Context: Loss-of-function mutations of CYP24A1 (which encodes the 25-OH-D₃-24-hydroxylase) have recently been reported to cause hypercalcemia. Objectives: The aims of this study were: 1) to evaluate the frequency of CYP24A1 mutations in patients with medical history of hypercalcemia; 2) to show the clinical utility of a simultaneous assay of serum 25-hydroxyvitamin D₃ (25-OH-D₃) and 24,25-dihydroxyvitamin D₃ (24,25-[OH]₂D₃) by liquid chromatography tandem mass spectrometry (LC-MS/MS); and 3) to investigate biochemical parameters in heterozygous gene carriers with CYP24A1 mutations. Patients and Methods: We screened for CYP24A1 mutations in 72 patients with serum calcium levels > 2.6 mmol/L and PTH levels < 20 pg/mL and recruited 24 relatives after genetic counseling for subsequent investigations. Vitamin D metabolite concentrations were assessed in a subset of patients by LC-MS/MS and results expressed as a ratio (R) of 25-OH-D₃:24,25-(OH)₂D₃. Results: Twenty-five patients with hypercalcemia (35%) harbored CYP24A1 variations. Twenty (28%) had biallelic variations, mostly found in subjects with nephrocalcinosis or renal stones (19/20). Five patients, all neonates, were heterozygous, without renal disease. We describe 15 new variations leading to loss-of-function according to pathogenicity prediction programs, and we functionally characterized 5 of them in vitro. A dramatic increase of R, usually >80, was found in patients harboring biallelic mutations providing evidence in vivo for the loss of CYP24A1 activity. In contrast, R value remains <25 in patients without CYP24A1 mutations. Subjects carrying one mutant allele, hypercalcemic individuals, as well as gene-carrier relatives, had a detectable 24,25-(OH)₂D₃ level and R < 25, indicating normal 24-hydroxylase activity. Conclusion: CYP24A1 biallelic mutations are frequently found in patients presenting with hypercalcemia, low PTH, and renal disease. We confirm the accuracy and effectiveness of a novel blood test estimating the ratio between relevant vitamin D metabolites as a useful screening tool for CYP24A1 mutations. Haploinsufficiency is not associated with CYP24A1 deficiency.

Patients with pseudohypoparathyroidism type Ib (PHP-1b) develop resistance toward PTH, leading to hypocalcemia and hyperphosphatemia. PHP-1b is an imprinted human disorder associated with methylation changes at one or several differentially methylated regions at the GNAS locus. This complex locus gives rise to several different transcripts with different patterns of imprinted expression depending on promoter methylation. They can be either coding [Gαs, XLαs, and neuroendocrine secretory protein-55 (NESP55)] or nontranslated (A/B and AS). The paternal AS transcript lies antisense to nesp55. Define the genetic defect in a new family with three patients presenting autosomal dominant PHP-1b. We used methylation analysis, comparative genomic hybridization, and genotyping to characterize the defect. AS expression was studied in two patients and their unaffected mothers. A novel deletion of 18,988 bp that removes NESP55 and a large part of its counterpart GNAS AS intron 4 was discovered. On maternal transmission, this deletion causes loss of A/B methylation without affecting XL/AS imprint. On paternal transmission, there are no methylation anomalies. The deletion creates a cryptic exon contained within AS intron 4, which is expressed from the mutated allele, be it paternal or maternal. This new deletion suggests that NESP55 is an additional imprinting control region that directs A/B methylation in humans. We bring arguments in support of the theory of reciprocal inhibition between the expression of NESP and AS. However, determining whether loss of methylation at the A/B differentially methylated region is a consequence of the loss of NESP expression or of the expression of AS requires additional investigations.

