X-linked spinal and bulbar muscular atrophy (Kennedy’s disease): the first case described in the Brazilian Amazon

<div><p>ABSTRACT The X-linked spinal and bulbar muscular atrophy (Kennedy’s disease) is a rare X-linked, recessive, lower motor neuron disease, characterized by weakness, atrophy, and fasciculations of the appendicular and bulbar muscle. The disease is caused by an expansion of the CAG repetition in the androgen receptor gene. Patients with Kennedy’s disease have more than 39 CAG repetitions. We report a case of 57-year-old man, resident of Monte Dourado (PA, Brazil) who complained of brachiocrural paresis evolving for 3 years along with fasciculations and tremors of extremities. In addition, he also developed dysarthria, dysphagia, and sexual dysfunction. The patient clinical picture included gait impairment, global hyporeflexia, proximal muscle atrophy of upper limbs, deviation of the uvula to right during phonation and tongue atrophy with fasciculations. The patient reported that about 30 years ago he had undergone gynecomastia surgery. His electroneuromyography suggested spinal muscular atrophy, and nuclear magnetic resonance imaging showed tapering of the cervical and thoracic spinal cord. Patient’s creatine kinase level was elevated. In view of the findings, an exam was requested to investigate Kennedy’s disease. The exam identified 46 CAG repetitions in the androgen receptor gene, which confirmed the diagnostic suspicion. This was the first case of Kennedy’s disease diagnosed and described in the Brazilian Amazon. To our knowledge only other four papers were published on this disease in Brazilian patients. A brief review is also provided on etiopathogenic, clinical and diagnostic aspects.</p></div

Nonmuscle myosin II and tension are required for molecular actin ordering during myofibril initiation.

<p>(A) Actin molecular disorder angles ΔΨ for <i>kon</i> and <i>sqh</i> knock-down muscles at 32-h and 90-h APF, relative to wild-type 90-h APF. Error bars represent SD. Note the higher actin disorder ΔΨ after <i>kon</i> or <i>sqh</i> knock-down at 32-h APF; however, notice the largely preserved actin order buildup at 90-h APF. <i>p</i>-values: **<i>P</i> < 0.01; ***<i>P</i> < 0.001 (nonparametric Mann-Whitney U test; see <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.2004718#pbio.2004718.s003" target="_blank">S1 Data</a>). Error bars represent SD. (B) Molecular actin order angle profiles along the averaged sarcomere length for <i>kon</i> knock-down (light blue), <i>sqh</i> knock-down (slate blue), and wild-type control (black) at 90-h APF. Values are relative to the minimum of the wild-type profile. See <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.2004718#pbio.2004718.s003" target="_blank">S1 Data</a> for primary data. APF, After Puparium Formation; <i>kon</i>, <i>kon-tiki</i>; <i>sqh</i>, <i>spaghetti-squash</i>.</p

Simultaneous order buildup during myofibril initiation and subsarcomeric order during sarcomere maturation.

<p>(A) Flight muscle at 32-h APF stained with rhodamine-Alexa488. Overlaid longitudinal orange lines (from one fiber end to the other) indicate positions at which molecular actin order was determined. Scale bar represents 50 μm. (B) Molecular actin order angle longitudinal profile for flight muscles at 32-h APF. Each color line corresponds to an average profile obtained on an individual pupa (the orange one corresponds to the pupa shown in panel A; the others are displayed in <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.2004718#pbio.2004718.s001" target="_blank">S1 Fig</a>). Bars indicate the mean values of 6 different pupae and error bars the SDs. Note that there is no significant difference between the center and the ends of the muscle fiber. Values are relative to the averaged value per pupa. (C–E) Grey maps showing the actin intensity of an averaged sarcomere with the Z-disc (green arrowhead) shown in the middle and the M-lines (red arrowhead) at 48-h (panel C), 72-h (panel D), and 90-h APF (panel E) as shown in <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.2004718#pbio.2004718.g002" target="_blank">Fig 2G–2I</a>. Red sticks represent the local actin orientation angle ρ. Stick angles have been amplified 50 times relative to horizontal axis to visualize the small differences. Scale bar represents 1 μm. (F) Molecular actin order angle profiles along the averaged sarcomere length at 48-h (light grey), 72-h (dark grey), and 90-h APF (black). Values are relative to the minimum of the 90-h APF profile. Red arrows show M-line positions and green ones the Z-disc position. Note the relatively higher actin order at the developing Z-disc of 48-h APF sarcomeres. **<i>P</i> < 0.01; (Wilcoxon signed-rank test; see <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.2004718#pbio.2004718.s003" target="_blank">S1 Data</a> for primary data). APF, After Puparium Formation.</p

Polarization-resolved microscopy—A method to determine actin order in biological systems.

