PMC8727435_01
The profile of the index patient. The patient, a 7-month-old boy indicated by an arrow in the pedigree diagram (A), had sparse hair and eyebrows and a depressed nasal bridge (B). Flow cytometric analysis of peripheral blood revealed normal CD40 ligand (CD154) expression (C) and decreased NEMO expression in CD3+ cells and CD14+ cells (D). Sequencing of the IKBKG gene revealed hemizygous mutation c.262_264delGAG in exon 3 (red arrow) (E). NEMO, nuclear factor kappa beta essential modulator.
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Family history was significant for two maternal uncles who had died at ages 4 and 7 months ( Figure 1A ). He had dry skin and features of ectodermal dysplasia, including sparse hair and eyebrows, a depressed nasal bridge, no eruption of the deciduous teeth, and decreased sweating indicated by starch-iodine test ( Figure 1B ). B lymphocytes were present in peripheral blood (CD19+ cells: 17%) ( Supplementary Figure 1 ). Flow cytometric analysis of peripheral blood revealed normal expression of CD40 ligand ( Figure 1C ) and decreased NEMO expression in CD3+ cells and CD14+ cells ( Figure 1D ). Analysis of the IKBKG gene by long-range PCR and the Sanger sequence revealed a novel in-frame deletion of a codon (c.262_264delGAG), which resulted in one amino acid microdeletion ( E88) in exon 3 ( Figure 1E ).
Findings for the mother. Flow cytometric analysis of maternal peripheral blood revealed mixed expression of normal and mutated NEMO (A). Sequencing of the IKBKG gene revealed the heterozygous variant c.262_264delGAG (red arrow) (B). The mother's left arm exhibited linear hyperpigmentation following the Blaschko line (blue arrows) (C). NEMO, nuclear factor kappa beta essential modulator.
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Flow cytometric analysis of maternal peripheral blood showed mixed expression of normal and mutated NEMO ( Figure 2A ). The same heterozygous variant (c.262_264delGAG) was also found in the mother ( Figure 2B ). She was ultimately diagnosed with IP on the basis of hypodontia and linear hyperpigmentation that followed the Blaschko line on her left arm ( Figure 2C ). Although each variant was identified in a heterozygote because of the existence of a pseudogene, both samples were confirmed to be from the uncles by confirming the presence of male-specific SRY gene ( Supplementary Table  and  Supplementary Figure 2 ).
Functional analysis of the variant. NF-kappaB reporter gene analysis using NEMO-deficient HEK293 cells revealed a loss of activity of the IKBKG variant  E88, as compared with the wild type, and no response against TNF-alpha. Hypomorphic variants already reported, i.e., E390RfsX5 and P398S, were also included to the analysis to ensure the accuracy of this experiment. Data are shown as the mean and standard error of triplicate measurements. NF-kappaB, nuclear factor kappa beta; NEMO, nuclear factor kappa beta essential modulator; TNF-alpha, tumor necrosis factor-alpha.
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An NF-kappaB reporter gene analysis measuring the activity of IKBKG variants using NEMO-deficient HEK293 cells showed loss of activity of the IKBKG variant  E88, as compared with the wild type, and no response against tumor necrosis factor-alpha (TNF-alpha) ( Figure 3 ). In contrast to the index patient, the mother, and two maternal uncles, sequencing of the IKBKG gene using the peripheral blood lymphocytes did not reveal any variants in exon 3 ( Supplementary Figure 3 ).
Preserved umbilical cords from deceased relatives. A-I is from the older uncle (Uncle III-2), and A-II is from the younger uncle (Uncle III-3) (A). Sequencing of the IKBKG gene revealed the heterozygous variant c.262_264delGAG in both uncles (B).
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Because their preserved umbilical cords ( Figure 4A ) were available, we extracted DNA with ZR-DuetTM DNA/RNA MiniPrep Plus kit (Zymo Research), in accordance with the manufacturer's instructions, and performed sequencing with the Sanger sequencing method ( Figure 4B ).