Role of genetic mutations in vascular access thrombosis among hemodialysis patients waiting for renal transplantation

Role of genetic mutations in vascular access thrombosis among hemodialysis patients waiting for renal transplantation

DIALYTIC THERAPIES Role of Genetic Mutations in Vascular Access Thrombosis Among Hemodialysis Patients Waiting for Renal Transplantation B. Atac¸, U¨...

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DIALYTIC THERAPIES

Role of Genetic Mutations in Vascular Access Thrombosis Among Hemodialysis Patients Waiting for Renal Transplantation B. Atac¸, U¨. Yakupog˘lu, N. O¨zbek, F.N. O¨zdemir, and N. Bilgin

A

RTERIOVENOUS fistula (AVF) is a type of vascular access used for hemodialysis therapy in patients with end-stage renal failure. Thrombosis, infections, pseudoaneurysm, diabetes mellitus, and hypotension may cause dysfunction of the fistula. The recent discovery of hereditary conditions that affect coagulation has had a major impact on our understanding of thrombosis, which is now viewed as a complex disease in which interactions between genetic and environmental components contribute to clinical phenotype.1,2 Among the mutations in several genes that are reportedly associated with thromboembolism, point mutations in the factor V and prothrombin genes have been identified as the most frequent genetic determinants.1 Resistance to activated protein C (APC) is now regarded as the most prevalent coagulation abnormality associated with venous thrombosis. In this condition, a missense mutation is induced by the replacement of guanine with adenine at nucleotide 1691 in exon 10 of the factor V gene. The resulting point mutation at nucleotide 1691 causes the replacement of arginine to glutamine at amino acid 506, located at one of the sites where factor V is recognized, cleaved, and inactivated by APC. The altered primary structure of the protein results in a predisposition to thrombosis because it produces an inefficiently inactivated clotting factor known as factor V Leiden (FVL).1–3 The replacement of guanine with adenine at nucleotide 20210 in the 3⬘ untranslated region of the prothrombin gene; a change called as prothrombin 20210 (Pt20210) is the second most common molecular determinant of inherited thromboembolism.1 In this case, elevated plasma prothrombin concentration is associated with higher risk of throm0041-1345/02/$–see front matter PII S0041-1345(02)02840-3 2030

bosis. For both mutations, heterozygotes as well as homozygotes are predisposed to thromboembolism.1,2,4,5 The incidences of the FVL and Pt20210 mutations are known to differ considerably among certain ethnic groups. In our population, the reported incidences of heterozygotes for both mutations are relatively high, at 7.3% and 2.3%, respectively.5,6 The aim of this study was to investigate possible associations between AVF thrombosis and both the FVL and Pt20210 mutations.

METHODS Patients In this study, 90 chronic renal failure patients on hemodialysis therapy in Bas¸kent University were sampled. The study group consisted of 46 patients with more than three episodes of AVF thrombosis, frequent clotting of the dialyzer on hemodialysis therapy despite high heparin doses, and acute thrombosis with therapeutically elevated prothrombin times. The other 44 patients had not experienced these problems and served as the control group. None of the patients in this study had diabetes mellitus, atherosclerosis, or vasculitis. The mean age and duration of hemodialysis for the study group were 47 years and 65 months, respectively; the corresponding figures for the controls were 42 years and 56 months, respectively Bas¸kent University Faculty of Medicine, Ankara, Turkey. Address reprint requests to N. Bilgin, Bas¸kent University Faculty of Medicine, Departments of Nephrology, Molecular Biology, Pediatric Hematology and General Surgery, Ankara, Turkey. 1. Cad No:77, Bahcelievler, 06490 Ankara, Turkey. E-mail: [email protected] © 2002 by Elsevier Science Inc. 655 Avenue of the Americas, New York, NY 10010 Transplantation Proceedings, 34, 2030 –2032 (2002)

GENETIC MUTATIONS All patients had bicarbonate dialysis using a cuprophane dialyzer with an average blood flow rate of 300 to 350 mL/min and mean Kt/V maintained at 1.42 ⫾ 0.3 during each treatment. Prior to initiation of dialysis, a baseline whole blood clotting time or whole blood activated clotting time was performed to determine the heparin requirements. Unfractioned heparin (2500 IU) was given to high-risk patients. The whole blood activated clotting time was measured in every 30 minutes; when it decreased below 6 minutes, additional boluses of heparin (up to 1000 IU) were administered.

