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Diagnosis Of Rare, Genetic Muscle Disease Improved By New Approach

It's not easy to distinguish between the dozens of subtypes of limb girdle muscular dystrophy—a rare, genetic muscle disease characterized by weakness in the hips and shoulders that causes difficulty walking and lifting the arms. Until now, determining the subtype has not been critical in caring for patients, because no specific treatments have been available. But gene therapies are on the horizon, and such therapies are targeted to specific genetic variants, so pinpointing the genetic roots of each patient's disease has taken on a new importance.

In new research, a team at Washington University School of Medicine in St. Louis has developed an approach that could help doctors make more precise diagnoses. The study is published in the Journal of Clinical Investigation.

Hundreds of genes are associated with limb girdle muscular dystrophy. While genetic testing may identify a handful of rare genetic variants in each patient with the condition, there's no way to know without painstaking, time-consuming additional experiments which, if any, of those variants is responsible for a patient's symptoms. Unfortunately, no comprehensive catalog exists yet of all the variants of all the genes linked to limb girdle muscular dystrophy, and whether each of those variants can cause disease or is harmless.

"There are people emailing me all the time saying, 'I have this variant. Am I eligible for this experimental therapy?' and much of the time I don't have an answer," said co-author Conrad "Chris" Weihl, MD, Ph.D., a professor of neurology. Weihl is chief of the neuromuscular diseases 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 limb girdle muscular dystrophy are in this position. It's critical that we resolve their diagnoses so we can get them access to necessary therapies as soon as they become available."

Weihl and colleagues at Washington University have taken an important step toward creating a catalog that could help resolve inconclusive diagnoses. For one gene commonly involved in the disease, the researchers created the protein that would be made from that gene's instructions. Then, they made every possible protein variant that could be formed by swapping out one amino acid for another, analyzed the functions of the variants and classified each as 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 the 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, vastly simplifying and speeding up the process of diagnosing this complex disease.

"People conflate knowing that there is a variant with knowing the cause of a disease," said corresponding author Gabriel Haller, Ph.D., an assistant professor of neurosurgery. "That isn't necessarily the case. In this study, most of the variants of unknown significance turned out to be benign. If you find a variant but you don't know its significance, you haven't figured out the answer."

Resolving variants of unknown significance is particularly beneficial to members of underrepresented groups. Genetic databases are dominated by people of western European background and reflect the genetic diversity found in that population. People of other backgrounds are more likely to carry genetic variations that have no match in reference databases and thus get the label "variant of unknown significance."

For this study, the researchers did a comprehensive analysis of sarcoglycan beta, one of the genes most often linked to limb girdle muscular dystrophy. Sarcoglycan beta forms a complex with three other proteins on the surface of muscle cells. For muscles to contract effectively, the complex must form properly and in the right place on the cell.

First author Chengcheng Li, Ph.D., a staff scientist in Weihl's lab, created all 6,340 possible variants of sarcoglycan beta protein. Then, she assessed where each variant protein was located on muscle cells, using a fluorescent antibody. Those variants capable of forming the correct complex at the correct spot—in other words, variants with normal functional activity—fluoresced brightly. Those that failed to form the complex properly or were located at the wrong place—i.E., less functional variants—fluoresced more dimly.

The correlation was perfect. All variants known to cause disease scored low in functional activity, while all known benign variants scored high. Not only that, the degree of functional activity correlated with severity of disease. People with the most severe symptoms of limb girdle muscular dystrophy may need to start using wheelchairs as young as age 7, while people with milder symptoms may not need one until decades later, if ever. In the study, variants with lower functional activity more often appeared in patients with more severe symptoms.

"Twenty percent of the variants of unknown significance turned out to be pathogenic, which means 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 amenable to this type of therapy,' and we can start them on the best therapy right away. 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."

More information: Chengcheng Li et al, Comprehensive functional characterization of SGCB coding variants predicts pathogenicity in limb-girdle muscular dystrophy type R4/2E, Journal of Clinical Investigation (2023). DOI: 10.1172/JCI168156

Citation: Diagnosis of rare, genetic muscle disease improved by new approach (2023, June 15) retrieved 25 June 2023 from https://medicalxpress.Com/news/2023-06-diagnosis-rare-genetic-muscle-disease.Html

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FDA Approves First Gene Therapy For Duchenne Muscular Dystrophy

For genetic conditions like Duchenne muscular dystrophy, there is little doctors can do to slow or treat the condition other than trying to manage symptoms, since only addressing the genetic changes responsible can help halt the disease.

