Importance of Variant Interpretation in Whole-Exome Molecular Autopsy: Population-Based Case Series.

BACKGROUND: Potentially lethal cardiac channelopathies/cardiomyopathies may underlie a substantial portion of sudden unexplained death in the young (SUDY). The whole-exome molecular autopsy represents the latest approach to postmortem genetic testing for SUDY. However, proper variant adjudication in the setting of SUDY can be challenging. METHODS: From January 2012 through December 2013, 25 consecutive cases of SUDY from 1 to 40 years of age (average age at death 27±5.7 years; 13 white, 12 black) from Cook County, Illinois, were referred after a negative (n=16) or equivocal (n=9) conventional autopsy. A whole-exome molecular autopsy with analysis of 99 sudden death-susceptibility genes was performed. The predicted pathogenicity of ultrarare, nonsynonymous variants was determined using the American College of Medical Genetics guidelines. RESULTS: Overall, 27 ultrarare nonsynonymous variants were seen in 16/25 (64%) victims of SUDY. Among black individuals, 9/12 (75%) had an ultrarare nonsynonymous variant compared with 7/13 (54%) white individuals. Of the 27 variants, 10 were considered pathogenic or likely pathogenic in 7/25 (28%) individuals in accordance with the American College of Medical Genetics guidelines. Pathogenic/likely pathogenic variants were identified in 5/16 (31%) of autopsy-negative cases and in 2/6 (33%) victims of SUDY with equivocal findings of cardiomyopathy. Overall, 6 pathogenic/likely pathogenic variants in 4/25 (16%) cases were congruent with the phenotypic findings at autopsy and therefore considered clinically actionable. CONCLUSIONS: Whole-exome molecular autopsy with gene-specific surveillance is an effective approach for the detection of potential pathogenic variants in SUDY cases. However, systematic variant adjudication is crucial to ensure accurate and proper care for surviving family members

Next-generation sequencing of AV nodal reentrant tachycardia patients identifies broad spectrum of variants in ion channel genes.

Atrioventricular nodal reentry tachycardia (AVNRT) is the most common form of regular paroxysmal supraventricular tachycardia. This arrhythmia affects women twice as frequently as men, and is often diagnosed in patients <40 years of age. Familial clustering, early onset of symptoms and lack of structural anomaly indicate involvement of genetic factors in AVNRT pathophysiology. We hypothesized that AVNRT patients have a high prevalence of variants in genes that are highly expressed in the atrioventricular conduction axis of the heart and potentially involved in arrhythmic diseases. Next-generation sequencing of 67 genes was applied to the DNA profile of 298 AVNRT patients and 10 AVNRT family members using HaloPlex Target Enrichment System. In total, we identified 229 variants in 60 genes; 215 missenses, four frame shifts, four codon deletions, three missense and splice sites, two stop-gain variants, and one start-lost variant. Sixty-five of these were not present in the Exome Aggregation Consortium (ExAC) database. Furthermore, we report two AVNRT families with co-segregating variants. Seventy-five of 284 AVNRT patients (26.4%) and three family members to different AVNRT probands had one or more variants in genes affecting the sodium handling. Fifty-four out of 284 AVNRT patients (19.0%) had variants in genes affecting the calcium handling of the heart. We furthermore find a large proportion of variants in the HCN1-4 genes. We did not detect a significant enrichment of rare variants in the tested genes. This could be an indication that AVNRT might be an electrical arrhythmic disease with abnormal sodium and calcium handling

Analysis of buccal mucosa as a prognostic tool in children with arrhythmogenic cardiomyopathy

Background The diagnosis of arrhythmogenic cardiomyopathy (ACM) is challenging especially in children at risk of adverse events. Analysis of cardiac myocyte junctional protein distribution may have diagnostic and prognostic implications, but its utility is limited by the need for a myocardial sample. We previously reported that buccal mucosa cells show junctional protein redistribution similar to that seen in cardiac myocytes of adult patients with ACM. Objectives We aimed to determine when junctional protein distribution abnormalities first occur in children with ACM variants and whether they correlate with progression of clinically apparent disease. Methods We analyzed buccal mucosa samples of children and adolescents with a family history of ACM (n = 13) and age-matched controls (n = 13). Samples were immunostained for plakoglobin, desmoplakin, plakophilin-1 and connexin43 and analyzed by confocal microscopy. All participants were swabbed at least twice with an average interval of 12–18 months between samplings. Results Junctional protein re-localization in buccal mucosa cells did not correlate with the presence of ACM-causing variants but instead occurred with clinical onset of disease. No changes in protein distribution were seen unless and until there was clinical evidence of disease. In addition, progressive shifts in the distribution of key proteins correlated with worsening of the disease phenotype. Finally, we observed restoration of junctional signal for Cx43 in patient with a favorable response to anti-arrhythmic therapy. Conclusions Due to ethical concerns about obtaining heart biopsies in children with no apparent disease, it has not been possible to analyze molecular changes in cardiac myocytes with the onset/progression of clinical disease. Using buccal smears as a surrogate for the myocardium may facilitate future studies of mechanisms and pathophysiological consequences of junctional protein redistribution in ACM. Buccal cells may also be a safe and inexpensive tool for risk stratification and potentially monitoring response to treatment in children bearing ACM variants

