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Previous spatio-temporal COVID-19 prediction models have focused on the prediction of subsequent number of cases, and have shown varying accuracy and lack of high geographical resolution. We aimed to predict trends in COVID-19 test positivity, an important marker for planning local testing capacity and accessibility. We included a full year of information (June 29, 2020-July 4, 2021) with both direct and indirect indicators of transmission, e.g. mobility data, number of calls to the national healthcare advice line and vaccination coverage from Uppsala County, Sweden, as potential predictors. We developed four models for a 1-week-window, based on gradient boosting (GB), random forest (RF), autoregressive integrated moving average (ARIMA) and integrated nested laplace approximations (INLA). Three of the models (GB, RF and INLA) outperformed the naïve baseline model after data from a full pandemic wave became available and demonstrated moderate accuracy. An ensemble model of these three models slightly improved the average root mean square error to 0.039 compared to 0.040 for GB, RF and INLA, 0.055 for ARIMA and 0.046 for the naïve model. Our findings indicate that the collection of a wide variety of data can contribute to spatio-temporal predictions of COVID-19 test positivity., (© 2022. The Author(s).)

Background: The immunohistochemical analysis of programmed cell death ligand 1 (PD-L1) expression in tumor tissue of non-small-cell lung cancer patients has now been integrated in the diagnostic workup. Analysis is commonly done on small tissue biopsy samples representing a minimal fraction of the whole tumor. The aim of the study was to evaluate the correlation of PD-L1 expression on biopsy specimens with corresponding resection specimens., Materials and Methods: In total, 58 consecutive cases with preoperative biopsy and resected tumor specimens were selected. From each resection specimen 2 tumor cores were compiled into a tissue microarray (TMA). Immunohistochemical staining with the antibody SP263 was performed on biopsy specimens, resection specimens (whole sections), as well as on the TMA., Results: The proportion of PD-L1-positive stainings were comparable between the resection specimens (48% and 19%), the biopsies (43% and 17%), and the TMAs (47% and 14%), using cutoffs of 1% and 50%, respectively (P > .39 all comparisons). When the resection specimens were considered as reference, PD-L1 status differed in 16%/5% for biopsies and in 9%/9% for TMAs (1%/50% cutoff). The sensitivity of the biopsy analysis was 79%/82% and the specificity was 90%/98% at the 1%/50% cutoff. The Cohens κ value for the agreement between biopsy and tumor. was 0.70 at the 1% cutoff and 0.83 at the 50% cutoff., Conclusion: The results indicate a moderate concordance between the analysis of biopsy and whole tumor tissue, resulting in misclassification of samples in particular when the lower 1% cutoff was used. Clinicians should be aware of this uncertainty when interpreting PD-L1 reports for treatment decisions., (Copyright © 2019. Published by Elsevier Inc.)

Disease epidemiology during ageing shows a transition from cancer to degenerative chronic disorders as dominant contributors to mortality in the old. Nevertheless, it has remained unclear to what extent molecular signatures of ageing reflect this phenomenon. Here we report on the identification of a conserved transcriptomic signature of ageing based on gene expression data from four vertebrate species across four tissues. We find that ageing-associated transcriptomic changes follow trajectories similar to the transcriptional alterations observed in degenerative ageing diseases but are in opposite direction to the transcriptomic alterations observed in cancer. We confirm the existence of a similar antagonism on the genomic level, where a majority of shared risk alleles which increase the risk of cancer decrease the risk of chronic degenerative disorders and vice versa. These results reveal a fundamental trade-off between cancer and degenerative ageing diseases that sheds light on the pronounced shift in their epidemiology during ageing.

Biological control is a promising approach to reduce plant diseases caused by nematodes. We tested the effect of the fungus Clonostachys rosea strain IK726 inoculation on nematode community composition in a naturally nematode infested soil in a pot experiment, and the effect of C. rosea on plant health. The numbers of plant-parasitic nematode genera extracted from soil and plant roots decreased by 40 to 73% when C. rosea was applied, while genera of nonparasitic nematodes were not affected. Soil inoculation of C. rosea increased fresh shoot weight and shoot length of wheat plants by 20 and 24%, respectively, while only shoot dry weight increased by 48% in carrots. Light microscopy of in vitro C. rosea-nematode interactions did not reveal evidence of direct parasitism. However, culture filtrates of C. rosea growing in potato dextrose broth, malt extract broth and synthetic nutrient broth exhibited toxicity toward nematodes and immobilized 57, 62, and 100% of the nematodes, respectively, within 48 h. This study demonstrates that C. rosea can control plant-parasitic nematodes and thereby improve plant growth. The most likely mechanism responsible for the antagonism is antibiosis through production of nematicidal compounds, rather than direct parasitism.

