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Using Molecular Genotyping Methods to Investigate of the Genetic Diversity of Mycobacterium tuberculosis Strains in Taiwan

Peter Chin

NDSRA

Mycobacteria Reference Laboratory

Division of Laboratory Research and Development

Center for Disease Control, Kun-Yang Office

Nankang, Taipei, Taiwan

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Section I. Introduction

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• Non-motile, non-pigmented

• Acid-Fast stain, rod-like, 0.4x3.0 micron

• Obligate aerobic, slow grower, need special media for culture

• Reduce nitrate, produce catalase

• Produce long chain fatty acids

• Genome of H37Rv sequenced in 1998

• Person to person transmission

through aerosol droplet

Mycobacterium tuberculosis

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Global TB Disease Burden – WHO Report 2002

Reprinted from www.who.int/entity/tb/publications/ global_report/2004/en/annex5.pdf

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Genome of Mycobacterium tuberculosis H37Rv

• more than 4.4 million base

pairs

• 3924 genes detected initially, 13 more genes uncovered through protemics and compatative genomics

• more than 25 genetic markers

identified for typing

Cole ST et al.

Nature 1998; 393: 537.

Reprinted from

http://www.cbs.dtu.dk/services/GenomeAtlas/atlasdata/Bacteria/Mtuberculosis_H37Rv_

Main/Mtuberculosis_H37Rv_Main.ZDNAatlas.nfp.png

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Section II. Mycobacterial Genotyping

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Purpose of genotyping of Mycobacterium tuberculosis

• To investigate biodiversity and population structure

of Mycobacterium tuberculosis

• To define predominant genotype

• To trace the origin of strains and their potential

spatial relatedness

• To built-up genomic database

• To initiate systematic TB molecular epidemiological

studies

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Genotyping Methodology

• Non-DNA Typing Methods

– Phage Typing

– Antibiotics Typing

– Serological Typing

– Biochemical Typing

• DNA-based Typing Methods

– Restriction Fragment Length Polymorphism w/o Hybridization

– Pulsed Field Gel Electrophoresis

– Transposable Element Typing (DR, IR, IS…etc. )

– 16s/32s rDNA(RNA) Typing

– Minisatellite-based Typing

• Polymorphic GC-rich Repetitive Sequence

• Major Polymorphic Tandem Repeat

• Variable Number Tandem Repeat

• Mycobacterial Interspersed Repeat Units

疾病管制局 Specimen Collection

Decontamination and Concentration

Microscopy GenotypingMolecular DiagnosisCulture

Acid-Fast stain L-J MGIT

Biochemical tests I

Genotyping:

RFLP,VNTR-MIRU

Spoligotyping

TB complex NTM

Drug susceptibility tests PCR-RFLP Biochemical tests II

PCR

PCR-RFLP

Real-time

PCR

spoligotyping

GenProbe VNTR

MIRU

Spoligotyping

Culture (+)

rpoB gene sequence

analysis

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RFLP Principle

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Heat Inactivation

DNA ExtractionTB Culture

DNA Digestion

Gel Electrophoresis Overnight GelMembrane Blotting

Hybridization with Probes

Detection & Analysis

Sample and Internal marker probe

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• a PCR based method

• DNA polymorphism at “Direct Repeat” (DR) region of chromosomal locus

Spoligotyping Principle

A

B

C

D

E

DR:36bp DVRNon-repetitive

DNA spacer

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Apply Oligos in an array using a miniblotter

