Re-evaluation of a reported increased micronucleus frequency in lymphocytes of workers occupationally exposed to formaldehyde

A replicate evaluation of increased micronucleus (MN) frequencies in peripheral lymphocytes of workers occupationally exposed to formaldehyde (FA) was undertaken to verify the observed effect and to determine scoring variability. May–Grünwald–Giemsa-stained slides were obtained from a previously performed cytokinesis-block micronucleus test (CBMNT) with 56 workers in anatomy and pathology laboratories and 85 controls. The first evaluation by one scorer (scorer 1) had led to a highly significant difference between workers and controls (3.96 vs 0.81 MN per 1000 cells). The slides were coded before re-evaluation and the code was broken after the complete re-evaluation of the study. A total of 1000 binucleated cells (BNC) were analysed per subject and the frequency of MN (in ‰) was determined. Slides were distributed equally and randomly between two scorers, so that the scorers had no knowledge of the exposure status. Scorer 2 (32 exposed, 36 controls) measured increased MN frequencies in exposed workers (9.88 vs 6.81). Statistical analysis with the two-sample Wilcoxon test indicated that this difference was not significant (p = 0.17). Scorer 3 (20 exposed, 46 controls) obtained a similar result, but slightly higher values for the comparison of exposed and controls (19.0 vs 12.89; p = 0.089). Combining the results of the two scorers (13.38 vs 10.22), a significant difference between exposed and controls (p = 0.028) was obtained when the stratified Wilcoxon test with the scorers as strata was applied. Interestingly, the re-evaluation of the slides led to clearly higher MN frequencies for exposed and controls compared with the first evaluation. Bland–Altman plots indicated that the agreement between the measurements of the different scorers was very poor, as shown by mean differences of 5.9 between scorer 1 and scorer 2 and 13.0 between scorer 1 and scorer 3. Calculation of the intra-class correlation coefficient (ICC) revealed that all scorer comparisons in this study were far from acceptable for the reliability of this assay. Possible implications for the use of the CBMNT in human biomonitoring studies are discussed

Non-coding RNA ANRIL and the number of plexiform neurofibromas in patients with NF1microdeletions

BACKGROUND: Neurofibromatosis type-1 (NF1) is caused by mutations of the NF1 gene at 17q11.2. In 95% of non-founder NF1 patients, NF1 mutations are identifiable by means of a comprehensive mutation analysis. 5-10% of these patients harbour microdeletions encompassing the NF1 gene and its flanking regions. NF1 is characterised by tumours of the peripheral nerve sheaths, the pathognomonic neurofibromas. Considerable inter- and intra-familial variation in expressivity of the disease has been observed which is influenced by genetic modifiers unrelated to the constitutional NF1 mutation. The number of plexiform neurofibromas (PNF) in NF1 patients is a highly heritable genetic trait. Recently, SNP rs2151280 located within the non-coding RNA gene ANRIL at 9p21.3, was identified as being strongly associated with PNF number in a family-based association study. The T-allele of rs2151280, which correlates with reduced ANRIL expression, appears to be associated with higher PNF number. ANRIL directly binds to the SUZ12 protein, an essential component of polycomb repressive complex 2, and is required for SUZ12 occupancy of the CDKN2A/CDKN2B tumour suppressor genes as well as for their epigenetic silencing. METHODS: Here, we explored a potential association of PNF number and PNF volume with SNP rs2151280 in 29 patients with constitutional NF1 microdeletions using the exact Cochran-Armitage test for trends and the exact Mann–Whitney–Wilcoxon test. Both the PNF number and total tumour volume in these 29 NF1 patients were assessed by whole-body MRI. The NF1 microdeletions observed in these 29 patients encompassed the NF1 gene as well as its flanking regions, including the SUZ12 gene. RESULTS: In the 29 microdeletion patients investigated, neither the PNF number nor PNF volume was found to be associated with the T-allele of rs2151280. CONCLUSION: Our findings imply that, at least in patients with NF1 microdeletions, PNF susceptibility is not associated with rs2151280. Although somatic inactivation of the NF1 wild-type allele is considered to be the PNF-initiating event in NF1 patients with intragenic mutations and patients with NF1 microdeletions, both patient groups may differ with regard to tumour progression because of the heterozygous constitutional deletion of SUZ12 present only in patients with NF1 microdeletions

