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.

Identification of a truncated ß1-chimaerin variant that inactivates nuclear Rac1.

ß1-chimaerin belongs to the chimaerin family of GTPase-activating proteins (GAPs) and is encoded by the CHN2 gene, which also encodes the ß2- and ß3-chimaerin isoforms. All chimaerin isoforms have a C1 domain that binds diacylglycerol as well as tumor-promoting phorbol esters and a catalytic GAP domain that inactivates the small GTPase Rac. Nuclear Rac has emerged as a key regulator of various cell functions, including cell division, and has a pathological role by promoting tumorigenesis and metastasis. However, how nuclear Rac is regulated has not been fully addressed. Here, using several approaches, including siRNA-mediated gene silencing, confocal microscopy, and subcellular fractionation, we identified a nuclear variant of ß1-chimaerin, ß1-Δ7p-chimaerin, that participates in the regulation of nuclear Rac1. We show that ß1-Δ7p-chimaerin is a truncated variant generated by alternative splicing at a cryptic splice site in exon 7. We found that, unlike other chimaerin isoforms, ß1-Δ7p-chimaerin lacks a functional C1 domain and is not regulated by diacylglycerol. We found that ß1-Δ7p-chimaerin localizes to the nucleus via a nuclear localization signal in its N terminus. We also identified a key nuclear export signal in ß1-chimaerin that is absent in ß1-Δ7p-chimaerin, causing nuclear retention of this truncated variant. Functionally analyses revealed that ß1-Δ7p-chimaerin inactivates nuclear Rac and negatively regulates the cell cycle. Our results provide important insights into the diversity of chimaerin Rac-GAP regulation and function and highlight a potential mechanism of nuclear Rac inactivation that may play significant roles in pathologies such as cancer.

Mis-splicing in breast cancer: identification of pathogenic BRCA2 variants by systematic minigene assays.

Splicing disruption is a common mechanism of gene inactivation associated with germline variants of susceptibility genes. To study the role of BRCA2 mis-splicing in hereditary breast/ovarian cancer (HBOC), we performed a comprehensive analysis of variants from BRCA2 exons 2-9, as well as the initial characterization of the regulatory mechanisms of such exons. A pSAD-based minigene with exons 2-9 was constructed and validated in MCF-7 cells, producing the expected transcript (1016-nt/V1-BRCA2_exons_2-9-V2). DNA variants from mutational databases were analyzed by NNSplice and Human Splicing Finder softwares. To refine ESE-variant prediction, we mapped the regulatory regions through a functional strategy whereby 26 exonic microdeletions were introduced into the minigene and tested in MCF-7 cells. Thus, we identified nine spliceogenic ESE-rich intervals where ESE-variants may be located. Combining bioinformatics and microdeletion assays, 83 variants were selected and genetically engineered in the minigene. Fifty-three changes impaired splicing: 28 variants disrupted the canonical sites, four created new ones, 10 abrogated enhancers, eight created silencers and three caused a double-effect. Notably, nine spliceogenic-ESE variants were located within ESE-containing intervals. Capillary electrophoresis and sequencing revealed more than 23 aberrant transcripts, where exon skipping was the most common event. Interestingly, variant c.67G>A triggered the usage of a noncanonical GC-donor 4-nt upstream. Thirty-six variants that induced severe anomalies (>60% aberrant transcripts) were analyzed according to the ACMG guidelines. Thus, 28 variants were classified as pathogenic, five as likely pathogenic and three as variants of uncertain significance. Interestingly, 13 VUS were reclassified as pathogenic or likely pathogenic variants. In conclusion, a large fraction of BRCA2 variants (∼64%) provoked splicing anomalies lending further support to the high prevalence of this disease-mechanism. The low accuracy of ESE-prediction algorithms may be circumvented by functional ESE-mapping that represents an optimal strategy to identify spliceogenic ESE-variants. Finally, systematic functional assays by minigenes depict a valuable tool for the initial characterization of splicing anomalies and the clinical interpretation of variants. © 2019 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

Alternative splicing and ACMG-AMP-2015-based classification of PALB2 genetic variants: an ENIGMA report.

