Fanconi Anemia Type Research Articles

Sustained Engraftment in Fanconi Anemia (FA) Mice Using in Utero Hematopoietic Cell Transplantation (IUHCT)

Introduction FA is the most common inherited bone marrow failure syndrome caused by a mutation in one of 23 genes involved in DNA repair; the disease manifests as hematologic abnormalities by age seven with near uniform bone marrow failure in adult patients. The gold standard therapy is bone marrow transplantation (BMT), which portends best outcomes when performed at a young age from Human Leukocyte Antigen (HLA)-matched sibling donors. However, due to the underlying pathophysiology of the disease, FA patients are especially sensitive to the chemoradiation conditioning regimens used with BMT, leading to high short-term morbidity and an increased long-term cancer risk. Due to these challenges of conventional BMT in FA, IUHCT is an attractive non-myeloablative and non-immunosuppressive option capable of inducing donor-specific tolerance when in utero testing has identified pathogenic FA mutations. Here we analyzed whether non-conditioned IUHCT confers fetal engraftment as a potential therapy in the FA mouse disease model. Methods Fanconi anemia type C homozygous mice (Fancc-/-) underwent IUHCT on gestation day 14 (E14) via vitelline vein injections of 1x107 T-cell depleted bone marrow cells from green fluorescent protein (GFP)-expressing congenic donor mice. Littermate wild-type mice served as controls. Donor cell engraftment in peripheral blood and bone marrow was assessed by flow cytometry. To confirm long-term donor HSC engraftment, we performed primary and secondary transplantation of an equivalent number of BM mononuclear cells from either Fancc -/- or wild-type mice that had undergone IUHCT. Statistical analysis was performed via Welch's t-test. P values <0.05 were considered statistically significant; for graphical purposes ***p<0.001; **p<0.002; *p<0.033; nonsignificant, ns. Results Fancc-/- homozygous mice had significantly higher donor GFP+ cell engraftment than wild-type controls at one, three, and six months post-transplantation (Figure 1). This higher engraftment was maintained across cell types in peripheral blood including CD3+ T cells (0.7% vs 3.4%), B220+ B cells (1.2% vs 6.9%), GR1+ granulocytes (1.7% vs 10.6%), and CD11B+ myeloid cells (1.9% vs 11.5%). Analysis of the bone marrow of these mice at 6 months post-transplant similarly showed a trend toward increased donor cell engraftment of progenitor cell populations including LSK, HSPC, LTHSC, STHSC, MPP2, MPP3, MPP4, CLP, CMP, GMP, and MEP cells in Fancc -/- recipients compared to wild-type mice. At all timepoints after primary and secondary transplants, recipient mice engrafted with BM mononuclear cells from prenatally transplanted Fancc -/- mice had higher peripheral blood GFP+ chimerism than those engrafted with BM mononuclear cells from prenatally transplanted wild-type mice (Figure 2). Conclusion Fancc-/- homozygous mice have higher sustained donor cell engraftment compared with wild-type littermates, indicating successful engraftment and possible early selection for wild-type donor cells in this preclinical model. These initial studies support the potential of IUHCT as a therapy for FA and highlight the need for additional studies to identify approaches to enhance donor cell engraftment to more clinically relevant levels. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Genotype-Phenotype Associations in Patients with Fanconi Anemia: National Cancer Institute Cohort

