Most genome-wide methylation studies (EWAS) of multifactorial disease traits use targeted arrays or enrichment methodologies preferentially covering CpG-dense regions, to characterize sufficiently ...large samples. To overcome this limitation, we present here a new customizable, cost-effective approach, methylC-capture sequencing (MCC-Seq), for sequencing functional methylomes, while simultaneously providing genetic variation information. To illustrate MCC-Seq, we use whole-genome bisulfite sequencing on adipose tissue (AT) samples and public databases to design AT-specific panels. We establish its efficiency for high-density interrogation of methylome variability by systematic comparisons with other approaches and demonstrate its applicability by identifying novel methylation variation within enhancers strongly correlated to plasma triglyceride and HDL-cholesterol, including at CD36. Our more comprehensive AT panel assesses tissue methylation and genotypes in parallel at ∼4 and ∼3 M sites, respectively. Our study demonstrates that MCC-Seq provides comparable accuracy to alternative approaches but enables more efficient cataloguing of functional and disease-relevant epigenetic and genetic variants for large-scale EWAS.
Pancreatic ductal adenocarcinoma (PDAC) is generally divided in two subtypes, classical and basal. Recently, single-cell RNA sequencing has uncovered the coexistence of basal and classical cancer ...cells, as well as intermediary cancer cells, in individual tumors. The latter remains poorly understood; here, we sought to characterize them using a multimodal approach.
We performed subtyping on a single-cell RNA sequencing dataset containing 18 human PDAC samples to identify multiple intermediary subtypes. We generated patient-derived PDAC organoids for functional studies. We compared single-cell profiling of matched blood and tumor samples to measure changes in the local and systemic immune microenvironment. We then leveraged longitudinally patient-matched blood to follow individual patients over the course of chemotherapy.
We identified a cluster of KRT17-high intermediary cancer cells that uniquely express high levels of CXCL8 and other cytokines. The proportion of KRT17high/CXCL8+ cells in patient tumors correlated with intratumoral myeloid abundance, and, interestingly, high protumor peripheral blood granulocytes, implicating local and systemic roles. Patient-derived organoids maintained KRT17high/CXCL8+ cells and induced myeloid cell migration in a CXCL8-dependent manner. In our longitudinal studies, plasma CXCL8 decreased following chemotherapy in responsive patients, while CXCL8 persistence portended worse prognosis.
Through single-cell analysis of PDAC samples, we identified KRT17high/CXCL8+ cancer cells as an intermediary subtype, marked by a unique cytokine profile and capable of influencing myeloid cells in the tumor microenvironment and systemically. The abundance of this cell population should be considered for patient stratification in precision immunotherapy. See related commentary by Faraoni and McAllister, p. 2297.
Background
Most individuals with mild to moderate reductions in plasma von Willebrand factor (VWF) levels do not demonstrate increased bleeding. However, some patients with plasma VWF levels of ...30–50 IU/dl do have a significant bleeding phenotype. Management of these “low VWF” patients, who may have significant bleeding scores >10, around times of elective procedures continues to pose a common clinical challenge because of a lack of evidence.
Objective
To investigate the safety and efficacy of different periprocedural management options for adult patients with low VWF.
Methods
Treatment and outcomes were retrospectively reviewed for 160 invasive procedures performed in 60 patients with well characterized low VWF enrolled in the previously described Low Von Willebrand factor Ireland Cohort study.
Results
We demonstrate that 1‐desamino‐8‐D‐arginine vasopressin is efficacious in preventing bleeding for both minor or major elective procedures in adult low VWF patients, even in those with significant bleeding histories. In addition, tranexamic acid alone is effective for low VWF patients undergoing nondental minor procedures. Importantly, age‐related increases in plasma VWF:antigen levels above 50 IU/dl were not necessarily associated with complete correction of bleeding phenotype. Procedure‐related bleeding complications were increased in low VWF patients who did not receive any hemostatic cover before their procedure.
Conclusion
Elective procedures in adult patients with low VWF should be managed in liaison with a comprehensive care tertiary referral center so that personalized treatment plans may be implemented before all minor or major elective procedures.
