Ricardo Fujita and Anand Swaroop
Departments of Ophthalmology and Human Genetics
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W.K. Kellogg Eye Center, University of Michigan
Ann Arbor, MI 48105
Fax: (313) 647 0228
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After a long and frustrating battle that lasted several years, researchers finally have identified the first retinitis pigmentosa (RP) gene by the positional cloning strategy. In the May 1996 issue of Nature Genetics, a team led by Drs. Thomas Meitinger and Alan Wright described the cloning of the RPGR (Retinitis Pigmentosa GTPase Regulator) gene from the region of X-linked RP locus RP3 (18). The uphill struggle involved in the RP3 gene cloning was complicated by the genetic heterogeneity in XLRP and by the chromosomal deletion in a patient BB that misled the search. Identification of RPGR should have far-reaching implications on the study of X-linked retinal dystrophies and for understanding basic biological pathways in the retinal cells.
RP is a group of debilitating disorders that are characterized by nightblindness, progressive loss of peripheral vision, and pigmentary alterations in the retina with the appearance of "bone-spicules" (13). More than 30 distinct genes that can cause RP and/or related retinal dystrophies have been assigned to human chromosomes (S.P. Daiger, personal communication). The estimates of the prevalence of various forms (collectively about 1 in 3500) is believed to vary in different populations. X-linked RP (XLRP) has been recognized as a clinical entity in the medical literature since as early as the 1930s (16). It is perhaps the most devastating form of RP because of the severity and an early onset of the disease, and may account for as much as 25% of RP families (26). Most males with XLRP show early onset of visual symptoms with night blindness before the age of 20 (9). A wide variation in the clinical profile of heterozygous carrier females, which many times display a "tapetal-like reflex," probably is due to the pattern of X-inactivation (5, 11).
Table 1 shows the milestones in the genetic and molecular studies of XLRP. In the early 80s, the XLRP locus RP2 was the second inherited disease gene to be localized to a chromosomal region by linkage to a polymorphic DNA marker (3). RP2 still remains unidentified among tens, if not hundreds, of genes in a 5 cM region at Xp11.3 (A. Hardcastle and S.S. Bhattacharya, 1996 ARVO). Soon after, linkage analysis and cytogenetic techniques confirmed at least two genetic loci for XLRP on the short arm of the X chromosome (Xp). Based on recombinations identified by haplotype analysis and map positions of deletions in XLRP patients, RP3 was mapped to a relatively small region at Xp21.1 between the flanking markers OTC and DXS1110 (19, 26). Genetic analysis has indicated the presence of two other XLRP loci (RP6 and RP15) in distal Xp. RP6 was postulated by statistical analysis of 62 families, although no individual XLRP pedigree has been linked to this locus (23). RP15 was localized in a phenotypically distinct family with early-onset pigmentary retinopathy and cone-rod degeneration (15).
Table 1: Milestones in Genetic and Molecular Studies of XLRP 1984 RP2 Mapped by Linkage to L1.28 at Xp11.3 (3) 1985 Genetic Heterogeneity in XLRP. Suggestion of Another Locus at Xp21. Large Chromosomal Deletion in a Patient BB (DMD, CGD, McLeod Phenotype and RP) (10, 22) 1988 RP3 location confirmed at Xp21 (20, 25) 1990 The RP6 locus suggested by statistical analysis, distal to DMD at Xp21 (23) 1995 Another locus RP15 mapped at Xp22. Is it same as RP6? (15) 1996 First XLRP gene, RPGR, identified (18)
Different XLRP subtypes cannot be identified clinically. Attempts to define RP2 or RP3 using the onset of myopia, night blindness, or tapetal reflex remain inconclusive. Genetic analysis provides the only means for distinguishing between the two major XLRP subtypes. The clustering of loci at Xp also makes it difficult to genetically discriminate among the RP loci in XLRP families, especially between the two prevalent subtypes RP3 and RP2. It is often necessary to obtain large number of samples >from an extended family in search of a key recombination. Nevertheless, when the genetic distinction is possible, it appears that RP3 is the predominant form (>70%) of XLRP in different Caucasian populations (23, 24, unpublished data from our laboratory).
Positional cloning is the strategy used for isolating a gene responsible for an inherited disorder without any clue of the biochemical function of the gene product (6). This labor-intensive approach requires the genetic analysis of affected families with polymorphic markers to determine the chromosomal location of the disease locus. This study, however, is greatly facilitated by gross chromosomal rearrangements, which can provide a focus for molecular analysis by pointing to a specific genomic region. Although the task to isolate an X-linked recessive disease gene is simplified by its mode of inheritance (i.e., transmission of the disease from phenotypically normal female carriers to male children; no male to male transmission), there are still more than two hundred million base pairs and several thousand genes to sort through. An interstitial deletion within the Xp21 region of a male patient (BB) with Duchenne muscular dystrophy (DMD), chronic granulomatous disease (CGD), McLeod phenotype, and retinitis pigmentosa (10), has been instrumental in cloning the genes for cytochrome b5 (CYBB) responsible for CGD, dystrophin for DMD and XK for the McLeod phenotype. The XLRP locus RP3 was believed to be in the proximal portion of the BB deletion because of its coincidence with the critical region delineated by linkage analysis. The two genetic markers DXS1110 and OTC that flank the RP3 locus encompass a genomic region of <1 Mb (21); the proximal breakpoint of deletion in BB is 40 kb centomeric to DXS1110. We and others, therefore, have been searching for the transcribed sequences in this region surrounding the proximal breakpoint for several years. However, causative mutations could not be identified in any gene.
