A JOURNAL OF NEUROLOGY
Multiple chronic pain states are associated
with a common amino acid–changing allele
Michael Costigan,1,* Inna Belfer,2,* Robert S. Griffin,1,* Feng Dai,2Lee B. Barrett,1
Giovanni Coppola,3Tianxia Wu,4Carly Kiselycznyk,5Minakshi Poddar,2Yan Lu,6
Luda Diatchenko,7Shad Smith,7Enrique J. Cobos,1Dmitri Zaykin,8Andrew Allchorne,1
Pei-Hong Shen,5Lone Nikolajsen,9Jaro Karppinen,10Minna Ma ¨nnikko ¨,10Anthi Kelempisioti,10
David Goldman,5William Maixner,7Daniel H. Geschwind,3Mitchell B. Max,2,zZe’ev Seltzer6,†
and Clifford J. Woolf1,†
1 FM Kirby Neurobiology Centre, Children’s Hospital Boston and Harvard Medical School, Boston, MA 02115, USA
2 Molecular Epidemiology of Pain Program, Department of Anaesthesiology, University of Pittsburgh, Pittsburgh, PA 15261, USA
3 Department of Neurology, David Geffen School of Medicine, University of California at Los Angeles, CA 90095, USA
4 Centre for Information Technology, National Institute of Health, Bethesda, MD 20892, USA
5 Laboratory of Neurogenetics, National Institute on Alcohol Abuse and Alcoholism, National Institutes of Health, Rockville, MD 20892, USA
6 Comparative Pain Phenomics and Genomics Lab, Centre for the Study of Pain, Faculties of Dentistry and Medicine, University of Toronto, ON,
M5G 1G6, Canada
7 Centre for Neurosensory Disorders, University of North Carolina at Chapel Hill, NC, 27599 USA
8 National Institute of Environmental Health Sciences, Research Triangle Park, Durham, NC 27709, USA
9 Danish Pain Research Centre in Aarhus University Hospital, Noerrebrogade 44, Aarhus DK-8000, Denmark
10 Department of Medical Biochemistry and Molecular Biology, University of Oulu, Aapistie 5A 90220, Oulu, 90014, Finland
*These authors contributed equally to this work.
†These authors contributed equally to this work.
Correspondence to: Inna Belfer,
Department of Anesthesiology,
Molecular Epidemiology of Pain Program,
University of Pittsburgh,
3550 Terrace Street, Scaife Hall A-1310,
Pittsburgh, PA 15261, USA
Not all patients with nerve injury develop neuropathic pain. The extent of nerve damage and age at the time of injury are two of
the few risk factors identified to date. In addition, preclinical studies show that neuropathic pain variance is heritable. To define
such factors further, we performed a large-scale gene profiling experiment which plotted global expression changes in the rat
dorsal root ganglion in three peripheral neuropathic pain models. This resulted in the discovery that the potassium channel alpha
subunit KCNS1, involved in neuronal excitability, is constitutively expressed in sensory neurons and markedly downregulated
following nerve injury. KCNS1 was then characterized by an unbiased network analysis as a putative pain gene, a result
confirmed by single nucleotide polymorphism association studies in humans. A common amino acid changing allele, the ‘valine
risk allele’, was significantly associated with higher pain scores in five of six independent patient cohorts assayed (total of
1359 subjects). Risk allele prevalence is high, with 18–22% of the population homozygous, and an additional 50% heterozygous.
doi:10.1093/brain/awq195 Brain 2010: 133; 2519–2527 |
Received May 11, 2010. Revised June 4, 2010. Accepted June 5, 2010. Advance Access publication August 18, 2010
? The Author (2010). Published by Oxford University Press on behalf of the Guarantors of Brain. All rights reserved.
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At lower levels of nerve damage (lumbar back pain with disc herniation) association with greater pain outcome in homozygote
patients is P=0.003, increasing to P=0.0001 for higher levels of nerve injury (limb amputation). The combined P-value for pain
association in all six cohorts tested is 1.14E?08. The risk profile of this marker is additive: two copies confer the most, one
intermediate and none the least risk. Relative degrees of enhanced risk vary between cohorts, but for patients with lumbar back
pain, they range between 2- and 3-fold. Although work still remains to define the potential role of this protein in the pathogenic
process, here we present the KCNS1 allele rs734784 as one of the first prognostic indicators of chronic pain risk. Screening for this
allele could help define those individuals prone to a transition to persistent pain, and thus requiring therapeutic strategies or
lifestyle changes that minimize nerve injury.
