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Department of Medicine (P.J.M., D.F.), Stanford University School of Medicine, Stanford, California 94305-5103; and Department of Molecular and Cellular Physiology (J.W.P.), University of Cincinnati, Cincinnati, Ohio 45267
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Abstract |
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 Top Abstract I. The Syndrome of...II. Vitamin D PhysiologyIII. 1,25-Dihydroxyvitamin D...IV. Cellular Basis of...V. The VDR Gene...VI. HVDRR Mutations Causing...VII. HVDRR Mutations Causing...VIII. Additional Mutations In...IX. HVDRR Mouse ModelX. Treatment of HVDRRXI. Analysis, Summary, and...References |
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-Hydroxylase deficiency |
I. The Syndrome of Hereditary 1,25-Dihydroxyvitamin D-Resistant Rickets (HVDRR) |
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TopAbstractI. The Syndrome of...II. Vitamin D PhysiologyIII. 1,25-Dihydroxyvitamin D...IV. Cellular Basis of...V. The VDR Gene...VI. HVDRR Mutations Causing...VII. HVDRR Mutations Causing...VIII. Additional Mutations In...IX. HVDRR Mouse ModelX. Treatment of HVDRRXI. Analysis, Summary, and...References |
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,25-dihydroxyvitamin
D, [1,25(OH)2D] (this notation will be used to signify
either D2 or D3), mediates its actions by
binding with high affinity to specific vitamin D receptors (VDRs)
located in the nucleus of target cells. Hereditary vitamin D-resistant
rickets (HVDRR) is a rare genetic disorder caused by a generalized
resistance to 1,25(OH)2D action (1, 2, 3). Heterogeneous
mutations in the VDR that alter the function of the receptor are the
molecular basis of HVDRR. A variety of mutations have been identified,
some of which render the VDR nonfuctional, imparting a complete
hormone-resistant state, while other mutations reduce VDR activity,
causing a hyporesponsive state. In this review, we will describe the
clinical manifestations of HVDRR, the VDR and its gene, the mechanism
of vitamin D action, and the genetic defects in the VDR that result in
this hormone-resistant syndrome. Other recent reviews of these subjects
can be found in the recently published volume entitled "Vitamin D"
and the references therein (4).
A. Historical
In 1978, Brooks et al. (5) described a patient with
osteomalacia who exhibited hypocalcemia, hypophosphatemia, and
secondary hyperparathyroidism. Interestingly, the patient had markedly
increased serum levels of 1,25(OH)2D. Brooks et
al. (5) suggested that the rickets was due to impaired
responsiveness of target organs to 1,25(OH)2D. They termed
this disease vitamin D-dependent rickets type II (VDDR-II) to
distinguish it from a closely related syndrome known as vitamin
D-dependent rickets type I (VDDR-I), which is due to a defect in an
enzyme leading to the synthesis of 1,25(OH)2D (see
below, Section II.B). Later the same year, Marx et
al. (6) reported similar findings in two children, and the authors
again suggested that the disease was due to end-organ resistance to
1,25(OH)2D. Since these initial studies, there have been
many reports of patients with apparent target organ resistance to
1,25(OH)2D. Over the years a number of different terms have
been used to describe this syndrome. As noted, the original reports
referred to this entity as vitamin D-resistant rickets type II. The
disease also has been called pseudo-vitamin D deficiency rickets type
II (PDDR-II), calcitriol-resistant rickets, vitamin D-resistant
rickets, and hereditary hypocalcemic vitamin D-resistant rickets. We
prefer the designation hereditary vitamin D-resistant rickets (HVDRR)
as a simple and accurate description of this syndrome caused by genetic
resistance to vitamin D.
B. Clinical features of HVDRR
The major clinical findings in patients with HVDRR,
hypocalcemia and rickets, are due to defective intestinal calcium
absorption leading to impaired mineralization of newly forming bone and
preosseous cartilage. The rickets is often severe and is usually
exhibited within months of birth.
Patients suffer from bone pain, muscle weakness, hypotonia, and
occasionally convulsions from hypocalcemia. Children are often growth
retarded, and in some cases they develop severe dental caries or
exhibit hypoplasia of the teeth (7, 8, 9, 10, 11, 12, 13). Some infants have died from
pneumonia caused by poor respiratory movement due to severe rickets of
the chest wall (8, 11, 14). Many children with HVDRR have sparse body
hair, and some have total scalp and body alopecia, including eyebrows
and in some cases eyelashes (Fig. 1
). Alopecia will be discussed in
more detail below (Section I.D).
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. The
abnormalities include low concentrations of calcium and phosphate and
elevated serum alkaline phosphatase activity. The hypocalcemia leads to
secondary hyperparathyroidism with elevated PTH levels and
hypophosphatemia. The 25(OH)D values are normal and, importantly, the
1,25-(OH)2D levels are elevated. This clinical feature
distinguishes HVDRR from 1-hydroxylase deficiency (VDDR-I or PDDR
discussed below, Section II.B) since the serum
1,25-(OH)2D values in the latter syndrome are depressed. In
the cases in which it has been measured, 24,25(OH)2D levels
have been normal or low (8, 11, 15, 16, 17, 18, 19, 20, 21). Unlike patients with
1-hydroxylase deficiency, most HVDRR individuals are resistant to
supraphysiological doses of all forms of vitamin D therapy (Table 1).
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(22).
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D. Alopecia
A clinical feature that is generally found in patients with HVDRR
is alopecia totalis (Fig. 1
). The majority of HVDRR patients have
sparse body hair, and some exhibit total scalp and body alopecia (17, 25, 26). Children with extreme alopecia often lack eyebrows and in some
cases eyelashes. Hair loss may be evident at birth or it occurs during
the first few months of life. An analysis of HVDRR patients shows that
there is some correlation between the severity of rickets and the
presence of alopecia (26). Patients with alopecia generally have more
severe resistance to calcitriol than those without alopecia. In
families with a prior history of the disease, the absence of scalp hair
in newborns provides initial diagnostic evidence for HVDRR. The
mechanism causing alopecia is unknown, but VDRs are present in the hair
follicle (27, 28). Skin biopsy has revealed apparently normal follicles
with no hair shaft present. The lack of 1,25-(OH)2D action
during a critical stage of hair follicle development is the suspected
cause of alopecia.
E. 1,25-(OH)2D action and HVDRR
The biological actions of 1,25-(OH)2D in tissues and
cells are orchestrated through complex changes in gene expression (29, 30). These changes lead to cell-specific alterations in the level of
proteins directly responsible for a myriad of differentiated cell
functions, as well as in proteins that act as transcription factors or
as signaling molecules to regulate secondary and tertiary levels of
gene expression (31). In the latter case, these molecules may function
directly within the cell or indirectly via additional cellular
signaling pathways in either autocrine or paracrine fashion. As
indicated earlier, most, if not all, of the molecular actions of
1,25-(OH)2D in the nucleus are mediated by the VDR. The
classic role of 1,25-(OH)2D is to regulate mineral
homeostasis, achieved through its coordinated actions on intestine,
kidney, bone, and parathyroid gland (32, 33). It is not surprising,
therefore, that initial evidence for the existence of the VDR derived
from early investigations in these tissues (34, 35, 36, 37, 38, 39, 40, 41, 42). Interestingly, the
VDR is expressed in a wide variety of tissues, including kidney, skin,
liver, pancreas, muscle, breast, prostate, adrenal, thyroid, and cells
of mesenchymal or hematopoietic origin (27, 28, 43, 44, 45, 46, 47). Although the
VDR in these tissues appears to arise from the same chromosomal gene,
its role in cellular function is not homeostatic in nature but rather
pleiotropic. Whereas the classic actions of vitamin D are to regulate
calcium homeostasis, the expanded scope of vitamin D pleiotropic
actions include stimulation of differentiation, inhibition of cell
proliferation, and suppression of the immune response (45, 46, 47, 48). In
addition, the regulation of cellular proliferation and differentiation
by 1,25-(OH)2D appears to be a common feature in many
tissues examined, and it is likely that this regulatory feature is a
fundamental component of all biological responses to
1,25-(OH)2D. Notwithstanding the complexity and diversity
of biological responses elicited by 1,25-(OH)2D, the
profound skeletal abnormalities demonstrated in patients with HVDRR
emphasizes the fundamental and essential role of
1,25-(OH)2D in calcium homeostasis.
Although there are multiple pleiotropic tissue responses regulated by 1,25-(OH)2D, children with HVDRR appear relatively normal except for the constellation of features that relate to their calcium deficiency, rickets, and alopecia. VDRs have been found in endocrine glands such as pituitary, pancreas, parathyroid, gonads, and placenta, and 1,25-(OH)2D3 regulates hormone synthesis and secretion from these glands (29, 45, 46, 49, 50, 51). VDRs have also been found in hematolymphopoietic cells, and 1,25-(OH)2D3 regulates cell differentiation and the production of interleukins and cytokines (52). Hochberg et al. (53) examined hormone secretion in patients with HVDRR and found no abnormalities in insulin, TSH, PRL, GH, and testosterone levels in serum. Even et al. (54) showed that urinary cAMP and renal excretion of potassium, phosphorous, and bicarbonate were normal in HVDRR patients treated with PTH. However, PTH failed to decrease urinary calcium and sodium excretion in these patients to the extent found in controls. This suggests that 1,25-(OH)2D may selectively modulate the renal response to PTH and facilitate the PTH-induced reabsorption of calcium and sodium (54). Although minor aberrations have been noted in the fungicidal activity of neutrophils from HVDRR patients (55), the patients do not exhibit any clinically apparent immunological defects. In the light of the diverse actions of 1,25-(OH)2D demonstrated in many nonosteogenic tissues, the absence of related findings in children with HVDRR suggests that the pleiotropic responses regulated by 1,25-(OH)2D in these nonosteogenic tissues are redundant and that other factors or compensatory mechanisms subsume the role of vitamin D in such a way that abnormalities are not clinically manifested. Similarly, the VDR knockout mouse displays the same phenotypic and physiological patterns as patients with HVDRR (56, 57). The VDR knockout mouse model can be used to analyze the abnormalities caused by the loss of VDR action in detail not possible in the HVDRR patients (see Section IX).
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II. Vitamin D Physiology |
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TopAbstractI. The Syndrome of...II. Vitamin D PhysiologyIII. 1,25-Dihydroxyvitamin D...IV. Cellular Basis of...V. The VDR Gene...VI. HVDRR Mutations Causing...VII. HVDRR Mutations Causing...VIII. Additional Mutations In...IX. HVDRR Mouse ModelX. Treatment of HVDRRXI. Analysis, Summary, and...References |
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) (58). Vitamins D2 and
D3 are essentially biologically inactive and must be
converted to hydroxylated metabolites to gain hormonal activity. Upon
entering the circulation, vitamin D (D2 or D3)
binds to the vitamin D-binding protein, a 58-kDa plasma -globulin
(59, 60). In the liver, vitamin D is hydroxylated at the carbon-25
position to form 25-hydroxyvitamin D [25(OH)D] by the enzyme
25-hydroxylase (61). This enzyme, also known as CYP27, is a
multifunctional cytochrome P450 oxidase that also hydroxylates
cholesterol and bile acids. In the kidney the enzyme 25-hydroxyvitamin
D-1-hydroxylase (1-hydroxylase), also a P450 oxidase, adds a
second hydroxyl group to 25(OH)D to form 1,25-(OH)2D, the
hormonally active form of vitamin D.
