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1
Taniguchi, Toshiyasu, and Alan D. D'Andrea. "The Fanconi anemia protein, FANCE, promotes the nuclear accumulation of FANCC." Blood 100, no. 7 (October 1, 2002): 2457–62. http://dx.doi.org/10.1182/blood-2002-03-0860.
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Fanconi anemia is an autosomal recessive disorder characterized by aplastic anemia, cancer susceptibility, and cellular sensitivity to mitomycin C. The 6 known Fanconi anemia gene products (FANCA, FANCC, FANCD2, FANCE, FANCF, and FANCG proteins) interact in a common pathway. The monoubiquitination and nuclear foci formation of FANCD2 are essential for the function of this pathway. FANCA, FANCC, FANCG, and FANCF proteins form a multisubunit nuclear complex (FA complex) required for FANCD2 monoubiquitination. Because FANCE and FANCC interact in vitro and FANCE is required for FANCD2 monoubiquitination, we reasoned that FANCE is a component of the FA complex in vivo. Here we demonstrate that retroviral transduction of Fanconi anemia subtype E (FA-E) cells with the FANCE cDNA restores the nuclear accumulation of FANCC protein, FANCA–FANCC complex formation, monoubiquitination and nuclear foci formation of FANCD2, and mitomycin C resistance. Hemagglutinin (HA)-tagged FANCE protein localizes diffusely in the nucleus. In normal cells, HA-tagged FANCE protein coimmunoprecipitates with FANCA, FANCC, and FANCG but not with FANCD2. Our data indicate that FANCE is a component of the nuclear FA complex in vivo and is required for the monoubiquitination of FANCD2 and the downstream events in the FA pathway.
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Guardiola, Ph, P. Ladne, J. Soulier, F. Siclon, J. Delrow, E. Gluckman, F. Sigaux, and J. P. Radich. "Involvement of Fanconi Anemia (FA) Genes FANCG and FANCA in Human DNA Replication, Mitosis and Chromosome Segregation: A Gene Expression Profiling Study." Blood 104, no. 11 (November 16, 2004): 2836. http://dx.doi.org/10.1182/blood.v104.11.2836.2836.
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Abstract FA is an autosomal recessive disorder with diverse clinical symptoms, including developmental anomalies, progressive bone marrow failure, and a predisposition to the development of malignancies. FA cells are hypersensitive to DNA cross-linking agents, and display chromosomal instability and defective DNA repair. FANCG-deficient pts accounts for 10% of all FA pts, and are a high risk FA population for acute myeloblastic leukemia. FANCG protein is part of the FA nuclear core complex, interacting with FANCA and FANCF. It can also interact with FANCD1, RAD51, and CYP2E1, suggesting roles in homologous recombination DNA repair and oxidative DNA-damage protection. However, FANCG lack apparent homology to other proteins involved in such processes. To address whether FANCG mRNA expression level was associated with some microarray-based functional signatures using Affymetrix HG-U133A Genechips, we first used a dataset including 111 T-acute lymphoblastic leukemia (T-ALL) samples. This large series was selected because FA pts do not usually develop T-ALL (risk of FANC gene mutation/inactivation low), FANCG is highly expressed in thymus and lymphoblasts, FANCG gene expression was significantly varying across these samples. We first selected samples having the lowest (FANCGlow) and highest (FANCGhigh) FANCG expression levels (n=15 per group, median expression values: 36 vs. 329), and identified genes differentially expressed between these 2 groups using SAM (fold change>3; qvalue<1%). Among the 368 probesets (308 known genes) significantly up-regulated in FANCGhigh group (likely to be co-expressed/regulated with FANCG), 55 were correlated to FANCG expression profile (>.75) when analyzing all 111 T-ALL samples. These genes were mainly involved in cell proliferation, mitotic cell cycle and its regulation (DNA replication, traversing Start control point, G2/M transition, chromosome segregation and cytokinesis), and were also representing cellular components involved in such processes (kinesin complex, spindle, and kinetochore). FANCA, RAD51, RAD51C, and PIR51 were also among the genes having expression patterns close to FANCG one. In addition, 11 of these 55 genes, all involved in DNA replication, were also correlated with FANCA (>.75). A robust regression analysis identified CKS1B, CCNB2, RFC5, MELK, STK18, and CENPF as the genes most significantly associated with FANCG expression profile (p<.0001, power=100%). Finally, we compared normal fibroblasts (CCL153) to primary fibroblasts from 2 FANCG-deficient pts grown under normal conditions or with MMC 100 nM for 36 hours (PD829, PD352). Fifty-eight of the 585 probesets down-regulated in FANCG-deficient fibroblasts (fold change>2; qvalue<1%) were also present in the FANCGhigh list of genes, and were mainly involved in DNA replication, mitotic chromosomal positioning and segregation, as well as in cytokinesis. Among these genes, GMNN (DNA replication inhibition by preventing MCM complex incorporation), MCM10 (DNA replication initiation), POLE2 (DNA replication and repair), and RRM2 (DNA synthesis) were also correlated with FANCA expression pattern. Of note, FANCC, FANCE, FANCF, FANCD1/BRCA2, and FANCL expression patterns were not significantly correlated with the one of FANCG or FANCA. Conclusion: these results suggest that the FA core complex, and especially FANCG and FANCA, is involved in DNA replication and mitosis.
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Ali, Abdullah M., Thiyam R. Singh, and Ruhikanta A. Meetei. "Identification and Partial Characterization of Fanconi Anemia Associated Polypeptides (FAAPs) Using a Versatile Multiprotein-Complex Purification Method." Blood 108, no. 11 (November 16, 2006): 989. http://dx.doi.org/10.1182/blood.v108.11.989.989.
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Abstract Fanconi Anemia (FA) is an autosomal recessive and X-linked disorder characterized by congenital abnormalities, progressive bone marrow failure, and a high incidence of hematological (acute leukemia) and non-hematological malignancies (squamous cell carcinomas of the head and neck or gynecologic system). FA is genetically heterogeneous disease and to date 12 complementation groups are known of which 11 gene products have been identified (FANC- A, B, C, D1, D2, E, F, G, J, L, M). Eight of the FA gene products, FANCA, FANCB, FANC, FANCE, FANCF, FANCG, FANCL and FANCM form a multiprotein FA core complex. This complex is required for the monoubiquitination of FANCD2 upon DNA damage by various genotoxic agents. The other two FA proteins; FANCD1/BRCA2 and FANCJ are believed to act “downstream” of FANCD2. In order to understand the role of FA proteins in DNA repair pathway it is necessary to find all the FA genes and their interacting partners. We have established a two-step purification method using 6XHis and FLAG tags for the biochemical and functional characterization of the FA core complex proteins. In an attempt to isolate interacting partners of FANCM and FANCL proteins; we have established two different HeLa cell lines; HeLa-HF-FANCM and HeLa-HF-FANCL, stably expressing HF-FANCM and HF-FANCL recombinant proteins respectively. Two step affinity purification was carried out to isolate the complexes from the extracts prepared from stable cell lines. Two polypeptides, namely, FAAP16 and FAAP100 were identified by mass-spectrometry as major interacting partners of FANCM and FANCL respectively. The interaction of FAAP16 and FAAP100 with other FA core complex proteins was confirmed by reciprocal affinity purification coupled mass-spectrometry using HeLa cells stably expressing HF-FAAP16 and HF-FAAP100 proteins. Furthermore, suppression of FAAP16 and FAAP100 in HeLa cells using siRNA resulted in a reduced MMC-induced FANCD2 monoubiquitination. Studies are being carried out to understand the precise role of these proteins in the FA core complex. These data suggest additional proteins interact with FA core complex members and demonstrate the utility of the purification method in delineating interacting proteins involved in FA.
4
Reuter, Tanja, Sabine Herterich, Oliver Bernhard, Holger Hoehn, and Hans J. Gross. "Strong FANCA/FANCG but weak FANCA/FANCC interaction in the yeast 2-hybrid system." Blood 95, no. 2 (January 15, 2000): 719–20. http://dx.doi.org/10.1182/blood.v95.2.719.
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Three of at least 8 Fanconi anemia (FA) genes have been cloned (FANCA, FANCC, FANCG), but their functions remain unknown. Using the yeast 2-hybrid system and full-length cDNA, the authors found a strong interaction between FANCA and FANCG proteins. They also obtained evidence for a weak interaction between FANCA and FANCC. Neither FANCA nor FANCC was found to interact with itself. These results support the notion of a functional association between the FA gene products.
5
Galimi, Francesco, Meenakshi Noll, Yoshiyuki Kanazawa, Timothy Lax, Cindy Chen, Markus Grompe, and Inder M. Verma. "Gene therapy of Fanconi anemia: preclinical efficacy using lentiviral vectors." Blood 100, no. 8 (October 15, 2002): 2732–36. http://dx.doi.org/10.1182/blood-2002-04-1245.
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Fanconi anemia (FA) is an inherited cancer susceptibility syndrome caused by mutations in a DNA repair pathway including at least 6 genes(FANCA, FANCC, FANCD2, FANCE, FANCF, and FANCG). The clinical course of the disease is dominated by progressive, life-threatening bone marrow failure and high incidence of acute myelogenous leukemia and solid tumors. Allogeneic bone marrow transplantation (BMT) is a therapeutic option but requires HLA-matched donors. Gene therapy holds great promise for FA, but previous attempts to use retroviral vectors in humans have proven ineffective given the impaired proliferation potential of human FA hematopoietic progenitors (HPCs). In this work, we show that using lentiviral vectors efficient genetic correction can be achieved in quiescent hematopoietic progenitors from Fanca−/− andFancc−/−mice. Long-term repopulating HPCs were transduced by a single exposure of unfractionated bone marrow mononuclear cells to lentivectors carrying the normal gene. Notably, no cell purification or cytokine prestimulation was necessary. Resistance to DNA- damaging agents was fully restored by lentiviral transduction, allowing for in vivo selection of the corrected cells with nonablative doses of cyclophosphamide. This study strongly supports the use of lentiviral vectors for FA gene therapy in humans.
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Yarde, Danielle N., Lori A. Hazlehurst, Vasco A. Oliveira, Qing Chen, and William S. Dalton. "Bortezomib Enhances Melphalan Response by Altering Fanconi Anemia (FA)/BRCA Pathway Expression and Function." Blood 108, no. 11 (November 16, 2006): 840. http://dx.doi.org/10.1182/blood.v108.11.840.840.
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Abstract The FA/BRCA pathway is involved in DNA damage repair and its importance in oncogenesis has only recently been implicated. Briefly, 8 FA/BRCA pathway family members facilitate the monoubiquitination of FANCD2. Upon monoubiquitination, FANCD2 translocates to the DNA repair foci where it interacts with other proteins to initiate DNA repair. Previously, we reported that the FA/BRCA pathway is upregulated in multiple myeloma cell lines selected for resistance to melphalan (Chen, et al, Blood 2005). Further, reducing FANCF in the melphalan resistant 8226/LR5 myeloma cell line partially reversed resistance, whereas overexpressing FANCF in the drug sensitive 8226/S myeloma line conferred resistance to melphalan. Others have reported, and we have also verified, that bortezomib enhances melphalan response in myeloma cells; however, the mechanism of enhanced melphalan activity in combination with bortezomib has not been reported. Based on our observation that the FA/BRCA pathway confers melphalan resistance, we hypothesized that bortezomib enhances melphalan response by targeting FA/BRCA DNA damage repair pathway genes. To investigate this hypothesis, we first analyzed FA/BRCA gene expression in 8226/S and 8226/LR5 cells treated with bortezomib, using a customized microfluidic card (to detect BRCA1, BRCA2, FANCA, FANCC, FANCD2, FANCE, FANCF, FANCG, FANCL, RAD51 and RAD51C) and q-PCR. Interestingly, we found that low dose (5nM) bortezomib decreased many FA/BRCA pathway genes as early as 2 hours, with maximal decreases seen at 24 hours. Specifically, 1.5- to 2.5-fold decreases in FANCA, FANCC, FANCD2, FANCE and RAD51C were seen 24 hours post bortezomib exposure. Moreover, pre-treatment of myeloma cells with low dose bortezomib followed by melphalan treatment revealed a greater than 2-fold reduction in FANCD2 gene expression levels. We also found that melphalan treatment alone enhanced FANCD2 protein expression and activation (monoubiquitination), whereas the combination treatment of bortezomib followed by melphalan decreased activation and overall expression of FANCD2 protein. Taken together, these results suggest that bortezomib enhances melphalan response in myeloma by targeting the FA/BRCA pathway. Further understanding of the role of the FA/BRCA pathway in determining melphalan response may allow for more customized and effective treatment of myeloma.
7
Heinrich, Michael C., Kirsten V. Silvey, Stacie Stone, Amy J. Zigler, Diana J. Griffith, Michelle Montalto, Lin Chai, Yu Zhi, and Maureen E. Hoatlin. "Posttranscriptional cell cycle–dependent regulation of human FANCC expression." Blood 95, no. 12 (June 15, 2000): 3970–77. http://dx.doi.org/10.1182/blood.v95.12.3970.
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Abstract The Fanconi Anemia (FA) Group C complementation group gene (FANCC) encodes a protein, FANCC, with a predicted Mr of 63000 daltons. FANCC is found in both the cytoplasmic and the nuclear compartments and interacts with certain other FA complementation group proteins as well as with non-FA proteins. Despite intensive investigation, the biologic roles of FANCC and of the other cloned FA gene products (FANCA and FANCG) remain unknown. As an approach to understanding FANCC function, we have studied the molecular regulation of FANCC expression. We found that although FANCCmRNA levels are constant throughout the cell cycle, FANCC is expressed in a cell cycle-dependent manner, with the lowest levels seen in cells synchronized at the G1/S boundary and the highest levels in the M-phase. Cell cycle–dependent regulation occurred despite deletion of the 5′ and 3′ FANCC untranslated regions, indicating that information in the FANCC coding sequence is sufficient to mediate cell cycle–dependent regulation. Moreover, inhibitors of proteasome function blocked the observed regulation. We conclude that FANCC expression is controlled by posttranscriptional mechanisms that are proteasome dependent. Recent work has demonstrated that the functional activity of FA proteins requires the physical interaction of at least FANCA, FANCC, and FANCG, and possibly of other FA and non-FA proteins. Our observation of dynamic control of FANCC expression by the proteasome has important implications for understanding the molecular regulation of the multiprotein complex.
