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Journal of Pediatric Gastroenterology & Nutrition:
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Research Agenda for Pediatric Gastroenterology, Hepatology and Nutrition: Molecular Basis of Gastrointestinal Diseases: Report of the North American Society for Pediatric Gastroenterology, Hepatology and Nutrition for the Children's Digestive Health and Nutrition Foundation

Perlmutter, David H.; Lopez, M. James; Martin, Martin; Rand, Elizabeth

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Pittsburgh

Dallas

Los Angeles

Philadelphia

Address requests for reprints to: Executive Director, Children's Digestive Health and Nutrition Foundation, PO Box 6, Flourtown, PA, 19031, U.S.A. (e-mail: NASPGHAN@naspghan.org).

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RATIONALE

Sophisticated computer software technology has recently been developed to provide data from the human genome project as well as from microarray and proteonomic studies. Initial studies using this technology have demonstrated the potential for major advances in several areas of biomedical research.

Deoxyribonucleic acid (DNA) microarray techniques may be used to characterize profiles of changes in gene expression in unprecedented detail. For example, there have been studies of changes in the expression of thousands of genes in specific cells or tissues in response to aging, tissue injury, caloric restriction, drug treatment or alteration of a specific gene (e.g., mutation, and expression of an oncogenic transcription factor). Novel classification systems for leukemias, which have been generated by gene expression profiles, can potentially lead to optimal, targeted treatments. Gene expression profiles also have been used to characterize pathogenic factors in microorganisms. Together with newly developed laser microdissection techniques, which permit isolation of specific structures and even cell types from a tissue specimen, microarray techniques are beginning to be used for characterization of gene expression profiles within different components of tumors or inflamed tissues.

DNA microarray techniques also may be used for large-scale studies of sequence variation. This type of application has the potential to revolutionize genetic mapping and, in large population studies, to identify genetic alterations that determine disease susceptibility.

In addition, DNA microarray techniques may be used to characterize interactions between gene products. For example, microarrays have been applied to screens designed to identify inhibitors of specific biological response pathways. This type of application will be used in the future to identify substrates of enzymes, substrates for chaperones and ligands for receptors. It may be especially useful for identification of antisense oligonucleotides or antisense ribozymes for pharmacologic interventions.

Finally, DNA microarray techniques have been used to screen for the function of products of disrupted genes in model systems. This type of application will be particularly important for understanding disease pathogenesis and for drug discovery projects.

Proteonomics, or the analysis of complete sets of gene products at the protein level, has not been used widely, but these techniques may prove even more powerful for the identification of interactions between gene products. Thus, they may be designed for identification of substrates, inhibitors, ligands, agonists, antagonists, transcriptional activators and repressors.

Application of these technologies to studies of the molecular basis of gastrointestinal (GI) health and disease in children may produce major advances relatively rapidly. The technology can be applied to almost all disorders affecting the GI tract, liver, biliary tract and pancreas. It can potentially facilitate the elucidation of genes altered in congenital anomalies or in inherited disorders that are monogenic, as well as the elucidation of genes that modify the clinical phenotype of monogenic disorders. Genetic traits that contribute to the development of polygenic diseases or determine susceptibility to infections, inflammatory diseases, toxins, drug reactions and developmentally determined or physiologic stressors also may be more rapidly identified with these technological advances.

Application of these technologies may be particularly useful in understanding host responses to disease and injury, including intestinal adaptation, liver regeneration, responses to intestinal or biliary obstruction, host inflammatory response and host stress response. Indeed, these responses are thought to be critical determinants of the clinical phenotype and severity of many GI disorders.

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AREAS OF EMPHASIS

Elucidate the Genetic Basis of Single-Gene Disorders and Genetic Determinants of Polygenic Diseases
Research Goals

Here we need to identify the genes altered in single-gene disorders, such as microvillus inclusion disease, neonatal iron storage disease, Alpers' disease and Shwachman syndrome. We also need to identify genes altered in polygenic diseases, such as inflammatory bowel disease and gluten-sensitive enteropathy. Finally, the genetic disorders that account for some general syndromes, such as intractable diarrhea of infancy and idiopathic neonatal hepatitis, need to be elucidated.

