A novel heterozygous OPA3 mutation located in the mitochondrial target sequence results in altered steady-state levels and fragmented mitochondrial network.

BACKGROUND: Mutations in OPA3 have been reported in patients with autosomal dominant optic atrophy plus cataract and Costeff syndrome. Here, we report the results of a comprehensive study on OPA3 mutations, including the mutation spectrum and its prevalence in a large cohort of OPA1-negative autosomal dominant optic atrophy (ADOA) patients, the associated clinical phenotype and the functional characterisation of a newly identified OPA3 mutant. METHODS: Mutation analysis was carried out in a patient cohort of 121 independent ADOA patients. To characterise a novel OPA3 mutation, we analysed the mitochondrial import, steady-state levels and the mitochondrial localisation of the mutated protein in patients' fibroblasts. Furthermore, the morphology of mitochondria harbouring the mutated OPA3 was monitored. RESULTS: We identified four independent cases (representing families with multiple affected members) with OPA3 mutations. Besides the known p.Q105E mutation, we observed a novel insertion, c.10_11insCGCCCG/p.V3_G4insAP which is located in the mitochondrial presequence. Detailed functional analysis of mitochondria harbouring this novel mutation demonstrates a fragmented mitochondrial network with a decreased mitochondrial mass in patient fibroblasts. In addition, quantification of the OPA3 protein reveals decreased steady-state levels of the mutant protein compared with the native one. Comparison of the clinical phenotypes suggests that OPA3 mutations can additionally evoke hearing loss and by that extend the clinical manifestation of OPA3-associated optic atrophy. This finding is supported by expression analysis of OPA3 in murine cochlear tissue. CONCLUSIONS: In summary, our study provides new insights into the clinical spectrum and the pathogenesis of dominant optic atrophy caused by mutations in the OPA3 gene.

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.

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.

ABCA4 gene analysis in patients with autosomal recessive cone and cone rod dystrophies.

The ATP-binding cassette (ABC) transporters constitute a family of large membrane proteins, which transport a variety of substrates across membranes. The ABCA4 protein is expressed in photoreceptors and possibly functions as a transporter for N-retinylidene-phosphatidylethanolamine (N-retinylidene-PE), the Schiff base adduct of all-trans-retinal with PE. Mutations in the ABCA4 gene have been initially associated with autosomal recessive Stargardt disease. Subsequent studies have shown that mutations in ABCA4 can also cause a variety of other retinal dystrophies including cone rod dystrophy and retinitis pigmentosa. To determine the prevalence and mutation spectrum of ABCA4 gene mutations in non-Stargardt phenotypes, we have screened 64 unrelated patients with autosomal recessive cone (arCD) and cone rod dystrophy (arCRD) applying the Asper Ophthalmics ABCR400 microarray followed by DNA sequencing of all coding exons of the ABCA4 gene in subjects with single heterozygous mutations. Disease-associated ABCA4 alleles were identified in 20 of 64 patients with arCD or arCRD. In four of 64 patients (6%) only one mutant ABCA4 allele was detected and in 16 patients (25%), mutations on both ABCA4 alleles were identified. Based on these data we estimate a prevalence of 31% for ABCA4 mutations in arCD and arCRD, supporting the concept that the ABCA4 gene is a major locus for various types of degenerative retinal diseases with abnormalities in cone or both cone and rod function.

Oligocone trichromacy: clinical and molecular genetic investigations.

PURPOSE: To describe the phenotype and genotype of patients with a diagnosis of oligocone trichromacy (OT). METHODS: Six unrelated patients had a detailed ophthalmic examination including color vision testing, a Goldmann visual field test, fundus photography, and full-field electroretinography (ffERG). Five patients also underwent multifocal (mf)ERG, autofluorescence recording, and optical coherence tomography (OCT). Genetic analysis included sequencing of all coding regions and flanking introns of CNGA3, CNGB3, GNAT2, KCNV2, and PDE6C. RESULTS: All patients had subnormal visual acuity, a history of congenital nystagmus, and subjectively normal or near-normal color vision; five patients reported photophobia. Clinical examinations revealed largely normal fundi, normal Goldmann visual field results with the IV/4e target, and normal color discrimination or mild color vision deficiency. Electrophysiological investigations showed either complete absence of recordable cone responses or severely reduced amplitudes. All retinal layers were identifiable by OCT, which also showed thinning of the peripheral retina. Genetic analysis revealed two causative CNGB3 mutations in one patient and single heterozygous mutations of unknown significance in CNGB3 and PDE6C in two other patients. CONCLUSIONS: Oligocone trichromacy is a heterogeneous condition with respect to both phenotypic appearance and genetic background. The finding of mutations in genes known to be involved in complete and incomplete achromatopsia supports the notion that some forms of OT is an extreme form of incomplete achromatopsia with preferential loss of peripheral cones.