Diagnostic yield of genetic testing in a multinational heterogeneous cohort of 2088 DCM patients.

Background: Familial dilated cardiomyopathy (DCM) causes heart failure and may lead to heart transplantation. DCM is typically a monogenic disorder with autosomal dominant inheritance. Currently disease-causing variants have been reported in over 60 genes that encode proteins in sarcomeres, nuclear lamina, desmosomes, cytoskeleton, and mitochondria. Over half of the patients undergoing comprehensive genetic testing are left without a molecular diagnosis even when patient selection follows strict DCM criteria. Methods and results: This study was a retrospective review of patients referred for genetic testing at Blueprint Genetics due to suspected inherited DCM. Next generation sequencing panels included 23-316 genes associated with cardiomyopathies and other monogenic cardiac diseases. Variants were considered diagnostic if classified as pathogenic (P) or likely pathogenic (LP). Of the 2,088 patients 514 (24.6%) obtained a molecular diagnosis; 534 LP/P variants were observed across 45 genes, 2.7% (14/514) had two diagnostic variants in dominant genes. Nine copy number variants were identified: two multigene and seven intragenic. Diagnostic variants were observed most often in TTN (45.3%), DSP (6.7%), LMNA (6.7%), and MYH7 (5.2%). Clinical characteristics independently associated with molecular diagnosis were: a lower age at diagnosis, family history of DCM, paroxysmal atrial fibrillation, absence of left bundle branch block, and the presence of an implantable cardioverter-defibrillator. Conclusions: Panel testing provides good diagnostic yield in patients with clinically suspected DCM. Causative variants were identified in 45 genes. In minority, two diagnostic variants were observed in dominant genes. Our results support the use of genetic panels in clinical settings in DCM patients with suspected genetic etiology.

Genetic resiliency associated with dominant lethal TPM1 mutation causing atrial septal defect with high heritability.

Analysis of large-scale human genomic data has yielded unexplained mutations known to cause severe disease in healthy individuals. Here, we report the unexpected recovery of a rare dominant lethal mutation in TPM1, a sarcomeric actin-binding protein, in eight individuals with large atrial septal defect (ASD) in a five-generation pedigree. Mice with Tpm1 mutation exhibit early embryonic lethality with disrupted myofibril assembly and no heartbeat. However, patient-induced pluripotent-stem-cell-derived cardiomyocytes show normal beating with mild myofilament defect, indicating disease suppression. A variant in TLN2, another myofilament actin-binding protein, is identified as a candidate suppressor. Mouse CRISPR knock-in (KI) of both the TLN2 and TPM1 variants rescues heart beating, with near-term fetuses exhibiting large ASD. Thus, the role of TPM1 in ASD pathogenesis unfolds with suppression of its embryonic lethality by protective TLN2 variant. These findings provide evidence that genetic resiliency can arise with genetic suppression of a deleterious mutation.

Prevalence of RPGR-Mediated Retinal Dystrophy in an Unselected Cohort of Over 5000 Patients.

Purpose: Comprehensive genetic testing for inherited retinal dystrophy (IRD) is challenged by difficult-to-sequence genomic regions, which are often mutational hotspots, such as RPGR ORF15. The purpose of this study was to evaluate the diagnostic contribution of RPGR variants in an unselected IRD patient cohort referred for testing in a clinical diagnostic laboratory. Methods: A total of 5201 consecutive patients were analyzed with a clinically validated next-generation sequencing (NGS)-based assay, including the difficult-to-sequence RPGR ORF15 region. Copy number variant (CNV) detection from NGS data was included. Variant interpretation was performed per the American College of Medical Genetics and Genomics guidelines. Results: A confirmed molecular diagnosis in RPGR was found in 4.5% of patients, 24.0% of whom were females. Variants in ORF15 accounted for 74% of the diagnoses; 29% of the diagnostic variants were in the most difficult-to-sequence central region of ORF15 (c.2470-3230). Truncating variants made up the majority (91%) of the diagnostic variants. CNVs explained 2% of the diagnostic cases, of which 80% were one- or two-exon deletions outside of ORF15. Conclusions: Our findings indicate that high-throughput, clinically validated NGS-based testing covering the difficult-to-sequence region of ORF15, in combination with high-resolution CNV detection, can help to maximize the diagnostic yield for patients with IRD. Translational Relevance: These results demonstrate an accurate and scalable method for the detection of RPGR-related variants, including the difficult-to-sequence ORF15 hotspot, which is relevant given current and emerging therapeutic opportunities.

