Results 1 comparison for 5 systems containing the SNP at postion 1 to 5 of the up-stream probe systems 2-5 work well, system 3 offers most stable & reliable

Results 1• comparison for 5 systems containing the SNP at postion 1 to 5 of the up-stream probe

systems 2-5 work well, system 3 offers most stable & reliable discrimination (Fig.4)

• concordance between sequencing results & QPCR measurements based on system 3

• same results with patient DNA from tissue, whole blood & leukocytes

Objectives

• single nucleotide polymorphisms (SNPs) are frequently associated with the onset or progression of

diseases including cancers

• SNPs in the promoter region of the vascular endothelial growth factor (VEGF) are reported to influence

VEGF expression which is increased in several tumor types including renal cell carcinoma (RCC)

• analysis of SNP distribution supports risk assessment and prediction of cancer development

• determination of SNP variants by sequencing, array analysis or quantitative PCR (QPCR)

• QPCR approaches include melting curve analyses or the use of SNP-specific probes

• aim of the study:

establishment of a novel QPCR detection format for real-time PCR suitable for SNP detection

improved or same sensitivity, specificity, flexibility, and robustness compared to available formats

no need for external licenses (interest of the cooperating company AJ Roboscreen)

development of the development of the TRIPLEHYB probe formatTRIPLEHYB probe format & its validation in a clinico-experimental application & its validation in a clinico-experimental application

Role of VEGF C(-460)T single nucleotide polymorphismRole of VEGF C(-460)T single nucleotide polymorphismin the development of renal cell carcinomain the development of renal cell carcinoma

S. FüsselS. Füssel11, S. Schneider, S. Schneider11, A. Lohse-Fischer, A. Lohse-Fischer11, S. Tomasetti, S. Tomasetti11, S. Fuessel, S. Fuessel11, T. Köhler, T. Köhler22, , A. RostA. Rost22, A. Meye, A. Meye11, M.P. Wirth, M.P. Wirth11

11Department of Urology, Medical Faculty, Technical University of Dresden & Department of Urology, Medical Faculty, Technical University of Dresden & 22AJ Roboscreen LeipzigAJ Roboscreen Leipzig

granted by technology support with financial sources of the European Regional Development Fund and the State of Saxonygranted by technology support with financial sources of the European Regional Development Fund and the State of Saxony

Fig. 1 TRIPLEHYB probe format: basic principle

D1 – D4: positions for the attachment of different dyes

D1 fluorescent dye D2 quencher

3‘-

downstream probeupstream probe

- 5‘

5‘- - 3‘

D1

D2 D3

D4

3‘ 5‘

stem structure

target sequence–complementary probe subsequence

Fig. 3 Genotyping of VEGF–C460T at position 3 of the up-stream probe

An fluorescence signal is only expected in case of perfect match: homozygous mut & heterozygous wt/mut positive in FAM channel homozygous wt & heterozygous wt/mut positive in ROX channel

homozygous wild-type

3`5`

5`

Q

R1

3`5`

5`

Q

R1

3`5`

5`

Q

R1

3`5`

5`

Q

R2

heterozygous homozygous mutant

3`5`

5`

Q

R2

3`5`

5`

Q

R2

R1 = FAM R2 = ROX Q = BHQ1 / BHQ2

Fig. 4 Optimization of the SNP-detection assay

TT Du-145CC patientCT LNCaP

TT Du-145CC patientCT LNCaP

FAM (for T-variant = mut) ROX (for C-variant = wt)

• plasmids and cell lines as standards and controls for all variants

• concordance with sequencing results (for 30 patient samples)

3-step PCR (45 cycles; LC480): 95°C/15s; 45°C/1s; 59°C/40s;primers: 0.5µM each; probes: 0.3µM ROX-up/0.4µM FAM-up + 1.2µM do;

Universal Master Mix + add. 5mM MgCl2 (ABI); template: 10 / 20 / 50 / 100ng DNA

Material & Methods

• selection of the SNP C/T at postion -460 of the VEGF promoter as model system since it is supposed to

alter VEGF expression analysis of this SNP in patients with clear-cell RCC

• C corresponds to wildtype (wt) & T to mutant (mut)

