Yonago Acta medica 2000;43:27–38

Association between Vitamin D Receptor Gene Polymorphisms andRenal Osteodystrophy in Patients on Maintenance Hemodialysis
Kiyotaka Kohama, Jiro Uemasu, Hironaka Kawasaki, Eiji Nanba* and Akihide
Tokumoto†
Second Department of Internal Medicine, Faculty of Medicine, *Gene Research Center, Tottori
University, Yonago 683-0826 and †Division of Nephrology, Sanin Rosai Hospital, Yonago 6830002, Japan
We examined the possible involvement of vitamin D receptor (VDR) gene polymorphisms
in patients on maintenance hemodialysis and further investigated the relation between
VDR genotypes and bone histology. Two hundred and nine patients undergoing regularhemodialysis (male/female ratio, 124/85) were included in this study. DNA was extracted
from peripheral blood leukocytes. VDR genotypes were analyzed as restriction fragment
length polymorphisms, by using BsmI, ApaI, TaqI and FokI. Lumbar bone mineraldensity was measured by dual-energy X-ray absorptiometry, and expressed as a Zscore. Serum 1,25(OH)2D3, osteocalcin and intact-parathyroid hormone (i-PTH) were
determined by an immunoradiometric assay. In 97 patients, bone biopsy was performed
and the histology was divided into osteitis fibrosa, mild lesion, adynamic bone disease
and osteomalacia. Serum levels of osteocalcin, i-PTH and bone mineral density weresignificantly lower in the presence of B, A, t and f alleles. However, in this study, we did
not find any association between bone histology and the four VDR genotypes. We
concluded that renal osteodystrophy in dialysis patients was modified by environmental
factors such as medication with active vitamin D, age, gender and duration of chronic
renal failure, and that the impact of the VDR allelic effect may play a small role indetermining on bone histology.
Key words: bone mineral density; hemodialysis; renal osteodystrophy; vitamin D receptor gene
polymorphism

80% of bone mass features in individuals. However, the search for a gene which determinesbone mass has long been unsuccessful.
In 1994, Morrison and others (1992) demonstrated that common allelic polymorphisms
in the gene encoding the vitamin D receptor(VDR) were significantly correlated with bonedensity in healthy individuals. Since then, aconsiderable number of studies were reported toconfirm this interesting finding. However, theresults have been controversial to date.
Recently, VDR has become a focus of research interest not only as a candidate gene controlling bone density, but also as a gene associ-Children obviously bear a striking likeness totheir parents, and bone density has been considered a genetic determinant in the inheritanceof appearance from parents to children. Smithand colleagues (1973) assessed bone density on
the midshaft radius in monozygotic twins anddizygotic twins to prove for the first time that asignificantly larger variation in intrapair differences was observed in dizygotic twins compared to that in monozygotic twins. Later investigations based on twin studies and epidemiologic surveys all favored this finding, indicatingthat there is an inheritable component related tobone mass which may be associated with 50 toAbbreviations: bp, base pair; BMD, bone mineral density; IGF, insulin-like growth factor; i-PTH, intactparathyroid hormone; kb, kilobase; nVDRE, negative vitamin D responsive element; RFLP, restriction
fragment length polymorphism; VDR, vitamin D receptor

27

K. Kohama et al.

Table 1. Patient characteristics
Number of patientsAge (year)

Chronic glomerulonephritis
123 (74/49)
55 ± 11*Diabetes mellitus
48 (30/18)
63 ± 11*
Rheumatoid arthritis
12 ( 2/10)
58 ± 9Polycystic kidney disease
8 ( 5/ 3)
59 ± 19
Others18 (13/ 5)
56 ± 24
Values are expressed as mean ± SD.
( ), male/female ratio.
* P < 0.05; chronic glomerulonephritis versus diabetes mellitus.

ated with bone turnover. Patients with chronic
renal failure are generally accompanied byrenal osteodystrophy caused by anomalies inbone turnover. Due to the successful spread ofmaintenance hemodialysis, patients withchronic renal failure have attained considerable
improvement in survival time, but at the sametime patients are increasingly left confronted
with renal osteodystrophy. Thus, renal osteodystrophy is an important factor influencing thequality of life of patients on maintenance hemodialysis. To date, however, there have been a
few reports describing the relationship between
the prevalence of renal osteodystrophy and
VDR polymorphisms. In the present study, we
assessed a possible correlation between VDRpolymorphisms and renal osteodystrophy inpatients undergoing maintenance hemodialysis
by measuring bone mineral density (BMD) andmarkers for bone turnover. We also assessedsuch correlation in patients on maintenancehemodialysis by performing a bone biopsy.

