International Fanconi Anemia Registry Research Articles

Characterization and Genotype-Phenotype Correlation of Fanconi Anemia Patients in Israel

Background: Fanconi Anemia (FA) is an inherited bone marrow failure syndrome in which defects in the DNA repair pathway lead to chromosomal instability. The clinical phenotype includes multiple congenital anomalies, aplastic anemia, an increased tendency to develop malignancies, and extreme sensitivity to alkylating agents. To date, mutations in 21 different genes have been detected in FA patients. A clear genotype-phenotype correlation has not been established, with the exception of patients with FANCD1 (BRCA2) and FANCN mutations, in which early onset of malignancy is almost invariably present. One study found that patients with FANCG mutations have a higher risk for leukemia (Faivre et al., 2000), but this was not confirmed in the International Fanconi Anemia Registry (Kutler et al., 2003). Other evidence suggests that the type of mutation correlates better with the phenotype than the specific gene. For example, one report found that patients with FANCA null mutations present with a more severe phenotype than other types of FANCA mutations (Faivre at al., 2000). Conversely, in a separate report, no functional or clinical difference was found between null and missense mutations (Castella et al., 2011). It is possible that specific mutations best correlate with the phenotype (reviewed in Neveling et al., 2009). However, even for a specific mutation, there is a variable phenotypic severity in different ethnicities and among siblings, suggesting a role for gene modifiers and environmental factors.Objective: To gain insight into the natural history of FA patients in Israel and shed light on genotype-phenotype correlations in this population.Methods: Patients with FA were registered by their treating physicians as part of the Israeli inherited bone marrow failure registry (I-IBMFR). Follow-up surveys were performed every year. For all patients for whom DNA was available, a genetic analysis was performed.Results: One hundred and eleven (111) FA patients diagnosed between 1980 and 2016 were registered to the I-IBMFR and followed for a median of 9.7 years. Analysis showed a slight male predominance (53% males, 47% females) and a near-even split between those of Jewish descent (42%) and those of Muslim descent (38%) with a smaller representation of Bedouins (11%), Druze (6%) and Christians (2%). Over 60% of the patients were offspring of consanguineous parents. Overall, 83% of the patients had confirmed mutations in genes encoding DNA repair proteins, the majority detected in the FANCA gene (67%) with the rest in FANCC (13%), FANCG (14%), FANCJ (3%) and FANCD1 (2%). Only 2 patients who underwent genetic analysis remained undiagnosed.Of the 111 patients, 90% had at least one congenital anomaly. No significant association was found between a specific congenital anomaly and the affected Fanconi gene or the type of mutation. Nearly one third of the patients developed myelodysplastic syndrome (MDS), leukemia or a solid tumor. The prevalence of MDS was higher in FANCC patients and lower in FANCG patients, reaching statistical significance (p=0.0078). There was a trend towards a higher prevalence of cancer in FANCD1 patients.Over half of the FA patients in the registry underwent a hematopoietic stem cell transplant (HSCT), without any correlation to the genotype; 64% of the transplanted patients are alive, while the rest died of transplant-related complications or malignancy. Of the 10 patients who developed solid tumors; 4 were post-HSCT with a solid tumor diagnosed at a range of 2.5-18 years post-transplant. Fifty six percent (56%) of the patients who did not undergo a transplant are alive; mortality was mostly due to malignancy or sepsis. Currently, 68 FA patients from the registry (61%) are alive, with a median age of 17 years.Conclusion: We hereby present a large cohort of FA patients in Israel, unique due to ethnic diversity combined with a high degree of consanguinity, the majority genetically diagnosed. Genotypic and clinical features of FA patients from the I-IBMFR correlate well with trends seen in larger previously published FA registries but also bring to light possible new links. Interestingly, MDS was more prevalent in FANCC patients and less in FANCG patients; a finding that will need further confirmation in future studies. Continuation of this registry may help to unravel further genotype-phenotype trends and enhance our understanding of the disease. DisclosuresNo relevant conflicts of interest to declare.