Keywords: neuropathic pain; phenotype; molecular genetics; axonal injury; gene expression
Abbreviations: DRG = dorsal root ganglia; SNP = single nucleotide polymorphism
Not all individuals with nerve injury develop neuropathic pain.
Neuropathic pain is the consequence of maladaptive changes in
the nervous system that lead to spontaneous pain and pain hyper-
sensitivity (Costigan et al., 2009b). Although the risk is higher with
more extensive injuries (Kehlet et al., 2006) and with increased
age at the time of injury (Kristensen et al., 2009)—which we have
recently suggested is connected to control of the immune re-
sponse (Costigan et al., 2009a)—it is not possible to predict
who is more or less susceptible among those with a similar risk
exposure and age. This hinders development, investigation and
application of therapies to prevent the establishment of persistent
pain. Because inbred mouse strain studies indicate a large (50%)
heritable component of neuropathic pain sensitivity (Mogil et al.,
1999), it is likely that genetic risk factors are important. This
cannot be teased out by traditional genetic family history studies
due to the rarity of neuropathic pain-inducing events.
An alternative approach is to look for associations between al-
lelic variations in genes and the degree of pain experienced by
cohorts of patients with neuropathic pain versus controls, using
single nucleotide polymorphism (SNP) association. As yet, no suc-
cessful genome-wide association studies have been performed be-
cause of the complexity of the pain phenotype. However, a
strategy that uses preclinical studies to identify gene candidates
and then tests these for SNP associations in patients has proved
effective. GTP cyclohydrolase 1, the rate limiting enzyme in the
tetrahydrobiopterin synthetic pathway, was identified in injured rat
dorsal root ganglia (DRG) neurons by expression profiling, fol-
lowed by identification of a loss-of-function common haplotype
of GTP cyclohydrolase 1 in humans associated with reduced
post-surgical chronic low back pain (Tegeder et al., 2006), as
well as lowered experimental pain sensitivity in at least three
healthy volunteer cohorts (Tegeder et al., 2006; Naylor et al.,
We have now used the same approach, first identifying a novel
pain-related gene by mining expression profiling data in rodent
neuropathic pain models, and then searching for associations be-
tween polymorphisms in the gene and pain phenotypes in human
cohorts. From an analysis of global gene expression profiles in the
rat DRG, across three distinct neuropathic pain models over five
time points, we have identified KCNS1, a potassium channel
modulatory subunit (also called Kv9.1) as a gene regulated in all
neuropathic pain models tested. KCNS1, by an unbiased network
analysis of the expression profiles, defines a group of genes that
are co-regulated in a number of pain models, many of which are
related to sensory neuron signalling and pain. We then found an
association between a common amino acid–altering KCNS1 poly-
morphism and pain phenotype in five of six independent cohorts.
The combination of a bioinformatic analysis of transcriptional
changes in rodent models and human gene polymorphism associ-
ation studies provides, therefore, a useful strategy to identify pu-
tative pain modulating genes that influence the risk of developing
Materials and methods
Array methods, including details of producing and phenotyping the
pain in the rat models of neuropathy, tissue preparation, RNA extrac-
tion and chip hybridization, have been described previously (Griffin
et al., 2007). Spared nerve injury, chronic constriction injury and
spinal nerve ligation injury were each carried out on three separate
groups of rats in accordance with the Massachusetts General Hospital/
Childrens Hospital Boston animal care regulations. L4 and L5 DRGs
ipsilateral to the nerve injury were dissected. Each cRNA probe was
prepared using pooled tissues from five rats; for each time point three
biologically independent hybridizations were performed (Costigan
et al., 2002).
An iteratively re-weighted least squares outlier-resistant regression
method was used to estimate gene expression levels across each
time point within each nerve injury model (Griffin et al., 2007).
Sammon’s nonlinear mapping was done to display the Euclidean dis-
tance matrix between pairs of conditions in a 2D space. Bootstrap
P-values were calculated. The threshold P-value consistent with a
false discovery rate near 5% was identified as 0.01 (Storey and
Tibshirani, 2003). This yielded a false discovery rate for the spared
nerve injury of 3.7, 7.2% for the chronic constriction injury,
and 1.5% for the spinal nerve ligation. A threshold fold change of
1.25 was imposed for the expression levels averaged across all post-
operative time points, relative to naı ¨ve rat expression levels.
Brain 2010: 133; 2519–2527M. Costigan et al.