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Research Strategies

This goal will require a combination of genetic mapping, microarray analysis, proteonomic analysis, and classical biochemical studies. A combined strategy was recently used to identify and characterize the ABC1 lipid transporter that is mutated in Tangier disease.

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Projected Timetable and Funding Requirements

This area will be optimally addressed by investigator-initiated grants (e.g., RO1 grants). Center grants and program projects also will be effective strategies, particularly because they could permit the establishment of core facilities for computer software and microarray and proteonomic analysis. The initiative should be started immediately to take advantage of the new data being generated by the human genome project.

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Analyze Genotype-Phenotype Relationships and Genetic Modifiers of Single-Gene Disorders
Research Goals

Here we need to identify the genetic traits that determine the clinical phenotype of disorders in which the primary genetic abnormalities have already been identified. Cystic fibrosis and α1-antitrypsin deficiency are examples of monogenic disorders in which there is wide variation in phenotypic expression of target organ injury. Although rare, hereditary hemorrhagic telangiectasia is an example of a disorder with a striking genetically determined phenotypic variation. It is also very important to learn how childhood GI, hepatobiliary and pancreatic diseases can lead to carcinoma during the adult years. Examples include hereditary polyposis syndromes, inflammatory bowel disease and metabolic liver disease. Detailed information on tumorigenesis and cell survival (i.e., apoptosis pathways) is important for our understanding of the pathobiology of these diseases. This information also is important for the development and use of novel therapeutic strategies. For example, in the case of metabolic liver disease, transplanted hepatocytes have a selective advantage for growth and proliferation in the liver of the c14CoS albino mouse (a murine model of hereditary tyrosinemia) and will replace most of the damaged liver. However, it is not known whether a small number of residual dysplastic liver cells will become malignant and thereby limit the potential success of this type of novel therapy.

Proteonomic techniques may be particularly useful for studying how GI diseases can lead to carcinoma. For example, proteonomic analysis was recently used to identify the expression of 43,302 proteins in normal human breast cells in anticipation of a comparison with protein expression in cells from breast cancer specimens.

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Research Strategies

This goal will require a focus on basic biologic and pathobiologic work and should utilize genetic mapping, microarray and proteonomic techniques together with classical biochemical and cell biological studies. It will also require large-scale multicenter collaborative studies and registries to carefully characterize patients from a clinical perspective and provide material from these patients for genotyping.

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Projected Timetable and Funding Requirements

This research should start immediately and will depend on investigator-initiated grants, program projects and center grants. The development of a registry for specific single-gene disorders, as defined by the other task force subcommittees, is strongly recommended. Funding should be solicited from several institutes at the National Institutes of Health (NIH) as well as from private foundations. A contract mechanism could work particularly well for the registries.

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Develop New Animal Models
Research Goals

It is recommended that the most sophisticated genetic engineering strategies be used to generate new animal models of GI disease. These strategies include transgenesis, targeted gene disruption, targeting mutagenesis, and conditional and inducible expression systems. It will be very important to ascertain the effect of genetic background in each animal model so that genetic modifiers of disease phenotype can be identified. Use of inducible or conditional expression systems will permit examination of the effects of developmental stage on disease phenotype. These studies are likely to be particularly informative for animal models of diseases that are affected by development. For example, in α1-antitrypsin deficiency, there is often clinical evidence of liver injury early in infancy and thereafter most patients enter a period in which there is marked lessening of liver injury. In some of these patients, liver disease recurs during adolescence. Some α1-antitrypsin-deficient individuals develop liver disease with or without hepatocellular carcinoma later during adult life. An animal model in which the hepatotoxic condition, retention of the mutant ZZ α1-antitrypsin molecule in the endoplasmic reticula of liver cells, is induced or abrogated at specific times during development may provide critical information about the mechanism underlying the effects of this disorder on the liver at different stages of development. Use of tissue-specific promoters will permit examination of the role of specific tissues in determining disease phenotype.

