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• W2026551512 abstract "Paroxysmal extreme pain disorder (PEPD), previously known as familial rectal pain (FRP, or OMIM 167400), is an inherited condition characterized by paroxysms of rectal, ocular, or submandibular pain with flushing. A genome-wide linkage search followed by mutational analysis of the candidate gene SCN9A, which encodes hNav1.7, identified eight missense mutations in 11 families and 2 sporadic cases. Functional analysis in vitro of three of these mutant Nav1.7 channels revealed a reduction in fast inactivation, leading to persistent sodium current. Other mutations in SCN9A associated with more negative activation thresholds are known to cause primary erythermalgia (PE). Carbamazepine, a drug that is effective in PEPD, but not PE, showed selective block of persistent current associated with PEPD mutants, but did not affect the negative activation threshold of a PE mutant. PEPD and PE are allelic variants with distinct underlying biophysical mechanisms and represent a separate class of peripheral neuronal sodium channelopathy. Paroxysmal extreme pain disorder (PEPD), previously known as familial rectal pain (FRP, or OMIM 167400), is an inherited condition characterized by paroxysms of rectal, ocular, or submandibular pain with flushing. A genome-wide linkage search followed by mutational analysis of the candidate gene SCN9A, which encodes hNav1.7, identified eight missense mutations in 11 families and 2 sporadic cases. Functional analysis in vitro of three of these mutant Nav1.7 channels revealed a reduction in fast inactivation, leading to persistent sodium current. Other mutations in SCN9A associated with more negative activation thresholds are known to cause primary erythermalgia (PE). Carbamazepine, a drug that is effective in PEPD, but not PE, showed selective block of persistent current associated with PEPD mutants, but did not affect the negative activation threshold of a PE mutant. PEPD and PE are allelic variants with distinct underlying biophysical mechanisms and represent a separate class of peripheral neuronal sodium channelopathy. Inherited disorders of ion channels are now known to account for a wide spectrum of human diseases characterized by paroxysmal dysfunction of excitable tissues. Understanding the molecular basis of these disorders has provided remarkable insights into normal physiological mechanisms (Ashcroft, 2000Ashcroft F.M. Ion channels and disease. Academic Press, San Diego, USA2000Google Scholar, Meisler and Kearney, 2005Meisler M.H. Kearney J.A. Sodium channel mutations in epilepsy and other neurological disorders.J. Clin. Invest. 2005; 115: 2010-2017Crossref PubMed Scopus (373) Google Scholar). The first such channelopathy to be described, hyperkalemic periodic paralysis, is caused by defects in the voltage-gated sodium channel of skeletal muscle Nav1.4 (Ptacek et al., 1991Ptacek L.J. George Jr., A.L. Griggs R.C. Tawil R. Kallen R.G. Barchi R.L. Robertson M. Leppert M.F. Idenitification of a mutation in the gene causing hyperkalemic periodic paralysis.Cell. 1991; 67: 1021-1027Abstract Full Text PDF PubMed Scopus (326) Google Scholar, Rojas et al., 1991Rojas C.V. Wang J. Schwartz L.S. Hoffman E.P. Powell B.R. Brown Jr., R.H. A Met-to-Val mutation in the skeletal muscle Na+ channel α-subunit in hyperkalaemic periodic paralysis.Nature. 1991; 354: 387-389Crossref PubMed Scopus (278) Google Scholar). Since this description, mutations in sodium channels have also been shown to cause epilepsy and inherited disorders of cardiac rhythm. Here we describe a study of the molecular basis of Paroxysmal extreme pain disorder (PEPD), previously known as familial rectal pain (Fertleman and Ferrie, 2006Fertleman C.R. Ferrie C. What's in a name - Familial Rectal Pain Syndrome (FRPS) becomes Paroxysmal Extreme Pain Disorder (PEPD).The Journal of Neurology, Neurosurgery and Psychiatry with Practical Neurology. 2006; 77: 1294-1295Crossref PubMed Scopus (31) Google Scholar), an autosomal dominant paroxysmal disorder of pain and autonomic dysfunction first identified by Hayden and Grossman, 1959Hayden R. Grossman M. Rectal, ocular and submaxillary pain.Am. J. Dis. Child. 1959; 97: 479-482Crossref PubMed Scopus (39) Google Scholar. Several further families have since been described (Mann and Cree, 1972Mann T.P. Cree J.E. Familial rectal pain.Lancet. 