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Heinrich, Michael C., Kirsten V. Silvey, Stacie Stone, Amy J. Zigler, Diana J. Griffith, Michelle Montalto, Lin Chai, Yu Zhi, and Maureen E. Hoatlin. "Posttranscriptional cell cycle–dependent regulation of human FANCC expression." Blood 95, no. 12 (June 15, 2000): 3970–77. http://dx.doi.org/10.1182/blood.v95.12.3970.012k33_3970_3977.
Full textAbstract:
The Fanconi Anemia (FA) Group C complementation group gene (FANCC) encodes a protein, FANCC, with a predicted Mr of 63000 daltons. FANCC is found in both the cytoplasmic and the nuclear compartments and interacts with certain other FA complementation group proteins as well as with non-FA proteins. Despite intensive investigation, the biologic roles of FANCC and of the other cloned FA gene products (FANCA and FANCG) remain unknown. As an approach to understanding FANCC function, we have studied the molecular regulation of FANCC expression. We found that although FANCCmRNA levels are constant throughout the cell cycle, FANCC is expressed in a cell cycle-dependent manner, with the lowest levels seen in cells synchronized at the G1/S boundary and the highest levels in the M-phase. Cell cycle–dependent regulation occurred despite deletion of the 5′ and 3′ FANCC untranslated regions, indicating that information in the FANCC coding sequence is sufficient to mediate cell cycle–dependent regulation. Moreover, inhibitors of proteasome function blocked the observed regulation. We conclude that FANCC expression is controlled by posttranscriptional mechanisms that are proteasome dependent. Recent work has demonstrated that the functional activity of FA proteins requires the physical interaction of at least FANCA, FANCC, and FANCG, and possibly of other FA and non-FA proteins. Our observation of dynamic control of FANCC expression by the proteasome has important implications for understanding the molecular regulation of the multiprotein complex.
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Ciccone, Samantha, Anna Pulliam, Xiaohong Li, Yue Si, Attilio Orazi, Grover C. Bagby, and D. Wade Clapp. "A Model of Clonal Evolution and Myelodysplasia (MDS) on Mice with Genetic Disruption of Both Fancc and Fancg." Blood 108, no. 11 (November 16, 2006): 2627. http://dx.doi.org/10.1182/blood.v108.11.2627.2627.
Full textAbstract:
Abstract Fanconi anemia (FA) is a rare inherited chromosomal instability syndrome characterized by bone marrow failure and a high relative risk of MDS. Eight FA proteins associate in a core nuclear complex and function at least in part to catalyze the monoubiquitination of the downstream target protein, FANCD2 in response to DNA damage. In this nuclear pathway the FA proteins are epistatic in the activation of FANCD2 since inactivation of any one of the eight FA proteins results in failure of FANCD2 monoubiquitination and hypersensitivity to cross-linking agents. Although biochemical studies have attributed additional survival signaling functions to the FA proteins, these functions have not been evaluated using a genetic model. Murine models of FA have been established using homologous recombination for gene disruption. Although all strains of knockout mice are hypersensitive to mitomycin c, none of the single gene knockout mice display bone marrow failure, MDS, or myeloid leukemia. Seeking to develop such a model, we utilized a genetic intercross to generate mice that harbor disruptions in both Fancc and Fancg. Genetic disruption of both Fancc and Fancg predispose Fancc−/−;Fancg −/− mice or recipients adoptively transferred with Fancc −/−; Fancg −/− hematopoietic stem cells to MDS analogous to the disease phenotype in FA patients as defined histologically and by cytogenetic analysis. Genome wide transcriptomal analysis and hierarchical clustering by genotypic group of bone marrow cells from wild type, single knockout, and double knockout mice (n=3 each) confirmed substantial differences between hematopoietic cells of Fancc, Fancg and double knockout (DKO) mice. Serial pairwise analysis and gene pattern analyses (GeneSifter) showed that of the 1190 genes expressed differentially (by a factor of >1.5, FDR adjusted p<0.05) in Fancc−/− marrow cells only 134 were differentially expressed in Fancg−/− cells. Of the 524 genes expressed differentially in Fancg−/− marrow compared to WT, 277 were not expressed differentially in Fancc−/− marrow compared to WT. In pairwise analysis of Fancc−/− vs. Fancg−/− gene expression, ontologies of those genes more highly expressed in Fancc −/− cells included responses to biotic stress, defense and immune response. The most over-represented ontological category of those genes more highly expressed in Fancg−/− cells was response to oxidative stress. Since these genes are not epistatic in regards to the hematopoietic phenotype, and the transcriptomal consequences of their loss-of-function in marrow cells are significantly different, this genetic model confirms that the Fancc and Fancg proteins are multi-functional. Transcriptosomal analyses were conducted on DKO mice that contained MDS and DKO mice with no overt disease. The transcriptome of DKO marrow cells was unique in that 152 suppressed and 687 activated gene products relative to WT samples were not found in either Fancc−/− or Fancg−/− samples. Furthermore, there are distinct transcriptomal differences between the DKO mice with MDS and those that do not have MDS. These data suggest that some of these changes may be adaptive and involved in the molecular pathogenesis of MDS. The DKO model provides the first preclinical platform to systematically evaluate the molecular pathogenesis of bone marrow failure and myelodysplasia in the setting of Fanconi anemia.
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Mori, Minako, Asuka Hira, Kenichi Yoshida, Hideki Muramatsu, Yusuke Okuno, Michiko Anmae, Kazuo Tamura, et al. "Characterization of Pathogenic Variants and Clinical Phenotypes in 117 Japanese Fanconi Anemia Patients." Blood 132, Supplement 1 (November 29, 2018): 3860. http://dx.doi.org/10.1182/blood-2018-99-110362.
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Abstract Objective: Fanconi anemia (FA) is the most common inherited bone marrow failure syndrome associated with multiple congenital abnormalities and predisposition to malignancies, resulting from mutations in one of the 22 known FA genes (FANCA to W). The proteins encoded by these genes participate in DNA repair pathway (the FA pathway) for endogenous aldehyde damage. Compared to the situation in the US or Europe, the number of Japanese FA patients with genetic diagnosis was relatively limited. In this study, we reveal the genetic subtyping and the characteristics of mutated FA genes in Japanese population and clarify the genotype-phenotype correlations. Results: We studied 117 Japanese FA patients from 103 families (1996 to 2018). The diagnosis of FA was confirmed on the basis of chromosomal breakage tests and clinical features. Molecular diagnosis was obtained in 107 (91.5%) of the 117 patients through direct sequencing of FANCA and FANCG, MLPA analysis for FANCA, targeted exome sequencing (targeted-seq), and whole exome sequencing (WES) analysis (Figure 1). To provide genetic subtyping for the 10 unclassified cases, we tried to apply various technologies. Array CGH revealed large deletions in two FA-B and one FA-T cases. Whole genome sequencing and RNA-sequencing analysis identified splicing site or aberrant splicing mutations among three cases (one FA-B, one FA-C, and one FA-N). Collectively, 113 (97%) of Japanese 117 FA patients were successfully subtyped and a total of 219 mutated alleles were identified. FA-A and FA-G accounted for the disease in 58% and 25% of FA patients, respectively, whereas each of the other complementation groups accounted for less than 5% of FA cases. FANCB was the third most common complementation group (n=4) and only one FA-C case was identified in Japanese FA patients. In the 68 FA-A patients, we identified 130 mutant alleles that included 55 different FANCA variants (17 nucleotide substitutions, 16 small deletions/insertions, 12 large deletions, 1 large duplication and 9 splice site mutation). FANCA c.2546delC was the most prevalent (41/130 alleles; 32%). In the 29 FA-G patients, 57 mutant alleles were identified and seven different FANCG variants were detected. FANCG c.307+1G>C and 1066C>T accounted for most of FANCG mutant alleles (49/57; 88%) in the Japanese FA-G patients. The three hotspot mutations (FANCA c.2546delC, FANCG c.307+1G>C and c.1066C>T) existed at low prevalence (0.04-0.1%) in the whole-genome reference panel of 3554 Japanese individuals (3.5KJPN, Tohoku Megabank). Consistent with the paucity of the FA-C patients as opposed to the previous report (Blood 2000), the FANCC IVS4+4A mutation was absent in the 3.5KJPN database. We were able to examine the hematological outcomes in a subset of our cases (52 FA-A and 23 FA-G). Interestingly, the FA-G patients developed bone marrow failure (BMF) at a significantly younger age than FA-A patients (median age at onset of BMF: 3.1 years vs 5 years). Furthermore, the patients with the FANCA c.2546delC mutation had an increased risk of developing myelodysplastic syndrome (MDS) and acute myeloid leukemia (AML), compared to FA-A patients without the mutation. In the rare complementation groups of FA, two FA-B cases with complete loss of FANCB gene and one FA-I patient with N-terminal premature termination codons revealed severe somatic abnormalities, consistent with VACTERL-H association. Two FANCD1 (BRCA2) patients and one FANCN (PALB2) patients did not experience bone marrow failure but developed early-onset malignancies (immature teratoma, T-lymphoblastic lymphoma, adenosquamous lung carcinoma, Wilms tumor). Conclusion: This is the largest series of subtyped Japanese FA patients to date and the results would be useful for future clinical management. To provide molecular diagnosis for FA in Japan, we suggest to start with PCR-direct sequencing of the three common mutations (FANCA c.2546delC, FANCG c.307+1G>C and FANCG c.1066C>T) along with MLPA assay for FANCA. These analyses would enable the identification of about 50% of the mutant alleles. For the rest of the cases, WES or targeted-seq analysis should be useful, however, large deletions and aberrant splicing need to be kept in mind. Disclosures Takaori-Kondo: Pfizer: Honoraria; Novartis: Honoraria; Celgene: Honoraria, Research Funding; Bristol-Myers Squibb: Honoraria; Janssen Pharmaceuticals: Honoraria.
11
Yang, Yi, Yanan Kuang, Rocio Montes De Oca, Tobias Hays, Lisa Moreau, Naifang Lu, Brian Seed, and Alan D. D'Andrea. "Targeted disruption of the murine Fanconi anemia gene,Fancg/Xrcc9." Blood 98, no. 12 (December 1, 2001): 3435–40. http://dx.doi.org/10.1182/blood.v98.12.3435.
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Abstract Fanconi anemia (FA) is a human autosomal recessive cancer susceptibility disorder characterized by cellular sensitivity to mitomycin C and ionizing radiation. Six FA genes (corresponding to subtypes A, C, D2, E, F, and G) have been cloned, and the encoded FA proteins interact in a common cellular pathway. To further understand the in vivo role of one of these human genes (FANCG), we generated a targeted disruption of murine Fancg and bred mice homozygous for the targeted allele. Similar to the phenotype of the previously described Fancc−/− andFanca−/− mice, theFancg−/− mice had normal viability and no gross developmental abnormalities. Primary splenic lymphocytes, bone marrow progenitor cells, and murine embryo fibroblasts from theFancg−/− mice demonstrated spontaneous chromosome breakage and increased sensitivity to mitomycin C and, to a lesser extent, ionizing radiation. Fancg−/−lymphocytes had a defect in the FA pathway, based on their failure to activate the monoubiquitination of the downstream Fancd2protein in response to IR. Finally,Fancg−/− mice had decreased fertility and abnormal gonadal histology. In conclusion, disruption of theFancg gene confirms the role of Fancg in the FA pathway. The Fancg−/− mouse may be useful as an animal model for future gene therapy and cancer susceptibility studies.
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Si, Yue, Samantha Ciccone, Feng-Chun Yang, Jin Yuan, Daisy Zeng, Shi Chen, Henri J. van de Vrugt, et al. "Continuous in vivo infusion of interferon-gamma (IFN-γ) enhances engraftment of syngeneic wild-type cells in Fanca–/– and Fancg–/– mice." Blood 108, no. 13 (December 15, 2006): 4283–87. http://dx.doi.org/10.1182/blood-2006-03-007997.
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Abstract Fanconi anemia (FA) is a heterogeneous genetic disorder characterized by bone marrow (BM) failure and cancer susceptibility. Identification of the cDNAs of FA complementation types allows the potential of using gene transfer technology to introduce functional cDNAs as transgenes into autologous stem cells and provide a cure for the BM failure in FA patients. However, strategies to enhance the mobilization, transduction, and engraftment of exogenous stem cells are required to optimize efficacy prior to widespread clinical use. Hypersensitivity of Fancc–/– cells to interferon-gamma (IFN-γ), a nongenotoxic immune-regulatory cytokine, enhances engraftment of syngeneic wild-type (WT) cells in Fancc–/– mice. However, whether this phenotype is of broad relevance in other FA complementation groups is unresolved. Here we show that primitive and mature myeloid progenitors in Fanca–/– and Fancg–/– mice are hypersensitive to IFN-γ and that in vivo infusion of IFN-γ at clinically relevant concentrations was sufficient to allow consistent long-term engraftment of isogenic WT repopulating stem cells. Given that FANCA, FANCC, and FANCG complementation groups account for more than 90% of all FA patients, these data provide evidence that IFN-γ conditioning may be a useful nongenotoxic strategy for myelopreparation in FA patients.
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Pagano, Giovanni, Annarita Aiello Talamanca, Giuseppe Castello, Federico V. Pallardó, Adriana Zatterale, and Paolo Degan. "Oxidative stress in Fanconi anaemia: from cells and molecules towards prospects in clinical management." Biological Chemistry 393, no. 1-2 (January 1, 2012): 11–21. http://dx.doi.org/10.1515/bc-2011-227.
