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. 2013 Nov 7;93(5):957-66.
doi: 10.1016/j.ajhg.2013.09.016. Epub 2013 Oct 24.

Gain-of-function mutations in SCN11A cause familial episodic pain

Xiang Yang Zhang  1 , Jingmin Wen, Wei Yang, Cheng Wang, Luna Gao, Liang Hong Zheng, Tao Wang, Kaikai Ran, Yulei Li, Xiangyang Li, Ming Xu, Junyu Luo, Shenglei Feng, Xixiang Ma, Hongying Ma, Zuying Chai, Zhuan Zhou, Jing Yao, Xue Zhang, Jing Yu Liu
Affiliations

Gain-of-function mutations in SCN11A cause familial episodic pain

Xiang Yang Zhang et al. Am J Hum Genet. .

Abstract

Many ion channel genes have been associated with human genetic pain disorders. Here we report two large Chinese families with autosomal-dominant episodic pain. We performed a genome-wide linkage scan with microsatellite markers after excluding mutations in three known genes (SCN9A, SCN10A, and TRPA1) that cause similar pain syndrome to our findings, and we mapped the genetic locus to a 7.81 Mb region on chromosome 3p22.3-p21.32. By using whole-exome sequencing followed by conventional Sanger sequencing, we identified two missense mutations in the gene encoding voltage-gated sodium channel Nav1.9 (SCN11A): c.673C>T (p.Arg225Cys) and c.2423C>G (p.Ala808Gly) (one in each family). Each mutation showed a perfect cosegregation with the pain phenotype in the corresponding family, and neither of them was detected in 1,021 normal individuals. Both missense mutations were predicted to change a highly conserved amino acid residue of the human Nav1.9 channel. We expressed the two SCN11A mutants in mouse dorsal root ganglion (DRG) neurons and showed that both mutations enhanced the channel's electrical activities and induced hyperexcitablity of DRG neurons. Taken together, our results suggest that gain-of-function mutations in SCN11A can be causative of an autosomal-dominant episodic pain disorder.

