Genetic factors determine extent of bone loss in inflammatory bowel disease

GASTROENTEROLOGY2000;119:909-920

Genetic Factors Determine Extent of Bone Loss in Inflammatory Bowel Disease CLAUDIA M. S. SCHULTE,* AXEL U. DIGNASS,{ HARALD GOEBELL,~ HANS-DIETRICH ROHER,§ and KLAUS-MARTIN SCHULTE§ Divisions of *Endocrinology and tGastroenterology, Department of Internal Medicine, University of Essen, Essen; and §Clinic for General Surgery, Heinrich-Heine-Universit~t D0sseldorf, D0sseldorf, Germany

Background &Aims: Although bone loss and osteoporosis are well-known long-term sequelae of inflammatory bowel disease (IBD), the risk factors for increased bone loss have not been identified. Balances of pro- and anti-inflammatory cytokines influence mechanisms of both chronic inflammation and bone resorption. The aim of this study was to identify genetic risk factors for rapid bone loss in IBD patients as a model of disease- and inflammation-associated bone loss. Methods: Multiple clinical parameters, biochemical markers of bone metabolism (vitamin D, parathyroid hormone, N-terminal telopeptide of type-I collagen, desoxypyridinoline, bone alkaline phosphatase), and bone mineral density were prospectively assessed in 83 IBD patients over 1.6 -+ 0.3 years. Eighty-six healthy bone marrow donors served as controls for allelotyping. The allele status of the interleukin 1 receptor antagonist (IL-lra), IL-6, heat shock protein 70-2 (hsp 70-2), and heat shock protein 70-hom (hsp horn) genes was typed and correlated with clinical course of IBD and extent of bone loss. Results: The extent of bone loss was not correlated to clinical severity of disease or application of corticosteroids. Noncarriage of the 240-base pair allele of the IL-lra gene and carriage of the 130-base pair allele of IL-6 were independently associated with increased bone loss. Genetic variations of the hsp genes were not associated with degree of bone loss. The combined presence of the named risk factors was significantly associated with increasing bone loss. Conclusions: Genetic variations in the IL-6 and IL-lra gene identify IBD patients at risk for increased bone loss.

one loss and osteoporosis are long-term sequelae of inflammatory bowel disease (IBD). 1 Although the increased prevalence of osteopenia and osteoporosis in patients with IBD is well recognized, knowledge about the pathophysiology, origin, and temporal course of bone loss in these patients is sparse. Multiple clinical parameters such as disease activity, corticosteroid use, changes in bone metabolism, or the patient's age have failed to predict the extent of bone loss in the setting of systemic inflammatory disease such as IBD. 2

B

Ulcerative colitis and Crohn's disease are immunemediated disorders characterized by a chronic, relapsing inflammatory response) Although the cause of IBD is not clearly known,4 the interplay of several cytokines with immunoregulatory and proinflammatory activities such as interleukin (IL)-I 5 and IL-6,6 and counteracting anti-inflammatory cytokines such as IL-1 receptor antagonist (IL-lra), may contribute to the pathogenesis of ongoing inflammation. Independently from the systemic inflammatory setting, IL-1 and IL-6 have a central role in the paracrine stimulation of osteoclast development and regulation of the process of bone resorption, v,8 Heat shock proteins (HSPs) play an important role in intracellular trafficking and conformation of proteins by acting as molecular chaperones, thus being involved in diseases with changes in immune regulation like IBD. 9-11 Different molecular chaperones have recently also been recognized as potent inducers of bone resorption and possibly play a role in the normal and pathologic remodeling of bone. ~2,13 Allelotyping is a powerful tool in the assessment of interindividual variations in the genetic equipment. Because the underlying functional changes are not often known, the first approach for identification of candidate genes often consists in analysis of hypervariable regions located within or around such a candidate gene. Genetic variations of cytokines with a key position in regulation of the inflammatory response and bone resorption might play a role in the regulation of inflammation-associated rapid bone loss. We consider bone as a metabolic compartment reacting to inflammatory stress. 1<15 We therefore typed the allele status of the IL-lra, IL-6, and the heat shock proteins hsp 70-2 and hsp 70-horn genes in Abbreviations used in this paper: BMD, bone mineral density; BW, body weight; CV, coefficients of variation; HSP, heat shock protein; IL, interleukin; IL-lra, interleukin I receptor antagonist; PCR, polymerase chain reaction. © 2000 by the American Gastroenterological Association 0016-5085/00/$10.00 doi:10.1053/gast.2000.18158

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I B D patients to analyze its correlation to the clinical course of inflammatory disease and longitudinal changes in bone mineral density (BMD) prospectively assessed by osteodensitometry. W e characterized a group of 83 patients with I B D with regard to clinical course, BMD, and biochemical markers of bone metabolism at entry to the study and prospectively followed t h e m for 1.6 years. W e analyzed the correlation of genetic variations with the following: (1) retrospectively assessed clinical course of I B D since diagnosis of disease; (2) clinical course of I B D prospectively observed over 1.6 years; (3) B M D and biochemical markers of bone metabolism at entry to the study; and (4) bone loss prospectively observed over 1.6 years.

M a t e r i a l s and M e t h o d s Subjects The study group consisted of 83 patients with IBD. Sixty (72%) patients had Crohn's disease, and 23 (28%) patients had ulcerative colitis. The diagnosis of IBD was established on the basis of standard clinical, radiologic, endoscopic, and pathologic criteria. Exclusion criteria included borderline manifestations; unclear subtype or uncertain diagnosis; former organ transplantation; other systemic inflammatory disease; medication with bone-toxic agents (exept steroids) such as heparin, thyroxine, and cyclosporin A; specific bone disease; and other endocrine metabolic or nutritional disease. All analyses were done either when the patients were seen regularly in the outpatient clinic or during hospital stays. The study was performed according to the principles of the Declaration of Helsinki, and patients gave informed consent. At baseline, all patients were interviewed by 2 examiners using a standardized questionnaire for the assessment of the clinical course of disease, accompanying diseases, medication for IBD and other medication, smoking, dietary habits, and occurrence of osteoporotic fractures. The cumulative steroid dose at baseline (expressed in prednisolone equivalent) was estimated with the help of patient information and patient chart. Steroid dose was expressed in mean daily dose of prednisolone equivalent per day of known disease activity (mg/day). To even out differences in body weight (BW), steroid dose was also expressed in milligrams prednisolone equivalent per day of known disease activity per kilogram of BW (mg • day -1 • kg BW 1). We planned to reevaluate patients 18 months after the baseline measurement. At follow-up, patients underwent the same assessment. Steroid doses applied between baseline and follow-up were evaluated by means of patient information and patient chart. Dosage again was expressed in prednisolone equivalent in mg/day and mg • day -1 • kg BW -1.

