Next-generation sequencing for the diagnosis of MYH9-RD: Predicting pathogenic variants.

The heterogeneous manifestations of MYH9-related disorder (MYH9-RD), characterized by macrothrombocytopenia, Döhle-like inclusion bodies in leukocytes, bleeding of variable severity with, in some cases, ear, eye, kidney, and liver involvement, make the diagnosis for these patients still challenging in clinical practice. We collected phenotypic data and analyzed the genetic variants in more than 3,000 patients with a bleeding or platelet disorder. Patients were enrolled in the BRIDGE-BPD and ThromboGenomics Projects and their samples processed by high throughput sequencing (HTS). We identified 50 patients with a rare variant in MYH9. All patients had macrothrombocytes and all except two had thrombocytopenia. Some degree of bleeding diathesis was reported in 41 of the 50 patients. Eleven patients presented hearing impairment, three renal failure and two elevated liver enzymes. Among the 28 rare variants identified in MYH9, 12 were novel. HTS was instrumental in diagnosing 23 patients (46%). Our results confirm the clinical heterogeneity of MYH9-RD and show that, in the presence of an unclassified platelet disorder with macrothrombocytes, MYH9-RD should always be considered. A HTS-based strategy is a reliable method to reach a conclusive diagnosis of MYH9-RD in clinical practice.

Rare variants in GP1BB are responsible for autosomal dominant macrothrombocytopenia.

The von Willebrand receptor complex, which is composed of the glycoproteins Ibα, Ibß, GPV, and GPIX, plays an essential role in the earliest steps in hemostasis. During the last 4 decades, it has become apparent that loss of function of any 1 of 3 of the genes encoding these glycoproteins (namely, GP1BA, GP1BB, and GP9) leads to autosomal recessive macrothrombocytopenia complicated by bleeding. A small number of variants in GP1BA have been reported to cause a milder and dominant form of macrothrombocytopenia, but only 2 tentative reports exist of such a variant in GP1BB By analyzing data from a collection of more than 1000 genome-sequenced patients with a rare bleeding and/or platelet disorder, we have identified a significant association between rare monoallelic variants in GP1BB and macrothrombocytopenia. To strengthen our findings, we sought further cases in 2 additional collections in the United Kingdom and Japan. Across 18 families exhibiting phenotypes consistent with autosomal dominant inheritance of macrothrombocytopenia, we report on 27 affected cases carrying 1 of 9 rare variants in GP1BB.

A gain-of-function variant in DIAPH1 causes dominant macrothrombocytopenia and hearing loss.

Macrothrombocytopenia (MTP) is a heterogeneous group of disorders characterized by enlarged and reduced numbers of circulating platelets, sometimes resulting in abnormal bleeding. In most MTP, this phenotype arises because of altered regulation of platelet formation from megakaryocytes (MKs). We report the identification of DIAPH1, which encodes the Rho-effector diaphanous-related formin 1 (DIAPH1), as a candidate gene for MTP using exome sequencing, ontological phenotyping, and similarity regression. We describe 2 unrelated pedigrees with MTP and sensorineural hearing loss that segregate with a DIAPH1 R1213* variant predicting partial truncation of the DIAPH1 diaphanous autoregulatory domain. The R1213* variant was linked to reduced proplatelet formation from cultured MKs, cell clustering, and abnormal cortical filamentous actin. Similarly, in platelets, there was increased filamentous actin and stable microtubules, indicating constitutive activation of DIAPH1. Overexpression of DIAPH1 R1213* in cells reproduced the cytoskeletal alterations found in platelets. Our description of a novel disorder of platelet formation and hearing loss extends the repertoire of DIAPH1-related disease and provides new insight into the autoregulation of DIAPH1 activity.

Dynamic analysis of platelet deposition to resolve platelet adhesion receptor activity in whole blood at arterial shear rate.

