Recent progress in positional cloning of the RP3 and RP2 genes

A comprehensive review of all published linkage data (Aldred et aL, 1994a), incorporating data of Bergen et aL, (1995a) suggests that 36% of European families are RP2, almost two and three times that found in Australia (22%) and the USA (14%). Overall, 30% of families are RP2, consistent with the heterogeneity analyses of Ott et aL,

(1990) and Teague et aL, (1994). Within Europe, RP2 appears particularly prevalent in the UK. Although this may reflect the large numbers of British families reported, this may be a consequence of a founder effect. The genetic origin and drift of distinct RP mutations within a country or region may considerably influence the probability of encountering a particular XLRP subtype on DNA diagnosis, with important implications for genetic counselling. The following sections summarise recent mapping progress on the two XLRP lod for which genetic evidence is undeniable, RP3 and RP2.

1.8.7.3.1 RP3 - Physical mapping studies and isolation of candidate genes

Fine mapping of deletion patients has provided the ultimate route towards identification of the RP3 disease-causative gene. The RP3 gene was believed to lie in the proximal portion of the BB deletion (section l.S.7.1) because of its coinddence with the critical region delineated by linkage analysis. The two genetic markers, DXSlllO and OTC that flank the RP3 locus span a physical distance of -'520kb (Nelson et al, 1995); the proximal end of the BB deletion is 40kb centromeric to DXSlllO. Several groups have been searching for transcribed sequences in this region surrounding the proximal breakpoint for several years.

The BB deletion junction was initially doned by Musarella et aL, (1991) using a DMD cDNA as a starting point from which to isolate genomic clones and create a long- range physical map of the proximal part of the deletion. PFGE studies restricted the RP3 locus to a 205kb Sfil fragment within which a CpG island was identified. Segments of genomic DNA adjacent to the CpG island hybridised to discrete bands in digested DNA from several spedes, indicating evolutionary conservation, and these segments were subsequently used to isolate a cDNA of ubiquitous expression from retinal cDNA libraries (Roux et aL, 1994). Given its high similarity to the murine tctex-1 gene (thought to be involved in spermatogenesis) the function of the novel human gene (TCTEIL) was speculative, but its location in Xp21 and complete deletion in patient BB prompted investigation as a candidate for RP3. However, no disease-assodated changes in the coding portion of the gene were found in 20 RP3 patients (Roux et aL, 1994).

At this point re-evaluation of the contiguous deletions assodated with syndromes in Xp21.1 suggested that the deletion observed in patient BB may have misdirected the search for RP3. A deletion in patient NF who suffered from DMD and

CGD but did not have RP symptoms, extended proximal to that of BB, essentially excluding the proximal part of the BB deletion from containing RP3 (Brown et ah, 1996). Gene cloning efforts were thus directed to an extended interval between the TCTEIL locus and the SB proximal deletion breakpoint 50kb distal to OTC (Meindl et ah, 1995a).

In the autumn of 1995, two groups simultaneously reported the cloning of a candidate RP3 gene, SRPX/ETX-1, mapping within this interval (Meindl et ah, 1995a; Dry et ah, 1995). To identify transcribed sequences, the strategy adopted by Meindl et ah,

(1995a) was to screen retinal cDNA libraries with sub-fragments of a YAC containing both OTC and CYBB, paralleled by genomic sequencing of putative CpG islands and hybridisation of cosmid digests with a splice-site consensus sequence. Dry et ah, (1995) used YACs covering the CYBB-OTC interval to isolate cosmids upon which to perform exon amplification (section 1.5.5). The SRPX/ETX-1 transcript showed highest expression in retina and heart, encodes a putative cell surface protein and contains sushi repeats similar to selectin cell adhesion proteins. SRPX/ETX-1 was found to lie 250kb proximal to the BB deletion junction (Meindl et ah, 1995a) but was considered a candidate gene for RP3 on the grounds that large chromosomal re-arrangements may affect the expression of nearby genes (position effect; Bedell et ah, 1996) or be associated with secondary deletions remote from the major site. The gene was completely deleted in XLRP patient SB, and a microdeletion was deleted in XLRP patient MO encompassing the promoter region and exon 1 of SRPX (Meindl et ah, 1995a). However, no further functionally significant mutations were detected by SSCP screening of all exons in a panel of unrelated XLRP patients nor by full-length RT-PCR sequencing in two RP3 families (Meindl et ah, 1995a). The role of this highly conserved retinally expressed gene in the pathogenesis of RP therefore remains to be determined.

