Anthony T. Moore

Effect of Gene Therapy on Visual Function in Leber's Congenital Amaurosis

Early-onset, severe retinal dystrophy caused by mutations in the gene encoding retinal pigment epithelium-specific 65-kD protein (RPE65) is associated with poor vision at birth and complete loss of vision in early adulthood. We administered to three young adult patients subretinal injections of recombinant adeno-associated virus vector 2/2 expressing RPE65 complementary DNA (cDNA) under the control of a human RPE65 promoter. There were no serious adverse events. There was no clinically significant change in visual acuity or in peripheral visual fields on Goldmann perimetry in any of the three patients. We detected no change in retinal responses on electroretinography. One patient had significant improvement in visual function on microperimetry and on dark-adapted perimetry. This patient also showed improvement in a subjective test of visual mobility. These findings provide support for further clinical studies of this experimental approach in other patients with mutant RPE65. (ClinicalTrials.gov number, NCT00643747 [ClinicalTrials.gov].).

OPA1, encoding a dynamin-related GTPase, is mutated in autosomal dominant optic atrophy linked to chromosome 3q28

Autosomal dominant optic atrophy (ADOA) is the most prevalent hereditary optic neuropathy resulting in progressive loss of visual acuity, centrocoecal scotoma and bilateral temporal atrophy of the optic nerve with an onset within the first two decades of life. The predominant locus for this disorder (OPA1; MIM 165500) has been mapped to a 1.4-cM interval on chromosome 3q28–q29 flanked by markers D3S3669 and D3S3562 (ref. 3). We established a PAC contig covering the entire OPA1 candidate region of approximately 1 Mb and a sequence skimming approach allowed us to identify a gene encoding a polypeptide of 960 amino acids with homology to dynamin-related GTPases. The gene comprises 28 coding exons and spans more than 40 kb of genomic sequence. Upon sequence analysis, we identified mutations in seven independent families with ADOA. The mutations include missense and nonsense alterations, deletions and insertions, which all segregate with the disease in these families. Because most mutations probably represent null alleles, dominant inheritance of the disease may result from haploinsufficiency of OPA1. OPA1 is widely expressed and is most abundant in the retina. The presence of consensus signal peptide sequences suggests that the product of the gene OPA1 is targeted to mitochondria and may exert its function in mitochondrial biogenesis and stabilization of mitochondrial membrane integrity.

Smoking and age related macular degeneration: the number of pack years of cigarette smoking is a major determinant of risk for both geographic atrophy and choroidal neovascularisation

Background/aims: There is evidence that smoking is a risk factor for age related macular degeneration (AMD). However, not all studies have demonstrated this association and several key questions about the role of smoking in AMD have still to be determined. The aim of this study was to further investigate this relation for both choroidal neovascularisation (CNV) and geographic atrophy (GA). Methods: To investigate the relation between smoking and the risk of developing age related macular degeneration (AMD) in white people, 435 cases with end stage AMD were compared with 280 controls. All subjects had graded stereoscopic colour fundus photography and AMD was defined as the presence of GA or CNV. Smoking history was assessed using multiple parameters in a detailed questionnaire. Results: Comparison of current and former smokers with non-smokers was consistent with smoking being a risk factor for AMD but did not reach statistical significance. There was a strong association between AMD and pack years of cigarette smoking (p = 0.002), the odds ratio increasing with the amount smoked; for subjects with more than 40 pack years of smoking the odds ratio was 2.75 (95% CI 1.22 to 6.20) compared with non-smokers. Both types of AMD showed a similar relation; smoking more than 40 pack years of cigarettes was associated with an odds ratio of 3.43 (95% CI 1.28 to 9.20) for GA and 2.49 (95% CI 1.06 to 5.82) for CNV. Stopping smoking was associated with reduced odds of AMD and the risk in those who had not smoked for over 20 years was comparable to non-smokers. The risk profile was similar for males and females. Passive smoking exposure was associated with an increased risk of AMD (OR 1.87; 95% CI 1.03 to 3.40) in non-smokers. Conclusions: The authors have demonstrated a strong association between the risk of both GA and CNV and pack years of cigarette smoking. This provides support for a causal relation between smoking and AMD. They also show an increased risk for AMD in non-smokers exposed to passive smoking. Stopping smoking appears to reduce the risk of developing AMD.

