Diminished expression of the type II receptor for TGFβ (TGFβRII) in T lymphocytes from patients with Sezary syndrome is not due to mutations in the receptor's poly-A tract: Limitations of the standard RT-PCR in cDNA sequence analysis of homopolymeric base stretches

Peripheral blood lymphocytes from patients with Sezary syndrome (SzS) frequently demonstrate decreased surface expression of transforming growth factor β receptor H (TGFβRII). The mechanism of this low TGFβRII expression remains unknown. Because mutations within the poly-A tract of the TGFβRII sequence (nucleotides 709-718) were shown to result in diminished TGFβRII expression in other types of malignant tumors, we examined the sequence of the TGFβRII poly-A tract in two SzS-derived cell lines and in peripheral blood SzS cells from 17 SzS patients and 4 control, healthy individuals using DNA sequencing and single-stranded conformation polymorphism (SSCP) analysis. A standard bidirectional, automated sequence analysis of the RT-PCR-generated cDNA TGFβRII fragment showed a heterogenous population of the normal length, 10-, with admixed, shortened, 9-base poly-A stretches. Surprisingly, this mixture was present not only in the cells from 5 SzS patients and 2 SzS cell lines, but also in cells from 2 healthy control individuals. Importantly, the proportion of the shortened, 9-base fragments was markedly reduced or practically eliminated when the procedure was modified by usage of high-fidelity DNA polymerase, labeled primers and/or cloned RT-PCR products, which indicates that the presence of the shortened, 9-base fragments represented a procedural phenomenon rather than a true deletional mutation within an allele of the TGFβRII gene. Accordingly, SSCP analysis of genomic DNA did not reveal any mutations within the poly-A tract-containing region. These results indicate that a mechanism different from mutations in the polyadenine tract underlies the diminished TGFβRII expression in SzS cells and that the results of an unmodified, direct sequence analysis of homopolymeric base streaches in RT-PCR-derived cDNA should be interpreted with caution.