Genomic investigations of unexplained acute hepatitis in children

Since its first identification in Scotland, over 1000 cases of unexplained pediatric hepatitis in children have been reported worldwide, including 278 cases in the UK 1. Here we report investigation of 38 cases, 66 age-matched immunocompetent controls and 21 immunocompromised comparator subjects, using a combination of genomic, transcriptomic, proteomic and immunohistochemical methods. We detected high levels of adeno-associated virus 2 (AAV2) DNA in liver, blood, plasma or stool from 27/28 cases. We found low levels of Adenovirus (HAdV) and Human Herpesvirus 6B (HHV-6B), in 23/31 and 16/23 respectively of the cases tested. In contrast, AAV2 was infrequently detected at low titre in blood or liver from control children with HAdV, even when profoundly immunosuppressed. AAV2, HAdV and HHV-6 phylogeny excluded emergence of novel strains in cases. Histological analyses of explanted livers showed enrichment for T-cells and B-lineage cells. Proteomic comparison of liver tissue from cases and healthy controls, identified increased expression of HLA class 2, immunoglobulin variable regions and complement proteins. HAdV and AAV2 proteins were not detected in the livers. Instead, we identified AAV2 DNA complexes reflecting both HAdV and HHV-6B-mediated replication. We hypothesize that high levels of abnormal AAV2 replication products aided by HAdV and in severe cases HHV-6B, may have triggered immune-mediated hepatic disease in genetically and immunologically predisposed children

Towards the characterization of the eukaryotic selenoproteome: a computational approach

Although the genome sequence and gene content are available for an increasing number of organisms, eukaryotic selenoproteins remain poorly characterized. In these proteins, selenium (Se) is incorporated in the form of selenocysteine(Sec), the 21st amino acid. Selenocysteine is cotranslationally inserted in response to UGA codons (a stop signal in the canonical genetic code). The alternative decoding is mediated by a stem-loop structure in the 3'UTR of selenoprotein mRNAs (the SECIS element). Selenium is implicated in male infertility, cancer and heart diseases, viral expression and ageing. In addition, most selenoproteins have homologues in which Sec is replaced by cysteine (Cys).Genome biologists rely on the high-quality annotation of genomes to bridge the gap from the sequence to the biology of the organism. However, for selenoproteins, which mediate the biological functions of selenium, the dual role of the UGA codon confounds both the automatic annotation pipelines and the human curators. In consequence, selenoproteins are misannotated in the majority of genome projects. Furthermore, the finding of novel selenoprotein families remains a difficult task in the newly released genome sequences.In the last few years, we have contributed to the exhaustive description of the eukaryotic selenoproteome (set of eukaryotic selenoproteins) through the development of a number of ad hoc computational tools. Our approach is based on the capacity of predicting SECIS elements, standard genes and genes with a UGA codon in-frame in one or multiple genomes. Indeed, the comparative analysis plays an essential role because 1) SECIS sequences are conserved between close species (eg. human-mouse); and 2) sequence conservation across a UGA codon between genomes at further phylogenetic distance strongly suggests a coding function (eg. human-fugu). Our analysis of the fly, human and Takifugu and Tetraodon genomes have resulted in 9 novel selenoprotein families. Therefore, 20 distinct selenoprotein families have been described in eukaryotes to date. Most of these families are widely (but not uniformly) distributed across eukaryotes, either as true selenoproteins or Cys-homologues.The correct annotation of selenoproteins is thus providing insight into the evolution of the usage of Sec. Our data indicate a discrete evolutionary distribution of selenoprotein in eukaryotes and suggest that, contrary to the prevalent thinking of an increase in the number of selenoproteins from less to more complex genomes, Sec-containing proteins scatter all along the complexity scale. We believe that the particular distribution of each family is mediated by an ongoing process of Sec/Cys interconversion, in which contingent events could play a role as important as functional constraints. The characterization of eukaryotic selenoproteins illustrates some of the most important challenges involved in the completion of the gene annotation of genomes. Notably among them, the increasing number of exceptions to our standard theory of the eukaryotic gene and the necessity of sequencing genomes at different evolutionary distances towards such a complete annotation

