Publications

The GeneCaRNA human gene database is a member of the GeneCards Suite. It presents ~280,000 human non-coding RNA genes, identified algorithmically from ~690,000 RNAcentral transcripts. This expands by ~tenfold the ncRNA gene count relative to other sources. GeneCaRNA thus contains ~120,000 long non-coding RNAs (LncRNAs, >200 bases long), including ~100,000 novel genes. The latter have sparse functional information, a vast terra incognita for future research. LncRNA genes are uniformly represented on all nuclear chromosomes, with 10 genes on mitochondrial DNA. Data obtained from MalaCards, another GeneCards Suite member, finds 1547 genes associated with 1 to 50 diseases. About 15% of the associations portray experimental evidence, with cancers tending to be multigenic. Preliminary text mining within GeneCaRNA discovers interactions of lncRNA transcripts with target gene products, with 25% being ncRNAs and 75% proteins. GeneCaRNA has a biological pathways section, which at present shows 131 pathways for 38 lncRNA genes, a basis for future expansion. Finally, our GeneHancer database provides regulatory elements for ~110,000 lncRNA genes, offering pointers for co-regulated genes and genetic linkages from enhancers to diseases. We anticipate that the broad vista provided by GeneCaRNA will serve as an essential guide for further lncRNA research in disease decipherment.

Background: The Critical Assessment of Genome Interpretation (CAGI) aims to advance the state-of-the-art for computational prediction of genetic variant impact, particularly where relevant to disease. The five complete editions of the CAGI community experiment comprised 50 challenges, in which participants made blind predictions of phenotypes from genetic data, and these were evaluated by independent assessors. Results: Performance was particularly strong for clinical pathogenic variants, including some difficult-to-diagnose cases, and extends to interpretation of cancer-related variants. Missense variant interpretation methods were able to estimate biochemical effects with increasing accuracy. Assessment of methods for regulatory variants and complex trait disease risk was less definitive and indicates performance potentially suitable for auxiliary use in the clinic. Conclusions: Results show that while current methods are imperfect, they have major utility for research and clinical applications. Emerging methods and increasingly large, robust datasets for training and assessment promise further progress ahead.

In this Consensus Statement, the authors present results from an international multi-stakeholder consensus conducted by the PIONEER consortium to identify the most important questions in the field of prostate cancer that could be addressed using big data.PIONEER is a European network of excellence for big data in prostate cancer consisting of 37 private and public stakeholders from 9 countries across Europe. Many progresses have been done in prostate cancer management, but unanswered questions in the field still exist, and big data could help to answer these questions. The PIONEER consortium conducted a two-round modified Delphi survey aiming at building consensus between two stakeholder groups - health-care professionals and patients with prostate cancer - about the most important questions in the field of prostate cancer to be answered using big data. Respondents were asked to consider what would be the effect of answering the proposed questions on improving diagnosis and treatment outcomes for patients with prostate cancer and to score these questions on a scale of 1 (not important) to 9 (critically important). The mean percentage of participants who scored each of the proposed questions as critically important was calculated across the two stakeholder groups and used to rank the questions and identify the highest scoring questions in the critically important category. The identification of questions in prostate cancer that are important to various stakeholders will help the PIONEER consortium to provide answers to these questions to improve the clinical care of patients with prostate cancer.

Objectives: Genome-wide association studies have revealed over 200 genetic susceptibility loci for prostate cancer (PCa). By combining them, polygenic risk scores (PRS) can be generated to predict risk of PCa. We summarize the published evidence and conduct meta-analyses of PRS as a predictor of PCa risk in Caucasian men. Patients and methods: Data were extracted from 59 studies, with 16 studies including 17 separate analyses used in the main meta-analysis with a total of 20,786 cases and 69,106 controls identified through a systematic search of ten databases. Random effects meta-analysis was used to obtain pooled estimates of area under the receiver-operating characteristic curve (AUC). Meta-regression was used to assess the impact of number of single-nucleotide polymorphisms (SNPs) incorporated in PRS on AUC. Heterogeneity is expressed as I² scores. Publication bias was evaluated using funnel plots and Egger tests. Results: The ability of PRS to identify men with PCa was modest (pooled AUC 0.63, 95% CI 0.62-0.64) with moderate consistency (I² 64%). Combining PRS with clinical variables increased the pooled AUC to 0.74 (0.68-0.81). Meta-regression showed only negligible increase in AUC for adding incremental SNPs. Despite moderate heterogeneity, publication bias was not evident. Conclusion: Typically, PRS accuracy is comparable to PSA or family history with a pooled AUC value 0.63 indicating mediocre performance for PRS alone.

A widespread dogma asserts that life could not have emerged without biopolymers RNA and proteins. However, the widely acknowledged implausibility of a spontaneous appearance and proliferation of these complex molecules in primordial messy chemistry casts doubt on this scenario. A proposed alternative is \u201cLipid-First\u201d, based on the evidence that lipid assemblies may spontaneously emerge in heterogeneous environments, and are shown to undergo growth and fission, and to portray autocatalytic self-copying. What seems undecided is whether lipid assemblies have protein-like capacities for stereospecific interactions, a sine qua non of life processes. This Viewpoint aims to alleviate such doubts, pointing to growing experimental evidence that lipid aggregates possess dynamic surface configurations capable of stereospecific molecular recognition. Such findings help support a possible key role of lipids in seeding life's origin.

Circulating inflammatory markers are essential to human health and disease, and they are often dysregulated or malfunctioning in cancers as well as in cardiovascular, metabolic, immunologic and neuropsychiatric disorders. However, the genetic contribution to the physiological variation of levels of circulating inflammatory markers is largely unknown. Here we report the results of a genome-wide genetic study of blood concentration of ten cytokines, including the hitherto unexplored calcium-binding protein (S100B). The study leverages a unique sample of neonatal blood spots from 9,459 Danish subjects from the iPSYCH initiative. We estimate the SNP-heritability of marker levels as ranging from essentially zero for Erythropoietin (EPO) up to 73% for S100B. We identify and replicate 16 associated genomic regions (p −9), of which four are novel. We show that the associated variants map to enhancer elements, suggesting a possible transcriptional effect of genomic variants on the cytokine levels. The identification of the genetic architecture underlying the basic levels of cytokines is likely to prompt studies investigating the relationship between cytokines and complex disease. Our results also suggest that the genetic architecture of cytokines is stable from neonatal to adult life.

Prostate Cancer Diagnosis and Treatment Enhancement Through the Power of Big Data in Europe (PIONEER) is a European network of excellence for big data in prostate cancer, consisting of 32 private and public stakeholders from 9 countries across Europe. Launched by the Innovative Medicines Initiative 2 and part of the Big Data for Better Outcomes Programme (BD4BO), the overarching goal of PIONEER is to provide high-quality evidence on prostate cancer management by unlocking the potential of big data. The project has identified critical evidence gaps in prostate cancer care, via a detailed prioritization exercise including all key stakeholders. By standardizing and integrating existing high-quality and multidisciplinary data sources from patients with prostate cancer across different stages of the disease, the resulting big data will be assembled into a single innovative data platform for research. Based on a unique set of methodologies, PIONEER aims to advance the field of prostate cancer care with a particular focus on improving prostate-cancer-related outcomes, health system efficiency by streamlining patient management, and the quality of health and social care delivered to all men with prostate cancer and their families worldwide.Author Correction: The originally published article
contained errors in Figure 1 and did not reflect the current organization of
the PIONEER consortium. The figure has been corrected in the HTML and PDF
versions of the manuscript to reflect the correct organization of PIONEER.

"The Lipid World" was published in 2001, stemming from a highly effective collaboration with David Deamer during a sabbatical year 20 years ago at the Weizmann Institute of Science in Israel. The present review paper highlights the benefits of this scientific interaction and assesses the impact of the lipid world paper on the present understanding of the possible roles of amphiphiles and their assemblies in the origin of life. The lipid world is defined as a putative stage in the progression towards life's origin, during which diverse amphiphiles or other spontaneously aggregating small molecules could have concurrently played multiple key roles, including compartment formation, the appearance of mutually catalytic networks, molecular information processing, and the rise of collective self-reproduction and compositional inheritance. This review brings back into a broader perspective some key points originally made in the lipid world paper, stressing the distinction between the widely accepted role of lipids in forming compartments and their expanded capacities as delineated above. In the light of recent advancements, we discussed the topical relevance of the lipid worldview as an alternative to broadly accepted scenarios, and the need for further experimental and computer-based validation of the feasibility and implications of the individual attributes of this point of view. Finally, we point to possible avenues for exploring transition paths from small molecule-based noncovalent structures to more complex biopolymer-containing proto-cellular systems.

Large-scale genome sequencing is poised to provide a substantial increase in the rate of discovery of disease-associated mutations, but the functional interpretation of such mutations remains challenging. Here we show that deletions of a sequence on human chromosome 16 that we term the intestine-critical region (ICR) cause intractable congenital diarrhoea in infants(1,2). Reporter assays in transgenic mice show that the ICR contains a regulatory sequence that activates transcription during the development of the gastrointestinal system. Targeted deletion of the ICR in mice caused symptoms that recapitulated the human condition. Transcriptome analysis revealed that an unannotated open reading frame (Percc1) flanks the regulatory sequence, and the expression of this gene was lost in the developing gut of mice that lacked the ICR. Percc1-knockout mice displayed phenotypes similar to those observed upon ICR deletion in mice and patients, whereas an ICR-driven Percc1 transgene was sufficient to rescue the phenotypes found in mice that lacked the ICR. Together, our results identify a gene that is critical for intestinal function and underscore the need for targeted in vivo studies to interpret the growing number of clinical genetic findings that do not affect known protein-coding genes.

We studied the simulated replication and growth of prebiotic vesicles composed of 140 phospholipids and cholesterol using our R-GARD (Real Graded Autocatalysis Replication Domain) formalism that utilizes currently extant lipids that have known rate constants of lipid-vesicle interactions from published experimental data. R-GARD normally modifies kinetic parameters of lipid-vesicle interactions based on vesicle composition and properties. Our original R-GARD model tracked the growth and division of one vesicle at a time in an environment with unlimited lipids at a constant concentration. We explore here a modified model where vesicles compete for a finite supply of lipids. We observed that vesicles exhibit complex behavior including initial fast unrestricted growth, followed by intervesicle competition for diminishing resources, then a second growth burst driven by better-adapted vesicles, and ending with a final steady state. Furthermore, in simulations without kinetic parameter modifications (invariant kinetics), the initial replication was an order of magnitude slower, and vesicles' composition variability at the final steady state was much lower. The complex kinetic behavior was not observed either in the previously published R-GARD simulations or in additional simulations presented here with only one lipid component. This demonstrates that both a finite environment (inducing selection) and multiple components (providing variation for selection to act upon) are crucial for portraying evolution-like behavior. Such properties can improve survival in a changing environment by increasing the ability of early protocellular entities to respond to rapid environmental fluctuations likely present during abiogenesis both on Earth and possibly on other planets. This in silico simulation predicts that a relatively simple in vitro chemical system containing only lipid molecules might exhibit properties that are relevant to prebiotic processes.