Outcomes of Cardiac Transplantation in Highly Sensitized Pediatric Patients

Despite aggressive immunosuppressive therapy, pediatric orthotopic heart transplant (OHT) candidates with elevated pre-transplant panel reactive antibody (PRA) carry an increased risk of rejection and early graft failure following transplantation. This study has aimed to more specifically evaluate the outcomes of transplant candidates stratified by PRA values. Records of pediatric patients listed for OHT between April 2004 and July 2008 were reviewed (n = 101). Survival analysis was performed comparing patients with PRA < 25 to those with PRA > 25, as well as patients with PRA < 80 and PRA > 80. Patients with PRA > 25 had decreased survival compared with those with PRA < 25 after listing (P = 0.004). There was an even greater difference in survival between patients with PRA > 80 and those with PRA < 80 (P = 0.002). Similar analyses for the patients who underwent successful transplantation showed no significant difference in post-transplant survival between patients with a pre-transplant PRA > 25 and those with PRA < 25 (P = 0.23). A difference approaching significance was noted for patients with PRA > 80 compared with PRA < 80 (P = 0.066). Patients with significantly elevated pre-transplant PRAs at the time of listing have a significantly worse outcome compared to those with moderately increased PRA values or non-sensitized patients. Further study is necessary to guide physician and family treatment decisions at the time of listing

E-Glass Fiber Reinforced Composites in Dental Applications

Fiber reinforced composites (FRCs) are more and more widely applied in dentistry to substitute for metallic restorations: periodontal splints, fixed partial dentures, endodontic posts, orthodontic appliances, and some other indirect restorations. In general in FRCs, the fiber reinforcement provides the composite structure with better biomechanical performance due to their superior properties in tension and flexure. Nowadays, the E-glass fiber is most frequently used because of its chemical resistance and relatively low cost. Growing interest is being paid to enhance its clinical performance. Moreover, various techniques are utilized to reinforce the adhesion between the fiber and the matrix. Oral conditions set special requirements and challenges for the clinical applications of FRCs. The biomechanical properties of dental materials are of high importance in dentistry, and given this, there is on-going scientific interest to develop E-glass fiber reinforced composite systems. FRCs are generally biocompatible and their toxicity is not a concern. © 2011 The Author(s).published_or_final_versionSpringer Open Choice, 21 Feb 201

Versatility and Stereotypy of Free-Tailed Bat Songs

In mammals, complex songs are uncommon and few studies have examined song composition or the order of elements in songs, particularly with respect to regional and individual variation. In this study we examine how syllables and phrases are ordered and combined, ie “syntax”, of the song of Tadarida brasiliensis, the Brazilian free-tailed bat. Specifically, we test whether phrase and song composition differ among individuals and between two regions, we determine variability across renditions within individuals, and test whether phrases are randomly ordered and combined. We report three major findings. First, song phrases were highly stereotyped across two regions, so much so that some songs from the two colonies were almost indistinguishable. All males produced songs with the same four types of syllables and the same three types of phrases. Second, we found that although song construction was similar across regions, the number of syllables within phrases, and the number and order of phrases in songs varied greatly within and among individuals. Last, we determined that phrase order, although diverse, deviated from random models. We found broad scale phrase-order rules and certain higher order combinations that were highly preferred. We conclude that free-tailed bat songs are composed of highly stereotyped phrases hierarchically organized by a common set of syntactical rules. However, within global species-specific patterns, songs male free-tailed bats dynamically vary syllable number, phrase order, and phrase repetitions across song renditions

Radiolabeled CCK/gastrin peptides for imaging and therapy of CCK2 receptor-expressing tumors

Cholecystokinin (CCK) receptors are overexpressed in numerous human cancers, like medullary thyroid carcinomas, small cell lung cancers and stromal ovarian cancers. The specific receptor-binding property of the endogenous ligands for these receptors can be exploited by labeling peptides with a radionuclide and using these as carriers to guide the radioactivity to the tissues that express the receptors. In this way, tumors can be visualized using positron emission tomography and single photon emission computed tomography imaging. A variety of radiolabeled CCK/gastrin-related peptides has been synthesized and characterized for imaging. All peptides have the C-terminal CCK receptor-binding tetrapeptide sequence Trp-Met-Asp-Phe-NH2 in common or derivatives thereof. This review focuses on the development and application of radiolabeled CCK/gastrin peptides for radionuclide imaging and radionuclide therapy of tumors expressing CCK receptors. We discuss both preclinical studies as well as clinical studies with CCK and gastrin peptides