Weighted gene coexpression network
The weighted gene co-expression network analysis was performed as
described (Oldham et al., 2006, 2008). Briefly, after selecting genes
present in at least five samples, the absolute Pearson correlation co-
efficients between one gene and every other screened gene were
computed, weighted and used to determine the topological overlap,
a measure of connection strength, or ‘neighbourhood sharing’ in the
network. A pair of nodes in a network is said to have high topological
overlap if they are both strongly connected to the same group of
nodes. In gene networks, genes with high topological overlap have
been found to have an increased chance of being part of the same
tissue, cell type or biological pathway. Network Neighbourhood
Analysis provides a set neighbourhood for an initial seed or node.
Using KCNS1 (Affymetrix probe Y17606) as the chosen seed, the
top 30 nearest neighbours were selected using topological overlap as
a measure of connection strength. We also included the microglial
marker MHC class II alpha (U31598_at) as a control seed. Visual
C++ implementation of the multinodeTOM software can be found
Evidence of population stratification was assayed for in the Maine
lumbar root pain cohort and the Israeli post-amputation stump and
phantom limb pain cohort by Pritchard’s Structure 2.1 using 178 an-
cestry informative markers (AIMs) (Pritchard et al., 2000; Enoch et al.,
Maine chronic lumbar root pain cohort
We collected DNA from peripheral blood samples of 151 Caucasian
adults who had participated in a prospective observational study of
surgical discectomy for persistent lumbar root pain caused by interver-
tebral disc herniation (Atlas et al., 1996, 2001). For phenotyping
methods and socio-demographic details of this cohort see Tegeder
et al. (2006). Briefly, we specified the following single primary end-
point: persistent leg pain over the first postoperative year, as a reflec-
tion of ongoing neuropathic pain and designated it the pain phenotype
for genetic association analysis. Leg pain was assessed on 13 occasions
(at baseline, followed by 3, 6 and 12 months post-surgery, and then
annually through to Year 10), using the following: frequency of ‘leg
pain’ and of ‘leg pain after walking’ in the week preceding data col-
lection, as well as improvements in ‘leg pain’ or in ‘leg pain after
walking’ since surgery (Tegeder et al., 2006). For each patient, we
calculated an area-under-the-curve score for every pain variable in
the first year, and converted these to a z-score by comparing the
patient with the rest of the cohort. The primary pain outcome variable
for association analysis was the mean of these four z-scores per
patient. Genotype–phenotype analysis was done using a prespecified
regression equation, incorporating our assumption that one or
two copies of the rare allele would affect the pain score in an additive
model, and adjusted by the following covariates: sex, age, worker’s
compensation status, delay in surgery after enrolment and the
Short Form-36 General Health subscale. This study, Institutional
Review Board (Atlas et al., 1996), has been approved by the
National Instituteof Dentaland
Israel limb amputation pain cohort
The study was approved by the Institutional Review Boards at Sheba
Medical Centre (Ramat Gan, Israel) and Beit Levinshtein Hospital
(Tel Aviv, Israel) and the Ministry of Health, Israel. DNA and chronic
pain data were collected from 199 Israelis of Jewish origin who had
undergone limb amputations. Of the amputees, 79 had suffered trau-
matic battle-related amputations 10–35 years prior to joining the study
and 120 had a leg amputated for medical reasons, mostly vascular
insufficiency and cancer, between 1 and 5 years before the study.
Each subject was asked to rate the typical intensity of their phantom
limb pain and stump pain episodes and these values were used for
genotype–phenotype association analyses. Israeli Jews originate from
two major ethnicities: ‘Ashkenazi’ (i.e. North- and Eastern-European)
and non-Ashkenazi (‘Sephardi’: North African, South European and
Middle Eastern). The participants of this cohort were of one ethnicity
or the other, none were of mixed origin. To minimize a possible effect
that genetic differences among these ethnicities could introduce into
the association analysis, we modelled the ethnicity as a covariate.
Informed consent was collected from all participants.
Finland sciatica pain cohort
This group consisted of 195 patients referred to the Oulu University
Hospital (Finland) due to sciatica symptoms (Virtanen et al., 2007).
Inclusion criteria to the study were unilateral pain radiating from the
lower back down to below the knee. All patients had MRI-based con-
firmation of having a lumbar disc herniation concordant with sciatica
pain. The primary outcome for the association analysis was leg pain
intensity at baseline, determined with a visual analogue scale, using a
10cm horizontal line and the anchor ‘no pain’ associated with the left
end of the line, and the anchor ‘the highest imaginable pain’ with the
right end. This outcome was adjusted for the following covariates: age,
sex and work compensation. Informed consent was collected from all
participants. The research protocol was approved by the Ethics
Committee of the University Hospital of Oulu, Finland.
