title: Detecting novel genetic variants associated with isoniazid-resistant Mycobacterium tuberculosis.
abstract: BACKGROUND
  Isoniazid (INH) is a highly effective antibiotic central for the treatment of Mycobacterium tuberculosis (MTB). INH-resistant MTB clinical isolates are frequently mutated in the katG gene and the inhA promoter region, but 10 to 37% of INH-resistant clinical isolates have no detectable alterations in currently known gene targets associated with INH-resistance. We aimed to identify novel genes associated with INH-resistance in these latter isolates.
  
  
  RESULTS
  INH-resistant clinical isolates of MTB were pre-screened for mutations in the katG, inhA, kasA and ndh genes and the regulatory regions of inhA and ahpC. Twelve INH-resistant isolates with no mutations, and 17 INH-susceptible MTB isolates were subjected to whole genome sequencing. Phylogenetically related variants and synonymous mutations were excluded and further analysis revealed mutations in 60 genes and 4 intergenic regions associated with INH-resistance. Sanger sequencing verification of 45 genes confirmed that mutations in 40 genes were observed only in INH-resistant isolates and not in INH-susceptible isolates. The ratios of non-synonymous to synonymous mutations (dN/dS ratio) for the INH-resistance associated mutations identified in this study were 1.234 for INH-resistant and 0.654 for INH-susceptible isolates, strongly suggesting that these mutations are indeed associated with INH-resistance.
  
  
  CONCLUSIONS
  The discovery of novel targets associated with INH-resistance described in this study may potentially be important for the development of improved molecular detection strategies.
authors.affiliation: Division of Medical Sciences, National Cancer Centre, Singapore, Singapore.
authors.forename: Sandhya
authors.initials: S
authors.lastname: Shekar
authors.affiliation: Division of Medical Sciences, National Cancer Centre, Singapore, Singapore.
authors.forename: Zhen Xuan
authors.initials: ZX
authors.lastname: Yeo
authors.affiliation: Division of Medical Sciences, National Cancer Centre, Singapore, Singapore.
authors.forename: Joshua C L
authors.initials: JC
authors.lastname: Wong
authors.affiliation: Division of Medical Sciences, National Cancer Centre, Singapore, Singapore.
authors.forename: Maurice K L
authors.initials: MK
authors.lastname: Chan
authors.affiliation: Division of Medical Sciences, National Cancer Centre, Singapore, Singapore.
authors.forename: Danny C T
authors.initials: DC
authors.lastname: Ong
authors.affiliation: Division of Medical Sciences, National Cancer Centre, Singapore, Singapore.
authors.forename: Pumipat
authors.initials: P
authors.lastname: Tongyoo
authors.affiliation: Department of Infectious Diseases, Singapore General Hospital, Singapore, Singapore.
authors.forename: Sin-Yew
authors.initials: SY
authors.lastname: Wong
authors.affiliation: Division of Medical Sciences, National Cancer Centre, Singapore, Singapore; Office of Clinical & Academic Faculty Affairs, Duke-NUS Graduate Medical School, Singapore, Singapore; Department of Physiology, Yong Loo Lin School of Medicine, National University of Singapore, Singapore, Singapore.
authors.forename: Ann S G
authors.initials: AS
authors.lastname: Lee
mesh_terms: D000995:Antitubercular Agents / Q000494:pharmacology*; D024881:Drug Resistance, Bacterial / Q000235:genetics*; D007538:Isoniazid / Q000494:pharmacology*; D009154:Mutation; D009169:Mycobacterium tuberculosis / Q000187:drug effects* / Q000235:genetics; D010802:Phylogeny; D011401:Promoter Regions, Genetic
chemical_list: D000995:Antitubercular Agents; D007538:Isoniazid