Anna Caciotti

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.

Primary and Secondary Elastin-Binding Protein Defect Leads to Impaired Elastogenesis in Fibroblasts from GM1-Gangliosidosis Patients

G(M1)-gangliosidosis is a lysosomal storage disorder caused by acid beta-galactosidase deficiency. Aside from the lysosomal beta-galactosidase enzyme, the beta-galactosidase gene also encodes the elastin-binding protein (EBP), deficiency in which impairs elastogenesis. Using expression studies and Western blots of COS-1 cells, we identified and characterized four new and two known beta-galactosidase gene mutations detected in G(M1)-gangliosidosis patients with infantile, juvenile, or adult forms of disease. We then focused on impaired elastogenesis detected in fibroblasts from patients with infantile and juvenile disease. The juvenile patient showed connective-tissue abnormalities, unusual urinary keratan sulfate excretion, and an EBP reduction, despite mutations affecting only beta-galactosidase. Because galactosugar-bearing moieties may alter EBP function and impair elastogenesis, we assessed infantile and juvenile patients for the source of altered elastogenesis. We confirmed that the infantile patients impaired elastogenesis arose from a primary EBP defect, according to molecular analysis. We examined the juveniles fibroblasts by immunohistochemistry, addition of keratanase, soluble/insoluble elastin assay, and radiolabeling of tropoelastin. These experiments revealed that the juveniles impaired elastogenesis likely arose from secondary EBP deficiency caused by keratan sulfate accumulation. Thus, impaired elastogenesis in G(M1)-gangliosidosis can arise from primary or secondary EBP defects in fibroblasts from infantile and juvenile patients, respectively.

The potential action of galactose as a "chemical chaperone": increase of beta galactosidase activity in fibroblasts from an adult GM1-gangliosidosis patient.

BACKGROUND The glycosphingolipid storage disorder GM1-gangliosidosis is a severe neurodegenerative condition for which no therapy is currently available. Protein misfolding in lysosomal defects may have the potential to be corrected by chemical chaperones: in vitro and clinical approaches are being investigated. AIMS We investigated the in vitro effect of galactose on some lysosomal hydrolases, and its in vitro efficacy as a chemical chaperone in GM1-gangliosidosis. METHODS Galactose was added to the culture medium of fibroblasts from patients, controls and transfected COS-1 cells. Enzyme assays of lysosomal hydrolases, beta galactosidase in particular, were performed. RESULTS Our data show that galactose alters selectively alpha and beta galactosidases. A significant increase (2,5 fold) in beta galactosidase activity occurred when galactose was added to the cultured fibroblasts of an adult patient. Chemical chaperone therapy requires the presence of residual enzyme activity. The adult patient here reported is heterozygous for the p.T329A mutation that showed no beta galactosidase activity, and for the p.R442Q mutation with residual enzyme activity. The p.R442Q mutation was therefore selected as a potential target for the galactose chaperone; after the addition of galactose, COS-1 cells transfected with this mutation showed an increase in beta galactosidase activity from 6.9% to 12% of control values. CONCLUSIONS These results suggest that galactose or its derivatives with potential chaperone properties could be used in the development of non-invasive therapies for GM1-gangliosidosis.

Molecular testing of 163 patients with Morquio A (Mucopolysaccharidosis IVA) identifies 39 novel GALNS mutations.

Morquio A (Mucopolysaccharidosis IVA; MPS IVA) is an autosomal recessive lysosomal storage disorder caused by partial or total deficiency of the enzyme galactosamine-6-sulfate sulfatase (GALNS; also known as N-acetylgalactosamine-6-sulfate sulfatase) encoded by the GALNS gene. Patients who inherit two mutated GALNS gene alleles have a decreased ability to degrade the glycosaminoglycans (GAGs) keratan sulfate and chondroitin 6-sulfate, thereby causing GAG accumulation within lysosomes and consequently pleiotropic disease. GALNS mutations occur throughout the gene and many mutations are identified only in single patients or families, causing difficulties both in mutation detection and interpretation. In this study, molecular analysis of 163 patients with Morquio A identified 99 unique mutations in the GALNS gene believed to negatively impact GALNS protein function, of which 39 are previously unpublished, together with 26 single-nucleotide polymorphisms. Recommendations for the molecular testing of patients, clear reporting of sequence findings, and interpretation of sequencing data are provided.

Severe prognosis in a large family with hypokalemic periodic paralysis.

Hypokalemic periodic paralysis (HypoPP) is a channel disorder caused primarily by mutations in the human skeletal muscle α1 subunit (CACNA1S) of the dihydropyridine‐sensitive calcium channel. Molecular, clinical, and biochemical studies were aimed at establishing genotype/phenotype correlations in a large Italian family affected by a severe form of HypoPP. Whereas patients with HypoPP usually show a normal life span, in this family three male patients died young, one of them from anesthetic complications resembling malignant hyperthermia. Our patients carried the c1583G>A genetic lesion (R528H), which has been associated with a mild phenotype and with incomplete penetrance in women. Surprisingly, the R528H amino acid substitution in the family presented here correlated with an unfavorable prognosis in both male and female patients. We conclude that genetic characterization is an important requirement to alert physicians about the management of similar patients, especially when anesthesia is considered. Muscle Nerve 27: 165–169, 2003

Morquio A Syndrome-Associated Mutations: A Review of Alterations in the GALNS Gene and a New Locus-Specific Database

Morquio A syndrome (mucopolysaccharidosis IVA) is an autosomal recessive disorder that results from deficient activity of the enzyme N‐acetylgalactosamine‐6‐sulfatase (GALNS) due to alterations in the GALNS gene, which causes major skeletal and connective tissue abnormalities and effects on multiple organ systems. The GALNS alterations associated with Morquio A are numerous and heterogeneous, and new alterations are continuously identified. To aid detection and interpretation of GALNS alterations, from previously published research, we provide a comprehensive and up‐to‐date listing of 277 unique GALNS alterations associated with Morquio A identified from 1,091 published GALNS alleles. In agreement with previous findings, most reported GALNS alterations are missense changes and even the most frequent alterations are relatively uncommon. We found that 48% of patients are assessed as homozygous for a GALNS alteration, 39% are assessed as heterozygous for two identified GALNS alterations, and in 13% of patients only one GALNS alteration is detected. We report here the creation of a locus‐specific database for the GALNS gene (http://galns.mutdb.org/) that catalogs all reported alterations in GALNS to date. We highlight the challenges both in alteration detection and genotype–phenotype interpretation caused in part by the heterogeneity of GALNS alterations and provide recommendations for molecular testing of GALNS.

Galactosialidosis: review and analysis of CTSA gene mutations

BackgroundMutations in the CTSA gene, that encodes the protective protein/cathepsin A or PPCA, lead to the secondary deficiency of β-galactosidase (GLB1) and neuraminidase 1 (NEU1), causing the lysosomal storage disorder galactosialidosis (GS). Few clinical cases of GS have been reported in the literature, the majority of them belonging to the juvenile/adult group of patients.MethodsThe correct nomenclature of mutations for this gene is discussed through the analysis of the three PPCA/CTSA isoforms available in the GenBank database. Phenotype-genotype correlation has been assessed by computational analysis and review of previously reported single amino acid substitutions.ResultsWe report the clinical and mutational analyses of four cases with the rare infantile form of GS. We identified three novel nucleotide changes, two of them resulting in the missense mutations, c.347A>G (p.His116Arg), c.775T>C (p.Cys259Arg), and the third, c.1216C>T, resulting in the p.Gln406* stop codon, a type of mutation identified for the first time in GS. An Italian founder effect of the c.114delG mutation can be suggested according to the origin of the only three patients carrying this mutation reported here and in the literature.ConclusionsIn early reports mutations nomenclature was selected according to all CTSA isoforms (three different isoforms), thus generating a lot of confusion. In order to assist physicians in the interpretation of detected mutations, we mark the correct nomenclature for CTSA mutations. The complexity of pathology caused by the multifunctions of CTSA, and the very low numbers of mutations (only 23 overall) in relation to the length of the CTSA gene are discussed.In addition, the in silico functional predictions of all reported missense mutations allowed us to closely predict the early infantile, late infantile and juvenile phenotypes, also disclosing different degrees of severity in the juvenile phenotype.

