Arleen D. Auerbach

Fanconi anaemia (FA), a model syndrome of genome instability, is caused by a deficiency in DNA interstrand crosslink repair resulting in chromosome breakage^1,2,3. The FA repair pathway protects against endogenous and exogenous carcinogenic aldehydes^4,5,6,7. Individuals with FA are hundreds to thousands fold more likely to develop head and neck (HNSCC), oesophageal and anogenital squamous cell carcinomas⁸ (SCCs). Molecular studies of SCCs from individuals with FA (FA SCCs) are limited, and it is unclear how FA SCCs relate to sporadic HNSCCs primarily driven by tobacco and alcohol exposure or infection with human papillomavirus⁹ (HPV). Here, by sequencing genomes and exomes of FA SCCs, we demonstrate that the primary genomic signature of FA repair deficiency is the presence of high numbers of structural variants. Structural variants are enriched for small deletions, unbalanced translocations and fold-back inversions, and are often connected, thereby forming complex rearrangements. They arise in the context of TP53 loss, but not in the context of HPV infection, and lead to somatic copy-number alterations of HNSCC driver genes. We further show that FA pathway deficiency may lead to epithelial-to-mesenchymal transition and enhanced keratinocyte-intrinsic inflammatory signalling, which would contribute to the aggressive nature of FA SCCs. We propose that the genomic instability in sporadic HPV-negative HNSCC may arise as a result of the FA repair pathway being overwhelmed by DNA interstrand crosslink damage caused by alcohol and tobacco-derived aldehydes, making FA SCC a powerful model to study tumorigenesis resulting from DNA-crosslinking damage.

Fanconi anemia (FA), a model syndrome of genome instability, is caused by a deficiency in DNA interstrand crosslink (ICL) repair resulting in chromosome breakage. The FA repair pathway comprises at least 22 FANC proteins including BRCA1 and BRCA2 and protects against carcinogenic endogenous and exogenous aldehydes. Individuals with FA are hundreds to thousands-fold more likely to develop head and neck (HNSCC), esophageal and anogenital squamous cell carcinomas (SCCs) with a median onset age of 31 years. The aggressive nature of these tumors and poor patient tolerance of platinum and radiation-based therapy have been associated with short survival in FA. Molecular studies of SCCs from individuals with FA (FA SCCs) have been limited, and it is unclear how they relate to sporadic HNSCCs primarily driven by tobacco and alcohol exposure or human papillomavirus (HPV) infection. Here, by sequencing FA SCCs, we demonstrate that the primary genomic signature of FA-deficiency is the presence of a high number of structural variants (SVs). SVs are enriched for small deletions, unbalanced translocations, and fold-back inversions that arise in the context of TP53 loss. The SV breakpoints preferentially localize to early replicating regions, common fragile sites, tandem repeats, and SINE elements. SVs are often connected forming complex rearrangements. Resultant genomic instability underlies elevated copy number alteration (CNA) rates of key HNSCC-associated genes, including PIK3CA, MYC, CSMD1, PTPRD, YAP1, MXD4, and EGFR. In contrast to sporadic HNSCC, we find no evidence of HPV infection in FA HNSCC, although positive cases were identified in gynecologic tumors. A murine allograft model of FA pathway-deficient SCC was enriched in SVs, exhibited dramatic tumor growth advantage, more rapid epithelial-to-mesenchymal transition, and enhanced autonomous inflammatory signaling when compared to an FA pathway-proficient model. In light of the protective role of the FA pathway against SV formation uncovered here, and recent findings of FA pathway insufficiency in the setting of increased formaldehyde load resulting in hematopoietic stem cell failure and carcinogenesis, we propose that high copy-number instability in sporadic HNSCC may result from functional overload of the FA pathway by endogenous and exogenous DNA crosslinking agents. Our work lays the foundation for improved FA patient treatment and demonstrates that FA SCC is a powerful model to study tumorigenesis resulting from DNA crosslinking damage. Citation Format: Andrew L. Webster, Mathijs A. Sanders, Krupa Patel, Ralf Dietrich, Raymond J. Noonan, Francis P. Lach, Ryan R. White, Audrey M. Goldfarb, Kevin Hadi, Matthew M. Edwards, Frank X. Donovan, Moonjung Jung, Sunandini Sridhar, Olivier Fedrigo, Huasong Tian, Joel Rosiene, Thomas Heineman, Jennifer Kennedy, Lorenzo Bean, Rasim O. Rosti, Rebecca Tryon, Ashlyn-Maree Gonzalez, Allana Rosenberg, Ji-Dung Luo, Thomas Carrol, Eunike Velleuer, Jeff C. Rastatter, Susanne I. Wells, Jordi Surrallés, Grover Bagby, Margaret L. MacMillan, John E. Wagner, Maria Cancio, Farid Boulad, Theresa Scognamiglio, Roger Vaughan, Amnon Koren, Marcin Imielinski, Settara Chandrasekharappa, Arleen D. Auerbach, Bhuvanesh Singh, David Kutler, Peter J. Campbell, Agata Smogorzewska. Fanconi anemia pathway deficiency drives copy number variation in squamous cell carcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 6196.