Background: A key challenge in the realm of human disease research is next generation sequencing (NGS) interpretation, whereby identified filtered variant-harboring genes are associated with a patient's disease phenotypes. This necessitates bioinformatics tools linked to comprehensive knowledgebases. The GeneCards suite databases, which include GeneCards (human genes), MalaCards (human diseases) and PathCards (human pathways) together with additional tools, are presented with the focus on MalaCards utility for NGS interpretation as well as for large scale bioinformatic analyses. Results: VarElect, our NGS interpretation tool, leverages the broad information in the GeneCards suite databases. MalaCards algorithms unify disease-related terms and annotations from 69 sources. Further, MalaCards defines hierarchical relatedness-aliases, disease families, a related diseases network, categories and ontological classifications. GeneCards and MalaCards delineate and share a multi-tiered, scored gene-disease network, with stringency levels, including the definition of elite status-high quality gene-disease pairs, coming from manually curated trustworthy sources, that includes 4500 genes for 8000 diseases. This unique resource is key to NGS interpretation by VarElect. VarElect, a comprehensive search tool that helps infer both direct and indirect links between genes and user-supplied disease/phenotype terms, is robustly strengthened by the information found in MalaCards. The indirect mode benefits from GeneCards' diverse gene-to-gene relationships, including SuperPaths-integrated biological pathways from 12 information sources. We are currently adding an important information layer in the form of "disease SuperPaths", generated from the gene-disease matrix by an algorithm similar to that previously employed for biological pathway unification. This allows the discovery of novel gene-disease and disease-disease relationships. The advent of whole genome sequencing necessitates capacities to go beyond protein coding genes. GeneCards is highly useful in this respect, as it also addresses 101,976 non-protein-coding RNA genes. In a more recent development, we are currently adding an inclusive map of regulatory elements and their inferred target genes, generated by integration from 4 resources. Conclusions: MalaCards provides a rich big-data scaffold for in silico biomedical discovery within the gene-disease universe. VarElect, which depends significantly on both GeneCards and MalaCards power, is a potent tool for supporting the interpretation of wet-lab experiments, notably NGS analyses of disease. The GeneCards suite has thus transcended its 2-decade role in biomedical research, maturing into a key player in clinical investigation.

Congenital general anosmia (CGA) is a neurological disorder entailing a complete innate inability to sense odors. While the mechanisms underlying vertebrate olfaction have been studied in detail, there are still gaps in our understanding of the molecular genetic basis of innate olfactory disorders. Applying whole-exome sequencing to a family multiply affected with CGA, we identified three members with a rare X-linked missense mutation in the TENM1 (teneurin 1) gene (ENST00000422452:c.C4829T). In Drosophila melanogaster, TENM1 functions in synaptic-partner-matching between axons of olfactory sensory neurons and target projection neurons and is involved in synapse organization in the olfactory system. We used CRISPR-Cas9 system to generate a Tenm1 disrupted mouse model. Tenm1^−/− and point-mutated Tenm1^A/A adult mice were shown to have an altered ability to locate a buried food pellet. Tenm1^A/A mice also displayed an altered ability to sense aversive odors. Results of our study, that describes a new Tenm1 mouse, agree with the hypothesis that TENM1 has a role in olfaction. However, additional studies should be done in larger CGA cohorts, to provide statistical evidence that loss-of-function mutations in TENM1 can solely cause the disease in our and other CGA cases.

Pemphigus vulgaris (PV) is a life-threatening autoimmune mucocutaneous blistering disease caused by disruption of intercellular adhesion due to auto-antibodies directed against epithelial components. Treatment is limited to immunosuppressive agents, which are associated with serious adverse effects. The propensity to develop the disease is in part genetically determined. We therefore reasoned that the delineation of PV genetic basis may point to novel therapeutic strategies. Using a genome-wide association approach, we recently found that genetic variants in the vicinity of the ST18 gene confer a significant risk for the disease. Here, using targeted deep sequencing, we identified a PV-associated variant residing within the ST18 promoter region (p

GeneCards, the human gene compendium, enables researchers to effectively navigate and inter-relate the wide universe of human genes, diseases, variants, proteins, cells, and biological pathways. Our recently launched Version 4 has a revamped infrastructure facilitating faster data updates, better-targeted data queries, and friendlier user experience. It also provides a stronger foundation for the GeneCards suite of companion databases and analysis tools. Improved data unification includes gene-disease links via MalaCards and merged biological pathways via PathCards, as well as drug information and proteome expression. VarElect, another suite member, is a phenotype prioritizer for next-generation sequencing, leveraging the GeneCards and MalaCards knowledgebase. It automatically infers direct and indirect scored associations between hundreds or even thousands of variant-containing genes and disease phenotype terms. Var- Elects capabilities, either independently or within TGex, our comprehensive variant analysis pipeline, help prepare for the challenge of clinical projects that involve thousands of exome/genome NGS analyses.

Background TECPR2 was first described as a disease causing gene when the c.3416delT frameshift mutation was found in five Jewish Bukharian patients with similar features. It was suggested to constitute a new subtype of complex hereditary spastic paraparesis (SPG49). Results We report here 3 additional patients from unrelated non-Bukharian families, harboring two novel mutations (c.1319delT, c.C566T) in this gene. Accumulating clinical data clarifies that in addition to intellectual disability and evolving spasticity the main disabling feature of this unique disorder is autonomic-sensory neuropathy accompanied by chronic respiratory disease and paroxysmal autonomic events. Conclusion We suggest that the disease should therefore be classified as a new subtype of hereditary sensory-autonomic neuropathy. The discovery of additional mutations in non-Bukharian patients implies that this disease might be more common than previously appreciated and should therefore be considered in undiagnosed cases of intellectual disability with autonomic features and respiratory symptoms regardless of demographic origin.

Systems medicine provides insights into mechanisms of human diseases, and expedites the development of better diagnostics and drugs. To facilitate such strategies, we initiated MalaCards, a compendium of human diseases and their annotations, integrating and often remodeling information from 64 data sources. MalaCards employs, among others, the proven automatic data-mining strategies established in the construction of GeneCards, our widely used compendium of human genes. The development of MalaCards poses many algorithmic challenges, such as disease name unification, integrated classification, gene-disease association, and disease-targeted expression analysis. MalaCards displays a Web card for each of >19,000 human diseases, with 17 sections, including textual summaries, related diseases, related genes, genetic variations and tests, and relevant publications. Also included are a powerful search engine and a variety of categorized disease lists. This unit describes two basic protocols to search and browse MalaCards effectively. Curr. Protoc. Bioinform. 47:1.24.1-1.24.19.

Autophagy dysfunction has been implicated in a group of progressive neurodegenerative diseases, and has been reported to play a major role in the pathogenesis of these disorders. We have recently reported a recessive mutation in TECPR2, an autophagy-implicated WD repeat-containing protein, in five individuals with a novel form of monogenic hereditary spastic paraparesis (HSP). We found that diseased skin fibroblasts had a decreased accumulation of the autophagy-initiation protein MAP1LC3B/LC3B, and an attenuated delivery of both LC3B and the cargorecruiting protein SQSTM1/p62 to the lysosome where they are subject to degradation. The discovered TECPR2 mutation reveals for the first time a role for aberrant autophagy in a major class of Mendelian neurodegenerative diseases, and suggests mechanisms by which impaired autophagy may impinge on a broader scope of neurodegeneration.

Olfactory receptors (ORs) constitute the largest gene family in the mammalian genome. The existence of these proteins underlies the nature of, and variability in, odorant perception. The Human Olfactory Receptor Data Explorer (HORDE, http://genome.weizmann.ac.il/horde/ ) is a free online resource, which presents a complete compendium of all OR genes and pseudogenes in the genome of human and four other vertebrates. HORDE includes three parts: (1) an automated pipeline, which mines OR gene and pseudogene sequences out of complete genomes, and generates gene symbols based on sequence similarity; (2) a card generator that obtains and displays annotative information on individual ORs retrieved from external databases and relevant studies; and (3) a search engine that allows user retrieval of OR information. For human ORs, HORDE specifically addresses the universe of interindividual variation, as obtained from several sources, including whole genome sequences made possible by next-generation sequencing. This encompasses single nucleotide polymorphisms (SNP) and copy number variation (CNV), including deleterious mutational events. HORDE also hosts a number of tools designed specifically to assist in the study of OR evolution and function. In this chapter, we describe the status of HORDE (build #43). We also discuss plans for future enhancements and a road map for HORDE to become a better community-based bioinformatics tool. We highlight HORDE's role as a major research tool in the study of an expanding cohort of OR repertoires.

Genetic variations in olfactory receptors likely contribute to the diversity of odorant-specific sensitivity phenotypes. Our working hypothesis is that genetic variations in auxiliary olfactory genes, including those mediating transduction and sensory neuronal development, may constitute the genetic basis for general olfactory sensitivity (GOS) and congenital general anosmia (CGA). We thus performed a systematic exploration for auxiliary olfactory genes and their documented variation. This included a literature survey, seeking relevant functional in vitro studies, mouse gene knockouts and human disorders with olfactory phenotypes, as well as data mining in published transcriptome and proteome data for genes expressed in olfactory tissues. In addition, we performed next-generation transcriptome sequencing (RNA-seq) of human olfactory epithelium and mouse olfactory epithelium and bulb, so as to identify sensory-enriched transcripts. Employing a global score system based on attributes of the 11 data sources utilized, we identified a list of 1,680 candidate auxiliary olfactory genes, of which 450 are shortlisted as having higher probability of a functional role. For the top-scoring 136 genes, we identified genomic variants (probably damaging single nucleotide polymorphisms, indels, and copy number deletions) gleaned from public variation repositories. This database of genes and their variants should assist in rationalizing the great interindividual variation in human overall olfactory sensitivity (http://genome.weizmann.ac.il/GOSdb).

We studied five individuals from three Jewish Bukharian families affected by an apparently autosomal-recessive form of hereditary spastic paraparesis accompanied by severe intellectual disability, fluctuating central hypoventilation, gastresophageal reflux disease, wake apnea, areflexia, and unique dysmorphic features. Exome sequencing identified one homozygous variant shared among all affected individuals and absent in controls: a 1 bp frameshift TECPR2 deletion leading to a premature stop codon and predicting significant degradation of the protein. TECPR2 has been reported as a positive regulator of autophagy. We thus examined the autophagy-related fate of two key autophagic proteins, SQSTM1 (p62) and MAP1LC3B (LC3), in skin fibroblasts of an affected individual, as compared to a healthy control, and found that both protein levels were decreased and that there was a more pronounced decrease in the lipidated form of LC3 (LC3II). siRNA knockdown of TECPR2 showed similar changes, consistent with aberrant autophagy. Our results are strengthened by the fact that autophagy dysfunction has been implicated in a number of other neurodegenerative diseases. The discovered TECPR2 mutation implicates autophagy, a central intracellular mechanism, in spastic paraparesis.