Denmark phantom limb pain cohort
Saliva, for analysis of DNA and pain data, was collected from
100 amputees (66 males and 34 females, mean age 59 years) following
the approval of the Central Denmark Region Committee on Biomedical
Research Ethics, Denmark. Of the amputees, 43 had suffered traumatic
amputations and 57 had amputations for medical reasons, mostly vascu-
lar insufficiency and cancer. Nineteen were upper limb amputees, 80
were lower limb amputees, and one had undergone amputation of
both an upper and a lower limb. Visual analogue scale scores of phantom
pain intensity during the typical episode was the primary outcome for the
association analysis, adjusted for covariates (age and sex).
Experimental pain sensitivity in
We genotyped 185 normal volunteers who had previously been phe-
notyped for ratings of experimental pain (Shabalina et al., 2009). The
subjects were all pain-free Caucasian females, 18–34 years of age,
taken from a larger prospective cohort study designed to examine
putative risk factors for the development of temporomandibular joint
disorder. All subjects gave informed consent following protocols
approved by the University of North Carolina (UNC) Committee on
Investigations using Human Subjects. Volunteers were phenotyped
KCNS1 as a marker for pain risk in humansBrain 2010: 133; 2519–2527 |
with respect to their sensitivity to 16 experimental pain procedures cor-
responding to multiple pain modalities, including pressure pain, heat
pain, ischaemic pain and temporal summation of heat pain (i.e.
windup). To obtain a general sensitivity measure for the present ana-
lysis, we converted the raw phenotype values to z-scores and summed
them to create a single, aggregate pain score.
Israel post-mastectomy pain cohort
This study was approved by the Institutional Review Boards at Sheba
Medical Centre (Ramat Gan, Israel), Hadassah University Hospital (Ein
Kerem, Jerusalem, Israel) and the Israel Ministry of Health. The study
group included 529 Israeli women of Jewish origin, some of Ashkenazi
and some of Sephardi ethnicities (none was of mixed ethnicity). They
had unilateral breast cancer and underwent surgical removal of the ma-
lignancy by unilateral radical mastectomy (removal of the whole breast
but not including the underlying chest muscles) or breast-conserving
surgery (lumpectomy) at least one year prior to joining the study. This
operation was accompanied, in all women, by auxiliary lymph node
dissection followed by a combination of adjuvant radiotherapy,
chemotherapy and hormonal therapy. Patients ranged from 22 to 80
years and were on average 52.9 years old. Breast (and auxiliary) sur-
gery resulted in post-mastectomy pain syndrome in ?50% of the
women. Similar rates of post-mastectomy pain syndrome were re-
ported previously for other cohorts, including the higher rates of
chronic pain following lumpectomy compared with mastectomy.
Using a self-administered questionnaire similar to that for the leg am-
putees, the typical chronic pain intensity was assessed on a numerical
rating scale. Informed consent was collected from all participants.
Covariates used in the regression model associating genotype with
phenotype included: surgery type (mastectomy versus lumpectomy),
age at surgery, years since the operation, type of adjuvant treatment
Combining association results
The Truncated Product Method (Zaykin et al., 2002) was used to
combine association P-values for six cohorts. This method takes the
product of P-values that are smaller than a pre-defined threshold (set
to the significance level) and evaluates the distribution of the product
under the null hypothesis. One-sided P-values for an association with
the valine allele were combined, and the result was doubled (Overall
and Rhoades, 1986).
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 (Fig. 1A)
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 Fig. 1B, and their time
course in Fig. 1C. Of the global total of 1238 regulated genes,
124 were co-regulated in all three pain models (Fig. 1B), 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-regulatedgenes, annotatedasparticipating in
neurotransmission and neuronal excitability, were selected for fur-
ther analysis (Fig. 1D, Supplementary Fig. 2), as they may be
directly involved in changes in the somatosensory pathway that
result in pain.
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 sub-
unit 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., 2007; Moss et al.,
2008; Xu et al., 2008). 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., 2004), while
RAB3C modulates synaptic vesicle release (Schluter et al., 2004).
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, mem-
bers of the Kv9 group are electrically silent when expressed alone
but modulate channel properties when forming heteromers with
other K(+) channels (Gutman et al., 2005). The KCNS1 transcript
is expressed in naı ¨ve rats at high levels in a subset of DRG neu-
rons, 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 tran-
scriptome related to changes in sensory function, we performed an
unbiased network analysis of the microarray expression profiles
(Fig. 2) (Oldham et al., 2006, 2008). 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 signal-
ling molecules whose injury-induced regulation may contribute to
the pain phenotype.
