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Atypical Forms of Incontinentia Pigmenti in Male Individuals Result from Mutations of a Cytosine Tract in Exon 10 of NEMO (IKK-γ)

2001; Elsevier BV; Volume: 68; Issue: 3 Linguagem: Inglês

10.1086/318806

1537-6605

Swaroop Aradhya, Gilles Courtois, Aleks Rajkovic, Richard A. Lewis, Moise L. Levy, Alain Israël, David L. Nelson,

Cancer and Skin Lesions

Familial incontinentia pigmenti (IP [MIM 308310]), or Bloch-Sulzberger syndrome, is an X-linked dominant and male-lethal disorder. We recently demonstrated that mutations in NEMO (IKK-γ), which encodes a critical component of the NF-κB signaling pathway, were responsible for IP. Virtually all mutations eliminate the production of NEMO, causing the typical skewing of X inactivation in female individuals and lethality in male individuals, possibly through enhanced sensitivity to apoptosis. Most mutations also give rise to classic signs of IP, but, in this report, we describe two mutations in families with atypical phenotypes. Remarkably, each family included a male individual with unusual signs, including postnatal survival and either immune dysfunction or hematopoietic disturbance. We found two duplication mutations in these families, at a cytosine tract in exon 10 of NEMO, both of which remove the zinc (Zn) finger at the C-terminus of the protein. Two deletion mutations were also identified in the same tract in additional families. However, only the duplication mutations allowed male individuals to survive, and affected female individuals with duplication mutations demonstrated random or slight skewing of X inactivation. Similarly, NF-κB activation was diminished in the presence of duplication mutations and was completely absent in cells with deletion mutations. These results strongly indicate that male individuals can also suffer from IP caused by NEMO mutations, and we therefore urge a reevaluation of the diagnostic criteria. Familial incontinentia pigmenti (IP [MIM 308310]), or Bloch-Sulzberger syndrome, is an X-linked dominant and male-lethal disorder. We recently demonstrated that mutations in NEMO (IKK-γ), which encodes a critical component of the NF-κB signaling pathway, were responsible for IP. Virtually all mutations eliminate the production of NEMO, causing the typical skewing of X inactivation in female individuals and lethality in male individuals, possibly through enhanced sensitivity to apoptosis. Most mutations also give rise to classic signs of IP, but, in this report, we describe two mutations in families with atypical phenotypes. Remarkably, each family included a male individual with unusual signs, including postnatal survival and either immune dysfunction or hematopoietic disturbance. We found two duplication mutations in these families, at a cytosine tract in exon 10 of NEMO, both of which remove the zinc (Zn) finger at the C-terminus of the protein. Two deletion mutations were also identified in the same tract in additional families. However, only the duplication mutations allowed male individuals to survive, and affected female individuals with duplication mutations demonstrated random or slight skewing of X inactivation. Similarly, NF-κB activation was diminished in the presence of duplication mutations and was completely absent in cells with deletion mutations. These results strongly indicate that male individuals can also suffer from IP caused by NEMO mutations, and we therefore urge a reevaluation of the diagnostic criteria. Familial incontinentia pigmenti (IP [MIM 308310]), or Bloch-Sulzberger syndrome, is a rare genodermatosis that segregates as an X-linked dominant trait with prenatal male lethality (Landy and Donnai Landy and Donnai, 1993Landy SJ Donnai D Incontinentia pigmenti (Bloch-Sulzberger syndrome).J Med Genet. 1993; 30: 53-59Crossref PubMed Scopus (303) Google Scholar). The hallmark sign of IP is the presence of skin lesions in affected female newborns. These lesions progress through various stages and result in hypopigmented patches along lines of X inactivation (Spitz Spitz, 1996Spitz JL Genodermatoses: a full color clinical guide to genetic skin disorders. Williams & Wilkins, New York1996: 66-67Google Scholar, pp. 66–67). The name of the disorder derives from the observation of incontinence of melanin from the epidermis into the dermis. The most significant medical problem in IP is blindness due to hypervascularization and consequent retinal detachment. Other manifestations include alopecia, hypodontia or anodontia, eosinophilia, nail dystrophy, and neurological complications. These medical problems, together with the skin-pigmentation abnormality, constitute the typical IP phenotype. The IP gene is thought to be essential for viability, since affected hemizygous male individuals die in utero and female patients survive with the mutant X chromosome selectively inactivated (Parrish et al. Parrish et al., 1996Parrish JE Scheuerle AE Lewis RA Levy ML Nelson DL Selection against mutant alleles in blood leukocytes is a consistent feature in Incontinentia Pigmenti type 2.Hum Mol Genet. 