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-hydroxylase activity is tightly regulated
by PTH (62). Patients with hyperparathyroidism have elevated serum
levels of 1,25-(OH)2D, whereas patients with
hypoparathyroidism show reduced levels of the hormone (62, 63). Other
factors, including phosphate and 1,25-(OH)2D and possibly
phosphatonin, also regulate 1-hydroxylase activity. High phosphate
levels in the serum lead to a suppression of 1-hydroxylase activity
while low phosphate levels tend to increase the amount of enzyme
activity. 1,25-(OH)2D regulates its own production, both by
suppressing PTH secretion and feedback inhibition of renal
1-hydroxylase activity (62). In the feedback inhibition loop, low
1,25-(OH)2D levels lead to increased 1-hydroxylase
activity and high 1,25-(OH)2D levels inhibit the enzyme
activity in the kidney. 1-Hydroxylase activity is also influenced by
the concentration of calcium in the serum. The calcium acts indirectly
by regulating PTH levels via the calcium-sensing receptor, thereby
determining 1-hydroxylase activity. The catabolism of 1,25-(OH)2D involves a series of enzymatic steps, the first of which is catalyzed by the enzyme 25-hydroxyvitamin D-24-hydroxylase (24-hydroxylase) (64). 24-Hydroxylation generates the trihydroxy form of vitamin D, 1,24,25(OH)3D, a biologically less active form of 1,25-(OH)2D, that is further subjected to additional hydroxylations and oxidations leading to the production of calcitroic acid, which is excreted in the urine (65, 66). Interestingly, 1,25-(OH)2D induces 24-hydroxylase activity in a number of cells and, therefore, regulates the rate of its own metabolism. The genes encoding the rat and human 24-hydroxylase (CYP24) have been cloned (67, 68, 69), and a vitamin D-response element (VDRE) (see below, Section III.C) has been identified in the regulatory region of these genes (70, 71). Regulation of 24-hydroxylase gene expression or enzyme activity is a very useful marker of 1,25-(OH)2D responsiveness in the evaluation of HVDRR (16). In humans, the 24-hydroxylase gene is found on chromosome 20 at 20q13 (69).
In addition to hydroxylating 1,25-(OH)2D, 24-hydroxylase can convert 25(OH)D to 24,25(OH)2D. The biological role of 24,25(OH)2D has not been established with certainty. However, recent studies with a 24-hydroxylase knockout mouse model strongly suggest that 24,25(OH)2D may have some biological activity distinct from 1,25-(OH)2D, especially on cartilage cells (72).
B. 1-Hydroxylase deficiency
We will discuss this disease entity briefly since it has some
elements in common with HVDRR and must be distinguished from it (Table 2). Decreased production of
1,25-(OH)2D is found in patients with 1-hydroxylase
deficiency. In 1961, Prader et al. (73) described a patient
with a genetic form of vitamin D-resistant rickets that ultimately was
due to a deficiency of renal 1-hydroxylase. This disease has been
known as vitamin D-dependent rickets type I (VDDR-I) and also as pseudo
vitamin D deficiency rickets (PDDR) (74). Genetic linkage studies
indicate that the mutation causing 1-hydroxylase deficiency is
linked to chromosome 12 at 12q14 (75, 76, 77). Recently, several groups
have cloned the 1-hydroxylase gene (78, 79, 80, 81, 82, 83), and it has been
confirmed that the locus on chromosome 12 is the 1-hydroxylase gene
and that VDDR-I (or PDDR) is due to mutations in the gene encoding the
1-hydroxylase. In several cases, mutations in the 1-hydroxylase
gene have been elucidated (78, 83).
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-Hydroxylase deficiency is an autosomal recessive disease that is
manifested at an early age, presenting with hypotonia, muscle weakness,
growth failure, and rickets. Hypocalcemia, elevated PTH levels,
increased alkaline phosphatase activity, and low urine calcium
excretion are also found. Tetany and convulsions may occur with severe
hypocalcemia. These symptoms are also characteristic of HVDRR. Patients
with 1-hydroxylase deficiency have normal or elevated 25(OH)D levels
but low 1,25-(OH)2D levels due to various defects in the
1-hydroxylase enzyme. Patients with this condition are
treated with physiological doses of
1,25-(OH)2D3 (0.25–2 µg/day) that bypass the
defective enzyme and restore serum calcium concentrations to
normal. The low serum levels of 1,25-(OH)2D and the
therapeutic response to physiological doses of exogenous
1,25-(OH)2D3 distinguish 1-hydroxylase
deficiency from HVDRR (Table 2). |
III. 1,25-Dihydroxyvitamin D Action Mediated by the Vitamin D Receptor (VDR) |
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TopAbstractI. The Syndrome of...II. Vitamin D PhysiologyIII. 1,25-Dihydroxyvitamin D...IV. Cellular Basis of...V. The VDR Gene...VI. HVDRR Mutations Causing...VII. HVDRR Mutations Causing...VIII. Additional Mutations In...IX. HVDRR Mouse ModelX. Treatment of HVDRRXI. Analysis, Summary, and...References |
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B. The domain structure of the VDR
1. Overview. The VDR exhibits a modular domain structure
generally similar to that of other members of the nuclear receptor gene
family (see Fig. 4). Since the mutations within the VDR gene, which
will be detailed subsequently in this review, are distributed across
several of its domains, we will describe the structural organization of
the functional protein. While the structural domains were initially
deduced from the gene sequence, it is important to note that functional
correlations with these domains have evolved from extensive examination
of receptor activities of natural mutations in patients with HVDRR as
well as site-directed mutagenesis of the cDNA and recombinant
expression in host mammalian cell types. Recent crystallographic
studies for several of the receptors support the predicted
structure-function relationships (see below).
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)
(96). While a considerable degree of sequence and structural homology
exists across the superfamily for several of the receptor domains,
others exhibit little or no sequence homology and in some instances
domains may be abbreviated or absent (92). This diversity is manifest
most strongly in regions A/B, D, and F. Segment A/B includes all
residues that extend amino terminal to the DNA-binding domain (DBD) of
the receptor. Its size is highly variable, ranging from hundreds of
amino acids (aa) in the progesterone receptor (PR), for example, to
approximately 24 aa in the VDR. Region C comprises the highly conserved
DBD of the receptor gene family and represents the hallmark feature of
this group of proteins, which will be described in detail below. The D
domain, which is least conserved among the nuclear receptors, appears
to serve as a hinge between the DBD and the E region. The D region
within the VDR is 50 residues longer than that found in the other
steroid receptors and is likely the result of an additional exon within
the VDR chromosomal gene (97). Consistent with other receptors, this
region in the VDR exhibits very limited species conservation,
particularly at the level of the inserted exon. The hinge region is
also a site of serine phosphorylation (98, 99) and may play additional
functional roles. The E region encodes the ligand-binding domain (LBD)
of the hormone-activated receptors and exhibits several important
functional activities, which will be discussed below. The small F
region is not conserved within the nuclear receptor family and is, in
fact, absent in the VDR as well as in several other members of the
family.
2. DBD. The DBD of the VDR (aa residues 24–90)
contains nine highly conserved cysteine residues and consists of two
similar motifs each comprised of a zinc-coordinated finger structure
(Fig. 5). Each zinc atom is tetrahedrally
coordinated through four of the highly conserved cysteine residues that
serve to stabilize the finger structure itself. These finger modules
are structurally unrelated to the multiple zinc fingers found in
transcription factor IIIA wherein the zinc atom is coordinated
through two cysteines and two histidines (100, 101). Interestingly,
although the two zinc modules of the VDR appear highly related
structurally, they are not topologically equivalent (100). This lack of
equivalency is consistent with the fact that each module serves a
different function within the protein. The amino-terminal module,
comprised of an -helix known as the P box (aa residues 41–46),
functions to direct specific DNA-binding in the major groove of the
DNA-binding site. The carboxy-terminal module, on the other hand, which
also contains an -helix known as the D box (aa residue 61–65),
serves as a dimerization interface for interaction with a partner
protein (see below) (102, 103). This region may serve a lesser function
in the VDR as a result of the asymmetric nature of the interaction of
VDR with its partner protein, retinoid X receptor (RXR), when bound to
a cognate DNA response element. Indeed, modeling studies suggest that
several residues (aa residues 91 and 92) located in an extended
-helix immediately downstream of the second zinc finger (aa residues
90–101) and termed the T box, provide key interactions with partner
proteins. This T box region also likely makes minor groove contacts
with nucleotides located between the two DNA half-sites, thus
strengthening the interaction of the VDR with its DNA-binding elements.
Posttranslational modification of VDR by phosphorylation of Ser51
inhibits its ability to complex with the VDRE and may serve as a
negative regulator of VDR activity (104, 105). When the
three-dimensional structure of the VDR DBD emerges, either as a result
of nuclear magnetic resonance spectroscopy or x-ray crystallography
studies, our understanding of the structural organization of these
modules, as well as the mechanisms through which they function to
interact with DNA, will be enhanced. Recent successes using these
techniques with the estrogen receptor (ER), glucocorticoid receptor
(GR), retinoic acid receptor (RAR), and RXR DBDs have provided
significant insights into the structure of this domain (106, 107, 108, 109, 110, 111). As
will be discussed below, mutations in critical amino acids within both
zinc finger modules have rendered the VDR nonfunctional and have caused
HVDRR presumably by interfering with VDR binding to DNA.
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4. Structural modeling. Recently, the three-dimensional
structure of the LBD of RXR (124), RAR
(125), and thyroid
receptor (TR1) (126), all RXR partners, as well as ER (127) and PR
(127, 128), have been elucidated. We will focus our analysis of the LBD
on the RXR partners since they are more relevant to the VDR. RAR and
TR1 receptors were crystallized in the presence of ligand
(holodomains), whereas the RXR structure was determined in the
absence of ligand (apodomain). Twelve -helices (H1-H12) arranged as
an antiparallel -helical sandwich comprise the bulk of the LBD of
each of the receptors (129). It is likely that the VDR will be arranged
in a structurally similar manner, and a model of the VDR helical
structure based on the canonical structure of nuclear receptors is
shown in Figs. 6 and 7. These
three-dimensional structures support the idea that H9 and H10 are
essential for the formation of RAR, VDR, TR, or peroxisome
proliferator-activating receptor (PPAR) heterodimers with a common RXR
subunit. While functional studies support the requirement of these
helical sequences in heterodimer formation, H9 and H10 may be
insufficient for VDR and PPAR dimerization. This conclusion is
experimentally supported by a complete evaluation of the dimerization
properties of the carboxy-terminal E region of the VDR (130).
