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A strategy for single nucleotide polymorphism analysis of chimerism for somatic cell therapy.

Abstract：

BACKGROUND AIMS: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.

journal_name

Cytotherapy

journal_title

Cytotherapy

authors

Chen X,Hofmann TJ,Otsuru S,Jethva R,Lind C,Monos D,Horwitz EM

doi

10.3109/14653241003774029

subject

Has Abstract

pub_date

2010-12-01 00:00:00

pages

1035-43

issue

8

eissn

1465-3249

issn

1477-2566

pii

S1465-3249(10)70472-2

journal_volume

12

pub_type

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