Ulrich Kellner

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.

Revised Recommendations on Screening for Chloroquine and Hydroxychloroquine Retinopathy

BACKGROUND The American Academy of Ophthalmology recommendations for screening of chloroquine (CQ) and hydroxychloroquine (HCQ) retinopathy were published in 2002, but improved screening tools and new knowledge about the prevalence of toxicity have appeared in the ensuing years. No treatment exists as yet for this disorder, so it is imperative that patients and their physicians be aware of the best practices for minimizing toxic damage. RISK OF TOXICITY New data have shown that the risk of toxicity increases sharply toward 1% after 5 to 7 years of use, or a cumulative dose of 1000 g, of HCQ. The risk increases further with continued use of the drug. DOSAGE The prior recommendation emphasized dosing by weight. However, most patients are routinely given 400 mg of HCQ daily (or 250 mg CQ). This dose is now considered acceptable, except for individuals of short stature, for whom the dose should be determined on the basis of ideal body weight to avoid overdosage. SCREENING SCHEDULE A baseline examination is advised for patients starting these drugs to serve as a reference point and to rule out maculopathy, which might be a contraindication to their use. Annual screening should begin after 5 years (or sooner if there are unusual risk factors). SCREENING TESTS Newer objective tests, such as multifocal electroretinogram (mfERG), spectral domain optical coherence tomography (SD-OCT), and fundus autofluorescence (FAF), can be more sensitive than visual fields. It is now recommended that along with 10-2 automated fields, at least one of these procedures be used for routine screening where available. When fields are performed independently, even the most subtle 10-2 field changes should be taken seriously and are an indication for evaluation by objective testing. Because mfERG testing is an objective test that evaluates function, it may be used in place of visual fields. Amsler grid testing is no longer recommended. Fundus examinations are advised for documentation, but visible bulls-eye maculopathy is a late change, and the goal of screening is to recognize toxicity at an earlier stage. COUNSELING Patients should be aware of the risk of toxicity and the rationale for screening (to detect early changes and minimize visual loss, not necessarily to prevent it). The drugs should be stopped if possible when toxicity is recognized or strongly suspected, but this is a decision to be made in conjunction with patients and their medical physicians.

Mutations in a human homologue of Drosophila crumbs cause retinitis pigmentosa (RP12)

Retinitis pigmentosa (RP) comprises a clinically and genetically heterogeneous group of diseases that afflicts approximately 1.5 million people worldwide. Affected individuals suffer from a progressive degeneration of the photoreceptors, eventually resulting in severe visual impairment. To isolate candidate genes for chorioretinal diseases, we cloned cDNAs specifically or preferentially expressed in the human retina and the retinal pigment epithelium (RPE) through a novel suppression subtractive hybridization (SSH) method. One of these cDNAs (RET3C11) mapped to chromosome 1q31–q32.1, a region harbouring a gene involved in a severe form of autosomal recessive RP characterized by a typical preservation of the para-arteriolar RPE (RP12; ref. 3). The full-length cDNA encodes an extracellular protein with 19 EGF-like domains, 3 laminin A G-like domains and a C-type lectin domain. This protein is homologous to the Drosophila melanogaster protein crumbs (CRB), and denoted CRB1 (crumbs homologue 1). In ten unrelated RP patients with preserved para-arteriolar RPE, we identified a homozygous AluY insertion disrupting the ORF, five homozygous missense mutations and four compound heterozygous mutations in CRB1. The similarity to CRB suggests a role for CRB1 in cell-cell interaction and possibly in the maintenance of cell polarity in the retina. The distinct RPE abnormalities observed in RP12 patients suggest that CRB1 mutations trigger a novel mechanism of photoreceptor degeneration.

