Association of Vitamin A Supplementation With Disease Course in Children With Retinitis Pigmentosa.

Importance: While oral vitamin A supplementation is considered to potentially slow loss of retinal function in adults with retinitis pigmentosa and normal liver function, little data from children with this disease are available. Objective: To compare disease courses in children with retinitis pigmentosa taking or not taking vitamin A supplementation. Design, Setting, and Participants: Retrospective, nonrandomized comparison of vitamin A and control cohorts followed up for a mean of 4 to 5 years by the Electroretinography Service of the Massachusetts Eye and Ear Infirmary. The study included children with different genetic types of typical retinitis pigmentosa: 55 taking vitamin A and 25 not taking vitamin A. The dates for patient evaluations ranged from June 1976 to July 2016, and the data analysis occurred in October 2016. Interventions: Age-adjusted dose of oral vitamin A palmitate (≤15 000 IU/d). Main Outcomes and Measures: Mean exponential rates of change of full-field cone electroretinogram amplitude to 30-Hz flashes estimated by repeated-measures longitudinal regression without and with adjusting for potential confounders. Results: Of the 55 children in the vitamin A cohort, 38 (69%) were male; the mean [SD] age was 9.1 [1.9] years; and 48 (87%) were white , 6 (11%) were Asian, and 1 (2%) was black. Of the 25 members of the control cohort, 19 (76%) were male; the mean [SD] age was 9.2 [1.7] years; and 25 (100%) were white. The estimated mean rates of change with the unadjusted model were -0.0713 loge unit/y (-6.9% per year) for the vitamin A cohort and -0.1419 loge unit per year (-13.2% per year) for the control cohort (difference, 0.0706 loge unit per year; 95% CI for the difference, 0.0149-0.1263 loge unit per year; P = .01). The adjusted model confirmed a slower mean rate of decline in the vitamin A cohort (difference, 0.0771 loge-unit per year; 95% CI for the difference, 0.0191-0.1350 loge-unit per year; P = .009). With respect to ocular safety, the mean exponential rates of change of visual field area and visual acuity and the incidences of falling to a visual field diameter of 20° or less or a visual acuity of 20/200 or less in at least 1 eye did not differ by cohort. Conclusions and Relevance: A vitamin A palmitate supplement was associated with a slower loss of cone electroretinogram amplitude in children with retinitis pigmentosa. Although the relatively small-sample, retrospective, nonrandomized design does not allow a test of causation and is subject to possible biases, these findings support consideration of an age-adjusted dose of vitamin A in the management of most children with the common forms of retinitis pigmentosa.

The risk of pedestrian collisions with peripheral visual field loss.

Patients with peripheral field loss complain of colliding with other pedestrians in open-space environments such as shopping malls. Field expansion devices (e.g., prisms) can create artificial peripheral islands of vision. We investigated the visual angle at which these islands can be most effective for avoiding pedestrian collisions, by modeling the collision risk density as a function of bearing angle of pedestrians relative to the patient. Pedestrians at all possible locations were assumed to be moving in all directions with equal probability within a reasonable range of walking speeds. The risk density was found to be highly anisotropic. It peaked at ≈45° eccentricity. Increasing pedestrian speed range shifted the risk to higher eccentricities. The risk density is independent of time to collision. The model results were compared to the binocular residual peripheral island locations of 42 patients with forms of retinitis pigmentosa. The natural residual island prevalence also peaked nasally at about 45° but temporally at about 75°. This asymmetry resulted in a complementary coverage of the binocular field of view. Natural residual binocular island eccentricities seem well matched to the collision-risk density function, optimizing detection of other walking pedestrians (nasally) and of faster hazards (temporally). Field expansion prism devices will be most effective if they can create artificial peripheral islands at about 45° eccentricities. The collision risk and residual island findings raise interesting questions about normal visual development.

Visual Function in Carriers of X-Linked Retinitis Pigmentosa.

