Pedigree, clinical features, and protein expression for the patient. (A) Pedigree of patient (P) with a NEMO mutation in position 311 (P, D311G). The different generations are indicated by roman numerals (I and II), and each individual is indicated by an Arabic numeral. Male subjects are represented by squares and female subjects by circles. Solid (black) symbols indicate affected individuals; open (white) symbols, healthy individuals; ⊡, female carriers. Individuals whose genetic status could not be determined are indicated by the symbol “E?” (B) Photograph and radiograph of P taken at 15 years, showing the agenesis of 3 adult teeth (14, 12, and 22). (C) Automated sequencing profile showing the NEMO D311G mutation in cDNA extracted from EBV-B cells from the proband (P) and comparison with the sequence obtained from a control subject (C⁺). The A→G mutation led to the replacement at residue 311 of aspartic acid (D) by glycine (G). NEMO protein was detected by Western blotting with the FL-419 antibody (D) in monocytes and granulocytes from a healthy control subject (C⁺) and the proband [P (D311G)]; in EBV-B cells from a healthy control subject (C⁺), a 110_111insC NEMO patient already reported to produce smaller than normal amounts of NEMO protein, an NEMO EDA-ID patient with another mutation in position 311 (D311N) and the proband [P (D311G)]; and in SV40-transformed fibroblasts from a healthy control subject (C+), an XD-IP patient (IP), and the proband [P (D311G)]. (E) In SV40-transformed fibroblasts from the XD-IP NEMO patient transfected or not transfected (IP) with expression vectors carrying NEMO alleles (wild-type [IP+NEMO+WT], D311G [IP+NEMO+D311G], D311N [IP+NEMO+D311N]) or with insert-free vector (IP+Empty), cells were cotransfected with cyan fluorescent (CFP) vector, as a control for transfection efficiency control. GAPDH antibody was used to normalize protein levels in panels D and E. These results are representative of 2 independent experiments. WB indicates Western blot.