DNA Repair and Cancer Susceptibility:

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DNA Repair and Cancer Susceptibility A
Molecular Epidemiology Approach Qingyi Wei,
M.D., Ph.D. Departments of Epidemiology
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Houston and Texas Medical Center
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Cancer Epidemiology
Risk Factors
Targets
Chronic Inflammation
Lung
Breast
Tobacco
Prostate
Colorectal
Alcohol
Hematologic
Diet
Stomach
Xenobiotics
Ovarian
Familial
Pancreas
Liver
Obesity
Esophagus
Hormones
Skin
Viral
H N
Asbestos
Uterine Cervix
Bladder-Kidney
Ion. Radiation
Brain
UV Radiation
Bone
Adapted from ISPO
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Cancer Epidemiology
Environment
Host
Chronic Inflammation
Lung
Breast
Tobacco
Prostate
Colorectal
Alcohol
Hematologic
Diet
Stomach
Xenobiotics
Ovarian
Gene-Environment Interaction
Familial
Pancreas
Liver
Obesity
Esophagus
Hormones
Skin
Viral
H N
Asbestos
Uterine Cervix
Bladder-Kidney
Ion. Radiation
Brain
UV Radiation
Bone
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Molecular Epidemiology
Biomarkers
Host
Metabolism
Lung
Breast
DNA adducts
Prostate
Colorectal
Mutagen sensitivity
Hematologic
Polymorphisms
Stomach
DNA repair
Ovarian
Gene-Gene Interactions
Cell cycle
Pancreas
Liver
Apoptosis
Esophagus
Methylation
Skin
imprinting
H N
Telomerase
Uterine Cervix
Bladder-Kidney
Angiogenesis
Brain
Genomic instability
Bone
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Molecular Epidemiology

• 2004
• DNA repair
• Cell cycle control
• Apoptosis
• Inflammation
• Viral infection

? seeks to identify human cancer risk and
carcinogenic mechanisms to improve cancer
prevention strategies ? is multi-disciplinary
and translational, going from the bench to
the field and back ? uses biomarkers and
state-of-art technologies to gain mechanistic
information from epidemiological studies
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Promises of Molecular Epidemiology

• Biomarker-based
• Individualized risk assessment
• - phenotypic vs genotypic
• - gene-gene interaction
• - gene-environment interaction
• Individualized early diagnosis
• - susceptibility vs tumor markers
• Individualized treatment / therapies
• - drug metabolic genes
• - cell growth markers

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DNA Damage-Response Pathway

p53 Protein Accumulation
Modified from Harris, 1994
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Biomarker Assays

• Cell culture-based assays
• DNA repair capacity (HCR)
• In vitro mutagen sensitivity
• Induced DNA adducts
• Chromosome instability (FISH)
• Apoptotic capacity
• Telomerase activity

• Gene expression assays
• Multiplex RT-PCR
• Real-time PCR
• Methylation
• Protein microarray

• Genotyping assays
• Metabolic genes (GSTs and folate-related)
• DNA repair genes (BER, NER, BSBR)
• Cell cycle genes
• Apoptosis genes

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Phenotype Analysis

• DNA Repair Capacity (Since 1993)
• UV-light ? skin cancers
• (Wei et al, PNAS, 1993 JNCI, 2003)
• BPDE ? lung cancer breast cancer
• (Wei et al, Cancer Res, 1996 JNCI, 2000)
• (Shi et al, Carcinogenesis, 2004)
• NNK ? lung adenocarcinoma brain tumors
• (to be published)
• 4-ABP ? bladder cancer
• (unpublished)
• PHIP ? prostate cancer
• (unpublished)
• MMC ? breast cancer brain tumors
• (unpublished)

