All experiments conformed to the guidelines in the National Institutes of Health (NIH) Guide for the Care and Use of Laboratory Animals. Behavioral measurements and gene expression data were collected at the Genomics Institute of the Novartis Research Foundation (GNF) and all procedures were approved by the GNF Institutional Animal Care and Use Committee. Animals were housed in an SPF barrier colony and were maintained on a 12-h light: 12-h dark cycle, housed in groups of 2–4 in standard high efficiency particulate air-filtered polycarbonate mouse cages containing a layer of Bed-o-cob bedding and one cotton nestlet. Food (Pico rodent chow 20; Purina, St Louis, MO, USA) and water were made available ad libitum. Animals used for q-PCR and ELISA experiments were housed under similar conditions as those described above and sacrificed according to guidelines set by the Institutional Animal Care and Use Committee (IACUC) at UNC Chapel Hill.
The 29 strains tested were: 129S1/SvImJ, A/J, AKR/J, BALB/cByJ, BTBR T+ tf/J, BUB/BnJ, C3H/HeJ, C57BL/6J, C57BR/cdJ, C58/J, CBA/J, CE/J, DBA/2J, FVB/NJ, I/LnJ, KK/HIJ, MA/MyJ, MRL/MpJ, NOD/LtJ, NON/LtJ, NZO/HILTJ, NZW/LacJ, P/J, PL/J, RIIIS/J, SJL/J, SM/J, SWR/J, and WSB/EiJ.
Six to eight week old male and female mice of each strain were purchased from the Jackson Laboratory (Bar Harbor, ME, USA). The mice were habituated in quarantine for approximately 6 weeks until they were transferred to the main barrier colony. Mice were habituated to the main colony holding room for at least one week prior to behavioral testing. All behavioral testing was conducted during the light part of the light/dark cycle and occurred between the hours of 8:00AM – 12:00PM. On the day of testing, mice were transported to a quiet anteroom adjacent to the testing room and acclimated for at least one to two hours prior to the start of testing. All testing equipment was cleaned with a light bleach solution (0.1%) in between each animal. Mice were tested in two behavioral cohorts that each went through a separate series of tests. One group of mice were tested in the open field and a second group were tested on the elevated plus maze and light/dark test. Mice were experimentally naïve for both open field and elevated plus maze testing. Animals tested in the light/dark assay had previously been tested in the elevated plus maze.
Open Field Assay (OF)
The open field is a 17″ × 17″ × 13″ arena with a white Plexiglas floor and clear Plexiglas walls (ENV-515-16; Med Associates, St Albans, VT, USA), which is surrounded by infrared detection beams on the x-, y- and z-axes that track the animals' position and activity over the course of the experiment. The apparatus is isolated within a 73.5 × 59 × 59 cm testing chamber fitted with overhead fluorescent lighting (lux level 14). Animals were removed from their home cage, immediately placed in the corner of the open field arena and allowed to freely explore the apparatus for a test interval of 10 min. Animals were scored for a number of behaviors in the open field, including total distance traveled (in cm), ambulatory episodes (number of times animal breaks user-defined number of beams before coming to rest), percent time resting, average velocity (in cm per second), number of rearings and percent time spent in the center of arena (defined as nine square-inch central part of arena). These data were recorded during testing and scored in post-session analyses using commercially available software (Activity Monitor 5.1, Med Associates). We have found in previous studies (Bailey et al., 2008
, Eisener-Dorman et al.
, 2010) that factor analysis can reduce the data from the OF assay to two distinct classes of behaviors - activity-related behaviors and anxiety-related behaviors and rearing behavior represents a separate behavior. For subsequent analyses, we included only one representative behavior from each class in our analysis; total distance (activity), percent time in the center (anxiety) and rearing behavior.
Elevated Plus Maze (EPM)
The elevated plus maze (7001-0336; San Diego Instruments, San Diego, CA, USA) consists of two open arms and two closed arms [26.5″ × 2.5″] that are directly opposing each other. The walls of the enclosed arms completely surround the end of the runway and are 6″ high. The top of the enclosed arms is open to the testing room. The entire apparatus is 15″ high and is placed on the floor for testing. A video camera above the maze captures the animal's location in the maze. Data is collected and analyzed with Actimetrics LimeLight software (Actimetrics, Wilmette, IL, USA). The animals are placed in the center of the maze and allowed to investigate the maze for 5 minutes. Data recorded include distance traveled in each region and percent time spent in the open arms of the maze.
