It’s hard to tell the difference between the dozens of subtypes of limb girdle muscular dystrophy, a rare genetic muscle disease characterized by weakness in the hips and shoulders that makes it difficult to walk and raise the arms. Until now, subtyping has not been critical in patient care because no specific treatments have been available. But with gene therapies on the horizon targeting specific genetic variants, pinpointing the genetic roots of each patient’s disease has taken on new importance.
Hundreds of genes are associated with limb and girdle muscular dystrophy. While genetic testing can identify several rare genetic variants in each patient with the disease, there is no way to know which, if any, of these variants are responsible for a patient’s symptoms without careful and time-consuming additional experiments. Unfortunately, there is still no comprehensive catalog of all variants of all genes associated with limb girdle muscular dystrophy and whether each of these variants can cause the disease or is harmless.
“I get people emailing me all the time saying, ‘I have this option. Am I eligible for this experimental therapy?’ and most of the time I don’t have an answer,” said co-author Conrad “Chris” Weihl, MD, PhD, professor of neurology. Weihl is chief of the neuromuscular disease section and treats people with muscular dystrophy at Barnes-Jewish Hospital. “These patients are in limbo.
We can’t get them into clinical trials until they have a diagnosis. More than half of all patients with muscular dystrophy of the limb girdle are in this position. It is important that we resolve their diagnoses so that they can access the necessary therapies as they become available.”
Weihl and colleagues at the University of Washington have taken an important step toward creating a catalog that could help resolve inconclusive diagnoses. For one gene commonly involved in disease, scientists created a protein that would be made from the instructions of that gene. They then created all the possible protein variants that could be created by replacing one amino acid with another, analyzed the functions of the variants and classified each as either harmful or benign.
Now, if a patient has a variant of this one gene, doctors can determine its pathogenicity simply by looking up the variant in a catalog. In principle, the same approach could be used to resolve variants of unknown significance for many other genes associated with limb-girdle muscular dystrophy, greatly simplifying and speeding up the process of diagnosing this complex disease.
“People confuse knowing that there is a variant with knowing the cause of the disease,” said corresponding author Gabriel Haller, PhD, assistant professor of neurosurgery. “That’s not necessarily the case. In this study, most variants of unknown significance were found to be benign. If you find a variant but don’t know its meaning, you haven’t come up with the answer.”
Solving variants of unknown meaning is especially beneficial for members of underrepresented groups. Genetic databases are dominated by people of Western European descent and reflect the genetic diversity within this population. People from other backgrounds are more likely to have genetic variations that do not have a match in the reference databases and are therefore labeled “variant of unknown significance”.
For this study, the researchers comprehensively analyzed sarcoglycan beta, one of the genes most commonly associated with limb girdle muscular dystrophy. Sarcoglycan beta forms a complex with three other proteins on the surface of muscle cells. In order for muscles to contract effectively, the complex must form correctly and in the correct place in the cell. First author Chengcheng Li, PhD, a research scientist in the Weihl lab, created all 6,340 possible variants of the sarcoglycan beta protein.
She then used a fluorescent antibody to assess where each variant protein was located on the muscle cells. Those variants capable of forming the correct complex at the correct site in other words, variants with normal functional activity fluoresced brightly. Those that failed to form the complex correctly or were placed in the wrong place i.e. less functional variants fluoresced more weakly.
The correlation was perfect. All variants known to cause disease have low functional activity, while all known benign variants have high scores. Not only that, the degree of functional activity was correlated with the severity of the disease.
People with the most severe symptoms of limb-girdle muscular dystrophy may need to start using wheelchairs as early as age 7, while people with milder symptoms may need one decades later, if at all. In the study, variants with lower functional activity appeared more often in patients with more severe symptoms.
“Twenty percent of variants of unknown significance were shown to be pathogenic, meaning they could be amenable to potential therapies,” Weihl said. “My dream is that one day we’ll be able to give people a genetic test report that says, ‘You have this variant and it’s suitable for this type of therapy,’ and we can immediately start them on the best therapy.” Or we tell them, ‘Your variant is benign,’ and we keep looking until we find the variant that is responsible for the patient’s disease.”
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