Vitamin D receptor gene polymorphisms in Turkish children with vitamin D deficient rickets.
ABSTRACT Vitamin D deficient rickets is prevalent in Turkey and a considerable number of children are at risk of growth retardation, impaired bone formation and fracture. In order to check whether vitamin D receptor (VDR) gene polymorphism relates to the vitamin D deficient rickets, we analyzed VDR gene FokI, TaqI and ApaI polymorphisms in 24 Turkish vitamin D deficient rickets patients and 100 healthy controls. We found that "A" (ApaI) allele is more abundant in patients than controls (83 vs 57%, p = 0.002) but there were no significant differences for FokI (p = 0.693) and TaqI (p = 0.804) allele frequencies between patients and controls. We also showed that the frequency of Tt and Aa genotypes was significantly decreased in patients. Our results indicated that VDR gene polymorphisms might be an important factor for genetic susceptibility to vitamin D deficient rickets in the Turkish population.
- SourceAvailable from: ncbi.nlm.nih.gov[show abstract] [hide abstract]
ABSTRACT: The epidemic scourge of rickets in the 19th century was caused by vitamin D deficiency due to inadequate sun exposure and resulted in growth retardation, muscle weakness, skeletal deformities, hypocalcemia, tetany, and seizures. The encouragement of sensible sun exposure and the fortification of milk with vitamin D resulted in almost complete eradication of the disease. Vitamin D (where D represents D2 or D3) is biologically inert and metabolized in the liver to 25-hydroxyvitamin D [25(OH)D], the major circulating form of vitamin D that is used to determine vitamin D status. 25(OH)D is activated in the kidneys to 1,25-dihydroxyvitamin D [1,25(OH)2D], which regulates calcium, phosphorus, and bone metabolism. Vitamin D deficiency has again become an epidemic in children, and rickets has become a global health issue. In addition to vitamin D deficiency, calcium deficiency and acquired and inherited disorders of vitamin D, calcium, and phosphorus metabolism cause rickets. This review summarizes the role of vitamin D in the prevention of rickets and its importance in the overall health and welfare of infants and children.Journal of Clinical Investigation 09/2006; 116(8):2062-72. · 12.81 Impact Factor
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ABSTRACT: The role in skeletal metabolism of the steroid hormone Vitamin D and its nuclear receptor (VDR) is well known. In addition, however, Vitamin D is also involved in a wide variety of other biological processes including modulation of the immune response and regulation of cell proliferation and differentiation. Variations in the Vitamin D endocrine system have thus been linked to several diseases, including osteoarthritis, diabetes, cancer, cardiovascular disease and tuberculosis. Evidence to support this pleiotropic character of Vitamin D has included epidemiological studies on circulating Vitamin D hormone levels, but also genetic epidemiological studies. Genetic studies provide excellent opportunities to link molecular insights with epidemiological data and have therefore gained much interest. DNA sequence variations which occur frequently in the population are referred to as "polymorphisms" and are usually suspected of having only modest and subtle effects. Recent studies have indicated many polymorphisms to exist in the VDR gene, but the influence of VDR gene polymorphisms on VDR protein function are largely unknown. Sofar, three adjacent restriction fragment length polymorphisms (RFLP) for BsmI, ApaI and TaqI, respectively, at the 3' end of the VDR gene have been the most frequently studied sofar. But because these polymorphisms are probably non-functional, linkage disequilibrium (LD) with one or more truly functional polymorphisms elsewhere in the VDR gene is assumed to explain the associations observed. Research is therefore focussed on documenting additional polymorphisms across the VDR gene to verify this hypothesis, and on trying to understand the functional consequences of the variations. Substantial progress has been made including the discovery of novel polymorphisms in the large promoter region of the VDR gene. Eventually, results of this research will deepen our understanding of variability in the Vitamin D endocrine system and might find applications in risk-assessment of disease and in predicting response-to-treatment.The Journal of Steroid Biochemistry and Molecular Biology 06/2004; 89-90(1-5):187-93. · 3.98 Impact Factor
