Murine model of autosomal dominant retinitis pigmentosa generated by targeted deletion at codon 307 of the rds-peripherin gene

We introduced a targeted single base deletion at codon 307 of the rds-peripherin gene in mice, similar mutations being known to cause autosomal dominant retinitis pigmentosa (RP) in man. Histopathological and electroretinographic analysis indicate that the retinopathy in mice homozygous for the codon 307 mutation appears more rapid than that in the naturally occurring null mutant, the rds(-/-) mouse, suggesting that the rds-307 mutation displays a dominant negative phenotype in combination with that due to haplosufficiency. RP is the most prevalent cause of registered visual handicap in those of working age in developed countries, the 50 or so mutations so far identified within the RDS-peripherin gene accounting for up to 10% of dominant cases of the disease. Given the sequence homologies that exist between the murine rds-peripherin and the human RDS-peripherin gene, this disease model, the first to be generated for peripherin-based RP using gene targeting techniques, should in principle be of value in the work-up in mice of therapeutics capable of targeting transcripts derived from the human gene.

Autosomal dominant retinitis pigmentosa:a new multi-allelic marker (D3S621) genetically linked to the disease locus (RP4)

We report the characterization of a new eight-allele microsatellite (D3S621) isolated from a human chromosome 3 library. Two-point and multi-locus genetic linkage analysis have shown D3S621 to co-segregate with the previously mapped RP4 (theta m = 0.12, Zm = 4.34) and with other genetic markers on the long arm of the chromosome, including D3S14 (R208) (theta m = 0.00, Zm = 15.10), D3S47 (C17) (theta m = 0.11, Zm = 4.95), Rho (theta m = 0.07, Zm = 1.37), D3S21 (L182) (theta m = 0.07, Zm = 2.40) and D3S19 (U1) (theta m = 0.13, Zm = 2.78). This highly informative marker, with a polymorphic information content of 0.78, should be of considerable value in the extension of linkage data for autosomal dominant retinitis pigmentosa with respect to locii on the long arm of chromosome 3.

Autosomal dominant retinitis pigmentosa:linkage to rhodopsin and evidence for genetic heterogeneity

Retinitis pigmentosa (RP) is the most prevalent human retinopathy of genetic origin. Chromosomal locations for X-linked RP and autosomal dominant RP genes have recently been established. Multipoint analyses with ADRP and seven markers on the long arm of chromosome 3 demonstrate that the gene for rhodopsin, the pigment of the rod photoreceptors, cosegregates with the disease locus with a maximum lod score of approximately 19, implicating rhodopsin as a causative gene. Recent studies have indicated the presence of a point mutation at codon 23 in exon 1 of rhodopsin which results in the substitution of histidine for the highly conserved amino acid proline, suggesting that this mutation is a cause of rhodopsin-linked ADRP. This mutation is not present in the Irish pedigree in which ADRP has been mapped close to rhodopsin. Another mutation in the rhodopsin gene or in a gene closely linked to rhodopsin may be involved. Moreover, the gene in a second ADRP pedigree, with Type II late onset ADRP, does not segregate with chromosome 3q markers, indicating that nonallelic as well as perhaps allelic genetic heterogeneity exists in the autosomal dominant form of this disease.

Autosomal dominant retinitis pigmentosa (ADRP):localization of an ADRP gene to the long arm of chromosome 3

Members of a large pedigree of Irish origin presenting with early onset Type I autosomal dominant retinitis pigmentosa (ADRP) have been typed for D3S47 (C17), a polymorphic marker from the long arm of chromosome 3. Significant, tight linkage of ADRP to D3S47, with a lod score of 14.7 maximizing at 0.00 recombination, has been obtained, hence localizing the ADRP gene (RP1) segregating in this pedigree to 3q.

The story of RP10: A positional cloning approach to the identification of an autosomal dominant retinitis pigmentosa gene

