Idiopathic Infantile Hypercalcemia Research Articles

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

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.

IDIOPATHIC INFANTILE HYPERCALCEMIA – CASE REPORT

Idiopathic Infantile Hypercalcemia (IIH) is a rare genetic disease which cardinal features is failure to thrive, polyuria, dehydration, vomiting, seizures, lethargy, comma and if unrecognized and untreated may result in death. Laboratory investigations show hypercalcemia, hypercalciuria, elevated values of 1, 25 (OH) D3 and suppressed levels of parathormon.  Ultrasound study usually reveals bilateral medullary echogenicity typical for nephrocalcinosis. Emergency treatment consists from large hydration, prednisolone, calcitonin and biphosphonates, which were used with success. In this report we present a 6 month old female infant who came to our attention due to growth delay as a consequence of  polyuria, vomiting and poor apetite.Dietary management with withdrawal of vitamin D and reduction of calcium intake significantly improved  the general health status and normalized the serum biochemistry. The diagnosis was established with mutational analysis of the CYP24A1 gene.   Key words: idiopathic infantile hypercalcemia, CYP24A1, vitamin D, nephrocalcinosis.

Probing the Scope and Mechanisms of Calcitriol Actions Using Genetically Modified Mouse Models.

ABSTRACTGenetically modified mice have provided novel insights into the mechanisms of activation and inactivation of vitamin D, and in the process have provided phenocopies of acquired human disease such as rickets and osteomalacia and inherited diseases such as pseudovitamin D deficiency rickets, hereditary vitamin D resistant rickets, and idiopathic infantile hypercalcemia. Both global and tissue‐specific deletion studies leading to decreases of the active form of vitamin D, calcitriol [1,25(OH)2D], and/or of the vitamin D receptor (VDR), have demonstrated the primary role of calcitriol and VDR in bone, cartilage and tooth development and in the regulation of mineral metabolism and of parathyroid hormone (PTH) and FGF23, which modulate calcium and phosphate fluxes. They have also, however, extended the spectrum of actions of calcitriol and the VDR to include, among others: modulation, jointly and independently, of skin metabolism; joint regulation of adipose tissue metabolism; cardiovascular function; and immune function. Genetic studies in older mice have also shed light on the molecular mechanisms underlying the important role of the calcitriol/VDR pathway in diseases of aging such as osteoporosis and cancer. In the course of these studies in diverse tissues, important upstream and downstream, often tissue‐selective, pathways have been illuminated, and intracrine, as well as endocrine actions have been described. Human studies to date have focused on acquired or genetic deficiencies of the prohormone vitamin D or the (generally inactive) precursor metabolite 25‐hyrodxyvitamin D, but have yet to probe the pleiotropic aspects of deficiency of the active form of vitamin D, calcitriol, in human disease. © 2020 American Society for Bone and Mineral Research © 2020 The Authors. JBMR Plus published by Wiley Periodicals LLC on behalf of American Society for Bone and Mineral Research.

Presymptomatic diagnosis of CYP24A1-related infantile idiopathic hypercalcemia: A case report

Vitamin D plays an important role in calcium homeostasis and bone mineralization. Inefficient inactivation of vitamin D leads to a condition called idiopathic infantile hypercalcemia (IIH). In the last decade mutations in CYP24A1, the gene responsible for vitamin D inactivation, were described as the main molecular cause of IIH.In this study, we present a family with two daughters diagnosed with IIH due to two different mutations in CYP24A1 gene. Based on next-generation sequencing (NGS), the elder daughter was diagnosed as carrying the mutations CYP24A1: c.1186C > T; (p.Arg396Trp) and c.428_430del; (p.Glu143del). Within this context, we were able to presymptomatically diagnose her newborn sister using Sanger sequencing technique.Screening for CYP24A1 mutations in families with IIH history helps preventing disease manifestations in newborn siblings. Thus, NGS combined with Sanger sequencing validation opens up the perspective of preventive medicine with great impact on IIH management, where stopping vitamin D administration is enough to prevent disease manifestation, in most cases.

