Five rare variants in BRIP1/FANCJ, initially identified in ovarian cancer (OC) or breast cancer (BC) cases by the adult hereditary cancer clinics, were investigated for their candidacy as clinically relevant variants. These variants were investigated genetically in a population exhibiting genetic drift and molecularly assayed for biological impact. Using in silico tools, population-based genetic databases and other resources, three of the five reported BRIP1 variants were likely to be damaging: c.797C>T; p.Thr266Met, c.2087C>T; p.Pro696Leu and c.2990_2993delCAAA; p.Thr997ArgfsTer61. The carrier frequencies ranged from 0 to 0.7% in ancestry-defined cancer groups comprising 47 OC families, 49 hereditary breast and ovarian cancer syndrome families, 142 hereditary breast cancer syndrome families, 435 sporadic OC cases and 563 sporadic BC cases and 0-0.2% in 1025 population-matched controls. Multiple carriers of the these variants were identified in additional population-matched cancer cases. Of the five reported BRIP1 variants, p.Thr266Met, p.Pro696Leu and c.2990_2993delCAAA; p.Thr997ArgfsTer61, which were predicted to be damaging, conferred cellular sensitivity to mitomycin C and cisplatin unlike p.Ser139Ala and p.Ala406Ser. Collectively, our investigation implicates BRIP1 c.797C>T; p.Thr266Met, c.2087C>T; p.Pro696Leu and c.2990_2993delCAAA; p.Thr997ArgfsTer61 as deleterious variants in OC and BC.

As gene-panels expand to include candidate breast cancer predisposing genes (CPGs) involved in diverse DNA repair pathways, we investigated an index breast cancer (BC) case from 56 BRCA1/BRCA2 implicated high-risk hereditary BC families using gene-based approach for co-occurring, rare germline variants predicted to be damaging and clinically relevant in 276 DNA repair genes (DRGs). Using whole exome sequencing analyses, we identified a total of 287 variants in 55% of DRGs, of which 24 loss-of-function and 36 other predicted damaging variants were identified in 72% and 76% of BRCA1 and BRCA2 implicated cases, respectively. We also identified 60 variants of clinical interest in various known CPGs, including those involved in risk to other cancers, in 72% of BRCA1 and 60% of BRCA2 cases. The number of variants predicted to be deleterious in diverse DRGs raises questions about the interpretability of findings as panel testing expands beyond clinically established breast CPGs.

Ovarian and endometrial cancers come within the top-4 for incident cancers as well as deaths in North American women. Cure rates have not improved in 30 years as high-grade subtypes continue to be diagnosed in Stage III/IV. Attempts at early diagnosis have failed because high-grade cancer cells exfoliate and metastasize while the primary cancer is small and undetectable by existing tests based on imaging and blood-based tumour markers. DOvEEgene (Detecting Ovarian and Endometrial cancers Early using genomics) is a genomic uterine pap test developed by a McGill team to screen and detect these cancers while they are confined to the gynecologic organs and curable by surgery. The test identifies pathogenic somatic mutations in uterine brush samples A high sensitivity error-reducing capture technology (DOvEEgene-SureSelectHS) utilizing duplex sequencing interrogates the exons of 23 genes involved in the development of sporadic and hereditary ovarian and endometrial cancers. We apply a combination of germline gene panel testing on saliva samples with deep duplex sequencing to detect somatic mutations at <0.1% VAF, interrogation of microsatellite loci for instability and low coverage WGS for copy number analysis of uterine brush samples. Currently, DOvEEgene is the only test that can discriminate ovarian and endometrial cancers in peri- and postmenopausal women from benign gynecologic diseases common in that age group. This is important because pathogenic somatic driver mutations are also associated with increasing age and benign disease. DOvEEgene incorporates a deep machine-learning derived classifier that can discriminate the mutational signature of these cancers from benign disease aiming for a sensitivity of 70% and a specificity of 100% in a population with high background mutational burden. Here we tested the Onso system, a highly accurate sequencing technology from PacBio in order to potentially increase sensitivity while driving down sequencing costs by reducing required sequencing depth vs the current NGS standard. We sequenced 15 duplex Illumina sequencing libraries produced using the DovEE assay at PE 100bp mode and compared Onso data in non- duplex sequencing mode as well as duplex sequencing mode to the original duplex sequencing method. Here, we present this comparison and highlight the benefits of high accuracy sequencing for the detection of very low frequency (<0.1%) somatic mutations. Citation Format: Jiannis Ragoussis, Nairi Pezeshkian, Lucy Gilbert. Improved detection of low frequency mutations in ovarian and endometrial cancers by utilizing a highly accurate sequencing platform [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 6522.

