Molecular Genetic Testing in Hemostasis and Thrombosis: The Past, the Present, and the Future

 

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Welcome to this special issue of Seminars in Thrombosis and Hemostasis. Characteristically, each issue of Seminars in Thrombosis and Hemostasis is theme-driven, with each new issue devoted to a particular theme of relevance to thrombosis and hemostasis. The current issue of Seminars in Thrombosis and Hemostasis carries the theme of genetic testing in hemostasis and thrombosis. This issue represents a rather more global and broad look into thrombosis and hemostasis than just a single specific topic. This issue is intended to represent a comprehensive update on the role of genetic analysis in the diagnosis of common and major hemostatic and thrombotic disorders, including up-to-date knowledge of our understanding of the genetic basis of these disorders. Along with updating current methodologies, pathophysiologies, molecular diagnosis, and interpretation and reporting, there is also included considerable discussion of some controversial and ethical issues related to these diagnoses and as well future research perspectives. The guest editor hopes that you make the most of this “dose” of genetic diagnostics and find this update both profitable and enjoyable.

The first article by Goodeve describes recent advances in methods of genetic analysis for the most common bleeding disorder, hemophilia, as well as the interpretation and reporting of genetic tests. This review discusses various types of mutations in severe, moderate, and mild hemophilia and updates the diagnostic applications. Polymerase chain reaction (PCR)-based amplification and mutation screening followed by DNA sequencing represent the current strategy for analysis of the factor VIII encoding (F8) gene after excluding common inversions using long and inverse PCR. Computerized analysis now helps reduce the time and effort required for mutation identification. Linkage analysis via common polymorphisms and polymorphism combinations is also informative. Advances in prenatal and preimplantation genetic diagnosis extend the reproductive options available to hemophilia carriers. F8 gene nomenclature based on the HGVS Web site and the specific GenBank reference sequence (RefSeq)[1] for F8 (cDNA, NM_000132.2; and protein, NP_000123.1) should be followed for consistency and correctness, and an integrated external quality assurance system is essential for the most appropriate molecular testing and reporting in hemophilia.

In the second article, James and Lillicrap provide a concise and useful evaluation of the role of genetic testing in diagnosis of von Willebrand disease (VWD) in view of both cumulative and specific experience in genetic testing of different types of VWD. The increased availability and improved feasibility of PCR-based methodologies as well as DNA sequence analysis have contributed to the increased integration of genetic testing in the diagnosis of VWD and have also helped clarification of diagnostic certainty in some subtypes. The article discusses pros and cons of the genetic analysis with respect to various types of VWD in addition to the role of phenotypic analysis. Correct and reliable interpretation of genetic analysis of the von Willebrand factor gene (VWF) requires care and experience in the choice of methodology and in the details of applications. The genetic analysis for types 3,[2] 2B (and the differentiation from the phenotypically similar platelet-type VWD),[3] 2M,[4] and 2N[5] VWD provides for additional diagnostic value. However, genetic testing for type 2A VWD is rather complicated,[6] and that for type 1 VWD still poses a challenge.[7] A recent addition to type 1 VWD patient identification on a basis of reduced von Willebrand factor (VWF) survival by Haberichter et al was an assay of the VWF propeptide in conjunction with plasma VWF levels.[8] Generally, James and Lillicrap recommend the gradual integration of genetic testing as an adjunctive approach for VWD diagnosis.

