Incontinentia Pigmenti (IP) is an X-linked genodermatosis that is lethal for males and present in females with abnormal skin pigmentation and high variable clinical signs, including retinal detachment, anodontia, alopecia, nail dystrophy and nervous system defects. The NF-κB essential modulator (NEMO) gene, responsible for IP, encodes the regulatory subunit of the IκB kinase (IKK) complex required for nuclear factor κB (NF-κB) activation. We analyzed the NEMO gene in 122 IP patients and identified mutations in 83 (36 familiar and 47 sporadic cases). The recurrent NEMO exon 4–10 deletion that is the major cause of the disease was present in 73 females (59.8%). In addition 10 point alterations (8.2% of females) were identified: three frameshift, three nonsense, three missense and one in-frame deletion of a single amino acid. We measured the effects of these NEMO point-mutations on NF-κB signaling in nemo(−/−) deficient murine pre-B cells. A mutation in the N-terminal domain, required for IKK assembly, reduced but did not abolish NF-κB activation following lipopolysaccharide stimulation. Mutations that disrupt the C-terminal domain, required for the recruitment of upstream factors, showed lower or no NF-κB activation. A phenotype score based on clinical features of our IP patients was applied for summarizing disease severity. The score did not correlate with mutation type or domain affected indicating that other factors influence the severity of IP. Such a factor is likely to be X-inactivation. Indeed, 64% of our patients have extremely skewed X-inactivation pattern (≥80 : 20). Overall IP pathogenesis thus depends on a combination of X-inactivation and protein domain that recruit upstream factors and activate NF-κB.

Mutations in the inhibitor of kappa light polypeptide gene enhancer in B-cells, kinase gamma (IKBKG), also called nuclear factor-kappaB (NF-kB) essential modulator (NEMO), gene are the most common single cause of incontinentia pigmenti (IP) in females and anhydrotic ectodermal dysplasia with immunodeficiency (EDA-ID) in males. The IKBKG gene, located in the Xq28 chromosomal region, encodes for the regulatory subunit of the inhibitor of kappaB (IkB) kinase (IKK) complex required for the activation of the NF-kB pathway. Therefore, the remarkably heterogeneous and often severe clinical presentation reported in IP is due to the pleiotropic role of this signaling transcription pathway. A recurrent exon 4_10 genomic rearrangement in the IKBKG gene accounts for 60 to 80% of IP-causing mutations. Besides the IKBKG rearrangement found in IP females (which is lethal in males), a total of 69 different small mutations (missense, frameshift, nonsense, and splice-site mutations) have been reported, including 13 novel ones in this work. The updated distribution of all the IP- and EDA-ID-causing mutations along the IKBKG gene highlights a secondary hotspot mutation in exon 10, which contains only 11% of the protein. Furthermore, familial inheritance analysis revealed an unexpectedly high incidence of sporadic cases (>65%). The sum of the observations can aid both in determining the molecular basis of IP and EDA-ID allelic diseases, and in genetic counseling in affected families. Hum Mutat 29(5), 595–604, 2008.

We report here on the building-up of a database of information related to 386 cases of Incontinentia Pigmenti collected in a thirteen-year activity (2000–2013) at our centre of expertise. The database has been constructed on the basis of a continuous collection of patients (27.6/year), the majority diagnosed as sporadic cases (75.6%). This activity has generated a rich source of information for future research studies by integrating molecular/clinical data with scientific knowledge. We describe the content, architecture and future utility of this collection of data on IP to offer comprehensive anonymous information to the international scientific community.

Incontinentia pigmenti (IP) is an X‐linked‐dominant Mendelian disorder caused by mutation in the IKBKG/NEMO gene, encoding for NEMO/IKKgamma, a regulatory protein of nuclear factor kappaB (NF‐kB) signaling. In more than 80% of cases, IP is due to recurrent or nonrecurrent deletions causing loss‐of‐function (LoF) of NEMO/IKKgamma. We review how the local architecture of the IKBKG/NEMO locus with segmental duplication and a high frequency of repetitive elements favor de novo aberrant recombination through different mechanisms producing genomic microdeletion. We report here a new microindel (c.436_471delinsT, p.Val146X) arising through a DNA‐replication‐repair fork‐stalling‐and‐template‐switching and microhomology‐mediated‐end‐joining mechanism in a sporadic IP case. The LoF mutations of IKBKG/NEMO leading to IP include small insertions/deletions (indel) causing frameshift and premature stop codons, which account for 10% of cases. We here present 21 point mutations previously unreported, which further extend the spectrum of pathologic variants: 14/21 predict LoF because of premature stop codon (6/14) or frameshift (8/14), whereas 7/21 predict a partial loss of NEMO/IKKgamma activity (two splicing and five missense). We review how the analysis of IP‐associated IKBKG/NEMO hypomorphic mutants has contributed to the understanding of the pathophysiological mechanism of IP disease and has provided important information on affected NF‐kB signaling. We built a locus‐specific database listing all IKBKG/NEMO variants, accessible at http://IKBKG.lovd.nl.

