A preliminary case series evaluating the safety and immediate to short-term clinical benefits of joint mobilization in hemophilic arthritis of the lower limb.

OBJECTIVES: Arthritis resulting from recurrent intra-articular bleeding in individuals with hemophilia can be severely debilitating due to joint pain and stiffness with subsequent loss of mobility and function. Very limited studies have investigated the potential benefits of joint mobilization for this condition. This case series is a preliminary investigation of safety, as well as immediate and short-term clinical benefits, associated with gentle knee and ankle joint mobilization in people with hemophilic arthropathy. METHODS: A single intervention of joint mobilization was applied to the affected knees and/or ankles of 16 individuals with severe or moderate hemophilia within a public hospital setting. Adverse events, as well as immediate (pain-free passive joint range, Timed Up and Go Test with maximum pain numerical rating scale) and short-term (Lower Extremity Functional Scale) effects of the intervention were evaluated with a repeated measures ANOVA. RESULTS: There were no adverse events. An immediate significant increase was observed in pain-free passive ankle joint range of motion (p < 0.05) following the joint mobilization intervention. DISCUSSION: The findings of this case series suggest that gentle joint mobilization techniques may be safely considered as part of a multimodal management approach for hemophilic arthropathy.

A rising titan: TTN review and mutation update.

The 364 exon TTN gene encodes titin (TTN), the largest known protein, which plays key structural, developmental, mechanical, and regulatory roles in cardiac and skeletal muscles. Prior to next-generation sequencing (NGS), routine analysis of the whole TTN gene was impossible due to its giant size and complexity. Thus, only a few TTN mutations had been reported and the general incidence and spectrum of titinopathies was significantly underestimated. In the last months, due to the widespread use of NGS, TTN is emerging as a major gene in human-inherited disease. So far, 127 TTN disease-causing mutations have been reported in patients with at least 10 different conditions, including isolated cardiomyopathies, purely skeletal muscle phenotypes, or infantile diseases affecting both types of striated muscles. However, the identification of TTN variants in virtually every individual from control populations, as well as the multiplicity of TTN isoforms and reference sequences used, stress the difficulties in assessing the relevance, inheritance, and correlation with the phenotype of TTN sequence changes. In this review, we provide the first comprehensive update of the TTN mutations reported and discuss their distribution, molecular mechanisms, associated phenotypes, transmission pattern, and phenotype-genotype correlations, alongside with their implications for basic research and for human health.

Desmopressin therapy to assist the functional identification and characterisation of von Willebrand disease: differential utility from combining two (VWF:CB and VWF:RCo) von Willebrand factor activity assays?

We performed a retrospective audit of desmopressin (DDAVP) usage to assist in the functional characterisation of von Willebrand disease (VWD). Data was evaluated for 208 patients, comprising those with VWD (Type 1 [n=160], Type 2A [n=19], Type 2M [n=10]), plus 19 individuals with haemophilia or carriers of haemophilia. Laboratory testing comprised pre- and post-DDAVP evaluation of factor VIII (FVIII:C), von Willebrand factor (VWF) antigen (VWF:Ag), VWF ristocetin cofactor (VWF:RCo) activity, VWF collagen binding (VWF:CB) activity, and in one laboratory an alternate VWF activity assay. In brief, combined usage of VWF:RCo and VWF:CB appears to provide improved functional characterisation and/or 'classification' of VWD types, in particular better differentiation of Type 2A and 2M VWD, and clearer validation of a Type 1 VWD diagnosis. Thus, (i) Type 1 VWD displayed generally good absolute and relative rises in all test parameters, although relative rises were greatest for FVIII:C and VWF:CB, and CB/Ag ratio increases overshadowed those for RCo/Ag; (ii) Type 2A VWD patients showed good absolute and relative rises in both FVIII:C and VWF:Ag, but poor absolute rises in both VWF:CB and VWF:RCo; although small rises in both CB/Ag and RCo/Ag were also observed, both ratios tended to remain below 0.7; (iii) finally, Type 2 M VWD patients generally showed good absolute and relative rises in FVIII:C, VWF:Ag and VWF:CB, but a poor absolute and relative rise in VWF:RCo; thus, there were good rises in CB/Ag ratios but little change in RCo/Ag, which tended to remain below 0.7. Future multi-centre prospective investigations are warranted to validate these findings and to investigate their therapeutic implications.

