Diepoxybutane Research Articles

Cytogenetic analysis of fanconi anemia patients: An hospital based study

: Fanconi anemia is a rare genetic disorder caused by mutations in genes whose protein products are involved in replication, cell cycle control and DNA repair and is associated with a very high frequency of bone marrow failure and many other manifestations including, but not restricted to, severe birth defects.The diagnosis of FA is confirmed by a specific test known as chromosomal breakage study, a differential technique in which clastogenic substances, such as DEB (diepoxy butane) or MMC (mitomycin C), lead to sections of the chromosome being deleted, added, or rearranged.In this retrospective study, peripheral blood smears of patients with Aplastic Anemia were analyzed to diagnose Fanconi Anemia.: A total of 135 cases of Aplastic anemia were analyzed and screened by chromosomal breakage analysis for ruling in/out Fanconi anemia.: A total of 9 (6.66%) out of 135 patients showed a significant increase in the number of chromosomal breaks in comparison to their control. An analysis of the variable clinical manifestations was also done and correlated to the diagnosis of Fanconi Anemia. : This study throws light on the importance of cytogenetic analysis as being the most classical test for FA which involves detection of chromosomal breakage or aberrations in metaphase spreads. This relatively inexpensive assay may be useful for screening patients for whom FA is in the differential diagnosis, such as those with radial ray anomalies, short stature, hypogonadism, or café au lait spots, or for population-based FA incidence studies.

Diepoxybutane induces the p53-dependent transactivation of the CCL4 gene that mediates apoptosis in exposed human lymphoblasts.

Diepoxybutane (DEB) is the most toxic metabolite of the environmental chemical 1,3-butadiene. We previously demonstrated the occurrence of DEB-induced p53-mediated apoptosis in human lymphoblasts. The p53 protein functions as a master transcriptional regulator in orchestrating the genomic response to a variety of stress signals. Transcriptomic analysis indicated that C-C chemokine ligand 4 (CCL4) gene expression was elevated in a p53-dependent manner in DEB-exposed p53-proficient TK6 cells, but not in DEB-exposed p53-deficient NH32 cells. Thus, the objective of this study was to determine whether the CCL4 gene is a transcriptional target of p53 and deduce its role in DEB-induced apoptosis in human lymphoblasts. Endogenous and exogenous wild-type p53 transactivated the activity of the CCL4 promoter in DEB-exposed lymphoblasts, but mutant p53 activity on this promoter was reduced by ∼80% under the same experimental conditions. Knockdown of the upregulated CCL4 mRNA levels in p53-proficient TK6 cells inhibited DEB-induced apoptosis by ∼45%-50%. Collectively, these observations demonstrate for the first time that the CCL4 gene is upregulated by wild-type p53 at the transcriptional level, and this upregulation mediates apoptosis in DEB-exposed human lymphoblasts.

Clinical Characterization, Cancer Incidence and Long-Term Outcomes of Fanconi Anemia Patients : A Single Center Analysis of 97 Patients

