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Mutations of CARD11 but not TNFAIP3 may activate the NF-B pathway in primary CNS lymphoma

Supplementary information

Manuel Montesinos-Rongen, Ph.D.1, Roland Schmitz, Ph.D.2,5, Anna Brunn, M.D.1, Stefan Gesk, M.D.3, Julia Richter, Ph.D.3, Ke Hong, Ph.D.1, Otmar D. Wiestler, M.D.4, Reiner Siebert, M.D.3, Ralf Küppers, Ph.D.2, and Martina Deckert, M.D.1

1: Department of Neuropathology, University Hospital Cologne, Cologne, Germany

2: Institute of Cell Biology (Cancer Research), Medical School, University of Duisburg-Essen, Essen, Germany

3: Institute of Human Genetics, Christian-Albrechts University Kiel, University Hospital Schleswig-Holstein, Campus Kiel, Kiel, Germany

4: German Cancer Research Center, Heidelberg, Germany

5: present address National Cancer Institute, National Institute of Health, Bethesda, MD 20892, U.S.A.

Corresponding author:

Manuel Montesinos-Rongen, Ph.D.

Department of Neuropathology, University Hospital Cologne

Kerpener Str. 62

50924 Cologne, Germany

Tel.:+49-221-478 5260

Fax:+49-221-478 7237

E-mail:

PCR amplification and sequencing of the CARD11 and the TNFAIP3 gene

Primer mix for first round CARD11 PCR (exon 4 to 10):

CARD11_ex4_ffGCCTACGTGGTTCATTTTGG

CARD11_ex5_ffTGTGAAATCCTCACGGTCTG

CARD11_ex6_ffCTGTCCCACCTGACAAAGGT

CARD11_ex7_ffTCTGTTTCAGAGCCGAGGAT

CARD11_ex8_ffATTGCACCAGCTTAGCACCT

CARD11_ex9_ffGGTGAGAGGATGGGTTCAGA

CARD11_ex10_ffGTGGCAGATGAGATGGAAGG

CARD11_ex4_rGAACCATGCCACTACTGAGGAC[2]

CARD11_ex5_rGTCACCCTGGCGGAGTAGCC[2]

CARD11_ex6_rACACCCTGGCAGGTTCATC[2]

CARD11_ex7_rCCCAGGCCCTCATCTGGTTG[2]

CARD11_ex8_rGCCTGTGACTTCCAAAAAAGCC[2]

CARD11_ex9_rCAAAGGACAAGGAGCCATTCATTG[2]

CARD11_ex10_rAGCGAGTCGCAGGATTTCCA[2]

PCR conditions were 1x 95°C for 1 min, 59°C for 30 sec, 72°C for 30 sec, followed by 19x 95°C for 30 sec, 59°C for 30 sec, 72°C for 30 sec, terminated by 72°C for 5 min and stored by 4°C until used for semi-nested PCR. Beside standard PCR conditions 2 mM MgCl2 and 1 M betaine was used.

Second round of CARD11 PCR consists of individual semi-nested:

CARD11_ex4_fGATTTACGTCATCTGGGCTCC[2]

CARD11_ex4_rGAACCATGCCACTACTGAGGAC[2]

CARD11_ex5_fCAGTGCCTCGTGGGCAGAGT[2]

CARD11_ex5_rGTCACCCTGGCGGAGTAGCC[2]

CARD11_ex6_fCTGGAGAAGGTTTCTTGGAGC[2]

CARD11_ex6_rACACCCTGGCAGGTTCATC[2]

CARD11_ex7_fCCCAGGATACGCCCAAGCAA[2]

CARD11_ex7_rCCCAGGCCCTCATCTGGTTG[2]

CARD11_ex8_fTCCCCTATGTTACCTGGTCTGTAGTG[2]

CARD11_ex8_rGCCTGTGACTTCCAAAAAAGCC[2]

CARD11_ex9_fCCTCAGTGCCCTCATCTGTAAAATG[2]

CARD11_ex9_rCAAAGGACAAGGAGCCATTCATTG[2]

CARD11_ex10_fCCAGAAGCCTGGGAGGAGGA[2]

CARD11_ex10_rAGCGAGTCGCAGGATTTCCA[2]

PCR conditions for all 7 semi-nested PCR were identical to the first round of PCR.

Primer mixes for first round of TNFAIP3 PCR. Due to overlapping PCR products (exon 7, part a and b), two individual PCR are applied.

