High-throughput microfluidic blood testing to phenotype genetically linked platelet disorders: an aid to diagnosis.

Linking the genetic background of patients with bleeding diathesis and altered platelet function remains challenging. We aimed to assess how a multiparameter microspot-based measurement of thrombus formation under flow can help identify patients with a platelet bleeding disorder. For this purpose, we studied 16 patients presenting with bleeding and/or albinism and suspected platelet dysfunction and 15 relatives. Genotyping of patients revealed a novel biallelic pathogenic variant in RASGRP2 (splice site c.240-1G>A), abrogating CalDAG-GEFI expression, compound heterozygosity (c.537del, c.571A>T) in P2RY12, affecting P2Y12 signaling, and heterozygous variants of unknown significance in the P2RY12 and HPS3 genes. Other patients were confirmed to have Hermansky-Pudlak syndrome type 1 or 3. In 5 patients, no genetic variant was found. Platelet functions were assessed via routine laboratory measurements. Blood samples from all subjects and day controls were screened for blood cell counts and microfluidic outcomes on 6 surfaces (48 parameters) in comparison with those of a reference cohort of healthy subjects. Differential analysis of the microfluidic data showed that the key parameters of thrombus formation were compromised in the 16 index patients. Principal component analysis revealed separate clusters of patients vs heterozygous family members and control subjects. Clusters were further segregated based on inclusion of hematologic values and laboratory measurements. Subject ranking indicated an overall impairment in thrombus formation in patients carrying a (likely) pathogenic variant of the genes but not in asymptomatic relatives. Taken together, our results indicate the advantages of testing for multiparametric thrombus formation in this patient population.

Health-related quality of life in children, adolescents and adults with hereditary and acquired bleeding disorders.

BACKGROUND: To better understand self-reported health-related quality-of-life (HrQoL) in children and adults with chronic hemostatic conditions compared with healthy controls. METHODS/PATIENTS/RESULTS: Group 1 consisted of 74 children/adolescents aged 8-18years with hereditary bleeding disorders (H-BD), 12 siblings and 34 peers. Group 2 consisted of 82 adult patients with hereditary/acquired bleeding disorders (H/A-BD), and group 3 of 198 patients with deep venous thrombosis (DVT) on anticoagulant therapy. Adult patients were compared to 1011 healthy blood donors. HrQoL was assessed with a 'revised KINDer Lebensqualitaetsfragebogen' (KINDL-R)-questionnaire adapted to adolescents and adults. No differences were found in multivariate analyses of self-reported HrQoL in children with H-BD. In contrast, apart from family and school-/work-related wellbeing in female patients with DVT the adult patients showed significantly lower HrQoL sub-dimensions compared to heathy control subjects. Furthermore, adults with H/A-BD disorders reported better friend-related HrQoL compared to patients with DVT, mainly due to a decreased HrQoL subscale in women on anticoagulation. CONCLUSION: In children with H-BD, HrQoL was comparable to siblings and peers. In adults with H/A-BD HrQoL was comparable to patients with DVT while healthy blood donors showed better HrQoL. The friend-related HrQoL subscale was significantly reduced in female compared to male patients.

Rare genetic variants in SMAP1, B3GAT2, and RIMS1 contribute to pediatric venous thromboembolism.

Recent genome-wide association studies (GWAS) have confirmed known risk mutations for venous thromboembolism (VTE) and identified a number of novel susceptibility loci in adults. Here we present a GWAS in 212 nuclear families with pediatric VTE followed by targeted next-generation sequencing (NGS) to identify causative mutations contributing to the association. Three single nucleotide polymorphisms (SNPs) exceeded the threshold for genome-wide significance as determined by permutation testing using 100 000 bootstrap permutations (P < 10-5). These SNPs reside in a region on chromosome 6q13 comprising the genes small ARF GAP1 (SMAP1), an ARF6 guanosine triphosphatase-activating protein that functions in clathrin-dependent endocytosis, and ß-1,3-glucoronyltransferase 2 (B3GAT2), a member of the human natural killer 1 carbohydrate pathway. Rs1304029 and rs2748331 are associated with pediatric VTE with unpermuted/permuted values of P = 1.42 × 10-6/2.0 × 10-6 and P = 6.11 × 10-6/1.8 × 10-5, respectively. Rs2748331 was replicated (P = .00719) in an independent study sample coming from our GWAS on pediatric thromboembolic stroke (combined P = 7.88 × 10-7). Subsequent targeted NGS in 24 discordant sibling pairs identified 17 nonsynonymous coding variants, of which 1 located in SMAP1 and 3 in RIMS1, a member of the RIM family of active zone proteins, are predicted as damaging by Protein Variation Effect Analyzer and/or sorting intolerant from tolerant scores. Three SNPs curtly missed statistical significance in the transmission-disequilibrium test in the full cohort (rs112439957: P = .08326, SMAP1; rs767118962: P = .08326, RIMS1; and rs41265501: P = .05778, RIMS1). In conjunction, our data provide compelling evidence for SMAP1, B3GAT2, and RIMS1 as novel susceptibility loci for pediatric VTE and warrant future functional studies to unravel the underlying molecular mechanisms leading to VTE.

