HLA-DRB1*07:01-HLA-DQA1*02:01-HLA-DQB1*02:02 haplotype is associated with a high risk of asparaginase hypersensitivity in acute lymphoblastic leukemia

Hypersensitivity reactions are the most frequent dose-limiting adverse reactions to Escherichia coli-derived asparaginase in pediatric acute lymphoblastic leukemia (ALL) patients. The aim of the present study was to identify associations between sequence-based Human Leukocyte Antigen Class II region alleles and asparaginase hypersensitivity in a Hungarian ALL population. Four-digit typing of HLA-DRB1 and HLA-DQB1 loci was performed in 359 pediatric ALL patients by using next-generation sequencing method. Based on genotypic data of the two loci, haplotype reconstruction was carried out. In order to investigate the possible role of the HLA-DQ complex, the HLA-DQA1 alleles were also inferred. Multivariate logistic regression analysis and a Bayesian network-based approach were applied to identify relevant genetic risk factors of asparaginase hypersensitivity. Patients with HLA-DRB1*07:01 and HLA-DQB1*02:02 alleles had significantly higher risk of developing asparaginase hypersensitivity compared to non-carriers [P=4.56×10−5; OR=2.86 (1.73–4.75) and P=1.85×10−4; OR=2.99 (1.68–5.31); n=359, respectively]. After haplotype reconstruction, the HLA-DRB1*07:01-HLA-DQB1*02:02 haplotype was associated with an increased risk. After inferring the HLA-DQA1 alleles the HLA-DRB1*07:01–HLA-DQA1*02:01–HLA-DQB1*02:02 haplotype was associated with the highest risk of asparaginase hypersensitivity [P=1.22×10−5; OR=5.00 (2.43–10.29); n=257]. Significantly fewer T-cell ALL patients carried the HLA-DQB1*02:02 allele and the associated haplotype than did pre-B-cell ALL patients (6.5%; vs. 19.2%, respectively; P=0.047). In conclusion, we identified a haplotype in the Human Leukocyte Antigen Class II region associated with a higher risk of asparaginase hypersensitivity. Our results confirm that variations in HLA-D region might influence the development of asparaginase hypersensitivity

Candidate gene association study in pediatric acute lymphoblastic leukemia evaluated by Bayesian network based Bayesian multilevel analysis of relevance

Background: We carried out a candidate gene association study in pediatric acute lymphoblastic leukemia (ALL) to identify possible genetic risk factors in a Hungarian population. Methods: The results were evaluated with traditional statistical methods and with our newly developed Bayesian network based Bayesian multilevel analysis of relevance (BN-BMLA) method. We collected genomic DNA and clinical data from 543 children, who underwent chemotherapy due to ALL, and 529 healthy controls. Altogether 66 single nucleotide polymorphisms (SNPs) in 19 candidate genes were genotyped. Results: With logistic regression, we identified 6 SNPs in the ARID5B and IKZF1 genes associated with increased risk to B-cell ALL, and two SNPs in the STAT3 gene, which decreased the risk to hyperdiploid ALL. Because the associated SNPs were in linkage in each gene, these associations corresponded to one signal per gene. The odds ratio (OR) associated with the tag SNPs were: OR = 1.69, P = 2.22x10-7 for rs4132601 (IKZF1), OR = 1.53, P = 1.95x10-5 for rs10821936 (ARID5B) and OR = 0.64, P = 2.32x10-4 for rs12949918 (STAT3). With the BN-BMLA we confirmed the findings of the frequentist-based method and received additional information about the nature of the relations between the SNPs and the disease. E.g. the rs10821936 in ARID5B and rs17405722 in STAT3 showed a weak interaction, and in case of T-cell lineage sample group, the gender showed a weak interaction with three SNPs in three genes. In the hyperdiploid patient group the BN-BMLA detected a strong interaction among SNPs in the NOTCH1, STAT1, STAT3 and BCL2 genes. Evaluating the survival rate of the patients with ALL, the BN-BMLA showed that besides risk groups and subtypes, genetic variations in the BAX and CEBPA genes might also influence the probability of survival of the patients. Conclusions: In the present study we confirmed the roles of genetic variations in ARID5B and IKZF1 in the susceptibility to B-cell ALL. With the newly developed BN-BMLA method several gene-gene, gene-phenotype and phenotype-phenotype connections were revealed. We showed several advantageous features of the new method, and suggested that in gene association studies the BN-BMLA might be a useful supplementary to the traditional frequentist-based statistical method

A Novel Epigenetic Silencing Pathway Involving the Highly Conserved 5'-3' Exoribonuclease Dhp1/Rat1/Xrn2 in Schizosaccharomyces pombe.