Platelet activation is traditionally quantified using turbidimetric aggregometry, which reflects integrin αIIbß3 activity, an important determinant of platelet function during pathophysiological thrombus formation. However, aggregometry does not recreate the shear conditions prevailing during thrombosis in vivo. Here we describe novel whole-frame analysis of real-time video microscopy to quantify platelet adhesion receptor activity under shear in parallel-plate flow chambers. We demonstrate that the rate of change of surface coverage (designated Platelet Population Mobility, PM) quantifies platelet mobility. On collagen, PM falls exponentially to a low level, corresponding to firm platelet adhesion, while on other substrates, PM remains high. Different receptor-specific thrombogenic surfaces reveal that the PM time constant reflects real-time changes in integrins αIIbß3 and α2ß1 activity. This ensemble kinetic analysis has the potential to provide valuable diagnostic information about platelet thrombus formation with both academic and clinical applications.

The UK National External Quality Assessment Scheme for heritable bleeding disorders.

Molecular genetic analysis of families with hemophilia and other heritable bleeding disorders is a frequently requested laboratory investigation. In the United Kingdom, laboratories undertaking genetic testing must participate in a recognized external quality assessment scheme for formal accreditation. The UK National External Quality Assessment Scheme (UK NEQAS) for heritable bleeding disorders was established in its current format in 2003, and currently has 27 registered participants in the United Kingdom, the European Union (EU), and the non-EU countries. Two exercises per annum are circulated to participants comprising either whole blood or DNA isolated from cell lines, and laboratories are allowed 6 weeks to analyze the samples and generate a report. Reports are assessed by a panel comprising clinicians and scientists with expertise in this area. Samples to date have involved analysis of the F8 gene (10 exercises), the F9 gene (4 exercises), and the VWF gene (3 exercises) and have comprised a wide spectrum of mutations representing the routine workload encountered in the molecular genetics laboratory. The majority of laboratories in each exercise passed, but a small number did not and reasons for failing included clerical errors, genotyping inaccuracies, and a failure to correctly interpret data. Overall we have seen an improvement in quality of reports submitted for assessment, with a more concise format that will be of value to referring clinicians and counsellors. Informal feedback from participants has been very positive.

Plasma IGF-1 and IGFBP-3 may be imprecise surrogates for breast concentrations: an analysis of healthy women.

We investigated insulin-like growth factor (IGF)-1 and IGF binding protein (IGFBP)-3 concentrations in histologically normal breast tissues and assessed their association with plasma concentrations, and breast cancer risk factors. IGF-1 and IGFBP-3 were assessed in plasma and breast tissues of 90 women with no history of any cancer and undergoing reduction mammoplasty. Pearson correlations and ANOVAs were used to describe plasma-breast associations and biomarker differences by breast cancer risk factors, respectively. Multivariable regression models were used to determine associations between risk factors, and breast IGF-1 and IGFBP-3. The mean age of the study sample was 37.3 years, 58 % were white, and generally these women were obese (mean BMI = 30.8 kg/m(2)). We observed no plasma-breast correlation for IGF-1, IGFBP-3, or IGF-1/IGFBP-3 (r = -0.08, r = 0.14, and r = 0.03, respectively; p-values >0.05). Through age- and BMI-adjusted analysis, BMI and years of oral contraceptive (OC) use were inversely associated with breast IGF-1 (p-values = 0.02 and 0.003, respectively) and age was associated with breast IGFBP-3 (p = 0.01), while breast IGF-1/IGFBP-3 was higher in blacks than whites (1.08 vs. 0.68, p = 0.04) and associated with age and BMI (p-values = 0.03 and 0.002, respectively). In multivariable-adjusted models, some breast cancer risk factors studied herein explained 24, 10, and 15 % of the variation in breast IGF-1, IGFBP-3, and IGF-1/IGFBP-3, respectively. While reasons for the lack of plasma-breast hormone correlations in these cancer-free women are unknown, several factors were shown to be associated with breast concentrations. The lack of correlation between blood and tissue IGF-1 and IGFBP-3 suggests that studies of breast cancer risk assessing blood IGF-1 and IGFBP-3 may have important limitations in understanding their role in breast carcinogenesis.

Point-of-care testing in haemostasis.

