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

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.

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.

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.

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.

Keywords: alternative splicing; chloroquine resistance transporters; malaria.

Figure 1.

Amplification strategy of the pfcrt gene employed in this study. (a) Amplification of the PF3D7_0709000 gDNA of the pfcrt gene, exons (black boxes), introns (solid grey lines) and UTR (grey boxes with black fill). Fragment 1 (solid black line) and fragment 2 (solid grey line) are indicated beneath the gene map. PCR primer pairs used to amplify the whole gene (in both gDNA and cDNA) are indicated by horizontal arrows. Primers NG6_REV and NG7_FOR were used to sequence the UTR. Locations of Q10K and D24Y polymorphisms are indicated with arrows. Intron deletion region in gDNA is indicated. (b) cDNA amplified from the pfcrt gene. Upper transcript map is the reference PF3D7_0709000 pfcrt cDNA. Lower map is the variant pfcrt cDNA amplified in sample 24. Black rectangles represent the sequenced transcribed exons. Asterisks represent the non-transcribed (spliced) exons. Grey rectangles represent the putative mRNA downstream from codon 220; this region was not sequenced from cDNA but inferred from gDNA sequence.

Figure 2.

(a) Ethidium-bromide-stained agarose gels of RT–PCR products of N-terminus of pfcrt mRNA. Lane 1, Hyper Ladder IV (100 bp); lanes 2–24, clinical samples. Arrows indicate samples 18 and 24 in which two RT–PCR products were observed, the expected 827 bp and the 521 bp truncated product. (b) Predicted amino acid sequence of abnormally spliced pfcrt mRNA from patient 24. The amino acid sequence of exons 1 and 2 is shown. The arrow indicates the splice site at the end of exon 2, followed by the sequence at the beginning of exon 5.

Figure 3.

Clustal alignment of predicted amino acid sequences of nine distinct pfcrt haplotypes identified by sequencing cDNA generated directly from peripheral blood parasite mRNA. Contributing data were derived from two New Halfa isolates with identical splice variant transcripts (Sp1), 26 full-length cDNA sequences from New Halfa and Gedaref, Sudan, and 35 full-length cDNA sequences from Fukayosi, Tanzania. Haplotype labels indicate predicted chloroquine resistance status (CQS, susceptible; CQR, resistant). Vertical gaps in the alignment indicate invariant sequence tracts not included. Labels under the alignment refer to variant positions (shaded).

Figure 4.

Frequency of pfcrt haplotypes in cDNA from isolates in eastern Sudan (n = 49) and Tanzania (n = 40). Number of observations of each haplotype defined at codons 74–76 and 220 (amino-terminus and full-length data) and 271, 326 and 371 (full-length data only). Data from isolates with multiple genotypes at the pfcrt locus were not included.

Figure 5.

A possible linear arrangement of transmembrane regions in the full-length CRT protein (a) and putative truncated CRT protein encoded by alternative-spliced pfcrt mRNA from Sudanese malaria patients (b). Transmembrane regions are depicted as rectangles; blue denotes presence in putative truncated form of CRT and white denotes presence only in full-length CRT. Freeform blue line denotes extramembrane polypeptide tracts in the cytosol or inside the digestive vacuole lumen. Transverse yellow bars denote site of splicing event and loss of contiguous nucleotide/amino acid sequence. Polymorphic residues are depicted by yellow or orange (codons 76 and 220) dots. Amino terminal polymorphisms at codons 10 and 24 are not shown.