Original paper
Prevalence of vitamin D receptor gene Fok I polymorphism in patients with systemic lupus erythematosus – a preliminary report

Introduction

Patients with systemic lupus erythematosus (SLE) are

at high risk of osteoporosis [1, 2]. The frequency of osteoporosis

in SLE is estimated at 18% and increases up to

68% in patients undergoing systemic glucocorticoid therapy

[1, 2]. Reasons for bone loss include limitation of locomotor

activity, impaired function of kidneys, chronic

inflammation, actions of cytokines and other mediators

of inflammation, insufficient vitamin D status, earlier

menopause, and also undertaken therapy, especially with

glucocorticoids [3, 4]. Van Staa et al. showed that the risk

of fractures was increased proportionally to the dose of

oral glucocorticoids [5]. In patients treated with a daily

dose of prednisolone < 2.5 mg, the risk of fractures was

1.55 times higher than in the control group of untreated

persons. The daily dose of prednisolone in the range of

2.5-7.5 mg was associated with two times higher risk, and

at the dose > 7.5 mg over five times higher. This means

that in patients taking the equivalent of prednisolone

> 7.5 mg a day within a period of 3 months, the probability

of a fracture of the spine is over five times higher than

in the control group [5]. The risk of osteoporotic fracture

in patients treated with glucocorticoids through the period

of 5-10 years affects over 30% of them, and one needs

to take into account that in older patients the femoral

neck fracture is connected with approximately 35% risk

of death within a period of 1 year [6]. On the other hand,

one ought to remember that the average daily consumption

of calcium by an average Polish man is evaluated

at 400 mg, whereas the recommended intake is about

Aim

To assess frequency of the nuclear receptor of vita -

min D (VDR) gene polymorphism Fok I in SLE patients in

comparison to the control group

Material and methods

The study covered 56 patients with SLE, including

50 women and 6 men, at the age of 44.18 ±11.71 years,

treated at the Department of Dermatology and Venereology

of the Medical University of Lodz. The control group

comprised 65 blood samples received from healthy blood

donors.

The DNA isolation from the full blood was performed

using Boom's technology on silicone magnetic particles

in NucliSens® miniMAG™, followed by real-time polymerase

chain reaction (RT-PCR) with the Simple Probe for

Fok I. The probe specifically hybridizes with the sequence

of DNA containing the selected polymorphism. This procedure

enables one to identify a single change of mononucleotide

in a sequence of DNA of the VDR gene Fok I (single-

nucleotide polymorphism – SNP). The genotype TT

represents the polymorphism ff, TC represents Ff, and

CC represents FF. The following reagents were used: lysis

buffer from bioMerieux (catalogue No. #200292), a set of

extraction reagents from bioMerieux (catalogue

No. #200293), LightCycler® 480 Probes Master from Roche

(catalogue No. #04-707-494-001), and the probe Light

SNiP rs2228570 (Fok I) Hu VDR from TIB MOLBIOL.

The study was approved by the local Ethics Committee

(No. RNN/67/08/KE).

Results

The frequencies of Fok I genotypes in SLE patients and

in the control group are shown in Table I. 14.29% of SLE

patients were ff homozygotes, 60.71% were Ff heterozygotes,

and 25% were FF homozygotes. In the control

group, ff homozygotes accounted for 15.38% of persons,

Ff heterozygotes for 61.54%, and FF homozygotes for

23.08%. The comparison of the frequency of Fok I based

on the analysis with the χ2 test did not reveal any significant

difference between SLE patients and the control

group (Fig. 1).

Discussion

The product of gene expression of VDR is a transcription

factor that binds with calcitriol. This enables the

expression of approximately 200 genes to be launched.

Molecular characteristics and cloning of the human VDR

was undertaken by Baker et al. in 1988 [10]. The VDR contains

a sequence of 427 amino acids [10, 11]. Up to now

there is only one study addressing the Fok I VDR polymorphism

in patients with SLE [12]. Huang et al. did not find

significant differences in the frequency of Fok I between

52 patients with SLE and with the control group [12].