Introduction: Fanconi Anemia (FA) is a rare inherited condition that may present with bone marrow failure (BMF), constitutional anomalies and high risk of developing cancer. A significant number of FA patients are seen in regions with higher rate of consanguinity and large family sizes, as in the Middle East. Large numbers of FA patients are referred to and treated in KFSHRC. We Studied the clinical characteristics and outcomes of adolescent and young adult (AYA) patients with a diagnosis of FA treated at our institution. Methods: A prospective study of Saudi patients with FA that presented to KFSHRC was obtained from an institutionally approved BMF database and hematopoietic stem cell transplant (HSCT) database. Cases were enrolled only if they were older than 14 years of age at the time of diagnosis or under the care of adult hematology with a clinical diagnosis of FA based on the presence of BMF and abnormal CB, or clinical phenotype with the presence of homozygous FA related genes. Family pedigrees were constructed based on history. In addition, patients presenting with BMF were enrolled onto an institutionally approved study investigating proteomic biomarkers and genomics of BMF syndromes. Consented peripheral blood samples and/or extracted DNA were subject to either panel based next generation sequencing (NGS) testing as part of the Saudi Genome Project or subjected to whole exome sequencing (WES) by external lab. Result: Patients and clinical features: a total of 97 patients (43 M, 54 F) in 60 families were identified and characterized. The median age of diagnosis was 14 years. The parental consanguinity among patients was 51%.The most frequent anomaly was short stature (45 patients, 65%), skin changes (11, 15%), urogenital abnormalities (21, 30%), dysmorphism/craniofacial abnormalities (25, 36%);A significant minority of patients (29%) had no other physical anomaly and presented with bone marrow failure or hematological malignancy. Diagnostic Tests: 68 patients (70%) had a positive CB analysis with diepoxybutane (DEB) or mitomycin-C (MMC) testing; in 5 patients (5%) DEB testing was borderline and 1 (1%) had a normal CBA but had a diagnostic phenotype/- family history and presence of a homozygous mutation in a known FA related gene. 33 patients had cytogenetic abnormalities and abnormalities involving chromosome 1 were the most frequent (50%). Mutation Analysis: Mutational analysis was available for 20 (21%) cases;a farther 15 cases had a presumed mutation.FANCA (23 patients/8 families); BRIP1 (2/2); FANCP (1/1); FANCD2 (5/2) FANCI(2/1);FANCG(2/2). Malignancies: 30 patients (31%) developed a malignancy either before or after diagnosis of FA: solid tumors in 8 (27%), AML and/or MDS in 17 (57%); 5 (16%) of these patients had both solid tumors and AML/MDS.The median age of cancer diagnosis was 27 years (range:7-48).Among patients developed solid tumor (46%) had head and neck SCC,(15%) had HCC,(8%) had lung cancer,(8%) had colon cancer , (8%) had cervical cancer and(8%) had esophageal cancer. Treatment outcomes: 74 out of 97 patients (76%) received HSCT. Actuarial survival of HSCT (n=56) and non-HSCT (n=11) patients was 75% and 58%, respectively. Treatment details were not available on 3 (3%). (11 %) Received FLU/CY /ATG.Low dose TBI was incorporated in 67%. Survival: Overall survival at 5 years was 37.8 %. The median overall survival Among non-transplanted patients was 28.8 months (95%CI 26.3-31.4) and it was 41.6 months (95% CI 28.1-55.03) among transplanted patients. (27%) died of solid tumor post-transplant, (22%) developed SCC and( 5%) developed HCC. CONCLUSION: We report long term outcomes of a large cohort of AYA patients with FA in Saudi Arabia.FANCA is the most frequent gene affected. FA patients have a significant risk of malignancies that reduces survival. Early detection and screening of malignancies is crucial. Further progress is required in improving outcomes in transplanted patients with reduced toxicity and less genotoxic therapies , including gene therapy. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Somatic mosaicism in patients with Fanconi anaemia: Proposal of alternative tissue for inconclusive diagnoses.

Fanconi anaemia (FA) is a rare genetic disorder marked by progressive bone marrow failure, chromosomal fragility, and increased cancer susceptibility. Laboratory diagnosis includes chromosomal instability test and mutation investigation. A total of 15%-25% of all patients may have somatic mosaicism, characterized by two distinct haematopoietic cell populations, one resistant and one sensitive to agents that induce chromosomal breakage, which complicates the diagnosis by a high incidence of reverted cells leading to inconclusive or false-negative results. The study aimed to evaluate the use of bone marrow stromal mesenchymal cells (BM-MSCs) as an alternative, non-haematopoietic tissue for diagnosis. Bone marrow mesenchymal stromal cells from 12 patients with positive diepoxybutane (DEB) tests were cultivated and analysed by cytogenetics and mutation investigation. The DEB test was performed at 0.1 and 0.01 μg/ml concentrations, with an index ranging from 0.24 to 1.00. At higher concentration, the metaphases number was lower, probably due to toxicity. Regarding the molecular investigation, all the mutations previously found in peripheral blood were identified on BM-MSC. This study demonstrated the possibility of using BM-MSCs as an alternative tissue for cytogenetic and molecular investigation. Future tests using an intermediate DEB concentration may lead to an optimal protocol that could be non-toxic to cells but provides conclusive results.

Pancytopenia and thrombosis defects in zebrafish mutants of Fanconi anemia genes

Progressive pancytopenia is a common feature observed in DNA crosslink repair deficiency disorder, Fanconi anemia (FA). However, this phenotype has not been recapitulated in single FA gene knockout animal models. In this study, we analyzed hematological characteristics in zebrafish null mutants for two FA genes, fanca and fanco. In adult mutants, we demonstrate age-associated reduction in blood cell counts for all lineages, resembling progressive pancytopenia in FA patients. In larval mutants, we demonstrate vascular injury-induced thrombosis defects, particularly upon treatment with crosslinking agent diepoxybutane (DEB), indicating DNA damage induced inefficiency of thrombocytes.

TGF-β1 Production By Bone Marrow Mesenchymal Stem Cells of FANCD2 Differs from Other Fanconi Anemia Complementation Groups

