A Review of the Repair of DNA Double Strand Breaks in the Development of Oral Cancer.

We explore the possibility that defects in genes associated with the response and repair of DNA double strand breaks predispose oral potentially malignant disorders (OPMD) to undergo malignant transformation to oral squamous cell carcinoma (OSCC). Defects in the homologous recombination/Fanconi anemia (HR/FA), but not in the non-homologous end joining, causes the DNA repair pathway to appear to be consistent with features of familial conditions that are predisposed to OSCC (FA, Bloom's syndrome, Ataxia Telangiectasia); this is true for OSCC that occurs in young patients, sometimes with little/no exposure to classical risk factors. Even in Dyskeratosis Congenita, a disorder of the telomerase complex that is also predisposed to OSCC, attempts at maintaining telomere length involve a pathway with shared HR genes. Defects in the HR/FA pathway therefore appear to be pivotal in conditions that are predisposed to OSCC. There is also some evidence that abnormalities in the HR/FA pathway are associated with malignant transformation of sporadic cases OPMD and OSCC. We provide data showing overexpression of HR/FA genes in a cell-cycle-dependent manner in a series of OPMD-derived immortal keratinocyte cell lines compared to their mortal counterparts. The observations in this study argue strongly for an important role of the HA/FA DNA repair pathway in the development of OSCC.

A minimal Fanconi Anemia complex in early diverging fungi.

Fanconi Anemia (FA) pathway resolves DNA interstrand cross links (ICL). The FA pathway was initially recognized in vertebrates, but was later confirmed in other animals and speculated in fungi. FA proteins FANCM, FANCL and FANCJ are present in Saccharomyces cerevisiae but, their mechanism of interaction to resolve ICL is still unclear. Unlike Dikarya, early diverging fungi (EDF) possess more traits shared with animals. We traced the evolutionary history of the FA pathway across Opisthokonta. We scanned complete proteomes for FA-related homologs to establish their taxonomic distribution and analyzed their phylogenetic trees. We checked transcription profiles of FA genes to test if they respond to environmental conditions and their genomic localizations for potential co-localization. We identified fungal homologs of the activation and ID complexes, 5 out of 8 core proteins, all of the endonucleases, and deubiquitination proteins. All fungi lack FANCC, FANCF and FANCG proteins responsible for post-replication repair and chromosome stability in animals. The observed taxonomic distribution can be attributed to a gradual degradation of the FA pathway from EDF to Dikarya. One of the key differences is that EDF have the ID complex recruiting endonucleases to the site of ICL. Moreover, 21 out of 32 identified FA genes are upregulated in response to different growth conditions. Several FA genes are co-localized in fungal genomes which also could facilitate co-expression. Our results indicate that a minimal FA pathway might still be functional in Mucoromycota with a gradual loss of components in Dikarya ancestors.

Germline biallelic BRCA2 pathogenic variants and medulloblastoma: an international cohort study.

Constitutional heterozygous pathogenic variants in genes coding for some components of the Fanconi anemia-BRCA signaling pathway, which repairs DNA interstrand crosslinks, represent risk factors for common cancers, including breast, ovarian, pancreatic and prostate cancer. A high cancer risk is also a main clinical feature in patients with Fanconi anemia (FA), a rare condition characterized by bone marrow failure, endocrine and physical abnormalities. The mainly recessive condition is caused by germline pathogenic variants in one of 21 FA-BRCA pathway genes. Among patients with FA, the highest cancer risks are observed in patients with biallelic pathogenic variants in BRCA2 or PALB2. These patients develop a range of embryonal tumors and leukemia during the first decade of life, however, little is known about specific clinical, genetic and pathologic features or toxicities. Here, we present genetic, clinical, pathological and treatment characteristics observed in an international cohort of eight patients with FA due to biallelic BRCA2 pathogenic variants and medulloblastoma (MB), an embryonal tumor of the cerebellum. Median age at MB diagnosis was 32.5 months (range 7-58 months). All patients with available data had sonic hedgehog-MB. Six patients received chemotherapy and one patient also received proton radiation treatment. No life-threatening toxicities were documented. Prognosis was poor and all patients died shortly after MB diagnosis (median survival time 4.5 months, range 0-21 months) due to MB or other neoplasms. In conclusion, MB in patients with biallelic BRCA2 pathogenic variants is a lethal disease. Future experimental treatments are necessary to help these patients.

