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Rhodopsin ( RHO ) mutations such as Pro23His are the leading cause of dominantly inherited retinitis pigmentosa in North America. As with other dominant retinal dystrophies, these mutations lead to production of a toxic protein product, and treatment will require knockdown of the mutant allele. The purpose of this study was to develop a CRISPR-Cas9-mediated transcriptional repression strategy using catalytically inactive Staphylococcus aureus Cas9 (dCas9) fused to the Krüppel-associated box (KRAB) transcriptional repressor domain. Using a reporter construct carrying green fluorescent protein (GFP) cloned downstream of the RHO promoter fragment (nucleotides -1403 to +73), we demonstrate a ∼74-84% reduction in RHO promoter activity in RHOp CRISPRi-treated versus plasmid-only controls. After subretinal transduction of human retinal explants and transgenic Pro23His mutant pigs, significant knockdown of rhodopsin protein was achieved. Suppression of mutant transgene in vivo was associated with a reduction in endoplasmic reticulum (ER) stress and apoptosis markers and preservation of photoreceptor cell layer thickness.

In humans, mutations in the beta subunit of cGMP-phosphodiesterase type 6 (PDE6B) cause autosomal recessive retinitis pigmentosa (RP), which typically has an aggressive clinical course of early-onset severe vision loss due to rapid photoreceptor degeneration. In this study, we describe the generation of a novel Pde6b-deficient rat model using CRISPR-Cas9 genome editing. We characterize the model at multiple time points using clinical imaging modalities as well as histology with immunohistochemistry to show rapid photoreceptor degeneration compared to wild-type and heterozygous animals. We describe the manufacture of two different adeno-associated viral (AAV) vectors (AAV2/1, AAV2/5) under current Good Manufacturing Practices (cGMP) and demonstrate their ability to drive human PDE6B expression in vivo. We further demonstrate the ability of AAV-mediated subretinal gene therapy to delay photoreceptor loss in Pde6b-deficient rats compared to untreated controls. However, severe progressive photoreceptor loss was noted even in treated eyes, likely due to the aggressive nature of the disease. These data provide useful preclinical data to guide the development of potential human gene therapy for PDE6B-associated RP. In addition, the rapid photoreceptor degeneration of the Pde6b-deficient rat with intact inner retina may provide a useful model for the study of cell replacement strategies., (© 2022. The Author(s), under exclusive licence to Springer Nature Limited.)

By combining next generation whole exome sequencing and induced pluripotent stem cell (iPSC) technology we found that an Alu repeat inserted in exon 9 of the MAK gene results in a loss of normal MAK transcript and development of human autosomal recessive retinitis pigmentosa (RP). Although a relatively rare cause of disease in the general population, the MAK variant is enriched in individuals of Jewish ancestry. In this population, 1 in 55 individuals are carriers and one third of all cases of recessive RP is caused by this gene. The purpose of this study was to determine if a viral gene augmentation strategy could be used to safely restore functional MAK protein as a step toward a treatment for early stage MAK-associated RP. Patient iPSC-derived photoreceptor precursor cells were generated and transduced with viral vectors containing the MAK transcript. One week after transduction, transcript and protein could be detected via rt-PCR and western blotting respectively. Using patient-derived fibroblast cells and mak knockdown zebra fish we demonstrate that over-expression of the retinal MAK transgene restored the cells ability to regulate primary cilia length. In addition, the visual defect in mak knockdown zebrafish was mitigated via treatment with the retinal MAK transgene. There was no evidence of local or systemic toxicity at 1-month or 3-months following subretinal delivery of clinical grade vector into wild type rats. The findings reported here will help pave the way for initiation of a phase 1 clinical trial for the treatment of patients with MAK-associated RP., (© 2021. The Author(s).)

Loss of photoreceptor cells is a primary feature of inherited retinal degenerative disorders including age-related macular degeneration and retinitis pigmentosa. To restore vision in affected patients, photoreceptor cell replacement will be required. The ideal donor cells for this application are induced pluripotent stem cells (iPSCs) because they can be derived from and transplanted into the same patient obviating the need for long-term immunosuppression. A major limitation for retinal cell replacement therapy is donor cell loss associated with simple methods of cell delivery such as subretinal injections of bolus cell suspensions. Transplantation with supportive biomaterials can help maintain cellular integrity, increase cell survival, and encourage proper cellular alignment and improve integration with the host retina. Using a pig model of retinal degeneration, we recently demonstrated that polycaprolactone (PCL) scaffolds fabricated with two photon lithography have excellent local and systemic tolerability. In this study, we describe rapid photopolymerization-mediated production of PCL-based bioabsorbable scaffolds, a technique for loading iPSC-derived retinal progenitor cells onto the scaffold, methods of surgical transplantation in an immunocompromised rat model and tolerability of the subretinal grafts at 1, 3, and 6 months of follow-up ( n = 150). We observed no local or systemic toxicity, nor did we observe any tumor formation despite extensive clinical evaluation, clinical chemistry, hematology, gross tissue examination and detailed histopathology. Demonstrating the local and systemic compatibility of biodegradable scaffolds carrying human iPSC-derived retinal progenitor cells is an important step toward clinical safety trials of this approach in humans.

Purpose: To investigate receptor and post-receptor function in KCNV2 retinopathy [cone dystrophy with supernormal rod electroretinogram (ERG)], using the pupillary light reflex (PLR) and the ERG., Methods: Two unrelated patients (1 male and 1 female) with molecularly confirmed KCNV2 retinopathy underwent full-field two-color pupillometry testing in one eye, with monitoring of the stimulated eye by an infrared digital camera. Pupillometry stimuli consisted of 1-s duration, short-wavelength (465-nm, blue) and long-wavelength (642-nm, red) stimuli. Pupillometry intensity series were performed under both a dark-adapted condition and a light-adapted condition (on a 0.76-log cd m -2 blue background). The transient PLR, defined as the maximum constriction following flash onset, was measured under all conditions. The melanopsin-mediated sustained constriction was measured 5-7 s following flash offset for the highest flash luminance presented in the dark. Both patients were also tested in one eye with the full-field ERG, including a dark-adapted intensity series and ISCEV standard stimuli., Results: Dark-adapted PLRs were markedly attenuated or extinguished for low-luminance stimuli, but the responses to higher-luminance blue stimuli were within normal limits. Light-adapted PLRs to blue stimuli were generally within normal limits, exceeding the responses to photopically matched red stimuli. Thus, light-adapted responses were consistent with either rod or S-cone mediation of the PLR. Melanopsin-mediated sustained PLRs were within normal limits. ERG showed the characteristic findings previously reported in this condition. Cone-mediated ERG responses were markedly decreased in amplitude. Rod-mediated ERG responses were absent for low-luminance stimuli (- 3 log cd s m -2 ), but had normal amplitude for stimuli of - 2 log cd s m -2 and above (although none were "supernormal"). The b-wave for the dark-adapted ISCEV standard - 2 log cd s m -2 stimulus was markedly delayed, whereas the b-wave timing was generally normal for higher flash luminances., Conclusions: The abnormalities measured by pupillometry have a similar pattern to the outer-retinal abnormalities measured by ERG in KCNV2 retinopathy. These findings as well as the normal sustained PLR suggest that inner-retinal function may be preserved in KCNV2 retinopathy and highlight the potential for therapies designed to restore outer-retinal function in these individuals.