To further refine the location of RP3 and to ascertain the disease locus in XLRP patients, we have been pursuing genetic analysis of more than 30 pedigrees (mostly from the U.S.) with several new polymorphic markers at both sides of the BB breakpoint. It was a surprise when a recombination event in one of our XLRP families indicated the location of the RP3 mutation outside the BB deletion, closer to the OTC locus (12). The finding of the RPGR gene 400 kb centromeric to the BB proximal breakpoint confirms the linkage data from our laboratory.
The RP3 cloning triumph was accomplished by the arduous work of the groups led by Thomas Meitinger from the University of Munich and Alan Wright from the MRC at Edinburgh. They reevaluated the "establishment" of the contiguous deletions associated with syndromes in Xp21.1 and found evidence that contradicted the presence of RP3 in the BB deletion. A patient, SB, had XLRP as well as CGD and the McLeod phenotype (7), but the deletion in this patient extended 400 kb centromeric to the proximal breakpoint of the BB deletion. The deletion in another patient, NF, who had DMD and CGD but not RP, spanned the complete proximal part of the BB deletion and extended a few kb more towards OTC (4). The key to RPGR cloning was a patient, MO, who had RP and was reported as having a 75 kb deletion overlapping the proximal portion of SB (17). At this stage, the two groups described the identification of a candidate gene ETX-1/SRPX, which was partly deleted in MO DNA (8, 17); however, no causative mutation in this gene was revealed in the DNA of other RP3 patients.
It is of interest to note that the search for more genes in the MO deletion region by cDNA selection methods and exon amplification (commonly used for positional cloning) was unsuccessful. In collaboration with Michele D'Urso in Italy , Meitinger and Wright then initiated the daunting task of sequencing 85 kb of DNA from the two cosmid clones spanning the MO deletion. GRAIL analysis to search potential exons yielded seven sequences; two of these identified expressed sequence tags (ESTs) derived from random clones in cDNA libraries. One of the ESTs was from a collection of 3' partial cDNAs sequences. This EST provided the rope to catch the new gene RPGR. Longer versions of the cDNA were then obtained by RT-PCR using primers >from the 3' EST and potential exons from the 5' region. A single cDNA sequence of 2784 bp was then obtained. This RPGR cDNA sequence encoded a putative protein of 815 amino acids. Northern and RNA-PCR analysis revealed a ubiquitous pattern of expression; surprisingly however, RPGR was shown to be expressed at very low levels in the retina and retinal pigment epithelium. This is particularly bizarre since the clinical manifestation of the XLRP disease is limited to the retina, and results in photoreceptor cell death.
The putative RP3 gene was called RPGR (RP GTPase Regulator) because in the amino-terminal region it contains six tandem repeats similar to RCC1, a regulator of the ubiquitous small nuclear GTPase- Ran (Ras-related nuclear protein). The RCC1 protein has a series of tandemly arranged repeats that are the catalytic site for the exchange of guanine nucleotides. The presence of this motif in the RPGR protein argues for its involvement in regulating small GTP-binding proteins of the ras family. The high turnover of retinal photoreceptors and RPE membranes suggests that RPGR may participate in this process. Studies on subcellular localization of RPGR will provide valuable insights into its physiological function and its relationship with other proteins implicated in RP.
Analysis of XLRP patients revealed that RPGR is the elusive RP3 gene; so far, two deletions and five point mutations (two nonsense and three missense) that are not observed in normal controls have been detected in XLRP families from U.K. and Germany. However, 67 out of the 74 XLRP patient samples did not reveal any abnormality in the coding sequence of RPGR. Since RP3 causes more than 70% of XLRP, a proportion larger than 7 out of 74 was expected. Possible explanations are that the common RPGR mutations are not detectable by the SSCP technique used for the analysis, or they may be present in the promoter or an as yet unidentified part of the gene or in an alternatively-spliced transcript. It is conceivable that RPGR is not responsible for most cases of RP3, and another novel RP3 gene(s) remains unidentified. A systematic analysis of RPGR sequences in genetically well-defined RP3 patients and cloning of the full-length RPGR cDNAs from the retina should help in distinguishing among these possible hypotheses.
In spite of the low number of mutations identified so far, a profile of genetic variability is emerging. It appears that most RP3 mutations are of independent origin. Our recent genetic analysis of XLRP families using polymorphic markers from the RP3 region shows that the majority of the independent patients have different genetic backgrounds. These results would suggest that the RP3 gene(s) has a high mutation rate, which may also account for an unexpectedly high number of sporadic cases of RP.
It is still puzzling why BB had a set of ophthalmologic symptoms similar to XLRP (see ref. 12 for further discussion), particularly since the RPGR gene is 400 kb from the proximal breakpoint of BB deletion. Molecular studies in this patient should test for an RPGR mutation independent of the neighboring deletion, a second unidentified chromosomal aberration in BB, a long-range disruption of the RPGR function by position effect, or another RP3 gene in the region.
Genetic loci for at least two related retinal dystrophies have also been mapped in the RP3 genomic region; these include progressive cone dystrophy (COD1) (1, 14) and congenital stationary night blindness (CSNB) (2). It will be now be possible to determine whether mutations in the RPGR gene can also cause these diseases.
As in the case of all the genes assigned to hereditary diseases, identification of the RPGR gene itself and of its product holds a promise for direct prenatal and presymptomatic diagnosis and for gene-based treatment or therapies. A long road is still ahead since mutations in most XLRP families are not revealed yet, and RPGR has to demonstrate that it holds the key to the mysterious world of XLRP.
The research in our laboratory is supported by grants from the National Institutes of Health EY07961 and from The Foundation Fighting Blindness, Hunt Valley, MD. We also wish to acknowledge NIH grants EY07003 (CORE) and M01-RR00042 (General Clinical Research Center).
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