Identification of potential non-invasive biomarkers in diastrophic dysplasia

Abstract

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.

Keywords: Biomarker; Collagen X; Diastrophic dysplasia; Glycosaminoglycan; Sulfation; Urine.

Fig. 1.

Correlation between the relative amount of non-sulfated disaccharide of chondroitin sulfate extracted from urine and age in controls. (A) Percentage of non-sulfated disaccharide for males and females plotted separately vs. age. Best fit linear regression lines were overlaid. (B) Male and female data were combined; best fit linear regression line was overlaid.

Fig. 2.

Urinary GAG sulfation analysis in controls and affected individuals grouped by age: 0–4, 4–8 and 8–12 years old. In controls, a decrease in the percentage of non-sulfated disaccharide was observed. The difference was significant between the 0–4 years old group and the other two groups. In the DTD study population, the percentage of non-sulfated disaccharide was higher than controls across the three age groups, but the difference was significant in the 4–8 and 8–12 years old groups. Data are reported as mean ± SD; 0–4 years old controls (n = 8) and DTD group (n = 6), 4–8 years old controls (n = 6) and DTD group (n = 5) and 8–12 years old controls (n = 13) and DTD group (n = 5). *P < 0.05; **P < 0.01; ***P < 0.001.

Fig. 3.

Z-score of the percentage of non-sulfated disaccharide in urine of affected individuals in relation with the clinical phenotype. For each individual the z-score was calculated considering mean and standard deviation of the corresponding age control group and the disease phenotype was classified in mild, moderate and severe based on the clinical features reported in Table 1. The highest z-scores were observed among 4–12 years old DTD individuals. Interestingly, DTD individuals with high z-scores presented a severe phenotype, while a mild phenotype was observed in DTD individuals with low z-scores, suggesting a possible correlation between urinary GAG sulfation and disease phenotype.

Fig. 4.

Z-scores of collagen X marker (CXM) in individuals with DTD grouped by sex, (A) males and (B) females. Individual z-scores were compared with general population reference data.