Mol Biol Rep DOI 10.1007/s11033-014-3040-x
Juvenile idiopathic arthritis patients and their skeletal status: possible role of vitamin D receptor gene polymorphism M. M. Kostik • A. M. Smirnov • G. S. Demin L. A. Scheplyagina • V. I. Larionova
•
Received: 28 October 2012 / Accepted: 4 January 2014 Ó Springer Science+Business Media Dordrecht 2014
Abstract We evaluated bone mineralization and metabolism changes related to vitamin D receptor (VDR) polymorphic genotypes in children with juvenile idiopathic arthritis. One hundred and ninety eight children (82 boys and 116 girls) were included in our study. Bone mineral density (BMD) was measured by lumbar spine DXA. Osteocalcin, CTX, parathyroid hormone, total and ionized calcium, inorganic phosphate, total alkaline phosphatase activity was utilized for assessment of bone metabolism. Molecular testing: TaqI (rs731236) and Cdx2 (rs11568820) polymorphisms of VDR were detected by RFLP. No differences in TaqI and Cdx2 haplotypes, genotypes and alleles distribution related with normal and low BMD (Zscore \-2SD) were found. Children with low linear growth (\10th percentile) had more allele T-contained genotypes of TagI VDR (p = 0.037), compare with
M. M. Kostik (&) Hospital Pediatric Department, Saint-Petersburg State Pediatric Medical University, Saint-Petersburg, Russian Federation e-mail: [email protected] A. M. Smirnov Department of Molecular Diagnostics, Gene, Ltd., Saint-Petersburg, Russian Federation G. S. Demin Department of Molecular Diagnostics, Genetic Systems, Ltd., Saint-Petersburg, Russian Federation L. A. Scheplyagina Moscow Scientific and Research Clinical Institute Named M.F. Vladimirskiy, Moscow, Russian Federation V. I. Larionova Department of Molecular Diagnostics, Turner’s Scientific and Research Institute for Children’s Orthopedics, Saint-Petersburg, Russian Federation
medium or high linear growth children. Children with high linear growth ([90th percentile) had the highest frequency of allele A-contained genotypes (GA?AA) of Cdx2 VDR (p = 0.009). Girls with TT TaqI VDR, who never been treated by glucocorticoides had lower BMD-Zscore than C allele carriers (TT = -0.94SD [IQR: -2.1;-0.5], TC?CC = -0.62SD [IQR: -1.26;0.39], p = 0.03). Girls with Tanner I with TT had higher total and ionized Ca level than carriers of C allele (Ca: TT = 2.43 ± 0.15 mmol/l, TC?CC = 2.28 ± 0.2 mmol/l, p = 0.024; Ca2?: TT = 1.15 ± 0.08 mmol/l, TC?CC = 1.06 ± 0.13 mmol/l, p = 0.026). Presence of TT genotype negatively correlated with BMD-Zscore (r = -0.28, p = 0.04), and positively with frequency of LBMD (r = 0.3, p = 0.037). Boy with GG Cdx2 genotype had lower total Ca (GG = 2.3 ± 0.17 mmol/l, GA?AA = 2.43 ± 0.17 mmol/l, p = 0.004) compare with carriers of A allele. Pubertal boys (Tanner IV–V) with GG had higher CTX (GG = 1.75 ± 0.11 ng/ml, GA?AA = 1.06 ± 0.07 ng/ml, p = 0.04. TT genotype of TaqI and GG genotype of Cdx2 VDR is a negative factor impact bone mineralization metabolism and linear growth. Keywords Juvenile idiopathic arthritis (JIA) Bone mineral density (BMD) Linear growth Vitamin D receptor gene polymorphism (VDR)
Introduction Child bone health provides optimal linear growth, risk of fractures and bone quality during adult life. Peak of bone mass occurs during childhood with maximum in the end of puberty [1]. Rheumatoid arthritis as well as glucocorticoids intake are the known risk factors of osteoporosis and bone
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fragility [2, 3]. The studies about bone mineralization in children and young adults with juvenile idiopathic arthritis (JIA) are few [4–6]. Vitamin D is one of the key points which interacts both immune system and bone. Active metabolites of vitamin D are known as D-hormone, besides immune regulation is responsible for calcium and phosphate absorption, bone mineralization and maintain calcium and phosphate homeostasis. Active form of vitamin D—1,25(OH)2D3 interacts with its nuclear receptors in different types of cells and the complex ligand-receptor binds with specific response elements of dependent genes. Vitamin D effects bone metabolism throw response-elements in genes of transepithelial calcium channels in intestinal and kidney epitheliocytes and also in RANKL gene, responsible for osteoclasts maturation, activation and survival [7]. Biological effects of vitamin D mediate the functional condition of its receptor. More than 200 single nucleotide polymorphisms are detected in vitamin D receptor gene, regulated its activity. VDR polymorphic genotypes are risk factor of celiac disease, insulin-dependent diabetes mellitus, rheumatoid arthritis, systemic lupus erythematosus, bronchial asthma and many others [8–11]. Polymorphic vitamin D receptor (VDR) genotypes take in important role in bone mineralization and metabolism in adults and children. Some polymorphisms are involved in calcium kinetics and bone mineral accrual during puberty [12]. The aim of our study was to evaluate association of polymorphic VDR genotypes with skeletal status in JIA patients.
