Supplemental Note In order to obtain all articles relevant to this study, key search terms were used in various combinations with the AND operator. Search terms included: pyrazinamide, resistance, Mycobacterium tuberculosis, pnca, mutations, and pza. Titles and abstracts were read and if the article seemed to include all necessary inclusion criteria based solely on the title and abstract, the entire article was read. Importantly, once an article was found that met inclusion criteria, the references were analysed for other potential articles. The same procedure was used for articles found in references of relevant papers as those found through a search in PubMed. Supplemental Figure 1. Number of pyrazinamide-resistant Mycobacterium tuberculosis isolates with each type of mutation in pnca open reading frame or its promoter (Note: Figure only reports consequential mutations, i.e. does not include synonymous mutations in pnca open reading frame.) Notation: SUB = Isolates harboring a single point (includes synonymous and nonsynonymous changes); DEL = Single deletion (of varying length); INS = Single insertion of varying length; numerals: Indicate the number of distinct mutations of a certain type (e.g. 3 SUBs = three distinct substitutions in a single isolate); + : indicates the combination of more than one type of mutation in an isolate (e.g. 2 SUBs + DEL = all isolates harboring two distinct consequential substitutions and one deletion); Supplemental Table 1. Country of origin of the samples collected for the studies included in this systematic review. Authors PubMed ID Published Date Geographic Location Number Resistant Clinical Sequenced Number Susceptible Clinical Sequenced Alexander et al. 22895038 2012 56 77 *not noted* Ando et al. 19832709 2010 19 17 Japan Aono et al. 24867972 2014 49 31 Japan Aragon et al. 17615155 2007 20 30 Spain
Barco et al. 16971417 2006 24 6 Brazil Bishop et al. 11605890 2001 15 6 Gauteng, South Africa Brown et al. 10882091 2000 17 5 Turkey, England Cheng et al. 10681313 2000 57 *not noted* Canada, United States, South Korea Chiu et al. 21910892 2011 36 30 Taiwan Clemente et al. 18687558 2008 8 0 Brazil Cuevas-Cordoba 23321280 2013 42 85 Mexico et al. Daum et al. 22972833 2012 9 17 South Africa Davies et al. 11015384 2000 6 0 South Africa Doustar et al. 19857130 2009 34 15 Iran Endoh et al. 12002553 2002 5 0 Japan Feuerriegel et al. 22646308 2012 10 47 Sierra Leone Hannan et al. 11158123 2001 3 6 United States Hirano et al. 9692180 1997 33 135 Myanmar, Thailand, Yemen, Nepal, India, Korea, Indonesia, Malaysia, Phillipines, Bangladesh, Canada Hoffner et al. 24125455 2014 17 35 *not noted* Hou et al. 10813147 2000 35 30 China Huang et al. 14576145 2003 17 59 Taiwan Huang et al. 24238524 2013 46 86 China Jnawali et al. 23561273 2013 119 31 South Korea Jonmalung et al. 20727143 2010 52 98 Thailand Jureen et al. 18316515 2008 32 34 *not noted* Lee et al. 12044302 2002 11 20 Singapore Lemaitre et al. 10390238 1999 19 16 France Marttila et al. 10390239 1999 36 8 Russia McCammon et al. 15917514 2005 31 1 United States, Mexico Mestdagh et al. 10471589 1999 23 39 Bangladesh, Azerbaijan, Siberia, Belgium, Abkhazia, Rwanda, Congo- Brazzaville, Kazakhstan, Canada, Scotland Miotto et al. 25336456 2014 843 1107 Various locations not noted Miyagi et al. 14972380 2004 26 0 Japan Morlock et al. 10952570 2000 35 18 United States
Mphalele et al. 18753350 2008 41 87 South Africa Muthaiah et al. 20717529 2010 39 11 India Pandey et al. 19900109 2009 24 0 New Zealand Park et al. 11429043 2001 22 6 Korea Pholwat et a. 24246607 2014 55 41 Thailand, Tanzania, United States Portugal et al. 15215139 2004 55 8 Portugal Rodrigues et al. 15616332 2005 40 19 Brazil Sachais et al. 10089282 1998 6 10 United States Scorpio & 8640557 1996 5 4 United States Zhang Scorpio et al. 9055989 1997 34 20 *not noted* Sekiguchi et al. 17108078 2007 17 121 Japan, Poland Sekiguchi et al. 17596354 2007 30 228 Japan, Poland Sheen et al. 19249243 2009 13 0 Sheen et al. 19535526 2009 110 53 Peru Shenai et al. 19810590 2009 33 30 India Simons et al. 22090409 2012 21 159 Netherlands Somoskovi et al. 17135430 2007 83 211 *not noted* Sreevatsan et al. 9056006 1997 67 51 *not noted* Stoffels et al. 22825123 2012 60 78 Belgium, Georgia, Chechnia, Rwanda Suzuki et al. 11825963 2002 30 17 Japan Therese et al. 23183465 2012 8 9 Tracevska et al. 15273154 2004 28 0 Latvia Wade et al. 15183857 2004 46 9 Canada, United States, South Korea Watcharasamph 24163999 2013 8 14 *not noted* ankul et al. Werngern et al. 22203587 2012 21 25 Sweden Yuksel et al. 19579692 2009 10 2 Turkey Zhang et al. 19930847 2009 47 0 China Zhou et al. 22102918 2011 22 27 China Supplemental Table 2. List of mutations discovered in PZase or the pnca promoter region of PZA R Mtb isolates with varying pyrazinamide MIC Levels. Multiple Lines for a Mutation Indicate Differing MIC Levels Reported by Different Studies. Mutation Number Resistant MIC Level -16AACGTA-11--> GGCAGTT 1 400 A-11G 1 12.5 A-11G 1 300
T-7C 1 100 11bp DEL 1 1 400 8bp DEL 1 1 600 Met1Ile 3 >400 11-bp DEL at Start 2 >900 Ala3Pro 1 400 Ala3Pro 1 1000 Ala3Glu 5 1600 Ala3Glu 1 >400 Leu4Ser 1 800 Leu4Ser 1 >400 C INS 16 1 25 Ile5Asn 1 >400 Ile5Ser 1 >400 Ile5Ser 1 >900 Ile6Thr 1 256 Ile6Thr 1 >100 <300 Val7Ala 1 400 Val7Gly 2 400 Val9Gly 1 1600 Gln10Arg 1 300 Gln10Pro 1 400 C DEL 28 2 600 Gln10Pro 1 800 Gln10Pro 2 >1600 Gln10Pro 1 >400 Gln10STOP 1 >800 C DEL 28 1 >900 Asp12Ala 1 400 Asp12Gly 1 400 Asp12Ala 1 600 Asp12Ala 1 >100 Asp12Ala 1 >300 Asp12Asn 1 >900 Phe13Ser 1 >2000 Cys14Arg 1 >800 Gly17Asp 2 >800 G INS 52 1 >100 2bp INS 54 1 >400 Leu19Pro 1 600
