Comprehensive splicing analysis of the alternatively spliced CHEK2 exons 8 and 10 reveals three enhancer/silencer-rich regions and 38 spliceogenic variants.

Splicing is controlled by a large set of regulatory elements (SREs) including splicing enhancers and silencers, which are involved in exon recognition. Variants at these motifs may dysregulate splicing and trigger loss-of-function transcripts associated with disease. Our goal here was to study the alternatively spliced exons 8 and 10 of the breast cancer susceptibility gene CHEK2. For this purpose, we used a previously published minigene with exons 6-10 that produced the expected minigene full-length transcript and replicated the naturally occurring events of exon 8 [Δ(E8)] and exon 10 [Δ(E10)] skipping. We then introduced 12 internal microdeletions of exons 8 and 10 by mutagenesis in order to map SRE-rich intervals by splicing assays in MCF-7 cells. We identified three minimal (10-, 11-, 15-nt) regions essential for exon recognition: c.863_877del [ex8, Δ(E8): 75%] and c.1073_1083del and c.1083_1092del [ex10, Δ(E10): 97% and 62%, respectively]. Then 87 variants found within these intervals were introduced into the wild-type minigene and tested functionally. Thirty-eight of them (44%) impaired splicing, four of which (c.883G>A, c.883G>T, c.884A>T, and c.1080G>T) induced negligible amounts (<5%) of the minigene full-length transcript. Another six variants (c.886G>A, c.886G>T, c.1075G>A, c.1075G>T, c.1076A>T, and c.1078G>T) showed significantly strong impacts (20-50% of the minigene full-length transcript). Thirty-three of the 38 spliceogenic variants were annotated as missense, three as nonsense, and two as synonymous, underlying the fact that any exonic change is capable of disrupting splicing. Moreover, c.883G>A, c.883G>T, and c.884A>T were classified as pathogenic/likely pathogenic variants according to ACMG/AMP (American College of Medical Genetics and Genomics/Association for Molecular Pathology)-based criteria. © 2024 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of The Pathological Society of Great Britain and Ireland.

Systematic Minigene-Based Splicing Analysis and Tentative Clinical Classification of 52 CHEK2 Splice-Site Variants.

BACKGROUND: Disrupted pre-mRNA splicing is a frequent deleterious mechanism in hereditary cancer. We aimed to functionally analyze candidate spliceogenic variants of the breast cancer susceptibility gene CHEK2 by splicing reporter minigenes. METHODS: A total of 128 CHEK2 splice-site variants identified in the Breast Cancer After Diagnostic Gene Sequencing (BRIDGES) project (https://cordis.europa.eu/project/id/634935) were analyzed with MaxEntScan and subsetted to 52 variants predicted to impact splicing. Three CHEK2 minigenes, which span all 15 exons, were constructed and validated. The 52 selected variants were then genetically engineered into the minigenes and assayed in MCF-7 (human breast adenocarcinoma) cells. RESULTS: Of 52 variants, 46 (88.5%) impaired splicing. Some of them led to complex splicing patterns with up to 11 different transcripts. Thirty-four variants induced splicing anomalies without any trace or negligible amounts of the full-length transcript. A total of 89 different transcripts were annotated, which derived from different events: single- or multi-exon skipping, alternative site-usage, mutually exclusive exon inclusion, intron retention or combinations of the abovementioned events. Fifty-nine transcripts were predicted to introduce premature termination codons, 7 kept the original open-reading frame, 5 removed the translation start codon, 6 affected the 5'UTR (Untranslated Region), and 2 included missense variations. Analysis of variant c.684-2A > G revealed the activation of a non-canonical TG-acceptor site and exon 6 sequences critical for its recognition. CONCLUSIONS: Incorporation of minigene read-outs into an ACMG/AMP (American College of Medical Genetics and Genomics/Association for Molecular Pathology)-based classification scheme allowed us to classify 32 CHEK2 variants (27 pathogenic/likely pathogenic and 5 likely benign). However, 20 variants (38%) remained of uncertain significance, reflecting in part the complex splicing patterns of this gene.

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.

Minigene-based splicing analysis and ACMG/AMP-based tentative classification of 56 ATM variants.

