Phenotypic correction of primary Fanconi anemia T cells with retroviral vectors as a diagnostic tool

Hanenberg, Helmut; Batish, Sat Dev; Pollok, Karen E.; Vieten, Lydia; Verlander, Peter C.; Leurs, Cordula; Cooper, Ryan; Göttsche, Kerstin; Haneline, Laura S.; Clapp, D. Wade; Lobitz, Stephan; Williams, David A.; Auerbach, Arleen D.

doi:10.1016/s0301-472x(02)00782-8

Cited by 88 publications

(73 citation statements)

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“…The transfected cell cultures, treated and untreated with MMC and DEB as above, were assayed as previously described. 28,29 Retroviral mediated correction of hypersensitivity to MMC and DEB was inferred if the percent of chromosome breaks and radials/cell detected was Ͻ10% using either DEB or MMC. All samples were blinded to us regarding previous genetic results.…”

Section: Cytogenetic Analysismentioning

confidence: 99%

“…[11][12][13][14][15][16][17][18][19][20][21][22][23][24][25][26][27] Cloning of the FANC genes has provided a means, via complementation group analysis, to routinely pinpoint the patient's specific FA gene harboring the mutations, thus greatly simplifying subsequent molecular analysis. 28,29 Although retroviral-mediated complementation analysis of FA patient-derived cells is commonly used to identify the patient's genetic subtype as a prerequisite for mutation screening, such analysis has not been formally validated for clinical use. Previous studies have considered Fanconi complementation Group A (FA-A) assignments, but none have performed comprehensive sequencing to verify complementation assignment and/or evaluated complementation assignments performed using retroviral-mediated rescue as evidenced by correction of MMC and DEB-induced chromosome breakage and radial formation.…”

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confidence: 99%

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Validation of Fanconi anemia complementation Group A assignment using molecular analysis

Moghrabi¹,

Johnson²,

Yoshitomi³

et al. 2009

Genetics in Medicine

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Purpose: Fanconi anemia is a genetically heterogeneous chromosomal breakage disorder exhibiting a high degree of clinical variability. Clinical diagnoses are confirmed by testing patient cells for increased sensitivity to crosslinking agents. Fanconi anemia complementation group assignment, essential for efficient molecular diagnosis of the disease, had not been validated for clinical application before this study. The purpose of this study was (1) confirmation of the accuracy of Fanconi anemia complementation group assignment to Group A (FANCA) and (2) development of a rapid mutation detection strategy that ensures the efficient capture of all FANCA mutations. Methods: Using fibroblasts from 29 patients, diagnosis of Fanconi anemia and assignment to complementation Group A was made through breakage analysis studies. FANCA coding and flanking sequences were analyzed using denaturing high pressure liquid chromatography, sequencing, and multiplex ligation-dependent probe amplification. Patients in which two mutations were not identified were analyzed by cDNA sequencing. Patients with no mutations were sequenced for mutations in FANCC, G, E, and F. Results: Of the 56 putative mutant alleles studied, 89% had an identifiable FANCA pathogenic mutation. Eight unique novel mutations were identified. Conclusion: Complementation assignment to Group A was validated in a clinical laboratory setting using our FANCA rapid molecular testing strategy. Genet anconi anemia (FA; MIM no. 227650), the most common inherited bone marrow disorder, has an overall prevalence of 1-5 per million and an estimated carrier frequency of 1 in 200 to 1 in 300 in most populations. 1,2 Demonstrating either an autosomal or X-linked recessive mode of inheritance, FA is characterized by childhood progressive bone marrow failure and predisposition to acute myelogenous leukemia; older patients are at increased risk for squamous cell carcinomas of the head, neck, and genitourinary tract. [3][4][5][6][7] Congenital abnormalities are present in approximately 70% of FA patients and may include radial ray defects; café au lait spots or hypopigmentation; short stature; microphthalmia; malformations of kidneys, gastrointestinal tract, and heart; mental retardation; and hearing defects. 8 Because of the high degree of phenotypic variability exhibited by FA patients, diagnosis may be difficult on the basis of clinical manifestations alone. Because FA patient-derived lymphocytes and fibroblasts exhibit hypersensitivity to DNA crosslinking agents such as diepoxybutane (DEB) 9 and mitomycin C (MMC), 10 resulting in a high rate of chromosomal breakage and radial formation, analysis on the basis of this hypersensitivity has been routinely used to confirm clinical diagnosis.Molecular diagnosis of FA has been challenging because of the genetic heterogeneity associated with the disease; FA is multigenic, with 13 complementation groups and associated genes having been characterized (A, B, C, D1, D2, E, F, G, I, J, L, M, N). [11][12][13][14][15][16][17][18][19][20][21][...

