Study identifies 51 amyotrophic lateral sclerosis-associated mutations that could help diagnose the disease microbiologystudy

Using sequencing techniques, researchers associated 51 mutations in mitochondrial DNA with amyotrophic lateral sclerosis (ALS), an incurable degenerative neurological disorder that leads to muscle atrophy and paralysis. Thirteen of the mutations increased the risk of ALS, while 38 were protective. The study suggests these mutations may be important for future tests and research on ALS.

The study is published in the journal Muscle & Nerve.

It is worth recalling that mitochondria, the organelles that produce energy for cells, have their own DNA (mtDNA), which is inherited solely from the mother. Mutations in mtDNA can cause several diseases, almost all of which affect neuromuscular processes.

The researchers analyzed 1,965 genomes of ALS patients and 2,547 non-ALS controls. The ALS patient genomes were supplied by the New York Genome Center’s ALS Consortium, a network of clinicians, basic scientists, geneticists and computational biologists from 45 institutions in several countries who apply whole genome sequencing and functional genomics to the study of ALS.

“We noted that our study was the first to associate mitochondrial genome mutations with ALS. We conducted an interesting analysis based on a quantitative approach. It’s hard to obtain a large sample for ALS. Our data was from patients in the United States who mainly had white ancestors. The incidence of ALS is highest among whites, according to the scientific literature. Among people with African ancestry, despite low incidence, the disease usually takes the most severe form, which is bulbar ALS,” Marcelo Briones, first author of the article, told Agência FAPESP. He is a professor at the Federal University of São Paulo’s Medical School (EPM-UNIFESP) in Brazil and a specialist in genomics and molecular biology.

Briones, who is co-corresponding author alongside James Broach, a researcher at Penn State College of Medicine in the U.S., stressed the importance of their findings. “We aren’t saying these mutations cause the disease, just that they’re associated and should be part of the diagnostic panel. In the future, they could be useful for gene therapy studies. We were rigorous with the data and used high values of statistical significance,” he said.

Multidisciplinary approach

The group analyzed mtDNA, which is unique because it is inherited solely from the mother, whereas nuclear DNA is inherited from both parents. Hereditary genetic factors are the main cause of ALS cases—only 10% are due to a genetic defect. In practice, the patient’s neurons degrade or die, ceasing to send messages to the muscles.

In Brazil, few epidemiological studies of ALS exist. The prevalence of the disease is estimated at 0.9-1.5 cases per 100,000 inhabitants per year. Symptoms typically appear after the age of 55.

The researchers used an approach known as genome-wide association study (GWAS) to identify small genetic variations called single nucleotide variants (SNVs). They compared SNV frequency and concluded that a specific SNV could be associated with ALS if it was significantly more common in patients than controls.

As mentioned above, 13 SNVs were associated with an increased risk of ALS. They were located in ten genes—HV1, HV2, HV3, RNR1, ND1, CO1, CO3, ND5, ND6 and CYB. The 38 protective mutations were located in the genes HV1, HV2, HV3, RNR1, RNA2, ND1, ND2, CO2, ATP8, ATP6, CO3, ND3, ND4, ND5, ND6 and CYB. All SNVs had p-values lower than 10^-7 (0.0000001).

The SNVs associated with increased risk of ALS had odds ratios higher than 1; the protective SNVs had odds ratios lower than 1. The odds ratio is a statistical parameter used in epidemiology to estimate the odds (probability) of an event for an exposed group.

P-value (probability value) is the probability of obtaining the sampling results due to chance. The smaller the p-value, the stronger the evidence that the results are significant and not due to chance. In biology, a p-value of less than 0.05 is considered significant.

Mendelian inheritance is not always observed in ALS, where extranuclear or cytoplasmic inheritance, especially via mutations in mtDNA, is frequent. This is another reason for the relevance of the study.

“Because we were looking for associations, we used GWAS to establish a panel of candidates as a starting point for anyone interested in identifying targets for therapy. A study of causal relations is then conducted,” said Fernando Antoneli, a professor of applied mathematics at UNIFESP and a co-author of the article.

With João Henrique Campos, then a Ph.D. candidate, and biologist Renata Carmona e Ferreira, the group opted to use GWAS in the study.

“When João joined the lab, we started looking at new techniques, including GWAS, which was being widely used at the time. João is a trained nurse and learned to use these tools, which we decided to leverage in the study. We’ve worked as a genuinely multidisciplinary team since 2000,” Antoneli said.

The researchers now want to analyze the available data using artificial intelligence. They also plan to sequence samples from a cohort of Brazilian patients and plot the results against the 51 mutations detected in the study.