Optimizing the molecular diagnosis of GALNS: novel methods to define and characterize Morquio-A syndrome-associated mutations

Morquio A syndrome (MPS IVA) is a systemic lysosomal storage disorder caused by the deficiency of N‐acetylgalactosamine‐6‐sulfatase (GALNS), encoded by the GALNS gene. We studied 37 MPS IV A patients and defined genotype–phenotype correlations based on clinical data, biochemical assays, molecular analyses, and in silico structural analyses of associated mutations. We found that standard sequencing procedures, albeit identifying 14 novel small GALNS genetic lesions, failed to characterize the second disease‐causing mutation in the 16% of the patients’ cohort. To address this drawback and uncover potential gross GALNS rearrangements, we developed molecular procedures (CNV [copy‐number variation] assays, QF‐PCRs [quantitative fluorescent‐PCRs]), endorsed by CGH‐arrays. Using this approach, we characterized two new large deletions and their corresponding breakpoints. Both deletions were heterozygous and included the first exon of the PIEZO1 gene, which is associated with dehydrated hereditary stomatocitosis, an autosomal‐dominant syndrome. In addition, we characterized the new GALNS intronic lesion c.245‐11C>G causing m‐RNA defects, although identified outside the GT/AG splice pair. We estimated the occurrence of the disease in the Italian population to be approximately 1:300,000 live births and defined a molecular testing algorithm designed to help diagnosing MPS IVA and foreseeing disease progression.

Clinical relevance of short-chain acyl-CoA dehydrogenase (SCAD) deficiency: Exploring the role of new variants including the first SCAD-disease-causing allele carrying a synonymous mutation

Short-chain acyl-coA dehydrogenase deficiency (SCADD) is an autosomal recessive inborn error of mitochondrial fatty acid oxidation caused by ACADS gene alterations. SCADD is a heterogeneous condition, sometimes considered to be solely a biochemical condition given that it has been associated with variable clinical phenotypes ranging from no symptoms or signs to metabolic decompensation occurring early in life. A reason for this variability is due to SCAD alterations, such as the common p.Gly209Ser, that confer a disease susceptibility state but require a complex multifactorial/polygenic condition to manifest clinically. Our study focuses on 12 SCADD patients carrying 11 new ACADS variants, with the purpose of defining genotype–phenotype correlations based on clinical data, metabolite evaluation, molecular analyses, and in silico functional analyses. Interestingly, we identified a synonymous variant, c.765G > T (p.Gly255Gly) that influences ACADS mRNA splicing accuracy. mRNA characterisation demonstrated that this variant leads to an aberrant splicing product, harbouring a premature stop codon. Molecular analysis and in silico tools are able to characterise ACADS variants, identifying the severe mutations and consequently indicating which patients could benefit from a long term follow- up. We also emphasise that synonymous mutations can be relevant features and potentially associated with SCADD.

Sudden unexpected infant death (SUDI) in a newborn due to medium chain acyl CoA dehydrogenase (MCAD) deficiency with an unusual severe genotype

Medium chain acyl CoA dehydrogenase deficiency (MCAD) is the most common inborn error of fatty acid oxidation. This condition may lead to cellular energy shortage and cause severe clinical events such as hypoketotic hypoglycemia, Reye syndrome and sudden death. MCAD deficiency usually presents around three to six months of life, following catabolic stress as intercurrent infections or prolonged fasting, whilst neonatal-onset of the disease is quite rare. We report the case of an apparently healthy newborn who suddenly died at the third day of life, in which the diagnosis of MCAD deficiency was possible through peri-mortem blood-spot acylcarnitine analysis that showed very high concentrations of octanoylcarnitine. Genetic analysis at the ACADM locus confirmed the biochemical findings by demonstrating the presence in homozygosity of the frame-shift c.244dup1 (p.Trp82LeufsX23) mutation, a severe genotype that may explain the unusual and very early fatal outcome in this newborn. This report confirms that inborn errors of fatty acid oxidation represent one of the genetic causes of sudden unexpected deaths in infancy (SUDI) and underlines the importance to include systematically specific metabolic screening in any neonatal unexpected death.