Is the prothrombin 19911A>G polymorphism a functional noncoding variant?

A noncoding polymorphism in the last intron of the prothrombin gene 19911A>G is associated with slightly elevated plasma prothrombin levels. When present in combination with the 20210A mutation on the other allele, it seems to contribute to the risk of thrombosis.^1,2  Recently, von Ahsen and Oellerich³  described that splicing efficiency was increased in constructs carrying the 19911G allele, supporting a functional role for the 19911A>G variant. However, they also presented data that contradict these findings. Using constructs in which the genomic fragment containing prothrombin exons 13 and 14 separated by intron 13 was cloned immediately downstream from the luciferase cDNA, protein expression was lower for the 19911G constructs than for the corresponding 19911A constructs.^3(Fig2) As a possible explanation for these contrasting findings, the authors suggest that more distant single-nucleotide polymorphisms (SNPs) on the 19911G-20210G allele, which are absent in the constructs, may be responsible for the increased expression of this allele. Although at present this cannot be excluded, we feel that there might be another explanation for their findings. One of the assumptions made by the authors is that the luciferase activity of the different fusion proteins is identical. More specifically, the luciferase protein translated from the nonspliced mRNA has been elongated by 22 amino acids encoded by exon 13 of the prothrombin gene and 20 additional amino acids translated from the 5′ region of intron 13 (Figure 1A), whereas the luciferase protein translated from the spliced mRNA has been elongated with the amino acids from prothrombin exon 13 and 14 (Figure 1B). This results in 2 luciferase molecules with different prothrombin-derived tails, depending on whether or not they have been spliced (Figure 1). The authors do not provide any evidence that these different tails do not affect luciferase activity differently. If the longer exon 13-exon 14 tail affects luciferase activity more negatively than the exon 13-intron 13 tail, increased splicing may be “punished” in this model system by a reduced specific activity. In other words, the 19911G variant may result in increased splicing, and increased protein expression, but the latter is masked by the differences in specific activity of the 2 luciferase fusion proteins. In this context it should be reminded that it has been shown that the C-terminal region of luciferase is important for its activity.⁴  Similar considerations as presented apply to the β-galactosidase activity of the double reporter constructs.^3(Fig6) Correct splicing results in a β-galactosidase-luciferase fusion protein, whereas absence of splicing results in a β-galactosidase protein alone. Because β-galactosidase activity is used for the normalization of luciferase activity, differences in β-galactosidase activity between the constructs influence the luc-gal ratio, possibly leading to a misinterpretation of results.

In conclusion, we believe that the authors should have included experiments with control constructs expressing the relevant fusion proteins for normalization of their luciferase activity assays. Furthermore, analysis of the mRNA species derived from the various constructs would have been helpful to further support the hypothesis that 19911G results in more efficient splicing.