scholarly journals Diagnosis of Fanconi Anemia: Mutation Analysis by Multiplex Ligation-Dependent Probe Amplification and PCR-Based Sanger Sequencing

Anemia ◽  
2012 ◽  
Vol 2012 ◽  
pp. 1-13 ◽  
Author(s):  
Johan J. P. Gille ◽  
Karijn Floor ◽  
Lianne Kerkhoven ◽  
Najim Ameziane ◽  
Hans Joenje ◽  
...  
Keyword(s):  
Fanconi Anemia ◽  
Sanger Sequencing ◽  
Bone Marrow Failure ◽  
Genetic Diagnosis ◽  
Cost Effective ◽  
Inherited Disease ◽  
Dna Testing ◽  
Founder Mutations ◽  
Chromosomal Breakage ◽  
Developmental Defects

Fanconi anemia (FA) is a rare inherited disease characterized by developmental defects, short stature, bone marrow failure, and a high risk of malignancies. FA is heterogeneous: 15 genetic subtypes have been distinguished so far. A clinical diagnosis of FA needs to be confirmed by testing cells for sensitivity to cross-linking agents in a chromosomal breakage test. As a second step, DNA testing can be employed to elucidate the genetic subtype of the patient and to identify the familial mutations. This knowledge allows preimplantation genetic diagnosis (PGD) and enables prenatal DNA testing in future pregnancies. Although simultaneous testing of all FA genes by next generation sequencing will be possible in the near future, this technique will not be available immediately for all laboratories. In addition, in populations with strong founder mutations, a limited test using Sanger sequencing and MLPA will be a cost-effective alternative. We describe a strategy and optimized conditions for the screening ofFANCA, FANCB, FANCC, FANCE, FANCF,andFANCGand present the results obtained in a cohort of 54 patients referred to our diagnostic service since 2008. In addition, the follow up with respect to genetic counseling and carrier screening in the families is discussed.

scholarly journals Diagnosis of Fanconi Anemia: Mutation Analysis by Next-Generation Sequencing

Anemia ◽  
2012 ◽  
Vol 2012 ◽  
pp. 1-7 ◽  
Author(s):  
Najim Ameziane ◽  
Daoud Sie ◽  
Stefan Dentro ◽  
Yavuz Ariyurek ◽  
Lianne Kerkhoven ◽  
...  
Keyword(s):  
Fanconi Anemia ◽  
Clinical Symptoms ◽  
Massively Parallel Sequencing ◽  
Bone Marrow Failure ◽  
Genetic Diagnosis ◽  
Chromosomal Breakage ◽  
Developmental Defects ◽  
Pathogenic Mutations ◽  
High Cancer Risk ◽  
Generation Sequencing

Fanconi anemia (FA) is a rare genetic instability syndrome characterized by developmental defects, bone marrow failure, and a high cancer risk. Fifteen genetic subtypes have been distinguished. The majority of patients (≈85%) belong to the subtypes A (≈60%), C (≈15%) or G (≈10%), while a minority (≈15%) is distributed over the remaining 12 subtypes. All subtypes seem to fit within the “classical” FA phenotype, except for D1 and N patients, who have more severe clinical symptoms. Since FA patients need special clinical management, the diagnosis should be firmly established, to exclude conditions with overlapping phenotypes. A valid FA diagnosis requires the detection of pathogenic mutations in a FA gene and/or a positive result from a chromosomal breakage test. Identification of the pathogenic mutations is also important for adequate genetic counselling and to facilitate prenatal or preimplantation genetic diagnosis. Here we describe and validate a comprehensive protocol for the molecular diagnosis of FA, based on massively parallel sequencing. We used this approach to identifyBRCA2,FANCD2,FANCIandFANCLmutations in novel unclassified FA patients.

scholarly journals Diagnostic Overlap between Fanconi Anemia and the Cohesinopathies: Roberts Syndrome and Warsaw Breakage Syndrome

Anemia ◽  
2010 ◽  
Vol 2010 ◽  
pp. 1-7 ◽  
Author(s):  
Petra van der Lelij ◽  
Anneke B. Oostra ◽  
Martin A. Rooimans ◽  
Hans Joenje ◽  
Johan P. de Winter
Keyword(s):  
Fanconi Anemia ◽  
Bone Marrow Failure ◽  
Clinical Manifestations ◽  
Nijmegen Breakage Syndrome ◽  
Inherited Disease ◽  
Chromosomal Breakage ◽  
Metaphase Chromosomes ◽  
Roberts Syndrome ◽  
Premature Centromere Division ◽  
Multiple Symptoms

