Biallelic loss-of-function variants in PLD1 cause congenital right-sided cardiac valve defects and neonatal cardiomyopathy.

Congenital heart disease is the most common type of birth defect, accounting for one-third of all congenital anomalies. Using whole-exome sequencing of 2718 patients with congenital heart disease and a search in GeneMatcher, we identified 30 patients from 21 unrelated families of different ancestries with biallelic phospholipase D1 (PLD1) variants who presented predominantly with congenital cardiac valve defects. We also associated recessive PLD1 variants with isolated neonatal cardiomyopathy. Furthermore, we established that p.I668F is a founder variant among Ashkenazi Jews (allele frequency of ~2%) and describe the phenotypic spectrum of PLD1-associated congenital heart defects. PLD1 missense variants were overrepresented in regions of the protein critical for catalytic activity, and, correspondingly, we observed a strong reduction in enzymatic activity for most of the mutant proteins in an enzymatic assay. Finally, we demonstrate that PLD1 inhibition decreased endothelial-mesenchymal transition, an established pivotal early step in valvulogenesis. In conclusion, our study provides a more detailed understanding of disease mechanisms and phenotypic expression associated with PLD1 loss of function.

Immune-Mediated Fetal Complete Atrioventricular Block: Can Dexamethasone Therapy Revert the Process?

BACKGROUND: Fetal complete atrioventricular block (CAVB) is usually autoimmune mediated. The risk of developing CAVB is 2% to 3% in anti-Ro/SS-A seropositive pregnancies and it increases 10 times after previous CAVB in siblings. Despite being a rare complication, CAVB carries a 20% mortality rate and substantial morbidity, as about 65% of newborns will eventually need life-long pacing. Once found, fetal CAVB is almost always irreversible, despite aggressive immunotherapy. This poor outcome prompted some research groups to address this situation. All groups followed anti-Ro/SS-A seropositive pregnancies on a weekly basis during the second trimester of pregnancy and tried to detect first degree atrioventricular block (AVB) using accurate echocardiographic tools, assuming they may characterize the initiation of the immune damage to the A-V conduction system, at which point the process might still be reversible. Some of the groups treated fetuses with first degree AVB with maternal oral fluorinated steroids. We summarized the results of all groups, including our group. We describe a case of a fetus that developed CAVB 6 days after normal sinus rhythm (NSR), who under aggressive dexamethasone therapy gradually reverted to NSR. This fetus had a previous sibling with CAVB. We assumed the immune damage to the conduction system in this small group of fetuses with a previous CAVB sibling may have occurred more quickly than usual. We therefore recommend a twice-weekly follow-up with these fetuses.

CFAP45 deficiency causes situs abnormalities and asthenospermia by disrupting an axonemal adenine nucleotide homeostasis module.

Axonemal dynein ATPases direct ciliary and flagellar beating via adenosine triphosphate (ATP) hydrolysis. The modulatory effect of adenosine monophosphate (AMP) and adenosine diphosphate (ADP) on flagellar beating is not fully understood. Here, we describe a deficiency of cilia and flagella associated protein 45 (CFAP45) in humans and mice that presents a motile ciliopathy featuring situs inversus totalis and asthenospermia. CFAP45-deficient cilia and flagella show normal morphology and axonemal ultrastructure. Proteomic profiling links CFAP45 to an axonemal module including dynein ATPases and adenylate kinase as well as CFAP52, whose mutations cause a similar ciliopathy. CFAP45 binds AMP in vitro, consistent with structural modelling that identifies an AMP-binding interface between CFAP45 and AK8. Microtubule sliding of dyskinetic sperm from Cfap45-/- mice is rescued with the addition of either AMP or ADP with ATP, compared to ATP alone. We propose that CFAP45 supports mammalian ciliary and flagellar beating via an adenine nucleotide homeostasis module.

Homozygous loss-of-function mutations in MNS1 cause laterality defects and likely male infertility.

