Analyzing Plasmodium falciparum erythrocyte membrane protein 1 gene expression by a next generation sequencing based method

Plasmodium falciparum is responsible for most cases of severe malaria and causes >1 million deaths every year. The particular virulence of this Plasmodium species is highly associated with the expression of certain members of the Plasmodium falciparum erythrocyte membrane protein 1(PfEMP1) family, encoded by ~60 highly variable 'var' genes per haploid genome.

PfEMP1 is exported to the surface of infected erythrocytes and is thought to be fundamental to immune evasion by adhesion to host and parasite factors. The highly variable nature has constituted a roadblock in var expression studies aimed at identifying PfEMP1 features associated with high virulence.

Here we present the first effective method for sequence analysis of var genes expressed in field samples: a sequential PCR and next generation sequencing based technique applied on expressed var sequence tags and subsequently on long range PCR amplicons of the expressed vars. The results obtained with this method supports quantitative PCR data showing PfEMP1 of the group A and domain cassettes 8 and 13 types being expressed at particularly high levels in severe childhood malaria.