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Supplementary Information for:
Genetically determined P2X7 receptor pore formation
regulates variability in chronic pain sensitivity
Robert E. Sorge, Tuan Trang, Ruslan Dorfman, Shad B. Smith, Simon Beggs,
Jennifer Ritchie, Jean-Sebastien Austin, Dmitri V. Zaykin, Heather Vander Meulen,
Michael Costigan, Teri A. Herbert, Merav Yarkoni-Abitbul, David Tichauer,
Jessica Livneh, Edith Gershon, Ming Zheng, Keith Tan,
Sally L. John, Gary D. Slade, Joanne Jordan, Clifford J. Woolf,
Gary Peltz, William Maixner, Luda Diatchenko, Ze’ev Seltzer,
Michael W. Salter and Jeffrey S. Mogil
Nature Medicine doi:10.1038/nm.2710
Supplementary Fig. 1. Mean (±S.E.M.) stimulus intensity (Threshold; g) at which mice withdrew from a monofilament pressed against the plantar hind paw, at baseline (BL) and 1, 4, 7, 14, 21 and 28 days after nerve injury (SNI) in 18 inbred mouse strains. For clarity, only ipsilateral data are shown.
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Supplementary Table 1. Top 10 genome-wide associations with SNI-induced mechanical allodynia. Gene p value Protein Gene Expressiona Relevanceb cSNP?c P2rx7 0.00056 purinergic receptor P2X, ligand-gated ion channel, 7 immune cells, microglia, 2 + osteoclasts, uterus Arntl 0.0013 aryl hydrocarbon receptor nuclear translocator-like intestines, cerebellum 0 – Pttg1 0.0019 pituitary tumor-transforming gene 1 B-cells, testes 0 –d Kcnk2 0.0021 potassium channel, subfamily K, member 2 (TREK-1) pituitary, nervous system 2 – Clcn4-2 0.0023 chloride channel 4-2 nervous system 1 –d Gabrg2 0.0025 gamma-aminobutyric acid (GABA) A receptor, subunit γ2 nervous system 1 – Slco3a1 0.0025 solute carrier organic anion transporter family, member 3a1 widespread 1 – Scn8a 0.0028 sodium channel, voltage-gated, type VIII, α (Nav1.6) nervous system 2 – Clpb 0.0028 ClpB caseinolytic peptidase B (E. coli) testes 0 – Ctgf 0.0032 connective tissue growth factor widespread 1 –e aLoci displaying highest relative expression, according to BioGPS gene expression database (http://biogps.gnf.org). bExisting evidence for involvement in pain, based on a literature search of December 20, 2011: 0 - no evidence, 1 – potential involvement, 2 – demonstrated involvement. cNon-synonymous coding SNP (cSNP) causing one or more amino acid changes in strains of different haplotype groups. dAltered amino acid only in DBA/2 strain. eAltered amino acid only in C57BL/6 strain.
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Supplementary Fig. 2. Chromosomal position (in bp) and haplotypes of SNPs in the chromosome 5 haplotype block within the mouse P2rx7gene showing high correlation to neuropathic mechanical allodynia (see main text). Of the 24 SNPs, 19 are intronic and four produce synonymous changes; only P451L (highlighted in yellow) produces a non-synonymous (i.e., amino acid) change.
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Supplementary Fig. 3. Mechanical allodynia after SNI in a set of 15 inbred mouse strains (n = 8–16 mice/strain; all male) common to those in Fig. 1a (“McGill”) but tested in a different laboratory at Massachusetts General Hospital (Dr. Clifford Woolf; “MGH”). Symbols in a represent mean ± S.E.M. threshold (g) to withdraw from von Frey fibers applied to the hind paw (only ipsilateral data shown) before (at -7 and -4 days) and 5 and 7 days after SNI. Bars in b represent the mean ± S.E.M. average percent change (i.e., decrease) in threshold of the two post-operative measures from the two baseline measures. The scatterplot in c shows the statistically significant correlation of strain means (percent allodynia versus average percent change, respectively) in the McGill and MGH cohorts.