Background: The liver is the major site for alcohol metabolism in the body and therefore the primary target organ for ethanol (EtOH)-induced toxicity. In this study, we investigated the in vitro response of human liver cells to different EtOH concentrations in a perfused bioartificial liver device that mimics the complex architecture of the natural organ., Methods: Primary human liver cells were cultured in the bioartificial liver device and treated for 24 hours with medium containing 150 mM (low), 300 mM (medium), or 600 mM (high) EtOH, while a control culture was kept untreated. Gene expression patterns for each EtOH concentration were monitored using Affymetrix Human Gene 1.0 ST Gene chips. Scaled expression profiles of differentially expressed genes (DEGs) were clustered using Fuzzy c-means algorithm. In addition, functional classification methods, KEGG pathway mapping and also a machine learning approach (Random Forest) were utilized., Results: A number of 966 (150 mM EtOH), 1,334 (300 mM EtOH), or 4,132 (600 mM EtOH) genes were found to be differentially expressed. Dose-response relationships of the identified clusters of co-expressed genes showed a monotonic, threshold, or nonmonotonic (hormetic) behavior. Functional classification of DEGs revealed that low or medium EtOH concentrations operate adaptation processes, while alterations observed for the high EtOH concentration reflect the response to cellular damage. The genes displaying a hormetic response were functionally characterized by overrepresented "cellular ketone metabolism" and "carboxylic acid metabolism." Altered expression of the genes BAHD1 and H3F3B was identified as sufficient to classify the samples according to the applied EtOH doses., Conclusions: Different pathways of metabolic and epigenetic regulation are affected by EtOH exposition and partly undergo hormetic regulation in the bioartificial liver device. Gene expression changes observed at high EtOH concentrations reflect in some aspects the situation of alcoholic hepatitis in humans., (Copyright © 2017 by the Research Society on Alcoholism.)

Hydrogen sulfide (H2S) is a gaseous signalling molecule involved in many physiological and pathological processes. There is increasing evidence that H2S is implicated in aging and lifespan control in the diet-induced longevity models. However, blood sulfide concentration of naturally long-lived species is not known. Here we measured blood sulfide in the long-lived naked mole-rat and five other mammalian species considerably differing in lifespan and found a negative correlation between blood sulfide and maximum longevity residual. In addition, we show that the naked mole-rat cystathionine β-synthase (CBS), an enzyme whose activity in the liver significantly contributes to systemic sulfide levels, has lower activity in the liver and is activated to a higher degree by S-adenosylmethionine compared to other species. These results add complexity to the understanding of the role of H2S in aging and call for detailed research on naked mole-rat transsulfuration., (Copyright © 2016 The Authors. Published by Elsevier B.V. All rights reserved.)

Background: Cellular senescence is induced either internally, for example by replication exhaustion and cell division, or externally, for example by irradiation. In both cases, cellular damages accumulate which, if not successfully repaired, can result in senescence induction. Recently, we determined the transcriptional changes combined with the transition into replicative senescence in primary human fibroblast strains. Here, by γ-irradiation we induced premature cellular senescence in the fibroblast cell strains (HFF and MRC-5) and determined the corresponding transcriptional changes by high-throughput RNA sequencing., Results: Comparing the transcriptomes, we found a high degree of similarity in differential gene expression in replicative as well as in irradiation induced senescence for both cell strains suggesting, in each cell strain, a common cellular response to error accumulation. On the functional pathway level, "Cell cycle" was the only pathway commonly down-regulated in replicative and irradiation-induced senescence in both fibroblast strains, confirming the tight link between DNA repair and cell cycle regulation. However, "DNA repair" and "replication" pathways were down-regulated more strongly in fibroblasts undergoing replicative exhaustion. We also retrieved genes and pathways in each of the cell strains specific for irradiation induced senescence., Conclusion: We found the pathways associated with "DNA repair" and "replication" less stringently regulated in irradiation induced compared to replicative senescence. The strong regulation of these pathways in replicative senescence highlights the importance of replication errors for its induction.

Sepsis is the systemic inflammatory host response to infection. Cystathionine beta-synthase (CBS)-dependent homocysteine (Hcy) pathway was demonstrated to affect disease severity and mortality in patients with severe sepsis/septic shock. Independent studies identified a single-nucleotide polymorphism (SNP, rs6586282, hg19 chr21:g.44478497C>T) in intron 14 of the CBS-coding gene (CBS) associated with Hcy plasma levels. We aimed to describe the association of this SNP and variants of a splice donor-affecting variable-number tandem repeat (VNTR, NG&#95;008938.1:g.22763&#95;22793[16&#95;22]) 243 bp downstream of rs6586282 with severe human sepsis. We analyzed the VNTR structure and genotyped variants of rs6586282 and a neighboring SNP (rs34758144, hg19 chr21:g.44478582G>A) in two case-control studies including patients with severe sepsis/septic shock from Germany (n=168) and Greece (n=237). In both studies, we consistently observed an association of CBS VNTR alleles with sepsis susceptibility. Risk linearly increased with number of tandem repeats (per allele odds ratio in the adjusted analysis 1.34; 95% confidence interval (CI)=1.17-1.55; P<0.001). Association had also been shown for rs34758144 whose risk allele is in linkage disequilibrium with one long VNTR allele (19 repeat). In contrast, we observed no evidence for an effect on 28-day survival in patients with severe sepsis/septic shock (per allele hazard ratio in the adjusted analysis for VNTR 1.10; 95% CI=0.95-1.28; P=0.20). In a minigene approach, we demonstrated alternative splicing in distinct VNTR alleles, which, however, was independent of the number of tandem units. In conclusion, there is no ordinary conjunction between human CBS and severe sepsis/septic shock, but CBS genotypes are involved in disease susceptibility.

