Transcript Life Technologies

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
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