High-throughput sequencing reveals novel features of immunoglobulin gene rearrangements in Burkitt lymphoma

Abstract

We thank Ralf Küppers for his interest in our article 1 and for his thoughtful commentary. 2 We address each of the points he raises below. First, Küppers claims that, in contrast to what has been demonstrated in mice,3 not all normal human B-cell progenitors undergo concurrent biallelic D H J H rearrangement during B-cell development, and he cites an analysis performed with Southern blot techniques of 26 B-cell lines derived from 3 individuals to support this claim. 4 We analyzed publicly available IGH high-throughput sequencing (HTS) data derived from analysis of B-cell genomic DNA from 60 healthy individuals (available at https://clients.adaptivebiotech. com/immuneaccess), and we conclude that the data strongly suggest that a very high fraction of normal human B cells do indeed undergo biallelic D H J H rearrangement. These IGH HTS data were acquired using the same sequencing platform used for our studies. 5 Second, Küppers points out that it is physiological for human B cells, and not an abnormal feature of Burkitt lymphoma (BL) cells, that nonproductive V H D H J H rearrangements undergo somatic hypermutation (SHM) as efficiently as productive V H D H J H rearrangements. He reports that this observation is attributable to the fact that nonproductive rearrangements are transcribed and that transcription is a prerequisite for being targeted by somatic hypermutation. In our study, however, we observed evidence of comparable hypermutation of incomplete D H J H rearrangements, which are not transcribed. Hypermutation on incomplete D H J H rearrangements has not been reported previously, to our knowledge. We interpret this novel finding as evidence of an aberrant mutational process that is broadly active across the immunoglobulin loci in BL tumors.Third, while one early report suggested that MYC translocation into the IGH locus may occur preferentially within the J H gene segment in endemic BL and preferentially within the S m switch region in sporadic BL, 6 subsequent studies have demonstrated that the translocation breakpoints in both endemic and sporadic BL regularly occur in the switch and J H regions.7-10 Therefore, the possible breakpoint locations on chromosome 14 span a genomic interval of ;280 kb, from the J H region through the switch region. For the V H D H J H or D H J H rearrangement carried on the IGH allele into which MYC is translocated to be undetectable by our HTS platform, the translocation breakpoint would have to occur within the ,200-bp interval that is amplified and sequenced by our sequencing strategy. If the breakpoint occurs outside this extremely narrow ,200-bp window, our sequencing strategy would still detect the IGH rearrangement on this allele, even if it is adjacent to MYC. While disruption of the polymerase chain reaction (PCR) target region by the MYC; IGH translocation is theoretically 11 JULY 2017 x VOLUME 1, NUMBER 16 1261 possible, it is a highly unlikely event, and it is therefore improbable that it could explain the lack of detection of a second rearranged IGH allele in 85...

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“…Another study showed that while the leukemic clone could be identified in both DNA and RNA, the transcriptional clonal frequencies were generally lower [56]. Caution is warranted because not all rearrangements are transcribed, as shown by several studies [57][58][59]. As RNA-sequencing has often been performed using shorter reads in its more general application, the robustness becomes apparent from the results presented by Blachly et.…”