Single linkage group per chromosome genetic linkage map for the horse, based on two three-generation, full-sibling, crossbred horse reference families

A genetic linkage map of the horse consisting of 742 markers, which comprises a single linkage group for each of the autosomes and the X chromosome, is presented. The map has been generated from two three-generation full-sibling reference families, sired by the same stallion, in which there are 61 individuals in the F2 generation. Each linkage group has been assigned to a chromosome and oriented with reference to markers mapped by fluorescence in situ hybridization. The average interval between markers is 3.7 cM and the linkage groups collectively span 2772 cM. The 742 markers comprise 734 microsatellite and 8 gene-based markers. The utility of the microsatellite markers for comparative mapping has been significantly enhanced by comparing their flanking sequences with the human genome sequence; this enabled conserved segments between human and horse to be identified. The new map provides a valuable resource for genetically mapping traits of interest in the horse.

EXPLORATION, NORMALIZATION, AND GENOTYPE CALLS OF HIGH DENSITY OLIGONUCLEOTIDE SNP ARRAY DATA

In most microarray technologies, a number of critical steps are required to convert raw intensity measurements into the data relied upon by data analysts, biologists and clinicians. These data manipulations, referred to as preprocessing, can influence the quality of the ultimate measurements. In the last few years, the high-throughput measurement of gene expression is the most popular application of microarray technology. For this application, various groups have demonstrated that the use of modern statistical methodology can substantially improve accuracy and precision of gene expression measurements, relative to ad-hoc procedures introduced by designers and manufacturers of the technology. Currently, other applications of microarrays are becoming more and more popular. In this paper we describe a preprocessing methodology for a technology designed for the identification of DNA sequence variants in specific genes or regions of the human genome that are associated with phenotypes of interest such as disease. In particular we describe methodology useful for preprocessing Affymetrix SNP chips and obtaining genotype calls with the preprocessed data. We demonstrate how our procedure improves existing approaches using data from three relatively large studies including one in which large number independent calls are available. Software implementing these ideas are avialble from the Bioconductor oligo package.

Genomics of liver fibrosis and cirrhosis

Hepatic fibrosis is the response to chronic injury from viral, toxic, metabolic, cholestatic, or autoimmune liver injury. However, only a minority of affected individuals develop advanced fibrosis or cirrhosis, suggesting that modifiers determine the individual risk. Aside from well-established progression factors including gender, duration of exposure to the disease, and ethnicity, unknown host genetic factors are likely to influence disease progression and prognosis. Potential genetic susceptibility loci are single nucleotide polymorphisms in fibrosis-associated genes, point mutations, microsatellites, and haplotype blocks composed of genes pivotal for fibrosis development. However, the study of complex polygenetic diseases poses numerous pitfalls in contrast to the elucidation of monogenetic (i.e., Mendelian) diseases. Many publications on the role of certain genetic variants in modulating the progression of hepatic fibrosis have been limited by inadequate study design and low statistical power. At present, powerful research strategies are being developed that allow the application of knowledge derived from the successful sequencing of the human genome that will help to translate our newly acquired insight into practical improvements for research activities and medical practice.

