Download POS gs 2007

The Human Genome
23 Pairs of Chromosomes
About 30,000 Genes
FAP (<1%)
MYH
(<1%)
HNPCC
~2-3%
Sporadic
~75%
Familial
(gene(s)) not
known 15-20%
~20-25%
Practical Colorectal Cancer
Genetics
100 new colorectal cancer patients
1 with FAP
2 with HNPCC
17 with FH
of polyps
or cancer
80 with no
FH of polyps
or cancer
Adenomatous Polyposis Coli Gene
(APC)
Exons 1-14
Exon 15
1309
AAPC
AAPC
Profuse Colorectal Polyposis
Severe Desmoids
Wild Type
Truncated Mutant Proteins
19 yo with Colon Cancer
Tumor Testing for
Microsatellite Instability
1
2
3
N T
N T
+
LOH
D17S250
N
T
–
The Canadian Contribution Family C
Hereditary Nonpolyposis
Colorectal Cancer
Germline mutations in mismatch repair genes
MSH2, MLH1, PMS1, PMS2, MSH6
hMSH2
3’
5’
hMLH1
3’
5’
Promoter
Exon
Mutation
hMSH2
3’
5’
hMLH1
3’
5’
Promoter
Exon
DNA Mismatch Repair & MSI Colorectal Cancer
MLH1
MSH2
Cancer
A
Cancer
B
Courtesy: Aaron Pollett
d. 80
3
Colon dx~50
Colon dx ?
d. 64
Bowel dx 55
pancreas,
prostate,
liver
d. 55
Colon dx 55
MSH2
84
Colon dx ?
MSH2
d. 55
52
Endo. dx 31
Rectal dx 34
Cecum dx 51
Transverse colon dx 52
Duodenum dx 53
28
MSH2 Immunohistochemistry
Duodenal Adenoma
Duodenal Cancer
Courtesy: Aaron Pollett
Kids with Colorectal Cancer
Both sides: Afganistan
d.90
CRC dx 60s
3
7
d.62
d. Lung ca
55
d. childbirth
0.06250
7
36
d.12
Duodenal ca dx 11
Mets
CAL spots
d.70
Gastric dx 60s
34
30
d.18-19
d.war
9
6
CRC dx 8
CAF spots
+ 3 dysplastic polyps Plexiform neurofibroma (tongue)
Aux. freckling
ATCG
A
C
C
ATCG
G
C
C
III - 2
III - 1
A
C
C
IV - 2
IV - 1
IV - 3
ATCG
A
C
C
ATCG
ATCG
A
C
C
G
C
C
Familial Adenomatous
Polyposis (FAP) - APC
Gene
Inherited Colorectal
Cancer - It’s ‘Easy’
Hereditary
Nonpolyposis
Colorectal Cancer
(HNPCC) - mismatch
repair genes
What have we learned from Hereditary
Colorectal Cancer Syndromes?
Germline Mutation
Familial Adenomatous
Polyposis (FAP)
Hereditary Nonpolyposis
Colorectal Cancer (HNPCC)
Sporadic Colorectal Cancer
APC gene
Mismatch Repair
Genes
……..
Somatic Mutation
APC gene
Mismatch Repair
Genes
APC gene
Mismatch Repair
Genes
Hypermethylation of MLH1 Promoter as an Epigenetic Cause
of MLH1 Inactivation in
SPORADIC COLORECTAL CANCER
hMLH1
3’
5’
Promoter
Exon
Hypermethylation
MSS vs MSI-H
colorectal
cancer
Survival by
Stage
Hazard Ratio
0.45 (0.30-0.68)
P<0.001
Gryfe et al, NEJM 342:69-77;2000
Adjuvant 5FU and Colon Cancer Survival
N Engl J Med 345(15) 1091-1097, 2001
Chemotherapy and
Mismatch Repair Deficiency
HCT 116
HCT 116 + chromosome 3
Meyers et al; 61:5193, 2001
MULTISITE COLLABORATION
No. Cases
National Cancer Institute of Canada
North Central Cancer Treatment Group
Protocol 784852
Protocol 874651
Gastrointestinal Intergroup, NCI
292
66
34
143
Fondation Francaise de Cancerologie Digestive
Total
35
570
Colorectal Cancer Genetics & 5-FU
Ribic CM NEJM 2003
Hazard Ratio
MSS
MSI
0.69 (0.50-0.94)
p=0.02
2.17 (0.84-5.55)
p=0.10
CPT11
Oxaliplatin
Avastin
Erbitux
?
