varAmpliCNV: analyzing variance of amplicons to detect CNVs in targeted NGS data.

MOTIVATION: Computational identification of copy number variants (CNVs) in sequencing data is a challenging task. Existing CNV-detection methods account for various sources of variation and perform different normalization strategies. However, their applicability and predictions are restricted to specific enrichment protocols. Here, we introduce a novel tool named varAmpliCNV, specifically designed for CNV-detection in amplicon-based targeted resequencing data (Haloplex™ enrichment protocol) in the absence of matched controls. VarAmpliCNV utilizes principal component analysis (PCA) and/or metric dimensional scaling (MDS) to control variances of amplicon associated read counts enabling effective detection of CNV signals. RESULTS: Performance of VarAmpliCNV was compared against three existing methods (ConVaDING, ONCOCNV and DECoN) on data of 167 samples run with an aortic aneurysm gene panel (n = 30), including 9 positive control samples. Additionally, we validated the performance on a large deafness gene panel (n = 145) run on 138 samples, containing 4 positive controls. VarAmpliCNV achieved higher sensitivity (100%) and specificity (99.78%) in comparison to competing methods. In addition, unsupervised clustering of CNV segments and visualization plots of amplicons spanning these regions are included as a downstream strategy to filter out false positives. AVAILABILITY AND IMPLEMENTATION: The tool is freely available through galaxy toolshed and at: https://hub.docker.com/r/cmgantwerpen/varamplicnv. Supplementary Data File S1: https://tinyurl.com/2yzswyhh; Supplementary Data File S2: https://tinyurl.com/ycyf2fb4. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

Novel pathogenic SMAD2 variants in five families with arterial aneurysm and dissection: further delineation of the phenotype.

BACKGROUND: Missense variants in SMAD2, encoding a key transcriptional regulator of transforming growth factor beta signalling, were recently reported to cause arterial aneurysmal disease. OBJECTIVES: The aims of the study were to identify the genetic disease cause in families with aortic/arterial aneurysmal disease and to further define SMAD2 genotype-phenotype correlations. METHODS AND RESULTS: Using gene panel sequencing, we identified a SMAD2 nonsense variant and four SMAD2 missense variants, all affecting highly conserved amino acids in the MH2 domain. The premature stop codon (c.612dup; p.(Asn205*)) was identified in a marfanoid patient with aortic root dilatation and in his affected father. A p.(Asn318Lys) missense variant was found in a Marfan syndrome (MFS)-like case who presented with aortic root aneurysm and in her affected daughter with marfanoid features and mild aortic dilatation. In a man clinically diagnosed with Loeys-Dietz syndrome (LDS) that presents with aortic root dilatation and marked tortuosity of the neck vessels, another missense variant, p.(Ser397Tyr), was identified. This variant was also found in his affected daughter with hypertelorism and arterial tortuosity, as well as his affected mother. The third missense variant, p.(Asn361Thr), was discovered in a man presenting with coronary artery dissection. Variant genotyping in three unaffected family members confirmed its absence. The last missense variant, p.(Ser467Leu), was identified in a man with significant cardiovascular and connective tissue involvement. CONCLUSION: Taken together, our data suggest that heterozygous loss-of-function SMAD2 variants can cause a wide spectrum of autosomal dominant aortic and arterial aneurysmal disease, combined with connective tissue findings reminiscent of MFS and LDS.

Cardiogeneticsbank@UZA: A Collection of DNA, Tissues, and Cell Lines as a Translational Tool.

Cardiogeneticsbank@UZA is an academic hospital integrated biobank that collects aortic tissue, blood, cell lines (fibroblasts, vascular smooth muscle cells, peripheral blood mononuclear cells, and induced pluripotent stem cells), and DNA from patients with cardiogenetic disorders, for both diagnostic and research purposes. We adhere to a quality management system and have established standard protocols for the sampling and processing of all cardiogenetic patient related materials. Cardiogeneticsbank@UZA is embedded in the Biobanking and Biomolecular Resources Research Infrastructure Belgium (BBMRI.be) and samples from this biobank are available for commercial and academic researchers, through an established access procedure. Currently, the extremely valuable cardiogenetics collection consists of more than 8,700 DNA samples, 380 tissue samples, and 500 cell lines of 7,578 patients, and is linked with extensive clinical data. Some interesting potential research applications are discussed.

Severe Phenotype of Cutis Laxa Type 1B with Antenatal Signs due to a Novel Homozygous Nonsense Mutation in EFEMP2.

EFEMP2 mutations are known to be responsible for autosomal recessive cutis laxa type 1B (ARCL1B), a rare multisystem disease affecting skin, skeleton, and vascular structures. We report 2 additional related cases of ARCL1B of particular severity leading to termination of pregnancy. Cardinal signs of this connective tissue disease were already seen during the second trimester of pregnancy, then confirmed and clarified at autopsy. Anomalies included cutis laxa, arachnodactyly, clubfoot, wormian bones, moderate bowing of long bones with slender bone trabeculae, rib fractures, undermuscularized diaphragm, hiatal hernia, and arterial tortuosity with thick vascular walls and disorganized elastic fibers. Sequencing of the EFEMP2 gene revealed a novel homozygous nonsense mutation: c.639C>A (p.Cys213*). We performed a thorough histological analysis and discuss differential diagnoses, genotype-phenotype correlations, and the challenge of prenatal diagnosis of this disease.

