Depending on clinical characteristics, population frequency, and methods of measurement for specific trait(s), research designs may be either observational or
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(c) son caugiroi cssuo^ior sen experimental (Table 4.1). Experimental methods can be employed to study groups of individuals (e.g. clinical trial, cohort),18 animal models (e.g. knock-out, knock-in, knock-down, knock-up, transgenic animal models),19 cells (e.g. cell culture),20 or specific proteins (e.g. biochemical assay). Observational methods usually involve groups of individuals (e.g. case-control, cohort, family-based).5'18 Given the uncertainty surrounding the accuracy of characterizing a trait in terms of measurement of the phenotype (e.g. self-report of pain intensity), a combination of approaches is often pursued. For example, animal models are used to study the pathophysiology of a pain condition and to identify potential genes and pathways. These findings are then used to guide the design and execution of studies in humans.
Epidemiology is the study of factors that contribute to health and disease in the population with the goal of disease prevention.18 Genetic epidemiology focuses on the identification of genetic and environmental risk factors that predispose individuals in families and populations to disease.5 Genetic epidemiology generally follows three steps:
1. providing evidence that a genetic component exists for the trait of interest;
2. estimating the relative size of said genetic component in relation to other factors that influence the trait (e.g. environmental factors, other genetic factors);
3. identifying the gene(s) that underlie the genetic component of the disease.
These three steps can be pursued by either population studies (termed association studies) or family studies which can involve genetic risk, segregation, linkage, or association analyses.
Genetic studies, step 1: evidence of heritability
Genetic risk studies evaluate the contribution of genetics to a trait as compared to the environment and are pursued by family-based, twin, or adoption studies.21 Twin studies compare the degree to which monozygotic twins (who share 100 percent of their genes) are concordant for a specific trait as compared to dizygotic twins (who share 50 percent of their genes in common). The greater the similarity between monozygotic twins compared to dizygotic twins, the greater the evidence of a heritable component to a medical condition.
Genetic studies, step 2: estimating the pattern of inheritance
Segregation analysis employs multigenerational families to fit a model for the genetic component of interest (e.g. autosomal recessive, X-linked, or environmental (no evidence of genetic component)).21 It is most useful in Mendelian disorders. It is a less powerful technique in the analysis of complex traits when multiple genes each with multiple alleles are involved. Segregation analysis is a prerequisite for parametric linkage analyses.
Genetic studies, step 3: identifying genes that cosegregate with a phenotype
Initially, linkage studies can be used in a hypothesis-based evaluation of the cosegregation of gene variations in "candidate genes'' with a trait.22 A candidate gene is defined as a gene that may contribute to a trait based on
Figure 4.5 A pedigree is a diagram of a family's members and their relationship to the member who was identified as having a genetic trait of interest (the proband). Typically, three generations or more are collected by a medical geneticist, advanced practice nurse, genetic counselor, or geneticist. (a) A pedigree of autosomal recessive inheritance. In this example, two unaffected parents each carry one copy of a gene mutation (they are each heterozygous) for an autosomal recessive disorder. They have one affected child (homozygous) and three unaffected children, two of which carry one copy of the gene mutation (again, they are heterozygous). Each offspring's risk of receiving a recessive allele is one half from each parent. Therefore, each offspring of two carriers has a 25 percent chance of being affected, a 50 percent of chance of being a carrier, and a 25 percent chance of inheriting neither mutant allele. Both genders are equally likely to be affected. (b) A pedigree of autosomal dominant inheritance. In this example, a man with an autosomal dominant disorder has two affected children and two unaffected children. In a typical autosomal dominant inheritance, every affected individual in a pedigree has an affected parent, who also has an affected parent. It also affects several generations. Both sexes are equally likely to be affected and a male-to-male transmission exists. Each offspring of an affected parent has a 50 percent chance of being affected. (c) A pedigree of X-linked recessive inheritance. In this example, an unaffected woman carries one copy of a gene mutation for an X-linked recessive disorder. She has an affected son, an unaffected daughter who carries one copy of the mutation, and two unaffected children who do not have the mutation. There is no male-to-male transmission. All daughters of an affected male are carriers. Sons of a carrier mother have a 50 percent chance of being affected. Daughters of a carrier mother have a 50 percent chance of being carriers. Reprinted from US National Library of Medicine. Handbook: Help Me Understand Genetics. Bethesda, MD, USA: US National Library of Medicine. Available from: http://ghr.nlm.nih.gov/handbook/inheritance.
