Mutation spectrum and clinical investigation of achromatopsia patients with mutations in the GNAT2 gene.

Achromatopsia (ACHM) is a hereditary cone photoreceptor disorder characterized by the inability to discriminate colors, nystagmus, photophobia, and low-visual acuity. Six genes have been associated with this rare autosomal recessively inherited disease, including the GNAT2 gene encoding the catalytic α-subunit of the G-protein transducin which is expressed in the cone photoreceptor outer segment. Out of a cohort of 1,116 independent families diagnosed with a primary clinical diagnosis of ACHM, we identified 23 patients with ACHM from 19 independent families with likely causative mutations in GNAT2, representing 1.7% of our large ACHM cohort. In total 22 different potentially disease-causing variants, of which 12 are novel, were identified. The mutation spectrum also includes a novel copy number variation, a heterozygous duplication of exon 4, of which the breakpoint matches exactly that of the previously reported exon 4 deletion. Two patients carry just a single heterozygous variant. In addition to our previous study on GNAT2-ACHM, we also present detailed clinical data of these patients.

Cardiovascular Risk Factors in Central Retinal Artery Occlusion: Results of a Prospective and Standardized Medical Examination.

PURPOSE: To analyze the underlying risk factors in patients with nonarteritic central retinal artery occlusion (CRAO) in a well-defined and homogenous group of patients enrolled in the European Assessment Group for Lysis in the Eye (EAGLE) study. DESIGN: Analysis of the cardiovascular risk factors in a prospective, randomized clinical trial. PARTICIPANTS: Seventy-seven EAGLE patients with nonarteritic CRAO. METHODS: Analysis of vascular risk factors and underlying diseases detected by questionnaire and standardized physical examination within 1 month after occlusion. MAIN OUTCOME MEASURES: The standardized physical examination included carotid Doppler ultrasonography, echocardiography, electrocardiography, blood pressure monitoring, pulse rate, urine analysis, body mass index analysis, and laboratory tests. RESULTS: Seventy-seven of 84 patients had complete datasets for analysis. Fifty-two (67%) patients had cardiovascular risk factors in their medical history, and comprehensive phenotyping identified at least 1 new risk factor in 60 patients (78%; 95% confidence interval, 67%-87%). Thirty-one (40%) had carotid artery stenosis of at least 70%. Eleven patients experienced a stroke, 5 of those within 4 weeks after the CRAO occurred. Arterial hypertension was found in 56 (73%) patients and was newly diagnosed in 12 (16%) study participants. Cardiac diseases were also highly prevalent (22% coronary artery disease, 20% atrial fibrillation, and 17% valvular heart disease). CONCLUSIONS: Previously undiagnosed vascular risk factors were found in 78% of all CRAO patients. The most meaningful risk factor was ipsilateral carotid artery stenosis. A comprehensive and prompt diagnostic work-up is mandatory for all CRAO patients.

Novel insights into the molecular pathogenesis of CYP4V2-associated Bietti's retinal dystrophy.

Bietti's crystalline dystrophy (BCD) is a rare, autosomal recessive retinal degenerative disease associated with mutations in CYP4V2. In this study, we describe the genetic and clinical findings in 19 unrelated BCD patients recruited from five international retinal dystrophy clinics. Patients underwent ophthalmic examinations and were screened for CYP4V2 mutations by Sanger sequencing and quantitative polymerase chain reaction (qPCR) copy number variation screening. Eight CYP4V2 mutations were found in 10/19 patients, including three patients in whom only monoallelic mutations were detected. Four novel mutations were identified: c.604G>A; p.(Glu202Lys), c.242C>G; p.(Thr81Arg), c.604+4A>G; p.(?), and c.1249dup; p.(Thr417Asnfs*2). In addition, we identified a heterozygous paternally inherited genomic deletion of at least 3.8 Mb, encompassing the complete CYP4V2 gene and several other genes, which is novel. Clinically, patients demonstrated phenotypic variability, predominantly showing choroidal sclerosis, attenuated vessels, and crystalline deposits of varying degrees of severity. To our knowledge, our study reports the first heterozygous CYP4V2 deletion and hence a novel mutational mechanism underlying BCD. Our results emphasize the importance of copy number screening in BCD. Finally, the identification of CYP4V2-negative patients with indistinguishable phenotypes from CYP4V2-positive patients might suggest the presence of mutations outside the coding regions of CYP4V2, or locus heterogeneity, which is unreported so far.