C DEL 59 1 800 C DEL 59 2 >1600 Gly24Asp 1 200 G DEL 71 1 900 G DEL 71 3 >800 G DEL 71 1 >900 Ala26Gly 1 >900 Tyr34Asp 1 100 Tyr34Asp 4 400 Tyr34STOP 1 1600 Tyr34Asp 2 >400 Tyr34Ser 2 >800 C DEL 104 1 >900 CG INS 107 1 >100 <300 5.3kb DEL 106 1 800 Tyr41His 1 900 Tyr41STOP 2 900 Tyr41STOP 1 >800 CG DEL 129 1 1600 His43Pro 1 >800 Ala46Val 6 >300 Ala46Glu 1 >300 Ala46Val 1 >800 G INS 136 1 >800 Thr47Ala 1 64 Thr47Pro 1 900 Lys48Thr 1 49 Asp49Asn 1 400 His51Pro 1 800 His51Gln 1 1600 His51Gln 1 >1024 His51Arg 1 >800 His51Arg 1 >800 His51Pro 1 >800 His51Arg 1 >800 His51Gln 1 >900 Asp53Ala 1 >300 Asp63Gly 1 >800 Pro54Leu 1 512 Pro54Thr 1 900
Pro54Ser 1 >1024 Pro54Leu 2 >1600 Pro54Arg 1 >400 G DEL 162 1 >500 Pro54Gln 1 >800 Pro54Thr 1 >900 His57Asp 1 >400 His57Arg 1 >800 Phe58Leu 1 128 Phe58Leu 1 512 Phe58Leu 1 >800 Ser59Pro 1 1600 Ser59Pro 1 >800 Thr61Pro 1 500 Pro62Gln 2 800 Pro62Leu 2 >400 Pro62Arg 1 >800 2bp INS 188 1 >400 Asp63His 1 >500 A INS 193 2 >300 Ser67Pro 1 >800 Trp68STOP 1 >300 Trp68Leu 2 >800 Pro69Arg 1 1000 Pro69Leu 1 >2000 Pro69Arg 2 >300 His71Tyr 1 1600 His71Arg 1 >1600 His71Glu 1 >300 His71Arg 1 >800 Cys72Arg 1 1000 29bp INS 216 1 >2000 Cys72Arg 1 >800 Cys72Arg 1 >900 Thr76Pro 1 200 Thr76Pro 1 >800 Thr76Pro 1 >800 Thr76Pro 1 >900 Gly78Cys 1 100 Gly78Asp 1 >800 His82Arg 1 >500
Leu85Pro 1 1000 Leu85Arg 1 >400 Leu85Arg 1 >500 Leu85Pro 2 >800 Leu85Pro 3 >900 Thr87Met 1 1500 AC INS 261 4 1600 2bp INS 261 2 >400 Ser88STOP 1 1000 Phe94Leu 1 400 Phe94Ser 1 >300 Lys96Thr/G DEL 436 1 1000 Lys96Asn 2 1200 Lys96Gln 1 >1600 G DEL 288 1 >400 G DEL 288 1 >500 Lys96Thr 1 >800 T INS 287 1 >800 Lys96Asn 1 >900 Gly97Asp 1 400 Gly97Ser 1 400 Gly97Asp 1 1600 Gly97Asp 1 >300 Gly97Asp 1 >400 Gly97Ser 2 >500 Gly97Cys 1 >900 Tyr99STOP 1 >800 Tyr99STOP 1 >800 A INS >300 1 1200 G DEL >300 1 >500 Tyr103STOP 2 100 C INS 307 1 1500 Tyr103Cys 1 1500 Ser104Arg 1 >800 Gly105Asp 2 >800 C INS 314 2 >800 Gly108Arg 1 >800 1355bp INS 342 1 1000 G DEL 341 1 >500 Thr114Pro 1 >800 Leu116Pro 1 100
Asn118Thr 1 1500 7bp DEL 352 1 >300 Leu120Arg 1 >800 Arg121Pro 1 1500 8bp INS 366 1 >1600 Gln122STOP 1 >900 Arg123Pro 1 256 AG INS 368 1 1500 Arg123Leu 1 >900 Val125Phe 1 >1024 16bp DEL 374 1 >1600 8bp DEL 382 1 >400 Val130Gly/GG INS 428 1 >500 Val130Gly 1 >800 9bp INS 388 (>800) 1 >800 Val130Gly 1 >800 GG INS 391 1 1000 G INS 392 1 >1600 GG INS 392 2 >1600 G DEL 391 1 >900 Gly132Ala 1 256 Gly132Asp 1 1000 Gly132Ser 1 1024 Gly132Asp 1 >800 17bp DEL 395 1 >800 Gly132Ser 2 >900 Ile133Asn 1 800 G INS 397 1 >300 Ile133Ser 1 >800 TT DEL 398 1 >900 Ala134Val 1 >500 Ala134Val 1 >800 CC INS 403 1 1800 Thr135Pro 1 >800 Thr135Pro 1 >800 His137Pro 1 >1600 His137Arg 2 >800 His137Pro 1 >900 Cys138Tyr 1 >2000 Cys138Arg 1 >400 Cys138Ser 1 >500
Cys138Ser 1 >500 Cys138Tyr 1 >800 Cys138Tyr 1 >900 Val139Leu 1 100 Val139Leu 1 >2000 Val139Ala 2 >2000 Val139Gly 1 >800 Val139Ala 1 >800 Val139Leu 2 >900 TG DEL 416 1 >900 Val139Met 1 >900 68bp DEL 420 1 1000 GC INS 420 1 >900 Arg140Ser/8bp DEL 446 21 >900 G INS 420 2 >900 Gln141Pro 1 >1600 Gln141Pro 1 >500 Gln141Pro 1 >900 Gln141Pro 1 >900 Thr142Lys 3 300 Thr142Lys 1 >1024 Thr142Ala 1 >800 Thr142Lys 2 >800 Thr142Lys 1 >900 Thr142Met 1 >900 GG INS 428 1 >900 Ala146Val 1 >800 G DEL 443 1 >900 8-bpDEL 446 1 >900 Thr153Asn 1 800 Val155Gly 1 1024 Val155Gly 1 1500 Val155Gly 1 >1024 Val155Gly 2 >300 Val155Glu 1 >800 Val155Gly 1 >900 C INS 475 1 >900 9bp INS 484/2bp DEL 494 1 >400 Ser164Pro 1 >800 C INS 495 1 >800 1bp INS 497 1 >400
CG INS >500 1 900 Thr168Asn 1 >900 Ala171Thr 1 >800 Ala171Pro 1 >900 Leu172Pro 2 200 Leu172Pro 1 1024 Leu172Pro 1 >800 5-bp INS 518 1 >900 G INS 522 1 >1600 Met175Thr 1 >1024 A INS 193 1 1000 Leu182Ser 1 2000 AG DEL 94/GA DEL 97 1 >1600 T-7C/Thr114Ala 2 >1600 Supplemental Table 3. List of media and concentrations used for pyrazinamide drug susceptibility testing for each study included in this systematic review. Authors PubMed ID Published Date DST/MIC (Media used) Alexander et al. 22895038 2012 DST (BACTEC (BACTEC 460TB) Ando et al. 19832709 2010 (BACTEC Aono et al. 24867972 2014 DST (BACTEC MIC (BACTEC Aragon et al. 17615155 2007 DST (BACTEC 460) (BacT/ALERT PZA kit) Barco et al. 16971417 2006 MIC (MABA) (LJ Proportion) Bishop et al. 11605890 2001 MIC (BACTEC 460TB) Brown et al. 10882091 2000 DST (BACTEC 460TB Concentration(s) (Susceptibility Cutoff) *not noted* (usually 100mg/L) *not noted* (usually 100mg/L) 100 ug/ml 200, 300, 400, 800, 1600 ug/ml *not noted* 12.5-1600mg/L 12.5-800mg/L (100 mg/l) (100mg/L) (100mg/L)
Cheng et al. 10681313 2000 MIC (BACTEC 460TB) (Lowenstein- Jensen) (100mg/L), 300, 900 100,500mg/L Chiu et al. 21910892 2011 DST (100mg/L) (BACTECT Clemente et al. 18687558 2008 DST (BACTEC 460) *not noted* Cuevas-Cordoba et al. 23321280 2013 DST (BACTECT (100mg/L) Daum et al. 22972833 2012 DST (BACTEC *not noted* (usually 100mg/L) Davies et al. 11015384 2000 DST (BACTEC (100mg/L) 460TB) Doustar et al. 19857130 2009 DST (7H9 broth) (100mg/L) Endoh et al. 12002553 2002 MIC (7H10) 25mg/l, 50mg/l, (100 mg/l) (7H9) Feuerriegel et al. 22646308 2012 DST (BACTEC 460) MIC (BACTEC Hannan et al. 11158123 2001 DST (BACTEC 460TB) Hirano et al. 9692180 1997 MIC (Middlebrook 7H11) Hoffner et al. 24125455 2014 DST (BACTEC MIC (BACTEC Hou et al. 10813147 2000 DST MIC (BACTEC 460TB) (Proportion Method) Huang et al. 14576145 2003 MIC (BACTEC 100mg/L, 400mg/L (100mg/L) 100, 50, 25, 12.5, 6.3mg/L (100mg/L) 25-800mg/L 100ug/ml 200-800 ug/ml (100mg/L),300, 400, 500, 600, 800, 900, 1000, 1200, 1500, 2000 25mg/L (100mg/L), 300mg/L Huang et al. 24238524 2013 DST (LJ) (100mg/L) Jnawali et al. 23561273 2013 DST (Proportion (100mg/L) Method) Jonmalung et al. 20727143 2010 DST (BACTEC (100mg/L) Jureen et al. 18316515 2008 DST (BACTEC (100mg/L) 460) Lee et al. 12044302 2002 DST (BACTEC *not noted* (usually
