Pro-347-Arg mutation of the rhodopsin gene in autosomal dominant retinitis pigmentosa.

It has been shown recently that autosomal dominant retinitis pigmentosa may be caused by point mutations of the rhodopsin gene in a portion of families. In this communication, a large six-generation family with autosomal dominant RP is described. Molecular analysis by PCR amplification followed by restriction digestion or heteroduplex analysis suggested a point mutation in codon 347, in which two different mutations (Pro-347-Ser and Pro-347-Leu) have already been reported. Direct sequencing of the patients' DNA revealed a previously undescribed CCG----CGG transversion in codon 347 predicting a Pro----Arg substitution. Ophthalmological data of the patients are summarized and compared to those of patients with other mutations in the rhodopsin gene.     

A 3-bp deletion in the rhodopsin gene in a family with autosomal dominant retinitis pigmentosa.

Autosomal dominant retinitis pigmentosa (ADRP) has recently been linked to locus D3S47 (probe C17), with no recombination, in a single large Irish family. Other ADRP pedigrees have shown linkage at zero recombination, linkage with recombination, and no linkage, demonstrating genetic heterogeneity. The gene encoding rhodopsin, the rod photoreceptor pigment, is closely linked to locus D3S47 on chromosome 3q. A point mutation changing a conserved proline to histidine in the 23d codon of the gene has been demonstrated in affected members of one ADRP family and in 17 of 148 unrelated ADRP patients. We have sequenced the rhodopsin gene in a C17-linked ADRP family and have identified in the 4th exon and in-frame 3-bp deletion which deletes one of the two isoleucine monomers at codons 255 and 256. This mutation was not found in 30 other unrelated ADRP families. The deletion has arisen in the sequence TCATCATCAT, deleting one of a run of three x 3-bp repeats. The mechanism by which this occurred may be similar to that which creates length variation in so-called mini- and microsatellites. Thus ADRP is an extremely heterogeneous disorder which can result from a range of defects in rhodopsin and which can have a locus or loci elsewhere in the genome.     

Suppression of wild-type rhodopsin maturation by mutants linked to autosomal dominant retinitis pigmentosa

Autosomal dominant retinitis pigmentosa (ADRP) has been linked to mutations in the gene encoding rhodopsin. Most RP-linked rhodopsin mutants are unable to fold correctly in the endoplasmic reticulum, are degraded by the ubiquitin proteasome system, and are highly prone to forming detergent-insoluble high molecular weight aggregates. Here we have reported that coexpression of folding-deficient, but not folding-proficient, ADRP-linked rhodopsin mutants impairs delivery of the wild-type protein to the plasma membrane. Fluorescence resonance energy transfer and co-precipitation studies revealed that mutant and wild-type rhodopsins form a high molecular weight, detergent-insoluble complex in which the two proteins are in close (<70 A) proximity. Co-expression of ARDP-linked rhodopsin folding-deficient mutants resulted in enhanced proteasome-mediated degradation and steady-state ubiquitination of the wild-type protein. These data suggested a dominant negative effect on conformational maturation that may underlie the dominant inheritance of ARDP.     

Mapping of locus for autosomal dominant retinitis pigmentosa on chromosome 6q23

Retinitis pigmentosa (RP) is a genetically heterogeneous group of retinal degenerative disorders resulting in severe visual loss and blindness that have remained incurable till date. We report the mapping of the disease locus in a 3-generation family of Indian origin with autosomal dominant RP (ADRP). Diagnosis of RP and recruitment was made after a complete clinical evaluation of all members. Manifestations of the disease included night blindness with blurred central vision in some cases, loss of peripheral vision, and diffuse degeneration of the retinal pigment epithelium. Linkage analysis using microsatellite markers was carried out on 34 members (14 affected). After testing for linkage to known retinal dystrophy loci as well as a subsequent genome-wide analysis, we detected linkage to markers on chromosome 6q23: D6S262 at 130 cM, D6S457 (130 cM) and D6S1656 (131 cM) gave significant 2-point LOD scores of 3.0–3.8. Multipoint LOD scores of ≥3.0 were obtained for markers between 121 and 130 cM. Haplotype analysis with several markers in the same region on chromosome 6 shows a disease-cosegregating region of about 25 Mb between 109 and 135 Mb. There are no known RP genes in this interval, which contains >100 genes. This study provides evidence for a novel ADRP locus on chromosome 6q23.     