Fanconi anemia (FA) is a recessively inherited disease characterized by multiple symptoms including growth retardation, skeletal abnormalities, and bone marrow failure. The FA diagnosis is complicated due to the fact that the clinical manifestations are both diverse and variable. A chromosomal breakage test using a DNA cross-linking agent, in which cells from an FA patient typically exhibit an extraordinarily sensitive response, has been considered the gold standard for the ultimate diagnosis of FA. In the majority of FA patients the test results are unambiguous, although in some cases the presence of hematopoietic mosaicism may complicate interpretation of the data. However, some diagnostic overlap with other syndromes has previously been noted in cases with Nijmegen breakage syndrome. Here we present results showing that misdiagnosis may also occur with patients suffering from two of the three currently known cohesinopathies, that is, Roberts syndrome (RBS) and Warsaw breakage syndrome (WABS). This complication may be avoided by scoring metaphase chromosomes—in addition to chromosomal breakage—for spontaneously occurring premature centromere division, which is characteristic for RBS and WABS, but not for FA.

scholarly journals Diagnosis of Fanconi Anemia: Chromosomal Breakage Analysis

Anemia ◽  
2012 ◽  
Vol 2012 ◽  
pp. 1-9 ◽  
Author(s):  
Anneke B. Oostra ◽  
Aggie W. M. Nieuwint ◽  
Hans Joenje ◽  
Johan P. de Winter
Keyword(s):  
Mitomycin C ◽  
Fanconi Anemia ◽  
Autosomal Recessive ◽  
Clinical Symptoms ◽  
Bone Marrow Failure ◽  
Blood Cultures ◽  
Chromosomal Breakage ◽  
Developmental Defects ◽  
Cycle Arrest ◽  
Laboratory Protocol

Fanconi anemia (FA) is a rare inherited syndrome with diverse clinical symptoms including developmental defects, short stature, bone marrow failure, and a high risk of malignancies. Fifteen genetic subtypes have been distinguished so far. The mode of inheritance for all subtypes is autosomal recessive, except for FA-B, which is X-linked. Cells derived from FA patients are—by definition—hypersensitive to DNA cross-linking agents, such as mitomycin C, diepoxybutane, or cisplatinum, which becomes manifest as excessive growth inhibition, cell cycle arrest, and chromosomal breakage upon cellular exposure to these drugs. Here we provide a detailed laboratory protocol for the accurate assessment of the FA diagnosis as based on mitomycin C-induced chromosomal breakage analysis in whole-blood cultures. The method also enables a quantitative estimate of the degree of mosaicism in the lymphocyte compartment of the patient.

Unexpected Fanconi Anemia Diagnosis in Patients with Bone Marrow Failure.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 1056-1056
Author(s):  
Fernando O. Pinto ◽  
Thierry Leblanc ◽  
Gwenaelle Le Roux ◽  
Helene Dastot ◽  
Moema Santos ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Aplastic Anemia ◽  
Clinical Features ◽  
Fanconi Anemia ◽  
Bone Marrow Failure ◽  
Somatic Mosaicism ◽  
Chromosome Breakage ◽  
Large Set ◽  
Chromosomal Breakage ◽  
Group A

Abstract Early diagnosis of Fanconi Anemia (FA) in patients with bone marrow failure is critical for optimal clinical management. However, the remarkably high clinical variability and the potential emergence of revertant hematopoietic cells (somatic mosaicism) can obscure and delay the diagnosis of FA. Here we addressed FA diagnosis in a prospective series of adult and pediatric patients who presented with bone marrow failure without clear overall clinical picture of FA. Sixty-six patients were classified into three groups: (1) bone marrow failure likely to be congenital, based on dysmorphic features or a family history [n=18], (2) aplastic anemia likely to be idiopathic [n=32], (3) patients with intermediate clinical features not classified into the former groups [n=16]. Of note, FA patients with typical clinical features were not included in the present study. FA diagnosis was evaluated using chromosome breakage test and FANCD2 immunoblot in PHA-stimulated-PBL. In addition, skin primary fibroblasts were analysed in order to overcome potential hematopoietic FA reversion. For that purpose, and considering that chromosome breakage tests are barely efficient in fibroblasts, we used FANCD2 immunoblot and also developped a new flow cytometry test based on MMC-sensitivity in fibroblasts (to detect downstream FA/BRCA groups). Using these approaches, we detected FA in 4 previously undiagnosed patients: a 35-years old patient from the congenital-like group; a 10-years old patient presenting as an idiopathic aplastic anemia without any FA signs; and two patients from the intermediate group: a 10-years old patient with an isolated thrombocytopenia, and a 50-years old patient presenting with pancytopenia/MDS and complete hematopoietic reversion. Importantly, FA diagnosis was definitely excluded in all other patients. In conclusion, we could identify a few unexpected FA cases in a series of patients with bone marrow failure. Therefore, the comprehensive use of a large set of tests is useful for accurate FA diagnosis. Classical chromosomal breakage tests in PBL appeared to be sufficient to exclude FA in idiopathic aplastic anemia, whereas fibroblast analysis can be necessary to definitely diagnose or exclude FA in other patients.