TGF-β secretion from cells in the bone marrow (BM) niche affects hematopoietic stem cell (HSC) fate and has a cardinal role in HSC quiescence. As a component of the BM niche, BM- mesenchymal stem cells (BM-MSCs) may produce abnormal levels of TGF-β in Fanconi anemia (FA) as obtained for HSCs previously and may play a role in bone marrow failure.Herein, we present the molecular and cellular characterization of FA BM-MSCs by addressing their immunophenotypes, proliferation- and differentiation capacities, as well as expression and secretion levels of transforming growth factor beta (TGF-β) isoforms. Proliferative potency of FA cells was determined between P3 to P7 and compared with healthy donors by calculating cumulative population doubling (cPD) at each passage. Senescence of FA patient BM-MSCs was evaluated at P3 and P6 by SA-β-gal activity and compared with donor BM-MSCs. Diepoxybutane (DEB) effect on TGF-β1 expression of BM-MSCs from FA patients and donors was also assessed.In ten FA patients, mutations were classified as FANCA (n=7), FANCG (n=1) and FANCD2 (n=2). Of six were novel mutations, five in FANCA and one in FANCD2 . BM-MSCs immunophenotype and differentiation capacity of FA BM-MSCs were similar to healthy donors.The CD200 (OX-2) levels were slightly lower in FA MSCs compared to donors. The percentage of CD106 (VCAM-1) positive cells were slightly high especially in FANCD2 cells. FA BM-MSCs showed arrest in dip G2 following DEB treatment. BM-MSCs derived from FANCA- or FANCG -deficient patients displayed similar ex vivo proliferative capacity as healthy donor cells from P3 to P7. However, FANCD2-deficient cells presented lower proliferative capacity. FANCA BM-MSCs had notably increased β-gal positivity at P6, while cells displaying FANCD2 mutation had higher activity at P3 indicating premature aging of these cells. Relative gene expression of TGF-β1 was similar between donor and FA MSCs and did not change by DEB treatment. All cells did not secrete TGF-β2 and -β3. Since the variation of TGF-β1 secretion by FANCA and donor cells was high, a significant difference between the groups could not be obtained. On the other hand, FANCD2 -deficient cells were lack of TGF-β1 secretion. Our data suggest that absence of TGF-β1 secretion may lead to early onset of senescence of the FA-D2 BM-MSCs and may be of value to understand the Fanconi anaemia bone marrow dysfunction in humans. DisclosuresNo relevant conflicts of interest to declare.

Generating New FANCA-Deficient HNSCC Cell Lines by Genomic Editing Recapitulates the Cellular Phenotypes of Fanconi Anemia.

Fanconi anemia (FA) patients have an exacerbated risk of head and neck squamous cell carcinoma (HNSCC). Treatment is challenging as FA patients display enhanced toxicity to standard treatments, including radio/chemotherapy. Therefore, better therapies as well as new disease models are urgently needed. We have used CRISPR/Cas9 editing tools in order to interrupt the human FANCA gene by the generation of insertions/deletions (indels) in exon 4 in two cancer cell lines from sporadic HNSCC having no mutation in FA-genes: CAL27 and CAL33 cells. Our approach allowed efficient editing, subsequent purification of single-cell clones, and Sanger sequencing validation at the edited locus. Clones having frameshift indels in homozygosis did not express FANCA protein and were selected for further analysis. When compared with parental CAL27 and CAL33, FANCA-mutant cell clones displayed a FA-phenotype as they (i) are highly sensitive to DNA interstrand crosslink (ICL) agents such as mitomycin C (MMC) or cisplatin, (ii) do not monoubiquitinate FANCD2 upon MMC treatment and therefore (iii) do not form FANCD2 nuclear foci, and (iv) they display increased chromosome fragility and G2 arrest after diepoxybutane (DEB) treatment. These FANCA-mutant clones display similar growth rates as their parental cells. Interestingly, mutant cells acquire phenotypes associated with more aggressive disease, such as increased migration in wound healing assays. Therefore, CAL27 and CAL33 cells with FANCA mutations are phenocopies of FA-HNSCC cells.

Genetic background and diagnosis of Fanconi anemia

Fanconi anemia (FA) is a rare genetic disease caused by mutations in genes whose protein products are involved in important cell processes such as replication, cell cycle control and repair of DNA damage. FA is characterized by congenital malformations, bone marrow failure and high risk of cancer. Phenotypic symptoms, present in about 75% of patients, most often include such abnormalities as short stature, microcephaly, thumb and radial side of the limb defects, abnormal skin pigmentation, gastrointestinal and genitourinary defects. Progressive bone marrow failure occurs in the first decade of life, often initially with leukopenia or thrombocytopenia. The most common cancers occurring in patients with FA are myelodysplastic syndromes and acute myeloid leukemia, as well as solid tumors of the head and neck, skin, gastrointestinal system and genitourinary system. So far, 22 genes of Fanconi anemia (FANC) have been identified, which are located on the autosomal chromosomes, except for FANCB, which is located on the X chromosome. Protein products of FANC genes are the elements of Fanconi anemia pathway, which regulates DNA damage repair systems. Genetic diagnostics of Fanconi anemia should start by testing crosslinking agents: mitomycin C (MMC) or diepoxybutane (DEB) assuring differential diagnosis of chromosome instability syndromes. In patients with Fanconi anemia, an increased number of chromosomal gaps and breaks as well as specific radial structures are observed. In order to detect a mutation underlying Fanconi anemia, molecular techniques should be used, preferentially next generation sequencing (NGS).

