Leffler 2017

RESEARCH ARTICLES
Cite as: E. M. Leffler et al., Science

10.1126/science.aam6393 (2017).
Resistance to malaria through structural variation of red

blood cell invasion receptors
Ellen M. Leffler,1,2 Gavin Band,1,2 George B. J. Busby,1 Katja Kivinen,2 Quang Si Le,1 Geraldine M. Clarke,1 Kalifa
A. Bojang,3 David J. Conway,3,4 Muminatou Jallow,3,5 Fatoumatta Sisay-Joof,3 Edith C. Bougouma,6 Valentina D.
Mangano,7 David Modiano,7 Sodiomon B. Sirima,6 Eric Achidi,8 Tobias O. Apinjoh,9 Kevin Marsh,10,11 Carolyne
M. Ndila,10 Norbert Peshu,10 Thomas N. Williams,10,12 Chris Drakeley,13,14 Alphaxard Manjurano,13,14,15 Hugh
Reyburn,13,14 Eleanor Riley,14 David Kachala,16 Malcolm Molyneux,16,17 Vysaul Nyirongo,16 Terrie Taylor,18,19
Nicole Thornton,20 Louise Tilley,20 Shane Grimsley,20 Eleanor Drury,2 Jim Stalker,2 Victoria Cornelius,1
Christina Hubbart,1 Anna E. Jeffreys,1 Kate Rowlands,1 Kirk A. Rockett,1,2 Chris C. A. Spencer,1* Dominic P.
Downloaded from http://science.sciencemag.org/ on May 18, 2017

Kwiatkowski,1,2* Malaria Genomic Epidemiology Network†
1
Wellcome Trust Centre for Human Genetics, University of Oxford, Roosevelt Drive, Oxford OX3 7BN, UK. 2Wellcome Trust Sanger Institute, Hinxton, Cambridge CB10 1SA,
UK. 3Medical Research Council Unit, Atlantic Boulevard, Fajara, Post Office Box 273, The Gambia. 4Department of Pathogen Molecular Biology, London School of Hygiene
and Tropical Medicine, Keppel Street, London WC1E 7HT, UK. 5Royal Victoria Teaching Hospital, Independence Drive, Post Office Box 1515, Banjul, The Gambia. 6Centre
National de Recherche et de Formation sur le Paludisme (CNRFP), 01 BP 2208 Ouagadougou 01, Burkina Faso. 7University of Rome La Sapienza, Piazzale Aldo Moro 5,
00185 Rome, Italy. 8Department of Medical Laboratory Sciences, University of Buea, Post Office Box 63, Buea, South West Region, Cameroon. 9Department of Biochemistry
and Molecular Biology, University of Buea, Post Office Box 63, Buea, South West Region, Cameroon. 10Kenyan Medical Research Institute (KEMRI)–Wellcome Trust
Research Programme, Post Office Box 230-80108, Kilifi, Kenya. 11Nuffield Department of Medicine (NDM), NDM Research Building, Roosevelt Drive, Headington, Oxford
OX3 7FZ, UK. 12Faculty of Medicine, Department of Medicine, Imperial College, Exhibition Road, London SW7 2AZ, UK. 13Joint Malaria Programme, Kilimanjaro Christian
Medical Centre, Post Office Box 2228, Moshi, Tanzania. 14Faculty of Infectious and Tropical Diseases, London School of Hygiene and Tropical Medicine, Keppel Street,
London WC1E 7HT, UK. 15National Institute for Medical Research, Mwanza Research Centre, Mwanza City, Tanzania. 16Malawi-Liverpool–Wellcome Trust Clinical Research
Programme, Queen Elizabeth Central Hospital, College of Medicine, Post Office Box 30096, Chichiri, Blantyre 3, Malawi. 17Liverpool School of Tropical Medicine, Pembroke
Place, Liverpool L3 5QA, UK. 18Blantyre Malaria Project, Queen Elizabeth Central Hospital, College of Medicine, Post Office Box 30096, Chichiri, Blantyre 3, Malawi. 19College
of Osteopathic Medicine, Michigan State University, East Lansing, MI 48824, USA. 20International Blood Group Reference Laboratory, National Health Service (NHS) Blood
and Transplant, 500 North Bristol Park, Filton, Bristol BS34 7QH, UK.
†For information about the Malaria Genomic Epidemiology Network (MalariaGEN) see www.malariagen.net.
*Corresponding author. Email: spencer@well.ox.ac.uk (C.C.A.S.); dominic.kwiatkowski@sanger.ac.uk (D.P.K.)
The malaria parasite Plasmodium falciparum invades human red blood cells via interactions between host
and parasite surface proteins. By analyzing genome sequence data from human populations, including
1269 individuals from sub-Saharan Africa, we identify a diverse array of large copy number variants
affecting the host invasion receptor genes GYPA and GYPB. We find that a nearby association with severe
malaria is explained by a complex structural rearrangement involving the loss of GYPB and gain of two
GYPB-A hybrid genes, which encode a serologically distinct blood group antigen known as Dantu. This
variant reduces the risk of severe malaria by 40% and has recently risen in frequency in parts of Kenya,
yet it appears to be absent from west Africa. These findings link structural variation of red blood cell
invasion receptors with natural resistance to severe malaria.
Malaria parasites cause human disease by invading and cause of malaria in Africa, P. falciparum, has an expanded
replicating inside red blood cells, which can lead to life- family of erythrocyte binding ligands that target a different
threatening complications that are a major cause of set of human receptors, most of which appear not to be
childhood mortality in Africa (1, 2). The invasion of red required for invasion (5–7). Two such invasion receptors are
blood cells is orchestrated by the specific binding of parasite the glycophorins GYPA and GYPB, which are abundantly
ligands to erythrocyte receptors (3), a stage at which genetic expressed on the erythrocyte surface and underlie the MNS
variation could influence the progression of infection. blood group system (6, 8–10). The antigenic complexity of
Indeed, a human genetic variant that prevents erythrocytic this system as well as rates of amino acid substitution and
expression of the Duffy antigen receptor for chemokines levels of diversity in African populations have led to
(DARC), which is essential for invasion by Plasmodium speculation that this locus is under evolutionary selection
vivax, is thought to have undergone a selective sweep due to malaria (8, 11–13).
resulting in the present-day absence of P. vivax malaria In a recent genome-wide association study (GWAS), we
across most of sub-Saharan Africa (4). In contrast the main identified alleles associated with protection from severe ma-
First release: 18 May 2017 www.sciencemag.org (Page numbers not final at time of first release) 1
laria on chromosome 4, between FREM3 and the cluster of duplicate units presents a challenge for short read sequence
genes encoding GYPE, GYPB and GYPA (14). Although the analysis due to ambiguous mapping (Fig. 1A) (21). We there-
association signal did not extend to these genes and a func- fore focused on changes in read depth at sites of high map-
tional variant was not identified, interpretation and further pability and developed a hidden Markov model (HMM) to
analysis of the association signal was inhibited by several infer the underlying copy number state for each individual
factors. First, the GWAS samples were collected at multiple in 1600 bp windows. We grouped individuals carrying simi-
locations in sub-Saharan Africa, where levels of human ge- lar copy number paths to define copy number variants
netic diversity are higher than in other parts of the world. (CNVs) and assign individual CNV genotypes.
This diversity remains underrepresented in genome varia- Across the 3,269 samples, we identified eight deletions
tion reference panels. Second, the glycophorin genes are in a and eight duplications that were found in ≥2 unrelated in-
region of segmental duplication that is difficult to character- dividuals (referred to below as non-singleton CNVs), as well
ize due to high levels of paralogy. Notably, the region is as at least 11 singleton variants (Fig. 1B and fig. S3) (22). For
known to harbor structural variation that contributes to the reference, we label these variants by copy number type (DEL

