Genomics of platelet disorders.

Genetic diagnosis in families with inherited platelet disorders (IPD) is not performed widely because of the genetic heterogeneity of this group of disorders and because in most cases, it is not possible to select single candidate genes for analysis using clinical and laboratory phenotypes. Next-generation sequencing (NGS) technology has revolutionized the scale and cost-effectiveness of genetic testing, and has emerged as a valuable tool for IPD. This review examines the potential utility of NGS as a diagnostic tool to streamline detection of causal variants in known IPD genes and as a vehicle for new gene discovery.

Rare platelet GPCR variants: what can we learn?

Platelet-expressed GPCRs are critical regulators of platelet function. Pharmacological blockade of these receptors forms a powerful therapeutic tool in the treatment and prevention of arterial thrombosis associated with coronary atherosclerosis and ischaemic stroke. However, anti-thrombotic drug therapy is associated with high inter-patient variability in therapeutic response and adverse bleeding side effects. In order to optimize the use of existing anti-platelet drugs and to develop new therapies, more detailed knowledge is required relating to the molecular mechanisms that regulate GPCR and therefore platelet function. One approach has been to identify rare, function-disrupting mutations within key platelet proteins in patients with bleeding disorders. In this review, we describe how an integrated functional genomics strategy has contributed important structure-function information about platelet GPCRs with specific emphasis upon purinergic and thromboxane A2 receptors. We also discuss the potential implications these findings have for pharmacotherapy and for understanding the molecular basis of mild bleeding disorders.

Diagnosis of suspected inherited platelet function disorders: results of a worldwide survey.

BACKGROUND: Diagnosis of inherited platelet function disorders (IPFDs) is important for appropriate management and to improve epidemiologic and clinical knowledge. However, there remains a lack of consensus on the diagnostic approach. OBJECTIVES: To gain knowledge on the current practices for the diagnosis of IPFD worldwide. METHODS: A 67-item questionnaire was distributed to the ISTH members and to the members of several national hemostasis and thrombosis societies. RESULTS: A total of 202 laboratories from 37 countries participated in the survey. The most frequent criterion to define patients with a suspected IPFD was a history of mucocutaneous bleeding and no acquired cause, but heterogeneity on the identification criteria was evident. Only 64.5% of respondents performed a direct clinical interview. On average, each laboratory studied 72 patients per year. The most commonly used laboratory equipment were the light-transmission aggregometer, the Platelet Function Analyzer-100, and the flow cytometer. Screening tests were platelet count, peripheral blood smear, light-transmission aggregometry, and Platelet Function Analyzer-100. Second-step tests were flow cytometry, molecular genetic analysis, and electron microscopy. Methodologies varied widely. In total, ~ 14,000 patients were investigated yearly and 60% turned out to not have a defect. Of the remaining 40%, only 8.7% received a diagnosis at a molecular level. CONCLUSIONS: Many laboratories worldwide are involved in the diagnosis of IPFD. A large fraction of the patients studied remain without a diagnosis. A high variability in the diagnostic approaches is evident.

How should we test for nonsevere heritable platelet function disorders?

Heritable platelet function disorders (HPFD) are a heterogeneous group of bleeding disorders with diverse clinical and laboratory characteristics. In contrast to the severe phenotype disorders, Glanzmann thrombasthenia and Bernard-Soulier syndrome, most nonsevere HPFD are incompletely characterized. This is a consequence of the poor standardization of diagnostic tests and of the lack of consensus about diagnostic criteria for the different subgroups of nonsevere HPFD. Distinguishing patients who have a nonsevere HPFD from those who do not is an essential first step in diagnosis which may be aided by bleeding assessment tools and screening tests such as the Platelet Function Analyser-100. However, high diagnostic accuracy can only be achieved with both light transmission aggregation (LTA) and secretion tests, for which streamlined agonist panels may be of similar utility to extended panels. Standardization of the methodology of these tests and quality assurance are essential for robust diagnosis. Identification of which platelet pathway is defective in patients with nonsevere HPFD is also usually possible with LTA and secretion tests. This strategy also sometimes enables exact diagnosis by implicating a single candidate protein and gene. Next-generation sequencing may offer a rapid approach to diagnosis of nonsevere HPFD, although rigorous strategies must be adopted to distinguish causative gene defects from bystander variations.

