Peculiar combinations of individually non-pathogenic missense mitochondrial DNA variants cause low penetrance Leber's hereditary optic neuropathy.

We here report on the existence of Leber's hereditary optic neuropathy (LHON) associated with peculiar combinations of individually non-pathogenic missense mitochondrial DNA (mtDNA) variants, affecting the MT-ND4, MT-ND4L and MT-ND6 subunit genes of Complex I. The pathogenic potential of these mtDNA haplotypes is supported by multiple evidences: first, the LHON phenotype is strictly inherited along the maternal line in one very large family; second, the combinations of mtDNA variants are unique to the two maternal lineages that are characterized by recurrence of LHON; third, the Complex I-dependent respiratory and oxidative phosphorylation defect is co-transferred from the proband's fibroblasts into the cybrid cell model. Finally, all but one of these missense mtDNA variants cluster along the same predicted fourth E-channel deputed to proton translocation within the transmembrane domain of Complex I, involving the ND1, ND4L and ND6 subunits. Hence, the definition of the pathogenic role of a specific mtDNA mutation becomes blurrier than ever and only an accurate evaluation of mitogenome sequence variation data from the general population, combined with functional analyses using the cybrid cell model, may lead to final validation. Our study conclusively shows that even in the absence of a clearly established LHON primary mutation, unprecedented combinations of missense mtDNA variants, individually known as polymorphisms, may lead to reduced OXPHOS efficiency sufficient to trigger LHON. In this context, we introduce a new diagnostic perspective that implies the complete sequence analysis of mitogenomes in LHON as mandatory gold standard diagnostic approach.

Carbonic anhydrase inhibitors in patients with X-linked retinoschisis: effects on macular morphology and function.

Background: Currently there is no medical treatment for X-linked retinoschisis (XLRS). In many retinal dystrophies, carbonic anhydrase inhibitors (CAIs) are effectively used to reduce cystoid macular edema. Prospective studies investigating the effect of CAIs in patients with XLRS are needed for the evaluation of their efficacy in this disease. The purpose of our work is to investigate the effects on macular morphology and function of oral CAIs used for the treatment of foveal lesions in patients with XLRS. Methods: Nineteen patients with a clinical diagnosis of XLRS were enrolled and prescribed oral CAIs for six months. We evaluated the therapeutic effect of CAIs with: best-corrected visual acuity (BCVA), spectral-domain optical coherence tomography, microperimetry (MP) and multifocal electroretinography (mfERG). Results: We observed a significant improvement of BCVA (p-value = 0.013), central retinal thickness (p-value = 0.004) and macular sensitivity (p-value<0.001). Moreover, in regards to mfERG responses, an increase of P1 wave amplitude was observed in three of the six rings. Conclusions: Our data supports the efficacy of oral CAIs for the treatment of macular cyst-like lesions in XLRS patients. The recovery of a normal retinal anatomy by means of oral CAIs could be useful to create the optimal circumstances for gene therapy. The increase in macular sensitivity and in P1 wave amplitude confirmed that MP and mfERG provide with an unbiased and more sensitive understanding of how macular function may respond to the use of CAIs. Therefore, we recommend the use of MP and mfERG to assess the effect of therapy in XLRS.

An Italian family affected by autosomal dominant microcephaly with chorioretinal degeneration.

PURPOSE: We studied an Italian family affected by the autosomal dominant form of microcephaly and chorioretinal degeneration that was characterized by various degrees of clinical expression. METHODS: An ophthalmologic examination, including visual acuity, visual field testing, an electroretinogram, and fundus photography, and a neurologic examination, including neurodevelopmental status and neuroimaging studies, were performed for all subjects. Skeletal radiography, chromosome studies, and serologic investigations were also performed. RESULTS: In this family, only two of the six affected members had an association of microcephaly, myopia, and chorioretinal degeneration. The other family members showed microcephaly, slight mental retardation, and short stature, but not chorioretinopathy. CONCLUSIONS: The significant finding in members from this dominant pedigree of microcephaly was the association of short stature and high myopia, heretofore seen only in families with recessive microcephaly. These findings could be useful for genetic counseling in the apparently isolated forms of microcephaly with chorioretinopathy.

Evaluation of Italian patients with leber congenital amaurosis due to AIPL1 mutations highlights the potential applicability of gene therapy.

