Originalien Z Rheumatol 2015 · 74:346–350 DOI 10.1007/s00393-015-1582-5 Published online: 16. April 2015 © Springer-Verlag Berlin Heidelberg 2015

Redaktion

U. Müller-Ladner, Bad Nauheim U. Lange, Bad Nauheim

Osteoporosis is a skeletal disorder characterised by compromised bone strength that predisposes one to an increased risk for fracture and is a major public health problem worldwide [1]. Osteoporosis affects mainly postmenopausal females, but also males. Several factors contribute to the development of osteoporosis, including clinical, medical, behavioural, nutritional and genetic variables [2]. Most multifactorial diseases that show a clear genetic component, including osteoporosis, are often called polygenic diseases, to emphasise their determination by multiple genetic factors. Given the complex biology of the skeleton, it is likely that bone mass is under the control of a large number of genes, many of which exert relatively small effects on bone mineral density (BMD), whereas a few contribute substantially to variation in this trait [3]. In addition, several epidemiological studies have demonstrated that a positive family history of osteoporosis is a risk factor for developing osteoporotic fractures [4, 5]. Inflammation can increase bone resorption, decrease bone formation but most commonly impacts both of these processes and patients with chronic inflammatory diseases are at high risk of systemic bone loss and osteoporosis [6]. The local renin–angiotensin system (RAS) in the vessel walls plays a crucial role in the endothelial control of vascular tonus and contributes to the inflammatory process by stimulating cytokine production [7]. Angiotensin-converting enzyme (ACE) is a regulatory component of the RAS and plays an important role in inflammatory and immune-related disor-

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Zeitschrift für Rheumatologie 4 · 2015

B. Cakmak1 · A. Inanir2 · N. Karakus3 · O. Ates3 · S. Yigit3 1 Department of Obstetrics and Gynecology, Gaziosmanpasa University School of Medicine, Tokat 2 Department of Physical Therapy and Rehabilitation, Gaziosmanpasa University School of Medicine, Tokat 3 Department of Medical Biology, Gaziosmanpasa University School of Medicine, Tokat

Association between the ACE gene I/D polymorphism and osteoporosis in a Turkish population ders, but the most important task is catalysing conversion of angiotensin I to angiotensin II, which is a short-lived potent vasoconstrictor, a stimulant of aldosterone release and a regulator of the expression of growth factors, cytokines, chemokines and adhesion molecules involved in cell growth, apoptosis, fibrosis and inflammation [8]. The ACE gene is located on the long arm of chromosome 17 and is expressed in multiple tissues [9]. This gene contains a polymorphism characterised by either insertion (I) or deletion (D) of a 287-base-pair ALU repetitive sequence in intron 16. The ACE I/D polymorphism determines functional variants of the ACE gene with a major impact on plasma ACE concentrations [10, 11, 12]. The ACE I/D polymorphism has been studied in several diseases characterised by inflammation and vascular conditions. It appears to be associated with Alzheimer’s disease, Behçet’s disease, contact dermatitis and osteoarthritis [13, 14, 15, 16]. In this study, we investigated the relationship between the ACE gene I/D polymorphism genotype and osteoporosis.

Materials and methods Subjects The study group consisted of 238 females with osteoporosis and 124 healthy controls. All patients were recruited at the Department of Physical Medicine and Rehabilitation of Gaziosmanpasa University Tokat, Turkey. All individuals were women of postmenopausal status and were from the central region of Turkey. Postmenopaus-

al status was defined as menstruation that had stopped completely for at least 1 year. Exclusion criteria were premature menopause, surgical menopause, presence of malignancy, presence of systemic disease (such as hyperthyroidism, parathyroid disorders, Crohn’s disease and chronic inflammatory and autoimmune rheumatic conditions) and administration of drugs (such as corticosteroid, ACE inhibitors or L-thyroxine) that might affect bone metabolism. Users of hormone replacement therapy (oestrogens/progesterone) and anti-osteoporotic drugs were excluded to provide homogeneity between groups. All subjects were evaluated for BMD. Lumbar spine (L2–L4), femoral neck and total femur BMD were measured by dual energy X-ray absorptiometry. BMD values were measured as kg/cm2 and revised to the T-score. The osteoporosis T-score is the number of standard deviations (SDs) above or below the mean bone mass values for young normal adults of the same sex. According to the World Health Organisation criteria, osteoporosis was defined as BMD >2.5 SD (T-score) below the mean value of young adults, i.e. peak bone mass [17]. Patients were divided into osteoporosis and control groups based on the BMD results. Selection criteria for the healthy controls included non-osteoporotic BMD (Tscore >−2.5 SD) and similar demographic characteristics to the osteoporosis group, such as age, menopausal age, weight, height, body mass index (BMI), presence of systemic disease, admission of drug use and smoking. This study protocol was approved by the Local Ethics Committee

Tab. 1  Baseline demographical characteristics of the patients and controls Characteristics Age (years) Age at menopause (years) Years since menopause BMI (kg/m2) T-score – Femoral neck – Total femoral – Lumbar spine Family history, n (%) Fractures, n (%) – Vertebral fracture – Hip fracture – All Calcium supplementation, n (%) Ever smoking, n (%)

