Final Report Summary - PAINGENES (Heritability of chronic neuropathic pain)
Chronic pain undermines the health and welfare of millions of EU citizens and carries with it enormous personal, social and financial costs. A recent large survey estimates that 19% of European adults suffer from one or another chronic pain condition (Breivik et al. 2006). Individual variability in the burden of pain has traditionally been attributed to psychosocial factors. However, new data indicate that there are also important inheritable predispositions to pain, particularly to the development of chronic pain after neural injury (neuropathic pain). Pain susceptibility genes are intrinsically hard to detect in human lineages and populations, particularly genes related to pain in acquired neuropathies such as those that may follow nerve trauma or infection. This STREP adopts an alternative approach.
We have exploited new rodent models of neuropathy to uncover pain-related genetic loci and associated neurobiological processes in inbred mouse strains that show high versus low pain susceptibility. Linkage analysis and positional cloning, together with gene expression arrays and a variety electrophysiological and neurochemical methods were applied to primary sensory neurons. The strategy was to identify genetic and cellular variables that co-vary with behavioural pain phenotype across mouse strains. Overall, very satisfying progress was made. The project has yielded ten articles published in the scientific literature, with additional publications anticipated. Highlights of the scientific results are as follows:
Genetic crosses of two mouse strains that are high and low, respectively, in susceptibility to a major type of neuropathic pain confirmed that the pain trait is heritable. The pattern of segregation observed suggests that the trait is passed by one gene of major effect together with minor modifier genes.
We identified a genetic locus on mouse chromosome 15 that contains the pain susceptibility gene. This quantitative trait locus (QTL), termed pain1, is the only significant QTL detected in a whole genome scan, and is therefore likely to be the sought-after gene of major effect in this system.
Application of a variety of genetic and bioinformatic techniques led us from the QTL to the identity of the pain susceptibility gene that apparently underlies the difference in neuropathic pain phenotype in the mouse strains investigated. This sets the stage for determining whether polymorphisms in this gene also affect neuropathic pain phenotype in human populations.
Environmental (apparently psychosocial) and sex factors affecting pain behaviour are sufficiently powerful that they can obscure the presence of genetic linkage to pain1. This observation serves as an alert for future searches for pain genes.
We studied a second pain trait, the mouse's sensitivity to noxious heat stimuli on the skin, using a second pair of pain sensitive and pain insensitive mice. The basis for the difference in pain sensitivity was found to be in the sensory receptor neurons that innervate the skin, rather than differences in brain processing.
Functional analysis suggests that the difference in the heat-sensitive sensory neurons results from differential expression of a gene that codes for the neuroactive peptide CGRP. A variety of physiological differences were found in the behaviour of primary sensory neurons in the mouse strain pair studies suggesting genetic pleiotropy.
In the event of nerve injury, injured sensory neurons change their response properties thus contributing to pain hypersensitivity. Neighbouring uninjured neurons may also be affected, but the story with them is more complex. Using the excised skin-nerve preparation essentially no spontaneous ectopic firing was detected, and there was no change in tactile sensitivity of fine afferents. Differences in evoked CGRP release from skin were detected, and appear to be sex specific.
A new technology, oligonucleotide arrays (gene chips), allows one to monitor the expression of thousands of genes simultaneously. We developed a technique for doing this by using very small quantities of neural tissue, individual mouse DRGs. This technique has yielded higher resolution expression data than any other published to date, and provide a rich database for correlational analysis.
This method was used to compare changes in gene expression in mouse strains with high and low in pain susceptibility, respectively. The expression of most genes changes in the same way in the two mouse strains. However, a small number of genes are differentially regulated, suggesting that they may be responsible for the heritable difference in pain experience. Bioinformatic methods were applied which identified transcripts whose expression correlates highly with behavioural pain phenotype.
In addition to comparisons of mouse strains, the method proved sensitive enough to permit correlation of the DRG expression profile of individual mice against readings of their individual pain behaviour.
While array technology efficiently detects strain differences in the expression of large numbers of genes expressed in the DRG, it cannot determine which types of cells are involved (neurons or glia, and if neurons, nociceptors of low threshold afferents). Immunocytochemistry and in situ hybridization were used to make this level of analysis for a variety of key molecular targets of interest. Several significant correlations with behavioural pain phenotype were identified, notably with ion channel transcripts.
Time-of-onset of ectopic spontaneous discharge was found to correspond to the time of onset of tactile allodynia in the Chung model of neuropathic pain.
On the other hand, there was little indication of an across-strain correlation between the level of ectopic spontaneous discharge and pain phenotype in the neuroma model of neuropathic pain.
In contrast a promising across strains correlation was found between neuropathic pain phenotype and sequelae of nerve injury expressed at the level of the spinal cord, notably readouts of central sensitization and neuropathic pain phenotype.
We were unable to replicate earlier published data on across-strain difference in the Chung model of neuropathic pain, but we did corroborate the across-strain pattern in the neuroma model.
Tactile hypersensitivity after ischemic nerve injury showed a high correlation with spontaneous pain scores in the neuroma model, but not with tactile hypersensitivity in the Chung model.
