The family pedigree is shown in Fig. 1. Six family members (two unaffected and four affected) were available for clinical and genetic studies approved by the Yale Human Investigations Committee.
The proband, a 13-year-old boy (II-1), was the product of an unremarkable pregnancy and delivery. He was diagnosed with cerebral palsy because of leg stiffness and delayed walking at 18 months old. Cognitive development was normal; his medical history was significant for esotropia. At 4 years old, he was hospitalized with increasing leg pain, stiffness, and inability to walk during the course of a flu-like illness. Creatine kinase (CK) was elevated at 520 U/l acutely (normal <195 U/l), but was normal when repeated interictally. Examination showed periorbital myokymia, mild abdominal and leg muscle hypertrophy, leg stiffness, spastic gait, hyperreflexia, and bilateral Babinski sign. Magnetic resonance imaging (MRI) of the brain and spine were normal. Metabolic studies including electrolytes, amino and organic acids, carnitine, ammonia, lactate, and thyroid functions were normal. Routine studies of cerebral spinal fluid and an electroencephalogram were normal. Needle EMG of deltoid and iliopsoas muscles showed irregular, polyphasic continuous motor unit discharges with normal interference pattern.
Additional family members
Six family members were evaluated. Four affected members (mother and three sons) had myokymia on clinical examination, EMG examination, or both. Nerve conduction velocities were normal. There was no history of seizures or episodic ataxia in any of them. The mother (I-2, Fig. 1) is a 40-year-old woman who had had muscle twitching since childhood, hyperreflexia, bilateral Babinski sign, and tendency to run on her toes. Her husband is unaffected and had a normal neurological exam. A 12-year-old son (II-2) also had delayed walking, leg stiffness, and was diagnosed with cerebral palsy. At age 3 years, he had an episode of prolonged generalized paralysis with no muscle response to tetanic stimulation after general anesthesia for esotropia surgery. He recovered within 1 day. MRI of the brain and spine were normal. An 8-year-old daughter (II-3) is unaffected. A 5-year-old son (II-4) had symptoms identical to his brothers and was hospitalized twice for episodes of increased muscle stiffness with presumed viral gastroenteritis, elevated CK during both episodes (606 U/l and 997 U/l) but had normal CKs measured while well. Carbamazepine treatment resulted in marked improvement of muscle symptoms in three of the brothers who remain asymptomatic. The mother, with milder symptoms, declined treatment. Three family members with myokymia had esotropia; one patient required three eye surgeries.
Genomic DNA was extracted from blood samples or buccal swabs. The coding region of the single-exon 1,448-bp gene KCNA1 (GenBank sequence NM_000217) was amplified. Polymerase chain reactions (PCRs) were performed in a 25-μl reaction volume containing 50 ng genomic DNA, 50 ng of each primer, 1.5 mM MgCl2, 200 μM dNTPs, 1× PCR buffer (Finnzyme), and 2.5 U of Finnzyme using a GeneAmp PCR System 9700 (Applied Biosystems, Foster City, CA). Cycling parameters consisted of a modified touchdown (stepdown) protocol as follows: an initial hot start denaturation at 94°C for 5 min (hot start); 94°C for 45 s, annealing temperature (three cycles each at 70, 67, 64, 61,and 58°C) for 45 s, and extension at 72°C for 2 min for a total of 15 cycles followed by 25 cycles of 94°C for 45 s, 55°C for 45 s, 72°C for 1 min; a final extension step of 72°C for 10 min followed by a 4°C hold. PCR products were separated by electrophoresis on a 1% agarose gel and visualized by ethidium bromide staining. PCR products were purified by spin column (Qiagen, Valencia, CA) or by enzymatic method (ExoSAP-IT, USB, Cleveland, OH) and analyzed for mutations by automated sequencing (Applied Biosystems, Foster City, CA). The c.1355A>C transversion resulted in the creation of a DdeI restriction site, so the presence of the sequence variant was tested by sequencing and confirmed with restriction digest in all subjects. Restriction digests were performed with DdeI (New England Biolabs, Beverly, MA) at 37°C for 2 h.
Oocyte expression and electrophysiological studies
Human Kv1.1 cDNA was subcloned into Kpn1 and HindIII sites of psGEM. The Kv1.1-T226K mutation was introduced by site-directed mutagenesis and confirmed by automated sequencing. cRNAs were synthesized using an mMessage mMachine kit (Ambion, Austin, TX) and quantified by spectroscopy.
Oocytes were isolated from Xenopus laevis and defolliculated by collagenase treatment. Each oocyte was injected with either 5 ng of human Kv1.1-WT, 5 ng Kv1.1-T226K, or 2.5 ng WT plus 2.5 ng T226K cRNA. Whole-oocyte currents were measured with two electrode voltage clamp techniques (Oocyte Clamp, Warner Instruments, Hamden, CT) with constant perfusion (1 ml/min, solution exchange\3 s) after 16 h. Data were sampled at 1 kHz and filtered at 0.25 kHz. Standard bath solution was ND-96 (in mM): 96 NaCl, 2 KCl, 1 MgCl2, 1.8 CaCl2, and 5 HEPES/NaOH, pH 7.5.