Background: Information on nucleotide diversity along completely sequenced human genomes has increased tremendously over the last few years. This makes it possible to reassess the diversity status of distinct receptor proteins in different human individuals. To this end, we focused on the complete inventory of human olfactory receptor coding regions as a model for personal receptor repertoires.Results: By performing data-mining from public and private sources we scored genetic variations in 413 intact OR loci, for which one or more individuals had an intact open reading frame. Using 1000 Genomes Project haplotypes, we identified a total of 4069 full-length polypeptide variants encoded by these OR loci, average of ~10 per locus, constituting a lower limit for the effective human OR repertoire. Each individual is found to harbor as many as 600 OR allelic variants, ~50% higher than the locus count. Because OR neuronal expression is allelically excluded, this has direct effect on smell perception diversity of the species. We further identified 244 OR segregating pseudogenes (SPGs), loci showing both intact and pseudogene forms in the population, twenty-six of which are annotatively " resurrected" from a pseudogene status in the reference genome. Using a custom SNP microarray we validated 150 SPGs in a cohort of 468 individuals, with every individual genome averaging 36 disrupted sequence variations, 15 in homozygote form. Finally, we generated a multi-source compendium of 63 OR loci harboring deletion Copy Number Variations (CNVs). Our combined data suggest that 271 of the 413 intact OR loci (66%) are affected by nonfunctional SNPs/indels and/or CNVs.Conclusions: These results portray a case of unusually high genetic diversity, and suggest that individual humans have a highly personalized inventory of functional olfactory receptors, a conclusion that might apply to other receptor multigene families.

Paenibacillus dendritiformis is a Gram-positive, soil-dwelling, spore-forming social microorganism. An intriguing collective faculty of this strain is manifested by its ability to switch between different morphotypes, such as the branching (T) and the chiral (C) morphotypes. Here we report the 6.3-Mb draft genome sequence of the P. dendritiformis C454 chiral morphotype.

Many reports in different populations have demonstrated linkage of the 10q24-q26 region to schizophrenia, thus encouraging further analysis of this locus for detection of specific schizophrenia genes. Our group previously reported linkage of the 10q24-q26 region to schizophrenia in a unique, homogeneous sample of Arab-Israeli families with multiple schizophrenia-affected individuals, under a dominant model of inheritance. To further explore this candidate region and identify specific susceptibility variants within it, we performed re-analysis of the 10q24-26 genotype data, taken from our previous genome-wide association study (GWAS) (Alkelai et al, 2011). We analyzed 2089 SNPs in an extended sample of 57 Arab Israeli families (189 genotyped individuals), under the dominant model of inheritance, which best fits this locus according to previously performed MOD score analysis. We found significant association with schizophrenia of the TCF7L2 gene intronic SNP, rs12573128, (p = 7.01×10^-6) and of the nearby intergenic SNP, rs1033772, (p = 6.59×10^-6) which is positioned between TCF7L2 and HABP2. TCF7L2 is one of the best confirmed susceptibility genes for type 2 diabetes (T2D) among different ethnic groups, has a role in pancreatic beta cell function and may contribute to the comorbidity of schizophrenia and T2D. These preliminary results independently support previous findings regarding a possible role of TCF7L2 in susceptibility to schizophrenia, and strengthen the importance of integrating linkage analysis models of inheritance while performing association analyses in regions of interest. Further validation studies in additional populations are required.

While the use of population-based samples is a common strategy in genome-wide association studies (GWASs), family-based samples have considerable advantages, such as robustness against population stratification and false-positive associations, better quality control, and the possibility to check for both linkage and association. In a genome-wide linkage study of schizophrenia in Arab-Israeli families with multiple affected individuals, we previously reported significant evidence for a susceptibility locus at chromosome 6q23.2-q24.1 and suggestive evidence at chromosomes 10q22.3-26.3, 2q36.1-37.3 and 7p21.1-22.3. To identify schizophrenia susceptibility genes, we applied a family-based GWAS strategy in an enlarged, ethnically homogeneous, Arab-Israeli family sample. We performed genome-wide single nucleotide polymorphism (SNP) genotyping and single SNP transmission disequilibrium test association analysis and found genome-wide significant association (best value of P=1.22×10 ^-11) for 8 SNPs within or near highly reasonable functional candidate genes for schizophrenia. Of particular interest are a group of SNPs within and flanking the transcriptional factor LRRFIP1 gene. To determine replicability of the significant associations beyond the Arab-Israeli population, we studied the association of the significant SNPs in a German case-control validation sample and found replication of associations near the UGT1 subfamily and EFHD1 genes. Applying an exploratory homozygosity mapping approach as a complementary strategy to identify schizophrenia susceptibility genes in our Arab Israeli sample, we identified 8 putative disease loci. Overall, this GWAS, which emphasizes the important contribution of family based studies, identifies promising candidate genes for schizophrenia.

Purpose: For diseases with complex phenotype such as diabetic nephropathy (DN), integration of multiple Omics sources promises an improved description of the disease pathophysiology, being the basis for novel diagnostics and therapy, but equally important personalization aspects. Experimental design: Molecular features on DN were retrieved from public domain Omics studies and by mining scientific literature, patent text and clinical trial specifications. Molecular feature sets were consolidated on a human protein interaction network and interpreted on the level of molecular pathways in the light of the pathophysiology of the disease and its clinical context defined as associated biomarkers and drug targets. Results: About 1000 gene symbols each could be assigned to the pathophysiological description of DN and to the clinical context. Direct feature comparison showed minor overlap, whereas on the level of molecular pathways, the complement and coagulation cascade, PPAR signaling, and the renin-angiotensin system linked the disease descriptor space with biomarkers and targets. Conclusion and clinical relevance: Only the combined molecular feature landscapes closely reflect the clinical implications of DN in the context of hypertension and diabetes. Omics data integration on the level of interaction networks furthermore provides a platform for identification of pathway-specific biomarkers and therapy options.

Copy-number variations (CNVs) are widespread in the human genome, but comprehensive assignments of integer locus copy-numbers (i.e., copy-number genotypes) that, for example, enable discrimination of homozygous from heterozygous CNVs, have remained challenging. Here we present CopySeq, a novel computational approach with an underlying statistical framework that analyzes the depth-of-coverage of high-throughput DNA sequencing reads, and can incorporate paired-end and breakpoint junction analysis based CNV-analysis approaches, to infer locus copy-number genotypes. We benchmarked CopySeq by genotyping 500 chromosome 1 CNV regions in 150 personal genomes sequenced at low-coverage. The assessed copy-number genotypes were highly concordant with our performed qPCR experiments (Pearson correlation coefficient 0.94), and with the published results of two microarray platforms (95-99% concordance). We further demonstrated the utility of CopySeq for analyzing gene regions enriched for segmental duplications by comprehensively inferring copy-number genotypes in the CNV-enriched >800 olfactory receptor (OR) human gene and pseudogene loci. CopySeq revealed that OR loci display an extensive range of locus copy-numbers across individuals, with zero to two copies in some OR loci, and two to nine copies in others. Among genetic variants affecting OR loci we identified deleterious variants including CNVs and SNPs affecting ~15% and~20% of the human OR gene repertoire, respectively, implying that genetic variants with a possible impact on smell perception are widespread. Finally, we found that for several OR loci the reference genome appears to represent a minor-frequency variant, implying a necessary revision of the OR repertoire for future functional studies. CopySeq can ascertain genomic structural variation in specific gene families as well as at a genome-wide scale, where it may enable the quantitative evaluation of CNVs in genome-wide association studies involving high-throughput sequencing.

Association with schizophrenia of the Abelson Helper Integration Site 1 (AHI1) gene on chromosome 6q23 and the adjacent primate-specific gene, C6orf217, was demonstrated in an inbred, Arab Israeli family sample and replicated in an Icelandic case control sample. Further support was provided by a second replication in a large European sample and a meta-analysis that supported association with schizophrenia of all seven alleles overtransmitted to affected subjects in the original study. We examined constitutive expression of AHI1 and C6orf217 in immortalized lymphoblasts of patients from the Arab Israeli family sample in which the association with schizophrenia was originally discovered and population-matched normal controls, and in post-mortem brain of patients with schizophrenia and bipolar (BP) disorder and control subjects from the Stanley Medical Research Institute Collection. We found a significant effect of diagnostic group in the lymphoblast sample (F = 5.72; df = 2,39; p = 0.006). Patients with early age of onset had higher AHI1 expression than controls and later onset patients (p = 0.002; 0.03 respectively). C6orf217 expression in lymphoblasts was too low to measure. We found no difference in brain expression of AHI1 in schizophrenia or BP patients compared to controls. However, there was a genotypic difference in AHI1 expression for SNP rs9321501, which was strongly associated with schizophrenia in the original study. Genotypes that included the undertransmitted C allele (CC/AC) showed lower expression than the homozygous AA genotype (F = 4.73, df = 2,83; p = 0.028). There was no significant difference in brain expression of C6orf217 between patients and controls and no genotypic effect. This study provides further evidence for involvement of AHI1 in susceptibility to schizophrenia. (C) 2010 Elsevier B.V. All rights reserved.

The Graded Autocatalysis Replication Domain (GARD) model describes an origin of life scenario which involves non-covalent compositional assemblies, made of monomeric mutually catalytic molecules. GARD constitutes an alternative to informational biopolymers as a mechanism of primordial inheritance. In the present work, we examined the effect of mutations, one of the most fundamental mechanisms for evolution, in the context of the networks of mutual interaction within GARD prebiotic assemblies. We performed a systematic analysis analogous to single and double gene deletions within GARD. While most deletions have only a small effect on both growth rate and molecular composition of the assemblies, ~10% of the deletions caused lethality, or sometimes showed enhanced fitness. Analysis of 14 different network properties on 2,000 different GARD networks indicated that lethality usually takes place when the deleted node has a high molecular count, or when it is a catalyst for such node. A correlation was also found between lethality and node degree centrality, similar to what is seen in real biological networks. Addressing double knockout mutations, our results demonstrate the occurrence of both synthetic lethality and extragenic suppression within GARD networks, and convey an attempt to correlate synthetic lethality to network node-pair properties. The analyses presented help establish GARD as a workable alternative prebiotic scenario, suggesting that life may have begun with large molecular networks of low fidelity, that later underwent evolutionary compaction and fidelity augmentation.