Specimen Collection Genomic DNA was isolated from EDTA-coagulated blood samples using the standard phenol-chloroform extraction method. The isolated DNA samples were resuspended at a concentration of 100 mg/L in Tris (pH 7.4) containing 0.1 mmol/L EDTA. The samples were kept at ⫺20°C until they were tested.

Rapid Real-Time PCR Amplification PCR was performed in a Light Cycler (Roche Diagnostics) according to the manufacturer’s instruction by using the the light– cycler Factor V Leiden (Roche, Cat. No. 2212161) and Prothrombin G20210A (Roche - Cat. No. 2236842) detection kits, which were specifically adapted for PCR in glass capillaries.

Fluorometric DNA Melting Curve Analysis Differentiation of the two alleles (FVL and prothrombin genes) was performed by determining the melting curves after PCR, according to the instructions of the manufacturer. Hybridization was performed with two different short oligonucleotides for two adjacent internal sequences of the amplified PCR fragment that hybridize during the annealing phase of the PCR cycles. One probe was labelled at the 5⬘ end with a light cycler red fluorophore and phosphorylated at the 3⬘ end. The second probe was labeled with fluorescein. After hybridization, probes in close proximity produce fluorescence energy transfer (FRET) between the two fluorophores. During FRET, fluorescein, the donor fluorophore, is stimulated by the light source of the instrument and part of the energy is transferred to light cycler red, the accepter fluorophore. The emitted fluorescence of the light– cycler red fluorophore is quantified by plotting the negative derivative of fluorescence with respect to time versus temperature (⫺dF/dt versus T).

Statistical Analysis Statistical analysis was performed by using SPSS for Windows 9.05. Student t-test was used to compare the means of the two groups and statistical significance was accepted at P ⬍ .05.

RESULTS

There were no significant differences between the study and control groups regarding age, gender, and duration of hemodialysis (P ⬎ .05). Among the 46 patients in the study group, six (13%) were heterozygotes for FVL and four (8.7%) were heterozygotes for Pt20210. In the control group, two patients (4.3%) were heterozygous for FVL, and none had the Pt20210 point mutation (Table 1). DISCUSSION

AVF is the most common form of vascular access for treatment of hemodialyzed end-stage renal failure pa-

2031 Table 1. Incidence of Genetic Mutations by Groups

Study group (n ⫽ 46) Control group (n ⫽ 44)

FVL Mutation

Pt20210 Mutation

6 (13%) 2 (4.3%)

4 0

tients.9 –11 Thrombosis is the leading cause of AVF dysfunction and hospitalization. It is responsible for a significant proportion of the morbidity and treatment cost.12 Recent studies linked a clotting predisposition to changes in factors involved in vascular proliferation, coagulation, anticoagulation, and fibrinolysis. In one study, elevated serum levels of cytokines that regulate proliferation of vascular smooth muscle cells (monocyte chemoattractant protein-1, and IL-6); elevated levels of hemostasis-derived factors (factor VII and plasminogen activator inhibitor [PAI] type 1); hyperinsulinemia and hyperlipidemia were shown to be independent risk factors for survival of the AVF.13 The same investigators found increased levels of prothrombin fragment 1⫹2 (PF1⫹2) and a rise in d-dimer levels in patients who had experienced AVF thrombosis. These findings reflect activation of coagulation associated with accelerated fibrinolysis. Another study noted a predominate thrombotic state and enhanced fibrinolysis in the systemic circulation of endstage renal disease patients with AVF.14 The authors also compared the concentrations of factors involved in coagulation, fibrinolysis, and fibrinolysis inhibition in blood drawn from the AVF to that drawn from systemic circulation. Surprisingly, this analysis revealed significantly higher levels of procoagulant activation indicators (PF1⫹2 and thrombin– antithrombin complexes) and fibrinolytic factors (plasminogen, tissue-plasminogen activator, and urokinase-plasminogen activator) in the AVF-derived blood. They concluded that the AVF itself may play an important role in modulating the coagulation and fibrinolytic cascade. Other investigators recorded longer euglobulin fibrinolysis time and elevated PAI levels in patients with AVF thrombosis after veno-occlusion compared to the levels of these factors in the same patients before thrombosis, and in individuals who had not encountered AVF thrombosis.15 However, the same study showed no differences between patients who had and did not have AVF thrombosis regarding hematocrit; levels of fibrinogen, factor VII, anti-thrombin, and protein C; and platelet number and function. Research on other hemodialysis patients with AVF thrombosis has also shown insignificant changes in other factors that would be expected to raise thrombosis risk.16,17 All the studies mentioned suggest that the procoagulant activation and enhanced fibrinolysis observed in patients with AVF may be partly due to effect(s) of the AVF itself. Thrombotic disease may develop under conditions of low (venous) or high (arterial) blood flow and pressure. The composition of the thrombus (fibrin rich in venous and platelet rich in arterial) and the causes are different in each case. Hereditary thrombophilic disorders are the most

