Abstract
Structural variation in the human genome can affect risk of disease. An example is a complex structural variant of the human glycophorin gene cluster, called DUP4, which is associated with a clinically significant level of protection against severe malaria. The human glycophorin gene cluster harbours at least 23 distinct structural variants, and accurate genotyping of this complex structural variation remains a challenge. Here, we use a polymerase chain reaction-based strategy to genotype structural variation at the human glycophorin gene cluster, including the alleles responsible for the U- blood group. We validate our approach, based on a triplex paralogue ratio test, on publically available samples from the 1000 Genomes project. We then genotype 574 individuals from a longitudinal birth cohort (Tori-Bossito cohort) using small amounts of DNA at low cost. Our approach readily identifies known deletions and duplications, and can potentially identify novel variants for further analysis. It will allow exploration of genetic variation at the glycophorin locus, and investigation of its relationship with malaria, in large sample sets at minimal cost, using standard molecular biology equipment.
Highlights
The malaria parasite Plasmodium falciparum is a major cause of childhood mortality in Africa (Liu et al.2016; Snow et al 2017; Whitty and Ansah 2019)
Genome-wide association studies have identified multiple alleles that increase protection from severe malaria symptoms; understanding the mechanistic basis of this protection, the precise phenotype affected by the allelic variation, and the evolutionary history of the protective alleles is an important area of current research
In this study we designed three cis-paralogue ratio test (PRT), each one targeting a different region of the glycophorin repeat, with test and reference distinguished on the bases of product size by electrophoresis
Summary
The malaria parasite Plasmodium falciparum is a major cause of childhood mortality in Africa (Liu et al.2016; Snow et al 2017; Whitty and Ansah 2019). 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 (Leffler et al 2017; Algady et al 2018). There is extensive structural variation at this locus resulting in at least 8 distinct deletions of one or two repeat units, 13 distinct duplications of one or two repeat units, and two more complex variants (Leffler et al 2017)((Louzada et al 2019)). It would allow access to large cohorts with limited DNA, where whole genome amplification is both prohibitively costly and known to introduce bias in copy number measurement (Pugh et al 2008; Veal et al 2012) It would allow laboratories in resourcelimited situations to genotype glycophorin structural variation, as only standard molecular genetic laboratory equipment, and an instrument suitable for DNA fragment analysis, are needed. In this report we describe a simple triplex paralogue ratio test system based on a single PCR of 10ng DNA that can distinguish other structural variants of the glycophorin gene cluster
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