3' Untranslated Region Structural Elements in CYP24A1 Are Associated With Infantile Hypercalcemia Type 1.

Loss-of-function mutations in the CYP24A1 protein-coding region causing reduced 25 hydroxyvitamin D (25OHD) and 1,25 dihydroxyvitamin D (1,25(OH)2 D) catabolism have been observed in some cases of infantile hypercalcemia type 1 (HCINF1), which can manifest as nephrocalcinosis, hypercalcemia and adult-onset hypercalciuria, and renal stone formation. Some cases present with apparent CYP24A1 phenotypes but do not exhibit pathogenic mutations. Here, we assessed the molecular mechanisms driving apparent HCINF1 where there was a lack of CYP24A1 mutation. We obtained blood samples from 47 patients with either a single abnormality of no obvious cause or a combination of hypercalcemia, hypercalciuria, and nephrolithiasis as part of our metabolic and stone clinics. We used liquid chromatography tandem mass spectrometry (LC-MS/MS) to determine serum vitamin D metabolites and direct sequencing to confirm CYP24A1 genotype. Six patients presented with profiles characteristic of altered CYP24A1 function but lacked protein-coding mutations in CYP24A1. Analysis upstream and downstream of the coding sequence showed single nucleotide variants (SNVs) in the CYP24A1 3' untranslated region (UTR). Bioinformatics approaches revealed that these 3' UTR abnormalities did not result in microRNA silencing but altered the CYP24A1 messenger RNA (mRNA) secondary structure, which negatively impacted translation. Our experiments showed that mRNA misfolding driven by these 3' UTR sequence-dependent structural elements was associated with normal 25OHD but abnormal 1,25(OH)2 D catabolism. Using CRISPR-Cas9 gene editing, we developed an in vitro mutant model for future CYP24A1 studies. Our results form a basis for future studies investigating structure-function relationships and novel CYP24A1 mutations producing a semifunctional protein. © 2023 The Authors. Journal of Bone and Mineral Research published by Wiley Periodicals LLC on behalf of American Society for Bone and Mineral Research (ASBMR).

The Effect of Short-Term Vitamin D Supplementation on Calcium Status in Vitamin D Insufficient Renal Transplant Recipients at Risk of Hypercalcemia.

OBJECTIVE: Vitamin D insufficiency is highly prevalent among renal transplant recipients and in observational studies is associated with adverse outcomes. Hypercalcemia, usually due to persistent hyperparathyroidism, also commonly occurs in this population and often coexists with vitamin D insufficiency. However, concern that vitamin D supplementation might exacerbate the pre-existing hypercalcemia often leads clinicians to avoid vitamin D supplementation in such patients. This feasibility study aimed to quantify the effect on serum calcium of short-term low-dose cholecalciferol supplementation in a group of renal transplant recipients with a recent history of serum calcium levels >10 mg/dL. DESIGN: A 2-week, single arm, open-label trial. SETTING: Renal transplant follow-up clinic in an Irish University Hospital. SUBJECTS: Eighteen vitamin D-insufficient adult patients with a functioning renal allograft (estimated glomerular filtration rate > 30 mL/minute/1.73 m2) and a recent history of serum calcium >10 mg/dL. INTERVENTION: Two weeks of treatment with 1,000 IU cholecalciferol/day. MAIN OUTCOME MEASURE: Change in ionized calcium and urine calcium:creatinine ratio at follow-up compared with baseline. RESULTS: Mean (standard deviation [SD]) baseline 25 (OH) vitamin D (25 (OH) D) concentration was 15.9 (5.97) ng/mL and mean (SD) baseline serum calcium was 10.50 (0.6) mg/dL. Following the 2-week intervention, median (interquartile range [IQR]) change in serum calcium from baseline was -0.08 (-3.6 to 0.08) mg/dL, P = .3. Mean (SD) ionized calcium decreased from 5.24 (0.32) mg/dL at baseline to 5.16 (0.28) mg/dL, P = .05. Median (IQR) change in the urinary calcium:creatinine ratio was 0.001 (-0.026 to 0.299) mg/mg, P = .88. Median (IQR) change in 25 (OH) D was 3.6 (2.9-6.2) ng/mL, P < .05. CONCLUSIONS: In vitamin D-insufficient renal transplant recipients at risk of hypercalcemia, low-dose short-term oral cholecalciferol supplementation improves 25 (OH) D concentrations without exacerbating hypercalcemia or increasing the urinary calcium:creatinine ratio.

Retrospective evaluation of a local protocol used to enhance laboratory savings through minimizing the performance of alkaline phosphatase isoenzyme analysis.

BACKGROUND: Alkaline phosphatase isoenzyme analysis is an expensive and time-consuming laboratory test. We evaluated the effect of a locally derived screening algorithm for alkaline phosphatase isoenzyme requests on the number of alkaline phosphatase isoenzyme analyses performed, laboratory cost and patient care. METHOD: A total of 110 alkaline phosphatase isoenzyme analysis requests from the year 2015 were reviewed and subsequent alkaline phosphatase concentrations were monitored over a two-year period, to determine if the decision of performing/not performing alkaline phosphatase isoenzyme analysis, based on the algorithm, had an impact on patient care and laboratory cost. All alkaline phosphatase isoenzyme analysis requests with two consecutive elevated alkaline phosphatase concentrations (>upper limit of reference interval) were screened and, subject to the gamma glutamyl transferase being within the reference interval, either Bone alkaline phosphatase or 25 hydroxyvitamin D was measured depending on the age of the patient. RESULTS: Compliance with this algorithm led to the normalization of subsequent serum alkaline phosphatase in 97% of patients without requiring alkaline phosphatase isoenzyme analysis. The cost of performing Bone alkaline phosphatase and 25 hydroxyvitamin D in-house was £778.50, while the cost of performing alkaline phosphatase isoenzyme analysis would have been £3040. This resulted in a laboratory saving of £2261.50. CONCLUSIONS: Implementation of this algorithm led to a significant reduction in alkaline phosphatase isoenzyme analysis, without compromising patient care. Total savings could be increased if 25 hydroxyvitamin D was used as a first-line test, for all patients with an elevated alkaline phosphatase and a normal gamma glutamyl transferase regardless of age. This algorithm is cost-effective and can be implemented in laboratories with 25 hydroxyvitamin D assay.