Safety and Efficacy of a Flexible Dosing Regimen of Ranibizumab in Neovascular Age-Related Macular Degeneration: The SUSTAIN Study

OBJECTIVE To evaluate the safety and efficacy of individualized ranibizumab treatment in patients with neovascular age-related macular degeneration. DESIGN Twelve-month, phase III, multicenter, open-label, single-arm study. PARTICIPANTS A total of 513 ranibizumab-naïve SUSTAIN patients. INTERVENTION Three initial monthly injections of ranibizumab (0.3 mg) and thereafter pro re nata (PRN) retreatment for 9 months based on prespecified retreatment criteria. Patients switched to 0.5 mg ranibizumab after approval in Europe. MAIN OUTCOME MEASURES Frequency of adverse events (AEs), monthly change of best-corrected visual acuity (BCVA) and central retinal thickness (CRT) from baseline, the time to first re-treatment, and the number of treatments were assessed. RESULTS A total of 249 patients (48.5%) reported ocular AEs, and 8 (1.5%) deaths, 5 (1.2%) patients with ocular serious AEs of the study eye (retinal hemorrhage, cataract, retinal pigment epithelial tear, reduced visual acuity [VA], vitreous hemorrhage), and 19 (3.7%) patients with arteriothromboembolic events were observed. Most frequent AEs in the study eye were reduced VA (18.5%), retinal hemorrhage (7.2%), increased intraocular pressure (7.0%), and conjunctival hemorrhage (5.5%). The average number of re-treatments from months 3 to 11 was 2.7. Mean best-corrected visual acuity increased steadily from baseline to month 3 to reach +5.8 letters, decreased slightly from month 3 to 6, and remained stable from month 6 to 12, reaching +3.6 at month 12. Mean change in CRT was -101.1 μm from baseline to month 3 and -91.5 μm from baseline to month 12. CONCLUSIONS The safety results are comparable to the favorable tolerability profile of ranibizumab observed in previous pivotal clinical studies; individualized treatment with less than monthly re-treatments shows a similar safety profile as observed in previous randomized clinical trials with monthly ranibizumab treatment. Efficacy outcomes were achieved with a low average number of re-treatments. Visual acuity in SUSTAIN patients with individualized re-treatment based on VA/optical coherence tomography assessment reached on average a maximum after the first 3 monthly injections, decreased slightly under PRN during the next 2 to 3 months, and was then sustained throughout the treatment period.

CNGA3 Mutations in Hereditary Cone Photoreceptor Disorders

We recently showed that mutations in the CNGA3 gene encoding the alpha-subunit of the cone photoreceptor cGMP-gated channel cause autosomal recessive complete achromatopsia linked to chromosome 2q11. We now report the results of a first comprehensive screening for CNGA3 mutations in a cohort of 258 additional independent families with hereditary cone photoreceptor disorders. CNGA3 mutations were detected not only in patients with the complete form of achromatopsia but also in incomplete achromats with residual cone photoreceptor function and (rarely) in patients with evidence for severe progressive cone dystrophy. In total, mutations were identified in 53 independent families comprising 38 new CNGA3 mutations, in addition to the 8 mutations reported elsewhere. Apparently, both mutant alleles were identified in 47 families, including 16 families with presumed homozygous mutations and 31 families with two heterozygous mutations. Single heterozygous mutations were identified in six additional families. The majority of all known CNGA3 mutations (39/46) are amino acid substitutions compared with only four stop-codon mutations, two 1-bp insertions and one 3-bp in-frame deletion. The missense mutations mostly affect amino acids conserved among the members of the cyclic nucleotide gated (CNG) channel family and cluster at the cytoplasmic face of transmembrane domains (TM) S1 and S2, in TM S4, and in the cGMP-binding domain. Several mutations were identified recurrently (e.g., R277C, R283W, R436W, and F547L). These four mutations account for 41.8% of all detected mutant CNGA3 alleles. Haplotype analysis suggests that the R436W and F547L mutant alleles have multiple origins, whereas we found evidence that the R283W alleles, which are particularly frequent among patients from Scandinavia and northern Italy, have a common origin.

