False-Negative Factor V Leiden Genetic Testing in a Patient With Recurrent Deep Venous Thrombosis

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1 American Journal of Hematology 81: (2006) False-Negative Factor V Leiden Genetic Testing in a Patient With Recurrent Deep Venous Thrombosis Edward N. Libby, 1 * Jessica K. Booker, 2 Margaret L. Gulley, 2 David Garcia, 1 and Stephan Moll 3 1 Department of Internal Medicine, University of New Mexico Health Sciences Center, Albuquerque, New Mexico 2 Department of Pathology, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina 3 Department of Internal Medicine, Division of Hematology-Oncology, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina False-negative genetic testing of the factor V Leiden (fvl) mutation is unusual. We report a case of a young woman with a history of deep venous thrombosis tested for the fvl at four separate laboratories on four separate dates. Two laboratories reported the patient to be heterozygous for the fvl, while the other two reported no evidence of a mutation. Testing methods of the various laboratories were reviewed, and additional testing was performed on stored and newly drawn DNA samples, including sequencing of the fvl gene segment. The preponderance of evidence indicates the patient to be heterozygous for the fvl mutation. Dissection of data suggests that either sample misidentification or faulty allele specific amplification methods could have led to false-negative results in two laboratories. In one of the two laboratories, misinterpretation of results and clerical error could not be excluded. There is a need for standardization of optimized fvl genetic testing methods. Further education of ordering physicians on the limitations of genetic testing is necessary. Am. J. Hematol. 81: , VC 2006 Wiley-Liss, Inc. Key words: Factor V Leiden; fvl; genetic testing; laboratory error; polymerase chain reaction; misclassification INTRODUCTION The factor V Leiden (fvl) mutation is a 1691G?A mutation in the factor V gene leading to an R506Q substitution in the factor V protein. It is the most common inherited thrombophilic condition, found in approximately 5% of Caucasians in the U.S. [1]. Heterozygous mutation increases the risk of primary venous thrombosis 3- to 7-fold, while the homozygous mutation increases the risk approximately 80- fold [2]. Although the fvl mutation is frequently tested for, there is lack of agreement on the best laboratory method for its detection [3,4]. Paucity of FDAapproved tests for the fvl mutation contributes to a lack of standardized methods for genetic testing among clinical laboratories. We report a case of a young woman with a history of deep venous thrombosis (DVT) tested for the fvl mutation at four separate laboratories on four separate dates. Two laboratories reported the patient VC 2006 Wiley-Liss, Inc. to be heterozygous for the fvl mutation, while the other two reported no evidence of mutation. Upon review of testing methods and additional testing on stored and newly drawn DNA samples, including sequencing of the relevant segment of the factor V gene, the preponderance of evidence indicates the patient to be heterozygous for fvl. Dissection of data suggests that either clerical errors at both laboratories with false-negative results occurred or that the allelespecific amplification method used by the laboratories *Correspondence to: Edward N. Libby, M.D., UNM Cancer Research and Treatment Center, 900 Camino de Salud NE, MSC # , Hematology/Oncology, Albuquerque, NM elibby@salud.unm.edu Received for publication 19 February 2005; Accepted 21 August 2005 Published online in Wiley InterScience ( DOI: /ajh.20543