Retinitis pigmentosa (RP) is an inherited retinal degenerative disease that is the leading cause of inherited blindness worldwide. Characteristic features of the disease include night blindness, progressive loss of visual fields, and deposition of pigment on the retina in a bone spicule-like pattern. RP is marked by extreme genetic heterogeneity with at least 19 autosomal dominant, autosomal recessive and X-linked loci identified. RP10, which maps to chromosome 7q, was the fifth autosomal dominant RP locus identified, and accounts for the early-onset disease in two independent families. Extensive linkage and haplotype analyses have been performed in these two families which have allowed the assignment of the disease locus to a 5-cM region on chromosome 7q31.3. In collaboration with Dr. Eric Green (National Center for Human Genome Research, National Institutes of Health), a well-characterized physical map of the region was constructed which includes YAC, BAC and cosmid coverage. The entire RP10 critical region resides within a 9-Mb well-characterized YAC contig. These physical maps not only provided the resources to undertake the CAIGES (cDNA amplification for identification of genomic expressed sequences) procedure for identification of retinal candidate genes within the critical region, but also identified a number of candidate genes, including transducin-$\gamma$ and blue cone pigment genes. All candidate genes examined were excluded. In addition, a number of ESTs were mapped within the critical region. EST20241, which was isolated from an eye library, corresponded to the 3$\sp\prime$ region of the ADP-ribosylation factor (ARF) 5 gene. ARF5, with its role in vesicle transport and possible participation in the regulation of the visual transduction pathway, became an extremely interesting candidate gene. Using a primer walking approach, the entire 3.2 kb genomic sequence of the ARF5 gene was generated and developed intronic primers to screen for coding region mutations in affected family members. No mutations were found in the ARF5 gene, however, a number of additional ESTs have been mapped to the critical region, and, as the large-scale sequencing projects get underway, megabases of raw sequence data from the RP10 region are becoming available. These resources will hasten the isolation and characterization of the RP10 gene. ^

CHROMOSOMAL MAPPING, LINKAGE RELATIONSHIPS, AND EVOLUTIONARY STUDIES OF THE HUMAN ANONYMOUS DNA CLONE D1S1 (IN SITU HYBRIDIZATION, PRIMATES, GENE MAPPING, AUTOSOMAL DOMINANT RETINITIS PIGMENTOSA, GENE DUPLICATION)

D1S1, an anonymous human DNA clone originally called (lamda)Ch4-H3 or (lamda)H3, was the first single copy mapped to a human chromosome (1p36) by in situ hybridization. The chromosomal assignment has been confirmed in other laboratories by repeating the in situ hybridization but not by another method. In the present study, hybridization to a panel of hamster-human somatic cell hybrids revealed copies of D1S1 on both chromosomes 1 and 3. Subcloning D1S1 showed that the D1S1 clone itself is from chromosome 3, and the sequence detected by in situ hybridization is at least two copies of part of the chromosome 3 copy. This finding demonstrates the importance of verifying gene mapping with two methods and questions the accuracy of in situ hybridization mapping.^ Non-human mammals have only one copy of D1S1, and the non-human primate D1S1 map closely resembles the human chromosome 3 copy. Thus, the human chromosome 1 copies appear to be part of a very recent duplication that occurred after the divergence between humans and the other great apes.^ A moderately informative HindIII D1S1 RFLP was mapped to chromosome 3. This marker and 12 protein markers were applied to a linkage study of autosomal dominant retinitis pigmentosa (ADRP). None of the markers proved linkage, but adding the three families examined to previously published data raises the ADRP:Rh lod score to 1.92 at (THETA) = 0.30. ^

Investigating the pathogenicity of mutations in two ubiquitously expressed housekeeping genes that cause autosomal dominant retinitis pigmentosa

The purpose of this dissertation research was to investigate potential mechanisms through which mutations in two ubiquitously expressed genes, inosine monophosphate dehydrogenase 1 (IMPDH1) and pre-mRNA processing factor 31 (PRPF31), cause autosomal dominant retinitis pigmentosa (adRP) but have no other apparent clinical consequences. Basic properties of the gene and gene product, such as expression and protein levels, were examined. The purpose of our research is to understand the genetic basis of inherited retinopathies such as retinitis pigmentosa (RP). RP is a heterogeneous retinal dystrophy that affects approximately one in 3,700 individuals, making it the most common heritable retinal degenerative disease worldwide. Currently, mutations in 35 genes are known to cause RP and additional loci have been mapped but the underlying gene is not yet known. Often the genes associated with RP are integral to the biological processes underlying vision, making their role in retinal disease easy to explain. However, the mechanisms by which other genes cause RP are not apparent, especially widely-expressed genes. For IMPDH1, this research characterized the enzymatic properties of retinal isoforms. Results show that the retinal isoforms have enzymatic functions similar to the previously known canonical IMPDH1 whether or not an adRP pigmentosa mutation is included in the protein. For PRPF31, this research tested the hypothesis that functional haploinsufficiency is the cause of disease and relates to nonpenetrance in some individuals. Studies in patients with known mutations show that haploinsufficiency is the likely cause of disease, however, we did not confirm that non-penetrant individuals are protected from disease via increased expression of the wild type allele. Information gleaned from these functional studies, and the testing methods developed in tandem, will contribute to future research on disease mechanism related to adRP. ^

