ORIGINAL ARTICLE Maternal and infantile hypercalcemia caused by vitamin-D-hydroxylase mutations and vitamin D intake Dganit Dinour & Miriam Davidovits & Shraga Aviner & Liat Ganon & Leonid Michael & Dalit Modan-Moses & Iris Vered & Haim Bibi & Yaacov Frishberg & Eli J. Holtzman Received: 1 January 2014 /Revised: 12 June 2014 /Accepted: 13 June 2014 /Published online: 7 September 2014 # IPNA 2014 Abstract Background Hypercalcemia is caused by many different con- ditions and may lead to severe complications. Loss-of- function mutations of CYP24A1, encoding vitamin D-24-hy- droxylase, have recently been identified in idiopathic infantile hypercalcemia and in adult kidney stone disease. The aim of this study was to investigate the genetics and clinical features of both infantile and maternal hypercalcemia. Methods We studied members of four unrelated Israeli fami- lies with hypercalcemia, namely, one woman during pregnan- cy and after delivery and three infants. Clinical and biochemical data were obtained from probands’ medical charts. Genomic DNA was isolated from peripheral blood and CYP24A1 was sequenced. Results Typical symptoms of hypercalcemia associated with the intake of recommended doses of vitamin D developed in the infants and pregnant woman. Four different loss-of- function CYP24A1 mutations were identified, two of which are reported here for the first time (p.Trp134Gly and p.Glu315*). The infants from families 1 and 2, respectively, were found to be compound heterozygotes, and the infant from family 3 and the pregnant woman were found to be homozygous. Conclusions This is the first report of maternal hypercalcemia caused by a CYP24A1mutation, showing that not only infants are at risk for this complication. Our findings emphasize the importance of recognition, genetic diagnosis and proper treat- ment of this recently identified hypercalcemic disorder in this era of widespread vitamin D supplements. Keywords CYP24A1 . Hypercalciuria . Infantile hypercalcemia .Maternal hypercalcemia . Nephrocalcinosis . Nephrolithiasis . VitaminD Introduction Hypercalcemia is caused by many different conditions, in- cluding primary hyperparathyroidism, cancer, granulomatous diseases (e.g. sarcoidosis), drugs (e.g. thiazides) and a high intake of calcium and/or vitamin D. In the early 1950s, approximately 200 cases of infants with unexplained hypercalcemia were reported in the UK within only 2 years. These infants presented with failure to thrive, vomiting, dehydration, spikes of fever, nephrocalcinosis and suppressed parathyroid hormone (PTH) levels [1, 2]. Some of the affected children had a complex phenotype that was later D. Dinour (*) : L. Ganon : E. J. Holtzman Department of Nephrology and Hypertension, The Chaim Sheba Medical Center, Tel-Hashomer, Ramat-Gan 52621, Israel e-mail:
[email protected] M. Davidovits Institute of Pediatric Nephrology, Schneider Children’s Medical Center, Petah Tiqwa, Israel S. Aviner :H. Bibi Department of Pediatrics and the Faculty of Health Sciences, Barzilai Medical Center Campus of the Ben Gurion University of the Negev, Ashkelon, Israel L. Michael : I. Vered Department of Endocrinology, The Chaim Sheba Medical Center, Tel-Hashomer, Ramat-Gan, Israel D. Modan-Moses Pediatric Endocrinology and Diabetes Unit, Chaim Sheba Medical Center, Tel-Hashomer, Ramat-Gan, Israel Y. Frishberg Division of Pediatric Nephrology, Shaare Zedek Medical Center and the Hebrew University School of Medicine, Jerusalem, Israel D. Dinour : L. Ganon : L. Michael :D. Modan-Moses : I. Vered : E. J. Holtzman The Sackler School ofMedicine, Tel Aviv University, Tel Aviv, Israel Pediatr Nephrol (2015) 30:145–152 DOI 10.1007/s00467-014-2889-1 identified as the Williams–Beuren syndrome, presenting with supravalvular aortic stenosis, multiple peripheral pulmonary arterial stenoses, ‘elfin face,’ psychomotor and growth delay, characteristic dental malformations and occasionally infantile hypercalcemia (OMIM, #194050). However, most of these infants with hypercalcemia did not have this syndrome, and the condition was considered idiopathic. Although