Epigenetic gene silencing plays a critical role in regulating gene expression and contributes to organismal development and cell fate acquisition in eukaryotes. In fission yeast, Schizosaccharomyces pombe, heterochromatin-associated gene silencing is known to be mediated by RNA processing pathways including RNA interference (RNAi) and a 3'-5' exoribonuclease complex, the exosome. Here, we report a new RNA-processing pathway that contributes to epigenetic gene silencing and assembly of heterochromatin mediated by 5'-3' exoribonuclease Dhp1/Rat1/Xrn2. Dhp1 mutation causes defective gene silencing both at peri-centromeric regions and at the silent mating type locus. Intriguingly, mutation in either of the two well-characterized Dhp1-interacting proteins, the Din1 pyrophosphohydrolase or the Rhn1 transcription termination factor, does not result in silencing defects at the main heterochromatic regions. We demonstrate that Dhp1 interacts with heterochromatic factors and is essential in the sequential steps of establishing silencing in a manner independent of both RNAi and the exosome. Genomic and genetic analyses suggest that Dhp1 is involved in post-transcriptional silencing of repetitive regions through its RNA processing activity. The results describe the unexpected role of Dhp1/Rat1/Xrn2 in chromatin-based silencing and elucidate how various RNA-processing pathways, acting together or independently, contribute to epigenetic regulation of the eukaryotic genome

Structural basis for 5'-ETS recognition by Utp4 at the early stages of ribosome biogenesis.

Eukaryotic ribosome biogenesis begins with the co-transcriptional assembly of the 90S pre-ribosome. The 'U three protein' (UTP) complexes and snoRNP particles arrange around the nascent pre-ribosomal RNA chaperoning its folding and further maturation. The earliest event in this hierarchical process is the binding of the UTP-A complex to the 5'-end of the pre-ribosomal RNA (5'-ETS). This oligomeric complex predominantly consists of β-propeller and α-solenoidal proteins. Here we present the structure of the Utp4 subunit from the thermophilic fungus Chaetomium thermophilum at 2.15 Å resolution and analyze its function by UV RNA-crosslinking (CRAC) and in context of a recent cryo-EM structure of the 90S pre-ribosome. Utp4 consists of two orthogonal and highly basic β-propellers that perfectly fit the EM-data. The Utp4 structure highlights an unusual Velcro-closure of its C-terminal β-propeller as relevant for protein integrity and potentially Utp8 recognition in the context of the pre-ribosome. We provide a first model of the 5'-ETS RNA from the internally hidden 5'-end up to the region that hybridizes to the 3'-hinge sequence of U3 snoRNA and validate a specific Utp4/5'-ETS interaction by CRAC analysis

Dhp1 is required for effective heterochromatin maintenance.

<p>(A) A map of the mating type locus shows the <i>cenH</i>-containing <i>K</i> region, a nucleation site from which repressive chromatin is initiated and spread. After the initial establishment of heterochromatin, deletion of the <i>K</i> region does not affect the chromatin structure and the reporter gene (<i>ade6</i>^<i>+</i>) remains silenced due to maintenance. Disruption of maintenance results in derepression of the reporter gene. (B) Representative images of grid-based assays from which the percent of colonies grouped by <i>ade6</i>^<i>+</i> expression were calculated. (C) Graph displays the percent of colonies grouped by relative expression of <i>ade6</i>^<i>+</i> as visually determined by colony color. (D) qRT-PCR shows the relative expression compared to wt of the <i>ade6</i>^<i>+</i> reporter gene in <i>dhp1-1</i> and <i>swi6Δ</i>. (E) H3K9me₂ qChIP was carried out on the indicated strains. * <i>p</i> ≤ 0.05 as determined by Student’s <i>t</i> test comparing the indicated sample values with the wt values for qRT-PCR and qChIP.</p

The catalytic activity of Dhp1 is required for silencing.