Z-score–0.105 ± 1.341
0.331 ± 1.042–0.233 ± 0.681
0.167 ± 1.150–0.750 ± 1.666

RFLP analysis of VDR locus
Prior to trial, informed consent was individuallyobtained from patients and their relatives by theattending physicians following full explanations of the aim of the research, and guarantees
of privacy. Two-milliliter aliquots of peripheral blood samples were collected from thepatients and stored in the presence of EDTA.
Genomic DNA was extracted from leukocytes
obtained from the blood samples with the conventional extraction method using phenol/
chloroform followed by ethanol precipitation.
So far, the VDR gene, mapped to the longarm of human chromosome 12, was considered
to contain 9 exons. In 1997, Miyamoto and coworkers (1996, 1997) identified two previously
unreported exons located 20 kilobase (kb) upstream from the originally reported exon 1; subsequently designated them exon 1A and exon
1B, and renamed exon 1 as exon 1C. Thus, theVDR gene is comprised of 11 exons which, together with intervening introns, span approximately 75 kb, where the translation start codon
is located in exon 2 as reported by Baker andothers (1998) (Fig. 1). A restriction fragment
length polymorphism (RFLP) involving the lossof an initiation codon (ATG) by a single basesubstitution from T to C (ACG) starts translation from 9-base pair (bp) downstream, raising
the possibility that the polypeptides translated
from the two alleles differ by three amino acidresidues.
To amplify a 265-bp sequence in exon 2, a
10-µL reaction mixture containing 50 ng oftemplate DNA, 1 µmol/L each of the primers
VDR2a: 5'-AGCTGGCCCTGGCACTGA

Materials and Methods

Patients
The subjects consisted of 209 patients (124males and 85 females) with chronic renal failure on maintenance hemodialysis. Primary diseases included chronic glomerulonephritis,
diabetes mellitus, rheumatoid arthritis, autosomal dominant polycystic kidney disease andother miscellaneous diseases in 123, 48, 12, 8
and 18 patients, respectively (Table 1).

28

Relation between VDR and ROD in HD patients

CTCTGCTCT-3' and VDR2b: 5'-ATGGAAACACCTTGCTTCTTC TCCCTC-3', 10mmol/L Tris-HCl (pH 8.3), 50-mmol/L KCl,
1.5-mmol/L MgCl2, 0.2 mmol/L each of deoxyribonucleotide triphosphates and 0.25 U of Taq
DNA polymerase (Perkin-Elmer Co., Foster
City, CA) were incubated in a microtube using
a thermal cycler (Touch Down, Hybaid Ltd.,
Ashford, Middlesex, United Kingdom) (Grosset al., 1996). A reaction cycle consisting ofsequential incubations for denaturation at 94°C
for 45 s, for annealing at 60°C for 45 s, and forextension at 72°C for 45 s was repeated 35
times, except that denaturation at 94°C for an
additional 7 min was included in the first cycle.
Five-microlitter aliquots of the polymerase
chain reaction (PCR) product was incubated at
37°C for 3 h in a 10-µL reaction mixture containing 50-mmol/L NaCl, 10-mmol/L Tris-HCl(pH 7.5), 10-mmol/L MgCl2, 1-mmol/L 1,4-dithiothreitol and 4 units of the restriction endonuclease FokI (Nippon Gene, Toyama, Japan).
Aliquots of the FokI digest of PCR products
were electrophoresed for 30 min at 100 V througha gel (1% agarose S plus 1% agarose X) containing 0.4-µg/mL ethidium bromide preparedin a buffer containing 89-mmol/L Tris-borate
and 2-mmol/L EDTA. As DNA size markers,
φX174/HaeIII digest and φX174/Hinc-II digest(Nippon Gene) were used.

DNA bands on the gel were visualized under 312-nm UV lamp (ATTO Bioinstrument,
Tokyo, Japan) and fluorescent DNA bands were
photographed with a Polaroid camera for RFLP
assessment. Upon digestion by FokI, the 165bp PCR products derived from an allele having
a FokI site in exon 2, designated f, were split
into two bands, 69 bp and 196 bp, respectively,
while those derived from an allele not having a
FokI site in the corresponding sequence, designated F, remained as a single band. Thus,
genotypes of the PCR products derived from individual subjects were presented as ff, Ff or FF.
Genotyping was similarly performed regarding RFLPs involving BsmI and ApaI sites
in intron 8, and RFLP involving the TaqI site inexon 9 (Tokita et al., 1996), using PCR primers
described by Morrison and others (1992). The
825-bp PCR products derived from an allele
having a BsmI site in intron 8, designated b,
were split into two bands, 650 bp and 175 bp,
respectively, upon digestion with BsmI, while
those derived from an allele not having a BsmI
site in the corresponding sequence, designatedB, remained a single band.
The 740-bp PCR products derived from anallele having both an ApaI and a TaqI site between intron 8 and exon 9, designated a, weresplit into two bands, 529 bp and 211 bp, respectively, upon digestion with ApaI, while thoseqpChromosome 12

ATG

TGA

3' VDR gene

5'
Exon 1A 1B

1C 2

3

4

Fok I

5

6

Bsm I

5'

7

8

Apa I

9

Taq I Taq I (invariant site)

UTR
Exon 2

Exon 7

Exon 8

3'

Exon 9Fig. 1. Genomic organization and restriction map of the vitamin D receptor (VDR) gene.