It needs to be underlined that the patients and the

control group of the aforementioned study were recru -

ited among a Chinese population [12]. 21.2% of SLE

patients were FF homozygotes, 65.4% Ff heterozygotes,

and 13.4% ff homozygotes. In the control group, 23.3% of

persons were FF homozygotes, 47.8% Ff heterozygotes,

and 28.9% ff homozygotes [12]. In our study, the analysis

of Fok I was undertaken in the Polish population. In accordance

with the results of the Chinese authors, we did not

reveal significant differences in the frequency of Fok I

between SLE patients and healthy individuals. Ascertained

frequencies undoubtedly result from the polymorphism,

as the criterion that differentiates a polymorphism from

a mutation is precisely the frequency. If in the population

a change of a sequence of nucleotides occurs more often

than in 1%, it is a polymorphism. The study of Ozaki et al.

based on the polymerase chain reaction method (PCR) and

restriction fragment length polymorphism (RFLP) investigated

the frequency of another VDR gene polymorphism

BsmI in 58 pa tients with SLE [13]. Allele B in BsmI polymorphism

represents in the RFLP method longer fragments

of DNA (with greater molecular mass), whereas allele b represents

shorter fragments. Ozaki et al. found that the genotype

BB occurred more often in SLE patients in comparison

to the control group [13]. These results were in accordance

with those of Huang et al., that revealed higher frequency

of the genotype BB in patients with SLE than in the control

group [14]. Genotype bb is profitable for bone parameters,

while the occurrence of allele B in VDR is connected with

decreased bone mass [8]. The density of bone mass in the

lumbar spine in individuals with genotype bb was significantly

higher than in the case of BB, and the difference

appeared to represent approximately 10 years of life. The

genotype bb occurs in the Japanese population more than

twice as often as in Caucasians (respectively 77% and 33%)

[8]. On the other hand, there are reports indicating a lack

of correlation between the VDR genotype and bone mineral

density as well as showing populations in which the

bb genotype was connected with lower bone mass. Reasons

for the discrepancies are suspected to involve linkage

disequilibrium between the VDR gene and another gene

located in proximity, that may also influence the bone mass

[8]. Fok I polymorphism is due to exchange of nucleotides,

i.e. transition T to C in exon 2, defined as letter F, that results

in elimination of the origination site of translation, and it

in turn leads to elimination of 3 amino acids in the VDR

molecule (Fig. 2). Laboratory studies show that shortened

VDR is characterized by greater transcriptional activity as

a consequence of enhanced binding to transcription factor

IIB [15]. Unquestionably, the detection of one or several

genes determining a broad spectrum of phenotypic features

of osseous tissue, including both parameters of

endurance, bone turnover, and susceptibility to fractures,

would constitute a step forward in diagnosis and prophylaxis

of osteoporosis in patients with systemic lupus erythematosus.

However, much more probable is the involvement

of many different genes, from which each may

separately influence the expression of a given feature.

Therefore, it seems that a group of genetic factors influence

bone metabolism. Moreover, expression of a given

allele may be connected with other genes and alleles [8].

Despite the great diversity of variants of the VDR gene,

determining whether the occurrence of a given polymorphism

may dispose to the development of SLE or given

symptoms encounters difficulties. This results from the fact

that up to now the exact role played by VDR polymorphism

is not fully understood. For instance, Oakley-Girvan

et al. ascertained that in FF homozygotes of Afro-

American origin, the risk of prostate cancer is 1.9 times

increased [16]. On the other hand, Chen et al. found

1.34 times higher risk of breast cancer in ff homozygotes

in comparison to the FF genotype [17], and in the study

of a Chinese population ff homozygotes were found to be

at 2.3 times increased risk of tuberculosis [18]. All these

results indicate the importance of the role of VDR polymorphism

and the necessity of further investigations on

large groups of patients [19-21].

Conclusions

Our preliminary report indicates that the distribution

of Fok I VDR gene polymorphism in patients with SLE is

not significantly different from that of the general population.

This work was supported by the Medical University of

Lodz, Poland, grant No. 503/1-152-01/503-01.

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