First Report of Non-Genotoxic Conditioning with JSP191 (anti-CD117) and Hematopoietic Stem Cell Transplantation in a Newly Diagnosed Patient with Severe Combined Immune Deficiency

Successful hematopoietic stem cell transplantation (HSCT) requires vacating recipient hematopoietic stem cell (HSC) niches in the bone marrow to permit donor HSC engraftment that can provide life-long hematopoietic and immune function. Currently, HSCT in SCID relies on DNA damaging chemotherapy to eliminate recipient HSC and achieve niche clearance. We have pursued a non-toxic approach to target and deplete HSC using a humanized monoclonal antibody, JSP191, that binds human CD117 (c-Kit). We previously showed the safety and successful HSC engraftment in a Phase 1 trial of the first 6 patients with severe combined immunodeficiency (SCID), who underwent a second transplant because of HSC engraftment failure and poor immunity after their first transplantation. In these re-transplant patients even a low level of stringently measured myeloid chimerism resulted in significant and sustained generation of naive T cells and clinical improvement. Based on these results, the study of JSP191 (NCT#02963064)has opened a cohort of newly diagnosed infants with SCID. Here we report data from the first patient in this cohort, a SCIDX1 patient who received a primary HSCT with haploidentical CD34+ cells after conditioning with JSP 191. The patient had a c.270-15A>G variant in the IL2RG gene, which is predicted to cause a null phenotype. Besides a T- B+ NK- phenotype typical of SCIDX1 including dysfunctional B cells, the patient had anemia and intermittent neutropenia and thrombocytopenia. Despite evidence of maternal T cell engraftment, the patient had no clinical graft-versus-host disease (GVHD). The patient was initially enrolled in a trial of lentiviral gene therapy, but harvested bone marrow cells died in vitro during transduction and culture. The patient also mobilized poorly with G-CSF/Plerixafor. Further investigation revealed heterozygosity for loss-of-function mutations in two genes involved in DNA repair, BRCA1 and RAD51; Diepoxybutane (DEB) breakage study showed greater than normal pathologic chromosomal breaks, but less than that seen in Fanconi anemia. Because of concern for possible hypersensitivity to alkylating agent-based conditioning, the patient was referred for transplant with JSP191 conditioning. The patient received a CD34+ peripheral blood HSCT from his father after conditioning with 0.3 mg/kg of JSP 191 antibody intravenously over an hour on Day -8 and rATG (Thymoglobulin) on Day -5, -4, -3 and -2 (3.5 mg/kg total) to prevent rejection by the maternal T cells. The cryopreserved donor CD34+ cells were administered after sufficient clearance of the JSP191 serum level. The antibody infusion was well tolerated without toxicity, and the post-transplant course was uneventful without acute toxicities or GVHD. As a surrogate marker for HSC engraftment, CD15+ myeloid cells from peripheral blood were stringently sorted by flow cytometry and donor levels were quantified by short-tandem repeat (STR) analysis. Progressive levels of myeloid engraftment were observed beginning at Week 4. The level of donor chimerism at 12 weeks was 8% in the sorted CD15+ blood cells, and a marrow aspirate showed 25% donor CD34+ cells. By 3 months pre-existing abnormal CD19-CD20+ host B lymphocytes were significantly reduced, and CD19+ donor-derived B lymphocytes were emerging. At 2 months, CD4+ recent thymic emigrant and naïve T lymphocytes were observed, and by 3 months, overall T and NK lymphocyte numbers were 390/uL and 117/uL, respectively. Normal blastogenic responses to the T cell mitogen PHA were observed at 3 months. These first-in-class results provide proof of concept of the safety and efficacy of the use of JSP191 antibody to clear host marrow niche space to enable sufficient donor HSC engraftment and immune reconstitution as primary therapy of SCID. Non-genotoxic conditioning with JSP191 may replace conventional conditioning for newly diagnosed infants with SCID, thereby avoiding toxicities of chemotherapy. Disclosures Kohn: Allogene Therapeutics: Consultancy, Membership on an entity's Board of Directors or advisory committees; Orchard Therapeutics: Consultancy, Patents & Royalties, Research Funding. De Oliveira:Orchard Therapeutics: Research Funding; bluebird bio, Inc.: Research Funding. Czechowicz:Rocket Pharmaceuticals, Inc.: Research Funding. Brown:Merck: Membership on an entity's Board of Directors or advisory committees; Ansun: Membership on an entity's Board of Directors or advisory committees; Cidara: Membership on an entity's Board of Directors or advisory committees; Allogene: Membership on an entity's Board of Directors or advisory committees; Cellerant Therapeutics: Membership on an entity's Board of Directors or advisory committees. Shizuru:Jasper Therapeutics, Inc: Current equity holder in private company, Membership on an entity's Board of Directors or advisory committees.