PURPOSE: To determine the frequency and severity of visual function loss in female carriers of X-linked retinitis pigmentosa (XLRP). DESIGN: Case series. PARTICIPANTS: Two hundred seventy-six XLRP carriers with cross-sectional data (n = 242) and longitudinal data (n = 34; median follow-up, 16 years; follow-up range, 3-37 years). Half of the carriers were from RPGR- or RP2-genotyped families. METHODS: Retrospective medical records review. MAIN OUTCOME MEASURES: Visual acuities, visual field areas, final dark adaptation thresholds, and full-field electroretinography (ERG) responses to 0.5-Hz and 30-Hz flashes. RESULTS: In genotyped families, 40% of carriers showed a baseline abnormality on at least 1 of 3 psychophysical tests. There was a wide range of function among carriers. For example, 3 of 121 (2%) genotyped carriers were legally blind because of poor visual acuity, some as young as 35 years. Visual fields were less affected than visual acuity. In all carriers, the average ERG amplitude to 30-Hz flashes was approximately 50% of normal, and the average exponential rate of amplitude loss over time was half that of XLRP males (3.7%/year vs. 7.4%/year, respectively). Among obligate carriers with affected fathers, sons, or both, 53 of 55 (96%) had abnormal baseline ERG results. Some carriers who initially had completely normal fundi in both eyes went on to experience moderately decreased vision, although not legal blindness. Among carriers with RPGR mutations, those with mutations in ORF15, compared with those in exons 1-14, had worse final dark adaptation thresholds and lower 0.5-Hz and 30-Hz ERG amplitudes. CONCLUSIONS: Most carriers of XLRP had mildly or moderately reduced visual function but rarely became legally blind. In most cases, obligate carriers could be identified by ERG testing. Carriers of RPGR ORF15 mutations tended to have worse visual function than carriers of RPGR exon 1 through 14 mutations. Because XLRP carrier ERG amplitudes and decay rates over time were on average half of those of affected men, these observations were consistent with the Lyon hypothesis of random X-inactivation.

Two specific mutations are prevalent causes of recessive retinitis pigmentosa in North American patients of Jewish ancestry.

PURPOSE: Retinitis pigmentosa is a Mendelian disease with a very elevated genetic heterogeneity. Most mutations are responsible for less than 1% of cases, making molecular diagnosis a multigene screening procedure. In this study, we assessed whether direct testing of specific alleles could be a valuable screening approach in cases characterized by prevalent founder mutations. METHODS: We screened 275 North American patients with recessive/isolate retinitis pigmentosa for two mutations: an Alu insertion in the MAK gene and the p.Lys42Glu missense in the DHDDS gene. All patients were unrelated; 35 reported Jewish ancestry and the remainder reported mixed ethnicity. RESULTS: We identified the MAK and DHDDS mutations homozygously in only 2.1% and 0.8%, respectively, of patients of mixed ethnicity, but in 25.7% and 8.6%, respectively, of cases reporting Jewish ancestry. Haplotype analyses revealed that inheritance of the MAK mutation was attributable to a founder effect. CONCLUSION: In contrast to most mutations associated with retinitis pigmentosa-which are, in general, extremely rare-the two alleles investigated here cause disease in approximately one-third of North American patients reporting Jewish ancestry. Therefore, their screening constitutes an alternative procedure to large-scale tests for patients belonging to this ethnic group, especially in time-sensitive situations.

Targeted exon sequencing in Usher syndrome type I.