The clinical spectrum of ciliopathies affecting motile cilia spans impaired mucociliary clearance in the respiratory system, laterality defects including heart malformations, infertility and hydrocephalus. Using linkage analysis and whole exome sequencing, we identified two recessive loss-of-function MNS1 mutations in five individuals from four consanguineous families: 1) a homozygous nonsense mutation p.Arg242* in four males with laterality defects and infertility and 2) a homozygous nonsense mutation p.Gln203* in one female with laterality defects and recurrent respiratory infections additionally carrying homozygous mutations in DNAH5. Consistent with the laterality defects observed in these individuals, we found Mns1 to be expressed in mouse embryonic ventral node. Immunofluorescence analysis further revealed that MNS1 localizes to the axonemes of respiratory cilia as well as sperm flagella in human. In-depth ultrastructural analyses confirmed a subtle outer dynein arm (ODA) defect in the axonemes of respiratory epithelial cells resembling findings reported in Mns1-deficient mice. Ultrastructural analyses in the female carrying combined mutations in MNS1 and DNAH5 indicated a role for MNS1 in the process of ODA docking (ODA-DC) in the distal respiratory axonemes. Furthermore, co-immunoprecipitation and yeast two hybrid analyses demonstrated that MNS1 dimerizes and interacts with the ODA docking complex component CCDC114. Overall, we demonstrate that MNS1 deficiency in humans causes laterality defects (situs inversus) and likely male infertility and that MNS1 plays a role in the ODA-DC assembly.

Mutations in PPCS, Encoding Phosphopantothenoylcysteine Synthetase, Cause Autosomal-Recessive Dilated Cardiomyopathy.

Coenzyme A (CoA) is an essential metabolic cofactor used by around 4% of cellular enzymes. Its role is to carry and transfer acetyl and acyl groups to other molecules. Cells can synthesize CoA de novo from vitamin B5 (pantothenate) through five consecutive enzymatic steps. Phosphopantothenoylcysteine synthetase (PPCS) catalyzes the second step of the pathway during which phosphopantothenate reacts with ATP and cysteine to form phosphopantothenoylcysteine. Inborn errors of CoA biosynthesis have been implicated in neurodegeneration with brain iron accumulation (NBIA), a group of rare neurological disorders characterized by accumulation of iron in the basal ganglia and progressive neurodegeneration. Exome sequencing in five individuals from two unrelated families presenting with dilated cardiomyopathy revealed biallelic mutations in PPCS, linking CoA synthesis with a cardiac phenotype. Studies in yeast and fruit flies confirmed the pathogenicity of identified mutations. Biochemical analysis revealed a decrease in CoA levels in fibroblasts of all affected individuals. CoA biosynthesis can occur with pantethine as a source independent from PPCS, suggesting pantethine as targeted treatment for the affected individuals still alive.

Postmortem Computer Tomography Appearance of the Aortic Arch in Children: What Is Considered Normal?

BACKGROUND: Virtual autopsies by computer tomography (CT) or magnetic resonance imaging can be valuable in cases of unexplained infant death. The radiologist must be familiar with the normal appearance of all the segments of the thoracic aorta in normal and deceased children. A thorough review of the literature revealed no prior articles describing CT changes in the ascending aorta or the aortic arch in pediatric virtual autopsies. OBJECTIVES: To compare the CT appearance of the thoracic aorta in deceased children and in those younger than 3 years of age. METHODS: Hospital registries were searched for cases of unexpected deaths in children younger than 3 years old, with a postmortem CT available, as well as for clinically indicated chest CT in children of the same age during a 5 year period. The ascending aorta (AA), aortic arch (arch), and the descending aorta (DA) diameters were measured. Student's t-tests and Mann-Whitney U-tests were used to compare the two groups. RESULTS: A total of 64 scans were reviewed: 35 postmortem and 29 performed on living patients. The differences in the diameter and the ratios of the diameter between the AA and the arch, as well as between the arch and the DA in the postmortem and living groups were statistically significant (P < 0.05). CONCLUSIONS: On postmortem CT scans, we found focal tapering of the aortic caliber at the level of the arch between the origin of the brachiocephalic artery and left subclavian artery. This finding should not be misinterpreted as a hypoplastic aortic arch.

Roles of the cilium-associated gene CCDC11 in left-right patterning and in laterality disorders in humans.