Materials and methods Subjects Approval was obtained from the Saint-Petersburg State Pediatric Medical Academy committee on the ethics of research on human beings. Blood samples were obtained after informed written consent. All specimens for the labs were collected at the same time as the DNA specimens. One hundred and ninety eight children (82 boys and 116 girls) with JIA were enrolled in the study. Each child was a patient of the Saint-Petersburg State Pediatric Medical Academy rheumatology clinic. We enrolled all patients who fulfilled the ILAR JIA criteria and agreed to take part in our study. Age of study subjects ranged from 1.5 to 17 years, with a mean age of 10.96 ± 4.6 years. All patients were Caucasians. Only one child per family was enrolled. The median age of disease onset was 7 (3.7; 12) years. The median duration of the disease at the time of inclusion in our study was 2 (0.5; 5) years. Pubertal stage was evaluated by Tanner method. All patients were divided
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into three groups according to arthritis course: oligoarticular 112 (56.6 %), polyarticular 68 (34.3 %), and systemic arthritis 18 (9.12 %). Extended oligoarticular and systemic patients without systemic features were referred to polyarticular course. For assessment of JIA activity we used onset age, duration of morning stiffness, number of active joints, white blood cells count, Westergren erythrocyte sedimentation rate, C-reactive protein. Physician global assessment of disease activity, measured on a 10-cm visual analog scale (VAS) where 0 = no activity and 10 = maximum activity; parent/patient global assessment of well-being, measured on a 10-cm VAS where 0 = very well and 10 = very poor. We utilized combined indexes for assessment disease activity—DAS28, JADAS71, CDAI. Assessment of bone mineralization and bone metabolism Bone mineralization was measured in all children by dualenergy X-ray absorptiometry (DXA) of lumbar spine (LS) at L1-L4 (Hologic QDR 4500C densitometer with reference pediatric database). Densitometry parameters, such as bone area (BA [cm2]), bone mineral content (BMC [g]) and bone mineral density (BMD, measured in [g/cm2] and in Zscore, SD) were all evaluated. Low bone mineral density (LBMD) for chronological age was defined by Zscore \2SD, according to the recommendation of the International Society for Clinical Densitometry, 2007. For assessment of bone metabolism the following measures were used: osteocalcin (bone gla-protein, a marker of osteosynthesis), carboxyterminal telopeptide of type I collagen (CTX -products of collagen I type degradation and a marker of bone resorption) and parathyroid hormone (PTH) by routine imunoenzyme methods. Also levels of total calcium (Ca), ionized calcium (Ca??), inorganic phosphate (Pi) and total alkaline phosphatase (ALP) were determined. Genetic analysis Molecular testing: TaqI (rs731236) and Cdx2 (rs11568820) polymorphisms of VDR were detected by polymerase chain reaction with restriction fragment length polymorphism according manufacture’s instruction manual of the kit «OsteoGene-M» (Gene, Ltd., Saint-Petersburg, Russian Federation). 192 and 189 patients were analyzed for TaqI and Cdx2 polymorphisms respectively. Data collection Informed consent was obtained from all study participants with explanation of the study protocol prior to examination,
Mol Biol Rep Table 1 TaqI and Cdx2 VDR polymorphic genotypes and alleles distribution in JIA children according to their mineralization
Table 2 Differences in linear growth in JIA patients related to Tag I and Cdx2 VDR gene polymorphisms
Population
Genotype
LBMD
Genotypes
TaqI, n (%)
150 (100)
42 (100)
Normal linear growth (25th– 75th percentile)
High linear growth ([90th percentile)
p
All JIA patients (n = 192)
Low linear growth (\10th percentile)
‘‘TT’’
65 (43.3)
20 (47.6)
‘‘TC’’ ‘‘CC’’
72 (48) 13 (8.7)
16 (38.1) 6 (14.3)
TaqI, n (%)
12 (100.0)
115 (100.0)
19 (100.0)
0.11
Cdx2, n (%)
41 (100)
148 (100)
GG
29 (70.7)
107 (72.3)
GA
12 (29.3)
35 (23.7)
AA
0 (0.0)
6 (4.0)
All JIA patients (n = 189)
NBMD
p 0.39
0.35
Total, n (%)
41 (100)
TTGG
13 (31.7)
49 (33.3)
TTGA
7 (17.1)
14 (9.5)
TTAA
147 (100)
5 (41.7)
46 (40.0)
8 (42.1)
‘‘TC’’
7 (58.3)
42 (36.5)
10 (52.6)
‘‘CC’’
0 (0.0)
27 (23.5)
1 (5.3)
11 (100.0)
95 (100.0)
19 (100.0)
GGb
7 (63.6)
73 (76.8)
8 (42.1)
GA
3 (27.3)
20 (21.1)
11 (57.9)
0.48
AA
1 (9.1)
2 (2.1)
0 (0.0)
a
0 (0)
a
Cdx2, n (%)
Haplotypes All JIA patients (n = 188)
‘‘TT’’
0.037a 0.01
0.009b
Allele T-contained genotypes (TT?TC) genotype compare with CC
b
Allele A-contained genotypes (GA?AA) compare with GG genotype
2 (1.4)
TCGG
11 (26.8)
50 (34.0)
TCGA
4 (9.7)
17 (11.6)
TCAA
0 (0)
4 (2.7)
CCGG
5 (12.2)
7 (4.8)
CCGA
1 (2.5)
4 (2.7)
LBMD low bone mineral density (Zscore \-2SD), NBMD normal bone mineral density (Zscore [-2SD), JIA juvenile idiopathic arthritis
densitometry and obtaining blood samples. Demographics, anthropometry and data about diagnosis and disease activity, GC and DMARD use, BA, BMC, BMD–Zscore, osteocalcin, CTX, PTH, Ca, Ca2?, Pi, ALP were recorded for analysis. According of linear growth to chronological age children were divided into three groups: low (\10th percentile), medium (25th–75th) and high linear growth ([90th percentile). Because we had a restricted number of JIA children with CC TaqI and AA Cdx2 genotypes we compared genotypes with allele C (TC ? CC) compare with TT carriers (no C allele) for TaqI, and genotypes with A allele (GA ? AA) compare with GG cariers (no A allele) for Cdx2. We have obtained several haplotypes consisted of VDR polymorphisms: TTGG, TTGA, TTAA, TCGG, TCGA, TCAA, CCGG and CCGA in the group of 188 JIA children due to missing data (data in Table 1). Statistical analysis We utilized, t test, Chi square test, Fisher’s exact test, Mann-Whitny test, Spearman’s and Pearson’s correlation analysis. In the cases of normal distribution data were
recorded as means (M) ± standard deviation (SD) and in the cases of non-normal distribution we indicated median (Me) and interquartil range (Me; 25 %; 75 %). P values \0.05 were considered to indicate a significant difference.
