Abstract
Background/Aims: Hypercalcemia can result in nephrocalcinosis/nephrolithiasis and may lead to renal failure. Idiopathic infantile hypercalcemia is caused by mutations of the CYP24A1 gene, which regulates vitamin D activity. Classically infants present with hypercalcemia. Recently, a number of individuals have been reported with late onset clinical manifestation or late diagnosis in adulthood. All these patients are believed to show hypercalciuria. Methods: We report a 24 year old patient of healthy consanguine parents. Genetic analysis was performed by Sanger sequencing of the CYP24A1 gene in the index patient and targeted exon 2 analysis of all other family members. Results: The patient was hospitalized with severe malaise during an acute EBV-infection. He showed hypercalcemia > 3mmol/l and acute, hypovolemic renal failure with profound nephrocalcinosis, but no hypercalciuria. Genetic workup revealed a homozygous loss-of-function mutation p.E143del in the CYP24A1 gene. His clinically asymptomatic brother showed nephrocalcinosis of lesser degree. Repeatedly, low parathyroid hormone levels were detected in both brothers. Conclusion: This family displays the highly variable phenotype of CYP24A1 biallelic mutation carriers. CYP24A1 associated disease is an important differential diagnosis for the workup and counseling of infants as well as adults with hypercalcemia since a proper genetic diagnosis may result in therapeutic consequences.
Introduction
Nephrocalcinosis (NC) is characterized by the deposition of calcium in the kidney parenchyma and tubules, which is usually situated in the medulla and visible on routine clinical imaging, such as ultrasound and computed tomography (CT), and if severe also on conventional abdominal X-ray. In 20% of premature infants, NC is a coincident finding upon abdominal ultrasound [1]. NC is also noted in about 1.7% of autopsies [2] and in approximately 50% of cases this finding can be attributed to malignancy. The etiology of the rest remains mostly unclear, yet clarification of its underlying cause is important, since progressive disease can lead to renal insufficiency and also bears the risk of extrarenal calcification. Histopathologically calcium oxalate (oxalosis; seen in primary and secondary hyperoxaluria) needs to be distinguished from calcium precipitates free of oxalate. Renal calcium deposition as NC and/or recurrent urolithiasis typically occurs in disorders of disturbed calcium or phosphate homeostasis with increased urinary excretion of calcium or phosphate.
Causes for hypercalcemia associated NC can be genetic or acquired . Important examples of the former are Williams-Beuren syndrome (WBS); elastin gene deletions, familial isolated hyperparathyroidism (FIHP), multiple endocrine neoplasia (MEN1) and mutations in the calcium sensing receptor (CASR). Examples of the latter are malignancy, sarcoidosis, drugs (e.g. thiazide diuretics) and vitamin D therapy. Differential diagnosis of disorders associated with hypercalcemia are listed in Table 1.
Clinical workup for NC routinely includes the acid-base status, vitamin D levels, parathyroid hormone (PTH), urinary excretion of calcium, phosphate, oxalate and citrate, as well as the measurement of serum calcium and phosphate. The interpretation of any of these parameters may be hampered by renal insufficiency, as well as dietary influences and a high diurnal variation of urinary excretion parameters. Therefore, careful clinical assessment of extrarenal manifestation, age at onset of symptoms and the family history may pose important clues towards clarification of the disease.
Among the growing list of possible causes of NC, mutations in the cytochrome P450 24-hydroxylase gene (CYP24A1, location 20q13.2, OMIM 143880) have been recently recognized as the major cause of autosomal recessive idiopathic infantile hypercalcemia (IHH) [3].
The CYP24A1 gene encodes the mitochondrial enzyme CYP24A1 which contains a cytochrome P450 domain responsible for inactivation of 1α,25(OH)2D3 and 25OHD3, resulting in1α,24,25(OH)3D3 and 24,25 (OH)2D3.The CYP24A1 enzyme is inhibited in a feedback mechanism by 1α,24,25(OH)3D3 and stimulated by 1α,25(OH)2D3 and PTH [3]. Biallelic mutations of CYP24A1 cause accumulation of the active form of vitamin D resulting in hypercalcemia (and PTH suppression), increased filtration and renal tubular reabsorption of calcium, and finally deposition of calcium precipitates in the urogenital tract.
In addition to IHH CYP24A1 mutations are increasingly identified in older patients with a subclinical course or late manifestation of disease which is not always found in relation to vitamin D intake (Table 2).
We report here two discordant brothers bearing a homozygous CYP24A1 in frame deletion, without apparent hypercalciuria, but with severe nephrocalcinosis, chronic renal failure and serious hypercalcemia in the index patient. Remarkably, despite documented vitamin D supplementation in infancy none of the brothers came to medical attention in childhood. This report emphasizes the importance of a precise molecular diagnosis with regard to proper treatment and counseling (family, life style and diet) in the era of widespread vitamin D supplementation.