Platelet dysfunction associated with the novel Trp29Cys thromboxane A2 receptor variant.

BACKGROUND: Genetic variations that affect the structure of the thromboxane A2 receptor (TP receptor) provide insights into the function of this key platelet and vascular receptor, but are very rare in unselected populations. OBJECTIVES: To determine the functional consequences of the TP receptor Trp29Cys (W29C) substitution. PATIENTS/METHODS: We performed a detailed phenotypic analysis of an index case (P1) with reduced platelet aggregation and secretion responses to TP receptor pathway activators, and a heterozygous TP receptor W29C substitution. An analysis of the variant W29C TP receptor expressed in heterologous cells was performed. RESULTS: Total TP receptor expression in platelets from P1 was similar to that of controls, but there was reduced maximum binding and reduced affinity of binding to the TP receptor antagonist [(3) H]SQ29548. HEK293 cells transfected with W29C TP receptor cDNA showed similar total TP receptor expression to wild-type (WT) controls. However, the TP receptor agonist U46619 was less potent at inducing rises in cytosolic free Ca(2+) in HEK293 cells expressing the W29C TP receptor than in WT controls, indicating reduced receptor function. Immunofluorescence microscopy and cell surface ELISA showed intracellular retention and reduced cell surface expression of the W29C TP receptor in HEK293 cells. Consistent with the platelet phenotype, both maximum binding and the affinity of binding of [(3) H]SQ29548 to the W29C TP receptor were reduced compared to WT controls. CONCLUSION: These findings extend the phenotypic description of the very rare disorder TP receptor deficiency, and show that the W29C substitution reduces TP receptor function by reducing surface receptor expression and by disrupting ligand binding.

Marked discrepancy between coagulometric protein C activity assays with the pro-thrombotic protein C Asn2Ile substitution.

INTRODUCTION: Protein C (PC) Asn2Ile is a rare pro-thrombotic variant in which loss of anticoagulant function in vivo is likely to result from defective PC binding to the endothelial PC receptor and phospholipid. METHODS: To characterize the consequences of this functional defect on the diagnostic performance of commercial PC activity assays, we performed one antigen, three chromogenic and three coagulometric assays on plasma from a subject who was heterozygous for the PC Asn2Ile substitution. RESULTS: As anticipated, the PC antigen (96.2 IU/dl) and chromogenic PC activity assays (98.4 IU/dl) were insensitive to PC Asn2Ile, which has intact enzymatic activity and is secreted normally. There was a marked reduction in apparent PC activity by STACLOT coagulometric assay (50.4 IU/dl). However, there was only a small reduction in apparent PC activity by CRYOcheck Clot C assay (69.5 IU/dl) and by HemosIL ProClot assay, the PC activity was within the laboratory reference range (91 IU/dl). This discrepancy between coagulometric assays could not be explained by lupus anticoagulant, activated PC resistance or elevated plasma factor VIII activity. CONCLUSION: We demonstrate that coagulometric assays are not equally sensitive to clinically important functional defects of PC and that multiple assays may be required to identify all variants.

Purpura fulminans: recognition, diagnosis and management.

Purpura fulminans (PF) is a haematological emergency in which there is skin necrosis and disseminated intravascular coagulation. This may progress rapidly to multi-organ failure caused by thrombotic occlusion of small and medium-sized blood vessels. PF may complicate severe sepsis or may occur as an autoimmune response to otherwise benign childhood infections. PF may also be the presenting symptom of severe heritable deficiency of the natural anticoagulants protein C or protein S. Early recognition and treatment of PF is essential to reduce mortality and to prevent major long-term health sequelae. However, management strategies require accurate identification of the underlying cause. This review focuses on the clinical features, differential diagnosis and laboratory features of the range of PF disorders and includes expert consensus opinion about immediate and on-going management.

Familial multiple coagulation factor deficiencies - chance associations and distinct clinical disorders.

