digital snp analysis of cancers -...
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Digital SNP Analysis of Cancers
Hsueh-Wei Chang1 張學偉
Email: [email protected]://genomed.dlearn.kmu.edu.tw
1. Department of Biomedical Science and Environmental Biology, Kaohsiung Medical University
SNP = Single Nucleotide Polymorphism
(read in SNiP)
SNPs are an abundant form of common genome variation, distinguished from rare variations by a requirement for the least abundant allele to have a frequency of 1% or more.
4 states of a SNP are possible (A,T,C,G)
TCATCTGTGGGAAGGGAGTCCCTGGCTAATACTGCCCCCACCACAGCTCCTCATCTGTGGGAAGGGAGTCCCTGGCTTATACTGCCCCCACCACAGCTCCTCATCTGTGGGAAGGGAGTCCCTGGCTCATACTGCCCCCACCACAGCTCCTCATCTGTGGGAAGGGAGTCCCTGGCTGATACTGCCCCCACCACAGCTCCTCATCTGTGGGAAGGGAGTCCCTGGCTAATACTGCCCCCACCACAGCTCCTCATCTGTGGGAAGGGAGTCCCTGGCTTATACTGCCCCCACCACAGCTCCTCATCTGTGGGAAGGGAGTCCCTGGCTCATACTGCCCCCACCACAGCTCCTCATCTGTGGGAAGGGAGTCCCTGGCTGATACTGCCCCCACCACAGCTCCTCATCTGTGGGAAGGGAGTCCCTGGCTCATACTGCCCCCACCACAGCTCCTCATCTGTGGGAAGGGAGTCCCTGGCTCATACTGCCCCCACCACAGCTCCTCATCTGTGGGAAGGGAGTCCCTGGCTTATACTGCCCCCACCACAGCTCCTCATCTGTGGGAAGGGAGTCCCTGGCTCATACTGCCCCCACCACAGCTCCTCATCTGTGGGAAGGGAGTCCCTGGCTTATACTGCCCCCACCACAGCTCCTCATCTGTGGGAAGGGAGTCCCTGGCTCATACTGCCCCCACCACAGCTCCTCATCTGTGGGAAGGGAGTCCCTGGCTCATACTGCCCCCACCACAGCTCCTCATCTGTGGGAAGGGAGTCCCTGGCTTATACTGCCCCCACCACAGCTCC
diffe
rent
alle
les
TCATCTGTGGGAAGGGAGTCCCTGGCTTATACTGCCCCCACCACAGCTCCTCATCTGTGGGAAGGGAGTCCCTGGCTTATACTGCCCCCACCACAGCTCCTCATCTGTGGGAAGGGAGTCCCTGGCTCATACTGCCCCCACCACAGCTCCTCATCTGTGGGAAGGGAGTCCCTGGCTTATACTGCCCCCACCACAGCTCCTCATCTGTGGGAAGGGAGTCCCTGGCTTATACTGCCCCCACCACAGCTCCTCATCTGTGGGAAGGGAGTCCCTGGCTCATACTGCCCCCACCACAGCTCCTCATCTGTGGGAAGGGAGTCCCTGGCTTATACTGCCCCCACCACAGCTCCTCATCTGTGGGAAGGGAGTCCCTGGCTCATACTGCCCCCACCACAGCTCCTCATCTGTGGGAAGGGAGTCCCTGGCTCATACTGCCCCCACCACAGCTCCTCATCTGTGGGAAGGGAGTCCCTGGCTCATACTGCCCCCACCACAGCTCCTCATCTGTGGGAAGGGAGTCCCTGGCTTATACTGCCCCCACCACAGCTCCTCATCTGTGGGAAGGGAGTCCCTGGCTCATACTGCCCCCACCACAGCTCCTCATCTGTGGGAAGGGAGTCCCTGGCTTATACTGCCCCCACCACAGCTCCTCATCTGTGGGAAGGGAGTCCCTGGCTCATACTGCCCCCACCACAGCTCCTCATCTGTGGGAAGGGAGTCCCTGGCTCATACTGCCCCCACCACAGCTCCTCATCTGTGGGAAGGGAGTCCCTGGCTTATACTGCCCCCACCACAGCTCC
diffe
rent
alle
les
But the diallelic type is the most common SNP (e.g. T->C)
SNPs will be evenly distributed across the human genome
~ 25,000 non-synonymous cSNPs
~ 50,000 synonymous cSNPs
~ 25,000 regulatory region SNPs
~ 50,000 intragenic non-coding SNPs
~ 50,000 distributed intergenic SNPs
SNP Analysis
Linkage & Association Analysis