460) 100mg/L) Lemaitre et al. 10390238 1999 MIC (7H9 (500mg/L),50mg/L liquid) Marttila et al. 10390239 1999 DST (BACTEC (100mg/L) 460TB) McCammon et al. 15917514 2005 DST (100mg/L) (BACTEC460) Mestdagh et al. 10471589 1999 DST (BACTEC (100mg/L) 460TB) Miotto et al. 25336456 2014 DST (BACTEC "according to manufacturer's instructions" = 100mg/L Miyagi et al. 14972380 2004 MIC (7H9 broth) (100), 200, 250, 400mg/L Morlock et al. 10952570 2000 DST (7H10 agar) (25mg/L) (100mg/L) MIC (BACTEC 460TB) Mphalele et al. 18753350 2008 DST (BACTEC (100mg/L) 460) Muthaiah et al. 20717529 2010 DST (Pportion (300mg/L) Mhod) Pandey et al. 19900109 2009 DST (BACTEC (100mg/L) (BACTEC 460TB) Park et al. 11429043 2001 DST (Pportion (100mg/L), 500mg/L Mhod) Pholwat et a. 24246607 2014 DST (BACTEC (100mg/L) Portugal et al. 15215139 2004 DST (BACTEC (100mg/L) Rodrigues et al. 15616332 2005 DST(Pportion *not noted* Mod) Sachais et al. 10089282 1998 DST (BACTEC 460TB) Scorpio & Zhang 8640557 1996 MIC (7H9 liquid) Scorpio et al. 9055989 1997 MIC (BACTEC 460TB) Sekiguchi et al. 17108078 2007 DST (BACTEC Sekiguchi et al. 17596354 2007 DST (BACTEC Sheen et al. 19249243 2009 Sheen et al. 19535526 2009 DST (BACTEC 460TB) (7H9 broth) *not noted* (usually 100mg/L) 50,(100mg/L),200,500,1000 (100mg/L), 300mg/L, 900mg/L *not noted* (usually 100mg/L) *not noted* (usually 100mg/L) (100mg/L) (200mg/L)
Shenai et al. 19810590 2009 DST (BACTEC (100mg/L) Simons et al. 22090409 2012 DST (BACTECT *not noted* (usually 100mg/L) Somoskovi et al. 17135430 2007 DST (BACTEC (100mg/L) 460) Sreevatsan et al. 9056006 1997 DST (7H10 agar) (BACTEC (25mg/L) *not noted* (usually 100mg/L) 460TB) Stoffels et al. 22825123 2012 DST (BACTEC *not noted* (usually 100mg/L) MIC (BACTEC Suzuki et al. 11825963 2002 DST (7H10) (100mg/L) Therese et al. 23183465 2012 DST (BACTEC (100mg/L) 460TB) Tracevska et al. 15273154 2004 DST (BACTEC) *not noted* (usually 100mg/L) Wade et al. 15183857 2004 MIC (100mg/L) (determined in Cheng et al, Scorpio et al.) Watcharasamphankul 24163999 2013 DST (BACTEC (100mg/L) et al. MGIT) Werngern et al. 22203587 2012 DST (BACTEC 460) (100mg/L) 8-100 OR 512-1024 MIC (BACTEC Yuksel et al. 19579692 2009 DST (BACTEC 460) *not noted* (usually 100mg/L) Zhang et al. 19930847 2009 DST & MIC 25, 50, (100), (200), 400, Zhou et al. 22102918 2011 DST (BACTEC 800mg/L (100mg/L) Supplemental Table 4. PRISMA checklist Section/topic # Checklist item TITLE Reported on page #
Title 1 Identify the report as a systematic review, Meta-analysis, or both. ABSTRACT Structured summary 2 Provide a structured summary including, as applicable: background; objectives; data sources; study eligibility criteria, participants, and interventions; study appraisal and synthesis Methods; results; limitations; conclusions and implications of key findings; systematic review registration number. INTRODUCTION Rationale 3 Describe the rationale for the review in the context of what is already known. Objectives 4 Provide an explicit statement of questions being addressed with reference to participants, interventions, comparisons, outcomes, and study design (PICOS). MHODS Protocol and registration 5 Indicate if a review Protocol exists, if and where it can be accessed (e.g., Web address), and, if available, Provide registration information including registration number. Eligibility criteria 6 Specify study characteristics (e.g., PICOS, length of followup) and report characteristics (e.g., years considered, language, publication status) used as criteria for eligibility, giving rationale. Information sources 7 Describe all information sources (e.g., databases with dates of coverage, contact with study authors to identify additional studies) in the search and date last searched. Search 8 Present full electronic search strategy for at least one database, including any limits used, such that it could be repeated. Study selection 9 State the Process for selecting studies (i.e., screening, eligibility, included in systematic review, and, if applicable, included in the Ma-analysis). Data collection Process 10 Describe Method of data extraction from reports (e.g., piloted forms, independently, in duplicate) and any Processes for obtaining and confirming data from investigators. Data items 11 List and define all variables for which data were sought (e.g., PICOS, funding sources) and any assumptions and simplifications made. Risk of bias in individual studies 12 Describe Methods used for assessing risk of bias of individual studies (including specification of whether this was done at the study or outcome level), and how this information is to be used in any data synthesis. Summary measures 13 State the principal summary measures (e.g., risk ratio, difference in means). Synthesis of results 14 Describe the Methods of handling data and combining results of studies, if done, including measures of consistency (e.g., I 2 ) for each Ma-analysis. Risk of bias across studies 15 Specify any assessment of risk of bias that may affect the cumulative evidence (e.g., publication bias, selective reporting within studies). 1 1 2-4 4 4 4 4 4 4 6 6, 16 5-6 6 5-6 5-6, 14-15
Additional analyses 16 Describe Mhods of additional analyses (e.g., sensitivity or subgroup analyses, Ma-regression), if done, indicating which were pre-specified. RESULTS Study selection 17 Give numbers of studies screened, assessed for eligibility, and included in the review, with reasons for exclusions at each stage, ideally with a flow diagram. Study characteristics 18 For each study, present characteristics for which data were extracted (e.g., study size, PICOS, follow-up period) and Provide the citations. Risk of bias within studies 19 Present data on risk of bias of each study and, if available, any outcome level assessment (see item 12). Results of individual studies 20 For all outcomes considered (benefits or harms), present, for each study: (a) simple summary data for each intervention group (b) effect estimates and confidence intervals, ideally with a forest plot. Synthesis of results 21 Present results of each Ma-analysis done, including confidence intervals and measures of consistency. Risk of bias across studies 22 Present results of any assessment of risk of bias across studies (see Item 15). Additional analysis 23 Give results of additional analyses, if done (e.g., sensitivity or subgroup analyses, Ma-regression [see Item 16]). DISCUSSION Summary of evidence 24 Summarize the main findings including the strength of evidence for each main outcome; consider their relevance to key groups (e.g., healthcare Providers, users, and policy makers). Limitations 25 Discuss limitations at study and outcome level (e.g., risk of bias), and at review-level (e.g., incomplete retrieval of identified research, reporting bias). Conclusions 26 Provide a general interpretation of the results in the context of other evidence, and implications for future research. FUNDING Funding 27 Describe sources of funding for the systematic review and other support (e.g., supply of data); role of funders for the systematic review. 6 6-7 7 7, 15-16 8-9 9-10 9-10 7-10 10-15 15-16 16-17 17