As a continuation of this theme, the next article by Novelli and Ragni reviews specifically the genetics of inherited bleeding disorders in women, focusing on bleeding manifestations, diagnostic methodologies, and management. Menorrhagia is a common and important health problem among women, and bleeding is common within reproductive and perimenopausal age. Although it is estimated that 30% of women complain of heavy periods, only around 5% seek medical care.[9] Menorrhagia is a presenting symptom in more than 70% of VWD cases, and VWD is diagnosed in 13% of cases with menorrhagia. The appropriate assessment of menorrhagia is critical to avoid overestimation or underestimation of the degree of bleeding and currently relies on more objective methodologies.[10] The integration of a bleeding score can be also helpful.[11] This article also discusses the non-VWD causes of bleeding disorders in women, namely (1) platelet disorders such as Bernard-Soulier syndrome, Glanzmann thrombasthenia, platelet-type VWD (PT-VWD), and platelet storage disease; (2) hemophilia carrier state and rare factor deficiencies such as those of factors II, V, VII, XIII, and combined factor V and VIII deficiency; (3) vessel wall disorders such as hereditary hemorrhagic telangiectasia and Ehlers-Danlos syndrome. In all non-VWD causes, the responsible genes are identified, and genetic testing is currently available. However, knowledge about the pathophysiology and the clinical suspicion of these disorders are critical to making an initial diagnosis before genetic testing can be initiated.

VWD will continue to occupy a large area on the hemostasis research map for several years to come, and this partly explains why three articles dealing with different aspects of this bleeding disorder have been included in this issue of Seminars in Thrombosis and Hemostasis. Not only is VWD the most common mild bleeding disorder, but also critical and specific interactions between the VWF protein and various molecules and cells in the endothelial/platelet interphase and also the uniqueness of such behavior in the normal and injured vessel characterize primary hemostasis. The article by Othman and Favaloro concerning type 2B VWD aims to highlight the idea that VWF/platelet binding is still not fully understood. In diagnostic evaluations, the classic picture of type 2B VWD is not always realized, and the complexity of the phenotype extends to beyond just identifying VWF and /or platelet GP1BA gene abnormalities that respectively cause type 2B VWD or its “twin,” PT-VWD. After an extensive literature review of atypical cases of 2B VWD, the authors discuss a list of phenotype modifiers that will likely expand with further understanding of the complex interaction.[12] VWF multimer analysis is useful when undertaking a phenotypic diagnosis of 2B VWD but does not always show the typical picture of an absence of high-molecular-weight multimers. Platelet size and morphology can also vary with different type 2B VWD mutations, adding complexity to the phenotype and often leading to its misdiagnosis as idiopathic thrombocytopenia (ITP). The role of the endothelium requires further investigation, and more about a disintegrin and metalloproteinase with a thrombospondin type 1 motif, member B (ADAMTS13) interactions remain to be revealed. Platelet integrins may have a role in sizing platelets in different type 2B variants, and a role of megakaryocytopoiesis cannot be excluded from this scenario. Understanding the role of these possible modifiers can help in the appropriate assessment of the type 2B VWD phenotype, explain its “atypicality,” and aid in its diagnostic clarification. Though appreciating the historical description about the disorder (paraphrased “2B or not 2B”), I would now like to add the concept of “tricky 2B” to help explain our current understanding of this complex and evolving subtype of VWD. Assessment of 2B VWD requires, in addition to genetic analysis of both the VWF and GP1BA genes, a careful and thoughtful phenotypic assessment including platelet counts and morphology, functional and antigenic determination of VWF, and if possible ristocetin-induced platelet agglutination (RIPA)/RIPA mixing studies and assessment of ADAMTS13.

Moving on to other areas of hemostasis and thrombosis, an update on platelet proteomics and genomics is provided in the article by Senzel et al. Transcriptomic studies began in 2003, and techniques have developed considerably since then. Molecular signatures in normal and diseased platelets are now possible and contribute to our understanding and the classification of prothrombotic patient phenotypes in disorders such as essential thrombocythemia, atherosclerotic disease, and sickle cell disease. Recent proteomic approaches have uncovered novel signaling pathways and identified proteins involved in platelet activation and inhibition, some of which may carry potential therapeutic applications. Proteomic data on platelets, its membrane, its granules, as well as microparticles are now available. The final platelet transcript list contains 432 genes, but only around 300 released proteins can be identified from activated platelets.[13] This discordance can be related to limitation of current proteomic methods and/or a lack of translation of mRNA in some cases. Technical advances currently allow platelet microarray studies to be performed using peripheral blood as starting material compared with previous needs for platelet apheresis.

