Novel Mutation m.10372A>G in MT-ND3 Causing Sensorimotor Axonal Polyneuropathy

ObjectiveTo investigate the pathogenicity of a novel MT-ND3 mutation identified in a patient with adult-onset sensorimotor axonal polyneuropathy and report the clinical, morphologic, and biochemical findings.MethodsClinical assessments and morphologic and biochemical investigations of skeletal muscle and cultured myoblasts from the patient were performed. Whole-genome sequencing (WGS) of DNA from skeletal muscle and Sanger sequencing of mitochondrial DNA (mtDNA) from both skeletal muscle and cultured myoblasts were performed. Heteroplasmic levels of mutated mtDNA in different tissues were quantified by last-cycle hot PCR.ResultsMuscle showed ragged red fibers, paracrystalline inclusions, a significant reduction in complex I (CI) respiratory chain (RC) activity, and decreased adenosine triphosphate (ATP) production for all substrates used by CI. Sanger sequencing of DNA from skeletal muscle detected a unique previously unreported heteroplasmic mutation in mtDNA encoded MT-ND3, coding for a subunit in CI. WGS confirmed the mtDNA mutation but did not detect any other mutation explaining the disease. Cultured myoblasts, however, did not carry the mutation, and RC activity measurements in myoblasts were normal.ConclusionsWe report a case with adult-onset sensorimotor axonal polyneuropathy caused by a novel mtDNA mutation in MT-ND3. Loss of heteroplasmy in blood, cultured fibroblasts and myoblasts from the patient, and normal measurement of RC activity of the myoblasts support pathogenicity of the mutation. These findings highlight the importance of mitochondrial investigations in patients presenting with seemingly idiopathic polyneuropathy, especially if muscle also is affected.

Transcriptome sequencing and delimitation of cryptic Oscarella species (O. carmela and O. pearsei sp. nov) from California, USA

The homoscleromorph sponge Oscarella carmela, first described from central California, USA is shown to represent two morphologically similar but phylogenetically distant species that are co-distributed. We here describe a new species as Oscarella pearsei, sp. nov. and redescribe Oscarella carmela; the original description was based upon material from both species. Further, we correct the identification of published genomic/transcriptomic resources that were originally attributed to O. carmela, and present new Illumina-sequenced transcriptome assemblies for each of these species, and the mitochondrial genome sequence for O. pearsei sp. nov. Using SSU and LSU ribosomal DNA and the mitochondrial genome, we report the phylogenetic relationships of these species relative to other Oscarella species, and find strong support for placement of O. pearsei sp. nov. in a clade defined by the presence of spherulous cells that contain paracrystalline inclusions; O. carmela lacks this cell type and is most closely related to the Western Pacific species, O. malakhovi. Oscarella pearsei sp. nov and O. carmela can be tentatively distinguished based upon gross morphological differences such as color, surface texture and extent of mucus production, but can be more reliably identified using mitochondrial and nuclear barcode sequencing, ultrastructural characteristics of cells in the mesohyl, and the morphology of the follicle epithelium which surrounds the developing embryo in reproductively active individuals. Usually, cryptic species are very closely related to each other, but in this case and in sponges generally, cryptic species may be very distantly related because sponges can be difficult to identify based upon gross morphological characteristics.

Formation of Hirano bodies in Dictyostelium and mammalian cells induced by expression of a modified form of an actin-crosslinking protein

We report the serendipitous development of the first cultured cell models of Hirano bodies. Myc-epitope-tagged forms of the 34 kDa actin bundling protein (amino acids 1-295) and the CT fragment (amino acids 124-295) of the 34 kDa protein that exhibits activated actin binding and calcium-insensitive actin filament crosslinking activity were expressed in Dictyosteliumand mammalian cells to assess the behavior of these modified forms in vivo. Dictyostelium cells expressing the CT-myc fragment: (1) form ellipsoidal regions that contain ordered assemblies of F-actin, CT-myc, myosin II, cofilin and α-actinin; (2) grow and develop more slowly than wildtype, but produce normal morphogenetic structures; (3) perform pinocytosis and phagocytosis normally; and (4) produce a level of total actin equivalent to wildtype, but a higher level of F-actin. The paracrystalline inclusions bear a striking resemblance to Hirano bodies, which are associated with a number of pathological conditions. Furthermore, expression of the CT fragment in murine L cells results in F-actin rearrangements characterized by loss of stress fibers, accumulation of numerous punctate foci, and large perinuclear aggregates, the Hirano bodies. Thus, failure to regulate the activity and/or affinity of an actin crosslinking protein can provide a signal for formation of Hirano bodies. More generally, formation of Hirano bodies is a cellular response to or a consequence of aberrant function of the actin cytoskeleton. The results reveal that formation of Hirano bodies is not necessarily related to cell death. These cultured cell models should facilitate studies of the biochemistry, genetics and physiological effects of Hirano bodies.

Mesothelial Paracrystalline Inclusions in Continuous Ambulatory Peritoneal Dialysis Patients

♦ Objective Uremia is known to be followed by changes in the serous membranes of pleura, pericardium, and peritoneum. During continuous ambulatory peritoneal dialysis (CAPD), the peritoneum is exposed to altered body conditions as well as to the influence of dialysate. The aim of the present study was to examine the ultrastructure of the mesothelial cells in CAPD patients, and to compare the findings with those from studies of the peritoneum in uremic controls. Paracrystalline intracytoplasmic inclusions in mesothelial cells were objects of special interest. ♦ Methods Biopsies of human parietal peritoneum were studied. These were taken from 12 uremic patients during catheter implantation before the start of CAPD, and from 7 CAPD patients during catheter removal for infection or malfunction. The samples were prepared in the standard way to be studied by transmission electron microscopy (TEM). ♦ Results Paracrystalline intracytoplasmic inclusions were seen in mesothelial cells only by TEM. They appear as filamentous structures at the outer part of the inclusions, and as pearl-like structures at the core of the inclusions. Sacculate dilatations of rough endoplasmic reticulum cisternae with partly destroyed membranes and only few ribosomes were also seen, with and without densely osmiophilic filaments within the cisternae. We have found paracrystalline intracytoplasmic inclusions in mesothelial cells from uremic and CAPD patients both. According to the literature, these changes are present in one third of biopsies from uremic patients. Until now, however, they have not been mentioned in CAPD patients.