SelenoDB 1.0 : a database of selenoprotein genes, proteins and SECIS elements

Selenoproteins are a diverse group of proteins/nusually misidentified and misannotated in sequence/ndatabases. The presence of an in-frame UGA (stop)/ncodon in the coding sequence of selenoprotein/ngenes precludes their identification and correct/nannotation. The in-frame UGA codons are recoded/nto cotranslationally incorporate selenocysteine,/na rare selenium-containing amino acid. The development/nof ad hoc experimental and, more recently,/ncomputational approaches have allowed the efficient/nidentification and characterization of the/nselenoproteomes of a growing number of species./nToday, dozens of selenoprotein families have been/ndescribed and more are being discovered in recently/nsequenced species, but the correct genomic annotation/nis not available for the majority of these/ngenes. SelenoDB is a long-term project that aims to/nprovide, through the collaborative effort of experimental/nand computational researchers, automatic/nand manually curated annotations of selenoprotein/ngenes, proteins and SECIS elements. Version 1.0 of/nthe database includes an initial set of eukaryotic/ngenomic annotations, with special emphasis on the/nhuman selenoproteome, for immediate inspection/nby selenium researchers or incorporation into more/ngeneral databases. SelenoDB is freely available at/nhttp://www.selenodb.org

The impact of genetic adaptation on chimpanzee subspecies differentiation

Chimpanzees, humans' closest relatives, are in danger of extinction. Aside from direct human impacts such as hunting and habitat destruction, a key threat is transmissible disease. As humans continue to encroach upon their habitats, which shrink in size and grow in density, the risk of inter-population and cross-species viral transmission increases, a point dramatically made in the reverse with the global HIV/AIDS pandemic. Inhabiting central Africa, the four subspecies of chimpanzees differ in demographic history and geographical range, and are likely differentially adapted to their particular local environments. To quantitatively explore genetic adaptation, we investigated the genic enrichment for SNPs highly differentiated between chimpanzee subspecies. Previous analyses of such patterns in human populations exhibited limited evidence of adaptation. In contrast, chimpanzees show evidence of recent positive selection, with differences among subspecies. Specifically, we observe strong evidence of recent selection in eastern chimpanzees, with highly differentiated SNPs being uniquely enriched in genic sites in a way that is expected under recent adaptation but not under neutral evolution or background selection. These sites are enriched for genes involved in immune responses to pathogens, and for genes inferred to differentiate the immune response to infection by simian immunodeficiency virus (SIV) in natural vs. non-natural host species. Conversely, central chimpanzees exhibit an enrichment of signatures of positive selection only at cytokine receptors, due to selective sweeps in CCR3, CCR9 and CXCR6 -paralogs of CCR5 and CXCR4, the two major receptors utilized by HIV to enter human cells. Thus, our results suggest that positive selection has contributed to the genetic and phenotypic differentiation of chimpanzee subspecies, and that viruses likely play a predominate role in this differentiation, with SIV being a likely selective agent. Interestingly, our results suggest that SIV has elicited distinctive adaptive responses in these two chimpanzee subspecies

Nematode selenoproteome: the use of the selenocysteine insertion system to decode one codon in an animal genome?

Selenocysteine (Sec) is co-translationally inserted into selenoproteins in response to codon UGA with the help of the selenocysteine insertion sequence (SECIS) element. The number of selenoproteins in animals varies, with humans having 25 and mice having 24 selenoproteins. To date, however, only one selenoprotein, thioredoxin reductase, has been detected in Caenorhabditis elegans, and this enzyme contains only one Sec. Here, we characterize the selenoproteomes of C.elegans and Caenorhabditis briggsae with three independent algorithms, one searching for pairs of homologous nematode SECIS elements, another searching for Cys- or Sec-containing homologs of potential nematode selenoprotein genes and the third identifying Sec-containing homologs of annotated nematode proteins. These methods suggest that thioredoxin reductase is the only Sec-containing protein in the C.elegans and C.briggsae genomes. In contrast, we identified additional selenoproteins in other nematodes. Assuming that Sec insertion mechanisms are conserved between nematodes and other eukaryotes, the data suggest that nematode selenoproteomes were reduced during evolution, and that in an extreme reduction case Sec insertion systems probably decode only a single UGA codon in C.elegans and C.briggsae genomes. In addition, all detected genes had a rare form of SECIS element containing a guanosine in place of a conserved adenosine present in most other SECIS structures, suggesting that in organisms with small selenoproteomes SECIS elements may change rapidly.This study was supported by NIH grants GM061603 and/nGM065204 and by grant BIO2000-1358-C02-02 from Plan/nNacional de I+D (Spain). Funding to pay the Open Access/npublication charges for this article was provided by/nGM061603

Nematode selenoproteome: the use of the selenocysteine insertion system to decode one codon in an animal genome?

