Comprehensive genotype–phenotype analysis in 230 patients with tetralogy of Fallot

Background Tetralogy of Fallot (ToF), the most frequent cyanotic congenital heart disease, is associated with a wide range of intra- and extracardiac phenotypes. In order to get further insight into genotype–phenotype correlation, a large cohort of 230 unselected patients with ToF was comprehensively investigated. Methods and results 230 patients with ToF were studied by karyotyping, comprehensive 22q11.2 deletion testing and sequencing of TBX1, NKX2.5 and JAG1, as well as molecular karyotyping in selected patients. Pathogenic genetic aberrations were found in 42 patients (18%), with 22q11.2 deletion as the most common diagnosis (7.4%), followed by trisomy 21 (5.2%) and other chromosomal aberrations or submicroscopic copy number changes (3%). Mutations in JAG1 were detected in three patients with Alagille syndrome (1.3%), while NKX2.5 mutations were seen in two patients with non-syndromic ToF (0.9%). One patient showed a recurrent polyalanine stretch elongation within TBX1 which represents a true mutation resulting in loss of transcriptional activity due to cytoplasmatic protein aggregation. Conclusion This study shows that 22q11.2 deletion represents the most common known cause of ToF, and that the associated cardiac phenotype is distinct for obstruction of the proximal pulmonary artery, hypoplastic central pulmonary arteries and subclavian artery anomalies. Atrioventricular septal defect associated with ToF is very suggestive of trisomy 21 and almost excludes 22q11.2 deletion. We report a further patient with a recurrent polyalanine stretch elongation within TBX1 and for the first time link TBX1 cytoplasmatic protein aggregation to congenital heart defects

Need for high-resolution genetic analysis in iPSC: results and lessons from the ForIPS consortium

Genetic integrity of induced pluripotent stem cells (iPSCs) is essential for their validity as disease models and for potential therapeutic use. We describe the comprehensive analysis in the ForIPS consortium: an iPSC collection from donors with neurological diseases and healthy controls. Characterization included pluripotency confirmation, fingerprinting, conventional and molecular karyotyping in all lines. In the majority, somatic copy number variants (CNVs) were identified. A subset with available matched donor DNA was selected for comparative exome sequencing. We identified single nucleotide variants (SNVs) at different allelic frequencies in each clone with high variability in mutational load. Low frequencies of variants in parental fibroblasts highlight the importance of germline samples. Somatic variant number was independent from reprogramming, cell type and passage. Comparison with disease genes and prediction scores suggest biological relevance for some variants. We show that high-throughput sequencing has value beyond SNV detection and the requirement to individually evaluate each clone

Benchmarking whole exome sequencing in the German Network for Personalized Medicine

Introduction Whole Exome Sequencing (WES) has emerged as an efficient tool in clinical cancer diagnostics to broaden the scope from panel-based diagnostics to screening of all genes and enabling robust determination of complex biomarkers in a single analysis. Methods To assess concordance, six formalin-fixed paraffin-embedded (FFPE) tissue specimens and four commercial reference standards were analyzed by WES as matched tumor-normal DNA at 21 NGS centers in Germany, each employing local wet-lab and bioinformatics investigating somatic and germline variants, copy-number alteration (CNA), and different complex biomarkers. Somatic variant calling was performed in 494 diagnostically relevant cancer genes. In addition, all raw data were re-analyzed with a central bioinformatic pipeline to separate wet- and dry-lab variability. Results The mean positive percentage agreement (PPA) of somatic variant calling was 76% and positive predictive value (PPV) 89% compared a consensus list of variants found by at least five centers. Variant filtering was identified as the main cause for divergent variant calls. Adjusting filter criteria and re-analysis increased the PPA to 88% for all and 97% for clinically relevant variants. CNA calls were concordant for 82% of genomic regions. Calls of homologous recombination deficiency (HRD), tumor mutational burden (TMB), and microsatellite instability (MSI) status were concordant for 94%, 93%, and 93% respectively. Variability of CNAs and complex biomarkers did not increase considerably using the central pipeline and was hence attributed to wet-lab differences. Conclusion Continuous optimization of bioinformatic workflows and participating in round robin tests are recommend