1996; 5: 1777-1783Crossref PubMed Scopus (86) Google Scholar; Woffendin et al. Woffendin et al., 1999Woffendin H Jakins T Jouet M Stewart H Landy S Haan E Harris A Donnai D Read A Kenwrick S X-inactivation and marker studies in three families with incontinentia pigmenti: implications for counseling and gene localisation.Clin Genet. 1999; 55: 55-60Crossref PubMed Scopus (15) Google Scholar). On the basis of these characteristics, we screened a gene called “NEMO” and identified mutations in multiple IP patients (International IP Consortium International IP Consortium, 2000International IP Consortium Genomic rearrangement in NEMO impairs NF-kappaB activation and is a cause of incontinentia pigmenti.Nature. 2000; 405: 466-472Crossref PubMed Scopus (559) Google Scholar). NEMO (IKK-γ) encodes a regulatory subunit of the IKK complex, which is central to activation of the NF-κB pathway (DiDonato et al. DiDonato et al., 1997DiDonato JA Hayakawa M Rothwarf DM Zandi E Karin M A cytokine-responsive IkappaB kinase that activates the transcription factor NF-kappaB.Nature. 1997; 388: 548-554Crossref PubMed Scopus (1857) Google Scholar; Israel Israël, 2000Israël A The IKK complex: an integrator of all signals that activate NF-kappaB?.Trends Cell Biol. 2000; 10: 129-133Abstract Full Text Full Text PDF PubMed Scopus (347) Google Scholar). NF-κB is sequestered in the cytoplasm by IκB inhibitory proteins, but, on stimulation by cytokines, IKK phosphorylates IκB and targets it for ubiquitin-mediated degradation. Release from IκB allows NF-κB to translocate into the nucleus and activate transcription of target genes. Knockout models for NF-κB subunits or components of the signaling cascade have shown that NF-κB is responsible for preventing apoptosis, particularly in response to TNF-α (Barkett and Gilmore Barkett and Gilmore, 1999Barkett M Gilmore TD Control of apoptosis by Rel/NF-kappaB transcription factors.Oncogene. 1999; 18: 6910-6924Crossref PubMed Scopus (1043) Google Scholar; Gerondakis et al. Gerondakis et al., 1999Gerondakis S Grossmann M Nakamura Y Pohl T Grumont R Genetic approaches in mice to understand Rel/NF-kappaB and IkappaB function: transgenics and knockouts.Oncogene. 1999; 18: 6888-6895Crossref PubMed Scopus (262) Google Scholar). Hence, loss of NEMO in cells of IP patients most likely results in apoptosis, which can explain both the male lethality and the skewing of X inactivation in female individuals. The C-terminus of NEMO is indispensable to activate NF-κB downstream, although the exact residues necessary for this function have yet to be determined (Rothwarf et al. Rothwarf et al., 1998Rothwarf DM Zandi E Natoli G Karin M IKK-γ is an essential regulatory subunit of the IkappaB kinase complex.Nature. 1998; 395: 297-300Crossref PubMed Scopus (815) Google Scholar; Yamaoka et al. Yamaoka et al., 1998Yamaoka S Courtois G Bessia C Whiteside ST Weil R Agou F Kirk HE Kay RJ Israel A Complementation cloning of NEMO, a component of the IkappaB kinase complex essential for NF-kappaB activation.Cell. 1998; 93: 1231-1240Abstract Full Text Full Text PDF PubMed Scopus (927) Google Scholar). Most cases (∼85%) of IP are due to a recurrent deletion within NEMO that removes exons 4–10 (International IP Consortium International IP Consortium, 2000International IP Consortium Genomic rearrangement in NEMO impairs NF-kappaB activation and is a cause of incontinentia pigmenti.Nature. 2000; 405: 466-472Crossref PubMed Scopus (559) Google Scholar). All reported mutations, including the rearrangement type, cause typical IP. However, while its penetrance approaches 100%, this disorder shows variable expressivity, even within the same family. To add to this complexity, although IP has traditionally been classified as a male-lethal disorder, a few cases have been reported in male patients (Kirchman et al. Kirchman et al., 1995Kirchman TT Levy ML Lewis RA Kanzler MH Nelson DL Scheuerle AE Gonadal mosaicism for incontinentia pigmenti in a healthy male.J Med Genet. 1995; 32: 887-890Crossref PubMed Scopus (25) Google Scholar; Scheuerle Scheuerle, 1998Scheuerle AE Male cases of incontinentia pigmenti: case report and review.Am J Med Genet. 1998; 77: 201-218Crossref PubMed Scopus (64) Google Scholar). The first NEMO mutation was recently found in a surviving male patient with abnormalities of the skin, teeth, and eyes, but also with osteopetrosis and immune dysfunction (International IP Consortium International IP Consortium, 2000International IP Consortium Genomic rearrangement in NEMO impairs NF-kappaB activation and is a cause of incontinentia pigmenti.Nature. 