A genome scan for schizophrenia related loci in Arab Israeli families by Lerer et al. [Lerer et al. (2003); Mol Psychiatry 8:488-498] detected significant evidence for linkage at chromosome 6q23. Subsequent fine mapping [Levi et al. (2005); Eur J Hum Genet 13:763-771], association [Amann-Zalcenstein et al. (2006); Eur J Hum Genet 14:1111-1119] and replication studies [Ingason et al. (2007); Eur J Hum Genet 15:988-991] identified AHI1 as a putative susceptibility gene. The same genome scan revealed suggestive evidence for a schizophrenia susceptibility locus in the 10q23-26 region. Genes at these two loci may act independently in the pathogenesis of the disease in our homogeneous sample of Arab Israeli families or may interact with each other and with other factors in a common biological pathway. The purpose of our current study was to test the hypothesis of genetic interaction between these two loci and to identify the type of interaction between them. The initial stage of our study focused on the 10q23-q26 region which has not been explored further in our sample. The second stage of the study included a test for possible genetic interaction between the 6q23.3 locus and the refined 10q24.33-q26.13 locus. A final candidate region of 19.9 Mb between markers D10S222 (105.3 Mb) and D10S587 (125.2 Mb) was found on chromosome 10 by non-parametric and parametric linkage analyses. These linkage findings are consistent with previous reports in the same chromosomal region. Two-locus multipoint linkage analysis under three complex disease inheritance models (heterogeneity, multiplicative, and additive models) yielded a best maximum LOD score of 7.45 under the multiplicative model suggesting overlapping function of the 6q23.3 and 10q24.33-q26.13 loci.

RGS2 (regulator of G-protein signaling 2) modulates dopamine receptor signal transduction. Functional variants in the gene may influence susceptibility to extrapyramidal symptoms (EPS) induced by antipsychotic drugs. To further investigate our previous report of association of the RGS2 gene with susceptibility to antipsychotic-induced EPS, we performed a replication study. EPS were rated in 184 US patients with schizophrenia (115 African Americans, 69 Caucasian) treated for at least a month with typical antipsychotic drugs (n = 45), risperidone (n = 46), olanzapine (n = 50) or clozapine (n = 43). Six single nucleotide polymorphisms (SNPs) within or flanking RGS2 were genotyped (rs1933695, rs2179652, rs2746073, rs4606, rs1819741 and rs1152746). Odds ratios (ORs) and 95% confidence intervals (CIs) were calculated by logistic regression. Our results indicate association of SNP rs4606 with antipsychotic-induced parkinsonism (AIP), as measured by the Simpson Angus scale, in the overall sample and in the African-American subsample, the G (minor) allele having a protective effect. ORs for AIP among rs4606 G-allele carriers were 0.23 (95% CI 0.10-0.54, P=0.001) in the overall sample, and 0.20 (0.07-0.57, P = 0.003) in the African-American subsample. In the previously studied Israeli sample the OR was 0.31 (0.11-0.84, P=0.02). We completely sequenced the RGS2 gene in nine patients with AIP and nine patients without, from the Israeli sample. No common coding polymorphisms or additional regulatory variants were revealed, suggesting that association of the rs4606 C/G polymorphism with AIP is biologically meaningful and not a consequence of linkage disequilibrium with another functional variant. Taken together, the findings of the current study support the association of RGS2 with AIP and focus on a possible protective effect of the minor G allele of SNP rs4606. This SNP is located in the 3-regulatory region of the gene, and is known to influence RGS2 mRNA levels and protein expression.

Previous work suggests that young women who smoke cigarettes regularly, or did so in the past, manifest a neurocognitive profile that is characterized by small but significant impairments of response inhibition and attention. The present study sought to determine whether variation in nicotinic cholinergic receptor (nAchR) genes impacts upon cognitive function in these domains by overall or differential effects on the performance of current, former and non-smokers. The study sample consisted of 100 female college students, current or past smokers, and 144 who had never smoked. All performed a computerized neurocognitive test battery and were genotyped for 39 single nucleotide polymorphisms in 11 nAchR genes. The results, derived from linear or logistic regression, show significant direct and interactive relationships between single nucleotide polymorphisms and haplotypes in several nAchR genes and performance on the Matching Familiar Figures Test (MFFT) Stroop test, Continuous Performance Task (CPT) and Tower of London (TOL) test. Response inhibition (MFFT, Stroop, CPT Loading Phase, TOL) was associated with variants in CHRNA2, CHRNA4, CHRNA5, CHRNA7, CHRNA9, CHRNA10, CHRNB2 and CHRNB3. Selective attention (Stroop) was associated with CHRNA4, CHRNA5, CHRNA9 and CHRNB2. Sustained attention (CPT Boring Phase) was associated with CHRNA4, CHRNA5, CHRNA7, CHRNA10 and CHRNB3. Up to 37% of the variance among the smokers and up to 47% of the variance among the non-smokers on the test measures was explained. Differences between smokers and non-smokers in neurocognitive function, putatively implicated in susceptibility to nicotine dependence, may be modulated by variants in nAchR genes, with potential implications for prevention and treatment.

The genetic basis of odorant-specific variations in human olfactory thresholds, and in particular of enhanced odorant sensitivity (hyperosmia), remains largely unknown. Olfactory receptor (OR) segregating pseudogenes, displaying both functional and nonfunctional alleles in humans, are excellent candidates to underlie these differences in olfactory sensitivity. To explore this hypothesis, we examined the association between olfactory detection threshold phenotypes of four odorants and segregating pseudogene genotypes of 43 ORs genome-wide. A strong association signal was observed between the single nucleotide polymorphism variants in OR11H7P and sensitivity to the odorant isovaleric acid. This association was largely due to the low frequency of homozygous pseudogenized genotype in individuals with specific hyperosmia to this odorant, implying a possible functional role of OR11H7P in isovaleric acid detection. This predicted receptor-ligand functional relationship was further verified using the Xenopus oocyte expression system, whereby the intact allele of OR11H7P exhibited a response to isovaleric acid. Notably, we also uncovered another mechanism affecting general olfactory acuity that manifested as a significant inter-odorant threshold concordance, resulting in an overrepresentation of individuals who were hyperosmic to several odorants. An involvement of polymorphisms in other downstream transduction genes is one possible explanation for this observation. Thus, human hyperosmia to isovaleric acid is a complex trait, contributed to by both receptor and other mechanisms in the olfactory signaling pathway.

Five common assumptions about the first cells are challenged by the pre-biotic ecology model and are replaced by the following propositions: firstly, early cells were more complex, more varied and had a greater diversity of constituents than modern cells; secondly, the complexity of a cell is not related to the number of genes it contains, indeed, modern bacteria are as complex as eukaryotes; thirdly, the unit of early life was an 'ecosystem' rather than a 'cell'; fourthly, the early cell needed no genes at all; fifthly, early life depended on non-covalent associations and on catalysts that were not confined to specific reactions. We present here the outlines of a theory that connects findings about modern bacteria with speculations about their origins.

OBJECTIVES: To investigate the role of genes encoding regulators of G protein signaling in early therapeutic response to antipsychotic drugs and in susceptibility to drug-induced extrapyramidal symptoms. As regulators of G protein signaling and regulators of G protein signaling-like proteins play a pivotal role in dopamine receptor signaling, genetically based, functional variation could contribute to interindividual variability in therapeutic and adverse effects. METHODS: Consecutively hospitalized, psychotic patients with Diagnostic and Statistical Manual of Mental Disorder-IV schizophrenia (n=121) were included in the study if they received treatment with typical antipsychotic medication (n=72) or typical antipsychotic drugs and risperidone (n=49) for at least 2 weeks. Clinical state and adverse effects were rated at baseline and after 2 weeks. Twenty-four single nucleotide polymorphisms were genotyped in five regulators of G protein signaling genes. RESULTS: None of the single nucleotide polymorphisms were related to clinical response to antipsychotic treatment at 2 weeks. Five out of six single nucleotide polymorphisms within or flanking the RGS2 gene were nominally associated with development or worsening of parkinsonian symptoms (PARK+) as measured by the Simpson Angus Scale, one of them after correction for multiple testing (rs4606, P=0.002). A GCCTG haplotype encompassing tagging single nucleotide polymorphisms within and flanking RGS2 was significantly overrepresented among PARK+ compared with PARK- patients (0.23 vs. 0.08, P=0.003). A second, 'protective', GTGCA haplotype was significantly overrepresented in PARK- patients (0.13 vs. 0.30, P=0.009). Both haplotype associations survive correction for multiple testing. CONCLUSIONS: Subject to replication, these findings suggest that genetic variation in the RGS2 gene is associated with susceptibility to extrapyramidal symptoms induced by antipsychotic drugs.

The MDM2 protein is an ubiquitin ligase that plays a critical role in regulating the levels and activity of the p53 protein, which is a central tumor suppressor. A SNP in the human MDM2 gene (SNP309 T/G) occurs at frequencies dependent on demographic history and has been shown to have important differential effects on the activity of the MDM2 and p53 proteins and to associate with altered risk for the development of several cancers. In this report, the haplotype structure of the MDM2 gene is determined by using 14 different SNPs across the gene from three different population samples: Caucasians, African Americans, and the Ashkenazi Jewish ethnic group. The results presented in this report indicate that there is a substantially reduced variability of the deleterious SNP309 G allele haplotype in all three populations studied, whereas multiple common T allele haplotypes were found in all three populations. This observation, coupled with the relatively high frequency of the G allele haplotype in both and Caucasian and Ashkenazi Jewish population data sets, suggests that this haplotype could have undergone a recent positive selection sweep. An entropy-based selection test is presented that explicitly takes into account the correlations between different SNPs, and the analysis of MDM2 reveals a significant departure from the standard assumptions of selective neutrality.

The existence of osteoarthritis susceptibility loci on chromosome 6 for individuals suffering from hip and knee osteoarthritis has been suggested. We determined whether radiographic hand osteoarthritis in a demographically homogeneous population of European origin can be linked to loci on chromosome 6p12.3-p12.1. Nine single nucleotide polymorphisms (SNPs) were genotyped in 764 individuals (members of 189 nuclear and more complex two- or three-generation families). Radiographic hand osteoarthritis was characterized by two traits: (1) the total individual osteoarthritis score (PC1-OA) and (2) the osteophytes score (PC1-OS), obtained from the principal components analysis of sums of the Kellgren and Lawrence grade and of the osteophyte grades, respectively, for 14 joints on each hand. The contribution of genetic and environmental factors and of covariates such as age and body mass index to hand osteoarthritis was evaluated by variance components analysis. The association between the studied traits and selected DNA markers was evaluated by three types of transmission disequilibrium tests. The parent-offspring and sib-sib correlations were statistically significant for all studied traits. The additive genetic effects for PC1-OA and PC1-OS were estimated to be 43% and 37.9%, respectively. Transmission disequilibrium tests consistently revealed a statistically significant association (p values ranged from 0.017 to 0.030) between SNP rs1508632 and PC1-OS. In the tested cohort the putative genetic factors are influential enough to determine interindividual differences regarding the extent of hand osteoarthritis. SNP rs1508632 lies in immediate proximity to the TINAG gene, implicating it as a possible hand osteoarthritis susceptibility gene.

