Einar Andreas Rødland

RNAmmer: consistent and rapid annotation of ribosomal RNA genes

The publication of a complete genome sequence is usually accompanied by annotations of its genes. In contrast to protein coding genes, genes for ribosomal RNA (rRNA) are often poorly or inconsistently annotated. This makes comparative studies based on rRNA genes difficult. We have therefore created computational predictors for the major rRNA species from all kingdoms of life and compiled them into a program called RNAmmer. The program uses hidden Markov models trained on data from the 5S ribosomal RNA database and the European ribosomal RNA database project. A pre-screening step makes the method fast with little loss of sensitivity, enabling the analysis of a complete bacterial genome in less than a minute. Results from running RNAmmer on a large set of genomes indicate that the location of rRNAs can be predicted with a very high level of accuracy. Novel, unannotated rRNAs are also predicted in many genomes. The software as well as the genome analysis results are available at the CBS web server.

miRNA-mRNA Integrated Analysis Reveals Roles for miRNAs in Primary Breast Tumors

Introduction Few studies have performed expression profiling of both miRNA and mRNA from the same primary breast carcinomas. In this study we present and analyze data derived from expression profiling of 799 miRNAs in 101 primary human breast tumors, along with genome-wide mRNA profiles and extensive clinical information. Methods We investigate the relationship between these molecular components, in terms of their correlation with each other and with clinical characteristics. We use a systems biology approach to examine the correlative relationship between miRNA and mRNAs using statistical enrichment methods. Results We identify statistical significant differential expression of miRNAs between molecular intrinsic subtypes, and between samples with different levels of proliferation. Specifically, we point to miRNAs significantly associated with TP53 and ER status. We also show that several cellular processes, such as proliferation, cell adhesion and immune response, are strongly associated with certain miRNAs. We validate the role of miRNAs in regulating proliferation using high-throughput lysate-microarrays on cell lines and point to potential drivers of this process. Conclusion This study provides a comprehensive dataset as well as methods and system-level results that jointly form a basis for further work on understanding the role of miRNA in primary breast cancer.

Cancer incidence and survival in Lynch syndrome patients receiving colonoscopic and gynaecological surveillance: first report from the prospective Lynch syndrome database.

Objective Estimates of cancer risk and the effects of surveillance in Lynch syndrome have been subject to bias, partly through reliance on retrospective studies. We sought to establish more robust estimates in patients undergoing prospective cancer surveillance. Design We undertook a multicentre study of patients carrying Lynch syndrome-associated mutations affecting MLH1, MSH2, MSH6 or PMS2. Standardised information on surveillance, cancers and outcomes were collated in an Oracle relational database and analysed by age, sex and mutated gene. Results 1942 mutation carriers without previous cancer had follow-up including colonoscopic surveillance for 13 782 observation years. 314 patients developed cancer, mostly colorectal (n=151), endometrial (n=72) and ovarian (n=19). Cancers were detected from 25 years onwards in MLH1 and MSH2 mutation carriers, and from about 40 years in MSH6 and PMS2 carriers. Among first cancer detected in each patient the colorectal cancer cumulative incidences at 70 years by gene were 46%, 35%, 20% and 10% for MLH1, MSH2, MSH6 and PMS2 mutation carriers, respectively. The equivalent cumulative incidences for endometrial cancer were 34%, 51%, 49% and 24%; and for ovarian cancer 11%, 15%, 0% and 0%. Ten-year crude survival was 87% after any cancer, 91% if the first cancer was colorectal, 98% if endometrial and 89% if ovarian. Conclusions The four Lynch syndrome-associated genes had different penetrance and expression. Colorectal cancer occurred frequently despite colonoscopic surveillance but resulted in few deaths. Using our data, a website has been established at http://LScarisk.org enabling calculation of cumulative cancer risks as an aid to genetic counselling in Lynch syndrome.

The Pfaffian Calabi-Yau, its Mirror, and their link to the Grassmannian G(2,7)

The rank 4 locus of a general skew-symmetric 7 × 7 matrix gives the Pfaffian variety in P20 which is not defined as a complete intersection. Intersecting this with a general P6 gives a Calabi–Yau manifold. An orbifold construction seems to give the 1-parameter mirror-family of this. However, corresponding to two points in the 1-parameter family of complex structures, both with maximally unipotent monodromy, are two different mirror-maps: one corresponding to the general Pfaffian section, the other to a general intersection of G(2,7) ⊂ P20 with a P13. Apparently, the Pfaffian and G(2,7) sections constitute different parts of the A-model (Kähler structure related) moduli space, and, thus, represent different parts of the same conformal field theory moduli space.

Combining gene signatures improves prediction of breast cancer survival.