GRINL1A Complex Transcription Unit Containing GCOM1, MYZAP, and POLR2M Genes Associates with Fully Penetrant Recessive Dilated Cardiomyopathy.

Background: Familial dilated cardiomyopathy (DCM) is a monogenic disorder typically inherited in an autosomal dominant pattern. We have identified two Finnish families with familial cardiomyopathy that is not explained by a variant in any previously known cardiomyopathy gene. We describe the cardiac phenotype related to homozygous truncating GCOM1 variants. Methods and Results: This study included two probands and their relatives. All the participants are of Finnish ethnicity. Whole-exome sequencing was used to test the probands; bi-directional Sanger sequencing was used to identify the GCOM1 variants in probands' family members. Clinical evaluation was performed, medical records and death certificates were obtained. Immunohistochemical analysis of myocardial samples was conducted. A homozygous GCOM1 variant was identified altogether in six individuals, all considered to be affected. None of the nine heterozygous family members fulfilled any cardiomyopathy criteria. Heart failure was the leading clinical feature, and the patients may have had a tendency for atrial arrhythmias. Conclusions: This study demonstrates the significance of GCOM1 variants as a cause of human cardiomyopathy and highlights the importance of searching for new candidate genes when targeted gene panels do not yield a positive outcome.

Diagnostic utility of next-generation sequencing-based panel testing in 543 patients with suspected skeletal dysplasia.

BACKGROUND: Skeletal dysplasia is typically diagnosed using a combination of radiographic imaging, clinical examinations, and molecular testing. Identifying a molecular diagnosis for an individual with a skeletal dysplasia can lead to improved clinical care, guide future medical management and treatment, and inform assessment of risk for familial recurrence. The molecular diagnostic utility of multi-gene panel testing using next-generation sequencing (NGS) has not yet been characterized for an unselected population of individuals with suspected skeletal dysplasia. In this study, we retrospectively reviewed patient reports to assess the diagnostic yield, reported variant characteristics, impact of copy number variation, and performance in prenatal diagnostics of panel tests for variants in genes associated with skeletal dysplasia and growth disorders. RESULTS: Clinical reports of consecutive patients with a clinical indication of suspected skeletal dysplasia who underwent panel testing were examined. The 543 patients included in the study submitted samples for diagnostic genetic testing with an indication of suspected skeletal dysplasia or growth disorder and received one of three nested panel tests. A molecular diagnosis was established in 42.0% of patients (n = 228/543). Diagnostic variants were identified in 71 genes, nearly half of which (n = 35, 49.3%) contributed uniquely to a molecular diagnosis for a single patient in this cohort. Diagnostic yield was significantly higher among fetal samples (58.0%, n = 51/88) than postnatal samples (38.9%, n = 177/455; z = 3.32, p < 0.0009). Diagnostic variants in fetal cases were identified across 18 genes. Thirteen diagnostic CNVs were reported, representing 5.7% of diagnostic findings and ranging in size from 241-bp to whole chromosome aneuploidy. Additionally, 11.4% (36/315) of non-diagnostic patient reports had suspicious variants of unknown significance (VUS), in which additional family studies that provide segregation data and/or functional characterization may result in reclassification to likely pathogenic. CONCLUSIONS: These findings demonstrate the utility of panel testing for individuals with a suspected skeletal dysplasia or growth disorder, with a particularly high diagnostic yield seen in prenatal cases. Pursuing comprehensive panel testing with high-resolution CNV analysis can provide a diagnostic benefit, given the considerable phenotype overlap amongst skeletal dysplasia conditions.

