Scripps scientists unravel mystery of genetic mutation in CMT disease

LA JOLLA, Calif.— Scientists at the Scripps ResearchInstitute have made an important discovery regarding the mutation of a geneassociated with Charcot-Marie-Tooth (CMT) disease, and they hope their findingswill lead to the development of new therapies for the genetic nerve disease aswell as other conditions.

CMT disease, also known as hereditary motor and sensoryneuropathy, was named after three physicians who first described the disease in1886: Jean-Martin Charcot, Pierre Marie and Howard Henry Tooth. The disease ischaracterized by loss of muscle tissue and touch sensation in body extremities,predominantly in the feet and legs, but also in the hands and arms. Incurable—butnot fatal—the disease is one of the most common inherited neurologicaldisorders, affecting one in 2,500 people.

CMT disease is caused by mutations that cause defects inneuronal proteins. Nerve signals are conducted by an axon with a myelin sheathwrapped around it. Most mutations in CMT disease affect the myelin sheath. Someaffect the axon. To date, researchers have identified mutations in 39 genes,and have classified CMT disease into two subtypes according to these mutations:Type 1 primarily affects the myelin sheath, and is either dominant, recessiveor X-linked. Type 2 primarily affects the axon, and is either dominant or recessive.The two types can also be mixed.

In a further classified CMT disease type 2D, which is causedby mutations in the GARS gene, a person inherits only one faulty copy of theGARS gene from one parent. The GARS gene holds the instructions for producingan enzyme called glycyl-tRNA synthase (GlyRS), which is vital to the process bywhich amino acids are attached to one another during protein synthesis. Althoughprevious research has resulted in the identification of 11 different kinds ofmutations in the GARS gene that cause type 2D, scientists have not understoodwhy some of the mutations affect the protein-building function of the GlyRSenzyme, but others don't—until now.

Publishing their study of this puzzle this month in anonline edition of Proceedings of theNational Academies of the Sciences (PNAS), the Scripps team suggests thatin fact, a change in enzyme activity is not what causes CMT disease at all.Rather, GARS mutations cause a structural opening in the resulting mutantprotein which then gain a new function that is toxic to nerve cells, saysScripps Research Associate Prof. Xiang-Lei Yang, the senior author of thestudy.

"Does the mutation result in a gain of function, or a lossof function? That question is at the core of our study," Yang says.

Thus, scientists have been trying to find a common featureshared by all GlyRS mutants that might explain the disease mechanism. Thiscommon feature shared by all GlyRS mutants that might explain the diseasemechanism, Yang adds.

To find this common feature, the Scripps team in 2007examined the three-dimensional structure of GlyRS and published the X-raycrystal structure of the wild-type protein and one of the mutants. However,finding no dramatic conformational change between the structures, theresearchers hypothesized that the differences may actually be found in thecrystal packing.

"We thought maybe because the molecules in the crystal areso tightly packed against each other, this packing somehow suppressed theconformation of change," Yang explains. "That is why we turned tohigh-resolution X-ray defraction."

In collaboration with Hui-Min Zhang, a postdoctoral fellowin the laboratory at the National High Magnetic Field Laboratory at FloridaState University, Yang and a graduate student, Weiwei He, usedhydrogen–deuterium exchange to obtain the structures of five different GlyRSmutants and the wild-type protein in solution. This method gave informationabout which parts of a protein are in touch with a solution. In collaborationwith Min Guo, assistant professor at Scripps Florida, small-angle X-rayscattering was used to measure changes in the overall shape of the proteinstructure in solution.

Significantly, the scientists observed that each of fivespatially dispersed mutations induced the same conformational opening of aconsensus area that is mostly buried in the wild-type protein. By becoming moreopen to potential new partners, the GlyRS mutants may gain a new function thatis toxic to nerve cells. That would explain why type 2D is inherited in adominant fashion, Yang says.

The identified neomorphic surface is thus a candidate for makingCMT-associated pathological interactions, and a target for disease correction,the researchers say.

"This was very exciting for us," she says. "While our lab isfocused on understanding the multifunctionality of the GARS gene, at the sametime, we are trying to understand the disease connection of these mutations.The intent is that we can develop drugs to fit into the opening, blocking itsaccess to other proteins. We would like to be able to come up with a monoclonalantibody that can block the surface and see in mice if that can reverse the CMTphenotype. This would be a very nice proof of concept."

Scripps already has a commercial partner in mind to takethese findings to the next level, and the end result could also haveimplications for other mutation-induced human diseases, such as amyotrophiclateral sclerosis (ALS), also known as Lou Gehrig's disease.

"Some proteins may be relatively unstable and can be easilytriggered into another conformation by different types of mutations," explainsYang. "This example deals with the GlyRS protein, but the general idea can beapplied to many other mutation-induced human diseases."

The paper, "Dispersed disease-causing neomorphic mutationson a single protein promote the same localized conformational opening," wassupported by grants from the U.S. National Institutes of Health, the NationalScience Foundation and the state of Florida.