Safety and Efficacy of Recombinant Fusion Protein Linking Coagulation Factor IX with Albumin (rIX-FP) in Previously Untreated Patients with Hemophilia B.

Introduction Recombinant fusion protein linking coagulation factor IX (FIX) with albumin (rIX-FP) has been shown to be an effective, well-tolerated treatment for patients with severe hemophilia B who had previously received factor replacement therapy. This study investigated the safety and efficacy of rIX-FP in previously untreated patients (PUPs). Methods Patients with moderately severe/severe hemophilia B (≤2% FIX) previously untreated with FIX replacement products received rIX-FP (25-75 IU/kg) prophylaxis weekly or on-demand treatment over ≥50 exposure days (EDs). Primary outcomes were the number of patients who developed FIX inhibitors and mean incremental recovery (IR) following a 50 IU/kg dose of rIX-FP. Secondary outcomes included incidence of adverse events (AEs) and annualized bleeding rates (ABRs). Results In total, 12 PUPs with a median age of 0 years (range, 0-11 years) were treated with rIX-FP for a median of 50 EDs (6/12 prophylaxis; 6/12 on-demand then prophylaxis). Overall, 11/12 patients did not develop FIX inhibitors; one 11-year-old patient developed an inhibitor against FIX after 8 EDs and was ultimately withdrawn. Mean (standard deviation) IR was 1.2 (0.4, n = 8) (IU/dL)/(IU/kg). Of the 137 treatment-emergent AEs recorded, five were attributed to rIX-FP. On the prophylaxis regimen, median ABR was 1.0 (range, 0-3.9, n = 12). No thromboembolic events or deaths occurred during the study. Conclusion This study provides data to support the safety and efficacy of rIX-FP in PUPs requiring on-demand or prophylactic treatment for moderately severe/severe hemophilia B, consistent with results in previously treated patients. Overall, 1/12 patients developed an inhibitor against FIX.

Biallelic deleterious germline SH2B3 variants cause a novel syndrome of myeloproliferation and multi-organ autoimmunity.

SH2B3 is a negative regulator of multiple cytokine receptor signalling pathways in haematopoietic tissue. To date, a single kindred has been described with germline biallelic loss-of-function SH2B3 variants characterized by early onset developmental delay, hepatosplenomegaly and autoimmune thyroiditis/hepatitis. Herein, we described two further unrelated kindreds with germline biallelic loss-of-function SH2B3 variants that show striking phenotypic similarity to each other as well as to the previous kindred of myeloproliferation and multi-organ autoimmunity. One proband also suffered severe thrombotic complications. CRISPR-Cas9 gene editing of zebrafish sh2b3 created assorted deleterious variants in F0 crispants, which manifest significantly increased number of macrophages and thrombocytes, partially replicating the human phenotype. Treatment of the sh2b3 crispant fish with ruxolitinib intercepted this myeloproliferative phenotype. Skin-derived fibroblasts from one patient demonstrated increased phosphorylation of JAK2 and STAT5 after stimulation with IL-3, GH, GM-CSF and EPO compared to healthy controls. In conclusion, these additional probands and functional data in combination with the previous kindred provide sufficient evidence for biallelic homozygous deleterious variants in SH2B3 to be considered a valid gene-disease association for a clinical syndrome of bone marrow myeloproliferation and multi-organ autoimmune manifestations.

Functional interaction between compound heterozygous TERT mutations causes severe telomere biology disorder.