To investigate if KCNS1 plays a role in determining pain thresh-
olds and chronicity in humans, we genotyped a seven SNP panel
spanning a 15-kb segment of chromosome 20q12, which com-
pletely encompasses the Kv9.1 gene (Fig. 3A). We first investi-
gated a potential association of KCNS1 haplotypes with leg pain
during the first postoperative year in 151 lumbar discectomy pa-
tients from the Maine Lumbar Spine Study (Atlas et al., 2005).
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 un-
common allele was also associated with greater pain (P=0.03)
(Fig. 3B). In comparison with Ile homozygotes, the relative risk
of failing to achieve a 1-year pain improvement following discec-
tomy 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).
Brain 2010: 133; 2519–2527 M. Costigan et al.
SNP rs734784 accounted for 4.6% of the variance in the pain
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 4.4kb section of DNA in the middle of the KCNS1
coding region which contains both of the positive SNPs (Fig. 3 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., 2000; Enoch
et al., 2006), there was no evidence that population stratification
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 nerve
ligation (blue triangles) are shown with time points as indicated. (B) Venn diagram showing the number of regulated genes meeting fold
difference and statistical thresholds in each pain model (spared nerve injury, chronic constriction injury or spinal nerve ligation).
(C) Temporal expression patterns of genes regulated in these neuropathic pain models within the DRG. Each gene was normalized to mean
0, SD 1 and subjected to k-means clustering. Increased relative expression level is shown by increasing darkness. (D) Genes related to
neurotransmission and neuronal excitability regulated in the DRG. Data shown for each gene are for spared nerve injury (red circles),
chronic constriction injury (green squares), or spinal nerve ligation (blue triangles) post-injury. Each plot is on a log2scale, with the origin at
zero equivalent to 1-fold (i.e. non-regulation). The rat gene symbol, maximum difference from origin on the log2scale, and in parentheses
the maximum linear difference, are indicated. Genes are sorted from maximum downregulation to maximum upregulation. SNI=spared
nerve injury; CCI=chronic constriction injury; SNL=spinal nerve ligation.
KCNS1 as a marker for pain risk in humansBrain 2010: 133; 2519–2527 |
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
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 by red links, ion channels identified as purple, receptors and membrane signalling in pink. (B) Heat map showing regulation of
the genes in the KCNS1 30 nearest neighbours grouped by hierarchical clustering for differential expression. Red on this plot represents
upregulated, with green representing downregulated. To the left are gene names highlighted for function (as above), orange indicates
genes have a published link to pain.
Brain 2010: 133; 2519–2527M. Costigan et al.
significantly associated with this locus (P=0.98 and 0.12, respect-
ively). 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 thresh-
olds in healthy individuals, we genotyped Kv9.1 SNPs rs734784 in
a group of female volunteers subjected to experimental pain
stimuli (Shabalina et al., 2009). 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 (Fig. 4).
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% trunca-
tion threshold, was 1.14E?08.
Rodent and human studies suggest that neuropathic pain suscep-
tibility is genetically linked (Diatchenko et al., 2007; Lacroix-Fralish
and Mogil, 2009; Costigan et al., 2009b). We used a convergent
experimental approach that implicates KCNS1 as a gene marking
the risk of developing neuropathic pain. First, we identified
co-regulated genes within the DRG across three partial peripheral
nerve injury models. KCNS1 was among those involved in neuro-
transmission or neuronal excitability, and was the most downre-
gulated gene in this functional category. KCNS1 expression and
function in the somatosensory system has not been described
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 this study. **Most associated SNP, *lesser
associated SNP. (B) Association of Val allele of KCNS1 with
persistent sciatica after discectomy (Maine chronic lumbar root
pain cohort). At one year after surgery, the proportion of pa-
tients describing their leg pain as improved falls from 90%, for
those with no copies of the Val allele, to 73% of those homo-
zygous for Val. (C) Association of Val allele of KCNS1 with
phantom limb pain following leg amputation (Israeli limb am-
putation pain cohort). Association of Val allele of KCNS1 with
proportion of amputee patients reporting no phantom pain falls
from 45% for those with no copies of Val to 22% of those
homozygous for Val.
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 homo-
zygous for the Val allele the most sensitive and those homozy-
gous for the Ile allele the least. *P50.05.
KCNS1 as a marker for pain risk in humans Brain 2010: 133; 2519–2527 |
previously. This prompted our focus on this gene as a possible
Potassium channels have many functions in neurons, including
setting the membrane resting potential and controlling action po-
tential shape and frequency. Voltage-gated potassium channels
are formed by alpha and beta subunit tetramers. Alpha subunits
are numerous, with at least twelve families (Kv1–12) containing
many members (Gutman et al., 2005). There are three Kv9 sub-
units (KCNS1, KCNS2, KCNS3), but each is incapable of forming
functional homo-multimeric channels in heterologous expression
systems (Stocker et al., 1999). Instead, Kv9 subunits modulate
potassium channel subunits from other families as heteromers.
