In this study, we have shown that a mutation in a highly conserved amino acid residue of the SCN9A sodium channel alpha subunit is associated with a wide clinical spectrum of seizure phenotypes in a single large family. These phenotypes include simple FS, self-limited afebrile seizures, and temporal lobe epilepsy. The SCN9A-N641Y segregating mutation in our large K4425 FS family, and the significantly reduced seizure threshold and enhanced kindling acquisiton rate phenotypes conferred uniquely by the same mutation introduced into the Scn9a-N641Y knockin mouse, provide strong evidence that SCN9A has a role in central excitability and is disease-causing in this family.
In addition, we find supporting evidence for a multifactorial etiology of Dravet syndrome by uncovering concurrent variants in both SCN9A and SCN1A in a subset of our patients. Our findings of numerous variants in separate FS and Dravet syndrome cohorts are statistically significant (p<0.05) when the frequency of the combined specific altered residues found in patients is compared to those same residues in controls, but not statistically significant when all variants found in FS and Dravet syndrome patients are compared to all variants found in controls. While our findings provide highly suggestive evidence of the role of SCN9A in FS and Dravet syndrome, replication in multiple cohorts, combined with functional studies, is needed to confirm a hyperexcitable role of SCN9A in unrelated epilepsy patients. The multifactorial etiology of Dravet syndrome proposed by many investigators suggests that it is very likely that genes responsible for Dravet syndrome will far outnumber SCN1A and SCN9A.
The corneal stimulation paradigm is a reliable and reproducible measure for inducing seizures to test the efficacy of anticonvulsant drugs 
. A Kcnq3
and two separate Kcnq2
mouse models as well as the models for the Wolf-Hirschhorn deletion syndrome have helped to validate the corneal stimulation paradigm as a robust seizure susceptibility test 
. Here, we show that homozygous knockin Scn9aN641Y/N641Y
mice are significantly more susceptible than wild-type mice to seizures that activate either the forebrain or hindbrain. Both the clonic and tonic-clonic generalized seizures characteristic of FS patients are induced by lower stimulation currents in mice that harbor the p.N641Y mutation in Scn9a
. In the corneal kindling model, repeated application of an initially subconvulsive electrical stimulus results in progressive escalation of the stimulus-induced epileptic activity, culminating in a partial seizure that secondarily generalizes 
. This model of partial epilepsy successfully validated a first-in-class neurotherapeutic agent based on galanin for treating pharmacoresistant epilepsy 
and differentiated knockin mutations known to cause childhood epilepsy. Indeed, mutations in the Kcnq2
subunits that underlie M-current channels were recently shown to significantly increase the rate of corneal kindling 
. Our model adds to the growing list of other specific human epilepsy knockin mice, including the Gabrg2 
, Kcnq2 
, Kcnq3 
, Scn1a 
and Chrna4 
mice, to report a clear-cut genotype to phenotype seizure susceptibility.
In multiple published studies, some Dravet syndrome patients inherit SCN1A
mutations from asymptomatic or mildly affected parents, making multiple mutations in this syndrome a likely finding 
. Furthermore, modifying alleles may preferentially be found in Dravet syndrome patients with SCN1A
mutations that are less deleterious when compared to complete heterozygous loss of function mutations. This is indeed that case for the majority of our Dravet syndrome patients with SCN9A variants. Six out of seven of our Dravet syndrome patients with SCN9A
variants harbor either missense or splice site mutations in SCN1A
while a sizable portion of published SCN1A
mutations are predicted to lead to truncated proteins 
. Our results support the idea that some SCN9A
variants when found alone might be asymptomatic or cause infrequent febrile seizures due to incomplete penetrance and variable expressivity, but likely contribute in a multifactorial fashion to Dravet syndrome. Indeed, a recent finding of almost 100 unique missense SCN1A
mutations challenges the previously held notion that haploinsufficient SCN1A
mutations alone are responsible for Dravet syndrome because many of these missense mutations likely confer only partial, rather than complete, heterozygous loss of function 
. Our results now suggest that Dravet syndrome may be included in the list of disorders with “modifier” genes that include Huntington's disease and cystic fibrosis 
. Additional new functional data that examines the two gene mutations will be required to test if the “two-hit” hypothesis is valid in certain Dravet syndrome patients.
None of our FS or Dravet syndrome variants overlaps with the SCN9A
disease-associated changes found in the extreme pain or insensitivity to pain disorders 
. Furthermore, in all published studies of PE, PEPD and CIP, an increased incidence of seizures is not reported in patients with SCN9A
. After follow-up questioning, none of the 21 affected members of K4425 reported the easily recognized extreme pain phenotypes associated with some SCN9A
missense mutations. PEPD is often misdiagnosed as epilepsy because tonic non-epileptic seizures are a particular feature in infancy and early childhood. However the “slow-flat-slow” ictal EEG pattern associated with profound syncope in PEPD patients is clearly not epileptiform, whereas EEGs in K4425 patients are epileptiform 
. Another distinguishing feature is that PEPD attacks are provoked by physical stimulation and not by hyperthermia as seen in FS.
The notion that dysfunction in the same ion channel can be associated in distinct paroxysmal phenotypes is already known for SCN9A
. In 17 of 18 patients with SCN9A
missense mutations published to date, the rectal, ocular and mandibular pain seen in PEPD does not overlap with the severe burning hand and foot pain characteristic of PE 
. We now extend the tissue specificity of paroxysmal Nav
1.7 malfunction to the central nervous system. Additional support for discrete phenotypes resulting from the same ion channel protein comes from the identification of SCN1A
mutations in either epilepsy or familial hemiplegic migraine 
, and CACNA1A
mutations in familial hemiplegic migraine, episodic ataxia and spinocerebellar ataxia 
. Experimentally, the Nav
1.7 p.L858H PE mutation causes hyperexcitable sensory neurons and hypoexcitable sympathetic neurons, and these opposing electrical properties are a result of neuron specific physiologically coupled proteins 
. A unique complement of Nav
1.7 interacting proteins or second messenger pathways in the central nervous system may also explain how the same gene previously implicated with peripheral pain can also be associated with an epilepsy phenotype, but this hypothesis will require further study.