ORIGINAL ARTICLE A novel Leu153Ser mutation of the Fanconi anemia FANCD2 gene is associated with severe chemotherapy toxicity in a pediatric T-cell acute lymphoblastic leukemia A Borriello 1 , A Locasciulli 2 , AM Bianco 3 , M Criscuolo 1 , V Conti 3 , P Grammatico 4 , S Cappellacci 4 , A Zatterale 5 , F Morgese 6 , V Cucciolla 1 , D Delia 7 , F Della Ragione 1 and A Savoia 6 1 Department of Biochemistry and Biophysics ‘F Cedrangolo’ II University of Naples, Naples, Italy; 2 Bone Marrow Transplantation Centre, ‘S Camillo – Forlanini’ Hospital, Rome, Italy; 3 Telethon Institute of Genetics and Medicine (TIGEM), Naples, Italy; 4 Department of Experimental Medicine and Pathology, University ‘La Sapienza’ of Rome, Rome, Italy; 5 Department of Genetics, ‘Elena d’Aosta Hospital’, ASL Napoli 1, Naples, Italy; 6 Medical Genetics, Department of Reproductive and Developmental Sciences, IRCCS Burlo Garofolo Hospital, University of Trieste, Trieste, Italy and 7 Department of Experimental Oncology, National Institute of Cancer, Milan, Italy Fanconi anemia (FA) is an autosomal recessive disease characterized by pancitopenia, congenital malformations, pre- disposition to cancers and chromosomal instability. We report the clinical and molecular features of a patient initially identified as a potential FA case only because of chemotherapy toxicity during the treatment of a T-lineage acute lymphoblastic leukemia (ALL). Cells from this patient showed a moderate chromosomal instability, increasing sensitivity to DNA cross- linking agents but normal response to ionizing radiation. The analysis of FA proteins demonstrated a marked reduction of FANCD2 (495%), but normal levels of FANCA or FANCG. Interestingly, this defect was associated with a homozygous missense mutation of FANCD2, resulting in a novel amino-acid substitution (Leu153Ser) at residue Leu153, which is highly conserved through evolution. The FANCD2 L153S protein, whose reduced expression was not due to impaired transcription, was detected also in its monoubiquitinated form in the nucleus, suggesting that the mutation does not affect post-translation modifications or subcellular localization but rather the stability of FANCD2. Therefore, the hypomorphic Leu153Ser mutation represents the first example of a FANCD2 defect that might promote clonal progression of tumors, such as T-ALL, and severe chemotherapy toxicity in patients without any clinical manifestations typical of FA. Leukemia (2007) 21, 72–78. doi:10.1038/sj.leu.2404468; published online 9 November 2006 Keywords: Fanconi anemia; T-lineage acute lymphoblastic leukemia; FANCD2 gene Introduction Fanconi anemia (FA) is an autosomal recessive disease characterized by a variety of clinical symptoms, including congenital malformations, progressive bone marrow failure and high predisposition to develop cancers. FA cells show sponta- neous chromosomal instability and an elevated sensitivity to crosslinking agents, such as diepoxybutane (DEB) and mito- mycin C (MMC). 1 Although the function of the eleven cloned FA genes is largely unknown, the proteins participate in a common pathway known as FA/BRCA. 1 Briefly, the majority, FANCA, FANCB, FANCC, FANCE, FANCF, FANCG, FANCL and FANCM, interact in a nuclear complex required for monoubiquitination of the down- stream FANCD2 protein. 2–5 The FANCL gene, also a component of the FA complex, is likely to function as a putative E3 ubiquitin ligase for the FANCD2 post-translational modification. 6 Mono- ubiquitinated FANCD2 is targeted to nuclear foci, where it colocalizes and interacts with several components of the DNA repair machinery, including breast cancer susceptibility 1 (BRCA1), DNA repair protein RAD51 homolog 1 and nibrin (NBS1) of the Nijmegen breakage syndrome. 1 Moreover, biallelic disruption of the BRCA2 gene also causes FA, as detected in patients of the FA-D1 complementation group. 7 BRCA2, which functions further downstream of the FA pathway, is implicated in DNA repair by homologous recombination directly binding the DNA repair protein RAD51. 8 Even the most recently identified gene FANCJ, a DNA-dependent ATPase and 5 0 -to-3 0 DNA helicase that directly binds to BRCA, is not required for FANCD2 monoubiquitination. 9–11 A critical role of FA proteins in carcinogenesis has been reported in literature. First, FA patients are highly susceptible to hematological malignancies (especially acute myelogenous leukemia (AML)) and solid tumors of the head and neck, gynecological system and other organs. 12 Second, FANCD2- deficient mice have an increased incidence of epithelial cancers (i.e. breast, ovarian and liver tumors). 13 Finally, defects of the FA genes may contribute to cancer progression. For instance, in 18% of primary ovarian epithelial cancers, the FANCF gene is methylated and silenced. 14 Germline FANCC or FANCG mutations have also been found in patients with inherited pancreatic cancers. 15 Moreover, acquired dysfunction of FAN- CA may promote cytogenetic instability and clonal progression in patients with AML. 16–18 Acute leukemias, mostly AML and rarely acute lymphoblastic leukemia (ALL), and myelodisplastic syndromes may follow clinically overt aplastic anemia or develop without a detectable antecedent. 19 Moreover, patients can be clinically suspected to be affected by FA – even failing the typical malformations – when they manifest an inexplicable marked sensitivity to chemotherapeutic agents with devastating and unpredictable clinical consequences that can affect both short- and long-term prognosis. 13,20 In this paper, we report the case of a patient affected by ALL without the clinical features specific for FA. Because of severe chemotherapy toxicity, occurring with several episodes of marrow aplasia leading to life-threatening infections, a suspi- cion of FA diagnosis led us to perform the DEB test, which was Received 30 July 2006; revised 20 September 2006; accepted 28 September 2006; published online 9 November 2006 Correspondence: Professor A Savoia, Medical Genetics, Department of Reproductive and Developmental Sciences, IRCCS Burlo Garofolo Hospital, University of Trieste, via dell’Istria, 65/1 – 34137 Trieste, Italy. E-mail: savoia@burlo.trieste.it Leukemia (2007) 21, 72–78 & 2007 Nature Publishing Group All rights reserved 0887-6924/07 $30.00 www.nature.com/leu