MNS blood group system but has not been characterized by for deletion, DUP for duplication), and number them in or-
next generation sequence data (15, 16). Here we aim to cap- der of frequency. To validate the CNV calls we analyzed
ture additional variation in sub-Saharan African popula- transmission in family trios and observed segregation as
tions, including structural variation, to determine the expected with few exceptions (table S4) (22). We also com-
underlying architecture of the association signal in this re- pared the CNV calls with the 1000 Genomes Project struc-
gion. tural variant analysis (23), and found highly consistent copy
number inference (98.8% of individuals have the same over-
An African-enriched reference panel in the glycophorin all copy number call) but substantial improvements to indi-
region vidual genotypes in our analysis (fig. S4) (22). Validation of
We constructed a reference panel with improved represen- the breakpoint of the most common variant (DEL1) by
tation of sub-Saharan African populations from countries Sanger sequencing further confirmed the accuracy of our
where malaria is endemic. We performed genome sequenc- method (figs. S5 and S6 and table S5) (22).
ing of 765 individuals from 10 ethnic groups in the Gambia, The variants ranged in length from 3.2 kb (the minimum
Burkina Faso, Cameroon and Tanzania, including 207 family possible with our method) to >200 kb and included dele-
trios (100 bp paired end (PE) reads, mean coverage 10x; ta- tions and duplications of entire genes. Loss of GYPB was a
bles S1 and S2). We focused on a region surrounding the common feature, with five different forms of GYPB deletion
observed association signal (chr4:140Mb-150Mb; GRCh37 among the non-singleton CNVs (Fig. 1B). Hybrid gene struc-
coordinates). Genotypes at single nucleotide polymorphisms tures were another common feature, with two non-singleton
(SNPs) and short indels in the region were called and com- CNVs predicted to generate GYPB-A or GYPE-A hybrids (fig.
putationally phased (17–19) and combined with Phase 3 of S7). Some variants are predicted to correspond to known
the 1000 Genomes Project (20) to obtain a reference panel MNS blood group antigens while others have not previously
of 3,269 individuals, including 1,269 Africans and a further been reported (table S6) (22). Of the non-singleton CNVs,
157 individuals with African ancestry (fig. S1 and tables S1 half (8/16) had a single pair of breakpoints in homologous
and S3). We imputed variants from this panel into the pub- parts of the segmental duplication, consistent with for-
lished severe malaria GWAS dataset comprising 4,579 cases mation via non-allelic homologous recombination (NAHR;
of severe malaria and 5,310 population controls from the Fig. 1C). Of these, four share a breakpoint position, which
Gambia, Kenya and Malawi and tested for association as coincides with a double-strand break (DSB) hotspot active
described previously (14). The signal of association, formerly in a PRDM9 C allele carrier (Fig. 1C and fig. S8) (24).
identified and replicated at SNPs lying between FREM3 and CNVs in the glycophorin region were observed more fre-
GYPE, extends over a region of at least 700 kb, and includes quently in Africa than other parts of the world (Fig. 2). In
linked variants within GYPA and GYPB where association is this dataset, the combined allele frequency of glycophorin
only apparent with the additional African reference data CNVs in African populations was 11% compared to 1.1% in
(fig. S2). non-African populations, and most of the non-singleton
CNVs (13/16) were identified in individuals of African ances-
Identification of copy number variants try. Among fourteen different ethnic groups sampled in Af-
We next assessed copy number variation in the glycophorin rica, the estimated frequency ranged from 4.7-21% with the
region (defined here as the segmental duplication within highest frequencies in west African populations.
which the three genes lie) for the sequenced reference panel
individuals. The high level of sequence identity between the Association with severe malaria
We sought to incorporate CNVs into the phased reference DUP4 present among the 4791 Gambian individuals in the
panel with the aim of imputing into our GWAS dataset. GWAS. This large frequency difference places DUP4 as an
Computational phasing of CNVs is challenging, as published outlier compared with imputed variants at a similar fre-
methods do not model CNV mutational mechanisms or non- quency in the Gambia or in Kenya genome-wide (P=1.7x10−3
diploid copy number at smaller variants within CNVs. To and P=5x10−3, respectively, based on the empirical distribu-
work around this, we excluded SNPs and short indels within tion of frequencies; Fig. 5A). DUP4 also varies in frequency
the glycophorin region and relied on the trio structure of within east Africa (Fig. 5B). Computation of haplotype ho-
sequenced individuals to resolve haplotype phase between mozygosity provides evidence that DUP4 is carried on an
CNVs and flanking SNPs (18, 25). Haplotype clustering and extended haplotype (P=0.012 for iHS (26, 27) based on the
cross-validation predict good imputation performance for empirical distribution for variants of similar frequency ge-
the three highest frequency CNVs, DEL1, DEL2, and DUP1 nome-wide; Fig. 5, C and D) that may have risen to its cur-
as well as for DUP4 (figs. S9 to S11). rent frequency in Kenya relatively recently. We note that
We used this panel to impute CNVs into the severe ma- DUP4 is also absent from all but two of the reference panel

laria GWAS samples (fig. S12) and tested for association as populations (Fig. 2).
before. One of the imputed CNVs, DUP4, is associated with
decreased risk of severe malaria (odds ratio, OR=0.60; 95% The physical structure of DUP4
CI 0.50-0.72; Padditive = 9.9x10−8; computed under an additive The copy number profile of DUP4 is complex, with a total of
model of association using fixed-effect meta-analysis; Fig. six copy number changes that cannot have arisen by a single
3A). Across populations, evidence for association at DUP4 is unequal crossover event from reference-like sequences (Figs.
among the strongest of any variant in our data. Moreover, 1B and 4B). At the gene level, this copy number profile cor-
conditioning on the imputed genotypes at DUP4 in the sta- responds to duplication of GYPE, deletion of the 3′ end of
tistical association model removes signal at all other strong- GYPB, duplication of the 5′ end of GYPB and triplication of
ly associated variants including the previously reported the 3′ end of GYPA. To begin to understand the functional
markers of association (e.g., conditional Padditive=0.32 at consequences of DUP4, we sought to reconstruct the physi-
rs186873296; Fig. 3B and fig. S13). DUP4 has an estimated cal arrangement of this variant by pooling data across the
heterozygous relative risk of 0.61 (95% CI 0.50-0.75) and its nine carriers in the sequenced reference panel (eight
genetic effect appears to be consistent with an additive Wasambaa individuals from Tanzania including three par-
model, although the low frequency of homozygotes makes it ent-child pairs, and a single African Caribbean individual
difficult to distinguish the extent of dominance (homozy- from Barbados). First, analysis of coverage along a multiple
gous relative risk 0.31; 95% CI 0.09-1.06; n=24 homozygotes; sequence alignment of the segmental duplication corrobo-
Fig. 3C). Analysis of different clinical forms of severe malar- rated the location of the six copy number changes from the
ia showed that DUP4 reduced the risk of both cerebral ma- HMM, with two pairs of breakpoints at homologous loca-
laria and severe malarial anemia to a similar degree (table tions in the alignment (fig. S15).
S7). We noted some evidence of additional associations in Next, we looked for sequenced read pairs spanning CNV
the region, including a possible protective effect of DEL2 breakpoints, which provide direct evidence of the structure
(OR=0.63; 95%CI=0.42-0.94, Padditive=0.02), but no evidence of the underlying DNA. We identified read pairs that were
of association with the more common DEL1, or with GYPB mapped near breakpoints but with discordant positions
deletion status overall (P>0.1 using logistic regression). (MQ>=1, absolute insert size > 1000 bp), including longer
These results are compatible with a primary signal of asso- read data we generated for the 1000 Genomes individual
ciation that is well explained by an additive effect of DUP4. who carries DUP4 (HG02554; 300 bp PE reads on Illumina
DUP4 is imputed with high confidence in both east Afri- MiSeq to 13x coverage). Discordant read pairs supported the
can populations (Fig. 3D), where it is at substantially higher connection between each pair of homologous breakpoints as
frequency than in the reference panel (fig. S12). To inde- well as between the remaining two breakpoints, which lie in
pendently confirm the imputed DUP4 genotypes, we ana- non-homologous sequence (Fig. 6A and fig. S16). On the ba-
lyzed SNP microarray data for intensity patterns indicative sis of the combined evidence from copy number changes,
of copy number variation (Fig. 4) using a Bayesian cluster- discordant read pairs, and homology between inferred
ing model informed by the sequenced DUP4 carriers (fig. breakpoints, we generated a model of the DUP4 chromo-
S14 and table S8) (22). Classification of GWAS samples was some that contains five glycophorin genes (Fig. 6B).
highly concordant with the imputed DUP4 genotypes in the A prominent functional change on this structure is the
east African populations (r2=0.96 in Kenya; r2=0.88 in Ma- presence of two GYPB-A hybrid genes, supported by several
lawi; table S9). Surprisingly, both imputation and the mi- read pairs within intron 4 of GYPA and GYPB and the copy
croarray intensity analysis suggest there may be no copies of number profile. We confirmed the hybrid sequence by PCR-
based Sanger sequencing of a 4.1 kb segment spanning the ment are likely to drive the underlying causal mechanism
breakpoint (Fig. 6B, figs. S17 and S18, and table S10) (22). for resistance to severe malaria. DUP4 was not present in
These data localize the breakpoint to a 184 bp section of the 1000 Genomes Phase 1 reference panel (used in (14)),
GYPA and GYPB where the two genes have identical se- and exists as a singleton in the 1000 Genomes Phase 3 refer-
quence (fig. S19). If translated, the encoded protein would ence panel. Thus, as previously observed at the sickle cell
join the extracellular domain of GYPB to the transmem- locus (34), mapping of the association signal by imputation
brane and intracellular domains of GYPA, creating a peptide was only possible with the inclusion of additional individu-
sequence at their junction that is characteristic of the Dantu als in the reference panel.
antigen in the MNS blood group system (28, 29). Moreover, Through additional sequencing, we have shown that
like DUP4, the most common Dantu variant (termed NE DUP4 corresponds to the variant encoding the Dantu+ (NE
type, here referred to as Dantu NE) is reported to have two type) blood group phenotype, thus linking the predicted
such hybrid genes and lack a full GYPB gene (30). We se- hybrid genes to a serologically distinct hybrid protein that is
quenced genomic DNA from an individual serologically de- expressed on the red blood cell (29, 35). The few existing