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(No Transcript)
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Specific DNA Repair Pathways
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Human DNA Repair Pathways
Type Genes Damage
Base excision repair DNA ligase
(LIG3), Single base damage repair DNA
glycosylase (MBD4, MPG, MYH, NTH1, OGG1,
SMUG1, TDG, UNG), APE1, APE2,
XRCC1, ADPRT, ADPRTL2, ADPRTL3 ...
Nucleotide excision XPA, XPC, XPE, XPF/ERCC4,
Bulky nucleotide damage
repair XPG/ERCC5, ERCC1, LIG1, including UV
photoproducts CSB/ERCC6,CSA/CKN1, XAB2, and
chemical TFIIH (XPB/ERCC3 XPD/ERCC2,
carcinogen-induced adducts GTF2H1, GTF2H2\1,
GTF2H3, GTF2H4, CDK7, CCNH, MNAT), DDB1,
DDB2, MMS19, CENN2, AD23A, RAD23B, RPA1,
RPA2, RPA3 ...
Mismatch repair MSH2, MHS3, MSH6,
MSH4, Base mismatch MSH5, MLH1, MLH3, PMS1,
PMS2, PMS2L3, PMS2L4 ...
Recombinational RAD50, RAD51, RAD51B,
RAD51C, Double strand breaks
repair RAD51D, RAD54L, RAD54B,
V(D)J recombination
RAD52, DMC1, MRE11A, NBS1, ERCC1,
XPF/ERCC4, XRCC2 XRCC3, XRCC4,
XRCC5, XRCC6 XRCC7, XRCC8, BRCA1,
BRCA2 ...
Wood et al, Science, 2001
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DNA Repair Genes and Cancer
Gene Cancer
XP(A) Skin XP(B) Skin XP(C) Ski
n XP(D) Skin XP(E-G) Skin
hMLH1 Colon hPSM1/2 Colon hMSH2 Colon
hMSH3 Colon hMSH6 Colon pRB
Retinoblastoma P53, hCHK2
Li-Fraumeni Syndrome P16 Melanoma ATM
Breast BRCA1/2 Breast
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Age of Onset of Skin Cancer in Normal and XP Skin
Cancer Patients
Skin cancers in XP population
Skin cancers in normal population
XP xeroderma pigmentosum
Kraemer, PNAS, 1997
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Theoretical Distribution of Relative DNA Repair
Capacity
Normal
Super
Deficient
General Population
XP
Relative DNA Repair Capacity
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Selection of Biological Materials for Biomarker
Assays

• Target tissue
• Specificity exposure/etiology
• Biological relevance tumor development
• No easy access/invasive
• Somatic changes/effect of exposure
• Costly

• Surrogate tissue/lymphocoytes
• Easy access/non-invasive
• Immortalization
• Affordable
• Good for genetic susceptibility biomarker
• Mass screening

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Study Design

• Cases patients with newly diagnosed,
  histopathologically confirmed incident cancer at
  MDACC no previous radiotherapy or chemotherapy,
  and no previous cancer history and blood
  transfusion
• Controls hospital-based, cancer-free subjects
  recruited from managed-care organization,
  hospital visitors unrelated to the cases,
  frequency matched on age, sex, and ethnicity
• Informed consent, questionnaire, and blood
  drawing
• Statistical analysis Student t test, Chi-square
  test and multivariate logistic regression

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Blood Sample Processing and Biomarker Assay Flow
Chart
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Example 1
Hypothesis
UV light Skin
cancers
Suboptimal DNA Repair ?
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DNA Repair and Skin Cancer

• Non-melanoma skin cancer
• (NMSK, including BCC and SCC)
• over one million new NMSK cases in 2004 and
  95curable
• Suggested risk factors
• UV radiation
• Other ionizing radiation
• Chemical exposure
• Deficient DNA repair
• Dose of UV-exposure

• Melanoma
• 55,100 melanoma cases and 7,910 deaths in 2004
• Suggested risk factors
• Family history
• UV radiation ?
• Other ionizing radiation
• Deficient DNA repair ?
• Dose of UV-exposure ?