Молекулярно-генетические технологии в диагностике моногенных форм уролитиаза: клинические наблюдения

Kidney stone disease (KSD) is an actual problem of modern health care. By now, more than 80 monogenic forms of urolithiasis have been described. To diagnose such forms of KSD different molecular genetic technologies are used. In the current article 5 clinical cases of KSD among the patients aged 1-9 years old are presented. All of them underwent comprehensive instrumental, clinical, laboratory and molecular genetic investigations. DNA analysis was carried out by Next Generation Sequencing method (NGS) (target NGS-panels and Whole Exome Sequencing). In all cases the molecular genetic cause of the disease was found - idiopathic infantile hypercalcemia type 1 (gene CYP24A1 - 3 cases) and cystinuria (gene SLC7A9 - 2 case). Several unknown genetic variants were found in CYP24A1 (c.1379G>T, c.1156A>T, c.1286T>C) and SLC7A9 (c.920T>A). The importance of genetic testing and the role of genetic counseling for patients with KSD were shown.

SUN-096 Incidentally Found Severe Hypercalcemia in a Pediatric Patient, Diagnostic Challenge

Introduction: Idiopathic infantile hypercalcemia is an intriguing feature of Williams syndrome (WS), occurring in ~15% of diagnoses and is typically not clinically severe. Symptomatic hypercalcemia usually resolves during childhood, but lifelong abnormalities of calcium(Ca) and vitamin D metabolism may persist. The cause of the abnormality in Ca metabolism is still unknown. Hypercalciuria generally accompanies hypercalcemia, but isolated hypercalciuria, especially after infancy, can also occur. Nephrocalcinosis is relatively rare, found in less than 10% of patients undergoing renal ultrasonography. We report a 13-month-old female infant with a history of peripheral pulmonary stenosis and constipation, who presented with severe hypercalcemia that led to a new diagnosis of WS. Case presentation: A 13-month-old girl with a history of peripheral pulmonary stenosis, global developmental delay, and constipation presented to the neurology clinic for evaluation of gross motor delay. She was found to have upper body part hypotonia, decreased reflexes, and on laboratory evaluation, severe hypercalcemia with Ca level of 15.0 mg/dL (8.7 - 10.7). The patient was admitted for management of severe hypercalcemia. Physical exam was also remarkable for subtle features of WS: a happy baby, very social, with prominent eyes, full cheeks, flat nasal bridge, round nasal tip, full lips, and a wide smile. Repeated Ca level on admission was 15.9 mg/dL, with normal albumin level of 4.6 g/dL (2.9-5.5), elevated ionized calcium (iCal) of 1.99 mmol/L (0.95 - 1.32), and intact parathyroid hormone (PTH) of <4.0 pg/mL (8.0 - 85.0). Further evaluation revealed a normal 25 hydroxy-vitamin D:41 ng/mL (30-80) and low 1,25-dihydroxy vitamin D:10pg/mL (31-87). Further evaluation revealed elevated urine calcium to creatinine ratio of 0.7 (normal for age <0.56) and renal ultrasound remarkable for medullary nephrocalcinosis. The patient had a complete blood count within normal limits and a PTH related protein of 26 pg/mL (14-27), ruling out malignancy. Hypercalcemia responded well to intravenous fluids and diuretics, the patient being discharged home after two days on furosemide and potassium supplements with close electrolytes monitoring. The patient required calcium reducing therapy for four months to maintain Ca levels within 9-12 range. The medication was decreased gradually based on calcium and ical levels. The patient is currently doing well, with a normal calcium level, and has being off medication for the past three months. Conclusion: This is a rare case of severe hypercalcemia, which led to the diagnosis of WS. Although idiopathic infantile hypercalcemia occurs in 15% of patients with WS, usually the presentation is mild, and the patients do not require medical interventions. Our patient presented with severe hypercalcemia and subtle physical features of WS that led to genetic testing and final diagnosis.

