Iveta Valášková

Towards clinical molecular diagnosis of inherited cardiac conditions: a comparison of bench-top genome DNA sequencers.

Background Molecular genetic testing is recommended for diagnosis of inherited cardiac disease, to guide prognosis and treatment, but access is often limited by cost and availability. Recently introduced high-throughput bench-top DNA sequencing platforms have the potential to overcome these limitations. Methodology/Principal Findings We evaluated two next-generation sequencing (NGS) platforms for molecular diagnostics. The protein-coding regions of six genes associated with inherited arrhythmia syndromes were amplified from 15 human samples using parallelised multiplex PCR (Access Array, Fluidigm), and sequenced on the MiSeq (Illumina) and Ion Torrent PGM (Life Technologies). Overall, 97.9% of the target was sequenced adequately for variant calling on the MiSeq, and 96.8% on the Ion Torrent PGM. Regions missed tended to be of high GC-content, and most were problematic for both platforms. Variant calling was assessed using 107 variants detected using Sanger sequencing: within adequately sequenced regions, variant calling on both platforms was highly accurate (Sensitivity: MiSeq 100%, PGM 99.1%. Positive predictive value: MiSeq 95.9%, PGM 95.5%). At the time of the study the Ion Torrent PGM had a lower capital cost and individual runs were cheaper and faster. The MiSeq had a higher capacity (requiring fewer runs), with reduced hands-on time and simpler laboratory workflows. Both provide significant cost and time savings over conventional methods, even allowing for adjunct Sanger sequencing to validate findings and sequence exons missed by NGS. Conclusions/Significance MiSeq and Ion Torrent PGM both provide accurate variant detection as part of a PCR-based molecular diagnostic workflow, and provide alternative platforms for molecular diagnosis of inherited cardiac conditions. Though there were performance differences at this throughput, platforms differed primarily in terms of cost, scalability, protocol stability and ease of use. Compared with current molecular genetic diagnostic tests for inherited cardiac arrhythmias, these NGS approaches are faster, less expensive, and yet more comprehensive.

Novel FOXF1 Mutations in Sporadic and Familial Cases of Alveolar Capillary Dysplasia with Misaligned Pulmonary Veins Imply a Role for its DNA Binding Domain

Alveolar capillary dysplasia with misalignment of pulmonary veins (ACD/MPV) is a rare and lethal developmental disorder of the lung defined by a constellation of characteristic histopathological features. Nonpulmonary anomalies involving organs of gastrointestinal, cardiovascular, and genitourinary systems have been identified in approximately 80% of patients with ACD/MPV. We have collected DNA and pathological samples from more than 90 infants with ACD/MPV and their family members. Since the publication of our initial report of four point mutations and 10 deletions, we have identified an additional 38 novel nonsynonymous mutations of FOXF1 (nine nonsense, seven frameshift, one inframe deletion, 20 missense, and one no stop). This report represents an up to date list of all known FOXF1 mutations to the best of our knowledge. Majority of the cases are sporadic. We report four familial cases of which three show maternal inheritance, consistent with paternal imprinting of the gene. Twenty five mutations (60%) are located within the putative DNA‐binding domain, indicating its plausible role in FOXF1 function. Five mutations map to the second exon. We identified two additional genic and eight genomic deletions upstream to FOXF1. These results corroborate and extend our previous observations and further establish involvement of FOXF1 in ACD/MPV and lung organogenesis.

A familial case of alveolar capillary dysplasia with misalignment of pulmonary veins supports paternal imprinting of FOXF1 in human

Alveolar capillary dysplasia with misalignment of pulmonary veins (ACD/MPV) is a rare developmental lung disorder that is uniformly lethal. Affected infants die within the first few weeks of their life despite aggressive treatment, although a few cases of late manifestation and longer survival have been reported. We have shown previously that mutations and deletions in FOXF1 are a cause of this disorder. Although most of the cases of ACD/MPV are sporadic, there have been infrequent reports of familial cases. We present a family with five out of six children affected with ACD/MPV. DNA analysis identified a missense mutation (c.416G>T; p.Arg139Leu) in the FOXF1 gene that segregated in the three affected siblings tested. The same variant is also present as a de novo mutation in the mother and arose on her paternally derived chromosome 16. The two tested affected siblings share the same chromosome 16 haplotype inherited from their maternal grandfather. Their single healthy sibling has a different chromosome 16 haplotype inherited from the maternal grandmother. The results are consistent with paternal imprinting of FOXF1 in human.

