A strategy for single nucleotide polymorphism analysis of chimerism for somatic cell therapy.
OBJECTIVE
Chimerism is an important outcome measure in hematopoietic cell transplantation as well as somatic cell therapy. Commonly used methods to estimate chimerism are restricted by either gender or inefficient sensitivity. In principle, real-time polymerase chain reaction (PCR)-based assays can be used to assess single nucleotide polymorphisms (SNP), which are a vast resource of molecular markers, and such assays demonstrate a substantially higher sensitivity (0.001%), but the specificity is unclear because of a low-level signal from mismatched sequences.


METHODS
In this study, we cloned 14 pairs of SNP selected from the SNP HapMap database and examined the specificity and sensitivity of their detection by real-time PCR using two primer/fluorescent probe pairs to allow genotyping of the two possible variant alleles. Clinical donor-recipient pairs from 18 families were used to explore the efficacy of using SNP assays to measure chimerism.


RESULTS
We found that the polymorphic nucleotide influences the ability to distinguish the signal generated by the target and mismatched sequences. Moreover, the specific fluorescent reporter probe can affect the difference in signal intensity between the target and mismatched sequences. Real-time PCR SNP assays can attain a sensitivity of 0.1-0.5% with 100% specificity. When comparing possible clinical donor-recipient pairs, we found an average 3.3 out of 14 SNP were informative.


CONCLUSIONS
By optimal selection of the polymorphic sequences and fluorescent reporter, the real-time PCR SNP assay is superior to the short-tandem repeat chimerism assay and broadly applicable. This strategy may be applied in future clinical trials of bone marrow cell therapy.
Chen
Xiaohua
X
Department of Pediatrics/Oncology, The Children's Hospital of Philadelphia and University of Pennsylvania School of Medicine, Philadelphia, Pennsylvania, USA.
Hofmann
Ted J
TJ
Otsuru
Satoru
S
Jethva
Reena
R
Lind
Curt
C
Monos
Dimitri
D
Horwitz
Edwin M
EM
D000293:Adolescent; D000328:Adult; D002648:Child; D002675:Child, Preschool; D046528:Chimerism; D004252:DNA Mutational Analysis; D005260:Female; D017930:Genes, Reporter / Q000235:genetics; D018380:Hematopoietic Stem Cell Transplantation; D006648:Histocompatibility / Q000235:genetics; D006801:Humans; D007223:Infant; D007231:Infant, Newborn; D008297:Male; D008875:Middle Aged; D020641:Polymorphism, Single Nucleotide; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods; D012680:Sensitivity and Specificity