Gene synteny comparisons between different vertebrates provide new insights into breakage and fusion events during mammalian karyotype evolution

<p>Abstract</p> <p>Background</p> <p>Genome comparisons have made possible the reconstruction of the eutherian ancestral karyotype but also have the potential to provide new insights into the evolutionary inter-relationship of the different eutherian orders within the mammalian phylogenetic tree. Such comparisons can additionally reveal (i) the nature of the DNA sequences present within the evolutionary breakpoint regions and (ii) whether or not the evolutionary breakpoints occur randomly across the genome. Gene synteny analysis (E-painting) not only greatly reduces the complexity of comparative genome sequence analysis but also extends its evolutionary reach.</p> <p>Results</p> <p>E-painting was used to compare the genome sequences of six different mammalian species and chicken. A total of 526 evolutionary breakpoint intervals were identified and these were mapped to a median resolution of 120 kb, the highest level of resolution so far obtained. A marked correlation was noted between evolutionary breakpoint frequency and gene density. This correlation was significant not only at the chromosomal level but also sub-chromosomally when comparing genome intervals of lengths as short as 40 kb. Contrary to previous findings, a comparison of evolutionary breakpoint locations with the chromosomal positions of well mapped common fragile sites and cancer-associated breakpoints failed to reveal any evidence for significant co-location. Primate-specific chromosomal rearrangements were however found to occur preferentially in regions containing segmental duplications and copy number variants.</p> <p>Conclusion</p> <p>Specific chromosomal regions appear to be prone to recurring rearrangement in different mammalian lineages ('breakpoint reuse') even if the breakpoints themselves are likely to be non-identical. The putative ancestral eutherian genome, reconstructed on the basis of the synteny analysis of 7 vertebrate genome sequences, not only confirmed the results of previous molecular cytogenetic studies but also increased the definition of the inferred structure of ancestral eutherian chromosomes. For the first time in such an analysis, the opossum was included as an outgroup species. This served to confirm our previous model of the ancestral eutherian genome since all ancestral syntenic segment associations were also noted in this marsupial.</p

Immune phenotypes predict survival in patients with glioblastoma multiforme

Background: Glioblastoma multiforme (GBM), a common primary malignant brain tumor, rarely disseminates beyond the central nervous system and has a very bad prognosis. The current study aimed at the analysis of immunological control in individual patients with GBM. Methods: Immune phenotypes and plasma biomarkers of GBM patients were determined at the time of diagnosis using flow cytometry and ELISA, respectively. Results: Using descriptive statistics, we found that immune anomalies were distinct in individual patients. Defined marker profiles proved highly relevant for survival. A remarkable relation between activated NK cells and improved survival in GBM patients was in contrast to increased CD39 and IL-10 in patients with a detrimental course and very short survival. Recursive partitioning analysis (RPA) and Cox proportional hazards models substantiated the relevance of absolute numbers of CD8 cells and low numbers of CD39 cells for better survival. Conclusions: Defined alterations of the immune system may guide the course of disease in patients with GBM and may be prognostically valuable for longitudinal studies or can be applied for immune intervention

Consideration of the haplotype diversity at nonallelic homologous recombination hotspots improves the precision of rearrangement breakpoint identification