Supplemental Table 5. Sensitivity and specificity calculated based on four different criteria for variants associated with PZA resistance in Mycobacterium tuberculosis clinical isolates. Resistance Association Criteria Sensitivity Specificity All mutations observed in PZA resistant isolates are associated with PZA resistance 83% 91% Only mutations that exclusively appear in resistant isolates (never appear in susceptible isolates) are associated with resistance 59% 100% Consider mutations associated with resistance 70% of the time (at least 70% of isolates that harbour these mutations are resistant) 63% 94% Consider mutations associated with resistance 60% of the time (at least 70% of isolates that harbour these mutations are resistant) 70% 91% Consider mutations associated with resistance 50% of the time (at least 70% of isolates that harbour these mutations are resistant) 78% 84% Supplemental Table 6. Statistical analysis of all resistance Associated mutations published in the included literature for diagnostic testing. Positive coordinates are amino acid numbers in PZase while negative coordinates indicate the position of the mutation in the promoter of pnca. Codon Mutation -16AACGTA-11--> GGCAGTT Number of Isolates Sensitivity Specificity Positive Predicti ve Value Negative Predicti ve Value 1 0.04% 100% 100% 55% G-13T 1 0.04% 100% 100% 55% T DEL -12 1 0.04% 100% 100% 55% T-12G 2 0.07% 100% 100% 55% T-12C 3 0.11% 100% 100% 55% A-11G 36 1.30% 100% 90% 55% A-11C 2 0.07% 100% 50% 55% A-11 6 0.22% 100% 100% 55% T-10C 1 0.04% 100% 100% 55% T-7C 2 0.07% 100% 100% 55% G DEL -5 2 0.07% 100% 100% 55% C INS -3 1 0.04% 100% 50% 55% 11bp DEL 1 1 0.04% 100% 100% 55% 8bp DEL 1 2 0.07% 100% 100% 55% Met1Ile 5 0.18% 100% 83% 55% Met1Lys 1 0.04% 100% 100% 55% Met1Thr 3 0.11% 100% 100% 55% 11bp DEL START 1 0.04% 100% 100% 55% Ala3Glu 12 0.43% 100% 100% 55% Ala3Pro 4 0.14% 100% 100% 55% Ala3Ser 1 0.04% 100% 100% 55% Ala3Gln 1 0.04% 100% 100% 55% Leu4Ser 12 0.43% 100% 100% 55%
Leu4Trp 2 0.07% 100% 100% 55% 6bp DEL 14 1 0.04% 100% 100% 55% C INS 15 1 0.04% 100% 100% 55% Ile5Asn 2 0.07% 100% 100% 55% Ile5Ser 6 0.22% 100% 100% 55% C INS 16 1 0.04% 100% 100% 55% Ile6Thr 7 0.25% 100% 100% 55% Ile6Ser 2 0.07% 100% 100% 55% G INS 16 1 0.04% 100% 100% 55% Ile6Leu 8 0.29% 96% 6% 54% Val7Ala 1 0.04% 100% 100% 55% Val7Gly 11 0.40% 100% 92% 55% Val7Phe 2 0.07% 100% 100% 55% Asp8Gly 13 0.47% 100% 100% 55% Asp8Asn 6 0.22% 100% 100% 55% Asp8Tyr 2 0.07% 100% 100% 55% Asp8Ala 2 0.07% 100% 100% 55% Asp8Glu 7 0.25% 100% 100% 55% TCG INS 23 1 0.04% 100% 100% 55% Val9Gly 2 0.07% 100% 100% 55% Gln10Pro 36 1.30% 100% 100% 55% Gln10Lys 4 0.14% 100% 100% 55% Gln10STOP 6 0.22% 100% 100% 55% Gln10Arg 8 0.29% 100% 89% 55% C DEL 28 3 0.11% 100% 100% 55% IS6110 INS 28 4 0.14% 100% 100% 55% Asp12Ala 31 1.12% 100% 91% 55% Asp12Asn 9 0.33% 100% 100% 55% Ala12Gly 1 0.04% 100% 100% 55% Asp12Glu 2 0.07% 100% 100% 55% Asp12Gly 13 0.47% 100% 93% 55% G INS 35 2 0.07% 100% 100% 55% Phe13Ser 2 0.07% 100% 67% 55% GACT INS 37 1 0.04% 100% 100% 55% Phe13Leu 3 0.11% 100% 75% 55% Cys14Arg 13 0.47% 100% 93% 55% Cys14STOP 3 0.11% 100% 100% 55% Cys14Trp 1 0.04% 100% 100% 55% Cys14Tyr 2 0.07% 100% 100% 55% Cys14Gly 2 0.07% 100% 40% 55% C INS 44 1 0.04% 100% 100% 55% 11bp DEL 48 1 0.04% 100% 100% 55% Gly16Ser 1 0.04% 100% 100% 55%
Gly17Asp 2 0.07% 100% 100% 55% Gly17Ser 3 0.11% 100% 75% 55% G DEL 49 1 0.04% 100% 100% 55% C INS 51 1 0.04% 100% 100% 55% G INS 50 1 0.04% 100% 100% 55% Met175Ile 11 0.40% 100% 85% 55% G INS 52 1 0.04% 100% 100% 55% 2bp INS 54 1 0.04% 100% 100% 55% Ser18Pro 1 0.04% 100% 100% 55% Val188Leu 1 0.04% 100% 100% 55% Leu19Pro 6 0.22% 100% 100% 55% Leu19Arg 1 0.04% 100% 100% 55% 234bp DEL 56 1 0.04% 100% 100% 55% C DEL 59 3 0.11% 100% 100% 55% Ala20STOP 1 0.04% 100% 100% 55% G DEL 60 1 0.04% 100% 100% 55% Val21Ala 1 0.04% 100% 25% 55% Val21Gly 2 0.07% 100% 100% 55% G DEL 61 1 0.04% 100% 100% 55% A DEL 64 1 0.04% 100% 100% 55% Gly23Ala 1 0.04% 100% 100% 55% G DEL 67 1 0.04% 100% 100% 55% G DEL 71 6 0.22% 100% 100% 55% Gly24Asp 6 0.22% 100% 100% 55% G DEL 70 21 0.76% 100% 100% 55% C DEL 74 1 0.04% 100% 100% 55% 5bp DEL 74 1 0.04% 100% 100% 55% Ala26Gly 3 0.11% 100% 100% 55% GC INS 76 2 0.07% 100% 100% 55% G INS codon 77 2 0.07% 100% 100% 55% Leu27Pro 7 0.25% 100% 88% 55% Ala28Val 1 0.04% 100% 100% 55% C DEL 84 0 0.00% 100% #DIV/0! 55% C DEL 85 1 0.04% 100% 100% 55% C INS 85 1 0.04% 100% 100% 55% 24bp DEL 88 1 0.04% 100% 100% 55% Ile31Ser 4 0.14% 100% 57% 55% lle31thr 4 0.14% 100% 100% 55% Ile31Thr 8 0.29% 100% 100% 55% Asp33Ala 1 0.04% 100% 50% 55% C DEL 99 1 0.04% 100% 100% 55% Tyr34Asp 11 0.40% 100% 100% 55% Tyr34STOP 7 0.25% 100% 100% 55%