Background: Quantitative variation in gene expression has been proposed to underlie phenotypic variation among human individuals. A facilitating step towards understanding the basis for gene expression variability is associating genome wide transcription patterns with potential cis modifiers of gene expression. Description: EXPOLDB, a novel Database, is a new effort addressing this need by providing information on gene expression levels variability across individuals, as well as the presence and features of potentially polymorphic (TG/CA)_n repeats. EXPOLDB thus enables associating transcription levels with the presence and length of (TG/CA)_n repeats. One of the unique features of this database is the display of expression data for 5 pairs of monozygotic twins, which allows identification of genes whose variability in expression, are influenced by non-genetic factors including environment. In addition to queries by gene name, EXPOLDB allows for queries by a pathway name. Users can also upload their list of HGNC (HUGO (The Human Genome Organisation) Gene Nomenclature Committee) symbols for interrogating expression patterns. The online application 'SimRep' can be used to find simple repeats in a given nucleotide sequence. To help illustrate primary applications, case examples of Housekeeping genes and the RUNX gene family, as well as one example of glycolytic pathway genes are provided. Conclusion: The uniqueness of EXPOLDB is in facilitating the association of genome wide transcription variations with the presence and type of polymorphic repeats while offering the feature for identifying genes whose expression variability are influenced by non genetic factors including environment. In addition, the database allows comprehensive querying including functional information on biochemical pathways of the human genes.

Background: Mammalian olfactory receptor (OR) genes reside in numerous genomic clusters of up to several dozen genes. Whole-genome sequence alignment nets of five mammals allow their comprehensive comparison, aimed at reconstructing the ancestral olfactory subgenome. Results: We developed a new and general tool for genome-wide definition of genomic gene clusters conserved in multiple species. Syntenic orthologs, defined as gene pairs showing conservation of both genomic location and coding sequence, were subjected to a graph theory algorithm for discovering CLICs (clusters in conservation). When applied to ORs in five mammals, including the marsupial opossum, more than 90% of the OR genes were found within a framework of 48 multi-species CLICs, invoking a general conservation of gene order and composition. A detailed analysis of individual CLICs revealed multiple differences among species, interpretable through species-specific genomic rearrangements and reflecting complex mammalian evolutionary dynamics. One significant instance involves CLIC #1, which lacks a human member, implying the human-specific deletion of an OR cluster, whose mouse counterpart has been tentatively associated with isovaleric acid odorant detection. Conclusion: The identified multi-species CLICs demonstrate that most of the mammalian OR clusters have a common ancestry, preceding the split between marsupials and placental mammals. However, only two of these CLICs were capable of incorporating chicken OR genes, parsimoniously implying that all other CLICs emerged subsequent to the avian-mammalian divergence.

Of all five senses, olfaction is the most complex molecular mechanism, as it comprises hundreds of receptor proteins enabling it to detect and discriminate thousands of odorants. Until lately, the understanding of this highly sophisticated sensory neuronal pathway has been rather sketchy. The sequencing of the human genome and the consequent advent of new genomic tools have opened new opportunities to better understand this multifaceted biological system. Here, we present the relevant progresses made in the last decade and highlight the possible genetic mechanisms of human olfactory variability.

While genetic factors clearly play a role in conferring breast cancer risk, the contribution of ATM gene mutations to breast cancer is still unsettled. To shed light on this issue, ATM haplotypes were constructed using eight SNPs spanning the ATM gene region (142 kb) in ethnically diverse non-Ashkenazi Jewish controls (n = 118) and high-risk (n = 142) women. Of the 28 haplotypes noted, four were encountered in frequencies of 5% or more and accounted for 85% of all haplotypes. Subsequently, ATM haplotyping of high-risk, non-Ashkenazi Jews was performed on 66 women with breast cancer and 76 asymptomatic. One SNP (rs228589) was significantly more prevalent among breast cancer cases compared with controls (P = 4 × 10^-9), and one discriminative ATM haplotype was significantly more prevalent among breast cancer cases (33.3%) compared with controls (3.8%), (P ≤ 10^-10). There was no significant difference in the SNP and haplotype distribution between asymptomatic high-risk and symptomatic women as a function of disease status. We conclude that a specific ATM SNP and a specific haplotype are associated with increased breast cancer risk in high-risk non-Ashkenazi Jews.

Despite the health hazards, cigarette smoking is disproportionately frequent among young women. A significant contribution of genetic factors to smoking phenotypes is well established. Efforts to identify susceptibility genes do not generally take into account possible interaction with environment, life experience and psychological characteristics. We recruited 501 female Israeli students aged 20-30 years, obtained comprehensive background data and details of cigarette smoking and administered a battery of psychological instruments. Smoking initiators (n = 242) were divided into subgroups with high (n = 127) and low (n = 115) levels of nicotine dependence based on their scores on the Fagerstrom Tolerance Questionnaire and genotyped with noninitiators (n = 142) for single nucleotide polymorphisms (SNPs) in 11 nicotinic cholinergic receptor genes. We found nominally significant (P -14, Nagelkerke r² = 0.30). For severity of nicotine dependence, two SNPs in CHRNA7 (rs1909884 and rs883473), one SNP in CHRNA5 (rs680244) and the interaction of a SNP in CHRNA7 (rs2337980) with neuroticism, were included in the model (P = 2.24 × 10^-7, Nagelkerke r² = 0.40). These findings indicate that background factors, psychological characteristics and genetic variation in nicotinic cholinergic receptors contribute independently or interactively to smoking initiation and to severity of nicotine dependence in young women.

Gene duplication and alternative splicing are distinct evolutionary mechanisms that provide the raw material for new biological functions. We explored their relationships in human and mouse and found an inverse correlation between the size of a gene's family and its use of alternatively spliced isoforms. A cross-organism analysis suggests that selection for genome-wide genic proliferation might be interchangeably met by either evolutionary mechanism.

We have systematically examined the domain composition across a comprehensive set of tissue-specific, midrange and housekeeping genes as defined by their mode of expression in 52 normal mouse tissues. We show a definite correlation between the number of domains and the degree of tissue specificity. This trend is further supported by a novel analysis involving the time of origin of each domain. Genes containing metazoan-specific domains are more prevalent in signal transduction and cell-communication pathways, and are depleted in primary metabolism. Our analyses suggest that highly modular gene products have been recruited for tissue-specific functions that are required in complex organisms.

While genetic factors clearly play a key role in colorectal cancer (CRC) pathogenesis and in determining its phenotypic features, the precise genes that involved are largely unknown. To gain insight into these genes, consecutive Israeli CRC patients were genotyped using SNPs from within candidate genes: APC, β-Catenin, K-RAS, DCC, P16, PTEN, RB1, P15, APOE, ERCC2, P53, MTHFR and hMSH2. Genotyping of consecutive, unselected colorectal cancer patients was done mostly by utilizing the MassARRAY technology (Sequenom) and to a lesser extent DGGE, ARMS and direct DNA sequencing. Correlation of genotypes with specific phenotypic features was carried out for all patients and separately for the Ashkenazim. Overall, 456 patients were analyzed, the majority (64.25%) being of Ashkenazi origin; mean age at diagnosis was 65.6 ± 14 (range 25-90 years), and the mean follow-up was 4.7 ± 0.28 (range 0-30 years). Statistically significant associations were noted between SNPs in β-catenin and APOE and a positive family history of cancer (β-catenin: p=0.034, APOE: p=0.033); tumor location and a DCC SNP (p=0.038) and the P53 R72P mutation and survival (p=0.0336). In Ashkenazi patients, ERCC2 and MTHFR genes' SNPs were associated with age at diagnosis (ERCC2: p=0.025, MTHFR: p=0.0005); a P53 polymorphism, APOE and Rb SNPs with a family history of cancer (P53 p=0.034; APOE p=0.04, Rb p=0.022); DCC SNP with tumor location (p=0.014); and p15 SNP with tumor grade (p=0.032). This preliminary study shows that genetic factors play a role in determining CRC phenotypic features and that a larger cohort with longer follow-up is clearly needed.

Systems Biology constitutes tools and approaches aimed at deciphering complex biological entities. It is assumed that such complexity arose gradually, beginning from a few relatively simple molecules at life's inception, and culminating with the emergence of composite multicellular organisms billions of years later. The main point of the present paper is that very early in the evolution of life, molecular ensembles with high complexity may have arisen, which are best described and analyzed by the tools of Systems Biology. We show that modeled prebiotic mutually catalytic pathways have network attributes similar to those of present-day living cells. This includes network motifs and robustness attributes. We point out that early networks are weighted (graded), but that using a cutoff formalism one may probe their degree distribution and show that it approximate that of a random network. A question is then posed regarding the potential evolutionary mechanisms that may have led to the emergence of scale-free networks in modem cells.

Autoimmune diseases seem to have strong genetic attributes, and are affected to some extent by shared susceptibility loci. The latter potentially amount to hundreds of candidate genes (CG), creating the need for a prioritization strategy in genetic association studies. To form such a strategy, 26 autoimmune-related CG were genotyped for a total of 72 single nucleotide polymorphisms (SNPs) in three distinct Israeli ethnic populations: Ashkenazi Jews, Sephardic Jews and Arabs. Four quantitative criteria reflecting population stratification were analyzed: allele frequencies, haplotype frequencies, the F_st statistic for homozygotes distribution and linkage disequilibrium extents. According to the consequent interpopulation genomic diversity profiles, the genes were classified into conserved, intermediate and diversified gene groups. Our results demonstrate a correlation between the biological role of autoimmune-related CG and their interpopulation diversity profiles as classified by the different analyses. Annotation analysis suggests that genes more readily influenced by environmental conditions, such as immunological mediators, are 'population specific'. Conversely, genes showing genetic conservation across all populations are characterized by apoptotic and cleaving functions. We suggest a research strategy by which CG association studies should focus first on likely conserved gene categories, to increase the likelihood of attaining significant results and promote the development of gene-based therapies.

We identified 971 olfactory receptor (OR) genes in the dog genome, estimated to constitute similar to80% of the canine OR repertoire. This was achieved by directed genomic DNA cloning of olfactory sequence tags as well as by mining the Celera canine genome sequences. The dog OR subgenome is estimated to have 12% pseudogenes, suggesting a functional repertoire similar to that of mouse and considerably larger than for humans. No novel OR families were discovered, but as many as 34 gene subfamilies were unique to the dog. "Fish-like" Class I ancient ORs constituted 18% of the repertoire, significantly more than in human and mouse. A set of 122 dog-human-mouse ortholog triplets was identified, with a relatively high fraction of Class I ORs. The elucidation of a large portion of the canine olfactory receptor gene superfamily, with some dog-specific attributes, may help us understand the unique chemosensory capacities of this species. (C) 2003 Elsevier Inc. All rights reserved.

Olfactory receptor (OR) genes constitute the molecular basis for the sense of smell and are encoded by the largest gene family in mammalian genomes. Previous studies suggested that the proportion of pseudogenes in the OR gene family is significantly larger in humans than in other apes and significantly larger in apes than in the mouse. To investigate the process of degeneration of the olfactory repertoire in primates, we estimated the proportion of OR pseudogenes in 19 primate species by surveying randomly chosen subsets of 100 OR genes from each species. We find that apes, Old World monkeys and one New World monkey, the howler monkey, have a significantly higher proportion of OR pseudogenes than do other New World monkeys or the lemur (a prosimian). Strikingly, the howler monkey is also the only New World monkey to possess full trichromatic vision, along with Old World monkeys and apes. Our findings suggest that the deterioration of the olfactory repertoire occurred concomitant with the acquisition of full trichromatic color vision in primates.