Background Several gene sets for prediction of breast cancer survival have been derived from whole-genome mRNA expression profiles. Here, we develop a statistical framework to explore whether combination of the information from such sets may improve prediction of recurrence and breast cancer specific death in early-stage breast cancers. Microarray data from two clinically similar cohorts of breast cancer patients are used as training (n = 123) and test set (n = 81), respectively. Gene sets from eleven previously published gene signatures are included in the study. Principal Findings To investigate the relationship between breast cancer survival and gene expression on a particular gene set, a Cox proportional hazards model is applied using partial likelihood regression with an L2 penalty to avoid overfitting and using cross-validation to determine the penalty weight. The fitted models are applied to an independent test set to obtain a predicted risk for each individual and each gene set. Hierarchical clustering of the test individuals on the basis of the vector of predicted risks results in two clusters with distinct clinical characteristics in terms of the distribution of molecular subtypes, ER, PR status, TP53 mutation status and histological grade category, and associated with significantly different survival probabilities (recurrence: p = 0.005; breast cancer death: p = 0.014). Finally, principal components analysis of the gene signatures is used to derive combined predictors used to fit a new Cox model. This model classifies test individuals into two risk groups with distinct survival characteristics (recurrence: p = 0.003; breast cancer death: p = 0.001). The latter classifier outperforms all the individual gene signatures, as well as Cox models based on traditional clinical parameters and the Adjuvant! Online for survival prediction. Conclusion Combining the predictive strength of multiple gene signatures improves prediction of breast cancer survival. The presented methodology is broadly applicable to breast cancer risk assessment using any new identified gene set.

Methods that remove batch effects while retaining group differences may lead to exaggerated confidence in downstream analyses.

Abstract Removal of, or adjustment for, batch effects or center differences is generally required when such effects are present in data. In particular, when preparing microarray gene expression data from multiple cohorts, array platforms, or batches for later analyses, batch effects can have confounding effects, inducing spurious differences between study groups. Many methods and tools exist for removing batch effects from data. However, when study groups are not evenly distributed across batches, actual group differences may induce apparent batch differences, in which case batch adjustments may bias, usually deflate, group differences. Some tools therefore have the option of preserving the difference between study groups, e.g. using a two-way ANOVA model to simultaneously estimate both group and batch effects. Unfortunately, this approach may systematically induce incorrect group differences in downstream analyses when groups are distributed between the batches in an unbalanced manner. The scientific community seems to be largely unaware of how this approach may lead to false discoveries.

Genetic Interactions of DNA Repair Pathways in the Pathogen Neisseria meningitidis

The current increase in the incidence and severity of infectious diseases mandates improved understanding of the basic biology and DNA repair profiles of virulent microbes. In our studies of the major pathogen and model organism Neisseria meningitidis, we constructed a panel of mutants inactivating genes involved in base excision repair, mismatch repair, nucleotide excision repair (NER), translesion synthesis, and recombinational repair pathways. The highest spontaneous mutation frequency among the N. meningitidis single mutants was found in the MutY-deficient strain as opposed to mutS mutants in Escherichia coli, indicating a role for meningococcal MutY in antibiotic resistance development. Recombinational repair was recognized as a major pathway counteracting methyl methanesulfonate-induced alkylation damage in the N. meningitidis. In contrast to what has been shown in other species, meningococcal NER did not contribute significantly to repair of alkylation-induced DNA damage, and meningococcal recombinational repair may thus be one of the main pathways for removal of abasic (apurinic/apyrimidinic) sites and strand breaks in DNA. Conversely, NER was identified as the main meningococcal defense pathway against UV-induced DNA damage. N. meningitidis RecA single mutants exhibited only a moderate decrease in survival after UV exposure as opposed to E. coli recA strains, which are extremely UV sensitive, possibly reflecting the lack of a meningococcal SOS response. In conclusion, distinct differences between N. meningitidis and established DNA repair characteristics in E. coli and other species were identified.

Interplay of choline metabolites and genes in patient-derived breast cancer xenografts