Tyr34Ser 2 0.07% 100% 100% 55% A INS 102 1 0.04% 100% 100% 55% Tyr34Cys 1 0.04% 100% 100% 55% 167bp DEL 102 3 0.11% 100% 100% 55% C DEL 103 1 0.04% 100% 100% 55% Leu35Pro 8 0.29% 100% 89% 55% Leu35Arg 1 0.04% 100% 33% 55% C DEL 104 1 0.04% 100% 100% 55% G INS codon 104 1 0.04% 100% 100% 55% CG INS 107 1 0.04% 100% 100% 55% C DEL 108 1 0.04% 100% 100% 55% 5.3kb DEL 106 0 0.00% 100% #DIV/0! 55% Ala36Ala 1 0.04% 100% 100% 55% Glu37stop 1 0.04% 100% 100% 55% Tyr41STOP 7 0.25% 100% 100% 55% Tyr41His 1 0.04% 100% 100% 55% T INS 122 1 0.04% 100% 100% 55% CG DEL 129 1 0.04% 100% 100% 55% His43Pro 4 0.14% 100% 100% 55% Val44Gly 2 0.07% 100% 100% 55% GG INS 131 1 0.04% 100% 100% 55% Val45Ala 3 0.11% 100% 75% 55% Val45Gly 1 0.04% 100% 100% 55% Ala46Val 12 0.43% 100% 80% 55% Ala46Pro 1 0.04% 100% 100% 55% Ala46Glu 1 0.04% 100% 100% 55% Val46Asn 1 0.04% 100% 100% 55% G INS 136 2 0.07% 100% 100% 55% Ala46Ser 1 0.04% 100% 100% 55% 7bp INS 141 1 0.04% 100% 100% 55% Thr47Ala 18 0.65% 100% 67% 55% Thr47Pro 4 0.14% 100% 100% 55% Thr47Ser 1 0.04% 100% 100% 55% Lys48Glu 3 0.11% 100% 60% 55% Lys48stop 1 0.04% 100% 100% 55% Lys48Thr 8 0.29% 100% 100% 55% Lys48Arg 1 0.04% 100% 100% 55% Asp49Gly 21 0.76% 100% 100% 55% Asp49Ala 5 0.18% 100% 100% 55% Asp49Asn 30 1.09% 100% 100% 55% Asp49Val 1 0.04% 100% 100% 55% Asp49Tyr 1 0.04% 100% 100% 55% A DEL 146 1 0.04% 100% 100% 55%
80bp DEL 150 1 0.04% 100% 100% 55% G INS 150 1 0.04% 100% 100% 55% T DEL 150 1 0.04% 100% 100% 55% His51Arg 24 0.87% 100% 92% 55% His51Asp 1 0.04% 100% 100% 55% His51Gln 23 0.83% 100% 100% 55% T INS 152 2 0.07% 100% 100% 55% His51Pro 10 0.36% 100% 91% 55% His51Tyr 4 0.14% 100% 100% 55% His51Asn 1 0.04% 100% 100% 55% His51Leu 1 0.04% 100% 100% 55% Asp53Ala 1 0.04% 100% 100% 55% Asp53Asn 1 0.04% 100% 100% 55% 44bp DEL 158 1 0.04% 100% 100% 55% Pro54Arg 3 0.11% 100% 100% 55% Pro54Leu 25 0.91% 100% 93% 55% Pro54Gln 7 0.25% 100% 88% 55% Pro54Thr 3 0.11% 100% 100% 55% Pro54Ser 3 0.11% 100% 100% 55% G DEL 162 1 0.04% 100% 100% 55% G DEL 164 1 0.04% 100% 100% 55% C INS 168 1 0.04% 100% 100% 55% His57Arg 16 0.58% 100% 100% 55% His57Asp 29 1.05% 100% 100% 55% His57Pro 7 0.25% 100% 100% 55% T INS 170 1 0.04% 100% 100% 55% His57Gln 1 0.04% 100% 100% 55% His57Tyr 7 0.25% 100% 88% 55% Phe58Leu 13 0.47% 100% 81% 55% T INS 174 1 0.04% 100% 100% 55% Phe58Ser 1 0.04% 100% 100% 55% Ser59Pro 7 0.25% 100% 100% 55% Thr61Pro 1 0.04% 100% 100% 55% 5bp INS 182 3 0.11% 100% 100% 55% 2bp DEL 182 1 0.04% 100% 100% 55% Pro62Gln 4 0.14% 100% 100% 55% Pro62Leu 8 0.29% 100% 100% 55% Pro62His 1 0.04% 100% 100% 55% 4bp INS 185 1 0.04% 100% 100% 55% A INS 186 1 0.04% 100% 100% 55% C DEL 186 3 0.11% 100% 100% 55% Pro62Arg 3 0.11% 100% 100% 55% Pro62Thr 3 0.11% 100% 100% 55%
T INS 184 1 0.04% 100% 100% 55% Asp63Gly 9 0.33% 100% 100% 55% Asp63Ala 6 0.22% 100% 100% 55% 2bp INS 188 1 0.04% 100% 100% 55% T INS 188 1 0.04% 100% 100% 55% Asp63His 1 0.04% 100% 100% 55% GG INS 188 1 0.04% 100% 100% 55% A INS 192 1 0.04% 100% 100% 55% Tyr64stop 3 0.11% 100% 100% 55% Tyr64Asp 1 0.04% 100% 100% 55% Ser65Ser 14 0.51% 99% 36% 55% A INS 193 8 0.29% 100% 100% 55% 7bp ins 193 1 0.04% 100% 100% 55% Ser65Pro 1 0.04% 100% 33% 55% Ser65Ser/Frameshift 1 0.04% 100% 100% 55% 14BP DEL 196 1 0.04% 100% 100% 55% Ser66Pro 2 0.07% 100% 100% 55% Ser67Pro 10 0.36% 100% 100% 55% 8bp INS 200 1 0.04% 100% 100% 55% Trp68Leu 4 0.14% 100% 100% 55% Trp68STOP 3 0.11% 100% 100% 55% Trp68Gly 16 0.58% 100% 94% 55% Trp68Arg 13 0.47% 100% 93% 55% Trp68Cys 6 0.22% 100% 100% 55% Pro69Arg 9 0.33% 100% 100% 55% Pro69Leu 1 0.04% 100% 100% 55% His71Tyr 8 0.29% 100% 100% 55% His71Arg 9 0.33% 100% 75% 55% His71Glu 1 0.04% 100% 100% 55% His71Asn 2 0.07% 100% 100% 55% 14BP INS 212 1 0.04% 100% 100% 55% 3bp DEL 211 1 0.04% 100% 100% 55% His71Asp 9 0.33% 100% 100% 55% His71Gln 4 0.14% 100% 100% 55% His71Thr 2 0.07% 100% 100% 55% T DEL 213 1 0.04% 100% 100% 55% His71Pro 1 0.04% 100% 100% 55% Cys72Arg 10 0.36% 100% 100% 55% Cys72STOP 2 0.07% 100% 100% 55% Cys72Tyr 2 0.07% 100% 100% 55% 29bp INS 216 1 0.04% 100% 100% 55% Cys72Trp 5 0.18% 100% 100% 55%
10bp INS 218 1 0.04% 100% 100% 55% G INS 218 1 0.04% 100% 100% 55% G INS 221 2 0.07% 100% 100% 55% Thr76Ile 13 0.47% 100% 100% 55% Thr76Pro 40 1.45% 100% 98% 55% Thr76Ala 1 0.04% 100% 100% 55% 251bp DEL 226 2 0.07% 100% 100% 55% IS 6110 INS 230 1 0.04% 100% 100% 55% C DEL 234 1 0.04% 100% 100% 55% 4bp INS 233 1 0.04% 100% 100% 55% G INS 234 1 0.04% 100% 100% 55% Gly78Asp 1 0.04% 100% 100% 55% Gly78Cys 3 0.11% 100% 100% 55% Gly78Val 1 0.04% 100% 100% 55% Gly78Gly 1 0.04% 100% 100% 55% A DEL 239 1 0.04% 100% 100% 55% Phe81Val 2 0.07% 100% 67% 55% Phe81Cys 1 0.04% 100% 100% 55% His82Arg 13 0.47% 100% 100% 55% His82Asp 2 0.07% 100% 67% 55% His82Leu 1 0.04% 100% 100% 55% CA INS 246 1 0.04% 100% 100% 55% Pro83Arg 1 0.04% 100% 100% 55% C INS 250 4 0.14% 100% 100% 55% Leu85Gln 2 0.07% 100% 100% 55% Leu85Arg 11 0.40% 100% 100% 55% Leu85Pro 41 1.49% 100% 98% 55% 14bp DEL 257 2 0.07% 100% 100% 55% GG INS 257 1 0.04% 100% 100% 55% 18bp INS 258 1 0.04% 100% 100% 55% 1bp DEL 260 1 0.04% 100% 100% 55% AC INS 261 6 0.22% 100% 100% 55% Thr87Met 2 0.07% 100% 100% 55% 2bp INS 261 2 0.07% 100% 100% 55% AC INS 260 1 0.04% 100% 100% 55% 68bp DEL 195-263 1 0.04% 100% 100% 55% Ser88STOP 2 0.07% 100% 100% 55% Ile90Ser 5 0.18% 100% 100% 55% Glu91STOP 2 0.07% 100% 100% 55% Val93Leu 1 0.04% 100% 100% 55% 106bp INS 279 1 0.04% 100% 100% 55% Phe94Ser 5 0.18% 100% 100% 55% Phe94Cys 5 0.18% 100% 83% 55%
Phe94Leu 14 0.51% 100% 93% 55% Phe94Pro 2 0.07% 100% 100% 55% Tyr95stop 2 0.07% 100% 100% 55% CA DEL 285 2 0.07% 100% 100% 55% Lys96Gln 2 0.07% 100% 100% 55% Lys96Thr 8 0.29% 100% 100% 55% Lys96Arg 5 0.18% 100% 100% 55% G DEL 288 2 0.07% 100% 100% 55% T INS 287 1 0.04% 100% 100% 55% Lys96Asn 7 0.25% 100% 100% 55% A DEL 286 2 0.07% 100% 100% 55% Lys96Glu 13 0.47% 100% 93% 55% 33bp INS 288 4 0.14% 100% 100% 55% T INS 288 2 0.07% 100% 100% 55% Lys96STOP 1 0.04% 100% 100% 55% Gly97Asp 13 0.47% 100% 93% 55% Gly97Cys 4 0.14% 100% 100% 55% Gly97Ser 16 0.58% 100% 100% 55% Gly97Ala 2 0.07% 100% 100% 55% T DEL 291 1 0.04% 100% 100% 55% G DEL 290 1 0.04% 100% 100% 55% T INS 290 1 0.04% 100% 100% 55% Gly97Arg 1 0.04% 100% 100% 55% 10bp DEL 293 1 0.04% 100% 100% 55% Tyr99STOP 6 0.22% 100% 100% 55% Thr100Pro 4 0.14% 100% 100% 55% Thr100Ala 1 0.04% 100% 100% 55% G DEL 301 1 0.04% 100% 100% 55% A INS 301 1 0.04% 100% 100% 55% A DEL 303 1 0.04% 100% 100% 55% Ala102Val 5 0.18% 100% 56% 55% Ala102Thr 2 0.07% 100% 100% 55% Als102Val 2 0.07% 100% 100% 55% Tyr103Asp 3 0.11% 100% 100% 55% Tyr103Cys 3 0.11% 100% 75% 55% Tyr103His 8 0.29% 100% 100% 55% C INS 307 1 0.04% 100% 100% 55% Tyr103STOP 19 0.69% 100% 95% 55% Tyr103Ser 1 0.04% 100% 100% 55% Ser104Cys 1 0.04% 100% 100% 55% Ser104Arg 9 0.33% 100% 100% 55% Ser104Ile 1 0.04% 100% 100% 55% C INS 314 2 0.07% 100% 100% 55%
