25-Hydroxyvitamin D-24-hydroxylase (CYP24A1): Its important role in the degradation of vitamin D

Perceived knowledge among patients cared for by nephrologists about chronic kidney disease and end-stage renal disease therapies

A Decade After the KDOQI CKD Guidelines: Impact on the United States and Global Public Policy

The differential diagnosis of endogenous hypercalcemia typically involves an assessment of the hormones and paracrine factors, such as PTH, PTH-related protein, and 1,25-dihydroxyvitamin D, whose oversecretion can pathologically activate bone resorption and/or intestinal absorption of calcium to an extent that overrides the usual homeostatic mechanisms. With our focus thus on investigating hormone oversecretion, we endocrinologists typically give little thought to disorders of hormone catabolism that might also result in hypercalcemia. It is in this context that an elegant clinical study in this issue by Dauber et al. (1) is both thought-provoking and illuminating. Dauber et al. (1) define a genetic defect and characterize a critical pathophysiology in a patient with severe infantile hypercalcemia. The patient, a product of a consanguineous marriage in which the parents were fifth-degree relatives, presented at 10 months of age with failure to thrive, increased urination, and refusal of solid foods. At presentation, the patient was markedly hypercalcemic, with a serum calcium of 15.3 mg/dl (3.8 mmol/liter), and inpatient evaluation revealed suppressed PTH, undetectable PTH-related protein, normal levels of 25-hydroxyvitamin D and 1,25-dihydroxyvitamin D, and normal thyroid and renal function. The infant was found to have severe hypercalciuria and bilateral nephrocalcinosis but no evidence of rickets or bone demineralization on imaging studies. Furthermore, no chromosome 7 deletion was found on genetic analysis to support a diagnosis of Williams syndrome, a rare cause of infantile hypercalcemia. With suppression of PTH, the investigators suspected an absorptive pathophysiology for the hypercalcemia, and indeed metabolic studies employing oral and parenteral administration of stable calcium isotopes along with a defined calcium diet demonstrated a marked intestinal hyperabsorption of calcium in this infant. Dauber et al. (1) then pursued an unbiased genome-wide investigation for the cause of the patient's hypercalcemia which, given the history of parental consanguinity, was suspected to be an autosomal recessive inherited genetic disorder. Recent advances in exome sequencing, in which the subset of the human genome that carries the protein-coding information is targeted for sequencing, make it an efficient and cost-effective way to elucidate the genetic basis of metabolic and other Mendelian disorders (2, 3). Whole exome sequencing of the patient's DNA revealed 1277 single nucleotide variants and 367 indels (alterations in DNA sequence arising from insertion or deletion of nucleotides) not included in the latest compendium of human nucleotide polymorphisms (1). Of these novel variants, seven single nucleotide changes encoding missense alterations at the protein level and seven indels mapped to one of three large blocks of genetic homozygosity on chromosomes 7, 16, and 20. Within the patient's DNA sequence on chromosome 20, a homozygous 3-bp in-frame deletion in the CYP24A1 gene was discovered. Both parents were proven to be heterozygous mutation carriers. This deletion in CYP24A1 is predicted to excise an evolutionarily highly conserved glutamate residue at position 143 in the vitamin D 24-hydroxylase enzyme (E143del). This finding captured the attention of Dauber et al. (1) because biallelic germline inactivation of the orthologous mouse gene, Cyp24a1, produces a phenotype of hypercalcemia and nephrocalcinosis reminiscent of the findings in the human infant (4–6). That vitamin D 24-hydroxylase is critically responsible for oxidation and degradation of bioactive vitamin D is supported by studies of such Cyp24a1 null mice demonstrating markedly impaired catabolism and clearance of 25-hydroxyvitamin D and 1,25-dihydroxyvitamin D (5, 6). The present study by Dauber et al. (1) complements and extends other recent clinical reports linking germline CYP24A1 loss of function mutation with clinically relevant hypercalcemia (7, 8). Using a candidate gene approach, Schlingmann et al. (7) demonstrated germline biallelic inactivation of CYP24A1 in four infant probands with idiopathic infantile hypercalcemia, medullary nephrocalcinosis, and suspected autosomal recessive inheritance. One of the probands described by Schlingmann et al. (7) was the product of a consanguineous marriage and was homozygous for a frameshift mutation in CYP24A1, whereas the remaining three were compound heterozygotes for mutations in CYP24A1, including two who carried the E143del mutant allele (7). The case of a patient who presented in young adulthood with nephrolithiasis and mild hypercalcemia, subsequently became asymptomatic, then re-presented 20 yr later with unexplained non-parathyroid-mediated hypercalcemia was described by Streeten et