Expression of Kv9.1 and Kv9.3 suppress the currents mediated
by Kv2 and Kv3 alpha-subunit families (Salinas et al., 1997;
Shepard and Rae, 1999; Stocker et al., 1999).
The effect of a decrease in KCNS1 in injured neurons would
depend on which K(+) channel KCNS1 heteromerizes with, their
inactivation kinetics and changes in expression of other K(+) chan-
nel transcripts after injury. Several studies have noted a reduction
in potassium channel transcript expression in the DRG after per-
ipheral nerve lesions (Ishikawa et al., 1999; Kim et al., 2002; Yang
et al., 2004). Potassium channel openers such as retigabine, a
KCNQ (Kv7.2–7.5) potassium channel opener, and BMS-204352,
a K(Ca) and KCNQ potassium channel opener, are analgesic in
neuropathicpain in animalmodels
Jensen, 2003; Dost et al., 2004).
To determine which genes are most closely co-regulated with
KCNS1 we performed a neighbourhood network analysis, where a
candidate gene is used to seed a network comprising its closest
neighbours in the global expression profile by an iterative process,
to uncover structure in the transcriptome (Oldham et al., 2006,
2008). The genes most closely related to KCNS1 are overwhelm-
ingly involved in membrane signalling, including many ion chan-
nels with published functional links to pain (45%). Together,
the preclinical data point to a possible role for KCNS1 in neuro-
Significant SNP associations with an altered pain phenotype may
occur by chance or be due to population stratification or linkage to
a neighbouring causative gene. However, we argue that this is not
the case for KCNS1 because associations were tested across mul-
tiple independent cohorts and replication was found in five of six
studies. Support for an association is reflected by the study-wide
P-value for association with pain phenotype of 1.14E?08, a value
low enough to be considered significant, even in genome-wide
analyses. The sodium channel subunit Nav1.7 has also recently
been shown to contain a risk marker allele (rs6746030) originally
identified by Estacion et al. (2009). Null mutations within this gene
cause complete loss of pain (Fischer and Waxman, 2010); how-
ever, this more subtle function changing SNP identified across
some of the same cohorts as used in this study (n=1277)
achieved a combined P-value of only 1.00E?04 (Reimann et al.,
2010), highlighting the relative predictive strength of the current
findings. Lack of replication in the post-mastectomy cohort may
be due to the difference in aetiology and pain phenotype, differ-
ent outcome measures or differences in patient treatment proto-
cols (Belfer and Dai, 2010). False positives due to population
stratification are unlikely, as we performed an analysis of
population structure and found no evidence of stratification
(Pritchard et al., 2000; Enoch et al., 2006). Three further associ-
ations obtained in ethnically diverse cohorts minimize the possibil-
ity that these data represent false positives due to this confounder.
Finally, the possibility that the significant associations with KCNS1
SNPs flag a neighbouring gene is unlikely because multiple haplo-
typic analyses find the two SNPs identified in this study contained
within a 4.4kb block completely encompassed within the KCNS1
gene. Further work is required to define if, and how, the ‘valine
risk’ allele alters KCNS1 function or if another change in the
haplo-block is responsible, and how this silent potassium channel
modulates endogenous potassium currents in sensory neurons fol-
lowing nerve injury. In any event, we have found a common allele
occurring in homozygous form in ?20% of the populations
assayed (predominantly Caucasian) that strongly associates with
pain following nerve injury and should prove useful in shaping
treatment strategies (Kehlet et al., 2006). Individuals at higher
risk for developing neuropathic pain need special effort to avoid
nerve damage at surgery, as well as aggressive early treatment in
the presence of an unavoidable nerve lesion, to prevent a transi-
tion from acute to chronic pain.
The authors would like to dedicate this manuscript to the memory
of their friend and collaborator Mitchell Max.
Intramural Research Programme of the National Institutes of
Health (NIH), National Institute of Environmental Health Sciences
to D.Z.; Spanish Ministry for Science and Innovation/Fulbright
programme to E.J.C.; NIH
NS058870 (C.J.W.); Canada Research Chair Programme to Z.S.
Supplementary material is available at Brain online.
Atlas SJ, Deyo RA, Keller RB, Chapin AM, Patrick DL, Long JM, et al.