Intraocular biopsy using special forceps: a new instrument and refined surgical technique.

AIM: The aim was to investigate the Essen biopsy forceps as a new instrument and surgical approach for biopsy of intraocular tumours. Biopsy is indicated for assessment of any uncertain intraocular process or confirmation for presumed diagnosis before treatment. There is increasing interest for further genetic and immunocytological information in order to characterise the neoplasm, especially grading and prognosis of micrometastasis in uveal melanoma. The authors have developed a new surgical technique using special intraocular biopsy forceps. METHODS: Twenty patients with uncertain intraocular subretinal tumour underwent biopsies carried out using the special Essen biopsy forceps. Biopsies were obtained through sutureless 23-gauge three-port vitrectomy. A small retinotomy tumour specimen was taken by the forceps branches. For further processing, the specimens were flushed out into a sterile tube and then sent to pathologists. RESULTS: The prebioptical tumour had a mean thickness of 3.48 mm (1.1 to 9.8 mm). In all cases (n=20) biopsies (0.3-2.1 mm in size) were obtained, in 19 cases (95%) allowing precise histological and immunohistochemical typing of the lesions following cytoblock embedding. Uveal melanoma was diagnosed in 50% (n=10), choroidal metastasis in 15% (n=3) and choroidal naevus in 15% (n=3); other diagnoses (n=3) included choroidal haemangioma, B cell lymphoma and old subretinal haemorrhage. Apart from three patients with temporary punctual bleeding on the surface, there were no intra- and postoperative complications. CONCLUSIONS: Biopsy using special forceps is a promising new approach and precise surgical procedure. Especially for small intraocular tumours, this technique has the advantage in providing enough tissue for improved histological examination and presenting a low risk for complications.

Decreased catalytic activity and altered activation properties of PDE6C mutants associated with autosomal recessive achromatopsia.

Mutations in the gene encoding the catalytic subunit of the cone photoreceptor phosphodiesterase (PDE6C) have been recently reported in patients with autosomal recessive inherited achromatopsia (ACHM) and early-onset cone photoreceptor dysfunction. Here we present the results of a comprehensive study on PDE6C mutations including the mutation spectrum, its prevalence in a large cohort of ACHM/cone dysfunction patients, the clinical phenotype and the functional characterization of mutant PDE6C proteins. Twelve affected patients from seven independent families segregating PDE6C mutations were identified in our total patient cohort of 492 independent families. Eleven different PDE6C mutations were found including two nonsense mutations, three mutations affecting transcript splicing as shown by minigene assays, one 1 bp-insertion and five missense mutations. We also performed a detailed functional characterization of six missense mutations applying the baculovirus system to express recombinant mutant and wildtype chimeric PDE6C/PDE5 proteins in Sf9 insect cells. Purified proteins were analyzed using Western blotting, phosphodiesterase (PDE) activity measurements as well as inhibition assays by zaprinast and Pγ. Four of the six PDE6C missense mutations led to baseline PDE activities and most likely represent functional null alleles. For two mutations, p.E790K and p.Y323N, we observed reduction in PDE activity of approximately 60% and 80%, respectively. We also observed differences for Pγ inhibition. The p.E790K mutant, with an IC50 value of 2.7 nm is 20.7-fold more sensitive for Pγ inhibition, whereas the p.Y323N mutant with an IC50 of 158 nm is 3-fold less sensitive when compared with the wildtype control.

