Transcriptomic analyses reveal three distinct molecular subgroups of Merkel cell carcinoma with differing prognoses.

Merkel cell carcinoma (MCC) is a cutaneous neuroendocrine malignancy with a poor prognosis and an unknown cell of origin. Proffered cells of origin include epithelial stem cells of the hair follicle or interfollicular epidermis, dermal stem cells and pro/pre- or pre-B cells. MCC has also been proposed to have more than one cell of origin and indeed to represent more than one type of carcinoma, currently grouped together due to phenotypic similarities. We explored the heterogeneous nature of MCC by studying the most variably expressed genes with the goal of identifying gene expression patterns that are either clinically relevant or have implications regarding the cell(s) of origin. We performed RNA sequencing on primary tumor samples from 102 patients and identified the top 200 most variably expressed genes. These genes and the tumor samples were hierarchically clustered based on their expression. The functions of three gene clusters exhibiting clearly divergent expression between samples were studied by cross-referencing the lists of genes with online databases. High expression of a gene cluster related to embryonic developmental processes and low expression of a gene cluster related to neuroendocrine processes distinguished Merkel cell polyomavirus (MCPyV)-negative tumors from MCPyV-positive tumors. Furthermore, two prognostically relevant subgroups of MCPyV-positive MCC were identified based on dichotomic expression of genes related to epidermal structures and processes. We identified three distinct molecular subgroups of MCC with prognostic relevance. We propose that the dichotomic expression of epidermis-related genes might reflect both an epidermal and a nonepidermal origin for MCPyV-positive MCC.

Classification of unknown primary tumors with a data-driven method based on a large microarray reference database.

We present a new method to analyze cancer of unknown primary origin (CUP) samples. Our method achieves good results with classification accuracy (88% leave-one-out cross validation for primary tumors from 56 categories, 78% for CUP samples), and can also be used to study CUP samples on a gene-by-gene basis. It is not tied to any a priori defined gene set as many previous methods, and is adaptable to emerging new information.

LGI2 truncation causes a remitting focal epilepsy in dogs.

One quadrillion synapses are laid in the first two years of postnatal construction of the human brain, which are then pruned until age 10 to 500 trillion synapses composing the final network. Genetic epilepsies are the most common neurological diseases with onset during pruning, affecting 0.5% of 2-10-year-old children, and these epilepsies are often characterized by spontaneous remission. We previously described a remitting epilepsy in the Lagotto romagnolo canine breed. Here, we identify the gene defect and affected neurochemical pathway. We reconstructed a large Lagotto pedigree of around 34 affected animals. Using genome-wide association in 11 discordant sib-pairs from this pedigree, we mapped the disease locus to a 1.7 Mb region of homozygosity in chromosome 3 where we identified a protein-truncating mutation in the Lgi2 gene, a homologue of the human epilepsy gene LGI1. We show that LGI2, like LGI1, is neuronally secreted and acts on metalloproteinase-lacking members of the ADAM family of neuronal receptors, which function in synapse remodeling, and that LGI2 truncation, like LGI1 truncations, prevents secretion and ADAM interaction. The resulting epilepsy onsets at around seven weeks (equivalent to human two years), and remits by four months (human eight years), versus onset after age eight in the majority of human patients with LGI1 mutations. Finally, we show that Lgi2 is expressed highly in the immediate post-natal period until halfway through pruning, unlike Lgi1, which is expressed in the latter part of pruning and beyond. LGI2 acts at least in part through the same ADAM receptors as LGI1, but earlier, ensuring electrical stability (absence of epilepsy) during pruning years, preceding this same function performed by LGI1 in later years. LGI2 should be considered a candidate gene for common remitting childhood epilepsies, and LGI2-to-LGI1 transition for mechanisms of childhood epilepsy remission.

Alignment of gene expression profiles from test samples against a reference database: New method for context-specific interpretation of microarray data.