FANCI was recently identified as a new candidate ovarian cancer (OC)-predisposing gene from the genetic analysis of carriers of FANCI c.1813C>T; p.L605F in OC families. Here, we aimed to investigate the molecular genetic characteristics of FANCI, as they have not been described in the context of cancer. We first investigated the germline genetic landscape of two sisters with OC from the discovery FANCI c.1813C>T; p.L605F family (F1528) to re-affirm the plausibility of this candidate. As we did not find other conclusive candidates, we then performed a candidate gene approach to identify other candidate variants in genes involved in the FANCI protein interactome in OC families negative for pathogenic variants in BRCA1, BRCA2, BRIP1, RAD51C, RAD51D, and FANCI, which identified four candidate variants. We then investigated FANCI in high-grade serous ovarian carcinoma (HGSC) from FANCI c.1813C>T carriers and found evidence of loss of the wild-type allele in tumour DNA from some of these cases. The somatic genetic landscape of OC tumours from FANCI c.1813C>T carriers was investigated for mutations in selected genes, copy number alterations, and mutational signatures, which determined that the profiles of tumours from carriers were characteristic of features exhibited by HGSC cases. As other OC-predisposing genes such as BRCA1 and BRCA2 are known to increase the risk of other cancers including breast cancer, we investigated the carrier frequency of germline FANCI c.1813C>T in various cancer types and found overall more carriers among cancer cases compared to cancer-free controls (p = 0.007). In these different tumour types, we also identified a spectrum of somatic variants in FANCI that were not restricted to any specific region within the gene. Collectively, these findings expand on the characteristics described for OC cases carrying FANCI c.1813C>T; p.L605F and suggest the possible involvement of FANCI in other cancer types at the germline and/or somatic level.

Not all familial ovarian cancer (OC) cases are explained by pathogenic germline variants in known risk genes. A candidate gene approach involving DNA repair pathway genes was applied to identify rare recurring pathogenic variants in familial OC cases not associated with known OC risk genes from a population exhibiting genetic drift. Whole exome sequencing (WES) data of 15 OC cases from 13 families tested negative for pathogenic variants in known OC risk genes were investigated for candidate variants in 468 DNA repair pathway genes. Filtering and prioritization criteria were applied to WES data to select top candidates for further analyses. Candidates were genotyped in ancestry defined study groups of 214 familial and 998 sporadic OC or breast cancer (BC) cases and 1025 population-matched controls and screened for additional carriers in 605 population-matched OC cases. The candidate genes were also analyzed in WES data from 937 familial or sporadic OC cases of diverse ancestries. Top candidate variants in ERCC5, EXO1, FANCC, NEIL1 and NTHL1 were identified in 5/13 (39%) OC families. Collectively, candidate variants were identified in 7/435 (1.6%) sporadic OC cases and 1/566 (0.2%) sporadic BC cases versus 1/1025 (0.1%) controls. Additional carriers were identified in 6/605 (0.9%) OC cases. Tumour DNA from ERCC5, NEIL1 and NTHL1 variant carriers exhibited loss of the wild-type allele. Carriers of various candidate variants in these genes were identified in 31/937 (3.3%) OC cases of diverse ancestries versus 0-0.004% in cancer-free controls. The strategy of applying a candidate gene approach in a population exhibiting genetic drift identified new candidate OC predisposition variants in DNA repair pathway genes.