Table 4.1 Examples of pain research that used different study designs.
Experimental Clinical trial Hudcova et al.6 To conduct a meta-analysis to evaluate the efficacy of patient-controlled studies analgesia (PCA) versus conventional analgesia (e.g. a nurse administering an analgesic upon a patient's request) for postoperative pain control
Huas et al.7 To evaluate the impact of using pain assessment scales on the management of musculoskeletal chronic pain employing a cluster-randomized controlled multicenter trial with practices randomized by region before patient recruitment
Linde et al.8 To compare patient characteristics and outcomes between a randomized controlled trial and an observational study of acupuncture treatment in patients with migraine
Cohort Ives et al.9 To estimate the incidence and risk factors for opioid misuse in patients with chronic pain in a prospective cohort study of patients enrolled in a chronic pain disease management program within an academic internal medicine practice
Animal model Lichtman et al.10 To test whether anandamide and other non-cannabinoid fatty amides modulate nociception in a fatty acid amide hydrolase FAAH deficient mouse model as compared to FAAH positive mice employing a series of tests (i.e. tail immersion, hot plate, formalin tests, thermal hyperalgesia in the carrageenan, chronic constriction injury) Al-Khrasani, et al.11 To examine the antinociceptive effects of peripheral micro-opioid receptor agonists (e.g. 14-O-methyloxymorphone, DAMGO and morphine) were evaluated in a mouse model of visceral pain In vitro Huang et al.12 To understand whether the proton-sensing G-protein-coupled receptors
(PS-GPCR) are expressed in nociceptors, four PS-GPCR (i.e. OGR1, GPR4, G2A, TDAG8) were cloned, their tissue distribution examined, and their localization in pain-relevant loci (i.e. the dorsal root ganglion) determined
Observational Case-control Kang et al.13 To investigate the association between an estrogen receptor alpha studies polymorphism and pain susceptibility in a case-control study of female symptomatic temporomandibular joint osteoarthritis Cohort Diatchenko et al.14 In order to identify genes that contribute to interindividual variability on pain sensitivity a cohort of healthy pain-free females were assessed for pain perception thresholds and genotyped for candidate genes Gansky et al.15 To assess the distribution of widespread pain, tender points, and fibromyalgia in young African American and Caucasian women in a community population of young women Family-based Indo et al.16 To identify mutations in the nerve growth factor receptor tyrosine kinase
(NTRK1) that underlie congenital insensitivity to pain in nine families Animal model Chesler et al.17 To examine the heritability of sensitivity to analgesia from gabapentin and pregabalin as a precursor to linkage mapping efforts, 11 inbred mouse strains were tested for inhibition of nociception by gabapentin or pregabalin in two different preclinical assays of inflammatory pain (i.e. formalin test, zymosan thermal hyperalgesia on the paw-withdrawal test)
the biochemical properties of its gene product. Alternatively, one can identify genes in a hypothesis-free manner by screening a set of genetic markers that span the entire genome or discrete chromosomal segments. Linkage studies attempt to locate gene(s) that underlie the trait(s) of interest.5 Linkage approaches can be used in any organism for which pedigrees can be collected and in which a genetic marker set of sufficient density is available (e.g. human, mouse,23 dog,24 fruit fly,25'26 zebrafish27'28) to interrogate the genome.
Linkage analysis examines the cosegregation of genetic markers (e.g. SNPs) within families whereas association is meant to provide information on the involvement of specific alleles in a trait of interest in a group of unrelated individuals.21 One potential weakness of association studies is the fact that cryptic relatedness or population substructure (e.g. self-reported ethnicity may not adequately capture different subpopulations that may self-identify as the same ethnicity and could result in chance differences in the proportions of such subpopulations between cases and controls) thereby confounding the results. Although association studies are susceptible to confounding due to differences in population substructure, they have several advantages over linkage analysis, including:
• the need to recruit multigenerational families where the contribution of family members to the analysis is difficult to ascertain a priori is obviated;
• the power to detect alleles with weak effects, and the ability to provide estimates of the relative magnitude of the effects of multiple alleles.
Several variations and mixtures of the above-mentioned study designs and statistical genetic analyses exist, but are beyond the scope of this chapter. For an in-depth review of these methods and analyses, the reader is directed to Analysis of human genetic linkage.29 An in-depth exploration of the use of murine models is provided by Lee Silver.30 An excellent description of the use of mammalian models in the study of the genetics of pain is available.23
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