Pain and rheumatology: Thinking outside the joint

No abstract.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/61864/1/24326_ftp.pd

cDNA amplification by SMART-PCR and suppression subtractive hybridization (SSH)-PCR.

The comparison of two RNA populations that differ from the effects of a single-independent variable, such as a drug treatment or a specific genetic defect, can identify differences in the abundance of specific transcripts that vary in a population-dependent manner. There are a variety of methods for identifying differentially expressed genes, including microarray, SAGE, qRT-PCR, and DDGE. This protocol describes a potentially less sensitive yet relatively easy and cost-effective alternative that does not require prior knowledge of the transcriptomes under investigation and is particularly applicable when minimal levels of starting material, RNA, are available. RNA input can often be a limiting factor when analyzing RNA from, for example, rigorously purified blood cells. This protocol describes the use of SMART-PCR to amplify cDNA from sub-microgram levels of RNA. The amplified cDNA populations under comparison are then subjected to suppression subtractive hybridization (SSH-PCR), a technique that couples subtractive hybridization with suppression PCR to selectively amplify fragments of differentially expressed genes. The final products are cDNA populations enriched for significantly over-represented transcripts in either of the two input RNA preparations. These cDNA populations may then be cloned to make subtracted cDNA libraries and/or used as probes to screen subtracted cDNA, global cDNA, or genomic DNA libraries

Somatization and psychological distress among women with vulvar vestibulitis syndrome

ObjectiveTo investigate the distribution of psychological characteristics and pain reporting among women with vulvar vestibulitis syndrome (VVS).MethodsIn this exploratory study, 109 women with VVS completed a battery of questionnaires to assess pain with intercourse and psychological characteristics (e.g. somatization, anxiety, distress). The distribution of these characteristics was compared, first with a conventional binary classification schema (primary and secondary) and subsequently with a 3â category schema (primary, latent primary, secondary).ResultsSeverity of pain with intercourse did not differ among the subgroups using either classification schema. Women with primary VVS consistently showed higher levels of somatization, anxiety, and distress compared with those with secondary VVS. Using a 3â tiered classification system, we found no difference between latent primary diagnosis and the other 2 groups (primary and secondary).ConclusionThis study highlights the critical need for research on subtype definition and the role of psychological factors in VVS.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/135448/1/ijgo38.pd

Painful Temporomandibular Disorder: Decade of Discovery from OPPERA Studies

In 2006, the OPPERA project (Orofacial Pain: Prospective Evaluation and Risk Assessment) set out to identify risk factors for development of painful temporomandibular disorder (TMD). A decade later, this review summarizes its key findings. At 4 US study sites, OPPERA recruited and examined 3,258 community-based TMD-free adults assessing genetic and phenotypic measures of biological, psychosocial, clinical, and health status characteristics. During follow-up, 4% of participants per annum developed clinically verified TMD, although that was a “symptom iceberg” when compared with the 19% annual rate of facial pain symptoms. The most influential predictors of clinical TMD were simple checklists of comorbid health conditions and nonpainful orofacial symptoms. Self-reports of jaw parafunction were markedly stronger predictors than corresponding examiner assessments. The strongest psychosocial predictor was frequency of somatic symptoms, although not somatic reactivity. Pressure pain thresholds measured at cranial sites only weakly predicted incident TMD yet were strongly associated with chronic TMD, cross-sectionally, in OPPERA’s separate case-control study. The puzzle was resolved in OPPERA’s nested case-control study where repeated measures of pressure pain thresholds revealed fluctuation that coincided with TMD’s onset, persistence, and recovery but did not predict its incidence. The nested case-control study likewise furnished novel evidence that deteriorating sleep quality predicted TMD incidence. Three hundred genes were investigated, implicating 6 single-nucleotide polymorphisms (SNPs) as risk factors for chronic TMD, while another 6 SNPs were associated with intermediate phenotypes for TMD. One study identified a serotonergic pathway in which multiple SNPs influenced risk of chronic TMD. Two other studies investigating gene-environment interactions found that effects of stress on pain were modified by variation in the gene encoding catechol O-methyltransferase. Lessons learned from OPPERA have verified some implicated risk factors for TMD and refuted others, redirecting our thinking. Now it is time to apply those lessons to studies investigating treatment and prevention of TMD

Structural Mechanism of S-Adenosyl Methionine Binding to Catechol O-Methyltransferase

Methyltransferases possess a homologous domain that requires both a divalent metal cation and S-adenosyl-L-methionine (SAM) to catalyze its reactions. The kinetics of several methyltransferases has been well characterized; however, the details regarding their structural mechanisms have remained unclear to date. Using catechol O-methyltransferase (COMT) as a model, we perform discrete molecular dynamics and computational docking simulations to elucidate the initial stages of cofactor binding. We find that COMT binds SAM via an induced-fit mechanism, where SAM adopts a different docking pose in the absence of metal and substrate in comparison to the holoenzyme. Flexible modeling of the active site side-chains is essential for observing the lowest energy state in the apoenzyme; rigid docking tools are unable to recapitulate the pose unless the appropriate side-chain conformations are given a priori. From our docking results, we hypothesize that the metal reorients SAM in a conformation suitable for donating its methyl substituent to the recipient ligand. The proposed mechanism enables a general understanding of how divalent metal cations contribute to methyltransferase function

Haplotype associations with quantitative traits in the presence of complex multilocus and heterogeneous effects

In genetic mapping of complex traits, scored haplotypes are likely to represent only a subset of all causal polymorphisms. At the extreme of this scenario, observed polymorphisms are not themselves functional, and only linked to causal ones via linkage disequilibrium (LD). We will demonstrate that due to such incomplete knowledge regarding the underlying genetic mechanism, the variance of a trait may become different between the scored haplotypes. Thus, unequal variances between haplotypes may be indicative of additional functional polymorphisms affecting the trait. Methods accounting for such haplotype-specific variance may also provide an increased power to detect complex associations. We suggest ways to estimate and test these haplotypic variance contrasts, and incorporate them into the haplotypic tests for association. We further extend this approach to data with unknown gametic phase via likelihood-based simultaneous estimation of haplotypic effects and their frequencies. We find our approach to provide additional power, especially under the following types of models: (a) where scored and unobserved variants are epistatically interacting with each other; and (b) under heterogeneity models, where multiple unobserved mutations are linked to nonfunctional observed polymorphisms via LD. An illustrative example of usefulness of the method is discussed, utilizing analysis of multilocus effects within the catechol-O-methyl transferase (COMT) gene