This article was originally published Dec. 5.
The University of Washington's Laboratory Medicine department has launched a high-throughput sequencing-based genetic test for mutations in genes associated with inherited colon cancer.
The test, known as ColoSeq, relies on custom oligonucleotide baits to capture genomic regions of interest, which can then be sequenced to high coverage. It was developed to simultaneously look for a range of mutations in the coding, non-coding, and flanking regions of seven genes with established roles in Lynch syndrome, familial adenomatous polyposis, MUTYH-associated polyposis, or other heritable syndromes known to elevate colon cancer risk.
"They are the most commonly mutated genes in hereditary colon cancer; that's how we picked these," the test's lead developer, Colin Pritchard, a laboratory medicine researcher at the University of Washington and associate director of its Clinical Molecular Genetics Laboratory, told Clinical Sequencing News. "These are genes that are firmly established to be clinically important."
The genes are "already being tested routinely, one at a time or in a small panel basis," he added, "so we don't anticipate that there will be issues with insurance reimbursement, especially when the cost of the test is going to be so much less than on a one-at-a-time basis."
"My own prediction is that this kind of testing, with dedicated [gene] panels for particular diseases, will become very widespread very quickly," University of Washington genome sciences and medical genetics researcher Mary-Claire King told CSN.
Pritchard and his colleague, University of Washington genome sciences researcher Tom Walsh, "just did a beautiful job of sorting this out for the inherited colon cancers," she said.
King, Walsh, and several other University of Washington researchers last year published a study in the Proceedings of the National Academy of Sciences describing a similar capture-based sequencing method for finding inherited mutations in 21 breast and ovarian cancer genes, including BRCA1 and BRCA2, using DNA from patient blood samples.
For that proof-of-principle study, the researchers captured genes of interest using custom Agilent oligonucleotides that corresponded to the coding, non-coding, and flanking sequences of the 21 genes and sequenced them by paired-end sequencing with the Illumina Genome Analyzer IIx. The approach helped them detect not only point mutations and small insertions and deletions, but also larger duplicated or deleted pieces of DNA.
The reagent cost of the breast and ovarian gene screening was less than $1,500 when researchers ran one sample per lane on the Illumina GAIIx. By barcoding the samples and running multiple samples per lane, they have since brought the reagent cost of the same method down to around $200 per sample, Walsh told CSN.
That method, dubbed BROCA, is still being used for research purposes and was featured in a recent PNAS study involving 360 women who were screened for inherited mutations related to ovarian, fallopian tube, or peritoneal cancer risk. In that study, Walsh, King, and co-authors uncovered 85 germline loss-of-function mutations in a dozen genes and found an example of a germline mosaic mutation in the tumor suppressor-coding gene TP53.
The university is not offering the BROCA panel as a commercially available genetic test, however, given the uncertainty surrounding the state of Myriad Genetics' BRCA1 and BRCA2 gene patents. The American Civil Liberties Union and other plaintiffs have challenged the patentability of the BRCA1 and BRCA2 genes in a case that many believe will eventually find its way to the Supreme Court (PGx Reporter 10/12/2011).
"For BRCA1 and BRCA2, until the patent situation is regularized, we will not be offering any commercial testing on those genes," King said.
The BROCA method served as an inspiration for Pritchard and others, who decided to take the same capture sequencing-based approach and develop a clinically available test for inherited colon cancer risk genes, which are not subject to the same restrictive intellectual property policies as the BRCA genes are.
The University of Washington Medical Center began offering the ColoSeq test on Nov. 1 at a list price of $2,650. Pritchard noted that while the reagent cost for the test is only around $200 per sample, that is only part of the expense of running the test, which also includes about a week of technician labor per sample and other expenses such as wear and tear on the instrument, overhead for running the lab, and so on.
Once a physician or genetic counselor orders the test, the request is sent to the university's laboratory medicine department, where members of Prichard's lab will prepare the samples for sequencing. The sequencing itself is performed by a clinical medical technologist on an Illumina HiSeq 2000 instrument in King's CLIA-certified laboratory.
Pritchard explained that it's possible to use an Agilent index barcoding system to run up to 96 samples on a single HiSeq lane and still get an average of around 320 times coverage of the targeted sequences.