A Human Homolog of Yeast Pre-mRNA Splicing Gene, PRP31, Underlies Autosomal Dominant Retinitis Pigmentosa on Chromosome 19q13.4 (RP11)

We report mutations in a gene (PRPF31) homologous to Saccharomyces cerevisiae pre-mRNA splicing gene PRP31 in families with autosomal dominant retinitis pigmentosa linked to chromosome 19q13.4 (RP11; MIM 600138). A positional cloning approach supported by bioinformatics identified PRPF31 comprising 14 exons and encoding a protein of 499 amino acids. The level of sequence identity to the yeast PRP31 gene indicates that PRPF31 is also likely to be involved in pre-mRNA splicing. Mutations that include missense substitutions, deletions, and insertions have been identified in four RP11-linked families and three sporadic RP cases. The identification of mutations in a pre-mRNA splicing gene implicates defects in the splicing process as a novel mechanism of photoreceptor degeneration.

Missense mutations in MIP underlie autosomal dominant ‘polymorphic’ and lamellar cataracts linked to 12q

Human inherited cataract is both clinically diverse and genetically heterogeneous. Here we report the identification of the first mutations affecting the major intrinsic protein of the lens, MIP, encoded by the gene MIP on 12q14. MIP is a member of the aquaporin family of membrane-bound water channels. The mutations identified are predicted to disturb water flux across the lens cell membrane.

Long-term effect of gene therapy on Leber's congenital amaurosis.

BACKGROUND Mutations in RPE65 cause Lebers congenital amaurosis, a progressive retinal degenerative disease that severely impairs sight in children. Gene therapy can result in modest improvements in night vision, but knowledge of its efficacy in humans is limited. METHODS We performed a phase 1-2 open-label trial involving 12 participants to evaluate the safety and efficacy of gene therapy with a recombinant adeno-associated virus 2/2 (rAAV2/2) vector carrying the RPE65 complementary DNA, and measured visual function over the course of 3 years. Four participants were administered a lower dose of the vector, and 8 were administered a higher dose. In a parallel study in dogs, we investigated the relationship among vector dose, visual function, and electroretinography (ERG) findings. RESULTS Improvements in retinal sensitivity were evident, to varying extents, in six participants for up to 3 years, peaking at 6 to 12 months after treatment and then declining. No associated improvement in retinal function was detected by means of ERG. Three participants had intraocular inflammation, and two had clinically significant deterioration of visual acuity. The reduction in central retinal thickness varied among participants. In dogs, RPE65 gene therapy with the same vector at lower doses improved vision-guided behavior, but only higher doses resulted in improvements in retinal function that were detectable with the use of ERG. CONCLUSIONS Gene therapy with rAAV2/2 RPE65 vector improved retinal sensitivity, albeit modestly and temporarily. Comparison with the results obtained in the dog model indicates that there is a species difference in the amount of RPE65 required to drive the visual cycle and that the demand for RPE65 in affected persons was not met to the extent required for a durable, robust effect. (Funded by the National Institute for Health Research and others; ClinicalTrials.gov number, NCT00643747.).

Two infant vision screening programmes: prediction and prevention of strabismus and amblyopia from photo- and videorefractive screening.

Two infant vision screening programmes on total populations in the Cambridge Health District have been designed to identify manifest strabismus and strabismogenic and amblyogenic refractive errors at 7–9 months of age. The first, completed, programme used the isotropic photorefractor with cycloplegia together with a standard orthoptic examination. The second, current, programme uses the VRP-1 isotropic videorefractor to identify infants with accommodative lags which are followed up by refraction under cycloplegia. Both programmes show good agreement between infants identified at screening and retinoscopic refractions at follow-up, showing that photo- and videorefraction (with or without cycloplegia) can be effective methods for screening for ametropia in infants and young children. In each programme 5–6% of infants showed abnormal levels of hyperopia (≥3.5 D in any meridian), less than 1% showed anisometropia ≥1.5 D; very few infants (0.25%) showed −3 D myopia or greater. Less than 1% showed manifest strabismus. Hyperopic and anisometropic children entered a randomised controlled trial of partial refractive correction. All children identified at screening, alongside appropriate control groups, are extensively followed up to age 4 years. The first programme has found that children who were hyperopic in infancy were 13 times more likely to become strabismic, and 6 times more likely to show measurable acuity deficits by 4 years, compared with controls. Wearing a partial spectacle correction reduced these risk ratios to 4:1 and 2.5:1 respectively. The impaired acuity can be attributed, in part, to meridional amblyopia resulting from persisting astigmatism. Both hyperopic and myopic infants showed refractive changes in the direction of emmetropia between 9 months and 4 years. Wearing a partial spectacle correction did not affect this process of emmetropisation, but does provide the possibility of reducing the incidence of common pre-school vision problems.

