GM1 gangliosidosis and Morquio B disease: an update on genetic alterations and clinical findings.

GM1 gangliosidosis and Morquio B syndrome, both arising from beta-galactosidase (GLB1) deficiency, are very rare lysosomal storage diseases with an incidence of about 1:100,000-1:200,000 live births worldwide. Here we report the beta-galactosidase gene (GLB1) mutation analysis of 21 unrelated GM1 gangliosidosis patients, and of 4 Morquio B patients, of whom two are brothers. Clinical features of the patients were collected and compared with those in literature. In silico analyses were performed by standard alignments tools and by an improved version of GLB1 three-dimensional models. The analysed cohort includes remarkable cases. One patient with GM1 gangliosidosis had a triple X syndrome. One patient with juvenile GM1 gangliosidosis was homozygous for a mutation previously identified in Morquio type B. A patient with infantile GM1 gangliosidosis carried a complex GLB1 allele harbouring two genetic variants leading to p.R68W and p.R109W amino acid changes, in trans with the known p.R148C mutation. Molecular analysis showed 27 mutations, 9 of which are new: 5 missense, 3 microdeletions and a nonsense mutation. We also identified four new genetic variants with a predicted polymorphic nature that was further investigated by in silico analyses. Three-dimensional structural analysis of GLB1 homology models including the new missense mutations and the p.R68W and p.R109W amino acid changes showed that all the amino acid replacements affected the resulting protein structures in different ways, from changes in polarity to folding alterations. Genetic and clinical associations led us to undertake a critical review of the classifications of late-onset GM1 gangliosidosis and Morquio B disease.

Critical aspects of detection of sperm DNA fragmentation by TUNEL/flow cytometry.

Despite the many studies documenting an increase of sperm DNA damage in subfertile and infertile subjects, determining whether this parameter is relevant for the clinic is still an open question. Indeed, results of clinical investigations on sperm DNA damage and outcome of ART procedures are often conflicting as many factors affect the predictive power of these tests. The techniques used to reveal such damage is one of these factors. Techniques detecting sperm DNA damage are indeed numerous and heterogeneous. In addition, it is not obvious that they reveal the same type of DNA damage and that their results are equivalent. One of the available methods to detect sperm DNA damage is the TUNEL assay. Since it was developed in the 1990s the TUNEL assay has been widely employed, becoming one of the most popular strategies to investigate the origin, the mechanism, and the clinical meaning of sperm DNA damage. Our group has used TUNEL coupled to flow cytometry to investigate sperm DNA fragmentation for about 10 years. According to our experience, this technique presents some pitfalls and limitations in the accuracy and reproducibility of the measures of sperm DNA fragmentation. In this review, we discuss several technical features of TUNEL and report the solutions adopted by our group to overcome some of its limitations.