EXPERIMENTAL AND CLINICAL TRANSPLANTATION

Objectives: Liver transplant in patients with preexisting portal vein thrombosis involves complicated surgical procedures and increased blood loss, indicating the need for further surgical innovations to overcome these difficulties. Patients who are at high risk of developing portal vein thrombosis may benefit from prophylactic anticoagulant therapy while on the transplant wait list. Homozygosity for C677T polymorphism in the methylenetetrahydrofolate reductase gene has been associated with a high plasma homocysteine concentration, which is a risk factor for venous thrombosis. This study investigated the association between C677T polymorphism in the methylenetetrahydrofolate reductase gene and preexisting portal vein thrombosis in patients with liver cirrhosis undergoing liver transplant.

Materials and Methods: C677T polymorphism in the methylenetetrahydrofolate reductase gene was investigated in 48 patients who underwent liver transplant at Nagoya University.

Results: Of 48 patients, 7 (14.6%) had preexisting portal vein thrombosis confirmed at transplant. The frequency of methylenetetrahydrofolate reductase gene C677T genotype in recipients was significantly associated with preexisting portal vein thrombosis (P = .009, with P < .013 considered significant). Logistic regression analysis showed that the TT genotype of the methylenetetrahydrofolate reductase gene was significantly associated with a higher incidence of preexisting portal vein thrombosis compared with the CC and CT genotypes (odds ratio of 14.6, 95% confidence interval, 1.86-115.21; P = .011).

Conclusions: The TT genotype of the methylenetetrahydrofolate reductase gene may be associated with a higher incidence of preexisting portal vein thrombosis, as confirmed at liver transplant. Identification of this genotype in patients with liver cirrhosis at the time of placement on a wait list for liver transplant may enable preventive therapy for portal vein thrombosis in these patients, reducing the complexity of surgical procedures.

Key words : Complication, Genotype, Liver cirrhosis, Risk factor

Portal vein thrombosis (PVT) is an important complication of liver cirrhosis. The incidence of nonneoplastic PVT in patients with cirrhosis is unknown, although its prevalence has been reported to range from 0.6% to 16%.^1-4 Because PVT is one of the leading causes of morbidity and mortality in patient with liver cirrhosis, early detection and treatment of de novo thrombosis is important, especially for patients on a wait list for liver transplant.⁵ Compared with liver transplant in patients without PVT, liver transplant in patients with preexisting PVT involves more complicated surgical procedures and increased blood loss, suggesting the need for additional surgical innovations to overcome these difficulties.⁶ Patients at high risk of developing PVT may benefit from prophylactic anticoagulant therapy while on a transplant wait list. Homozygosity for C677T polymorphism in the methylenetetrahydrofolate reductase (MTHFR) gene has been associated with high plasma homocysteine concentration, a risk factor for venous thrombosis.^7-10 To date, however, the relation between this MTHFR C677T polymorphism and PVT confirmed at liver transplant has not been determined. This study therefore investigated whether this polymorphism was associated with preexisting PVT in patients with liver cirrhosis who underwent liver transplant.

Materials and Methods

Patients
Of the 65 adult patients who underwent liver transplant at Nagoya University between 2004 and 2009, 17 underwent liver transplant for noncirrhotic liver diseases, including acute liver failure; patients with acute-on-chronic liver failure and familial amyloid polyneuropathy were excluded. The procedures for patients receiving a liver transplant have been previously described.^11,12 This study was approved by the ethics committees of Nagoya University School of Medicine (approval No. 290). Signed informed consent was obtained from all participating patients. Portal vein thrombosis was diagnosed preoperatively from a computed tomography scan and confirmed at surgery.

Genotyping
Blood samples were obtained from each patient, mixed with sodium EDTA, and centrifuged, and DNA was extracted from the buffy coat fraction. The MTHFR C677T polymorphism was genotyped by polymerase chain reaction with confronting two-pair primers.¹³ Each 25-μL reaction tube contained 50 to 80 ng of DNA, 0.12 mM dNTP, 12.5 pmol of each primer, 0.5 U Amplitaq Gold (Perkin-Elmer, Foster City, CA), and 2.5 μL of 10× polymerase chain reaction buffer containing 15 mM MgCl₂. The polymerase chain reaction plus confronting 2-pair primer amplification procedure consisted of an initial denaturation at 95°C for 10 minutes, 35 cycles of denaturation at 95°C for 1 minute, annealing at 56°C for 1 minute, and extension at 72°C for 1 minute, followed by a final extension at 72°C for 5 minutes. The primers were 5′-AGC CTC TCC TGA CTG TCA TCC-3′ and 5′-TGC GTG ATG ATG AAA TCG G-3′ (forward and reverse) and 5′-GAG AAG GTG TCT GCG GGA GT-3′ and 5′-CAT GTC GGT GCA TGC CTT-3′ (forward and reverse). The amplified DNA fragments were 128 bp for the C allele, 93 bp for the T allele, and 183 bp for the common band.¹⁴

Statistical analyses
Categorical variables were compared using chi-squared tests, and continuous variables were compared using t tests. The association between C677T genotype and the risk of preexisting PVT was analyzed using a logistic regression model and reported as odds ratios and 95% confidence intervals. P < .05 was considered statistically significant unless stated otherwise.