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-helix
back upon the hydrophobic core and upstream -helices in the E domain
in response to ligand binding (Fig. 7).
This repositioning likely "locks"
1,25-(OH)2D3 into its binding pocket and leads
to the formation of a complex high-affinity protein surface capable of
interacting with specific comodulators such as SRC-1 and others. This
requisite interaction between H12 and more upstream -helices likely
accounts for the observed loss of VDR transcriptional capacity after
mutation of residues located in either region of the receptor molecule
(132, 133). The three-dimensional structure of the VDR will, however,
have to be solved directly to confirm these predictions.
C. The regulation of gene expression by the VDR
The proof of the regulation of gene expression by VDR was
initially developed by studies of HVDRR where natural mutations in the
VDR gene prevented 1,25-(OH)2D3 induction of
target genes such as 24-hydroxylase (16, 18, 134). An understanding of
the molecular mechanisms through which
1,25-(OH)2D3 and its receptor modulate gene
expression is outlined in Fig. 3 and emerged initially through
examination of the promoters for genes known to be regulated by this
hormone. A major focus of these studies was to demonstrate that
1,25-(OH)2D3 could regulate promoter activity,
that specific DNA sequences within the promoter were required, and that
a functional VDR was an essential component of the transactivation
machinery. These principles of direct regulation of gene expression
by 1,25-(OH)2D3 were established using human
(135, 136, 137) and rat (138, 139, 140) osteocalcin gene promoters as models and
were strengthened through subsequent investigation of the osteopontin
gene (141), the calbindin genes (142, 143), and the 25-hydroxyvitamin
D3 24-hydroxylase genes (70, 144, 145). HVDRR mutant VDRs
were shown to be incapable of activating such promoter constructs, both
supporting the critical role of functional VDR in transactivation as
well as defining the defect causing HVDRR (146, 147, 148).
1. Direct regulation of transcription by 1,25-(OH)2D3. Initial investigations by McDonnell et al. (149) demonstrated that 1,25-(OH)2D3 stimulated transcription from the human osteocalcin gene promoter in a dose-dependent fashion consistent with its actions on the expression of the endogenous gene. The sensitivity of this promoter to 1,25-(OH)2D3 enabled subsequent definition of the DNA sequence element within the promoter responsible for mediating hormone action (see below). Equally important, the ability of 1,25-(OH)2D3 to stimulate this transcriptional activation was dependent upon the presence of intact VDR. Thus, while the osteocalcin promoter was unresponsive to 1,25-(OH)2D3 when introduced into a VDR-negative cell line or cotransfection with an HVDRR mutant VDR, cointroduction of the wild-type VDR via a recombinant expression vector restored hormonal responsiveness. The establishment of this VDR- and hormone-dependent assay in cells enabled a determination of the functional domains of the VDR. As will be seen in subsequent sections of this review, this assay has become essential in establishing the inactivating nature of numerous mutations found in the VDR chromosomal gene in patients with HVDRR.
2. VDREs. Deletion analysis of the human osteocalcin promoter
led to the identification of a cis-acting element that was
located approximately 500 bp upstream of the transcriptional start site
and that mediated 1,25-(OH)2D3 induction (135).
This study and one by Ozono et al. (137) resulted in
definition of the first VDRE, a directly repeated hexanucleotide
sequence separated by 3 bp. Parallel studies using the rat osteocalcin
gene promoter led to similar conclusions regarding the organizational
motif of the VDRE (138, 139, 140). Definitive VDREs have since been
localized in the mouse osteopontin gene (141), the rat and human
24-hydroxylase genes (70, 144, 145), and the human p21 gene (150). As
seen in Fig. 8, each of these elements is
comprised of two directly repeated hexanucleotide half-sites and, like
the human osteocalcin VDRE, the half-sites are separated by a 3-bp
spacer. Studies on the mouse calbindin D9K (143) and the
rat calbindin D28K (142) genes have also revealed
apparent VDREs, although these sequences mediate rather weak
1,25-(OH)2D3 induction in their natural
promoter environments and do not exhibit structural similarity to the
more well characterized VDREs. One possibility is that the VDR binds to
these sites, perhaps in combination with a nonconventional protein
partner(s) (see below). The exact mechanism whereby the VDR interacts
with its VDRE, which is described in the next section, is particularly
relevant since a large majority of the mutations found within the VDR
gene that lead to HVDRR are found within the VDR DBD and their
mechanism is to interfere with DNA binding.
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4. Subunit structure of the active VDR heterodimer. The
observation that VDREs are comprised of two half-sites led to the
prediction that the VDR might bind to these sites as a functional
dimer. Indeed, the interaction of each of the classic steroid receptors
with their respective hormone response elements (HREs) are known to
occur via homodimerization. It was a surprise, therefore, to observe
that the VDR did not associate with DNA as a homodimer, but rather as a
heterodimer (Fig. 8). This finding, made by Liao et al.
(159) and Sone et al. (152, 160), demonstrated that DNA
binding of recombinant VDR produced in yeast or in vitro
could only be achieved after reconstitution with mammalian cellular
extracts. The factor supplied by the extract, whose presence was
confirmed by several additional groups (161, 162), was termed
nuclear accessory factor or NAF. NAF was found to be widely distributed
in cells and tissues and hypothesized to be an unknown nuclear
receptor. Similar heterodimeric activities were identified for the
thyroid hormone receptor (TR) and RAR. In 1991 and 1992, four
groups of investigators demonstrated that this activity derived from
three related members of the nuclear receptor gene family termed
retinoid X receptor (RXR, RXRß, and RXR) (163, 164, 165, 166). It was
concluded that NAF was a single manifestation of a mixture of one or
more of the RXRs expressed in any given cell type. True dimerization
between the VDR and RXR has been confirmed through definition of the
VDR dimerization domain (130). Utilizing an extensive series of
internal deletions of the VDR, two regions located within the E domain
were shown to be essential for interaction with RXR. These regions
coincide with two subregions within the E domain that are moderately
conserved within the entire nuclear receptor gene family. Perlmann
et al. (167) suggested recently that a small region of 40 aa
lying within the second E/F homology domain (corresponding to H9 and
H10 of the crystal structure of the RXR LBD) is sufficient within RXR
to form dimers with RAR and TR. This same region is apparently not
sufficient, however, to permit formation of RXR-VDR heterodimers,
suggesting that the domains responsible for interaction between RXR and
other signaling partners may be different.
Interestingly, in view of the asymmetric nature of the VDRE (two
directly repeated half-sites) and the heterodimeric nature of the VDR
modulatory unit (RXR and VDR), distinct polarity must also exist with
respect to the half-site-receptor interaction. Indeed, Jin et
al. (130) and Freedman and co-workers (153) demonstrated that RXR
binds to the upstream or 5'-half-element and that VDR binds to the
downstream half-element on consensus type VDREs (Fig. 8). Whether this
polarity is maintained on all VDREs remains to be proven. However, this
organization is consistent with that noted for both RXR-TR and RXR-RAR
heterodimers bound to their respective HREs (168, 169). Irrespective of
the mechanism, the existence of a general permissive partner protein
that functions as a central regulator for several endocrine systems
suggests the potential for considerable cross-talk between the systems.
5. The transactivation domain of the VDR. As indicated
earlier, two regions are believed to mediate the transactivation
capacity of the VDR, a carboxy-terminal AF-2 region comprised of H12,
which includes residues 416–424, and the E1 region located in the
midregion of the receptor comprising H3 and H4, which includes residues
from 232 to 272 (30) (Figs. 4 and 6). Additional helices downstream of
H4 may be involved as well. Based upon the crystal structure of apo-RXR
LBD and holo-RAR LBD (124, 125, 126), it is likely that interaction of
1,25-(OH)2D3 with the VDR leads to the folding
of H12 back against the hydrophobic core of the E domain and helices
therein. This repositioning completes the high-affinity binding site
for 1,25-(OH)2D3 and creates a complex surface
capable of additional protein-protein interactions (Fig. 7). Indeed,
mutations in each of these two regions can selectively abrogate ligand
binding, transactivation, or both.
Does the AF-2 domain of RXR contribute to transactivation by the VDR? It is clear that RXR has the potential to function as a signaling partner when complexed to orphan receptors, such as LXR and NGF-IB (167, 170). Additional studies suggest that the AF-2 function of the silent partner is required for activation by the signaling partner (171, 172). This supports the idea that the AF-2s of the silent and the signaling receptors both participate in a common protein surface essential for activation of transcription. Perhaps activation of the silent partner occurs via the process of dimerization with the signaling partner and/or after DNA binding (173). Evidence is rapidly accumulating for such regulatory events.
6. Transcriptional comodulators and cofactors. The
transactivation domain of most nuclear receptors interacts directly
with additional classes of proteins termed comodulators. These proteins
include the positive modulators or coactivators SRC-1 (116),
GRIP-1/TIF2 (174, 175), and ACTR (176) and the negative regulators or
corepressors NCoR (177) and SMRT (119, 120). Indeed, the AF-2 of
the VDR is found to be essential for interaction with SRC-1 (117, 118)
and likely is essential for other comodulator interactions as well
(Fig. 9). After recruitment of a
comodulator into the promoter region by a nuclear receptor, these
proteins both induce chromatin structural changes enzymatically or
recruit additional proteins capable of similar actions or both.
Coactivators appear to exhibit histone acetyltransferase activity (176)
and recruit additional histone acetyltransferases such as P/CAF
(178) and CBP/p300 (179), each of which functions to
enzymatically modify histones such that nucleosomes are destabilized
and transcription is facilitated. The ability of CBP to interact
simultaneously with a number of unrelated transcription factors also
suggests an additional function for these large proteins—that of
integrating multiple inputs on a gene promoter so as to produce a
coherent output (180, 181). In contrast, corepressors apparently served
to recruit deacetylases such as HDAC1 (182, 183, 184). Histone deacetylase
activity serves to stabilize chromatin and repress transcription (111).
In this paradigm, nuclear receptors appear to function, at least in
part, to recruit the enzymatic machinery necessary for carrying out the
changes in chromatin structure essential for the effective regulation
of transcription. Nuclear receptors are, however, capable of additional
important contacts. Two groups have shown that a domain within the VDR
hinge region makes important contacts with TFIIB (121, 122), a member
of the initiation complex per se. This interaction, one of
several made by nuclear receptors, appears to enhance transcription
through direct stabilization of the transcriptional machinery.
