Abstract
The plasminogen activator inhibitor 1 (PAI-1) molecule is like a double-edged sword: it can act in physiological as well as in pathological processes. One important physiological process PAI-1 takes part in is fibrinolysis. In this plasminogen activator (PA) system, the zymogen plasminogen is activated by either the tissue-type PA (tPA) or urokinase-type PA (uPA) to the broad-specificity proteinase plasmin. Both PAI-1 and PAI-2 are inhibitors of this event; and, since PAI-1 is the more important inhibitor of both, it is no surprise that high levels of PAI-1 are associated with acute diseases such as sepsis and myocardial infarction (MI); or with chronic disorders such as cancer, fibrosis, and atherosclerosis. 1
Several variations in the PAI-1 gene are known to affect the function of the resulting protein. From these, the 4G/5G deletion/insertion promoter polymorphism has been the one which was investigated most widely as to its physiopathological and clinical significance. In vitro experiments first demonstrated that the 4G allele exhibited increased messenger RNA (mRNA) transcription in comparison with the 5G allele. 2 Later it has also been shown that this polymorphism determines PAI-1 levels, with the 4G genotype being associated with higher plasma concentrations. 3,4 For these reasons, the 4G/5G promoter polymorphism has been investigated in numerous studies as a risk factor for arterial thrombotic disease, but the results were contradicting. Although many studies claimed that the 4G genotype contributes to the risk of coronary thrombosis in patients with cardiovascular disease, there were also many studies that claimed it was not. 5–10 Nevertheless, the latest meta-analysis study indicated that the PAI-1 4G/5G promoter polymorphism influences cardiovascular risk via a mechanism that is not simply related to its plasma levels alone. 11
We recently found 2 new variations at the same point of the PAI-1 gene that overlaps with the 4G/5G promoter polymorphism by changing the first guanine nucleotide (at position –679) of the possible 4 or 5 guanines either into a thymine (G-679T) or into a cytosine (G-679C) nucleotide in 3 unrelated Turkish cases with different clinical histories (Figure 1 ).
A, Real-time polymerase chain reaction (PCR). For the genetic analysis of the PAI-1 4G/5G deletion/insertion promoter polymorphism by real-time PCR, we use the commercial Human PAI-1 4G/5G Lightmix Kit (TIB MOLBIOL, Germany). By this method, we can distinguish the deletion (4G) and insertion (5G) allelles by the specific melting temperatures (Tm) of the resulting amplicons after a melting curve analysis, which is 54°C for the 4G and 60°C for the 5G allele (blue curve). In the 3 cases described in this article, we were able to find beside the known 5G allele another new allele according to its different Tm value, which was 49°C (red curve). A sample without DNA was used as negative control (green line). B, PAI-1 G-679T Variation. and C, PAI-1 G-679C Variation. After an initial PCR reaction, sequencing and screening for new variations of the PAI-1 4G/5G promoter region was accomplished. Twenty microliter of PCR reaction contained 0.5 μmol/L of each primer (F: 3′-TGTTTCTATCCCTTTTCCCCT-5′ and R: 5′-ATAACCTCCATCAAAACGTG-3′), 5 ng/mL template DNA, 2.5 U FastStart Taq DNA polymerase (Roche Applied Science, Germany), 200 μmol/L deoxynucleotide triphosphates (dNTPs), 1 mmol/L MgCl2, and sterile distilled water. The PCR procedure was performed on a thermal cycler and programmed with initial denaturation at 95°C for 10 minutes, followed by 35 cycles of 94°C for 30 seconds, 55°C for 45 seconds, and 72°C for 1 minutes. The final extension step was performed at 72°C for 7 minutes. PCR products were checked by 2% agarose gel electrophoresis and purified with ethanol precipitation before they were prepared using the BigDye Terminator Kit and analyzed on the 3100 DNA Analyzer (Applied Biosystems, Foster City). Sequencing results were compared with the corresponding entries in the NCBI GenBank (GenBank accession: PAX9, NC_000014.837126773–37147012; MSX1, NG_008121.1; AXIN2, NG_012142.1).
