DNA mismatch repair deficiency drives microsatellite instability (MSI). Cells with MSI accumulate numerous frameshift mutations. Frameshift mutations affecting cancer-related genes may promote tumorigenesis and, therefore, are shared among independently arising MSI tumors. Consequently, such recurrent frameshift mutations can give rise to shared immunogenic frameshift peptides (FSPs) that represent ideal candidates for a vaccine against MSI cancer. Pathogenic germline variants of mismatch repair genes cause Lynch syndrome (LS), a hereditary cancer syndrome affecting approximately 20-25 million individuals worldwide. Individuals with LS are at high risk of developing MSI cancer. Previously, we demonstrated safety and immunogenicity of an FSP-based vaccine in a phase I/IIa clinical trial in patients with a history of MSI colorectal cancer. However, the cancer-preventive effect of FSP vaccination in the scenario of LS has not yet been demonstrated.

DNA mismatch repair-deficient colorectal cancers (CRCs) accumulate numerous frameshift mutations at repetitive sequences recognized as microsatellite instability (MSI). When coding mononucleotide repeats (cMNRs) are affected, tumors accumulate frameshift mutations and premature termination codons (PTC) potentially leading to truncated proteins. Nonsense-mediated RNA decay (NMD) can degrade PTC-containing transcripts and protect from such faulty proteins. As it also regulates normal transcripts and cellular physiology, we tested whether NMD genes themselves are targets of MSI frameshift mutations. A high frequency of cMNR frameshift mutations in the gene was found in MSI CRC cell lines (67.7%), MSI colorectal adenomas (55%) and carcinomas (63%). In normal colonic crypts, UPF3A expression was restricted to single chromogranin A-positive cells. SILAC-based proteomic analysis of KM12 CRC cells revealed UPF3A-dependent down-regulation of several enzymes involved in cholesterol biosynthesis. Furthermore, reconstituted UPF3A expression caused alterations of 85 phosphosites in 52 phosphoproteins. Most of them (38/52, 73%) reside in nuclear phosphoproteins involved in regulation of gene expression and RNA splicing. Since UPF3A mutations can modulate the (phospho)proteomic signature and expression of enzymes involved in cholesterol metabolism in CRC cells, UPF3A may influence other processes than NMD and loss of UPF3A expression might provide a growth advantage to MSI CRC cells.

Different DNA mismatch repair (MMR)-deficient mouse strains have been developed as models for the inherited cancer predisposing Lynch syndrome. It is completely unresolved, whether coding mononucleotide repeat (cMNR) gene mutations in these mice can contribute to intestinal tumorigenesis and whether MMR-deficient mice are a suitable molecular model of human microsatellite instability (MSI)-associated intestinal tumorigenesis. A proof-of-principle study was performed to identify mouse cMNR-harboring genes affected by insertion/deletion mutations in MSI murine intestinal tumors. Bioinformatic algorithms were developed to establish a database of mouse cMNR-harboring genes. A panel of five mouse noncoding mononucleotide markers was used for MSI classification of intestinal matched normal/tumor tissues from MMR-deficient (Mlh1(-/-) , Msh2(-/-) , Msh2(LoxP/LoxP) ) mice. cMNR frameshift mutations of candidate genes were determined by DNA fragment analysis. Murine MSI intestinal tumors but not normal tissues from MMR-deficient mice showed cMNR frameshift mutations in six candidate genes (Elavl3, Tmem107, Glis2, Sdccag1, Senp6, Rfc3). cMNRs of mouse Rfc3 and Elavl3 are conserved in type and length in their human orthologs that are known to be mutated in human MSI colorectal, endometrial and gastric cancer. We provide evidence for the utility of a mononucleotide marker panel for detection of MSI in murine tumors, the existence of cMNR instability in MSI murine tumors, the utility of mouse subspecies DNA for identification of polymorphic repeats, and repeat conservation among some orthologous human/mouse genes, two of them showing instability in human and mouse MSI intestinal tumors. MMR-deficient mice hence are a useful molecular model system for analyzing MSI intestinal carcinogenesis. © 2014 Wiley Periodicals, Inc.

