Addition of human melanopsin renders mammalian cells photoresponsive.

A small number of mammalian retinal ganglion cells act as photoreceptors for regulating certain non-image forming photoresponses. These intrinsically photosensitive retinal ganglion cells express the putative photopigment melanopsin. Ablation of the melanopsin gene renders these cells insensitive to light; however, the precise role of melanopsin in supporting cellular photosensitivity is unconfirmed. Here we show that heterologous expression of human melanopsin in a mouse paraneuronal cell line (Neuro-2a) is sufficient to render these cells photoreceptive. Under such conditions, melanopsin acts as a sensory photopigment, coupled to a native ion channel via a G-protein signalling cascade, to drive physiological light detection. The melanopsin photoresponse relies on the presence of cis-isoforms of retinaldehyde and is selectively sensitive to short-wavelength light. We also present evidence to show that melanopsin functions as a bistable pigment in this system, having an intrinsic photoisomerase regeneration function that is chromatically shifted to longer wavelengths.

Mutations in the pre-mRNA splicing factor gene PRPC8 in autosomal dominant retinitis pigmentosa (RP13).

Retinitis pigmentosa (RP) is a genetically heterogeneous disorder characterized by progressive degeneration of the peripheral retina leading to night blindness and loss of visual fields. With an incidence of approximately 1 in 4000, RP can be inherited in X-linked, autosomal dominant or autosomal recessive modes. The RP13 locus for autosomal dominant RP (adRP) was placed on chromosome 17p13.3 by linkage mapping in a large South African adRP family. Using a positional cloning and candidate gene strategy, we have identified seven different missense mutations in the splicing factor gene PRPC8 in adRP families. Three of the mutations cosegregate within three RP13 linked families including the original large South African pedigree, and four additional mutations have been identified in other unrelated adRP families. The seven mutations are clustered within a 14 codon stretch within the last exon of this large 7 kb transcript. The altered amino acid residues at the C-terminus exhibit a high degree of conservation across species as diverse as humans, Arabidopsis and trypanosome, suggesting that some functional significance is associated with this part of the protein. These mutations in this ubiquitous and highly conserved splicing factor offer compelling evidence for a novel pathway to retinal degeneration.

Temporal and spatial expression patterns of the CRX transcription factor and its downstream targets. Critical differences during human and mouse eye development.

Cone--rod homeobox (CRX), a paired-like homeobox transcription factor, plays a major role in photoreceptor development and maintenance of the retina. Fifteen different mutations in the CRX gene have been identified as a cause of blinding retinal dystrophy. As a step towards characterizing the underlying pathophysiology of disease, temporal and spatial gene expression patterns during human and mouse eye development were investigated for CRX and for downstream retinally expressed genes, postulated to be transactivated by CRX. We found that human CRX was expressed at 10.5 weeks post-conception (p.c.). This was significantly later than observed in mouse development. Immunocytochemistry in human retina showed that CRX protein was not detected until >4 weeks later at 15 weeks p.c., implying that it would be unable to transactivate PDEB, IRBP and arrestin, which were all expressed before 15 weeks. These data therefore eliminate CRX as the major transcriptional activator of these three genes from a wide group of retinal genes that can be transactivated by CRX in vitro. Additionally, PDEB was expressed 2 weeks before CRX whereas murine Pdeb was expressed after Crx, highlighting a potential difference for the role of PDEB in human eye development. Previous data had shown CRX expression in the adult human retina to be photoreceptor-specific; however, we demonstrate that this gene is also expressed in the inner nuclear layer (INL) of the human and mouse retina by in situ hybridization and immunocytochemistry. INL localization of murine Crx was confirmed in rd/rd,cl mice, as in this mouse model the photoreceptors are absent. We have found important differences in the temporal expression of this gene in human and mouse retina, although spatial expression of the CRX gene appears to be conserved. In addition, downstream targets of CRX in vitro might not represent in vivo function during development. These data support concerns about the extent to which we can extrapolate from rodent models regarding embryonic development and disease pathophysiology.

Molecular genetic heterogeneity in autosomal dominant drusen.

OBJECTIVE: Autosomal dominant drusen is of particular interest because of its phenotypic similarity to age related macular degeneration. Currently, mutation R345W of EFEMP1 and, in a single pedigree, linkage to chromosome 6q14 have been causally related to the disease. We proposed to investigate and quantify the roles of EFEMP1 and the 6q14 locus in dominant drusen patients from the UK and USA. DESIGN: Molecular genetic analysis. PARTICIPANTS: Ten unrelated families and 17 young drusen patients. MAIN OUTCOME MEASURES: Exons 1 and 2 of EFEMP1 were characterised by 5' rapid amplification of cDNA ends and direct sequencing. Exons 1-12 of EFEMP1 were then investigated for mutation by direct sequencing. A HpaII restriction digest test was constructed to detect the EFEMP1 R345W mutation. Marker loci spanning the two dominant drusen linked loci were used to generate haplotype data. RESULTS: Only seven of the 10 families (70%) and one of the 17 sporadic patients (6%) had the R345W mutation. The HpaII restriction digest test was found to be a reliable and quick method for detecting this. No other exonic or splice site mutation was identified. Of the three families without EFEMP1 mutation, two were linked to the 2p16 region. CONCLUSIONS: EFEMP1 R345W accounts for only a proportion of the dominant drusen phenotype. Importantly, other families linked to chromosome 2p16 raise the possibility of EFEMP1 promoter sequence mutation or a second dominant drusen gene at this locus. Preliminary haplotype data suggest that the disease gene at the 6q14 locus is responsible for only a minority of dominant drusen cases.