1972; 1: 1016-1017Abstract PubMed Scopus (8) Google Scholar, Schubert and Cracco, 1992Schubert R. Cracco J.B. Familial rectal pain. A type of reflex epilepsy?.Ann. Neurol. 1992; 32: 824-826Crossref PubMed Scopus (21) Google Scholar), and there is evidence that this condition may be underdiagnosed (Elmslie et al., 1996Elmslie F.V. Wilson J. Rossiter M.A. Familial rectal pain: is it under-diagnosed?.J. R. Soc. Med. 1996; 89: 290P-291PPubMed Google Scholar). The distinctive features of PEPD are paroxysmal episodes of burning pain in the rectal, ocular, and mandibular areas accompanied by autonomic manifestations such as skin flushing. The anti-epilepsy drug carbamazepine is effective in many patients. A genome-wide linkage search mapped the PEPD gene to a region of chromosome 2 encompassing SCN9A, which encodes the Nav1.7 voltage-gated sodium channel. The expression pattern and function of this channel rendered SCN9A an excellent functional and positional candidate. Additionally, mutations in this channel were shown to underlie primary erythermalgia (PE) (Drenth et al., 2005Drenth J.P.H. te Morsche R.H.M. Guillet G. Taieb A. Kirby R.L. Jansen J.B.M.J. SCN9A mutations define primary erythermalgia as a neuropathic disorder of voltage gated sodium channels.J. Invest. Dermatol. 2005; 124: 1333-1338Crossref PubMed Scopus (146) Google Scholar, Yang et al., 2004Yang Y. Wang Y. Li S. Xu Z. Li H. Ma L. Fan J. Bu D. Liu B. Fan Z. et al.Mutations in SCN9A, encoding a sodium channel alpha subunit, in patients with primary erythermalgia.J. Med. Genet. 2004; 41: 171-174Crossref PubMed Scopus (559) Google Scholar), a disease associated with episodic burning pain, flushing of the extremities, inflammation, and swelling. Identification of a distinct repertoire of SCN9A mutations in PEPD, together with detailed functional analysis, suggest that these two disorders are allelic variants that are not only clinically but also mechanistically distinct, and that differences in the effect of the mutations on sodium channel activity account for the clear differences in the clinical phenotypes and in their differential responses to sodium channel-blocking drugs. We conducted a genome-wide linkage search in one large pedigree with PEPD, family 7 (Figure 1). We genotyped DNA from 13 affected and 13 unaffected family members using 455 microsatellite markers. The marker D2S2330 on chromosome 2q gave a LOD score of 4.99 at θ = 0. Haplotype analysis (Figure 1) identified critical recombinants at D2S142 and D2S335, defining a 16 cM critical region containing a cluster of sodium channel genes. One of these, SCN9A (Klugbauer et al., 1995Klugbauer N. Lacinova L. Flockerzi V.H.F. Structure and functional expression of a new member of the tetrodotoxin-sensitive voltage-activated sodium channel family from human neuroendocrine cells.EMBO J. 1995; 14: 1084-1090Crossref PubMed Scopus (262) Google Scholar, Toledo-Aral et al., 1997Toledo-Aral J.J. Moss B.L. He Z.J. Koszowski A.G. Whisenand T. Levinson S.R. Wolf J.J. Silos-Santiago I. Halegoua S. Mandel G. Identification of PN1, a predominant voltage-dependent sodium channel expressed principally in peripheraláneurons.Proc. Natl. Acad. Sci. USA. 1997; 94: 1527-1532Crossref PubMed Scopus (424) Google Scholar), encoding the hNav1.7 voltage-gated sodium channel α-subunit, is expressed only in the peripheral nervous system, in both sympathetic neurons and the sensory neurons of the dorsal root ganglion (DRG) (Sangameswaran et al., 1997Sangameswaran L. Fish L.M. Koch B.D. Rabert D.K. Delgado S.G. Ilnicka M. Jakeman L.B. Novakovic S. Wong K. Sze P. et al.A Novel Tetrodotoxin-sensitive, Voltage-gated Sodium Channel Expressed in Rat and Human Dorsal Root Ganglia.J. Biol. Chem. 1997; 272: 14805-14809Crossref PubMed Scopus (240) Google Scholar). Nav1.7 mediates a tetrodotoxin (TTX) -sensitive, fast-inactivating current and has a role in acute and inflammatory pain (Nassar et al., 2004Nassar M.A. Stirling L.C. Matthews E.A. Forlani G. Baker M.D. Dickinson A.H. Mandel G. Wood J.N. Nociceptor-specific gene deletion reveals a major role for Nav1.7 (PN1) in acute and inflammatory pain.Proc. Natl. Acad. Sci. USA. 2004; 101: 12706-12711Crossref PubMed Scopus (485) Google Scholar). We therefore investigated SCN9A as a positional and functional candidate. We designed intronic primers flanking the 26 exons of SCN9A to amplify genomic DNA from the probands of family 7 (individual IV:4, Figure 1), ten additional pedigrees (in family 15, only DNA from the