HUMAN MUTATION 28(12), 1198^1206, 2007 RESEARCH ARTICLE Double Complex Mutations Involving F8 and FUNDC2 Caused by Distinct Break-Induced Replication Campbell R. Sheen, 1Ã Ursula R. Jewell, 2 Christine M. Morris, 2,3 Stephen O. Brennan, 1 Claude Fe ´rec, 4,5 Peter M. George, 1 Mark P. Smith, 6 and Jian-Min Chen 4,5 1 Molecular Pathology Laboratory, Canterbury Health Laboratories, Christchurch, New Zealand; 2 Cancer Genetics Research Group, Christchurch School of Medicine and Health Sciences, University of Otago, Christchurch, New Zealand; 3 Cytogenetics Unit, Canterbury Health Laboratories, Christchurch, New Zealand; 4 Institut National de la Sante ´ et de la Recherche Me ´dicale (INSERM), U613, Brest, France; 5 Etablissement Franc - ais du Sang-Bretagne, Brest, France; 6 Haematology Service, Canterbury District Health Board, Christchurch, New Zealand Communicated by Haig H. Kazazian, Jr. Genomic rearrangements are a well-recognized cause of genetic disease and can be formed by a variety of mechanisms. We report a complex rearrangement causing severe hemophilia A, identified and further characterized using a range of PCR-based methods, and confirmed using array–comparative genomic hybridization (array-CGH). This rearrangement consists of a 15.5-kb deletion/16-bp insertion located 0.6 kb from a 28.1-kb deletion/263-kb insertion at Xq28 and is one of the most complex rearrangements described at a DNA sequence level. We propose that the rearrangement was generated by distinct but linked cellular responses to double strand breakage, namely break-induced replication (BIR) and a novel model of break-induced serial replication slippage (SRS). The copy number of several genes is affected by this rearrangement, with deletion of part of the Factor VIII gene (F8, causing hemophilia A) and the FUNDC2 gene, and duplication of the TMEM185A, HSFX1, MAGEA9, and MAGEA11 genes. As the patient exhibits no clinically detectable phenotype other than hemophilia A, it appears that the biological effects of the other genes involved are not dosage-dependent. This investigation has provided novel insights into processes of DNA repair including BIR and the first description of SRS during repair in a pathological context. Hum Mutat 28(12), 1198–1206, 2007. r r 2007 Wiley-Liss, Inc. KEY WORDS: break-induced replication; complex genomic rearrangement; F8; Factor VIII; FUNDC2; hemophilia A; serial replication slippage INTRODUCTION Hemophilia A (MIM] 306700) is caused by mutations in the F8 gene, which is located approximately 1 Mb from the telomere of Xq. Its 26 exons span 186 kb and to date, some 943 distinct F8 gene mutations have been reported, of which 120 (13%) involve deletions of Z50 bp (HAMSTeRS database; http://euro- pium.csc.mrc.ac.uk/webpages/main/main.htm). However, of these large deletions, only 10 have been fully characterized at the nucleotide level; and they can be divided into simple (n 5 8) or complex (n 5 2) deletions, the latter having additional sequence inserted into the breakpoint junction. Simple deletions have been postulated to result from either nonhomologous end-joining (NHEJ) [Woods-Samuels et al., 1991] or unequal homologous Alu-mediated recombination [Shibata et al., 2000; Vidal et al., 2002; Nakaya et al., 2004; Rossetti et al., 2004] whereas the two reported insertion/deletions involving F8 appeared to involve different mechanisms: a 38-bp insert associated with a 20.7-kb deletion within the F8 gene [Van de Water et al., 1998]is likely to involve long interspersed nuclear element-1 (LINE-1) retro- transposon-mediated DNA repair [Morrish et al., 2002]; and a 6-bp insert into a 316-bp deletion in the F8 gene [Tavassoli et al., 1999] has recently been explained by a model of serial replication slippage (SRS) in cis [Chen et al., 2005a]. In addition, nonallelic homologous recombination (NAHR)- mediated inversions cause approximately half of severe hemophilia A: while the most common inversion occurs as a consequence of recombination between a sequence in intron 22 and one of two further telomeric copies of the repeat [Lakich et al., 1993], the less common inversion occurs between a tract of intron 1 and a further copy located telomeric to F8 [Bagnall et al., 2002]. In this report, we present a previously undescribed type of genomic rearrangement in a patient with severe hemophilia A. This mutational event consists of two adjacent complex deletion/ Published online 7 August 2007 in Wiley InterScience (www. interscience.wiley.com). DOI 10.1002/humu.20591 The Supplementary Material referred to in this article can be accessed at http://www.interscience.wiley.com/jpages/1059-7794/ suppmat. Received 5 April 2007; accepted revised manuscript 5 June 2007. Grant sponsors: Bayer Haemophilia Awards Special Project Grant; New Zealand Child Health Research Foundation; Child Cancer Foundation; Lottery Health Research. Ã Correspondence to: Campbell Sheen, Molecular Pathology Laboratory, Canterbury Health Laboratories, P.O. Box 151, Christch- urch, New Zealand. E-mail: campbell.sheen@chmeds.ac.nz r r 2007 WILEY-LISS, INC.