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.

Real-World Use of Highly Sensitive Liquid Biopsy Monitoring in Metastatic Breast Cancer Patients Treated with Endocrine Agents after Exposure to Aromatase Inhibitors.

Endocrine-resistant, hormone receptor-positive, and HER2-negative (HR+/HER2-) metastatic breast cancer (mBC) is largely governed by acquired mutations in the estrogen receptor, which promote ligand-independent activation, and by truncal alterations in the PI3K signaling pathway, with a broader range of gene alterations occurring with less prevalence. Circulating tumor DNA (ctDNA)-based technologies are progressively permeating the clinical setting. However, their utility for serial monitoring has been hindered by their significant costs, inter-technique variability, and real-world patient heterogeneity. We interrogated a longitudinal collection of 180 plasma samples from 75 HR+/HER2- mBC patients who progressed or relapsed after exposure to aromatase inhibitors and were subsequently treated with endocrine therapy (ET) by means of highly sensitive and affordable digital PCR and SafeSEQ sequencing. Baseline PIK3CA and TP53 mutations were prognostic of a shorter progression-free survival in our population. Mutant PIK3CA was prognostic in the subset of patients receiving fulvestrant monotherapy after progression to a CDK4/6 inhibitor (CDK4/6i)-containing regimen, and its suppression was predictive in a case of long-term benefit with alpelisib. Mutant ESR1 was prognostic in patients who did not receive concurrent CDK4/6i, an impact influenced by the variant allele frequency, and its early suppression was strongly predictive of efficacy and associated with long-term benefit in the whole cohort. Mutations in ESR1, TP53, and KRAS emerged as putative drivers of acquired resistance. These findings collectively contribute to the characterization of longitudinal ctDNA in real-world cases of HR+/HER2- mBC previously exposed to aromatase inhibitors and support ongoing studies either targeting actionable alterations or leveraging the ultra-sensitive tracking of ctDNA.

Germline gain-of-function MMP11 variant results in an aggressive form of colorectal cancer.

Matrix metalloproteinase-11 (MMP11) is an enzyme with proteolytic activity against matrix and nonmatrix proteins. Although most MMPs are secreted as inactive proenzymes and are later activated extracellularly, MMP11 is activated intracellularly by furin within the constitutive secretory pathway. It is a key factor in physiological tissue remodeling and its alteration may play an important role in the progression of epithelial malignancies and other diseases. TCGA colon and colorectal adenocarcinoma data showed that upregulation of MMP11 expression correlates with tumorigenesis and malignancy. Here, we provide evidence that a germline variant in the MMP11 gene (NM_005940: c.232C>T; p.(Pro78Ser)), identified by whole exome sequencing, can increase the tumorigenic properties of colorectal cancer (CRC) cells. P78S is located in the prodomain region, which is responsible for blocking MMP11's protease activity. This variant was detected in the proband and all the cancer-affected family members analyzed, while it was not detected in healthy relatives. In silico analyses predict that P78S could have an impact on the activation of the enzyme. Furthermore, our in vitro analyses show that the expression of P78S in HCT116 cells increases tumor cell invasion and proliferation. In summary, our results show that this variant could modify the structure of the MMP11 prodomain, producing a premature or uncontrolled activation of the enzyme that may contribute to an early CRC onset in these patients. The study of this gene in other CRC cases will provide further information about its role in CRC development, which might improve patient treatment in the future.

Characterization of the NRAMP Gene Family in the Arbuscular Mycorrhizal Fungus Rhizophagus irregularis.

Transporters of the NRAMP family are ubiquitous metal-transition transporters, playing a key role in metal homeostasis, especially in Mn and Fe homeostasis. In this work, we report the characterization of the NRAMP family members (RiSMF1, RiSMF2, RiSMF3.1 and RiSMF3.2) of the arbuscular mycorrhizal (AM) fungus Rhizophagus irregularis. Phylogenetic analysis of the NRAMP sequences of different AM fungi showed that they are classified in two groups, which probably diverged early in their evolution. Functional analyses in yeast revealed that RiSMF3.2 encodes a protein mediating Mn and Fe transport from the environment. Gene-expression analyses by RT-qPCR showed that the RiSMF genes are differentially expressed in the extraradical (ERM) and intraradical (IRM) mycelium and differentially regulated by Mn and Fe availability. Mn starvation decreased RiSMF1 transcript levels in the ERM but increased RiSMF3.1 expression in the IRM. In the ERM, RiSMF1 expression was up-regulated by Fe deficiency, suggesting a role for its encoded protein in Fe-deficiency alleviation. Expression of RiSMF3.2 in the ERM was up-regulated at the early stages of Fe toxicity but down-regulated at later stages. These data suggest a role for RiSMF3.2 not only in Fe transport but also as a sensor of high external-Fe concentrations. Both Mn- and Fe-deficient conditions affected ERM development. While Mn deficiency increased hyphal length, Fe deficiency reduced sporulation.

