Phenotypes

392 siblings of 108 families resident in West Africa were followed over a period of 31 wk, which covered an entire rainy season. Prevalences of P. falciparum blood trophozoites, parasite densities, and interim or present malaria fever episodes were monitored weekly and anemia as indicated by the packed blood-cell volume (PCV) was determined biweekly. Compliance was as follows; 98.8% of 12,152 parasitemia assessments, 95.4% of 6,272 PCV assessments, and 98.5% of 12,152 assessments for malaria fever episodes were recorded with a maximum of data missing per planned visits of single participants of 13/31, 6/16, and 18/31 records, respectively. Results from regression models for analyzing the effect of age, bednet use, and intake of antimalarials on the various phenotypes are summarized in Figure 1. Gender had no significant effect on any of the phenotypes, and therefore was not included in the final regression models used for phenotype corrections.

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Figure 1. Phenotype Adjustments of the Study Group (392 Siblings)

(A) Presence of asexual blood forms of P. falciparum in 31 weekly blood smears.

(B) Log of the 75th percentile of 31 parasite density values per individual.

(C) Median of 16 PCV assessments per individual.

(D) Malaria fever episodes were defined following WHO recommendations whereby multiple malaria attacks within 3 wk were considered recrudescences and counted as one episode (see Materials and Methods).

^#Gender did not significantly influence any of the phenotypes. For the phenotype of parasite density, the effects of antimalarial treatments were addressed by exclusion of density values following 3 wk after treatment (see Materials and Methods). The phenotype of fever episodes was not corrected for the number of antimalarial treatments because of the direct causal relationship between disease episodes and treatments.

^§31 weekly regression models.

^*Anemia (PCV) was normally distributed, Shapiro-Wilk W test, p > 0.2.

https://doi.org/10.1371/journal.pgen.0030048.g001

Genotyping

Based on a ranking that favored high levels of parasite densities in conjunction with high intrafamilial variability (see Materials and Methods), 377 individuals of 68 families were selected for genotyping, including 136 parental individuals and 241 siblings, who formed 330 sib-pairs. Applying the Affymetrix Human Mapping 10K array yielded an overall autosomal calling fraction of 94.5% for the raw genotypes. These were defined as SNPs for which definitive genotypes were obtained. After application of the quality control procedure, 1,524 autosomal markers (15.2%) were excluded from further analysis. The remaining markers yielded a mean information content of 0.976 (SD ± 0.029, range 0.510–1.000).

Linkage Analysis

The nonparametric linkage analysis (NPL) and Haseman-Elston multipoint linkage analysis (HE) were applied (Figure 2A–2D). Parasite prevalence, parasite density, fever episodes, and anemia were analyzed as quantitative phenotypes. The most prominent result was a linkage signal for malaria fever episodes on Chromosome 10p15.3–10p14, which reached statistical significance in both the NPL and HE analyses. NPL showed an asymptotic z score of 4.37 (empirical p-value = 4.0 × 10⁻⁵) between SNP markers rs952153 and rs1964428 marking the interval of 5.9–12.0 cM and 2.5–3.5 Mb of the genetic and physical chromosomal maps, respectively (Figure 2D). HE showed a maximum asymptotic logarithm of odds (LOD) score of 3.03 (empirical p-value = 2.1 × 10⁻⁴) at marker rs1964428 corresponding to 12.0 cM/3.5 Mb (Figure 2D). The locus-specific heritability was estimated to be 37.7% (95% confidence interval, 15.7%– 59.7%) at 11.2 cM. The linkage region was termed PFFE-1 for P. falciparum-fever episode 1. The signal was robust to variations in data analysis, including the use of a raw phenotype without adjustments for covariates (z score of 4.52), or the use of a wider definition of malaria fever episodes that included afebrile malaria episodes diagnosed by the study physicians (z score of 4.04). The 2.2 z score support interval (corresponding to a 1-LOD support) encompassed a 27.4 cM/11.0 Mb distance containing 71 annotated or hypothetical genes. Functional candidates include genes encoding a platelet-type phosphofructokinase (PFKP) also expressed in red blood cells, an inducible 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase (iPFK-2/PFKFB3), the alpha chain of the interleukin-15 receptor (IL15RA), the alpha chain of the interleukin 2 receptor (IL2RA), protein kinase C theta (PRKCQ), the GATA-binding protein 3 (GATA3), and a gene similar to that of the interleukin 9 receptor precursor (LOC439945).

