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Pediatr Emerg Care. Author manuscript; available in PMC 2007 November 19. Published in final edited form as: | PMCID: PMC2080853 NIHMSID: NIHMS33892 |
Understanding Genomics: Implications for the Emergency Medicine Physician and the Treatment of Asthma Robert J. Freishtat, MD, MPH* and Stephen J. Teach, MD, MPH† * Assistant Professor of Pediatrics and Emergency Medicine, Division of Emergency Medicine, Children’s National Medical Center, Washington, DC † Associate Chief of Emergency Medicine, Children’s National Medical Center, Washington, DC TARGET AUDIENCE Physicians, nurse practitioners, and physician assistants who evaluate and care for children with minor illnesses. Specialists including pediatricians, emergency physicians, pediatric emergency physicians, family practitioners, and pediatric nurse practitioners will find this information particularly useful. LEARNING OBJECTIVES After completion of this article, the reader will be able to: - Explain the role of single-nucleotide polymorphisms and haplotypes in the development of asthma and asthma exacerbations.
- Describe what is currently known about how environmental influences interact with genotype to produce an asthmatic phenotype.
- Describe the 3 possible effects of a single-nucleotide polymorphism or haplotype on pharmacology in the context of asthma.
Keywords: gene, asthma, linkage, microarray In 2015, a 7-year-old African-American boy presents in your emergency department with a 2-day history of cough and wheeze. The symptoms began in the setting of a mild upper respiratory tract infection and were worsened by exercise. He is known to have chronic asthma as do his father and younger sister. His respiratory rate is 32 breaths per minute, and he is wheezing bilaterally and diffusely with moderate retractions. How will your evaluation and management begin? If recent trends in other fields are predictive, you will begin with a review of your patient’s genetic profile. Although that profile may guide a portion of or your entire approach, some facts about asthma will remain the same. It will still be a multifactorial chronic disorder, 1–3 highly prevalent among children and adolescents, 4,5 with a strong environmental component 1,2,4,6–8 and genetic predilection. 9–11 In addition, the pathophysiology will likely not have changed. As Sir William Osler wrote more than 100 years ago in The Principles and Practice of Medicine, “Asthma is a term which has been applied to various conditions associated with dyspnea. Of the numerous theories, the following are most important: (1) that it is due to spasm of bronchial muscles, (2) that the attack is due to swelling of the bronchial mucous membrane, (3) and that, in many cases, it is a special form of inflammation of the smaller bronchioles.” 12During the past century, our understanding of the pathophysiology of asthma has advanced slowly, without significant change in Sir Osler’s basic paradigm. Now, however, with a completed “map” of the human genome in hand, new developments in the field of asthma pathobiology are arriving more quickly and often involve the identification of specific “asthma genes.” Some of these asthma genes hold promise as potential predictors of the clinical variability of disease and even response to therapy. The advanced technologies used to make these discoveries will continue to slowly find their way into clinical practice. PRIMER ON THE NEW GENOMICS DNA as the Precursor of Phenotype Genomic DNA, the genotype, is the chemical code for the assembly of protein via a RNA intermediate. DNA is a long series of triplets of various combinations of 4 nucleotides (adenine, thymine, cytosine, and guanine). Each triplet, a codon, is transcribed into RNA and then translated into 1 of 22 amino acids that are ultimately assembled to form proteins. DNA is the basis for the hereditary passage of traits from parent to child, but misperceptions persist that a given genotype directly leads to a given phenotype. In reality, there are 3 components to phenotypic development: genotype, environmental influences, and the interaction between the two. This is the basis for the multifactorial etiology for asthma and other similarly complex diseases, which greatly complicates their study. Mutations Versus Single-Nucleotide Polymorphisms Phenotypic differences between individuals stemming solely from genotypic changes are attributed to spontaneous alterations or deletions in one or more base pairs of DNA. When one of these changes can be found in only 1% or less of the population and when it is deterministic (ie, necessarily results in phenotypic modification), it can be classified as a mutation. As causative of phenotypic change, a mutation must occur in coding regions (exons) of DNA. Because of the close relationship of the mutation with the disease, family pedigrees and chromosomal analysis allow for fairly routine identification of the causative genetic error. Far more frequently, alterations in a single nucleotide occur in more than 1% of the population and cannot be identified as singly causative of any illness or trait. Unlike mutations, these changes can be found throughout the human genome, both in coding (exons) and noncoding (introns) regions of DNA. These are called single-nucleotide polymorphisms (SNPs), and because of their distribution throughout the genome, they are probabilistic (ie, do not always lead to phenotypic variation). For example, the nucleotide change may occur in a noncoding region of DNA or may not ultimately alter the translated amino acid because multiple codons can denote a single amino acid (). However, many of more than 3 million recorded SNPs do have known or suspected functionality in various diseases, including several for asthma. 13Overall, SNPs are relatively frequent, occurring once every 300 base pairs. 