Results Table 1 shows no differences in TaqI and Cdx2, genotypes and alleles distribution related with normal and LBMD with no deviation from Hardy–Weinberg equilibrium. Also no differences were observed in haplotypes distribution according to normal or LBMD. No differences in demographics, disease subtypes, clinical and laboratorial signs activity, glucocorticoids and DMARD treatment according to polymorphic genotypes. Table 2 describes that children with low linear growth (less than 10th percentile) had more allele T-contained genotypes of TagI VDR (p = 0.037), compare with medium or high linear growth children. Children with high linear growth (more than 90th percentile) had the highest frequency of allele A-contained genotypes (GA ? AA) of Cdx2 VDR (p = 0.009) compare with GG genotypes. In the total group with JIA no differences in skeletal size (LS), bone mineralization and metabolism related to polymorphic genotypes were found (Table 3). However, girls with TT TaqI VDR, who never been treated by GC had lower BMD-Zscore than C allele carriers, without fractures prevalence. Pre-pubertal girls (Tanner I) with TT had higher total and ionized Ca level than carriers of C allele. TT genotypes negatively correlated with BMD-Zscore (r = -0.28, p = 0.04), and
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Mol Biol Rep Table 3 Bone mineralization and metabolism status in JIA patients related to Tag I and Cdx2 VDR gene polymorphisms TagI
Cdx2
Genotypes
TT
TC?CC
GG
Parameters
(n = 85)
(n = 107)
(n = 136)
GA?AA (n = 53)
Weight (kg)
40.0 ± 18.0
41.2 ± 18.9
42.1 ± 18.8
38.3 ± 18.2
Height (cm)
144.5 ± 25.9
144.4 ± 25.8
145.5 ± 25.5
143.2 ± 27.0
BA (cm2)
42.7 ± 13.5
44.1 ± 14.4
44.27 ± 14.6
42.3 ± 14.1
BMC (g)
28.7 (16.0; 45.7)
31.6 (16.2; 48.3)
30.6 (16.2; 49.0)
31.6 (14.9; 43.5)
BMD (g/cm2)
0.67 ± 0.2
0.7 ± 0.2
0.69 ± 0.2
0.68 ± 0.2
BMD-Zscore, SD
-1.2 ± 1.2
-1.0 ± 1.1
-1.0 ± 1.3
-1.1 ± 1.2
Ca, mmol/l
2.4 ± 0.2
2.3 ± 0.2
2.3 ± 0.2
2.4 ± 0.2
Ca2? (mmol/l)
1.1 ± 0.1
1.1 ± 0.1
1.1 ± 0.1
1.1 ± 0.1
Pi, mmol/l ALP (U/l)
1.6 ± 0.2 362,0 (263.0; 442.2)
1.6 ± 0.2 341.0 (250.0; 423.0)
1.6 ± 0.2 343.5 (244.0; 436.0)
1.6 ± 0.2 350.0 (266.0; 442.0)
Osteocalcin (ng/ml)
101.4 (74.1; 146.2)
98.1 (50.2; 128.8)
99.0 (50.0; 136.5)
98.5 (80.5; 147.2)
CTX (ng/ml)
1.3 ± 0.5
1.1 ± 0.4
1.1 ± 0.5
1.2 ± 0.4
PTH, pmol/ml
2.2 (1.7; 2.8)
2.0 (1.4; 2.7)
2.2 (1.5; 2.8)
1.88 (1.4; 2.3)
LBMD, n (%)
20 (23.5)
22 (20.6)
29 (21.3)
12 (22.6)
Fractures, n (%)
13 (15.3)
16 (15.0)
22 (16.2)
6 (11.3)
2?
BA bone area, BMC bone mineral content, BMD bone mineral density, Ca serum total calcium, Ca serum ionized calcium, Pi serum inorganic phosphate, ALP total alkaline phosphatase, CTX serum carboxyterminal telopeptide of type I collagen, PTH parathyroid hormone, LBMD low bone mineral density (Zscore \-2SD)
positively with BMD deficiency (r = 0.3, p = 0.03) and presence of LBMD (r = 0.3, p = 0.037). Cdx2 polymorphic genotypes of VDR were related only with changes in markers of bone metabolism without changes of bone mineralization. Boy with GG genotype had lower total Ca and pubertal boys (Tanner IV–V) with GG had higher CTX compare with carriers of A allele (Table 4).