Fanconi anemia (FA) is a predominantly autosomal recessive disorder resulting from mutations in one of &amp;gt;22 genes involved in the FA/BRCA DNA repair pathway. FA is characterized by multiple congenital abnormalities, progressive bone marrow failure (BMF) and cancer predisposition. Genetic heterogeneity and diverse clinical presentations challenge early diagnosis and optimal management. We previously reviewed the genotype-phenotype associations in FA from literature cases (Fiesco-Roa MO et al. Blood Rev. 2019). We now report the results from the NCI cohort. We studied 147 patients with FA in the NCI inherited bone marrow failure syndromes Cohort Study (ClinicalTrials.gov, NCT00027274) to explore genotype phenotype associations by genes, location in the FA/BRCA pathway (upstream, ID complex, downstream), and compare information on the clinic cohort (CC) and field cohort (FC) patients. 57 patients (CC) were evaluated at the NIH Clinical Center between 2002 and 2020. Details on 90 patients in the FC were obtained from the review of medical records. The sex ratio (M:F) was similar (0.6:1 and 0.8:1). Patients in the FC were younger than in the CC (p=0.004) with median ages 27 (3-68) years for the CC and 19 (0-57) for the FC. The main genotypes in the CC were 59% FANCA, 17% FANCC, 6% FANCI and in the FC were 60% FANCA, 13% FANCC and 8% FANCG. At least one FA type physical abnormality was present in all CC patients and 73/79 (92%) FC patients (phenotype data not reported on 11 FC patients). &amp;gt;3/8 VACTERL-H features (Vertebral, Anal, Cardiac, Tracheo-esophageal fistula (TEF), Esophageal or duodenal atresia, Renal, upper Limb (radial ray) and Hydrocephalus) were present in 32% of CC patients and 16% of FC (p=0.04). At least 4/6 PHENOS features (skin Pigmentation, small Head, small Eyes, other central Nervous system (CNS) anomalies, Otology and Short stature) were present in 54% of CC patients and 34% FC (p=0.02). The types and frequencies of phenotypic abnormalities are shown in figure 1. 17 patients in the CC (30%) and 10 in the FC (13%) had both VACTERL-H and PHENOS (p=0.01). We excluded patients with unknown genotype or phenotype from further analysis. In the CC, cardiac abnormalities were more common in patients with FANCI or ID complex gene variants than in all others (p=0.02 and 0.001, respectively) as were VACTERL-H and structural CNS abnormalities in patients with ID complex variants (p=0.03 and 0.006, respectively). In the FC, VACTERL-H, imperforate anus and hydrocephalus were more common in patients with FANCD1 genotype (p=0.03, 0.009 and 0.004, respectively) and downstream pathway gene variants (p=0.004, &amp;lt;0.001 and 0.03, respectively). PHENOS, renal and neurodevelopmental abnormalities were less common in patients with upstream genes variants (p=0.001, 0.009 and &amp;lt;0.001, respectively). Upper limb abnormalities were less common in patients with FANCC genotype (p=0.007). BMF was present in 121/147 (88%) patients; 33% had been transfusion-dependent and 26% received androgen therapy. Clonal cytogenetic abnormalities were seen in 30%; 17% developed myelodysplastic syndrome at a median age of 17 (1.4-44) years and 6 patients developed acute myeloid leukemia at a median age of 19 (12-29) years. 72 (49%) patients underwent bone marrow transplant at a median age of 9.5 (1.5-44) years for BMF, MDS or leukemia. There was no significant difference between the FC and CC. The median survival age of our cohort is 38 (95% CI 34-43) years and at least 80% of our patients are &amp;gt;18 years of age. Kaplan-Meier survival estimates are presented in figure 2. Solid tumors developed in 30/135 (22%) patients with available data; median age at first cancer was 30 (2-44) years. The most common tumor was head and neck squamous cell carcinoma (n=15 patients), followed by skin (n=8) and anogenital cancers (n=6); many patients developed multiple cancers. Detailed hematologic, cancer, endocrine outcomes and survival analyses are ongoing. Overall, renal and upper limb abnormalities were reported in most of the patients in both CC and FC, as shown previously (Alter BP et al. Mol Syndromol. 2013). Data from the CC were more complete than from the review of charts from the FC highlighting that the clinical in person evaluation of patients provides detailed characterization of FA phenotypes and more accurate assessment of genotype-phenotype associations. This will facilitate timely diagnosis, surveillance and clinical management of patients with FA. Disclosures No relevant conflicts of interest to declare.

Two truncating variants in FANCC and breast cancer risk

Fanconi anemia (FA) is a genetically heterogeneous disorder with 22 disease-causing genes reported to date. In some FA genes, monoallelic mutations have been found to be associated with breast cancer risk, while the risk associations of others remain unknown. The gene for FA type C, FANCC, has been proposed as a breast cancer susceptibility gene based on epidemiological and sequencing studies. We used the Oncoarray project to genotype two truncating FANCC variants (p.R185X and p.R548X) in 64,760 breast cancer cases and 49,793 controls of European descent. FANCC mutations were observed in 25 cases (14 with p.R185X, 11 with p.R548X) and 26 controls (18 with p.R185X, 8 with p.R548X). There was no evidence of an association with the risk of breast cancer, neither overall (odds ratio 0.77, 95%CI 0.44–1.33, p = 0.4) nor by histology, hormone receptor status, age or family history. We conclude that the breast cancer risk association of these two FANCC variants, if any, is much smaller than for BRCA1, BRCA2 or PALB2 mutations. If this applies to all truncating variants in FANCC it would suggest there are differences between FA genes in their roles on breast cancer risk and demonstrates the merit of large consortia for clarifying risk associations of rare variants.