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.

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.

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.

BRIP1, a Gene Potentially Implicated in Familial Colorectal Cancer Type X.

Familial colorectal cancer Type X (FCCTX) comprises a heterogeneous group of families with an increased risk of developing colorectal cancer and other related tumors, but with mismatch repair-proficient, microsatellite-stable (MSS) tumors. Unfortunately, the genetic basis underlying their cancer predisposition remains unknown. Although pathogenic germline variants in BRIP1 increase the risk of developing hereditary ovarian cancer, the involvement of BRIP1 in hereditary colorectal cancer is still not well known. In order to identify new BRIP1 variants associated with inherited colorectal cancer, affected and nonaffected individuals from 18 FCCTX or high-risk MSS colorectal cancer families were evaluated by whole-exome sequencing, and another 62 colorectal cancer patients from FCCTX or high-risk MSS colorectal cancer families were screened by a next-generation sequencing (NGS) multigene panel. The families were recruited at the Genetic Counseling Unit of Hospital Clínico San Carlos of Madrid. A total of three different BRIP1 mutations in three unrelated families were identified. Among them, there were two frameshift variants [c.1702_1703del, p.(Asn568TrpfsTer9) and c.903del, p.(Leu301PhefsTer2)] that result in the truncation of the protein and are thus classified as pathogenic (class 5). The remaining was a missense variant [c.2220G>T, p.(Gln740His)] considered a variant of uncertain significance (class 3). The segregation and loss-of-heterozygosity studies provide evidence linking the two BRIP1 frameshift variants to colorectal cancer risk, with suggestive but not definitive evidence that the third variant may be benign. The results here presented suggest that germline BRIP1 pathogenic variants could be associated with hereditary colorectal cancer predisposition.Prevention Relevance: We suggest that BRIP1 pathogenic germline variants may have a causal role in CRC as moderate cancer susceptibility alleles and be associated with hereditary CRC predisposition. A better understanding of hereditary CRC may provide important clues to disease predisposition and could contribute to molecular diagnostics, improved risk stratification, and targeted therapeutic strategies.

Contribution of New Adenomatous Polyposis Predisposition Genes in an Unexplained Attenuated Spanish Cohort by Multigene Panel Testing.

Attenuated adenomatous polyposis (AAP) is a heterogeneous syndrome in terms of clinical manifestations, heritability and etiology of the disease. Genetic heterogeneity and low penetrance alleles are probably the best explanation for this variability. Certainly, it is known that APC and MUTYH are high penetrance predisposition genes for adenomatous polyposis, but they only account for 5-10% of AAP. Other new predisposition genes, such as POLE, POLD1, NTHL1, AXIN2 or MSH3, have been recently described and have been associated with AAP, but their relative contribution is still not well defined. In order to evaluate the genetic predisposition to AAP in a hospital based population, germline DNAs from 158 AAP subjects were screened for genetic variants in the coding regions and intron-exon boundaries of seven associated genes through a next-generation sequencing (NGS) custom gene panel. Splicing, segregation studies, somatic mutational screening and RNA quantitative expression assays were conducted for selected variants. In four of the probands the adenoma susceptibility could be explained by actionable mutations in APC or MUTYH, and one other patient was a double carrier of two truncating variants in both POLE and NTHL1. Furthermore, 16 additional patients harbored uncertain significance variants in the remaining tested genes. This report gives information about the contribution of the newly described adenomatous polyposis predisposition genes in a Spanish attenuated polyposis cohort. Our results highly support the convenience of NGS multigene panels for attenuated polyposis genetic screening and reveals POLE frameshift variants as a plausible susceptibility mechanism for AAP.

