Fanconi anemia (FA) is an autosomal recessive disorder of hematopoiesis characterized by hypersensitivity to DNA crosslinkers such as mitomycin C (MMC). There is growing evidence for a model of the FA pathway, wherein a nuclear multiprotein complex of five FA proteins (FANCA, C, E, F and G) regulates activation of FANCD2 into a monoubiquitinated form, which, collaborating with the BRCA1 machinery, affects cellular response to DNA damage. However, the role of the FA pathway in defective DNA damage response caused by various mutant forms of FA proteins has not been fully assessed. In the present study, 21 patient-derived FANCA mutants with a missense or a small in-frame deletion were expressed in FANCA-deficient fibroblasts and examined for complementation of MMC sensitivity and for reconstitution of the FA pathway: FANCA phosphorylation, interaction with FANCC, FANCF and FANCG and nuclear localization and FANCD2 monoubiquitination. The altered FANCA proteins complemented MMC sensitivity at different grades: five proteins (group I) behaved like wild-type FANCA, whereas the other proteins were either mildly (group II, n=4) or severely (group III, n=12) impaired. Group I proteins showed an apparently normal reconstitution of the FA pathway, thus they may be pathogenic by reducing endogenous expression or possibly benign polymorphisms. Reconstitution of the FA pathway by group II and III mutants closely correlated with cellular sensitivity to MMC. The different activation of the FA pathway may partly account for the phenotypic variation seen in FA patients.
ABSTRACThad a high risk of bias due to their study design and were conducted more than 10 years ago and may not be applicable to the standard of care of today.11 Updated evidence to aid treatment decisions in pediatric SAA is, therefore, required.In children, the choice of an appropriate treatment is particularly influenced by the long-term sequelae of the disease and its therapy. Thus, failure-free survival is much more important than survival alone when analyzing the long-term outcomes of children with aplastic anemia. Lack of response, relapse, and clonal evolution are problematic in the IST setting, whereas graft failure, acute and chronic graft-versus-host disease (GVHD), and infectious complications limit the success of BMT. In the present study, we compared the outcomes of children with SAA who received IST or BMT from an MFD as first-line treatment using data from nationwide IST and BMT registries.
Methods
PatientsBetween 1992 and 2009, a total of 599 consecutive children (younger than 17 years) with acquired SAA underwent BMT from an MFD or received IST as first-line treatment in Japan; 213 patients with an MFD underwent BMT and were registered in the Transplant Registry Unified Management Program (TRUMP) conducted by the Japanese Society for Hematopoietic Cell Transplantation, and 386 patients without an MFD were enrolled in two consecutive prospective multicenter trials (AA-92/97) conducted by the Japanese Childhood Aplastic Anemia Study Group and were initially treated with IST (Table 1). The disease severities were defined as previously reported.12,13 Underlying inherited marrow failure disorders were excluded clinically and by chromosome fragility testing. Marrow cytogenetic studies were performed for all patients, and patients with clonal cytogenetic abnormalities were excluded from this study. Patients with paroxysmal nocturnal hemoglobinuria with clinical symptoms and positive findings on the Ham test/sucrose test were also excluded from this analysis. All treatments were performed after obtaining written informed consent from patients or their parents in accordance with the Declaration of Helsinki.
Immunosuppressive therapy and bone marrow transplantation proceduresThe characteristics of the treatment procedures are detailed in Table 2. Three hundred and eighty-six patients were enrolled in the AA-92 (n=84) and AA-97 (n=302) trials, and all the patients were initially treated with a combination of antithymocyte globulin and cyclosporine A. Response to IST and disease relapse were evaluated as previously reported.12 Transplantation data were collected with the use of standardized forms provided by the TRUMP. A total of 213 patients underwent BMT from an MFD as first-line treatment following the local protocols for conditioning regimens and GVHD prophylaxis. Patients who did not reach neutrophil counts >0.5×10 9 /L for 3 consecutive days after transplantation were considered to have had primary graft failure. Patients with initial engraftment in whom absolute neutrophil counts subsequently declin...
We developed a multiplex real-time PCR assay using 6-carboxyfluorescein, 6-carboxy-4,5-dichloro-2,7-dimethoxyfluorescein, and carbocyanine 5-labeled probes to simultaneously quantify Epstein-Barr virus (EBV), cytomegalovirus (CMV), and human herpesvirus 6 (HHV-6) DNA. When previously tested and stored DNA samples were examined, results of the multiplex real-time PCR assay were as sensitive and specific as those of a single real-time PCR assay. The multiplex assay was used to quantify the EBV, CMV, and HHV-6 DNA in 46 transplant recipients. A total of 303 whole-blood and plasma specimens were collected and analyzed. According to the results of the multiplex assay, the detection rates for viral DNA in whole blood and plasma were 23.8% and 5.9% for EBV, 11.2% and 5.3% for CMV, and 12.5% and 2.0% for HHV-6, respectively. All forms of viral DNA were detected more frequently in whole blood than in plasma. During the symptomatic period, EBV DNA was detected in all whole-blood specimens but not in all plasma specimens. Furthermore, the EBV DNA load in whole blood was higher during the symptomatic period than during the asymptomatic period, whereas the EBV DNA load in plasma was similar for both periods. These results demonstrate that whole blood is more suitable for the quantification of EBV DNA in transplant patients. However, a cutoff value with clinical relevance still needs to be determined.
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