The genetic background of inflammatory bowel disease

Available evidence indicates that genetic factors are essential in providing the susceptibility to the majority of the various forms of inflammatory bowel disease occurring in man. It is also clear that the genetic susceptibility to these diseases is complex, and that more than one gene may predispose (the concept of multilocus/oligogenic inheritance), and likely in different etiologic combinations (the concept of genetic heterogeneity). Paradigms are now available that should lead to the identification of a number of these predisposing genes. These paradigms include the candidate gene approach, systematic genome wide scans, and mouse human synteny. While genome wide scans are currently limited to multiplex family linkage studies, both candidate genes and mouse human synteny can be approached in either linkage or association paradigms. Eventually whole genome association studies will be available as well. Identification of inflammatory bowel disease predisposing genes should lead to their incorporation in studies of natural history, investigation of environmental risk factors, and especially utilization of genetic markers in clinical trials. This will allow us to identify the best therapy available for the individual patient based on their unique genetic constitution. With advances in molecular technology, the search for genes influencing traits and diseases with a complex genetic background, such as the inflammatory bowel diseases, has become a realistic task. Although exogenous or infectious agents may contribute to the pathogenesis or may trigger the onset of disease, and the immune system almost certainly mediates the tissue damage, it is clear from available data that genetic factors determine the susceptibility of a given individual to inflammatory bowel disease (reviewed below). Thus, genetic studies are essential for the delineation of the basic etiologies of the various forms of inflammatory bowel disease and thus can aid in the development of radically new and specific therapies. In this review, we will discuss the importance and complexity of genetic factors in inflammatory bowel disease, methods and problems in the genetic dissection of complex traits, and future directions of genetic studies in inflammatory bowel disease.     

Applications of molecular genetics to gastrointestinal and liver diseases. I. Technical approaches

Recent developments in recombinant DNA techniques have allowed an understanding of the molecular genetics of many diseases, some affecting the gastrointestinal tract and liver. DNA probes which detect sequences within or near disease genes can be selected by direct approaches, if the gene product or primary gene function is known, or by indirect methods when the chromosomal location is known. Such probes have resulted in extensive family studies which can now define risks to family members of developing a genetic disease. The development of the polymerase chain reaction will also be of considerable use in clinical genetics and in the diagnosis of some infectious diseases. The techniques are summarized and examples of their use are given. A glossary of terms is also provided.     

Precise gene localization by phenotypic assay of radiation hybrid cells

A high resolution map of the human genome previously has been constructed by using the G3 panel of human/hamster radiation hybrid cell lines and >15,000 unique human genetic markers. By determining whether human DNA sequences are present or absent in each of the hybrids, localization of single genes may routinely be achieved at approximately 250-kb resolution. In this paper we have tested whether similarly precise localization might be achieved by phenotypic screening of the hybrids to facilitate positional cloning of unknown genes. We assayed the susceptibility of each of the hybrid cell lines to transduction by retroviral vectors bearing different retroviral envelope proteins that recognize receptors present on human but not on hamster cells. The results for each of the retroviral vectors were informative and allowed precise localization of the receptor genes for the RD114 cat endogenous retrovirus, xenotropic murine leukemia virus, and type C feline leukemia virus. After cloning of the receptors for these retroviruses, we found that standard genotypic mapping by PCR gave results that were nearly identical to those from phenotypic mapping. These experiments show that precise gene localization by phenotypic assay of radiation hybrids is practical and was not appreciably impacted by the known instability of such hybrid cells. This technique should be applicable to many other human genes having discernible phenotypes in hamster cells and, with completion of the human genome project, will allow rapid identification of unknown genes on the basis of phenotype.     

Modifier genes and sickle cell anemia

PURPOSE OF REVIEW: With the completion of the human genome project and HapMap, previously unknown genetic polymorphisms associated with disease have been observed. This review highlights genetic polymorphisms that have provided insight into the pathophysiology underlying the many phenotypes of sickle cell disease. RECENT FINDINGS: The phenotypes of sickle cell disease are likely to be modulated by polymorphisms in genes that are involved in inflammation, cell-cell interaction, and nitric oxide biology. Case-control studies are beginning to define the relationships between single-nucleotide polymorphisms in candidate genes and the many subphenotypes of sickle cell anemia. A common theme emerging from these studies is that single-nucleotide polymorphisms in genes of the transforming growth factor-beta/bone morphogenetic protein and a few other genes such as Klotho are associated with several subphenotypes of sickle cell disease. SUMMARY: Genomic medicine is merging with clinical practice as our understanding of the structure and variability of the human genome increases. Patients with diseases caused by identical mutations in a single gene - sickle cell anemia is a prime example - can have clinical courses very different from one another, and when environmental influences are removed the phenotypic heterogeneity of mendelian single-gene disorders is best explained by single-nucleotide polymorphisms in genes that modulate the disease phenotype. As this field expands, insights will be gained into complex epistatic factors that influence the clinical presentation of sickle cell disease, enabling physicians to better predict and manage the many complications of this disease.     