Axial determination occurs during early stages of embryogenesis. Flaws in laterality patterning result in abnormal positioning of visceral organs, as manifested in heterotaxy syndrome, or complete left-right inversion as in situs inversus totalis. These malformations are often associated with ciliopathies, as seen in primary ciliary dyskinesia. We have recently described a novel mutation in the Coiled-Coil Domain-Containing 11 (CCDC11) gene associated with laterality disorders in a consanguineous family of Arab-Muslim origin with two affected siblings presenting with diverse phenotypes, one with heterotaxy syndrome and the other with non-primary ciliary dyskinesia situs inversus totalis. This study further characterizes the roles of CCDC11 and the implications of the identified mutation on left-right axial patterning in patient-derived cells and in the frog embryo as a model organism. We analyzed patient-derived cells and manipulated Ccdc11 levels in Xenopus laevis frog embryos. Cilia length in patient cells was longer than in controls, and CCDC11 was localized to the centriole and the actin cytoskeleton. Mutated truncated protein accumulated and was also localized to the centriole and actin cytoskeleton. In frog embryos, Ccdc11 was regulated downstream of FoxJ1, and overexpression of the full-length or truncated protein, or downregulation of the gene resulted in severe disruption of embryonic left-right axial patterning. Taken together, our initial description of the deleterious mutation in CCDC11 in patients, the current results and more recent supportive studies highlight the important role of CCDC11 in axial patterning.

Congenital valvular defects associated with deleterious mutations in the PLD1 gene.

BACKGROUND: The underlying molecular aetiology of congenital heart defects is largely unknown. The aim of this study was to explore the genetic basis of non-syndromic severe congenital valve malformations in two unrelated families. METHODS: Whole-exome analysis was used to identify the mutations in five patients who suffered from severe valvular malformations involving the pulmonic, tricuspid and mitral valves. The significance of the findings was assessed by studying sporulation of yeast carrying a homologous Phospholipase D (PLD1) mutation, in situ hybridisation in chick embryo and echocardiography and histological examination of hearts of PLD1 knockout mice. RESULTS: Three mutations, p.His442Pro, p.Thr495fs32* and c.2882+2T>C, were identified in the PLD1 gene. The mutations affected highly conserved sites in the PLD1 protein and the p.His442Pro mutation produced a strong loss of function phenotype in yeast homologous mutant strain. Here we show that in chick embryos PLD1 expression is confined to the forming heart (E2-E8) and homogeneously expressed all over the heart during days E2-E3. Thereafter its expression decreases, remaining only adjacent to the atrioventricular valves and the right ventricular outflow tract. This pattern of expression follows the known dynamic patterning of apoptosis in the developing heart, consistent with the known role of PLD1 in the promotion of apoptosis. In hearts of PLD1 knockout mice, we detected marked tricuspid regurgitation, right atrial enlargement, and increased flow velocity, narrowing and thickened leaflets of the pulmonic valve. CONCLUSIONS: The findings support a role for PLD1 in normal heart valvulogenesis.

Variable phenotypic presentation of a novel FOXF1 missense mutation in a single family.

BACKGROUND: Heterozygous mutations in the FOXF1 transcription factor gene are implicated in alveolar capillary dysplasia with misalignment of pulmonary veins (ACDMPV), a developmental disorder of the lungs classically presenting with pulmonary hypertension and early demise. Evidence has suggested haploinsufficiency and partial paternal imprinting. We present a family with several affected members with an extremely variable phenotype. PATIENTS: The index patient presented several hours after birth with severe pulmonary hypertension. She is now 3-years old, thriving on maximal pulmonary hypertension therapy, chronic steroids, and oxygen. One of the patient's siblings died at 16 days with pulmonary hypertension and an annular pancreas, consistent with classical ACDMPV. METHODS: Whole exome sequencing was performed in the index case. The identified variant was confirmed by Sanger sequencing, and tested in the remaining family members. Parental origin was determined by PCR amplification and cloning, sequencing, and identification of adjacent single nucleotide polymorphisms. Echocardiography was performed in the asymptomatic carriers. RESULTS: Whole exome analysis revealed a novel, predictably pathogenic heterozygous missense mutation, g.chr16:86544406 C>A NM_001451, c.C231A, p.F77L, in the FOXF1 gene. The mutation arose in the father, de novo, early postzygotically, with 70% somatic mosaicism in the blood, on the grandpaternal chromosome. It was also present in the proband's asymptomatic sister, found to have partial anomalous pulmonary venous return. CONCLUSION: FOXF1 mutations may have an extremely variable phenotype, possibly as a result of somatic mosaicism and complex gene regulation including unorthodox imprinting of the gene locus. The prolonged survival of the proband suggests the need for aggressive treatment. Pediatr Pulmonol. 2016; 51:921-927. © 2016 Wiley Periodicals, Inc.

Hepatopulmonary Syndrome in Patients With Cystic Fibrosis and Liver Disease.