Current status of the genetic susceptibility in attenuated adenomatous polyposis.

Adenomatous polyposis (AP) is classified according to cumulative adenoma number in classical AP (CAP) and attenuated AP (AAP). Genetic susceptibility is the major risk factor in CAP due to mutations in the known high predisposition genes APC and MUTYH. However, the contribution of genetic susceptibility to AAP is lower and less understood. New predisposition genes have been recently proposed, and some of them have been validated, but their scarcity hinders accurate risk estimations and prevalence calculations. AAP is a heterogeneous condition in terms of severity, clinical features and heritability. Therefore, clinicians do not have strong discriminating criteria for the recommendation of the genetic study of known predisposition genes, and the detection rate is low. Elucidation and knowledge of new AAP high predisposition genes are of great importance to offer accurate genetic counseling to the patient and family members. This review aims to update the genetic knowledge of AAP, and to expound the difficulties involved in the genetic analysis of a highly heterogeneous condition such as AAP.

MUC1 expression in colorectal carcinoma: Clinicopathological correlation and prognostic significance / Expresión de MUC1 en el carcinoma colorectal: correlación clinicopatológica y valor pronóstico

Introduction: MUC1 overexpression has been linked to cancer development and has been associated with a higher stage at diagnosis and presence of lymph node or distant metastases. However, its prognostic significance is still unclear. We aimed to evaluate the relationship between MUC1 expression and prognosis of colorectal carcinoma. Materials and methods: Immunohistochemical expression of MUC1 in 96 colorectal carcinomas with analysis of potential prognostic influence. Results: 55.2% of patients were women and the mean age was 65.9 years. Tumors were more frequently located in rectum or sigmoid colon (60.4% and 21.9%). Most tumors were T3 (60.3%). 36.9% of patients showed lymph node metastases and 30.2% showed distant metastasis at the time of diagnosis. MUC1 was intensely positive in 46% and negative in 37.9% of tumors. Overall, 61% of patients recurred and 40.4% died during follow-up. 58.5% of tumors of surviving patients were intensely positive for MUC1 and 29.5% were negative, as compared with 28.5% (intense positivity) and 51.4% (negativity) in the group of patients who died (p=0.022). 65% of tumors of patients without recurrences showed intense positivity for MUC1 and 23% of them were negative as compared with 33.9% (intense positivity) and 47% (negativity) in the group of patients who recurred (p=0.019). Conclusions: Loss of MUC1 expression was more frequent in cases with disease recurrence or death, as compared with patients with stable disease, in whom intense positivity was more frequently seen. These findings disagree with the majority of previous studies, indicating the need for further investigation

Introducción: La sobreexpresión de MUC1 se ha asociado al desarrollo de cáncer, mayor estadio al diagnóstico y la presencia de metástasis ganglionares o a distancia. Sin embargo, su valor pronóstico es dudoso. Nuestro objetivo es evaluar la relación entre la expresión inmunohistoquímica de MUC1 y el pronóstico del carcinoma colorrectal (CRC). Material y métodos: Expresión inmunohistoquímica de MUC1 en 96 CRC y análisis de su influencia pronóstica. Resultados: El 55,2% de los pacientes eran mujeres, con edad media de 65,9 años. Los tumores se localizaban principalmente en recto o sigma (60,4 y 21,9%). El 60,3% de los CRC fueron estadio T3. El 36,9% de pacientes mostraron metástasis ganglionares y el 30,2% a distancia. MUC1 fue intensamente positivo en el 46% y negativo en el 37,9% de los casos. En el 61% de los pacientes se observaron recurrencias y el 40,4% falleció. Un 58,5% de tumores de pacientes vivos mostró positividad intensa y un 29,5% negatividad para MUC1, en comparación con un 28,5 y 51,4% de positividad intensa y negatividad en los pacientes que murieron (p=0,022). El 65% de los tumores de pacientes sin recurrencias fueron intensamente positivos para MUC1 y el 23% fue negativo, en comparación con un 33,9 y 47% de positividad intensa y negatividad en pacientes con recurrencias (p=0,019). Conclusiones: La pérdida de expresión de MUC1 fue más frecuente en pacientes con recurrencias o fallecidos que en pacientes estables. Estos resultados son contrarios a la mayoría de estudios previos y subrayan la necesidad de realización de más estudios

MUC1 expression in colorectal carcinoma: Clinicopathological correlation and prognostic significance.