2 Case Report: False-Negative fvl in a Patient With Recurrent DVT 285 was insensitive to mutation detection. This case is presented to (a) demonstrate the need for standardization of fvl genetic test methods, (b) stress the importance of optimal clerical handling of samples and test results, and (c) illustrate the need to educate physicians about the limitations of genetic testing. episodes of superficial thrombophlebitis. Because of her thrombotic problems and the discrepant fvl test results, the patient was referred to our tertiary-care center. Review of the patient s records indicated that the repeat fvl tests had been ordered either because the treating physicians did not have access to earlier data or to clarify discrepant results. CASE REPORT In November 1995, an 18-year-old woman developed an acute ileofemoral and popliteal deep venous thrombosis (DVT) of the left leg several weeks after initiating oral contraceptives. Her oral contraceptives were discontinued, and she was treated for 6 months with warfarin. In June of 1996, the patient developed recurrent DVT of the left leg, and warfarin was reinitiated for several months. Factor V mutation testing was performed (Laboratory 1, 1996), and the patient was reported to be heterozygous for the fvl mutation. In 1999, another fvl mutation test (Laboratory 2, 1999) was reported to be negative for the mutation. In 2000, a third fvl test was ordered (Laboratory 3, 2000), and the patient was reported to be negative for the mutation. In June 2001, the patient was attempting to conceive. Based on the recent negative fvl result, a reproductive medicine specialist felt it was safe to start clomiphene to induce ovulation. Clomiphene is an ovarian stimulant with estrogenic and anti-estrogenic properties that has been reported to increase the risk of venous thromboembolism. Two months later, the patient presented with extensive right-leg DVT. The patient was anticoagulated. In 2001, a fourth fvl mutation test (Laboratory 4, 2001) was reported to show heterozygosity for fvl. In early 2002, the warfarin was stopped, and subsequently the patient had four MATERIALS AND METHODS The Human Research Review Committee from the submitting author s institution reviewed this report and approved its submission for publication. All four laboratories that had performed fvl mutation analysis on the patient s DNA were contacted and asked to review the results of their earlier work and to describe the methods used. Two of the laboratories still had residual DNA available for retesting. Pertinent data related to the genetic tests are summarized in Table I. RESULTS Laboratory 1 had tested a blood sample in 1996 by polymerase chain reaction (PCR) followed by digestion with HindIII restriction endonuclease (which cuts the mutant but not the wild-type allele) and gel electrophoresis, and reported the patient to be heterozygous for the fvl mutation. A second blood sample was sent to the same laboratory in 2003 as part of the current investigation. Both the new sample and the original sample from 1996 were then tested by the method described above and also by a second method relying on analyte specific reagents from Roche Molecular Biochemicals and a Roche Light- Cycler instrument (Roche Diagnostics Corporation, TABLE I. Factor V Leiden Test Dates, Methods, and Results by Laboratory* Testing site Type of laboratory Accreditation Sample date Test date Method Result Lab. 1 Academic medical center (UNC) CLIA PCR, HindIII digestion Heterozygous CAP 2003 Roche ASR on LightCycler 1 Heterozygous PCR, HindIII digestion Heterozygous 2003 Roche ASR on LightCycler 1 Heterozygous 2003 DNA Sequencing Heterozygous Lab. 2 Academic medical center CLIA Allele-specific PCR Wild-type CAP 2003 Multiplex PCR, TaqI digestion Heterozygous 2003 DNA Sequencing Heterozygous Lab. 3 Commercial CLIA Allele-specific PCR Wild-type 1996 a 2003 Allele-specific PCR Heterozygous 2003 a 2003 Allele-specific PCR Heterozygous Lab. 4 Commercial CLIA Promega READIT 1 Heterozygous CAP *Abbreviations: UNC, University of North Carolina; PCR, polymerase chain reaction; ASR, analyte-specific reagent. a These samples were aliquots from DNA that was extracted by Laboratory 1.