PAX6 haploinsufficiency causes cerebral malformation and olfactory dysfunction in humans.

PAX6 is widely expressed in the central nervous system. Heterozygous PAX6 mutations in human aniridia cause defects that would seem to be confined to the eye. Magnetic resonance imaging (MRI) and smell testing reveal the absence or hypoplasia of the anterior commissure and reduced olfaction in a large proportion of aniridia cases, which shows that PAX6 haploinsuffiency causes more widespread human neuro developmental anomalies.

Alpha-B crystallin gene (CRYAB) mutation causes dominant congenital posterior polar cataract in humans.

Congenital cataracts are an important cause of bilateral visual impairment in infants. In a four-generation family of English descent, we mapped dominant congenital posterior polar cataract to chromosome 11q22-q22.3. The maximum LOD score, 3.92 at recombination fraction 0, was obtained for marker D11S898, near the gene that encodes crystallin alpha-B protein (CRYAB). By sequencing the coding regions of CRYAB, we found in exon 3 a deletion mutation, 450delA, that is associated with cataract in this family. The mutation resulted in a frameshift in codon 150 and produced an aberrant protein consisting of 184 residues. This is the first report of a mutation, in this gene, resulting in isolated congenital cataract.

Phenotypic expression in von Hippel-Lindau disease: correlations with germline VHL gene mutations.

Von Hippel-Lindau disease is an autosomal dominantly inherited familial cancer syndrome predisposing to retinal and central nervous system haemangioblastomas, renal cell carcinoma, and phaeochromocytoma. VHL disease shows variable expression and interfamilial differences in predisposition to phaeochromocytoma. In a previous study of 65 VHL kindreds with defined VHL mutations we detected significant differences between VHL families with and without phaeochromocytoma such that missense mutations were more common and large deletions or protein truncating mutations less frequent in phaeochromocytoma positive families. To investigate the significance and cause of this association further, we studied 138 VHL kindreds for germline mutations and calculated the age related tumour risks for different classes of VHL gene mutations. Using SSCP, heteroduplex and Southern analysis we identified a germline VHL gene mutation in 101 families (73%). Direct sequencing of the VHL coding region further increased the mutation detection rate to 81%. In addition to precise presymptomatic diagnosis, identification of a VHL gene mutation can provide an indication of the likely phenotype. We found that large deletions and mutations predicted to cause a truncated protein were associated with a lower risk of phaeochromocytoma (6% and 9% at 30 and 50 years, respectively) than missense mutations (40% and 59%, respectively) and that missense mutations at codon 167 were associated with a high risk of phaeochromocytoma (53% and 82% at ages 30 and 50 years). Cumulative probabilities of renal cell carcinoma did not differ between the two groups (deletion/ truncation mutations: 8% and 60%, and missense mutations: 10% and 64% at ages 30 and 50 years, respectively). Age related risks for haemangioblastoma were similar in the two mutation groups, with the age related risks of cerebellar haemangioblastoma slightly less (35% and 64% v 38% and 75% at ages 30 and 50 years) and retinal haemangioblastoma slightly higher (45% and 72% v 37% and 64% at ages 30 and 50 years) in the missense mutation group than in the deletion/protein truncation group. These results provide valuable data for counselling VHL families and indicate that specific VHL mutations may be associated with different tumour susceptibility risks. There was no evidence of a generalised increase in age related tumour risks for missense mutations, suggesting that missense mutations predisposing to phaeochromocytoma have tissue specific effects, possibly because the VHL protein has several functions, the importance of which varies from tissue to tissue, or because the proteins which interact with VHL differ between different tissues.