In a screen of 1,000 consecutively ascertained families, we recently found that mutations in the gene RPGR are the third most common cause of all inherited retinal disease. As the two most frequent disease-causing genes, ABCA4 and USH2A , are far too large to fit into clinically relevant adeno-associated virus (AAV) vectors, RPGR is an obvious early target for AAV-based ocular gene therapy. In generating plasmids for this application, we discovered that those containing wild-type RPGR sequence, which includes the highly repetitive low complexity region ORF15, were extremely unstable ( i.e. , they showed consistent accumulation of genomic changes during plasmid propagation). To develop a stable RPGR gene transfer vector, we used a bioinformatics approach to identify predicted regions of genomic instability within ORF15 ( i.e. , potential non-B DNA conformations). Synonymous substitutions were made in these regions to reduce the repetitiveness and increase the molecular stability while leaving the encoded amino acid sequence unchanged. The resulting construct was subsequently packaged into AAV serotype 5, and the ability to drive transcript expression and functional protein production was demonstrated via subretinal injection in rat and pull-down assays, respectively. By making synonymous substitutions within the repetitive region of RPGR , we were able to stabilize the plasmid and subsequently generate a clinical-grade gene transfer vector (IA-RPGR). Following subretinal injection in rat, we demonstrated that the augmented transcript was expressed at levels similar to wild-type constructs. By performing in vitro pull-down experiments, we were able to show that IA-RPGR protein product retained normal protein binding properties ( i.e. , analysis revealed normal binding to PDE6D, INPP5E, and RPGRIP1L). In summary, we have generated a stable RPGR gene transfer vector capable of producing functional RPGR protein, which will facilitate safety and toxicity studies required for progression to an Investigational New Drug application.

Retinitis pigmentosa (RP) is an inherited retinal degenerative disease with severe vision impairment leading to blindness. About 10-15% of RP cases are caused by mutations in the RPGR gene, with RPGR mutations accounting for 70% of X-linked RP cases. The mechanism by which RPGR mutations cause photoreceptor cell dysfunction is not well understood. In this study, we show that the two isoforms of RPGR (RPGR 1-19 and RPGR ORF15 ) interact with endogenous PDE6D, INPP5E, and RPGRIP1L. The RPGR 1-19 isoform contains two PDE6D binding sites with the C-terminal prenylation site being the predominant PDE6D binding site. The C terminus of RPGR 1-19 that contains the prenylation site regulates its interaction with PDE6D, INPP5E, and RPGRIP1L. Only the RPGR 1-19 isoform localizes to cilia in cultured RPE1 cells. Missense variations found in RPGR patients disrupt the interaction between RPGR isoforms and their endogenous interactors INPP5E, PDE6D, and RPGRIP1L. We evaluated a RPGR missense variation (M58K) found in a family with X-linked retinitis pigmentosa (XLRP) and show that this missense variation disrupts the interaction of RPGR isoforms with their endogenous interactors. The M58K variation also disrupts the ciliary localization of the RPGR 1-19 isoform. Using this assay, we also show that some of the RPGR missense variants reported in the literature might not actually be disease causing. Our data establishes an in vitro assay that can be used to validate the potential pathogenicity of RPGR missense variants., Competing Interests: The authors declare no conflict of interest.

Purpose: GUCY2D has been associated with autosomal recessive Leber congenital amaurosis and autosomal dominant cone-rod dystrophy. This report expands the phenotype of autosomal recessive mutations to congenital night blindness, which may slowly progress to mild retinitis pigmentosa., Design: Retrospective case series., Methods: Multicenter study of 5 patients (3 male, 2 female)., Results: All patients presented with night blindness since childhood. Age at referral was 9-45 years. Length of follow-up was 1-7 years. Best-corrected visual acuity at presentation ranged from 20/15 to 20/30 and at most recent visit averaged 20/25. No patient had nystagmus or high refractive error. ISCEV standard electroretinography revealed nondetectable dark-adapted dim flash responses and reduced amplitude but not electronegative dark-adapted bright flash responses with similar waveforms to the reduced-amplitude light-adapted single flash responses. The 30 Hz flicker responses were relatively preserved. Macular optical coherence tomography revealed normal lamination in 3 patients, with abnormalities in 2. Goldmann visual fields were normal at presentation in children but constricted in 1 adult. One child showed loss of midperipheral fields over time. Fundus appearance was normal in childhood; the adult had sparse bone spicule-like pigmentation. Full-field stimulus testing (FST) revealed markedly decreased retinal sensitivity to light. Dark adaptation demonstrated lack of rod-cone break. Two patients had tritanopia. All 5 had compound heterozygous mutations in GUCY2D. Three of the 5 patients harbor the Arg768Trp mutation reported in GUCY2D-associated Leber congenital amaurosis., Conclusions: Autosomal recessive GUCY2D mutations may cause congenital night blindness with normal acuity and refraction, and unique electroretinography. Progression to mild retinitis pigmentosa may occur., (Copyright © 2018 Elsevier Inc. All rights reserved.)

Retinitis pigmentosa (RP) is a heterogeneous group of monogenic disorders characterized by progressive death of the light-sensing photoreceptor cells of the outer neural retina. We recently identified novel hypomorphic mutations in the tRNA Nucleotidyl Transferase, CCA-Adding 1 (TRNT1) gene that cause early-onset RP. To model this disease in vitro, we generated patient-specific iPSCs and iPSC-derived retinal organoids from dermal fibroblasts of patients with molecularly confirmed TRNT1-associated RP. Pluripotency was confirmed using rt-PCR, immunocytochemistry, and a TaqMan Scorecard Assay. Mutations in TRNT1 caused reduced levels of full-length TRNT1 protein and expression of a truncated smaller protein in both patient-specific iPSCs and iPSC-derived retinal organoids. Patient-specific iPSCs and iPSC-derived retinal organoids exhibited a deficit in autophagy, as evidenced by aberrant accumulation of LC3-II and elevated levels of oxidative stress. Autologous stem cell-based disease modeling will provide a platform for testing multiple avenues of treatment in patients suffering from TRNT1-associated RP., (Copyright © 2017. Published by Elsevier B.V.)

Whether we are driving to work or spending time with loved ones, we depend on our sense of vision to interact with the world around us. Therefore, it is understandable why blindness for many is feared above death itself. Heritable diseases of the retina, such as glaucoma, age-related macular degeneration, and retinitis pigmentosa, are major causes of blindness worldwide. The recent success of gene augmentation trials for the treatment of RPE65-associated Leber congenital amaurosis has underscored the need for model systems that accurately recapitulate disease. With the advent of patient-specific induced pluripotent stem cells (iPSCs), researchers are now able to obtain disease-specific cell types that would otherwise be unavailable for molecular analysis. In the present review, we discuss how the iPSC technology is being used to confirm the pathogenesis of novel genetic variants, interrogate the pathophysiology of disease, and accelerate the development of patient-centered treatments. Significance: Stem cell technology has created the opportunity to advance treatments for multiple forms of blindness. Researchers are now able to use a person's cells to generate tissues found in the eye. This technology can be used to elucidate the genetic causes of disease and develop treatment strategies. In the present review, how stem cell technology is being used to interrogate the pathophysiology of eye disease and accelerate the development of patient-centered treatments is discussed., (©AlphaMed Press.)