Fanconi Anemia Type C: Results of Population Carrier Testing [2A

INTRODUCTION: Fanconi Anemia Type C (FAC) is an autosomal recessive disorder characterized by skeletal malformations, bone marrow failure, increased risk of malignancy, and severe aplastic anemia. Currently, carrier testing for FAC is recommended for the Ashkenazi Jewish (AJ) population. A joint ACOG/ACMG statement highlighted the increasingly multi-ethnic society as consideration for pan-ethnic carrier testing, and recent ACOG committee opinion states that pan-ethnic testing is a reasonable strategy. Our current study assesses how many non-AJ individuals screened positive for FAC when pan-ethnic carrier testing was implemented. METHODS: Retrospective database analysis of individuals that received expanded carrier testing utilizing a genotyping panel for three pathologic variants of FAC (c.322delG, IVS4 c.456(+4)A&gt;T, p.R548X) was performed. Expected number of positive carriers in AJ and non-AJ groups were calculated and compared to observed rates. Chi-square analysis was performed to assess for statistical significance (p &lt; 0.01). RESULTS: A total of 71,235 individuals were tested for FAC. A higher than expected number of non-AJ individuals tested positive for FAC (56 observed vs 8.89 expected, p&lt;0.001). The number of AJ individuals that screened positive was consistent with expected values (10 observed vs 11.11 expected, p&lt;0.73). CONCLUSION: Non-AJ carriers of FAC were more common than expected in this cohort. Pan-ethnic expanded carrier testing will increase the detection of carriers for FAC compared with current ethnicity based screening recommendations. Importantly, carriers of FAC who would be missed by ethnicity based testing convention, will be identified allowing for more complete genetic counseling and family planning options for those who choose testing.

Abstract P4-12-16: Teasing out the PALB2 phenotype

Abstract Background: Biallelic mutations in PALB2 (Partner and Localizer of BRCA2) are known to cause Fanconi Anemia Type N. Multiple reports have demonstrated an increased risk for cancer in individuals heterozygous for PALB2 mutations. For example, a recent study by Antoniou et al reported a 33-58% lifetime risk for breast cancer in PALB2 mutation carriers, with 30% of carriers reporting triple negative breast cancer (TNBC). Other studies have suggested associations between PALB2 heterozygosity and pancreatic cancer, ovarian cancer, male breast cancer, and prostate cancer as well. We aimed to better define PALB2 phenotypes by assessing clinical history of TNBC, pancreatic, ovarian, and prostate cancers amongst PALB2 mutation carriers identified via multigene panel testing. Methods: We reviewed clinical histories of 11,007 individuals who underwent PALB2 sequence and deletion/duplication analysis as part of a multigene hereditary cancer panel. Descriptive statistics were utilized for clinical histories of PALB2 carriers, and chi square analysis was used to compare clinical histories of PALB2 mutation carriers to mutation-negative controls. Individuals with mutations in other cancer susceptibility genes were excluded from analysis. Results: A total of 98 PALB2 mutation carriers identified among 9610 individuals were included in our analysis. The majority of mutation carriers were Caucasian (80%) and female (92.8%). All identified mutations were truncating (nonsense, frameshift, or gross deletions). No pathogenic missense mutations were identified in this cohort. 77.6% (n=76) of mutation carriers had breast cancer, diagnosed at a mean age of 48. Hormone receptor status was available for 48 mutation carriers and 2469 controls. 37.5% (18/48) of breast cancers in mutation carriers were reported as triple negative, compared to 17.1% (423/2469) of breast cancers in controls (OR: 2.9 ; p= 0.0002). 7.8% (n= 8) of PALB2 mutation carriers had ovarian cancer. There was no significant difference in the incidence of ovarian cancer between PALB2 mutation carriers and controls (OR: 0.65 ; p= 0.25). Additionally, mutation carriers were significantly less likely to have a family history of ovarian cancer than controls (OR: 0.5; p= 0.02). 5.9% (n=6) of mutation carriers had pancreatic cancer, diagnosed at a mean age of 57.8, compared to 61 for controls. PALB2 mutation carriers were 1.3 times more likely to have personal and/or family history of pancreatic cancer, although this was not statistically significant (p= 0.22). Similarly, PALB2 mutation carriers were 1.5 times more likely to have a family history of prostate cancer, although this was not statistically significant (p= 0.09). Conclusions: Our data supports existing literature associating PALB2 mutations with TNBC. We did not observe significant associations between PALB2 carrier status and a clinical history of pancreatic, prostate, or ovarian cancers. However, this data should be interpreted with caution, as it is possible that unidentified genetic factors contributed to the clinical history of cancer in our mutation-negative controls. Investigation of PALB2-associated cancer risks in an unselected prospective cohort would help to further elucidate the PALB2 phenotype. Citation Format: Emily K Dalton, Rachel McFarland, Holly Laduca, Shuwei Li, Chia-Ling Gau. Teasing out the PALB2 phenotype [abstract]. In: Proceedings of the Thirty-Seventh Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2014 Dec 9-13; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2015;75(9 Suppl):Abstract nr P4-12-16.

Whole exome sequencing reveals concomitant mutations of multiple FA genes in individual Fanconi anemia patients.