Constitutive elevation of serum alpha-fetoprotein in Fanconi anemia

Blood ◽  
2000 ◽  
Vol 96 (3) ◽  
pp. 859-863 ◽  
Author(s):  
Bruno Cassinat ◽  
Philippe Guardiola ◽  
Sylvie Chevret ◽  
Marie-Hélène Schlageter ◽  
Marie-Elisabeth Toubert ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Fanconi Anemia ◽  
Bone Marrow Failure ◽  
Allogeneic Bone Marrow Transplantation ◽  
Cost Effective ◽  
Alpha Fetoprotein ◽  
Allogeneic Bone ◽  
Bone Marrow Failure Syndromes ◽  
Chromosomal Breaks ◽  
Serum Alpha Fetoprotein

The diagnosis of Fanconi anemia (FA) is based on the association of congenital malformations, bone marrow failure syndrome, and hypersensitivity to chromosomal breaks induced by cross-linking agents. In the absence of typical features, the diagnosis is not easy to establish because there is no simple and cost-effective test; thus, investigators must rely on specialized analyses of chromosomal breaks. Because we observed elevated serum alpha-fetoprotein (sAFP) levels in FA patients, we investigated this parameter as a possible diagnostic tool. Serum AFP levels from 61 FA patients and 27 controls with acquired aplastic anemia or other inherited bone marrow failure syndromes were analyzed using a fluoroimmunoassay based on the TRACE technology. Serum AFP levels were significantly more elevated (P < .0001) in FA than in non-FA aplastic patients. In the detection of FA patients among patients with bone marrow failure syndromes, this assay had a sensitivity of 93% and a specificity of 100%. This elevation was not explained by liver abnormalities. Levels of sAFP were unchanged during at least 4 years of follow-up, and allogeneic bone marrow transplantation did not modify sAFP levels. Three of 4 FA patients with mosaicism as well as 5 of 6 FA patients with myelodysplastic syndrome were detected by this test. Heterozygous parents of FA patients had normal sAFP levels. Measurement of sAFP levels with this automated, cost-effective, and reproducible fluoroimmunoassay could be proposed for the preliminary diagnosis of FA whenever this disorder is suspected.

scholarly journals Knockdown of Fanconi anemia genes in human embryonic stem cells reveals early developmental defects in the hematopoietic lineage

Blood ◽  
2010 ◽  
Vol 115 (17) ◽  
pp. 3453-3462 ◽  
Author(s):  
Asmin Tulpule ◽  
M. William Lensch ◽  
Justine D. Miller ◽  
Karyn Austin ◽  
Alan D'Andrea ◽  
...  
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
Embryonic Stem Cells ◽  
Human Embryonic Stem Cells ◽  
Fanconi Anemia ◽  
Bone Marrow Failure ◽  
Embryonic Stem ◽  
Developmental Defects ◽  
Hematopoietic Development

Abstract Fanconi anemia (FA) is a genetically heterogeneous, autosomal recessive disorder characterized by pediatric bone marrow failure and congenital anomalies. The effect of FA gene deficiency on hematopoietic development in utero remains poorly described as mouse models of FA do not develop hematopoietic failure and such studies cannot be performed on patients. We have created a human-specific in vitro system to study early hematopoietic development in FA using a lentiviral RNA interference (RNAi) strategy in human embryonic stem cells (hESCs). We show that knockdown of FANCA and FANCD2 in hESCs leads to a reduction in hematopoietic fates and progenitor numbers that can be rescued by FA gene complementation. Our data indicate that hematopoiesis is impaired in FA from the earliest stages of development, suggesting that deficiencies in embryonic hematopoiesis may underlie the progression to bone marrow failure in FA. This work illustrates how hESCs can provide unique insights into human development and further our understanding of genetic disease.