it became evident that vitamin D supplementation plays a role in the pathogenesis of idiopathic infantile hypercalcemia, it took 60 years to identify the genetic defect in vitamin D metabo- lism that causes this disorder. In 2011, Schlingmann et al. identified loss-of-function mutations in the cytochrome P450 24A1 gene (CYP24A1), which encodes vitamin D-24-hydroxylase, the enzyme that degrades the active form of vitamin D, as the cause of idio- pathic infantile hypercalcemia [3]. This finding was later supported by additional studies [4, 5]. Our group and other researchers have since shown that loss-of-function mutations of CYP24A1 may also affect adults, causing long-standing kidney stone disease and nephrocalcinosis that can even lead to chronic kidney injury [6, 7]. During pregnancy and lactation several physiologic adap- tations are invoked to supply the required calcium for the offspring. Intestinal calcium absorption more than doubles during pregnancy, and bone resorption occurs to provide calcium for breast milk [8]. Nevertheless, symptomatic hyper- calcemia in pregnancy is very rare. We describe here four unrelated subjects who developed symptomatic hypercalcemia due to loss-of-function mutations of CYP24A1. Three infants presented with typical features of “idiopathic infantile hypercalcemia.” The fourth case is a woman with a long-standing kidney stone disease who devel- oped severe hypercalcemia during pregnancy. This is the first report attributing maternal hypercalcemia to a mutated vita- min D-24-hydroxylase. Our findings emphasize the importance of recognition, genetic diagnosis and proper treatment of CYP24A1 defects in infantile as well as maternal hypercalcemia, especially in an era when both expectant mothers and infants are given vitamin D supplements around the world. Patients and methods Patients We studied four unrelated individuals of Israeli descent who suffered from symptomatic hypercalcemia. the probands were three infants and a woman who developed hypercalcemia during pregnancy and post-partum.Data on clinical symptoms and biochemical tests were obtained from medical charts. The study protocol was approved by the institutional review board of the Chaim Sheba Medical Center, Israel. Informed consent regarding genetic analysis and medical evaluation was obtain- ed from all of the study subjects. Consent for the infants was given by their parents. Genetic studies Genomic DNA was isolated from peripheral blood cells using the ArchivePure DNA Blood kit (5 PRIME, Gaithersburg, MD) according to the manufacturer’s in- structions. The coding sequence and splice-sites of CYP24A1 were amplified by PCR using intronic primers. Primers for CYP24A1 have been described previously [7]. All PCR products were sequenced directly (ABI Prism 3100; Applied Biosystems, Foster City, CA). Results Clinical findings Family 1 A 5-month-old male infant was hospitalized for repeated vomiting after meals and weight loss during the preceding 2 weeks without fever or diarrhea. The patient was the first child of healthy unrelated parents: a 28-year-old mother of Jewish- Ashkenazi origin and a 34-year-old father of Jewish-Tunisian origin. He was born at term after a normal pregnancy and delivery. Development was normal, and past medical history was unremarkable. He was fed a standard milk formula (Similac; Abbott Laboratories, Abbott Park, IL) and was given the usual vitamin D supplement of 400 IU/day. Physical examination at admission revealed no abnormalities except for a closed anterior fontanel. Blood pressure was 90/ 55 mmHg. Laboratory investigation revealed hypercalcemia, with a serum total calcium level of 13.5 (normal for age 8.8– 11.2) mg/dl and ionized calcium level of 1.8 (normal 1.12– 1.32) mmol/l; the phosphate level was normal (4.7 mg/dl). Kidney function was preserved (serum creatinine 0.2 mg/dl), and levels of serum albumin, glucose, sodium, potassium, magnesium and bicarbonate were within normal range. Urine calcium excretion was markedly elevated; the urinary calcium (mg/dl)/creatinine (mg/dl) ratio was 3.03 (normal for