Loss of oligodendrocytic Cx47 and Cx32 expression in chronic lesions of MS (case MS-5).

<p>KB staining in a chronic inactive lesion of the optic nerve (A). Higher magnification view of the lesion boundary area (B–I, corresponding to <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0072919#pone-0072919-g005" target="_blank">Figure 5A</a>, square). Immunoreactivities for Cx47 and Cx32 are diminished beyond the demyelinated area (B, C) as revealed by immunostaining for MOG, OSP and MAG (D–F). This lesion is classified as pattern D for Cx43 and pattern A for Cx47/Cx32. In contrast, MLC1, AQP4 and Cx43 expression are up-regulated because of astrogliosis (G–I). MLC1 is localized at perivascular foot processes even in chronic gliotic tissues (G). Scale Bar = 1 mm(A); 200 µm (B–I).</p

Connexin 43 Astrocytopathy Linked to Rapidly Progressive Multiple Sclerosis and Neuromyelitis Optica

<div><p>Background</p><p>Multiple sclerosis (MS) and neuromyelitis optica (NMO) occasionally have an extremely aggressive and debilitating disease course; however, its molecular basis is unknown. This study aimed to determine a relationship between connexin (Cx) pathology and disease aggressiveness in Asian patients with MS and NMO.</p> <p>Methods/Principal Findings</p><p>Samples included 11 autopsied cases with NMO and NMO spectrum disorder (NMOSD), six with MS, and 20 with other neurological diseases (OND). Methods of analysis included immunohistochemical expression of astrocytic Cx43/Cx30, oligodendrocytic Cx47/Cx32 relative to AQP4 and other astrocytic and oligodendrocytic proteins, extent of demyelination, the vasculocentric deposition of complement and immunoglobulin, and lesion staging by CD68 staining for macrophages. Lesions were classified as actively demyelinating (n=59), chronic active (n=58) and chronic inactive (n=23). Sera from 120 subjects including 30 MS, 30 NMO, 40 OND and 20 healthy controls were examined for anti-Cx43 antibody by cell-based assay. Six NMO/NMOSD and three MS cases showed preferential loss of astrocytic Cx43 beyond the demyelinated areas in actively demyelinating and chronic active lesions, where heterotypic Cx43/Cx47 astrocyte oligodendrocyte gap junctions were extensively lost. Cx43 loss was significantly associated with a rapidly progressive disease course as six of nine cases with Cx43 loss, but none of eight cases without Cx43 loss regardless of disease phenotype, died within two years after disease onset (66.7% vs. 0%, <i>P</i>=0.0090). Overall, five of nine cases with Cx43 loss and none of eight cases without Cx43 loss had distal oligodendrogliopathy characterized by selective myelin associated glycoprotein loss (55.6% vs. 0.0%, <i>P</i>=0.0296). Loss of oligodendrocytic Cx32 and Cx47 expression was observed in most active and chronic lesions from all MS and NMO/NMOSD cases. Cx43-specific antibodies were absent in NMO/NMOSD and MS patients.</p> <p>Conclusions</p><p>These findings suggest that autoantibody-independent astrocytic Cx43 loss may relate to disease aggressiveness and distal oligodendrogliopathy in both MS and NMO.</p> </div

Analysis of IDH1 mutation in an inappropriate tissue sample.

<p>Analysis of a tumor tissue showing a sampling discrepancy. Whereas wild-type calls were obtained by SS and conventional HRM for the first sampling, the mutation was detected using our present method based on a heteroduplex-derived curve that presented a positive HRM-MI value (first column). The second sampling confirmed the mutation, with consistent results among the three approaches (second column).</p

HRM analysis using mixed samples.

<p>A) Distribution plots of HRM-MI results obtained using an HRM analysis with mixed samples. The <i>x</i>-axis shows the fraction of tumor DNA in the mixture. The <i>y</i>-axis shows the HRM-MI value. Each dot represents the HRM-MI value for each assay, performed with multiple replicate across the 6 different mixing ratios. Green and blue lines indicate mean and SD HRM-MI values, respectively. B) Mutation detection ratios for HRM-MI (red line) and conventional HRM analyses (blue line). At a mixing ratio of 20% (i.e., 20% tumor DNA and 80% nontumor DNA), the sensitivities of the conventional HRM method and HRM-MI were 30% and 100%, respectively. C) The representative -<i>d</i>² curves of samples with tumor-to-nontumor DNA ratios. Even the 10% tumor DNA sample presented an interpretable heteroduplex-derived peak.</p

Distal oligodendrogliopathy and astrocytopathy in anti-AQP4 antibody-seropositive NMO (case NMO-10).

