Diminished expression of the type II receptor for TGFbeta (TGFbetaRII) 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 beta receptor II (TGFbetaRII). The mechanism of this low TGFbetaRII expression remains unknown. Because mutations within the poly-A tract of the TGFbetaRII sequence (nucleotides 709-718) were shown to result in diminished TGFbetaRII expression in other types of malignant tumors, we examined the sequence of the TGFbetaRII 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 TGFbetaRII 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 TGFbetaRII 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 TGFbetaRII 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.
Zhang
Qian
Q
Department of Pathology and Laboratory Medicine, University of Pennsylvania Medical Center, Philadelphia, PA 19104, USA.
Capocasale
Renold J
RJ
Fox
Floyd E
FE
Bedian
Vahe
V
Vonderheid
Eric C
EC
Rook
Alain
A
Moore
Jonni S
JS
Nowell
Peter C
PC
Haines
Dale S
DS
Wasik
Mariusz A
MA
D001483:Base Sequence; D018076:DNA, Complementary / Q000737:chemistry*; D006801:Humans; D009154:Mutation; D011061:Poly A / Q000235:genetics*; D018807:Polymorphism, Single-Stranded Conformational; D017346:Protein Serine-Threonine Kinases; D000077294:Receptor, Transforming Growth Factor-beta Type II; D018125:Receptors, Transforming Growth Factor beta / Q000032:analysis* / Q000235:genetics*; D020133:Reverse Transcriptase Polymerase Chain Reaction; D012751:Sezary Syndrome / Q000276:immunology*; D013601:T-Lymphocytes / Q000737:chemistry*; D014407:Tumor Cells, Cultured
D018076:DNA, Complementary; D018125:Receptors, Transforming Growth Factor beta; D011061:Poly A; D017346:Protein Serine-Threonine Kinases; D000077294:Receptor, Transforming Growth Factor-beta Type II