The article by Curtis discusses an uncommonly visited topic when considering genetic testing in thrombosis and hemostasis: platelet genotyping (methodology, value, and applications). The first description of a platelet gene polymorphism was made in 1989, and currently six biallelic human platelet antigen (HPA) systems are known and can be typed using genomic DNA. The platelet is a valuable tool in confirming platelet antigen specificities of alloantibodies detected in patient sera. This information complements the clinical history in the diagnosis of immune-mediated platelet disorders like fetal and neonatal alloimmune thrombocytopenia (FNAIT), posttransfusion purpura (PTP), and multiplatelet transfusion refractoriness (MPTR). Serology has always been, and still remains, the main method of assessing these antigens. However, the introduction and advances of PCR assays including PCR with sequence-specific primers, melting curve analysis by LightCycler, and 5′-nuclease assays and multiplex PCR has resulted in some shift from serology and has allowed for the development of high-throughput assays for genotyping large numbers of patients and blood donors. Genotyping has superiority over serotyping in detection of low-frequency alleles, as specific typing sera are rare, which may be more significant than originally appreciated in alloimmune platelet disorders such as FNAIT. There is no need for platelet samples, and genomic DNA can be extracted from white blood cells or buccal swabs of the cheek. Limitations include the presence of unknown single nucleotide polymorphisms (SNPs) located near the HPA polymorphism of interest affecting PCR results, and contamination of fetal samples by maternal blood is an additional challenge. Expression of specific platelet SNPs can help also in nonimmune disorders. Association with risk for atherothrombosis and myocardial infarction has been documented,[13] and a large screening study looking into the importance of different platelet SNPs in various hemostatic disorders is in progress.[14] Combined serology and genotyping allows for powerful diagnosis of immune platelet disorders and aids prenatal diagnosis of the fetus in suspected cases. Curtis predicts a future value and application for platelet genotyping as an aid in determining the relative risk of patients for various thrombotic disorders, particularly with further improvement in methodology.

The article by Varga et al provides an update on genetic testing for inherited thrombotic disorders, so-called thrombophilia. The increased knowledge regarding the contribution of genetic predisposition to thrombosis has helped to expand the diagnosis and management of these disorders, and up to 70% of patients with thrombotic problems can now be linked to genetically mediated risk factors. However, debate still exists about whether or not individuals with a personal or family history of thrombosis should be screened for thrombophilia and the degree of impact that this information has on clinical utility. According to guidelines by the College of American Pathologists (CAP) and the American College of Medical Genetics (ACMG), thrombophilia testing is offered to individuals with first venous thromboembolism (VTE) before age 50; recurrent VTE; VTE at any age with a strong family history of thrombotic disease; VTE in an unusual site at any age; and to women suffering VTE in association with pregnancy, the immediate postpartum period, or oral contraceptive use.[15] Although these guidelines are not very different from those of the British Committee for Standards in Hematology (BCSH),[16] such guidelines are only appropriately followed in a minor percentage of cases reflecting either a limited understanding or controversy among physicians regarding the utility and indications for thrombophilia screening. This raises a fundamental question: Why are we performing these tests? Is testing performed to explain why an individual develops thrombosis or to optimize treatment decision? And therefore how much value should we place on whether testing patients with VTE for laboratory evidence of thrombophilia will have a significant predictive value?[17] [18] The article moves on to discuss elegantly several critical issues beyond genetic analysis including psychosocial benefits of testing and impact on lifestyle, the influence on a woman's choices about hormone therapy and contraception, the critical impact on modification of treatment decision (intensity and duration of anticoagulation) once a thrombophilic risk factor is identified, and insurance and employment discrimination. The article concludes that decisions regarding the utility of thrombophilia testing should be taken carefully with serious consideration of all issues. Once the decision is made, a thorough understanding of the pathophysiology and genetic basis would help the most appropriate choice of testing methodology matched to the clinical scenario to maximize the management impact.