The Maine Lumbar Spine Study, Part II. 1-year outcomes of
surgical and nonsurgical management of sciatica. Spine 1996; 21:
Atlas SJ, Keller RB, Chang Y, Deyo RA, Singer DE. Surgical and nonsur-
gical management of sciatica secondary to a lumbar disc herniation:
five-year outcomes from the Maine Lumbar Spine Study. Spine 2001;
Atlas SJ, Keller RB, Wu YA, Deyo RA, Singer DE. Long-term outcomes of
surgical and nonsurgical management of sciatica secondary to a
lumbar disc herniation: 10 year results from the maine lumbar spine
study. Spine 2005; 30: 927–35.
Belfer I, Dai F. Phenotyping and genotyping neuropathic pain. Curr Pain
Headache Rep 2010.
Brain 2010: 133; 2519–2527M. Costigan et al.
Blackburn-Munro G, Jensen BS. The anticonvulsant retigabine attenuates
nociceptive behaviours in rat models of persistent and neuropathic
pain. Eur J Pharmacol 2003; 460: 109–16.
Brumovsky P, Shi TS, Landry M, Villar MJ, Hokfelt T. Neuropeptide
tyrosine and pain. Trends Pharmacol Sci 2007; 28: 93–102.
Costigan M, Befort K, Karchewski L, Griffin RS, D’Urso D, Allchorne A,
et al. Replicate high-density rat genome oligonucleotide microarrays
reveal hundreds of regulated genes in the dorsal root ganglion after
peripheral nerve injury. BMC Neurosci 2002; 3: 16.
Costigan M, Moss A, Latremoliere A, Johnston C, Verma-Gandhu M,
Herbert TA, et al. T-cell infiltration and signaling in the adult dorsal
spinal cord is a major contributor to neuropathic pain-like hypersensi-
tivity. J Neurosci 2009a; 29: 14415–22.
Costigan M, Scholz J, Woolf CJ. Neuropathic pain: a maladaptive
response of the nervous system to damage. Annu Rev Neurosci
2009b; 32: 1–32.
Diatchenko L, Nackley AG, Tchivileva IE, Shabalina SA, Maixner W.
Genetic architecture of human pain perception. Trends Genet 2007;
Dickinson T, Fleetwood-Walker SM. VIP and PACAP: very important in
pain? Trends Pharmacol Sci 1999; 20: 324–9.
Dost R, Rostock A, Rundfeldt C. The anti-hyperalgesic activity of retiga-
bine is mediated by KCNQ potassium channel activation. Naunyn
Schmiedebergs Arch Pharmacol 2004; 369: 382–390.
Enna SJ, McCarson KE. The role of GABA in the mediation and percep-
tion of pain. Adv Pharmacol 2006; 54: 1–27.
Enoch MA, Shen PH, Xu K, Hodgkinson C, Goldman D. Using
population stratification. J Psychopharmacol 2006; 20: 19–26.
Estacion M, Harty TP, Choi JS, Tyrrell L, Dib-Hajj SD, Waxman SG. A
sodium channel gene SCN9A polymorphism that increases nociceptor
excitability. Ann Neurol 2009; 66: 862–6.
Fischer TZ, Waxman SG. Familial pain syndromes from mutations of the
NaV1.7 sodium channel. Ann N Y Acad Sci 2010; 1184: 196–207.
Griffin RS, Costigan M, Brenner GJ, Ma CH, Scholz J, Moss A, et al.
Complement induction in spinal cord microglia results in anaphylatoxin
C5a-mediated pain hypersensitivity. J Neurosci 2007; 27: 8699–708.
Gutman GA, Chandy KG, Grissmer S, Lazdunski M, McKinnon D,
Pardo LA,et al.International
Nomenclature and molecular relationships of voltage-gated potassium
channels. Pharmacol Rev 2005; 57: 473–508.
Hruska-Hageman AM, Benson CJ, Leonard AS, Price MP, Welsh MJ.
PSD-95 and Lin-7b interact with acid-sensing ion channel-3 and
have opposite effects on H+- gated current. J Biol Chem 2004; 279:
Ishikawa K, Tanaka M, Black JA, Waxman SG. Changes in expression
of voltage-gated potassium channels in dorsal root ganglion neurons
following axotomy. Muscle Nerve 1999; 22: 502–7.
Kehlet H, Jensen TS, Woolf CJ. Persistent postsurgical pain: risk factors
and prevention. Lancet 2006; 367: 1618–25.
