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 acids 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.

We investigated two unrelated children with an isolated defect of mitochondrial complex III activity. The clinical picture was characterized by a progressive encephalopathy featuring early-onset developmental delay, spasticity, seizures, lactic acidosis, brain atrophy and MRI signal changes in the basal ganglia. Both children were compound heterozygotes for novel mutations in the human bc1 synthesis like (BCS1L) gene, which encodes an AAA mitochondrial protein putatively involved in both iron homeostasis and complex III assembly. The pathogenic role of the mutations was confirmed by complementation assays, using a DeltaBcs1 strain of Saccharomyces cerevisiae. By investigating complex III assembly and the structural features of the BCS1L gene product in skeletal muscle, cultured fibroblasts and lymphoblastoid cell lines from our patients, we have demonstrated, for the first time in a mammalian system, that a major function of BCS1L is to promote the maturation of complex III and, more specifically, the incorporation of the Rieske iron-sulfur protein into the nascent complex. Defective BCS1L leads to the formation of a catalytically inactive, structurally unstable complex III. We have also shown that BCS1L is contained within a high-molecular-weight supramolecular complex which is clearly distinct from complex III intermediates.

Background Lack of functional evidence hampers variant interpretation, leaving a large proportion of individuals with a suspected Mendelian disorder without genetic diagnosis after whole genome or whole exome sequencing (WES). Research studies advocate to further sequence transcriptomes to directly and systematically probe gene expression defects. However, collection of additional biopsies and establishment of lab workflows, analytical pipelines, and defined concepts in clinical interpretation of aberrant gene expression are still needed for adopting RNA sequencing (RNA-seq) in routine diagnostics. Methods We implemented an automated RNA-seq protocol and a computational workflow with which we analyzed skin fibroblasts of 303 individuals with a suspected mitochondrial disease that previously underwent WES. We also assessed through simulations how aberrant expression and mono-allelic expression tests depend on RNA-seq coverage. Results We detected on average 12,500 genes per sample including around 60% of all disease genes—a coverage substantially higher than with whole blood, supporting the use of skin biopsies. We prioritized genes demonstrating aberrant expression, aberrant splicing, or mono-allelic expression. The pipeline required less than 1 week from sample preparation to result reporting and provided a median of eight disease-associated genes per patient for inspection. A genetic diagnosis was established for 16% of the 205 WES-inconclusive cases. Detection of aberrant expression was a major contributor to diagnosis including instances of 50% reduction, which, together with mono-allelic expression, allowed for the diagnosis of dominant disorders caused by haploinsufficiency. Moreover, calling aberrant splicing and variants from RNA-seq data enabled detecting and validating splice-disrupting variants, of which the majority fell outside WES-covered regions. Conclusion Together, these results show that streamlined experimental and computational processes can accelerate the implementation of RNA-seq in routine diagnostics.

The spectrum of brain and spine MRI abnormalities in Hunter disease is extremely wide and requires a thorough evaluation. WMAs, atrophy/communicating hydrocephalus and spinal stenosis progress over time and might represent possible disease severity markers for new treatment efficacy assessment.