Next-generation sequencing in childhood-onset epilepsies: Diagnostic yield and impact on neuronal ceroid lipofuscinosis type 2 (CLN2) disease diagnosis.

Epilepsy is one of the most common childhood-onset neurological conditions with a genetic etiology. Genetic diagnosis provides potential for etiologically-based management and treatment. Existing research has focused on early-onset (<24 months) epilepsies; data regarding later-onset epilepsies is limited. The goal of this study was to determine the diagnostic yield of a clinically available epilepsy panel in a selected pediatric epilepsy cohort with epilepsy onset between 24-60 months of life and evaluate whether this approach decreases the age of diagnosis of neuronal ceroid lipofuscinosis type 2 (CLN2). Next-generation sequencing (NGS)-based epilepsy panels, including genes associated with epileptic encephalopathies and inborn errors of metabolism (IEMs) that present with epilepsy, were used. Copy-number variant (CNV) detection from NGS data was included. Variant interpretation was performed per American College of Medical Genetics and Genomics (ACMG) guidelines. Results are reported from 211 consecutive patients with the following inclusion criteria: 24-60 months of age at the time of enrollment, first unprovoked seizure at/after 24 months, and at least one additional finding such as EEG/MRI abnormalities, speech delay, or motor symptoms. Median age was 42 months at testing and 30 months at first seizure onset; the mean delay from first seizure to comprehensive genetic testing was 10.3 months. A genetic diagnosis was established in 43 patients (20.4%). CNVs were reported in 25.6% diagnosed patients; 27.3% of CNVs identified were intragenic. Within the diagnosed cohort, 11 (25.6%) patients were diagnosed with an IEM. The predominant molecular diagnosis was CLN2 (14% of diagnosed patients). For these patients, diagnosis was achieved 12-24 months earlier than reported by natural history of the disease. This study supports comprehensive genetic testing for patients whose first seizure occurs ≥ 24 months of age. It also supports early application of testing in this age group, as the identified diagnoses can have significant impact on patient management and outcome.

Diagnostic yield of genetic testing in a heterogeneous cohort of 1376 HCM patients.

BACKGROUND: Genetic testing in hypertrophic cardiomyopathy (HCM) is a published guideline-based recommendation. The diagnostic yield of genetic testing and corresponding HCM-associated genes have been largely documented by single center studies and carefully selected patient cohorts. Our goal was to evaluate the diagnostic yield of genetic testing in a heterogeneous cohort of patients with a clinical suspicion of HCM, referred for genetic testing from multiple centers around the world. METHODS: A retrospective review of patients with a suspected clinical diagnosis of HCM referred for genetic testing at Blueprint Genetics was undertaken. The analysis included syndromic, myopathic and metabolic etiologies. Genetic test results and variant classifications were extracted from the database. Variants classified as pathogenic (P) or likely pathogenic (LP) were considered diagnostic. RESULTS: A total of 1376 samples were analyzed. Three hundred and sixty-nine tests were diagnostic (26.8%); 373 P or LP variants were identified. Only one copy number variant was identified. The majority of diagnostic variants involved genes encoding the sarcomere (85.0%) followed by 4.3% of diagnostic variants identified in the RASopathy genes. Two percent of diagnostic variants were in genes associated with a cardiomyopathy other than HCM or an inherited arrhythmia. Clinical variables that increased the likelihood of identifying a diagnostic variant included: an earlier age at diagnosis (p < 0.0001), a higher maximum wall thickness (MWT) (p < 0.0001), a positive family history (p < 0.0001), the absence of hypertension (p = 0.0002), and the presence of an implantable cardioverter-defibrillator (ICD) (p = 0.0004). CONCLUSION: The diagnostic yield of genetic testing in this heterogeneous cohort of patients with a clinical suspicion of HCM is lower than what has been reported in well-characterized patient cohorts. We report the highest yield of diagnostic variants in the RASopathy genes identified in a laboratory cohort of HCM patients to date. The spectrum of genes implicated in this unselected cohort highlights the importance of pre-and post-test counseling when offering genetic testing to the broad HCM population.