Impact of high risk thrombophilia status on recurrence among children and adults with VTE: An observational multicenter cohort study.

BACKGROUND: Antithrombin [AT]-, protein C [PC]- or protein S [PS]-deficiency [D] constitutes a major risk factor for venous thromboembolism [VTE]. Primary study objective was to evaluate if the clinical presentation at first VTE onset differs between children and adults and to compare the individual recurrence risk among patients with respect to age at onset and their thrombophilia status ATD, PCD or PSD. METHODS/PATIENTS/RESULTS: In 137 of 688 consecutively enrolled pediatric and adult VTE patients we calculated the absolute risk of VTE recurrence and event-free-survival adjusted for thrombophilia and positive family VTE history. At first VTE children manifested i) with a lower rate of pulmonary embolism, ii) a higher rate of cerebral vascular events or multiple VTEs, and iii) showed a higher proportion of unprovoked VTE compared to adolescents and adults. Adult patients reported more often a positive VTE history compared to younger study participants. The adjusted odds of recurrence in adults was 2.05 compared to children. CONCLUSION: At disease manifestation children and adults differ with respect to i) thrombotic locations, ii) percentage of unprovoked versus provoked VTE, and iii) different rates of positive VTE family histories. Furthermore, adults showed a two-fold increase risk of VTE recurrence compared to children.

Impact of high-risk thrombophilia status on recurrence among children with a first non-central-venous-catheter-associated VTE: an observational multicentre cohort study.

Deficiency of antithrombin (AT), protein C (PC) or protein S (PS) constitutes a major risk factor for venous thromboembolism (VTE). Individuals at high risk for recurrence who benefit from screening need to be identified. The primary study objective was to determine the individual recurrence risk among children with a first non-central-venous-catheter-associated VTE with respect to their thrombophilia status and to evaluate if the clinical presentation at first VTE onset differs between children with AT, PC or PS deficiency versus no thrombophilia. We calculated the absolute risk of VTE recurrence and event-free-survival adjusted for thrombophilia, age, sex and positive family VTE history in 161 consecutively enrolled paediatric VTE patients. The presence of a deficiency relative to no thrombophilia was evaluated as a potential predictor of recurrence. Predictors for recurrence were AT deficiency (hazard ratio/95% CI: 6·5/2·46-17·2) and female gender (2·6/1·1-6·35). The annual recurrence rates (95% CIs) were 5·4% (2·6-10) in AT-deficient children, 1·3% (0·3-3·8) in patients with PC deficiency, 0·7% (0·08-2·4) in the PS-deficient cohort and 0·9% (0·4-1·8) in patients with no thrombophilia. Positive family VTE history or combined thrombophilias did not predict recurrence. Given the overall annual incidence rate of recurrence of 1·5% we suggest screening for AT deficiency in children with VTE.

Health-Related Quality of Life in Children and Adolescents with Hereditary Bleeding Disorders and in Children and Adolescents with Stroke: Cross-Sectional Comparison to Siblings and Peers.

