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Molecular Testing in Coagulation


John Bartlett, Director of Diagnostic Development at the Ontario Institute for Cancer Research writes that “accurate and appropriate diagnosis is fundamental to the successful treatment of disease.” With the introduction and evolution of new technologies, we can evaluate coagulopathy at a molecular level like never before. Nevertheless, we must avoid ordering expensive and sometimes expansive molecular tests for rare disorders when alternative methods of diagnosis are available and recommended. The Core Laboratory (in-house or commercial) provides most tools for making many of these diagnoses, and it is only under certain circumstances, for example, with equivocal values or when we require additional information that may be important for the patient, the patient’s family, or family planning, which we should proceed with molecular testing.

Although testing guidelines for certain coagulopathies, such as factor V Leiden (FVL) and prothrombin 20210A mutations, have been well established, testing for other conditions, such as von Willebrand disease (VWD), are more complicated, and with respect to methylenetetrahydrofolate reductase C677T (MTHFR) are generally refuted. In this discussion, we present two tables ( Tables 161.1 and 161.2 ) which, along with the gold standard of clinical correlation, should serve as a molecular reference guide for identifying the etiology of either thrombosis or bleeding.

Table 161.1
Inherited Prothrombotic Disorders
Gene, Location, Most Common Mutation (SNP Identification) Heterozygous Genotype Frequency or Prevalence Homozygous Genotype Frequency or Prevalence Odds Ratio Is Molecular Testing Available? Notes
Prothrombin mutation G20210A, heterozygous F2; 11p11.2; 20210G>A (rs1799963) (A; G), 0.006 – 2.80 Yes; Molecular analysis is the test of choice. Accounts for 40%–50% of inherited thrombophilia
Prothrombin mutation G20210A, homozygous – (A; A), 0.0004 6.74
Factor V Leiden, heterozygous F5 ; 1q24.2; R506Q (rs6025) (C; T), 0.011 – 4.38 Yes, although activated protein C (APC) resistance is the coagulation test of choice. Present almost exclusively in Caucasian population. Small risk of arterial thrombosis.
Factor V Leiden, homozygous – (T; T); 0.0004 11.45
Dysfibrinogenemia (F1) FGA (exon 2) and FGG (exon 8); 4q35 Core/Commercial Laboratory testing; identified with FGA, FGB, and FGG sequencing. Multiple SNPs are reported in the literature, with odds ratios <1.5 and minor allele frequencies >0.3
Factor XI F11 Not routinely evaluated. Multiple SNPs are reported in the literature, with odds ratios <1.5 and minor allele frequencies >0.25
Protein C deficiency PROC ; 2q14.3; 0.14%–1.5% (5%–9% of patients with VTE) Homozygous or compound heterozygotes—seen with severe hereditary protein C deficiency (1 in 4 million newborns); milder manifestations have been observed 2- to 11-fold increased risk; also increased risk of arterial thrombosis Core/Commercial Laboratory testing; molecular testing is generally unnecessary. Account for <10% of inherited thrombophilia; many variations of mutations have been identified throughout these genes and thus, only if necessary, sequencing of the entire gene would be recommended
Protein S deficiency PROS1 ; 3q11.1 0.1% (2% of patients with VTE) – See above discussion Core/Commercial Laboratory measurement of free protein S is generally recommended, and molecular analysis is considered unnecessary.
Antithrombin deficiency SERPINC1 ; 1q25.1; autosomal dominant pattern of inheritance 0.02%–0.17% (0.5%–5% of patients with VTE) – 5- to 50-fold increased risk of VTE Core/Commercial Laboratory testing; molecular testing is generally unnecessary.
MTHFR/Hyperhomocysteinemia MTHFR ; 1p36.22; 677C>T (rs1801133) and 1298A>C (rs1801131) 677C>T: approximately 20%–40% of Caucasian and Hispanic patients (1%–2% in African Americans) 677C>T: >25% Hispanics, 10%–15% North American Caucasians – Not recommended Mutation identified in 5%–7% of general population with mildly elevated homocysteine levels
Prevalence and allele frequency data are provided using general population–based statistics. Many of these variants have different prevalence depending on patient ethnicity. Additionally, in the setting of thrombophilia, the table represents relative risk of a single risk factor, and that in combination with these relative risk scores could portend a far higher risk of thromboembolism. Finally, the “Most Common” mutation is the one, which is most prevalent, not necessarily the mutation, which confers the greatest risk. For information regarding additional mutations, which may or may not confer varying risk from the abovementioned variant, consultation with your local coagulation expert is recommended.

Table 161.2
Bleeding Disorders
Worldwide Prevalence Gene, Location Number of Associated Mutations Is Molecular Testing Available? Notes
Hemophilia A 1:10,000 F8 ; Xq28; ∼45% of severe disease due to recurring intron 22 inversion >2100 Yes, although Core/Commercial Laboratory testing is usually sufficient. Molecular testing may be recommended in severe disease, but this should be individualized and discussed with a coagulation expert.

  • Genetic analysis of F8 and F9 genes results in identification of causative mutations in >95% of patients

  • Identifying multidomain mutations are at higher risk of inhibitor development

  • Identifying multidomain mutations in Hemophilia B also has been associated with the potential for anaphylactic reactions to FIX concentrate infusion.

  • Hemophilia B Leiden is caused by a group of single-nucleotide substitutions in the area around the transcription start site of FIX and results in severe hemophilia in childhood that progresses to the normal range after puberty.

Hemophilia B 1:30,000 F9; Xq27.1 >1100
von Willebrand disease 1:100,000 Chromosome 12 ∼400 Yes, although Core/Commercial Laboratory testing is usually sufficient for the vast majority of patients; molecular testing should be individualized and discussed with a coagulation expert.