Biallelic loss-of-function in NRAP is a cause of recessive dilated cardiomyopathy.

BACKGROUND: Familial dilated cardiomyopathy (DCM) is typically a monogenic disorder with dominant inheritance. Although over 40 genes have been linked to DCM, more than half of the patients undergoing comprehensive genetic testing are left without molecular diagnosis. Recently, biallelic protein-truncating variants (PTVs) in the nebulin-related anchoring protein gene (NRAP) were identified in a few patients with sporadic DCM. METHODS AND RESULTS: We determined the frequency of rare NRAP variants in a cohort of DCM patients and control patients to further evaluate role of this gene in cardiomyopathies. A retrospective analysis of our internal variant database consisting of 31,639 individuals who underwent genetic testing (either panel or direct exome sequencing) was performed. The DCM group included 577 patients with either a confirmed or suspected DCM diagnosis. A control cohort of 31,062 individuals, including 25,912 individuals with non-cardiac (control group) and 5,150 with non-DCM cardiac indications (Non-DCM cardiac group). Biallelic (n = 6) or two (n = 5) NRAP variants (two PTVs or PTV+missense) were identified in 11 unrelated probands with DCM (1.9%) but none of the controls. None of the 11 probands had an alternative molecular diagnosis. Family member testing supports co-segregation. Biallelic or potentially biallelic NRAP variants were enriched in DCM vs. controls (OR 1052, p<0.0001). Based on the frequency of NRAP PTVs in the gnomAD reference population, and predicting full penetrance, biallelic NRAP variants could explain 0.25%-2.46% of all DCM cases. CONCLUSION: Loss-of-function in NRAP is a cause for autosomal recessive dilated cardiomyopathy, supporting its inclusion in comprehensive genetic testing.

Publisher Correction: Relevance of Titin Missense and Non-Frameshifting Insertions/Deletions Variants in Dilated Cardiomyopathy.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

DSP p.(Thr2104Glnfs*12) variant presents variably with early onset severe arrhythmias and left ventricular cardiomyopathy.

BACKGROUND: Dilated cardiomyopathy (DCM) is a condition characterized by dilatation and systolic dysfunction of the left ventricle in the absence of severe coronary artery disease or abnormal loading conditions. Mutations in the titin (TTN) and lamin A/C (LMNA) genes are the two most significant contributors in familial DCM. Previously mutations in the desmoplakin (DSP) gene have been associated with arrhythmogenic right ventricular cardiomyopathy (ARVC) and more recently with DCM. METHODS: We describe the cardiac phenotype related to a DSP mutation which was identified in ten unrelated Finnish index patients using next-generation sequencing. Sanger sequencing was used to verify the presence of this DSP variant in the probands' relatives. Medical records were obtained, and clinical evaluation was performed. RESULTS: We identified DSP c.6310delA, p.(Thr2104Glnfs*12) variant in 17 individuals of which 11 (65%) fulfilled the DCM diagnostic criteria. This pathogenic variant presented with left ventricular dilatation, dysfunction and major ventricular arrhythmias. Two patients showed late gadolinium enhancement (LGE) and myocardial edema on cardiac magnetic resonance imaging (MRI) that may suggest inflammatory process at myocardium. CONCLUSIONS: The patients diagnosed with DCM showed an arrhythmogenic phenotype as well as SCD at young age supporting the recently proposed concept of arrhythmogenic cardiomyopathy. This study also demonstrates relatively low penetrance of truncating DSP variant in the probands' family members by the age of 40. Further studies are needed to elucidate the possible relations between myocardial inflammation and pathogenic DSP variants.

2019 updated consensus statement on the diagnosis and treatment of pediatric pulmonary hypertension: The European Pediatric Pulmonary Vascular Disease Network (EPPVDN), endorsed by AEPC, ESPR and ISHLT.