Discussion This study is about role of two genetic polymorphisms of VDR and its relation to BMD in JIA. Cdx2 polymorphism located in promoter part of VDR gene associated with capability of gene transcription in intestinal epitheliocytes and influences on calcium intestinal absorption [13]. The same authors in consequent study have revealed that G allele associated with lower transcriptional activity and present only 70 % of activity inherent to A homozygote. In their study postmenopausal women with AA genotype had 12 % higher BMD than carriers of GG. In premenopausal women no differences were found and authors consider the protective role of estrogens in premenopausal women [14]. In the study from Aberdeen the role of 5 VDR polymorphisms (ApaI, TaqI, BsmI, FokI and Cdx-2) on BMD were evaluated in 3,100 women during 10 year period. Women
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with AA Cdx2 genotype had lower PTH and annual femoral neck bone loss compare with GG and GA carriers despite similar vitamin D status. Women with AA genotype, who were low calcium intakes (lower 25th percentile), had higher femoral neck BMD than other genotypes. Authors suggested that A allele results in more efficient calcium absorption than G allele [15]. There are ethnic differences in genotypes distribution between populations. In our study the frequency of A allele of Cdx2 VDR was 15.6 %, that was similar to data of Aberdeen study (19.0 %), but in African people the frequency of this allele reaches up to 74.0 %, that can explain why BMD in Africans prevalence the Europeans despite low calcium intake [16]. Black adolescents had higher calcium absorption and low calcium excretion than white adolescent despite the similar diet [17]. The same changes with BsmI and TaqI VDR polymorphisms were observed. Women with low calcium intake with bb genotype BsmI had higher lumbar spine and femoral neck BMD and lower rate of bone loss in LS than women with allele B. Low calcium intake women with TT genotype had higher femoral neck BMD than carriers of t allele [15]. In our study children with A allele had also higher calcium level as described above and suggested that high transcription activity of A allele leads to high calcium absorption. Our patients with high linear growth had more frequently A allele which correlated with data about high
Mol Biol Rep Table 4 Parameters of bone metabolism, mineralization and mineral turnover related to pubertal stage an Tag I and Cdx2 VDR gene polymorphisms Population
Parameter
Taq I
p
‘‘TT’’
‘‘TC’’ and ‘‘CC’’
Girls without GC (n = 53)
BMD-Zscore
-0.94 SD (-2.1; -0.5)
-0.62 SD (-1.26; 0.39)
0.03
Girls in Tanner stage I
Ca (mmol/l)
2.43 ± 0.15
2.28 ± 0.2
0.024
(n = 34)
Ca2? (mmol/l)
1.15 ± 0.08
1.06 ± 0.13
0.026
Cdx 2 ‘‘GG’’
‘‘GA’’ and ‘‘AA’’
Boys (n = 79)
Ca (mmol/l)
2.3 ± 0.17
2.43 ± 0.17
0.004
Boys in Tanner stage IV–V (n = 23)
CTX (ng/ml)
1.75 ± 0.11
1.06 ± 0.07
0.04
GC glucocorticoids, BMD bone mineral density, Ca serum total calcium, Ca2? serum ionized calcium, CTX serum carboxyterminal telopeptide of type I collagen
calcium absorption and higher serum calcium level characteristic for this polymorphism. Four VDR polymorphisms (BsmI, FokI, ApaI and TaqI) were studied in 395 children from Poland aged 6–18. Only a allele of ApaI VDR polymorphism was associated with higher BMD and BMC in children, whom BMD-Zscore ranged -1.1 up to -2.0 SD [18]. Five VDR polymorphisms (BsmI, FokI, ApaI, TaqI and Cdx2) were detected in 90 children with insulin-dependent diabetes mellitus. No significant associations between bone mineralization, bone metabolic markers and polymorphic VDR genotypes were found [19]. In Netherland relation anthropometry and bone mineralization with BsmI, ApaI, TaqI polymorphic VDR and Sp1 polymorphic genotypes of aI chain I type of collagen (COL1A1) were studied in 148 children and young adults during 4 year period. No relation of discovered single nucleotide polymorphisms with BMD were found, but haplotype bAT had positive influence on linear growth and vertebrae width. Also increased number of risk alleles in haplotype were associated with linear growth delay (p = 0.006) and delay of vertebrae width (p = 0.001) [20]. In Danish study with 224 girls aged 11–12 there were no influence of FokI and TaqI VDR gene polymorphisms on bone mineralization and metabolism. Only ff genotype was associated with higher linear growth. The role of TaqI remained unknown [21]. In the study of Australian (n = 3,906) and Netherland (n = 1,689) twins significant influence of BsmI-, FokI-, TaqI- and (-1521) VDR polymorphisms on linear growth no found [22]. Several studies were devoted the role of VDR polymorphisms in bone metabolism disturbance in adults with rheumatoid arthritis (RA). Bone mineralization was studied annual during 3 years in 232 adults with early RA (160 women and 72 men) and VDR TaqI polymorphism was identified. Allele distribution in RA patients was similar to
population. At the end of the study women with of t allele had more rapid LS bone loss (4.9 %) and femoral neck (FN) (9.6 %) compare with carriers of TT genotype (0.1 % for LS and 3.9 % for FN, p \ 0.05 and \0.01, respectively). Interesting that this effect did not dependent of other diseases characteristics and authors suggested that t allele was associated with accelerated bone loss in women with RA [23]. In the study of 64 adult RA patients the carriers of bb genotype BsmI VDR had higher BMD (p \ 0.05), lower urinary deoxypyridinoline (p \ 0.05) and urinary calcium excretion (p \ 0.05) than carriers of BB and Bb genotypes [24]. In several studies there were no changes in bone metabolism and mineralization related to VDR genotypes and alleles. In study of 62 women with RA associations between BsmI-, TaqI-, FokI VDR genotypes and bone metabolic markers were not found [25]. In 155 Korean patients with RA, no influence of BsmI- and TaqI polymorphic genotypes in development of bone erosions [26]. In the single study of 50 JIA and 80 controls TT genotype of Alu I polymorphism of calcitonin receptor gene and ff genotype of FokI VDR were associated with lower BMD (p = 0.04 and 0.02, relatively) in the LS [27]. In our study TT genotypes TaqI VDR shows contrary effects: it associated with lower BMD in JIA girls, who never been treated by GCS and in mid-puberty boys. Also children with low linear growth (less than 10th percentile) more often had allele T-contained genotypes. Association between linear growth delay and low bone mineralization is well-known and shows negative effect of T allele (risk allele) on children’s bone health. These data are contrast to results of described above studies, with exclusion the prepubertal girls with TT genotype associated with higher total and ionized calcium level. We have not found any associations between VDR haplotypes distribution and presence of LBMD, possibly
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because we have small number of patients with different haplotypes. These differences also could be explained by small sample size and its peculiarities.
Conclusion In whole JIA group no differences in TaqI and Cdx2 haplotypes, genotypes and alleles distribution related with normal and low BMD (Zscore \-2SD) were found. Association VDR molecular markers with bone mineralization and metabolism disturbances, related to gender, puberty stage, GC treatment was found. TT genotype of TaqI is a negative factor impact bone mineralization and linear growth. GG genotype of Cdx2 negatively influences bone metabolism in JIA children. In contrast, A allele of the same molecular marker associated with high linear growth and could be mentioned as a bone protective marker. Conflict of interests The authors declare that they have no conflict of interests.