Parameters of Disease Activity The following parameters were used to describe disease activity. Duration of disease since diagnosis in years was de-

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fined as the time since clinical diagnosis. Only a few patients had a difference longer than 1 year between first disease symptoms and clinical diagnosis of disease. Patients were asked about acute phases of the disease since diagnosis, which made acute-phase therapy with corticosteroids and/or hospital stays necessary. Furthermore, the patient's chart was evaluated for analysis of disease activity. Where available, the Colitis Activity Index for ulcerative colitis and the Crohn's Disease Activity Index for Crohn's disease were used to complete analysis of active disease phases. In many patients, long disease periods had to be evaluated, which made estimation of the number of flare-ups necessary. Every acute phase, which made either acute-phase therapy with steroids or a hospital stay necessary, was called an acute flare-up. The integral of all remembered and documented flare-ups was defined as the number of acute flare-ups at baseline. The number of flares was divided by the number of years since diagnosis and defined as acute flares per year since diagnosis. For the time period between baseline and follow-up, evaluation of disease activity was easier, because all disease events could be exactly proven by the patient chart including Colitis Activity Index and Crohn's Disease Activity Index as disease activity indices.

Healthy Controls Eighty-six patients who had recently received a bone marrow transplant from healthy donors served as control for genotyping. BMD Assessment

BMD was measured by dual-energy x-ray absorptiometry (model DPX-L; Lunar Corp., Madison, WI) at the lumbar spine (second, third, and fourth vertebrae) and at the proximal left femur (femoral neck, trochanter, Ward's triangle). BMD was expressed as the number of standard deviations (SDs) from normal values of age- and sex-matched controls (Z-score). Reference values for men and women were supplied by Lunar Corporation, referring to a normal white north European population (2588 white women and men aged 2 0 - 7 9 years). A quality assurance test was performed daily with a standard block of tissue-equivalent material with 3 bone-simulating chambers of known bone mineral content and weekly with an anthropomorphic spine phantom (Hologic Inc., Waltham, MA). The reproducibility of the method was >99%. Analysis of spine BMD may be hampered by major sclerosis of the aorta, osteophytes, and scoliosis. Results of spine BMD from patients presenting one of these problems were therefore excluded. Correct analysis of BMD at the femoral neck must take into account considerable interobserver mistakes because positional variations of the rotated leg cause mistakes in the analysis. To reduce interobserver variability, 3 examiners operated the Lunar DPX-L only. All analyses were checked by one investigator (C.S.). Bone loss data are given as annualized values calculated as difference in BMD between baseline and follow-up 1.6 years later. Positive values for change in bone mass indicate gain of bone mass, and negative values indicate loss of bone mass.

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Assessment of Total Body Composition Total body composition was measured by dual-energy x-ray absorptiometry (Lunar DPX-L) as a total body scan. Fat mass was expressed in percent of total BW.

Biochemical Assessment Biochemical assessment was performed at baseline only. A urine sample (not the first morning urine) was taken between 9 aM and 1 eM. On the same day blood samples were obtained. The subjects had no dietary restrictions. Blood was immediately centrifuged, and serum, plasma, and urine were stored at - 3 0 ° C until assay performance. Serum bone-specific alkaline phosphatase (normal range, 8 - 2 4 U/L) as a marker of bone formation was measured by an enzyme-linked immunoassay using a monoclonal anti-bonespecific alkaline phosphatase antibody (Alkphase; Metra, Mountain View, CA). The intra- and interassay coefficients of variation (CV) were < 1 0 % ; the sensitivity was 2 U/L. N-terminal-telopeptide of type I collagen (normal range, 5 - 6 0 mmol/L per mmol/L creatinine) as a marker of bone resorption was measured by an enzyme-linked immunoassay (Osteomark; Ostex Inc., Seattle, WA) using a monoclonal antibody directed against the N-telopeptide-to-helix intermolecular cross-linking domain of type I collagen isolated from human urine. This monoclonal antibody does not recognize free cross-links. 16 The intra- and interassay CV were < 9 % ; the sensitivity was 25 nmol/L. Free desoxypyridinoline (normal range, 1.5-6. l mmol/L per mmol/L creatinine) as a marker of bone resorption was measured by an enzyme-linked immunosorbent assay using a monoclonal antibody (Pyrilinks-D; Metra) with < 2 . 5 % crossreactivity with free pyridinoline and 10% with cross-linked peptides. The intra- and interassay CV were 8% and 9%, respectively; the sensitivity was 25 nmol/L. Serum concentrations of 25-(OH)-vitamin D (normal range, 10-55 ng/mL) were measured by a radioimmunoassay using an antibody with specificity to 25-(OH)-vitamin D (25-Hydroxyvitamin D-Iodine-125 RIA Kit; Incstar, Stillwater, MN). Plasma concentrations of intact parathyroid hormone (PTH[1-84]) (normal range, 1 0 - 6 0 pg/mL) were measured by a radioimmunoassay using an antibody with specificity to human parathyroid hormone (PTH[1-84]; Nichols Institute, San Juan Capistrano, CA). Serum concentrations of transferrin (normal range, 2 3 0 430 mg/dL) were measured using a nephelometer analyzer (Behring Co., Marburg, Germany). Intra-assay CV was 2.6%, and interassay CV was 2.7%. Serum concentrations of prealbumin (normal range, 2 5 - 4 5 mg/dL) were measured using a nephelometer analyzer (Behring).

Preparation of Genomic DNA D N A was isolated from 200 b~L venous EDTA-blood using a commercial kit (QIAamp D N A blood extraction kit; Qiagen, Hilden, Germany) yielding about 1 b~g of DNA.

GENETICFACTORSAND BONELOSSIN IBD 911

D N A quality and quantity were assessed by spectrometry at 260/280 nm and agarose gel electrophoresis with subsequent ethidium bromide staining (1 ~g/mL). Suitability of isolated D N A for polymerase chain reaction (PCR) was controlled by use in various amplification systems. Genomic D N A was isolated and analyzed from unrelated patients with IBD (n = 83) and healthy controls (n = 86).