Mutations and genetic variability affect gene expression and lifespan, but the impact of variations in gene expression within individuals on their aging-related mortality is poorly understood. We performed a longitudinal study in the short-lived killifish, Nothobranchius furzeri, and correlated quantitative variations in gene expression during early adult life with lifespan. Shorter- and longer-lived individuals differ in their gene expression before the onset of aging-related mortality; differences in gene expression are more pronounced early in life. We identified mitochondrial respiratory chain complex I as a hub in a module of genes whose expression is negatively correlated with lifespan. Accordingly, partial pharmacological inhibition of complex I by the small molecule rotenone reversed aging-related regulation of gene expression and extended lifespan in N. furzeri by 15%. These results support the use of N. furzeri as a vertebrate model for identifying the protein targets, pharmacological modulators, and individual-to-individual variability associated with aging., (Copyright © 2016 The Authors. Published by Elsevier Inc. All rights reserved.)

Acid sphingomyelinase (ASM or sphingomyelin phosphodiesterase, SMPD) activity engages a critical role for regulation of immune response and development of organ failure in critically ill patients. Beside genetic variation in the human gene encoding ASM (SMPD1), alternative splicing of the mRNA is involved in regulation of enzymatic activity. Here we show that the patterns of alternatively spliced SMPD1 transcripts are significantly different in patients with systemic inflammatory response syndrome and severe sepsis/septic shock compared to control subjects allowing discrimination of respective disease entity. The different splicing patterns might contribute to the better understanding of the pathophysiology of human sepsis.

Glucose restriction mimicked by feeding the roundworm Caenorhabditis elegans with 2-deoxy-D-glucose (DOG) - a glucose molecule that lacks the ability to undergo glycolysis - has been found to increase the life span of the nematodes considerably. To facilitate understanding of the molecular mechanisms behind this life extension, we analyzed transcriptomes of DOG-treated and untreated roundworms obtained by RNA-seq at different ages. We found that, depending on age, DOG changes the magnitude of the expression values of about 2 to 24 percent of the genes significantly, although our results reveal that the gross changes introduced by DOG are small compared to the age-induced changes. We found that 27 genes are constantly either up- or down-regulated by DOG over the whole life span, among them several members of the cytochrome P450 family. The monotonic change with age of the temporal expression patterns of the genes was investigated, leading to the result that 21 genes reverse their monotonic behaviour under impaired glycolysis. Put simply, the DOG-treatment reduces the gross transcriptional activity but increases the interconnectedness of gene expression. However, a detailed analysis of network parameters discloses that the introduced changes differ remarkably between individual signalling pathways. We found a reorganization of the hubs of the mTOR pathway when standard diet is replaced by DOG feeding. By constructing correlation based difference networks, we identified those signalling pathways that are most vigorously changed by impaired glycolysis. Taken together, we have found a number of genes and pathways that are potentially involved in the DOG-driven extension of life span of C. elegans. Furthermore, our results demonstrate how the network structure of ageing-relevant signalling pathways is reorganised under impaired glycolysis.

The single nucleotide polymorphism rs2071746 and a (GT)n microsatellite within the human gene encoding heme oxygenase-1 (HMOX1) are associated with incidence or outcome in a variety of diseases. Most of these associations involve either release of heme or oxidative stress. Both polymorphisms are localized in the promoter region, but previously reported correlations with heme oxygenase-1 expression remain not coherent. This ambiguity suggests a more complex organization of the 5' gene region which we sought to investigate more fully. We evaluated the 5' end of HMOX1 and found a novel first exon 1a placing the two previously reported polymorphisms in intronic or exonic positions within the 5' untranslated region respectively. Expression of exon 1a can be induced in HepG2 hepatoma cells by hemin and is a repressor of heme oxygenase-1 translation as shown by luciferase reporter assays. Moreover, minigene approaches revealed that the quantitative outcome of alternative splicing within the 5' untranslated region is affected by the (GT)n microsatellite. This data supporting an extended HMOX1 gene model and provide further insights into expression regulation of heme oxygenase-1. Alternative splicing within the HMOX1 5' untranslated region contributes to translational regulation and is a mechanistic feature involved in the interplay between genetic variations, heme oxygenase-1 expression and disease outcome.

