Hypoxanthine-guanine phosphoribosyltransferase reporter gene mutation for analysis of in vivo clonal amplification in patients with HTLV type 1-associated Myelopathy/Tropical spastic paraparesis.
We tested a surrogate selection approach utilizing mutation at a reporter gene [hypoxanthine-guanine phosphoribosyltransferase (hprt)] as a probe for in vivo cell division, for detection of clonal T cell expansion in human T lymphotropic (HTLV-1) carriers. Peripheral blood samples from HTLV-1-infected individuals with HTLV-1-associated myelopathy/tropical spastic paraparesis (HAM/TSP) were tested to determine the hprt mutant frequency (Mf). Wild-type and hprt mutant T cell clones were isolated, and clonal identity determined by multiplex PCR and DNA sequencing of T cell receptor (TCR) variable region beta-chain (TCR BV) and third complementarity determining regions (CDR3). Seven samples from HAM/TSP patients were tested, and Mfs were within the normal range for adults (mean 11.3 x 10(-6), max 22.4 x 10(-6), min 5.6 x 10(-6)). The frequency of HTLV-1 infection in wild-type and hprt mutant T cells from HAM/TSP patients was determined to identify enrichment in the mutant fraction of cells. This analysis was performed on 196 isolates from 6 individuals with HAM/TSP. In each case, there is enrichment for virally infected cells in the hprt mutant fraction of isolates. Ten mutant and eight wild-type isolates from sample LS42A (Mf 8.4 x 10(-6)) were tested for clonality by TCR BV PCR and sequencing. Of the 10 hprt mutants, there were two in vivo-expanded clones (four isolates with two identical TCRs, or 80% unique TCR sequences). These studies may provide new insights into the precise mechanism of HTLV-1 leukemogenesis, and aid in the study of mutator phenotypes generated by a combination of Tax-mediated in vivo expansion and mutagenesis.
Albertini
R J
RJ
Toxicology Laboratory, University of Vermont, Burlington, Vermont, USA.
Ardell
S K
SK
Judice
S A
SA
Jacobson
S
S
Allegretta
M
M
D002999:Clone Cells; D017930:Genes, Reporter; D015368:Human T-lymphotropic virus 1 / Q000472:pathogenicity*; D006801:Humans; D007041:Hypoxanthine Phosphoribosyltransferase / Q000235:genetics* / Q000378:metabolism; D009154:Mutation; D015493:Paraparesis, Tropical Spastic / Q000503:physiopathology* / Q000821:virology; D016133:Polymerase Chain Reaction / Q000379:methods; D016693:Receptors, Antigen, T-Cell, alpha-beta / Q000235:genetics / Q000378:metabolism; D013601:T-Lymphocytes / Q000201:enzymology / Q000502:physiology* / Q000821:virology*; D016662:Virus Integration
D016693:Receptors, Antigen, T-Cell, alpha-beta; D007041:Hypoxanthine Phosphoribosyltransferase