Innovations in Molecular Epidemiology · (Microsoft PowerPoint - Ppt0000001 [S\363lo lectura]) Author: lhinojos Created Date: 12/9/2009 2:41:43 PM ...

$Page 1: Innovations in Molecular Epidemiology · (Microsoft PowerPoint - Ppt0000001 [S\363lo lectura]) Author: lhinojos Created Date: 12/9/2009 2:41:43 PM ...$
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




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





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





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





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------------





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

Innovations in Molecular Epidemiology · (Microsoft PowerPoint - Ppt0000001 [S\363lo lectura]) Author: lhinojos Created Date: 12/9/2009 2:41:43 PM ...

Documents