The Clinical and Hematologic Course of Fanconi Anemia Amongst Affected Siblings

Fanconi anemia is the most common inherited bone marrow failure syndrome characterized by impaired DNA repair pathway. Patients with Fanconi anemia often present with congenital anomalies and up to 70% of patients develop bone marrow failure (BMF) during the first decade of life, with high risk of progression to myelodysplastic syndromes (MDS) and acute myeloid leukemia (AML). While high penetrance of BMF phenotype is a key characteristic of Fanconi anemia, patients may have variable onset and severity. Despite extensive research, there are no available data whether affected siblings with Fanconi anemia have a similar or different clinical course.First, we reviewed medical records of 9 sets of probands and affected siblings with Fanconi anemia who were seen at the Memorial Sloan Kettering Cancer Center (MSKCC) in the last 30 years (Figure 1). As longitudinal and detailed clinical information was available for these sibling pairs, we collected data by retrospective chart review on the onset of BMF, MDS or AML, time to hematopoietic stem cell transplantation (HSCT), the presence of any congenital anomaly, microcephaly, renal anomaly, low birth weight, short stature, learning disability and frequent infections between older and younger siblings. After observing a signal of positive correlation in BMF onset between siblings, we moved onto reviewing additional sibling pairs in the International Fanconi Anemia Registry (IFAR), where additional 62 sibling pairs with available hematologic onset information were registered. IFAR defines hematologic onset as the onset of BMF, MDS or AML.The Spearman rank correlation coefficient was computed to assess correlation between sibling pair's clinical manifestation for those seen at MSKCC. We observed a positive correlation in the time to bone marrow failure, defined by development of any peripheral blood cytopenias, between sibling pairs, although the p value was borderline due to small sample size (correlation coefficient r=0.75, p=0.05, number of pairs = 7). We excluded two pairs of siblings in which one of the siblings was known to have blood mosaicism. The sample size was too small to compute statistics for the time to MDS/AML or time to HSCT. We also calculated the Cohen's kappa coefficient to determine whether a particular clinical event was likely to occur in both siblings or neither of siblings. We found that kidney anomaly (kappa coefficient = 0.5714; 95% CI 0.0982 - 1) and microcephaly (kappa coefficient = 1; 95% CI 1-1) showed good agreement between siblings, whereas we did not find good agreement between siblings in skeletal anomaly, cardiac anomaly, low birth weight, short stature, growth retardation, learning disability or frequent infections. To assess correlation of hematologic onset in all available sets of probands and affected siblings, the Spearman's rank correlation was calculated on total 69 sibling pairs. We found a positive correlation in the hematologic onset, defined as time to bone marrow failure, MDS or AML, whichever comes first (correlation coefficient r=0.6767, p <0.0001; Figure 2).In summary, affected siblings with Fanconi anemia tend to develop hematologic manifestations at similar ages with a median age difference of 2.18 years (range 0 - 15.36 years; 95% CI 2.61 - 4.17 years). Among other clinical manifestations, kidney anomaly and microcephaly appeared to occur concordantly between siblings. We plan to reach out to other institutions to enhance collaboration and increase number of research participants. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Abstract B23: The role of bone marrow transplantation in patients with Fanconi anemia and head and neck squamous cell carcinoma receiving radiotherapy: An examination of the International Fanconi Anemia (IFAR) Registry

Abstract Introduction: Fanconi anemia (FA) patients have a high predilection for developing aggressive head and neck squamous cell carcinoma (HNSCC) at an early age. Because of their aggressive HNSCC, these patients frequently require multiple modality treatment, including radiation therapy. However, their underlying chromosomal instability makes giving radiation to these patients difficult because of increased radiation-induced complications. In order to better understand this problem, we evaluated a large population of FA patients with HNSCC who received radiation therapy for HNSCC. Hypothesis: FA patients who have undergone prior bone marrow transplant (BMT) and present with head and neck squamous cell carcinoma (HNSCC) demonstrate decreased relative risk of radiation-induced complications compared to FA patients who have not received BMT. Study Design: Retrospective Cohort Study Methods: Demographic information, genotypic data, prognostic factors, therapeutic management, and survival outcomes for FA patients enrolled in the International Fanconi Anemia Registry (IFAR) who developed HNSCC from 1981-2017 were analyzed. Results: Data were collected from the IFAR registry for n=50 patients. The mean age at time of HNSCC diagnosis was 30.5 + 9.0 years, and 36% of total patients were male. 42% of the patients received adjuvant or neoadjuvant radiotherapy for HNSCC, with the most common subsites involving the oral tongue (n=8), followed by alveolus (n=3) and supraglottis (n=2). The average dose given was 48.8 + 16.9 Gy over a 28- to 70-day period. Among patients receiving radiotherapy, 24% had received a bone marrow transplant. No patients who had a prior BMT were noted to develop pancytopenia as a radiation-related complication, while 68% of those without prior BMT developed pancytopenia from radiation. 6/16 patients who did not undergo transplant completed the full course of radiation, while 4/5 BMT patients were able to complete treatment. Both mucositis and sepsis were secondary complications more common in the non-BMT group (56% vs. 40%, and 12% vs. 0%, respectively). The relative risk of pancytopenia among patients receiving BMT and adjuvant radiation was 0.15 (95% CI 0.01-2.2); however, this was not statistically significant given the small BMT-group sample size (p=0.17). Interestingly, 80% of patients in the BMT group exhibited genetic mutations of the Fanconi complementation group C. Among patients who received a BMT and adjuvant radiotherapy, 5 patients died of XRT-related complications, while only 1 patient died from these complications in the BMT group. Conclusion: While it is well known that FA patients have a high predilection for developing aggressive HNSCC at an early age, among the patients who received preradiation bone marrow transplant the incidence of radiation-induced pancytopenia was reduced. This reduction suggests that FA patients undergoing radiotherapy for HNSCC may exhibit less adverse side effects of radiation treatment if they received a prior BMT. Citation Format: Ade Obayemi, K.R. Patel, O. Rosti, A. Smogorzewska, D. Kutler. The role of bone marrow transplantation in patients with Fanconi anemia and head and neck squamous cell carcinoma receiving radiotherapy: An examination of the International Fanconi Anemia (IFAR) Registry [abstract]. In: Proceedings of the AACR-AHNS Head and Neck Cancer Conference: Optimizing Survival and Quality of Life through Basic, Clinical, and Translational Research; 2019 Apr 29-30; Austin, TX. Philadelphia (PA): AACR; Clin Cancer Res 2020;26(12_Suppl_2):Abstract nr B23.

