Molecular Diagnostics in Hemostatic Disorders

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The use of molecular diagnostic techniques in clinical and research hemostasis laboratories is increasing as genetic factors that affect the procoagulant and anticoagulant systems are identified. Many of these molecular alterations are associated with thrombotic tendencies, whereas others tip the hemostatic balance in favor of bleeding. In either scenario, molecular testing may serve as a primary diagnostic modality or may provide information that complements clot-based “functional” assays. The clinical application of DNA-based testing continues to expand since the discoveries of the factor V Leiden and prothrombin G20210A gene mutations. Indications for genetic testing continue to evolve as the underlying causes of hemostatic disorders are better understood. Further development of molecular assays depends on their proved utility in the clinical management and treatment of these complex multifactorial disorders.

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Molecular testing for inherited thrombophilia

A predisposition to thrombosis, or thrombophilia, may be genetically predetermined based on overexpression of procoagulant proteins or underexpression of anticoagulant proteins. Thrombophilia is termed acquired when it is associated with external risk factors for thrombosis, including oral contraceptive or estrogen therapy, pregnancy or postpartum state, antiphospholipid syndrome, cancer, chemotherapy, and so forth. Major anticoagulant proteins include PC, AT, and PS. The serine protease

Indications for genetic testing of hemostatic disorders

The clinical indications for genetic testing in coagulation disorders are constantly evolving. Professional organizations have made recommendations for such testing in thrombotic disease. There is general agreement that a DNA-based assay for the FVL and PT gene mutations is indicated in the following circumstances: (1) venous thromboembolism (VTE) at an age younger than 50 years, (2) recurrent VTE, (3) VTE when occurring at an unusual site, (4) unprovoked VTE, (5) VTE in patient with a family

Strengths of molecular testing for hemostatic disease

Molecular testing is extremely useful for resolving patients as heterozygous or homozygous for a particular mutation associated with a coagulation disorder. Because clot-based assays have relatively wide analytic variability and carrier levels may overlap the “normal” ranges, this cannot be reliably done by assays based on factor levels. In addition, activity levels of homozygotes and heterozygotes may overlap. Other difficulties with factor assays, activity-based and antigenic, are that test

Practical issues in molecular hemostasis testing

Pathology laboratories that perform molecular diagnostic testing for coagulation disorders are faced with the same practical and ethical issues that are encountered in other types of genetic testing.26 Informed consent is typically required for all genetic testing. A recognized exception includes FVL testing; the American College of Medical Genetics (ACMG) recommends that formal informed consent not be required for FVL testing and that laboratories assume that informal consent was obtained.21

Pharmacogenomics in treating thrombosis: molecular testing for “personalized” Coumadin dosing

One purpose of “personalized medicine” is to identify genetic differences that affect the metabolism or action of a drug. Ideally, these differences would be detected before prescribing a medication, allowing drug doses to be tailored to an individual's genetic composition. One such example is using cytochrome P450 (2C9) and vitamin K epoxide reductase (VKORC1) genotyping to select a warfarin dose. Warfarin has a narrow therapeutic range, and adverse drug reactions are common.31 Drug

Hemophilia A

Hemophilia A (HA) is a well-characterized X-linked recessive bleeding disorder resulting from deficient or defective factor VIII (F8) protein. Clinical disease severity correlates with residual F8 activity, ranging from less than 1% for severe, 1% to 5% for moderate, and 5% to 40% (IU/dL) for mild HA. Most carriers of an HA mutation have F8 levels of greater than 35%; thus, they may not be detected by F8 activity assays that have wide reference ranges (∼50%–150%). Only approximately 10% of

Hemophilia B

Hemophilia B (HB) is an X-linked recessive bleeding disorder caused by a deficiency of coagulation factor IX (F9). The F9 gene is located on Xq27.1 to Xq27.2 and contains eight exons. The HB bleeding disorder is clinically indistinguishable from HA. A database encompassing known mutations in the F9 gene is also available.73 Notable entries include several promoter mutations (HB Leyden 1, 2, 3) that are associated with a phenotype characterized by severe disease at birth and significant

von Willebrand disease

The most commonly diagnosed inherited bleeding disorder, vWD, is caused by a qualitative or quantitative abnormality of the von Willebrand factor (vWF), a multimeric glycoprotein that plays a critical role in primary hemostasis and serves as a protective carrier protein for F8. After binding to the platelet glycoprotein Ib receptor, vWF forms a bridge between the platelet surface and subendothelial collagen exposed by vascular injury. vWF consists of low, intermediate, and high molecular weight

Genomics and proteomics in hemostasis

The information gleaned from the Human Genome Project has not yet translated into a better understanding of the hemostatic system at the molecular level. Presumably, a detailed human genetic map would facilitate discovery of new genetic markers of bleeding and thrombophilia. Such knowledge could then be used more precisely to estimate hemostatic risk and to refine treatment of coagulation disorders.84 Mutations in regions that are not routinely assessed, such as deep intronic portions of genes,

Future of genetic testing in hemostasis

As knowledge of the human genome expands, layers of complexity beyond DNA and RNA sequences may be defined to the point at which analysis is useful in a clinical context. Epigenetic factors (changes in the genome that do not involve alterations in the DNA sequence, per se [eg, methylation of bases, modifications of proteins associated with the DNA]) and expression of microRNAs (short single-stranded regulatory RNA molecules) may prove useful in thrombophilia diagnostics. Furthermore, mutations

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