Supplementary Table 2: Primer sequences and DHPLC conditions on the Wave®
Nucleic Acid Fragment Analysis System 4500 (Applied Biosystems, Darmstadt,
Germany) for TRAF3 mutational analysis.
Exon1 Direction
3
4
5
6
7
8
9
10
11
12
Sequence
f
r
f
r
f
r
f
r
f
r
f
r
f
r
f
r
5´-CTCAGGCACTTTTGCTTTCC-3´
5´-CGGAGGCTTTTATGGACTTC-3´
5´-CTGTGAGCCACTGTGCAGAC-3´
5´-TGAGAGAATGTGTTGTGCCAG-3´
5´-TGCCTTGTCCAAAGTAGCAGC-3´
5´-TGGTCTTAAAGTGCCACATCC-3´
5´- TCCCAATTAAGAACATTGAATGGT-3´
5´-CATATAGGAATTGAGTGGAAGGC-3´
5´-GATGGTGAGCAGAGCCATTC-3´
5´-CATGACTCTCACGAACTGGG-3´
5´-CACACCTGTAGCGATAAACACC-3´
5´-CTTTCGTCACCCATGCAAC-3´
5´-CCCCTCTTTGTGTTTAGTGCTG-3´
5´-TTGATAGTTTCTTTTATCCAAAACCC-3´
5´-AAGCTAACAGAAGGCCTATATTGTG-3´
5´-GCAAAGGTCAAGGACTCAGC-3´
f
r
f
r
5´-GCCTCTGACTGTTCTGCTCC-3´
5´-AGTCATCACACCTCTGCGTG-3´
5´-GTGCCAGGGTCTACCTGAAC-3´
5´-TTCCTCCGTCTCACAAGGTC-3´
Annealing
DHPLC
Product
temperature2 temperature
size [bp]
[°C]
[°C]
475
65 °C →
60 °C
58,1
62,0
263
65 °C →
60 °C
57,7
278
63 °C →
58 °C
248
65 °C →
60 °C
296
65 °C →
60 °C
340
65 °C →
60 °C
307
65 °C →
60 °C
347
65 °C →
60 °C
55,0
58,0
55,3
60
60,5
61,8
57,0
59,0
55,8
62,0
51,9
54,9
55,9
430
65 °C →
60 °C
59,1
456
65 °C →
60 °C
55,0
59,7
f: forward; r: reverse
1
The exons of the known TRAF3 isoforms are numbered according to their genomic position from 1-12.
The non-coding exons 1 and 2 have not been analyzed.
2
The annealing temperature decreased during the first ten cycles 0.5 °C per cycle followed by 15 cycles
at the final annealing temperature.
Quantitative reverse transcription PCR:
RNA was extracted from patients and controls using the RNeasy mini kit (Qiagen,
Hilden, Germany) and transcribed into cDNA with the QuantiTect Rev. Transcription Kit
(Qiagen). Quantitative TRAF3 mRNA expression was calculated by comparative Ct
method (3). Standardization was carried out by subtraction of corresponding Ct-values
of the housekeeping genes hypoxanthine phosphoribosyltransferase 1 (HPRT1) and
glucose-6-phosphat-1-dehydrogenase (G6PD). Standardized TRAF3 expression of the
tumor samples was compared to the standardized TRAF3 expression in “Normal
Peripheral Blood B Cells cDNA (CD19+)” provided by AllCells (Emeryville, CA, USA).
The PCR assays were obtained from Qiagen:
Quantitect® Primer Assay Hs_TRAF3_1_SG
Quantitect® Primer Assay Hs_HPRT1_1_SG
Quantitect® Primer Assay Hs_G6PD_1_SG
Nine cases with del(14)(q24.1q32.33) but without homozygous TRAF3 deletion were
compared to eleven B-CLLs with the typical chromosomal changes for CLL including trisomy
12 (n=3), 13q-deletion (n=3), 11q-deletion (n=3) and 17p-deletion (n=2).
Methylation-specific PCR:
Methylation-specific PCR was performed using the AccuPrimeTM Taq DNA polymerase
system (Invitrogen, Karlsruhe, Germany). Primers were obtained from biomers.net
(Ulm, Germany) (Supplementary Table 3).
Supplementary Table 3: Description of the methylation specific PCR assays.
Assay
MSP assay 1
methylated
Direction
Sequence
Fragment size
(bp)
f
TTAGTCGGCGGTAGTCGCGGC
109
r
AAAACGCCGAACGCGTCCTCCG
MSP assay 1
f
TTTTTAGTTGGTGGTAGTTGTGGT
111
unmethylated
r
AAAACACCGAACACATCCTCCA
MSP assay 2
f
CGGTATTTCGCGGGGTCGC
74
methylated
r
AACAACGCGCTCGACCACG
MSP assay 2
f
GGATTTGGTATTTTGTGGGGTTGT
83
unmethylated
r
CTCAAACAACACACTCAACCACA
Abbreviations: bp, base pairs; f, forward; MSP, methylation specific PCR; r, reverse
Annealing
temperature [°C]
60
63
65
63
Array CGH:
Array CGH on case 1 was performed applying the NimbleGen Array 385k (Roche
NimbleGen, Madison, WI, USA) whereas case 2 was analyzed using the Human
Genome Microarray 244A platform (Agilent, Santa Clara, USA). The experimental
procedures were performed according to the protocols provided by the
manufacturers. Slides were scanned with a GenePix® 4000B microarray reader (Axon
Instruments, Toronto, Canada) at a scan resolution of 5 µm. Signal intensities from the
generated images were measured and evaluated with NimbleScan v2.3 and the
SignalMap v1.9 software using the segMNT algorithm (Roche NimbleGen) in case of
the NimbleGen Array 385k or with the Feature Extraction 9.5.3 and CGH Analytics
v3.5.14 software (Agilent) applying the Aberration Detection Method-2 (ADM-2)
algorithm with a threshold of 6.0 in case of the Human Genome Microarray 244A.
References
1
2
3
Martin-Subero JI, Harder L, Gesk S, Schlegelberger B, Grote W, Martinez-Climent JA,
et al. Interphase FISH assays for the detection of translocations with breakpoints in
immunoglobulin light chain loci. Int J Cancer 2002; 98: 470-474.
Arnold N, Gross E, Schwarz-Boeger U, Pfisterer J, Jonat W, Kiechle M. A highly
sensitive, fast, and economical technique for mutation analysis in hereditary breast
and ovarian cancers. Hum Mutat 1999; 14: 333-339.
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: 402-408.
Supplementary Figure 1: TRAF3 mutations shown by DNA sequence analysis and Surveyor
nuclease mutation detection. For description of genomic variants, the reference sequence
NM_145725 (TRAF3 isoform 1) was used. (a) Partial DNA forward and reverse sequence of
case 3, exon 8 indicating the deletion c.669delA. (b) Partial DNA sequences of exon 4 from
case 4 and a control from normal peripheral blood. The PCR product of case 4 was cloned
with the TOPO TA cloning kit (Invitrogen) before sequencing. The clones 3 (c.296delA) and 17
(c.288_297+6del15) are representatively shown. (c) Partial DNA sequences of case 5 exon 3
(c.157T>C) and exon 10 (c.820-1G>A). The mutation in exon 10 was confirmed with the
Surveyer nuclease mutation detection system according to the manufacturers protocol
(Transgenomic, Elancourt, France).