Hepatopulmonary syndrome (HPS) is a liver-induced lung disorder defined as a triad of liver disease, pulmonary vascular dilatation, and a defect in oxygenation. It can complicate chronic liver disease of any etiology, but is most commonly associated with portal hypertension. Severe liver disease with portal hypertension is present in 2% to 8% of patients with cystic fibrosis (CF), but to date, to our knowledge, only one patient with CF has been reported to suffer from HPS. Here, we describe two patients with CF diagnosed with HPS, one subsequent to unresolved hypoxemia and the other following screening for HPS performed in our center. We speculate that HPS is underdiagnosed in patients with CF because of their coexisting respiratory morbidity, and we advocate routine screening for every patient with CF who has liver disease and portal hypertension.

A human laterality disorder caused by a homozygous deleterious mutation in MMP21.

BACKGROUND: Laterality in the vertebrate embryo is determined by left-right asymmetric gene expression driven by the flow of extraembryonic fluid across the embryonic node. Defects in these processes cause heterotaxy, the abnormal formation and arrangement of visceral organs that can range from complete inversion of symmetry to the selective misarrangement of organs. However, our understanding of the genetic causality for laterality defects in human beings remains relatively limited. METHODS: We performed whole exome sequencing in a consanguineous family with heterotaxia. To interrogate the pathogenic potential of the discovered variant, we used an in vivo system in which the potential of the candidate gene to induce L-R asymmetry was tested by transient suppression and CRISPR/Cas9-induced deletions. We also used in vitro assays to test a possible link between our exome-derived candidate and Notch signaling. RESULTS: We identified a homozygous 2 bp deletion in MMP21, encoding matrix metalloproteinase-21, as the sole coding mutation that segregated with the phenotype. Transient suppression or CRISPR/Cas9-mediated deletion of mmp21 in zebrafish embryos induced cardiac looping defects, with concomitant disruption of laterality markers in the lateral plate mesoderm and disrupted notch signalling in vitro and in vivo. CONCLUSIONS: Our data implicate loss of MMP21 as a cause of heterotaxy in humans with concomitant defects in Notch signaling. In support of this finding, a homozygous missense mutation in MMP21 was identified previously in mice with N-Ethyl-N-Nitrosourea (ENU)-induced heterotaxy. Taken together, these observations suggest a role of matrix metalloproteinases in the establishment of asymmetric organ development, likely through the regulation of morphogenetic signals.

A human laterality disorder associated with a homozygous WDR16 deletion.

The laterality in the embryo is determined by left-right asymmetric gene expression driven by the flow of extraembryonic fluid, which is maintained by the rotary movement of monocilia on the nodal cells. Defects manifest by abnormal formation and arrangement of visceral organs. The genetic etiology of defects not associated with primary ciliary dyskinesia is largely unknown. In this study, we investigated the cause of situs anomalies, including heterotaxy syndrome and situs inversus totalis, in a consanguineous family. Whole-exome analysis revealed a homozygous deleterious deletion in the WDR16 gene, which segregated with the phenotype. WDR16 protein was previously proposed to play a role in cilia-related signal transduction processes; the rat Wdr16 protein was shown to be confined to cilia-possessing tissues and severe hydrocephalus was observed in the wdr16 gene knockdown zebrafish. The phenotype associated with the homozygous deletion in our patients suggests a role for WDR16 in human laterality patterning. Exome analysis is a valuable tool for molecular investigation even in cases of large deletions.

Conotruncal malformations and absent thymus due to a deleterious NKX2-6 mutation.

BACKGROUND: Truncus arteriosus (TA) accounts for ~1% of congenital heart defects. The aetiology of isolated TA is largely unknown but when occurring as part of a syndrome, it is mostly associated with chromosome 22q11 deletion. Vice versa, the most common congenital heart defects associated with chromosome 22q11 deletion are conotruncal malformations. In this study we investigated the cause of multiple conotruncal malformations accompanied by athymia in a consanguineous family. METHODS AND RESULTS: Whole exome analysis revealed a homozygous deleterious mutation in the NKX2-6 gene. CONCLUSIONS: NKX2-6 encodes a homeobox-containing protein which is expressed in mouse embryo at E8.0-E9.5 at the caudal pharyngeal arches and the outflow tract. A single missense mutation was previously implicated in the aetiology of familial isolated TA; however, null mice are entirely normal. The clear phenotype associated with a homozygous deleterious mutation in the present report, falls well within the spectrum of the cardiac defects seen in DiGeorge syndrome, is in agreement with NKX2-6 downstream location in the TBX1 signalling pathway and confirms NKX2-6 role in human cardiogenesis.