A timely collaborative effort between the two groups in searching for further transcripts mapping within the MO deletion has since resulted in the cloning of a gene which is mutated in a proportion of RP3 patients (Meindl et ah, 1996). The gene is evolutionarily conserved and is thought to be a housekeeping gene' on the basis of its ubiquitous expression and 5' CpG island (Dry et ah, 1996). Interestingly, this gene shows very low levels of expression in the retina and RPE, which explains why it could not be isolated through direct screening of retinal cDNA libraries (Meindl et ah, 1996). The gene was eventually identified by systematically subcloning and sequencing two cosmids covering the proximal part of the MO deletion, and detection of gene sequences by computational analysis (section 1.5.4.8). Mutations were found in highly conserved residues which segregated with disease in 7 XLRP families from a pool of 74 unrelated patients, providing evidence that this gene underlies at least a percentage of RP3. The predicted protein contains a tandem repeat structure similar to RCC-1 (regulator of chromosome condensation) which regulates the GTPase Ran (Ras-related nuclear protein), known to play a role in cell cycle progression, membrane transport and RNA

processing. The high rates of membrane turnover in the retina and RPE (see section 1.6.1.2) have led to speculation that this novel gene, termed RPGR (retinitis pigmentosa GTPase regulator) acts to regulate this process (Meindl et aL, 1996). It is interesting that in another X-linked eye disorder, choroideremia, the defective gene plays a role in the geranylgeranylation of different Rab proteins, another family of Ras-related GTPases (Seabra et aL, 1993; van Bokhoven et al, 1994). In view of the clinical similarities between choroideremia and RP3 (Bird 1975), it is speculated that RPGR may be a guanine- nucleotide exchange factor for retina-specific Rab proteins.

Shortly after the publication of the RPGR gene, Roepman et aL, (1996) cloned the same gene via a novel method called 'YAC representation hybridisation' and found disease-assodated mutations in 5 out of 28 XLRP patients. It is surprising that so few of the XLRP patients screened, about 20% (Buraczynska et aL, 1997)) revealed disease- assodated mutations when RP3 accounts for -70% of all XLRP cases (section l.S.7.1). Perhaps mutations may lie in unidentified parts of the gene or alternative transcripts, common mutations were not identified by SSCP analysis and/or there is heterogeneity within RP3, with the major locus still to be identified. Fujita et aL, (1996) have recently reported a recombination event in a large RP3 family which localises the causative mutation proximal to the BB deletion. This was further supported by Brown et aL, (1996) whose results suggested that RP3 lies outside the BB and NF deletions and within a 380-kb region between the proximal NF and SB deletion breakpoint junctions, from which region the RPGR gene was isolated (Meindl et aL, 1996). If RPGR is the major RP3 gene, questions remain as to the presence of XLRP in patient BB. It is feasible that his large deletion may indude another RP3 locus, or an RPGR regulatory element situated some distance from the gene. Other likely explanations indude a long-range chromosomal position effect, a small secondary rearrangement and without a family history, it is also difficult to exdude the possibility that patient BB may have had an autosomal form of RP.

Studies are currently underway to determine the predse location and function of RPGR in the retina and eluddate the mechanism by which defects within it lead to retinal degeneration. This will enhance our understanding of normal retina function and may provide dues as to the cause of other inherited retinopathies, in particular RP2. Investigation of the physiological function of the RPGR protein and generation of a mouse model of X-linked RP has recently been attempted by doning and characterising the full-length and variant cDNA isoforms derived from the mouse homolog of the human RPGR gene; designated mRpgr (Van et aL, 1998). The discovery of RPGR will undoubtedly benefit women in families segregating the gene who request carrier testing, and will diagnose XLRP in a proportion of sporadic patients enabling more appropriate counselling and prognosis. Recent mutation screening of 10 CSNBX pedigrees has disdosed an RPGR mutation segregating in one family which is absent from 170 control

X chromosomes (Hermann et aL, 1996). This provides evidence for the postulated allelism between XLRP and CSNB (section 1.8.2) and may assist functional analysis of the gene product.

