TYROSINE KINASE GENE POLYMORPHISMS ASSOCIATE WITH FRESH SEMEN QUALITY FROM DAIRY BULL FRIESIAN HOLSTEIN

This research aims to identify polymorphisms in TEK genes to identify any related possibility to fresh semen quality of FH bull using the PCR method. A total of 14 samples of bull’s whole blood were collected and also the quality of each bull’s fresh semen. DNA amplification was carried out using primer Forward (TEK_F) 5'-TAGATTGTCGCTTGCCTGGG-3 'and Reverse (TEK_R) 5'-CCTGTGCCGACAGGTTTACT-3'. Analysis of the DNA sequence results was carried out using BioEdit and NCBI BLAST software. The results showed that of the 7 samples producing 262 bp and found polymorphisms in the TEK gene sequence in 23 gene bank databases. In the analysis of the relationship between the motility of individual spermatozoa with mutations, r count> r table (0.806> 0.754) or significance value <5% significance level (0.029 <0.050). In the analysis of the relationship between semen concentration and mutation, r count> r table (0.897> 0.754) or significance level <5% significance level (0.006 <0.050) is obtained.

HGG-28. ONCOGENIC TYROSINE KINASE GENE FUSIONS IN SUPRATENTORIAL HIGH GRADE GLIOMAS OF YOUNG CHILDREN - COMPARISON OF RNA- AND DNA-BASED METHODS FOR THEIR RELIABLE DETECTION

High-grade diffuse gliomas in early childhood are characterized by a more favorable outcome compared to older children. We have shown in previous studies that these tumors are characterized by stable genomes. The occurrence of tyrosine kinase gene fusions in high-grade gliomas of infancy may represent therapeutic targets. 50 glioblastomas (GBM) with supratentorial location occurring in children younger than four years were retrieved from the archives of the Brain Tumor Reference Center, Institute of Neuropathology, Bonn University. DNA and RNA were extracted from FFPE tumor samples. Gene fusions were identified on the DNA level by FISH using break-apart probes for ALK, NTRK1, -2, -3, ROS1 and MET and Molecular Inversion Probe (MIP) methodology. On the RNA level, fusion transcripts were detected by targeted RNA sequencing as well as Nanostring assay with fusion-specific probes. 37 supratentorial GBM occurred in the first year of life, 13 GBM between one and four years. 18 cases showed fusions of ALK to different partners; all occurred in the first year of life (18/37, 48.6%). Fusions of ROS1 were found in 5, MET in 3, NTRK1, -2, -3 in 10 cases. 12 cases showed no and two cases novel fusions. The different methods led to comparable results. Only recurrent fusions with known fusion partners were detectable with fusion sequence-specific Nanostring probes and library construction for targeted RNA sequencing failed in a fraction of cases. Break-apart FISH led to reliable results on the next day, and MIP technology represented the most sensitive method for analysis of FFPE samples. Gene fusions involving the tyrosine kinase genes ALK, MET, ROS1 and NTRK1, -2, -3 occurred in 72% of glioblastomas of young children; most frequent were ALK fusions occurring in infant GBM. DNA-based MIP technology represented the most robust and sensitive assay. A combination of RNA- and DNA-based methods to detect these fusions with high reliability is recommended.

A multi-enhancer RET regulatory code is disrupted in Hirschsprung disease

In Hirschsprung disease (HSCR; congenital colonic aganglionosis), three GWAS-identified common variants residing in three distinct enhancers of the RET receptor tyrosine kinase gene reduce its gene expression and are causal disease risk variants. Further, their combined effects significantly dysregulate the expression of other functionally-related genes defining the RET gene regulatory network (GRN). In this study, we asked how many variants in how many distinct RET enhancers affect HSCR risk by reducing RET gene expression? We demonstrate that 22 additional HSCR-associated polymorphisms, both independent and associated, reside within multiple candidate RET enhancers and among which 7 display differential allelic enhancer activities. Of these 7 RET enhancers, two bind PAX3 and extend the known RET-EDNRB GRN. Therefore, sequence variants within a minimum of 10 RET enhancers affect HSCR risk, revealing a diverse regulatory code modulating complex disease risk even at a single locus.