Enriched whole genome sequencing identified compensatory mutations in the RNA polymerase gene of rifampicin-resistant Mycobacterium leprae strains

Figures & data

Table 1 Demographic and clinical details of rifampicin-resistant cases (A). Demographic and clinical details of rifampicin-sensitive cases (B).

A
Patients’ identification	507	42	80	1027	439
Age at diagnosis (years)	23	31	18	18	18
Sex	Male	Male	Female	Male	Male
Start of MDT (month/year)	10/2011	05/2013	02/2012	11/2011	2013
Completion of MDT (month/year)	10/2011	05/2014	02/2013	11/2012	2014
Relapse date	08/2016	05/2015	12/2015	01/2015	07/2015
RJ Classification
At diagnosis	BB	BT	BL	BL	BL
At relapse	BB	BL	BL	LL	LL
Bacillary index
First diagnosis	0.66+	4.33+	3.0+	2.66+	NA
Relapse	2.0+	3.0+	3.0+	2.0+	6.0+
Clinical complication at the time of relapse	None	Type 1 reaction	Type 2 reaction	Type 2 reaction with concomitant pulmonary tuberculosis infection	None
B
Patients’ identification	1,335	834	730	SM	728
Age at diagnosis (years)	38	19	33	40	34
Sex	Male	Male	Male	Male	Female
Start of MDT (month/year)	11/2008	2005	04/2013	2005	2008
Completion of MDT (month/year)	10/2009	2007	04/2014	2007	2009
Relapse date	12/2015	10/2010	12/2015	04/2017	10/2010
RJ Classification
At diagnosis	BL	LL		NA	LL
At relapse	LL	LL	BL	BL	LL
Bacillary index
First diagnosis	3.0+	2.0+	1.0+	2.0+	4.33+
Relapse	4.0+	2.66+	2.0+	3.0+	4.33+
Clinical complication at the time of relapse	Infiltration with raised reddish lesions	Infiltration all over the body	Infiltration with Type 1 reaction	Infiltration with new lesions	Multiple ENLs on face

Table 2 Mutations in rpoB and rpoC genes by WGS in molecularly characterized rifampicin-resistant strains

Patient ID	Resistance	Geographical area	rpoB mutations (WGS)*	rpoC mutations (WGS)
1027	RIF	Delhi	Arg1168Leu; Arg505Trp	Va1517Leu
507	RIF	Chhattisgarh	Iso433ser; leu436 Met	NM
42	RIF	Delhi	Leu458Phe	NM
80	RIF	Delhi	Tyr-cys	NM
439	RIF	Chhattisgarh	Tyr-cys	Leu807Met; Leu244Phe

Note: *Amino acid.

Abbreviations: RIF, rifampicin; NM, no mutation; WGS, whole genome sequencing.

Table 3 List of unique 16 SNPs present in resistant strains

Position	Name of the gene	TN base (reference)	Variant	SNP effect	Amino acid change	Type
80206	gltB	C	T	Non-synonymous coding	T/M	Probable ferredoxin-dependent glutamate synthase
83120	gltD	G	A	Non-synonymous coding	G/E	Probable NADH-dependent glutamate synthase
61866	esxB	C	A	Non-synonymous coding	G/C	Possible 10 KDA culture filtrate antigen homolog ESXB (lhp) (cfp10)
257506	ML0I92	C	A	Non-synonymous coding	Q/H	Conserved membrane protein
626352	ML05I4	C	A	Non-synonymous coding	P/Q	Probable conserved membrane protein
666171	priA	C	A	Non-synonymous coding	H/N	Putative primosomal protein N’ PriA (Replication factor Y)
672897	ribG	C	A	Non-synonymous coding	L/M	Probable bifunctional riboflavin-specific deaminase/reductase
140830	embA	G	A	Non-synonymous coding	P/L	Probable integral membrane indolylacetylinositol arabinosyltransferase EmbA (arabinosylindolylacetylinositol synthase)
145310	embC	G	A	Non-synonymous coding	T/I	Probable integral membrane indolylacetylinositol arabinosyltransferase Embc (arabinosylindolylacetylinositol synthase)
2271147	rpoC	C	A	Non-synonymous coding	V/L	DnA-directed RnA polymerase (beta’ chain)
190301	mmpL7	G	A	Non-synonymous coding	S/L	Conserved transmembrane transport protein MmpL7
2600317	ML2I86		+CAG	codon change plus codon insertion	R/RR	Possible transcriptional regulatory protein
241842	ML0I75	G	T	Non-synonymous coding	R/L	Probable two component sensor kinase MprB
148945	ML0107	C	A	synonymous coding	P/P	Probable conserved transmembrane protein
3237399	ponA		-A	Frame shift	-
623908	alaS		+AcAG	Frame shift	-/T	Probable alanyl-tRNA synthetase AlaS (Alanine-tRNA ligase) and AlaRS (Alanine translase)

Abbreviation: SNP, single-nucleotide polymorphism.

Table S1 Concentration of DNA from biopsies and after library preparation

Serialnumber	Sample ID	DNA concentration by Qubit (ng/μL)	Library concentration (ng/μL)	Volume
1.	570	56.7	26.7	13
2.	507	93.2	0.62	13
3.	42	85.6	0.74	13
4.	80	35.6	0.38	13
5.	1027	204	0.54	13
6.	281	33.1	0.36	13
7.	439	25.6	0.41	13
8.	4923	15.3	78.5	13
9.	395	20.5	0.32	13
10.	834	432	0.37	13
11.	588	235	0.41	13
12.	SM	45.4	0.33	13
13.	728	24.9	0.57	13
14.	1335	25.4	2.85	13
15.	730	33.2	0.34	13
16.	BH	64.2	1.33	13

Abbreviations: BB, borderline leprosy; BL, borderline lepromatous; BT, borderline tuberculoid; LL, lepromatous leprosy; MDT, multidrug therapy; NA, not available; RJ, Ridley–Jopling.