Neocerebellar Crus I Abnormalities Associated with a Speech and Language Disorder Due to a Mutation in FOXP2

Demographic details of participants in each of the three time points are outlined in Table 1. All of the affected KE members who were available and eligible for brain imaging had been originally scanned at time point 1 (n = 10). At time points 2 (delay from time point 1: mean = 9.17; SD = 0.45 years) and 3 (delay from time point 1: mean = 11.50; SD = 0.58 years), subsets of those individuals (time point 2: n = 6; time-point 3: n = 4) were available for recruitment.

We combined cerebellum-specific voxel-based morphometry (VBM) and lobular volumetry using SUIT (v. 3.1; http://www.icn.ucl.ac.uk/motorcontrol/imaging/suit.htm; [37]) in SPM12 (v. 6225; Wellcome Department of Cognitive Neurology, London, UK) running in Matlab 2015a (The MathWorks, Inc., Natick, MA, USA). Compared with normalization to the MNI whole-brain template, SUIT provides stronger contrast for the cerebellum, improving fissure overlap among subjects by reducing spatial variance to 1/3.

T₁-weighted images were re-orientated so that the origin coordinates lay over the anterior commissure and segmented into grey matter (GM), white matter (WM) and CSF, using the unified segmentation procedure [45]. The cerebellum and brainstem were isolated and a mask was created per scan, which was manually corrected using MRICron [46] (http://​www.​mccauslandcenter​.​sc.​edu/​mricro/​mricron) as non-cerebellar regions (e.g. transverse sinus, bone marrow) are occasionally misclassified as cerebellar GM [37]. Using SUIT’s DARTEL-algorithm, the cerebellum was deformed to fit the probability maps of cortical GM and WM to an atlas template. Nonlinear deformation was applied to GM segmentation maps, which were modulated to compensate for volume changes during normalization, by multiplying the intensity value in each voxel with Jacobian determinants. The amount of GM signal in normalized images was thus preserved, with VBM statistics reflecting GM volume differences [47, 48]. Images were smoothed with an isotropic Gaussian kernel of 4 mm full-width at half-maximum (FWHM), in line with previous cerebellar VBM studies [49, 50]. GM differences between groups were assessed by voxel-wise t test analyses. Whenever participants in the groups were not individually matched, sex and age were entered as between-subjects covariates (ANCOVA). Comparisons were conducted separately for each subset/time point. Given the small smoothing kernels employed for spatial precision in these analyses, we applied stringent corrections for multiple comparisons (voxel peak- or cluster-level familywise error (FWE)-correction: p < .005) and non-stationary smoothness [51] over an individual voxel threshold of p < .001.

The procedure involved cropping and isolating the cerebellum, SUIT normalization, re-slicing the cerebellar atlas into subject space using the deformation parameters from normalization and calculating the number of voxels in each lobule in the re-sliced images. One of the authors (GPDA) corrected the cerebellar isolation masks and the re-sliced cerebellar atlas while blind to the participant’s identity. This process resulted in volumetric measurements of cerebellar lobules (left, right I–IV, V; left, medial [vermal], or right hemispheric VI, VIIa Crus I, VIIa Crus II, VIIb, VIIIa, VIIIb, IX and X). For all comparisons, volumes were expressed both in cc and as a proportion of cerebellar cortical volume. As in VBM, ANCOVAs were used with age and sex as covariates. We predicted a main effect of group for Crus I volume, and a group × lobule interaction. Comparisons were conducted separately for each time-point. Significance for two-tailed tests was set at p < .05, using Tukey’s honestly significant difference (HSD) to correct for multiple comparisons. Violations of the sphericity assumption (tested by Mauchly’s W) were followed by a Huynh-Feldt correction of degrees of freedom. Hemispheric asymmetry was calculated for Crus I using the formula (right − left/right + left hemisphere) [52].