BACKGROUND: PALB2 monoallelic loss-of-function germ-line variants confer a breast cancer risk comparable to the average BRCA2 pathogenic variant. Recommendations for risk reduction strategies in carriers are similar. Elaborating robust criteria to identify loss-of-function variants in PALB2-without incurring overprediction-is thus of paramount clinical relevance. Towards this aim, we have performed a comprehensive characterisation of alternative splicing in PALB2, analysing its relevance for the classification of truncating and splice site variants according to the 2015 American College of Medical Genetics and Genomics-Association for Molecular Pathology guidelines. METHODS: Alternative splicing was characterised in RNAs extracted from blood, breast and fimbriae/ovary-related human specimens (n=112). RNAseq, RT-PCR/CE and CloneSeq experiments were performed by five contributing laboratories. Centralised revision/curation was performed to assure high-quality annotations. Additional splicing analyses were performed in PALB2 c.212-1G>A, c.1684+1G>A, c.2748+2T>G, c.3113+5G>A, c.3350+1G>A, c.3350+4A>C and c.3350+5G>A carriers. The impact of the findings on PVS1 status was evaluated for truncating and splice site variant. RESULTS: We identified 88 naturally occurring alternative splicing events (81 newly described), including 4 in-frame events predicted relevant to evaluate PVS1 status of splice site variants. We did not identify tissue-specific alternate gene transcripts in breast or ovarian-related samples, supporting the clinical relevance of blood-based splicing studies. CONCLUSIONS: PVS1 is not necessarily warranted for splice site variants targeting four PALB2 acceptor sites (exons 2, 5, 7 and 10). As a result, rare variants at these splice sites cannot be assumed pathogenic/likely pathogenic without further evidences. Our study puts a warning in up to five PALB2 genetic variants that are currently reported as pathogenic/likely pathogenic in ClinVar.

Functional classification of DNA variants by hybrid minigenes: Identification of 30 spliceogenic variants of BRCA2 exons 17 and 18.

Mutation screening of the breast cancer genes BRCA1 and BRCA2 identifies a large fraction of variants of uncertain clinical significance (VUS) whose functional and clinical interpretations pose a challenge for genomic medicine. Likewise, an increasing amount of evidence indicates that genetic variants can have deleterious effects on pre-mRNA splicing. Our goal was to investigate the impact on splicing of a set of reported variants of BRCA2 exons 17 and 18 to assess their role in hereditary breast cancer and to identify critical regulatory elements that may constitute hotspots for spliceogenic variants. A splicing reporter minigene with BRCA2 exons 14 to-20 (MGBR2_ex14-20) was constructed in the pSAD vector. Fifty-two candidate variants were selected with splicing prediction programs, introduced in MGBR2_ex14-20 by site-directed mutagenesis and assayed in triplicate in MCF-7 cells. Wild type MGBR2_ex14-20 produced a stable transcript of the expected size (1,806 nucleotides) and structure (V1-[BRCA2_exons_14-20]-V2). Functional mapping by microdeletions revealed essential sequences for exon recognition on the 3' end of exon 17 (c.7944-7973) and the 5' end of exon 18 (c.7979-7988, c.7999-8013). Thirty out of the 52 selected variants induced anomalous splicing in minigene assays with >16 different aberrant transcripts, where exon skipping was the most common event. A wide range of splicing motifs were affected including the canonical splice sites (15 variants), novel alternative sites (3 variants), the polypyrimidine tract (3 variants) and enhancers/silencers (9 variants). According to the guidelines of the American College of Medical Genetics and Genomics (ACMG), 20 variants could be classified as pathogenic (c.7806-2A>G, c.7806-1G>A, c.7806-1G>T, c.7806-1_7806-2dup, c.7976+1G>A, c.7977-3_7978del, c.7977-2A>T, c.7977-1G>T, c.7977-1G>C, c.8009C>A, c.8331+1G>T and c.8331+2T>C) or likely pathogenic (c.7806-9T>G, c.7976G>C, c.7976G>A, c.7977-7C>G, c.7985C>G, c.8023A>G, c.8035G>T and c.8331G>A), accounting for 30.8% of all pathogenic/likely pathogenic variants of exons 17-18 at the BRCA Share database. The remaining 8 variants (c.7975A>G, c.7977-6T>G, c.7988A>T, c.7992T>A, c.8007A>G, c.8009C>T, c.8009C>G, and c.8072C>T) induced partial splicing anomalies with important ratios of the full-length transcript (≥70%), so that they remained classified as VUS. Aberrant splicing is therefore especially prevalent in BRCA2 exons 17 and 18 due to the presence of active ESEs involved in exon recognition. Splicing functional assays with minigenes are a valuable strategy for the initial characterization of the splicing outcomes and the subsequent clinical interpretation of variants of any disease-gene, although these results should be checked, whenever possible, against patient RNA.