ABSTRACT Five rare variants in BRIP1/FANCJ , initially reported in ovarian (OC) or breast (BC) cancer cases by the adult hereditary cancer clinics, were investigated for their candidacy as clinically relevant variants. These variants were investigated genetically in a population exhibiting genetic drift and molecularly assayed for biological impact. Using in silico tools, population-based genetic databases and other resources, three of the five reported BRIP1 variants were likely to be damaging: c.797C>T; p.Thr266Met, c.2087C>T; p.Pro696Leu and c.2990_2993delCAAA; p.Thr997ArgfsTer61. The carrier frequencies ranged from 0-0.7% in ancestry defined cancer groups comprised of 47 OC families, 49 hereditary breast and ovarian cancer syndrome families, 142 hereditary breast cancer syndrome families, 435 sporadic OC cases and 563 sporadic BC cases and 0-0.2% in 1025 population-matched controls. Multiple carriers of the same variants were identified in additional population-matched cancer cases. Of the five reported BRIP1 variants, p.Thr266Met, p.Pro696Leu and p.Thr997ArgfsTer61, which were predicted to be damaging, conferred cellular sensitivity to mitomycin C and cisplatin unlike p.Ser139Ala and p.Ala406Ser. Collectively, our investigation implicates BRIP1 c.797C>T; p.Thr266Met, c.2087C>T; p.Pro696Leu and p.Thr997ArgfsTer61 as deleterious variants in OC and BC.

Five rare variants in BRIP1/FANCJ , initially reported in ovarian (OC) or breast (BC) cancer cases by the adult hereditary cancer clinics, were investigated for their candidacy as clinically relevant variants. These variants were investigated genetically in a population exhibiting genetic drift and molecularly assayed for biological impact. Using in silico tools, population-based genetic databases and other resources, three of the five reported BRIP1 variants were likely to be damaging: c.797C>T; p.Thr266Met, c.2087C>T; p.Pro696Leu and c.2990_2993delCAAA; p.Thr997ArgfsTer61. The carrier frequencies ranged from 0-0.7% in ancestry defined cancer groups comprised of 47 OC families, 49 hereditary breast and ovarian cancer syndrome families, 142 hereditary breast cancer syndrome families, 435 sporadic OC cases and 563 sporadic BC cases and 0-0.2% in 1025 population-matched controls. Multiple carriers of the same variants were identified in additional population-matched cancer cases. Of the five reported BRIP1 variants, p.Thr266Met, p.Pro696Leu and p.Thr997ArgfsTer61, which were predicted to be damaging, conferred cellular sensitivity to mitomycin C and cisplatin unlike p.Ser139Ala and p.Ala406Ser. Collectively, our investigation implicates BRIP1 c.797C>T; p.Thr266Met, c.2087C>T; p.Pro696Leu and p.Thr997ArgfsTer61 as deleterious variants in OC and BC.### Competing Interest StatementThe authors have declared no competing interest.### Funding StatementThe work was supported in part by the Department of Medicine, McGill University to P.N.T.; Saudi Arabian Cultural Bureau to P.N.T. and W.M.A.; The Canadian Institute for Health Research (CIHR) operating grants (PCC-156736 to P.N.T., C.M.T.G. and J.R.) and (PJT-156124 to P.N.T. and J.R.); Cancer Research Society and Ovarian Cancer Canada partnership grant (21123 to P.N.T.); Department of Medicine, McGill University Grant to P.N.T.; the Fond de la recherche du Quebec en Sante (FRQS) and Quebec Breast Cancer Foundation network grants to P.N.T.; Compute Canada resource allocation project wst-164 and Genome Canada Genome Technology Platform award to J.R.; CIHR foundation grant to J.Y.M (DN-388879); Gen3G has been supported over the time by FRQS grant (20697 to M.F.H.); CIHR grant (MOP-115071 to M.F.H.) and (PJT-152989 to L.B.); American Diabetes Association (ADA) accelerator award (1-15-ACE-26 to M.F.H.). LB is a senior research scholar from FRQS. The sequencing of the Gen3G offspring has been sponsored by Fonds de recherche du Quebec, McGill University and Universite de Sherbrooke. Ovarian tumour banking was supported by the Banque de tissus et de donnees of the Reseau de recherche sur le cancer of the FRQS affiliated with the Canadian Tumour Repository Network (CTRNet). The RI-MUHC and CRCHUM receive support from the FRQS. J.-Y.M. is a Tier I Canada Research Chair in DNA repair and cancer therapeutics. W.M.A. was supported by Taibah University Scholarship Award and C.T.F. was supported in part by RI-MUHC Scholarship Award and James O and Maria Meadows Award.### Author DeclarationsI confirm all relevant ethical guidelines have been followed, and any necessary IRB and/or ethics committee approvals have been obtained.YesThe details of the IRB/oversight body that provided approval or exemption for the research described are given below:This project was conducted with approval and in accordance with the guidelines of The McGill University Health Centre Research Ethics Board (MP-37-2019-4783).I confirm that all necessary patient/participant consent has been obtained and the appropriate institutional forms have been archived, and that any patient/participant/sample identifiers included were not known to anyone (e.g., hospital staff, patients or participants themselves) outside the research group so cannot be used to identify individuals.YesI understand that all clinical trials and any other prospective interventional studies must be registered with an ICMJE-approved registry, such as ClinicalTrials.gov. I confirm that any such study reported in the manuscript has been registered and the trial registration ID is provided (note: if posting a prospective study registered retrospectively, please provide a statement in the trial ID field explaining why the study was not registered in advance).YesI have followed all appropriate research reporting guidelines, such as any relevant EQUATOR Network research reporting checklist(s) and other pertinent material, if applicable.YesAll data produced in the present study are available upon reasonable request to the authors