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Juvenile idiopathic arthritis patients and their skeletal status: possible role of vitamin D receptor gene polymorphism M. M. Kostik • A. M. Smirnov • G. S. Demin L. A. Scheplyagina • V. I. Larionova
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Received: 28 October 2012 / Accepted: 4 January 2014 Ó Springer Science+Business Media Dordrecht 2014
Abstract We evaluated bone mineralization and metabolism changes related to vitamin D receptor (VDR) polymorphic genotypes in children with juvenile idiopathic arthritis. One hundred and ninety eight children (82 boys and 116 girls) were included in our study. Bone mineral density (BMD) was measured by lumbar spine DXA. Osteocalcin, CTX, parathyroid hormone, total and ionized calcium, inorganic phosphate, total alkaline phosphatase activity was utilized for assessment of bone metabolism. Molecular testing: TaqI (rs731236) and Cdx2 (rs11568820) polymorphisms of VDR were detected by RFLP. No differences in TaqI and Cdx2 haplotypes, genotypes and alleles distribution related with normal and low BMD (Zscore \-2SD) were found. Children with low linear growth (\10th percentile) had more allele T-contained genotypes of TagI VDR (p = 0.037), compare with
M. M. Kostik (&) Hospital Pediatric Department, Saint-Petersburg State Pediatric Medical University, Saint-Petersburg, Russian Federation e-mail: [email protected] A. M. Smirnov Department of Molecular Diagnostics, Gene, Ltd., Saint-Petersburg, Russian Federation G. S. Demin Department of Molecular Diagnostics, Genetic Systems, Ltd., Saint-Petersburg, Russian Federation L. A. Scheplyagina Moscow Scientific and Research Clinical Institute Named M.F. Vladimirskiy, Moscow, Russian Federation V. I. Larionova Department of Molecular Diagnostics, Turner’s Scientific and Research Institute for Children’s Orthopedics, Saint-Petersburg, Russian Federation
medium or high linear growth children. Children with high linear growth ([90th percentile) had the highest frequency of allele A-contained genotypes (GA?AA) of Cdx2 VDR (p = 0.009). Girls with TT TaqI VDR, who never been treated by glucocorticoides had lower BMD-Zscore than C allele carriers (TT = -0.94SD [IQR: -2.1;-0.5], TC?CC = -0.62SD [IQR: -1.26;0.39], p = 0.03). Girls with Tanner I with TT had higher total and ionized Ca level than carriers of C allele (Ca: TT = 2.43 ± 0.15 mmol/l, TC?CC = 2.28 ± 0.2 mmol/l, p = 0.024; Ca2?: TT = 1.15 ± 0.08 mmol/l, TC?CC = 1.06 ± 0.13 mmol/l, p = 0.026). Presence of TT genotype negatively correlated with BMD-Zscore (r = -0.28, p = 0.04), and positively with frequency of LBMD (r = 0.3, p = 0.037). Boy with GG Cdx2 genotype had lower total Ca (GG = 2.3 ± 0.17 mmol/l, GA?AA = 2.43 ± 0.17 mmol/l, p = 0.004) compare with carriers of A allele. Pubertal boys (Tanner IV–V) with GG had higher CTX (GG = 1.75 ± 0.11 ng/ml, GA?AA = 1.06 ± 0.07 ng/ml, p = 0.04. TT genotype of TaqI and GG genotype of Cdx2 VDR is a negative factor impact bone mineralization metabolism and linear growth. Keywords Juvenile idiopathic arthritis (JIA) Bone mineral density (BMD) Linear growth Vitamin D receptor gene polymorphism (VDR)
Introduction Child bone health provides optimal linear growth, risk of fractures and bone quality during adult life. Peak of bone mass occurs during childhood with maximum in the end of puberty [1]. Rheumatoid arthritis as well as glucocorticoids intake are the known risk factors of osteoporosis and bone
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fragility [2, 3]. The studies about bone mineralization in children and young adults with juvenile idiopathic arthritis (JIA) are few [4–6]. Vitamin D is one of the key points which interacts both immune system and bone. Active metabolites of vitamin D are known as D-hormone, besides immune regulation is responsible for calcium and phosphate absorption, bone mineralization and maintain calcium and phosphate homeostasis. Active form of vitamin D—1,25(OH)2D3 interacts with its nuclear receptors in different types of cells and the complex ligand-receptor binds with specific response elements of dependent genes. Vitamin D effects bone metabolism throw response-elements in genes of transepithelial calcium channels in intestinal and kidney epitheliocytes and also in RANKL gene, responsible for osteoclasts maturation, activation and survival [7]. Biological effects of vitamin D mediate the functional condition of its receptor. More than 200 single nucleotide polymorphisms are detected in vitamin D receptor gene, regulated its activity. VDR polymorphic genotypes are risk factor of celiac disease, insulin-dependent diabetes mellitus, rheumatoid arthritis, systemic lupus erythematosus, bronchial asthma and many others [8–11]. Polymorphic vitamin D receptor (VDR) genotypes take in important role in bone mineralization and metabolism in adults and children. Some polymorphisms are involved in calcium kinetics and bone mineral accrual during puberty [12]. The aim of our study was to evaluate association of polymorphic VDR genotypes with skeletal status in JIA patients.