Objectives. To investigate self-reported health-related quality of life (HrQoL) in children and adolescents with chronic medical conditions compared with siblings/peers. Methods. Group 1 (6 treatment centers) consisted of 74 children/adolescents aged 8-16 years with hereditary bleeding disorders (HBD), 12 siblings, and 34 peers. Group 2 (one treatment center) consisted of 70 children/adolescents with stroke/transient ischemic attack, 14 siblings, and 72 peers. HrQoL was assessed with the "revised KINDer Lebensqualitätsfragebogen" (KINDL-R) questionnaire. Multivariate analyses within groups were done by one-way ANOVA and post hoc pairwise single comparisons by Student's t-tests. Adjusted pairwise comparisons were done by hierarchical linear regressions with individuals nested within treatment centers (group 1) and by linear regressions (group 2), respectively. Results. No differences were found in multivariate analyses of self-reported HrQoL in group 1, while in group 2 differences occurred in overall wellbeing and all subdimensions. These differences were due to differences between patients and peers. After adjusting for age, gender, number of siblings, and treatment center these differences persisted regarding self-worth (p = .0040) and friend-related wellbeing (p < .001). Conclusions. In children with HBD, HrQoL was comparable to siblings and peers. In children with stroke/TIA HrQoL was comparable to siblings while peers, independently of relevant confounder, showed better self-worth and friend-related wellbeing.

Role of protein S deficiency in children with venous thromboembolism. An observational international cohort study.

Venous thromboembolism [TE] is a multifactorial disease, and protein S deficiency [PSD] constitutes a major risk factor. In the present study the prevalence of PSD and the clinical presentation at TE onset in a cohort of children is reported. In 367 unselected paediatric patients with TE (age 0.1-18 years) recruited between July 1996 and December 2013, a comprehensive thrombophilia screening was performed along with recording of anamnestic data. Thirty of 367 paediatric patients (8.2 %) derived from 27 families had PSD. Mean age at first TE onset was 14.5 years (range 0.1 to 18). Thrombotic locations were cerebral veins (n=8), calf vein TE (n=3) deep veins (DVT) of the leg (n=12), DVT & pulmonary embolism (n=5) and intra-cardiac veins (n=1) or purpura fulminans (n=1). PSD co-occurred with the factor 5 mutation at rs6025 or the homozygous factor 2 susceptibility variant at rs1799963 in one case each. The Heerlen polymorphism detected in five children presented with milder PSD. In 18 patients (60 %) a concomitant risk factor for TE was identified. A second TE event within primarily healthy siblings occurred in three of 27 PSD families (11.0 %). In this cohort of children with symptomatic TE, the prevalence of PSD adjusted for family status was 7.4 %. Given its clinical implication for patients and family members, thrombophilia testing should be performed and the benefit of medical or educational interventions should be evaluated in this high-risk population.

Inherited thrombophilia in children with venous thromboembolism and the familial risk of thromboembolism: an observational study.

Screening for inherited thrombophilia (IT) is controversial; persons at high risk for venous thromboembolism (VTE) who benefit from screening need to be identified. We tested 533 first- and second-degree relatives of 206 pediatric VTE patients for IT (antithrombin, protein C, protein S, factor V G1691A, factor II G20210A) and determined the incidence of symptomatic VTE relative to their IT status. The risk for VTE was significantly increased among family members with, versus without, IT (hazard ratio = 7.6; 95% confidence interval [CI], 4.0-14.5; P < .001) and highest among carriers of antithrombin, protein C, or protein S deficiency (hazard ratio = 25.7; 95% CI, 12.2-54.2; P < .001). Annual incidences of VTE were 2.82% (95% CI, 1.63%-4.80%) among family members found to be carriers of antithrombin, protein C, or protein S deficiency, 0.42% (0.12%-0.53%) for factor II G202010A, 0.25% (0.12%-0.53%) for factor V G1691A, and 0.10% (0.06%-0.17%) in relatives with no IT. Given the high absolute risk of VTE in relatives with protein C, protein S, and antithrombin deficiency, we suggest screening for these forms of hereditary thrombophilia in children with VTE and their relatives. Interventional studies are required to assess whether thromboembolism can be prevented in this high-risk population.

Safety and efficacy of low molecular weight heparins in children: a systematic review of the literature and meta-analysis of single-arm studies.

