Rare deletions at 16p13.11 predispose to a diverse spectrum of sporadic epilepsy syndromes.

Deletions at 16p13.11 are associated with schizophrenia, mental retardation, and most recently idiopathic generalized epilepsy. To evaluate the role of 16p13.11 deletions, as well as other structural variation, in epilepsy disorders, we used genome-wide screens to identify copy number variation in 3812 patients with a diverse spectrum of epilepsy syndromes and in 1299 neurologically-normal controls. Large deletions (> 100 kb) at 16p13.11 were observed in 23 patients, whereas no control had a deletion greater than 16 kb. Patients, even those with identically sized 16p13.11 deletions, presented with highly variable epilepsy phenotypes. For a subset of patients with a 16p13.11 deletion, we show a consistent reduction of expression for included genes, suggesting that haploinsufficiency might contribute to pathogenicity. We also investigated another possible mechanism of pathogenicity by using hybridization-based capture and next-generation sequencing of the homologous chromosome for ten 16p13.11-deletion patients to look for unmasked recessive mutations. Follow-up genotyping of suggestive polymorphisms failed to identify any convincing recessive-acting mutations in the homologous interval corresponding to the deletion. The observation that two of the 16p13.11 deletions were larger than 2 Mb in size led us to screen for other large deletions. We found 12 additional genomic regions harboring deletions > 2 Mb in epilepsy patients, and none in controls. Additional evaluation is needed to characterize the role of these exceedingly large, non-locus-specific deletions in epilepsy. Collectively, these data implicate 16p13.11 and possibly other large deletions as risk factors for a wide range of epilepsy disorders, and they appear to point toward haploinsufficiency as a contributor to the pathogenicity of deletions.

Minipig and beagle animal model genomes aid species selection in pharmaceutical discovery and development.

Improving drug attrition remains a challenge in pharmaceutical discovery and development. A major cause of early attrition is the demonstration of safety signals which can negate any therapeutic index previously established. Safety attrition needs to be put in context of clinical translation (i.e. human relevance) and is negatively impacted by differences between animal models and human. In order to minimize such an impact, an earlier assessment of pharmacological target homology across animal model species will enhance understanding of the context of animal safety signals and aid species selection during later regulatory toxicology studies. Here we sequenced the genomes of the Sus scrofa Göttingen minipig and the Canis familiaris beagle, two widely used animal species in regulatory safety studies. Comparative analyses of these new genomes with other key model organisms, namely mouse, rat, cynomolgus macaque, rhesus macaque, two related breeds (S. scrofa Duroc and C. familiaris boxer) and human reveal considerable variation in gene content. Key genes in toxicology and metabolism studies, such as the UGT2 family, CYP2D6, and SLCO1A2, displayed unique duplication patterns. Comparisons of 317 known human drug targets revealed surprising variation such as species-specific positive selection, duplication and higher occurrences of pseudogenized targets in beagle (41 genes) relative to minipig (19 genes). These data will facilitate the more effective use of animals in biomedical research.

Transcription and pathway analysis of the superior temporal cortex and anterior prefrontal cortex in schizophrenia.

The molecular basis of schizophrenia is poorly understood; however, different brain regions are believed to play distinct roles in disease symptomology. We have studied gene expression in the superior temporal cortex (Brodmann area 22; BA22), which may play a role in positive pathophysiology, and compared our results with data from the anterior prefrontal cortex (BA10), which shows evidence for a role in negative symptoms. Genome-wide mRNA expression was determined in the BA22 region in 23 schizophrenics and 19 controls and compared with a BA10 data set from the same subjects. After adjustments for confounding sources of variation, we carried out GeneGO pathway enrichment analysis in each region. Significant differences were seen in age-related transcriptional changes between the BA22 and the BA10 regions, 21.8% and 41.4% of disease-associated transcripts showing age association, respectively. After removing age associated changes from our data, we saw the highest enrichment in processes mediating cell adhesion, synaptic contact, cytoskeletal remodelling, and apoptosis in the BA22 region. For the BA10 region, we observed the strongest changes in reproductive signalling, tissue remodelling, and cell differentiation. Further exploratory analysis also identified potentially disease-relevant processes that were undetected in our more stringent primary analysis, including autophagy in the BA22 region and the amyloid process in the BA10 region. Collectively, our analysis suggests disruption of many common pathways and processes underpinning synaptic plasticity in both regions in schizophrenia, whereas individual regions emphasize changes in certain pathways that may help to highlight pathway-specific therapeutic opportunities to treat negative or positive symptoms of the disease.