PharmacogenomicsDiagnosis
Personalized Medicine
Genetic Instability
Genetic instability &Microsatellite markers
Figure 9.8
Primers
Microsatellite markerParents
heterozygous
Parentsdelete
Normal Delete
Primer
Microsatellite marker
Parentsdelete Primers
destroy
Normal Delete
Microsatellite marker
Lose of heterozygosity(LOH)
Microsatellite marker needs the long template (~200 to 350 bp)
Intact DNA
Degraded DNA No PCR[degrade partly]
false-positive for LOH
Normal Cancer Mixed
Oral Oncology 36 (2000) 484-490
DNA released from non-neoplastic cells, canmask allelic imbalance (AI) because it is difficult to quantify the allelic ratio usingmicrosatellite markers.
Jump from Analogue
Microsatellite marker
SNP marker
to Digital
Principle of Digital SNP Analysis
Why called it “ Digital ”
Microsatellite markers occur less frequent than SNP
Molecular Beacons : Structure
Sanjay Tyagi and Fred Kramer (1996) Nature Biotechnology 14: 303-308
fluorophore quencher
SNP-specific Molecular Beacon
Ch SNPs Forward Primer Reverse Primer Molecular Beacon-green (flouorescein) Molecular Beacon-red (Hex)1p 8118 CAGGGCAAGACGCTGTGGT AACAGAATGTGCTTCCCTCCC CACGCTGCCCAGCGCACGGCCGTG CACGCTGCCCAGTGCACGGCCGTG7q 273 AGGGCTAGAGTATGAGAAGTCC GTAATTTAGGTGAGCTATCCAGAG CACGGTTTTTTTTCCCTATAACGTG CACGGTTTTTTTTTCCTATAACGTG8p 1085 CACTGAATGCTCTGCCATGA AACCTGTCCTTGTGGGTGAT CACGATGAGCCACAAGCAGCACGTG CACGATGAGCCGCAAGCAGCCGTG12p 852 TGATCTGCTTCTCCCACGA TGGAGTCCCAGACATTGCA CACGATCACGTCCGTGGCCTTCCGTG CACGATCACGTCTGTGGCCTTCCGTG15q 1861 ACAGCCATTTATTATGTTTACTTGG AGAATAATTGTGATAAGAATTCCCC CACGAGCCAACACGGAGGTGACGTG CACGAGCCAACATGGAGGTGACGTG17p p53-e4 AAGACCCAGGTCCAGATGA GGTGTAGGAGCTGCTGGTG CACGGCTCCCCCCGTGGCCCGTG CACGGCTCCCCGCGTGGCCCGTG18q 1468 AGCGAGCATCAGAATCACCT CGGGACAAGCAGCATCT CACGTGGGGCTTACAAATTAGTATCGTG CACGTGGGGCTTACGAATTAGTATCGTG
Ch: chromosomal arm; SNPs: single nucleotide polymorphisms
Primers and probes used for digital SNP analysis
DNA diluted to 384-well plate
DNA templates become all-or-nonein each well of 384-well plate
PCR / hybrid with MB
Digital PCR pattern all-or-none
PNAS February 27, 2001 vol. 98 no. 5 p.2640–2645
1 2 3 4 5 6 7 8 9 10 11 12 Average Sample
10.9 13.0 0.9 14.6 14.8 22.3 1.3 6.7 11.1 5.6
1.2 1.0 18.5 1.3 8.4 0.3 1.6 10.0 0.4 0.7 16.2 11.6 49.2 10.1 11.7 0.8 10.2 0.5 1.6 1.2 1.5 0.9
1.7 9.5 14.6 12.8 11.29.5 11.2 0.9 0.9 14.8 13.7
6.2 0.4 0.7 1.4 1.1 1.2 18.7
10.2 1.0 1.8 1.7 0.8 11.4 1.1 0.7 0.6 0.8
1.1 11.1 0.8 1.2 10.5 12.6 1.1 1.1 11.4 13.2 1.6
4.5 15.2 11.0 9.3 11.2 0.69.6 2.7 30.9 9.90.9 10.8