al. (8). The patient was found at age 47 to be homozygous for the CYP24A1 E143del mutant allele (8). In the present case described by Dauber et al. (1), evidence for functional deficiency of vitamin D 24-hydroxylase activity was strongly supported by documentation of undetectable serum levels of 24,25-dihydroxyvitamin D, a major vitamin D metabolite whose levels in control subjects were proportionate to vitamin D stores as reflected in 25-hydroxyvitamin D levels (1). The adult CYP24A1 E143del homozygote described by Streeten et al. (8) also had low serum 24,25-dihydroxyvitamin D levels. The study by Schlingmann et al. (7) did not report serum 24,25-dihydroxyvitamin D levels in their patient cohort, but did analyze the function of CYP24A1 by transfection and documented a striking lack of 24-hydroxylated vitamin D metabolites when patient-derived CYP24A1 mutants were tested in vitro with 1,25-dihydroxyvitamin D as substrate. The determination of the crystal structure of rat Cyp24a1 has provided a structural template to allow molecular modeling studies that probe the effect of patient-derived mutations on the function of human CYP24A1 by computer simulation (9). On the basis of such molecular-modeling simulation, it was found that of four CYP24A1 missense mutations reported by Schlingmann et al. (7), only one was predicted to weaken substrate-enzyme binding, whereas three appeared to disrupt interactions between the CYP24A1 enzyme and its heme prosthetic moiety (10). Despite the convincing correlation of homozygous CYP24A1 mutation, hypercalcemia, and intestinal hyperabsorption of calcium in the present case, a number of questions remain when considering the findings of Dauber et al. (1) in the context of other recent reports linking CYP24A1 loss of function to hypercalcemia. A major question is the prevalence of CYP24A1 mutation among patients with apparently idiopathic infantile hypercalcemia. Although Dauber et al. (1) found no germline CYP24A1 missense mutation or deletions among 27 patients with infantile hypercalcemia referred to a pediatric calcium disorder specialty clinic, Schlingmann et al. (7) identified biallelic germline inactivating mutations in four of four kindreds with idiopathic infantile hypercalcemia. Whether this difference reflects occult clinical heterogeneity or ascertainment bias in the population of patients with apparently idiopathic infantile hypercalcemia under study, or whether some CYP24A1 mutation-negative patients nevertheless harbor defects in other molecular components that diminish vitamin D catabolism or enhance vitamin D activation (for example, activating mutations in the CYP27B1 25-hydroxyvitamin D 1-α-hydroxylase) remains to be determined. Another interesting question posed by these cases concerns the inconsistency of serum 1,25-dihydroxyvitamin D elevation in patients with biallelic CYP24A1 inactivation despite documented hypercalcemia. For example, the patient described by Dauber et al. (1) often had normal 1,25-dihydroxyvitamin D levels when tested over time, even while hypercalcemic. Three of the four index cases presented by Schlingmann et al. (7) also had normal circulating 1,25-dihydroxyvitamin D levels despite marked hypercalcemia. The patient described by Streeten et al. (8), in contrast, had clearly elevated 25-hydroxyvitamin D and 1,25-dihydroxyvitamin D levels accompanying his hypercalcemia. A possibly related issue is the variability of hypercalcemia over time in patients with biallelic CYP24A1 inactivation, such as in the present case in which the patient was able to resume a normal calcium diet without recurrence of hypercalcemia after 3 yr of age (1). The observation that Cyp24a1 null mice exhibit an apparently compensatory down-regulation of renal Cyp27b1, the 25-hydroxyvitamin D 1-α-hydroxylase, and have lower circulating 1,25-dihydroxyvitamin D levels than control mice may be relevant to humans in this regard (5, 6). The possibility that serum levels of 1,25-dihydroxyvitamin D may not always reflect hormone activity in target tissues, such as intestine, bone, or parathyroid cells, for example, because of local variability in vitamin D homeostasis must also be considered (1, 8). The present work by Dauber et al. (1) and the related clinical studies cited here, as well as findings from the Cyp24a1-null mouse model (5, 6), underscore the importance of CYP24A1 and vitamin D catabolism in calcium homeostasis as well as the potential pathophysiological importance of this pathway in hypercalcemia. It remains to be seen whether nonsynonymous “benign polymorphisms” in the coding region or pathological variants in the intronic or promoter regions of CYP24A1 might also affect the catabolism of vitamin D and have the potential to induce hypercalcemia, either at baseline or upon bolus loading with vitamin D2 or D3. In the investigation of hypercalcemia, particularly in “idiopathic” cases with suppressed PTH, we endocrinologists clearly have a new mechanism to ponder and should consider adding a screening 24,25-dihydroxyvitamin D determination to our workup as clinically warranted.

