Severe retinitis pigmentosa mapped to 4p15 and associated with a novel mutation in the PROM1 gene

Mutation in the PROM1 gene previously has been identified in one family with retinal degeneration for which neither ERG recordings nor detailed information about visual impairment is available. A large family with multiple individuals affected by retinal degeneration was ascertained in the Punjab province of Pakistan. The visual acuity of all affected patients in the family was severely compromised beginning in early childhood. The retinal disease in this family is a severe form of retinitis pigmentosa (RP) accompanied by macular degeneration. Fundus changes advanced with age. Choriocapillaris atrophy and posterior RPE atrophy were obvious allowing visualization of the large choroidal vessels in patients over 40 years of age. Rod and cone responses on ERG recordings were extinguished in patient’s teens. A genome-wide scan mapped the disease to a 34.7 cM region of chromosome 4p14–p16 between D4S1599 and D4S405. A maximum lod score of 3.96 with D4S403 and D4S391 is seen at θ = 0. Sequence analysis of PROM1 located in the linkage interval identified a c.1726C>T homozygous transition in exon 15: resulting in p.Gln576X in the translated protein. This mutation is found in a homozygous state in all six affected individuals and was heterozygous in five of the six unaffected family members examined. The mutation was not detected in 192 chromosomes of unrelated control individuals of the same ethnicity and from the same region. This delineates the phenotypic characteristics of retinopathy caused by mutations in PROM1. Qingjiong Zhang, Fareeha Zulfiqar, Xueshan Xiao, Sheikh Riazuddin and J. Fielding Hejtmancik contributed equally.     

Autosomal recessive retinitis pigmentosa and E150K mutation in the opsin gene

Retinitis pigmentosa (RP) is a heterogeneous group of hereditary disorders of the retina caused by mutation in genes of the photoreceptor proteins with an autosomal dominant (adRP), autosomal recessive (arRP), or X-linked pattern of inheritance. Although there are over 100 identified mutations in the opsin gene associated with RP, only a few of them are inherited with the arRP pattern. E150K is the first reported missense mutation associated with arRP. This opsin mutation is located in the second cytoplasmic loop of this G protein-coupled receptor. E150K opsin expressed in HEK293 cells and reconstituted with 11-cis-retinal displayed an absorption spectrum similar to the wild type (WT) counterpart and activated G protein transducin slightly faster than WT receptor. However, the majority of E150K opsin showed a higher apparent molecular mass in SDS-PAGE and was resistant to endoglycosidase H deglycosidase. Instead of being transported to the plasma membrane, E150K opsin is partially colocalized with the cis/medial Golgi compartment markers such as GM130 and Vti1b but not with the trans-Golgi network. In contrast to the endoplasmic reticulum-retained adRP mutant, P23H opsin, Golgi-retained E150K opsin did not influence the proper transport of the WT opsin when coexpressed in HEK293 cells. This result is consistent with the recessive pattern of inheritance of this mutation. Thus, our study reveals a novel molecular mechanism for retinal degeneration that results from deficient export of opsin from the Golgi apparatus.     

The role of mislocalized phototransduction in photoreceptor cell death of retinitis pigmentosa

Most of inherited retinal diseases such as retinitis pigmentosa (RP) cause photoreceptor cell death resulting in blindness. RP is a large family of diseases in which the photoreceptor cell death can be caused by a number of pathways. Among them, light exposure has been reported to induce photoreceptor cell death. However, the detailed mechanism by which photoreceptor cell death is caused by light exposure is unclear. In this study, we have shown that even a mild light exposure can induce ectopic phototransduction and result in the acceleration of rod photoreceptor cell death in some vertebrate models. In ovl, a zebrafish model of outer segment deficiency, photoreceptor cell death is associated with light exposure. The ovl larvae show ectopic accumulation of rhodopsin and knockdown of ectopic rhodopsin and transducin rescue rod photoreceptor cell death. However, knockdown of phosphodiesterase, the enzyme that mediates the next step of phototransduction, does not. So, ectopic phototransduction activated by light exposure, which leads to rod photoreceptor cell death, is through the action of transducin. Furthermore, we have demonstrated that forced activation of adenylyl cyclase in the inner segment leads to rod photoreceptor cell death. For further confirmation, we have also generated a transgenic fish which possesses a human rhodopsin mutation, Q344X. This fish and rd10 model mice show photoreceptor cell death caused by adenylyl cyclase. In short, our study indicates that in some RP, adenylyl cyclase is involved in photoreceptor cell death pathway; its inhibition is potentially a logical approach for a novel RP therapy.     