Association Between Bat Vitamin D Receptor 3' Haplotypes and Vitamin D Levels at Baseline and a Lower Response After Increased Vitamin D Supplementation and Exposure to Sunlight.

Objective: The aim of this study was to evaluate the relationship between vitamin D levels at baseline and after 12 weeks of supplementation/exposure to sunlight and VDR genotypes (BsmI, TaqI and ApaI) and haplotypes in a homogeneous population of postmenopausal women. Methods: We made a prospective study in which 151 women were randomized to two groups: One with 1000 mg of calcium and 800 IU vitamin D supplementation (102 women) and a placebo group with neither calcium or vitamin D supplementation (49 women). The follow-up was from May to September 2012.Vitamin D was determined by chemiluminescent immunoassay. Genotypes were determined using the Sequenomi Plexplatform and haplotypes using PHASE software. Results: Baseline (25 ± 10 ng/mlvs.23 ± 9 ng/ml, p > 0.05) and 12-week (32 ± 8 ng/mlvs.29 ± 10 ng/ml, p > 0.05) vitamin D levels were similar between the two groups. The genetic study was made in the total population. There were no differences in baseline and final levels of vitamin D in terms of genotypes and haplotypes, except for the Bat haplotype, whose baseline values were lower (25OHD: 21 ± 10 ng/mlvs. 21 ± 10 ng/ml, p = 0.038). The rate of nonresponders in this group was 15 % (p = 0.001), compared with 9 %, 2 % and 3 % in the other groups. Conclusions: The Bat haplotype was associated with lower baseline levels of vitamin D and a worse response to supplementation and, therefore, may be a risk factor for vitamin D deficiency.

Characterization of spliceogenic variants located in regions linked to high levels of alternative splicing: BRCA2 c.7976+5G > T as a case study.

Many BRCA1 and BRCA2 (BRCA1/2) genetic variants have been studied at mRNA level and linked to hereditary breast and ovarian cancer due to splicing alteration. In silico tools are reliable when assessing variants located in consensus splice sites, but we may identify variants in complex genomic contexts for which bioinformatics is not precise enough. In this study, we characterize BRCA2 c.7976 + 5G > T variant located in intron 17 which has an atypical donor site (GC). This variant was identified in three unrelated Spanish families and we have detected exon 17 skipping as the predominant transcript occurring in carriers. We have also detected several isoforms (Δ16-18, Δ17,18, Δ18, and ▼17q224 ) at different expression levels among carriers and controls. This study remarks the challenge of interpreting genetic variants when multiple alternative isoforms are present, and that caution must be taken when using in silico tools to identify potential spliceogenic variants located in GC-AG introns.

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.

Functional classification of BRCA2 DNA variants by splicing assays in a large minigene with 9 exons.

Numerous pathogenic DNA variants impair the splicing mechanism in human genetic diseases. Minigenes are optimal approaches to test variants under the splicing viewpoint without the need of patient samples. We aimed to design a robust minigene construct of the breast cancer gene BRCA2 in order to investigate the impact of variants on splicing. BRCA2 exons 19-27 (MGBR2_ex19-27) were cloned in the new vector pSAD. It produced a large transcript of the expected size (2,174 nucleotides) and exon structure (V1-ex19-27-V2). Splicing assays showed that 18 (17 splice-site and 1 silencer variants) out of 40 candidate DNA variants induced aberrant patterns. Twenty-four anomalous transcripts were accurately detected by fluorescent-RT-PCR that were generated by exon-skipping, alternative site usage, and intron-retention events. Fourteen variants induced major anomalies and were predicted to disrupt protein function so they could be classified as pathogenic. Furthermore, minigene mimicked previously reported patient RNA outcomes of seven variants supporting the reproducibility of minigene assays. Therefore, a relevant fraction of variants are involved in breast cancer through splicing alterations. MGBR2_ex19-27 is the largest reported BRCA2 minigene and constitutes a valuable tool for the functional and clinical classification of sequence variations.

