SMAD4 mosaicism in juvenile polyposis: Essential contribution of somatic analysis in diagnosis.

Juvenile polyposis syndrome (JPS) is a rare disease characterized by multiple hamartomatous polyps in the gastrointestinal tract, associated with pathogenic variants of BMPR1A and SMAD4. We present the description of SMAD4 mosaicism in a 30-year-old man who had caecum adenocarcinoma, 11 juvenile colon polyps and epistaxis since childhood. We conducted NGS polyposis and CRC panel analysis on DNA extracted from two polyps, revealing a likely pathogenic SMAD4 variant: NM_005359.5:c. 1600C>T, p.(Gln534*). This variant was then identified at a very low frequency on blood and normal colonic tissue, by targeted visualization of previously obtained NGS data. These findings support the presence of a likely pathogenic mosaic SMAD4 variant that aligns with the patient's phenotype. Given the relatively frequent occurrence of de novo SMAD4 mutations, somatic mosaicism could account for a significant proportion of sporadic JPS patients with unidentified pathogenic variants. This case underscores the diagnosis challenge of detecting mosaicism and emphasizes the importance of somatic analyses.

Predicting the impact of rare variants on RNA splicing in CAGI6.

Variants which disrupt splicing are a frequent cause of rare disease that have been under-ascertained clinically. Accurate and efficient methods to predict a variant's impact on splicing are needed to interpret the growing number of variants of unknown significance (VUS) identified by exome and genome sequencing. Here, we present the results of the CAGI6 Splicing VUS challenge, which invited predictions of the splicing impact of 56 variants ascertained clinically and functionally validated to determine splicing impact. The performance of 12 prediction methods, along with SpliceAI and CADD, was compared on the 56 functionally validated variants. The maximum accuracy achieved was 82% from two different approaches, one weighting SpliceAI scores by minor allele frequency, and one applying the recently published Splicing Prediction Pipeline (SPiP). SPiP performed optimally in terms of sensitivity, while an ensemble method combining multiple prediction tools and information from databases exceeded all others for specificity. Several challenge methods equalled or exceeded the performance of SpliceAI, with ultimate choice of prediction method likely to depend on experimental or clinical aims. One quarter of the variants were incorrectly predicted by at least 50% of the methods, highlighting the need for further improvements to splicing prediction methods for successful clinical application.

Germline HPF1 retrogene insertion in RB1 gene involved in cancer predisposition.

About half of the human genome is composed of repeated sequences derived from mobile elements, mainly retrotransposons, generally without pathogenic effect. Familial forms of retinoblastoma are caused by germline pathogenic variants in RB1 gene. Here, we describe a family with retinoblastoma affecting a father and his son. No pathogenic variant was identified after DNA analysis of RB1 gene coding sequence and exon-intron junctions. However, RB1 mRNA analysis showed a chimeric transcript with insertion of 114 nucleotides from HPF1 gene inside RB1 gene. This chimeric transcript led to an insertion of 38 amino acids in functional domain of retinoblastoma protein. Subsequent DNA analysis in RB1 intron 17 revealed the presence of a full-length HPF1 retrogene insertion in opposite orientation. Functional assay shows that this insertion has a deleterious impact on retinoblastoma protein function. This is the first report of a full-length retrogene insertion involved in human Mendelian disease leading to a chimeric transcript and a non-functional chimeric protein. Some retrogene insertions may be missed by standard diagnostic genetic testing, so contribution of retrogene insertions to human disease may be underestimated. The increasing use of whole genome sequencing in diagnostic settings will help to get a more comprehensive view of retrogenes.

Disequilibrium between BRCA1 and BRCA2 Circular and Messenger RNAs Plays a Role in Breast Cancer.

Breast cancer is a frequent disease for which the discovery of markers that enable early detection or prognostic assessment remains challenging. Circular RNAs (circRNAs) are single-stranded structures in closed loops that are produced by backsplicing. CircRNA and messenger RNA (mRNA) are generated co-transcriptionally, and backsplicing and linear splicing compete against each other. As mRNAs are key players in tumorigenesis, we hypothesize that a disruption of the balance between circRNAs and mRNAs could promote breast cancer. Hence, we developed an assay for a simultaneous study of circRNAs and mRNAs, which we have called splice and expression analyses by exon ligation and high-throughput sequencing (SEALigHTS). Following SEALigHTS validation for BRCA1 and BRCA2, our hypothesis was tested using an independent research set of 95 pairs from tumor and adjacent normal breast tissues. In this research set, ratios of BRCA1 and BRCA2 circRNAs/mRNAs were significantly lower in the tumor breast tissue compared to normal tissue (p = 1.6 × 10-9 and p = 4.4 × 10-5 for BRCA1 and BRCA2, respectively). Overall, we developed an innovative method to study linear splicing and backsplicing, described the repertoire of BRCA1 and BRCA2 circRNAs, including 15 novel ones, and showed for the first time that a disequilibrium between BRCA1 and BRCA2 circRNAs and mRNAs plays a role in breast cancer.