3 286 Case Report: Libby et al. Indianapolis, IN) in which PCR followed by melting curve analysis was done. Results demonstrated that both samples were heterozygous for the fvl mutation by both methods. Heterozygosity was confirmed by gene sequencing, with no sequence variants identified that could potentially interfere with interpretation of any of the assays used by the four laboratories. Laboratory 2 had tested a blood sample in 1999 by simultaneous allele-specific PCR using one primer set targeting the normal allele and another targeting the mutant allele, followed by gel electrophoresis, and reported the patient to be negative for the fvl mutation. The same sample was retested in 2003 using the laboratory s current method, which is a multiplex assay for the simultaneous detection of the fvl and prothrombin mutations by PCR amplification, digestion with TaqI restriction endonuclease (which cuts the wild-type but not the mutant factor V allele), and gel electrophoresis. Results from the current method identified the patient as heterozygous for the fvl mutation. Heterozygosity was further confirmed in that laboratory by DNA sequencing, with no evidence of sequence variants that might have interfered with interpretation of their assays. The results from 1999 were reviewed, and neither misinterpretation of the raw data nor clerical transcription error was identified. Laboratory 3 tested a sample in 2000 by allelespecific amplification followed by gel electrophoresis and reported the patient to be negative for the fvl mutation. The original sample was no longer available for retesting, but both samples from Laboratory 1 were provided to Laboratory 3, and the same method that was used in 2000 was applied and identified both samples from Laboratory 1 to be heterozygous for the fvl mutation. Laboratory 4 tested a sample in 2001 by Promega s READIT 1 SNP Genotyping System (Promega Corp., Madison, WI) and reported the patient as heterozygous for the fvl mutation. The primer and interrogation oligonucleotide sequences were considered to be proprietary and were not made available for this study. DISCUSSION The preponderance of evidence suggests that the patient is heterozygous for the fvl mutation. The strongest supportive evidence comes from DNA sequencing performed independently by Laboratory 1 and Laboratory 2, both of which identified the 1691G?A mutation. Because both wild-type and mutant alleles were identified, it cannot be argued that the sequencing strategy preferentially favored one allele over the other. It is interesting that the two laboratories that reported the patient as false negative for the fvl mutation used nearly identical primers (Fig. 1). Sequence variants within the targeted primer-binding region can theoretically lead to false-negative fvl results. However, no such variant was found upon sequencing studies done in laboratories 1 and 2. Laboratory 2 correctly identified the mutation that it first missed, using the same DNA sample with a different method. This demonstrates that if a sample misidentification occurred, it was after sample collection and DNA extraction. In other words, we cannot exclude sample misidentification during assay set-up as the cause for the false-negative results from Laboratory 2. Alternatively, it is possible that the 1999 allele-specific amplification method was insensitive to mutation detection. Laboratory 3 no longer had the original DNA sample or the original gel photograph, making it impossible to determine whether the error was due to sample misidentification, failure of the assay, incorrect interpretation of the results, or clerical error transcribing the results. However, the same assay method was applied to patient samples provided by Laboratory 1, and these samples were correctly identified as heterozygous, suggesting that the initial false-negative interpretation was not a reproducible defect in the PCR step. This would argue for a sample misidentification, incorrect interpretation of test results, or clerical error as a cause of the falsenegative result from laboratory 3. Mutation testing is not foolproof and is subject to operator errors in technique and interpretation. Recent publications have reported a % accuracy in detecting the heterozygous fvl mutation [5,6]. Error is also reported for the 20210G?A prothrombin gene mutation [6,7]. False-negative genetic testing has been found with other genetic diseases, such as familial adenomatous polyposis involving the APC gene [8]. Common reasons for incorrect laboratory reports are shown in Table II. Perhaps the most common reason is sample misidentification occurring at the time of collection or in the laboratory [9]. From the time of sample collection to the time data are used in clinical decision making, there are multiple points where a misidentification can occur. Because the number of manual specimen transfers is relatively high in molecular laboratories, technologists must be meticulous in maintaining sample integrity and identity. In molecular genetic testing, sequence variants beyond the specific mutation being tested for can

