Evaluating threshold for donor fraction cell-free DNA using clinically available assay for rejection in pediatric and adult heart transplantation.

BACKGROUND: The aims of the study were to assess the performance of a clinically available cell-free DNA (cfDNA) assay in a large cohort of pediatric and adult heart transplant recipients and to evaluate performance at specific cut points in detection of rejection. METHODS: Observational, non-interventional, prospective study enrolled pediatric and adult heart transplant recipients from seven centers. Biopsy-associated plasma samples were used for cfDNA measurements. Pre-determined cut points were tested for analytic performance. RESULTS: A total of 487 samples from 160 subjects were used for the analysis. There were significant differences for df-cfDNA values between rejection [0.21% (IQR 0.12-0.69)] and healthy samples [0.05% (IQR 0.01-0.14), p < .0001]. The pediatric rejection group had a median df-cfDNA value of 0.93% (IQR 0.28-2.84) compared to 0.09% (IQR 0.04-0.23) for healthy samples, p = .005. Overall negative predictive value was 0.94 while it was 0.99 for pediatric patients. Cut points of 0.13% and 0.15% were tested for various types of rejection profiles and were appropriate to rule out rejection. CONCLUSION: The study suggests that pediatric patients with rejection show higher levels of circulating df-cfDNA compared to adults and supports the specific cut points for clinical use in pediatric and adult patients with overall acceptable performance.

Correction: Integrative analysis of genomic variants reveals new associations of candidate haploinsufficient genes with congenital heart disease.

[This corrects the article DOI: 10.1371/journal.pgen.1009679.].

Integrative analysis of genomic variants reveals new associations of candidate haploinsufficient genes with congenital heart disease.

Numerous genetic studies have established a role for rare genomic variants in Congenital Heart Disease (CHD) at the copy number variation (CNV) and de novo variant (DNV) level. To identify novel haploinsufficient CHD disease genes, we performed an integrative analysis of CNVs and DNVs identified in probands with CHD including cases with sporadic thoracic aortic aneurysm. We assembled CNV data from 7,958 cases and 14,082 controls and performed a gene-wise analysis of the burden of rare genomic deletions in cases versus controls. In addition, we performed variation rate testing for DNVs identified in 2,489 parent-offspring trios. Our analysis revealed 21 genes which were significantly affected by rare CNVs and/or DNVs in probands. Fourteen of these genes have previously been associated with CHD while the remaining genes (FEZ1, MYO16, ARID1B, NALCN, WAC, KDM5B and WHSC1) have only been associated in small cases series or show new associations with CHD. In addition, a systems level analysis revealed affected protein-protein interaction networks involved in Notch signaling pathway, heart morphogenesis, DNA repair and cilia/centrosome function. Taken together, this approach highlights the importance of re-analyzing existing datasets to strengthen disease association and identify novel disease genes and pathways.

Altered contractility, Ca2+ transients, and cell morphology seen in a patient-specific iPSC-CM model of Ebstein's anomaly with left ventricular noncompaction.

Patients with two congenital heart diseases (CHDs), Ebstein's anomaly (EA) and left ventricular noncompaction (LVNC), suffer higher morbidity than either CHD alone. The genetic etiology and pathogenesis of combined EA/LVNC remain largely unknown. We investigated a familial EA/LVNC case associated with a variant (p.R237C) in the gene encoding Kelch-like protein 26 (KLHL26) by differentiating induced pluripotent stem cells (iPSCs) generated from affected and unaffected family members into cardiomyocytes (iPSC-CMs) and assessing iPSC-CM morphology, function, gene expression, and protein abundance. Compared with unaffected iPSC-CMs, CMs containing the KLHL26 (p.R237C) variant exhibited aberrant morphology including distended endo(sarco)plasmic reticulum (ER/SR) and dysmorphic mitochondria and aberrant function that included decreased contractions per minute, altered calcium transients, and increased proliferation. Pathway enrichment analyses based on RNASeq data indicated that the "structural constituent of muscle" pathway was suppressed, whereas the "ER lumen" pathway was activated. Taken together, these findings suggest that iPSC-CMs containing this KLHL26 (p.R237C) variant develop dysregulated ER/SR, calcium signaling, contractility, and proliferation.NEW & NOTEWORTHY We demonstrate here that iPSCs derived from patients with Ebstein's anomaly and left ventricular noncompaction, when differentiated into cardiomyocytes, display significant structural and functional changes that offer insight into disease pathogenesis, including altered ER/SR and mitochondrial morphology, contractility, and calcium signaling.

