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 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.

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.

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.

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.

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 International Fanconi Anemia Registry: relation of clinical symptoms to diepoxybutane sensitivity

Blood ◽  
1989 ◽  
Vol 73 (2) ◽  
pp. 391-396
Author(s):  
AD Auerbach ◽  
A Rogatko ◽  
TM Schroeder-Kurth
Keyword(s):  
Fanconi Anemia ◽  
Clinical Symptoms ◽  
Clinical Manifestations ◽  
Chromosomal Breakage ◽  
Scoring Method ◽  
Cellular Sensitivity ◽  
Progressive Pancytopenia ◽  
Clastogenic Effect ◽  
Blood Specimens ◽  
Stepwise Multivariate Logistic Regression

Fanconi anemia (FA) is characterized clinically by a progressive pancytopenia, diverse congenital abnormalities and increased predisposition to malignancy. Although a variable phenotype makes accurate diagnosis on the basis of clinical manifestations difficult in some patients, study of cellular sensitivity to the clastogenic effect of DNA cross-linking agents such as diepoxybutane (DEB) has been used to facilitate the diagnosis. Data from DEB-induced chromosomal breakage studies of 328 peripheral blood specimens from patients considered at risk for FA were analyzed using a stepwise multivariate logistic regression, in order to determine which method of representing the data best discriminated between DEB-sensitive (DEB+) and DEB-insensitive (DEB-) cases. Similar methods were applied to the data from the International Fanconi Anemia Registry (IFAR) to determine whether DEB+ and DEB- cases may be considered as distinct clinical entities, and if so, which variables provide the best discrimination between the two groups. We conclude that hypersensitivity to the clastogenic effect of DEB is a useful discriminator for FA. A simplified scoring method for classifying patients on the basis of eight clinical manifestations that are the best predictors for FA is presented. Our data indicate that the clinical diversity in FA is more widespread than previously recognized.

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.

scholarly journals Diagnostic Utility of Targeted Next Generation Sequencing in Patients with Vascular Anomalies

Blood ◽  
2020 ◽  
Vol 136 (Supplement 1) ◽  
pp. 8-9
Author(s):  
Whitney Eng ◽  
Sophie Dilek ◽  
Abigail Ward ◽  
Harry PW Kozakewich ◽  
Alyaa Al-Ibraheemi ◽  
...  
Keyword(s):  
Clinical Trials ◽  
Next Generation Sequencing ◽  
Dna Sequences ◽  
Cancer Genomics ◽  
Massively Parallel Sequencing ◽  
Genetic Diagnosis ◽  
Vascular Anomalies ◽  
Next Generation ◽  
Targeted Next Generation Sequencing ◽  
Generation Sequencing

Background: Vascular anomalies are diverse entities and can range in severity from self-limiting to life-threatening. Diagnosis and care of these patients is challenging due to overlapping clinical and histologic features. Recently, it has been established that many vascular anomalies arise from somatic mutations in cancer genes (PIK3CA, AKT, NRAS). Use of cancer genomics in patients with vascular anomalies may establish a genetic diagnosis and expand use of targeted medical therapies. We evaluated the utility of targeted next generation sequencing for vascular anomalies patients at a single pediatric center. Methods: Using OncoPanel, a hybrid-capture and massively parallel sequencing assay that surveys DNA sequences of 447 genes implicated in cancer, we analyzed genetic variants in lesional tissue from vascular anomalies patients evaluated at Boston Children's Hospital between 5/2/2017 and 3/23/2020. Results: A total of 276 patients were consented and sequenced under the Dana Farber Cancer Institute Profile protocols DFCI 11-104 (n= 68) and DFCI 17-000 (n= 208). Clinical diagnoses prior to testing were varied and 11 patients (7%) had an unknown diagnosis. Tissue was analyzed for 138 patients. Targeted sequencing resulted in diagnostically significant alterations in 80 of 138 (57%) of patients and therapeutically significant alterations in 58 of 138 (42%) patients. To date, 18 patients in our cohort have been treated with medical therapy informed by their genetic diagnosis. Several more await enrollment on clinical trials. For patients with diagnoses previously categorized as unknown (n=11), sequencing led to identification of a genetic variant in 6 patients (54%). Additionally, 8/138 patients had variants requiring further evaluation for potential germline involvement. Discussion: Next generation sequencing in vascular anomalies patients identified actionable variants in a large proportion of the patients in our cohort. The mTOR inhibitor sirolimus has been used to treat a variety of vascular anomalies, but not all patients respond to this treatment. Targeted therapies based on specific genotypes hold promise as clinical trials in vascular anomalies are emerging. Additionally, sequencing in this cohort identified several variants suggesting a germline cancer predisposition requiring follow-up. Use of next generation sequencing has clinical utility and increased use of this testing may improve diagnosis, prognosis, and treatment for patients with vascular anomalies. Disclosures Adams: Novartis: Consultancy; Venthura: Consultancy. OffLabel Disclosure: Sirolimus is used off-label for the treatment of vascular anomalies.

Non-ocular Stickler Syndrome With a Novel Mutation in COL11A2 Diagnosed by Massively Parallel Sequencing in Japanese Hearing Loss Patients

2015 ◽  
Vol 124 (1_suppl) ◽  
pp. 111S-117S ◽  
Author(s):  
Yoh-ichiro Iwasa ◽  
Hideaki Moteki ◽  
Mitsuru Hattori ◽  
Ririko Sato ◽  
Shin-ya Nishio ◽  
...  
Keyword(s):  
Hearing Loss ◽  
Clinical Symptoms ◽  
Massively Parallel Sequencing ◽  
Novel Mutation ◽  
Genetic Diagnosis ◽  
Analysis Data ◽  
Massively Parallel ◽  
Stickler Syndrome ◽  
Parallel Sequencing ◽  
First Case

Objectives: This study aims to document the clinical features of patients with COL11A2 mutations and to describe the usefulness of massively parallel sequencing. Methods: One thousand one hundred twenty (1120) Japanese hearing loss patients from 53 ENT departments nationwide participated in this study. Massively parallel sequencing of 63 genes implicated in hearing loss was performed to identify the genetic causes in the Japanese hearing loss patients. Results: A novel mutation in COL11A2 (c.3937_3948delCCCCCAGGGCCA) was detected in an affected family, and it was segregated in all hearing loss individuals. The clinical findings of this family were compatible with non-ocular Stickler syndrome. Orofacial features of mid-facial hypoplasia and slowly progressive mild to moderate hearing loss were also presented. Audiological examinations showed favorable auditory performance with hearing aid(s). Conclusion: This is the first case report of the genetic diagnosis of a non-ocular Stickler syndrome family in the Japanese population. We suggest that it is important to take both genetic analysis data and clinical symptoms into consideration to make an accurate diagnosis.

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)

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