The 4830C>A polymorphism within intron 5 affects the pattern of alternative splicing occurring within exon 6 of the thrombopoietin gene

• Published in: Experimental hematology Vol. 31; no. 6; pp. 488 - 494
• Main Authors: Webb, Karen E, Martin, John F, Cotton, James, Erusalimsky, Jorge D, Humphries, Steve E
• Format: Journal Article
• Language: English
• Published: Netherlands 01.06.2003
• Subjects: Alternative Splicing; Exons; Gene Expression Regulation - genetics; Humans; Introns - genetics; Myocardial Infarction - genetics; Polymorphism, Single Nucleotide - genetics; Promoter Regions, Genetic; Risk Factors; Thrombopoietin - genetics; Transcription, Genetic - genetics; Tumor Cells, Cultured
• ISSN: 0301-472X
• DOI: 10.1016/S0301-472X(03)00065-1

A common variant in intron 5 of the thrombopoietin (TPO) gene (4830C>A) has been associated with risk of myocardial infarction (MI). To explore the molecular mechanism of this association, the ability of the intron to act as a transcription enhancer and to influence mRNA splicing was tested. In HepG2 cells the presence of intron 5 upstream of the TPO promoter decreased promoter activity to between 60% and 30%. This effect was orientation dependent; in the reverse orientation, intron 5 caused a twofold greater decrease in promoter activity compared to the forward orientation. However, the effects were similar with either the C or the 4830A allele. An in vitro exon trapping system was used to study the effect of the polymorphism on splicing events in exon 6. The full-length (TPO-1) and three previously reported splice variants (TPO-2, TPO-3, and TPO-5) were identified. The 4830A allele resulted in a small but statistically significant increase in production of the TPO-3 splice variant relative to the full-length transcript (10.6%+/-0.6%) compared to the 4830C allele (8.3%+/-0.6%) (p=0.02). Generation of TPO-5 was also slightly increased, but this did not reach significance. The identification of a potential "silencer" sequence in intron 5 of the TPO gene demonstrates the complexity of control of expression of the gene. Although the precise role of the different splice variants is not known, the finding that the 4830C>A sequence change alters their relative amounts, suggests a possible molecular mechanism whereby TPO genotype may influence the risk of MI.