Génétique de la susceptibilité au paludisme: possibles approches épidemiologiques sur le terrain - Présentation de la 1ere édition du Cours international « Atelier Paludisme » - MODIANO David - Università "La Sapienza", Rome - david.modiano@uniroma1.it

1. Génétique de la susceptibilité au paludisme:
approches épidémiologiques sur le terrain
David Modiano
Département de Sciences de Santé Publique
Université de Rome “La Sapienza” Italie
World Health Organization Collaborating Centre
for Malaria Epidemiology and Control
david.modiano@uniroma1.it
Atelier paludisme 2004

2. Satellite-derived predictions of Entomological
Inoculation Rates (EIR) in sub-Saharan Africa
Source: Rogers DJ et al. Nature. 2002. 415: 710-715.

3. Source: Miller LH et al. Nature. 2002. 415: 673-679.
Source: Greenwood B et al.
Parasitology Today. 1991. 7: 277-
281

4. Genes studied for association with malaria I
ABO Lell 99 The role of red blood cell polymorphisms in resistance and susceptibility to malaria
CD36 Aitman 00 Malaria susceptibility and CD36 mutation
Pain 01 A non-sense mutation in Cd36 gene is associated with protection from severe malaria
CR1 Rowe 97 P. falciparum rosetting mediated by a parasite-variant erythrocyte membrane protein and complement-receptor 1
Bellamy 98 Absence of an association between intercellular adhesion molecule 1, complement receptor 1 and interleukin 1 receptor antagonist gene
polymorphisms and severe malaria in a West African population
FCGR2A Omi 02 Fcgamma receptor IIA and IIIB polymorphisms are associated with susceptibility to cerebral malaria
FY Miller 76 The resistance factor to Plasmodium vivax in blacks. The Duffy-blood- group genotype, FyFy
Chitnis 94 Identification of the erythrocyte binding domains of Plasmodium vivax and Plasmodium knowlesi proteins involved in erythrocyte invasion
Tournamille 95 Disruption of a GATA motif in the Duffy gene promoter abolishes erythroid gene expression in Duffy-negative individuals
G6PD Gilles 67 Glucose-6-phosphate-dehydrogenase deficiency, sickling, and malaria in African children in South Western Nigeria
Ruwende 95 Natural selection of hemi- and heterozygotes for G6PD deficiency in Africa by resistance to severe malaria
Ruwende 98 Glucose-6-phosphate dehydrogenase deficiency and malaria
GYPC Patel 01 The association of the glycophorin C exon 3 deletion with ovalocytosis and malaria susceptibility in the Wosera, Papua New Guinea
HBA1 Allen 93 A prospective study of the influence of alpha thalassaemia on morbidity from malaria and immune responses to defined Plasmodium
falciparum antigens in Gambian children
Williams 96 High incidence of malaria in alpha-thalassaemic children
Allen 97 alpha+-Thalassemia protects children against disease caused by other infections as well as malaria
HBB Neel 49 (Science: 110, 64-6) The inheritance of sickle cell anemia
Pauling 49 (Science: 110, 543-8) Sickle cell anemia, a molecular disease
Gilles 67 Glucose-6-phosphate-dehydrogenase deficiency, sickling, and malaria in African children in South Western Nigeria
Hill 88 Beta thalassemia in Melanesia: association with malaria and characterization of a common variant (IVS-1 nt 5 G----C)
Hill 91 Common west African HLA antigens are associated with protection from severe malaria
Allen 92 Morbidity from malaria and immune responses to defined Plasmodium falciparum antigens in children with sickle cell trait in The Gambia
Stirnadel 99 Malaria infection and morbidity in infants in relation to genetic polymorphisms in Tanzania
Agarwal 00 HBC associated with protection from severe malaria in the dogon of mali, a westafrican population with a low prevalence of hemoglobin S
Modiano 01 Haemoglobin C protects against clinical Plasmodium falciparum malaria
Modiano 01 The lower susceptibility to Plasmodium falciparum malaria of Fulani of Burkina Faso (west Africa) is associated with low frequencies of
classic malaria-resistance genes
HLA-B Hill 91 Common west African HLA antigens are associated with protection from severe malaria
Hill 92 (Parasitology Today: 8, 57) HLA and malaria: on the mechanisms of dominant protective associations [letter].
Hill 92 Molecular analysis of the association of HLA-B53 and resistance to severe malaria
Bennett 93 Human leucocyte antigen (HLA) and malaria morbidity in a Gambian community
Gilbert 98 Association of malaria parasite population structure, HLA, and immunological antagonism
Modiano 01 HLA class I in three West African ethnic groups: genetic distances from sub-Saharan and Caucasoid populations
SOURCE: http://www.gmap.net/topics/malaria.htm

