|Molecular Vision 2004;
Received 1 July 2003 | Accepted 26 April 2004 | Published 15 June 2004
Confirmation of linkage and refinement of the RP28 locus for autosomal recessive retinitis pigmentosa on chromosome 2p14-p15 in an Indian family
Arun Kumar,1 Jyoti
Shetty,2 Bharath Kumar,2 Susan
1Department of Molecular Reproduction, Development and Genetics, Indian Institute of Science, Bangalore, India; 2Bangalore West Lions Eye Hospital and Cornea Grafting Center, Bangalore, India; 3Department of Pediatrics, University of Virginia Health Science Center, Charlottesville, VA
Correspondence to: Dr. Arun Kumar, Department of Molecular Reproduction, Development and Genetics, Indian Institute of Science, Sir C. V. Raman Avenue, Bangalore 560 012, India; Phone: 80-2 346 5523; FAX: 80-2 360 0999; email: firstname.lastname@example.org
Purpose: To report the linkage analysis of retinitis pigmentosa (RP) in an Indian family.
Methods: Individuals were examined for symptoms of retinitis pigmentosa and their blood samples were withdrawn for genetic analysis. The disorder was tested for linkage to known 14 adRP and 22 arRP loci using microsatellite markers.
Results: Seventeen individuals including seven affecteds participated in the study. All affected individuals had typical RP. The age of onset of the disease ranged from 8-18 years. The disorder in this family segregated either as an autosomal recessive trait with pseudodominance or an autosomal dominant trait. Linkage to an autosomal recessive locus RP28 on chromosome 2p14-p15 was positive with a maximum two-point lod score of 3.96 at theta=0 for D2S380. All affected individuals were homozygous for alleles at D2S2320, D2S2397, D2S380, and D2S136. Recombination events placed the minimum critical region (MCR) for the RP28 gene in a 1.06 cM region between D2S2225 and D2S296.
Conclusions: The present data confirmed linkage of arRP to the RP28 locus in a second Indian family. The RP28 locus was previously mapped to a 16 cM region between D2S1337 and D2S286 in a single Indian family. Haplotype analysis in this family has further narrowed the MCR for the RP28 locus to a 1.06 cM region between D2S2225 and D2S296. Of 15 genes reported in the MCR, 14 genes (KIAA0903, OTX1, MDH1, UGP2, VPS54, PELI1, HSPC159, FLJ20080, TRIP-Br2, SLC1A4, KIAA0582, RAB1A, ACTR2, and SPRED2) are either expressed in the eye or retina. Further study needs to be done to test which of these genes is mutated in patients with RP linked to the RP28 locus.
Retinitis pigmentosa (RP) is a clinically and genetically heterogeneous group of retinal degenerations characterized by abnormalities of the photoreceptors or retinal pigment epithelium (RPE) leading to progressive loss of vision. Patients typically present with night blindness, followed by constriction of the peripheral visual fields. In the advance stages of the disease, the retina develops intraretinal and preretinal clumps of black melanin pigments appearing like bone spicules, attenuated retinal vessels, loss of RPE and paleness of the optic nerves . The age of onset of the initial clinical symptom varies from childhood to middle age . The incidence of RP is estimated to be 1 in 4,000 people in western populations . The actual incidence of RP is not known in the Indian population. RP exhibits a variety of modes of inheritance. It can be inherited as an autosomal dominant, autosomal recessive, X-linked recessive, X-linked dominant or, in rare cases, as a digenic trait. Autosomal dominant RP (adRP) and autosomal recessive RP (arRP) each accounts for approximately 20% of RP cases, while X-linked RP accounts for 10% of RP cases. Approximately 50% of all RP patients are simplex cases, which include sporadic RP and arRP [4-6]. To date, a total of 22 loci for arRP and 14 loci for adRP have been reported (RetNet). In a majority of the cases, the linkage has been reported in either a single family or only a few families. To date, genes for a total of 17 arRP and 12 adRP loci have been isolated (RetNet). Gu et al.  have reported linkage of a consanguineous Indian family with arRP to the RP28 locus on chromosome 2p11.2-p15. We report here confirmation of this linkage in a second consanguineous Indian family. Gu et al.  have previously mapped this locus to a 16 cM region between D2S1337 and D2S286. Haplotype analysis in the present family has further narrowed this region to a 1.06 cM region between markers D2S2225 and D2S296.