proband's affected father was available), and two sporadic cases (families 8 and 9). We found a total of eight missense mutations in 8/13 families (Figure 2A) (See the Supplemental Data online for additional pedigrees and electropherograms). All patients are heterozygous for the respective mutations that cosegregated with the disorder in all affected members from whom DNA was available. In family 12, the proband is a compound heterozygote, having inherited R996C from his affected father, but also having a de novo mutation (V1298D). This individual is more severely affected than his father, indicating that R996C may represent a subtle mutation. Family 4, in which R996C is the only mutation, also appears to have a less severe phenotype not requiring medication. None of these mutations were present in 192 ethnically matched control chromosomes (data not shown). All represent nonconservative amino acid changes likely to have an effect on channel function. Moreover, 7/8 mutations alter amino acids that are conserved across other human voltage-gated sodium channel α-subunits as well as those from other species (Figure 2B). Two mutations (I1461T, F1462V) occur within the highly conserved IFM amino acid motif in the linker region between domains III and IV (Figure 2Biii). Site-directed mutagenesis studies show that this motif forms the inactivation gate and plays a pivotal role in fast inactivation (West et al., 1992West J.W. Patton D.E. Scheuer T. Wang X. Goldin A.L. Catterall W.A. A cluster of hydrophobic amino acid residues required for fast Na+ channel inactivation.Proc. Natl. Acad. Sci U.S.A. 1992; 89: 10910-10914Crossref PubMed Scopus (635) Google Scholar, Kellenberger et al., 1997Kellenberger S. West J.W. Scheuer T. Catterall W.A. Molecular analysis of the putative inactivation particle in the inactivation gate of brain type IIA Na+ channels.J. Gen. Physiol. 1997; 109: 589-605Crossref PubMed Scopus (64) Google Scholar). The T1464I mutation changes a completely conserved threonine residue immediately adjacent to the IFM domain (Figure 2Biii), and substitutions at this residue have also previously been shown to disrupt inactivation by substantially altering the stability of the inactivated state (Kellenberger et al., 1997Kellenberger S. West J.W. Scheuer T. Catterall W.A. Molecular analysis of the putative inactivation particle in the inactivation gate of brain type IIA Na+ channels.J. Gen. Physiol. 1997; 109: 589-605Crossref PubMed Scopus (64) Google Scholar). The residues V1298 and V1299 are also completely conserved (Figure 2Bii) and occur in the loop between domain III S4-S5, believed to interact with the domain III-IV inactivation motif (Smith and Goldin, 1997Smith M.R. Goldin A.L. Interaction between the sodium channel inactivation linker and domain III S4-S5.Biophys. J. 1997; 73: 1885-1895Abstract Full Text PDF PubMed Scopus (141) Google Scholar). The effect of mutations in the S4-S5 linker in domain IV has previously been investigated in brain, heart, and skeletal muscle (McPhee et al., 1998McPhee J.C. Ragsdale D.S. Scheuer T. Catterall W.A. A critical role for the S4-S5 intracellular loop in Domain IV of the sodium channel α-subunit in fast inactivation.J. Biol. Chem. 1998; 273: 1121-1129Crossref PubMed Scopus (149) Google Scholar). The M1627K mutation alters a conserved methionine residue (Figure 2Biv) that may form part of the inactivation gate receptor, as previous mutagenesis studies in heart (Nav1.5) and skeletal muscle (Nav1.4) channels suggest a role in fast inactivation (Filatov et al., 1998Filatov G.N. Nguyen T.P. Kraner S.D. Barchi R.L. Inactivation and secondary structure in the D4/S4-5 region of the SkM1 sodium channel.J. Gen. Physiol. 1998; 111: 703-715Crossref PubMed Scopus (44) Google Scholar, Lerche et al., 1997Lerche H. Peter W. Fleischhauer R. Pika-Hartlaub U. Malina T. Mitrovic N. Lehmann-Horn F. Role in fast inactivation of the IV/S4-S5 loop of the human muscle Na+ channel probed by cysteine mutagenesis.J. Physiol. 1997; 505: 345-352Crossref PubMed Scopus (67) Google Scholar, McPhee et al., 1998McPhee J.C. Ragsdale D.S. Scheuer T. Catterall W.A. A critical role for the S4-S5 intracellular loop in Domain IV of the sodium channel α-subunit in fast inactivation.J. Biol. Chem. 1998; 273: 1121-1129Crossref PubMed Scopus (149) Google Scholar, Tang et al., 1996Tang L. Kallen R.G. Horn R. Role of an S4-S5 linker in sodium channel inactivation probed by mutagenesis and a peptide blocker.J. Gen. Physiol. 