A paper receiving advance online release in Nature Medicine described a strategy for meeting one of today’s most significant challenges in genomic medicine – determining whether a specific DNA variant in the non-protein-coding genome is the actual
disease-causing variant of an associated disease risk. The report from a multi-institutional team study led by investigators at Dana-Farber Cancer Institute, Massachusetts General Hospital (MGH), and the Keck School of Medicine at the University of
Southern California described a procedure called CAUSEL (Characterization of Alleles Using Editing of Loci) that uses epigenome- and genome-editing tools to determine functional causality of disease-associated variants in the non-coding genome and
to study the mechanisms by which those variants contribute to disease.
Co-senior author Matthew Freedman, MD, of the Dana-Farber Center for Cancer Genome Discovery and Center Functional Cancer Epigenetics explained that the effort to determine the precise DNA variants that increase disease
risk and how they do so is extremely complicated. While the genetic mapping approach known as linkage analysis has enabled the identification of DNA variants in protein-coding genes – like the BRCA1 and 2 genes that, when mutated, cause a clearly
increased, inherited risk of breast and ovarian cancer – those variants account for approximately 5 percent of cases of inherited disease risk. The other 95 percent appears to be predominantly influenced by variants mapping to non-protein-coding regulatory
elements that control the levels at which protein-coding genes are expressed.
“This is a good example of how the intersection of cutting-edge genetic and epigenetic profiling with the latest genome- and epigenome-editing technologies can be used to advance our understanding of how sequence variants can impact diseases,” said J.
Keith Joung, MD, PhD, associate chief for Research in the MGH Department of Pathology and a co-senior author of the report. “We believe that this is an important next frontier for advancing diagnosis and treatment of diseases influenced by genetics
and that CAUSEL provides a blueprint for how to proceed with these types of studies.”
“Now the question is how can we identify the actual pathogenic variant that is driving disease and determine its functional consequences” Freedman said. “The reason that is difficult to answer is that we are not used to working in the non-protein-coding
genome. The genetic code – an elegant rule book for how specific DNA sequences code for the amino acids that make up proteins – has been understood since the 1960s, but there is no such code for the vastly greater portion of our genome that does not
code for proteins. The genome-wide association studies (GWASs) that are being performed worldwide to find associations with everything from eye color to disease susceptibility usually discover that many variants are correlated with a condition, but
it’s very hard to isolate which variant is actually driving the trait.”
The CAUSEL procedure or ‘pipeline’ consists of five steps:
- genetic fine mapping to identify candidate variants,
- epigenomic profiling, in which the candidate variants are intersected with epigenetic data to identify which are most likely to cause the condition,
- epigenomic editing, in this case using reagents developed in Joung’s laboratory, to confirm whether or not the candidate variants may possess regulatory capacity,
- genome editing, to create cell lines with all possible genotypes of the candidate variants,
- phenotypic analysis of those cell lines, to evaluate functional differences relevant to the disease or condition of interest.
The researchers tested this pipeline on a region of chromosome 6 that previous studies have associated with increased prostate cancer risk, probably by controlling expression levels of RFX6, a regulatory protein involved with tumor-associated properties.
Fine-mapping that region identified 27 candidate variants, all associated with increased prostate cancer risk, and epigenomic profiling identified one as most likely to be relevant. Epigenomic editing performed at that site, called rs339331, confirmed
that the region it lies in could potentially regulate RFX6 expression, so the investigators created three cell lines – one with two copies of the disease-associated variant or allele, one with two copies of the ‘normal’ version, and one with a copy
of both versions.
Analysis of these cell lines revealed that, while cells with two normal alleles had the appearance of normal cells, both lines containing the cancer-associated variant had an appearance more typical of cancer cells. Cells carrying two cancer-related alleles
were more likely to adhere to surfaces, a property typical of cancer cells, and those cells lines also exhibited changes in the expression of genes involved with androgen signaling, a pathway known to be critical in the risk for and treatment of prostate
Joung noted that, while his team used epigenome- and genome-editing reagents based on engineered transcription activator-like effector (TALE) technology, other approaches, such as the easier to use CRISPR-Cas9 platform, should also work for these steps,
making the CAUSEL approach accessible to most laboratories. “As the number of gene variants associated with disease expands, it will become more and more important to identify which ones actually contribute to disease development. When sequence variants
are identified by CAUSEL as functional, we can envision that pathologists might rapidly develop tests for those variants, which could then be used to impact clinical care,” he says.
Simon Gayther, PhD, of the Keck School and Cedars Sinai Medical Center, co-senior author of the manuscript explained, “This study and the pipeline it describes represent something of a Holy Grail for the GWAS community, which has been incredibly successful
at identifying thousands of novel susceptibility alleles associated with disease but has not yet been able to show how these risk variants cause disease at the cellular level. This pipeline opens up a new-world opportunity to assign biological and
clinical significance to risk variants that are, at least in part, responsible for a multitude of cancers and other traits.”
Added Freedman, who is also an associate member of the Broad Institute, “There is no reason this technology couldn’t be applied to non-cancer-related variants as well. Of the approximately 17,000 gene variants that have been associated with diseases or
other conditions, less than 0.1 percent of these associations have rigorously been identified as causal variants. For the more than 99.9 percent that still need causal variant identification, we hope that finding the right cell type and applying our
pipeline will close that gap. Now we need to improve the efficiency of our steps and deploy CAUSEL in examining these and the many other variants that are being identified by labs around the world. If this works the way we anticipate, I do believe
that the impact will be transformative.”
Joung is a professor of Pathology and Freedman an associate professor of Medicine at Harvard Medical School. The co-lead authors Nature Medicine paper are Sándor Spisák, PhD, Dana-Farber; Kate Lawrenson, PhD, Cedars Sinai Medical Center, and Yanfang
Fu, PhD, MGH Pathology. Support for this study includes National Institutes of Health grants R01 GM107427, DP1 GM105378, R01CA193910, U19 CA148112 and U19 CA148537, the H.L. Snyder Medical Foundation, and the Prostate Cancer Foundation.