Identification of potential non-invasive biomarkers in diastrophic dysplasia.

Diastrophic dysplasia (DTD) is a recessive chondrodysplasia caused by pathogenic variants in the SLC26A2 gene encoding for a cell membrane sulfate/chloride antiporter crucial for sulfate uptake and glycosaminoglycan (GAG) sulfation. Research on a DTD animal model has suggested possible pharmacological treatment approaches. In view of future clinical trials, the identification of non-invasive biomarkers is crucial to assess the efficacy of treatments. Urinary GAG composition has been analyzed in several metabolic disorders including mucopolysaccharidoses. Moreover, the N-terminal fragment of collagen X, known as collagen X marker (CXM), is considered a real-time marker of endochondral ossification and growth velocity and was studied in individuals with achondroplasia and osteogenesis imperfecta. In this work, urinary GAG sulfation and blood CXM levels were investigated as potential biomarkers for individuals affected by DTD. Chondroitin sulfate disaccharide analysis was performed on GAGs isolated from urine by HPLC after GAG digestion with chondroitinase ABC and ACII, while CXM was assessed in dried blood spots. Results from DTD patients were compared with an age-matched control population. Undersulfation of urinary GAGs was observed in DTD patients with some relationship to the clinical severity and underlying SLC26A2 variants. Lower than normal CXM levels were observed in most patients, even if the marker did not show a clear pattern in our small patient cohort because CXM values are highly dependent on age, gender and growth velocity. In summary, both non-invasive biomarkers are promising assays targeting various aspects of the disorder including overall metabolism of sulfated GAGs and endochondral ossification.

Phenotypic Characterization of Immortalized Chondrocytes from a Desbuquois Dysplasia Type 1 Mouse Model: A Tool for Studying Defects in Glycosaminoglycan Biosynthesis.

The complexity of skeletal pathologies makes use of in vivo models essential to elucidate the pathogenesis of the diseases; nevertheless, chondrocyte and osteoblast cell lines provide relevant information on the underlying disease mechanisms. Due to the limitations of primary chondrocytes, immortalized cells represent a unique tool to overcome this problem since they grow very easily for several passages. However, in the immortalization procedure the cells might lose the original phenotype; thus, these cell lines should be deeply characterized before their use. We immortalized primary chondrocytes from a Cant1 knock-out mouse, an animal model of Desbuquois dysplasia type 1, with a plasmid expressing the SV40 large and small T antigen. This cell line, based on morphological and biochemical parameters, showed preservation of the chondrocyte phenotype. In addition reduced proteoglycan synthesis and oversulfation of glycosaminoglycan chains were demonstrated, as already observed in primary chondrocytes from the Cant1 knock-out mouse. In conclusion, immortalized Cant1 knock-out chondrocytes maintained the disease phenotype observed in primary cells validating the in vitro model and providing an additional tool to further study the proteoglycan biosynthesis defect. The same approach might be extended to other cartilage disorders.

Improvement of the skeletal phenotype in a mouse model of diastrophic dysplasia after postnatal treatment with N-acetylcysteine.

Diastrophic dysplasia (DTD) is a recessive chondrodysplasia caused by mutations in the SLC26A2 gene encoding for a sulfate/chloride transporter. When SLC26A2 is impaired intracellular level of sulfate is reduced leading to the synthesis of undersulfated proteoglycans. In normal chondrocytes, the main source of intracellular sulfate is the extracellular uptake through SLC26A2, but a small amount comes from the catabolism of sulfur-containing amino acids and other thiols. Here N-acetylcysteine (NAC), an extensively used drug, is proposed as alternative source of intracellular sulfate in an animal model of DTD (dtd mouse). Mutant and wild type mice were treated twice a day with hypodermic injections of 250 mg NAC/kg body weight for one week after birth. At the end of the treatment, an improvement trend in cartilage proteoglycan sulfation and in the skeletal phenotype of treated dtd mice were observed. Thus, a longer treatment lasted three weeks starting from birth was performed. Treated mutant mice showed a significant increase of cartilage proteoglycan sulfation and a relevant improvement of the skeletal phenotype based on measurements of several bony elements and bone quality by DEXA and micro CT. Moreover, the amelioration of the overall growth plate morphology in treated dtd mice suggested a partial rescue of the endochondral ossification process. Overall, the results prove that NAC is an effective source of intracellular sulfate for dtd mice in the postnatal period. This finding paves the way for a potential pharmacological treatment of DTD patients taking advantage from a drug repositioning strategy.

Skeletal Dysplasias Caused by Sulfation Defects.

Proteoglycans (PGs) are macromolecules present on the cell surface and in the extracellular matrix that confer specific mechanical, biochemical, and physical properties to tissues. Sulfate groups present on glycosaminoglycans, linear polysaccharide chains attached to PG core proteins, are fundamental for correct PG functions. Indeed, through the negative charge of sulfate groups, PGs interact with extracellular matrix molecules and bind growth factors regulating tissue structure and cell behavior. The maintenance of correct sulfate metabolism is important in tissue development and function, particularly in cartilage where PGs are fundamental and abundant components of the extracellular matrix. In chondrocytes, the main sulfate source is the extracellular space, then sulfate is taken up and activated in the cytosol to the universal sulfate donor to be used in sulfotransferase reactions. Alteration in each step of sulfate metabolism can affect macromolecular sulfation, leading to the onset of diseases that affect mainly cartilage and bone. This review presents a panoramic view of skeletal dysplasias caused by mutations in genes encoding for transporters or enzymes involved in macromolecular sulfation. Future research in this field will contribute to the understanding of the disease pathogenesis, allowing the development of targeted therapies aimed at alleviating, preventing, or modifying the disease progression.