Characterization of the Non-Coding IGH J-Regions in Follicular Lymphoma and Chronic Lymphocytic Leukemia
Janice M Spence, W Richard Burack. University of Rochester School of Medicine and Dentistry, Rochester, NY
Background: The IGH locus undergoes Somatic Hypermutation (SHM) when B cells reside in the Germinal Center (GC). While the rate of SHM is highest in the IGH V regions (Vh), it affects many genomic loci and it appears that this aberrant SHM contributes to lymphomagenesis. Numerous studies report the Vh mutation status to estimate SHM in B cells and lymphoid tumors. However, mutations in Vh are limited by the need to encode a functional immunoglobulin. To overcome this constraint, we examined the intronic, non-coding IGH J-regions to identify mutations that could not be tolerated in Vh coding regions.
Design: The clone-specific IGH was identified in 38 specimens (12 follicular lymphoma (FL), 10 CLL, and 16 normal hyperplastic tonsils (NL)). CLL and FL clones were isolated by heteroduplex analysis and gel purification of the PCR product generated with BIOMED-2 primers. Gene specific Vh and IGH J6 primers were then used to amplify the Vh-J6 region. Normal IGH genomic regions were cloned from DNA sequences derived from hyperplastic LN from 2 individuals using Vh family leader primers and IGH J6 primer. IgBLAST (NCBI) was used to determine formation of a functional VDJ junction and Vh mutation rate. Non-coding J-results were analyzed for local homology using Needleman-Wunsch algorithm. Pre-germinal center (GC) was defined as Vh with >98% identity to reference sequence. Insertions/deletions (indels) were defined as >4 base alteration.
Results: 38 unique in-frame IGH sequences containing intronic J regions were analyzed; 15 were pre-germinal center (GC) and 23 were post GC (12/12 FL, 5/10 CLL, 6/16 NL). Indels within the J-intron were found in 12/12 FL, 5/5 post-GC CLL, 2/6 post-GC NL. The number of indels in each IGH J-intron ranged from 1 to 7 and their size varied from 6 to 300 bp. More than one indel per IGH was found in all FL and 2/5 post-GC CLL, but not in any of 6 post-GC NL (p<0.05 Fisher exact).
Conclusions: The intronic J-regions of the clonal IGH from FL and CLL patients are severely mutated, typically with multiple indel events of a size that would not be tolerated in coding regions. These events likely reflect AID exposure, as they are only found in post-GC IGH. The range of damage in any given tumor varied significantly among both CLL and FL patients. Mutation analysis of the Jh region is greatly simplified compared to Vh. The unbiased nature of mutations to non-coding J regions coupled with ease of analysis may make intronic Jh a robust marker for genome wide damage.
Tuesday, March 20, 2012 1:15 PM
Platform Session: Section G, Tuesday Afternoon