The European Pediatric Pulmonary Vascular Disease Network is a registered, non-profit organization that strives to define and develop effective, innovative diagnostic methods and treatment options in all forms of pediatric pulmonary hypertensive vascular disease, including pulmonary hypertension (PH) associated with bronchopulmonary dysplasia, PH associated with congenital heart disease (CHD), persistent PH of the newborn, and related cardiac dysfunction. The executive writing group members conducted searches of the PubMed/MEDLINE bibliographic database (1990-2018) and held face-to-face and web-based meetings. Ten section task forces voted on the updated recommendations, based on the 2016 executive summary. Clinical trials, meta-analyses, guidelines, and other articles that include pediatric data were searched using the term "pulmonary hypertension" and other keywords. Class of recommendation (COR) and level of evidence (LOE) were assigned based on European Society of Cardiology/American Heart Association definitions and on pediatric data only, or on adult studies that included >10% children or studies that enrolled adults with CHD. New definitions by the World Symposium on Pulmonary Hypertension 2018 were included. We generated 10 tables with graded recommendations (COR/LOE). The topics include diagnosis/monitoring, genetics/biomarkers, cardiac catheterization, echocardiography, cardiac magnetic resonance/chest computed tomography, associated forms of PH, intensive care unit/lung transplantation, and treatment of pediatric PH. For the first time, a set of specific recommendations on the management of PH in middle- and low-income regions was developed. Taken together, these executive, up-to-date guidelines provide a specific, comprehensive, detailed but practical framework for the optimal clinical care of children and young adults with PH.

Relevance of Titin Missense and Non-Frameshifting Insertions/Deletions Variants in Dilated Cardiomyopathy.

Recent advancements in next generation sequencing (NGS) technology have led to the identification of the giant sarcomere gene, titin (TTN), as a major human disease gene. Truncating variants of TTN (TTNtv) especially in the A-band region account for 20% of dilated cardiomyopathy (DCM) cases. Much attention has been focused on assessment and interpretation of TTNtv in human disease; however, missense and non-frameshifting insertions/deletions (NFS-INDELs) are difficult to assess and interpret in clinical diagnostic workflow. Targeted sequencing covering all exons of TTN was performed on a cohort of 530 primary DCM patients from three cardiogenetic centres across Europe. Using stringent bioinformatic filtering, twenty-nine and two rare TTN missense and NFS-INDELs variants predicted deleterious were identified in 6.98% and 0.38% of DCM patients, respectively. However, when compared with those identified in the largest available reference population database, no significant enrichment of such variants was identified in DCM patients. Moreover, DCM patients and reference individuals had comparable frequencies of splice-region missense variants with predicted splicing alteration. DCM patients and reference populations had comparable frequencies of rare predicted deleterious TTN missense variants including splice-region missense variants suggesting that these variants are not independently causative for DCM. Hence, these variants should be classified as likely benign in the clinical diagnostic workflow, although a modifier effect cannot be excluded at this stage.

Genetic Basis of Severe Childhood-Onset Cardiomyopathies.

BACKGROUND: Childhood cardiomyopathies are progressive and often lethal disorders, forming the most common cause of heart failure in children. Despite severe outcomes, their genetic background is still poorly characterized. OBJECTIVES: The purpose of this study was to characterize the genetics of severe childhood cardiomyopathies in a countrywide cohort. METHODS: The authors collected a countrywide cohort, KidCMP, of 66 severe childhood cardiomyopathies from the sole center in Finland performing cardiac transplantation. For genetic diagnosis, next-generation sequencing and subsequent validation using genetic, cell biology, and computational approaches were used. RESULTS: The KidCMP cohort presents remarkable early-onset and severe disorders: the median age of diagnosis was 0.33 years, and 17 patients underwent cardiac transplantation. The authors identified the pathogenic variants in 39% of patients: 46% de novo, 34% recessive, and 20% dominantly-inherited. The authors report NRAP underlying childhood dilated cardiomyopathy, as well as novel phenotypes for known heart disease genes. Some genetic diagnoses have immediate implications for treatment: CALM1 with life-threatening arrhythmias, and TAZ with good cardiac prognosis. The disease genes converge on metabolic causes (PRKAG2, MRPL44, AARS2, HADHB, DNAJC19, PPA2, TAZ, BAG3), MAPK pathways (HRAS, PTPN11, RAF1, TAB2), development (NEK8 and TBX20), calcium signaling (JPH2, CALM1, CACNA1C), and the sarcomeric contraction cycle (TNNC1, TNNI3, ACTC1, MYH7, NRAP). CONCLUSIONS: Childhood cardiomyopathies are typically caused by rare, family-specific mutations, most commonly de novo, indicating that next-generation sequencing of trios is the approach of choice in their diagnosis. Genetic diagnoses may suggest intervention strategies and predict prognosis, offering valuable tools for prioritization of patients for transplantation versus conservative treatment.

