In this study, we report an association of GM6 and KM genotypes on the risk of PM in HIV-1 infected and uninfected pregnant women. Although none of the individual GM6 and KM genotypes were by themselves significantly associated with PM, the combination of homozygous GM6(+) and KM3 was significantly associated with decreased risk of PM infection in HIV negative women. In HIV-1 positive women, the combination of heterozygous GM6(+/−) with either KM1-3 or KM1 revealed an increased risk of PM. These results suggest that GM6 and KM genotypes, two unlinked genetic loci, could interact to influence the susceptibility to PM infection in HIV-1 negative and positive women by regulation of disease-specific humoral responses.
The combination of GM6(+) and KM3 homozygotes may possibly affect susceptibility to PM infection in HIV-1 negative women through immune regulation to influence the specificity, subclass switch and titer of anti-malarial antibody responses. GM markers are located on the constant region and there is evidence for the involvement of these regions in antibody specificity with the variable region, probably through the formation of idiotypic determinants, modulation of antibody binding affinity and linkage disequilibrium with the variable epitopes 
. A previous study conducted in non-pregnant Sudanese (all ages) showed that the carriers of GM1,17 (present on IgG1); 5,13,14,6 (present on IgG3) phenotype were associated with higher incidence of malaria and higher baseline levels of total IgG and non-cytophilic IgG subclasses than the non-carriers 
. This same study further suggested that the above-mentioned implication of GM 1,17; 5,13,14,6 phenotype might be mainly due to the GM6 allotype involvement. Although there was an indication of limited involvement of KM allotype in susceptibility to malaria infection and antibody responses in this previous study 
, the current study showed that the combination GM6(+) and KM3(+) homozygote plays a role in the decreased risks of PM infection in HIV negative women. This could be due to the difference in the study models used, pregnant women vs. general population. In addition, a more recent study in Beninese children showed an age-related impact of GM 5,6,13,14;1,17 or KM1 phenotype on malaria specific cytophilic IgG responses 
. The interactive effect of GM and KM phenotypes in the clearance and persistence of hepatitis C infection has also been reported 
, suggesting that Ig GM and KM allotypes are important immuno-genetic factors in infectious diseases. Functionally, the γ and κ chains carrying specific GM and KM allotypes by nonrandom pairing could form a paratope with the necessary structure for recognition 
of the malaria antigen epitopes. Importantly, GM allotypes may further interact with Fcγ receptors (FcγR) to affect disease risk or protection. All GM epitopes including GM6, with the exception of G1M3 and 17, are found on the Fc-portion of the IgG molecule 
. It is possible that particular FcγR and GM6 alleles epistatically interact 
to influence the susceptibility to PM infection. Our previous study showed that FcγRIIa-H/H131 receptor for immunoglobulin G is associated with susceptibility to PM 
. An in vitro
study conducted by others reported that pregnancy-associated VSA-specific cytophilic IgGs promote phagocytic clearance of parasite infected erythrocytes, suggesting the contribution of this immune effector mechanism in the decrease of parasite load in the placenta 
. It is known that IgG opsonized phagocytosis of P. falciparum
infected erythrocytes could be mediated by FcγRI and FcγRIIa 
. In addition, the potential effect of the cytophlic IgG3 involving Fcγ receptors in protection against P. falciparum
malaria through phagocytosis of merozoites, neutrophil respiratory bursts and monocyte-mediated growth inhibition have been established in naturally acquired immunity 
In HIV-1 positive women the combination of heterozygous GM6(+/−) with either KM1-3 or KM1 was associated with susceptibility to PM infection. This suggests that HIV-1 infection could influence the relationship between GM and KM allotypes and PM infection, possibly by altering malaria-specific antibody responses. Indeed, several studies in pregnant women have reported that HIV infection decreases frequency and concentration of malaria-specific antibodies and/or changes balance of IgG subclasses against various antigens including pre-erythrocytic and blood stage antigens as well as pregnancy-associated VSA with impairment of opsonic phagocytic clearance of parasites 
. This could lead to the increased risk of PM infection in HIV-1 positive women. In this study, we also observed that the combination of heterozygous GM6(+/−) again with different KM, homozygous KM3(+), was associated with maternal anemia (Hb<7 g/dL) in HIV-1 positive women. Although the actual mechanism by which the combination of GM6(+/−) and KM3 would regulate anemia in pregnant women is not clear, the result indicates that GM6(+/−) could play a role in the risk of severe maternal anemia, one symptom of malaria during pregnancy.
We also measured departure from HWE in GM6 and KM allele frequencies and F(is) in the study population to evaluate possible selection 
by malaria on GM6 and KM as malaria has a strong selection on human genes 
. HWE tests clearly showed that there was no deviation for KM genotype frequency distribution in the study population. In contrast, an overall significant deviation of GM6 genotype frequency distribution was observed in the same population, showing significant positive F(is). Positive F(is) indicates a deficit on heterozygotes in the population 
. Most importantly, when further stratified by PM and HIV status there were still significant positive F(is) for GM6 in the HIV-1 negative women regardless of PM and in the HIV-1 positive women without PM while there was no significant deviation from HWE in HIV-1 positive women with PM. Tests of departure from HWE and F(is) have been used for different purposes; some have used the tests for searching disease-susceptibility gene loci and detecting selection, others have used them for genotyping quality control 
. However, HWE and F(is) results from our study with distinct patterns between GM6 and KM and with different strength among disease groups for GM6 were unlikely due to genotype errors as we employed different quality control measures for GM6 genotyping (see method section). In addition, our study was not a typical case-control study; rather, a diseases-stratified enrollment was used (see method section). Most likely, GM6 showing overall significant positive F(is) indicates low heterozygote fitness in the population possibly shaped by selection over time 
. The results of no significant departure from HWE in GM6 genotype frequency in HIV-1 positive women with PM but strong positive F(is) in other three groups further suggest that the group without low heterozygote fitness has less potential for resistance to malaria and/or HIV-1 infection. This notion could be partially explained by the results from our association analysis above showing GM6(+) involvement in the association with decreased risk of PM (homozygote advantage) while GM6(+/−) was associated with increased risk of PM (heterozygote disadvantage) regardless of HIV-1 status. As such, GM6 has fitness value likely selected by malaria infection. The assertion that GM6 is under selection by malaria is also supported by the fact that the geographic distribution of GM6 related haplotypes coincides with the region of high falciparum malaria and sickle-cell allele prevalence 
Taken together, this study highlights the association between GM6 and KM genotypes with PM in HIV-1 negative and HIV-1 positive women. These findings suggest that the combination of homozygous GM6(+) and KM3 may protect against PM in HIV-1 negative women, while the HIV-1 positive women with heterozygous GM6(+/−) combined with KM1-3 or KM1 may be more susceptible to PM. In addition, the result of a deficit in heterozygotes for GM6 in our study population (Kenyan pregnant women) suggests that low GM6 heterozygous fitness could be due to selection mainly by malaria infection. However, more studies in other populations and in different malaria endemic areas are needed to validate this finding.
Presentation of Results
This result was presented in part at the 59th Annual ASTMH meeting, November 3–7, 2010, in Atlanta, Georgia, USA.