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ABSTRACT: The effect of a T-C transition polymorphism at the translation initiation codon of the human vitamin D receptor (VDR) gene on the biological function of the encoded protein was investigated. Of 239 Japanese women volunteers subjected to genotype analysis for this polymorphism, 32 (13%) were genotype MM (the M allele is ATG at the putative translation start site), 75 (31%) were genotype mm (the m allele is ACG at the putative translation start site), and 132 (55%) were genotype Mm. The bone mineral density (BMD) in the lumbar spine (L2–L4) was determined for 110 healthy premenopausal women from the volunteers and was shown to be 12.0% greater (p < 0.05) for mm homozygotes than for MM homozygotes. Synthesis of the proteins by the M and m alleles from the cloned cDNAs in vitro and in transfected COS-7 cells revealed them to have a size of 50 and 49.5 kD, respectively, as determined by sodium dodecyl sulfate polyacrylamide gel electrophoresis. This size difference is consistent with initiation of translation of the M allele-encoded protein from an ATG codon located at nucleotides +10 to +12 in the conventional open reading frame. The extent of vitamin D–dependent transcriptional activation of a reporter construct under the control of a vitamin D response element in transfected HeLa cells was ∼1.7-fold greater for the m type VDR than for the M type protein. These results suggest that the polymorphism at the translation start site of the VDR gene may modulate BMD in premenopausal Japanese women.Journal of bone and mineral research: the official journal of the American Society for Bone and Mineral Research 05/1997; 12(6):915 - 921. · 6.04 Impact Factor
The Turkish Journal of Pediatrics 2008; 50: 30-33Original
Vitamin D receptor gene polymorphisms in Turkish children
with vitamin D deficient rickets
Gamze Bora1, Behzat Özkan2, Didem Dayangaç-Erden1
Hayat Erdem-Yurter1, Turgay Coşkun3
Departments of 1Medical Biology, and 3Pediatrics, Nutrition and Metabolism Unit, Hacettepe University Faculty of
Medicine, Ankara, and 2Pediatrics, Atatürk University Faculty of Medicine, Erzurum, Turkey
SUMMARY: Bora G, Özkan B, Dayangaç-Erden D, Erdem-Yurter H, Coşkun
T. Vitamin D receptor gene polymorphisms in Turkish children with vitamin
D deficient rickets. Turk J Pediatr 2008; 50: 30-33.
Vitamin D deficient rickets is prevalent in Turkey and a considerable number of
children are at risk of growth retardation, impaired bone formation and fracture.
In order to check whether vitamin D receptor (VDR) gene polymorphism
relates to the vitamin D deficient rickets, we analyzed VDR gene FokI, TaqI
and ApaI polymorphisms in 24 Turkish vitamin D deficient rickets patients
and 100 healthy controls. We found that “A” (ApaI) allele is more abundant
in patients than controls (83 vs 57%, p=0.002) but there were no significant
differences for FokI (p=0.693) and TaqI (p=0.804) allele frequencies between
patients and controls. We also showed that the frequency of Tt and Aa
genotypes was significantly decreased in patients. Our results indicated that
VDR gene polymorphisms might be an important factor for genetic susceptibility
to vitamin D deficient rickets in the Turkish population.
Key words: vitamin D receptor gene polymorphisms, vitamin D deficient rickets.
Vitamin D deficiency rickets is prevalent today
in many developing countries while it is on the
rise in the developed ones. Hence, Turkey is
of no exception, with a frequency of rickets of
6% among children under three years according
to one recent study. Not only infants but also
a considerable number of adolescent girls,
pregnants and nursing mothers are at risk of
vitamin D deficiency in Turkey1.
Among the factors responsible for the high
prevalence of vitamin D deficiency in developing
countries and its resurgence in developed
countries are: limited sunshine exposure due
to individuals’ spending more time indoors
to watch television and work on computer or
their avoiding sunshine intentionally for fear
of air pollution and skin cancer development.
Traditional clothing (covered dress) further limits
the exposure time to sunshine and thus decreases
the endogenous synthesis of vitamin D1,2.