BackgroundFanconi anemia (FA) is a rare inherited genetic syndrome with highly variable clinical manifestations. Fifteen genetic subtypes of FA have been identified. Traditional complementation tests for grouping studies have been used generally in FA patients and in stepwise methods to identify the FA type, which can result in incomplete genetic information from FA patients.MethodsWe diagnosed five pediatric patients with FA based on clinical manifestations, and we performed exome sequencing of peripheral blood specimens from these patients and their family members. The related sequencing data were then analyzed by bioinformatics, and the FANC gene mutations identified by exome sequencing were confirmed by PCR re-sequencing.ResultsHomozygous and compound heterozygous mutations of FANC genes were identified in all of the patients. The FA subtypes of the patients included FANCA, FANCM and FANCD2. Interestingly, four FA patients harbored multiple mutations in at least two FA genes, and some of these mutations have not been previously reported. These patients’ clinical manifestations were vastly different from each other, as were their treatment responses to androstanazol and prednisone. This finding suggests that heterozygous mutation(s) in FA genes could also have diverse biological and/or pathophysiological effects on FA patients or FA gene carriers. Interestingly, we were not able to identify de novo mutations in the genes implicated in DNA repair pathways when the sequencing data of patients were compared with those of their parents.ConclusionsOur results indicate that Chinese FA patients and carriers might have higher and more complex mutation rates in FANC genes than have been conventionally recognized. Testing of the fifteen FANC genes in FA patients and their family members should be a regular clinical practice to determine the optimal care for the individual patient, to counsel the family and to obtain a better understanding of FA pathophysiology.

Sleeping Beauty‐mediated correction of Fanconi anemia type C

The Sleeping Beauty (SB) transposon system can insert defined sequences into chromosomes to direct the extended expression of therapeutic genes. Our goal is to develop the SB system for nonviral complementation of Fanconi anemia (FA), a rare autosomal recessive disorder accompanied by progressive bone marrow failure. We used a CytoPulse electroporation system (CytoPulse, Glen Burnie, MD, USA) to introduce SB transposons into human lymphoblastoid cells (LCL) derived from both Fanconi anemia type C (FA-C) defective and normal patients. Correction of the FA-C defect was assessed by resistance to mitomycin C, a DNA-crosslinking agent. Culture of both cell types with the antioxidant N-acetyl- l-cysteine improved cell viability after electroporation. Co-delivery of enhanced green fluorescent protein (GFP) transposon with SB100X transposase-encoding plasmid supported a 50- to 90-fold increase in stable GFP expression compared to that observed in the absence of SB100X for normal LCL, but in FA-C defective LCL SB100X enhancement of stable GFP-expression was a more moderate five- to 13-fold. SB-mediated integration and expression of the FA-C gene was demonstrated by the emergence of a mitomycin C-resistant population bearing characteristic transposon-chromosome junction sequences and exhibiting a mitomycin dose response identical to that of normal LCL. The SB transposon system achieved stable expression of therapeutic FA-C genes, complementing the genetic defect in patient-derived cells by nonviral gene transfer.

Development of Genomic DNA Reference Materials for Genetic Testing of Disorders Common in People of Ashkenazi Jewish Descent

Many recessive genetic disorders are found at a higher incidence in people of Ashkenazi Jewish (AJ) descent than in the general population. The American College of Medical Genetics and the American College of Obstetricians and Gynecologists have recommended that individuals of AJ descent undergo carrier screening for Tay Sachs disease, Canavan disease, familial dysautonomia, mucolipidosis IV, Niemann-Pick disease type A, Fanconi anemia type C, Bloom syndrome, and Gaucher disease. Although these recommendations have led to increased test volumes and number of laboratories offering AJ screening, well-characterized genomic reference materials are not publicly available. The Centers for Disease Control and Prevention-based Genetic Testing Reference Materials Coordination Program, in collaboration with members of the genetic testing community and Coriell Cell Repositories, have developed a panel of characterized genomic reference materials for AJ genetic testing. DNA from 31 cell lines, representing many of the common alleles for Tay Sachs disease, Canavan disease, familial dysautonomia, mucolipidosis IV, Niemann-Pick disease type A, Fanconi anemia type C, Bloom syndrome, Gaucher disease, and glycogen storage disease, was prepared by the Repository and tested in six clinical laboratories using three different PCR-based assay platforms. A total of 33 disease alleles was assayed and 25 different alleles were identified. These characterized materials are publicly available from Coriell and may be used for quality control, proficiency testing, test development, and research.

Distinct roles of stress-activated protein kinases in Fanconi anemia type C–deficient hematopoiesis

