Search

PALB2 loss-of-function variants confer high risk of developing breast cancer. Here, we present a systematic functional analysis of PALB2 splice-site variants detected in ~113,000 women of the large-scale sequencing project BRIDGES (Breast Cancer After Diagnostic Gene Sequencing; https://bridges-research.eu/). Eighty-two PALB2 variants at the intron-exon boundaries were analyzed with MaxEntScan. Forty-two variants were selected for the subsequent splicing functional assays. For this purpose, three splicing reporter minigenes comprising exons 1-12 were constructed. The 42 potential spliceogenic variants were introduced into the minigenes by site-directed mutagenesis and assayed in MCF-7/MDA-MB-231 cells. Splicing anomalies were observed in 35 variants, 23 of which showed no traces or minimal amounts of the expected full-length transcripts of each minigene. More than 30 different variant-induced transcripts were characterized, 23 of which were predicted to truncate the PALB2 protein. The pathogenicity of all variants was interpreted according to an in-house adaptation of the ACMG/AMP (American College of Medical Genetics and Genomics and the Association for Molecular Pathology) variant classification scheme. Up to 23 variants were classified as Pathogenic/Likely Pathogenic. Remarkably, three ±1,2 variants (c.49-2A>T, c.108+2T>C and c.211+1G>A) were classified as variants of unknown significance since they produced significant amounts of either in-frame transcripts of unknown impact on the PALB2 protein function or the minigene full-length transcripts. In conclusion, we have significantly contributed to the ongoing effort of identifying spliceogenic variants in the clinically relevant PALB2 cancer susceptibility gene. Moreover, we suggest some approaches to classify the findings in accordance with the ACMG/AMP rationale.

This research was supported by Madrid Salud (Ayuntamiento de Madrid) with the participation of Centre VISAVET, ATHISA and TRAGSEGA., Feral pigeons have increased in urban settings worldwide becoming a potential health risk for humans and other animals. Control and surveillance programs are essential to prevent the possible transmission of zoonotic pathogens carried by pigeons. A surveillance program was carried out in Madrid City (Spain) during 2005–2014 to determine the role of urban pigeons as carriers of zoonotic agents comparing these results with studies performed elsewhere in the last fifteen years. A total of 1372 pigeons were randomly captured and tested for detection of Antimicrobial susceptibility and genetic heterogeneity of Campylobacter and Salmonella isolates were determined. During the first phase (August 2005–July 2010), 428 animals were analyzed individually, while in the second period (August 2010–December 2014), 944 pigeons were analyzed in pools (n = 2–3 in 2010 and n = 5–6 in 2013 and 2014). The most prevalent pathogen during the first phase was Campylobacter spp., (6.57%, 95% confidence interval 3.05–12.10%) followed by Salmonella spp. (4.41%, 95% CI: 2.30–7.58%) and C. psittaci (2.56%, 95% CI: 0.70–6.53%)]. The PCR techniques, used during the 2010–2014 phase of the study, confirmed the presence of Campylobacter spp. (prevalence of 0–14.83%) and C. psittaci (0–12,94%) among pigeons of Madrid. Antimicrobial susceptibility testing suggested low levels of resistance. Presence of zoonotic agents in feral pigeons highlights the importance of surveillance programs on this species, although the relative low prevalence found suggests a limited risk to Public and Animal Health in Madrid., Madrid Salud. Ayuntamiento de Madrid, Depto. de Sanidad Animal, Centro de Vigilancia Sanitaria Veterinaria (VISAVET), Fac. de Veterinaria, TRUE, pub

[Purpose]: Promoter mutations may affect transcription and can be associated with human diseases. However, the promoters of the breast cancer (BC) genes are not regularly screened. Our goal was to investigate the BRCA2 promoter in order to study a possible correlation between impaired transcription and disease. [Methods]: The proximal and core promoter of the BRCA2 gene was sequenced in 95 high-risk BC patients. A BRCA2-promoter insert [- 938 to + 312 from the transcription start site (TSS)] was generated and cloned into the firefly luciferase vector pGL4.10. Promoter variants and deletions were introduced by site-directed mutagenesis and quantified by Dual-Luciferase assays and semi-quantitative RT-PCR. [Results]: Three different variants were detected in high-risk BC patients: rs3092989, rs206118, and rs563971900. Functional mapping of 13 overlapping deletions revealed four down-regulating segments (TSS positions): -59_-10del/µdel3 (16% of activity of the wild-type construct), -104_-55del/µdel4 (62%), -239_-190del/µdel7 (39%), -464_-415/µdel12 (78%), suggesting the presence therein of putative transcriptional activator motifs. Additionally, six microdeletions rendered luciferase overexpression: +32_+81del/µdel1 (356%), -14_+36del/µdel2 (180%), -194_-145del/µdel6 (154%), -284_-235del/µdel8 (168%), -329_-280del/µdel9 (111%), and -509_-460del/µdel13 (139%), which is indicative of repressor elements. Functional assays of 15 promoter variants (including those detected in patients) showed that ten of them significantly altered expression with seven up-regulating (113-163%) and three down-regulating (rs551887850_G, rs570548398_T, rs55880202_T; 72-83%) SNPs. Eight of them were located in an ENCODE-DNase Hypersensitive Cluster (TSS - 185 to + 105) where most active transcriptional motifs are known to be placed. [Conclusions]: BRCA2 expression is highly sensitive to promoter variations as most of them induced relevant changes. Moreover, we mapped critical regions of the BRC

The distribution of virulence factors (VFs) typical of diarrheagenic Escherichia coli and the antimicrobial resistance (AMR) profiles were assessed in 780 isolates from healthy pigs, broilers, and cattle from Spain. VF distribution was broader than expected, although at low prevalence for most genes, with AMR being linked mainly to host species., Unión Europea. FP7, Comunidad de Madrid, Centro de Vigilancia Sanitaria Veterinaria (VISAVET), TRUE, pub