5. Genes studied for association with malaria II
HLA-DRB1 Hill 91 Common west African HLA antigens are associated with protection from severe malaria
HP Elagib 98 Association of the haptoglobin phenotype (1-1) with falciparum malaria in Sudan
Quaye 00 Haptoglobin 1-1 is associated with susceptibility to severe Plasmodium falciparum malaria
Hunt 01 Haptoglobin and malaria
Aucan 02 Haptoglobin genotypes are not associated with resistance to severe malaria in The Gambia
ICAM1 Fernandez-Reyes 97 A high frequency African coding polymorphism in the N-terminal domain of ICAM-1 predisposing to cerebral malaria in
Kenya
Bellamy 98 Absence of an association between intercellular adhesion molecule 1, complement receptor 1 and interleukin 1 receptor antagonist gene
polymorphisms and severe malaria in a West African population
Kun 99 Association of the ICAM-1Kilifi mutation with protection against severe malaria in Lambarene, Gabon
IFNGR1 Koch 02 IFNGR1 Gene Promoter Polymorphisms and Susceptibility to Cerebral Malaria
IL12B Morahan 02 A promoter polymorphism in the gene encoding interleukin-12 p40 (IL12B) is associated with mortality from cerebral malaria and with
reduced nitric oxide production
IL4 Luoni 01 Antimalarial antibody levels and IL4 polymorphism in the Fulani of West Africa
MBL2 Bellamy 98 Mannose binding protein deficiency is not associated with malaria, hepatitis B carriage nor tuberculosis in Africans
Luty 98 Mannose-binding lectin plasma levels and gene polymorphisms in Plasmodium falciparum malaria
NOS2A Burgner 98 Inducible nitric oxide synthase polymorphism and fatal cerebral malaria
Kun 98 Polymorphism in promoter region of inducible nitric oxide synthase gene and protection against malaria
Levesque 99 Nitric oxide synthase type 2 promoter polymorphisms, nitric oxide production, and disease severity in Tanzanian children with malaria
Kun 01 Nitric oxide synthase 2(Lambarene) (G-954C), increased nitric oxide production, and protection against malaria
Hobbs 02 A new NOS2 promoter polymorphism associated with increased nitric oxide production and protection from severe malaria in Tanzanian and
Kenyan children
SLC4A1 Foo 92 Ovalocytosis protects against severe malaria parasitemia in the Malayan aborigines
Genton 95 Ovalocytosis and cerebral malaria
Allen 99 Prevention of cerebral malaria in children in Papua New Guinea by southeast Asian ovalocytosis band 3
TNF McGuire 94 Variation in the TNF-alpha promoter region associated with susceptibility to cerebral malaria
Knight 99 A polymorphism that affects OCT-1 binding to the TNF promoter region is associated with severe malaria
McGuire 99 Severe malarial anemia and cerebral malaria are associated with different tumor necrosis factor promoter alleles
Stirnadel 99 Malaria infection and morbidity in infants in relation to genetic polymorphisms in Tanzania
Wattavidanage 99 TNFalpha*2 marks high risk of severe disease during Plasmodium falciparum malaria and other infections in Sri Lankans
Meyer 02 TNFalpha-308A associated with shorter intervals of Plasmodium falciparum reinfections
Knight 03 In vivo characterization of regulatory polymorphisms by allele-specific quantification of RNA polymerase loading
SOURCE: http://www.gmap.net/topics/malaria.htm

6. Geographic distribution of malaria resistance genes
Hemoglobin S α-thalassemia
β-thalassemia G6PD deficiency

7. Geographic distribution of haemoglobin C
Source: Cavalli Sforza, Paolo Menozzi, Alberto Piazza. The history
and geography of human genes. 1994. Princeton University Press

8. Host Factors – Genetics
Two complementary populational
approaches/models:
- intra-ethnic
- inter-ethnic

9. Intra-ethnic approach I
Comparison of genotype and allele frequencies of candidate resistance/susceptibility genes
between the general healthy population and hospitalized malaria patients (severe malaria,
non complicated malaria)
vs

10. Intra-ethnic approach II
Comparison of genotype and allele frequencies of candidate resistance/susceptibility genes
among groups belonging to the same genetic background but living in endemic and non endemic
areas
The inverse relationship between altitude, Thal heterozygotes (left) and GD-
allele (right) distribution in Sardinia, Italy. Shaded areas > 500 m a.s.l.
Source: Modiano G. et al, 1986. Atti Accademia Nazionale dei Lincei, Serie VIII – Volume 18, Sezione III,
Fascicolo 4

