Quality in Molecular Biology Testing for Inherited Thrombophilia Disorders

Abstract

As the understanding of the genetic basis of the inherited thrombophilias has increased over recent years, their routine diagnostic genetic analysis has also matured. This review considers methods used to test for the factor V (F5) Leiden mutation and prothrombin 20210A (F2 c.*97G>A) allele, and analysis of the SERPINC1, PROC, and PROS1 genes in cases of antithrombin, protein C (PC), and protein S (PS) deficiency, respectively. Issues relating to quality are explored, highlighting where analytical and sample handling errors may occur. Detection of the factor V Leiden mutation and the prothrombin c.*97G>A allele are best performed using real-time polymerase chain reaction analysis as this relatively simple technique allows their discrimination from rare variants of neighboring nucleotides; not possible using the more time-consuming restriction digestion assays. With the advent of low-cost and high-throughput sequence analysis, direct sequencing has become the first-line method to provide a definitive diagnosis of inherited, rather than acquired, deficiencies. Large cohort studies have shown that antithrombin and PC mutations are identified in between 61 and 87% of patients, whereas the detection rate in PS deficiency is substantially lower in around 40% of patients. Large gene deletions make up between 7 and 10% of PS and antithrombin mutations and only 1% of PC mutations, but it is suggested that dosage analysis techniques such as multiplex ligation-dependent probe amplification should be used for all three genes as part of routine analysis to ensure mutations are not missed. Best practice guidelines are available from EuroGentest covering a wide variety of the issues raised in this review and all laboratories should participate in appropriate external quality assurance schemes to ensure they continue to offer high quality service.

Distinct frequencies and mutation spectrums of genetic thrombophilia in Korea in comparison with other Asian countries both in patients with thromboembolism and in the general population

“…47,48 None of the population group had large dosage mutations. A total of 16 novel mutations were identified in this study, two in PROC, seven in PROS1, and seven in SERPINC1 (Table 1).…”

“…Large dosage mutations were frequent in AT deficiency and PS deficiency in our group of patients (18.2% in SERPINC1 and 14.3% in PROS1) (Figure 2), and they were more frequent than in previous studies (9.1% and 6.4%, respectively). 47, 48 Although we did not perform MLPA experiments for PROC, the number of cases with large dosage mutations causing PC deficiency would have been quite limited when considering the rarity of these mutations in PROC (0.6%). 47,48 None of the population group had large dosage mutations.…”

“…47, 48 Although we did not perform MLPA experiments for PROC, the number of cases with large dosage mutations causing PC deficiency would have been quite limited when considering the rarity of these mutations in PROC (0.6%). 47,48 None of the population group had large dosage mutations. A total of 16 novel mutations were identified in this study, two in PROC, seven in PROS1, and seven in SERPINC1 (Table 1).…”

Age-specific onset and distribution of the natural anticoagulant deficiency in pediatric thromboembolism

“…Large deletions are reported to be 3-6% in these deficiencies (5). Cooper et al (23) revealed that large deletions make up between 7 and 10% of PS and AT mutations and only 1% of PC mutations. The rare occurrence of thrombosis and the wide range of factor activities impeded to define the standard values of "true healthy" children who develop no thromboembolism until the forties.…”

Laboratory hemostasis: milestones in Clinical Chemistry and Laboratory Medicine

“…Second line assays are typically those designed to provide further insights into abnormalities of screening tests, or used to monitor more accurately some antithrombotic therapies, and thereby include clotting factors assays [27] , ristocetin-induced platelet agglutination and VWF antigen tests [28] , anticardiolipin (aCL) IgG and IgM, anti-β (2) glycoprotein I (anti-β (2) GPI) antibodies IgG and IgM and phospholipid-dependent coagulation assays [29,30] , platelet function tests such as Platelet Function Analyzer-100 (PFA-100) and aggregometry [31,32] , assays for heparin-induced thrombocytopenia [33,34] , additional tests for thrombophilia screening including resistance to activated protein C, antithrombin, proteins C and S, and genetic polymorphisms/mutations (e.g., prothrombin G20210A and factor V Leiden) [35,36] along with ecarin clotting time, chromogenic anti-factor Xa and dilute Russell viper venom time (dRVVT) for monitoring novel anticoagulants [37,38] . Both first and second line tests might be available to most clinical laboratories, whereas third line tests -which are intended to troubleshoot the most challenging conditions and encompass analyses such as VWF collagen binding, VWF ristocetin cofactor assay, VWF-FVIII binding assay, multimer and molecular analysis for the precise classification of VWD [39,40] , coagulation factors inhibitors testing [41,42] , analyses of rare thrombophilic mutations [43] , rare platelet functional disorders [44] , pharmacogenetics testing [45,46] -are occasionally used and typically available in specialized laboratories. Quality in laboratory diagnostics is irrecusable, since spurious results obtained on unsuitable specimens may negatively bias the clinical decision-making and jeopardize patient safety.…”