13 To date, there are estimated to be approximately 11 million SNPs in the approximately 3 billion base pairs of the human genome, making them the most common form of human genetic variation. 14 Their frequency, however, is variable according to race and ethnicity. 15 Evidence suggests that approximately half of SNPs are silent, meaning that there is ultimately no change in the amino acid coded for by the SNP-containing codon. 16 In addition, most SNPs in humans are unequally distributed through the genome. This is the notion of linkage disequilibrium. This concept derives from the tendency for closely approximated SNPs in one region of a gene to remain clustered together over many generations because their close proximity decreases their chances of being separated when genes recombine during fertilization. Each variant of these clusters (eg, A ATGGT GT vs A CTGGT AT) is called a haplotype and represents one possible combination of SNPs in a region of a gene. Thus, although there are 3 million identified SNPs, because of the frequency of linkage disequilibrium, there are only 300,000 identified haplotypes. 13Genomewide Linkage Analysis and Association Studies The standard theory underlying the asthmatic phenotype is that many genes, both beneficial and detrimental, interact with each other and the environment, ultimately leading to one of several asthmatic phenotypes. To better understand the contribution of specific genes toward the development and response to treatment of asthma, researchers can undertake linkage analysis studies, which are essentially pedigree studies looking at affected and unaffected family members. The affected family members’ genotypes are examined for markers that track through their pedigree but do not track through that of the unaffected members. Log-of-the-odds scores determine which markers (ie, SNPs and/or haplotypes) are most likely to cluster among affected members, and in turn, those markers may be located proximally to genes that are important in asthma pathogenesis. After the identification of potentially biologically significant candidate gene segments, an association study (essentially a case-control investigation) can be used to determine the specific shared DNA regions among affected individuals. In this type of study, matched asthmatic and healthy controls are compared for prevalence of specific SNPs and haplotypes. A more prevalent SNP or haplotype in the asthmatics suggests a higher risk of asthma among patients with that genotype. Using these techniques, there are now several successful genomewide linkage studies for asthma that have identified a number of chromosomal regions as containing possible candidate genes for roles in asthma pathogenesis, the so-called “asthma genes.” 7,17,18 These include chromosome 5q, chromosome 11q (β chain of Fc εRI), chromosome 12q (interferon γ, STAT-6), and chromosome 16p (interleukin 4R). 19Incorporation of Environmental Factors Although these genomic advances have identified potential “asthma genes,” environmental influences will continue to interact with these genotypes to determine the pathogenesis of asthma. Because patients and their environmental exposures vary widely, these interactions have been difficult to study. However, a recent publication identified 3 regions of DNA in a genomewide linkage analysis study that may contain candidate genes for the development of asthma in patients with environmental tobacco smoke exposure: regions of chromosome 1p, chromosome 5q, and chromosome 9q. 6 In addition, other genes with potential interaction with tobacco smoking and risk for asthma exacerbations have been identified in candidate gene linkage approaches: 20 interleukin 10, 21 interleukin 1β, 22 and matrix metalloproteinases 1 and 12. 23RNA as the Mediator of Phenotype: Microarray Technology Although DNA holds the information for cellular construction and processes, it is RNA that mediates the transition between DNA and proteins, where phenotype is expressed. As such, the study of RNA expression using nucleic acid sequence microarrays made them the first genomewide high-throughput genomic technologies to undergo widespread use by scientists in many areas of biology. The basis for microarray technology is that messenger RNA (mRNA) from study tissue is reverse transcribed into complementary DNA and then transcribed back into fluorescently labeled complementary RNA before being hybridized (ie, annealed by a chemical- and heat-induced reaction) to millions of pieces of RNA (oligonucleotide probe sets) which have been bound onto a glass slide in an array pattern. The degree of hybridization is measured by a signal detection apparatus and computer software allowing for the calculation of the original relative concentration of mRNA in the study tissue (). These genome-anchored arrays are available for many species, including human, mouse, and rat, enabling their use in animal models of asthma. The expense of preparation and analysis of microarrays has prompted a cooperative approach to data resulting from these studies. Most journal-published microarray data are Web-accessible, and much of it searchable and/or analyzable on the Web by researchers without access to specialized equipment in their laboratories. This has been the case since the Minimal Information About a Microarray Experiment guidelines ( http://www.mged.org) were presented in 2001. 24 An example of this is the Public Expression Profiling Resource portal ( http://pepr.cnmcresearch.org/home.do) of the Research Center for Genetic Medicine at Children’s National Medical Center in Washington, DC. It allows any internet user to analyze data within our laboratory’s database which represents the largest single center data set of microarrays in the United States. This data set encompasses many diseases, including arrays from humans with and animal models of asthma. 