Retinitis pigmentosa (RP) is a highly heterogeneous group of disorders characterized by degeneration of the retinal photoreceptor cells and progressive loss of vision. While hundreds of mutations in more than 100 genes have been reported to cause RP, discovering the causative mutations in many patients remains a significant challenge. Exome sequencing in an individual affected with non-syndromic RP revealed two plausibly disease-causing variants in TRNT1, a gene encoding a nucleotidyltransferase critical for tRNA processing. A total of 727 additional unrelated individuals with molecularly uncharacterized RP were completely screened for TRNT1 coding sequence variants, and a second family was identified with two members who exhibited a phenotype that was remarkably similar to the index patient. Inactivating mutations in TRNT1 have been previously shown to cause a severe congenital syndrome of sideroblastic anemia, B-cell immunodeficiency, recurrent fevers and developmental delay (SIFD). Complete blood counts of all three of our patients revealed red blood cell microcytosis and anisocytosis with only mild anemia. Characterization of TRNT1 in patient-derived cell lines revealed reduced but detectable TRNT1 protein, consistent with partial function. Suppression of trnt1 expression in zebrafish recapitulated several features of the human SIFD syndrome, including anemia and sensory organ defects. When levels of trnt1 were titrated, visual dysfunction was found in the absence of other phenotypes. The visual defects in the trnt1-knockdown zebrafish were ameliorated by the addition of exogenous human TRNT1 RNA. Our findings indicate that hypomorphic TRNT1 mutations can cause a recessive disease that is almost entirely limited to the retina., (© The Author 2015. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.)

Purpose: To phenotypically and genotypically characterize a large Puerto Rican kindred with X-linked retinitis pigmentosa associated with a novel RP GTPase regulator (RPGR) genotype., Methods: A total of 100 family members of a single kindred with X-linked RP were evaluated with ophthalmic examinations and blood DNA analysis. Visual fields, OCT, and full-field ERG were obtained on all affected males and carriers., Results: Of the 100 family members examined, 13 were affected males and 18 were carriers. A deletion of 2 base pair of the RPGR gene in the ORF15 region at position c.2267-2268 (Lys756del2aaAG hemi) was identified with the affected and carriers. Best eye visual acuity was correlated with age (Spearman coefficient = 0.95) with hand-motion acuity by age 35 and light perception to no light perception by age 50-60. Visual fields were minimally plottable by age 40, and ERG responses reached non-detectable levels by late teens. Carriers had no or mild visual symptoms. All carriers had visual acuity of at least 20/50 or better in one eye, and the amount of retinal degeneration was variable with ERG responses ranging from severely impaired to normal., Conclusions: Profound visual loss occurred by the second decade of life with progression to near no light perception by age 60 in this kindred of X-linked RP associated with the RPGR genotype. Female carriers maintained visual acuity with age and were identifiable by clinical and ERG examination. The information from this study is important to determine the optimal age for intervention, as new RP treatments are being developed and tested.

Variants in ABCA4 are responsible for autosomal-recessive Stargardt disease and cone-rod dystrophy. Sequence analysis of ABCA4 exons previously revealed one causative variant in each of 45 probands. To identify the "missing" variants in these cases, we performed multiplex ligation-dependent probe amplification-based deletion scanning of ABCA4. In addition, we sequenced the promoter region, fragments containing five deep-intronic splice variants, and 15 deep-intronic regions containing weak splice sites. Heterozygous deletions spanning ABCA4 exon 5 or exons 20-22 were found in two probands, heterozygous deep-intronic variants were identified in six probands, and a deep-intronic variant was found together with an exon 20-22 deletion in one proband. Based on ophthalmologic findings and characteristics of the identified exonic variants present in trans, the deep-intronic variants V1 and V4 were predicted to be relatively mild and severe, respectively. These findings are important for proper genetic counseling and for the development of variant-specific therapies., (© 2014 WILEY PERIODICALS, INC.)

Vision is the most important human sense. It facilitates every major activity of daily living ranging from basic communication, mobility and independence to an appreciation of art and nature. Heritable diseases of the retina, such as age-related macular degeneration and retinitis pigmentosa, are the leading cause of blindness in the developed world, collectively affecting as many as one-third of all people over the age of 75, to some degree. For decades, scientists have dreamed of preventing vision loss or of restoring the vision of patients affected with retinal degeneration through some type of drug, gene or cell-based transplantation approach. In this review, we will discuss the current literature pertaining to retinal transplantation. We will focus on the use of induced pluripotent stem cells for interrogation of disease pathophysiology, analysis of drug and gene therapeutics and as a source of autologous cells for cell replacement., (© The Author 2014. Published by Oxford University Press.)

Next-generation and Sanger sequencing were combined to identify disease-causing USH2A mutations in an adult patient with autosomal recessive RP. Induced pluripotent stem cells (iPSCs), generated from the patient's keratinocytes, were differentiated into multi-layer eyecup-like structures with features of human retinal precursor cells. The inner layer of the eyecups contained photoreceptor precursor cells that expressed photoreceptor markers and exhibited axonemes and basal bodies characteristic of outer segments. Analysis of the USH2A transcripts of these cells revealed that one of the patient's mutations causes exonification of intron 40, a translation frameshift and a premature stop codon. Western blotting revealed upregulation of GRP78 and GRP94, suggesting that the patient's other USH2A variant (Arg4192His) causes disease through protein misfolding and ER stress. Transplantation into 4-day-old immunodeficient Crb1 (-/-) mice resulted in the formation of morphologically and immunohistochemically recognizable photoreceptor cells, suggesting that the mutations in this patient act via post-developmental photoreceptor degeneration. DOI:http://dx.doi.org/10.7554/eLife.00824.001.

Purpose: Autosomal recessive retinitis pigmentosa (ARRP) is a genetically heterogeneous condition characterized by progressive loss of retinal photoreceptor cells. In order to gain new insights into the pathogenesis of ARRP, we evaluated the morphological, biochemical, and gene expression changes in eyes from a human donor with ARRP due to mutations in the ABCA4 gene., Methods: Eyes were obtained postmortem from a donor with end-stage retinitis pigmentosa. The coding sequences of the RDS, RHO, and ABCA4 genes were screened for disease-causing mutations. Morphological changes in different regions of the retina were examined histologically, and levels of lipofuscin-associated bisretinoids were measured. Gene expression was examined in retinal/choroidal tissue using microarray analysis, and all parameters were compared to those in unaffected control donors., Results: Genetic analysis of the donor's DNA identified two mutations in the ABCA4 gene, IVS14+1G > C and Phe1440del1 cT, each on a separate allele. Morphological evaluation revealed complete loss of the outer nuclear layer, remodeling of the inner retina, loss of retinal vasculature, and regional neovascularization. The retinal pigment epithelium and choriocapillaris exhibited regional preservation. Microarray analysis revealed loss of photoreceptor cell-associated transcripts, with preservation of multiple genes expressed specifically in inner retinal neurons., Conclusions: The persistence of transcripts expressed by inner retinal neurons suggests that despite significant plasticity that occurs during retinal degeneration, bipolar cells and ganglion cells remain at least partially differentiated. Findings from this study suggest that some forms of therapy currently under investigation may have benefit even in advanced retinal degeneration.