SUN-LB13 Idiopathic Infantile Hypercalcemia Secondary to CYP24A1 Mutation: A Rare Case Without Exogenous Vitamin D Supplementation

Background: Mutations in CYP24A1, which encodes 24-hydroxylase, the key enzyme for Vitamin D breakdown, cause symptomatic hypercalcemia and nephrocalcinosis in infants on Vitamin D supplementation. New, symptomatic diagnoses of idiopathic infantile hypercalcemia without exogenous supplementation are rare. Previous case reports describe a seasonal effect with worsening hypercalcemia and hypercalciuria during summertime, attributed to increased sun exposure and endogenous Vitamin D production. Clinical Case: A 10-month-old female presented to endocrine care with hypercalcemia and nephrocalcinosis, detected on renal ultrasound (US) due to history of UTI. Her first renal US and serum calcium (Ca) at 3mo of age were normal. Subsequent renal US at 6mo and 9mo of age demonstrated nephrocalcinosis, prompting nephrology and endocrine evaluation. History was significant for failure to thrive. She was born in the fall, with worsening hypercalcemia and nephrocalcinosis during the summer. Diet consisted of standard infant formula and age appropriate solid foods with no added Vitamin D supplementation (~300 IU/day in her formula). She had no family history of nephrocalcinosis, nephrolithiasis, bone disease, or disorders of Ca regulation. Initial labs were notable for Ca corrected for albumin 11.5 (7.8-11.1 mg/dL), PTH <4 (8.7-77.1 pg/mL), 25-OH-Vitamin D 81 (30-96 ng/mL), 1,25-OH-Vitamin D 23.1 (26.1-95 pg/mL), Urine Ca/creatinine ratio of 0.9 mg/mg (<0.81), normal chromosomal microarray, and normal thyroid function tests. She was started on reduced mineral formula PM 60/40. One week later, repeat Ca level increased to Ca corrected 14.2 (7.8-11.1 mg/dL). She was admitted for IV fluids and pamidronate, and was transitioned to a low Ca and Vitamin D formula (Calcilo), with improvement in Ca levels. Testing revealed an increased ratio of 25-OH-Vitamin D to 24,25-OH-Vitamin D of 192 (normal <25), and genetic testing showed 2 pathogenic missense mutations in CYP24A1 genes: c.1226T>C p.(Leu409Ser) and c.1186C>T p.(Arg396Trp). The Leu409Ser mutation has shown a small amount of 24-hydroxylase activity in previous in vitro analysis. She has continued a low Ca diet with stable Ca corrected of 10.7-10.8 (8.7-9.8 mg/dL) and significantly improved weight gain. Conclusion: This is one of the few documented cases of symptomatic idiopathic infantile hypercalcemia secondary to CYP24A1 mutation in an infant without exogeneous Vitamin D supplementation. Her nephrocalcinosis and hypercalcemia worsened over the summer, suggesting increased sun exposure may have been a contributing factor. This case demonstrates that 1,25-OH-Vitamin D levels may be normal or low in this condition, particularly for individuals with the Leu409Ser mutation who may retain partial 24-hydroxylase function.

SAT-368 CYP24A1 Mutation Masking Malignancy Mediated Hypercalcemia

Background: Mutations in CYP24A1, resulting in reduced conversion of 1,25(OH)2D to its inactive metabolite 24,25-(OH)2D3,are rare causes of parathyroid hormone (PTH)-independent hypercalcemia. While manifestations may range from the severe idiopathic infantile hypercalcemia due to biallelic mutations, heterozygous loss-of-function mutations causing milder phenotypes are increasingly reported in adults. Elevated 1,25(OH)2D and hypercalciuria often accompanied by a history of nephrolithiasis are characteristic. Worsening hypercalcemia, under conditions such as pregnancy or sunlight exposure that enhance 1,25(OH)2D and 25(OH)D production, respectively, has been described in CYP24A1 mutations. We describe a patient with hypercalcemia and a history of lymphoma who was found to have elevated 25-OH-D3 and low 24,25-(OH)2D3 levels, suggesting a mutation in CYP24A1. Clinical Case: An 80 year-old Caucasian male with history of indolent non-Hodgkin lymphoma diagnosed in 2016,well controlled after several courses of chemotherapy,was referred for recurrent hypercalcemia. Laboratory studies showed levels of 1,25(OH)2D of 78 (18–72 pg/mL) and PTH 22 (10–65 pg/mL) with calcium ranging from 10.3 to 12.6 (8.5–10.1mg/dL), an undetectable PTHrP, 25(OH)D level of 32.9 (30–100 ng/mL), and 24-hour urinary calcium of 378mg. He was treated with high dose prednisone for presumed 1,25(OH)2D-mediated hypercalcemia. Despite initial good response, hypercalcemia became progressively difficult to control requiring escalating doses of steroids. Repeat 1,25(OH)2D levels improved to 30–40 pg/mL, but subsequently rebounded to >150. Oncologic re-evaluation found low-grade follicular lymphoma in inguinal lymph nodes,which were thought to be the source of 1,25(OH)2D overproduction. Detailed history and records review, however, revealed that onset of hypercalcemia dated back to 2006, concurrent with the development of several episodes obstructive uropathy due to stones. These events preceded the diagnosis of lymphoma by a decade, and resulted in CKD stage 4. Family history is notable for nephrolithiasis in his fatherand son. We suspected CYP 24A1 mutation. Vitamin D metabolite analysis demonstrated a 25-OH-D3 of 27 (20-50ng/mL) and 24,25-(OH)2D3 of 0.56 ng/mL with a ratio elevated to 48.21 (<25), indicative of a defect in vitamin D degradation that potentially exacerbates oversupply of 1,25(OH)2D, mediated via its reduced metabolism. Genetic evaluation is in progress. After initiation of treatment with Rituximab, his serum calcium levels declined along with regression of his lymphoma. Conclusion: Although mutations in CYP24A1 are an uncommon cause of hypercalcemia, they should be considered in the differential diagnosis of elevated 1,25(OH)2D levels without a clear source, as confirming this diagnosis strongly impacts treatment decisions and clinical outcome.