2000; 405: 466-472Crossref PubMed Scopus (559) Google Scholar). This patient died of tuberculosis at age 2.5 years. We have characterized two additional male cases, to test the hypothesis that male IP patients survive due to milder (hypomorphic) mutations. Analysis of these two families now reveals the genetic basis for the occurrence of IP in male individuals. Family XL320 was described elsewhere (Roberts et al. Roberts et al., 1998Roberts JL Morrow B Vega-Rich C Salafia CM Nitowsky HM Incontinentia pigmenti in a newborn male infant with DNA confirmation.Am J Med Genet. 1998; 75: 159-163Crossref PubMed Scopus (22) Google Scholar), but the diagnosis of IP was complicated by substantial phenotypic variation among the affected female family members. A male infant (XL320-04), who had been carried to term, died from severe hemorrhage 24 h after birth (table 1). Prenatal testing by linkage analysis, using nearby informative markers, in a subsequent pregnancy, predicted another affected male fetus (XL320-09). This pregnancy was terminated, and the mother (XL320-01) experienced severe bleeding during the procedure, further suggesting that the mutation in this family disrupted hemostasis. A second family, XL344, also included female members with typical signs of IP (table 1). An affected male individual (XL344-04) exhibited skin pigmentation and dental problems but also demonstrated immune dysfunction. He suffered multiple episodes of infection, including meningitis and pneumonia, due to poor lymphocyte function and remarkably low levels of circulating IgG. He also exhibited heat intolerance with hyperthermia, anhidrosis, eczema, and fine sparse hair, which led to a diagnosis of ectodermal dysplasia (ED) (X-linked anhidrotic ectodermal dysplasia [MIM 305100], autosomal recessive hypohidrotic ectodermal dysplasia [MIM 224900], and autosomal dominant hidrotic ectodermal dysplasia [MIM 129500]). Now, at age 3 years, he receives routine supplements of IgG, to prevent recurrent infections. He is currently losing weight, complains of abdominal pains, and tests positive for hepatosplenomegaly. He has also contracted mycobacterium avium intracellulare, an infection common among patients with AIDS.Table 1Exon-10 Cytosine Tract Mutations, Corresponding Phenotypes, X-Inactivation Status, and NEMO ActivityFamilyPhenotype in Female SubjectsPhenotype in Male SubjectsMutationPredicted Amino AcidsX-Inactivation StatusAverage NEMO Activity Relative to Wild TypeaBy genetic complementation assay in a NEMO-mutant cell line. All values relative to wild type (100%).XL213TypicalbTypical of IP—skin pigmentation, conical teeth, retinal detachment, alopecia, CNS problems. Not all signs demonstrated in families.1 spontaneous abortionΔC1161P389fsX60c60 novel amino acids: RSHLTSAVPSASIRPLIWTPCRYMSWSALSRAGQCKATACRGRARDRAVCAFLSRLPSPGX.Skewed10% = backgroundXL267TypicalNo male subjects in pedigreedupC1161P389fsX4Random34%XL287Typical1 spontaneous abortionΔC1161P389fsX60c60 novel amino acids: RSHLTSAVPSASIRPLIWTPCRYMSWSALSRAGQCKATACRGRARDRAVCAFLSRLPSPGX.Skewed10% = backgroundXL320TypicaldFemale family members show variation in expression of phenotype.Death after 1 d, from hemorrhagingdup1166-78P393fsX4Slight skewing19%XL344TypicalImmune dysfunction; skinbTypical of IP—skin pigmentation, conical teeth, retinal detachment, alopecia, CNS problems. Not all signs demonstrated in families.; teethbTypical of IP—skin pigmentation, conical teeth, retinal detachment, alopecia, CNS problems. Not all signs demonstrated in families.dupC1161P389fsX4Random34%XL345TypicalNo male subjects in pedigreeΔC1161P389fsX60c60 novel amino acids: RSHLTSAVPSASIRPLIWTPCRYMSWSALSRAGQCKATACRGRARDRAVCAFLSRLPSPGX.Skewed10% = backgroundXL374Typical3 spontaneous abortionsΔ1163-75S387fsX58e58 novel amino acids: HLTSAVPSASIRPLIWTPCRYMSWSALSRAGQCKATACRGRARDRAVCAFLSRLPSPGX.SkewedNot donea By genetic complementation assay in a NEMO-mutant cell line. All values relative to wild type (100%).b Typical of IP—skin pigmentation, conical teeth, retinal detachment, alopecia, CNS problems. Not all signs demonstrated in families.c 60 novel amino acids: RSHLTSAVPSASIRPLIWTPCRYMSWSALSRAGQCKATACRGRARDRAVCAFLSRLPSPGX.d Female family members show variation in expression of phenotype.e 58 novel amino acids: HLTSAVPSASIRPLIWTPCRYMSWSALSRAGQCKATACRGRARDRAVCAFLSRLPSPGX. Open table in a new tab DNA samples from these two families (XL320 and XL344) were examined for all coding exons of NEMO by conformation-sensitive gel electrophoresis (CSGE) (Ganguly et al. Ganguly et al., 1993Ganguly A Rock MJ Prockop DJ Conformation-sensitive gel electrophoresis for rapid detection of