Molecular genetics and prospects for therapy of the inherited retinal dystrophies.

More than 60 genes responsible for human retinal dystrophies have been identified. Those recently isolated include the transcription factor genes NRL and NR2E3, RDH5 (retinol dehydrogenase), EFEMP1 (which encodes an extracellular matrix protein), CRB1, PROML1, RP1, AIPL1 and USH1C (harmonin). The ABCR protein has been identified as a critical transporter in the recycling of retinal (vitamin A). At present, a number of novel therapeutic strategies are being evaluated including pharmacological treatments, cell transplantation and gene therapy. The progress made with such approaches now offers hope to patients with these incurable forms of blindness.

Spectrum of mutations in USH2A in British patients with Usher syndrome type II.

Usher syndrome (USH) is a combination of a progressive pigmentary retinopathy, indistinguishable from retinitis pigmentosa, and some degree of sensorineural hearing loss. USH can be subdivided in Usher type I (USHI), type II (USHII) and type III (USHIII), all of which are inherited as autosomal recessive traits. The three subtypes are genetically heterogeneous, with six loci so far identified for USHI, three for USHII and only one for USHIII. Mutations in a novel gene, USH2A, encoding the protein usherin, have recently been shown to be associated with USHII. The gene encodes a protein with partial sequence homology to both laminin epidermal growth factor and fibronectin motifs. We analysed 35 British and one Pakistani Usher type II families with at least one affected member, for sequence changes in the 20 translated exons of the USH2A gene, using heteroduplex analysis and sequencing. Probable disease-causing mutations in USH2A were identified in 15 of 36 (41.7%) Usher II families. The most frequently encountered mutation (11/15 families or 11/18 mutated alleles) was del2299G in exon 13, resulting in a frameshift and premature stop codon. Other mutations include insertions and point mutations, of which two are previously unreported. Five different polymorphisms were also detected. Our results indicate that mutations in this gene are responsible for disease in a large proportion of British Usher type II patients. Moreover, if screening for mutations in USH2A is considered, it is sensible to screen for the del2299G mutation first.

NRL S50T mutation and the importance of 'founder effects' in inherited retinal dystrophies.

The aim of this work was to identify NRL mutations in a panel of 200 autosomal dominant retinitis pigmentosa (adRP) families. All samples were subjected to heteroduplex analysis of the three exons of the NRL gene, and HphI restriction digest analysis of exon 2 (to identify the S50T mutation). Families found to have the S50T mutation, and six additional larger pedigrees (which had previously been excluded from the other nine adRP loci) underwent linkage analysis using polymorphic markers located in the region of 14q11. HphI restriction analysis followed by direct sequencing of the amplified NRL exon 2 product demonstrated the presence of the NRL S50T sequence change in three adRP families. Comparison of marker haplotypes in affected individuals from these families with those of affected members of the original 14q11 linked family revealed a common disease haplotype for markers within the adRP locus. Recombination events observed in these families define an adRP critical interval of 14.9 cM between D13S72 and D14S1041. Linkage analysis enabled all six of the larger adRP pedigrees to be excluded from the 14q11 locus. The NRL S50T mutation represents another example of a 'founder effect' in a dominantly inherited retinal dystrophy. Identification of such 'founder effects' may greatly simplify diagnostic genetic screening and lead to better prognostic counselling. The exclusion of several adRP families from all ten adRP loci indicates that at least one further adRP locus remains to be found.

Genetic analysis of the guanylate cyclase activator 1B (GUCA1B) gene in patients with autosomal dominant retinal dystrophies.