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Sunlight Exposure and Incidence Rate of Skin
Cancer
SCC gt
BCC gt
Melanoma
SCC
Incidence Rate
BCC
Melanoma
Sunlight Exposure
Adapted from Armstrong Kricker, 2001
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UV-Induced Photoproducts and Repair
UV
UV
UV Photoproducts
Nucleotide Excision Repair
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Recombinant Plasmid DNA Expression Vectors Used
in the Host-Cell Reactivation Assay

UV
UV
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Transfection Efficiency in the Host-Cell
Reactivation Assay

Cheng et al., BioTechniques, 1997
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DNA repair capacity dose-response curve by
lymphoblastoid cells
DRC ()
UV Dose to Plasmids (J/M2)
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Theoretical Distribution of DRC in the General
Population
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Theoretical Distribution of DRC in the General
Population
Normal
Cancer
Median
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Selected Risk Factors for SC
Variable Odd Ratio (95 CI) BCC
SCC CMM (N185) (N135)
(N312)
Skin color Dark 1.00 1.00 1.00
Brown or fair 2.01 (1.30-3.27) 1.94
(1.05-3.58) 2.18 (1.53-3.10) Tanning ability
Good 1.00 1.00 1.00 Poor
1.48 (0.91-2.40) 2.63 (1.38-4.98) 1.96
(1.40-2.73) Lifetime sunburns None
1.00 1.00 1.00 gt1 1.08
(0.68-1.70) 2.35 (1.24-4.49) 2.13 (1.51-3.00)


Adjusted for age and sex, compared with 324
controls, unpublished data
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Risk of Skin Cancer Associated with Sun Light
Exposure and Suboptimal DRC
Melanoma
SCC
Incidence Rate
BCC
BCC
Melanoma
SCC
High Low
Sunlight Exposure
DRC ()
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Revised Hypothesis
UV exposure ?
SCC
DNA Damage Mutations
BCC
Melanoma
Suboptimal DNA Repair ?
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Example 2
Hypothesis
Lung and HN cancers
Smoking
Suboptimal DNA Repair ? Genetic variants of NER
genes?
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Shared Cancer Deaths
Perinatal factors growth 5
Others 7
Immunity 5
Tobacco 30
Genetic factors 5
Adult diet Obesity 30
Alcohol 5
Radiation 3
Viral Infection 5
Sedentary lifestyle 5
Harvard School of Public Health, 1998
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Smoking kill different populations in different
ways
China
USA
Peto J, Nature, 2001
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Smoking and Cancer
Snapshot No. (in
millions) US population 200.0
Smokers 46.5 All cancer cases
1.3 Lung cancer 0.5 Head and
neck cancer 0.04
Genetic susceptibility plays a role !
ACS, 2004
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Nucleotide Excision Repair of Tobacco-Induced
DNA Damage
ERCC1 XPA XPB/ERCC3 XPC XPD/ERCC2 XPE/DDB1/2 XPE/
ERCC4 XPG/ERCC5
NER Core Proteins
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Recombinant Plasmid DNA Expression Vectors Used
in the Host-Cell Reactivation Assay

BPDE
BPDE
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Risk of Lung Cancer Associated with Low DRC
HCR Assay (plasmid DNA)
Cases 316 Controls 316
Trend test P lt 0.001
Odds Ratio
Wei et al., JNCI, 2000
In Vitro Adduct Assay (genomic DNA)
Cases 221 Controls 229
Trend test P lt 0.001
High
Low
DRC () by Quartile
Li et al, Cancer Res., 2001
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Studies on DRC and Cancer
Cancer Case/Control OR
(95CI) Reference
Skin BCC 88/123 2.3 (1.2-4.5) Wei et al,
1993 BCC 76/87 1.1 (0.9-1.2) Hall et
al, 1994 BCC 49/68 1.2 (0.5-2.7)
DErrico et al, 1999 SCC 25/57 0.9 (0.8-1.2)
Hall et al, 1994
SCC/BCC 280/177 3.8 (2.3-5.7) Matta et al,
2002 Melanoma 132/145 1.0 (0.6-1.7)
Landi et al, 2002 312/324 2.0 (1.5-2.8)
Wei et al, 2002 Lung 51/56 5.7 (2.1-15.7)
Wei et al, 1996 467/488 1.9 (1.4-2.4)
Shen et al, 2003 Head Neck 55/61 2.2
(1.0-4.8) Cheng et al, 1998 228/244 2.6
(1.6-4.2) Unpublished Prostate 140/96 2.1
(1.2-3.9) Hu et al, 2004 Breast
69/79 3.4 (1.2-9.8) Shi et al, 2004
39/37 ??? Ramos et al., 2004
For the low tertile DRC and others for the
low-median DRC Recalculated based on published
data
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Factors that have an effect on DRC

