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Overview research topics and techniques
1. clinical, biochemical and molecular studies of
hereditary connective tissue disorders
2. genetic studies of neurofibromatosis type I,
BPES and familial breast cancer
3. genetic studies of pediatric tumors
(incl neuroblastoom) and Merkel cell carcinoma,
genetic study of leukaemia
http://www.come.to/neuroblastoma
Nadine Van Roy
Mireille Van Gele
Jo Vandesompele
Katleen De Preter
Jasmien Hoebeeck
Filip Pattyn
Bruce Poppe
Stefan Vermeulen
Overview research topics and techniques
1. basic technology for DNA and RNA work biochemistry of collagen
2. karyotyping
3. FISH : detection of DNA targets in
cytological preparations)
one or two DNA targets
CGH (comparative genomic hybridisation) : detection of
genomic imbalances of chromosomal regions
fiber FISH : FISH on stretched chromatin fibers
FICTION : FISH combined with immunophenotyping
M-FISH (multicolor FISH) : 24 or more DNA targets
MYCN copy number
Fluorescence in situ hybridisation
DNA
Commercial Cot1 DNA
Labeling: nick translation
Mix
10ml culture
Denaturation and incubation at 37°C
Denaturation
Wash, detection
and counterstain
Hybridisatie o/n
f
Comparative genomic hybridisation - CGH
Tumor DNA
Normaal DNA
Normale metaphase
Groen
Rood
Fluorescentie
intensiteit
Ratio
1.0
Normal
Amplified
Loss
Comparative Genomic Hybridization (CGH)
to Chromosomes and Arrays
Test Genomic DNA
Reference Genomic DNA
Cot-1 DNA
Position on Chromosome
Position on Sequence
Comparative genomic hybridisation - CGH
1
2
3
6
7
8
13
14
15
19
20
9
21
4
5
10
11
12
16
17
18
22
x
y
Comparative genomic hybridisation - CGH
n =2
n =2
1
n =2
n =2
2
n =2
6
n =2
n =2
7
n =2
13
n =2
n =2
8
n =2
9
n =2
10
n =2
15
n =2
21
5
n =2
11
n =2
16
n =2
20
n =2
4
n =2
14
n =2
19
n =2
3
12
n =2
17
18
n =2
22
n =0
x
y
CGH profiles for chromosomes 1, 6, 8, 15 en 17
in a neuroblastoma case
Comparative Genomic Hybridization (CGH)
to Chromosomes and Arrays
Test Genomic DNA
Reference Genomic DNA
Cot-1 DNA
Position on Chromosome
Position on Sequence
Low and High Resolution Arrays for the Analysis of
Copy Number Changes on 20q in Breast Tumors
Low resolution scanning array of chromosome 20
q arm
p arm
D20S776
STSG25428
WI-11116E
WI-15702E
D20S120
D20S609
WI19642
WI-9227
afm 224zd12
D20S720
931h6
P4 0 1 0
B4 1 8 8
P4 0 3 9
B4 0 8 7
P4 0 2 8
B4 1 3 0
B4 1 6 6
D20S913
D20S1044
D20S876
D20S211
WI-16697
SGC31010
D20S854
D20S183
RMC20C001
D20S1020
D20S902
D20S480
820f5
845f3
847g7
D20S708E
~1 Mb
High resolution array of 20q13.2
B4 0 9 0
B4 0 1 8
A4 1 8 5
P4 0 0 9
dJ 1 1 9 1 N1 6
B4 1 9 8
P4 0 1 6
B4 1 2 1
B4 1 9 5
B4 1 3 2
B4 2 0 8
P4 0 0 7
P4 1 8 7
P4 0 6 9
P4 0 4 1
B4 2 0 1
B4 1 0 9
P4 0 6 7
P4 0 3 0
B4 1 0 3
B4 0 9 7
B4 1 0 4 , B4 1 2 3
B4 0 9 9
GT 849 CRP

100 Kb
ZNF218
20q::22q
ZNF217
L39 
PIC 1- like
NABC1*
NABC1
Proc. Natl. Acad. Sci . USA, 95, 8703-8708 (1998)
Human Array CGH with ~ 1 Mb Resolution
0.5 mg DNA
~2500 BACs
Random Prime
Triplicate spots
Cy3 and Cy5
130 mm centers
16 hr Hyb.
