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Clinical Biochemistry 47 (2014) 1337–1340
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Clinical Biochemistry
j ourna l homepage: www.e lsev ie r .com/ locate /c l inb iochem
Short Communication
Discordant diagnoses obtained by different approaches in antithrombinmutation analysis
Søren Feddersen, Mads Nybo ⁎Department of Clinical Biochemistry and Pharmacology, Odense University Hospital, Denmark
⁎ Corresponding author at: Dept. of Clinical BiochemisUniversity Hospital, Sdr. Boulevard 29, DK-5000 Odense, D
E-mail address: [email protected] (M. Nybo).
http://dx.doi.org/10.1016/j.clinbiochem.2014.06.0130009-9120/© 2014 The Canadian Society of Clinical Chem
a b s t r a c t
a r t i c l e i n f oArticle history:
Received 2 May 2014Received in revised form 10 June 2014Accepted 11 June 2014Available online 20 June 2014Keywords:AntithrombinMutation analysisRisk assessment
Objectives: In hereditary antithrombin (AT) deficiency it is important to determine the underlying mutationsince the future risk of thromboembolism varies considerably betweenmutations. DNA investigations are in gen-eral thought of as flawless and irrevocable, but the diagnostic approach can be critical. We therefore investigatedmutation results in the AT gene, SERPINC1, with two different approaches.
Design and methods: Sixteen patients referred to the Centre for Thrombosis and Haemostasis, OdenseUniversity Hospital, with biochemical indications of AT deficiency, but with a negative denaturing high-performance liquid chromatography (DHPLC) mutation screening (routine approach until recently) were in-cluded. As an alternative mutation analysis, direct sequencing of all exons and exon–intron boundaries withoutpre-selection by DHPLC was performed.
Results:Out of sixteen patients with a negative DHPLCmutation screening, discordant results were found inten patients (62.5%) when using direct sequencing: Eight had the Basel mutation (c.218CNT), while two had theCambridge II mutation (c.1246GNT). For seven of the ten patients this meant an altered clinical risk-assessmentfor future thromboses.
Conclusions: Awareness must be drawn to the possibility of differences in DNA diagnostics in general andadvances when using newer techniques in particular. One should consider re-analysis of results obtained byearlier sequencing strategies, as clinically important information can be overlooked.
© 2014 The Canadian Society of Clinical Chemists. Published by Elsevier Inc. All rights reserved.
Introduction
It is well-known that hereditary antithrombin (AT) deficiencypredisposes to venous and arterial thromboses [1], and examinationof AT deficiency is an important part of thrombophilia testing. AT isa member of the serpin superfamily with the physiological functionto inhibit thrombin and coagulation factor Xa (and to lesser extentfactors IXa, XIa and XIIa) [2]. Hereditary AT deficiency is inheritedas an autosomal dominant trait, where most cases are heterozygousand found in 4% of families with inherited thrombophilia, 1% ofpatients with a first-time diagnosis of deep venous thrombosis(DVT), and in 0.02% of healthy individuals [3]. SERPINC1, the geneencoding AT, is localised on chromosome 1q23–25.1 and consistsof seven exons spanning 13.4 kb [4]. The most frequent genetic de-fects responsible for hereditary AT deficiency are missense muta-tions, but also nonsense mutations, splice-site mutations, deletions
try and Pharmacology, Odenseenmark. Fax: +45 6541 1911.
ists. Published by Elsevier Inc. All rig
and insertions have been reported (please see the Human GeneMutation Database, www.hgmd.org).
First-line investigation of AT deficiency depends on functional assays,but significant inter-laboratory, inter-individual and intra-individual var-iability impedes the diagnosis [5]. Also, different types can be identified(type I characterized by lowprotein levels and type II characterized by re-duced activity), where type II AT deficiency caused by defects in theheparin-binding site has been found less thrombogenic [3]. Therefore, ge-netic testing is important to disclose the exact reason for AT deficiencyand to determine a correct prophylactic strategy. Furthermore, detectionof a mutation causing AT deficiency may provide useful information forrelatives of index patients.
