In this study we have used a composite diagnostic experimental approach incorporating QST, genetic sequencing, and perfusion imaging to study a patient presenting with pain and erythema of the extremities. Genetic testing showed that these symptoms were due to the painful channelopathy IEM (due to a L858F Nav1.7 mutation). QST demonstrated hypersensitivity to heat and confirmed the temperature dependence of her tonic pain. Finally, a novel perfusion imaging approach was used to investigate the neural correlates of the patient’s pain vs phases of cooling relief.
This patient has a severe form of IEM; she was noted to have erythematous feet at birth and although onset of this disorder has previously been described as early as 1–2 years of age, we have not seen any previous reports of congenital onset 
. She has always complained of bilateral foot pain exacerbated by warmth and relieved by cooling. Initially her symptoms were episodic, however, from her second decade she developed constant foot pain at room temperature that could only be relieved by cooling. IEM occurs as a consequence of gain of function mutations in the voltage-gated sodium channel Nav
. The patient described here was found to have a substitution of the amino acid phenylalanine for leucine at codon 858 of Nav
1.7. This is within the S4/5 linker of domain 2 of the channel. This mutation has previously been described and is associated with a severe erythromelalgia phenotype with a young age of onset in a Canadian 
and a Chinese kindred 
1.7 mutations causing IEM are highly penetrant and give rise to increased dorsal root ganglion cell hyperexcitability (and hence pain) via a number of alterations in channel function, including: a hyperpolarizing shift in channel activation, slowed deactivation, and an enhanced response to slow ramp-like stimuli (ramp currents) 
. Particular mutations have distinct effects on channel function and there is a complex relationship between these changes and the clinical severity: the greater the hyperpolarizing shift in activation, the earlier the age of onset 
. However, mutations that also enhance slow inactivation reduce channel availability and lead to a milder phenotype 
. The L858F mutation present in our patient has been reported to cause a large hyperpolarizing shift in voltage-dependent activation with no effect on slow inactivation. These biophysical changes therefore correlate with the severe phenotype [9,16]
. Interestingly, cooling has been shown to shift the activation midpoint of L858F in a depolarizing direction, bringing the threshold of activation of the mutant channel closer to wild-type Nav
. The patient reported here demonstrated noxious heat hypersensitivity on QST of the feet, and the level of her tonic pain was strongly modulated by ambient temperature (being rapidly relieved by cooling). We exploited this phenomenon to investigate the imaging correlates of her tonic pain.
The patient showed robust activation of key pain sensory and pain-affect regions during phases of IEM-related pain that ceased during cooling relief. Pain was related to increased perfusion in the bilateral thalamus, S1, inferior frontal gyrus, anterior, mid and posterior cingulate, right putamen, right caudate, and the left anterior insula. There was a lateral dominance of activity in several regions that was contralateral to her greatest foot pain, as would be expected. Our data align with previous tonic pain studies using ASL and positron emission tomography. For example, regions such as the insula, thalamus, putamen, cingulate, and S1 have all been shown previously to play a role in ongoing pain states. While the present study is unable to interrogate the mechanisms of tonic pain processing, these data provide exciting additional evidence that many regions activated in well-known BOLD fMRI studies of acute pain are also found to be active during a chronic pain state [20,21]
Because of the robust relieving action induced by cooling at the site of the erythromelalgia pain, it was possible to image the attenuation of brain regions constituent of a multidimensional pain network. Cooling relief caused a cessation of activation in these regions. No additional regions were observed to be more active during cooling compared to pain. While this could be due to low signal-to-noise related to the single-subject nature of the case, it is clear that the cooling stimulus acting at the site of the erythema is a major component driving pain relief. This would be consistent with the effects of cooling on mutant L858P Nav
1.7 function and presumably correlates with reduced ongoing nociceptor activity following cooling. Microneurography has been performed in a cohort of patients with primary erythromelalgia, and this demonstrated altered C-fibre function, including enhanced activity-dependent slowing of afferent units and increased spontaneous activity 
. The effects of cooling on C-fibre function were not investigated in this report.
Interestingly, pain relief in the form of cessation or reduction of a noxious stimulus can be thought of as a reward. Occasionally, this sensation is pleasurable. Previous work by Becerra et al. (2001, 2006) and Baliki et al. (2010) provide strong evidence that activity within the nucleus accumbens (NAc) tracks phases of pleasurable relief related to pain offset [3–5]
. Similarly, because of the block design used in the current study, we predict that increased activation within the NAc during phases of cooling would be visible as the cooling blocks reflect a kind of pleasurable pain-offset triggered by reduced nociceptor function. While the present investigation was unable to observe significant perfusion changes from a whole-brain analysis during cooling relief; it is possible that subsequent investigations focused purely on the NAc may provide insight into the higher cognitive processes underlying the experience of pleasure related to cooling-induced relief of erythromelalgia-associated pain.
The present study confirms the temperature dependence of the L858F Nav
1.7 mutation and further demonstrates the related effects on pain processing in the cortex by using a pCASL fMRI approach. Recent advances in ASL pulse sequence programming have improved the technique such that pCASL benefits from greatly enhanced signal-to-noise ratio and reproducibility compared to other commonly used ASL approaches [8,15]
. However, it is possible that the technique is still ill suited to do single-subject investigations without the capacity to do multiple repeat scans within the subject, as we have done here. Follow-up studies may further benefit by defining a priori hypotheses about specific brain regions of interest such that more region-of-interest-driven analyses may be used that do not necessitate whole-brain corrections.
A key goal of this work was to employ a composite investigative approach to study a chronic pain condition. From previous pain neuroimaging studies, it is well known that pain is a complex, subjective experience constituent of sensory, psychological, cognitive and neuropharmacological factors. This is made even more complex in the context of chronic pain, where unique combinations of structural and functional abnormalities could possibly be used to define different types of pain disease states 
. The success of future human pain studies relies partly on the capacity to encapsulate this complexity. As a case study, the present work attempted to do this by employing a multivariate investigative approach that interrogated the pain from the level of ion channel dysfunction on peripheral nociceptors to higher cortical pain processing in the brain. Interpreting the brain imaging data, although in a single subject, it is clear that the optimised ASL approach used is capable of imaging the effect of chronic pain and relief across a whole brain. Additionally, our data provide evidence that in the case of erythromelalgia, it appears as though cooling relief produces analgesia via attenuation of peripheral inputs rather than via top-down brain mechanisms related to pleasant relief.
We hope that future studies employing a composite, “molecule-to-man” approach as we have done here will help develop a mechanism-based understanding of chronic pain in patients.