Mix 1 (exon 2, 3, 7 (part a), and 8):

A20-01TGCCTACAGATCAGGGTAATGACAAG

A20-03AGCTTCATGAATGGGGATCCAGCAG

A20-05ACCATTCAGTCCCCTAGAATAGCAG

A20-07TATGCCCACCATGGAGCTCTGTTAG

A20-15GGTTCTACAATTCTTGCCATAATCCAC

A20-17CAAAATCCGTTGTGCTGCACATTCAG

A20-22ATGAGGAGACAGAACCTGGCAGAG

A20-23ACTGTCAGCATCTCTGTATCGGTG

Mix 2 (exon 4/5, 6, 7 (part b), and 9):

A20-09TGAATAATTGTAGAGTGATGTCAGAATGAC

A20-11GGAAAACCCTGATGTTTCAGTGTCTAG

A20-12AATCACTCTACTGTTGAGCTTCAGG

A20-13TGAGATCTACTTACCTATGGCCTTG

A20-18CAGTTCTGCCTGACTGCCTACATG

A20-20CTCTCGGGGAGAAGCCTATGAGC

A20-25GTAGACTCCACACTCTCCAATGAG

A20-28TAGCACCATGATGACTGACAGCTCG

PCR conditions were 1x 95°C for 5 min, 61°C for 30 sec, 72°C for 90 sec, followed by 24x 95°C for 30 sec, 61°C for 30 sec, 72°C for 90 sec, terminated by 72°C for 5 min and stored by 4°C until used for semi-nested PCR. Beside standard PCR conditions 3 mM MgCl2 and 1 M betaine was used.

Primer for 8 individual semi-nested second rounds ofTNFAIP3 PCR:

Exon 2:

A20-02GTTTCCTGCAGGCAGCTATAGAGG

A20-03AGCTTCATGAATGGGGATCCAGCAG

Exon 3:

A20-06ACCTTTGCTGGGTCTTACATGCAG

A20-07TATGCCCACCATGGAGCTCTGTTAG

Exon 4/5:

A20-10TACAGGGAGTACAGGATACATTCAAGC

A20-11GGAAAACCCTGATGTTTCAGTGTCTAG

Exon 6:

A20-13TGAGATCTACTTACCTATGGCCTTG

A20-14TCAGGTGGCTGAGGTTAAAGACAG

Exon 7 (part a):

A20-16GAGCTAATGATGTAAAATCTTGTGTGTG

A20-17CAAAATCCGTTGTGCTGCACATTCAG

Exon 7 (part b):

A20-18CAGTTCTGCCTGACTGCCTACATG

A20-19GTGGAACCCTGAGGAGTCCACTGC

Exon 8:

A20-23ACTGTCAGCATCTCTGTATCGGTG

A20-24TGTCACTGTCGGTAGAAAACGCTC

Exon 9:

A20-26GTGCTCTCCCTAAGAAATGTGAGC

A20-28TAGCACCATGATGACTGACAGCTCG

PCR conditions for all 8 semi-nested PCR were identical to the first round PCRs, insteadof use of 2 mM MgCl2. All primers are derived from Schmitz et al. [3].

References

1.Chanudet E, Huang Y, Ichimura K, et al. (2010) A20 is targeted by promoter methylation, deletion and inactivating mutation in MALT lymphoma. Leukemia 24:483-487

2.Lenz G, Davis RE, Ngo VN, et al. (2008) Oncogenic CARD11 mutations in human diffuse large B cell lymphoma. Science 319:1676-1679

3.Schmitz R, Hansmann ML, Bohle V, et al. (2009) TNFAIP3 (A20) is a tumor suppressor gene in Hodgkin lymphoma and primary mediastinal B cell lymphoma. J Exp Med 206:981-989

Figure Legends

Figure S1: Bisulfite pyrosequencing in PCNSL and spinal DLBCL

In 40 of 42 cases (except case #13 and #28) suffcient DNA for bisulfite-conversion was available. In these tumors pyrosequencing of the TNFAIP3 promoter region according to Chanudet et al. [1]was performed. Data of the individual tumors are shown.

Figure S2: qPCR for TNFAIP3 in PCNSL and spinal DLBCL

For all cases, in which mRNA material was available (21/42) Taqman qRT-PCR assay for TNFAIP3 was performed (Applied Biosystems). RQ-values with RQ= 2-ddCt are shown. Data show that PCNSL and spinal DLBCL show increased TNFAIP3 mRNA levels as compared to tonsillar tissue.