Gly105Asp 7 0.25% 100% 100% 55% G INS 315 4 0.14% 100% 100% 55% Gly105Val 1 0.04% 100% 100% 55% CT INS 317 1 0.04% 100% 100% 55% Glu107STOP 1 0.04% 100% 100% 55% Gly108Arg 10 0.36% 100% 100% 55% Gly108STOP 1 0.04% 100% 100% 55% Gly108Val 1 0.04% 100% 100% 55% A DEL 333 1 0.04% 100% 100% 55% Glu111STOP 2 0.07% 100% 100% 55% Asn112Tyr 3 0.11% 100% 100% 55% G INS 339 1 0.04% 100% 100% 55% G DEL 337 1 0.04% 100% 100% 55% G DEL 341 1 0.04% 100% 100% 55% Thr114Pro 2 0.07% 100% 100% 55% Tyr114Ser 1 0.04% 100% 100% 55% 1355bp INS 342 1 0.04% 100% 100% 55% IS6110 INS codon 114 1 0.04% 100% 100% 55% C DEL 342 2 0.07% 100% 100% 55% Leu116Pro 5 0.18% 100% 100% 55% Leu116Arg 8 0.29% 100% 100% 55% Leu116Gly 1 0.04% 100% 100% 55% C INS 350 2 0.07% 100% 100% 55% T INS 350 1 0.04% 100% 100% 55% Asn118Thr 1 0.04% 100% 100% 55% 7bp DEL 352 1 0.04% 100% 100% 55% Trp119Arg 6 0.22% 100% 100% 55% Trp119Cys 2 0.07% 100% 67% 55% Trp119Gly 1 0.04% 100% 100% 55% Trp119Ser 1 0.04% 100% 100% 55% Trp119Stop 1 0.04% 100% 100% 55% Trp119Leu 1 0.04% 100% 100% 55% Leu120Arg 5 0.18% 100% 100% 55% Leu120Pro 27 0.98% 100% 100% 55% Leu120Gln 2 0.07% 100% 100% 55% 81bp DEL 359 2 0.07% 100% 100% 55% C DEL 361 1 0.04% 100% 100% 55% Arg121Pro 4 0.14% 100% 100% 55% 8bp INS 366 1 0.04% 100% 100% 55% Gln122STOP 3 0.11% 100% 100% 55% Gln122Gln 1 0.04% 100% 100% 55% C INS 365 1 0.04% 100% 100% 55%
Arg123Leu 1 0.04% 100% 100% 55% Arg123Pro 4 0.14% 100% 100% 55% AG INS 368 1 0.04% 100% 100% 55% 16bp DEL 374 2 0.07% 100% 100% 55% 14bp DEL 375 1 0.04% 100% 100% 55% C INS 375 1 0.04% 100% 100% 55% Val125Phe 6 0.22% 100% 100% 55% 9BP INS 373 2 0.07% 100% 100% 55% Val125Asp 3 0.11% 100% 100% 55% 15bp DEL 375 1 0.04% 100% 100% 55% Val125Gly 10 0.36% 100% 100% 55% Val125Leu 1 0.04% 100% 100% 55% GA INS 377 1 0.04% 100% 100% 55% 10bp DEL 378 1 0.04% 100% 100% 55% 30bp DEL 379 1 0.04% 100% 100% 55% 9bp DEL 380 1 0.04% 100% 100% 55% Glu127Lys 1 0.04% 100% 100% 55% Glu127Cys 1 0.04% 100% 100% 55% 11BP DEL 380 1 0.04% 100% 100% 55% Glu127STOP 1 0.04% 100% 100% 55% 8bp DEL 382 1 0.04% 100% 100% 55% Val128Gly 15 0.54% 100% 94% 55% AG INS 382 1 0.04% 100% 100% 55% 9bp DEL 382 1 0.04% 100% 100% 55% 9bp DEL codon 128 1 0.04% 100% 100% 55% 5BP DEL 385 1 0.04% 100% 100% 55% Asp129Tyr 1 0.04% 100% 100% 55% 3bp DEL 386 1 0.04% 100% 100% 55% CG INS 385 1 0.04% 100% 100% 55% 11-bpDEL 379-389 1 0.04% 100% 100% 55% GG INS 390 1 0.04% 100% 100% 55% Val130Gly 4 0.14% 100% 100% 55% Val130Ala 4 0.14% 100% 80% 55% GG INS 389 3 0.11% 100% 100% 55% 9bp INS 388 0 0.00% 100% #DIV/0! 55% G INS 389 1 0.04% 100% 100% 55% GG INS codon 130 1 0.04% 100% 100% 55% 9-bp INS 388 4 0.14% 100% 100% 55% GG INS 392 9 0.33% 100% 100% 55% G INS 392 2 0.07% 100% 100% 55% G INS 391 5 0.18% 100% 100% 55% GG INS 391 10 0.36% 100% 100% 55% Val131Phe 1 0.04% 100% 100% 55%
G INS 393 1 0.04% 100% 100% 55% GG INS 393 1 0.04% 100% 100% 55% GT INS 393 1 0.04% 100% 100% 55% T INS 393 1 0.04% 100% 100% 55% G DEL 391 1 0.04% 100% 100% 55% 9bp INS 395 1 0.04% 100% 100% 55% Gly132Asp 8 0.29% 100% 100% 55% Gly132Ser 16 0.58% 100% 100% 55% Gly132Val 3 0.11% 100% 100% 55% 17bp DEL 395 1 0.04% 100% 100% 55% Gly132Ala 5 0.18% 100% 100% 55% 6bp INS 394 1 0.04% 100% 100% 55% Gly132Cys 2 0.07% 100% 100% 55% Gly132STOP 1 0.04% 100% 100% 55% Ile133Ser 1 0.04% 100% 100% 55% TT DEL 398 1 0.04% 100% 100% 55% Ile133Thr 16 0.58% 100% 70% 55% G INS 397 1 0.04% 100% 100% 55% 163bp DEL codon 133 1 0.04% 100% 100% 55% T INS 398 1 0.04% 100% 100% 55% Ile133Asn 1 0.04% 100% 100% 55% T INS 397 2 0.07% 100% 100% 55% Ala134Val 8 0.29% 100% 100% 55% CC INS 403 1 0.04% 100% 100% 55% Thr135Pro 21 0.76% 100% 95% 55% Thr135Asn 3 0.11% 100% 100% 55% C INS 403 1 0.04% 100% 100% 55% A INS 408 4 0.14% 100% 100% 55% A INS 407 1 0.04% 100% 100% 55% Asp136Gly 6 0.22% 100% 75% 55% Asp136His 2 0.07% 100% 100% 55% C INS 407 1 0.04% 100% 100% 55% Asp136Tyr 4 0.14% 100% 100% 55% GC INS 407 1 0.04% 100% 100% 55% His137Pro 9 0.33% 100% 100% 55% G INS 411 1 0.04% 100% 100% 55% His137Arg 5 0.18% 100% 100% 55% His137Asp 1 0.04% 100% 100% 55% Cys138Ser 2 0.07% 100% 100% 55% Cys138Tyr 8 0.29% 100% 100% 55% Cys138STOP 2 0.07% 100% 100% 55% Cys138Arg 5 0.18% 100% 100% 55%
Cys138Trp 1 0.04% 100% 100% 55% Cys138Cys 2 0.07% 100% 100% 55% T DEL 412 1 0.04% 100% 100% 55% Val139Gly 16 0.58% 100% 94% 55% Val139Met 1 0.04% 100% 100% 55% Val139Ala 21 0.76% 100% 81% 55% Val139Leu 14 0.51% 100% 100% 55% G INS 417 1 0.04% 100% 100% 55% TG DEL 416 1 0.04% 100% 100% 55% GC INS 420 1 0.04% 100% 100% 55% G INS 420 3 0.11% 100% 100% 55% G DEL 418 1 0.04% 100% 100% 55% 68bp DEL 420 1 0.04% 100% 100% 55% Arg140Pro 1 0.04% 100% 100% 55% Arg140Gly 1 0.04% 100% 100% 55% Gln141Pro 24 0.87% 100% 100% 55% 10bp INS codon 141 1 0.04% 100% 100% 55% 13bp INS 423 1 0.04% 100% 100% 55% Gln141stop 4 0.14% 100% 100% 55% C INS 422 1 0.04% 100% 100% 55% Thr142Ala 6 0.22% 100% 100% 55% Thr142Lys 10 0.36% 100% 100% 55% Thr142Met 15 0.54% 100% 100% 55% GG INS 428 1 0.04% 100% 100% 55% Ala143Pro 2 0.07% 100% 100% 55% Ala143Thr 1 0.04% 100% 25% 55% Ala143Gly 1 0.04% 100% 100% 55% GAG DEL 430 1 0.04% 100% 100% 55% Ala146Thr 9 0.33% 100% 100% 55% Ala146Glu 2 0.07% 100% 100% 55% Ala146Val 3 0.11% 100% 100% 55% Ala146Pro 1 0.04% 100% 100% 55% T INS 440 1 0.04% 100% 100% 55% CG INS 439 3 0.11% 100% 100% 55% Arg148Ser 3 0.11% 100% 100% 55% 12bp INS 444 1 0.04% 100% 100% 55% 12bp INS 444 4 0.14% 100% 100% 55% G DEL 443 2 0.07% 100% 100% 55% 8-bpDEL 446 1 0.04% 100% 100% 55% GC INS 448 2 0.07% 100% 100% 55% DEL 449 1 0.04% 100% 100% 55% Leu151Ser 6 0.22% 100% 100% 55%