Purpose: To evaluate and compare the protective effect of tauroursodeoxycholic acid (TUDCA) on photoreceptor degeneration in different models of retinal degeneration (RD) in mice., Methods: Bbs(M390R/M390R) mice were injected subcutaneously twice a week, from P40 to P120, and rd10 mice were injected every 3 days from P6 to P38 with TUDCA or vehicle (0.15 M NaHCO(3)). Rd1 and rd16 mice were injected daily from P6 to P30 with TUDCA or vehicle. Retinal structure and function were determined at multiple time points by electroretinography (ERG), optical coherence tomography (OCT), and histology., Results: The amplitude of ERG b-waves was significantly higher in TUDCA-treated Bbs1 and rd10 animals than in controls. Retinal thickness on OCT was slightly greater in treated Bbs1 animals than in the controls. Histologically, outer segments were preserved, and the outer nuclear layer was significantly thicker in the treated Bbs1 and rd10 mice than in the controls. Bbs1(M390R/M390R) mice developed less obesity than the control Bbs1(M390R/M390R) while receiving TUDCA. The Rd1 and rd16 mice showed no improvement with TUDCA treatment, and the rd1 mice did not have normal weight gain during treatment., Conclusions: TUDCA treatment preserved ERG b-waves and the outer nuclear layer in Bbs1(M390R/M390R) mice, and prevented obesity assessed at P120. TUDCA treatment preserved ERG b-waves and the outer nuclear layer in the rd10 mice to P30. TUDCA is a prime candidate for treatment of humans with retinal degeneration, especially those with Bardet-Biedl syndrome, whom it may help not only with the vision loss, but with the debilitating obesity as well.

Purpose: To determine the disease expression in autosomal recessive (ar) retinitis pigmentosa (RP) caused by mutations in the MAK (male germ cell-associated kinase) gene., Methods: Patients with RP and MAK gene mutations (n = 24; age, 32-77 years at first visit) were studied by ocular examination, perimetry, and optical coherence tomography (OCT)., Results: All but one MAK patient were homozygous for an identical truncating mutation in exon 9 and had Ashkenazi Jewish heritage. The carrier frequency of this mutation among 1207 unrelated Ashkenazi control subjects was 1 in 55, making it the most common cause of heritable retinal disease in this population and MAK-associated RP the sixth most common Mendelian disease overall in this group. Visual acuities could be normal into the eighth decade of life. Kinetic fields showed early loss in the superior-temporal quadrant. With more advanced disease, superior and midperipheral function was lost, but the nasal field remained. Only a central island was present at late stages. Pigmentary retinopathy was less prominent in the superior nasal quadrant. Rod-mediated vision was abnormal but detectable in the residual field; all patients had rod>cone dysfunction. Photoreceptor layer thickness was normal centrally but decreased with eccentricity. At the stages studied, there was no evidence of photoreceptor ciliary elongation., Conclusions: The patterns of disease expression in the MAK form of arRP showed some resemblance to patterns described in autosomal dominant RP, especially the form caused by RP1 mutations. The similarity in phenotypes is of interest, considering that there is experimental evidence of interaction between Mak and RP1 in the photoreceptor cilium.

Retinitis pigmentosa (RP) is a genetically heterogeneous heritable disease characterized by apoptotic death of photoreceptor cells. We used exome sequencing to identify a homozygous Alu insertion in exon 9 of male germ cell-associated kinase (MAK) as the cause of disease in an isolated individual with RP. Screening of 1,798 unrelated RP patients identified 20 additional probands homozygous for this insertion (1.2%). All 21 affected probands are of Jewish ancestry. MAK encodes a kinase involved in the regulation of photoreceptor-connecting cilium length. Immunohistochemistry of human donor tissue revealed that MAK is expressed in the inner segments, cell bodies, and axons of rod and cone photoreceptors. Several isoforms of MAK that result from alternative splicing were identified. Induced pluripotent stem cells were derived from the skin of the proband and a patient with non-MAK-associated RP (RP control). In the RP control individual, we found that a transcript lacking exon 9 was predominant in undifferentiated cells, whereas a transcript bearing exon 9 and a previously unrecognized exon 12 predominated in cells that were differentiated into retinal precursors. However, in the proband with the Alu insertion, the developmental switch to the MAK transcript bearing exons 9 and 12 did not occur. In addition to showing the use of induced pluripotent stem cells to efficiently evaluate the pathogenicity of specific mutations in relatively inaccessible tissues like retina, this study reveals algorithmic and molecular obstacles to the discovery of pathogenic insertions and suggests specific changes in strategy that can be implemented to more fully harness the power of sequencing technologies.

Purpose: To correlate visual acuity of patients with Leber's congenital amaurosis (LCA) and early childhood-onset retinitis pigmentosa (RP) with mutations in underlying LCA genes., Design: Multicentered retrospective observational study., Participants: After exclusion of 28 subjects, 169 patients with the diagnosis of LCA and 27 patients with early childhood-onset RP were included in the study because the underlying mutations in AIPL1, GUCY2D, RDH12, RPE65, CRX, CRB1, RPGRIP1, CEP290, LCA5, and TULP1 genes could be identified in this cohort of patients., Methods: We collected data on best-corrected visual acuity as recorded at the time of the patient's most recent visit to one of the participating ophthalmology departments. The median and range of visual acuities for each genetic subtype were calculated separately for the LCA and early childhood-onset RP groups., Main Outcome Measures: The range and median best-corrected visual acuities for each genetic subtype and age-related mean visual acuities for each genetic subtype., Results: A wide variation in visual acuity was observed in patients with LCA and RPE65, RDH12, and CRB1 mutations, whereas AIPL1, GUCY2D, CRX, and RPGRIP1 gene mutations were associated with severely decreased visual acuities beginning within the first year of life. It was also noted that patients with either an RPE65 or CRB1 mutation have progressive visual loss with advancing age. Onset of visual symptoms after infancy was associated with a relatively better visual prognosis., Conclusions: The data obtained from this study will help clinicians provide counseling on visual prognosis to patients with known mutations in LCA genes and be of value in future studies aimed at the treatment of LCA and early childhood-onset RP., (Copyright 2010 American Academy of Ophthalmology. Published by Elsevier Inc. All rights reserved.)

Retinitis pigmentosa is a genetically heterogeneous group of inherited ocular disorders characterized by progressive photoreceptor cell loss, night blindness, constriction of the visual field, and progressive visual disability. Homozygosity mapping and gene expression studies identified a 2 exon gene, C2ORF71. The encoded protein has no homologs and is highly expressed in the eye, where it is specifically expressed in photoreceptor cells. Two mutations were found in C2ORF71 in human RP patients: A nonsense mutation (p.W253X) in the first exon is likely to be a null allele; the second, a missense mutation (p.I201F) within a highly conserved region of the protein, leads to proteosomal degradation. Bioinformatic and functional studies identified and validated sites of lipid modification within the first three amino acids of the C2ORF71 protein. Using morpholino oligonucleotides to knockdown c2orf71 expression in zebrafish results in visual defects, confirming that C2ORF71 plays an important role in the development of normal vision. Finally, localization of C2ORF71 to primary cilia in cultured cells suggests that the protein is likely to localize to the connecting cilium or outer segment of photoreceptor cells., (Copyright (c) 2010 The American Society of Human Genetics. Published by Elsevier Inc. All rights reserved.)