Substrate 1,25(OH)2D3 and Adrenodoxin exert a coordinated change in CYP24A1 structure

The mitochondrial cytochrome P450 24A1 (CYP24A1) is involved in deactivation of bioactive 1,25‐(OH)2‐vitD by side chain hydroxylation. Mutated CYP24A1 leads to idiopathic infantile hypercalcemia, characterized by elevated calcium, failure to thrive, vomiting, dehydration, and nephrocalcinosis. Deactivation relies on the protein‐protein interaction with the redox partner, Adrenodoxin (Adx), housing the iron‐sulfur cluster which provides two electrons for the hydroxylation step through reduction of heme iron in CYP24A1. We have previously used solution nuclear magnetic resonance (NMR) to demonstrate the long‐range allostery of CYP24A1 in response to redox partner recognition. Here we report a comprehensive multidisciplinary approach using chemical cross‐linking coupled to mass spectrometry, site‐directed mutagenesis, NMR, functional assays and molecular dynamics simulations. Chemical crosslinking using 1‐ethyl‐3‐(3‐dimethylaminopropyl)carbodiimide hydrochloride (EDC) traps a 1:1 CYP24A1‐Adx complex by way of reaction with the carboxylate side chains of Adx and the primary amines of CYP24A1. Through chymotrypsin digest and mass spectrometry analysis of this product, we’ve identified a set of CYP24A1 peptides that are differentially modified, consistent with rearrangement of salt bridges in the presence or absence of substrate and Adrenodoxin. Affected sites include regions of the F‐G helices, the A‐helix, and the C/D elbow of the proximal surface. Interestingly, substrate‐induced differential cross‐linking at these sites only occurs when Adx is already present, suggesting a coordinated effect on CYP24A1 structure. This approach is combined with data from additional biophysical methods in order to inform a model of the functional 1:1 P450‐Adx complex that is required for metabolism of vitamin‐D.

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

Loss of function mutations in the CYP24A1 gene, involved in vitamin D catabolism and in calcium homeostasis, are known to be the genetic drivers of both idiopathic infantile hypercalcemia (IIH) and adult renal stone disease. Recently, also defects in the SLC34A1 gene, encoding for the renal sodium-phosphate transporter NaPi-IIa, were associated with the disease. IIH typically affects infants and pediatric patients with a syndrome characterized by severe hypercalcemia, hypercalciuria, suppressed parathyroid hormone level and nephrolithiasis. In SLC34A1 mutated carriers, hypophosphatemia is also a typical biochemical tract. IIH may also persist undiagnosed into adulthood, causing an increased risk of nephrocalcinosis and renal complication. To note, a clinical heterogeneity characterizes IIH manifestation, principally due to the controversial gene-dose effect and, to the strong influence of environmental factors. The present review is aimed to provide an overview of the current molecular findings on the IIH disorder, giving a comprehensive description of the association between genotype and biochemical and clinical phenotype of the affected patients. We also underline that patients may benefit from genetic testing into a targeted diagnostic and therapeutic workflow.