As mutation diagnostics evolves, new, more thorough methodsare emerging. As recognised in other areas this can eventually leadto alternating results, which is problematic as DNA investigationsin general are considered flawless and irrevocable. We here reporton discordant diagnoses from two different investigation ap-proaches, namely detection of AT sequence variation with denatur-ing high-performance liquid chromatography (DHPLC) followed bysequencing of pre-selected regions in contrast to direct sequencingof all SERPINC1 exons and exon–intron boundaries without pre-selection by DHPLC.
hts reserved.
Table 1Biochemical and genetic findings in 16 patients initially found negative for mutations in the AT gene (SERPINC1) by DHPLC and sequencing. Numbers in brackets are reference intervals.
Antithrombin activityc Type of ATdeficiency
Detection ofmutationsby DHPLCa
Detection ofmutationsby sequencingb
Disease-relatedmutation
Amino-acidchange
Referreddue to
FIIa:Ld
(0.80–1.20)FIIa:Se
(0.80–1.20)FXaf
(0.80–1.20)
Female 0.99 (0.99/0.98) 0.71 (0.72/0.70) 0.85 (0.82/0.88) IIb No Yes c.218CNT p.P73L DVT × 360 years oldFemale 1.05 (1.02/1.07) 0.70 (0.70/0.69) 0.88 (0.79/0.97) IIb No Yes c.218CNT p.P73L Arterial thrombosis42 years oldFemale 1.10 (1.13/1.07) 0.68 (0.62/0.74) 0.76 (0.77/0.75) IIb No Yes c.218CNT p.P73L DVT × 234 years oldMale 0.92 (0.90/0.94) 0.68 (0.72/0.64) 0.74 (0.74/0.73) IIb No Yes c.218CNT p.P73L Family history33 years oldFemale 0.91 (0.90/0.92) 0.69 (0.61/0.77) 0.72 (0.75/0.69) IIb No Yes c.218CNT p.P73L Family history52 years oldMale 0.77 (0.74/0.80) 0.75 (0.71/0.79) 0.90 (0.82/0.98) IIa No Yes c.1246GNT p.A416S Venous thrombosis50 years oldFemale 0.97 (0.99/0.94) 0.72 (0.78/0.66) 1.01 (0.95/1.07) No No Family history41 years oldFemale 0.80 (0.74/0.86) 0.70 (0.74/0.66) 0.98 (0.92/1.04) IIa No Yes c.1246GNT p.A416S DVT × 338 years oldFemale 0.77 (0.72/0.82) 0.64 (0.53/0.74) 0.61 (0.58/0.64) No No Family history62 years oldMale 0.79 (0.79/0.78) 0.79 (0.82/0.76) 0.75 (0.77/0.72) No No DVT43 years oldMale 0.66 (0.56/0.75) 0.75 (0.78/0.72) 0.72 (0.72/0.72) IIb No Yes c.218CNT p.P73L Family history40 years oldMale 0.71 (0.65/0.77) 0.69 (0.70/0.67) 0.76 (0.69/0.83) No No DVT36 years oldFemale 0.67 (0.68/0.65) 0.67 (0.57/0.76) 0.63 (0.66/0.60) No No Arterial thrombosis52 years oldMale 0.62 (0.61/0.62) 0.66 (0.61/0.71) 0.77 (0.77/0.76) No No Venous thrombosis61 years oldFemale 0.60 (0.56/0.63) 0.72 (0.73/0.70) 1.00 (1.00/1.01) IIb No Yes c.218CNT p.P73L Family history51 years oldFemale 0.63 (0.60/0.65) 0.69 (0.68/0.69) 1.03 (1.01/1.05) IIb No Yes c.218CNT p.P73L Arterial thrombosis29 years old
a DHPLC; DNA sequence variation in SERPINC1 detected by DHPLC followed by sequence analysis of amplicons which appear heterozygous.b Sequencing; DNA sequence variation detected by sequence analysis of all SERPINC1 exons without pre-analysis by DHPLC.c Mean of two measurements (shown in brackets).d FIIa:L; AT activity measured using a FIIa-inhibition method with long incubation time, see Materials and methods.e FIIa:S: AT activity measured using a FIIa-inhibition method with short incubation time, see Materials and methods.f FXa; AT activity measured using a Xa-inhibition method, see Materials and methods.