We have ascertained 17 members of a four-generation consanguineous family with RP from the state of Karnataka, India (Figure 1). There are two consanguinous matings in the pedigree. Individuals III-4 and III-5 are first cousins. Individuals II-1 and II-2 are known to be related; however, the exact relationship is unknown. A total of seven individuals were diagnosed with RP. The age of onset of nightblindness ranged from 8 to 18 years. Funduscopy revealed the typical features of RP including attenuated blood vessels, bone spicule pigmentation, waxy pallor of the disc and optic atrophy. Informed consent was obtained for research following the guidelines of the Indian Council of Medical Research, New Delhi.
Genotyping and linkage analyses
For genotyping, total genomic DNA sample was extracted from peripheral blood samples using a WizardTM Genomic DNA Purification kit (Promega, Madison, WI). To determine if this family was linked to one of the known adRP or arRP loci, 2 to 4 microsatellite markers were selected from the candidate region of each of these loci and used to genotype the family. Microsatellite markers were synthesized commercially according to data from the Genome Database. Marker order and genetic distances were obtained from the Marshfield Medical Research Foundation web site. Amplification of microsatellite markers was performed as reported in Kumar et al. . Radiolabelled PCR products were separated on a 6% denaturing polyacrylamide gels and subjected to either Phosphor Image analysis (Fuji Inc., Kanagawa, Japan) or exposed to X-ray films.
Two-point lod scores were calculated using the MLINK program from the LINKAGE package version 5.1 , under the assumption of autosomal recessive mode of inheritance and a disease gene frequency of 0.0001. Population specific allele frequencies are not available for the Indian population; therefore equal marker allele frequencies were assumed for linkage analysis for most of the markers. Allele frequencies were calculated for markers showing homozygosity using 30 control individuals. However, varying the allele frequencies did not substantially change the linkage results.
All affected individuals from the family IIS-2 had typical RP including bone spicules and attenuated vessels. The age of onset of the disease (night blindness) ranged from 8-18 years (Table 1). Individual III-4 had completely lost his vision by the age of 40 years and had advanced stage RP involving the macula with bone spicule pigmentation all around the fundus. His perception of light was positive in both eyes. Individuals IV-1 and IV-7 had typical RP with early consecutive optic atrophy with atrophic macular degeneration. Individual IV-2 had typical RP with macular degeneration. Individual IV-3 had typical RP with perivascular bone spicule pigmentation, sheathed vessels with a tapetal reflex at the macula. Individual IV-4 had typical RP with a normal macula. Individual IV-5 had typical RP with early consecutive optic atrophy and normal macula. In addition to RP, individuals IV-2, IV-3, IV-4, IV-5, and IV-7 were also myopic.
Visual inspection of the pedigree suggested that RP is either segregating as an autosomal dominant trait or an autosomal recessive trait with pseudodominance in the family IIS-2 (Figure 1). Linkage analysis was carried out in this family using primers from candidate regions of each of the 14 adRP and 22 arRP loci. Linkage was excluded for all except arRP locus RP28 on chromosome 2p11.2-p15 (data not shown). Linkage to RP28 locus on chromosome 2p11.2-p15 was positive with a maximum two-point lod score of 3.96 at theta=0 for marker D2S380 (Table 2). All the affected individuals were homozygous for alleles for the markers D2S2320, D2S2397, D2S380, and D2S136 (Figure 1). None of the normal individuals were found to be homozygous for alleles at these marker loci. A disease haplotype 1-1-1-1 for these markers was segregated with the disease in this family. The telomeric boundary of the homozygous region was set by heterozygosity for the marker D2S2225 in affected individuals III-4, IV-1, IV-3, and IV-4. Similarly, the centromeric boundary was delineated by heterozygosity for the marker D2S296 in individuals III-4, IV-1, IV-3, and IV-4. The homozygous region of 1.06 cM between D2S2225 and D2S296 at 2p14-p15 most probably represents the chromosomal segment homozygous by descent (Figure 2).