• design of a probe system based on the TRIPLEHYB format (Fig.1) consisting of:

the same forward & reverse primers used for both SNP variants

two up-stream probes each specific for one SNP variant

one down-stream probe matching to both up-stream probes

each one half of the up- & down-stream probes is complementary to the template and one half to the

other forming a stem structure (Fig. 1)

labeling with fluorescent dyes (D1) and quencers (D2) specific combination for each SNP variant:

ROX + BHQ2 for wt and FAM + BHQ1 for mut (Fig.2)

fluorescence signal only expected in case of perfect match between up-stream probe and template

parallel detection of amplification signals in both channels homozygous wt & heterozygous wt/mut

positive in ROX channel, homozygous mut & heterozygous wt/mut positive in FAM channel (Fig.3)

• design and testing of different probe systems containing the nucleotides substitution at postions 1-5 of the

up-stream probe reliable discrimination of both SNP variants possible?

• testing on cloned model plasmids for each SNP variant using the LightCylcer 480 (Roche) and the

Universal Master Mix (Applied Biosystems) supplemented with 5mM MgCl2

• use of DNA from cell lines or patients with known SNP-status as positive controls for each variant

• validation of the system 3 (with SNP at pos. 3 of the up-stream probe) on DNA samples from 30 patients

with known SNP status (sequenced at AJ Innuscreen, Berlin)

• parallel determination of SNP status in patient DNA samples originating from tumor tissue, whole blood

and leukocytes (isolated by standard protocols)

• SNP analysis on DNA (50-100ng per reaction) from leukocytes of 99 patients with clear-cell RCC

• comparison with SNP data from 116 healthy controls

• calculation of Odds ratios for different SNP variant as indicator of an increased chance to develop RCC

Fig. 2 C(-460)T polymorphism of the VEGF promoter

• SNP in pos. 3 of up-stream probe no binding to template no signal

• model system: SNP CT at position -460 of the VEGF promoter

• labeling with fluorescent dyes (FAM, ROX) and quenchers (BHQ1+2)

3`5`

5`

Q

R1

3`5`

5`

Q

R2

5 -̀AC CCCTCAACC...CCACACGCACA- 3`

GACT

CTGA

----

....

....A

3 -̀........TG GGGAGTTGG..GGTGTGCGTGT........-5`T

R1


GACT

CTGA

----

....

....

G3 -̀........TG GGGAGTTGG..GGTGTGCGTGT........-5`C

R2

mt

wt

R1 = FAM R2 = CFO or YY Q = BHQ1

3`5`

5`

Q

R1

3`5`

5`

Q

R2


GACT

CTGA

----

....

....A


R1


GACT

CTGA

----

....

....


R2

mt

wt

R1 = FAM R2 = CFO or YY Q = BHQ1

3`5`

5`

QQQ

R1R1

3`5`

5`

QQQ

R2R2R2


GACT

CTGA

----

....

....A


R1

5 -̀AC CCCTCAACC...CCACACGCACA- 3`- 3`

GACT

CTGA

----

....

....

GACT

CTGA

----

....

....A

3 -̀........TG GGGAGTTGG..GGTGTGCGTGT........-5`-5`T

R1R1


GACT

CTGA

----

....

....


R2

5 -̀AC CCCTCAACC...CCACACGCACA- 3`- 3`

GACT

CTGA

----

....

....

GACT

CTGA

----

....

....

G3 -̀........TG GGGAGTTGG..GGTGTGCGTGT........-5`-5`C

R2R2R2

mt

wt

R1 = FAM R2 = CFO or YY Q = BHQ1Q = BHQ1 or BHQ2 R2 = ROX

Results 2• analysis of DNA from 99 pts with primary clear cell RCC

• median age: 65 yrs (37-83 yrs), 62 male & 37 female

• SNP-distribution in clear cell RCC: 22.2 % CC / 55.6 % CT / 22.2 % TT

• first comparison with data from 116 healthy controls from the HapMap-study

(CEU - people from Utah with ancestry from northern & western Europe):

19.0 % CC / 46.6 % CT / 34.5 % TT

• comparison of CT vs. TT: OR = 1.85 prevalence for RCC

• next steps:

analysis of more ccRCC patients

analysis of matched healthy controls with this assay

dependence on tumor stage / grade / outcome

haplotype analysis of further VEGF-promoter SNPs

http://urologie.uniklinikum-dresden.de / [email protected]

Documents

Results 1 comparison for 5 systems containing the SNP at postion 1 to 5 of the up-stream probe systems 2-5 work well, system 3 offers most stable & reliable