C. Kapsalis1, P. J. Matheson1, J. Hata2, J. Shepherd1, L. M. Bond1, J. W. Smith1, C. D. Downard1 1University Of Louisville,Surgery,Louisville, KY, USA 2Kosair Children’s Hospital,Pathology,Louisville, KY, USA
Introduction: NEC affects premature infants and is a leading gastrointestinal cause of infant death. Formula-fed infants have a 6 to 10X greater risk of developing NEC compared to breast fed infants. Relaxin, a hormone of pregnancy found at high concentrations in maternal milk compared to that of blood, might play an important role in preventing the intestinal sequelae in patients at risk for NEC. Thus, decreased expression of relaxin-family receptor (RXFP) or relaxin protein (RLN) might be a critical event in the development of the disease. We hypothesized that gene expression of gut RXFP might be decreased in infants with clinically diagnosed NEC.
Methods: IRB approval was obtained to collect paraffin-embedded gut tissue samples from patients with pathologist-graded NEC and Controls. All study personal were blinded to patient groups. Qiagen RNeasy FFPE was performed to isolate total RNA from tissue samples. mRNA was quantified and analyzed for Quality Control (QC). Total cDNA was obtained using a NuGen Ovation RNA Amplification V2 kit. Biotin-labeled aRNA was created for analysis on an Illumina HumanHT-12 v4 BeadChip to measure the entire human genome in each sample. Ingenuity Pathway software was used to analyze the results.
Results: Twenty-four (24) tissue samples were obtained and processed yielding an average of 1,230 ng mRNA per sample. QC demonstrated a 5S marker peak, which is consistent with mRNA extracted from paraffin-embedded samples. The Illumina HumanHT-12 v4 BeadChip demonstrated increased relaxin 4 gene expression (1.54x compared to Control*P<0.05). Other relaxin receptor and relaxin isoforms were not significantly different.
Conclusion: QC and mRNA levels from FFPE extraction were consistent with expected results and biotin-labeled RNA for the BeadChip was sufficient for the genome evaluation. Ingenuity Pathway analysis showed RXFP-4 upregulation suggesting altered relaxin family gene expression in NEC. We have previously shown that RXFP protein levels are altered as early as 48 hours prior to histologic changes of NEC. These results support those prior animal studies.