Cancer risks by sex and variant type in PTEN hamartoma tumor syndrome.

BACKGROUND: PTEN Hamartoma Tumor Syndrome (PHTS) is a rare syndrome with a broad phenotypic spectrum, including increased risks of breast (BC, 67%-78% at age 60 years), endometrial (EC, 19%-28%), and thyroid cancer (TC, 6%-38%). Current risks are likely overestimated due to ascertainment bias. We aimed to provide more accurate and personalized cancer risks. METHODS: This was a European, adult PHTS cohort study with data from medical files, registries, and/or questionnaires. Cancer risks and hazard ratios were assessed with Kaplan-Meier and Cox regression analyses, and standardized incidence ratios were calculated. Bias correction consisted of excluding cancer index cases and incident case analyses. RESULTS: A total of 455 patients were included, including 50.5% index cases, 372 with prospective follow-up (median 6 years, interquartile range = 3-10 years), and 159 of 281 females and 39 of 174 males with cancer. By age 60 years, PHTS-related cancer risk was higher in females (68.4% to 86.3%) than males (16.4% to 20.8%). Female BC risks ranged from 54.3% (95% confidence interval [CI] = 43.0% to 66.4%) to 75.8% (95% CI = 60.7% to 88.4%), with two- to threefold increased risks for PTEN truncating and approximately twofold for phosphatase domain variants. EC risks ranged from 6.4% (95% CI = 2.1% to 18.6%) to 22.1% (95% CI = 11.6% to 39.6%) and TC risks from 8.9% (95% CI = 5.1% to 15.3%) to 20.5% (95% CI = 11.3% to 35.4%). Colorectal cancer, renal cancer, and melanoma risks were each less than 10.0%. CONCLUSIONS: Females have a different BC risk depending on their PTEN germline variant. PHTS patients are predominantly at risk of BC (females), EC, and TC. This should be the main focus of surveillance. These lower, more unbiased and personalized risks provide guidance for optimized cancer risk management.

SPiP: Splicing Prediction Pipeline, a machine learning tool for massive detection of exonic and intronic variant effects on mRNA splicing.

Modeling splicing is essential for tackling the challenge of variant interpretation as each nucleotide variation can be pathogenic by affecting pre-mRNA splicing via disruption/creation of splicing motifs such as 5'/3' splice sites, branch sites, or splicing regulatory elements. Unfortunately, most in silico tools focus on a specific type of splicing motif, which is why we developed the Splicing Prediction Pipeline (SPiP) to perform, in one single bioinformatic analysis based on a machine learning approach, a comprehensive assessment of the variant effect on different splicing motifs. We gathered a curated set of 4616 variants scattered all along the sequence of 227 genes, with their corresponding splicing studies. The Bayesian analysis provided us with the number of control variants, that is, variants without impact on splicing, to mimic the deluge of variants from high-throughput sequencing data. Results show that SPiP can deal with the diversity of splicing alterations, with 83.13% sensitivity and 99% specificity to detect spliceogenic variants. Overall performance as measured by area under the receiving operator curve was 0.986, better than SpliceAI and SQUIRLS (0.965 and 0.766) for the same data set. SPiP lends itself to a unique suite for comprehensive prediction of spliceogenicity in the genomic medicine era. SPiP is available at: https://sourceforge.net/projects/splicing-prediction-pipeline/.

Detecting inversions in routine molecular diagnosis in MMR genes.

Inversions, i.e. a change in orientation of a segment of DNA, are a recognized cause of human diseases which remain overlooked due to their balanced nature. Inversions can have severe or more subtle impacts on gene expression. We describe two families that exemplify these aspects and underline the need for inversion detection in routine diagnosis. The first family (F1) displayed a sibship with two constitutional mismatch repair deficiency patients and a family history of colon cancer in the paternal branch. The second family (F2) displayed a severe history of Lynch syndrome. These families were analyzed using a whole gene panel (WGP) strategy i.e. including colon cancer genes with their intronic and flanking genomic regions. In F1, a PMS2 inversion encompassing the promoter region to intron 1 and a PMS2 splice variant were found in the maternal and paternal branch, respectively. In F2, we described the first MSH6 inversion, involving the 5' part of MSH6 and the 3' part of the nearby gene ANXA4. Inversion detection mandates genomic sequencing, but makes a valuable contribution to the diagnostic rate. WGP is an attractive strategy as it maximizes the detection power on validated genes and keeps sufficient depth to detect de novo events.

