Base-Pair Resolution of Somatic and Germline-Derived Genome Rearrangement Breakpoints in Follicular Lymphoma.

Follicular lymphoma (FL) is the most common indolent lymphoid malignancy in North America with approximately 20,000 new cases of this incurable cancer diagnosed each year. In approximately 85% of patients, FL is associated with the reciprocal translocation t(14;18)(q32;q21), which results in a fusion between IGH and BCL2 genes and consequent over-expression of the anti-apoptotic protein BCL2. This translocation likely represents an initiating event for FL, requiring additional mutational events for the onset of clinical disease. To investigate the relationship between genome rearrangements and FL we identified rearrangement locations in the genome followed by detailed, fine-structure analysis of the rearrangements to ascertain their effects on genes and other features of biological interest.

We used a whole-genome bacterial artificial chromosome (BAC) fingerprint-based approach, termed Fingerprint Profiling (FPP, Krzywinski, M. et al. 2007), to detect genome rearrangements relative to the reference human genome in neoplastic B cells purified from 24 FL patient biopsies. Analysis of 2,640,707 BAC fingerprints revealed 721 candidate genomic rearrangements. To validate these observations and provide base-pair resolution of the rearrangement breakpoints we performed paired-end massively parallel sequencing, on the Illumina Genome Analyzer II platform, of the breakpoint-containing regions captured in the BAC clones. Sequence reads were assembled into contigs using our in-house de novo assembly algorithm ABySS (Assembly By Short Sequences, Simpson, J. et al. 2009) then aligned to the reference human genome. Following manual annotation of the breakpoint junctions PCR primers were designed to assay patient tumour and matched constitutional DNA and thus determine whether the observed genome rearrangements were somatic (acquired) or germline in origin.

727 BACs with apparent large-scale genome rearrangements, representing 354 distinct genome rearrangements across 20 patients, were sequenced in 95 pools, generating 72 Gbp of sequence. The 354 distinct events include 163 deletions, 71 inversions, 27 insertions, 83 translocations and 10 duplications, ranging in size from 3 kb to 67 Mb. PCR assays for 194 of the distinct events have been performed thus far identifying 80 distinct somatic and 114 germline-derived structural variations at base-pair resolution. Of the somatic events 5 are present in two or more of the 20 patients analyzed including a 720 kb inversion of 3q27.3 that results in expression of a BCL6-ST6GAL1 fusion transcript. Identification at base-pair resolution of breakpoint sequences enabled a detailed study of breakpoint and fusion mechanisms. We classified breakpoint junctions into 4 groups; those with microhomology (48%), those with sequence additions (28%), those with blunt fusions (20%) and those with flanking low copy repeats (4%). We were particularly interested in establishing the origin of the observed nucleotide sequence additions in 97 breakpoint junctions. The sequence additions ranged in size from a single nucleotide to 454 bp. In one case we have unambiguously mapped a 53 bp sequence, lying within one of the 3q27.3 inversion breakpoints, to chromosome 5q12.3. This finding is consistent with the recently proposed fork stalling and template switching (FoSTeS) DNA replication-based mechanism and thus represents a novel mechanism in FL lymphomagenesis.

We have successfully employed high-throughput clone fingerprinting and sequencing to identify numerous novel somatic and germline genome rearrangements from FL primary tumour samples. Furthermore, base-pair resolution of rearrangement breakpoints provides mechanistic insights. With the complete inventory of somatic and germline events in hand we will be able to propose recurrent structurally altered genes in FL patients for validation in independent datasets and improve our understanding of FL biology. Pathway analyses to identify emerging themes from somatic mutations are also being performed. The PCR assays we have developed will also be of utility in identifying germline predisposition alleles in larger FL patient cohorts.

No relevant conflicts of interest to declare.