4 Case Report: False-Negative fvl in a Patient With Recurrent DVT 287 Fig. 1. Primers used by three of four laboratories to test for the G1691A factor V Leiden mutation (arrow). The factor V gene exon 10 sequence is in upper case letters, and the flanking intronic sequences are in lower case letters. Laboratory 2 used two different assays, an allele-specific PCR utilizing four primers, and a multiplex PCR using two primers for factor V and two not shown targeting the Prothrombin gene. The underlined nucleotides indicate a change made to introduce a restriction site. Sequence information was not available on the proprietary analyte specific reagents used by Laboratory 4 (Promega) and Laboratory 1 (Roche). interfere with the assay, depending on the specific variant and the method used. In an assay that relies on a restriction endonuclease recognition sequence to distinguish between the presence or absence of a mutation, a sequence variant can alter the expected restriction digestion pattern. In amplification assays, a sequence variant present within the primer region can interfere with the efficiency of amplification of the allele carrying the variant and, in some instances, can entirely prevent amplification of that allele. Even DNA sequencing, which is generally considered to be the definitive method, can be misleading due to preferential amplification of a given allele. In addition, sequencing cannot differentiate between the presence of one or two identical alleles and therefore cannot detect a deletion of one allele in the sequenced region. It is within these limitations that all laboratory results must be interpreted, and it is why the polymorphism rate should be investigated when designing such assays. Many different approaches to detecting a mutation are employed in clinical laboratories [10]. Some investigators assert that PCR is the most practical method for clinical use, and most laboratories utilize some variation of PCR [4,5]. The spectrum of testing commonly used by clinical laboratories includes (i) PCR amplification of the region encompassing the mutation followed by restriction enzyme digestion and gel electrophoresis, (ii) PCR using primermediated restriction endonuclease site generation followed by restriction enzyme digestion and gel electrophoresis, (iii) allele-specific PCR followed by gel electrophoresis, (iv) real-time PCR followed by melt curve analysis using fluorochrome-labeled internal probes, and (v) proprietary methods marketed by commercial vendors. Nearly all of these alternative

5 288 Case Report: Libby et al. TABLE II. Contributors to Incorrect or Conflicting Genotype Results Misidentification or Clerical Error Sample mislabeling at the time of collection Sample misidentification during sequential steps of test procedure Error in recording result on laboratory worksheet Improper data entry or transmission Procedural Explanations Suboptimal extraction and purification of nucleic acid Imperfect analytic sensitivity or specificity Inhibitor of amplification reaction Amplicon contamination Random preferential amplification of one allele DNA polymerase replication error Inadequate restriction enzyme digestion Inappropriate quality control or quality assurance Technical error not detected by internal or external control assays Failure to compare with previous result (delta check) Faulty interpretation of raw data Patient-Specific or Clinical Characteristics Polymorphism interfering with hybridization of primers or probes Polymorphism interfering with restriction enzyme digestion Mosaicism (genotype differs by anatomic site) Allogeneic transplantation causing chimerism Transfusion of nucleated cells (granulocyte unit or platelet unit rich in white cells) methods were used among the four clinical laboratories surveyed in this study, emphasizing the lack of standardization and the potential problem that this causes in patient care. Consensus statements regarding fvl genetic testing published by the American College of Medical Genetics (ACMG) and the College of American Pathologists (CAP) [3,4] do not favor a specific assay for fvl testing. The ACMG recommends that the factor V Leiden mutation test should be performed using any of the accepted technical approaches as long as they have been properly validated by the laboratory, while adhering to current ACMG/CAP quality assurance guidelines for molecular genetic testing. Cases like this one suggest that studying large cohorts of patients might reveal subtle differences in assay performance that could be used to further define optimized methods. FDA approval of optimized assays contributes to standardization of laboratory practice. Currently only one fvl mutation test kit is FDA approved, and it was not yet in existence at the time that our patient was tested. In the United States, federal law requires that laboratories performing genetic testing undergo twice-yearly proficiency testing. Most testing laboratories subscribe to a CAP proficiency survey in which blinded challenges are mailed out for fvl analysis, and submitted results are subsequently compared among all laboratories in relation to the analytic method that was employed. When deciding which laboratory will perform patient testing, it is