HLA-haploidentical stem cell transplantation in children with inherited bone marrow failure syndromes: A retrospective analysis on behalf of EBMT severe aplastic Anemia and pediatric diseases working parties.

Haploidentical stem cell transplantation (haplo-SCT) represents the main alternative for children with inherited bone marrow failure syndrome (I-BMF) lacking a matched donor. This retrospective study, conducted on behalf of the EBMT SAAWP and PDWP, aims to report the current outcomes of haplo-SCT in I-BMFs, comparing the different in vivo and ex vivo T-cell depletion approaches. One hundred and sixty-two I-BMF patients who underwent haplo-SCT (median age 7.4 years) have been registered. Fanconi Anemia was the most represented diagnosis (70.1%). Based on different T-cell depletion (TCD) approaches, four categories were identified: (1) TCRαß+/CD19+-depletion (43.8%); (2) T-repleted with post-transplant Cyclophosphamide (PTCy, 34.0%); (3) In-vivo T-depletion with ATG/alemtuzumab (14.8%); (4) CD34+ positive selection (7.4%). The cumulative incidences (CI) of neutrophil and platelet engraftment were 84% and 76% respectively, while that of primary and secondary graft failure was 10% and 8% respectively. The 100-day CI of acute GvHD grade III-IV(95% CI) was 13%, while the 24-month CI of extensive chronic GvHD was 4%. After a median follow-up of 43.4 months, the 2-year overall survival(OS) and GvHD/Rejection-free Survival (GRFS) probabilities are 67% and 53%, respectively. The TCR CD3+αß+/CD19+ depletion group showed a significantly lower incidence of both acute and chronic GvHD and higher OS (79%; p0.013) and GRFS (71%; p < .001), while no significant differences in outcomes have been observed by different diagnosis and conditioning regimens. This large retrospective study supports the safety and feasibility of haplo-SCT in I-BMF patients. TCRαß+/CD19+ depletion offers higher chances of patients' survival, with a significantly lower risk of severe a- and c-GvHD in I-BMFs compared to other platforms.

Pancancer analysis of the prognostic and immunological role of FANCD2: a potential target for carcinogenesis and survival.

Recent evidence has shed light on the significant role of FANCD2 in cancer initiation, development, and progression. However, a comprehensive pan-cancer analysis of FANCD2 has been lacking. In this study, we have conducted a thorough investigation into the expression profiles and prognostic significance of FANCD2, as well as its correlation with clinicopathological parameters and immune cell infiltration, using advanced bioinformatic techniques. The results demonstrate that FANCD2 is significantly upregulated in various common cancers and is associated with prognosis. Notably, higher expression levels of FANCD2 are linked to poor overall survival, as indicated by Cox regression and Kaplan-Meier analyses. Additionally, we have observed a decrease in the methylation of FANCD2 DNA in some cancers, and this decrease is inversely correlated with FANCD2 expression. Genetic alterations in FANCD2 predominantly manifest as mutations, which are associated with overall survival, disease-specific survival, disease-free survival, and progression-free survival in certain tumor types. Moreover, FANCD2 exhibits a strong correlation with infiltrating cell levels, immune checkpoint genes, tumor mutation burden (TMB), and microsatellite instability (MSI). Enrichment analysis further highlights the potential impact of FANCD2 on Fanconi anemia (FA) pathway and cell cycle regulation. Through this comprehensive pan-cancer analysis, we have gained a deeper understanding of the functions of FANCD2 in oncogenesis and metastasis across different types of cancer.

Umbilical Cord Blood Transplantation for Fanconi Anemia With a Special Focus on Late Complications: a Study on Behalf of Eurocord and SAAWP-EBMT.

