To the Editor,
The diagnosis of graft rejection is one of the fundamental aspects of caring for patients with heart transplantation. Endomyocardial biopsy (EMB) has important limitations, as it is an invasive procedure, with variable interpretation and limited sensitivity.1-3 Thus, it would be of interest to have a noninvasive diagnostic method that facilitates follow-up of patients with heart transplantation.
The gene expression patterns of peripheral blood lymphocytes may represent a highly sensitive, highly specific tool that would identify patients who present rejection and avoid the inconvenience of EMB. We present the preliminary results of a study performed to determine a set of candidate genes for use in a noninvasive molecular expression test with peripheral blood to detect cardiac graft rejection.
The experiments to analyze gene expression were conducted with 6 peripheral blood samples taken from heart transplant recipients on the day of EMB. Rejection was diagnosed according to the International Society of Heart and Lung Transplantation (ISHLT) classification.4 Three of the samples corresponded to patients with rejection (ISHLT Grade ≥2 ISHLT rejection) and three to patients without rejection (ISHLT Grade 0 rejection). The blood samples were collected in tubes containing a solution for RNA stabilization (PAXgene Blood RNA Tubes, Qiagen) and stored at 80oC until the time of analysis.
Genetic tests were performed with Affymetrix microarrays (GeneChip Human Genome U133 Bonus 2.0 Array). Differences in the expression of 262 genes were found when using the GeneSpring comparison algorithm. Two-dimensional hierarchical cluster allows verification of the presence of differential expression patterns consisting of samples with rejection compared to the control samples. Of these 262 genes, Table lists the candidate genes.
Five metabolic pathways with at least 2 genes of modified expression level were found. These metabolic pathways were: apoptosis (4 genes), MAP-kinase pathway (5 genes), B cell receptor signaling pathway (2 genes), hematopoietic cell lineage (2 genes), signaling pathway, and T-cell receptor signaling pathway (2 genes).
The results are consistent with the hypothesis of immune system stimulation implicated in the rejection of transplanted organs. Horwitz et al5 used a similar method, finding that CFLAR, a gene implicated in the apoptotic pathway, was altered in patients with transplant rejection. Similar results were obtained by Deng et al,6 who found alterations in the expression of genes related to the MAP-kinase pathway, apoptosis, and T-cell receptors.
In conclusion, this exploratory study in heart transplant patients found differences in gene expression between patients with and without rejection. Of the five pathways showing genes with altered expression, the apoptotic and MAP-kinase pathways appeared to have the highest number of affected genes. The low number of study samples limits the interpretation of the findings. Nevertheless, we consider that the results of this preliminary work justify the expectations placed in this promising strategy, which may contribute to the development of a molecular test to monitor immunosuppressive therapy in patients who have undergone transplantation.
This study was funded by a grant from the Heart Failure Section of the Sociedad Española de Cardiología (Spanish Society of Cardiology), 2003 (Title: "Análisis de expresión génica en células mononucleares circulantes mediante Real-time RT-PCR en pacientes con trasplante cardiaco. Estudio de su posible relación con el rechazo cardiaco") [Analysis of gene expression in circulating mononuclear cells by real-time PCR (RT-PCR) in heart transplant recipients. Study of its possible relationship to cardiac rejection]) and by the Red Nacional de Investigación Cardiovascular, RECAVA C03/01.