Pedigrees of six Japanese familial episodic pain syndrome in Japanese families.

<p>(A) Some ^a)Family 2 and ^b)Family 3 members have been reported previously [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0154827#pone.0154827.ref013" target="_blank">13</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0154827#pone.0154827.ref014" target="_blank">14</a>]. Black and white symbols indicate affected and unaffected individuals, respectively. Gray symbols indicate individuals with unknown phenotypic status. Squares and circles indicate males and females, respectively. Slashes indicate deceased individuals. “P” indicates probands. Blue arrows indicate exome sequenced individuals. * indicates linkage analysis performed individuals. The genotype of <i>SCN11A</i> p.R222H (Family 1, 2, 4, 5 and 6) or <i>SCN11A</i> p.R222S (Family 3) for each individual is illustrated. (B) Sequence chromatography of the identified <i>SCN11A</i> mutations.</p

Three subtypes of clinical manifestations.

<p>Three subtypes of clinical manifestations.</p

Exome analysis filtering process in the three Japanese familial episodic pain syndrome families.

<p>Exome analysis was performed for three affected members in Family 1, four affected and four unaffected members in Family 2, and two affected and one unaffected member in Family 3. Exome data was processed through seven filtering steps: (1) non-synonymous, (2) read depth ≥ 8, (3) not registered in dbSNP135, (4) MAF < 0.01 in Japanese patients from 1000 Genomes database, (5) heterozygote in affected members and not present in unaffected members, (6) located on 3p22 linkage region, and (7) variants in the same gene among all three families. Numbers in boxes represent the numbers of variants after each filtering step.</p

Genome-wide linkage analysis in two Japanese familial episodic pain syndrome families.

<p>Genome-wide linkage analysis was performed for eight affected and five unaffected members in Family 1, and four affected and four unaffected members in Family 2. Parametric linkage analysis was performed using 386 genetic markers (including 382 microsatellite genetic markers) that were 10 cM apart and covered 22 autosomes, as well as additional SNP markers. GeneHunter software was used.</p

Characteristics of Nav1.9-overexpressing ND7/23 cells.

<p>(A) The typical traces of Na⁺ currents under 3 μM TTX treatment among Nav1.9-overexpressing ND7/23 cells (Control, WT, R222S, and R222H) were selected at step pulses from −80–0 mV for 100 ms with 20-mV increments for clarity. Control indicates ND7/23 cells without Nav1.9 transfection. The data were obtained at 28°C. Red-colored traces were obtained at −20 mV step pulse. (B) Current-voltage relationships for each Nav1.9-overexpressing ND7/23 cell (Control <i>n</i> = 5, WT <i>n</i> = 5, R222S <i>n</i> = 5, and R222H <i>n</i> = 4). Step pulses were applied from −120–30 mV for 100 ms in 10-mV increments. (C) Comparison of activation of each Nav1.9-overexpressing ND7/23 cell. The Boltzmann fit correspond to V_1/2 (WT: −44.83 ± 2.44 mV, <i>n</i> = 5, R222S: −39.5 ± 2.09 mV, <i>n</i> = 5, R222H: −42.22 ± 2.91 mV, <i>n</i> = 4).</p

R222S mutation increases excitability in DRG neurons.

<p>(A) Small DRG neuron (< 25μm) responses to 500-ms depolarizing current steps of 10, 110, and 210 pA in WT and R222S mice. The parameter of the first AP obtained during current injections of 210 pA, showing calculated maximum rate of rise (B) and fall (C) of AP firing. Open and closed circles represent WT (<i>n</i> = 4) and R222S mice (<i>n</i> = 5), respectively. (D) Input impedance was measured at an injection current of 10 pA. Open and closed columns represent WT (<i>n</i> = 6) and R222S mice (<i>n</i> = 5), respectively. (E) Comparison of repetitive action potentials between WT and R222S mice. Open and closed circles represent WT (<i>n</i> = 6) and R222S mice (<i>n</i> = 5), respectively. The range of 500-ms-step current injections was 10–210 pA. Data are presented as mean ± S.E.M. (*<i>p</i> < 0.05; two-sided Student’s <i>t</i> test).</p

Three different haplotypes carrying R222H in five families.

<p>Haplotypes in Family 1, 2, 4, 5, and 6 are shown for five <i>SCN11A</i> variants (R222H, P308L, C546C, V909I, and T1609I) and two microsatellite markers flanking <i>SCN11A</i> (D3S1277 [Position: 34,614,226 bp, NCBI build 38.2] and D3S1289 [Position: 54,445,451 bp, NCBI build 38.2]; determined in Family 1 and 2). Variant genotypes were determined by exome analysis or direct sequencing. Microsatellite marker genotypes were determined using the ABI Prism Linkage Mapping Set. Three different haplotypes carrying R222H are represented in orange, blue, and green boxes. Red characters show the minor R222H allele. Blue characters show genotypes not shared by the other families.</p