For decades, researchers have been slowly uncovering the genetic causes of congenital heart disease (CHD). About 45 percent of CHD cases have an identifiable cause, such as chromosomal abnormalities, genetic mutations that affect protein-coding genes, or environmental factors. What about the rest of CHD?

Although non-coding DNA elements have long been thought to be involved in CHD, few variants have been identified as causative. A new study from the laboratory of cardiologist William Pugh, MD, at Boston Children’s Hospital provides the strongest evidence yet that non-coding DNA contributes to CHD. It also establishes a pipeline for finding and validating variants.

“It’s very difficult to determine which non-coding variants are actually involved in heart disease and which are just innocent bystanders,” says Boston Children’s Hospital Basic and Translational Cardiovascular Research. said Pugh, vice chair of the board. “The average person has about 70 mutations in non-coding regions. Also (I don’t share it with my parents). And when you compare people with and without heart disease, there are the same number of variants. ”

As explained in natural genetics, Pu and colleagues used data from the multicenter Pediatric Cardiac Genomics Consortium (PCGC), led by Jane Neuberger, MD, MPH, at Boston Children’s Hospital. Amy Roberts, MD. and Wendy Chan, MD. The consortium enrolls more than 13,000 of his CHD patients (about one-fifth of them at Boston Children’s Hospital) and more than 18,000 of her family members. More than 3,000 patients have had their complete genome sequenced, and Pu’s lab has obtained whole-genome sequence data for 750 of her patients and their parents.

Narrow down the causative variant

To eliminate non-coding mutations that were likely to be background “noise” rather than the cause, the researchers applied statistical filters. They also focused on: Also Mutations that are more likely to be the cause.

This left nearly 7,000 mutations in non-coding DNA regions in CHD patients. To investigate the function of the mutants, the team created a high-throughput system to test the effects of the mutants on the expression of neighboring genes.

“Of the 7,000 mutations we tested, we found 403 mutations that affected the activity of transcriptional enhancers,” Pu said. “These would be good candidates for variants that may actually contribute to congenital heart disease.”

They then painstakingly introduced the 10 non-coding variants into the appropriate locations within the genome of normal human stem cells. When the stem cells were instructed to form cardiomyocytes, four of the 10 mutants changed the expression of adjacent genes.

“These genes were already known to cause congenital heart disease when mutated,” Pugh said. “Mutants either cause the enhancer to no longer perform its original function, resulting in a drop in gene expression levels, or they change a region that should not be an enhancer into an enhancer, activating the gene at the wrong time or in the wrong location. . Both scenarios can lead to heart disease.”

As a second test, we performed single-cell sequencing of cardiomyocytes derived from stem cells carrying the four mutations. These cells also have altered gene expression profiles, consistent with their potential to cause CHD.

Future directions of CHD genetics

Pu believes that hundreds of additional non-coding mutations may be involved in structural heart disease and help explain many cases of CHD that are negative on exome sequencing. Co-lead author Sarah Morton, MD, of the Department of Neonatal Medicine, specializes in researching the genetic causes of CHD. Using data from her study, she created an algorithm to prioritize the variants most likely to be associated with her CHD for further investigation.

“When evaluating coding gene variants, such as missense variants that change a single amino acid, we want to know how likely they are to alter protein expression,” Morton says. “There are relatively few tools to predict the functional impact of non-coding mutations, especially those that may be associated with pediatric disease.”

The researchers hope to expand their search beyond cardiomyocytes to other cell types, such as neural crest cells and endocardial cells.

In the future, the results of this study could be used to stratify patient risk, predict surgical outcomes, and even more distantly, to predict structural changes during pregnancy and after birth. It may even be possible to change the trajectory of heart disease.

“It’s completely unexplored territory,” Pugh said. “What we found in this study is just the tip of the iceberg.”

Dr. Feng Xiao and Dr. Xiaoran Zhang of Boston Children’s Hospital are the study’s other lead authors.

Visit the Pu Lab to learn more about research and innovation at the Boston Pediatric Benderson Family Heart Center.