RAPID COMMUNICATION Two Cases of Misinterpretation of Molecular Results in Incontinentia Pigmenti, and a PCR- Based Method to Discriminate NEMO/IKKc Gene Deletion Tiziana Bardaro, 1 Geppino Falco, 1 Angela Sparago, 1 Vincenzo Mercadante, 1 Esther Gean Molins, 2 Enrico Tarantino, 3 Matilde Valeria Ursini, 1 and Michele D’Urso 1n 1 Institute of Genetics and Biophysics, Adriano Buzzati Traverso-CNR, Naples, Italy; 2 Hospital Sant Joan Deu, Barcelona, Spain; 3 Servizio di Genetica Medica, Clinica Pediatrica I, Pisa, Italy Communicated by Arnold Munnich Familial incontinentia pigmenti (IP) is a rare X-linked dominant disorder that affects ectodermal tissues. Over 90% of IP carrier females have a recurrent genomic deletion of exons 4–10 of the NEMO (IKBKG- IKKc) gene, which encodes a regulatory component of the IkB kinase complex, required to activate the NF-kB pathway. In IP, mutations in NEMO lead to the complete loss of NF-kB activation creating a susceptibility to cellular apoptosis in response to TNF-a. This condition is lethal for males during embryogenesis while females, who are mosaic as a result of X-inactivation, can survive. Recently, a second nonfunctional copy of the gene, DNEMO, was identified, opposite in direction to NEMO in a 35.5-kb duplicated sequence tract. PCR-based detection of the NEMO deletion is diagnostic for IP disease. However, we present instances in which ex 4–10 DNEMO pseudogene deletion occurs in unaffected parents of two females with clinically characteristic IP. These were missed by the currently standard PCR- based method, but can be easily discriminated by a new PCR-based test reported here that permits unambiguous molecular diagnosis and proper familial genetic counseling for IP. Hum Mutat 21:8–11, 2002. r 2002 Wiley-Liss, Inc. KEY WORDS: NEMO; IKBKG; IKKg; NF-kB essential modulator; incontinentia pigmenti; IP; X-inactivation; molecular diagnosis DATABASES: NEMO – OMIM: 300248, 308310 (IP); GenBank: AJ271718 (NEMO), AL596249 (DNEMO pseudogene) INTRODUCTION Familial incontinentia pigmenti (IP; MIM# 308310) is a rare X-linked genodermatosis that affects ectodermal tissues [Francis and Sybert, 1997]. We recently showed that IP results from mutations in the NEMO/IKBKG,IKKg gene (NF-kB essential modula- tor; MIM# 300248) [Smahi et al., 2000], that disrupt NF-kB activity and lead to increased apoptosis. The complete loss of NF-kB activation is lethal in males during embryogenesis, while females can survive based on X-chromosome mosaicism—there is selection for cells carrying an active normal X after X-inactivation, a feature often used to confirm the diagnosis [Parrish et al., 1996]. The sequencing of BAC clones mapped to the Xq28 locus reveals the presence of a nonfunctional second copy of the NEMO gene (DNEMO), located 31.6 kb from exon 10 (Fig. 1) [Aradhya et al., 2001a]. DNEMO maps within a 35.5-kb duplicated fragment opposite to NEMO in orientation. This duplication spans from 282 bp upstream of exon 3 to 9.9 kb downstream of exon 10. Notably, we found 45 single nucleotide differences over the entire 35.5 kb, only seven of which are intronic sequence differences between the NEMO copies. Received 17 June 2002; accepted revised manuscript 16 September 2002. n Correspondence to: M. D’Urso, Institute of Genetics and Bio- physics, Adriano BuzzatiTraverso-CNR, 12-80125 Via Marconi, Naples, Italy. E-mail: durso@iigb.na.cnr.it Grant sponsors: CNR-PS Molecular Genetics; BioGeM; Grant sponsor:Telethon-Italy; Grant number: GP0414Y02. DOI 10.1002/humu.10150 Published online in Wiley InterScience (www.interscience.wiley. com). r r2002 WILEY-LISS, INC. HUMAN MUTATION 21:8^11 (2002)