Prevalence of persistent superior vena cava and association with congenital heart anomalies.

A contralateral persistent superior vena cava (PSVC) can occur in a normal child or in association with congenital heart defects (CHDs). Its prevalence has been demonstrated in relatively small cohorts. We aim to assess the frequency of a PSVC in a large cohort of children with and without CHDs. To estimate its significance, we have searched for a PSVC in all children referred for echocardiography in our institution during a 16.5-year period. A group of 17,219 children comprised 8,140 children with a structural heart anomaly and 9,079 children with a structurally normal heart. Association between a PSVC and specific classes of CHD were looked for. A total of 288 children (1.7%) had a PSVC; 0.56% (51 of 9,079) in the normal heart group and 2.9% (237 of 8,140) in the congenital heart anomalies group. Odds ratio for having heart anomaly in the presence of PSVC was 5.2 (95% confidence interval 3.7 to 7.0). A PSVC was above all associated with atrioventricular septal defects, conotruncal malformations, and left-sided defects. The odds ratio of having PSVC in the aforementioned malformations compared with the normal heart group was 23.8, 13.6, and 11.0, respectively. In conclusion, although present in normal subjects, PSVC was more often associated with congenital heart and other anomalies, especially with atrioventricular septal defects, conotruncal malformations, and left-sided defects.

A brain abscess of probable odontogenic origin in a child with cyanotic heart disease.

The purpose of the present report was to present a rare case of a brain abscess in a child with heterotaxy syndrome, severe cardiac anomalies, and extensive dental caries. The pathogen was Streptococcus intermedius isolated from the cerebrospinal fluid. The source of the pathogen was probably an infection of a primary molar with a dentoalveolar abscess involving the bud of the permanent successor. After a long course of antibiotic regimens followed by a craniotomy with abscess drainage, a shunt, and comprehensive dental treatment, the patient was discharged from the hospital without any neurological sequel. At home, she completed an additional 3 months of oral antibiotics. This is the only known documented case of a toddler with a brain abscess of probable odontogenic origin without previous dental intervention. It emphasizes the importance of collaboration between cardiologists and pediatric dentists, especially in referring children with congenital heart defects for early dental checkups.

A human laterality disorder associated with recessive CCDC11 mutation.

BACKGROUND: Significant advancements in understanding the molecular pathophysiology of laterality determination were recently made. However, there are large gaps in our knowledge of the initial processes that lead to laterality defects, such as heterotaxy syndrome (HS, also known as situs ambiguous) and situs inversus totalis (SIT). The former refers to abnormal distribution of visceral organs, and the latter refers to a complete laterality inversion of both abdominal and thoracic viscera. METHODS: In order to identify a mutated gene in SIT and HS patients, the authors performed homozygosity mapping in a consanguineous family with laterality disorders identified in two siblings. RESULTS: A homozygous deleterious mutation in the CCDC11 gene was identified in the patients. The mutation resulted in an abnormally smaller protein in the patient's skin fibroblasts. The parents and five healthy siblings were heterozygous for the mutation, which was not present in 112 anonymous controls. CONCLUSIONS: Few genes have been associated with both SIT and HS, usually accompanied by other abnormalities. The authors suggest that CCDC11 is associated with autosomal recessive laterality defects of diverse phenotype resulting in SIT in one individual family member who is otherwise healthy, and in complex laterality anomalies (HS) in another member. This report underscores the importance of CCDC11 in laterality determination.

Cardiac, lung, and brain thrombosis in a child with obstructive sleep apnea.

A 3-year-old boy with failure to thrive and severe adenotonsillar hypertrophy with a clinical presentation of prolonged obstructive sleep apnea (OSA), was referred to the emergency room due to severe respiratory distress and anasarca. Echocardiography revealed right heart failure, a cystic lesion in the right ventricle and severe pulmonary hypertension. D-dimer was elevated but spiral computerized tomography (CT) and lung scan did not show any perfusion defects. Excision of the cardiac lesion during open-heart surgery, lung biopsy, and adenotonsillectomy were performed. Pathological examination showed an intracadiac organized thrombus and eccentric intimal fibrosis of the pulmonary arteries-which is a pathognomonic of pulmonary arterial microemboli. Brain CT revealed vein thrombosis of the left sigmoid sinus. Blood tests for inherited thrombophilia were normal. Today, 5 years after adenotonsillectomy, the child is normally developed, completely asymptomatic, free of any medications, and has a normal echocardiography. This case report may indicate that prolonged OSA can be a procoagulant state which can cause severe cardiovascular morbidity in children.