29

K. Kohama et al.

derived from an allele not having an ApaI site in the corresponding sequence, designatedA, remained a single band.
In exon 9, there is an additional invariant TaqI site that is
not involved in generating
RFLP. Thus, the above 740-bp
PCR products derived from anallele having an RFLP-associated TaqI site in exon 9, designated t, were split into threebands, 291 bp, 247 bp and 202
bp, respectively, upon digestion with TaqI, while those derived from an allele not having
an RFLP-associated TaqI site in
the corresponding sequence,designated T, were split into
two bands, 493 bp and 247 bp,
respectively (Fig. 2).

Measurements of boneturnover markers and BMD

Bsm I

Apa I
(bp)

(bp)

825
650

740
529
211

175

M

BB

Bb

bb

M

Taq I

AA

Aa

aa

Fok I
(bp)

(bp)

265
196
69

493
291
247
202

M

TT

Tt

tt

M

FF

Ff

ff

Fig. 2. Restriction fragment length plymorphism analysis of poly-morphic vitamin D receptor (VDR) alleles. bp, base pair.
Serum 1,25(OH) 2 D 3 , osteocalcin and intact-parathyroid
hormone (i-PTH) were determined as bone biopsy was performed within 7 days after adturnover markers. Patients with fracture, scoli- ministration of the second label. Before boneosis or lumbar osteoarthritis upon X-ray biopsy was performed, diazepam and ketamine
examination were excluded from evaluation of were given. Under local anesthesia with lidoBMD, since bones with these anomalies could caine, bone specimens were taken verticallyprovide abnormal BMD values. BMD at the from the right iliac crest with a manually driven
lumbar spine (L2–L4) was measured by dual- drill attached to Kitasato’s trephine. During
energy X-ray absorptiometry (model XR-26, this biopsy procedure, no patients complained
Norland, Fort Atkinson, WI). For age- and of severe pain. None of the serious complicagender-matched comparisons, BMD values tions were experienced. Biopsy specimens
were expressed as Z-scores.
were fixed in dehydrated ethanol, embedded inmethylmethacrylate and left for 7 days for complete solidification. Sections (4–5 µm) were cut
Bone biopsy
on a microtome, mounted on glass slides andBone biopsy was performed in 97 patients with stained with Villanueva-Goldner and aluminon.
chronic renal failure on maintenance hemo- The bone turnover rate was quantitatively asdialysis. To determine the mineralization rate, sessed by bone histomorphometry. According
bones were subjected to tetracycline double- to the classification described by Sherrard andlabeling: tetracycline hydrochloride was given others (1993), bone tissues were histopathoat a dose of 750 mg/24 h for 2 days, and after a logically diagnosed as osteitis fibrosa, osteocessation for the following 10 days, the drug malacia and mild lesion and adynamic bone
was further given during the next 4 days. Bone disease (Table 2).

30

Relation between VDR and ROD in HD patients

Table 2. Histologic classification of renal osteodystrophy
Area of osteoid(%)

Area of fibrosis(%)

Bone formation rate(µm2/mm2 of tissue area/day)

Osteitis fibrosa
< 15
> 0.5
Increased
Osteomalacia
> 15
< 0.5
Decreased
Mixed*> 15> 0.5
NormalMild lesion
< 15
< 0.5
Normal (> 108)
Adynamic bone disease
< 15
< 0.5
Decreased (< 108)
Area of osteoid, osteoid volume/bone volume; area of fibrosis, fibrosis volume/tissue volume; bone formation rate, bone formation rate/bone surface.
* Mixture of osteitis fibrosa and osteomalacia.
The table is cited from Sherrard et al. (1993).

the study of a sample of European population(Uitterlinden et al., 1996), the present value of
1.0% was significantly lower, suggesting an
ethnic variation in allele frequencies. The
above allele frequencies for the BsmI-RFLP did
not significantly differ among patients of different ages, or among those showing different
serum values for Ca, P, alkaline phosphatase or
active vitamin D. Values for serum osteocalcin
were 43.5 ± 3.5 mg/dL, 54.9 ± 30.6 mg/dL and
78.0 ± 66.4 mg/dL for BB, Bb and bb, respectively, indicating that the values in individuals
possessing the b haplotype were significantly
higher (P < 0.05). Individuals possessing the bhaplotype also showed significantly higher
values for i-PTH: 130 ± 55 pg/mL, 149 ± 144
pg/mL and 278 ± 260 pg/mL for BB, Bb and bb,
respectively (P < 0.05). The age- and gendermatched Z-scores for BMD were also significantly higher in individuals possessing b haplotype (P < 0.01): –2.50 ± 0.14, –0.50 ± 1.44 andStatistical analysis
The results were assessed by the analysis ofvariance, contingency table test and regression
analysis. P values < 0.05 were regarded as significant.