Oligos are bound covalently in line pattern to membrane

Rotate filter 90° apply biotin-labeled

PCR products and hybridize

Incubate with HRP-conjugate and substrate and expose X-ray film

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Heat Inactivation

DNA ExtractionTB Culture

PCR

Sample Application

Hybridization

Detection

Spoligotyping

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MIRU Principle

• Application of MTB H37Rv Genome Project

• Mid-short tandem repeat around the genome, so

called minisatellite genetic marker

• Multi nucleotide repeat unit

– ex. (ATCG)n

R. Frothingham and W.A. Meeker-O’Connell, 1998

P. Supply et al. 2000

疾病管制局 MIRU Primer SetPanel Oligo Name Sequence(5'→3') Modification

Panel-A-Locus-4-F GCGCGAGAGCCCGAACTGC 5' FAM

Panel-A-Locus-4-R GCGCAGCAGAAACGTCAGC

Panel-A-Locus-26-F TAGGTCTACCGTCGAAATCTGTGAC

Panel-A-Locus-26-R CATAGGCGACCAGGCGAATAG 5' HEX

Panel-A-Locus-40-F GGGTTGCTGGATGACAACGTGT 5' TAMRA

Panel-A-Locus-40-R GGGTGATCTCGGCGAAATCAGATA

Panel-B-Locus-10-F GTTCTTGACCAACTGCAGTCGTCC

Panel-B-Locus-10-R GCCACCTTGGTGATCAGCTACCT 5' FAM

Panel-B-Locus-16-F TCGGTGATCGGGTCCAGTCCAAGTA

Panel-B-Locus-16-R CCCGTCGTGCAGCCCTGGTAC 5' HEX

Panel-B-Locus-31-F ACTGATTGGCTTCATACGGCTTTA

Panel-B-Locus-31-R GTGCCGACGTGGTCTTGAT 5' TAMRA

Panel-C-Locus-2-F TGGACTTGCAGCAATGGACCAACT

Panel-C-Locus-2-R TACTCGGACGCCGGCTCAAAAT 5' FAM

Panel-C-Locus-23-F CTGTCGATGGCCGCAACAAAACG 5' HEX

Panel-C-Locus-23-R AGCTCAACGGGTTCGCCCTTTTGTC

Panel-C-Locus-39-F CGCATCGACAAACTGGAGCCAAAC

Panel-C-Locus-39-R CGGAAACGTCTACGCCCCACACAT 5' TAMRA

Panel-D-Locus-20-F TCGGAGAGATGCCCTTCGAGTTAG 5' FAM

Panel-D-Locus-20-R GGAGACCGCGACCAGGTACTTGTA

Panel-D-Locus-24-F CGACCAAGATGTGCAGGAATACAT

Panel-D-Locus-24-R GGGCGAGTTGAGCTCACAGAA 5' HEX

Panel-D-Locus-27-F TCGAAAGCCTCTGCGTGCCAGTAA

Panel-D-Locus-27-R GCGATGTGAGCGTGCCACTCAA 5' TAMRA

Panel-E-ETR-A-F AAATCGGTCCCATCACCTTCTTAT 5' FAM

Panel-E-ETR-A-R CGAAGCCTGGGGTGCCCGCGATTT

Panel-E-ETR-B-F GCGAACACCAGGACAGCATCATG 5' HEX

Panel-E-ETR-B-R GGCATGCCGGTGATCGAGTGG

Panel-E-ETR-C-F GTGAGTCGCTGCAGAACCTGCAG

Panel-E-ETR-C-R GGCGTCTTGACCTCCACGAGTG 5' TAMRAE

A

B

C

D

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Heat Inactivation

DNA ExtractionTB Culture Sample Dilution

MultiplexingPCRDetection

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Multiplex PCR With Dye-labeling Primer by Cap EP

FAM

TAMRA

HEX

MIRU 4

MIRU 40

MIRU 26

P

P

P

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LASER Scanner

Cap

(-) (+)

Time/bp

intensity

Multiplex PCR With Dye-labeling Primer by Cap EP

Injection Parameter: 3KV for 45 secs

Running Parameter: 10KV for 100 mins

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Allelic Calling

P. Supply et al. 2000, 2001

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Allelic Calling

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Section III. Study Proposal

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Objective

• Evaluating the genotyping efficiency between four method we performed

• Finding the proper balance between accuracy, throughput and timing

• Choosing the selective genetic markers

• Cutting down the cost

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Approach For MIRU Typing

• PCR each 15 locus

– Simply

– Lowest throughput, difficult to analysis

– Heavy loading, time-cost and expensive (15 PCR reactions/sample)