Clinical implications of omics and systems medicine: focus on predictive and individualized treatment

Many patients with common diseases do not respond to treatment. This is a key challenge to modern health care, which causes both suffering and enormous costs. One important reason for the lack of treatment response is that common diseases are associated with altered interactions between thousands of genes, in combinations that differ between subgroups of patients who do or do not respond to a given treatment. Such subgroups, or even distinct disease entities, have been described recently in asthma, diabetes, autoimmune diseases and cancer. High‐throughput techniques (omics) allow identification and characterization of such subgroups or entities. This may have important clinical implications, such as identification of diagnostic markers for individualized medicine, as well as new therapeutic targets for patients who do not respond to existing drugs. For example, whole‐genome sequencing may be applied to more accurately guide treatment of neurodevelopmental diseases, or to identify drugs specifically targeting mutated genes in cancer. A study published in 2015 showed that 28% of hepatocellular carcinomas contained mutated genes that potentially could be targeted by drugs already approved by the US Food and Drug Administration. A translational study, which is described in detail, showed how combined omics, computational, functional and clinical studies could identify and validate a novel diagnostic and therapeutic candidate gene in allergy. Another important clinical implication is the identification of potential diagnostic markers and therapeutic targets for predictive and preventative medicine. By combining computational and experimental methods, early disease regulators may be identified and potentially used to predict and treat disease before it becomes symptomatic. Systems medicine is an emerging discipline, which may contribute to such developments through combining omics with computational, functional and clinical studies. The aims of this review are to provide a brief introduction to systems medicine and discuss how it may contribute to the clinical implementation of individualized treatment, using clinically relevant examples.     

Applications of CRISPR systems in respiratory health: Entering a new ‘red pen’ era in genome editing

Respiratory diseases, such as influenza infection, acute tracheal bronchitis, pneumonia, tuberculosis, chronic obstructive pulmonary disease, asthma, lung cancer and nasopharyngeal carcinoma, continue to significantly impact human health. Diseases of the lung and respiratory tract are influenced by environmental conditions and socio‐economic factors; however, many of these serious respiratory disorders are also rooted in genetic or epigenetic causes. Clustered regularly interspaced palindromic repeats (CRISPR) and CRISPR‐associated (Cas) proteins, isolated from the immune system of prokaryotes, provide a tool to manipulate gene sequences and gene expression with significant implications for respiratory research. CRISPR/Cas systems allow preclinical modelling of causal factors involved in many respiratory diseases, providing new insights into their underlying mechanisms. CRISPR can also be used to screen for genes involved in respiratory processes, development and pathology, identifying novel disease drivers or drug targets. Finally, CRISPR/Cas systems can potentially correct genetic mutations and edit epigenetic marks that contribute to respiratory disorders, providing a form of personalized medicine that could be used in conjunction with other technologies such as stem cell reprogramming and transplantation. CRISPR gene editing is a young field of research, and concerns regarding its specificity, as well as the need for efficient and safe delivery methods, need to be addressed further. However, CRISPR/Cas systems represent a significant step forward for research and therapy in respiratory health, and it is likely we will see the breakthroughs generated from this technology continue.     

A Strategy for the Identification of Candidate Genes for Alcohol-Related Phenotypes and Other Human Disorders Using Rapid Polymerase Chain Reaction Mapping of Gene-Based Sequence-Tagged Sites

We describe a method for the rapid identification and mapping of human genes, including those possibly contributing to disease and alcohol-related phenotypes. New human genes are identified from cDNA libraries through single-pass sequencing into the 3' untranslated (3'UT) regions of human brain cDNAs. Primers derived from the 3'UT region sequences [representing gene-based, sequence-tagged sites (STSs)] are used for polymerase chain reaction (PCR) analyses of the CEPH megabase insert yeast artificial chromosome (YAC) DNA pools. With this approach, ∼18,000 megabase YACs can be screened and a single YAC identified using only 52 PCR reactions. The YAC localization in conjunction with other mapping techniques, such as PCR mapping to human chromosomes using somatic cell hybrids, allows identification of chromosomal band locations. In this manner, each gene can be associated with its own STS, which in turn specifies both a corresponding genomic clone and specific location in the genome. These locations can be compared with the purported locations of disease genes. The locations of the STSs can also be compared with those of Quantitative Trait Loci implicated for quantitative traits (e.g., alcohol-related phenotypes) on the basis of synteny between the mouse and human genes. Using this strategy, we found candidates for 78 human disease/syndrome genes among the first 220 genes mapped.     

