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Chapter 3 Genomic Approaches to Hematology 29

TUMOR

NORMAL

Fig. 3.2 GENOME DELETION IN A PATIENT WITH DIFFUSE LARGE B-CELL LYMPHOMA
(DLBCL). Genome sequencing of a patient with DLBCL revealed a clear region of genome deletion within
the TNFRS14 gene, as visualized in the Integrative Genomics Viewer. The gray bars indicate the extent of the
sequence read, with this region being interrogated multiple times. The white block in the middle (bracketed
by arrows) indicates the region of genome deletion captured by all of the reads in the tumor but in none of
the reads from the matched normal DNA sample (bottom portion of figure).

region in B cells. For example, in follicular lymphoma, translocations expression at that locus. Widespread methylation appears frequently
frequently involve juxtaposition of the antiapoptotic gene BCL2 to in cancer and may serve as an important mechanism of silencing
the immunoglobulin heavy chain enhancer region, leading to massive tumor suppressor genes. Massively parallel sequencing, coupled with
overexpression of BCL2 RNA and protein. bisulfite sequencing approaches, allows for genome-wide assessment
Translocations are best detected by either whole-genome sequenc- of DNA methylation in development and disease. DNA methylation
ing or RNA sequencing (RNA-Seq), although their detection requires is an effective mechanism of silencing genes, for example, as is
advanced computational analysis to distinguish them from artefactual required to specify cell type, but with relatively few dynamic changes
errors in aligning sequence reads to a reference genome. For reasons over time. In contrast, open chromatin regions may be analyzed by
that remain unclear, some tumors contain few, if any, translocations, technologies that are based on sequencing of DNA regions that are
but others contain hundreds, often involving multiple complex rear- accessible for certain DNA-cutting enzymes such as DNase, micro-
rangements. A particularly interesting phenomenon, termed chro- coccal nuclease, or transposase that preferentially cut at open chro-
mothripsis, involves extensive complex genome rearrangements matin regions. Several large-scale profiling efforts (e.g., through the
thought to occur via a single “big bang” genomic catastrophe National Institutes of Health ENCODE project) have used these
(Fig. 3.4). It has been speculated that chromothripsis may represent technologies to annotate cancer cell lines and normal human and
a mechanism by which a cell can acquire multiple oncogenic events murine tissues, including hematopoietic subsets.
required for cellular transformation in a single event rather than in a Modifications to histones are orchestrated and tightly regulated
stepwise manner. by a group of enzymes called chromatin regulators. Perhaps one of the
most striking results derived from genome-wide sequencing analyses
in cancer is the frequency of somatic mutations in chromatin regula-
SEQUENCING APPROACHES TO EPIGENOMICS tors, which account for up to 25% of all cancer drivers. Detailed
mechanistic analysis of epigenetic modifications and the contribution
Sequencing approaches to epigenomics include chromatin immuno- of individual chromatin regulators to these modifications have long
precipitation followed by sequencing (ChIP-Seq), micrococcal been hindered by a lack of effective technologies. With the use
nuclease (MNase) sequencing, DNAse sequencing (DNAse-Seq), of next-generation sequencing techniques combined with chroma-
bisulfite sequencing and assay for transposase-accessible chromatin tin immunoprecipitation, it is now possible to comprehensively
with high-throughput sequencing (ATAC-Seq). Although the major- investigate the molecular mechanisms of epigenetic alterations and
ity of information encoded in the genome is thought to emanate from define their disease relevance. ChIP-Seq can be used to map histone
its primary DNA sequence, the importance of epigenetic gene regula- modifications that are associated with actively transcribed regions,
tory mechanisms has become increasingly evident over the past few repressed regions, or regions found at distal regulatory elements.
years. Epigenetic modifications play a critical role in the regulation Optimized technologies now allow for ChIP-Seq with small input of
of transcription, DNA repair, and replication. For example, DNA samples or with FFPE samples, which has significant implications for
methylation can occur, particularly in CpG-rich regions of the translating this technology to approaches for the analysis of clinical
genome, and such methylation can lead to the silencing of gene specimens.