Development of a new diagnostic algorithm for the study of diseases characterized by high genetic heterogeneity

2013/2014Next Generation Sequencing (NGS) technologies, such the Ion Torrent platform, could allow to simplify the diagnostic process of diseases characterized by an high genetic and phenotypic heterogeneity, because of the possibility to sequence simultaneously more genes and more patients in a single sequencing run. In order to develop a new diagnostic algorithm for rapid molecular diagnosis of these disorders, we have applied the Ion Torrent technology on two different genetically heterogeneous diseases, Fanconi anemia (FA) and inherited thrombocytopenias (IT). Since FA is a disorder better characterized than ITs, we first validated the Ion torrent technology on 30 samples (2 wild type and 28 FA), 25 of which were already analyzed with Sanger sequencing. Because of their low sequencing quality, we have excluded from this type of analysis 2 of the 28 FA samples. Then, comparing Ion Torrent and Sanger sequencing data, we have evaluated the sensitivity (95%) and the specificity (100%) of Ion Torrent technology. Moreover, in order to detect copy number variations (CNVs) in FA genes, we have improved a statistical analysis based on coverage sequencing data, confirming the presence of large intragenic deletions on FANCA in 5 patients. In summary we have characterized 25 of the 26 FA patients analyzed, identifying also 4 mutant alleles in the rare complementation group FANCL and FANCF and 10 mutations in loci different from genes causing the disease. Since we cannot exclude that new genes are involved in FA, the only patient without any mutation identified is suitable for whole exome analysis. Taking advantage from these good sequencing data, we have developed a diagnostic algorithm that combines the identification of both point mutations and CNVs. In order to verify if this new diagnostic process could be applied also to other genetically heterogeneous diseases, we have analyzed 21 IT patients, already characterized by Sanger sequencing. Among the 2225 variants identified by Ion torrent technology, using this new approach, we have select those (N=75, 56 different) potentially pathogenetic because of their frequency (MAF<0.01), or of their presence in IT mutation database o because of bioinformatics analysis. Thirty of these variants were confirmed by Sanger sequencing, 14 (12 different) of which localized in loci different from the gene causing the disease. It would be interesting to carry out functional studies on these additional variants to unravel the molecular basis of ITs. In summary we were able to characterized 17 of the 21 IT patients, including 2 patients with deletions in RBM8A (Thrombocytopenia and Absent Radii syndrome, TAR). The remaining 4 mutant alleles were not detected because of a low sequencing coverage. In conclusion, according to our data, we can consider the Ion Torrent technology and in particular the diagnostic algorithm proposed in our study, as a feasible approaches for the study of diseases characterized by high genetic and phenotypic heterogeneity. RIASSUNTO Le tecnologie di Next Generation Sequencing (NGS) consentono di analizzare più geni e più campioni contemporaneamente. In questo modo potrebbe essere possibile ridurre i tempi e i costi di analisi di tutte quelle patologie caratterizzate da elevata eterogeneità genetica e fenotipica, la cui caratterizzazione risulta essere