Back to basics--how the evolution of the extracellular matrix underpinned vertebrate evolution.

The extracellular matrix (ECM) is a complex substrate that is involved in and influences a spectrum of behaviours such as growth and differentiation and is the basis for the structure of tissues. Although a characteristic of all metazoans, the ECM has elaborated into a variety of tissues unique to vertebrates, such as bone, tendon and cartilage. Here we review recent advances in our understanding of the molecular evolution of the ECM. Furthermore, we demonstrate that ECM genes represent a pivotal family of proteins the evolution of which appears to have played an important role in the evolution of vertebrates.

Drug discovery in the extracellular matrix.

The extracellular matrix (ECM) is an organised mesh of secreted proteins that provides structure, organisation and orientation to tissues and influences a spectrum of cell behaviours of direct relevance to disease and drug discovery. Many drugs currently in development target components of the ECM, yet most drug discovery teams perceive the ECM as a barrier to efficacious drug action, rather than a therapeutic target. Here we review current therapeutic approaches and consider potentially novel druggable opportunities to target the ECM, taking into account the factors that make it both unique and challenging, including its evolutionary history and innate multi-dimensional complexity.

LRRCE: a leucine-rich repeat cysteine capping motif unique to the chordate lineage.

BACKGROUND: The small leucine-rich repeat proteins and proteoglycans (SLRPs) form an important family of regulatory molecules that participate in many essential functions. They typically control the correct assembly of collagen fibrils, regulate mineral deposition in bone, and modulate the activity of potent cellular growth factors through many signalling cascades. SLRPs belong to the group of extracellular leucine-rich repeat proteins that are flanked at both ends by disulphide-bonded caps that protect the hydrophobic core of the terminal repeats. A capping motif specific to SLRPs has been recently described in the crystal structures of the core proteins of decorin and biglycan. This motif, designated as LRRCE, differs in both sequence and structure from other, more widespread leucine-rich capping motifs. To investigate if the LRRCE motif is a common structural feature found in other leucine-rich repeat proteins, we have defined characteristic sequence patterns and used them in genome-wide searches. RESULTS: The LRRCE motif is a structural element exclusive to the main group of SLRPs. It appears to have evolved during early chordate evolution and is not found in protein sequences from non-chordate genomes. Our search has expanded the family of SLRPs to include new predicted protein sequences, mainly in fishes but with intriguing putative orthologs in mammals. The chromosomal locations of the newly predicted SLRP genes would support the large-scale genome or gene duplications that are thought to have occurred during vertebrate evolution. From this expanded list we describe a new class of SLRP sequences that could be representative of an ancestral SLRP gene. CONCLUSION: Given its exclusivity the LRRCE motif is a useful annotation tool for the identification and classification of new SLRP sequences in genome databases. The expanded list of members of the SLRP family offers interesting insights into early vertebrate evolution and suggests an early chordate evolutionary origin for the LRRCE capping motif.

The evolution of the vertebrate metzincins; insights from Ciona intestinalis and Danio rerio.

