ABSTRACT.Purpose: Scleral biomechanical weakness and thinning is known to be one of the main factors in the pathogenesis of progressive myopia. We tried to strengthen rabbit sclera by cross-linking scleral collagen using ultraviolet A (UVA) and the photosensitizer riboflavin. Methods: Circumscribed 10 · 10 mm sectors of the posterior -equatorial sclera of six chinchilla rabbit eyes were treated in vivo using a UVA double diode with 4.2 mW/cm 2 UVA at 370 nm and applying 0.1% riboflavin-5-phosphate drops as photosensitizer for 30 min. 1 day postoperatively biomechanical stressÀstrain measurements of three treated scleral strips were performed using a microcomputercontrolled biomaterial testing device and compared to non-treated contralateral control sclera. In addition, three treated eyes were examined histologically by light microscopy, TUNEL staining and electron microscopy to evaluate side-effects. Results: Following the cross-linking treatment, the ultimate stress was 11.87 -1.8 MPa versus 3.63 -0.40 in the controls (increase of 227.9%, p = 0.014), Young's modulus 27.67 -4.16 MPa versus 4.9 -2.15 MPa in the controls (increase of 464.7%, p = 0.021) and ultimate strain 92.2 -7.43% versus 165.63 -19.09% in the controls (decrease of 54.52%, p = 0.012). Histologically, serious side-effects were found in the entire posterior globe with almost complete loss of the photoreceptors, the outer nuclear layer and the retinal pigment epithelium (RPE). Conclusions: Our new method of scleral collagen cross-linking proved very effective in increasing the scleral mechanical strength; the new treatment may represent an option for strengthening scleral tissue in progressive myopia. However, serious sideeffects were observed in the outer retina. In future studies these side-effects could be avoided by reducing the irradiation dose below the cytotoxic level of the retina. Before its clinical application, the new method should be tested in a myopia animal model.

Mutations in the gene encoding the phosphoinositide phosphatase myotubularin 1 protein (MTM1) are usually associated with severe neonatal X-linked myotubular myopathy (XLMTM). However, mutations in MTM1 have also been recognized as the underlying cause of "atypical" forms of XLMTM in newborn boys, female infants, female manifesting carriers and adult men. We reviewed systematically the biopsies of a cohort of patients with an unclassified form of centronuclear myopathy (CNM) and identified four patients presenting a peculiar histological alteration in some muscle fibers that resembled a necklace ("necklace fibers"). We analyzed further the clinical and morphological features and performed a screening of the genes involved in CNM. Muscle biopsies in all four patients demonstrated 4-20% of fibers with internalized nuclei aligned in a basophilic ring (necklace) at 3 microm beneath the sarcolemma. Ultrastructurally, such necklaces consisted of myofibrils of smaller diameter, in oblique orientation, surrounded by mitochondria, sarcoplasmic reticulum and glycogen granules. In the four patients (three women and one man), myopathy developed in early childhood but was slowly progressive. All had mutations in the MTM1 gene. Two mutations have previously been reported (p.E404K and p.R241Q), while two are novel; a c.205_206delinsAACT frameshift change in exon 4 and a c.1234A>G mutation in exon 11 leading to an abnormal splicing and the deletion of nine amino acids in the catalytic domain of MTM1. Necklace fibers were seen neither in DNM2- or BIN1-related CNM nor in males with classical XLMTM. The presence of necklace fibers is useful as a marker to direct genetic analysis to MTM1 in CNM.

Congenital myasthenic syndromes (CMSs) are a heterogeneous group of diseases caused by genetic defects affecting neuromuscular transmission. Mutations of DOK7 have recently been described in recessive forms of CMS. Dok-7 is a cytoplasmic post-synaptic protein co-activator of the muscle-specific receptor-tyrosine kinase (MuSK) involved in neuromuscular synaptogenesis and maintenance. We report clinical, morphological and molecular data on 15 patients with mutations in DOK7. Eleven different mutations (5 novel) were identified and all patients but one were found to carry at least the common c.1124_1127dupTGCC mutation. Patients with DOK7 mutations have a particular limb-girdle pattern, without tubular aggregates but a frequent lipidosis on the muscle biopsy. Changes in pre- and post-synaptic compartments of the neuromuscular junction were also observed in muscle biopsies: terminal axons showed defective branching which resulted in a unique terminal axon contacting en passant postsynaptic cups. Clinical features, muscle biopsy findings or response to therapy were confusing in several patients. Characterization of this distinct phenotype is essential to provide clues for targeted genetic screening and to predict the therapeutic response to anticholinesterase treatments or ephedrine as has been suggested.