Results
Regarding patient characteristics (Table 1),
average age and BMD values did not significantly differ among patients with regard to primary diseases, except that the average age wasapproximately 8 years older in the diabetes
mellitus group than in the chronic glomerulonephritis group.
Allele and genotype frequencies for BsmIRFLP were 1.0%, 18.2% and 80.9% for BB, Bb
and bb, respectively (Table 3). Compared to thevalue of approximately 20% reported for BB in

Table 3. Relationship between bone turnover markers and Bsm I genotypes of VDRGenotype
Bb

BB

P*
bb

Number of subjects
2 (1.0%)38 (18.2%)
169 (80.9%)
Age(year)
77.5 ± 2.1
56.0 ± 15.8
57.0 ± 13.9Ca
(mg/dL)6.1 ± 3.3
7.4 ± 2.1
7.8 ± 2.2P
(mg/dL)
6.5 ± 1.6
5.6 ± 2.0
5.7 ± 1.6
Alkaline phosphatase (IU/L)
158 ± 102149 ± 96186 ± 307(pg/mL)6.9 ± 5.7
8.9 ± 5.8
8.0 ± 5.6
1,25 (OH)2D3
Osteocalcin(mg/dL)43.5 ± 3.5
54.9 ± 30.6
78.0 ± 66.4
i-PTH
(pg/mL)130 ± 55
149 ± 144278 ± 260
Z-score–2.50 ±0.14–0.50 ± 1.44
0.43 ± 1.58
* Significant difference in P values among the three genotypes.
i-PTH, intact parathyroid hormone; NS, not significant; VDR, vitamin D receptor.

31

NS
NS
NS
NS
NS
< 0.05< 0.05< 0.01

K. Kohama et al.

Table 4. Relationship between bone turnover markers and Apa I genotypes of VDRGenotype
Aa

AA

P*
aa

Number of subjects
23 (11.0%)
79 (37.8%)
107 (51.2%)
Age(year)
55.9 ± 16.7
56.7 ± 14.5
57.7 ± 13.0Ca
(mg/dL)
7.4 ± 2.2
7.3 ± 2.2
8.1 ± 2.2P
(mg/dL)5.7 ± 2.0
5.8 ± 1.7
5.6 ± 1.5
Alkaline phosphatase (IU/L)
164 ± 120
194 ± 375
170 ± 217
(pg/mL)6.1 ± 1.6
10.6 ± 7.3
6.7 ± 3.8
1,25 (OH)2D3
Osteocalcin(mg/dL)56.5 ± 30.0
65.4 ± 60.4
82.9 ± 58.7
i-PTH
(pg/mL)129 ± 118262 ± 207273 ± 258
Z-score–0.68 ± 1.50
0.32 ± 1.73
0.36 ± 1.50
* Significant difference in P values among the three genotypes.
i-PTH, intact parathyroid hormone; NS, not significant; VDR, vitamin D receptor.

NS
NS
NS
NS
NS
NS
< 0.05< 0.05

Table 5. Relationship between bone turnover markers and Taq I genotypes of VDRGenotype
Tt

TT

P*
tt

Number of subjects
169 (80.9%)
35 (16.7%)
5 (2.4%)Age(year)
57.7 ± 13.6
54.3 ± 17.0
57.4 ± 19.8
Ca
(mg/dL)
7.9 ± 2.2
7.3 ± 2.4
6.7 ± 2.5P
(mg/dL)5.8 ± 1.6
5.5 ± 1.9
5.5 ± 1.8
Alkaline phosphatase (IU/L)
185 ± 309
144 ± 103
201 ± 87
(pg/mL)
7.5 ± 4.5
11.0 ± 8.6
6.2 ± 0.6
1,25 (OH)2D3
Osteocalcin(mg/dL)77.1 ± 66.0
58.3 ± 37.1
55.4 ± 26.9
i-PTH
(pg/mL)273 ± 98
182 ± 172129 ± 123
Z-score0.45 ± 1.58
–0.56 ± 1.39
–1.55 ± 1.58
* Significant difference in P values among the three genotypes.
i-PTH, intact parathyroid hormone; NS, not significant; VDR, vitamin D receptor.

