7

Positive Selection at Complement Receptor 1 in Che Wong
The extended haplotype homozygosity (EHH) of the chromosome 1 region between 206.96 and
210.84 Mb, encapsulating the complement receptor (CR1) gene, at a coding SNP rs6691117
(Val

Ile) for the derived guanine allele, did not decay below 0.25 even after two megabases
(Figure S1A). Variants in the CR1 gene have previously been reported consistently for conferring a strong protection against falciparum-driven cerebral malaria, where the guanine allele at a coding SNP rs2274567 (His

Arg) conferred a 3-fold protection for the heterozygotes
and up to 38-fold protection for the homozygotes (Panda et al. 2012; Rout et al. 2011); and an
intronic SNP rs11118133 (HindIII A>T) was reported to confer a 5-fold and 25-fold protection to cerebral malaria for heterozygote and homozygote carriers of the thymine allele in Asian
populations (Rout et al. 2011). While both rs2274567 and rs11118133 were not present in our
dataset, it was observed that these two SNPs were located in a region of high linkage disequilibrium with our index SNP rs6691117 (Figure S1B), and that the linkage disequilibrium between the two guanine alleles in our index SNP rs6691117 and rs2274567 was around r 2 =
0.90 for East and Southeast Asian populations with population-level sequencing data (Table
S24). The guanine allele at the nonsynonymous rs6691117 had also been previously reported to increase erythrocyte sedimentation rate (ESR) in a genome-wide association study on ESR in
Europeans (Kullo et al. 2011) and Sardinians (Naitza et al. 2012), and was reported with a
perfect LD (r 2 = 1) with rs2274567 in the Sardinians (r 2 = 0.74 in the Europeans). The extended haplotype form identified by haploPS in this region for the Che Wong carried the protective guanine allele at rs6691117 (Figure S1C), but there was no evidence of long haplotypes in the
Jakun and Negrito (Figure S1D).
Positive selection at TNF/LTA in Negrito
HaploPS identified strong evidence of positive selection in both the Negrito (haploPS P unadjusted
=
7.47 × 10 -8 , P adjusted
= 0.0036) and Jakun (haploPS P unadjusted
= 1.20 × 10 -6 , P adjusted
= 0.023) where the extended haplotypes encompassed both TNF and LTA genes. Previous studies have identified six polymorphisms, including five SNPs in the promoter region of TNF and one SNP in the 5’UTR, that are involved in malaria-related phenotypes (Table S24), including rs1800750
(promoter SNP in TNF, -376A>G) where the guanine allele conferred between 3- to 5-fold
protection against cerebral malaria onset in Africans (Knight et al. 1999). In addition, the AGGG
haplotype with SNPs rs3093662 (TNF -851), TNF -238, rs1800629 (TNF -308) and rs1800750
(TNF -376) were reported to confer a 5-fold protection against cerebral malaria in samples from
Myanmar (Ubalee et al. 2001). The extended haplotype forms identified by haploPS in both
Jakun and Negrito were perfectly identical across overlapping sites (Haplotype Similarity Index,
HSI = 1.00) and carried exactly the AGG configuration on rs3093662, rs1800629 and rs1800750

(Figure S2). The identity between the extended haplotype forms from Jakun and Negrito suggests that if malaria were responsible for driving the evolutionary adaptation, this would have happened prior to the split of the two populations.

Table S24. The LD between rs2274567, rs11118133 and index SNP rs6691117 in East and
Southeast Asian populations with population-level sequencing data
1KG CHB
1KG CHS
1KG JPT
SSM rs2274567
0.883519
0.94152
0.929663
0.928828 rs11118133
0.883519
0.94152
0.964881
0.951227

Table S24. SNPs in TNF previously reported to be associated with malaria-related phenotypes.
SNP Function Risk Protective Phenotype OR rs1800750
(TNF 376) rs1800629
(TNF 308)
Promoter
Promoter
A
A
G
G
Cerebral malaria
Severe malaria(SM) and cerebral malaria (CM)
Gambian 4.3 (1.5-12.8)
Kenyan 4.6 (1.3-15.7) 1
Gambian (SM)
1.1(1.01-1.23) 3
Gambian (CM)
1.29 (1.1-12.4) 1
Indian 1.87 (1.03-3.4) 2 rs1800630
(TNF 863) rs1799964
(TNF 1031)
Promoter
Promoter
A
C
C
T
Susceptibility to severe malaria
Susceptibility to severe malaria Indian 1.84 (1.01-3.38) 2 rs3093662
(TNF 851)
Intron G A Haplotype association with SM 3 rs361525 Promoter A G Haplotype association with SM 3
(TNF 238)
1 Knight JC, Udalova I, Hill AV, Greenwood BM, Peshu N, Marsh K, Kwiatkowski D: A polymorphism that affects OCT-1 binding to the TNF promoter region is associated with severe malaria. Nat Genet 1999, 22(2):145-150.
2 Sinha S, Mishra SK, Sharma S, Patibandla PK, Mallick PK, Sharma SK, Mohanty S, Pati SS, Mishra SK, Ramteke BK, Bhatt R, Joshi H, Dash AP, Ahuja RC, Awasthi S,
Indian Genome Variation Consortium, Venkatesh V, Habib S: Polymorphisms of TNF-enhancer and gene for FcgammaRIIa correlate with the severity of falciparum malaria in the ethnically diverse Indian population. Malar J 2008, 7:13.
3 Clark TG, Diakite M, Auburn S, Campino S, Fry AE, Green A, Richardson A, Small K, Teo YY, Wilson J, Jallow M, Sisay-Joof F, Pinder M, Griffiths MJ, Peshu N,
Williams TN, Marsh K, Molyneux ME, Taylor TE, Rockett KA, Kwiatkowski DP: Tumor necrosis factor and lymphotoxin-alpha polymorphisms and severe malaria in African populations. J Infect Dis 2009, 199(4):569-575

Figure S1. Positive selection at CR1 in the Che Wong
(A) Decay of the extended haplotype homozygosity (EHH) for both the ancestral allele (blue dashed line) and derived allele (red solid line) around the focal SNP rs6691117. (B) Pairwise linkage disequilibrium (LD) between 21 SNPs in the CR1 gene region. The numbers in the squares represent the LD values between every possible pairs of SNPs as measured by r 2 . The target SNP rs6691117 is indicated by the red box, while the green and blue arrows represent the locations of rs2274567 and rs11118133 respectively. (C) Identifying the selected haplotype form surrounding CR1, where the extended haplotype form identified by haploPS is illustrated
(bottom panel). The specific section that overlapped with CR1 is further zoomed into (top panel), and rs6691117 is indicated by the red line. (D) The longest haplotype forms identified by haploPS in Che Wong (red), Jakun (blue) and Negrito (green), between a haplotype frequency of
5% to 65%. At frequencies below 30%, the longest haplotype forms in Che Wong are significantly longer than those present in the Jakun and Negrito, which agree with the identification of a positive selection signal by multiple selection metrics in the Che Wong but not in the Jakun and Negrito.

Figure S2. Positive selection at TNF/LTA in Negrito. Extended haplotype forms around TNF in both the Jakun and the Negrito. The zoomed-in region illustrates the specific allelic variants reported to confer malaria protection.

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