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IV. Cellular Basis of HVDRR |
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TopAbstractI. The Syndrome of...II. Vitamin D PhysiologyIII. 1,25-Dihydroxyvitamin D...IV. Cellular Basis of...V. The VDR Gene...VI. HVDRR Mutations Causing...VII. HVDRR Mutations Causing...VIII. Additional Mutations In...IX. HVDRR Mouse ModelX. Treatment of HVDRRXI. Analysis, Summary, and...References |
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. The HVDRR cases are referred to
throughout this review as F1, F2, etc., where the F denotes the family
number (8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 25, 134, 186, 187, 188, 189, 190, 191, 192, 193, 194, 195, 196, 197, 198, 199, 200, 201, 202, 203, 204, 205, 206, 207, 208, 209). The clinical findings in children
affected with HVDRR, along with their failure to respond to the
administration of physiological and even supraphysiological doses of
vitamin D, suggest that the disease is caused by end-organ resistance
to vitamin D. The identification and characterization of the VDR as the
mediator of 1,25-(OH)2D3 action and its
presence in the major target tissues of vitamin D action led
investigators to suspect that the cause of HVDRR was due to a genetic
defect in the VDR (6, 7, 8, 9). Studies of VDR from HVDRR cases began after
the demonstration that VDRs were present in skin (27, 43, 210) and
could be studied in cultures derived from human skin biopsies (211).
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Studies by Griffin and Zerwekh (190) and Liberman et al. (191, 192) also used 24-hydroxylase activity to demonstrate 1,25-(OH)2D3 resistance. On the other hand, Clemens et al. (189) showed that fibroblasts from HVDRR patients were not growth-arrested after hormone treatment in contrast to fibroblasts from healthy individuals that were growth-arrested. These early observations showed that cells from HVDRR patients were resistant to 1,25-(OH)2D3 and that a variety of abnormalities in the VDR existed.
1. Ligand-binding negative phenotype. As the number of reports on HVDRR increased, the heterogeneous nature of the defects in the VDR became more apparent. Hochberg et al. (17, 25) reported clinical findings in four patients from two unrelated families of Arab origin (F11, F18) who exhibited HVDRR and alopecia. Fibroblasts from three of these patients and several of their parents, as well as an additional unrelated family from Germany (F17), were studied by Chen et al. (134). In these studies, HVDRR fibroblasts exhibited negligible [3H]1,25-(OH)2D3-binding, and hormonal treatment failed to induce 24-hydroxylase enzyme activity. These cases were designated as representing the ligand-binding negative phenotype. Interestingly, of the five parents in the study, only the father in family F18 exhibited a phenotype theoretically expected of a heterozygote. His fibroblasts contained half of the normal amount of [3H]1,25-(OH)2D3-binding and also showed a half-maximal response to 1,25-(OH)2D3. The fibroblasts of the other four parents showed a normal complement of VDR and a normal response to 1,25-(OH)2D3 treatment, which has been the usual pattern found in fibroblasts of heterozygotes.
In 1982, after the development of a monoclonal antibody to the VDR (87, 212, 213), the presence of VDR in some patients with the ligand binding-negative phenotype (11, 15) was assessed by Pike et al. (214). Using a radioligand immunoassay (215), an immunoreactive protein was detected in cell extracts from fibroblasts of ligand binding-negative HVDRR patients (214). The authors speculated that the defect in the VDR was due to a structural abnormality in the LBD preventing [3H]1,25-(OH)2D3 from binding to the receptor and not from defective synthesis of the VDR protein (214).
2. Ligand binding-positive phenotype. A ligand binding-positive case was described by Hirst et al. (18) who studied the VDR in fibroblasts from a Haitian family (F19) with HVDRR. Cultured fibroblasts from two sisters with HVDRR were unresponsive to 1,25-(OH)2D3 treatment despite normal [3H]1,25-(OH)2D3-binding and VDR abundance. In addition, sucrose gradient sedimentation studies revealed a VDR of normal size. However, the protein exhibited a decreased ability to form aggregates in low-salt conditions as compared with normal receptor. Using DNA-cellulose chromatography, the authors demonstrated that the VDR from the HVDRR fibroblasts exhibited a significant decrease in affinity for heterologous DNA. The normal receptor eluted from the DNA-cellulose at 170–173 mM KCl, while the mutant receptor eluted at 105–109 mM KCl. A second HVDRR family (F31), studied by Malloy et al. (203), exhibited a similar defect in the DNA binding properties of the receptor. The VDR from the affected individuals displayed normal [3H]1,25-(OH)2D3 binding and was normal in size as shown by Western blotting. However, DNA-cellulose chromatography clearly revealed that the VDR had a low affinity for DNA eluting at 100 mM KCl in contrast to the wild-type VDR, which eluted at 200 mM KCl. The patients from F19 and F31 families were therefore categorized as having the ligand binding-positive phenotype and, in addition, had a VDR that exhibited a low affinity for DNA. When the VDR from the parents’ cells were subjected to DNA-cellulose chromatography, two forms of the receptor were found. One receptor form eluted at 200 mM KCl, indicating that it had a high affinity for DNA similar to the wild-type receptor, while the other form eluted at 100 mM KCl, demonstrating a low affinity for DNA similar to the HVDRR patient. This was the first clear evidence showing the heterozygous state of the HVDRR parents. It was suspected that the defects in these cases would likely be due to point mutations in the DBD (18, 203), which was later proved correct (146).
Liberman et al. (197) also described four cases (F1, F3, F5, F20) of ligand binding-positive resistance to 1,25-(OH)2D3. Two of the cases (F5, F20) exhibited VDRs with a low affinity for DNA similar to the F19 and F31 families. Gamblin et al. (194) examined 1,25-(OH)2D3 induction of 24-hydroxylase activity in F5 and F20 fibroblasts and demonstrated complete hormone resistance. In the other cases, F1 and F3, Liberman et al. (197) demonstrated that the VDRs had a reduced ability to localize to the nucleus despite showing a normal affinity for DNA. Gamblin et al. (194) further showed that the F1 and F3 fibroblasts exhibited 24-hydroxylase activity when exposed to high concentrations of 1,25-(OH)2D3. Patients F1 and F3, whose fibroblasts showed a response to high concentrations of 1,25-(OH)2D3 in vitro, also exhibited a calcemic response to high doses of calciferols in vivo.
Castells et al. (196) also described a ligand binding-positive patient (F22) who had sparse hair, rickets, and high circulating 1,25-(OH)2D3 levels. Studies of the VDR from the patient’s fibroblasts showed that the receptor had a decreased affinity for [3H]1,25-(OH)2D3. The patient responded with a marked improvement in the disease after treatment with extremely high doses of 1,25-(OH)2D3, apparently overcoming the low-affinity binding abnormality in the VDR.
B. Studies in other cells
In addition to cultured skin fibroblasts and bone cells, studies
of the VDR from patients with HVDRR have been carried out in a number
of other cell types including peripheral mononuclear cells (195),
phytohemagglutinin (PHA)-stimulated lymphocytes (200, 206), myeloid
progenitor cells (201), Epstein-Barr virus (EBV)-immortalized B
lymphoblasts (22, 146, 147, 203), and HTLV-1 virus-immortalized T
lymphoblasts (205). It is interesting to note that EBV-immortalized B
lymphoblasts, from normal subjects that express wild-type VDR, do not
exhibit induction of 24-hydroxylase activity or growth inhibition in
response to 1,25-(OH)2D3 (22). On the other
hand, PHA-stimulated lymphocytes and HTLV-1-immortalized T lymphoblasts
from normal subjects are capable of responding to
1,25-(OH)2D3 (205, 216). In PHA-stimulated
lymphocytes, the absence of an inhibitory effect of
1,25-(OH)2D3 on DNA synthesis or lack of
induction of 24-hydroxylase activity have been used as markers to
rapidly diagnose HVDRR (200, 206). In addition, Takeda et
al. (206) showed that PHA-stimulated lymphocytes from parents of
children with HVDRR expressed intermediate levels of 24-hydroxylase
when treated with 1,25-(OH)2D3.
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V. The VDR Gene and the Molecular Basis of HVDRR |
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TopAbstractI. The Syndrome of...II. Vitamin D PhysiologyIII. 1,25-Dihydroxyvitamin D...IV. Cellular Basis of...V. The VDR Gene...VI. HVDRR Mutations Causing...VII. HVDRR Mutations Causing...VIII. Additional Mutations In...IX. HVDRR Mouse ModelX. Treatment of HVDRRXI. Analysis, Summary, and...References |
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A. The VDR chromosomal gene
The human VDR is the product of a single chromosomal gene located
on chromosome 12 at 12q13–14 (76). Initial efforts to determine its
structure resulted in the identification and relative spatial
organization of eight exons that comprise the entire coding region of
the human protein (146). As outlined diagrammatically in Figs. 6 and 10, the first of these exons is
designated exon 2, which contains the most proximal 3 bp of the
5'-noncoding sequence, the translation start site, and nucleotide
sequence that encodes the first zinc finger module. Exon 3 lies
approximately 15 kb downstream and encodes the second zinc finger
module. Exons 4, 5, and 6 encode the D region or hinge. Exon 5 may
represent an insertion. Exons 6, 7, 8, and 9 encode a portion of the
hinge and the carboxy-terminal E regions as well as approximately 3200
nucleotides of 3'-noncoding sequence. These exons span approximately 50
kb of genomic DNA. Sequence determination of each of the exons as well
as the exon-intron boundaries (and in some cases extended portions of
the introns) enabled subsequent examination of DNA from patients with
HVDRR using gene amplification techniques and detection of the
mutations in the VDR gene described in this review.
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). These noncoding exons
appear to be selectively spliced to produce at least three different
human VDR mRNA transcripts, each of which contain exons 2–9. Type I
transcripts contain exons 1a and 1c, type II transcripts contain exons
1a, 1b, and 1c, and type III transcripts contain only exon 1a fused
directly to exon 2. The human VDR promoter lies immediately upstream of
exon 1a. In contrast to numerous genes, this promoter is TATA-less and
correspondingly GC rich, a feature of a number of nuclear receptor
genes including those for progesterone (217) and glucocorticoids (218).
Five potential GC boxes located in the proximal region of the VDR
promoter suggest that the transcription factor SP-1 may play an
important role in its expression (97). Indeed, deletion of these
elements results in a dramatic reduction in the activity of this
promoter after introduction into cultured cell lines. Interestingly,
the mouse VDR gene shows a similar, although not identical,
organizational profile at the level of the promoter, suggesting a
similar mode of regulation (219). Additional studies are in progress to
evaluate and identify further important molecular determinants of VDR
gene expression. Thus far, no cases of HVDRR have been shown to be
caused by mutations in the 5'-prime regulatory region of the VDR gene.
C. Polymorphisms of the VDR gene
Mutations in the coding regions of the VDR gene lead to
significant functional consequences, as will be discussed extensively
in the next section on HVDRR. On the other hand, two sets of normal
variants or polymorphisms have been noted in the VDR gene, and their
effects on VDR function are worthy of discussion. The first class
represents a set of polymorphisms at the 3'-end of the VDR in intron J
(between exons 8 and 9) and exon 9 (Fig. 11). Although multiple polymorphisms
have been identified in this region (220), the commonly studied ones
are defined by the restriction enzymes BsmI,
ApaI, and TaqI. These polymorphisms have been
associated with variations in bone mineral density (BMD) in a number of
human studies and, although controversial, are hypothetically
predictive of osteoporosis risk (220). A meta-analysis of a number of
studies concluded that there was a small difference in BMD that was
associated with these polymorphisms (221). A poly A microsatellite of
variable length found in the 3'-untranslated region in exon 9 is
linked to the three sites (220), although the linkage is of variable
tightness, depending upon ethnicity (222). Interestingly, the
polymorphisms have also been associated with other diseases such as
osteoarthritis, hyperparathyroidism, and prostate cancer (223, 224, 225, 226, 227, 228, 229).