PURPOSE: Patients with Usher syndrome type I (USH1) have retinitis pigmentosa, profound congenital hearing loss, and vestibular ataxia. This syndrome is currently thought to be associated with at least six genes, which are encoded by over 180 exons. Here, we present the use of state-of-the-art techniques in the molecular diagnosis of a cohort of 47 USH1 probands. METHODS: The cohort was studied with selective exon capture and next-generation sequencing of currently known inherited retinal degeneration genes, comparative genomic hybridization, and Sanger sequencing of new USH1 exons identified by human retinal transcriptome analysis. RESULTS: With this approach, we were able to genetically solve 14 of the 47 probands by confirming the biallelic inheritance of mutations. We detected two likely pathogenic variants in an additional 19 patients, for whom family members were not available for cosegregation analysis to confirm biallelic inheritance. Ten patients, in addition to primary disease-causing mutations, carried rare likely pathogenic USH1 alleles or variants in other genes associated with deaf-blindness, which may influence disease phenotype. Twenty-one of the identified mutations were novel among the 33 definite or likely solved patients. Here, we also present a clinical description of the studied cohort at their initial visits. CONCLUSIONS: We found a remarkable genetic heterogeneity in the studied USH1 cohort with multiplicity of mutations, of which many were novel. No obvious influence of genotype on phenotype was found, possibly due to small sample sizes of the genotypes under study.

The relationship of central foveal thickness to urinary iodine concentration in retinitis pigmentosa with or without cystoid macular edema.

IMPORTANCE: Current treatments for cystoid macular edema (CME) in retinitis pigmentosa (RP) are not always effective, may lead to adverse effects, and may not restore visual acuity. The present research lays the rationale for evaluating whether an iodine supplement could reduce CME in RP. OBJECTIVE: To determine whether central foveal thickness (CFT) in the presence of CME is related to dietary iodine intake inferred from urinary iodine concentration (UIC) in nonsmoking adults with RP. DESIGN, SETTING, AND PARTICIPANTS: We performed a cross-sectional observational study of 212 nonsmoking patients aged 18 to 69 years referred to our institution for RP with visual acuity of no worse than 20/200 in at least 1 eye. EXPOSURE: Retinitis pigmentosa with or without CME. MAIN OUTCOMES AND MEASURES: With the eye as the unit of analysis, the relationship of log CFT measured by optical coherence tomography to UIC measured from multiple spot samples and represented as a 3-level classification variable (<100, 100-199, and ≥200 µg/L), assigning greater weight to patients with more reliable UIC estimates. RESULTS: Analyses were limited to 199 patients after excluding 11 who failed to return urine samples for measuring UIC and 2 outliers for UIC. Of the 199 patients, 36.2% had CME in 1 or both eyes. Although log CFT was inversely related to UIC based on findings from all eyes (P = .02), regression of log CFT on UIC separately for eyes with and without CME showed a strong inverse significant relationship for the former group (P < .001) and no significant relationship for the latter group (P = .66) as tested. For the eyes with CME, CFT ranged from a geometric mean of 267 µm for a median UIC of less than 100 µg/L to a geometric mean of 172 µm for a median UIC of 200 µg/L or greater. In contrast, we found no significant association between CME prevalence and UIC based on the entire sample as tested (odds ratio, 1.01 [95% CI, 0.38-2.67]; P = .99). CONCLUSIONS AND RELEVANCE: A higher UIC in nonsmoking adults with RP was significantly associated with less central foveal swelling in eyes with CME. Additional study is required to determine whether an iodine supplement can limit or reduce the extent of CME in patients with RP.

Mutational screening of splicing factor genes in cases with autosomal dominant retinitis pigmentosa.