reasonable to inquire about accreditation status. In the United States, laboratory inspection and accreditation are typically in the purview of the Clinical Laboratory Improvement Act (CLIA), the Joint Commission on Accreditation of Healthcare Organizations (JCAHO), and the College of American Pathologists (CAP). It is not uncommon for the ordering physician to be unaware of the limitations of a test that they are requesting. Some physicians naively believe that genetic testing is infallible, and, as a consequence, the results may not be questioned [11]. We found only one prior case report of discrepant fvl test in the medical literature [12]. The case involved a patient whose blood genotype changed after stem cell transplant for acute myelogenous leukemia. Other discrepant test results have been published in relation to quality assurance work. In Italy, a qualityassurance performance survey of 30 clinical laboratories found that 5% of them failed to detect heterozygous fvl, 3.4% homozygous fvl, 5.2% heterozygous prothrombin 20210G?A, and 17.2% homozygous prothrombin 20210G?A [6]. In the United Kingdom, a quality assurance exercise involving 47 participants determined that 3 6% of challenges failed to detect fvl or prothrombin gene mutation [13]. In the Italian survey, poor sensitivity was associated with laboratory tests developed inhouse as opposed to using commercial kits. No false-positive fvl test results were reported. The etiology of the discrepant results in our patient remains uncertain. Neither transplantation nor transfusion is implicated in her case. Sample misidentification at various steps of the laboratory testing process is probably the most common cause for incorrect genotyping results, and is a possibility for the discrepant results in our patient. However, it would be an unusual coincidence that laboratories 2 and 3 were both plagued by this source of error. We were unable to find any analytic explanation for the errors. However, one thread in common among the two problematic assays was that they were both allelespecific PCRs that rely on a single 3 0 mismatch to distinguish normal from mutant sequences. Further work is needed to determine if this type of assay is more prone to error than the other analytic strategies commonly used by clinical laboratories. In general, genotyping is safe and effective when performed in an accredited laboratory using a validated assay with appropriate controls. Nevertheless, false results may occur. This is not unique to fvl. To improve fvl testing, standardization of fvl genetic test methods is desirable. Also, strict adherence to optimal clerical handling of samples

6 and results is critical. In addition, clinicians need to understand the limitations of genetic testing. Repeat confirmatory testing may be appropriate if testing results are unexpected or do not seem to make sense from an inheritance pattern point of view. Finally, the phenomenon of false-negative and false-positive test results outside the setting of routine proficiency testing should be studied more extensively, and a larger number of laboratories should be surveyed. REFERENCES 1. Ridker PM, Miletich JP, Hennekens CH, Buring JE. Ethnic Distribution of Factor V Leiden in 4047 men and women. Implications for venous thromboembolism screening. JAMA 1997;277: Federman DG, Kirsner RS. An update on hypercoagulable disorders. Arch Intern Med 2001;161: Grody WW, Griffin JH, Taylor AK, Korf BR, Heit JA. American College of Medical Genetics Consensus Statement on factor V Leiden mutation testing. Genet Med 2001;3: Press RD, Bauer KA, Kujovich JL, Heit JA. Clinical utility of factor V Leiden (R506Q) testing for the diagnosis and management of thromboembolic disorders. Arch Pathol Lab Med 2002; 126: Case Report: False-Negative fvl in a Patient With Recurrent DVT Lutz CT, Foster PA, Noll WW, et al. Multicenter evaluation of PCR methods for the detection of factor V Leiden (R506Q) genotypes. Clin Chem 1998;44: Tripodi A, Peyvandi F, Chantarangkul V, Menegatti M, Manucci PM. Relatively poor performance of clinical laboratories for DNA analyses in the detection of two thrombophilic mutations- a cause for concern. Thromb Haemost 2002;88: McGlennen RC, Key NS. Clinical and laboratory management of the prothrombin G20210A mutation. Arch Pathol Lab Med 2002;126: Giardiello FM, Brensinger JD, Petersen GM, et al. The use and interpretation of commercial APC gene testing for familial adenomatous polyposis. N Engl J Med 1997;336: Bonini P, Plebani M, Ceriotti F, Rubboli F. Errors in laboratory medicine. Clin Chem 2002;48: Voelkerding KV. Resistance to activated protein C and a novel factor V gene mutation. Clin Lab Med 1996;16: Freidman LC, Plon SE, Cooper HP, Weinberg AD. Cancer genetics survey of primary care physicians attitudes and practices. J Cancer Educ 1997;12: Crookston KP, Henderson R, Chandler WL. False-negative factor V Leiden assay following allogenic stem cell transplant. Br J Haematol 1998;100: Preston FE, Kitchen S, Jennings I, Woods TAL. A UK National External Quality Assessment Scheme (UK Neqas) for molecular genetic testing for the diagnosis of familial thrombophilia. Thromb Haemost 1999;82: