Mapping Cortical Degeneration in ALS with Magnetization Transfer Ratio and Voxel-Based Morphometry

<div><p>Pathological and imaging data indicate that amyotrophic lateral sclerosis (ALS) is a multisystem disease involving several cerebral cortical areas. Advanced quantitative magnetic resonance imaging (MRI) techniques enable to explore in vivo the volume and microstructure of the cerebral cortex in ALS. We studied with a combined voxel-based morphometry (VBM) and magnetization transfer (MT) imaging approach the capability of MRI to identify the cortical areas affected by neurodegeneration in ALS patients. Eighteen ALS patients and 18 age-matched healthy controls were examined on a 1.5T scanner using a high-resolution 3D T1 weighted spoiled gradient recalled sequence with and without MT saturation pulse. A voxel-based analysis (VBA) was adopted in order to automatically compute the regional atrophy and MT ratio (MTr) changes of the entire cerebral cortex. By using a multimodal image analysis MTr was adjusted for local gray matter (GM) atrophy to investigate if MTr changes can be independent of atrophy of the cerebral cortex. VBA revealed several clusters of combined GM atrophy and MTr decrease in motor-related areas and extra-motor frontotemporal cortex. The multimodal image analysis identified areas of isolated MTr decrease in premotor and extra-motor frontotemporal areas. VBM and MTr are capable to detect the distribution of neurodegenerative alterations in the cortical GM of ALS patients, supporting the hypothesis of a multi-systemic involvement in ALS. MT imaging changes exist beyond volume loss in frontotemporal cortices.</p></div

Regional MTr decrease surviving correction for local atrophy in ALS.

<p>Results of the between group MTI analysis adjusted for atrophy [p<0.05 FWE corrected for multiple comparisons] superimposed on volume rendering template in the right and upper views reveals clusters (in red) of reduced MTr mainly affecting premotor and frontotemporal cortex. MTI changes exist beyond volume loss in these cortical.</p

Next-Generation Sequencing Identifies Transportin 3 as the Causative Gene for LGMD1F

<div><p>Limb-girdle muscular dystrophies (LGMD) are genetically and clinically heterogeneous conditions. We investigated a large family with autosomal dominant transmission pattern, previously classified as LGMD1F and mapped to chromosome 7q32. Affected members are characterized by muscle weakness affecting earlier the pelvic girdle and the ileopsoas muscles. We sequenced the whole exome of four family members and identified a shared heterozygous frame-shift variant in the Transportin 3 (<i>TNPO3</i>) gene, encoding a member of the importin-β super-family. The <i>TNPO3</i> gene is mapped within the LGMD1F critical interval and its 923-amino acid human gene product is also expressed in skeletal muscle. In addition, we identified an isolated case of LGMD with a new missense mutation in the same gene. We localized the mutant TNPO3 around the nucleus, but not inside. The involvement of gene related to the nuclear transport suggests a novel disease mechanism leading to muscular dystrophy.</p></div

Total and Shared Variants in Patients with LGMD1F.

<p>Total and Shared Variants in Patients with LGMD1F.</p

Indirect immunofluorescence analysis of the wt-hTNPO3 compared with delA p.X924C -hTNPO3.

<p>Following transient transfections, HeLa cells were incubated for 48 h with normal DMEM and detected by anti-HA immunofluorescence. Nuclei are stained with DAPI (blue). The endogenous protein is recognized using a rabbit monoclonal anti-TNPO3 antibody (green), while the transfected TNPO3 proteins were HA-tagged (red). <b>a</b>) An accumulation around the nucleus is usually observed using the mutant delA p.X924C -hTNPO3. <b>b</b>) The typical intranuclear staining pattern can be observed in cells transfected with wt-hTNPO3 (in red) or <b>c)</b> in non transfected HeLa cells.</p

LGMD1F family pedigree.

<p>Squares represent male; circles represent female; white figures symbolize normal individuals; black figures indicate individuals with clinical muscular dystrophy. The original LGMD1F family has been extended from subject II,2 and now includes 64 LGMD patients of both sexes and five non-penetrant carriers (IV-4, V-26, V-29, V-33, and VI-68). The whole-exome sequencing was performed in four patients indicated by arrows (V-28, VI-36, VI-53, VII-5).</p

Primer and probe sequences used for the real-time detection of β-actin.

<p>Primer and probe sequences used for the real-time detection of β-actin.</p

mRNA quantification of VEGF relative to the age and clinical conditions of the patients.

<p>(A) Comparison of the level of VEGF mRNA in PBMCs from HD pre-manifest, manifest patients and age- and sex-matched healthy controls.Age 27–40: Controls = 15; pre-manifest = 12; manifest = 13; Age 43–58: Controls = 26; pre-manifest = 8; manifest = 42; Age 59–84: Controls = 13; manifest = 35; ** p<0.01; *** p<0.001. (B) Comparison of the level of VEGF mRNA in PBMCs of patients with different stages of the disease: pre-manifest = 20; early-manifest = 17; manifest = 55; late-manifest = 18;* p<0.05. The mean values ± SD of three independent assays are shown.</p

Primers used to evaluate the number of CAG repeats for the molecular diagnosis of HD.

<p>Primers used to evaluate the number of CAG repeats for the molecular diagnosis of HD.</p

RNA quantification of PMCA1, SERCA2, SERCA3 and VEGF in PBMCs from healthy controls and HD patients divided according to sex.

<p>Subjects analysed: 10 healthy male controls and 10 healthy female controls age-matched ± 3 years; 10 males HD, mean age of 44.5 ± 10.1; 10 females HD, mean age of 53.4 ± 9.6. *** p<0.005. Each column represents the relative mean value of three independent experiments ± SD.</p