Alternatively spliced transcripts and novel pseudogenes of the Plasmodium falciparum resistance-associated locus pfcrt detected in East African malaria patients.


Gadalla, NB; Malmberg, M; Adam, I; Oguike, MC; Beshir, K; Elzaki, SE; Mukhtar, I; Gadalla, AA; Warhurst, DC; Ngasala, B; Mårtensson, A; El-Sayed, BB; Gil, JP; Sutherland, CJ; (2015) Alternatively spliced transcripts and novel pseudogenes of the Plasmodium falciparum resistance-associated locus pfcrt detected in East African malaria patients. The Journal of antimicrobial chemotherapy, 70 (1). pp. 116-23. ISSN 0305-7453 DOI: 10.1093/jac/dku358

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Abstract

OBJECTIVES: Polymorphisms in the lysosomal transporter encoded by the pfcrt gene directly impact on Plasmodium falciparum susceptibility to aminoquinolines. The Lys76Thr mutation is the critical change conferring chloroquine resistance in vitro and in vivo, but always occurs with additional non-synonymous changes in the pfcrt coding sequence. We sought to better describe pfcrt polymorphisms distal to codon 76.<br/> METHODS: Small-volume samples (≤ 500 μL) of parasite-infected blood collected directly from malaria patients presenting for treatment in Sudan and Tanzania were immediately preserved for RNA extraction. The pfcrt locus was amplified from cDNA preparations by nested PCR and sequenced directly to derive full-length mRNA sequences.<br/> RESULTS: In one of two sites in Sudan, two patients were found with an unorthodox spliced form of pfcrt mRNA in which two exons were skipped, but it was not possible to test for the presence of the putative protein products of these aberrant transcripts. Genomic DNA sequencing from dried blood spots collected in parallel confirmed the presence of spliced pfcrt pseudogenes in a minority of parasite isolates. Full-length cDNA from conventionally spliced mRNA molecules in all study sites demonstrated the existence of a variety of pfcrt haplotypes in East Africa, and thus provides evidence of intragenic recombination.<br/> CONCLUSIONS: The presence of pseudogenes, although unlikely to have any direct public health impact, may confound results obtained from simple genotyping methods that consider only codon 76 and the adjacent residues of pfcrt.<br/>

Item Type: Article
Faculty and Department: Faculty of Infectious and Tropical Diseases > Dept of Immunology and Infection
Faculty of Infectious and Tropical Diseases > Dept of Pathogen Molecular Biology
Research Centre: Antimicrobial Resistance Centre (AMR)
Malaria Centre
PubMed ID: 25253286
Web of Science ID: 350210800017
URI: http://researchonline.lshtm.ac.uk/id/eprint/1987648

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