L. Blázquez

CD24 V/V is an allele associated with the risk of developing multiple sclerosis in the Spanish population.

The allele C in the CD24 gene has been related to multiple sclerosis (MS). In this work we check this single nucleotide polymorphism (SNP) in a population of 135 patients and 285 controls. Our results confirm the association between the V/V genotype at aa 57 of this gene and MS and highlight the importance of taking into account the origin of the subjects to avoid a population bias.

Apolipoprotein E ɛ4 allele in familial and sporadic Parkinson's disease

Abstract Parkinsons disease (PD) is the second most common age-related neurodegenerative disease after Alzheimers disease (AD). Common risk factors for both diseases have been explored to study potential etiologic interactions between these two neurodegenerative disorders. The APOEɛ4 allele, previously associated with AD, has also been associated with risk of PD and with the presence of some clinical features in PD patients. However, the role of APOE ɛ4 allele in risk of PD remains unclear. We studied the distribution of APOE alleles in 276 unrelated familial and sporadic PD patients and in 212 controls. Patients and controls were classified by ethnicity. No genetic heterogeneity between Basques and people from other regions of Spain was found. No significant differences in APOE allele distribution between PD patients and controls were found; however, lower ɛ4 allele frequency was observed when the sporadic PD group was analyzed separately. By contrast, an increase in ɛ4 allele frequency was found in familial PD patients with cognitive decline. We conclude that the APOE ɛ4 allele may be associated with the risk of developing PD in isolated cases and that it is linked to the presence of cognitive decline in familial PD in our sample.

Genes related to iron metabolism and susceptibility to Alzheimer's disease in Basque population.

Alzheimers disease (AD) is the most common dementing disorder and presents with a progressive and irreversible cognitive decline of gradual onset. To date, several reports have involved iron in AD physiopathology. In this study, we have analysed TFC2 variant and HFE mutations (H63D and C282Y) in 211 AD patients and 167 controls recruited from an area of the Basque Country. Furthermore, we have studied APOE genotype as it is a well-known risk factor for AD. APOE epsilon 4 allele was associated with an increased risk of AD and an earlier age at onset, whereas no association was found between TFC2 or HFE C282Y mutation and disease susceptibility. The frequency of H63D mutation was higher in control population (29.9%) than in AD patients (18%), suggesting a protective role of this allele on AD either due to the presence of the mutation itself or through the effect of other related genes in the ancestral haplotype in which it is included.

Characterization of novel CAPN3 isoforms in white blood cells: an alternative approach for limb-girdle muscular dystrophy 2A diagnosis.

Limb-girdle muscular dystrophy type 2A (LGMD2A) is an autosomal recessive disorder caused by mutations in the CAPN3 gene. Its definitive diagnosis is laborious, since the clinical phenotype is often similar to other types of muscular dystrophy and since the CAPN3 gene encompasses a large genomic region with more than 300 pathogenic mutations described to date. In fact, it is estimated that nearly 25% of the cases with a phenotype suggestive of LGMD2A do not have mutations in the CAPN3 gene and that, in up to 22% of the cases, only one mutation is identified. In the present work, we have characterised CAPN3 messenger RNA (mRNA) expression in peripheral blood, and we have performed a retrospective diagnostic study with 26 LGMD2A patients, sequencing a transcript of CAPN3 present in white blood cells (WBCs). The 25% of the mutations presented in this paper (7/28) act modifying pre-mRNA splicing of the CAPN3 transcript, including the first deep-intronic mutation described to date in the CAPN3 gene. Our results determine that the sequencing of CAPN3 transcripts present in WBCs could be applied as a new approach for LGMD2A diagnosis. This method improves and simplifies diagnosis, since it combines the advantages of mRNA analysis in a more accessible and rapidly regenerated tissue. However, the lack of exon 15 in the CAPN3 isoforms present in blood, and the presence of mRNA degradation make it necessary to combine mRNA and DNA analyses in some specific cases.

In Vitro Correction of a Pseudoexon‐Generating Deep Intronic Mutation in LGMD2A by Antisense Oligonucleotides and Modified Small Nuclear RNAs

Limb‐girdle muscular dystrophy type 2A (LGMD2A) is the most frequent autosomal recessive muscular dystrophy. It is caused by mutations in the calpain‐3 (CAPN3) gene. The majority of the mutations described to date are located in the coding sequence of the gene. However, it is estimated that 25% of the mutations are present at exon–intron boundaries and modify the pre‐mRNA splicing of the CAPN3 transcript. We have previously described the first deep intronic mutation in the CAPN3 gene: c.1782+1072G>C mutation. This mutation causes the pseudoexonization of an intronic sequence of the CAPN3 gene in the mature mRNA. In the present work, we show that the point mutation generates the inclusion of the pseudoexon in the mRNA using a minigene assay. In search of a treatment that restores normal splicing, splicing modulation was induced by RNA‐based strategies, which included antisense oligonucleotides and modified small‐nuclear RNAs. The best effect was observed with antisense sequences, which induced pseudoexon skipping in both HeLa cells cotransfected with mutant minigene and in fibroblasts from patients. Finally, transfection of antisense sequences and siRNA downregulation of serine/arginine‐rich splicing factor 1 (SRSF1) indicate that binding of this factor to splicing enhancer sequences is involved in pseudoexon activation.

Increased in vivo inhibition of gene expression by combining RNA interference and U1 inhibition

Inhibition of gene expression can be achieved with RNA interference (RNAi) or U1 small nuclear RNA—snRNA—interference (U1i). U1i is based on U1 inhibitors (U1in), U1 snRNA molecules modified to inhibit polyadenylation of a target pre-mRNA. In culture, we have shown that the combination of RNAi and U1i results in stronger inhibition of reporter or endogenous genes than that obtained using either of the techniques alone. We have now used these techniques to inhibit gene expression in mice. We show that U1ins can induce strong inhibition of the expression of target genes in vivo. Furthermore, combining U1i and RNAi results in synergistic inhibitions also in mice. This is shown for the inhibition of hepatitis B virus (HBV) sequences or endogenous Notch1. Surprisingly, inhibition obtained by combining a U1in and a RNAi mediator is higher than that obtained by combining two U1ins or two RNAi mediators. Our results suggest that RNAi and U1i cooperate by unknown mechanisms to result in synergistic inhibitions. Analysis of toxicity and specificity indicates that expression of U1i inhibitors is safe. Therefore, we believe that the combination of RNAi and U1i will be a good option to block damaging endogenous genes, HBV and other infectious agents in vivo.

Does the severity of the LGMD2A phenotype in compound heterozygotes depend on the combination of mutations

Introduction: Limb‐girdle muscular dystrophy type 2A (LGMD2A) is caused by a deficiency of calpain‐3/p94. Although the symptoms in most LGMD2A patients are generally homogeneous, some variation in the severity and progression of the disease has been reported. Methods: We describe 2 patients who carry the same combination of compound heterozygous mutations (pG222R/pR748Q) and whose symptoms are exceptionally benign compared to homozygotes with each missense mutation. Results: The benign phenotype observed in association with the combined pG222R and pR748Q mutations suggested that it may result from a compensatory effect of compound heterozygosity rather than the individual mutations themselves. Our analyses revealed that these two mutations exert different effects on the protease activity of calpain‐3, suggesting “molecular complementation” in these patients. Conclusion: We propose several hypotheses to explain how this specific combination of mutations may rescue the normal proteolytic activity of calpain‐3, resulting in an exceptionally benign phenotype. Muscle Nerve, 2011