Recurrent rearrangements of chromosome 1q21.1 and variable pediatric phenotypes

BACKGROUND: Duplications and deletions in the human genome can cause disease or predispose persons to disease. Advances in technologies to detect these changes allow for the routine identification of submicroscopic imbalances in large numbers of patients. METHODS: We tested for the presence of microdeletions and microduplications at a specific region of chromosome 1q21.1 in two groups of patients with unexplained mental retardation, autism, or congenital anomalies and in unaffected persons. RESULTS: We identified 25 persons with a recurrent 1.35-Mb deletion within 1q21.1 from screening 5218 patients. The microdeletions had arisen de novo in eight patients, were inherited from a mildly affected parent in three patients, were inherited from an apparently unaffected parent in six patients, and were of unknown inheritance in eight patients. The deletion was absent in a series of 4737 control persons (P=1.1x10(-7)). We found considerable variability in the level of phenotypic expression of the microdeletion; phenotypes included mild-to-moderate mental retardation, microcephaly, cardiac abnormalities, and cataracts. The reciprocal duplication was enriched in nine children with mental retardation or autism spectrum disorder and other variable features (P=0.02). We identified three deletions and three duplications of the 1q21.1 region in an independent sample of 788 patients with mental retardation and congenital anomalies. CONCLUSIONS: We have identified recurrent molecular lesions that elude syndromic classification and whose disease manifestations must be considered in a broader context of development as opposed to being assigned to a specific disease. Clinical diagnosis in patients with these lesions may be most readily achieved on the basis of genotype rather than phenotype.

Inherited epithelial transporter disorders-an overview

In the late 1990s, the identification of transporters and transporter-associated genes progressed substantially due to the development of new cloning approaches such as expression cloning and, subsequently, to the implementation of the human genome project. Since then, the role of many transporter genes in human diseases has been elucidated. In this overview, we focus on inherited disorders of epithelial transporters. In particular, we review genetic defects of the genes encoding glucose transporters (SLC2 and SLC5 families) and amino acid transporters (SLC1, SLC3, SLC6 and SLC7 families).

Gene expression in cultured endometrium from women with different outcomes following IVF

Estradiol and progesterone are crucial for the acquisition of receptivity and the change in transcriptional activity of target genes in the implantation window. The aim of this study was to differentiate the regulation of genes in the endometrium of patients with recurrent implantation failure (IF) versus those who became pregnant after in vitro fertilization (IVF) treatment. Moreover, the effect of embryo-derived factors on endometrial transcriptional activity was studied. Nine women with known IVF outcome (IF, M, miscarriage, OP, ongoing pregnancy) and undergoing hysteroscopy with endometrial biopsy were enrolled. Biopsies were taken during the midluteal phase. After culture in the presence of embryo-conditioned IVF media, total RNA was extracted and submitted to reverse transcription, target cDNA synthesis, biotin labelling, fragmentation and hybridization using the Affymetrix Human Genome U133A 2.0 Chip. Differential expression of selected genes was re-analysed by quantitative PCR, in which the results were calculated as threshold cycle differences between the groups and normalized to Glyceraldehyde phosphate dehydrogenase and beta-actin. Differences were seen for several genes from endometrial tissue between the IF and the pregnancy groups, and when comparing OP with M, 1875 up- and 1807 down-regulated genes were returned. Real-time PCR analysis confirmed up-regulation for somatostatin, PLAP-2, mucin 4 and CD163, and down-regulation of glycodelin, IL-24, CD69, leukaemia inhibitory factor and prolactin receptor between Op and M. When the different embryo-conditioned media were compared, no significant differential regulation could be demonstrated. Although microarray profiling may currently not be sensitive enough for studying the effects of embryo-derived factors on the endometrium, the observed differences in gene expression between M and OP suggest that it will become an interesting tool for the identification of fertility-relevant markers produced by the endometrium.

Patentability and Scope of Protection for DNA Sequence

Since the mapping of the human genome and the technical innovations in the field of biotechnology, patent law has gone through great controversies. Protection is required for an investor to make an investment but how broad should the given protection be? Whether the invention is a mi- cro-organism capable of dissolving crude oil, or the gene of a soya plant, the genetic engineering required for their production entails vast amounts of capi- tal. The policy in that respect is tailored by legislative acts and judicial decisions, ensuring a fair balance be- tween the interests of patent right holders and third parties. However, the policy differs from jurisdiction to jurisdiction, thus creating inconsistencies with re- gards to the given protection to the same invention, and as a result this could deter innovation and pro- mote stagnation. The most active actors shaping the patent policy on an international level are the patent offices of the United States of America, Japan and the European Patent Organization. These three patent offices have set up a cooperation programme in order to promote and improve efficiency with regards to their patent policies on a global scale. However, recent judicial de- velopments have shown that the policy in respect to the field of biotechnology differs between the patent regimes of the United States of America and the two- layer system of the European Patent Organisation/ the European Union.