Telomere biology disorders (TBDs) are a spectrum of multisystem inherited disorders characterized by bone marrow failure, resulting from mutations in the genes encoding telomerase or other proteins involved in maintaining telomere length and integrity. Pathogenicity of variants in these genes can be hard to evaluate, because TBD mutations show highly variable penetrance and genetic anticipation related to inheritance of shorter telomeres with each generation. Thus, detailed functional analysis of newly identified variants is often essential. Herein, we describe a patient with compound heterozygous variants in the TERT gene, which encodes the catalytic subunit of telomerase, hTERT. This patient had the extremely severe Hoyeraal-Hreidarsson form of TBD, although his heterozygous parents were clinically unaffected. Molecular dynamic modeling and detailed biochemical analyses demonstrate that one allele (L557P) affects association of hTERT with its cognate RNA component hTR, whereas the other (K1050E) affects the binding of telomerase to its DNA substrate and enzyme processivity. Unexpectedly, the data demonstrate a functional interaction between the proteins encoded by the two alleles, with wild-type hTERT rescuing the effect of K1050E on processivity, whereas L557P hTERT does not. These data contribute to the mechanistic understanding of telomerase, indicating that RNA binding in one hTERT molecule affects the processivity of telomere addition by the other molecule. This work emphasizes the importance of functional characterization of TERT variants to reach a definitive molecular diagnosis for patients with TBD, and, in particular, it illustrates the importance of analyzing the effects of compound heterozygous variants in combination, to reveal interallelic effects.

Final results of the PUPs B-LONG study: evaluating safety and efficacy of rFIXFc in previously untreated patients with hemophilia B.

PUPs B-LONG evaluated the safety and efficacy of recombinant factor IX Fc fusion protein (rFIXFc) in previously untreated patients (PUPs) with hemophilia B. In this open-label, phase 3 study, male PUPs (age <18 years) with hemophilia B (≤2 IU/dL of endogenous factor IX [FIX]) were to receive treatment with rFIXFc. Primary end point was occurrence of inhibitor development, with a secondary end point of annualized bleed rate (ABR). Of 33 patients who received ≥1 dose of rFIXFc, 26 (79%) were age <1 year at study entry and 6 (18%) had a family history of inhibitors. Twenty-eight patients (85%) received prophylaxis; median dosing interval was 7 days, with an average weekly dose of 58 IU/kg. Twenty-seven patients (82%) completed the study. Twenty-one (64%), 26 (79%), and 28 patients (85%) had ≥50, ≥20, and ≥10 exposure days (EDs) to rFIXFc, respectively. One patient (3.03%; 95% confidence interval, 0.08% to 15.76%) developed a low-titer inhibitor after 11 EDs; no high-titer inhibitors were detected. Twenty-three patients (70%) had 58 treatment-emergent serious adverse events; 2 were assessed as related (FIX inhibition and hypersensitivity in 1 patient, resulting in withdrawal). Median ABR was 1.24 (interquartile range, 0.00-2.49) for patients receiving prophylaxis. Most (>85%) bleeding episodes required only 1 infusion for bleed resolution. In this first study reporting results with rFIXFc in pediatric PUPs with hemophilia B, rFIXFc was well tolerated, with the adverse event profile as expected in a pediatric hemophilia population. rFIXFc was effective, both as prophylaxis and in the treatment of bleeding episodes. This trial was registered at www.clinicaltrials.gov as #NCT02234310.

A Practical, One-Clinic Visit Protocol for Pharmacokinetic Profile Generation with the ADVATE myPKFiT Dosing Tool in Severe Hemophilia A Subjects.

Standard pharmacokinetic (PK) assessments are demanding for persons with hemophilia A, requiring a 72-hour washout and 5 to 11 timed blood samples. A no-washout, single-clinic visit, sparse sampling population PK (PPK) protocol is an attractive alternative. Here, we compared PK parameters obtained with a traditional washout, 6-sampling time point PPK protocol with a no-washout, single-clinic visit, reverse 2-sampling time point PPK protocol in persons with severe hemophilia A (SHA) receiving ADVATE. A total of 39 inhibitor-negative males with SHA (factor VIII activity [FVIII:C] < 2%) were enrolled in a prospective sequential design PK study. Participants completed a washout, 6-sampling time point PPK protocol as well as a no-washout, reverse 2-sampling time point protocol, with samples taken during a single 3-hour clinic visit 24 hours post home infusion of FVIII and then 3 hours post infusion in clinic. FVIII:C levels were analyzed by one-stage and chromogenic assays; blood group and von Willebrand factor antigen (VWF:Ag) were determined; and PK parameters were analyzed using the ADVATE myPKFiT dosing tool. There was moderate to almost perfect agreement for the PK parameters obtained with the 2- and the 6- point PPK protocols using a one-stage FVIII:C assay and a substantial to almost perfect agreement using a chromogenic FVIII:C assay. Significant associations between specific PK parameters and blood group and VWF:Ag were observed. The no-washout, single-clinic visit, reverse 2-sampling time point PPK protocol can be used in the routine clinical setting since it demonstrates sufficient accuracy compared with the more demanding and less practical washout, 6-sampling time point PPK protocol in persons with SHA receiving ADVATE.