The ataxia telangiectasia-mutated (ATM) protein is a major coordinator of the DNA damage response pathway. ATM loss-of-function variants are associated with 2-fold increased breast cancer risk. We aimed at identifying and classifying spliceogenic ATM variants detected in subjects of the large-scale sequencing project BRIDGES. A total of 381 variants at the intron-exon boundaries were identified, 128 of which were predicted to be spliceogenic. After further filtering, we ended up selecting 56 variants for splicing analysis. Four functional minigenes (mgATM) spanning exons 4-9, 11-17, 25-29, and 49-52 were constructed in the splicing plasmid pSAD. Selected variants were genetically engineered into the four constructs and assayed in MCF-7/HeLa cells. Forty-eight variants (85.7%) impaired splicing, 32 of which did not show any trace of the full-length (FL) transcript. A total of 43 transcripts were identified where the most prevalent event was exon/multi-exon skipping. Twenty-seven transcripts were predicted to truncate the ATM protein. A tentative ACMG/AMP (American College of Medical Genetics and Genomics/Association for Molecular Pathology)-based classification scheme that integrates mgATM data allowed us to classify 29 ATM variants as pathogenic/likely pathogenic and seven variants as likely benign. Interestingly, the likely pathogenic variant c.1898+2T>G generated 13% of the minigene FL-transcript due to the use of a noncanonical GG-5'-splice-site (0.014% of human donor sites). Circumstantial evidence in three ATM variants (leakiness uncovered by our mgATM analysis together with clinical data) provides some support for a dosage-sensitive expression model in which variants producing ≥30% of FL-transcripts would be predicted benign, while variants producing ≤13% of FL-transcripts might be pathogenic. © 2022 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of The Pathological Society of Great Britain and Ireland.

Minigene Splicing Assays Identify 20 Spliceogenic Variants of the Breast/Ovarian Cancer Susceptibility Gene RAD51C.

RAD51C loss-of-function variants are associated with an increased risk of breast and ovarian cancers. Likewise, splicing disruptions are a frequent mechanism of gene inactivation. Taking advantage of a previous splicing-reporter minigene with exons 2-8 (mgR51C_ex2-8), we proceeded to check its impact on the splicing of candidate ClinVar variants. A total of 141 RAD51C variants at the intron/exon boundaries were analyzed with MaxEntScan. Twenty variants were selected and genetically engineered into the wild-type minigene. All the variants disrupted splicing, and 18 induced major splicing anomalies without any trace or minimal amounts (<2.4%) of the minigene full-length (FL) transcript. Twenty-seven transcripts (including the wild-type and r.904A FL transcripts) were identified by fluorescent fragment electrophoresis; of these, 14 were predicted to truncate the RAD51C protein, 3 kept the reading frame, and 8 minor isoforms (1.1−4.7% of the overall expression) could not be characterized. Finally, we performed a tentative interpretation of the variants according to an ACMG/AMP (American College of Medical Genetics and Genomics/Association for Molecular Pathology)-based classification scheme, classifying 16 variants as likely pathogenic. Minigene assays have been proven as valuable tools for the initial characterization of potential spliceogenic variants. Hence, minigene mgR51C_ex2-8 provided useful splicing data for 40 RAD51C variants.

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.

Validity, reliability, and calibration of the physical activity unit 7 item screener (PAU-7S) at population scale.

BACKGROUND: Validation of self-reported tools, such as physical activity (PA) questionnaires, is crucial. The aim of this study was to determine test-retest reliability, internal consistency, and the concurrent, construct, and predictive validity of the short semi-quantitative Physical Activity Unit 7 item Screener (PAU-7S), using accelerometry as the reference measurement. The effect of linear calibration on PAU-7S validity was tested. METHODS: A randomized sample of 321 healthy children aged 8-16 years (149 boys, 172 girls) from the nationwide representative PASOS study completed the PAU-7S before and after wearing an accelerometer for at least 7 consecutive days. Weight, height, and waist circumference were measured. Cronbach alpha was calculated for internal consistency. Test-retest reliability was determined by intra-class correlation (ICC). Concurrent validity was assessed by ICC and Spearman correlation coefficient between moderate to vigorous PA (MVPA) derived by the PAU-7S and by accelerometer. Concordance between both methods was analyzed by absolute agreement, weighted kappa, and Bland-Altman statistics. Multiple linear regression models were fitted for construct validity and predictive validity was determined by leave-one-out cross-validation. RESULTS: The PAU-7S overestimated MVPA by 18%, compared to accelerometers (106.5 ± 77.0 vs 95.2 ± 33.2 min/day, respectively). A Cronbach alpha of 0.76 showed an acceptable internal consistency of the PAU-7S. Test-retest reliability was good (ICC 0.71 p < 0.001). Spearman correlation and ICC coefficients of MVPA derived by the PAU-7S and accelerometers increased from 0.31 to 0.62 and 0.20 to 0.62, respectively, after calibration of the PAU-7S. Between-methods concordance improved from a weighted kappa of 0.24 to 0.50 after calibration. A slight reduction in ICC, from 0.62 to 0.60, yielded good predictive validity. Multiple linear regression models showed an inverse association of MVPA with standardized body mass index (ß - 0.162; p < 0.077) and waist to height ratio (ß - 0.010; p < 0.014). All validity dimensions were somewhat stronger in boys compared to girls. CONCLUSION: The PAU-7S shows a good test-retest reliability and acceptable internal consistency. All dimensions of validity increased from poor/fair to moderate/good after calibration. The PAU-7S is a valid instrument for measuring MVPA in children and adolescents. TRIAL REGISTRATION: Trial registration number ISRCTN34251612 .