The ABCs of membrane transporters in health and disease (SLC series): introduction

The field of transport biology has steadily grown over the past decade and is now recognized as playing an important role in manifestation and treatment of disease. The SLC (solute carrier) gene series has grown to now include 52 families and 395 transporter genes in the human genome. A list of these genes can be found at the HUGO Gene Nomenclature Committee (HGNC) website (see www.genenames.org/genefamilies/SLC). This special issue features mini-reviews for each of these SLC families written by the experts in each field. The existing online resource for solute carriers, the Bioparadigms SLC Tables (www.bioparadigms.org), has been updated and significantly extended with additional information and cross-links to other relevant databases, and the nomenclature used in this database has been validated and approved by the HGNC. In addition, the Bioparadigms SLC Tables functionality has been improved to allow easier access by the scientific community. This introduction includes: an overview of all known SLC and "non-SLC" transporter genes; a list of transporters of water soluble vitamins; a summary of recent progress in the structure determination of transporters (including GLUT1/SLC2A1); roles of transporters in human diseases and roles in drug approval and pharmaceutical perspectives.

Antimicrobial effects of murine mesenchymal stromal cells directed against Toxoplasma gondii and Neospora caninum: role of immunity-related GTPases (IRGs) and guanylate-binding proteins (GBPs).

Mesenchymal stromal cells (MSCs) have a multilineage differentiation potential and provide immunosuppressive and antimicrobial functions. Murine as well as human MSCs restrict the proliferation of T cells. However, species-specific differences in the underlying molecular mechanisms have been described. Here, we analyzed the antiparasitic effector mechanisms active in murine MSCs. Murine MSCs, in contrast to human MSCs, could not restrict the growth of a highly virulent strain of Toxoplasma gondii (BK) after stimulation with IFN-γ. However, the growth of a type II strain of T. gondii (ME49) was strongly inhibited by IFN-γ-activated murine MSCs. Immunity-related GTPases (IRGs) as well as guanylate-binding proteins (GBPs) contributed to this antiparasitic effect. Further analysis showed that IFN-γ-activated mMSCs also inhibit the growth of Neospora caninum, a parasite belonging to the apicomplexan group as well. Detailed studies with murine IFN-γ-activated MSC indicated an involvement in IRGs like Irga6, Irgb6 and Irgd in the inhibition of N. caninum. Additional data showed that, furthermore, GBPs like mGBP1 and mGBP2 could have played a role in the anti-N. caninum effect of murine MSCs. These data underline that MSCs, in addition to their regenerative and immunosuppressive activity, function as antiparasitic effector cells as well. However, IRGs are not present in the human genome, indicating a species-specific difference in anti-T. gondii and anti-N. caninum effect between human and murine MSCs.