age At this stage, the results of an extended workup were received. PTH was suppressed to 6.2 (normal 12–87) pg/ ml, and vitamin D was at the upper limit of normal, with the 25-hydroxy-vitamin D3 (25-(OH)D3) level at 59 (nor- mal 10–60) ng/ml and the 1,25-dihydroxyvitamin D3 [1,25-(OH)2D3] level at 64 (normal 20–67) pg/ml (Table 1). Genetic analysis for William’s syndrome was negative. As sarcoidoisis was suspected, angiotensin- converting enzyme blood levels were measured and found to be normal. With the exclusion of other causes of PTH-independent hypercalcemia, disturbed vitamin D metabolism was suspected, and genetic analysis for CYP24A1 mutations was performed. In the meantime, treatment with glucocor- ticoids (prednisone) was started, and formula feeding (containing 47 IU vitamin D/100 ml) was replaced with cow milk without vitamin D supplement until a modified formula without any vitamin D content (Calcilo XD; Abbott Laboratories) could be provided. During this peri- od of changes to the feeding schedule and the addition of solid foods, calcium levels normalized, and treatment with calcitonin could be stopped. The patient was discharged in good clinical condition, without vomiting and with some weight gain. The blood calcium level at discharge was normal at 10.1 mg/dl, and the ionized calcium measure- ment was 1.27 mmol/l. After discharge, prednisone therapy was gradually tapered off. At the 1-month follow-up examination, the patient was in good health and was not receiving any medication. He continues with the Calcilo XD formula and solid foods. The blood calcium level is currently 9.6 mg/dl. Family 2 A 9-month-old female baby was admitted for a 2-day history of fever and cough. Urinalysis was positive for white blood cells (WBC) and nitrites. She is the product of a normal full-term pregnancy and delivery, the first child of healthy unrelated parents—a 29-year-old father of Tunisian origin and a 27-year-old mother of Georgian origin. She was fed a standard milk-based formula and received the usual supplement of 400 IU/day vitamin D. She had poor weight gain, dropping from the 50th per- centile to the third percentile during the 4 months preced- ing admission. Replacement of the milk-based formula to Nutramigen® (hydrolysate of casein, containing 33.8 IU vitamin D/100 ml; Mead Johnson & Co., Glenview, IL ) resulted in a mild improvement in weight gain. Physical examination was normal, except for left otitis media. Laboratory investigation revealed hypercalcemia of 11.2– 11.8 (normal 8.8–11.2) mg/dl, ionized calcium 1.51 (normal 1.09–1.3) mmol/l and phosphate 3.5 (normal 2.5–4.5) mg/dl. Serum creatinine, albumin, glucose, electrolytes, magnesium and bicarbonate were within normal limits. Urine calcium excretion was elevated, with a urinary calcium (mg/dl)/creat- inine (mg/dl) ratio of 0.87 (normal for age otitis and suspected urinary tract infection but urinary culture remained sterile. The serum 25-(OH)D3 level was relatively high at 53.2 (normal 10–60) ng/ml, the 1,25-(OH)2D3 level was 35 (nor- mal 20–67) pg/ml and PTH was suppressed to Genetic findings We identified four different loss-of-function CYP24A1 mutations in four unrelated Israeli families with hypercalcemia. In family 1 the affected infant was found to be compound heterozygous for two missense CYP24A1 mutations: p.Arg396Trp (c.1186C>T) inherited from the mother and p.Trp134Gly (c.400 T>G) inherited from the father (Fig. 1a). The p.Arg396Trp mutation has already been report- ed in infantile hypercalcemia and shown in in vitro studies to create loss of this enzyme activity [3]. The second mutation, p.Trp134Gly, has not yet been reported but has already been studied using mammalian cell expression system and shown to reduce enzyme activity to 1.5–3 % of the wild-type (M. Kaufmann, G. Jones G, personal communication). The affected infant of family 2 was also found to be a compound heterozygote carrying the p.Trp134Gly mutation inherited from his father and a novel truncating mutation, p.Glu315* (c.943G>T), inherited from his mother. This mu- tation leads to the