SmartCube found different degrees of separation between 16p11.2 df/+ and Cntnap2 -/- as compared to their control littermates.

<p>A & C: To build a 2D representation of the multidimensional space in which the two groups are best separated, we first find statistically independent combinations of the original features, pick the two new composite features (drf 1 and 2) that best discriminate between the two groups, and used them as x- and y-axes (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0134572#pone.0134572.s014" target="_blank">S4 Methods</a>). As in Principal Component Analyses, these two axes represent the combinations of uncorrelated feature transforms that account for most of the variance. Each dot represents either a WT (blue) or a mutant (red) mouse. The center, small and large ellipses are the mean, standard error and standard deviation of the composite features for each group. The overlap between the groups is used to calculate the discrimination index, which measures how reliably a classifier can be trained to discriminate between the two groups (the more overlap, the worse the discrimination). B & D: To estimate how likely it is that such separation is simply due to chance, the obtained classifier is challenged many times with correctly labeled samples (“WT” and “mutant”; see the green distribution) or with randomized labels (blue distribution). The overlap between these two distributions (in red) represents the probability of obtaining the observed discrimination by chance. A: The 16p11.2 df/+ standard deviation ellipse overlaps considerably with the WT control ellipse. Note the spread and position variability of the individual mice; B: A 65% discrimination between 16p11.2 df/+ and WT mice could be found by chance with p < .12; C: The Cntnap2 -/- model separates well from the WT group; D: A 88% discrimination index for the Cntnap2 -/- versus WT comparison can be found by chance with p < .0002.</p

Number of marbles and activity in the marble burying test.

<p>In the marble burying test mice are presented with a novel diggable medium. Marbles are placed on the surface to help quantify the amount of digging shown by the mice. A: Number of marbles buried was not different between the mutants and their respective WT controls; B: Locomotor activity in 16p11.2 df/+ mice was greater than that WT control mice, whereas the Cntnap2 -/- mice were as active as the corresponding WT mice. Data shown are means ± SEM. (*p < .05).</p

Activity, vocalizations and marking in the urine-exposure open field.

<p>Female-experienced male mice are first habituated to an arena and then presented with a small quantity of urine from a female in estrous. A: During the baseline session 16p11.2 df/+ mice explored the center of the arena more than the WT mice, whereas Cntnap2 -/- showed no differences from its WT control; B: during the exposure session all groups explored the center similarly; C: Ultrasonic vocalizations emitted during the exposure session showed deficits in the Cntnap2 -/- mice but not in the 16p11.2 df/+ as compared to their WT controls; D: Similarly, Cntnap2 -/-, but not 16p11.2 df/+ mice, showed deficits in scent marking during both baseline and urine exposure. Data shown are means ± SEM. (*p < .05; **p < .01).</p

Proportion of total time sniffing a stimulus in the three-chamber social test.

<p>In the three-chamber social test the proportion of the total time sniffing the novel mouse or an alternative (social or not) can be used as a measure of preference, with a dashed line at 50% indicating a lack of preference. A: All groups showed a strong preference for the social stimulus without any genotype effects; B: All groups showed a strong preference for the novel social stimulus, with no genotype effects. Sniffing was scored manually. Data shown are means ± SEM.</p

Mouse preference for a social stimulus versus a non-social stimulus (sociability) and recognition of a social stimulus in the three-chamber test.

<p>A: During the sociability phase the percent time spent in each of the three chambers showed that all groups spent more time in the social chamber as shown by a significant Chamber Type main effect (in a 2-way mixed model Genotype x Chamber Type ANOVA). The Cntnap2 -/- mice spent less time in the side chambers compared to the WT control mice as shown by a significant Genotype main effect (with no significant Genotype x Chamber Type interaction). B: During the sociability phase all groups sniffed more the social container as shown by a significant Stimulus Type main effect (in a 2-way mixed model Genotype x Stimulus Type ANOVA). The Cntnap2 -/- mice spent overall less time sniffing than the WT control mice as shown by a significant Genotype main effect (with no significant Genotype x Stimulus Type interaction). C: During the recognition phase the percent time spent in each of the three chambers showed that all groups spent more time in the novel mouse chamber than in the familiar mouse chamber. The Cntnap2 -/- mice again spent less time in the side chambers compared to the WT control mice (analysis as in A). D. During the recognition phase all groups sniffed more the novel social container. The Cntnap2 -/- mice again spent overall less time sniffing than the WT control mice (analysis as in B). Time in each chamber was recorded automatically, whereas sniffing time was scored manually Data shown are means ± SEM. (Chamber or Stimulus Type main effect: ^###p < .001; Genotype main effect: *p < .05, **p < .01).</p

Time-Course of Tests for Cohort 1.

<p>Time-Course of Tests for Cohort 1.</p

SmartCube found the behavioral features that showed larger contributions to an overall discrimination.

<p>A: Both 16p11.2 df/+ mice and Cntnap2 -/- tended to freeze less than their corresponding WT mice; B: Both 16p11.2 df/+ and Cntnap2 -/- mice tended to approach a stimulus faster than their corresponding WT mice; C: Cntnap2 -/- mice showed considerably more abrupt movements than WT mice; D: Cntnap2 -/- showed more locomotion bouts as compared to the WT mice.</p

Motor coordination and thermoregulation during the neonatal test.

<p>In the righting test mice are placed upside down three times and allowed to right until they are on all four limbs. During the isolation test the number of times mice fall on their sides or roll is used as another measure of motor coordination. Thermoregulation is measured by taking axillary temperature before and after the isolation test. A: Number of righting successes; B: righting latency; C: Number of rolls on a side; D: Difference between the axillary temperature measurements before and after the isolation test. Data shown are means ± SEM. (*p < .05).</p

NeuroCube Results at P30 and P60.

<p>Analysis of NeuroCube data showed that the 16p11.2 df/+ mice were significantly different from the WT control mice overall, and, in particular, for paw imaging and position features. Gait was different only at P60. The Cntnap2 -/- mice were also different overall and, in particular, in their speed and gait features. The numbers shown are the maximal discrimination found between the two groups.</p><p>*Indicates that the associated <i>p</i>-value was less than .05.</p><p>NeuroCube Results at P30 and P60.</p

Body weight during neonatal (A) and postnatal (B) periods.

<p>Simple main effect analysis following a significant Genotype x Age interaction showed significant reductions as compared to corresponding WT controls. Note errors bars are hidden by the graphing symbols. Data shown are means ± SEM. (**p < .01, ***p < .001).</p