Whereas most hemostasis research typically entertains the coagulation side rather than the fibrinolytic side, the article by Asselbergs et al describes the genetics of two major components in the fibrinolytic system, namely tissue plasminogen activator (t-PA) and its inhibitor plasminogen activator inhibitor-1 (PAI-1). The plasma variations in these components have been associated with cardiovascular and thrombotic disorders. The physiology of t-PA and PAI-1 and the heritability of the related genes is discussed, and an update of the genetic sequence variations is provided. The influence of genetic variations from other loci such as the renin-angiotensin and bradykinin systems[19] represents another important path for investigation. The impact of environmental factors like obesity and alcohol on individual variation of plasma levels is now documented. The authors conclude that the genetic architecture of t-PA and its inhibitor PAI-1 is complex, and more effort should be directed to studying gene-to-gene and gene-to-environment interaction, as well as investigating other higher order proteins and genes from unknown pathways.

The final article in this issue of Seminars in Thrombosis and Hemostasis represents a rather special review of experience with respect to molecular genetics in relation to diagnosis, patient care, as well as research in three developing countries (Brazil, Colombia, and Iran) from three authors from these locations. Information about developing countries is not always easy for us to find. It was therefore believed critical that authors be found with specific interest and knowledge to enable a full discussion of this topic. From Brazil, a clinician Ph.D. scientist who has experience in diagnosis and treatment of hemophilia and other bleeding disorders was contacted, as were an Iranian Ph.D. laboratory scientist with experience in laboratory diagnosis of routine hematologic disorders and a Colombian specialist with more than 20 years of experience in laboratory analysis of hematologic and other disorders, who also founded in 1985 and is treasurer of Liga Antioqueña de Hemofílicos (Hemophilia League) in Antioquia, Colombia. These individuals responded enthusiastically to the invitation to contribute to this article. The article highlights a considerable variation in structure and infrastructure in the health care systems within these countries and more importantly the differences in ethnicity, culture, and socioeconomic status. In Brazil, a highly heterogenous genetic pool provides particular characteristics and raises some scientific interest to study these characteristics. A completion of a bacterial genome project in the beginning of the millennium was a huge step forward for genetic work in Brazil, and the organization of academic study groups and collaborative research networks has contributed toward the improvement of diagnosis and research in hemophilia and thrombophilia. In Colombia, external investigators have helped to promote diagnosis and molecular characterization of hemophilia, but there still is no national registry for bleeding disorders. Currently, VWD diagnosis is based on personal and family history of bleeding and a single VWF measurement with ~40 cases waiting to be confirmed and typed. Iran, with its population of about 70 million with 50% below 20 years of age, poses a current and future challenge to the health care system. Consanguinity contributes to an increased percentage of genetic disorders in general, and “rare” bleeding disorders are much more common than is identified in developed countries. Specific sociocultural features govern the outcome of prenatal diagnosis, and comprehensive hemophilia care is widely available throughout the country. Experience with and facilities for molecular diagnosis and research, however, are only available in large cities. PCR-based methods are available in most of these laboratories, and a recent improvement of public education is also evident.

I sincerely thank all the contributors to this special issue of Seminars in Thrombosis and Hemostasis for their excellent contributions and collaboration during the process of generation of the manuscripts. A very special thanks to Dr. Emmanuel J. Favaloro for his kind invitation to prepare this issue of Seminars in Thrombosis and Hemostasis and for his thoughtful and generous help with this issue, which represents my first editorial experience. His mentorship and valuable and expert advice has supported this project considerably. Finally, on behalf of all the contributors, I sincerely hope that you, the reader, enjoy this update in genetic testing in hemostasis and thrombosis.

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Maha OthmanM.D. Ph.D. 

Assistant Professor, Department of Pathology and Molecular Medicine

Queen's University, Kingston, Ontario K7L 3N6, Canada

Email: othman@queensu.ca