PURPOSE: Mutations in genes encoding proteins from the tri-snRNP complex of the spliceosome account for more than 12% of cases of autosomal dominant retinitis pigmentosa (adRP). Although the exact mechanism by which splicing factor defects trigger photoreceptor death is not completely clear, their role in retinitis pigmentosa has been demonstrated by several genetic and functional studies. To test for possible novel associations between splicing factors and adRP, we screened four tri-snRNP splicing factor genes (EFTUD2, PRPF4, NHP2L1, and AAR2) as candidate disease genes. METHODS: We screened up to 303 patients with adRP from Europe and North America who did not carry known RP mutations. Exon-PCR and Sanger methods were used to sequence the NHP2L1 and AAR2 genes, while the sequences of EFTUD2 and PRPF4 were obtained by using long-range PCRs spanning coding and non-coding regions followed by next-generation sequencing. RESULTS: We detected novel missense changes in individual patients in the sequence of the genes PRPF4 and EFTUD2, but the role of these changes in relationship to disease could not be verified. In one other patient we identified a novel nucleotide substitution in the 5' untranslated region (UTR) of NHP2L1, which did not segregate with the disease in the family. CONCLUSIONS: The absence of clearly pathogenic mutations in the candidate genes screened in our cohort suggests that EFTUD2, PRPF4, NHP2L1, and AAR2 are either not involved in adRP or are associated with the disease in rare instances, at least as observed in this study in patients of European and North American origin.

Molecular genetics of FAM161A in North American patients with early-onset retinitis pigmentosa.

Retinitis pigmentosa (RP) is a hereditary disease that leads to the progressive degeneration of retinal photoreceptor cells and to blindness. It is caused by mutations in several distinct genes, including the ciliary gene FAM161A, which is associated with a recessive form of this disorder. Recent investigations have revealed that defects in FAM161A represent a rather prevalent cause of hereditary blindness in Israel and the Palestinian territories, whereas they seem to be rarely present within patients from Germany. Genetic or clinical data are currently not available for other countries. In this work, we screened a cohort of patients with recessive RP from North America to determine the frequency of FAM161A mutations in this ethnically-mixed population and to assess the phenotype of positive cases. Out of 273 unrelated patients, only 3 subjects had defects in FAM161A. A fourth positive patient, the sister of one of these index cases, was also identified following pedigree analysis. They were all homozygous for the p.T452Sfx3 mutation, which was previously reported as a founder DNA variant in the Israeli and Palestinian populations. Analysis of cultured lymphoblasts from patients revealed that mutant FAM161A transcripts were actively degraded by nonsense-mediated mRNA decay. Electroretinographic testing showed 30 Hz cone flicker responses in the range of 0.10 to 0.60 microvolts in all cases at their first visit (age 12 to 23) (lower norm  =  50 µV) and of 0.06 to 0.32 microvolts at their most recent examination (age 27 to 43), revealing an early-onset of this progressive disease. Our data indicate that mutations in FAM161A are responsible for 1% of recessive RP cases in North America, similar to the prevalence detected in Germany and unlike the data from Israel and the Palestinian territories. We also show that, at the molecular level, the disease is likely caused by FAM161A protein deficiency.

Whole genome sequencing in patients with retinitis pigmentosa reveals pathogenic DNA structural changes and NEK2 as a new disease gene.

We performed whole genome sequencing in 16 unrelated patients with autosomal recessive retinitis pigmentosa (ARRP), a disease characterized by progressive retinal degeneration and caused by mutations in over 50 genes, in search of pathogenic DNA variants. Eight patients were from North America, whereas eight were Japanese, a population for which ARRP seems to have different genetic drivers. Using a specific workflow, we assessed both the coding and noncoding regions of the human genome, including the evaluation of highly polymorphic SNPs, structural and copy number variations, as well as 69 control genomes sequenced by the same procedures. We detected homozygous or compound heterozygous mutations in 7 genes associated with ARRP (USH2A, RDH12, CNGB1, EYS, PDE6B, DFNB31, and CERKL) in eight patients, three Japanese and five Americans. Fourteen of the 16 mutant alleles identified were previously unknown. Among these, there was a 2.3-kb deletion in USH2A and an inverted duplication of ~446 kb in EYS, which would have likely escaped conventional screening techniques or exome sequencing. Moreover, in another Japanese patient, we identified a homozygous frameshift (p.L206fs), absent in more than 2,500 chromosomes from ethnically matched controls, in the ciliary gene NEK2, encoding a serine/threonine-protein kinase. Inactivation of this gene in zebrafish induced retinal photoreceptor defects that were rescued by human NEK2 mRNA. In addition to identifying a previously undescribed ARRP gene, our study highlights the importance of rare structural DNA variations in Mendelian diseases and advocates the need for screening approaches that transcend the analysis of the coding sequences of the human genome.

