Splicing Analysis of 16 PALB2 ClinVar Variants by Minigene Assays: Identification of Six Likely Pathogenic Variants.

PALB2 loss-of-function variants are associated with significant increased risk of breast cancer as well as other types of tumors. Likewise, splicing disruptions are a common mechanism of disease susceptibility. Indeed, we previously showed, by minigene assays, that 35 out of 42 PALB2 variants impaired splicing. Taking advantage of one of these constructs (mgPALB2_ex1-3), we proceeded to analyze other variants at exons 1 to 3 reported at the ClinVar database. Thirty-one variants were bioinformatically analyzed with MaxEntScan and SpliceAI. Then, 16 variants were selected for subsequent RNA assays. We identified a total of 12 spliceogenic variants, 11 of which did not produce any trace of the expected minigene full-length transcript. Interestingly, variant c.49-1G > A mimicked previous outcomes in patient RNA (transcript ∆(E2p6)), supporting the reproducibility of the minigene approach. A total of eight variant-induced transcripts were characterized, three of which (∆(E1q17), ∆(E3p11), and ∆(E3)) were predicted to introduce a premature termination codon and to undergo nonsense-mediated decay, and five (▼(E1q9), ∆(E2p6), ∆(E2), ▼(E3q48)-a, and ▼(E3q48)-b) maintained the reading frame. According to an ACMG/AMP (American College of Medical Genetics and Genomics/Association for Molecular Pathology)-based classification scheme, which integrates mgPALB2 data, six PALB2 variants were classified as pathogenic/likely pathogenic, five as VUS, and five as likely benign. Furthermore, five ±1,2 variants were catalogued as VUS because they produced significant proportions of in-frame transcripts of unknown impact on protein function.

Splicing predictions, minigene analyses, and ACMG-AMP clinical classification of 42 germline PALB2 splice-site variants.

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 approximately 113,000 women in the large-scale sequencing project Breast Cancer After Diagnostic Gene Sequencing (BRIDGES; 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 American College of Medical Genetics and Genomics and the Association for Molecular Pathology (ACMG-AMP) 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, as 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. © 2021 The Authors. The Journal of Pathology published by John Wiley & Sons, Ltd on behalf of The Pathological Society of Great Britain and Ireland.

A ten-year-surveillance program of zoonotic pathogens in feral pigeons in the City of Madrid (2005-2014): The importance of a systematic pest control.

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.

Minigene Splicing Assays Identify 12 Spliceogenic Variants of BRCA2 Exons 14 and 15.

A relevant fraction of BRCA2 variants is associated with splicing alterations and with an increased risk of hereditary breast and ovarian cancer (HBOC). In this work, we have carried out a thorough study of variants from BRCA2 exons 14 and 15 reported at mutation databases. A total of 294 variants from exons 14 and 15 and flanking intronic sequences were analyzed with the online splicing tools NNSplice and Human Splicing Finder. Fifty-three out of these 294 variants were selected as candidate splicing variants. All variants but one, were introduced into the minigene MGBR2_ex14-20 (with exons 14-20) by site-directed mutagenesis and assayed in MCF-7 cells. Twelve of the remaining 52 variants (23.1%) impaired splicing at different degrees, yielding from 5 to 100% of aberrant transcripts. Nine variants affected the natural acceptor or donor sites of both exons and three affected putative enhancers or silencers. Fluorescent capillary electrophoresis revealed at least 10 different anomalous transcripts: (E14q5), Δ (E14p10), Δ(E14p246), Δ(E14q256), Δ(E14), Δ(E15p12), Δ(E15p13), Δ(E15p83), Δ(E15) and a 942-nt fragment of unknown structure. All transcripts, except for Δ(E14q256) and Δ(E15p12), are expected to truncate the BRCA2 protein. Nine variants induced severe splicing aberrations with more than 90% of abnormal transcripts. Thus, according to the guidelines of the American College of Medical Genetics and Genomics, eight variants should be classified as pathogenic (c.7008-2A > T, c.7008-1G > A, c.7435+1G > C, c.7436-2A > T, c.7436-2A > G, c.7617+1G > A, c.7617+1G > T, and c.7617+2T > G), one as likely pathogenic (c.7008-3C > G) and three remain as variants of uncertain clinical significance or VUS (c.7177A > G, c.7447A > G and c.7501C > T). In conclusion, functional assays by minigenes constitute a valuable strategy to primarily check the splicing impact of DNA variants and their clinical interpretation. While bioinformatics predictions of splice site variants were accurate, those of enhancer or silencer variants were poor (only 3/23 spliceogenic variants) which showed weak impacts on splicing (∼5-16% of aberrant isoforms). So, the Exonic Splicing Enhancer and Silencer (ESE and ESS, respectively) prediction algorithms require further improvement.

Genetic dissection of the BRCA2 promoter and transcriptional impact of DNA variants.

