CYP24A1-mediated abnormal calcium handling' /> CYP24A1-mediated abnormal calcium handling' /> 3&#146; UTR structural elements are associated with <em>CYP24A1</em>-mediated abnormal calcium handling | SFEBES2022 | Society for Endocrinology BES 2022 | Endocrine Abstracts
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Endocrine Abstracts (2022) 86 OP5.1 | DOI: 10.1530/endoabs.86.OP5.1

1University of East Anglia, Norwich, United Kingdom; 2John Innes Centre, Norwich, United Kingdom; 3School of Biology, Norwich, United Kingdom; 4Clinical Biochemistry Norfolk and Norwich University Hospital, Norwich, United Kingdom

Hypomorphic CYP24A1 protein coding mutations causing inappropriate 1,25(OH)2D concentrations are associated with idiopathic infantile hypercalcemia and adult-onset hypercalciuria and nephrolithiasis. It is unclear why some cases present with CYP24A1-mediated abnormal calcium handling lack protein-coding CYP24A1 mutations. Non-coding region mutations, e.g. the 3’ UTR, impacting messenger RNA (mRNA) structure have rarely been studied in patients. RNAs fold into complex structures critical for their function and regulation including post-transcriptional modifications, localisation, translation and degradation. Non-coding variants altering CYP24A1 mRNA structure may be the fundamental mechanism behind cases with absent protein coding pathogenic mutations. Biochemical profiling, next generation sequencing, bioinformatics, proteomic and molecular cytogenetic approaches were used to examine CYP24A1 in a patient cohort of four adults with hypercalciuria and nephrolithiasis and two infants with nephrocalcinosis. We identified inappropriate 1,25(OH)2D concentrations (mean±SD=247.3±189.3pmol/l [range=55-139 pmol/l]) in our cohort, associated with elevated 25OHD:24,25(OH)2D (32 [range 7-23]) in one adult and both 25OHD:24,25(OH)2D (35) and 1,25(OH)2D:24,25(OH)2D (176 [range=11-62]) in one infant. CYP24A1 direct sequencing revealed single nucleotide variants located in the 3’ UTR of each patient (c.1993C>T, c.2083T>C, c.2512T>A, c.2658C>G and c.2691G>A) causing mRNA misfolding in silico. These mRNA structural abnormalities are associated with significant increase in CYP24A1 retention (P<0.05), while biochemical profiles suggest compromised functionality. We generated a CRISPR-Cas 9 model cell line containing CYP24A1 3’ UTR mRNA structural alterations for in vitro investigations into non-canonical CYP24A1 pathogenesis. Single molecule fluorescence in situ hybridisation (smFISH) in this model revealed no significant effect on CYP24A1 mRNA cellular localisation and abundance. We present insights into novel non-coding 3’UTR CYP24A1 hypomorphic variants that alter mRNA folding, associated with CYP24A1 biological function. These results will improve knowledge of structure-function relationships affecting RNA translation and protein expression, expanding our understanding of the molecular basis of disease pathogenesis in patients lacking protein coding abnormalities.

Volume 86

Society for Endocrinology BES 2022

Harrogate, United Kingdom
14 Nov 2022 - 16 Nov 2022

Society for Endocrinology 

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