termined to be Dantu positive, and of NE type (150 bp PE studies of Dantu+ (NE type) erythrocytes indicate high lev-
reads on Illumina HiSeq to 18x coverage) and analyzed it els of the hybrid GYPB-A protein and lower levels of GYPA
using our HMM. The coverage profile and HMM-inferred than wild type cells (35, 36). A single study reports parasite
copy number path, indistinguishable from those of DUP4 growth to be impaired in Dantu+ cells (37), making Dantu
carriers, confirm identification of DUP4 as the molecular NE one of many glycophorin variants that have been hy-
basis of Dantu NE (Fig. 6, C and D). pothesized to influence malaria susceptibility or shown to
In addition to duplicate GYPB-A hybrid genes, these data have an effect in vitro (12, 37–40). Our results regarding a
reveal the full structure of this Dantu variant, including a specific protective effect of DUP4, and the lack of evidence
duplicated copy of GYPE and the precise location of six for other protective CNVs, suggest the relevance of these
breakpoints. Either complex mutational events or a series of effects in natural populations may be complicated. We cau-
at least four unequal crossover events are needed to account tion that many of the other CNVs are rare, such that larger
for the formation of this variant (confirmed by simulation; sample sizes and direct typing may be required to test their
fig. S20) (22). However, we find no potential intermediates effect in vivo.
and no obvious relationship between DUP4 and other struc- These findings then raise the question of how DUP4 pro-
tural variant haplotypes in the present dataset (fig. S9) (22). tects against malaria. GYPA and GYPB are exclusively ex-
Further analysis of discordant read pairs identifies a num- pressed on the erythrocyte surface and are targeted by
ber of shorter discrepancies relative to the reference se- parasites during invasion (6, 7). P. falciparum EBA175 binds
quence that are consistent with gene conversion events (fig. to the extracellular portion of GYPA (41), which is preserved
S21) and could be functionally relevant (e.g., fig. S22). in DUP4. P. falciparum EBL1 binds to the extracellular por-
tion of GYPB (42) which is duplicated in DUP4 but joined to
Discussion intracellular GYPA. The significance of the extra copy of
Here we use whole-genome sequence data to identify at GYPE or the absence of full GYPB in DUP4 is uncertain,
least 27 CNVs in the glycophorin region that segregate in since GYPE is not known to be expressed at the protein level
global populations. In this study, 14% of sub-Saharan Afri- (8, 43), and we do not observe evidence that absence of
can individuals carry a variant that affects the genic copy GYPB alone confers protection (Fig. 3A). GYPA and GYPB
number relative to the reference assembly. Our description are known to form homodimers as well as heterodimers in
of these variants complements the existing literature on an- the red cell membrane (33), so these copy number changes
tigenic variation associated with the MNS blood group sys- could have complex functional effects. There are physical
tem and offers additional insights. For example, the interactions between GYPA and band 3 (encoded by
frequency of GYPB deletion is broadly commensurate with SLC4A1) at the red cell surface (44) and parasite binding to
previous surveys of the S−s−U− blood group phenotype GYPA appears to initiate a signal leading to increased mem-
linked to absence of the GYPB protein, but the GYPB dele- brane rigidity (45). Thus the GYPB-A hybrid proteins seen in
tions in our data differ from the reported molecular variant DUP4 could potentially affect both receptor-ligand interac-
(fig. S23) (16, 22, 31–33). tions and the physical properties of the red cell membrane.
Of the array of glycophorin CNVs identified, one (DUP4) Previous surveys of the Dantu blood group antigen have
is associated with resistance to severe malaria and explains indicated that it is rare (table S11) (33, 46–48). We find that
the previously reported signal of association (14). While DUP4 is absent or at very low frequency outside parts of
there may be other functional mutations on this haplotype, east Africa, with a frequency difference and extended haplo-
we propose that the direct consequences of this rearrange- type consistent with a recent rise in frequency in Kenya. In
contrast, the malaria-protective variant causing sickle-cell reads to an average of 10x coverage. Reads were mapped to
anemia (rs334 in HBB), which is thought to be under bal- the GRCh37 human reference genome with additional se-
ancing selection, has a similar frequency in both the Gambia quences as modified by the 1000 Genomes Project
and Kenya (Fig. 5A). One possibility for why DUP4 is not (hs37d5.fa; (20)), using BWA (52) with base quality score
more widespread, given its strong protective effect against recalibration (BQSR) and local realignment around known
malaria, is that it has arisen recently without time for gene indels as implemented in GATK (53, 54).
flow to facilitate its dispersion. Alternatively, this frequency
distribution could be consistent with balancing selection, Sequence data curation
for example if it protects only against certain strains of P. We used GATK HaplotypeCaller to compute an initial set of
falciparum that are specific to east Africa. The glycophorin genotype likelihoods across samples at a genome-wide set of
region is near a signal of long-term balancing selection, and variants, including polymorphic sites from 1000 Genomes
measures of polymorphism in both the human glycophorins Phase 3 (55). We computed average coverage across the ge-
and P. falciparum EBA175 have been suggestive of diversify- nome for each individual using BEDTools genomecov (56)