<p>In active lesions of the cerebral white matter, KB staining and MOG immunostaining show remaining myelin (A, B). Patterns of preferential MAG loss and marked loss of GFAP immunoreactivity are seen (C, D). Higher magnification reveals sharply demarcated, prominent MAG loss in this lesion with infiltration of numerous CD68-positive macrophages, whereas immunoreactivity for MOG, MBP and OSP is preserved in the lesion (E–I). Immunoreactivity for Cx47 is diminished compared with non-affected white matter (J). Complete loss of AQP4 and Cx43 in degenerative, GFAP-positive astrocytes (K–M) and complement deposition are observed around blood vessels with perivascular cell cuffing (N, O). Complement components are present within foamy macrophages in this lesion (P). Nogo-A-positive oligodendrocytes are markedly decreased in this lesion and some remaining oligodendrocytes show nuclear condensation, suggesting apoptotic changes (Q). This lesion is classified as pattern A for Cx43 and pattern B for Cx47/Cx32. Scale Bar = 4 mm (A–D); 200 µm (E–M); 50 µm (N, O); 20 µm (P, Q).</p

Coexistence of distal oligodendrogliopathy in active NMO lesions in case NMO-4

<p>(A–L) CD68 immunostaining demonstrates massive infiltration of macrophages in the cerebral peduncle (A, arrows). Immunoreactivity for MOG is relatively preserved but is completely lost for MAG (B, C, arrows). Higher magnification of the lesion (D–L). Immunoreactivity for MOG is relatively preserved in contrast to complete loss of MAG in this lesion (D, E). Cx47 expression is diminished compared with non-affected white matter (F). Lesion boundary areas (G–I). A dotted line indicates the boundary. Immunostainings for Cx47 and Cx32 are slightly diminished but preserved inside the lesion (G, H). Immunostaining of Nogo-A is markedly decreased and apoptotic nuclear condensation of oligodendrocytes is present (I, insert). Immunoreactivities for AQP4 and Cx43 within highly degenerative GFAP-positive astrocytes are completely lost (J–L). This lesion is classified as pattern B for Cx43 and pattern B for Cx47/Cx32. Scale Bar = 4 mm(A-C); 0.5 mm (D–F); 50 µm (G, H); 100 µm (I); 200 µm (J–L).</p

HRM analysis using different amplicon lengths.

<p>Multiple amplicon lengths (90, 129, and 212 bp) were tested for mutation analyses in order to optimize the HRM analysis. First row: -<i>d</i>¹ curve; second row: -<i>d</i>² curve. The 90-bp amplicon showed the most interpretable heteroduplex-derived peaks.</p

Differential calculus analysis of HRM data.

<p>A) Representative results of differential calculus analyses of HRM for <i>IDH1</i>^<i>R132</i>. First row: Sanger sequencing; second row: fluorescence intensity curve; third row: -<i>d</i>¹ curve; fourth row: -<i>d</i>² curve. First column: A result of sequence-wild type DNA containing no heteroduplex-derived peaks in either the -<i>d</i>¹ or -<i>d</i>² curves. Second column: A result of sequence-mutant DNA. Whereas the heteroduplex-derived peak is recognized in the -<i>d</i>¹ curve as a slight change of shape, the -<i>d</i>² curve demonstrates a more distinct peak formation. B) Distributions of run-to-run variability in the low-temperature melting transition (LTMT) (blue) and high-temperature melting transition (HTMT) (red) positions from <i>Tm</i>. Bars indicate the number of observations within the bins of width 0.01°C. The curved black line shows the approximate normal distributions of LTMT and HTMT, in which the standard deviations were 0.076°C and 0.048°C, respectively.</p

Precise Detection of <i>IDH1/2</i> and <i>BRAF</i> Hotspot Mutations in Clinical Glioma Tissues by a Differential Calculus Analysis of High-Resolution Melting Data

<div><p>High resolution melting (HRM) is a simple and rapid method for screening mutations. It offers various advantages for clinical diagnostic applications. Conventional HRM analysis often yields equivocal results, especially for surgically obtained tissues. We attempted to improve HRM analyses for more effective applications to clinical diagnostics. HRM analyses were performed for <i>IDH1</i>^<i>R132</i> and <i>IDH2</i>^<i>R172</i> mutations in 192 clinical glioma samples in duplicate and these results were compared with sequencing results. <i>BRAF</i>^<i>V600E</i> mutations were analyzed in 52 additional brain tumor samples. The melting profiles were used for differential calculus analyses. Negative second derivative plots revealed additional peaks derived from heteroduplexes in PCR products that contained mutations; this enabled unequivocal visual discrimination of the mutations. We further developed a numerical expression, the HRM-mutation index (MI), to quantify the heteroduplex-derived peak of the mutational curves. Using this expression, all <i>IDH1</i> mutation statuses matched those ascertained by sequencing, with the exception of three samples. These discordant results were all derived from the misinterpretation of sequencing data. The effectiveness of our approach was further validated by analyses of <i>IDH2</i>^<i>R172</i> and <i>BRAF</i>^<i>V600E</i> mutations. The present analytical method enabled an unequivocal and objective HRM analysis and is suitable for reliable mutation scanning in surgically obtained glioma tissues. This approach could facilitate molecular diagnostics in clinical environments.</p></div

Relationship of inflammatory components with patterns of Cx43 loss in MS and NMO lesions.

<p>(A, B) Positivity rates of perivascular lymphocytic cuffing or perivascular deposition of complement and immunoglobulin in active (A) or chronic active (B) lesions of MS cases according to Cx43 patterns. (C, D) Positivity rates of perivascular lymphocytic cuffing or perivascular deposition of complement and immunoglobulin in active (C) or chronic active (D) lesions of NMO/NMOSD cases according to Cx43 patterns. Patterns N lesions are excluded in this figure.</p