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Supplementary data include a table with the primer sequences and three figures showing the validation and segregation study of the families carrying the BRIP1 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., PD, MPGV, DFE, MdlH and EAV received funding from the European Union's Horizon 2020 research and innovation program under grant agreement no. 634935. The EAV lab is supported by grants from the Spanish Ministry of Science and Innovation, Plan Nacional de I+D+I 2013-2016, ISCIII (PI17/00227 and PI20/00225) co-funded by the FEDER from Regional Development European Funds (European Union) and from the Consejería de Educación, Junta de Castilla y León, ref. CSI242P18 (actuación cofinanciada P.O. FEDER 2014-2020 de Castilla y León). The MdlH lab is supported by a grant from the Spanish Ministry of Science and Innovation, Plan Nacional de I+D+I 2013-2016, ISCIII (PI20/00110) co-funded by FEDER from Regional Development European Funds (European Union). Programa Estratégico Instituto de Biología y Genética Molecular (IBGM), Escalera de Excelencia, Junta de Castilla y León (Ref. CLU-2019-2002). EB-M is a postdoctoral researcher funded by the University of Valladolid (POSTDOC-UVA05, 2022-2025). AV-P and IL-B are supported by predoctoral fellowships from the Consejería de Educación, Junta de Castilla y León. LS-M is supported by a predoctoral fellowship from the AECC-Scientific Foundation, Sede Provincial de Valladolid (2019–2023). AE-S is supported through the Operational Program for Youth Employment and Youth Employment Initiative (YEI), called by the Community of Madrid in 2020, and co-financed by the European Social Fund.

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.

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.49-2AT, c.108+2TC, and c.211+1GA) 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 WileySons, Ltd on behalf of The Pathological Society of Great Britain and Ireland.

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.

Pathogenic sequence variants (PSV) in BRCA1 or BRCA2 (BRCA1/2) are associated with increased risk and severity of prostate cancer. We evaluated whether PSVs in BRCA1/2 were associated with risk of overall prostate cancer or high grade (Gleason 8+) prostate cancer using an international sample of 65 BRCA1 and 171 BRCA2 male PSV carriers with prostate cancer, and 3,388 BRCA1 and 2,880 BRCA2 male PSV carriers without prostate cancer. PSVs in the 3′ region of BRCA2 (c.7914+) were significantly associated with elevated risk of prostate cancer compared with reference bin c.1001-c.7913 [HR = 1.78; 95% confidence interval (CI), 1.25–2.52; P = 0.001], as well as elevated risk of Gleason 8+ prostate cancer (HR = 3.11; 95% CI, 1.63–5.95; P = 0.001). c.756-c.1000 was also associated with elevated prostate cancer risk (HR = 2.83; 95% CI, 1.71–4.68; P = 0.00004) and elevated risk of Gleason 8+ prostate cancer (HR = 4.95; 95% CI, 2.12–11.54; P = 0.0002). No genotype–phenotype associations were detected for PSVs in BRCA1. These results demonstrate that specific BRCA2 PSVs may be associated with elevated risk of developing aggressive prostate cancer. Significance: Aggressive prostate cancer risk in BRCA2 mutation carriers may vary according to the specific BRCA2 mutation inherited by the at-risk individual.

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.

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.

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.

e17551 Background: Few studies have addressed the carcinogenic process of laryngeal cancer from its premalignant phases. Our aim was to compare mutational status of laryngeal dysplasias (LD) evolving to laryngeal cancer (ELC) and not evolving to LC (NELC). Methods: Retrospective study of LD diagnosed between 2007 and 2011. A customized 15-gene NGS panel (DICER, IRF6, NOTCH1, NOTCH2, NOTCH3, NOTCH2NLA, PIK3CA, PTEN, RB1, RIPK4, SYNE1, SYNE2, TP53, TP63, CASP8) was used for mutational analysis with Truseq Custom Amplicon (Illumina) performed in FFPE LD samples and results compared between LD-ELC and LD-NELC. Results: Sixty-four patients (pts) with LD were identified. LD-ELC (N = 23) and LD-NELC (N = 41) were scored as mild (N = 40; NELC: 32 / ELC:8), moderate (N = 8; NELC: 4/ ELC: 4) and severe (N = 16; NELC:5 / ELC: 11). Prior or current moderate-to-heavy tobacco smoking ( > 10 pack-year): 53/64 (83%). Males: N = 50 (ELC: 21; NELC: 29); Female: N = 14 (ELC: 2; NELC: 12). Median time to cancer among 23 LD-ELC: 8 m (range: 0-46). Forty-seven gene variants were detected in ELC not found in NELC, of which 27 were pathogenic/likely pathogenic: Frameshift: 8 (1 in NOTCH2, 1 in PTEN, 2 in RB1, 2 in SYNE1 y 2 in SYNE2); Stop-gained: 3 (1 in NOTCH2NL, 2 in SYNE2); Splicing: 3 (1 in RB1, 1 in RIPK4 y 1 in TP53); Missense: 12 (1 in IRF6, 2 in NOTCH1, 1 in RB1, 2 in RIPK4, 6 in SYNE1). Conclusions: LD from pts ELC showed a different mutational profile than LD-NELC. These results show promise for identifying pts at higher risk for developing LC and should be validated in a larger, prospective study.