The guanylate cyclase activator proteins (GCAP1 and GCAP2) are calcium binding proteins which by activating Ret-GC1 play a key role in the recovery phase of phototransduction. Recently a mutation in the GUCA1A gene (coding for GCAP1) mapping to the 6p21.1 region was described as causing cone dystrophy in a British family. In addition mutations in Ret-GC1 have been shown to cause Leber congenital amaurosis and cone-rod dystrophy. To determine whether GCAP2 is involved in dominant retinal degenerative diseases, the GCAP2 gene was screened in 400 unrelated subjects with autosomal dominant central and peripheral retinal dystrophies. A number of changes involving the intronic as well as the coding sequence were observed. In exon 1 a T to C nucleotide change was observed leaving the tyrosine residue 57 unchanged. In exon 3 a 1 bp intronic insertion, a single nucleotide substitution G to A in the intron 3' of this exon, and a GAG to GAT change at codon 155 were observed. This latter change results in a conservative change of glutamic acid to aspartic acid. In exon 4 a 7 bp intronic insertion, a single nucleotide A to G substitution in the intron 5' of this exon, and a single base pair change C to G in the intron 3' of exon 4 were seen. None of these changes would be expected to affect correct splicing of this gene. All these changes were observed in controls. The results of this study do not show any evidence so far that GCAP2 is involved in the pathogenesis of autosomal dominant retinal degeneration in this group of patients. All the changes detected were found to be sequence variations or polymorphisms and not disease causing.

Clinical features of codon 172 RDS macular dystrophy: similar phenotype in 12 families.

OBJECTIVE: To report the phenotype associated with the codon 172 RDS (gene for retinal degeneration slow) mutation in 11 separate families with an arginine-to-tryptophan substitution with common ancestry, and 1 family with an arginine-to-glutamine transition. PATIENTS: Screening for RDS gene mutations was performed in 400 subjects with autosomal dominant retinal degeneration. Twelve families were identified with a mutation in codon 172. Haplotype analysis was performed. Full ophthalmic evaluation was performed, including electrophysiologic and psychophysical investigation and imaging of autofluorescence using confocal laser scanning ophthalmoscopy. RESULTS: Haplotype analysis demonstrated that the 11 families were ancestrally related. All 12 families showed a common phenotype of macular dysfunction, with the deficit increasing with age. Abnormally high autofluorescence predated loss of visual acuity or visual field changes. Pattern electroretinographic (PERG) findings were affected early in disease. There was high intrafamilial and interfamilial consistency of phenotype. CONCLUSION: These families demonstrate a striking conformity of symptoms and signs. CLINICAL RELEVANCE: In the codon 172 RDS mutation, unlike disease resulting from other RDS mutations, prediction of approximate age of onset and progression of visual deficit is possible. This should assist diagnosis and counseling.

Phenotype of autosomal recessive congenital microphthalmia mapping to chromosome 14q32.

BACKGROUND: Congenital microphthalmia (OMIM: 309700) may occur in isolation or in association with a variety of systemic malformations. Isolated microphthalmia may be inherited as an autosomal dominant, an autosomal recessive, or an X linked trait. METHODS: Based on a whole genome linkage analysis, in a six generation consanguineous family with autosomal recessive inheritance, the first locus for isolated microphthalmia was mapped to chromosome 14q32. Eight members of this family underwent clinical examination to determine the nature of the microphthalmia phenotype associated with this locus. RESULTS: All affected individuals in this family suffered from bilateral microphthalmia in association with anterior segment abnormalities, and the best visual acuity achieved was "perception of light". Corneal changes included partial or complete congenital sclerocornea, and the later development of corneal vascularisation and anterior staphyloma. Intraocular pressure, as measured by Schiotz tonometry, was greatly elevated in many cases. CONCLUSIONS: This combination of ocular defects suggests an embryological disorder involving tissues derived from both the neuroectoderm and neural crest. Other families with defects in the microphthalmia gene located on 14q32 may have a similar ocular phenotype aiding their identification.

Severe autosomal dominant retinitis pigmentosa caused by a novel rhodopsin mutation (Ter349Glu). Mutations in brief no. 208. Online.

