Intracranial haemorrhage in children with inherited bleeding disorders in the UK 2003-2015: A national cohort study.

INTRODUCTION: Intracranial haemorrhage in children with inherited bleeding disorders is a potentially life-threatening complication and presents a significant therapeutic challenge. AIM: To define the characteristics, management and outcomes of intracranial haemorrhage presenting in UK children ≤16 years of age with inherited bleeding disorders from 2003 to 2015. METHOD: Retrospective analysis of children treated at UK haemophilia centres. RESULTS: Of 66 children presenting with Intracranial haemorrhage (ICH), 82% had haemophilia A or B, 3% VWD and 15% a rare IBD. The IBD was a severe phenotype in 91%. The rates of ICH were 6.4 and 4.2 per 1000 patient years for haemophilia A and B, respectively. Median age at presentation was 4 months (33% neonates; 91% children <2 years of age). In neonates, delivery was spontaneous vaginal (SV) in 11, instrumental in 6, caesarean in 4 and unknown in 1. In children with haemophilia, the risk of ICH after instrumental delivery was 10.6 times greater than after SV delivery. Trauma was more common in children >2 years (67%) than in children 1 month to 2 years (18%; P = .027). Prior to ICH, only 4.5% of children were on prophylaxis. 6% of haemophiliacs had an inhibitor. The median duration of initial replacement therapy was 15 days. Mortality was 13.5%. Neurological sequelae occurred in 39% of survivors, being more common following intracerebral bleeding. In haemophilia survivors, 52% subsequently developed a FVIII inhibitor. CONCLUSION: Intracranial haemorrhage occurs most frequently in children with severe IBDs, during the first 2 years of life and in children not receiving prophylaxis. Intracranial haemorrhage often occurs without documented trauma.

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.

On-demand treatment of bleeds in haemophilia patients with inhibitors: strategies for securing and maintaining predictable efficacy with recombinant activated factor VII.

On-demand therapy with recombinant activated factor VII (rFVIIa) can provide effective haemostasis for spontaneous bleeds in haemophilia patients with inhibitors. However, treatment approaches vary amongst physicians, positively or negatively affecting outcomes. A panel of physicians proposed recommendations for securing and maintaining predictable efficacy with rFVIIa, comparing these with 'real-life' patient management, using a questionnaire circulated to other expert physicians from haemophilia care centres in Europe and the United States. For rFVIIa treatment of spontaneous bleeds in inhibitor patients, early intervention with the highest appropriate dose is recommended. Home-based therapy can facilitate early intervention. If additional rFVIIa therapy is required after the initial dose, rFVIIa 90 µg kg(-1) may be administered at 2-3 h intervals. Treatment should be tailored to bleed site/severity, recognizing the advantages of appropriate adjunct therapy. Questionnaire results suggested that many respondents adopted strategies in line with the recommendations. Most (36/46) recommended initial therapy within 1 h of bleed onset. rFVIIa 270 µg kg(-1) was the most frequently prescribed/recommended initial dose for paediatric (aged ≤ 15 years; 22/44 respondents) and adult (aged > 15 years; 23/44 respondents) patients. However, there may be opportunity for improved bleed management on occasion, with regard, for instance, to dosing and dose interval. To secure and maintain predictable efficacy with rFVIIa, judicious dose selection and treatment timing are important, together with adjunct therapy where necessary. As inhibitor patients present with different bleeding scenarios, a tailored treatment approach should be adopted.

Bacterial community structure and bamA gene diversity in anaerobic degradation of toluene and benzoate under denitrifying conditions.

AIM: To characterize the microbial community structure and bamA gene diversity involved in anaerobic degradation of toluene and benzoate under denitrifying conditions. METHODS AND RESULTS: Nitrate-reducing enrichment cultures were established on either toluene, benzoate or without additional substrate. Bacterial community structures were characterized by 16S rRNA gene-based PCR-DGGE analysis. bamA gene diversity was analysed using DGGE and cloning methods. The results showed that bamA gene related to bamA of Thauera chlorobenzoica was dominant in toluene and benzoate cultures. However, a greater diversity of sequences was obtained in benzoate cultures. Low diversity of bamA gene was observed, and some similarities of bamA were also found between active cultures and backgrounds, suggesting that potential natural attenuation of aromatic compounds might occur. CONCLUSIONS: The combined analysis of 16S rRNA and bamA genes suggests that the species related to genera Thauera dominated toluene- and benzoate-degrading cultures. The combination of multiple methods (DGGE and cloning) provides a more complete picture of bamA gene diversity. SIGNIFICANCE AND IMPACT OF THE STUDY: To our knowledge, this is the first report of bamA gene in denitrifying enrichments using DGGE and cloning analysis.

A systematic review of the management and outcomes of pregnancy in Glanzmann thrombasthenia.