MON-255 A Novel Variant of SLC34A1 Gene in an Infant with Idiopathic Infantile Hypercalcemia

Bacground & objective: Idiopathic infantile hypercalcemia is one of rare diseases characterizing hypercalcemia in infancy. Renal phosphate absorption in proximal tubules plays a very important role in the phosphate and calcium homeostasis. SLC34A1 is known a key regulator of renal phosphate reabsorption. SLC34A1 gene mutation is one of very uncommon causes of idiopathic infantile hypercalcemia. We have experienced a case of idiopathic infantile hypercalcemia caused by a homozygous novel variant c.1483C>T(p.Arg495Cys) of SLC34A1 gene. Materials & Methods: A study patient was 5 months-old boy. His medical records are reviewed retrospectively. Results: A 5-month-old boy was transferred to Kyungpook National University Children's Hospital because of sustained hypercalcemia with hypercalciuria. Laboratory investigations revealed a serum calcium level of 12.6 mg/dL (normal range: 9.0-10.6 ), phosphate level of 3.7 mg/dL (normal range: 4.8-8.2), serum magnesium level of 2.0 mEq/L (normal range: 1.44-3.12), intact PTH level of 0.6 pg/mL (normal range: 10-65), PTHrP <1.1pmol/L (normal range: <2.0), 25(OH)vitamin D3 level of 65 ng/mL (normal range: 8.0-51.9) and 1,25(OH) vitamin D3 level of 79 pg/mL (normal range: 25-65). Spot urine calcium/urinary creatinine ratio (mg%: mg%) was elevated 1.4 (normal level: <0.8 for infant). Targeted exome sequencing in the patient was performed, resulting in a homozygous novel variant c.1483C>T(p.Arg495Cys) of SLC34A1 gene confirmed by Sanger sequencing. Consulsions: We report a case with idiopathic infantile hypercalcemia caused by a novel variant of SLC34A1 gene mutation

Infantile hypercalcemia with novel compound heterozygous mutation in SLC34A1 encoding renal sodium-phosphate cotransporter 2a: a case report.

Idiopathic infantile hypercalcemia is characterized by hypercalcemia, dehydration, vomiting, and failure to thrive, and it is due to mutations in 24-hydroxylase (CYP24A1). Recently, mutations in sodium-phosphate cotransporter (SLC34A1) expressed in the kidney were discovered as an additional cause of idiopathic infantile hypercalcemia. This report describes a female infant admitted for evaluation of nephrocalcinosis. She presented with hypercalcemia, hypercalciuria, low intact parathyroid hormone level, and high 1,25-dihydroxyvitamin D3 level. Exome sequencing identified novel compound heterozygous mutations in SLC34A1 (c.1337G>A, c.1483C>T). The patient was treated with fluids for hydration, furosemide, a corticosteroid, and restriction of calcium/vitamin D intake. At the age of 7 months, the patient's calcium level was within the normal range, and hypercalciuria waxed and waned. Renal echogenicity improved on the follow-up ultrasonogram, and developmental delay was not noted. In cases of hypercalcemia with subsequent hypercalciuria, DNA analysis for SLC34A1 gene mutations and CYP24A1 gene mutations should be performed. Further studies are required to obtain long-term data on hypercalciuria and nephrocalcinosis.

Calcioic acid: In vivo detection and quantification of the terminal C24-oxidation product of 25-hydroxyvitamin D3 and related intermediates in serum of mice treated with 24,25-dihydroxyvitamin D3

