Angela Yetman

A direct comparison of next generation sequencing enrichment methods using an aortopathy gene panel- clinical diagnostics perspective

BackgroundAortopathies are a group of disorders characterized by aneurysms, dilation, and tortuosity of the aorta. Because of the phenotypic overlap and genetic heterogeneity of diseases featuring aortopathy, molecular testing is often required for timely and correct diagnosis of affected individuals. In this setting next generation sequencing (NGS) offers several advantages over traditional molecular techniques.MethodsThe purpose of our study was to compare NGS enrichment methods for a clinical assay targeting the nine genes known to be associated with aortopathy. RainDance emulsion PCR and SureSelect RNA-bait hybridization capture enrichment methods were directly compared by enriching DNA from eight samples. Enriched samples were barcoded, pooled, and sequenced on the Illumina HiSeq2000 platform. Depth of coverage, consistency of coverage across samples, and the overlap of variants identified were assessed. This data was also compared to whole-exome sequencing data from ten individuals.ResultsRead depth was greater and less variable among samples that had been enriched using the RNA-bait hybridization capture enrichment method. In addition, samples enriched by hybridization capture had fewer exons with mean coverage less than 10, reducing the need for followup Sanger sequencing. Variants sets produced were 77% concordant, with both techniques yielding similar numbers of discordant variants.ConclusionsWhen comparing the design flexibility, performance, and cost of the targeted enrichment methods to whole-exome sequencing, the RNA-bait hybridization capture enrichment gene panel offers the better solution for interrogating the aortopathy genes in a clinical laboratory setting.

Stentless porcine valves in the right ventricular outflow tract: improved durability?

OBJECTIVE Stentless porcine valves are commonly used for aortic valve replacement in adults, yet their long-term performance in the right ventricular (RV) outflow tract is unknown. We evaluated intermediate-term performance of stentless porcine valves in the RV outflow tract in 150 children and adults over a 10-year period. METHODS We retrospectively reviewed data on all patients undergoing placement of a pulmonary valve or RV-PA conduit with a stentless porcine prosthesis (>/=19 mm) from 1998 to 2008. Valvar function was assessed with echocardiography. Freedom from reintervention (explantation or catheter-based intervention) was determined by actuarial methods. RESULTS A stentless porcine prosthesis was placed in the pulmonary position in 150 patients with a median weight and age of 50.1 kg (range 9.8-127) and 15.8 years (range 1.4-55), respectively. There were three early deaths (2%) and no late deaths. Actuarial freedom from reintervention was 100% at 1 year and 95.5% at 5 years. Peak transvalvar gradient at 1 and 5 years was 13+/-12 mmHg and 25+/-11 mmHg, respectively. At last follow-up no patient had severe insufficiency (PI), five patients had moderate PI and the remainder mild or no PI. CONCLUSIONS Stentless porcine valves function well in the pulmonary position over the intermediate-term and are associated with low rates of reintervention in patients requiring a >19 mm valve or valved conduit. Longer-term follow-up and comparison with other alternatives will be necessary to determine if these valves are superior to commonly used allograft or bovine jugular venous valved conduits.

Characterization of large genomic deletions in the FBN1 gene using multiplex ligation-dependent probe amplification