DYSREGULATION OF MEOX2 FOLLOWING WT1 MUTATION IN KIDNEY DEVELOPMENT AND WILMS TUMORIGENESIS

Wilms tumor (WT) is a childhood tumor of the kidney and a productive model for understanding the role of genetic alteration and interactions in tumorigenesis. The Wilms tumor gene 1 (WT1) is a transcriptional factor and one of the few genes known to have genetic alterations in WT and has been shown be inactivated in 20% of WTs. However, the mechanisms of how WT1 mutations lead to Wilms tumorigenesis and its influence on downstream genes are unknown. Since it has been established that WT1 is a transcriptional regulator, it has been hypothesized that the loss of WT1 leads to the dysregulation of downstream genes, in turn result in the formation of WTs. To identify the dysregulated downstream genes following WT1 mutations, an Affymetrix GeneChip Human Genome Array was previously conducted to assess the differentially expressed genes in the WT1-wildtype human and WT1-mutant human WTs. Approximately 700 genes were identified as being significantly dysregulated. These genes were further prioritized based on their statistical significance, fold change, chromosomal region, spatial pattern of gene expression and known or putative cellular functions. Mesenchyme homeobox 2 (MEOX2) was one of the most significantly upregulated genes in WT1-mutant WT. MEOX2 is known to play a role in cell proliferation, apoptosis, and differentiation. In addition to its biological roles, it is expressed during early kidney development in the condensed mesenchyme similar to WT1. Furthermore, the use of the Match® web-based tool from the BIOBASE Biological Data base identified a significant predicted WT1 binding site within the first intron of MEOX2. The similarity in spatial gene expression in the developing kidney and the significant predicted WT1 binding site found in the first intron of MEOX2 lead to the development of my hypothesis that MEOX2 is upregulated via a WT1-dependent manner. Here as a part of my master’s work, I have validated the Affymetrix GeneChip Human Genome Array data using an independent set of Wilms tumors. MEOX2 remained upregulated in the mutant WT1 Wilms tumor by 41-fold. Wt1 and Meox2 gene expression were assessed in murine newborn kidney; both Wt1 and Meox2 were expressed in the condensed, undifferentiated metanephric mesenchyme. I have shown that the in vivo ablation of Wt1 during embryonic development at embryonic day (E) 13.5 resulted in the slight increase of Meox2 gene expression by two fold. In order to functionally demonstrate the effect of the loss of Wt1 on Meox2 gene expression in undifferentiated metanephric mesenchyme, I have generated a kidney mesenchymal cell line to genetically ablate Wt1 in vitro by adenoviral infection. The ablation of Wt1 in the kidney mesenchymal cell line resulted in the upregulation of Meox2 by 61-fold. Moreover, the upregulation of Meox2 resulted in the significant induction of p21 and Itgb5. In addition to the dysregulation of these genes the ablation of Wt1 in the kidney mesenchymal cells resulted in decrease in cell growth and loss of cellular adherence. However, it is uncertain whether the upregulation of Meox2 caused this particular cellular phenotype. Overall, I have demonstrated that the upregulation of Meox2 is Wt1-dependent during early kidney development.

Chromosomal control of the dominant suppression of {\it ras\/}-mediated transformation

The role of tumor suppressor function in the multistep process of carcinogenesis was studied in the human teratocarcinoma cell line PA-1. Early passage PA-1 cells ($<$P100) are preneoplastic while late passage ($>$P100) PA-1 cells are spontaneously transformed. Previous work demonstrated a causal role for the N-ras oncogene in the neoplastic transformation of this cell line and the gene was cloned. A clonal cell line established at passage 40 has been shown to suppress the neoplastic transformation potential of the PA-1 N-ras oncogene in gene transfer experiments. This phenotype has been termed SRT+ for suppression of ras transformation. A clonal cell line established at passage 63 is neoplastically transformed by the N-ras in similar gene transfer experiments and is regarded as srt$-$. Somatic cell hybrids were formed between the SRT+ cell and two different N-ras transformed srt$-$ cells. The results indicate that five of the seven independent hybrid clones, and all 14 subclones, failed to form tumors in the nude mouse tumor assay. Chromosomal analysis of rare neoplastic segregants which arose from suppressed hybrid populations demonstrate that the general loss of chromosomes correlates with the reemergence of neoplastic transformation. Karyotype analyses demonstrate a statistically correlative loss of chromosomes 1, 4, 19, and to a lesser extent 11, 14, and 16. DNA hybridization analysis demonstrates a single copy of the intact N-ras oncogene in parental cells, suppressed hybrids, and neoplastically transformed hybrids. These results indicate that functional ras transformation suppression is a trans-dominant trait which may be controlled by sequences residing on particular chromosomes in the human genome. Furthermore, the suppression of ras transformation results from a unique step in the multistep process of carcinogenesis that is different from the induction of immortality. Thus, the neoplastic process of the PA-1 cell line involves at least three steps: (1) induction of immortality, (2) activation of the N-ras oncogene, and (3) loss of tumor suppressor function. ^