Fanconi anaemia (FA) is a genetic disorder due to mutations in any of the 22 FANC genes (FANCA–FANCW) and has high phenotypic variation. Siblings may have similar clinical outcome because they share the same variants; however, such association has not been reported. We present the detailed phenotype and clinical course of 25 sibling sets with FA from two institutions. Haematological progression significantly correlated between siblings, which was confirmed in an additional 55 sibling pairs from the International Fanconi Anemia Registry. Constitutional abnormalities were not concordant, except for a moderate degree of concordance in kidney abnormalities and microcephaly.

Summary: DNA interstrand crosslinks (ICLs) are a form of DNA damage that requires the interplay of a number of repair proteins including those of the Fanconi anemia (FA) and the homologous recombination (HR) pathways. Pathogenic variants in the essential gene BRCA2/FANCD1, when monoallelic, predispose to breast and ovarian cancer, and when biallelic, results in a severe subtype of Fanconi anemia. BRCA2 function in the FA pathway is attributed to its role as a mediator of the RAD51 recombinase in HR repair of the programmed DNA double strand breaks (DSB). BRCA2 and RAD51 functions are also required to protect stalled replication forks from nucleolytic degradation during response to hydroxyurea (HU). While RAD51 has been shown to be necessary in the early steps of ICL repair to prevent aberrant nuclease resection, the role of BRCA2 in this process has not been described. Here, based on the analysis of BRCA2 DNA binding domain (DBD) mutants discovered in FA patients presenting with atypical FA-like phenotypes, we establish that BRCA2 is necessary for protection of DNA at an ICL. Cells carrying DBD BRCA2 mutations are sensitive to ICL inducing agents but resistant to HU treatment consistent with relatively high HR repair in these cells. BRCA2 function at an ICL protects against DNA2-WRN nuclease-helicase complex and not the MRE11 nuclease implicated in the resection of HU-stalled replication forks. Our results also indicate that unlike the processing at HU-stalled forks, function of the SNF2 translocases (SMARCAL1, ZRANB3, or HLTF), implicated in fork reversal, are not an integral component of the ICL repair, pointing to a different mechanism of fork protection at different DNA lesions.

Fanconi anemia (FA) is a rare genetic disorder characterized by bone marrow failure, predisposition to cancer, and congenital abnormalities. FA is caused by pathogenic variants in any of 22 genes involved in the DNA repair pathway responsible for removing interstrand crosslinks. FANCL, an E3 ubiquitin ligase, is an integral component of the pathway, but patients affected by disease‐causing FANCL variants are rare, with only nine cases reported worldwide. We report here a FANCL founder variant, anticipated to be synonymous, c.1092G>A;p.K364=, but demonstrated to induce aberrant splicing, c.1021_1092del;p.W341_K364del, that accounts for the onset of FA in 13 cases from South Asia, 12 from India and one from Pakistan. We comprehensively illustrate the pathogenic nature of the variant, provide evidence for a founder effect, and propose including this variant in genetic screening of suspected FA patients in India and Pakistan, as well as those with ancestry from these regions of South Asia.