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Abstract Fanconi anaemia (FA) is a genetic disease featuring bone marrow failure, proneness to malignancies, and chromosomal instability. A line of studies has related FA to oxidative stress (OS). This review attempts to evaluate the evidence for FA-associated redox abnormalities in the literature from 1981 to 2010. Among 2170 journal articles on FA evaluated, 162 related FA with OS. Early studies reported excess oxygen toxicity in FA cells that accumulated oxidative DNA damage. Prooxidant states were found in white blood cells and body fluids from FA patients as excess luminol-dependent chemiluminescence, 8-hydroxy-deoxyguanosine, reduced glutathione/oxidized glutathione imbalance, and tumour necrosis factor-α. Some FA gene products involved in redox homeostasis can be summarized as follows: (a) FANCA, FANCC, and FANCG interact with cytochrome P450-related activities and/or respond to oxidative damage; (b) FANCD2 in OS response interacts with forkhead box O3 and ataxia telangiectasia mutated protein; (c) FANCG is found in mitochondria and interacts with PRDX3, and FA-G cells display distorted mitochondria and decreased peroxidase activity; (d) FANCJ (BACH1/BRIP1) is a repressor of haeme oxygenase-1 gene and senses oxidative base damage; (e) antioxidants, such as tempol and resveratrol decrease cancer incidence and haematopoietic defects in Fancd2-/- mice. The overall evidence for FA-associated OS may suggest designing chemoprevention studies aimed at delaying the onset of OS-related clinical complications.
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Shain, Kenneth H., Vasco Oliveira, Danielle Yarde, Linda Mathews, and William S. Dalton. "A Novel Upregulation of Fanconi Anemia/BRCA DNA Repair Pathway Is Observed Upon Co-Culture of Bone Marrow Stroma with Multiple Myeloma Cell Lines: A Potential New Participant(s) in Environment Mediated Drug Resistance." Blood 114, no. 22 (November 20, 2009): 2798. http://dx.doi.org/10.1182/blood.v114.22.2798.2798.
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Abstract Abstract 2798 Poster Board II-774 Multiple Myeloma (MM) is an incurable malignancy of mature clonal B cells. The refractory nature of MM has long been attributed to the acquisition of drug resistance. Traditionally, mechanisms of drug resistance have been defined by acquired changes in the expression or function of specific gene products. To this end, we have recently demonstrated that selected resistance to the cytotoxic agent melphalan correlated with increased expression of components of the Fanconi Anemia (FA)/BRCA DNA repair pathway and a concomitant increase in repair of DNA interstrand cross-links (ICLs).(Hazlehurst et al Cancer Res 2003; Chen et al Blood 2005) Further, the exogenous expression of specific FANC components in RPMI 8226 cell lines enhanced ICL repair, favored the release from melphalan-induced S-phase delay, and rendered these cells partially resistant to melphalan treatment. Together, these results suggest a causal relationship between increased expression of FA DNA repair components, increased DNA repair, and acquired resistance to melphalan. Over the past decade a large body of evidence has emerged demonstrating that in addition to drug resistance mechanisms intrinsic to the cancer cell, there exist dynamic, de novo mechanisms coordinated by the tumor microenvironment resulting in an environment-mediated drug resistance (EM-DR). As such, we examined the potential role of the microenvironment in regulating the FA/BRCA DNA repair pathway. FA pathway protein expression was evaluated with anti-sera to FANCD1/BRCA2, FANCC, FANCD2, FANCI, FANCG and BRCA1 in drug sensitive RPMI 8226 cells and melphalan resistant 8226/LR5 cells in co-culture with the HS-5 bone marrow stromal cell line. With these preliminary results we present three novel findings. First, we demonstrate that expression of FA/BRCA pathway components is regulated by intracellular interactions in both MM cells and bone marrow stromal cells (BMSCs). Second, we show that the acquisition of drug resistance alters FANC protein expression profiles upon co-culture. Third, in the HS-5 BMSCs, mono-ubiquitinated FANCD2 is observed in the absence of detectable FANCG. In RPMI 8226 cells, Western blot analysis demonstrated an acute (within 30minutes) and prolonged (up to 48hours) time-dependent increase in expression of FANCD2/BRCA2, FANCC, FANCD2, and BRCA1 upon incubation with BMSCs relative to MM cells incubated alone. However, no appreciated increases in FANCI or FANCG were noted under the same conditions. Incubation of 8226/LR5s with HS-5 BMSCs demonstrated a slightly different up-regulation of FA/BRCA pathway protein expression with addition of increased FANCI expression and no increase in FANCD2 or FANCC expression. We also examined FANC protein expression in the HS-5 cells. Interestingly, in the BMSCs significant differences were noted in the FANC expression profiles. Co-culture of RPMI 8226 cells with HS-5 cells demonstrated only modest elevations in FANCD2; however, co-culture with drug-resistant 8226/LR5s resulted in increased levels of FANCD2, FANCI and BRCA1. These data indicate that different tumor cells may alternately influence FA/BRCA-mediated DNA repair and potentially drug resistance in juxtaposed bone marrow stroma. Curiously, we also observed mono-ubiquitinated FANCD2 in the absence of any detectable levels of FANCG protein under co-culture conditions. As the FA/BRCA DNA repair pathway has been associated with cell cycle progression, we evaluated cell cycle kinetics under the co-culture conditions. The results of BrdU analysis demonstrated that the observed changes in FA/BRCA protein expression in MM and BMSC could not be fully explained by cell-cycle distribution. Therefore, within this report we demonstrate for the first time that microenvironmental interactions can modulate the FA/BRCA DNA repair pathway in MM and BMSCs. These results suggest that the FA/BRCA DNA damage repair pathway may be an important modulatory component of EM-DR. Importantly, the potential de novo drug resistance likely involves both the MM tumor cell and adjacent stromal cells. Current and future studies will attempt to examine a causal relationship between increased FANC expression and melphalan (and other drug) resistance seen in co-culture conditions, as well as to identify specific signaling molecules and mechanisms controlling the enhanced expression in both cell models. Disclosures: No relevant conflicts of interest to declare.
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von Lindern, Marieke, Emile van den Akker, Henri van de Vrugt, Godfrey Grech, Fre Arwert, Bob Lowenberg, and Ivo Touw. "Hyperoxia Induces Defective Epo-Induced Signal Transduction and Loss of Expansion Capacity in Fanca- and Fancg-Deficient Erythroid Progenitors." Blood 104, no. 11 (November 16, 2004): 2837. http://dx.doi.org/10.1182/blood.v104.11.2837.2837.
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Abstract Fanconi anemia (FA) is characterized by chromosomal instability and bone marrow hypoplasia. To explore the mechanism of FA-related bone marrow failure, we studied erythropoietin (Epo)-dependent proliferation and differentiation of erythroid progenitors expanded from mice lacking the Fanca or Fancg gene. Erythroid progenitors can be expanded in serum-free medium supplemented with Epo, Stem Cell Factor (SCF) and glucocorticoids. The expansion capacity of Fanca−/− and Fancg−/− erythroid progenitors was reduced and the progenitors differentiated to mature erythrocytes instead after 10 days of culture. When Epo-induced signal transduction was compared between progenitors expanded for 6 days from fetal livers of Fanca−/−, Fancg−/− and wt littermates, Epo-induced phosphorylation of STAT5 and ERK1/2 appeared to be severely reduced and delayed in progenitors lacking Fanca or Fancg. Both in wt, Fanca−/− and Fancg−/− progenitors the phosphorylation of STAT5 and ERK1/2 increased with rising Epo-concentrations, but at low concentrations 4-fold more Epo was required to obtained comparable Stat5 phosphorylation in FancA−/− or FancG−/− cells, while efficient phosphorylation at high concentrations was never achieved in Fanca or Fancg-deficient cells. As a consequence, the regulation of Epo-target genes like p21WAF, Gilz, SOCS3, Spi2.1 and BclXL was impaired. In contrast to downstream signaling intermediates, Epo-induced phosphorylation of the Epo-receptor and its associated kinase JAK2 was not affected. Stem Cell Factor-induced c-Kit activation and downstream Erk1/2 phosphorylation was also equally efficient in wt, Fanca−/− and Fancg−/− progenitors. Thus, the signaling defect specifically seems to affect targets downstream of Jak2. Surprisingly, Epo-induced activation of Stat5 was unaffected in freshly isolated bone marrow cells. Comparison of Epo-induced Stat5 phosphorylation in erythroid progenitors cultured at atmospheric (20%) and physiologic (3%) oxygen revealed that Stat5 phosphorylation was only lost upon expansion of the progenitors at atmospheric (hyperoxic) conditions. Hyperoxia can damage many biomolecules among which DNA. In contrast to wt cells, Fanca−/− and Fancg−/− progenitors may not be able to repair DNA-damage induced by atmospheric oxygen. To examine whether increased DNA damage could impair Epo-induced signal transduction, we expanded erythroid progenitors from fetal livers of mice lacking the DNA-repair enzyme Ercc1. Ercc1 is directly involved in repair of DNA crosslinks, while a defective FA pathway renders cells hypersensitive to DNA-crosslinks. Epo-induced phosphorylation of Stat5 was impaired in Ercc1−/− progenitors, indicating that deficient repair of DNA damage, rather than FA-pathway specific mechanisms may cause the observed signaling defect. To substantiate this finding we treated wt and p53−/− progenitors with increasing concentrations of mitomycin C (MMC). Sublethal concentrations of MMC abrogated the renewal capacity and impaired Epo-induced phosporylation of Stat5 and Erk1/2 in wt but not in p53−/− erythroid progenitors. Together the data suggest that impaired progenitor expansion and Stat5 phosphorylation is not a specific aspect of cells lacking the fanconi pathway, but a general aspect of a p53-dependent response to genomic stress.
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Munkhjargal, Anudari, Myung-Jin Kim, Da-Yeon Kim, Young-Jun Jeon, Young-Hoon Kee, Lark-Kyun Kim, and Yong-Hwan Kim. "Promyelocytic Leukemia Proteins Regulate Fanconi Anemia Gene Expression." International Journal of Molecular Sciences 22, no. 15 (July 21, 2021): 7782. http://dx.doi.org/10.3390/ijms22157782.
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Promyelocytic leukemia (PML) protein is the core component of subnuclear structures called PML nuclear bodies that are known to play important roles in cell survival, DNA damage responses, and DNA repair. Fanconi anemia (FA) proteins are required for repairing interstrand DNA crosslinks (ICLs). Here we report a novel role of PML proteins, regulating the ICL repair pathway. We found that depletion of the PML protein led to the significant reduction of damage-induced FANCD2 mono-ubiquitination and FANCD2 foci formation. Consistently, the cells treated with siRNA against PML showed enhanced sensitivity to a crosslinking agent, mitomycin C. Further studies showed that depletion of PML reduced the protein expression of FANCA, FANCG, and FANCD2 via reduced transcriptional activity. Interestingly, we observed that damage-induced CHK1 phosphorylation was severely impaired in cells with depleted PML, and we demonstrated that CHK1 regulates FANCA, FANCG, and FANCD2 transcription. Finally, we showed that inhibition of CHK1 phosphorylation further sensitized cancer cells to mitomycin C. Taken together, these findings suggest that the PML is critical for damage-induced CHK1 phosphorylation, which is important for FA gene expression and for repairing ICLs.
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Yamamoto, Kazuhiko, Masamichi Ishiai, Nobuko Matsushita, Hiroshi Arakawa, Jane E. Lamerdin, Jean-Marie Buerstedde, Mitsune Tanimoto, Mine Harada, Larry H. Thompson, and Minoru Takata. "Fanconi Anemia FANCG Protein in Mitigating Radiation- and Enzyme-Induced DNA Double-Strand Breaks by Homologous Recombination in Vertebrate Cells." Molecular and Cellular Biology 23, no. 15 (August 1, 2003): 5421–30. http://dx.doi.org/10.1128/mcb.23.15.5421-5430.2003.
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ABSTRACT The rare hereditary disorder Fanconi anemia (FA) is characterized by progressive bone marrow failure, congenital skeletal abnormality, elevated susceptibility to cancer, and cellular hypersensitivity to DNA cross-linking chemicals and sometimes other DNA-damaging agents. Molecular cloning identified six causative genes (FANCA, -C, -D2, -E, -F, and -G) encoding a multiprotein complex whose precise biochemical function remains elusive. Recent studies implicate this complex in DNA damage responses that are linked to the breast cancer susceptibility proteins BRCA1 and BRCA2. Mutations in BRCA2, which participates in homologous recombination (HR), are the underlying cause in some FA patients. To elucidate the roles of FA genes in HR, we disrupted the FANCG/XRCC9 locus in the chicken B-cell line DT40. FANCG-deficient DT40 cells resemble mammalian fancg mutants in that they are sensitive to killing by cisplatin and mitomycin C (MMC) and exhibit increased MMC and radiation-induced chromosome breakage. We find that the repair of I-SceI-induced chromosomal double-strand breaks (DSBs) by HR is decreased ∼9-fold in fancg cells compared with the parental and FANCG-complemented cells. In addition, the efficiency of gene targeting is mildly decreased in FANCG-deficient cells, but depends on the specific locus. We conclude that FANCG is required for efficient HR-mediated repair of at least some types of DSBs.
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Gruber, Joshua James, Anosheh Afghahi, Alyssa Hatton, Danika Scott, Alex McMillan, James M. Ford, and Melinda L. Telli. "Talazoparib beyond BRCA: A phase II trial of talazoparib monotherapy in BRCA1 and BRCA2 wild-type patients with advanced HER2-negative breast cancer or other solid tumors with a mutation in homologous recombination (HR) pathway genes." Journal of Clinical Oncology 37, no. 15_suppl (May 20, 2019): 3006. http://dx.doi.org/10.1200/jco.2019.37.15_suppl.3006.
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3006 Background: Talazoparib, a PARP inhibitor, is active in germline BRCA1/2 mutant advanced HER2-negative breast cancer, but its activity beyond BRCA1/2 is unknown. We conducted a single institution phase II trial to evaluate talazoparib in patients (pts) with advanced HER2-negative breast cancer or other solid tumors with a germline (g) or somatic (s) alteration in HR pathway genes not including BRCA1/2. Methods: Eligible pts had measurable disease, lacked a germline or somatic mutation in BRCA1/2, received at least one prior therapy for advanced HER2-negative breast cancer or other solid tumor and had a HR pathway gene mutation: PALB2, CHEK2, ATM, NBN, BARD1, BRIP1, RAD50, RAD51C, RAD51D, MRE11, ATR, PTEN, FANCA, FANCC, FANCD2, FANCE, FANCF, FANCG, FANCL. Pts with no progression on or within 8 weeks of their last platinum dose were eligible. Pts were treated with talazoparib 1 mg po daily until disease progression. Response was assessed every 8 +/- 1 weeks. If 2 or more responses were observed in 10 pts in stage I, the study would proceed to stage II and enroll 10 additional pts. The null hypothesis of a ≤ 5% objective response rate would be rejected if at least 3 of 20 respond. Results: Twenty pts were enrolled; 13 breast cancer (12 HR+/HER2-, 1 TNBC) and 7 non-breast cancer (pancreas, colon, uterine, testicular, parotid salivary). Median age was 54 years. Of 12 response evaluable pts with breast cancer, 3 had a RECIST response (ORR = 25%, 2 g PALB2, 1 g CHEK2/g FANCA/sPTEN) and 3 additional pts (g PALB2, s ATR, s PTEN) had SD ≥ 6 months (CBR = 50%). No responses were seen in non-breast tumors; 2 (g CHEK2 testicular, g ATM colon) had SD ≥ 6 months. Talazoparib was well tolerated; 5 patients required dose reduction for hematologic toxicity. Results of tumor HR deficiency status assessment from metastatic biopsies and serial ctDNA profiling will be presented. Conclusions: In this proof-of-concept phase II study, single agent talazoparib demonstrated activity in HER2-negative advanced breast cancer pts with a HR pathway mutation beyond BRCA1/2. Further evaluation of talazoparib in this population is warranted. Clinical trial information: NCT02401347.