Cancer Core Europe: A translational research infrastructure for a European mission on cancer.

Cancer Core Europe is a European legal alliance consisting of seven leading cancer centres - most of them Comprehensive Cancer Centres (CCCs) - with a single portal system to engage in various research projects with partners. Cancer Core Europe was established to create a sustainable, high-level, shared research infrastructure platform hosting research collaborations and task forces (data sharing, clinical trials, genomics, immunotherapy, imaging, education and training, and legal and ethical issues), with a controlled expansion agenda. Translational cancer research covers the cancer research continuum from basic to preclinical to early clinical, late clinical, and outcomes research. Basic-preclinical research serves as the 'engine' for early clinical research by bridging the early translational research gap and is the primary and current focus of the consortium as exemplified by the launching of the Basket of Baskets trial, Europe's largest precision cancer medicine trial. Inspired by the creation of Cancer Core Europe, the prevention community established Cancer Prevention Europe, a consortium of ten cancer prevention centres aimed at supporting the complete prevention research continuum. Presently, Cancer Core Europe and Cancer Prevention Europe are integrating therapeutics and prevention strategies to address in partnership the widening cancer problem. By providing innovative approaches for cancer research, links to healthcare systems, development of quality-assured multidisciplinary cancer care, and assessment of long-term outcomes, the virtual infrastructure will serve as a hub to connect and interact with other centres across Europe and beyond. Together, Cancer Core Europe and Cancer Prevention Europe are prepared to function as a central engine to tackle, in collaboration with various partners, a potential 'mission on cancer' addressing the cancer burden.

Cancer Core Europe: A European cancer research alliance realizing a research infrastructure with critical mass and programmatic approach to cure cancer in the 21st century.

Translational cancer research covers the whole cancer research continuum from basic to preclinical to early clinical, late clinical and outcomes research. Basic-preclinical research is the "engine" for early clinical research bridging the early translational research gap. Cancer Core Europe has been created to construct a sustainable, high level, shared research infrastructure platform with research collaborations and taskforces (data sharing, clinical trials, genomics, immunotherapy, imaging, legal & ethical problems, and education & training) having representatives from all seven member centres, in a controlled expansion model. In parallel, a consortium of ten cancer prevention centres was established, Cancer Prevention Europe, to support the complete cancer prevention research continuum. Cancer Core Europe is launching at present the Basket of Baskets trial, which is the largest personalized cancer medicine trial effort in Europe. At present, Cancer Core Europe and Cancer Prevention Europe are in the process of integrating therapeutics and prevention strategies to address in partnership the increasing cancer problem. By offering innovative approaches for cancer research, links to the healthcare systems, development of quality-assured multidisciplinary cancer care, as well as the assessment of long-term outcomes, the infrastructure is expected to serve as a hub to connect with other centres in Europe as well as on other continents. In this manner Cancer Core Europe and Cancer Prevention Europe prepare to tackle the "Mission on Cancer", with infrastructure and proofs of concept for therapeutics and prevention, research for assessment of effectiveness, health economics and added value for patients and the healthcare systems.

Demonstration of a novel Xp22.2 microdeletion as the cause of familial extreme skewing of X-inactivation utilizing case-parent trio SNP microarray analysis.