Hematopoietic cell transplantation (HCT) remains the sole available curative treatment for Fanconi anemia (FA), with particularly favorable outcomes reported after matched sibling donor (MSD) HCT. This study aimed to describe outcomes, with a special focus on late complications, of FA patients who underwent umbilical cord blood transplantation (UCBT). In this retrospective analysis of allogeneic UCBT for FA performed between 1988 and 2021 in European Society for Blood and Marrow Transplantation (EBMT)-affiliated centers, a total of 205 FA patients underwent UCBT (55 related and 150 unrelated) across 77 transplant centers. Indications for UCBT were bone marrow failure in 190 patients and acute leukemia/myelodysplasia in 15 patients. The median age at transplantation was 9 years (range, 1.2 to 43 years), with only 20 patients aged >18 years. Among the donor-recipient pairs, 56% (n = 116) had a 0 to 1/6 HLA mismatch. Limited-field radiotherapy was administered to 28% (n = 58) and 78% (n = 160) received a fludarabine (Flu)-based conditioning regimen. Serotherapy consisted of antithymocyte globulin (n = 159; 78%) or alemtuzumab (n = 12; 6%). The median follow-up was 10 years for related UCBT and 7 years for unrelated UCBT. Excellent outcomes were observed in the setting of related UCBT, including a 60-day cumulative incidence (CuI) of neutrophil recovery of 98.1% (95% confidence interval [CI], 93.9% to 100%), a 100-day CuI of grade II-IV acute graft-versus-host disease (GVHD) of 17.3% (95% CI, 9.5% to 31.6%), and a 5-year CuI of chronic GVHD (cGVHD) of 22.7% (95% CI, 13.3% to 38.7%; 13% extensive). Five-year overall survival (OS) was 88%. In multivariate analysis, none of the factors included in the model predicted a better OS. In unrelated UCBT, the 60-day CuI of neutrophil recovery was 78.7% (95% CI, 71.9% to 86.3%), the 100-day CuI of grade II-IV aGVHD was 31.4% (95% CI, 24.6% to 40.2%), and the 5-year CuI of cGVHD was 24.3% (95% CI, 17.8% to 32.2%; 12% extensive). Five-year OS was 44%. In multivariate analysis, negative recipient cytomegalovirus serology, Flu-based conditioning, age <9 years at UCBT, and 0 to 1/6 HLA mismatch were associated with improved OS. A total of 106 patients, including 5 with acute leukemia/myelodysplasia, survived for >2 years after UCBT. Nine of these patients developed subsequent neoplasms (SNs), including 1 donor-derived acute myelogenous leukemia and 8 solid tumors, at a median of 9.7 years (range, 2.3 to 21.8 years) post-UCBT (1 related and 8 unrelated UCBT). In a subset of 49 patients with available data, late nonmalignant complications affecting various organ systems were observed at a median of 8.7 years (range, 2.7 to 28.8 years) post-UCBT. UCB is a valid source of stem cells for transplantation in patients with FA, with the best results observed after related UCBT. After unrelated UCBT, improved survival was observed in patients who underwent transplantation at a younger age, with Flu-based conditioning, and with better HLA parity. The incidence of organ-specific complications and SNs was relatively low. The incidence of SNs, mostly squamous cell carcinoma, increases with time. Rigorous follow-up and lifelong screening are crucial in survivors of UCBT for FA.

Novel compound heterozygous variants in FANCI cause premature ovarian insufficiency.

Premature ovarian insufficiency (POI) is a common reproductive aging disorder due to a dramatic decline of ovarian function before 40 years of age. Accumulating evidence reveals that genetic defects, particularly those related to DNA damage response, are a crucial contributing factor to POI. We have demonstrated that the functional Fanconi anemia (FA) pathway maintains the rapid proliferation of primordial germ cells to establish a sufficient reproductive reserve by counteracting replication stress, but the clinical implications of this function in human ovarian function remain to be established. Here, we screened the FANCI gene, which encodes a key component for FA pathway activation, in our whole-exome sequencing database of 1030 patients with idiopathic POI, and identified two pairs of novel compound heterozygous variants, c.[97C > T];[1865C > T] and c.[158-2A > G];[c.959A > G], in two POI patients, respectively. The missense variants did not alter FANCI protein expression and nuclear localization, apart from the variant c.158-2A > G causing abnormal splicing and leading to a truncated mutant p.(S54Pfs*5). Furthermore, the four variants all diminished FANCD2 ubiquitination levels and increased DNA damage under replication stress, suggesting that the FANCI variants impaired FA pathway activation and replication stress response. This study first links replication stress response defects with the pathogenesis of human POI, providing a new insight into the essential roles of the FA genes in ovarian function.

Deregulated protein homeostasis constrains fetal hematopoietic stem cell pool expansion in Fanconi anemia.