AbstractThe underlying molecular mechanisms that promote bone marrow failure in Fanconi anemia are incompletely understood. Evidence suggests that enhanced apoptosis of hematopoietic precursors is a major contributing factor. Previously, enhanced apoptosis of Fanconi anemia type C–deficient (Fancc−/−) progenitors was shown to involve aberrant p38 MAPK activation. Given the importance of c-Jun N-terminal kinase (JNK) in the stress response, we tested whether enhanced apoptosis of Fancc−/− cells also involved altered JNK activation. In Fancc−/− murine embryonic fibroblasts, tumor necrosis factor α (TNF-α) induced elevated JNK activity. In addition, JNK inhibition protected Fancc−/− murine embryonic fibroblasts and c-kit+ bone marrow cells from TNF-α-induced apoptosis. Importantly, hematopoietic progenitor assays demonstrated that JNK inhibition enhanced Fancc−/− colony formation in the presence of TNF-α. Competitive repopulation assays showed that Fancc−/− donor cells cultured with the JNK inhibitor had equivalent levels of donor chimerism compared with Fancc−/− donor cells cultured with vehicle control. In contrast, culturing Fancc−/− cells with a p38 MAPK inhibitor significantly increased repopulating ability, supporting an integral role of p38 MAPK in maintaining Fancc−/− hematopoietic stem cell function. Taken together, these data suggest that p38 MAPK, but not JNK, has a critical role in maintaining the engraftment of Fancc−/−-reconstituting cells under conditions of stress.

The Role of Stress Activated Protein Kinases in Promoting Fanconi Anemia Hematopoietic Stem and Progenitor Cell Dysfunction

The underlying molecular mechanisms that promote bone marrow failure in Fanconi anemia (FA) are incompletely understood. Evidence from our lab and others suggest that enhanced oxidant and TNF-α mediated apoptosis of hematopoietic stem and progenitor cells is a major contributing factor. Previously, we showed that enhanced apoptosis of FA type C deficient (Fancc −/−) progenitors involves aberrant activation of the stress kinase, p38MAPK. Given the importance of c-Jun N-terminal kinase (JNK) in the stress response, we hypothesized that enhanced TNF-α-induced apoptosis in Fancc −/− cells would also involve altered JNK activation. The aims of the current study were to determine whether Fancc −/− cells exhibit altered JNK activation and to examine whether inhibition of JNK and/or p38MAPK kinase would enhance Fancc −/− hematopoietic stem cell (HSC) repopulating ability. In Fancc −/− murine embryonic fibroblasts (MEFs), TNF-α induced a 2-fold increase in JNK in vitro kinase activity compared to WT (n=4, p<0.05). Use of either a JNK inhibitor (n=3, p<0.003) or knockdown of JNK1/JNK2 expression using siRNAs (n=3, p<0.001) protected Fancc −/− MEFs from TNF-α induced apoptosis. Importantly, TNF-α induced apoptosis of Fancc −/− ckit+ low-density bone marrow cells was also restored to WT levels by culturing with a JNK inhibitor (n=3, p<0.01), and clonogenic hematopoietic progenitor assays demonstrated that JNK inhibition improved Fancc −/− colony formation in the presence of TNF-α (n=3, p<0.002). Taken together these data suggest that the predisposition of Fancc −/− MEFs and hematopoietic progenitors to TNF-α induced apoptosis involves JNK activation. Based on these data and previous studies demonstrating a role for p38MAPK in enhanced Fancc −/− progenitor apoptosis, competitive repopulation assays were conducted to examine whether culturing Fancc −/− cells with JNK and/or p38MAPK inhibitors would enhance HSC reconstituting function. Surprisingly, in two separate experiments Fancc −/− donor cells cultured with the JNK inhibitor had levels of donor chimerism that were equivalent to Fancc −/− donor cells cultured with the vehicle control. In contrast, culturing Fancc −/− cells with a p38MAPK inhibitor (SB203580) significantly increased repopulating ability compared to Fancc −/− cells cultured with vehicle control in two separate transplants (total n=12–14 recipients/transplant group, p<0.01). Interestingly, a similar increase in donor chimerism was observed in WT donor cells cultured with the p38MAPK inhibitor (total n=12–14 recipients/transplant group, p<0.002), supporting an integral role of p38MAPK in maintaining HSC function. The improvement in reconstitution was sustained over 12 months, and multilineage analysis revealed enhanced lymphoid and myeloid reconstitution, supporting an increase in HSC function with inhibition of p38MAPK. Twelve months post-transplantation all mice exhibited normal peripheral blood counts, BM and spleen histology. Taken together these data suggest that p38MAPK, but not JNK has a critical role in maintaining survival of WT and Fancc −/− reconstituting cells under conditions of stress.

Carrier frequency of autosomal‐recessive disorders in the Ashkenazi Jewish population: should the rationale for mutation choice for screening be reevaluated?

Ashkenazi Jewish (AJ) population is at increased risk for several recessive inherited diseases. Therefore, carrier testing of AJ members is important in order to identify couples at risk of having offspring with an autosomal recessive disorder. In the present study, a database containing the results of 28 410 genotyping assays was screened. Ten thousand seventy eight nonselected healthy members of the AJ population were tested for carrier status for the following diseases; Gaucher disease (GD), cystic fibrosis (CF), Familial dysautonomia (FD), Alpha 1 antitrypsin (A1AT), Mucolipidosis type 4 (ML4), Fanconi anemia type C (FAC), Canavan disease (CD), Neimann-Pick type 4 (NP) and Bloom syndrome (BLM). The results demonstrated that 635 members were carriers of one mutation and 30 members were found to be carriers of two mutations in the different genes related to the development of the above mentioned diseases. GD was found to have the highest carrier frequency (1:17) followed by CF (1:23), FD (1:29), A1AT (1:65), ML4 (1:67) and FAC (1:77). The carrier frequency of CD, NP and BLM was 1:82, 1:103 and 1:157, respectively. The frequency of the disease-causing mutations screened routinely among the AJ population indicated that there are rare mutations with very low frequencies. The screening policy of the disease-causing mutations should be reevaluated and mutations with a high frequency should be screened, while rare mutations with a lower frequency may be tested in partners of carriers.