High frequency of transversion mutations in the rice (Oryza sativa L.) mutant population produced by diepoxybutane mutagenesis

Discovering new genetic mutations is vital for expanding genetic resources for exploring the functions and breeding applications of genes. In this study, we created new mutant populations of rice and evaluated its effectiveness by using a chemical mutagen diepoxybutane (DEB) with the expectation of manageably causing small-scale deletions that induce knock-out mutations. Compared with the more common mutagen, ethyl methanesulfonate (EMS), DEB exhibited approximately 160 times the adverse impact on the early growth of rice. At 0.3 mM, which was 1/160 the concentration of EMS in the control population, the heading date in the DEB-mutated population showed dispersion, albeit with a small standard deviation. Therefore, similar to EMS, DEB has been shown to induce DNA mutations. According to the screening of waxy mutants using the Targeting Induced Local Lesions in Genomes (TILLING) method, DEB-mutated populations had nearly similar mutation frequencies to those of EMS-mutated populations. Therefore, we successfully isolated five mutant lines from the DEB-mutated population. Some of these mutants exhibited a low-amylose phenotype, which is applicable to breeding leading to enhanced taste evaluation. To utilize these mutated alleles, we developed co-dominant DNA markers. In this study, EMS induced transition mutations, as previously reported. In contrast, DEB specifically induced transversion mutations rather than small-scale deletions contrary to our initial expectations. These results demonstrate that DEB has a novel point of action for mutation and is useful for expanding genetic resources for crops.

Diepoxybutane induces the expression of a novel p53-target gene XCL1 that mediates apoptosis in exposed human lymphoblasts.

Diepoxybutane (DEB) is the most potent active metabolite of the environmental chemical 1,3-butadiene (BD). BD is a human carcinogen that exhibits multiorgan systems toxicity. Our previous studies demonstrated that the X-C motif chemokine ligand 1 (XCL1) gene expression was upregulated 3.3-fold in a p53-dependent manner in TK6 lymphoblasts undergoing DEB-induced apoptosis. The tumor-suppressor p53 protein is a transcription factor that regulates a wide variety of cellular processes, including apoptosis, through its various target genes. Thus, the objective of this study was to determine whether XCL1 is a novel direct p53 transcriptional target gene and deduce its role in DEB-induced toxicity in human lymphoblasts. We utilized the bioinformatics tool p53scan to search for known p53 consensus sequences within the XCL1 promoter region. The XCL1 gene promoter region was found to contain the p53 consensus sequences 5'-AGACATGCCTAGACATGCCT-3' at three positions relative to the transcription start site (TSS). Furthermore, the XCL1 promoter region was found, through reporter gene assays, to be transactivated at least threefold by wild-type p53 promoter in DEB-exposed human lymphoblasts. Inactivation of the XCL1 promoter p53-binding motif located at -2.579 kb relative to TSS reduced the transactivation function of p53 on this promoter in DEB-exposed cells by 97%. Finally, knockdown of XCL1 messenger RNA with specific small interfering RNA inhibited DEB-induced apoptosis in human lymphoblasts by 50%. These observations demonstrate, for the first time, that XCL1 is a novel DEB-induced direct p53 transcriptional target gene that mediates apoptosis in DEB-exposed human lymphoblasts.

A NEW METHODOLOGY FOR DIAGNOSIS OF FANCONI ANEMIA BASED ON BIOLOGICAL DOSIMETRY

Fanconi Anemia (FA) is a syndrome associated with chromosomal fragility. Current laboratory tests to diagnose this disease are based on the scoring of chromosomal aberrations induced in peripheral blood lymphocytes by clastogenic chemical agents, mainly: diepoxybutane (DEB) or mitomycin C (MMC). This study evaluated an alternative test for the diagnosis of FA, in which ionizing radiation replaces DEB/MMC. Two groups were studied: normal and DEB-sensitive individuals. From each individual, samples of peripheral blood were irradiated using an electron linear accelerator. Following lymphocyte cultures, and slide preparation, metaphases were scored based on the same methodology for biological dosimetry, according to recommendations of the International Atomic Energy Agency. Our results emphasized a pattern of distribution of dicentrics, fragments, as well as abnormal chromosomal arrangements. The methodology of analysis here proposed permitted to distinguish normal from DEB-sensitive subjects.

