Malaria genomic epidemiology research
Dr. Alyssa Barry
Malaria Genomic Epidemiology Lab., Centre for
Population Health
What is Malaria?
• A disease caused by infection with Plasmodium spp. parasites
• Carried from person to person by anopheline mosquitoes
• Six species of Plasmodium cause malaria
– P. vivax, P.falciparum, P. malariae P. ovale curtisii, P. ovale
wallikeri, P. knowlesii
– P.falciparum causes most morbidity and mortality
• Symptoms include fever, nausea, vomiting, diarrhoea, tissue
damage, multiple organ failure, severe anaemia, coma (cerebral
Malaria), death
The Malaria Parasite Lifecycle – human host
The Burden of Malaria
•
•
•
~ 50% of the global
population at risk of malaria
Half a billion clinical attacks
each year
At least 1 million deaths
each year
Two or three people die of
malaria every minute !
Who are Most at Risk?
• Children under 5yrs old
– In the top 5 causes of death
• Pregnant women
– 400million births/yr in
malaria affected areas
• Other non-immunes
–
–
–
–
natural disaster
war
environmental change
climate change
Effects of Malaria
Besides direct morbidity
and mortality:
– Reduced school
attendance
– Lower productivity
– Impaired intellectual
development
– Developmental
abnormalities
– 2% less GDP growth in
malarious countries
– Costs Africa about US$12
billion a year
Malaria Genomic Epidemiology…
• Genomic epidemiology (Def’n): The systematic
investigation of how variation in the human genome, and
in the genomes of human pathogens, affect the
occurrence and clinical outcome of disease
• We are investigating patterns of genomic diversity within
natural malaria parasite populations to:
– Monitor patterns and routes of transmission (molecular
epidemiology, population genetics, ecology)
– Design malaria vaccines (what strains circulate?)
– Understand parasite evolution (changes over time, immune
selection, interactions with host molecules)
– Understand how humans naturally acquire immunity to
diverse malaria parasites
Malaria parasite diversity
Variant specific antibodies
Vaccine
A diverse parasite population will be more resilient to interventions
Partial efficacy of single strain vaccines. A malaria vaccine may need to
contain multiple variants.
Rapid evolution of drug resistance and other advantageous traits
Malaria parasite population structure
C
Gene flow
Variability in
allele
frequencies
A
B
Unique
alleles
Movement of different strains between populations
D
Population specific approaches to malaria control (e.g. tailored
vaccines, efforts targeted to specific foci)
Speed and direction of the dissemination of advantageous traits
Polymorphism 101
•
Derived from the Greek language
– Poly = many πολύ
– Morph = form
μορφή
•
The occurrence in a population (or among populations) of several phenotypic forms
associated with alleles (variants, types) of one gene
•
Genetic polymorphism: the occurrence together in the same population of one or
more allele or genetic marker (e.g. nucleotide or string of nucleotides) at the same
locus (position in the genome)
•
Therefore: Genetic variation results in the occurrence of several different forms or
types of individuals among the members of a single species (diversity)
•
e.g. Humans: blood group, hair colour, eye colour, disease status in
•
e.g. microorganisms: drug sensitivity/resistance, growth characteristics, antigenic
diversity (strains)
•
Caused by mutation
Types of polymorphism
Fragment size/pattern analysis (electrophoresis):
• AFLP: Amplified Fragment Length Polymorphism
• RFLP: Restriction Fragment Length Polymorphism
• SSLP: Short Sequence Length Polymorphism
– Microsatellites: tandem repeats (2-3 bp)
– Minisatellites:tandem repeats (>3 bp)
Sequence analysis (sequencing, but also RFLP, SSLP):
• SNP : single nucleotide polymorphism
• Indel: Insert or Deletion
• Simple sequence repeats: polynucleotides (AAAA), microsatellites
(TATATA) etc…
Microsatellites
TA TA TA TA TA TA TA TA
•Arrays of short tandem repeats 1-4 bp long
•A class of variable number tandem repeat (VNTR) used in DNA
fingerprinting
•Also known as simple sequence repeats (SSR)
•Abundant and rapidly evolving
•Highly polymorphic
•Detected by size variation
•Fairly evenly spaced through the genome
•Cheap to analyze
Chromatin Binding Protein
Intronic microsatellite polymorphism (TA)n
3D7(GB)
3D7
HB3
W2
Muz12
Muz37
Muz51
#61
#61
#61
#61
#61
#61
#61
AATTAAATAG
AATTAAATAG
AATTAAATAG
AATTAAATAG
AATTAAATAG
AATTAAATAG
AATTAAATAG
GATTAAAATA
GATTAAAATA
GATTAAAATA
GATTAAAATA
GATTAAAATA
GATTAAAATA
GATTAAAATA
ATTGTCATAA
ATTGTCATAA
ATTGTCATAA
ATTGTCATAA
ATTGTCATAA
ATTGTCATAA
ATTGTCATAA
AAAAAATTAT
AAAAAATTAT
AAAAAATTAT
AAAAAATTAT
AAAAAATTAT
AAAAAATTAT
AAAAAATTAT
ATATACTTGA
ATATACTTGA
ATATACTTGA
ATATACTTGA
ATATACTTGA
ATATACTTGA
ATATACTTGA
AAAAGCAAAT
AAAAGCAAAT