We examined the correlation (Pearson’s r; SPSS, v. 22) of Crus I volume and asymmetry with the behavioural measures reported to fully dissociate affected from unaffected members [42] and to correlate with the affected members’ caudate volume (time point 1; [8]): (i) Non-word repetition: Participants heard and repeated 40 non-words [53], half of which contained consonant clusters (‘complex’ non-words). The dependent measure was the number of complex non-words accurately repeated; (ii) Non-verbal orofacial praxis: Α rating scale was used to assess the performance of orofacial movements [42]. We predicted that the number of accurately repeated complex non-words and the orofacial movement ratings would correlate with Crus I volumetric measures from the same time-point.

After realignment, preprocessing of EPIs followed the same steps as in cerebellar VBM, apart from modulation. We compared task (non-word repetition) vs. baseline (listening to white noise bursts), using the fixed-effects analysis employed in [11], reporting regions active in the group as a whole (time point 3). We identified regions that were both activated in controls (n = 4; inclusive mask threshold for ‘task > baseline: p < .01) and less/more active in affected members (n = 4). The same stringent FWE corrections were applied as those in our VBM analyses.

In order to explore the structural covariance of the caudate nucleus with lobules that were structurally/functionally abnormal in affected KE family members, we first examined the correlation of the previously measured volumes of the caudate nuclei corrected for total intracranial volume (TICV) from time point 1 [8] with GM volume across the whole brain (VBM regression). The pre-processing pipeline was the same as that for cerebellar VBM, with the exception that GM images were normalized to MNI space by generating a group-specific whole-brain template (DARTEL), and an 8-mm³ FWHM smoothing kernel was used. Age and sex were entered as covariates and total caudate volume as a main effect regressor. We expected to identify clusters in the caudate nuclei bilaterally, given that volumetry and VBM measure the same effects [54], but also in structures the volume of which would covary with the caudate nucleus, probably due to common experience-related plasticity or mutually trophic influences, driven by environmental and genetic factors [48]. We subsequently focused on bivariate correlations (Pearson’s r) between TICV-corrected left/right/total caudate nucleus volumes and TICV-corrected left/right hemispheric/medial/total volumes of lobules that showed structural/functional abnormalities consistently across time-points.

Across time points, affected members showed smaller Crus I volume relative to unaffected members and unrelated controls (Fig. 1; Table 3; time point 1 = − 18%; time point 2 = − 21%; time point 3 = − 21%), but did not differ consistently in any other lobule (Table S2). Results were replicated with (a) age and sex as between-subjects covariates (Table S3); (b) when left, right hemispheric and medial volumes were compared separately (Fig. S1); (c) when volume was expressed as percent cerebellar cortex (Table S3; Fig. S2). Unaffected KE members did not differ from unrelated controls in any lobule (time points 1 and 2).

The VBM analyses showed a number of regions with reduced GM volume in affected KE members relative to unrelated controls and unaffected KE members. These included medial IV–VI and hemispheric portions of VIIb–VIIIb. Nevertheless, the only discrepancy consistent across time points and comparison groups was the GM volume reduction in HVIIa Crus I of affected members (Fig. 2; Tables S4–6; Fig. S3), shown to be bilateral in most comparisons.

The number of accurately repeated complex non-words and the orofacial praxis ratings correlated negatively with VIIa Crus I volumetric measures (Fig. 3), but not with any other lobular volumes (all ps, p > .15). The same negative correlations have already been reported for the right and total caudate nucleus volume [8]. Using a series of partial correlation analyses, we thus sought to examine whether right/total HVIIa Crus I volumes would correlate with these scores above and beyond right/total caudate nucleus volumes, and vice versa. As expected, no significant partial correlation was shown, given the strong positive volumetric correlations of these two structures in affected KE members (Table S7).

There was reduced activity in HVI/HVIIa Crus I bilaterally in affected KE members relative to matched controls during non-word repetition compared with the noise-burst baseline (Fig. 4).

Crus I and caudate nuclei showed structural covariance in affected KE members. This was seen in whole-brain VBM regression, where total caudate volume expectedly correlated bilaterally with GM volume in the caudate nuclei (Fig. S4), but also with regions in the right HVIIa Crus I and the left supplementary motor area (SMA) (Fig. 5). The same relationship was seen between the previously measured volumes of the caudate nuclei [8] and those of Crus I (Fig. 6).