ing selection (11, 12, 49, 50). Although apparently not direct- and excluded seven Bantu individuals from Cameroon with
ly related to these signals, current selection on DUP4 may less than 2x coverage across the genome, and one Wollof
represent a snapshot of the long-term evolutionary process- individual from the Gambia with less than 6x coverage and
es acting at this locus. Mapping the allele frequency of greater than 10% missing call rate in the GATK analysis. All
DUP4 across additional populations could help clarify the further analyses described here are based on the 765 non-
nature of selection. excluded individuals.
Recent GWAS have confirmed three other loci associated We inferred the sex of sequenced samples based on the
with severe malaria (HBB, ABO, ATP2B4), all of which are ratio of X chromosome coverage to autosomal coverage. To
also related to red blood cell function (14, 51). However, only infer family relationships, we used lcMLkin (57) to compute
the association with GYPA and GYPB directly involves varia- maximum likelihood pairwise kinship estimates from the
tion in invasion receptors. These receptors have been found GATK-estimated genotype likelihoods at a thinned set of
to be non-essential in experimental models (7, 9), yet this ~26,000 SNPs genome-wide. We then ran PRIMUS (58) to
result indicates important functional roles in natural popu- infer pedigrees from the kinship estimates and compared
lations. Intriguingly, there is marked variation among P. the inferred and reported relationships. Based on this we
falciparum strains in preference for different invasion manually curated the family structure of sequenced samples
pathways in vitro (7); field studies that account for parasite by removing relationships incompatible with trio structure
heterogeneity and tests for genetic interactions may there- (IBD1 < 0.9 for parent-child relationship), swapping three
fore be important in determining how DUP4 affects parasite individuals between trios with clear sample mixups, and
invasion. The discovery that a specific alteration of these exchanging parental labels in two trios to be consistent with
invasion receptors confers substantial protection provides a the genetic sex of the parents. The curated dataset contains
foundation for experimental studies on the precise func- 207 trios, 16 duos, and 115 individuals without nominal close
tional mechanism, and may lead us toward novel parasite relationships. All trios and duos are unrelated to each other
vulnerabilities that can be utilized in future interventions except for one extended family in the Wollof from the Gam-
against this deadly disease. bia, which consists of a quad (two parents and two children,
here encoded as two trios), where one of the children is a
Materials and methods parent in an additional trio.
Sample collection and sequencing
Sequencing of African individuals 1000 Genomes sequence data
Blood samples from a total of 773 healthy individuals from The 2,504 individuals from 26 populations in the 1000 Ge-
10 ethnic groups in four countries in sub-Saharan Africa nomes Phase 3 release (20) were analyzed. Bam files con-
were collected by MalariaGEN partners taining reads mapped to GRCh37 were downloaded from
(www.malariagen.net) and the MRC Unit in the Gambia the 1000 Genomes FTP site
(http://www.cggh.org/collaborations/mrc-unit-the-gambia) (ftp://ftp.1000genomes.ebi.ac.uk/vol1/ftp/data/; fig. S1B and
as part of ongoing projects (fig. S1A and tables S1 and S2). table S3).
Individuals were from the general population, with most
collected in family trios except in Burkina Faso where indi- Overview of the glycophorin region
viduals are unrelated. Genomic DNA was extracted and se- The glycophorin gene cluster on chromosome 4 results from
quencing was performed on Illumina HiSeq 2000 at the segmental duplication events in the ancestor to African
Wellcome Trust Sanger Institute with 100 bp paired end great apes (59) and is not related in sequence to GYPC on
chromosome 2. By aligning segments of the reference se- of genotype calls at these variants using BEAGLE 4.0 (bea-
quence against one another, we identified the region of gle.r1399; (17)) without specifying family information. Based
segmental duplication, here referred to as the glycophorin on the initial calls, we removed variants with more than five
region, as chr4:144,706,830-145,069,066, and the three pa- mendelian errors in the 207 trios, strong evidence for devia-
ralogous units of the segmental duplication as: GYPE unit, tion from Hardy-Weinberg equilibrium (PHWE < 1x10−3 in the
chr4:144,706,830-144,837,481; GYPB unit, chr4:144,837,482- 542 individuals without parents in the dataset) or more
144,947,716; GYPA unit, chr4:144,947,717-145,069,066. Each than one alternate allele. We then re-ran BEAGLE on the
gene occupies ~30 kb toward the end of its ~120 kb repeat remaining set of 111,167 variants, including family infor-
unit. We generated a multiple sequence alignment of the mation, to produce genotype calls. We phased these geno-
reference sequence for the three units by running kalign types using SHAPEIT2, specifying 400 selected and 200
(60) with default parameters, and calculated pairwise iden- random conditioning states, an effective population size of
tity in 1600 bp windows along this alignment (Fig. 1A). The 17,469, and 10 burn-in, 8 pruning and 50 main iterations,
glycophorin genes are transcribed on the negative strand, and including trio information.

and we adopt the convention of numbering exons and in- Finally, to form a joint reference panel across all indi-
trons as they occur in the GYPA transcript (exon 3 is a viduals, we merged the phased haplotypes with the 1000
pseudoexon in GYPB and exons 3 and 4 are pseudoexons in Genomes Phase 3 haplotypes (20) at the overlapping set of
GYPE). We focus on the three protein-coding genes, but note variants. The merged reference panel, which contains 96,676
that a long noncoding RNA is annotated between GYPE and variants in the region chr4:139.5-150.5Mb, is available for
GYPB (LOC101927636). The coordinates here, and through- use in other studies (see
out the paper, are given with respect to GRCh37. https://www.malariagen.net/resource/23).
Construction of a regional reference panel Imputation and association testing at SNPs and short
Identification of polymorphic sites and genotype likelihood indels
computation Imputation and association testing
To construct a regional reference panel we focused on the 10 We used both the 1000 Genomes Phase 3 reference panel
Mb region chr4:140-150Mb, including a 500 kb margin at (20) and the merged reference panel described above to im-
either end. We first assembled a list of previously identified pute genotypes into the previously published sets of severe
SNPs and short indels from the 1000 Genomes Phase 3 (20), malaria cases and population controls from the Gambia,
the Illumina Omni 2.5M array, the ExAC project (61) and the Kenya and Malawi (14). In brief, we ran IMPUTE2 (62, 63)
European Variation Archive (downloaded on 24th February in 2 Mb chunks, using Illumina Omni 2.5M genotype calls as
2016; www.ebi.ac.uk/eva/), totaling 421,670 variants. We previously described. A total of 5,621 (the Gambia), 5,203
then used freebayes v1.0.2 (19) to calculate genotype likeli- (Malawi), and 5,583 (Kenya) genotyped variants were in-
hoods at these sites as well as at newly identified putatively cluded in the 11 Mb region considered. We specified 1,000
polymorphic sites, across the 3,269 sequenced individuals. copying states (-k_hap 1000), an effective population size of
Freebayes was run in 10 kb chunks across the region. We 20,000 (-Ne 20000) as recommended for African popula-
filtered freebayes output to include all previously identified tions, and a buffer region of 500 kb. We removed reference
variants and high quality novel variants, i.e., novel variants panel individuals with parents in the panel before imputa-
with quality (QUAL) > 1, presence of supporting reads on tion.
both strands (SAF > 0 and SAR > 0) and both sides of the We tested for association in each population under addi-
variant (RPL>1 and RPR>1), and high quality per alternate tive, dominant, recessive and heterozygote models using a
observation (QUAL/AO>10). In total, the filtered output logistic regression model as implemented in the program
contained 424,909 variants, of which 412,795 were among SNPTEST, including five principal components to account
the previously identified variants. for population structure. We restricted analysis here to se-
vere malaria cases with nonzero parasitaemia, as measured
Genotype calling and phasing by blood slide at the time of admission, as this is likely to be
We next aimed to generate a high-quality set of genotype the most accurate phenotype. For downstream meta-
calls in the 765 individuals collected by MalariaGEN part- analysis, we omitted results for variants at low frequency
ners. We focused on sites with combined mean coverage (MAF < 0.5%) or with low imputation quality (IMPUTE info
between 7.5x and 13.5x, excluding approximately 4% and 1% < 0.75) in each population.
of variants with depth below or above this range respective- We used fixed-effect meta-analysis to compute an esti-
ly, and restricted to variants with allele count at least 2, mate of the odds ratio across populations, along with a
leaving a total of 117,531 variants. We produced an initial set standard error and P-value, for each model. For additive
model we refer to the resulting P-value as Padditive. Addition- proportional to how much individuals with no copy number
ally, we computed a model-averaged Bayes factor (BFavg) variation (k=2) are above or below their mean in that win-
reflecting the overall evidence for association as previously dow. These define the emission probabilities for the HMM.
described (14). Meta-analysis results under the two imputa- We used a fixed transition matrix that assumes a low rate of
tion reference panels, as well as for the previously published switching with approximately 0.999 probability of remain-
1000 Genomes Phase 1 reference panel imputation, are ing in the current copy number state; 1x10−4 for leaving the
shown in fig. S2. diploid state; 0.001 probability of returning to diploid (k=2)
from a non-diploid state; and 1x10−5 probability of switching
Functional annotation of variants among non-diploid states.
We used Variant Effect Predictor (VEP; (64)) to annotate the We estimated μi, σi and wj by starting with an initial
functional consequences of all variants with evidence of as- guess assuming everyone to be diploid across the region,
sociation (BFavg>1). We noted one variant with VEP IMPACT and then running the Viterbi algorithm separately for each
rating ‘moderate’ with some evidence of association individual. We then recalculated μi and σi for each individu-

