Optimised molecular genetic diagnostics of Fanconi anaemia by whole exome sequencing and functional studies.

PURPOSE: Patients with Fanconi anaemia (FA), a rare DNA repair genetic disease, exhibit chromosome fragility, bone marrow failure, malformations and cancer susceptibility. FA molecular diagnosis is challenging since FA is caused by point mutations and large deletions in 22 genes following three heritability patterns. To optimise FA patients' characterisation, we developed a simplified but effective methodology based on whole exome sequencing (WES) and functional studies. METHODS: 68 patients with FA were analysed by commercial WES services. Copy number variations were evaluated by sequencing data analysis with RStudio. To test FANCA missense variants, wt FANCA cDNA was cloned and variants were introduced by site-directed mutagenesis. Vectors were then tested for their ability to complement DNA repair defects of a FANCA-KO human cell line generated by TALEN technologies. RESULTS: We identified 93.3% of mutated alleles including large deletions. We determined the pathogenicity of three FANCA missense variants and demonstrated that two FANCA variants reported in mutations databases as 'affecting functions' are SNPs. Deep analysis of sequencing data revealed patients' true mutations, highlighting the importance of functional analysis. In one patient, no pathogenic variant could be identified in any of the 22 known FA genes, and in seven patients, only one deleterious variant could be identified (three patients each with FANCA and FANCD2 and one patient with FANCE mutations) CONCLUSION: WES and proper bioinformatics analysis are sufficient to effectively characterise patients with FA regardless of the rarity of their complementation group, type of mutations, mosaic condition and DNA source.

Generating New FANCA-Deficient HNSCC Cell Lines by Genomic Editing Recapitulates the Cellular Phenotypes of Fanconi Anemia.

Fanconi anemia (FA) patients have an exacerbated risk of head and neck squamous cell carcinoma (HNSCC). Treatment is challenging as FA patients display enhanced toxicity to standard treatments, including radio/chemotherapy. Therefore, better therapies as well as new disease models are urgently needed. We have used CRISPR/Cas9 editing tools in order to interrupt the human FANCA gene by the generation of insertions/deletions (indels) in exon 4 in two cancer cell lines from sporadic HNSCC having no mutation in FA-genes: CAL27 and CAL33 cells. Our approach allowed efficient editing, subsequent purification of single-cell clones, and Sanger sequencing validation at the edited locus. Clones having frameshift indels in homozygosis did not express FANCA protein and were selected for further analysis. When compared with parental CAL27 and CAL33, FANCA-mutant cell clones displayed a FA-phenotype as they (i) are highly sensitive to DNA interstrand crosslink (ICL) agents such as mitomycin C (MMC) or cisplatin, (ii) do not monoubiquitinate FANCD2 upon MMC treatment and therefore (iii) do not form FANCD2 nuclear foci, and (iv) they display increased chromosome fragility and G2 arrest after diepoxybutane (DEB) treatment. These FANCA-mutant clones display similar growth rates as their parental cells. Interestingly, mutant cells acquire phenotypes associated with more aggressive disease, such as increased migration in wound healing assays. Therefore, CAL27 and CAL33 cells with FANCA mutations are phenocopies of FA-HNSCC cells.

Elevated levels of IL-1beta in Fanconi anaemia group A patients due to a constitutively active phosphoinositide 3-kinase-Akt pathway are capable of promoting tumour cell proliferation.

FA (Fanconi anaemia) is a hereditary disease characterized by congenital malformations, progressive bone marrow failure and an extraordinary elevated predisposition to develop cancer. In the present manuscript we describe an anomalous high level of the proinflammatory cytokine IL-1beta (interleukin-1beta) present in the serum of FA patients. The elevated levels of IL-1beta were completely reverted by transduction of a wild-type copy of the FancA cDNA into FA-A (FA group A) lymphocytes. Although the transcription factor NF-kappaB (nuclear factor-kappaB) is a well established regulator of IL-1beta expression, our experiments did not show any proof of elevated NF-kappaB activity in FA-A cells. However, we found that the overexpression of IL-1beta in FA-A cells is related to a constitutively activated PI3K (phosphoinositide 3-kinase)-Akt pathway in these cells. We provide evidence that the effect of Akt on IL-1beta activation is mediated by the inhibition of GSK3beta (glycogen synthase kinase 3beta). Finally, our data indicate that the levels of IL-1beta produced by FA-A lymphoblasts are enough to promote an activation of the cell cycle in primary glioblastoma progenitor cells. Together, these results demonstrate that the constitutive activation of the PI3K-Akt pathway in FA cells upregulates the expression of IL-1beta through an NF-kappaB-independent mechanism and that this overproduction activates the proliferation of tumour cells.