Association of clinical severity with FANCB variant type in Fanconi anemia

AbstractFanconi anemia (FA) is the most common genetic cause of bone marrow failure and is caused by inherited pathogenic variants in any of 22 genes. Of these, only FANCB is X-linked. We describe a cohort of 19 children with FANCB variants, from 16 families of the International Fanconi Anemia Registry. Those with FANCB deletion or truncation demonstrate earlier-than-average onset of bone marrow failure and more severe congenital abnormalities compared with a large series of FA individuals in published reports. This reflects the indispensable role of FANCB protein in the enzymatic activation of FANCD2 monoubiquitination, an essential step in the repair of DNA interstrand crosslinks. For FANCB missense variants, more variable severity is associated with the extent of residual FANCD2 monoubiquitination activity. We used transcript analysis, genetic complementation, and biochemical reconstitution of FANCD2 monoubiquitination to determine the pathogenicity of each variant. Aberrant splicing and transcript destabilization were associated with 2 missense variants. Individuals carrying missense variants with drastically reduced FANCD2 monoubiquitination in biochemical and/or cell-based assays tended to show earlier onset of hematologic disease and shorter survival. Conversely, variants with near-normal FANCD2 monoubiquitination were associated with more favorable outcome. Our study reveals a genotype-phenotype correlation within the FA-B complementation group of FA, where severity is associated with level of residual FANCD2 monoubiquitination.

Fanconi Anemia: A Paradigm for Understanding DNA Repair During Replication.

Fanconi anemia, the most common hereditary bone marrow failure disorder, results from defective repair of DNA interstrand crosslinks (ICLs), which covalently link complementary DNA strands causing replication stalling. Mutations in 22 different genes (FANCA-FANCW) have been shown to result in Fanconi anemia. Their protein products work at different stages of DNA repair leading to considerable heterogeneity in human phenotypes. The majority of the FANC gene mutations are recessively inherited with the exceptions of FANCB and FANCR/RAD51. FANCB is X-linked, and all FANCR/RAD51 mutations arise de novo, affect only one allele, and the mutant protein acts as a dominant negative against the wild type protein. Despite advances in the molecular diagnosis of Fanconi anemia, if Fanconi anemia is suspected, chromosome breakage (DEB or MMC) testing on patient cells is essential. We have seen a number of patients referred to the International Fanconi Anemia Registry (http://lab.rockefeller.edu/smogorzewska/ifar/) who are misdiagnosed with Fanconi anemia based solely on the presence of a FANC gene variant in gene panel or whole exome sequencing. Conversely, blood mosaicism may lead to a negative blood chromosome breakage test. If there is a high suspicion of Fanconi anemia, but blood breakage results are negative, breakage test on patient fibroblasts should be performed. Diagnosis of Fanconi anemia should also be entertained in young adults presenting with squamous cell carcinoma of the aerodigestive tract, since this may be their initial presentation of Fanconi anemia and conventional chemotherapy dose would precipitate bone marrow failure in these patients. In my talk, I will discuss the mechanism of the Fanconi anemia repair pathway during DNA replication. Then, I will concentrate on the mechanism of bone marrow failure and tumorigenesis in Fanconi anemia. I will explore the hypothesis that the endogenously produced aldehydes including some that are still unknown, contribute to disease development. Fanconi anemia-deficient hematopoietic stem cells have an autonomous DNA repair defect. Accumulation of DNA damage leads to apoptosis due to the activation of p53. If cells escape death, mutagenesis may lead to the development of leukemia. The sources of endogenous DNA damage are poorly understood. Cell cycle induction of Fanconi anemia pathway-deficientmouse hematopoietic stem cells results in DNA damage and bone marrow failure, which implies that the DNA lesions encountered during replication are the culprit. There is mounting evidence that the endogenous aldehydes, including acetaldehyde and formaldehyde,may cause those DNA lesions. To identify other metabolites that may induce bone marrow failure in Fanconi anemia, we used a library of CRISPR guides to target Cas9 to metabolic genes to screen for and identify synthetic lethality with Fanconi anemia deficiency. We have identifiedALDH9A1as the most significantly depleted gene in FANCD2-/- cells. The synthetically lethal interaction was validated using single gene editing in human umbilical cord-derived hematopoietic stem progenitor cells. We propose a model in which aldehydes that are metabolized by ALDH9A1 accumulate in the absence of this enzyme and cause DNA damage that requires the Fanconi anemia pathway proteins for repair, survival, and suppression of tumorigenesis. We are testing this model using Fanca-/-Aldh9a1-/-mice. Disclosures No relevant conflicts of interest to declare.