11. Intra-ethnic approach III
Family studies: comparison of genotype and allele frequencies of candidate
resistance/susceptibility genes among families showing different capacity to control
parasite density
Abel 92 Segregation analysis detects a major gene controlling blood infection levels in human malaria
Garcia 98 Genetic control of blood infection levels in human malaria: evidence for a complex genetic model
Rihet 98 Human malaria: segregation analysis of blood infection levels in a suburban area and a rural area in Burkina Faso
Stirnadel 99 Heritability and segregation analysis of immune responses to specific malaria antigens in Papua New Guinea
Chromosomal regions linked to malaria
Human chromosome 5
Garcia 98 Linkage analysis of blood Plasmodium falciparum levels: interest of the 5q31-q33 chromosome region
Rihet 98 Malaria in humans: Plasmodium falciparum blood infection levels are linked to chromosome 5q31-q33
Human chromosome 6
Jepson 97 Genetic linkage of mild malaria to the major histocompatibility complex in Gambian children: study of affected sibling pairs
Jepson 97 Quantification of the relative contribution of major histocompatibility complex (MHC) and non-MHC genes to human
immune responses to foreign antigens
Flori 03 Linkage of mild malaria to the major histocompatibility complex in families living in Burkina Faso

12. Inter-ethnic approach
Comparison of genotype and allele frequencies of candidate resistance/susceptibility genes
among ethnic groups with different genetic background and different susceptibility to
malaria living in the same epidemiological context, i.e. exposed to the same transmission
levels and to the same parasite strains
vs
Fulani Mossi-Rimaibé

13. CONDITIONS NEEDED FOR INTER-ETHNIC
APPROACH
Fulani
ETHNIC GROUPS must show:
- similar exposure to malaria (higher the
level, lower the risk of confounders)
- similar access to anti-malaria drugs
- similar use of control methods (ex. ITN)
vs
- different genetic background
- different susceptibility (parasite rates, Mossi -
parasite densities, incidence and of clinical Rimaibé
malaria)

14. Intra-ethnic approach

15. INTRA-ETHNIC APPROACH
β globin genotype and allele frequencies in healthy subjects and in malaria patients
from Burkina Faso, West Africa (Source: Modiano D, et al., Nature, 2001: 414: 305-308)
Sample N Relative and (absolute) genotype frequencies Relative and (absolute) allele frequencies
AA AC AS CC SC SS βA βC βS
Healthy subjects 3513 0.6641 0.2172 0.0954 0.0165 0.0065 0.0003 0.8204 0.1284 0.0512
(2333) (763) (335) (58) (23) (1) (5764) (902) (360)
Severe malaria 359 0.8078 0.1755 0.0111 0.0028 0.0028 0 0.9011 0.0919 0.0070
(290) (63) (4) (1) (1) (0) (647) (66) (5)
Non-complicated malaria 476 0.8004 0.1555 0.0399 0 0.0042 0 0.8981 0.0798 0.0221
(381) (74) (19) (0) (2) (0) (855) (76) (21)
Malaria patients (total) 835 0.8036 0.1641 0.0275 0.0012 0.0036 0 0.8994 0.0850 0.0156
(671) (137) (23) (1) (3) (0) (1502) (142) (26)
Comparisons Odds ratio (95% confidence interval) and P values *
Healthy subjects vs. Malaria patients 2.07 Ω 0.71 ψ 0.27 φ 0.07 δ
(1.71-2.50) (0.58-0.87) (0.17-0.42) (0.00-0.48) n.s. n.s. <<0.001 <<0.001 <<0.001
<<0.001 0.0008 <<0.001 0.0011
Severe malaria vs. non-severe malaria n.s. n.s 0.27 ϕ n.s. n.s. n.s. n.s. n.s. 0.023
(0.08-0.86)
0.021
v
s
Healthy subjects Severe malaria patients

16. Geographic distribution of haemoglobin C
“the fact that βC protects mainly in the
homozygous state may suggest a
straightforward explanation for its very
localized occurrence in West Africa: its
selective advantage would be
proportional to the allele frequency so
that homozygous βC would progressively
fade out with increasing distance from the
epicentre”

17. Inter-ethnic approach

18. INTER-ETHNIC APPROACH
Fulani
Km
MALI NIGER
Kaya 0 0.5 1
Study area
F
vs
BURKINA
Ouagadougou FASO
F
BENIN F
Bobo
Dioulasso GHANA F
Barkoundouba F F
Km R
CÔTE F
R
D'IVOIRE 100 F F
R
F R R
R FR
F
F
Mossi -
Ouagadougou Rimaibé
M M R R
R
M M M R
M M R R
M F = Fulani
M MM Barkoumbilen
M = Mossi
R = Rimaibé

19. Entomological inoculation rates in the Barkoundouba area, Burkina
Faso
Source: Modiano D, et al., 1996. PNAS; 93: 13206-
13211

20. Gene frequencies of 17 HLA I alleles showing significant
differences between Fulani and Mossi + Rimaibé
Source: Modiano D, et al., Tissue Antigens; 2001; 57:128-137