0.43 ± 1.58 for BB, Bb and bb, respectively.
Regarding ApaI-RFLP, allele and genotype
frequencies were 11.0%, 37.8% and 51.2% for
AA, Aa and aa, respectively (Table 4). Again,

NS
NS
NS
NS
NS
NS
< 0.05< 0.01the frequencies differed from those reported in
the European population, but less significantly
than in cases of BsmI-RFLP. Allele frequencies
for ApaI-RFLP did not significantly differ among

Table 6. Relationship between bone turnover markers and FokI-genotypes of VDRGenotype
Ff

FF

P*
ff

Number of subjects
82 (39.2%)
93 (44.5%)
34 (16.3%)
Age(year)
58.3 ± 14.8
56.4 ± 14.5
56.5 ± 13.2
Ca
(mg/dL)8.4 ± 2.1
7.4 ± 2.3
7.4 ± 2.2P
(mg/dL)5.6 ± 1.6
5.8 ± 1.6
5.7 ± 1.7
Alkaline phosphatase (IU/L)
179 ± 257
197 ± 344
131 ± 84
(pg/mL)9.1 ± 6.4
6.7 ± 3.0
8.4 ± 6.7
1,25 (OH)2D3
Osteocalcin(mg/dL)80.8 ± 52.3
75.7 ±74.3
49.2 ± 34.5
i-PTH
(pg/mL)278 ± 236
260 ± 214172 ± 112
Z-score0.40 ± 1.60
0.29 ± 1.66
–0.37 ± 1.42
* Significant difference in P values among the three genotypes.
i-PTH, intact parathyroid hormone; NS, not significant; VDR, vitamin D receptor.

32

NS
NS
NS
NS
NS
< 0.05NS< 0.05

Relation between VDR and ROD in HD patients(mg/dL)

50

50

BB

Bb

bb

AA

Aa

[2]

[38] [169][23][79] [107](mg/dL)

150

150

100
50

P = 0.01
P = 0.03

100
50

Tt

tt

FF

Ff

[169] [35]

[5]

[82]

[93] [34]

TT

400
200

BB

Bb

[2]

[38] [169]

400
200

AA

(pg/mL)(pg/mL)
600

200

aa

400
200
0

Tt

tt

FF

Ff

ff

[169] [35]

[5]

[82]

[93]

[34]

TT

ff

Aa

[23] [79] [107]

600
400

P = 0.02
P = 0.04

0

bb

0

0

0

600

0

aa

i-PTH

(mg/dL)

Osteocalcin

Osteocalcin

100

0

0

(pg/mL)P = 0.008

600

i-PTH

Osteocalcin

Osteocalcin

100(pg/mL)

150

i-PTH

P = 0.04

i-PTH(mg/dL)
150

Fig. 3. Serum osteocalcin levels compared among
polymorphic vitamin D receptor (VDR) genotypes.
Values shown are mean ± SE. [ ], number ofsubjects.

Fig. 4. Serum intact-parathyroid hormone (i-PTH)levels compared among polymorphic vitamin D
receptor ( VDR) genotypes. Values shown are mean
± SE. [ ], number of subjects.

patients of different ages, or among those showing different serum values for Ca, P, alkaline
phosphatase, 1,25(OH)2D3 or osteocalcin. However, values for i-PTH and Z-scores representing BMD were significantly higher in individuals possessing the a haplotype.
Allele and genotype frequencies for TaqIRFLP were 80.9%, 16.7% and 2.4% for TT, Tt
and tt, respectively (Table 5), indicating thatgenotype frequency for tt was extremely low.
Z-scores for BMD significantly varied among
patients having different genotypes; that is, thehighest score was obtained for TT, followed bythose for Tt and tt in that order.
Allele and genotype frequencies for the
FokI-RFLP were 39.2%, 44.5% and 16.3% for
FF, Ff and ff, respectively (Table 6). Values forosteocalcin and Z-scores significantly variedamong patients having different genotypes; i.e.,the highest value was obtained for FF, followedby those for Ff and ff in that order.
It is noted that allele and genotype frequencies for RFLPs obtained with chronic renal
failure in the present study did not differ fromthose reported for healthy subjects.
Distribution of serum osteocalcin levels
were presented with respect to genotyping for

BsmI, ApaI, TaqI and FokI RFLPs (Fig. 3).
Distribution of serum i-PTH levels (Fig. 4) andZ-scores (Fig. 5) were similarly presented.
Low levels of osteocalcin and i-PTH were notably correlated with B, A, t and f haplotypes,

P = 0.007
P = 0.01

2

2

1

1

Z-scoreZ-scoreP = 0.01
P = 0.002

0
–1
–2

0
–1
–2

–3

BB

Bb

[2]

[38] [169]

–3

bb

AA

2

1

1

–1
–2

aa

P = 0.032

Z-scoreZ-scoreP = 0.01
P = 0.001

0Aa[23] [79] [107]

0
–1
–2

–3

–3

Tt

tt

FF

Ff

ff

[169] [35]

[5]

[82]

[93]

[34]

TT

Fig. 5. Z-scores compared among polymorphic
vitamin D receptor (VDR) genotypes. Values shown
are mean ± SE. [ ], number of subjects.