Inactivation of p53 and amplification of MYCN gene in a terminal lymphoblastic relapse in a chronic lymphocytic leukemia patient

B-cell chronic lymphocytic leukemia (CLL) is an incurable disease with a highly variable clinical course. A proportion of patients eventually progress to a higher stage of malignancy. A recent association has been observed between the presence of aberrant somatic hypermutations in leukemic cells (hypermutations occurring outside of the immunoglobulin locus) and the transformation to a diffuse large B-cell lymphoma or prolymphocytic leukemia. In this study, we report on the rarely observed blastic transformation in a CLL patient who had previously been shown to harbor aberrant somatic hypermutations in the TP53 tumor-suppressor gene (Mol Immunol 2008;45:1525-29). The enzyme responsible, the activation-induced cytidine deaminase, was still active within the transformation, as evidenced by the ongoing class-switch recombination of cytoplasmic immunoglobulins. The transformation was accompanied by a complete p53 inactivation, as well as complex karyotype changes including prominent amplification of MYCN oncogene. Our case-study supports the view that the aberrant somatic hypermutation is associated with transformation of CLL to a more aggressive malignancy.

Clinical Characteristics and Mutational Analysis of the RyR2 Gene in Seven Czech Families with Catecholaminergic Polymorphic Ventricular Tachycardia

Background: Catecholaminergic polymorphic ventricular tachycardia (CPVT) is a rare hereditary arrhythmia. The onset of clinical symptoms usually occurs during childhood, and is typically related to exercise. The aim of our study was to describe the clinical characteristics of seven Czech families with CPVT and the results of mutational analysis of the RyR2 gene in these families.

WS11.1 Development of a comprehensive workflow for the analysis of CFTR gene using next generation sequencing technology

I. Valaskova1,2,3, J. Necasova1, R.M. Selingerova1, R. Spesna1, A. Holcikova4, L. Homola4, E. Pokojova5, R. Gaillyova1,2,3. 1University Hospital, Medical Genetics, Brno, Czech Republic; 2Masaryk University, Faculty of Medicine, Department of Biology, Brno, Czech Republic; 3Masaryk University, Faculty of Medicine, Department of Laboratory Methods, Brno, Czech Republic; 4University Hospital, Clinic of Pediatric Infectious Diseases, Brno, Czech Republic; 5University Hospital, Department of Pulmonary Diseases and Tuberculosis, Brno, Czech Republic

Identification of a Novel Ryanodine Receptor Mutation Causing Malignant Hyperthermia

Malignant hyperthermia (MH) is a potentially fatal autosomal dominant disorder of the skeletal muscle and is triggered in susceptible individuals by commonly used inhalation anaesthetics and depolarizing muscle relaxants. Around 80% of the affected family are linked to the ryanodine receptor (RYR1) gene. More than 300 mutations in RYR1 have been associated with the MH-susceptible phenotype. Here we report the identification by two independent methods of a novel mutation associated with the MH-susceptible phenotype in the RYR1 gene.

Newborn screening for cystic fibrosis (NBSCF) in region Moravia, Czech Republic

Review of newborn screening for cystic fibrosis in Moravia region from the begining in 2009

Patients homozygous for F508del are not different at the beginning

Retrospective 10 year analysis of child cystic fibrosis onset. Relation between genotype and symptoms and time of onset are evaluated for 40 children. The results shows no major difference betwenn two main genotype groups (homozygous F508del and the others). First symptoms are ussualy failure to thrive and starts until 1 year of age.