Increase in nuclear cell-free DNA is associated with major adverse events in adult and pediatric heart transplant recipients.

BACKGROUND: Cell-free DNA is an emerging biomarker. While donor fraction may detect graft events in heart transplant recipients, the prognostic value of total nuclear cell-free DNA (ncfDNA) itself is largely unexplored. OBJECTIVE: Explore the relationship between ncfDNA and clinical events in heart transplant recipients. METHODS: We conducted a multi-center prospective study to investigate the value of cell-free DNA in non-invasive monitoring following heart transplantation. Over 4000 blood samples were collected from 388 heart transplant patients. Total ncfDNA and donor fraction were quantified. Generalized linear models with maximum likelihood estimation for repeated measures with subjects as clusters were used to explore the relationship of ncfDNA and major adverse events. Receiver operating characteristic curves were used to help choose cutpoints. RESULTS: A ncfDNA threshold (50 ng/ml) was identified that was associated with increased risk of major adverse events. NcfDNA was elevated in patients who suffered cardiac arrest, required mechanical circulatory support or died post heart transplantation as well as in patients undergoing treatment for infection. CONCLUSIONS: Elevated ncfDNA correlates with risk for major adverse events in adult and pediatric heart transplant recipients and may indicate a need for enhanced surveillance after transplant.

Relationship between donor fraction cell-free DNA and clinical rejection in heart transplantation.

BACKGROUND: Clinical rejection (CR) defined as decision to treat clinically suspected rejection with change in immunotherapy based on clinical presentation with or without diagnostic biopsy findings is an important part of care in heart transplantation. We sought to assess the utility of donor fraction cell-free DNA (DF cfDNA) in CR and the utility of serial DF cfDNA in CR patients in predicting outcomes of clinical interest. METHODS: Patients with heart transplantation were enrolled in two sequential, multi-center, prospective observational studies. Blood samples were collected for surveillance or clinical events. Clinicians were blinded to the results of DF cfDNA. RESULTS: A total of 835 samples from 269 subjects (57% pediatric) were included for this analysis, including 28 samples associated with CR were analyzed. Median DF cfDNA was 0.43 (IQR 0.15, 1.36)% for CR and 0.10 (IQR 0.07, 0.16)% for healthy controls (p < .0001). At cutoff value of 0.13%, the area under curve (AUC) was 0.82, sensitivity of 0.86, specificity of 0.67, and negative predictive value of 0.99. There was serial decline in DF cfDNA post-therapy, however, those with cardiovascular events (cardiac arrest, need for mechanical support or death) showed significantly higher levels of DF cfDNA on Day 0 (2.11 vs 0.31%) and Day 14 (0.51 vs 0.22%) compared to those who did not have such an event (p < .0001). CONCLUSION: DF cfDNA has excellent agreement with clinical rejection and, importantly, serial measurement of DF cfDNA predict clinically significant outcomes post treatment for rejection in these patients.

Early changes in cell-free DNA levels in newly transplanted heart transplant patients.