Detectable clonal mosaicism in blood as a biomarker of cancer risk in Fanconi anemia.

Detectable clonal mosaicism for large chromosomal events has been associated with aging and an increased risk of hematological and some solid cancers. We hypothesized that genetic cancer predisposition disorders, such as Fanconi anemia (FA), could manifest a high rate of chromosomal mosaic events (CMEs) in peripheral blood, which could be used as early biomarkers of cancer risk. We studied the prevalence of CMEs by single-nucleotide polymorphism (SNP) array in 130 FA patients' blood DNA and their impact on cancer risk. We detected 51 CMEs (4.4-159 Mb in size) in 16 out of 130 patients (12.3%), of which 9 had multiple CMEs. The most frequent events were gains at 3q (n = 6) and 1q (n = 5), both previously associated with leukemia, as well as rearrangements with breakpoint clustering within the major histocompatibility complex locus (P = 7.3 × 10-9). Compared with 15 743 age-matched population controls, FA patients had a 126 to 140 times higher risk of detectable CMEs in blood (P < 2.2 × 10-16). Prevalent and incident hematologic and solid cancers were more common in CME carriers (odds ratio [OR] = 11.6, 95% confidence interval [CI] = 3.4-39.3, P = 2.8 × 10-5), leading to poorer prognosis. The age-adjusted hazard risk (HR) of having cancer was almost 5 times higher in FA individuals with CMEs than in those without CMEs. Regarding survival, the HR of dying was 4 times higher in FA individuals having CMEs (HR = 4.0, 95% CI = 2.0-7.9, P = 5.7 × 10-5). Therefore, our data suggest that molecular karyotyping with SNP arrays in easy-to-obtain blood samples could be used for better monitoring of bone marrow clonal events, cancer risk, and overall survival of FA patients.

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

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 signal-regulated kinase (ERK) responded to the proper stimuli in FA-A cells. We also demonstrate that the early growth-response factor-1 (Egr-1), a JNK downstream target gene that is normally induced by genotoxic stress, is not upregulated in UV-treated FA-A cells. Moreover, FA-A cells are more sensitive to apoptosis than control lymphoblasts. Both JNK and Egr-1 may be part of a pathway triggered by FA proteins, because functional correction of FA-A cells by gene transfer restores, at least in part, JNK activation and Egr-1 expression after UV exposure. Together, our data suggest that activation of JNK and expression of Egr-1 gene in B lymphoblasts mediate a cellular response to genotoxic agents that may be induced by FA proteins.

In vitro phenotypic correction of hematopoietic progenitors from Fanconi anemia group A knockout mice.

Fanconi anemia (FA) is a rare autosomal recessive disease, characterized by bone marrow failure and cancer predisposition. So far, 8 complementation groups have been identified, although mutations in FANCA account for the disease in the majority of FA patients. In this study we characterized the hematopoietic phenotype of a Fanca knockout mouse model and corrected the main phenotypic characteristics of the bone marrow (BM) progenitors using retroviral vectors. The hematopoiesis of these animals was characterized by a modest though significant thrombocytopenia, consistent with reduced numbers of BM megakaryocyte progenitors. As observed in other FA models, the hematopoietic progenitors from Fanca(-/-) mice were highly sensitive to mitomycin C (MMC). In addition, we observed for the first time in a FA mouse model a marked in vitro growth defect of Fanca(-/-) progenitors, either when total BM or when purified Lin(-)Sca-1(+) cells were subjected to in vitro stimulation. Liquid cultures of Fanca(-/-) BM that were stimulated with stem cell factor plus interleukin-11 produced low numbers of granulocyte macrophage colony-forming units, contained a high proportion of apoptotic cells, and generated a decreased proportion of granulocyte versus macrophage cells, compared to normal BM cultures. Aiming to correct the phenotype of Fanca(-/-) progenitors, purified Lin(-)Sca-1(+) cells were transduced with retroviral vectors encoding the enhanced green fluorescent protein (EGFP) gene and human FANCA genes. Lin(-)Sca-1(+) cells from Fanca(-/-) mice were transduced with an efficiency similar to that of samples from wild-type mice. More significantly, transductions with FANCA vectors corrected both the MMC hypersensitivity as well as the impaired ex vivo expansion ability that characterized the BM progenitors of Fanca(-/-) mice.