Background: Detecting pathogenic intronic variants resulting in aberrant splicing remains a challenge in routine genetic testing. We describe germline whole-exome sequencing (WES) analyses and apply in silico predictive tools of familial ovarian cancer (OC) cases reported clinically negative for pathogenic BRCA1 and BRCA2 variants. Methods: WES data from 27 familial OC cases reported clinically negative for pathogenic BRCA1 and BRCA2 variants and 53 sporadic early-onset OC cases were analyzed for pathogenic variants in BRCA1 or BRCA2. WES data from carriers of pathogenic BRCA1 or BRCA2 variants were analyzed for pathogenic variants in 10 other OC predisposing genes. Loss of heterozygosity analysis was performed on tumor DNA from variant carriers. Results: BRCA1 c.5407-25T>A intronic variant, identified in two affected sisters and one sporadic OC case, is predicted to create a new splice effecting transcription of BRCA1. WES data from BRCA1 c.5407-25T>A carriers showed no evidence of pathogenic variants in other OC predisposing genes. Sequencing the tumor DNA from the variant carrier showed complete loss of the wild-type allele. Conclusions: The findings support BRCA1 c.5407-25T>A as a likely pathogenic variant and highlight the importance of investigating intronic sequences as causal variants in OC families where the involvement of BRCA1 is highly suggestive.

To identify candidate variants in RAD51C and RAD51D ovarian cancer (OC) predisposing genes by investigating French Canadians (FC) exhibiting unique genetic architecture. Candidates were identified by whole exome sequencing analysis of 17 OC families and 53 early-onset OC cases. Carrier frequencies were determined by the genetic analysis of 100 OC or HBOC families, 438 sporadic OC cases and 1025 controls. Variants of unknown function were assayed for their biological impact and/or cellular sensitivity to olaparib. RAD51C c.414G>C;p.Leu138Phe and c.705G>T;p.Lys235Asn and RAD51D c.137C>G;p.Ser46Cys, c.620C>T;p.Ser207Leu and c.694C>T;p.Arg232Ter were identified in 17.6% of families and 11.3% of early-onset cases. The highest carrier frequency was observed in OC families (1/44, 2.3%) and sporadic cases (15/438, 3.4%) harbouring RAD51D c.620C>T versus controls (1/1025, 0.1%). Carriers of c.620C>T (n = 7), c.705G>T (n = 2) and c.137C>G (n = 1) were identified in another 538 FC OC cases. RAD51C c.705G>T affected splicing by skipping exon four, while RAD51D p.Ser46Cys affected protein stability and conferred olaparib sensitivity. Genetic and functional assays implicate RAD51C c.705G>T and RAD51D c.137C>G as likely pathogenic variants in OC. The high carrier frequency of RAD51D c.620C>T in FC OC cases validates previous findings. Our findings further support the role of RAD51C and RAD51D in hereditary OC.