Identical mutation in a novel retinal gene causes progressive rod-cone degeneration in dogs and retinitis pigmentosa in humans

Progressive rod-cone degeneration (prcd) is a late-onset, autosomal recessive photoreceptor degeneration of dogs and a homolog for some forms of human retinitis pigmentosa (RP). Previously, the disease-relevant interval was reduced to a 106-kb region on CFA9, and a common phenotype-specific haplotype was identified in all affected dogs from several different breeds and breed varieties. Screening of a canine retinal EST library identified partial cDNAs for novel candidate genes in the disease-relevant interval. The complete cDNA of one of these, PRCD, was cloned in dog, human, and mouse. The gene codes for a 54-amino-acid (aa) protein in dog and human and a 53-aa protein in the mouse; the first 24 aa, coded for by exon 1, are highly conserved in 14 vertebrate species. A homozygous mutation (TGC --> TAC) in the second codon shows complete concordance with the disorder in 18 different dog breeds/breed varieties tested. The same homozygous mutation was identified in a human patient from Bangladesh with autosomal recessive RP. Expression studies support the predominant expression of this gene in the retina, with equal expression in the retinal pigment epithelium, photoreceptor, and ganglion cell layers. This study provides strong evidence that a mutation in the novel gene PRCD is the cause of autosomal recessive retinal degeneration in both dogs and humans.     

A diffusible factor from normal retinal cells promotes rod photoreceptor survival in an in vitro model of retinitis pigmentosa

Transgenic mice expressing a dominant mutation in the gene for the phototransduction molecule rhodopsin undergo retinal degeneration similar to that experienced by patients with the retinal degenerative disease, retinitis pigmentosa (RP). Although the mutation is thought to cause photoreceptor degeneration in a cell‐autonomous manner, the fact that rod photoreceptor degeneration is slowed in chimeric wild‐type/mutant mice suggests that cellular interactions are also important for maintaining photoreceptor survival. To more fully characterize the nature of the cellular interactions important for rod degeneration in the RP mutant mice, we have used an in vitro approach. We found that when the retinas of the transgenic mice were isolated from the pigmented epithelium and cultured as explants, the rod photoreceptors underwent selective degeneration with a similar time course to that observed in vivo. This selective rod degeneration also occurred when the cells were dissociated and cultured as monolayers. These data indicate that the mutant rod photoreceptors degenerate when removed from their normal cellular relationships and without contact with the pigmented epithelium, thus confirming the relative cell autonomy of the mutant phenotype. We next tested whether normal retinal cells could rescue the mutant photoreceptors in a coculture paradigm. Coculture of transgenic mouse with wild‐type mouse or rat retinal cells significantly enhanced transgenic rod photoreceptor survival; this survival‐promoting activity was diffusible through a filter, was heat labile, and not present in transgenic retinal cells. Several peptide growth factors known to be present in the retina were tested as the potential survival‐promoting molecule responsible for the effects of the conditioned medium; however, none of them promoted survival of the photoreceptors expressing the Pro23His mutant rhodopsin. Nevertheless, we were able to demonstrate that the mutant photoreceptors could be rescued by an antagonist to a retinoic acid receptor, suggesting that the endogeneous survival‐promoting activity may function through this pathway. These data thus confirm and extend the findings of previous work that local trophic interactions are important in regulating rod photoreceptor degeneration in retinitis pigmentosa. A diffusible factor found in normal but not transgenic retinal cells has a protective effect on the survival of rod photoreceptors from Pro23His mutant rhodopsin mice. © 1999 John Wiley & Sons, Inc. J Neurobiol 39: 475–490, 1999     

Lysosomal membrane permeabilization and autophagy blockade contribute to photoreceptor cell death in a mouse model of retinitis pigmentosa