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Figure 2. Quantitative-Trait, Multipoint Linkage Analysis of the Key Phenotypes of Uncomplicated Human Malaria

(A and B) Parasitological phenotypes. (C and D) Clinical phenotypes. A 10k-Affymetrix SNP array was used for genotyping 377 individuals comprising 241 siblings of 68 families. Results obtained by NPL are presented in the upper panels (z scores [Z] on the left axis) and those of HE are presented in the lower panels (LOD scores [LOD] on the left axis). Empirical significance levels (p-values indicated by dotted lines) were estimated by 100,000-fold Monte-Carlo permutations.

https://doi.org/10.1371/journal.pgen.0030048.g002

A further region with evidence for linkage was found using parasite density as the phenotype. The NPL analysis yielded a signal on Chromosome 13q with a maximum asymptotic z score of 3.73 (empirical p-value = 2.3 × 10⁻⁴) between rs2147363 at chromosomal position 55.0 cM/51.4 Mb and rs726540 at 55.5 cM/52.3 Mb (Figure 2B). HE resulted in a LOD score of 1.19 at this position (Figure 2B). The locus-specific heritability was estimated to be 33.7% (95% confidence interval, 9.8%–57.6%). The region was termed PFPD-2 for P. falciparum-parasite density 2, whereby another linkage region with parasite density had previously been reported [17–19]. The 2.2-z support interval of PFPD-2 encompassed 24.2 cM/32.4 Mb containing 158 annotated or hypothetical genes. Possible functional candidates include genes encoding the lymphocyte cytosolic protein 1 (LCP1), S-formylglutathione hydrolase (esterase D, ESD), the cysteinyl leukotriene receptor 2 (CYSLTR2), and the endothelin receptor, nonselective type, (EDNRB).

Furthermore, a signal on Chromosome 1p36 at 18 cM/9 Mb provided evidence for linkage with both parasite prevalence (LOD score of 2.31; empirical p-value = 5.3 × 10⁻⁴), and PCV (LOD score of 2.45; empirical p-value = 3.9 × 10⁻⁴) at adjacent marker positions (rs205474 and rs966134, respectively; Figure 2A and 2C). The NPL z scores were low in both instances (2.75 and 2.36, respectively).

Finally, no evidence was obtained for linkage of parasite density or malaria fever episodes to 5q31-q33 and to the MHC region on 6q23, respectively, which had previously been reported. In the present study, weak evidence was obtained that malarial anemia might be linked to 5q31-q33 (z score = 2.7, LOD score = 1.8) (Figure 2C).

Participants.

The study was conducted in the Asante Akim North District, Ashanti Region of Ghana, West Africa, a region classified as hyperendemic for malaria by a cross-sectional prevalence of 0.54 for P. falciparum.

Ethical approval was obtained from the Committee for Research, Publications and Ethics of the School of Medical Sciences, Kwame Nkrumah University of Science and Technology, Kumasi, Ghana. All procedures were explained in the local language, and consent was obtained from both parents. Parents of 2,551 families were recruited who had three or more children below the age of 12 y and agreed to participate. Venous blood samples of 2 ml were obtained from both parents and preserved by addition of an equal volume of 8 M urea. The genetic variants of hemoglobin (Hb)S, HbC, alpha⁺ thalassemia deletion 3.7, and glucose-6-phosphate-dehydrogenase (G6PD) deficiency A-, which were considered to possibly influence susceptibility to P. falciparum parasitemia and mild malaria, were determined, and 346 (13.6%) families were identified not segregating any of the traits.

Of the 346 families, a study group of 392 siblings of 108 families was selected based on (i) the logistic criterion that their homes clustered in 16 of the 30 villages included in the initial survey and (ii) that they did not live in homes equipped with window screens, which a posteriori were found to significantly reduce parasite prevalences from 0.54 to 0.35 (p < 0.001) and other parameters of malaria infection intensity. All families belonged to the ethnic group of Akan.