Purpose: To define molecular and ophthalmic features of a rare phenotype in autosomal dominant (ad) retinitis pigmentosa (RP)., Methods: A 32-year-old woman (proband) with adRP and the low-frequency damped electroretinographic (ERG) wavelet phenotype and her mother were studied with optical coherence tomography (OCT), chromatic perimetry and ERG. A previously reported adRP patient with this ERG phenotype (Lam et al) was also studied with OCT. Genotype in the two families was determined with DNA sequencing., Results: ERGs from the proband were identical to those reported previously. Chromatic perimetry and ERG stimulus intensity series indicated that there can be severely reduced rod function in addition to substantial cone dysfunction. A heterozygous deletion in peripherin/RDS (Met152del3 atGAA) was present in the patient and the affected mother. There were foveal cystoid changes and pericentral splitting of the inner nuclear layer. ONL thickness and vision tapered with eccentricity, and 'blind' regions without discernible ONL showed a thickened, delaminated inner retina. Similar OCT findings were present in the reported adRP patient with this ERG; the patient was heterozygous for a 4-bp deletion (Leu107del4 ctGAGT) in PRPF31., Conclusions: The low-frequency damped ERG wavelet phenotype is genetically heterogeneous. Inner retinal structural abnormalities are also present in this rare disease expression.

Objective: To identify the gene causing retinitis pigmentosa (RP) in an autosomal dominant pedigree., Methods: Family members with RP were studied with linkage analysis using single-nucleotide polymorphism and short tandem repeat polymorphic markers. Candidate genes in the linked region were evaluated with DNA sequencing., Results: Nineteen family members had a mild form of RP. Multipoint linkage analysis of single-nucleotide polymorphism genotypes yielded a maximum nonparametric linkage score of 19.97 with markers located on chromosome 14q. LOD scores higher than 3.0 were obtained with 20 short tandem repeat polymorphic markers, and recombinants defined a 21.7-centimorgan locus on chromosome 14q. The retinol dehydrogenase 12 (RDH12) gene lies within this locus and was evaluated as a candidate gene. A frameshift mutation (776delG) was detected in all affected family members and was not detected in 158 control subjects., Conclusions: Heterozygous mutations in RDH12 can cause autosomal dominant RP with a late onset and relatively mild severity. This phenotype is dramatically different from the other disease associated with mutation in this gene, autosomal recessive Leber congenital amaurosis., Clinical Relevance: The demonstration that mutations in a gene previously associated with recessive Leber congenital amaurosis can also cause dominant RP illustrates the wide phenotypic variability of retinal degeneration genes.

Purpose: To determine the retinal phenotype of Usher syndrome type III (USH3A) caused by clarin-1 (CLRN1) gene mutations in a non-Finnish population., Methods: Patients with USH3A (n = 13; age range, 24-69) representing 11 different families were studied and the results compared with those from patients with USH2A (n = 24; age range, 17-66). The patients were evaluated by ocular examination, kinetic and static perimetry, near-infrared autofluorescence, and optical coherence tomography (OCT)., Results: Ten of 11 families had Ashkenazi Jewish origins and the N48K CLRN1 mutation. Rod function was lost in the peripheral field in the first two decades of life, but central rod function could be retained for another decade. Peripheral cone function was detectable into the third decade of life. Central cone function had a slower decline that extended for decades. Photoreceptor layer loss and features of retinal remodeling were present in retinal regions with severe visual dysfunction, even at the youngest ages tested. Central retinal structure could be normal in younger patients but structural integrity was lost in older patients. RPE disease generally paralleled photoreceptor degeneration. Comparisons between USH3A and USH2A suggested a common rod and cone phenotype but a more accelerated time course of rod loss in USH3A., Conclusions: USH3A and USH2A share patterns of rod and cone dysfunction and retinal structural abnormalities. Peripheral function measurements showed USH3A to be more rapidly progressive than USH2A.

Purpose: To determine the underlying retinal micropathology in subclasses of autosomal dominant retinitis pigmentosa (ADRP) caused by rhodopsin (RHO) mutations., Methods: Patients with RHO-ADRP (n = 17, ages 6-73 years), representing class A (R135W and P347L) and class B (P23H, T58R, and G106R) functional phenotypes, were studied with optical coherence tomography (OCT), and colocalized visual thresholds were determined by dark- and light-adapted chromatic perimetry. Autofluorescence imaging was performed with near-infrared light. Retinal histology in hT17M-rhodopsin mice was compared with the human results., Results: Class A patients had only cone-mediated vision. The outer nuclear layer (ONL) thinned with eccentricity and was not detectable within 3 to 4 mm of the fovea. Scotomatous extracentral retina showed loss of ONL, thickening of the inner retina, and demelanization of RPE. Class B patients had superior-inferior asymmetry in function and structure. The superior retina could have normal rod and cone vision, normal lamination (including ONL) and autofluorescence of the RPE melanin; laminopathy was found in the scotomas. With Fourier-domain-OCT, there was apparent inner nuclear layer (INL) thickening in regions with ONL thinning. Retinal regions without ONL had a thick hyporeflective layer that was continuous with the INL from neighboring regions with normal lamination. Transgenic mice had many of the laminar abnormalities found in patients., Conclusions: Retinal laminar abnormalities were present in both classes of RHO-ADRP and were related to the severity of colocalized vision loss. The results in human class B and the transgenic mice support the following disease sequence: ONL diminution with INL thickening; amalgamation of residual ONL with the thickened INL; and progressive retinal remodeling with eventual thinning.

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.

Aim: To describe the clinical characteristics and disease course of a large family with retinitis pigmentosa (RP) from an Arg135Leu change in rhodopsin., Methods: 29 patients in this family were evaluated. Goldmann visual fields were performed on 14 affected individuals, Ganzfeld electroretinography (ERG) on eight individuals (11-56 years), and blood samples collected on 10 individuals (11-58 years). Patient visual field data were compared with previously reported patients with different rhodopsin mutations using linear regression., Results: An Arg135Leu mutation was identified in rhodopsin. Distinct stages of clinical evolution were identified for this family ranging from normal, white dots, classic bone spicules and, finally, ending with extensive retinal pigment epithelium (RPE) atrophy. 9/16 patients over the age of 20 years also demonstrated marked macular atrophy. All patients who underwent full field ERG testing demonstrated non-recordable ERGs. The overall regression model comparing solid angles of visual fields from patients with rhodopsin mutations (Pro23His, Pro347Ala, Arg135Leu) shows significant effects for age (p = 0.0005), mutation (p = 0.0014), and interaction between age and mutation (p = 0.018) with an R(2) of 0.407., Conclusions: An Arg135Leu change in rhodopsin results in a severe form of RP that evolves through various fundus appearances that include white dots early in life and classic appearing RP later. This transmembrane change in rhodopsin proves to be more severe than in a family with an intradiscal change and a family with a cytoplasmic change.

