RESEARCH ARTICLE Genetic Instability of the Tumor Suppressor Gene FHIT in Normal Human Cells Elisa Palumbo †,1 , Elena Tosoni ‡,1 , Laura Matricardi § , Antonella Russo * Department of Biology,University of Padova,Via U.Bassi 58/b, 35131, Padova, Italy Common fragile sites are hotspots for chromosome instability and co-localize to cancer genomic rearrangements. Whether these loci may be considered stable in human subjects under physiological conditions remains an open question. Here we show by molecular combing that a small but significant percentage of normal human cells carry an abnormal sequence pattern within the tumor suppressor gene FHIT (3p14.2) at FRA3B. Each sequence variation represents a unique pattern within a normal cell population, and therefore it would remain undetected or not interpreted by genome-wide analyses. Remarkably, the region is the same as in FHIT rearrangements described in tumors. By analyses on several normal cell lines (proliferating and resting primary lymphocytes, primary fibroblasts, lymphoblastoid cells including clonal cell cul- tures) we verified that: (a) each cell type displays altered sequence patterns at FHIT; (b) the presence of abnormal sequence patterns is specific for the FHIT locus; and (c) FHIT instability occurs de novo during cell proliferation, and hetero- geneous sequence variants progressively accumulate in the cell populations. FHIT has been widely investigated in cancer cells, but to our knowledge this is the first direct evidence of spontaneous and recurrent occurrence of genomic instability at this gene in human subjects, at the same region involved in cancer rearrangements. Our results suggest that common fragile site activity is not restricted to in vitro cell culture and that genomic instability may pre-exist in normal cells in the absence of exogenous replication stress. V C 2013 Wiley Periodicals, Inc. INTRODUCTION FRA3B (3p14.2), the most actively expressed common fragile site of the human genome, encom- passes a large region (over 4 Mb) of chromosomal instability (Rassool et al., 1996; Becker et al., 2002) harboring several genes including the Fragile His- tidine Triad gene (FHIT). FHIT is a large gene that extends over about 1.5 Mb. Its large size is related to the presence of very large intronic sequences, a typical feature of genes mapping within common fragile sites (Smith et al., 2006). In particular, introns 4 and 5 are large, respectively, 285 kb and 523 kb; the core of fragility is centered on exon 5 (Ohta et al., 1996; Zimonjic et al., 1997). FHIT encodes a protein involved in intracellular signaling and DNA damage response (Okumura et al., 2009; Saldivar et al., 2010, 2012). An increas- ing number of studies indicates that the FHIT protein is absent or reduced in most tumors, including many of the digestive system (from the oral cavity and oesophagus to the colon), and those affecting the pancreas and liver (Okumura et al., 2009). Because FHIT protein appears necessary for correct DNA damage response, the loss of this gene may be the first step on a genomic instability pathway ultimately leading to cell transformation (Saldivar et al., 2010, 2012). Several chromosomal alterations and rearrange- ments in cancer cells involve the FHIT locus (Okumura et al., 2009), but this observation does not imply a direct causative role of the fragile site in cell transformation, because cancer cells are sometimes genetically unstable per se. Moreover, because of the tumor suppressor activity of FHIT, the finding of rearrangements in its genomic sequence could simply reflect a preferential posi- tive selection of cells that have lost FHIT function. About 30 years after the hypothesis was put for- ward (Yunis and Soreng, 1984) of a possible involvement of common fragile sites in cancer Additional Supporting Information may be found in the online version of this article. 1 E. Palumbo and E. Tosoni contributed equally to this work. † Present address: Familial Cancer Clinic and Oncoendocrinology, Veneto Institute of Oncology (IOV-IRCCS); Department of Medi- cal and Surgical Sciences, University of Padova, Via Gattamelata 64, Padova 35128, Italy ‡ Present address: Department of Molecular Medicine, University of Padova, Via A. Gabelli, 63, 35121, Padova, Italy. § Present address: Veneto Institute of Oncology (IOV-IRCCS), Via Gattamelata 64 35128, Padova, Italy. Supported by: University of Padova, Department of Biology. *Correspondence to: Antonella Russo, Department of Biology, University of Padova, Via U. Bassi 58/b, 35131 Padova, Italy. E-mail: antonella.russo@unipd.it Received 23 April 2013; Accepted 10 May 2013 DOI 10.1002/gcc.22079 Published online in Wiley Online Library (wileyonlinelibrary.com). V V C 2013 Wiley Periodicals, Inc. GENES, CHROMOSOMES & CANCER 00:00–00 (2013)