11.9 11.9 0.7 10.5 12.4 13.7 10.8 13.2 11.8 8.7 14.0 0.4 11.0 0.9 10.9 6.1 0.8 10.4 9.5 19.3 1.1 15.0 11.2 8.0
1.0 10.2 14.0 9.8 15.2 13.2 9.9 11.1 12.6 9.1 8.8 11.8 10.5 13.7 17.5 11.8 Allel G
1.0 1.2 1.3 1.3 1.1 1.1 0.4 1.3 0.9 0.8 1.0 Allel A 0 H2O
6.9
9.2
Normal
Adenoma
Normal Tumor
G:R = 32:34allelic balance
G:R = 62:16allelic imbalance
Assess genetic abnormality by counting alleles
40%
50%
60%
70%
80%
90%
100%
0 50 100 150 200
Allele Count
Alle
le R
atio
Allelic Imbalance
Allelic Balance
Tumor DNA
Normal DNA
Sequencial probability ratio test (SPRT)determines allelic imbalance
The paternal or maternal alleles within a DNA sample are individually counted, thus allowing a quantitative measure of allelic imbalance in the presence of normal DNA.
The advantages of Digital SNP analysis
in assessing allelic imbalance
1. As compared with microsatellite markers the PCR products derivedfrom the two SNP alleles at every locus are of the same size and therefore their analysis is not biased by the preferential DNA degradation of larger alleles.
2. The Digital SNP approach, which amplifies single allele templates in the PCR reaction, can precisely determine the number of allelesexamined in each experiment.
Accordingly, SNP genotyping is “digital” involving the detection or absence of a specific allele rather than “analogue” as is microsatellite genotyping that measures the length of microsatellites.
3. A statistical method, SPRT, can be employed to conclude whether allelic imbalance is present in the background DNA.
Indeed, it has been shown that allelic imbalance can be demonstrated inmuch higher percentage in colorectal carcinomas using Digital SNP analysis than the traditional methods using microsatellite markers.
[Science. 244: 207-11, 1989. & Nat Biotechnol. 19: 78-81., 2001.]
Digital PCR transforms the exponential, analog signals obtained from conventional PCR to linear, digital signals, allowing statistical analysisof the PCR product.
Digital SNP analysis is able to discriminate different alleles (e.g., wild type vs. mutant or paternal vs. maternal alleles).
Digital PCR has been applied in detection of allelic imbalance in clinical specimens
providing a promising molecular diagnostic tool for cancer detection.
Summary for Digital SNP analysis
Digital SNP analysis for Ovarian Cancer
J National Cancer Institute 94 (22), 1697-1703. (2002)
Allelic Imbalance (AI)
• represents losses or gains of defined chromosomal regions
• is useful in elucidating the molecular basis of cancer as well as cancer detection.