Glanzmann Thrombasthenia (GT) is a rare autosomal recessive disorder which usually manifests as severe mucocutaneous bleeding and is caused by deficiency of the platelet glycoprotein IIb-IIIa. Pregnancy in women with GT presents particular challenges as there is increased risk of both maternal and foetal bleeding. To improve understanding and clarify the optimum management of pregnancy in this disorder, we performed a systematic review of the world literature of pregnancy and GT. This identified three single-centre case series of patients with GT that included brief descriptions of women in pregnancy and 31 detailed case reports of 40 pregnancies in 35 women that resulted in 38 live births. Among the detailed case reports, ante-natal bleeding was described in 50% of pregnancies but was usually mild and occurred at mucocutaneous sites. Primary postpartum haemorrhage (PPH) was reported in 34% of pregnancies and secondary PPH in 24%. PPH was frequently severe and occurred up to 20 days after delivery. There was a wide variation in approach to prevention and treatment of PPH but most women received platelet transfusion, sometimes with additional recombinant FVIIa and anti-fibrinolytics. Maternal alloimmunization against platelet antigens was reported in 73% of pregnancies and was associated with four neonatal deaths. These data emphasize the need for multidisciplinary management of pregnancy in women with GT. Delivery plans should recognize the need for prevention and aggressive treatment of PPH and should minimize foetal bleeding risk in pregnancies complicated by alloimmunization.

F8 and F9 mutations fail to co-segregate in a family with co-incident haemophilia A and B.

Haemophilia A and B in one individual may arise from co-incident inheritance of independent mutations in the F8 and F9 genes. However, this association is rare and has been studied poorly at a genetic level. We report a male patient with abnormal bleeding and reduced factor VIII:C (26 IU dL(-1)) and factor IX:C (35 IU dL(-1)). This index case harboured a F8 c.979C>G transversion (predictive of p.Leu327Val) and a F9 c.845A>G transition (predictive of p.His282Arg) which have been previously associated with mild haemophilia A and B, respectively. Identical F8 and F9 mutations were identified in the mother and maternal grandmother. However, an affected maternal uncle showed only the F8 c.979C>G mutation, indicating haemophilia A alone. The sister of the index case was heterozygous only for F9 c.845A>G, indicating carriership of haemophilia B alone. The non-Mendelian inheritance of F8 c.979C>G and F9 c.845A>G in this kindred is consistent with recombination between F8 and F9 and illustrates the large recombination distance between these loci. Recognition of this phenomenon was essential for accurate genetic counselling in this kindred.

FEIBA prophylaxis in haemophilia patients: a clinical update and treatment recommendations.

In patients with severe haemophilia and inhibitors, regular factor VIII inhibitor bypassing activity (FEIBA) prophylaxis has been shown to reduce the frequency of bleeding by up to 85% and to improve patient quality of life. FEIBA is well tolerated; the incidence of thrombotic events and of allergic reactions is extremely low. The concept of prophylaxis in haemophilia patients with inhibitors is relatively new and some clinicians may be unsure of how to use FEIBA in this context. These treatment recommendations, based on published evidence plus the collective experience of a group of haematologists (with practical knowledge of managing inhibitor patients with FEIBA prophylaxis), are intended to provide guidance to clinicians considering initiating and maintaining patients on FEIBA prophylaxis with specific focus on practical aspects of patient selection, dosing, monitoring and stop criteria.

Phenotypic approaches to gene mapping in platelet function disorders - identification of new variant of P2Y12, TxA2 and GPVI receptors.

Platelet number or function disorders cause a range of bleeding symptoms from mild to severe. Patients with platelet dysfunction but normal platelet number are the most prevalent and typically have mild bleeding symptoms. The study of this group of patients is particularly difficult because of the lack of a gold-standard test of platelet function and the variable penetrance of the bleeding phenotype among affected individuals. The purpose of this short review is to discuss the way in which this group of patients can be investigated through platelet phenotyping in combination with targeted gene sequencing. This approach has been used recently to identify patients with mutations in key platelet activation receptors, namely those for ADP, collagen and thromboxane A2 (TxA2). One interesting finding from this work is that for some patients, mild bleeding is associated with heterozygous mutations in platelet proteins that are co-inherited with other genetic disorders of haemostasis such as type 1 von Willebrand's disease. Thus, the phenotype of mild bleeding may be multifactorial in some patients and may be considered to be a complex trait.

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.

Identification of critical members in a sulfidogenic benzene-degrading consortium by DNA stable isotope probing.

Stable isotope probing (SIP) was used to identify the active members in a benzene-degrading sulfidogenic consortium. SIP-terminal restriction fragment length polymorphism analysis indicated that a 270-bp peak incorporated the majority of the (13)C label and is a sequence closely related to that of clone SB-21 (GenBank accession no. AF029045). This target may be an important biomarker for anaerobic benzene degradation in the field.