About 15% of human colorectal cancers and, at varying degrees, other tumor entities as well as nearly all tumors related to Lynch syndrome are hallmarked by microsatellite instability (MSI) as a result of a defective mismatch repair system. The functional impact of resulting mutations depends on their genomic localization. Alterations within coding mononucleotide repeat tracts (MNRs) can lead to protein truncation and formation of neopeptides, whereas alterations within untranslated MNRs can alter transcription level or transcript stability. These mutations may provide selective advantage or disadvantage to affected cells. They may further concern the biology of microsatellite unstable cells, e.g. by generating immunogenic peptides induced by frameshifts mutations. The Selective Targets database (http://www.seltarbase.org) is a curated database of a growing number of public MNR mutation data in microsatellite unstable human tumors. Regression calculations for various MSI-H tumor entities indicating statistically deviant mutation frequencies predict TGFBR2, BAX, ACVR2A and others that are shown or highly suspected to be involved in MSI tumorigenesis. Many useful tools for further analyzing genomic DNA, derived wild-type and mutated cDNAs and peptides are integrated. A comprehensive database of all human coding, untranslated, non-coding RNA- and intronic MNRs (MNR_ensembl) is also included. Herewith, SelTarbase presents as a plenty instrument for MSI-carcinogenesis-related research, diagnostics and therapy.

Glycosyl epitopes have been identified as tumor-specific markers in colorectal tumors and various lines of evidence indicate the significance of altered synthesis, transport, and secretion of glycoproteins in tumorigenesis. However, aberrant glycosylation has been largely ignored in microsatellite unstable (MSI-H) colorectal tumors. Therefore, we analyzed mutation frequencies of genes of the cellular glycosylation machinery in MSI-H tumors, focusing on frameshift mutations in coding MNRs (cMNRs). Among 28 candidate genes, LMAN1/ERGIC53, a mannose-specific lectin mediating endoplasmatic reticulum (ER)-to-Golgi transit of glycosylated proteins, showed high mutation frequency in MSI-H colorectal cancer cell lines (52%; 12 of 23), carcinomas (45%; 72 of 161), and adenomas (40%; 8 of 20). Biallelic mutations were observed in 17% (4 of 23) of MSI-H colorectal cancer cell lines. LMAN1 was found to be transcribed but truncated protein remained undetectable in these LMAN1-mutant cell lines. Immunohistochemical and molecular analysis of LMAN1-mutated carcinomas and adenomas revealed regional loss of LMAN1 expression due to biallelic LMAN1 cMNR frameshift mutations. In LMAN1-deficient colorectal cancer cell lines, secretion of the LMAN1 client protein alpha-1-antitrypsin (A1AT), an inhibitor of angiogenesis and tumor growth, was significantly impaired but could be restored upon LMAN1 re-expression. These results suggest that LMAN1 mutational inactivation is a frequent and early event potentially contributing to MSI-H tumorigenesis.

ABSTRACT: BACKGROUND: Protein tyrosine phosphatases (PTPs) like their antagonizing protein tyrosine kinases are key regulators of signal transduction thereby assuring normal control of cellular growth and differentiation. Increasing evidence suggests that mutations in PTP genes are associated with human malignancies. For example, mutational analysis of the tyrosine phosphatase (PTP) gene superfamily uncovered genetic alterations in about 26 % of colorectal tumors. Since in these studies tumors have not been stratified according to genetic instability status we hypothesized that colorectal tumors characterized by high-level of microsatellite instability (MSI-H) might show an increased frequency of frameshift mutations in those PTP genes that harbor long mononucleotide repeats in their coding region (cMNR). RESULTS: Using bioinformatic analysis we identified 16 PTP candidate genes with long cMNRs that were examined for genetic alterations in 19 MSI-H colon cell lines, 54 MSI-H colorectal cancers, and 17 MSI-H colorectal adenomas. Frameshift mutations were identified only in 6 PTP genes, of which PTPN21 show the highest mutation frequency at all in MSI-H tumors (17 %). CONCLUSIONS: Although about 32 % of MSI-H tumors showed at least one affected PTP gene, and cMNR mutation rates in PTPN21, PTPRS, and PTPN5 are higher than the mean mutation frequency of MNRs of the same length, mutations within PTP genes do not seem to play a common role in MSI tumorigenesis, since no cMNR mutation frequency reached statistical significance and therefore, failed prediction as a Positive Selective Target Gene.