Light/Dark Assay (LD)
The light/dark enclosure (ENV-511; Med Associates) inserts into the Med-Associates open field apparatus and is a light-impermeable box that covers one third of the area of the open field. The dark box has a classic “mouse hole” entry for the animal to enter and exit the box. The animals were placed in the center of the open field directly in front of and facing the dark enclosure and allowed to explore the arena for ten minutes. Transitions between light and dark quadrants and time spent in each quadrant were assessed.
Gene Expression Studies
Eight to ten week old male and female mice of each strain were purchased from the Jackson Laboratory (Bar Harbor, ME, USA). The mice were habituated for one week prior to tissue collection. Mice were sacrificed by cervical dislocation without anesthesia to avoid gene expression differences in response to anesthetic. All dissections were conducted between the hours of 9:00AM to 11:30AM. Prefrontal cortex, amygdala, hypothalamus, hippocampus, nucleus accumbens, striatum and pituitary were dissected as follows: Immediately following euthanasia, mice were decapitated and the whole brain was removed from the skull. The hypothalamus was lifted from the ventral surface of the brain using curved forceps. The pituitary was lifted from the sella tursica in the base of the skull using microforceps. The brain was then transferred, ventral side up, to an ice-cold brain matrix with 0.5mm spacing (505C Braintree Scientific, Braintree, MA, USA). A single razor blade was placed into the first space on the brain matrix and the rostral surface of the brain was placed in the matrix and touching this blade. Thin, double-edged razor blades were placed in the twelve most anterior spaces. Following removal from the matrix, the 0.5 mm brain slices were placed flat onto an ice-cold dissection stage and specific regions dissected using anatomical landmarks as described below. The prefrontal cortex was taken from the slice corresponding to approximately 2.5 mm to 2.0 mm anterior to Bregma. To do so, a “V-shaped” wedge was made just medial to the corpus callosum with the apex terminating at about the lateral ventricle (viewed from the caudal side of the slice). Nucleus accumbens and striatum were taken from the adjacent slice approximately 2.0mm to 1.5mm anterior to Bregma. To isolate nucleus accumbens, 1mm diameter punches were taken just ventromedial to the anterior commissure (AC). For striatum, 1mm diameter punches were taken dorsolateral to the AC, midway between the AC and corpus collosum, and lateral ventricle and corpus collosum. Amygdala was dissected from the slices corresponding to approximately 0.5mm to 1.5mm posterior to Bregma, ventromedial to the ventral boundary of the external capsule and directly below the caudate putamen. Hippocampus was dissected from slices at approximately 1.0mm to 2.0mm posterior to Bregma. The dissected regions included the following - Prefrontal cortex consists of the piriform cortex, secondary motor cortex, cingulate cortex area 1 and medial orbital cortex; nucleus accumbens consists of the anterior portion of the nucleus accumbens core and shell; striatum is a one mm diameter punch of striatum only; amygdala contains all amygdaloid nuclei plus 50% of the piriform cortex and the entire ventral endopiriform nucleus; hippocampus contains hippocampus only as it is easily peeled away from the surrounding tissue.
Gene expression analysis
After dissection tissues were immediately frozen on dry ice and stored in a -80 freezer until RNA preparation. Tissue was pulverized to a fine powder while frozen to obtain a mixture of homogeneous tissue. A small (~10mg) aliquot was homogenized using a rotor-stator (Omni TH polytron, Omni International, USA) homogenizer in Trizol (Invitrogen, Carlsbad, CA, USA) and total RNA purified with RNeasy columns (Qiagen, Valencia, CA, USA). RNA quality was tested using an Agilent Bioanalyzer (Santa Clara, CA, USA) and high quality RNA was pooled from three animals per strain to run one microarray. Male and female samples were pooled separately and processed on separate microarrays. Gene expression analysis was performed according to standard procedures (Su et al., 2004
). RNA was amplified and labeled using the Affymetrix one-cycle target labeling kit. Samples were hybridized to Affymetrix GeneChip 430v2 whole-genome mouse arrays and data were processed using the gcRMA algorithm (Wu et al., 2008
). Raw data were deposited in GEO (http://ncbi.nih.gov/geo
) under series accession GSE20160. In this study, data were filtered to remove probe sets whose expression was either undetectable (maximum expression across strains <200) or invariant across strains (ratio of maximum expression to minimum expression across strains). Although summarization algorithms are designed to be robust to single-probe outliers, the presence of SNPs in the probe sequence could theoretically lead to spurious detection of cis-eQTL. An analysis performed after removing all probes overlapping a SNP in dbSNP from the CDF file resulted in qualitatively similar results.