Heart transplantation is a well-established therapy for end-stage heart failure in children and young adults. The highest risk of graft loss occurs in the first 60 days post-transplant. Donor fraction of cell-free DNA is a highly sensitive marker of graft injury. Changes in cell-free DNA levels have not previously been studied in depth in patients early after heart transplant. A prospective study was conducted among heart transplant recipients at a single pediatric heart center. Blood samples were collected from children and young adult transplant patients at three time points within 10 days of transplantation. DF and total cell-free DNA levels were measured using a targeted method (myTAIHEART ). In 17 patients with serial post-transplant samples, DF peaks in the first 2 days after transplant (3.5%, [1.9-10]%) and then declines toward baseline (0.27%, [0.19-0.52]%) by 6-9 days. There were 4 deaths in the first year among the 10 patients with complete sample sets, and 3 out of 4 who died had a late rise or blunted decline in donor fraction. Patients who died trended toward an elevated total cell-free DNA at 1 week (41.5, [34-65] vs 13.6, [6.2-22] P = .07). Donor fraction peaks early after heart transplant and then declines toward baseline. Patients without sustained decline in donor fraction and/or elevated total cell-free DNA at 1 week may have worse outcomes.

GSEPD: a Bioconductor package for RNA-seq gene set enrichment and projection display.

BACKGROUND: RNA-seq, wherein RNA transcripts expressed in a sample are sequenced and quantified, has become a widely used technique to study disease and development. With RNA-seq, transcription abundance can be measured, differential expression genes between groups and functional enrichment of those genes can be computed. However, biological insights from RNA-seq are often limited by computational analysis and the enormous volume of resulting data, preventing facile and meaningful review and interpretation of gene expression profiles. Particularly, in cases where the samples under study exhibit uncontrolled variation, deeper analysis of functional enrichment would be necessary to visualize samples' gene expression activity under each biological function. RESULTS: We developed a Bioconductor package rgsepd that streamlines RNA-seq data analysis by wrapping commonly used tools DESeq2 and GOSeq in a user-friendly interface and performs a gene-subset linear projection to cluster heterogeneous samples by Gene Ontology (GO) terms. Rgsepd computes significantly enriched GO terms for each experimental condition and generates multidimensional projection plots highlighting how each predefined gene set's multidimensional expression may delineate samples. CONCLUSIONS: The rgsepd serves to automate differential expression, functional annotation, and exploratory data analyses to highlight subtle expression differences among samples based on each significant biological function.

Human genotyping and an experimental model reveal NPR-C as a possible contributor to morbidity in coarctation of the aorta.

Coarctation of the aorta (CoA) is a common congenital cardiovascular (CV) defect characterized by a stenosis of the descending thoracic aorta. Treatment exists, but many patients develop hypertension (HTN). Identifying the cause of HTN is challenging because of patient variability (e.g., age, follow-up duration, severity) and concurrent CV abnormalities. Our objective was to conduct RNA sequencing of aortic tissue from humans with CoA to identify a candidate gene for mechanistic studies of arterial dysfunction in a rabbit model of CoA devoid of the variability seen with humans. We present the first known evidence of natriuretic peptide receptor C (NPR-C; aka NPR3) downregulation in human aortic sections subjected to high blood pressure (BP) from CoA versus normal BP regions (validated to PCR). These changes in NPR-C, a gene associated with BP and proliferation, were replicated in the rabbit model of CoA. Artery segments from this model were used with human aortic endothelial cells to reveal the functional relevance of altered NPR-C activity. Results showed decreased intracellular calcium ([Ca2+]i) activity to C-type natriuretic peptide (CNP). Normal relaxation induced by CNP and atrial natriuretic peptide was impaired for aortic segments exposed to elevated BP from CoA. Inhibition of NPR-C (M372049) also impaired aortic relaxation and [Ca2+]i activity. Genotyping of NPR-C variants predicted to be damaging revealed that rs146301345 was enriched in our CoA patients, but sample size limited association with HTN. These results may ultimately be used to tailor treatment for CoA based on mechanical stimuli, genotyping, and/or changes in arterial function.

Ciliopathy variant burden and developmental delay in children with hypoplastic left heart syndrome.