Point-of-care testing (POCT) in haematology has seen a significant increase in both the spectrum of tests available and the number of tests performed annually. POCT is frequently undertaken with the belief that this will reduce the turnaround time for results and so improve patient care. The most obvious example of POCT in haemostasis is the out-of-hospital monitoring of the International Normalized Ratio in patients receiving a vitamin K antagonist, such as warfarin. Other areas include the use of the Activated Clotting Time to monitor anticoagulation for patients on cardio-pulmonary bypass, platelet function testing to identify patients with apparent aspirin or clopidogrel resistance and thrombelastography to guide blood product replacement during cardiac and hepatic surgery. In contrast to laboratory testing, POCT is frequently undertaken by untrained or semi-trained individuals and in many cases is not subject to the same strict quality control programmes that exist in the central laboratory. Although external quality assessment programmes do exist for some POCT assays these are still relatively few. The use of POCT in haematology, particularly in the field of haemostasis, is likely to expand and it is important that systems are in place to ensure that the generated results are accurate and precise.

Piriform spider silk sequences reveal unique repetitive elements.

Orb-weaving spider silk fibers are assembled from very large, highly repetitive proteins. The repeated segments contain, in turn, short, simple, and repetitive amino acid motifs that account for the physical and mechanical properties of the assembled fiber. Of the six orb-weaver silk fibroins, the piriform silk that makes the attachment discs, which lashes the joints of the web and attaches dragline silk to surfaces, has not been previously characterized. Piriform silk protein cDNAs were isolated from phage libraries of three species: A. trifasciata , N. clavipes , and N. cruentata . The deduced amino acid sequences from these genes revealed two new repetitive motifs: an alternating proline motif, where every other amino acid is proline, and a glutamine-rich motif of 6-8 amino acids. Similar to other spider silk proteins, the repeated segments are large (>200 amino acids) and highly homogenized within a species. There is also substantial sequence similarity across the genes from the three species, with particular conservation of the repetitive motifs. Northern blot analysis revealed that the mRNA is larger than 11 kb and is expressed exclusively in the piriform glands of the spider. Phylogenetic analysis of the C-terminal regions of the new proteins with published spidroins robustly shows that the piriform sequences form an ortholog group.

Rare case of hepatocellular carcinoma metastasising to the pituitary and cavernous sinus causing panhypopituitarism and bilateral ophthalmoplegia.

Pituitary metastases, especially from a primary hepatocellular carcinoma (HCC), are rare. Review of the literature revealed only few cases reporting pituitary metastases complicated by panhypopituitarism from HCC. Calvarial metastases from HCC are even more rare. Here, we present a unique case of primary HCC with metastases to both the calvarium and the pituitary causing panhypopituitarism and bilateral ophthalmoplegia, respectively. To our knowledge, this is the first reported case of two unique and rare complications from metastatic HCC.

Structural analysis of eight novel and 112 previously reported missense mutations in the interactive FXI mutation database reveals new insight on FXI deficiency.

Factor XI (FXI) functions in blood coagulation. FXI is composed of four apple (Ap) domains and a serine protease (SP) domain. Deficiency of FXI leads to an injury-related bleeding disorder, which is remarkable for the lack of correlation between bleeding symptoms and FXI coagulant activity (FXI:C). The number of mutations previously reported in our interactive web database (http://www.FactorXI.org) is now significantly increased to 183 through our new patient studies and from literature surveys. Eight novel missense mutations give a total of 120 throughout the FXI gene (F11). The most abundant defects in FXI are revealed to be those from low-protein plasma levels (Type I: CRM-) that originate from protein misfolding, rather than from functional defects (Type II: CRM+). A total of 70 Ap missense mutations were analysed using a consensus Ap domain structure generated from the FXI dimer crystal structure. This showed that all parts of the Ap domain were affected. The 47 SP missense mutations were also distributed throughout the SP domain structure. The periphery of the Ap beta-sheet structure is sensitive to structural perturbation caused by residue changes throughout the Ap domain, yet this beta-sheet is crucial for FXI dimer formation. Residues located at the Ap4:Ap4 interface in the dimer are much less directly involved. We conclude that the abundance of Type I defects in FXI results from the sensitivity of the Ap domain folding to residue changes within this, and discuss how structural knowledge of the mutations improves our understanding of FXI deficiencies.

Laboratory assay measurement of modified clotting factor concentrates: a review of the literature and recommendations for practice.