DNA mismatch repair deficiency is observed in about 15% of human colorectal, gastric, and endometrial tumors and in lower frequencies in a minority of other tumors thereby causing insertion/deletion mutations at short repetitive sequences, recognized as microsatellite instability (MSI). Evolution of tumors, including those with MSI, is a continuous process of mutation and selection favoring neoplastic growth. Mutations in microsatellite-bearing genes that promote tumor cell growth in general (Real Common Target genes) are assumed to be the driving force during MSI carcinogenesis. Thus, microsatellite mutations in these genes should occur more frequently than mutations in microsatellite genes without contribution to malignancy (ByStander genes). So far, only a few Real Common Target genes have been identified by functional studies. Thus, comprehensive analysis of microsatellite mutations will provide important clues to the understanding of MSI-driven carcinogenesis. Here, we evaluated published mutation frequencies on 194 repeat tracts in 137 genes in MSI-H colorectal, endometrial, and gastric carcinomas and propose a statistical model that aims to identify Real Common Target genes. According to our model nine genes including BAX and TGFbetaRII were identified as Real Common Targets in colorectal cancer, one gene in gastric cancer, and three genes in endometrial cancer. Microsatellite mutations in five additional genes seem to be counterselected in gastrointestinal tumors. Overall, the general applicability, the capacity to unlimited data analysis, the inclusion of mutation data generated by different groups on different sets of tumors make this model a useful tool for predicting Real Common Target genes with specificity for MSI-H tumors of different organs, guiding subsequent functional studies to the most likely targets among numerous microsatellite harboring genes.

HGVbase (Human Genome Variation database; http://hgvbase.cgb.ki.se, formerly known as HGBASE) is an academic effort to provide a high quality and non-redundant database of available genomic variation data of all types, mostly comprising single nucleotide polymorphisms (SNPs). Records include neutral polymorphisms as well as disease-related mutations. Online search tools facilitate data interrogation by sequence similarity and keyword queries, and searching by genome coordinates is now being implemented. Downloads are freely available in XML, Fasta, SRS, SQL and tagged-text file formats. Each entry is presented in the context of its surrounding sequence and many records are related to neighboring human genes and affected features therein. Population allele frequencies are included wherever available. Thorough semi-automated data checking ensures internal consistency and addresses common errors in the source information. To keep pace with recent growth in the field, we have developed tools for fully automated annotation. All variants have been uniquely mapped to the draft genome sequence and are referenced to positions in EMBL/GenBank files. Data utility is enhanced by provision of genotyping assays and functional predictions. Recent data structure extensions allow the capture of haplotype and genotype information, and a new initiative (along with BiSC and HUGO-MDI) aims to create a central repository for the broad collection of clinical mutations and associated disease phenotypes of interest.

Microsatellite instability (MSI) caused by defective DNA mismatch repair (MMR) is a hallmark of hereditary nonpolyposis colorectal cancers (HNPCC) but also occurs in about 15% of sporadic tumors. If instability affects microsatellites in coding regions, translational frameshifts lead to truncated proteins often marked by unique frameshift peptide sequences at their C-terminus. Since MSI tumors show enhanced lymphocytic infiltration and our previous analysis identified numerous coding mono- and dinucleotide repeat-bearing candidate genes as targets of genetic instability, we examined the role of frameshift peptides in triggering cellular immune responses. Using peptide pulsed autologous CD40-activated B cells, we have generated cytotoxic T lymphocytes (CTL) that specifically recognize HLA-A2.1-restricted peptides derived from frameshift sequences. Among 16 frameshift peptides predicted from mutations in 8 different genes, 3 peptides conferred specific lysis of target cells exogenously loaded with cognate peptide. One peptide derived from a (-1) frameshift mutation in the TGFbetaIIR gene gave rise to a CTL bulk culture capable of lysing the MSI colorectal cancer cell line HCT116 carrying this frameshift mutation. Given the huge number of human coding microsatellites and assuming only a fraction being mutated and encoding immunologically relevant peptides in MSI tumors, frameshift protein sequences represent a novel subclass of tumor-specific antigens. It is tempting to speculate that a frameshift peptide-directed vaccination approach not only could offer new treatment modalities for existing MSI tumors but also might benefit asymptomatic at-risk individuals in HNPCC families by a prophylactic vaccination strategy.

Microsatellite instability (MSI) caused by deficient DNA mismatch-repair functions is a hallmark of cancers associated with the hereditary nonpolyposis colorectal cancer (HNPCC) syndrome but is also found in about 15% of all sporadic tumors. Most affected microsatellites reside in untranslated intergenic or intronic sequences. However, recently few genes with coding microsatellites were also shown to be mutational targets in MSI-positive cancers and might represent important mutation targets in their pathogenesis. The systematic identification of such genes and the analysis of their mutation frequency in MSI-positive cancers might thus reveal major clues to their functional role in MSI-associated carcinogenesis. We therefore initiated a systematic database search in 33,595 distinctly annotated human genes and identified 17,654 potentially coding mononucleotide repeats (cMNRs) and 2,028 coding dinucleotide repeats (cDNRs), which consist of n > or = 6 and n > or = 4 repeat units, respectively. Expression pattern and mutation frequency of 19 of these genes with the longest repeats were compared between DNA mismatch repair-deficient (MSI(+)) and proficient (MSS) cancer cells. Instability frequencies in these coding microsatellite genes ranged from 10% to 100% in MSI-H tumor cells, whereas MSS cancer cells did not show mutations. RT-PCR analysis further showed that most of the affected genes (10/15) were highly expressed in tumor cells. The approach outlined here identified a new set of genes frequently affected by mutations in MSI-positive tumor cells. It will lead to novel and highly specific diagnostic and therapeutic targets for microsatellite unstable cancers.