BACKGROUND: The metzincins are a large gene superfamily of proteases characterized by the presence of a zinc protease domain, and include the ADAM, ADAMTS, BMP1/TLL, meprin and MMP genes. Metzincins are involved in the proteolysis of a wide variety of proteins, including those of the extracellular matrix. The metzincin gene superfamily comprises eighty proteins in the human genome and ninety-three in the mouse. When and how the level of complexity apparent in the vertebrate metzincin gene superfamily arose has not been determined in detail. Here we present a comprehensive analysis of vertebrate metzincins using genes from both Ciona intestinalis and Danio rerio to provide new insights into the complex evolution of this gene superfamily. RESULTS: We have identified 19 metzincin genes in the ciona genome and 83 in the zebrafish genome. Phylogenetic analyses reveal that the expansion of the metzincin gene superfamily in vertebrates has occurred predominantly by the simple duplication of pre-existing genes rather than by the appearance and subsequent expansion of new metzincin subtypes (the only example of which is the meprin gene family). Despite the number of zebrafish metzincin genes being relatively similar to that of tetrapods (e.g. man and mouse), the pattern of gene retention and loss within these lineages is markedly different. In addition, we have studied the evolution of the related TIMP gene family and identify a single ciona and four zebrafish TIMP genes. CONCLUSION: The complexity seen in the vertebrate metzincin gene families was mainly acquired during vertebrate evolution. The metzincin gene repertoire in protostomes and invertebrate deuterostomes has remained relatively stable. The expanded metzincin gene repertoire of extant tetrapods, such as man, has resulted largely from duplication events associated with early vertebrate evolution, prior to the sarcopterygian-actinopterygian split. The teleost repertoire of metzincin genes in part parallels that of tetrapods but has been significantly modified, perhaps as a consequence of a teleost-specific duplication event.

The TriTryp phosphatome: analysis of the protein phosphatase catalytic domains.

BACKGROUND: The genomes of the three parasitic protozoa Trypanosoma cruzi, Trypanosoma brucei and Leishmania major are the main subject of this study. These parasites are responsible for devastating human diseases known as Chagas disease, African sleeping sickness and cutaneous Leishmaniasis, respectively, that affect millions of people in the developing world. The prevalence of these neglected diseases results from a combination of poverty, inadequate prevention and difficult treatment. Protein phosphorylation is an important mechanism of controlling the development of these kinetoplastids. With the aim to further our knowledge of the biology of these organisms we present a characterisation of the phosphatase complement (phosphatome) of the three parasites. RESULTS: An ontology-based scan of the three genomes was used to identify 86 phosphatase catalytic domains in T. cruzi, 78 in T. brucei, and 88 in L. major. We found interesting differences with other eukaryotic genomes, such as the low proportion of tyrosine phosphatases and the expansion of the serine/threonine phosphatase family. Additionally, a large number of atypical protein phosphatases were identified in these species, representing more than one third of the total phosphatase complement. Most of the atypical phosphatases belong to the dual-specificity phosphatase (DSP) family and show considerable divergence from classic DSPs in both the domain organisation and sequence features. CONCLUSION: The analysis of the phosphatome of the three kinetoplastids indicates that they possess orthologues to many of the phosphatases reported in other eukaryotes, including humans. However, novel domain architectures and unusual combinations of accessory domains, suggest distinct functional roles for several of the kinetoplastid phosphatases, which await further experimental exploration. These distinct traits may be exploited in the selection of suitable new targets for drug development to prevent transmission and spread of the diseases, taking advantage of the already extensive knowledge on protein phosphatase inhibitors.

On the origins of the extracellular matrix in vertebrates.

Extracellular matrix (ECM) is a key metazoan characteristic. In addition to providing structure and orientation to tissues, it is involved in many cellular processes such as adhesion, migration, proliferation and differentiation. Here we provide a comprehensive analysis of ECM molecules focussing on when vertebrate specific matrices evolved. We identify 60 ECM genes and 20 associated processing enzymes in the genome of the urochordate Ciona intestinalis. A comparison with vertebrate and protostome genomes has permitted the identification of both a core set of metazoan matrix genes and vertebrate-specific innovations in the ECM. We have identified a few potential cases of de novo vertebrate ECM gene innovation, but the majority of ECM genes have resulted from duplication of pre-existing genes present in the ancestral vertebrate. In conclusion, the modern complexity we see in vertebrate ECM has come about largely by duplication and modification of pre-existing matrix molecules. Extracellular matrix genes and their processing enzymes appear to be over-represented in the vertebrate genome suggesting that these genes played an active role enabling and underpinning the evolution of vertebrates.

Identification of multiple integrin beta1 homologs in zebrafish (Danio rerio).