Mutations in the rhodopsin gene are reported to be responsible for approximately 25% of all cases of autosomal dominant Retinitis pigmentosa (adRP). Affected individuals from a large family with an unusually severe form of adRP were screened for mutations in the rhodopsin gene. Direct sequencing of exon 5 revealed a TAA to GAA transversion at nucleotide 5276/codon 349, which was confirmed by Dde1 restriction digest analysis. This change would replace the normal termination codon with a glutamic acid residue (Ter-349-Glu, or X349E). The next predicted termination codon (TAA) lies 153bp downstream at nucleotides 5429 to 5431. Termination of transcription at this point would add an additional 51 amino-acid residues to the carboxy terminus of the rhodopsin molecule. This mutation is unique in producing a mutant rhodopsin in which all of the normal 348 amino-acid residues remain intact. It produces one of the most severe adRP phenotypes ever observed in a family with a rhodopsin mutation. In view of this the Ter-349-Glu mutation is worthy of further investigation to determine how the presence of this particular mutant opsin leads to rod photoreceptor degeneration.

The management of dislocated lens material after phacoemulsification.

PURPOSE: To determine the visual outcome, prognostic factors, and effect of timing of surgical intervention in patients with retained lens fragments after phacoemulsification. METHODS: A retrospective review was carried out of the notes of 44 consecutive patients who suffered posterior dislocation of lens fragments during phacoemulsification, of whom 34 underwent vitrectomy. RESULTS: The presence of severe uveitis at presentation was a significant predictor of a final visual acuity of less than 6/60 (p = 0.002). A raised intraocular pressure at presentation (> 29 mmHg) significantly increased the risk of chronic glaucoma (p = 0.0279), but there was no association between glaucoma and delay in vitrectomy. Patients operated on within 1 week of cataract surgery obtained a better visual outcome (64.7%) of patients achieved 6/12 or better visual acuity) than those operated on later than 1 week (41% obtained 6/12), but this difference did not reach statistical significance. CONCLUSIONS: The trend towards a better visual outcome with early vitrectomy was not statistically significant. A large prospective trial is indicated to determine the optimum time for vitrectomy in these patients.

Further refinement of the Usher 2A locus at 1q41.

Usher syndrome (USH) is characterised by congenital sensorineural hearing loss and progressive pigmentary retinopathy. All three subtypes (USH1, USH2, and USH3) are inherited as recessive traits. People with Usher type 2 (USH2) have normal vestibular responses and moderate to severe hearing loss. These syndromes have been found to be genetically heterogeneous, with a single locus for USH2 at 1q41 (USH2A), six loci for USH1, and one for USH3. Some USH2 families have been excluded from the 1q41 locus suggesting that a second, as yet unidentified, locus (USH2B) must exist. Linkage studies suggest that around 90% of USH2 families are USH2A. Four USH2 families were analysed for linkage to markers flanking the USH2A locus. In one of these families a recombination event was observed in an affected subject which excludes the USH2A gene from proximal to the marker AFM143XF10 and defines this as the new centromeric flanking marker for the USH2A locus. A further recombination event in another patient from this family confirmed AFM144XF2 as the telomeric flanking marker. The interval between these polymorphic markers is estimated to be 400 kb. This region is completely contained in each of three YACs from the CEPH library: 867g9, 919h3, and 848b9. This refinement more than halves the critical genetic interval and will greatly facilitate positional cloning of the USH2A gene.

A locus for autosomal recessive congenital microphthalmia maps to chromosome 14q32.

Congenital microphthalmia (CMIC) (OMIM 309700) may occur in isolation or in association with a variety of systemic malformations. Isolated CMIC may be inherited as an autosomal dominant, an autosomal recessive, or an X-linked trait. On the basis of a whole-genome linkage analysis, we have mapped the first locus for isolated CMIC, in a five-generation consanguineous family with autosomal recessive inheritance, to chromosome 14q32. All affected individuals in this family have bilateral CMIC. Linkage analysis gave a maximum two-point LOD score of 3.55 for the marker D14S65. Surrounding this marker is a region of homozygosity of 7.3 cM, between the markers D14S987 and D14S267, within which the disease gene is predicted to lie. The genes for several eye-specific transcription factors are located on human chromosome 14q and in the syntenic region of mouse chromosome 12. However, both CHX10 (14q24.3), mutations of which give rise to CMIC in mouse models, and OTX2 (14q21-22) can be excluded as candidates for autosomal recessive congenital microphthalmia (arCMIC), since they map outside the critical disease region defined by recombination events. This suggests that arCMIC is caused by defects in a novel developmental gene that may be important or even essential in eye development.

