Genome-wide multilocus analysis for immune-mediated complex diseases

In postgenomic era, searching and identification of disease genes associated with complex diseases are still one of the great challenge for dissecting human complex diseases. To improve the disease gene localization for complex diseases, a group of closely immune-mediated disease loci were overlapped on each chromosome based on previously reported genome-wide scanning data. Interestingly, five overlapping chromosomal regions (1q21, 2q33, 5q31.1-q33.1, 6p21, and 11q13) were identified by co-localizing disease loci for the following diseases: diabetes, asthma, atopic dermatitis, osteoporosis, and inflammatory bowel disease. The development of specific disease was associated with different combinations of disease loci among five overlapped chromosomal regions. Therefore, the analysis of multiple genetic loci should be considered to determine the effects of multiple genes responsible for complex diseases resulting from the influence of multiple genes.     

Recent advances in molecular genetics of glaucoma

Glaucoma represents a heterogeneous group of optic neuropathies, with different genetic bases. It can affect all ages generally with a rise in intra-ocular pressure. Three major types of glaucoma have been reported: primary open angle glaucoma (POAG), primary acute closed angle glaucoma (PACG) and primary congenital glaucoma (PCG), as well as a few others associated with developmental abnormalities. In recent years impressive progress has been made in the molecular genetic studies of POAG and PCG. These include the discovery of three genes – Myocilin, Optineurin and CYP1B1 – defects in which results in Mendelian transmission of glaucoma. Identification of single nucleotide polymorphisms in multiple other genes that are associated with glaucoma and alteration of drug sensitivity are enriching our knowledge regarding the complex nature of the disease. This review attempts to present the recent progress made in the molecular genetics of glaucoma.     

A search for susceptibility genes in multiple sclerosis

Multiple sclerosis is a chronic inflammatory, putatively autoimmune disease characterized by multifocal demyelination in the central nervous system. Two main strategies are used to identify genes influencing the susceptibility to multiple sclerosis: (i) elucidation of the role of a candidate gene chosen on the basis of the possible function of the encoded protein in etiology and/or pathogenesis of the disease, and (ii) complete genomic screen using a panel of anonymous genetic markers for identification of the chromosome regions involved in the disease development. The complete genomic search revealed multiple loci for multiple sclerosis on thirteen chromosomes, and analysis of the candidate genes added three more chromosomes to this list. The combined data prove the polygenic nature of this complex disease. Detection of individual genes responsible for susceptibility to multiple sclerosis is complicated by the genetic heterogeneity of analyzed populations and families, which is determined both by the ethnic heterogeneity and the peculiarity of clinical forms of the disease. However, it seems highly probable that HLA and non-HLA genes of the major histocompatibility complex, as well as some unidentified genes on chromosomes 5p and 17q, are involved in the disease development. In addition to HLA, some authors have also shown that a role in the disease development is played by the genes encoding other components of the trimolecular complex involved in antigen presentation: those of the T-cell receptor and the best studied autoantigen, the myelin basic protein. The most promising for further studies of the genetic susceptibility to multiple sclerosis are approaches that combine the candidate-gene strategy with the complete genomic search as well as distinguish the genetically differentiated forms of the disease.     

The Fanconi anemia gene network is conserved from zebrafish to human

Fanconi anemia (FA) is a complex disease involving nine identified and two unidentified loci that define a network essential for maintaining genomic stability. To test the hypothesis that the FA network is conserved in vertebrate genomes, we cloned and sequenced zebrafish (Danio rerio) cDNAs and/or genomic BAC clones orthologous to all nine cloned FA genes (FANCA, FANCB, FANCC, FANCD1, FANCD2, FANCE, FANCF, FANCG, and FANCL), and identified orthologs in the genome database for the pufferfish Tetraodon nigroviridis. Genomic organization of exons and introns was nearly identical between zebrafish and human for all genes examined. Hydrophobicity plots revealed conservation of FA protein structure. Evolutionarily conserved regions identified functionally important domains, since many amino acid residues mutated in human disease alleles or shown to be critical in targeted mutagenesis studies are identical in zebrafish and human. Comparative genomic analysis demonstrated conserved syntenies for all FA genes. We conclude that the FA gene network has remained intact since the last common ancestor of zebrafish and human lineages. The application of powerful genetic, cellular, and embryological methodologies make zebrafish a useful model for discovering FA gene functions, identifying new genes in the network, and identifying therapeutic compounds.     