A novel cause of DKC1-related bone marrow failure: Partial deletion of the 3' untranslated region.

Telomere biology disorders (TBDs), including dyskeratosis congenita (DC), are a group of rare inherited diseases characterized by very short telomeres. Mutations in the components of the enzyme telomerase can lead to insufficient telomere maintenance in hematopoietic stem cells, resulting in the bone marrow failure that is characteristic of these disorders. While an increasing number of genes are being linked to TBDs, the causative mutation remains unidentified in 30-40% of patients with DC. There is therefore a need for whole genome sequencing (WGS) in these families to identify novel genes, or mutations in regulatory regions of known disease-causing genes. Here we describe a family in which a partial deletion of the 3' untranslated region (3' UTR) of DKC1, encoding the protein dyskerin, was identified by WGS, despite being missed by whole exome sequencing. The deletion segregated with disease across the family and resulted in reduced levels of DKC1 mRNA in the proband. We demonstrate that the DKC1 3' UTR contains two polyadenylation signals, both of which were removed by this deletion, likely causing mRNA instability. Consistent with the major function of dyskerin in stabilization of the RNA subunit of telomerase, hTR, the level of hTR was also reduced in the proband, providing a molecular basis for his very short telomeres. This study demonstrates that the terminal region of the 3' UTR of the DKC1 gene is essential for gene function and illustrates the importance of analyzing regulatory regions of the genome for molecular diagnosis of inherited disease.

Simplifying surgery in haemophilia B: Low factor IX consumption and infrequent infusions in surgical procedures with rIX-FP.

INTRODUCTION: Long-acting recombinant factor IX (FIX) products may simplify the surgical treatment of haemophilia B patients. The impact of rIX-FP, a recombinant FIX fused to recombinant albumin, on FIX consumption and surgical management was assessed in patients with haemophilia B. MATERIALS AND METHODS: Male patients, ≤65 years old with severe haemophilia B (FIX activity ≤2%) requiring non-emergency surgery were enrolled in the surgical substudy of PROLONG-9FP. Dosing was based on World Federation of Hemophilia guidelines and patients' pharmacokinetics. Haemostatic efficacy was assessed on a 4-point scale. rIX-FP consumption and safety were monitored throughout the perioperative period. RESULTS: This updated dataset reports on thirty (8 minor and 22 major) surgeries conducted in 21 patients. A single preoperative bolus was used in 96.7% (n = 29) of surgeries. After minor surgery, patients received a median (range) of 0 (0-3) infusions with a median (range) consumption of 0 (0-178.89) IU/kg in the 14-day postoperative period. In patients who underwent major surgery (including 15 patients undergoing joint replacement surgery), the median (range) number of infusions in the 14-day postoperative period was 5 (0-11) and median consumption was 221.7 (0-444.07) IU/kg. Haemostatic efficacy was rated as excellent or good in 87.5% (7/8) of minor surgeries and 95.5% (21/22) of major surgeries. CONCLUSION: Surgical procedures can be performed using a single preoperative bolus of rIX-FP in nearly all patients. During postoperative care, use of rIX-FP necessitated infrequent infusions and low FIX consumption. Overall, data suggest rIX-FP simplifies perioperative care in patients with haemophilia B.

ALPK1 missense pathogenic variant in five families leads to ROSAH syndrome, an ocular multisystem autosomal dominant disorder.

