New GAA mutations destabilize key enzyme in Pompe disease: Study
Small molecules partly restored enzyme activity, pointing to possible treatment
Written by |
Two newly identified mutations in the GAA gene, along with a previously known variant of unclear significance, were found to reduce the activity of the enzyme whose deficiency causes Pompe disease, a study has shown.
The three variants appeared to produce unstable forms of the enzyme that were prone to being destroyed inside cells. Small molecules designed to stabilize proteins, called chaperons, partly restored enzyme activity in some cell models, pointing to a possible strategy “for facilitating the treatment of Pompe disease,” researchers wrote.
The study, “Two novel GAA missense variants and a known VUS in pompe disease: Functional characterization and pharmacological chaperone therapy,” was published in Genes & Diseases.
Mapping of GAA mutation spectrum vital for early diagnosis, treatment
Pompe is caused by mutations in the GAA gene, which provides instructions for making acid alpha-glucosidase (GAA), an enzyme that helps break down glycogen — the body’s stored form of sugar — inside lysosomes, the cell compartments responsible for recycling waste.
When GAA is missing or defective, glycogen builds up to harmful levels in cells, especially muscle cells, damaging tissues and leading to Pompe symptoms. Because different GAA mutations can leave varying levels of residual enzyme activity, symptoms and disease severity can vary widely from person to person.
Thus, “comprehensive characterization and mapping of the GAA mutation spectrum are crucial for early diagnosis, treatment and prognosis,” the researchers wrote.
With this in mind, a team in China reviewed 39 unique GAA variants identified in 42 samples collected between 2017 and 2020. Some individuals were suspected to have Pompe disease based on genetic testing and clinical features; others were classified as GAA variant carriers, meaning they carried a genetic change in the gene but did not necessarily develop the disease.
The variants included 24 missense variants, which change a single amino acid, one of the building blocks of a protein; nine frameshift variants, which shift how a gene’s instructions are read and often disrupt the resulting protein; three nonsense variants, which introduce an early stop signal and produce a shortened protein; and three splicing variants, which interfere with how genetic instructions are processed before a protein is made.
After reassessing the variants using standard genetic classification guidelines, the researchers categorized 11 as pathogenic (disease-causing), 13 as likely pathogenic, and 15 as variants of uncertain significance (VUS), or genetic changes whose role in Pompe remains unclear.
All three variants led to sharply reduced enzyme activity
The researchers then focused on three missense variants for closer study. These included p.E174G, previously classified as a VUS, and two newly identified variants, p.G461C and p.W621G. All three affected highly conserved parts of the GAA protein, meaning regions that have remained largely unchanged through evolution and are usually important for normal function. Computer analysis consistently predicted that the variants would impair the enzyme activity.
To test those predictions, the researchers introduced both normal and mutant forms of the GAA gene into several laboratory-grown cell lines and measured enzyme activity. Compared with normal GAA, all three variants led to sharply reduced enzyme activity. Cells carrying the p.E174G variant retained more enzyme activity than those carrying the p.G461C or p.W621G variant.
The researchers next wanted to understand the reasons behind the reduced enzyme activity.
For GAA to function properly, the enzyme must undergo several processing steps within the cell before reaching lysosomes, where it becomes fully active. In cells carrying the variants, mature GAA protein was barely detectable despite normal levels of precursor protein, suggesting that the enzyme was failing to complete the maturation process.
Further experiments helped explain why.
We functionally characterized three missense variants in GAA, two of which are novel, expanding the GAA mutation spectrum.
Instead of successfully traveling to their intended destination, the faulty enzymes accumulated inside the endoplasmic reticulum — a cellular compartment involved in producing and folding proteins — while showing little evidence of moving through the Golgi apparatus, where proteins are normally modified before reaching their final destination. As a result, delivery to lysosomes was markedly impaired.
Further analysis showed that faulty enzymes proved largely unstable and, rather than completing their journey through the cell, were largely destroyed through endoplasmic reticulum-associated degradation (ERAD), a cellular quality-control system that identifies and destroys misfolded proteins.
Finally, the researchers explored whether chaperones could rescue enzyme function.
Treatment with 1-deoxynojirimycin, also called duvoglustat, and N-butyl-deoxynojirimycin, also known as miglustat — the stabilizing component used in the combination therapy Pombiliti + Opfolda — improved GAA activity in some cell models, though not all. These findings suggest treatment responses may vary depending on the cellular environment.
“We functionally characterized three missense variants in GAA, two of which are novel, expanding the GAA mutation spectrum,” the researchers concluded.