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Section: Cytogenetic Analysismentioning

confidence: 99%

mentioning

confidence: 99%

Validation of Fanconi anemia complementation Group A assignment using molecular analysis

Moghrabi¹,

Johnson²,

Yoshitomi³

et al. 2009

Genetics in Medicine

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Section: Cell Culturementioning

confidence: 99%

“…To determine the complementation group of these patients, cells were transduced with retroviral vectors expressing FANCA, FANCC, FANCF, and FANCG proteins (kindly provided by H. Hanenberg, Heinrich-Heine University, Düsseldorf, Germany) and then exposed to increasing concentrations of MMC. 20 All patients studied so far belong to FA complementation group A. Epstein-Barr virus (EBV)-transformed lymphoblasts from FA-A patients and healthy donors were maintained in RPMI media (Biochrom, Berlin, Germany) supplemented with 15% heat-inactivated fetal calf serum (FCS) and antibiotics.Cells were seeded in 6-well plates with 3 mL culture medium and exposed to a UV dose of 700 J/m 2 , unless otherwise indicated, or treated with 10 M MMC or 1 M phorbol-12-myrisate-13-acetate (PMA) (both from Sigma, St Louis, MO) and then analyzed for MAPK activity.Cell viability was determined by the trypan blue exclusion test by counting at least 200 cells from each individual culture.Apoptosis was assessed by a quantitative analysis of histone-associated DNA fragments using an enzyme immunoassay kit (Roche, Mannheim, Germany) and further confirmed by Western blot, detecting the 85-kDa cleavage fragment of poly (adenosine diphosphate [ADP] ribose) polymerase (PARP). …”

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confidence: 99%

“…PIPAON 20 Analysis of transduced cells (about 35% were green fluorescent protein [EGFP]-positive) revealed a partial correction of the sensitivity to MMC. By 5 days of treatment with 100 nM MMC, more than 60% of control vector-transduced cells were dead as assessed by the uptake of propidium iodide, whereas 81% of FANCA-transduced cells were viable (data not shown).…”

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confidence: 99%

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Jun N-terminal kinase activity and early growth-response factor-1 gene expression are down-regulated in Fanconi anemia group A lymphoblasts

Pipaón

Casado

Bueren

et al. 2004

Blood

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Fanconi anemia (FA) is an autosomal recessive cancer susceptibility syndrome characterized by cellular sensitivity to genotoxic agents. In recent years, FA proteins have been associated with different molecules involved in signal transduction, which has raised the interest in FA-dependent signaling pathways. Here, we report that the c-Jun N-terminal kinase (JNK) fails to phosphorylate in response to UV radiation and treatment with mitomycin C in FA lymphoblast cells derived from type A patients (FA-A). Furthermore, defective kinase activity seems to be specific for JNK, because extracellular signalregulated kinase (ERK) responded to the proper stimuli in FA-A cells. We also demonstrate that the early growthresponse factor-1 (Egr-1), a JNK downstream target gene that is normally induced by genotoxic stress, is not upregulated in UV IntroductionFanconi anemia (FA) is an autosomal recessive disease characterized by developmental abnormalities, progressive bone marrow failure, and cancer susceptibility. At the cellular level, FA cells are hypersensitive to DNA cross-linking agents such as mitomycin C (MMC) or diepoxybutane. 1,2 FA is a genetically heterogeneous disease, comprising at least 8 complementation groups: FA-A, -B, -C, -D1, -D2, -E, -F and -G, the FA-A group being the most frequently represented in these patients. 3,4 Seven FA-associated genes have been cloned to date, and their products were found to interact with DNA damage-response proteins, including BRCA1, ATM, and NBS1. [5][6][7] In line with this, it has been described that FA proteins may be involved in the process of repairing chromosome defects that occur during homologous recombination. 5 A complex of 5 FA proteins (FANCA, FANCC, FANCE, FANCF, and FANCG) triggers the monoubiquitination of the FANCD2 protein and its interaction with BRCA1 in response to DNA damage. 6 BRCA1 plays an important role in mediating cell cycle arrest, apoptosis, and cellular responses to DNA damage signals. 8,9 Recently it has been shown that BRCA1 is cleaved by caspase-3 during UV-induced apoptosis, which produces a C-terminal fragment that triggers an apoptotic response through activation of c-Jun N-terminal kinase (JNK) and GADD45. 10 Consistently, phosphorylation activity of JNK is abrogated in breast cancer cells after blockade of BRCA1 expression with specific ribozymes, indicating that JNK is a downstream target of BRCA1. 11 Additionally, FANC proteins exhibit functions besides their role in the complex. To this end, a number of FANCC-dependent molecular mechanisms have been suggested to ensure the survival of hematopoietic cells. Notably, FANCC is involved in the activation of signal transducer and activator of transcription-1 (STAT1) through growth factor receptors, 12 it cooperates with heat-shock protein 70 (Hsp70) to suppress the activity of proapoptotic interferon-inducible double-stranded RNA-dependent protein kinase (PKR), 13 and binds to glutathione S-transferase P1-1 (GSTP1), inducing the detoxification of by-products of oxidative stress. 14 Interest...