PURPOSE: To identify the molecular cause in five unrelated families with a distinct autosomal dominant ocular systemic disorder we called ROSAH syndrome due to clinical features of retinal dystrophy, optic nerve edema, splenomegaly, anhidrosis, and migraine headache. METHODS: Independent discovery exome and genome sequencing in families 1, 2, and 3, and confirmation in families 4 and 5. Expression of wild-type messenger RNA and protein in human and mouse tissues and cell lines. Ciliary assays in fibroblasts from affected and unaffected family members. RESULTS: We found the heterozygous missense variant in the ɑ-kinase gene, ALPK1, (c.710C>T, [p.Thr237Met]), segregated with disease in all five families. All patients shared the ROSAH phenotype with additional low-grade ocular inflammation, pancytopenia, recurrent infections, and mild renal impairment in some. ALPK1 was notably expressed in retina, retinal pigment epithelium, and optic nerve, with immunofluorescence indicating localization to the basal body of the connecting cilium of the photoreceptors, and presence in the sweat glands. Immunocytofluorescence revealed expression at the centrioles and spindle poles during metaphase, and at the base of the primary cilium. Affected family member fibroblasts demonstrated defective ciliogenesis. CONCLUSION: Heterozygosity for ALPK1, p.Thr237Met leads to ROSAH syndrome, an autosomal dominant ocular systemic disorder.

Updated Australian consensus statement on management of inherited bleeding disorders in pregnancy.

INTRODUCTION: There have been significant advances in the understanding of the management of inherited bleeding disorders in pregnancy since the last Australian Haemophilia Centre Directors' Organisation (AHCDO) consensus statement was published in 2009. This updated consensus statement provides practical information for clinicians managing pregnant women who have, or carry a gene for, inherited bleeding disorders, and their potentially affected infants. It represents the consensus opinion of all AHCDO members; where evidence was lacking, recommendations have been based on clinical experience and consensus opinion. MAIN RECOMMENDATIONS: During pregnancy and delivery, women with inherited bleeding disorders may be exposed to haemostatic challenges. Women with inherited bleeding disorders, and their potentially affected infants, need specialised care during pregnancy, delivery, and postpartum, and should be managed by a multidisciplinary team that includes at a minimum an obstetrician, anaesthetist, paediatrician or neonatologist, and haematologist. Recommendations on management of pregnancy, labour, delivery, obstetric anaesthesia and postpartum care, including reducing and treating postpartum haemorrhage, are included. The management of infants known to have or be at risk of an inherited bleeding disorder is also covered. CHANGES IN MANAGEMENT AS A RESULT OF THIS STATEMENT: Key changes in this update include the addition of a summary of the expected physiological changes in coagulation factors and phenotypic severity of bleeding disorders in pregnancy; a flow chart for the recommended clinical management during pregnancy and delivery; guidance for the use of regional anaesthetic; and prophylactic treatment recommendations including concomitant tranexamic acid.

Recombinant activated factor VII in neonatal cardiac surgery.

OBJECTIVES: The aim of this study is to determine whether recombinant activated factor VII (rVIIa) was associated with thrombus formation in neonates undergoing cardiac surgery. METHODS: This is a retrospective study of neonates undergoing surgical repair of congenital cardiac lesions during a 9-year period. RESULTS: In our study, 416 cardiac operations requiring cardiopulmonary bypass (CPB) were performed on 414 neonates. The overall intravascular thrombus (thrombus) frequency for all operations was 45 of 416 (11%). A thrombus developed in 17 of 287 (6%) operations when rVIIa was not given. rVIIa was administered in 129 of 416 (31%) operations. Thrombus formation occurred in 28 of 129 (22%) operations when rVIIa was administered. There was an association between rVIIa use and thrombus formation [odds ratio (OR) 4.4, 95% confidence interval (CI) 2.3-8.4; P < 0.0001]. Patients with thrombus formation had an increased length of stay compared to those without thrombus. Neonates who underwent the Norwood procedures and received rVIIa and developed thrombus were more likely to be supported with extracorporeal membrane oxygenation (ECMO) and had a higher mortality compared to Norwood patients without thrombus. Logistic analysis adjusted for the paediatric index of mortality 2 score, the risk adjustment for congenital heart surgery and the use of ECMO demonstrated a strong association between rVIIa administration and thrombus formation (OR 3.5, 95% CI 1.7-6.9; P = 0.0004). However, there was no effect of the risk adjustment for congenital heart surgery-1 category or the paediatric index of mortality 2 score on thrombus formation. CONCLUSIONS: In neonates who underwent CPB surgery, administration of rVIIa was associated with an increased occurrence of intravascular thrombus formation compared to neonates not given rVIIa. In the Norwood population, thrombus formation was associated with a higher mortality.