Screening of a large cohort of leber congenital amaurosis and retinitis pigmentosa patients identifies novel LCA5 mutations and new genotype-phenotype correlations.

This study was undertaken to investigate the prevalence of sequence variants in LCA5 in patients with Leber congenital amaurosis (LCA), early-onset retinal dystrophy (EORD), and autosomal recessive retinitis pigmentosa (arRP); to delineate the ocular phenotypes; and to provide an overview of all published LCA5 variants in an online database. Patients underwent standard ophthalmic evaluations after providing informed consent. In selected patients, optical coherence tomography (OCT) and fundus autofluorescence imaging were possible. DNA samples from 797 unrelated patients with LCA and 211 with the various types of retinitis pigmentosa (RP) were screened by Sanger sequence analysis of all LCA5 exons and intron/exon junctions. Some LCA patients were prescreened by APEX technology or selected based on homozygosity mapping. In silico analyses were performed to assess the pathogenicity of the variants. Segregation analysis was performed where possible. Published and novel LCA5 variants were collected, amended for their correct nomenclature, and listed in a Leiden Open Variation Database (LOVD). Sequence analysis identified 18 new probands with 19 different LCA5 variants. Seventeen of the 19 LCA5 variants were novel. Except for two missense variants and one splice site variant, all variants were protein-truncating mutations. Most patients expressed a severe phenotype, typical of LCA. However, some LCA subjects had better vision and intact inner segment/outer segment (IS/OS) junctions on OCT imaging. In two families with LCA5 variants, the phenotype was more compatible with EORD with affected individuals displaying preserved islands of retinal pigment epithelium. One of the families with a milder phenotype harbored a homozygous splice site mutation; a second family was found to have a combination of a stop mutation and a missense mutation. This is the largest LCA5 study to date. We sequenced 1,008 patients (797 with LCA, 211 with arRP) and identified 18 probands with LCA5 mutations. Mutations in LCA5 are a rare cause of childhood retinal dystrophy accounting for ∼2% of disease in this cohort, and the majority of LCA5 mutations are likely null. The LCA5 protein truncating mutations are predominantly associated with LCA. However, in two families with the milder EORD, the LCA5 gene analysis revealed a homozygous splice site mutation in one and a stop mutation in combination with a missense mutation in a second family, suggesting that this milder phenotype is due to residual function of lebercilin and expanding the currently known phenotypic spectrum to include the milder early onset RP. Some patients have remaining foveal cone structures (intact IS/OS junctions on OCT imaging) and remaining visual acuities, which may bode well for upcoming treatment trials.

Spinocerebellar ataxia type 7: clinical course, phenotype-genotype correlations, and neuropathology.

