Genetic Defects In Vitamin D Metabolism and Action

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Chapter 11

Genetic Defects In Vitamin D Metabolism and Action

Vitamin D is a key regulator of mineral homeostasis1,2 and bone development,3 and perturbation of the vitamin D endocrine system leads to rickets and/or osteomalacia (see Chapter 15). In order to gain a complete perspective of the clinical manifestations of genetic anomalies involving vitamin D endocrine function, this chapter will first present a short overview of the salient aspects of the vitamin D metabolic pathway. (An in-depth discussion of vitamin D metabolism and bone development and remodeling can be found in Chapters 3 and 4). The concepts presented here will lay the groundwork for the discussion of the clinical, pathophysiologic, and molecular aspects of hereditary rickets involving the vitamin D endocrine system.

Overview of Vitamin D Metabolism

Following exposure to sunlight, both plants and animals are able to synthesize vitamin D. Vitamin D2 is generated in yeast and plants; vitamin D3 is produced in fish and mammals. The slight differences in the chemical structure of the two compounds do not affect function or metabolism. The generic term of vitamin D (without subscript) will be used hereafter.

In humans, a sizeable proportion of vitamin D requirements can be produced endogenously in the skin upon exposure to ultraviolet (UV) light (sunlight). It has been shown, however, that at latitudes where vitamin D synthesis is reduced or absent during winter months, there is a seasonal variation in the photosynthesis of vitamin D.4 People who receive an ample supply of sunlight during the rest of the year are not at risk of developing vitamin D deficiency, because excess cutaneously produced vitamin D is stored in fat and muscle and released at times of need.5 Dietary sources such as fish, plants, and grains can help meet vitamin D requirements. In the absence of any exposure to sunlight, however (such as in elderly people), a daily multivitamin that contains 400 IU of vitamin D is indicated.6,7

Ultraviolet B (UVB) photons penetrate the epidermis and photolize 7-dehydrocholesterol into previtamin D, which rapidly becomes a more thermodynamically stable molecule, vitamin D. Vitamin D then exits the keratinocyte cells and enters the dermal capillary bed, where it becomes bound to the vitamin D binding protein, DBP. Once associated with DBP in the circulation, vitamin D is transported to the liver, where cytochrome P450 25-hydroxylase enzymes (CYP27A1 and/or CYP2R1) add a hydroxyl group on carbon 25 to produce 25-hydroxyvitamin D [25(OH)D]. Early studies using perfused rat liver revealed kinetics of vitamin D metabolism that supported two 25-hydroxylase activities: a microsomal high-affinity, low-capacity enzyme and a mitochondrial low-affinity, high-capacity form.8 The mitochondrial enzyme is the bifunctional CYP27A1 sterol, 27-hydroxylase, that derives its name from its ability to both 27-hydroxylate the side chains of cholesterol-derived intermediates involved in bile acid biosynthesis and 25-hydroxylate vitamin D.9 The microsomal, high-affinity vitamin D 25-hydroxylase was recently identified as CYP2R1, using an elegant expression-based screening strategy.10 Based on specific activity determination, it is estimated that the microsomal enzyme is responsible for about 30% of the total 25-hydroxylation activity, while the mitochondrial enzyme is responsible for the remaining 70%.11

The 25(OH)D metabolite also circulates in the bloodstream bound to DBP. It is an abundant but relatively inactive vitamin D metabolite. Its circulating concentration provides the most readily available evaluation of the vitamin D status in a given individual. 25(OH)D must be further hydroxylated at a different site in the convoluted and straight portions of the proximal kidney tubule to gain hormonal bioactivity. Hydroxylation at position 1α by the mitochondrial cytochrome P450 enzyme 25-hydroxyvitamin D 1α-hydroxylase (CYP27B1) converts 25(OH)D to 1α,25-dihydroxyvitamin D [1,25(OH)2D], the active, hormonal form of vitamin D that plays an essential role in mineral homeostasis, bone growth, and cellular differentiation.1214