Abstract The potentially pathogenic variant (PPV), FANCI c.1813C>T; p.L605F, in a new candidate ovarian cancer (OC) predisposing gene was discovered by whole exome sequencing (WES) of familial OC cases from the founder French Canadian (FC) population for discovering new OC predisposing genes. Modeling this variant in cellulo suggested this variant encodes an unstable protein. FANCI is an essential member upstream of the homologous recombination DNA repair pathway and intersects BRCA1 and BRCA2 function, proteins encoded by genes involved in hereditary OC. Investigating the FC founder population facilitates the discovery of PPVs as they are more likely to harbor recurrent variants due to common ancestors, increasing the likelihood of identifying candidate genes in cancer families. To further support the candidacy of FANCI as a new OC predisposing gene, we investigated the germline landscape of c.1813C>T carrier FC OC cases for co-occurring PPVs in known or proposed new (emerging) OC predisposing genes or other genes involved in similar pathways (DNA repair). Using WES of peripheral blood lymphocyte DNA, the genomic landscape of 10 FANCI carriers were investigated for heterozygous PPVs in 276 DNA repair pathways genes, which included known (BRCA1, BRCA2, MSH2, MLH1, MSH6, PMS2) and emerging (BRIP1, RAD51C, RAD51D) OC predisposing genes. Top ranking candidate variants (minor allele frequency <1%) were identified using 11 different in silico tools that assessed amino acid conversation or potential pathogenicity. Pathogenicity of known and emerging OC predisposing genes was assessed using BRCAExchange (www.brcaexchange.org) and ClinVar (www.ncbi.nlm.nih.gov/clinvar/). A similar analysis was done with WES data from 13 FC OC cases harboring pathogenic BRCA1/BRCA2 variants. We identified 31 variants in 27 genes in FANCI c.1813C>T carriers. A previously known carrier of a pathogenic BRCA1 variant (c.2836_2837del; p.Ile946GlnfsTer5) was also identified in a familial case. No carriers of other pathogenic variants in known or emerging OC predisposing genes were found. However, FANCI carriers were found to carry at least one other PPV in a DNA repair pathway gene (range 2-9; average=4.1). There were no other carriers of variants in common among all OC cases, though at least 2 cases carried a variant in the same gene. FC OC cases harboring BRCA1/BRCA2 variants carried at least one other PPV in a DNA repair pathway gene (range 2-7; average 4). It is possible that the identified variants influence or modify risk in conjunction with FANCI, though no PPV was identified in all carriers. As new cancer predisposing genes are identified it will become increasingly important to characterize the genetic context in which variants are identified. This will allow for further insight to clinical translatability once penetrance has been established. Citation Format: Caitlin Fierheller, Wejdan M Alenezi, Corinne Serruya, Timothée Revil, Javad Nadaf, Anne-Marie Mes-Masson, Diane Provencher, William D Foulkes, Zaki El Haffaf, Celia M T Greenwood, Jean-Yves Masson, Jiannis Ragoussis, Patricia N Tonin. The genomic landscape of carriers of rare variants in FANCI, a new candidate ovarian cancer predisposing gene [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2056.