Environmental signatures and effects of an oil and gas wastewater spill in the Williston Basin, North Dakota.

Wastewaters from oil and gas development pose largely unknown risks to environmental resources. In January 2015, 11.4ML (million liters) of wastewater (300g/L TDS) from oil production in the Williston Basin was reported to have leaked from a pipeline, spilling into Blacktail Creek, North Dakota. Geochemical and biological samples were collected in February and June 2015 to identify geochemical signatures of spilled wastewaters as well as biological responses along a 44-km river reach. February water samples had elevated chloride (1030mg/L) and bromide (7.8mg/L) downstream from the spill, compared to upstream levels (11mg/L and <0.4mg/L, respectively). Lithium (0.25mg/L), boron (1.75mg/L) and strontium (7.1mg/L) were present downstream at 5-10 times upstream concentrations. Light hydrocarbon measurements indicated a persistent thermogenic source of methane in the stream. Semi-volatile hydrocarbons indicative of oil were not detected in filtered samples but low levels, including tetramethylbenzenes and di-methylnaphthalenes, were detected in unfiltered water samples downstream from the spill. Labile sediment-bound barium and strontium concentrations (June 2015) were higher downstream from the Spill Site. Radium activities in sediment downstream from the Spill Site were up to 15 times the upstream activities and, combined with Sr isotope ratios, suggest contributions from the pipeline fluid and support the conclusion that elevated concentrations in Blacktail Creek water are from the leaking pipeline. Results from June 2015 demonstrate the persistence of wastewater effects in Blacktail Creek several months after remediation efforts started. Aquatic health effects were observed in June 2015; fish bioassays showed only 2.5% survival at 7.1km downstream from the spill compared to 89% at the upstream reference site. Additional potential biological impacts were indicated by estrogenic inhibition in downstream waters. Our findings demonstrate that environmental signatures from wastewater spills are persistent and create the potential for long-term environmental health effects.

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.

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.

Biogeochemical environments of streambed-sediment pore waters with and without arsenic enrichment in a sedimentary rock terrain, New Jersey Piedmont, USA.

Release of arsenic (As) from sedimentary rocks has resulted in contamination of groundwater in aquifers of the New Jersey Piedmont Physiographic Province, USA; the contamination also may affect the quality of the region's streamwater to which groundwater discharges. Biogeochemical mechanisms involved in the release process were investigated in the streambeds of Six Mile Run and Pike Run, tributaries to the Millstone River in the Piedmont. At Six Mile Run, streambed pore water and shallow groundwater were low or depleted in oxygen, and contained As at concentrations greater than 20 µg/L. At Pike Run, oxidizing conditions were present in the streambed, and the As concentration in pore water was 2.1 µg/L. The 16S rRNA gene and the As(V) respiratory reductase gene, arrA, were amplified from DNA extracted from streambed pore water at both sites and analyzed, revealing that distinct bacterial communities that corresponded to the redox conditions were present at each site. Anaerobic enrichment cultures were inoculated with pore water from gaining reaches of the streams with acetate and As(V). As(V) was reduced by microbes to As(III) in enrichments with Six Mile Run pore water and groundwater, whereas no reduction occurred in enrichments with Pike Run pore water. Cloning and sequencing of the arrA gene indicated 8 unique operational taxonomic units (OTUs) at Six Mile Run and 11 unique OTUs at Pike Run, which may be representative of the arsenite oxidase gene arxA. Low-oxygen conditions at Six Mile Run have favored microbial As reduction and release, whereas release was inhibited by oxidizing conditions at Pike Run.

Use of next-generation sequencing and candidate gene analysis to identify underlying defects in patients with inherited platelet function disorders.

BACKGROUND: Inherited platelet function disorders (PFDs) are heterogeneous, and identification of the underlying genetic defects is difficult when based solely on phenotypic and clinical features of the patient. OBJECTIVE: To analyze 329 genes regulating platelet function, number, and size in order to identify candidate gene defects in patients with PFDs. PATIENTS/METHODS: Targeted analysis of candidate PFD genes was undertaken after next-generation sequencing of exomic DNA from 18 unrelated index cases with PFDs who were recruited into the UK Genotyping and Phenotyping of Platelets (GAPP) study and diagnosed with platelet abnormalities affecting either Gi signaling (n = 12) or secretion (n = 6). The potential pathogenicity of candidate gene defects was assessed using computational predictive algorithms. RESULTS: Analysis of the 329 candidate PFD genes identified 63 candidate defects, affecting 40 genes, among index cases with Gi signaling abnormalities, while 53 defects, within 49 genes, were identified among patients with secretion abnormalities. Homozygous gene defects were more commonly associated with secretion abnormalities. Functional annotation analysis identified distinct gene clusters in the two patient subgroups. Thirteen genes with significant annotation enrichment for 'intracellular signaling' harbored 16 of the candidate gene defects identified in nine index cases with Gi signaling abnormalities. Four gene clusters, representing 14 genes, with significantly associated gene ontology annotations were identified among the cases with secretion abnormalities, the most significant association being with 'establishment of protein localization.' CONCLUSION: Our findings demonstrate the genetic complexity of PFDs and highlight plausible candidate genes for targeted analysis in patients with platelet secretion and Gi signaling abnormalities.