The familial multiple coagulation factor deficiencies (FMCFDs) are a group of rare haemostatic disorders of genetic origin in which there is reduced plasma activity of more than one coagulation factor. FMCFDs may arise from co-incidental inheritance of separate coagulation factor deficiencies or from a single genetic or cytogenetic defect. All the FMCFDs present significant challenges in diagnosis and management yet there is little systematic evidence with which to guide clinical practice. This review summarizes the historical literature that describes the FMCFDs and introduces a refined classification of these disorders. The clinical and laboratory characteristics of the most common FMCFDs are considered in detail.

Pharmacodynamic resistance to warfarin is associated with nucleotide substitutions in VKORC1.

BACKGROUND: Vitamin K epoxide reductase subunit 1 (VKORC1) is the molecular target of coumarin anticoagulants and mutations in VKORC1 have been identified previously in individuals who required high warfarin doses. OBJECTIVE: Detailed characterization of the relationship between variation in VKORC1 and the warfarin resistance phenotype. PATIENTS AND METHODS: Serum warfarin concentration and coagulation parameters were determined in 289 subjects who required warfarin doses >20 mg day(-1). The VKORC1 sequence was studied in selected study subjects. RESULTS: Twenty-eight out of 289 (10%) subjects had serum warfarin >2.3 mg L(-1) during stable therapeutic anticoagulation indicating pharmacodynamic warfarin resistance. Detailed analysis of 15 subjects from this group showed that eight out of 15 (53%) had nucleotide substitutions in VKORC1 predictive of p.V66M, p.L128R, p.V54L or p.D36Y. VKORC1 was normal in the remaining seven out of 15 (47%) subjects and in nine out of nine (100%) subjects with high warfarin dose requirement not caused by pharmacodynamic resistance. At referral, subjects with VKORC1 mutations received a median warfarin dose of 32 mg day(-1) (range 22-55) and had a median serum warfarin concentration of 4.6 mg L(-1) (range 2.6-9.0). VKORC1 substitutions were associated with a requirement for high warfarin doses but not with adverse clinical events. Family members with VKORC1 nucleotide substitutions and not receiving warfarin had undetectable PIVKA-II and K(1) epoxide (K(1)O). CONCLUSIONS: Nucleotide variations in VKORC1 are a common cause of pharmacodynamic warfarin resistance but are not associated with adverse outcome during anticoagulation. Mutations associated with warfarin resistance do not cause a discernible defect in VKORC1 reductase function.

Hereditary hyperferritinemia cataract syndrome: ocular, genetic, and biochemical findings.

PURPOSE: To describe the cataract morphology and genetic and biochemical findings in a four-generation family with hereditary hyperferritinemia cataract syndrome (HHCS). METHODS: Family members of the proband with HHCS were investigated. DNA sequencing was carried out to identify the iron responsive element (IRE) of the L-ferritin gene in affected and non-affected family members. Molecular modeling allowed prediction of the structure of the mutant IRE in affected cases. Serum ferritin and transferrin saturation were determined using standard methods. All family members underwent slit lamp examination by an ophthalmologist to document presence of cataract or lens status. Cataract morphology was documented where present. RESULTS: This family with HHCS had the genetic heterozygous mutation G32C in the IRE of the L-ferritin mRNA. Lens opacities were detectable in young members of the family, and morphology of cataracts was consistent with previous reports. Biochemical testing demonstrated high serum ferritin levels in affected individuals. CONCLUSIONS: The morphology of cataracts in HHCS seems to be similar in all cases. In the heterozygous G32C mutation, the age at onset of cataracts is very early. Greater awareness of this condition among ophthalmologists will lead to effective family counseling of those affected, by genetic testing or simple biochemical tests. Serum ferritin levels can be effectively used to screen for this condition in suspected families.

The rare coagulation disorders--review with guidelines for management from the United Kingdom Haemophilia Centre Doctors' Organisation.

The rare coagulation disorders are heritable abnormalities of haemostasis that may present significant difficulties in diagnosis and management. This review summarizes the current literature for disorders of fibrinogen, and deficiencies of prothrombin, factor V, FV + VIII, FVII, FX, the combined vitamin K-dependent factors, FXI and FXIII. Based on both collective clinical experience and the literature, guidelines for management of bleeding complications are suggested with specific advice for surgery, spontaneous bleeding, management of pregnancy and the neonate. We have chosen to include a section on Ehlers-Danlos Syndrome because haematologists may be consulted about bleeding manifestations in such patients.

