Myotubularin, a phosphatase deficient in myotubular myopathy, acts on phosphatidylinositol 3-kinase and phosphatidylinositol 3-phosphate pathway

Myotubular myopathy (MTM1) is an X-linked disease, characterized by severe neonatal hypotonia and generalized muscle weakness, with pathological features suggesting an impairment in maturation of muscle fibres. The MTM1 gene encodes a protein (myotubularin) with a phosphotyrosine phosphatase consensus. It defines a family of at least nine genes in man, including the antiphosphatase hMTMR5/Sbf1 and hMTMR2, recently found mutated in a recessive form of Charcot-Marie-Tooth disease. Myotubularin shows a dual specificity protein phosphatase activity in vitro. We have performed an in vivo test of tyrosine phosphatase activity in Schizosaccharomyces pombe, indicating that myotubularin does not have a broad specificity tyrosine phosphatase activity. Expression of active human myotubularin inhibited growth of S.pombe and induced a vacuolar phenotype similar to that of mutants of the vacuolar protein sorting (VPS) pathway and notably of mutants of VPS34, a phosphatidylinositol 3-kinase (PI3K). In S.pombe cells deleted for the endogenous MTM homologous gene, expression of human myotubularin decreased the level of phosphatidylinositol 3-phosphate (PI3P). We have created a substrate trap mutant which shows relocalization to plasma membrane projections (spikes) in HeLa cells and was inactive in the S.pombe assay. This mutant, but not the wild-type or a phosphatase site mutant, was able to immunoprecipitate a VPS34 kinase activity. Wild-type myotubularin was also able to directly dephosphorylate PI3P and PI4P in vitro. Myotubularin may thus decrease PI3P levels by down-regulating PI3K activity and by directly degrading PI3P.     

Three families with high expression of a fragile site at Xq27.3, lack of anomalies at the FMR-1 CpG island, and no clear phenotypic association.

We report on 3 families where the presence and segregation at high frequency of a fragile Xq27.3 site is not associated with the mutations and methylation anomalies typically seen in the fragile X [Fra(X)] syndrome. In one family, a folate insensitive fragile site was associated with Robin sequence in the propositus. In a second family a fra(X) negative mother has two fra(X) positive sons (one mentally retarded and the other newborn). The third family presents very high expression of a folate sensitive site, unlinked to mental retardation, and was described previously by Voelckel et al. [1989]. The fragile sites in these or similar families recently described must be different from the one associated with the fra(X) syndrome. Their association with a clinical phenotype or with mental retardation is certainly not consistent, and may represent an ascertainment bias. However, the relatively high frequency with which they have been found among previously diagnosed fra(X) families suggests that, at least in some cases, the association with mental impairment may be significant. In two families reported up to now, a male with high expression of such variant fra(X) site failed to transmit it to his daughter, which may reflect an imprinting effect. Previously diagnosed families should be reinvestigated before direct DNA analysis is used for prenatal or carrier diagnosis of the fra(X) syndrome.     

Selection in blood cells from female carriers of the fragile X syndrome: inverse correlation between age and proportion of active X chromosomes carrying the full mutation.

We have studied the patterns of mutation and X inactivation in female carriers of a fragile X mutation, to try to correlate them with various phenotypic features. We used a simple assay, which shows simultaneously the size of the mutation, its methylation status, and DNA fragments that represent the normal active and inactive X chromosomes. We have observed an age dependent process, whereby the 'full' fragile X mutation is found preferentially on the inactive X in leucocytes in adult females, but not in younger ones. This phenomenon was not observed in female carriers of a 'premutation', who have little phenotypic expression. Preliminary data suggest that young females who show preferential presence of a full mutation on the active X in leucocytes may be at increased risk for mental retardation. We have also obtained preliminary evidence for an age dependent decrease in the somatic heterogeneity of full mutations, possibly owing to selection for smaller mutated fragments. If confirmed, the latter phenomenon might account for the known decrease with age of the expression of the fragile site. Our observations suggest that a gene whose expression is affected by the presence of a full mutation (possibly the FMR-1 gene) has a cell autonomous function in leucocytes, leading to a slowly progressive selection for cells where the mutation is on the inactive X chromosome.     