Within the last two decades low molecular weight heparins (LMWH) have gained increasing widespread use as anticoagulants in children. The use of LMWH has been implemented into clinical care even though there is a lack of firm evidence on the efficacy and safety of LMWH in this population due to the absence of sufficiently powered randomized controlled trials. In the absence of clinical trials, we performed a meta-analysis of available single-arm studies using LMWH in children. A systematic search of electronic databases (Medline, EMBASE, OVID, Web of Science, The Cochrane Library) for studies published from 1980 to 2010 was conducted using keywords in combination both as MeSH terms and text words. Two authors independently screened citations and those meeting a priori defined inclusion criteria were retained. Data on year of publication, study design, country of origin, number of patients, ethnicity, venous thromboembolic events type, and frequency of recurrence and major bleedings were abstracted. Pooled incidence rates (IR) including 95% confidence intervals (95% CIs) on efficacy and safety data of LMWH administration on primary prophylaxis, as well as on secondary prophylaxis in children following symptomatic thromboembolism (TE) were shown. We included 2251 pediatric patients derived from 35 single-arm studies from 12 study countries who were eligible for analysis in the present systematic review. Pooled incidence rates (95% CI) to develop first TE on primary prophylaxis, further TE event on LMWH secondary prophylaxis, or a major bleeding event on LMWH were 0.047 (0.023 to 0.091), 0.052 (0.037 to 0.073) for efficacy, and 0.054 (0.039 to 0.074) for safety (treatment data only), respectively. Efficacy and safety data are comparable with adult data. The present systematic review suggests that use of LMWH in children as primary prophylaxis and in treatment of symptomatic thrombosis is effective and safe. However, properly designed randomized controlled trials are needed.

Health-related quality of life in children and adolescents with stroke, self-reports, and parent/proxies reports: cross-sectional investigation.

OBJECTIVE: Limited data are available on health-related quality of life (HR-QoL) in pediatric stroke survivors. The aim of the present study was to assess HR-QoL by self-assessment and parent/proxy-assessment in children and adolescents who survived a first stroke episode. METHODS: We investigated HR-QoL in pediatric stroke survivors (71 preschool children [G1] and 62 school children/adolescents [G2]) and in 169 healthy controls. HR-QoL was assessed in patients and parents/proxies with the generic KINDL-R questionnaire exploring overall well-being and 6 well-being subdimensions (physical, psychological, self-esteem, family-related, friend-related, and school-related). In pediatric stroke survivors the neurological long-term outcome was measured with the standardized Pediatric Stroke Outcome Measure. RESULTS: Of stroke survivors, 65% exhibited at least 1 neurologic disability. Pediatric stroke survivors reported lower overall well-being compared with healthy controls. In G2 stroke patients, friend-related well-being respectively emotional well-being was significantly reduced compared with healthy controls (73.0 vs 85.0 points; p < 0.001 respectively 80.2 vs 84.5 points; p = 0.049). Parents/proxies of both stroke survivors rated the overall well-being and all subdimensions (except family-related and school-related well-being in G1 and G2 stroke survivors and physical functioning in G2 stroke survivors) lower compared with parents/proxies of healthy children/adolescents. Overall well-being was significantly reduced in children with moderate/severe neurological deficits compared with normal/mildly affected patients (75.5 vs 83.3 points, p = 0.01). Neonatal stroke survivors reported a significantly better neurological long-term outcome compared to childhood stroke survivors (82.0 vs 75.0 points; p = 0.005). INTERPRETATION: Pediatric stroke survivors compared with healthy controls are strongly affected regarding their overall well-being and older children/adolescents regarding their well-being with peers.

Long-term secondary prophylaxis in children, adolescents and young adults with von Willebrand disease. Results of a cohort study.

In patients with von Willebrand disease (VWD) replacement therapy with factor VIII/von Willebrand (VWF) concentrates is increasingly applied as prophylactic regimen. Since 2000, 82 consecutively enrolled patients with clinically relevant bleeding episodes (spontaneous, peri- or postoperative) were diagnosed with VWD [type 1: 42/82; type 2: 24/82; type 3: 13/82; acquired: 3/82]. In all patients, decision for initiating prophylaxis was based on a bleeding score > 2 prior to diagnosis, concomitant with recurrent bleeds associated with anaemia in patients with on-demand VWD therapy. We report results on secondary prophylactic VWF replacement therapy applied in 32 patients [children n=13; adolescents n=7; adults n=12] with VWD [type 1: 4; type 2: 15; type 3: 13], 15 of which were females, and nine of these at the reproductive period. Eight patients were treated with Humate P® or Wilate® (n=24). Median [min-max] dose [vWF:RCo] was 40 [20-47] IU/kg, 23 patients were given substitution therapy twice weekly, seven patients three times a week, and two children four times per week. Within a 12-month-period haemoglobin concentrations returned to normal values. Median duration of prophylaxis was three years. Recurrent bleeding episodes stopped in 31 of 32 patients, whereas inhibitors developed in one. Following a 12-month observation period the monthly bleeding frequency and the bleeding score was significantly reduced [3 vs. 0.07; 3 vs. 0: p< 0.001], compared to the pre-prophylaxis/pre-diagnostic values. The use of secondary prophylactic VWF replacement therapy is an effective tolerated treatment modality, highly beneficial for patients with VWD, who present with recurrent bleeding events during on-demand therapy.