Material and Methods
Patients
We studied two related patients from a consanguineous family of Eastern Mediterranean origin. All reported individuals gave informed, written consent to genetic analysis and scientific publication under strictly anonymous fashion.
Clinical, imaging and laboratory studies
All studies reported used the routine diagnostic facilities of two hospitals. Genetic analysis was performed by standard Sanger sequencing of the entire CYP24A1 gene in the index patient and targeted exon 2 analysis of all other family members.
Results
Patient's history
The 24-year-old index patient presented generally unwell with fever, odynophagia, diarrhea and volume depletion. He appeared agitated, yet was fully orientated and showed no neurological abnormalities. The preclinical medical history was unremarkable, yet two months before admission the patient presented at his general practitioner with mild arterial hypertension, microscopic hematuria and mild proteinuria on dipstick testing, as well as sterile leucocyturia.
Clinical findings and laboratory results
The patient showed renal impairment with a serum creatinine of 2.34 mg/dl and the serum calcium was significantly elevated at 2.92 mmol/l initially, increasing to 3.1 mmol/l on follow up. The abdominal ultrasound showed pronounced signal intense kidneys, most strongly at the renal medulla (Figure 1). The decision was made to admit him for further investigations, because of renal failure of unknown cause with hypercalcemia together with signs of infection.
Pedigree and clinical key findings. The index patient (arrow) is homozygous for the CYP24A1 mutation, as is his 28 year old brother (p.E143del/p.E143del). The parents are heterozygous (WT/p. E143del) for the CYP24A1 mutation and the third brother is not affected, displaying the wild-type allele homozygously (WT/WT). Both affected brothers show suppressed PTH levels (normal value 15-65 pg/ml), but hypercalcemia is an inconsistent finding. The mildly affected brother shows moderate medullary enhancement on ultrasound, whereas the index patient has profound medullary calcifications.
Pedigree and clinical key findings. The index patient (arrow) is homozygous for the CYP24A1 mutation, as is his 28 year old brother (p.E143del/p.E143del). The parents are heterozygous (WT/p. E143del) for the CYP24A1 mutation and the third brother is not affected, displaying the wild-type allele homozygously (WT/WT). Both affected brothers show suppressed PTH levels (normal value 15-65 pg/ml), but hypercalcemia is an inconsistent finding. The mildly affected brother shows moderate medullary enhancement on ultrasound, whereas the index patient has profound medullary calcifications.
The extended laboratory workup showed no abnormal findings for c and pANCA, ANA, dsDNA antibodies, angiotensin converting enzyme, Quantiferon test. Urine sediment repeatedly showed abacterial leucocyturia without acanthocytes and any crystals. The serology for Epstein Barr virus was suggestive of reactivated infectious mononucleosis, as suspected cause for the recent infection.
PTH was suppressed and 1α,25(OH)2D3 was within normal limits (42 ng/l). The patient displayed normal calciuria, and no abnormal excretion of oxalate or citrate (for detailed laboratory parameters see Table 3). He negated the intake of vitamin D and / or calcium containing dietary supplements.
Abdominal x-ray and CT displayed symmetric calcification of the medullary pyramids of both kidneys, typical for Grade III nephrocalcinosis (Figure 2). No solitary kidney stones were found.
Imaging studies displaying the index patient's nephrocalcinosis. (A) Distinct medullary calcification in conventional abdominal x-ray. (B) Abdominal computed tomography (CT) without contrast medium showing the calcified medullary pyramids.
Imaging studies displaying the index patient's nephrocalcinosis. (A) Distinct medullary calcification in conventional abdominal x-ray. (B) Abdominal computed tomography (CT) without contrast medium showing the calcified medullary pyramids.
Under treatment with intravenous Pamidronate and loop diuretics with fluid load the patient's creatinine slightly improved and the serum calcium moderately decreased. Thus, the patient was dismissed with a serum calcium of 2.5 mmol/l, continuing the next few months with Pamidronate and recommendations for dietary restriction of calcium and vitamin D. Two years after initial presentation abacterial leucocyturia and light proteinuria were persistent and serum creatinine increased to 1.7 mg/dl, which would be classified as chronic kidney disease (CKD) stage 3 (estimated GFR 49 ml/min). Serum calcium was mildly elevated at 2.7 mmol/l.
Family studies and genetic analysis
The consanguineous family history was suspicious for an autosomal recessive disease. The clinical picture with NC, hypercalcemia and suppressed PTH primarily led us to analyze for IHH. Direct Sanger sequencing of the CYP24A1 gene revealed the previously described [4] c.428_430delAAG (p. E143del) mutation in homozygous state in the index patient and his clinically asymptomatic older brother who demonstrated milder medullary NC (Grade I) and persistently low PTH levels in the absence of hypercalcemia and hypercalciuria (Table 3, Figure 1).