ColoSeq is currently validated to find inherited colon cancer-related mutations in 209,000 bases of sequence, covering the exons, introns, and flanking sequences of five Lynch syndrome-associated genes — MLH1, MSH2, MSH6, PMS2, EPCAM — and two genes tied to polyposis: APC and MUTYH.
The lab is performing sequence capture for the test with custom oligonucleotides and the Agilent SureSelect system.
To sift through variants in the ColoSeq panel genes, the team is taking advantage of the bioinformatics pipeline developed to deal with BROCA assay data. The analysis methods allow for the detection of small genetic changes, such as single nucleotide variants and small indels, as well as larger duplications and deletions.
The team is using normalized read depth data to identify copy number variations — a strategy that Walsh, King, and others used to find CNVs as small as a few dozen base pairs in a BMC Genomics study published earlier this year.
"It fills in that gap that people run into with exome sequencing, in which one can pick up small indels and one can pick up very large CNVs, but there's a gap in the few hundred to few [thousand base] range," King said.
The ability to simultaneously test for single nucleotide changes and small indels as well as copy number changes using a next-generation sequencing-based test is expected to be important in a clinical setting, Prichard added, since a significant proportion of mutations in colon cancer-related risk genes are predicted to involve large deletions and duplications.
In addition to the bioinformatics strategies that are already in place to analyze capture gene sequence data, Pritchard's department wrote additional scripts designed to make it more straightforward to return ColoSeq results in a clinical setting.
"Our scripts annotate each variant via official [Human Genome Variation Society] nomenclature, give us columns that tell us something about the predicted function of each variant … and [also tell] us the predicted protein change for every variant we see."
The software also makes it possible to keep tabs on the frequency of specific mutations detected with the panel — information that should eventually help in interpreting the functional importance of these changes.
Any positive pathogenic mutations and variants of uncertain significance that are detected by ColoSeq will be validated in Pritchard's lab by Sanger sequencing or multiplex ligation-dependent probe amplification, in the case of copy number changes.
The laboratory medicine group will then provide an interpretive report to the oncologist or genetic counselor authorized to receive the patient's information.
For his part, Pritchard believes ColoSeq it will be particularly useful for patients who have a few colonic polyps and a family history that points to a possible inherited colon cancer syndrome, such as a first-degree relative with colon cancer or another Lynch syndrome-related cancer.
In such situations, he noted, clinicians often start by either testing Lynch syndrome genes or polyposis syndrome genes one at a time or in small panels.
For instance, Myriad offers a test called Colaris that looks for disease-related mutations in the Lynch syndrome genes MLH1, MSH2, and MSH6. The company's Colaris AP test, meanwhile, assesses the mutation status of genes implicated in several polyposis syndromes.
Prichard noted that depending on an individual's condition, it sometimes takes multiple rounds of testing to track down a disease-related mutation.
"We see patients that maybe have three, four, or five rounds of testing before they get a diagnosis for this relatively common presentation of having some colonic polyps, but not hundreds," he said. "We think that a cost-effective test where you can get information on all of these genes up front is going to be helpful."
"I'm not aware of any other commercial testing for tumor suppressor genes that has this kind of depth of coverage," King added. "What it allows the patient to have is complete information, accurately and quickly and at much less cost."
For validation studies of ColoSeq, the researchers tested more than 100 samples from several different cohorts, including samples from individuals with known mutations in one of the seven genes tested, from individuals with clinical symptoms that warranted ColoSeq testing, and from individuals not expected to harbor mutations in the genes.
Cell lines with known mutations and samples collected through HapMap were also tested as part of the assay's validation.
So far, ColoSeq has only been validated using DNA from peripheral blood samples, Pritchard noted, but he and his team are looking at ways of applying the assay using DNA from formalin-fixed tissue samples.
Researchers involved in developing the genetic test plan to include additional, less frequently mutated inherited colon cancer-related genes in the ColoSeq panel as they are clinically validated.
"We decided to start sort of conservatively with these core seven genes," Pritchard said. "We have the clinical expertise to know what mutations mean in these genes and we have the validation specimens to know that we are accurately detecting mutations in all of these genes."
Additional information on ColoSeq is available here.
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