Corrigendum: Candidate Gene Resequencing in a Large Bicuspid Aortic Valve-Associated Thoracic Aortic Aneurysm Cohort: SMAD6 as an Important Contributor.

[This corrects the article on p. 400 in vol. 8, PMID: 28659821.].

Candidate Gene Resequencing in a Large Bicuspid Aortic Valve-Associated Thoracic Aortic Aneurysm Cohort: SMAD6 as an Important Contributor.

Bicuspid aortic valve (BAV) is the most common congenital heart defect. Although many BAV patients remain asymptomatic, at least 20% develop thoracic aortic aneurysm (TAA). Historically, BAV-related TAA was considered as a hemodynamic consequence of the valve defect. Multiple lines of evidence currently suggest that genetic determinants contribute to the pathogenesis of both BAV and TAA in affected individuals. Despite high heritability, only very few genes have been linked to BAV or BAV/TAA, such as NOTCH1, SMAD6, and MAT2A. Moreover, they only explain a minority of patients. Other candidate genes have been suggested based on the presence of BAV in knockout mouse models (e.g., GATA5, NOS3) or in syndromic (e.g., TGFBR1/2, TGFB2/3) or non-syndromic (e.g., ACTA2) TAA forms. We hypothesized that rare genetic variants in these genes may be enriched in patients presenting with both BAV and TAA. We performed targeted resequencing of 22 candidate genes using Haloplex target enrichment in a strictly defined BAV/TAA cohort (n = 441; BAV in addition to an aortic root or ascendens diameter ≥ 4.0 cm in adults, or a Z-score ≥ 3 in children) and in a collection of healthy controls with normal echocardiographic evaluation (n = 183). After additional burden analysis against the Exome Aggregation Consortium database, the strongest candidate susceptibility gene was SMAD6 (p = 0.002), with 2.5% (n = 11) of BAV/TAA patients harboring causal variants, including two nonsense, one in-frame deletion and two frameshift mutations. All six missense mutations were located in the functionally important MH1 and MH2 domains. In conclusion, we report a significant contribution of SMAD6 mutations to the etiology of the BAV/TAA phenotype.

TGF-ß signalopathies as a paradigm for translational medicine.

This review focusses on impact of a better knowledge of pathogenic mechanisms of Marfan and related disorders on their treatment strategies. It was long believed that a structural impairment formed the basis of Marfan syndrome as deficiency in the structural extracellular matrix component, fibrillin-1 is the cause of Marfan syndrome. However, the study of Marfan mouse models has revealed the strong involvement of the transforming growth factor-ß signalling pathway in the pathogenesis of Marfan. Similarly, this pathway was demonstrated to be key in the pathogenesis of Loeys-Dietz and Shprintzen-Goldberg syndrome. The elucidation of the underlying pathogenic mechanisms has led to new treatment strategies, targeting the overactive TGF-ß pathway. Various clinical trials are currently investigating the potential new treatment options. A meta-analysis will contribute to a better understanding of the various trial results.

Mutations in a TGF-ß ligand, TGFB3, cause syndromic aortic aneurysms and dissections.

BACKGROUND: Aneurysms affecting the aorta are a common condition associated with high mortality as a result of aortic dissection or rupture. Investigations of the pathogenic mechanisms involved in syndromic types of thoracic aortic aneurysms, such as Marfan and Loeys-Dietz syndromes, have revealed an important contribution of disturbed transforming growth factor (TGF)-ß signaling. OBJECTIVES: This study sought to discover a novel gene causing syndromic aortic aneurysms in order to unravel the underlying pathogenesis. METHODS: We combined genome-wide linkage analysis, exome sequencing, and candidate gene Sanger sequencing in a total of 470 index cases with thoracic aortic aneurysms. Extensive cardiological examination, including physical examination, electrocardiography, and transthoracic echocardiography was performed. In adults, imaging of the entire aorta using computed tomography or magnetic resonance imaging was done. RESULTS: Here, we report on 43 patients from 11 families with syndromic presentations of aortic aneurysms caused by TGFB3 mutations. We demonstrate that TGFB3 mutations are associated with significant cardiovascular involvement, including thoracic/abdominal aortic aneurysm and dissection, and mitral valve disease. Other systemic features overlap clinically with Loeys-Dietz, Shprintzen-Goldberg, and Marfan syndromes, including cleft palate, bifid uvula, skeletal overgrowth, cervical spine instability and clubfoot deformity. In line with previous observations in aortic wall tissues of patients with mutations in effectors of TGF-ß signaling (TGFBR1/2, SMAD3, and TGFB2), we confirm a paradoxical up-regulation of both canonical and noncanonical TGF-ß signaling in association with up-regulation of the expression of TGF-ß ligands. CONCLUSIONS: Our findings emphasize the broad clinical variability associated with TGFB3 mutations and highlight the importance of early recognition of the disease because of high cardiovascular risk.