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Kennedy, R. D., P. Stuckert, E. Archila, M. De LaVega, C. Chen, L. Moreau, and A. D'Andrea. "Sensitivity of tumor cells deficient in the fanconi anemia pathway to inhibition of ataxia telangiectasia mutated (ATM)." Journal of Clinical Oncology 25, no. 18_suppl (June 20, 2007): 10509. http://dx.doi.org/10.1200/jco.2007.25.18_suppl.10509.
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10509 Loss of the fanconi anemia (FA) pathway function has been described in a number of sporadic tumor types including breast, ovarian, pancreatic, head and neck and hematological malignancies. Functionally, the FA pathway responds to stalled DNA replication following DNA damage. Given the importance of the FA pathway in the response to DNA damage, we hypothesized that cells deficient in this pathway may become hyper-dependent on alternative DNA damage response pathways in order to respond to endogenous genotoxic stress such as occurs during metabolism. Therefore, targeting these alternative pathways could offer therapeutic strategies in FA pathway deficient tumors. To identify new therapeutic targets we treated FA pathway competent and deficient cells with a DNA damage response siRNA library, that individually knocked out 230 genes. We identified a number of gene targets that were specifically toxic to FA pathway deficient cells, amongst which was the DNA damage response kinase Ataxia Telangiectasia Mutated (ATM). To test the requirement for ATM in FA pathway deficient cells, we interbred Fancg ± Atm± mice. Consistent with the siRNA screen result, Fancg-/- Atm-/- mice were non viable and Fancg± Atm-/- and Fancg-/- Atm ± progeny were less frequent that would have been expected. Several human cell lines with FA gene mutations were observed to have constitutive activation of ATM which was markedly reduced on correction with the appropriate wild-type FA gene. Interestingly, FA pathway deficient cells, including the FANCC mutant and FANCG mutant pancreatic cancer cell lines, were selectively sensitive to monotherapy with the ATM inhibitor KU55933, as measured by dose inhibition and colony count assays. FA pathway deficient cells also demonstrated an increased level of chromosomal breakage, cell cycle arrest and apoptosis following KU55933 treatment when compared to FA pathway corrected cells. We conclude that FA pathway deficient cells have an increased requirement for ATM activation in order to respond to sporadic DNA damage. This offers the possibility that monotherapy with ATM inhibitors could be a therapeutic strategy for tumors that are deficient for the FA pathway. No significant financial relationships to disclose.
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Ramirez, M. H., C. Adelfalk, M. Kontou, M. Hirsch-Kauffmann, and M. Schweiger. "The Cellular Control Enzyme PolyADP Ribosyl Transferase Is Eliminated in Cultured Fanconi Anemia Fibroblasts at Confluency." Biological Chemistry 384, no. 1 (January 27, 2003): 169–74. http://dx.doi.org/10.1515/bc.2003.018.
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AbstractFanconi anemia (FA) is a hereditary disease of unknown pathogenic mechanisms, although mutations in seven different genes can be causative. Six of these genes have been cloned and sequenced. Only slight homology to the DNA of any other known gene has been found with the exception of FANCG which is identical to XRCC9. The function of these genes, including XRCC9, is presently unknown. Since pADP ribosyl transferase (pADPRT) plays a role in apoptosis, and apoptosis is affected in FA cells, we studied the correlation between pADPRT and FA cells. We reinvestigated the previously reported lack of pADPRT activity in fibroblasts from patients with Fanconi anemia. Here we describe the role of the lower redox potential of FA cells and demonstrate that this is an efficient strategy in the prevention of cell death due to the lack of energy under oxidative stress. This strategy is advantageous for the cells under the nonreplicative condition of confluency in which the risk of mutation is low and the prevention of apoptosis permits cell survival. pADPRT is not diminished to the same extent in all complementation groups of FA. It is prominent in FANCA, FANCG and FANCF cells, indicating that these genes control pADPRT diminution. Our experiments suggest that the pADPRT level is linked with the oxidoreduction reactions seen in FA.
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Mateo, Joaquin, Nuria Porta, Ursula Brigid McGovern, Tony Elliott, Robert J. Jones, Isabel Syndikus, Christy Ralph, et al. "TOPARP-B: A phase II randomized trial of the poly(ADP)-ribose polymerase (PARP) inhibitor olaparib for metastatic castration resistant prostate cancers (mCRPC) with DNA damage repair (DDR) alterations." Journal of Clinical Oncology 37, no. 15_suppl (May 20, 2019): 5005. http://dx.doi.org/10.1200/jco.2019.37.15_suppl.5005.
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5005 Background: We previously reported the antitumor activity of olaparib (400mg BID) against molecularly unselected mCRPC (TOPARP-A; Mateo et al NEJM 2015). We now report TOPARP-B, a phase II trial for patients with mCRPC preselected for putatively pathogenic DDR alterations. Methods: Patients with mCRPC progressing after ≥ 1 taxane chemotherapy underwent targeted sequencing of tumor biopsies and were deemed eligible when alterations (germline or somatic; mono- or bi-allelic) in any DDR gene were detected. Patients were randomized 1:1 under a “pick-the-winner” design to 400mg or 300mg of olaparib BID, aiming to exclude ≤30% response rate (RR) in either arm. The primary endpoint RR was defined as radiological response (RECIST 1.1) and/or PSA50% fall and/or CTC count conversion (Cellsearch; ≥5 to < 5), confirmed after 4-weeks. Analyses of RR per gene alteration subgroup was pre-planned. Secondary endpoints included progression-free survival (PFS), tolerability. Results: Overall, 98 patients (median age 67.6y) were randomized, with 92 patients treated and evaluable for the primary endpoint (70 RECIST-evaluable; 89 PSA50%-evaluable; 55 CTC-evaluable). All had progressed on ADT; 99% were post-docetaxel, 90% post-abiraterone/enzalutamide, 38% post-cabazitaxel. The overall RR was 54% (95%CI 39-69%, meeting threshold for primary endpoint) in the 400mg cohort and 37% (95%CI 23-53%) in the 300mg cohort. With a median follow-up of 17.6 months (mo), the overall median PFS (mPFS) was 5.4 mo. Subgroup analyses per altered gene identified indicated response rates for: BRCA1/2 of 80% (24/30; mPFS 8.1mo); PALB2 57% (4/7; mPFS 5.3mo); ATM 37% (7/19; mPFS 6.1mo); CDK12 25% (5/20; mPFS 2.9mo); others [ATRX, CHEK1, CHEK2, FANCA, FANCF, FANCG, FANCI, FANCM, RAD50, WRN] 20% (4/20; mPFS 2.8mo). The highest PSA50% response rates were observed in the BRCA1/2 (22/30; 73%) and PALB2 (4/6; 67%) subgroups. Conclusions: Olaparib has antitumor activity against heavily pre-treated mCRPC with DDR gene defects, with BRCA1/2 aberrant tumors being most sensitive but with confirmed responses in patients with other DDR alterations. Clinical trial information: NCT01682772.
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Levitus, Marieke, Hans Joenje, and Johan P. de Winter. "The Fanconi Anemia Pathway of Genomic Maintenance." Analytical Cellular Pathology 28, no. 1-2 (January 1, 2006): 3–29. http://dx.doi.org/10.1155/2006/974975.
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Fanconi anemia (FA), a recessive syndrome with both autosomal and X-linked inheritance, features diverse clinical symptoms, such as progressive bone marrow failure, hypersensitivity to DNA cross-linking agents, chromosomal instability and susceptibility to cancer. At least 12 genetic subtypes have been described (FA-A, B, C, D1, D2, E, F, G, I, J, L, M) and all except FA-I have been linked to a distinct gene. Most FA proteins form a complex that activates the FANCD2 protein via monoubiquitination, while FANCJ and FANCD1/BRCA2 function downstream of this step. The FA proteins typically lack functional domains, except for FANCJ/BRIP1 and FANCM, which are DNA helicases, and FANCL, which is probably an E3 ubiquitin conjugating enzyme. Based on the hypersensitivity to cross-linking agents, the FA proteins are thought to function in the repair of DNA interstrand cross-links, which block the progression of DNA replication forks. Here we present a hypothetical model, which not only describes the assembly of the FA pathway, but also positions this pathway in the broader context of DNA cross-link repair. Finally, the possible role for the FA pathway, in particular FANCF and FANCB, in the origin of sporadic cancer is discussed.
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Ma, Ke, Hao Rong, Changbin Zhu, Huachuan Zhang, Jintao He, Bo Xiao, Jiang Zhu, and Jun Peng. "Clinical characters of somatic DNA damage repair (DDR) gene mutations in Chinese NSCLC patients." Journal of Clinical Oncology 38, no. 15_suppl (May 20, 2020): e13665-e13665. http://dx.doi.org/10.1200/jco.2020.38.15_suppl.e13665.
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e13665 Background: Chinese NSCLC patients displayed unique genetic profile. NSCLC patients with DNA damage repair gene mutations showed distinct clinical appearance as well as superior response to immunotherapy. This study aims to disclose distribution of DNA damage mutations as well as their clinical characters in Chinese NSCLC patients. Methods: One hundred and ten patients with pathologically confirmed NSCLC were consecutively recruited from 2016-2018 in our center. Genomic DNA was captured and sequenced for 508 cancer related genes by BGI-seq 500. Raw data was processed followed by variant calling via in-house developed pipeline. Results: 89 (of 110) patients with median age of 55 years old (33-74 years) were included into final analysis due to complete follow-up as well as sufficient FFPE tissue for successful genomic profiling. Among them, 12 patients were squamous NSCLC, 72 patients were non-squamous NSCLC. Among 89 patients, 22 patients (24.7%) carried 36 mutations in 21 DDR genes (BRCA2 n = 6, BAP1 n = 3, ARID1A n = 2, ATM n = 2, ATR n = 2, BLM n = 2, FANCF n = 2, PARP1 n = 2,BARD1 n = 1,BRCA1 n = 1,CHEK2 n = 1, FANCD2 n = 1,FANCE n = 1,FANCG n = 1,FANCM N = 1,NBN n = 1,PARP3 n = 1,POLQ n = 1,RAD50 n = 1,RAD51C n = 1), most of them belongs to homologous recombination repair pathway (n = 19, 21.34%).8 patients carried compound mutations in DDR genes. No difference was found in age of initial diagnosis between DDR mutated and DDR wild type patients. While, ever smokers (RR (95%CI): 2.12 (1.37-3.20),P = 0.003), squamous NSCLC (RR(95%CI):4.47 (1.62-12.03), P = 0.006), male (RR (95%CI): 1.77(1.23-2.44),P = 0.006) displayed significantly higher prevalence of DDR gene mutations. Only one stage I patient carried DDR mutation(P = 0.008). Conclusions: In this study, mutation spectrum of DDR genes was proposed in Chinese NSCLC patients. DDR gene mutations more possibly occurred in male, higher disease stage as well as in squamous NSCLC and smokers. These findings show potential relevance to disease prognosis which needs further investigation.
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Brauner, Raja, Joelle Bignon-Topalovic, Anu Bashamboo, and Ken McElreavey. "Pituitary stalk interruption syndrome is characterized by genetic heterogeneity." PLOS ONE 15, no. 12 (December 3, 2020): e0242358. http://dx.doi.org/10.1371/journal.pone.0242358.
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Pituitary stalk interruption syndrome is a rare disorder characterized by an absent or ectopic posterior pituitary, interrupted pituitary stalk and anterior pituitary hypoplasia, as well as in some cases, a range of heterogeneous somatic anomalies. A genetic cause is identified in only around 5% of all cases. Here, we define the genetic variants associated with PSIS followed by the same pediatric endocrinologist. Exome sequencing was performed in 52 (33 boys and 19 girls), including 2 familial cases single center pediatric cases, among them associated 36 (69.2%) had associated symptoms or syndromes. We identified rare and novel variants in genes (37 families with 39 individuals) known to be involved in one or more of the following—midline development and/or pituitary development or function (BMP4, CDON, GLI2, GLI3, HESX1, KIAA0556, LHX9, NKX2-1, PROP1, PTCH1, SHH, TBX19, TGIF1), syndromic and non-syndromic forms of hypogonadotropic hypogonadism (CCDC141, CHD7, FANCA, FANCC, FANCD2, FANCE, FANCG, IL17RD, KISS1R, NSMF, PMM2, SEMA3E, WDR11), syndromic forms of short stature (FGFR3, NBAS, PRMT7, RAF1, SLX4, SMARCA2, SOX11), cerebellum atrophy with optic anomalies (DNMT1, NBAS), axonal migration (ROBO1, SLIT2), and agenesis of the corpus callosum (ARID1B, CC2D2A, CEP120, CSPP1, DHCR7, INPP5E, VPS13B, ZNF423). Pituitary stalk interruption syndrome is characterized by a complex genetic heterogeneity, that reflects a complex phenotypic heterogeneity. Seizures, intellectual disability, micropenis or cryptorchidism, seen at presentation are usually considered as secondary to the pituitary deficiencies. However, this study shows that they are due to specific gene mutations. PSIS should therefore be considered as part of the phenotypic spectrum of other known genetic syndromes rather than as specific clinical entity.