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Figures

Figure 1
Figure 1
Pedigree Structures, Genotypic, and Restriction Analysis in Two Chinese Families with Episodic Pain Individuals with pain disease are indicated by solid squares (males) or solid circles (females). Unaffected individuals are indicated by open symbols. Deceased individuals are indicated by slashes (/). The proband is indicated by an arrow. (A) Microsatellite haplotypes spanning the linkage region on chromosome 3p22.3–p21.32 identified in the whole-genome scan from the JLTH family. Genotypes for markers D3S1277, D3S3623, D3S1298, D3S3521, D3S3517, D3S3685, and D3S1581 are shown below each symbol. The black vertical bar (including markers D3S3623, D3S1298, D3S3521, and D3S3517) indicates the haplotype cosegregating with the disease. (B) The NciI restriction analysis showing full segregation of the c.673C>T mutation with the disease phenotype in the JLTH family. (C) Linkage analysis from the HBBJ family showed the markers D3S1298, D3S3521, and D3S3685 cosegregating with the disease, but D3S1581 did not. (D) The HindIII restriction analysis showing full segregation of the c.2423C>G mutation with the disease phenotype in the HBBJ family.
Figure 2
Figure 2
Diagram of Chromosome 3 Showing the Critical Region and Identification of SCN11A Mutations from the JLTH and HBBJ Families (A) The linkage intervals and flanking markers of the critical region are indicated. (B and C) DNA sequence chromatograms showing the different heterozygous mutations in SCN11A identified in our study families. (D) Schematic structure diagram of the Nav1.9 protein with a summary of pain-associated mutations.
Figure 3
Figure 3
Alterations p.Arg225Cys and p.Ala808Gly Increase Current Densities of hNav1.9 Channels and Leave Voltage Dependence for Activation Unchanged when Overexpressed in Mouse DRG Neurons through Electroporation (A) Representative whole-cell recordings evoked by voltage for wild-type channels (left) and mutant channels p.Arg225Cys (middle) and p.Ala808Gly (right), respectively. Currents were elicited by 200 ms test pulses stepped from −90 to −40 mV with 5 mV increments. Note the pipette solutions contained 135 mM fluoride. Vh = −120 mV. The recording was performed at least 10 min after achieving whole-cell recording with fluoride to reduce the time-dependent shift of the parameters of current properties. (B) Current-voltage relationships of p.Arg225Cys and p.Ala808Gly versus the wild-type for data in (A). The peak current density (normalized by membrane capacitance) is plotted (n = 8–15, p > 0.05). The endogenous responses from mouse DRG neurons activated by the same voltage protocol are displayed in hexagons (6 of 16 cells). Data are presented as mean ± SEM. (C) Mean activation time constant fit by a single exponential equation is plotted against voltage (n = 8–15). There was no significant change for the time-to-peak values from each other (p > 0.05). (D) Summary plot of relative current at the voltage ranging from −60 mV to −40 mV for WT and two substitutions. For the relative currents, persistent sodium currents (measured at the end of stimulation) were normalized by the respective maximum activation currents (the peak amplitude for activation). (E) Normalized conductance-voltage relationships. Boltzmann fits correspond to V1/2 = −59 ± 0.7 mV and k = 5.1 ± 0.5 mV for wild-type (n = 16), to V1/2 = −57.7 ± 1.4 mV, k = 5.7 ± 0.7 mV for p.Arg225Cys (n = 20), and to V1/2 = −60.4 ± 0.9 mV, k = 4.8 ± 0.6 mV for p.Ala808Gly (n = 18), respectively. (F) Steady-state inactivation curves of wild-type, p.Arg225Cys, and p.Ala808Gly are plotted. Steady-state availability data were derived by measuring the peak current amplitude evoked by −50 mV after 500 ms pre-pulse to voltages ranging from −120 mV to 0 mV. The smooth lines are fits to a Boltzmann equation giving values of V1/2 of −57.3 ± 0.7 mV, −60.6 ± 0.9 mV, and −56.5 ± 0.9 mV for wild-type, p.Arg225Cys, and p.Ala808Gly, respectively (p > 0.05). All experiments in the figure were carried out in the presence of 135 mM fluoride in the pipette solutions and 1 μM TTX in the bath solutions.
Figure 4
Figure 4
Both Alterations p.Arg225Cys and p.Ala808Gly Increase the Excitability of DRG Neurons (A) Current-clamp responses of DRG neurons expressed with wild-type, p.Arg225Cys, and p.Ala808Gly channels to 500 ms current pulse injection, respectively. Amplitude of injected current is indicated on the top of each panel. (B) Statistics plot showing no significant changes for RMP (resting membrane potential) and Vthreshold (the threshold at which action potential take-off occurs) from DRG neurons expressed with wild-type channels. RMP of neurons expressed with WT (−46.5 ± 0.8 mV, n = 40), p.Arg225Cys (−45.6 ± 1.3 mV, n = 20; p > 0.05), and p.Ala808Gly (−42.8 ± 1.2 mV, n = 25; p > 0.05) was not significantly different. Voltage threshold of DRG neurons expressed with WT (−21.4 ± 0.5 mV, n = 40), p.Arg225Cys (−23.5 ± 0.6 mV, n = 20; p > 0.05), and p.Ala808Gly (−23.7 ± 0.6 mV, n = 25; p > 0.05) was not significantly altered, either. (C) Comparison of averaged frequency of action potential (AP) firing (numbers of action potential firing) evoked by 500 ms current injection ranging from 0 to 225 pA in DRG neurons untransfected or overexpressed with empty pcDNA3.1 vector, wild-type, p.Arg225Cys, and p.Ala808Gly channels (∗p < 0.05 as compared to wild-type).
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