qPCR Validation of Affymetrix GeneChip array
Microarray data was validated by q-PCR in cDNA prepared from pituitary and cortex RNA from male animals. The first round of q-PCR used RNA remaining from the original samples prepared for the microarray. The sixteen strains (from 48 male animals) used in the first round of q-PCR validation were: BTBR T+ tf/J, C3H/HeJ, C57BL/6J, C57BR/cdJ, CBA/J, CE/J, KK/HIJ, MRL/MpJ, NOD/LtJ, NON/LtJ, NZO/HILTJ, NZW/LacJ, P/J, PL/J, RIIIS/J and SM/J. The most significant change in gene expression was validated by an ABI custom designed Taqman assay in the 3′UTR interval (data not shown). The primer and probe sequences are: Forward primer 5′AAACCCCTCACGGTGAATCC3′, Probe 5′TCTGCACCCAAGAACA3′, Reverse primer 5′CATCTCACCAGTCCCCCTTTTT3′, (Applied Biosystems Incorporated, Foster City, CA, USA). The second round of validation used cortex from four 10 week old males from strains AKR/J, BALB/cByJ, C3H/HeJ and SJL/J. The material was not pooled in the second q-PCR assay. Data was collected from sixteen individual animals, from each of four strains. Animals were housed and sacrificed as previously described. ABI TaqMAN probe Mm00514377_m1, which measures transcript in the coding region, was used for the cortex q-PCR.
Identification of B2 SINE element
BAC clone sequence for 129S6/SvEvTac (AC012399) and C57BL6/J (AC133487) was aligned with Sequencher software. A 234 bp insert was discovered in the 3′UTR. This sequence was run through Repeat Masker (http://www.repeatmasker.org
), which identified it as a B2 SINE element. PCR primers flanking the region (forward primer 5
′ and reverse primer 5
′) were designed using publically available Primer3 software (http://frodo.wi.mit.edu/primer3/
). PCR was performed on both DNA and cDNA. Liver and whole brain cDNA was sequenced and referenced against Comt1 cDNA transcript NM_001111063.1 in 129S1/SvImJ, BALB/cByJ, C57BL/6J, CBA/J and WSB/EiJ.
Development of COMT ELISA assay
COMT1 protein was measured from three pooled brain regions (two male animals), determined by proximity: region HHA (hypothalamus, hippocampus and amygdala), region AS (nucleus accumbens and striatum) and region C (cortex). To detect the levels of COMT1 in brain lysates, the Meso Scale discovery (MSD) electrochemiluminescence (ECL) assay was performed using a MSD Sector Imager 2400 according to the manufacturer's protocol (Meso Scale Discovery, Gaithersburg, MD, USA). The MSD assay is based on a sandwich immunoassay that utilizes ECL to measure protein levels. Frozen brain regions were pulverized to a powder in liquid nitrogen. The pulverized tissue was mixed with RIPA buffer (Pierce, Thermo Fisher Scientific, Rockford, IL, USA, Cat. # PIH9901) and protease inhibitor cocktail (Pierce, Cat. # PI-78430) and were then centrifuged at 13,000 RPM for 30 min at 4 °C to obtain the supernatant. Coomassie Plus (Pierce, Cat. # PI-23200) assays were performed according to manufacturer's protocol to determine the protein concentrations in the lysates. 10 μl of primary antibody at a 1:100 concentration (Abcam, Cambridge, CA, USA Cat. #36144) was spotted in each well of a 96-well plate (MSD, Cat. # MA2400), and allowed to dry at room temperature overnight. The plate was incubated for 1 h with blocking buffer containing 3% bovine serum albumin at room temperature, then 25 mg/ml protein lysates in 50 μl lysis buffer were added to the ELISA plates. The plate was incubated at 4 °C overnight, and washed three times with 150 μl of the MSD wash buffer (50 mM Tris pH 7.5, 1.5 M NaCl, 0.2% Tween-20). 25 μl of the secondary antibody (Abcam, Cat. #51984), diluted 1:10 and labeled with the MSD SULFO-TAG detection antibody solution was added to the wells and the plates were incubated for 2 h at room temperature, while rocking. The plates were again washed three times with the wash buffer and 150 μl of Buffer T from MSD was added to each well. The plates were analyzed with the SECTOR Imager 2400. ECL intensities obtained from the assay were normalized by the blank wells of the secondary antibody to the bovine serum albumin-coated spot with no lysate.