A subset of 377 members of 68 families were selected from the study group by excluding siblings who were absent at more than two assessments and by a ranking that favored high levels of parasite densities of P. falciparum in conjunction with high intrafamilial variability. The ranking score was based on the product of the mean of log parasite densities of P. falciparum within sib-ships multiplied by the standard deviation of log parasite densities within sibships. Families with highest scores were selected until 377 individuals were identified for genotyping. The genetic study group comprised 136 parents with 241 children, 52.5% boys and 47.5% girls, who, with a mean of 3.54 siblings per family, formed 330 sib pairs. Their median age was 5 y (range 0.5–11 y; IQR 3–8 y).

Phenotype assessments.

The children were phenotyped from May 20 to December 20, 2002. Weekly assessments by the visit of a trained physician included a medical history, measurement of body temperature by an infrared ear thermometer, a blood sample by finger prick or heel prick (approximately 100 μl), and in case of disease symptoms, a physical examination. The installation of window screens in homes and the use of bed-nets were recorded.

Weekly malaria smears were prepared at the study site, and in the laboratory they were stained with Giemsa and examined [34]. Parasite species were assessed, and parasite counts were recorded per 200 leukocytes (if >10 parasites/200 leukocytes) or 500 leukocytes (if ≤10 parasites/200 leukocytes) by two independent examiners. Parasite densities were calculated assuming a leukocyte count of 8,000/μl [34]. If the densities as determined in the two counts differed by a factor of three or more, a third independent count was obtained. The median parasite density of two or three counts was included in the analysis.

Weekly point prevalences of malaria parasitemias showed a median of 54% (range 40%–61%) yielding a cumulative period prevalence of 99%. Contributing 98% of all parasitemias, P. falciparum was the predominant species; Plasmodium ovale or Plasmodium malariae were found in 19% and in 90% of these in combination with P. falciparum. For the assessments of parasite prevalences and parasite densities, only P. falciparum and only the disease-causing asexual forms were included, which showed a median point prevalence of 53.1% and a period prevalence of 99%. Parasite densities of P. falciparum ranged from 0–317,360 parasites per μl, with an overall median of 32 and a 75th percentile of 680 in 12,011 assessments.

Anemia was on the spot assessed as PCV by capillary hematocrit centrifugation using 70 μl EDTA anticoagulated capillary tubes (Becton Dickinson, Germany) and mobile centrifuges. To reduce iron-deficiency as a possible confounder, prior to phenotyping, all children were treated against hookworm infection with 400 mg albendazole followed by oral iron supplementation of 2 mg/kg Fe²⁺ over 6 wk. The median PCV value of 16 biweekly assessments was used as the phenotype in all cases because none of the children showed evidence for any other disease causing substantial anemia. As determined by 16 biweekly PCV measurements, mild anemia defined by a PCV of <33% in the age group of 0.5–6 y and of <36% in the age group of 6–14 y [11] was found at 1,625 of 5,986 assessments (27%) affecting 326 (66%) of the children.

Mild malaria attacks were defined following WHO recommendations [9], first, by either assessing fever by a tympanic temperature of >37.7 °C, corresponding to a rectal temperature of >37.6 °C [35], or reported fever within the previous 4 d and second, a blood smear positive for asexual forms of P. falciparum at any density. The number of fever episodes during the observation period of 31 wk was counted for each individual, whereby multiple episodes within 3 wk were considered recrudescences and counted as one episode [36].

Applying this definition resulted in the identification of 504 malaria fever episodes affecting 257 children, which corresponds to a period prevalence of 66%. In addition, 34 febrile states of other etiologies including upper respiratory tract infections, lower respiratory tract infections, pneumonia, measles, and urinary tract infections were diagnosed by the study physicians. Suspected malaria attacks were treated by a standard dose of chloroquine or amodiaquine following national guidelines, other illnesses as deemed appropriate.

In greater detail, a tympanic temperature of >37.7 °C was measured at 365 visits. Subsequent malaria smears showed that 248 (68%) of the fever attacks occurred in conjunction with P. falciparum parasitemias (and four with other malaria parasites). Fever attacks reported by parents or guardians to have occurred between two examinations were recorded at 870 visits, whereby blood smears at the time of examination were positive for P. falciparum at 522 of these instances, and 133 of the attacks had reportedly been treated with antimalarials. The study physicians at 587 instances suspected malaria attacks at the time of examination, 389 (66%) of which were retrospectively supported by the presence of P. falciparum parasitemia. Of 631 mild malaria attacks as defined by WHO recommendations, 57% were in agreement with malaria diagnoses made by the study physicians, whereas 3% were classified by the study physicians as a febrile illness other than malaria.