• Multiplex PCR With Dye-labeling Primer by Gel EP

– More complex

– Mid throughput, but easy data handling

– Heavy loading

• Multiplex PCR With Dye-labeling Primer by Cap EP

– Most complex and most expensive

– High throughput and easy data handling

– Lowest Labor Loading

– Bottle neck :upstream DNA extraction

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Stage Progress Period

2003

六月 七月 八月 九月

1 88dConstructing The TB Ref. LAB. Facility

2 88dEstablishment of VNTR Typing Protocol

3 21dEstablishment of MIRU Typing Protocol

4 133dSetup Automation and HTP Protocol

2004

十月 一月 二月 三月 四月 五月 六月 七月 八月 九月

5 23dFirst-stage Data Analysis

2004年10月7日

頁面 1

Progress Chart for The High-Throughput TB Genotyping Methodology Establishment

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Section IV. Result

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• Time interval: 2002~2004^

• Space: northern, central, southern and eastern area in Taiwan

• Criteria: AND (RFLP, Spoligotyping, VNTR, MIRU)

• Sample size: 479

Description of Sample Model

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Strain Selection

54%

16%

22%

8%

Northern

Central

Southern

Eastern

Description of Sample Model

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Age Distribution

1%18%

29%

49%

3% <20

21<x<40

41<x<60

>60

Unknown

Description of Sample Model

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Gender Distribution

67%

30%

3%

Male

Female

Unknown

Description of Sample Model

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Genotype Distribution

43%

53%

4%

B

NB

BL

Description of Sample Model

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Locus Discrimination power

1 2 3 4 5 6

Locus-2 0.241 0.02 0.080 0.140 0.020 0.077 Low

Locus-4 0.479 0.35 0.220 0.280 0.500 0.281 Low

Locus-10 0.617 0.69 0.440 0.700 0.710 0.639 Medium

Locus-16 0.526 0.52 0.420 0.280 0.310 0.300 Medium

Locus-20 0.205 0.29 0.090 0.080 0.030 0.061 Low

Locus-23 0.656 0.58 0.120 0.540 0.420 0.307 Low

Locus-24 0.445 0.24 0.160 0.000 0.350 0.160 Low

Locus-26 0.688 0.67 0.540 0.590 0.730 0.757 High

Locus-27 0.124 0.19 0.090 0.140 0.210 0.159 Low

Locus-31 0.647 0.37 0.470 0.550 0.640 0.690 High

Locus-39 0.394 0.34 0.220 0.380 0.600 0.535 Medium

Locus-40 0.797 0.74 0.630 0.650 0.540 0.453 Medium

ETR-A 0.756 N/A N/A N/A N/A 0.586 Medium

ETR-B 0.530 N/A N/A N/A N/A 0.560 Medium

ETR-C 0.584 N/A N/A N/A N/A 0.128 Low

Table. Allelic Diversity for each MIRU Locus

h

1. C. Sola. et al. 2003. Infect. Genet Evolution. 3:125–133

2. Mazars. et al. 2001. PNAS 98:1901-1906

3. Cowan. et al. 2002. J. Clin. Microbiol. 40:1592-1602

4. Supply. et al. 2003. Mol. Microbiol. 47:529-538

5. Sun. et al. 2004. J. Clin. Microbiol. 42: 1986-1993

6. This study

Allelic Diversity (h)

n: Sample size

xi: frequency of the ith

allele at the locus

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1. This study

2. C. Sola et al. 2003. Infect. Genet Evolution. 3 125–133

3. Robin et al. 2002. Microbiology. 148:519-528

4. Roring et al. 2002. J Clin. Microbiol. 40:2126-2133

5. Rachael et al. 2001. J Clin. Microbiol. 39:2453-2457

6. Sola et al. 2001. J Clin. Microbiol.39:1559–1565

Hunter-Gaston Discrimination Index (HGDI)