Long-acting recombinant fusion protein linking coagulation factor IX with albumin (rIX-FP) in children. Results of a phase 3 trial.

A global phase 3 study evaluated the pharmacokinetics, efficacy and safety of a recombinant fusion protein linking coagulation factor IX with albumin (rIX-FP) in 27 previously treated male children (1-11 years) with severe and moderately severe haemophilia B (factor IX [FIX] activity ≤2 IU/dl). All patients received routine prophylaxis once every seven days for up to 77 weeks, and treated any bleeding episodes on-demand. The mean terminal half-life of rIX-FP was 91.4 hours (h), 4.3-fold longer than previous FIX treatment and clearance was 1.11 ml/h/kg, 6.4-fold slower than previous FIX treatment. The median (Q1, Q3) annualised spontaneous bleeding rate was 0.00 (0.00, 0.91) and was similar between the <6 years and ≥6 years age groups, with a weekly median prophylactic dose of 46 IU/kg. In addition, patients maintained a median trough level of 13.4 IU/dl FIX activity on weekly prophylaxis. Overall, 97.2 % of bleeding episodes were successfully treated with one or two injections of rIX-FP (95 % CI: 92 % to 99 %), 88.7 % with one injection, and 96 % of the treatments were rated effective (excellent or good) by the Investigator. No patient developed FIX inhibitors and no safety concerns were identified. These results indicate that rIX-FP is safe and effective for preventing and treating bleeding episodes in children with haemophilia B with weekly prophylaxis. Routine prophylaxis with rIX-FP at treatment intervals of up to 14 days are currently being investigated in children with severe and moderately severe haemophilia B. Clinicaltrials.gov (NCT01662531).

A novel 33-Gene targeted resequencing panel provides accurate, clinical-grade diagnosis and improves patient management for rare inherited anaemias.

Accurate diagnosis of rare inherited anaemias is challenging, requiring a series of complex and expensive laboratory tests. Targeted next-generation-sequencing (NGS) has been used to investigate these disorders, but the selection of genes on individual panels has been narrow and the validation strategies used have fallen short of the standards required for clinical use. Clinical-grade validation of negative results requires the test to distinguish between lack of adequate sequencing reads at the locations of known mutations and a real absence of mutations. To achieve a clinically-reliable diagnostic test and minimize false-negative results we developed an open-source tool (CoverMi) to accurately determine base-coverage and the 'discoverability' of known mutations for every sample. We validated our 33-gene panel using Sanger sequencing and microarray. Our panel demonstrated 100% specificity and 99·7% sensitivity. We then analysed 57 clinical samples: molecular diagnoses were made in 22/57 (38·6%), corresponding to 32 mutations of which 16 were new. In all cases, accurate molecular diagnosis had a positive impact on clinical management. Using a validated NGS-based platform for routine molecular diagnosis of previously undiagnosed congenital anaemias is feasible in a clinical diagnostic setting, improves precise diagnosis and enhances management and counselling of the patient and their family.

Human plasma-derived FVIII/VWD concentrate (Biostate): a review of experimental and clinical pharmacokinetic, efficacy and safety data.

Human plasma-derived factor VIII/von Willebrand factor complex concentrates are used to control bleeding in patients with von Willebrand disease (VWD) or haemophilia A (HA). The properties of these haemostatic factor concentrates vary widely, which can have significant clinical implications. This review provides an extensive overview of the molecular properties, in addition to pharmacokinetic, efficacy and safety data, and case studies of clinical experience of one such concentrate, Biostate. These data are discussed in the context of various therapeutic applications and compared with other factor concentrate products. Data are presented from data on file from the manufacturer; product information and published experimental and clinical pharmacokinetic, safety and efficacy study data; and example case studies of clinical experience. The data discussed herein demonstrate that Biostate has well-established efficacy profiles in the treatment of patients with VWD or HA, with the control of bleeding rated as 'excellent', 'good' or 'moderate' in >90% of patients. In an immune-tolerance induction setting, 73% of patients achieved a complete response following treatment with Biostate. Biostate was generally well tolerated in patients with HA or VWD, with infrequent minor adverse events reported and no reported cases of clinically relevant thrombosis.