Learning from Small Fry: The Zebrafish as a Genetic Model Organism for Aquaculture Fish Species

In recent years, the zebrafish has become one of the most prominent vertebrate model organisms used to study the genetics underlying development, normal body function, and disease. The growing interest in zebrafish research was paralleled by an increase in tools and methods available to study zebrafish. While zebrafish research initially centered on mutagenesis screens (forward genetics), recent years saw the establishment of reverse genetic methods (morpholino knock-down, TILLING). In addition, increasingly sophisticated protocols for generating transgenic zebrafish have been developed and microarrays are now available to characterize gene expression on a near genome-wide scale. The identification of loci underlying specific traits is aided by genetic, physical, and radiation hybrid maps of the zebrafish genome and the zebrafish genome project. As genomic resources for aquacultural species are increasingly being generated, a meaningful interaction between zebrafish and aquacultural research now appears to be possible and beneficial for both sides. In particular, research on nutrition and growth, stress, and disease resistance in the zebrafish can be expected to produce results applicable to aquacultural fish, for example, by improving husbandry and formulated feeds. Forward and reverse genetics approaches in the zebrafish, together with the known conservation of synteny between the species, offer the potential to identify and verify candidate genes for quantitative trait loci (QTLs) to be used in marker-assisted breeding. Moreover, some technologies from the zebrafish field such as TILLING may be directly transferable to aquacultural research and production.     

Molecular complexity of primary open angle glaucoma: current concepts

Glaucoma is a group of heterogeneous optic neuropathies with complex genetic basis. Among the three principle subtypes of glaucoma, primary open angle glaucoma (POAG) occurs most frequently. Till date, 25 loci have been found to be linked to POAG. However, only three underlying genes (Myocilin, Optineurin and WDR36) have been identified. In addition, at least 30 other genes have been reported to be associated with POAG. Despite strong genetic influence in POAG pathogenesis, only a small part of the disease can be explained in terms of genetic aberration. Current concepts of glaucoma pathogenesis suggest it to be a neurodegenerative disorder which is triggered by different factors including mechanical stress due to intra-ocular pressure, reduced blood flow to retina, reperfusion injury, oxidative stress, glutamate excitotoxicity, and aberrant immune response. Here we present a mechanistic overview of potential pathways and crosstalk between them operating in POAG pathogenesis.     

Genetic studies in rat models: insights into cardiovascular disease

PURPOSE OF REVIEW: Limited to 2003-2004 publications, this review focuses on 'big picture' concepts learned from rat genetic studies of cardiovascular disease. RECENT DEVELOPMENTS: Analysis reveals insights into pathogenic paradigms, as well as experimental perspectives into rat-based systems of analyses of complex cardiovascular disease. Key concepts are forwarded. Multiple susceptibility genes underlie several quantitative trait loci for blood pressure suggesting a 'quantitative trait loci cluster' concept; hypertension end-organ disease quantitative trait loci are distinct from blood pressure quantitative trait loci indicating differential susceptibility paradigms for hypertension and each complication (stroke, renal disease, cardiac hypertrophy); distinct blood pressure quantitative trait loci are found in males and females indicating gender-specific susceptibility; and genetic subtypes comprise polygenic hypertension in rat models suggesting a genetic basis for clinical heterogeneity of human essential hypertension. Gender specific genetic susceptibility plays a key role in coronary artery disease susceptibility; multiple distinct quantitative trait loci underlie hyperlipidemia and type-2 diabetes, indicating multiple susceptibilities in risk factors for cardiovascular disease. Studies in transgenic inbred rat-strain models demonstrate value for serial, complex, cardiovascular pathophysiological analyses within a genetic context. SUMMARY: Cognizant of the limitations of animal model studies, observations from rat genetic studies provide insight into respective modeled human cardiovascular diseases and risk factor susceptibility, as well as systematically dissect the multifaceted complexities apparent in human complex cardiovascular disease. Given the recapitulation of many features of human cardiovascular disease, the value of rat model-based genetic studies for complex cardiovascular disease is unequivocal, thus mandating the expansion of resources for maximization of rat-based genetic studies.     