Two problems for Traditional Methods1. plasma DNA is a mixture of neoplastic and non-neoplastic
DNA
2. plasma DNA is often degraded to a variable extent
Pla
sma
DN
A (n
g/m
l)
0
500
1000
1500
2000
2500
600065007000
n = 380% + n = 26
7.7% +
n = 7229.2% +
n = 2433.3% +
n = 9047.8% +
Normal
Non-tu
mor dis
ease
Benign
solid
tumor
Leuk
emia
Malign
ant s
olid t
umor
Plasma DNA concentrations in different physiological situations
Diagnosis Patients (n) n > 50 ng/ml % > 50 ng/ml
Healthy normal 44 0 0%Non-neoplastic Dx 164 33 20% Anemia 8 2 25%
AIDS 10 4 40%
Musculoskeletal Dx 10 3 30%
Diabetes Mellitus 7 1 14%
Cardiovascular Dx 13 2 15%
Asthma and pulmonary Dx 5 1 20%
Essential hypertension 6 1 17%
Infectious Dx 19 5 26%
Autoimmune Dx 10 4 40%
Status post transplant 23 3 13%
Liver Dx 9 1 11%
Trauma 8 0 0%
Neurological disorder 12 2 17%
Gynecological Dx 10 1 10%
Drug abuse 8 2 25%
Others 8 1 13%
Neoplastic Dx 122 64 52% Ovarian tumor 54 30 56%
Endometrium/cervix CA 10 5 50%
Head & Neck CA 11 8 73%
Sarcoma 4 1 25%
Breast CA 8 4 50%
Lung CA 11 6 55%
Gastrointenstinal CA 11 5 45%
Brain tumor 5 2 40%
Others 6 3 50%
CA: carcinoma; Dx: disease
Plasma DNA concentration in healthy normal individuals and patients.
Ch SNPs Forward Primer Reverse Primer Molecular Beacon-green (flouorescein) Molecular Beacon-red (Hex)1p 8118 CAGGGCAAGACGCTGTGGT AACAGAATGTGCTTCCCTCCC CACGCTGCCCAGCGCACGGCCGTG CACGCTGCCCAGTGCACGGCCGTG7q 273 AGGGCTAGAGTATGAGAAGTCC GTAATTTAGGTGAGCTATCCAGAG CACGGTTTTTTTTCCCTATAACGTG CACGGTTTTTTTTTCCTATAACGTG8p 1085 CACTGAATGCTCTGCCATGA AACCTGTCCTTGTGGGTGAT CACGATGAGCCACAAGCAGCACGTG CACGATGAGCCGCAAGCAGCCGTG12p 852 TGATCTGCTTCTCCCACGA TGGAGTCCCAGACATTGCA CACGATCACGTCCGTGGCCTTCCGTG CACGATCACGTCTGTGGCCTTCCGTG15q 1861 ACAGCCATTTATTATGTTTACTTGG AGAATAATTGTGATAAGAATTCCCC CACGAGCCAACACGGAGGTGACGTG CACGAGCCAACATGGAGGTGACGTG17p p53-e4 AAGACCCAGGTCCAGATGA GGTGTAGGAGCTGCTGGTG CACGGCTCCCCCCGTGGCCCGTG CACGGCTCCCCGCGTGGCCCGTG18q 1468 AGCGAGCATCAGAATCACCT CGGGACAAGCAGCATCT CACGTGGGGCTTACAAATTAGTATCGTG CACGTGGGGCTTACGAATTAGTATCGTG
Ch: chromosomal arm; SNPs: single nucleotide polymorphisms
Primers and probes used for digital SNP analysis
40%
50%
60%
70%
80%
90%
100%
0 50 100 150 200
Allele Count
Alle
le R
atio
Allelic Imbalance
Allelic Balance
Plasma DNA
Lymphocyte DNA
SPRT analysis
Allelic status in plasma DNA of ovarian cancer patients
Allelic balanceAllelic imbalance Homozygous (non-informative)
Undetermined
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27
1 1 1 1 1 1 1 1 1 1 1 1 2 2 2 3 3 3 3 3 3 3 3 3 3 3 3
1p 8118
5q 1756
7p 273
8p 1085
12q 852
15q 1861
17p p53
18p 331
18
266 65
199 38 3
1309
264 15 8 17
186 23 42 61 92
210 26 6 10 94
746 36 28
131 8
110
0.3
N/A
N/A
0.5
0.3
0.4
0.3
0.4
0.3
0.3
0.5
0.3
0.4
N/A
N/A
0.7
0.7
0.4
N/A
0.5
0.3
0.7
0.3
0.3
0.3
0.3
0.9
X
167 X 18 25 X
580 11 19 15 46 28
115 8 X
1260 76 36 X
220
875 X 9 30
270 16
3185
28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54
3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 4 4 4
1p 8118
5q 1756
7p 273
8p 1085
12q 852
15q 1861
17p p53
18p 331
2
146 38 3 67
149 5
338
491
103 92
147 65
285 20
538 39 25 40 14 78 35
895
2481 18
504
1190
0.4
0.4
0.4
N/A
0.5
0.7
0.4
0.6
0.4
0.6
0.6
0.7
0.7
0.7
0.4
0.9
0.3
0.5
0.3
0.3
0.4
0.6
0.7
0.9
0.4
0.7
0.7
8 84 X 29
600 75 15
250
377 66
979 13
979
209 19
231
643
319
788 X X
580
365 66 38
2397
304
Fraction of tumor DNA
CA125 (U/ml)
Stage
Chrom
osome
SN
P
Plasma DNA (ng/ml)
Plasma DNA (ng/ml)
Fraction of tumor DNA
CA125 (U/ml)
Patient No.