Capillary electrophoresis analysis of conventional splicing assays: IARC analytical and clinical classification of 31 BRCA2 genetic variants.

Rare sequence variants in "high-risk" disease genes, often referred as unclassified variants (UVs), pose a serious challenge to genetic testing. However, UVs resulting in splicing alterations can be readily assessed by in vitro assays. Unfortunately, analytical and clinical interpretation of these assays is often challenging. Here, we explore this issue by conducting splicing assays in 31 BRCA2 genetic variants. All variants were assessed by RT-PCR followed by capillary electrophoresis and direct sequencing. If assays did not produce clear-cut outputs (Class-2 or Class-5 according to analytical International Agency for Research on Cancer guidelines), we performed qPCR and/or minigene assays. The latter were performed with a new splicing vector (pSAD) developed by authors of the present manuscript (patent #P201231427 CSIC). We have identified three clinically relevant Class-5 variants (c.682-2A>G, c.7617+1G>A, and c.8954-5A>G), and 27 analytical Class-2 variants (not inducing splicing alterations). In addition, we demonstrate that rs9534262 (c.7806-14T>C) is a BRCA2 splicing quantitative trait locus.

Severe alpha-1 antitrypsin deficiency in composite heterozygotes inheriting a new splicing mutation QOMadrid.

BACKGROUND: Severe Alpha-1 Antitrypsin (AAT) deficiency is a hereditary condition caused by mutations in the SERPINA1 gene, which predisposes to lung emphysema and liver disease. It is usually related to PI*Z alleles, and less frequent to rare and null (QO) alleles. Null-AAT alleles represent the end of a continuum of variants associated with profound AAT deficiency and extremely increased risk of emphysema. METHODS: A family with severe AAT deficiency was analyzed to achieve genetic diagnosis. The complete exons and introns of the SERPINA1 gene were sequenced and transcriptional analysis by RT-PCR was performed to characterize the effect of splicing variants found in the patients. In addition, a minigene MGserpa1_ex1b-1c was cloned into the pSAD vector to in vitro investigate the independent impact of variants on splicing process. RESULTS: We report a new identified null allele (PI*QOMadrid) in two adult siblings with practically no detectable serum AAT. The PI*QOMadrid allele consist of a duplication of the thymine (T) in position +2 of the donor splice site of exon 1C (+2dupT). In these two subjects, PI*QOMadrid occurred in compound heterozygote combination with the previously described variant PI*QOPorto. Both QOMadrid and QOPorto variants are located very close together in a regulatory region of the SERPINA1 gene. Analysis of transcripts revealed that QOMadrid variant prevented the expression of transcripts from exon 1C, and then normally spliced RNA products are not expected in the liver of these patients. In addition, aberrant splicing patterns of both variants were clearly distinguished and quantified by functional in vitro assays lending further support to their pathogenicity. CONCLUSION: Finding pathogenic mutations in non-coding regions of the SERPINA1 highlight the importance that regulatory regions might have in the disease. Regulatory regions should be seriously considered in discordant cases with severe AAT deficiency where no coding mutations were found.

The highly prevalent BRCA2 mutation c.2808_2811del (3036delACAA) is located in a mutational hotspot and has multiple origins.

BRCA2-c.2808_2811del (3036delACAA) is one of the most reported germ line mutations in non-Ashkenazi breast cancer patients. We investigated its genetic origin in 51 Spanish carrier families that were genotyped with 11 13q polymorphic markers. Three independent associated haplotypes were clearly distinguished accounting for 23 [west Castilla y León (WCL)], 20 [east Castilla y León (ECL)] and 6 (South of Spain) families. Mutation age was estimated with the Disequilibrium Mapping using Likelihood Estimation software in a range of 45-68 and 45-71 generations for WCL and ECL haplotypes, respectively. The most prevalent variants, c.2808_2811del and c.2803G > A, were located in a double-hairpin loop structure (c.2794-c.2825) predicted by Quikfold that was proposed as a mutational hotspot. To check this hypothesis, random mutagenesis was performed over a 923 bp fragment of BRCA2, and 86 DNA variants were characterized. Interestingly, three mutations reported in the mutation databases (c.2680G > A, c.2944del and c.2957dup) were replicated and 20 affected the same position with different nucleotide changes. Moreover, five variants were placed in the same hairpin loop of c.2808_2811del, and one affected the same position (c.2808A > G). In conclusion, our results support that at least three different mutational events occurred to generate c.2808_2811del. Other highly prevalent DNA variants, such as BRCA1-c.68_69delAG, BRCA2-c.5946delT and c.8537delAG, are concentrated in hairpin loops, suggesting that these structures may represent mutational hotspots.

