Discrepancy between phenotype and genotype on screening for factor V Leiden after transplantation

Factor V (FV) Leiden is the most common genetic defect found in persons of European descent with venous thromboembolism.1The 1691G>A transition determines the R506Q substitution.2 This suppresses a cleavage site for activated protein C (APC) on FVa, resulting in resistance to APC in a functional coagulation-based assay.3 FV Leiden accounts for over 90% cases of APC resistance, but this phenomenon may also be associated with pregnancy, oral contraceptive use, lupus anticoagulant, elevated FVIII levels, and rarely mutations other than FV Leiden.4 5 Although APC resistance may occur in the absence of FV Leiden, the converse is usually not true. We describe 2 patients who, following bone marrow and liver transplantation respectively, developed anomalies between APC resistance and FV Leiden genotype.

A 38-year-old female presented with a right-sided iliofemoral deep-vein thrombosis (DVT) secondary to venous obstruction by inguinal lymphadenopathy. She was diagnosed to have stage IV low-grade follicular B-cell non-Hodgkin lymphoma (NHL) and received combination chemotherapy. After a second relapse, she underwent sibling allogeneic progenitor cell transplantation with busulphan (16 mg/kg) and cyclophosphamide (200 mg/kg) conditioning. After transplantation, she developed menopausal symptoms and was considered for hormone-replacement therapy (HRT). Further questioning revealed that, although her stem cell donor was well, her other sibling had previously suffered a spontaneous popliteal DVT. In view of this history and the 2- to 4-fold increased risk of venous thrombosis associated with HRT,6 thrombophilia screening was undertaken. Resistance to APC in FV-deficient plasma was normal. FV Leiden genotyping by PCR and Hind III restriction enzyme analysis, however, demonstrated heterozygosity for FV Leiden (G/A). In view of these unexpected results, mutation analysis of stored pretransplantation DNA was performed. Because this revealed a normal FV genotype (G/G), we surmised that the mutant allele had originated from the donor, and given the history of spontaneous DVT, the other sibling was also likely to be a carrier of FV Leiden. These predictions were confirmed by testing both siblings (Table 1).

The second patient was a 41-year-old female who had undergone orthotopic liver transplantation (OLT) for cirrhosis secondary to Wilson disease. She became pregnant 1 year after OLT. At 20 weeks' gestation she developed progressive hepatocellular failure, and thrombophilia screening was performed to investigate possible hepatic vaso-occlusion. Although her APC-sensitivity ratio (in FV-deficient plasma) of 1.5 would have been consistent with heterozygosity for FV Leiden, genotyping was normal. But DNA analysis is routinely performed on peripheral blood leukocytes, which may not be representative of donor liver genotype. Analysis of donor-liver biopsy samples for FV Leiden demonstrated heterozygosity (G/A), confirming that the mutant FV giving rise to APC resistance originated from the donor liver.

The FV Leiden mutation results in resistance to APC and may be associated with a 3- to 8-fold increased risk of venous thrombosis in heterozygotes and an 80-fold increased risk in homozygotes.1 Screening for FV Leiden involves a combination of coagulation and genetic assays. The conventional APC-resistance assay will detect not only mutant factor V but also several conditions associated with the APC-resistant phenotype that may also pose a thrombotic risk. These conditions may be excluded by use of the modified APC-resistance assay in which samples are prediluted in FV-deficient plasma.7 Confirmatory genetic analysis usually employs DNA extracted from peripheral blood leukocytes. Although these would have the same genotype as hepatic tissue in normal subjects, this might not be the case after transplantation. We highlight this possible discrepancy between genotype and phenotype also previously reported in 2 subjects who, in a reversal of the situation described here, were heterozygous for FV Leiden and received bone marrow and liver transplants, respectively, from FV wild-type donors.8 

In humans, circulating FV is synthesized primarily by hepatocytes with a smaller pool originating from megakaryocytes.8 9 In our first patient, circulating FV synthesized by recipient liver would be of wild type, despite heterozygosity for FV Leiden in donor-derived hemopoietic cells. The risk of venous thromboembolism would not, therefore, be significantly increased, and she was able to receive HRT. In contrast, production of mutant FV by donor hepatocytes indicates that the second patient might have an increased risk of thrombosis despite a normal FV Leiden genotype. We therefore elected to give her thromboprophylaxis in the postpartum period. These cases illustrate the difficulties of thrombophilia testing after tissue transplantation and emphasize the need for evaluating both FV phenotype and genotype in order to accurately assess thrombotic risk in such patients.