Molecular cloning of illegitimate 3′ switch γ rearrangement from a case of CD5⁺ DLBCL.

(A) Left- hand panel: Southern blot using an IGH 3′Sγ probe. Tumor DNA (lane 1) showed a 3′Sγ rearranged fragment of 4.2 kb that did not comigrate with a 5′Sγ probe (data not shown). Lane 2 contained germline control DNA. The 4.2-kb-SphI rearranged fragment was amplified by LDI-PCR. Right-hand panel: The LDI-PCR target for IGH 3′Sγ yielded a 1.8-kb product that corresponded to the 4.2-kb fragment seen on Southern blot. The LDI-PCR product was subcloned and sequenced. (B) Left-hand panel: Southern blot of tumor and normal DNA digested with BglII and hybridized with chromosome 6p21 breakpoint probe, showing single rearrangement in the tumor DNA. Comigrating rearranged bands are shown with an arrow. Right-hand panel: Same filter rehybridized with IGH 3′Sγ, showing comigration of rearranged 6p21 and IGH sequences (arrows). (C) Sequence of the der(14)t(6;14)(p21;q32) breakpoint from case 1 (Accession No. AF388309). Bold letters denote breakpoint sequence; above, IGHSγ2 sequence (gi 1 066 109 gb U39 934.1 HSU39 934); and below, 6p21.1 BAC clone RP11-533O20, which contained both the breakpoint andCCND3. (D) Ideogram to show the localization of chromosome 6p21.1 breakpoints. All 3 cloned breakpoints fell 5′ (centromeric) of the cyclin D3/CCND3 gene, in regions containing repetitive DNA sequences. Within the BAC clone RP11-533O20, the breakpoints were located at nucleotides 79567 (case 1, AF388309), 84057 (myeloma cell line KMM-1, AF364073),29 and 93510 (case 2, AF388310). Horizontal arrow denotes transcriptional orientation ofCCND3.