Emerging infections and newly-recognised pathogens

Clinicians and microbiologists have for many years relied on growth and characterisation of micro-organisms in the laboratory as the major method for their detection and identification, but reliance upon microbial growth in the laboratory has probably significantly limited our ability to recognise important pathogenic micro-organisms. The traditional methods are often slow, non-specific and insensitive, and sometimes discriminate poorly among microbial species and strains. It is now known that the evolutionary ancestry and interrelationships of all living organisms can be reliably inferred from sequences in their genetic material. Highly conserved sequences characterise broad phylogenetic groups and variable sequences allow specific identification. Sequence-based methods combined with DNA amplification methods, such as the polymerase chain reaction (PCR), have led to powerful molecular identification techniques such as consensus nucleic acid amplification and representational difference analysis. These methods allow one to detect and isolate informative gene sequences from occult microbial pathogens in human tissues. Sequence-based methods are often quicker, more sensitive and more specific than traditional methods not only in detecting known microbial pathogens, but also in identifying previously-uncharacterised micro-organisms. Widespread, organised use of these methods will reveal new emerging microbial pathogens, implicate microbes in the aetiology of poorly-understood chronic inflammatory diseases and significantly expand our understanding of microbial diversity.     

Improved Methods for Extracting RNA from Exfoliated Human Colonocytes in Stool and RT-PCR Analysis

In order to diagnose colon cancer at an earlier, more localized stage, there is a need to develop diagnostic markers (genes) which can detect early patterns of gene expression in exfoliated colonocytes shed in the stool during routine screening for this disease. An RNA-based detection is more pertinent than either a DNA-based or a protein-based method as a screening procedure, but it has not been widely used as a cancer screen because of the difficulty of handling and stabilizing the RNA molecule. We describe a method that permits extraction of intact nondegraded total RNA from human colonocytes in stool and from normal and malignant colon tissues (which were employed for comparison with stool). Because it utilizes commercially available kits, this method is simpler than other published methods and does not require isolation of messenger (m)RNA, thereby reducing the chances of contaminating the preparations with degrading nucleases, and even a small amount of isolated total RNA can be adequately reverse transcribed, making high-quality copy (c) DNA. This is followed by PCR (either qualitative end point or semiquantitative real-time) using colon cancer-specific gene primers. By routinely and systematically being able to perform quantitative gene expression measurements on noninvasive samples, the goal of this pilot work is to lay the groundwork for conducting a large clinical study to identify groups of selected genes whose expression is consistently altered at an early stage in the neoplastic process. Such work will permit noninvasive monitoring of at-risk patients through the analysis of their stool samples. Correct diagnosis will allow for surgical and/or other interventions before the tumor is well established and, thus, should decrease mortality from this preventable disease.     

Gene therapy: Recombinant adeno-associated virus vectors

Gene transfer using recombinant adeno-associated virus (rAAV) vectors shows great promise for human gene therapy. The broad host range, low level of immune response, and longevity of gene expression observed with these vectors in numerous disease paradigms has enabled the initiation of a number of clinical trials using this gene delivery system. This review presents an overview of the current developments in the field of AAV-mediated gene delivery. Such developments include the establishment of new production methods allowing the generation of high titer preparations, improved purification methods, the use of alternative AAV serotypes, and the generation of trans-splicing rAAV genomes. Together, these developments have improved results interpretation, host range, and the coding capacity of rAAV vectors. Furthermore, the recent identification of regions within the viral capsid that are amenable to modification has begun to address the issue of direct rAAV vector targeting, which could potentially allow targeted gene delivery to specific cell populations. The versatility shown by this vector has enabled new diseases to be realistically considered for therapeutic intervention and considerably broadened the scope of gene therapy.     