Demand-adjusted and cell type specific rates of protein synthesis represent an important safeguard for fate and function of long-term hematopoietic stem cells. Here, we identify increased protein synthesis rates in the fetal hematopoietic stem cell pool at the onset of hematopoietic failure in Fanconi Anemia, a prototypical DNA repair disorder that manifests with bone marrow failure. Mechanistically, the accumulation of misfolded proteins in Fancd2-/- fetal liver hematopoietic stem cells converges on endoplasmic reticulum stress, which in turn constrains midgestational expansion. Restoration of protein folding by the chemical chaperone tauroursodeoxycholic acid, a hydrophilic bile salt, prevents accumulation of unfolded proteins and rescues Fancd2-/- fetal liver long-term hematopoietic stem cell numbers. We find that proteostasis deregulation itself is driven by excess sterile inflammatory activity in hematopoietic and stromal cells within the fetal liver, and dampened Type I interferon signaling similarly restores fetal Fancd2-/- long-term hematopoietic stem cells to wild type-equivalent numbers. Our study reveals the origin and pathophysiological trigger that gives rise to Fanconi anemia hematopoietic stem cell pool deficits. More broadly, we show that fetal protein homeostasis serves as a physiological rheostat for hematopoietic stem cell fate and function.

Oral cancer and oral potentially malignant disorders in patients with Fanconi anemia - A systematic review.

The purpose of the present study was to perform a systematic review focusing on oral squamous cell carcinoma (OSCC) and oral potentially malignant disorders (OPMD) in Fanconi anemia (FA) individuals. Electronic searches were undertaken in five databases supplemented by manual scrutiny and gray literature. Case reports and/or cases series were included. The searches yielded 55 studies describing 112 cases of OSCC (n = 107) and/or OPMD (n = 5) in FA individuals. The mean age at diagnosis of OSCC/OPMD was 27.1 (±9.6) years, and females (51.8 %) were slightly more affected. Ulcer (n = 37) or mass (n = 25) were described as clinical presentations for OSCC and OPMD. White lesions (n = 4) were the most common manifestation in OPMD. Tongue (47.2 %) was the most frequent location. Sixty-one (54.5 %) individuals underwent HSCT. Surgical resection (n = 75) was the main treatment adopted. The estimated rate of OPMD malignant transformation was 1.8 % and recurrences following OSCC excision occurred in 26.8 % of individuals. Overall, at 60 months of follow-up, the probability of survival fell to 25.5 % and at 64 months the probability of recurrence increased to 63.2 %. The present data support the need for strict surveillance of patients with FA, even in the absence of OPMD, for early OSCC detection and reduction of mortality.

HLA-haploidentical T-cell receptor αßT/B-cell-depleted stem cell transplantation for Fanconi anemia.

HLA-haploidentical stem cell transplantation (haplo-SCT) using post-transplant high-dose cyclophosphamide (PT-CY) is an alternative choice when a suitable donors is unavailable. However, PT-CY is difficult in patients with Fanconi anemia (FA) due to their high vulnerability to alkylating agents. For FA, we prefer haplo-SCT by T-cell receptor αßT-cell and B-cell depletion (αßT/B-depleted haplo-SCT), which can reduce the risks of PT-CY-related complications and graft-versus-host disease (GVHD). An 11-year-old boy with diagnosed FA (FANCG mutation) and bone marrow failure was to receive αßT/B-depleted haplo-SCT from his father (HLA 4/8 allele matched) due to absence of an HLA-matched donors. αßT/B-depleted peripheral blood stem cells (CD34 + cell count, 1.17 × 107/kg; αß + T-cell count, 1.3 × 105/kg) were infused following conditioning consisting of fludarabine (150 mg/m2), cyclophosphamide (40 mg/kg), anti-thymocyte globulin (5 mg/kg), rituximab (375 mg/m2), and thoraco-abdominal irradiation (3 Gy). Tacrolimus was used for GVHD prophylaxis until day + 30. Neutrophil engraftment was achieved on day + 9, and complete chimerism was confirmed on days + 28 and + 96. At 12-month post-SCT, the patient was well without GVHD or any other complications. αßT/B-depleted haplo-SCT is a good choice not only for patients unsuitable for PT-CY, but also for all pediatric recipients to reduce SCT-related complications.

Aberrant R-loop-mediated immune evasion, cellular communication, and metabolic reprogramming affect cancer progression: a single-cell analysis.