1996; 108: 89-104Crossref PubMed Scopus (83) Google Scholar). In contrast, the arginine at position 996 is not conserved (Figure 2Bi), although the substitution by cysteine is not a conservative one and may have a subtle effect on channel function. This would be consistent with the less severe nature of the phenotype in family 4 and in the father in family 12. The highly conserved nature of seven of the eight amino acids mutated in these patients supports a causative role in the disorder, and their location makes it likely that channel inactivation would be affected. Therefore, we investigated the electrophysiological properties of three of these mutations (I1461T, T1464I, M1627K) using cDNA constructs of hNav1.7 transfected into HEK293 cells (Figure 3). Wild-type hNaV1.7 expressed in HEK293 cells exhibits normal inactivation (Figures 3A–3C). All three mutant channels exhibited altered inactivation (Figures 3D–3F) with associated persistent currents, which were maintained for more than several hundred milliseconds in the two inactivation gate mutants (Figures 3D and 3E). There is a depolarizing (rightward) shift in steady-state inactivation (h∞) curves for all three mutants (Figures 3G–3I) and inactivation is incomplete for I1461T and T1464I. For M1627K, the greater shift and reduction in voltage-dependence would widen the range of potentials over which “window current” (activation-inactivation gating overlap) could operate. We further investigated the properties of T1464I and the in vitro effects of carbamazepine, as symptoms in patients with this mutation are effectively controlled using this drug. There was a depolarizing shift in the voltage-dependence of activation in this mutant (6.8 mV compared with wild-type, Figure 3C). The average inactivation time constant (τh) was less voltage-dependent and the kinetics of this partial inactivation were rapid (see the Supplemental Data available with this article online). The reduced sensitivity of inactivation rate to membrane potential is seen in other sodium channels with mutations in the domain III-IV linker (Cannon, 2000Cannon S.C. Spectrum of sodium channel disturbances in the nondystrophic myotonias and periodic paralyses.Kidney Int. 2000; 57: 772-779Crossref PubMed Scopus (59) Google Scholar). In response to long depolarizing steps, the T1464I persistent current was maintained (Figure 4A). In the presence of carbamazepine, this current was reduced in a concentration-dependent manner (Figures 4A–4C, and the Supplemental Data) while the transient current evoked in the same protocol was spared. The dissociation constant (Kd) obtained from individual data sets is 32.7 ± 8.2 μM (n = 4), assuming that complete block is possible, a value within the range of therapeutic plasma concentrations observed in patients (Breton et al., 2005Breton H. Cociglio M. Bressolle F. Peyriere H. Blayac J.P. Hillaire-Buys D. Liquid chromatography-electrospray mass spectrometry determination of carbazepine, oxcarbazepine and eight of their metabolites in human plasma.J. Chromatogr. B. Analyt. Technol. Biomed. Life Sci. 2005; 828 (Published online October 5, 2005): 80-90https://doi.org/10.1016/j.jchromb.2005.09.019Crossref PubMed Scopus (124) Google Scholar). Although these observations indicate that this mutant channel is sensitive to carbamazepine in vitro (as is the I1461T mutant, see the Supplemental Data), the therapeutic effect in vivo may also depend on its effects on the other sodium channels, including Nav1.8, expressed in the same sensory neurons. The negative activation potential of a PE mutant, I848T (Yang et al., 2004Yang Y. Wang Y. Li S. Xu Z. Li H. Ma L. Fan J. Bu D. Liu B. Fan Z. et al.Mutations in SCN9A, encoding a sodium channel alpha subunit, in patients with primary erythermalgia.J. Med. Genet. 2004; 41: 171-174Crossref PubMed Scopus (559) Google Scholar) (Figure 4D), was unaffected by exposure to 100 μM carbamazepine, demonstrating the molecular basis for the difference in drug effectiveness in PEPD and PE. We have identified eight SCN9A mutations in families with PEPD. All are missense, indicating a gain-of-function or dominant-negative mechanism for the autosomal dominant inheritance displayed in familial cases. PEPD is the second inherited pain disorder to be shown to be attributable to mutations in SCN9A. Nine mutations have recently been shown to be associated with PE (Dib-Hajj et al., 2005Dib-Hajj S.D. Rush A.M. Cummins T.R. Hisama F.M. Novella S. Tyrrell L. Marshall L. Waxman S.G. Gain-of-function mutation in Nav1.7 in familial erythromelalgia induces bursting of sensory neurons.Brain. 