<p>(A) Sequence alignment of the catalytic domains between <i>K</i>. <i>lactis</i> and <i>S</i>. <i>pombe</i>. The position of D55 and E207 of Dhp1 in <i>S</i>. <i>pombe</i> are highlighted. (B) A ten-fold serial dilution assay shows <i>dhp1-1</i> cells carrying indicated plasmids grown on standard SC-leucine medium at 30°C or 37°C. (C-D) qRT-PCR showing relative <i>dg</i> (C) and <i>cenH</i> (D) transcript levels in <i>dhp1-1</i> cells carrying indicated plasmids. *<i>P</i> ≤ 0.05 as determined by student’s <i>t</i> test comparing the indicated sample values with the WT for qRT-PCR.</p

Dhp1-mediated silencing occurs independently of RNAi and the exosome.

<p>(A-B) qRT-PCR analysis of silenced repeat regions in the centromere (A) and mating type locus (B) shows negative genetic interactions between <i>dhp1-1</i> and <i>ago1Δ</i> or <i>rrp6Δ</i>. (C-D) H3K9me₂ ChIPs show the enrichments of H3K9me mark at indicated strains. Relative enrichments of H3K9me₂ at indicated genomic regions were normalized to <i>leu1</i>^<i>+</i>. * <i>p</i> ≤ 0.05 as determined by Student’s <i>t</i> test comparing the indicated sample values with the wt values for qRT-PCR and qChIP. Significance between the single mutants and double mutants is indicated by horizontal lines linking the compared samples.</p

Dhp1 contributes to heterochromatin spreading.

<p>(A) Insertion of the <i>cenH</i> nucleation site adjacent to the <i>ade6</i>^<i>+</i> reporter gene in an ectopic euchromatic locus (<i>ura4</i>^<i>+</i>) will result in the silencing of the reporter gene through heterochromatin spreading. Gray circles: H3K9me. (B) Above, representative images of grid-based assays used to calculate the percentage of cells exhibiting distinct <i>ade6</i>^<i>+</i> expression on low adenine medium. Below: 100% stacked column graph represents the observed phenotypes of these colonies. (C) H3K9me₂ qChIP shows relative enrichment at the <i>ade6</i>^<i>+</i> reporter gene and its surrounding regions normalized to <i>leu1</i>^<i>+</i>. The genomic positions for oligos used for qPCR are indicated by gray boxes. * <i>p</i> ≤ 0.05 as determined by Student’s <i>t</i> test comparing the indicated sample values with wt values.</p

Dhp1 represents a novel RNA processing mechanism which mediates epigenetic silencing independent of both RNAi and the exosome.

<p>(A) Aberrant transcripts produced by RNAPII are subject to degradation via three distinct PTGS pathways. RNAi involves the degradation of the transcript by Dcr1 or Triman (Tri1), producing siRNAs. The exosome degrades transcripts in a 3’ to 5’ direction with guidance from its cofactors, the TRAMP and MTREC complexes. Dhp1 mediates a distinct third pathway of post-transcriptional gene silencing via its 5’ to 3’ exoribonuclease activity. Together, these mechanisms function to initiate and maintain the epigenetic silencing of repeat RNAs. (B) RNA processing-mediated mechanisms in epigenetic silencing. We propose that Dhp1/Xrn2-mediated silencing is a novel RNAi- and exosome-independent processing mechanism in gene silencing.</p

Dhp1 does not affect the binding of RNAPII to repeat transcripts.

<p>(A) Cross-linking and analysis of cDNA (CRAC) followed by expression profiling reveals RNAPII targets on a genome-wide scale. (B) CRAC was carried out using a carboxyl-terminal HTP-tagged subunit of RNAPII, Rpb2-HTP. The difference plot maps the relative enrichments (Mutant/wt) of RNAPII substrates averaged across the genome for each mutant in relation to the transcriptional termination site (TTS). RNAPII substrates are relatively enriched after the normal termination site of transcripts in <i>dhp1-1</i> due to a transcriptional termination defect. (C) No dramatic changes in enrichment of RNAPII substrates were observed across the centromeric regions and the mating type locus between wt and <i>dhp1-1</i> cells, suggesting that Dhp1 does not affect silencing by TGS, but likely through a PTGS mechanism. The chromosome position of the genomic region is displayed below the figure (Fwd, forward strand; Rev, reverse strand).</p