A recurrent deletion in the ubiquitously expressed NEMO (IKK-γ) gene accounts for the vast majority of incontinentia pigmenti mutations

Swaroop Aradhya 2 Hayley Woffendin 6 Tracy Jakins 6 Tiziana Bardaro 4 5 Teresa Esposito 5 Asmae Smahi 3 Christine Shaw 2 Moise Levy 1 Arnold Munnich 3 Michele D'Urso 5 Richard A. Lewis 0 2 Sue Kenwrick 6 David L. Nelson 2 0 Department of Ophthalmology and the Cullen Eye Institute, Baylor College of Medicine , Houston, TX 77030, USA 1 Department of Dermatology 2 Department of Molecular and Human Genetics 3 Department of Genetics, Unit des Recherches sur les Handicaps Gntiques de l'Enfant INSERM-393 , Hopital Necker-Enfants Malades, 75015 Paris, France 4 BioGem, Naples, Italy 5 International Institute of Genetics and Biophysics , Area di Ricerca del CNR di Napoli, Naples, Italy 6 Wellcome Trust Centre for Molecular Mechanisms of Disease and University of Cambridge Department of Medicine, Addenbrooke's Hospital , Hills Road, Cambridge CB2 2XY, UK - Incontinentia pigmenti (IP) is an X-linked dominant disorder characterized by abnormal skin pigmentation, retinal detachment, anodontia, alopecia, nail dystrophy and central nervous system defects. This disorder segregates as a male lethal disorder and causes skewed X-inactivation in female patients. IP is caused by mutations in a gene called NEMO, which encodes a regulatory component of the IB kinase complex required to activate the NF-B pathway. Here we report the identification of 277 mutations in 357 unrelated IP patients. An identical genomic deletion within NEMO accounted for 90% of the identified mutations. The remaining mutations were small duplications, substitutions and deletions. Nearly all NEMO mutations caused frameshift and premature protein truncation, which are predicted to eliminate NEMO function and cause cell lethality. Examination of families transmitting the recurrent deletion revealed that the rearrangement occurred in the paternal germline in most cases, indicating that it arises predominantly by intrachromosomal misalignment during meiosis. Expression analysis of human and mouse NEMO/Nemo showed that the gene becomes active early during embryogenesis and is expressed ubiquitously. These data confirm the involvement of NEMO in IP and will help elucidate the mechanism underlying the manifestation of this disorder and the in vivo function of NEMO. Based on these and other recent findings, we propose a model to explain the pathogenesis of this complex disorder. Familial incontinentia pigmenti (IP; BlochSulzberger syndrome; MIM 308300) is a rare genodermatosis (14) that occurs in approximately 1 of 50 000 newborns. The most conspicuous sign of IP is a progressive skin pigmentation abnormality which begins with vesicular lesions containing apoptotic cells and infiltration of eosinophils. These lesions eventually heal and lead to hyperpigmentation due to incontinence of melanin from the superficial epidermis into the dermis. The clearance of melanin by macrophages finally leaves patients with linear or reticular hypopigmented patches along lines of X-inactivation. Although the skin phenotype can be quite dramatic, the most significant medical problems in IP are blindness due to retinal detachment and central nervous system defects, which cause mental retardation or seizures (1,3). A few minor signs include hair loss, conical or absent teeth and nail dystrophy. IP demonstrates complete penetrance, but its phenotypic expression is highly variable, even among related patients with the same mutation. Affected IP male conceptuses typically fail to survive past the second trimester and thus, this disorder has earned recognition as a classic male-lethal condition. Concordant with this observation, female individuals with IP mutations survive because of dizygosity for the X chromosome and selection against cells expressing the mutant X chromosome. Thus, female IP patients exhibit skewed X-inactivation (5,6), a feature that is often used to confirm diagnosis. We recently demonstrated that cells in IP patients lack NF-B function due to mutations of an upstream activator called NEMO (NF-B essential modulator) (7). In an initial screening of 50 patients, most had an identical deletion (hereafter termed NEMO 410) within the NEMO gene that eliminated exons 410 and consequently abolished protein function. The *To whom correspondence should be addressed. Tel: +1 713 798 4787; Fax: +1 713 798 5386; Email: The authors are members of the International IP Consortium The authors wish it to be known that, in their opinion, Swaroop Aradhya, Hayley Woffendin, Tiziana Bardaro and Asmae Smahi should be regarded as joint First Authors deletion alters sequence after nucleotide 399 (from ATG) in the NEMO mRNA and leads to a truncated protein containing the first 133 N-terminal amino acids. This recurrent rearrangement occurs between two identical, 878 bp MER67B repeats, the first of which is located in intron 3 and the second 4 kb telomeric to the gene. NEMO is a 23 kb gene composed of 10 exons (GenBank accession no. AJ271718) (7). The 48 kDa NEMO protein has two coiled-coil motifs and a leucine zipper which are required for dimerization and proteinprotein interactions, and a zinc finger at the C-terminus that appears to be necessary for posttranslational stability (810). It has also been shown that the C-terminus of NEMO is indispensable for function. NEMO is the regulatory component of IB kinase (IKK), a central activator of the NF-B transcriptional signaling pathway (11,12). In response to various cytokines, IKK phosphorylates the inhibitory IB molecules, which sequester NF-B in the cytoplasm. The removal of IB allows NF-B to translocate into the nucleus and activate transcription of various genes. Through this mechanism, NF-B regulates immune and inflammatory responses, and prevents apoptosis in response to TNF-. Therefore, NEMO mutations eliminate NF-B activity and cause potentially widespread disruption of downstream cellular responses, although the exact downstream effects are only now being elucidated. With respect to IP, loss-of-function mutations in NEMO create a susceptibility to cellular apoptosis in response to TNF- (7). This phenomenon explains the male lethality and skewing of X-inactivation in female patients. Our goal was to determine whether NEMO mutations could explain all cases of IP and to assess the mutation spectrum and the genotypephenotype correlations in IP patients. The analysis was performed in a cohort of 357 unrelated IP patients. We also examined the expression pattern of NEMO in an effort to relate its presence to the pathogenesis of IP. Based on this work and other recent findings, we propose a model to explain the basis for the complicated IP phenotype. A recurrent mutation in NEMO Analysis of the coding sections of NEMO (exons 210) in 357 unrelated patients revealed 277 mutations (Fig. 1 and Tables 1 and 2). Mutations in 50 of these patients have been reported previously (7). Among the 277 patients with mutations, 248 (90%) demonstrated the recurrent IP deletion (NEMO 410), which was detectable (...truncated)