Case 1
Our first case is a 30-year-old male patient who was taken to the emergency service of our hospital with the diagnosis of primary sclerosing cholangitis. Since our routine thrombophilia genetic screening panel also includes 4 mutations/polymorphisms other than the PAI-1 4G/5G promoter polymorphism, we were analyzing these risk factors as well. The patient did not carry the factor V Leiden (FVL) G1691A mutation, prothrombin (Factor II [FII]) G20210A mutation, and methylenetetrahydrofolate reductase (MTHFR) C677T gene polymorphism; but was heterozygous for the new PAI-1 G-679T gene variant and the factor XII (FXII) C46T gene polymorphism. This frequent C/T polymorphism in the 5′-untranslated region of the FXII gene destroys a consensus sequence, resulting in lower translation efficiency and decrease in FXII plasma levels. 12 As it is for the PAI-1 4G/5G promoter polymorphism, studies with the FXII C46T gene polymorphism are also controversial. While some studies claimed that it contributes to the risk of thrombophilia, others did not. 13,14 Unfortunately, we could not deepen our studies with this patient since we could not reach him through his contact address.
Case 2
The second case is a 55-year-old female patient who admitted to hospital with complaint of splenomegaly when she was pregnant with her first child and found to have portal vein thrombosis. After giving birth, she was treated with Coumadin and did not have any kind of thrombosis again. Recent blood analysis showed that her homocysteine level is normal, antithrombin III level is low, proteins C and S at their sublevels, and C-reactive protein (CRP) <0.1. During the following period, she also became diabetic and hypertensive. Her mother is still alive and in healthy condition, her father had skin cancer before he died. Through our thrombophilia genetic screening panel, we could identify that this patient, like our first case, was heterozygous for the new PAI-1 G-679T gene variant but also carried the FVL mutation heterozygously. We could analyze the genomic DNA of her 2 younger brothers (47 and 42 years) who were also heterozygous for the new PAI-1 G-679T gene variant but did not carry the FVL mutation; and her son (29 years) was also evaluated and found that he did not carry the mutation or variation. All 3 are clinically healthy.
Case 3
Our last case is a 56-year-old male patient who had been diagnosed for systemic lupus erythematosus 20 years ago. As a complication due to his corticosteroid treatment he was operated for cataract in the year 1993 and then for the diagnosis of an avascular necrosis of the femoral head in 1995. In 2008, he applied to a Health Care Institution because of swelling and erythema of his right foot and difficulties in breathing which started afterward. He was found to have deep vein thrombosis (DVT) and pulmonary embolism. After his treatment was established he was advised to undergo a checkup in a hematology clinic. Proteins S and C and homocystein levels were found to be normal. He was also negative for immunologic markers like antinuclear antibody (ANA), antiphospholipid antibodies (APL), and Crithidia luciliae. His blood sedimentation and serum were normal, as was his CRP level. Although, no MI or cerebrovascular accident was present in his family history at young ages, his father died of MI at the age of 87 and his mother had MI at the age of 86 but is healthy and alive today after she became a coronary stent. His eldest sister (66 years) is diabetic but has no history of thrombosis or miscarriage; another sister (63 years) had a stillbirth of unknown reason but again no history of thrombosis; and his youngest sister is healthy. Through our thrombophilia genetic screening panel, we could identify that besides being heterozygous for the second new PAI-1 G-679C gene variant, this patient was also heterozygous for the FVL mutation, which could explain the reasons for DVT and pulmonary embolism. We analyzed the genomic DNA of his daughter (30 years) and son (28 years) and found that although his daughter was neither carrying the FVL mutation nor the PAI-1 G-679T gene variant, his son inherited the PAI-1 G-679T gene variant but is clinically healthy.
In summary, this is the first time that these 2, PAI-1 G-679T and G-679C, gene variations have ever been reported. These variations have been found in 3 unrelated cases with different clinical histories. Noteworthy, one common thing of them was that they were all coming from the same region; which is the West Coast of Turkey close to the city of Izmir. It might be possible that this variation evolved some time ago regionally and was therefore not found and described by others until now. We do not think that this variation predisposes risk to thrombophilia alone, since the relatives that carried just this variation do not show any clinical symptoms and are healthy. But, we do suggest that these 2 new PAI-1 G-679T and G-679C gene variations, together with one of the other well-known risk factors like FVL mutation or FXII C46T gene polymorphism, could contribute to increased risk of thrombophilia. But for this further functional analyses have do be done.