Allelotyping at the Ik-lra Gene Oligonucleotide primers for amplification of a fragment of the IL-lra gene were derived from published genomic sequences. Primers for amplification of IL-lra were forward 5' ctc agc aac act cct at (position 8 8 6 8 - 8 8 8 4 ) and reverse 5' tcc tgg tct gca ggt aa (position 9279-9263) (GenBank accession no. X64532). The reaction mix for PCR contained 25-75 ng D N A and 10 pmol oligonucleotides. Reactions were performed in a 1X solution of TaqMasterMix (Qiagen). Amplification was accomplished by initial incubation at 95°C for 5 minutes followed by 36 cycles of denaturation at 94°C for 30 seconds, annealing at 55°C for 30 seconds, and extension at 72°C for 1 minute. The reaction was completed by a final incubation at 72°C for 8 minutes. This generated the following amplimers according to the variable number of tandem repeats: A 1 , 4 1 0 base pairs (bp); A 2 , 2 4 0 bp; A 3 , 5 0 0 bp; A4, 325 bp; and A5, 595 bp. Amplimers were separated by electrophoresis on 1.5% (wt/vol) agarose gels with subsequent staining in ethidium bromide solution (1 Dg/mL) for 15 minutes. Size was determined by comparison to a molecularweight-standard (1 kilobase plus; Gibco, Paisley, Scotland).

PCR and Electrophoresis of the D 7 S 6 2 9 Microsatellite Neighboring the IL-6 Gene Primers for amplification of D7S62917 were forward 5' ttc tac atg aca gca gaa cac (position 189-209) and reverse 5' tct gtg gga aag tat atg tgc (position 320-300) (GenBank accession no. Z23419). The forward primer was labeled with cybergreen at the 5' nucleotide. The reaction mix for PCR contained 25-75 ng D N A and 10 pmol oligonucleotides in a 1 × solution of TaqMasterMix (Qiagen). Amplification was accomplished by initial incubation at 94°C for 5 minutes followed by 30 cycles of denaturation at 94°C for 30 seconds, annealing at 55°C for 30 seconds, and extension at 72°C for 45 seconds. The reaction was completed by a final incubation at 72°C for 7 minutes. This generated amplimers of 124-134 bp in size: A 1 , 1 3 4 bp; A 2 , 1 3 2 bp; A 3 , 1 3 0 bp; A4, 128 bp; AS, 126 bp; and A6, 124 bp. Amplimers were separated by electrophoresis on 8% polyacrylamide sequencing gels. Preparation of the gel matrix was performed using premixed highresolution sequencing gels containing UV-activated linkers for polymerization (ReproGel High Resolution; Amersham Pharmacia Biotech, Uppsala, Sweden) and initiation of the polymerization process by a ReproSet (Amersham Pharmacia Biotech). Fluorescence-labeled D N A adducts were detected by a laser-activated system (ALF express II; Pharmacia, Uppsala, Sweden) on electrophoresis at 1500 V, 60 mA, and 30 W at 55°C in a 0.5× Tris-borate-EDTA buffer system. Exact size

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determination of amplimers was achieved using dye-amidite molecular weight standards: 100, 200, and 300 bp as external standard and 100 bp and 150 bp in each lane added to the sample before denaturation. Calibration was performed using the Fragment analyzer software (Pharmacia).

Restriction Fragment Length Polymorphism Analysis of the HSP 70-2 and HSP-hom Genes Oligonucleotide primers for amplification of fragments of the HSP 70-2 and HSP-hom gene were derived from published D N A sequences. Primers for amplification of HSP 70-2 were forward 5' gtg ctc cga cct gtt ccg aag c (position 1400-1421) and reverse 5' egg agt agg tgg tga aga tct g (position 1782-1761) (GenBank accession no. M59830 and M34269). The reaction mix for PCR contained 25-75 ng D N A and 10 pmol oligonucleotides in a 1 × solution of TaqMasterMix (Qiagen). Amplification was accomplished by initial incubation at 94°C for 5 minutes, followed by 30 cycles of denaturation at 94°C for 30 seconds, annealing at 57°C for 30 seconds, and extension at 72°C for 1 minute. The reaction was completed by a final incubation at 72°C for 10 minutes. This amplified a fragment of 383 bp containing an A/G polymorphism in position 1538 generating a PST I restriction cleavage site. For restriction fragment length polymorphism analysis of the hsp 70-2 gene, amplimers were digested by the restriction endonuclease PstI (Boehringer Mannheim, Mannheim, Germany) at 37°C in 1 × one phor all buffer (Boehringer) for 2 hours and subjected to electrophoresis on 1.5% wt/vol agarose gels with subsequent staining by ethidium bromide (1 ~g/mL). When a Pst I site was present, restriction digest produced 2 fragments of 244 and 139 bp. Size was determined by comparison with a molecular-weightstandard. Primers for amplification of HSP 70-hom were forward 5' gat cca ggt gta tga ggg (position 2282-3103) and reverse 5' gta act tag att cag gtc tgg (position 2988-2968) (GenBank accession no. M59829 and M34268). The reaction mixes for PCR contained 25-75 ng DNA, 10 pmol oligonucleotides, and a 1 X solution of TaqMasterMix (Qiagen). Amplification was accomplished by initial incubation at 94°C for 5 minutes followed by 30 cycles of denaturation at 94°C for 30 seconds, annealing at 55°C, and extension at 72°C for 1 minute followed by a final incubation at 72°C for 10 minutes. This amplified a fragment of 706 bp containing a C/T polymorphism in position 2437 generating an NcoI restriction cleavage site. In the presence of this site, restriction digest produced 2 fragments of 550 and 155 bp. Amplimers were digested by the restriction endonuclease NcoI (Boehringer) at 37°C in 1× One Phor All Buffer (Boehringer) for 2 hours. Fragments were separated by electrophoresis on 1.0% wt/vol agarose gels with subsequent staining in ethidium bromide solution (1 gtg/mL) for 15 minutes. Size was determined by comparison with a molecular-weight-standard 1 kb plus (GIBCO).

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Statistical Analysis Results are expressed as the mean - 1 SD. Statistical analysis was performed using the JMP statistical software (SA8 Institute, Cary, NC). The variation in BMD between baseline and follow-up was given as the annualized change in BMD. Differences between groups were analyzed by using the Student unpaired t test and analysis of variance for normally distributed values and the M a n n - W h i t n e y U test for not normally distributed values. The Fisher exact test was used in comparison between categorial data. Significance was assumed when P values were <0.05.