spesso complessa e dispendiosa. Al fine di elaborare un nuovo algoritmo diagnostico che consenta la rapida elaborazione di una diagnosi molecolare di tali patologie, abbiamo deciso di validare una tra le più innovative tecnologie NGS attualmente in commercio, la metodica Ion Torrent, su due differenti malattie, entrambe caratterizzate da eterogeneità genetica. l’anemia di Fanconi (FA) e le piastrinopenie ereditarie (IT). Siccome la FA è una patologia meglio caratterizzata rispetto alle IT, durante la prima fase di questo lavoro di tesi abbiamo analizzato 30 campioni (25 dei quali già precedentemente analizzati con sequenziamento Sanger), di cui 2 wild type e 28 affetti. In seguito all’esclusione dalla nostra analisi di 2 campioni FA a causa di una bassa qualità di sequenziamento, abbiamo determinato la sensibilità (95%) e la specificità (100%) della nuova metodica confronto i dati di sequenziamento Ion Torrent e quelli Sanger a nostra disposizione. Inoltre, utilizzando i dati di copertura della sequenza, abbiamo messo a punto un’analisi statistica volta all’identificazione delle Copy Number Variation (CNV), confermando le delezioni a carico del gene FANCA presenti in 5 pazienti. Abbiamo quindi caratterizzato 25 dei 26 pazienti analizzati, identificando inoltre 2 casi con mutazioni nei rari gruppi di complementazione FANCF e FANCL e 10 mutazioni in loci differenti dai geni causativi. Poiché non escludiamo la possibilità che un nuovo gene possa essere coinvolto nella patologia, riteniamo che l’unico paziente ancora privo di diagnosi molecolare possa essere un buon candidato per lo studio dell’esoma. Infine, avvalendoci dei buoni risultati ottenuti, abbiamo elaborato un nuovo processo diagnostico con il quale identificare in modo semplice e rapido sia le mutazioni sia le CNV a carico dei 16 geni coinvolti nella FA. Nella seconda parte del nostro studio, abbiamo verificato se l’applicazione di tale algoritmo possa essere estesa anche ad altre patologie ad elevata eterogeneità genetica. Per questo motivo abbiamo analizzato 21 campioni affetti da piastrinopenie ereditarie, già precedentemente analizzati mediante sequenziamento Sanger. Grazie all’algoritmo proposto abbiamo potuto selezionare tra le 2225 varianti identificate le 75 (56 differenti) che sono risultate essere potenzialmente patogenetiche in base alla loro frequenza nella popolazione (MAF<0.01), alla loro presenza nei database di mutazione e all’analisi bioinformatica di patogenicità. Trenta (27 differenti) di queste varianti sono state confermate mediante sequenziamento Sanger, di cui in particolare 14 (12 differenti) presenti in geni diversi da quelli causativi. Alla luce di questo dato si rendono necessari studi funzionali su tali varianti al fine di comprendere i meccanismi molecolari alla base delle piastrinopenie ereditarie. Infine, utilizzando l’algoritmo proposto, è stato possibile confermare la diagnosi molecolare in 17 dei 21 pazienti IT, compresi i 2 affetti da trombocitopenia con assenza del radio (TAR) e portatori di una delezione sul cromosoma 1q21.1. I restanti 4 alleli mutati non sono stati identificati a causa di una bassa copertura di sequenziamento. In conclusione, in base ai dati raccolti sui campioni affetti da FA e IT, possiamo affermare che la tecnologia di sequenziamento Ion Torrent e l’algoritmo diagnostico da noi proposto sono degli strumenti utili per ottenere una diagnosi molecolare completa, veloce ed economica.XXVII Ciclo198

