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 g ene [hypoxanthine-guanine phosphoribosyltransferase (hprt)] as a probe for ill vivo cell division, For detection of clonal T cell expansion in human T lymphotropic (HTLV-1) carriers. Peripheral blood samples from HTLV-1-infec ted individuals with HTLV-1-associated myelopathy/tropical spastic parapare sis (HAM/TSP) were tested to determine the hprt mutant frequency (Mf), Wild -type and hprt mutant T cell clones were isolated, and clonal identity dete rmined by multiplex PCR and DNA sequencing of T cell receptor (TCR) variabl e region beta -chain (TCR BV) and third complementarity determining regions (CDR3). Seven samples from HAM/TSP patients were tested, and Mfs were with in 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 muta nt T cells from HAM/TSP patients was determined to identify enrichment in t he mutant fraction of cells. This analysis was performed on 196 isolates fr om 6 individuals with HAM/TSP, In each case, there is enrichment for virall y infected cells in the hprt mutant fraction of isolates, Ten mutant and ei ght 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 prec ise mechanism of HTLV-1 leukemogenesis, and aid in the study of mutator phe notypes generated by a combination of Tax-mediated in vivo expansion and mu tagenesis.