• Environmental
• Therapeutic
• Genetic

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Research Question
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Exposure
Cancer
DNA Repair Phenotype?
mRNA expression?
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Multiplex RT-PCR Assays
NER Gene Panel (ERCC1, ERCC3, ERCC5, ERCC6, XPC)
MMR Gene Panel (hMSH3, hMSH2, hPMS2, hMSH6,
hMLH1, hPMS2)
Cheng et al., CEBP, 1998
Wei et al., Pathobiology, 1997
HRR Gene Panel (XRCC7, XRCC6, XRCC1, ATM, RAD51,
XRCC4)
Cell Cycle Gene Panel (p21, PCNA, p27, pRB, bcl2)
Shen et al., Mol Carcinogenesis, 2000
Zou et al., Cancer Lett, in press
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Real-Time PCR for Gene Expression
Liu et al., Mol Carcinogenesis, 2003
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Low DNA Repair Gene Expression and Risk of SCCHN
Phenotype (mRNA) (MMR genes) hMLH1 hMSH2 hGTBP/hM
SH6 hPMS1 hPMS2 (NER genes) ERCC1 XPB/ERCC3 XPG/
ERCC5 CSB/ERCC6 XPC
Cases 78 Controls 86
9.06
Wei Q et al., CEBP, 1998
Cases 57 Controls 105
15.7
7.90
0.0 1.0 2.0 3.0
4.0 5.0 6.0
7.0
Odds Ratio
Cheng et al, Cancer, 2002
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Low DNA Repair Gene Expression and Risk of
Cancers
Phenotype (mRNA) (MMR genes) hMLH1 hMSH2 hGTBP/hM
SH6 hPMS1 hPMS2 (NER genes) ERCC1 XPB/ERCC3 XPG/
ERCC5 CSB/ERCC6 XPC
Colon Cancer
Cases 31 Controls 47
7.65
7.76
Soliman et al., IJO, 1998
Lung Cancer
Cases 79 Controls 95
Cheng et al, Carcino, 2000
0.0 1.0 2.0 3.0
4.0 5.0 6.0
7.0
Odds Ratio
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Research Question
?
Exposure
Cancer
DNA Repair Phenotype?
Protein expression?
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Reversed Protein Array
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NER Repair Protein Expression and Risk of SCCHN
Relative expression ERCC1 XPA XPC XPD/ERCC2 XPF/E
RCC4 XPG/ERCC5 (compared to beta-actin)
Cases 57 Controls 63
0.0 1.0 2.0 3.0
4.0 5.0 6.0
7.0
Odds Ratio
Unpublished data
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Research Question
?
Exposure
Cancer
DNA Repair Phenotype?
DNA Repair Genotypes?
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Human Genome Project
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Electropherograms produced by fluorescence-based
sequencing using an ABI 3700
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Genotyping Assays
XPD/ERCC2 Exon 10 (G23591A Sty I )
XPD/ERCC2 Exon 6 (C22541A Tfi I )
AA CA CC CA CA CA
GG GA
GA AA
XPC Poly(AT) Polymorphism
XPD/ERCC2 Exon 23 (A35931CPst I )
PAT- PAT
CC CC AC AC CC AA AA
CCND1
A Allele G Allele
Genotype AA GG GA AA GG
GG GA GA AA GA
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Correlation between DRC Phenotype (UV-damage)
and Genotype in Cancer-free Subjects
N 102 P 0.02
None Hetero Homozygous
Three Variant Genotype of XPD and XPC Genes
Qiao et al., Mutat Res, 2003
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DNA Repair Phenotype/Genotypes and Risk of SCCHN
Phenotype Marker (reference) DRC Induced BPDE
adducts ERCC1 RNA XPB/ERCC3 RNA XPG/ERCC5
RNA CSB/ERCC6 RNA XPC RNA
Variant genotype (reference) XPD 22541 CA/AA XRD
312DN/NN XRD 751LQ/QQ XPC PAT-//PAT/ ERCC1
8092 CC
0.0 1.0 2.0 3.0
4.0 5.0 6.0
7.0
Odds Ratio
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?
Exposure
Cancer
DNA Repair Phenotype?
Cell Cycle Control?
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Dysfunctional Signaling Pathways in Cancer
Exposure
Suppressor/Oncogene Network
Growth Factor Receptor
Phase I / II (exonibiotics seonsors)
p85
Nuclear membrane
PI3K
Damage sensors
p110
Cell membrane
Akt
PTEN
ARF
ATM/ATR
P16
Rb Pathway
cAbl
CDK4/6
CHk1
NBS1
CHk2
MDM2
BRCA1
P53/p73
cdc25
Rb
p21
CDK
p27
E2F
Apoptosis
DNA Repair
Cell cycle Arrest by DNA damage Checkpoints
Transcription
G0
G1/S Phase transition
DNA Damage Response Pathway
G1
S
G2
Modified from The Scientist, 09/03
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SNPS of Cell Cycle Control Genes and Cancer Risk
of SCCHN
Gene Comparison
Cases/Controls Risk
HN Cancer 703/1085 p21 Ex2C98A
CC vs CAAA 1.3 (1.0-1.6) Ex3C70T
CC vs CTTT 1.4 (1.1-1.8) p27
EX1T150G TT vs GG 1.2
(0.8-1.8) p73 G4C14/A4T14 GC/GC vs
GC/ATAT/AT 1.3 (1.1-1.7) Lung
Cancer 1054/1139 p73 G4C14/A4T14
GC/GC vs GC/AT 1.3 (1.1-1.6) GC/GC
vs AT/AT 1.5 (1.1-2.3)
(Unpublished data)
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Genes involved in folate metabolism
Folate Metabolism Pathway
Modified from Ulrich CM, CEBP, 2000
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Functional Polymorphisms in Folate-Related
Metabolic Genes
Gene Polymorphism
Effect on tHcy

