Polymorphisms of collagen 1A1 (COL1A1) gene and their relation to bone mineral density in postmenopausal women

P R A C E O R Y G I N A L N E Polymorphisms of collagen 1A1 (COL1A1) gene and their relation to bone mineral density in postmenopausal women Polimorfizm genu kolagenu 1A1 (COL1A1) i jego związek z gęstością mineralną kości u kobiet po menopauzie 1 Department of Rheumatology and Rehabilitation, Poznan University of Medical Sciences, Poznan, Poland 2 Laboratory of Experimental Pharmacogenetics, Department of Clinical Pharmacy and Biopharmacy, University of Medical Sciences, Poznan, Poland 3 Division of Perinatology and Women s Diseases, Poznan University of Medical Sciences, Poznan, Poland 4 Division of Gynecology and Obstetrics, Podhale Multidisciplinary Hospital, Nowy Targ, Poland 5 Department of Stem Cells and Regenerative Medicine, Institute of Natural Fibres and Medicinal Plants, Poznan, Poland 6 Laboratory of Molecular Biology in Division of Perinatology and Women s Diseases, Poznan University of Medical Sciences, Poznan, Poland 7 Department of General Pharmacology and Pharmacoeconomics, Pomeranian Medical University, Szczecin, Poland 8 Clinical Division of Anaesthesiology and Intensive Care of Adults and Children, Pomeranian Medical University, Szczecin, Poland 9 Department of Pharmacology and Phytochemistry, Institute of Natural Fibres and Medicinal Plants, Poznan, Poland Abstract Objectives: The goal of this study was to evaluate the frequency of Sp1 +1245G>T (rs 1800012) and -1997G>T (rs 1107946) COL1A1 gene polymorphisms in postmenopausal women with osteoporosis and osteopenia as well as assessing their relations with the clinical parameters and parameters of bone turnover. Study design: The study included 538 (236 postmenopausal and 302 healthy reproductive) Polish women. The postmenopausal group included women with osteoporosis (n=90), osteopenia (n=90), as well as healthy individuals (n=56). All women of reproductive age were healthy. BMD was marked in the L2-L4 lumbar region of the spine using dual energy X-ray absorptiometry (DXA). Genomic DNA was isolated from peripheral blood, the genotype frequency of investigated polymorphisms was determined by PCR-RFLP technique. Results: The frequency of Sp1 +1245G>T and -1997G>T polymorphisms of COL1A1 gene showed no statistically significant differences between group with osteoporosis, osteopenia and correct T-score and women of reproductive age. In postmenopausal women it was found that osteopenia and osteoporosis were correlated with age, birth weight, age of last menses occurrence, height, body weight and BMI value. Clinical parameters in all groups of women did not show any statistically significant correlation with frequency of Sp1 +1245G>T and -1997G>T COL1A1 polymorphisms. Conclusions: An evaluation of Sp1 +1245G>T (rs1800012) and -1997G>T (rs 1107946) COL1A1 polymorphisms showed any influence of these genetic variants on osteoporosis development in Polish postmenopausal women. The presented correlation between osteoporosis and age, birth weight, age of last menses occurrence, height, body weight and BMI value confirms the important role of environmental factors in disease etiology. Key words: polymorphism / collagen 1A1 / bone mineral density / Corresponding author: Agnieszka Seremak-Mrozikiewicz Division of Perinatology and Woman s Diseases, Poznań Poland, 60-535 Poznań, Polna 33 tel. + 48 61 84196 13, fax: +48 61 8474651 e-mail: asm@data.pl Otrzymano: 03.03.2015 Zaakceptowano do druku: 01.04.2015 Nr 12/2015 Polskie Towarzystwo Ginekologiczne 907