Purpose: To define ophthalmic findings in a family with autosomal dominant retinitis pigmentosa and a novel IMPDH1 gene mutation., Design: Genetic and observational family study., Participants: Sixteen affected members of a family with autosomal dominant retinitis pigmentosa., Methods: Ophthalmic examination, including best-corrected visual acuity (VA), slit-lamp biomicroscopy, direct and indirect ophthalmoscopy, Goldmann kinetic perimetry, and electroretinography were performed. Deoxyribonucleic acid single-strand conformation polymorphism (SSCP) analysis was done. Abnormal polymerase chain reaction products identified by SSCP analysis were sequenced bidirectionally., Results: All affected patients had the onset of night blindness within the first decade of life. Ocular findings were characterized by diffuse retinal pigmentary degenerative changes, marked restriction of peripheral visual fields, severe loss of VA, nondetectable electroretinography amplitudes, and a high frequency of posterior subcapsular lens opacities. Affected members were observed to harbor a novel IMPDH1 gene mutation., Conclusion: A novel IMPDH1 gene mutation (Arg231Pro) was associated with a severe form of autosomal dominant retinitis pigmentosa. Families affected with a severe form of this genetic subtype should be investigated for a mutation in the IMPDH1 gene.

Purpose: The Arg677ter mutation in the RP1 gene is one of the most common causes of autosomal dominant retinitis pigmentosa (RP). In the current study, a de novo Arg677ter RP1 gene mutation was identified in a patient with RP., Methods: RP1 gene mutation screening was performed in probands with simplex RP. In one proband with the RP1 mutation, paternity was established by analyzing 24 short tandem repeat polymorphisms. Additional candidate RP genes, including rhodopsin, RDS/peripherin, RP2, and RPGR, were also examined in this proband. Phenotype was characterized with psychophysics, electroretinography, and optical coherence tomography., Results: An RP1 (Arg677ter) mutation was identified in one of the patients with simplex RP, but the sequence change was not detected in his parents. Parentage was confirmed, and other candidate genes were negative for mutations. Retinal function and cross-sectional imaging studies in the patient indicated greater rod than cone dysfunction with a photoreceptor basis for the abnormalities., Conclusions: The de novo origin of an RP1 (Arg677ter) mutation in a patient with simplex RP suggests that this common autosomal dominant RP mutation can arise independently in the population and supports the hypothesis of a mutational hotspot in the RP1 gene.

Purpose: To examine the difference in expression of retinitis pigmentosa from mutations at codon 23 and codon 347 or rhodopsin; to report a novel mutation in rhodopsin., Methods: Goldmann perimetry (solid angle of I4e isopter) and electroretinographic amplitudes (square root transform of a response ratio) were analyzed for 24 patients with mutations at codon 347 (15 with Pro347Ala, 2 with Pro347Gln, 6 with Pro347Leu, and 1 with a novel Pro347Cys change) and 41 patients with mutations at codon 23 (6 with Pro23Ala; 35 with Pro23His)., Results: When all patients with mutations at codons 347 and 23 were compared, loss of visual fields was significantly worse in patients with codon 347 changes (P =.0003). Only rod responses of the electroretinograms were significantly different between the two groups (P =.048). Specific comparison of Pro347Ala with Pro23Ala using regression analysis demonstrated significant differences in severity between codon 23 and codon 347 patients for b-wave amplitudes of rod (P =.0069), cone (P =.039) and maximum combined response (P =.049). The solid angle of the I4e isopter was also significantly different (P =.025) between the groups after controlling for age. Modeling age by group for Pro347Ala comparison produced an R(2) of.44., Conclusion: We reconfirmed that rhodopsin-related retinitis pigmentosa from mutations involving codon 347 produces a more severe phenotype than that involving codon 23. Accurate modeling of disease was shown to be possible by incorporating the effects of a patient's age and specific genotype. Therefore, both of these variables must be considered in prognostic counseling and subject recruitment for future therapeutic trials.

Purpose: To characterize the genomic organization of human IMPG2, the gene encoding the retinal interphotoreceptor matrix (IPM) proteoglycan IPM 200, to evaluate its relationship to IPM 150, and to evaluate its involvement in inherited retinopathies, such as age-related macular degeneration, retinitis pigmentosa, and Leber congenital amaurosis., Methods: After isolation of human genomic clones, the structure of IMPG2 was determined by sequence analysis. Mutational analyses were conducted on genomic DNA isolated from 316 probands using single-strand conformation polymorphism analysis., Results: The IMPG2 gene is organized into 19 exons, and the structure of the gene is highly similar to that of the IMPG1 gene, which encodes another retinal proteoglycan, IPM 150. Mutational analyses indicate that the observed sequence changes are present at approximately equal rates in donors with and without retinal disease. Additional data derived from RT-PCR and Northern blot analysis show that IMPG2 is processed in the human retina into multiple alternatively sized transcripts that may represent splicing isoforms., Conclusions: Analysis of the overall relationship of human IMPG2 (located on chromosome 3q12.2-12.3) to human IMPG1 (located on chromosome 6q14) suggests that these genes have evolved from a common ancestral gene. Although this is an excellent candidate gene for hereditary retinopathies, single-strand conformation polymorphism analyses provided no evidence that variations in IMPG2 coding region are responsible for the inherited retinopathies examined.

Progressive concentric (centripetal) loss of vision is one pattern of visual field loss in retinitis pigmentosa. This study provides the first clinicopathologic correlations for this form of retinitis pigmentosa. A family with autosomal dominant concentric retinitis pigmentosa was examined clinically and with visual function tests. A post-mortem eye of an affected 94 year old family member was processed for histopathology and immunocytochemistry with retinal cell specific antibodies. Unrelated simplex/multiplex patients with concentric retinitis pigmentosa were also examined. Affected family members of the eye donor and patients from the other families had prominent peripheral pigmentary retinopathy with more normal appearing central retina, good visual acuity, concentric field loss, normal or near normal rod and cone sensitivity within the preserved visual field, and reduced rod and cone electroretinograms. The eye donor, at age 90, had good acuity and function in a central island. Grossly, the central region of the donor retina appeared thinned but otherwise normal, while the far periphery contained heavy bone spicule pigment. Microscopically the central retina showed photoreceptor outer segment shortening and some photoreceptor cell loss. The mid periphery had a sharp line of demarcation where more central photoreceptors were near normal except for very short outer segments and peripheral photoreceptors were absent. Rods and cones showed abrupt loss of outer segments and cell death at this interface. It is concluded that concentric retinitis pigmentosa is a rare but recognizable phenotype with slowly progressive photoreceptor death from the far periphery toward the central retina. The disease is retina-wide but shows regional variation in severity of degeneration; photoreceptor death is severe in the peripheral retina with an abrupt edge between viable and degenerate photoreceptors. Peripheral to central gradients of unknown retinal molecule(s) may be defective or modify photoreceptor degeneration in concentric retinitis pigmentosa.