PURPOSE: To test the hypothesis that patients with hypoplastic left heart syndrome (HLHS) and developmental delay will have a higher average summative C-score in ciliopathy genes than patients with HLHS without developmental delay. METHODS: Ciliopathy gene variant burden was determined utilizing a summative C-score for 14 ciliopathy genes in children with HLHS (n = 24). Mean summative C-scores were compared between children with and without developmental delay. Genome-wide randomizing gene sets were evaluated as a scoring control. RESULTS: Children with developmental delay had a mean summative C-score of 4.05 in ciliopathy genes as compared to a mean summative C-score of 2.02 for children without developmental delay. This difference in means was higher than 99.1% (empirical P value <0.01) of 2 million random lists of 14 genes. CONCLUSION: Genetically complex disorders such as ciliopathies can be assessed to determine phenotypic risk with summative C-score in appropriately chosen gene sets. If these results are replicated in subsequent cohorts, a diagnostic gene panel could identify risk for developmental delay and other ciliopathy-related comorbidities in infants with congenital heart disease.Genet Med advance online publication 27 October 2016Genetics in Medicine (2016); doi:10.1038/gim.2016.167.

Impact of MYH6 variants in hypoplastic left heart syndrome.

Hypoplastic left heart syndrome (HLHS) is a clinically and anatomically severe form of congenital heart disease (CHD). Although prior studies suggest that HLHS has a complex genetic inheritance, its etiology remains largely unknown. The goal of this study was to characterize a risk gene in HLHS and its effect on HLHS etiology and outcome. We performed next-generation sequencing on a multigenerational family with a high prevalence of CHD/HLHS, identifying a rare variant in the α-myosin heavy chain (MYH6) gene. A case-control study of 190 unrelated HLHS subjects was then performed and compared with the 1000 Genomes Project. Damaging MYH6 variants, including novel, missense, in-frame deletion, premature stop, de novo, and compound heterozygous variants, were significantly enriched in HLHS cases (P < 1 × 10-5). Clinical outcomes analysis showed reduced transplant-free survival in HLHS subjects with damaging MYH6 variants (P < 1 × 10-2). Transcriptome and protein expression analyses with cardiac tissue revealed differential expression of cardiac contractility genes, notably upregulation of the ß-myosin heavy chain (MYH7) gene in subjects with MYH6 variants (P < 1 × 10-3). We subsequently used patient-specific induced pluripotent stem cells (iPSCs) to model HLHS in vitro. Early stages of in vitro cardiomyogenesis in iPSCs derived from two unrelated HLHS families mimicked the increased expression of MYH7 observed in vivo (P < 1 × 10-2), while revealing defective cardiomyogenic differentiation. Rare, damaging variants in MYH6 are enriched in HLHS, affect molecular expression of contractility genes, and are predictive of poor outcome. These findings indicate that the etiology of MYH6-associated HLHS can be informed using iPSCs and suggest utility in future clinical applications.

Effect of 22q11.2 deletion on bleeding and transfusion utilization in children with congenital heart disease undergoing cardiac surgery.

BACKGROUND: Postsurgical bleeding causes significant morbidity and mortality in children undergoing surgery for congenital heart defects (CHD). 22q11.2 deletion syndrome (DS) is the second most common genetic risk factor for CHD. The deleted segment of chromosome 22q11.2 encompasses the gene encoding glycoprotein (GP) Ibß, which is required for expression of the GPIb-V-IX complex on the platelet surface, where it functions as the receptor for von Willebrand factor (VWF). Binding of GPIb-V-IX to VWF is important for platelets to initiate hemostasis. It is not known whether hemizygosity for the gene encoding GPIbß increases the risk for bleeding following cardiac surgery for patients with 22q11.2 DS. METHODS: We performed a case-control study of 91 pediatric patients who underwent cardiac surgery with cardiopulmonary bypass from 2004 to 2012 at Children's Hospital of Wisconsin. RESULTS: Patients with 22q11.2 DS had larger platelets and lower platelet counts, bled more excessively, and received more transfusion support with packed red blood cells in the early postoperative period relative to control patients. CONCLUSION: Presurgical genetic testing for 22q11.2 DS may help to identify a subset of pediatric cardiac surgery patients who are at increased risk for excessive bleeding and who may require more transfusion support in the postoperative period.