Retinitis pigmentosa is a group of hereditary retinal dystrophies that normally result in photoreceptor cell death and vision loss both in animal models and in affected patients. The rd10 mouse, which carries a missense mutation in the Pde6b gene, has been used to characterize the underlying pathophysiology and develop therapies for this devastating and incurable disease. Here we show that increased photoreceptor cell death in the rd10 mouse retina is associated with calcium overload and calpain activation, both of which are observed before the appearance of signs of cell degeneration. These changes are accompanied by an increase in the activity of the lysosomal protease cathepsin B in the cytoplasm of photoreceptor cells, and a reduced colocalization of cathepsin B with lysosomal markers, suggesting that lysosomal membrane permeabilization occurs before the peak of cell death. Moreover, expression of the autophagosomal marker LC3-II (lipidated form of LC3) is reduced and autophagy flux is blocked in rd10 retinas before the onset of photoreceptor cell death. Interestingly, we found that cell death is increased by the induction of autophagy with rapamycin and inhibited by calpain and cathepsin inhibitors, both ex vivo and in vivo. Taken together, these data suggest that calpain-mediated lysosomal membrane permeabilization underlies the lysosomal dysfunction and downregulation of autophagy associated with photoreceptor cell death.Autophagy is a cellular self-degradative pathway that mediates the recycling of damaged or disposable cell components and is activated in situations of nutritional, oxidative and other forms of stress.¹ This process begins with the formation of the autophagosome, an intracellular double-membrane organelle that surrounds parts of the cytoplasm containing organelles and protein aggregates. Autophagosomes subsequently fuse with lysosomes to initiate the degradation of the engulfed cellular components. Autophagy dysfunction has been implicated in many pathological conditions including infections, cancer and muscular and degenerative diseases.² In the nervous system, autophagy has a key role in preventing intracellular accumulation of misfolded and/or aggregated proteins, and its pharmacological upregulation through the administration of rapamycin and other drugs exerts protective effects against a wide range of proteinopathies.³ Moreover, defects in different stages of the autophagy pathway, including autophagosome formation, cargo recognition and lysosomal fusion and degradation, have been often implicated in neurodegeneration.⁴In addition to their degradative role, lysosomes are emerging as key regulators of cellular homeostasis, acting as nutritional sensors or actively participating in cell death.^{5, 6, 7} Lysosomal alterations including increases in lysosomal pH and lysosomal membrane permeabilization (LMP) have been demonstrated in Alzheimer''s and Parkinson''s diseases,^{8, 9} and mutations in lysosomal enzymes cause defects in autophagy, inducing a marked neurodegenerative phenotype in patients with lysosomal storage disorders.¹⁰ LMP induces the selective translocation of cathepsins to the cytoplasm, triggering caspase-dependent and -independent cell death.^{11, 12, 13} LMP has been implicated in mammary gland involution in physiological conditions,¹⁴ indicating that lysosomal-mediated cell death is not merely a consequence of accidental lysosomal damage. As in vivo administration of cathepsin inhibitors attenuates cell death in this model, a similar approach could hold therapeutic potential for the treatment of diseases associated with LMP, including Parkinson''s disease, Niemann–Pick disease type A and stroke.^{7, 10, 15} Oxidative stress and calpain activation are some of the many stimuli that can induce LMP, and have been observed both in vitro and in vivo.⁷ Several pathological processes in the nervous system associated with cell death, including excitotoxicity and ischaemia–reperfusion, have been linked to increased calpain activation.¹⁶ Calpains have also been shown to cleave many intracellular substrates including autophagy and lysosomal proteins,^{17, 18} suggesting links between calcium levels, calpain activation, lysosomal damage and autophagy blockade.Recent findings have begun to shed light on the role of autophagy in the retina. We previously reported decreased autophagy flux in the retinas of aged mice,¹⁹ and demonstrated photoreceptor cell death and decreased dim-light vision in the neuronal-specific Atg5-deficient mouse, a phenotype that closely resembles that observed during physiological aging.¹⁹ We have also demonstrated the essential cytoprotective role of autophagy in vivo in response to retinal ganglion cell damage in experimental models of glaucoma.²⁰ A recent study described lysosomal basification and decreased autophagic flux in travecular meshwork cells in response to chronic oxidative stress, with important implications for the pathogenesis of glaucoma.²¹ Furthermore, specific Atg5 deletion in pigment epithelium leads to reduced levels of visual pigments and vision alterations,²² indicating that autophagy has an important role in sustaining retinal pigment epithelium function.Retinitis pigmentosa is a large group of genetic disorders that normally involves photoreceptor cell death and leads to vision loss in both animal models and affected patients. To date, no treatment for this devastating disease has been developed to clinic. The study of animal models is thus essential to unravel the mechanisms of photoreceptor degeneration involved in these disorders and to identify therapeutic targets. The rd10 mouse, which harbours a mutation in the rod-specific phosphodiesterase gene Pde6b, is a suitable model of human retinitis pigmentosa.^{23, 24} This mutation results in reduced enzymatic function leading to increased cGMP and rod cell death, peaking around postnatal day 25 (P25), with only residual vision remaining after P30.^{24, 25} Here we show that rd10 mice exhibit massive intracellular calcium accumulation and m-calpain (calpain-2) activation at early ages, before the peak of photoreceptor cell death, that correlate with the blockade of autophagic flux. Moreover, we demonstrate an increase in cathepsin B activity in the cytoplasm of rd10 photoreceptors that correlates with the activation of DNAse II-dependent cell death. Induced calcium overload in wild-type (Wt) retinal explants phenocopies the degenerative features seen in rd10 retinas: lysosomal damage, cathepsin translocation and cell death. Finally, we show that calpain and cathepsin inhibitors attenuate cell death both in vitro, ex vivo and in vivo. Taken together, these data suggest that calpain-mediated LMP underlies the lysosomal dysfunction and downregulation of autophagy associated with photoreceptor cell death.     