Patient No.
Stage
Chrom
osome
SN
P
An ROC curve (Receiver-operating characteristic) is a graphic representation of the sensitivity plotted against the false-positive rate (i.e., 1 minus specificity).
The area under the ROC curve is a measure of the overall ability of a diagnostic test with multiple cutoffs to distinguish between diseased and control individuals.
Conclusion
These findings suggest that measurement ofplasma DNA levels may not be sensitive or specific for cancer diagnosis but detection of allelic imbalance in plasma DNA using the Digital SNP analysis holds a great promise for the early detection of cancer.
mesothelial cells & inflammatory cells
ovarian serous carcinoma cell
40%
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100%
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60%
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100%
0 50 100 150 200
T1
T2
T3
T4
T5
T6
T7
T8
T9
T10
T11
T12
T13
T14
T15
T16
T17
T18
T19
T20
Tumor
40%
60%
80%
100%
0 50 100 150 200
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60%
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100%
0 50 100 150 200
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60%
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100%
0 50 100 150 200
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60%
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100%
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60%
80%
100%
0 50 100 150 200
40%
60%
80%
100%
0 50 100 150 200
4 0 %
6 0 %
8 0 %
1 0 0 %
0 5 0 1 0 0 1 5 0 2 0 0
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60%
80%
100%
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80%
100%
0 50 100 150 200
4 0 %
6 0 %
8 0 %
1 0 0 %
0 5 0 1 0 0 1 5 0 2 0 0
40%
60%
80%
100%
0 50 100 150 200
N1
N2
N3
N4
N5
N6
N7
N8
N9
N10
N11
N12
N13
N14
N15
N16
N17
N18
N19
N20
Normal
8118 486 273 2952 331 852 1085 8118 486 273 2952 331 852 1085
T1 OVCA 45 1388 N1 NFT 49 10
T2 OVCA 55 127 N2 NFT 48 168
T3 OVCA 71 240 N3 NFT 58 2
T4 OVCA 73 606 N4 NFT 73 8
T5 OVCA 44 31 N5 NFT 55 22
T6 OVCA 60 405 N6 NFT 41 2
T7 OVCA 53 42 N7 NFT 62 15
T8 OVCA 36 1430 N8 NFT 44 9
T9 OVCA 45 550 N9 NFT 62 16
T10 Colon CA 69 2711 N10 NFT 41 25
T11 OVCA 48 180 N11 NFT 75 19
T12 OVCA 51 58 N12 NFT 38 293
T13 Pancrease CA 53 37 N13 NFT 53 7
T14 OVCA 53 399 N14 NFT 44 4
T15 Colon CA 46 289 N15 NFT 34 1141
T16 OVCA 53 880 N16 Ovarian cyst 45 10
T17 OVCA 74 5774 N17 Ovarian cyst 65 6
T18 OVCA 59 40 N18 NFT 63 208
T19 Pancrease CA 63 1195 N19 NFT 32 49
T20 Pancrease CA 56 26 N20 NFT 37 16
SNP markersCase No. Diagnosis Age DNA conc. Age DNA conc.