INTRODUCTION: MUC1 overexpression has been linked to cancer development and has been associated with a higher stage at diagnosis and presence of lymph node or distant metastases. However, its prognostic significance is still unclear. We aimed to evaluate the relationship between MUC1 expression and prognosis of colorectal carcinoma. MATERIALS AND METHODS: Immunohistochemical expression of MUC1 in 96 colorectal carcinomas with analysis of potential prognostic influence. RESULTS: 55.2% of patients were women and the mean age was 65.9 years. Tumors were more frequently located in rectum or sigmoid colon (60.4% and 21.9%). Most tumors were T3 (60.3%). 36.9% of patients showed lymph node metastases and 30.2% showed distant metastasis at the time of diagnosis. MUC1 was intensely positive in 46% and negative in 37.9% of tumors. Overall, 61% of patients recurred and 40.4% died during follow-up. 58.5% of tumors of surviving patients were intensely positive for MUC1 and 29.5% were negative, as compared with 28.5% (intense positivity) and 51.4% (negativity) in the group of patients who died (p=0.022). 65% of tumors of patients without recurrences showed intense positivity for MUC1 and 23% of them were negative as compared with 33.9% (intense positivity) and 47% (negativity) in the group of patients who recurred (p=0.019). CONCLUSIONS: Loss of MUC1 expression was more frequent in cases with disease recurrence or death, as compared with patients with stable disease, in whom intense positivity was more frequently seen. These findings disagree with the majority of previous studies, indicating the need for further investigation.

Novel genetic mutations detected by multigene panel are associated with hereditary colorectal cancer predisposition.

Half of the high-risk colorectal cancer families that fulfill the clinical criteria for Lynch syndrome lack germline mutations in the mismatch repair (MMR) genes and remain unexplained. Genetic testing for hereditary cancers is rapidly evolving due to the introduction of multigene panels, which may identify more mutations than the old screening methods. The aim of this study is the use of a Next Generation Sequencing panel in order to find the genes involved in the cancer predisposition of these families. For this study, 98 patients from these unexplained families were tested with a multigene panel targeting 94 genes involved in cancer predisposition. The mutations found were validated by Sanger sequencing and the segregation was studied when possible. We identified 19 likely pathogenic variants in 18 patients. Out of these, 8 were found in MMR genes (5 in MLH1, 1 in MSH6 and 2 in PMS2). In addition, 11 mutations were detected in other genes, including high penetrance genes (APC, SMAD4 and TP53) and moderate penetrance genes (BRIP1, CHEK2, MUTYH, HNF1A and XPC). Mutations c.1194G>A in SMAD4, c.714_720dup in PMS2, c.2050T>G in MLH1 and c.1635_1636del in MSH6 were novel. In conclusion, the detection of new pathogenic mutations in high and moderate penetrance genes could contribute to the explanation of the heritability of colorectal cancer, changing the individual clinical management. Multigene panel testing is a more effective method to identify germline variants in cancer patients compared to single-gene approaches and should be therefore included in clinical laboratories.

Role of GALNT12 in the genetic predisposition to attenuated adenomatous polyposis syndrome.

The involvement of GALNT12 in colorectal carcinogenesis has been demonstrated but it is not clear to what extent it is implicated in familial CRC susceptibility. Partially inactivating variant, NM_024642.4:c.907G>A, p.(D303N), has been previously detected in familial CRC and proposed as the causative risk allele. Since phenotypes of the described carrier families showed not only CRC but also a polyp history, we hypothesized that GALNT12 could be involved in adenoma predisposition and consequently, in hereditary polyposis CRC syndromes. For that purpose, we have screened the GALNT12 gene in germline DNA from 183 unrelated attenuated polyposis patients. c.907G>A, p.(D303N) was detected in 4 cases (MAF = 1.1%) and no other candidate variants were found. After segregation studies, LOH analyses, glycosylation pattern tests and case-control studies, our results did not support the role of c.907G>A, p.(D303N) as a high-penetrance risk allele for polyposis CRC.