Clinical Severity Correlates with in Vitro Residual Function of FANCB Missense Variants

Fanconi anemia (FA) is the most common inherited bone marrow failure syndrome, characterized by congenital anomalies, bone marrow failure and cancer predisposition. FA patients with mutations in FANCB exhibit X-linked inheritance pattern. FANCB patients have been frequently associated with a severe phenotype, resembling VACTERL association with hydrocephalus, although heterogeneity in these patients still exists and is not well understood. To determine the underlying pathogenesis of clinical heterogeneity, we investigated phenotype-genotype correlations in 19 patients from 16 families with FANCB pathogenic variants. We also investigated functional properties of five FANCB missense variants to determine their possible contribution to clinical manifestations.Patients were accrued at the Rockefeller University Hospital in the International Fanconi Anemia Registry. Among the 16 families with FANCB pathogenic variants, three carry nonsense variants, five have indels, two with large deletions, one has an intragenic duplication, two with canonical splice site variants that should result in aberrant splicing, and five display missense variants. One patient carried both an indel and a missense variant. Patients with variants leading to FANCB truncations (including nonsense variants, indels, large deletions/duplications, and splice variants) tend to show shorter survival and earlier hematologic onset, than patients with missense variants. However, the differences did not reach statistical significance due to small sample size.Functional properties of five missense variants were examined by overexpression of variant cDNA (HA-tagged) in FANCB-null fibroblasts, followed by assessment of FANCD2 ubiquitination, mitomycin C (MMC) sensitivity, FANCD2 foci formation and HA-FANCB localization. We found that expression of p.W479G and p.L676P FANCB variants leads to near-normal FANCD2 ubiquitination upon MMC exposure, and MMC sensitivity. Conversely, p.L43S-expressing cells behaved like FANCB-null fibroblasts. Expression of p.L329P and p.G750V resulted in low levels of FANCD2 ubiquitination upon MMC exposure and intermediate MMC sensitivity. Expression of p.L43S resulted in smaller-sized and lowest quantity of FANCD2 foci with predominant cytoplasmic localization of HA-FANCB, while other variants showed intermediate phenotype. We quantified the nuclear expression of HA-FANCB, which confirmed the lowest relative nuclear expression of p.L43S variant, suggesting a defect in nuclear localization necessary for the interstrand crosslink repair. We also assessed splicing defects in the patient primary cells by reverse transcription PCR. Majority of the transcripts in the patient carrying c.1435T>G (predicted to express p.W479G) variant showed aberrant splicing, exon 7 skipping, which should result in an out-of-frame truncated protein. Although the expression level of c.1435T>G encoding the missense variant p.W479G is lower than the splicing variant, its functional characteristics were near-normal, thus reflecting relatively milder phenotype in this patient. Overall patient survival correlated with residual function of their FANCB variants as assessed by in vitro assays described above.In summary, we report clinical course and mutation types of 19 patients with FANCB pathogenic variants. We also report that FANCB missense variants can have varying degree of residual function, which correlates with patient survival. Our data suggest that variant-level analysis may be a useful prognostic marker of patient outcome. In vitro assays evaluating the residual function of proteins with missense variants may help to predict patient outcome. DisclosuresNo relevant conflicts of interest to declare.

A comprehensive approach to identification of pathogenic FANCA variants in Fanconi anemia patients and their families.

Fanconi anemia (FA) is a rare recessive DNA repair deficiency resulting from mutations in one of at least 22 genes. Two-thirds of FA families harbor mutations in FANCA. To genotype patients in the International Fanconi Anemia Registry (IFAR) we employed multiple methodologies, screening 216 families for FANCA mutations. We describe identification of 57 large deletions and 261 sequence variants, in 159 families. All but seven families harbored distinct combinations of two mutations demonstrating high heterogeneity. Pathogenicity of the 18 novel missense variants was analyzed functionally by determining the ability of the mutant cDNA to improve the survival of a FANCA-null cell line when treated with MMC. Overexpressed pathogenic missense variants were found to reside in the cytoplasm, and nonpathogenic in the nucleus. RNA analysis demonstrated that two variants (c.522G>C and c.1565A>G), predicted to encode missense variants, which were determined to be nonpathogenic by a functional assay, caused skipping of exons 5 and 16, respectively, and are most likely pathogenic. We report 48 novel FANCA sequence variants. Defining both variants in a large patient cohort is a major step toward cataloging all FANCA variants, and permitting studies of genotype-phenotype correlations.

Abstract A27: Assessing somatic tumor-associated RAD51 mutations and screening for novel dominant-interfering RAD51 proteins