production of a truncated protein with 314 of the 518 amino acids (Fig. 1b). The affected infant of family 3 was found to be homozy- gous for the known loss-of -funct ion p.E143del (c.427_429delGAA) mutation [3, 4, 7]. Both parents were heterozygous carriers of this deletion (Fig. 1c). In family 4, the adult woman with nephrolithiasis and nephrocalcinosis who developed hypercalcemia during preg- nancy, as well as her asymptomatic brother, were found to be homozygous carriers of the p.E143del mutation. Their parents are heterozygous for the mutation (Fig. 1d). The woman’s four children, as expected, were also found to be heterozygous for the mutation. Discussion 24-Hydroxylase, a mitochondrial CYP enzyme encoded by CYP24A1, is involved in both 24-hydroxylation and 23- hydroxylation on the side chain of 25-(OH)D3 and 1,25-(OH)2D3. This enzyme catalyzes a 5-step pathway of 24-hydroxylation from 1,25-(OH)2D3 to calcitroic acid (Fig. 2), which is secreted into the bile, as well as a similar pathway of 23-hydroxylation, the final product of which is 1,25-(OH)2D3-26,23-lactone. It also catalyzes 25(OH)D3 to 24,25-(OH)2D [9]. It has been suggested that the role of 24- hydroxylase is to regulate and attenuate 1,25-(OH)2D3 action on target cells. In CYP24A1 null mice, 1,25-(OH)2D3 clearance is dramat- ically decreased, and its half life increases by tenfold [10]. Of note, these mice are particularly sensitive to the acute or chronic administration of 1,25-(OH)2D3, responding with high serum levels of 1,25-(OH)2D3 with slow clearance [11]. Infantile hypercalcemia is a syndrome characterized by hypercalcemia presenting with failure-to-thrive, vomiting, de- hydration and nephrocalcinosis. Recent reports have identi- fied CYP24A1 mutations as the cause of infantile hypercalce- mia [3, 4]. Schlingmann et al. [3] studied two groups of infants with CYP24A1 mutations: the first group developed symp- tomatic hypercalcemia 1–3 weeks after receiving a very high dose of oral vitamin D (600,000 IU), but the second group presented at the age of 6–8 months with a similar phenotype after receiving only 500 IU vitamin D per day. The mutations identified in both groups were shown in in vitro studies to markedly reduce the enzyme’s catabolic activity. We describe here four unrelated families with hypercalce- mia caused by four different loss-of-function CYP24A1 mu- tations, including two not previously reported mutations (p.Trp134Gly and p.Glu315*). It is interesting to note that in families 1 and 2 both infants’ fathers, though not known to be related, are from Tunisian decent and carry the same hetero- zygous mutation: – p.Trp134Gly. The infants presented with typical symptomatic infantile hypercalcemia at the age of 5 (2 infants) and 9 months (1 infant). All three were given the recommended oral dose of 400 IU vitamin D per day and fed on formulas which also contain vitamin D. When hypercalce- mia was diagnosed, supplemental vitamin D was immediately stopped, and their diet was changed accordingly. The fourth patient, homozygous for the p.E143del muta- tion, is the first described case of maternal hypercalcemia caused by a CYP24A1 mutation. Her four heterozygous chil- dren were not treated with vitamin D. Although they all have relatively high serum calcium levels, none of them have hypercalciuria (Table 2) and they are all asymptomatic. In heterozygous carriers of CYP24A1 mutations, as in the chil- dren of family 4, the implications of vitamin D supplements are still unknown. All our patients had hypercalcemia and low PTH levels (Table 1), but the. However, 25-hydroxyvitamin D and 1,25- hydroxyvitamin D levels varied. PTH is suppressed in hyper- calcemia and, therefore, the level of 1,25-hydroxyvitamin D is expected to be low in these patients. In our patients, except for infant 2, the level of 1,25-hydroxyvitamin D was much higher than expected in view of the low PTH level, and 25- hydroxyvitamin D levels were mostly on the upper level of the normal range. The variability in 1,25-hydroxyvitamin D and 25-hydroxyvitamin D levels may depend on the timing of the