Morquio B disease: From pathophysiology towards diagnosis.

Morquio B disease is an attenuated phenotype within the spectrum of beta galactosidase (GLB1) deficiencies. It is characterised by dysostosis multiplex, ligament laxity, mildly coarse facies and heart valve defects due to keratan sulphate accumulation, predominantly in the cartilage. Morquio B patients have normal neurological development, setting them apart from those with the more severe GM1 gangliosidosis. Morquio B disease, with an incidence of 1:250.000 to 1:1.000.000 live births, is very rare. Here we report the clinical-biochemical data of nine patients. High amounts of keratan sulfate were detected using LC-MS/MS in the patients' urinary samples, while electrophoresis, the standard procedure of qualitative glycosaminoglycans analysis, failed to identify this metabolite in any of the patients' samples. We performed molecular analyses at gene, gene expression and protein expression levels, for both isoforms of the GLB1 gene, lysosomal GLB1, and the cell-surface expressed Elastin Binding Protein. We characterised three novel GLB1 mutations [c.75 + 2 T > G, c.575A > G (p.Tyr192Cys) and c.2030 T > G (p.Val677Gly)] identified in three heterozygous patients. We also set up a copy number variation assay by quantitative PCR to evaluate the presence of deletions/ insertions in the GLB1 gene. We propose a diagnostic plan, setting out the specific clinical- biochemical and molecular features of Morquio B, in order to avoid misdiagnoses and improve patients' management.

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.

Vascular endothelial growth factor (VEGF-a) in Fabry disease: association with cutaneous and systemic manifestations with vascular involvement.

INTRODUCTION: Fabry disease is an X-linked inherited metabolic disorder characterized by the deficiency of lysosomal α-galactosidase A enzyme. This leads to the accumulation, into lysosomes through the body, of glycosphingolipids, mainly Gb3. Skin involvement and progressive multi-organ failure are usually observed. Endothelium is the preferential target of the Gb3 storage that determines endothelial dysfunction and vasculopathy leading to the clinical manifestations of the disease. The serum levels of Vascular Endothelial Growth Factor-A (VEGF-A), a specific endothelial cell mitogen, were analyzed in Fabry patients to explore a possible association to the clinical manifestations with vascular involvement. METHODS: Thirty-five patients with a biochemical and genetic diagnosis of Fabry disease, along with an age-gender-matched healthy control group, were enrolled. Serum samples were collected and analyzed by ELISA. The genetic mutations, the specific organ dysfunction, and the cardiovascular risk factors such as dyslipidaemia, diabetes, smoking habits and hypertension were evaluated in Fabry patients. RESULTS: The mean serum level of VEGF-A in Fabry patients group was significantly higher than in the control group (P=0.006). A statistical significant association, between VEGF-A levels and the skin manifestation including angiokeratomas, sweating abnormalities and Fabry Facies was found. An association was also found between high VEGF-A and specific GLA mutations, the male gender, the renal and neurological manifestations, the presence of eye vessels tortuosity, smoking habit and hypertension. CONCLUSIONS: We detected increased VEGF-A levels in patients with Fabry disease compared to the controls, and we hypothesized that this could be a response to the vascular damage characterising this lysosomal disorder. However, further studies are necessary to clarify the role of VEGF-A in Fabry.

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.

IDUA mutational profiling of a cohort of 102 European patients with mucopolysaccharidosis type I: identification and characterization of 35 novel α-L-iduronidase (IDUA) alleles.

Mutational analysis of the IDUA gene was performed in a cohort of 102 European patients with mucopolysaccharidosis type I. A total of 54 distinct mutant IDUA alleles were identified, 34 of which were novel including 12 missense mutations, 2 nonsense mutations, 12 splicing mutations, 5 micro-deletions, 1 micro-duplication 1 translational initiation site mutation, and 1 'no-stop' change (p.X654RextX62). Evidence for the pathological significance of all novel mutations identified was sought by means of a range of methodological approaches, including the assessment of evolutionary conservation, RT-PCR/in vitro splicing analysis, MutPred analysis and visual inspection of the 3D-model of the IDUA protein. Taken together, these data not only demonstrate the remarkable mutational heterogeneity characterizing type 1 mucopolysaccharidosis but also illustrate our increasing ability to make deductions pertaining to the genotype-phenotype relationship in disorders manifesting a high degree of allelic heterogeneity.

The Use of Chitotriosidase as a Marker of Active Sarcoidosis and in the Diagnosis of Fever of Unknown Origin (FUO).

