Evaluation of a gene expression biomarker to identify operationally tolerant liver transplant recipients: the LITMUS trial.

LITMUS was a single-centre, Phase 2a study designed to investigate whether the gene biomarker FGL2/IFNG previously reported for the identification of tolerance in murine models could identify operationally tolerant liver transplant recipients. Multiplex RT-PCR was used to amplify eight immunoregulatory genes in peripheral blood mononuclear cells (PBMC) from 69 adult liver transplant recipients. Patients with PBMC FGL2/IFNG ≥ 1 and a normal liver biopsy underwent immunosuppression (IS) withdrawal. The primary end point was the development of operational tolerance. Secondary end points included correlation of tolerance with allograft gene expression and immune cell markers. Twenty-eight of 69 patients (38%) were positive for the PBMC tolerance biomarker and 23 proceeded to IS withdrawal. Nine of the 23 patients had abnormal baseline liver biopsies and were excluded. Of the 14 patients with normal biopsies, eight (57%) have achieved operational tolerance and are off IS (range 12-57 months). Additional studies revealed that all of the tolerant patients and only one non-tolerant patient had a liver gene ratio of FOXP3/IFNG ≥ 1 prior to IS withdrawal. Increased CD4+ T regulatory T cells were detected both in PBMC and livers of tolerant patients following IS withdrawal. Higher expression of SELE (gene for E-selectin) and lower expression of genes associated with inflammatory responses (GZMB, CIITA, UBD, LSP1, and CXCL9) were observed in the pre-withdrawal liver biopsies of tolerant patients by RNA sequencing. These results suggest that measurement of PBMC FGL2/IFNG may enrich for the identification of operationally tolerant liver transplant patients, especially when combined with intragraft measurement of FOXP3/IFNG. Clinical Trial Registration: ClinicalTrials.gov (LITMUS: NCT02541916).

Multicapillary gel electrophoresis based analysis of genetic variants in the WFS1 gene.

The WFS1 gene is one of the thoroughly investigated targets in diabetes research, variants of the gene were suggested to be the genetic components of the common forms (type 1 and type 2) of diabetes. Our project focused on the analysis of polymorphisms (rs4689388, rs148797429, rs4273545) localized in the WFS1 promoter region. Although submarine gel electrophoresis based approaches were also employed in the genetic tests, it was demonstrated that multicapillary electrophoresis offers a state of the art approach for reliable high-throughput SNP and VNTR analysis. Association studies were carried out in a case-control setup. Luciferase reporter assay was employed to test the effect of the investigated loci on the activity of gene expression in vitro. Significant association could be demonstrated between all three polymorphisms and type 2 diabetes in both allele- and genotype-wise settings even using Bonferroni correction. It is notable; however, that the three loci were in strong linkage disequilibrium, thus the observed associations cannot be considered as separate effects. Molecular analyses showed that the rs4273545 GT SNP played a role in the regulation of transcription in vitro. However, this effect took place only in the presence of the region including the rs148797429 site, although this latter locus did not have its own impact on the regulation of gene expression. The paper provides genotyping protocols readily applicable in any multiplex SNP and VNTR analyses, moreover confirms and extends previous results about the role of WFS1 polymorphisms in the genetic risk of diabetes mellitus.

ArrayPlex SA: a turn-key automated gene expression target preparation system.

Automated target preparation for gene expression analysis eliminates the time-consuming and labor-intensive manual process, which is error prone and diverts scientists from value added activities. Target preparation methods were developed, on the fully integrated ArrayPlex SA system, based on the field proven Biomek FX, which streamlined the target preparation procedure allowing up to 96 samples to be processed in less than 36 h. The process is comprised of three functional methods, cDNA synthesis, in vitro transcription, and fragmentation, providing the users with the ability to consolidate runs for optimal use of instrument time and minimize reagent cost. Starting with sets of eight tRNA samples, the cDNA synthesis method synthesizes the first and second strand DNA followed by a cDNA clean-up step using an ultrafiltration plate. The in vitro transcription method then amplifies and biotin-labels the cDNA to cRNA in 6 h at 37 degrees C, and purifies the product using a solid support extraction plate. Finally, the fragmentation method quantifies the cRNA, adjusts the concentration to the recommended 0.625 microg/mL and fragments the cRNA prior to an off-line hybridization. Universal human reference RNA with concentration ranging from 1 to 7.5 microg were prepared on the ArrayPlex SA, compared against a manual method and scanned using Affymetrix human genome U133 Plus 2.0 array GeneChip cartridges. Nested analysis of variance was then performed to identify sources of variability between the automated and manual methods.