IntroductionDysregulated choline metabolism is a well-known feature of breast cancer, but the underlying mechanisms are not fully understood. In this study, the metabolomic and transcriptomic characteristics of a large panel of human breast cancer xenograft models were mapped, with focus on choline metabolism.MethodsTumor specimens from 34 patient-derived xenograft models were collected and divided in two. One part was examined using high-resolution magic angle spinning (HR-MAS) MR spectroscopy while another part was analyzed using gene expression microarrays. Expression data of genes encoding proteins in the choline metabolism pathway were analyzed and correlated to the levels of choline (Cho), phosphocholine (PCho) and glycerophosphocholine (GPC) using Pearson’s correlation analysis. For comparison purposes, metabolic and gene expression data were collected from human breast tumors belonging to corresponding molecular subgroups.ResultsMost of the xenograft models were classified as basal-like (N = 19) or luminal B (N = 7). These two subgroups showed significantly different choline metabolic and gene expression profiles. The luminal B xenografts were characterized by a high PCho/GPC ratio while the basal-like xenografts were characterized by highly variable PCho/GPC ratio. Also, Cho, PCho and GPC levels were correlated to expression of several genes encoding proteins in the choline metabolism pathway, including choline kinase alpha (CHKA) and glycerophosphodiester phosphodiesterase domain containing 5 (GDPD5). These characteristics were similar to those found in human tumor samples.ConclusionThe higher PCho/GPC ratio found in luminal B compared with most basal-like breast cancer xenograft models and human tissue samples do not correspond to results observed from in vitro studies. It is likely that microenvironmental factors play a role in the in vivo regulation of choline metabolism. Cho, PCho and GPC were correlated to different choline pathway-encoding genes in luminal B compared with basal-like xenografts, suggesting that regulation of choline metabolism may vary between different breast cancer subgroups. The concordance between the metabolic and gene expression profiles from xenograft models with breast cancer tissue samples from patients indicates that these xenografts are representative models of human breast cancer and represent relevant models to study tumor metabolism in vivo.

Incidence of and survival after subsequent cancers in carriers of pathogenic MMR variants with previous cancer: a report from the prospective Lynch syndrome database

Objective Today most patients with Lynch syndrome (LS) survive their first cancer. There is limited information on the incidences and outcome of subsequent cancers. The present study addresses three questions: (i) what is the cumulative incidence of a subsequent cancer; (ii) in which organs do subsequent cancers occur; and (iii) what is the survival following these cancers? Design Information was collated on prospectively organised surveillance and prospectively observed outcomes in patients with LS who had cancer prior to inclusion and analysed by age, gender and genetic variants. Results 1273 patients with LS from 10 countries were followed up for 7753 observation years. 318 patients (25.7%) developed 341 first subsequent cancers, including colorectal (n=147, 43%), upper GI, pancreas or bile duct (n=37, 11%) and urinary tract (n=32, 10%). The cumulative incidences for any subsequent cancer from age 40 to age 70 years were 73% for pathogenic MLH1 (path_MLH1), 76% for path_MSH2 carriers and 52% for path_MSH6 carriers, and for colorectal cancer (CRC) the cumulative incidences were 46%, 48% and 23%, respectively. Crude survival after any subsequent cancer was 82% (95% CI 76% to 87%) and 10-year crude survival after CRC was 91% (95% CI 83% to 95%). Conclusions Relative incidence of subsequent cancer compared with incidence of first cancer was slightly but insignificantly higher than cancer incidence in patients with LS without previous cancer (range 0.94–1.49). The favourable survival after subsequent cancers validated continued follow-up to prevent death from cancer. The interactive website http://lscarisk.org was expanded to calculate the risks by gender, genetic variant and age for subsequent cancer for any patient with LS with previous cancer.

Characterization of the major formamidopyrimidine–DNA glycosylase homolog in Mycobacterium tuberculosis and its linkage to variable tandem repeats

The ability to repair DNA damage is likely to play an important role in the survival of facultative intracellular parasites because they are exposed to high levels of reactive oxygen species and nitrogen intermediates inside phagocytes. Correcting oxidative damage in purines and pyrimidines is the primary function of the enzymes formamidopyrimidine (faPy)–DNA glycosylase (Fpg) and endonuclease VIII (Nei) of the base excision repair pathway, respectively. Four gene homologs, belonging to the fpg/nei family, have been identified in Mycobacterium tuberculosis H37Rv. The recombinant protein encoded by M. tuberculosis Rv2924c, termed Mtb-Fpg1, was overexpressed, purified and biochemically characterized. The enzyme removed faPy and 5-hydroxycytosine lesions, as well as 8-oxo-7,8-dihydroguanine (8oxoG) opposite to C, T and G. Mtb-Fpg1 thus exhibited substrate specificities typical for Fpg enzymes. Although Mtb-fpg1 showed nearly complete nucleotide sequence conservation in 32 M. tuberculosis isolates, the region upstream of Mtb-fpg1 in these strains contained tandem repeat motifs of variable length. A relationship between repeat length and Mtb-fpg1 expression level was demonstrated in M. tuberculosis strains, indicating that an increased length of the tandem repeats positively influenced the expression levels of Mtb-fpg1. This is the first example of such a tandem repeat region of variable length being linked to the expression level of a bacterial gene.