Idiopathic infantile hypercalcaemia (IIH) is an autosomal recessively inherited disease, presented in the first year of life with hypercalcaemia, precipitated by normal amounts of vitamin D supplementation. Recently loss-of-function mutations in the CYP24A1 gene, which encodes the vitamin D-metabolizing enzyme 24-hydroxylase, have been found in these patients. We describe a young man homozygous for a novel missense mutation (c.628T>C) of the CYP24A1 gene. He had suffered from severe hypercalcaemia in early childhood. At age 29 he presented with medullary nephrocalcinosis, chronic kidney disease (CKD) stage 2, microalbuminuria, mild hypertension and nephrogenic diabetes insipidus. He had mild hypercalcaemia and moderate hypercalciuria. As a novel finding, fibroblast growth factor 23 (FGF23) was elevated.

[This corrects the article on p. 211 in vol. 6, PMID: 24175086.].

A Decade After the KDOQI CKD Guidelines: Impact on Primary Care

Background and Aims The CYP24A1 gene plays a key role in the metabolism of calcitriol and calcium homeostasis. Variants in this gene have been implicated in conditions such as idiopathic infantile hypercalcemia (IIH) and recurrent nephrolithiasis. While biallelic mutations are commonly associated with IIH, monoallelic variants can also lead to a wide spectrum of clinical presentations, including nephrolithiasis, osteoporosis, renal cysts, and mild hypercalcemia. We report two families with kidney stones with mild metabolic activity and renal cysts, carrying novel heterozygous missense variants in the CYP24A1 gene, expanding the phenotypic spectrum of this genetic disorder. Method Genetic analysis was performed using exome sequencing, and novel variants were assessed for pathogenicity according to ACMG criteria. Results Family 1 (Fig. 1a): We evaluated a 58-year-old woman (patient 1a) with hypertension, normal renal function, and a history of recurrent stone disease since the age of 40, producing approximately one stone per year. Stone composition was: 30% calcium oxalate, 60% struvite, and 10% apatite. She also has small bilateral renal cysts and liver cysts. The patient has two sisters: one is 53 years old (patient 1b) with mild renal insufficiency, bilateral renal cysts, and one liver cyst. Although she has no significant history of stone disease, urine analysis shows crystalluria. The other sister, aged 47, has had hypertension since the age of 33, along with type 2 diabetes and mild renal insufficiency. Their deceased father had a history of recurrent bilateral nephrolithiasis that began at age 20 and chronic kidney failure, which was diagnosed at age 80, ultimately requiring hemodialysis. He also had bilateral renal cysts. Given the family history of chronic kidney disease, stone disease, and renal cysts, the patient and her sisters underwent genetic analysis via exome sequencing. The exome sequencing has identified the heterozygous missense variant c.1405 G>C in CYP24A1, classified by ACMG guidelines as probably pathogenic. The variant is also identified in the sister with renal cysts, crystalluria and mild renal insufficiency. No variants associated with autosomal dominant polycystic kidney disease are identified by exome sequencing or PKD1/2 sanger sequencing. Family 2 (Fig. 1b): The proband, a 23-year-old male, presented to our clinic with kidney stones, a paternal family history of nephrolithiasis and kidney cysts (father and grandfather), macrohematuria and renal insufficiency. His father exhibits a mild form of nephrolithiasis and he has small bilateral parapelvic renal