3' UTR structural elements in CYP24A1 are associated with Infantile hypercalcaemia type 1.

Loss-of-function mutations in the CYP24A1 protein-coding region causing reduced 25OHD and 1,25(OH) 2 D catabolism have been observed in some cases of Infantile hypercalcaemia type 1 (HCINF1), which can manifest as nephrocalcinosis, hypercalcaemia 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 up- 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, 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 semi-functional protein. This article is protected by copyright. All rights reserved.