High resolution melting analysis of deletion/insertion polymorphisms: A new method for the detection and quantification of mixed chimerism in allogeneic stem cell transplantation.
Increasing mixed chimerism after allogeneic stem cell transplantation has been associated with a high risk of relapse and probable graft failure in patient with hematological malignancies as well as non-malignant conditions. We evaluated a new method for chimerism detection, based on the quantitative High Resolution Melting Analysis (HRMA) of deletion/insertion polymorphisms (DIPs). The study consisted in the selection of a panel of DIPs, all generating genotype-specific melting curves, and in the use of samples containing opposite molecular species (homozygous INS/INS and DEL/DEL) mixed in different percentages to create a standard curve for each polymorphism. The detection of mixed chimerism with the HRMA attained a sensitivity of <1%, as well as good accuracy and precision with Percent Errors and Coefficients of Variation not exceeding 30% in reconstruction experiments with DNA mixtures. The present approach provides accurate and precise estimates of mixed chimerism and makes the method open to evaluation for its use in clinical practice.
Department of Clinical and Experimental Biomedical Sciences, Clinical Physiopathology Unit, University of Florence, Viale Gaetano Pieraccini, 6, I-50139 Florence, Italy.
Chiara
C
Gerini
Maurizio
M
Dal Canto
Berardino
B
Porfirio
D004247:DNA / Q000032:analysis*; D014644:Genetic Variation; D015894:Genome, Human; D060005:Genotyping Techniques; D018380:Hematopoietic Stem Cell Transplantation; D006801:Humans; D054643:INDEL Mutation; D018895:Microsatellite Repeats; D016133:Polymerase Chain Reaction; D011110:Polymorphism, Genetic; D012680:Sensitivity and Specificity; D044366:Transition Temperature; D018183:Transplantation Chimera / Q000235:genetics*; D014184:Transplantation, Homologous
D004247:DNA
CV; DEL; DIPs; Deletion/insertion polymorphisms; HRMA; High resolution melting; INS; Mixed chimerism; Q-D-HRMA; SCT; SD; SNPs; coefficient of variation; deletion; deletion/insertion polymorphisms; double stranded DNA; dsDNA; high resolution melting analysis; insertion; quantitative differential HRMA; single nucleotide polymorphisms; standard deviation; stem cell transplantation