Splicing generates mature transcripts from genes in pieces in eukaryotic cells. Overwhelming evidence has accumulated that alternative routes in splicing are possible for most human and mammalian genes, thereby allowing formation of different transcripts from one gene. No function has been assigned to the majority of identified alternative splice forms, and it has been assumed that they compose inert or tolerated waste from aberrant or noisy splicing. Here we demonstrate that five human transcription units (WT1, NOD2, GNAS, RABL2A, RABL2B) have constant splice-isoform ratios in genetically diverse lymphoblastoid cell lines independent of the type of alternative splicing (exon skipping, alternative donor/acceptor, tandem splice sites) and gene expression level. Even splice events that create premature stop codons and potentially trigger nonsense-mediated mRNA decay are found at constant fractions. The analyzed alternative splicing events were qualitatively but not quantitatively conserved in corresponding chimpanzee cell lines. Additionally, subtle splicing at tandem acceptor splice sites (GNAS, RABL2A/B) was highly constrained and strongly depends on the upstream donor sequence content. These results also demonstrate that unusual and unproductive splice variants are produced in a regulated manner., (© 2011 by the Genetics Society of America)

Pheochromocytomas and abdominal paragangliomas are adrenal and extra-adrenal catecholamine-producing tumours. They arise due to heritable cancer syndromes, or more frequently occur sporadically due to an unknown genetic cause. The majority of cases are benign, but malignant tumours are observed. Previous comparative genomic hybridization (CGH) and loss of heterozygosity studies have shown frequent deletions of chromosome arms 1p, 3q and 22q in pheochromocytomas. We applied high-resolution whole-genome array CGH on 53 benign and malignant pheochromocytomas and paragangliomas to narrow down candidate regions as well as to identify chromosomal alterations more specific to malignant tumours. Minimal overlapping regions (MORs) were identified on 16 chromosomes, with the most frequent MORs of deletion (> or = 32%) occurring on chromosome arms 1p, 3q, 11p/q, 17p and 22q, while the chromosome arms 1q, 7p, 12q and 19p harboured the most common MORs of gain (> or = 14%). The most frequent MORs (61-75%) in the pheochromocytomas were identified at 1p, and the four regions of common losses encompassed 1p36, 1p32-31, 1p22-21 and 1p13. Tumours that did not show 1p loss generally demonstrated aberrations on chromosome 11. Gain of chromosomal material was significantly more frequent among the malignant cases. Moreover, gain at 19q, trisomy 12 and loss at 11q were positively associated with malignant pheochromocytomas, while 1q gain was commonly observed in the malignant paragangliomas. Our study revealed novel and narrow recurrent chromosomal regions of loss and gain at several autosomes, a prerequisite for identifying candidate tumour suppressor genes and oncogenes involved in the development of adrenal and extra-adrenal catecholamine-producing tumours.

Breast cancer is a major cause of morbidity and mortality in women and its metastatic spread is the principal reason behind the fatal outcome. Metastasis-related research of breast cancer is however underdeveloped when compared with the abundant literature on primary tumors. We applied an unexplored approach comparing at high resolution the genomic profiles of primary tumors and synchronous axillary lymph node metastases from 13 patients with breast cancer. Overall, primary tumors displayed 20% higher number of aberrations than metastases. In all but two patients, we detected in total 157 statistically significant differences between primary lesions and matched metastases. We further observed differences that can be linked to metastatic disease and there was also an overlapping pattern of changes between different patients. Many of the differences described here have been previously linked to poor patient survival, suggesting that this is a viable approach toward finding biomarkers for disease progression and definition of new targets useful for development of anticancer drugs. Frequent genetic differences between primary tumors and metastases in breast cancer also question, at least to some extent, the role of primary tumors as a surrogate subject of study for the systemic disease.

Urinary bladder cancer is a heterogeneous disease with tumors ranging from papillary noninvasive (stage Ta) to solid muscle infiltrating tumors (stage T2+). The risk of progression and death for the most frequent diagnosed type, Ta, is low, but the high incidence of recurrences has a significant effect on the patients' quality of life and poses substantial costs for health care systems. Consequently, the purpose of this study was to search for predictive factors of recurrence on the basis of genetic profiling. A clinically well characterized cohort of Ta bladder carcinomas, selected by the presence or absence of recurrences, was evaluated by an integrated analysis of DNA copy number changes and gene expression (clone-based 32K, respectively, U133Plus2.0 arrays). Only a few chromosomal aberrations have previously been defined in superficial bladder cancer. Surprisingly, the profiling of Ta tumors with a high-resolution array showed that DNA copy alterations are relatively common in this tumor type. Furthermore, we observed an overrepresentation of focal amplifications within high-grade and recurrent cases. Known (FGFR3, CCND1, MYC, MDM2) and novel candidate genes were identified within the loci. For example, MYBL2, a nuclear transcription factor involved in cell-cycle progression; YWHAB, an antiapoptotic protein; and SDC4, an important component of focal adhesions represent interesting candidates detected within two amplicons on chromosome 20, for which DNA amplification correlated with transcript up-regulation. The observed overrepresentation of amplicons within high-grade and recurrent cases may be clinically useful for the identification of patients who will benefit from a more aggressive therapy.

