PKLR intronic risk variants and allelic specific expression. (A) PKLR gene with associated variants in intron 2 and coding variant R596R. Line representation of the PKLR gene with the associated intron variants (6 in intron 2 and 1 in intron 4). rs1052176 (R596R) is the synonymous single-nucleotide polymorphism in exon 11 that was used as a marker for allele-specific expression analysis. (B) PKLR allelic-specific expression analysis. Fifty-two heterozygous R569R individuals were selected: 23 without and 29 heterozygous for the associated PKLR intron 2 risk haplotype. Allele-specific expression analysis was carried out using the Bio-Rad droplet digital polymerase chain reaction system (QX200 Droplet Digital PCR System; Bio-Rad Laboratories) and analyzed with QuantSoft software. Ratios of the 2 R596R alleles in complementary DNA (cDNA) and genomic DNA (gDNA), respectively, were derived for each sample. The absolute difference in the ratios between the cDNA and gDNA samples was then calculated for each participant. The average expression ratio in those heterozygous for the variant intron 2 haplotype was 0.2073 (± standard deviation [SD], 0.0135) compared with an average expression ratio of 0.1239 (± SD, 0.0682) in those without the variant intron 2 (homozygous wild type). Wilcoxon rank sum test P = .0297.