Phenotypic variability in a French family with a novel mutation in the BEST1 gene causing multifocal best vitelliform macular dystrophy.

AIMS: To describe genetic and clinical findings in a French family affected by best vitelliform macular dystrophy (BVMD). METHODS: We screened eight at-risk members of a family, including a BVMD-affected proband, by direct sequencing of 11 bestrophin-1 (BEST1) exons. Individuals underwent ophthalmic examination and autofluorescent fundus imaging, indocyanine green angiography, electro-oculogram (EOG), electroretinogram (ERG), multifocal ERG, optical coherence tomography (OCT), and where possible, spectral domain OCT. RESULTS: The sequence analysis of the BEST1 gene revealed one previously unknown mutation, c.15C>A (p.Y5X), in two family members and one recently described mutation, c.430A>G (p.S144G), in five family members. Fundus examination and electrophysiological responses provided no evidence of the disease in the patient carrying only the p.Y5X mutation. Three patients with the p.S144G mutation did not show any preclinical sign of BVMD except altered EOGs. Two individuals of the family exhibited a particularly severe phenotype of multifocal BVMD-one individual carrying the p.S144G mutation heterozygously and one individual harboring both BEST1 mutations (p.S144G inherited from his mother and p.Y5X from his father). Both of these family members had multifocal vitelliform autofluorescent lesions combined with abnormal EOG, and the spectral domain OCT displayed a serous retinal detachment. In addition, ERGs demonstrated widespread retinal degeneration and multifocal ERGs showed a reduction in the central retina function, which could be correlated with the decreased visual acuity and visual field scotomas. CONCLUSIONS: A thorough clinical evaluation found no pathological phenotype in the patient carrying the isolated p.Y5X mutation. The patients carrying the p.S144G variation in the protein exhibited considerable intrafamilial phenotypic variability. Two young affected patients in this family exhibited an early onset, severe, multifocal BVMD with a diffuse distribution of autofluorescent deposits throughout the retina and rapid evolution toward the loss of central vision. The other genetically affected relatives had only abnormal EOGs and displayed no or extremely slow electrophysiological evolution.

ATM localization and gene expression in the adult mouse eye.

PURPOSE: High levels of metabolism and oxygen consumption in most adult murine ocular compartments, combined with exposure to light and ultraviolet (UV) radiation, are major sources of oxidative stress, causing DNA damage in ocular cells. Of all mammalian body cells, photoreceptor cells consume the largest amount of oxygen and generate the highest levels of oxidative damage. The accumulation of such damage throughout life is a major factor of aging tissues. Several multiprotein complexes have recently been identified as the major sensors and mediators involved in the maintenance of DNA integrity. The activity of these complexes initially seemed to be restricted to dividing cells, given their ultimate role in major cell cycle checkpoints. However, it was later established that they are also active in post-mitotic cells. Recent findings demonstrate that the DNA damage response (DDR) is essential for the development, maintenance, and normal functioning of the adult central nervous system. One major molecular factor in the DDR is the protein, ataxia telangiectasia mutated (ATM). It is required for the rapid induction of cellular responses to DNA double-strand breaks. These cytotoxic DNA lesions may be caused by oxidative damage. To understand how ATM prevents oxidative stress and participates in the maintenance of genomic integrity and cell viability of the adult retina, we determined the ATM expression patterns and studied its localization in the adult mouse eye. METHODS: Atm gene expression was analyzed by RT-PCR experiments and its localization by in situ hybridization on adult mouse ocular and cerebellar tissue sections. ATM protein expression was determined by western blot analysis of proteins homogenates extracted from several mouse tissues and its localization by immunohistochemistry experiments performed on adult mouse ocular and cerebellar tissue sections. In addition, subcellular localization was realized by confocal microscopy imaging of ocular tissue sections, with a special focus on retinal cells. RESULTS: Using RT-PCR, we detected a band of the expected size, with its sequence matching the amplified Atm cDNA sequence. Atm mRNA was detected in most cell bodies of the adult mouse eye by in situ hybridization of ocular tissue sections with specific digoxigenin-labeled PCR-amplified cDNA probes. Western blotting with different specific antibodies revealed bands corresponding to the expected sizes of ATM and its active forms (ATMp). These bands were not observed in the analysis of protein homogenates from Atm-deficient mouse tissues. ATM immunoreactivity was detected in the nucleus of all adult mice retinal cells and in most non-neuronal ocular cell types. The active phosphorylated form of ATM was also present in the retina as well as in non-neuronal cells of the adult mouse eye. However, its subcellular localization differed as a function of the cell type examined. A major finding of this study was that ATMp immunostaining in photoreceptor cells was exclusively in the cytoplasm, whereas ATM immunostaining was only in the nucleus of these cells. Furthermore, the specific and distinct ATM and ATMp immunolabeling patterns in photoreceptor cells were identical to those observed in the adult mouse cerebellar granule cells. CONCLUSIONS: We report the expression profile of Atm gene and protein in the adult mouse eye. In particular, we observed a difference between the localization patterns of the active and inactive forms of ATM in photoreceptor cells. These localization patterns suggest that ATM and its phosphorylated activated form may be involved in both the protection of cells from oxidative damage and the maintenance of ocular cell structure and function. The protection mechanisms mediated by the two forms of ATM appear to be particularly important in maintaining photoreceptor integrity.