Enzymatic analysis of brain lysates
Whole brains from two 10 week old males from strains 129S1/SvImJ, C3H/HeJ, C57BL/6J, C57BLKS/J, NZB/BlNJ, NZW/LacJ, and PL/J were purchased from Jax Laboratories. Animals were killed by cervical dislocation, brains removed, frozen on dry ice, shipped, and thawed for our dissection of the frontal cortex. Tissue was pooled by strain, refrozen on dry ice, pulverized, and homogenized in 0.1mM CDTA (Sigma Chemical Company, St. Louis, MO, USA) with a 16 gauge syringe. A second round of experiments used non-pooled material from four 10 week old males from strains AKR/J, BALB/cByJ C3H/HeJ, and SJL/J. Animals were housed and sacrificed as previously described. Cortex was dissected from fresh tissue, and frozen on dry ice. Samples were then pulverized and homogenized in 0.1mM CDTA as for the first experiment. In both experiments, homogenized brain lysate was centrifuged at 2000g for 10 minutes and filtrate quantified with the Pierce BCA assay (Thermo Fisher Scientific, Rockford, IL, USA) in accordance with the manufacture's recommendation.
COMT1 activity was assessed with the Normetanephrine ELISA kit (RE59171, IBL, Hamburg, Germany as described (Nackley & Diatchenko 2010
, Nackley et al., 2006
). In this method, a known amount of substrate, L-norepinephrine, is added to a biological lysate. The measure of COMT1 enzymatic activity is the amount of product, normetanephrine (NMN), produced in the reaction of lysate and added substrate. Briefly: after lysates were normalized to equal protein concentrations, 8 μl was incubated with 200μM S-adenosyl-L-methionine (SAMe; ICN Chemical, Aurora OH, USA), 7.5 mM L-norepinephrine (NE; Sigma Chemical Co.) and 2mM MgCL2
in 50mM phosphate buffered saline for 1 hr at 37 C°, final volume 21μl. The reaction was terminated using 20μl of 0.4M hydrochloric acid and 1μl of 330 mM EDTA. 10μl of the halted reaction mixture was then used in the Normetanephrine ELISA kit in accordance with the manufacture's recommendation. Lysates from cortex regions of brains in four strains of the -SINE haplotype and 6 strains of the +SINE haplotype were assayed for COMT1 enzymatic activity. Four technical replicates were conducted per animal or per strain in two separate experiments. C3H/HeJ was run in both experiments and data were normalized to C3H/HeJ COMT1 activity.
A Comt1 cDNA clone in expression vector pCMV-SPORT6 was purchased from the I.M.A.G.E. Consortium (ATCC, Manassas, VA, USA, clone ID 4210097). The clone contained the full length 5′UTR and aligned to the Comt1 NM_001111063 transcript. The full length 3′UTR was not present in the clone. To construct the +SINE and -SINE expression vectors, the truncated 3′UTR was excised by a double digestion of Not1 and Bsu36I. Primers to genomic DNA were designed flanking the 3′UTR genomic region to be ligated into the double digested expression construct. The 5′ primer was 5′ of the Bsu36I cut site, and the 3′ primer had a Not1 linker 3′ of the sequence aligning to genomic sequence. C57BL6/J and WSB/J genomic DNA was PCR amplified for the 3′UTR, resulting in +SINE and -SINE 3′UTR fragment. The amplimer was double digested with Not1 and Bsu36I, gel purified, and ligated into the double digested expression vector. Clones were verified by sequencing the entire insert in both directions.
Transient transfection of Comt1 cDNA clones
This transfection assay has been previously described (Nackley et al., 2007
). A rat pheochromocytoma cell line (PC12) was transiently transfected in 35-mm six well plates using FuGENE 6 Transfection Reagent (Roche, Indianapolis, IN, USA) according to manufacturer's recommendations. The amount of +SINE or -SINE construct was at a concentration of 0.9 μg/ml of media. To account for transfection efficiency, pSV-βGalactosidase vector (Promega, Madison, WI, USA) was co-transfected at 0.1 μg/ml of media. Transfection with empty vector was done for each experiment. Cell lysates were collected approximately 24 hours post-transfection.