P R A C E O R Y G I N A L N E Streszczenie Cel pracy: Celem pracy była ocena częstości występowania genotypów polimorfizmów Sp1 +1245G>T (rs 1800012) i -1997G>T (rs 1107946) genu COL1A1 u kobiet po menopauzie z osteoporozą i osteopenią oraz ocena związku pomiędzy analizowanymi polimorfizmami a parametrami klinicznymi i parametrami obrotu kostnego. Materiały i metody: Do badania włączono 538 (236 po menopauzie i 302 zdrowych kobiet w wieku rozrodczym) kobiet z populacji polskiej. Grupa kobiet po menopauzie podzielona została na kobiety z osteoporozą (n=90), osteopenią (n=90) oraz kobiety o prawidłowych wartościach T-score (n=56). BMD oznaczano w odcinku lędźwiowym kręgosłupa (L2-L4) za pomocą absorpcjometrii podwójnej energii promieniowania rentgenowskiego (DXA). Genomowy DNA izolowano z krwi obwodowej, częstość genotypów badanych polimorfizmów oznaczano z zastosowaniem techniki PCR-RFLP. Wyniki: Częstość występowania genotypów polimorfizmów SP1 +1245G>T i -1997G>T genu COL1A1 w grupie kobiet po menopauzie z osteoporozą, osteopenią i prawidłowym T-score oraz kobiet wieku rozrodczym nie wykazała istotnych statystycznie różnic i była zgodna z prawem Hardy-Weinberga. U kobiet po menopauzie stwierdzono, że występowanie osteopenii i osteoporozy było skorelowane z wiekiem, masą urodzeniową, masą ciała kobiet, wiekiem wystąpienia ostatniej miesiączki i wartością BMI. Parametry kliniczne we wszystkich grupach kobiet nie wykazały istotnego związku z występowaniem obydwu polimorfizmów SP1 +1245G>T i -1997G>T genu COL1A1. Wnioski: Analiza polimorfizmów Sp1 +1245G>T (rs1800012) oraz -1997G>T (rs 1107946) COL1A1 nie pokazała żadnego wpływu tych wariantów genetycznych na rozwój osteoporozy w grupie polskich kobiet po menopauzie. Prezentowana korelacja pomiędzy rozwojem osteoporozy a wiekiem, masą urodzeniową, wiekiem wystąpienia ostatniej miesiączki, wysokością, masą ciała i wskaźnikiem BMI potwierdza ważną rolę czynników środowiskowych w etiologii osteoporozy także w grupie polskich kobiet. Słowa kluczowe: / kobiety / menopauza / osteoporoza / osteopenia / Introduction Material and methods 908 Polskie Towarzystwo Ginekologiczne Nr 12/2015

P R A C E O R Y G I N A L N E MscEcoR31 p Results Table I. Demographic and clinical data of the studied population. Age - women at the postmenopausal age; mean ± SD Age - women at the reproductive age; mean ± SD Weight - women at the postmenopausal age; mean ± SD 58.5±5.9 31.0±4.4 67.0±11.8 Weight - women at the reproductive age; mean ± SD 61.9±11.0 Area of living, 538 (100.0) 55.0 Rural () 45.0 538 (100.0) Primary educatio 1.2 Vocational educatio 5.0 15.0 Licentiate () 9.5 Higher educatio 69.3 Number of children, mean ± SD 1.6±1.1 Nutritional diet knowledge score, 538 (100.0) Vegetaria 3.1 Meat diet () 0.6 Standard () 84.5 11.8 538 (100.0) Yes () 48.4 No () 51.6 Nr 12/2015 Polskie Towarzystwo Ginekologiczne 909

P R A C E O R Y G I N A L N E Table II. Primer sequences for PCR and the length of PCR products. +1245G>T Sp1-1997G>T 262 pz 257 pz Table III. The sequences and restriction sites of Msc I and EcoR31I enzymes. MscI Sp1 EcoR31I Enzyme hydrolizes, when T is present, and doesn t when G GG (262pz) GT (262pz, 242pz, 20pz) TT (242pz, 20pz) Enzyme hydrolizes, when G is present, and doesn t when T TT (257pz) GT (257pz, 176pz, 81pz) GG (176pz, 81pz) Discussion 910 Polskie Towarzystwo Ginekologiczne Nr 12/2015

P R A C E O R Y G I N A L N E Table IV. The frequency of genotypes and alleles of SP1 +1245G>T COL1A1 polymorphism in women before menopause and after menopause. COL1A1 +1245G/T Osteopenia Osteoporosis reproductive age SS 59 (65.6) 64.0 66 (73.3) 74.2 39 (69.7) 66.0 164 (69.5) 68.2 215 (71.2) 70.7 Ss 26 (28.9) 32.0 23(25.6) 23.9 13 (23.2) 30.5 62 (26.3) 28.8 78 (25.8) 26.8 ss 5 (5.5) 4.0 1 (1.1) 1.9 4 (7.1) 3.5 10 (4.2) 3.0 9 (3.0) 2.5 90 (100.0) 100.0 90 (100.0) 100.0 56 (100.0) 100.0 236 (100.0) 100.0 302 (100.0) 100.0 Alleles S 144 (80.0) - 155 (86.1) - 91 (81.3) - 390 (82.6) - 508 (84.1) - s 36 (20.0) - 25 (13.9) - 21 (18.7) - 82 (17.4) - 96 (15.9) - 180 (100.0) - 180 (100.0) - 112 (100.0) - 472 (100.0) - 604 (100.0) - Table V. The frequency of genotypes and alleles of -1997G>T polymorphism in women before menopause and after menopause. COL1A1 Osteopenia Osteoporosis 47 (52.2) 55.4 53 (58.9) 62.2 36 (64.3) 63.2 136 (57.6) 59.8 202 (66.9) 67.2 40 (44.4) 38.1 36 (40.0) 33.3 17 (30.4) 32.6 93 (39.4) 35.1 91 (30.1) 29.6 3 (3.4) 6.5 1 (1.1) 4.5 3(5.3) 4.2 7 (3.0) 5.1 9 (3.0) 3.2 90 (100.0) 100.0 90 (100.0) 100.0 56 (100.0) 100.0 538 (100.0) 100.0 302 (100.0) 100.0 Alleles 134 (74.4) - 142 (78.9) - 89 (79.5) - 365 (77.3) - 495 (81.9) - 46 (25.6) - 38 (21.1) - 23 (20.5) - 107 (22.7) - 109 (18.1) - 180 (100.0) - 180 (100.0) - 12 (100.0) - 472 (100.0) - 604 (100.0) - Nr 12/2015 Polskie Towarzystwo Ginekologiczne 911