Results Study Population The characteristics of the study population at baseline are summarized in Table 1. In the group of 9 postmenopausal women, 4 patients received hormone replacement therapy. At baseline, 14% of the patients received immunosuppressive therapy with azathioprine. Thirty-two patients (38%) were on current medication with steroids at baseline. The follow-up visit and second BMD measurement were performed 1.6 years (589 days; SD, 110 days;

Table 1. Characteristics of the Study Population of 8 3 Patients With IBD at Baseline

n Type of IBD Crohn's disease Involvement of Colon Distal small bowel Upper GI tract Resection of lleocecal valve Colon (subtotal or total) Upper GI tract Ulcerative colitis Pancolitis Left-sided colitis Proctosigmoiditis Sex Male Female Pre/postmenopausal

Age (yr) Duration of disease (yr) Acute flare-ups/yr of disease Prednisolone/day since diagnosis (mg/day) Prednisolone/day since diagnosis/kg BW (mg. day - I • kg BW-I)

%

60 50 45 10

83 75 17

29 11 4 23 10 6 7

48 19 7 43.5 26 30.5

45 38 29/9

54 46

Mean

SD

37 10.8 2.1

14 6.7 3

6.3

7.4

3.2

1-33

0.1

0.12

0.04

0-0.43

n, number of patients; GI, gastrointestinal,

Median 33 11 1

Range

17-75 1-33 0.1-16

October 2 0 0 0

median, 573 days; range, 412-911 days) after baseline assessment. Fifty-five patients (67%) did not receive corticosteroids in the prospective observation interval, whereas 28 (33%) patients did receive corticosteroids with a mean dose of 7.8 + 5.6 mg prednisolone equivalent per day (0.12 - 0.1 mg • day -1 • kg B W - I ) . Averaged for all patients, mean prednisolone equivalent applied per day between baseline and follow-up assessment was 2.6 mg/day (SD, 4.9; median, 0; range, 0 - 2 4 ) , and mean prednisolone equivalent applied per day and B W was 0.04 mg ° day -1 • kg B W -1 (SD, 0.08; median, 0; range, 0 - 0 . 4 4 ) . At baseline 14% and at follow-up 22% of the patients received i mmunosuppressive therapy with azathioprine.

Retrospective Assessment: Previous Disease Activity, BMD, and Biochemical Markers of Bone Metabolism at Baseline According to the World Health Organization definition of osteoporosis, 18 41 patients (49%) had normal BMD at the spine and femoral neck. Twenty-eight patients (34%) showed an osteopenic result at the spine, and 33 patients (40%) at the femoral neck. Fourteen patients showed spinal osteoporosis and 9 patients (11%) osteoporosis of the femoral neck. To analyze the influence of clinical course of the IBD on BMD as an integral over time, we compared the 14 patients whose BMD result at baseline showed spinal osteoporosis (BMD < - 2 . 5 SD of the sex-matched healthy control group) with 69 patients with a normal spinal BMD (BMD > - 2 . 5 SD of the sex- and agematched healthy control group). The spinal T-score was -3.1 -+ 0.4 in the osteoporosis and - 0 . 6 + 1.2 in the normal BMD group. The following parameters were considered indicators of clinical course of IBD: age at diagnosis of disease, duration of disease, the number of acute flares per year since diagnosis, immunosuppressive therapy with azathioprine, cumulative steroid dose expressed as prednisolone equivalent per day and prednisolone equivalent per day per kilogram BW, body mass index, percent of fat tissue in body composition, and levels of transferrin and prealbumin. Patients with osteoporosis were different from those with normal BMD with regard to a few of the abovenamed parameters: in osteoporotic patients, duration of disease was longer (14.4 + 7 vs. 10 + 6.5 years; P = 0.026) and the body mass index was lower (20.9 - 3.1 vs. 23 -+- 3.27 kg/m2; P = 0.03). Bone alkaline phosphatase at baseline was significantly suppressed in the 32 patients under current steroid medication compared with the 51 patients not receiving steroids (16 - 4 vs. 22 -+ 10.7 U/L; P = 0.01). All other

GENETIC FACTORS AND BONE LOSS IN IBD

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biochemical bone markers (N-terminal telopeptide of type I collagen, free desoxypyridinoline, vitamine D, parathyroid hormone) did not differ between current steroid users and nonusers.

Prospective Assessment: Disease Activity, Biochemical Markers of Bone Metabolism, and Their Relation to Bone Loss During Prospective Follow-up We analyzed the influence of the clinical course of IBD and bone metabolism at baseline on bone loss observed over the study interval (1.6 + 0.3 years). Thirty patients who had lost bone mass at the spine ( - 1.7% + 1.9% per year) were compared with 53 patients who had gained bone mass ( + 2 . 5 % + 2.8% per year) during study interval. Neither the amount nor intensity of immunosuppressive therapy as a measure of disease activity during follow-up, nor any disease parameter shown to influence baseline BMD (duration of disease, number of acute flares, BMI), differed between patients who lost and patients who gained bone mass during prospective follow-up (Table 2). Similarly, BMD and biochemical markers of bone metabolism at baseline were of little value to predict prospective bone loss.

Influence of the Immunosuppressive Therapy Applied During Study Interval on Prospectively Observed Bone Loss The interindividual variability in changes of bone mass over 1.6 years was large at all sites of measurement: spine, 1.08%/yr (SD, 3.3; median 0.4; range - 4 to +5); femoral neck, 0.52%/yr (SD, 5.0; median, 0.1; r a n g e , - 6 to +7); and Ward's triangle, 1.44%/yr (SD, 7; median, 0.23; range, - 6 to +7). Differences in the immunosuppressive therapy could not explain this huge variability. Twenty-eight patients receiving steroids during follow-up (average daily dose, 7.8 + 5.6 mg) did not show more bone loss at any site than 55 patients not receiving steroids: spine, - 0 . 3 1 % + 2.6% v s . - 1 . 5 % + 3.7%/yr (P = 0.14); femoral neck, 1.34% + 6% v s . - 0 . 8 % + 6.1%/yr (P = 0.16); and Ward's triangle, - 0 . 8 3 % + 8% vs. - 2 . 4 6 % + 7%/yr (P = 0.08). In the group of steroid users, there was no dose-effect relation between steroids and bone loss at spine (r = - 0 . 0 6 ) , neck (r = - 0 . 1 2 ) , and Ward's triangle (r = - 0 . 1 ) . Steroids plus azathioprine were administered to 12.5% of all patients, steroids alone to 21%, azathioprine alone to 7.5%, and neither steroids nor azathioprine to 59% during the 1.5-year follow-up. There was no significant difference in bone loss at any site between 35 patients (41%) who received immunosuppressive therapy and 48 patients (59%) who did not.