Platelet metabolism is linked to platelet hyper- and hypoactivity in numerous human diseases. Developing a detailed understanding of the link between metabolic shifts and platelet activation state is ...integral to improving human health. Here, we show the first application of isotopically nonstationary 13C metabolic flux analysis to quantitatively measure carbon fluxes in both resting and thrombin activated platelets. Metabolic flux analysis results show that resting platelets primarily metabolize glucose to lactate via glycolysis, while acetate is oxidized to fuel the tricarboxylic acid cycle. Upon activation with thrombin, a potent platelet agonist, platelets increase their uptake of glucose 3-fold. This results in an absolute increase in flux throughout central metabolism, but when compared to resting platelets they redistribute carbon dramatically. Activated platelets decrease relative flux to the oxidative pentose phosphate pathway and TCA cycle from glucose and increase relative flux to lactate. These results provide the first report of reaction-level carbon fluxes in platelets and allow us to distinguish metabolic fluxes with much higher resolution than previous studies.
•Instationary isotope assisted metabolic flux analysis was applied for the first time to human platelets.•Central carbon metabolic fluxes were determined for both resting and activated platelets.•Resting platelets primarily convert glucose to lactate via glycolysis and acetate is oxidized in the TCA cycle.•Activated platelets increase glucose consumption 3-fold and redistribute carbon dramatically.
Critical clinical questions remain unanswered regarding diagnosis and management of patients with low von Willebrand factor (VWF) levels (30-50 IU/dL). To address these questions, the Low VWF Ireland ...Cohort (LoVIC) study investigated 126 patients registered with low VWF levels. Despite marginally reduced plasma VWF levels, International Society of Thrombosis and Haemostasis Bleeding Assessment Tool (ISTH BAT) confirmed significant bleeding phenotypes in the majority of LoVIC patients. Importantly, bleeding tendency did not correlate with plasma VWF levels within the 30 to 50 IU/dL range. Furthermore, bleeding phenotypes could not be explained by concurrent hemostatic defects. Plasma factor VIII to VWF antigen (VWF:Ag) ratios were significantly increased in LoVIC patients compared with controls (P < .0001). In contrast, VWF propeptide to VWF:Ag ratios >3 were observed in only 6% of the LoVIC cohort. Furthermore, platelet-VWF collagen binding activity levels were both significantly reduced compared with controls (P < .05). In response to 1-desamino-8-D-arginine vasopressin (DDAVP), peak VWF:Ag levels exceeded 100 IU/dL in 88% of patients and was sustained >100 IU/dL after 4 hours in 72% of subjects. In conclusion, our novel data suggest that low VWF levels can be associated with significant bleeding and are predominantly due to reductions in VWF synthesis and/or constitutive secretion. Although enhanced VWF clearance may contribute to the pathophysiology in some individuals, the absolute reduction in VWF plasma half-life is usually mild and not sufficient to significantly impact upon the duration of DDAVP-induced VWF response. This trial was registered at www.clinicaltrials.gov as #NCT03167320.
•Patients registered with low VWF have significant bleeding phenotypes that cannot be explained by concomitant bleeding disorders.•Low VWF levels in the range of 30 to 50 IU/dL are predominantly due to reduced VWF synthesis/secretion rather than enhanced clearance.