Olfactory receptors (ORs) constitute the largest gene-family in the vertebrate genome. We have attempted to provide a comprehensive view of the OR universe through diverse tools of bioinformatics and computational biology. Among others, we have constructed the Human Olfactory Receptor Data Exploratorium (HORDE, http:// bioportal.weizmann.ac.il/HORDE/) as a free online resource, which integrates information on ORs from different species. We studied the genomic organization of 853 human ORs and divided the repertoire into 135 clusters, accessible through our new cluster viewer feature. An analysis of intact and pseudogenized ORs in different clusters, as well as of OR expression patterns, is provided, relevant to OR transcription control. Coding single nucleotide polymorphisms were integrated; these are to be used for genotype-phenotype correlation studies. HORDE allows a unique opportunity for discerning protein structural and functional information of the individual OR proteins. By applying novel data analysis strategies to the >3000 OR genes of mouse, dog and human within HORDE, we have generated a set of refined rhodopsin-based homology models for ORs. For model improvement, we employed a novel analysis of specific positions along the seven transmembrane helices at which prolines generate helix-breaking kinks. The model shows family-specific structural features, including idiosyncratic kink patterns, which lead to significant differences in the inferred odorant binding site structure. Such analyses form a basis for a comprehensive sequence-based classification of OR proteins in terms of potential odorant binding specificities.

Molecular Chirality is of central interest in biological studies because enantiomeric compounds, while indistinguishable by most inanimate systems, show profoundly different properties in biochemical environments. Enantioselective separation methods, based on the differential recognition of two optical isomers by a chiral selector, have been amply documented. Also, great effort has been directed towards a theoretical understanding of the fundamental mechanisms underlying the chiral recognition process. Here we report a comprehensive data examination of enantioseparation measurements for over 72,000 chiral selector-selectand pairs from the chiral selection compendium CHIRBASE. The distribution of α = k_D/k_L values was found to follow a power law, equivalent to an exponential decay for chiral differential free energies. This observation is experimentally relevant in terms of the number of different individual or combinatorial selectors that need to be screened in order to observe α values higher than a preset minimum. A string model for enantiorecognition (SMED) formalism is proposed to account for this observation on the basis of an extended Ogston three-point interaction model. Partially overlapping molecular interaction domains are analyzed in terms of a string complementarity model for ligand-receptor complementarity. The results suggest that chiral selection statistics may be interpreted in terms of more general concepts related to biomolecular recognition.

We propose a new feature extraction method for use with chemical sensors. It is based on fitting a parametric analytic model of the sensor's response over time to the measured signal, and taking the set of best-fitting parameters as the features. The process of finding the features is fast and robust, and the resulting set of features is shown to significantly enhance the performance of subsequent classification algorithms. Moreover, the model that we have developed fits equally well to sensors of different technologies and embeddings, suggesting its applicability to a diverse repertoire of sensors and analytic devices.

Groups of related genes abound in large eukaryotic genomes. In such 'subgenomes', homology modeling carried out for a few genes will probably have relevance to the entire group. Subgenomes also afford unique ways of determining protein structural information. In addition to analyses based on the quantification of residue variability in paralogs, two-way comparisons, both within and among species, help to disclose functional amino acids. Comparative studies of gene families throughout the mammalian genome will also help elucidate the functional significance of single nucleotide polymorphisms in coding regions.

We propose a setup for an odor communication system. Its different parts are described, and ways to realize them are outlined. Our scheme enables an output device - the whiffer - to release an imitation of an odorant read in by an input device - the sniffer - upon command. The heart of the system is the novel algorithmic scheme that makes the scheme feasible. We are currently at work researching and developing some of the components that constitute the algorithm, and we hope that the description of the overall scheme in this paper will help to get other groups to join in this effort.

Olfactory receptor (OR) genes constitute the basis for the sense of smell and are encoded by the largest mammalian gene superfamily of >1,000 genes. In humans, >60% of these are pseudogenes. In contrast, the mouse OR repertoire, although of roughly equal size, contains only ≈20% pseudogenes. We asked whether the high fraction of nonfunctional OR genes is specific to humans or is a common feature of all primates. To this end, we have compared the sequences of 50 human OR coding regions, regardless of their functional annotations, to those of their putative orthologs in chimpanzees, gorillas, orangutans, and rhesus macaques. We found that humans have accumulated mutations that disrupt OR coding regions roughly 4-fold faster than any other species sampled. As a consequence, the fraction of OR pseudogenes in humans is almost twice as high as in the non-human primates, suggesting a human-specific process of OR gene disruption, likely due to a reduced chemosensory dependence relative to apes.

[No abstract available]

A novel data set, GeneNote (Gene Normal Tissue Expression), was produced to portray complete gene expression profiles in healthy human tissues using the Affymetrix GeneChip HG-U95 set, which includes 62 839 probe-sets. The hybridization intensities of two replicates were processed and analyzed to yield the complete transcriptome for twelve human tissues. Abundant novel information on tissue specificity provides a baseline for past and future expression studies related to diseases. The data is posted in GeneNote (http://genecards.weizmann.ac.il/genenote/), a widely used compendium of human genes (http://bioinfo.weizmann.ac.il/genecard).

The goal of pharmacogenetics is to identify "genetic fingerprints" that may predict a patient's response to pharmaceutical treatment. The use of pharmacogenetics replaces the trial-and-error strategy, which governs much of our clinical decision-making regarding treatment allocation in current medical practice, with individually tailored therapy. We review a pharmacogenetic research model, which implements high-throughput single nucleotide polymorphism technology to establish the correlation between drug-responsiveness and genetic polymorphisms of Copaxone®-treated multiple sclerosis patients. Implementation of similar pharmacogenetic approaches may promote the development of personalized medicine in multiple sclerosis as well as in other diseases.

We developed a novel efficient scheme, DEFOG (for "deciphering families of genes"), for determining sequences of numerous genes from a family of interest. The scheme provides a powerful means to obtain a gene family composition in species for which high-through-put genomic sequencing data are not available. DEFOG uses two key procedures. The first is a novel algorithm for designing highly degenerate primers based on a set of known genes from the family of interest. These primers are used in PCR reactions to amplify the members of the gene family. The second combines oligofingerprinting of the cloned PCR products with clustering of the clones based on their fingerprints. By selecting members from each cluster, a low-redundancy clone subset is chosen for sequencing. We applied the scheme to the human olfactory receptor (OR) genes. OR genes constitute the largest gene superfamily in the human genome, as well as in the genomes of other vertebrate species. DEFOG almost tripled the size of the initial repertoire of human ORs in a single experiment, and only 7% of the PCR clones had to be sequenced. Extremely high degeneracies, reaching over a billion combinations of distinct PCR primer pairs, proved to be very effective and yielded only 0.4% nonspecific products.

We investigated the population differences in patterns of single nucleotide polymorphisms (SNPs) for a 400 kb olfactory receptor (OR) gene cluster on human chromosome 17p13.3. Samples were drawn from 35 individuals, of four different ethnogeographical origins: Pygmies, Bedouins, Yemenite Jews and Ashkenazi Jews. Of the 74 SNPs identified, two segregated between pseudogenized and intact ORs, while a third involved a change in a highly conserved motif proposed to mediate ligand-induced signal transduction. Linkage disequilibrium (LD) was computed based on phase inference across the cluster using Clark's haplotype subtraction algorithm. We also calculated LD directly from the genotypes using the expectation-maximization (EM) algorithm. Both methods yielded very similar results. Our analyses revealed substantial differences in nucleotide diversity, haplotype distribution and LD patterns among the different human populations. In particular, the two Jewish populations had low haplotype diversity and negligible decay of LD across the entire genomic region. Intriguingly, the three functional SNPs segregated at different frequencies in the different ethnogeographical groups, with the Pygmies having higher frequencies of the intact OR genes. Our data suggests that OR genes may have evolved to create different functional repertoires in distinct human populations.

Keywords: Cardiac & Cardiovascular Systems; Cell Biology

The RUNX3 gene belongs to the runt domain family of transcription factors that act as master regulators of gene expression in major developmental pathways. In mammals the family includes three genes, RUNX1, RUNX2 and RUNX3. Here, we describe a comparative analysis of the human chromosome 1p36.1 encoded RUNX3 and mouse chromosome 4 encoded Runx3 genomic regions. The analysis revealed high similarities between the two genes in the overall size and organization and showed that RUNX3/Runx3 is the smallest in the family, but nevertheless exhibits all the structural elements characterizing the RUNX family. It also revealed that RUNX3/Runx3 bears a high content of the ancient mammalian repeat MIR. Together, these data delineate RUNX3/Runx3 as the evolutionary founder of the mammalian RUNX family. Detailed sequence analysis placed the two genes at a GC-rich H3 isochore with a sharp transition of GC content between the gene sequence and the downstream intergenic region. Two large conserved CpG islands were found within both genes, one around exon 2 and the other at the beginning of exon 6. RUNX1, RUNX2 and RUNX3 gene products bind to the same DNA motif, hence their temporal and spatial expression during development should be tightly regulated. Structure/function analysis showed that two promoter regions, designated P1 and P2, regulate RUNX3 expression in a cell type-specific manner. Transfection experiments demonstrated that both promoters were highly active in the GM1500 B-cell line, which endogenously expresses RUNX3, but were inactive in the K562 myeloid cell line, which does not express RUNX3.

Olfactory receptors likely constitute the largest gene superfamily in the vertebrate genome. Here we present the nearly complete human olfactory subgenome elucidated by mining the genome draft with gene discovery algorithms. Over 900 olfactory receptor genes and pseudogenes (ORs) were identified, two-thirds of which were not annotated previously. The number of extrapolated ORs is in good agreement with previous theoretical predictions. The sequence of at least 63% of the ORs is disrupted by what appears to be a random process of pseudogene formation. ORs constitute 17 gene families, 4 of which contain more than 100 members each. "Fish-like" Class I ORs, previously considered a relic in higher tetrapods, constitute as much as 10% of the human repertoire, all in one large cluster on chromosome II. Their lower pseudogene fraction suggests a functional significance. ORs are disposed on all human chromosomes except 20 and Y, and nearly 80% are found in clusters of 6-138 genes. A novel comparative cluster analysis was used to trace the evolutionary path that may have led to OR proliferation and diversification throughout the genome. The results of this analysis suggest the following genome expansion history: first, the generation of a "tetrapod-specific" Class II OR cluster on chromosome II by local duplication, then a single-step duplication of this cluster to chromosome I, and finally an avalanche of duplication events out of chromosome 1 to most other chromosomes. The results of the data mining and characterization of ORs can be accessed at the Human Olfactory Receptor Data Exploratorium Web site (http://bioinfo.weizmann.ac.il/HORDE).