Polymorphisms in the glucocerebrosidase gene and pseudogene urge caution in clinical analysis of Gaucher disease allele c.1448T&gt;C (L444P)

BackgroundGaucher disease is a potentially severe lysosomal storage disorder caused by mutations in the human glucocerebrosidase gene (GBA). We have developed a multiplexed genetic assay for eight diseases prevalent in the Ashkenazi population: Tay-Sachs, Gaucher type I, Niemann-Pick types A and B, mucolipidosis type IV, familial dysautonomia, Canavan, Bloom syndrome, and Fanconi anemia type C. This assay includes an allelic determination for GBA allele c.1448T>C (L444P). The goal of this study was to clinically evaluate this assay.MethodsBiotinylated, multiplex PCR products were directly hybridized to capture probes immobilized on fluorescently addressed microspheres. After incubation with streptavidin-conjugated fluorophore, the reactions were analyzed by Luminex IS100. Clinical evaluations were conducted using de-identified patient DNA samples.ResultsWe evaluated a multiplexed suspension array assay that includes wild-type and mutant genetic determinations for Gaucher disease allele c.1448T>C. Two percent of samples reported to be wild-type by conventional methods were observed to be c.1448T>C heterozygous using our assay. Sequence analysis suggested that this phenomenon was due to co-amplification of the functional gene and a paralogous pseudogene (ΨGBA) due to a polymorphism in the primer-binding site of the latter. Primers for the amplification of this allele were then repositioned to span an upstream deletion in the pseudogene, yielding a much longer amplicon. Although it is widely reported that long amplicons negatively impact amplification or detection efficiency in recently adopted multiplex techniques, this assay design functioned properly and resolved the occurrence of false heterozygosity.ConclusionAlthough previously available sequence information suggested GBA gene/pseudogene discrimination capabilities with a short amplified product, we identified common single-nucleotide polymorphisms in the pseudogene that required amplification of a larger region for effective discrimination.

Targeted Disruption of FANCC and FANCG in Human Cancer Provides a Preclinical Model for Specific Therapeutic Options

How specifically to treat pancreatic and other cancers harboring Fanconi anemia gene mutations has raised great interest recently, yet preclinical studies have been hampered by the lack of well-controlled human cancer models. We endogenously disrupted FANCC and FANCG in a human adenocarcinoma cell line and determined the impact of these genes on drug sensitivity, irradiation sensitivity, and genome maintenance. FANCC and FANCG disruption abrogated FANCD2 monoubiquitination, confirming an impaired Fanconi anemia pathway function. On treatment with DNA interstrand-cross-linking agents, FANCC and FANCG disruption caused increased clastogenic damage, G2/M arrest, and decreased proliferation. The extent of hypersensitivity varied among agents, with ratios of inhibitory concentration 50% ranging from 2-fold for oxaliplatin to 14-fold for melphalan, a drug infrequently used in solid tumors. No hypersensitivity was observed on gemcitabine, etoposide, 3-aminobenzamide, NU1025, or hydrogen peroxide. FANCC and FANCG disruption also resulted in increased clastogenic damage on irradiation, but only FANCG disruption caused a subsequent decrease in relative survival. Finally, FANCC and FANCG disruption increased spontaneous chromosomal breakage, supporting the role of these genes in genome maintenance and likely explaining why they are mutated in sporadic cancer. Our human cancer cell model provides optimal controls to elucidate fundamental biologic features of individual Fanconi anemia gene defects and facilitates preclinical studies of therapeutic options. The impact of Fanconi gene defects on drug and irradiation sensitivity renders these genes promising targets for a specific, genotype-based therapy for individual cancer patients, providing a strong rationale for clinical trials.