Calcitroic acid, the excretory form of vitamin D, is the terminal product of a 5-step pathway catalyzed by CYP24A1, commencing with C24-hydroxylation of 1,25-dihydroxyvitamin D3 (1,25-(OH)2D3). Catabolism of 25-hydroxyvitamin D3 (25-OH-D3) proceeds via analogous steps culminating in calcioic acid; however this C23-truncated acid has not been reported in the circulation. It has recently been shown that 24,25-dihydroxyvitamin D3 (24,25-(OH)2D3) is an important factor in optimal bone fracture healing acting via an effector molecule FAM57B2 to produce lactosylceramide. Administration of 24,25-(OH)2D3 was found to restore normal fracture repair in Cyp24a1−/− mice devoid of 24,25-(OH)2D3. We set out to study the multi-step catabolism of D3 metabolites in vivo using LC–MS/MS methods in vehicle or 24,25-(OH)2D3-treated mice. Vehicle-treated Cyp24a1+/- mice possessed normal levels of serum 24,25-(OH)2D3 (7 ng/mL) and 25−OH-D3-26,23-lactone (4 ng/mL). We also detected 24-oxo-25−OH-D3 (3 ng/mL) and 24-oxo-23,25-(OH)2D3 (0.4 ng/mL); which were not detectable in vehicle-treated Cyp24a1−/− mice. In 24,25-(OH)2D3-treated Cyp24a1+/- mice, serum 24,25-(OH)2D3 rose to 200 ng/mL while 25-OH-D3-26,23-lactone remained unchanged in comparison to vehicle-treated Cyp24a1+/- mice Concentration of serum 24-oxo-25-OH-D3 and 24-oxo-23,25-(OH)2D3 rose by 10-fold, when Cyp24a1+/- mice were treated with 24,25-(OH)2D3 Calcioic acid was increased to 0.030 ng/mL for 24,25-(OH)2D3-treated Cyp24a1+/- mice. In 24,25-(OH)2D3-treated Cyp24a1−/− mice, serum 24,25-(OH)2D3 rose further to a striking 830 ng/mL due to lack of catabolism of the 24,25-(OH)2D3 dose. Serum 1,25-(OH)2D3 levels were suppressed in 24,25-(OH)2D3-treated Cyp24a1+/- and Cyp24a1−/− mice. Circulating 1,24,25-(OH)3D3 rose from 73 pg/mL to 106 pg/mL when Cyp24a1+/- mice were treated with 24,25-(OH)2D3. While undetectable in vehicle-treated Cyp24a1−/− mice, 1,24,25-(OH)3D3 rose unexpectedly to 153 pg/mL in 24,25-(OH)2D3-treated nulls suggesting conversion of 24,25-(OH)2D3 to 1,24,25-(OH)3D3 via 1-hydroxylation. Taken together, amplification of 24,25-(OH)2D3 catabolism by exogenous doses of this metabolite have enabled detection of downstream C24-oxidation pathway products in vivo, including calcioic acid; and provides a platform for studying alternative routes of vitamin D metabolism that may occur in pathological states including hypervitaminosis D and idiopathic infantile hypercalcemia caused by mutations of CYP24A1.

External Quality Assessment of 24,25-dihydroxyvitamin D3 (24,25(OH)2D3) assays

The discovery that mutations of the CYP24A1 gene are a cause of idiopathic infantile hypercalcemia (IIH) has revived interest in measuring serum 24,25(OH)2D3. Several studies have also suggested that a high 25-hydroxyvitamin D3(25-OHD3):24,25(OH)2D3 ratio might provide additional diagnostic information in the investigation of vitamin D deficiency. Measurement of 24,25(OH)2D3 is necessarily restricted to laboratories with mass spectrometry methods although cross reactivity of the metabolite in immunoassays for 25-OHD is a potential cause of misleading results. The international External Quality Assessment (EQA) scheme for vitamin D metabolites (DEQAS) was set up in 1989. In 2013 DEQAS became an accuracy based EQA for 25-OHD with ‘target values’ assigned by the National Institute of Standards and Technology (NIST) Reference Measurement Procedure (RMP). A pilot scheme for serum 24,25(OH)2D3 was started in 2015 and participants were asked to measure the metabolite on each of the 5 samples sent out for 25-OHD. Inter-laboratory agreement was poor but this may reflect methodological differences, in particular different approaches to assay standardization. An important potential contribution to reducing variability among assays was the development by NIST of a 24,25(OH)2D3 RMP and its use in assigning values to SRMs 972a, 2973 and 2971, supported by the NIH Office of Dietary Supplements (ODS) as part of the Vitamin D Standardization Program (VDSP) effort.