T DEL 451 1 0.04% 100% 100% 55% T INS 451 1 0.04% 100% 100% 55% Thr153Asn 1 0.04% 100% 100% 55% Thr153Ile 2 0.07% 100% 100% 55% Arg154Gly 6 0.22% 100% 75% 55% Val155Met 5 0.18% 100% 100% 55% Val155Glu 1 0.04% 100% 100% 55% Val155Gly 19 0.69% 100% 100% 55% Val155Ala 1 0.04% 100% 20% 55% CT INS codon 155 1 0.04% 100% 100% 55% T INS 465 1 0.04% 100% 100% 55% Leu156Pro 2 0.07% 100% 100% 55% Leu156Gln 1 0.04% 100% 100% 55% Val157Ala 2 0.07% 100% 100% 55% Val157Gly 1 0.04% 100% 50% 55% Val158Gly 1 0.04% 100% 100% 55% Leu159Pro 7 0.25% 100% 100% 55% Leu159Arg 17 0.62% 100% 100% 55% C INS 475 1 0.04% 100% 100% 55% Thr160Lys 2 0.07% 100% 100% 55% Thr160Pro 5 0.18% 100% 100% 55% 6bp INS codon 160 1 0.04% 100% 100% 55% Thr160Ala 1 0.04% 100% 50% 55% Ala161Pro 3 0.11% 100% 100% 55% Gly162Ser 2 0.07% 100% 100% 55% Gly162Asp 4 0.14% 100% 100% 55% GT DEL 485 1 0.04% 100% 100% 55% Gly162Val 1 0.04% 100% 100% 55% 11bp DEL 486 1 0.04% 100% 100% 55% 10bp DEL 487 1 0.04% 100% 100% 55% 1bp DEL 487 1 0.04% 100% 100% 55% Val163Ala 1 0.04% 100% 50% 55% Val163Arg 1 0.04% 100% 100% 55% Ser164Pro 3 0.11% 100% 100% 55% T DEL 490 5 0.18% 100% 100% 55% C INS 495 1 0.04% 100% 100% 55% C INS 493 1 0.04% 100% 100% 55% 1bp INS 497 1 0.04% 100% 100% 55% CG INS 501 1 0.04% 100% 100% 55% Thr167Pro 1 0.04% 100% 100% 55% Thr168Pro 4 0.14% 100% 100% 55% Thr168Asn 1 0.04% 100% 100% 55% 6bp DEL 504 1 0.04% 100% 100% 55%
Val169Ala 1 0.04% 100% 100% 55% C DEL 512 1 0.04% 100% 100% 55% Ala171Val 5 0.18% 100% 71% 55% Ala171Thr 3 0.11% 100% 75% 55% Ala171Pro 3 0.11% 100% 100% 55% GC DEL 513 1 0.04% 100% 100% 55% Ala171Glu 4 0.14% 100% 100% 55% Leu172Pro 22 0.80% 100% 96% 55% Leu172Ala 2 0.07% 100% 100% 55% C DEL 514 2 0.07% 100% 100% 55% CG INS 516 1 0.04% 100% 100% 55% T DEL 515 1 0.04% 100% 100% 55% G INS 516 1 0.04% 100% 100% 55% Leu172Arg 1 0.04% 100% 100% 55% 5-bp INS 518 1 0.04% 100% 100% 55% G INS 522 1 0.04% 100% 100% 55% Gln174STOP 2 0.07% 100% 100% 55% GG INS 520 1 0.04% 100% 100% 55% T INS 521 1 0.04% 100% 100% 55% Met175Val 12 0.43% 100% 100% 55% Met175Arg 1 0.04% 100% 100% 55% Met175Thr 3 0.11% 100% 100% 55% Thr177Pro 2 0.07% 100% 100% 55% 6bp DEL 531 3 0.11% 100% 100% 55% CG INS codon 177 1 0.04% 100% 100% 55% C DEL 530 1 0.04% 100% 100% 55% 6bp DEL 533 6 0.22% 100% 100% 55% Ser179Arg 2 0.07% 100% 100% 55% Ser179Ile 1 0.04% 100% 100% 55% Ser179Cys 1 0.04% 100% 100% 55% Val180Phe 7 0.25% 100% 100% 55% Val180Ala 1 0.04% 100% 100% 55% Val180Gly 4 0.14% 100% 100% 55% A DEL 542 1 0.04% 100% 100% 55% 4bp DEL 542 1 0.04% 100% 100% 55% Leu182Ser 4 0.14% 100% 100% 55% Cys184Tyr 1 0.04% 100% 100% 55% STOP187Gly 1 0.04% 100% 100% 55% Arg191Glu 1 0.04% 100% 100% 55% T DEL 582 1 0.04% 100% 100% 55% Leu4Trp/Ser65Ser 1 0.04% 100% 100% 55% Asp8Tyr/Leu34Arg 1 0.04% 100% 100% 55%
Gln10Arg/Ser65Ser 1 0.04% 100% 100% 55% Ile5Met/TG INS codon 4 1 0.04% 100% 100% 55% Gln10Pro/Val128Phe 1 0.04% 100% 100% 55% Thr100Ala/Arg148Cys 1 0.04% 100% 100% 55% Val180Ala/C DEL codon 100 1 0.04% 100% 100% 55% A-11G/Ala161Ala 1 0.04% 100% 100% 55% A-11G/A DEL 535 1 0.04% 100% 100% 55% A-11G/Ser65Ser 1 0.04% 100% 100% 55% G DEL -115/Val128Gly 1 0.04% 100% 100% 55% G INS -117/His137Arg 1 0.04% 100% 100% 55% C DEL -125/Ser65Ser/GC INS 528 C DEL - 125/Ser65Ser/Trp68Arg 1 0.04% 100% 100% 55% 1 0.04% 100% 100% 55% Val130Gly/GG INS 428 1 0.04% 100% 100% 55% Arg140Cys/T DEL 415 1 0.04% 100% 100% 55% Cys14Trp/Tyr103His 1 0.04% 100% 100% 55% Cys14STOP/A DEL 535 1 0.04% 100% 100% 55% Cys14Arg/Ser65Ser 2 0.07% 100% 100% 55% Arg140Ser/8bp DEL 446 37 1.34% 100% 100% 55% A-15C/Leu4Ser 1 0.04% 100% 100% 55% T DEL 43/Ser65Ser 1 0.04% 100% 100% 55% Ala152Gly/7bp DEL 506 1 0.04% 100% 100% 55% Thr153Ile/Asp159Glu 1 0.04% 100% 100% 55% Arg154Met/Thr160Ala 1 0.04% 100% 100% 55%
9bp INS 484/2bp DEL 494 1 0.04% 100% 100% 55% Asp49His/Ala102Val 1 0.04% 100% 100% 55% 3bp DEL 57/4bp DEL 62/5bp 1 0.04% 100% 100% 55% DEL 70 Gly78Ser/DEL 59 1 0.04% 100% 100% 55% Val21Glu/Gln122Leu/Arg140 Gly/Asp145His/Gly150Asp/V al155met/ala165asp/ala178 Asp/Ser179Cys/Val183Phe 1 0.04% 100% 100% 55% Ala3Glu/Arg140His 1 0.04% 100% 100% 55% AG DEL 94/GA DEL 97 1 0.04% 100% 100% 55% Tyr34Asp/Ala134Ser 1 0.04% 100% 100% 55% Leu35Arg/His82Asp 1 0.04% 100% 100% 55% Ala39Ala/His57Asp 1 0.04% 100% 100% 55% Ala165Thr/2bp DEL 129 1 0.04% 100% 100% 55% Thr47Ala/Arg148Cys 1 0.04% 100% 100% 55% His51Gln/Ala165Thr 1 0.04% 100% 100% 55% Ile52Ile/Leu182Phe 1 0.04% 100% 100% 55% Asp53Asn/5bp INS 349 1 0.04% 100% 100% 55% Asp56Cys/His57Pro/Phe58Le 1 0.04% 100% 100% 55% u Thr61Ser/Val139Ala 1 0.04% 100% 100% 55% Thr61Arg/Trp68Leu 1 0.04% 100% 100% 55% Ser65Ser/Gly105Asp/ A DEL 1 0.04% 100% 100% 55% 535 Ser65Ser/A DEL 329 1 0.04% 100% 100% 55%
Ser65Ser/Asp110Tyr/A DEL 1 0.04% 100% 100% 55% 535 Ser65Ser/Val128Gly 2 0.07% 100% 100% 55% Ser67Leu/GG INS 390 1 0.04% 100% 100% 55% Ser65Ser/G INS 391 1 0.04% 100% 100% 55% Ser65Ser/G INS 408 1 0.04% 100% 100% 55% Ser65Ser/Asp136Asn 1 0.04% 100% 100% 55% Ser65Ser/Gln141Pro 1 0.04% 100% 100% 55% Ser65Ser/Thr142Pro 2 0.07% 100% 100% 55% Ser65Ser/Leu172Pro 1 0.04% 100% 100% 55% Ser65Ser/GC INS 528 1 0.04% 100% 100% 55% Ser65Ser/Ser179Arg 1 0.04% 100% 100% 55% Ser65Ser/A DEL 535 1 0.04% 100% 100% 55% Ser65Ser/His82Asp 1 0.04% 100% 100% 55% Ser65Ser/2bp DEL 282 1 0.04% 100% 100% 55% Thr135Ala/Arg154Thr/ACC 1 0.04% 100% 100% 55% DEL 207 T-7C/Thr114Ala 4 0.14% 100% 100% 55% Ala79Val/T INS 360 1 0.04% 100% 100% 55% His82Arg/Val130Ala 1 0.04% 100% 100% 55% Lys96Thr/G DEL 436 1 0.04% 100% 100% 55% GENE DEL 1 0.04% 100% 100% 55% 200+bp DEL 3 0.11% 100% 100% 55% Note: Mutations within isolates with multiple mutations in pnca were considered together and exclusive from other isolates with the same mutations. Isolates with multiple mutations are denoted with a slash ( / ) between the different mutations.