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Table 2. Influence o f D i s e a s e Activity and M a r k e r s o f B o n e M e t a b o l i s m on C h a n g e s in B o n e M a s s Bone mass loss (n = 30)

Bone mass gain (n = 53)

- 1 . 7 _+ 1.9 n = 30

+ 2 . 5 +_ 2.8 n = 53

0.000

3,8 + 5

2.3 _+ 5

NS

P value

Change of spinal bone mass

(%/yr) Medication during prospective follow-up Steroids applied (mg/day) Steroids normalized to BW

(mg. da• 1 • kg BI/V 1)

0.05 _+ 0.07

0.04 _+ 0.09

NS

50

40

NS

9 ± 6 1.23 _+ 1.4 23 + 3

12 _+ 7.3 2 _+ 2.6 22.2 _+ 3

NS NS NS

- 0 . 6 9 _+ 1.2 - 0 . 6 8 _+ 1.4

NS

% of patients on steroids and/or azathioprine Disease activity at baseline Duration of disease at baseline (yr) Acute flares/yr at baseline BMI at baseline (kg/m 2)

Bone metabolism at baseline Z-score, spine NTX (mmol/L per mmol/L

creatinine)

56 _+ 44

62 ± 52

NS

6.5 + 3.5 20 + 10.8 17 _+ 12

NS NS NS

DPD (mmol/L per mmol/L

creatinine)

5.9 + 3.7 18.5 _+ 6.7 19 + 13

BAP (U/L) Vitamin D (ng/mL) Parathyroid hormone

(pg/mL)

40 ± 22

39 ± 18

NS

NOTE. Patients were categorized according to the change of spinal bone mass during follow-up into those who lost (n = 30) and those who gained (n = 53) bone mass. Neither the immunosuppressive medication as a measure of disease activity during follow-up nor disease activity or bone metabolism at baseline were of value to predict future bone loss. BAP, bone-specific alkaline phosphatase; DPD, free desoxypyridinoline; NS, not significant; NTX, N-terminal telopeptide of type I collagen.

Genotyping Analysis of the IL-lra gene polymorphism identified 4 of 5 possible alleles: A1 (410 bp, 4 repeats), A2

(240 bp, 2 repeats), A3 (500 bp, 5 repeats), and A4 (325 bp, 3 repeats) (Figure 1). The 2 alleles of the hsp 70-2, corresponding to the presence (2 fragments of 244 and 139 bp) or absence (1 fragment of 383bp) of the Pstl site, are referred as hspP1 and hspP2 (Figure 2). The 2 alleles of the hsp 70-horn, referred to as hspN1 and hspN2, correspond to the presence (2 restriction fragments of 550 and 155 bp) and absence of the Ncol site (amplimer of 705 bp). Allele frequency and genotype were comparable for IL-lra, hsp 70-2, and hsp 70-horn in IBD patients and controls (Table 3). Eight alleles were identified for the IL-6 gene: 118 (1.2%), 120 (11.6%), 122 (27.5%), 124 (26%), 126 (3.7%), 128 (23%), 130 (5.5%), and 134 (1.2%) (Figure 3). Nineteen different haplotypes were identified; the 6 most frequent are given in descending order: 122/124 (17%), 122/128 (13.5%), 120/122 (11%), 128/128 (11%), 124/124 (10%), and 124/128 (7.3%). Allele and genotype frequencies were comparable for the IL-6-related polymorphism D7S629 in IBD patients and controls (data not given).

Analysis of Associations Between the IL-lra, IL-6, hsp 70-2, and hsp 70-horn Polymorphisms and Different Variables The following alleles and baplotypes were tested in statistical analysis regarding an association with the below named variables: 1. IL-lra: alleles 240 and 410, the 3 haplotypes 240/240, 240/410, and 410/410. 2. IL-6: the frequent alleles 120, 122, 124, 128, and 130 and the frequent haplotypes 120/122, 122/ 124, 122/128, 124/124, and 128/128. 3. hsp 70-2 and hsp 70-horn: the frequent haplotypes of homozygotes for the restriction sites (hsp

500bp A3 410bp A1 240bp A2

1

2

3 4

5

6

7

8

Figure 1. Amplified variable number tandem repeat polymorphism of IL-lra gene. The products of 7 patients after electrophoresis in 0.8% agarose. Lanes 1, 4, and 7, homozygotes 4 1 0 / 4 1 0 ; lane 2, heterozygote 2 4 0 / 5 0 0 ; lane 3, heterozygote 2 4 0 / 4 1 0 ; lane 5, heterozygote 4 1 0 / 5 0 0 ; lane 6, homozygote 2 4 0 / 2 4 0 ; lane 8, molecular weight marker.

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915

383bp hspP2 244bp hspP1

Figure 2, Fragment length polymorphism in amplimers of hsp 70-2 gene. The hsp 70-2 PCR products were digested with Pstl. The hsp P1 allele corresponds to the 244- and 139-bp fragments (cleavage of the 383-bp fragment). The hsp P2 allele corresponds to the 383-bp fragment (absence of the Pstl site). The products of digestion reactions after electrophoresis in 1.0% agarose are shown. Lane M, molecular weight marker; lane 1, homozygotes P1/P1; lanes 2 and 3, heterogygotes P1/P2.

139bp hspP1

1

2

70-2 P1/P1 and hsp 70-horn N1/N1) and heterozygotes (hsp 70-2 P1/P2 and hsp 70-hom NI/N2).