PURPOSE: To evaluate the suitability of gene delivery-based approaches as potential treatment of Leber congenital amaurosis 4 (LCA4) due to AIPL1 mutations. METHODS: Genomic DNA from patients was analyzed using a microarray chip and direct sequencing. A detailed clinical evaluation including fundus autofluorescence (FAF) and optical coherence tomography (OCT) was performed in patients with AIPL1 mutations. Aipl1 null mice and porcine eyes were subretinally injected with adeno-associated viral (AAV) vectors harboring the human AIPL1 coding sequence. RESULTS: We identified 10 LCA4 patients with mutations in AIPL1. The p.W278X sequence variation was the one most frequently found. Clinical assessment revealed common features including diffuse retinal dystrophies and maculopathy. However, optical coherence tomography showed partially retained photoreceptors in extramacular regions at all ages. The fundus autofluorescence was elicitable at the posterior pole and absent in the fovea. AAV-mediated gene transfer in Aipl1 -/- mice was associated with restoration of AIPL1 and ßPDE expression in photoreceptors and protection from degeneration. Administration of a clinically relevant dose of AAV2/8-AIPL1 to the preclinical large porcine retina resulted in high level of AIPL1 photoreceptor expression in the absence of toxicity. CONCLUSIONS: Using advanced imaging diagnostics we showed that maculopathy is a main feature of LCA4. We identified retinal areas at the posterior pole with surviving photoreceptors present even in adult LCA4 patients, which could be the target of gene therapy. The possible use of gene therapy for LCA4 is additionally supported by the protection from photoreceptor degeneration observed in Aipl 1-/- mice and by the high levels of photoreceptor transduction in the absence of toxicity observed after AAV2/8 delivery to the large porcine retina.

Genotype-phenotype correlation in Italian families with Stargardt disease.

Autosomal recessive Stargardt disease (STGD) has been associated with substantial genetic and phenotypic heterogeneity. By systematic clinical analyses of STGD patients with complete genetic data (i.e. identified mutations on both alleles of the ABCA4 gene), we set out to determine phenotypic subtypes and to correlate these with specific ABCA4 alleles. Twenty-eight patients from 18 families with STGD/fundus flavimaculatus were investigated. All patients were submitted to complete ophthalmologic examination, electrophysiology, fluorescein angiography and ABCA4 gene chip analysis. Two main clinical phenotypes were observed among the examined patients. The severe phenotype was characterized by the onset of the disease <20 years and reduced ERG response, whereas the mild phenotype presented with later onset of the disease and a normal ERG response. Genetic analysis of the ABCA4 gene revealed, in the severe group, more frequently deletions, stop codons and insertions as compared to the mild phenotype group (p=0.0113 by Fisher's exact test). Moreover, the compound heterozygous mutations G1961E/5018+2T-->C found in 7 patients from 3 unrelated STGD families were associated with a mild phenotype in all subjects, except 1. This study documented variability of the clinical expression of STGD in relation to the age of onset of the disease, fundus appearance and the ERG response and allowed to subdivide patients into a severe and a mild phenotype group. These findings suggest that an extensive and comprehensive genetic analysis of STGD patients combined with thorough clinical evaluation, including the careful recording of the age of onset of the disease, would allow a more precise prognostic evaluation.

Association of a homozygous nonsense mutation in the ABCA4 (ABCR) gene with cone-rod dystrophy phenotype in an Italian family.

Genetic variation in the ABCA4 (ABCR) gene has been associated with several distinct retinal phenotypes, including Stargardt disease/fundus flavimaculatus (STGD/FFM), cone-rod dystrophy (CRD), retinitis pigmentosa (RP) and age-related macular degeneration. The current model of genotype/phenotype association suggests that patients harboring deleterious mutations in both ABCR alleles would develop RP-like retinal pathology. Here we describe ABCA4-associated phenotypes, including a proband with a homozygous nonsense mutation in a family from Southern Italy. The proband had been originally diagnosed with STGD. Ophthalmologic examination included kinetic perimetry, electrophysiological studies and fluorescein angiography. DNA of the affected individual and family members was analyzed for variants in all 50 exons of the ABCA4 gene by screening on the ABCR400 microarray. A homozygous nonsense mutation 2971G>T (G991X) was detected in a patient initially diagnosed with STGD based on funduscopic evidence, including bull's eye depigmentation of the fovea and flecks at the posterior pole extending to the mid-peripheral retina. Since this novel nucleotide substitution results in a truncated, nonfunctional, ABCA4 protein, the patient was examined in-depth for the severity of the disease phenotype. Indeed, subsequent electrophysiological studies determined severely reduced cone amplitude as compared to the rod amplitude, suggesting the diagnosis of CRD. ABCR400 microarray is an efficient tool for determining causal genetic variation, including new mutations. A homozygous protein-truncating mutation in ABCA4 can cause a phenotype ranging from STGD to CRD as diagnosed at an early stage of the disease. Only a combination of comprehensive genotype/phenotype correlation studies will determine the proper diagnosis and prognosis of ABCA4-associated pathology.