Dysregulation of R-loop homeostasis is closely related to various human diseases, including cancer. However, the causality of aberrant R-loops in tumor progression remains unclear. In this study, using single-cell RNA-sequencing datasets from lung adenocarcinoma (LUAD), we constructed an R-loop scoring model to characterize the R-loop state according to the identified R-loop regulators related to EGFR mutations, tissue origins, and TNM stage. We then evaluated the relationships of the R-loop score with the tumor microenvironment (TME) and treatment response. Furthermore, the potential roles of FANCI-mediated R-loops in LUAD were explored using a series of in vitro experiments. Results showed that malignant cells with low R-loop scores displayed glycolysis and epithelial-mesenchymal transition pathway activation and immune escape promotion, thereby hampering the antitumor therapeutic effects. Cell communication analysis suggested that low R-loop scores contributed to T cell exhaustion. We subsequently validated the prognostic value of R-loop scores by using bulk transcriptome datasets across 33 tumor types. The R-loop scoring model well predicted patients' therapeutic response to targeted therapy, chemotherapy, or immunotherapy in 32 independent cohorts. Remarkably, changes in R-loop distribution mediated by FANCI deficiency blocked the activity of Ras signaling pathway, suppressing tumor-cell proliferation and dissemination. In conclusion, this study reveals the underlying molecular mechanism of metabolic reprogramming and T cell exhaustion under R-loop score patterns, and the changes in R-loops mediated by R-loop regulators resulting in tumor progression. Therefore, incorporating anticancer methods based on R-loop or R-loop regulators into the treatment schemes of precision medicine may be beneficial.

FANCD2 deficiency sensitizes SHH medulloblastoma to radiotherapy via ferroptosis.

Radiotherapy is one of the standard therapeutic regimens for medulloblastoma (MB). Tumor cells utilize DNA damage repair (DDR) mechanisms to survive and develop resistance during radiotherapy. It has been found that targeting DDR sensitizes tumor cells to radiotherapy in several types of cancer, but whether and how DDR pathways are involved in the MB radiotherapy response remain to be determined. Single-cell RNA sequencing was carried out on 38 MB tissues, followed by expression enrichment assays. Fanconi anemia group D2 gene (FANCD2) expression was evaluated in MB samples and public MB databases. The function of FANCD2 in MB cells was examined using cell counting assays (CCK-8), clone formation, lactate dehydrogenase activity, and in mouse orthotopic models. The FANCD2-related signaling pathway was investigated using assays of peroxidation, a malondialdehyde assay, a reduced glutathione assay, and using FerroOrange to assess intracellular iron ions (Fe2+ ). Here, we report that FANCD2 was highly expressed in the malignant sonic hedgehog (SHH) MB subtype (SHH-MB). FANCD2 played an oncogenic role and predicted worse prognosis in SHH-MB patients. Moreover, FANCD2 knockdown markedly suppressed viability, mobility, and growth of SHH-MB cells and sensitized SHH-MB cells to irradiation. Mechanistically, FANCD2 deficiency led to an accumulation of Fe2+ due to increased divalent metal transporter 1 expression and impaired glutathione peroxidase 4 activity, which further activated ferroptosis and reduced proliferation of SHH-MB cells. Using an orthotopic mouse model, we observed that radiotherapy combined with silencing FANCD2 significantly inhibited the growth of SHH-MB cell-derived tumors in vivo. Our study revealed FANCD2 as a potential therapeutic target in SHH-MB and silencing FANCD2 could sensitize SHH-MB cells to radiotherapy via inducing ferroptosis. © 2024 The Pathological Society of Great Britain and Ireland.

Liver abnormalities are frequent and persistent in patients with Fanconi anemia.

ABSTRACT: Liver disease has not been well described in patients with Fanconi anemia (FA). Improvements in outcomes of transplant mean that more individuals with FA are reaching adulthood and new features of the FA phenotype are being discovered. We performed a retrospective review of liver function in a cohort of 97 patients with FA followed-up for at least 10 years at a single center. We identified a high frequency of transaminitis (n = 31, 32%) without elevation of bilirubin and with no evidence of structural hepatic abnormality in patients with FA. Transaminitis was persistent in many cases, sometimes lasting more than a decade without clinical manifestation, although 2 patients with prolonged transaminitis are deceased from liver failure, indicating important long-term clinical consequences. Transaminitis was found in patients who had and had not received transplant but was more frequent in recipients of transplant. Exposure to total body irradiation increased risk (odds ratio, 15.5 [95% confidence interval, 2.44-304.54]; P = .01), whereas treatment with androgens did not. Review of limited numbers of liver biopsies and autopsy material showed a cholestatic pattern of liver injury, with progressive fibrosis, in the majority of patients. Occurrence in cases without transplant as well as cases with transplant argues against a potential diagnosis of atypical liver graft-versus-host disease. Limited data regarding therapy suggest no benefit from treatment with steroids or other immune suppressive medications or ursodeoxycholic acid. Our data show that liver disease is common in patients with FA, and because most children with FA now reach adulthood, end-stage liver disease in young adulthood means systematic testing of potential therapies is urgently needed.