Upon reaching a target tissue, 1,25(OH)2D binds a specific receptor (vitamin D receptor [VDR]) that is a member of the nuclear hormone receptor superfamily.15 The VDR is considered a class II nuclear hormone receptor because it needs to form a heterodimer with the retinoid X receptor (RXR) to bind specific DNA sequence elements with high affinity. These target sequences are termed vitamin D response elements (VDRE), and the best characterized of these binding sites consist of two tandemly repeated hexanucleotide sequences separated by 3 base pairs (bp).15 Transcriptional coactivators and components of the basal transcriptional machinery interact with the liganded, DNA-bound VDR-RXR heterodimer to activate the transcription of vitamin D target genes responsible for carrying out the physiologic actions of 1,25(OH)2D.13,15 Among several target genes, the 1,25(OH)2D hormone induces in target cells the expression of the gene encoding the key effector of its catabolic breakdown: 25-hydroxyvitamin D 24-hydroxylase (CYP24A1).16,17 This insures attenuation of the 1,25(OH)2D biological signal inside target cells and helps regulate vitamin D homeostasis.

Rickets and Osteomalacia

The term rickets is often used to describe all of the skeletal abnormalities associated with defective mineralization in the growing skeleton, but it is more precise to restrict the term to changes in the growth plate and adjacent metaphysis. When mineralization is impaired, the accumulation of unmineralized osteoid at sites other than the growing metaphysis should be referred to as osteomalacia, not as rickets. Thus, defective mineralization can lead to both rickets and osteomalacia in the growing skeleton but only to osteomalacia in the mature skeleton.

Rickets is characterized by the inadequate calcification of the growth plate and adjacent metaphysis. The impaired mineralization of the growth plate cartilage in the zone of provisional calcification prevents this zone from being resorbed. As the cartilage continues to be formed but not resorbed, the growth plate begins to widen. Simultaneously, the trabecular bone directly underneath the cartilage fails to mineralize properly, and vascularization of this tissue becomes aberrant. These defects are accompanied by similar abnormalities in cortical bone, leading to the full spectrum of skeletal symptoms associated with the pathology (see following).

Vitamin D–Deficiency Rickets

It is evident from the brief overview presented earlier that vitamin D metabolism can be affected at several levels: inadequate exposure to sunlight; inadequate dietary intake; malabsorption of dietary vitamin D; impaired hepatic 25-hydroxylation; defects in renal 1α-hydroxylation; or defects in receptor function (Fig. 11-1). Reviews on the nonhereditary disorders of the vitamin D endocrine system have been published previously.18 Only defects in renal 1α-hydroxylation or receptor activity have been associated with several specific mutations in genes involved in regulating vitamin D metabolism and action; these will be discussed in this chapter. There is a single report19 of mutations in both alleles of CYP2R1, the microsomal hepatic 25-hydroxylase,10 in a patient with low serum calcium concentrations, low circulating levels of 25(OH)D, and rickets.20 While obviously rare, this finding confirms CYP2R1 as a physiologically relevant vitamin D 25-hydroxylase and demonstrates that selective 25-hydroxylase deficiency can also cause a hereditary defect in vitamin D metabolism.

The prime metabolic consequence of vitamin D deficiency is reduced net intestinal absorption of calcium.1,2 Calcium malabsorption leads to a fall in plasma calcium, secondary hyperparathyroidism, reduced renal tubular reabsorption of phosphate, hypophosphatemia, and thus a reduction in the calcium X phosphate product. Eventually, deposition of mineral in osteoid is impaired because the supply of the relevant ions is reduced. The impaired mineralization triggers the development of the rachitic and/or osteomalacic phenotype.

Clinical Features of Pseudo–Vitamin D–Deficiency Rickets (Vitamin D–Dependent Rickets Type I)

The clinical symptoms of pseudo–vitamin D–deficiency rickets (PDDR), also referred to as vitamin D–dependent rickets type I, are similar to those of common vitamin D–deficiency rickets, including failure to thrive, hypotonia, and growth retardation. Affected babies lie supine because of severe muscle weakness and bone pain. At this age, gross skeletal deformities are rare; however, if diagnosis and treatment are delayed, severe deformities of the spine and long bones occur, together with generalized muscle weakness simulating myopathy. Motor problems translate into regression in head control and ability to stand. In some patients, the initial event is generalized convulsions, tremulations and Bravais-Jacksonian fits, or tetany. Pathologic fractures may occur (Fig. 11-2). The onset in most cases occurs early during the third trimester of life; the patients look healthy at birth.