2005; 1281: 1847-1854Crossref Scopus (338) Google Scholar, Drenth et al., 2005Drenth J.P.H. te Morsche R.H.M. Guillet G. Taieb A. Kirby R.L. Jansen J.B.M.J. SCN9A mutations define primary erythermalgia as a neuropathic disorder of voltage gated sodium channels.J. Invest. Dermatol. 2005; 124: 1333-1338Crossref PubMed Scopus (146) Google Scholar, Michiels et al., 2005Michiels J.J. te Morsche R.H.M. Jansen J.B.M.J. Drenth J.P.H. Autosomal Dominant erythermalgia associated with a Novel mutation in the voltage-gated sodium channel α-subunit Nav1.7.Arch. Neurol. 2005; 62: 1587-1590Crossref PubMed Scopus (85) Google Scholar, Yang et al., 2004Yang Y. Wang Y. Li S. Xu Z. Li H. Ma L. Fan J. Bu D. Liu B. Fan Z. et al.Mutations in SCN9A, encoding a sodium channel alpha subunit, in patients with primary erythermalgia.J. Med. Genet. 2004; 41: 171-174Crossref PubMed Scopus (559) Google Scholar). In contrast to phenotypes associated with mutations in SCN1A (Meisler and Kearney, 2005Meisler M.H. Kearney J.A. Sodium channel mutations in epilepsy and other neurological disorders.J. Clin. Invest. 2005; 115: 2010-2017Crossref PubMed Scopus (373) Google Scholar), sporadic cases with haplo-insufficiency have not been observed in these SCN9A-related disorders, and variable phenotypic expression is limited within families, suggesting that modifier genes are less important in channelopathies of peripheral nerves than in those of central neurons. Our results suggest that around two-thirds of families with PEPD have mutations in SCN9A, suggesting the existence of locus heterogeneity, as in other sodium channelopathies (George, 2005George Jr., A.L. Inherited disorders of voltage-gated sodium channels.J. Clin. Invest. 2005; 115: 1990-1999Crossref PubMed Scopus (270) Google Scholar). Altered function of hNav1.7 in nociceptive and sympathetic neurons is likely to underlie the pain and flushing common to both PEPD and PE. However, there are marked clinical differences between the phenotypes, as well as differential responses to medication. PEPD is essentially a visceral pain condition, often unprovoked, with marked cranial nerve involvement, whereas PE is a condition primarily affecting the extremities in which the triggers, such as exercise and temperature change, are well documented. To what extent can these differences, including the responses to sodium channel blockers, be accounted for by the functional effects caused by the mutations studied? Three PE mutations have been studied. Two, I848T and L858H (Yang et al., 2004Yang Y. Wang Y. Li S. Xu Z. Li H. Ma L. Fan J. Bu D. Liu B. Fan Z. et al.Mutations in SCN9A, encoding a sodium channel alpha subunit, in patients with primary erythermalgia.J. Med. Genet. 2004; 41: 171-174Crossref PubMed Scopus (559) Google Scholar), cause a significant hyperpolarizing shift in activation, slow deactivation, and increased response to small ramp depolarizations (Cummins et al., 2004Cummins T.R. Dib-Hajj S.D. Waxman S.G. Electrophysiological properties of mutant Nav1.7 sodium channels in a painful inherited neuropathy.J. Neurosci. 2004; 24: 8232-8236Crossref PubMed Scopus (294) Google Scholar). A third mutation, F1449V, produces a smaller hyperpolarizing shift in activation and a depolarizing shift in steady-state inactivation, leading to a lowering of the threshold for single action potentials and high-frequency firing in DRG neurons (Dib-Hajj et al., 2005Dib-Hajj S.D. Rush A.M. Cummins T.R. Hisama F.M. Novella S. Tyrrell L. Marshall L. Waxman S.G. Gain-of-function mutation in Nav1.7 in familial erythromelalgia induces bursting of sensory neurons.Brain. 2005; 1281: 1847-1854Crossref Scopus (338) Google Scholar). These effects are likely to reduce the activation threshold for sensory and nociceptive neurons, causing the chronic hyperalgesia, inflammation, and central sensitization associated with activation through nonnoxious stimulation, e.g. walking. In marked contrast to the functional effects of PE mutations, the three PEPD mutants studied here display marked deficits in sodium channel fast inactivation. Similar effects on fast inactivation have been described for muscle sodium channels in myotonia (Cannon, 2000Cannon S.C. Spectrum of sodium channel disturbances in the nondystrophic myotonias and periodic paralyses.Kidney Int. 2000; 57: 772-779Crossref PubMed Scopus (59) Google Scholar, Wu et al., 2005Wu F.f. Gordon E. Hoffman E.P. Cannon S.C. A C-terminal skeletal muscle sodium channel mutation associated with myotonia disrupts fast inactivation.J. Physiol. 