P R A C E O R Y G I N A L N E Tabela VI. Investigated parameter in postmenopausal women with osteopenia, osteoporosis and correct T-score. n Mean ±SD Min Max p Z-score Age [years] [g] occurence [years] Age of last occurence [years] Reproduction years Years after pregnancies Height osteopenia 90-1,8354,4139-2,47-1,14 osteoporosis 90-3,1662,5524-4,73-2,50 correct T-score 56,1395,9667 -,97 3,13 Total 236-1,8743 1,4161-4,73 3,13 osteopenia 90 -,8039,5916-1,97,77 osteoporosis 90-3,0324 13,3367-128,00,98 correct T-score 56,6445 1,0306-1,85 2,65 Total 236-1,3101 8,3599-128,00 2,65 osteopenia 90 54,4 7,8 31,0 77,0 osteoporosis 90 57,6 7,9 37,0 78,0 correct T-score 56 53,9 8,5 28,0 71,0 Total 236 55,5 8,2 28,0 78,0 osteopenia 26 3216,2 400,6 2500,0 4500,0 osteoporosis 16 3141,3 536,3 2470,0 4500,0 correct T-score 18 3633,3 494,3 2460,0 5100,0 Total 60 3321,3 504,9 2460,0 5100,0 osteopenia 90 13,2 2,2 9,0 18,0 osteoporosis 90 13,2 2,1 9,0 18,0 correct T-score 56 13,3 2,2 3,0 16,0 Total 236 13,2 2,2 3,0 18,0 osteopenia 90 49,7 4,3 38,0 60,0 osteoporosis 90 48,2 4,8 34,0 58,0 correct T-score 56 50,6 3,7 41,0 58,0 Total 236 49,3 4,5 34,0 60,0 osteopenia 90 36,4 4,8 23,0 49,0 osteoporosis 90 35,6 4,8 24,0 47,0 correct T-score 56 37,1 4,6 27,0 48,0 Total 236 36,3 4,8 23,0 49,0 osteopenia 90 8,6 5,8,0 25,0 osteoporosis 90 10,3 5,2 1,0 22,0 correct T-score 56 8,9 5,3 1,0 23,0 Total 236 9,3 5,5,0 25,0 osteopenia 90 1,9 1,1,0 6,0 osteoporosis 90 1,9 1,3,0 7,0 correct T-score 56 1,9 1,3,0 6,0 Total 236 1,9 1,2,0 7,0 osteopenia 90 65,2 9,6 41,0 90,0 osteoporosis 90 60,9 9,1 43,0 85,0 correct T-score 56 68,9 12,4 50,0 100,0 Total 236 64,4 10,6 41,0 100,0 osteopenia 90 162,8 4,8 153,0 175,0 osteoporosis 90 159,9 5,2 150,0 175,0 correct T-score 56 163,1 5,8 152,0 176,0 Total 236 161,8 5,4 150,0 176,0 0,026 0,009 0,005 0,942 0,004 0,177 0,094 0,889 912 Polskie Towarzystwo Ginekologiczne Nr 12/2015

