Figure 6. In vitro and in vivo analysis of genomic region F1–F2 and SNPs associated with conduction.

(A) Luciferase assays performed in Cos7 showed that rs6801957 was involved in T-box factor response. Representative human major (G) alleles Maj1 and Maj2 responded to stimulation by TBX5 and repression by TBX3, whereas this effect on the minor (A) allele Min1 was significantly less. Mutating rs6801957 in these alleles showed a similar effect. *P < 0.01. Error bars represent SD. See Supplemental Table 10 for information on variants per allele. (B) EMSA showing relative oligonucleotide binding of MBP-TBX3 (+) compared with MBP only (–). MBP-TBX3 fusion associated well with both Nppa probe and human Maj1, but less with Min1 and Mut (containing a 3-bp mutation in the proposed T-box binding element). See Supplemental Figure 9 for quantification and statistical analysis. (C) Representative image of the zebrafish heart (dotted outline) containing ZED-Maj1, showing expression in the ventricle (V). A, atrium. Scale bar: 100 μm. (D) Percent GFP expression in hearts of zebrafish containing the major or minor allele for rs6801957. ZED represents the vector without genomic region F1–F2. Representative human major alleles Maj1 and Maj3 showed GFP expression in 60%–70% of the hearts of zebrafish containing these constructs. When major alleles were mutated into minor alleles (Maj1Mut and Maj3Mut), a significant reduction in GFP expression was found. The presence of rs6795970 in Maj3, also linked to conduction disease, had no significant effect on GFP expression in the heart.