High Incidence of Fanconi Anemia in Patients with a Morphological Picture Consistent with Refractory Cytopenia of Childhood

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 2780-2780
Author(s):  
Ayami Yoshimi ◽  
Charlotte M. Niemeyer ◽  
Irith Baumann ◽  
Stephan Schwarz-Furlan ◽  
Detlev Schindler ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Fanconi Anemia ◽  
Conflicts Of Interest ◽  
Bone Marrow Failure ◽  
Test Group ◽  
Clinical Feature ◽  
Chromosomal Breakage ◽  
Mild Phenotype ◽  
European Working ◽  
Chromosome Breaking

Abstract Abstract 2780 Introduction: Refractory cytopenia in childhood (RCC) is the most common subtype of myelodysplastic syndrome (MDS) in children. Differential diagnosis from inherited bone marrow failure (IBMF) such as Fanconi anemia (FA) remains an intriguing challenge, because most patients with RCC have a hypocellular bone marrow (BM) and dysplastic features in haematopoiesis are observed in both RCC and IBMF. Moreover the spectrum of phenotypic findings in FA is extremely wide. Some FA patients have a mild phenotype without malformation. The purpose of this study is to estimate the incidence of FA in an RCC cohort without a full clinical feature of FA, but subsequently diagnosed by chromosome breaking test. Patients and Methods: Between 01/2007 and 12/2010 reference pathologists of the European Working Group of MDS in Childhood (EWOG-MDS) provided a morphological report consistent with RCC in 137 children studied in Germany. Seventeen patients with hypercellular BM or abnormal karyotype, 2 patients, in whom dyskeratosis congenital was diagnosed after initial inclusion and one patient, in whom chromosome breaking test was not performed, were excluded. Results: Seven of remaining 117 patients had facial and/or skeletal anomalies typically noted in FA and one patient had a brother with FA. In these 8 patients, FA had been suspected by their local physicians (group FA-1). Nine patients (8.3%) without these typical anomalies were subsequently diagnosed of FA by chromosome breakage test (group FA-2). The diagnosis of RCC was finally made in the remaining 100 patients with negative chromosomal breakage test (group RCC). The clinical features of patients in each group are summarized in the Table. The mean corpuscular volume of red cells (MCV) was elevated (> +2SD) for ages in all patients with FA, but only 42 % in patient with RCC. In some children of group FA-2 additional non-haematological abnormalities were also observed. However, they were not evident and or typical to prompt the treating physicians to suspect FA. A few patients in the group RCC also had some physical anomalies, not specific for any of the known IBMF disorders. Possibly that other known or not yet described IBMF disorders remain uncovered in children with “de novo” RCC. Conclusion: Our results illustrate that the same haematological features and congenital anomalies can be noted in FA and RCC. More importantly, they indicate that the exclusion of FA by a chromosomal breakage test or other methods is mandatory in all patients prior to diagnosis RCC. Chromosomal breakage analysis may identify patients with FA in 8% of patients with a morphological description of RCC without a full clinical picture of FA. Disclosures: No relevant conflicts of interest to declare.

Fanconi anemia type C and p53 cooperate in apoptosis and tumorigenesis

Blood ◽  
2003 ◽  
Vol 102 (12) ◽  
pp. 4146-4152 ◽  
Author(s):  
Brian Freie ◽  
Xiaxin Li ◽  
Samantha L. M. Ciccone ◽  
Kathy Nawa ◽  
Scott Cooper ◽  
...  
Keyword(s):  
Fanconi Anemia ◽  
Bone Marrow Failure ◽  
Developmental Defects ◽  
Atm Gene ◽  
Type C ◽  
Cellular Stresses ◽  
Brca1 Brca2 ◽  
Induced Apoptosis ◽  
Factor Α ◽  
Tumor Types

Abstract Fanconi anemia (FA) is a recessive genomic instability syndrome characterized by developmental defects, progressive bone marrow failure, and cancer. FA is genetically heterogeneous, however; the proteins encoded by different FA loci interact functionally with each other and with the BRCA1, BRCA2, and ATM gene products. Although patients with FA are highly predisposed to the development of myeloid leukemia and solid tumors, the alterations in biochemical pathways responsible for the progression of tumorigenesis in these patients remain unknown. FA cells are hypersensitive to a range of genotoxic and cellular stresses that activate signaling pathways mediating apoptosis. Here we show that ionizing radiation (IR) induces modestly elevated levels of p53 in cells from FA type C (Fancc) mutant mice and that inactivation of Trp53 rescues tumor necrosis factor α-induced apoptosis in myeloid cells from Fancc-/- mice. Further, whereas Fancc-/- mice failed to form hematopoietic or solid malignancies, mice mutant at both Fancc and Trp53 developed tumors more rapidly than mice mutant at Trp53 alone. This shortened latency was associated with the appearance of tumor types that are found in patients with FA but not in mice mutant at Trp53 only. Collectively, these data demonstrate that p53 and Fancc interact functionally to regulate apoptosis and tumorigenesis in Fancc-deficient cells. (Blood. 2003;102:4146-4152)

scholarly journals Bone Marrow Failure in the Fanconi Anemia Group C Mouse Model After DNA Damage