Human genome polymorphism is expected to play a key role in defining the etiologic basis of phenotypic differences between individuals in aspects such as drug responses and common disease predisposition. Relevant functional DNA changes will probably be located in or near to transcribed sequences, and include many single nucleotide polymorphisms. To aid the future analysis of such genome variation, HGBASE (Human Genic Bi-Allelic SEquences) was constructed as a means to gather human gene-linked polymorphisms from all possible public sources, and show these as a non-redundant set of records in a standardized and user-friendly database endowed with text and sequence based search facilities. After 1 year of presence on the WWW, the HGBASE project has compiled data for over 22 000 records, and this number continues to triple every 6-12 months with data harvested or submitted from all major public genome databases and published literature from the previous decade. Extensive annotation enhancement, internal consistency checking and manual review of every record is undertaken to address potential errors and deficiencies sometimes present in the original source data. The fully polished and comprehensive database is made freely available to all at http://hgbase.cgr.ki.se

Four years after the original sequence submission, we have re-annotated the genome of Mycoplasma pneumoniae to incorporate novel data. The total number of ORFss has been increased from 677 to 688 (10 new proteins were predicted in intergenic regions, two further were newly identified by mass spectrometry and one protein ORF was dismissed) and the number of RNAs from 39 to 42 genes. For 19 of the now 35 tRNAs and for six other functional RNAs the exact genome positions were re-annotated and two new tRNA(Leu) and a small 200 nt RNA were identified. Sixteen protein reading frames were extended and eight shortened. For each ORF a consistent annotation vocabulary has been introduced. Annotation reasoning, annotation categories and comparisons to other published data on M.pneumoniae functional assignments are given. Experimental evidence includes 2-dimensional gel electrophoresis in combination with mass spectrometry as well as gene expression data from this study. Compared to the original annotation, we increased the number of proteins with predicted functional features from 349 to 458. The increase includes 36 new predictions and 73 protein assignments confirmed by the published literature. Furthermore, there are 23 reductions and 30 additions with respect to the previous annotation. mRNA expression data support transcription of 184 of the functionally unassigned reading frames.

SUMMARY: Network Analysis Interface for Linking HMMER results (NAIL) is a web-based tool for the analysis of results from a HMMER protein database-search. NAIL facilitates the selection of protein hits and the creation of an alignment, which can be used for a new sequence similarity search.

Predicting function from sequence using computational tools is a highly complicated procedure that is generally done for each gene individually. This review focuses on the added value that is provided by completely sequenced genomes in function prediction. Various levels of sequence annotation and function prediction are discussed, ranging from genomic sequence to that of complex cellular processes. Protein function is currently best described in the context of molecular interactions. In the near future it will be possible to predict protein function in the context of higher order processes such as the regulation of gene expression, metabolic pathways and signalling cascades. The analysis of such higher levels of function description uses, besides the information from completely sequenced genomes, also the additional information from proteomics and expression data. The final goal will be to elucidate the mapping between genotype and phenotype.

Homology search techniques based on the iterative PSI-BLAST method in combination with various filters for low sequence complexity are applied to assign folds to all Mycoplasma genitalium proteins. The resulting procedure (implemented as a web server) is able to predict at least one domain in 37% of these proteins automatically, with an estimated accuracy higher than 98%. Taking structural features such as coiled coil or transmembrane regions aside, folds can be assigned to more than half of the globular proteins in a bacterium just by iterative sequence comparison.

MOTIVATION: Numerous homologous sequences from diverse species can be retrieved from databases using programs such as BLAST. However, due to multigene families, evolutionary relationship often cannot be easily determined and proper functional assignment becomes difficult. Thus, discrimination between orthologues and paralogues within BLAST output lists of homologous sequences becomes more and more important. RESULT: We therefore developed a method that attempts to construct a reconciled tree from a gene tree of selected sequences and its corresponding phylogenetic tree of the species involved (species tree). An interface on the Web is developed to enable users to analyse the BLAST result. BLAST outputs are parsed and, for the selected sequences, multiple alignments are constructed either globally or for local regions. Bootstrapped trees are returned and compared with the expected species tree. In cases of discrepancies, gene duplications are assumed and a reconciled tree is computed. The reconciled tree shows probable orthologues and paralogues as predicted.