1.8.7.3.2 RP2 - further genetic mapping studies to confirm and refine the RF2 locus and recent isolation of a candidate gene

The lack of associated cytogenetic abnormalities for RP2 has hampered localisation of this gene and its precise location by linkage analysis has been less well- defined. Ott et aL, (1990) localised RP2 to a broad region extending from DXS7 in Xpll.3 to the centromere. Multipoint linkage analysis by Wright et aL, (1991) on the large British kindred of Bhattacharya et aL, (1984) gave a maximum likelihood location for RP2 close to DXS255 in Xpll.22 and TIMP-1 in Xpll.23 in an area extending from 2cM proximal to DXS7 to IcM distal to DXS14. This is supported by the study of Teague et aL, (1994), where RP2 had a maximum likelihood location 6cM proximal to DXS7. The recent heterogeneity analysis of Bergen et aL, (1995a) produced a most likely location for RP2 at DXS255, with a confidence interval extending firom DXS7 to DXS14.

In summary, all multipoint linkage studies have given differing most likely locations for RP2, albeit with varying confidence intervals which overlap to some degree. This may reflect detection of linkage by DXS7 to both RP2 and RP3 lod (only /~10-15cM separates them). More importantly, the relative rarity of the RP2-type family (30% of most XLRP populations) reduces the number of informative recombination events necessary for fine genetic localisation of the gene.

At the outset of this study, overall data indicated a location for the RP2 locus between DXS7 and DXS255 (Wright et aL, 1991; Friedrich et al, 1992), a genetic interval of 13-18cM (Mahtani et aL, 1991) which was refined to 5cM between markers DXS8083 and DXS6616 in this laboratory (Thiselton et aL, 1996). Thus there is still scope for refining the localisation of RP2 further by genetic mapping, but given the problems of classifying families and obtaining large pedigrees with informative recombinants, other approaches are being pursued. Since a physical map was being constructed in the laboratory using YACs centred around the DXS426 locus, then cDNA selection and isolation strategies using these YAC reagents to generate new ESTs was conducted (Chapters 4 & 5). Also investigation of candidate genes mapping to the critical interval was carried out (Chapter 6) . Families segregating CSNBX mapping to X pll may also be useful for refining the localisation of RP2 if these represent allelic disorders.

Aldred et aL, (1994b) have described a family in which XLRP cosegregates with mental retardation and which appears to be RP2 by linkage analysis (maximum likelihood location 0.5cM distal to TIMP). This may represent a new genetic syndrome due to a locus that is fortuitously located in the same region, or, more interestingly,

raises the possibility of a contiguous gene deletion syndrome involving the RP2 gene in this family, which could significantly reduce the region of search for the RP2 gene.

Recently a novel gene RP2, which is mutated in approximately 18% of the patients with X-linked retinitis pigmentosa 2 was isolated (Schwahn et aL, 1998). The gene was identified by positional cloning from a 5-cM linkage interval in Xpll.3, through the detection of an LINEl retrotransposition in intron 1 of the RP2 gene. This was found in 1 of 26 patients screened with the YAC representation hybridisation (YRH) technique, which was used previously for the identification of a 6.4-kb microdeletion that was instrumental in the isolation of the RP3 gene (Roepman et aL, 1996a). In addition to the LI retrotransposition, two nonsense, two frameshift and one missense mutation as well as one in-frame deletion have also been identified (Schwahn et aL,

1998). Through expression studies and RT-PCR the RP2 gene is found to be expressed ubiquitously and subject to X chromosome inactivation. A domain of 151 aa within the N-terminal portion of the RP2 polypeptide revealed significant homology with cofactor C, a protein involved in the biogenesis of the p-tubulin molecule, which could suggest a possible role of the RP2 gene in tubulin folding. Thus, RP2 may be due to a novel mechanism not previously implicated in the pathogenesis of RP.