Ernesto Rinaldi

Clinical features of X linked juvenile retinoschisis associated with new mutations in the XLRS1 gene in Italian families.

Aims: To describe the clinical phenotype of X linked juvenile retinoschisis in eight Italian families with six different mutations in the XLRS1 gene. Methods: Complete ophthalmic examinations, electroretinography and A and B-scan standardised echography were performed in 18 affected males. The coding sequences of the XLRS1 gene were amplified by polymerase chain reaction and directly sequenced on an automated sequencer. Results: Six different XLRS1 mutations were identified; two of these mutations Ile81Asn and the Trp122Cys, have not been previously described. The affected males showed an electronegative response to the standard white scotopic stimulus and a prolonged implicit time of the 30 Hz flicker. In the families with Trp112Cys and Trp122Cys mutations we observed a more severe retinoschisis (RS) clinical picture compared with the other genotypes. Conclusion: The severe RS phenotypes associated with Trp112Cys and to Trp122Cys mutations suggest that these mutations determine a notable alteration in the function of the retinoschisin protein.

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.

A normal electro-oculography in a family affected by best disease with a novel spontaneous mutation of the BEST1 gene.

Aims: To describe clinical and genetic findings in an Italian family affected by Best disease. Methods: Five related patients underwent a complete ophthalmological assessment; genetic testing was performed by single-strand conformation polymorphism analysis and direct sequencing of the BEST1 gene. Results: In three of five family members, the sequence analysis of the BEST1 gene revealed a single Phe-to-Leu transition at nucleotide 305 associated with clinical evidence of Best disease. Surprisingly, the electro-oculogram was normal in all affected patients. Conclusion: This study reveals a de novo mutation in the BEST1 gene never described before, sustaining the autosomal-dominant pattern of inheritance of the disease. Clinical evaluation showed phenotypic variability between affected members. In addition, these data suggest that a normal electro-oculography (EOG) does not rule out a diagnosis of Best disease, supporting instead the crucial role of molecular analysis.

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.

Characterization of MPP4, a gene highly expressed in photoreceptor cells, and mutation analysis in retinitis pigmentosa.

Membrane-associated guanylate kinase (MAGUK) proteins are cell-cell contact organizing molecules that mediate targeting, clustering and anchoring of proteins at synapses and other cell junctions. MAGUK proteins may contain multiple protein-protein interaction motifs including PDZ, SH3 and guanylate kinase (GuK) domains. In this study, we performed a detailed analysis of the expression pattern of MPP4, a recently described member of the MAGUK protein family. We confirmed that this gene is highly expressed in retina, and demonstrate that it is also present, at lower levels, in brain. We identified a new retina specific isoform encoding a predicted protein lacking 71 amino acids. This protein region contains a newly identified L27 domain, another module playing a role in protein-protein interaction. By RNA in situ hybridization, Mpp4 expression was found to be localized to photoreceptor cells in postnatal retina. The MPP4 gene is localized to chromosome 2, in band 2q31-33, where a locus for autosomal recessive retinitis pigmentosa (RP26) has been mapped. Mutation analysis of the entire open reading frame of the MPP4 gene in a RP26 family revealed no pathologic mutations. In addition, we did not identify mutations in a panel of 300 unrelated patients with retinitis pigmentosa.

Identification and characterization of C1orf36, a transcript highly expressed in photoreceptor cells, and mutation analysis in retinitis pigmentosa

By means of computational methods, we identified an uncharacterized human transcript, Chromosome 1 open reading frame 36 (C1orf36), that is expressed in the retina and that maps to 1q32.3. The cDNA contains an open reading frame of 585bp that encodes a 195-aminoacid protein with a predicted mass of 22.7kDa. An alternatively spliced transcript in a retinoblastoma cell line, encoding for a truncated peptide, was also identified. PCR experiments performed using human cDNA from several sources indicate that C1orf36 has a preferential expression in the retina. Accordingly, in situ hybridization experiments, performed using as probe a murine C1orf36 cDNA fragment, detected a hybridization signal on mouse retinal adult sections. The C1orf36 protein shares homology with putative proteins in Mus musculus and Fugu rubripes, suggesting evolutionary conservation of its function. Additional sequence analysis of the C1orf36 gene product predicts its subcellular mitochondrial localization and the presence of both evolutionary conserved phosphorylation sites and regions adopting a coiled-coil conformation. We also defined the genomic structure of the gene. This enabled us to perform a mutational analysis of the C1orf36 coding region of about 300 patients affected by retinitis pigmentosa. No pathological mutations were detected in this analysis.