Detection of partial cDNA sequences differentially expressed in patients with myelodysplasia.
The most common chromosomal aberrations in myelodysplastic syndromes (MDS) are complete or partial loss of chromosomes 5 and 7, and trisomy 8. To identify genes important in the pathogenesis of this disease that could be associated with these gross chromosomal defects, we have employed the differential display PCR (DDPCR) procedure developed by Liang and Pardee. This method allows simultaneous comparison of several cDNA sources for the presence of differentially expressed genes. Polymorphonuclear cells (PMNs) from two MDS patients, containing a 5q deletion or a trisomy 8, and three healthy controls were used. Initial screening resulted in the identification of five and three partial cDNA sequences, respectively that were either differentially expressed in both patient samples or in individual patients, as compared with the controls. The authenticity of aberrant expression was verified by reanalyzing the same primer combinations on newly prepared cDNA. Differential expression of the three remaining fragments was subsequently checked on a larger panel of MDS patients, using amplicon-specific primer sets. These were obtained by cloning and sequencing of the fragments. For one partial cDNA (DC3), the original expression pattern, i.e., decreased expression in individual MDS patients, was confirmed. These results demonstrate the utility of the DDPCR procedure to isolate differentially expressed sequences in primary patient samples where the availability of cells is a limiting factor.
Kroef
M J
MJ
Laboratory of Experimental Hematology, Leiden University Hospital, The Netherlands.
Jansen
J H
JH
Willemze
R
R
Landegent
J E
JE
D000328:Adult; D000368:Aged; D001483:Base Sequence; D002869:Chromosome Aberrations; D002895:Chromosomes, Human, Pair 5; D002897:Chromosomes, Human, Pair 7; D002898:Chromosomes, Human, Pair 8; D018076:DNA, Complementary / Q000235:genetics; D005260:Female; D006801:Humans; D008297:Male; D008969:Molecular Sequence Data; D009190:Myelodysplastic Syndromes / Q000235:genetics*; D016133:Polymerase Chain Reaction
D018076:DNA, Complementary