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Research Strategies

This goal will require a collaborative effort between investigators, genetically altered mouse core facilities and their personnel, anatomists, pathologists, embryologists and physiologists. Characterization of the animal models will require microarrays and proteonomics. In fact, it is likely that at some point in the future, companies will be making the genetically altered mice and supplying them to investigators for this type of characterization. The development of a database for accessing information on all animal models is strongly recommended.

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Projected Timetable and Funding Requirements

Program projects and center grants with core facilities would greatly facilitate this type of work. Consideration should be given to encouraging and facilitating collaboration between industry and academic research programs.

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Develop Novel Prophylactic/Therapeutic Interventions for GI Disease
Research Goals

It is recommended that proteonomic analytical systems be used to identify novel agonists and antagonists. In addition, three novel strategies for the prevention and treatment of genetically determined conditions should be studied in detail:

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Cell transplantation.

Recent studies have shown that normal adult hepatocytes can replicate and replace much of the liver parenchyma, but only when transplanted into the background of an injured liver—specifically a liver injured by a metabolic defect, hereditary tyrosinemia. This form of therapy is therefore applicable to liver diseases in which the defect is cell-autonomous (e.g., many of the childhood metabolic liver diseases). In fact, recent studies have shown that hepatocyte transplantation may be effective in the treatment of type I Crigler-Najjar syndrome. There is reason to believe that cell transplantation strategies also can be used for injuries in other organs. Such studies will need to address the changes that occur in the diseased tissues, whether it is regenerative signals that permit cell replication, and how the cells that are to be transplanted can be optimally manipulated ex vivo (or in vivo after transplantation) using state-of-the-art genetic and molecular techniques.

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Chemical chaperones.

The use of chemical chaperones for chemoprophylaxis of metabolic liver disease also is deserving of more detailed study. This class of compounds, which includes glycerol, trimethylamine oxide, deuterated water and 4-phenylbutyric acid (PBA), has shown to reverse the cellular mislocalization or misfolding of mutant membrane and lysosomal, nuclear, cytoplasmic and secretory proteins, including mutant CFTRΔF508 and ZZ α1-antitrypsin. In fact, PBA has been shown to have positive biochemical effects in an animal model of α1 -antitrypsin deficiency and in humans with cystic fibrosis. Recent studies have also suggested that competitive antagonists may have chaperone effects on mutant enzymes. One drug in this class, 1-deoxy-galactonojirimycin, a competitive antagonist of the lysosomal enzyme galactosidase A, mediates partial correction of the defect in localization of this enzyme in one type of Fabry disease. Recent studies have also shown that imino sugar compounds, which inhibit oligosaccharide side-chain trimming of glycoproteins, can partially reverse the mislocalization of mutant proteins such as ZZ α1-antitrypsin. Taken together, these compounds may have broad applicability in a variety of metabolic and genetic diseases of the liver, biliary tract, pancreas and GI tract, including α1-antitrypsin deficiency, cystic fibrosis, Wilson's disease, hemochromatosis, Gaucher's disease, Niemann-Pick disease and carbohydrate-deficient glycoprotein syndrome.

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Chimeric oligonucleotides.

The use of chimeric ribonucleic acid (RNA)/DNA oligonucleotides for the prevention of tissue injury in genetic diseases is deserving of further investigation. Recent studies have shown that chimeric RNA/DNA oligonucleotides, based on the sequence of coagulation factor IX complex with lactose so that it could be taken up by asialoglycoprotein receptor-mediated endocytosis, are delivered to hepatocytes with high efficiency after intravenous administration. Moreover, the oligonucleotide complexes mended the mutation in more than 90% of the liver parenchymal cells in an animal model of factor IX deficiency.

Studies are strongly recommended of genetic polymorphisms in drug metabolism, drug disposition, drug transporters and drug targets.

