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The presenilin proteins (PS1 and PS2) with their partners (NCT, Aph1, and Pen2) are the major components of the high molecular weight gamma-secretase complex which facilitates the intramembraneous cleavage of various type 1 transmembrane proteins, including the amyloid-beta precursor protein (APP) and the Notch receptor. Additional gamma-secretase complex components may be involved in regulation of its activity and specificity. A recent investigation indicated that the Crumbs protein is a negative regulator of Notch signaling and may act by repressing gamma/epsilon-secretase activity in Drosophila [Herranz, H., Stamataki, E., Feiguin, F., and Milan, M. (2006) EMBO Rep. 7, 297-302]. To address this question, we investigated potential functional interactions between the human Crumbs homologues (CRB1, CRB2, and CRB3) and presenilin complexes which mediate gamma/epsilon-secretase cleavage of APP and Notch. We found no evidence for direct interaction between CRB1, CRB2, or CRB3 and presenilin complex components. Furthermore, overexpression of human CRB1 and related isoforms, CRB2 and CRB3, had no effect on the levels of presenilin complex components, on NCT maturation or on PS endoproteolysis, and did not alter Abeta AICD or NICD production. These results suggest that, in mammalian cells at least, Crumbs is unlikely to be a significant direct modulator of presenilin-dependent gamma/epsilon-secretase activity.

The paired-like homeodomain (HD) protein Chx10 is distinguished by the presence of the CVC domain, a conserved 56 amino acid sequence C-terminal to the HD. In mammals, Chx10 is essential both for the proliferation of retinal progenitor cells and for the formation or survival of retinal bipolar interneurons. We describe the cloning and characterization of a mouse Chx10 homologue, Vsx1; phylogenetic analysis suggests that Vsx1 and its putative vertebrate orthologues have evolved rapidly. Vsx1 expression in the adult is predominantly retinal. Whereas Chx10 is expressed both in retinal progenitors in the developing eye and apparently in all bipolar cells of the mature retina, Vsx1 expression is first detected in the eye at postnatal day 5, where it is restricted to cone bipolar cells.

The homologous membrane proteins Rom-1 and peripherin-2 are localized to the disk rims of photoreceptor outer segments (OSs), where they associate as tetramers and larger oligomers. Disk rims are thought to be critical for disk morphogenesis, OS renewal and the maintenance of OS structure, but the molecules which regulate these processes are unknown. Although peripherin-2 is known to be required for OS formation (because Prph2-/- mice do not form OSs; ref. 6), and mutations in RDS (the human homologue of Prph2) cause retinal degeneration, the relationship of Rom-1 to these processes is uncertain. Here we show that Rom1-/- mice form OSs in which peripherin-2 homotetramers are localized to the disk rims, indicating that peripherin-2 alone is sufficient for both disk and OS morphogenesis. The disks produced in Rom1-/- mice were large, rod OSs were highly disorganized (a phenotype which largely normalized with age) and rod photoreceptors died slowly by apoptosis. Furthermore, the maximal photoresponse of Rom1-/- rod photoreceptors was lower than that of controls. We conclude that Rom-1 is required for the regulation of disk morphogenesis and the viability of mammalian rod photoreceptors, and that mutations in human ROM1 may cause recessive photoreceptor degeneration.

Mutations in the retinal-expressed gene CRX (cone-rod homeobox gene) have been associated with dominant cone-rod dystrophy and with de novo Leber congenital amaurosis. However, CRX is a transcription factor for several retinal genes, including the opsins and the gene for interphotoreceptor retinoid binding protein. Because loss of CRX function could alter the expression of a number of other retinal proteins, we screened for mutations in the CRX gene in probands with a range of degenerative retinal diseases. Of the 294 unrelated individuals screened, we identified four CRX mutations in families with clinical diagnoses of autosomal dominant cone-rod dystrophy, late-onset dominant retinitis pigmentosa, or dominant congenital Leber amaurosis (early-onset retinitis pigmentosa), and we identified four additional benign sequence variants. These findings imply that CRX mutations may be associated with a wide range of clinical phenotypes, including congenital retinal dystrophy (Leber) and progressive diseases such as cone-rod dystrophy or retinitis pigmentosa, with a wide range of onset.

To examine the role of ROM1, a homologue of peripherin/RDS, in autosomal dominant retinitis pigmentosa (adRP), we screened 224 adRP and 29 simplex RP probands for ROM1 mutations. Four ROM1 alleles were designated as potentially pathogenic because they were found only in RP patients but not in 50-100 controls nor in 249 other RP probands. The substitutions P60T and T108M were present in a single allele in a subject with typical adRP, and this allele cosegregated with the disease in the small family. The putative null allele L114 [1 bp] was present in an individual with atypical RP but not in three unaffected siblings. This insertion has been previously reported to cause RP only when accompanied by a peripherin/RDS mutation, but no peripherin/RDS mutations were found in any of the four probands reported here. Two substitutions (G75D, R242Q) were present in two other probands with simplex RP. These data suggest that potentially pathogenic ROM1 mutations occur in 1% or less of patients with adRP or simplex RP. The absence of detectable peripherin/RDS mutations in these families suggests either that: (i) mutations in other digenic partners are required for pathogenic ROM1 alleles to cause retinal degeneration; (ii) these ROM1 mutations do not cause RP; or (iii) peripherin/RDS mutations are present but were not identified in these patients.