Use of combination chemotherapy for treatment of granulomatous and lymphocytic interstitial lung disease (GLILD) in patients with common variable immunodeficiency (CVID).

PURPOSE: A subset of patients with common variable immunodeficiency (CVID) develops granulomatous and lymphocytic interstitial lung disease (GLILD), a restrictive lung disease associated with early mortality. The optimal therapy for GLILD is unknown. This study was undertaken to see if rituximab and azathioprine (combination chemotherapy) would improve pulmonary function and/or radiographic abnormalities in patients with CVID and GLILD. METHODS: A retrospective chart review of patients with CVID and GLILD who were treated with combination chemotherapy was performed. Complete pulmonary function tests (PFTs) and high-resolution computed tomography (HRCT) scans of the chest were done prior to therapy and >6 months later. HRCT scans of the chest were blinded, randomized, and scored independently (in pairs) by two radiologists. The differences between pre- and post-treatment HRCT scores and PFT parameters were analyzed. RESULTS: Seven patients with CVID and GLILD met inclusion criteria. Post-treatment increases were noted in both FEV1 (p=0.034) and FVC (p=0.043). HRCT scans of the chest demonstrated improvement in total score (p=0.018), pulmonary consolidations (p=0.041), ground-glass opacities (p=0.020) nodular opacities (p=0.024), and both the presence and extent of bronchial wall thickening (p=0.014, 0.026 respectively). No significant chemotherapy-related complications occurred. CONCLUSIONS: Combination chemotherapy improved pulmonary function and decreased radiographic abnormalities in patients with CVID and GLILD.

Nuclear Cell-Free DNA Predicts Adverse Events After Pediatric Cardiothoracic Surgery.

BACKGROUND: Preoperative risk stratification in cardiac surgery includes patient and procedure factors that are used in clinical decision-making. Despite these tools, unidentified factors contribute to variation in outcomes. Identification of latent physiologic risk factors may strengthen predictive models. Nuclear cell-free DNA (ncfDNA) increases with tissue injury and drops to baseline levels rapidly. The goal of this investigation is to measure and to observe ncfDNA kinetics in children undergoing heart operations with cardiopulmonary bypass (CPB), linking biomarkers, organ dysfunction, and outcomes. METHODS: This is a prospective observational study of 116 children <18 years and >3 kg undergoing operations with CPB. Plasma ncfDNA samples were collected and processed in a stepwise manner at predefined perioperative time points. The primary outcome measure was occurrence of postoperative cardiac arrest or extracorporeal membrane oxygenation. RESULTS: Data were available in 116 patients (median age, 0.9 years [range, 0-17.4 years]; median weight, 7.8 kg [range, 3.2-98 kg]). The primary outcome was met in 6 of 116 (5.2%). Risk of primary outcome was 2% with ncfDNA <20 ng/mL and 33% with ncfDNA >20 ng/mL (odds ratio, 25; CI, 3.96-158; P = .001). Elevated ncfDNA was associated with fewer hospital-free days (P < .01). CONCLUSIONS: This study analyzes ncfDNA kinetics in children undergoing operations with CPB for congenital heart disease. Elevated preoperative ncfDNA is strongly associated with postoperative arrest and extracorporeal membrane oxygenation. Further studies are needed to validate this technology as a tool to predict morbidity in children after cardiac surgical procedures.

3D-bioprinting of patient-derived cardiac tissue models for studying congenital heart disease.

Introduction: Congenital heart disease is the leading cause of death related to birth defects and affects 1 out of every 100 live births. Induced pluripotent stem cell technology has allowed for patient-derived cardiomyocytes to be studied in vitro. An approach to bioengineer these cells into a physiologically accurate cardiac tissue model is needed in order to study the disease and evaluate potential treatment strategies. Methods: To accomplish this, we have developed a protocol to 3D-bioprint cardiac tissue constructs comprised of patient-derived cardiomyocytes within a hydrogel bioink based on laminin-521. Results: Cardiomyocytes remained viable and demonstrated appropriate phenotype and function including spontaneous contraction. Contraction remained consistent during 30 days of culture based on displacement measurements. Furthermore, tissue constructs demonstrated progressive maturation based on sarcomere structure and gene expression analysis. Gene expression analysis also revealed enhanced maturation in 3D constructs compared to 2D cell culture. Discussion: This combination of patient-derived cardiomyocytes and 3D-bioprinting represents a promising platform for studying congenital heart disease and evaluating individualized treatment strategies.