The olfactory receptor (OR) subgenome harbors the largest known gene family in mammals, disposed in clusters on numerous chromosomes. One of the best characterized OR clusters, located at human chromosome 17p13.3, has previously been studied by us in human and in other primates, revealing a conserved set of 17 OR genes. Here, we report the identification of a syntenic OR cluster in the mouse and the partial DNA sequence of many of its OR genes. A probe for the mouse M5 gene, orthologous to one of the OR genes in the human cluster (OR17-25), was used to isolate six PAC clones, all mapping by in situ hybridization to mouse chromosome 11B3-11B5, a region of shared synteny with human chromosome 17p13.3. Thirteen mouse OR sequences amplified and sequenced from these PACs allowed us to construct a putative physical map of the OR gene cluster at the mouse Olfr1 locus. Several points of evidence, including a strong similarity in subfamily composition and at least four cases of gene orthology, suggest that the mouse Olfr1 and the human 17p13.3 clusters are orthologous. A detailed comparison of the OR sequences within the two clusters helps trace their independent evolutionary history in the two species. Two types of evolutionary scenarios are discerned: cases of "true orthologous genes" in which high sequence similarity suggests a shared conserved function, as opposed to instances in which orthologous genes may have undergone independent diversification in the realm of "free reign" repertoire expansion.

The gene MCOLN1 is mutated in Mucolipidosis type IV (MLIV), a neurodegenerative, recessive, lysosomal storage disorder. The disease is found in relatively high frequency among Ashkenazi Jews due to two founder mutations that comprise 95% of the MLIV alleles in this population [Bargal et al., 2000]. In this report we complete the mutation analysis of Jewish and non-Jewish MLIV patients whose DNA were available to us. Four novel mutations were identified in the MCOLN1 gene of severely affected patients: two missense, T232P and F465L; a nonsense, R322X; and an 11-bp insertion in exon 12. The nonsense mutation (R322X) was identified in two unrelated patients with different haplotypes in the MCOLN1 chromosomal region, indicating a mutation hotspot in this CpG site. An in-frame deletion (F408del) was identified in a patient with unusual mild psychomotor retardation. The frequency of MLIV in the general Jewish Ashkenazi population was estimated in a sample of 2,000 anonymous, unrelated individuals assayed for the two founder mutations. This analysis indicated a heterozygotes frequency of about 1/100. A preferred nucleotide numbering system for MCOLN1 mutations is presented and the issue of a screening program for the detection of high-risk families in the Jewish Ashkenazi population is discussed.

The continuity of abiotically formed bilayer membranes with similar structures in contemporary cellular life, and the requirement for microenvironments in which large and small molecules could be compartmentalized, support the idea that amphiphilic boundary structures contributed to the emergence of life. As an extension of this notion, we propose here a 'Lipid World' scenario as an early evolutionary step in the emergence of cellular life on Earth. This concept combines the potential chemical activities of lipids and other amphiphiles, with their capacity to undergo spontaneous self-organization into supramolecular structures such as micelles and bilayers. In particular, the documented chemical rate enhancements within lipid assemblies suggest that energy-dependent synthetic reactions could lead to the growth and increased abundance of certain amphiphilic assemblies. We further propose that selective processes might act on such assemblies, as suggested by our computer simulations of mutual catalysis among amphiphiles. As demonstrated also by other researchers, such mutual catalysis within random molecular assemblies could have led to a primordial homeostatic system displaying rudimentary life-like properties. Taken together, these concepts provide a theoretical framework, and suggest experimental tests for a Lipid World model for the origin of life.

Catecholamine-induced polymorphic ventricular tachycardia (PVT) is characterized by episodes of syncope, seizures, or sudden death, in response to physical activity or emotional stress. Two modes of inheritance have been described: autosomal dominant and autosomal recessive. Mutations in the ryanodine receptor 2 gene (RYR2), which encodes a cardiac sarcoplasmic reticulum (SR) Ca²⁺-release channel, were recently shown to cause the autosomal dominant form of the disease. In the present report, we describe a missense mutation in a highly conserved region of the calsequestrin 2 gene (CASQ2) as the potential cause of the autosomal recessive form. The CASQ2 protein serves as the major Ca²⁺ reservoir within the SR of cardiac myocytes and is part of a protein complex that contains the ryanodine receptor. The mutation, which is in full segregation in seven Bedouin families affected by the disorder, converts a negatively charged aspartic acid into a positively charged histidine, in a highly negatively charged domain, and is likely to exert its deleterious effect by disrupting Ca²⁺ binding.

The genomic and cDNA structures were studied for eight human olfactory receptor (OR) genes within the chromosome 17p13.3 cluster. A common gene structure was revealed, which included an ~1-kb intronless coding region terminated by a signal for polyadenylation add a variable number of upstream noncoding exons. The latter were found to be alternatively spliced, giving rise to different isoforms of OR mRNA. While the initial exons mostly agreed with previous computer predictions and were conserved within OR subfamilies, other upstream exons were novel and idiosyncratic. In some cases, repetitive sequences were involved in the generation of splice sites and putative transcription control elements. Such gene structure is consistent with early repertoire enhancement by retrogene generation, which was likely followed by extensive genomic duplication. Each OR gene had a unique signature of transcription factor elements, consistent with a combinatorial expression control mechanism. (C) 2000 Academic Press.

Mucolipidosis type IV (MLIV) is an autosomal recessive, neurodegenerative, lysosomal storage disorder characterized by psychomotor retardation and ophthalmological abnormalities including corneal opacities, retinal degeneration and strabismus. Most patients reach a maximal developmental level of 12-15 months. The disease was classified as a mucolipidosis following observations by electron microscopy indicating the lysosomal storage of lipids together with water-soluble, granulated substances. Over 80% of the MLIV patients diagnosed are Ashkenazi Jews, including severely affected and mildly affected patients. The gene causing MLIV was previously mapped to human chromosome 19p13.2-13.3 in a region of approximately 1 cM (ref. 7). Haplotype analysis in the MLIV gene region of over 70 MLIV Ashkenazi chromosomes indicated the existence of two founder chromosomes among 95% of the Ashkenazi MLIV families: a major haplotype in 72% and a minor haplotype in 23% of the MLIV chromosomes (ref. 7, and G.B., unpublished data). The remaining 5% are distinct haplotypes found only in single patients. The basic metabolic defect causing the lysosomal storage in MLIV has not yet been identified. Thus, positional cloning was an alternative to identify the MLIV gene. We report here the identification of a new gene in this human chromosomal region in which MLIV-specific mutations were identified.

The primordial emergence of biopolymers, agents of the genetic machinery in modern cells, is not less enigmatic than the emergence of the genetic code itself. Here we discuss how potential early replicating protocellular systems based on a rudimentary form of inheritance, a "compositional genome", could evolve towards the emergence of "alphabetic" polymers, predating the genetic code. A computer simulated evolutionary process based on our previously proposed kinetic model may help understand the appearance of chemical combinatorics through early natural selection.

The olfactory receptor (OR) gene cluster on human chromosome 17p13.3 was subjected to mixed shotgun automated DNA sequencing. The resulting 412 kb of genomic sequence include 17 OR coding regions, 6 of which are pseudogenes. Six of the coding regions were discovered only upon genomic sequencing, while the others were previously reported as partial sequences. A comparison of DNA sequences in the vicinity of the OR coding regions revealed a common gene structure with an intronless coding region and at least one upstream noncoding exon. Potential gene control regions including specific pyrimidine:purine tracts and Olf-1 sites have been identified. One of the pseudogenes apparently has evolved into a CpG island. Four extensive CpG islands can be discerned within the cluster, not coupled to specific OR genes. The cluster is flanked at its telomeric end by an unidentified open reading frame (C17orf2) with no significant similarity to any known protein. A high proportion of the cluster sequence (about 60%) belongs to various families of interspersed repetitive elements, with a clear predominance of LINE repeats. The OR genes in the cluster belong to two families and seven subfamilies, which show a relatively high degree of intermixing along the cluster, in seemingly random orientations. This genomic organization may be best accounted for by a complex series of evolutionary events. (C) 2000 Academic Press.

The olfactory receptor (OR) subgenome harbors the largest known gene family in mammals, disposed in clusters on numerous chromosomes. We have carried out a comparative evolutionary analysis of the best characterized genomic OR gene cluster, on human chromosome 17p13. Fifteen orthologs from chimpanzee (localized to chromosome 19p15), as well as key OR counterparts from other primates, have been identified and sequenced. Comparison among orthologs and paralogs revealed a multiplicity of gene conversion events, which occurred exclusively within OR subfamilies. These appear to lead to segment shuffling in the odorant binding site, an evolutionary process reminiscent of somatic combinatorial diversification in the immune system. We also demonstrate that the functional mammalian OR repertoire has undergone a rapid decline in the past 10 million years: while for the common ancestor of all great apes an intact OR cluster is inferred, in present-day humans and great apes the cluster includes nearly 40% pseudogenes.

In order to elicit an olfactory response, a substance has to partition into the gas phase and diffuse into the nose. Such odorant molecules, usually low molecular-mass hydrophobic compounds, encounter the ciliated endings of sensory neuronal dendrites, which protrude into a mucus layer at the surface of the olfactory epithelium in the nasal cavity. Embedded in the membranes of such cilia are olfactory receptor (OR) proteins, which recognize odorants and elicit a transduction cascade that underlies the nerve cell response. The sensory axons project to the olfactory bulb in the brain, where they converge into synaptic structures called glomeruli. The specific convergence patterns of olfactory axons, which depend on OR expression, provide a model system for neuronal network development. Here, initial processing of odour information occurs, which is followed by additional analysis in higher olfactory brain centres.

A thorough outlook on the origin of life needs to delineate a chemically rigorous, self-consistent path from highly heterogeneous, random ensembles of relatively simple organic molecules, to an entity that has rudimentary life-like characteristics. Such entity should be endowed with a capacity to express variation, undergo mutation-like changes and manifest a simple evolutionary process. For simulating such system we developed the Graded Autocatalysis Replication Domain (GARD) model for explicit kinetic analysis of mutual catalysis in sets of random oligomers derived from energized precursor monomers. The kinetic properties of the GARD model are based on vesicle enclosure and expansion. With the additional assumption of spontaneous vesicle splitting, a GARD evolution scenario is envisaged as a consequence of pure chemical kinetics. Here we show how the GARD model can serve as a platform for investigating the dynamics of self-organization mechanisms in molecular evolutionary processes. (C) 1998 Elsevier Science B.V. All rights reserved.