Novel rhodopsin mutation in an autosomal dominant retinitis pigmentosa family: phenotypic variation in both heterozygote and homozygote Val137Met mutant patients

A family affected with autosomal dominant retinitis pigmentosa (RP) is presented. Two clinically affected patients (mother and daughter) were heterozygous for the same novel missense mutation (Val137Met) of the rhodopsin gene (RHO). Both heterozygous and homozygous cases were observed among their few symptomatic relatives. Wide clinical variation was exhibited among the individuals with mutations in this family. None of the controls showed this change in RHO, nor has it been previously reported in other RP families. No other RHO mutation was observed. Additional genetic or environmental factors could play a role in modulating the penetrance and clinical expression of this RHO mutation. Received: 20 February 1995 / Revised: 1 September 1995, 27 November 1995, 3 February 1996     

Autosomal dominant retinitis pigmentosa: Absence of the rhodopsin proline→histidine substitution (codon 23) in pedigrees from Europe

In exon 1 at codon 23 of the rhodopsin gene, a mutation resulting in a proline-to-histidine substitution has previously been observed in approximately 12% of American autosomal dominant retinitis pigmentosa (ADRP) patients. The region around the site of this mutation in the rhodopsin gene has been amplified and analyzed in affected individuals from 91 European ADRP pedigrees. The codon 23 mutation has been found to be absent in all cases, including a large Irish pedigree in which the disease gene has previously been shown to be closely linked to the rhodopsin locus. This indicates the presence of either allelic or nonallelic heterogeneity in ADRP.     

Gene mapping and mutation screeneng in candidate genes in a Chinese family of autosomal dominant retinitis pigmentosa

We wanted to find the gene defect in a Chinese pedigree with autosomal dominant form of retinitis pigmentosa (ADRP). A small Chinese family with retinitis pigmentosa was collected. The genetic analysis of the family suggested an autosomal dominant pattern. Microsatellite (STR) markers tightly linked to candidate genes for ADRP were selected for linkage analysis. We got a maximum LOD score of 0.87 between markers D19S210 and D19S418. Precursor mRNA-processing factor (PRPF) 31, 3, 8, rhodopsin (RHO), peripherin 2 (PRPH2 or RDS), rod outer segment protein 1 (ROM1), neural retina leucine zipper (NRL), cone-rod homeobox-containing (CRX), inosine-5-prime-monophosphate dehydrogenase, type I (IMPDH1) and retinitis pigmentosa 1 (RP1) were amplified by polymerase chain reaction (PCR) and screened by direct sequencing. One new sequence variation was found. It was the missence mutation c.148G > C (D50H) occurred in exon 1 of RDS gene which existed in all the effected individuals and one unaffected family member. The DNA sequence variation didn’t cosegregate with the RP disease. We considered this transition was one new polymorphism which we speculate involved in the pathogenesis of ADRP and increased the risk of ADRP. Further study should be conducted to confirm the causative gene of this family.     

Gene mapping and mutation screening in candidate genes in a Chinese family of autosomal dominant retinitis pigmentosa

We wanted to find the gene defect in a Chinese pedigree with autosomal dominant form of retinitis pigmentosa (ADRP). A small Chinese family with retinitis pigmentosa was collected. The genetic analysis of the family suggested an autosomal dominant pattern. Microsatellite (STR) markers tightly linked to candidate genes for ADRP were selected for linkage analysis. We got a maximum LOD score of 0.87 between markers D19S210 and D19S418. Precursor mRNA-processing factor (PRPF) 31, 3, 8, rhodopsin (RHO), peripherin 2 (PRPH2 or RDS), rod outer segment protein 1 (ROM1), neural retina leucine zipper (NRL), cone-rod homeobox-containing (CRX), inosine-5-prime-monophosphate dehydrogenase, type I (IMPDH1) and retinitis pigmentosa 1 (RPI) were amplified by polymerase chain reaction (PCR) and screened by direct sequencing. One new sequence variation was found. It was the missence mutation c.148G > C (D50H) occurred in exon 1 of RDS gene which existed in all the effected individuals and one unaffected family member. The DNA sequence variation didn't cosegregate with the RP disease. We considered this transition was one new polymorphism which we speculate involved in the pathogenesis of ADRP and increased the risk of ADRP. Further study should be conducted to confirm the causative gene of this family.     

Probing mechanisms of photoreceptor degeneration in a new mouse model of the common form of autosomal dominant retinitis pigmentosa due to P23H opsin mutations

Rhodopsin, the visual pigment mediating vision under dim light, is composed of the apoprotein opsin and the chromophore ligand 11-cis-retinal. A P23H mutation in the opsin gene is one of the most prevalent causes of the human blinding disease, autosomal dominant retinitis pigmentosa. Although P23H cultured cell and transgenic animal models have been developed, there remains controversy over whether they fully mimic the human phenotype; and the exact mechanism by which this mutation leads to photoreceptor cell degeneration remains unknown. By generating P23H opsin knock-in mice, we found that the P23H protein was inadequately glycosylated with levels 1-10% that of wild type opsin. Moreover, the P23H protein failed to accumulate in rod photoreceptor cell endoplasmic reticulum but instead disrupted rod photoreceptor disks. Genetically engineered P23H mice lacking the chromophore showed accelerated photoreceptor cell degeneration. These results indicate that most synthesized P23H protein is degraded, and its retinal cytotoxicity is enhanced by lack of the 11-cis-retinal chromophore during rod outer segment development.     