(rs147343123; nonsynonymous in exon 1 of GYPA, predicted al, only including windows in which the copy number is
deleterious; association test BFavg=17.34, Padditive=1.4x10−3 for inferred to be 2, and wj for each window only including in-
1000 Genomes imputation; BFavg=121.3, Padditive=1.5x10−4 for dividuals in which the copy number is inferred to be 2. We
full panel imputation) (fig. S2). We also noted that the non- iterated this algorithm until no further changes in the in-
synonymous variant in FREM3 exon 1 previously found to ferred underlying copy number paths were observed for any
have evidence of association (rs181620317; see (14)), is im- individuals.
puted at much lower frequency using both the 1000 Ge-
nomes Phase 3 panel and the full reference panel (e.g., CNV calling in the 3,269 sequenced individuals
frequency = 0.2% in Kenya using the full reference panel), We applied the HMM method described above to the full set
and shows no evidence for association using genotype im- of 3,269 individuals with sequence data in an 850 kb region
puted from these panels. including the glycophorin genes (chr4:144.35-145.20Mb).
After inferring the copy number state paths for each indi-
Identification of copy number variation vidual, we then considered variants to be the same across
Method to call copy number variation individuals if the direction of copy number change was the
To identify large CNVs across the glycophorin region, we same and both end points were within three bins of each
implemented a hidden Markov model (HMM) to infer the other. Heterozygous triplications and homozygous duplica-
underlying copy number state from the observed read tions were differentiated by looking across individuals; vari-
counts. The input to the HMM is read depth, averaged over ants that were always found in copy number state 4 were
sites in windows of fixed length (here, 1600 bp) for each attributed as triplications. We excluded copy number varia-
individual. To reduce the problems with mapping in the ble segments that covered only a single bin or were outside
region, we included only (i) reads with at least a mapping the segmental duplication. We then considered copy num-
quality (MQ) of 20; (ii) mappable sites, defined as sites with ber variable segments that were always found together to be
mappability >0.9, where mappability of a site is the mean a single CNV, and manually refined the few other copy
value of the CRG mappability track for all 100-mers over- number variable segments that were not found separately
lapping that site (21); (iii) windows with ≥25% of sites ful- from other CNVs (22). This process identified 16 non-
filling this criterion; windows with fewer mappable sites singleton variants and 28 singleton variants (Fig. 1B and fig.
were considered uninformative. S3), although we note several caveats about the singleton
We modeled the mean depth of coverage for individual i CNVs including some that likely correspond to more com-
at window j, di,j, as normally distributed with mean and var- mon variants (22).
iance dependent on the assumed underlying copy number k To validate the CNVs and genotype calls, we assessed in-
(k=2 is the normal diploid state): heritance in the MalariaGEN trios and compared genotype
k 2 calls for the 1000 Genomes individuals with those released
k  
d i , j ~ Norm  w j mi ,  σ i   k ∈ {0,1, 2, 3, 4} in the 1000 Genomes Phase 3 paper on structural variants
2  2  
 (table S4 and fig. S4) (22, 23). We also designed PCR pri-
For copy number k=0 (homozygous deletion), we used a mers on either side of the DEL1 variant and generated
truncated normal distribution (truncated at 0) and assigned Sanger sequence that confirmed and localized this break-
a variance of 0.04 to allow for spurious mapping. To ac- point (figs. S5 and S6 and table S5) (22).
count for systematic variation in coverage along the ge-
nome, we estimated a window-specific factor (wj) Phasing and imputation of CNVs
Initial phasing of CNVs phasing we observed that haplotypes carrying several of
We investigated whether CNVs could be accurately phased these clustered with the corresponding common variant
relative to surrounding SNP variation in the regional refer- (DEL9 with DEL1; DEL11, DEL12 and DEL14 with DEL2;
ence. Collectively, non-singleton CNVs in our dataset cover a DUP15 and DUP18 with DUP1). We reasoned that these are
total of 350 kb (Fig. 1B), which extends over most of the re- likely to represent the same variant, with differences in call-
gion of segmental duplication surrounding the glycophorin ing potentially due to noise in coverage profiles or variation
genes. In principle, inference of CNV haplotype phase might in the other chromosome. We merged these singletons with
benefit from copy-number-aware genotype calls at smaller the corresponding non-singleton CNV and repeated the pro-
variants within CNVs. However, implementing this is chal- cedure described above, using SHAPEIT2 and MVNCALL to
lenging as the state-of-the art phasing methods assume re-phase CNVs and flanking short variants then merging the
samples are diploid. A further possible issue is that read two reference panels. We also noted that haplotypes carry-
mapping, and hence the quality of SNP genotype calls, is ing DEL4 cluster with DUP1, which shares a similar break-
likely to be impaired in such regions of segmental duplica- point (Fig. 1, B and C). A plausible explanation for this is

tion. that DUP1 arose by NAHR on a DEL4 background (fig. S24)
Motivated by these observations, we took the following (22).
approach to phasing CNVs which leverages the family trio For subsequent imputation steps we restricted the com-
structure of sequenced individuals to infer accurate phase, bined panel to the set of haplotypes from unrelated individ-
using both SHAPEIT2 (18) and MVNCALL (25). First, we uals (i.e., removing the children of family trios and duos).
focused on the 765 sequenced individuals collected by Ma- Three individuals in the 1000 Genomes data carry CNVs
lariaGEN partners. We removed variants within the region that are not singletons in the overall dataset but are private
of segmental duplication (here taken as chr4:144.7- to that individual within the 1000 Genomes data (HG01986,
145.07Mb) from the reference panel, and replaced these with carrying DEL4; HG02554, carrying DUP4, and HG02585,
CNV genotype calls, to form a single file with genotypes at carrying DUP5). Of these, we noted in particular that
the CNVs and flanking SNPs and indels. SHAPEIT2 requires HG02554 appears to have a switch error in the 1000 Ge-
each variant to be assigned a single genomic position. For nomes (explaining clustering of opposite haplotypes with
each CNV longer than 10 kb we included the genotypes for other DUP4 haplotypes on either side of the region; fig. S9).
that variant at the start-, mid- and endpoints of the CNV; Because our approach phases variants in the 1000 Genomes
for variants less than 10 kb we used the midpoint. We then separately and these are singletons in that dataset, we also
ran SHAPEIT2 with parameters as for SNP/indel phasing to excluded these three individuals from the panel used for
produce phased genotype calls, treating each CNV as a sepa- imputation.
rate, biallelic variant. Next, to phase CNVs into the 1000
Genomes Project individuals, we extracted the Omni 2.5M Cross-validation of CNV imputation in the reference panel
sites with allele frequency > 1% from the 1000 Genomes Pro- To evaluate how well CNVs are likely to be imputed in our
ject phased haplotypes and removed the region of segmental GWAS dataset, we performed a cross-validation experiment
duplication. We used MVNCALL to phase each non- using the African reference panel individuals as follows. For
singleton CNV into this scaffold, again placing each CNV each individual, we removed the individual and his/her fam-
greater than 10 kb in length at its start-, mid-, and end posi- ily members (if present) from the phased reference panel
tions. haplotypes to form a subsetted panel. We also extracted
We assessed accuracy of phasing by considering patterns genotype calls for that individual at variants on the Illumina
of LD between CNVs and variants in the left and right flank- Omni 2.5M array from the reference panel genotypes, ex-
ing regions (figs. S9 and S10). We noted high LD between cluding variants within the glycophorin region. We used
some CNVs and variants to the left of the region, including these genotypes and the subsetted panel to re-impute CNVs
for DEL1, DEL2, DUP1 and DUP4. LD estimated from for that individual.
phased haplotypes captured most of the correlation between We evaluated CNV re-imputation by computing the cor-
genotypes across the region, as expected in an outbred pop- relation between HMM-based genotype calls and genotype
ulation if haplotype phase is accurate. The small deviations dosages from the re-imputation (fig. S11) and by direct com-
observed may be due to the presence of a small number of parison of HMM and re-imputed calls. We note that DEL4
switch errors, or potentially to population substructure. carriers were imputed with some confidence as carrying
DUP1, consistent with the shared haplotype for these vari-
Haplotype-based curation and re-phasing of CNVs ants and the higher frequency of DUP1. Given the function-
Some singleton CNVs have similar copy number profiles to ally distinct nature of these variants, this affects
more common CNVs in the HMM-based calls (22) and after interpretation of imputed DUP1 genotypes.
Among CNVs >10 kb in length, we noted little variation this analysis.
between the three imputation locations (leftmost break-
point, midpoint, and rightmost breakpoint of the CNV), ex- Association with gene content
cept for DUP4, where the right endpoint had slightly higher To test for association with overall copy number of each
imputation performance (fig. S11). For all analyses presented glycophorin gene, we used the imputed genotype probabili-
in this paper we refer to the midpoint imputation of each ties and the copy number profile of non-singleton CNVs
CNV. (Fig. 1B) to compute the expected number of copies of each
gene in each GWAS sample. Since overlap of some CNVs
Imputation of CNVs with genes is partial, we measured copy number separately
We used the combined panel to impute CNVs into the three at the transcription start and end sites. We also computed
GWAS datasets. Imputation settings were as described the expected number of hybrid genes as formed by DUP2,
above. To ensure imputation was based on flanking SNPs, DUP4 and DUP8. We tested for association with each copy
we removed SNPs within the glycophorin region from the number measure by fitting a logistic regression model