Comprehensive splicing functional analysis of DNA variants of the BRCA2 gene by hybrid minigenes.

INTRODUCTION: The underlying pathogenic mechanism of a large fraction of DNA variants of disease-causing genes is the disruption of the splicing process. We aimed to investigate the effect on splicing of the BRCA2 variants c.8488-1G > A (exon 20) and c.9026_9030del (exon 23), as well as 41 BRCA2 variants reported in the Breast Cancer Information Core (BIC) mutation database. METHODS: DNA variants were analyzed with the splicing prediction programs NNSPLICE and Human Splicing Finder. Functional analyses of candidate variants were performed by lymphocyte RT-PCR and/or hybrid minigene assays. Forty-one BIC variants of exons 19, 20, 23 and 24 were bioinformatically selected and generated by PCR-mutagenesis of the wild type minigenes. RESULTS: Lymphocyte RT-PCR of c.8488-1G > A showed intron 19 retention and a 12-nucleotide deletion in exon 20, whereas c.9026_9030del did not show any splicing anomaly. Minigene analysis of c.8488-1G > A displayed the aforementioned aberrant isoforms but also exon 20 skipping. We further evaluated the splicing outcomes of 41 variants of four BRCA2 exons by minigene analysis. Eighteen variants presented splicing aberrations. Most variants (78.9%) disrupted the natural splice sites, whereas four altered putative enhancers/silencers and had a weak effect. Fluorescent RT-PCR of minigenes accurately detected 14 RNA isoforms generated by cryptic site usage, exon skipping and intron retention events. Fourteen variants showed total splicing disruptions and were predicted to truncate or eliminate essential domains of BRCA2. CONCLUSIONS: A relevant proportion of BRCA2 variants are correlated with splicing disruptions, indicating that RNA analysis is a valuable tool to assess the pathogenicity of a particular DNA change. The minigene system is a straightforward and robust approach to detect variants with an impact on splicing and contributes to a better knowledge of this gene expression step.

Detection of a large rearrangement in PALB2 in Spanish breast cancer families with male breast cancer.

It has been demonstrated that monoallelic PALB2 (Partner and Localizer of BRCA2) gene mutations predispose to familial breast cancer. Some of the families reported with germline PALB2 mutations presented male breast cancer as a characteristic clinical feature. Therefore, we wanted to investigate the contribution of germline PALB2 mutations in a set of 131 Spanish BRCA1/BRCA2-negative breast/ovarian cancer families with at least one male breast cancer case. The analysis included direct sequencing of all coding exons and intron/exon boundaries as well as a Multiplex Ligation-dependent Probe Amplification-based analysis of genomic rearrangements. For the first time we have identified a genomic rearrangement of PALB2 gene involving a large deletion from exon 7 to 11 in a breast cancer family. We have also identified several PALB2 variants, but no other obvious deleterious PALB2 mutation has been found. Thus, our study does not support an enrichment of PALB2 germline mutations in the subset of breast cancer families with male breast cancer cases. The identification of intronic and exonic variants indicates the necessity of assessing the implications of variants that do not lead to PALB2 truncation in the pathoghenicity of the PALB2 gene.

Germline Variants of CYBA and TRPM4 Predispose to Familial Colorectal Cancer.