Blood ◽  
1998 ◽  
Vol 91 (8) ◽  
pp. 2737-2744 ◽  
Author(s):  
Madeleine Carreau ◽  
Olga I. Gan ◽  
Lili Liu ◽  
Monica Doedens ◽  
Colin McKerlie ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Bone Marrow ◽  
Dna Damage ◽  
Mouse Model ◽  
Fanconi Anemia ◽  
Bone Marrow Failure ◽  
Blood Parameters ◽  
Cross Linking ◽  
Inherited Disease

Fanconi anemia (FA) is a pleiotropic inherited disease that causes bone marrow failure in children. However, the specific involvement of FA genes in hematopoiesis and their relation to bone marrow (BM) failure is still unclear. The increased sensitivity of FA cells to DNA cross-linking agents such as mitomycin C (MMC) and diepoxybutane (DEB), including the induction of chromosomal aberrations and delay in the G2 phase of the cell cycle, have suggested a role for the FA genes in DNA repair, cell cycle regulation, and apoptosis. We previously reported the cloning of the FA group C gene (FAC) and the generation of a Fac mouse model. Surprisingly, the Fac −/− mice did not show any of the hematologic defects found in FA patients. To better understand the relationship of FA gene functions to BM failure, we have analyzed the in vivo effect of an FA-specific DNA damaging agent in Fac −/− mice. The mice were found to be highly sensitive to DNA cross-linking agents; acute exposure to MMC produced a marked BM hypoplasia and degeneration of proliferative tissues and caused death within a few days of treatment. However, sequential, nonlethal doses of MMC caused a progressive decrease in all peripheral blood parameters of Fac −/− mice. This treatment targeted specifically the BM compartment, with no effect on other proliferative tissues. The progressive pancytopenia resulted from a reduction in the number of early and committed hematopoietic progenitors. These results indicate that the FA genes are involved in the physiologic response of hematopoietic progenitor cells to DNA damage.

scholarly journals The Fanconi Anemia Polypeptide, FAC, Binds to the Cyclin-Dependent Kinase, cdc2

Blood ◽  
1997 ◽  
Vol 90 (3) ◽  
pp. 1047-1054 ◽  
Author(s):  
Gary M. Kupfer ◽  
Takayuki Yamashita ◽  
Dieter Naf ◽  
Ahmed Suliman ◽  
Shigetaka Asano ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Fanconi Anemia ◽  
Mammalian Cells ◽  
Cell Cycle Progression ◽  
Cancer Susceptibility ◽  
Bone Marrow Failure ◽  
Autosomal Recessive Disorder ◽  
Cyclin Dependent Kinase ◽  
Binding Interaction ◽  
Developmental Defects

Fanconi anemia (FA) is an autosomal recessive disorder characterized by developmental defects, bone marrow failure, and cancer susceptibility. Cells derived from FA patients are sensitive to crosslinking agents and have a prolonged G2 phase, suggesting a cell cycle abnormality. Although transfection of type-C FA cells with the FAC cDNA corrects these cellular abnormalities, the molecular function of the FAC polypeptide remains unknown. In the current study we show that expression of the FAC polypeptide is regulated during cell cycle progression. In synchronized HeLa cells, FAC protein expression increased during S phase, was maximal at the G2 /M transition, and declined during M phase. In addition, the FAC protein coimmunoprecipitated with the cyclin-dependent kinase, cdc2. We next tested various mutant forms of the FAC polypeptide for binding to cdc2. A patient-derived mutant FAC polypeptide, containing a point mutation at L554P, failed to bind to cdc2. The FAC/cdc2 binding interaction therefore correlated with the functional activity of the FAC protein. Moreover, binding of FAC to cdc2 was mediated by the carboxyl-terminal 50 amino acids of FAC in a region of the protein required for FAC function. Taken together, our results suggest that the binding of FAC and cdc2 is required for normal G2 /M progression in mammalian cells. Absence of a functional interaction between FAC and cdc2 in FA cells may underlie the cell cycle abnormality and clinical abnormalities of FA.

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