title: Independent origins of loss-of-function mutations conferring oxamniquine resistance in a Brazilian schistosome population.
abstract: Molecular surveillance provides a powerful approach to monitoring the resistance status of parasite populations in the field and for understanding resistance evolution. Oxamniquine was used to treat Brazilian schistosomiasis patients (mid-1970s to mid-2000s) and several cases of parasite infections resistant to treatment were recorded. The gene underlying resistance (SmSULT-OR) encodes a sulfotransferase required for intracellular drug activation. Resistance has a recessive basis and occurs when both SmSULT-OR alleles encode for defective proteins. Here we examine SmSULT-OR sequence variation in a natural schistosome population in Brazil ∼40years after the first use of this drug. We sequenced SmSULT-OR from 189 individual miracidia (1-11 per patient) recovered from 49 patients, and tested proteins expressed from putative resistance alleles for their ability to activate oxamniquine. We found nine mutations (four non-synonymous single nucleotide polymorphisms, three non-coding single nucleotide polymorphisms and two indels). Both mutations (p.E142del and p.C35R) identified previously were recovered in this field population. We also found two additional mutations (a splice site variant and 1bp coding insertion) predicted to encode non-functional truncated proteins. Two additional substitutions (p.G206V, p.N215Y) tested had no impact on oxamniquine activation. Three results are of particular interest: (i) we recovered the p.E142del mutation from the field: this same deletion is responsible for resistance in an oxamniquine selected laboratory parasite population; (ii) frequencies of resistance alleles are extremely low (0.27-0.8%), perhaps due to fitness costs associated with carriage of these alleles; (iii) that four independent resistant alleles were found is consistent with the idea that multiple mutations can generate loss-of-function alleles.
authors.affiliation: Texas Biomedical Research Institute, Department of Genetics, P.O. Box 760549, San Antonio, TX 78245-0549, USA. Electronic address: fcheval@txbiomed.org.
authors.forename: Frédéric D
authors.initials: FD
authors.lastname: Chevalier
authors.affiliation: Texas Biomedical Research Institute, Department of Genetics, P.O. Box 760549, San Antonio, TX 78245-0549, USA.
authors.forename: Winka
authors.initials: W
authors.lastname: Le Clec'h
authors.affiliation: Texas Biomedical Research Institute, Department of Genetics, P.O. Box 760549, San Antonio, TX 78245-0549, USA.
authors.forename: Nina
authors.initials: N
authors.lastname: Eng
authors.affiliation: Department of Biochemistry, University of Texas Health Science Center, San Antonio, TX 78229-3900, USA; Department of Pathology, University of Texas Health Science Center, San Antonio, TX 78229-3900, USA.
authors.forename: Anastasia R
authors.initials: AR
authors.lastname: Rugel
authors.affiliation: Centro de Pesquisas René Rachou, Fundação Oswaldo Cruz, Av. Augusto de Lima, 1715, Belo Horizonte, Minas Gerais 30190-002, Brazil.
authors.forename: Rafael Ramiro de
authors.initials: RR
authors.lastname: Assis
authors.affiliation: Centro de Pesquisas René Rachou, Fundação Oswaldo Cruz, Av. Augusto de Lima, 1715, Belo Horizonte, Minas Gerais 30190-002, Brazil; Vale Institute of Technology, Rua Boaventura da Silva, 955, Belém, Pará 66055-090, Brazil.
authors.forename: Guilherme
authors.initials: G
authors.lastname: Oliveira
authors.affiliation: Department of Biochemistry, University of Texas Health Science Center, San Antonio, TX 78229-3900, USA; Department of Pathology, University of Texas Health Science Center, San Antonio, TX 78229-3900, USA.
authors.forename: Stephen P
authors.initials: SP
authors.lastname: Holloway
authors.affiliation: Department of Biochemistry, University of Texas Health Science Center, San Antonio, TX 78229-3900, USA; Department of Pathology, University of Texas Health Science Center, San Antonio, TX 78229-3900, USA.
authors.forename: Xiaohang
authors.initials: X
authors.lastname: Cao
authors.affiliation: Department of Biochemistry, University of Texas Health Science Center, San Antonio, TX 78229-3900, USA; Department of Pathology, University of Texas Health Science Center, San Antonio, TX 78229-3900, USA; Department of Veterans Affairs, South Texas Veterans Health Care System, San Antonio, TX 78229, USA.
authors.forename: P John
authors.initials: PJ
authors.lastname: Hart
authors.affiliation: Department of Biochemistry, University of Texas Health Science Center, San Antonio, TX 78229-3900, USA; Department of Pathology, University of Texas Health Science Center, San Antonio, TX 78229-3900, USA.
authors.forename: Philip T
authors.initials: PT
authors.lastname: LoVerde
authors.affiliation: Texas Biomedical Research Institute, Department of Genetics, P.O. Box 760549, San Antonio, TX 78245-0549, USA. Electronic address: tanderso@txbiomed.org.
authors.forename: Timothy J C
authors.initials: TJ
authors.lastname: Anderson
mesh_terms: D000483:Alleles; D000818:Animals; D001938:Brazil; D002648:Child; D002675:Child, Preschool; D004351:Drug Resistance / Q000235:genetics; D005091:Exons / Q000235:genetics; D005243:Feces / Q000469:parasitology; D005787:Gene Frequency; D055106:Genome-Wide Association Study; D006801:Humans; D007223:Infant; D008968:Molecular Conformation; D009154:Mutation; D010073:Oxamniquine / Q000494:pharmacology*; D016133:Polymerase Chain Reaction; D020641:Polymorphism, Single Nucleotide; D012550:Schistosoma mansoni / Q000187:drug effects* / Q000235:genetics; D012552:Schistosomiasis / Q000469:parasitology*; D012556:Schistosomicides / Q000494:pharmacology*
chemical_list: D012556:Schistosomicides; D010073:Oxamniquine
keywords: Biochemical assay; Loss-of-function; Oxamniquine resistance; Schistosoma mansoni; Soft selective event; Sulfotransferase