Association Between the IL-lra, IL-6, hsp 70-2, and hsp 70-horn Polymorphisms and Clinical Course of Disease Until Baseline The following parameters were again regarded as indicators of disease activity before entry to the study: age at diagnosis of disease, duration of disease, average number of acute flare-ups per year since diagnosis, immunosuppressive

Table 3. Allele Frequencies and Genotypes of IL-lra, HSP 70-2, and HSP 70-horn in Healthy Controls and Patients With IBD, Subanalyzed for Crohn's Disease and Ulcerative Colitis HC (n = 85) IL-lra A1 (410 bp) A2 (240 bp) Haplotype 410/410 240/410 240/240 HSP 70-2 P1 (Pstl digest) P2 (no Pstl digest) Haplotype P1/P1 P1/P2 P2/P2 HSP 70-hom N1 (Ncol digest) N2 (no Ncol digest) N1/N1 N1/N2 N2/N2

All IBD (n = 82)

CD (n = 60)

UC (n = 22)

79 19

77 23

77 23

77 23

62 34 1.2

63 27 10

63 27 10

64 27 9

61 39

56 44

57 43

55 45

23 75 2

14 84 2.5

15 83 1.7

9 86 4.5

74 26 58 32 10

85 15 68 32 0

82 18 63 37 0

91 9 82 18 0

NOTE. Results are in percentage. CD, Crohn's disease; HC, healthy controls; UC, ulcerative colitis.

3

M

therapy with azathioprine, cumulative steroid dose expressed as prednisolone equivalent per day and prednisolone equivalent per day per kilogram of BW, body mass index, percent of fat tissue in body composition, and transferrin and prealbumin levels. Statistically relevant differences were observed for the following associations: 1. Twenty-seven heterozygous hsp 70-hom N1/N2 patients had received less cumulative steroid dose since diagnosis than 56 homozygous hsp 70-hom N I / N 1 patients: prednisolone equivalent/day, 3.8 + 5.8 vs. 7.6 -+ 7.8 mg/day (P = 0.03); prednisolone equivalent per day per kg BW, 0.06 --- 0.1 vs. 0.12 + 0.12 mg • day -1 • kg BW -1 (P = 0.03). 2. Ten patients with the 130-bp allele of IL-6 had experienced more flare-ups per year than 73 patients not carrying the 130-bp allele (4.6 - 6.8 vs. 1.8 -+ 2.3 flare-ups/yr; P = 0.01). This suggested a more complicated course of disease in patients with the 130-bp allele neighboring the IL-6 locus, although it was not accompanied by differences in steroid or azathioprine intakes.

Association Between the IL-lra, IL-6, hsp 70-2, and hsp 70-hom Polymorphisms and Bone Metabolism at Baseline The Z-score for BMD at spine, femoral neck, and Ward's triangle and the biochemical markers N-terminal telopeptide of type I collagen, DPD, bone-specific alkaline phosphatase, vitamin D, and parathyroid hormone at baseline were considered as markers of bone metabolism. There was no association between the named parameters and the above-named alleles and haplotypes.

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internal ] size marker 122

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size bp Figure 3.

Amplified CG-tandem repeat of the IL-6 gene. A product separated by electrophoresis on 8% polyacrylamide sequencing gels. The a r r o w indicates the internal standard of 100 bp; the product is a heterozygote 1 2 0 / 1 2 2 .

Association Between the IL-lra, IL-6, hsp 70-2, and hsp 70-horn Polymorphisms and Disease Activity During Prospective Follow-up The percentage of patients undergoing immunosuppressive therapy with azathioprine and/or steroids, the cumulative steroid dose applied since baseline (expressed in prednisolone equivalent per day and prednisolone equivalent per day per kilogram BW), and the absolute change of fat tissue in the body composition from baseline were regarded as parameters of clinical course since baseline. There was no association between the named parameters and the above-named alleles and haplotypes.

Association Between the IL-lra, IL-6, hsp 70-2, and hsp 70-hom Polymorphisms and Bone Loss During Study Interval Analysis of changes in bone mass revealed noncarriage of the A2 (240 bp) allele of the IL-lra as a risk factor for increased bone loss at the femoral neck and

carriage of the 130-bp IL-6 allele as a risk factor for increased bone loss at the spine (Table 4).

Disease Activity of IBD and Extent of Bone Loss in Dependence of the Number of Genetic Risk Factors Analysis had shown noncarriage of the A2 (240 bp) allele of the IL-lra and carriage of the 130-bp IL-6 allele as risk factors for an increased bone loss. We defined noncarriage of one A2 allele as 1 risk factor, noncarriage of two A2 alleles as 2 risk factors, carriage of one 130-bp IL-6 allele as 1 risk factor, and carriage of two 130-bp IL-6 alleles as 2 risk factors. According to this definition, 7 patients had no risk factor, 22 patients had 1 risk factor, 46 patients had 2 risk factors, 7 patients had 3 risk factors, and 1 patient had 4 risk factors. For further analysis, we stratified patients into 3 risk groups according to the number of existing risk factors (Table 5). An increasing number of risk factors was significantly associated with an increasing amount of bone loss. The risk groups did not differ with regard to the immunosuppressive therapy applied during baseline and follow-up.

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T a b l e 4. N o n c a r r i a g e o f t h e A2 Allele o f t h e I L - l r a G e n e and Carriage o f t h e 1 3 0 - b p Allele o f t h e IL-6 G e n e Are A s s o c i a t e d With I n c r e a s e d Bone L o s s Change of bone mass (%/yr) a No. of cases Spine Femoral neck Ward's triangle

IL-lra (240 yes) 30 1,6 _+ 4 2,4 + 4.9 4.4 _+ 7.26

IL-lra (240 no) 53 0.8 _+ 2.8 - 0 . 4 3 4- 6 - 0 . 3 4 4- 7

P value

IL-6 (130 yes)

IL-6 (130 no)

P value

0.3 0.04 0.0096

10 - 1 . 9 5 + 3.5 - 2 _+ 5 - 1 . 7 4- 7

73 1.44 + 3 1.17 + 5.5 2 + 7

0.0056 0.1 0.14

apositive values indicate gain of bone mass; negative values indicate loss of bone mass.