The human olfactory subgenome represents several hundred olfactory receptor (OR) genes in a dozen or more clusters on several chromosomes. One OR gene cluster on human chromosome 17 has been characterized by us in detail. Based on a large-scale DNA sequence analysis, we have identified events of gene duplication and fusion as well as the generation of pseudogenes. The latter instances of 'gene death' could underlie the widespread phenomenon of human specific anosmias. Sixteen OR coding regions were found on this cluster, and six of them are pseudogenes. One of these pseudogenes, OR17-23, was found to be an intact open reading frame in an old world monkey. This may be a reflection of an OR repertoire diminution in man. A homology model of the OR protein was constructed by utilizing the rich information available on ~ 200 OR sequences. The putative odorant complementarity determining regions (CDR) was found to consist of 20 hypervariable residues facing an interior caving defined by transmembrane helices 3, 4 and 5. Such a model could be useful in analyzing additional OR gene sequences in the human genome in terms of odorant binding.

The superfamily of olfactory receptor genes, whose products are thought to be activated by odorant ligands, is critical for odor recognition. Two olfactory receptors, olp4 from rat and OR17-4 From human, were overexpressed in Sf9 insect cells. The presence of the proteins in cell membranes was monitored by immunoblotting with peptide-specific polyclonal antibodies directed against the C-terminal sequences of these receptors and with a mAb against an N-terminal octapeptide epitope tag. A DNA sequence that codes for a His₆ tag, which binds tightly to a Ni²⁺-chelate-affinity column, was incorporated into the N-termini of both genes. The expressed olfactory receptors were found mainly in the cell-membrane fraction. The proteins were difficult to solubilize by many detergents and only lysophosphatidylcholine was found to be both suitable for efficient solubilization of the overexpressed olfactory receptors anti compatible with the purification system used. After solubilization, the olfactory receptors were purified to near homogeneity by affinity chromatography on nickel nitrilotriacetic acid resin and by cation-exchange chromatography. Electrophoresis of the purified proteins and visualization with Coomassie Blue staining or by immunoblotting with specific antibodies, revealed bands of 32, 69 and 94 kDa, which were identified as the monomeric, dimeric and trimeric forms of the receptor proteins. The oligomeric forms were resistant to reduction and alkylation, and are therefore thought to be held together by non-covalent hydrophobic interactions that are resistant to SDS. This finding is similar to previous observations for other guanine-nucleotide-binding-regulatory-protein-coupled receptors. Reconstitution in phospholipid vesicles showed that the purified olfactory receptors insert specifically into the lipid bilayer. This provides a means to study functional reconstitution with putative transduction components such as olfactory guanine-nucleotide-binding-regulatory protein.

A gene superfamily of olfactory receptors (ORs) has recently been identified in a number of species. These receptors share a seven transmembrane domain structure with many neurotransmitter and hormone receptors, and are likely to underlie the recognition and G-protein-mediated transduction of odorant signals. Previously, OR genes cloned in different species were from random locations in the respective genomes. We report here the cloning of 16 human OR genes, air from chromosome 17(17p13.3). The intronless coding regions are physically mapped (on 35 cosmids) in one 0.35Mb long contiguous cluster, with an average intergenic separation of 15kb. The human OR genes in the cluster belong to four different gene subfamilies, displaying as much sequence variability as any randomly selected group of ORs. This suggests that the cluster identified may be one of several copies of an ancestral OR gene repertoire whose existence may predate the divergence of mammals. The latter may have duplicated in some species to form the present mammalian OR gene repertoire, with several hundred genes. The human chromosome 17 OR gene cluster may thus be a good model for understanding human olfaction, as well as the ontogeny and phylogeny of the OR gene superfamily.

The molecular components of olfactory reception and regulation are expressed in a tissue-specific manner. The functional attributes mediated by some of these proteins have been previously shown to display a well-defined developmental emergence during the last week of rat gestation. To gain a better understanding of the relations between chemosensory function and neuronal development, we studied the ontogeny of 7 olfactory-specific genes by quantitative PCR. Relative levels of expression during rat development were determined for each gene, starting at embryonic day 15 (E15) and ending at postnatal day 35 (P35). In addition, the level of expression of the different genes was quantified in juvenile rats. The onset of expression for olfactory receptors and the olfactory cation channel at embryonic day 19 (E19) coincides with the functional maturation of the sensory neurons. Olfactory G-protein and adenylyl cyclase are expressed earlier (∼ E16) while olfactory biotransformation enzymes appear later (E20-E21), just before birth. The sequence of developmental expression of olfactory receptor genes has possible implications to the establishment of neuronal connectivity in this sensory pathway.

A generalized phenomenological model is presented for stereospecific recognition between biological receptors and their ligands. We ask what is the distribution of binding constants ψ(K) between an arbitrary ligand and members of a large receptor repertoire, such as immunoglobulins or olfactory receptors. For binding surfaces with B potential subsite and S different types of subsite configurations, the number of successful elementary interactions obeys a binomial distribution. The discrete probability function ψ(K) is then derived with assumptions on α, the free energy contribution per elementary interaction. The functional form of ψ(K) may be universal, although the parameter values could vary for different ligand types. An estimate of the parameter values of ψ(K) for iodovanillin, an analog of odorants and immunological haptens, is obtained by equilibrium dialysis experiments with nonimmune antibodies. Based on a simple relationship, predicted by the model, between the size of a receptor repertoire and its average maximal affinity toward an arbitrary ligand, the size of the olfactory receptor repertoire (N_olf) is calculated as 300-1000, in very good agreement with recent molecular biological studies. A very similar estimate, N_olf = 500, is independently derived by relating a theoretical distribution of maxima for ψ(K) with published human olfactory threshold variations. The present model also has implications to the question of olfactory coding and to the analysis of specific anosmias, genetic deficits in perceiving particular odorants. More generally, the proposed model provides a better understanding of ligand specificity in biological receptors and could help in understanding their evolution.

Olfactory thresholds for four odorants were determined in groups of monozygotic and dizygotic human twins. Odorants were presented in an ascending dilution series in odorless solvent, using a three-way forced choice method. For two of the tested odorants, 5α-androst-16-en-3-one and isoamyl acetate, the thresholds showed a strong genetic component. This was demonstrated by respective values of 0.78 and 0.73 for the intraclass correlation difference, and of z = 3.69 and z = 2.71 in a within-pair difference analysis. The results for isoamyl acetate are novel, and suggest that genetic polymorphism in the affinity of odorant receptor proteins contributes to the (nearly normal) threshold distribution for this odorant.

The complete amino-acid sequence of the bovine olfactory epithelium adenosine 3',5'cyclic monophosphate (cAMP)-gated channel has been determined by cloning and sequencing its cDNA. It exhibits a high degree of sequence homology with the cGMP-gated channel of rod photoreceptors, suggesting that cyclic nucleotide-gated channels fall into a new family of genetically related proteins.

We have characterized the adenylyl cyclase activity in a newly developed preparation of isolated olfactory cilia from the bovine chemosensory neuroepithelium. Like its counterparts from frog and rat, the ciliary enzyme was stimulated by guanine nucleotides, by forskolin, and by a variety of odorants in the presence of GTP. The main difference between the bovine olfactory cilia preparation and the frog and rat olfactory cilia preparation is that odorant stimulation of the bovine olfactory adenylyl cyclase is strongly inhibited by submillimolar concentrations of dithiothreitol. This inhibition is a consequence of a concomitant increase in the GTP-stimulated level and the decrease of the odorant stimulation of the enzyme. Nasal respiratory cilia have a much lower level of adenylyl cyclase activity and show no odorant stimulation. Owing to the large quantities of material available, the bovine olfactory cilia preparation is advantageous for studies of the proteins involved in chemosensory transduction.

We isolated cDNA clones for cytochrome P-450 genes expressed in the olfactory neuroepithelium by screening a corresponding rat cDNA library. Sequence analysis and RNA blot hybridization revealed a new cytochrome P-450, designated cytochrome P-450olf1, which is the first reported cytochrome P-450 mRNA uniquely expressed in the chemosensory organ. Cytochrome P-450olf1 shows intermediate level of sequence similarity (38-53% identity) to several liver cytochrome P-450 enzymes, suggesting that it belongs to the cytochrome P-450II family, but defines a new subfamily (cytochrome P-450IIG) within it. Cytochrome P-450II enzymes are known to process diverse organic compounds, including odorants. This, together with the specificity of cytochrome P-450olf1 to the sensory neuroepithelium, may indicate a role for this protein in olfactory reception.

In order to assess the dependence of perceived odor quality on odorant concentration, we studied 21 subjects. For eight subjects all possible pairs from a pool of six odorants at three decimal dilutions were presented, and subjects were requested to state whether members of the pair were qualitatively 'similar' or 'different' It was found that while pairs with the same odorant at identical concentrations were judged 'similar' in >90% of the cases by all subjects, scores went down to ≤10% 'similar' judgements in some cases when the same odorant was presented at a 100-fold concentration difference. Large time-invariable differences were found among subjects and among odorants. For the additional 13 subjects, all possible pairs from a pool of four odorants at three decimal dilutions were presented. Subjects were instructed to state whether members of the pair were qualitatively 'same' or 'different', and were also requested to rank the degree of difference on a visual analogue scale. Results for this group were, in general, similar to the results of the former group of subjects and good agreement between the two tasks was found. The results suggest that variations in olfactory stimulus magnitude may be perceived as quality differences, as previously shown for vision and audition.

The short-term and long-term effects of total inferior turbinectomy on smell acuity was assessed in two groups of patients. Olfactory thresholds were determined by a three-way forced-choice method, using four odorants. Resection of obstructive inferior turbinates resulted in a decrease in olfactory thresholds in 22 of 24 tested patients. No deleterious effect on smell acuity was observed in 16 patients tested 2½ years or more after surgery. Subjective assessment of olfactory acuity is unreliable. It is our intention to focus attention on an aspect of intranasal surgery not frequently reported. (Arch Otolaryngol Head Neck Surg 1986;112:195-197)

The affinity values of antibodies to the random synthetic antigen poly-(Tyr,Glu)-poly(dl Ala)-poly(Lys) [known as (T,G)-A-L] and to the ordered (Tyr-Tyr-Glu-Glu)-poly(dlAla)-poly(Lys) [known as (T-T-G-G)-A-L] were measured. Determinations of the association constants were performed by antigen binding capacity assay, ABC, using the whole antigen, and by equilibrium dialysis, using a radioactive conjugate of the ordered peptide T-T-G-G which was found to represent the major determinant of the random (T,G)-A-L. The affinity values of antibodies elicited by high responder mice to (T,G)-A-L and to (T-T-G-G)-A-L were found to be similar. However, a difference of two orders of magnitude was found between the values obtained by the two methods. This difference is partially explained by probability analysis. Upon immunizing low responder (H-2^k) mice with complexes of the antigens with methylated bovine serum albumin (MBSA), an increase in their antibody response was observed. The affinity values of these antibodies were found to be similar to those of high responder antibodies after immunization with either the antigens complexed to MBSA or with the antigens alone. On the basis of these data we conclude that low responder (H-2^k) B cells have the potential to produce antibodies of the same specificity and quality as the high responders to the random (T,G)-A-L and to the ordered (T-T-G-G)-A-L, and that the genetic defect in these mice should be located at the level of the B-cell acceptor for the T-cell signal.