BACKGROUND: We report a kindred referred for molecular investigation of severe hemophilia A in a young female in which extremely skewed X-inactivation was observed in both the proband and her clinically normal mother. METHODS: Bidirectional Sanger sequencing of all F8 gene coding regions and exon/intron boundaries was undertaken. Methylation-sensitive restriction enzymes were utilized to investigate skewed X-inactivation using both a classical human androgen receptor (HUMARA) assay, and a novel method targeting differential methylation patterns in multiple informative X-chromosome SNPs. Illumina Whole-Genome Infinium microarray analysis was performed in the case-parent trio (proband and both parents), and the proband's maternal grandmother. RESULTS: The proband was a cytogenetically normal female with severe hemophilia A resulting from a heterozygous F8 pathogenic variant inherited from her similarly affected father. No F8 mutation was identified in the proband's mother, however, both the proband and her mother both demonstrated completely skewed X-chromosome inactivation (100%) in association with a previously unreported 2.3 Mb deletion at Xp22.2. At least three disease-associated genes (FANCB, AP1S2, and PIGA) were contained within the deleted region. CONCLUSIONS: We hypothesize that true "extreme" skewing of X-inactivation (≥95%) is a rare occurrence, but when defined correctly there is a high probability of finding an X-chromosome disease-causing variant or larger deletion resulting in X-inactivation through a survival disadvantage or cell lethal mechanism. We postulate that the 2.3 Mb Xp22.2 deletion identified in our kindred arose de novo in the proband's mother (on the grandfather's homolog), and produced extreme skewing of X-inactivation via a "cell lethal" mechanism. We introduce a novel multitarget approach for X-inactivation analysis using multiple informative differentially methylated SNPs, as an alternative to the classical single locus (HUMARA) method. We propose that for females with unexplained severe phenotypic expression of an X-linked recessive disorder trio-SNP microarray should be undertaken in combination with X-inactivation analysis.

Comparison of the pharmacokinetics of two von Willebrand factor concentrates [Biostate and AHF (High Purity)] in people with von Willebrand disorder. A randomised cross-over, multi-centre study.

Plasma-derived factor concentrates are important in the management of von Willebrand disorder (VWD). In our geographic locality, a single viral inactivation step concentrate (AHF [High Purity]), has been replaced with one using a double viral inactivation step (Biostate). The aim of this study was to compare the pharmacokinetics of von Willebrand factor (VWF) and factor VIII (FVIII) after administration of AHF (High Purity) and Biostate. This study was a single-blind, randomised cross-over, multi-centre investigation in twelve people with VWD, comprising four type 3, two type 2B, one type 2M and five type 1 VWD. The subjects received a single infusion of 60 IU/kg ristocetin cofactor activity (VWF:RCo) of either AHF (High Purity) or Biostate, and after a minimum 15-day wash-out period they received the alternative product. Blood samples were collected for up to 48 hours after each dose for assay of FVIII coagulant activity (FVIII:C) and VWF by VWF:RCo, collagen binding capacity (VWF:CB) and antigen (VWF:Ag). As a measure of delivered VWF 'functionality' we calculated the area-under-the-concentration-time-curve (AUC) ratios of VWF:RCo to VWF:Ag and VWF:CB to VWF:Ag. The effect on platelet adhesiveness by PFA-100 closure times (CTs) was measured prior to and 30 minutes post infusion. VWF multimers were also assessed pre and post infusion. Pharmacokinetic parameters after AHF (High Purity) and Biostate were in close agreement for VWF:RCo (confirming dosing equivalence). Parameters for other study markers were also similar, although Biostate tended to yield relatively lower VWF:Ag and higher VWF:CB levels. Although AHF (High Purity) and Biostate resulted in similar levels of high-molecular-weight (HMW) multimers post-infusion, the relative level of HMW to low-molecular-weight (LMW) multimers were determined to be higher following Biostate. The relative levels of functional VWF (i.e. VWF:CB and VWF:RCo) to VWF:Ag were also higher in Biostate compared to AHF (High Purity). With both study products, PFA-100 CTs 30 minutes post infusion showed minor improvement for only some subjects. In conclusion, the pharmacokinetics of FVIII:C and VWF are not significantly different after administration of AHF (High Purity) and Biostate. Study parameters considered as 'in-vitro' markers of VWF 'functionality' or potential clinical efficacy (i.e. VWF:CB and VWF:RCo relative to VWF:Ag, level of HMW VWF relative to LMW-VWF) were determined to be higher for Biostate than AHF (High Purity). PFA-100 CTs did not adequately reflect changes in these VWF parameters. Based on these results, one would expect Biostate to be at least as effective, if not superior to AHF (High Purity) for the treatment of VWD.