Precise characterization of nonallelic homologous recombination (NAHR) breakpoints is key to identifying those features that influence NAHR frequency. Until now, analysis of NAHR-mediated rearrangements has generally been performed by comparison of the breakpoint-spanning sequences with the human genome reference sequence. We show here that the haplotype diversity of NAHR hotspots may interfere with breakpoint-mapping. We studied the transmitting parents of individuals with germline type-1 NF1 deletions mediated by NAHR within the paralogous recombination site 1 (PRS1) or paralogous recombination site 2 (PRS2) hotspots. Several parental wild-type PRS1 and PRS2 haplotypes were identified that exhibited considerable sequence differences with respect to the reference sequence, which also affected the number of predicted PRDM9-binding sites. Sequence comparisons between the parental wild-type PRS1 or PRS2 haplotypes and the deletion breakpoint-spanning sequences from the patients (method #2) turned out to be an accurate means to assign NF1 deletion breakpoints and proved superior to crude reference sequence comparisons that neglect to consider haplotype diversity (method #1). The mean length of the deletion breakpoint regions assigned by method #2 was 269-bp in contrast to 502-bp by method #1. Our findings imply that paralog-specific haplotype diversity of NAHR hotspots (such as PRS2) and population-specific haplotype diversity must be taken into account in order to accurately ascertain NAHR-mediated rearrangement breakpoints

SVA retrotransposon insertion-associated deletion represents a novel mutational mechanism underlying large genomic copy number changes with non-recurrent breakpoints

Background: Genomic disorders are caused by copy number changes that may exhibit recurrent breakpoints processed by nonallelic homologous recombination. However, region-specific disease-associated copy number changes have also been observed which exhibit non-recurrent breakpoints. The mechanisms underlying these non-recurrent copy number changes have not yet been fully elucidated. Results: We analyze large NF1 deletions with non-recurrent breakpoints as a model to investigate the full spectrum of causative mechanisms, and observe that they are mediated by various DNA double strand break repair mechanisms, as well as aberrant replication. Further, two of the 17 NF1 deletions with non-recurrent breakpoints, identified in unrelated patients, occur in association with the concomitant insertion of SINE/variable number of tandem repeats/Alu (SVA) retrotransposons at the deletion breakpoints. The respective breakpoints are refractory to analysis by standard breakpoint-spanning PCRs and are only identified by means of optimized PCR protocols designed to amplify across GC-rich sequences. The SVA elements are integrated within SUZ12P intron 8 in both patients, and were mediated by target-primed reverse transcription of SVA mRNA intermediates derived from retrotranspositionally active source elements. Both SVA insertions occurred during early postzygotic development and are uniquely associated with large deletions of 1 Mb and 867 kb, respectively, at the insertion sites. Conclusions: Since active SVA elements are abundant in the human genome and the retrotranspositional activity of many SVA source elements is high, SVA insertion-associated large genomic deletions encompassing many hundreds of kilobases could constitute a novel and as yet under-appreciated mechanism underlying large-scale copy number changes in the human genome

T‐cell prolymphocytic leukemia is associated with deregulation of oncogenic microRNAs on transcriptional and epigenetic level

Deregulation of micro(mi)-RNAs is a common mechanism in tumorigenesis. We investigated the expression of 2083 miRNAs in T-cell prolymphocytic leukemia (T-PLL). Compared to physiologic CD4+ and CD8+ T-cell subsets, 111 miRNAs were differentially expressed in T-PLL. Of these, 33 belonged to miRNA gene clusters linked to cancer. Genomic variants affecting miRNAs were infrequent with the notable exception of copy number aberrations. Remarkably, we found strong upregulation of the miR-200c/-141 cluster in T-PLL to be associated with DNA hypomethylation and active promoter marks. Our findings suggest that copy number aberrations and epigenetic changes could contribute to miRNA deregulation in T-PLL

SVA retrotransposon insertion-associated deletion represents a novel mutational mechanism underlying large genomic copy number changes with non-recurrent breakpoints

Background: Genomic disorders are caused by copy number changes that may exhibit recurrent breakpoints processed by nonallelic homologous recombination. However, region-specific disease-associated copy number changes have also been observed which exhibit non-recurrent breakpoints. The mechanisms underlying these non-recurrent copy number changes have not yet been fully elucidated. Results: We analyze large NF1 deletions with non-recurrent breakpoints as a model to investigate the full spectrum of causative mechanisms, and observe that the