Central retinal artery occlusion: local intra-arterial fibrinolysis versus conservative treatment, a multicenter randomized trial.

PURPOSE: The reported outcomes of central retinal artery occlusion (CRAO) with or without treatment vary considerably. Although local intra-arterial fibrinolysis (LIF) using recombinant tissue plasminogen activator (rtPA) is a promising treatment, outcomes have not been compared in randomized trials. DESIGN: Prospective randomized multicenter clinical trial (the European Assessment Group for Lysis in the Eye Study) to compare treatment outcome after conservative standard treatment (CST) and LIF for acute nonarteritic CRAO. PARTICIPANTS: Between 2002 and 2007, 9 centers in Austria and Germany recruited 84 patients (40 received CST, 44 received LIF), and data for 82 patients were analyzed. METHODS: Patients (age 18-75 years) with CRAO, symptoms for 20 hours or less, and best-corrected visual acuity (BCVA) <0.5 logarithm of the minimum angle of resolution (logMAR) were randomized to the CST or LIF group. MAIN OUTCOME MEASURES: The primary end point was BCVA after 1 month; the secondary end point was safety. RESULTS: The mean interval between first symptoms and therapy was 10.99+/-5.49 hours (CST) and 12.78+/-5.77 hours (LIF). The mean BCVA (logMAR) improved significantly in both groups (CST: -0.44 [standard deviation 0.55]; LIF: -0.45 [standard deviation 0.55]; both P < 0.0001) and did not differ between groups (P=0.69). Clinically significant visual improvement (> or = 0.3 logMAR) was noted in 60.0% (CST) and 57.1% (LIF) of patients. Two patients in the CST group (4.3%) and 13 patients in the LIF group (37.1%) had adverse reactions. Because of apparently similar efficacy and the higher rate of adverse reactions in the LIF group, the study was stopped after the first interim analysis at the recommendation of the data and safety monitoring committee. CONCLUSIONS: In light of these 2 therapies' similar outcomes and the higher rate of adverse reactions associated with LIF, we cannot recommend LIF for the management of acute CRAO. FINANCIAL DISCLOSURE(S): The author(s) have no proprietary or commercial interest in any materials discussed in this article.

A homologous genetic basis of the murine cpfl1 mutant and human achromatopsia linked to mutations in the PDE6C gene.

Retinal cone photoreceptors mediate fine visual acuity, daylight vision, and color vision. Congenital hereditary conditions in which there is a lack of cone function in humans cause achromatopsia, an autosomal recessive trait, characterized by low vision, photophobia, and lack of color discrimination. Herein we report the identification of mutations in the PDE6C gene encoding the catalytic subunit of the cone photoreceptor phosphodiesterase as a cause of autosomal recessive achromatopsia. Moreover, we show that the spontaneous mouse mutant cpfl1 that features a lack of cone function and rapid degeneration of the cone photoreceptors represents a homologous mouse model for PDE6C associated achromatopsia.

Multifocal electroretinography in patients with exudative amd and intravitreal treatment with pegaptanib sodium.