Glioblastomas (GBs) are malignant CNS tumors often associated with devastating symptoms. Patients with GB have a very poor prognosis, and despite treatment, most of them die within 12 months from diagnosis. Several pathways, such as the RAS, tumor protein 53 (TP53), and phosphoinositide kinase 3 (PIK3) pathways, as well as the cell cycle control pathway, have been identified to be disrupted in this tumor. However, emerging data suggest that these aberrations represent only a fraction of the genetic changes involved in gliomagenesis. In this study, we have applied a 32K clone-based genomic array, covering 99% of the current assembly of the human genome, to the detailed genetic profiling of a set of 78 GBs. Complex patterns of aberrations, including high and narrow copy number amplicons, as well as a number of homozygously deleted loci, were identified. Amplicons that varied both in number (three on average) and in size (1.4 Mb on average) were frequently detected (81% of the samples). The loci encompassed not only previously reported oncogenes (EGFR, PDGFRA, MDM2, and CDK4) but also numerous novel oncogenes as GRB10, MKLN1, PPARGC1A, HGF, NAV3, CNTN1, SYT1, and ADAMTSL3. BNC2, PTPLAD2, and PTPRE, on the other hand, represent novel candidate tumor suppressor genes encompassed within homozygously deleted loci. Many of these genes are already linked to several forms of cancer; others represent new candidate genes that may serve as prognostic markers or even as therapeutic targets in the future. The large individual variation observed between the samples demonstrates the underlying complexity of the disease and strengthens the demand for an individualized therapy based on the genetic profile of the patient.

Neurofibromatosis Type I (NF1) is an autosomal dominant disorder characterized by the development of both benign and malignant tumors. The lifetime risk for developing a malignant peripheral nerve sheath tumor (MPNST) in NF1 patients is approximately 10% with poor survival rates. To date, the molecular basis of MPNST development remains unclear. Here, we report the first genome-wide and high-resolution analysis of DNA copy number alterations in MPNST using the 32K bacterial artificial chromosome microarray on a series of 24 MPNSTs and three neurofibroma samples. In the benign neurofibromas, apart from loss of one copy of the NF1 gene and copy number polymorphisms, no other changes were found. The profiles of malignant samples, however, revealed specific loss of chromosomal regions including 1p35-33, 1p21, 9p21.3, 10q25, 11q22-23, 17q11, and 20p12.2 as well as gain of 1q25, 3p26, 3q13, 5p12, 5q11.2-q14, 5q21-23, 5q31-33, 6p23-p21, 6p12, 6q15, 6q23-q24, 7p22, 7p14-p13, 7q21, 7q36, 8q22-q24, 14q22, and 17q21-q25. Copy number gains were more frequent than deletions in the MPNST samples (62% vs. 38%). The genes resident within common regions of gain were NEDL1 (7p14), AP3B1 (5q14.1), and CUL1 (7q36.1) and these were identified in >63% MPNSTs. The most frequently deleted locus encompassed CDKN2A, CDKN2B, and MTAP genes on 9p21.3 (33% cases). These genes have previously been implicated in other cancer conditions and therefore, should be considered for their therapeutic, prognostic, and diagnostic relevance in NF1 tumorigenesis., ((c) 2009 Wiley-Liss, Inc.)

Background: DNA methylation is a major epigenetic modification important for regulating gene expression and suppressing spurious transcription. Most methods to scan the genome in different tissues for differentially methylated sites have focused on the methylation of CpGs in CpG islands, which are concentrations of CpGs often associated with gene promoters., Results: Here, we use a methylation profiling strategy that is predominantly responsive to methylation differences outside of CpG islands. The method compares the yield from two samples of size-selected fragments generated by a methylation-sensitive restriction enzyme. We then profile nine different normal tissues from two human donors relative to spleen using a custom array of genomic clones covering the euchromatic portion of human chromosome 1 and representing 8% of the human genome. We observe gross regional differences in methylation states across chromosome 1 between tissues from the same individual, with the most striking differences detected in the comparison of cerebellum and spleen. Profiles of the same tissue from different donors are strikingly similar, as are the profiles of different lobes of the brain. Comparing our results with published gene expression levels, we find that clones exhibiting extreme ratios reflecting low relative methylation are statistically enriched for genes with high expression ratios, and vice versa, in most pairs of tissues examined., Conclusion: The varied patterns of methylation differences detected between tissues by our methylation profiling method reinforce the potential functional significance of regional differences in methylation levels outside of CpG islands.