Immune tolerance induction using a factor VIII/von Willebrand factor concentrate (BIOSTATE), with or without immunosuppression, in Australian paediatric severe haemophilia A patients with high titre inhibitors: a multicentre, retrospective study.

INTRODUCTION: It has been postulated that factor VIII (FVIII) products containing von Willebrand factor (VWF) may improve immune tolerance induction (ITI) success rate in patients with haemophilia A and poor prognostic factors. MATERIALS AND METHODS: We conducted a retrospective cohort analysis of a FVIII/VWF concentrate (BIOSTATE) for ITI in paediatric patients with severe haemophilia A (SHA) and inhibitors, from January 2003 to December 2011 at 3 paediatric-only Haemophilia Treatment Centres in Australia. Response to ITI was assessed at or before 33 months and at completion of ITI. Fifteen male patients with SHA were included in the analysis. RESULTS: BIOSTATE was used for primary ITI in 8 patients (2 years, range 1.1-11.5 years) and for salvage ITI in 7 patients (9.9 years, range 1.1-15.4). At the end of the observation period there were 11 patients who achieved a complete response with BIOSTATE after a median duration of 21 months (range 5-85 months); a partial response was achieved in 2 patients in whom ITI is ongoing. Therefore, the overall response rate was 86.6%. Two patients were deemed treatment failures: one due to non-compliance after 18 months of ITI and another in whom a partial response had not been achieved after 22 months of ITI. CONCLUSION: BIOSTATE was well-tolerated and effective when used for primary or salvage ITI in this cohort of paediatric patients with SHA and a high-level inhibitor.

Association between physical activity and risk of bleeding in children with hemophilia.

CONTEXT: Vigorous physical activity is thought to increase risk of bleeds in children with hemophilia, but the magnitude of the risk is unknown. OBJECTIVE: To quantify the transient increase in risk of bleeds associated with physical activity in children with hemophilia. DESIGN, SETTING, AND PARTICIPANTS: A case-crossover study nested within a prospective cohort study was conducted at 3 pediatric hemophilia centers in Australia between July 2008 and October 2010. A total of 104 children and adolescent boys aged 4 through 18 years with moderate or severe hemophilia A or B were monitored for bleeds for up to 1 year. Following each bleed, the child or parent was interviewed to ascertain exposures to physical activity preceding the bleed. Physical activity was categorized according to expected frequency and severity of collisions. The risk of bleeds associated with physical activity was estimated by contrasting exposure to physical activity in the 8 hours before the bleed with exposures in two 8-hour control windows, controlling for levels of clotting factor in the blood. MAIN OUTCOME MEASURES: Association of physical activity and factor level with risk of bleeding. RESULTS: The participants were observed for 4839 person-weeks during which time 436 bleeds occurred. Of these, 336 bleeds occurred more than 2 weeks after the preceding bleed and were used in the primary analysis of risk. Compared with inactivity and category 1 activities (eg, swimming), category 2 activities (eg, basketball) were associated with a transient increase in the risk of bleeding (30.6% of bleed windows vs 24.8% of first control windows; odds ratio, 2.7; 95% CI, 1.7-4.8, P < .001). Category 3 activities (eg, wrestling) were associated with a greater transient increase in risk (7.0% of bleed windows vs 3.4% of first control windows; odds ratio, 3.7; 95% CI, 2.3-7.3, P < .001). To illustrate absolute risk increase, for a child who bleeds 5 times annually and is exposed on average to category 2 activities twice weekly and to category 3 activities once weekly, exposure to these activities was associated with only 1 of the 5 annual bleeds. For every 1% increase in clotting factor level, bleeding incidence was lower by 2% (95% CI, 1%-3%; P = .004). CONCLUSIONS: In children and adolescents with hemophilia, vigorous physical activity was transiently associated with a moderate relative increase in risk of bleeding. Because the increased relative risk is transient, the absolute increase in risk of bleeds associated with physical activity is likely to be small.