SNP markers
OVCA: ovarian carcinoma; CA: carcinoma; NFT: negative for tumor; Conc: concentration; NI: not informative;
Case No. Diagnosis
Tumor Normal
Allelic balanceAllelic imbalance Homozygous (non-informative)
Undetermined
negative-cytology
positive allelic imbalance in ascitic fluid who was known to have stage III ovarian carcinoma at the time of sample collection
Am J Pathol 2002, 160:1223–1228
S B T (Serous borderline tumor)
1p5q8p
18q 22q
Xp
1p5q8p
18q 22q
Xp
1p5q8p
18q 22q
Xp
Conventional serous carcinoma
Invasive MPSC
Noninvasive MPSC
Paraffin tissue DNA
Allelic balanceAllelic imbalanceHomozygous (non-informative)
1p5q8p
18q 22q
Xp
Primer dimer
PCR product
Urine DNA for PCR (Work in taiwan)
Conclusion I
Digital SNP analysis can count the allelic imbalance in samples from plasma, ascites, paraffin, urine …etc.
in the presence of background (normal) DNA.
Digital SNP analysis for Bladder Cancer
FP-TDI stands for template directed dye terminator incorporation assay with detection by fluorescence polarization
FP–TDI Assay
Validate the Taiwanese SNP of interests
by
Body fluid-based Digital SNP analysis
Adapted from Genome Research
9:492-498, 1999
Adapted from Genome Research
9:492-498, 1999
Fluororescence Polarization
C/C
C/TT/T
Control
Good SNP candidate
Bad SNP candidate
Not SNP
Not SNP
Typicalresults
SNP Analysis
Linkage association Study
PharmacogenomicsDiagnosis
Personalized Medicine
Genetic Instability
Gene 234 (1999) 177–186
LD (Linkage disequilibrium)
Strategies for association analysis
Genotype Analysis Using Human Hair Shaft
Cancer Epidemiology, Biomarkers & Prevention 11: 925–929, 2002
Hsueh-Wei Chang, Ching-Yu Yen, Shyun-Yui Liu, Gad Singer, and Ie-Ming Shih*
Apolipoprotein E4: an allele associated with many diseases
Ann Med.32(2): 118-127, 2000 . Smith, Jonathan D
TTTCCC
1 9 17 25 33 412 10 18 26 34 423 11 19 27 35 434 12 20 28 36 445 13 21 29 37 456 14 22 30 38 467 15 23 31 39 478 16 24 32 40 48
APO-E 112 APO-E 158
C825T sample panel
SNP Genotyping
APO-E158
CC
TC
TT
CCTGCAGAAGCGCCTGGC
CCTGCAGAAGTGCCTGGC
CCTGCAGAAG GCCTGGC TC
CC TC TT
T allele
C allele
72bp
39bp + 33bp
PCR-RFLP
Populations n
E2/E2 E2/E3 E3/E3 E2/E4 E3/E4 E4/E4 ε2 ε3 ε4
Taiwanese 48 2.1 14.6 68.8 0.0 12.5 2.1 9.4 82.3 8.3ns
Chinese22 141 1.4 12.1 70.9 0.0 14.9 0.7 7.4 84.4 8.2ns
European19 590 0.8 9.3 57.3 1.9 27.3 3.4 6.4 75.6 18.0*
ns = not significant; * p < 0.03
APO-E
Allele (%)Genotype (%)
Genotype and allele frequency of APO-E
Populations n
TT TC CC
Taiwanese 48 23.0ns 41.0 36.0
Chinese12 960 22.4ns 50.6 27.0
European12 277 10.0* 43.7 46.2
ns = not significant; *p < 0.001
Genotype (%)
C825T
Genotyping of C825T
Conclusion II
Digital SNP analysis can apply for High throughput genotyping
SNP Genotype & Haplotype
2003-01-21: Conceptual Web Site up2003-5-27 : International HapMap Meeting at CSHL2003-06-25: Allele frequencies submitted to dbSNP2003-07-01: Quality control SNPs2003-11-01: First major public data release!
Expression of P-glycoprotein and Genotype (MDR1 3435)
IncreasedC C
NormalC T
ReducedT T
P-gp Expression P-gp Genotype
Stage I Stage II Stage III
Cancer Progression
Digital SNP Data of Cancers(AI for certain chromosomes)
TRENDS in Biotechnology Vol.19 No.12 December 2001
personalized medicine