Trends in the association between smoking history and general/central obesity in Catalonia, Spain (1992-2003).

OBJECTIVES: To examine trends in the relationship between smoking history and both general and central fatness in adults from a Mediterranean setting. Materials and methods: The ENCAT 1992-1993 and 2002-2003 surveys were used; samples consisted of 482 men, 589 women from 1992-1993, and 515 men, 613 women from 2002-2003, aged 25-60 years. Measured anthropometry and self-reported data on smoking habits, diet, lifestyle and SES were collected. General fatness was defined as WHO's BMI overweight and obesity, and central fatness was defined as WHO's Increased-Risk-for-metabolic-complications Waist Circumference (IR WC) and Substantially-Increased-Risk WC (SIR WC). Simple logistic regression was used to estimate multivariate-adjusted associations between general/central fatness and smoking history. RESULTS: By 2002-2003, most associations observed in 1992-1993 had been strongly attenuated: only male current-heavy-smoking remained associated with IR/SIR WC (three-fold) and female current-moderate-smokers were 0.57 times less likely to have an IR/SIR WC (p < 0.10). CONCLUSIONS: Although causality cannot be established, results suggest a positive association between heavy smoking and central fatness among men, but no association between former smoking and general/central fatness; findings strengthen arguments for promoting smoking cessation to reduce smoking -and obesity- associated morbidity and mortality.

Plant food supplement (PFS) market structure in EC Member States, methods and techniques for the assessment of individual PFS intake.

The popularity of herbal products, especially plant food supplements (PFS) and herbal medicine is on the rise in Europe and other parts of the world, with increased use in the general population as well as among specific subgroups encompassing children, women or those suffering from diseases such as cancer. The aim of this paper is to examine the PFS market structures in European Community (EC) Member States as well as to examine issues addressing methodologies and consumption data relating to PFS use in Europe. A revision of recent reports on market data, trends and main distribution channels, in addition an example of the consumption of PFS in Spain, is presented. An overview of the methods and administration techniques used to assess individual food consumption as a starting point, including their uses and limitations, as well as some examples of studies that collect Food Supplement (FS) information, including herbal/botanical/plant-derived products are also discussed. Additionally, the intake estimation process of food nutrients is described and used to propose the PFS ingredients intake estimation process. Nationally representative PFS consumption data is scarce in Europe. The majority of studies have been conducted in Scandinavia and the UK. However the heterogeneity of definitions, study design and objectives make it difficult to compare results and extrapolate conclusions.

[Mediterranean diet. Characteristics and health benefits]. / Dieta Mediterránea: características y beneficios para la salud.

The purpose of this article is to define the concept of Mediterranean Diet or Diets and to describe the associated health benefits recognised by the scientific community. The characteristics of the Mediterranean Diet are described as well as the effects the foods comprising it have on the most common pathologies such as cardiovascular disease and cancer. The Spanish Society of Community Nutrition's consensus based Healthy Diet Pyramid, along with Greece's pyramid for food guidelines for the adult population (from the Greek Ministry of Health), are presented and compared. They are the graphic representation of the food and physical activity guides of two typically Mediterranean countries. Nutritional and sociological trends are also discussed and their impact on the evolution of the Mediterranean Diet.

Dieta Mediterránea: características y beneficios para la salud / Mediterranean diet characteristics and health benefits

El objetivo de este artículo es definir el concepto de Dieta Mediterránea o "Dietas Mediterráneas" y describir el impacto en la salud que la comunidad científica actualmente te reconoce. En él se describen las características de la Dieta Mediterránea y se identifican los efectos de sus componentes sobre patologías principales tales como las enfermedades cardiovasculares y el cáncer. Por otro lado, se presentan y comparan la Pirámide de la Dieta Saludable, fruto del concenso de la Sociedad Española de Nutrición Comunitaria, y la Pirámide de la Dieta para la Población Adulta Griega, del Ministerio de Salud de Grecia. Ambas pirámides son la representación gráfica de las recomendaciones o guías alimentarias y de actividad física de las poblaciones de dos países típicamente mediterráneos. Por último, se exponen las tendencias nutricionales y sociológicas de la Dieta Mediterránea y su impacto en la evolución de la Dieta Mediterránea