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Next Generation Sequencing for NF1 molecular analysis Dominique Vidaud

Next Generation Sequencing for NF1 molecular analysis Dominique Vidaud Eric Pasmant - Franck Letourneur Department of Biochemistry and Molecular Genetics Cochin Hospital, Paris. ESHG, june 2013, Paris. Neurofibromatosis type 1 ( von Recklinghausen disease).

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Next Generation Sequencing for NF1 molecular analysis Dominique Vidaud

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  1. Next Generation Sequencing for NF1 molecular analysis Dominique Vidaud Eric Pasmant - Franck Letourneur Department of Biochemistry and Molecular Genetics Cochin Hospital, Paris ESHG, june 2013, Paris

  2. Neurofibromatosis type 1 (von Recklinghausen disease) Autosomal dominant transmission 50% of cases are sporadic Fullypenetrantdisease Important phenotypic variations Cancer predisposition syndrome Affects # 1 in 3000 people In France # 20 000 Neurofibromas (2 or more) Lisch nodules (2 or more) NF1 Café au lait spots (6 or more) Axillary or inguinal Freckling Bone lesions One first degreeaffected parent Opticglioma National Institut of Health Consensus Development Conference Neurofibromatosis. Conference statement. Arch Neurol1988:45, 575-578

  3. RAS/MAPK pathway and neurofibromatosis type 1 Neurofibromatosis type 1 RASOPATHIES : NCFC SYNDROMES HAMARTOMATOSIS LEOPARD syndrome Noonan syndrome CFC syndrome Costello syndrome Cowden syndrome McCune-Albrigh syndrome GPCR X NF1 PIP2 PIP3 PY PY PY PY Gs AC NRAS KRAS HRAS PI3K PY PY PTEN PDK AMPc ARAF BRAF CRAF SHP2 Grb2 X Gab1 PKA Spred1 SOS1 MEK1 MEK2 AKT3 AKT1 Legius syndrome ERK1 ERK2 mTOR TSC1/2 RSK Rheb Carney Complex Cytoplasmic substrates STK11 HIF1α vHL Proliferation Cell survival Apoptosis Tuberous Sclerosis Peutz-Jeghers syndrome Von-Hippel Lindau syndrome Proteus syndrome

  4. NF1 : 17q11.2, ~ 350 kb, 60 exons ubiquitously expressed in most tissues 12-kb mRNA 49

  5. NF1 : several pseudogenes located on chromosomes 2, 12, 14, 15, 18, 21, 22

  6. ~5% ~5% NF1 mutational spectrum 90% Intragenic mutations (but no hot spot) Large deletions No mutation identified Sabbagh et al. Hum Mol Genet. 2009

  7. Molecular analysis of the NF1 gene : DNA and RNA analysis Genomic DNA extracted from blood leucocytes RNA extracted from blood leucocytes (PAXgene) 2 1 Four intragenic microsatellites genotyping pre-screening Homozygosity Heterozygosity Real-Time PCR-Based gene dosage Deletion - cDNA sequencing Mutation - Mutation + Deletion + Custom high resolution Array CGH MLPA P081/P082 Confirmation with genomic DNA + familial segregation Mutation - Mutation + 4,3% NF1 total deletion 0,5% NF1 large partial deletion 89,4% Custom high resolution Array CGH Coding exons sequencing Mutation - Mutation + 3,7% No NF1 mutation identification Confirmation with cDNA + familial segregation 3 0,3% 3,4% From Parfait B. et al. Human Mutation, in press

  8. Cochin NGS platform Ion PGM™ Sequencer Ion Proton™ System 5500 SOLiD™ System

  9. NF1 sequencing using Ampliseq Library Kit ™ 2.0  NF1/SPRED1custom primer panel - 2 primer pools - 197 amplicons - NF1 and SPRED1 genes : #20 kb of coding sequences and UTRs - theoritical coverage : 90.76%

  10. Ion Workflow Ion AmpliSeq Library Kit 2.0 20 ng DNA 10 10 Clonal amplification Prepare library Isolate positive Ion Sphere particules Load Chip and Sequence Data analysis 10 10 DNA Sequencing Data Analysis Sample Preparation 2.5 days

  11. The target : NF1 coding sequences and UTRs : ~15 kb  48 samples mixed on one 316 chip 48 samples 24 samples

  12. Ion Workflow Ion AmpliSeq Library Kit 2.0 15 ng DNA Data analysis Clonal amplification Prepare library Isolate positive Ion Sphere particules Load Chip and Sequence 10 10 10 10 DNA Sequencing Sample Preparation 2 days (48 samples)

  13. igv_2.3.3 Reads displaying between exons 38 and 57

  14. Exon 41

  15. Sanger sequencing Exon 50 : c.7285C>T (NM_000267.3), p.Arg2429* (NP_000258.1)

  16. Sanger sequencing Exon 16, c.1842_1846delTAAGG (NM_000267.3),p.Lys874Phefs*3(NP_000258.1)

  17. Run TIN_89, may 21 2013

  18. NF1 negative samples MLPA (deletion or insertion of few exons) ? Positive Negative mRNA sequencing (deep intronic mutations) Negative Positive Phenotype Sequence with a greater depth Mosaicism Not NF1

  19. Conclusion • The NGS is a real improvement in our department • NF1 : - validation on 50 samples previously characterized • - #200 samples to analyze  end of 2013 • - analysis of mosaïcism •  Development of many other targets for the analysis of : • - Duchenne mulecular dystrophy • - cystic fibrosis • - haemopilia A and B • - vitreoretinopathies • - hypogonadotrophic hypogonadisms • - ….

  20. Department of Genetics Audrey BRIAND Engineer Catherine DODE MCUPH Philippe GOUSSARD Technician Chrystel LEROY Technician Eric PASMANT AHU Michel VIDAUD PUPH Franck LETOURNEUR’s team « Association Neurofibromatoses et Recklinghausen »

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