Highly Sensitive Detection Method of Retinoblastoma Genetic Predisposition and Biomarkers.

Retinoblastoma is a malignant tumor of the infant retina. Nearly half of patients are predisposed to retinoblastoma by a germline RB1 pathogenic variant. Nonhereditary retinoblastoma is mainly caused by inactivation of both RB1 alleles at a somatic level. Several polymorphisms have been reported as biomarkers of retinoblastoma risk, aggressiveness, or invasion. The most informative genetic testing is obtained from tumor DNA. Historically, access to tumor DNA has been warranted by the frequent indication of enucleation, which has decreased because of advances in conservative approaches. Recent studies showed that tumor cell-free DNA can be analyzed in aqueous humor from retinoblastoma patients. This report describes a next-generation sequencing method relying on unique molecular identifiers for a highly sensitive detection of retinoblastoma genetic predisposition and biomarkers in a single analysis. It is the first use of unique molecular identifiers for retinoblastoma genetics. This gene panel enables the detection of RB1 point variants, large genome rearrangements, and loss of heterozygosity. It is adapted for genomic DNA extracted from blood or tumor DNA extracted from tumor fragment, aqueous humor, or plasma. The access to tumor cell-free DNA improves the diagnosis of genetic predisposition in case of conservative ocular therapy and provides access to biomarkers guiding the treatment strategy. The analysis of a gene panel is cost-effective and can be easily implemented in diagnostic laboratories.

A high-risk retinoblastoma subtype with stemness features, dedifferentiated cone states and neuronal/ganglion cell gene expression.

Retinoblastoma is the most frequent intraocular malignancy in children, originating from a maturing cone precursor in the developing retina. Little is known on the molecular basis underlying the biological and clinical behavior of this cancer. Here, using multi-omics data, we demonstrate the existence of two retinoblastoma subtypes. Subtype 1, of earlier onset, includes most of the heritable forms. It harbors few genetic alterations other than the initiating RB1 inactivation and corresponds to differentiated tumors expressing mature cone markers. By contrast, subtype 2 tumors harbor frequent recurrent genetic alterations including MYCN-amplification. They express markers of less differentiated cone together with neuronal/ganglion cell markers with marked inter- and intra-tumor heterogeneity. The cone dedifferentiation in subtype 2 is associated with stemness features including low immune and interferon response, E2F and MYC/MYCN activation and a higher propensity for metastasis. The recognition of these two subtypes, one maintaining a cone-differentiated state, and the other, more aggressive, associated with cone dedifferentiation and expression of neuronal markers, opens up important biological and clinical perspectives for retinoblastomas.

TUMOSPEC: A Nation-Wide Study of Hereditary Breast and Ovarian Cancer Families with a Predicted Pathogenic Variant Identified through Multigene Panel Testing.

Assessment of age-dependent cancer risk for carriers of a predicted pathogenic variant (PPV) is often hampered by biases in data collection, with a frequent under-representation of cancer-free PPV carriers. TUMOSPEC was designed to estimate the cumulative risk of cancer for carriers of a PPV in a gene that is usually tested in a hereditary breast and ovarian cancer context. Index cases are enrolled consecutively among patients who undergo genetic testing as part of their care plan in France. First- and second-degree relatives and cousins of PPV carriers are invited to participate whether they are affected by cancer or not, and genotyped for the familial PPV. Clinical, family and epidemiological data are collected, and all data including sequencing data are centralized at the coordinating centre. The three-year feasibility study included 4431 prospective index cases, with 19.1% of them carrying a PPV. When invited by the coordinating centre, 65.3% of the relatives of index cases (5.7 relatives per family, on average) accepted the invitation to participate. The study logistics were well adapted to clinical and laboratory constraints, and collaboration between partners (clinicians, biologists, coordinating centre and participants) was smooth. Hence, TUMOSPEC is being pursued, with the aim of optimizing clinical management guidelines specific to each gene.

Molecular diagnosis of retinoblastoma by circulating tumor DNA analysis.