BACKGROUND: To objectively investigate central retinal function in patients with choroidal neovascularization (CNV) due to age-related macular degeneration (AMD) before and after treatment with pegaptanib sodium (PS). METHODS: Patients with CNV due to exudative AMD received intravitreal injections of 0.3 mg PS every sixth week if angiographic activity was evident. Longitudinal observation included recordings with multifocal electroretinography (mfERG) before the first treatment and before each injection at follow-up intervals. RESULTS: During the observation period of 30.5 +/- 8 weeks (mean +/- SD) a mean number of 5.3 +/- 1.3 injections were applied. Final mean log(MAR) visual acuity decreased, statistically nonsignificant, from 0.67 +/- 0.3 at baseline to 0.74 +/- 0.16. mfERG recordings in 12 patients after 25 +/- 9 weeks evinced a decrease in response density which was statistically significant in the central 5 degrees . Mean P1-amplitudes of ring 1, 2, and 3 were reduced by 66%, 39% and 30%, respectively. During follow-up, implicit times of the P1 components remained stable within 4% of baseline. In three of four patients with vision loss of 2 lines or more, P1-response amplitudes decreased substantially at least 6 weeks prior vision loss. CONCLUSION: Treatment with PS resulted in a decrease of central retinal function more obvious in mfERG than in VA longitudinal testing. Good correlations were seen between changes in mean vision and changes in mfERG response density components. As a decline in P1-response amplitudes anteceded vision loss in this study, our results indicate a possible role of mfERG to predict vision loss during intravitreal pharmacotherapy.

A novel gene for Usher syndrome type 2: mutations in the long isoform of whirlin are associated with retinitis pigmentosa and sensorineural hearing loss.

Usher syndrome is an autosomal recessive condition characterized by sensorineural hearing loss, variable vestibular dysfunction, and visual impairment due to retinitis pigmentosa (RP). The seven proteins that have been identified for Usher syndrome type 1 (USH1) and type 2 (USH2) may interact in a large protein complex. In order to identify novel USH genes, we followed a candidate strategy, assuming that mutations in proteins interacting with this "USH network" may cause Usher syndrome as well. The DFNB31 gene encodes whirlin, a PDZ scaffold protein with expression in both hair cell stereocilia and retinal photoreceptor cells. Whirlin represents an excellent candidate for USH2 because it binds to Usherin (USH2A) and VLGR1b (USH2C). Genotyping of microsatellite markers specific for the DFNB31 gene locus on chromosome 9q32 was performed in a German USH2 family that had been excluded for all known USH loci. Patients showed common haplotypes. Sequence analysis of DFNB31 revealed compound heterozygosity for a nonsense mutation, p.Q103X, in exon 1, and a mutation in the splice donor site of exon 2, c.837+1G>A. DFNB31 mutations appear to be a rare cause of Usher syndrome, since no mutations were identified in an additional 96 USH2 patients. While mutations in the C-terminal half of whirlin have previously been reported in non-syndromic deafness (DFNB31), both alterations identified in our USH2 family affect the long protein isoform. We propose that mutations causing Usher syndrome are probably restricted to exons 1-6 that are specific for the long isoform and probably crucial for retinal function. We describe a novel genetic subtype for Usher syndrome, which we named USH2D and which is caused by mutations in whirlin. Moreover, this is the first case of USH2 that is allelic to non-syndromic deafness.

Photodynamic therapy using verteporfin for choroidal neovascularization associated with angioid streaks--long-term effects.

AIM: To investigate the safety and efficacy of photodynamic therapy with verteporfin (PDT) in patients with choroidal neovascularization associated with angioid streaks (CNVAS). METHODS: A nonrandomized, prospective clinical investigation of 12 patients with CNVAS was performed. PDT was based on the criteria concerning the treatment of age-related macular degeneration. RESULTS: The mean follow-up was 41.75 months (range 24-60). The mean number of (re)treatments was 3.3 (range 2-7). Visual acuity improved by at least 1 line in 42%, was stable within +/-2 lines in 33%, decreased by at least 1 line in 58% and by >3 lines in 25% of the patients. The mean visual acuity was 0.30 (range 0.2-0.5) prior to and 0.17 (range 0.03-0.6) after the final PDT. The mean visual acuity of the contralateral eye was 0.1. 75% of contralateral eyes and 25% of the treated eyes had a final visual acuity of < or =0.1 (20/200). At the final follow-up, a significant enlargement of the lesion size was noted in 92% of the cases. CONCLUSION: Using the current (re)treatment criteria, PDT does not appear to limit the growth of CNVAS. Compared to the aggressive natural course and to the limited treatment options, PDT may at least in part help to stabilize macular function over a limited period of time.