33

K. Kohama et al.

[n]

[n]

50

50

50

40

40

40

40

30

30

30

30

20

20

10

10

17

gen

e

8
4

33

VDR Aa
gen
otyp

Apa

LP

0
10

20

AA

11

aa

e

I-RF

OM
ADB
ML

psy

psy

OF

bb

otyp

I-RF

1

4
4

bio

1

Bb

VDR

2

0

OM
15
ADB
47
ML

ne

BB

Bsm

1

10

Bo

1

5

0

10

bio

0

0

0

20
0

OF

ne

0

10

Bo

20

50

LP

[n]

[n]
50

50

40

40

40

40

30

30

30

30

20

20

10

10

1

1

Tt

gen

1

tt

otyp

I-RF

e

psy

0

VDR

OF

8

28
6

LP

4

11

VDR

Fok

1

21

FF

bio

0

0

8

0

OM
ADB
ML

ne

9

TT

Taq

0

4

Bo

47
18

10

8

Ff

2

gen

ff

otyp

I-RF

e

OM
ADB
ML

psy

16

0

20
0

bio

0

10

OF

ne

20

50

Bo

50

LP

Fig. 6. Histologic comparison of bone biopsy in terms of polymorphic vitamin D receptor (VDR) genotypes.
ADB, adynamic bone disease; ML, mild lesion; OF, osteitis fibrosa; OM, osteomalacia; RFLP, restriction
fragment length polymorphism.

sions and 5 adynamic bone diseases in thosehaving genotype Bb. These constant distribution patterns of bone diseases among RFLPs
generated by different restriction endonucleases
were consistently demonstrated by the contingency table analysis (Fig. 6).
With respect to a possible correlation between serum levels of bone turnover markers
and histologic types of bone diseases, both
osteocalcin and i-PTH levels were the highestin osteitis fibrosa, followed by mild lesion, andthe values were lowest in adynamic bone disease. There was a positive correlation between
osteocalcin and i-PTH, indicating that these twomarkers were both useful in predicting renal
osteodystrophy (Fig. 7).

suggesting that VDR polymorphisms are correlated with BMD via bone turnover markers.
Histopathological diagnosis of bone biopsy
specimens revealed osteitis fibrosa, mild lesion,adynamic bone disease and osteomalacia in 19,
57, 20 and 1 cases, respectively. Distribution of
the incidences of these bone diseases among
RFLP genotypes did not differ by the restriction
endonuclease used: in incidence, mild lesion
was the highest, osteitis fibrosa and adynamic
bone disease were almost equal, and osteomalacia was the lowest in all studies. For example,
in BsmI-RFLP, 17 osteitides fibrosa, 47 mild
lesions, 15 adynamic bone diseases and 1 osteomalacia were found among patients having
genotype bb, and 1 osteitis fibrosa, 10 mild le-34

Relation between VDR and ROD in HD patients(pg/mL)1500

F =19.16
P < 0.0001

F = 6.729
P = 0.0004
200

i-PTH

Osteocalcin(mg/dL)
300

100

500

0

0

OF

ML

ADB

OM

OF

(mg/dL)
600

Osteocalcin

1000

ADB

OM

ADB adynamic bone disease
i-PTH intact-parathyroid hormone
ML mild lesion
OF osteitis fibrosa
OM osteomalacia

y = 42.38 + 0.123xr = 0.535
P < 0.001

400

ML

200

Fig. 7. Serum levels of bone turnover markers
evaluated with respect to the histology of bonebiopsy.

0
0

500

1000

1500

2000
(pg/mL)

i-PTH

alleles. These findings were consistent with thepositive correlation observed between particular VDR polymorphic alleles and serum i-PTH
in patients with chronic renal failure in thepresent study. There is a negative vitamin D
responsive element (nVDRE) on the PTH geneto which 1,25(OH)2D3 acts negatively by binding to the nVDRE (Demay et al., 1992). Taken
together, it seems that VDR polymorphisms
affect nVDRE-mediated regulation of the PTH
gene expression.
Vitamin D and PTH primarily act on osteoblasts by binding to respective cellular receptors.
Subsequently, the activated osteoblasts stimulate bone resorption through the monocytemacrophage colony-stimulating factor and theosteoclast differentiation factor. At the sametime, however, vitamin D stimulates normal
calcification of bone matrix including type I
collagen, alkaline phosphatase and osteocalcin
produced by osteoblasts. PTH also enhances
calcification by stimulating the release of
insulin-like growth factor (IGF)-I and -II fromosteoblast cells, and stimulates the accumula-