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 BRCA2 promoter that may constitute potential targets for regulatory variants. Three SNPs moderately decreased luciferase activity, but confirmation of its potential pathogenicity requires further analysis. These data reinforce the need to screen the promoter regions of breast cancer genes with a view to discovering novel deleterious mutations.

Efficacy of a typing scheme for Campylobacter based on the combination of true and questionable CRISPR.

This study evaluates an improved scheme for Campylobacter genotyping based on the combination of true and questionable CRISPR (clustered regularly interspaced short palindromic repeats) elements. A total of 180 Campylobacter strains (Campylobacter jejuni n=93 and Campylobacter coli n=87), isolated from neck skin and caecal content of broilers, poultry meat and sewage water were analysed. Another 97 C. jejuni DNA samples from cases of human campylobacteriosis were assessed. Sixty-three genotypes were found in C. jejuni considering only true CRISPR, and 16 additional genotypes were identified when questionable CRISPR were also taken into account. Likewise in C. coli the number of genotypes increased from eight for only true CRISPR to 14 after including questionable CRISPR elements. The number of typeable C. jejuni and C. coli isolates was 115 (60.5%) and 17 (19.5%) respectively considering only true CRISPR. These percentages increased to 92.7% (n=176) and 39.1% (n=34) respectively when both true and questionable CRISPR were considered. 60.9% of the C. coli isolates were non-typeable by CRISPR due to the lack of any PCR amplifiable CRISPR loci, which raises questions about CRISPR analysis as an appropriate method for C. coli typing. However the assessment of true and questionable CRISPR has proved to be fairly useful for typing C. jejuni due to its high discriminatory power (Simpson's index=0.960) and typeability (92.7%) values. The results of the present work show that our genotyping method based on the combination of true and questionable CRISPR elements may be used as a suitable complementary tool to existing C. jejuni genotyping methods.

Detection of virulence-associated genes characteristic of intestinal Escherichia coli pathotypes, including the enterohemorrhagic/enteroaggregative O104:H4, in bovines from Germany and Spain.

Cattle are reservoirs of enterohemorrhagic Escherichia coli; however, their role in the epidemiology of other pathogenic E. coli remains undefined. A new set of quantitative real-time PCR assays for the direct detection and quantification of nine virulence-associated genes (VAGs) characteristic of the most important human E. coli pathotypes and four serotype-related genes (wzxO104 , fliCH4 , rbfO157 , fliCH7 ) that can be used as a surveillance tool for detection of pathogenic strains was developed. A total of 970 cattle fecal samples were collected in slaughterhouses in Germany and Spain, pooled into 134 samples and analyzed with this tool. stx1, eae and invA were more prevalent in Spanish samples whereas bfpA, stx2, ehxA, elt, est and the rbfO157 /fliCH7 combination were observed in similar proportions in both countries. Genes characteristic of the hybrid O104:H4 strain of the 2011 German outbreak (stx2/aggR/wzxO104 /fliCH4 ) were simultaneously detected in six fecal pools from one German abattoir located near the outbreak epicenter. Although no isolate harboring the full stx2/aggR/wzxO104 /fliCH4 combination was cultured, sequencing of the aggR positive PCR products revealed 100% homology to the aggR from the outbreak strain. Concomitant detection by this direct approach of VAGs from a novel human pathogenic E. coli strain in cattle samples implies that the E. coli gene pool in these animals can be implicated in de novo formation of such highly-virulent strains. The application of this set of qPCRs in surveillance studies could be an efficient early-warning tool for the emergence of zoonotic E. coli in livestock.

Methicillin resistant Staphylococcus aureus (MRSA) carriage in different free-living wild animal species in Spain.

Methicillin-resistant Staphylococcus aureus (MRSA) is a life-threatening pathogen in humans and its presence in animals is a public health concern. The aim of this study was to measure the prevalence of MRSA in free-living wild animals. Samples from red deer (n=273), Iberian ibex (n=212), Eurasian Griffon vulture (n=40) and wild boar (n=817) taken from different areas in Spain between June 2008 and November 2011 were analyzed. Characterization of the isolates was performed by spa typing, multi-locus sequence typing (MLST) and antimicrobial susceptibility testing. A low prevalence of MRSA was found with 13 isolates obtained from 12 animals (0.89%; 95% CI: 0.46-1.56). All MRSA sequence types belonged to ST398 (t011 and t1451) and ST1 (t127). Genotypes and antimicrobial susceptibility patterns (tetracycline resistance in ST398 and clindamycin-erythromycin-tetracycline resistance in ST1) suggest that the MRSA found probably originated in livestock (ST398) or humans (ST1). This is the first report of MRSA carriers in free-living wild animals in Europe. Although our data showed that MRSA prevalence is currently low, free-living wild animals might act as reservoir and represent a potential risk for human health.