Epithelial debridement for secondary hyperopia following myopic excimer laser photorefractive keratectomy.

BACKGROUND: To evaluate epithelial debridement for the treatment of persistent hyperopia in eyes that had photorefractive keratectomy (PRK). SETTING: Optimax Laser Eye Clinics, Manchester, London, Bristol, England. METHODS: Epithelial debridement was performed on 46 eyes to reduce the hypermetropia following excimer laser PRK. RESULTS: Mean age of the patients was 43 years +/- 9.7 (SD). Mean refractive change was -0.51 diopter (D) +/- 0.76 (range +0.75 to -2.50 D). Mean change in best corrected visual acuity (BCVA) was 0.00 Logmar units (range +0.40 to -0.20 units), although 33% of eyes lost one line or more of Logmar BCVA. Mean follow-up after debridement was 61.0 +/- 26.9 weeks (range 26 to 140 weeks). CONCLUSIONS: Epithelial debridement is an unpredictable procedure to treat secondary hyperopia after PRK, producing a small mean change in spherical equivalent with a wide range of results. A significant number of eyes lost one line or more of Logmar BCVA. We therefore do not advocate epithelial debridement after PRK.

A mutation in guanylate cyclase activator 1A (GUCA1A) in an autosomal dominant cone dystrophy pedigree mapping to a new locus on chromosome 6p21.1.

We report a mutation (Y99C) in guanylate cyclase activator 1A (GUCA1A), the gene for guanylate cyclase activating protein (GCAP1), in a family with autosomal dominant cone dystrophy. Linkage analysis excluded all the known cone and cone-rod dystrophy loci, except the chromosome 6p21.1 region. This is known to contain the RDS gene, which is associated with dominant cone-rod dystrophy. Screening of the RDS gene by heteroduplex analysis and direct sequencing failed to demonstrate sequence changes in the coding region of this gene. The gene for GCAP1, a calcium binding protein which is highly expressed in photoreceptor outer segments, is also located in 6p21.1. It was screened for mutations, and all affected individuals showed a single base pair missense mutation (A-->G) at codon 99 in exon 2 of this gene generating a tyrosine-to-cysteine change in the GCAP1 protein. This change was absent from 206 unrelated normal controls. We propose that this change would at least disrupt the EF3handof GCAP1 thereby preventing calcium binding and consequently interfere with activation. The resulting effect on cGMP production would predictably modify the number of open cGMP gated cation channels, and could explain the ultimate demise of cone photoreceptor cells.

Astigmatism induced by spherical photorefractive keratectomy corrections.

PURPOSE: The purpose of the study is to evaluate the induced astigmatism after spherical photorefractive keratectomy on the Summit Omnimed (Summit Instruments, Waltham, MA) and the Nidek EC-5000 (Nidek Co. Ltd, Aichi, Japan) excimer lasers. METHODS: A total of 4269 eyes of 3289 patients were treated with a 5-mm optical zone using the Summit Omnimed excimer laser and 1825 eyes of 1303 patients treated with the Nidek EC-5000 excimer laser. The final astigmatic refractive outcome was compared with the initial refraction by vector analysis (Alpin and Jaffe method). RESULTS: Subjective astigmatic refraction for the Summit laser reduced from a mean of -0.39 diopter (D) +/- standard deviation (SD) 0.33 D (range, 0 to -2.50 D) to -0.33 D +/- SD 0.41 D (range, 0 to -3.00 D). Surgically induced astigmatism (SIA) had a mean of 0.42 +/- SD 0.34 D (range, 0 to 2.89 D). Mean SIA increased with increasing preoperative astigmatism by 0.60 D SIA for every 1.00 D of preoperative cylinder. For the Nidek laser, subjective astigmatic refraction changed from a mean of -0.18 D +/- SD 0.21 D (range, 0 to -1.25 D) to -0.30 D +/- SD 0.33 D (range, 0 to -3.00 D). Surgically induced astigmatism had a mean of -0.32 D +/- SD 0.29 (range, 0 to 3.05 D). Mean SIA increased with increasing preoperative astigmatism by 0.47 D SIA for every 1.00 D of preoperative cylinder. CONCLUSIONS: The authors show that spherical photorefractive keratectomy corrections can induce significant astigmatic change, particularly if a large amount of preoperative astigmatism is present.