We have reported the linkage of a consanguineous family from India segregating with autosomal recessive RP to the RP28 locus on chromosome 2p14-p15. This confirms the mapping of the RP28 locus in a second Indian family. Gu et al.  have previously reported that the minimum critical region (MCR) for the RP28 locus spans 16 cM between D2S1337 and D2S286 in a single Indian family. Haplotype analysis in the family IIS-2 has further narrowed this region to a 1.06 cM region defined by markers D2S2225 and D2S296 (Figure 2). To the best of our knowledge, this family is from a different ethnic background and is not related to the family reported by Gu et al. .
Of the 22 known loci for arRP, genes for 17 loci have been isolated and characterized so far (Table 3; RetNet). However, genes for five loci (RP28, RP26, RP29, RP25, and RP22) have not been identified yet (Table 3). A total of 15 known genes have been reported between the markers D2S2225 and D2S296 (Table 4; UCSC Genome Bioinformatics Site) which span the RP28 locus. While none of these genes show any homology with known arRP genes, 14 of these 15 genes (KIAA0903, OTX1, MDH1, UGP2, VPS54, PELI1, HSPC159, FLJ20080, TRIP-Br2, SLC1A4, KIAA0582, RAB1A, ACTR2, and SPRED2) are either expressed in eye and/or retina (Table 4). It is possible that mutations in one of these genes result in the disease phenotype in the families linked to the RP28 locus.
In summary, we have confirmed linkage of arRP to the RP28 locus on chromosome 2p14-p15 in a second consanguineous Indian family. Haplotype analysis in the family has further narrowed the MCR of the RP28 locus to a 1.06 cM region between the markers D2S2225 and D2S296. These data will be useful in the isolation and characterization of the gene, mutations in which result in arRP in families linked to the RP28 locus. This will eventually lead to a proper patient management and genetic diagnosis in future.
Financial support from DST and UGC, New Delhi is gratefully acknowledged. We thank the family members for participating in this study. We also thank Anju K. Jaishwal for drawing the figures.
1. Pagon RA. Syndromic retinal dystrophy. In: Rinaldi E, editor. Retinitis pigmentosa: Present knowledge and outlook. Napoli: Liviana Medicina; 1993. p.151-166.
2. Gu S, Kumaramanickavel G, Srikumari CR, Denton MJ, Gal A. Autosomal recessive retinitis pigmentosa locus RP28 maps between D2S1337 and D2S286 on chromosome 2p11-p15 in an Indian family. J Med Genet 1999; 36:705-7.
3. Bird AC. X-linked retinitis pigmentosa. Br J Ophthalmol 1975; 59:177-99.
4. Boughman JA, Conneally PM, Nance WE. Population genetic studies of retinitis pigmentosa. Am J Hum Genet 1980; 32:223-35.
5. Jay M. On the heredity of retinitis pigmentosa. Br J Ophthalmol 1982; 66:405-16.
6. Haim M. Retinitis pigmentosa: problems associated with genetic classification. Clin Genet 1993; 44:62-70.
7. Kumar A, Becker LA, Depinet TW, Haren JM, Kurtz CL, Robin NH, Cassidy SB, Wolff DJ, Schwartz S. Molecular characterization and delineation of subtle deletions in de novo "balanced" chromosomal rearrangements. Hum Genet 1998; 103:173-8.
8. Lathrop GM, Lalouel JM, Julier C, Ott J. Strategies for multilocus linkage analysis in humans. Proc Natl Acad Sci U S A 1984; 81:3443-6.