Validation of donor fraction cell-free DNA with biopsy-proven cardiac allograft rejection in children and adults.

OBJECTIVES: Donor-specific cell-free DNA shows promise as a noninvasive marker for allograft rejection, but as yet has not been validated in both adult and pediatric recipients. The study objective was to validate donor fraction cell-free DNA as a noninvasive test to assess for risk of acute cellular rejection and antibody-mediated rejection after heart transplantation in pediatric and adult recipients. METHODS: Pediatric and adult heart transplant recipients were enrolled from 7 participating sites and followed for 12 months or more with plasma samples collected immediately before all endomyocardial biopsies. Donor fraction cell-free DNA was extracted, and quantitative genotyping was performed. Blinded donor fraction cell-free DNA and clinical data were analyzed and compared with a previously determined threshold of 0.14%. Sensitivity, specificity, negative predictive value, positive predictive value, and receiver operating characteristic curves were calculated. RESULTS: A total of 987 samples from 144 subjects were collected. After applying predefined clinical and technical exclusions, 745 samples from 130 subjects produced 54 rejection samples associated with the composite outcome of acute cellular rejection grade 2R or greater and pathologic antibody-mediated rejection 2 or greater and 323 healthy samples. For all participants, donor fraction cell-free DNA at a threshold of 0.14% had a sensitivity of 67%, a specificity of 79%, a positive predictive value of 34%, and a negative predictive value of 94% with an area under the curve of 0.78 for detecting rejection. When analyzed independently, these results held true for both pediatric and adult cohorts at the same threshold of 0.14% (negative predictive value 92% and 95%, respectively). CONCLUSIONS: Donor fraction cell-free DNA at a threshold of 0.14% can be used to assess for risk of rejection after heart transplantation in both pediatric and adult patients with excellent negative predictive value.

Human gene copy number spectra analysis in congenital heart malformations.

The clinical significance of copy number variants (CNVs) in congenital heart disease (CHD) continues to be a challenge. Although CNVs including genes can confer disease risk, relationships between gene dosage and phenotype are still being defined. Our goal was to perform a quantitative analysis of CNVs involving 100 well-defined CHD risk genes identified through previously published human association studies in subjects with anatomically defined cardiac malformations. A novel analytical approach permitting CNV gene frequency "spectra" to be computed over prespecified regions to determine phenotype-gene dosage relationships was employed. CNVs in subjects with CHD (n = 945), subphenotyped into 40 groups and verified in accordance with the European Paediatric Cardiac Code, were compared with two control groups, a disease-free cohort (n = 2,026) and a population with coronary artery disease (n = 880). Gains (≥200 kb) and losses (≥100 kb) were determined over 100 CHD risk genes and compared using a Barnard exact test. Six subphenotypes showed significant enrichment (P ≤ 0.05), including aortic stenosis (valvar), atrioventricular canal (partial), atrioventricular septal defect with tetralogy of Fallot, subaortic stenosis, tetralogy of Fallot, and truncus arteriosus. Furthermore, CNV gene frequency spectra were enriched (P ≤ 0.05) for losses at: FKBP6, ELN, GTF2IRD1, GATA4, CRKL, TBX1, ATRX, GPC3, BCOR, ZIC3, FLNA and MID1; and gains at: PRKAB2, FMO5, CHD1L, BCL9, ACP6, GJA5, HRAS, GATA6 and RUNX1. Of CHD subjects, 14% had causal chromosomal abnormalities, and 4.3% had likely causal (significantly enriched), large, rare CNVs. CNV frequency spectra combined with precision phenotyping may lead to increased molecular understanding of etiologic pathways.