Discussion Our study shows that variations of the IL-6 and ILl-ra genes, but not the hsp 70 gene, are independent determinants of bone loss in the setting of IBD. The IL-6 and ILl-ra genes have been identified as independent predictors of bone loss in the setting of postmenopausal osteoporosis. That they function in a similar way in our study group of IBD patients may signify that they specifically determine the response of bone to different stressors such as the postmenopausal status or systemic inflammation. Inflammation is assumed to be associated with increased bone loss. 19 IBD is a paradigm of chronic inflammatory disease, and there is substantial evidence for an increased incidence of osteopenia and osteoporosis in IBD. 2°-22 IBD is under discussion as a model of inflammation-associated bone loss, 14 and it has repeatedly been suggested that patients with high inflammatory disease activity of IBD and intake of high amounts of steroids are prone to associated bone disease. This conclusion has been drawn from many studies that assessed bone mass

mainly in cross-sectional study designs. Our data show that bone mass itself is not the relevant point to study because it is subject to many factors that are only partly related to the disease. It is rather the change in bone mass, herein also addressed as bone loss, which reflects dynamic processes in the bone during active phases of disease. Only a few attempts have been made to identify risk factors of bone loss in a prospective, longitudinal design. 2,23-27 These studies showed huge variations in individual annual bone loss. 2,24,26 Preventive bone protective therapy is demanded for patients at risk, but risk factors for a rapid bone loss have not been clearly identified. The question to what extent the systemic inflammatory process itself causes bone loss in IBD patients has been addressed in several animal studies, 15,28 but remains finally unresolved. Like several other studies, we could again show that bone loss in IBD patients is highly variable among individuals (Table 5). W e attempted to answer the question whether this variability is caused by interindividual variability in the extent of systemic inflammation, as previously postulated, or by variations in the individual reaction of the bone.

T a b l e 5. I n c r e a s i n g N u m b e r o f G e n e t i c Risk Factors Are A s s o c i a t e d With I n c r e a s i n g A m o u n t o f B o n e L o s s and Is T h e r e f o r e V a l u a b l e f o r Risk Stratification o f IBD Pat i ent s

All patients (n = 82) Change of bone mass (%/yr) a Spine Femoral neck Ward's triangle Clinical data Steroids applied until baseline (mg/day) ( m g . day -1 • kg BW - I ) During follow-up (mg/day) ( m g . day -1 • kg BI4#1) Immunosuppression b

1.08 4- 3.3 0.52 + 5 1.44 +_ 7

Group A 0-1 risk factors (n = 29) 1.62 + 4 2.5 + 4.9 4.6 _+ 7

Group B 2 risk factors (n = 46) 1.3 + 2.4 - 0 . 7 5 4- 7 - 1 . 2 4- 7

6.3 + 4.9 0.1 + 0.12

6.4 _+ 7.4 0.1 + 0.11

6.3 _+ 7.6 0,1 + 0.13

2.6 + 4.9 0.04 _+ 0.08 41

2.9 4- 4.9 0.04 _+ 0.06 46

2.1 + 4 0.04 _+ 0.07 38

Group C 3 or more risk factors (n = 8)

P value A vs. C

B vs. C

A vs. B

- 1 . 9 5 + 3.4 - 2 . 5 + 5.7 -2.9 + 7

0.03 0.027 0.02

0.002 NS NS

NS 0.01 0.04

5.5 _+ 6.9 0.1 _+ 0.1

NS NS

NS NS

NS NS

4.6 + 8.6 0.08 _+ 0.15 37

NS NS NS

NS NS NS

NS NS NS

NOTE. Genetic risk factors are the carriage of each individual 130-bp allele of the IL-6 gene and the noncarriage of each individual IL-lra gene. Patients were categorized into 3 groups according to the number of genetic risk factors present. NS, not significant. apositive values indicate gain of bone mass; negative values indicate loss of bone mass. bpercentage of patients receiving immunosuppressive medication with steroids and/or azathioprine.

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W e defined 1 study group and assessed 3 categories of parameters in this study group: (1) those reflecting the clinical severity of disease, (2) those reflecting the degree of bone loss, and (3) those reflecting a potential genetic predisposition for either an increased systemic inflammatory reaction or an increased local bone remodeling process in response to stress. W e obtained the following answers. There is change of bone mass in a relatively short time span of 1.6 years in our collective, which can be shown in 3 different bone compartments using osteodensitometry. While its degree is moderate in average, there is a large interindividual variability. There is substantial bone loss in a subset of patients. The observed bone loss cannot be predicted by single or even combinations of multiple biochemical parameters in our study group of IBD patients. Such biochemical parameters were suggested to be useful in menopausal women. They have repeatedly been used to predict future b o n e loss 29-31 and response to estrogen replacement therapy. 32 Patients with increased bone resorption are assumed to suffer from rapid bone loss and to profit most from early therapy. The extent of bone loss does not correlate with the extent of inflammation in IBD (Table 2). The clinical course of disease activity, analyzed retrospectively and prospectively, was taken as a surrogate for the extent of the inflammatory process. Neither the number of acute flare-ups of disease nor duration of disease nor the mode of immunosuppressive therapy showed correlation with the degree of bone loss. Our data consolidate findings of previous studies that no combination o f classic epidemiologic risk factors and/or biochemical markers of bone turnover is useful as a predictor of bone loss in I B D . 2,25,26 In our study group, dosage of corticosteroids was moderate. Heterogeneity of study populations with large variations, e.g., in steroid use from low average steroid dose of 3.2 + 6.8 mg/day 2 to high average daily dose of about 24 + 15 mg/day, 26 makes the comparison of data of different study populations difficult. Compared with other patient populations, our patient group in average had mild disease activity expressed by moderate steroid dosage and low prevalence of osteopenia. Only 10 of our ulcerative colitis patients had extended pancolitis. Further evaluation in patient groups with high disease activity and extended bowel involvement has to be performed to further illuminate the interrelation between inflammation and bone disease. However, as in these previous studies, 2,26,27,33 we did not find the expected dose-response curve of the bone toxic effect of steroids. In summary, our data do not support

