Fig. 2

The upstream region of ACVR2A affects ACVR2A expression. a Allelic-imbalance test for the level of ACVR2A mRNA expression in heterozygotes. cDNA from primary dermal fibroblasts and brain, and gDNA from heterozygous animals were amplified using primers to BovineHD4100001198 (48,443,632 bp), which is located in an intron of ACVR2A (Table 1). The PCR product was directly sequenced. Peak heights at the SNP site were quantified using PeakPicker 2 software [30]. The Y-axis shows the ratio of the peak height of the Q allele to that of the q allele in gDNA (n = 16, mean = 1.035), cDNA from primary dermal fibroblasts (n = 13, mean = 1.32), or cDNA from brains (n = 9, mean = 1.29). Red bars show the mean. The P values for the difference between cDNA and gDNA were 0.009 in primary dermal fibroblasts and 0.044 in brain, respectively, as determined by performing t tests. b Schematic representation of the positions of variants in the 5′ upstream region of ACVR2A. “SNP (−716)” and “3 indel (−737 to −740)” represent g.48476925C > T and g.48476943_48476946insGGC, respectively. “ATG (+1)” and “TSS (−522)” represent the start codon and the predicted transcriptional start site, respectively. A Q and q haplotype sequence alignment of the upstream region of ACVR2A encompassing 48,476,920 and 48,476,964 bp is shown. c Luciferase reporter assays for the 5′ upstream region of ACVR2A-derived Q and q haplotypes. The 5′ upstream region (814 bp upstream from the start codon) derived from the Q and q haplotypes was cloned into the firefly luciferase pGL3-Q and pGL3-q plasmids, respectively. The Firefly-to-Renilla luminescence ratios observed after cotransfecting HeLa cells were measured to evaluate the effects of the 5′ upstream region. Bars represent the mean ± SEM obtained in triplicate from 3 independent experiments. P values determined by t tests are shown