Earlier today (June 22), the U.S. Food and Drug Administration (FDA) approved the first such intervention, a gene therapy called Elevidys, from Sarepta Therapeutics, a Massachusetts-based biotech company. The approval applies only to children ages 4 and 5, which reflects the conflicting opinions within the agency about the quality of data supporting the gene therapy's effectiveness, which delayed the decision from the original anticipate date, May 29.

Duchenne is an inherited genetic disorder, more common among males, that affects the muscles, including those not only in the limbs but in the heart and respiratory system as well, causing them to progressively weaken and waste away. The disease is caused by a genetic variant in the dystrophin gene that reduces the amount of dystrophin protein in muscles, making them stiffer and less pliable, eventually causing muscles to contracted too tightly and become less mobile. Over time, scarring and fibrosis further limit muscle function.

Most people born with the inherited genetic disorder start to show symptoms of muscle weakness as early as age 2, and as the disease worsens, typically lose their ability to walk and eventually to breathe on their own. There is no cure, but doctors can prescribe medications to improve muscle strength, particularly for the heart and lungs, or recommend surgery to treat severe contractions that can affect posture to help patients live to their 20s or 30s.

A new pioneering gene therapy

Elevidys works by replacing the Duchenne variant of dystrophin with man-made version based on a version found in a patient with a milder form of the disease, called Becker muscular dystrophy. That patient remained mobile and ambulatory into his 60s. His dystrophin gene was significantly shorter than normal versions, a key feature that made the gene therapy possible, since the dystrophin gene is the largest human gene, making it nearly impossible to pack into small enough molecular vehicles to deliver it into cells safely.

Dr. Jerry Mendell, a neurologist at Nationwide Children's Hospital, and Dr. Louise Rodino-Klapac, then a post doctoral fellow in Mendell's lab and now chief scientific officer at Sarepta, spent years figuring out which parts of the Becker dystrophin gene were essential to conserving muscle function. That genetic construct is the backbone of Sarepta's gene therapy for Duchenne, which patients receive in a single injection.

Before the researchers could license the technology to the company, they had to solve the problem of how to deliver the carefully selected, improved version of the dystrophin gene to as many muscles throughout the body as possible. They eventually settled on a cold virus vector that can easily find muscle cells, and added a genetic sequence that was activated when it bound to muscle cells, ensuring the genetic payload was preferentially delivered to muscles and not to other types of cells.

In the first human studies of the gene therapy, in four boys ages 4 to 7 years old in 2018, the new dystrophin gene found its targets and the amount of healthier dystrophin they produced was "higher than we anticipated," says Mendell. "We are at the five year mark for those kids and they haven't declined; that's very encouraging."

Inconclusive results in a larger study

In the next, larger study of patients however, the results were less conclusive. Mendell faults the study design, which involved a broader range of patients including those who might have been too far along in their disease to show benefit from the therapy. Some FDA officials questioned whether the increased dystrophin levels seen in lab tests translated to real-world benefits such as greater muscle strength, or extended time during which the patients could walk independently.

That uncertainty is likely the reason why the FDA decided to limit its approval to 4- and 5-year old patients.

But for boys that age, the results look good: that data, says Dr. Barry Byrne, chief medical advisor for the Muscular Dystrophy Association (MDA) and director of the Powell Gene Therapy Center at the University of Florida, show "that are doing things that untreated boys could never do. One of the simplest things is to run. Boys who are not treated have trouble putting even one foot on the floor, [much] less both feet which is required to run. And running is a fundamental part of childhood and playing."

Indeed, the first patients who received the gene therapy appear to have stabilized—which "buys time," says Sharon Hesterlee, chief research officer at MDA, since there are other drugs in development that can reduce other disease processes, such as muscle scarring. And if the gene therapy can keep muscles functioning longer, some people who receive it early enough may never develop the more severe forms of the condition.

A potential price tag of more than $1 million

The next challenge for patients and their families will be access. Sarepta has priced the treatment at $3.2 million, which, it says, as a one-time therapy, is still cost effective compared to standard therapy. In the company's own analysis, it determined that the treatment would be cost effective if priced anywhere from $5 million to $13 million. It's now up to insurers to determine if they agree, and will reimburse for the therapy at that cost.