Phenotype definitions and adjustments.

Regression models were used for adjusting the raw phenotypes for influences of covariates (Figure 1). Continuous covariates were modeled using multivariable fractional polynomials [37,38] at the nominal 5% test level. The choice of the 5% test level has been discussed in [38]. Specifically, we looked for non-linearity by fitting a second-order fractional polynomial to the data. The best power transformation x^p of covariate x is found, with the power p chosen from −2, −1, −0.5, 0, +0.5, +1, +2, +3, where x⁰ denotes ln x. For example, for p = 0, 0.5 the model is b₀ + b₁ ln(x) + b₂ √x. The set includes the straight line, i.e., no transformation p = 1, and the reciprocal, logarithmic, square root, square, and cubic transformations. Even though the set is small, the powers offer considerable flexibility. The test is performed by comparing the difference in model deviances with a χ² distribution on 1 degree of freedom. The resulting p-value is approximate and is justified by statistical arguments [37]. Pearson residuals from logistic regression and ordinary residuals from linear and Tobit regressions [39] were used for further analyses.

The difference between the sum of observed weekly P. falciparum parasitemias (1 or 0) minus the sum of predicted probabilities for parasitemias of an individual were calculated and used as phenotype. The predictions were derived from the study population by logistic regression models for parasitemia for each week's set of data with respect to the influences of age, time period since the last use of antimalarials, and the use of bed-nets (Figure 1).

Parasite densities recorded within 2 wk following the administration of antimalarials were found to be significantly lower than parasite densities determined at other time points (Wilcoxon rank sums tests, p < 0.05) and were therefore excluded. Since the 31 weekly parasite-density values (or their log-transformations) of an individual deviated from a normal distribution, quantiles were used. The 75^th percentiles were chosen because they were substantially more informative than the median values, which would have been zero in nearly half the assessments. The 75^th percentiles were log-transformed, whereby half the detection threshold of 16 parasites per μl was added before taking the logarithm. The phenotype was adjusted to age by calculating residuals from a Tobit regression model, whereby 74 of 392 observations were left censored (Figure 1).

The median values of 16 biweekly PCV assessments were adjusted using a multiple regression model which included the variables of age, number of antimalarial treatments, and use of bednets. Age was modeled as a second-degree fractional polynomial with exponents of −2, 2, the number of antimalarial treatments were considered untransformed, and bednet use was coded as a dummy variable (Figure 1).

The numbers of malaria fever episodes of each individual were adjusted for age and bednet use by calculating residuals from a Poisson regression model. Age was modeled as a second-degree fractional polynomial with exponents of −0.5, −0.5, bednet use was coded as a dummy variable. To avoid outliers for HE, phenotypic values above the 95th percentile were winsorized [40] (Figure 1).

Data analysis showed that siblings of families using mosquito protection by window screens had significantly reduced parasite prevalences, parasite densities, and numbers of fever episodes. As this indicated a marked reduction in exposure, the siblings were excluded a posteriori. The use of bednets was associated with slightly increased parasite prevalences, anemia, and numbers of fever episodes. As bednet use was heterogeneous within one family, all phenotypes were corrected accordingly. Intuitively, the associations appear paradoxical but may be explained by hypothesizing that households living under particularly high exposure, which implies particular molestation by mosquito bites, might use bednets preferentially, albeit with insufficient effectiveness [21].

Genotyping.