single-base differences in double-stranded PCR products and DNA fragments: evidence for solvent-induced bends in DNA heteroduplexes.Proc Natl Acad Sci USA. 1993; 90: 10325-10329Crossref PubMed Scopus (603) Google Scholar). PCR products from exon 10 demonstrated altered band sizes in both families (fig. 1a). Sequencing of exon-10 PCR products from XL320-01 and XL320-04 revealed a 13-base duplication (dup1166-78) (fig. 1b and 1c) at the end of a cytosine tract (the “C7 tract”). This duplication would cause frameshift after amino acid P393 and protein truncation after addition of four novel amino acids (fig. 1d and table 1). Sequence analysis of all NEMO exons in affected male subject XL320-04, including the noncoding exon 1, failed to identify other base changes. Exon-10 PCR products from family XL344 (fig. 1a) contained a duplication of one cytosine (dupC1161), also within the C7 tract (fig. 1b). A frameshift after amino acid P389 causes addition of the same four novel amino acids as are found in the XL320 mutant protein (fig. 1c). Despite the addition of identical novel amino acids, the phenotypes of the male subjects in these two families were entirely different (table 1). One additional family (XL267) demonstrated the same base duplication, dupC1161, as in family XL344, but no male offspring have been reported in this pedigree (table 1). The shorter protein sizes predicted from these mutations were demonstrated by in vitro translation experiments (fig. 2b).Figure 2Analysis of NEMO activity by means of a genetic complementation assay in a NEMO-minus cell line. A, Expression analyses with various NEMO cDNA constructs were performed in six trials; the average luciferase-expression values are shown. Specific mutations introduced into the NEMO cDNA are indicated below the chart. The ΔC1161 deletion shows complete absence of complementation activity (same as background). In contrast, the duplication mutations, dupC1161 and dup1166-78, show diminished and residual activities. B, In vitro translation experiment confirms both the expected smaller protein sizes due to the duplication mutations and a larger protein size with the ΔC1161 mutation.View Large Image Figure ViewerDownload Hi-res image Download (PPT) Given the nature of the mutations in families XL320 and XL344, we expected that reciprocal mutations—namely, a 13-base deletion and a single-cytosine deletion—might also be found. A 13-base deletion (Δ1163-75) at the C7 tract was identified in family XL374 (fig. 1a–c). However, this mutation was not the precise reciprocal of the XL320 mutation but was shifted 5' by two bases. This mutation predicts a frameshift after amino acid S387 and protein truncation after addition of 58 novel amino acids to the C-terminus of the protein (fig. 1d and table 1). Three additional families exhibited a deletion of a single cytosine (ΔC1161) within the C7 tract (fig. 1a–c and table 1), which is also expected to cause a frameshift at amino acid P389 and protein truncation after addition of 60 novel amino acids (fig. 1d and table 1). This larger protein size, caused by ΔC1161, was demonstrated by in vitro translation experiments (fig. 2b). Notably, after the first two residues, both deletion mutations add the same novel amino acid sequence to the truncated protein. We analyzed all available family members for the duplication and deletion mutations, and altered band sizes were detected exclusively in affected members (fig. 1a). Moreover, CSGE analysis of 50 normal female subjects (100 X chromosomes) failed to reveal variations, confirming that the base alterations we found were not common polymorphisms (data not shown). During preparation of this report, another group reported that the mutation in family XL320 was a deletion that removes the first exon (Makris et al. Makris et al., 2000Makris C Godfrey VL Krahn-Senftleben G Takahashi T Roberts JL Schwarz T Feng L Johnson RS Karin M Female mice heterozygous for IKKγ/NEMO deficiencies develop a dermatopathy similar to the human X-linked disorder incontinentia pigmenti.Mol Cell. 2000; 5: 969-979Abstract Full Text Full Text PDF PubMed Scopus (324) Google Scholar). We have analyzed the entire NEMO gene in XL320 and have found a mutation in exon 10, with complete preservation of exon 1, as described above. Southern blot analysis also failed to identify aberrant bands indicative of potential genomic rearrangements within NEMO. We hypothesized that some male individuals survive because their mutations are “milder” (hypomorphic) and not cell lethal. Thus, we expected female individuals with the same mutations to show random or only minimal skewing of X inactivation, rather than the complete skewing observed in female patients with classic IP (Parrish et al. Parrish et al., 1996Parrish JE Scheuerle AE Lewis RA Levy ML Nelson DL Selection against mutant alleles in blood leukocytes is a consistent feature in Incontinentia Pigmenti type 2.Hum Mol Genet. 