Comet assay and cytogenetic findings in differential diagnosis of Fanconi anemia

Fanconi anemia (FA) is a complex genetic disease with a variety of congenital and hematological symptoms, including the predisposition for cancer development. The main hallmark of FA cells, an increased chromosomal fragility, in the presence of the DNA-interstrand cross-linking chemicals, mitomycin C or diepoxybutane (DEB), makes the diagnosis of FA much easier. Cytogenetic method can detect the FA patients with highly elevated chromosomal breakage, but also some of the patients with borderline sensitivity to DEB no matter if they have FA or not. These particular circumstances lead us to introduce comet assay along with cytogenetic analysis, in order to determine DNA lesions and chromosomal fragility in untreated and DEB-treated lymphocytes of full blood from seven patients with clinical features of FA. Highly elevated DEB induced chromosomal sensitivity confirmed the diagnosis in five patients (FA group: 0.48-4.47 breaks/cell vs control group: 0.00-0.08 breaks/cell). Borderline DEB sensitivity (FA* group: 0.15-0.44 breaks/cell) was found in the remaining two patients. Results of the comet assay showed higher baseline and DEB-induced DNA damage values (Olive tail moment and tail intensity) in all five FA and one FA

From exome analysis in idiopathic azoospermia to the identification of a high-risk subgroup for occult Fanconi anemia

PurposeIn about 10% of patients affected by Fanconi anemia (FA) the diagnosis is delayed until adulthood, and the presenting symptom in these “occult” FA cases is often a solid cancer and cancer treatment–related toxicity. Highly predictive clinical parameter(s) for diagnosing such an adult-onset cases are missing. Methods(1) Exome sequencing (ES), (2) Sanger sequencing of FANCA, (3) diepoxybutane (DEB)-induced chromosome breakage test. ResultsES identified a pathogenic homozygous FANCA variant in a patient affected by Sertoli cell–only syndrome (SCOS) and in his azoospermic brother. Although they had no overt anemia, chromosomal breakage test revealed a reverse somatic mosaicism in the former and a typical FA picture in the latter. In 27 selected SCOS cases, 1 additional patient showing compound heterozygous pathogenic FANCA variants was identified with positive chromosomal breakage test. ConclusionWe report an extraordinarily high frequency of FA in a specific subgroup of azoospermic patients (7.1%). The screening for FANCA pathogenic variants in such patients has the potential to identify undiagnosed FA before the appearance of other severe clinical manifestations of the disease. The definition of this high-risk group for “occult” FA, based on specific testis phenotype with mild/borderline hematological alterations, is of unforeseen clinical relevance.

PKNOX2 expression and regulation in the bone marrow mesenchymal stem cells of Fanconi anemia patients and healthy donors.

HOX and TALE transcription factors are important regulators of development and homeostasis in determining cellular identity. Deregulation of this process may drive cancer progression. The aim of this study was to investigate the expression of these transcription factors in the bone marrow derived mesenchymal stem cells (BM-MSCs) of Fanconi anemia (FA) patients, which is a cancer-predisposing disease. Expression levels of HOX and TALE genes in BM-MSCs were obtained from FA patients and healthy donors by RT-qPCR and highly conserved expression levels were observed between patient and donor cells, except PKNOX2, which is a member of TALE class. PKNOX2 was significantly downregulated in FA cells compared to donors (P < 0.05). PKNOX2 expression levels did not change with diepoxybutane (DEB), a DNA crosslinking agent, in either donor or FA cells except one patient's with a truncation mutation of FANCA. A difference of PKNOX2 protein level was not obtained between FA patient and donor BM-MSCs by western blot analysis. When human TGF-β1 (rTGF-β1) recombinant protein was provided to the cultures, PKNOX2 as well as TGF-β1 expression increased both in FA and donor BM-MSCs in a dose dependent manner. 5ng/mL rTGF-β stimulation had more dominant effect on the gene expression of donor BM-MSCs compared to FA cells. Decreased PKNOX2 expression in FA BM-MSCs may provide new insights into the molecular pathophysiology of the disease and TGF-β1 levels of the microenvironment may be the cause of PKNOX2 downregulation.

Severe Aplastic Anemia (SAA) in Colombia: Characteristics and Treatment in 6 Centers