GASTROENTEROLOGY Vol. 119, No. 4

the common idea of inflammation-mediated osteopenia in IBD patients. They do not support the hypothesis that the degree of inflammation is the critical determinant for the degree of bone loss. The degree of systemic inflammation does not correlate with genetic variations assessed by allelotyping of 4 genes, IL-lra, IL-6, hsp 70-2, and 70-hom, assumed to be involved in the inflammatory response. An imbalance of pro- and anti-inflammatory cytokines has been postulated as a pathogenic factor in IBD. Serum IL-6 levels were shown to be increased in IBD patients compared with controls. 34.35 There is no information in the literature that allelic variations in the examined D7S629 microsatellite neighboring the IL-6 gene correlate with the systemic IL-6 production. Our study did examine the question whether the pathogenesis of IBD is related to systemic functioning of IL-6. If we suppose such a hypothesis, our data do not confer evidence that analysis of D7S629 yields information on systemic IL-6 regulation. Like others, 3<~7 we found no significant differences in carriage rates of markers in the IL-l[3 and IL-lra genes between patients and healthy controls. IL-lra and IL-6 allele status was independent from the systemic course of IBD. We could not confirm the recently published data that polymorphisms of the hsp 70-2 are associated with clinical disease phenotype. 11 In summary, our data do not support a hypothesis implying a systemic immune modulation of IBD course related to the allele status of IL-6, IL-lra, hsp 70-2, and hsp 70-hom genes. BMD at baseline and at follow-up 1.6 years later does not correlate with any of the above genetic variations. This is because multiple factors determine this parameter. Sex, age, nutritional status, immobilization, and many more factors are evidentially associated with bone mass in healthy individuals and IBD patients. 38 Assessment of BMD at any point in the clinical course of a patient enables us to define a risk for future pathologic fractures but not for the extent of future bone loss. This is the limit of all cross-sectional data, which by now make up the majority of available data in IBD patients.21.39 -42 The extent of bone loss in IBD correlates with specific genetic variations assessed by allelotyping. The cytokines IL-lra and IL-643 and the molecular chaperones hsp 70-2 and hsp 70-hom 12.~3 are known to be involved in the regulation of bone remodeling. Bone loss prospectively followed over 1.6 years was significantly associated with the allele status of the IL- 1 ra and the IL-6 gene but not with allele status or haplotypes of the HSPs 70-2 and 70-hom. Noncarriage of the 240-bp allele of the IL-lra and carriage of the 130-bp allele of IL-6 were signifi-

October 2000

cantly associated with risk of bone loss. W e even identified a concerted action of such risk factors: the more risk factors, the more bone loss (Table 5). Recently, genetic variations of the IL-lra and IL-6 gene recently were shown to participate in the determination of bone loss in postmenopausal women. Carriage of the 134-bp allele of the IL-6 gene is associated with decreased bone mass. 44 Carriage of the A2 allele of the IL-lra gene is associated with reduced bone loss. 45 We feel that the characterization of genetic variation in identical genes as risk factors for increased bone loss in 2 entirely different clinical setting is of general interest. Perimenopause and IBD share increased bone loss as a common feature. The mechanisms accused of causing this bone loss are very different. In the situation of perimenopause, estrogen withdrawal is the main factor, whereas bone loss in IBD is looked at as multifactorial, implying inflammation, corticosteroid use, vitamin D deficiency, and malnutrition. Similar to bone loss in IBD, perimenopausal bone loss 46,47 is characterized by large interindividual variability, 48-5° making the identification of a patient at risk difficult. 51 Clinical reality teaches us that it is wrong to describe every perimenopausal woman and every IBD patient as a patient at risk for bone loss. This constellation of arguments supports a hypothesis that different systemic stress factors may afflict bone as a metabolic compartment. The effect of such stress factors, however, is largely determined by the way in which bone reacts to them. An intrinsic property of the bone, i.e., its genetic equipment, determines the bone's vulnerability, no matter whether the stressing influence is estrogen withdrawal or inflammation. Our results help to identify patients at risk for increased bone loss and consecutive increased risk of osteoporosis and osteoporotic fractures. They urge us to prospectively follow and to study treatment in such identified high-risk patients on a long-term basis. We do not confer information on the mechanism by which genetic variations of these genes are related to the process of bone loss. This topic merits intensive further study because identification of the effector pathways may open ways for novel specific treatment options.

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40. Silvennoinen J, Risteli L, Karttunen T, Risteli J. Increased degradation of type I collagen in patients with inflammatory bowel disease. Gut 1996;38:223-228. 41. Jahnsen J, Falch JA, Aadland E, Mowinckel P. Bone mineral density is reduced in patients with Crohn's disease but not in patients with ulcerative colitis: a population based study. Gut 1997;40:313-319. 42. Bjarnason I, Macpherson A, Mackintosh C, Buxton-Thomas M, Forgacs I, Moniz C. Reduced bone density in patients with bowel disease. Gut 1 9 9 7 ; 4 0 : 2 2 8 - 2 3 3 . 43. Rifas L. Bone and cytokines: beyond IL-1, 11-6 and TNF-alpha. Calcif Tissue Int 1999;64:1-7. 44. Tsukamoto K, Yoshida H, Watanabe S, Miyao M, Hosoi T, Orimo H, Emi M. Association of radial bone mineral density with CA repeat polymorphism at the interleukin 6 locus in postmenopausal Japanese women. J Hum Genet 1 9 9 9 ; 4 4 : 1 4 8 - 1 5 1 . 45. Keen RW, Woodford-Richens KL, Lachbury JS, Spector TD. Allelic variation at the Interleukin-1 receptor antagonist gene is associated with early postmenopausal bone loss at the spine. Bone 1998;23:367-371. 46. Hansen MA, Overgaard K, Christiansen C. Spontaneous postmenopausal bone loss in different skeletal areas: followed up for 15 years. J Bone Miner Res 1995;10:205-210. 47. Pouilles JM, Tremollieres F, Ribot C. The effects of menopause on longitudinal bone loss from the spine. Calcif Tissue Int 1993; 52:340-343. 48. Ribot C, Tremollieres F, Pouilles JM, Bonneu M, Germain F, Louvet JV. Obesity and postmenopausal bone loss. The influence of obesity on vertebral density and bone turnover in postmenopausal women. Bone 1988;8:327-331. 49. Tremollieres FA, Pouilles J-M, Ribot C. Vertebral postmenopausal bone loss is reduced in overweight women: a longitudinal study in 155 early postmenopausal women. J Clin Endocrinol Metab 1993;77:683-686. 50. Pouilles JM, Tremollieres F, Ribot C. Variability of vertebral and femoral postmenopausal bone loss: a longitudinal study. Osteoporos Int 1 9 9 6 ; 6 : 3 2 0 - 3 2 4 . 51. Rosen CJ, Chesnut CH III, Mallinek NJS. The predictive value of biochemical markers of bone turnover for bone mineral density in early postmenopausal women treated with hormone replacement or calcium supplementation. J Clin Endocrinol Metab 1997;82: 1904-1910.

Received November 15, 1999. Accepted May 10, 2000. Address requests for reprints to: Claudia M. S. Schulte, M.D., Division of Endocrinology, Department of Internal Medicine, University of Essen, Hufelandstrasse 55, 45122 Essen, Germany. e-mail: claudia. [email protected]; fax: (49) 201-268988.