Factor V I359T: a novel mutation associated with thrombosis and resistance to activated protein C.

We report a kindred in which two siblings suffered spontaneous venous thromboses in the second decade of life. Further investigation showed reduced coagulation factor V (FV) activity and activated protein C resistance (APCR) ratio but no other thrombophilic abnormalities. The reduction in APCR ratio persisted in a modified APCR assay in which FV activity was normalized between test and control plasmas. Analysis of the FV gene showed that the thrombotic individuals had a complex genotype that included two novel point mutations c.529G>T and c.1250T>C resulting in FV E119X and FV I359T substitutions inherited on different alleles. Individuals in the kindred with FV E119X or FV I359T substitutions alone were asymptomatic. We suggest that the FV I359T substitution confers pro-thrombotic risk and APCR, but that this is only clinically manifest when co-inherited with the FV E119X allele. The FV I359T substitution creates a new consensus sequence for N-linked glycosylation within the FV heavy chain and we speculate that this abnormal glycosylation may disrupt activated protein C-mediated proteolysis of the variant FV and FVa.

Marked elevation of thrombin generation in patients with elevated FVIII:C and venous thromboembolism.

Elevated plasma factor VIII coagulant activity (FVIII:C, > 150 IU/dl) is a risk factor for venous thromboembolism (VTE). We hypothesized that increased FVIII:C may exert a prothrombotic effect by increasing basal thrombin generation. To test this hypothesis we have measured prothrombin fragment 1 + 2 (F1 + 2) and thrombin-antithrombin complex (TAT) in three groups: (i) patients with objectively confirmed VTE and elevated FVIII:C; (ii) patients with VTE and no detectable thrombophilia; and (iii) healthy age- and sex-matched control subjects. In the group of patients with elevated FVIII:C, TAT and F1 + 2 levels were increased in 85% and 78% of individuals respectively. This frequency of coagulation activation is dramatically higher than that reported for other recognized constitutional thrombophilias. In the group of patients with VTE but no proven thrombophilia, increased thrombin generation was present in 30% of individuals. Basal thrombin generation was significantly higher in patients with elevated FVIII:C compared with individuals with VTE but no documented thrombophilia (median TAT = 8.65 microg/l versus 2.95 microg/l, median F1 + 2 = 1.5 nmol/l versus 0.87 nmol/l; P < 0.0001, P < 0.001). Overall FVIII:C levels were strongly correlated with levels of thrombin generation (r= 0.5, P < 0001). The clinical significance of such markedly increased F1 + 2 and TAT levels in patients with high FVIII:C levels remains unclear.

Factor VII deficiency and the FVII mutation database.

Factor VII (FVII) is a zymogen for a vitamin K-dependent serine protease essential for the initiation of blood coagulation. It is synthesized primarily in the liver and circulates in plasma at a concentration of approximately 0.5 microg/ml (10 nmol/L). The FVII gene (F7) is located on chromosome 13 (13q34), consists of 9 exons, and spans approximately 12kb. It encodes a mature protein of 406 amino acids, which has an N-terminal domain (Gla) post-translationally modified by gamma-carboxylation of glutamic acid residues, two domains with homology to epidermal growth factor (EGF1 and 2), and a C-terminal serine protease domain. The single chain zymogen is activated by proteolytic cleavage at Arg152-Ile153. There are 238 individuals described in the world literature with mutations in their F7 genes (FVII mutation database; europium.csc. mrc.ac.uk). Complete absence of FVII activity in plasma is usually incompatible with life, and individuals die shortly after birth due to severe hemorrhage. The majority of individuals with mutations in their F7 gene(s), however, are either asymptomatic or the clinical phenotype is unknown. In general, a severe bleeding phenotype is only observed in individuals homozygous for a mutation in their F7 genes with FVII activities (FVII:C) below 2% of normal, however, a considerable proportion of individuals with a mild-moderate bleeding phenotype have similar FVII:C by in vitro assay. The failure of in vitro tests to differentiate between these groups may be due to lack of sensitivity in the assays to the very low amounts of FVII:C, which are sufficient to initiate coagulation in vivo. A number of polymorphisms have been identified in the F7 gene and some have been shown to influence plasma FVII antigen levels.