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

Gene flow C

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 3D7(GB) 3D7 HB3 W2 Muz12 Muz37 Muz51 3D7(GB) 3D7 HB3 W2 Muz12 Muz37 Muz51 #61 #61 AATTAAATAG GATTAAAATA ATTGTCATAA AAAAAATTAT ATATACTTGA AAAAGCAA AT AATTAAATAG GATTAAAATA ATTGTCATAA AAAAAATTAT ATATACTTGA AAAAGCAA AT #61 AATTAAATAG GATTAAAATA ATTGTCATAA AAAAAATTAT ATATACTTGA AAAAGCAA AT #61 AATTAAATAG GATTAAAATA ATTGTCATAA AAAAAATTAT ATATACTTGA AAAAGCAA AT #61 AATTAAATAG GATTAAAATA ATTGTCATAA AAAAAATTAT ATATACTTGA AAAAGCAA AT #61 AATTAAATAG GATTAAAATA ATTGTCATAA AAAAAATTAT ATATACTTGA AAAAGCAA AT #61 AATTAAATAG GATTAAAATA ATTGTCATAA AAAAAATTAT ATATACTTGA AAAAGCAA AT 5’ Regulatory Domain #121 #121 #121 #121 #121 #121 #121 GACTGATTTT TTAAG gtatg aataaaatga atataatata tatatatat: :::::::att GACTGATTTT TTAAG Gtatg aataaaatga atataatata tatatatat: :::::::att GACTGATTTT TTAAG Gtatg aataaaatga atataatata tatatatat: :::::::att GACTGATTTT TTAAG Gtatg aataaaatga atataatata tatatat::: :::::::att GACTGATTTT TTAAG Gtatg aataaaatga atataatata tatatatat: :::::::att GACTGATTTT TTAAG Gtatg aataaaatga atataatata tatatatat: :::::::att GACTGATTTT TTAAG Gtatg aataaaatga atataatata tatatatata tatat::att Exon I Intron I #181 #181 #181 #181 #181 #181 #181 taacctaaga tatatatgtt ttttcatata atagttaata taatataaac aaaatatatt taacctaaga tatatatgtt ttttcatata atagttaata taatataaac aaaatatatt taacctaaga tatatatgtt ttttcatata atagttaata taatataaac aaaatatatt taacctaaga tatatatgtt ttttcatata atagttaata taatataaac aaaatatatt taacctaaga tatatatgtt ttttcatata atagttaata taatataaac aaaatatatt taacctaaga tatatatgtt ttttcatata atagttaata taatataaac aaaatatatt taacctaaga tatatatgtt ttttcatata atagttaata taatataaac aaaatatatt 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 – Vaccine candidate antigens • inform vaccine design • Novel vaccine candidates - immune selection?

non-coding – 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 Blood Pic of Ivo here Isolate 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 CACACACACACACACACACA GTGTGTGTGTGTGTGTGTGT 20 bp 50 + 50 + 20 bp = 120 bp PCR product = 10 repeat units • 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 number of loci that can be analyzed in a single run and the sensitivity of the assay and different c o l o u r e d dyes increase the Sequencing gel

Isolates

Chromatogram 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

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

Harvard School of Public Health

Caroline Buckee

Funding Burnet Institute Lee Schultz

Pilate Ntsuke Johanna Wapling John Reeder