Genome-Wide Association Study Identifies a Novel Canine Glaucoma Locus

Glaucoma is an optic neuropathy and one of the leading causes of blindness. Its hereditary forms are classified into primary closed-angle (PCAG), primary open-angle (POAG) and primary congenital glaucoma (PCG). Although many loci have been mapped in human, only a few genes have been identified that are associated with the development of glaucoma and the genetic basis of the disease remains poorly understood. Glaucoma has also been described in many dog breeds, including Dandie Dinmont Terriers (DDT) in which it is a late-onset (>7 years) disease. We designed clinical and genetic studies to better define the clinical features of glaucoma in the DDT and to identify the genetic cause. Clinical diagnosis was based on ophthalmic examinations of the affected dogs and 18 additionally investigated unaffected DDTs. We collected DNA from over 400 DTTs and a genome wide association study was performed in a cohort of 23 affected and 23 controls, followed by a fine mapping, a replication study and candidate gene sequencing. The clinical study suggested that ocular abnormalities including abnormal iridocorneal angles and pectinate ligament dysplasia are common (50% and 72%, respectively) in the breed and the disease resembles human PCAG. The genetic study identified a novel 9.5 Mb locus on canine chromosome 8 including the 1.6 Mb best associated region (p = 1.63×10⁻¹⁰, OR = 32 for homozygosity). Mutation screening in five candidate genes did not reveal any causative variants. This study indicates that although ocular abnormalities are common in DDTs, the genetic risk for glaucoma is conferred by a novel locus on CFA8. The canine locus shares synteny to a region in human chromosome 14q, which harbors several loci associated with POAG and PCG. Our study reveals a new locus for canine glaucoma and ongoing molecular studies will likely help to understand the genetic etiology of the disease.     

Zebrafish: a model for the study of addiction genetics

Drug abuse and dependence are multifaceted disorders with complex genetic underpinnings. Identifying specific genetic correlates is challenging and may be more readily accomplished by defining endophenotypes specific for addictive disorders. Symptoms and syndromes, including acute drug response, consumption, preference, and withdrawal, are potential endophenotypes characterizing addiction that have been investigated using model organisms. We present a review of major genes involved in serotonergic, dopaminergic, GABAergic, and adrenoreceptor signaling that are considered to be directly involved in nicotine, opioid, cannabinoid, and ethanol use and dependence. The zebrafish genome encodes likely homologs of the vast majority of these loci. We also review the known expression patterns of these genes in zebrafish. The information presented in this review provides support for the use of zebrafish as a viable model for studying genetic factors related to drug addiction. Expansion of investigations into drug response using model organisms holds the potential to advance our understanding of drug response and addiction in humans.     

Combinatorial Wnt control of zebrafish midbrain-hindbrain boundary formation

Wnt signaling is known to be required for the normal development of the vertebrate midbrain and hindbrain, but genetic loss of function analyses in the mouse and zebrafish yield differing results regarding the relative importance of specific Wnt loci. In the zebrafish, Wnt1 and Wnt10b functionally overlap in their control of gene expression in the ventral midbrain-hindbrain boundary (MHB), but they are not required for the formation of the MHB constriction. Whether other wnt loci are involved in zebrafish MHB development is unclear, although the expression of at least two wnts, wnt3a and wnt8b, is maintained in wnt1/wnt10b mutants. In order to address the role of wnt3a in zebrafish, we have isolated a full length cDNA and examined its expression and function via knockdown by morpholino antisense oligonucleotide (MO)-mediated knockdown. The expression pattern of wnt3a appears to be evolutionarily conserved between zebrafish and mouse, and MO knockdown shows that Wnt3a, while not uniquely required for MHB development, is required in the absence of Wnt1 and Wnt10b for the formation of the MHB constriction. In zebrafish embryos lacking Wnt3a, Wnt1 and Wnt10b, the expression of engrailed orthologs, pax2a and fgf8 is not maintained after mid-somitogenesis. In contrast to acerebellar and no isthmus mutants, in which midbrain and hindbrain cells acquire new fates but cell number is not significantly affected until late in embryogenesis, zebrafish embryos lacking Wnt3a, Wnt1 and Wnt10b undergo extensive apoptosis in the midbrain and cerebellum anlagen beginning in mid-somitogenesis, which results in the absence of a significant portion of the midbrain and cerebellum. Thus, the requirement for Wnt signaling in forming the MHB constriction is evolutionarily conserved in vertebrates and it is possible in zebrafish to dissect the relative impact of multiple Wnt loci in midbrain and hindbrain development.     

