Neurofibromatosis type 1 (NF1), the most common tumor-predisposing disorder in humans, is caused by defects in the NF1 tumor-suppressor gene. Comprehensive mutation analysis applying RNA-based techniques complemented with FISH analysis achieves mutation detection rates of approximately 95% in NF1 patients. The majority of mutations are minor lesions, and approximately 5% are total gene deletions. We found 13 single- and/or multiexon deletions/duplications out of 1,050 detected mutations using our RNA-based approach in a cohort of 1,100 NF1 patients and confirmed these changes using multiplex ligation-dependent probe amplification (MLPA). With MLPA, we found another 12 novel multiexon deletion/duplications in 55 NF1 patients for whom analysis with multiple assays had not revealed a NF1 mutation, including 50 previously analyzed comprehensively. The extent of the 22 deletions and 3 duplications varied greatly, and there was no clustering of breakpoints. We also evaluated the sensitivity of MLPA in identifying deletions in a mosaic state. Furthermore, we tested whether the MLPA P122 NF1 area assay could distinguish between type I deletions, with breakpoints in low-copy repeats (NF1-LCRs), and type II deletions, caused by aberrant recombination between the JJAZ gene and its pseudogene. Our study showed that intragenic deletions and/or duplications represent only approximately 2% of all NF1 mutations. Although MLPA did not substantially increase the mutation detection rate in NF1 patients, it was a useful first step in a comprehensive mutation analysis scheme to quickly pinpoint patients with single- or multiexon deletions/duplications as well as patients with a total gene deletion who will not need full sequencing of the complete coding region.

Purpose To characterize a canine model of autosomal recessive RP due to a PDE6A gene mutation. Methods Affected and breed- and age-matched control puppies were studied by electroretinography (ERG), light and electron microscopy, immunohistochemistry and by assay for retinal PDE6 levels and enzymatic activity. Results The mutant puppies failed to develop normal rod-mediated ERG responses and had reduced light-adapted a-wave amplitudes from an early age. The residual ERG waveforms originated primarily from cone-driven responses. Development of photoreceptor outer segments was halted and rod cells were lost by apoptosis. Immunohistochemistry demonstrated a marked reduction in rod-opsin immunostaining outer segments and relative preservation of cones early in the disease process. With exception of rod bipolar cells that appeared to be reduced in number relatively early in the disease process other inner retinal cells were preserved in the early stages of the disease although there was marked and early activation of Müller glia. Western blotting showed that the PDE6A mutation not only resulted in a lack of PDE6A protein but the affected retinas also lacked the other PDE6 subunits, suggesting expression of PDE6A is required for normal expression of PDE6B and PDE6G. Affected retinas lacked PDE6 enzymatic activity. Conclusions This represents the first characterization of a PDE6A model of autosomal recessive retinitis pigmentosa and the PDE6A mutant dog shows promise as a large animal model for investigation of therapies to rescue mutant rod photoreceptors and to preserve cone photoreceptors in the face a rapid loss of rod cells.

To understand the factors controlling expression of the cGMP phosphodiesterase type 6 (PDE6) genes, we have characterized the promoter of the human PDE6A gene that encodes the catalytic ␣-subunit. In vivo DNase I hypersensitivity assays revealed two sites immediately upstream of the PDE6A core promoter region. Transient transfection assay in Y79 cells of constructs containing varying lengths of the promoter region showed a decrease in promoter activity with increasing length. The most active segment contained a 177-bp upstream sequence including apparent Crx and Nrl transcription factor binding sites. Both Crx and Nrl transactivated the PDE6A promoter in HEK293 cells and showed a >100-fold increase when coexpressed. Coexpression of a dominant negative inhibitor of Nrl abolished Nrl transactivation but had no effect on Crx. DNase I footprinting assays identified three potential Crx binding sites within a 55-bp segment beginning 29 bp upstream of the transcription start point. Mutation of two of these sites reduced reporter gene activity by as much as 69%. Gel shifts showed that all three Crx sites required a TAAT sequence for efficient binding. Consistent with a requirement for Crx and Nrl in Pde6a promoter activity, Pde6a mRNA is reduced by 87% in the retina of Crx ؊/؊ mice and is undetectable in Nrl ؊/؊ mice at postnatal day 10. These results establish that both Nrl and Crx are required for full transcriptional activity of the PDE6A gene.

BackgroundThe ferret (Mustela putorius furo) represents an attractive animal model for the study of respiratory diseases, including influenza. Despite its importance for biomedical research, the number of reagents for molecular and immunological analysis is restricted. We present here a parallel sequencing effort to produce an extensive EST (expressed sequence tags) dataset derived from a normalized ferret cDNA library made from mRNA from ferret blood, liver, lung, spleen and brain.ResultsWe produced more than 500000 sequence reads that were assembled into 16000 partial ferret genes. These genes were combined with the available ferret sequences in the GenBank to develop a ferret specific microarray platform. Using this array, we detected tissue specific expression patterns which were confirmed by quantitative real time PCR assays. We also present a set of 41 ferret genes with even transcription profiles across the tested tissues, indicating their usefulness as housekeeping genes.ConclusionThe tools developed in this study allow for functional genomic analysis and make further development of reagents for the ferret model possible.

Retinal membrane guanylyl cyclase (retGC) and Ca2+/Mg2+ -sensor proteins, GCAPs1, control the recovery of the photoresponse in vertebrate photoreceptors, through their molecular interactions that remain rather poorly understood and controversial. Here we have determined the main retGC isozyme (RetGC1):GCAP1 binding stoichiometry at saturation in cyto, using fluorescently labeled RetGC1 and GCAP1 co-expressed in HEK293 cells. In a striking manner, the equimolar binding of RetGC1 with GCAP1 in transfected HEK293 cells typical for the wild type RetGC1 was eliminated by a substitution, D639Y, in the kinase homology domain of RetGC1 found in a patient with a severe form of retinal dystrophy, Leber congenital amaurosis (LCA). Similar effect was observed with another LCA-related mutation, R768W, in the same domain of RetGC1. In contrast to the completely suppressed binding and activation of RetGC1 by Mg2+-liganded GCAP1, neither of these two mutations eliminated the GCAP1-independent activity of retGC stimulated by Mn2+. These results directly implicate the D639 (and possibly R768) -containing portion of the RetGC1 kinase homology domain in its primary recognition by the Mg2+-bound activator form of GCAP1.