RESEARCH ARTICLE OFFICIAL JOURNAL www.hgvs.org Identification of Large NF1 Duplications Reciprocal to NAHR-Mediated Type-1 NF1 Deletions Hildegard Kehrer-Sawatzki, 1 ∗ † Kathrin Bengesser, 1 † Tom Callens, 2 Fady Mikhail, 3 Chuanhua Fu, 2 Morten Hillmer, 1 Martha E. Walker, 4 Howard M. Saal, 4 Yves Lacassie, 5 David N. Cooper, 6 and Ludwine Messiaen 2 ∗ 1 Institute of Human Genetics, University of Ulm, Ulm, Germany; 2 Medical Genomics Laboratory, Department of Genetics, University of Alabama at Birmingham, Birmingham, Alabama; 3 Cytogenetics Laboratory, Department of Genetics, University of Alabama at Birmingham, Birmingham, Alabama; 4 Division of Human Genetics, Cincinnati Children’s Hospital Medical Center, Cincinnati, Ohio; 5 Louisiana State University Health Sciences Center and Children’s Hospital, New Orleans, Louisiana; 6 Institute of Medical Genetics, School of Medicine, Cardiff University, Cardiff, UK Communicated by Claude Ferec Received 7 July 2014; accepted revised manuscript 28 August 2014. Published online 9 September 2014 in Wiley Online Library (www.wiley.com/humanmutation). DOI: 10.1002/humu.22692 ABSTRACT: Approximately 5% of all patients with neu- rofibromatosis type-1 (NF1) exhibit large deletions of the NF1 gene region. To date, only nine unrelated cases of large NF1 duplications have been reported, with none of the affected patients exhibiting multiple caf´ e au lait spots (CALS), Lisch nodules, freckling, or neurofibro- mas, the hallmark signs of NF1. Here, we have charac- terized two novel NF1 duplications, one sporadic and one familial. Both index patients with NF1 duplications ex- hibited learning disabilities and atypical CALS. Addition- ally, patient R609021 had Lisch nodules, whereas patient R653070 exhibited two inguinal freckles. The mother and sister of patient R609021 also harbored the NF1 dupli- cation and exhibited cognitive dysfunction but no CALS. The breakpoints of the nine NF1 duplications reported previously have not been identified and hence their un- derlying generative mechanisms have remained unclear. In this study, we performed high-resolution breakpoint analysis that indicated that the two duplications studied were mediated by nonallelic homologous recombination (NAHR) and that the duplication breakpoints were lo- cated within the NAHR hotspot paralogous recombina- tion site 2 (PRS2), which also harbors the type-1 NF1 deletion breakpoints. Hence, our study indicates for the first time that NF1 duplications are reciprocal to type-1 NF1 deletions and originate from the same NAHR events. Hum Mutat 35:1469–1475, 2014. C  2014 Wiley Periodicals, Inc. Additional Supporting Information may be found in the online version of this article. † These authors contributed equally to this work. ∗ Correspondence to: Hildegard Kehrer-Sawatzki, Institute of Human Genetics, Uni- versity of Ulm, Albert-Einstein-Allee 11, Ulm 89081, Germany. E-mail: hildegard.kehrer- sawatzki@uni-ulm.de; Ludwine Messiaen, Medical Genomics Laboratory, Department of Genetics, University of Alabama at Birmingham, Kaul Human Genetics Building, 720 20th Street South, Birmingham, Alabama 35294-0024. E-mail: lmessiaen@uabmc.edu Contract grant sponsors: Deutsche Forschungsgemeinschaft (grants no. KE/12-1 and KE/7-2); Children’s Tumor Foundation; UAB Medical Genomics Laboratory. KEY WORDS: neurofibromatosis type 1; NF1 microdele- tions; nonallelic homologous recombination; mutational mechanism; microduplication Introduction Neurofibromatosis type 1 (NF1; MIM #162200) is an autosomal- dominant disorder characterized by neurofibromas, caf´ e au lait spots (CALS), axillary and/or inguinal freckling, and Lisch nod- ules. In 5% of all patients with NF1, large deletions of the NF1 gene and its flanking regions are observed [Kluwe et al., 2004]. These large NF1 deletions frequently cause a severe manifestation of the disease associated with a higher risk for the development of malignant peripheral nerve sheath tumors (MPNSTs) as com- pared with NF1 individuals exhibiting intragenic NF1 mutations [De Raedt et al., 2003; Mautner et al., 2010]. Whereas the lifetime risk of an MPNST in all NF1 individuals is 8%–13% [Evans et al., 2002], patients with large NF1 deletions have an estimated lifetime risk for an MPNST of 16%–26% [De Raedt et al., 2003]. Further- more, patients with large NF1 deletions frequently exhibit severe developmental delay and considerable cognitive impairment as well as dysmorphic facial features [Mautner et al., 2010; and references therein]. Different types of large NF1 deletion have been reported that are distinguishable by virtue of their breakpoint position and causative mutational mechanism [reviewed by Kehrer-Sawatzki and Cooper, 2012]. Most frequent is the type-1 NF1 deletion that is observed in 70%–80% of all patients with large NF1 deletions [Pasmant et al., 2010; Messiaen et al., 2011]. Type-1 NF1 deletions encompass 1.4-Mb, exhibit breakpoints located within the low-copy repeats (LCRs) NF1-REPa and NF1-REPc and are mediated by nonallelic homologous recombination (NAHR). High-sequence similarity of the recombining sequences is a prerequisite for NAHR to occur. Although NF1-REPa and NF1-REPc show sequence identity of 98% over 51-kb, the type-1 deletion breakpoints cluster within specific recombination hotspots termed paralogous recombination sites 1 and 2 (PRS1 and PRS2) [Forbes et al., 2004; De Raedt et al., 2006]. An estimated 80% of all type-1 NF1 deletion breakpoints are located within the PRS2 NAHR hotspot that spans only 2.2-kb [De Raedt et al., 2006; Bengesser et al., 2014]. C  2014 WILEY PERIODICALS, INC.