Physical examination reveals a small, hypotonic child with wide anterior fontanel, frontal bossing, and frequent craniotabes (easy depression of the softened parieto-occipital region). Tooth eruption is delayed, and erupted teeth show evidence of enamel hypoplasia. A rachitic rosary is either visible or palpable. In limbs, widening of the metaphyseal areas is evidenced by enlargement of wrists and ankles, and there is a variable degree of deformity (bowing) of long-bone diaphyses. Deformities of the thorax may interfere with ventilation and predispose to pulmonary infection; infant death by pulmonary infections was not infrequent in the past, when the diagnosis was either missed (confused with a neurologic or respiratory condition) or made too late. The Chvostek sign (twitching of the upper lip upon light finger tapping of the facial nerve) reflects nerve irritability, a consequence of a rapid fall in serum calcium.

X-ray features include diffuse osteopenia (mild to severe hypomineralization of the skeleton) and classic rachitic metaphyseal changes: fraying, cupping, widening, and fuzziness of the zone of provisional calcification immediately under the growth plate (see Fig. 11-2). These changes are seen better and detected earlier in the most active growth plates—namely, the distal ulna and femur and the proximal and distal tibia. Changes in the diaphyses may not be evident when metaphyseal changes are first detected. However, they will appear a few weeks later as rarefaction, coarse trabeculation, cortical thinning, and subperiosteal erosion. Looser-Milkman’s pseudofractures and curvature of the shafts of long bones may be observed, especially in children older than 1 to 2 years.

Hypocalcemia is the main biochemical feature in PDDR. A rapid decrease in serum calcium concentration may give rise to tetany and convulsions, which may occur prior to any radiologic evidence of rickets. Prolonged hypocalcemia triggers secondary hyperparathyroidism and hyperaminoaciduria.21 Urinary calcium excretion is very low, whereas fecal calcium is high as a consequence of impaired intestinal calcium absorption. Elevated urinary cyclic adenosine monophosphate (cAMP) is not a consistent finding, and normal values have been measured in PDDR patients, despite high circulating parathyroid hormone (PTH) levels.22

Serum phosphate concentrations may be normal or low. When reduced, the hypophosphatemia is usually of a lesser degree than that measured in X-linked hypophosphatemic rickets.23 It results from the combination of impaired intestinal absorption and increased urinary loss induced by the secondary hyperparathyroidism. Serum alkaline phosphatase activity is consistently elevated, and its increase precedes the appearance of clinical symptoms.

Patients with PDDR have normal serum levels of 25(OH)D after exposure to sunlight or oral intake of small doses of vitamin D; the concentrations increase if higher doses are given.22 Circulating levels of 24,25-dihydroxyvitamin D [24,25(OH)2D] are normal and correlate with those of 25(OH)D.24 Serum levels of 1,25(OH)2D are low in untreated patients.22,25 This is evident immediately after birth, months before any clinical evidence of rickets appears. Even when patients are treated with high doses of vitamin D, causing major increases in circulating levels of 25(OH)D, the blood concentration of 1,25(OH)2D does not reach the normal range. These characteristic features of serum vitamin D metabolites have provided key insight into the pathogenesis of PDDR.

Clinical Features of Hereditary Vitamin D–Resistant Rickets (Vitamin D–Dependent Rickets Type II)

Many of the clinical findings in patients with hereditary vitamin D–resistant rickets (HVDRR), also termed vitamin D–dependent rickets type II, are identical to those described for PDDR, including bone pain, muscle weakness, hypotonia, and occasional convulsions.26 Children are often growth retarded, and hypoplasia of the teeth is observed. The radiologic features of rickets are present. A major difference is that many children with HVDRR have sparse body hair, and some have total scalp and body alopecia, sometimes even including eyebrows and eyelashes. Hair loss may be evident at birth or occur during the first months of life. Patients with alopecia generally have more severe resistance to vitamin D. In families with a prior history of the disease, the absence of scalp hair in newborns can provide initial diagnostic clues for HVDRR. A defect in epithelial-mesenchymal communication that is required for normal hair cycling has been shown to be the cause of the alopecia in an animal model of HVDRR.27