Abstract The responsiveness of cancer cells to chemotherapy and targeted treatment, as well as the development of resistance, is determined by the state of DNA damage response pathways. Homology-directed repair (HDR) is crucial for error-free repair of DNA double-strand breaks that arise during replication stress or are induced by exogenous genotoxins. Therefore, HDR efficacy status in cancer cells could potentially be utilized as an indicator to predict tumor responsiveness to standard chemotherapies and to poly(ADP-ribose) polymerases (PARP) inhibitors. We are interested in understanding the influence of different levels of functional activity of the HDR pathway on treatment responses. To this end, we have investigated the functional consequence of RAD51 point mutations identified in tumors. RAD51 is an effector of the key tumor suppressor BRCA2 and contributes to genome integrity in actively dividing cells. RAD51 mutations are identified from Memorial Sloan Kettering clinical sequencing cohort (MSK-IMPACT), the Catalogue of Somatic Mutations in Cancer (COSMIC) and the International Fanconi Anemia Registry (IFAR). To understand the implications of these altered residues on RAD51 function in HDR, we tested repair efficiency using a previously established DR-GFP reporter assay in mammalian cells. Of eighteen novel RAD51 mutants examined, ten displayed decreased HDR efficiency, three had wild-type levels of HDR, and five had increased HDR levels. To test whether the HDR-deficient RAD51 mutants lead to sensitivity to genotoxins, we assayed the survival of corn smut U. maydis that expressed these RAD51 mutants after treatment with DNA damaging agents ultraviolet light, diepoxybutane, or methylmethane sulfonate. All ten HDR-defective mutants were found to be sensitive to one or more genotoxins. In addition, six were found to promote sensitivity to DNA damaging agents when they were expressed in wild-type cells—i.e., a dominant negative phenotype. Interestingly, these dominant negative mutants confer selective sensitivity to specific DNA damaging agent(s). The underlying mechanism is still under investigation; nevertheless, these mutations can be used as biomarkers for HDR efficiency and to identify novel interactions of RAD51. The identification of these somatic tumor-associated RAD51 mutants warrants further testing for sensitivity towards chemotherapy drugs such as PARP inhibitors and gemcitabine. Furthermore, a random mutagenesis screening is in progress to identify more novel dominant negative RAD51 mutations. The functional analysis of RAD51 is critical as readouts for HDR efficiency, which impacts personalized chemotherapeutic choices. The result of this study will hopefully expand the benefits of using PARP inhibitors not just on BRCA1/2- and RAD51 paralog-deficient tumors but also on tumors with RAD51 mutations. Citation Format: Pei Xin Lim, Jeanette Sutherland, Raymond Noonan, Alexandra Dananberg, William Holloman, Agata Smogorzewska, Maria Jasin. Assessing somatic tumor-associated RAD51 mutations and screening for novel dominant-interfering RAD51 proteins [abstract]. In: Proceedings of the AACR Special Conference on DNA Repair: Tumor Development and Therapeutic Response; 2016 Nov 2-5; Montreal, QC, Canada. Philadelphia (PA): AACR; Mol Cancer Res 2017;15(4_Suppl):Abstract nr A27.

Natural history and management of Fanconi anemia patients with head and neck cancer: A 10-year follow-up.

To describe the management and outcomes of Fanconi anemia (FA) patients with head and neck squamous cell carcinoma. Cohort study. Demographic information, prognostic factors, therapeutic management, and survival outcomes for FA patients enrolled in the International Fanconi Anemia Registry who developed head and neck squamous cell carcinoma (HNSCC) were analyzed. Thirty-five FA patients were diagnosed with HNSCC at a mean age of 32 years. The most common site of primary cancer was the oral cavity (26 of 35, 74%). Thirty patients underwent surgical resection of the cancer. Sixteen patients received radiation therapy with an average radiation dose of 5,050 cGy. The most common toxicities were high-grade mucositis (9 of 16, 56%), hematologic abnormalities (8 of 16, 50%), and dysphagia (8 of 16, 50%). Three patients received conventional chemotherapy and had significant complications, whereas three patients who received targeted chemotherapy with cetuximab had fewer toxicities. The 5-year overall survival rate was 39%, with a cause-specific survival rate of 47%. Fanconi anemia patients have a high risk of developing aggressive HNSCC at an early age. Fanconi anemia patients can tolerate complex ablative and reconstructive surgeries, but careful postoperative care is required to reduce morbidity. The treatment of FA-associated HNSCC is difficult secondary to the poor tolerance of radiation and chemotherapy. However, radiation should be used for high-risk cancers due to the poor survival in these patients. 4.

Massively parallel sequencing, aCGH, and RNA-Seq technologies provide a comprehensive molecular diagnosis of Fanconi anemia

AbstractCurrent methods for detecting mutations in Fanconi anemia (FA)–suspected patients are inefficient and often miss mutations. We have applied recent advances in DNA sequencing and genomic capture to the diagnosis of FA. Specifically, we used custom molecular inversion probes or TruSeq-enrichment oligos to capture and sequence FA and related genes, including introns, from 27 samples from the International Fanconi Anemia Registry at The Rockefeller University. DNA sequencing was complemented with custom array comparative genomic hybridization (aCGH) and RNA sequencing (RNA-seq) analysis. aCGH identified deletions/duplications in 4 different FA genes. RNA-seq analysis revealed lack of allele specific expression associated with a deletion and splicing defects caused by missense, synonymous, and deep-in-intron variants. The combination of TruSeq-targeted capture, aCGH, and RNA-seq enabled us to identify the complementation group and biallelic germline mutations in all 27 families: FANCA (7), FANCB (3), FANCC (3), FANCD1 (1), FANCD2 (3), FANCF (2), FANCG (2), FANCI (1), FANCJ (2), and FANCL (3). FANCC mutations are often the cause of FA in patients of Ashkenazi Jewish (AJ) ancestry, and we identified 2 novel FANCC mutations in 2 patients of AJ ancestry. We describe here a strategy for efficient molecular diagnosis of FA.