Attempts to define differences in VDR function between the polymorphic
variants have not been successful, so that a rationale for disease
association is not obvious (230, 231, 232).
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The system used to number the amino acids in the VDR and thus to identify the site of a mutation in HVDRR cases has been somewhat confusing due to variations in the VDR length caused by the SCP (233). Depending upon the nucleotide sequence at this polymorphic site, the VDR can be comprised of 424 (F variant) or 427 (f variant) aa. Most of the earlier reports of HVDRR cases based their numbering on 424 aa since this is the most common genotype, and the HVDRR patients and normal subjects usually exhibited the SCP that resulted in the loss of the first 3 aa at the N terminus of the VDR (3). However, since researchers have universally adopted the 427-aa numbering system of Baker et al. (90), the numbering used in this review to describe mutations previously numbered using 424 aa has been adjusted by 3 to correspond to that of the published sequence (90).
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VI. HVDRR Mutations Causing the Ligand Binding-Positive Phenotype |
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TopAbstractI. The Syndrome of...II. Vitamin D PhysiologyIII. 1,25-Dihydroxyvitamin D...IV. Cellular Basis of...V. The VDR Gene...VI. HVDRR Mutations Causing...VII. HVDRR Mutations Causing...VIII. Additional Mutations In...IX. HVDRR Mouse ModelX. Treatment of HVDRRXI. Analysis, Summary, and...References |
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In 1988, Hughes et al. (146) used PCR to amplify exons of
the VDR gene in DNA samples from the F19 and F31 families that
exhibited the ligand binding-positive, but DNA binding-defective
phenotype (18, 203). In family F19 (18), a missense mutation (CAA to
CGA) was found in exon 3, which encodes the second zinc module of the
DBD. The mutation substitutes a polar uncharged glutamine for a
positively charged arginine at amino acid 73 (Arg73Gln) (Fig. 12). In family F31 (203), a missense
mutation (GGC to GAC) was identified in exon 2, which encodes the first
zinc module of the DBD. This mutation results in the replacement of a
glycine with a highly charged aspartic acid at amino acid 33 (Gly33Asp)
(Fig. 12). Both a normal and mutant allele were found in the parents’
DNA from both families, which confirmed the genetic transmission and
recessive nature of the disease. Using site-directed mutagenesis
each mutation was recreated in the wild-type VDR cDNA, and the
properties of the mutant VDR were analyzed after expression in COS-1
cells. Both F19 and F31 mutant VDRs exhibited normal
[3H]1,25-(OH)2D3 binding, and
each exhibited low-affinity binding to calf thymus DNA (146, 148).
These experiments confirmed that the mutations gave rise to the ligand
binding-positive, DNA binding-defective phenotype seen in the
patients’ fibroblasts (18, 203). In addition, Sone et al.
(148) demonstrated that the mutant VDRs were transcriptionally inactive
in cotransfection experiments in CV-1 cells. Using an osteocalcin-CAT
reporter plasmid, the authors showed that CAT activity could be induced
by the wild-type VDR but not by the two mutant VDRs, proving that these
mutations were the cause of vitamin D resistance (148). The VDR
mutations described by Hughes et al. (146) were the first
natural disease-causing mutations identified in the entire
steroid-thyroid-retinoid receptor gene superfamily. Mutations have
since been found in many of the classic receptors including TR (158),
AR (239), ER (240), GR (241), and mineralocorticoid receptor (MR)
(242).
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and tabulated in
Table 4. Sone et al. (243)
examined the VDR from two unrelated patients (F5 and F20) previously
shown to exhibit a ligand binding-positive and low-affinity DNA binding
phenotype by Liberman et al. (191, 197). In both patients, a
G-to-A missense mutation (CGG to CAG) was identified in exon 3. This
mutation replaces arginine with a glutamine at amino acid 80 located in
the second zinc finger module (Arg80Gln). The recreated Arg80Gln mutant
receptor bound [3H]1,25-(OH)2D3
normally and exhibited low-affinity binding to calf thymus DNA. In
addition, the Arg80Gln mutant receptor was unable to activate gene
transcription from a reporter plasmid, demonstrating that this
molecular defect is the cause of HVDRR in these cases (243). The same
Arg80Gln mutation was also identified in two siblings with HVDRR (F49)
by Malloy et al. (21). The F49 family and the families (F5,
F20) described by Sone et al. (243) both had origins in
North Africa; however, no genetic relationship between these families
could be established.
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A His35Gln mutation in the first zinc module of the VDR DBD was identified by Yagi et al. (20). This mutation changed a positively charged amino acid to a neutral one. The VDR from the patient’s cells (F45) exhibited normal 1,25-(OH)2D3 binding but, consistent with the location of the mutation in the DBD, the receptors exhibited low-affinity binding to DNA. The patient’s fibroblasts were transiently transfected with a VDRE-CAT reporter plasmid to test for 1,25-(OH)2D3 responsiveness. The transformed cells were unable to induce gene transcription. However, when the patient’s fibroblasts were cotransformed with the wild-type VDR cDNA and the reporter plasmid, the cells acquired the ability to respond to hormone.
Two mutations in the VDR DBD were reported by Rut et al. (244). One patient (F47) described in a previous report by Lin and Uttley (209) had an A-to-G mutation in exon 2. The missense mutation resulted in lysine at amino acid 45 being replaced by glutamic acid (Lys45Glu). In the same report, Rut et al. (244) examined the VDR gene in a patient (F44) described in a previous report by Simonin et al. (208). They identified a unique T-to-C base change in exon 2 that resulted in phenylalanine at amino acid 47 being replaced by isoleucine (Phe47Ile). The recreated mutant VDRs exhibited normal [3H]1,25-(OH)2D3 binding but were transcriptionally inactive (244).
A patient with HVDRR from a Moroccan family (F46) was examined for mutations in the VDR gene by Wiese et al. (245). At the cellular level, this patient exhibited a ligand binding-negative phenotype, suggesting that the mutation would lie in the LBD. The authors discovered an opal mutation (CGA to TGA) in which a C-to-T substitution introduced a premature stop codon at amino acid 73. The Arg73 stop mutation truncates the receptor in the middle of the second zinc finger module, resulting in the production of a 72-aa polypeptide. No evidence for the presence of a truncated VDR in the patient’s cells was demonstrated since both the LBD and monoclonal antibody-binding sites were deleted in the mutant protein. The Arg73 stop mutation occurs in the same codon that causes the Arg73Gln mutation in the D family (F19) (146). In the Arg73 stop mutation, CGA is mutated to TGA, while in the Arg73Gln mutation the CGA is mutated to CAA. The Arg73 stop mutation has also been identified in a young boy (F56) from Greece, who had HVDRR with alopecia (246).
Lin et al. (247) examined the VDR gene for mutations in a patient (F23) with HVDRR previously described by Sakati et al. (199). DNA sequencing uncovered a unique G-to-A base change in exon 2. This mutation resulted in a glycine at amino acid 46 being changed to an aspartic acid (Gly46Asp). The recreated Gly46Asp mutant VDR exhibited the characteristics of a DBD mutation in that the mutant receptor bound [3H]1,25-(OH)2D3 normally but displayed a reduced affinity for DNA. The mutant receptor was also shown to be transcriptionally inactive in reporter gene assays. The authors demonstrated that the patient was homozygous for the mutation and the patient’s father was a carrier of the mutant allele using PCR and a restriction fragment length polymorphism (RFLP) generated by the mutation. In contrast to the other DBD mutations described above, the mutation at Gly46 occurs in an amino acid that is not well conserved in the steroid-thyroid-retinoid receptor superfamily. However, Gly46 is conserved among receptors that form heterodimers with RXR, proteins such as TR and RAR.
A young boy (F53) of French-Canadian origin with HVDRR and alopecia has been reported by Zhu et al. (248). The patient’s fibroblasts lacked specific [3H]1,25-(OH)2D3 binding and failed to exhibit 24-hydroxylase mRNA induction after treatment with up to 100 nM 1,25-(OH)2D3. Northern blotting showed that the cells expressed a normal sized VDR mRNA, but Western blotting failed to detect any protein. A C-to-T base substitution was located in exon 2, which changed the codon for arginine (CAG) at amino acid 30 to an opal stop codon (TAG) (Arg30 stop). The 29-aa polypeptide represents the shortest truncated protein produced by a premature stop mutation in the VDR. The mutation eliminated 398 aa including the LBD, the monoclonal antibody epitope, the second zinc finger module, and a portion of the first zinc finger module.
The same Arg30 stop mutation was also identified in two children with HVDRR from a family (F54) living in Brazil (249). One child died at 4 yr of age due to cardiorespiratory insufficiency. Interestingly, the parents, who were first cousins, were phenotypically normal but had slightly elevated levels of serum 1,25-(OH)2D. The mean value for the father was 73 pg/ml and for the mother, 93 pg/ml (normal range 20–80 pg/ml). The elevated 1,25-(OH)2D values raise the possibility of mild vitamin D resistance in the heterozygotic parents, a finding that has not been documented previously in other parents of HVDRR children.
C. Structural analysis of DBD mutations
As noted above, the crystal structure of the VDR has not been
reported at this time. However, crystallographic studies of the GR
(108), RXR, and TR (111) DBD structures have been elucidated and, based
on these studies, one can extrapolate the alterations created by the
mutations to the VDR DBD (250, 251). Crystallographic analyses of the
GR demonstrate that amino acids 457–469 (corresponding to residues
38–50 in the VDR) form an -helix that joins the two zinc finger
modules. This -helix packs perpendicularly with a second -helix
at the base of the second zinc finger. Together, the hydrophobic
residues of these two -helices comprise the hydrophobic core of the
DBD. Lys45Glu and Gly46Asp mutations are located in the P box (aa
residues 41–46), a region of the receptor likely important in
contacting the DNA bases and determining the specificity of the
receptor for specific VDREs (Figs. 5 and 12). Rut et al.
(244) proposed that the Lys45Glu mutation would disturb the hydrogen
bonding between Lys45 and a guanine nucleoside in the VDRE half-site.