Background Familial ovarian cancer (OC) cases not harbouring pathogenic variants in either of the BRCA1 and BRCA2 OC-predisposing genes, which function in homologous recombination (HR) of DNA, could involve pathogenic variants in other DNA repair pathway genes. Methods Whole exome sequencing was used to identify rare variants in HR genes in a BRCA1 and BRCA2 pathogenic variant negative OC family of French Canadian (FC) ancestry, a population exhibiting genetic drift. OC cases and cancer-free individuals from FC and non-FC populations were investigated for carrier frequency of FANCI c.1813C>T; p.L605F, the top-ranking candidate. Gene and protein expression were investigated in cancer cell lines and tissue microarrays, respectively. Results In FC subjects, c.1813C>T was more common in familial (7.1%, 3/42) than sporadic (1.6%, 7/439) OC cases ( P = 0.048). Carriers were detected in 2.5% (74/2950) of cancer-free females though female/male carriers were more likely to have a first-degree relative with OC (121/5249, 2.3%; Spearman correlation = 0.037; P = 0.011), suggesting a role in risk. Many of the cancer-free females had host factors known to reduce risk to OC which could influence cancer risk in this population. There was an increased carrier frequency of FANCI c.1813C>T in BRCA1 and BRCA2 pathogenic variant negative OC families, when including the discovery family, compared to cancer-free females (3/23, 13%; OR = 5.8; 95%CI = 1.7–19; P = 0.005). In non-FC subjects, 10 candidate FANCI variants were identified in 4.1% (21/516) of Australian OC cases negative for pathogenic variants in BRCA1 and BRCA2 , including 10 carriers of FANCI c.1813C>T. Candidate variants were significantly more common in familial OC than in sporadic OC ( P = 0.04). Localization of FANCD2, part of the FANCI-FANCD2 (ID2) binding complex in the Fanconi anaemia (FA) pathway, to sites of induced DNA damage was severely impeded in cells expressing the p.L605F isoform. This isoform was expressed at a reduced level, destabilized by DNA damaging agent treatment in both HeLa and OC cell lines, and exhibited sensitivity to cisplatin but not to a poly (ADP-ribose) polymerase inhibitor. By tissue microarray analyses, FANCI protein was consistently expressed in fallopian tube epithelial cells and only expressed at low-to-moderate levels in 88% (83/94) of OC samples. Conclusions This is the first study to describe candidate OC variants in FANCI , a member of the ID2 complex of the FA DNA repair pathway. Our data suggest that pathogenic FANCI variants may modify OC risk in cancer families.

AbstractSome familial ovarian cancer (OC) could be due to rare risk alleles in genes that each account for a relatively small proportion of cases not due toBRCA1andBRCA2, major risk genes in the homologous recombination (HR) DNA repair pathway. We report a new candidate OC risk allele,FANCIc.1813C>T in a Fanconi anemia (FA) gene that plays a role upstream of the HR DNA repair pathway. This variant was identified by whole exome sequencing of aBRCA1andBRCA2mutation-negative French Canadian (FC) OC family from a population exhibiting founder effects. In FCs, the c.1813C>T allele was detected in 7% (3/43) of familial and 1.6% (7/439) of sporadic OC cases; and in 3.7% (3/82) of familial breast cancer (BC) cases with a family history of OC and in 1.9% (3/158) of BC only families. This allele was significantly associated with FCBRCA1andBRCA2mutation-negative OC families (OR=5.6; 95%CI=1.6-19; p=0.006). AlthoughFANCIc.1813C>T was detected in 2.5% (74/2950) of cancer-free FC females, carriers had a personal history of known OC risk reducing factors, and female/male carriers were more likely to have reported a first-degree relative with OC (ρ=0.037; p=0.011). Eight rare potentially pathogenicFANCIvariants were identified in 3.3% (17/516) of Australian OC cases, including 10 carriers ofFANCIc.1813C>T. Potentially pathogenicFANCIvariants were significantly more common in AUS OC cases with a family history of OC than in isolated OC cases (p=0.027). The odds ratios (OR) were >3 for carriers of any of the seven rarestFANCIalleles, and 1.5 for c.1813C>T. Data from the OC Association Consortium revealed that the ORs for the c.1813C>T allele were highest for the most common OC subtypes. Localization of FANCD2, part of the FANCI-FANCD2 (ID2) binding complex in the FA pathway, to sites of induced DNA damage was severely impeded in cells expressing the p.L605F isoform. This isoform was expressed at a reduced level; unstable by formaldehyde or mitomycin C treatment; and exhibited sensitivity to cisplatin but not to olaparib (a poly [ADP-ribose] polymerase inhibitor). By tissue microarray analyses, FANCI protein was robustly expressed in fallopian tube epithelial cells but expressed at low-to-moderate levels in 88% (83/94) of high-grade serous carcinoma OC samples. This is the first study to describe potentially pathogenic variants in OC in a member of the ID2 complex of the FA DNA repair pathway. Our data suggest that potentially pathogenicFANCIvariants may modify OC risk in cancer families.