IKBKG/NEMO gene mutations cause an X-linked, dominant neuroectodermal disorder named Incontinentia Pigmenti (IP). Located at Xq28, IKBKG/NEMO has a unique genomic organization, as it is part of a segmental duplication or low copy repeat (LCR1–LCR2, >99% identical) containing the gene and its pseudogene copy (IKBKGP). In the opposite direction and outside LCR1, IKBKG/NEMO partially overlaps G6PD, whose mutations cause a common X-linked human enzymopathy. The two LCRs in the IKBKG/NEMO locus are able to recombine through non-allelic homologous recombination producing either a pathological recurrent exon 4–10 IKBKG/NEMO deletion (IKBKGdel) or benign small copy number variations. We here report that the local high frequency of micro/macro-homologies, tandem repeats and repeat/repetitive sequences make the IKBKG/NEMO locus susceptible to novel pathological IP alterations. Indeed, we describe the first two independent instances of inter-locus gene conversion, occurring between the two LCRs, that copies the IKBKGP pseudogene variants into the functional IKBKG/NEMO, causing the de novo occurrence of p.Glu390ArgfsX61 and the IKBKGdel mutations, respectively. Subsequently, by investigating a group of 20 molecularly unsolved IP subjects using a high-density quantitative polymerase chain reaction assay, we have identified seven unique de novo deletions varying from 4.8 to ∼115 kb in length. Each deletion removes partially or completely both IKBKG/NEMO and the overlapping G6PD, thereby uncovering the first deletions disrupting the G6PD gene which were found in patients with IP. Interestingly, the 4.8 kb deletion removes the conserved bidirectional promoterB, shared by the two overlapping IKBKG/NEMO and G6PD genes, leaving intact the alternative IKBKG/NEMO unidirectional promoterA. This promoter, although active in the keratinocytes of the basal dermal layer, is down-regulated during late differentiation. Genomic analysis at the breakpoint sites indicated that other mutational forces, such as non-homologous end joining, Alu-Alu-mediated recombination and replication-based events, might enhance the vulnerability of the IP locus to produce de novo pathological IP alleles.

The Arp2/3 complex is essential for actin filament nucleation in a variety of cellular processes. The activation of the Arp2/3 complex is mediated by nucleation-promoting factors, such as the Wiskott-Aldrich syndrome family proteins, which share a WCA (WH2 domain, central region, acidic region) catalytic module at the C-terminal region, required for Arp2/3 activation, but diverge at the N-terminal region, required for binding to specific activators. Here, we report the characterization of WASH, a new member of the WAS family that has nucleation-promoting factor activity and recently has been demonstrated to play a role in endosomal sorting. We found that overexpression of the WASH-WCA domain induced disruption of the actin cytoskeleton, whereas overexpression of full-length WASH in mammalian cells did not affect stress fiber organization. Furthermore, our analysis has revealed that nerve growth factor treatment of PC12 cells overexpressing full-length WASH leads to disruption of the actin cytoskeleton. We have also found that WASH interacts through its N-terminal region with BLOS2, a centrosomal protein belonging to the BLOC-1 complex that functions as a scaffolding factor in the biogenesis of lysosome-related organelles. In addition to BLOS2, WASH also interacts with centrosomal gamma-tubulin and with pallidin, an additional component of the BLOC-1 complex. Collectively, our data propose that WASH is a bimodular protein in which the C terminus is involved in Arp2/3-mediated actin nucleation, whereas the N-terminal portion is required for its regulation and localization in the cells. Moreover, our data suggest that WASH is also a component of the BLOC-1 complex that is associated with the centrosomes.