Two major types of genetic variation are known: single nucleotide polymorphisms (SNPs), and a more recently discovered structural variation, involving changes in copy number (CNVs) of kilobase- to megabase-sized chromosomal segments. It is unknown whether CNVs arise in somatic cells, but it is, however, generally assumed that normal cells are genetically identical. We tested 34 tissue samples from three subjects and, having analyzed for each tissue < or =10(-6) of all cells expected in an adult human, we observed at least six CNVs, affecting a single organ or one or more tissues of the same subject. The CNVs ranged from 82 to 176 kb, often encompassing known genes, potentially affecting gene function. Our results indicate that humans are commonly affected by somatic mosaicism for stochastic CNVs, which occur in a substantial fraction of cells. The majority of described CNVs were previously shown to be polymorphic between unrelated subjects, suggesting that some CNVs previously reported as germline might represent somatic events, since in most studies of this kind, only one tissue is typically examined and analysis of parents for the studied subjects is not routinely performed. A considerable number of human phenotypes are a consequence of a somatic process. Thus, our conclusions will be important for the delineation of genetic factors behind these phenotypes. Consequently, biobanks should consider sampling multiple tissues to better address mosaicism in the studies of somatic disorders.

Motivation: Copy number profiling methods aim at assigning DNA copy numbers to chromosomal regions using measurements from microarray-based comparative genomic hybridizations. Among the proposed methods to this end, Hidden Markov Model (HMM)-based approaches seem promising since DNA copy number transitions are naturally captured in the model. Current discrete-index HMM-based approaches do not, however, take into account heterogeneous information regarding the genomic overlap between clones. Moreover, the majority of existing methods are restricted to chromosome-wise analysis., Results: We introduce a novel Segmental Maximum A Posteriori approach, SMAP, for DNA copy number profiling. Our method is based on discrete-index Hidden Markov Modeling and incorporates genomic distance and overlap between clones. We exploit a priori information through user-controllable parameterization that enables the identification of copy number deviations of various lengths and amplitudes. The model parameters may be inferred at a genome-wide scale to avoid overfitting of model parameters often resulting from chromosome-wise model inference. We report superior performances of SMAP on synthetic data when compared with two recent methods. When applied on our new experimental data, SMAP readily recognizes already known genetic aberrations including both large-scale regions with aberrant DNA copy number and changes affecting only single features on the array. We highlight the differences between the prediction of SMAP and the compared methods and show that SMAP accurately determines copy number changes and benefits from overlap consideration.

To further explore the extent of structural large-scale variation in the human genome, we assessed copy number variations (CNVs) in a series of 71 healthy subjects from three ethnic groups. CNVs were analyzed using comparative genomic hybridization (CGH) to a BAC array covering the human genome, using DNA extracted from peripheral blood, thus avoiding any culture-induced rearrangements. By applying a newly developed computational algorithm based on Hidden Markov modeling, we identified 1,078 autosomal CNVs, including at least two neighboring/overlapping BACs, which represent 315 distinct regions. The average size of the sequence polymorphisms was approximately 350 kb and involved in total approximately 117 Mb or approximately 3.5% of the genome. Gains were about four times more common than deletions, and segmental duplications (SDs) were overrepresented, especially in larger deletion variants. This strengthens the notion that SDs often define hotspots of chromosomal rearrangements. Over 60% of the identified autosomal rearrangements match previously reported CNVs, recognized with various platforms. However, results from chromosome X do not agree well with the previously annotated CNVs. Furthermore, data from single BACs deviating in copy number suggest that our above estimate of total variation is conservative. This report contributes to the establishment of the common baseline for CNV, which is an important resource in human genetics.

The exploration of copy-number variation (CNV), notably of somatic cells, is an understudied aspect of genome biology. Any differences in the genetic makeup between twins derived from the same zygote represent an irrefutable example of somatic mosaicism. We studied 19 pairs of monozygotic twins with either concordant or discordant phenotype by using two platforms for genome-wide CNV analyses and showed that CNVs exist within pairs in both groups. These findings have an impact on our views of genotypic and phenotypic diversity in monozygotic twins and suggest that CNV analysis in phenotypically discordant monozygotic twins may provide a powerful tool for identifying disease-predisposition loci. Our results also imply that caution should be exercised when interpreting disease causality of de novo CNVs found in patients based on analysis of a single tissue in routine disease-related DNA diagnostics.