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common underlying causes of both venous and arterial thrombi; however, disturbance of hemostasis by various environmental factors (hormone ingestion, oral contraceptive pills, immobilization, and surgery) are the main features that promote clot development in both vessel types. Among the hereditary thrombophilic disorders, FVL mutation is the most common cause of venous thrombosis, whereas the Pt20210 mutation usually causes arterial thrombosis.18 The AVF is an artificial system that includes both venous and arterial components; thus, risk factors for both systems can affect fistula function. Changes in blood flow and pressure at the AVF site can alter hemostasis. In addition, it seems that the AVF itself may actually increase the risk of developing a thrombus.14 In our study, we found a significantly higher frequency of both FVL and Pt20210 mutations in patients with AVF thrombosis than in those who did not have this complication. In the control group, the number of patients with the FVL mutation was significantly lower and none in the group exhibited the Pt20210 mutation. Interestingly, the frequencies of both mutations in the affected group were approximately twice the prevalence that has been noted in our population.5,6 We believe that, in patients with inherited thrombophilia, the changes introduced by AVF, the hemodynamic changes that occur in the immediate fistula area, may combine to produce more frequent thrombosis. We conclude that hereditary thrombophilic disorders are important risk factors in development of AVF thrombosis, and that the fistula itself induces local hemostatic disturbances that may further increase the likelihood of thrombosis. In terms of future work, studies on other thrombophilic disorders in these patients may also reveal valuable information. The increased tendency toward thrombosis may cause another important problem for these patients, all of whom

are on transplantation waiting lists: thrombosis is known to be one of the most serious complications leading to increased morbidity and graft dysfunction after renal transplantation. Finally, we suggest that patients who encounter frequent AVF thrombosis should be screened for FVL and Pt20210 mutations; this situation cannot be corected by transplantation and can lead to serious thrombosis complications during posttransplantation follow up. REFERENCES 1. Bertina RM, Rosendal FR: N Engl J Med 338:1840, 1998 2. Vooberg J, Roelse J, Koopman R, et al: Lancet 348:1535, 1994 3. Bertina RM, Koelman BPC, Koster T, et al: Nature 369:64, 1994 4. Lane D, Grande P: Blood 95:1517, 2000 5. Gurgey A, Mesci L: Turk J Pediatr 39:313, 1997 6. Akar N, Misirlioglu M, Akar E, et al: Am J Hematol 58:249, 1998 7. D’Angelo A, Sehub J: Blood 90:1, 1997 8. De Stefano V, Zappacosta B, Persichilli S: Br J Haematol 341:801, 1999 9. Albers FJ: Adv Ren Replace Ther 1:107, 1994 10. Porile J, Richter MP: J Am Soc Nephrol 4:997, 1993 11. Goldwaseer P, Avram MM, Collier J, et al: Am J Kidney Dis 24:785, 1994 12. Chazan JA, London MR, Pono LM: Am J Kidney Dis 6:523, 1992 13. De Machi SD, Falleti E, Giacomello R, et al: J Am Soc Nephrol 7:1169, 1996 14. Erdem Y, Haznedaroglu I, Celik A, et al: Nephrol Dial Trans 11:1299, 1996 15. Charvat J, Kestlerova M, Jarosova H, et al: Cas Lek Cesk 133:242, 1994 16. Manns BJ, Burgess ED, Parsons HG, et al: Kidney Int 55:315, 1999 17. Shand BI, Buttimore AL, Hurrell MA, et al: Nephron 64:53, 1993 18. Lane DA, Grant PJ: Blood 95:1517, 2000