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Supplementary Fig. 4. Reversal of SNI (a) and CFA (b) mechanical allodynia by a TAT-P451 but not a TAT-451L peptide in A/J but not B10.D2 mice. Symbols represent mean ± S.E.M. threshold (g) to withdraw from increasing pressure applied to the ipsilateral hind paw (electronic von Frey test). Mice were tested at baseline (BL), at the presumed peak of allodynia (Day 7 after SNI; 24 h after CFA), and 15 and 60 min post-peptide injection. Data presented are from a subset of mice of both strains such that initial allodynia levels (on Day 7 after SNI surgery, and 24 h after CFA injection) are equivalent, but the same results were obtained when all subjects are considered. *P<0.05, **P<0.01 compared to pre-injection baseline on Day 7 (SNI) or 24 h (CFA) by Student’s t-test.
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Supplementary Table 2. Results of the statistical association between pain intensity and genotypes of 3 of the 30 studied SNPs (top) in the PMP cohort (middle) and OA cohort (bottom).
SNP ID rs208294 rs208296 rs7958311 SNP position (bp) 121600253 121600953 121605355 Amino acid change p.His155Tyr c.533+630C>T p.Arg270His Genotypes G/G A/G A/A C/C T/C T/T G/G A/G A/A
Protein types HH HY YY Intronic RR RH HH
PMP Cohort
N 93 191 70 146 171 37 171 160 23 Mean Rating (±SEM) (0–20 NRS)
2.7
(0.47)
3.6
(0.36)
4.9
(0.69)
4.4
(0.19)
3.2
(0.37)
2.2
(0.25)
4.3
(0.21)
3.0
(0.08)
2.4
(0.13)
Beta 1.17 -1.21 -1.19
P value 0.003 0.003 0.006
OA Cohort
N 378 672 279 620 572 136 743 505 81 Freq. of Cases (%) 57.7 55.5 54.5 55.3 55.2 61.0 58.3 52.3 56.8
OR (adjusted) (95% CI) 0.94 (0.79-1.10) 1.05 (0.88-1.25) 0.79 (0.65-0.95)
P value 0.43 0.60 0.015
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Supplementary Fig. 5. Human 270H (rs7958311)-containing P2X7Rs are hypofunctional compared with R270-containing P2X7Rs. 1321N1 cells were transfected with human R270 or 270H containing P2X7Rs, or with transfection reagent only (mock transfection) for 24 h. Cells were then loaded with the dye YO-PRO and following 5 min stable baseline readings, treated with saline or BzATP (300 mM). Pore formation was assessed by YO-PRO dye uptake. (a) Time course of YO-PRO dye uptake upon BzATP stimulation. (b) Rate of YO-PRO dye uptake following 7.5-min treatment with saline or BzATP. BBG (5 mM) was pretreated 5 min prior to application of saline or BzATP. Each experimental group represents n=10. ***P<0.001.
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Supplementary Table 3. Association between haplotypes of the SNPs rs208294 (p.H155Y), rs208296 (c.533+630C>T), and rs7958311 (p.R270H) and PMP (top) and OA (bottom) pain. Significantly associated haplotypes are shown in bold.
aFreq.: haplotype frequency in cohort. bSTAT: coefficient t-statistic. cOR: odds ratio.