Complex phenotypes in the haemoglobinopathies: recommendations on screening and DNA testing.

This document considers a number of scenarios involving complex haemoglobinopathies and provides 28 recommendations at both the clinical and laboratory levels on how these should be managed.

D2 dopamine receptor gene polymorphism: paroxetine and social functioning in posttraumatic stress disorder.

This study examined whether allelic status of the D2 dopamine receptor (DRD2) gene was associated with response to a selective serotonin reuptake inhibitor, paroxetine, in the treatment of posttraumatic stress disorder (PTSD). Sixty-three Caucasian war veterans with combat-related PTSD were treated with paroxetine for 8 weeks. Patients were assessed at baseline and at follow-up using the General Health Questionnaire-28 (GHQ). TaqI A DRD2 alleles were determined by PCR. Before paroxetine treatment, patients with the DRD2 A1+ allele (A1A2 genotype) compared to those with the A1- allele (A2A2 genotype) had higher total GHQ psychopathological scores (P=0.040) and higher GHQ subscale scores for anxiety/insomnia (0.046), social dysfunction (P=0.033) and depression (P=0.011). In an intention-to-treat analysis, paroxetine was associated with significant improvement in total GHQ scores (P=0.014) and in the factor scores of social dysfunction (P=0.033), anxiety (P=0.009) and depression (P=0.026). Furthermore, there was a significant allele by time interaction on the social dysfunction scale, with A1+ allelic patients showing significant improvement in social functioning compared to A1- allelic patients (P=0.031), an effect independent of changes in depression or anxiety. This suggests changes in social functioning induced by paroxetine may be, in part, mediated via D2 dopamine receptors. The DRD2 A1 allele may prove to be a useful marker to assist clinicians in predicting which patients with PTSD are likely to obtain improvements in social functioning with paroxetine treatment.

Variability of master gutta-percha cones.

The common technique to hermetically fill prepared root canals involves the use of "standardised" gutta-percha cones that are selected to fit the apical portion of the prepared canal space. These gutta-percha cones are manufactured to conform to a standard size and taper which should correspond to the size and taper of standard root canal instruments. Clinical observation of commercially available gutta-percha cones seemed to indicate that there is wide variation in the diameter and taper of "standardised" gutta-percha cones within the size range 25-35. The present study was undertaken to determine how closely current commercially available gutta-percha cones sizes 25, 30 and 35 conformed to the current ISO standard, and was initiated by the above clinical observation. It was not the purpose of this study to compare the results from different brands or manufacturers, but rather to establish whether commercially available gutta-percha cones collectively conformed to expected standardised sizes. One phial of cones for each of the sizes 25, 30 and 35 of eight different brands was selected for examination. The diameter of each of ten cones for each size from each brand was measured at two points, at 1 mm and at 6 mm from the tip of the cone. The results obtained for each size and each brand were tabulated and compared with ISO 6877:1995 for dental root canal obturating cones. This study demonstrated wide variability for cones from all brands, for all sizes, when individual cones of the same size were compared. While collectively the arithmetic means showed a closer correlation to the ISO Standard, irrespective of the brand size of the cone, or whether the cone was measured at 1 mm or 6 mm, many individual cones showed a great variation from the ideal. The need for less variability is discussed. It is concluded that ISO standard 6877:1995 is inappropriate- and allows for too much variation in the size of "standardised" gutta-percha cones.