testing and/or the effects of other modifying genes and in any case were found to be high given the low PTH level. Both the 1,25-hydroxyvitamin D and 25-hydroxyvitamin D levels in our study are similar to those found in previously reported patients [3]. Hollis et al. [12] suggested that the British experience with idiopathic infantile hypercalcemia in the 1950s had a profound effect on vitamin D supplementation not only during infancy but also during pregnancy. A common Pediatr Nephrol (2015) 30:145–152 149 assumption is that an increased intake of calcium and vitamin D is required during pregnancy and lactation to maintain maternal and child health. However, com- pared to other nutrients, such as folic acid, there has been limited clinical investigation of the adequate dietary vitamin D supplementation required during pregnancy. Over the last 10 years, there has been increasing interest in the health benefits of vitamin D to the general population. Numerous studies suggest that the importance of vitamin D goes beyond bone health and the associated vitamin D defi- ciency, with many acute and chronic illnesses, including au- toimmune diseases, cancer, type 2 diabetes mellitus, cardio- vascular disease and infectious diseases [13]. Fig. 1 Pedigree and sequencing results of four unrelated Israeli families with hypercalcemia. a Family 1—the proband is compound heterozygous for the p.Trp134Gly and p.Arg396Trp mutations, b family 2—the proband is compound heterozygous for the p.Trp134Gly and p.Glu315* mutations. Black arrow The proband patient, red arrows indicate mutations, green symbols the paternal allele, red symbols the maternal allele. c, d Pedigrees of families 3 and 4. Arrow The proband patient. Solid symbols, half-solid symbols The genotype of the homozygous patients and heterozygous patients, respectively, for the p.E143del mutation 150 Pediatr Nephrol (2015) 30:145–152 In November 2010, the United State Institute of Medicine (IOM) released its new guidelines for vitamin D and calcium, updating them from 1997. While the recommendations for calcium intake showed little change, the recommendations for vitamin D were increased (www.iom.edu/vitamind). The new guidelines with respect to pregnant women define the estimated average requirement and recommended dietary allowance to be 400 and 600 IU/day, respectively. The IOM also increased the tolerable upper intake limit to 4,000 IU/day. For infants from birth to 1 year, the recommendation of vitamin D doubled from 200 to 400 IU per day, with the upper intake limit set at 1,000 and 1,500 IU per day for infants aged 0–6 months and 6–12 months, respectably. Two recent large randomized controlled studies con- firmed the safety of vitamin D supplementation during pregnancy. In the first study 502 pregnant women were allocated to treatment with 400 IU, 2,000 IU or 4,000 IU of vitamin D supplement. The authors claimed that “Not a single adverse event was attributed to vitamin D supple- ment” [12]. In the second study, 257 pregnant women were given either 2,000 or 4,000 IU of vitamin D supplement. Neither dose group experienced any instance of hypercal- cemia or hypercalciuria [14]. Hypercalcemia during pregnancy or after delivery is uncommon and mostly related to primary hyperparathy- roidism [15]. About 200 cases of primary hyperparathy- roidism in pregnancy have been reported in the English literature [8]. In a single tertiary medical center in Israel, with a total of 10,000 deliveries per year, only five cases of hypercalcemia were identified during a 10- year period (1998–2007); all were due to hyperparathy- roidism [16]. Another rare cause for maternal hypercal- cemia is elevated PTH-related proteins either associated with malignant or benign tumors or produced during lactation in the puerperal period [17]. Finally, several cases of hypercalcemia in pregnancy, diagnosed as Milk-alkali syndrome, have been reported. The diagno- sis was based on a medical history of calcium carbon- ate, milk and/or vitamin D intake, depressed PTH levels and exclusion of other known cases for hypecalcemia. In