title: Neurofibromatosis type 1 molecular diagnosis: what can NGS do for you when you have a large gene with loss of function mutations?
abstract: Molecular diagnosis of neurofibromatosis type 1 (NF1) is challenging owing to the large size of the tumour suppressor gene NF1, and the lack of mutation hotspots. A somatic alteration of the wild-type NF1 allele is observed in NF1-associated tumours. Genetic heterogeneity in NF1 was confirmed in patients with SPRED1 mutations. Here, we present a targeted next-generation sequencing (NGS) of NF1 and SPRED1 using a multiplex PCR approach (230 amplicons of ∼150 bp) on a PGM sequencer. The chip capacity allowed mixing 48 bar-coded samples in a 4-day workflow. We validated the NGS approach by retrospectively testing 30 NF1-mutated samples, and then prospectively analysed 279 patients in routine diagnosis. On average, 98.5% of all targeted bases were covered by at least 20X and 96% by at least 100X. An NF1 or SPRED1 alteration was found in 246/279 (88%) and 10/279 (4%) patients, respectively. Genotyping throughput was increased over 10 times, as compared with Sanger, with ∼90[euro ] for consumables per sample. Interestingly, our targeted NGS approach also provided quantitative information based on sequencing depth allowing identification of multiexons deletion or duplication. We then addressed the NF1 somatic mutation detection sensitivity in mosaic NF1 patients and tumours.
authors: 1] Service de Biochimie et Génétique Moléculaire, Hôpital Cochin, Assistance Publique-Hôpitaux de Paris, Paris, France [2] EA7331, Université Paris Descartes, Sorbonne Paris Cité, Faculté des Sciences Pharmaceutiques et Biologiques, Paris, France.
authors: Eric
authors: E
authors: Pasmant
authors: 1] Service de Biochimie et Génétique Moléculaire, Hôpital Cochin, Assistance Publique-Hôpitaux de Paris, Paris, France [2] EA7331, Université Paris Descartes, Sorbonne Paris Cité, Faculté des Sciences Pharmaceutiques et Biologiques, Paris, France.
authors: Béatrice
authors: B
authors: Parfait
authors: 1] Service de Biochimie et Génétique Moléculaire, Hôpital Cochin, Assistance Publique-Hôpitaux de Paris, Paris, France [2] EA7331, Université Paris Descartes, Sorbonne Paris Cité, Faculté des Sciences Pharmaceutiques et Biologiques, Paris, France.
authors: Armelle
authors: A
authors: Luscan
authors: Service de Biochimie et Génétique Moléculaire, Hôpital Cochin, Assistance Publique-Hôpitaux de Paris, Paris, France.
authors: Philippe
authors: P
authors: Goussard
authors: 1] Service de Biochimie et Génétique Moléculaire, Hôpital Cochin, Assistance Publique-Hôpitaux de Paris, Paris, France [2] EA7331, Université Paris Descartes, Sorbonne Paris Cité, Faculté des Sciences Pharmaceutiques et Biologiques, Paris, France.
authors: Audrey
authors: A
authors: Briand-Suleau
authors: EA7331, Université Paris Descartes, Sorbonne Paris Cité, Faculté des Sciences Pharmaceutiques et Biologiques, Paris, France.
authors: Ingrid
authors: I
authors: Laurendeau
authors: Service de Biochimie et Génétique Moléculaire, Hôpital Cochin, Assistance Publique-Hôpitaux de Paris, Paris, France.
authors: Corinne
authors: C
authors: Fouveaut
authors: Service de Biochimie et Génétique Moléculaire, Hôpital Cochin, Assistance Publique-Hôpitaux de Paris, Paris, France.
authors: Chrystel
authors: C
authors: Leroy
authors: 1] Service de Biochimie et Génétique Moléculaire, Hôpital Cochin, Assistance Publique-Hôpitaux de Paris, Paris, France [2] EA7331, Université Paris Descartes, Sorbonne Paris Cité, Faculté des Sciences Pharmaceutiques et Biologiques, Paris, France.
authors: Annelore
authors: A
authors: Montadert
authors: Département de Dermatologie, Centre de référence des neurofibromatoses, Hôpital Henri-Mondor, Université Paris Est Créteil (UPEC), Créteil, France.
authors: Pierre
authors: P
authors: Wolkenstein
authors: 1] Service de Biochimie et Génétique Moléculaire, Hôpital Cochin, Assistance Publique-Hôpitaux de Paris, Paris, France [2] EA7331, Université Paris Descartes, Sorbonne Paris Cité, Faculté des Sciences Pharmaceutiques et Biologiques, Paris, France.
authors: Michel
authors: M
authors: Vidaud
authors: 1] Service de Biochimie et Génétique Moléculaire, Hôpital Cochin, Assistance Publique-Hôpitaux de Paris, Paris, France [2] EA7331, Université Paris Descartes, Sorbonne Paris Cité, Faculté des Sciences Pharmaceutiques et Biologiques, Paris, France.
authors: Dominique
authors: D
authors: Vidaud
mesh_terms: D048868:Adaptor Proteins, Signal Transducing; D019295:Computational Biology / Q000379:methods; D005091:Exons; D020440:Gene Duplication; D016514:Genes, Neurofibromatosis 1; D059014:High-Throughput Nucleotide Sequencing; D006801:Humans; D047908:Intracellular Signaling Peptides and Proteins / Q000235:genetics; D008565:Membrane Proteins / Q000235:genetics; D009030:Mosaicism; D009154:Mutation; D009456:Neurofibromatosis 1 / Q000175:diagnosis* / Q000235:genetics*; D015203:Reproducibility of Results; D012189:Retrospective Studies; D017384:Sequence Deletion
chemical_list: D048868:Adaptor Proteins, Signal Transducing; D047908:Intracellular Signaling Peptides and Proteins; D008565:Membrane Proteins; C504812:SPRED1 protein, human