864 well plates
12 mm
MPE600 Chromosome 1 Copy Number
Profiles Measured by Chromosome and
Array CGH
2
log2ratio
1.5
1
0.5
0
-0.5
-1
-1.5
-2
0
50000
100000
150000
kb
200000
250000
300000
Assembly of microarrays for genome-wide
measurement of DNA copy number
Snijders et al. Nature Genetics november , 263-264
Low and High Resolution Arrays for the Analysis of
Copy Number Changes on 20q in Breast Tumors
Low resolution scanning array of chromosome 20
q arm
p arm
D20S776
STSG25428
WI-11116E
WI-15702E
D20S120
D20S609
WI19642
WI-9227
afm 224zd12
D20S720
931h6
P4 0 1 0
B4 1 8 8
P4 0 3 9
B4 0 8 7
P4 0 2 8
B4 1 3 0
B4 1 6 6
D20S913
D20S1044
D20S876
D20S211
WI-16697
SGC31010
D20S854
D20S183
RMC20C001
D20S1020
D20S902
D20S480
820f5
845f3
847g7
D20S708E
~1 Mb
High resolution array of 20q13.2
B4 0 9 0
B4 0 1 8
A4 1 8 5
P4 0 0 9
dJ 1 1 9 1 N1 6
B4 1 9 8
P4 0 1 6
B4 1 2 1
B4 1 9 5
B4 1 3 2
B4 2 0 8
P4 0 0 7
P4 1 8 7
P4 0 6 9
P4 0 4 1
B4 2 0 1
B4 1 0 9
P4 0 6 7
P4 0 3 0
B4 1 0 3
B4 0 9 7
B4 1 0 4 , B4 1 2 3
B4 0 9 9
GT 849 CRP

100 Kb
ZNF218
20q::22q
ZNF217
L39 
PIC 1- like
NABC1*
NABC1
Proc. Natl. Acad. Sci . USA, 95, 8703-8708 (1998)
Human Array CGH with ~ 1 Mb Resolution
0.5 mg DNA
~2500 BACs
Random Prime
Triplicate spots
Cy3 and Cy5
130 mm centers
16 hr Hyb.
864 well plates
12 mm
MPE600 Chromosome 1 Copy Number
Profiles Measured by Chromosome and
Array CGH
2
log2ratio
1.5
1
0.5
0
-0.5
-1
-1.5
-2
0
50000
100000
150000
kb
200000
250000
300000
Assembly of microarrays for genome-wide
measurement of DNA copy number
Snijders et al. Nature Genetics november , 263-264
Overview research topics and techniques
'real time' quantitative PCR using SYBR I Green:
quantification of gene expression
laser 'capture' microdissectie (LCM):
dissectie van cellen uit weefselcoupes
'suppression subtractive hybridisation (SSH):
comparative gene expression analysis for two
cell populations
microarray-analysis:
'high throughput' gene expression analysis
laser 'capture' microdissectie (LCM):
isolation of single cells or small islets of cells
from cytological preparation
laser 'capture' microdissectie (LCM):
laser 'capture' microdissectie (LCM):
laser 'capture' microdissection (LCM):
Lab-on-Chip - Bioanalyser Agilent technologies
for analysis of minute amounts of DNA, RNA or protein
SK-N-BE
SJNB-10
N206
NMB
NGP
LA-N-6
SK-N-AS
NBL-S
SK-N-SH
SJNB-1
SK-N-FI
CLB-GA
CHP 901
TR-14
UHG-NP
LA-N-1
SMS-KAN
SK-N-MC
SJNB-6
IMR-32
SJNB-12
SJNB-8
CHP 134
STA-NB-3
NLF
LA-N-5
STA-NB-10
STA-NB-12
SMS-KCNR
H brain
H fetal brain
H uterus
H mammary gland
H lung
H trachea
H kidney
H liver
H heart
STA-NB-9
NN-1
NB-3
STA-NB-8
GI-ME-N
BCAT1
ASCL1
MYCN
NCL
LAP18
GAP43
NEUROD1
VHL
CHD3
SAM68
KPNA2
HMG2
CDC25B
TBP_run1b
TBP_run1a
TBP_run2
NPY
TMOD
CALM2
RGS4
HAND2
NTRK1
MAB21L1
HPRT1_run1b
HPRT1_run1a
HPRT1_run2
S100A6
TGFBI
MME
UBC_run1a
UBC_run1b
18s_rRNA_run1b
18s_rRNA_run1a
RLP11
DDOST
CDC42
Hs.12842
Hs.120931
NAG
DDX1
PHB
HMBS_run1a
HMBS_run1a
HMBS_run2
RPL13A
RPS25
TNFRSF10B
GAPD_run2
Hs.28462
CCND1
MEIS2
ATBF1-A
ATBF1-B
MEIS1
TCF12
G10D12
DVL2
DKK3
MMP2
Hs.182594
MSTPO32
RGS5
IGF2
G4D5
UBC_run2
ACTB_run2
ACTB_run1a
ACTB_run1b
CD44
PMX1
MYC
LTBP1
HXB
SRPR
INHBA
TGM2
SOS1
YWHAZ_run2
ID2
SDHA_run2
NTRK2
CTNNB1
PIM1
B2M
HOXA9
microarrays
microarras
microarrays
Alizadeh et al. Nature
Sestri Levante 2000