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Research Strategies

This goal will require not only continued innovative cell transplantation gene transfer and pharmacology research, but also continued basic research. The basic research will focus on the developmental biology of the GI tract and liver, the biology of tissue response to injury and the biology of tissue regeneration. Presumably, the success of cell transplantation in treating genetic disease will depend on the development and differentiation of the cells to be transplanted, the ability to manipulate them ex vivo (and presumably in vivo after transplantation) using gene transduction techniques, the signals being generated by the response of the affected tissue to injury, and the need for the affected tissues to regenerate. Moreover, one would suspect that the response to injury and regeneration in the native tissue would depend on the developmental stage of the organ. Proteonomic techniques will be particularly useful for the identification of novel agonists and antagonists that bind to key cellular target molecules. This goal will also require large-scale, high-throughput screening methods to identify candidate drugs and genomic screens for the identification of polymorphisms in drug-metabolizing enzymes, transporters and targets.

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Projected Timetable and Funding Requirements

This goal will require intensive partnerships with industry; in particular large pharmaceutical companies.

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Study the Basic Biology of Intestinal, Hepatic and Pancreatic Development and Responses to Infection, Inflammation, Tissue Injury and Other Physiologic/Environmental Stressors (e.g., Intestinal Adaptation and Liver Regeneration)
Research Goals

An enhanced understanding is required of the molecules involved in development and differentiation of the GI, hepatobiliary and pancreatic systems. In addition, how these tissues specifically respond to conditions associated with the expression of clinical disease is required. The intestinal adaptation response and liver regenerative response to injury are examples of signal transduction systems that are fundamental to determining whether a host will develop clinical disease and how severely the subject will be affected.

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Research Strategies

Microarray and proteonomic techniques are particularly well suited to address this type of basic research. These state-of-the-art techniques, together with data from the ongoing genome projects, are likely to produce significant fundamental advances.

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Projected Timetable and Funding Requirements

This requires immediate action. Investigator-initiated grants should be used, but sponsorship by industry for this type of research also should be sought.

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Develop Tools for Diagnostic and Population Biology Issues (e.g., Screening Programs)
Research Goals

A combination of programs is recommended, including a basic research program designed to identify new methods for diagnostic and population screening assays and a clinical research program in which these assays can be applied. There are many potential applications of such methods, including:

* Diagnostic tests for GI diseases in which the diagnosis currently involves invasive procedures or sophisticated equipment and technology, such as celiac disease, glycogen storage diseases, hereditary fructosemia and disorders of fatty acid oxidation

* Diagnostic tests that can be used in large populations for screening programs and population biology research

* Diagnostic tests to identify markers of tissue injury, such as fibrosis, cirrhosis, graft rejection and risk for cancer

* Assays for modifying genes

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Research Strategies

This goal will require basic research to develop diagnostic tools and methods based on DNA, microarray and proteonomic techniques. For example, in cystic fibrosis, genome scanning methods could be used to study the inheritance of microsatellite markers in multigenerational kindreds. The resulting linkage data could lead to identification of additional modifying genes. This goal will also require applied research in the design of screening studies, such as have been undertaken for Tay-Sachs disease and α1-antitrypsin deficiency, and population biology studies.

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Projected Timetable and Funding Requirements

Immediate action is recommended via nationally funded peer-reviewed grant mechanisms. We also recommend partnerships with industry to develop diagnostic assays and sponsor screening studies. A planning committee should be convened to identify key areas and to plan large-scale multicenter collaborative efforts for the development of screening programs and population biology studies. For this effort, multiple NIH institutes as well as foundations should be approached for financial support.

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HEALTH AND ECONOMIC OUTCOMES

A greater understanding of the molecular basis of GI disease and improved methods for diagnosis, prophylaxis and treatment will have a major impact on the clinical outcome for affected children and on the associated costs of health care. This section of the Research Agenda has described the potential use of data from genome projects, as well as use of microarray and proteonomic techniques, to achieve these goals. How the recommended research programs will influence health outcome and cost of care is described in the following sections focusing on specific disorders and organ systems.

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© 2002 Lippincott Williams & Wilkins, Inc.

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