BackgroundConnective tissue diseases characterized by aortic aneurysm, such as Marfan syndrome, Loeys-Dietz syndrome and Ehlers Danlos syndrome type IV are heterogeneous and despite overlapping phenotypes, the natural history, clinical manifestations and interventional course for each diagnosis can be quite unique. The majority of mutations involved in the etiology of these disorders are missense and nonsense mutations. However, large deletions and duplications undetected by sequencing may be implicated in their pathogenesis, and may explain the apparent lack of genotype-phenotype correlation in a subset of patients. The objective of this study was to search for large pathogenic deletions and/or duplications in the FBN1, TGFβR1, and TGFβR2 genes using multiplex-ligation dependent probe amplification (MLPA) in patients with aortopathy, in whom no mutations in the FBN1, TGFβR1, and TGFβR2 genes were identified by sequencing.MethodsThe study included 14 patients from 11 unrelated families with aortic aneurysm. Of those, six patients (including 3 first-degree relatives), fulfilled the revised Ghent criteria for Marfan syndrome, and eight had predominantly aortic aneurysm/dilatation with variable skeletal and craniofacial involvement. MLPA for FBN1, TGFβR1, and TGFβR2 was carried out in all patients. A 385 K chromosome 15 specific array was used in two patients with a deletion of the entire FBN1 in order to define its size and boundaries.ResultsWe identified two novel large deletions in the FBN1 gene in four patients of two unrelated families who met clinical diagnostic criteria for Marfan syndrome. One patient was found to have a FBN1 deletion encompassing exons 1-5. The other three patients had a 542 Kb deletion spanning the whole FBN1 gene and five additional genes (SLC24A5, MYEF2, CTXN2, SLC12A1, DUT) in the chromosome 15.ConclusionsOur findings expand the number of large FBN1 deletions, and emphasize the importance of screening for large genomic deletions in connective tissue disorders featuring aortopathies, especially for those with classic Marfan phenotype.

Pulmonary vascular remodelling after heart transplantation in patients with cavopulmonary connection

OBJECTIVES Pulmonary vascular resistance (PVR) after heart transplantation (HT) is an important predictor of postoperative outcomes. We hypothesize that PVR and pulmonary capillary wedge pressure (PCWP) will exhibit favourable pulmonary vascular remodelling in patients with failing cavopulmonary connection (CPC) after HT. METHODS Retrospective analysis of patients with superior CPC (SCPC) and total CPC (TCPC) who have undergone HT was performed. Patient data, including age, underlying congenital heart defect, timing of CPC surgery and timing of HT, were reviewed. Right heart catheterization data, including PCWP (mmHg) and PVR indexed (PVRi, Woods Units) from preoperative, at 1 month, 6 months and 12 months after HT, were collected. Paired data were analysed using Students t-test. RESULTS Among 21 patients with failing CPC who underwent HT, 10 had SCPC and 11 had TCPC. Average age at HT was 13.3 ± 8 years. Average time after CPC to HT was 8.5 ± 6.2 years. PVRi was noted to trend down over time after HT (PVRi pre-HT versus 6 months after HT, 2.75 vs 2.06, P = 0.06 and pre-HT versus 12 months after HT, 2.79 vs 2.27, P = 0.09). There was a statistically significant decrease in PCWP at 6 months (pre-HT versus 6 months after HT, 12.6 vs 10.8, P = 0.01) and 12 months (pre-HT versus 12 months after HT, 12.9 vs 10.1, P = 0.01) after HT. CONCLUSIONS Pulmonary vascular changes occur gradually after HT in patients with CPC similar to those shown after HT in patients with cardiomyopathy. However, larger studies are needed to investigate correlation between outcomes and the presence or absence of pulmonary vascular changes after HT in CPC patients.

PREDICTION OF CORONARY ARTERY DISEASE AMONG ADULT CONGENITAL HEART DISEASE PATIENTS USING FRAMINGHAM RISK SCORE

Risk assessment tools to predict coronary artery disease (CAD), such as the Framingham Risk Score (FRS), have been developed to assess various populations. The FRS evaluates cardiovascular (CV) risk factors including age, gender, BP, smoking status, diabetes, and lipid profile. The CAD risk among

GASTROINTESTINAL COMPLICATIONS IN PATIENTS WITH A FONTAN CIRCUIT

Methods: The electronic Adult Congenital and Pediatric Cardiac databases from 2 tertiary facilities serving a 3-state region were reviewed and all Fontan survivors > 18 years old were identified. Patients were followed in either a pediatric cardiology clinic or subspecialized ACHD clinic. Demographic, radiographic, laboratory, and surgical data were reviewed to determine whether screening for hepatic dysfunction during adolescence or adulthood was performed, the type of screening procedure used, and the results of such testing. Cirrhosis was noted to be present or absent based on the results of radiographic imaging or biopsy specimen. Surgical and clinical correlates of cirrhosis were sought.