Synthesis and characterization of a novel retinylamine analog inhibitor of constitutively active rhodopsin mutants found in patients with autosomal dominant retinitis pigmentosa

Two different mutations of the active-site Lys-296 in rhodopsin, K296E and K296M, have been found to cause autosomal dominant retinitis pigmentosa (ADRP). In vitro studies have shown that both mutations result in constitutive activation of the protein, suggesting that the activated state of the receptor may be responsible for retinal degeneration in patients with these mutations. Previous work has highlighted the potential of retinylamine analogs as active-site directed inactivators of constitutively active mutants of rhodopsin with the idea that these or related compounds might be used therapeutically for cases of ADRP involving mutations of the active-site Lys. Unfortunately, however, amine derivatives of 11-cis-retinal, although highly effective against a K296G mutant of rhodopsin, were without affect on the two naturally occurring ADRP mutants, presumably because of the greater steric bulk of Glu and Met side chains in comparison to Gly. For this reason we synthesized a retinylamine analog one carbon shorter than the parent 11-cis-retinal and show that this compound is indeed an effective inhibitor of both the K296E and K296M mutants. The 11-cis C19 retinylamine analog 1 inhibits constitutive activation of transducin by these mutants and their constitutive phosphorylation by rhodopsin kinase, and it does so in the presence of continuous illumination from room lights.

Deficiency of rds/peripherin causes photoreceptor death in mouse models of digenic and dominant retinitis pigmentosa

Retinitis pigmentosa (RP) is a group of inherited blinding diseases caused by mutations in multiple genes including RDS. RDS encodes rds/peripherin (rds), a 36-kDa glycoprotein in the rims of rod and cone outer-segment (OS) discs. Rom1 is related to rds with similar membrane topology and the identical distribution in OS. In contrast to RDS, no mutations in ROM1 alone have been associated with retinal disease. However, an unusual digenic form of RP has been described. Affected individuals in several families were doubly heterozygous for a mutation in RDS causing a leucine 185 to proline substitution in rds (L185P) and a null mutation in ROM1. Neither mutation alone caused clinical abnormalities. Here, we generated transgenic/knockout mice that duplicate the amino acid substitutions and predicted levels of rds and rom1 in patients with RDS-mediated digenic and dominant RP. Photoreceptor degeneration in the mouse model of digenic RP was faster than in the wild-type and monogenic controls by histological, electroretinographic, and biochemical analysis. We observed a positive correlation between the rate of photoreceptor loss and the extent of OS disorganization in mice of several genotypes. Photoreceptor degeneration in RDS-mediated RP appears to be caused by a simple deficiency of rds and rom1. The critical threshold for the combined abundance of rds and rom1 is ≈60% of wild type. Below this value, the extent of OS disorganization results in clinically significant photoreceptor degeneration.

Next Generation Sequencing of Pooled Samples Reveals New SNRNP200 Mutations Associated with Retinitis Pigmentosa

The gene SNRNP200 is composed of 45 exons and encodes a protein essential for pre-mRNA splicing, the 200 kDa helicase hBrr2. Two mutations in SNRNP200 have recently been associated with autosomal dominant retinitis pigmentosa (adRP), a retinal degenerative disease, in two families from China. In this work we analyzed the entire 35-Kb SNRNP200 genomic region in a cohort of 96 unrelated North American patients with adRP. To complete this large-scale sequencing project, we performed ultra high-throughput sequencing of pooled, untagged PCR products. We then validated the detected DNA changes by Sanger sequencing of individual samples from this cohort and from an additional one of 95 patients. One of the two previously known mutations (p.S1087L) was identified in 3 patients, while 4 new missense changes (p.R681C, p.R681H, p.V683L, p.Y689C) affecting highly conserved codons were identified in 6 unrelated individuals, indicating that the prevalence of SNRNP200-associated adRP is relatively high. We also took advantage of this research to evaluate the pool-and-sequence method, especially with respect to the generation of false positive and negative results. We conclude that, although this strategy can be adopted for rapid discovery of new disease-associated variants, it still requires extensive validation to be used in routine DNA screenings. (C) 2011 Wiley-Liss, Inc.