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Diagnosis of Fanconi Anemia by Diepoxybutane Analysis

Auerbach

2003

CP Human Genetics

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107

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Fanconi anemia (FA) is a genetically and phenotypically heterogeneous disorder characterized by congenital malformations, progressive bone marrow failure, and predisposition to cancer, particularly hematological malignancies and solid tumors of the head and neck. The main role of FA proteins is in the repair of DNA interstrand crosslinks (ICLs). FA results from pathogenic variants in at least 16 distinct genes, causing genomic instability. Although the highly variable phenotype makes accurate diagnosis on the basis of clinical manifestations difficult in some patients, diagnosis based on a profound sensitivity to DNA crosslinking agents can be used to identify the pre-anemia patient as well as patients with aplastic anemia or leukemia who may or may not have the physical stigmata associated with the syndrome. Diepoxybutane (DEB) analysis is the preferred test for FA because other agents have higher rates of false-positive and falsenegative results. KeywordsFanconi anemia; DEB test; DNA interstrand crosslink repair; genomic instability Diagnosis of Fanconi anemia (FA) based on clinical manifestations alone is often difficult and unreliable because of the considerable overlap of the FA phenotype with that of a variety of genetic and nongenetic diseases (Table 8.7.1). It is currently considered standard of care that testing for crosslink sensitivity to rule out FA and testing for telomere length to rule out DKC should be part of the workup before treatment of pediatric acquired aplastic anemia (Williams et al., 2014).Although most FA patients exhibit abnormalities in growth parameters and skin pigmentation, approximately one third of the patients do not have any major birth defects (Giampietro et al., 1993(Giampietro et al., , 1997. Extensive genetic heterogeneity has been shown in FA patients, by use of various strategies for gene identification including functional and positional cloning, and more recently by mass spectrometric analysis of isolated protein complexes (Meetei et al., 2004), candidate gene sequencing in FA patient DNA (Kim et al., 2011), and by whole exome sequencing (Bogliolo et al., 2013). Sixteen unique genes (212) (Chandrasekharappa et al. 2013;Flynn et al., 2014), in order to individualize treatment for the patient, and provide genetic counseling regarding cancer risk for family members.FA affects approximately one in 100,000 live births. The average life expectancy is about 20 years, but some patients may not be diagnosed until the 3 rd decade of life or even later. Hematopoietic progenitor cells in FA are genetically unstable, leading eventually to bone marrow failure, which is the main life-threatening problem in these patients. This defect also predisposes to an increased frequency of clonal cytogenetic abnormalities, myelodysplastic syndrome (MDS), and acute leukemia. Development of any of these conditions is associated with poorer survival. Allogeneic hematopoietic stem cell transplantation (HSCT) is the only curative treatment currently for bone marrow failure in patients with...

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Phenotypic correction of primary Fanconi anemia T cells with retroviral vectors as a diagnostic tool

Cited by 88 publications

References 63 publications

Validation of Fanconi anemia complementation Group A assignment using molecular analysis

Validation of Fanconi anemia complementation Group A assignment using molecular analysis

Jun N-terminal kinase activity and early growth-response factor-1 gene expression are down-regulated in Fanconi anemia group A lymphoblasts

Diagnosis of Fanconi Anemia by Diepoxybutane Analysis

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