Deficiency of Bruton's tyrosine kinase in B cell precursor leukemia cells -- Feldhahn et al. 102 (37): 13266 Data Supplement - HTML Page - index.htslp -- Proceedings of the National Academy of Sciences

Feldhahn et al. 10.1073/pnas.0505196102.

Supporting Information

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Supporting Table 1
Supporting Figure 6
Supporting Figure 7

Supporting Figure 6

Fig. 6. Bruton’s tyrosine kinase (BTK) splice variants in B cell precursor leukemia. BTK isoform expression was analyzed by RT-PCR in 29 cases of B cell precursor leukemia carrying BCR-ABL1, E2A-PBX1, MLL-AF4, TEL-AML1, or TEL-PDGFRB gene rearrangements. Fourteen aberrant BTK isoforms could be identified in addition to full-length BTK. None of these BTK isoforms carry a functional kinase domain resulting from the usage of regular or cryptic splice sites leading to large deletion and/or the generation of preterminal translation-stops. Protein domains (Upper) and mRNA exons (Lower) are depicted. Untranslated regions are indicated by hatched boxes. Sequence data of all splice variants are available from EMBL/GenBank under accession nos. AM051275-AM051286.

Supporting Figure 7

Fig. 7. Ligation-mediated (LM)-PCR amplification of DNA double-strand-break intermediates at Jk1 and Jl7 recombination signal sequences. Ig light-chain gene rearrangement involves the introduction of DNA double-strand breaks at recombination signal sequences (RSS) flanking V and J segments. DNA double-strand breaks at RSS can be ligated to double-stranded DNA linker molecules. To detect DNA double-strand breaks preceding V-J recombination at the IGK or IGL loci, linker-ligated DNA can be amplified by using Jk-RSS or Jl-RSS-specific primers together with a linker-specific primer.

Table 1. Sequence analysis of aberrant splice variants of BTK in preB acute lymphoblastic leukemia

Gene rearrangement	Cases*	Exons involved	Splice site	Transcript	Translation	Western blot	Frequency
Recurrent BTK splice variants
MLL-AF4 BCR-ABL1 E2A-PBX1 TEL-AML1 TEL-PDGFRB	SEM, RS411, BEL1, IX, X, XI, XIV, BV173, NALM1, SUP-B15, SD1, KASUMI2, MHH- CALL3, NALM6	Exons 15 and 16^†	Regular	D Exons 15, 16	In-frame deletion in KD	65-kD protein	14/29





BCR-ABL1	IX, X, XI, XIV, BV173, NALM1, SUP-B15, SD1	Exon 15^‡	Regular	D Exon 15	Truncated KD	52 kD protein	8/29


MLL-AF4 BCR-ABL1	VII and IX	Exons 14 and 15	Cryptic	D Exon 15, D41-bp exon 14	Truncated KD	ND	2/29
MLL-AF4 BCR-ABL1	VII and IX
Unique BTK splice variants
MLL-AF4	V	Exon 18	Cryptic	D 33 bp	In-frame deletion in KD	ND
MLL-AF4	SEM	Exons 16 and 17	Cryptic	D Exons 16, 17, D119-bp exon 15, D136-bp exon 18	Truncated KD	ND
BCR-ABL1	X	Exons 16^†	Regular	D Exon 16	Truncated KD	ND
	XI	Exons 15 to 18	Cryptic, Slippage	D Exons 15-18, D79-bp exon 14, Ins 3 bp, D24-bp exon 19	Truncated KD	ND
	XV	Exon 14	Cryptic	D Exon 14, D62-bp exon 13, Ins 8 bp, D77-bp exon 15	Truncated SH2 and KD	ND
TEL-AML1	XVIII	Exons 13 to 18	Cryptic, Slippage	D Exons 14-18, D31-bp exon 12, D 22-bp exon 13, 212-bp in exon 19 and 3’UTR	Truncated SH2 and KD	ND
	XVIII	Exons 14 to 16	Regular	D Exons 14-16	Truncated KD	47-kD protein
	XIX	Exons 13 to 18	Cryptic, Slippage	D Exons 13-18, Ins of a 106-bp fragment of Intron18	Truncated SH2 and KD	ND.
	XIX	Exons 14 to 17	Regular	Loss of exons 14-17	In-frame deletion in KD	56-kD protein
E2A-PBX1	KASUMI2	Exons 15 to 17	Cryptic, Slippage	D Exons 15-17, D18-bp exon 14, Ins 1-bp, D34-bp exon 18	Truncated KD	ND
E2A-PBX1	MHH-CALL3	Exons 13 to 17	Cryptic	D Exons 13-17, D12-bp exon 12, D92-bp exon 18	Truncated KD	ND

Sequence data are available from EMBL/GenBank under accession nos. AMO51275-AMO51286. SH2, SRC homology domain 2; KD, kinase domain; Ins, Insertion; ND, not done.

*Roman numerals indicate primary cases of B cell precursor leukemia; cell lines are indicated by their names.

^†These splice variants were previously described by Goodman et al. [Goodman, P. A., Wood, C. M., Vassilev, A. O., Mao, C. & Uckun, F. M. (2003) Leukemia Lymphoma 44, 1011-1008]. ^‡The specific function of this splice variant in BCR-ABL1⁺ preB lymphoblastic leukemia was investigated by Feldhahn et al. [Feldhahn, N., Klein, F., Mooster, J. L., Hadweh, P., Sprangers, M., Wartenberg, M., Bekhite, M. M., Hofmann, W. K., Herzog, S., Jumaa, H., et al. (2005) J. Exp. Med. 201, 1837-1852].