1338 S. Feddersen, M. Nybo / Clinical Biochemistry 47 (2014) 1337–1340
Materials and methods
Patients
Sixteen patients with a personal or family history of venous or arte-rial thrombosis andwith decreased antithrombin activity were enrolledin this study (Table 1). All patients were initially (i.e. using the DHPLCpre-selection approach) found negative for mutations in the AT gene(SERPINC1).
Measurement of antithrombin activity
AT activity was measured using three different methods: Twobased on factor IIa (FIIa)-inhibition and one based on factor Xa(FXa)-inhibition. The FIIa-inhibitor methods will be referred to asFIIa:Long (FIIa:L) and FIIa:Short (FIIa:S). In the FIIa:L method, ATactivity was measured on a STAR coagulometer (Diagnostica Stago,Amsieres-Sur-Seine, France) using the STA Antithrombin III re-agents as described by the manufacturer. Pre-incubation time was60 s. In the FIIa:S method, AT activity was measured as describedearlier [6]. Pre-incubation time was 10 s. For the FXa-inhibitionmethod AT activity was also measured on a STAR coagulometerusing the Coamatic antithrombin reagents (Chromogenix, Mölndal,Sweden) as described by the manufacturer.
Antithrombin gene analyses
Blood samples were collected into EDTA tubes and genomic DNA(gDNA) was extracted from whole blood using a Maxwell 16 BloodDNA Purification Kit (Promega Corporation, Madison, WI, USA). Toidentify sequence variation in the AT gene all seven SERPINC1 exonswere amplified (primer sequences are available upon request) fromgDNA and amplicons were subjected to DHPLC on a WAVE® System(Transgenomic, Inc., Omaha, NE, USA). Amplicons, which by DHPLCappeared heterozygous at one or more of the applied temperatures,were subsequently sequenced bi-directionally on an ABI 3730xl DNAAnalyser (Applied Biosystems, Foster City, CA, USA) using BigDyeTerminator v3.1 Cycle sequencing kit (Applied Biosystems, Foster City,CA, USA). In addition to this approach all samples were analysed bydirect sequencing of all amplified SERPINC1 exons without any initialDHPLC pre-selection step. As described for the DHPLC approach am-plified exons were bi-directionally sequenced on the ABI 3730xl DNAAnalyser.
Results
All patients had biochemistry measurements indicating AT defi-ciency (Table 1). Interestingly, all had decreased AT activity whenmeasured with a short incubation period (IIa:S), while only seven
1339S. Feddersen, M. Nybo / Clinical Biochemistry 47 (2014) 1337–1340
had decreased values with the long incubation period. When usingthe Xa-based assay, eight patients had decreased activity. Differ-ences in these AT measurements were however unrelated to themutations found.
As described, no mutations were found by the DHPLC pre-selectionapproach. By direct sequencing, SERPINC1 mutations were found inten out of 16 patients: Eight had the Basel mutation (c.218CNT), whiletwo had the Cambridge II mutation (c.1246GNT). For seven of theten patients this meant an altered clinical risk-assessment for futurethromboses. There was no significant relation between the finding ofan un-revealed mutation and the presence of clinical symptoms and/or a positive family history.
Discussion
As DNA analyses becomes more feasible and cheaper, they are in-creasingly used to pin-point a final diagnosis. Mutation analyses are ingeneral regarded as safe, utterly correct and irrefutable. However, theyare often an algorithmic process consisting of different steps in orderto align the analytical process and to diminish expensive labour time.Therefore, different approaches to a mutation analysis can introducesources of error that is not evident.Wehere show that ten out of 16 per-sons with a biochemical antithrombin deficiency, but a negative ATmutation analysis, came out positive when investigated by direct se-quencing omitting pre-selection by DHPLC. This is important informa-tion since DHPLC for a long period of time has been used in detectionof AT mutations [7].