Impact of inherited thrombophilia on venous thromboembolism in children: a systematic review and meta-analysis of observational studies.

BACKGROUND: The aim of the present study was to estimate the impact of inherited thrombophilia (IT) on the risk of venous thromboembolism (VTE) onset and recurrence in children by a meta-analysis of published observational studies. METHODS AND RESULTS: A systematic search of electronic databases (Medline, EMBASE, OVID, Web of Science, The Cochrane Library) for studies published from 1970 to 2007 was conducted using key words in combination as both MeSH terms and text words. Citations were independently screened by 2 authors, and those meeting the inclusion criteria defined a priori were retained. Data on year of publication, study design, country of origin, number of patients/controls, ethnicity, VTE type, and frequency of recurrence were abstracted. Heterogeneity across studies was evaluated, and summary odds ratios and 95% CIs were calculated with both fixed-effects and random-effects models. Thirty-five of 50 studies met inclusion criteria. No significant heterogeneity was discerned across studies. Although >70% of patients had at least 1 clinical risk factor for VTE, a statistically significant association with VTE onset was demonstrated for each IT trait evaluated (and for combined IT traits), with summary odds ratios ranging from 2.63 (95% CI, 1.61 to 4.29) for the factor II variant to 9.44 (95% CI, 3.34 to 26.66) for antithrombin deficiency. Furthermore, a significant association with recurrent VTE was found for all IT traits except the factor V variant and elevated lipoprotein(a). CONCLUSIONS: The present meta-analysis indicates that detection of IT is clinically meaningful in children with, or at risk for, VTE and underscores the importance of pediatric thrombophilia screening programs.

Effects of primary and secondary prophylaxis on the clinical expression of joint damage in children with severe haemophilia A. Results of a multicenter non-concurrent cohort study.

Patients with severe haemophilia A (HA) can either be treated by regular FVIII infusions twice or three times per week (prophylaxis), or only in case of bleeding episodes (on-demand). Whereas prophylaxis reduces the number of bleeding episodes and may therefore prevent the development of haemophilic arthropathy, there is still a lot of controversy surrounding recommendations on age and dose at start of prophylactic regimens. The present database study was performed to investigate the role of primary versus secondary prophylaxis in HA children. The outcome variable was imaging-proven haemophilic joint damage. Forty-two children were initially treated with primary prophylaxis following the first bleeding episode, and were frequency-matched (year of birth, catchment area) to 67 patients receiving "on-demand" therapy with an early switch to "secondary prophylaxis". In multivariate analysis adjusted for the HA mutation type and the presence or absence of thrombophilia, the Pettersson score investigated at a median age of 12.5 years in joints with at least one documented bleeding episode was not significantly different between the two patient groups (p = 0.944), and no statistically significant differences were found in patients with target joints (p = 0.3), nor in children in whom synovitis had occurred (p = 0.77). No conclusion can be drawn from the data presented herein whether primary prophylaxis or an early start of secondary prophylaxis is superior with respect to joint outcome in children with severe HA.

Effects of the factor V G1691A mutation and the factor II G20210A variant on the clinical expression of severe hemophilia A in children--results of a multicenter studys.

The present multicenter cohort study of 107 pediatric PUPs was performed to determine whether the concomitant inheritance of the factor (F) V G1691A or the F II G20210A mutation influences the clinical expression of severe hemophilia A (HA). Carriers of the FV and FII mutations had a significantly lower annual bleeding frequency (ABF) than non-carriers (p=0.012). Joint damage (Pettersson score) was significantly less severe in patients with thrombophilia (p=0.022). A protective effect of thrombophilic risk factors was shown for ABF (OR [CIs]: 0.7[0.5-0.9]; p=0.02) and the severity of the hemophilic arthropathy (OR [CIs]: 0.06[0.01-0.3]; p=0.0009).