Malaria genomic epidemiology research

Dr. Alyssa BarryMalaria 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

Vaccine

Variant specific antibodies

Partial efficacy of single strain vaccines. A malaria vaccine may need to contain multiple variants.

Rapid evolution of drug resistance and other advantageous traits

A diverse parasite population will be more resilient to interventions

Malaria parasite population structure

A B

C

Variability in allele frequencies

D

Unique

alleles

Gene flow

Population specific approaches to malaria control (e.g. tailored vaccines, efforts targeted to specific foci) Speed and direction of the dissemination of advantageous traits

Movement of different strains between populations

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

•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

TA TA TA TA TA TA TA TA

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 tatat::att

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

Intron IIntron I

Intron IIntron I

Exon IExon I

5’ Regulatory Domain5’ Regulatory Domain

Chromatin Binding ProteinIntronic microsatellite polymorphism (TA)n

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”

• 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?

coding

non-coding

• 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?

Population biology of P. falciparum in PNG

Blood

Volunteers

Extractgenomic DNA

Analysis

Isolate

Collecting samples

Pic of Ivo here

“Wet” lab. methodsgDNA

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 infectionsn ~ 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

= 10 repeat units

20 bp

CACACACACACACACACACAGTGTGTGTGTGTGTGTGTGT

• 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

TA TA TA TA TA TA TA TA TA TA TA TA TA

TA TA TA TA TA TA TA TA TA TA TA TA TA

Fluorescent dye is incorporated into PCR product

Dye attached to the 5’ end of primer

Microsatellite genotyping

Locus 1

Isolates

Sequencing gel Chromatogram

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

Locus 7 2 1

“In silico” analysis

Population biology of P. falciparum in PNG

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

Factors that may influence the distribution of parasites:

-Malala boarding school

-Mugil/Karkar Is. ferry

-vector spp. -language groups-human genetics

Acknowledgements

PNGIMRPNGIMRPeter SibaPeter SibaIvo MuellerIvo MuellerNicholas SennNicholas SennLivingstone TavulLivingstone TavulOre ToporuaOre ToporuaBenson KiniboroBenson KiniboroJoe NaleJoe NaleThomas AdigumaThomas AdigumaElias NamoshaElias Namosha

Burnet Institute Burnet Institute Lee SchultzLee SchultzPilate NtsukePilate NtsukeJohanna WaplingJohanna WaplingJohn ReederJohn Reeder

Funding

Harvard School of Public HealthHarvard School of Public HealthCaroline BuckeeCaroline Buckee

PNG Communities and PNG Communities and VolunteersVolunteers