21. A: Plasmodium falciparum parasite rate; B: log10
of positive parasite density in three sympatric
ethnic groups from Burkina Faso, West Africa
Source: Modiano D, et al., 1996. PNAS; 93: 13206-13211

22. Percentage (± s.e.) of negative individuals at the first survey (Aug. 94),
and rate of persistence of negativity in the four successive cross-
sectional surveys
Only individuals aged more than 10 years and examined in all five surveys of are considered. The comparison
does not include the 0-10 years age-groups since their negativity rates were too low for this type of analysis
Source: Modiano D, et al., 1996. PNAS; 93: 13206-13211

23. Incidence of non complicated malaria in three sympatric ethnic groups of Burkina
Faso, West Africa
Ethnic groups N Geom mean Average of clinical episodes with
of PD different Pf PD thresholds
>0 >6400 >12800 >25600
Mossi M 95 3304 0.579 0.250 0.195 0.098
Rimaibé (Barkoumbilen) RB 92 3069 0.497 0.243 0.162 0.065
Rimaibé (Barkoundouba) RD 21 4345 0.618 0.265 0.206 0.118
Mossi + Rimaibé NF 208 3289 0.543 0.248 0.180 0.084
Fulani F 51 955 0.495 0.136 0.058 0.010
Comparisons
M vs RB 0.85 0.32 0.76 0.25 0.25
M vs RD 0.68 0.72 0.80 0.73 0.57
RB vs RD 0.64 0.33 0.66 0.28 0.17
M vs F 0.004 0.38 0.09 0.005 0.009
RB vs F 0.004 0.97 0.15 0.052 0.06
RD vs F 0.021 0.36 0.16 0.011 0.004
NF vs F 0.001 0.56 0.08 0.009 0.016
Source: Modiano D, et al., 1996. PNAS; 93: 13206-13211

24. Prevalence (A) and levels (B)
of antibodies to the
Plasmodium falciparum
circumsporozoite protein, in
three sympatric ethnic groups
of Burkina Faso, West Africa
Source: Modiano D, et al., 1999. AJTMH; 61: 663- 667

25. Anti-Pf155/RESA (EENV)6 and anti-Pf332 (SVTEEIAEEDK)2
antibody prevalences and levels by age and ethnic group
Source: Modiano D, et al., AJTMH; 1998; 58:220-224

26. Gene frequencies of malaria related genes in three
sympatric ethnic groups of Burkina Faso, West Africa
Source: Modiano D. et al., 2001. Transactions of the Royal Society of Tropical Medicine and Hygiene. 95: 149-152

27. The chromosome 5q31-q33
1M b
region contains numerous
U B P G D F 9 K IF 3 A il- 4 il- 1 3 RAD 50 il- 5 il- 3 il- 9
candidate genes encoding
immunological molecules such
as cytokines, growth factors, and
150K b
growth-factor receptors
IL -4 C N S -1 IL -1 3 R A D 50 IL -5
C -5 2 4 T C -1 1 1 2 T
A -7 4 6 G
C +33T
C +1923T
G +2044A

28. Entomological inoculation rates in the Barkoundouba area,
Burkina Faso from August 1994 to October 1996.
Permethrin-impregnated curtains were installed in June
1996.
Source: Modiano D, et al., AJTMH; 1998; 59:336-340

29. Impact of permethrin-impregnated curtains on Plasmodium falciparum
infection rates in three sympatric ethnic groups from Burkina Faso, West
Africa
Source: Modiano D, et al., AJTMH; 1998; 59:336-340

30. Dipartimento di Scienze di Sanità Pubblica, Sezione di Parassitologia, Università “La
Sapienza” Rome, Italy
D. Modiano, G. Luoni, F. Verra, G.Paganotti, V. Petrarca, M. Coluzzi
Dipartimento di Biologia, Università “Tor Vergata” Rome, Italy;
G. Modiano, F. Pompei, G. Bancone, BM Ciminelli
Associazione Nazionale Lotta contro le Microcitemie, Rome, Italy;
I. Bianco, P. Grisanti, E. Foglietta
Tissue Antigen Lab., Imperial Cancer Research Found,London, UK;
J. Bodmer
Department of Immunology Stockholm University, Sweden;
P Perlmann, H Perlmann, M Troye Blomberg
Institute of Cell, Animal and Population Biology, Edinburgh, UK;
D Walliker, H Babiker
Wellcome Trust Center for Human Genetics, Oxford, UK;
D. Kwiatkowski
Centre National de Récherche et Formation sur le Paludisme, Ministère de la Santé,
Burkina Faso;
BS Sirima, I Nebié, D Diallo, A Konaté, J Simporé
Hopital Yalgado Ouedraogo, Ouagadougou, Burkina Faso
A. Sawadogo, I Sanou
Ecole de Medecine et Pharmacie, Université de Bamako, Mali
O Doumbo, A Dolo