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Appendix e-1
Supplementary Methods

Patients

The muscle impairment was measured by using a modified form of the muscular disability rate based (MDRS) on a five-point scale as grade 1: no muscular impairment; grade 2: minimal signs (myotonia, facial weakness); grade 3: mild muscle weakness (no external help in every day tasks); grade 4: moderate muscle weakness (external help in every day tasks needed); grade 5: severe muscle weakness (wheelchair required). The MDRS was originally validated for DM1 so that we had to modify it to receive a comparable muscle score also for DM2.

Neuropsychological investigations

To test global cognitive functions, we used the mini-mental state examination (MMSE),e1 the WORLD test using the German word “SANFT”,e2 the clock test (part of the original MMSE) and the vocabulary test (WST).e3 To evaluate visual-constructional abilities in addition to the clock test, we used the copy of the Rey-Osterrieth figure (ROFI)e4,e5 Non-verbal episodic memory was tested by the recall of the Rey-Osterrieth figure (ROFII) and a revised form of the Wechsler-Memory-Scale (WMS).e6 WMSI was a direct re-call after presenting the figures in terms of a working memory test, WMSII was the long-term recall testing the non-verbal memory. For assessing frontal functions, we used the Spatial span with Corsi`s blocks (BS),e7 the verbal fluency test (VF; part of CERAD, consortium for the establishment of a registry for Alzheimer’s disease), the digit span (ZS)e8 and the Wisconsin card-sorting test (WCST).e9 Verbal episodic memory was evaluated by the verbal-learn-and-memory-test part 1 (short term recall) and 2 (long term recall) (VLMT).e10 To assess psychomotor speed and alertness, we used the trail making test (TMT)e11 and a computer-assisted alertness test (with [Al-wW] or without [Al-oW] a warning tone before recognizing a cross, or both a tone and a cross recognizing system [GA], BeriSoft Cooperation, Frankfurt/Main, Germany). Depression was assessed using Beck`s depression inventory (BDI)e12 and a self-estimated depression rating scale (BfS; Befindlichkeitsskala).e13

Data were analyzed by using the SPSS statistical software package Version 8.0 and nonparametric tests. A P-value < 0.05 was considered as significant.

Magnetic resonance imaging of the brain using with voxel based morphometry

The 3-dimensional MRI data were analyzed using the „optimized“ voxel based morphometry protocol (VBM)e14using statistical parametric mapping, SPM2.e15 All images were normalized to a common template space using linear and non-linear transformations. The images were segmented into gray-matter, white-matter and CSF-compartments, and then modulated to preserve the quantity of tissue that was deformed during normalisation, so that the resulting analysis more correctly represents atrophy. Finally, the images were smoothed with an isotropic Gaussian kernel of 8 mm to reduce interindividual variation in gyration.

After the preprocessing, we compared the groups of patients with those of controls. The statistical analysis was performed using threshold masking (0.8). Contrasts were defined for revealing areas of increased and decreased gray matter. The results of the whole brain analysis were corrected for multiple comparisons using the False Discovery Rate of 5%e16with an extended threshold searching for clusters equal to or greater than 100 voxels. Based on previous neuropsychological findings showing specific deficits in episodic memory we assumed an involvement of the mesial temporal lobe and assessed it separately without correction for multiple comparisons using a significance level p = 0.01.

In order to further determine if there was any correlation between certain neuropsychological parameters of episodic memory (as well as the clinical score developed for this study) and atrophy in the mesial temporal lobe, we performed a correlation analysis using the T1-3D-MRI datasets from the patients of both disease groups respectively for which the neuropsychological data was here available. Given the low number of patients with complete data sets we set the significance level p = 0.05 which therefore only represents a trend.

[18F] Fluorodeoxyglucose positron emission tomography scans (FDG-PET)

The dynamic image acquisition was immediately started after 18FDG injection and continued until 50 min after injection using the following time frames: six 20-s scans, three 60-s scans, two 150-s scans, two 300-s scans und three 600-s scans. 17 arterialized blood samples were collected from the peripheral vein before injection and at the midpoint of each time frame. Parametric images were generated by determining the cerebral metabolic rate for glucose (CMRglc). The data were iteratively reconstructed based on the measured attenuation-corrected images. For evaluation of the individual regions showing a relative hypometabolism, statistical parametric mapping (SPM2, Wellcome Department of Imaging Neuroscience Group, London, UK) was implemented. The data were normalized into the standard stereotaxic space of Talairach and Tournoux and then compared with a group of normal controls (n = 26; mean age 51 years, range 24-73) by means of an ANCOVA-based subtraction approach. The scanner protocol for the controls was same as used for the patients.

In order to minimise the effect of cortical atrophy on the results, we applied partial volume correction for those 15 DM1 patients, 9 DM2 patients and 13 healthy controls, for whom we could analyze both the FDG-PET images and the 3D-T1-MP-RAGE images. We used the methods descibed by Müller-Gärtnere17and Roussete18with an integrated software package (PVEout)e19and repeated the above analysis with these groups of patients and controls.

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