Long-term effect of acetazolamide treatment of patients with uveitic chronic cystoid macular edema is limited by persisting inflammation.

PURPOSE: To assess the long-term effect of acetazolamide treatment on patients with cystoid macular edema (CME) in the course of intermediate or posterior chronic uveitis and to define those patients who may particularly benefit from the drug. METHODS: Fifty-two eyes (45 patients) with chronic uveitic CME were treated with acetazolamide at an initial dosage of 500 mg/d. The effect of treatment was assessed by fluorescein angiography, ophthalmoscopy, visual acuity, and Amsler testing. Therapy was withdrawn when CME did not improve at 3 weeks. In cases with CME improvement, the dosage was gradually tapered. RESULTS: The mean follow-up was 3.1 years (minimum, 1.5 years). Two subgroups were identified: group 1, quiescence of uveitis with acetazolamide as the single therapeutic agent (33 eyes); and group 2, chronically active uveitis requiring additional systemic antiinflammatory drugs (19 eyes). In both groups, visual acuity improvement was statistically significant (group 1, P = 0.012; group 2, P = 0.025). In 12 patients with a stable visual acuity gain, the medication dose could be tapered off completely without any recurrent edema shown by fluorescein angiography after a minimum follow-up of 1 year. Sixteen patients required a maintenance dosage, ranging from 125 to 500 mg daily. No major adverse effects of the medication were observed. CONCLUSIONS: During long-term follow-up, low-dose acetazolamide can be a useful therapeutic option for chronic CME in uveitis. The effect was better in patients with quiescence of uveitis than in those with chronically active uveitis. Permanent therapy is not imperative in every case.

CNGB3 mutations account for 50% of all cases with autosomal recessive achromatopsia.

Achromatopsia is a congenital, autosomal recessively inherited disorder characterized by a lack of color discrimination, low visual acuity (<0.2), photophobia, and nystagmus. Mutations in the genes for CNGA3, CNGB3, and GNAT2 have been associated with this disorder. Here, we analyzed the spectrum and prevalence of CNGB3 gene mutations in a cohort of 341 independent patients with achromatopsia. In 163 patients, CNGB3 mutations could be identified. A total of 105 achromats carried apparent homozygous mutations, 44 were compound (double) heterozygotes, and 14 patients had only a single mutant allele. The derived CNGB3 mutation spectrum comprises 28 different mutations including 12 nonsense mutations, eight insertions and/or deletions, five putative splice site mutations, and three missense mutations. Thus, the majority of mutations in the CNGB3 gene result in significantly altered and/or truncated polypeptides. Several mutations were found recurrently, in particular a 1 bp deletion, c.1148delC, which accounts for over 70% of all CNGB3 mutant alleles. In conclusion, mutations in the CNGB3 gene are responsible for approximately 50% of all patients with achromatopsia. This indicates that the CNGB3/ACHM3 locus on chromosome 8q21 is the major locus for achromatopsia in patients of European origin or descent.

Mutation analysis of NR2E3 and NRL genes in Enhanced S Cone Syndrome.

Ten new and seventeen previously reported Enhanced S Cone Syndrome (ESCS) subjects were used to search for genetic heterogeneity. All subjects were diagnosed with ESCS on the basis of clinical, psychophysical and/or electroretinography testing using published criteria. Mutation analysis was performed on the NR2E3 nuclear receptor gene by single strand conformation analysis and direct sequencing, which revealed either homozygous (N=13) or compound heterozygous (N=11) mutations in 24 subjects (89%), heterozygous mutations in 2 subjects (7%) and no mutations in 1 subject (4%). Fifteen different mutations were identified, including six not previously reported. The subject (Patient A) with no detected NR2E3 mutation had features not usually associated with ESCS, in particular moderate rod photoreceptor function in peripheral retina and an abnormally thick retinal nerve fibre layer. Mutation analysis of the NRL, CRX, NR1D1 and THRB genes in this individual revealed a heterozygous one base-pair insertion in exon 2 of the NRL gene, which results in a predicted truncation of the NRL protein. Loss-of-function NRL alleles have not been described previously in humans, but since the same mutation was present in unaffected family members, it raises the possibility that the abnormal ESCS phenotype in Patient A may result from a digenic mechanism, with a heterozygous NRL mutation and a mutation in another unknown gene.