Tuberculosis contact investigation with a new, specific blood test in a low-incidence population containing a high proportion of BCG-vaccinated persons

BACKGROUND: BCG-vaccination can confound tuberculin skin test (TST) reactions in the diagnosis of latent tuberculosis infection. METHODS: We compared the TST with a Mycobacterium tuberculosis specific whole blood interferon-gamma assay (QuantiFERON(®)-TB-Gold In Tube; QFT-G) during ongoing investigations among close contacts of sputum smear positive source cases in Hamburg, Germany. RESULTS: During a 6-month period, 309 contacts (mean age 28.5 ± 10.5 years) from a total of 15 source cases underwent both TST and QFT-G testing. Of those, 157 (50.8%) had received BCG vaccination and 84 (27.2%) had migrated to Germany from a total of 25 different high prevalence countries (i.e. >20 cases/100,000). For the TST, the positive response rate was 44.3% (137/309), whilst only 31 (10%) showed a positive QFT-G result. The overall agreement between the TST and the QFT-G was low (κ = 0.2, with 95% CI 0.14.-0.23), and positive TST reactions were closely associated with prior BCG vaccination (OR 24.7; 95% CI 11.7–52.5). In contrast, there was good agreement between TST and QFT-G in non-vaccinated persons (κ = 0.58, with 95% CI 0.4–0.68), increasing to 0.68 (95% CI 0.46–0.81), if a 10-mm cut off for the TST was used instead of the standard 5 mm recommended in Germany. CONCLUSION: The QFT-G assay was unaffected by BCG vaccination status, unlike the TST. In close contacts who were BCG-vaccinated, the QFT-G assay appeared to be a more specific indicator of latent tuberculosis infection than the TST, and similarly sensitive in unvaccinated contacts. In BCG-vaccinated close contacts, measurement of IFN-gamma responses of lymphocytes stimulated with M. tuberculosis-specific antigen should be recommended as a basis for the decision on whether to perform subsequent chest X-ray examinations or to start treatment for latent tuberculosis infection

Methods to study splicing from high-throughput RNA Sequencing data

The development of novel high-throughput sequencing (HTS) methods for RNA (RNA-Seq) has provided a very powerful mean to study splicing under multiple conditions at unprecedented depth. However, the complexity of the information to be analyzed has turned this into a challenging task. In the last few years, a plethora of tools have been developed, allowing researchers to process RNA-Seq data to study the expression of isoforms and splicing events, and their relative changes under different conditions. We provide an overview of the methods available to study splicing from short RNA-Seq data. We group the methods according to the different questions they address: 1) Assignment of the sequencing reads to their likely gene of origin. This is addressed by methods that map reads to the genome and/or to the available gene annotations. 2) Recovering the sequence of splicing events and isoforms. This is addressed by transcript reconstruction and de novo assembly methods. 3) Quantification of events and isoforms. Either after reconstructing transcripts or using an annotation, many methods estimate the expression level or the relative usage of isoforms and/or events. 4) Providing an isoform or event view of differential splicing or expression. These include methods that compare relative event/isoform abundance or isoform expression across two or more conditions. 5) Visualizing splicing regulation. Various tools facilitate the visualization of the RNA-Seq data in the context of alternative splicing. In this review, we do not describe the specific mathematical models behind each method. Our aim is rather to provide an overview that could serve as an entry point for users who need to decide on a suitable tool for a specific analysis. We also attempt to propose a classification of the tools according to the operations they do, to facilitate the comparison and choice of methods.Comment: 31 pages, 1 figure, 9 tables. Small corrections adde

Multiple populations in globular clusters. Lessons learned from the Milky Way globular clusters

Recent progress in studies of globular clusters has shown that they are not simple stellar populations, being rather made of multiple generations. Evidence stems both from photometry and spectroscopy. A new paradigm is then arising for the formation of massive star clusters, which includes several episodes of star formation. While this provides an explanation for several features of globular clusters, including the second parameter problem, it also opens new perspectives about the relation between globular clusters and the halo of our Galaxy, and by extension of all populations with a high specific frequency of globular clusters, such as, e.g., giant elliptical galaxies. We review progress in this area, focusing on the most recent studies. Several points remain to be properly understood, in particular those concerning the nature of the polluters producing the abundance pattern in the clusters and the typical timescale, the range of cluster masses where this phenomenon is active, and the relation between globular clusters and other satellites of our Galaxy.Comment: In press (The Astronomy and Astrophysics Review