PMP Cohort Alleles Beta Freq.a STATb P ATA 155Y; T; 270H 1.58 0.01 1.76 0.186
GTA 155H; T; 270H -0.71 0.20 8.92 0.003 ACA 155Y; C; 270H -0.48 0.05 0.86 0.354 GCA 155H; C; 270H 0.08 0.02 0.01 0.922 GTG 155H; T; 270R -0.41 0.12 1.57 0.211 ACG 155Y; C; 270R 0.70 0.41 10.9 0.001 GCG 155H; C; 270R 0.06 0.18 0.06 0.803
OA Cohort Alleles ORc Freq. STAT P
GTA 155H; T; 270H 0.91 0.19 0.65 0.422 ACA 155Y; C; 270H 0.49 0.04 7.69 0.005 GCA 155H; C; 270H 0.43 0.02 5.34 0.021 ATG 155Y; T; 270R 0.91 0.01 0.04 0.831 GTG 155H; T; 270R 1.30 0.11 3.53 0.060 ACG 155Y; C; 270R 1.02 0.40 0.06 0.807 GCG 155H; C; 270R 1.10 0.22 0.88 0.348
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Supplementary Fig. 6. Prevention (a,c) and reversal (b,d) of nerve injury- (SNI; a,b) and chronic inflammation- (CFA; c,d) induced mechanical allodynia by the P2X7R antagonist, BBG (4 or 120 mg/kg, i.v.). Timing of nerve injury and injections is indicated by the vertical dotted lines. Symbols represent mean ± S.E.M. threshold (g) to withdraw from increasing pressure applied to the ipsilateral hind paw (electronic von Frey test). In every case, a drug-by-repeated measures ANOVA revealed a significant interaction at values of p<0.01). *p<0.05, **p<0.01, ***p<0.001 compared to vehicle at same time point by Student’s t-test. Lower BBG doses were investigated (data not shown), but found to be ineffective at reversal of already developed SNI and CFA allodynia.
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Supplementary Methods
Subjects. Inbred mice of 18 strains (129S1, A, A/He, AKR, B10.D2-H2/oSn, BALB/c,
BALB/cBy, BUB/Bn, C3H/He, C57BL/6, DBA/2, FVB/N, LG, LP, MRL/Mp, NZB/BIn,
NZW/LaC, and SM; all “J” substrains; n=5–6 mice/strain) were obtained from The Jackson
Laboratory (Bar Harbor, ME). A second strain survey, performed independently but using
similar protocols at Massachusetts General Hospital (MGH), included 15 of the strains listed
above (n=8–26 mice/strain). Mice bred in-house were weaned at 18–21 days and housed with
their same-sex littermates; purchased mice were housed same-sex and habituated to the vivarium
for at least one week before testing. Mice were housed in standard shoebox cages, 2–4 per cage,
maintained in a temperature-controlled (20±1° C) environment (14:10 h light cycle; with lights
on at 07:00 h), and fed (Harlan Teklad 2920X) and watered ad lib. All procedures were
approved by local animal care and use committees and were consistent with national guidelines.
von Frey testing. Mice were placed individually in transparent Plexiglas cubicles (5 cm wide ×
8.5 cm long × 6 cm high; each cubicle was separated from the other by an opaque divider) placed
upon a perforated metal floor (with 5 mm diameter holes placed 7 mm apart), and habituated for
at least 2 h before behavioral testing began. Nylon monofilaments (Stoelting Touch Test
Sensory Evaluator Kit #2 to #9, corresponding to 0.015–1.3 g bending force; calibrated weekly
using a microbalance) were firmly applied to the plantar surface of the hindpaw (alternating the
side of the body being tested) until they bowed for 5 s. In subsequent experiments, an automated
von Frey test was used (UgoBasile Dynamic Plantar Aesthesiometer). In this assay, pressure is
gradually increased by the device until the mouse withdraws its hind paw; the maximal pressure
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at that point is displayed. We have found this method to feature less variability than the up-down
technique. Relative strain sensitivities are preserved using both methods (unpublished data).
Haplotype mapping. In HapMapper, SNPs are organized into haplotype blocks; only a limited
number of haplotypes—typically 2, 3 or 4—are present within a haplotype block. The
haplotype-based computational analysis identifies haplotype blocks in which the haplotypic
strain grouping within the block correlates with the distribution of phenotypic data among the
inbred strains analyzed. To do this, a P value for a test that assesses the likelihood that genetic
variation within each block could underlie the observed distribution of phenotypes among the
inbred strains is calculated. The haplotype blocks are then ranked based upon the calculated P
value. A genetic effect score is also calculated from the ANOVA, as: (SSB – (k-1)*MSE) /
(SStotal + MSE), where SSB is the between-group sum-of-squares of the ANOVA model,
SStotal is the total sum-of-squares, k is the number of groups and MSE is within-group
sum-of-squares divided by (n–k), where n is the total number of objects in the model.
