Guido Pieles

VACTERL/caudal regression/Currarino syndrome-like malformations in mice with mutation in the proprotein convertase Pcsk5

We have identified an ethylnitrosourea (ENU)-induced recessive mouse mutation (Vcc) with a pleiotropic phenotype that includes cardiac, tracheoesophageal, anorectal, anteroposterior patterning defects, exomphalos, hindlimb hypoplasia, a presacral mass, renal and palatal agenesis, and pulmonary hypoplasia. It results from a C470R mutation in the proprotein convertase PCSK5 (PC5/6). Compound mutants (Pcsk5(Vcc/null)) completely recapitulate the Pcsk5(Vcc/Vcc) phenotype, as does an epiblast-specific conditional deletion of Pcsk5. The C470R mutation ablates a disulfide bond in the P domain, and blocks export from the endoplasmic reticulum and proprotein convertase activity. We show that GDF11 is cleaved and activated by PCSK5A, but not by PCSK5A-C470R, and that Gdf11-deficient embryos, in addition to having anteroposterior patterning defects and renal and palatal agenesis, also have a presacral mass, anorectal malformation, and exomphalos. Pcsk5 mutation results in abnormal expression of several paralogous Hox genes (Hoxa, Hoxc, and Hoxd), and of Mnx1 (Hlxb9). These include known Gdf11 targets, and are necessary for caudal embryo development. We identified nonsynonymous mutations in PCSK5 in patients with VACTERL (vertebral, anorectal, cardiac, tracheoesophageal, renal, limb malformation OMIM 192350) and caudal regression syndrome, the phenotypic features of which resemble the mouse mutation. We propose that Pcsk5, at least in part via GDF11, coordinately regulates caudal Hox paralogs, to control anteroposterior patterning, nephrogenesis, skeletal, and anorectal development.

Role of the Transcription Factor Sox4 in Insulin Secretion and Impaired Glucose Tolerance

OBJECTIVES— To identify, map, clone, and functionally validate a novel mouse model for impaired glucose tolerance and insulin secretion. RESEARCH DESIGN AND METHODS— Haploinsufficiency of the insulin receptor and associated mild insulin resistance has been used to sensitize an N-ethyl-N-nitrosourea (ENU) screen to identify novel mutations resulting in impaired glucose tolerance and diabetes. The new impaired glucose tolerance 4 (IGT4) model was selected using an intraperitoneal glucose tolerance test and inheritance of the phenotype confirmed by generation of backcross progeny. Segregation of the phenotype was correlated with genotype information to map the location of the gene and candidates sequenced for mutations. The function of the SRY-related high mobility group (HMG)-box 4 (Sox4) gene in insulin secretion was tested using another ENU allele and by small interfering RNA silencing in insulinoma cells. RESULTS— We describe two allelic autosomal dominant mutations in the highly conserved HMG box of the transcription factor Sox4. Previously associated with pancreas development, Sox4 mutations in the adult mouse result in an insulin secretory defect, which exhibits impaired glucose tolerance in association with insulin receptor+/−–induced insulin resistance. Elimination of the Sox4 transcript in INS1 and Min6 cells resulted in the abolition of glucose-stimulated insulin release similar to that observed for silencing of the key metabolic enzyme glucokinase. Intracellular calcium measurements in treated cells indicate that this defect lies downstream of the ATP-sensitive K+ channel (KATP channel) and calcium influx. CONCLUSIONS— IGT4 represents a novel digenic model of insulin resistance coupled with an insulin secretory defect. The Sox4 gene has a role in insulin secretion in the adult β-cell downstream of the KATP channel.

microMRI-HREM pipeline for high-throughput, high-resolution phenotyping of murine embryos.