Lack of Autoimmune Serological Reactions in Rodent Models of Insulin Dependent Diabetes Mellitus

Spontaneous insulitis with insulin-dependent diabetes mellitus (IDDM) in rodent models, the BB rat and NOD mouse, has clarified the pathogenesis of and guided decisions on interventional therapy for human IDDM. However, the occurrence in such models of a standard marker of human IDDM, autoantibodies to β islet cell constituents, has been controversial. Hence we assessed diabetes-prone rodents for the frequencies of raised levels of auto-antibodies to glutamic acid decarboxylase GAD (anti-GAD), insulin and heat shock protein 65 (HSP-65) in relation to levels in non-diabetes-prone animals and levels in human diabetic sera. Assays were performed sequentially at various ages of life. The immunoassays used for anti-GAD and anti-insulin were those validated for sensitivity and specificity for detection of the corresponding autoantibodies in human IDDM sera at international workshops. Positive controls included human IDDM sera with reactivity with GAD or insulin and, for mouse anti-GAD, the highly reactive monoclonal antibody, GAD-6. The results were that levels of autoantibodies in diabetes-prone BB rats or NOD mice to the ‘IDDM-relevant’ autoantigens in our panel did not exceed levels in control rats or mice, and were much lower than levels in humans with IDDM. We conclude that the BB rat and NOD mouse represent a model, but not a facsimile, of human IDDM and that therapeutic successes in such models should be interpreted with caution in relation to interventional therapy for human IDDM.     

Anti-Interleukin-6 Therapy Decreases Hip Synovitis and Bone Resorption and Increases Bone Formation Following Ischemic Osteonecrosis of the Femoral Head

Legg-Calvé-Perthes disease (LCPD) is a juvenile form of ischemic femoral head osteonecrosis, which produces chronic hip synovitis, permanent femoral head deformity, and premature osteoarthritis. Currently, there is no medical therapy for LCPD. Interleukin-6 (IL-6) is significantly elevated in the synovial fluid of patients with LCPD. We hypothesize that IL-6 elevation promotes chronic hip synovitis and impairs bone healing after ischemic osteonecrosis. We set out to test if anti-IL-6 therapy using tocilizumab can decrease hip synovitis and improve bone healing in the piglet model of LCPD. Fourteen piglets were surgically induced with ischemic osteonecrosis and assigned to two groups: the no treatment group (n = 7) and the tocilizumab group (15 to 20 mg/kg, biweekly intravenous injection, n = 7). All animals were euthanized 8 weeks after the induction of osteonecrosis. Hip synovium and femoral heads were assessed for hip synovitis and bone healing using histology, micro-CT, and histomorphometry. The mean hip synovitis score and the number of synovial macrophages and vessels were significantly lower in the tocilizumab group compared with the no treatment group (p < .0001, p = .01, and p < .01, respectively). Micro-CT analysis of the femoral heads showed a significantly higher bone volume in the tocilizumab group compared with the no treatment group (p = .02). The histologic assessment revealed a significantly lower number of osteoclasts per bone surface (p < .001) in the tocilizumab group compared with the no treatment group. Moreover, fluorochrome labeling showed a significantly higher percent of mineralizing bone surface (p < .01), bone formation rate per bone surface (p < .01), and mineral apposition rate (p = .04) in the tocilizumab group. Taken together, tocilizumab therapy decreased hip synovitis and osteoclastic bone resorption and increased new bone formation after ischemic osteonecrosis. This study provides preclinical evidence that tocilizumab decreases synovitis and improves bone healing in a large animal model of LCPD. © 2020 American Society for Bone and Mineral Research (ASBMR).     

Ontogeny of J chain expression in pig lymphocytes

The ontogeny of lymphocytes expressing J chain in the cytoplasm (J+) was studied in pig foetuses by the immunofluorescent technique. Peripheral blood lymphocytes were the first J+ cells in prenatal life. The spleen and lymph nodes contained J+ cells in the last days of gestation. J+ cells were found in the lamina propria of the gut and some glands of conventional but not of germ-free piglets. J chain was not detected on or in cell membranes at any developmental stage.