Venous blood samples of 2 ml were obtained at the end of the study period and preserved by the addition of urea. DNA was extracted by a standard procedure (NucleoMag 96 Blood, Macherey-Nagel, Germany). HbS, HbC [41], and alpha⁺ thalassemia [42] were determined as described. G6PD variants A (A376G), and A- (A376G, G202A) [43] were typed following a PCR-flourescence-resonance-energy-transfer hybridization method [41], whereby forward and reverse primers (F, R) as well as fluorescein (FL) or LC640red (LC640) (Roche, Germany) labeled anchor (A) and sensor (S) oligonucleotides were used: Variant A376G was typed with F: 5′-tgtgtgtctgtctgtccgtgt-3′, R: 5′-aacggcaagccttacatctg-3′, A: 5′-FL-cgatgatgcagcctcctaccagcgc-3′, S: 5′-LC640-tcaacagccacatggatgccctcc-3′ and variant G202A with F: 5′-cagctgccctgccctcag-3′, R: 5′-cttgaagaagggctcactctgtttg-3′, A: 5′-LC640-cagaaggccatcccggaacagcc-phosphate-3′, S: 5′-gcatagcccacgatgaaggtgttttc-FL-3′. Allele frequencies of HbS, HbC, alpha⁺ thalassemia, and G6PD A- were estimated to be 0.071, 0.058, 0.175, and 0.063, respectively. G6PD variant A, which was previously reported to show only a slightly decreased enzyme activity of 80% compared to the normal variant B (A376) [44], was retrospectively found not to show a significant influence on any of the phenotypes (Wilcoxon rank sums tests of genotypes AA or A, AB, and BB or B on the phenotypes parasite prevalence (p = 0.13), parasite density (p = 0.55), anemia (p = 0.36), and fever episodes (p = 0.80); therefore children being homozygous, heterozygous, or hemizygous for G6PD B and/or A, were included in the study group. Paternities and maternities were assessed by typing the short tandem repeat markers D1S2782, D6S273 (Genethon, www.genethon.fr), D5S816, D7S2212, D11S1984, D17S1299, and D17S1294 (The Cooperative Human Linkage Center, http://lpgws.nci.nih.gov/CHLC/), in(TG)_n of CD36 [45], and IL10G [46], all showing heterozygosity of ≥0.6 in the study population.

A high density SNP genome scan was performed using a whole-genome sampling analysis (WGSA) approach [32] with the Affymetrix GeneChip Human Mapping 10K v2 Array (early access) comprising 10,660 SNP markers with an average heterozygosity in Caucasians of 38% and a mean spacing of 258 kb/0.36 cM (Affymetrix , NetAffx Annotation files, http://www.affymetrix.com). Mapping order and genetic distances of markers were obtained from Affymetrix, the genetic position of 86 markers was unavailable, 295 were X-linked, and 10,279 were from autosomes. Allele frequencies were estimated from 134 founders. The physical positions of the markers were aligned to human DNA sequence information available from NCBI/NIH (http://www.ncbi.nlm.nih.gov/mapview/maps.cgi).

Statistical analysis.

In a first step, gender of participants was verified by investigating heterozygosity and hemizygosity, respectively, at X-linked markers. The relationships between individuals were confirmed by using Graphical Relationship Representation [47]. Genotypes incompatible with Mendelian inheritance were identified with PedCheck [48] and removed in members of the respective families. Unlikely genotypes, e.g., double recombinants, were investigated with Merlin [49], and apparent errors were resolved by deleting the respective genotypes of all family members. The genotypes of two participants showing <80% called genotypes were completely removed from the data. In a second step, SNP markers were excluded if they showed either (i) a deviation from Hardy-Weinberg equilibrium in founders at the nominal p < 0.001 test level, (ii) a genotype calling fraction of <80%, or (iii) a heterozygosity of <5%.

A quantitative trait locus autosomal linkage analysis was performed using the model-free nonparametric linkage method (NPL) [50] and the Haseman-Elston method (HE) [51], assuming an additive genetic model. In order to allow the simultaneous analysis of all SNP loci on a given chromosome in a multipoint approach, we adapted GENEHUNTER [52] to a 64 bit version [53]. Asymptotic z scores from NPL and LOD scores from HE are reported. Additionally, empirical p-values were determined using 100,000 replicates on the basis of the mean information content in the multipoint SNP analysis, which was 0.976 (SD ± 0.029, range 0.510–1.000). Specifically, a single marker was simulated with 20 equally frequent alleles, thus yielding a heterozygosity of 0.95. For the simulation, family structures and phenotypes were left unchanged. Details on this Monte-Carlo approach can be found, e.g., in [54]. A linkage signal of p < 10⁻⁴ in either NPL or HE was considered significant [55]. In addition, we describe two linkage signals which were below the threshold of significance, one signal of p < 5·10⁻⁴ and another signal of p < 10⁻³ obtained at the same genomic region with two largely independent phenotypes. We calculated locus-specific heritabilities of additive effects using the regression approach of Sham and colleagues [56]. Applying this method, we estimated the mean and variance from the data and fixed the heritability to 0.5. Decreasing the overall heritability led to moderate increases of the locus specific heritability. Candidate genes were defined as genes relevant to red blood cell structure, red blood cell metabolism or the inflammatory response.