The 48 patients who underwent liver transplant included 27 male (56.3%) and 21 female patients (43.7%), with median age of 49 years at transplant (range, 1 to 68 years), including 4 who received liver transplants from donors after brain death. Indications for liver transplant included hepatitis B virus-associated liver cirrhosis in 15 patients, hepatitis C virus-associated liver cirrhosis in 12 patients, primary biliary cirrhosis in 9 patients, cryptogenic liver cirrhosis in 3 patients, and primary sclerosing cholangitis in 3 patients. Other indications included Wilson disease, alcoholic liver disease, biliary atresia, and Budd-Chiari syndrome in 6 patients. Seven patients (14.6%) had preexisting PVT confirmed at transplant. Intraoperative assessment of the degree and extent of PVT¹⁵ showed that 2 patients had grade 2 disease, 3 had grade 3a, and 2 had grade 3b. The genotype frequencies of the MTHFR C677T in patients with end-stage liver disease were similar to those shown in living liver donors, who are regarded as healthy (Table 1). Demographic variables were comparable in liver transplant recipients with and without preexisting PVT (Table 2). As shown in Table 3, the MTHFR C677T genotype frequency in the liver transplant recipients was significantly associated with preexisting PVT (P = .009, with P < .013 considered significant). Logistic regression analyses showed that, after adjustment for patient age, sex, and presence of hepatocellular carcinoma, the MTHFR TT genotype was significantly associated with a higher incidence of preexisting PVT than the CC and CT genotypes (odds ratio of 14.6; 95% confidence interval, 1.86-115.21; P = .011) (Table 4).

This study investigated the association between the C677T polymorphism in the MTHFR gene and preexisting PVT in liver transplant recipients. Logistic regression analyses showed that the MTHFR TT genotype was associated with a significantly higher incidence of preexisting PVT as confirmed at transplant.

Portal vein thrombosis is a complication of decompensated cirrhosis and is more likely to occur during late-stage liver cirrhosis.¹⁶ Because of surgical innovations, preexisting PVT is not a contraindication for liver transplant at most centers. However, the procedure can be complicated by the more severe preoperative condition and extensive collaterals in PVT patients.^6,17,18 Technical difficulties, especially prominent during living-donor liver transplant in patients with preexisting PVT, include the necessity of distal dissection of the vascular pedicle of the hilum and restricted availability of a vein graft.19 Therefore, identifying risk factors for PVT development in patients with liver cirrhosis on a transplant wait list may enhance outcomes in these patients. Studies of the causes of PVT in patients with liver cirrhosis have improved our understanding of the natural history of this condition.^1,20 Male sex, previous abdominal surgery including splenectomy and portocaval shunts, encephalopathy, ascites, previous history of bleeding varices, low platelet count, and Child-Pugh class C have been considered predisposing factors for PVT in patients with liver cirrhosis.^1,5,21,22

Genetic polymorphisms may increase the likelihood of venous thrombosis and thromboembolism. These polymorphisms may predispose patients to venous thrombosis and thromboembolic phenomena, even in the absence of any other predisposing factors.²³

The MTHFR C677T polymorphism is a C-to-T transition at base pair 677 (exon 4), leading to an amino acid substitution (alanine to valine) at codon 222 of the MTHFR protein.²⁴ Serum homocysteine concentrations have been found to be significantly higher in TT than in CC homozygotes. Hyperhomocysteinemia has been reported to be associated with venous thrombotic disorders,^8,10 suggesting that this polymorphism may be a genetic risk factor for venous thromboembolism. A recent large meta-analysis, including 27 studies assessing the association between homocysteine concentration and venous thrombosis and 53 studies assessing the association between MTHFR 677TT genotype and venous thrombosis, revealed that hyperhomocysteinemia was associated with a 27% to 60% higher risk of venous thrombosis and that the MTHFR 677TT genotype was associated with a 20% higher risk of venous thrombosis compared with the MTHFR 677CC genotype.¹⁰ Several previous studies^25-27 have reported that the MTHFR C677T gene polymorphism is associated with PVT diagnosed by imaging modalities, including computed tomography scan or Doppler ultrasonography. Our results are in agreement with these previous findings, in that we showed that the MTHFR C677T gene polymorphism was associated with PVT confirmed during liver transplant.

The optimal treatment of PVT in patients with cirrhosis has not been determined. Although the factors associated with recanalization or the extent of thrombosis has been investigated, the actual effect of PVT treatment on the natural history of cirrhosis has not been determined.²⁸ Stratification of patients with liver cirrhosis according to risk of PVT may enable those at greater risk to be provided preventive therapy for PVT. A randomized controlled trial evaluating the safety and efficacy of low-molecular-weight heparin in prevention of PVT in patients with liver cirrhosis found that the actuarial probability of PVT was lower in patients who were treated with enoxaparin than those who were not.²⁹

One limitation of this study was the small number of patients, thus precluding any definitive conclusions. Further studies in larger numbers of patients are required to confirm whether the MTHFR C677T gene polymorphism is associated with PVT in patients with liver cirrhosis requiring liver transplant.

In conclusion, this study showed that the MTHFR TT genotype may be associated with an increased incidence of preexisting PVT, as confirmed at liver transplant. Further studies in larger patient populations are required to confirm whether this genotype can be one of the risk factors for development of PVT in patients with liver cirrhosis.

Volume: 14 Issue: 3 June 2016

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