Molecular characterization of a recurrent 10.9 kb CYP24A1 deletion in Idiopathic Infantile Hypercalcemia

Loss-of-function mutations in CYP24A1 (MIM 126065 20q13.2), the gene encoding the 24-hydroxylase responsible for 25-OH-D and 1,25-(OH)2D degradation, are identified in about 20% of patients presenting Idiopathic Infantile Hypercalcemia (IIH) (MIM 143880). Common features of this autosomal recessive condition included hypercalcemia with hypercalciuria, suppressed PTH and a high 25-OH-D3:24,25-(OH)2D3 ratio. Medical care mainly relies on sun protection and life-long contraindication of vitamin D to avoid complications such as early nephrocalcinosis and renal failure.Molecular diagnosis therefore keeps a crucial place in the diagnosis of IIH, and genetic counseling should be systematically recommended to prevent vitamin D administration in affected siblings.In this report is described the molecular characterization of a CYP24A1 deletion identified in two unrelated families. This highlights the potential role of CYP24A1 copy number variations (CNV) in IIH. Considering the presence of CNV affecting CYP24A1 in public databases, CNV analysis should be systematically added to the sequencing studies in IIH. Targeted Massively Parallel Sequencing (MPS) study coupled with a CNV detection tool called CovCop is a powerful method to detect genic rearrangement and improve genetic analysis.

Vitamin D Toxicity-A Clinical Perspective.

Confusion, apathy, recurrent vomiting, abdominal pain, polyuria, polydipsia, and dehydration are the most often noted clinical symptoms of vitamin D toxicity (VDT; also called vitamin D intoxication or hypervitaminosis D). VDT and its clinical manifestation, severe hypercalcemia, are related to excessive long-term intake of vitamin D, malfunctions of the vitamin D metabolic pathway, or the existence of coincident disease that produces the active vitamin D metabolite locally. Although VDT is rare, the health effects can be serious if it is not promptly identified. Many forms of exogenous (iatrogenic) and endogenous VDT exist. Exogenous VDT is usually caused by the inadvertent or improper intake of extremely high doses of pharmacological preparations of vitamin D and is associated with hypercalcemia. Serum 25-hydroxyvitamin D [25(OH)D] concentrations higher than 150 ng/ml (375 nmol/l) are the hallmark of VDT due to vitamin D overdosing. Endogenous VDT may develop from excessive production of an active vitamin D metabolite – 1,25(OH)2D in granulomatous disorders and in some lymphomas or from the reduced degradation of that metabolite in idiopathic infantile hypercalcemia. Endogenous VDT may also develop from an excessive production of 25(OH)D and 1,25(OH)2D in congenital disorders, such as Williams–Beuren syndrome. Laboratory testing during routine clinical examinations may reveal asymptomatic hypercalcemia caused by the intake of vitamin D even in doses recommended for the general population and considered safe. That phenomenon, called hypersensitivity to vitamin D, reflects dysregulated vitamin D metabolism. Researchers have proposed many processes to explain VDT. Those processes include elevated activity of 1α-hydroxylase or inhibited activity of 24-hydroxylase, both leading to increased concentration of 1,25(OH)D; increased number of vitamin D receptors; and saturation of the capacity of vitamin D binding protein. Increased public awareness of vitamin D–related health benefits might increase the risk of VDT due to self-administration of vitamin D in doses higher then recommended for age and body weight or even higher than the established upper limit intake values. Consequently, the incidence of hypercalcemia due to hypervitaminosis D might increase.

Juvenile onset IIH and CYP24A1 mutations

The term Idiopathic infantile hypercalcemia (IIH) was first introduced almost 70 years ago when symptomatic hypercalcemia developed in children after receiving high doses of vitamin D for the prevention of rickets. The underlying pathophysiology remained unknown until recessive mutations in CYP24A1 encoding Vitamin D3-24-hydroxylase were discovered. The defect in vitamin D degradation leads to an accumulation of active 1,25(OH)2D3 with subsequent hypercalcemia. Enhanced renal calcium excretions lead to hypercalciuria and nephrocalcinosis. Meanwhile, the phenotypic spectrum associated with CYP24A1 mutations has significantly broadened. Patients may present at all age groups with symptoms originating from increased serum calcium levels as well as from increased urinary calcium excretions, i.e. kidney stones. Possible long term sequelae comprise chronic renal failure as well as cardiovascular disease. Here, we present a family with two affected siblings with differing clinical presentation as an example for the phenotypic variability of CYP24A1 defects.