genotype data before imputation. We evaluated imputation across the three populations, including the genic dosage and
performance in the GWAS data by comparing the overall five principal components in each population. Overall genic
expected allele frequency against the IMPUTE info metric copy number was significantly associated with severe malar-
and another metric of confidence in imputed CNV call ia status (e.g., P=3x10−7 for dosage at transcription start
probabilities, the proportion of expected frequency of CNV sites), as was the expected number of hybrid genes. Howev-
heterozygote or homozygote that is due to genotypes with at er, these effects were well explained by the effect of DUP4 (P
least 90% probability (Fig. 3D and fig. S12, A and B). We also > 0.14 after conditioning on the expected dosage of DUP4).
compared the expected frequency in control samples with We note that several of these predictors are strongly corre-
the frequency in the geographically nearest reference panel lated with DUP4 dosage; this analysis could not distinguish
population (fig. S12C). between an effect of DUP4 and that of copy number of
GYPE, copy number of the GYPA transcription end site, or
Analysis of association at CNVs the total copy number of hybrid genes (P > 0.13 for either
Association with severe malaria effect in a joint model).
We tested for association with each CNV in each population We also specifically tested whether the number of delet-
using logistic regression including five principal compo- ed copies of GYPB, or the presence of one or two deleted
nents as covariates, and computed both fixed-effect and copies, was associated with severe malaria status, but saw
Bayesian meta-analyses as described above for SNPs and no evidence of association (P>0.48).
indels. To directly estimate the effect of heterozygote and
homozygote genotypes, we modified SNPTEST to fit the lo- Population genetic analysis
gistic regression model with a separate parameter for heter- Frequency differentiation
ozygote and homozygote genotypes in a missing data We used estimated minor allele frequencies (MAF) at both
likelihood framework that integrates over imputation uncer- typed and imputed variants from (14) for the 2,490 popula-
tainty. To assess evidence for association after accounting tion controls from the Gambia and 1,498 population con-
for DUP4, we used QCTOOL v2 trols from Kenya to investigate the extent to which the
(http://www.well.ox.ac.uk/~gav/qctool_v2/) to extract the observed frequency difference of DUP4 is extreme relative to
additive and heterozygote imputed dosages of DUP4 for other variants genome-wide. For this comparison, we in-
each individual, and repeated the association test and meta- cluded all autosomal variants having IMPUTE info >0.75
analysis at SNPs, indels and CNVs conditioning on these and estimated frequency ≥0.5% in at least one of the popu-
dosages (Fig. 3B and fig. S13). lations (14,973,426 variants in total). We binned variants
into 0.5% MAF bins and noted the frequency estimates for
Association with clinical subtypes DUP4 (fKenya=0.0895 and fGambia=0.0003) and, for compari-
Severe malaria-affected children in our data are recorded as son, the sickle cell anemia-causing allele (rs334:T;
either having cerebral malaria (CM), severe malarial anemia MAF=0.0853 in Kenya and 0.0766 in the Gambia based on
(SMA), or other nonspecific severe malaria phenotype direct Sequenom typing of this SNP in these samples (65)).
(OTHER). To assess the association of DUP4 with these To quantify the extent to which DUP4 is an outlier, we
subphenotypes, we fit a multinomial logistic regression computed two empirical P-values based on the marginal
model with these outcome levels using population controls distributions. Specifically, we computed PGambia|Kenya as the
as a baseline (table S7). A small number of individuals are proportion of variants with MAF less than or equal to fGambia
annotated as both CM and SMA and were excluded from in the Gambia, among all variants with MAF within 1% of
fKenya in Kenya (empirical PGambia|Kenya<1.2x10−6; 0 of 831,956 generated 300 bp PE sequence data on Illumina MiSeq at
variants in this bin). Similarly, we computed PKenya|Gambia as the High Throughput Genomics core at the Wellcome Trust
the proportion of variants with MAF equal to or greater Centre for Human Genetics at the University of Oxford. We
than fKenya in Kenya, among all variants with MAF within 1% mapped these reads to the same GRCh37 human reference
of fGambia in the Gambia (empirical PKenya|Gambia=1.7x10−3; 2,851 (hs37d5.fa) with bwa mem, yielding 13x genomic coverage.
of 1,710,922 variants in this bin). For all re-mapped bam files, we marked duplicates with Pi-
We note that the computation of PGambia|Kenya is sensitive card MarkDuplicates and excluded duplicate reads from
to the frequency of DUP4 in the Gambia, which may be un- further analysis.
derestimated due to poor imputation performance. To ac- We then used samtools to pull out read pairs where both
count for this we recomputed PGambia|Kenya assuming 1% reads had a primary alignment to the glycophorin region
frequency in the Gambia (adjusted empirical PGam- with MQ≥1 and an absolute insert size ≥1 kb. For the 300 bp
bia|Kenya=5x10 ) and refer to this value in the main text.
−3
data, we allowed one of the reads in the pair to be mapped
to the glycophorin region with MQ=0. Across samples, there

