Popula'on genomic approaches toward understanding anthelmin'c resistance in Onchocerca volvulus Stephen Doyle La Trobe University, Melbourne, Australia
Onchocerciasis (River blindness) Onchocerca volvulus
• • • •
Adults are long living: < 15 yrs Intermediate host: Blackfly Prepatent period: 12-‐18 mths Causes severe morbidity
Onchocerciasis: Treatment Ivermec?n (IVM) • Target: Glutamate-‐gated chloride channels • Effect: Does not kill adult worms…. • >99% of microfilaria disappear • Inhibits reproduc'on • Interrupts transmission
HOWEVER 1977
2002
Evidence for varia'on in IVM response Evidence • Phenotypic: increase in microfilariae load & repopula'on rate • Gene4c: β-‐tubulin & p-‐glycoprotein
Unknown • What is the mechanism and is there truly a gene?c component? • What is the poten&al for spread? • How might poor response impact the treatment of onchocerciasis and its eradica'on? Churcher et al (2009) PNAS v106 n39
Defining the response phenotype Responder status:
Suscep'ble or sub-‐op'mal responder (SOR) to IVM Sub-‐op?mal response -‐ High skin microfilaria density -‐ Faster repopula'on rates -‐ High embryograms
Ivermec?n
Suscep?ble response -‐ Low skin microfilaria density -‐ Slower repopula'on rates -‐ Low embryograms
Experimental Outline Ghana Suscep?ble
SOR
Cameroon Naive
Suscep?ble
SOR
Naive
Sequencing: 15-‐20 worms per pool, 20-‐30X coverage for each pool of worms (8 lanes GAII, ~280 x 106 reads) Analysis: variant read frequency ≈ allele frequency, and characterise variants where suscep'ble ≠ resistant
Gene'c diversity between groups -‐ How is the gene'c diversity distributed between groups and is any of it shared? 1.0
FST
0.8 0.6 0.4 0.2 0 Naive v Suceptible
Naive v SOR
Cameroon
SOR v Suceptible
Naive v Suceptible
Naive v SOR
SOR v Suceptible
Ghana
-‐ Naive and SOR pools maintain diversity and are significantly more similar to each other than others
Correla'on between countries
12
1.00
Ghana: SOR v susceptible (FST)
Ghana: SOR v susceptible (-log10(p))
-‐ Is there a correla'on between variants that differen'ate suscep'ble and SOR between countries?
9
6
3
0
0.75
0.50
0.25
0.00 0
3
6
9
12
Cameroon: SOR v susceptible (-log10(p))
0.00
0.25
0.50
0.75
1.00
Cameroon: SOR v susceptible (FST)
-‐ Significant varia'on within each country but liile/no correla'on between
Genome-‐wide analysis of IVM response -‐ Where are variants that differen'ate suscep'ble and SOR found in the genome? Cameroon
1.0
A FST
0.8 0.6
Mean Fst + 3 SD
0.4 0.2 0.0 1.0
B FST
0.8
Ghana
0.6 0.4
Mean Fst + 3 SD
0.2 0.0 OM1a
OM1b
OM2
OM3
OM4
OM5
OVOC.Scaffolds
-‐ 30 regions in total; only one shared between countries
Linkage disequilibrium by response • What degree of linkage is found between variants, and is it different between suscep'ble and SOR? LD_ghana_GR+LR 1.0
GR LR
Suscep?ble SOR
0.8
R2
0.6
0.4
0.2
0.0
0
100000
200000
300000
400000
500000
Distance (bp)
• Increase in LD ~50-‐100kb, but not significantly different between suscep'ble and SOR
Candidate IVM-‐response genes • Do candidate IVM response genes/markers show differen'a'on between suscep'ble and resistant pools? • glutamate gated chloride channels
Cameroon Ghana
Candidate IVM-‐response genes • Do candidate IVM response genes/markers show differen'a'on between suscep'ble and resistant pools? • p-‐glycoproteins
Cameroon Ghana
Candidate IVM-‐response genes • Do candidate IVM response genes/markers show differen'a'on between suscep'ble and resistant pools? • Other candidates...?