BACKGROUND: Gene expression microarray data have been organized and made available as public databases, but the utilization of such highly heterogeneous reference datasets in the interpretation of data from individual test samples is not as developed as e.g. in the field of nucleotide sequence comparisons. We have created a rapid and powerful approach for the alignment of microarray gene expression profiles (AGEP) from test samples with those contained in a large annotated public reference database and demonstrate here how this can facilitate interpretation of microarray data from individual samples. METHODS: AGEP is based on the calculation of kernel density distributions for the levels of expression of each gene in each reference tissue type and provides a quantitation of the similarity between the test sample and the reference tissue types as well as the identity of the typical and atypical genes in each comparison. As a reference database, we used 1654 samples from 44 normal tissues (extracted from the Genesapiens database). RESULTS: Using leave-one-out validation, AGEP correctly defined the tissue of origin for 1521 (93.6%) of all the 1654 samples in the original database. Independent validation of 195 external normal tissue samples resulted in 87% accuracy for the exact tissue type and 97% accuracy with related tissue types. AGEP analysis of 10 Duchenne muscular dystrophy (DMD) samples provided quantitative description of the key pathogenetic events, such as the extent of inflammation, in individual samples and pinpointed tissue-specific genes whose expression changed (SAMD4A) in DMD. AGEP analysis of microarray data from adipocytic differentiation of mesenchymal stem cells and from normal myeloid cell types and leukemias provided quantitative characterization of the transcriptomic changes during normal and abnormal cell differentiation. CONCLUSIONS: The AGEP method is a widely applicable method for the rapid comprehensive interpretation of microarray data, as proven here by the definition of tissue- and disease-specific changes in gene expression as well as during cellular differentiation. The capability to quantitatively compare data from individual samples against a large-scale annotated reference database represents a widely applicable paradigm for the analysis of all types of high-throughput data. AGEP enables systematic and quantitative comparison of gene expression data from test samples against a comprehensive collection of different cell/tissue types previously studied by the entire research community.

First analysis of a bacterial collagen-binding protein with collagen Toolkits: promiscuous binding of YadA to collagens may explain how YadA interferes with host processes.

The Yersinia adhesin YadA mediates the adhesion of the human enteropathogen Yersinia enterocolitica to collagens and other components of the extracellular matrix. Though YadA has been proposed to bind to a specific site in collagens, the exact binding determinants for YadA in native collagen have not previously been elucidated. We investigated the binding of YadA to collagen Toolkits, which are libraries of triple-helical peptides spanning the sequences of type II and III human collagens. YadA bound to many of them, in particular to peptides rich in hydroxyproline but with few charged residues. We were able to block the binding of YadA to collagen type IV with the triple-helical peptide (Pro-Hyp-Gly)(10), suggesting that the same site in YadA binds to triple-helical regions in network-forming collagens as well. We showed that a single Gly-Pro-Hyp triplet in a triple-helical peptide was sufficient to support YadA binding, but more than six triplets were required to form a tight YadA binding site. This is significantly longer than the case for eukaryotic collagen-binding proteins. YadA-expressing bacteria bound promiscuously to Toolkit peptides. Promiscuous binding could be advantageous for pathogenicity in Y. enterocolitica and, indeed, for other pathogenic bacteria. Many of the tightly binding peptides are also targets for eukaryotic collagen-binding proteins, and YadA was able to inhibit the interaction between selected Toolkit peptides and platelets. This leads to the intriguing possibility that YadA may interfere in vivo with host processes mediated by endogenous collagen-binding proteins.

CD14 and TNfa promoter polymorphisms in patients with acute arthritis. Special reference to development of chronic spondyloarthropathy.

OBJECTIVE: To examine CD14 and TNFalpha gene polymorphisms in early arthritis in relation to clinical outcome. METHODS: We studied 141 Caucasians who had had early arthritis 10 to 38 years earlier. We analysed CD14 (-159) and TNFalpha (-238, -308, -376) polymorphisms using a novel cycle minisequencing method. DNA pools from 370 Caucasian blood donors served as controls. RESULTS: CD14 (-159)C-->T allele frequencies were comparable among patients and controls (39% vs 40%). Fifty men and 42 women had recovered while 24 men and six women had chronic spondyloarthropathy (SpA). Mutant T allele frequency was higher in the chronic SpA group than in the recovered group in women (75% vs 32%, relative risk 1.3, 95% confidence limit 1.1 to 1.6, P = 0.011), but not in men (38% vs 44%). All female patients with chronic SpA had CD14 (-159)T allele and none had a possibly protective TNFalpha (-308)G-->A allele. CONCLUSIONS: Possession of CD14 (-159)T allele does not increase risk of ReA but may increase susceptibility of female patients to development of chronic SpA.