Identification of new biomarkers for phenotypical characterization in peripheral neuropathies

Abstract

The Charcot-Marie-Tooth disease (CMT; ORPHA166) is the most common hereditary neuropathy. CMT disease contains a heterogeneous group of disorders with similar clinical, neurophysiological, genetic and pathological features. The clinical expression and individual phenotype is the consequence of a progressive axonal loss of both motor and sensory nerves. The most frequent genetic defect underlying CMT is an intrachromosomal duplication on human chromosome 17p12, the locus defining CMT subtype 1a (CMT1A). In this duplicated region, the gene encoding the peripheral myelin protein of 22 kDa (PMP22) is located. PMP22 is a specific integral membrane protein expressed by the myelinating Schwann cells of peripheral neurons at elevated levels and it has been involved in myelin formation and maintenance. Furthermore, PMP22 has been associated with the formation of intercellular junctions of epithelial cells, and cell proliferation and apoptosis regulation. Friedreich’s ataxia (FRDA; OMIM 229300), an autosomal recessive neurodegenerative mitochondrial disease, is the most prevalent hereditary ataxia. This rare, childhood-onset disease is characterized by a progressive loss of sensory neurons in the dorsal root ganglia (DRG) and posterior columns. The cerebellar dentate nucleus is also affected. Other non-neurological features of FRDA are scoliosis, diabetes and cardiac hypertrophy. The last is the primary cause of death in these patients. FRDA is most often caused by a homozygous GAA repeat expansion mutation (typically between 600 and 1200 repeats) in the first intron of the frataxin gene (FXN), which have been proposed as causes of decreased expression of the mitochondrial protein frataxin. The principal function of Frataxin is not clear yet, but it has been described its role in iron homeostasis and Iron-Sulfur Cluster formation. Oxidative stress and proteomic analysis would provide differences between mild CMT patients, severe CMT patients, and controls that could be used as new biomarkers of disease progression. On the other hand, miRNAs analysis would provide different expression profile signatures between patients and controls that may help in the identification of different phenotypes in FRDA’s patients. Furthermore, the miRNome characterization in cellular models of the disease, may help to the comprehension of the special function of differential expressed miRNAs in the physiopathology of Friedreich ataxia. Carbonylation and nitrosylation of proteins, malondialdehide levels, total antioxidant capacity, and glutathione levels did not show differences between mild CMT patients, severe CMT patients, therefore any oxidative stress biomarker was identified for this disease. However, in our 2D-DIGE proteomic analysis we found seventeen proteins (Plasminogen, Complement factor B, Gelsolin, Antithrombin-III, Apolipoprotein A-IV, Haptoglobin, Clusterin, Complement component C6, Fibrinogen gamma chain, Vitronectin, Serotransferrin, Kininogen-1, Alpha-1- antitrypsin, Alpha-2-HS-glycoprotein, Vitamin D-binding protein, Apolipoprotein E, and Apolipoprotein A-I) with a close phenotypic relationship with PMP22 and differential expression compared to controls. One of these candidate proteins, gelsolin, was validated by Enzyme-linked InmunoSorbent Assay (ELISA). However, no differences were found between the three groups analysed. On the other hand, small-RNA sequencing and bioinformatic miRNAs analysis provides different expression miRNA’s profile between plasma samples from patients or cellular models of the disease compared to their respective controls. In the smallRNA-seq analysis in cellular models, we found selected miRNAs shared in at least two of three cell models, which had a relevant role in pathways that were affected in Friedreich’s ataxia. In case of plasma samples, we found different expression profile of miRNAs (hsa-miR-128-3p, hsa-miR-625-3p, hsa-miR-130b-5p, hsa- miR-151a-5p, hsa-miR-330-3p, hsa-miR-323a-3p, and hsa-miR-142-3p) between plasma samples of patients and those obtained from healthy subjects. In addition, we found that hsa-miR323a- 3p was a marker for phenotypic differentiation in FRDA patients suffering from cardiomyopathy and may play a role in Ca+2 regulation in heart. Finally, we found that some circulating miRNAs detected in plasma were also detected in some of the cellular models analysed, reinforcing the hypothesis that these miRNAs play a relevant role in the pathophysiology of FRDA. Taking all together this study, we improve the knowledge about physiopathology of these neuromuscular diseases. In addition, we provide different possible proteomic biomarkers that were previously associated with other neurodegenerative diseases, and should be validated in CMT. Furthermore, we propose miR-323a-3p as biomarker of cardiomyopathy in patients of FRDA.