Patients n=238 57.0±6.5 46.3±3.7 3.7±2.5 30.8±4.02

Controls n=124 56.3±9.0 46.5±3.5 4.1±2.4 31.4±4.6

p

−2.77±0.66 −1.52±1.00 −0.70±0.96 18 (7.6)

0.00±0.89 0.01±0.87 0.46±1.09 9 (7.3)

0.000 0.000 0.000 0.917

6 (2.5) – 20 (8.4) 76 (31.9) 9 (3.8)

– – – 43 (34.7) 7 (5.6)

0.098 – 0.000 0.598 0.428

0.068 0.750 0.238 0.175

Data were analysed by analysis of variance and χ2 test. Mean plus standard deviation values are presented for all variables, except family history, fractures, calcium supplementation and ever smoking ACE Angiotensinconverting enzyme, BMI Body mass index.

Tab. 2  Clinical and demographical characteristics of patients stratified according to ACE

gene I/D polymorphism Characteristic Age (years) Age at disease onset (years) Disease duration (years) Age at menopause (years) Years since menopause BMI (kg/m2) T-score – Femoral neck – Total femoral – Lumbar spine Family history, n (%) Fractures, n (%) – Vertebral fracture – Hip fracture – All Calcium supplementation, n (%) Ever smoking, n (%)

Total n=238 57.90±6.505 53.32±4.691 4.95±5.343 46.3±3.7 3.7±2.5 30.84±4.022

DD n=106 57.75±6.144 52.76±3.932 5.40±6.028 46.5±3.6 3.8±2.6 31.24±4.202

ID n=98 58.28±6.719 54.08±5.247 4.46±4.802 46.0±3.9 3.6±2.4 30.33±4.023

II n=34 57.29±7.082 52.81±4.889 5.03±4.687 46.7±3.7 3.7±2.6 31.06±3.327

P value 0.713 0.133 0.490 0.661 0.868 0.261

−2.77±0.66 −1.52±1.00 −0.70±0.96 18 (7.6)

−2.75±0.67 −1.55±1.01 −0.56±0.93 6 (5.7)

−2.77±0.72 −1.57±0.97 −0.82±0.98 9 (9.2)

−2.86±0.43 −1.25±1.03 −0.78±0.97 3 (8.8)

0.710 0.231 0.145 0.608

6 (2.5) – 20 (8.4) 76 (31.9)

2 (1.9) – 8 (7.5) 40 (37.7)

2 (2.1) – 8 (8.2) 27 (27.6)

2 (5.9) – 4 (12.1) 9 (26.1)

0.404 – 0.705 0.226

9 (3.8)

6 (5.7)

3 (3.1)

–

0.286

Data were analyzed by analysis of variance and χ2 test. Mean plus standard deviation values are presented for all variables, except family history, fractures, calcium supplementation and ever smoking ACE Angiotensinconverting enzyme, BMI Body mass index.

of Gaziosmanpasa University, Faculty of Medicine, and written informed consent was obtained from the study participants.

Genotyping Genomic DNA was isolated from whole venous blood samples using a commer-

cial DNA isolation kit (Sigma-Aldrich, Taufkirchen, Germany). The ACE gene I/D polymorphism was analysed by polymerase chain reaction (PCR). PCR was performed in a 25-µl reaction mixture containing 100 ng of genomic DNA, 2.5µl 10× PCR buffer, 200 µM dNTP, 10 pM each primer and 1 unit of Taq DNA poly-

merase. Amplification was carried out using the following primers: 5’-CTG GAGACCACT CCCATC CTT TCT-3’ and 5’-GAT GTG GCC ATC ACATTC GTC AGAT-3’ with an initial melting step of 5 min at 94°C, followed by 30 cycles of 1 min at 94°C, 1 min 45 s at 60°C, 1 min 30 s at 72°C and a final elongation step of 5 min at 72°C. PCR products were resolved on 2% agarose gels after ethidium bromide staining. In the absence of the 287 bp in intron 16 of the ACE gene, this PCR method resulted in a 190-bp product (D allele) but resulted in a 477-bp product (I allele) in the presence of 287 bp. Two bands (477 and 190 bp) were detected in heterozygous samples. A second PCR was performed to confirm samples with ambiguous results.

Statistical analysis Statistical analyses were performed using SPSS, version 20 (SPSS Inc., Chicago IL, USA) and OpenEpi Info version 3.01 software package (http://www.openepi.com). Results are given as means ± SD. The chisquare (χ2) test was used to evaluate the Hardy–Weinberg equilibrium for the genotype distributions of the patients and controls. The relationships between the I/ D polymorphism and the clinical and demographic features of patients were analysed using the χ2 test or analysis of variance. The χ2 test was used to compare categorical variables, and odds ratios (ORs) and 95% confidence intervals (CIs) were calculated to assess risk. All p-values were two-tailed, and values