What about….?
Palliative Rx NEJM 2000
Aduvant Rx NEJM 2004
Palliative Rx NEJM 2004
Genetic Basis of Colorectal Cancers
with Microsatellite Instability
1 MLH1 germline mutation
2 HNPCC
1 MSH2 germline mutation
15 MSI-H
100 CRC
13 Sporadic
85 MSS (1 FAP)
13 MLH1 promoter methylation
Attenuated Polyposis
Family N - Autosomal Recessive Colorectal
Cancer?
multiple polyps multiple polyps
multiple polyps
DNA Base Excision Repair
Slupska et al, J Bacteriol. 178, 3885, 1996
Functionally Important MYH Mutations
tyrosine
Exon 7 (codon 165) …GGGCTACTATT…
cysteine
Exon 7 (codon 165) …GGGCTGCTATT…
glycine
Exon 13 (codon 382) …ctcaGGTCTGC…
aspartic acid
Exon 13 (codon 382)… ctcaGATCTGC…
MYH Associated Polyposis (MAP)
Autosomal Recessive Colorectal Cancer
Y165C
Y165C
G382D
multiple polyps
Y165C
G382D
multiple polyps
G382D
Y165C
G382D
multiple polyps
How did it happen?
Pancreatic cancer: Causes
Environmental factors
• Smoking
• Alcohol
• Coffee
• Diet
• Chemicals
Host factors
• Past medical
history
• Pancreatitis
Genetic predisposition
Familial
Aggregation
of cases
Familial cancer
syndromes
Somatic Mutations in
Pancreatic Cancer
Gene or Region
Frequency of Alteration (% of tumors)
K-ras
>90
p16
>95
p53
50 - 75
DPC4
55
Chromosome 19q/AKT2
10 - 20
Chromosome 6q/MYB
10
Chromosome 20q/AIB1
10
BRCA2
7 – 10
LKB1/STK11
4
MKK4
4
TGF-β R-I or R-II
<5
RB1
<5
Kern S. Molecular genetic alterations in ductal pancreatic adenocarcinomas. Med Clin North Am 2000(84): 691-695.
Familial Cancer Syndromes
Familial Melanoma
(p16)
Familial Breastovarian cancer
syndrome
HNPCC
PANCREATIC
CANCER
(BRCA1, BRCA2)
Peutz-Jeghers, Li-Fraumeni etc.
(less common)
(hMSH2, hMLH1
& other
mismatch repair
genes)
Melanoma + Pancreas Cancer
Breast/Ovarian Cancer + Pancreas Cancer
Lifetime Risk of Pancreas Cancer
General Population
~ 0.2-0.5 %
BRCA2, PJS, HNPCC,
p16, Familial Pancreatitis
~ 5-10 %
Familial Pancreas Cancer
~ 20-30%
High Risk Pancreas Cancer
Screening Program
Who?
BRCA2, p16, Familial Pancreas Cancer, PeutzJeghers, FAP, Hereditary NonPolyposis
Colorectal Cancer, Familial Pancreatitis
What?
Yearly MRI, Ultrasound, Blood
collection/banking
What Joe General Surgeon
should know
BRCA1/2, MMR genes, APC, MYH, RET, p16,
VHL
Keep your eyes and ears open for new ones!
Microsatellite instability/18q LOH in colorectal
cancer
Somatic genetics of Wilms, neuroblastoma
Molecular based therapies - eg Herceptin,
Gleevec
Non-cancer genetic syndromes
FAP (<1%)
MYH
(<1%)
HNPCC
~2-3%
Sporadic
~75%
Familial
(gene(s)) not
known 15-20%
~20-25%
Is it ALL genetic? If it is, how do we figure it out?
