NIH scientists are developing a breath test for methylmalonic acidemia

Researchers at the National Institutes of Health have developed a breath test that measures how well patients with methylmalonic acidemia (MMA) respond to a liver or combined liver and kidney transplant. The researchers also used the test to assess the severity of the disease in people and determine whether they would benefit from surgical or experimental genomic therapies that target the liver. The study results were published in Genetics in Medicine. Scientists from the National Institute for Human Genome Research (NHGRI) led the project team with staff from the National Institute for Diabetes and Digestive and Kidney Diseases and the National Institute for Mental Health.

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The breath test for methylmalonic acidemia measures the severity of the disease and the success of liver transplantation in patients. Photo credit: Ernesto del Aguila III, NHGRI.

MMA is a rare genomic disease that affects the body’s ability to metabolize certain proteins and fats. This leads to the formation of toxic substances that can lead to kidney disease, pancreatitis, movement disorders, mental impairment, complications in many organs and, in severe cases, death. One in 80,000 children born in the United States will be diagnosed with MMA on newborn screenings. Currently, MMA is incurable, but people with MMA manage their symptoms through dietary restrictions and vitamin supplements. In extreme cases, patients receive liver or combined liver and kidney transplants, which help restore normal levels of metabolic proteins.

“Large fluctuations in the metabolism of a patient’s body make it difficult for us to say whether treatments like genome editing and transplants are likely to be successful,” said Dr. Charles P. Venditti, senior author and senior researcher in the NHGRI Medical Genomics and Metabolic Genetics Branch. “Instead of looking at the values, we decided to measure the metabolism ourselves.”

Large fluctuations in metabolic substances in patients’ bodies make it difficult for us to say whether treatments such as genome editing and transplants are likely to be successful. Instead of looking at the values, we decided to measure the metabolism ourselves.

One form of MMA is caused by mutations in the methylmalonyl-CoA mutase gene (MMUT), which codes for the MMUT protein. People with this form of MMA are deficient in MMUT protein, which plays a crucial role in metabolism. The protein is involved in the biological steps that help break down foods, fats, cholesterol, and amino acids.

MMUT helps break down food into a chemical by-product called propionate, which is followed by an integral process in metabolism called oxidation. Through oxidation, a healthy body converts propionate into energy and carbon dioxide, which is exhaled. However, this process is flawed for people with MMA.

Because MMUT protein function is impaired in people with MMA, Venditti and his team studied how well the MMUT protein contributed to the breakdown of propionate in both patients who received treatment or not. The researchers believed that this would act as a substitute for how much oxidation was going on in a patient’s body.

“We wanted to measure exhaled carbon dioxide because we wanted to use a breath test to track propionate oxidation in a non-invasive way,” said Dr. med. Irini Manoli, co-author and associate investigator at NHGRI Medical’s Genomics and Metabolic Genetics branch. “The trick was to sort of ‘tag’ the carbon dioxide so we could see which patients were unable to oxidize propionate due to a faulty MMUT protein.”

Usually, the carbon dioxide we breathe out from the breakdown of propionate in the body contains a lighter, more common form of carbon, carbon 12. But because carbon dioxide, which contains carbon 12, is simply released through various metabolic processes in the human body, the measurement of the amount of MMA -Patients of exhaled carbon dioxide would not show how well MMUT contributed to the oxidation of propionate.

To see if the MMUT protein was working properly, the researchers gave the patients a dose of the heavier, less common version of carbon – carbon 13 – via a commercially available food additive.

The team recruited 57 study participants, including 19 MMA patients who had received transplants (liver, kidney, or both) and 16 healthy volunteers. The researchers gave the participants a dose of the carbonaceous food additive 13 via a drink or a feeding tube and collected their breath samples after waiting two minutes.

The researchers measured how much of the exhaled carbon dioxide contained the usual carbon 12 compared to the added carbon 13. As suspected, MMA patients who received no treatment had lower carbon levels 13 than healthy volunteers. In contrast, liver transplant MMA patients had a higher carbon content 13, similar to the healthy volunteers. This result indicated that the MMUT protein aided in the oxidation of the carbon-13 molecules by binding to inhaled oxygen molecules.

Higher levels of carbon-13 oxidation also correlated with better clinical outcomes, such as improved cognition and slower decline in kidney function.

Currently, the test can only be used at the NIH Clinical Center. However, the researchers hope that it will soon be widely used for clinical and research purposes.

“Our next goal is to see if this particular breath test can detect an increase in carbon-13 propionate oxidation following gene, mRNA, or genome editing therapies,” Venditti said. “In this way we can also use this test to measure how effective these treatments are in restoring MMUT function.”

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