Spinocerebellar ataxia type 7 is a neurodegenerative polyglutamine disease characterized by ataxia and retinal degeneration. The longitudinal course is unknown, and relationships between repeat expansion, clinical manifestations, and neuropathology remain uncertain. We followed 16 affected individuals of a 61-member kindred over 27 years with electroretinograms, neurological examinations including the Brief Ataxia Rating Scale, neuroimaging in five, and autopsy in four cases. We identified four stages of the illness: Stage 0, gene-positive but phenotypically silent; Stage 1, no symptoms, but hyperreflexia and/or abnormal electroretinograms; Stage 2, symptoms and signs progress modestly; and Stage 3, rapid clinical progression. CAG repeat length correlated inversely with age of onset of visual or motor signs (r = -0.74, p = 0.002). Stage 3 rate of progression did not differ between cases (p = 0.18). Electroretinograms correlated with Brief Ataxia Rating Scale score and were a biomarker of disease onset and progression. All symptomatic patients developed gait ataxia, extremity dysmetria, dysarthria, dysrhythmia, and oculomotor abnormalities. Funduscopy revealed pale optic discs and pigmentary disturbances. Visual acuity declined to blindness in those with longer CAG expansions. Hyperreflexia was present from Stage 1 onwards. Restless legs syndrome and sensory impairment were common. Neuropathological hallmarks were neuronal loss in cerebellar cortex, deep cerebellar nuclei, inferior olive, and anterior horns of the spinal cord, and axonal loss in spinocerebellar tracts, dorsal nerve roots, and posterior columns. Retinal pathology included photoreceptor degeneration and disruption of retinal pigment epithelium. Spinocerebellar ataxia type 7 evolves through four clinical stages; neuropathological findings underlie the clinical presentation; electroretinograms are a potential biomarker of disease progression.

NMNAT1 mutations cause Leber congenital amaurosis.

Leber congenital amaurosis (LCA) is an infantile-onset form of inherited retinal degeneration characterized by severe vision loss(1,2). Two-thirds of LCA cases are caused by mutations in 17 known disease-associated genes(3) (Retinal Information Network (RetNet)). Using exome sequencing we identified a homozygous missense mutation (c.25G>A, p.Val9Met) in NMNAT1 that is likely to be disease causing in two siblings of a consanguineous Pakistani kindred affected by LCA. This mutation segregated with disease in the kindred, including in three other children with LCA. NMNAT1 resides in the previously identified LCA9 locus and encodes the nuclear isoform of nicotinamide mononucleotide adenylyltransferase, a rate-limiting enzyme in nicotinamide adenine dinucleotide (NAD(+)) biosynthesis(4,5). Functional studies showed that the p.Val9Met alteration decreased NMNAT1 enzyme activity. Sequencing NMNAT1 in 284 unrelated families with LCA identified 14 rare mutations in 13 additional affected individuals. These results are the first to link an NMNAT isoform to disease in humans and indicate that NMNAT1 mutations cause LCA.

Genotype-phenotype correlations in Bardet-Biedl syndrome.

OBJECTIVE: To determine whether mutations in different Bardet-Biedl syndrome (BBS) genes result in different ocular phenotypes. METHODS: Thirty-seven patients from 31 families were enrolled who met the clinical criteria for BBS and for whom a BBS mutation had been identified. Seventeen patients harbored mutations in BBS1, 10 in BBS10, and 10 in other genes (BBS2, BBS3, BBS5, BBS7, and BBS12). All the patients underwent ocular examination; 36 patients had computerized full-field electroretinograms (ERGs). RESULTS: Visual acuity was significantly better in BBS1 patients than in patients with other BBS mutations (P=.01), and a larger proportion of BBS1 patients had good (≥20/50) visual acuity (P=.01). The ERG amplitudes were significantly higher in BBS1 patients than in patients with other BBS mutations in response to 0.5-Hz and 30-Hz flashes (P<.001 for both). All the BBS1 patients harbored at least 1 missense mutation compared with only 45% of patients with mutations in other BBS genes (P<.001); the rest harbored only null alleles. However, multivariate analysis demonstrated that visual acuity or ERG amplitude did not depend on the type of mutation present (missense or null) when controlling for BBS gene. Prevalences of bone spicule pigmentation and cataract were comparable in BBS subtypes. CONCLUSIONS: Patients with BBS1 mutations had a milder phenotype than did patients with mutations in other BBS genes. Clinically, this manifested as significantly better visual acuity and larger ERG amplitudes. CLINICAL RELEVANCE: These phenotypic differences can help guide genetic testing and genetic counseling for patients with this syndrome.

ω-3 intake and visual acuity in patients with retinitis pigmentosa receiving vitamin A.