Idiopathic Infantile Hypercalcemia Presenting in Childhood but Diagnosed in Adulthood

<h2>ABSTRACT</h2> <b>Objective:</b> Hypercalcemia can be caused by a variety of pathologies and factors. Vitamin D plays a central role in calcium homeostasis, where tight control of its metabolism is necessary. Inadequate 25-hydroxyvitamin D-24-hydroxylase (CYP24A1) enzyme activity leads to failure of 25-hydroxyvitamin D and 1,25-dihydroxyvitamin D catabolism, resulting in hypercalcemia. <b>Methods:</b> This study consists of a case report and brief review of the literature. A young woman with persistent hypercalcemia of unknown origin was investigated by measuring phosphate, parathyroid hormone, and vitamin D metabolites, as well as by evaluating kidney and bone involvement. <b>Results:</b> The patient was asymptomatic and had a cystitis-like syndrome during childhood. The patient exhibited hypercalcemia and hypercalciuria with suppressed PTH. Liquid chromatography with tandem mass spectrometry analysis of vitamin D metabolites revealed elevated 1,25-dihydroxyvitamin D₃ (301 pg/mL) and an elevated ratio of 25-hydroxyvitamin D₃ to 24,25-dihydroxyvitamin D₃ of 390. Screening for <i>CYP24A1</i> gene mutations was performed. Bi-allelic mutations in the coding region of <i>CYP24A1</i> were identified (Arg396Trp/Leu409S), confirming the diagnosis of hypercalcemia due to <i>CYP24A1</i> mutation. The patient's family members were also studied. <b>Conclusion:</b> This particular case of hypercalcemia due to a mutation in <i>CYP24A1</i> emphasizes 2 main concerns: vitamin D-related hypercalcemia should be considered at any age in patients with hypercalcemia, and asymptomatic patients with <i>CYP24A1</i> mutations are at risk of developing hypercalcemia arising from vitamin D supplementation. <b>Abbreviations: 1,25-(OH)</b>₂<b>D</b>₃ 1,25-dihydroxyvitamin D3 <b>25-OH-D</b>₃ 25-hydroxyvitamin D₃ <b>24,25-(OH)</b>₂<b>D</b>₃ 24,25-dihydroxyvitamin D₃ <b>CYP24A1</b> 25-hydroxyvitamin D-24-hydroxylase <b>FGF23</b> fibroblast growth factor 23 <b>IIH</b> idiopathic infantile hypercalcemia <b>LC-MS/MS</b> liquid chromatography with tandem mass spectrometry <b>PTH</b> parathyroid hormone

The When, What & How of Measuring Vitamin D Metabolism in Clinical Medicine.

We now have the ability to measure a number of different vitamin D metabolites with very accurate methods. The most abundant vitamin D metabolite, 25-hydroxyvitamin D, is currently the best marker for overall vitamin D status and is therefore most commonly measured in clinical medicine. The added value of measuring metabolites beyond 25-hydroxyvitamin D, like 1,25-, and 24,25-dihydroxyvitamin D is not broadly appreciated. Yet, in some more complicated cases, these metabolites may provide just the information needed for a legitimate diagnosis. The problem at present, is knowing when to measure, what to measure and how to measure. For 25-hydroxyvitamin D, the most frequently used automated immunoassays do not meet the requirements of today’s standards for certain patient groups and liquid chromatography-tandem mass spectrometry is the desired method of choice in these individuals. The less frequently measured 1,25-dihydroxyvitamin D metabolite enables us to identify a number of conditions, including 1α-hydroxylase deficiency, hereditary vitamin D-resistant rickets and a number of granulomatous diseases or lymphoproliferative diseases accompanied by hypercalcaemia. Furthermore, it discriminates between the FGF23-mediated and non-FGF23-mediated hypophosphatemic syndromes. The 24,25-dihydroxyvitamin D metabolite has proven its value in the diagnosis of idiopathic infantile hypercalcaemia and has the potential of having value in identifying other diseases. For both metabolites, the understanding of the origin of differences between assays is limited and requires further attention. Nonetheless, in every way, appropriate measurement of vitamin D metabolism in the clinical laboratory hinges eminently on the comprehension of the value of the different metabolites, and the importance of the choice of method.

A case of Idiopathic Infantile Hypercalcaemia (IIH) persisting into adulthood, caused by compound heterozygous mutations of 1,25-dihydroxyvitamin D2 24-hydroxylase (CYP24A1)

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