Hereditary AT deficiency is a serious risk factor for thromboembolicdisease, but risk assessment highly depends upon the type of mutationcausing the AT deficiency. As described, themutations foundusing a dif-ferent analysis approach altered the clinical risk-assessment for a largeproportion of the patients in this study. So if DHPLC has been used forscreening of SERPINC1, re-analysis of earlier patients with AT deficiencyought to be considered. Importantly, we cannot exclude that the lack ofsensitivity observed in this study is specifically linked to our DHPLCsetup. DHPLC has been used for mutation screening of numerousdisease genes including TP53 [8], ABCD1 [9] and CFTR [10] and hasbeen shown to be very sensitive with potential detection rates ofN95–100% [11]. But our study underscores that thorough validationis necessary when using DHPLC for mutation screening and the out-come of mutation analysis may indeed depend on the methodologyused.
Two different missense mutations (p.P73L in exon 2 and p.A416Sin exon 6) missed detection by DHPLC in our study although PCRsamples were run at several different column temperatures: exon2 PCR samples were run at 59.0 °C, 59.6 °C, 60.0 °C and 61.8 °Cand exon 6 PCR samples were run at 54.4 °C, 57.0 °C, 57.8 °C and60.5 °C. Since the GC content of the region near a mismatch may in-fluence the analysis temperature necessary to detect a certainhetero-duplex by DHPLC [12], we examined the GC content of theregion near the p.P73L and the p.A416S mutations: The GC contentof the region within 20 nucleotides of the p.P73L and the p.A416Smutations were 65% and 55%, respectively. In comparison, the GCcontent of the region within 20 nucleotides of the p.R45W (inexon 2) and the p.S426L (in exon 6) mutations were 70% and 65%,respectively. Since the p.R45W and the p.S426L mutations were pre-viously detected by DHPLC in our laboratory, since their GC contentis relatively high and since they are situated close to the p.P73L andthe p.A416S mutations, missed detection of the p.P73L and thep.A416S mutations by DHPLC does not seem to be caused by ahigh GC content. The reason for missed detection of the p.P73L andthe p.A416S mutations by DHPLC is currently unknown, and generalprecaution must therefore be taken when using DHPLC for AT muta-tion analysis. This can have implications for AT deficiency in specific,but also for mutation analyses in general: As an example, ProteinS (PS) analysis has a well-known, sad reputation for not displaying
a detectable mutation in almost 2/3 of the patients with biochemicalPS deficiency [13]. Of note, PS deficiency can be caused by many fac-tors and is not necessarily inherited. But the more important is thecertainty of the mutation analysis, and it would therefore be inter-esting to elucidate whether former experiences with poor mutationfrequencies could be due to the mutation analysis approach.
Type II AT deficiency can be sub-classified into three types: IIa,IIb and IIc. Of these, types IIa and IIb were observed in this study(Table 1). Types IIa and IIb are caused bymutations that affect the reac-tive domain and the heparin-binding domain of AT, respectively [14].Notably, the two patients with the type IIa variant presented with nor-mal AT activity when using the FXa inhibition-activity assay (Table 1).This is in agreement with a previous report showing that normal AT ac-tivity values may occur in patients with type IIa mutations when apply-ing an FXa inhibition-based test [15]. Moreover, two of the patientswitha type IIb mutation also presented with normal AT activity levels usingthe FXa inhibition-activity assay (Table 1) suggesting that this methodmay be affected also by other type II mutations. Altogether, this sup-ports previous findings indicating that there is a risk of not detectingall type II deficiencies when using FXa inhibition-based methods alone[14,15].
When discussing routine AT genetic testing it is importantto mention large rearrangements since special tests are needed todetect these. In a recent study, 188 patients with low AT proteinlevels underwent AT mutation testing including test for largedeletions and duplications by multiplex ligation-dependent probeamplification (MLPA). Interestingly, the MLPA analysis revealedeight patients (4%) with large deletions [16], suggesting that testsdetecting large rearrangements should be implemented in routineAT genetic testing. Again, a novel approach seems to refine DNAtesting.