Over the past several years, novel modified clotting factor concentrates (CFCs) have been introduced into practice and are now widely prescribed in the countries where they are licensed. These products allow for less frequent infusions of CFC, thereby providing improved convenience and/or higher trough levels. They have been extensively studied for prophylaxis, episodic treatment of bleeding and for surgical prophylaxis. One issue that has emerged regarding the clinical application of these products revolves around the measurement of infused CFC in the clinical coagulation laboratory. Recent studies have demonstrated significant problems with the measurement of correct FVIII/IX levels following infusion of novel CF VIII/IX concentrates. The source of this problem appears to be related to the tremendous variability of the APTT reagents that are used in the one-stage clotting assay, the most commonly used assay for determining factor levels. More specifically, the issue is related to the type of activator used in the reagents. Depending on the combination of the CFC and the APTT activator, the observed results may be either under- or overestimated to degrees that would be clinically relevant. Recommendations based on a review of published information regarding the potential for incorrect measurements of factor VIII/IX levels following infusion of recently developed, novel factor VIII/IX CFCs are presented for the clinician to use in clinical practice.

Long-term safety and efficacy of extended-interval prophylaxis with recombinant factor IX Fc fusion protein (rFIXFc) in subjects with haemophilia B.

The safety, efficacy, and prolonged half-life of recombinant factor IX Fc fusion protein (rFIXFc) were demonstrated in the Phase 3 B-LONG (adults/adolescents ≥12 years) and Kids B-LONG (children <12 years) studies of subjects with haemophilia B (≤2 IU/dl). Here, we report interim, long-term safety and efficacy data from B-YOND, the rFIXFc extension study. Eligible subjects who completed B-LONG or Kids B-LONG could enrol in B-YOND. There were four treatment groups: weekly prophylaxis (20-100 IU/kg every 7 days), individualised prophylaxis (100 IU/kg every 8-16 days), modified prophylaxis (further dosing personalisation to optimise prophylaxis), and episodic (on-demand) treatment. Subjects could change treatment groups at any point. Primary endpoint was inhibitor development. One hundred sixteen subjects enrolled in B-YOND. From the start of the parent studies to the B-YOND interim data cut, median duration of rFIXFc treatment was 39.5 months and 21.9 months among adults/adolescents and children, respectively; 68/93 (73.1 %) adults/adolescents and 9/23 (39.1 %) children had ≥100 cumulative rFIXFc exposure days. No inhibitors were observed. Median annualised bleeding rates (ABRs) were low in all prophylaxis regimens: weekly (≥12 years: 2.3; <6 years: 0.0; 6 to <12 years: 2.7), individualised (≥12 years: 2.3; 6 to <12 years: 2.4), and modified (≥12 years: 2.4). One or two infusions were sufficient to control 97 % (adults/adolescents) and 95 % (children) of bleeding episodes. Interim data from B-YOND are consistent with data from B-LONG and Kids B-LONG, and confirm the long-term safety of rFIXFc, absence of inhibitors, and maintenance of low ABRs with prophylactic dosing every 1 to 2 weeks.

Plasma and plasma products in the treatment of massive haemorrhage.

Massive haemorrhage requires the use of plasma products when it is accompanied by a coagulopathy or when the more than one blood volume has been lost and intractable bleeding continues. The coagulopathy results from haemorrhagic shock, hypothermia, and activation, consumption and dilution of coagulation factors. Plasma products have a critical role in maintaining sufficient levels of coagulation proteins to ensure haemostasis can occur. Fresh frozen plasma is a source of all coagulation proteins and is required when the prothrombin time and activated partial thromboplastin time exceed 1.5 times the normal control. Cryoprecipitate is the plasma product of choice if fibrinogen, the most critical coagulation protein, is required rapidly and to maintain levels at >1g/L. Prothrombin complex concentrates, monocomponent factor therapy and fibrin sealants each have a role in specific clinical settings. Recombinant factor VIIa has now been shown to have a role in massive haemorrhage. Randomised controlled trials are currently underway to determine the optimal dose and timing of its administration. The physiology and management of the coagulation disturbance using plasma products in the massive haemorrhage of specific clinical situations are described.

The UK National External Quality Assessment Scheme (UK NEQAS) for molecular genetic testing in haemophilia.

