Mutational Spectrum of <b><i>CYP24A1</i></b> Gene in a Cohort of Italian Patients with Idiopathic Infantile Hypercalcemia

Abstract

Background/Aims: Loss-of-function mutations in the CYP24A1 gene, which encodes the vitamin D-24 hydroxylase, have been recognized as a cause of elevated 1,25-dihydroxyvitamin D concentrations, hypercalcemia, hypercalciuria, nephrocalcinosis and nephrolithiasis in infants and adults. As only a case report describing 2 adult patients has been reported in Italian population, we report here the mutation analysis of CYP24A1 gene in an Italian cohort of 12 pediatric and adult patients with idiopathic infantile hypercalcemia (IIH). Methods: We performed mutational screening of CYP24A1 gene in a cohort of 12 Italian patients: 8 children with nephrocalcinosis, hypercalcemia and PTH levels <10 pg/ml and 4 adult patients with nephrolithiasis, mild hypercalcemia and PTH levels <10 pg/ml from 11 unrelated Italian families. Clinical and biochemical data were collected. Genomic DNA was extracted from peripheral blood leucocytes using standard methods, and whole coding sequence of CYP24A1 gene was analysed in all patients and family members by polymerase chain reaction and direct sequencing. The potential pathogenicity of the newly identified missense mutations was evaluated by 3 different in silico approaches (Sorting Intolerant from Tolerant, Polyphen and Mutation Taster) and by comparative analysis in 14 different species using ClustalW software. Results:CYP24A1 bi-allelic mutations were found in 8 individuals from 7 Italian families (7/11; 64%). Overall, 6 different CYP24A1 mutations, including one small deletion (p.Glu143del), 4 missense mutations (p.Leu148Pro; p.Arg396Trp; p.Pro503Leu; p.Glu383Gln) and one nonsense mutation (p.Tyr220*) were identified. Two out of 6 mutations (p.Tyr220* and p.Pro503Leu) were not previously described. Moreover, a new CYP24A1 variant was identified by genetic screening of asymptomatic controls. Conclusion: To the best of our knowledge, this is the first report of a CYP24A1 molecular analysis performed in an Italian cohort of adult and pediatric Italian patients. This study (1) confirms that CYP24A1 plays a causal role in some but not all cases of IIH (64%); (2) expands the spectrum of known CYP24A1 pathogenic mutations; (3) describes 2 hotspots detected in 50% of all Italian cases; and (4) emphasizes the importance of recognition and genetic diagnosis of CYP24A1 defects in infantile as well as adult hypercalcemia.

Hypercalciuria caused by CYP24A1 mutation: Fourteen years of the patient’s follow-up

“…It has been demonstrated that CYP24A1 knockout (-/-) mice suffer from increased sensitivity to exogenous vitamin D intake and approximately half of them die due to severe hypercalcemia [11]. In humans, CYP24A1 mutations can cause idiopathic infantile hypercalcemia (IIH) [12][13][14][15][16][17][18][19], idiopathic hypercalciuria [9], nephrocalcinosis, and possibly reduced bone density [20]. In patients with IHH due to CYP24A1 mutations, even small doses of vitamin D, as prescribed for vitamin D prophylaxis, may provoke symptomatic hypercalcemic crisis which need treatment by acute hemodiafiltration [16].…”

CYP24A1 and SLC34A1 genetic defects associated with idiopathic infantile hypercalcemia: from genotype to phenotype

“…This variant within exon 6 of the CYP24A1 gene involves the highly conserved amino acid residue occurring in the polar region of the F helix: it is annotated with a very low MAF (=0.00045). Gigante et al detected this genetic variant in homozygote status in an asymptomatic child during genetic screening [82]. This Italian proband (14 years old) showed biochemical findings within the normal range: nevertheless, the putative role of this variant in a late onset manifestation of the disease or in a latent phenotype was hypothesized.…”

“…p.Glu383Gln) and d) the protein folding (e.g. p.Glu322Lys) [2,53,82,110]. In particular, the c.476G>A (p.Arg159Gln) and the c.1186C>T (p.Arg396Trp) variants influence the heme binding site due to the destruction of the hydrogen bond between the heme propionate group and the arginine residues [110].…”

“…Moreover, Masuda et al demonstrated that the substitutions of the residues Trp134 and Leu148, together with Met246 and Ile131, affect the substrate orientation into the enzyme channel and lead to the decrease of the enzymatic activity [113]. Among the CYP24A1 genetic variants reported in the Table 1, the small deletion c.428_430delAAG (p.Glu143del) is one of the most common in IIH patients, being that it has been described in many case reports [2,48,51,53,59,60,65,82,85,112]. This variant represents a mutational hot spot of the CYP24A1 gene, together with the c.1226T>C All the mutations in CYP24A1 associated with IIH phenotype are reported.…”

“…In the remaining cases, the specific reference studies are indicated. (p.Leu409Ser) in the French population [59] and the c.1186C>T (p.Arg396Trp) in Italians [82]. The deletion of this highly conserved amino acid has a great impact on the enzyme function, destabilizing the B′/C loop and causing a reorientation of the B′-helix.…”