Identification of point mutations and large intragenic deletions in Fanconi anemia using next-generation sequencing technology

Fanconi anemia (FA) is a rare bone marrow failure disorder characterized by clinical and genetic heterogeneity with at least 17 genes involved, which make molecular diagnosis complex and time-consuming. Since next-generation sequencing technologies could greatly improve the genetic testing in FA, we sequenced DNA samples with known and unknown mutant alleles using the Ion PGMTM system (IPGM). The molecular target of 74.2 kb in size covered 96% of the FA-coding exons and their flanking regions. Quality control testing revealed high coverage. Comparing the IPGM and Sanger sequencing output of FANCA, FANCC, and FANCG we found no false-positive and a few false-negative variants, which led to high sensitivity (95.58%) and specificity (100%) at least for these two most frequently mutated genes. The analysis also identified novel mutant alleles, including those in rare complementation groups FANCF and FANCL. Moreover, quantitative evaluation allowed us to characterize large intragenic deletions of FANCA and FANCD2, suggesting that IPGM is suitable for identification of not only point mutations but also copy number variations

Hypomorphic FANCA mutations correlate with mild mitochondrial and clinical phenotype in Fanconi anemia

Fanconi anemia is a rare disease characterized by congenital malformations, aplastic anemia, and predisposition to cancer. Despite the consolidated role of the Fanconi anemia proteins in DNA repair, their involvement in mitochondrial function is emerging. The purpose of this work was to assess whether the mitochondrial phenotype, independent of genomic integrity, could correlate with patient phenotype. We evaluated mitochondrial and clinical features of 11 affected individuals homozygous or compound heterozygous for p.His913Pro and p.Arg951Gln/Trp, the two residues of FANCA that are more frequently affected in our cohort of patients. Although p.His913Pro and p.Arg951Gln proteins are stably expressed in cytoplasm, they are unable to migrate in the nucleus, preventing cells from repairing DNA. In these cells, the electron transfer between respiring complex I-III is reduced and the ATP/AMP ratio is impaired with defective ATP production and AMP accumulation. These activities are intermediate between those observed in wild-type and FANCA-/- cells, suggesting that the variants at residues His913 and Arg951 are hypomorphic mutations. Consistent with these findings, the clinical phenotype of most of the patients carrying these mutations is mild. These data further support the recent finding that the Fanconi anemia proteins play a role in mitochondria, and open up possibilities for genotype/phenotype studies based on novel mitochondrial criteria

Molecular analysis of Fanconi anemia: the experience of the Bone Marrow Failure Study Group of the Italian Association of Pediatric Onco-Hematology

Fanconi anemia is an inherited disease characterized by congenital malformations, pancytopenia, cancer predisposition, and sensitivity to cross-linking agents. The molecular diagnosis of Fanconi anemia is relatively complex for several aspects including genetic heterogeneity with mutations in at least 16 different genes. In this paper, we report the mutations identified in 100 unrelated probands enrolled into the National Network of the Italian Association of Pediatric Hematoly and Oncology. In approximately half of these cases, mutational screening was carried out after retroviral complementation analyses or protein analysis. In the other half, the analysis was performed on the most frequently mutated genes or using a next generation sequencing approach. We identified 108 distinct variants of the FANCA, FANCG, FANCC, FANCD2, and FANCB genes in 85, 9, 3, 2, and 1 families, respectively. Despite the relatively high number of private mutations, 45 of which are novel Fanconi anemia alleles, 26% of the FANCA alleles are due to 5 distinct mutations. Most of the mutations are large genomic deletions and nonsense or frameshift mutations, although we identified a series of missense mutations, whose pathogenetic role was not always certain. The molecular diagnosis of Fanconi anemia is still a tiered procedure that requires identifying candidate genes to avoid useless sequencing. Introduction of next generation sequencing strategies will greatly improve the diagnostic process, allowing a rapid analysis of all the genes

Molecular diagnosis of thrombocytopenia-absent radius syndrome using next-generation sequencing

Introduction: Thrombocytopenia-absent radius (TAR) syndrome is a rare autosomal recessive disease. Patients are compound heterozygotes for a loss-of-function allele, which in most cases is a large genomic deletion on chromosome 1q21.1 containing the RBM8A gene, and a noncoding variant located in the 5′UTR (rs139428292) or intronic (rs201779890) regions of RBM8A. As the molecular genetic testing in TAR requires multiple techniques for detection of copy-number variations (CNV) and nucleotide substitutions, we tested whether a next-generation sequencing (NGS) approach could identify both alterations. Methods: Two unrelated families were analyzed with Ion PGM sequencing using a target panel of genes responsible for different forms of inherited thrombocytopenia. A statistical quantitative evaluation of amplicon coverage was performed to detect CNV, in particular those on the RBM8A gene. Results: All the probands were apparently homozygous for the rare allele inherited by the father at the rs139428292 locus, suggesting the presence of a deletion on the maternal chromosome. The statistical analysis confirmed the hemizygous condition of RBM8A. Conclusion: We concluded that NGS approaches could be used as a cost-effective method for molecular investigation of TAR as they could simultaneously detect CNV and point mutations. © 2016 John Wiley & Sons Lt