Kim DS, Choi JO, Rim HD, Cho HJ. Downregulation of voltage-gated
potassium channel alpha gene expression in dorsal root ganglia
following chronic constriction injury of the rat sciatic nerve. Brain
Res Mol Brain Res 2002; 105: 146–52.
Kristensen AD, Pedersen TA, Hjortdal VE, Jensen TS, Nikolajsen L.
Chronic pain in adults after thoracotomy in childhood or youth. Br J
Anaesth 2009; 104: 75–9.
define populationsand detect
Lacroix-Fralish ML, Mogil JS. Progress in genetic studies of pain and
analgesia. Annu Rev Pharmacol Toxicol 2009; 49: 97–121.
Mogil JS, Wilson SG, Bon K, Lee SE, Chung K, Raber P, et al. Heritability
of nociception I: responses of 11 inbred mouse strains on 12 measures
of nociception. Pain 1999; 80: 67–82.
Moss A, Ingram R, Koch S, Theodorou A, Low L, Baccei M, et al.
Origins, actions and dynamic expression patterns of the neuropeptide
VGF in rat peripheral and central sensory neurones following
peripheral nerve injury. Mol Pain 2008; 4: 62.
Naylor AM, Pojasek KR, Hopkins AL, Blagg J. The tetrahydrobiopterin
pathway and pain. Curr Opin Investig Drugs 2010; 11: 19–30.
Oldham MC, Horvath S, Geschwind DH. Conservation and evolution
of gene coexpression networks in human and chimpanzee brains.
Proc Natl Acad Sci USA 2006; 103: 17973–8.
Oldham MC, Konopka G, Iwamoto K, Langfelder P, Kato T, Horvath S,
et al. Functional organization of the transcriptome in human brain.
Nat Neurosci 2008; 11: 1271–82.
Overall JE, Rhoades HM. Beware of a half-tailed test. Psychol Bull 1986;
Pritchard JK, Stephens M, Rosenberg NA, Donnelly P. Association
mapping in structured populations. Am J Hum Genet 2000; 67:
Reimann F, Cox JJ, Belfer I, Diatchenko L, Zaykin DV, McHale DP, et al.
Pain perception is altered by a nucleotide polymorphism in SCN9A.
Proc Natl Acad Sci USA 2010; 107: 5148–53.
Salinas M, Duprat F, Heurteaux C, Hugnot JP, Lazdunski M. (1997) New
modulatory alpha subunits for mammalian Shab K+ channels. J Biol
Chem 1997; 272: 24371–9.
Schluter OM, Schmitz F, Jahn R, Rosenmund C, Sudhof TC. A complete
genetic analysis of neuronal Rab3 function. J Neurosci 2004; 24:
Shabalina SA, Zaykin DV, Gris P, Ogurtsov AY, Gauthier J, Shibata K,
et al. Expansion of the human mu-opioid receptor gene architecture:
novel functional variants. Hum Mol Genet 2009; 18: 1037–51.
Shepard AR, Rae JL. Electrically silent potassium channel subunits from
human lens epithelium. Am J Physiol 1999; 277: C412–24.
Stocker M, Hellwig M, Kerschensteiner D. Subunit assembly and domain
analysis of electrically silent K+ channel alpha-subunits of the rat Kv9
subfamily. J Neurochem 1999; 72: 1725–34.
Storey JD, Tibshirani R. Statistical significance for genomewide studies.
Proc Natl Acad Sci USA 2003; 100: 9440–5.
Tegeder I, Costigan M, Griffin RS, Abele A, Belfer I, Schmidt H, et al.
GTP cyclohydrolase and tetrahydrobiopterin regulate pain sensitivity
and persistence. Nat Med 2006; 12: 1269–77.
Virtanen IM, Song YQ, Cheung KM, Ala-Kokko L, Karppinen J, Ho DW,
et al. Phenotypic and population differences in the association
between CILP and lumbar disc disease. J Med Genet 2007; 44: 285–8.
Xu XJ, Hokfelt T, Wiesenfeld-Hallin Z. Galanin and spinal pain
mechanisms: where do we stand in 2008? Cell Mol Life Sci 2008;
Yang EK, Takimoto K, Hayashi Y, de Groat WC, Yoshimura N. Altered
expression of potassium channel subunit mRNA and alpha-dendrotoxin
sensitivity of potassium currents in rat dorsal root ganglion neurons
after axotomy. Neuroscience 2004; 123: 867–74.
Zaykin DV, Zhivotovsky LA, Westfall PH, Weir BS. Truncated product
method for combining P-values. Genet Epidemiol 2002; 22: 170–185.
KCNS1 as a marker for pain risk in humansBrain 2010: 133; 2519–2527 |