Discussion
In the present study, a significant correlation
was demonstrated between patients with chronic renal failure having the RFLP haplotypes B,
A, t and f and patients showing lower Z-scoresfor BMD. This fact suggests that particular
RFLP haplotypes can be used as a risk factor
predictive of osteoporosis. Indeed, RFLP
haplotypes B, A, t and f were associated withlow levels of serum osteocalcin and i-PTH;
polymorphic VDR genes could determine dynamic features of bone remodeling via actionsof these bone turnover markers.
Carling and colleagues (1997) assessed
extracellular Ca2+-mediated suppression of iPTH secretion in 62 patients with primary
hyperparathyroidism due to parathyroid adenoma using dispersed adenoma cells in vitro, andshowed that Ca2+-mediated PTH inhibition produced a higher median effective dose and reduced suppression in adenoma cells from patients who were homozygous for b, a and T
35

K. Kohama et al.

tion of IGF-I and transforming growth factor-β
in the bone. Thus, PTH seems to be one of thepotent regulators of dynamic bone remodeling(Christakos et al., 1989; Linkhart et al., 1989),
and VDR polymorphism may be associatedwith the mechanism of bone remodeling.
Since a reduction in BMD is due to a failure
in normal bone remodeling, a VDR polymorphism could be associated with BMD at thelevel of the bone remodeling process.
There remains the important question of
how BsmI- and ApaI-RFLPs, due to a nucleotide change in introns, and TaqI-RFLP, due to asynonymous codon change (ATC/ATT) in exon
9, lead to a functional change in VDR proteins.
Although an answer to the question of theinvolvement of changes in 3'UTR, poly(A) tail,transcription efficiency and messenger RNA
stability has been proposed (Gross et al., 1998),
there has been no conclusive evidence which
can explain this problem.
Regarding FokI-RFLP, the nucleotidechange in the VDR gene was reported to alter
not only the translation initiation site but alsotranscription efficiency as demonstrated byluciferase assay, and change the translation efficiency determined in vitro (Arai et al., 1997).
These changes should all affect BMD.
As Sainz and coworkers (1997) clearly demonstrated, VDR gene polymorphisms and bonedensity were significantly correlated with eachother in 100 prepubertal American girls of
Mexican descent in California. The success of
their findings may be due to the recruitment offemales at puberty when bone density reaches
the maximum; during the puberty, genetic factors may most predominantly affect bone density control, while environmental factors such as
nutrition and exercise may play more influential
roles at later ages (Cooper et al., 1996).
Histopathological diagnosis of bone biopsy
revealed osteitis fibrosa, mild lesion, adynamic
bone disease and osteomalacia in 19, 57, 20 and1 cases, respectively. The incidence distribution of these bone diseases did not vary whendifferent restriction endonucleases were used
for VDR genotyping. A cyclical process, called
remodeling, maintains a dynamic steady stateof bone structure density without changing thesize and shape, through sequential resorption
and formation of a small amount of bone at thesame site. Renal osteodystrophy can be defined
as a failure of normal bone remodeling (Hruska,
1997). Upon the introduction of hemodialysis,
patients with end stage renal failure tend to havecomplications with hyperphosphatemia, hypocalcemia, acidosis and a moderate increase in iPTH due to the retarded elimination of serum
phosphates and impaired activation of vitamin
D. These signs are mainly associated with mild
lesion. Complication with severe secondary
hyperthyroidism is associated with osteitis fibrosa. When patients are medicated with calcium carbonate as a precipitant for aluminum accumulation or for hypocalcemia, or have complications with diabetes mellitus, adynamic bonedisease develops. In the case of 1,25(OH)2D3
deficiency and hypocalcemia, osteomalacia
develops due to insufficient calcification. A
spectrum of bone diseases developing into renal
osteodystrophy may be partly influenced byvarious factors including duration of chronic
renal failure, primary disease, age, complications, nutrition, extent of exercise, aluminum
accumulation and effectiveness of medication.
In the present study, although the pathogenesisof renal osteodystrophy may well be affected by
VDR polymorphism at the stage of i-PTH
secretion and activation of osteoblasts associated with remodeling, the incidence distributionof bone diseases did not vary depending on different restriction endonucleases used for VDR
genotyping. This discrepancy may be explained by assuming that the haplotype-dependent
genetic consequences may have been obscuredby the prominent improvement of bone diseaseinduced by medication with active vitamin D
and calcium pharmaceuticals.
Bone turnover markers such as i-PTH andosteocalcin have proven useful in predicting
renal osteodystrophy. Malluche and others (1984)
first claimed osteocalcin as a useful marker reflecting the state of bone formation by demonstrating that there was a significant correlation
between osteocalcin and cellular and noncellular parameters of bone formation includingbone turnover. In general, i-PTH values below
60–65 pg/mL reflect adynamic bone disease,36

