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New Tuberculosis Drug Resistance Mutations Emerge From GWAS

NEW YORK (GenomeWeb) – Using a genome-wide association study-based approach, researchers have uncovered known and novel resistance mutations within Mycobacterium tuberculosis.

About 10.4 million people around the world became ill with tuberculosis in 2016, according to the World Health Organization. Of those, the agency estimated, 600,000 cases were resistant to the first-line drug rifampicin and 490,000 were multidrug resistant TB (MDR-TB). The WHO further calculated that about 6.2 percent of the MDR-TB cases were extensively drug-resistant TB (XDR-TB), meaning they didn't respond to the most effective second-line drugs.

An international team of researchers collected nearly 6,500 clinical isolates of M. tuberculosis to search for mutations within their genomes that confer drug resistance. As the team reported in Nature Genetics today, its approach, which combined a GWAS with phylogenetic analysis, uncovered known resistance loci like folC and drfA-thyA, but also novel ones like ethA and katG.

"Improved tools are needed to guide treatment of patients with multidrug-resistant disease, where personalized treatment offers improved rates of cure," co-senior author Taane Clark from the London School of Hygiene and Tropical Medicine and his colleagues wrote in their paper. "Next-generation sequencing offers a comprehensive assessment and may be used to guide treatment.

He and his colleagues sequenced 6,465 clinical isolates of M. tuberculosis that were collected from more than 30 geographic locations. These isolates, they noted, represent the four major Mtb lineages. Among these samples, the researchers identified more than 102,000 SNPs, 11,000 indels, and nearly 300 large deletions.

In addition, nearly a third of these isolates were resistant to at least one anti-TB drug, while 15.1 percent were deemed to be MDR-TB and 4.3 percent were XDR-TB.

To tease out loci that confer resistance, the researchers conducted a GWAS of these samples within a mixed-regression framework, in which they first looked for genes associated with either the MDR-TB or XDR-TB phenotype and then conducted a phylogenetics-based test for independent mutations. Clark and his colleagues also repeated their analysis for 14 drugs individually.

Among the MDR-TB isolates, the researchers found that the most frequent resistance mutations — together accounting for 75 percent of resistance mutations — were located within the katG gene at codon 315 and provide resistance to isoniazid. At the same time, they uncovered resistance loci in rpoB, which confers resistance to rifampicin; in the Rv1482c–fabG1 operon, which provides resistance to isoniazid and ethionamide; and in folC, which gives resistance to para-aminosalicylic acid.

The XDR-TB isolates harbored additional mutations, such as in gyrA, rrs, and ethA, which confer fluoroquinolone, aminoglycoside, and ethambutol resistance, respectively.

When the researchers stratified their GWAS analysis by lineage the TB isolates belonged to, they found that most mutations occurred across lineages.

They also uncovered epistatic relationships between various loci pairs, and noted known compensatory relationships, such as between rpoB and rpoC, but also new ones, such as between pncA and pncB2 that affected pyrazinamide susceptibility.

Based on their findings, Clark and his colleagues also hypothesized that efflux pumps play a role in drug resistance phenotypes. Both drrA and Rv2688c encode ABC transporters, and mutations in drrA are linked to XDR-TB, while mutations in Rv2688c are linked to moxifloxacin and fluoroquinolone resistance.

"These loci warrant functional follow-up and characterization studies to fully elucidate their role in treatment failure," the researchers wrote of the novel loci they found, such as ethA and katG. "The associations identified may shed light on the molecular mechanisms underlying drug resistance and assist in the design of novel antibiotics."