In this study we describe three patients (two siblings, and a third, unrelated patient) housing the I228M variant of sodium channel NaV
1.7. One of these patients displayed a clinical phenotype that included pain in the face as well as in other parts of the body together with autonomic symptoms, with the diagnosis of SFN confirmed by demonstration of reduced IENFD on skin biopsy, and abnormal QST. The second patient gave a history of distal extremity pain and redness, triggered by warmth and relieved by cooling. While these symptoms are commonly reported in IEM [7
], she also reported autonomic symptoms including increased perspiration, gastrointestinal complaints and hot flashes, which are not characteristic of IEM. The third patient initially experienced discomfort and vasomotor instability over the occiput, which progressed to involve the distal extremities, together with abnormal perspiration, intermittent difficulties with micturition; skin biopsy and QST in this patient were both abnormal, confirming the diagnosis of SFN.
Because facial pain was a prominent part of the clinical picture in one of the patients described in this paper, we assessed the effect of the I228M mutation on excitability of trigeminal ganglion neurons. Our current-clamp analysis demonstrated that the I228M variant depolarizes resting membrane potential, reduces current threshold and enhances repetitive firing in these cells. The effect of only one other NaV
1.7 mutation has been assessed in trigeminal ganglion neurons. We previously reported that the A1632E NaV
1.7 mutation, from a patient who displayed a mixed clinical phenotype with features of both IEM and PEPD, produces hyperexcitability in trigeminal ganglion neurons [22
]. The A1632E mutation, however, produced hyperexcitability in these cells over the entire range of stimulus intensities, while I228M produces hyperexcitability only at low stimulus intensities. Whether other gain-of-function mutations of NaV
1.7 have similar effects on trigeminal ganglion neurons remains to be determined.
The I228M substitution is located within the fourth transmembrane segment (S4) within domain I of the NaV
1.7 channel. The S4 in each of the domains of sodium channels is an amphiphatic helix which is characterized by a repeat motif of positively charged amino acids at every third position [19
]. Non-charge-conserved mutations, S211P and F216S, in DI/S4 have been linked to IEM, and have been shown to shift voltage-dependence of activation in a hyperpolarizing direction, making it easier to open the mutant channels [23
]. The I228M substitution does not change the number of charges in the S4 segment, and reasonably conserves the hydrophobic nature of the side-chain of this residue, and thus might not have been predicted to have a functional effect. A link to function, however, is suggested by the conservation of the I228 residue at the equivalent position in all voltage-gated sodium channels sequenced to date (Figure ); I228 is substituted by the other branched side-chain residue, valine, in NaV
1.9. The functional effect of I228M might be related to the proximity of the I228 residue to the cytoplasmic end of the S4 segment, which could alter the local structure of the helix in a subtle manner affecting slow-inactivation but not activation. Notably, while the I228M variant produced hyperexcitability in both DRG and trigeminal ganglion neurons, only two of the three patients described here reported cranial pain, and it was experienced in the jaw and eyes in one, while it was focused on the scalp in the other.
Our results demonstrate phenotypic diversity in the pain syndromes associated with the I228M substitution in the NaV
1.7 channel in three different patients. Two of these patients were from the same family, which also includes patient 1's two asymptomatic sons who carry the I228M NaV
1.7 variant. Both of these asymptomatic carriers are younger than the age of onset of the three patients presented, and whether they will develop pain in the future is unclear. We have previously noted different ages of onset and different degrees of pain, and an asymptomatic carrier in members of a single family, all housing the G616R NaV
1.7 mutation [25
]. Whether this phenotypic variability is due to modifier genes, epigenetic factors, and/or environmental factors is not yet clear. The minor allele of the NaV
1.7 R1150W variant, which is known to produce hyperexcitability in DRG neurons [26
], has been associated with increased pain scores in a number of acquired pain syndromes (osteoarthritis, compressive radiculopathies, traumatic limb amputation), suggesting that environmental factors may, at least in some individuals, act as triggers or increase risk of developing pain [27
Most peripheral neuropathies present in a "stocking glove" distribution with sensory abnormalities and pain first appearing in the most distal parts of the limbs (feet, then hands). It has traditionally been held that longer nerve fibers, or the cells giving rise to them, are affected before shorter fibers or the cells giving rise to them. A number of potential mechanisms have been invoked for this length-dependent mode of progression of neuropathy, including impairment of axoplasmic transport [28
], increased probability of demyelination along longer nerve fibers [29
], or a higher probability of impairment of calcium homeostasis along longer nerve fibers [30
]. However, the present results show that the NaV
1.7 I228M variant, which impairs slow-inactivation, produces physiological changes in primary afferent neurons (trigeminal ganglion neurons) that innervate the relatively proximal sensory field of the face and scalp, as well as DRG neurons. While we do not know whether there was degeneration of small fibers innervating the face or scalp in these patients, both exhibited degeneration of the relatively long axons, as demonstrated by reduced IENFD on skin biopsy from the leg.
In summary, our results demonstrate phenotypic diversity in pain syndromes associated with the I228M gain-of-function variant of NaV1.7. Importantly, variability in clinical presentation was present not only when comparing patients from different families, but also for patients within a single family. Our findings also demonstrate that the I228M variant can increase excitability of trigeminal ganglion as well as DRG neurons. While the mechanism(s) responsible for this phenotypic diversity remain unexplained, our findings suggest that clinical studies, in patients who are carriers of functional variants of sodium channels, should be designed to take phenotypic variability, even within single families, into account.