Expression map of human chromosome region 17p13.3, spanning the RP13 dominant retinitis pigmentosa locus, the Miller-Dieker lissencephaly syndrome (MDLS) region, and a putative tumour suppressor locus.

Chromosome region 17p13.3 is rich in genes, with 223 expressed sequence tags (ESTs) within the last 15 cM (7 Mb) of chromosome 17p in the GeneMap database. Loci for dominant retinitis pigmentosa (RP13), central areolar choroidal dystrophy (CACD), anterior polar cataract (CTAA2), Miller-Dieker lissencephaly syndrome (MDLS), and a region of tumour loss of heterozygosity (LOH) distinct from TP53 all map into the region adjacent to the 17p telomere. To date, however, there is no physical map of the region, which has resisted the efforts of the CEPH and Whitehead physical mapping programmes to generate contiguous clones across it. We have created a physical map covering approximately 3.5 Mb (6 cM)in this region, spanning the RP13 interval and extending distally to the gene MDCR (formerly, LIS1), which, when deleted, leads to the MDLS phenotype. The region covered is also the point of maximum LOH in lung cancer and has been implicated in the pathogenesis of many other human cancers. The map orders 47 sequence tagged sites, including 32 genes or ESTs, nine genetic markers, four anonymous sequences, and two YAC end clones, and highlights new candidate ESTs for involvement in RP13, MDLS, CTAA2, and a tumour-susceptibility gene.

Cloning and characterization of a novel orphan G-protein-coupled receptor localized to human chromosome 2p16.

We report the identification and characterisation of a novel human orphan G-protein-coupled receptor (GPR) which maps to chromosome 2p16. We have determined the full-length coding sequence and genomic structure of a gene corresponding to the anonymous expressed sequenced tag, WI-31133. This gene encodes a novel protein that is 540 amino acids in length. Protein sequence analysis predicts the presence of seven transmembrane domains, a characteristic feature of GPRs. In situ hybridisation to human retina and Northern blot analysis of human retinal pigment epithelium (RPE) showed localisation of this transcript to the RPE and cells surrounding retinal arterioles. In contrast, the transcript was localised to the photoreceptor inner segments and the outer plexiform layer in mouse sections. Northern blot analysis demonstrated a 7 kb transcript highly expressed in the brain. No mutations were identified during a screen of patients suffering from Doyne's honeycomb retinal dystrophy (DHRD), an inherited retinal degeneration which maps to chromosome 2p16.

Refined genetic and physical positioning of the gene for Doyne honeycomb retinal dystrophy (DHRD).

Doyne honeycomb retinal dystrophy (DHRD) is a late-onset autosomal dominant disorder that causes degeneration of the retina and can lead to blindness. We have previously assigned DHRD to a 5-cM region of chromosome 2p16 between marker loci D2S2739 and D2S378. Using sequence-tagged sites (STSs), expressed sequence tags (ESTs) and polymorphic markers within the DHRD region, we have identified 18 yeast artificial chromosomes (YACs) encompassing the DHRD locus, spanning approximately 3 Mb. The YAC contig was constructed by STS content mapping of these YACs and incorporates 13 STSs, including four genes and six polymorphic marker loci. We also report the genetic mapping of two families with a dominant drusen phenotype to the DHRD locus, and genetic refinement of the disease locus to a critical interval flanked by microsatellite marker loci D2S2352 and D2S2251, a distance of approximately 700 kb. These studies exclude a number of candidate genes and provide a resource for construction of a transcriptional map of the region, as a prerequisite to identification of the DHRD disease-causing gene and genes for other diseases mapping in the region, such as Malattia leventinese and Carney complex.

A linkage survey of 20 dominant retinitis pigmentosa families: frequencies of the nine known loci and evidence for further heterogeneity.

Autosomal dominant retinitis pigmentosa (ADRP) is caused by mutations in two known genes, rhodopsin and peripherin/Rds, and seven loci identified only by linkage analysis. Rhodopsin and peripherin/Rds have been estimated to account for 20-31% and less than 5% of ADRP, respectively. No estimate of frequency has previously been possible for the remaining loci, since these can only be implicated when families are large enough for linkage analysis. We have carried out such analyses on 20 unrelated pedigrees with 11 or more meioses. Frequency estimates based on such a small sample provide only broad approximations, while the above estimations are based on mutation detection in much larger clinic based patient series. However, when markers are informative, linkage analysis cannot fail to detect disease causation at a locus, whereas mutation detection techniques might miss some mutations. Also diagnosing dominant RP from a family history taken in a genetic clinic may not be reliable. It is therefore interesting that 10 (50%) of the families tested have rhodopsin-RP, suggesting that, in large clearly dominant RP pedigrees, rhodopsin may account for a higher proportion of disease than had previously been suspected. Four (20%) map to chromosome 19q, implying that this is the second most common ADRP locus. One maps to chromosome 7p, one to 17p, and one to 17q, while none maps to 1cen, peripherin/Rds, 8q, or 7q. Three give exclusion of all of these loci, showing that while the majority of dominant RP maps to the known loci, a small proportion derives from loci yet to be identified.