PURPOSE: The analysis of circulating tumor DNA (ctDNA), a fraction of total cell-free DNA (cfDNA), might be of special interest in retinoblastoma patients. Because the accessibility to tumor tissue is very limited in these patients, either for histopathological diagnosis of suspicious intraocular masses (biopsies are proscribed) or for somatic RB1 studies and genetic counseling (due to current successful conservative approaches), we aim to validate the detection of ctDNA in plasma of non-hereditary retinoblastoma patients by molecular analysis of RB1 gene. EXPERIMENTAL DESIGN: In a cohort of 19 intraocular unilateral non-hereditary retinoblastoma patients for whom a plasma sample was available at diagnosis, we performed high-deep next-generation sequencing (NGS) of RB1 in cfDNA. Two different bioinformatics/statistics approaches were applied depending on whether the somatic RB1 status was available or not. RESULTS: Median plasma sample volume was 600 µL [100-1000]; median cfDNA plasma concentration was 119 [38-1980] and 27 [11-653] ng/mL at diagnosis and after complete remission, respectively. In the subgroup of patients with known somatic RB1 alterations (n = 11), seven of nine somatic mutations were detected (median allele fraction: 6.7%). In patients without identified somatic RB1 alterations (n = 8), six candidate variants were identified for seven patients. CONCLUSIONS: Despite small tumor size, blood-ocular barrier, poor ctDNA blood release and limited plasma sample volumes, we confirm that it is possible to detect ctDNA with high-deep NGS in plasma from patients with intraocular non-hereditary retinoblastoma. This may aid in diagnosis of suspicious cases, family genetic counseling or follow-up of residual intraocular disease.

5' Region Large Genomic Rearrangements in the BRCA1 Gene in French Families: Identification of a Tandem Triplication and Nine Distinct Deletions with Five Recurrent Breakpoints.

BACKGROUND: Large genomic rearrangements (LGR) in BRCA1 consisting of deletions/duplications of one or several exons have been found throughout the gene with a large proportion occurring in the 5' region from the promoter to exon 2. The aim of this study was to better characterize those LGR in French high-risk breast/ovarian cancer families. METHODS: DNA from 20 families with one apparent duplication and nine deletions was analyzed with a dedicated comparative genomic hybridization (CGH) array, high-resolution BRCA1 Genomic Morse Codes analysis and Sanger sequencing. RESULTS: The apparent duplication was in fact a tandem triplication of exons 1 and 2 and part of intron 2 of BRCA1, fully characterized here for the first time. We calculated a causality score with the multifactorial model from data obtained from six families, classifying this variant as benign. Among the nine deletions detected in this region, eight have never been identified. The breakpoints fell in six recurrent regions and could confirm some specific conformation of the chromatin. CONCLUSIONS: Taken together, our results firmly establish that the BRCA1 5' region is a frequent site of different LGRs and highlight the importance of the segmental duplication and Alu sequences, particularly the very high homologous region, in the mechanism of a recombination event. This also confirmed that those events are not systematically deleterious.

Further delineation of the NTHL1 associated syndrome: A report from the French Oncogenetic Consortium.

Biallelic pathogenic variants in the NTHL1 (Nth like DNA glycosylase 1) gene cause a recently identified autosomal recessive hereditary cancer syndrome predisposing to adenomatous polyposis and colorectal cancer. Half of biallelic carriers also display multiple colonic or extra-colonic primary tumors, mainly breast, endometrium, urothelium, and brain tumors. Published data designate NTHL1 as an important contributor to hereditary cancers but also underline the scarcity of available informations. Thanks to the French oncogenetic consortium (Groupe Génétique et Cancer), we collected NTHL1 variants from 7765 patients attending for hereditary colorectal cancer or polyposis (n = 3936) or other hereditary cancers (n = 3829). Here, we describe 10 patients with pathogenic biallelic NTHL1 germline variants, that is, the second largest NTHL1 series. All carriers were from the "colorectal cancer or polyposis" series. All nine biallelic carriers who underwent colonoscopy presented adenomatous polyps. For digestive cancers, average age at diagnosis was 56.2 and we reported colorectal, duodenal, caecal, and pancreatic cancers. Extra-digestive malignancies included sarcoma, basal cell carcinoma, breast cancer, urothelial carcinoma, and melanoma. Although tumor risks remain to be precisely defined, these novel data support NTHL1 inclusion in diagnostic panel testing. Colonic surveillance should be conducted based on MUTYH recommendations while extra-colonic surveillance has to be defined.