AQP4-dependent glioma cell features affect the phenotype of surrounding cells via extracellular vesicles

Background: Extracellular vesicles (EVs) are membrane-enclosed particles released systemically by all cells, including tumours. Tumour EVs have been shown to manipulate their local environments as well as distal targets to sustain the tumour in a variety of tumours, including glioblastoma (GBM).We have previously demonstrated the dual role of the glial water channel aquaporin-4 (AQP4) protein in glioma progression or suppression depending on its aggregation state. However, its possible role in communication mechanisms in the microenvironment of malignant gliomas remains to be unveiled.Results: Here we show that in GBM cells AQP4 is released via EVs that are able to affect the GBM microenvironment. To explore this role, EVs derived from invasive GBM cells expressing AQP4-tetramers or apoptotic GBM cells expressing orthogonal arrays of particles (AQP4-OAPs) were isolated, using a differential ultracentrifugation method, and were added to pre-seeded GBM cells. Confocal microscopy analysis was used to visualize the interaction and uptake of AQP4-containing EVs by recipient cells. Chemoinvasion and Caspase3/7 activation assay, performed on recipient cells after EVs uptake, revealed that EVs produced by AQP4-tetramers expressing cells were able to drive surrounding tumour cells toward the migratory phenotype, whereas EVs produced by AQP4-OAPs expressing cells drive them toward the apoptosis pathway.Conclusion: This study demonstrates that the different GBM cell phenotypes can be transferred by AQP4-containing EVs able to influence tumour cell fate toward invasiveness or apoptosis.This study opens a new perspective on the role of AQP4 in the brain tumour microenvironment associated with the EV-dependent communication mechanism

Mutations of cytochrome c identified in patients with thrombocytopenia THC4 affect both apoptosis and cellular bioenergetics.

Inherited thrombocytopenias are heterogeneous diseases caused by at least 20 genes playing different role in the processes of megakaryopoiesis and platelet production. Some forms, such as thrombocytopenia 4 (THC4), are very rare and not well characterized. THC4 is an autosomal dominant mild thrombocytopenia described in only one large family from New Zealand and due to a mutation (G41S) of the somatic isoform of the cytochrome c (CYCS) gene. We report a novel CYCS mutation (Y48H) in patients from an Italian family. Similar to individuals carrying G41S, they have platelets of normal size and morphology, which are only partially reduced in number, but no prolonged bleeding episodes. In order to determine the pathogenetic consequences of Y48H, we studied the effects of the two CYCS mutations in yeast and mouse cellular models. In both cases, we found reduction of respiratory level and increased apoptotic rate, supporting the pathogenetic role of CYCS in thrombocytopenia. \ua9 2013 Elsevier B.V

Mutations of cytochrome c identified in patients with thrombocytopenia THC4 affect both apoptosis and cellular bioenergetics

Inherited thrombocytopenias are heterogeneous diseases caused by at least 20 genes playing different role in the processes of megakaryopoiesis and platelet production. Some forms, such as thrombocytopenia 4 (THC4), are very rare and not well characterized. THC4 is an autosomal dominant mild thrombocytopenia described in only one large family from New Zealand and due to a mutation (G41S) of the somatic isoform of the cytochrome c (CYCS) gene. We report a novel CYCS mutation (Y48H) in patients from an Italian family. Similar to individuals carrying G41S, they have platelets of normal size and morphology, which are only partially reduced in number, but no prolonged bleeding episodes. In order to determine the pathogenetic consequences of Y48H, we studied the effects of the two CYCS mutations in yeast and mouse cellular models. In both cases, we found reduction of respiratory level and increased apoptotic rate, supporting the pathogenetic role of CYCS in thrombocytopenia. © 2013 Elsevier B.V