Selenocysteine (Sec) is co-translationally inserted into selenoproteins in response to codon UGA with the help of the selenocysteine insertion sequence (SECIS) element. The number of selenoproteins in animals varies, with humans having 25 and mice having 24 selenoproteins. To date, however, only one selenoprotein, thioredoxin reductase, has been detected in Caenorhabditis elegans, and this enzyme contains only one Sec. Here, we characterize the selenoproteomes of C.elegans and Caenorhabditis briggsae with three independent algorithms, one searching for pairs of homologous nematode SECIS elements, another searching for Cys- or Sec-containing homologs of potential nematode selenoprotein genes and the third identifying Sec-containing homologs of annotated nematode proteins. These methods suggest that thioredoxin reductase is the only Sec-containing protein in the C.elegans and C.briggsae genomes. In contrast, we identified additional selenoproteins in other nematodes. Assuming that Sec insertion mechanisms are conserved between nematodes and other eukaryotes, the data suggest that nematode selenoproteomes were reduced during evolution, and that in an extreme reduction case Sec insertion systems probably decode only a single UGA codon in C.elegans and C.briggsae genomes. In addition, all detected genes had a rare form of SECIS element containing a guanosine in place of a conserved adenosine present in most other SECIS structures, suggesting that in organisms with small selenoproteomes SECIS elements may change rapidly.This study was supported by NIH grants GM061603 and/nGM065204 and by grant BIO2000-1358-C02-02 from Plan/nNacional de I+D (Spain). Funding to pay the Open Access/npublication charges for this article was provided by/nGM061603

SelenoDB 2.0: annotation of selenoprotein genes in animals and their genetic diversity in humans

SelenoDB (http://www.selenodb.org) aims to provide high-quality annotations of selenoprotein genes, proteins and SECIS elements. Selenoproteins are proteins that contain the amino acid selenocysteine (Sec) and the first release of the database included annotations for eight species. Since the release of SelenoDB 1.0 many new animal genomes have been sequenced. The annotations of selenoproteins in new genomes usually contain many errors in major databases. For this reason, we have now fully annotated selenoprotein genes in 58 animal genomes. We provide manually curated annotations for human selenoproteins, whereas we use an automatic annotation pipeline to annotate selenoprotein genes in other animal genomes. In addition, we annotate the homologous genes containing cysteine (Cys) instead of Sec. Finally, we have surveyed genetic variation in the annotated genes in humans. We use exon capture and resequencing approaches to identify single-nucleotide polymorphisms in more than 50 human populations around the world. We thus present a detailed view of the genetic divergence of Sec- and Cys-containing genes in animals and their diversity in humans. The addition of these datasets into the second release of the database provides a valuable resource for addressing medical and evolutionary questions in selenium biology.This work was supported by the Plan Nacional and the Instituto Nacional de BIoinformatica (Spain

Genomic investigations of unexplained acute hepatitis in children.

Since its first identification in Scotland, over 1000 cases of unexplained pediatric hepatitis in children have been reported worldwide, including 278 cases in the UK 1. Here we report investigation of 38 cases, 66 age-matched immunocompetent controls and 21 immunocompromised comparator subjects, using a combination of genomic, transcriptomic, proteomic and immunohistochemical methods. We detected high levels of adeno-associated virus 2 (AAV2) DNA in liver, blood, plasma or stool from 27/28 cases. We found low levels of Adenovirus (HAdV) and Human Herpesvirus 6B (HHV-6B), in 23/31 and 16/23 respectively of the cases tested. In contrast, AAV2 was infrequently detected at low titre in blood or liver from control children with HAdV, even when profoundly immunosuppressed. AAV2, HAdV and HHV-6 phylogeny excluded emergence of novel strains in cases. Histological analyses of explanted livers showed enrichment for T-cells and B-lineage cells. Proteomic comparison of liver tissue from cases and healthy controls, identified increased expression of HLA class 2, immunoglobulin variable regions and complement proteins. HAdV and AAV2 proteins were not detected in the livers. Instead, we identified AAV2 DNA complexes reflecting both HAdV and HHV-6B-mediated replication. We hypothesize that high levels of abnormal AAV2 replication products aided by HAdV and in severe cases HHV-6B, may have triggered immune-mediated hepatic disease in genetically and immunologically predisposed children