Enzymatic analysis of cell constructs
Sample preparation and assay
After removing media, cells were washed once with ice cold 0.9% saline solution (1ml/35 mm well), and collected by scraping the wells (on ice) with 150μl/well of ice cold 0.1mM CDTA. The lysate was collected in 1.8 ml tubes and freeze/thawed (-80 C°/RT) twice. The tubes were centrifuged at 2000g for 10 min and filtrate removed. Filtrate was quantified with the Pierce BCA assay in accordance with the manufacture's recommendation and lysates normalized. The Normetanephrine ELISA kit was also used for the cell constructs, in the same manner as for brain lysates. COMT1 activity was determined after subtracting the amount of NMN produced by endogenous enzymatic assay (transfection with empty vector). COMT1 activity was then normalized for transfection efficiency by measuring the β-galactosidase activity for each lysate. β-galactosidase activity was determined by incubating 50μg of normalized lysate with 2× β-galactosidase buffer (39.3 mM NaH2PO4 •H2O, 154.84 mM Na2HPO4, 4.3 mM MgCl2, 4.45 mM O-Nitrophenyl β-D-galactopyranoside, 12.mM β-mercaptoethanol) in a 100μl reaction for 30 minutes at 37 C°. The lysates were then read with a luminometer at 405nM filter. The read for blank, 50μl 0.1mM CDTA and 50μl 2× β-galactosidase buffer, was subtracted from each lysate.
Genome-wide eQTL mapping
The detailed algorithm underlying the Haplotype Association Mapping (HAM) method has been previously described (McClurg et al., 2007
, McClurg et al., 2006
, Pletcher et al., 2004
). Briefly, HAM uses ANOVA to calculate the strength of genetic associations between an input phenotype and the ancestral haplotype structure (as inferred using a local window of three adjacent SNP alleles across the genome). A weighted bootstrap method was introduced to detect association peaks conditional on the population structure in the mouse diversity panel. At each genetic locus, the association score was represented as the negative log10-transformed P value. A score of −LogP=6 is a maximal score resultant from 106
permutations performed at each locus. This score is not corrected for genome-wide significance. HAM analysis was performed for Comt1
, across 29 strains using the web-based analysis SNPster (http://snpster.gnf.org/cgi-bin/snpster_ext.cgi
) with the expression phenotype transformed to log scale. The genomic mapping of all genes and SNPs was based on Mouse Genome NCBI Build 35 (mm7).
Analysis of B2 SINE element and Comt1 mRNA expression and enzymatic activity
Comt1 mRNA expression data (Affymetrix 430v2 probe set 1449183_at) was analyzed in seven brain regions using analysis of variance (ANOVA) (SPSS, v.16 for Mac, Chicago, IL USA) with sex and SINE status as independent variables.
Replicate enzymatic assays were performed and C3H/HeJ was included in all replicates. COMT1 enzymatic data were normalized to C3H/HeJ. An independent t-test (SPSS) was performed to determine differences in enzymatic activity between +SINE and -SINE strains.
Analysis of behavioral data
A total of 744 mice (355 females from 32 strains and 389 males from 31 strains) were tested in the OF assay and 223 mice (113 female and 110 males from 24 strains each) were tested in both the EPM and the LD assays as described above. Fifty-one percent of the mice tested in the OF were -SINE and forty-nine percent were +SINE. Thirty-nine percent of the mice tested in the EPM and LD assays were −SINE and sixty-one percent were +SINE. The complete data set is available on (http://www.jax.org/phenome
; Project: MPD: 214). The analyses described below were conducted on the behavioral data from individual mice. However, for those results that showed significance with individual test scores we also conducted the analysis using inbred strain means to avoid potential strain bias of uneven numbers of animals between strains. Only results that support significance by strain means as well as with individual behavioral scores were considered to have significant genotype-phenotype associations. Strain mean data and numbers of animals tested for each phenotype is listed in Supplemental Table 1
We used multivariate analysis to account for large numbers of data vectors (Marron, 2007
). This methodology allows us to test the single hypothesis that mice with the +SINE haplotype are behaviorally different from -SINE mice. All of the behavioral data from both cohorts of mice were used in single analyses in both male and female mice. This technique does not assign a P-value
to individual behavioral assays, and contributions from each behavioral assay are visualized by distance weighted discrimination (DWD) plots. After the data is plotted, hypothesis testing then confirms the impressions gained from the visualization of the data. This can be done without the loss of information entailed by classical dimensionality reduction, using a Direction Projection Permutation hypothesis test (DiProPerm) (Benito et al., 2004
, Hu et al., 2006
, Marron, 2007
DWD focuses on two-class discrimination in multi-dimensional space formed by the data. The objective is to find a direction, a loading vector, which best separates the two classes. The variables are the 11 behavioral test scores for all of the individual animals. The length of a bar corresponds to the importance in separating the two classes and a positive loading value indicates that the -SINE haplotype tends to have a higher level of the corresponding variable than the +SINE haplotype, while the negative loading value means the opposite.