This detailed genetic and molecular analysis of a three-generation family reveals a new form of autosomal dominant tumor susceptibility syndrome entitled familial multinodular goiter (MNG) with schwannomatosis. Biallelic alterations of the DGCR8 gene were found in all affected patients: First, all patients harbored the same heterozygous germline mutation p.E518K. Secondly, all thyroid nodules and all schwannomas analyzed from the patients showed somatic loss of the whole chromosome 22 in addition to the p.E518K germline mutation, resulting in loss of heterozygosity. DGCR8 is key element of the canonical microRNA production pathway by forming a trimeric nuclear complex called microprocessor. The biallelic alteration of DGCR8 at the tumor tissue levels caused disrupted microprocessor function, resulting in reduced precursor and mature microRNA expression due to disrupted cleavage of precursor microRNA from primary RNA.

The current study aimed to identify new breast and/or ovarian cancer predisposition genes. For that, whole-exome sequencing (WES) was performed in the germline DNA of 52 non-BRCA1/BRCA2/TP53 mutation carrier women at high-risk for hereditary breast and ovarian cancer (HBOC). All variants were classified using information from population and disease specific databases, in silico prediction tools and the American College of Medical Genetics and Genomics (ACMG) criteria. Loss of heterozygosity (LOH) of tumor samples and segregation analyses were performed whenever possible. The variants identified were investigated in a second, independent cohort of 17 BC cases. Pathogenic/Likely Pathogenic variants were identified in known cancer genes such as CHEK2, MUTYH, PMS2, and RAD51C. Rare and potentially pathogenic variants were identified in DNA repair genes (FAN1, POLQ, and RAD54L) and other cancer-related genes such as DROSHA and SLC34A2. Interestingly, the variant c.149T>G in the FAN1 gene was identified in two unrelated families, and exhibited LOH in the tumor tissue of one of them. In conclusion, this is the largest Brazilian WES study involving families at high-risk for HBOC which has brought novel insights into the role of potentially new genetic risk factors for hereditary breast and ovarian cancer.

We report changes in the genomic landscape in the development of head and neck squamous cell carcinomas HNSCC from potentially premalignant lesions (PPOLS) to malignancy and lymph node metastases. Likely pathological mutations predominantly involved a relatively small set of genes reported previously (TP53, KMT2D, CDKN2A, PIK3CA, NOTCH1 and FAT1) but also other predicted cancer drivers (MGA, PABPC3, NR4A2, NCOR1 and MACF1). Notably, all these mutations arise early and are present in PPOLs. The most frequent genetic changes, which follow acquisition of immortality and loss of senescence, are of consistent somatic copy number alterations (SCNAs) involving chromosomal regions enriched for genes in known and previously unreported cancer-related pathways. We mapped the evolution of SCNAs in HNSCC progression. One of the earliest SCNAs involved deletions of CSMD1 (8p23.2). CSMD1 deletions or promoter hypermethylation were present in all of the immortal PPOLs and occurred at high frequency in the immortal HNSCC cell lines. Modulation of CSMD1 in cell lines revealed significant suppression of proliferation and invasion by forced expression, and significant stimulation of invasion by knockdown of expression. Known cancer drivers NOTCH1, PPP6C, RAC1, EIF4G1, PIK3CA showed significant increase in frequency of SCNA in transition from PPOLs to HNSCC that correlated with their expression. In the later stages of progression, HNSCC with and without nodal metastases showed some clear differences including high copy number gains of CCND1, hsa-miR-548k and TP63 in the metastases group.

