Spinal muscular atrophy is an autosomal recessive disorder which affects about 1 in 10,000 individuals. The three clinical forms of SMA were mapped to the 5q13 region. Three candidate genes have been isolated and shown to be deleted in SMA patients: the Survival Motor Neuron gene (SMN), the Neuronal Apoptosis Inhibitory Protein gene (NAIP) and the XS2G3 cDNA. In this report we present the molecular analysis of the SMN exons 7 and 8 and NAIP exon 5 in 65 Spanish SMA families. NAIP was mostly deleted in type I patients (67.9%) and SMN was deleted in 92.3% of patients with severe and milder forms. Most patients who lacked the NAIP gene also lacked the SMN gene, but we identified one type II patient deleted for NAIP exon 5 but not for SMN exons 7 and 8. Two other patients carried deletions of NAIP exon 5 and SMN exon 7 but retained the SMN exon 8. Three polymorphic variants from the SMN gene, showing changes on the sequence of the centromeric (cBCD541) and telomeric copies of the SMN gene, were found. In addition, we show several genetic rearrangements of the telomeric SMN gene, which include duplication of this gene in one normal chromosome, and putative gene conversion events in affected and normal chromosomes. Altogether these results corroborate the high genetic variability of the SMA region. Finally, we have determined the ratio between the number of centromeric and telomeric copies of the SMN gene in parents of SMA patients, showing that the majority of parents of types II and III patients carried three or more copies of the cBCD541 gene; we suggest a relationship between the number of copies of cBCD541 and the disease phenotype.

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Aims: To explain the dominance of OTA-producing fungal species in sun-dried grapes for special wine production through an ecophysiological approach. Methods and Results: Grapes at different ripening stages, sun-dried grapes and raisins were analysed for fungal presence, and isolates identified. Aspergillus section Nigri incidence in grapes increased with grape maturation. In the ecophysiological study five isolates (Alternaria alternata, Cladosporium herbarum, Penicillium decumbens, Aspergillus carbonarius, A. niger aggregate and A. section Nigri uniseriate) were inoculated in SNM medium at four a w (0AE82-0AE97) and incubated at 20, 30 and 40°C for 18 d. Isolates were also inoculated in pairs to evaluate fungal interactions recording their growth rates and indexes of dominance. Aspergillus section Nigri grew in a wider range of temperature and a w , had higher growth rates than the others under most of the conditions tested and showed behaviour usually dominant. Conclusions: The presence of A. section Nigri is predominant in grapes during harvesting and sun-drying period likely because of a better adaptation to hot and humid environment. Significance and Impact of the Study: The duration of the drying period should be reduced as much as possible without compromising the quality of the final product, or drying the grapes in controlled chambers with dry hot air flow.

Neurofibromatosis type 1 (NF1) is a hereditary disorder caused by mutations in the NF1 gene. Detecting mutation in NF1 is hindered by the gene's large size, the lack of mutation hotspots, the presence of pseudogenes, and the wide variety of possible lesions. We developed a method for detecting germline mutations by combining an original RNA-based cDNA-PCR mutation detection method and denaturing high-performance liquid chromatography (DHPLC) with multiplex ligation-dependent probe amplification (MLPA). The protocol was validated in a cohort of 56 blood samples from NF1 patients who fulfilled NIH diagnostic criteria, identifying the germline mutation in 53 cases (95% sensitivity). The efficiency and reliability of this approach facilitated detection of different types of mutations, including single-base substitutions, deletions or insertions of one to several nucleotides, microdeletions, and changes in intragenic copy number. Because mutational screening for minor lesions was performed using cDNA and the characterization of mutated alleles was performed at both the RNA and genomic DNA level, the analysis provided insight into the nature of the different mutations and their effect on NF1 mRNA splicing. After validation, we implemented the protocol as a routine test. Here we present the overall unbiased spectrum of NF1 mutations identified in 93 patients in a cohort of 105. The results indicate that this protocol is a powerful new tool for the molecular diagnosis of NF1.