Molecular genetic analysis of families with haemophilia and other inherited bleeding disorders is now a common laboratory investigation. In contrast to phenotypic testing in which strict quality control is adhered to, in haemophilia molecular genetic testing there has been a lack of any external quality assurance schemes. In 1998 the UK National External Quality Assessment Scheme (UK NEQAS) established a pilot quality assurance scheme for molecular genetic testing in haemophilia. Results from three initial surveys highlighted problems with the quality of samples when used to screen for the intron 22 inversion within the F8 gene. The scheme was re-launched in 2003, and since that time there have been five exercises involving whole blood or immortalised cell line DNA. The results together with an overall summary of the exercise are subsequently returned to participants. Exercises to date have focused exclusively on haemophilia A and QA, material has included screening for the intron 1 and intron 22 inversions as well as sequence analysis. A paper exercise circulated in 2003 highlighted problems with the format of reports and, following feedback to participants, only a single error has been made in the subsequent four exercises. Participating laboratories now receive QA material every six months. Immortalised cell line material was introduced in 2005 and was shown to perform well. This will allow expansion of the scheme and a reduction in the dependence on blood donation.

Phase II trial of concurrent paclitaxel, carboplatin, and external-beam radiation followed by surgical resection in locally advanced non-small-cell lung cancer, protocol 99-444.

PURPOSE: We conducted a phase II study to evaluate the utility and outcomes of concurrent weekly low-dose chemotherapy with concurrent radiation in an effort to "downstage" patients with locally advanced non-small-cell lung cancer (NSCLC). PATIENTS AND METHODS: Eighteen patients with pathologically confirmed stage IIIA (T1-3 N2 or T3 N1) and 3 patients with stage IIB (T3 N0) NSCLC were enrolled. Seventeen of 18 patients with stage IIIA cancer had N2 disease. A chemotherapy/radiation schedule consisted of paclitaxel 50 mg/m(2 )and carboplatin administered at an area under the curve of 2 weekly for 5 weeks along with chest irradiation of 45 Gy. Patients with regressed or stable disease upon restaging were considered surgical candidates. Patients deemed inoperable were given additional radiation therapy. RESULTS: Twenty-one patients were enrolled from April 2000 to March 2004. Data from 21 patients were available for evaluation at the time of analysis. Grade 3/4 constitutional and pulmonary toxicity was

The effects of unfractionated heparin, low molecular weight heparin and danaparoid on the thromboelastogram (TEG): an in-vitro comparison of standard and heparinase-modified TEGs with conventional coagulation assays.

To investigate the effects of unfractionated heparin (UFH), low molecular weight heparin (LMWH) and danaparoid (DPD) added to whole blood in vitro on standard and heparinase-modified thromboelastogram (TEG) parameters compared with conventional assays of coagulation. The effects of UFH, LMWH and DPD on standard TEG parameters were compared with the prothrombin time, activated partial thromboplastin time, thrombin time and anti-activated factor X (anti-FXa) activity, at concentrations of these anticoagulants ranging from 0.025 to 1 U/ml. In the second part of the study, the effects of very low concentrations (0.005-0.05 U/ml) of UFH, LMWH and DPD on the difference between standard and heparinase-modified TEG parameters were compared with the prothrombin time, activated partial thromboplastin time, thrombin time and anti-FXa activity. Standard TEG parameters were outside the reference range at lower concentrations of UFH, LMWH and DPD than most conventional coagulation assays were able to detect. Only anti-FXa activity was more sensitive to the presence of these anticoagulants than the standard TEG alone. The lowest concentration of UFH, LMWH and DPD used in this study (0.005 U/ml) caused significant differences between the standard and heparinase-modified alpha-angles of the TEG. In addition, the difference between standard and heparinase-modified TEG parameters distinguished between low concentrations (0.005-0.05 U/ml) of UFH with greater sensitivity than anti-FXa activity, but were less sensitive to LMWH and DPD. The standard TEG is more sensitive to UFH, LMWH and DPD than most conventional coagulation tests, with the exception of anti-FXa activity. Calculation of the difference between standard and heparinase-modified TEG parameters greatly increases the sensitivity of the assay for the effects of these anticoagulants, and is more sensitive to very low quantities of UFH than anti-FXa activity.

A dominant gain-of-function mutation in universal tyrosine kinase SRC causes thrombocytopenia, myelofibrosis, bleeding, and bone pathologies.