BACKGROUND. DICER1 is the only miRNA biogenesis component associated with an inherited tumor syndrome, featuring multinodular goiter (MNG) and rare pediatric-onset lesions. Other susceptibility genes for familial forms of MNG likely exist. METHODS. Whole-exome sequencing of a kindred with early-onset MNG and schwannomatosis was followed by investigation of germline pathogenic variants that fully segregated with the disease. Genome-wide analyses were performed on 13 tissue samples from familial and nonfamilial DGCR8-E518K-positive tumors, including MNG, schwannomas, papillary thyroid cancers (PTCs), and Wilms tumors. miRNA profiles of 4 tissue types were compared, and sequencing of miRNA, pre-miRNA, and mRNA was performed in a subset of 9 schwannomas, 4 of which harbor DGCR8-E518K. RESULTS. We identified c.1552G>A;p.E518K in DGCR8, a microprocessor component located in 22q, in the kindred. The variant identified is a somatic hotspot in Wilms tumors and has been identified in 2 PTCs. Copy number loss of chromosome 22q, leading to loss of heterozygosity at the DGCR8 locus, was found in all 13 samples harboring c.1552G>A;p. E518K. miRNA profiling of PTCs, MNG, schwannomas, and Wilms tumors revealed a common profile among E518K hemizygous tumors. In vitro cleavage demonstrated improper processing of pre-miRNA by DGCR8-E518K. MicroRNA and RNA profiling show that this variant disrupts precursor microRNA production, impacting populations of canonical microRNAs and mirtrons. CONCLUSION. We identified DGCR8 as the cause of an unreported autosomal dominant mendelian tumor susceptibility syndrome: familial multinodular goiter with schwannomatosis.

the PPOL analyses was conined to metachronous lesions. A very recent study reported the status of SCNAs and common driver mutations in several keratinocyte cultures derived from metachronous normal and dysplastic lesions characterised for replicative lifespan 20 . Here we extend their indings by a combination of exome/targeted sequencing, methylation and SNP/CGH array analyses, using our unique panel of fully characterised mortal cultures and immortal cell lines derived from both PPOLS and HNSCC, to show that these genetic alterations are mostly associated with cellular immortalisation and increase with the stage of tumour progression in this class of SCC keratinocyte. he principal focus of this study is to characterise in detail changes in the evolution and progression of HNSCC rather than focussing on HNSCC as a group which have already been well characterised with respect to mutation and genomic landscapes. In doing so we identify common alterations of the candidate tumour suppressor gene CSMD1 not highlighted previously and provide preliminary evidence of its function. Furthermore, alterations of CSMD1 were common in PPOL cultures and therefore may be early events in SCC development. these SCNAs. Additionally, we show here that the earliest changes are oten characterised by focal SCNAs (for example, deletions at chromosome 8p23 involving CSMD1 ) with further selection for loss of whole arm or large region of arm during progression or evolution. his suggests that regions of SCNAs harbour multiple genes that collectively provide selective growth advantage. his is supported by demonstration of enrichment of genes in the cancer relevant pathways in regions of peak SCNAs identiied in HNSCC. Several of these genes have previously individually been associated with HNSCC but the present study is the irst to systematically identify these genes and others, in the context of cancer-relevant pathways and map them to cancer progression. In the present study, we conirm previous observations 17,24–26 , that loss-of-function of TP53 (primarily through sequence mutations) and CDKN2A (through SCNA, promoter hypermethylation and sequence mutations) are the earliest changes in HNSCC development. Furthermore, we demonstrate for the irst time that deleterious sequence mutations in previously identiied HNSCC cancer drivers KMT2D , PIK3CA , and NOTCH1 , are also present in PPOLs and represent early but less common changes. Our study also provides indication that other genes such as MGA , PABPC3 , NR4A2 , NCOR1 Q-CpG sotware. proportion alleles. sotware generated methylation values for each CpG site and also methylation percentage for all CpG sites analyzed.