BACKGROUND: Integrins comprise a large family of alpha,beta heterodimeric, transmembrane cell adhesion receptors that mediate diverse essential biological functions. Higher vertebrates possess a single beta1 gene, and the beta1 subunit associates with a large number of alpha subunits to form the major class of extracellular matrix (ECM) receptors. Despite the fact that the zebrafish (Danio rerio) is a rapidly emerging model organism of choice for developmental biology and for models of human disease, little is currently known about beta1 integrin sequences and functions in this organism. RESULTS: Using RT-PCR, complete coding sequences of zebrafish beta1 paralogs were obtained from zebrafish embryos or adult tissues. The results show that zebrafish possess two beta1 paralogs (beta1-1 and beta1-2) that have a high degree of identity to other vertebrate beta1 subunits. In addition, a third, more divergent, beta1 paralog is present (beta1-3), which may have altered ligand-binding properties. Zebrafish also have other divergent beta1-like transcripts, which are C-terminally truncated forms lacking the transmembrane and cytoplasmic domains. Together with beta1-3 these truncated forms comprise a novel group of beta1 paralogs, all of which have a mutation in the ADMIDAS cation-binding site. Phylogenetic and genomic analyses indicate that the duplication that gave rise to beta1-1 and beta1-2 occurred after the divergence of the tetrapod and fish lineages, while a subsequent duplication of the ancestor of beta1-2 may have given rise to beta1-3 and an ancestral truncated paralog. A very recent tandem duplication of the truncated beta1 paralogs appears to have taken place. The different zebrafish beta1 paralogs have varied patterns of temporal expression during development. Beta1-1 and beta1-2 are ubiquitously expressed in adult tissues, whereas the other beta1 paralogs generally show more restricted patterns of expression. CONCLUSION: Zebrafish have a large set of integrin beta1 paralogs. beta1-1 and beta1-2 may share the roles of the solitary beta1 subunit found in other vertebrates, whereas beta1-3 and the truncated beta1 paralogs may have acquired novel functions.

The integrins of the urochordate Ciona intestinalis provide novel insights into the molecular evolution of the vertebrate integrin family.

BACKGROUND: Integrins are a functionally significant family of metazoan cell surface adhesion receptors. The receptors are dimers composed of an alpha and a beta chain. Vertebrate genomes encode an expanded set of integrin alpha and beta chains in comparison with protostomes such as drosophila or the nematode worm. The publication of the genome of a basal chordate, Ciona intestinalis, provides a unique opportunity to gain further insight into how and when the expanded integrin supergene family found in vertebrates evolved. RESULTS: The Ciona genome encodes eleven alpha and five beta chain genes that are highly homologous to their vertebrate homologues. Eight of the alpha chains contain an A-domain that lacks the short alpha helical region present in the collagen-binding vertebrate alpha chains. Phylogenetic analyses indicate the eight A-domain containing alpha chains cluster to form an ascidian-specific clade that is related to but, distinct from, the vertebrate A-domain clade. Two Ciona alpha chains cluster in laminin-binding clade and the remaining chain clusters in the clade that binds the RGD tripeptide sequence. Of the five Ciona beta chains, three form an ascidian-specific clade, one clusters in the vertebrate beta1 clade and the remaining Ciona chain is the orthologue of the vertebrate beta4 chain. CONCLUSION: The Ciona repertoire of integrin genes provides new insight into the basic set of these receptors available at the beginning of vertebrate evolution. The ascidian and vertebrate alpha chain A-domain clades originated from a common precursor but radiated separately in each lineage. It would appear that the acquisition of collagen binding capabilities occurred in the chordate lineage after the divergence of ascidians.

The characterisation of six ADAMTS proteases in the basal chordate Ciona intestinalis provides new insights into the vertebrate ADAMTS family.

ADAMTS, constituting a recently discovered family of secreted zinc-dependent metalloproteases, have been shown to have critical physiological roles through identification of a number of natural animal and human gene mutations. The identification of six ADAMTS genes in the basal chordate Ciona intestinalis provides new insight into how, when and in what order the vertebrate orthologues have evolved. The phylogenetic assignments, based on sequences conserved across all genes, are supported by conserved domain structures within defined sub-families. The phylogeny and the frequent localisation of ADAMTS genes in paralogous regions of the genome are consistent with the vertebrate lineages having arisen by large scale or genome duplication. The high level of conservation in the protease active site of vertebrate orthologues within some sub-families suggests subfunctionalisation, whereas the greater divergence in others would favour the evolution of novel substrate specificities and these observations are borne-out where substrate-specificity is known. The expansion and sub-specialization of the ADAMTS family is a component of the increased complexity of extracellular matrix that is associated with the evolution of vertebrates.