Fanconi Anemia Complementary Group A (FANCA) Facilitates the Occurrence and Progression of Liver Hepatocellular Carcinoma.

BACKGROUND: Liver hepatocellular carcinoma (LIHC) is a serious liver disease worldwide, and its pathogenesis is complicated. AIMS: This study investigated the potential role of FANCA in the advancement and prognosis of LIHC. METHODS: Public databases, quantitative reverse transcription polymerase chain reaction (qRT-PCR), western blot (WB) and immunohistochemistry (IHC) were employed to measure FANCA expression between tumor and normal samples. The relationship between FANCA expression and prognosis of LIHC patients were examined. Functional enrichment of FANCA-related genes was performed. Furthermore, univariate and multivariate analyses were conducted to determine the independent prognosis value of FANCA in LIHC. Finally, influence of FANCA knockout on the proliferation, migration, and invasion of HepG2 cell was validated with cloning formation, CCK8, and Transwell assays. RESULTS: Expression analysis presented that FANCA had high expression level in LIHC tissues and cells. Receiver operating characteristic (ROC) curve analysis showed that FANCA was of great diagnosis value in LIHC. Clinicopathological analysis revealed that FANCA was significantly greater expressed in the advanced stage than in the early stage of LIHC. Univariate, multivariate, and Kaplan-Meier survival analysis confirmed that high expression of FANCA was strongly associated with poor survival of LIHC patients. In addition, high level of FANCA in LIHC showed a negative association with immunoinfiltrated B cells, T cells, and stromal scores. Moreover, Knockout of FANCA significantly inhibited HepG2 cell proliferative activity, migration, and invasion ability. CONCLUSIONS: Our data revealed that high level of FANCA was closely associated with LIHC malignant progression, suggesting its potential utility as a diagnostic, predictive indicator, and therapeutic target.

Male with an apparently normal phenotype carrying a BRCA1 exon 20 duplication in trans to a BRCA1 frameshift variant.

BACKGROUND: Reports of dual carriers of pathogenic BRCA1 variants in trans are extremely rare, and so far, most individuals have been associated with a Fanconi Anemia-like phenotype. METHODS: We identified two families with a BRCA1 in-frame exon 20 duplication (Ex20dup). In one male individual, the variant was in trans with the BRCA1 frameshift variant c.2475delC p.(Asp825Glufs*21). We performed splicing analysis and used a transcription activation domain (TAD) assay to assess the functional impact of Ex20dup. We collected pedigrees and mapped the breakpoints of the duplication by long- and short-read genome sequencing. In addition, we performed a mitomycin C (MMC) assay from the dual carrier using cultured lymphoblastoid cells. RESULTS: Genome sequencing and RNA analysis revealed the BRCA1 exon 20 duplication to be in tandem. The duplication was expressed without skipping any one of the two exon 20 copies, resulting in a lack of wild-type transcripts from this allele. TAD assay indicated that the Ex20dup variant has a functional level similar to the well-known moderate penetrant pathogenic BRCA1 variant c.5096G > A p.(Arg1699Gln). MMC assay of the dual carrier indicated a slightly impaired chromosomal repair ability. CONCLUSIONS: This is the first reported case where two BRCA1 variants with demonstrated functional impact are identified in trans in a male patient with an apparently normal clinical phenotype and no BRCA1-associated cancer. The results pinpoint a minimum necessary BRCA1 protein activity to avoid a Fanconi Anemia-like phenotype in compound heterozygous status and yet still predispose carriers to hormone-related cancers. These findings urge caution when counseling families regarding potential Fanconi Anemia risk. Furthermore, prudence should be taken when classifying individual variants as benign based on co-occurrence in trans with well-established pathogenic variants.