Vitelliform macular dystrophy (Best disease) is an autosomal dominant macular dystrophy which shares important clinical features with age-related macular degeneration, the most common cause of legal blindness in the elderly. Unfortunately, our understanding and treatment for this common age-related disorder is limited. Discovery of the gene which causes Best disease has the potential to increase our understanding of the pathogenesis of all types of macular degeneration, including the common age-related form. Best disease has recently been mapped to chromosome 11q13. The photoreceptor-specific protein ROM1 has also been recently mapped to this location, and the ROM1 gene is a candidate gene for Best disease. Using highly polymorphic markers, we have narrowed the genetic region which contains the Best disease gene to the 10-cM region between markers D11S871 and PYGM. Marker D11S956 demonstrated no recombinants with Best disease in three large families and resulted in a lod score of 18.2. In addition, a polymorphism within the ROM1 gene also demonstrated no recombinants and resulted in a lod score of 10.0 in these same three families. We used a combination of SSCP analysis, denaturing gradient gel electrophoresis, and DNA sequencing to screen the entire coding region of the ROM1 gene in 11 different unrelated patients affected with Best disease. No nucleotide changes were found in the coding sequence of any affected patient, indicating that mutations within the coding sequence are unlikely to cause Best disease.

Rom-1 is an integral membrane protein of the rod photoreceptor outer segment. The ROM1 gene is located on human chromosome 11q13, a region to which the loci of four degenerative retinopathies have been mapped. To identify alleles of ROM1, we have screened the DNA of 57 controls and 180 patients with inherited retinopathies. Six ROM1 polymorphisms were identified: two in non-coding sequences (C-7T, T insertion 966/967), two substitutions (Ala118Gly, Arg223Arg), and two RFLPs outside the transcription unit, detected with BcII and Hind III. One rare sequence variant (Arg229His) was found in two adRP probands; in the one family studied the allele was discordant with the disease. A second rare variant (Ala265Thr) was found in both an adRP family and a control; a third rare variant (Met271Thr) was present only in a control family. These polymorphisms will be useful in the evaluation of ROM1 as a candidate gene in inherited retinal diseases. The recognition of the rare variants will prevent their misassignment as disease-causing mutations.

Rom-1 and peripherin are related membrane proteins of the photoreceptor outer segments. Both proteins are located at the rims of the photoreceptor disks, where they may act jointly in disk biogenesis. Mutations in the gene (RDS) encoding peripherin cause autosomal dominant retinitis pigmentosa, autosomal dominant punctata albescens and butterfly macular degeneration in man, and retinal degeneration slow in mice. To facilitate ROM1 mutation and linkage analysis in inherited retinal diseases, we cloned and characterized the human and murine ROM1 genes. In both species, the ROM1 coding region is contained within approximately 1.8 kb of genomic DNA and is interrupted by only two introns. The structures of the ROM1 and RDS genes are similar, with perfect conservation of the intron splice sites. Putative transcription regulatory regions of the ROM1 locus, 5' to an apparent transcription start site, were identified by cloning the mouse Rom-1 gene and comparing the sequence to the human homologue. Alignment of the human and murine rom-1 predicted protein sequences with the peripherin polypeptides of four species reveals a high degree of conservation (47% overall identity between the six proteins) in the central hydrophilic domain of the two family members. Despite this conservation of sequence, the predicted pI's of only this region of rom-1 and peripherin differ substantially, being 5.2 and 8.2, respectively. The charge difference in this region may mediate the non-covalent association of these two proteins in vivo. The conserved genomic structure and sequence of ROM1 and RDS indicates that these genes evolved from a common ancestor by duplication event.

Rom-1 is a retinal integral membrane protein that, together with the product of the human retinal degeneration slow gene (RDS), defines a photoreceptor-specific protein family. The gene for rom-1 (HGM symbol: ROM1) has been assigned to human chromosome 11 and mouse chromosome 19 by Southern blot analysis of somatic cell hybrid DNAs. ROM1 was regionally sublocalized to human 11p13-11q13 by using three mouse-human somatic cell hybrids; in situ hybridization refined the sublocalization to human 11q13. Analysis of somatic cell hybrids suggested that the most likely localization of ROM1 is in the approximately 2-cM interval between human PGA (human pepsinogen A) and PYGM (muscle glycogen phosphorylase). ROM1 appears to be a new member of a conserved syntenic group whose members include such genes as CD5, CD20, and OSBP (oxysterol-binding protein), on human chromosome 11 and mouse chromosome 19. Localization of the ROM1 gene will permit the examination of its linkage to hereditary retinopathies in man and mouse.