The second step projects all behavioral test scores from the +SINE and -SINE haplotypes in a DWD direction to obtain a pair-wise t-statistic. The data projection plots a cloud of data points from each of the two classes, with the Gaussian distribution of both classes under the data points. The Gaussian distribution of all data points is represented above the two curves of +SINE and -SINE Gaussian distribution. A pair-wise t-statistic was then obtained.
The DiProPerm test is used to assess the significance of the t-statistic. All of the data from each behavioral test and each mouse was randomly re-labeled into two classes. This permutation was performed 100 times, and each time there was a new t-statistic. The empirical p-value corresponds to the proportion of the t-statistics of the permutated data at or above the t-statistic (http://www.stat.colostate.edu/~chihoon/FDA_ratPMdata.pdf
Genetic Correlation Analysis with Nociception Assays
To determine if the absence of the SINE element is genetically related to sensitivity in pain models of several fundamental nociceptive modalities, Comt1
SINE status was compared to sensitivity in 22 nociception and hypersensitivity assays previously collected in twelve inbred mouse strains. Six of the strains had the SINE element: A/J, AKR/J, BALB/cJ, C57BL/6J, C57BL/10J and SM/J. Six of the strains did not: 129P3/J, C3H/HeJ, C58/J, CBA/J, DBA/2J and RIIIS/J. Brief descriptions of the assays are found in , with greater detail available on The Jackson Laboratory's Mouse Phenome Database website (http://www.jax.org/phenome
) and in the original reports (Lariviere et al., 2002
, Mogil et al., 1999a
, Mogil et al., 1999b
). Multivariate statistical analyses were used as previously described (Lariviere et al., 2002
) to simultaneously assess the genetic correlations between absence of the SINE element in standard inbred strains and their sensitivity in: six inflammatory, six thermal and one mechanical nociception assay; one mechanical sensitivity assay; and eight mechanical, thermal and afferent-dependent hypersensitivity assays (see ). Pearson product-moment correlation coefficients were calculated between the strain means for each assay and the SINE status of the strain, with 1 = absence and 0 = presence due to the expectation of a negative correlation with pain sensitivity, and with strain means corrected by multiplication by -1 so that higher numbers indicated greater sensitivity. As such, a positive correlation indicates that absence of the SINE element is observed in strains more sensitive in the particular assay. Multidimensional scaling (MDS) and principal components analysis (PCA) were used to visualize all pairwise correlations simultaneously (Systat 13, Chicago, IL, USA). Briefly, in MDS, coordinates in two-dimensional space are reiteratively computed for a set of points representing SINE status and the assays to fit as closely as possible the measured similarities of Pearson correlations using a Kruskal loss function with monotonic regression. High positive correlations are represented as small distances between points, and high negative correlations are represented as large distances between points. Uncorrelated points have intermediate distances between them. In PCA, two linear combinations of the points are constructed, and the weights of the linear combinations are plotted in a two-dimensional space to produce a vector for each point. Highly positively correlated points are represented with vectors with angles close to 0° between them, and high negative correlations as angles close to 180° between vectors. The results can also be viewed in three dimensions to determine if the same groupings are observed as in the two-dimensional representation.
Table 3 Correlations between negative SINE status and strain means for 22 assays of sensation, nociception and hypersensitivity (corrected for sensitivity; positive correlation indicates absence of SINE haplotype associated with increased sensitivity in the assay). (more ...)