• MTHFR 677C?T (Ala222Val)
• TS/TYMS 2R?3R in enhancer
• MS/MTR 2756G?A (Gly919Asp)
• MTRR 66A?G (Ile22Met)
• SHMT1 1420T?C in TIS
• CBS 844ins68 (insertion)

tHcy total plasma homocysteine
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SNPS of Folate-Related Genes and Risk of SCCHN
Gene Comparison
Cases/Controls Risk
MTHFR 537/545 C667T CC vs CTTT
1.1 (0.9-1.4) A1298C CC vs AAAC
3.1 (1.5-6.5) G1793A GG vs GAAA
1.3 (0.8-2.1) TS/TYMS 704/1085 TS5ER
3R3R vs 3R2R/2R2R 1.2 (0.9-1.4) TS3UTR
6bp/6bp vs
6bp/0bp0bp/0bp 1.0 (0.8-1.3) MS/MTR A
2756G AA vs AGGG 1.3 (1.1-1.6) MTRR A66G
AA vs AGGG 1.5 (1.2-1.9) SHMT1
721/1234 C34761T CC vs CTTT 1.0
(0.9-1.3) C34840G CC vs CGGG 1.0
(0.9-1.2) C34859T CC vs CTTT 1.1
(0.9-1.3)
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Conclusions

• Suboptimal DRC is a risk factor for developing
  sunlight-induced skin cancer and tobacco-induced
  cancer such as lung and head and neck cancers

• Some genotypes of DNA repair or related genes are
  also risk factors for these cancers, but their
  correlation with DRC phenotype warrant further
  investigations

• Larger studies are needed to evaluate gene-gene
  and gene-environment interactions

• High-throughput genotyping/DNA repair phenotying
  should be integrated into risk assessment model

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Acknowledgments

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Acknowledgments

• Recruitment of subjects
• Administering questionnaires
• Processing of blood samples
• Extraction of DNA
• Cell culture
• Phenotyping/DRC
• Genotyping
• Data entry
• Data analysis
• Manuscripts