The conversion of Gly46 to aspartic acid, a bulky, charged amino acid,
probably leads to unfavorable electrostatic interactions with the
negatively charged phosphate backbone of the DNA helix, which may
prevent the receptor from contacting specific nucleotide bases in the
VDRE. Alternatively, the Gly46Asp mutation may eliminate the ability of
the VDR to specifically recognize VDREs (247). Similarly, the Gly33Asp
mutation is expected to have a repelling effect on the negatively
charged phosphate backbone due to the negatively charged aspartic acid
(244). On the other hand, the substitution of glycine for histidine in
the His35Gly mutation most likely eliminates a hydrogen bond donated
from the positively charged histidine to the phosphate of a guanine
nucleoside in the VDRE (244). The Phe47Ile mutation is a relatively
conserved substitution; however, the loss of the phenylalanine ring
structure may disrupt the integrity of the hydrophobic core of the DBD
and obstruct the formation of the proposed -helical structure at the
base of the first zinc finger, such that the VDR could not bind
normally to its VDRE (244).
It is interesting to note that four of the DBD mutations, Lys45Glu, Gly46Asp, Phe47Ile, and Arg50Gln, occur in a LysXxxPhePhe[Lys/Arg]Arg sequence motif that has been identified as a binding site for calreticulin (252, 253, 254). Calreticulin binds to the VDR (253), and cotransfection of calreticulin expression plasmids with a VDRE/RARE-luciferase reporter construct causes a decrease in the reporter gene activation by VDR in a dose-dependent manner (253). Since calreticulin may modulate VDR transactivation, disruption of the calreticulin-binding site may lead to a decrease in VDR function. The effects these mutations might exert to alter possible calreticulin actions on the VDR are unknown.
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VII. HVDRR Mutations Causing the Ligand Binding-Negative Phenotype |
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TopAbstractI. The Syndrome of...II. Vitamin D PhysiologyIII. 1,25-Dihydroxyvitamin D...IV. Cellular Basis of...V. The VDR Gene...VI. HVDRR Mutations Causing...VII. HVDRR Mutations Causing...VIII. Additional Mutations In...IX. HVDRR Mouse ModelX. Treatment of HVDRRXI. Analysis, Summary, and...References |
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and summarized in Table 4.
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). A total of
eight children from this kindred exhibited HVDRR with alopecia and the
same hormone binding-negative HVDRR phenotype. All of the affected
children were homozygous for the ochre stop mutation at Tyr295, and
their parents were heterozygous as determined by analysis of a
RsaI RFLP created by the mutation (22). Interestingly, the
30,000 mol wt truncated protein that is predicted to be produced by
this mutation could not be detected by Western blot analysis. In
addition, Northern blot analysis of RNA obtained from skin fibroblasts
or EBV-transformed lymphoblasts from the patients failed to detect any
mutant VDR mRNA in all but one case (F35). The absence of mRNA
transcripts has been reported in other genetic diseases where a
premature stop mutation has been found and, in this case, likely
accounts for the absence of the mutant-truncated VDR protein
(255, 256, 257).
B. Characterization of additional LBD mutations
Family F11, described in earlier papers, had two affected children
with HVDRR who exhibited the ligand binding-negative phenotype (16, 17, 25, 134). This family of Christian Arabs lives in the same town as the
extended kindred (F18,F34–39) described above who are Muslim Arabs.
Although there is no known genetic relationship between family F11 and
the large kindred, the Tyr295 stop mutation is the cause of HVDRR in
this family as well (258). The Tyr295 stop mutation was also identified
by Wiese et al. (245) in two related patients (F29, F30)
from Saudi Arabia who were previously studied by Bliziotes et
al. (13). These patients are apparently unrelated to the other
families with the same mutation.
An analysis of the VDR from the parents of children with the Tyr295 stop mutation led to an interesting observation. As mentioned previously, fibroblasts from the heterozygous parents, with the exception of the father in F18, were shown to have a normal abundance of VDR. In contrast, when the VDR was examined in EBV-transformed lymphoblasts from peripheral blood of the same individuals, the receptor abundance was half of normal controls, which is consistent with the heterozygous state of these individuals (22). The reason for the difference in the level of VDR expression in the two cell types remains unresolved. Interestingly, the VDR abundance in the lymphoblasts of the father in F18 was approximately one-fourth of the level in normal controls, which suggests that an additional problem related to VDR expression may have arisen in that individual.
The first mutation found in the VDR LBD that resulted in an amino acid substitution was described by both Rut et al. (259) and Kristjansson et al. (260). The patient from Kuwait (F21) had HVDRR without alopecia. Preliminary studies by Fraher et al. (14) on the patient’s fibroblasts showed absent [3H]1,25-(OH)2D3 binding. However, a later study by Rut et al. (259) showed that the fibroblasts contained normal amounts of [3H]1,25-(OH)2D3 binding but the affinity of the receptor for 1,25-(OH)2D3 was significantly reduced [dissociation constant (Kd) = 10 x 10-10 M] compared with normal controls (Kd = 0.7 x 10-10 M). 1,25-(OH)2D3 resistance was demonstrated by the failure of the patient’s fibroblasts to induce 24-hydroxylase activity when treated with the hormone (14, 259). Molecular analysis of the VDR gene identified a unique G-to-T missense mutation in exon 7 (259, 260). This mutation resulted in replacement of a positively charged arginine residue by a neutral charged leucine at amino acid 274. The recreated Arg274Leu mutant VDR was relatively resistant to vitamin D. However, the mutant VDR was able to activate gene transcription from a VDRE reporter plasmid but required 1,25-(OH)2D3 concentrations approximately 1,000-fold higher than the wild-type receptor (260).
A second missense mutation in the VDR LBD was described by Malloy et al. (261). The patient in this case exhibited three rare genetic disorders: HVDRR, congenital generalized lipoatrophic diabetes (Berardinelli-Seip syndrome), and persistent Müllerian duct syndrome (262). The patient, a Turkish boy (F51), had rickets and high 1,25-(OH)2D3 levels but did not have alopecia. He was treated with extremely high doses of calcitriol (Rocaltrol, 12.5 µg/day) which eventually normalized his serum calcium and ultimately improved his rickets. However, the child died of apparently unrelated problems. The patient’s fibroblasts had normal VDR abundance, but the affinity of the receptor for 1,25-(OH)2D3 was shown to be decreased by about 2-fold when assayed at 0 C. Induction of 24-hydroxylase mRNA in the patient’s cultured fibroblasts required approximately a 5-fold increase in 1,25-(OH)2D3 compared with control cells. Sequence analysis of the VDR gene uncovered a C-to-G missense mutation in exon 8. This mutation leads to replacement of histidine by glutamine at amino acid 305 (His305Gln). Interestingly, [3H]1,25-(OH)2D3-binding studies of the reconstructed mutant protein demonstrated an 8-fold lower affinity for 1,25-(OH)2D3 compared with the wild-type VDR when the assays were performed at 24 C. In gene transactivation assays, the His305Gln mutant VDR was approximately 5-fold less responsive to 1,25-(OH)2D3 compared with the wild-type VDR. RFLP analysis with AlwNI showed that a sibling with HVDRR was homozygous for the same mutation and that the parents were heterozygous. It is unclear how the HVDRR point mutation is related, if at all, to the two other genetic abnormalities present in this child. The boy’s sister, who also had HVDRR and the same mutation in the VDR, did not exhibit the other genetic defects.
Two novel missense mutations in the LBD have been characterized by Whitfield et al. (157). One patient, a girl (F4), had the classic symptoms of HVDRR but without alopecia. The patient’s fibroblasts were originally examined by Griffin and Zerwekh (190), who showed that the cells had normal 1,25-(OH)2D3 binding but had defective induction of 24-hydroxylase activity. Nucleotide sequencing of the VDR uncovered a T-to-G substitution in exon 8, which changed the codon for isoleucine to serine at amino acid 314 (Ile314Ser). In transactivation experiments, high concentrations of 1,25-(OH)2D3 were required to achieve normal activity. This patient showed a nearly complete cure when treated with pharmacological doses of 25-hydroxyvitamin D3.
The second patient in the study was a young girl (F52) who had HVDRR with alopecia. Sequencing showed that the patient had a C-to-T base change in exon 9, which converted an arginine to cysteine at amino acid 391 (Arg391Cys). In transactivation studies, the mutant VDR required high concentrations of 1,25-(OH)2D3, as well as increased levels of RXR to achieve normal gene transactivation. The "rescue" achieved by RXR supplementation demonstrates the importance of both 1,25-(OH)2D3 binding and heterodimerization with RXR in VDR-mediated gene activation (157).
Two siblings, a brother and sister from India (F57) that had HVDRR with alopecia, were studied by Cockerill et al. (246). The children’s parents were first cousins. Using cultured fibroblasts, [3H]1,25-(OH)2D3 binding was found to be normal, but 1,25-(OH)2D3 induction of 24-hydroxylase activity was absent. DNA sequence analysis revealed a single base alteration (A to C) that changed glutamine to proline at amino acid 259 (Gln259Pro). Transactivation experiments demonstrated that the recreated Gln259Pro mutant VDR was functionally inactive. In addition, to examine VDR protein-protein interactions and DNA binding, electrophoretic mobility shift assays were performed. Whereas the wild-type VDR formed two complexes (complex A and complex B) in the electrophoretic mobility shift assay, the Gln259Pro mutant VDR showed a reduction in the formation of complex B and an enhancement in complex A. The authors speculated that the Gln259Pro mutation in the VDR affected protein-protein interactions possibly by increasing the affinity of the receptor for an unidentified protein.
An additional mutation has been described in the VDR LBD by Thompson et al. (263). The HVDRR patient (F43) was shown to have a mutation in exon 5. In this case, a unique single base change (TGT to TGG) was found, which changed cysteine to tryptophan at amino acid 190 (Cys190Trp). Further details about this case were not included in this preliminary report.
C. Structural analysis of LBD mutations
From crystallographic studies of the holo- and/or apo-LBDs of RXR,
RAR, and TR and from sequence alignments of the nuclear receptors, a
generalized canonical structure of the LBD has been developed (129)
(see Fig. 13). In the model, the LBD is
composed of helixes H1 and H3–H10 with a variable length region
between H1 and H3 (loops 1–3). The majority of the conserved residues
in the LBD are located in a 34-aa cluster from the C terminus of H3 to
the middle of H5, including the E1 region. These conserved residues
form the hydrophobic core by holding together H3, H4, H5, H8, and H9
and loops 3–4 and 8–9. Residues in H1, H3, H5, ß-turn, loop 6–7,
H11, loop 11–12, and H12 form a hydrophobic ligand-binding pocket that
accommodates the hormone. Once a ligand enters the pocket, a lid formed
by H11 and H12 closes over the pocket.