Magnitude of effect
Allelic architecture and mapping
strategy
Unlikely to exist
Family-based
linkage studies
Association studies in
populations
Frequency in population
http://pgaedu.net/GtD_Presentations/snp_palmer.ppt
Slide thanks to D.
Altshuler
Common complex genetic diseases
Predisposing mutation
Normal cell
Disease
high penetrance
Cancer
BRCA1, BRCA2, others
APC, MMR genes, others
Mutant
Variant A
low penetrance
Cancer
Common complex genetic diseases
Predisposing mutation
Normal cell
Disease
high penetrance
Cancer
BRCA1, BRCA2, others
APC, MMR genes, others
Mutant
Variant A
SNP Example:
low penetrance
Cancer
Subject 1: GC GCT TAG A TTCCAG
GC GCT TAG A TTCCAG
Subject 2: GC GCT TAG G TTCCAG
GC GCT TAG A TTCCAG
Common complex genetic diseases
Predisposing mutation
Normal cell
Disease
high penetrance
Cancer
BRCA1, BRCA2, others
APC, MMR genes, others
Mutant
Variant A
Mutant
Variant B
low penetrance
Cancer
low penetrance
Cancer
Common complex genetic diseases
Predisposing mutation
Normal cell
Disease
high penetrance
Cancer
BRCA1, BRCA2, others
APC, MMR genes, others
Mutant
Variant A
low penetrance
Cancer
+
Mutant
Variant B
Gene:gene interactions
Common complex genetic diseases
Predisposing mutation
Normal cell
Disease
high penetrance
Cancer
BRCA1, BRCA2, others
APC, MMR genes, others
Mutant
Variant A
low penetrance
Cancer
+
Mutant
Variant B
Gene:environment interactions
Genome Wide Association Studies
Definition
Study of genetic variation across
the genome, designed to identify
genetic associations with
observable traits (eg. blood
pressure), or the presence or
absence of a disease (eg.
colorectal cancer)
Genome Wide Association Studies of
Common Multigenic Diseases
Risk Variants in an individual
Diabetes
Arthritis
Autism
Asthma
Colon Cancer
How many subjects do you need for a powerful GWAS?
Risch, Nature 2000
http://pgaedu.net/GtD_Presentations/snp_palmer.ppt
THE ARCTIC PROJECT
Assessment of Risk of Colo-Rectal Tumors in Canada
GWS Stage 1 Design:
•1200 Cases (Ontario)
•1200 Controls (Ontario)
• 1.4 billion genetic tests
Brent Zanke, Steve Gallinger, Celia
Greenwood, Michele Cotterchio, Tom Hudson
Progress to Date
Genotyping (1200 CRC and 1200 controls)
 1536 SNPs from candidate genes

 10K coding non synonymous SNPs.

Validation
 100K Affymetrix gene chip SNP array. 
 500K Affymetrix gene chip SNP array.  (In Analysis)
Affymetrix Centurion 100K+500K SNP chips
250 ng Genomic DNA
Xba
Xba
Xba
RE Digestion
PCR: One Primer
Amplification
Adaptor
Ligation
Complexity
Reduction
Fragmentation
and Labeling
Hyb & Wash
AA BB AB
Multi-Stage Analysis of ~100,000 SNPs
Stage 1: Ontario
1142 SNPs
1257 cases/1336 controls
99,632 SNPs
Stage 2: Seattle +
Newfoundland
1139 cases/1055 controls
Stage 3: Scotland
975 cases/1002 controls
76 SNPs
9 SNPs
Stage 4: Scotland
1910 cases/1985 controls
1 SNP confirmed
2 SNPs
Validation: EPIC & France
2199 cases/2401 controls
Outcome of Two Best SNPs
OR
95%CI
p-value
rs10505477
Stages 1-4
1.18
1.12-1.25
1.41E-08
French/EPIC
1.16
1.07-1.26
5.05E-04
All cohorts 1-7
1.17
1.12-1.23
3.16E-11
rs719725
Stages 1-4
1.14
1.07-1.20
1.32E-05
French/EPIC
0.98
0.90-1.06
0.61
All cohorts 1-7
1.08
1.01-1.15
0.023
rs10505477 (8q24 locus)
8q24 locus
Affymetrix Illumina