title: Alternatively spliced transcripts and novel pseudogenes of the Plasmodium falciparum resistance-associated locus pfcrt detected in East African malaria patients.
abstract: OBJECTIVE
  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.
authors.affiliation: Department of Immunology and Infection, London School of Hygiene and Tropical Medicine, London, UK Department of Epidemiology, Tropical Medicine Research Institute, Khartoum, Sudan.
authors.forename: Nahla B
authors.initials: NB
authors.lastname: Gadalla
authors.affiliation: Malaria Research, Infectious Diseases Unit, Department of Medicine Solna, Karolinska Institutet, Stockholm, Sweden.
authors.forename: Maja
authors.initials: M
authors.lastname: Malmberg
authors.affiliation: Faculty of Medicine, University of Khartoum, Khartoum, Sudan.
authors.forename: Ishag
authors.initials: I
authors.lastname: Adam
authors.affiliation: Department of Immunology and Infection, London School of Hygiene and Tropical Medicine, London, UK.
authors.forename: Mary C
authors.initials: MC
authors.lastname: Oguike
authors.affiliation: Department of Immunology and Infection, London School of Hygiene and Tropical Medicine, London, UK.
authors.forename: Khalid
authors.initials: K
authors.lastname: Beshir
authors.affiliation: Department of Epidemiology, Tropical Medicine Research Institute, Khartoum, Sudan.
authors.forename: Salah-Eldin
authors.initials: SE
authors.lastname: Elzaki
authors.affiliation: National Health Laboratory, Federal Ministry of Health, Khartoum, Sudan.
authors.forename: Izdihar
authors.initials: I
authors.lastname: Mukhtar
authors.affiliation: Department of Epidemiology, Tropical Medicine Research Institute, Khartoum, Sudan.
authors.forename: Amal A
authors.initials: AA
authors.lastname: Gadalla
authors.affiliation: Department of Pathogen Molecular Biology, London School of Hygiene and Tropical Medicine, London, UK.
authors.forename: David C
authors.initials: DC
authors.lastname: Warhurst
authors.affiliation: Department of Parasitology, Muhimbili University of Health and Allied Sciences, Dar es Salaam, Tanzania.
authors.forename: Billy
authors.initials: B
authors.lastname: Ngasala
authors.affiliation: Malaria Research, Infectious Diseases Unit, Department of Medicine Solna, Karolinska Institutet, Stockholm, Sweden Global Health (IHCAR), Department of Public Health Sciences, Karolinska Institutet, Stockholm, Sweden.
authors.forename: Andreas
authors.initials: A
authors.lastname: Mårtensson
authors.affiliation: Department of Epidemiology, Tropical Medicine Research Institute, Khartoum, Sudan.
authors.forename: Badria B
authors.initials: BB
authors.lastname: El-Sayed
authors.affiliation: Drug Resistance Unit, Division of Pharmacogenetics, Department of Physiology and Pharmacology, Karolinska Institutet, Stockholm, Sweden Drug Resistance and Pharmacogenetics, Center for Biodiversity, Functional and Integrative Genomics, Faculdade de Ciências, Universidade de Lisboa, Lisbon, Portugal The Harpur College of Arts and Sciences, Binghamton University, The State University of New York, Binghamton, NY, USA.
authors.forename: J Pedro
authors.initials: JP
authors.lastname: Gil
authors.affiliation: Department of Immunology and Infection, London School of Hygiene and Tropical Medicine, London, UK colin.sutherland@lshtm.ac.uk.
authors.forename: Colin J
authors.initials: CJ
authors.lastname: Sutherland
mesh_terms: D000328:Adult; D017398:Alternative Splicing; D000595:Amino Acid Sequence; D002648:Child; D002675:Child, Preschool; D018076:DNA, Complementary / Q000235:genetics / Q000302:isolation & purification; D005260:Female; D006801:Humans; D007223:Infant; D016778:Malaria, Falciparum / Q000469:parasitology*; D008297:Male; D026901:Membrane Transport Proteins / Q000235:genetics*; D008954:Models, Biological; D008958:Models, Molecular; D008969:Molecular Sequence Data; D010963:Plasmodium falciparum / Q000235:genetics* / Q000302:isolation & purification; D016133:Polymerase Chain Reaction; D011110:Polymorphism, Genetic; D011487:Protein Conformation; D015800:Protozoan Proteins / Q000235:genetics*; D011544:Pseudogenes; D012322:RNA Precursors / Q000378:metabolism*; D016053:RNA, Protozoan / Q000235:genetics / Q000302:isolation & purification; D017422:Sequence Analysis, DNA; D013397:Sudan; D013636:Tanzania
chemical_list: D018076:DNA, Complementary; D026901:Membrane Transport Proteins; C416369:PfCRT protein, Plasmodium falciparum; D015800:Protozoan Proteins; D012322:RNA Precursors; D016053:RNA, Protozoan
keywords: alternative splicing; chloroquine resistance transporters; malaria