Oligonucleotide microarrays were used to measure changes in mRNA expression in rat DRG in three models of neuropathic pain. Global expression profiles post-nerve injury ( A) showed that time was less important than type of nerve injury. The relative distribution of regulated genes (Supplementary Table 1
) across models is shown in B, and their time course in C. Of the global total of 1238 regulated genes, 124 were co-regulated in all three pain models (B), and therefore may potentially contribute to the phenotype common to the three models, i.e. mechanical and cold hypersensitivity (Supplementary Fig. 1
). To analyse the 124 genes, they were grouped into functional categories (Supplementary Fig. 2
). Ten co-regulated genes, annotated as participating in neurotransmission and neuronal excitability, were selected for further analysis (D, Supplementary Fig. 2
), as they may be directly involved in changes in the somatosensory pathway that result in pain.
Figure 1 Global and functional DRG expression profiles in three neuropathic pain models. (A) Multidimensional scaling plot of the similarities among the microarrays. Data post-spared nerve injury (red circles), chronic constriction injury (green squares) and spinal (more ...)
We considered the existing literature on these 10 candidate genes. All of the six upregulated genes in this functional class have previously been shown to undergo transcriptional regulation in response to nerve injury. These include the neuropeptides NPY, GAL, ADCYAP1 and VGF, the alpha 2 delta Ca(2+) channel subunit CACNA2D1 and the GDNF receptor GFRA1, all of which are linked to the pathogenesis of neuropathic pain (Dickinson and Fleetwood-Walker, 1999
; Brumovsky et al.
; Moss et al.
; Xu et al.
). Of the four downregulated genes, only a reduction in the GABA-A receptor GABRG1 is implicated in neuropathic pain hypersensitivity (Enna and McCarson, 2006
). LIN7B encodes a PDZ domain protein that modulates the acid sensing ion channel ASIC3 (Hruska-Hageman et al.
), while RAB3 C modulates synaptic vesicle release (Schluter et al.
). The remaining downregulated gene, which showed greatest relative decrease among co-regulated genes, is KCNS1 and has not previously been studied in pain.
KCNS1 encodes the K(+) channel subunit Kv9.1. In common with alpha subunits of the Kv5, Kv6 and Kv8 subfamilies, members of the Kv9 group are electrically silent when expressed alone but modulate channel properties when forming heteromers with other K(+) channels (Gutman et al.
). The KCNS1 transcript is expressed in naïve rats at high levels in a subset of DRG neurons, most of which are neurofilament 200 positive, but TrkA and peripherin negative (Supplementary Fig. 3
To see if regulation of KCNS1 reflects a structure in the transcriptome related to changes in sensory function, we performed an unbiased network analysis of the microarray expression profiles () (Oldham et al.
). We used KCNS1 as a seed, and identified its 30 nearest co-associated neighbours in the network. Among the neighbouring genes, 83% (24 of 29) were expressed by neurons, while 79% (23 of 29) were involved in membrane signalling. Furthermore, 45% (13 of 29) have a published link to pain (Supplementary Table 2
). This gene analysis protocol, therefore, places KCNS1 in a group of neuronal signalling molecules whose injury-induced regulation may contribute to the pain phenotype.
Figure 2 (A) Weighted gene co-expression network analysis/neighbourhood network analysis. KCNS1 was used as a seed and the 30 nearest neighbours were identified, using topological overlap as a measure of connection strength with directly connected genes identified (more ...)
To investigate if KCNS1 plays a role in determining pain thresholds and chronicity in humans, we genotyped a seven SNP panel spanning a 15-kb segment of chromosome 20q12, which completely encompasses the Kv9.1 gene ( A). We first investigated a potential association of KCNS1 haplotypes with leg pain during the first postoperative year in 151 lumbar discectomy patients from the Maine Lumbar Spine Study (Atlas et al.
). Associations with two SNPs were statistically significant: rs734784, in which the allele coding for valine was associated with greater pain than the alternative allele coding for isoleucine (P
= 0.003); and rs13043825, an adjacent synonymous SNP in which the uncommon allele was also associated with greater pain (P
= 0.03) (B). In comparison with Ile homozygotes, the relative risk of failing to achieve a 1-year pain improvement following discectomy was 2.4 for two copies of the Val allele [95% confidence interval (CI): 1.2–4.5] and 1.3 for one copy (95% CI: 0.7–2.6). SNP rs734784 accounted for 4.6% of the variance in the pain endpoint.
Figure 3 (A) Locations of seven genotyped SNPs on coding DNA strand of KCNS1. Coding exons are shown as solid blocks. The SNPs with significant association of pain phenotype are marked. Also marked in yellow is the position of the haplotype block identified in (more ...)
The two significant SNPs were then genotyped in a cohort of 199 amputees with phantom limb pain. The Val allele at rs734784 was again associated with the intensity of phantom limb pain (P = 0.00012) as well as stump pain (P = 0.0033). SNP rs734784 accounted for 7.8% of the variance in phantom limb pain and 6.3% in stump pain, respectively. The adjacent SNP, rs13043825, was not significant for stump pain (P = 0.056) or for phantom limb pain (P = 0.094).
Detailed analysis of the haplotype structure of the genome in and around the KCNS1 gene, using three different algorithms, all produced essentially the same result. There is a strongly co-inherited 4.4 kb section of DNA in the middle of the KCNS1 coding region which contains both of the positive SNPs ( and Supplementary Fig. 4
). We have therefore identified a KCNS1 haplotype variant associated with pain phenotype.
After characterizing each patient’s ethnic background by typing 186 ancestry informative markers (Pritchard et al.
; Enoch et al.
), there was no evidence that population stratification biases contributed to the results in the Maine or Israel limb pain cohorts (Supplementary Fig. 5
Further validation was obtained in two other independent neuropathic pain cohorts. In the first cohort, of patients with sciatica pain (the Finnish cohort), the Val allele at rs734784 was associated with more severe sciatica pain prior to discectomy (P = 0.04, adjusted for a significant gender effect); however, pain post-surgery or change in pain following surgery was not significantly associated with this locus (P = 0.98 and 0.12, respectively). In the second cohort, comprising Danish limb amputees, the rs734784 SNP was associated with more severe phantom limb pain (P = 0.01, adjusted for age and gender). We also genotyped SNP rs734784 (Val) in a post-mastectomy pain cohort, but found no evidence of an association with pain intensity (P = 0.74).
To investigate if KCNS1 plays a role in determining pain thresholds in healthy individuals, we genotyped Kv9.1 SNPs rs734784 in a group of female volunteers subjected to experimental pain stimuli (Shabalina et al.
). The scores of all 16 experimental pain measurements were normalized and an aggregate pain score was calculated for each subject. The effect of rs734784 genotype was significant (P
= 0.0360), using ANOVA with a genotypic model [F
(2,182) = 3.3851], with homozygotes for Val allele showing greater sensitivity to painful stimuli ().
Figure 4 Association of Val allele of KCNS1 with acute pain in healthy volunteers (UNC experimental pain cohort). Combined z-score of all experimental assays (18 measures) shows additive correlation of differences in pain thresholds with those homozygous for the (more ...)
We then tested the hypothesis that ‘the KCNS1 valine allele is associated with increased pain sensitivity’ over all six independent cohorts. To do this we combined the five positive associations with the negative post-mastectomy result to determine a study-wide P-value, using the truncated product method. The combined P-value for the six cohorts (1359 subjects), using the 5% truncation threshold, was 1.14 E−08.