Supplemental Table 7. List of Excluded Mutations Due to Incorrect Reporting of Reference Amino Acid in PZase in Mycobacterium tuberculosis clinical isolates Reference and Author PubMed ID Year Published Number of Pyrazina mide- Resistant Isolates Codon Number H37rv Amino Acid Mutation Found in the Correspon ding Study Wade et 15183857 2004 1 118 Trp Tyr118Arg al. Sachais et 10089282 1998 1 80 Asp Phe80Val al. Jonmalun 20727143 2010 1 174 Gln Thr174Ile g et al. Jonmalun 20727143 2010 1 74 Ser Gly74Arg g et al. Jonmalun 20727143 2010 1 122 Gln Gly122Ser g et al. Jnwali et al. 23561273 2013 1 114 Thr Tyr114Ser Note: H37rv reference is under GenBank Accession Number NC_000962.3. Supplemental Table 8. Statistical Analysis of the Coding Region or the Coding Region and the Promoter Region of pnca in Mycobacterium tuberculosis Pyrazinamide-Resistant Isolates for Molecular-Diagnostic Tests pnca Sensitivity Specificity Negative Predictive Value Positive Predictive Value Coding Region Only 79% 90% 88% 88% Coding + Promoter Regions 83% 91% 86% 88% Supplemental Table 9. List and frequency of PZase mutations found in phenotypically Pyrazinamide-Susceptible Mycobacterium tuberculosis isolates. Number of Unique Mutation Number of Susceptible Isolates Resistant with Mutation C DEL -125 1 0
A-11C 2 2 A-11G 4 36 A-11T 1 0 C INS -3 1 1 Met1Ile 1 5 Ile6Leu 120 8 Val7Gly 1 11 Gln10Arg 1 8 Asn11Asp 1 0 Asp12Gly 1 13 Asp12Ala 3 31 Phe13Leu 1 3 Phe13Ser 1 2 Cys14Arg 1 13 Cys14Gly 3 2 Val163Ala 1 1 Gly17Ser 1 3 Val21Ala 3 1 Leu27Pro 1 7 Ala28Thr 1 0 Ile31Ser 3 4 Asp33Ala 1 1 Leu35Pro 1 8 Leu35Arg 2 1 Ala38Ala 1 0 His43Tyr 1 0 Val45Ala 1 3 Ala46Val 3 12 Thr47Ala 9 18 Lys48Glu 2 3 Asp49Glu 1 0 His51Arg 2 24 His51Pro 1 10 Pro54Gln 1 7 Pro54Leu 2 25 His57Tyr 1 7 Phe58Leu 3 13 Gly60Gly 2 0 Tyr64Ser 1 0 Ser65Ser 25 14 Ser65Pro 2 1 TCG INS 199 1 0 Trp68Arg 1 13
Trp68Gly 1 16 His71Arg 3 9 Thr76Pro 1 40 Phe81Ser 1 0 Phe81Val 1 2 His82Asp 1 2 Leu85Pro 1 41 Phe94Cys 1 5 Phe94Leu 1 14 Lys96Glu 1 13 Gly97Asp 1 13 Thr100Ile 1 0 Ala102Arg 1 0 Ala102Pro 1 0 Ala102Val 4 5 Tyr103STOP 1 19 Tyr103Cys 1 3 Trp119Cys 1 2 Leu119Pro 1 0 Gly124Asp 1 0 Val128Gly 1 15 Val128Phe 1 0 Val130Ala 1 4 Ile133Thr 7 16 Thr135Ser 1 0 Thr135Pro 1 21 Asp136Gly 2 6 His137Cys 2 0 Val139Ala 5 21 Val139Gly 1 16 Arg140His 2 0 Ala143Thr 3 1 Ala143Val 1 0 Arg154Gly 2 6 Arg154Ser 3 0 Val155Ala 4 1 Val155Leu 1 0 Val157Gly 1 1 Leu159Val 1 0 Thr160Ala 1 1 Thr168Ile 2 0 Ala171Val 2 5 Ala171Thr 1 3
Leu172Pro 1 22 Met175Ile 2 11 Leu182Trp 1 0 STOP187Ala 1 0 Stop187Arg 2 0 Trp119stop/Thr168Ile 1 0 C DEL -124/Ser65Ser 1 0 Arg140His/Gly150Gly/Ala165Thr 1 0 Arg140His/Ala165Thr 2 0 Supplemental Table 10. Percentages of Pyrazinamide Resistant Mycobacterium tuberculosis with or without Detectable Pyrazinamidase Activity or PZase Mutations Resistant isolates with PZase activity and at least one mutation in PZase Resistant isolates with PZase activity and no mutation in PZase Resistant isolates with no PZase activity and no mutations in PZase 6%* 6%* 3%* *Percentage of 1979 PZA-resistant isolates that underwent enzymatic testing Supplemental Table 11. List of Mutations in PZase or the Promoter Region Found in Pyrazinamide-Resistant M. tuberculosis Isolates that Tested Positive or Negative for Pyrazinamidase Activity. PZA-Resistant Associated Mutations in Pzase Number of PZA- Resistant Isolates with Positive Pzase Activity Number of PZA-Resistant Isolates with Negative Pzase Activity T-10C 1 0 Ala3Ser 1 0 Asp12Gly 1 8 Asp12Asn 0 4 Asp12Ala 1 15 Gly23Ala 1 0 Gly24Asp 1 1 Ala28Val 1 0 lle31thr 4 0 Tyr34Asp 1 9 Leu35Pro 1 2 Leu35Arg 1 0
His43Pro 1 1 Lys48Thr 2 0 Asp49Asn 1 1 Pro54Leu 1 10 His57Pro 1 6 Pro62Arg 1 1 Pro62Gln 1 2 Pro62Leu 1 5 Asp63Ala 1 0 Ser65Pro 1 0 His71Thr 1 1 Thr76Pro 2 15 Thr76Ile 1 1 Gly78Cys 1 0 Phe81Val 1 1 His82Arg 1 0 Pro83Arg 1 0 Phe94Leu 1 0 Ala102Val 1 3 Asn112Tyr 1 2 Leu116Pro 1 2 G INS 392 1 0 Gly132Asp 1 2 Ile133Thr 1 3 A INS 407 1 0 Val139Ala 1 3 Ala143Gly 1 0 Leu151Ser 1 4 Thr160Ala 1 0 Gly162Ser 1 0 Ala171Val 1 1 Met175Val 1 1 Thr177Pro 2 0 Ser65Ser/Leu172Pro 1 0
Supplemental Figure 2. Observed stratification of clinical isolates included in this study and the conclusion (typical diagnostic decision) based on three phenotypic, genotypic, and enzymatic tests. Based on the three tests eight groups have been defined. Groups 2 and 7 include isolates that produced concordant results in all three tests. The remaining six groups contain isolates with the results of one test to be inconsistent with the other two. Notation: DST=Drug Susceptibility Testing; WT=Wild Type; Molecular Test (any sequencing technology, e.g. whole genome sequencing, Sanger, or other PCR based methods)