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.

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.

Point-of-care derived INR does not reliably detect significant coagulopathy following Australian snakebite.

INTRODUCTION: Point-of-care international normalised ratio (INR) has been suggested as a way to screen for venom-induced consumption coagulopathy following snakebite, but has not been validated for this. This study aimed to assess the diagnostic reliability of point-of-care INR for venom-induced consumption coagulopathy. METHODS: This was a prospective study of snakebite patients recruited between January 2011 and May 2012 where a point-of-care INR was done and compared to an INR done on a laboratory coagulation analyser, as part of a quality assurance exercise. Data was obtained for each patient, including demographics, information on the snake bite, the point-of-care INR results and any laboratory derived coagulation studies. Snake identification was confirmed by expert identification or venom specific enzyme immunoassay. RESULTS: There were 15 patients with a median age of 29 years (2 to 68 y) and 13 were male. Four of the 7 patients with venom-induced consumption coagulopathy had an abnormal point-of-care INR (3 false negatives) and 1 of the 7 non-envenomed patients had an abnormal point-of-care INR (1 false positive). The patient with a falsely elevated point-of-care INR was given antivenom prior to formal coagulation studies. The point-of-care INR was also negative in the patient with an anticoagulant coagulopathy. CONCLUSIONS: The study shows that point-of-care INR testing devices should not be used in suspected snakebite cases in Australia to diagnose venom-induced consumption coagulopathy.

Prevention of intraventricular haemorrhage: a role for recombinant activated factor VII?

As the survival from extreme prematurity continues to improve, focus on the quality of this survival becomes increasingly important. Prevention of intraventricular haemorrhage (IVH) and its potential long-term sequelae remains one of the major challenges in the early management of these infants. Recombinant activated factor VII (rVIIa), a novel haemostatic agent with an ever-expanding list of potential applications, warrants consideration for use in this setting. This review examines the pathogenesis and prevention of IVH, current concepts of haemostasis both in adults and neonates, and the postulated mechanism of action and various uses of rVIIa. Published data specifically relating to use of rVIIa in neonates is summarised. The hypothesis that early (prophylactic) administration of rVIIa to extremely preterm infants (<28 weeks) would reduce the incidence of severe IVH is explored.