G20210A is a functional mutation in the prothrombin gene; effect on protein levels and 3'-end formation.
BACKGROUND
The prothrombin G20210A mutation is associated with increased plasma prothrombin levels and risk of thrombosis. The mechanism by which this mutation leads to increased prothrombin expression is as yet unclear and still the subject of debate.


OBJECTIVE
The aim of this study was to investigate the effect of the G20210A mutation on mRNA and protein expression.


METHODS
We made a set of constructs containing the prothrombin 5'-regulatory region, the firefly luciferase reporter gene and the prothrombin 3'-UTR+ downstream region. The latter element contained either the 20210G or A allele and was inserted either as a single unit (constructs G1 and A1) or in tandem (A1A2, G1G2, A1G2, G1A2). Constructs were transiently expressed in HepG2 cells. Expression was evaluated by luciferase assays and reverse transcriptase-polymerase chain reaction (RT-PCR), followed by quantification of the products and determination of the ratio of poly(A)site usage. RT-PCR sequencing was used for determination of the actual site of polyadenylation in mRNAs from constructs G1 and A1 and from endogenous prothrombin mRNAs from HepG2 cells and human liver tissue.


RESULTS
The A1 constructs expressed 1.2-fold more protein than the G1 constructs. The double constructs expressed 1.4-fold more protein (A1A2 vs. G1G2). Similar results were found in a set of constructs in which an SV40 promoter replaced the prothrombin 5'-regulatory region. Ratios of poly(A) site usage (expressed as ratio poly(A) site 1 and 2) for the tandem constructs were similar for constructs with two Gs or As at both poly(A)sites; 2.92 (95% confidence interval 2.39-3.45) and 2.75 (2.55-2.95). pA1/pA2 ratios were 1.46 (1.11-1.81) for G1A2 and 6.29 (5.48-7.10) for A1G2 constructs with different poly(A) sites, indicating that the poly(A)site with the 20210A variant is favored over the normal site. In 20210G mRNAs, the G at 20210 was the last non-A nucleotide in the majority of mRNAs, whereas in most 20210A mRNAs, the last non-A nucleotide was the C at 20209. Over 70% of the prothrombin 20210G mRNAs from HepG2 cells and human liver tissue is polyadenylated at position 20210.


CONCLUSIONS
The 20210A variant has a more effective poly(A) site, leading to increased mRNA and protein expression, irrespective of the promoter and gene. It does not affect the position of poly(A) attachment.
Ceelie
H
H
Hemostasis and Thrombosis Research Center, Department of Hematology, Leiden University Medical Center, Leiden, The Netherlands. hceelie@lumc.nl
Spaargaren-van Riel
C C
CC
Bertina
R M
RM
Vos
H L
HL
D020413:3' Untranslated Regions; D001483:Base Sequence; D001665:Binding Sites / Q000235:genetics; D002460:Cell Line; D015870:Gene Expression; D005809:Genes, Regulator; D017930:Genes, Reporter; D014644:Genetic Variation; D006801:Humans; D008099:Liver / Q000378:metabolism; D008156:Luciferases / Q000235:genetics; D008969:Molecular Sequence Data; D010957:Plasmids / Q000235:genetics; D017354:Point Mutation; D011061:Poly A / Q000378:metabolism; D011516:Prothrombin / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D014162:Transfection; D020246:Venous Thrombosis / Q000097:blood / Q000209:etiology / Q000235:genetics
D020413:3' Untranslated Regions; D012333:RNA, Messenger; D011061:Poly A; D011516:Prothrombin; D008156:Luciferases