Rapid and precise phenotyping analysis of large numbers of wild‐type and mutant mouse embryos is essential for characterizing the genetic and epigenetic factors regulating embryogenesis. We present a novel methodology that permits precise high‐throughput screening of the phenotype of embryos with both targeted and randomly generated mutations. To demonstrate the potential of this methodology we show embryo phenotyping results produced in a large‐scale ENU‐mutagenesis study. In essence this represents an analysis pipeline, which starts with simultaneous micro‐magentic resonance imaging (µMRI) screening (voxel size: 25.4 × 25.4 × 24.4 µm) of 32 embryos in one run. Embryos with an indistinct phenotype are then cut into parts and suspect organs and structures are analysed with HREM (high‐resolution episcopic microscopy). HREM is an imaging technique that employs ‘positive’ eosin staining and episcopic imaging for generating three‐dimensional (3D) high‐resolution (voxel size: 1.07 × 1.07 × 2 µm) digital data of near histological contrast and quality. The results show that our method guarantees the rapid availability of comprehensive phenotype information for high numbers of embryos in, if necessary, histological quality and detail. The combination of high‐throughput µMRI with HREM provides an alternative screening pipeline with advantages over existing 3D phenotype screening methods as well as traditional histology. Thus, the µMRI‐HREM phenotype analysis pipeline recommends itself as a routine tool for analysing the phenotype of transgenic and mutant embryos.

Paediatric exercise training in prevention and treatment

Exercise training is an underused intervention in paediatric healthcare. This is surprising, since initial evidence demonstrates its effectiveness and safety; furthermore it confers socioeconomic benefits for healthcare systems. Pilot studies have assessed and confirmed the feasibility of exercise training in many paediatric disease settings. However, more research is needed to understand the pathophysiology, quantify treatment effects and monitor outcomes. A concerted effort from researchers, health professionals and police makers will be necessary to make exercise training an evidence-based and cost-effective intervention in paediatric care.

Electrical and structural adaptations of the paediatric athlete's heart: a systematic review with meta-analysis.

Aim To describe the electrocardiographic (ECG) and echocardiographic manifestations of the paediatric athlete’s heart, and examine the impact of age, race and sex on cardiac remodelling responses to competitive sport. Design Systematic review with meta-analysis. Data sources Six electronic databases were searched to May 2016: MEDLINE, PubMed, EMBASE, Web of Science, CINAHL and SPORTDiscus. Inclusion criteria (1) Male and/or female competitive athletes, (2) participants aged 6–18 years, (3) original research article published in English language. Results Data from 14 278 athletes and 1668 non-athletes were included for qualitative (43 articles) and quantitative synthesis (40 articles). Paediatric athletes demonstrated a greater prevalence of training-related and training-unrelated ECG changes than non-athletes. Athletes ≥14 years were 15.8 times more likely to have inferolateral T-wave inversion than athletes <14 years. Paediatric black athletes had significantly more training-related and training-unrelated ECG changes than Caucasian athletes. Age was a positive predictor of left ventricular (LV) internal diameter during diastole, interventricular septum thickness during diastole, relative wall thickness and LV mass. When age was accounted for, these parameters remained significantly larger in athletes than non-athletes. Paediatric black athletes presented larger posterior wall thickness during diastole (PWTd) than Caucasian athletes. Paediatric male athletes also presented larger PWTd than females. Conclusions The paediatric athlete’s heart undergoes significant remodelling both before and during ‘maturational years’. Paediatric athletes have a greater prevalence of training related and training-unrelated ECG changes than non-athletes, with age, race and sex mediating factors on cardiac electrical and LV structural remodelling.