cysts. His history of kidney stones was relatively mild, consisting of a single episode of renal colic and a total of two stones. Given his clinical presentation and the presence of proteinuria, a kidney biopsy was performed, which identifies a diagnosis of IgA nephropathy. However, due to the early onset of nephrolithiasis and the family history of recurrent kidney stones, genetic testing through exome sequencing was undertaken. This analysis identified the heterozygous missense variant c.1390 G>T in CYP24A1, inherited by the father and classified by ACMG guidelines as probably pathogenic. Conclusion We reported two families affected by a mild form of nephrolithiasis and renal cysts in which we identified two novel missense variants in the CYP24A1 gene that have not yet been described in the literature. These two novel heterozygous CYP24A1 variants expand the understanding of the gene's contribution to kidney stone disease and renal cysts. Although monoallelic variants are typically associated with milder kidney stone phenotypes, a high incidence of chronic kidney disease has been reported in these families. The findings highlight the clinical variability and importance of genetic screening in families with nephrolithiasis. Future research should focus on the functional implications of these variants and their interaction with other risk factors.

Race, Biomarkers, and Cardiovascular Disease in Patients With Chronic Kidney Disease.

Monogenic causes of chronic kidney disease (CKD) are more prevalent in adults than previously thought, as causative gene variants are found in almost 10% of unselected patients with CKD. Even so, genetic testing in patients with adult-onset CKD is uncommon in clinical practice and the optimal criteria for patient selection remain unclear. A family history of kidney disease emerges as one marker associated with a high diagnostic yield of genetic testing. We present 3 cases of adult-onset CKD with underlying monogenic causes exemplifying different modes of inheritance. Case 1 is a 60-year-old male with slowly progressive CKD initially ascribed to hypertension and diabetes despite a family history with several affected first-degree relatives. A pathogenic MUC1 variant was found, and thus we identified the first Danish family of MUC1-associated autosomal dominant tubulointerstitial kidney disease. Case 2 is a 40-year-old female with nephrocalcinosis, nephrolithiasis, and unexplainable hypercalcemia consistent with vitamin D intoxication. The family history indicated autosomal recessive inheritance, and genetic testing revealed 2 pathogenic CYP24A1 variants in compound heterozygous form associated with idiopathic infantile hypercalcemia. Case 3 is a 50-year-old male with microscopic hematuria, proteinuria, and hearing loss. Electron microscopy of renal biopsy showed thin basal membrane syndrome, and the family history indicated X-linked inheritance. A novel missense variant in COL4A5 was identified, suggesting an atypical late-onset form of X-linked Alport syndrome. This case series illustrates the heterogeneous presentations of monogenic kidney disease in adults and emphasizes the importance of family history for initiating genetic testing to identify underlying monogenic causation. Moreover, we discuss the potential impact of genetic diagnostics on patient management and genetic family counseling.

Too Many for Too Few: Finding Appropriate Nephrology Referrals for Patients With CKD That Optimize Outcomes

Management of Psychiatric Disorders in Patients with Chronic Kidney Diseases.