Haplotype homozygosity were 434 such read pairs. We grouped read pairs where
To assess haplotype homozygosity, we first used SHAPEIT2 both ends were mapped within 1 kb of each other and iden-
to phase imputed CNV genotypes onto haplotypes defined tified those near the HMM-inferred breakpoints.
by directly-typed SNPs in the region chr4:139.5-150.5Mb, To view how uniquely the discordant read pairs matched
excluding SNPs in the glycophorin region. We specified 400 each of the three possible homologous positions in the seg-
selected and 200 random copying states (–states 400,– mental duplication, we used the mapped position and the
states-random 200), an effective population size of 17,469 as cigar string to place each read into the multiple sequence
recommended for African populations, and included refer- alignment and then identified positions in the read with a
ence panel haplotypes to inform phasing. EHH (27) was match or mismatch to each of the three aligned reference
then computed around DUP4 using the rehh R package. We sequences using custom scripts in R (e.g., fig. S16).
used a custom script to compute an unstandardized inte-
grated haplotype score (uiHS) (26), using recombination Molecular assays and Sanger sequencing
rate estimates from the HapMap combined recombination Briefly, a 4.1 kb fragment spanning the GYPB-A hybrid
map (66). breakpoint (located between exons 4 and 5) was amplified
To generate a distribution of uiHS at SNPs with a similar by PCR using primers designed against GYPB and GYPA
frequency to DUP4, we computed uiHS at those genotyped sequences (fig. S17 and table S10). In practice, it is difficult
SNPs (14) where the human ancestral allele could be in- to design specific primers due to the high homology and the
ferred using the six primate EPO alignments assay amplifies both GYPA and GYPB-A hybrid sequences.
(ftp://ftp.1000genomes.ebi.ac.uk/vol1/ftp/phase1/analysis_re To separate these, we identified a restriction enzyme site
sults/supporting/ancestral_alignments/), and the derived (BlpI [5′…GC/TNAGC…3′]) that cleaves the GYPA se-
allele frequency was in the 2% frequency bin centered quence but not that of the hybrid. PCR products were di-
around fKenya (0.0795-0.0995). We computed an empirical P- gested and then separated on an agarose gel. We excised the
value as the proportion of SNPs with uiHS less than or 4.1 kb band and obtained Sanger sequence of the region
equal to that observed at DUP4 (P=0.0119; 996 of the 83,419 around the putative breakpoint (figs. S18 and S19). A full
variants in this bin). We note that the exclusion of the gly- description of the design and protocols is given in (22).
cophorin region from the estimate at DUP4 is likely to be
conservative, since in effect it adds a constant term equal to Sequencing and copy number analysis of a serologically-
the recombination length of the removed interval (approxi- typed Dantu+ individual
mately 0.15 cM) to both the numerator and denominator of We obtained DNA from the International Blood Group Ref-
the statistic. erence Laboratory in Bristol, UK from an archived reference
sample that had been serologically typed as Dantu+ (NE
Resolution of the structure of DUP4 type). This sample was collected in 1992 and the individual
Discordant read pair analysis was originally from Natal, South Africa. The DNA was se-
We began by remapping reads from each of the nine hetero- quenced with 150 bp PE reads on Illumina HiSeq 4000 by
zygous DUP4 carriers genome-wide using bwa mem, which the High Throughput Genomics core at the Wellcome Trust
has better performance for longer reads (67). Because even Centre for Human Genetics at the University of Oxford.
longer reads should facilitate more confident mapping Reads were mapped to the same GRCh37 human reference
around the breakpoints, we also obtained DNA from Coriell genome (hs37d5.fa) with bwa mem, yielding 18x coverage.
for HG02554, the 1000 Genomes sample carrying DUP4, and To generate CNV calls, we ran our HMM method on this
individual alone, without using the window-specific factors. Spencer, K. A. Rockett, C. C. A. Spencer, V. Cornelius, D. J. Conway, T. N.
Williams, T. Taylor, D. P. Kwiatkowski, D. J. Conway, K. A. Bojang, O. Doumbo, M.
A. Thera, D. Modiano, S. B. Sirima, M. D. Wilson, K. A. Koram, T. Agbenyega, E.
Simulations of DUP4 formation Achidi, T. N. Williams, K. Marsh, H. Reyburn, C. Drakeley, E. Riley, T. Taylor, M.
To determine possible routes to formation of DUP4, we im- Molyneux, M. Jallow, K. A. Bojang, D. J. Conway, M. Pinder, O. Doumbo, M. A.
plemented a computer program in C++ to iteratively gener- Thera, O. B. Toure, S. Konate, S. Sissoko, E. C. Bougouma, V. D. Mangano, D.
ate structural rearrangements via unequal crossing over, Modiano, S. B. Sirima, L. N. Amenga-Etego, A. K. Ghansah, A. V. O. Hodgson, K.
A. Koram, M. D. Wilson, T. Agbenyega, D. Ansong, A. Enimil, J. Evans, E. Achidi, T.
allowing breakpoints to occur at any of the six locations ob- O. Apinjoh, A. Macharia, K. Marsh, C. M. Ndila, C. Newton, N. Peshu, S. Uyoga, T.
served for DUP4 with no constraint based on homology. In N. Williams, C. Drakeley, A. Manjurano, H. Reyburn, E. Riley, D. Kachala, M.
brief, we encode the reference haplotype as a string of seven Molyneux, V. Nyirongo, T. Taylor, K. A. Rockett, K. Kivinen, D. Mead, E. Drury, S.
segments delineated by the coverage breakpoints (i.e., as the Auburn, S. G. Campino, B. MacInnis, J. Stalker, E. Gray, C. Hubbart, A. E. Jeffreys,
K. Rowlands, A. Mendy, R. Craik, K. Fitzpatrick, S. Molloy, L. Hart, R. Hutton, A.
string 0123456; Fig. 6A). We ran the program through three Kerasidou, K. J. Johnson, V. Cornelius, Malaria Genomic Epidemiology Network,
generations of events, where the first generation produced A novel locus of resistance to severe malaria in a region of ancient balancing
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Fukushima, D. R. Macer, E. Suda, C. N. Rotimi, C. A. Adebamowo, I. Ajayi, T. ACKNOWLEDGMENTS
Aniagwu, P. A. Marshall, C. Nkwodimmah, C. D. M. Royal, M. F. Leppert, M. Dixon, We thank all the study participants and the members of the MalariaGEN Consortial
A. Peiffer, R. Qiu, A. Kent, K. Kato, N. Niikawa, I. F. Adewole, B. M. Knoppers, M. Projects 1 and 3 (https://www.malariagen.net/projects). These MalariaGEN
W. Foster, E. W. Clayton, J. Watkin, R. A. Gibbs, J. W. Belmont, D. Muzny, L. Consortial Projects were supported by the Wellcome Trust (WT077383/Z/05/Z)
Nazareth, E. Sodergren, G. M. Weinstock, D. A. Wheeler, I. Yakub, S. B. Gabriel, R. and the Bill & Melinda Gates Foundation through the Foundations of the National
C. Onofrio, D. J. Richter, L. Ziaugra, B. W. Birren, M. J. Daly, D. Altshuler, R. K. Institutes of Health (566) as part of the Grand Challenges in Global Health
Wilson, L. L. Fulton, J. Rogers, J. Burton, N. P. Carter, C. M. Clee, M. Griffiths, M. Initiative. The Resource Centre for Genomic Epidemiology of Malaria is
C. Jones, K. McLay, R. W. Plumb, M. T. Ross, S. K. Sims, D. L. Willey, Z. Chen, H. supported by the Wellcome Trust (090770/Z/09/Z). This research was
Han, L. Kang, M. Godbout, J. C. Wallenburg, P. L’Archevêque, G. Bellemare, K. supported by the Medical Research Council (G0600718; G0600230;
Saeki, H. Wang, D. An, H. Fu, Q. Li, Z. Wang, R. Wang, A. L. Holden, L. D. Brooks, J. MR/M006212/1). Chris C.A. Spencer was supported by a Wellcome Trust Career
E. McEwen, M. S. Guyer, V. O. Wang, J. L. Peterson, M. Shi, J. Spiegel, L. M. Sung, Development Fellowship (097364/Z/11/Z). The Wellcome Trust also provides
L. F. Zacharia, F. S. Collins, K. Kennedy, R. Jamieson, J. Stewart, International core awards to The Wellcome Trust Centre for Human Genetics
HapMap Consortium, A second generation human haplotype map of over 3.1 (090532/Z/09/Z) and the Wellcome Trust Sanger Institute (098051). Eric
million SNPs. Nature 449, 851–861 (2007). doi:10.1038/nature06258 Medline Achidi received partial funding from the European Community's Seventh
67. H. Li, Aligning sequence reads, clone sequences and assembly contigs with BWA- Framework Programme (FP7/2007-2013) under grant agreement N° 242095 –
MEM. arXiv:1303.3997v2 [q-bio.GN] (26 May 2013). EVIMalaR and the Central African Network for Tuberculosis, HIV/AIDS and
68. A. G. Hinch, A. Tandon, N. Patterson, Y. Song, N. Rohland, C. D. Palmer, G. K. Malaria (CANTAM) funded by the European and Developing Countries Clinical
Chen, K. Wang, S. G. Buxbaum, E. L. Akylbekova, M. C. Aldrich, C. B. Ambrosone, Trials Partnership (EDCTP). Thomas N. Williams is funded by Senior Fellowships
from the Wellcome Trust (076934/Z/05/Z and 091758/Z/10/Z) and through
the European Community's Seventh Framework Programme (FP7/2007-2013)
under grant agreement N° 242095 – EVIMalaR. The KEMRI-Wellcome Trust
Programme is funded through core support from the Wellcome Trust. Carolyne
Ndila is supported through a strategic award to the KEMRI-Wellcome Trust
Programme by the Wellcome Trust (084538). Tanzania/KCMC/JMP received
funding from MRC grant number (G9901439). The Malawi-Liverpool-Wellcome
Trust Clinical Research Programme (MLW) is a Major Overseas Programme of
the Wellcome Trust. Malcolm Molyneux was funded by a Wellcome Trust
Research Leave Fellowship. Vysaul Nyirongo was supported on the MLW core
grant. V.D.M. was funded by Istituto Pasteur-Fondazione Cenci Bolognetti,
BioMalPar and Evimalar (European Community FP6,FP7). We thank the staff of
the WTSI Sample Logistics, Genotyping, Sequencing and Informatics facilities
and the WTCHG High-Throughput Genomics core for their contributions to
sample handling and generation and processing of sequence data. Author
contributions: Writing group: E.M.L., G.B., G.B.J.B., K.A.R., C.C.A.S., D.P.K.; Data

analysis: E.M.L., G.B., G.B.J.B., Q.S.L., D.P.K., G.M.C., K.A.R., C.C.A.S.; Study site
lead investigators: K.A.B., D.J.C., D.M., S.B.S., E.A., K.M., T.N.W., C.D., H.R., E.R.,
M.M., T.T.; Sample collection and curation: K.A.B., D.J.C., M.J., F.S-J., E.C.B.,
V.D.M., D.M., S.B.S., E.A., T.O.A., K.M., C.M.N., N.P., T.N.W., C.D., A.M., H.R., E.R.,
D.K., M.M., V.N., T.T.; Serotyped sample curation and handling: N.T., L.T., S.G.;
Sample processing, sequencing, data management and project coordination:
G.B., K.K, E.D, J.S, V.C., C.H., A.E.J., K.R., K.A.R.; Experimental design and
targeted assay development: E.M.L., G.B., C.H., A.E.J., K.R., K.A.R., C.C.A.S.,
D.P.K. The regional combined reference panel and association summary
statistics, multiple sequence alignment of the glycophorin segmental
duplication, and Sanger sequences are available at
https://www.malariagen.net/resource/23. The sequence data generated for
HG02554 and the Dantu+ (NE type) individual have been deposited in the
European Nucleotide Archive under study accession number PRJEB20081.
Accompanying scripts are available at
https://github.com/malariagen/glycophorin_cnvs.
SUPPLEMENTARY MATERIALS
www.sciencemag.org/cgi/content/full/science.aam6393/DC1
Supplementary Text
Figs. S1 to S24
Tables S1 to S11
References (70–79)
21 December 2016; accepted 8 May 2017