Vitamin D regulates calcium and phosphate
homeostasis in the body and has a positive
impact on bone mineralization3. The most
active form of vitamin D (a steroid hormone),
1,25 (OH)2 D, exerts its effect on target tissues
through the vitamin D receptor (VDR). VDR
is a ligand-dependent transcription factor
and belongs to the steroid hormone receptor
superfamily. The liganded VDR results in
dimerization of the receptor and it forms
homodimers or heterodimers with one of the
retinoid X receptors (RXRa, RXRb, and RXRg).
VDR homodimers or VDR-RXR heterodimers
bind to specific enhancer elements, referred to
as vitamin D response element, and activate
target gene transcription4,5. Many tissues
contain VDR and thus 1,25 (OH)2D is expected
to affect these tissues and cells like epidermis,
macrophages, prostate, breasts, pancreas, and
The emerging field in nutrition science, so-
called nutritional genomics (nutrigenomics),
draws attention to the fact that development
of certain conditions of diseases may be linked
to the polymorphisms that individuals carry.
Presence of certain polymorphisms renders
the host susceptible for certain diseases even
in the presence of recommended intake of
the offending nutrient. Whether this is so for
vitamin D and calcium is not yet clear. There
have been some studies conducted in Africa
indicating a possible link between certain VDR
polymorphisms and calciopenic rickets8.
The VDR protein is encoded by the VDR gene,
which is linked to 12q13.1. VDR gene is about
100kb, consists of 9 exons and has highly
polymorphic sites. Several polymorphisms
in the VDR gene have been reported so far,
including FokI, TaqI and ApaI. FokI, which
is a translation start codon polymorphism,
is located in exon 2, and due to the T to
C transition, translation initiates 3 amino
acid downstream of the first ATG. The other
polymorphism, which is localized in exon 9, is
TaqI and ATT codon is converted to ATC, but
either of them encodes isoleucine amino acid.
ApaI is an intronic polymorphism, which is a
G/T transition, localized in intron 89,10.
In this study, we therefore determined VDR
genotypes (FokI, TaqI and ApaI) of 24
Turkish children diagnosed with vitamin D
deficiency rickets and compared the allelic
frequencies of these polymorphisms with those
of normal children.
Material and Methods
The present study included 24 children with
vitamin D deficient rickets ranging in age
from 3 to 32 months and 100 children with
no history of rickets as controls. Patients were
recruited from the outpatient pediatric clinics
of Atatürk University Faculty of Medicine,
Erzurum, a province located in the East of
Turkey with a high frequency of rickets.
The diagnosis of rickets was confirmed both
biochemically and radiologically in children
who presented to the clinics with a different
combination of signs and symptoms like tetany,
craniotabes, rachitic rosary, Harrison’s groove,
delayed closure of anterior fontanelle, delayed
dentition, enlarged wrists and bowed legs.
Individual’s peripheral blood was obtained and
genomic DNA was extracted from leukocytes
by salting out method. Exon 2 and intron8/
exon9 of the VDR gene were amplified by
polymerase chain reaction (PCR). PCR products
were digested with FokI (37°C, 1.5h), TaqI
(65°C, 1h) and ApaI (37°C, 2h) and subjected
to electrophoresis in 3% agarose gel. Digested
fragments were visualized after staining with
ethidium bromide. VDR genotypes of each
subject were identified according to the
Distribution of VDR genotypes and allelic
frequencies of patients were calculated and
compared with healthy individuals (controls)
using chi-square (χ2) test. Conformance to
Hardy-Weinberg equilibrium was assessed using
χ2 test as well. P-values less than 0.05 were
considered to indicate statistical significance.
The present study was performed among 24
rickets patients and 100 healthy individuals.
The allelic frequencies of FokI, TaqI and
ApaI are shown in Table I. “F”, “T”, “A”
indicate the absence and “f”, “t”, “a” the
presence of digestion sites for FokI, TaqI
and ApaI, respectively. Our findings indicate
that “A” (ApaI) allele is more abundant in
patients than controls (83 vs 57%, p=0.002).
