POSTER NO: 402

The genetic IP defects: molecular analysis of NEMO gene and NF-kB related genes

Tiziana Bardaro, Geppino Falco, Angela Sparago, Vincenzo Mercadante, Matilde Valeria Ursini, Michele D'Urso
International Institute of Genetics and Biophysics, CNR, Via Marconi, 12 -80125- Naples Italy

In collaboration with International IP Consortium (IPIF) we recently demonstrated that 78% of Incontinentia Pigmenti (IP; MIM 308310) patients show an identical deletion within the NEMO/IKKg (NF-kB Essential MOdulator/IKKgamma) gene that eliminated exons 4 to 10 and consequently abolished protein function, required for the activation of the transcription factor NF-kB. This recurrent rearrangement occurs between two identical, 878-bp MER67B repeats: the first copy is located in intron 3 and the second is approximately 4kb telomeric to the gene. To determine the spectrum of mutations in IP families, we sequenced the complete NEMO locus (AJ271718). The 23 kb gene is composed of 10 exons with three alternative non-coding first exons (1a, 1b, 1c). The NEMO gene partially overlaps the G6PD gene and is transcribed in the opposite direction. Screening of RPCI11 BAC library with the NEMO cDNA we revealed that an incomplete copy of NEMO was present in the genome. deltaNEMO (AL596249) maps 75kb distal to NEMO and lacks exons 1 and 2. Mapping and sequence information bringing the total length of the duplication to 35.5kb and indicated that the duplicated regions are in opposite orientation and only 22 single nucleotide differences are present, making the duplications >99% identical. The high frequency of the rearrangement affecting NEMO locus allows for the molecular diagnosis of IP in the vast majority of cases by a simple PCR assay in the pre and post-natal studies. The complete loss of NF-kB activation is lethal for IP males during embryogenesis while IP females can survive, owing to mosaicism as a result of X-inactivation. However, males showing an IP female-like phenotype are rarely identified and they can be explained by a chromosomal abnormality (47, XXY), a genomic X-chromosomal mosaicism resulting from an early post-zygotic mutation or a gametic half-chromatid mutation. Hypomorphic mutations affecting NEMO gene impair but do not abolish NF-kB signaling and lead also to surviving males showing features of ostopetrosis, lymphoedema, EDA, and immunodeficiency (OL-ED-ID). In our study we performed the molecular genetic studies in 99 IP collected patients: 87 are females and 12 are male. There were 76 Italian, 20 Spanish, 1 Polish, 1 Turkish and 1 Indian patients. Analysing genomic DNA from the proband we revealed that the D4-10 deletion accounts for 56 cases including one 47,XXY and three males patients with X-chromosomal mosaicism. Although one of the three male shows a late post-zygotic mutation he has severe neurological involvement asociated with ocular abnormalities. Interestingly, a preliminary biochemical study revealed an impaired cellular response of proinflammatory cytokine in response to LPS cytokines related to immune modulation and apoptosis. Mutational analysis by DHPLC revealed 5 new small mutations and do not exhibit mutations in NEMO gene in 22 females and 2 male patients, although a typical IP phenotype has been ascertained on clinical presentation. The remaining 14, 6 of them are male with no family history of IP and with normal karyotype, show OL-ED-ID clinical features. Since multiple pathways impinge on the NF-kB transcription system, it is conceivable that combinations of mutations in the upstream and downstream genes could cause a phenotype similar to that produced by specific defects in NEMO. For this purpose, we are identifying and characterizing regulatory regions of the NEMO promoters and search for mutations in IP and OL-ED-ID patients, which still lack a molecular diagnosis. In the meantime, we are searching for other genes through the comparison of gene expression profiles in IP, ED-ID and EDA mice in order to determine the relative regulatory targets and pathological mechanisms. Those genes will be considered and processed as candidate for both IP and other EDs with immunodeficiency. These observations indicate that male individuals can suffer from IP and they have implications for clinical care, genetic counseling and prenatal diagnosis.