S161 from chromosome 9p. Additional markers from the 9p22-p13 region were used to confirm this finding. These markers (order is shown in Table 1) were selected from the GDB Human Genome Database (GDB, http://gdbwww.gdb.org/); PCR primers and conditions are available on request. This new locus has now been given the acronym RP31 by the HUGO Gene Nomenclature Committee (HGNC, http://www.gene.ucl.ac.uk/nomenclature/). Recombination events were observed with markers D9S285 (distally) and D9S1874 (proximally). Markers in-between did not show any recombination, thus indicating the limit of genetic refinement achievable in this family. The results of the two-point linkage analysis between adRP and each of the microsatellite markers from chromosome 9 are shown in Table 1. The highest two-point LOD score (Z) obtained was 6.3 at a recombination fraction (h) of zero with markers D9S161 and D9S746. No other suggestive or significant LOD scores were observed for markers from the other 21 autosomes. On the recent integrated map of ENSEMBL Human Genome Browser (http://www.ensembl.org/), the flanking markers D9S285 and D9S1874 are placed 22 Mb apart, and define the maximal physical size of the critical region of the RP31 locus. It is noted that further refinement of the distal (D9S285-D9S171) and proximal (D9S248-D9S1874) boundaries for the RP31 region may be feasible and will be the subject of future analysis. Within this region, approximately 130 annotated genes are available for study in order to subsequently identify the disease gene. We phenotyped 14 members with RP from this fourgeneration family (as shown in Fig. 1) whose ages ranged from 8–64 years. Onset of symptoms ranged from 10–50 years and differed between the generations. Visual acuities (range 20/20—count fingers) were well maintained in most patients as 13/14 had better than 20/40 and 9/14 had 20/20 acuities at the last visit. Visual field sizes ranged from 10 to 80 and ERG abnormalities were highly variable as well, with early rod defects followed by cone defects. The earliest sign of disease (found in three children in the bottom generation) was an unusual perivascular cuff of RPE atrophy found surrounding the superior and inferior arcades. This progressed to a diffuse pigmentary retinopathy with choroidal sclerosis. One of the patients with completely normal retinal appearance had ERG abnormalities similar to the symptomatic patients. Previously published data indicate the existence of additional adRP loci in the human genome (Inglehearn
et al. 1998). We have now identified a family with autosomal dominant retinitis pigmentosa that clearly demonstrates the presence of a new RP locus (RP31) on the short arm of chromosome 9, and is the only lineage of adRP known to map to this region. It is also worthy of note that there are no other known loci for retinal degenerations mapping on chromosome 9 to date. The identification of genes and molecular defects that underlie retinitis pigmentosa will help improve our understanding of the pathophysiology of the disease and increase our knowledge of the aetiology of retinal dystrophies in general. Such insight should provide a means of presymptomatic diagnosis and preventative therapy to family members at risk for these disorders.
Electronic-database information RetNet, http://www.sph.uth.tmc.edu/Retnet/ (Retinal Information Network for Cloned and/or Mapped Genes Causing Retinal Diseases) ISCEV International Society for Clinical Electrophysiology of Vision, http://www.iscev.org GDB Human Genome Database, http:// gdbwww.gdb.org/ (for marker sequences and conditions) HUGO Gene Nomenclature Committee, http:// www.gene.ucl.ac.uk/nomenclature/ (for genetic notations and symbols) ENSEMBL Human Genome Browser, http:// www.ensembl.org/ (for marker and gene positions) Acknowledgements We thank the family members for their participation in this study. We are grateful to Beverly Scott for excellent technical assistance. This research has been supported by grants from the European Union (HPRN-CT-2000-00098 and MRTNCT-2003-504003), Foundation Fighting Blindness—USA, The Special Trustees of Moorfields Eye Hospital, Foundation Fighting Blindness—Canada and Fonds de la Recherche en Sante´ Quebec (RKK).
References Berson EL (1993) Retinitis pigmentosa. The Friedenwald lecture. Invest Ophthalmol Vis Sci 34:1659–1676 Bundey S, Crews SJ (1984) A study of retinitis pigmentosa in the city of Birmingham-I. Prevalence. J Med Genet 21:417–420 Dryja TP, Li T (1995) Molecular genetics of retinitis pigmentosa. Hum Mol Genet 4:1739–1743 Himms MM, Daiger SP, Inglehearn CF (2003) Retinitis pigmentosa: genes, proteins and prospects. Dev Ophthalmol 37:109– 125 Inglehearn CF, Tarttelin EE, Plant C, Peacock RE, al-Maghtheh M, Vithana E, Bird AC, Bhattacharya SS (1998) A linkage survey of 20 dominant retinitis pigmentosa families: frequencies of the nine known loci and evidence for further heterogeneity. J Med Genet 35:1–5