Characterization of the human diacylglycerol kinase epsilon gene and its assessment as a candidate for inherited retinitis pigmentosa

Human diacylglycerol kinase epsilon (hDGK epsilon) displays high selectivity for arachidonate-containing substrates and may be essential in the termination of signals transmitted through arachidonoyl-diacylglycerol and/or the synthesis of phospholipids with defined fatty acid composition. We herein report the genomic structure, chromosomal mapping, and mutation screening of hDGK epsilon gene. hDGK epsilon gene contains at least 12 exons spanning approximately 30 kb of genomic sequence and was mapped to chromosome 17q22 by fluorescence in situ hybridization. A search for disease gene linkage revealed that a locus for autosomal dominant retinitis pigmentosa (adRP) known as RP17 resided in that region, and Northern blot analysis showed that hDGK epsilon was expressed in human retina. The hDGK epsilon gene was then localized to one of the YAC clones containing a STS marker for the RP17 locus by YAC contig mapping. Direct sequencing following PCR amplification of two affected DNA samples from that type of adRP patients, however, did not reveal any mutation in hDGK epsilon exons.     

Mutation analysis of codons 345 and 347 of rhodopsin gene in Indian retinitis pigmentosa patients

More than 100 mutations have been reported till date in the rhodopsin gene in patients with retinitis pigmentosa. The present study was undertaken to detect the reported rhodopsin gene point mutations in Indian retinitis pigmentosa patients. We looked for presence or absence of codon 345 and 347 mutations in exon 5 of the gene using the technique of allele-specific polymerase chain reaction by designing primers for each mutation. We have examined 100 patients from 76 families irrespective of genetic categories. Surprisingly, in our sample the very widely reported highly frequent mutations of codon 347 (P → S/A/R/Q/L/T) were absent while the codon 345 mutation V → M was seen in three cases in one family (autosomal dominant form) and in one sporadic case (total two families). This is the first report on codon 345 and 347 mutation in Indian retinitis pigmentosa subjects.     

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.     

A comprehensive mutation analysis of RP2 and RPGR in a North American cohort of families with X-linked retinitis pigmentosa

X-linked retinitis pigmentosa (XLRP) is a clinically and genetically heterogeneous degenerative disease of the retina. At least five loci have been mapped for XLRP; of these, RP2 and RP3 account for 10%-20% and 70%-90% of genetically identifiable disease, respectively. However, mutations in the respective genes, RP2 and RPGR, were detected in only 10% and 20% of families with XLRP. Mutations in an alternatively spliced RPGR exon, ORF15, have recently been shown to account for 60% of XLRP in a European cohort of 47 families. We have performed, in a North American cohort of 234 families with RP, a comprehensive screen of the RP2 and RPGR (including ORF15) genes and their 5' upstream regions. Of these families, 91 (39%) show definitive X-linked inheritance, an additional 88 (38%) reveal a pattern consistent with X-linked disease, and the remaining 55 (23%) are simplex male patients with RP who had an early onset and/or severe disease. In agreement with the previous studies, we show that mutations in the RP2 gene and in the original 19 RPGR exons are detected in <10% and approximately 20% of XLRP probands, respectively. Our studies have revealed RPGR-ORF15 mutations in an additional 30% of 91 well-documented families with X-linked recessive inheritance and in 22% of the total 234 probands analyzed. We suggest that mutations in an as-yet-uncharacterized RPGR exon(s), intronic changes, or another gene in the region might be responsible for the disease in the remainder of this North American cohort. We also discuss the implications of our studies for genetic diagnosis, genotype-phenotype correlations, and gene-based therapy.     

A gene mutated in nephronophthisis and retinitis pigmentosa encodes a novel protein,nephroretinin, conserved in evolution

Nephronophthisis (NPHP) comprises a group of autosomal recessive cystic kidney diseases, which constitute the most frequent genetic cause for end-stage renal failure in children and young adults. The most prominent histologic feature of NPHP consists of development of renal fibrosis, which, in chronic renal failure of any origin, represents the pathogenic event correlated most strongly to loss of renal function. Four gene loci for NPHP have been mapped to chromosomes 2q13 (NPHP1), 9q22 (NPHP2), 3q22 (NPHP3), and 1p36 (NPHP4). At all four loci, linkage has also been demonstrated in families with the association of NPHP and retinitis pigmentosa, known as “Senior-Løken syndrome” (SLS). Identification of the gene for NPHP type 1 had revealed nephrocystin as a novel docking protein, providing new insights into mechanisms of cell-cell and cell-matrix signaling. We here report identification of the gene (NPHP4) causing NPHP type 4, by use of high-resolution haplotype analysis and by demonstration of nine likely loss-of-function mutations in six affected families. NPHP4 encodes a novel protein, nephroretinin, that is conserved in evolution—for example, in the nematode Caenorhabditis elegans. In addition, we demonstrate two loss-of-function mutations of NPHP4 in patients from two families with SLS. Thus, we have identified a novel gene with critical roles in renal tissue architecture and ophthalmic function.     

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.     

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.