Disruptive Noncoding Mutations Found for Rare Disease

October 3, 2016
Disruptive Noncoding Mutations Found for Rare Disease
Source: Cell, http://dx.doi.org/10.1016/j.cell.2016.09.005

Scientists at Johns Hopkins University believe they are closer to understanding the genetic mechanism of a rare, complex, multiple-gene disorder called Hirschsprung's disease. The results from the latest study—entitled “Enhancer Variants Synergistically Drive Dysfunction of a Gene Regulatory Network In Hirschsprung Disease” and published in Cell—suggest that many patients develop the disease when multiple mutations in gene regulatory sequences of a particular gene combine to destroy the normal cooperative function of a whole network of genes.

The Hopkins team expects that similarly dysfunctional gene networks influence the onset and progress of other diseases that involve multiple genes and that dissecting these networks may eventually lead to improved diagnosis and treatment options for patients with disorders like cancer, diabetes, and high blood pressure.

"We tend to think of genes as operating on their own, but they can't and don't," explained senior study investigator Aravinda Chakravarti, Ph.D., professor of medicine and director of the Center for Complex Disease Genomics at Johns Hopkins, as well as pioneering researcher on the genetics of so-called multigenic and complex diseases. "Instead, they exist within 'families' of genes and regulatory elements that live in the same section of the chromosome and frequently interact, each having its job but all working together toward one goal: making a gene function. If you perturb one individual, everyone else is affected."

Hirschsprung's has been a model disease with complex genetic causes to study, as the disorder's wide range of symptoms affect only a single organ—the colon. Overall, the disease occurs when nerve cells fail to connect to the muscles of the colon during fetal development, leaving a child unable to pass stool. It affects about one in 5,000 newborns and can usually be resolved surgically, but 30 to 50 percent of patients still have complications throughout their life.

Over the past decade, Dr. Chakravarti and his team have performed several genome-wide association studies (GWAS) to identify common genetic variants associated with the disease. Further research showed that variations in the RET gene and its regulatory elements were standouts among patients, in that almost every person with Hirschsprung's disease has at least one RET-associated variant. However, because many healthy people without Hirschsprung's do too, the causal mechanism remained a mystery and led them to look for multifactorial causes.

The investigators decided to focus on eight single nucleotide polymorphisms (SNPs) known to occur in specific regions near the RET gene. These gene enhancers act like a rheostat, turning up or down the activity, or transcription, of nearby genes by binding and correctly positioning proteins called transcription factors.

The Hopkins team swapped out the RET gene for a reporter gene and ran a series of tests in a human nerve cell line that showed only three of the SNPs, each in a different enhancer, were capable of decreasing gene activity on their own by weakening the binding of that enhancer's transcription factor.

Interestingly, when the researchers tested the three SNPs in different combinations, they found that although none of the enhancers are known to interact with each other directly, a general trend appeared—the more enhancers were affected, the lower the reporter gene activity. When the team looked at the frequency of these regulatory variants among 346 patients with Hirschsprung's disease and 732 healthy controls, the trend was confirmed. Those with all three SNPs had a fourfold higher risk of having the disease.

"These findings help explain why RET-associated variants are necessary but not sufficient to cause Hirschsprung's disease," noted lead study author Sumantra Chatterjee, Ph.D., postdoctoral fellow in Dr. Chakravarti’s lab. "It provides a way forward for studying genetic synergy in the context of health disorders."

"We've been able to show that these genes are intimately related to each other, not because of similarities in sequence or function or even location in our genome,” Dr. Chakravarti added. “But because they all work together to execute one function, getting cells to become nerve cells for the colon."

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