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Can Deep Sequencing Cells Help Develop Targeted Therapies for Vascular Anomaly Patients?

Published on July 5, 2023 in Cornerstone Blog · Last updated 6 months ago


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Lymphatic system
Researchers utilized deep sequencing techniques to identify genetic variants related to diseases that were previously undescribed using conventional testing.

The Findings

Researchers from Children’s Hospital of Philadelphia expanded on a previous study that identified a mutation in the ARAF gene responsible for a vascular anomaly affecting the lymphatic system. Deep sequencing — a next-generation sequencing approach that reads a genomic nucleotide up to thousands of times, enabling the detection of mutations in <0.2% frequency of the original sample — helped scientists identify genetic variants related to diseases that were previously undescribed using conventional testing. In this study, researchers observed marked improvements in more than 60% of patients who were treated with targeted therapies related to their previously undetected genetic variants.

Why It Matters

Approximately 90% of genetic causes of vascular anomalies are acquired somatic mutations that only present themselves in certain cells or tissue types, often going undetected due to their low frequency — less than 1% in an individual’s DNA. Vascular anomalies can cause a variety of conditions in any part of the body, including the lungs, stomach, and blood, that result in physical deformities, organ dysfunction, and in some cases, death. These new findings will allow researchers to look directly into the low concentrations of potentially life-threatening mutations and treat these conditions with high precision using drugs informed by the genetic discovery.

Who Conducted The Study

CHOP researchers who contributed to the study include co-first authors Dong Li, PhD, junior faculty in the Center for Applied Genomics and Division of Human Genetics, and Sarah E. Sheppard, MD, PhD, MSTR, FAAP, FACMG, a research scientist in the Jill and Mark Fishman Center for Lymphatic Disorders and the Division of Human Genetics; and senior authors Denise Adams, MD, co-principal investigator of the Frontier Vascular Anomalies Program and director of the Comprehensive Vascular Anomalies Program; Yoav Dori, MD, PhD, pediatric cardiologist and director of the Pediatric Lymphatic Imaging and Interventions and Lymphatic Research; and Hakon Hakonarson, MD, PhD, director of the Center for Applied Genomics and co-principal investigator of the Frontier Vascular Anomalies Program.

How They Did It

Building on their previous findings, the current study deep-sequenced cells isolated from tissue samples and cell-free DNA collected from patients experiencing vascular anomalies to an unprecedented low frequency of 0.15% of the original sample.

Researchers obtained DNA from CD31+ cells or cell-free DNA isolated from lymphatic fluid or plasma from a cohort of 356 participants, 104 of which had primary complex lymphatic anomalies. These isolated cells underwent deep exome sequencing in which more than 60% of the samples tested positive for disease-causing genetic variants with between 2.3% and 44% allele frequency.

Scientists sequenced multiple samples to a depth of more than 1500x to determine if variant alleles could still be present at lower than 2% frequency. The remaining unresolved cases underwent ultra-deep panel sequencing that resulted in molecular diagnoses detected from variant alleles with a frequency as low as 0.15%.

Quick Thoughts

“This method is absolutely transformative for the field,” Dr. Hakonarson said. “It’s being performed at an unprecedented sequencing depth that we have mastered at CHOP. We are resolving more samples every day to help guide treatment of patients we previously could not.”

What’s Next

These findings will help inform decision making for currently available targeted therapies. More work is required to better understand why some of the same pathogenic variants can cause different conditions in individuals and to search for new, disease-causing variants not previously known to exist.

Where The Study Was Published

This study appeared in Nature Medicine.