Supplementary Material 1
Patients and Methods
Patients
The cohort comprised 68 probands from unrelated families selected for matching either
“classic” Li–Fraumeni Syndrome (LFS) [1], or one of the Li-Fraumeni Like (LFL) criteria [15] and negative for mutations on the TP53 gene [6]. All subjects were seen at the AC
Camargo Hospital, São Paulo, Brazil.
As control, a Brazilian group of 404 unrelated individuals from families investigated for
reasons other than cancer was used. DNA samples obtained from peripheral blood after
informed consents were provided by the Genetic Center of the Institute of Biosciences,
University of São Paulo, São Paulo, Brazil.
Array-CGH
DNA from patients and controls was obtained from peripheral blood. Investigation of copy
number changes was performed by array-CGH using the whole genome Agilent SurePrint G3
Human CGH Microarray (Agilent Technologies, Santa Clara, USA) containing ~180.000
oligonucleotides. Briefly, samples were labeled with Cy3- and Cy5-dCTPs by random
priming; purification, hybridization, and washing were carried out as recommended by the
manufacturer. Scanned images of the arrays were processed and analyzed using Feature
Extraction and Genomic Workbench software (both from Agilent Technologies), together
with the statistical algorithm ADM-2, and using a sensitivity threshold of 6.7. We used two
reversed labeled hybridizations for each sample. Gains or losses in copy number were
accepted when the log2 ratio of the Cy3/Cy5 intensities of a given genomic segment
encompassing at least three consecutive probes was > 0.3 or < -0.3, respectively; any
alterations not detected in both dye-swap experiments were disregarded. All detected
imbalances were compared to CNVs reported in the Database of Genomic Variants (DGV;
http://projects.tcag.ca/variation/ - freeze of March, 2011) and to data from our own group.
Gene annotation was performed using University of California Santa Cruz Genome Browser
(UCSC).
Copy number variation by Real time PCR
Gene dosage ratios were calculated for BAX and FTL genes using the methods of 2-∆∆Ct [7].
Primers for GAPDH intron 7 (12p13) and HPRT1 exon 3 (Xq26.1) were used as normalizer
(norm) genes. All assays were conducted in 96-well plates (MicroAmpOptical 96-Well
Reaction Plate, Applied Biosystems, Foster City, CA). As the reference sample or calibrator,
we used commercial human genomic DNA (Promega, Madison, USA). 20 ng of each DNA
sample were amplified in a 20 uL reaction, containing 0.3 µmol of each primer and 1X
SYBR® Green PCR MasterMix (Applied Biosystems). qPCR was performed on an ABI
Prism 7500 detection system (Applied Biosystems) and entailed a preincubation of 95°C for
10 minutes followed by 40 cycles of denaturation at 95°C for 15 seconds, annealing and
extension at 60°C for 1 minute. Samples were amplified in triplicate for both target and
normalizer genes. The average Ct of the duplicate was used in the gene dosage ratio
calculations. For each gene, two control individuals were used to calculate the average
control Ct. Gene dosage ratios were calculated using the following equation: 2-[∆Ct (target) - ∆Ct
(norm)]
where ∆Ct (target) equals the difference between the Ct values for the patient and the
control average for the target exon, and the ∆Ct (norm) equals the difference between CT
values for the patient and the control average for the normalizer gene. Ratios between 0.82
and 1.22 were considered normal, between 0.41 and 0.61 indicated deletions in one allele and
above 1.4 indicated amplifications.
DNA sequencing
Genomic DNA was prepared following standard DNA extraction procedures. Direct
sequencing for P16INK4, P14ARF, CDK4, KIT, PDGFRA and BAX genes were performed on
the peripheral blood DNA. Each exon was amplified individually using 50 ng of genomic
DNA. The PCR reactions contained 0.25 uM of each primer, 0.2 mM of each dNTPs, 2.0 mM
of MgCl2 and 0.5 U Platinum Taq DNA Polymerase (Invitrogen) in a final reaction volume
of 20 ul. PCR products were purified with ExoSAP-IT (USB) and sequenced in both
directions. Sequencing reactions were performed with Big Dye v.3.1 (Applied Biosystems)
and separated on ABI Prism 3500 (Applied Biosystems) and analyzed using CLC Bio
software. Primers sequences are available upon request.
References
1.
2.
3.
4.
5.
6.
Li FP, Fraumeni JF, Mulvihill JJ, Blattner WA, Dreyfus MG, Tucker MA, Miller
RW. A cancer family syndrome in twenty-four kindreds. Cancer Res 1988;
48(18):5358-5362.
Eeles RA. Germline mutations in the TP53 gene. Cancer Surv 1995; 25:101-124.
Birch JM, Heighway J, Teare MD, Kelsey AM, Hartley AL, Tricker KJ, Crowther D,
Lane DP, Santibáñez-Koref MF. Linkage studies in a Li-Fraumeni family with
increased expression of p53 protein but no germline mutation in p53. Br J Cancer
1994; 70(6):1176-1181.
Birch JM, Hartley AL, Tricker KJ, Prosser J, Condie A, Kelsey AM, Harris M, Jones
PH, Binchy A, Crowther D. Prevalence and diversity of constitutional mutations in
the p53 gene among 21 Li-Fraumeni families. Cancer Res 1994; 54(5):1298-1304.
Tinat J, Bougeard G, Baert-Desurmont S, Vasseur S, Martin C, Bouvignies E, Caron
O, Bressac-de Paillerets B, Berthet P, Dugast C et al. 2009 version of the Chompret
criteria for Li Fraumeni syndrome. J Clin Oncol 2009; 27(26):e108-109; author reply
e110.
Achatz MI, Olivier M, Le Calvez F, Martel-Planche G, Lopes A, Rossi BM, AshtonProlla P, Giugliani R, Palmero EI, Vargas FR et al. The TP53 mutation, R337H, is
associated with Li-Fraumeni and Li-Fraumeni-like syndromes in Brazilian families.
Cancer Lett 2007;245(1-2):96-102.
7.
Livak KJ, Schmittgen TD. Analysis of relative gene expression data using real-time
quantitative PCR and the 2(-Delta Delta C(T)) Method. Methods 2001; 25(4):402408.