The FANCI/FANCD2 complex links DNA damage response to R-loop regulation through SRSF1-mediated mRNA export.

Fanconi anemia (FA) is characterized by congenital abnormalities, bone marrow failure, and cancer susceptibility. The central FA protein complex FANCI/FANCD2 (ID2) is activated by monoubiquitination and recruits DNA repair proteins for interstrand crosslink (ICL) repair and replication fork protection. Defects in the FA pathway lead to R-loop accumulation, which contributes to genomic instability. Here, we report that the splicing factor SRSF1 and FANCD2 interact physically and act together to suppress R-loop formation via mRNA export regulation. We show that SRSF1 stimulates FANCD2 monoubiquitination in an RNA-dependent fashion. In turn, FANCD2 monoubiquitination proves crucial for the assembly of the SRSF1-NXF1 nuclear export complex and mRNA export. Importantly, several SRSF1 cancer-associated mutants fail to interact with FANCD2, leading to inefficient FANCD2 monoubiquitination, decreased mRNA export, and R-loop accumulation. We propose a model wherein SRSF1 and FANCD2 interaction links DNA damage response to the avoidance of pathogenic R-loops via regulation of mRNA export.

FANCA c.3624C>T (p.Ser1208=) is a hypomorphic splice variant associated with delayed onset of Fanconi anemia.

ABSTRACT: Fanconi anemia (FA) is a hereditary, DNA repair deficiency disorder caused by pathogenic variants in any 1 of 22 known genes (FANCA-FANCW). Variants in FANCA account for nearly two-thirds of all patients with FA. Clinical presentation of FA can be heterogeneous and include congenital abnormalities, progressive bone marrow failure, and predisposition to cancer. Here, we describe a relatively mild disease manifestation among 6 individuals diagnosed with FA, each compound heterozygous for 1 established pathogenic FANCA variant and 1 FANCA exon 36 variant, c.3624C>T. These individuals had delayed onset of hematological abnormalities, increased survival, reduced incidence of cancer, and improved fertility. Although predicted to encode a synonymous change (p.Ser1208=), the c.3624C>T variant causes a splicing error resulting in a FANCA transcript missing the last 4 base pairs of exon 36. Deep sequencing and quantitative reverse transcription polymerase chain reaction analysis revealed that 6% to 10% of the FANCA transcripts included the canonical splice product, which generated wild-type FANCA protein. Consistently, functional analysis of cell lines from the studied individuals revealed presence of residual FANCD2 ubiquitination and FANCD2 foci formation, better cell survival, and decreased late S/G2 accumulation in response to DNA interstrand cross-linking agent, indicating presence of residual activity of the FA repair pathway. Thus, the c.3624C>T variant is a hypomorphic allele, which contributes to delayed manifestation of FA disease phenotypes in individuals with at least 1 c.3624C>T allele.

Expression levels and clinical significance of ferroptosis-related genes in patients with myocardial infarction.

Myocardial infarction (MI) is the most serious type of cardiovascular disease and the leading cause of cardiac death.Ferroptosis is one of the newly discovered programmed cell death modes in MI, but its mechanism of action in MI has not been clarified.In this study, we analyzed the expression changes of ferroptosis-related genes in MI and explored the potential mechanisms of ferroptosis-related functions in myocardial infarction. Public data sets GSE19339, GSE97320 and GSE141512 were retrieved from the Gene Expression Omnibus (GEO) Datasets public database. After data preprocessing, differentially expressed genes were screened, and differentially expressed ferroptosis-related genes associated with myocardial infarction were obtained. The biological function and signaling pathway enrichment analysis were performed to establish the PPI interaction network specific to heart tissue, and the differential diagnosis significance of differentially expressed ferroptosis-related genes associated with myocardial infarction was analyzed by ROC curve and decision tree model.A total of 317 genes showed significant changes in expression levels in patients with myocardial infarction, including 205 down-regulated genes and 112 up-regulated genes.Gene Ontology (GO) enrichment analysis and functional classification of Kyoto Encyclopedia of Genes and Genomes (KEGG) signaling pathways showed that these genes were mainly involved in signaling pathways or biological functions related to inflammation and apoptosis.Five differentially expressed ferroptosis-related genes (SLC2A3, EPAS1, HMOX1, ATM, FANCD2) were obtained, all of which played key biological functions in cardiac tissue function. SLC2A3, EPAS1, HMOX1, ATM and FANCD2 genes all had good diagnostic value for myocardial infarction (P < 0.05). The increase of SLC2A3, EPAS1 and HMOX1 are risk factors for myocardial infarction, while ATM and FANCD2 are protective factors.Decision tree analysis showed that SLC2A3, HMOX1, ATM, FANCD2 gene had higher net yield in diagnosing myocardial infarction. In summary, the mechanism of ferroptosis is involved in the occurrence and progression of myocardial infarction. In this study, five differentially expressed ferroptosis-related genes associated with myocardial infarction were retrieved, which may be good biomarkers of ferroptosis after MI.These findings also suggest that the differential expression of ferroptosis-related genes associated with myocardial infarction has significant diagnostic significance for myocardial infarction.