Results of excimer laser retreatment of residual myopia after previous photorefractive keratectomy.

BACKGROUND: Nine percent to 30% of all patients who undergo a single excimer laser photorefractive keratectomy (PRK) do not achieve an unaided visual acuity of 20/ 40 or better and may require optical correction to obtain adequate vision. METHODS: The authors performed a retrospective analysis of the records of 164 patients who had undergone retreatment with the excimer laser for residual myopia after a previous PRK. Mean follow-up was 35.5 +/- 15.2 weeks (range, 26-104 weeks). RESULTS: The mean spherical equivalent (MSE) before retreatment was -2.59 +/- 1.36 diopter (D) (range, -0.50 to -7.75 D). The final MSE after reablation was -0.52 +/- 1.36 D (range, 2.50 to -5.50). Of the 164 patients, 107 (65.2%) obtained a final refraction within 1.00 D of emmetropia and 111 (67.3%) achieved an unaided visual acuity of 20/40 or better. Only 10 patients (6.1%) lost more than one Snellen line of best-corrected visual acuity. The final MSE result for the subgroup of patients who had a pre-retreatment myopia of between -0.50 and -1.90 D (-0.31 +/- 1.09 D) was significantly closer to emmetropia than that of the subgroup with a residual myopia of -4.00 to -7.75 D (-1.62 +/- 1.94 D). CONCLUSIONS: Excimer laser retreatment may provide a relatively safe and predictable method of correcting residual myopia after an earlier PRK with a 25% extra correction recommended for residual myopia.

Reduction in intraocular pressure after excimer laser photorefractive keratectomy. Correlation with pretreatment myopia.

PURPOSE: The authors relate the observed reduction in intraocular pressure (IOP) after excimer laser treatment to the degree of myopia treated. BACKGROUND: Intraocular pressure, measured by both Goldmann applanation and noncontact tonometry, has been reported to decrease after excimer laser photorefractive keratectomy (PRK). However, IOP readings after excimer laser PRK might be inaccurate as a consequence of changes in both the thickness and curvature of the cornea. METHODS: Baseline IOP readings were measured by noncontact tonometry in each eye of a group of 1320 patients at the time of their initial consultation. These were compared to readings obtained before treatment of the second eye, which took place a minimum of 4 months later. The untreated eyes served as controls. The paired Student's t test was used for statistical analysis. RESULTS: After PRK, a decrease was observed in the IOP of treated eyes that was related to the degree of myopia treated. A significant difference was observed between treated and untreated eyes (P < 0.0000). CONCLUSIONS: The IOP measured after PRK for myopia may be reduced because of changes in corneal thickness (absence of Bowman's membrane and central thinning) and topography. This is of particular relevance when monitoring the IOP of those patients who are given steroid drops to prevent regression. It also may be of importance in the management of any future glaucoma.

Management of strabismus due to orbital myositis.

We report on 5 consecutive patients seen at the botulinum toxin clinic at Moorfields Eye Hospital with an ocular motility disorder secondary to orbital myositis. CT scans demonstrated involvement of one or both of the medial recti in the inflammatory process in all 5 patients. In addition 1 patient had involvement of both the lateral recti and the right superior rectus. Two patients had been treated with oral steroids, 3 with non-steroidal anti-inflammatory agents, and 1 with orbital radiotherapy. Prior to toxin injection 3 patients had an esotropia (ranging from 4 delta to 30 delta) and two an exotropia (52 delta and 85 delta). A vertical imbalance was present in 3, and all 5 patients had symptomatic diplopia. A total of six injections were given to 5 patients, 2 of whom later went on to have surgery. Toxin injection reduced the angle of the deviation to less than 10 delta in 4 patients, all of whom are now asymptomatic. The fifth patient has persistent diplopia despite two operations to correct a large exotropia. We discuss the role of botulinum toxin and surgery in the management of strabismus due to orbital myositis.