Serum biochemistry includes low concentrations of calcium and phosphate and elevated alkaline phosphatase activity. Secondary hyperparathyroidism with elevated circulating PTH is measurable. The key difference concerns circulating levels of vitamin D metabolites. The 25(OH)D values are normal, and in the cases in which it has been measured, 24,25(OH)2D levels have been low. Importantly, serum levels of 1,25(OH)2D are elevated. This clinical feature clearly distinguishes HVDRR from PDDR, where circulating concentrations of 1,25(OH)2D are depressed. Additionally, patients with HVDRR are resistant to supraphysiologic doses of all forms of vitamin D therapy. Table 11-1 outlines the similarities and differences between the two forms of hereditary rickets involving the vitamin D endocrine system.

Pseudo–Vitamin D–Deficiency Rickets

Molecular Etiology

As previously mentioned, serum levels of 25(OH)D are normal in untreated patients with PDDR and elevated in patients receiving large daily amounts of vitamin D.22 These results indicate that intestinal absorption of vitamin D and its hydroxylation in the liver are not impaired in PDDR. Circulating levels of 24,25(OH)2D are also normal and highly correlated with those of 25(OH)D, indicating a fully functional 25(OH)D-24-hydroxylase enzyme.24 However, serum concentrations of 1,25(OH)2D are low in untreated patients and remain low even when they are treated with high doses of vitamin D.22,25 This clearly identifies defective activity of the 25(OH)D-1α-hydroxylase enzyme (CYP27B1; hereafter referred to as 1α-hydroxylase) as the basic abnormality in PDDR and differentiates it from HVDRR.

PDDR is inherited as a simple autosomal recessive trait.28 No phenotypic abnormalities are observed in heterozygotes.21 By taking advantage of the unusual frequency of PDDR in the French-Canadian population and the availability of sample material from relatively large kindreds, the PDDR locus was mapped to the region of band 14 on the long arm of chromosome 12 (12q13-14).29,30

Tremendous progress has been achieved in the study of the molecular etiology of PDDR through the cloning of the complementary DNA (cDNA) encoding for 1α-hydroxylase.3134 The human gene has also been cloned, sequenced, and mapped to 12q13.1-13.3 by fluorescence in situ hybridization,33,35,36 consistent with the earlier mapping of the disease to this locus by linkage analysis.

The ultimate proof that mutations in the 1α-hydroxylase gene were responsible for the PDDR phenotype required the identification of such mutations in PDDR patients and carriers of the disease. The first identified mutation was reported by Fu et al.31 in 1997; several additional mutations in various ethnic groups have since been published.3541 These findings unequivocally establish the molecular genetic basis of PDDR as inactivating mutation(s) in the 1α-hydroxylase gene (CYP27B1). Further proof was provided by developing valid animal models of the disease using targeted inactivation of the cyp27B1 gene in mice.42,43

25-Hydroxyvitamin D 1α-Hydroxylase


The 25(OH)D-1α-hydroxylase (CYP27B1; 1α-hydroxylase) enzyme catalyzes the addition of a hydroxyl group at position 1α of the secosteroid backbone of 25(OH)D. The 1α-hydroxylase is a mitochondrial cytochrome P450 enzyme that requires electrons from nicotinamide adenine dinucleotide phosphate (NADPH) to promote catalysis. These are delivered to the P450 moiety by the flavoprotein NADPH-ferredoxin reductase44 and the nonheme iron protein, ferredoxin.45 The expression of these cofactors is ubiquitous, and their genes were mapped to chromosomes 17 and 11,44,45 respectively, excluding them rapidly in the search for the PDDR mutations, since the PDDR locus was mapped early on to chromosome 12.30

The main site for the 1α-hydroxylation of 25(OH)D is the proximal tubule of the renal cortex.46 In the kidney, the expression of the 1α-hydroxylase gene is subject to complex regulation by PTH, calcitonin, calcium, phosphorus, and 1,25(OH)2D itself.4749 The 1α-hydroxylase gene exists in a single copy in the human genome and contains nine exons spanning 5 kb of sequence. The ferredoxin-binding domain is encoded by sequences contained in exons 6 and 7, while the heme-binding domain is contained in exon 8.50,51


To date, 35 different 1α-hydroxylase mutations have been described in PDDR patients and their parents (Table 11-2). All patients have mutations on both alleles, but a high frequency of compound heterozygosity (a different mutation on each allele) has been observed (23 compound heterozygotes out of 54 cases reported). Splice-site mutations, nucleotide deletions and duplications, and missense and nonsense mutations have been reported (see Table 11-2). The mutations are dispersed throughout the 1α-hydroxylase sequence, affecting all exons (Fig. 11-3).