1996; 5: 1777-1783Crossref PubMed Scopus (86) Google Scholar). We examined the X-inactivation status in families with the duplication mutations by means of a method established elsewhere, based on digesting genomic DNA with a methylation-sensitive enzyme, HpaII, and amplifying a polymorphism at the HUMARA locus (Allen et al. Allen et al., 1992Allen RC Zoghbi HY Moseley AB Rosenblatt HM Belmont JW Methylation of Hpall and Hhal sites near the polymorphic CAG repeat in the human androgen-receptor gene correlates with X chromosome inactivation.Am J Hum Genet. 1992; 51: 1229-1239PubMed Google Scholar; Parrish et al. Parrish et al., 1996Parrish JE Scheuerle AE Lewis RA Levy ML Nelson DL Selection against mutant alleles in blood leukocytes is a consistent feature in Incontinentia Pigmenti type 2.Hum Mol Genet. 1996; 5: 1777-1783Crossref PubMed Scopus (86) Google Scholar). In support of our hypothesis, blood leukocytes from female subjects carrying either the dup1166-78 or dupC1161 mutations showed slight skewing or random X inactivation, respectively (fig. 3b and 3c). Hence, these NEMO mutations were apparently not deleterious enough to cause lethality in cells with an active mutant X chromosome and are consistent with the survival to term of affected male individuals in these families. In contrast with family XL344, the slight skewing in family XL320 could account for the more-severe phenotype in the XL320-04 male patient. We also examined the X-inactivation status in families with the deletion mutations (Δ1163–75 and Δ1161) and showed that they result in complete skewing of X inactivation (fig. 3a and 3d). Hence, the deletion mutations appear to be cell lethal, similar to other IP mutations (International IP Consortium International IP Consortium, 2000International IP Consortium Genomic rearrangement in NEMO impairs NF-kappaB activation and is a cause of incontinentia pigmenti.Nature. 2000; 405: 466-472Crossref PubMed Scopus (559) Google Scholar). Moreover, all families with deletion mutations, except XL345, have reported spontaneous abortions, presumably caused by in utero demise of affected male fetuses (table 1). NEMO is central to activation of the NF-κB pathway, and its absence completely abolishes this signaling pathway (Yamaoka et al. Yamaoka et al., 1998Yamaoka S Courtois G Bessia C Whiteside ST Weil R Agou F Kirk HE Kay RJ Israel A Complementation cloning of NEMO, a component of the IkappaB kinase complex essential for NF-kappaB activation.Cell. 1998; 93: 1231-1240Abstract Full Text Full Text PDF PubMed Scopus (927) Google Scholar; International IP Consortium International IP Consortium, 2000International IP Consortium Genomic rearrangement in NEMO impairs NF-kappaB activation and is a cause of incontinentia pigmenti.Nature. 2000; 405: 466-472Crossref PubMed Scopus (559) Google Scholar; Makris et al. Makris et al., 2000Makris C Godfrey VL Krahn-Senftleben G Takahashi T Roberts JL Schwarz T Feng L Johnson RS Karin M Female mice heterozygous for IKKγ/NEMO deficiencies develop a dermatopathy similar to the human X-linked disorder incontinentia pigmenti.Mol Cell. 2000; 5: 969-979Abstract Full Text Full Text PDF PubMed Scopus (324) Google Scholar; Schmidt-Supprian et al. Schmidt-Supprian et al., 2000Schmidt-Supprian M Bloch W Courtois G Addicks K Israel A Rajewsky K Pasparakis M NEMO/IKK gamma-deficient mice model incontinentia pigmenti.Mol Cell. 2000; 5: 981-992Abstract Full Text Full Text PDF PubMed Scopus (355) Google Scholar). Therefore, concordant with X inactivation, we expected hypomorphic mutations to cause reduction, rather than elimination, of NF-κB activation. The dupC1161, dup1166-78, and ΔC1161 mutations were engineered into an expression vector and, to evaluate their genetic complementation ability by a method described elsewhere, were transiently transfected into NEMO-minus cells (Courtois et al. Courtois et al., 1997Courtois G Whiteside ST Sibley CH Israel A Characterization of a mutant cell line that does not activate NF-kappaB in response to multiple stimuli.Mol Cell Biol. 1997; 17: 1441-1449Crossref PubMed Google Scholar; Yamaoka et al. Yamaoka et al., 1998Yamaoka S Courtois G Bessia C Whiteside ST Weil R Agou F Kirk HE Kay RJ Israel A Complementation cloning of NEMO, a component of the IkappaB kinase complex essential for NF-kappaB activation.Cell. 1998; 93: 1231-1240Abstract Full Text Full Text PDF PubMed Scopus (927) Google Scholar; International IP Consortium International IP Consortium, 2000International IP Consortium Genomic rearrangement in NEMO impairs NF-kappaB activation and is a cause of incontinentia pigmenti.Nature. 