Abstract
Inherited activated protein C resistance (APCR) is an autosomal dominant condition caused by a single point mutation in exon 10 of the F5 gene (G to A transversion at nucleotide 1691) resulting in an arginine to glutamine substitution at amino acid 506 of the factor V protein (FV Leiden). 1 This missense mutation changes the sequence of one of the activated protein C cleavage sites, suppressing factor V inactivation in vivo, and the described mechanism explains the prothrombotic tendency of individuals carrying the mutant allele. 2 The effect can also be measured in vitro by a clotting assay 3 that forms the basis of the functional APCR diagnostic test. 4 Although polymerase chain reaction (PCR)-based FV Leiden mutation testing is considered the definitive diagnostic test for use in stratifying risk of deep vein thrombosis (DVT) and recurrent DVT, it remains unclear whether testing and the resultant management will improve outcomes in adults with venous thromboembolism. 5 The majority of individuals with APCR have inherited the FV Leiden mutation. 6 Less commonly, APCR is due to acquired conditions, such as lupus inhibitor, cancer, and pregnancy. 7
Here we describe 4 different cases referred for thrombophila workup at our regional reference laboratory where results from functional and molecular testing did not correlate. In all cases, the disparity was not due to acquired APCR but because all testing were performed on peripheral blood of patients with unusual clinical histories. Case 1 was an adult female from a family with quantitative factor V deficiency. Case 2 was a young male with congenital urea cycle disorder and homozygosity for FV Leiden. He underwent a maternal (living donor) liver transplant. Case 3 was an adult male with a history of allogeneic bone marrow transplantation (allo-BMT) for acute myeloid leukemia, multiple posttransplant pulmonary embolisms, and heparin-induced thrombocytopenia. Case 4 was an adult female with no personal history of thrombosis. She underwent allo-BMT for multiple myeloma.
Results from functional and molecular assays are presented in Table 1. The APCR assay was performed on platelet-poor plasma using a commercially available kit with factor V-deficient plasma as sample diluent (Beckman Coulter, Mississauga, Ontario, Canada) on an ACL Advance Coagulizer platform (Instrumentation Laboratory Co, Bedford, Massachusetts). This assay is highly sensitive and specific for the FV Leiden mutation. Calculated APCR ratio cutoff values of <1.4 (homozygous mutant), 1.4 to 2.0 (heterozygous), and ≥2.1 (normal) were previously established by correlation of APCR ratios with the corresponding genotyping data from 375 normal (G/G), 963 heterozygous (A/G), and 40 homozygous mutant (A/A) individuals.
Summary of Patient Results From Peripheral Blood Specimens
Abbreviations: APCR, activated protein C resistance; nt, nucleotide.
a In uncomplicated situations, relationship between APCR ratio and nt 1691 genotype: ≥2.1 for G/G (normal), 1.4 to 2.0 for A/G (FV Leiden heterozygous), and <1.4 for A/A (FV Leiden homozygous).
The FV Leiden molecular assay was performed on EDTA-anticoagulated peripheral blood aliquots using our previously described, whole blood allele-specific PCR assay. 8
In all cases, results from our calibrated APCR assay did not reflect the determined FV Leiden genotype. In cases 1 and 4, normal ratios (>2.1) were obtained, while gene testing showed heterozygosity (A/G) for the FV Leiden mutation. The individual in case 1 also had low factor V coagulant activity. In case 2, an APCR ratio of 1.8 was observed, but this individual was homozygous (A/A) for the FV Leiden mutation by molecular assay. Case 3 had an abnormal APCR ratio but genotyping demonstrated only the wild-type gene sequence (G/G).
Results presented can be correctly interpreted after careful consideration of the unusual clinical histories of these patients. In case 1, the phenotype can most likely be explained by heterozygosity for a rare F5 gene allele carrying both an as yet uncharacterized family specific, F5 gene null mutation and the FV Leiden mutation. In this kindred, expression of only the wild-type F5 allele in heterozygotes (pseudohomozygous normals) will result in a normal APCR ratio but with decreased factor V coagulant activity. The proband had a normal APCR ratio (2.6) and factor V activity level of 30 IU/dL. In her family, 5 other affected family members who were heterozygous for FV Leiden were also found to have normal APCR ratios (>2.1) and factor V clotting activities below the locally established normal cutoff (≥50 IU/dL), while the only available unaffected daughter with normal plasma FV activity (80 IU/dL) was homozygous for the wild-type F5 gene sequence at nucleotide 1691. Our case cannot be explained by a presence of previously described F5 1690 C→T termination mutation, which causes false positive results in factor V-deficient patients using FV Leiden molecular assays based on loss of a Mnl1 restriction site, 9 as our PCR assay is specific for the F5 1691 G→A mutation. 8 Instead, case 1 is an alternate to the more commonly reported double heterozygosity for a F5 null mutation and FV Leiden that results in pseudohomozygous FV Leiden 10 and similar to one other report of a F5 null/FV Leiden allele in a family with FV deficiency. 11
In case 2, the liver transplant patient would be expected to have the phenotype of the donor liver, where the circulating plasma factor V is synthesized. The calculated APCR ratio is consistent with a heterozygous FV Leiden liver donor who was also his biological mother and a carrier of the FV Leiden mutation. Three further cases of disparate results from posttransplant analyses have been identified, although we were unable to directly assess the FV Leiden genotype status of anonymous liver donors.
Cases 3 and 4 represent other instances where the medical treatment resulted in tissue-specific chimeras. In case 3, bone marrow from a donor subsequently proven homozygous for wild-type FV was used for allo-BMT of an individual heterozygous for FV Leiden. Case 4 illustrates the alternate situation, where the normal BMT recipient was transplanted with bone marrow from a donor subsequently proven heterozygous for FV Leiden. In both cases, the APCR assay of posttransplant patient plasma reflected the recipient liver-derived FV Leiden function, while the molecular assay on peripheral blood detected the genotypes of the engrafted donor hematopoietic cells. Retrospective analysis of 290 allo-BMT donor/recipient pairs from our patient cohort has identified 5 additional instances with mismatched FV Leiden genotypes.
Although rare, these examples illustrate the problem of interpreting molecular data in isolation. Due to the relatively high diagnostic and prognostic impact of molecular diagnostic assays, clinicians need to recognize the importance of providing all relevant clinical history to the testing laboratory. Additionally, we suggest that results from molecular testing based on DNA from peripheral blood should be interpreted with caution in individuals with complex medical conditions and always in the context of results from functional, coagulation-based assays.