2005; 565 (Published online March 17, 2005): 371-380https://doi.org/10.1113/jphysiol.2005.082909Crossref PubMed Scopus (34) Google Scholar). In PEPD such inactivation deficits may only become apparent after near-normal activation and would promote prolonged action potentials and repetitive firing in response to provoking stimuli, such as stretching and experiencing cold. In either case, it is likely that the role of Nav1.7 as a “threshold channel” is a factor, with Nav1.8 probably contributing to the regenerative current at the nerve ending (e.g., Strassman and Raymond, 1999Strassman A.M. Raymond S.A. Electrophysiological evidence for tetrodotoxin-resistant sodium channels in slowly conducting dural sensory fibers.J. Neurophysiol. 1999; 81: 413-424PubMed Google Scholar, Renganathan et al., 2001Renganathan M. Cummins T.R. Waxman S.G. Contribution of Nav1.8 sodium channels to action potential electrogenesis in DRG neurons.J. Neurophysiol. 2001; 86: 629-640Crossref PubMed Scopus (410) Google Scholar, Carr et al., 2002Carr R.W. Pianova S. Brock J.A. The effects of polarizing current on nerve terminal impulses recorded from polymodal and cold receptors in the guinea-pig cornea.J. Gen. Physiol. 2002; 120: 395-405Crossref PubMed Scopus (34) Google Scholar). Such allelic heterogeneity is not uncommon in the channelopathies. There are several well-documented examples of channels in which different mutational mechanisms lead to clinically distinct phenotypes. These include generalized epilepsy with febrile seizures plus (GEFS+) and severe myoclonic epilepsy of infancy in SCN1A; the skeletal muscle disorders, hyperkalaemic periodic paralysis (HYPP) and myotonia, caused by mutations in SCN4A; and the cardiac disorders long-QT and Brugada syndromes, caused by mutations in SCN5A (reviewed in detail in George, 2005George Jr., A.L. Inherited disorders of voltage-gated sodium channels.J. Clin. Invest. 2005; 115: 1990-1999Crossref PubMed Scopus (270) Google Scholar). It is of interest to note that GEFS+, long-QT, and HYPP, in a situation analogous to that of PEPD, are largely the result of gain-of-function mutations leading to persistent sodium current. That there are clear mechanistic differences between PEPD and PE is supported by their differential responses to drug therapy. Oral mexiletine and topical lidocaine, both local anesthetic/type 1b anti-arrhythmia drugs, have been reported to give relief in PE (Legroux-Crespel et al., 2003Legroux-Crespel E. Sassolas B. Guillet G. Kupfer I. Dupre D. Misery L. Treatment of familial erytheramalgia with the association of lidocaine and mexiletine.Ann. Dermatol. and Venereol. 2003; 130: 429-433PubMed Google Scholar), whereas many PEPD patients respond well to the anti-epilepsy drug carbamazepine. Mexiletine is also effective in skeletal muscle myotonias arising from mutations in hNav1.4 (Mohammadi et al., 2005Mohammadi B. Jurkat-Rott K. Alekov A. Dengler R. Bufler J. Lehmann-Horn F. Preferred mexiletine block of human sodium channels with IVS4 mutations and its pH-dependence.Pharmacogenet. Genomics. 2005; 15: 235-244Crossref PubMed Scopus (26) Google Scholar). It is noteworthy that certain myotonia mutations (e.g., substitutions of R1448) respond well to the drug, an effect ascribed to an increased probability of the channel being in closed-state inactivation and/or enhanced recovery from fast inactivation (Mohammadi et al., 2005Mohammadi B. Jurkat-Rott K. Alekov A. Dengler R. Bufler J. Lehmann-Horn F. Preferred mexiletine block of human sodium channels with IVS4 mutations and its pH-dependence.Pharmacogenet. Genomics. 2005; 15: 235-244Crossref PubMed Scopus (26) Google Scholar). A similar effect on channel kinetics is seen in the PE mutant F1449V, so the response to mexiletine may be mutation-specific. Carbamazepine also blocks sodium channels in a use-dependent manner and has long been established as a treatment for neuropathic pain (Backonja, 2002Backonja M.M. Use of anticonvulsants for treatment of neuropathic pain.Neurology. 2002; 59: S14-S17Crossref PubMed Google Scholar), but has little or no effect in PE (Waxman and Dib-Hajj, 2005Waxman S.G. Dib-Hajj S. Erythromelalgia: A hereditary pain syndrome enters the molecular era.Ann. Neurol. 