Heterozygous junctophilin-2 (JPH2) p.(Thr161Lys) is a monogenic cause for HCM with heart failure.

During the last two decades, mutations in sarcomere genes have found to comprise the most common cause for hypertrophic cardiomyopathy (HCM), but still significant number of patients with dominant HCM in the family are left without molecular genetic diagnosis. Next generation sequencing (NGS) does not only enable evaluation of established HCM genes but also candidate genes for cardiomyopathy are frequently tested which may lead to a situation where conclusive interpretation of the variant requires extensive family studies. We aimed to characterize the phenotype related to a variant in the junctophilin-2 (JPH2) gene, which is less known non-sarcomeric candidate gene. In addition, we did extensive review of the literature and databases about JPH2 variation in association with cardiac disease. We characterize nine Finnish index patients with HCM and heterozygous for JPH2 c.482C>A, p.(Thr161Lys) variant were included and segregation studies were performed. We identified 20 individuals affected with HCM with or without systolic heart failure and conduction abnormalities in the nine Finnish families with JPH2 p.(Thr161Lys) variant. We found 26 heterozygotes with the variant and penetrance was 71% by age 60 and 100% by age 80. Co-segregation of the variant with HCM phenotype was observed in six families. Main clinical features were left ventricular hypertrophy, arrhythmia vulnerability and conduction abnormalities including third degree AV-block. In some patients end-stage severe left ventricular heart failure with normal or mildly enlarged diastolic dimensions was detected. In conclusion, we propose that the heterozygous JPH2 p.(Thr161Lys) variant is a new Finnish mutation causing atypical HCM.

Case reports of two pedigrees with recessive arrhythmogenic right ventricular cardiomyopathy associated with homozygous Thr335Ala variant in DSG2.

BACKGROUND: Arrhythmogenic right ventricular cardiomyopathy (ARVC) is an inherited cardiac disease, involving changes in ventricular myocardial tissue and leading to fatal arrhythmias. Mutations in desmosomal genes are thought to be the main cause of ARVC. However, the exact molecular genetic etiology of the disease still remains largely inconclusive, and this along with large variabilities in clinical manifestations complicate clinical diagnostics. CASE PRESENTATION: We report two families (n = 20) in which a desmoglein-2 (DSG2) missense variant c.1003A > G, p.(Thr335Ala) was discovered in the index patients using next-generation sequencing panels. The presence of this variant in probands' siblings and children was studied by Sanger sequencing. Five homozygotes and nine heterozygotes were found with the mutation. Participants were evaluated clinically where possible, and available medical records were obtained. All patients homozygous for the variant fulfilled the current diagnostic criteria for ARVC, whereas none of the heterozygous subjects had symptoms suggestive of ARVC or other cardiomyopathies. CONCLUSIONS: The homozygous DSG2 variant c.1003A > G co-segregated with ARVC, indicating autosomal recessive inheritance and complete penetrance. More research is needed to establish a detailed understanding of the relevance of rare variants in ARVC associated genes, which is essential for informative genetic counseling and rational family member testing.

Reduced carboxylesterase 1 is associated with endothelial injury in methamphetamine-induced pulmonary arterial hypertension.

