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) Autosomal dominant transmission 50% of cases are sporadic Neurofibromas (2 or more) Lisch nodules (2 or more) Fully penetrant disease Important phenotypic variations Cancer predisposition syndrome NF1 Affects # 1 in 3000 people Café au lait spots (6 or more) In France # 20 000 Axillary or inguinal Freckling Bone lesions Optic glioma National Institut of Health Consensus Development Conference Neurofibromatosis. Conference statement. Arch Neurol 1988:45, 575-578 One first degree affected parent RAS/MAPK pathway and neurofibromatosis type 1 Neurofibromatosis type 1 RASOPATHIES : NCFC SYNDROMES LEOPARD syndrome Noonan syndrome CFC syndrome HAMARTOMATOSIS Costello syndrome Cowden syndrome McCune-Albrigh syndrome GPCR X NF1 PY PY PY PY PY PY NRAS KRAS HRAS PIP2 PIP3 Gs PI3K AC PTEN ARAF SHP2 Gab1 Grb2 SOS1 X Spred1 Legius syndrome BRAF PDK CRAF MEK1 MEK2 ERK1 ERK2 PKA AKT3 RSK Cytoplasmic substrates Proliferation AMPc TSC1/2 STK11 mTOR Carney Complex vHL Cell survival Peutz-Jeghers syndrome Rheb AKT1 HIF1α Apoptosis Tuberous Sclerosis Von-Hippel Lindau syndrome Proteus syndrome NF1 : 17q11.2, ~ 350 kb, 60 exons ubiquitously expressed in most tissues 12-kb mRNA Promoteur 60 1 68 76 158 174 123 75 30 142 4c 5 6 7 8 9 9a 10a 10a-2 144 84 195 103 2 3 4a 4b 45 135 114 10b 10c 80 124 158 250 74 84 441 140 123 84 117 11 12a 12b 13 14 15 16 17 18 19a 19b CSRD 182 212 20 21 162 104 136 63 22 23.1 23.2 23a GRD 159 98 147 147 110 24 25 26 27a 27b SEC14 OMG EVI2B EVI2A 433 341 203 194 141 280 28 29 30 31 32 33 215 68 115 102 141 127 132 136 158 34 35 36 37 38 39 40 41 42 158 131 101 143 47 217 54 43 44 45 46 47 48 48a 49 NF1 : several pseudogenes located on chromosomes 2, 12, 14, 15, 18, 21, 22 8 10a 10b 16 19b 10c 11 12b 14 18 21 23-1 23-2 24 26 Chr. 17 7 9 Chr. 2 Chr. 12 Chr. 14 (x6) Chr. 15 Chr. 18 Chr. 21 Chr. 22 12a 13 17 20 15 19a 22 23a 25 27a 27b NF1 mutational spectrum ~5% 90% ~5% Intragenic mutations (but no hot spot) Large deletions No mutation identified Sabbagh et al. Hum Mol Genet. 2009 Molecular analysis of the NF1 gene : DNA and RNA analysis Genomic DNA extracted from blood leucocytes RNA extracted from blood leucocytes (PAXgene) 2 Four intragenic microsatellites genotyping pre-screening 1 Homozygosity Heterozygosity Real-Time PCR-Based gene dosage Deletion - cDNA sequencing Mutation - Deletion + Custom high resolution Array CGH Mutation + MLPA P081/P082 Mutation - 4,3% NF1 total deletion 0,5% NF1 large partial deletion Mutation + Custom high resolution Array CGH Coding exons sequencing Mutation - Mutation + No NF1 mutation identification Confirmation with cDNA + familial segregation 3,4% 0,3% From Parfait B. et al. Human Mutation, in press Confirmation with genomic DNA + familial segregation 3,7% 3 89,4% Cochin NGS platform Ion PGM™ Sequencer Ion Proton™ System 5500 SOLiD™ System NF1 sequencing using Ampliseq Library Kit ™ 2.0 NF1/SPRED1 custom primer panel - 2 primer pools - 197 amplicons - NF1 and SPRED1 genes : #20 kb of coding sequences and UTRs - theoritical coverage : 90.76% Ion Workflow Ion AmpliSeq Library Kit 2.0 20 ng DNA Prepare library Clonal amplification Isolate positive Ion Sphere particules Sample Preparation 2.5 days Load Chip and Sequence Data analysis DNA Sequencing Data Analysis The target : NF1 coding sequences and UTRs : ~15 kb 48 samples mixed on one 316 chip 48 samples 24 samples Ion Workflow Ion AmpliSeq Library Kit 2.0 15 ng DNA Data analysis Prepare library Clonal amplification Sample Preparation Isolate positive Ion Sphere particules Load Chip and Sequence DNA Sequencing 2 days (48 samples) igv_2.3.3 Reads displaying between exons 38 and 57 Exon 41 Sanger sequencing Exon 50 : c.7285C>T (NM_000267.3), p.Arg2429* (NP_000258.1) Sanger sequencing Exon 16, c.1842_1846delTAAGG (NM_000267.3), p.Lys874Phefs*3 (NP_000258.1) Run TIN_89, may 21 2013 Number of samples 26 Sample BC 001 Run TIN_89 Exon / BC 002 TIN_89 10 1185+1delG, splice mutation BC 003 TIN_89 23 3049C>T, Gln1017* BC 004 TIN_89 1 61-1G>C , splice mutation BC 005 TIN_89 / BC 006 TIN_89 37 5203A>T, Lys1735* BC 007 TIN_89 9 1017_1018delCT, Ser340fs BC 008 TIN_89 / BC 009 TIN_89 54 BC 011 TIN_89 / BC 012 TIN_89 12 BC 013 TIN_89 / BC 015 TIN_89 32 4267T>A Lys1423* BC 017 TIN_89 3 234_235delTT, Asn78fs BC 019 TIN_89 23 3011_3012insA, Asn1004fs BC 020 TIN_89 35 4537C>T, Arg1513* BC 021 TIN_89 14 1545delG, Gly515fs BC 022 TIN_89 38 5498T>C, Leu1833Pro BC 023 TIN_89 / BC 024 TIN_89 / BC 025 TIN_89 33 4361+1G>A, splice mutation BC 026 TIN_89 40 5851_5852insA, Thr1951fs BC 028 TIN_89 6 592_593insT, Ala199fs, BC 029 TIN_89 12 1302T>A, Cys434* BC 030 TIN_89 52 7631_7633delCAC, Thr2544del BC 031 TIN_89 39 5839C>T, Arg1947* Positive PGM 19 (75%) Mutation 7846C>T, Arg2616* 1381C>T Arg461* 4 days Time mRNA sequencing (Sanger) 4 weeks 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 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 - …. Department of Genetics Audrey BRIAND Catherine DODE Philippe GOUSSARD Chrystel LEROY Eric PASMANT Michel VIDAUD Engineer MCUPH Technician Technician AHU PUPH Franck LETOURNEUR’s team « Association Neurofibromatoses et Recklinghausen »