Towards precision prevention: Technologies for identifying healthy individuals with high risk of disease

Abstract

The rise of advanced technologies for characterizing human populations at the molecular level, from sequence to function, is shifting disease prevention paradigms toward personalized strategies. Because minimization of adverse outcomes is a key driver for treatment decisions for diseased populations, developing personalized therapy strategies represent an important dimension of both precision medicine and personalized prevention. In this commentary, we highlight recently developed enabling technologies in the field of DNA damage, DNA repair, and mutagenesis. We propose that omics approaches and functional assays can be integrated into population studies that fuse basic, translational and clinical research with commercial expertise in order to accelerate personalized prevention and treatment of cancer and other diseases linked to aberrant responses to DNA damage. This collaborative approach is generally applicable to efforts to develop data-driven, individualized prevention and treatment strategies for other diseases. We also recommend strategies for maximizing the use of biological samples for epidemiological studies, and for applying emerging technologies to clinical applications.

Keywords: Comet; DNA damage; DNA damage response; DNA repair; H2AX; Host cell reactivation; Precision medicine.

Fig. 1

From traditional assays (center) to today’s tools for population studies (outer circle). The impact of new technologies emerges through their integration into population studies (orange circle).

Fig. 2

Major classes of DNA damage and major DNA repair pathways.

Fig. 3

CometChip uses photolithography to create a mold with micrometer scale pegs. A) The mold creates an array of microwells. B) Cells are loaded into the wells by gravity and excess cells are removed by shear force. C) Comet data from an undamaged cell (top) and a heavily damaged cell (bottom). D) An array of comets resulting from the CometChip.

Fig. 4

γ-H2AX yields and DNA repair kinetics as measured with the high-throughput fully-automated RABiT system [21,22], from a study of finger stick blood samples from 94 healthy individuals [23]. Experimental data and model fit (see [23]) pooled from 94 donors exposed ex vivo to 4 Gy gamma radiation, assayed at 0.5, 2, 4, 7 and 24 h post irradiation.

Fig. 5

Distributions of the DNA repair parameters F_res (residual DSBs) and K_dec (characteristic DSB decay time) derived from a recent study [23] of 94 healthy individuals using the high throughputfully-automated RABiT system [21,22]. The red curves show fits to biphasic normal distributions, showing evidence for distinct subpopulations with different DNA repair capacities.

Fig. 6

Schematic of FM-HCR. Fluorescence based multiplex host cell reactivatio (FM-HCR) assays use unique fluorescent reporter plasmids to measure repair capacity in multiple DNA repair pathways in parallel in live cells (Nagel et al. (2014) PNAS 111(18), E1823–32).

Fig. 7

DNA Repair Molecular Beacons – (A) Overall design of the DNA repair molecular beacons – a deoxyoligonucleotide containing a single base lesion with a 6-Carboxyfluorescein (6-FAM) moiety conjugated to the 5′ end and a Dabcyl moiety conjugated to the 3′ end of the oligonucleotide. (B) Schematic representation of utility of the DNA repair molecular beacon assay in 96- or 384-well plates for analysis of cell and tissue lysates or purtified proteins. (C) Modification of the DNA repair molecular beacon platform – microspheres or bead-based Beacons for increased multiplexing capacity.

Fig. 8

Schematic representation of the double-stranded CypherSeq construct. The CypherSeq construct includes all of the components necessary for sequencing on Illumina platforms, plus two 7-nucleotide double-stranded, randomly generated barcodes (flanking a blunt-SmaI restriction site). Sheared genomic DNA is ligated into the vector at the SmaI site. The library is then amplified (via E. coli transformation or PCR) and deep sequenced. The resulting sequence reads are filtered, computationally de-convoluted, and error-corrected via the double-stranded CypherSeq barcodes.

Fig. 9

Overview of rolling circle amplification (RCA) enrichment from CypherSeq libraries. A CypherSeq vector library is amplified by extension of biotinylated, target-specific primers using the strand displacement synthesis-proficient polymerase. Two primers, one targeting each of the complementary strands, must be used to achieve double-strand molecular barcoded error correction. Template CypherSeq vectors containing non- target sequences remain unamplified while templates containing the target sequence are amplified via RCA into long single-stranded products containing redundant copies of the target sequence and sequencing cassette. The RCA products are purified using magnetic streptavidin-coated beads, subjected to limited PCR with the library preparation primers, and sequenced. Reads are computationally compiled by barcode and a consensus is made for each barcode family independently. Substitutions occurring in < 90% of the reads within a family are rejected as artifacts, while substitutions present in all or nearly all (> 90%) of a family are accepted as true mutations.