Pulmonary arterial hypertension is a complication of methamphetamine use (METH-PAH), but the pathogenic mechanisms are unknown. Given that cytochrome P450 2D6 (CYP2D6) and carboxylesterase 1 (CES1) are involved in metabolism of METH and other amphetamine-like compounds, we postulated that loss of function variants could contribute to METH-PAH. Although no difference in CYP2D6 expression was seen by lung immunofluorescence, CES1 expression was significantly reduced in endothelium of METH-PAH microvessels. Mass spectrometry analysis showed that healthy pulmonary microvascular endothelial cells (PMVECs) have the capacity to both internalize and metabolize METH. Furthermore, whole exome sequencing data from 18 METH-PAH patients revealed that 94.4% of METH-PAH patients were heterozygous carriers of a single nucleotide variant (SNV; rs115629050) predicted to reduce CES1 activity. PMVECs transfected with this CES1 variant demonstrated significantly higher rates of METH-induced apoptosis. METH exposure results in increased formation of reactive oxygen species (ROS) and a compensatory autophagy response. Compared with healthy cells, CES1-deficient PMVECs lack a robust autophagy response despite higher ROS, which correlates with increased apoptosis. We propose that reduced CES1 expression/activity could promote development of METH-PAH by increasing PMVEC apoptosis and small vessel loss.

Systemic Dosing of Thymosin Beta 4 before and after Ischemia Does Not Attenuate Global Myocardial Ischemia-Reperfusion Injury in Pigs.

The use of cardiopulmonary bypass (CPB) and aortic cross-clamping causes myocardial ischemia-reperfusion injury (I-RI) and can lead to reduced postoperative cardiac function. We investigated whether this injury could be attenuated by thymosin beta 4 (TB4), a peptide which has showed cardioprotective effects. Pigs received either TB4 or vehicle and underwent CPB and aortic cross-clamping for 60 min with cold intermittent blood-cardioplegia and were then followed for 30 h. Myocardial function and blood flow was studied by cardiac magnetic resonance and PET imaging. Tissue and plasma samples were analyzed to determine the amount of cardiomyocyte necrosis and apoptosis as well as pharmacokinetics of the peptide. In vitro studies were performed to assess its influence on blood coagulation and vasomotor tone. Serum levels of the peptide were increased after administration compared to control samples. TB4 did not decrease the amount of cell death. Cardiac function and global myocardial blood flow was similar between the study groups. At high doses a vasoconstrictor effect on mesentery arteries and a vasodilator effect on coronary arteries was observed and blood clot firmness was reduced when tested in the presence of an antiplatelet agent. Despite promising results in previous trials the cardioprotective effect of TB4 was not demonstrated in this model for global myocardial I-RI.

Genetic testing and blood biomarkers in paediatric pulmonary hypertension. Expert consensus statement on the diagnosis and treatment of paediatric pulmonary hypertension. The European Paediatric Pulmonary Vascular Disease Network, endorsed by ISHLT and DGPK.

Childhood-onset pulmonary arterial hypertension (PAH) is considered complex and multifactorial, with relatively poor estimates of the natural history of the disease. Strategies allowing earlier detection, establishment of disease aetiology together with more accurate and sensitive biomarkers could enable better estimates of prognosis and individualise therapeutic strategies. Evidence is accumulating that genetic defects play an important role in the pathogenesis of idiopathic and hereditary forms of PAH. Altogether nine genes have been reported so far to be associated with childhood onset PAH suggesting that comprehensive multigene diagnostics can be useful in the assessment. Identification of disease-causing mutations allows estimates of prognosis and forms the most effective way for risk stratification in the family. In addition to genetic determinants the analysis of blood biomarkers are increasingly used in clinical practice to evaluate disease severity and treatment responses. As in genetic diagnostics, a multiplex approach can be helpful, as a single biomarker for PAH is unlikely to meet all requirements. This consensus statement reviews the current evidence for the use of genetic diagnostics and use of blood biomarkers in the assessment of paediatric patients with PAH.