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Innovations in Molecular Epidemiology
Molecular EpidemiologyMolecular Epidemiology
• Measure current rates of active transmission
• Determine whether recurrent tuberculosis is attributable to exogenous reinfection
• Determine whether all strains in a population exert similar epidemiological
characteristics
• Understand transmission dynamics
• Identify outbreaks or extensive transmission from apparently sporadic,
epidemiologically unrelated cases
Which approach to choose?Which approach to choose?
• stability of biomarker - the observed rate of
polymorphism
• the genetic diversity of strains in the population
The rate of change of the chosen biomarker
must be able to distinguish epidemiologically
unrelated strains and yet sufficiently slow
and stable to reliably link related cases
Genetic Variation Generation Strategies in Genetic Variation Generation Strategies in
BacteriaBacteria
Can be divided into just three categories:
1. Small local changes in nucleotide sequence of genome (e.g. single nucleotide polymorphisms or ‘SNPs’)
2. Intragenomic reshuffling and deletion of segments of genomic sequence
3. Acquisition of DNA sequences from another organism
Low resolution high resolution
Different levels of resolution are Different levels of resolution are
obtained from different markersobtained from different markers
SNPs LSPs SpoligoVNTR/
MIRUIS6110
1.1. SNPsSNPs
• Mutational generation of a completely novel biological function unlikely: None described in M. tuberculosis
• Stepwise improvement of already available biological functions: a study of 26 structural genes from 842 strains of Mtb showed 95% of mutations linked to antibiotic resistance (rpoB, gyrA )
• ‘Silent’ (Synonymous) SNPs that do not affect the biological function: <subject to selective pressure
Strain typing, speciation
Strain Strain typingtyping
Taken from Sreevatsan et al, 1997
Broad evolutionary scenario for M.
tuberculosis complex organisms
characterised by KatG codon 463
(Leu) and GyrA codon 95 (Thr) into
three Principal Genetic Groups
(PGG1-3).
SpeciationSpeciation
SNPs used:
RpoB ID short sequence assay
gyrB SNP position 675
gyrB SNP position 756
gyrB SNP position 1410
gyrB SNP position 1450
2. Intragenomic Reshuffling of 2. Intragenomic Reshuffling of
Segments of DNASegments of DNA
Occurs via recombination of related sequences and can lead to:
• Novel combination of capacities by the fusion of different functional
domains
• Reassortment of expression control signals with different reading
frames serving in protein production
• Duplication of DNA segments serving as substrates for evolution
• Deletion of DNA segments to remove inessential sequences
Low resolution high resolution
Different levels of resolution are Different levels of resolution are
obtained from different markersobtained from different markers
SNPs LSPs SpoligoVNTR/
MIRUIS6110
Taken from Brosch et al, 2002
Deletion analysisDeletion analysis
Large Sequence PolymorphismsLarge Sequence Polymorphisms
Gagneux’a global phylogeny of M.
tuberculosis complex, based on 89 concatenated gene sequences
in 108 strains. Coloured branches
indicate the main strain lineages
(from Hershberg et al. PLoS
Biology 2008).
Frequency of subtypes based on
40,000 spoligotypes in spoldb4
Beijing 11.3%EAI 8.8%
H 11.2%
LAM 13.4%T 25%
X 9%
Large Sequence PolymorphismsLarge Sequence Polymorphisms
Global phylogeography of M. tuberculosis
Taken from Gagneux S and Small PM, Lancet Infect Dis 7:328-337 2007
Low resolution high resolution
Different levels of resolution are Different levels of resolution are
obtained from different markersobtained from different markers
SNPs LSPs SpoligoVNTR/
MIRUIS6110
Spoligotyping
Spacer Regions 35 – 43
Direct Repeat Region
35 36 37 38 39 40 41 42 43
Extension Primer
Direct Repeat PCR Primer
PCR Amplification
G
Primer Hybridization and Extension
C
Detection
Spacer 38 (+)extension product detected
Spacer 03 (-)extension product not detected
unextended primer only
1-34
M. tuberculosis M. tuberculosis SpoligotypingSpoligotyping
Developed by
SEQUENOM®, slide
courtesy of Dr Christiane Honisch
Low resolution high resolution
Different levels of resolution are Different levels of resolution are
obtained from different markersobtained from different markers
SNPs LSPs SpoligoVNTR/
MIRUIS6110
VNTR/MIRUVNTR/MIRU
• Variable number tandem repeat (VNTR) variation is
thought to caused by slipped-strand mispairing.
• The peculiar tertiary structure of repetitive DNA allows mis-matching
of neighbouring repeats, and, depending on the strand orientation,
repeats can be inserted or deleted during replication.
• Variation in repeat numbers and sequence degeneracy can be
explained by DNA recombination between multiple loci consisting of
homologous repeat motifs.
Genesis of repeats in Genesis of repeats in M. tuberculosisM. tuberculosis
• Requires pre-existing small
repeats (‘seeds)
• Short stem-loop structure aids slipped-strand
mispairing
G C
C GG CC GC GG C
VNTR sequence alignmentVNTR sequence alignment
10 20 30 40 50 60
....|....|....|....|....|....|....|....|....|....|....|....|....|.