Genetic effects on susceptibility,clinical expression,and treatment outcome of type 1 autoimmune hepatitis

Currently, three genetic factors have been short-listed as possible modulators of susceptibility and severity in type 1 AIH. They are female sex, HLA DRB alleles encoding lysine at position DR beta 71, and the CTLA4*G allele. The fourth association (i.e., TNFRSF6) remains to be confirmed. There are many other candidates to investigate. Current hypotheses suggest that the autoimmune genotype will include multiple (some linked, others discrete) loci which make a permissive background. Not all "at risk" individuals will develop clinical disease, and selection will depend on the interaction of this "permissive gene pool" (i.e., the host) with the environment. The resulting autoimmune phenotype will depend on gene dose and gene interaction. The human genome project has presented medical science with the challenge to identify the genes that determine common human diseases, including autoimmunity [1]. Although type 1 AIH is considerably less common than diabetes or RA, it may serve as a useful model for other autoimmune diseases. Diagnosis depends on histologic findings, and liver biopsy examinations are part of the usual assessment strategy in type 1 AIH. The availability of these tissue specimens provides a clear basis for monitoring disease progression and may permit investigators to study the impact of genetic polymorphism on disease activity. The emergence of high throughput technologies will significantly enhance our ability to study the interactions between constellations of polymorphic genes and both disease expression and behavior. An abundance of polymorphism is found in the genome. In many diseases, functional studies and genome scanning have helped revise and reduce the list of candidates. Affected families are rare in type 1 AIH, and patients are at risk if corticosteroid treatment is withheld. Under these circumstances, genetic studies may be the most practical, low risk means to investigate the pathogenesis of type 1 AIH and many other autoimmune diseases.     

Genomic medicine in Mexico. Applications of gene therapy for cirrhosis reversion

Genomic medicine represents a powerful armamentarium to tackle down most of chronic diseases which have not, so far, defeated. Thus, this new and powerful biotechnologic set of weapons enable us to make use of molecular diagnostic to detect silent diseases, otherwise undetectable by conventional analysis. Moreover, elucidation of the complete and final draft of the human genome code will allow, although not in this decade, the design of specific farmaco-genetic treatments for patients on basis of their individual genetic code. Regarding new medical treatments, gene therapy as emerged as a true hope for treatment of many chronic diseases. 636 FDA-.approved clinical protocols are currently undergoing and sooner than later we ll be witness of the results     

Hepatology in the 21st century. Gene transfer, hepatocyte transplantation, DNA chips, cyberspace and ... a friendly hospital

What to expect for hepatology in the 21st century? If science is allowed to proceed at its current rate, expectations can hardly be underestimated. Bound by the present day's limitations we are only able to see a glimpse of what could be available 100 years from now. For the next few decades, the global eradication of viral hepatitis will be on the agenda. For the treatment of inherited and acquired metabolic, toxic and immune liver disease, targeted drugs, genes and antisense oligonucleotides will be added to our therapeutic repertoire. The completion of the human genome project in 2003 will have far-reaching consequences: the widespread use of prenatal diagnosis, using DNA chip technology, may be expected to cause a dramatic decrease in the incidence of inherited diseases. Liver cirrhosis, hepatocellular carcinoma and inborn errors of metabolism may be treated by gene transfer or gene repair therapy. Although eventually these developments may decrease the need for organ transplantation, this by no means is the case yet and no solution is available for an increased demand and a decreased supply of organs. In the long run, diseases caused by multi-drug-resistant infectious agents and diseases associated with the abuse of alcohol and drugs are expected to become major problems. The future of university-based research is uncertain. The staggering costs of research and limited career possibilities may force universities to the limited task of higher education, with as a result biotech companies, shareholders and corporate finance ruling the scientific waves in the next century. The 21st century patient will know the way in cyberspace and will go shopping for the best doctor, for the best treatment and for the best, or friendliest, hospital.     

Epigenetik in der Rheumatologie

The human genome comprises approximately 30000 genes needed for the formation and function of approximately 1 Million proteins in the human body. Differentiation leads to the deactivation of genes that are not needed in the specific tissues or cells. To regulate the cell specific gene expression in normal cells epigenetic modifications work in concert with genetic mechanisms. In contrast to genetic mutations, epigenetics encompasses the wide range of heritable changes in gene expression that do not result from alteration in the DNA sequence itself. A dysregulation of epigenetic modifications results in diseases such as cancer or autoimmune diseases. Since these epigenetic modifications of the DNA and the histones are reversible they are good targets for novel therapeutic intervention.