A previously detected copy number polymorphism (Ep CNP) in patients affected with neuroectodermal tumors led us to investigate its frequency and length in the normal population. For this purpose, a program called Sequence Allocator was developed and applied for the construction of an array that consisted of unique and duplicated fragments, allowing the assessment of copy number variation within regions of segmental duplications. The average resolution of this array was 11 kb and we determined the size of the Ep CNP to be 290 kb. Analysis of normal controls identified 7.7 and 7.1% gains in peripheral blood and lymphoblastoid cell line (LCL) DNA, respectively, while deletions were found only in the LCL group (7.1%). This array platform allows the detection of DNA copy number variation within regions of pronounced genomic complexity, which constitutes an improvement over available technologies.

Maintenance of CpG island methylation in the genome is crucial for cellular homeostasis and this balance is disrupted in cancer. Our rationale was to compare the methylation of CpG islands in tissues (tumor, healthy breast and blood) from patients with breast cancer. We studied 72 genes in 103 samples using microarray hybridization and bisulfite sequencing. We observed tumor specific hyper- or hypomethylation of five genes; COL9A1, MT1A, MT1J, HOXA5 and FLJ45983. A general drop of methylation in COL9A1 was apparent in tumors, when compared with blood and healthy breast tissue. Furthermore, one tumor displayed a complete loss of methylation of all five genes, suggesting overall impairment of methylation. The downstream, evolutionary conserved island of HOXA5 showed hypomethylation in 18 tumors and complete methylation in others. This CpG island also displayed a semimethylated state in the majority of normal breast samples, when compared to complete methylation in blood. Distinct methylation patterns were further seen in MT1J and MT1A, belonging to the metallothionein gene family. The CpG islands of these genes are spaced by 2 kb, which shows selective methylation of two structurally and functionally related genes. The promoters of FLJ45983 and MT1A were methylated above 25% in 18 primary and metastatic tumors. Concurrently, there was also >10% methylation of healthy breast tissue in 11 and 5 samples, respectively. This suggests that the methylation process for the latter two genes takes place already in normal breast cells. Our results also point to a considerable heterogeneity of epigenetic disturbance in breast cancer. This article contains Supplementary Material available at http://www.interscience.wiley.com/jpages/1045-2257/suppmat., ((c) 2006 Wiley-Liss, Inc.)

The International Human Genome Sequencing Consortium (IHGSC) recently completed a sequence of the human genome. As part of this project, we have focused on chromosome 8. Although some chromosomes exhibit extreme characteristics in terms of length, gene content, repeat content and fraction segmentally duplicated, chromosome 8 is distinctly typical in character, being very close to the genome median in each of these aspects. This work describes a finished sequence and gene catalogue for the chromosome, which represents just over 5% of the euchromatic human genome. A unique feature of the chromosome is a vast region of approximately 15 megabases on distal 8p that appears to have a strikingly high mutation rate, which has accelerated in the hominids relative to other sequenced mammals. This fast-evolving region contains a number of genes related to innate immunity and the nervous system, including loci that appear to be under positive selection--these include the major defensin (DEF) gene cluster and MCPH1, a gene that may have contributed to the evolution of expanded brain size in the great apes. The data from chromosome 8 should allow a better understanding of both normal and disease biology and genome evolution.

Schwannomatosis is characterized by multiple peripheral and cranial nerve schwannomas that occur in the absence of bilateral 8th cranial nerve schwannomas. The latter is the main diagnostic criterion of neurofibromatosis type 2 (NF2), which is a related but distinct disorder. The genetic factors underlying the differences between schwannomatosis and NF2 are poorly understood, although available evidence implicates chromosome 22 as the primary location of the gene(s) of interest. To investigate this, we comprehensively profiled the DNA copy number in samples from sporadic and familial schwannomatosis, NF2, and a large cohort of normal controls. Using a tiling-path chromosome 22 genomic array, we identified two candidate regions of copy number variation, which were further characterized by a PCR-based array with higher resolution. The latter approach allows the detection of minute alterations in total genomic DNA, with as little as 1.5 kb per measurement point of nonredundant sequence on the array. In DNA derived from peripheral blood from a schwannomatosis patient and a sporadic schwannoma sample, we detected rearrangements of the immunoglobulin lambda (IGL) locus, which is unlikely to be due to a B-cell specific somatic recombination of IGL. Analysis of normal controls indicated that these IGL rearrangements were restricted to schwannomatosis/schwannoma samples. In the second candidate region spanning GSTT1 and CABIN1 genes, we observed a frequent copy number polymorphism at the GSTT1 locus. We further describe missense mutations in the CABIN1 gene that are specific to samples from schwannomatosis and NF2 and make this gene a plausible candidate for contributing to the pathogenesis of these disorders., (Copyright 2005 Wiley-Liss, Inc.)

