Browsing by Author "Laner, Andreas"
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- Comparison of the ABC and ACMG systems for variant classificationPublication . Houge, Gunnar; Bratland, Eirik; Aukrust, Ingvild; Tveten, Kristian; Žukauskaitė, Gabrielė; Sansovic, Ivona; rea-Fernández, Alejandro J.B; Mayer, Karin; Paakkola, Teija; McKenna, Caoimhe; Wright, William; Markovic, Milica Keckarevic; Lildballe, Dorte L.; Konecny, Michal; Smol, Thomas; Alhopuro, Pia; Gouttenoire, Estelle Arnaud; Obeid, Katharina; Todorova, Albena; Jankovic, Milena; Lubieniecka, Joanna M.; Stojiljkovic, Maja; Buisine, Marie-Pierre; Haukanes, Bjørn Ivar; Lorans, Marie; Roomere, Hanno; Petit, François M.; Haanpää, Maria K.; Beneteau, Claire; Pérez, Belén; Plaseska-Karanfilska, Dijana; Rath, Matthias; Fuhrmann, Nico; Ferreira, Bibiana I.; Stephanou, Coralea; Sjursen, Wenche; Maver, Aleš; Rouzier, Cécile; Chirita-Emandi, Adela; Gonçalves, João; Kuek, Wei Cheng David; Broly, Martin; Haer-Wigman, Lonneke; Thong, Meow-Keong; Tae, Sok-Kun; Hyblova, Michaela; Dunnen, Johan T. den; Laner, AndreasThe ABC and ACMG variant classification systems were compared by asking mainly European clinical laboratories to classify variants in 10 challenging cases using both systems, and to state if the variant in question would be reported as a relevant result or not as a measure of clinical utility. In contrast to the ABC system, the ACMG system was not made to guide variant reporting but to determine the likelihood of pathogenicity. Nevertheless, this comparison is justified since the ACMG class determines variant reporting in many laboratories. Forty-three laboratories participated in the survey. In seven cases, the classification system used did not influence the reporting likelihood when variants labeled as “maybe report” after ACMG-based classification were included. In three cases of population frequent but disease-associated variants, there was a difference in favor of reporting after ABC classification. A possible reason is that ABC step C (standard variant comments) allows a variant to be reported in one clinical setting but not another, e.g., based on Bayesian-based likelihood calculation of clinical relevance. Finally, the selection of ACMG criteria was compared between 36 laboratories. When excluding criteria used by less than four laboratories (<10%), the average concordance rate was 46%. Taken together, ABC-based classification is more clear-cut than ACMG-based classification since molecular and clinical information is handled separately, and variant reporting can be adapted to the clinical question and phenotype. Furthermore, variants do not get a clinically inappropriate label, like pathogenic when not pathogenic in a clinical context, or variant of unknown significance when the significance is known.
- Escherichia coli-cloned CFTR loci relevant for human artificial chromosome therapyPublication . Rocchi, Lucia; Braz, Carla; Cattani, Sonja; Ramalho, Anabela; Christan, Sulith; Edlinger, Marlene; Ascenzioni, Fiorentina; Laner, Andreas; Kraner, Simone; Amaral, Margarida; Schindelhauer, DirkClassical gene therapy for cystic fibrosis has had limited success because of immune response against viral vectors and short-term expression of cDNA-based transgenes. These limitations could be overcome by delivering the complete genomic CFTR gene on nonintegrating human artificial chromosomes (HACs). Here, we report reconstruction of the genomic CFTR locus and analyze incorporation into HACs of three P1 phage-based and F factor bacteria-based artificial chromosomes (PACs/BACs) of various sizes: (1) 5A, a large, nonselectable BAC containing the entire wild-type CFTR locus extending into both adjacent genes (296.8-kb insert, from kb -58.4 to +51.4) containing all regulators; (2) CGT21, a small, selectable, telomerized PAC (134.7 kb, from kb -60.7 to + 2) containing a synthetic last exon joining exon 10, EGFP, exon 24, and the 3' untranslated region; and (3) CF225, a midsized, nonselectable PAC (225.3 kb, from kb -60.7 to +9.8) ligated from two PACs with optimized codons and a silent XmaI restriction variant to discriminate transgene from endogenous expression. Cotransfection with telomerized, blasticidin-S-selectable, centromere-proficient α-satellite constructs into HT1080 cells revealed a workable HAC formation rate of 1 per ∼25 lines when using CGT21 or 5A. CF225 was not incorporated into a de novo HAC in 122 lines analyzed, but integrants were expressed. Stability analyses suggest the feasibility of prefabricating a large, tagged CFTR transgene that stably replicates in the proximity of a functional centromere. Although definite conclusions about HAC-proficient construct configurations cannot be drawn at this stage, important transfer resources were generated and characterized, demonstrating the promise of de novo HACs as potentially ideal gene therapy vector systems.