Photodynamic therapy for symptomatic choroidal hemangioma: visual and anatomic results.

OBJECTIVE: To document the anatomic and functional outcome of photodynamic therapy (PDT) in symptomatic choroidal hemangioma DESIGN: Prospective, noncomparative, interventional case series. PARTICIPANTS: Fifteen patients with circumscribed choroidal hemangioma of the posterior pole presenting with progressive vision loss caused by exudation into the macular area. INTERVENTION: PDT using 6 mg/m(2) body surface area verteporfin and a light dose of 100 J/cm(2) at 692 nm was performed. One to four treatments with a single laser spot were applied in 6-week intervals. A standardized evaluation was provided before and at 6-week intervals after each treatment, at 3, 6, and 12 months, and a mean follow-up 19 months after the last application. MAIN OUTCOME MEASURES: Functional tests included best-refracted visual acuity (Early Treatment of Diabetic Retinopathy Study criteria) and scanning laser scotometry. Anatomic results were documented by ophthalmoscopy, fluorescein/indocyanine green angiography, and ultrasonography. RESULTS: A complete regression of the vascular mass was achieved in all eyes after the last course of one to four consecutive treatments. Tumors (mean height, 3.8 mm) responded with a reproducible decrease in size to each treatment, with the most intensive effect seen after the first application. Progressive occlusion of the angiomatous net without recanalization was documented angiographically. Two patients had stable vision with resolution of metamorphopsia; 13 patients demonstrated visual recovery. An overall visual acuity (VA) improvement of an average of 3 lines was documented, with a mean VA level of 20/125 before treatment and 20/80 after therapy. Visual fields showed withdrawal of central scotomas from the macula. No recurrence was seen during a follow-up for up to 50 months. CONCLUSIONS: PDT using verteporfin offers a safe and effective option to treat choroidal hemangiomas. Complete anatomic regression with persistent absence of leakage is associated with substantial improvements in vision.

Ten novel ORF15 mutations confirm mutational hot spot in the RPGR gene in European patients with X-linked retinitis pigmentosa.

RGPR was the first gene found to be mutated in XLRP, the subtype of RP displaying the most severe form of retinal degeneration with partial or complete blindness in the third or fourth decade of life. Despite the RP3 locus on Xp21.1 accounting for 60-90% of XLRP, only 10-20% of identified RPGR mutations were reported in earlier analyses. This discrepancy appeared to be resolved when Vervoort et al. identified a mutational hot spot in a new purine-rich 3' exon (ORF15) that accounted for 60% of their XLRP patients [Vervoort et al., 2000]. In our mutation screening of 37 unrelated European XLRP patients we identified two recently described deletions and 10 novel mutations in exon ORF15 of RPGR, 4 of which were nonsense and 6 frameshift mutations. The latter included one duplication and 5 deletion mutations, all of which lead to a downstream premature termination. No mutations were detected in the additionally screened new exon ORF14. The data reported here, together with previous findings, document a significant clustering of mutations as well as polymorphisms in ORF15 of RPGR. In our unselected XLRP patient population, ORF15 mutations constitute 32% of cases, a finding that contradicts the results of Vervoort and coworkers [Vervoort et al., 2000] but is in agreement with a more recent study on North American XLRP patients [Breuer et al., 2002]. The observed prevalence is sufficient to justify an initial mutation screening of ORF15 in the genetically heterogeneous group of XLRP.