2000; 405: 466-472Crossref PubMed Scopus (559) Google Scholar). The mutant cDNA constructs introduced into these cells produce NEMO proteins that activate NF-κB in varying degrees, depending on the mutations they contain. NF-κB then activates a reporter gene (luciferase) by binding target sites in the promoter, and, hence, luciferase levels reflect NEMO activity and NF-κB activation. In support of the X inactivation and phenotypic data, the deletion mutation completely failed to complement NEMO activity in these cells (fig. 2a). The duplication mutations resulted in residual (19.5%, with XL320 mutation) or reduced (34%, with XL344 mutation) activity relative to wild type (fig. 2a). The greater reduction in XL320 may explain the postnatal lethality of the male subject (XL344-04) carrying this mutation, although the 34% activity in XL344 is apparently sufficient for survival past infancy. These experiments measured activity in vitro, but these mutations may be of a different nature in vivo, with effects on protein structure and stability. Analysis of fibroblast cells from patients would have been useful to validate these results, but the death of the first male subject and difficult access to the second male subject prevented investigations in vivo. Although IP has been considered a male-lethal disease, this report now describes mutations in male patients that demonstrate atypical forms of IP. However, it is imperative to understand that affected female relatives of these male individuals exhibited classic IP signs. Another group recently reported mutations in male individuals diagnosed with ectodermal dysplasia and immune dysfunction (Zonana et al. Zonana et al., 2000Zonana J Elder ME Schneider LC Orlow SJ Moss C Golabi M Shapira SK Farndon PA Wara DW Emmal SA Ferguson BM A novel X-linked disorder of immune deficiency and hypohidrotic ectodermal dysplasia is allelic to incontinentia pigmenti and due to mutations in IKK-gamma.Am J Hum Genet. 2000; 67: 1555-1562Abstract Full Text Full Text PDF PubMed Scopus (403) Google Scholar). Although these families were described as having a novel syndrome in the form of ED and ID, it is likely that they are simply variants of IP, since they present clinical features associated with IP such as abnormal dentition, skin pigmentation abnormalities, and alopecia. The proband from the fourth pedigree (HED-ID 4), in this report, resembled the XL344 family described here. However, although the carrier female in family HED-ID 4 did not suffer from any medical problems, affected female individuals in family XL344 demonstrated typical IP skin-pigmentation and retinal abnormalities, a contrast that could be explained by variation in expression due to differences in X inactivation between the affected female individuals of these two families. Similarly, while the XL320 phenotype appears to be a novel disorder of IP combined with disrupted hemostasis, this should also be considered a variation of IP in male individuals. These variant phenotypes arise because of the full expression of mutations in male indiviuals, whereas the X inactivation in carrier female individuals prevents the complete phenotypic manifestation of the mutations, resulting in typical IP. These data provide a basis for understanding the function of the NEMO protein, especially the C-terminus, which is indispensable for function (Rothwarf et al. Rothwarf et al., 1998Rothwarf DM Zandi E Natoli G Karin M IKK-γ is an essential regulatory subunit of the IkappaB kinase complex.Nature. 1998; 395: 297-300Crossref PubMed Scopus (815) Google Scholar; Yamaoka et al. Yamaoka et al., 1998Yamaoka S Courtois G Bessia C Whiteside ST Weil R Agou F Kirk HE Kay RJ Israel A Complementation cloning of NEMO, a component of the IkappaB kinase complex essential for NF-kappaB activation.Cell. 1998; 93: 1231-1240Abstract Full Text Full Text PDF PubMed Scopus (927) Google Scholar). We have suggested elsewhere that removal of a Zn finger at the C-terminus is lethal, since nearly all mutations initially identified removed this domain (International IP Consortium International IP Consortium, 2000International IP Consortium Genomic rearrangement in NEMO impairs NF-kappaB activation and is a cause of incontinentia pigmenti.Nature. 2000; 405: 466-472Crossref PubMed Scopus (559) Google Scholar). In support of this interpretation, we have elsewhere described a male patient with IP with a stop codon mutation that resulted in the addition of 21 novel amino acids to the C-terminus, but this mutation preserved the Zn finger and allowed this patient to survive beyond birth (International IP Consortium International IP Consortium, 2000International IP Consortium Genomic rearrangement in NEMO impairs NF-kappaB activation and is a cause of incontinentia pigmenti.Nature. 