Selective analysis of cell-free DNA in maternal blood for evaluation of fetal trisomy.

OBJECTIVE: To develop a novel prenatal assay based on selective analysis of cell-free DNA in maternal blood for evaluation of fetal Trisomy 21 (T21) and Trisomy 18 (T18). METHODS: Two hundred ninety-eight pregnancies, including 39 T21 and seven T18 confirmed fetal aneuploidies, were analyzed using a novel, highly multiplexed assay, termed digital analysis of selected regions (DANSR™). Cell-free DNA from maternal blood samples was analyzed using DANSR assays for loci on chromosomes 21 and 18. Products from 96 separate patients were pooled and sequenced together. A standard Z-test of chromosomal proportions was used to distinguish aneuploid samples from average-risk pregnancy samples. DANSR aneuploidy discrimination was evaluated at various sequence depths. RESULTS: At the lowest sequencing depth, corresponding to 204,000 sequencing counts per sample, average-risk cases where distinguished from T21 and T18 cases, with Z statistics for all cases exceeding 3.6. Increasing the sequencing depth to 410,000 counts per sample substantially improved separation of aneuploid and average-risk cases. A further increase to 620,000 counts per sample resulted in only marginal improvement. This depth of sequencing represents less than 5% of that required by massively parallel shotgun sequencing approaches. CONCLUSION: Digital analysis of selected regions enables highly accurate, cost efficient, and scalable noninvasive fetal aneuploidy assessment.

Influence of temperature during transportation on cell-free DNA analysis.

OBJECTIVE: To determine the effect of shipping blood in Streck blood collection tubes (BCT) prior to processing on cell-free DNA (cf-DNA) levels. METHODS: Blood was collected in ethylenediaminetetraacetic acid (EDTA) and BCT tubes from 10 pregnant women carrying male fetuses. One set of each tube for each subject was processed to plasma immediately (standard cf-DNA protocol), whereas the other set was shipped by air courier and then processed. DNA was extracted and total and fetal DNA concentrations were measured by TaqMan multiplexed quantitative real-time PCR. RESULTS: No significant difference was observed in total cf-DNA in plasma between immediately processed EDTA (control) and immediately processed BCT samples. Moreover, no significant change in total cf-DNA was detected in plasma of BCT samples shipped at room temperature. Significant differences in total cf-DNA leading to a significantly decreased fetal fraction were found in shipped EDTA samples and BCT samples shipped at 4°C. DISCUSSION: BCT tubes are suitable for shipping whole blood prior to processing with respect to cf-DNA levels. However, care should be taken to ensure that samples are not exposed to extreme temperatures during shipment. This finding will benefit the development and clinical application of noninvasive methods of fetal diagnosis that utilize cf-DNA.

A Systematic Review of Ebstein's Anomaly with Left Ventricular Noncompaction.

Traditional definitions of Ebstein's anomaly (EA) and left ventricular noncompaction (LVNC), two rare congenital heart defects (CHDs), confine disease to either the right or left heart, respectively. Around 15-29% of patients with EA, which has a prevalence of 1 in 20,000 live births, commonly manifest with LVNC. While individual EA or LVNC literature is extensive, relatively little discussion is devoted to the joint appearance of EA and LVNC (EA/LVNC), which poses a higher risk of poor clinical outcomes. We queried PubMed, Medline, and Web of Science for all peer-reviewed publications from inception to February 2022 that discuss EA/LVNC and found 58 unique articles written in English. Here, we summarize and extrapolate commonalities in clinical and genetic understanding of EA/LVNC to date. We additionally postulate involvement of shared developmental pathways that may lead to this combined disease. Anatomical variation in EA/LVNC encompasses characteristics of both CHDs, including tricuspid valve displacement, right heart dilatation, and left ventricular trabeculation, and dictates clinical presentation in both age and severity. Disease treatment is non-specific, ranging from symptomatic management to invasive surgery. Apart from a few variant associations, mainly in sarcomeric genes MYH7 and TPM1, the genetic etiology and pathogenesis of EA/LVNC remain largely unknown.