Characterization of a UNC13D intron 1 mutation. (A) Evolutionary nucleotide conservation (gray bars) as predicted by the AlaMut algorithm relative to intronic (black line) and exonic (black boxes) regions of UNC13D. (B) The nucleotide exchange c.118-308C > T is located in a highly conserved region of intron 1. The c.118-308C > T mutation was identified in 7 patients (6 families) in a heterozygous state and in one patient in a homozygous state. (C) PBMCs from patient C and a healthy control were lysed, and protein content was analyzed by SDS-PAGE and Western blotting. Rabbit polyclonal antibodies for detection of Munc13-4 and ERK1 and 2 were used. (D) Nine different primer pairs were used for amplification of overlapping, consecutive UNC13D cDNA fragments. The amplified fragments from patient E, carrying the intron 1 mutation c.118-308C > T in a homozygous state, and a healthy control were separated by gel electrophoresis and are shown relative to their position in the UNC13D transcript.