In parallel with highly satisfying scientific progress, a considerable effort has been made to bring the message of pain biology and pain genetics to scientific and general audiences. Moreover, discovery of the gene underlying the pain1 QTL provides a novel and exciting new target for the study of pain genetics in humans, and ultimately for the future development of novel prognostic, diagnostic and treatment options for chronic pain sufferers. Avenues for commercial development of this discovery are currently being explored. Overall, we believe that the outcomes of the PainGenes STREP have contributed substantially to the body of knowledge on pain genetics, with ultimate benefits for pain sufferers, their families, employers and European economies.
We have exploited new rodent models of neuropathy to uncover pain-related genetic loci and associated neurobiological processes in inbred mouse strains that show high versus low pain susceptibility. Linkage analysis and positional cloning, together with gene expression arrays and a variety electrophysiological and neurochemical methods were applied to primary sensory neurons. The strategy was to identify genetic and cellular variables that co-vary with behavioural pain phenotype across mouse strains. Overall, very satisfying progress was made. The project has yielded ten articles published in the scientific literature, with additional publications anticipated. Highlights of the scientific results are as follows:
Genetic crosses of two mouse strains that are high and low, respectively, in susceptibility to a major type of neuropathic pain confirmed that the pain trait is heritable. The pattern of segregation observed suggests that the trait is passed by one gene of major effect together with minor modifier genes.
We identified a genetic locus on mouse chromosome 15 that contains the pain susceptibility gene. This quantitative trait locus (QTL), termed pain1, is the only significant QTL detected in a whole genome scan, and is therefore likely to be the sought-after gene of major effect in this system.
Application of a variety of genetic and bioinformatic techniques led us from the QTL to the identity of the pain susceptibility gene that apparently underlies the difference in neuropathic pain phenotype in the mouse strains investigated. This sets the stage for determining whether polymorphisms in this gene also affect neuropathic pain phenotype in human populations.
Environmental (apparently psychosocial) and sex factors affecting pain behaviour are sufficiently powerful that they can obscure the presence of genetic linkage to pain1. This observation serves as an alert for future searches for pain genes.
We studied a second pain trait, the mouse's sensitivity to noxious heat stimuli on the skin, using a second pair of pain sensitive and pain insensitive mice. The basis for the difference in pain sensitivity was found to be in the sensory receptor neurons that innervate the skin, rather than differences in brain processing.
Functional analysis suggests that the difference in the heat-sensitive sensory neurons results from differential expression of a gene that codes for the neuroactive peptide CGRP. A variety of physiological differences were found in the behaviour of primary sensory neurons in the mouse strain pair studies suggesting genetic pleiotropy.
In the event of nerve injury, injured sensory neurons change their response properties thus contributing to pain hypersensitivity. Neighbouring uninjured neurons may also be affected, but the story with them is more complex. Using the excised skin-nerve preparation essentially no spontaneous ectopic firing was detected, and there was no change in tactile sensitivity of fine afferents. Differences in evoked CGRP release from skin were detected, and appear to be sex specific.
A new technology, oligonucleotide arrays (gene chips), allows one to monitor the expression of thousands of genes simultaneously. We developed a technique for doing this by using very small quantities of neural tissue, individual mouse DRGs. This technique has yielded higher resolution expression data than any other published to date, and provide a rich database for correlational analysis.
This method was used to compare changes in gene expression in mouse strains with high and low in pain susceptibility, respectively. The expression of most genes changes in the same way in the two mouse strains. However, a small number of genes are differentially regulated, suggesting that they may be responsible for the heritable difference in pain experience. Bioinformatic methods were applied which identified transcripts whose expression correlates highly with behavioural pain phenotype.
In addition to comparisons of mouse strains, the method proved sensitive enough to permit correlation of the DRG expression profile of individual mice against readings of their individual pain behaviour.
While array technology efficiently detects strain differences in the expression of large numbers of genes expressed in the DRG, it cannot determine which types of cells are involved (neurons or glia, and if neurons, nociceptors of low threshold afferents). Immunocytochemistry and in situ hybridization were used to make this level of analysis for a variety of key molecular targets of interest. Several significant correlations with behavioural pain phenotype were identified, notably with ion channel transcripts.
Time-of-onset of ectopic spontaneous discharge was found to correspond to the time of onset of tactile allodynia in the Chung model of neuropathic pain.
On the other hand, there was little indication of an across-strain correlation between the level of ectopic spontaneous discharge and pain phenotype in the neuroma model of neuropathic pain.
In contrast a promising across strains correlation was found between neuropathic pain phenotype and sequelae of nerve injury expressed at the level of the spinal cord, notably readouts of central sensitization and neuropathic pain phenotype.
We were unable to replicate earlier published data on across-strain difference in the Chung model of neuropathic pain, but we did corroborate the across-strain pattern in the neuroma model.
Tactile hypersensitivity after ischemic nerve injury showed a high correlation with spontaneous pain scores in the neuroma model, but not with tactile hypersensitivity in the Chung model.
In parallel with highly satisfying scientific progress, a considerable effort has been made to bring the message of pain biology and pain genetics to scientific and general audiences. Moreover, discovery of the gene underlying the pain1 QTL provides a novel and exciting new target for the study of pain genetics in humans, and ultimately for the future development of novel prognostic, diagnostic and treatment options for chronic pain sufferers. Avenues for commercial development of this discovery are currently being explored. Overall, we believe that the outcomes of the PainGenes STREP have contributed substantially to the body of knowledge on pain genetics, with ultimate benefits for pain sufferers, their families, employers and European economies.