Loss-of-function mutations in the
(Kv1.1) gene cause episodic ataxia type 1 (EA1), a neurological disease characterized by cerebellar dysfunction, ataxic attacks, persistent myokymia with painful ...cramps in skeletal muscles, and epilepsy. Precision medicine for EA1 treatment is currently unfeasible, as no drug that can enhance the activity of Kv1.1-containing channels and offset the functional defects caused by
mutations has been clinically approved. Here, we uncovered that niflumic acid (NFA), a currently prescribed analgesic and anti-inflammatory drug with an excellent safety profile in the clinic, potentiates the activity of Kv1.1 channels. NFA increased Kv1.1 current amplitudes by enhancing the channel open probability, causing a hyperpolarizing shift in the voltage dependence of both channel opening and gating charge movement, slowing the OFF-gating current decay. NFA exerted similar actions on both homomeric Kv1.2 and heteromeric Kv1.1/Kv1.2 channels, which are formed in most brain structures. We show that through its potentiating action, NFA mitigated the EA1 mutation-induced functional defects in Kv1.1 and restored cerebellar synaptic transmission, Purkinje cell availability, and precision of firing. In addition, NFA ameliorated the motor performance of a
mouse model of EA1 and restored the neuromuscular transmission and climbing ability in
(Kv1.1) mutant
flies (
). By virtue of its multiple actions, NFA has strong potential as an efficacious single-molecule-based therapeutic agent for EA1 and serves as a valuable model for drug discovery.
We studied neurodevelopmental outcomes and behaviours in healthy 2-year old children (N = 1306) from Brazil, India, Italy, Kenya and the UK participating in the INTERGROWTH-21
Project. There was a ...positive independent relationship of duration of exclusive breastfeeding (EBF) and age at weaning with gross motor development, vision and autonomic physical activities, most evident if children were exclusively breastfed for ≥7 months or weaned at ≥7 months. There was no association with cognition, language or behaviour. Children exclusively breastfed from birth to <5 months or weaned at >6 months had, in a dose-effect pattern, adjusting for confounding factors, higher scores for "emotional reactivity". The positive effect of EBF and age at weaning on gross motor, running and climbing scores was strongest among children with the highest scores in maternal closeness proxy indicators. EBF, late weaning and maternal closeness, associated with advanced motor and vision maturation, independently influence autonomous behaviours in healthy children.
To describe the construction of the international INTERGROWTH-21st Neurodevelopment Assessment (INTER-NDA) standards for child development at 2 years by reporting the cognitive, language, motor and ...behaviour outcomes in optimally healthy and nourished children in the INTERGROWTH-21st Project.
Population-based cohort study, the INTERGROWTH-21st Project.
Brazil, India, Italy, Kenya and the UK.
1181 children prospectively recruited from early fetal life according to the prescriptive WHO approach, and confirmed to be at low risk of adverse perinatal and postnatal outcomes.
Scaled INTER-NDA domain scores for cognition, language, fine and gross motor skills and behaviour; vision outcomes measured on the Cardiff tests; attentional problems and emotional reactivity measured on the respective subscales of the preschool Child Behaviour Checklist; and the age of acquisition of the WHO gross motor milestones.
Scaled INTER-NDA domain scores are presented as centiles, which were constructed according to the prescriptive WHO approach and excluded children born preterm and those with significant postnatal/neurological morbidity. For all domains, except negative behaviour, higher scores reflect better outcomes and the threshold for normality was defined as ≥10th centile. For the INTER-NDA's cognitive, fine motor, gross motor, language and positive behaviour domains these are ≥38.5, ≥25.7, ≥51.7, ≥17.8 and ≥51.4, respectively. The threshold for normality for the INTER-NDA's negative behaviour domain is ≤50.0, that is, ≤90th centile. At 22-30 months of age, the cohort overlapped with the WHO motor milestone centiles, showed low postnatal morbidity (<10%), and vision outcomes, attentional problems and emotional reactivity scores within the respective normative ranges.
From this large, healthy and well-nourished, international cohort, we have constructed, using the WHO prescriptive methodology, international INTER-NDA standards for child development at 2 years of age. Standards, rather than references, are recommended for population-level screening and the identification of children at risk of adverse outcomes.