Assessment of Multiplex Digital Droplet RT-PCR as a Diagnostic Tool for SARS-CoV-2 Detection in Nasopharyngeal Swabs and Saliva Samples.

BACKGROUND: Reverse transcription-quantitative PCR on nasopharyngeal swabs is currently the reference COVID-19 diagnosis method but exhibits imperfect sensitivity. METHODS: We developed a multiplex reverse transcription-digital droplet PCR (RT-ddPCR) assay, targeting 6 SARS-CoV-2 genomic regions, and evaluated it on nasopharyngeal swabs and saliva samples collected from 130 COVID-19 positive or negative ambulatory individuals, who presented symptoms suggestive of mild or moderate SARS-CoV2 infection. RESULTS: For the nasopharyngeal swab samples, the results obtained using the 6-plex RT-ddPCR and RT-qPCR assays were all concordant. The 6-plex RT-ddPCR assay was more sensitive than RT-qPCR (85% versus 62%) on saliva samples from patients with positive nasopharyngeal swabs. CONCLUSION: Multiplex RT-ddPCR represents an alternative and complementary tool for the diagnosis of COVID-19, in particular to control RT-qPCR ambiguous results. It can also be applied to saliva for repetitive sampling and testing individuals for whom nasopharyngeal swabbing is not possible.

Calibration of Pathogenicity Due to Variant-Induced Leaky Splicing Defects by Using BRCA2 Exon 3 as a Model System.

BRCA2 is a clinically actionable gene implicated in breast and ovarian cancer predisposition that has become a high priority target for improving the classification of variants of unknown significance (VUS). Among all BRCA2 VUS, those causing partial/leaky splicing defects are the most challenging to classify because the minimal level of full-length (FL) transcripts required for normal function remains to be established. Here, we explored BRCA2 exon 3 (BRCA2e3) as a model for calibrating variant-induced spliceogenicity and estimating thresholds for BRCA2 haploinsufficiency. In silico predictions, minigene splicing assays, patients' RNA analyses, a mouse embryonic stem cell (mESC) complementation assay and retrieval of patient-related information were combined to determine the minimal requirement of FL BRCA2 transcripts. Of 100 BRCA2e3 variants tested in the minigene assay, 64 were found to be spliceogenic, causing mild to severe RNA defects. Splicing defects were also confirmed in patients' RNA when available. Analysis of a neutral leaky variant (c.231T>G) showed that a reduction of approximately 60% of FL BRCA2 transcripts from a mutant allele does not cause any increase in cancer risk. Moreover, data obtained from mESCs suggest that variants causing a decline in FL BRCA2 with approximately 30% of wild-type are not pathogenic, given that mESCs are fully viable and resistant to DNA-damaging agents in those conditions. In contrast, mESCs producing lower relative amounts of FL BRCA2 exhibited either null or hypomorphic phenotypes. Overall, our findings are likely to have broader implications on the interpretation of BRCA2 variants affecting the splicing pattern of other essential exons. SIGNIFICANCE: These findings demonstrate that BRCA2 tumor suppressor function tolerates substantial reduction in full-length transcripts, helping to determine the pathogenicity of BRCA2 leaky splicing variants, some of which may not increase cancer risk.

Association Between Genotype and Phenotype in Consecutive Unrelated Individuals With Retinoblastoma.