ADAMTSL3 as a candidate gene for schizophrenia: gene sequencing and ultra-high density association analysis by imputation.

We previously reported an association with a putative functional variant in the ADAMTSL3 gene, just below genome-wide significance in a genome-wide association study of schizophrenia. As variants impacting the function of ADAMTSL3 (a disintegrin-like and metalloprotease domain with thrombospondin type I motifs-like-3) could illuminate a novel disease mechanism and a potentially specific target, we have used complementary approaches to further evaluate the association. We imputed genotypes and performed high density association analysis using data from the HapMap and 1000 genomes projects. To review all variants that could potentially cause the association, and to identify additional possible pathogenic rare variants, we sequenced ADAMTSL3 in 92 schizophrenics. A total of 71 ADAMTSL3 variants were identified by sequencing, many were also seen in the 1000 genomes data, but 26 were novel. None of the variants identified by re-sequencing was in strong linkage disequilibrium (LD) with the associated markers. Imputation analysis refined association between ADAMTSL3 and schizophrenia, and highlighted additional common variants with similar levels of association. We evaluated the functional consequences of all variants identified by sequencing, or showing direct or imputed association. The strongest evidence for function remained with the originally associated variant, rs950169, suggesting that this variant may be causal of the association. Rare variants were also identified with possible functional impact. Our study confirms ADAMTSL3 as a candidate for further investigation in schizophrenia, using the variants identified here. The utility of imputation analysis is demonstrated, and we recommend wider use of this method to re-evaluate the existing canon of suggestive schizophrenia associations.

Joining forces in the quest for orthologs.

Better orthology-prediction resources would be beneficial for the whole biological community. A recent meeting discussed how to coordinate and leverage current efforts.

The collagens of hydra provide insight into the evolution of metazoan extracellular matrices.

A collagen-based extracellular matrix is one defining feature of all Metazoa. The thick sheet-like extracellular matrix (mesoglia) of the diploblast, hydra, has characteristics of both a basement membrane and an interstitial matrix. Several genes associated with mesoglea have been cloned including a basement membrane and fibrillar collagen and an A and B chain of laminin. Here we report the characterization of a further three fibrillar collagen genes (Hcol2, Hcol3, and Hcol5) and the partial sequence of a collagen gene with a unique structural organization consisting of multiple von Willebrand factor A domains interspersed with interrupted collagenous triple helices (Hcol6) from Hydra vulgaris. Hcol2 and -5 have major collagenous domains of classical length ( approximately 1020 amino acid residues), whereas the equivalent domain in Hcol3 is shorter (969 residues). The N-propeptide of Hcol2 contains a whey acid protein four-cysteine repeat (WAP) domain, and the equivalent domain of Hcol3 contains two WAP and two von Willebrand factor A domains. Phylogenetic analyses reveal that the hydra fibrillar collagen genes form a distinct clade that appears related to the protostome/deuterostome A clade of fibrillar collagens. Data base searches reveal Hcol2, -5, and -6 are highly conserved in Hydra magnipapillata, which also provided preliminary evidence for the expression of a B-clade fibrillar collagen. All four of the H. vulgaris collagens are expressed specifically by the ectoderm. The expression pattern for Hcol2 is similar to that previously reported for Hcol1 (Deutzmann, R., Fowler, S., Zhang, X., Boone, K., Dexter, S., Boot-Handford, R. P., Rachel, R., and Sarras, M. P., Jr. (2000) Development 127, 4669-4680) but distinct from the pattern shared by Hcol3 and Hcol5. The characterization of multiple collagen genes in relatively simple diploblastic organisms provides new insights into the molecular evolution of collagens and the origins of the collagen-based extracellular matrix found throughout the multicellular animal kingdom.