2005; 57: 785-788Crossref PubMed Scopus (58) Google Scholar). This is consistent with pain in PEPD being precipitated by persistent Na current (because carbamazepine preferentially suppresses persistent Na current, as demonstrated for two of the mutants studied—see the Supplemental Data) in comparison with the pain in PE being caused by lowered activation thresholds. In summary, these observations establish the existence of allelic heterogeneity in a class of sodium channelopathy affecting peripheral neurons. PEPD and PE are both inherited autosomal dominant inflammatory pain conditions, but have entirely distinct phenotypes. Our data demonstrate that these distinct phenotypes are the result of distinct repertoires of mutations leading to either lowered thresholds of activation (PE) or defective inactivation (PEPD) These data further emphasize the critical role of Nav1.7 in human inflammatory pain and explain the differential drug sensitivity of PEPD and PE. This study was approved by the joint UCL/UCLH committees on the ethics of human research. Informed consent was obtained from all participants or their parents. Twelve families and two sporadic cases (family 8 and 9) with PEPD were ascertained using the diagnostic criteria outlined below. PEPD is a rare, autosomal dominant paroxysmal disorder of pain and autonomic dysfunction that presents with four types of painful episode: (1) birth crisis, when babies are born red and stiff; (2) rectal crisis, which is triggered by defecation in infants and young children and by a variety of emotional factors in older children and adults; (3) ocular crisis, which may be provoked, but is more usually spontaneous, and (4) mandibular crisis, which is often triggered by eating and yawning. The distinctive features of this condition are paroxysmal episodes of burning pain in the rectal, ocular, and mandibular areas, accompanied by the autonomic manifestations such as skin flushing, reflex asystolic syncopal events, lacrimation, and rhinorrhoea. In between episodes, physical examination is normal, and investigations such as imaging or tissue biopsy are normal. Diagnosis therefore relies on the history and either direct observation or video of the episodes. The former is, of course, more helpful in adults and the latter in infants and children. Family 1 was originally referred with an erroneous diagnosis of hyperekplexia. Other families were ascertained from previous publications (Mann and Cree, 1972Mann T.P. Cree J.E. Familial rectal pain.Lancet. 1972; 1: 1016-1017Abstract PubMed Scopus (8) Google Scholar, Schubert and Cracco, 1992Schubert R. Cracco J.B. Familial rectal pain. A type of reflex epilepsy?.Ann. Neurol. 1992; 32: 824-826Crossref PubMed Scopus (21) Google Scholar) or by direct contact from those managing affected children. Family 7 is a recently ascertained large pedigree from Berkshire, UK and was selected for linkage analysis. Detailed clinical documentation of this family will be published elsewhere. In all cases, DNA was extracted from venous blood or buccal swabs from patients and unaffected family members using standard methods. Primers flanking a total of 455 polymorphic loci primarily identified from the Généthon map spanning the human genome were used in the genome search in family 7. Primers were obtained from MRC HGMP Resource Centre, Research Genetics, MWG Biotech, and Invitrogen. These microsatellites were genotyped using a fluorescent-based semi-automated method on automated DNA sequencing machines (ABI 373A, ABI 3100). Primers were designed for the mutation screen of SCN9A from the genomic sequence obtained from the Human Genome Browser (http://genome.ucsc.edu/). Intronic primers were designed to flank each exon (or groups of exons if separated by small introns). Exons from each of the eight probands and appropriate controls were amplified and sequenced on an automated DNA sequencer (ABI 373A or ABI 3100) and the sequences aligned and compared using Sequence Navigator v1.0.1. A full-length version of the human Nav1.7 cDNA was cloned into a modified version of the expression vector pcDNA3 (Klugbauer et al., 1995Klugbauer N. Lacinova L. Flockerzi V.H.F. Structure and functional expression of a new member of the tetrodotoxin-sensitive voltage-activated sodium channel family from human neuroendocrine cells.EMBO J. 1995; 14: 1084-1090Crossref PubMed Scopus (262) Google Scholar). For