Stochastic search gene suggestion: a Bayesian hierarchical model for gene mapping

Mapping the genes for a complex disease, such as diabetes or rheumatoid arthritis (RA), involves finding multiple genetic loci that may contribute to the onset of the disease. Pairwise testing of the loci leads to the problem of multiple testing. Looking at haplotypes, or linear sets of loci, avoids multiple tests but results in a contingency table with sparse counts, especially when using marker loci with multiple alleles. We propose a hierarchical Bayesian model for case-parent triad data that uses a conditional logistic regression likelihood to model the probability of transmission to a diseased child. We define hierarchical prior distributions on the allele main effects to model the genetic dependencies present in the human leukocyte antigen (HLA) region of chromosome 6. First, we add a hierarchical level for model selection that accounts for both locus and allele selection. This allows us to cast the problem of identifying genetic loci relevant to the disease into a problem of Bayesian variable selection. Second, we attempt to include linkage disequilibrium as a covariance structure in the prior for model coefficients. We evaluate the performance of the procedure with some simulated examples and then apply our procedure to identifying genetic markers in the HLA region that influence risk for RA. Our software is available on the website http://www.epigenetic.org/Linkage/ssgs-public/.     

Two genetic loci regulate T cell-dependent islet inflammation and drive autoimmune diabetes pathogenesis

Insulin-dependent diabetes mellitus (IDDM) is a polygenic disease caused by progressive autoimmune infiltration (insulitis) of the pancreatic islets of Langerhan, culminating in the destruction of insulin-producing beta cells. Genome scans of families with diabetes suggest that multiple loci make incremental contributions to disease susceptibility. However, only the IDDM1 locus is well characterized, at a molecular and functional level, as alleleic variants of the major histocompatibility complex (MHC) class II HLA-DQB1, DRB1, and DPB1 genes that mediate antigen presentation to T cells. In the nonobese diabetic (NOD) mouse model, the Idd1 locus was shown to be the orthologous MHC gene I-Ab. Inheritance of susceptibility alleles at IDDM1/Idd1 is insufficient for disease development in humans and NOD mice. However, the identities and functions of the remaining diabetes loci (Idd2-Idd19 in NOD mice) are largely undefined. A crucial limitation in previous genetic linkage studies of this disease has been reliance on a single complex phenotype-diabetes that displays low penetrance and is of limited utility for high-resolution genetic mapping. Using the NOD model, we have identified an early step in diabetes pathogenesis that behaves as a highly penetrant trait. We report that NOD-derived alleles at both the Idd5 and Idd13 loci regulate a T lymphocyte-dependent progression from a benign to a destructive stage of insulitis. Human chromosomal regions orthologous to the Idd5 and -13 intervals are also linked to diabetes risk, suggesting that conserved genes encoded at these loci are central regulators of disease pathogenesis. These data are the first to reveal a role for individual non-MHC Idd loci in a specific, critical step in diabetes pathogenesis-T cell recruitment to islet lesions driving destructive inflammation. Importantly, identification of intermediate phenotypes in complex disease pathogenesis provides the tools required to progress toward gene identification at these loci.     

Glaucoma in Costa Rica. Initial approaches

Glaucoma is the second most frequent cause of irreversible blindness worldwide. Genetic factors have been implicated in the development of the disease. So far six loci (GLC1A-GLC1F) and two genes (TIGR/MYOC and OPTN) are involved in the development of juvenile (JOAG) and adult onset or chronic primary open angle glaucoma (COAG), while two loci (GLC3A,GLC3B) and one gene (CYP1B1) are known for primary congenital glaucoma (PCG). Here we summarize the results of the first genetic studies of glaucoma in Costa Rica. Nine families: 1 with JOAG, 1 with PCG and 7 with COAG were screened for mutations at the known genes. A 10 bp duplication, 1546-1555dupTCATGCCACC, at the CYP1B1 gene, causes, in homozygous state, glaucoma in the consanguineous PCG family. This mutation has been found in different countries and generates an early stop codon that termitates protein synthesis 140 amino acids earlier than the normal allele. In exon 1 of the T1GR/MYOC the innocuous Arg76Lys variant was found in two of the COAG families. In the OPTN gene two variants in the coding region (Thr34Thr, Met 98Lys) and 7 intronic changes were found in other Costa Rican glaucoma patients. One of the COAG families was chosen for a genome scan with 379 microsatellite markers and linkage analysis. LOD scores "suggestive" of linkage were obtained for several chromosomal regions. Evidence indicates that hereditary glaucoma in Costa Rica is highly heterogeneous and that further studies in the country will probably disclose some up to now unknown genes responsible for the disease.     