Transcriptional regulation of the mouse $\alpha$2(I) collagen gene

The mouse $\alpha$2(I) collagen gene is specifically expressed in a limited number of cell types in the body including fibroblasts and osteoblasts. We had previously shown that a promoter containing the sequences between $-$350 and +54 bp was expressed at low levels in a cell- and tissue-specific fashion in transgenic mice. Further studies suggested that the sequence between $-$315 and $-$284 bp could mediate cell- and tissue-specific expression of reporter genes in cell culture and in transgenic mice. We report here characterization of the proteins binding to this segment and propose a model for the cell-specific expression conferred by this sequence. In this study we also identified a strong enhancer for the mouse $\alpha$2(I) collagen gene located approximately 13.5 to 19.5 kb upstream of the transcriptional start site. This enhancer segment is characterized by the presence of three cell-specific hypersensitive sites and can drive high levels of cell-specific expression of a heterologous 220-bp mouse $\alpha$1(I) collagen promoter. In the course of this study, we identified a novel zinc finger transcription factor (designated murine epithelial zinc finger, mEZF) which was transiently expressed in the mesenchymal cells which give rise to the skeletal primordia and the metanephric kidney during the early stages of embryogenesis. In newborn mice, the mEZF gene is expressed at high levels in differentiated epithelial cells of the skin, oral mucosa, tongue, esophagus, stomach and colon. Chromosomal mapping suggested that the mEZF gene mapped to mouse Chromosome 4 and that the human homolog of mEZF would likely map to human Chromosome 9q31. This region of the human genome contains tumor suppressor genes for basal cell carcinomas of the skin as well as for squamous cell carcinomas of various organs. We cloned and characterized the human homolog of mEZF and mapped its chromosomal position as a first step in determining whether or not this gene plays a role in the development of these tumors. ^

The story of RP10: A positional cloning approach to the identification of an autosomal dominant retinitis pigmentosa gene

Retinitis pigmentosa (RP) is an inherited retinal degenerative disease that is the leading cause of inherited blindness worldwide. Characteristic features of the disease include night blindness, progressive loss of visual fields, and deposition of pigment on the retina in a bone spicule-like pattern. RP is marked by extreme genetic heterogeneity with at least 19 autosomal dominant, autosomal recessive and X-linked loci identified. RP10, which maps to chromosome 7q, was the fifth autosomal dominant RP locus identified, and accounts for the early-onset disease in two independent families. Extensive linkage and haplotype analyses have been performed in these two families which have allowed the assignment of the disease locus to a 5-cM region on chromosome 7q31.3. In collaboration with Dr. Eric Green (National Center for Human Genome Research, National Institutes of Health), a well-characterized physical map of the region was constructed which includes YAC, BAC and cosmid coverage. The entire RP10 critical region resides within a 9-Mb well-characterized YAC contig. These physical maps not only provided the resources to undertake the CAIGES (cDNA amplification for identification of genomic expressed sequences) procedure for identification of retinal candidate genes within the critical region, but also identified a number of candidate genes, including transducin-$\gamma$ and blue cone pigment genes. All candidate genes examined were excluded. In addition, a number of ESTs were mapped within the critical region. EST20241, which was isolated from an eye library, corresponded to the 3$\sp\prime$ region of the ADP-ribosylation factor (ARF) 5 gene. ARF5, with its role in vesicle transport and possible participation in the regulation of the visual transduction pathway, became an extremely interesting candidate gene. Using a primer walking approach, the entire 3.2 kb genomic sequence of the ARF5 gene was generated and developed intronic primers to screen for coding region mutations in affected family members. No mutations were found in the ARF5 gene, however, a number of additional ESTs have been mapped to the critical region, and, as the large-scale sequencing projects get underway, megabases of raw sequence data from the RP10 region are becoming available. These resources will hasten the isolation and characterization of the RP10 gene. ^