Familial colorectal cancer (CRC) is only partially explained by known germline predisposing genes. We performed whole-genome sequencing in 15 Polish families of many affected individuals, without mutations in known CRC predisposing genes. We focused on loss-of-function variants and functionally characterized them. We identified a frameshift variant in the CYBA gene (c.246delC) in one family and a splice site variant in the TRPM4 gene (c.25-1 G > T) in another family. While both variants were absent or extremely rare in gene variant databases, we identified four additional Polish familial CRC cases and two healthy elderly individuals with the CYBA variant (odds ratio 2.46, 95% confidence interval 0.48-12.69). Both variants led to a premature stop codon and to a truncated protein. Functional characterization of the variants showed that knockdown of CYBA or TRPM4 depressed generation of reactive oxygen species (ROS) in LS174T and HT-29 cell lines. Knockdown of TRPM4 resulted in decreased MUC2 protein production. CYBA encodes a component in the NADPH oxidase system which generates ROS and controls, e.g., bacterial colonization in the gut. Germline CYBA variants are associated with early onset inflammatory bowel disease, supported with experimental evidence on loss of intestinal mucus barrier function due to ROS deficiency. TRPM4 encodes a calcium-activated ion channel, which, in a human colonic cancer cell line, controls calcium-mediated secretion of MUC2, a major component of intestinal mucus barrier. We suggest that the gene defects in CYBA and TRPM4 mechanistically involve intestinal barrier integrity through ROS and mucus biology, which converges in chronic bowel inflammation.

International distribution and age estimation of the Portuguese BRCA2 c.156_157insAlu founder mutation.

The c.156_157insAlu BRCA2 mutation has so far only been reported in hereditary breast/ovarian cancer (HBOC) families of Portuguese origin. Since this mutation is not detectable using the commonly used screening methodologies and must be specifically sought, we screened for this rearrangement in a total of 5,443 suspected HBOC families from several countries. Whereas the c.156_157insAlu BRCA2 mutation was detected in 11 of 149 suspected HBOC families from Portugal, representing 37.9% of all deleterious mutations, in other countries it was detected only in one proband living in France and in four individuals requesting predictive testing living in France and in the USA, all being Portuguese immigrants. After performing an extensive haplotype study in carrier families, we estimate that this founder mutation occurred 558 ± 215 years ago. We further demonstrate significant quantitative differences regarding the production of the BRCA2 full length RNA and the transcript lacking exon 3 in c.156_157insAlu BRCA2 mutation carriers and in controls. The cumulative incidence of breast cancer in carriers did not differ from that of other BRCA2 and BRCA1 pathogenic mutations. We recommend that all suspected HBOC families from Portugal or with Portuguese ancestry are specifically tested for this rearrangement.

RAD51D Aberrant Splicing in Breast Cancer: Identification of Splicing Regulatory Elements and Minigene-Based Evaluation of 53 DNA Variants.

RAD51D loss-of-function variants increase lifetime risk of breast and ovarian cancer. Splicing disruption is a frequent pathogenic mechanism associated with variants in susceptibility genes. Herein, we have assessed the splicing and clinical impact of splice-site and exonic splicing enhancer (ESE) variants identified through the study of ~113,000 women of the BRIDGES cohort. A RAD51D minigene with exons 2-9 was constructed in splicing vector pSAD. Eleven BRIDGES splice-site variants (selected by MaxEntScan) were introduced into the minigene by site-directed mutagenesis and tested in MCF-7 cells. The 11 variants disrupted splicing, collectively generating 25 different aberrant transcripts. All variants but one produced negligible levels (<3.4%) of the full-length (FL) transcript. In addition, ESE elements of the alternative exon 3 were mapped by testing four overlapping exonic microdeletions (≥30-bp), revealing an ESE-rich interval (c.202_235del) with critical sequences for exon 3 recognition that might have been affected by germline variants. Next, 26 BRIDGES variants and 16 artificial exon 3 single-nucleotide substitutions were also assayed. Thirty variants impaired splicing with variable amounts (0-65.1%) of the FL transcript, although only c.202G>A demonstrated a complete aberrant splicing pattern without the FL transcript. On the other hand, c.214T>C increased efficiency of exon 3 recognition, so only the FL transcript was detected (100%). In conclusion, 41 RAD51D spliceogenic variants (28 of which were from the BRIDGES cohort) were identified by minigene assays. We show that minigene-based mapping of ESEs is a powerful approach for identifying ESE hotspots and ESE-disrupting variants. Finally, we have classified nine variants as likely pathogenic according to ACMG/AMP-based guidelines, highlighting the complex relationship between splicing alterations and variant interpretation.

Two founder BRCA2 mutations predispose to breast cancer in young women.