Sarcoidosis is a multi-organ inflammatory granulomatosis with a lung-predominant involvement. The aim of this study was to investigate the use of serum chitotriosidase (CHIT1) in patients with fever of unknown origin (FUO); the patients with confirmed diagnosis of active sarcoidosis were compared with ones affected by inactive or treated sarcoidosis. CHIT1 activity was evaluated in 110 patients initially admitted at the hospital as FUOs. The overall performance of CHIT1 for active sarcoidosis diagnosis was assessed by performing an area under the receiver operating characteristic curve analysis (AUROC). The sarcoidosis patients were significantly older than the FUO patients not affected by sarcoidosis (p < 0.01). CHIT1 showed a good accuracy as a biomarker for active sarcoidosis in patients explored for FUO (AUROC 0.955; CI 95% 0.895-0.986; p < 0.001). A CHIT1 value >90.86 showed 96.8% sensitivity (84.2-99.9) and 85.5% specificity (75-92.8) in discriminating active sarcoidosis from other causes of FUO. CHIT1 significantly discriminated active versus inactive/under treatment sarcoidosis patients (with lower enzyme activity) (ROC analysis, sensitivity: 96.9%, specificity: 94.7%, value >83.01 nmol/mL/h, AUROC: 0.958, 0.862-0.994, p < 0.001) compared to ACE (ROC analysis, sensitivity: 25.8%, specificity: 93.7%, value >65 UI/L). In conclusion, CHIT1 is a reliable/sensitive biomarker of active sarcoidosis, with values significantly decreasing in remitted/treated patients. It significantly discriminates active sarcoidosis from FUO patients, providing a useful tool in the diagnosis-assessing process.

Functional and pharmacological evaluation of novel GLA variants in Fabry disease identifies six (two de novo) causative mutations and two amenable variants to the chaperone DGJ.

BACKGROUND: Allelic heterogeneity is an important feature of the GLA gene for which almost 900 known genetic variants have been discovered so far. Pathogenetic GLA variants cause alpha-galactosidase A (α-Gal A) enzyme deficiency leading to the X-linked lysosomal storage disorder Fabry disease (FD). Benign GLA intronic and exonic variants (e.g. pseudodeficient p.Asp313Tyr) have also been described. Some GLA missense variants, previously deemed to be pathogenetic (e.g. p.Glu66Gln and p.Arg118Cys), they have been reclassified as benign after re-evaluation by functional and population studies. Hence, the functional role of novel GLA variants should be investigated to assess their clinical relevance. RESULTS: We identified six GLA variants in 4 males and 2 females who exhibited symptoms of FD: c.159C>G p.(Asn53Lys), c.400T>C p.(Tyr134His), c.680G>C (p.Arg227Pro), c.815A>T p.(Asn272Ile), c.907A>T p.(Ile303Phe) and c.1163_1165delTCC (p.Leu388del). We evaluated their impact on the α-Gal A protein by bioinformatic analysis and homology modelling, by analysis of the GLA mRNA, and by site-directed mutagenesis and in vitro expression studies. We also measured their responsiveness to the pharmacological chaperone DGJ. CONCLUSIONS: The six detected GLA variants cause deficient α-Gal A activity and impairment or loss of the protein wild-type structure. We found p.Asn53Lys and p.Ile303Phe variants to be susceptible to DGJ.

Anderson-Fabry's Disease: A Rare but Treatable Case of Fever of Unknown Origin.

Anderson-Fabry's disease (AFD) is a rare, X-linked lysosomal storage disorder caused by the complete deficiency or attenuated activity of the enzyme α-galactosidase A, leading to progressive systemic intracellular accumulation of glycosphingolipids and subsequent cellular dysfunction, inflammation and fibrosis. Fever is a frequently misinterpreted symptom in the early stages of the disease, leading to diagnostic delay. We present the case of a 35-year-old man admitted to our Periodic Fever Research Centre for long-lasting recurrent episodes of fever of unknown origin. After extensive assessment, we diagnosed AFD associated with a novel GLA mutation. We started enzyme replacement therapy with clinical benefit and complete remission of fever. LEARNING POINTS: Anderson-Fabry's Disease (AFD) is an inherited lysosomal storage disorder, in which progressive multi-organ glycosphingolipid accumulation leads to multi-systemic dysfunction. Diagnosis requires a high level of suspicion as the clinical presentation can be very heterogeneous.As fever is an early uncommon symptom causing diagnostic delay, it is important to consider AFD in the differential diagnosis of recurrent fevers, particularly when febrile episodes are not associated with an increase in acute phase reactants and when other signs or symptoms suggestive of AFD are present.Prognosis depends on an early diagnosis because promptly initiation of enzyme replacement therapy (ERT) can prevent the progression of organ damage. In our case fever disappeared after ERT initiation, a finding not previously reported to our knowledge. Therefore, fever remission could be an early marker of response to ERT.

Clinical use of plasma chitotriosidase in severe sepsis.

Plasma chitotriosidase activity (ChT) was previously proposed to quantify severity of sepsis. In a complex surgical case, with prolonged sepsis and consistently high ChT, we found that the least increased values occurred in stages of extreme illness, with profound hypocholesterolemia. ChT needs better characterization before becoming a reliable biomarker of septic evolution.