  • Mutations for Type 1 and Type 3 VWD are spread throughout the entire VWF gene, whereas Type 2 mutations are predominantly identified within Exon 28.

α2-Antiplasmin deficiency ∼40 known cases; autosomal recessive inheritance SERPINF2 ; 17p13.3 – Yes, although Core/Commercial Laboratory testing is generally sufficient. Euglobulin lysis time and/or a specific assay for α2-antiplasmin deficiency can be performed. (Carpenter, 2008)

  • Homozygous patients may exhibit severe bleeding symptoms or may exhibit delayed bleeding.

  • Heterozygous patients may have milder bleeding or may be asymptomatic.

Plasminogen Activator Inhibitor Type I (PAI-1) <10 families with complete PAI-1 deficiency have been reported SERPINE1; 7q21.3-22 – Yes, although Core/Commercial Laboratory testing (PAI-1 antigen and PAI-1 activity) is the initial tests of choice. Association of PAI-1 deficiency with cardiac fibrosis.

  • Elevated levels of PAI-1: The SERPINE1 c.-820_-817G(4_5) (also known as 4G/5G variant) is a common insertion/deletion of four or five G-nucleotides in the promoter region of SERPINE1. Studies have shown that the 4G allele results in higher levels of PAI-1 activity. This leads to a state of decreased fibrinolysis and therefore increases one’s risk of developing arterial and venous thrombosis.

Factor I deficiency; fibrinogen disorders 1:1 million (AR), unknown AD FGA, FGB, GG >250 See above. Associated with both bleeding and thrombosis. These rare inherited coagulation factor deficiencies represent 3%–5% of coagulation factor deficiency–associated bleeding.

  • FVII and FXI deficiencies represent of 30% of these rare bleeding disorders.

  • Molecular testing may be useful when evaluating borderline factor deficiency states or with combined FV/FVIII deficiency disease.

Factor II deficiency 1:1–2 million F2; 11p11.2 >50 Yes, although as above, standard functional clotting assays are the diagnostic method of choice. If necessary, molecular testing should be individualized and discussed with a coagulation expert.
Factor V deficiency 1:1 million F5 ; 1q24.2 >130
Factor VII deficiency 1:0.5–1 million F7; 13q34 >240
Factor X deficiency 1:0.5–1 million F10; 13q34 >100
Factor XI deficiency 1:100,000–1,000,000 F11; 4q35 >220
Factor XIII deficiency 1:2 million F13A (6p24.2-p23) , F13B (1q31-q32.1) >120
Combined FV and FVIII deficiency ∼200 known cases; often associated with consanguinity; concentrated in the Mediterranean, Middle Eastern, and South Asian countries (Zheng, 2013) LMAN1 (18q.21) and MCFD2 (2p21) >20

Bleeding

A focused patient and family history of bleeding can aid in the determination of factor deficiency versus platelet/vessel interaction bleeding. In the appropriate clinical setting and after excluding acquired etiologies, we should consider inherited bleeding disorders. Evaluation of inherited bleeding disorders should include Hemophilia A and B, VWD, and then the more rare, inherited coagulation factor deficiencies, dysfibrinogenemia, antiplasmin deficiency, and Plasminogen Activator Inhibitor Type I.

As was alluded to above, phenotypic assays in the Core Laboratory are widely sufficient for assessing bleeding disorders; however, certain circumstances may necessitate molecular testing. The most common utilization of molecular testing in the setting of inherited bleeding disorders involves prenatal diagnosis of hemophilic pregnancies, and to a lesser extent, VWD Type III. Genetic analysis could also be used in determining a patient’s carrier status when he or she has a known family history of a defined inherited bleeding disorder. Finally, molecular testing could also expound on a phenotypic diagnosis when determination of the genotype can influence clinical management, most commonly, when concerned about inhibitor development in patients with hemophilia and possibly VWD Type III.

Thrombosis

Inherited or acquired thrombophilia should be evaluated from a multifactorial perspective. Although an inherited or acquired defect can rarely cause thrombosis without provocation, thromboembolism is more commonly the result of the interplay among multiple risk factors, including physiologic (i.e., pregnancy or obesity), environmental (i.e., smoking or oral contraceptive use), acquired (i.e., antiphospholipid syndrome), and inherited (i.e., FVL or prothrombin) disorders. Additionally, incomplete penetrance of many of the inherited thrombophilia disorders only generates more confusion when evaluating thrombophilia risk.

Evaluation of thrombophilias should consider hereditary and acquired conditions, with molecular testing pertaining mostly to hereditary coagulopathies. Hereditary conditions generally affect the quality or quantity of a coagulation protein with gain or loss of function mutations. The gain of function mutations includes the more common FVL and prothrombin-associated mutations, and the loss of function category includes antithrombin (AT), protein C, and protein S (PS) mutations.

Platelet Disorders

The most common platelet disorders to be considered in coagulopathy include Glanzmann thrombasthenia, Bernard–Soulier syndrome, platelet-type VWD, May–Hegglin syndrome, Hermansky–Pudlak syndrome, Gray platelet syndrome, Wiskott–Aldrich syndrome, and Quebec syndrome. Platelet disorders are generally evaluated in the Core/Commercial laboratory. Methods of platelet disorder testing include light transmission aggregometry, whole-blood aggregometry, secretion assays, and flow cytometry. When diagnostic confirmation is requested because phenotypic studies are equivocal, or when genetic information can be important for familial purposes, molecular analysis can be utilized (see Chapter 142 ). For more information on platelet-derived coagulopathic disorders, please see “Genetic Loci Associated with Platelet Traits and Platelet Disorders.”

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