In HVDRR, six single amino acid substitutions have been identified in
the VDR LBD that lead to 1,25-(OH)2D resistance (Figs. 13 and 14). One mutation, Cys190Trp,
occurs in loop 1–3, but the consequences of this alteration on the LBD
are not readily discernible from the LBD model. A second mutation,
Gln259Pro, occurs in H4. In RAR, this amino acid (Gln259 in RAR)
has been shown to stabilize the hydrophobic core by binding to the
main-chain NH groups in loop 8–9. Although Gln259Pro had no apparent
effect on ligand binding, there was evidence of impaired VDR-RXR-VDRE
formation (246). The Arg274Leu mutation occurs in H5. This arginine,
which is conserved in the retinoid-thyroid subgroup of nuclear
receptors, has been shown in RAR to be involved in hydrogen bonding
with the carboxylate group of the ligand. The Arg274Leu mutation lowers
the affinity of the VDR for 1,25-(OH)2D3, which
in vitro can be overcome by high concentrations of hormone
(260). The His305Gln mutation (261) occurs in loop 6–7, which causes a
decrease in hormone affinity, probably by increasing the flexibility of
the ligand-binding pocket. The Ile314Ser mutation occurs in H7 between
a ligand-binding contact observed in TR and a dimerization interface
in RXR. The mutation causes a subtle defect in heterodimerization
with RXR and a decreased response to
1,25-(OH)2D3 in transactivation assays (157).
The Arg391Cys mutation in H10 causes a strong heterodimerization defect
(157) consistent with the finding that H10 in RXR is the dimerization
helix (124). It is interesting to note that the location of three
mutations in the VDR (Gln259Pro, Arg274Leu, His305Gln) correspond to
the location of three mutations in the AR (Gln733His, Ala748Asp,
Met780Ile) that result in partial androgen insensitivity (239).
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VIII. Additional Mutations In The VDR Gene |
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TopAbstractI. The Syndrome of...II. Vitamin D PhysiologyIII. 1,25-Dihydroxyvitamin D...IV. Cellular Basis of...V. The VDR Gene...VI. HVDRR Mutations Causing...VII. HVDRR Mutations Causing...VIII. Additional Mutations In...IX. HVDRR Mouse ModelX. Treatment of HVDRRXI. Analysis, Summary, and...References |
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B. Splice site mutations
Hawa et al. (264) examined the molecular cause of HVDRR
with alopecia in a young Greek girl (F50). Using RT-PCR and DNA
sequencing, they showed that the patient’s RNA sequence diverged from
the wild-type sequence at nucleotide 147. The sequence from exon 4 was
deleted and the sequence that followed was from exon 5. Sequence
analysis of the VDR chromosomal gene found no mutations in the exons;
however, a G-to-C base change was found in the 5'-end of intron E (Fig. 10). This single nucleotide change converts the wild-type sequence from
GTAAGT to GTAACT and eliminates the 5'-donor
splice site [consensus sequence: GT(A/G)AGT]. The loss of the
5'-donor splice site caused exon 4 to be skipped in the processing of
the VDR transcript. The loss of exon 4 introduced a reading frameshift
that resulted in a premature stop codon in exon 5. The mutant protein
contains 92 aa of the wild-type sequence and an additional 6 aa due to
the frameshift (Glu92fs) (Fig. 12). The shortened VDR had no
[3H]1,25-(OH)2D3 binding and
failed to induce 24-hydroxylase activity.
A splice site mutation was also identified in a German patient (F56)
with HVDRR and alopecia (246). Studies of the patient’s fibroblasts
showed absent [3H]1,25-(OH)2D3
binding and failure to induce 24-hydroxylase activity with
1,25-(OH)2D3 treatment. In this case, a cryptic
5'-donor splice site was generated in exon 6. The mutation in this
case, a C-to-G transition, changed the sequence from GTCAGT
to GTGAGT. This single base change did not alter the amino
acid coding sequence in exon 6 but introduced a splice site that could
be recognized by the spliceosome complex during RNA processing. As a
result, the mutation caused a 56-bp deletion in exon 6 that led to a
frameshift 15 bases into exon 7. The mutant protein contains 233 aa of
the wild-type sequence and an additional 4 aa due to the frameshift
(Leu233fs) (Fig. 13). The mutation caused the truncation of 194 aa of
the VDR leading to a loss of 1,25-(OH)2D3
binding and hormone responsiveness.
C. Major structural mutations
There has been one case (F42) reported in which a major structural
defect in the VDR gene was found to cause HVDRR (263). The defect, a
deletion in the VDR gene, was identified by PCR and Southern blotting.
The deletion eliminated exons 7, 8, and 9. This is the only case thus
far reported in which a partial gene deletion has been shown to be the
cause of HVDRR.
D. Vitamin D resistance without a mutation in the VDR
Since the initial description of HVDRR as a genetic disorder,
mutations in the VDR gene were suspected to be the cause of
1,25-(OH)2D3 resistance exhibited by HVDRR
patients. In addition to mutations in the coding region of the VDR
gene, mutations may also be located in the promoter and noncoding
regions of the gene that could prevent VDR mRNA transcription or hinder
the translation of the VDR protein. However, although the VDR is the
principle determinant in the 1,25-(OH)2D3
action pathway, it is possible that target organ resistance to
1,25-(OH)2D3 may also result from mutations in
other proteins that are essential to the transactivation process. Some
of the likely candidates include transcription factors such as RXR, as
well as coactivators or corepressors of VDR activity. Defects in these
proteins may disturb the contact between the interacting protein and
the VDR and therefore prevent the VDR from binding to VDREs and/or
hinder VDR transactivation. Since RXR and other interacting proteins
are essential for the activity of many receptors in the
retinoid-thyroid subgroup, mutations in these proteins would be
expected to exhibit a more complex phenotype than simply vitamin D
resistance and HVDRR.
Hewison et al. (265) described a case of HVDRR in which a mutation could not be found in the VDR. The patient (F48), a young girl of English descent, exhibited all of the hallmarks of HVDRR including alopecia. The patient’s fibroblasts expressed a normal sized VDR mRNA and exhibited normal [3H]1,25-(OH)2D3 binding. However, no induction of 24-hydroxylase activity was observed when the fibroblasts were treated with up to 1 µM 1,25-(OH)2D3. Although the cells were clearly resistant to 1,25-(OH)2D3, no mutations were found within the coding region of the VDR gene. The patient’s VDR cDNA was recovered using RT-PCR and then expressed in CV-1 cells. In these cells cotransfected with a VDRE reporter plasmid, the patient’s VDR exhibited a normal transactivation response to 1,25-(OH)2D3. These results clearly demonstrated that the patient’s VDR was normal. The authors suggested that the tissue resistance was not due to a defect in the VDR and that the hormone resistance causing HVDRR was most likely the result of a mutation in an essential protein that participates in the 1,25-(OH)2D3 action pathway. However, the source of the defect causing vitamin D resistance in this case remains unknown.
In Cauca, Columbia, more than 200 patients have been diagnosed with a disease that somewhat resembles HVDRR without alopecia (266). The patients exhibit lower limb deformities due to rickets but are otherwise in good physical condition. Rickets limited to the lower extremities, as in these cases, has not been reported in other HVDRR families. The affected individuals have serum calcium levels that are within the normal range but their serum 1,25-(OH)2D levels are unusually high, suggesting target organ resistance. Analysis of 24-hydroxylase activity from fibroblasts of two of the most severely affected patients showed a low induction of enzyme activity by 1,25-(OH)2D3 when compared with normal controls. After RT-PCR, the VDR cDNA was obtained from these two patients and sequenced. No mutations were found. Since the cause of vitamin D resistance in this instance was not due to mutations in the VDR and a functional response to 1,25-(OH)2D3 was demonstrated, it has not been clearly ascertained whether this entity is a variant of HVDRR. However, these cases support the concept that target organ resistance may be due to mechanisms other than mutations in the VDR. The high prevalence of the disease in the population and the localized distribution of the rickets raises the possibility of an environmental cause.
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IX. HVDRR Mouse Model |
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TopAbstractI. The Syndrome of...II. Vitamin D PhysiologyIII. 1,25-Dihydroxyvitamin D...IV. Cellular Basis of...V. The VDR Gene...VI. HVDRR Mutations Causing...VII. HVDRR Mutations Causing...VIII. Additional Mutations In...IX. HVDRR Mouse ModelX. Treatment of HVDRRXI. Analysis, Summary, and...References |
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X. Treatment of HVDRR |
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TopAbstractI. The Syndrome of...II. Vitamin D PhysiologyIII. 1,25-Dihydroxyvitamin D...IV. Cellular Basis of...V. The VDR Gene...VI. HVDRR Mutations Causing...VII. HVDRR Mutations Causing...VIII. Additional Mutations In...IX. HVDRR Mouse ModelX. Treatment of HVDRRXI. Analysis, Summary, and...References |
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(OH)D3 and unfortunately later died of pneumonia as a
result of the disease (14). Fibroblasts from this patient were also
unresponsive to hormone treatment. Interestingly, when the recreated
Arg274Leu mutant VDR was examined in CV-1 cells for transactivation
activity, a positive response was observed at high doses of hormone
(260).
In general, HVDRR patients with alopecia appear to be more resistant to
treatment with vitamin D metabolites; however, a small number of these
patients have been treated successfully using vitamin D. Two patients
showed signs of improvement when given vitamin D or
1(OH)D3 (10, 187), and one patient responded to 25(OH)D
as well as 1(OH)D3 (11). 1(OH)D3 and
1,25-(OH)2D3 were also effective treatments in
other cases (18, 19, 186, 196, 204) including patients with the
Arg50Gln and Arg73Gln mutations (146, 233). Two siblings (F32), each
identified as having a Glu152 stop mutation, showed no increase in
serum calcium during high-dose vitamin D treatment, which raised their
circulating 1,25-(OH)2D levels to more than 100 times the
mean normal range. However, notwithstanding their low serum calcium
concentrations, healing of rickets and suppression of PTH was evident
(267). In one case, although vitamin D and
1,25-(OH)2D3 therapies were ineffective, the
patient did respond to oral phosphorus (7). The molecular cause of
HVDRR in this case has not been elucidated.
Although there are exceptions, our view of treatment responses to vitamin D fits the following general pattern. Patients who have mutations that result in a totally unresponsive VDR, such as those due to premature stop signals or those in the DBD that cause abnormal DNA binding, are unresponsive even to pharmacological doses of calcitriol. On the other hand, in patients with missense mutations in the LBD that result in a fully translated VDR, high doses of calcitriol are more likely to be effective. In cases that fail to respond to 1,25-(OH)2D3, intensive calcium therapy is indicated (as described below).
B. Calcium
Since hypocalcemia is a major manifestation of HVDRR, restoration
of serum calcium by the administration of calcium salts has been used
as a therapy for treating HVDRR patients. In one study by Sakati
et al. (199), high-dose oral calcium therapy was given to a
patient (F23) who had failed to respond to calciferols. The patient
received 3–4 g of elemental calcium orally per day and showed clinical
improvement during 4 months of therapy. The patient was later shown to
have the Gly46Asp mutation (247). In another study, Balsan et
al. (268) successfully used long term intravenous calcium
infusions in a child with HVDRR who failed prior treatment with large
doses of vitamin D derivatives and/or oral calcium (11). This therapy
apparently bypasses the calcium absorption defect in the intestine
caused by the mutant VDR. High doses of calcium were infused
intravenously during the nocturnal hours over a 9-month period.