Primary macrophage cultures. Peritoneal macrophages were isolated from adult male A/J or
B10.D2 mice. Mice were sacrificed using ether and the peritoneal cavity lavaged with
RPMI-1640 media (Invitrogen). The recovered media from 3–4 mice was pooled, washed twice
with RMPI-1640 media, and centrifuged at 1200 rpm for 10 min at 4 ºC. The cells were then
re-suspended in RPMI-1640 media supplemented with 10% fetal bovine serum (Invitrogen) and
1% penicillin-streptomycin (Invitrogen) and seeded onto poly-D-lysine coated cover-slips and
allowed to adhere for 24 h at 37 °C with 5% CO2 and 95% O2 prior to imaging experiments.
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Human P2X7R subcloning, mutagenesis and expression. Human P2X7R cDNA was obtained
from GeneCopoeia (EX-Z8319-M11) expressed in the pReceiver-M11 vector. Mutagenesis was
performed using PCR (Affymetrix Change-IT Multiple Mutation Site Directed Mutagenesis Kit)
using the primer CACTGCCATCCCAAATACAGTTTCCGTCGCCTTG to convert 809G>A
(R270 to 270H in the protein). All constructs were verified by sequencing. P2X7R R270 or
270H constructs were transfected separately into 1321N1 cells using protocols described
previously (C.J. Gallagher & M.W. Salter, J. Neurosci. 23:6728-6739, 2003). Cells were plated
onto 96-well plates and YO-PRO dye uptake measured as described below.
Calcein dye loss assay for P2X7R-mediated pore formation. Peritoneal macrophages were
incubated at room temperature for 30 min with calcein-AM (2.5 µM; Invitrogen) in extracellular
recording solution (ECS). Following incubation, cells were thoroughly rinsed with ECS without
calcein-AM and mounted on an inverted microscope (Diaphot-TMD, Nikon) and viewed using a
40x epifluorescence Fluor objective lens. Measurement of calcein fluorescence was performed
by means of single-photon counting from individual macrophages using a photomultiplier
detector and the number of photons measured for 3 s every 60 s. Calcein was excited by 490 nm
light generated from a compact xenon arc lamp (75 W). Emitted light was sent to the side
camera port of the microscope, where it entered a dual optical pass adapter (Nikon). Emitted
light (520 nm) was directed through a DM510 bandpass emission filter, after which the light
passed through a manually adjustable aperture and was detected by a photomultiplier tube in
single-photon counting mode (Photon Technologies). The output of the photomultiplier was
sampled at a rate of 5 Hz by an IBM-compatible computer with hardware and software from
Photon Technologies.
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YO-PRO dye uptake assay for P2X7R-mediated pore formation. Peritoneal macrophages were
plated onto a 96-well plate and allowed to adhere for 24 h at 37°C with 5% CO2 and 95% O2
prior to imaging experiments. To initiate P2X7R pore formation, cells were stimulated with
BzATP (300 µM). In the macrophage experiments, BzATP was applied with no YO-PRO dye
(2.5 µM; Invitrogen) present in the ECS. After 10 min of BzATP stimulation, YO-PRO was
then co-applied with TAT-P451 (25 µM), TAT-451L (25 µM), or saline, directly into the ECS.
Measurement of YO-PRO fluorescence was performed every 5 min for a total of 30 min using a
fluorescence plate reader (Molecular Devices). In the experiments using human P2X7Rs
expressed in 1321N1 cells, BzATP was applied after 5 min pre-incubation with YO-PRO and
fluorescence measurements were made every 30 s. YO-PRO was excited at 491 nm and emitted
fluorescence was detected at 509 nm.
