Submitted on September 17, 2007
Accepted on October 26, 2007
Interference of globin genes with biomarker discovery for allograft rejection in peripheral blood samples
Li Li1, Lihua Ying1, Maarten Naesens2, Wenzhong Xiao3, Tara Sigdel1, Sue Hsieh1, Jon Martin1, Rong Chen4, Kang Liu1, Michael N. Mindrinos5, Ron Davis3, and Minnie Sarwal1*
1 Pediatrics, Stanford University, Stanford, California, United States
2 Pediatrics, Stanford University, Stanford, California, United States; Nephrology and Renal Transplantation, University Hospitals Leuven, Leuven, Belgium
3 Stanford Genome Technology Center, Stanford University, Stanford, California, United States
4 Stanford Medical Informatics, Stanford University, 94305, California, United States
5 Stanford Genome Technology Center, Stanford University School of Medicine, Stanford, California, United States
* To whom correspondence should be addressed. E-mail: msarwal{at}stanford.edu.
Microarray technology is a powerful tool in the discovery of new biomarkers for disease. After solid organ transplantation, where the detection of rejection is usually made on invasive biopsies, it could be hypothesized that non-invasive transcriptional profiling of peripheral blood will reveal rejection-specific expression patterns from circulating immune cells. However, in kidney transplant rejection, the analysis of gene expression data in whole blood has proven difficult for detecting significant genes specific for acute graft rejection. Previous studies have demonstrated that the abundance of globin genes in whole blood may mask the underlying biological differences between whole blood samples. In the present study, we compared the gene expression profiles of peripheral blood of 9 stable renal allograft recipients with 7 matched patients having an ongoing acute renal transplant rejection, using four different protocols of preparation, amplification and synthesis of cRNA or cDNA and hybridization on the Affymetrix platform. We demonstrated that the globin reduction method is not sufficient to unmask clinically relevant rejection specific transcriptome profiles in whole blood. Applying an additional mathematical depletion of the globin genes improves the efficacy of globin reduction but cannot remove the confounding influence of globin gene hybridization. Sampling of peripheral blood leukocytes alone, without the confounding influence of globin mRNA, provides sensitive and specific peripheral signatures for graft rejection, many of these signals overlapping with rejection driven tissue (kidney) specific signatures from matched biopsies. Similar applications may lie for array-based biomarker discovery for other diseases associated with changes in leukocyte trafficking, activation or function.
