Distinct Kidins220 splice isoforms display specific cellular localisations.

<p>(A) Schematics of Kidins220 splice isoform m6 and Kidins220 ATE m6/C2 (from Exon 24 onwards) used for transfection of PC12 cells in Fig 7B. (B) PC12 cells were transfected with HA-tagged Kidins220 isoform m6, isoform m6/C2 or with Tet-ON pLVX vector only (control) and after 6 h stimulated with doxycycline and differentiated for 48 h with NGF. Full-length Kidins220 was detected using a polyclonal antibody directed against the carboxy-terminus of Kidins220 (GSC16 antibody; in green). An anti-HA antibody was used to stain Kidins220 isoforms m6 and m6/C2 (in red). The gain of the red channel was enhanced equally for cells overexpressing isoform m6/C2 and control cells, whilst it was tuned down for PC12 cells transfected with isoform m6 to adjust for the higher expression levels of this Kidins220 variant. Boxed areas of the merged images are magnified on the right. Representative pictures were chosen from three different experiments. Scale bars, 10 μm.</p

Kidins220 alternative splice isoforms are translated into protein isoforms in embryonic and adult mouse brain.

<p>Western blots of embryonic day 18.5 (E) and adult (A) brain lysates show evidence of protein translation of Kidins220 variants containing: amino-terminus (N-term), Kidins220 isoform 1 (m1), Kidins220 isoform 6 (m6) and exon 33 (C2). Actin was used a loading control.</p

Expression of alternative splice isoforms of Kidins220 in adult mouse and human tissues.

<p>(A-B) RT-PCR analyses for exons encoding the amino-terminus of Kidins220 and between exons 24 and 30 (24f-30r) were carried out on adult mouse (A) and human (B) tissue panels. Tissues are labelled by capital letters. N indicates PCR products obtained using primers designed to recognise exons 3 and 8 in mouse, and exon 9 and 13 in human. 24f-30r indicates samples obtained by amplification with primers recognising exons 24 and 30. Arrowheads point to samples in which a specific alternative splicing pattern was detected. (C-D) Schematics of Kidins220 splice isoforms identified in mouse heart and brain (C) and human brain (D) in the region encoded by exons 24 to 30.</p

Kidins220 alternative terminal exons in mouse and human tissue.

<p>(A-B) RT-PCR analysis for the carboxy-terminal endings of Kidins220 (exon 32 and exon 33) was carried out on an adult mouse (A) and human (B) tissue panels. Capital letters indicate the different tissues. 31f and 33 rev indicate amplification with primers against exons 31 and 33. Arrowheads point out samples where alternative terminal exon (ATE) splicing for Kidins220 is detected. (C) PCR products obtained using primers designed to recognise exons encoding the carboxy-terminus of Kidins220 (exons 31/32 and 32 in mouse and exon 31 and 32 in human).</p

BDNF accelerates the appearance of full-length Kidins220 in cortical and hippocampal primary neurons.

<p>(A-B) Primary cortical (A) and hippocampal (B) neurons were prepared from E18.5 mouse embryos and half of each culture was plated with medium containing 100 ng/ml BDNF. RNA was extracted at different time points and reverse transcribed. N indicates PCR products obtained using primers designed to recognise exons 3 and 8. 24f-30r indicates samples obtained by amplification with primers recognising exons 24 and 30. (A) Arrowheads point to early appearance of Kidins220 isoform m1 (full-length) in BDNF treated cultures compared to non-treated cultures.</p

Different neuronal populations express specific Kidins220 splice isoforms.

<p>(A-F) RNA extracted from cortical (A, D), hippocampal (B, E) and motor neuron (C, F) primary cultures was reverse transcribed into cDNA. N indicates PCR products obtained using primers designed to recognise exons 3 and 8 of Kidins220. 24f-30r indicates samples obtained by amplification with primers recognising exons 24 and 30 (A-C). C indicates PCR products obtained using primers designed to recognise exons 31/32 and 32. 31f-33r indicates samples obtained by amplification with primers recognising exons 31 and 33 (D-F). Note the absence of ATE splicing isoforms C1 and C2 in primary motor neuron cultures.</p

Table_1_The Diagnostic Value of MRI Pattern Recognition in Distal Myopathies.DOCX

<p>Objective: Distal myopathies are a diagnostically challenging group of diseases. We wanted to understand the value of MRI in the current clinical setting and explore the potential for optimizing its clinical application.</p><p>Methods: We retrospectively audited the diagnostic workup in a distal myopathy patient cohort, reassessing the diagnosis, whilst documenting the usage of MRI. We established a literature based distal myopathies MRI pattern template and assessed its diagnostic utility in terms of sensitivity, specificity, and potential impact on the diagnostic workup.</p><p>Results: Fifty-five patients were included; in 38 with a comprehensive set of data the diagnostic work-up was audited. The median time from symptoms onset to diagnosis was 12.1 years. The initial genetic diagnostic rate was 39%; 18% were misdiagnosed as neuropathies and 13% as inclusion body myositis (IBM). Based on 21 publications we established a MRI pattern template. Its overall sensitivity (50%) and specificity (32%) were low. However in some diseases (e.g., MYOT-related myopathy, TTN-HMERF) MRI correctly identified the causative gene. The number of genes suggested by MRI pattern analysis was smaller compared to clinical work up (median 1 vs. 9, p < 0.0001) but fewer genes were correctly predicted (5/10 vs. 7/10). MRI analysis ruled out IBM in all cases.</p><p>Conclusion: In the diagnostic work-up of distal myopathies, MRI is useful in assisting genetic testing and avoiding misdiagnosis (IBM). The overall low sensitivity and specificity limits its generalized use when traditional single gene test methods are applied. However, in the context of next generation sequencing MRI may represent a valuable tool for interpreting complex genetic results.</p