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Maguire, Alanna, Xianfeng Chen, Lee Wisner, Colleen Ramsower, Betty Glinsmann-Gibson, Michael S. McGrath, and Lisa M. Rimsza. "Enhanced DNA Repair and Genomic Stability in HIV(+) Diffuse Large B Cell Lymphoma of Germinal Center Origin." Blood 132, Supplement 1 (November 29, 2018): 1570. http://dx.doi.org/10.1182/blood-2018-99-116571.
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Abstract Introduction: HIV infected individuals are 17x more likely to receive a diagnoses of Diffuse Large B cell Lymphoma (DLBCL) compared to their uninfected counterparts. Moreover, DLBCL is more aggressive in HIV infected individuals, with up to 70% of patients being primary refractory to chemotherapeutic regimens. However, the molecular pathology driving the aggressive nature of HIV related DLBCL is poorly understood. Here, we have assessed the genomic and transcriptional differences between HIV(+) and HIV(-) DLBCL in order to identify the mechanisms driving the enhanced aggressive and refractory nature of HIV related DLBCL. Methods: A total of 66 cases, including 27 HIV(+) from the AIDS Cancer Specimen Resource Network (https://acsr.ucsf.edu/) and 39 HIV(-) institutional cases were included in this study. Fresh H&Es were reviewed by a hematopathologist to validate diagnosis and determine tumor content. Samples with <60% tumor content were macro-dissected. A total of 4x 5µm FFPE sections per sample were used for DNA and RNA extraction. DNA damage at the gross aberration level was assessed using array comparative genomic hybridization (aCGH). NanoString digital gene expression profiling (GEP) was used to perform the Lymph2Cx DLBCL Cell of Origin molecular subtyping assay (Germinal Center B-cell/ GCB, Activated B-Cell/ ABC, Unclassifiable/UNC) and to assess the differential expression of genes with known roles in cancer using the PanCancer Pathways panel. Results: Both the HIV(+) and HIV(-) cohorts were found to be GCB enriched with GCB-ABC-UNC distributions of 70%(19/27)-11%(3/27)-19%(5/27) and 54%(21/39)-33%(13/39)-13%(5/39) respectively. For both statistical and biological reasons, analysis was restricted to the GCB-DLBCL subtype only. Array CGH revealed the HIV(+) tumors have less DNA aberrations than their HIV(-) counterparts, indicative of enhanced genomic stability. GEP GSEA analysis revealed significant differences in the HIV(+) cohort compared to the HIV(-) cohort; including increases in DNA replication (MCM2, MCM4, MCM7) and Cell Cycle progression (CDC25A, CCNA2, CCNB1, E2F1) related genes as well as decreases in cell cycle negative regulators (CDKN1A, CDKN1B, CDKN2B, RB1), pro-apoptotic BCL2 genes (BAX, BIM, BMF, PUMA, BNIP3) and pro-survival BCL2 genes (BCL2, BCLW). Analyses also revealed significant increases in DNA repair related genes, particularly those with roles in the Fanconi Anemia-Homologous Recombination-Translesion Synthesis axis (FANCA, FANCD1/BRCA2, FANCE, FANCG, FANCR/RAD51, FANCS/BRCA1, FANCT, FANCV/MAD2L2). Conclusions: Our results show, for the first time, that GCB DLBCL arising in HIV infected individuals possesses a distinct molecular pathology to that which arises in uninfected individuals. These results indicate that the aggressive nature of HIV(+) GCB DLBCL is mediated by increased proliferation in conjunction with reduced cell cycle inhibitory capabilities and potentiated by heightened DNA repair that promotes genomic stability. Moreover, it is probable that the bolstered DNA repair capabilities confer an innate ability to repair DNA damage resulting from the administration of genotoxic chemotherapeutic agents and may be the mechanism underlying the high primary refractory rate to chemotherapeutics in HIV related DLBCL. Disclosures Rimsza: NanoString: Other: Inventor on the patent for the Lymph2Cx assay.
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Neviere, Zoé, Agathe Ricou, Dominique Vaur, Etienne Muller, Sophie Krieger, Flavie Boulouard, Laurent Castera, et al. "Outcomes of patients with metastatic castration resistant prostate cancer according to somatic sequencing of domage DNA repair genes." Journal of Clinical Oncology 38, no. 15_suppl (May 20, 2020): e17595-e17595. http://dx.doi.org/10.1200/jco.2020.38.15_suppl.e17595.
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e17595 Background: More than 20% of mCRPC present somatic DNA damage repair gene mutation (DDR). If some data are available for germline mutations, there are few studies describing the relation between somatic alterations and response to standard therapies in mCRPC. Methods: To describe outcomes of mCRPC patients treated with standard therapies according to somatic DDR status.A retrospective somatic analysis on a 69-gene panel was conducted among mCRPC patients. Progression-free survival (PFS) of first-line treatment was analyzed according to somatic DDR mutation as primary endpoint. First exposition to taxanes chemotherapy and PFS2 (time to the second event of disease progression) depending on therapeutic sequences (new-generation hormonotherapy [NGHT]/taxanes chemotherapy) were also analyzed. Results: Among 83 patients tested, 33 (40%) had a somatic DDR mutation, including 10 ATM, 5 BRCA2, 4 CHEK2, 3 CDK12, 3 FANCG, 1 BLM, 1 CHEK1, 1 FANCF, 1 FANCI, 1 FANCM, 1 MDC1, 1 MRE11A and 1 PALB2. In first-line treatment, 20 patients received taxanes and 63 NGHT. Median PFS was 9.8 months for mutated patients and 8.4 months for non-mutated patients (p = 0.9). For mutated patients, median PFS was 12.3 months in taxanes group and 9.8 months in NGHT group, (p = 0.74). One BRCA2-mutated patient treated by docetaxel was particularly long responder (PFS = 39.2 months). PFS of first exposition to taxanes was 8.2 months among mutated patients and 5.7 months among non-mutated patients (p = 0.31). Patients with BRCA1/BRCA2/ATM mutations had longer PFS compared to the others patients (10.6 months versus 5.5 months, p = 0.04). Ten patients received the chemotherapy-NGHT sequence (CHS) and 28 the NGHT-chemotherapy sequence (HCS). PFS2 were 16.5 months for mutated patients, whatever the sequence, and 11.7 months among non-mutated patients (p = 0.07). The 3 mutated patients (1 BRCA1, 1 BRCA2, 1 ATM mutation) had long PFS2 in the CHS (49.8 months). PFS2 of mutated patients in HCS was 15.6 months. Conclusions: Mutated mCRPC patients benefit from standard therapies, with long responders to taxanes among patients with BRCA1/2 and ATM mutations.
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Yang, Ping, Guanghui Ji, Qionghui Jiang, Zejun Lu, Yao Yu, Yadong Yang, and Ge Jin. "Mutation profiles in circulating tumor DNA (ctDNA) to predict the efficacy of sorafenib treatment in patients with advanced hepatocellular carcinoma (HCC)." Journal of Clinical Oncology 37, no. 15_suppl (May 20, 2019): e15648-e15648. http://dx.doi.org/10.1200/jco.2019.37.15_suppl.e15648.
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e15648 Background: To find biomarkers from blood samples through ctDNA and leucocyte genomic DNA sequencing to predict the efficacy of sorafenib. Methods: CtDNA and leucocyte genomic DNA were extracted from patients with advanced hepatocellular carcinoma for sequencing analysis before and after targeted therapy with sorafenib. Mutation profiles were obtained by deep sequencing of 620 selected genes for further bioinformatics analysis. Gene mutation frequency and status were further integrated with clinical data to dynamically monitor and predict the efficacy of sorafenib. Results: Twelve patients with advanced hepatocellular carcinoma admitted to our hospital from March 2017 to September 2018 were enrolled. A total of 27 blood samples were collected for further analysis. The results showed that there were significant differences in BRD4 and FANCG gene mutation abundance between therapeutic effect group and poor effect group (p = 0.014). The mutations of BRD4, FANCG and TP53 were associated with shorter PFS (BRD4 mutation type vs wild type: 12 weeks vs 32 weeks, p = 0.0011; FANCG mutation type vs wild type: 16 weeks vs 32 weeks, p = 0.0103; TP53 mutation type vs wild type: 8 weeks vs 32 weeks, p = 0.0095). Furthermore, we found another 19 genes (SDHA, OTUD7A, TMPRSS2, NAV3, AKT1, EIF2S2, DICER1, MAP3K1, RAD9A, TCF3, IRS1, PIK3C2G, CTCF, TSHZ2, ITPKB, KDM5C, STAG2, AMER1, CDC27) associated with therapeutic efficacy, and dynamic changes of these 19 genes mutation alleys could predict therapeutic efficacy. Conclusions: Our results showed that mutations in ctDNA could predict the efficacy and prognosis of sorafenib for advanced hepatocellular carcinoma, and that dynamic monitoring of mutation frequency in ctDNA of peripheral blood samples could be an additional method to monitor therapeutic efficacy of sorafenib. The clinical significance of the mutations of BRD4, FANCG, TP53 and another 19 genes we found needs to be confirmed on a larger scale.
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de Winter, Johan P., Quinten Waisfisz, Martin A. Rooimans, Carola G. M. van Berkel, Lucine Bosnoyan-Collins, Noa Alon, Madeleine Carreau, et al. "The Fanconi anaemia group G gene FANCG is identical with XRCC9." Nature Genetics 20, no. 3 (November 1998): 281–83. http://dx.doi.org/10.1038/3093.
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Durrani, Jibran, Hassan Awada, Wenyi Shen, Cassandra M. Kerr, Vera Adema, Sunisa Kongkiatkamon, Ashwin Kishtagari, et al. "FA Gene Carrier Status Predisposes to Myeloid Neoplasms and Bone Marrow Failure in Adults." Blood 134, Supplement_1 (November 13, 2019): 452. http://dx.doi.org/10.1182/blood-2019-130142.
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Background. As compared to somatic mutations, our understanding of germline (GL) variants' contribution to myeloid neoplasia (MN) is less advanced. To date, only a limited number of leukemogenic GL alterations have been identified in adult cases, but WHO Classification of Leukemias does distinguish a separate entity of MN with GL predisposition. Conceptually, pathogenetic variants can be sub-classified according to their penetrance ranging from "leukemia genes" (e.g., CEBPA, NPM1, RUNX1) to "leukemia risk genes" (e.g., DKC, FANCA, SBDS). Methods. Using publicly available data we compiled a cohort of 600 MN for which whole exome sequencing was available for both tumor and GL DNA. With the aid of an extensive calling pipeline we investigated the presence of pathogenic GL variants in 677 genes known to be involved in cancer. Application of a stringent pathogenicity score and somatic contamination filters allowed for identification of 5880 variants (with frequency <1% in general population) for further analysis. Results. Overall 14 rare allele homozygous alterations and compound heterozygotes were found in 7/23 Fanconi anemia (FA) genes (e.g., BRCA2, SLX4, FANCI, FANCD2, FANCA, ERCC4, and BRIP1). Biparental inheritance in FA genes occurred more than would be expected given the total number of rare variants in the patient cohort suggesting a disease predisposition for individuals with biparental rare FA gene variants constituting a non-clonal first hit with a long latency period. This finding prompted us to prospectively analyze the frequency of FA variants in an independent MN cohort of 729 [MDS, MDS/MPN and secondary AML (sAML)] and 268 marrow failure (BMF) cases (AA, PNH). We have defined Tier-1 GL variants as those known variants (described in various FA data bases), pathogenic variants according to ASHG and new frame-shift or missense variants scored deleterious by using >4/6 applied scoring algorithms. All other GL variants including possibly pathogenic according to ASHG were assigned to Tier-2 group. The analysis revealed a total of 39 mutations (Tier 1); 80 Tier 2 variants were not included in this stringent analysis. There were 13 frame-shifts and 17 stop-gain, and there was 1 case in which 2 genes or 2 alleles were affected. The most commonly affected gene was BRCA2 with 6/39 alterations (6 variants) followed my BRCA1 in 5/39 (5 variants), FANCD2 with 5/39 alterations (4 variants), FANCA/FANCB and FANCG each with 3/39 (3 variants), FANCC in 2/39 (2 variants to a total frequency of 3.2% (23/729) and 6% (16/268) in MN and BMF, respectively. If combined, the frequency of these variants in control cohort would be expected to be 0.09%. The average age of presentation of FA carriers was 62 vs. 67 yrs. in all other MN (P=.01). The average age of presentation in BMF with FA variant was 30 vs. 45 yrs (P=0.02) in the rest of BMF group. Remarkably, we have identified 33 patients with AA who progressed to MDS, mostly del(7q), within this group: 9/33 FA Tier-1 carriers were found among these patients vs. 16/238 in non-progressors. Conclusions. The prevalence of FA gene variants are enriched in patients with MN (primarily MDS and sAML) and in BMF (primarily AA and PNH) suggesting that the carrier status of these genes may not follow an autosomal recessive trait and that the presence of a healthy allele is not entirely protective. The impact of carrier configuration may be missed due to long latency, competing morbidities and the resultant incomplete penetrance. Mono-allelic forms under genomic insult and replicative stress conditions, such as emergent increased hematopoiesis, endogenous or exogenous DNA damaging toxins and other environmental factors, might be responsible for instability in DNA repair responses. The clinical implications of it includes responsiveness to chemotherapy and in fact the choice of chemotherapy, transplant conditioning and for tumor surveillance e.g., in patients with AA at risk for MDS progression. Disclosures Nazha: Tolero, Karyopharma: Honoraria; Incyte: Speakers Bureau; Abbvie: Consultancy; Novartis: Speakers Bureau; Jazz Pharmacutical: Research Funding; Daiichi Sankyo: Consultancy; MEI: Other: Data monitoring Committee. Saunthararajah:Novo Nordisk: Consultancy; EpiDestiny: Consultancy, Equity Ownership, Patents & Royalties. Sekeres:Millenium: Membership on an entity's Board of Directors or advisory committees; Syros: Membership on an entity's Board of Directors or advisory committees; Celgene: Membership on an entity's Board of Directors or advisory committees. Maciejewski:Alexion: Consultancy; Novartis: Consultancy.
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An, Wenbin, Ye Guo, Yumei Chen, Yao Zou, Xiaojuan Chen, Wenyu Yang, Lixian Chang, Yang Wan, and Xiaofan Zhu. "The Significance of the Next Generation Targeted Sequencing in the Precise Diagnosis of Pediatric Acquire Aplastic Anemia and Inherited Bone Marrow Failure Syndromes." Blood 128, no. 22 (December 2, 2016): 3908. http://dx.doi.org/10.1182/blood.v128.22.3908.3908.