Fanconi anemia complementation group D2 promotes sensitivity of endometrial cancer cells to chemotherapeutic agents by inhibiting the ferroptosis pathway.

BACKGROUND: Resistance can develop during treatment of advanced endometrial cancer (EC), leading to unsatisfactory results. Fanconi anemia complementation group D2 (Fancd2) has been shown to be closely related to drug resistance in cancer cells. Therefore, this study was designed to explore the correlation of Fancd2 with EC resistance and the mechanism of Fancd2. METHODS: Real-time quantitative PCR (RT-qPCR) was used to detect the expression of Fancd2 in EC tissues and cells. EC cells (Ishikawa) and paclitaxel-resistant EC cells (Ishikawa/TAX) were transfected to knock down Fancd2. In addition, the ferroptosis inhibitor Ferrostatin-1 was adopted to treat Ishikawa/TAX cells. The sensitivity of cancer cells to chemotherapeutic agents was observed via 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2-H-tetrazolium bromide (MTT) assay, and inhibitory concentration (IC)50 was calculated. Reactive oxygen species (ROS) levels were measured by flow cytometry, the activity of malondialdehyde (MDA) and the levels of glutathione (GSH) and Fe2+ in cells were detected by corresponding kits, and protein expression of solute farrier family 7 member 11 (SLC7A11) and glutathione peroxidase 4 (GPX4) was obtained through western blot. RESULTS: Compared with the normal tissues and endometrial epithelial cells, Fancd2 expression was significantly increased in EC tissues and Ishikawa cells, respectively. After knock-down of Fancd2, Ishikawa cells showed significantly increased sensitivity to chemotherapeutic agents. Besides, compared with Ishikawa cells, the levels of ROS, the activity of MDA, and the levels of GSH and Fe2+ were significantly decreased in Ishikawa/TAX cells, while the expression levels of SLC7A11 and GPX4 were significantly increased. Knock-down of Fancd2 significantly increased the ferroptosis levels in Ishikawa/TAX cells, but this effect could be reversed by Ferrostatin-1. CONCLUSION: Fancd2 increases drug resistance in EC cells by inhibiting the cellular ferroptosis pathway.

A murine model to evaluate immunotherapy effectiveness for human Fanconi anemia-mutated acute myeloid leukemia.

Fanconi anemia (FA)-mutated acute myeloid leukemia (AML) is a secondary AML with very poor prognosis and limited therapeutic options due to increased sensitivity to DNA-damaging agents. PD-1 immune checkpoint inhibitors upregulate T-cell killing of cancer cells and is a class of promising treatment for FA-AML. Here, we developed a novel FA-AML murine model that allows the study of human AML with a humanized immune system in order to investigate immunotherapeutic treatments in vivo. FA-AML1 cells and non-FA-mutated Kasumi-1 cells were injected into 8-10 week old NSG mice. Once leukemic engraftment was confirmed by HLA-DR expression in the peripheral blood, human peripheral blood mononuclear cells (hPBMCs) were injected into the mice. One week post-hPBMCs injection, Nivolumab (PD-1 inhibitor) or PBS vehicle control was administered to the mice bi-weekly. In our Nivolumab treated mice, FA-AML1, but not Kasumi-1-engrafted mice, had significantly prolonged overall survival. Both FA-AML1 and Kasumi-1 engrafted mice had decreased spleen weights. Higher leukemic infiltration into vital organs was observed in FA-AML1 engrafted mice compared to Kasumi-1 engrafted mice. In conclusion, our novel humanized murine model of FA-mutated AML is an attractive tool for supporting further studies and clinical trials using PD-1 inhibitors to treat FA-mutated AML.