However, there were no significant differences
for FokI (p=0.693) and TaqI (p=0.804) allele
frequencies between patients and controls.
Table I. VDR Allelic Frequencies of
Patients and Controls
Table II shows the distribution of VDR
genotypes between patients and controls. We
observed that the most common genotypes
were FF, TT, AA in patients and FF, Tt,
Aa in controls. The frequency of TT, tt and
AA genotypes was higher in patients than
controls, while the frequency of Tt and Aa was
significantly decreased in patients (p<0.001).
In contrast, the genotype distribution of FokI
was not significantly different between patients
and controls (p=0.793).
The genotype distributions of all polymorphisms
in controls and FokI in patients were in
Hardy-Weinberg equilibrium. There was a
departure from the equilibrium for TaqI and
Volume 50 • Number 1 VDR Polymorphisms in Nutritional Rickets 31
Recently, there have been many efforts to
investigate an association between VDR
polymorphisms and several diseases in different
populations11-13. In our study, we report VDR
gene polymorphisms (FokI, TaqI and ApaI) in
vitamin D deficient rickets for the first time
Multiple polymorphic variations exist in the VDR
gene, each of which could have different types
of consequences. VDR polymorphisms can affect
VDR mRNA/protein level, stability, translation
efficiency and protein-protein interactions14.
More than 25 different polymorphisms are
currently known to be present at the VDR
gene and most are in/near the regulatory areas
rather than coding sequences. The most studied
polymorphisms include FokI, TaqI and ApaI.
FokI is an exonic polymorphism, which leads
to T/C transition, and variant alleles generate
two VDR gene products that differ in length
by three amino acids. Some studies have
shown that “F” allele and FF genotype may be
more advantageous for bone mineralization15.
However, we did not observe differences in
the frequencies of “F” allele and FF genotypes
between patients and controls. In contrast
to our results, Lu et al.16 reported that “F”
alleles and FF genotypes were more common
in patients suffering from vitamin D deficient
rickets. Likewise, Fischer et al.8 indicated that
“F” allele was more abundant in rickets.
TaqI and ApaI polymorphisms are localized
in 3’ regulatory region and are in linkage
disequilibrium with 3’UTR. TaqI is another
exonic polymorphism that does not affect the
amino acid sequence of encoded protein. We
Table II. Distribution of VDR Genotypes
demonstrated a significant increase in TT and
tt and decrease in Tt genotypes in patients.
However, Fischer8 and Kaneko et al.17 reported
that neither allele nor genotype frequencies
of TaqI were significantly different among
rickets and controls in Nigeria and Mongolia
populations, respectively. ApaI, which is an
intronic polymorphism, affects neither splicing
site nor transcription factor binding site. We
showed that the frequencies of “A” allele and
AA genotype were increased and Aa genotype
was decreased in rickets compared to controls;
however, Wei-Ping et al.18 indicated that the
distribution of ApaI polymorphism between
rickets and controls was balanced. Although these
polymorphisms seem to be non-functional, they
can be used as a marker to detect a functional
allele due to the linkage disequilibrium. The
3’UTR region of the VDR gene is involved
in the regulation of gene expression, so these
polymorphisms may play an important role in
mRNA stability. It is possible that different
allelic frequencies and VDR genotypes among
populations can occur due to the gene-gene and
We demonstrated that VDR gene polymorphisms
might be an important factor for genetic
susceptibility to vitamin D deficient rickets in
the Turkish population. Further studies will be
needed to determine the functional consequences
of different VDR alleles. Considering the
ethnic background of the patients in different
countries, it may be normal to find different
allelic frequencies among patients. If a causal
relationship could be established between certain
polymorphisms and vitamin D deficient rickets,
carriers of these particular polymorphisms
might be supplemented with more vitamin D
32 Bora G, et al The Turkish Journal of Pediatrics • January - February 2008
(more than the recommended daily dose of
200-400 IU) and calcium in order to prevent
the development of poor bone mineralization
and rickets (personalized dietetic approach
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Volume 50 • Number 1 VDR Polymorphisms in Nutritional Rickets 33