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Rett Syndrome (RTT), caused by mutations in Methyl CPG binding protein-2 (MeCP2) gene, is a disorder of neuronal maturation and connections. Our aim was to prospectively examine fractional anisotropy by diffusion tensor imaging (DTI) and correlate with certain clinical features in patients with RTT.
Thirty-two RTT patients underwent neurological assessments and DTI. Thirty-seven age-matched healthy female controls were studied for comparison. Using a 1.5-Tesla MR unit, DTI data was acquired, and fractional anisotropy (FA), was evaluated to investigate multiple regional tract-specific abnormalities in RTT patients.
In RTT, significant reductions in FA were noted in the genu and splenium of corpus callosum and external capsule, with regions of significant reductions in the cingulate, internal capsule, posterior thalamic radiation, and frontal white matter. In contrast, FA of visual pathways was similar to controls. FA in superior longitudinal fasciculus, which is associated with speech, was equal to controls in RTT patients with preserved speech (phrases and sentences) (p=0.542) while FA was reduced in those RTT patients who were non-verbal or speaking only single words (p<.001). No correlation between FA values for tracts and clinical features like seizures, gross or fine motor skills and head circumference were identified.
DTI, a non-invasive technique of assessing white matter tract pathology, may add specificity to assessment of RTT clinical severity that is presently based on classifying MeCP2 gene mutation and X-inactivation.
Rett Syndrome (RTT), a neurodevelopmental disorder that predominantly affects females, is caused by mutations in the MeCP2 gene located at Xq281, 2. RTT occurs in one in 22,000 women and RTT patients appear to develop normally up to six months of life3, 4. Nonetheless, deceleration in the velocity of head growth occurs as early as four months of life up to two years leading to microcephaly in most patients5. They lose ability to use words, and develop poor hand use with cognitive and motor deficits.
Pathological studies show that brain weight in RTT is reduced when compared to controls 6. This decrease in weight of the brain is not generalized. It is more significant in the cerebral hemispheres and less prominent in the cerebellum. Brain volume measurements in vivo show regional alterations, with relative preservation in the posterior occipital and posterior temporal regions7. The regional reduction in volumes is not attributed to atrophy8 but due to poor neuronal maturation 9, 10 and their connections 11. Increased cell packing density due to poor dendritic arborization has been attributed to poor neuronal interconnectivity resulting in microcephaly 6, 11, 12,
Structure-specific pathology of white matter can be assessed non-invasively by diffusion tensor imaging (DTI) 13,14. Different quantitative measures can be derived from a DTI study. Our prospective study utilized the directionality of water diffusion, quantified by fractional anisotropy (FA)15, to investigate the tract-specific abnormalities and their correlations with some clinical features in patients with RTT.
Thirty-two girls with RTT syndrome were included in the study based on MeCP2 gene mutation analysis and clinical parameters. They underwent neuroimaging and neurological evaluation as part of the Natural History study in RTT being conducted at the Kennedy Krieger Institute. In-depth clinical assessments included neurological status, head circumference, history of seizures, respiratory irregularities, gait, and speech. Imaging and clinical tests were performed during the same admission. Thirty-seven age-matched normal female control subjects were included for comparison. There was no significant age difference between RTT patients vs. controls subjects (5.5±0.7 vs. 6.1 ±0.7 years, p =0.58). All RTT patients were sedated for MRI scan, and controls were not.
The Institutional Review Board at Johns Hopkins Medical Institutions approved the study, and informed consent was obtained from legal guardians.
A 1.5-Tesla MR unit (Philips Medical Systems, Best, The Netherlands) was used for data acquisition. Conventional MRI consisted of sagittal T1-weighted, axial T2 fast spin-echo and axial fluid-attenuated inversion recovery images. Conventional brain MR images were obtained in the RTT and control subjects. DTI data were acquired using a single-shot echo-planar imaging sequence with the sensitivity-encoding, or SENSE, parallel-imaging scheme (reduction factor, 2.5) 16. The field of view was 240 × 240 mm; 96 × 96 imaging matrix was zero filled to 256 × 256 pixels. Transverse sections of 2.5 mm in thickness were acquired parallel to the anterior commissure-posterior commissure line. Diffusion weighting was encoded along 30 independent orientations17, and the b value was 700 sec/mm2. Three DTI data sets were acquired for each participant, and the acquisition time per data set was approximately 6 minutes.