AAAAGCAAAT
AAAAGCAAAT
AAAAGCAAAT
AAAAGCAAAT
AAAAGCAAAT
tatatatat:
tatatatat:
tatatatat:
tatatat:::
tatatatat:
tatatatat:
tatatatata
:::::::att
:::::::att
:::::::att
:::::::att
:::::::att
:::::::att
tatat::att
taatataaac
taatataaac
taatataaac
taatataaac
taatataaac
taatataaac
taatataaac
aaaatatatt
aaaatatatt
aaaatatatt
aaaatatatt
aaaatatatt
aaaatatatt
aaaatatatt
5’ Regulatory Domain
3D7(GB)
3D7
HB3
W2
Muz12
Muz37
Muz51
#121
#121
#121
#121
#121
#121
#121
GACTGATTTT
GACTGATTTT
GACTGATTTT
GACTGATTTT
GACTGATTTT
GACTGATTTT
GACTGATTTT
TTAAGgtatg
TTAAGGtatg
TTAAGGtatg
TTAAGGtatg
TTAAGGtatg
TTAAGGtatg
TTAAGGtatg
aataaaatga
aataaaatga
aataaaatga
aataaaatga
aataaaatga
aataaaatga
aataaaatga
atataatata
atataatata
atataatata
atataatata
atataatata
atataatata
atataatata
Exon I
3D7(GB)
3D7
HB3
W2
Muz12
Muz37
Muz51
#181
#181
#181
#181
#181
#181
#181
taacctaaga
taacctaaga
taacctaaga
taacctaaga
taacctaaga
taacctaaga
taacctaaga
Intron I
tatatatgtt
tatatatgtt
tatatatgtt
tatatatgtt
tatatatgtt
tatatatgtt
tatatatgtt
ttttcatata
ttttcatata
ttttcatata
ttttcatata
ttttcatata
ttttcatata
ttttcatata
atagttaata
atagttaata
atagttaata
atagttaata
atagttaata
atagttaata
atagttaata
Intron I
Single Nucleotide Polymorphisms (SNPs)
• Point mutation, variation at a single nucleotide
position
– e.g. A/C, G/A etc…
• Clustered in rapidly evolving genes e.g. human MHC
genes, P. falciparum var, HIV env,
• A good SNP map is useful for population genetics and
linkage analysis
• Rapid, high throughput detection
possible but can be expensive
P. falciparum Erythrocyte Binding Antigen 175 (EBA175)
Population genetic markers for P. falciparum
• Different markers show different patterns
• The P. falciparum genome : SNP “islands”
coding
• Selected markers
non-coding
– Vaccine candidate antigens
• inform vaccine design
• Novel vaccine candidates - immune selection?
– Drug resistance genes and their genetic background
• Is it spreading (how fast, which direction) or multiple independent
origins?
• Neutral markers
– Genome wide microsatellites and SNPs
• Population biology e.g. how diverse (fit) is the parasite population?
gene flow? i.e. how difficult will the parasite population be to control?
Population biology of P. falciparum in PNG
•
•
•
•
Intense year round transmission of P. falciparum in the lowlands (50-60%),
epidemics in the highlands
Any spp. (~80%), P. vivax (~50%), P. malariae (~20%), P. ovale (~5%)
Diverse micro-epidemiology- Spatially variable transmission, host genetics,
vector species, malaria control (bednets)
Complex population genetics?
Collecting samples
Volunteers
Isolate
Blood
Pic of Ivo here
Extract
genomic
DNA
Analysis
“Wet” lab. methods
gDNA
n ~ 3000
Screen for P.falciparum infection (msp2 PCR, multiplex)
n ~1500
Count the number of msp2 bands
Mean MOI = 1.7 (1-13)
Whole genome amplification of single infections
n ~ 700
Microsatellite genotyping
Antigen gene PCR and sequencing
Data Analysis
Microsatellite protocol
50 + 50 + 16 bp = 116 bp PCR product
= 8 repeat units
50 + 50 + 20 bp = 120 bp PCR product
CACACACACACACACACACA
GTGTGTGTGTGTGTGTGTGT
= 10 repeat units
20 bp
• Small size difference (4bp) – cannot be detected by agarose gel
electrophoresis
• Solution: Sequencing, or for cheaper high throughput run PCR products
on an ABI
Sequencer
• The latter solution requires products to be fluorescently labeled
Approach:
Fragment analysis on an ABI Sequencer
Dye attached to the 5’ end of primer
TA TA TA TA TA TA TA TA TA TA TA TA TA
Fluorescent dye is incorporated into PCR product
TA TA TA TA TA TA TA TA TA TA TA TA TA
Microsatellite genotyping
Multilocus genotyping:
Different sizes and different coloured dyes increase the
number of loci that can be analyzed in a single run and the
sensitivity of the assay
Sequencing gel
Chromatogram
Isolates
Locus 1
Locus
7
2
1
The haplotype, a string of alleles (e.g. the number of repeats per
loci 15_12_6_8_10_6) is then determined for each isolate
“In silico” analysis
Allelic richness
12
10
8
6
4
2
0
Utu
Malala
Mugil
Wosera
Total
Population biology of P. falciparum in PNG
Factors that may influence
the distribution of
parasites:
-Mugil/Karkar Is. ferry
-Malala boarding school
-vector spp.
-language groups
-human genetics
High but variable diversity and population structure
Implications for control, elimination and the spread of vaccine
and drug resistance (mapping routes of transmission)
Currently sequencing several vaccine candidate antigens in
two of these populations to inform vaccine design
Acknowledgements
PNG Communities and
Volunteers
PNGIMR
Peter Siba
Ivo Mueller
Nicholas Senn
Livingstone Tavul
Ore Toporua
Benson Kiniboro
Joe Nale
Thomas Adiguma
Elias Namosha
Burnet Institute
Lee Schultz
Pilate Ntsuke
Johanna Wapling
John Reeder
Harvard School of Public Health
Caroline Buckee
Funding