The challenge for genetic epidemiologists: how to analyze large numbers of SNPs in relation to complex diseases

Background

Molecular genetic approaches have much to offer population biology. Despite recent advances, convenient techniques to develop and screen highly-resolving markers can be limiting for some applications and taxa. We describe an improved PCR-based, cloning-free, nuclear marker development procedure, in which single-stranded conformation polymorphism (SSCP) plays a central role. Sequence-variable alleles at putative nuclear loci are simultaneously identified and isolated from diploid tissues. Based on a multiple allele alignment, locus-specific primers are designed in conserved regions, minimizing 'null' alleles. Using two undescribed endemic Australian Collembola as exemplars, we outline a comprehensive approach to generating and validating suites of codominant, sequence-yielding nuclear loci for previously unstudied invertebrates.

Results

Six markers per species were developed without any baseline genetic information. After evaluating the characteristics of each new locus via SSCP pre-screening, population samples were genotyped on the basis of either DNA sequence, restriction site, or insertion/deletion variation, depending on which assay was deemed most appropriate. Polymorphism was generally high (mean of nine alleles per locus), and the markers were capable of resolving population structuring over very fine spatial scales (<100 km). SSCP coupled with targeted DNA sequencing was used to obtain genotypic, genic and genealogical information from six loci (three per species). Phylogeographic analysis identified introns as being most informative.

Conclusion

The comprehensive approach presented here feasibly overcomes technical hurdles of (i) developing suitably polymorphic nuclear loci for non-model organisms, (ii) physically isolating nuclear allele haplotypes from diploid tissues without cloning, and (iii) genotyping population samples on the basis of nuclear DNA sequence variation.     

Echocardiographic quantification of myocardial function using tissue deformation imaging, a guide to image acquisition and analysis using tissue Doppler and speckle tracking

Recent developments in the field of echocardiography have allowed the cardiologist to objectively quantify regional and global myocardial function. Regional deformation (strain) and deformation rate (strain-rate) can be calculated non-invasively in both the left and right ventricle, providing information on regional (dys-)function in a variety of clinical settings. Although this promising novel technique is increasingly applied in clinical and preclinical research, knowledge about the principles, limitations and technical issues of this technique is mandatory for reliable results and for implementation both in the clinical as well as the scientific field. In this article, we aim to explain the fundamental concepts and potential clinical applicability of strain and strain-rate for both tissue Doppler imaging (TDI) derived and speckle tracking (2D-strain) derived deformation imaging. In addition, a step-by-step approach to image acquisition and post processing is proposed. Finally, clinical examples of deformation imaging in hypertrophic cardiomyopathy (HCM), cardiac resynchronization therapy (CRT) and arrhythmogenic right ventricular dysplasia/cardiomyopathy (ARVD/C) are presented.     

Nucleotide sequence of the D-loop region of the sperm whale (Physeter macrocephalus) mitochondrial genome

We have amplified, by the polymerase chain reaction, and have sequenced the D-loop region of the mitochondrial DNA from the sperm whale (Physeter macrocephalus). The sperm whale D-loop was aligned with D- loop sequences from four other cetaceans (Commerson's dolphin, orca, fin whale, and minke whale) and an out-group (cow). This alignment showed the sperm whale sequence to be larger than that of other cetaceans. In addition, some sequence blocks were highly conserved among all six species, suggesting roles in the functioning of mitochondrial DNA. Other blocks that were previously reported to be well conserved among cetaceans showed little sequence conservation with the sperm whale D-loop, which argues against the functional importance of these sequence blocks in cetaceans.      

Immune responses to stress after stress management training in patients with rheumatoid arthritis

Introduction

Psychological stress may alter immune function by activating physiological stress pathways. Building on our previous study, in which we report that stress management training led to an altered self-reported and cortisol response to psychological stress in patients with rheumatoid arthritis (RA), we explored the effects of this stress management intervention on the immune response to a psychological stress task in patients with RA.

Methods

In this study, 74 patients with RA, who were randomly assigned to either a control group or a group that received short stress management training, performed the Trier Social Stress Test (TSST) 1 week after the intervention and at a 9-week follow-up. Stress-induced changes in levels of key cytokines involved in stress and inflammatory processes (for example, interleukin (IL)-6 and IL-8) were assessed.

Results

Basal and stress-induced cytokine levels were not significantly different in patients in the intervention and control groups one week after treatment, but stress-induced IL-8 levels were lower in patients in the intervention group than in the control group at the follow-up assessment.

Conclusions

In line with our previous findings of lower stress-induced cortisol levels at the follow-up of stress management intervention, this is the first study to show that relatively short stress management training might also alter stress-induced IL-8 levels in patients with RA. These results might help to determine the role of immunological mediators in stress and disease.

Trial registration

The Netherlands National Trial Register (NTR1193)     

Insects and light interact to mediate vine colonization of fast growing Microberlinia bisulcata tree seedlings in gaps of an African rain forest

Vines thrive in lowland tropical forests, yet the biotic factors underlying their colonization of host tree seedlings and saplings remain surprisingly understudied. Insect herbivores presumably could influence this process, especially where disturbance has opened the canopy (i.e., gaps)—temporary areas of higher primary productivity favoring the recruitment of vines and trees and invertebrates in forests—but their impact on vine colonization has never been experimentally tested. Using data from an insect herbivore exclusion (mesh-netting cages) experiment conducted in an African rain forest (Korup, Cameroon), I logistically modeled the probability of vines colonizing seedlings of three co-dominant species (Microberlinia bisulcata vs. Tetraberlinia bifoliolata and T. korupensis) in paired shaded understory and sunny gap locations (41 blocks across 80 ha, starting n = 664 seedlings) in a 1–2-yr period (2008–2009). Vine colonization occurred almost exclusively in gaps, occurring on 16% of seedlings there. Excluding herbivores in gaps doubled colonization of the light-demanding and faster growing M. bisulcata but had negligible effects on the two shade-tolerant, slower growing and less palatable Tetraberlinia species, which together were twice as susceptible to vines under natural forest gap conditions (controls). When protected from herbivores in gaps, more light to individual seedlings strongly increased vine colonization of M. bisulcata whereas its well-lit control individuals supported significantly fewer vines. These results suggest vines preferably colonize taller seedlings, and because light-demanding tree species grow faster in height with more light, they are more prone to being colonized in gaps; however, insect herbivores can mediate this process by stunting fast growing individuals so that colonization rates becomes more similar between co-occurring slow and fast growing tree species. Further influencing this process might be associational resistance or susceptibility to herbivores linked to host species’ leaf traits conferring shade-tolerant ability as seedlings or saplings. A richer understanding of how vines differentially influence forest regeneration and species composition may come from investigating vine–tree–herbivore interactions across light gradients, ideally via long-term studies and intercontinental comparisons. Abstract in French is available with online material.