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


RESULTS
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.


CONCLUSIONS
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.
Webb
Karen E
KE
Centres for Cardiovascular Genetics, British Heart Foundation Laboratories, Royal Free and University College Medical School, Rayne Building, 5 University Street, London WC1E 6JF, England.
Martin
John F
JF
Cotton
James
J
Erusalimsky
Jorge D
JD
Humphries
Steve E
SE
D017398:Alternative Splicing; D005091:Exons; D005786:Gene Expression Regulation / Q000235:genetics; D006801:Humans; D007438:Introns / Q000235:genetics*; D009203:Myocardial Infarction / Q000235:genetics; D020641:Polymorphism, Single Nucleotide / Q000235:genetics*; D011401:Promoter Regions, Genetic; D012307:Risk Factors; D013926:Thrombopoietin / Q000235:genetics*; D014158:Transcription, Genetic / Q000235:genetics; D014407:Tumor Cells, Cultured
D013926:Thrombopoietin