Meningiomas are common neoplasms of the meninges lining of the central nervous system. Deletions of 1p have been established as important for the initiation and/or progression of meningioma. The rationale of this array-CGH study was to characterize copy number imbalances of chromosome 1 in meningioma, using a full-coverage genomic microarray containing 2,118 distinct measurement points. In total, 82 meningiomas were analyzed, making this the most detailed analysis of chromosome 1 in a comprehensive series of tumors. We detected a broad range of aberrations, such as deletions and/or gains of various sizes. Deletions were the predominant finding and ranged from monosomy to a 3.5-Mb terminal 1p homozygous deletion. Although multiple aberrations were observed across chromosome 1, every meningioma in which imbalances were detected harbored 1p deletions. Tumor heterogeneity was also observed in three recurrent meningiomas, which most likely reflects a progressive loss of chromosomal segments at different stages of tumor development. The distribution of aberrations supports the existence of at least four candidate loci on chromosome 1, which are important for meningioma tumorigenesis. In one of these regions, our results already allow the analysis of a number of candidate genes. In a large series of cases, we observed an association between the presence of segmental duplications and deletion breakpoints, which suggests their role in the generation of these tumor-specific aberrations. As 1p is the site of the genome most frequently affected by tumor-specific aberrations, our results indicate loci of general importance for cancer development and progression.

We analyzed 137 kb covering human neurofibromatosis 2 ( NF2) tumor suppressor locus and orthologous loci from baboon, mouse, rat, and pufferfish Takifugu rubripes. A predominant feature of human-rodent conservation is a very similar distribution of conserved islands, regarding length, position, and degree of identity. By use of a threshold of 75% identity over > or =100 bp of gap-free alignment, comparisons of human-mouse sequences resulted in 3.58% for extra-exonic conservation, which can be compared to 4.5% of exonic sequence content within the human locus. We identified a duplication of neurofibromin 2 in pufferfish, which resulted in two putative proteins with 74% and 76% identity to the human protein. One distinct island (called inter 1), conserved between all analyzed species, was located between promoters of the NIPSNAP1 and NF2 genes. Inter 1 might represent a novel regulatory element, important for the function of this locus. The high level of intronic conservation in the NF2 locus suggests that a number of unknown regulatory elements might exist within this gene. These elements could be affected by disease-causing mutations in NF2 patients and NF2-associated tumors. Alternatively, this conservation might be explained by presence of not yet characterized transcriptional unit(s) within this locus.

Neurofibromatosis type 2 (NF2) is an autosomal dominant cancer syndrome caused by the biallelic inactivation of the neurofibromin 2 tumor suppressor gene ( NF2). Current molecular diagnostic methods for NF2 involve the detection of point mutations and/or microdeletions across the 100-kb locus from 22q12. Despite the fact that NF2 gene inactivating deletions occur in 25-30% of NF2 patients, the available approaches for high-resolution and high-throughput detection of deletions are underdeveloped. This need for improved methodology for gene copy number analysis is especially apparent when compared to a variety of methods available for accurate detection of point mutations. The microarray-based form of comparative genomic hybridization has been previously applied in the high-resolution analysis of gene copy number variation across large genomic regions. In this study we apply a PCR-based, strictly sequence-defined, repeat-free approach for the preparation of a diagnostic microarray for the detection of disease-causing deletions in the NF2 gene. The methodology is based on the preselection of target DNA by excluding redundant sequence within the NF2 locus using bioinformatics. This approach allows a significant increase in the resolution of deletion detection. The current average resolution of analysis across the NF2 locus is 23 kb. Therefore this NF2 gene-specific microarray is the first high-resolution tool for detection of diagnostically significant gene copy number aberrations. This microarray should now be applied in the analysis of an extensive series of NF2 patients, and hence we would like to call for such samples.

We have constructed the first comprehensive microarray representing a human chromosome for analysis of DNA copy number variation. This chromosome 22 array covers 34.7 Mb, representing 1.1% of the genome, with an average resolution of 75 kb. To demonstrate the utility of the array, we have applied it to profile acral melanoma, dermatofibrosarcoma, DiGeorge syndrome and neurofibromatosis 2. We accurately diagnosed homozygous/heterozygous deletions, amplifications/gains, IGLV/IGLC locus instability, and breakpoints of an imbalanced translocation. We further identified the 14-3-3 eta isoform as a candidate tumor suppressor in glioblastoma. Two significant methodological advances in array construction were also developed and validated. These include a strictly sequence defined, repeat-free, and non-redundant strategy for array preparation. This approach allows an increase in array resolution and analysis of any locus; disregarding common repeats, genomic clone availability and sequence redundancy. In addition, we report that the application of phi29 DNA polymerase is advantageous in microarray preparation. A broad spectrum of issues in medical research and diagnostics can be approached using the array. This well annotated and gene-rich autosome contains numerous uncharacterized disease genes. It is therefore crucial to associate these genes to specific 22q-related conditions and this array will be instrumental towards this goal. Furthermore, comprehensive epigenetic profiling of 22q-located genes and high-resolution analysis of replication timing across the entire chromosome can be studied using our array.