Determining the Role of Ancestry in Cancer Development and Response to Future Therapy

Growing up in a Native American family from Nevada, Dr. John Tyson McDonald noticed something about his culture: Native Americans suffered from unique health conditions that weren’t common in other cultures. Now an Assistant Professor in the Department of Physics and principal investigator at the Hampton University Cancer Research Center (HUCRC) in Virginia, McDonald knows firsthand that ancestry plays a role in genetic predisposition to certain conditions. It’s this knowledge that drives his research.

McDonald completed his undergraduate training as a nuclear engineer at the University of Michigan–Ann Arbor, and a PhD as a radiation biologist in the Biomedical Physics program at the University of California, Los Angeles. In 2013, Hampton University was home to one of only a handful of proton radiation therapy centers in the nation; so, combining his interests, McDonald joined the university to investigate the role ancestry plays in carcinogenesis and response to radiation therapy.

“We currently have a grant from the National Cancer Institute to look at genetic variants in African American women with breast cancer,” said McDonald. “We know that mutations in BRCA1 and BRCA2 confer the greatest risk of breast cancer in white women. We also know that in ethnically diverse populations, variants of unknown significance (VUSs) are found in 1 in 3 women with cancer. We have a unique set of samples that we can probe for these variants in pathways leading to cancer.”¹.

The “unique set of samples” was collected by McDonald’s collaborator and co–principal investigator Luisel Ricks-Santi of Howard University, and consists of over 300 frozen tissue and nucleic acid samples from African American women with breast cancer, plus 300 control samples. The sample set also includes material from family members of some of the women. According to McDonald, these samples are unique because most genome-wide association studies (GWAS) focus on European variants and don’t include data from family members.

How Is Sanger Sequencing Used?

As part of his research, McDonald uses Sanger sequencing to verify the presence of rare variants in his samples. He says he’s probably biased because he started his career as a researcher using Sanger sequencing, but he prefers it to other sequencing technologies.

“I trust Sanger,” he said. “It’s the tried-and-true method for sequencing. It’s been around for a while, and I think we’re using it in new ways, which is exciting.”

One of the ways McDonald is using Sanger sequencing is to determine editing efficiency following CRISPR-Cas9 gene editing. Once he has identified a variant in his samples, McDonald uses CRISPR-Cas9 gene editing to knock it into a cancer cell line, and Sanger sequencing to determine editing efficiency.

Using products from Thermo Fisher Scientific as part of his protocol, McDonald designs and employs small guide RNAs and synthetic DNA sequences as repair templates for the knock-in experiment. In collaboration with a former mentor, he is also knocking in a selection marker to improve positive identification of the particular variant.

While next-generation sequencing has higher throughput, McDonald says he uses Sanger sequencing to confirm variants because it is cost efficient, has a simple workflow, and is still the gold standard. He also appreciates the easy-to-use data analysis modules on Connect, Thermo Fisher’s cloud-based platform. “I used to try to distinguish dual peaks by hand. Now, I run everything through Peak Scanner. The computer algorithm just pulls it apart, and it generally matches our NGS results perfectly.” 

“Sanger sequencing is a wonderful tool at all stages, from plasmid design to preliminary identification of editing in the heterogeneous cell culture, and in the final positive identification of clones,” said McDonald.

Once he verifies the presence of the knock-in variant using Sanger sequencing, he then has a cell line that he can use to examine response to radiation therapy—but that’s still a way off, he says. “We haven’t found a particular variant that we think will make for a good model yet,” said McDonald.

He says that, in addition to potentially impacting treatment decisions, the future inclusion of these low-level variants on panels used for genetic counseling would greatly expand the populations for which the panels would be relevant, such as African Americans.

How is Sanger Sequencing Used In Animal Models?

In a recent study, McDonald used Sanger sequencing  to confirm three variants of high quality in canine signaling pathways and show that they are similar to those in humans with peripheral T cell lymphoma².

“Because our results show a prominent overlap in signaling pathways, implicating that T cell lymphoma varies in dogs in a similar way to humans, canines may be an ideal animal model to identify potential drug targets and proteomic biomarkers of human T cell lymphoma, and to predict therapeutic outcomes,” said McDonald.

In his spare time, McDonald enjoys the beach, learning Japanese, and improving on the piano to exercise the artistic side of his brain. In fact, he just had his first formal recital.

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1. Ricks-Santi L, McDonald JT, Gold B et al. (2017) Next generation sequencing reveals high prevalence of BRCA1 and BRCA2 variants of unknown significance in early-onset breast cancer in African American women. Ethn Dis 27(2):169-178.
2. McDonald JT, Kritharis A, Beheshti A et al. (2018) Comparative oncology DNA sequencing of canine T cell lymphoma via human hotspot panel. Oncotarget 9(32):22693-22702.

 

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