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Abstract Background Diagnosis of inherited bone marrow failure syndromes (IBMFs) depend on classic clinical manifestation including early onset, physical anomalies, family history of cancer and/or bone marrow failure and chromosome breakage testing (MMC and/or DEB), mutation analyses and bone marrow chromosome analyses. At present, more than 70 pathogenic gene mutations had been identified. However, in some patients, physical anomalies is absent or delayed, and were misdiagnosed as acquired aplastic anemia(AA). Genetic analysis is very important to establish a precise diagnosis, predict cancer risk, direct treatment and genetic counseling. In this study, we focus on the application of next generation targeted sequencing in precise diagnosis of pediatric acquired AA/IBMFs, and the association between genetic abnormalities and clinical and laboratory characteristics. Methods We designed a targeted sequencing assay to test a panel of 417 genes. The panel contain reported gene associated with IBMFs and other diseases need be differentiated. Pediatic patients (≤14 year old) with suspected diagnosis of AA/IBMFs were enrolled. Peripheral blood (PB) DNA was used to genetic analysis and oral epithelia cells or PB DNA from their parents were used to identify somatic mutations and unreported polymorphism. All the results were validated by Sanger sequencing. Results The average coverage of targeted region was 98.15%, and the average sequencing depth was 315.9×. Totally, 283 patients were enrolled, including 176 clinically diagnosed acquired AA, 51 Fanconi anemia (FA), 8 dyskeratosis congenital(DKC), 30 Diamond-Blanckfan anemia(DBA), 15 congenital neutropenia(CN), and 3 congenital thrombocytopenia. Totally, 19% subjects had IBMFs related genetic mutations. In the patients who were clinically diagnosed as acquired AA patients, about 7% had IBMFs related disease-causing genetic mutations. Finally, 7 patients were genetically diagnosed as FA, 2 were DKC, 1 was WAS and 1 was SDS. In patients who were clinically diagnosed as FA, 33.4% had FANC related gene mutations. Telomere associated gene mutations were detected in 75% of clinical diagnosed DKC. For patients clinically diagnosed as DBA and CN, 36.7% and 20% were detected disease-causing mutations. After genetic screening, 2 patients who had been diagnosed as FA were modified as WAS and 1 DBA was modified as SDS. Only 26% genetic diagnosed IBMFs patients had family history of bone marrow failure, leukemia, tumor or physical anomalies. Compared with acquired AA, patients with genetic diagnosed FA were more likely to have physical anomalies of short stature and development retardation, Cafe au lait spots and finger or toe malformation(P<0.001).However, 46% patients with IBMFs did not have any type of physical anomalies. Moreover, there were only 24% patients with genetic diagnosed IBMFs had positive results of MMC induced chromosome breakage test or SCGE, and both the examinations could not differentiate subtype of IBMFs. FANCD2 mono-ubiquitination test were performed recently. However, even in the genetically confirmed FA, the positive rate was only 18% (2/11). And, there were positive results in some acquired AA patients. For FA patients with definitely genetic mutations, 62.5%(15/24) were compound heterozygous mutations,37.5%(9/24) were homozygous mutations. Mutational frequencies of FANC were: FANCA 65%, FANCD2 23%, FANCG 9%, FANCI 9% and FANCB 4%. For the mutated type, the frequencies of missense, frameshift, nonsense, splicing mutation were 42%, 26%, 16%, 16%. In our study, there were 4 undetermined patients met the clinical diagnostic criteria of FA, and having heterozygous damaged mutations in FANC genes. Conclusion In conclusion, our IBMFs associated genes targeted sequencing assay is an effective strategy for precise diagnosis of bone marrow failure diseases, especially for those without family history or physical anomalies. However, nearly half of the clinically diagnosed IBMFs patients in our study were not detected the disease-causing mutations. This may be due to the mutations in the intron area, or large fragment deletion, which cannot be detected by targeted sequencing. And the novel gene involved in IBMFs need further study. Disclosures No relevant conflicts of interest to declare.
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Tricot, Guido, Fenghuang Zhan, Yongsheng Huang, Bart Barlogie, and John Shaughnessy. "DNA Repair Genes Are Upregulated in Multiple Myeloma (MM) Patients Relapsing after Tandem Transplantation." Blood 108, no. 11 (November 16, 2006): 3392. http://dx.doi.org/10.1182/blood.v108.11.3392.3392.
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Abstract Background: Repair of DNA interstrand crosslinks (ICLs) is considered the major mechanism by which resistance to high and low dose melphalan emerges (Spanswick et al. Blood2002; 100: 224). Enhanced ICL repair occurs via the Fanconi anemia (FA)/ BRCA pathway in myeloma cell lines (Chen et al. Blood2005; 106: 698). The aim of this study was to evaluate if DNA repair genes were indeed upregulated at relapse when compared to baseline in patients enrolled on our Total Therapy 2 protocol. Methods: We compared 51 paired baseline and relapse bone marrow samples for gene expression profiling of CD138 selected plasma cells using the Affimetrix U133 Plus 2.0 microarray. The microarrays were preprocessed using GCOS 1.1 software and normalized using conventional GCOS 1.1 scaling. Baseline and relapse gene expression was compared using the paired Student t test. Results: More than 30 genes related to DNA repair on the microarray were analyzed. We observed significantly higher expression at relapse of FANCA (p=.003), BRCA 1 (p<.001), CHEK1 (p=.006) and FANCG (p=0.03) (Figure 1). In contrast, FANCD2 was down-regulated at relapse (p=.006), while no significant changes in expression of FANCF or DNA mismatch repair genes were seen. Conclusion: Our data supports the concept that DNA repair genes in the FA/BRCA pathway contribute to acquired resistance to melphalan in patients after high dose melphalan transplants and that inhibition of DNA repair by bortezomib, gemcitabine or the CHEK1 kinase inhibitor UCN-01, may reverse such resistance if added to high dose melphalan. DNA Repair Genes in Paired Relapse Samples (N=51) DNA Repair Genes in Paired Relapse Samples (N=51)
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Li, Zhihui, Yongqiang Zhao, Yanzhi Song, Caiyan Zhang, Qinlong Zheng, Chunrong Tong, and Tong Wu. "Influence of Inherited and Acquired Hematological and Immunological Gene Variants on the Outcomes of Allogeneic Hematopoietic Stem Cell Transplantation in Patients with Hematological Malignancies." Blood 136, Supplement 1 (November 5, 2020): 46–47. http://dx.doi.org/10.1182/blood-2020-139220.
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Chromosome abnormalities and gene variants are important factors in prognosis of the patients with hematological malignancies. Our previous study showed that some germline gene mutations-related to hematological and immunological disorders may have negative impact on the complications of allogeneic hematopoietic stem cell transplantation (allo-HSCT). In current study, the influence of both hematological and immunological hereditary predisposition gene variants and tumor gene variants on the outcomes of allo-HSCT in patients with hematological malignancies was studied. Between January 2018 and June 2020, 164 patients with hematological malignancies who underwent allo-HSCT in our hospital were analyzed. The median age was 14 (1 to 67) years old. The diagnosis included acute myeloid leukemia (n=68, 41.5%), acute lymphoblastic leukemia (n=67, 40.8%) and non-Hodgkin's lymphoma (n=29, 17.7%). The disease status before transplant was CR1 in 49 cases (29.9%), CR2 in 63 cases (38.4%), PR in 22 cases (13.4%), and NR in 30 cases (18.3%). Donors were from haploidentical family members (n=125, 76.2%) or identical siblings (n=18, 11.0%) or unrelated volunteers (n=21, 12.8%). Myeloablative conditioning regimens with either total body irradiation/fludarabine-based or busulfan/fludarabine-based were applied. Anti-thymocyte globulin was used in haploidentical and unrelated transplants. Graft-versus-host disease (GVHD) prophylaxis was with cyclosporine, short-term methotrexate and mycophenolate mofetil. Before transplant, blood samples from patients, their parents and potential related donors were collected to analyze for more than 700 kinds of hematological and immunological hereditary predisposition genes with whole exon sequencing and validation by sanger sequencing. The average sequencing depth was 150×. At diagnosis or relapse, bone marrow samples from patients were obtained to detect for 339 kinds of tumor genes by Illumina sequencing. The average sequencing depth was more than 3000×. With the median follow-up 12.6 (11.2 to 17.0) months, 105 patients (64.0%) achieved durable remission after allo-HSCT. Forty-seven patients (28.7%) relapsed. Twelve patients (7.3%) died. Total 191 immunodeficiency-related hereditary predisposition gene variants were identified in this cohort (average 3.5 gene variants per patient; range, 0-10). Twenty-six of them were recurrent more than 6 times that including TYK2, IFIH1, CFTR, LRBA, IL7R, POLE, RNF31, NLRP12, TTC7A, ATM, CARD14, CHD7, NOD2, TNFRSF13B, BLB,CFB, EPG5, C8A, C8B, CFH, IRF, MSH6, NCF2, NFAT5, PMS2 and ST1M1.IFIH1and IL10RB gene variants were poor factors for relapse post-transplant (P=0.006, P=0.007). The functions of these genes involve in combined immunodeficiency, autoinflammatory disease, complement deficiency, immune deficiency, T-cell dysfunction and antibody deficiency. Total 153 tumor gene variants were identified in this patient series (average 1.93 gene variants per patient; range, 0-16). Seventeen of them were recurrent more than 4 times that including TP53, TPN11, KIT, FLT3, NRAS, NUDT15, STK11, CREBBP, NPM1, DNAH9, DNMT3A, IDH2, HEK2, KRAS, KMT2C, NOTCH1 and NR3C1. TP53, KRAS and NUDT15 gene variants were poor factors for relapse after allo-HSCT (P=0.000025, P=0.0082, P=0.000018). Hemophagocytic lymphohistiocytosis (HLH)-related genes variants were found in 9 patients (5.5%). Frequent HLH-related gene variants were in CD27, PRF1, STX11, and UNC13D. Fanconi anemia (FA)-related gene variants were seen in 11 patients (6.7%). Common FA-related gene variants were in BRCA1, BRCA2, BRIP1, FANCA, FANCC, FANCF, FANCM and FANCG. Significantly higher incidences of acute GVHD (aGVHD) and/or infections were noted in the patients with HLH and/or FA-related gene variants compared with those without HLH and FA-related gene variants (p=0.019). Our results have shown that both inherited and acquired hematological and immunological-related gene variant profiles in the patients with hematological malignancies and some recurrent gene variants (IFIH1 and IL10RB; TP53, KRAS and NUDT15) have negative impact on the outcomes of allo-HSCT including leukemia-free survival, aGVHD and infections. Keywords: allogeneic hematopoietic stem cell transplant, gene expression, relapse, hematological malignancy Disclosures No relevant conflicts of interest to declare.
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Demuth, Ilja, Marcin Wlodarski, Alex J. Tipping, Neil V. Morgan, Johan P. de Winter, Michaela Thiel, Sonja Gräsl, et al. "Spectrum of mutations in the Fanconi anaemia group G gene, FANCG/XRCC9." European Journal of Human Genetics 8, no. 11 (October 31, 2000): 861–68. http://dx.doi.org/10.1038/sj.ejhg.5200552.
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Aymun, Ujala, Saima Iram, Iram Aftab, Saba Khaliq, Nadir Ali, Nisar Ahmed, and Shahida Mohsin. "Screening for mutations in two exons of FANCG gene in Pakistani population." Biomedical Papers 161, no. 2 (June 14, 2017): 158–63. http://dx.doi.org/10.5507/bp.2017.030.
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Nakanishi, Koji, Anna Moran, Tobias Hays, Yanan Kuang, Edward Fox, Danielle Garneau, Rocio Montes de Oca, Markus Grompe, and Alan D. D'Andrea. "Functional analysis of patient-derived mutations in the Fanconi anemia gene, FANCG/XRCC9." Experimental Hematology 29, no. 7 (July 2001): 842–49. http://dx.doi.org/10.1016/s0301-472x(01)00663-4.
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Van De Vrugt, Henri J., Mireille Koomen, Mariska A. D. Berns, Yne De Vries, Martin A. Rooimans, Laura Van Der Weel, Eric Blom, et al. "Characterization, expression and complex formation of the murine Fanconi anaemia gene product Fancg." Genes to Cells 7, no. 3 (March 2002): 333–42. http://dx.doi.org/10.1046/j.1365-2443.2002.00518.x.
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Yamada, T., A. Tachibana, T. Shimizu, H. Mugishima, M. Okubo, and M. S. Sasaki. "Novel mutations of the FANCG gene causing alternative splicing in Japanese Fanconi anemia." Journal of Human Genetics 45, no. 3 (May 2000): 159–66. http://dx.doi.org/10.1007/s100380050203.
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Smogorzewska, Agata. "Fanconi Anemia: A Paradigm for Understanding DNA Repair During Replication." Blood 134, Supplement_1 (November 13, 2019): SCI—32—SCI—32. http://dx.doi.org/10.1182/blood-2019-121229.