Three new PAX6 mutations including one causing an unusual ophthalmic phenotype associated with neurodevelopmental abnormalities.

PURPOSE: The PAX6 gene was first described as a candidate for human aniridia. However, PAX6 expression is not restricted to the eye and it appears to be crucial for brain development. We studied PAX6 mutations in a large spectrum of patients who presented with aniridia phenotypes, Peters' anomaly, and anterior segment malformations associated or not with neurological anomalies. METHODS: Patients and related families were ophthalmologically phenotyped, and in some cases neurologically and endocrinologically examined. We screened the PAX6 gene by direct sequencing in three groups of patients: those affected by aniridia; those with diverse ocular manifestations; and those with Peters' anomaly. Two mutations were investigated by generating crystallographic representations of the amino acid changes. RESULTS: Three novel heterozygous mutations affecting three unrelated families were identified: the g.572T>C nucleotide change, located in exon 5, and corresponding to the Leucine 46 Proline amino-acid mutation (L46P); the g.655A>G nucleotide change, located in exon 6, and corresponding to the Serine 74 Glycine amino-acid mutation (S74G); and the nucleotide deletion 579delG del, located in exon 6, which induces a frameshift mutation leading to a stop codon (V48fsX53). The L46P mutation was identified in affected patients presenting bilateral microphthalmia, cataracts, and nystagmus. The S74G mutation was found in a large family that had congenital ocular abnormalities, diverse neurological manifestations, and variable cognitive impairments. The 579delG deletion (V48fsX53) caused in the affected members of the same family bilateral aniridia associated with congenital cataract, foveal hypolasia, and nystagmus. We also detected a novel intronic nucleotide change, IVS2+9G>A (very likely a mutation) in an apparently isolated patient affected by a complex ocular phenotype, characterized primarily by a bilateral microphthalmia. Whether this nucleotide change is indeed pathogenic remains to be demonstrated. Two previously known heterozygous mutations of the PAX6 gene sequence were also detected in patients affected by aniridia: a de novo previously known nucleotide change, g.972C>T (Q179X), in exon 8, leading to a stop codon and a heterozygous g.555C>A (C40X) recurrent nonsense mutation in exon 5. No mutations were found in patients with Peters' anomaly. CONCLUSIONS: We identified three mutations associated with aniridia phenotypes (Q179X, C40X, and V48fsX53). The three other mutations reported here cause non-aniridia ocular phenotypes associated in some cases with neurological anomalies. The IVS2+9G>A nucleotide change was detected in a patient with a microphthalmia phenotype. The L46P mutation was detected in a family with microphthalmia, cataract, and nystagmus. This mutation is located in the DNA-binding paired-domain and the crystallographic representations of this mutation show that this mutation may affect the helix-turn-helix motif, and as a consequence the DNA-binding properties of the resulting mutated protein. Ser74 is located in the PAX6 PD linker region, essential for DNA recognition and DNA binding, and the side chain of the Ser74 contributes to DNA recognition by the linker domain through direct contacts. Crystallographic representations show that the S74G mutation results in no side chain and therefore perturbs the DNA-binding properties of PAX6. This study highlights the severity and diversity of the consequences of PAX6 mutations that appeared to result from the complexity of the PAX6 gene structure, and the numerous possibilities for DNA binding. This study emphasizes the fact that neurodevelopmental abnormalities may be caused by PAX6 mutations. The neuro-developmental abnormalities caused by PAX6 mutations are probably still overlooked in the current clinical examinations performed throughout the world in patients affected by PAX6 mutations.

Sirt1 involvement in rd10 mouse retinal degeneration.

PURPOSE: Sirtuin1 (Sirt1) is an NAD(+)-dependent deacetylase involved in development, cell survival, stress resistance, energy metabolism, and aging. It is expressed in the mammalian central nervous system (CNS) and is activated during processes associated with neuroprotection. The retinal degeneration 10 (rd10) mouse model of retinitis pigmentosa (RP) was used to investigate the possible role of Sirt1 in this type of retinal degeneration. METHODS: Eyes from control and rd10 mice were used. Sirt1 mRNA was detected by in situ hybridization, and its abundance was estimated by semiquantitative RT-PCR. The presence of Sirt1 protein was investigated by immunohistofluorescence and Western blot analysis. The apoptosis of photoreceptor cells was analyzed by terminal dUTP transferase nick-end labeling (TUNEL). Immunolabeling for Sirt1, apoptosis-inducing factor (Aif), and caspase-12 (Casp-12) was performed on retinal tissue sections. RESULTS: Sirt1 mRNA and immunoreactivity were observed in normal adult mouse eyes. In the control retina, Sirt1 was immunolocalized mostly to the nucleus. In rd10 mice with retinal degeneration, changes in Sirt1 immunolabeling were observed only in the retinal outer nuclear layer (ONL). The pathologic pattern of Sirt1 immunoreactivity correlated with the start of retinal degeneration in rd10 mice. CONCLUSIONS: The results suggest a link between Sirt1 production and retinal degeneration in rd10 mice. The anti-apoptotic, neuroprotective role of Sirt1 in the mouse retina is based on the involvement of Sirt1 in double DNA strand-break repair mechanisms and in maintaining energy homeostasis in photoreceptor cells. The results suggest that the neuroprotective properties of Sirt1 may gradually weaken in rd10 mouse photoreceptor cells.