Materials and methods Subjects Approval was obtained from the Saint-Petersburg State Pediatric Medical Academy committee on the ethics of research on human beings. Blood samples were obtained after informed written consent. All specimens for the labs were collected at the same time as the DNA specimens. One hundred and ninety eight children (82 boys and 116 girls) with JIA were enrolled in the study. Each child was a patient of the Saint-Petersburg State Pediatric Medical Academy rheumatology clinic. We enrolled all patients who fulfilled the ILAR JIA criteria and agreed to take part in our study. Age of study subjects ranged from 1.5 to 17 years, with a mean age of 10.96 ± 4.6 years. All patients were Caucasians. Only one child per family was enrolled. The median age of disease onset was 7 (3.7; 12) years. The median duration of the disease at the time of inclusion in our study was 2 (0.5; 5) years. Pubertal stage was evaluated by Tanner method. All patients were divided
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into three groups according to arthritis course: oligoarticular 112 (56.6 %), polyarticular 68 (34.3 %), and systemic arthritis 18 (9.12 %). Extended oligoarticular and systemic patients without systemic features were referred to polyarticular course. For assessment of JIA activity we used onset age, duration of morning stiffness, number of active joints, white blood cells count, Westergren erythrocyte sedimentation rate, C-reactive protein. Physician global assessment of disease activity, measured on a 10-cm visual analog scale (VAS) where 0 = no activity and 10 = maximum activity; parent/patient global assessment of well-being, measured on a 10-cm VAS where 0 = very well and 10 = very poor. We utilized combined indexes for assessment disease activity—DAS28, JADAS71, CDAI. Assessment of bone mineralization and bone metabolism Bone mineralization was measured in all children by dualenergy X-ray absorptiometry (DXA) of lumbar spine (LS) at L1-L4 (Hologic QDR 4500C densitometer with reference pediatric database). Densitometry parameters, such as bone area (BA [cm2]), bone mineral content (BMC [g]) and bone mineral density (BMD, measured in [g/cm2] and in Zscore, SD) were all evaluated. Low bone mineral density (LBMD) for chronological age was defined by Zscore \2SD, according to the recommendation of the International Society for Clinical Densitometry, 2007. For assessment of bone metabolism the following measures were used: osteocalcin (bone gla-protein, a marker of osteosynthesis), carboxyterminal telopeptide of type I collagen (CTX -products of collagen I type degradation and a marker of bone resorption) and parathyroid hormone (PTH) by routine imunoenzyme methods. Also levels of total calcium (Ca), ionized calcium (Ca??), inorganic phosphate (Pi) and total alkaline phosphatase (ALP) were determined. Genetic analysis Molecular testing: TaqI (rs731236) and Cdx2 (rs11568820) polymorphisms of VDR were detected by polymerase chain reaction with restriction fragment length polymorphism according manufacture’s instruction manual of the kit «OsteoGene-M» (Gene, Ltd., Saint-Petersburg, Russian Federation). 192 and 189 patients were analyzed for TaqI and Cdx2 polymorphisms respectively. Data collection Informed consent was obtained from all study participants with explanation of the study protocol prior to examination,
Mol Biol Rep Table 1 TaqI and Cdx2 VDR polymorphic genotypes and alleles distribution in JIA children according to their mineralization
Table 2 Differences in linear growth in JIA patients related to Tag I and Cdx2 VDR gene polymorphisms
Population
Genotype
LBMD
Genotypes
TaqI, n (%)
150 (100)
42 (100)
Normal linear growth (25th– 75th percentile)
High linear growth ([90th percentile)
p
All JIA patients (n = 192)
Low linear growth (\10th percentile)
‘‘TT’’
65 (43.3)
20 (47.6)
‘‘TC’’ ‘‘CC’’
72 (48) 13 (8.7)
16 (38.1) 6 (14.3)
TaqI, n (%)
12 (100.0)
115 (100.0)
19 (100.0)
0.11
Cdx2, n (%)
41 (100)
148 (100)
GG
29 (70.7)
107 (72.3)
GA
12 (29.3)
35 (23.7)
AA
0 (0.0)
6 (4.0)
All JIA patients (n = 189)
NBMD
p 0.39
0.35
Total, n (%)
41 (100)
TTGG
13 (31.7)
49 (33.3)
TTGA
7 (17.1)
14 (9.5)
TTAA
147 (100)
5 (41.7)
46 (40.0)
8 (42.1)
‘‘TC’’
7 (58.3)
42 (36.5)
10 (52.6)
‘‘CC’’
0 (0.0)
27 (23.5)
1 (5.3)
11 (100.0)
95 (100.0)
19 (100.0)
GGb
7 (63.6)
73 (76.8)
8 (42.1)
GA
3 (27.3)
20 (21.1)
11 (57.9)
0.48
AA
1 (9.1)
2 (2.1)
0 (0.0)
a
0 (0)
a
Cdx2, n (%)
Haplotypes All JIA patients (n = 188)
‘‘TT’’
0.037a 0.01
0.009b
Allele T-contained genotypes (TT?TC) genotype compare with CC
b
Allele A-contained genotypes (GA?AA) compare with GG genotype
2 (1.4)
TCGG
11 (26.8)
50 (34.0)
TCGA
4 (9.7)
17 (11.6)
TCAA
0 (0)
4 (2.7)
CCGG
5 (12.2)
7 (4.8)
CCGA
1 (2.5)
4 (2.7)
LBMD low bone mineral density (Zscore \-2SD), NBMD normal bone mineral density (Zscore [-2SD), JIA juvenile idiopathic arthritis
densitometry and obtaining blood samples. Demographics, anthropometry and data about diagnosis and disease activity, GC and DMARD use, BA, BMC, BMD–Zscore, osteocalcin, CTX, PTH, Ca, Ca2?, Pi, ALP were recorded for analysis. According of linear growth to chronological age children were divided into three groups: low (\10th percentile), medium (25th–75th) and high linear growth ([90th percentile). Because we had a restricted number of JIA children with CC TaqI and AA Cdx2 genotypes we compared genotypes with allele C (TC ? CC) compare with TT carriers (no C allele) for TaqI, and genotypes with A allele (GA ? AA) compare with GG cariers (no A allele) for Cdx2. We have obtained several haplotypes consisted of VDR polymorphisms: TTGG, TTGA, TTAA, TCGG, TCGA, TCAA, CCGG and CCGA in the group of 188 JIA children due to missing data (data in Table 1). Statistical analysis We utilized, t test, Chi square test, Fisher’s exact test, Mann-Whitny test, Spearman’s and Pearson’s correlation analysis. In the cases of normal distribution data were
recorded as means (M) ± standard deviation (SD) and in the cases of non-normal distribution we indicated median (Me) and interquartil range (Me; 25 %; 75 %). P values \0.05 were considered to indicate a significant difference.