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Fanconi anemia, the most common hereditary bone marrow failure disorder, results from defective repair of DNA interstrand crosslinks (ICLs), which covalently link complementary DNA strands causing replication stalling. Mutations in 22 different genes (FANCA-FANCW) have been shown to result in Fanconi anemia. Their protein products work at different stages of DNA repair leading to considerable heterogeneity in human phenotypes. The majority of the FANC gene mutations are recessively inherited with the exceptions of FANCB and FANCR/RAD51. FANCB is X-linked, and all FANCR/RAD51 mutations arise de novo, affect only one allele, and the mutant protein acts as a dominant negative against the wild type protein. Despite advances in the molecular diagnosis of Fanconi anemia, if Fanconi anemia is suspected, chromosome breakage (DEB or MMC) testing on patient cells is essential. We have seen a number of patients referred to the International Fanconi Anemia Registry (http://lab.rockefeller.edu/smogorzewska/ifar/) who are misdiagnosed with Fanconi anemia based solely on the presence of a FANC gene variant in gene panel or whole exome sequencing. Conversely, blood mosaicism may lead to a negative blood chromosome breakage test. If there is a high suspicion of Fanconi anemia, but blood breakage results are negative, breakage test on patient fibroblasts should be performed. Diagnosis of Fanconi anemia should also be entertained in young adults presenting with squamous cell carcinoma of the aerodigestive tract, since this may be their initial presentation of Fanconi anemia and conventional chemotherapy dose would precipitate bone marrow failure in these patients. In my talk, I will discuss the mechanism of the Fanconi anemia repair pathway during DNA replication. Then, I will concentrate on the mechanism of bone marrow failure and tumorigenesis in Fanconi anemia. I will explore the hypothesis that the endogenously produced aldehydes including some that are still unknown, contribute to disease development. Fanconi anemia-deficient hematopoietic stem cells have an autonomous DNA repair defect. Accumulation of DNA damage leads to apoptosis due to the activation of p53. If cells escape death, mutagenesis may lead to the development of leukemia. The sources of endogenous DNA damage are poorly understood. Cell cycle induction of Fanconi anemia pathway-deficientmouse hematopoietic stem cells results in DNA damage and bone marrow failure, which implies that the DNA lesions encountered during replication are the culprit. There is mounting evidence that the endogenous aldehydes, including acetaldehyde and formaldehyde,may cause those DNA lesions. To identify other metabolites that may induce bone marrow failure in Fanconi anemia, we used a library of CRISPR guides to target Cas9 to metabolic genes to screen for and identify synthetic lethality with Fanconi anemia deficiency. We have identifiedALDH9A1as the most significantly depleted gene in FANCD2-/- cells. The synthetically lethal interaction was validated using single gene editing in human umbilical cord-derived hematopoietic stem progenitor cells. We propose a model in which aldehydes that are metabolized by ALDH9A1 accumulate in the absence of this enzyme and cause DNA damage that requires the Fanconi anemia pathway proteins for repair, survival, and suppression of tumorigenesis. We are testing this model using Fanca-/-Aldh9a1-/-mice. Disclosures No relevant conflicts of interest to declare.
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Savery, Laura C., Eliza Grlickova-Duzevik, Sandra S. Wise, W. Douglas Thompson, John M. Hinz, Larry H. Thompson, and John Pierce Wise. "Role of the Fancg gene in protecting cells from particulate chromate-induced chromosome instability." Mutation Research/Genetic Toxicology and Environmental Mutagenesis 626, no. 1-2 (January 2007): 120–27. http://dx.doi.org/10.1016/j.mrgentox.2006.09.005.
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Lamerdin, J. E. "Characterization of the hamster FancG/Xrcc9 gene and mutations in CHO UV40 and NM3." Mutagenesis 19, no. 3 (May 1, 2004): 237–44. http://dx.doi.org/10.1093/mutage/geh019.
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Nie, Daijing, Fang Wang, Jing Zhang, Xvxin Li, Lili Liu, Wei Zhang, Panxiang Cao, et al. "Fanconi Anemia Gene Associated Germline Predisposition in Aplastic Anemia and Hematologic Malignancies." Blood 136, Supplement 1 (November 5, 2020): 18–19. http://dx.doi.org/10.1182/blood-2020-142452.
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Introduction Fanconi anemia (FA) is the most common inherited bone marrow failure syndrome with a surged cancer incidence, especially in hematologic malignancies, which has been listed in the entity of myeloid neoplasms with germline predisposition in the 2016 revision to the World Health Organization classification of tumors of hematopoietic and lymphoid tissues.Whether FA heterozygotes are predisposed to bone marrow failure and hematologic neoplasm is crucial but unsettled. We therefore retrospectively analyzed rare possibly significant variations (PSVs) in the five most obligated FA genes, BRCA2, FANCA, FANCC, FANCD2, and FANCG, in 788 aplastic anemia (AA) and hematologic malignancies patients to address this issue. Methods Patients diagnosed as AA, myelodysplastic syndrome (MDS), acute myeloid leukemia (AML), and acute lymphocytic leukemia (ALL) from April 2015 to December 2018 in Hebei Yanda Lu Daopei Hospital were enrolled with the ones diagnosed as FA via chromosome breakage test and/or genetic test excluded. Detailed disease histories and workup files were retrieved from the electronic medical record. Amplicon-based high throughput sequencing of aforementioned five genes were performed. The significance of the germline missense variants was assessed by in silico prediction algorithms, including SIFT, Polyphen2, PROVEAN, FATHMM, MutationTaster, and MutationAssessor. Variant predicted as deleterious/possibly deleterious by ≥ 3/6 scoring tools was defined as rare PSV and further included in statistical analysis. For splice site mutations, GeneSplicer, Human Splicing Finder, NetGene2, and FSPLICE were employed and only when ≥2/4 algorithms predict to affect/possibly affect splicing, would the variant be regarded as possibly significant. Same criteria were adopted when stratifying variants recorded in the ExAC. All reported variants in this study were confirmed germline variants by Sanger sequencing with fingernail specimens and/or pedigree analysis. WFisher exact two-tailed test was adopted for variant frequency comparison. Development of disease was analyzed by cumulative incidence method and Kaplan-Meier method. p < 0.05 was considered as statistically significant. Results A total of 788 patients, who were all of the East Asian ancestry, were enrolled, including 341 females and 447 males (Figure 1A). The median age of onset was 10 (1-63), 27 (1-65), 14.5 (1-65), and 6 (1-53) years in AA, MDS, AML, and ALL subgroup, respectively. Sixty-eight variants were identified in 66 patients (8.38%). FANCA is the most frequently mutated gene (n = 29), followed by BRCA2 (n = 20) (Figure 1B). When compared to the ExAC East Asian dataset, there was an overall higher rare PSVs incidence in our cohort (p = 0.016). BRCA2 PSVs showed a higher frequency in ALL (p = 0.038), and FANCA PSVs were significantly enriched in AA and AML subgroups (p = 0.020; p = 0.008). The patients with FANCA heterozygotes also tended to show an increased risk for developing MDS (p = 0.075) (Table 1). No impact of FA-PSV status was found neither on cumulative disease incidence (Figure 1C). FA-PSV + MDS/AML patients have a heavier tumor mutation burden, higher rate of cytogenetic abnormalities, and less epigenetic regulation and spliceosome gene mutations than those of FA-PSV - MDS/AML patients (p = 0.024, p = 0.029, p = 0.024, and p = 0.013) (Figure 1D). Discussion The overall PSVs enrichment in our cohort buttresses that heterozygous mutations of FA genes abate the capacity of DNA homologous recombination repair pathway and contribute to hematopoietic failure. Furthermore, we present the first evidence that BRCA2 heterozygotes have a significantly higher risk of developing into ALL. Instead of all or nothing, the impact of different variant imposed on protein might be seen as a continuous variation. Therefore, contributions of the FA pathway defect could be latent and subtle but be profound as time goes by. We further firstly observed a higher incidence of cytogenetic abnormalities and somatic mutations with statistical significance, and lower frequency of epigenetic regulation and spliceosome gene mutations in FA-PSV + myeloid malignancies. This provides evidence that these FA-PSV carriers are prone to accumulate chromosomal structural abnormalities, and confer the congenital susceptibility of myeloid malignancies. Disclosures No relevant conflicts of interest to declare.
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Muramatsu, Hideki, Yusuke Okuno, Kenichi Yoshida, Sayoko Doisaki, Asahito Hama, Xinan Wang, Atsushi Narita, et al. "Diagnostic Efficacy of Whole-Exome Sequencing in 250 Patients with Congenital Bone Marrow Failure." Blood 124, no. 21 (December 6, 2014): 4385. http://dx.doi.org/10.1182/blood.v124.21.4385.4385.
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Abstract Introduction: Congenital bone marrow failure syndromes (CBMFSs) are a heterogeneous class of diseases with overlapping phenotypes. Therefore, a precise and comprehensive genetic diagnostic system is strongly warranted to arrive at appropriate clinical decisions to avoid ineffective therapies and/or lethal complications of allogeneic hematopoietic stem cell transplantation. However, a large panel of newly identified causative genes of CBMFSs have been identified in recent years; therefore, it is virtually impossible to establish a routine genetic diagnostic test using conventional Sanger sequencing. Whole-exome sequencing (WES) is a promising solution for the diagnosis of inherited diseases because it tests virtually all genes simultaneously. For the introduction of WES into clinical practice, it is necessary to clarify whether this technique has superior diagnostic efficacy to conventional clinical genetic tests. Methods: We performed WES in 250 patients with CBMFSs lacking genetic diagnoses. Exome capture was performed using the SureSelect® Human All Exon V3–5 kit (Agilent Technologies, Santa Clara, CA, USA), which covers all known coding exons, followed by massively parallel sequencing using the HiSeq 2000 Sequencing System (Illumina, San Diego, CA, USA). Our established pipeline for WES (genomon: http://genomon.hgc.jp/exome/) detected >20,000 candidate variants per patient. Diagnoses were based on variants of 130 genes with pathogenicities confirmed by published studies. Results: Genetic diagnoses were possible in 68 patients (27%). The best efficacy was achieved in patients with Fanconi anemia [35/73, 48%; FANCG (n = 17), FANCA (n = 14), FANCB (n = 1), FANCF (n = 1), SLX4 (n = 1), and BRCA2 (n = 1)], although Sanger sequencing was not applied because of the large sizes of its causative genes. Encouraging results were obtained in patients with Diamond–Blackfan anemia [11/ 61, 18%; RPS26 (n = 3), RPS7 (n = 2), RPS19 (n = 2), RPL5 (n = 2), RPL35A (n = 1), and RPL11 (n = 1)] and dyskeratosis congenita [7/29, 24%; TERT (n = 3), TINF2 (n = 2), and DKC1 (n = 2)]. Five genetic diagnoses (7%) were inconsistent with clinical diagnoses, possibly because of overlapping disease phenotypes. Conclusion: Relative to conventional genetic testing, WES was found to be effective for the diagnoses of CBMFSs. Furthermore, the efficacy of WES will increase as our knowledge of gene mutations expands. In conclusion, the use of WES in clinical practice is warranted. Disclosures No relevant conflicts of interest to declare.
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Labriola, Matthew, Jason Zhu, Rajan Gupta, Shannon McCall, Jennifer Jackson, James R. White, Elizabeth Weingartner, et al. "Characterization of tumor mutational burden (TMB), PD-L1, and DNA repair genes to assess correlation with immune checkpoint inhibitors (ICIs) response in metastatic renal cell carcinoma (mRCC)." Journal of Clinical Oncology 37, no. 15_suppl (May 20, 2019): e16079-e16079. http://dx.doi.org/10.1200/jco.2019.37.15_suppl.e16079.
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e16079 Background: ICIs have revolutionized treatment for mRCC; however there are limited predictive biomarkers for response to ICIs. PD-L1 status is still controversial, demonstrating little predictive utility in mRCC. TMB is predictive for response to ICIs in melanoma and non-small cell lung cancer (NSCLC), but has not been validated in mRCC. Here, we assess the correlations between TMB and PD-L1 status with outcomes to ICI treatment in mRCC. Methods: 34 patients (pts) with mRCC who had previously received ICIs at Duke Cancer Institute were identified. Tumor samples were retrospectively evaluated using a Personal Genome Diagnostics Assay for somatic variants across > 500 genes, as well as TMB and microsatellite status. PD-L1 status was tested via the Dako 28-8 PD-L1 IHC assay. Deidentified clinical information was extracted from the medical record and tumor response was evaluated based on RECIST criteria. Results: Pts were grouped by overall response following ICI therapy into either progressive disease (“PD”, n = 18) or disease control group (“DC”, n = 16), defined as either stable disease, partial response, or complete response. Pts displayed a TMB range from 0.36 to 12.24 mutations/Mb with a mean score of 2.83 muts/Mb, with no significant difference between the PD and DC groups (mean 3.01 muts/Mb vs. 2.63 muts/Mb, p > 0.05). 9 of 32 evaluable samples were PD-L1 positive, with 4 in the PD group and 5 in the DC group. Notably, the DC group displayed a significant enrichment of mutations in genes affiliated with DNA repair (including BRCA1, BRCA2, FANCA, FANCB, FANCG, FANCM, MSH3, MSH6, RAD50, RAD51C, RAD51D, RAD54B, RECQL4, and SLX4; p = 0.0444). DNA damage gene mutations were found in 8/10 (80%) metastatic tumor specimens and 14/24 (58%) primary tumors. Conclusions: Overall, in this mRCC cohort, neither TMB nor PD-L1 correlated with patient outcomes or with ICI response. Furthermore, high TMB was not significantly associated with PD-L1 expression within the samples. The higher frequency of mutations in DNA repair genes in the DC group suggests potential use as a predictive signature for ICI response, warranting future prospective studies. Further studies with matched primary-metastatic samples would be beneficial to determine if DNA repair mutations occur more frequently in metastatic versus primary tumor specimens.
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Auerbach, Arleen D., Jason Greenbaum, Kanan Pujara, Sat Dev Batish, Marco A. Bitencourt, Indira Kokemohr, Hildegard Schneider, et al. "Spectrum of sequence variation in the FANCG gene: An International Fanconi Anemia Registry (IFAR) study." Human Mutation 22, no. 3 (August 18, 2003): 255. http://dx.doi.org/10.1002/humu.10266.
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Hess, C. J., N. Ameziane, G. J. Schuurhuis, A. Errami, F. Denkers, G. J. L. Kaspers, J. Cloos, et al. "Hypermethylation of the FANCC and FANCL Promoter Regions in Sporadic Acute Leukaemia." Analytical Cellular Pathology 30, no. 4 (January 1, 2008): 299–306. http://dx.doi.org/10.1155/2008/506729.