Enhanced TNF-α–induced apoptosis in Fanconi anemia type C–deficient cells is dependent on apoptosis signal-regulating kinase 1

Fanconi anemia (FA) is a chromosomal instability disorder characterized by progressive bone marrow failure. Experimental evidence suggests that enhanced oxidant and myelosuppressive cytokine-mediated apoptosis of hematopoietic stem and progenitor cells contributes to the pathogenesis of marrow failure in FA. However, the molecular mechanisms responsible for the apoptotic phenotype in hematopoietic cells are incompletely understood. Recent data in Fancc-/- murine embryonic fibroblasts (MEFs) implicate increased oxidant-induced apoptotic signaling through the redox-dependent protein, apoptosis signal-regulating kinase 1 (Ask1). Here, we examined whether altered Ask1 signaling participated in the proapoptotic phenotype of primary Fancc-/- MEFs and hematopoietic progenitors treated with the myelosuppressive cytokine tumor necrosis factor-alpha (TNF-alpha). Our data indicate that TNF-alpha induces hyperactivation of Ask1 and the downstream effector p38 in Fancc-/- MEFs. In addition,Ask1 inactivation in Fancc-/- MEFs and hematopoietic progenitors restored survival to wild-type (WT) levels in the presence of TNF-alpha. Furthermore, targeting the Ask1 pathway by using either antioxidants or a p38 inhibitor protected Fancc-/- MEFs and c-kit+ cells from TNF-alpha-induced apoptosis. Collectively, these data argue that the predisposition of Fancc-/- hematopoietic progenitors to apoptosis is mediated in part through altered redox regulation and Ask1 hyperactivation.

Fanconi anemia type C–deficient hematopoietic cells are resistant to TRAIL (TNF-related apoptosis-inducing ligand)-induced cleavage of pro-caspase-8

The pathophysiology of bone marrow failure in Fanconi anemia (FA) patients is thought to involve excessive apoptosis involving signaling triggered by fas ligation and tumor necrosis factor (TNF)-alpha, or interferon (IFN)-gamma exposure. We investigated whether a new member of the TNF family, TRAIL (TNF-related apoptosis-inducing ligand), would similarly trigger preferential apoptotic cell death in FA phenotype cells. Hematopoietic cells from FANCC(-/-) transgenic mice and human FA-C lymphoblasts (HSC536N) as well as their phenotypically corrected counterparts (FANCC(+/+), HSC536/FA-Cneo) were compared for their response to apoptosis induction by TRAIL and fas ligation in the presence or absence of IFN-gamma. Cells were also studied for the protein and gene expression of TRAIL-receptors, caspase-8 and its inhibitory protein, FLIP. TRAIL exposure by itself or in combination with IFN-gamma did not lead to preferential apoptosis induction in human and murine FA-C phenotype hematopoietic cells. This resistance was unrelated to the expression of TRAIL receptors or FLIP isoforms, but correlated with absent cleavage of pro-caspase-8. Results were validated by those from gene expression profiling of relevant genes in the two lymphoblast cell lines. TRAIL, in contrast to fas ligation, does not induce preferential apoptosis in FA-C phenotype cells despite shared downstream signaling described in non-FA models. These data provide further insight into the complexity of FA-C-regulated apoptotic signaling.

Fanconi anemia type C and p53 cooperate in apoptosis and tumorigenesis

AbstractFanconi 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 theBRCA1, BRCA2, andATM 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 ofTrp53 rescues tumor necrosis factor α-induced apoptosis in myeloid cells fromFancc-/- mice. Further, whereasFancc-/- mice failed to form hematopoietic or solid malignancies, mice mutant at bothFancc andTrp53 developed tumors more rapidly than mice mutant atTrp53 alone. This shortened latency was associated with the appearance of tumor types that are found in patients with FA but not in mice mutant atTrp53 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)

Cloning, gene expression, and characterization of CP27, a novel gene in mouse embryogenesis.

We report the full-length sequencing, tissue-specific expression, and immunolocalization of cp27, a novel gene in mouse embryogenesis. The cp27 gene was isolated and cloned from a mouse E11 lambdagt11 library using a peptide antibody that recognized a distinct expression pattern in mouse craniofacial development. The cp27 gene contains an open reading frame of 295 amino acids corresponding to a predicted molecular mass of 33kDa. On Western blots, a polyclonal antibody against CP27 detected a single epitope at 27kDa. The putative CP27 protein has an isoelectric point of 4.75 and features a distinct helix-loop-helix structure according to prediction algorithms. We have cloned the human cp27 gene and mapped it to a locus on the human chromosome 16 which is in proximity to several loci associated with inherited craniofacial diseases such as fanconi anemia type A. Northern blot analysis of RNA from multiple mouse tissues demonstrated high levels of expression in developing mouse teeth, heart, lung, and liver of a single transcript of approx. 1. 8kbp. In situ hybridization using a radioactive RNA probe resulted in distinct signals in the developing neuroepithelium, cerebellum, heart, lung, liver, teeth, salivary glands, and periosteum of developing bones. Immunohistochemical staining of developing mouse tissues detected epitopes specific for CP27 in the mesenchyme surrounding the primary brain vesicles, in basement membranes, in the periosteum, in salivary glands, and in the stellate reticulum of teeth. Thus, CP27 represents a unique gene product involved in mouse embryogenesis.