OBJECTIVE: To evaluate whether a diet high in long chain ω-3 fatty acids can slow the rate of visual acuity loss among patients with retinitis pigmentosa receiving vitamin A palmitate. METHODS: We calculated dietary intake from questionnaires completed annually by 357 adult patients from 3 randomized trials who were all receiving vitamin A, 15 000 IU/d, for 4 to 6 years. Rates of visual acuity decline were compared between those with high (≥0.20 g/d) vs low (<0.20 g/d) ω-3 intake. Analyses took age into account. RESULTS: Mean rates of decline of acuity were slower among those with high ω-3 intake: Early Treatment Diabetic Retinopathy Study distance acuity: high intake=0.59 letter per year, low intake=1.00 letter per year,P=.001; Snellen retinal acuity: high intake=1.5% per year, low intake=2.8% per year, P=.03. CONCLUSIONS: We conclude that mean annual rates of decline in distance and retinal visual acuities in adults with retinitis pigmentosa receiving vitamin A, 15 000 IU/d,are slower over 4 to 6 years among those consuming a diet rich in ω-3 fatty acids. To our knowledge, this is the first report that nutritional intake can modify the rate of decline of visual acuity in retinitis pigmentosa.

Disease course of patients with unilateral pigmentary retinopathy.

PURPOSE: To evaluate the change in ocular function by eye in patients with unilateral pigmentary retinopathy. METHODS: Longitudinal regression was used to estimate mean exponential rates of change in Goldmann visual field area (V4e white test light) and in full-field electroretinogram (ERG) amplitudes to 0.5- and 30-Hz white flashes in 15 patients with unilateral pigmentary retinopathy. Snellen visual acuity was assessed case by case. RESULTS: Mean annual rates of change for the affected eyes were -4.9% for visual field area, -4.7% for ERG amplitude to 0.5-Hz flashes, and -4.6% for ERG amplitude to 30-Hz flashes. All three rates were faster than the corresponding age-related rates of change for the fellow normal eyes (P = 0.0006, P = 0.003, P = 0.03, respectively). An initial cone ERG implicit time to 30-Hz flashes in affected eyes ≥ 40 ms predicted a faster mean rate of decline of visual field area and of ERG amplitude to 0.5- and 30-Hz flashes (P < 0.0001 for all three measures). The visual acuity of affected eyes was more likely to decrease in patients presenting at >35 years of age than in patients presenting at a younger age (P = 0.0004). CONCLUSIONS: The affected eye in unilateral pigmentary retinopathy shows a progressive loss of peripheral retinal function that cannot be attributed to aging alone and that is faster in eyes with a more prolonged initial cone ERG implicit time. Patients presenting at >35 years of age are at greater risk for losing visual acuity.

Histopathology and functional correlations in a patient with a mutation in RPE65, the gene for retinol isomerase.

PURPOSE: Here the authors describe the structural features of the retina and retinal pigment epithelium (RPE) in postmortem donor eyes of a 56-year-old patient with a homozygous missense RPE65 mutation (Ala132Thr) and correlate the pathology with the patient's visual function last measured at age 51. METHODS: Eyes were enucleated within 13.5 hours after death. Representative areas from the macula and periphery were processed for light and electron microscopy. Immunofluorescence was used to localize the distribution of RPE65, rhodopsin, and cone arrestin. The autofluorescence in the RPE was compared with that of two normal eyes from age-similar donors. RESULTS: Histologic examination revealed the loss of rods and cones across most areas of the retina, attenuated retinal vessels, and RPE thinning in both eyes. A small number of highly disorganized cones were present in the macula that showed simultaneous labeling with cone arrestin and red/green or blue opsin. RPE65 immunoreactivity and RPE autofluorescence were reduced compared with control eyes in all areas studied. Rhodopsin labeling was observed in rods in the far periphery. The optic nerve showed a reduced number of axons. CONCLUSIONS: The clinical findings of reduced visual acuity, constricted fields, and reduced electroretinograms (ERGs) 5 years before death correlated with the small number of cones present in the macula and the extensive loss of photoreceptors in the periphery. The absence of autofluorescence in the RPE suggests that photoreceptor cells were probably missing across the retina for extended periods of time. Possible mechanisms that could lead to photoreceptor cell death are discussed.

