An SCN9A channelopathy causes congenital inability to experience pain

Abstract

The complete inability to sense pain in an otherwise healthy individual is a very rare phenotype. In three consanguineous families from northern Pakistan, we mapped the condition as an autosomal-recessive trait to chromosome 2q24.3. This region contains the gene SCN9A, encoding the alpha-subunit of the voltage-gated sodium channel, Na(v)1.7, which is strongly expressed in nociceptive neurons. Sequence analysis of SCN9A in affected individuals revealed three distinct homozygous nonsense mutations (S459X, I767X and W897X). We show that these mutations cause loss of function of Na(v)1.7 by co-expression of wild-type or mutant human Na(v)1.7 with sodium channel beta(1) and beta(2) subunits in HEK293 cells. In cells expressing mutant Na(v)1.7, the currents were no greater than background. Our data suggest that SCN9A is an essential and non-redundant requirement for nociception in humans. These findings should stimulate the search for novel analgesics that selectively target this sodium channel subunit.

Fig. 1. The families used to map the locus for channelopathy-associated insensitivity to pain.

Autozygosity mapping in families 1 and 2 on the left of the diagram enabled the identification of a shared 20 cM homozygous region on chromosome 2q24 (there were no other significant homozygous regions detected). The two-point LOD score was 3.2 at θ = 5 0, for AC064843GT21 and AC092641TG18, but greater for more informative markers, see Supplementary Table 1. Family 3, on the right of the diagram, was used to refine this region to an 11.7-Mb shared homozygous region flanked by the heterozygous markers D2S1353 and AC012594TG25 and defined by the homozygous markers AC064843GT21 and AC092641TG18. Affected individuals are indicated with filled symbols.

Fig. 2. Sequence chromatograms showing the mutations identified in families 1, 2 and 3.

The arrows indicate the site of the mutations.

Fig. 3. Schematic representation of Na_v1.7, the voltage-gated sodium channel α-subunit encoded by SCN9A, and the locations of the identified human mutations.

SCN9A encodes a plasma membrane protein: in the figure, the plasma membrane is shown in grey; the extracellular region is uppermost; and intracellular region below. Na_v1.7 is predicted to fold into four similar domains with each domain comprising six α-helical transmembrane segments (labelled 1–6). Transmembrane segments 5 and 6 are the pore-lining segments and the voltage sensor is located in transmembrane segment 4 of each domain (depicted by a plus symbol). The red arrows indicate the location of the nonsense mutation in each family.

Fig. 4. Patch-clamping experiments to investigate the voltage-gated sodium channel activity of wild-type and truncated Na_v1.7.

a, Constructs used in the patch-clamping experiments (see Supplementary Methods). We cloned wild-type SCN9A and then used site-directed mutagenesis to manufacture three further constructs each containing a family mutation. Each SCN9A construct was sequentially co-transfected with a plasmid containing the auxiliary sodium channel β₁ and β₂ subunits, and only cells clearly expressing DsRed2 (red fluorescence) and EGFP (green fluorescence) were measured for electrical activity. ECMV, encephalomyocarditis virus; IRES, internal ribosome entry site. b, A typical cell used in the patch-clamping experiments, showing both EGFP fluorescence (left) and DsRed2 fluorescence (middle), with the pipette attached in phase contrast (right). c, Left panel: initial current responses to 50-ms voltage steps of 5-mV increments between −70 and +40 mV from a holding potential of −100 mV, in a whole-cell voltage clamp recording applied at ∼0.5 Hz for a cell co-expressing wild-type (WT) Na_v1.7 (top) or Na_v1.7 W897X (bottom) with the β-subunits. The inset shows the voltage pulse protocol. Right panel: current–voltage relationship of the peak currents normalized for cell size (pA per pF) obtained using the experimental set-up shown on the left. Black squares, wild type (n = 13); red circles, I767X (n = 7); blue squares, W897X (n = 7); green diamonds, S459X (n = 5); white diamonds, β-subunits only (n = 5). The red line represents a fit of the wild-type data with a Boltzmann equation y = (A₂ + (A₁ − A₂)/(1 + exp((V_0.5 − x)/k)))(x − V_rev), where V_0.5 = 28.0 mV, k = 4.9 mV, V_rev = 64 mV. d, Left panel: voltage dependence of the steady-state inactivation of wild-type Na_v1.7 plus β-subunits was measured by holding the membrane potential for 500 ms at conditioning voltages from −120 to 0 mV (at 5-mV increments) before stepping to a test pulse at −10 mV for 50 ms. The inset shows the voltage pulse protocol, which was applied at 0.5 Hz. Only the current responses to the test pulse are shown. Right panel: the peak currents obtained as on the left were normalized to the maximum peak current, and plotted against the holding potential applied during the conditioning pulse. The red line represents a fit of the data with a Boltzmann equation y = (A₁ − A₂)/(1 + exp((x − V_0.5)/k)) + A₂, where V_0.5 = −71 mV, k = 5.9 mV, n = 13. Error bars in c and d represent standard errors.