Handout for the Department Meeting 20011120

Modiano D, Luoni G, Sirima BS, Simpore J, Verra F, Konate A, Rastrelli E, Olivieri A, Calissano C, Paganotti GM, D'Urbano L, Sanou I, Sawadogo A, Modiano G, Coluzzi M (2001) Haemoglobin C protects against clinical Plasmodium falciparum malaria. Nature, 414: 305-308.

Background

Genes with resistance to malaria:

Duffy (fy): RBC surface antigen refuse P.v. meroziote's invading
Thalassaemia: P.f. cannot grow within RBC due to hemolysis
HbS: P.f. cannot grow within RBC due to sickling
HLA Bw53: P.f. is killed by cell-mediated immunity
G6PD-deficiency: P.f. cannot grow in RBC due to oxidative stress
HbE: P.f. cannot grow in RBC (details are not known)

Previous reports on HbC ... contradictory

No correlation between HbC hetero and parasite rates/densities
Some clinical studies showed no protection from severe malaria
Some clinical studies showed protection from severe malaria
P.f. cannot proliferate in HbCC RBC ate's in vitro
P.f. parasitaemia with non-complicated malaria in HbCC case

None of above met sufficient statistical power to evaluate possible HbCC role to protect

Aim of the study

By large case-control study of P.f. malaria, evaluate it.

Subjects and Methods

3513 healthy Mossi (about 95% of Ouagadougou area is Mossi) controls (recruited at 12 primary schools), 835 Mossi malaria patients (recruited at Ouagadougou Univ. Hospital, among whom 359 were clinically severe) in Burkina Faso.

Genotypes and allele frequencies (A,C,S) for the b-globin gene were estimated by cellulose acetate electrophoresis for controls and by PCR-RFLP for cases.

The definition of severe malaria^*: (1) the presence of P.f. in thick blood slide (2) at least one of the following conditions:

(2-1) prostration (cannot sit without help), (2-2) unrousable coma (score between 0 and 2 on the Glasgow coma scale), (2-3) repeated generalized convulsions (more than 2 episodes in preceding 24h), (2-4) severe anaemia (Hb<5 g/dL), (2-5) hypoglycaemia (blood Glu<40 mg/dL), (2-6) pulmonary oedema/respiratory distress, (2-7) spontaneous bleeding and renal failure (plasma Cre<3 mg/dL)

The definition of non-severe malaria: (1) fever more than 37.5 degree C, (2) P.f. in thick blood slide.

^* The definition of severe malaria slightly differs by study.

Modified WHO criteria (1990) applied in the Dogon (Bandiagara, Mali) study by Agarwal et al. (2000). That is, (1) presence of asexual form of P.f. in blood slide, and (2-1) evidence of cerebral compromise (coma, obtundation, or witnessed convulsion without other explanation), (2-2) severe anemia (Hct<15%), (2-3) hyperparasitemia (parasite density [Ep]>5e+5 /mL), (2-4) hypoglycemia (serum Glu<40 mg/dL), (2-5) respiratory distress, (2-6) clinical shock, or (2-7) severe prostration.

Guinet et al. (1997) applied WHO criteria (1990) for 'severe and complicated malaria' in their study at Bamako, Mali. That is, (1) presence of P.f. in peripheral blood, and (2-1) cerebral malaria (seizures, obtundation or coma without other explanations), (2-2) severe anemia (Hct<15% or Hb<5g/dL without evidence of marasmus or kwashiorkor), (2-3) hyperparasitemia (Ep>1e+5 parasites /mL), (2-4) hypoglycemia (serum Glu<40 mg/dL), or (2-5) hyperpyrexia (fever > 40 degree C at mouth without other known cause of fever).

Results and Discussions

Results shown in Table 1 (statistics: Chi-square test with Yates' adjustment)

Genotype frequencies of AA, AC, and CC were in Hardy-Weinberg equilibrium^** (p>0.6) among controls but not in cases (p=0.0005), which depends on the only 1 HbCC in cases (expected HbCC was 13.8)
HbSC and HbSS were rare, not in H-W eq. among controls (HbSC, p<0.001; HbSS, p<0.01)
No age effect in both of cases and controls

^**Let p the gene frequency of A and q the gene frequency of C. Under no selective pressure, genotype frequencies of AA: AC: CC will converge into Hardy-Weinberg equilibrium, p²:2pq:q².

Results shown in Table 2

Protection against clinical malaria seen in HbAS, HbAC and HbCC (strongest!!)
HbC frequency did not differ by severity (maybe due to sampling bias)
Significantly lower bS allele and HbAS genotype in severe cases.

Other results

Ep did not differ between HbAA and HbAC but lower in HbAS (p=0.004 for HbAA, p=0.013 for HbAC)
No non-severe malaria cases with HbCC. Only 1 HbCC case had very low Ep (8/mL) with severe symptoms.

Conclusion

HbCC provides strong protection against clinical malaria (93% reduction), stronger than HbAC (29%), similar to HbAS (73%). Given the genotype frequencies of HbAA, HbAC and HbCC among healthy subjects, potential clinical malaria cases is prevented by HbC gene 0.29x0.2172+0.93x0.0165=0.0783. It's similar to 0.73x0.0954=0.0696 by HbAS. Since HbCC is not fatal, HbC may be replacing HbS. The reason why HbC's distribution is limited may be relatively recent appearance and that the protective effect is mainly by HbCC.