CYP24A1 mutations are now accepted as a cause of idiopathic infantile hypercalcemia (IIH). A rapid liquid-chromatography tandem mass spectrometry (LC-MS/MS)-based blood test enabling measurement of the 25-OH-D3 :24,25-(OH)2 D3 ratio (R) can identify IIH patients on the basis of reduced C24-hydroxylation of 25-OH-D3 by CYP24A1 in vivo. Although values of this ratio are significantly elevated in IIH, somewhat surprisingly, serum 24,25-(OH)2 D3 remains detectable. The current study explores possible explanations for this including: residual CYP24A1 enzyme activity in individuals with certain CYP24A1 genotypes, expression of alternative C24-hydroxylases, and the possibility of isobaric contamination of the 24,25-(OH)2 D3 peak on LC-MS/MS. We employed an extended 20-min run time on LC-MS/MS to study serum vitamin D metabolites in patients with IIH due to mutations of CYP24A1 or SLC34A1; in unaffected heterozygotes and dialysis patients; in patients with vitamin D deficiency; as well as in normal subjects exhibiting a broad range of 25-OH-D levels. We identified 25,26-(OH)2 D3 as a contaminant of the 24,25-(OH)2 D3 peak. In normals, the concentration of 24,25-(OH)2 D3 greatly exceeds 25,26-(OH)2 D3 ; however, 25,26-(OH)2 D3 becomes more significant in IIH with CYP24A1 mutations and in dialysis patients, where 24,25-(OH)2 D3 levels are low when CYP24A1 function is compromised. Mean R in 30 IIH-CYP24A1 patients was 700 (range, 166 to 2168; cutoff = 140) as compared with 31 in 163 controls. Furthermore, patients possessing CYP24A1 L409S alleles exhibited higher 24,25-(OH)2 D3 levels and lower R (mean R = 268; n = 8) than patients with other mutations. We conclude that a chromatographic approach which resolves 24,25-(OH)2 D3 from 25,26-(OH)2 D3 produces a more accurate R that can be used to differentiate pathological states where CYP24A1 activity is altered. The origin of the residual serum 24,25-(OH)2 D3 in IIH patients appears to be multifactorial. © 2017 American Society for Bone and Mineral Research.

Definition and Classification of CKD: The Debate Should Be About Patient Prognosis—A Position Statement From KDOQI and KDIGO

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

Idiopathic infantile hypercalcemia (IIH) is characterized by severe hypercalcemia with failure to thrive, vomiting, dehydration, and nephrocalcinosis. Recently, mutations in the vitamin D catabolizing enzyme 25-hydroxyvitamin D3-24-hydroxylase (CYP24A1) were described that lead to increased sensitivity to vitamin D due to accumulation of the active metabolite 1,25-(OH)2D3. In a subgroup of patients who presented in early infancy with renal phosphate wasting and symptomatic hypercalcemia, mutations in CYP24A1 were excluded. Four patients from families with parental consanguinity were subjected to homozygosity mapping that identified a second IIH gene locus on chromosome 5q35 with a maximum logarithm of odds (LOD) score of 6.79. The sequence analysis of the most promising candidate gene, SLC34A1 encoding renal sodium-phosphate cotransporter 2A (NaPi-IIa), revealed autosomal-recessive mutations in the four index cases and in 12 patients with sporadic IIH. Functional studies of mutant NaPi-IIa in Xenopus oocytes and opossum kidney (OK) cells demonstrated disturbed trafficking to the plasma membrane and loss of phosphate transport activity. Analysis of calcium and phosphate metabolism in Slc34a1-knockout mice highlighted the effect of phosphate depletion and fibroblast growth factor-23 suppression on the development of the IIH phenotype. The human and mice data together demonstrate that primary renal phosphate wasting caused by defective NaPi-IIa function induces inappropriate production of 1,25-(OH)2D3 with subsequent symptomatic hypercalcemia. Clinical and laboratory findings persist despite cessation of vitamin D prophylaxis but rapidly respond to phosphate supplementation. Therefore, early differentiation between SLC34A1 (NaPi-IIa) and CYP24A1 (24-hydroxylase) defects appears critical for targeted therapy in patients with IIH.