A case of mucopolysaccharidosis type VI in a polish family. Importance of genetic testing and genotype-phenotype relationship in the diagnosis of mucopolysaccharidosis.

BACKGROUND AND OBJECTIVES: Mucopolysaccharidosis type VI (MPS VI) is a rare, autosomal recessive lysosomal storage disorder caused by deficient enzymatic activity of N-acetyl galactosamine-4-sulphatase, which is caused by mutations in the arylsulphatase B (ARSB) gene. To date, 163 different types of mutations in the ARSB have been reported. However, the full mutation spectrum in the MPS VI phenotype is still not known. The aim of this study was to perform molecular testing of the ARSB gene in the patient and his family members to confirm MPS VI. METHODS: Molecular characterisation of the ARSB gene was performed using Sanger sequencing. We studied a child suspected of having MPS VI and 16 other relatives. RESULTS: We identified a C-to-T transition resulting in an exchange of the Arg codon 160 for a premature stop codon (R160*, in exon 2). The transition was in CpG dinucleotides. INTERPRETATION AND CONCLUSIONS: The study provided some insights into the genotype-phenotype relationship in MPS VI and the importance of genetic testing when diagnosing MPS, which is not a mandatory test for the diagnosis and only very occasionally performed. Additionally, we present here the history of a family with confirmed MPS VI, which is extremely rare especially in south-eastern Poland. What is more, the position where the mutation is located is very interesting because it is the region of CpG, which is the site of the methylation process. Thus, this opens the possibility of a new approach indicating the involvement of an epigenetic mechanism that should be examined in the context of the pathomechanism of MPS.

Case report of dysregulation of primary bile acid synthesis in a family with X-linked adrenoleukodystrophy.

RATIONALE: X-linked adrenoleukodystrophy (X-ALD) is a rare disorder caused by mutations in the ABCD1 gene, coding for peroxisomal membrane transporter adrenoleukodystrophy (ALD) protein. The disease is characterized by accumulation of very long chain fatty acids (VLCFAs) in tissues. Adult adrenomyeloneuropathy (AMN) and the cerebral inflammatory form of ALD are the main phenotypes presenting various symptoms. PATIENT CONCERNS: We report a case of 37-year-old patient with diagnosis of X-ALD, confirmed based on elevated VLCFA concentrations and genetic testing of ABCD1 gene. The complete clinical picture in the patient indicates AMN phenotype with cerebral involvement. DIAGNOSES: The reduced synthesis of unconjugated cholic and chenodeoxycholic acids, and the reduction to 28% to 29% of peroxisomal beta-oxidation of behenic acid and normal peroxisomal metabolism of pristanic and palmitic acid were observed in the X-ALD patient. Sanger sequencing of major genes involved in primary bile acid (BA) synthesis failed to identify pathogenic mutations of the investigated set of genes. INTERVENTIONS: Plasma concentrations of BAs, VLCFAs, and beta-oxidation of C22:0, C16:0, and pristanic acid were studied in primary skin fibroblasts of the patient. In addition, we performed sequencing of the ABCD1, ABCD3, CYP7A1, CYP7B1, CYP27A1, HSD3B7, AKR1D1, and SLC27A5 genes in the X-ALD family. OUTCOMES: In the Polish family affected with AMN a dysregulation of the primary BA synthesis pathway was found. LESSONS: We have demonstrated the coincidence of the adult form of X-ALD with abnormalities in BA synthesis. We suggest that decreased synthesis of BAs may be an additional dysfunction as a consequence of the ABCD1 c.659T>C, p.(Leu220Pro) mutation and may be further evidence that disturbed cholesterol metabolism is important in the pathology of ALD.

[Effect of free fatty acids on CYP19A1 (aromatase) gene expression in human adipose tissue stromal vascular fraction cells].

Aromatase plays an important role in the estrogen biosynthesis. Its gen (CYP19A1) is expressed in preadipocytes (stromal vascular fraction, SVF) of adipose tissue. Estrogens are found to be protective for metabolism homeostasis, and cardiovascular system. Disturbed dietary and endogenous fatty acids (FAs) turnover is responsible for development of metabolic syndrome and it complications. Aim of the work was to investigate the effect of physiological concentrations of acids: arachidonic (AA), oleic (OA), palmitynoic (PA) and eikozapentaenoic (EPA) on CYP19A1 expression in differentiating human SVF, able to form adipocytes as well as endothelial cells. Material and Methods: Human (n=38 healthy woman) SVF cells were isolated from subcutaneous adipose tissue harvested intrasurgery. SVF cells were incubated in proadipogenic or angiogenic media to obtain adipocytes (Adipo-SVF) or endothelial (Angio-SVF) cells (confirmed by microarray). Changes in the CYP19A1 expression induced by 24hs incubation in the presence of FAs (10 ­ 30 µM )were monitored by the Real time PCR (qRT -PCR). Results: The aromatase gene expression correlated positively with BMI of patients, but only in group of obese or overweight women. The negative correlation was found in the group of young, slim women. The highest expression of aromatase was found in the fresh, not differentiated SVF. In differentiating to endothelial cells (Angio - SVF) OA inhibited (p=0.008), when n-3 polyunsaturated AA activated (p=0.003) the CYP19A1 gene expression. In differentiating to preadipocytes (Adipo-SVF) AA significantly (p=0.031) inhibited CYP19A1 expression. Conclusion: The changes in the aromatase gene expression in differentiating SVF has been confirmed. The different effect of the dietary FA (OA vs. AA) on the aromatase gene expression argue for the role of the locally formed proangiogenic estrogens.

Connexin 43 and metabolic effect of fatty acids in stressed endothelial cells.

Changes in the inner mitochondrial membrane potential (∆ψ) may lead either to apoptosis or to protective autophagy. Connexin 43 (Cx43), a gap junction protein, is suggested to affect mitochondrial membrane permeability. The aim of our study was to analyze Cx43 gene expression, Cx43 protein localization and mitochondrial function in the human endothelial cells stressed by dietary-free fatty acids (FFA) and TNFα. Human endothelial cells (HUVECs) were incubated with (10-30 uM) palmitic (PA), oleic (OA), eicosapentaenoic (EPA) or arachidonic (AA) acids for 24 h. TNFα (5 ng/ml) was added at the last 4 h of incubation. The Cx43 gene expression was analyzed by the quantitative real-time PCR. The Cx43 protein concentrations in whole cells and in the isolated mitochondria were measured. Changes in ∆ψ and Cx43 localization were analyzed by flow cytometry or fluorescence microscopy. Generated ATP was measured by a luminescence assay. TNFα, PA and OA significantly decreased ∆ψ, while AA (P = 0.047) and EPA (P = 0.004) increased ∆ψ value. Preincubation with EPA or AA partially prevented the TNFα-induced decrease of ∆ψ. Incubation with AA resulted in up-regulation of the Cx43 gene expression. AA or PA significantly increased Cx43 protein content; however, presence of TNFα in general aggravated the negative effect of FFA. Only EPA was found to increase ATP generation in HUVECs. The fatty acid-specific induction of changes in Cx43 expression and protein concentration as well as the normalization of ∆ψ and increase of ATP generation seem to be the separate, independent mechanisms of FFA-mediated modulatory effect in the human endothelial cells pathology.