drp, A novel cell density regulated protein

Growing cells are continuously processing signals of all varieties and responding to these signals by changes in cellular gene expression. One signal that cells in close proximity relay to each other is cell-cell contact. Non-transformed cells respond to cell-cell contact by arrest of growth and entry into G$\sb0,$ a process known as contact inhibition. Transformed cells do not respond to contact inhibition and continue to grow to high cell density, forming foci when in cell culture and tumors in the living organism. The events surrounding the generation, transduction, and response to cellular contact are poorly understood. In the present study, a novel gene product, drp, is shown to be expressed at high levels in cultured cells at high cell density. This density regulated protein, drp, has an apparent molecular weight of 70 kDa. Northern analysis shows drp to be highly expressed in cardiac and skeletal muscle and least abundant in lung and kidney tissues. By homology to two independently derived sequence tagged sites (STSs) used in the human genome project, drp or a closely related sequence maps to human chromosome 12. Density-dependent increases in drp expression have been demonstrated in six different cell lines including NIH 3T3, Hela and a human teratocarcinoma cell line, PA-1. Cells exhibit increased drp expression both when they are plated at increasing concentrations per unit area, or plated at low density and allowed to grow naturally to higher cell density. Cells at high density can exhibit several phenotypes including growth arrest, accumulation of soluble factors in the media, and increased numbers of cell contacts. Growth arrest by serum starvation or TGF-$\beta$ treatment fails to produce an increase in drp expression. Similarly, treatment of low density cells with conditioned media from high density cells fails to elicit drp expression. These results argue that neither soluble factors accumulated or expressed at high density nor simple exit from the cell cycle is sufficient to produce an increase in drp expression. The expression of drp appears to be uniquely regulated by cell density alone. ^

Transcriptional analysis of left-sided colitis, pancolitis, and ulcerative colitis-associated dysplasia.

BACKGROUND It is unknown why patients with extensive ulcerative colitis (UC) have a higher risk of colorectal cancer compared with patients with left-sided UC. This study characterizes the inflammatory processes in left-sided UC, pancolitis, and UC-associated dysplasia at the transcriptional level to identify potential biomarkers and transcripts of importance for the carcinogenic behavior of chronic inflammation. METHODS The Affymetrix GeneChip Human Genome U133 Plus 2.0 was applied on colonic biopsies from UC patients with left-sided UC, pancolitis, dysplasia, and controls. Reverse transcription polymerase chain reaction and immunohistochemistry were performed for validating selected transcripts in the initial cohort and in 2 independent cohorts of patients with UC. Microarray data were analyzed by principal component analysis, and reverse transcription polymerase chain reaction and immunohistochemistry data by the Wilcoxon's rank-sum test. RESULTS The principal component analysis results revealed separate clusters for left-sided UC, pancolitis, dysplasia, and controls. Close clustering of dysplastic and pancolitic samples indicated similarities in gene expression. Indeed, 101 and 656 parallel upregulated and downregulated transcripts, respectively, were identified in specimens from dysplasia and pancolitis. Validation of selected transcripts hereof identified insulin receptor alpha (INSRA) and MAP kinase interacting serine/threonine kinase 2 (MKNK2) with an enhanced expression in dysplasia compared with left-sided UC and controls, whereas laminin γ2 (LAMC2) was found with a lower expression in dysplasia compared with the remaining 3 groups. CONCLUSIONS This study demonstrates pancolitis and left-sided UC as distinct inflammatory processes at the transcriptional level, and identifies INSRA, MKNK2, and LAMC2 as potential critical transcripts in the inflammation-driven preneoplastic process of UC.