IntroductionAcquired severe aplastic anemia (SAA) is a low frequency hematologic disease associated with significant morbidity and mortality. The destruction by immunological mechanisms of hematopoietic progenitor cells appears to play a fundamental role in the pathophysiology of the disease, therefore, immunosuppressive treatment (IST) is one of the pillars of management. In Colombia and Latin-America there is lack of information about the characteristics of patients with SAA and the effectiveness of IST and other treatments. The main objective of this study was to assess the outcomes of a group of patients treated in different centers in Colombia during the last five years.Materials and methodsThis is a retrospective observational study, carried out within 6 institutions of 3 Colombian cities (Bogota, Medellín, Bucaramanga) of patients treated from 2013 to 2018. An anonymized database was constructed. All patients diagnosed with SAA and treated with IST as first line of therapy were included.ResultsWe analyzed the data of 37 patients; 22 of them were women. The average age was 37 years old (with a range of 7-68 years). The median time since the onset of the symptoms to the time of diagnosis was 88 days (with a range of 7-655 days). Clastogenic effect of diepoxybutane (DEB) tests were available only in 9 patients (7 of which were positive); Paroxysmal Nocturnal Hemoglobinuria (PNH) clone was detected in 9 of the 27 tested, all of which were under 5%. Mean leukocytes, hemoglobin concentration, and platelets at the moment of diagnosis were 3.090, 8.5 and 22.000. Bacterial infection was the most important clinical manifestation at diagnosis (in 16 of the 37 patients); 19 out of 37 patients had more than 5 red blood cell transfusions before the first treatment. As first line therapy, 25 patients received IST (most by antithymocyte globulin (ATG) of which 10 were treated with rabbit ATG, 12 with horse ATG and 3 combined with Eltrombopag); 4 patients had cyclosporin and prednisone, 2 had allogeneic transplant and 6 received other therapies. Those treated with IST patients, 8 had a complete response (CR) (32%), 6 had partial response (PR) (24%), 8 had no response (32%). 5 received a second course of IST and 3 had CR. 3 patients were lost in follow up. At a mean follow up of 18.7 months (1.7-65), 44% were alive in CR, 26% in PR, 9% were in relapse and 21% died. Causes of death were SAA related infection in 5 patients, transplant related in 1 and non-disease related in 1.ConclusionsThis is the first multicenter report of SAA treatment in Colombia, it includes children and adults, resulting in a very heterogeneous population. This preliminary information shows that there is a significant delay in the diagnosis and initiation of treatment in patients, most patients have more than 5 transfusions before treatment. This could, in part, explain the apparent low response rates to IST in comparison to that of what have been reported in literature. A larger registry of hematologic pathologies is required so we can confirm these findings, start proposing strategies applicable in our country to achieve better outcomes in these patients and planning clinical trials to answer questions relevant to our population. DisclosuresNo relevant conflicts of interest to declare.

A Clinical, Genomic and Proteomic Approach for the Characterization of Fanconi Anemia in Adolescent and Young Adult (AYA) Patients : A Single Center Study of 55 Patients from a National Bone Marrow Failure Referral Center

Introduction:Fanconi Anemia (FA) is an autosomal recessive disorder characterized by bone marrow failure (BMF), constitutional anomalies and high risk of developing cancer. Distinguishing FA from severe aplastic anemia (SAA) can be challenging especially in asyndromic patients. We undertook a clinical and laboratory cohort study of adolescent and young adult (AYA) patients with a diagnosis of FA treated at our institution to characterize the clinical features in our population, and conducted a prospective translational study to explore integration of a genomic and proteomic approach for improved diagnosis and molecular characterization of FA.Methods:Data on FA patients was obtained from an institutionally approved BMF database and hematopoietic stem cell transplant (HSCT) database. Further data was obtained from a register of chromosomal breakage (CB) analysis results. Index cases were identified if they were older than 14 years of age at the time of diagnosis or under the care of adult hematology with a clinical diagnosis of FA based on the presence of BMF and abnormal CB, or clinical phenotype with the presence of homozygous FA related genes. Family pedigrees were constructed based on history.In addition, patients presenting with BMF were enrolled onto an institutionally approved study investigating proteomic biomarkers and genomics of BMF syndromes. Consented peripheral blood samples and/or extracted DNA were subject to either panel based next generation sequencing (NGS) testing as part of the Saudi Genome Project or subjected to whole exome sequencing (WES) by external lab.For proteomic analysis, peripheral blood plasma (PBP) samples from 6 patients with FA, 10 SAA patients and 7 normal controls were subjected to expression proteomics using liquid chromatography tandem mass spectrometry (LC-MS/MS).Result:Patients and clinical features: 55 patients (26 M, 29 F) in 30 families were identified. While 18 patients (32%) were referred with a diagnosis/suspicion of FA, in 26 (47%) FA was diagnosed at our institution. The most frequent anomaly was short stature (14 patients, 25%), skin changes (7, 12%), urogenital abnormalities (7, 12%), dysmorphism/craniofacial abnormalities (7, 12%), hands anomalies (4, 7%); 12 (22%) had no recorded anomalies. 18 patients (33%) developed a malignancy either before or after diagnosis of FA: solid tumors in 5 (9%), AML and/or MDS in 15 (27%); 3 (5%) of these patients had both solid tumors and AML/MDS.Diagnostic Tests: 35 patients (63.6%) had a positive CB analysis with diepoxybutane (DEB) or mitomycin-C (MMC) testing; in 5 patients (9%) DEB testing was borderline and 3 (5%) had a normal CBA but had a diagnostic phenotype+/- family history and presence of a homozygous mutation in a known FA related gene. 14 patients had cytogenetic abnormalities and abnormalities involving chromosome 1 were the most frequent (50%).Mutation Analysis: Mutational analysis was available for 12 (22%) cases; homozygous mutations in FA genes were identified in 10 patients (18%) in 7 families (23% of families): FANCA (5 patients/3 families); BRIP1 (2/2); FANCP (1/1); FANCD2 (2/1). In one case, post matched sibling (HSCT) blood sample revealed a known pathogenic heterozygous c.2632G>C,p.Glu878Gln mutation in FANCA, suggesting a carrier donor.Proteomic analysis: Over 1650 unique PBP protein species were identified of which 605 were significantly differentially expressed (≥ 2 to ∞ - fold change & p < 0.001) between SAA /FA/ normal control subjects (Fig 1a). DNMT3A, Kinase Insert Domain Receptor (KDR) and TGFB-1 was found to be highly expressed in SAA versus FA, while ATM and APOB were highly expressed in FA versus SAA (Fig.1b).Treatment outcomes: 36 out of 55 patients (65%) received HSCT. Actuarial survival of HSCT (n=37) and non-HSCT (n=14) patients was 70% and 77%, respectively. Treatment details were not available on 6 (11%).CONCLUSION:We report the first characterization of AYA patients with FA in Saudi Arabia. Our report emphasizes the need for a high index of suspicion of a diagnosis of FA in BMFs. CB may be falsely negative in cases, and panel based and/or WES based NGS testing increases diagnostic accuracy; in this cohort, mutations in FANCA were the most frequent (50%). Occurrence of hematological and solid tumors is a significant risk in these AYA patients. We also report proteomic panels as potential biomarkers that distinguish FA from SAA and may provide mechanistic insights. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

