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Charcot-Marie-Tooth disease is characterized by broad genetic heterogeneity with >50 known disease-associated genes. Mutations in some of these genes can cause a pure motor form of hereditary motor neuropathy, the genetics of which are poorly characterized. We designed a panel comprising 56 genes associated with Charcot-Marie-Tooth disease/hereditary motor neuropathy. We validated this diagnostic tool by first testing 11 patients with pathological mutations. A cohort of 33 affected subjects was selected for this study. The DNAJB2 c.352+1G>A mutation was detected in two cases; novel changes and/or variants with low frequency (<1%) were found in 12 cases. There were no candidate variants in 18 cases, and amplification failed for one sample. The DNAJB2 c.352+1G>A mutation was also detected in three additional families. On haplotype analysis, all of the patients from these five families shared the same haplotype; therefore, the DNAJB2 c.352+1G>A mutation may be a founder event. Our gene panel allowed us to perform a very rapid and cost-effective screening of genes involved in Charcot-Marie-Tooth disease/hereditary motor neuropathy. Our diagnostic strategy was robust in terms of both coverage and read depth for all of the genes and patient samples. These findings demonstrate the difficulty in achieving a definitive molecular diagnosis because of the complexity of interpreting new variants and the genetic heterogeneity that is associated with these neuropathies.

Charcot-Marie-Tooth (CMT) disease or hereditary motor and sensory neuropathy (HMSN) is a genetically heterogeneous group of conditions that affect the peripheral nervous system. The disease is characterized by degeneration or abnormal development of peripheral nerves and exhibits a range of patterns of genetic transmission. In the majority of cases, CMT first appears in infancy, and its manifestations include clumsiness of gait, predominantly distal muscular atrophy of the limbs, and deformity of the feet in the form of foot drop. It can be classified according to the pattern of transmission (autosomal dominant, autosomal recessive, or X linked), according to electrophysiological findings (demyelinating or axonal), or according to the causative mutant gene. The classification of CMT is complex and undergoes constant revision as new genes and mutations are discovered. In this paper, we review the most efficient diagnostic algorithms for the molecular diagnosis of CMT, which are based on clinical and electrophysiological data.

Transformations of surfactant after secretion are incompletely understood. To clarify them, we lavaged lungs in fetuses and in newborn rabbits, fractionated the lavage fluid by differential and density gradient centrifugation, and analyzed the distribution of surfactant protein (SP) phospholipids, SP-A, SP-B, and SP-C. Furthermore, we administered into trachea of newborn rabbits labeled surfactant and compared the alveolar clearance of SP-A, SP-B, SP-C and saturated phosphatidylcholine. We found that, in the fetus, secreted lamellar bodies contain all components of surfactant, except a small amount of SP-A. As breathing starts and new surfactant subtypes are generated, the proteins are mostly associated with dense subtypes, but SP-B and SP-C are especially concentrated in dense materials that contain minor amounts of phospholipids and SP-A. Furthermore, we found that, during breathing, alveolar surfactant is degraded into more than one type of remnant, that the lavage fluid contains a pool of SP-A not associated with membranes, and that SP-A, SP-B, and SP-C are all turned over at a faster rate than saturated phosphatidylcholine.

X-Adrenoleukodystrophy (X-ALD) and its adult-onset, most prevalent variant adrenomyeloneuropathy (AMN) are caused by mutations in the peroxisomal transporter of the very long-chain fatty acid ABCD1. AMN patients classically present spastic paraparesis that can progress over decades, and a satisfactory treatment is currently lacking. Oxidative stress is an early culprit in X-ALD pathogenesis. A combination of antioxidants halts the clinical progression and axonal damage in a murine model of AMN, providing a strong rationale for clinical translation. In this phase II pilot, open-label study, 13 subjects with AMN were administered a high dose of α-tocopherol, N-acetylcysteine, and α-lipoic acid in combination. The primary outcome was the validation of a set of biomarkers for monitoring the biological effects of this and future treatments. Functional clinical scales, the 6-minute walk test (6MWT), electrophysiological studies, and cerebral MRI served as secondary outcomes. Most biomarkers of oxidative damage and inflammation were normalized upon treatment, indicating an interlinked redox and inflammatory homeostasis. Two of the inflammatory markers, MCP1 and 15-HETE, were predictive of the response to treatment. We also observed a significant decrease in central motor conduction time, together with an improvement or stabilization of the 6MWT in 8/10 subjects. This study provides a series of biomarkers that are useful to monitor redox and pro-inflammatory target engagement in future trials, together with candidate biomarkers that may serve for patient stratification and disease progression, which merit replication in future clinical trials. Moreover, the clinical results suggest a positive signal for extending these studies to phase III randomized, placebo-controlled, longer-term trials with the actual identified dose. ClinicalTrials.gov Identifier: NCT01495260

Amiodarone may induce lung damage by direct toxicity or indirectly through inflammation. To clarify the mechanism of direct toxicity, we briefly exposed rabbit alveolar macrophages to amiodarone and analyzed their morphology, synthesis, and degradation of dipalmitoylphosphatidylcholine (DPPC); distribution of lysosomal enzymes; and uptake of diphtheria toxin and surfactant protein (SP) A used as tracers of the endocytic pathway. Furthermore, in newborn rabbits, we studied the clearance of DPPC and SP-A instilled into the trachea together with increasing amounts of amiodarone. We found that in vitro amiodarone decreases the surface density of mitochondria and lysosomes while increasing the surface density of inclusion bodies, increases the incorporation of choline into DPPC, modifies the distribution of lysosomal enzymes, and does not affect the uptake and processing of diphtheria toxin but inhibits the degradation of SP-A. In vivo amiodarone inhibits the degradation of SP-A but not of DPPC. We conclude that the acute exposure to amiodarone perturbs the endocytic pathway acting after the early endosomes, alters the traffic of lysosomal enzymes, and interferes with the turnover of SP-A.

In patients with idiopathic alveolar proteinosis, the alveoli are filled with materials rich in surfactant components, especially surfactant protein A (SP-A). The anomaly could be caused by either increased secretion, decreased clearance, or both. To clarify this point, we studied five patients who underwent therapeutic lavage and then were ventilated mechanically for 24 h. During the first 8 h of mechanical ventilation, a surfactant-depleted lung was lavaged at selected intervals, and the bronchoalveolar lavage fluid was analyzed. We observed that, after lavage, various surfactant components accumulated in the airways with different time courses. We also observed that SP-A increased until the second hour and then dropped rapidly, suggesting the existence of an efficient mechanism of removal. These findings suggest that idiopathic alveolar proteinosis might be caused by a primary defect in a slow mechanism of removal or by the presence of factor(s) that interfere with the clearance of surfactant and that can be removed by lavage. It seems clear, however, that an increased secretion rate is unlikely to be the major cause of idiopathic alveolar proteinosis.

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