Retinoids assist the cellular folding of the autosomal dominant retinitis pigmentosa opsin mutant P23H

The clinically common mutant opsin P23H, associated with autosomal dominant retinitis pigmentosa, yields low levels of rhodopsin when retinal is added following induction of the protein in stably transfected HEK-293 cells. We previously showed that P23H rhodopsin levels could be increased by providing a 7-membered ring, locked analog of 11-cis-retinal during expression of P23H opsin in vivo. Here we demonstrate that the mutant opsin is effectively rescued by 9- or 11-cis-retinal, the native chromophore. When retinal was added during expression, P23H rhodopsin levels were 5-fold (9-cis) and 6-fold (11-cis) higher than when retinal was added after opsin was expressed and cells were harvested. Levels of P23H opsin were increased approximately 3.5-fold with both compounds, but wild-type protein levels were only slightly increased. Addition of retinal during induction promoted the Golgi-specific glycosylation of P23H opsin and transport of the protein to the cell surface. P23H rhodopsins containing 9- or 11-cis-retinal had blue-shifted absorption maxima and altered photo-bleaching properties compared with the corresponding wild-type proteins. Significantly, P23H rhodopsins were more thermally unstable than the wild-type proteins and more rapidly bleached by hydroxylamine in the dark. We suggest that P23H opsin is similarly unstable and that retinal binds and stabilizes the protein early in its biogenesis to promote its cellular folding and trafficking. The implications of this study for treating retinitis pigmentosa and other protein conformational disorders are discussed.     

Identification of a novel rhodopsin mutation (Met-44-Thr) in a simplex case of retinitis pigmentosa

Retinitis pigmentosa (RP) is a group of genetically heterogeneous retinal degenerations that can be autosomal dominant (ADRP), autosomal recessive (ARRP), or X-linked. Approximately 30% of ADRP patients show point mutations or small deletions in the rhodopsin gene. However, over 50% of the RP patients are simplex cases (sporadic). Screening for mutations in the rhodopsin gene of 33 patients with simplex RP by denaturing gradient gel electrophoresis (DGGE) was carried out. One patient, with D-type (diffuse) RP and consanguineous parents, showed an altered electrophoretic pattern for the 5 half of exon 1. Direct sequencing revealed a new mutation ATG to ACG in codon 44; this predicts a change of Met-44-Thr in rhodopsin. The position and amino acid substitution suggest that this mutation causes the RP phenotype. Implications for genetic counselling are discussed.     

A rhodopsin mutant linked to autosomal dominant retinitis pigmentosa is prone to aggregate and interacts with the ubiquitin proteasome system

The inherited retinal degenerations are typified by retinitis pigmentosa (RP), a heterogeneous group of inherited disorders that causes the destruction of photoreceptor cells, the retinal pigmented epithelium, and choroid. This group of blinding conditions affects over 1.5 million persons worldwide. Approximately 30-40% of human autosomal dominant (AD) RP is caused by dominantly inherited missense mutations in the rhodopsin gene. Here we show that P23H, the most frequent RP mutation in American patients, renders rhodopsin extremely prone to form high molecular weight oligomeric species in the cytoplasm of transfected cells. Aggregated P23H accumulates in aggresomes, which are pericentriolar inclusion bodies that require an intact microtubule cytoskeleton to form. Using fluorescence resonance energy transfer (FRET), we observe that P23H aggregates in the cytoplasm even at extremely low expression levels. Our data show that the P23H mutation destabilizes the protein and targets it for degradation by the ubiquitin proteasome system. P23H is stabilized by proteasome inhibitors and by co-expression of a dominant negative form of ubiquitin. We show that expression of P23H, but not wild-type rhodopsin, results in a generalized impairment of the ubiquitin proteasome system, suggesting a mechanism for photoreceptor degeneration that links RP to a broad class of neurodegenerative diseases.     

Mice with a D190N mutation in the gene encoding rhodopsin: a model for human autosomal-dominant retinitis pigmentosa

Rhodopsin is the G protein-coupled receptor in charge of initiating signal transduction in rod photoreceptor cells upon the arrival of the photon. D190N (Rho(D190n)), a missense mutation in rhodopsin, causes autosomal-dominant retinitis pigmentosa (adRP) in humans. Affected patients present hyperfluorescent retinal rings and progressive rod photoreceptor degeneration. Studies in humans cannot reveal the molecular processes causing the earliest stages of the condition, thus necessitating the creation of an appropriate animal model. A knock-in mouse model with the D190N mutation was engineered to study the pathogenesis of the disease. Electrophysiological and histological findings in the mouse were similar to those observed in human patients, and the hyperfluorescence pattern was analogous to that seen in humans, confirming that the D190N mouse is an accurate model for the study of adRP.     

Deletions in exon 5 of the human rhodopsin gene causing a shift in the reading frame and autosomal dominant retinitis pigmentosa

By screening patients with autosomal dominant retinitis pigmentosa for mutations in the rhodopsin gene, two deletions (8bp and 1bp) have been identified in exon 5; these deletions cause a shift in the reading frame. The predicted proteins should be radically altered with translation continuing past the normal stop signal and resulting in a rhodopsin molecule that is, respectively, 1 and 10 amino acids longer. The clinical phenotype of the patients is described and is compared with that associated with other mutations in the same region of the gene.