HMGB1-facilitated p53 DNA binding occurs via HMG-Box/p53 transactivation domain interaction, regulated by the acidic tail.

Facilitated binding of p53 to DNA by high mobility group B1 (HMGB1) may involve interaction between the N-terminal region of p53 and the high mobility group (HMG) boxes, as well as HMG-induced bending of the DNA. Intramolecular shielding of the boxes by the HMGB1 acidic tail results in an unstable complex with p53 until the tail is truncated to half its length, at which point the A box, proposed to be the preferred binding site for p53(1-93), is exposed, leaving the B box to bind and bend DNA. The A box interacts with residues 38-61 (TAD2) of the p53 transactivation domain. Residues 19-26 (TAD1) bind weakly, but only in the context of p53(1-93) and not as a free TAD1 peptide. We have solved the structure of the A-box/p53(1-93) complex by nuclear magnetic resonance spectroscopy. The incipient amphipathic helix in TAD2 recognizes the concave DNA-binding face of the A box and may be acting as a single-stranded DNA mimic.

Potential supplementary utility of combined PFA-100 and functional von Willebrand factor testing for the laboratory assessment of desmopressin and factor concentrate therapy in von Willebrand disease.

We performed a retrospective audit of cross-laboratory testing of desmopressin and factor concentrate therapy to assess the potential utility of supplementary testing using the PFA-100 with functional von Willebrand factor (VWF) activity testing. Data were evaluated for a large number of patients with von Willebrand disease of type 1, type 2A or type 2M, as well as a comparative subset of individuals with haemophilia or carriers of haemophilia. Laboratory testing comprised pre and postdesmopressin, or pre and postconcentrate, evaluation of factor VIII, VWF antigen (VWF:Ag) and VWF ristocetin cofactor activity as traditionally performed, supplemented with collagen-binding (VWF:CB) testing and PFA-100 closure times. In brief, both therapies tended to normalize VWF test parameters and closure times in individuals with type 1 von Willebrand disease, with the level of correction in closure times related to the level of normalization of VWF, particularly the VWF:CB. However, although occasional correction of closure times was observed in patients with type 2A or type 2M von Willebrand disease, these did not in general normalize PFA-100 closure times either with desmopressin or factor concentrate therapy. In these patients, improvement in closure times was more likely in those in whom VWF:CB values normalized or when VWF:CB/VWF:Ag ratios normalized. This study confirms that there is a strong relationship between the presenting levels of plasma VWF and PFA-100 closure times, and that the supplementary combination of PFA-100 and VWF:CB testing might provide added clinical utility to current broadly applied testing strategies limited primarily to VWF:Ag, VWF ristocetin cofactor and factor VIII:coagulant. Future prospective investigations are warranted to validate these relationships and to investigate their therapeutic implications.

A consensus statement on the management of pregnancy and delivery in women who are carriers of or have bleeding disorders.

Pregnancy and delivery are critical times for women with bleeding disorders, with mothers, and possibly their affected infants, being exposed to a variety of haemostatic challenges. Management of women with bleeding disorders during pregnancy involves a multidisciplinary team including, but not limited to, an obstetrician, an anaesthetist and a haematologist. This consensus document from the Australian Haemophilia Centre Directors' Organisation (AHCDO) provides practical information for clinicians managing women with bleeding disorders during pregnancy. Included are: the expected physiological response in pregnancy in such women; management of pregnancy, labour and delivery, as well as obstetric anaesthesia issues, postpartum care, and reducing and treating postpartum haemorrhage; and management of infants at risk of a bleeding disorder and of bleeding in neonates. The guidelines were developed after extensive consultation, face-to-face meetings and revisions. The final document represents a consensus opinion of all AHCDO members. Where evidence is lacking, recommendations are based on clinical experience and consensus opinion.