The mutation spectrum of BRCA1 and BRCA2 presents a wide range of unique mutations in breast/ovarian cancer patients but recurrent mutations with founder effects have also been described. BRCA2 5344delAATA and 9538delAA are recurrent mutations in Castilla-León (Spain) representing 10.6% of BRCA2 positive families. By genotyping eleven chromosome 13 markers (4.3 Mb) we demonstrate that each mutation shows core haplotypes of 1.66 and 0.87 Mb, respectively, supporting a common ancestor in Castilla-León. Furthermore, both mutations are associated with earlier onset of breast cancer (5344delAATA: 37.4 years, P = 0.033; 9538delAA: 39.4 years, P = 0.008). The identification of founder effects improves the genetic screening strategy to be followed and facilitates the clinical management of asymptomatic carriers.

UGT1A1 Variants c.864+5G>T and c.996+2_996+5del of a Crigler-Najjar Patient Induce Aberrant Splicing in Minigene Assays.

A large fraction of DNA variants impairs pre-mRNA splicing in human hereditary disorders. Crigler-Najjar syndrome (CNS) is characterized by a severe unconjugated hyperbilirubinemia caused by variants in the UGT1A1 gene. We previously reported one CNS-type II patient with two splice-site variants in trans (c.864+5G>T and c.996+2_996+5del). According to MaxEntScan, both disrupt their corresponding donor sites (c.864+5G>T: 6.99 → 2.28; c.996+2_996+5del: 5.96 → -11.02), so they were selected for subsequent functional tests. Given the unavailability of patient RNA, we constructed an UGT1A1 splicing-reporter minigene with exons 1-4 to characterize the underlying splicing anomaly. The variant c.996+2_996+5del generated two aberrant transcripts, Δ(E2) (exon 2 skipping/64%) and ▼(E2q135) (intron retention of 135-nt/36%), which lead to the loss of 18 conserved amino-acids and the gain of 45 new ones of a critical functional domain, respectively. The c.864+5G>T variant mainly produced the aberrant transcript Δ(E1q141) (141-nt deletion/70.4%) and the full-length isoform (29.6%). Δ(E1q141) would provoke the loss of 47 amino-acids of the N-terminal domain that encodes for substrate specificity. Thus, the three anomalous transcripts are likely to inactivate UGT1A1. Moreover, this patient is also homozygous for the promoter variant A(TA)7TAA that decreases UGT1A1 expression by 70%, so the full-length transcript produced by c.864+5G>T would be even more reduced (<9%), thus supporting the diagnosis of CNS-type II. Therefore, minigenes represent valuable tools for the functional and clinical classifications of genetic variants.

Comprehensive Functional Characterization and Clinical Interpretation of 20 Splice-Site Variants of the RAD51C Gene.

Hereditary breast and/or ovarian cancer is a highly heterogeneous disease with more than 10 known disease-associated genes. In the framework of the BRIDGES project (Breast Cancer Risk after Diagnostic Gene Sequencing), the RAD51C gene has been sequenced in 60,466 breast cancer patients and 53,461 controls. We aimed at functionally characterizing all the identified genetic variants that are predicted to disrupt the splicing process. Forty RAD51C variants of the intron-exon boundaries were bioinformatically analyzed, 20 of which were selected for splicing functional assays. To test them, a splicing reporter minigene with exons 2 to 8 was designed and constructed. This minigene generated a full-length transcript of the expected size (1062 nucleotides), sequence, and structure (Vector exon V1- RAD51C exons_2-8- Vector exon V2). The 20 candidate variants were genetically engineered into the wild type minigene and functionally assayed in MCF-7 cells. Nineteen variants (95%) impaired splicing, while 18 of them produced severe splicing anomalies. At least 35 transcripts were generated by the mutant minigenes: 16 protein-truncating, 6 in-frame, and 13 minor uncharacterized isoforms. According to ACMG/AMP-based standards, 15 variants could be classified as pathogenic or likely pathogenic variants: c.404G > A, c.405-6T > A, c.571 + 4A > G, c.571 + 5G > A, c.572-1G > T, c.705G > T, c.706-2A > C, c.706-2A > G, c.837 + 2T > C, c.905-3C > G, c.905-2A > C, c.905-2_905-1del, c.965 + 5G > A, c.1026 + 5_1026 + 7del, and c.1026 + 5G > T.