Figure 1
(A) Clinical information of the patients. Pedigree of the family and genital appearance of patient 1 (at 2.4 years of age) and patient 2 (at 6 months of age) are shown. (B) The c.2254C>T variant and known pathogenic variants in MAP3K1. Black and blue arrows indicate missense substitutions and other variants respectively, which have been subjected to in vitro functional assays8,12,13. White and black boxes denote odd- and even-numbered exons, respectively. GEF, guanine exchange factor domain; SWIM, SWI2/SNF2 and MuDR domain; PHD, plant homeo domain; ARM, armadillo repeat domain; and Kinase, protein kinase domain. Asterisks in the chromatographs depict the mutated nucleotide. (C) Representative results of RT-PCR. The upper panel shows the results of the patients, the mother, and an unaffected individual. NTC, negative control; and CHX, cycloheximide. The middle panel shows chromatograms of the long and short PCR products. The long PCR product was wild-type MAP3K1, while the short product was a variant transcript containing a 117-bp deletion in exon 13. The lower panel shows mRNA structures of the wild-type and variant MAP3K1. The red arrow delineates the position of the c.2254C>T variant, and the red box indicates the 117-bp deletion. Arrowheads depict the position of primers. (D) Predicted structures of the wild-type and variant transcripts. The uppercase and lowercase letters indicate nucleotides in exons and introns, respectively. The underlined letters indicate consensus sequences for a splice donor site37. The shaded region in the long PCR product indicates the 117-bp deletion. The affected nucleotide is shown in red. (E) Representative results of western blotting analysis. Transient transfection of the wild-type and variant MAP3K1 yielded proteins of the expected size and amount. GAPDH was used as a loading control. (F) The results of APOD induction assay. Expression levels of APOD relative to GAPDH (mean ± SD) are shown. Genital skin fibroblasts of the patients and control individuals were treated with either DHT (+) or methanol (DHT −).