Purpose: To report a new phenotype in retinitis pigmentosa (RP) patients with CRB1 mutations at the RP12 locus., Patients: Thirty-seven patients from two Pakistani families with severe retinitis pigmentosa., Methods: Samples were screened with single-strand conformation polymorphism analysis followed by DNA sequencing of the coding sequence of the CRB1 gene., Results: Two novel CRB1 mutations were discovered. No patients had evidence of preservation of the para-arteriolar retinal pigment epithelium (PPRPE) that has been previously reported in all cases of RP associated with CRB1 mutations., Conclusions: Patients with severe autosomal recessive (or simplex) RP who lack the finding of PPRPE should not be excluded from molecular analysis of CRB1 purely because they lack the clinical feature of PPRPE. This report illustrates that RP at the RP12 locus is not clinically uniform. The absence of PPRPE cannot be used to exclude CRB1 as a potential molecular explanation for RP.

Objectives: To report the clinical characteristics of a family with autosomal dominant retinitis pigmentosa caused by a proline-to-alanine mutation at codon 23 (Pro23Ala) of the rhodopsin gene and to compare this phenotype with that associated with the more common proline-to-histidine mutation at codon 23 (Pro23His)., Methods: We examined 6 patients within a single pedigree. The electroretinograms (ERGs) of 35 patients with known Pro23His mutations and of 22 healthy individuals were reviewed. Scotopic dim flash-response amplitudes, maximum combined-response amplitudes, and photopic-response amplitudes from the ERGs of these patients were plotted against age. The ERG indices of 5 individuals in the Pro23Ala family were compared with those of the patients with Pro23His mutations and of healthy individuals. Multiple linear regression was performed to evaluate the effect of age and mutation type on amplitudes. Mutation detection was performed using single-strand conformation polymorphism analysis, followed by automated DNA sequencing., Results: Patients with the Pro23Ala mutation have a clinical phenotype characterized by onset of symptoms in the second to fourth decades of life, loss of superior visual field with relatively well-preserved inferior fields, and mild nyctalopia. Comparison with patients with the Pro23His mutation demonstrates statistically significant differences (P<.001) in responses to dim flash, maximum combined, and photopic responses between patients with these mutations after controlling for the effects of age. Patients with Pro23Ala mutations were less affected by ERG criteria than patients with Pro23His mutations. Patients with Pro23Ala mutations also differed significantly from healthy patients in all ERG indices examined (P<.001), after controlling for age., Conclusion: We describe a rare mutation in codon 23 of rhodopsin causing autosomal dominant retinitis pigmentosa. The retinal dystrophy associated with the Pro23Ala mutation is characteristically mild in presentation and course, with greater preservation of ERG amplitudes than the more prevalent Pro23His mutation. Arch Ophthalmol. 2000;118:1269-1276

Purpose: To determine the disease expression in heterozygotes for mutations in the RP1 gene, a newly identified cause of autosomal dominant retinitis pigmentosa (adRP)., Methods: Screening strategies were used to detect disease-causing mutations in the RP1 gene, and detailed studies of phenotype were performed in a subset of the detected RP1 heterozygotes using electroretinography (ERG), psychophysics, and optical coherence tomography (OCT)., Results: Seventeen adRP families had heterozygous RP1 changes. Thirteen families had the Arg677ter mutation, whereas four others had one of the following: Pro658 (1-bp del), Ser747 (1-bp del), Leu762-763 (5-bp del), and Tyr1053 (1-bp del). In Arg677ter RP1 heterozygotes, there was regional retinal variation in disease, with the far peripheral inferonasal retina being most vulnerable; central and superior temporal retinal regions were better preserved. The earliest manifestation of disease was rod dysfunction, detectable as reduced rod ERG photoresponse maximum amplitude, even in heterozygotes with otherwise normal clinical, functional, and OCT cross-sectional retinal imaging results. At disease stages when cone abnormalities were present, there was greater rod than cone dysfunction. Patients with the RP1 frameshift mutations showed similarities in phenotype to those with the Arg677ter mutation., Conclusions: Earliest disease expression of RP1 gene mutations causing adRP involves primarily rod photoreceptors, and there is a gradient of vulnerability of retinopathy with more pronounced effects in the inferonasal peripheral retina. At other disease stages, cone function is also affected, and severe retina-wide degeneration can occur. The nonpenetrance or minimal disease expression in some Arg677ter mutation-positive heterozygotes suggests important roles for modifier genes or environmental factors in RP1-related disease.

Purpose: The RHO C110Y mutation has been recently reported to cause a phenotypically unspecified form of autosomal dominant retinitis pigmentosa (adRP). The study of a family affected with this mutation allowed us to hereby describe the genotype/phenotype correlation associated with the RHO C110Y mutation., Methods: A six-generation pedigree cosegregating adRP and RHO C110Y in ten accessible individuals was ophthalmologically investigated. All family members affected with RP went through complete eye examination and ERG testing., Results: The disease first manifested with nyctalopia during adulthood and slowly progressed over the next decades towards tubular visual field defects and relatively preserved central vision. Ophthalmoscopically, the fundus remained almost unaltered until the end of the third decade of life, and then slowly progressed towards typical RP changes with minimal macular involvement by the eighth decade. Color vision remained unaltered. Earliest ERG alteration was limited to the rod system followed by a rod-cone pattern. Scotopic and photopic ERG were recordable until the fourth and sixth decades, respectively., Discussion: RHO C110Y-associated adRP is characterized by a late onset and a mild progression compatible with type 2 or regional RP with little intrafamilial phenotypic variability and complete penetrance. Characterization of genotype-phenotype correlations plays a role in the improvement of genetic and prognostic counselling.

Background: Mutations in the RPGR gene at the RP3 locus have been found to cause x-linked retinitis pigmentosa in some families., Objectives: To identify a previously undescribed 2-base pair insertion in codon 99 of the RPGR gene and to describe the phenotype in a well-characterized family with X-linked retinitis pigmentosa., Design: Case reports with clinical features, fluorescein angiography, kinetic perimetry, electrophysiological studies, and molecular genetics., Setting: University medical centers., Patients: Eight members of the family were screened for the codon 99 insertion in the RPGR gene., Results: Three affected males were found to be hemizygous for the 2-base pair insertion; 2 carriers were heterozygous. This insertion creates a frameshift that would be expected to cause a premature arrest of translation after only 132 amino acids (683 amino acids less than the normal protein). The affected males had typical retinitis pigmentosa with visual field contraction and abnormal findings on electroretinograms with little to no rod activity, profoundly subnormal residual cone responses to single flash and 30-Hz flicker stimuli, and prolonged b-wave implicit times. The electroretinogram of a 49-year-old carrier showed amplitudes that were roughly half of normal. Carrier women did not show a tapetallike fundus reflex but showed asymmetrical patchy pigmentary disturbances consistent with lyonization., Conclusion: A frameshifting 2-base pair insertion at codon 99 of the RPGR gene produced typical retinitis pigmentosa and carrier findings (but no tapetallike reflex) in this family.

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.