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Objective: Inactivation of the FA-BRCA pathway results in chromosomal instability. Fanconi anaemia (FA) patients have an inherited defect in this pathway and are strongly predisposed to the development of acute myeloid leukaemia (AML). Studies in sporadic cancers have shown promoter methylation of the FANCF gene in a significant proportion of various solid tumours. However, only a single leukaemic case with methylation of one of the FA-BRCA genes has been described to date, i.e. methylation of FANCF in cell line CHRF-288. We investigated the presence of aberrant methylation in 11 FA-BRCA genes in sporadic cases of leukaemia.Methods: We analyzed promoter methylation in 143 AML bone marrow samples and 97 acute lymphoblastic leukaemia (ALL) samples using methylation-specific multiplex ligation-dependent probe amplification (MS-MLPA). Samples with aberrant methylation were further analyzed by bisulphite sequencing and tested for mitomycin C sensitivity using Colony Forming Units assays.Results: MS-MLPA showed promoter methylation of FANCC in one AML and three ALL samples, while FANCL was found methylated in one ALL sample. Bisulphite sequencing of promoter regions confirmed hypermethylation in all cases. In addition, samples with hypermethylation of either FANCC or FANCL appeared more sensitive towards mitomycin C in Colony Forming Units assays, compared to controls.Conclusion: Hypermethylation of promoter regions from FA-BRCA genes does occur in sporadic leukaemia, albeit infrequently. Hypermethylation was found to result in hypersensitivity towards DNA cross-linking agents, a hallmark of the FA cellular phenotype, suggesting that these samples displayed chromosomal instability. This instability may have contributed to the occurrence of the leukaemia. In addition, this is the first report to describe hypermethylation of FANCC and FANCL. This warrants the investigation of multiple FA-BRCA genes in other malignancies.
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Smirnova, Natalya A., Peter J. Romanienko, Pavel P. Khil, and R. Daniel Camerini-Otero. "Gene expression profiles of Spo11−/− mouse testes with spermatocytes arrested in meiotic prophase I." Reproduction 132, no. 1 (July 2006): 67–77. http://dx.doi.org/10.1530/rep.1.00997.
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Spo11, a meiosis-specific protein, introduces double-strand breaks on chromosomal DNA and initiates meiotic recombination in a wide variety of organisms. Mouse null Spo11 spermatocytes fail to synapse chromosomes and progress beyond the zygotene stage of meiosis. We analyzed gene expression profiles in Spo11−/ −adult and juvenile wild-type testis to describe genes expressed before and after the meiotic arrest resulting from the knocking out of Spo11. These genes were characterized using the Gene Ontology data base. To focus on genes involved in meiosis, we performed comparative gene expression analysis of Spo11−/ −and wild-type testes from 15-day mice, when spermatocytes have just entered pachytene. We found that the knockout of Spo11 causes dramatic changes in the level of expression of genes that participate in meiotic recombination (Hop2, Brca2, Mnd1, FancG) and in the meiotic checkpoint (cyclin B2, Cks2), but does not affect genes encoding protein components of the synaptonemal complex. Finally, we discovered unknown genes that are affected by the disruption of the Spo11 gene and therefore may be specifically involved in meiosis and spermatogenesis.
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Altintas, Burak, Neelam Giri, Lisa J. McReynolds, and Blanche P. Alter. "Genotype-Phenotype Associations in Patients with Fanconi Anemia: National Cancer Institute Cohort." Blood 136, Supplement 1 (November 5, 2020): 4–5. http://dx.doi.org/10.1182/blood-2020-137200.
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Fanconi anemia (FA) is a predominantly autosomal recessive disorder resulting from mutations in one of >22 genes involved in the FA/BRCA DNA repair pathway. FA is characterized by multiple congenital abnormalities, progressive bone marrow failure (BMF) and cancer predisposition. Genetic heterogeneity and diverse clinical presentations challenge early diagnosis and optimal management. We previously reviewed the genotype-phenotype associations in FA from literature cases (Fiesco-Roa MO et al. Blood Rev. 2019). We now report the results from the NCI cohort. We studied 147 patients with FA in the NCI inherited bone marrow failure syndromes Cohort Study (ClinicalTrials.gov, NCT00027274) to explore genotype phenotype associations by genes, location in the FA/BRCA pathway (upstream, ID complex, downstream), and compare information on the clinic cohort (CC) and field cohort (FC) patients. 57 patients (CC) were evaluated at the NIH Clinical Center between 2002 and 2020. Details on 90 patients in the FC were obtained from the review of medical records. The sex ratio (M:F) was similar (0.6:1 and 0.8:1). Patients in the FC were younger than in the CC (p=0.004) with median ages 27 (3-68) years for the CC and 19 (0-57) for the FC. The main genotypes in the CC were 59% FANCA, 17% FANCC, 6% FANCI and in the FC were 60% FANCA, 13% FANCC and 8% FANCG. At least one FA type physical abnormality was present in all CC patients and 73/79 (92%) FC patients (phenotype data not reported on 11 FC patients). >3/8 VACTERL-H features (Vertebral, Anal, Cardiac, Tracheo-esophageal fistula (TEF), Esophageal or duodenal atresia, Renal, upper Limb (radial ray) and Hydrocephalus) were present in 32% of CC patients and 16% of FC (p=0.04). At least 4/6 PHENOS features (skin Pigmentation, small Head, small Eyes, other central Nervous system (CNS) anomalies, Otology and Short stature) were present in 54% of CC patients and 34% FC (p=0.02). The types and frequencies of phenotypic abnormalities are shown in figure 1. 17 patients in the CC (30%) and 10 in the FC (13%) had both VACTERL-H and PHENOS (p=0.01). We excluded patients with unknown genotype or phenotype from further analysis. In the CC, cardiac abnormalities were more common in patients with FANCI or ID complex gene variants than in all others (p=0.02 and 0.001, respectively) as were VACTERL-H and structural CNS abnormalities in patients with ID complex variants (p=0.03 and 0.006, respectively). In the FC, VACTERL-H, imperforate anus and hydrocephalus were more common in patients with FANCD1 genotype (p=0.03, 0.009 and 0.004, respectively) and downstream pathway gene variants (p=0.004, <0.001 and 0.03, respectively). PHENOS, renal and neurodevelopmental abnormalities were less common in patients with upstream genes variants (p=0.001, 0.009 and <0.001, respectively). Upper limb abnormalities were less common in patients with FANCC genotype (p=0.007). BMF was present in 121/147 (88%) patients; 33% had been transfusion-dependent and 26% received androgen therapy. Clonal cytogenetic abnormalities were seen in 30%; 17% developed myelodysplastic syndrome at a median age of 17 (1.4-44) years and 6 patients developed acute myeloid leukemia at a median age of 19 (12-29) years. 72 (49%) patients underwent bone marrow transplant at a median age of 9.5 (1.5-44) years for BMF, MDS or leukemia. There was no significant difference between the FC and CC. The median survival age of our cohort is 38 (95% CI 34-43) years and at least 80% of our patients are >18 years of age. Kaplan-Meier survival estimates are presented in figure 2. Solid tumors developed in 30/135 (22%) patients with available data; median age at first cancer was 30 (2-44) years. The most common tumor was head and neck squamous cell carcinoma (n=15 patients), followed by skin (n=8) and anogenital cancers (n=6); many patients developed multiple cancers. Detailed hematologic, cancer, endocrine outcomes and survival analyses are ongoing. Overall, renal and upper limb abnormalities were reported in most of the patients in both CC and FC, as shown previously (Alter BP et al. Mol Syndromol. 2013). Data from the CC were more complete than from the review of charts from the FC highlighting that the clinical in person evaluation of patients provides detailed characterization of FA phenotypes and more accurate assessment of genotype-phenotype associations. This will facilitate timely diagnosis, surveillance and clinical management of patients with FA. Disclosures No relevant conflicts of interest to declare.
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Yagasaki, Hiroshi, Tsukasa Oda, Daiki Adachi, Toshiaki Nakajima, Tatsutoshi Nakahata, Shigetaka Asano, and Takayuki Yamashita. "Two common founder mutations of the fanconi anemia group g gene FANCG/XRCC9 in the Japanese population." Human Mutation 21, no. 5 (May 2003): 555. http://dx.doi.org/10.1002/humu.9142.
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Pan, Jing, Tong Wu, Jun-Fang Yang, Jian-Ping Zhang, Yan-Li Zhao, Yang Zhang, Hong-Xing Liu, and Chun-Rong Tong. "Clinical Features and Outcome of 22 Leukemia Patients with TLS-ERG Fusion Gene." Blood 128, no. 22 (December 2, 2016): 3985. http://dx.doi.org/10.1182/blood.v128.22.3985.3985.
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Abstract TLS-ERG fusion gene resulting from translocation changes involving chromosomes 16 and 21 is a rare genetic event mostly observed in acute myeloid leukemia (AML). AML with TLS-ERG has unique clinical and morphological features and showed poor prognosis and a high relapse rate. However, many clinical aspects of this type of leukemia still remain unknown. Here, we report the clinical features and outcome of 22 leukemia patients (20 AML and 2 B-ALL) with TLS-ERG fusion gene. The median age of patients was 6 (2-35) years old. 12 patients were males and 10 were females. The median WBC count at diagnosis was 26.54 (1.3-110.8)X109/L. TLS-ERG is tightly associated with extramedullary disease (EMD), complex chromosome abnormalities, and high risk gene mutations. 9/22 (40.9%) cases had complex chromosome abnormalities, 8/22 (36.4%) cases had EMDs, and a large proportion of cases had hematological malignancies gene mutations such as IKZF1, TET2, WT1, PHF6 and NOTCH2. 9 cases in this study had been examined for congenital inherited diseases (Familial hemophagocytic lymphohistiocytosis, FHLH, Fanconi anemia, FA and Dyskeratosis congenital, DC) gene mutations such as FANCD2, FANCG, FANCA, STXBP2, UNC13D, LAMP1 and LYST, and 7/9 (77.8%) cases had positive results. The details are summarized in Table I. We firstly evaluated the general outcome of these patients. Excluding B-ALL patients, 7/20 (35%) cases achieved complete remission (CR) after induction. The median CR, complete molecular remission (CMR), and overall survival (OS) time of the 20 cases was 7 (0-16), 3 (0-14), and 12 (1-36) months. The 6 months relapse free survival (RFS) of EMD and non-EMD patients was 75% and 83.3% (P=0.017, Figure 1). We evaluated the impact of HCT on the outcome of these patients.11/20 AML received allo-HCT, but 9/20 did not. In non-HCT group, the median CR time was 8 (0-29) months. 7/9 cases achieved CR, 3/9 cases achieved CMR, and all cases had disease progression. The median OS time was 11 (1-76) months. In HCT group, 9/11 cases underwent haploidentical HCT (HID-HCT), and 2/11 underwent HLA-matched unrelated donor HCT (URD-HCT). The median OS time was 19 (9-36) months. 1 year OS in non-HCT and HCT group was 62.5% and 90%, respectively (P=0.026, Figure 2), but 6 month RFS in non-HCT and HCT group was 55.6% and 100% (P=0.192), without statistical significance. We analyzed the effect of diverse clinic factors on the outcome of HCT. At the time of HCT, 1/11 case was NR, 10/11 cases were CR, 7/10 CR cases were not detected blasts by FCM, and 6/10 CR cases were CMR. 6 months RFS of CR and NR case was 67.5% and 0, respectively (P=0.034). 6 months RFS of FCM+ and FCM- cases was 50% and 71.4% (P=0.403). 6 months RFS of CMR and non-CMR cases was 66.7% and 60% (P=0.671). There are 5 EMD cases among the 11 cases that underwent HCT, and 6 months RFS of HCT cases with or without EMDs was 33.3% and 71.4%, respectively (P=0.204). Among the 8 TLS-ERG+ AML patients with EMD, 6/8 cases achieved CR after induction, and 5/8 cases underwent HCT after CR, the 6 months RFS of EMDs with or without HCT was 100% and 33.3% (P=0.203). Among the 12 TLS-ERG+ AML patients without EMD, 10/12 cases achieved CR after induction, and 6/12 cases underwent HCT after CR, the 6 months RFS of them with or without HCT was 100% and 66.7%, respectively (P=0.176). In conclusion, we found TLS-ERG is tightly associated with complex chromosome abnormalities and high risk gene mutations including IKZF1, TET2, WT1, PHF6, FANCD2, FANCG, FANCA, which might partially explain the overall poor prognosis of TLS-ERG patients. The leukemia burden before HCT and EMD has negative impact on the outcome of TLS-ERG patients. HCT could prolong the OS of the patients, but could not overcome the poor prognosis of TLS-ERG. No matter the patients had EMD or not, the RFS could not be improved by HCT. Our study will be valuable for evaluating the therapeutic regimen and prognosis of AML patients with TLS-ERG fusion gene. Disclosures No relevant conflicts of interest to declare.
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Chen, Qing, Pieter C. Van der Sluis, Lori Hazlehurst, and William S. Dalton. "Enhanced DNA Repair Via Fanconi Anemia/BRCA Pathway Is Involved in Melphalan-Resistant Myeloma Cells." Blood 104, no. 11 (November 16, 2004): 284. http://dx.doi.org/10.1182/blood.v104.11.284.284.
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Abstract Melphalan, a DNA crosslinker, is one of the most widely used and effective drugs in the treatment of multiple myeloma (MM). Interstrand cross-links (ICL) are amongst the most toxic types of DNA damage; therefore, DNA cross-linking agents are important drugs in cancer treatment. Unfortunately, although most patients respond to standard and high dose melphalan therapy, eventually patients acquire drug resistance. Acquired melphalan resistance has been associated with reduced DNA crosslinks, elevated levels of glutathione and increased radiation survival. However, mechanisms associated with resistance are not well understood. Evidence has accumulated to suggest that ICL repair contributes to the melphalan resistance. In this study, we compared the gene expression profile (GEP) of the melphalan-resistant myeloma cell line, 8226/LR5 to the 8226/S drug sensitive cell line, and found genes involved in FANC/BRCA DNA cross-link repair pathway had increased expression in drug resistant cells. The aim of our study was to determine whether FANC-BRCA pathway affects the DNA cross link repair capacity and accounts for acquired melphalan-resistance. Using real time RT-PCR and Western Blotting, we examined the expression levels of FANC/BRCA pathway genes in two different drug sensitive and resistant cell lines: 8226/S 8226/LR5, and U266/S and U266/LR6. The results showed that increased expression of FANC/BRCA pathway genes correlated with the melphalan resistance. The formation of ICL at 5 hours after a 2 hour melphalan exposure was reduced in the LR5 compared to the drug sensitive cell 8226 using single cell comet assay. Using siRNA to knock down FANCL or FANCF in melphalan-resistant cell line LR5 reversed the drug resistance. Conversely, overexpression of FANCL or FANCF in the 8226/S drug sensitive cell line enhanced cell survival. These data show that enhanced DNA repair via Fanconi anemia/BRCA pathway is involved in melphalan-resistant myeloma cells.