Postoperative Clinical Radiosensitivity in Patients With Fanconi Anemia and Head and Neck Squamous Cell Carcinoma

To describe the complications and adverse effects of postoperative radiotherapy in patients with Fanconi anemia (FA). Cohort study. Patients with FA treated at community and tertiary care hospitals throughout the United States. The study included patients with FA who were enrolled in the International FA Registry (IFAR) and who developed head and neck squamous cell carcinoma and received postoperative radiotherapy. Demographics of patients with FA and adverse effects and dosages of radiotherapy. Twelve patients with FA (7 men and 5 women) were identified. They developed cancers at a mean age of 35.5 years (age range, 20-48 years). The sites of primary cancer were the oral cavity (n = 8), larynx (n = 2), pharynx (n = 1), and unknown (n = 1). The median radiation dose was 5590 cGy (range, 2500-7020 cGy). The most common adverse effects were mucositis (n = 9), dysphagia (n = 8), and pancytopenia (n = 6). Other complications included esophageal stenosis, laryngeal edema, and wound breakdown. Radiotherapy could not be completed in 5 cases. Overall, 8 patients died, 4 during the course of radiotherapy. The postoperative disease-free survival time ranged from 0 to 55 months. Patients with FA have a high rate of complications from radiotherapy. Common adverse effects, particularly mucositis, are especially prevalent and difficult to manage in this population. Pancytopenia is common and may lead to further complications, particularly bleeding and infection. Overall survival is poor. Further study of the response to radiotherapy in patients with FA should be attempted to establish appropriate dosages to balance treating disease while limiting adverse effects.

Genetic heterogeneity among Fanconi anemia heterozygotes and risk of cancer.

Fanconi anemia (FA) is a rare autosomal recessive disease characterized by a greatly increased risk of cancer among those diagnosed with the syndrome. The question as to whether FA heterozygotes are at increased risk for cancer is of great importance to those at risk for being a carrier. To address this question, we formed a cohort of grandparents of probands identified through the International Fanconi Anemia Registry. We obtained informed consent, a short questionnaire, and either blood or buccal swab DNA. After diagnosis of the proband was confirmed and complementation studies or DNA sequencing on the proband were completed, mutation analyses of the putative carriers and noncarriers was carried out. Standardized incidence ratios (SIR) were calculated to compare the observed cancer incidence of the grandparents and other relatives with the expected rates of cancer, using the Surveillance, Epidemiology, and End Results registries and the Connecticut Cancer registry. In the 944 study subjects who participated (784 grandparents and 160 other relatives), there was no suggestion of an increase in overall cancer incidence. On the other hand, a significantly higher rate of breast cancer than expected was observed among carrier grandmothers [SIR, 1.7; 95% confidence interval (95% CI), 1.1-2.7]. Among the grandmothers, those who were carriers of FANCC mutations were found to be at highest risk (SIR, 2.4; 95% CI, 1.1-5.2). Overall, there was no increased risk for cancer among FA heterozygotes in this study of Fanconi relatives, although there is some evidence that FANCC mutations are possibly breast cancer susceptibility alleles.

Hypomorphic Mutations in the Gene Encoding a Key Fanconi Anemia Protein, FANCD2, Sustain a Significant Group of FA-D2 Patients with Severe Phenotype

FANCD2 is an evolutionarily conserved Fanconi anemia (FA) gene that plays a key role in DNA double-strand-type damage responses. Using complementation assays and immunoblotting, a consortium of American and European groups assigned 29 patients with FA from 23 families and 4 additional unrelated patients to complementation group FA-D2. This amounts to 3%-6% of FA-affected patients registered in various data sets. Malformations are frequent in FA-D2 patients, and hematological manifestations appear earlier and progress more rapidly when compared with all other patients combined (FA-non-D2) in the International Fanconi Anemia Registry. FANCD2 is flanked by two pseudogenes. Mutation analysis revealed the expected total of 66 mutated alleles, 34 of which result in aberrant splicing patterns. Many mutations are recurrent and have ethnic associations and shared allelic haplotypes. There were no biallelic null mutations; residual FANCD2 protein of both isotypes was observed in all available patient cell lines. These analyses suggest that, unlike the knockout mouse model, total absence of FANCD2 does not exist in FA-D2 patients, because of constraints on viable combinations of FANCD2 mutations. Although hypomorphic mutations arie involved, clinically, these patients have a relatively severe form of FA.

Clinical Characteristics of Patients with Fanconi Anemia in Complementation Group J.