Relation between VDR and ROD in HD patientsMiner Res 1997;12:915–921.
2 Baker AR, McDonnel DP, Hughes M, Crissp
TM, Mangelsdorf DJ, Haussler MR, et al. Cloning and expression of full-length cDNA encoding
human vitamin D receptor. Proc Natl Acad Sci
USA 1998;85:3294–3298.
3 Carling T, Ridefelt P, Hellman P, Rastad J,
Akerstrom G. Vitamin D receptor polymorphisms
correlate to parathyroid cell function in primary
hyperparathyroidism. J Clin Endocrinol Metab
1997;82:1772–1775.
4 Christakos S, Gabrielides C, Rhoten WB. Vitamin
D dependent calcium binding proteins: chemistry,
distribution, functional considerations and molecular biology. Endocrine Rev 1989;10:3–26.
5 Cooper G, Umbach DM. Are vitamin D receptor
polymorphisms associated with bone mineraldensity? A meta-analysis. J Bone Miner Res
1996;11:1841–1849.
6 Demay MB, Kierman MS, DeLuca HF, Kronenberg
HM. Sequences in the human parathyroid hormone
gene that bind the 1,25-dihydroxy vitamin D 3
receptor and mediate transcriptional repression in
response to 1,25-dihydroxy-vitamin D3. Proc Natl
Acad Sci USA 1992;89:8097–8101.
7 Faugere MC, Qi QL, Geng ZP, Malluche HH.
What serum levels of parathyroid hormone discriminate between high vs normal or low boneturnover in dialyzed patients? J Am Soc Nephrol
1994;5:878.
8 Gross C, Musiol IM, Ross Eccleshall T, Malloy
PJ, Feldman D. Vitamin D receptor gene polymorphisms: analysis of ligand binding andhormone responsiveness in cultured skin fibroblasts. Biochen Biophys Res Comm 1998;242:
467–473.
9 Gross C, Ross Eccleshall T, Malloy PJ, Luz Villa
M, Marcus R, Feldman D. The presence of apolymorphism at the translation initiation site of
the vitamin D receptor gene is associated withlow bone mineral density in postmenopausal
Mexican-American women. J Bone Miner Res
1996;11:1850–1855.
10 Hruska KA. Renal osteodystrophy. Ballieres
Clin Endocrinol Metab 1997;11:165–194.
11 Linkhart TA, Mohan S. Parathyroid hormone
stimulates release of insulin-like growth factor(IGF-I) and IGF-II from neonatal mouse calvarina in organ culture. Endocrinology 1989;
125:1484–1491.
12 Malluche HH, Faugere MC, Fanti P, Price PA.
Plasma levels of bone Gla-protein reflect boneformation in patients on chronic maintenancedialysis. Kidney Int 1984;26:869–874.
13 Miyamoto K, Kesterson RA, Yamamoto H,
Taketani Y, Nishikawa E, Tatsumi S, et al.
Structural organization of the human vitamin D
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Mol Endocrinol 1997;11:1165–1179.

while those above 450–460 pg/mL reflect osteitis
fibrosa. Clearly, bone turnover markers arethus useful as noninvasive diagnostic markers
of renal osteodystrophy. However, bone biopsy
is necessary when i-PTH values between 65 and460 pg/mL reflect various histologic features ofrenal osteodystrophy as reported by Faugere
and others (1994).

Conclusion
Possible correlations between VDR polymorphic genotypes and factors involving BMD
were evaluated in 209 patients with chronic renal failure by assessing allele and genotype
frequencies of VDR polymorphisms, bone turnover markers and BMD. Individuals having B,
A, t and f haplotypes showed low levels of
serum osteocalcin and i-PTH and low BMD.
The histologic spectrum of bone diseases asassessed by biopsy did not significantly varydepending on different restriction endonucleases used for VDR genotyping. VDR polymorphism was shown to be one of the factors
determining BMD. Histologic features of bone
in renal osteodystrophpy failed to correlate with
VDR polymorphisms, probably due to bias
generated by clinical improvement due to medication. Further studies that exclude the distorting effects of active vitamin D and calcium supplementations are warranted to clarify this matter.

Acknowledgments: We are grateful to Prof. Keisuke
Satoh, Dept. of Pharmacology and to Prof. Ikuo
Miyagawa, Dept. of Urology, Faculty of Medicine,
Tottori University, for their useful comments on the
manuscript.

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(Received December 20, Accepted December 27, 1999)

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