The effect of the Fanconi anemia polypeptide, FAC, upon p53 induction and G2 checkpoint regulation

Fanconi anemia (FA) is an autosomal recessive disease marked by developmental defects, bone marrow failure, and cancer susceptibility. FA cells are hypersensitive to DNA cross-linking and alkylating agents and accumulate in the G2 phase of the cell cycle in response to these agents. FA cells also display genomic instability, suggesting a possible defect in the p53 pathway. To test the effect of heterologous expression of FAC cDNA on drug-induced cytotoxicity, G2 accumulation, and p53 induction in FA cells, we compared two isogenic FA cell lines: HSC536N (mock), a FA type C cell line sensitive to mitomycin C (MMC), and the same cell line transfected (corrected) with wild-type FAC cDNA (HSC536N [+FAC]). HSC536N (+FAC) cells showed a 30-fold increase in resistance to MMC concentration. Similarly, increases in resistance were observed following exposure to cisplatin, carboplatin, and cyclophosphamide. In addition, HSC536N (+FAC) cells showed a twofold lower G2 accumulation following MMC treatment. To analyze the possible interaction of FAC with the p53 pathway, we analyzed p53 induction in mock and corrected cell lines following exposure to MMC. HSC536N (mock) cells induced p53 at lower MMC concentrations than HSC536N (corrected). Caffeine, a known G2 checkpoint inhibitor, not only inhibited G2 accumulation seen in both cell lines but also caused the resistant HSC536N (+FAC) to become as sensitive to MMC as HSC536N (mock) cell line. We conclude that the FAC protein has a specific cytoprotective effect and may function as a cell cycle regulator of the G2 phase of the cell cycle.

The Effect of the Fanconi Anemia Polypeptide, FAC, Upon p53 Induction and G2 Checkpoint Regulation

Fanconi anemia (FA) is an autosomal recessive disease marked by developmental defects, bone marrow failure, and cancer susceptibility. FA cells are hypersensitive to DNA cross-linking and alkylating agents and accumulate in the G2 phase of the cell cycle in response to these agents. FA cells also display genomic instability, suggesting a possible defect in the p53 pathway. To test the effect of heterologous expression of FAC cDNA on drug-induced cytotoxicity, G2 accumulation, and p53 induction in FA cells, we compared two isogenic FA cell lines: HSC536N (mock), a FA type C cell line sensitive to mitomycin C (MMC), and the same cell line transfected (corrected) with wild-type FAC cDNA (HSC536N [+FAC]). HSC536N (+FAC) cells showed a 30-fold increase in resistance to MMC concentration. Similarly, increases in resistance were observed following exposure to cisplatin, carboplatin, and cyclophosphamide. In addition, HSC536N (+FAC) cells showed a twofold lower G2 accumulation following MMC treatment. To analyze the possible interaction of FAC with the p53 pathway, we analyzed p53 induction in mock and corrected cell lines following exposure to MMC. HSC536N (mock) cells induced p53 at lower MMC concentrations than HSC536N (corrected). Caffeine, a known G2 checkpoint inhibitor, not only inhibited G2 accumulation seen in both cell lines but also caused the resistant HSC536N (+FAC) to become as sensitive to MMC as HSC536N (mock) cell line. We conclude that the FAC protein has a specific cytoprotective effect and may function as a cell cycle regulator of the G2 phase of the cell cycle.

Correction of fanconi anemia Type C phenotypic abnormalities using a clinically suitable retroviral vector infection protocol

Fanconi anemia (FA) is a complex autosomal recessive disease with hematologic manifestations characterized by a progressive hypoplastic anemia, hypersensitivity to clastogenic agents, and an increased incidence of acute myelogenous leukemia. The cDNA that corrects one of four FA complementation subtypes, named Fanconi anemia Type C (FAC) has recently been identified. We constructed a a simplified recombinant retrovirus (vMFGFAC) encoding only the FAC cDNA, and tested its ability to correct the FAC defect in a lymphocytic cell line and primary mobilized blood progenitor cells. In addition, the gene transfer efficiency using a clinically applicable gene transfer protocol into normal primitive hematopoietic progenitor cells, high proliferating potential colony forming cells (HPP-CFC), derived from CD34+ purified cord blood cells was examined. The gene transfer efficiency was significantly enhanced when cells were transduced with supernatant while adherent to a 30/35 KD fragment of fibronectin, FN30/35, and was similar to efficiency obtained by coculture with retrovirus packaging cells. Transduction of an FAC deficient lymphoid cell line with vMFGFAC supernatant resulted in an enhanced cell viability, and G-CSF mobilized peripheral blood cells from an FAC-deficient patient transduced with the vMFGFAC virus demonstrated enhanced progenitor cell colony formation. These data indicate that the vMFGFAC virus allows functional complementation of FAC in lymphoblasts and primary hematopoietic progenitors, and that primitive cord blood hematopoietic stem/progenitor cells can be transduced at an efficiency comparable to protocols using cocultivation if adherent to FN 30/35 fragment.