Outcomes of Cardiac Screening in Adolescent Soccer Players

Background Reports on the incidence and causes of sudden cardiac death among young athletes have relied largely on estimated rates of participation and varied methods of reporting. We sought to investigate the incidence and causes of sudden cardiac death among adolescent soccer players in the United Kingdom. Methods From 1996 through 2016, we screened 11,168 adolescent athletes with a mean (±SD) age of 16.4±1.2 years (95% of whom were male) in the English Football Association (FA) cardiac screening program, which consisted of a health questionnaire, physical examination, electrocardiography, and echocardiography. The FA registry was interrogated to identify sudden cardiac deaths, which were confirmed with autopsy reports. Results During screening, 42 athletes (0.38%) were found to have cardiac disorders that are associated with sudden cardiac death. A further 225 athletes (2%) with congenital or valvular abnormalities were identified. After screening, there were 23 deaths from any cause, of which 8 (35%) were sudden deaths attributed to cardiac disease. Cardiomyopathy accounted for 7 of 8 sudden cardiac deaths (88%). Six athletes (75%) with sudden cardiac death had had normal cardiac screening results. The mean time between screening and sudden cardiac death was 6.8 years. On the basis of a total of 118,351 person‐years, the incidence of sudden cardiac death among previously screened adolescent soccer players was 1 per 14,794 person‐years (6.8 per 100,000 athletes). Conclusions Diseases that are associated with sudden cardiac death were identified in 0.38% of adolescent soccer players in a cohort that underwent cardiovascular screening. The incidence of sudden cardiac death was 1 per 14,794 person‐years, or 6.8 per 100,000 athletes; most of these deaths were due to cardiomyopathies that had not been detected on screening. (Funded by the English Football Association and others.)

The relationship between biventricular myocardial performance and metabolic parameters during incremental exercise and recovery in healthy adolescents

Background left ventricular (LV) and right ventricular (RV) myocardial reserve during exercise in adolescents has not been directly characterized. The aim of this study was to quantify myocardial performance response to exercise by using two-dimensional (2-D) speckle tracking echocardiography and describe the relationship between myocardial reserve, respiratory, and metabolic exercise parameters. A total of 23 healthy boys and girls (mean age 13.2 ± 2.7 yr; stature 159.1 ± 16.4 cm; body mass 49.5 ± 16.6 kg; BSA 1.47 ± 0.33 m(2)) completed an incremental cardiopulmonary exercise test (25 W · 3 min increments) with simultaneous acquisition of 2-D transthoracic echocardiography at rest, each exercise stage up to 100 W, and in recovery at 2 min and 10 min. Two-dimensional LV (LV Sl) and RV (RV Sl) longitudinal strain and LV circumferential strain (LV Sc) were analyzed to define the relationship between myocardial performance reserve and metabolic exercise parameters. Participants achieved a peak oxygen uptake (V̇o 2peak) of 40.6 ± 8.9 ml · kg(-1) · min(-1) and a work rate of 154 ± 42 W. LV Sl and LV Sc and RV Sl increased significantly across work rates (P < 0.05). LV Sl during exercise was significantly correlated to resting strain, V̇o 2peak, oxygen pulse, and work rate (0.530 ≤ r ≤ 0.784, P < 0.05). This study identifies a positive and moderate relationship between LV and RV myocardial performance and metabolic parameters during exercise by using a novel methodology. Relationships detected present novel data directly describing myocardial adaptation at different stages of exercise and recovery that in the future can help directly assess cardiac reserve in patients with cardiac pathology.

Tricuspid Atresia With Truncus Arteriosus: Successful Surgical Treatment

Tricuspid atresia and common truncus arteriosus are rare forms of congenital heart disease; the coexistence of both anomalies is therefore an extremely uncommon event. Without treatment, early mortality is the natural course so diagnostic and therapeutic management must be performed without delay. We report a case of a newborn with a postnatal diagnosis of coexistent tricuspid atresia and common arterial trunk in whom successful palliation was performed using a staged surgical approach.

Diagnostic accuracy and Bayesian analysis of new international ECG recommendations in paediatric athletes