Pulmonary embolism in children.

Unlike in adults, pulmonary embolism (PE) is an infrequent event in children. It has a marked bimodal distribution during the paediatric years, occurring predominantly in neonates and adolescents. The most important predisposing factors to PE in children are the presence of a central venous line (CVL), infection, and congenital heart disease. Clinical signs of PE are non-specific in children or can be masked by underlying conditions. Diagnostic testing is necessary in children, especially with the lack of clinical prediction rules. Recommendations for tests are derived from adult studies with ventilation/perfusion (V/Q) scintigraphy being well established. There exists an increasing role for computerised tomography pulmonary angiography (CTPA) and magnetic resonance pulmonary angiography (MRPA). Thrombotic events in children are initially treated with unfractionated heparin (UFH) or low molecular weight heparin (LMWH). For the extended anticoagulant therapy LMWH or vitamin K antagonists can be used with duration of treatment recommendations extrapolated from adult data. Mortality rates for PE in children are reported to be around 10%, with death usually related to the underlying disease processes. Exact data about recurrence risk in children is unknown. Because of the difference in aetiology, presentation, diagnostic methods and treatment between adults and children further research is necessary to assess the validity of recommendations for children.

Extended spectrum of human glucose-6-phosphatase catalytic subunit 3 deficiency: novel genotypes and phenotypic variability in severe congenital neutropenia.

OBJECTIVE: To delineate the phenotypic and molecular spectrum of patients with a syndromic variant of severe congenital neutropenia (SCN) due to mutations in the gene encoding glucose-6-phosphatase catalytic subunit 3 (G6PC3). STUDY DESIGN: Patients with syndromic SCN were characterized for associated malformations and referred to us for G6PC3 mutational analysis. RESULTS: In a cohort of 31 patients with syndromic SCN, we identified 16 patients with G6PC3 deficiency including 11 patients with novel biallelic mutations. We show that nonhematologic features of G6PC3 deficiency are good predictive indicators for mutations in G6PC3. Additionally, we demonstrate genetic variability in this disease and define novel features such as growth hormone deficiency, genital malformations, disrupted bone remodeling, and abnormalities of the integument. G6PC3 mutations may be associated with hydronephrosis or facial dysmorphism. The risk of transition to myelodysplastic syndrome/acute myeloid leukemia may be lower than in other genetically defined SCN subgroups. CONCLUSIONS: The phenotypic and molecular spectrum in G6PC3 deficiency is wider than previously appreciated. The risk of transition to myelodysplastic syndrome or acute myeloid leukemia may be lower in G6PC3 deficiency compared with other subgroups of SCN.

Megakaryocyte and platelet abnormalities in a patient with a W33C mutation in the conserved SH3-like domain of myosin heavy chain IIA.

Heterozygous mutations in MYH9, which encodes non-muscle myosin heavy chain IIA (MHC-IIA), result in autosomal dominant inherited MYH9-related disorders characterised by macro-thrombocytopenia, granulocyte inclusions, variable sensorineural deafness, cataracts and nephritis. MHC-IIA is assembled into a complex consisting of two pairs of light chains and two heavy chains, where the latter contain a neck region, SH3-like, motor and rod domains. We describe a patient with a Trp33Cys missense mutation in the SH3-like domain of MHC-IIA. Abnormal platelet function was observed using platelet aggregometry with the agonists epinephrine and adenosine diphosphate (ADP). Patient granulocytes and megakaryocytes, but not platelets, contained abnormal MHC-IIA inclusions visualised by confocal immunofluorescence or electron microscopy. Megakaryocytes grown in culture were smaller and contained hypolobulated nuclei compared to controls. Bone marrow-derived megakaryocytes revealed a preponderance of immature forms, the presence of structurally diverse inclusion bodies, and frequent emperipolesis as assessed by electron microscopy. Platelets and leukocytes contained indistinguishable amounts of total MHC-IIA determined by immunoblotting. Molecular modelling studies indicated that mutation of Trp33 destabilises the interface between the SH3-like and motor domain of MHC-IIA, which is close to previously described motor domain mutations, implying an important structural and/or functional role for this region in MHC-IIA.