A rare manifestation of Fanconi anemia

Fanconi anemia is an inherited pancytopenia, primarily inherited as autosomal recessive form. It occurs in all racial and ethnic groups. Majority of patients have both physical and haematological abnormalities, about one-third of patients will have normal physical features but abnormal haematological findings and unknown percentage have physical anomalies and normal haematological findings. The diagnosis is based on characteristic physical anomalies and abnormal haematological findings, which is confirmed with a lymphocytic chromosomal breakage study using Diepoxy butane (DEB). The report here is about a two and half years old female child who presented with physical features in the form of short stature, microcephaly, left hypoplastic thumb and congenital heart disease without haematological abnormalities. Chromosomal study was suggestive of Fanconi’s anemia.

Cross-linking by epichlorohydrin and diepoxybutane correlates with cytotoxicity and leads to apoptosis in human leukemia (HL-60) cells

The bifunctional alkylating agents epichlorohydrin (ECH) and diepoxybutane (DEB) have been linked to increased cancer risks in industrial workers. These compounds react with DNA and proteins, leading to genotoxic effects. We used the comet assay to monitor formation of cross-links in HL-60 cells treated with ECH, DEB, and the structurally related anti-cancer drug mechlorethamine (HN2). We report a time- and dose-dependent cytotoxicity that correlated with cross-linking activity, following the order HN2 > DEB > ECH. The rate of cross-link repair also varied with drug, with ECH-induced lesions the fastest to repair. High drug doses led to the formation of saturating amounts of HN2 cross-links that were repaired inefficiently. DEB and ECH produced fewer overall cross-links, but some were also resistant to repair. These persistent cross-links may activate cell-cycle arrest to allow repair of damage, with prolonged arrest triggering apoptosis. Quantitative reverse transcription polymerase chain reaction experiments revealed that treatment of HL-60 cells with DEB and ECH results in up-regulation of several genes involved in the intrinsic (mitochondrial) apoptosis pathway, including BAX, BAK1, CASP-9, APAF-1, and BCL-2. These findings contribute to our understanding of the principles underlying the carcinogenic potentials of these xenobiotics.

Diepoxybutane-induced apoptosis is mediated through the ERK1/2 pathway.

Diepoxybutane (DEB) is the most potent active metabolite of butadiene, a regulated air pollutant. We previously reported the occurrence of DEB-induced, p53-dependent, mitochondrial-mediated apoptosis in human lymphoblasts. The present study investigated the role of the extracellular signal-regulated protein kinases 1 and 2 (ERK1/2) pathway in DEB-induced apoptotic signaling in exposed human lymphoblasts. Activated ERK1/2 and mitogen-activated protein (MAP) kinase/ERK1/2 kinase (MEK) levels were significantly upregulated in DEB-exposed human lymphoblasts. The MEK inhibitor PD98059 and ERK1/2 siRNA significantly inhibited apoptosis, ERK1/2 activation, as well as p53 and phospho-p53 (serine-15) levels in human lymphoblasts undergoing DEB-induced apoptosis. Collectively, these results demonstrate that DEB induces apoptotic signaling through the MEK-ERK1/2-p53 pathway in human lymphoblasts. This is the first report implicating the activation of the ERK1/2 pathway and its subsequent role in mediating DEB-induced apoptotic signaling in human lymphoblasts. These findings contribute towards the understanding of DEB toxicity, as well as the signaling pathways mediating DEB-induced apoptosis in human lymphoblasts.