Duplex high resolution melting analysis (dHRMA) to detect two hot spot CYP24A1 pathogenic variants (PVs) associated to idiopathic infantile hypercalcemia (IIH)

Maria De Bonis,Maria Elisabetta Onori,Angelo Minucci,Pietro Ferrara,Elisa De Paolis,Pietro Manuel Ferraro,Andrea Urbani,Antonio Gatto,Giorgia Mazzuccato

doi:10.1007/s11033-021-06324-x

Abstract

Pathogenic variants (PVs) in CYP24A1 gene are associated with Idiopathic Infantile Hypercalcemia disease (IIH). The identification of CYP24A1 PVs can be a useful tool for the improvement of target therapeutic strategies. Aim of this study is to set up a rapid and inexpensive High Resolution Melting Analysis (HRMA)-based method for the simultaneous genotyping of two hot spot PVs in CYP24A1 gene, involved in IIH. A duplex-HRMA (dHRMA) was designed in order to detect simultaneously CYP24A1 c.428_430delAAG, p.(Glu143del) (rs777676129) and c.1186C > T, p.(Arg396Trp) (rs114368325), in peculiar cases addressed to our Laboratory. dHRMA was able to identify clearly and simultaneously both hot spot CYP24A1 PVs evaluating melting curve shape and melting temperature (Tm). This is the first dHRMA approach to rapidly screen the two most frequent CYP24A1 PVs in peculiar case, providing useful information for diagnosis and patient management in IIH disease.

Highlights

The term Idiopathic Infantile Hypercalcemia (IIH, OMIM 143880) first received attention almost 70 years ago in the UK, when symptomatic hypercalcemia was observed in infants after receiving high doses of vitamin D for the prevention of rickets [1, 2]
We report the case of a 6-years-old Italian male child with a personal history of bilateral nephrocalcinosis, severe hypercalcemia, increased urinary calcium/creatinine ratio and suppressed parathyroid hormone (PTH)
The major strength of the current study was to set up an efficient and robust molecular test based on duplex assay coupled with High Resolution Melting Analysis (HRMA) to screen two Italian hot spot PVS in CYP24A1 gene, implementing the previous our molecular diagnostic workflow for Infantile Hypercalcemia disease (IIH) disease [8]

Summary

Introduction

Pathogenic variants (PVs) in the CYP24A1 gene, involved in the degradation of vitamin-D, have been identified as being a relevant part of the IIH etiology [3]. The clinical and biological role of CYP24A1 gene, encoding the vitamin D-24-hydroxylase, lies in the metabolism of the 1,25(OH)2D, the physiologically active form of vitamin D. CYP24A1 enzyme is responsible of the 1,25(OH)2D catabolism and it enhances the turnover and elimination of the 25(OH)D, the abundant precursor metabolite and storage form of vitamin D [4]. PVs in the CYP24A1 gene can lead to elevated levels of 1,25(OH)2D, cause of pathological absorptive hypercalcemia and hypercalciuria. Calcium deposition in mitochondrial structures, with a consequent damage of metabolism, may lead to renal epithelium impairment and tubular necrosis, potentially resulting in chronic kidney disease [5,6,7]

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