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.

Elevation of FVIII: C in venous thromboembolism is persistent and independent of the acute phase response.

Recent literature has suggested a role for elevated FVIII:C in venous thromboembolic disease (VTED). However since FVIII:C is known to rise in response to an acute phase reaction, it is difficult to determine whether the increased FVIII:C precedes the thrombosis or represents a secondary reactive phenomenon. In an attempt to address this question, we followed 35 patients with confirmed VTED, raised FVIII:C level (>1.5 iu/ml) and no other thrombotic tendency. Serial measurements of FVIII:C, vWF:Ag, C-reactive protein and fibrinogen were performed. We hypothesized that a persistent increase in FVIII:C in the absence of any other measures of ongoing acute phase response, would support the idea that elevation of FVIII:C is a constitutional phenomenon. Of this initial group, 94% continued to have an elevated FVIII:C level throughout the period of follow up (median 8 months; range 3 to 39 months), with no significant difference between the FVIII:C levels determined at first estimation and those obtained during follow up (p = 0.58). Conversely, only 18% had evidence of an acute phase reaction when first assessed, and nonparametric ranking analysis demonstrated no correlation between FVIII:C and either C-reactive protein or fibrinogen (p = 0.315 and 0.425 respectively).We conclude that increased FVIII:C levels following VTED are persistent, independent of the acute phase reaction, and thus may represent a constitutional risk factor for VTED.

Lesion of the subfornical organ affects the haemotensive response to centrally administered relaxin in anaesthetized rats.

Experiments were performed on anaesthetized, lactating rats to investigate the acute central actions of relaxin on blood pressure and vasopressin release. When compared with saline and control injections of isotonic protein extract, administration of relaxin into either the lateral or dorsal portion of the third ventricle caused a significant and sustained rise in arterial blood pressure. In contrast, relaxin administered to the ventral portion of the third ventricle caused only a short-term rise in blood pressure. Injections of relaxin into the fourth ventricle were without significant effect, suggesting that the central actions of relaxin on blood pressure are mediated by receptors restricted to the diencephalon or mesencephalon. A similar ventricular specificity was noted for the central relaxin-induced stimulation of vasopressin release as judged by concentrations of the hormone in the peripheral plasma. It is unlikely that the stimulation of vasopressin release is wholly responsible for the observed pressor effect observed. Lesion of the subfornical organ negated the pressor effect to relaxin injected into the dorsal region of the third ventricle, but did not affect the pressor response observed after injection of relaxin into the ventral portion of the third ventricle. These results demonstrate a biphasic action of centrally administered relaxin, with the response to dorsally placed third ventricle relaxin being mediated by the subfornical organ, and the response to ventral injections associated with an unknown structure of the ventral third ventricle wall.

Porcine relaxin affects the release of luteinizing hormone in rats.

Abstract The effects of intravenous injection of porcine relaxin on the pulsatile secretion of luteinizing hormone (LH) were investigated in conscious rats. In untreated, ovariectomized animals, relaxin at doses 2.5 to 10/mug/rat caused a dose-dependent suppression of pulsatile release of LH. At 5mug relaxin, pulses were suppressed for approximately 60 min and there was a significant (P<0.05) fall in mean plasma LH levels. Pulses returned with the same frequency as the pretreatment period but amplitude and nadir of these pulses were significantly (P<0.05) reduced at doses >2.5mug/rat. In ovariectomized rats pretreated with either estradiol or progesterone alone, relaxin did not alter plasma LH levels. In contrast, injection of 5mug relaxin in rats primed with a combination of estradiol and progesterone caused a 90% increase in circulating LH levels. Intracerebroventricular infusion of a specific angiotensin II antagonist blocked the inhibitory effect of relaxin on LH release in untreated, ovariectomized rats and negated the stimulatory effect of relaxin on LH release in estradiol-progesterone-primed, ovariectomized rats. The results demonstrate that acute injections of porcine relaxin in ovariectomized rats suppress the pulsatile release of LH. This effect is blocked when the central angiotensinergic system is compromised suggesting that relaxin might act through the central angiotensin system. The findings are in agreement with other studies that indicate relaxin activates the central angiotensin system. It is also possible that relaxin may act at the level of the adenohypophysis to alter secretion of LH but data in the present study suggest that this may not be a significant site of relaxin action.