In addition, the current age requirement means access could become challenging if insurers strictly enforce the FDA approval recommendations for four- and five-year olds only, and families find themselves racing against an age deadline as affected children approach their sixth birthday.

While the treatment currently has an age-limited approval, Rodino-Klapac, Sarepta's chief scientific officer, says in the future it could lead to options for Duchenne patients of all ages. "The mechanism of action applies to all patients with Duchenne muscular dystrophy regardless of age or disease status," she says. "We may see differences in functional outcomes based on where they are in the course of their disease because it targets the muscle they have, but we believe it is applicable to all patients." Rodino-Klapac says there are studies currently underway that could provide data to support that by the end of this year.

Gene therapy is still a nascent technology, and approvals like this one will help more patients to take advantage of it, and allow researchers to learn more about how to optimize it and apply those lessons to other genetic conditions as well. For Duchenne patients, it's the beginning of a new era of potentially life-changing therapy. "This is the opening event," says Mendell, "the pioneering event,"

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New Approach Could More Accurately Diagnose Limb-Girdle Muscular Dystrophy

The advent of gene therapies, which can target specific variants, means pinpointing the genetic roots of each patient's disease has taken on new importance, researchers said.

A team of researchers developed an approach that may provide a more accurate diagnosis of limb-girdle muscular dystrophy (LGMD) type R4/2E, according to findings published in The Journal of Clinical Investigation.

LGMD is a rare genetic muscle disease that causes weakness in the hips and shoulders, which can lead to difficulty walking and lifting the arms. Individuals with the most severe symptoms of the disease may need to start using wheelchairs as young as age seven.

Although in the past it has not been crucial to differentiate between different subtypes of LGMD, the advent of gene therapies, which can target specific variants, means pinpointing the genetic roots of each patient's disease has taken on new importance, researchers said.

In an effort to help resolve inconclusive diagnoses, investigators at Washington University School of Medicine in St. Louis have taken a step toward creating a catalog of variants of genes linked with LGMD.

"Limb-girdle muscular dystrophy (LGMD) type R4/2E is caused by mutations in β-sarcoglycan (SGCB)," they explained. "β-sarcoglycan in association with α-, γ-, and δ-sarcoglycan form a four protein transmembrane complex (SGC) that localizes to the sarcolemma. Biallelic loss of function mutations in any subunit can lead to LGMD."

Specifically, researchers performed deep mutational scanning (DMS) of SGCB and assessed SGC cell-surface localization for all possible amino acid changes. From this, they found variants with less severe functional scores appeared more often in patients with slower disease progression, suggesting there may be a relationship between variant function and disease severity.

Variants known to cause LGMD also scored low in functional activity and all known benign variants scored high.

In addition, "positions intolerant to variation mapped to points of predicted SGC interactions, validated in silico structural models and enabled accurate prediction of pathogenic variants in other SGC genes," researchers wrote.

Results may not only be useful for the clinical interpretation of SGCB variants, but could also improve LGMD diagnoses, and potentially enable wider use of gene therapies.

"Twenty percent of the variants of unknown significance turned out to be pathogenic, which means they could be amenable to potential therapies," said co-author Conrad Weihl, MD, PhD, in a release.

The study marks the first time a full-length muscular dystrophy gene has undergone DMS. Findings also suggest pooled functional screens are a viable method of large-scale functional assessment of protein-coding genetic variation in other muscular dystrophy genes, authors added.

"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 amenable to this type of therapy,' and we can start them on the best therapy right away. 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," Weihl said.

More research could probe whether the functional scores measured in the current study correlate with more subtle muscle traits, like exercise intolerance, in the general population.

Investigators were unable to measure the effects of the variants on endogenous mRNA or protein levels, marking a limitation to the study. They also did not measure the effect of coding and non-coding genetic variation on splicing.

"In the future, splicing effects could be measured by mutagenizing splice regions in the endogenous locus and measuring cell-surface expression of the SGC complex in primary muscle cells," they wrote.

Their assay did not measure muscle integrity or atrophy over time.

Reference

Li C, Wilborn J, Pittman S, et al. Comprehensive functional characterization of SGCB coding variants predict pathogenicity in limb-girdle muscular dystrophy type R4/2E. J Clin Invest. Published online June 15, 2023. Doi:10.1172/JCI168156

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