Purpose: We identified genetic mutations and characterized their associated phenotypes in patients with retinitis pigmentosa., Methods: Patients with retinitis pigmentosa were prospectively examined and screened for genetic mutations., Results: A 46-year-old man with retinitis pigmentosa was found to have a heterozygous mutation in the peripherin/RDS gene (arginine-46-stop). He had late onset of symptoms and demarcated peripheral retinal atrophy. All five first-degree relatives including his parents had no detectable mutations or retinitis pigmentosa. Genotypic data were consistent with reported family structure., Conclusions: This study shows that new dominant mutations are a rare cause of isolated, or simplex, cases of retinitis pigmentosa. Identification of these mutations is helpful for genetic counseling.

Purpose: To investigate functional abnormalities in mutations in the peripherin (RDS) gene leading to different clinical types of autosomal dominant retinal disease--macular degeneration and retinitis pigmentosa., Methods: Patients from two families, one with a mutation in codon 167 (Gly167Asp) leading to macular degeneration and another with a mutation in codon 210 (Pro210Ser) leading to retinitis pigmentosa, were studied with clinical examinations and measurements of rod and cone sensitivities and dark adaptation, electroretinography, and rhodopsin levels., Results: Mildly affected patients had sizable rod and cone electroretinograms, reduced levels of rhodopsin, and minor losses of sensitivity. In both mutations, there were delays of rod and cone dark adaptation after bleaching, and the adaptational abnormalities were observed in peripheral and central retinal locations. Analysis of the kinetics of rod adaptation indicates that the underlying abnormalities are similar in both mutations and that the effects of the mutations are similar to those caused by mild systemic vitamin A deficiency., Conclusions: Patients with the Gly167Asp and Pro210Ser mutations in the peripherin/RDS gene have widely different clinical phenotypes but show the same abnormality, slowed dark adaptation, of rod and cone photoreceptor function. The similarities of the characteristics of the adaptational abnormalities in the two genotypes suggest that, in addition to the structural roles normally assumed for it, peripherin influences or participates in the function of the visual cycle.

Bardet-Biedl syndrome is an autosomal recessive disorder characterized by mental retardation, obesity, retinitis pigmentosa, polydactyly and hypogonadism. Other findings include hypertension, diabetes mellitus and renal and cardiovascular anomalies. We have performed a genome-wide search for linkage in a large inbred Bedouin family. Pairwise analysis established linkage with the locus D16S408 with no recombination and a lod score of 4.2. A multilocus lod score of 5.3 was observed. By demonstrating homozygosity, in all affected individuals, for the same allele of marker D16S408, further support for linkage is found, and the utility of homozygosity mapping using inbred families is demonstrated. In a second family, linkage was excluded at this locus, suggesting non-allelic genetic heterogeneity in this disorder.

Background and Objectives: Mutations of the peripherin/RDS gene have been reported in autosomal dominant retinitis pigmentosa, pattern macular dystrophy, and retinitis punctata albescens. We report herein the occurrence of three separate phenotypes within a single family with a novel 3-base pair deletion of codon 153 or 154 of the peripherin/RDS gene., Design: Case reports with clinical features, fluorescein angiography, kinetic perimetry, electrophysiological studies, and molecular genetics., Setting: University medical centers., Patients: A 75-year-old woman, her two daughters (aged 44 and 50 years), and her 49-year-old son were screened for peripherin/RDS mutations because of the presence of multiple phenotypes within the same family., Results: The mother presented at age 63 years with a profoundly abnormal electroretinogram (ERG) and adult-onset retinitis pigmentosa that progressed dramatically over 12 years, with marked loss of peripheral visual field. One daughter developed pattern macular dystrophy at age 31 years. At age 44 years, her ERG was moderately abnormal but her clinical disease was limited to the macula. Another daughter presented at age 42 years with macular degeneration and over 10 years developed the clinical picture of fundus flavimaculatus. Her peripheral visual field was preserved but her ERG was moderately abnormal. The son had onset of macular degeneration at age 44 years. Pericentral scotomas were present and the ERG was markedly abnormal. Fluorescein angiography revealed punctate pigment epithelial transmission defects., Conclusions: A 3-base pair deletion of codon 153 or 154 of the peripherin/RDS gene can produce clinically disparate phenotypes even within the same family.

Two members of a family with autosomal dominant retinitis pigmentosa were found to have a cytosine-to-thymine mutation in the second nucleotide of codon 267 in the rhodopsin gene that resulted in a proline-to-leucine change. Two members of another family with autosomal dominant retinitis pigmentosa showed a guanine-to-thymine mutation in the first nucleotide of codon 190 in the rhodopsin gene that resulted in an aspartate-to-tyrosine change. Three members from a third family with autosomal dominant retinitis pigmentosa were also found to have a mutation in codon 190; however, this guanine-to-adenine mutation in the first nucleotide of codon 190 resulted in an aspartate-to-asparagine change. The relatively less severe functional retinal impairment in our patients with a transmembrane codon 267 rhodopsin gene mutation is generally comparable with that observed in a previously described codon 58 transmembrane mutation. The two families with different intradiscal codon 190 mutations showed a considerable difference in severity of their disease.

Three members of one family and one person from another family were found to have a guanine-to-adenine transition mutation in the first nucleotide of codon 106 in the rhodopsin gene that results in a glycine-to-arginine change. All affected members presented with a similar phenotype that included a regional predilection for pigmentary changes to occur in the inferior retina as well as visual field impairment predominantly in the superior hemisphere. The distribution of pigmentary changes, pattern of visual field loss, and substantial remaining electroretinographic amplitudes with normal implicit times were consistent with a form of "sector" retinitis pigmentosa. We documented the association of a distinct phenotype of autosomal dominant retinitis pigmentosa with a better visual prognosis and a specific rhodopsin gene mutation.

Six members of a family with autosomal dominant retinitis pigmentosa were found to have a cytosine-to-thymine transition mutation in the second nucleotide of codon 17 in the rhodopsin gene that resulted in a threonine to methionine change. Three members from another family with autosomal dominant retinitis pigmentosa showed a guanine-to-adenine transition mutation in the first nucleotide of codon 182 in the rhodopsin gene that resulted in a glycine to serine change. Each of these two mutations presented with a similar phenotype because both showed a regional predilection for pigmentary changes to occur in the inferior part of the retina as well as field impairment predominantly in the superior hemisphere. Electroretinographic amplitudes were more substantial than usually encountered in other forms of retinitis pigmentosa, a finding consistent with the better visual prognosis in patients with either of these two mutations. This article documents the association of two similar phenotypes of autosomal dominant retinitis pigmentosa with specific gene defects at a molecular level.

Eight members of a family with autosomal dominant retinitis pigmentosa were found to have a cytosine-to-guanine (C-to-G) transversion mutation in the second nucleotide of codon 58 of the rhodopsin gene, causing a substitution of the amino acid arginine for threonine. Five of these individuals were examined clinically. There was a distinct phenotypic expression of the gene defect within this family that included a regional predilection for pigmentary changes in the inferior and inferonasal parts of the retina, as well as field impairment predominantly in the superior hemisphere. Characteristic electroretinographic recordings and psychophysical threshold profiles also helped to identify this phenotype that, on a relative basis, causes less severe photoreceptor cell functional impairment than often occurs in other subtypes of retinitis pigmentosa. This report documents the association of a clinically recognizable phenotype in a family with autosomal dominant retinitis pigmentosa and a specific gene defect at the molecular level.