Prediction and verification of the influence of the rs367781716 SNP on the interaction of the ТАТА-binding protein with the promoter of the human АВСА9 gene

Abstract

The high-throughput sequencing project 1000 Genomes made it possible to catalog and utilize genetic loci and single nucleotide polymorphisms (SNPs) in medicine. The analysis of SNP markers allows physicians to optimize treatment. However, tens of millions of unannotated SNPs correspond to a gigantic number of false positive (false negative) candidate SNP markers that are selected by computer methods for comparing their frequency in patients with that in healthy people. This approach contributes to the undervaluation of clinically relevant SNPs and to unnecessary computational expenses for the verification of neutral SNPs. The preclinical empirical verification of possible candidate SNP markers may eliminate neutral SNPs from the dataset. In the present study, using the SNP_TATA_Comparator web service, we found the unannotated SNP rs367781716: the substitution of ancestral T (health) with a minor C at position–37 before the transcription initiation site of the ABCA9 gene. This SNP significantly reduces the affinity of TATA-binding protein (TBP) for this gene’s promoter and corresponds to the deficiency (low protein level) of the ABCA9 gene product (the transporter ATP-binding cassette A9) in patients with the–37C allele. For preclinical empirical verification of rs367781716, we used an electrophoretic mobility shift assay (EMSA) to measure the rates of association (ka) and dissociation (kd) of the complexes of TBP with an oligonucleotide matching either allele–37C or–37T of the ABCA9 gene. We found that the rate of association (ka) of the TBP/TATA complex for the minor allele is lower by a factor of 2.4 than that for the ancestral allele. We calculated the empirical value of the change in the equilibrium constant of dissociation (KD = kd/ka), which characterizes the binding affinity of TBP for a promoter containing the TATA box. This empirical value matched the value predicted by the SNP_TATA_Comparator within the allowable margin of error of the measurements and calculations. We also determined the half-life and Gibbs free energy of the complex of TBP with the ABCA9 promoter. Possible phenotypic manifestations of the candidate SNP marker rs367781716 are discussed.