Objective Historically, electrocardiographic (ECG) interpretation criteria for athletes were only applicable to adults. New international recommendations now account for athletes ≤16 years, but their clinical appropriateness is unknown. We sought to establish the diagnostic accuracy of new international ECG recommendations against the Seattle criteria and 2010 European Society of Cardiology (ESC) recommendations in paediatric athletes using receiver operator curve analysis. Clinical context was calculated using Bayesian analysis. Methods 876 Arab and 428 black male paediatric athletes (11–18 years) were evaluated by medical questionnaire, physical examination, ECG and echocardiographic assessment. ECGs were retrospectively analysed according to the three criteria. Results Thirteen (1.0%) athletes were diagnosed with cardiac pathology that may predispose to sudden cardiac arrest/death (SCA/D) (8 (0.9%) Arab and (5 (1.2%) black)). Diagnostic accuracy was poor (0.68, 95% CI 0.54 to 0.82) for 2010 ESC recommendations, fair (0.70, 95% CI 0.54 to 0.85) for Seattle criteria and fair (0.77, 95% CI 0.61 to 0.93) for international recommendations. False-positive rates were 41.0% for 2010 ESC recommendations, 21.8% for Seattle criteria and 6.8% for international recommendations. International recommendations provided a positive (+LR) and negative (−LR) post-test likelihood ratio of 9.0 (95% CI 5.1 to 13.1) and 0.4 (95% CI 0.2 to 0.7), respectively. Conclusion In Arab and black male paediatric athletes, new international recommendations outperform both the Seattle criteria and 2010 ESC recommendations, reducing false positive rates, while yielding a ‘fair’ diagnostic accuracy for cardiac pathology that may predispose to SCA/D. In clinical context, the ‘chance’ of detecting cardiac pathology within a paediatric male athlete with a positive ECG (+LR=9.0) was 8.3%, whereas a negative ECG (−LR=0.4) was 0.4%.

Use of phase-contrast magnetic resonance angiography to measure adaptations of aortic and pulmonary artery flow during supine aerobic exercise

Abstract Background Cardiovascular adaptations to aerobic exercise are mediated through interactions of cardiac, hormonal, metabolic, and muscular mechanisms with a dominant role for increase in cardiac output to enhance oxygen delivery. However, increased demands for oxygen uptake and use during exercise depend on adequate adaptations of systemic and pulmonary vasculature. Assessment of exercise response is crucial in various cardiac and pulmonary diseases. Recent advances in MRI techniques allow for direct measurement of aortic and pulmonary blood flow with phase-contrast magnetic resonance angiography (PCMRA). We have studied aortic and pulmonary flow in healthy individuals to assess haemodynamic adaptations to exercise using PCMRA. Methods Nine adult healthy volunteers underwent PCMRA while doing heart-rate-targeted (180% of resting heart rate) aerobic supine leg exercise. Aortic flow was reassessed post exercise after 2 min rest. All flow variables were assessed by retrospective free-breathing pulse-gated PCMRA in the mid ascending aorta and main pulmonary artery (field of view [FOV] 320 mm, FOV phase 75%, base resolution 256, voxel 1·3 × 1·3 × 5·0 mm, TE 2·2 ms, TR 29·9 ms, slice thickness 5 mm, three averages, and 30 reconstructed phases, Venc 150 cm/s at rest, 300 cm/s during exercise). All images were assessed by three individuals trained in MRI flow evaluation and independently checked by an experienced cardiac radiologist. Flow sequence analysis was done with offline software (Argus, Siemens Medical Systems), by manually contouring the vessel of interest so that all flow was accounted for. Pulmonary and aortic blood flow was determined from the flow velocities in individual voxels in the region of interest. Findings Increase in heart rate during exercise (from a mean of 69 beats per min [SD 10] at rest to 120 [14] after exercise) resulted in increased cardiac output (mean 6·5 L/min [SD 1·4] to 12·4 [1·8]). All flow variables significantly increased with exercise compared with rest: aorta systolic peak velocity increased from mean 89 cm/s [SD 14] to 122 [34] (p=0·016); pulmonary artery systolic peak velocity 86 cm/s [18] to 140 [48] (p=0·007); aorta systolic peak flow rate 415 mL/s [83] to 550 [135] (p=0·002); and pulmonary artery systolic peak flow rate 410 mL/s [80] to 577 [180] (p=0·006). These variables showed a trend to normalisation at 2 min recovery. Interpretation Use of free-breathing pulse-gated PCMRA to measure aortic and pulmonary blood flow and velocity during exercise is feasible. Moderate aerobic exercise leads to a steep increase in blood flow and flow velocities in aorta and main pulmonary artery facilitating increased oxygen uptake. Exercise PCMRA could help in diagnosis, assessment of treatment response, and follow-up in many patient groups with pulmonary and cardiovascular disease. Funding Bristol NIHR Cardiovascular Biomedical Research Unit, NIHR Academic Clinical Lectureship to GEP