The mutations detected at the highest frequency are 958ΔG, common among French-Canadian patients (owing to a founder effect),29,36,38 and a mutation located at codons 438 to 442 in exon 8. These codons are composed of the duplicated 7-bp sequence 5′-CCCACCC CCCACCC-3′. In 11 families described to date,36,38,39,41 three rather than two copies of the 7-bp sequence are present, which alters the downstream reading frame (Fig. 11-4). Careful analysis of the correlation between ethnic origin, microsatellite haplotyping, and the presence of the 7-bp duplication mutation suggested that the mutation has arisen by several independent de novo events.36,38

Structure/Function Relationships

An important aspect of the identification of mutations in the 1α-hydroxylase gene was to correlate the genotype of the patients with their phenotype—that is, the severity of the disease and the circulating levels of 1,25(OH)2D. In several cases, although 1,25(OH)2D serum levels are low, they are not undetectable,22,25,37,40,52 suggesting some degree of residual 1α-hydroxylase activity. Presumably, some 1α-hydroxylase mutations affect the structural integrity of the enzyme, resulting in a modification of its kinetics. This reasoning cannot apply to the frameshift (deletions, insertions, and duplications) and nonsense mutations described to date. All such mutations eliminate the heme-binding site of the protein and thus completely abolish the 1α-hydroxylase enzymatic activity. The apparent residual 1α-hydroxylase activity observed in some patients could be attributable to missense mutations. Most of these missense mutations were entirely devoid of enzymatic activity in the assays used,38,40 except for the L343F mutation (retained 2.3% of wild-type activity) and the E189G mutation (retained 22% of wild-type activity).37 Thus some missense mutations contribute to the variable phenotype observed in patients with PDDR.

Early modeling efforts16,38 compared the sequence of the 1α-hydroxylase protein (a mitochondrial class I cytochrome P450) with the sequence of bacterial class I cytochrome P450s for which x-ray crystallographic data were available. The tertiary structures of these enzymes show remarkable conservation despite low amino acid sequence identity.53 These sequence alignments yielded improper predictions of the functions of the residues mutated in PDDR patients.54 Further modeling based on the first solved crystal structure of a eukaryotic cytochrome P450,55 combined with extensive structure/function analysis of recombinant 1α-hydroxylase proteins, identified the functions of many residues mutated in PDDR patients (see Table 11-2).54,56,57 Most mutations affect folding and conformation.54,56 Residue T321 is involved in oxygen activation, and amino acid R389 is essential for heme binding.54,56 Spectroscopic analysis of wild-type and mutant 1α-hydroxylase proteins identified residue T409 as critical for binding of the 25(OH)D substrate.57


Vitamin D2, at high doses, was initially used to treat PDDR. Under such treatment, circulating levels of 25(OH)D increase sharply, and it is likely that massive concentrations of 25(OH)D can bind to the VDR and induce the response of the target organs to normalize calcium homeostasis. The risk of overdose is high because vitamin D progressively accumulates in fat and muscle, and the therapeutic doses are close to the toxic doses, ultimately placing the patient at risk for nephrocalcinosis and impaired renal function. The use of 25(OH)D3 as a therapeutic agent in PDDR has been reported.58 The mechanism of action is likely to be similar to the one described earlier for vitamin D. The low availability and high cost of the metabolite have not encouraged its widespread use as long-term therapy for PDDR.

The treatment of choice is long-term (lifelong) replacement therapy with 1,25(OH)2D3.22,59 This results in rapid and complete correction of the abnormal phenotype, eliminating hypocalcemia, secondary hyperparathyroidism, and radiographic evidence of rickets. Strikingly, the myopathy disappears within days after initiation of therapy. The restoration of bone mineral content is equally rapid and histologic evidence of healing of the bone structure has been reported.22

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