Introduction For many years, there has been debate about how to optimally diagnose type 1 VWD. Initial guidelines proposed that only individuals with plasma VWF levels < 30 IU/dL should be diagnosed ...with ‘type 1 VWD’. This was revised in recent ASH/ISTH/WFH/NHF panel which recommended that patients with a significant bleeding history and plasma VWF of 30-50 IU/dL should also be diagnosed with type 1 VWD, as opposed to the discrete ‘Low VWF’ entity proposed in previous guidelines. In this study, we investigated whether Low VWF is a discrete clinical entity, or whether it is instead part of an age-dependent type 1 VWD evolving phenotype. Methods We utilized datasets from two renowned national cohort studies - the Low VWF in Ireland Cohort (LoVIC) and Willebrand in the Netherlands (WiN) studies. In the LoVIC study, patients had a personal bleeding history and historically lowest VWF levels in the 30-50 IU/dL range. In the WiN study, patients had either a personal bleeding history or positive family history, combined with historically lowest VWF levels ≤ 30 IU/dL. Based upon levels at study inclusion versus original diagnosis, WiN patients were categorized into three groups - (i) patients with persistent VWF levels <30 IU/dL; (ii) patients with partial correction in VWF levels into the range of 30-50 IU/dL; and (iii) patients with normalization of VWF levels > 50 IU/dL. The FVIII:C/VWF:Ag ratio was used to assess the synthesis/secretion of VWF and the VWFpp/VWF:Ag ratio was used to assess the clearance of VWF. Results In total, 565 patients were included (403 WiN patients with type 1 VWD and 162 LoVIC patients with Low VWF). Mean age at diagnosis was significantly increased in the LoVIC cohort compared to the three WiN subgroups (32.5 years versus 23.3 years, 26.5 years and 25 years respectively; p<0.001). Conversely, there was no difference in age at time of enrollment into both national studies (p=0.532). To assess the significance of age, we first investigated how plasma VWF:Ag varied over time in WiN type 1 VWD patients compared to the LoVIC cohort. Among the total WiN cohort (with initial VWF levels < 30 IU/dL), 47% of subjects (n=188) had VWF levels that remained < 30 IU/dL despite advancing age. Conversely, 30% of type 1 VWD patients (n=121) increased their plasma VWF levels into the Low VWF range (30-50 IU/dL) over time, whereas 23% (n=94) had age-dependent increments that led to complete normalization in VWF levels > 50 IU/dL. Similarly, 39% (n=63) of patients from the LoVIC study had normalization of VWF levels over time. Crucially, we observed that VWF:Ag in Low VWF patients clearly overlapped with those in normalized (> 50 IU/dL) type 1 VWD subjects, indicating that the LoVIC cohort is a subgroup within the WiN normalized subgroup (Figure 1). To further investigate this concept, we performed multiple regression analysis. Importantly, we observed that plasma VWF:Ag in the LoVIC cohort and the WiN normalized (> 50 IU/dL) subgroup would have not been different had they been diagnosed at the same age (difference of β=0.00 (95% CI -0.03 to 0.04)). Cumulatively, these results indicate that significant heterogeneity exists amongst type 1 VWD patients with respect to the effect of ageing on their plasma VWF levels. In addition, our findings demonstrate that because of age-dependent increases in plasma VWF:Ag, the majority of Low VWF patients would have been diagnosed with type 1 VWD had they undergone assessment earlier in life. Consistently, no difference in prevalence of VWF mutation (36.4% vs 22.6% respectively, p=1.000), FVIII:C/VWF:Ag ratio (1.24 ±0.24 vs 1.46 ±0.27 respectively, p=0.444) or VWFpp/VWF:Ag ratio (1.36 ±0.99 vs 1.75 ±0.38 respectively, p=1.000) was found between LoVIC and normalized WiN patients (Figure 2). Contrarily, WiN patients with persistent VWF levels <30 IU/dL had more often VWF gene variants (93.0%), higher FVIII:C/VWF:Ag ratio (2.65 ±1.62) and higher VWFpp/VWF:Ag ratio (6.14 ±7.05) compared to the other groups (Figure 2, p<0.001). Conclusion Our findings clearly demonstrate that Low VWF does not constitute a discrete clinical or pathological entity. Rather, it is part of an age-dependent type 1 VWD evolving phenotype, as demonstrated by the same VWF levels throughout life, genetic background and pathophysiology. This study has direct consequences for VWD diagnosis, the overall understanding of type 1 VWD as an evolving disease and for patients with low VWF/type 1 VWD.
PDF