Importance: Retinoblastoma (RB) is the most common pediatric intraocular neoplasm. RB is a complex model in which atypical pathogenic variants, modifier genes, imprinting, and mosaicism are known to be associated with the phenotype. In-depth understanding of RB therefore requires large genotype-phenotype studies. Objective: To assess the association between genotype and phenotype in patients with RB. Design, Setting, and Participants: This single-center, retrospective cohort study, conducted from January 1, 2000, to September 30, 2017, enrolled 1404 consecutive ascertained patients with RB who consulted an oncogeneticist. All patients had their genotype and phenotype recorded. Statistical analysis was performed from July 1, 2018, to December 31, 2018. Main Outcomes and Measures: RB1 germline and somatic pathogenic variant types, family history, and disease presentation characteristics (ie, age at diagnosis, sex, laterality, and International Intraocular Retinoblastoma Classification group). Results: Among 1404 patients with RB (734 [52.3%] female; mean [SD] age, 20.2 [21.2] months), 866 cases (61.7%) were unilateral and 538 cases (38.3%) were bilateral. Loss of function variants were found throughout the coding sequence, with 259 of 272 (95.2%) somatic pathogenic variants and 537 of 606 (88.6%) germline pathogenic variants (difference, 6.6%; 95% CI, 4.0%-9.2%; P < .001) after excluding tumor-specific pathogenic variants (ie, promoter methylation and loss of heterozygosity); a novel low-penetrance region was identified in exon 24. Compared with germline pathogenic variants estimated to retain RB protein expression, germline pathogenic variants estimated to abrogate RB protein expression were associated with an earlier mean (SD) age at diagnosis (12.3 [11.3] months among 457 patients vs 16.3 [13.2] months among 55 patients; difference, 4 months; 95% CI, 1.9-6.1 months; P = .01), more frequent bilateral involvement (84.2% among 452 patients vs 65.2% among 45 patients; difference, 18.9%; 95% CI, 14.5%-23.3%; P < .001), and more advanced International Intraocular Retinoblastoma Classification group (85.3% among 339 patients vs 73.9% among 34 patients; difference: 11.4%; 95% CI, 6.5%-16.3%; P = .047). Among the 765 nongermline carriers of an RB1 pathogenic variant, most were female (419 females [54.8%] vs 346 males [45.2%]; P = .008), and males were more likely to have bilateral RB (23 males [71.4%] vs 12 females [34.3%]; P = .01). Conclusions and Relevance: These results suggest that RB risk is associated with the germline pathogenic variant and with maintenance of RB protein and that there is a sex-linked mechanism for nongermline carriers.

Lack of evidence for CDK12 as an ovarian cancer predisposing gene.

CDK12 variants were investigated as a genetic susceptibility to ovarian cancer in a series of 416 unrelated and consecutive patients with ovarian carcinoma and who carry neither germline BRCA1 nor BRCA2 pathogenic variant. The presence of CDK12 variants was searched in germline DNA by massive parallel sequencing on pooled DNAs. The lack of detection of deleterious variants and the observed proportion of missense variants in the series of ovarian carcinoma patients as compared with all human populations strongly suggests that CDK12 is not an ovarian cancer predisposing gene.

Skipping Nonsense to Maintain Function: The Paradigm of BRCA2 Exon 12.

Germline nonsense and canonical splice site variants identified in disease-causing genes are generally considered as loss-of-function (LoF) alleles and classified as pathogenic. However, a fraction of such variants could maintain function through their impact on RNA splicing. To test this hypothesis, we used the alternatively spliced BRCA2 exon 12 (E12) as a model system because its in-frame skipping leads to a potentially functional protein. All E12 variants corresponding to putative LoF variants or predicted to alter splicing (n = 40) were selected from human variation databases and characterized for their impact on splicing in minigene assays and, when available, in patient lymphoblastoid cell lines. Moreover, a selection of variants was analyzed in a mouse embryonic stem cell-based functional assay. Using these complementary approaches, we demonstrate that a subset of variants, including nonsense variants, induced in-frame E12 skipping through the modification of splice sites or regulatory elements and, consequently, led to an internally deleted but partially functional protein. These data provide evidence, for the first time in a cancer-predisposition gene, that certain presumed null variants can retain function due to their impact on splicing. Further studies are required to estimate cancer risk associated with these hypomorphic variants. More generally, our findings highlight the need to exercise caution in the interpretation of putative LoF variants susceptible to induce in-frame splicing modifications. SIGNIFICANCE: This study presents evidence that certain presumed loss-of-function variants in a cancer predisposition gene can retain function due to their direct impact on RNA splicing.

The Spectrum of FANCM Protein Truncating Variants in European Breast Cancer Cases.

Germline protein truncating variants (PTVs) in the FANCM gene have been associated with a 2-4-fold increased breast cancer risk in case-control studies conducted in different European populations. However, the distribution and the frequency of FANCM PTVs in Europe have never been investigated. In the present study, we collected the data of 114 European female breast cancer cases with FANCM PTVs ascertained in 20 centers from 13 European countries. We identified 27 different FANCM PTVs. The p.Gln1701* PTV is the most common PTV in Northern Europe with a maximum frequency in Finland and a lower relative frequency in Southern Europe. On the contrary, p.Arg1931* seems to be the most common PTV in Southern Europe. We also showed that p.Arg658*, the third most common PTV, is more frequent in Central Europe, and p.Gln498Thrfs*7 is probably a founder variant from Lithuania. Of the 23 rare or unique FANCM PTVs, 15 have not been previously reported. We provide here the initial spectrum of FANCM PTVs in European breast cancer cases.