Fanconi anemia (FA) is an inherited disorder characterized by chromosomal instability, developmental abnormalities, progressive bone-marrow failure, and cancer susceptibility. To date, twelve complementation groups have been described for FA (A, B, C, D1, D2, E, F, G, I, J, L and M) and eleven associated genes have already been identified (FANCA, FANCB, FANCC, FANCD1/BRCA2, FANCD2, FANCE, FANCF, FANCG/XRCC9, BRIP1/FANCJ, FANCL and FANCM/Hef). The FA complementation group J (FA-J) was first reported in 2004. FA-A is the most common group, accounting for approximately 65% of all affected individuals while FA-J is a rare group, comprising only 1.6% of all FA patients in the International Fanconi Anemia Registry (IFAR). To the best of our knowledge 11 FA-J patients have been reported in the IFAR. Patients with Fanconi anemia show extreme clinical heterogeneity.Thus, it is of interest to associate the phenotype with the complemantation group, since this could provide predictive information on clinical outcome.So far, in the Hospital La Fe, 4 patients (two siblings and two unrelated patients) belong to scarce FA-J complementation group. This represents the 4.5 % of the FA patients included in the Spanish Fanconi Anemia Registry. Subtyping was done by viral complementation. Cells from these patients were not complemented with a battery of vectors with FA genes (FANCA, FANCC, FANCG, FANCE, FANCF, and FANCD2). However, the characteristic cellular hypersensitivity to mitomycin C was reverted when a lentiviral vector encoding for BRIP1/FANCJ was used. Moreover, mutational studies confirmed the subtyping of FA-J in these Fanconi patients. The clinical course of the 4 FA-J patienst are shown in the Table.In conclusion, its noteworthy the high frequency of the FA-J genotype in Spain. The 75% of our patients have major congenital malformations and severe endocrinopathy. All our patients had short stature and 50% were mycrosomic. None of our patients had skin abnormalities, although this is present in 80% of the patients in other registries. Although two of these patients developed aplasia very early (5 years old), FA-J siblings developed severe aplasia at the age of 20 years but none of them developed leukemia or solid tumors.Clinical course of patientsPatient1*2*34sexmalefemalemalefemaleAplasia onset (years)2420.54.33.5Survival (years)2831.56aliveShort statureYesYesYesYesMicrosomyNoYesYesNoSkin pigmentationNoNoNoNoEyes abnormalitiesNoNomicrophthalmiaEpiphoraSkeletal malformationsSyndactylyNoAbnormal thumbsAbnormal thumbsKidneysAgenesis, leftNoNoHorseshoeEndocrinopathyfailure pubertynot availablehypotiroidismhypopituitarysmGastrointestinal malformationsNoNoEsophageal atresiaOnphaloceleMalignaciesNoNoNoNoFamily cancersNot availableNot availableLiverLung, brains and breast* Sibling

Spectrum of sequence variations in the FANCA gene: An International Fanconi Anemia Registry (IFAR) study

Fanconi anemia (FA) is an autosomal recessive disorder that is defined by cellular hypersensitivity to DNA cross-linking agents, and is characterized clinically by developmental abnormalities, progressive bone-marrow failure, and predisposition to leukemia and solid tumors. There is extensive genetic heterogeneity, with at least 11 different FA complementation groups. FA-A is the most common group, accounting for approximately 65% of all affected individuals. The mutation spectrum of the FANCA gene, located on chromosome 16q24.3, is highly heterogeneous. Here we summarize all sequence variations (mutations and polymorphisms) in FANCA described in the literature and listed in the Fanconi Anemia Mutation Database as of March 2004, and report 61 novel FANCA mutations identified in FA patients registered in the International Fanconi Anemia Registry (IFAR). Thirty-eight novel SNPs, previously unreported in the literature or in dbSNP, were also identified. We studied the segregation of common FANCA SNPs in FA families to generate haplotypes. We found that FANCA SNP data are highly useful for carrier testing, prenatal diagnosis, and preimplantation genetic diagnosis, particularly when the disease-causing mutations are unknown. Twenty-two large genomic deletions were identified by detection of apparent homozygosity for rare SNPs. In addition, a conserved SNP haplotype block spanning at least 60 kb of the FANCA gene was identified in individuals from various ethnic groups.

Germline mutations in BRCA2: shared genetic susceptibility to breast cancer, early onset leukemia, and Fanconi anemia

AbstractThe breast cancer susceptibility geneBRCA2has recently been identified as identical to the Fanconi anemia (FA) geneFANCD1. Here we expand the clinical implications of this discovery. Notably, we identified 6 children in 5 kindreds exhibiting the co-occurrence ofBRCA2mutations, FA, and early onset acute leukemia. Leukemia occurred at a median of 2.2 years of age in theBRCA2patients in contrast to a median onset of 13.4 years in all other FA patients in the International Fanconi Anemia Registry (IFAR;P< .0001). Breast cancer was noted in 4 of the 5 kindreds. Of the 6 children with leukemia, 4 were treated with bone marrow transplantation and 2 are alive at 3 and 9 months after treatment. Our results suggest thatBRCA2testing should be considered in all patients with FA in whom the complementation group cannot be defined or in whom leukemia is diagnosed at or before 5 years of age. (Blood. 2004;103:3226-3229)

Spectrum of sequence variation in the FANCG gene: An International Fanconi Anemia Registry (IFAR) study

Spectrum of sequence variation in the FANCG gene: An International Fanconi Anemia Registry (IFAR) study