Published online 18 May 2017
10.1126/science.aam6393
Fig. 1. Copy number variants in the glycophorin region. (A) Mappability in the glycophorin region. The
number of mappable sites and the maximum pairwise identity between homologous locations in the
segmental duplication are shown in 1600 bp windows. Pairwise identity is inferred from a multiple
sequence alignment, with mean of 0.96 indicated with a red dashed line. The locations of protein-coding
genes and the segmentally duplicated units are indicated below, with sequences of at least 100 bp that are
unique to one or two out of the three segmentally duplicated units marked in black and gray, respectively.
(B) Sequence coverage in 1600 bp windows and copy number for non-singleton CNVs. Black dashes show
the mean normalized sequence coverage across heterozygous individuals not carrying another CNV. Only
windows input to the HMM are shown. The inferred CNVs are indicated with deletion in yellow, duplication
in light blue, and triplication in dark blue. A horizontal gray line indicates the expected coverage without
copy number variation and blue vertical lines mark the locations of the three genes. (C) Positions of
breakpoints, colored as the variant names in (B) and shaded by whether the variant has a single pair of
homologous breakpoints, a single pair of non-homologous breakpoints, or is a multi-segment CNV. The
recombination rate from LD-based recombination maps (66, 68) and locations of DSB hotspots (24) are
annotated below.
Fig. 2. Frequency of CNVs in the sampled populations. Populations
are grouped on the basis of geographical proximity; abbreviations can
be found in tables S1 and S3.
Fig. 3. Evidence of association. (A) The evidence for association at SNPs, short indels, and CNVs across the
glycophorin region. P-values are computed under an additive model of association using meta-analysis across
the three African populations included in our study. Points are colored by LD with DUP4 in east African
reference panel populations. Directly typed SNPs are denoted with black plusses, and CNVs with diamonds.
Black triangles represent SNPs where the association signal was previously reported and replicated in further
samples. CNV copy number profiles, as in Fig. 1B, and protein-coding genes are shown below. (B) Comparison
of unconditional association test P-values (y axis, as in panel (A)), and after additionally conditioning on
genotypes at DUP4 (x axis). (C) The evidence for association at DUP4. Colored circles and text show the
estimated allele frequency of DUP4 in population controls and severe malaria cases. To the right is the odds
ratio and 95% confidence interval for DUP4 heterozygotes (diamonds) and homozygotes (circles) relative to
non-carriers. The bottom two rows represent effect sizes computed by fixed-effect meta-analysis. Sample
sizes (number of controls/number of cases) are denoted to the left. (D) Comparison of IMPUTE info score and
expected imputed allele frequency for the four annotated CNVs.
Fig. 4. The effect of DUP4 on SNP array intensities. (A) Normalized Illumina Omni 2.5M intensity values at
selected SNP assays across the glycophorin region for reference panel individuals (top row; N=367 individuals
from Burkina Faso, Cameroon, and Tanzania) and Kenyan GWAS individuals (second row, N = 3,142). Blue and
yellow points represent individuals heterozygous or homozygous for DUP4 respectively, as determined by the
HMM in reference panel individuals and by imputation in Kenya (genotypes with posterior probability at least
0.75). Arrows denote the mapping location of these SNPs. (B) The copy number profile of DUP4. (C) Position
of the glycophorin genes and exons.
Fig. 5. DUP4 frequency and haplotype homozygosity. (A) The empirical joint allele-frequency spectrum for
population controls in the Gambia and Kenya, in 0.5% frequency bins between 0 and 20%. The frequencies of
DUP4 and rs334 are highlighted. Histograms show the frequency in the Gambia of SNPs in the DUP4
frequency bin in Kenya (8.5-9%, top) and the frequency in Kenya of SNPs in the DUP4 frequency bin in the
Gambia (0-0.5%, right). (B) The estimated frequency of DUP4 in east African populations, shown with 95%
confidence intervals and the number of haplotypes sampled. Estimates are from population controls in the
GWAS or from the HMM genotype calls in the reference panel (daggers). The dotted vertical line denotes the
overall frequency of DUP4 in Kenyan controls. (C) Extended haplotype homozygosity (EHH) computed
outward from the glycophorin region for DUP4 haplotypes and non-DUP4 haplotypes in Kenya, after excluding
other variants within the glycophorin region. Below, the empirical distribution of unstandardized iHS for all
typed SNPs within 1% frequency of DUP4 in Kenyan controls, with empirical P-value annotated. (D) The 272
haplotypes imputed to carry DUP4 and a random sample of 272 non-DUP4 haplotypes in Kenya. Haplotypes in
each panel are clustered on 1 Mb extending in either direction from the glycophorin region, which is shaded in
blue. The bar on the left depicts the population for each haplotype with colors as in panel (B). Recombination
rate estimates (66, 68) are shown above and protein-coding genes below.
Fig. 6. The structure of DUP4. (A) Discordant read pairs mapped near DUP4 copy number changes. Colored
arrows represent read pairs from DUP4 carriers, with paired reads shown on the same horizontal line and the
direction of the arrows depicting the strand and position as mapped to the human reference sequence. The
number of such read pairs and distinct carriers is given to the left. A schematic of the reference sequence is
below with colors indicating the segmentally duplicated units. Brackets delineate segments with different copy
number in DUP4, numbered and labeled with their length to the nearest kb. (B) The structure of DUP4, inferred
by connecting sequence at breakpoints based on sequence homology and discordant read pairs. Arrows
depict the concordant positions of the read pairs in (A) on this structure, and the order of reference segments
is shown below. Inset: detail of the inferred GYPB-A hybrid genes, indicating the positions of discordant read
pairs (arrows), PCR primers (vertical red lines) and the resulting product (horizontal red line). (C) Normalized
coverage in 1600 bp windows (black) and HMM path (red) for a DUP4 carrier (top) and for an individual
serotyped as Dantu+ (NE type; bottom), on the same x axis as (A). (D) Protein sequences of GYPA, GYPB, and
the Dantu hybrid within the cell membrane depicting the extracellular, transmembrane, and intracellular
domains as visualized with protter (69).
Resistance to malaria through structural variation of red blood cell
invasion receptors
Ellen M. Leffler, Gavin Band, George B. J. Busby, Katja Kivinen,
Quang Si Le, Geraldine M. Clarke, Kalifa A. Bojang, David J. Conway,
Muminatou Jallow, Fatoumatta Sisay-Joof, Edith C. Bougouma,
Valentina D. Mangano, David Modiano, Sodiomon B. Sirima, Eric
Achidi, Tobias O. Apinjoh, Kevin Marsh, Carolyne M. Ndila, Norbert
Peshu, Thomas N. Williams, Chris Drakeley, Alphaxard Manjurano,
Hugh Reyburn, Eleanor Riley, David Kachala, Malcolm Molyneux,
Vysaul Nyirongo, Terrie Taylor, Nicole Thornton, Louise Tilley, Shane
Grimsley, Eleanor Drury, Jim Stalker, Victoria Cornelius, Christina
Hubbart, Anna E. Jeffreys, Kate Rowlands, Kirk A. Rockett, Chris C. A.
Spencer, Dominic P. Kwiatkowski and Malaria Genomic Epidemiology
Network (May 18, 2017)
published online May 18, 2017
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Resistance to malaria through structural variation of red

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21 December 2016; accepted 8 May 2017

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