Vitamin D requires a two‐step activation by hydroxylation: The first step is catalyzed by hepatic 25‐hydroxylase (CYP2R1, 11p15.2) and the second one is catalyzed by renal 1α‐hydroxylase (CYP27B1, 12q13.1), which produces the active hormonal form of 1,25‐(OH)₂D. Mutations of CYP2R1 have been associated with vitamin D–dependent rickets type 1B (VDDR1B), a very rare condition that has only been reported to affect 4 families to date. We describe 7 patients from 2 unrelated families who presented with homozygous loss‐of‐function mutations of CYP2R1. Heterozygous mutations were present in their normal parents. We identified a new c.124_138delinsCGG (p.Gly42_Leu46delinsArg) variation and the previously published c.296T>C (p.Leu99Pro) mutation. Functional in vitro studies confirmed loss‐of‐function enzymatic activity in both cases. We discuss the difficulties in establishing the correct diagnosis and the specific biochemical pattern, namely, very low 25‐OH‐D suggestive of classical vitamin D deficiency, in the face of normal/high concentrations of 1,25‐(OH)₂D. Siblings exhibited the three stages of rickets based on biochemical and radiographic findings. Interestingly, adult patients were able to maintain normal mineral metabolism without vitamin D supplementation. One index case presented with a partial improvement with 1alfa‐hydroxyvitamin D₃ or alfacalcidol (1α‐OH‐D₃) treatment, and we observed a dramatic increase in the 1,25‐(OH)₂D serum concentration, which indicated the role of accessory 25‐hydroxylase enzymes. Lastly, in patients who received calcifediol (25‐OH‐D₃), we documented normal 24‐hydroxylase activity (CYP24A1). For the first time, and according to the concept of personalized medicine, we demonstrate dramatic improvements in patients who were given 25‐OH‐D therapy (clinical symptoms, biochemical data, and bone densitometry). In conclusion, the current study further expands the CYP2R1 mutation spectrum. We note that VDDR1B could be easily mistaken for classical vitamin D deficiency. © 2017 American Society for Bone and Mineral Research.

Context: Heterozygous GNAS inactivating mutations cause pseudohypoparathyroidism type Ia (PHP-Ia) when maternally inherited and pseudopseudohypoparathyroidism (PPHP)/progressive osseous heteroplasia (POH) when paternally inherited. Recent studies have suggested that mutations on the paternal, but not the maternal, GNAS allele could be associated with intrauterine growth retardation (IUGR) and thus small size for gestational age. Objectives: The aim of the study was to confirm and expand these findings in a large number of patients presenting with either PHP-Ia or PPHP/POH. Patients and Methods: We collected birth parameters (ie, gestational age, weight, length, and head circumference) of patients with either PHP-Ia (n = 29) or PPHP/POH (n = 26) with verified GNAS mutations. The parental allele carrying the mutation was assessed by investigating the parents or, when a de novo mutation was identified, through informative intragenic polymorphisms. Results: Heterozygous GNAS mutations on either parental allele were associated with IUGR. However, when these mutations are located on the paternal GNAS allele, IUGR was considerably more pronounced than with mutations on the maternal allele. Moreover, birth weights were lower with paternal GNAS mutations affecting exons 2–13 than with exon 1/intron 1 mutations. Conclusions: These data indicate that a paternally derived GNAS transcript, possibly XLαs, is required for normal fetal growth and development and that this transcript affects placental functions. Thus, similar to other imprinted genes, GNAS controls growth and/or fetal development.

Postsynaptic density (PSD) proteins have been implicated in the pathophysiology of neurodevelopmental and psychiatric disorders. Here, we present detailed clinical and genetic data for 20 patients with likely gene-disrupting mutations in TANC2—whose protein product interacts with multiple PSD proteins. Pediatric patients with disruptive mutations present with autism, intellectual disability, and delayed language and motor development. In addition to a variable degree of epilepsy and facial dysmorphism, we observe a pattern of more complex psychiatric dysfunction or behavioral problems in adult probands or carrier parents. Although this observation requires replication to establish statistical significance, it also suggests that mutations in this gene are associated with a variety of neuropsychiatric disorders consistent with its postsynaptic function. We find that TANC2 is expressed broadly in the human developing brain, especially in excitatory neurons and glial cells, but shows a more restricted pattern in Drosophila glial cells where its disruption affects behavioral outcomes.