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Malaria genomic epidemiology research Dr. Alyssa Barry Malaria Genomic Epidemiology Lab., Centre for Population Health

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Title: Malaria genomic epidemiology research Dr. Alyssa Barry Malaria Genomic Epidemiology Lab., Centre for Population Health


1
Malaria genomic epidemiology researchDr. Alyssa
BarryMalaria Genomic Epidemiology Lab., Centre
for Population Health
2
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

3
The Malaria Parasite Lifecycle human host
4
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 !

5
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

6
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 US12 billion a year

7
Malaria Genomic Epidemiology
  • Genomic epidemiology (Defn) 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

8
Malaria parasite diversity
A diverse parasite population will be more
resilient to interventions
9
Malaria parasite population structure
Gene flow
A
B
Movement of different strains between populations
10
Polymorphism 101
  • Derived from the Greek language
  • Poly many p???
  • Morph form µ??f?
  • 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

11
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)
  • Minisatellitestandem repeats (gt3 bp)
  • Sequence analysis (sequencing, but also RFLP,
    SSLP)
  • SNP single nucleotide polymorphism
  • Indel Insert or Deletion
  • Simple sequence repeats polynucleotides (AAAA),
    microsatellites (TATATA) etc

12
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

13
Chromatin Binding Protein Intronic microsatellite
polymorphism (TA)n
3D7(GB) 61 AATTAAATAG GATTAAAATA ATTGTCATAA
AAAAAATTAT ATATACTTGA AAAAGCAAAT 3D7 61
AATTAAATAG GATTAAAATA ATTGTCATAA AAAAAATTAT
ATATACTTGA AAAAGCAAAT HB3 61
AATTAAATAG GATTAAAATA ATTGTCATAA AAAAAATTAT
ATATACTTGA AAAAGCAAAT W2 61 AATTAAATAG
GATTAAAATA ATTGTCATAA AAAAAATTAT ATATACTTGA
AAAAGCAAAT Muz12 61 AATTAAATAG
GATTAAAATA ATTGTCATAA AAAAAATTAT ATATACTTGA
AAAAGCAAAT Muz37 61 AATTAAATAG GATTAAAATA
ATTGTCATAA AAAAAATTAT ATATACTTGA
AAAAGCAAAT Muz51 61 AATTAAATAG
GATTAAAATA ATTGTCATAA AAAAAATTAT ATATACTTGA
AAAAGCAAAT

3D7(GB) 121 GACTGATTTT TTAAGgtatg
aataaaatga atataatata tatatatat att
3D7 121 GACTGATTTT TTAAGGtatg aataaaatga
atataatata tatatatat att HB3 121
GACTGATTTT TTAAGGtatg aataaaatga atataatata
tatatatat att W2 121 GACTGATTTT
TTAAGGtatg aataaaatga atataatata tatatat
att Muz12 121 GACTGATTTT TTAAGGtatg
aataaaatga atataatata tatatatat att
Muz37 121 GACTGATTTT TTAAGGtatg
aataaaatga atataatata tatatatat att Muz51
121 GACTGATTTT TTAAGGtatg aataaaatga
atataatata tatatatata tatatatt
3D7(GB) 181 taacctaaga
tatatatgtt ttttcatata atagttaata taatataaac
aaaatatatt 3D7 181 taacctaaga tatatatgtt
ttttcatata atagttaata taatataaac aaaatatatt HB3
181 taacctaaga tatatatgtt ttttcatata
atagttaata taatataaac aaaatatatt W2 181
taacctaaga tatatatgtt ttttcatata atagttaata
taatataaac aaaatatatt Muz12 181 taacctaaga
tatatatgtt ttttcatata atagttaata taatataaac
aaaatatatt Muz37 181 taacctaaga tatatatgtt
ttttcatata atagttaata taatataaac
aaaatatatt Muz51 181 taacctaaga tatatatgtt
ttttcatata atagttaata taatataaac aaaatatatt

5 Regulatory Domain
Intron I
Exon I
Intron I
14
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

15
P. falciparum Erythrocyte Binding Antigen 175
(EBA175)
16
Population genetic markers for P. falciparum
  • Different markers show different patterns
  • The P. falciparum genome SNP islands
  • Selected markers
  • 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?

17
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?

18
Collecting samples
Pic of Ivo here
19
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

20
Microsatellite protocol
50 50 16 bp 116 bp PCR product 8 repeat
units
50 50 20 bp 120 bp PCR product 10
repeat units
CACACACACACACACACACA GTGTGTGTGTGTGTGTGTGT
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

21
ApproachFragment 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
22
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
Chromatogram
Sequencing gel
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
23
In silico analysis
24
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
25
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
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