Molecular analysis of Bardet-Biedl syndrome families: report of 21 novel mutations in 10 genes.

PURPOSE: Bardet-Biedl syndrome (BBS) is genetically heterogeneous with 15 BBS genes currently identified, accounting for approximately 70% of cases. The aim of our study was to define further the spectrum of BBS mutations in a cohort of 44 European-derived American, 8 Tunisian, 1 Arabic, and 2 Pakistani families (55 families in total) with BBS. METHODS: A total of 142 exons of the first 12 BBS-causing genes were screened by dideoxy sequencing. Cases in which no mutations were found were then screened for BBS13, BBS14, BBS15, RPGRIP1L, CC2D2A, NPHP3, TMEM67, and INPP5E. RESULTS: Forty-three mutations, including 8 frameshift mutations, 10 nonsense mutations, 4 splice site mutations, 1 deletion, and 20 potentially or probably pathogenic missense variations, were identified in 46 of the 55 families studied (84%). Of these, 21 (2 frameshift mutations, 4 nonsense mutations, 4 splice site mutations, 1 deletion, and 10 missense variations) were novel. The molecular genetic findings raised the possibility of triallelic inheritance in 7 Caucasian families, 1 Arabian family, and 1 Tunisian patient. No mutations were detected for BBS4, BBS11, BBS13, BBS14, BBS15, RPGRIP1L, CC2D2A, NPHP3, TMEM67, or INPP5E. CONCLUSIONS: This mutational analysis extends the spectrum of known BBS mutations. Identification of 21 novel mutations highlights the genetic heterogeneity of this disorder. Differences in European and Tunisian patients, including the high frequency of the M390R mutation in Europeans, emphasize the population specificity of BBS mutations with potential diagnostic implications. The existence of some BBS cases without mutations in any currently identified BBS genes suggests further genetic heterogeneity.

Next generation sequencing of pooled samples reveals new SNRNP200 mutations associated with retinitis pigmentosa.

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

A missense mutation in PRPF6 causes impairment of pre-mRNA splicing and autosomal-dominant retinitis pigmentosa.

Retinitis pigmentosa (RP) is an inherited form of retinal degeneration that leads to progressive visual-field constriction and blindness. Although the disease manifests only in the retina, mutations in ubiquitously expressed genes associated with the tri-snRNP complex of the spliceosome have been identified in patients with dominantly inherited RP. We screened for mutations in PRPF6 (NM_012469.3), a gene on chromosome 20q13.33 encoding an essential protein for tri-snRNP assembly and stability, in 188 unrelated patients with autosomal-dominant RP and identified a missense mutation, c.2185C>T (p.Arg729Trp). This change affected a residue that is conserved from humans to yeast and cosegregated with the disease in the family in which it was identified. Lymphoblasts derived from patients with this mutation showed abnormal localization of endogenous PRPF6 within the nucleus. Specifically, this protein accumulated in the Cajal bodies, indicating a possible impairment in the tri-snRNP assembly or recycling. Expression of GFP-tagged PRPF6 in HeLa cells showed that this phenomenon depended exclusively on the mutated form of the protein. Furthermore, analysis of endogenous transcripts in cells from patients revealed intron retention for pre-mRNA bearing specific splicing signals, according to the same pattern displayed by lymphoblasts with mutations in other PRPF genes. Our results identify PRPF6 as the sixth gene involved in pre-mRNA splicing and dominant RP, corroborating the hypothesis that deficiencies in the spliceosome play an important role in the molecular pathology of this disease.