For DTI processing, software (DtiStudio) built in-house was used (H. Jiang, S. Mori; Johns Hopkins University and Kennedy Krieger Institute, Baltimore MD, http://lbam.med.jhmi.edu) 18. Images were first realigned by using 12-mode affine transformation of the AIR program for co-registration and eddy-current distortion correction 19. The six elements of the diffusion tensor were calculated for each pixel by using multivariate linear fitting. 13 After the diagonalization, three eigenvalues and eigenvectors were obtained. For the anisotropy map, fractional anisotropy (FA) was used 15.
The image analysis was based on manually delineated regions of interest (ROIs). To enhance reproducibility of the structure identification, a method aided by a brain normalization scheme was adopted (a “hybrid approach” method) in our previous publication22. First, all brains were normalized to ICBM-DTI-81 atlas http://www.loni.ucla.edu/Atlases/Atlas_Detail.jsp?atlas_id=15) using 12-mode affine transformation. To drive the registration, non-diffusion-weighted images were used. This adjusted the overall brain sizes and orientations across the subjects so that 2D observation slices with consistent orientations and locations could be extracted for the ROI drawing. This initial brain registration was performed using Landmarker (www.mristudio.org) 22 After the registration, pre-defined 2D observation places (2 axial slice at z =82 and 65 and 2 coronal slices at y = 89 and 125 in the atlas space) were extracted. In this study, we placed multiple ROIs for large fiber bundles. These multiple ROIs on the same white matter structures do not always lead to the same observations. The FA values of the same white matter tracts vary along their path20. Individual axons may merge and exit a tract at many points. It is, therefore, anatomically possible that abnormal FAs are found only at a portion of white matter tracts20.
Figure 1 and Table 1 show all 45 ROIs placed at the following anatomic locations: genu of corpus callosum (GC), splenium of corpus callosum (SCC), body of the corpus callosum (CC), external capsule (EC), inferior fronto-occipital fasciculus (IFO), sagittal stratum (SS), superior longitudinal fasciculus (SLF), posterior corona radiata (PCR), anterior limb of internal capsule (ALIC), posterior limb of internal capsule (PLIC), posterior thalamic radiation (PTR), frontal white matter (FW), cerebral peduncle (CP), superior cerebellar peduncle (SCP), middle cerebellar peduncle (MCP), cingulate gyrus (CG1, CG3, CG4), cingulum (CG2), fornix/Stria terminalis (FX/ST). The shape of these ROIs followed White Matter Parcellation Map (WMPM) defined in the ICBM-DTI-81 atlas.
T-test and Mann Whitney test were used for comparison of diffusion parameters between RTT patients and age-matched female control subjects. A p value of 0.05 or less was considered statistically significant. To investigate the asymmetry between FA of the left and right hemispheres, two methods were used: a) comparison of left and right FA values, and b) Laterality Index (LI) for FA in all examined fiber tracts by utilizing the formula LI= (left − right)/[(left + right)]. Significance of the hemispheric differences was assessed with paired t-test for left to right comparison and unpaired t-test for LI comparison. Bonferroni correction was applied. All statistical analyses were done using STATA version 8.0 (STATA, College Station, TX).
32 RTT patients and 37 controls were age-matched when compared as one cohort (p=0.58), or in three sub-groups: a) under 5years old (2.8(SD 0.86) vs. 2.7(SD 1.45) p=0.9), b) 5–10 years old (7 (SD 1.09) vs. 7.6 (SD1.34) p=0.414), c) greater than 10 years old (13.5 (SD2.72) vs. 12.8(SD1.2) p=0.472). Conventional MRI imaging of RTT and control subjects were obtained and found to be unremarkable in control subjects. However, RTT patient were noted to have microcephaly without other abnormalities. Table 1 shows the comparisons of FA between RTT patients and controls. FA was not different in regions comprising the limbic system, namely fornix and posterior cingulate gyrus. However, in RTT patients, FA for anterior cingulum bundles (CG1) was significantly reduced. Of cerebellar tracts, only the right middle cerebellar tract showed trend of reduced FA. Significant reductions in FA were noted in the genu and splenium of corpus callosum, external capsule, with regional reductions in the anterior and posterior limbs of internal capsule, posterior thalamic radiation, and frontal white matter in RTT. In addition there was no difference in FA between RTT and controls in three of the four ROIs in PTR with significantly higher FA in posterior corona radiata of RTT patients (Table 1).
RTT patients were categorized by their capacity to speak: 1) Mute (n=13), 2) Single words (n=13), 3) Could speak in phrases or sentences (n=6). As shown in Figure 2, mean FA of SLF in controls and Group 3 patients was not different (0.38±0.04 vs 0.375±0.02, t=0.615, p=0.542). Similarly, there was no difference in the FA of Group 1 and Group 2 RTT patients (0.32±0.05 vs. 0.33±0.05, t=−0.089, p=0.929). But the Groups 1 and 2 with minimal speech were significantly different from the Group 3 and controls (Group 1 vs. group 3: t=−2.12, p=0.046, group 1 vs. control: t=3.77, p<0.001, Group 2 vs. Group 3: t=−2.03, p=0.055, Group 2 vs. control: t=3.58, p<0.001). This trend was not seen in any other region.
