Whole genome sequencing reveals large deletions and other loss of function mutations in Mycobacterium tuberculosis drug resistance genes

Abstract

Drug resistance in Mycobacterium tuberculosis , the causative agent of tuberculosis disease, arises from genetic mutations in genes coding for drug-targets or drug-converting enzymes. SNPs linked to drug resistance have been extensively studied and form the basis of molecular diagnostics and sequencing-based resistance profiling. However, alternative forms of functional variation such as large deletions and other loss of function (LOF) mutations have received much less attention, but if incorporated into diagnostics they are likely to improve their predictive performance. Our work aimed to characterize the contribution of LOF mutations found in 42 established drug resistance genes linked to 19 anti-tuberculous drugs across 32689 sequenced clinical isolates. The analysed LOF mutations included large deletions (n=586), frameshifts (n=4764) and premature stop codons (n=826). We found LOF mutations in genes strongly linked to pyrazinamide (pncA), isoniazid (katG), capreomycin (tlyA), streptomycin (e.g. gid) and ethionamide (ethA, mshA) (P<10−5), but also in some loci linked to drugs where relatively less phenotypic data is available [e.g. cycloserine, delaminid, bedaquiline, para-aminosalicylic acid (PAS), and clofazimine]. This study reports that large deletions (median size 1115 bp) account for a significant portion of resistance variants found for PAS (+7.1% of phenotypic resistance percentage explained), pyrazinamide (+3.5%) and streptomycin (+2.6%) drugs, and can be used to improve the prediction of cryptic resistance. Overall, our work highlights the importance of including LOF mutations (e.g. large deletions) in predicting genotypic drug resistance, thereby informing tuberculosis infection control and clinical decision-making.

A precision overview of genomic resistance screening in isolates ofMycobacterium tuberculosisusing web-based bioinformatics tools

“…In agreement with previous studies, we showed that 95.5% of ethambutol resistance is due to SNPs in embB (M306I/V, D354A, G406A/D/S), and 9.1% to the -16C>T substitution in the embA gene. Similarly, all the mutations we found related to pyrazinamide resistance (95.2% SNP and 26.6% Indels in pncA) coincided with studies pointing to the importance of the promoter and the pncA gene (4,46,51,75,76).…”

“…Similarly, all the mutations we found related to pyrazinamide resistance (95.2% SNP and 26.6% Indels in pncA) coincided with studies pointing to the importance of the promoter and the pncA gene (4,46,51,75,76).…”

“…Therefore, the key is to identify those SNPs which are responsible for or strongly associated with resistance (8,25,42,43). Previous studies have indicated canonical mutations associated with resistance (32,44–50) as well as deletions that affect gene functionality (51) or promote bacterial transmission (52). In our study, only 19 resistance-associated genes were identified among 42 candidate genes, involving a total of 73 mutations.…”

Resistance and tolerance of Mycobacterium tuberculosis to antimicrobial agents—How M. tuberculosis can escape antibiotics

“…Mutations in Mtb can also lead to drug resistance by reducing the accumulation of drugs in the bacteria or inactivating them (Alame Emane et al, 2021). Mutations can also impair the efficacy of the drugs to bind to target genes, and bacteria may develop drug resistance (Gomes et al, 2021; Le Chevalier et al, 2014).…”

Large-scale genomic analysis of Mycobacterium tuberculosis reveals extent of target and compensatory mutations linked to multi-drug resistant tuberculosis

“…To acquire resistance to anti-TB drugs, Mtb drug targets or activating proteins are often mutated 1 . As a consequence, the biological function of these proteins is impaired or sometimes completely lost 2 , causing the bacterium to incur a fitness cost. These costs can manifest as a phenotypic difference, such as reduced virulence or transmissibility.…”