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Purpose: We studied microRNAs as potential biomarkers for Pompe disease., Methods: We analyzed microRNA expression by small RNA-seq in tissues from the disease murine model at two different ages (3 and 9 months), and in plasma from Pompe patients., Results: In the mouse model we found 211 microRNAs that were differentially expressed in gastrocnemii and 66 in heart, with a different pattern of expression at different ages. In a preliminary analysis in plasma from six patients 55 microRNAs were differentially expressed. Sixteen of these microRNAs were common to those dysregulated in mouse tissues. These microRNAs are known to modulate the expression of genes involved in relevant pathways for Pompe disease pathophysiology (autophagy, muscle regeneration, muscle atrophy). One of these microRNAs, miR-133a, was selected for further quantitative real-time polymerase chain reaction analysis in plasma samples from 52 patients, obtained from seven Italian and Dutch biobanks. miR-133a levels were significantly higher in Pompe disease patients than in controls and correlated with phenotype severity, with higher levels in infantile compared with late-onset patients. In three infantile patients miR-133a decreased after start of enzyme replacement therapy and evidence of clinical improvement., Conclusion: Circulating microRNAs may represent additional biomarkers of Pompe disease severity and of response to therapy.

Carnitine palmitoyltransferase II (CPT II) deficiency is the most common inherited disorder of lipid metabolism characterized in its adult form by attacks of myalgia and myoglobinuria. We analyzed a cohort of 22 CPT II-deficient patients (representing 20 independent probands) to correlate clinical presentation and molecular data. The common p.Ser113Leu mutation was detected with an allelic frequency of 67.5% (27/40), in association with mild adult-onset phenotype. In addition to the p.Ser113Leu mutation, other 10 disease-causing mutations were identified, 5 of which were novel. They are a micro-insertion within exon 5, three aminoacid substitutions within the coding region, namely p.Arg151Trp, p.Asp576Gly, p.Arg247Trp and a truncating stop codon mutation (p.Arg554Ter). Our data expand the spectrum of CPT II mutations and help to evaluate possible correlations between genotypes and phenotypes.

We report a new missense mutation (Ala140Gly) in exon 5 of the Cu/Zn superoxide dismutase (SOD-1) gene in a 73-year-old man with familial amyotrophic lateral sclerosis (FALS). The enzymatic activity of mutated SOD-1 measured in erythrocyte lysate was 70% of control. This heterozygote mutation, which is associated with the late onset of the disease, is located in the active site of the enzyme.

The pathogenic mechanisms of the A1555G mitochondrial DNA mutation in the 12S rRNA gene, associated with maternally inherited sensorineural deafness, are largely unknown. Previous studies have suggested an involvement of nuclear factor(s). To address this issue cybrids were generated by fusing osteosarcoma cells devoid of mtDNA with enucleated fibroblasts from two genetically unrelated patients. Furthermore, to determine the contribution, if any, of the mitochondrial and nuclear genomes, separately or in combination, in the expression of the disease phenotype, transmitochondrial fibroblasts were constructed using control and patient's fibroblasts as nuclear donors and homoplasmic mutant or wild-type cybrids as mitochondrial donors. Detailed analysis of mutant and wild-type cybrids from both patients and transmitochondrial fibroblast clones did not reveal any respiratory chain dysfunction suggesting that, if nuclear factors do indeed act as modifier agents, they may be tissue-specific. However, in the presence of high concentrations of neomycin or paromomycin, but not of streptomycin, mutant cells exhibit a decrease in the growth rate, when compared to wild-type cells. The decrease did not correlate with the rate of synthesis or stability of mitochondrial DNA-encoded subunits or respiratory chain activity. Further studies are required to determine the underlying biochemical defect.

We identified a novel stop codon mutation in the myophosphorylase gene in a Greek patient with typical symptoms of McArdle's disease. This is the first genetic study of myophosphorylase deficiency in a Greek family, showing that the proband was a compound heterozygous for the common "caucasian" mutation (R49X) and a new nonsense mutation (Y52X), both within exon 1. The new point mutation, a C-to-G transversion at codon 52, converts an encoded tyrosine to a stop codon. Our study confirms that the R49X is also present in the Greek population. The Y52X may represent a private mutation or a common mutation among Greeks. Our data further expand the already remarkable genetic heterogeneity of McArdle's disease. The prevalence of the Y52X mutation in Greek patients with McArdle's disease remains to be determined.