2000; 405: 466-472Crossref PubMed Scopus (559) Google Scholar). However, although all the mutations reported here lead to removal of the Zn finger, only the deletion mutations were lethal in the embryonic period. Thus, male individuals may survive in spite of mutations that remove the Zn finger, indicating that the region within the C-terminus of NEMO that is indispensable for function must lie beyond the Zn finger domain. The specific role of the Zn finger is not known, and these mutations should be helpful in the determination of its function. With knowledge of NEMO’s role in the NF-κB pathway, how can we explain the unusual phenotypes observed in male subjects from families XL320 and XL344? A few reports have associated IP with immune problems (Dahl et al. Dahl et al., 1975Dahl MV Matula G Leonards R Tuffanelli DL Incontinentia pigmenti and defective neutrophil chemotaxis.Arch Dermatol. 1975; 111: 1603-1605Crossref PubMed Scopus (40) Google Scholar; Jessen et al. Jessen et al., 1978Jessen RT Van Epps DE Goodwin JS Bowerman J Incontinentia pigmenti. Evidence for both neutrophil and lymphocyte dysfunction.Arch Dermatol. 1978; 114: 1182-1186Crossref PubMed Scopus (29) Google Scholar; Ment et al. Ment et al., 1978Ment L Alper J Sirota RL Holmes LB Infant with abnormal pigmentation, malformations, and immune deficiency.Arch Dermatol. 1978; 114: 1043-1044Crossref PubMed Scopus (20) Google Scholar; Person Person, 1985Person JR Incontinentia pigmenti: a failure of immune tolerance?.J Am Acad Dermatol. 1985; 13: 120-124Abstract Full Text PDF PubMed Scopus (15) Google Scholar; Menni et al. Menni et al., 1990Menni S Piccinno R Biolchini A Plebani A Immunologic investigations in eight patients with incontinentia pigmenti.Pediatr Dermatol. 1990; 7: 275-277Crossref PubMed Scopus (26) Google Scholar), and knockout-mouse models for NEMO suggest that immune dysfunction in IP results from uncontrolled apoptosis in the thymus and spleen (Makris et al. Makris et al., 2000Makris C Godfrey VL Krahn-Senftleben G Takahashi T Roberts JL Schwarz T Feng L Johnson RS Karin M Female mice heterozygous for IKKγ/NEMO deficiencies develop a dermatopathy similar to the human X-linked disorder incontinentia pigmenti.Mol Cell. 2000; 5: 969-979Abstract Full Text Full Text PDF PubMed Scopus (324) Google Scholar). The phenotype of the male individual with the stop codon mutation was attributed to the addition of the novel amino acids to the end of the NEMO protein (International IP Consortium International IP Consortium, 2000International IP Consortium Genomic rearrangement in NEMO impairs NF-kappaB activation and is a cause of incontinentia pigmenti.Nature. 2000; 405: 466-472Crossref PubMed Scopus (559) Google Scholar). This may affect the stability of the protein or may mask the nearby leucine zipper domain, which is essential for dimerization. The immune dysfunction in this male subject, as well as in the XL344 male subject, may arise from cellular apoptosis in tissues critical to the immune system, including the thymus and spleen. Similarly, the XL320 mutation may affect hematopoietic or vascular development due to a deleterious effect on tissues involved in these processes. Vascular development is also thought to be the basis for the most significant medical problems in IP—namely, blindness caused by retinal detachment and mental retardation due to central nervous system malformations (Goldberg and Custis Goldberg and Custis, 1993Goldberg MF Custis PH Retinal and other manifestations of incontinentia pigmenti (Bloch-Sulzberger syndrome).Ophthalmology. 1993; 100: 1645-1654Abstract Full Text PDF PubMed Scopus (130) Google Scholar; Francis and Sybert Francis and Sybert, 1997Francis JS Sybert VP Incontinentia pigmenti.Semin Cutan Med Surg. 1997; 16: 54-60Crossref PubMed Scopus (30) Google Scholar). In this regard, a recent report demonstrated that primary congenital lymphedema (MIM 153100) results from mutations in vascular endothelial growth-factor receptor 3 (VEGFR-3), which implements its effects via the NF-κB pathway (Karkkainen et al. Karkkainen et al., 2000Karkkainen MJ Ferrell RE Lawrence EC Kimak MA Levinson KL McTigue MA Alitalo K Finegold DN Missense mutations interfere with VEGFR-3 signalling in primary lymphoedema.Nat Genet. 2000; 25: 153-159Crossref PubMed Scopus (507) Google Scholar). Knockout models for VEGFR-3 demonstrate absence of a primary vascular network and embryonic lethality (Dumont et al. Dumont et al., 1998Dumont DJ Jussila L Taipale J Lymboussaki A Mustonen T Pajusola K Breitman M Alitalo K Cardiovascular failure in mouse embryos deficient in VEGF receptor-3.Science. 