Western blot measurement of P2X7R protein expression. Whole brain tissue from A/J or B10.D2
mice was rapidly isolated and and homogenized in RIPA buffer (50 mM Tris-HCl, pH 8.0;
150 mM NaCl, 1 mM EDTA, 1% NP-40, 0.5% deoxycholic acid, 0.1% SDS, 1 mM Na3VO4,
1 U/ml aprotinin, 20 µg/ml leupetin, and 20 µg/ml pepstatin A) and centrifuged at 14,000 rpm for
30 min at 4 °C. The protein concentration was determined using a detergent-compatible protein
assay with a bovine serum albumin as standard. Whole brain homogenates were separated on a
precast sodium dodecyl sulfate polyacrylamide gradient gel (4–12% Tris-HCl; Bio-Rad) and
transferred onto nitrocellulose membrane. The membrane was probed with rabbit anti-P2X7R
(1:1000 Alomone) and mouse anti-actin (1:5,000 Sigma) antibodies. Membranes were washed
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and incubated for 1 h at room temperature in rabbit and mouse specific secondary antibody
before quantification with the Odyssey Licor system (USA).
Fluorescent measurement of intracellular [Ca2+]. Peritoneal macrophages were prepared as
described above. Prior to recording, cells were incubated at room temperature for 30 min with
the fluorescent Ca2+-indicator dye Fura-2 AM (2.5 µM; Molecular Probes) in ECS. Following
fluorophore loading, cells were thoroughly rinsed with ECS lacking Fura-2 AM, and mounted on
an inverted microscope (Diaphot-TMD, Nikon). As previously described in detail (M.W. Salter
& J.L. Hicks, J. Neurosci. 4:2961-2971, 1995), excitation light was generated from a 75 W
xenon arc lamp and passed in an alternating fashion through 340 or 380 nm bandpass filters
(Omega Optical). Emitted light was directed through a 510 nm bandpass filter and the light
collected by a photomultiplier tube detector. Data were acquired by computer at a rate of ∼2.5/s
and were stored on a hard disk. The fluorescence ratio of emitted light with 340 nm excitation to
that emitted with 380 nm excitation was calculated after baseline subtraction. The change in
fluorescence ratio over the initial fluorescence ratio (ΔF/F) was calculated offline. Hardware and
software for Ca2+ measurement were from Photon Technologies.
In vitro pharmacology. Drugs for in vitro experiments were prepared as stock solutions in saline
and stored as single-use aliquots at 4 °C or -20 °C as required. The control groups without drug
received the vehicle. For calcein dye efflux experiments and fluorescent measurement of
intracellular Ca2+ experiments, drugs were applied by bath in the ECS at room temperature
containing: NaCl 140 mM; KCl 5.4 mM; CaCl2 1.3 mM; HEPES 10 mM; and glucose 33 mM,
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pH 7.35, osmolarity315–320 mOsm. Cells were stimulated with 2′(3′)-O-(4-
Benzoylbenzoyl)adenosine 5′-triphosphate triethylammonium (BzATP) (300 µM; Sigma).
Cells were pretreated with TAT-P451 (5 µM) or TAT-451L (5 µM) 30 min prior to BzATP
stimulation. In other experiments, TAT-P451 (25 µM) or TAT-451L (25 µM) was co-applied
with YO-PRO dye 10 min following BzATP stimulation. TAT fusion peptides were obtained
from Advanced Protein Technology Centre Peptide Synthesis Facility (Toronto, ON).
In vivo pharmacology. Experiments using BBG were conducted in outbred, CD-1 mice.
Following baseline testing as described, mice were subjected to SNI surgeries or CFA injection
(see above). Immediately following the confirmation of allodynia development at post-operative
day 7 or 24 h post-injection, respectively, mice were injected intravenously (i.v.) with vehicle
(0.9% saline; 5 ml/kg) or BBG (4, 40 or 120 mg/kg) (n=4–8 mice/dose). We found that blocking
P2X7Rs with BBG both prevented and reversed the development of mechanical allodynia in
CD-1 mice, albeit at different doses.
Because only P451 strains are able to form the pore, if pore formation is responsible for the
greater allodynia in P451 strains, one would predict that only TAT-P451 given to A/J mice
would reverse allodynia. Immediately following the confirmation of SNI- or CFA-related
allodynia development at post-operative day 7 or 24 h post-injection, respectively, A/J and
B10.D2 mice were injected i.v. with saline (1 ml/g body weight), TAT-P451 (30 nmol) or
TAT-451L (30 nmol) (n=6–13 mice/strain/peptide). Data are expressed as a percentage of the
maximum possible anti-allodynia from the peptide at its peak effect, calculated as a ratio of the
pre- vs. post-peptide injection difference compared to the baseline vs. pre-injection
(postoperative day 7 for SNI; 24 h post-CFA).