Cameroon Ghana
What genes are in the clusters? • Known IVM sensi'vity alleles – che-‐3, unc-‐44, klp-‐11, inx-‐5
• Neurotransmission, par'cularly ACh – – – –
ion channels (acc-‐1, lgc-‐46, lgc-‐47, gtl-‐2) acetylcholine synthesis (cha-‐1), transport (unc-‐17, aex-‐3), and regula'on (pha-‐2, snb-‐1, emc-‐6)
• Lipid synthesis (acs-‐16, mecr-‐1, fard-‐1, fat-‐1) and regula'on and storage (obr-‐2, ech-‐4, tub-‐1, sms-‐1) • Suppression (sel-‐7, bre-‐5) or cleavage (pen-‐2, crb-‐1) of the LIN-‐12 receptor
Uncoupling popula'on structure from treatment response • To what extent does treatment shape popula'on structure? 0.2
Ghana
Cameroon
0.1
-0.2
-0.1
0.1
C1
-0.1
-0.2
Asubende (Ghana) Begbomdo (Ghana) Kyingakrom (Ghana) *Good Response (Ghana) Jagbenbendo (Ghana) New Longoro (Ghana) Wiae (Ghana) Littoral (Cameroon) Mbam (Cameroon) Mbam 1994 (Cameroon)
0.2-0.2
0.2
Unknown phenotype
(good response) Cameroon Susceptible Resistant (SOR)
Ghana 0.1
-0.1
0.1
C1
-0.1
-0.2
• Dis'nc'on between countries, but not between response types
0.2
Uncoupling popula'on structure from treatment response 0.10
A closer look in Ghana
0.05
Asubende (Ghana) Begbomdo (Ghana) Kyingakrom (Ghana) *Good Response (Ghana) Jagbenbendo (Ghana) New Longoro (Ghana) Wiae (Ghana) Littoral (Cameroon) Mbam (Cameroon) Mbam 1994 (Cameroon)
0.2
Ghana 0.1
-0.1
0.1
0.2
C1
-0.05
-0.1
AB2 AB1 ASU BAY -0.10 AB2 CHA AB1 JAG STUDY AREAS IN THE SELECTED DISTRICTS ASU KOJ BAY AB2 KYG CHA AB2 AB1 NLG JAG AB1 ASU NYR KOJ ASU AB2 BAY OHP KYG BAY AB1 AB2 AB2 CHA SEN NLG CHA ASU AB1 AB11 MANTUKWA SENYASE BEPOSO JAG [ TAK _ [ _ FAWOMAN_ [BAAYA [ _ [ _ _ [ASUBENDE NYR JAG AB2 BAY ASU BOLE DISTRICT [OHIAMPE _ KOJ WIAASU AB2 OHP KOJ AB1 CHA BAY BAY KYG AB1 SEN KYG JAG CHA CHA [ _ ASU ASU NLG TAK [ KOJ _ NLG_ JAG JAG [ [_ _ [BAY [ _ NYR BAY KINTAMPO [ [ [ _ WIA [_ [ [_ _ _ NYRCHA KYG [_ KOJ_ SOUTH [ _ KOJ [ _ CHA DISTRICT OHP OHPJAG NLG PRU DISTRICT KYG KYG SEN JAG TAIN DISTRICT SEN KOJ NYR NLG NLG TAK KOJ _ [ TAK KYGOHP NYR NYR KYG WIA WIA NLGSEN OHP OHP NLG éé TAK SEN SEN NYR NYR WIA TAK TAK OHP OHP WIA WIA SEN 1°40'0 "W
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Study Areas
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Towns
Major Roads
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1°40'0 "W
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River
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8°2 0'0"N
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Location Map of Ghana
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AB2 AB1 AB2 ASU AB1 BAY ASU CHA BAY JAG CHA AB2 KOJ JAG AB1 KYG KOJ ASU NLG KYG BAY NYR NLG CHA AB2 OHP NYR JAG AB1 SEN OHP AB2 KOJ ASU TAK SEN [ _ AB1 KYG BAY WIA KPANDAE DISTRICT TAK ASU NLG [_ _ CHA [ [ _ [ _ WIA BAY NYR JAG CHA OHP KOJ JAG SEN KYG KOJ TAK NLG KYG WIA NYR NLG OHP NYR SEN OHP TAK SEN WIA TAK
East – West grouping of samples
R Bole !
-0.2
0.10
-0.05
2°30 '0"W
9°10'0 "N
0.05
Bl ac
-0.2
-0.10
20
30
1:600,000
40
50 Kilometers
Volta Lake
District Boundary
Produced b y:
0°0'0"
MAPTECH SYSTEMS
P.M.B. 1AF Adenta Flats, Accra Tel: +233(0)20 8509305 / 026 3104095 Email:
[email protected]
7°30 '0 "N
Uncoupling popula'on structure from treatment response 0.10
By correc'on for popula'on structure....
0.05
-0.10
10-40
-0.05
0.05
-0.05
p-value
10-30 -0.10
10-20 10-10 100 1010
Genomic scaffold (97-Mb total)
... We begin to unravel likely true signals of response
0.10
Conclusions • Evidence of sop selec've sweeps that are popula'on specific • Signals of differen'a'on s'll point to a small number of pathways • (Popula'on structure + lifecycle + treatment) suggests processes such as gene'c drip rather than selec'on impacts rapid allelic change • Understanding underlying gene'c varia'on and its distribu'on is cri'cally important in drug response analyses
Work in progress • Defining heritability (h2) and selec'on coefficient (s) of response • Broader characteriza'on of transmission zones • Rate of change within and between zones
Acknowledgements La Trobe (Australia) • Warwick Grant • Sam Armoo • Ka'e Crawford • Sheila Nankoberanyi • Nathan Hall • Andrew Robinson McGill (Canada) • Roger Prichard • Catherine Bourguinat • Astrid Erber • Kathy Keller • Hua Che WHO • Anneie Kuesel MDSC (Burkina Faso) • Gilles Aime Adjami • Laurent Toe
Water Research Ins?tute (Ghana) • Mike Osei-‐Atweneboana • Daniel Boakye
Cameroon • Joseph Kamgno • Samuel Wanji • Hugues Nana Djeunga • Jonas Ouafo France • Michel Boussinesq • Sebas'en Pion Wellcome Trust Sanger Centre • Mai Berriman • Nancy Holroyd • James Coion • Eleanor Stanley