Intracardiac echogenic foci have no hemodynamic significance in the fetus.

Intracardiac echogenic foci (ECFs), probably representing microcalcifications of the papillary muscles, are a common finding in fetal ultrasonic screening examinations. Their significance is unclear, and their value as markers for chromosomal anomalies is debatable. It also is unknown whether ECFs predict abnormal cardiac performance. This prospective study analyzed and compared the systolic and diastolic properties of the heart in 28 fetuses with ECFs and 70 fetuses without ECFs using both conventional and novel myocardial deformation methods. The findings suggest that left-sided ECFs do not predict depressed left- or right-side systolic or diastolic properties in the fetus. A longitudinal study that would follow ECF fetuses into their childhood is warranted to confirm the findings of this study.

Analysis of segmental and global function of the fetal heart using novel automatic functional imaging.

BACKGROUND: Functional assessment of the fetal heart has always been a challenge. Automatic functional imaging (AFI), a novel non-Doppler methodology based on 2-dimensional acoustic markers tracking, measures myocardial deformation regardless of angle of interrogation. Thus, we studied the validity of AFI in segmental and global assessment of myocardial function in the fetus. METHODS: AFI-based myocardial deformation parameters including segmental tissue velocity, strain, and strain rate as well as biventricular global strain and strain rate were measured from raw scan-line data obtained from 28 normal fetuses (20-38, median 28 weeks of gestation). Interobserver and intraobserver variability was analyzed. AFI data were compared with analogous Doppler-derived tissue velocity imaging parameters measured in the same 28 fetuses. RESULTS: AFI was feasible in 94% of the fetuses studied with high reproducibility. AFI-based tissue velocity (3.9 +/- 1 cm/s) was comparable with tissue velocity imaging-based velocity (4 +/- 1.6 cm/s) in the right ventricle and in the left ventricle (AFI velocity 3.3 +/- 0.6 vs tissue velocity imaging 3.1 +/- 0.9 cm/s). Strain rate obtained by these two methods was also similar. Biventricular global strain and strain rate measured 16 +/- 4% and 1.6 +/- .5 seconds(-1), respectively. Tissue velocity increased whereas segmental strain rate decreased throughout gestation. Strain remained unchanged. Global strain rate significantly decreased with gestational age (r = -0.7). CONCLUSION: AFI, a novel non-Doppler methodology, allows fast and accurate quantification of segmental and global myocardial function in the fetus. AFI-based tissue velocity increases with gestational age whereas segmental and the new parameter global strain rate decrease throughout gestation.

N-terminal-proB-type natriuretic peptide as a marker for acute anthracycline cardiotoxicity in children.

BACKGROUND: Anthracyclines are widely used in the treatment of pediatric cancer but their use is associated with cardiotoxicity. The cardiotoxic effect may become clinically apparent many years after therapy, and no reliable method exists for early detection of cardiac damage while the patient is receiving the drug. The natriuretic peptides have been established as markers for anthracycline-induced cardiotoxicity in adults and markers for cardiac dysfunction in children. We examined whether N-terminal proB-type natriuretic peptide (NT-proBNP) may be used as a marker for anthracycline-induced cardiotoxicity in children. METHODS: Twenty-three consecutive pediatric patients with newly diagnosed cancer were enrolled in this study. All patients received anthracycline-containing chemotherapy. Fifty-four age-matched children served as controls. Serial measurements of plasma NT-proBNP levels were taken before and after each anthracycline-containing course. Echocardiograms were performed before initiation of treatment and at the end of the study. RESULTS: Plasma levels of NT-proBNP were within normal limits before treatment and increased significantly only after the first anthracycline dose (from 150+/-112 to 327+/-321 pg/mL, mean+/-SD, P=0.02) and not after subsequent doses. This increase was attributed mainly to a subgroup of patients who received more than 25 mg/m of doxorubicin. In 14 patients (61%), the highest NT-proBNP level occurred after the first anthracycline dose. All patients had normal echocardiograms and none developed heart failure. CONCLUSIONS: NT-proBNP increases significantly after the first anthracycline course in a subset of pediatric cancer patients. This increase is not associated with clinical or echocardiographic evidence of cardiac dysfunction. Anthracyclines may be more cardiotoxic in the first course than in subsequent courses. Longer follow-up of these patients is necessary to determine whether NT-proBNP can be used as an early marker for anthracycline-induced cardiotoxicity.