Results Table 1 shows no differences in TaqI and Cdx2, genotypes and alleles distribution related with normal and LBMD with no deviation from Hardy–Weinberg equilibrium. Also no differences were observed in haplotypes distribution according to normal or LBMD. No differences in demographics, disease subtypes, clinical and laboratorial signs activity, glucocorticoids and DMARD treatment according to polymorphic genotypes. Table 2 describes that children with low linear growth (less than 10th percentile) had more allele T-contained genotypes of TagI VDR (p = 0.037), compare with medium or high linear growth children. Children with high linear growth (more than 90th percentile) had the highest frequency of allele A-contained genotypes (GA ? AA) of Cdx2 VDR (p = 0.009) compare with GG genotypes. In the total group with JIA no differences in skeletal size (LS), bone mineralization and metabolism related to polymorphic genotypes were found (Table 3). However, girls with TT TaqI VDR, who never been treated by GC had lower BMD-Zscore than C allele carriers, without fractures prevalence. Pre-pubertal girls (Tanner I) with TT had higher total and ionized Ca level than carriers of C allele. TT genotypes negatively correlated with BMD-Zscore (r = -0.28, p = 0.04), and
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Mol Biol Rep Table 3 Bone mineralization and metabolism status in JIA patients related to Tag I and Cdx2 VDR gene polymorphisms TagI
Cdx2
Genotypes
TT
TC?CC
GG
Parameters
(n = 85)
(n = 107)
(n = 136)
GA?AA (n = 53)
Weight (kg)
40.0 ± 18.0
41.2 ± 18.9
42.1 ± 18.8
38.3 ± 18.2
Height (cm)
144.5 ± 25.9
144.4 ± 25.8
145.5 ± 25.5
143.2 ± 27.0
BA (cm2)
42.7 ± 13.5
44.1 ± 14.4
44.27 ± 14.6
42.3 ± 14.1
BMC (g)
28.7 (16.0; 45.7)
31.6 (16.2; 48.3)
30.6 (16.2; 49.0)
31.6 (14.9; 43.5)
BMD (g/cm2)
0.67 ± 0.2
0.7 ± 0.2
0.69 ± 0.2
0.68 ± 0.2
BMD-Zscore, SD
-1.2 ± 1.2
-1.0 ± 1.1
-1.0 ± 1.3
-1.1 ± 1.2
Ca, mmol/l
2.4 ± 0.2
2.3 ± 0.2
2.3 ± 0.2
2.4 ± 0.2
Ca2? (mmol/l)
1.1 ± 0.1
1.1 ± 0.1
1.1 ± 0.1
1.1 ± 0.1
Pi, mmol/l ALP (U/l)
1.6 ± 0.2 362,0 (263.0; 442.2)
1.6 ± 0.2 341.0 (250.0; 423.0)
1.6 ± 0.2 343.5 (244.0; 436.0)
1.6 ± 0.2 350.0 (266.0; 442.0)
Osteocalcin (ng/ml)
101.4 (74.1; 146.2)
98.1 (50.2; 128.8)
99.0 (50.0; 136.5)
98.5 (80.5; 147.2)
CTX (ng/ml)
1.3 ± 0.5
1.1 ± 0.4
1.1 ± 0.5
1.2 ± 0.4
PTH, pmol/ml
2.2 (1.7; 2.8)
2.0 (1.4; 2.7)
2.2 (1.5; 2.8)
1.88 (1.4; 2.3)
LBMD, n (%)
20 (23.5)
22 (20.6)
29 (21.3)
12 (22.6)
Fractures, n (%)
13 (15.3)
16 (15.0)
22 (16.2)
6 (11.3)
2?
BA bone area, BMC bone mineral content, BMD bone mineral density, Ca serum total calcium, Ca serum ionized calcium, Pi serum inorganic phosphate, ALP total alkaline phosphatase, CTX serum carboxyterminal telopeptide of type I collagen, PTH parathyroid hormone, LBMD low bone mineral density (Zscore \-2SD)
positively with BMD deficiency (r = 0.3, p = 0.03) and presence of LBMD (r = 0.3, p = 0.037). Cdx2 polymorphic genotypes of VDR were related only with changes in markers of bone metabolism without changes of bone mineralization. Boy with GG genotype had lower total Ca and pubertal boys (Tanner IV–V) with GG had higher CTX compare with carriers of A allele (Table 4).