A 20-year perspective on the International Fanconi Anemia Registry (IFAR)

Fanconi anemia (FA) is an autosomal recessive disorder characterized by cellular hypersensitivity to DNA cross-linking agents and cancer predisposition. Recent evidence for the interactions of ataxia-telangiectasia mutated protein ATM and breast cancer susceptibility proteins BRCA1 and BRCA2 (identified as FANCD1) with other known FA proteins suggests that FA proteins have a significant role in DNA repair/recombination and cell cycle control. The International Fanconi Anemia Registry (IFAR), a prospectively collected database of FA patients, allows us the unique opportunity to analyze the natural history of this rare, clinically heterogeneous disorder in a large number of patients. Of the 754 subjects in this study, 601 (80%) experienced the onset of bone marrow failure (BMF), and 173 (23%) had a total of 199 neoplasms. Of these neoplasms, 120 (60%) were hematologic and 79 (40%) were nonhematologic. The risk of developing BMF and hematologic and nonhematologic neoplasms increased with advancing age with a 90%, 33%, and 28% cumulative incidence, respectively, by 40 years of age. Univariate analysis revealed a significantly earlier onset of BMF and poorer survival for complementation group C compared with groups A and G; however, there was no significant difference in the time to hematologic or nonhematologic neoplasm development between these groups. Multivariate analysis of overall survival time shows that FANCC mutations (P =.007) and hematopoietic stem cell transplantation (P = <.0001) define a poor-risk subgroup. The results of this study of patients registered in the IFAR over a 20-year period provide information that will enable better prediction of outcome and aid clinicians with decisions regarding major therapeutic modalities.

Spectrum of sequence variation in the FANCG gene: an International Fanconi Anemia Registry (IFAR) study.

Fanconi anemia (FA) is a genetically heterogeneous autosomal recessive syndrome associated with chromosomal instability, hypersensitivity to DNA cross-linking agents, and predisposition to malignancy. The gene for FA complementation group G (FANCG) was the third FA gene to be cloned, and was found to be identical with human XRCC9, which maps to 9p13. The cDNA is predicted to encode a polypeptide of 622 amino acids, with no sequence similarities to any other known protein or motifs that could point to a molecular function for FANCG/XRCC9. We used single strand conformational polymorphism analysis (SSCP) to screen genomic DNA from a panel of 307 racially and ethnically diverse unrelated FA patients from the International Fanconi Anemia Registry (IFAR) for variants in FANCG. Twenty-seven abnormal SSCP patterns were found; 18 of these variants appear to be pathogenic mutations while nine are likely to be nonpathogenic polymorphisms. Direct sequencing of genomic DNA from seven FA-G probands with one mutant allele not detected in the SSCP study and three additional probands assigned to the FA-G complementation group by retroviral correction with FANCG resulted in the detection of nine additional pathogenic mutations and two common SNPs. Conditions for rapid screening for these mutations by DHPLC for use in a clinical laboratory setting were established. The most common FANCG mutations in the IFAR population were: IVS8-2A>G (seven Portuguese-Brazilian probands), IVS11+1G>C (seven French-Acadian probands), 1794_1803del10 (seven European probands), and IVS3+1G>C (five Korean or Japanese probands). Our data suggest that the Portuguese-Brazilian, French-Acadian, and Korean/Japanese mutations were likely to have been present in a founding member of each of these populations.

High incidence of head and neck squamous cell carcinoma in patients with Fanconi anemia.

Fanconi anemia (FA) is a rare autosomal recessive disorder characterized by a high degree of genomic instability and predisposition to cancer development. Recent evidence suggests that the incidence of head and neck squamous cell carcinoma (HNSCC) may be increased in patients with FA. To determine the cumulative incidence, tumor distribution, and outcome of HNSCC in patients with FA. We analyzed data from 754 subjects from the International Fanconi Anemia Registry, a prospectively collected database of patients with FA. Cumulative incidence of HNSCC and 2-year overall, relapse-free and disease-specific survival. Of the 754 patients in the International Fanconi Anemia Registry, 19 (3%) had HNSCC. This is a significantly higher incidence of HNSCC compared with that observed in the general population (standardized incidence ratio, 500; 95% confidence interval, 300-781) (P<.001). The patients' age ranged from 15 to 49 years (median, 31 years), and there was a 2:1 female predominance. Surgical treatment was well tolerated (n = 17); however, radiation therapy and chemotherapy were associated with significant morbidity and mortality. Of the 19 patients, 10 (53%) developed locoregional recurrences within a median of 16 months from diagnosis. The median follow-up was 29 months. The 2-year disease-specific, overall, and relapse-free survival rates were 49%, 49%, and 42%, respectively. The cumulative incidence of relapse by the age of 40 years was 50%. In patients with FA, there is a high incidence of aggressive HNSCC at a young age. Surgery remains the mainstay of treatment because patients with FA tolerate radiation therapy and chemotherapy poorly, with significant morbidity. An increased understanding of FA-associated malignancies is not only important in the clinical management of patients with FA but can also elucidate the role of chromosomal instability in the development of HNSCC in general.