1998; 282: 946-949Crossref PubMed Scopus (664) Google Scholar). Therefore, the vascular anomalies in IP may be due to disruption, via NEMO, of the signaling between VEGFR-3 and NF-κB. Since IP has always been considered a male-lethal disease, it is likely that numerous cases of IP in male patients have been misdiagnosed with other human disorders. Our data clearly demonstrate that male individuals can suffer from IP. This observation has implications for clinical care, genetic counseling, and prenatal diagnosis. In the past, linkage analysis was the only method for prenatal diagnosis, but with knowledge of the involvement of NEMO, prenatal DNA samples can now be quickly subjected to molecular analysis. Because of the formerly held belief that affected male individuals undergo spontaneous abortion, some families currently continue a pregnancy when a male fetus has inherited the mutant haplotype. Therefore, if male patients do not have the common rearrangement mutation, it is imperative to rule out exon-10 mutations, because a devastating phenotype is still possible. In this regard, we emphasize that IP male individuals may present additional complications not seen in classic IP, such as immune dysfunction or hemorrhaging. Such variant phenotypes often cause misdiagnosis in any disease, as illustrated by an original diagnosis of ectodermal dysplasia in family XL344. The genes responsible for X-linked and autosomal ectodermal dysplasia have been discovered, and they produce proteins similar to TNF and its receptor, TNFR (Kere et al. Kere et al., 1996Kere J Srivastava AK Montonen O Zonana J Thomas N Ferguson B Munoz F Morgan D Clarke A Baybayan P Chen EY Ezer S Saarialho-Kere U de la Chapelle A Schlessinger D X-linked anhidrotic (hypohidrotic) ectodermal dysplasia is caused by mutation in a novel transmembrane protein.Nat Genet. 1996; 13: 409-416Crossref PubMed Scopus (561) Google Scholar; Monreal et al. Monreal et al., 1999Monreal AW Ferguson BM Headon DJ Street SL Overbeek PA Zonana J Mutations in the human homologue of mouse dl cause autosomal recessive and dominant hypohidrotic ectodermal dysplasia.Nat Genet. 1999; 22: 366-369Crossref PubMed Scopus (315) Google Scholar). These proteins activate NF-κB through NEMO (Kumar et al. Kumar et al., 2000Kumar A Eby MT Sinha S Jasmin A Chaudhary PM Ectodermal dysplasia receptor activates the nuclear factor kappa B, c-Jun N-terminal kinase and cell death pathways and binds to ectodysplasmin A.J Biol Chem. 2000; (electronically published)Google Scholar) and will be useful in understanding the skin-pigmentation and hair abnormalities that are common to both ED and IP. However, since IP has been associated with immune deficiency but ED has not, and because the XL344-04 male subject was initially diagnosed with ED, we had predicted that, in cases of apparently variant forms of ED with immune deficiency, mutations in NEMO would be present. Indeed, this was demonstrated recently by another group (Zonana et al. Zonana et al., 2000Zonana J Elder ME Schneider LC Orlow SJ Moss C Golabi M Shapira SK Farndon PA Wara DW Emmal SA Ferguson BM A novel X-linked disorder of immune deficiency and hypohidrotic ectodermal dysplasia is allelic to incontinentia pigmenti and due to mutations in IKK-gamma.Am J Hum Genet. 2000; 67: 1555-1562Abstract Full Text Full Text PDF PubMed Scopus (403) Google Scholar). Similarly, variant phenotypes of Partington syndrome II (MIM 301220) and Goltz syndrome (MIM 305600), especially if they occur sporadically, and unexplained cases of skin abnormalities with immunodeficiency (Ment et al. Ment et al., 1978Ment L Alper J Sirota RL Holmes LB Infant with abnormal pigmentation, malformations, and immune deficiency.Arch Dermatol. 1978; 114: 1043-1044Crossref PubMed Scopus (20) Google Scholar; Ruiz-Maldonado and Orozco-Covarrubias, 1997Ruiz-Maldonado R Orozco-Covarrubias ML Postinflammatory hypopigmentation and hyperpigmentation.Semin Cutan Med Surg. 1997; 16: 36-43Crossref PubMed Scopus (118) Google Scholar) are good candidates to examine for NEMO mutations. The authors are members of the International IP Consortium, established with the encouragement and help of the National IP Foundation (NIPF), in New York. Susanne Emmerich, Director of NIPF, deserves special mention for arranging collaborative meetings. We thank all involved families for their invaluable and continuing participation in these research investigations. John Bargerstock, Laura Molinari, and Yumei Ying, of the BCM MRRC tissue culture core, provided expert technical assistance with cell lines. We also thank Kerry L. Wright for editing the manuscript. This work was supported, in part, by National Institutes of Health grants 5 R01 HD35617 and 2 P30 HD24064, to D.L.N., and by grants from Ligue Nationale contre le Cancer (“Equipe labellisee”), to A.I. R.A.L. is a Senior Scientific Investigator of The Foundation Fighting Blindness, Hunt Valley, Maryland.