CDC1551MIRU10 -------ATGGCGCCGCTCCTCCTCATCGCTGCGCTCTGCATCGTCGCCGGCGGTAGTTA------
MIRU 10 repeat 2 -------...................................................C.------
MIRU 10 repeat 3 -------...................................................C.------
MIRU 10 repeat 4 -------...................................................C.------
MIRU 10 repeat 5 -------..............................................CGG.GGTCAT---
CDC1551MIRU23 ---------T.....................T..............A......CG.C.C.------
MIRU 23 repeat 2 -------TCT.....................T..............A......CG.C.C.------
MIRU 23 repeat 3 -------TCT...........G.........T..............A......CG.C.C.------
MIRU 23 repeat 4 -------TCT...........G.........T..............A......CG.C.C.------
MIRU 23 repeat 5? -------TCT............-........T..............A......CGCA.GGTCAGCG
CDC1551MIRU26 --------AA...........................................--GAGGTCA----
MIRU 26 repeat 2 ---------A...........................................--GAGGTCA----
MIRU26 repeat 3 ---------A...........................................--GAGGTCA----
MIRU26 repeat 4 ---------A...........................................--GAGGTCA----
MIRU 26 repeat 5? ----------...........................................--GAGGTCACAGA
CDC1551MIRU16 ------------...................T...............T.....CGGT.C.------
MIRU16 repeat 2 -------CGA.....................T...............T.....CGGT.C.------
MIRU 16 repeat 3? -------CGA.....................T...............T.....CGGC.C.CGTGG-
CDC1551ETRE -----------------....................................CC.ACC.------
ETRE repeat 2 -------TCT...........................................CC.ACC.------
ETRE repeat 3 -------TCT...........................................CG.AGC.GCG---
CDC1551 MIRU2 --------A......................T....G...........T....CG...C.------
MIRU 2 repeat 2 -------TA......................T....G................CG...C.------
MIRU 2 repeat 3 -------TA..........TC..C.GCAAG..G.AGG...CC.CA.CT.ATGT..G..C.ACT---
CDC1551MIRU39 ----------..........................T................CGG.CCG------
MIRU 39 repeat 2 -------T............................T................CGG..C.------
CDC1551QUB5 -----------------------A.G..AGATT-.A.................CGG..C.------
QUB 5 repeat 2 aga -------C...............C.......TT....................CGG..C.CTGGC-
QUB5 repeat 3 -------C...............C.......TT....................CGG..C.------
QUB5 repeat 4 -------C...............C.......TT....................CGG..C.ATCG--
CDC1551MIRU24 repe TGCTTCG...............AG.............................C------------
Low resolution high resolution
Different levels of resolution are Different levels of resolution are
obtained from different markersobtained from different markers
SNPs LSPs SpoligoVNTR/
MIRUIS6110
Transposable Elements Transposable Elements –– ISIS61106110
Transposon enzyme generated
Transposon cut out and inserted
in new location (‘hotspot’)
Resulting in disrupted gene
Transposon mediated DNA rearrangements are often regarded as a major evolutionary
driving force, however repetitive DNA sequences also play a role. In M. tuberculosis the
role of repetitive sequences may be greater.
•1355 base pairs containing an ORF encoding a transposase
• PvuII restriction followed by southern blotting and hybridisation with IS6110 probe
•0-20 copies in M. tuberculosis.
•8% UK isolates, 40% India isolates and most M. bovis isolates possess only a single copy of IS6110
4.4Mbp
0 bp
ISIS61106110 typingtyping
The drawbacks of IS6110 RFLP are widely reported and include:
•The need for extended culturing
•time-consuming and can take weeks from start to completion
•lower throughput
•costly methodology
RFLPRFLP
Exact sizing of fragments is also an issue, especially when considering interlaboratory comparison of data.
ISIS61106110 FAFLP of FAFLP of M. tuberculosisM. tuberculosis
IS6110 primer
IS6110
‘blue’ labelled fragments
IS6110
Frequent cutter (TaqI)
Genomic DNA
TaqI adaptor-specific primer
In silicoIn silico (H37Rv)(H37Rv)
Expected fragments in this window:
75, 85, 87, 100, 127, 141, 163, 172, 190, 211
75.24
84.88
210.58198.6986.49 97.95 126.12 141.27 163.36 173.74 190.60
Sunderland
Sunderland
Preston
Preston
Example of strain comparisonExample of strain comparison
Highlighted area corresponds to the IS6110 element inserted in the DR region –
if spacer 24 is present then the FAFLP profile will contain the 318 bp fragment
Four colour ISFour colour IS61106110--based FAFLPbased FAFLP
DR based fragment
T lineage definedT lineage defined
H, PGG2X, PGG2
T, PGG3
T2Uganda, PGG2
LAM, PGG2S, PGG2
Beijing PGG1,
PGG2 outliers sharing no fragments
Evolutionary timelineEvolutionary timelineMapped using multiple markers
AcknowledgementsAcknowledgements
Sonia Borrell
Nikki Thorne
John Magee
Jason Evans
Saheer Gharbia
Chloe Mortimer
Christiane Honisch
HPA TB Diagnostics And Molecular Epidemiology Group