Isabelle Piec

Reference intervals for serum 24,25-dihydroxyvitamin D and the ratio with 25-hydroxyvitamin D established using a newly developed LC–MS/MS method

24,25(OH)2D is the product of 25(OH)D catabolism by CYP24A1. The measurement of serum 24,25(OH)2D concentration may serve as an indicator of vitamin D catabolic status and the relative ratio with 25(OH)D can be used to identify patients with inactivating mutations in CYP24A1. We describe a LC-MS/MS method to determine: (1) the relationships between serum 24,25(OH)2D and 25(OH)D; (2) serum reference intervals in healthy individuals; (3) the diagnostic accuracy of 24,25(OH)2D measurement as an indicator for vitamin D status; 4) 24,25(OH)2D cut-off value for clinically significant change between inadequate and sufficient 25(OH)D status. Serum samples of healthy participants (n=1996) from Army recruits and patients (n=294) were analysed. The LC-MS/MS assay satisfied industry standards for method validation. We found a positive, concentration-dependent relationship between serum 24,25(OH)2D and 25(OH)2D concentrations. The 25(OH)D:24,25(OH)2D ratio was significantly higher (P<.001) at 25(OH)D<50 nmol/L. The reference interval for 25(OH)D:24,25(OH)2D ratio in healthy subjects was 7-23. Measurement of serum 24,25(OH)2D can be used as predictor of vitamin D status, a concentration of>4.2 nmol/L was identified as a diagnostic cut-off for 25(OH)D replete status. One patient sample with an elevated 25(OH)D:24,25(OH)2D ratio of 32 and hypercalcaemia who on genetic testing confirmed to have a biallelic mutation of CYP24A1. Our study demonstrated the feasibility of a combined 24,25(OH)2D and 25(OH)D assessment profile. Our established cut-off value for 24,25(OH)2D and ratio reference ranges can be useful to clinicians in the investigation of patients with an impaired calcium/phosphate metabolism and may point towards the existence of CYP24A1 gene abnormalities.

MicroRNA expression in a phosphaturic mesenchymal tumour

Phosphaturic mesenchymal tumours are a heterogeneous set of bone and soft tissue neoplasms that can cause a number of paraneoplastic syndromes such as tumour induced osteomalacia. The term phosphaturic comes from the common finding that these tumours secrete high levels of fibroblast growth factor 23 which causes renal phosphate wasting leading to hypophosphatemia. Phosphaturic mesenchymal tumours are rare and diagnosis is difficult. A very active 68 year old male presented with bone pain and muscle weakness. He was hypophosphataemic and total alkaline phosphatase was markedly elevated. The patient was placed on vitamin D supplementation but his condition progressed. In the fifth year of presentation the patient required the use of a wheelchair and described “explosive” bone pain on physical contact. Serum 1,25 dihydroxyvitamin D was low and serum fibroblast growth factor 23 was significantly elevated, raising suspicion of a phosphaturic mesenchymal tumour. A lesion was detected in his left femoral head and the patient underwent a total hip replacement. The patient displayed a rapid improvement to his condition and during a three year follow up period he returned to an active lifestyle. As molecular testing may help provide a robust diagnosis and is particularly useful in rare diseases we took a next generation sequencing approach to identify a differential expression of small RNAs in the resected tumour. Small RNAs are non-coding RNA molecules that play a key role in regulation of gene expression and can be used as specific biomarkers. We found an upregulation of miR-197. We also found a downregulation of miR-20b, miR-144 and miR-335 which is a small RNA profile typical of osteosarcoma. MiR-21, the most frequently upregulated microRNA in cancer, was downregulated. We conclude that the specific small RNA profile is typical of osteosarcoma except for the downregulation of oncogenic miR-21. Transcriptional plasticity of miR-197, which is computationally predicted to target fibroblast growth factor 23 messenger RNA, may be upregulated in a cellular effort to correct the ectopic expression of the protein.