Clinical improvement accompanied by relief of bone pain was observed
within the first 2 weeks of the start of intravenous therapy. Within 7
months, the child gained both weight and height. Eventually, the serum
calcium normalized, the secondary hyperparathyroidism was reversed, and
the rickets was ultimately cured as assessed by x-ray and bone biopsy.
The syndrome recurred, however, when the intravenous infusions were
discontinued. Several other investigators have similarly demonstrated
beneficial effects of intravenous calcium infusion in HVDRR children
(13, 269, 270). In a study by Weisman et al. (269), two
patients treated with calcium infusion showed a decrease in serum
alkaline phosphatase activity and an increase in their serum calcium
and phosphate over a 1-yr period (269). x-Ray analysis showed
resolution of the rickets with the appearance of normal mineralization
of bone. After radiological healing of the rickets has been achieved
with intravenous calcium infusions, high-dose oral calcium therapy has
been shown to be effective in maintaining normal serum calcium
concentrations in some cases (270). For those HVDRR children that do
not respond to high-dose calcitriol, it seems reasonable to initiate
this two-step protocol at an early age.
The effectiveness of intravenous calcium to heal rickets suggests that the lack of calcium absorption caused by the mutant VDRs in the intestine is the major defect causing the bone disease. The intravenous calcium infusion bypasses this intestinal defect and provides mineral to the bone-forming site. Apparently, normal bone formation is achieved if the mineral supply is adequate, despite the lack of vitamin D action on the bone cells. Since some intestinal calcium absorption is vitamin D-independent, oral calcium in high dosage has been used as an effective treatment. Once healing of rickets has been achieved by intravenous calcium, the requirement for mineral is decreased, and maintenance calcium treatment by the oral route may adequately protect against rickets.
C. Prenatal diagnosis
A prenatal diagnosis of HVDRR is now possible in pregnant women
from high-risk families. Cultured cells from chorionic villus samples
or amniotic fluid have been used to determine whether the fetus has
HVDRR using [3H]1,25-(OH)2D3
binding, induction of 24-hydroxylase activity, and RFLP analyses of
known mutations (271, 272). A summary of the RFLPs generated by
mutations in the VDR gene is shown in Table 5.
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XI. Analysis, Summary, and Conclusions |
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TopAbstractI. The Syndrome of...II. Vitamin D PhysiologyIII. 1,25-Dihydroxyvitamin D...IV. Cellular Basis of...V. The VDR Gene...VI. HVDRR Mutations Causing...VII. HVDRR Mutations Causing...VIII. Additional Mutations In...IX. HVDRR Mouse ModelX. Treatment of HVDRRXI. Analysis, Summary, and...References |
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). Usually, the diagnosis of HVDRR has
been based on high circulating levels of
1,25-(OH)2D3 and resistance of cultured skin
fibroblasts to 1,25-(OH)2D3 treatment. Many
cases have been analyzed for
[3H]1,25-(OH)2D3 binding to the
VDR and/or transactivation of reporter genes by the VDR, which revealed
that the disease was caused by heterogeneous mutations in the VDR gene.
A number of cases of HVDRR have not yet been examined for mutations in
the VDR gene. Since some of these cases presented late in life, it is
possible they may have been due to nonhereditary or acquired resistance
to 1,25-(OH)2D3. Analysis of the syndrome of HVDRR provides many interesting insights into vitamin D physiology and the role of the VDR in mediating 1,25-(OH)2D3 action. Our analysis of the findings leads us to make the following observations.
At the time of this writing, eight missense mutations have been found in the VDR DBD that prevent the receptor from activating gene transcription even though 1,25-(OH)2D3 binding is normal. Six missense mutations have been identified in the LBD that cause reduced or complete hormone insensitivity either by decreasing hormone affinity and/or interfering with heterodimerization with RXR. Four mutations have been found that introduce premature termination codons, which truncate the VDR and lead to complete hormone resistance. Two unique splice site mutations have been demonstrated that also introduce premature termination codons. A partial deletion encompassing exons 7–9 of the VDR gene has also been described.
Despite the many pleiotropic processes regulated by 1,25-(OH)2D3, children with HVDRR exhibit only symptoms that relate to their calcium deficiency and/or alopecia. We conclude that in vivo, the pleiotropic actions of vitamin D can be compensated for by other mechanisms but the calcemic effects cannot.
The improvement in rickets after chronic intravenous calcium infusion or oral calcium raises interesting questions about the role of vitamin D in bone homeostasis. Correction of hypocalcemia and secondary hyperparathyroidism leads to healing of the rickets, as assessed by x-ray and bone biopsy. Thus, although there are many well defined actions of vitamin D on osteoblasts, the response to normalization of serum calcium suggests that 1,25-(OH)2D3 action on osteoblasts is not essential to form normal bone. The implication is that 1,25-(OH)2D3 action on bone is mainly due to its effects on intestinal mineral absorption to provide calcium and phosphate for bone formation. The same conclusion was reached by Underwood and DeLuca (273), who showed that the development of rickets is prevented in totally vitamin D-deficient rats by calcium and phosphate infusions in the absence of vitamin D. The ramifications for therapy are that normalization of serum calcium by intravenous and/or oral calcium supplements will bypass the defective intestinal VDR and heal the rickets.
Although 1,25-(OH)2D3 is an inhibitor of PTH production, in the HVDRR children, normalizing serum calcium by intravenous infusion is sufficient to suppress their PTH overproduction and does not require 1,25-(OH)2D3 action. In addition, intravenous calcium therapy without phosphate is adequate to correct all of the metabolic abnormalities in children with HVDRR. This suggests that the hypophosphatemia in these patients is mainly the result of secondary hyperparathyroidism and not inadequate intestinal phosphate absorption.
A number of interesting issues concerning alopecia and HVDRR are worth
noting. Since VDRs have been found in hair follicles (27, 28),
1,25-(OH)2D3 action through the VDR appears to
be essential for the differentiation of this structure during
embryogenesis. Also, Marx et al. (26) have shown that there
is some correlation between the severity of rickets and the presence of
alopecia. HVDRR patients with alopecia tend to be nonresponsive to
calciferols while those without alopecia tend to be responsive to high
doses of vitamin D. The alopecia or some degree of hair loss appears to
be associated primarily with DBD mutations or premature stop mutations,
which usually result in complete hormone unresponsiveness and total
resistance. A few patients with LBD mutations did not develop alopecia.
Interestingly, the patient from family F48 had alopecia despite not
having a mutation in the VDR. Alopecia remains unchanged in patients
that undergo successful therapy or that show spontaneous improvement.
In families with a history of HVDRR, the absence of body hair in
newborns provides initial evidence for the disease. Interestingly,
alopecia usually has not been found in other conditions related to
defective vitamin D action, including 1-hydroxylase deficiency (VDDR
I) and other forms of vitamin D deficiency. In HVDRR, the hair
follicles appear normal on microscopic examination, but without hair
shafts present. We suspect that 1,25-(OH)2D3
action is necessary for the establishment of the hair synthetic
machinery at a critical stage in development.
A prenatal diagnosis of HVDRR is now possible in pregnant women from high-risk families. Cultured cells from chorionic villus samples or amniotic fluid have been used to ascertain whether the fetus has HVDRR using [3H]1,25-(OH)2D3 binding, induction of 24-hydroxylase activity, and RFLP analyses (271, 272).
The mechanism for the spontaneous improvement in some HVDRR children as they get older is an interesting dilemma. One hypothesis that explains the normalization of the 1,25-(OH)2D3 endocrine system, in the face of inactive VDRs, is that some other transcription factor can substitute for the defective vitamin D system. Possibly RAR, RXR, or TR can substitute for a nonfunctional VDR and activate the appropriate target genes to reverse the hypocalcemia and restore the bones to normal. This hypothetical explanation remains untested. However, Whitfield et al. (157) have shown in vitro that addition of RXR can rescue mutant VDR with defects in the dimerization domain and restore hormone responsiveness.
HVDRR is a relatively rare disease compared with androgen (239)- and thyroid (158)-resistant syndromes, which occur more frequently in the population. On the other hand, only a few cases of glucocorticoid (241), mineralocorticoid (242), and estrogen (240) resistance have been reported, while progesterone resistance has not been described. It is therefore interesting to speculate on the reasons for these differences in prevalence of diseases caused by mutations in the steroid hormone receptors. We believe that HVDRR is relatively rare because it is a true recessive disease. Heterozygotes are asymptomatic and for these rare mutations to affect both alleles in an individual, consanguinity is usually required. On the other hand, the number of cases of androgen resistance (androgen insensitivity syndrome or AIS) is greater, in part, because the AR gene is on the X-chromosome and a single mutation would result in the disease in the hemizygous male population. Females with two copies of the AR gene appear phenotypically normal even when one allele is mutated. Males acquire the mutant AR gene from their asymptomatic, heterozygotic mothers and consanguinity is not required. Thyroid hormone resistance (generalized resistance to thyroid hormone or GRTH), on the other hand, is often caused by dominant negative mutations in which one defective allele inactivates the normal allele (158). Like androgen resistance, only a single mutant TR allele is necessary to cause thyroid resistance. In contrast, dominant negative mutations have not been described in HVDRR where heterozygotes exhibit a normal phenotype. Since TR and VDR are thought to act through a common heterodimerization partner, RXR, one might speculate that the difference between GRTH and HVDRR due to a dominant negative mechanism might be due to the ability of TRs to form homodimers while VDRs do not. Few reports of glucocorticoid and mineralocorticoid resistance have been described (241, 242). Total resistance is likely to be lethal while mild cases might be more prevalent in the population than one might expect but are not diagnosed because they are not as easily recognized as androgen insensitivity syndrome, GRTH, and HVDRR (241). Mutations in ER and PR are rarely recognized although a single case of defective ER has been described in a male patient (240). The rarity of clinical cases was originally thought to be due to lethality of the mutation but, since knockout mice survive, this may also be due to lack of ascertainment or interference with successful pregnancy.
In conclusion, the biochemical and genetic analysis of the VDR in the HVDRR syndrome has yielded important insights into the structure and function of the receptor in mediating 1,25-(OH)2D3 action. Similarly, studies of children affected with HVDRR continue to provide further insight into the biological role of 1,25-(OH)2D3 in vivo. A concerted investigative approach of HVDRR at the clinical, cellular, and molecular level has proven exceedingly valuable in understanding the mechanism of action of 1,25-(OH)2D3 and improving the diagnostic and clinical management of this rare genetic disease.
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Footnotes |
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1 Supported in part by NIH Grant DK-42482. 
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References |
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TopAbstractI. The Syndrome of...II. Vitamin D PhysiologyIII. 1,25-Dihydroxyvitamin D...IV. Cellular Basis of...V. The VDR Gene...VI. HVDRR Mutations Causing...VII. HVDRR Mutations Causing...VIII. Additional Mutations In...IX. HVDRR Mouse ModelX. Treatment of HVDRRXI. Analysis, Summary, and...References |
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-loop (loop 1–3)
flips under H6, which stabilizes H11 and H12 positions.
