Atypical forms of incontinentia pigmenti in male individuals result from mutations of a cytosine tract in exon 10 of NEMO (IKK-gamma)

Abstract

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-gamma), which encodes a critical component of the NF-kappaB 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-kappaB 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.

Figure 1

Mutation analysis of NEMO exon 10. A, CSGE bandshifts in affected members. B, Sequence analysis reveals mixed sequence (arrows indicate start point) due to mutations. C, DNA-sequence alignments between normal and mutant exon-10 sequences, with the cytosine tract at the left. Arrows point to mutations, and blue boxes mark the duplications. D, Protein-sequence alignments show the addition of novel amino acids, enclosed in blue boxes. Orange boxes indicate stop codon positions. E, Mutational mechanism at the cytosine tract, with duplications (1, 3) on top and deletions (2, 4) at the bottom.

Figure 2

Analysis 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.

Figure 3

X-inactivation analysis in pedigrees with exon-10 mutations. The methylation-based assay detects the active X chromosome after digestion with a methylation-sensitive enzyme, HpaII (H). Two bands in the “H” lanes indicate random X inactivation, and a single band suggests complete skewing of X inactivation. The RsaI (R) digest serves as control, and both alleles are amplified. A, Affected members with the ΔC1161 mutation show complete skewing of X inactivation. B, Patients with the dupC1161 mutation show random X inactivation. C, Affected female subjects with the dup1166-78 mutation in family XL320 show mild skewing (∼60%) of X inactivation. D, Family XL374 has the Δ1163-75 mutation, which causes complete skewing in affected members.