Multiple pathogenic and benign genomic rearrangements occur at a 35 kb duplication involving the NEMO and LAGE2 genes

Swaroop Aradhya 3 Tiziana Bardaro 2 3 Petra Galgczy 1 3 Takanori Yamagata 0 3 Teresa Esposito 2 3 Henry Patlan 3 Alfredo Ciccodicola 2 3 Arnold Munnich 3 5 Sue Kenwrick 3 4 Matthias Platzer 1 3 Michele D'Urso 2 3 David L. Nelson 3 0 Department of Pediatrics, Jichi Medical School , 3311-1 Yakushiji, Minamikawachi-machi, Tochigi 329-0433 , Japan 1 Department of Genome Analysis, Institute of Molecular Biotechnology , Beutenbergstrasse 11, 07445 Jena , Germany 2 International Institute of Genetics and Biophysics (IIGB) , Via G. Marconi 10, 80125 Naples , Italy 3 Department of Molecular and Human Genetics, Baylor College of Medicine , One Baylor Plaza 902E, Houston, TX 77030 , USA 4 Wellcome Trust Centre for Molecular Mechanisms of Disease and University of Cambridge Department of Medicine, Addenbrooke's Hospital , Hills Road, Cambridge CB2 2XY , UK 5 Department of Genetics, Unite des Recherches sur les Handicaps Genetiques de l'Enfant INSERM-393, Hopital Necker-Enfants Malades , 75015 Paris , France - The X-linked dominant and male-lethal disorder incontinentia pigmenti (IP) is caused by mutations in a gene called NEMO (IKK-). We recently reported the structure of NEMO and demonstrated that most IP patients carry an identical deletion that arises due to misalignment between repeats. Affected male abortuses with the IP deletion had provided clues that a second, incomplete copy of NEMO was present in the genome. We have now identified clones containing this truncated copy ( NEMO) and incorporated them into a previously constructed physical contig in distal Xq28. NEMO maps 22 kb distal to NEMO and only contains exons 310, confirming our proposed model. A sequence of 26 kb 3 of the NEMO coding sequence is also present in the same position relative to the NEMO locus, bringing the total length of the duplication to 35.5 kb. The LAGE2 gene is also located within this duplicated region, and a similar but unique LAGE1 gene is located just distal to the duplicated loci. Mapping and sequence information indicated that the duplicated regions are in opposite orientation. Analysis of the great apes suggested that the NEMO/LAGE2 duplication occurred after divergence of the lineage leading to present day humans, chimpanzees and gorillas, 1015 million years ago. Intriguingly, despite this substantial evolutionary history, only 22 single nucleotide differences exist between the two copies over the entire 35.5 kb, making the duplications >99% identical. This high sequence identity and the inverted orientations of the two copies, along with duplications of smaller internal sections within each copy, predispose this region to various genomic alterations. We detected four rearrangements that involved NEMO, NEMO or LAGE1 and LAGE2. The high sequence similarity between the two NEMO/LAGE2 copies may be due to frequent gene conversion, as we have detected evidence of sequence transfer between them. Together, these data describe an unusual and complex genomic region that is susceptible to various types of pathogenic and polymorphic rearrangements, including the recurrent lethal deletion associated with IP. Mutations in NEMO (IKBKG, IKK-) cause the X-linked dominant disorder, incontinentia pigmenti (IP) (1,2). This disorder is typically lethal in male individuals but female patients survive because cells expressing the mutant X chromosome are selectively eliminated. Thus, skewed X-inactivation is a characteristic of this disorder (3,4). As a regulatory component of IB kinase, NEMO is responsible for downstream activation of the NF-B transcription factor. By inducing the transcription of various target genes, the NF-B signaling pathway regulates immune and inflammatory reactions and prevents apoptosis (5,6). Disruption of NEMO or NF-B renders cells susceptible to apoptosis, leading to the IP-associated male lethality and skewed X-inactivation in female patients (2). Nearly 7080% of IP mutations are accounted for by an identical deletion within NEMO, which eliminates exons 410 (7). This mutation arises due to misalignment between two identical MER67B *To whom correspondence should be addressed. Tel: +1 713 798 4787; Fax: +1 713 798 5386; Email: +AF277315, AL596249 and AJ271718 The authors wish it to be known that, in their opinion, the first three authors should be regarded as joint First Authors sequences (termed int3h repeats); one copy is located in intron 3 and another 4 kb distal to the last exon of NEMO. When the recurrent IP deletion was first identified due to an aberrant fragment on a Southern blot, fragments of normal size were also present (2). This led us to propose that a second copy of NEMO ( NEMO) existed in the genome. In addition, PCR analysis of DNA samples from spontaneous male abortuses with the IP deletion yielded the expected amplification products from exon 2 to 3 and exon 3 to 4, but failed to amplify from exon 2 to 4. These observations supported a model that the second copy of NEMO was truncated, lacking the first four exons. The human genome contains numerous examples of gene duplications, several of which are involved in genomic disorders (8). Thus, it was conceivable that rearrangements could occur between the two NEMO copies and that such events may have a role in the genetics underlying IP or another human disease due to disruption of genes between them. Rearrangements would be especially likely if the two NEMO copies share significant homology, and a preliminary analysis of exons suggested complete identity between NEMO and NEMO. We recently constructed a high-density bacterial- and P1-artificial chromosome (BAC and PAC) contig to study the region between G6PD and Xqter (9). After another group mapped NEMO to Xq28, we sequenced the entire gene from a BAC clone in the contig and showed that it lies head-to-head with G6PD and is transcribed in the centromeric to telomeric direction (Fig. 1A and B) (2,10). The 23 kb NEMO gene contains 12 exons with three alternative primary exons that independently splice into exon 2, where the initiating ATG codon lies (GenBank accession no. AJ271718). In our initial BAC/PAC contig, a gap existed just distal to NEMO and efforts to close it with flanking probes had failed repeatedly. When the idea of a second copy of NEMO was proposed, we decided to search the gap region since duplicated copies of genes are often located close to the parent copy, as exemplified by the IDS gene in Xq28 (11). This report describes how we identified NEMO, when and how it originated, its current structure and the homology it shares with NEMO. The duplication boundaries were cloned and the entire region containing NEMO and NEMO was sequenced. Interestingly, NEMO was part of a larger duplication that originated during evolution of the great apes. The sequence, structure and evolution of this duplication provide significant insight into how the human genome evolves through mechanisms of structural alteration as well as sequence preservation. Isolatio (...truncated)