25,26 Two other large publicly available databases are the NCBI GEO Web site ( http://www.ncbi.nih.gov/geo) and the ArrayExpress site ( http://www.ebi.ac.uk/arrayexpress). The utility of microarray studies is readily apparent from a quick review of the literature 27,28 with the activation of the genes for arginase 16,29–31 and amphiregulin, a profibrotic, 32 in asthma identified by microarray studies. Microarrays can be especially effective when used as a hypothesis-generation tool along with novel software that incorporates the National Library of Medicine PubMed database of abstracts into microarray experimental analysis. Each gene transcript can be used much like another independent variable in these often longitudinal cohort studies. However, the importance of quality control and standard operating procedures are likely unsurpassed as small procedural errors can cause very significant changes in results. 33 Because of this, it is accepted practice to verify microarray findings with polymerase chain reaction of mRNA for select transcripts of interest. This technique has higher specificity. FUTURE DIRECTIONS: IMPLICATIONS FOR ASTHMA EVALUATION AND MANAGEMENT Polymorphisms in Asthma Earlier in this article, we touched on some “asthma genes” that may contribute to an individual’s risk for developing asthma and for incurring exacerbations. This has become an intense area of research. A search of the National Institutes of Health CRISP database showed 2004 studies with keywords of asthma and gene out of 7787 asthma studies in the prior 10 years. 34 Many of these are linkage analysis and association studies aimed at finding genes responsible in part for asthma risk. There are also quite a few microarray-based investigations targeted at determining novel pathways for asthma disease and exacerbation development. A possibly more compelling direction for asthma research stems from the concept that SNPs may alter the function of receptors important in the treatment of asthma. In doing so, SNPs can contribute to changes in the response to therapeutics in several ways. First, they can alter the metabolism of a drug (ie, pharmacokinetic effect). For example, HIV-positive children with an SNP in the multidrug-resistance transporter gene ( MDR1) had higher plasma levels of nelfinavir in the highly active antiretroviral therapy regimen. 35 Second, SNPs can cause an adverse reaction in certain patients. A prime example of this is the HIV drug, abacavir, hypersensitivity. This reaction has been found to be 29 times more likely with a particular SNP in HLA B. 36–42 Last, SNPs can alter the response to a drug at its target (ie, variant pharmacodynamic effects). This is most important in the concept of pharmacogenomics, where drug therapy is tailored toward the genotype of the patient. Although futuristic in some ways, pharmacogenomics is routinely practiced in oncology: cancers are genotyped and treated according to those results. With regard to asthma, pharmacogenomics is very much in its infancy. That said, there is promise that, someday, practitioners may be able to tailor their asthma medication regimens to a patient’s genotype. For example, SNPs and haplotypes in the β 2-agonist receptor gene are suspected to explain some of the variability in therapeutic response seen among asthmatics. 43–45 These findings bear further confirmation, but it is reasonable to suspect that there would be some culpability for genotype in this variability. In addition, SNPs are suspected to contribute to alterations in leukotriene metabolism 46,47 and glucocorticoid responsiveness. 48,49Despite what is likely to be a rapidly growing list of SNPs associated with asthma treatment efficacy, pharmacogenomics will require individuals’ genotypes to be available to direct clinicians as to how to alter therapy for patients whose physiology varies because of these SNPs. The technology for high-throughput genotyping is readily available in many centers; however, and its cost is decreasing. For example, in our laboratory, we can genotype 4000 samples in 1 day at approximately US$1 per sample (not including the initial cost of the sequencer). It is likely that chronically ill patients will be the first to benefit from this technology as they can be genotyped in their outpatient clinic and then that data would be available when they present acutely ill to the emergency department. The technology must improve to obtain real-time results (ie, in fewer than several hours) to impact not previously genotyped patients as they present to the emergency department. Even so, studies will need to be done to determine how to modify current treatments to most effectively treat patients with variant pharmacodynamics. In 2015, the 7-year-old chronic asthmatic mentioned at the beginning of the article has some genotype information known at the time of his presentation—it was collected at a pulmonary clinic visit the previous year after an ICU admission. Much like a history of a smoker at home, his parent may report that he has the “steroid gene” that you know means he carries a haplotype in the gene for one of his glucocorticoid receptors that makes him less sensitive to glucocorticoid treatment and thus requires higher doses and longer treatment. You adjust your therapy accordingly. This patient scenario becomes closer to reality each year as genomic technologies rapidly mature. The role of environment in the asthma epidemic cannot be discounted—its role is clear and important. However, despite the long-held knowledge that genes are important in asthma as well, the study of genetics in asthma had been limited by the lack of a means by which to examine it. Relatively, little had changed since Sir William Osler wrote about asthma 100 years ago. Now, we are on the verge of significant change. The authors thank Eric P. Hoffman, PhD, for his input during the preparation of this manuscript. 1. Brugge D, Vallarino J, Ascolillo L, et al. 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