Evidence for a major retinitis pigmentosa locus on 19q13.4 (RP11) and association with a unique bimodal expressivity phenotype.

Retinitis pigmentosa (RP) is the name given to a heterogeneous group of retinal degenerations mapping to at least 16 loci. The autosomal dominant form (ARP), accounting for approximately 25% of cases, can be caused by mutations in two genes, rhodopsin and peripherin/RDS, and by at least six other loci identified by linkage analysis. The RP11 locus for adRP has previously been mapped to chromosome 19q13.4 in a large English family. This linkage has been independently confirmed in a Japanese family, and we now report three additional unrelated linked U.K. families, suggesting that this is a major locus for RP. Linkage analysis in the U.K. families refines the RP11 interval to 5 cM between markers D19S180 and AFMc001yb1. All linked families exhibit incomplete penetrance; some obligate gene carriers remain asymptomatic throughout their lives, whereas symptomatic individuals experience night blindness and visual field loss in their teens and are generally registered as blind by their 30s. This "bimodal expressivity" contrasts with the variable-expressivity RP mapping to chromosome 7p (RP9) in another family, which has implications for diagnosis and counseling of RP11 families. These results may also imply that a proportion of sporadic RP, previously assumed to be recessive, might result from mutations at this locus.

A new family linked to the RP13 locus for autosomal dominant retinitis pigmentosa on distal 17p.

A form of autosomal dominant retinitis pigmentosa (ADRP) mapping to chromosome 17p has been reported in a single large South African family. We now report a new family with severe early onset ADRP which maps to 17p. Linkage and haplotype analysis in this family places the ADRP locus in the 5 cM interval between markers AFMc024za5 and D17S1845, confirming the data obtained in the South African family. The discovery of a second 17p linked family may imply that this is one of the more common loci for dominant RP. In addition, the confirmation of an RP diagnosis at this locus is of interest since loci for a dominant cone dystrophy and Leber's congenital amaurosis (LCA1) have recently been linked to the same markers. While the cone dystrophy locus may be allelic with RP, our data and that of Goliath et al show that distinct genes are responsible for dominant RP and Leber's congenital amaurosis on chromosome 17p.

Fine genetic mapping defines the genetic order of Pax9, Tcf3a, and Acrodysplasia (Adp).

We present here the fine genetic mapping of the proximal part of mouse Chromosome (Chr) 12 between D12Mit54 and D12Mit4. This chromosomal region contains three loci, Pax9, Tcf3a, and Acrodysplasia (Adp), which seem to play an important role in pattern formation during mouse embryogenesis. The Adp mutation, which was created by transgene integration, causes skull, paw, and tail deformities. Pax9, which is expressed in the face, paws, and tail, once qualified as a possible candidate for the Adp locus. We analyzed 997 interspecific backcross progeny for recombination between the markers D12Mit54 and D12Mit4; we recovered 117 recombinants, which were further typed for Pax9, Tcf3a, Adp, D12Mit88, D12Nds1, D12Mit36, and D12Mit34. This study represents the first instance in which all the above loci have been included in a single analysis, thereby allowing unambiguous determination of the genetic order and distance between D12Mit54 and D12Mit4. From our results, we conclude that the Adp locus is distinct from either Pax9 or Tcf3a.

The murine NF2 homologue encodes a highly conserved merlin protein with alternative forms.

The recently isolated gene for neurofibromatosis type 2 (NF2) encodes a 595 amino acid protein, named merlin, which is related to the cytoskeleton-associated proteins moesin, ezrin and radixin. To identify evolutionarily conserved regions and to provide sequence information necessary for the establishment of a mouse model for NF2, we have determined the cDNA sequence of the mouse NF2 tumor suppressor gene, and mapped it in the mouse genome. Mouse merlin is a 596 amino acid protein, 98% identical to human merlin, but one amino acid longer due to the insertion of a proline residue near the C-terminus. Of the nine amino acid differences between mouse and humans, seven occur in the C-terminal 20% of the protein, far from the protein 4.1 domain that defines this family. Two of the NF2 cDNA clones reveal evidence of alternative splicing events that alter the predicted merlin product, one removing a 45 amino acid segment from the middle section of the protein and the other changing the C-terminus. The existence of several different forms of merlin potentially with different primary roles will complicate the identification of the precise function that must be disrupted to cause the NF2-associated tumors. The mouse NF2 homologue maps to Chr 11, in a region homologous to human Chr 22, but devoid of any mouse mutations which could be models of the human disorder.