Allele-specific quantitative real-time PCR in heterozygous carriers of the UNC13D intron 1 mutation. (A) Allele-specific expression analysis of UNC13D was assessed in WBCs from healthy controls and heterozygous carriers of the intron 1 mutation, c.118-308C > T, using real-time PCR (standard curve method). Primers were designed for specific amplification of either the c.888G or the c.888C allele, a single nucleotide polymorphism located in exon 11. Both healthy controls (n = 3) and carriers of the intron 1 mutation (n = 4) carried the SNP, c.888G > C, in a heterozygous state. In all heterozygous carriers of the intron 1 mutation, c.118-308C > T, was located on the c.888C allele. (B) For evaluation of allele-specific transcription in different cell subsets, CD14⁺, CD4⁺, CD8⁺, and CD56⁺ cells were isolated consecutively by positive magnetic selection. Allele specific real-time PCR was performed in healthy controls (n = 4) and heterozygous carriers of the intron 1 mutation, c.118-308C > T (n = 4). Statistical significance was analyzed using the 2-tailed Mann-Whitney U test. *P < .05.