A novel 5q35.3 subtelomeric deletion syndrome

We observed a novel 3.5 Mb 5q subtelomeric deletion in a 3-year-old girl with developmental delay, hypotonia and multiple minor anomalies. Comparison of her phenotype with the few published patients with terminal 5q35 deletions revealed several overlapping features, but also showed remarkable differences such as shortness of stature versus macrosomia. After the report of 5q35.3 microdeletions in Sotos syndrome we integrated the published BACs into the public draft sequence and exactly mapped the deletion size in our patient by FISH analysis with 15 BAC probes. We demonstrated that the deletion in our patient is immediately adjacent to the reported Sotos syndrome deletion site. Subtracting the symptoms of Sotos syndrome from the published patients with larger 5q35.3 deletions allowed us to delineate a distinct phenotype of prenatal lymphedema with increased nuchal translucency, pronounced muscular hypotonia and delay of reaching motor milestones, but speech development within normal limits, wide fontanels, failure to thrive with postnatal short stature, and multiple minor anomalies such as mildly bell-shaped chest, minor congenital heart disease, and a distinct facial gestalt, associated with the novel 3.5 Mb cryptic deletion. We further showed in our patient that the deletion of the LCT(4) synthase gene results in a reduction of cysteinyl leukotriene synthesis to about 65% compared to normal values. The prenatal nuchal lymphedema associated with this deletion syndrome my be related to the deletion of the FLT4 gene causing autosomal dominant primary lymphedema and contributes to the differential diagnosis of increased fetal nuchal translucency

Nanoscale manipulation of the properties of solids at high pressure with relativistic heavy ions

High-pressure and high-temperature phases show unusual physical and chemical properties, but they are often difficult to \u27quench\u27 to ambient conditions. Here, we present a new approach, using bombardment with very high-energy, heavy ions accelerated to relativistic velocities, to stabilize a high-pressure phase. In this case, Gd 2 Zr 2 O 7, pressurized in a diamond-anvil cell up to 40 GPa, was irradiated with 20 GeV xenon or 45 GeV uranium ions, and the (previously unquenchable) cubic high-pressure phase was recovered after release of pressure. Transmission electron microscopy revealed a radiation-induced, nanocrystalline texture. Quantum-mechanical calculations confirm that the surface energy at the nanoscale is the cause of the remarkable stabilization of the high-pressure phase. The combined use of high pressure and high-energy ion irradiation provides a new means for manipulating and stabilizing new materials to ambient conditions that otherwise could not be recovered. © 2009 Macmillan Publishers Limited. All rights reserved

Comprehensive genotype–phenotype analysis in 230 patients with tetralogy of Fallot

Background Tetralogy of Fallot (ToF), the most frequent cyanotic congenital heart disease, is associated with a wide range of intra- and extracardiac phenotypes. In order to get further insight into genotype–phenotype correlation, a large cohort of 230 unselected patients with ToF was comprehensively investigated. Methods and results 230 patients with ToF were studied by karyotyping, comprehensive 22q11.2 deletion testing and sequencing of TBX1, NKX2.5 and JAG1, as well as molecular karyotyping in selected patients. Pathogenic genetic aberrations were found in 42 patients (18%), with 22q11.2 deletion as the most common diagnosis (7.4%), followed by trisomy 21 (5.2%) and other chromosomal aberrations or submicroscopic copy number changes (3%). Mutations in JAG1 were detected in three patients with Alagille syndrome (1.3%), while NKX2.5 mutations were seen in two patients with non-syndromic ToF (0.9%). One patient showed a recurrent polyalanine stretch elongation within TBX1 which represents a true mutation resulting in loss of transcriptional activity due to cytoplasmatic protein aggregation. Conclusion This study shows that 22q11.2 deletion represents the most common known cause of ToF, and that the associated cardiac phenotype is distinct for obstruction of the proximal pulmonary artery, hypoplastic central pulmonary arteries and subclavian artery anomalies. Atrioventricular septal defect associated with ToF is very suggestive of trisomy 21 and almost excludes 22q11.2 deletion. We report a further patient with a recurrent polyalanine stretch elongation within TBX1 and for the first time link TBX1 cytoplasmatic protein aggregation to congenital heart defects

Neocentric small supernumerary marker chromosomes (sSMC) – three more cases and review of the literature

Here we report on three new patients with neocentric small supernumerary marker chromosomes (sSMC) derived from chromosome 2, 13 and 15, respectively. The sSMC(13) and sSMC(15) had inverted duplicated shapes and the sSMC(2) a ring chromosome shape. All three cases were clinically severely abnormal. A review of the available sSMC literature revealed that up to the present 73 neocentric sSMC cases including these three new cases have been reported. Seven of these cases were not characterized morphologi- cally; in the remainder, 80% had an inverted duplication, 17% a ring and 3% a minute shape. 81% of the reported neocentric sSMC carriers showed severe, 12% moderate and 8% no clinical abnormalities. In summary, we report three more neocentric sSMC cases, provide a review on all up to now published cases, highlight their special characteristics and compare them to centric sSMC