A Novel Gene, FGA7, Is Fused to RUNX1/AML1 in a t(4;21)(q28;q22) in a Patient With T-Cell Acute Lymphoblastic Leukemia Fady M. Mikhail, 1,2 Lionel Coignet, 3 Nadia Hatem, 4 Zeinab I. Mourad, 1 Hala M. Farawela, 5 Dalal M. El Kaffash, 1 Nahla Farahat, 1 and Giuseppina Nucifora 2* 1 Department of Clinical Pathology, Faculty of Medicine, University of Alexandria, Alexandria, Egypt 2 Department of Pathology, University of Illinois at Chicago Medical Center, Chicago, Illinois 3 Department of Cancer Genetics, Roswell Park Cancer Institute, Buffalo, New York 4 Department of Pediatrics, Faculty of Medicine, University of Alexandria, Alexandria, Egypt 5 Department of Clinical Pathology, Faculty of Medicine, Cairo University, Cairo, Egypt AML1 is among the most frequent targets of chromosomal rearrangements in human leukemias. We report here the molecular analysis of a t(4;21)(q28;q22) that has disrupted AML1 in a patient with de novo T-cell acute lymphoblastic leukemia. By using 3'-RACE analysis, we show that this rearrangement results in the fusion of a novel gene immediately downstream of exon 5 or exon 6 of AML1, indicating that the AML1 breakpoint lies in intron 6 and that alternative fusion splice variants are generated. The sequence of the novel gene, located at 4q28, does not have any significant homology with any of the known genes in the human GenBank DNA database. However, the first 118 bases are identical to a part of a human ovarian EST. Also, its high homology with mouse and rat sequences suggests that this sequence most probably represents a part of a novel gene, which we named FGA7 (Fused Gene 7 to AML1). Following the AML1 open reading frame, the FGA7 sequence encodes an unknown protein of 27 amino acids. We isolated three bacterial artificial chromosome (BAC) clones that contain the FGA7 sequence and confirmed the breakpoint of the gene on the patient’s metaphase spreads by fluorescence in situ hybridization using these BACs as probes. RT-PCR and Northern blot analyses revealed that FGA7 is expressed in ovarian and skeletal muscle tissues. The predicted AML1-FGA7 chimeric proteins contained a limited number of residues fused to AML1 in a situation similar to that reported for the AML1-EAP fusion that is a product of t(3;21). It is possible that the expression of a constitutively shortened AML1 could compete with full-length AML1 and act as a dominant negative inhibitor of the promoters that the core binding factor activates. © 2003 Wiley-Liss, Inc. INTRODUCTION The AML1 gene (also known as RUNX1 or CBFA2), located in chromosome band 21q22, was originally cloned as one of the two genes rear- ranged in t(8;21)(q22;q22) associated with acute myeloid leukemia (AML)–M2. This translocation also disrupts the ETO gene (also known as MTG8 or CBFA2T1) on chromosome band 8q22 (Miyoshi et al., 1993). AML1 encodes a protein that belongs to a small family of transcription factors. Members of this family are homologous in a region of 128 amino acids at the N-terminus of the protein, designated the RUNT domain, first identified in the Drosoph- ila Runt gene (Ogawa et al., 1993; Levanon et al., 2001). This domain binds the DNA consensus se- quence TGT/cGGT in the promoter of target genes and is required for AML1 heterodimeriza- tion with the CBF protein, forming the functional transcription complex core binding factor (CBF) (Meyers et al., 1993). Upon CBF binding, a con- formational change takes place in the critical DNA- binding residues, resulting in increased DNA- binding affinity of the heterodimer (Tahirov et al., 2001). Heterodimerization of AML1 with CBF inhibits AML1 proteolysis by the ubiquitin–protea- some pathway, suggesting that this interaction pro- longs AML1 activities (Huang et al., 2001). AML1 also has a C-terminus transactivation domain (Nu- cifora and Rowley, 1995; Levanon et al., 2001). The DNA-binding activity of CBF is critical for the tissue-specific expression of several hematopoiesis- specific genes, including the genes encoding IL-3, Giuseppina Nucifora is a Scholar of the Leukemia and Lym- phoma Society. Supported by: Alexandria Faculty of Medicine; Egyptian Ministry of Higher Education Missions Department (FM); NIH-NCI; Grant numbers: CA67189 and CA72675 (to GN). *Correspondence to: Giuseppina Nucifora, Department of Pa- thology, University of Illinois at Chicago, MBRB (MC 737), 900 South Ashland Avenue, Chicago, IL 60607. E-mail: nucifora@uic.edu Received 27 May 2003; Accepted 12 September 2003 DOI 10.1002/gcc.10302 GENES, CHROMOSOMES & CANCER 39:110 –118 (2004) © 2003 Wiley-Liss, Inc.