<p>(A) Polarization-resolved microscopy provides information about F-actin molecular order Ψ and the average actin filament orientation ρ. The left image shows a 32-h APF flight muscle stained with phalloidin–Alexa488 and overlaid with an orange box representing the confocal volume. The middle image displays a hypothetical actin order in the confocal volume, with the different orientations of the phalloidin–Alexa488 dipoles shown by blue arrows. For each confocal volume, one value for the actin molecular order Ψ can be determined. The right diagram shows the average actin filament orientation angle ρ in blue and the actin molecular order angle Ψ in red for the hypothetical confocal volume shown. All order angle values Ψ are shifted by a term Ψ₀, as first, the Alexa488 dipole forms an undefined angle to the helicoidal actin filament (blue arrows) and second, the Alexa488 label is connected to phalloidin with a linker, allowing some molecular flexibility (represented by the solid light blue cones in the Fig). An order angle Ψ = Ψ₀ is thus a signature of perfect order, whereas a random distribution corresponds to Ψ = 180°. (B–E) Molecular actin order in different biological systems. Samples were fixed and stained with the same phalloidin–Alexa488 probe to reveal a highly disordered actin cytoskeleton in the developing <i>Drosophila</i> pupal eye (41-h APF; several ommatidia are shown) (panels B, B’; Ψ close to 180°), a more regular actin cytoskeleton in the furrow canal of early <i>Drosophila</i> embryos (panels C, C’; Ψ = 140°–150°), and in stress fibers of plated mouse NIH-3T3 fibroblasts (panels D, D’; Ψ = 130°–150°), compared with a very-high actin order in mature sarcomeres of 90-h APF flight muscles (panels E, E’; Ψ = 127°). The colors in the overlaid images (panels B–E) relate with the colors in the order tables (B’–E’); the bluer, the higher the molecular actin order. Scale bars represent 10 μm. See <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.2004718#pbio.2004718.s003" target="_blank">S1 Data</a> for primary data. APF, After Puparium Formation; F-actin, filamentous actin.</p

Nonmuscle myosin II is essential for myotube compaction.

<p>(A–F) Flight muscle fibers of wild-type 32-h APF pupae and of the indicated <i>sqh</i> and <i>zip</i> knock-down (hairpins expressed with <i>Mef2</i>-GAL4) were stained with phalloidin (red) and anti-Mhc (green) and observed by confocal microscopy (panels A, C, and E). Unattached rounded fibers after knock-down of <i>zip</i> are labeled with yellow asterisks (panel E). Red boxes indicate the positions of the high-magnification images that display the immature myofibrils. Scale bars represent 100 μm (panels A, C, and E) and 2 μm (panels B, D, and F). (G) Quantification of muscle fiber length at 32-h APF of the indicated genotypes. Plots represent the average sarcomere length per pupa shown within the whiskers (minimum to maximum), with 50% of the values shown within the boxplots (25% to 75%) and the median indicated as a line. <i>p</i>-values: *<i>P</i> < 0.05 (nonparametric Mann-Whitney U test; see <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.2004718#pbio.2004718.s003" target="_blank">S1 Data</a> for primary data). APF, After Puparium Formation; Mhc, Myosin heavy chain; <i>sqh</i>, <i>spaghetti-squash</i>; <i>zip</i>, <i>zipper</i>.</p

Nonmuscle myosin II is essential for ordered myofibrillogenesis at 90-h APF.

<p>(A–L) Hemithoraces of wild-type 90-h APF pupae and of the indicated <i>sqh</i> and <i>zip</i> knock-down genotypes (hairpins expressed with <i>Mef2</i>-GAL4) were stained with phalloidin (green) and anti-Mhc (red) and observed by confocal microscopy (panels A, C, E, G, I, and K). Missing flight muscle fibers after knock-down of <i>zip</i> are indicated with a yellow asterisk (panels I, K). Red boxes indicate the positions of the high-magnification images of the myofibrils. Actin accumulations are marked with yellow arrowheads and split myofibrils with white arrows. Scale bars represent 100 μm (panels A, C, E, G, I, and K) and 5 μm (panels B, D, F, H, J, and L). (M) Quantification of sarcomere length at 90-h APF of the indicated genotypes. Plots represent the average sarcomere length per pupa shown within the whiskers (minimum to maximum), with 50% of the values shown within the boxplots (25% to 75%) and the median indicated as a line. <i>p</i>-values: ***<i>P</i> < 0.001 (nonparametric Mann-Whitney U test; see <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.2004718#pbio.2004718.s003" target="_blank">S1 Data</a> for primary data). APF, After Puparium Formation; Mhc, Myosin heavy chain; <i>sqh</i>, <i>spaghetti-squash</i>; <i>zip</i>, <i>zipper</i>.</p

Polarization-resolved microscopy reveals the buildup of molecular actin order during sarcomerogenesis.

<p>(A–F) Flight muscle myofibrils at different developmental stages from 18-h to 90-h APF stained by Alexa488-labeled phalloidin and imaged with a spinning disc microscope. Scale bar represents 10 μm. (G–I) Averaged sarcomere actin concentration maps obtained after automated sarcomere detection. Arrowheads indicate M-line (red) and Z-disc (green) positions. Scale bar represents 1 μm. (J) Quantification of sarcomere length. <i>p</i>-values: **<i>P</i> < 0.01; ***<i>P</i> < 0.001 (nonparametric Mann-Whitney U test; see <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.2004718#pbio.2004718.s003" target="_blank">S1 Data</a>). Error bars represent SD. (K) Molecular actin order angles determined in fixed developing flight muscles at different pupal stages. The same samples as in Fig 2 were used. The molecular disorder ΔΨ relative to highest order value at 90-h APF is shown. Note the strong gain of order from 22-h to 32-h APF and from 32-h to 48-h APF. Error bars represent SD. <i>p</i>-values: **<i>P</i> < 0.01; ***<i>P</i> < 0.001 (nonparametric Mann-Whitney U test; see <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.2004718#pbio.2004718.s003" target="_blank">S1 Data</a> for primary data). APF, After Puparium Formation; ns, nonsignificant.</p