functional analysis, three mutated constructs were made (I1461T, T1464I, M1627K). These single base substitutions were introduced into this wild-type clone by PCR-based site-directed mutagenesis using the QuikChange II XL kit (Stratagene, La Jolla, CA). Correct introduction of these substitutions was confirmed by complete sequencing of the inserts. HEK293 cells were grown in sparse culture in 35 mm diameter Petri dishes. Transfections were carried out in serum-free medium by incubating with pcDNA3 incorporating Nav1.7 cDNA and mutant DNA 0.8–1.3 μg 3 μl lipofectamine 2000, 0.2 μg pBS500 for 2 hr at 37°C. The medium was then replaced with normal culture medium incorporating 5% serum. Recordings were made from 1–2 days later. Conventional voltage-clamp recordings in the whole-cell patch-clamp configuration were made from HEK293 cells 1–2 days following transfection, using an Axopatch 200B amplifier (Axon Instruments, Union City, California, USA) driven from a PC generating pulse protocols (Pclamp 9, Axon Instruments). The extracellular solution contained the following: 140 mM NaCl, 10 mM Tetraethylammonium Cl, 10 mM HEPES, 2.1 mM CaCl2, 2.12 mM MgCl2, 0.5 mM 4-Aminopyridine, 7.5 mM KCl, and 0.1 mM CdCl. The intracellular solution contained the following: 130 mM CsCl, 13 mM CsF, 3 mM EGTA (Na), 10 mM Tetraethylammonium Cl, 10 mM HEPES, 1.21 mM CaCl2, 3 mM ATP(Mg), and 500 μM GTP (Li). The external and internal solutions were buffered to pH 7.2–7.3 with the addition of NaOH and CsOH, respectively. Electrodes were made from thin-walled glass capillaries (Harvard Apparatus, Edenbridge, Kent, UK), with initial resistances of 1–2 MΩ when filled with recording solution. During recording, series-resistance compensation was always used and set to 70% or higher. Families of sodium currents were recorded in response to a series of incrementing depolarizing voltage-clamp steps from a holding potential of −90 mV. Steady-state inactivation protocols comprised an incrementally depolarizing prepulse, 60 or 100 ms in duration, followed immediately by a depolarizing step to +10 mV. Records were usually filtered at 5 KHz (4-pole Bessel) and sampled at 20 or 10 KHz. Current traces were generated by averaging three or more records. Leak subtraction was achieved using a P/N protocol, where five reverse polarity clamp-steps were used to produce the leakage record. TTX was applied to ascertain that the currents were indeed TTX-s sodium currents. The HEK293 cells failed to generate TTX-s currents unless they were transfected with an hNaV1.7 clone. Because HEK293 cells can generate nonlinear leakage currents, records of membrane currents were made both before and after the application of TTX, and the TTX-sensitive component was isolated by off-line digital subtraction. TTX (250 nM) and carbamazepine (up to 200 μM) were applied by local superfusion. Carbamazepine was made up as a stock solution of 50 mM or 200 mM in DMSO. Recordings were made at room temperature. Peak currents were converted to conductances, assuming that the sodium equilibrium potential (ENa) was +70 mV, allowing activation curves to be plotted. Boltzmann relations were drawn according to best-fit parameters (Sigma-Stat) for activation curves and h∞ curves. The maximal block in the presence of carbamazepine was derived from the asymptote of the h∞ curve or (as in the Supplemental Data) by measuring the residual current amplitude at 0 mV. Concentration block data for carbamazepine was described as a rectangular-hyperbola (Langmuir isotherm), assuming that 100% block was achievable, from which the apparent Kd for block could be derived. We wish to thank the numerous clinicians who have aided us in this study: J. Aicardi, N. Bednarek, J. Broomhall, G. Clayden, C. Ferrie, F. Gouthieres, D. Griesemer, M. Kirkpatrick, J. Kerrigan, M. Kyllerman, I. Malmros, D. Makki-Awada, N. Mann, P. Plouin, M. Pollitzer, V. Ramesh, E. Roulet-Perez, M. Rossiter, C. Sainsbury, R. Schubert, J. Stephenson, H. Testard, and V. Wong. This work was funded by The Wellcome Trust (Research Training Fellowship, C.R.F.) and MRC (UK). Download .pdf (.36 MB) Help with pdf files Document S1. Supplemental Figures" @default.
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• W2026551512 title "SCN9A Mutations in Paroxysmal Extreme Pain Disorder: Allelic Variants Underlie Distinct Channel Defects and Phenotypes" @default.
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