N: Sample size

S: Cluster number

nj: size of cluster belong to jth pattern

Pattern Sample Size Cluster No

1 2 3 4 5 6

RFLP 479 400 0.998 0.97 0.997

Spoligotyping 479 111 0.799 0.965 0.79 0.74 0.967

VNTR 479 57 0.866 0.959 0.81 0.74 0.938 0.939

MIRU 479 171 0.942 0.988

VNTR+MIRU 479 215 0.966

S+V+M 479 278 0.973

R+V+M 479 431 0.999

R+S+V+M 479 437 0.999

HGDI

Estimating HGDI For Each Genotyping Method And Combination Set

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0.975

0.98

0.985

0.99

0.995

1

1.005

0 2 4 6 8 10 12

Position Tolerance (%)

HG

DI

Position Tolerance 0.5 1.5 3 5 10

HGDI 0.999545776 0.998480097 0.997562914 0.99592072 0.980232528

HGDI Under Different PT Value

1 2

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Low Copy Number IS-6110 Genotyping Pattern:

RFLP Versus MIRU

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Section V. Allelic Diversity and Linkage Disequilibrium of Genetic Markers

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Introduction of Linkage Disequilibrium

• Disobeying Principle of Segregation

• Example

– 2 alleles (A & a) at locus A: P(A)=p

– 2 alleles (B & b) at locus A: P(B)=r

– 2 alleles (C & c) at locus A: P(C)=q

– D=P(AB)-prq

– D’=prq

• Useful marker for genome-wide scan analysis

– Pedigree analysis

– Sib-pair study

a

b

c C

A

B

C

b

A

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Phenomenon of Linkage Disequilibrium In M. tuberculosis

Sample 1

Locus-A 2 3 4 7

Locus-B 3 4 3 2

Locus-C 8 8 8 8

Locus-D 5 1 5 4

Locus-E 2 2 2 2

Sample 2 Sample 3 Sample n

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• Monte Carlo simulation

– Developed in 1948 by Los Alamos National Lab.

– Von Neumann, Fermi, Ulam and Metropolis

– Simulation of the H-bond explosion by resampling formula

– Calculating Physics, Chemistry, Biology, Economics

– Resampling the loci without replacement and computing a VD

value for each resampled data set

– Resampling for about 10000 times

• Parametric Method

– L = Ve + 1.654 √ Var(VD)

• Null hypothesis for Ve=VD

LIAN Version 3.1: Detecting LD in Multilocus Data

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Highly LD in Taiwan, Clonal Separation May Occur

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Conclusion

• Lower discrimination power compared with other publications

– Diverse genotype observed in Taiwan

• Different PT value can effect discrimination power obviously in

RFLP pattern

– Stability of IS6110, Electrophoresis Bias, Subjective Judgment of Data

• Linkage analysis showed highly clonal separation in Taiwan

– Need Further Surveillance

• Combination of VNTR & MIRU achieves compatible selective power

as RFLP

• Genotyping Strategy

– MIRU-VNTR as frontline, RFLP as confirmation and spoligotyping for

Beijing type surveillance

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Outlook

• Selection for appropriate LD markers

– Pairwise Association Analysis for Each Locus

• Redefinition the roles of RFLP, Spoligotyping, VNTR

and MIRU

– Flowchart or Pipeline for Genotyping Methodology

• Throughput Is Minority, Discrimination Power is

Majority

– More high-diversity markers (h>0.5)

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Acknowledgements

• Ruwen Jou- Principal Investigator

• Wei-Lun Huang- Assistant Research Fellow• Strain Identification, General Affair

• Su-Ying Chang- Medical Technician• Bacteria Culture, MDR surveillance

• Hsuan Liu- Research Assistant• RFLP, DNA Extraction

• Huang-Yau Chen- Research Assitant• Strain Identification, TB Virulence Research

• Meng-Hsun Chen- NDSRA• Spoligotyping, MDR Surveillance

• Ting-Yung Kou- Research Assistant• Proteomic, MDR Surveillance

• Pei-Ju Chin• MIRU-VNTR, TB susceptibility Research, Bioinformatics

Reprinted from http://profiles.nlm.nih.gov/VC/B/B/B/T/_/vcbbbt.jpg

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Thanks For Your Attention !