some cases, vitamin D levels were suppressed as expected [18, 19], but in other reports 25-(OH)2D3 and/or 1,25-(OH)2D3levels were either inappropriately elevated or unmeasured [20, 21]. We describe the first case of maternal hypercalcemia caused by a CYP24A1 homozygous mutation. Our patient Vitamin D3 25-Hydroxylase CYP2R1 CYP27A1 25-OH-D 3 1α-Hydroxylase Active 1,25-(OH )2 D 3 CYP27B1 24-Hydroxylase Calcitroic acid Vitamin D supplements Diet Sunlight 24,25-(OH )2 D 3 FGF23/ Klotho + + Fig. 2 The vitamin D metabolism pathway (based on Schlingmann et.al [3]; used with permission). Red arrows The effects of cytochrome P450 24A1 (CYP24A1) loss of function on vitamin D metabolism Table 2 Clinical and genetic data for the four children in family 4 heterozygous for the p.E143del mutation Clinical and genetic data Child 1 Child 2 Child 3 Child 4 Age 8 months 8 months 5 years 9 years Gender Male Male Male Female Origin Moroccan/Ashkenazi Moroccan/Ashkenazi Moroccan/Ashkenazi Moroccan/Ashkenazi Serum calcium (mg/dl) [8.5–10.5] 10.7 10.6 10.6 10.5 Serum iPTH (pg/ml) [4.1–57.1] 8.8 10.2 6.5 7.4 Serum 25-(OH)D3 (ng/ml) [10–60] 33 30.2 29.8 28.8 Serum 1,25-(OH)2D3 (pg/ml) [20–67] 107.5 43.3 Not available Not available Urine calcium (mg/dl)/creatinine (mg/dl) [ had suffered from kidney stones for many years, but hyper- calcemia developed only in pregnancies. During the last preg- nancy, which was complicated by severe and symptomatic hypercalcemia, she was advised to consume a vitamin D- containing supplement and was also taking over-the-counter calcium carbonate for heart burn. Of note, in a previous report of adult kidney stone disease due to CYP24A1 mutation, we mentioned the proband’s sister who developed significant hypercalcemia during pregnancy, but she refused genetic analysis [6]. Increased intake of calcium and vitamin D is commonly recommended during pregnancy and lactation. However, sev- eral adaptive mechanisms are invoked during these time- periods to provide the required needs of the fetus. Pregnancy is characterized by a doubling of intestinal calcium absorption, high calcitriol levels, increased PTH-related protein (PTHrP), depressed PTH and hypercalciuria. During lactation there is a further increase in PTHrP levels, and although intestinal cal- cium absorption returns to normal, bone resorption occurs [8]. In pregnant women with the CYP24A1 mutation, these mech- anisms, together with the recommended intake of calcium and vitamin D, may lead to severe hypercalcemia (Fig. 2). It is quite possible that previous “Milk-alkali” cases were actually caused by undiagnosed CYP24A1 mutations. The correct diagnosis of both infantile and maternal hyper- calcemia is crucial for appropriate treatment and diet modifi- cation. Diagnosed infants should be fed with a formula that does not contain vitamin D and calcium. All affected individ- uals should avoid sunlight exposure, as well as vitamin D and calcium supplementation. The clinical findings of hypercalce- mia, hypercalciuria, nephrocalcinosis or a history of kidney stones disease should indicate the need to investigate serum PTH and vitamin D levels. The combination of suppressed PTH with normal–high 25-(OH)D3 and 1,25-(OH)2D3 sug- gest a vitamin D-mediated disease. 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Picolos MK, Sims CR, Mastrobattista JM, Carroll MA, Lavis VR (2004) Milk-alkali syndrome in pregnancy. Obstet Gynecol 104: 1201–1204 20. Gordon MV, McMahon LP, Hamblin PS (2005) Life-threatening milk-alkali syndrome resulting from antacid ingestion during preg- nancy. Med J Aust 182:350–351 21. Morton A (2002) Milk-alkali syndrome in pregnancy, associated with elevated levels of parathyroid hormone-related protein. Intern Med J 32:492–493 152 Pediatr Nephrol (2015) 30:145–152 Maternal and infantile hypercalcemia caused �by vitamin-D-hydroxylase mutations and vitamin D intake Abstract Abstract Abstract Abstract Abstract Introduction Patients and methods Patients Genetic studies Results Clinical findings Family 1 Family 2 Family 3 Family 4 Genetic findings Discussion References