Fanconi anemia (FA) is the most common genetic cause of bone marrow failure, and is caused by inherited pathogenic variants in any of 22 genes. Of these, only FANCB is X-linked. We describe a cohort of 19 children with FANCB variants, from 16 families of the International Fanconi Anemia Registry (IFAR). Those with FANCB deletion or truncation demonstrate earlier than average onset of bone marrow failure, and more severe congenital abnormalities compared to a large series of FA individuals in the published reports. This reflects the indispensable role of FANCB protein in the enzymatic activation of FANCD2 monoubiquitination, an essential step in the repair of DNA interstrand crosslinks. For FANCB missense variants, more variable severity is associated with the extent of residual FANCD2 monoubiquitination activity. We used transcript analysis, genetic complementation, and biochemical reconstitution of FANCD2 monoubiquitination to determine the pathogenicity of each variant. Aberrant splicing and transcript destabilization was associated with two missence variants. Individuals carrying missense variants with drastically reduced FANCD2 monoubiquitination in biochemical and/or cell-based assays showed earlier onset of hematologic disease and shorter survival. Conversely, variants with near-normal FANCD2 monoubiquitination were associated with more favorable outcome. Our study reveals a genotype-phenotype correlation within the FA-B complementation group of FA, where severity is linked to the extent of residual FANCD2 monoubiquitination. KEY POINTS: X-linked FANCB pathogenic variants predominantly cause acute, early onset bone marrow failure and severe congenital abnormalities Biochemical and cell-based assays with patient variants reveal functional properties of FANCB that associate with clinical severity

Fanconi anemia (FA) is a genetically and phenotypically heterogeneous disorder characterized by congenital malformations, progressive bone marrow failure, and predisposition to cancer, particularly hematological malignancies and solid tumors of the head and neck. The main role of FA proteins is in the repair of DNA interstrand crosslinks (ICLs). FA results from pathogenic variants in at least sixteen distinct genes, causing genomic instability. Although the highly variable phenotype makes accurate diagnosis on the basis of clinical manifestations difficult in some patients, diagnosis based on a profound sensitivity to DNA‐crosslinking agents can be used to identify the pre‐anemia patient as well as patients with aplastic anemia or leukemia who may or may not have the physical stigmata associated with the syndrome. Diepoxybutane (DEB) analysis is the preferred test for FA because other agents have higher rates of false‐positive and false‐negative results. © 2015 by John Wiley & Sons, Inc.

Fanconi anemia (FA) is a rare recessive disease resulting from mutations in one of at least 16 different genes. Mutation types and phenotypic manifestations of FA are highly heterogeneous and influence the clinical management of the disease. We analyzed 202 FA families for large deletions, using high‐resolution comparative genome hybridization arrays, single‐nucleotide polymorphism arrays, and DNA sequencing. We found pathogenic deletions in 88 FANCA, seven FANCC, two FANCD2, and one FANCB families. We find 35% of FA families carry large deletions, accounting for 18% of all FA pathogenic variants. Cloning and sequencing across the deletion breakpoints revealed that 52 FANCA deletion ends, and one FANCC deletion end extended beyond the gene boundaries, potentially affecting neighboring genes with phenotypic consequences. Seventy‐five percent of the FANCA deletions are Alu–Alu mediated, predominantly by AluY elements, and appear to be caused by nonallelic homologous recombination. Individual Alu hotspots were identified. Defining the haplotypes of four FANCA deletions shared by multiple families revealed that three share a common ancestry. Knowing the exact molecular changes that lead to the disease may be critical for a better understanding of the FA phenotype, and to gain insight into the mechanisms driving these pathogenic deletion variants.