Our study did not find any correlation between FA values in the examined tracts and seizure status, head circumference, or walking ability. Hemispheric asymmetry analysis evaluating left/right differences showed that, in contrast to controls, asymmetry was not present in IFO (p>0.99), EC (p>0.99), SLF (p>0.99), and PTR (p>0.99) in patients with RTT. Additional LI analysis only showed significance trend in IFO tracts (p=0.082) when compared to controls. We noted hemispheric asymmetry in CG3 (posterior cingulate gyrus) of the RTT patients (p=0.044) when compared to controls who showed no asymmetry (p=0.505).
In the studied group of patients with RTT, FA was reduced in corpus callosum, internal capsule, and frontal white matter when compared to controls. In contrast, mean FA in most of the regions comprising the limbic system and cerebellar tracts was similar in both groups. Posterior corona radiata was the only region in the RTT girls where FA was greater than that of controls, and is in keeping with the observation in volumetric studies by Carter et al. Without histological results, it is difficult to conclude about the exact mechanism of the FA decrease observed in this patient population. Past studies have suggested axonal involvement to explain FA reduction 21, 22, but loss of myelin has not been shown to be a contributing factor23. In RTT patients’ axonal vulnerability must be an important contributor to the reduced FA, as shown by reduced NAA in MRSI24.
We noted three major correlations with clinical findings in RTT, the most significant of which was correlation of FA in SLF with ability to speak. SLF is one of the main association bundles that connect the external surface of temporo-parieto-occipital regions with the convexity of the frontal lobe and in part associated with phonologic speech25, 26. The left>right asymmetry in SLF, as seen in the controls, was not found in RTT patients.
Secondly, the presence of intact visual capabilities, which are utilized by some girls with RTT to communicate, may be in accordance with normal to increased FA in PCR. This observation was in contrast to FA that was significantly reduced by greater margins in all regions of the corpus callosum affecting inter-hemispheric connectivity probably due to immature neurons and their poor dendritic arborization27. However, other white matter structures that are known to be associated with the visual functions such as the sagittal stratum did not show increased FA. The increased FA of the PTR, therefore, needs careful interpretation with increased number of patients. Study data also argue that with few exceptions white matter tracts in RTT are diffusely affected in comparison to controls. Except for the right middle cerebellar tract, other cerebellar tracts were generally spared. Right-to-left differences in some brain regions of RTT patients suggest that neuronal connectivity may have become restricted before development of specific skills contributing to inter hemispheric asymmetry in these patients.
The third significant observation is that of reduced FA in anterior cingulate gyrus, which is particularly revealing in view of the characteristic mood and behavioral changes seen in RTT patients. Similarly, changes in ACG and other components of the limbic system are also reported in schizophrenic 28 and autistic 29 patients. In addition, posterior cingulate gyrus showed hemispheric asymmetry which was not present in the controls. Larger studies are required, however, to investigate the correlation between severe behavioral and emotional problems in RTT patients and limbic system components.
Volumetric MRI studies in RTT, have shown significantly reduced cerebral volume; affecting both gray and white matter 7, 30, 31. Magnetic resonance spectroscopy has shown significant decreases in average concentrations of N-acetylaspartate in both gray and white matter of frontal and parietal lobes and insula when compared to controls, demonstrating axonal involvement in RTT 24,23. Our in vivo observation confirms diffuse involvement of white matter tracts with areas of selective vulnerability in limbic system components.
Longitudinal evaluations and studies in older subjects are needed for better understanding of changes in FA and its relation to motor regression, and loss of speech noted in the older RTT girls. Increased sample size will also allow better correlation of function to FA measurements. Improved technology using 3 or higher Tesla MRI’s modalities may provide better definition of tracts.
Clinical variability is currently assessed by neurological exams and mutation type. DTI studies would add specificity to these observations and help to understand effects of individual mutations whose clinical effects are modified by age, and X-inactivation in females. Therapeutic benefits may also be ascertained by their effects on neuronal tracts either as improvement or lack of progressive reduction in FA.
Supported by P01 HD 24448, AG20012, RR15241, and UL1RR025005 Institute for Clinical and Translational Research grant. We thank Terri Brawner and Kathleen Kahl for their technical support in acquiring MRI’s as well as Carolyn Gillen for nursing assistance during MRI examinations.