Identification and functional characterization of a highly divergent N-acetylglucosaminyltransferase I (TbGnTI) in <em>Trypanosoma brucei</em>

Trypanosoma brucei expresses a diverse repertoire of N-glycans, ranging from oligomannose and paucimannose structures to exceptionally large complex N-glycans. Despite the presence of the latter, no obvious homologues of known β1–4-galactosyltransferase or β1–2- or β1–6-N-acetylglucosaminyltransferase genes have been found in the parasite genome. However, we previously reported a family of putative UDP-sugar-dependent glycosyltransferases with similarity to the mammalian β1–3-glycosyltransferase family. Here we characterize one of these genes, TbGT11, and show that it encodes a Golgi apparatus resident UDP-GlcNAc:α3-d-mannoside β1–2-N-acetylglucosaminyltransferase I activity (TbGnTI). The bloodstream-form TbGT11 null mutant exhibited significantly modified protein N-glycans but normal growth in vitro and infectivity to rodents. In contrast to multicellular organisms, where the GnTI reaction is essential for biosynthesis of both complex and hybrid N-glycans, T. brucei TbGT11 null mutants expressed atypical “pseudohybrid” glycans, indicating that TbGnTII activity is not dependent on prior TbGnTI action. Using a functional in vitro assay, we showed that TbGnTI transfers UDP-GlcNAc to biantennary Man(3)GlcNAc(2), but not to triantennary Man(5)GlcNAc(2), which is the preferred substrate for metazoan GnTIs. Sequence alignment reveals that the T. brucei enzyme is far removed from the metazoan GnTI family and suggests that the parasite has adapted the β3-glycosyltransferase family to catalyze β1–2 linkages

Low-frequency variation near common germline susceptibility loci are associated with risk of Ewing sarcoma

Background: Ewing sarcoma (EwS) is a rare, aggressive solid tumor of childhood, adolescence and young adulthood associated with pathognomonic EWSR1-ETS fusion oncoproteins altering transcriptional regulation. Genome-wide association studies (GWAS) have identified 6 common germline susceptibility loci but have not investigated low-frequency inherited variants with minor allele frequencies below 5% due to limited genotyped cases of this rare tumor. Methods We investigated the contribution of rare and low-frequency variation to EwS susceptibility in the largest EwS genome-wide association study to date (733 EwS cases and 1,346 unaffected controls of European ancestry). Results We identified two low-frequency variants, rs112837127 and rs2296730, on chromosome 20 that were associated with EwS risk (OR = 0.186 and 2.038, respectively;P-value < 5x10(-8)) and located near previously reported common susceptibility loci. After adjusting for the most associated common variant at the locus, only rs112837127 remained a statistically significant independent signal (OR = 0.200, P-value = 5.84x10(-8)). Conclusions: These findings suggest rare variation residing on common haplotypes are important contributors to EwS risk. Impact Motivate future targeted sequencing studies for a comprehensive evaluation of low-frequency and rare variation around common EwS susceptibility loci

Genomic EWS-FLI1 Fusion Sequences in Ewing Sarcoma Resemble Breakpoint Characteristics of Immature Lymphoid Malignancies

Chromosomal translocations between the EWS gene and members of the ETS gene family are characteristic molecular features of the Ewing sarcoma. The most common translocation t(11;22)(q24;q12) fuses the EWS gene to FLI1, and is present in 85–90% of Ewing sarcomas. In the present study, a specifically designed multiplex long-range PCR assay was applied to amplify genomic EWS-FLI1 fusion sites from as little as 100 ng template DNA. Characterization of the EWS-FLI1 fusion sites of 42 pediatric and young adult Ewing sarcoma patients and seven cell lines revealed a clustering in the 5′ region of the EWS-breakpoint cluster region (BCR), in contrast to random distribution of breakpoints in the FLI1-BCR. No association of breakpoints with various recombination-inducing sequence motifs was identified. The occurrence of small deletions and duplications at the genomic junction is characteristic of involvement of the non-homologous end-joining (NHEJ) repair system, similar to findings at chromosomal breakpoints in pediatric leukemia and lymphoma

Continuous therapy response references for BCR::ABL1 monitoring in pediatric chronic myeloid leukemia

Abstract Response to tyrosine kinase inhibitor (TKI) therapy in patients with chronic myeloid leukemia (CML) is monitored by quantification of BCR::ABL1 transcript levels. Milestones for assessing optimal treatment response have been defined in adult CML patients and are applied to children and adolescents although it is questionable whether transferability to pediatric patients is appropriate regarding genetic and clinical differences. Therefore, we analyzed the molecular response kinetics to TKI therapy in 129 pediatric CML patients and investigated whether response assessment based on continuous references can support an early individual therapy adjustment. We applied a moving quantiles approach to establish a high-resolution response target curve and contrasted the median responses in all patients with the median of the ideal target curve obtained from a subgroup of optimal responders. The high-resolution response target curve of the optimal responder group presents a valuable tool for continuous therapy monitoring of individual pediatric CML patients in addition to the fixed milestones. By further comparing BCR::ABL1 transcript levels with BCR::ABL1 fusion gene copy numbers, it is also possible to model the differential dynamics of BCR::ABL1 expression and cell number under therapy. The developed methodology can be transferred to other biomarkers for continuous therapy monitoring

Molecular Composition of Genomic TMPRSS2-ERG Rearrangements in Prostate Cancer

There is increasing interest in the use of cell-free circulating tumor DNA (ctDNA) as a serum marker for therapy assessment in prostate cancer patients. Prostate cancer is characterized by relatively low numbers of mutations, and, in contrast to many other common epithelial cancers, commercially available single nucleotide mutation assays for quantification of ctDNA are insufficient for therapy assessment in this disease. However, prostate cancer shares some similarity with translocation-affected mesenchymal tumors (e.g., leukemia and Ewing sarcoma), which are common in pediatric oncology, where chromosomal translocations are used as biomarkers for quantification of the tumor burden. Approximately 50% of prostate cancers carry a chromosomal translocation resulting in generation of the TMPRSS2-ERG fusion gene, which is unique to the tumor cells of each individual patient because of variability in the fusion breakpoint sites. In the present study, we examined the structural preconditions for TMPRSS2-ERG fusion sites in comparison with mesenchymal tumors in pediatric patients to determine whether the sequence composition is suitable for the establishment of tumor-specific quantification assays in prostate cancer patients. Genomic repeat elements represent potential obstacles to establishment of quantification assays, and we found similar proportions of repeat elements at fusion sites in prostate cancer to those reported for mesenchymal tumors, where genomic fusion sequences are established as biomarkers. Our data support the development of the TMPRSS2-ERG fusion gene as a noninvasive tumor marker for therapy assessment, risk stratification, and relapse detection to improve personalized therapy strategies for patients with prostate cancer