Discussion This study is about role of two genetic polymorphisms of VDR and its relation to BMD in JIA. Cdx2 polymorphism located in promoter part of VDR gene associated with capability of gene transcription in intestinal epitheliocytes and influences on calcium intestinal absorption [13]. The same authors in consequent study have revealed that G allele associated with lower transcriptional activity and present only 70 % of activity inherent to A homozygote. In their study postmenopausal women with AA genotype had 12 % higher BMD than carriers of GG. In premenopausal women no differences were found and authors consider the protective role of estrogens in premenopausal women [14]. In the study from Aberdeen the role of 5 VDR polymorphisms (ApaI, TaqI, BsmI, FokI and Cdx-2) on BMD were evaluated in 3,100 women during 10 year period. Women
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with AA Cdx2 genotype had lower PTH and annual femoral neck bone loss compare with GG and GA carriers despite similar vitamin D status. Women with AA genotype, who were low calcium intakes (lower 25th percentile), had higher femoral neck BMD than other genotypes. Authors suggested that A allele results in more efficient calcium absorption than G allele [15]. There are ethnic differences in genotypes distribution between populations. In our study the frequency of A allele of Cdx2 VDR was 15.6 %, that was similar to data of Aberdeen study (19.0 %), but in African people the frequency of this allele reaches up to 74.0 %, that can explain why BMD in Africans prevalence the Europeans despite low calcium intake [16]. Black adolescents had higher calcium absorption and low calcium excretion than white adolescent despite the similar diet [17]. The same changes with BsmI and TaqI VDR polymorphisms were observed. Women with low calcium intake with bb genotype BsmI had higher lumbar spine and femoral neck BMD and lower rate of bone loss in LS than women with allele B. Low calcium intake women with TT genotype had higher femoral neck BMD than carriers of t allele [15]. In our study children with A allele had also higher calcium level as described above and suggested that high transcription activity of A allele leads to high calcium absorption. Our patients with high linear growth had more frequently A allele which correlated with data about high
Mol Biol Rep Table 4 Parameters of bone metabolism, mineralization and mineral turnover related to pubertal stage an Tag I and Cdx2 VDR gene polymorphisms Population
Parameter
Taq I
p
‘‘TT’’
‘‘TC’’ and ‘‘CC’’
Girls without GC (n = 53)
BMD-Zscore
-0.94 SD (-2.1; -0.5)
-0.62 SD (-1.26; 0.39)
0.03
Girls in Tanner stage I
Ca (mmol/l)
2.43 ± 0.15
2.28 ± 0.2
0.024
(n = 34)
Ca2? (mmol/l)
1.15 ± 0.08
1.06 ± 0.13
0.026
Cdx 2 ‘‘GG’’
‘‘GA’’ and ‘‘AA’’
Boys (n = 79)
Ca (mmol/l)
2.3 ± 0.17
2.43 ± 0.17
0.004
Boys in Tanner stage IV–V (n = 23)
CTX (ng/ml)
1.75 ± 0.11
1.06 ± 0.07
0.04
GC glucocorticoids, BMD bone mineral density, Ca serum total calcium, Ca2? serum ionized calcium, CTX serum carboxyterminal telopeptide of type I collagen
calcium absorption and higher serum calcium level characteristic for this polymorphism. Four VDR polymorphisms (BsmI, FokI, ApaI and TaqI) were studied in 395 children from Poland aged 6–18. Only a allele of ApaI VDR polymorphism was associated with higher BMD and BMC in children, whom BMD-Zscore ranged -1.1 up to -2.0 SD [18]. Five VDR polymorphisms (BsmI, FokI, ApaI, TaqI and Cdx2) were detected in 90 children with insulin-dependent diabetes mellitus. No significant associations between bone mineralization, bone metabolic markers and polymorphic VDR genotypes were found [19]. In Netherland relation anthropometry and bone mineralization with BsmI, ApaI, TaqI polymorphic VDR and Sp1 polymorphic genotypes of aI chain I type of collagen (COL1A1) were studied in 148 children and young adults during 4 year period. No relation of discovered single nucleotide polymorphisms with BMD were found, but haplotype bAT had positive influence on linear growth and vertebrae width. Also increased number of risk alleles in haplotype were associated with linear growth delay (p = 0.006) and delay of vertebrae width (p = 0.001) [20]. In Danish study with 224 girls aged 11–12 there were no influence of FokI and TaqI VDR gene polymorphisms on bone mineralization and metabolism. Only ff genotype was associated with higher linear growth. The role of TaqI remained unknown [21]. In the study of Australian (n = 3,906) and Netherland (n = 1,689) twins significant influence of BsmI-, FokI-, TaqI- and (-1521) VDR polymorphisms on linear growth no found [22]. Several studies were devoted the role of VDR polymorphisms in bone metabolism disturbance in adults with rheumatoid arthritis (RA). Bone mineralization was studied annual during 3 years in 232 adults with early RA (160 women and 72 men) and VDR TaqI polymorphism was identified. Allele distribution in RA patients was similar to
population. At the end of the study women with of t allele had more rapid LS bone loss (4.9 %) and femoral neck (FN) (9.6 %) compare with carriers of TT genotype (0.1 % for LS and 3.9 % for FN, p \ 0.05 and \0.01, respectively). Interesting that this effect did not dependent of other diseases characteristics and authors suggested that t allele was associated with accelerated bone loss in women with RA [23]. In the study of 64 adult RA patients the carriers of bb genotype BsmI VDR had higher BMD (p \ 0.05), lower urinary deoxypyridinoline (p \ 0.05) and urinary calcium excretion (p \ 0.05) than carriers of BB and Bb genotypes [24]. In several studies there were no changes in bone metabolism and mineralization related to VDR genotypes and alleles. In study of 62 women with RA associations between BsmI-, TaqI-, FokI VDR genotypes and bone metabolic markers were not found [25]. In 155 Korean patients with RA, no influence of BsmI- and TaqI polymorphic genotypes in development of bone erosions [26]. In the single study of 50 JIA and 80 controls TT genotype of Alu I polymorphism of calcitonin receptor gene and ff genotype of FokI VDR were associated with lower BMD (p = 0.04 and 0.02, relatively) in the LS [27]. In our study TT genotypes TaqI VDR shows contrary effects: it associated with lower BMD in JIA girls, who never been treated by GCS and in mid-puberty boys. Also children with low linear growth (less than 10th percentile) more often had allele T-contained genotypes. Association between linear growth delay and low bone mineralization is well-known and shows negative effect of T allele (risk allele) on children’s bone health. These data are contrast to results of described above studies, with exclusion the prepubertal girls with TT genotype associated with higher total and ionized calcium level. We have not found any associations between VDR haplotypes distribution and presence of LBMD, possibly
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because we have small number of patients with different haplotypes. These differences also could be explained by small sample size and its peculiarities.
Conclusion In whole JIA group no differences in TaqI and Cdx2 haplotypes, genotypes and alleles distribution related with normal and low BMD (Zscore \-2SD) were found. Association VDR molecular markers with bone mineralization and metabolism disturbances, related to gender, puberty stage, GC treatment was found. TT genotype of TaqI is a negative factor impact bone mineralization and linear growth. GG genotype of Cdx2 negatively influences bone metabolism in JIA children. In contrast, A allele of the same molecular marker associated with high linear growth and could be mentioned as a bone protective marker. Conflict of interests The authors declare that they have no conflict of interests.
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