Positional candidate cloning of the RP10 form of retinitis pigmentosa

Retinitis pigmentosa (RP) is a genetically heterogeneous group of retinal degenerations that affects over one million people worldwide. To date, 11 autosomal dominant, 13 autosomal recessive, and 5 X-linked forms of retinitis pigmentosa have been identified through linkage analysis, but the disease-causing genes and mutations have been found for only half of these loci. My research uses a positional candidate cloning approach to identify the gene and mutations responsible for one type of autosomal dominant retinitis pigmentosa, RP10. The premise is that identifying the genes and mutations responsible for disease will provide insight into disease mechanisms and provide treatment options. Previous research mapped the RP10 locus to a 5cM region on chromosome 7q31 between markers D7S686 and D7S530. Linkage and fine-point haplotype analysis was used to reduce and refine the RP10 disease interval to a 4cM region located between D7S2471 and a new marker located 45,000bp telomeric of D7S461. In order to identify genes located in the RP10 interval, an extensive EST map was created of this region. Five EST clusters from this map were analyzed to determine if mutations in these genes cause the RP10 form of retinitis pigmentosa. The genomic structure of a known metabotrophic glutamate receptor, GRMS8, was determined first. DNA sequencing of GRM8 in RP10 family members did not identify any disease-causing mutations. Four other EST clusters (A170, A173, A189, and A258) were characterized and determined to be part of the same gene, UBNL1 (ubinuclein-like 1). The full-length mRNA sequence and genomic structure of UBNL1 was determined and then screened in patients. No disease-causing mutations were identified in any of the RP10 family members tested. Recent data made available with the release of the public and Celera genome assemblies indicates that UBNL1 is outside of the RP10 disease region. Despite this complication, characterization of UBNL1 is still important in the understanding of normal visual processes and it is possible that mutations in UBNL1 could cause other forms of retinopathy. The EST map and list of RP10 candidates will continue to aid others in the search for the RP10 gene and mutations. ^

Structure and function in rhodopsin: correct folding and misfolding in two point mutants in the intradiscal domain of rhodopsin identified in retinitis pigmentosa.

The rhodopsin mutants P23H and G188R, identified in autosomal dominant retinitis pigmentosa (ADRP), and the site-specific mutants D190A and DeltaY191-Y192 were expressed in COS cells from synthetic mutant opsin genes containing these mutations. The proteins expressed from P23H and D190A partially regenerated the rhodopsin chromophore with 11-cis-retinal and were mixtures of the correctly folded (retinal-binding) and misfolded (non-retinal-binding) opsins. The mixtures were separated into pure, correctly folded mutant rhodopsins and misfolded opsins. The proteins expressed from the ADRP mutant G188R and the mutant DeltaY191-Y192 were composed of totally misfolded non-retinal-binding opsins. Far-UV CD spectra showed that the correctly folded mutant rhodopsins had helical content similar to that of the wild-type rhodopsin, whereas the misfolded opsins had helical content 50-70% of the wild type. The near-UV CD spectra of the misfolded mutant proteins lack the characteristic band pattern seen in the wild-type opsin, indicative of a different tertiary structure. Further, whereas the folded mutant rhodopsins were essentially resistant to trypsin digestion, the misfolded opsins were degraded to small fragments under the same conditions. Therefore, the misfolded opsins appear to be less compact in their structures than the correctly folded forms. We suggest that most, if not all, of the point mutations in the intradiscal domain identified in ADRP cause partial or complete misfolding of rhodopsin.

Structure and function in rhodopsin: correct folding and misfolding in point mutants at and in proximity to the site of the retinitis pigmentosa mutation Leu-125-->Arg in the transmembrane helix C.

L125R is a mutation in the transmembrane helix C of rhodopsin that is associated with autosomal dominant retinitis pigmentosa. To probe the orientation of the helix and its packing in the transmembrane domain, we have prepared and studied the mutations E122R, I123R, A124R, S127R, L125F, and L125A at, and in proximity to, the above mutation site. Like L125R, the opsin expressed in COS-1 cells from E122R did not bind 11-cis-retinal, whereas those from I123R and S127R formed the rhodopsin chromophore partially. A124R opsin formed the rhodopsin chromophore (lambda max 495 nm) in the dark, but the metarhodopsin II formed on illumination decayed about 6.5 times faster than that of the wild type and was defective in transducin activation. The mutant opsins from L125F and L125A bound 11-cis-retinal only partially, and in both cases, the mixtures of the proteins produced were separated into retinal-binding and non-retinal-binding (misfolded) fractions. The purified mutant rhodopsin from L125F showed lambda max at 500 nm, whereas that from L125A showed lambda max at 503 nm. The mutant rhodopsin L125F showed abnormal bleaching behavior and both mutants on illumination showed destabilized metarhodopsin II species and reduced transducin activation. Because previous results have indicated that misfolding in rhodopsin is due to the formation of a disulfide bond other than the normal disulfide bond between Cys-110 and Cys-187 in the intradiscal domain, we conclude from the misfolding in mutants L125F and L125A that the folding in vivo in the transmembrane domain is coupled to that in the intradiscal domain.