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Human pain cohorts. The two cohorts —including 354 Israeli women having previously
undergone breast surgery to remove a malignant tumor and axillary lymph node dissection, and
743 osteoarthritis patients (plus 586 controls)—were genotyped. The studies were approved by
the ethics review board of the Israeli Ministry of Health and University of North Carolina,
respectively. In order to compare these cohorts directly, we made an a priori decision to analyze
only the most obviously comparable pain intensity measure in each cohort: intensity of a typical
pain episode in the post-mastectomy pain cohort and WOMAC pain subscale in the osteoarthritis
cohort (see below).
PMP Cohort. Subjects were recruited from two large oncology institutes at the Hadassah
Medical Center, Jerusalem, and the Sheba Medical Center, Ramat Gan, who had undergone
breast surgery to remove a malignant tumor and axillary lymph node dissection at least a year
prior to the study, and who were at least 6 months after the last radio-, chemo-, and hormonal
adjuvant therapy. The pain intensity outcome was rated on a numerical rating scale (NRS) that
ranges from 0 (‘no pain’) to 20 (‘maximal pain imaginable’) (mean NRS = 3.7; SD = 5.3). Using
these ratings we found that about half of the women have chronic PMP syndrome that
manifested as patient-specific episodes of pressing, piercing, crushing, pinching and electrical
shock-like pain in the missing breast and surgical scars, that may spread to the chest, arm and
shoulder.
OA Cohort. This cohort was made up of subjects of both sexes (66% female for cases, 55%
female for controls) extracted from the Johnston County Osteoarthritis Project, an ongoing
population-based prospective study of OA of the knee and hip in a rural North Carolina county.
Civilian, non-institutionalized African-American and Caucasian individuals 45 years of age and
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older were recruited by probability sampling of 6 townships in Johnston County, NC, between
May 1991 and December 1997. All participants had two interviewer-administered home
interviews, a limited clinical and functional examination, and X-rays of the knees; women over
the age of 50 years and all men also underwent hip radiography. Only mean scores from the five
pain-specific items (on a 5-point Likert scale) are reported (mean = 8.2; SD = 3.7).
Genotyping. For the PMP cohort, the genotype data was cleaned by removing samples with
>80% failed genotypes resulting in the call rate over 98%; none of the markers showed a
significant deviation from Hardy-Weinberg equilibrium. Multiple regression analysis was used
to identify significant covariates affecting pain levels using the GLM procedure in PLINK v1.07,
adjusting for covariates (i.e., age at surgery, and time since the operation, which robustly
affected pain traits in this cohort).
For the OA cohort, samples were filtered based on call rate <98%, inconsistency between
self-report and genotypic sex, race, and cryptic relatedness. SNP markers were removed for call
rate <98%, discordance across repeated samples>0, minor allele frequency <0.005%, and test of
Hardy-Weinberg equilibrium p<10-4. The final overall call rate for the study was 99.96%.
Association between SNP genotype and symptomatic OA (WOMAC pain subscale values >3)
was assessed by logistic regression in PLINK v1.07, using a model that included sex, age, BMI,
collection site, and genotyping batch as covariates.
Statistical analyses. A criterion α level of 0.05 was adopted in all cases. Behavioral data were
first analyzed by repeated measures ANOVA. In some cases, raw data were integrated over time
by calculating the area over the threshold x time curve using the trapezoidal method; from these
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areas, percentage of maximum possible allodynia was calculated. Data from three mice in the
strain survey were omitted from further analysis because they were identified as statistical
outliers (Studentized residual > 3). For in vitro experiments, statistical significance between
treatment groups was determined by ANOVA followed by a Student Newman-Keuls post hoc
test. Haploview 4.2 (Broad Institute, Cambridge MA) was used for linkage disequilibrium
analysis, using phased HapMap (Phase III) data. Proportion of variance explained by genetic
variants was calculated using the liability threshold model for the binary OA phenotype.
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