A Post-Developmental Genetic Screen for Zebrafish Models of Inherited Liver Disease

Nonalcoholic fatty liver disease (NAFLD) is one of the most common causes of chronic liver disease such as simple steatosis, nonalcoholic steatohepatitis (NASH), cirrhosis and fibrosis. However, the molecular pathogenesis and genetic variations causing NAFLD are poorly understood. The high prevalence and incidence of NAFLD suggests that genetic variations on a large number of genes might be involved in NAFLD. To identify genetic variants causing inherited liver disease, we used zebrafish as a model system for a large-scale mutant screen, and adopted a whole genome sequencing approach for rapid identification of mutated genes found in our screen. Here, we report on a forward genetic screen of ENU mutagenized zebrafish. From 250 F2 lines of ENU mutagenized zebrafish during post-developmental stages (5 to 8 days post fertilization), we identified 19 unique mutant zebrafish lines displaying visual evidence of hepatomegaly and/or steatosis with no developmental defects. Histological analysis of mutants revealed several specific phenotypes, including common steatosis, micro/macrovesicular steatosis, hepatomegaly, ballooning, and acute hepatocellular necrosis. This work has identified multiple post-developmental mutants and establishes zebrafish as a novel animal model for post-developmental inherited liver disease.     

Molecular genetics of atherosclerosis

Atherosclerosis is a complex multifocal arterial disease involving interactions of multiple genetic and environmental factors. Advances in techniques of molecular genetics have revealed that genetic polymorphisms significantly influence susceptibility to atherosclerotic vascular diseases. A large number of candidate genes, genetic polymorphisms and susceptibility loci associated with atherosclerotic diseases have been identified in recent years and their number is rapidly increasing. In this review we focus on some of the major candidate genes and genetic polymorphisms associated with human atherosclerotic vascular diseases.     

Finding clues to the riddle of sex determination in zebrafish

How sex is determined has been one of the most intriguing puzzles in biology since antiquity. Although a fundamental process in most metazoans, there seems to be myriad of ways in which sex can be determined – from genetic to environmental sex determination. This variation is limited mainly to upstream triggers with the core of sex determination pathway being conserved. Zebrafish has gained prominence as a vertebrate model system to study development and disease. However, very little is known about its primary sex determination mechanism. Here we review our current understanding of the sex determination in zebrafish. Zebrafish lack identifiable heteromorphic sex chromosomes and sex is determined by multiple genes, with some influence from the environment. Recently, chromosome 4 has been identified as sex chromosome along with few sex-linked loci on chromosomes 5 and 16. The identities of candidate sex-linked genes, however, have remained elusive. Sex in zebrafish is also influenced by the number of meiotic oocytes in the juvenile ovary, which appear to instruct retention of the ovarian fate. The mechanism and identity of this instructive signal remain unknown. We hypothesize that sex in zebrafish is a culmination of combinatorial effects of the genome, germ cells and the environment with inputs from epigenetic factors translating the biological meaning of this interaction.     

牛疾病相关基因的DNA变异及遗传控制

牛基因组中一些重要基因的DNA突变通过改变基因的表达和蛋白质功能来影响机体对疾病的抗性或易感性。控制牛疾病的DNA变异主要分为单基因座及多基因座两类。导致疾病的单基因座类型亦称因果突变,其遗传基础较简单,突变一般位于基因编码区或非编码区,多为单碱基或少数几个碱基的突变,这些突变导致氨基酸的错义突变、翻译提前终止或部分外显子缺失等。相比而言,多基因相关疾病的遗传基础较为复杂,遗传-病原体-环境间的互作是导致这类复杂疾病的主要原因。文章综述了由单基因座和多基因座遗传变异所控制的牛主要疾病的研究和应用现状,以及在牛育种及生产中为降低这些疾病的发生所采用的遗传控制策略。