Next-generation genetic testing for retinitis pigmentosa

Molecular diagnostics for patients with retinitis pigmentosa (RP) has been hampered by extreme genetic and clinical heterogeneity, with 52 causative genes known to date. Here, we developed a comprehensive next‐generation sequencing (NGS) approach for the clinical molecular diagnostics of RP. All known inherited retinal disease genes (n = 111) were captured and simultaneously analyzed using NGS in 100 RP patients without a molecular diagnosis. A systematic data analysis pipeline was developed and validated to prioritize and predict the pathogenicity of all genetic variants identified in each patient, which enabled us to reduce the number of potential pathogenic variants from approximately 1,200 to zero to nine per patient. Subsequent segregation analysis and in silico predictions of pathogenicity resulted in a molecular diagnosis in 36 RP patients, comprising 27 recessive, six dominant, and three X‐linked cases. Intriguingly, De novo mutations were present in at least three out of 28 isolated cases with causative mutations. This study demonstrates the enormous potential and clinical utility of NGS in molecular diagnosis of genetically heterogeneous diseases such as RP. De novo dominant mutations appear to play a significant role in patients with isolated RP, having major implications for genetic counselling. Hum Mutat 33:963–972, 2012.

Mutations in the Cone Photoreceptor G-Protein α-Subunit Gene GNAT2 in Patients with Achromatopsia

Achromatopsia is an autosomal recessively inherited visual disorder that is present from birth and that features the absence of color discrimination. We here report the identification of five independent families with achromatopsia that segregate protein-truncation mutations in the GNAT2 gene, located on chromosome 1p13. GNAT2 encodes the cone photoreceptor-specific alpha-subunit of transducin, a G-protein of the phototransduction cascade, which couples to the visual pigment(s). Our results demonstrate that GNAT2 is the third gene implicated in achromatopsia.

TRPM1 is mutated in patients with autosomal-recessive complete congenital stationary night blindness.

Night vision requires signaling from rod photoreceptors to adjacent bipolar cells in the retina. Mutations in the genes NYX and GRM6, expressed in ON bipolar cells, lead to a disruption of the ON bipolar cell response. This dysfunction is present in patients with complete X-linked and autosomal-recessive congenital stationary night blindness (CSNB) and can be assessed by standard full-field electroretinography (ERG), showing severely reduced rod b-wave amplitude and slightly altered cone responses. Although many cases of complete CSNB (cCSNB) are caused by mutations in NYX and GRM6, in approximately 60% of the patients the gene defect remains unknown. Animal models of human diseases are a good source for candidate genes, and we noted that a cCSNB phenotype present in homozygous Appaloosa horses is associated with downregulation of TRPM1. TRPM1, belonging to the family of transient receptor potential channels, is expressed in ON bipolar cells and therefore qualifies as an excellent candidate. Indeed, mutation analysis of 38 patients with CSNB identified ten unrelated cCSNB patients with 14 different mutations in this gene. The mutation spectrum comprises missense, splice-site, deletion, and nonsense mutations. We propose that the cCSNB phenotype in these patients is due to the absence of functional TRPM1 in retinal ON bipolar cells.

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.

Diagnostic Clinical Findings of a New Syndrome with Night Blindness, Maculopathy, and Enhanced S Cone Sensitivity

We studied eight patients who had night blindness, maculopathy (often cystoid), degenerative changes in the region of the vascular arcades, relatively mild visual field loss, and an unusual but characteristic electroretinogram. The dark-adapted electroretinogram showed no response to low-intensity stimuli that normally activate the rods, but large, slow responses to high-intensity stimuli. These large, slow waveforms persisted without change under light adaptation, and showed a striking mismatch to photopically balanced short and long wavelength stimuli (with sensitivity much greater to short than long wave-lengths). Since there is evidence from other studies that the electroretinogram and psychophysical responses represent hypersensitivity of short wavelength-sensitive (S or blue) cones, we propose that this disorder be called the enhanced S cone syndrome. There can be different degrees of severity in this syndrome, and progression appears to be slow.