The Src family kinase (SFK) member SRC is a major target in drug development because it is activated in many human cancers, yet deleterious SRC germline mutations have not been reported. We used genome sequencing and Human Phenotype Ontology patient coding to identify a gain-of-function mutation in SRC causing thrombocytopenia, myelofibrosis, bleeding, and bone pathologies in nine cases. Modeling of the E527K substitution predicts loss of SRC's self-inhibitory capacity, which we confirmed with in vitro studies showing increased SRC kinase activity and enhanced Tyr(419) phosphorylation in COS-7 cells overexpressing E527K SRC. The active form of SRC predominates in patients' platelets, resulting in enhanced overall tyrosine phosphorylation. Patients with myelofibrosis have hypercellular bone marrow with trilineage dysplasia, and their stem cells grown in vitro form more myeloid and megakaryocyte (MK) colonies than control cells. These MKs generate platelets that are dysmorphic, low in number, highly variable in size, and have a paucity of α-granules. Overactive SRC in patient-derived MKs causes a reduction in proplatelet formation, which can be rescued by SRC kinase inhibition. Stem cells transduced with lentiviral E527K SRC form MKs with a similar defect and enhanced tyrosine phosphorylation levels. Patient-derived and E527K-transduced MKs show Y419 SRC-positive stained podosomes that induce altered actin organization. Expression of mutated src in zebrafish recapitulates patients' blood and bone phenotypes. Similar studies of platelets and MKs may reveal the mechanism underlying the severe bleeding frequently observed in cancer patients treated with next-generation SFK inhibitors.

Factor XI deficiency database: an interactive web database of mutations, phenotypes, and structural analysis tools.

Factor XI (FXI) is the zymogen of a serine protease enzyme in the intrinsic pathway of blood coagulation and is an important factor in the creation of a stable fibrin clot. Deficiency of FXI leads to an injury-related bleeding disorder and is remarkable for the lack of correlation between bleeding symptoms and FXI coagulant activity (FXI:C). The FXI protein is composed of five domains: four tandem repeat domains of approximately 80 residues known as Apple (Ap) domains, and the catalytic serine protease (Sp) domain. A total of 65 mutations throughout the FXI gene (F11) have been reported in FXI deficient patients. An interactive web database of these mutations has been created (www.FactorXI.org) that integrates the phenotypic data with genetic data and structural homology models for the five FXI domains. The database provides a central repository for all reported genetic alterations within F11. With the use of recently developed visualization tools, each mutation can be highlighted on the structural models of the FXI domains together with an appropriate survey of patient data, such as FXI:C levels and FXI antigen levels. The database also enables new F11 mutations to be interpreted. The interactive design of this database will lead to a more comprehensive comparative understanding of the genetic factors that influence bleeding risk.

Human phenotype ontology annotation and cluster analysis to unravel genetic defects in 707 cases with unexplained bleeding and platelet disorders.

BACKGROUND: Heritable bleeding and platelet disorders (BPD) are heterogeneous and frequently have an unknown genetic basis. The BRIDGE-BPD study aims to discover new causal genes for BPD by high throughput sequencing using cluster analyses based on improved and standardised deep, multi-system phenotyping of cases. METHODS: We report a new approach in which the clinical and laboratory characteristics of BPD cases are annotated with adapted Human Phenotype Ontology (HPO) terms. Cluster analyses are then used to characterise groups of cases with similar HPO terms and variants in the same genes. RESULTS: We show that 60% of index cases with heritable BPD enrolled at 10 European or US centres were annotated with HPO terms indicating abnormalities in organ systems other than blood or blood-forming tissues, particularly the nervous system. Cases within pedigrees clustered closely together on the bases of their HPO-coded phenotypes, as did cases sharing several clinically suspected syndromic disorders. Cases subsequently found to harbour variants in ACTN1 also clustered closely, even though diagnosis of this recently described disorder was not possible using only the clinical and laboratory data available to the enrolling clinician. CONCLUSIONS: These findings validate our novel HPO-based phenotype clustering methodology for known BPD, thus providing a new discovery tool for BPD of unknown genetic basis. This approach will also be relevant for other rare diseases with significant genetic heterogeneity.