As expected both parents are heterozygous carriers of p.E143del, while the third brother being completely unremarkable with regard to clinical, biochemical and ultrasound findings is homozygous for the wild type allele (Figure 1).
Interestingly, all three brothers are reported to have received prophylactic oral and daily Vitamin D supplementation by the pediatrician during the first year of life.
Discussion
As described above, a reasonably large spectrum of diseases should be considered when encountering patients with hypercalcemia and nephrocalcinosis (NC). Important clues leading to the diagnosis in this case were consanguinity of the parents and consistent suppression of PTH in affected siblings. However, this interesting family shows clearly that the phenotype can be highly variable and thus the diagnosis missed when genetic testing is not performed. First, neither hypercalciuria, nor elevation of 1,25-dihydroxy vitamin D3 were seen in any sibling homozygous for the CYP24A1 mutation, where the former has also been reported in single cases by others ( [4,5]- see Table 2). Second, hypercalcemia is not a consistent finding, but may be influenced by alimentary factors and other stressors, such as infections and / or acute renal failure. Third, the two affected brothers show greatly differing clinical courses, where the older brother may not have been diagnosed during life-time, if family testing had not been performed. At least in this family, PTH appeared to be the most sensitive laboratory parameter and imaging with ultrasound and / or CT appeared helpful.
In view of the greatly variable phenotype of the disease, it is noteworthy that many patients may experience first clinical complications and diagnosis only in adulthood and not in infancy, as has been initially reported. About 30% of the cases published so far (Table 2) manifest or are diagnosed in adolescence or adulthood. Whether or not any given individual with biallelic inactivation of CYP24A1 encounters clinical consequences, may depend largely on dietary influences such as calcium and vitamin D intake, but also on other genetic variables, such as calcium receptor sensitivity. However, the type of mutation may also contribute to the variability of disease, in the sense of genotype-phenotype association. The mutation found in the current family p. E143del in exon 2 of the CYP24A1 gene affects the CYP450 domain and seems to alter the secondary structure of the substrate binding cavity by preventing a structural bond with the amino acid K283. The lost bondage destabilizes the B'/C loop and leads to a reorientation of the B'-helix (see supplemental figure of [3]) and a complete loss-of-function of the enzyme. A number of other mutations have been identified in other families, where most but not all are missense mutations (see Table 2). To what extent these mutations result in variation of enzyme activity and thereby contribute to disease severity is not known to date.
Interestingly, homozygous Cyp24a1 knockout mice that show elevated 1α, 25(OH)2D3 levels die before weaning [6]. Their homozygous littermates that survive weaning show even 3-fold reduced 1α25(OH)2D3 levels compared to wild type mice. Masuda et al. [7] suggest an unknown alternative pathway of Vitamin D metabolism or a reduced renal 1α,25(OH)2D3 production in these mice. Despite these uncertainties, knowledge of a risk genotype may be valuable in reducing life-time risks by relatively simple dietary restrictions, avoiding high amounts of calcium and Vitamin D. Since the content of one or the other can be surprisingly high in particular diets, we would emphasize the necessity of a professional dietary consultation.
Furthermore, in severely affected patients, treatment with the cytochrome 3A inhibitor ketoconazole may be beneficial, not only in reducing hypercalciuria and hypercalcemia, but also renal insufficiency [4,8]. However, frequent and significant liver toxicity may hamper long term use in these patients. Other treatment options may be bisphosphonates, as given to the index patient, or high volume intake with or without the use of loop diuretics. None of these strategies have been tested in a randomized fashion, but this may be worthwhile in the future.
Conclusion
Importantly, the disease termed idiopathic infantile hypercalcemia (IHH) may be much more frequent than thought or implicated by its name. Since a significant number of patients manifest late or are diagnosed in adulthood with CYP24A1 associated disease the term IHH should be seen as a misnomer. The allele frequency of inactivating CYP24A1 variants may be unexpectedly high [9]. Thus, this disease should also be suspected in respective non-consanguine families or sporadic cases, since compound heterozygous cases are described [10].
Furthermore, some heterozygous mutations may also result in a similar disease, following an autosomal dominant trait, however this mode of transmission has been an isolated finding so far [8]. Finally, the CYP24A1-associated disease may also result in kidney stones, which is considerably more frequent than IHH. For all these reasons it is pertinent to implement this disease in routine genetic consultations. Besides giving the patient a distinct diagnosis, this approach would improve our knowledge of the disease, the epidemiology and may help us ameliorate the clinical course of this potentially serious condition.
Disclosure Statement
The authors declare no conflict of interest.