The malaria-protective human glycophorin structural variant DUP4 shows somatic mosaicism and association with hemoglobin levels

Abstract

Glycophorin A and glycophorin B are red blood cell surface proteins that are both receptors for the parasite Plasmodium falciparum, which is the principal cause of malaria in sub-Saharan Africa. DUP4 is a complex structural genomic variant that carries extra copies of a glycophorin A - glycophorin B fusion gene, and has a dramatic effect on malaria risk by reducing the risk of severe malaria by up to 40%. Using fiber-FISH and Illumina sequencing, we validate the structural arrangement of the glycophorin locus in the DUP4 variant, and reveal somatic variation in copy number of the glycophorin A-glycophorin B fusion gene. By developing a simple, specific, PCR-based assay for DUP4 we show the DUP4 variant reaches a frequency of 13% in a village in south-eastern Tanzania. We genotype a substantial proportion of that village and demonstrate an association of DUP4 genotype with hemoglobin levels, a phenotype related to malaria, using a family-based association test. Taken together, we show that DUP4 is a complex structural variant that may be susceptible to somatic variation, and show that it is associated with a malarial-related phenotype in a non-hospitalized population.Significance statementPrevious work has identified a human complex genomic structural variant called DUP4, which includes two novel glycophorin A-glycophorin B fusion genes, is associated with a profound protection against severe malaria. In this study, we present data showing the molecular basis of this complex variant. We also show evidence of somatic variation in the copy number of the fusion genes. We develop a simple robust assay for this variant and demonstrate that DUP4 is at an appreciable population frequency in Tanzania and that it is associated with higher hemoglobin levels in a malaria-endemic village. We suggest that DUP4 is therefore protective against malarial anemia.

Evolutionary and functional analysis of RBMY1 gene copy number variation on the human Y chromosome

“…RP11-95B23 was labelled with biotin 16-dUTP; P1 with digoxigenin 11-dUTP; P2 with Cy5xx-dUTP; P3 with DNP 11-dUTP; P4 with digoxigenin 11-dUTP and DNP 11-dUTP; and P5 with DNP 11-dUTP and Cy5xx-dUTP (all from Jena Bioscience, Jena, Germany). Molecular combing fibres preparation and fibre-FISH experiments were carried out as described previously (30). The order of the probes on single-molecule DNA fibres allowed us to determine both the copy number and orientation of RBMY1 genes and neighbouring genomic regions and to identify other structural changes, for example, inversions.…”

Chromatin organization modulates the origin of heritable structural variations in human genome

“…Direct observation of association of NAHR with DSBs, as we showed, is unlikely to be observed because of the limitations of existing experimental approaches to identify DSBs in repeats flanking NAHR breakpoints. The predominant occurrence of NAHR deletions from errors in DNA repair implies that most of them happen either in germ cells prior to meiosis (mosaic in parent and de novo in children) or during early development (mosaic in children) (40–43).…”

Genotyping complex structural variation at the malaria‐associated human glycophorin locus using a PCR‐based strategy

“…2018). One of the association signals was shown to be due to a complex structural variant, called DUP4, involving the human glycophorin gene cluster on chromosome 4 (Algady et al., 2018; Leffler et al., 2017).…”

“…Some of these variants have been shown to be responsible for particular blood group phenotypes – for example the DUP4 variant codes for the Dantu blood group (Leffler et al., 2017), and homozygosity for either DEL1 and DEL2 variants results in the U– blood group as part of the S‐s‐U– phenotype observed in Africans (Gassner et al., 2020). These structural variants can be identified and genotyped using sequence read depth of mapped high‐throughput sequencing reads (Algady et al., 2018; Leffler et al., 2017). However, the ability to rapidly genotype the different structural variants at this locus using simple polymerase chain reaction (PCR) approaches would have a number of benefits.…”

“…Previously we published a PCR‐based approach to genotype the malaria‐protective DUP4 variant using a paralogue‐specific PCR targeting a junction unique to that variant. We used this PCR approach to genotype a Tanzanian cohort and demonstrate association of the DUP4 variant with haemoglobin levels in peripheral blood (Algady et al., 2018). In this report, we describe a simple triplex paralogue ratio test (PRT) system based on a single PCR of 10‐ng DNA that can distinguish other structural variants of the glycophorin gene cluster.…”