In conclusion, laboratories must be aware of the possibility of dif-ferences in DNA diagnostics in general and alternations when usingnew techniques in particular. Especially, an altered algorithmicapproach can cause discordant findings, which can by vital to thepatient. One should consider re-analysis of results obtained byearlier sequencing strategies, as clinically important informationcan be overlooked.
References
[1] Kuhle S, Lane DA, Jochmanns K, Male C, Quehenberger P, Lechner K, et al. Homozy-gous antithrombin deficiency type II (99 Leu to Phemutation) and childhood throm-boembolism. Thromb Haemost 2001;86:1007–11.
[2] Bafunno V, Margaglione M. Genetic basis of thrombosis. Clin Chem Lab Med2010;48(Suppl. 1):S41–51.
[3] Lane DA, Mannucci PM, Bauer KA, Bertina RM, Bochkov NP, Boulyjenkov V, et al.Inherited thrombophilia: part 1. Thromb Haemost 1996;76:651–62.
[4] Olds RJ, Lane DA, Chowdhury V, De Stefano V, Leone G, Thein SL. Complete nucleo-tide sequence of the antithrombin gene: evidence for homologous recombinationcausing thrombophilia. Biochemistry 1993;32:4216–24.
[5] CunninghamMT, Olson JD, Chandler WL, Van Cott EM, Eby CS, Teruya J, et al. Exter-nal quality assurance of antithrombin, protein C, and protein s assays: results of theCollege of American Pathologists proficiency testing program in thrombophilia. ArchPathol Lab Med 2011;135:227–32.
[6] Kristensen SR, Rasmussen B, Pedersen S, Bathum L. Detecting antithrombin defi-ciency may be a difficult task—more than one test is necessary. J Thromb Haemost2007;5:617–8.
[7] Wang LP, Huang SW, Wang SS, Wang C, Mou TY, Zhong M. Mutation screening ofthe AT-III gene in pregnant and post-partum women with venous thromboembo-lism by DHPLC. Lab Med 2010;41:667–71.
[8] Loyant V, Jaffré A, Breton J, Baldi I, Vital A, Chapon F, et al. Screening of TP53mutations by DHPLC and sequencing in brain tumours from patients withan occupational exposure to pesticides or organic solvents. Mutagenesis2005;20:365–73.
[9] Montagna G, Di Biase A, Cappa M, Melone MA, Piantadosi C, Colabianchi D,et al. Identification of seven novel mutations in ABCD1 by a DHPLC-basedassay in Italian patients with X-linked adrenoleukodystrophy. Hum Mutat2005;25:222.
[10] Ravnik-Glavac M, Atkinson A, Glavac D, Dean M. DHPLC screening of cystic fibrosisgene mutations. Hum Mutat 2002;19:374–83.
[11] Mitchell M, Cutler J. Denaturing HPLC for mutation screening. Methods Mol Biol2011;688:17–33.
1340 S. Feddersen, M. Nybo / Clinical Biochemistry 47 (2014) 1337–1340
[12] Yu B, Sawyer NA, Chiu C, Oefner PJ, Underhill PA. DNA mutation detection usingdenaturing high-performance liquid chromatography (DHPLC). Curr Protoc HumGenet 2006:7.10.1-14 (Chapter 7).
[13] Bathum L, Münster AM, Nybo M, Madsen JS, Larsen TB, Kristensen SR. Denaturinghigh-performance liquid chromatography mutation analysis in patients withreduced protein S levels. Clin Chim Acta 2008;390:76–81.
[14] Patnaik MM, Moll S. Inherited antithrombin deficiency: a review. Haemophilia2008;14:1229–39.
[15] Ungerstedt JS, Schulman S, Egberg N, Antovic J, Blombäck M. Discrepancybetween antithrombin activity methods revealed in Antithrombin Stockholm:do factor Xa-based methods overestimate antithrombin activity in some patients?Blood 2002;99:2271–2.
[16] Caspers M, Pavlova A, Driesen J, Harbrecht U, Klamroth R, Kadar J, et al.Deficiencies of antithrombin, protein C and protein S — practical expe-rience in genetic analysis of a large patient cohort. Thromb Haemost2012;108:247–57.