P R A C E O R Y G I N A L N E Tabela VI (cd). Investigated parameter in postmenopausal women with osteopenia, osteoporosis and correct T-score. n Mean ±SD Min Max p BMD L2-L4 [g/ BMD L2-L4 YA BMD L2-L4 [AM/] osteopenia 90 24,6 3,3 17,3 34,3 osteoporosis 90 23,6 3,1 17,1 31,6 correct T-score 56 25,9 4,7 18,3 37,2 Total 236 24,6 3,7 17,1 37,2 osteopenia 90,9733,0499,90 1,07 osteoporosis 90,8178,0715,63,90 correct T-score 56 1,2204,1046 1,08 1,47 Total 236,9726,1710,63 1,47 osteopenia 90 81,1444 4,1935 75,0 89,0 osteoporosis 90 68,2556 5,7914 53,0 75,0 correct T-score 56 102,1250 9,0193 90,0 123,0 Total 236 81,2076 14,3845 53,0 123,0 osteopenia 90 89,5444 6,6690 76,0 108,0 osteoporosis 90 78,6222 7,7613 60,0 92,0 correct T-score 56 110,5000 10,7788 92,0 133,0 Total 236 90,3517 14,7149 60,0 133,0 0,001 in vivo p Conclusions Oświadczenie autorów: 1. Marian Majchrzycki autor koncepcji i założeń pracy, analiza statystyczna wyników, przygotowanie manuskryptu i piśmiennictwa autor zgłaszający i odpowiedzialny za manuskrypt. 2. Joanna Bartkowiak-Wieczorek zebranie materiału, analiza statystyczna wyników, przygotowanie manuskryptu, korekta i aktualizacja literatury. 3. Hubert Wolski współautor tekstu pracy i protokołu, analiza statystyczna wyników, współautor protokołu, korekta i aktualizacja literatury. 4. Krzysztof Drews korekta i akceptacja ostatecznego kształtu manuskryptu. 5. Anna Bogacz zebranie materiału, analiza statystyczna wyników, przygotowanie manuskryptu. 6. Bogusław Czerny opracowanie wyników badań, współautor protokołu. 7. Edyta Zagrodnik-Ułan opracowanie protokołu badań, współautor tekstu pracy, aktualizacja literatury 8. Agnieszka Seremak-Mrozikiewicz autor koncepcji i założeń pracy, ostateczna weryfikacja i akceptacja manuskryptu. Źródło finansowania: Badania statutowe Kliniki Perinatologii i Chorób Kobiecych UM w Poznaniu. Konflikt interesów: Autorzy nie zgłaszają konfliktu interesów oraz nie otrzymali żadnego wynagrodzenia związanego z powstawaniem pracy. References 1. Mohammadi Z, Fayyazbakhsh F, Ebrahimi M, [et al.]. Association between vitamin D receptor gene polymorphisms (Fok1 and Bsm1) and osteoporosis: a systematic review. J Diabetes Metab Disord. 2014, 17, 13 (1), 98. doi: 10.1186/s40200-014-0098-x. 2. Shang M, Lin L, Cui H. Association of genetic polymorphisms of RANK, RANKL and OPG with bone mineral density in Chinese peri- and postmenopausal women. Clin Biochem. 2013, 46, 1493-1501. 3. Kaleta B, Walicka M, Sawicka A, [et al.]. Toll-Like receptor 4 gene polymorphism C1196T in Polish women with postmenopausal osteoporosis - preliminary investigation. Adv Clin Exp Med. 2015, 24, 239-243. 4. Boron D, Czerny B, Bartkowiak-Wieczorek J, [et al.]. Omentin polymorphism and its relations to bone mineral density in women. Arch Med Res. 2015, 46, 173-180. Nr 12/2015 Polskie Towarzystwo Ginekologiczne 913

P R A C E O R Y G I N A L N E 5. Wolski H, Drwęska-Matelska N, Seremak-Mrozikiewicz A, [et al.]. The role of Wnt/ -catenin pathway and LRP5 protein in metabolism of bone tissue and osteoporosis etiology. Ginekol Pol. 2015, 86, 311-314. 6. Jin H, Evangelou E, Ioannidis JP, [et al.]. Polymorphisms in the 5 flank of COL1A1 gene and osteoporosis: meta-analysis of published studies. Osteoporosis Int. 2011, 22, 911-921. 7. Raisz LG. Pathogenesis of osteoporosis: concepts, conflicts, and prospects. J Clin Invest. 2005, 115, 3318-3325. 8. Kleerekoper M. Osteoporosis prevention and therapy: preserving and building strength through bone quality. Osteoporos Int. 2006, 17, 1707-1715. 9. Yarbrough DE, Barrett-Connor E, Morton DJ. Birth weight as a predictor of adult bone mass in postmenopausal women: the Rancho Bernardo Study. Osteoporosis Int. 2000, 11, 626-630. 10. Antoniades L, MacGregor AJ, Andrew T, [et al.]. Association of birth weight with osteoporosis and osteoarthritis in adult twins. Rheumatology. 2003, 42, 791-796. 11. Martínez-Mesa J, Restrepo-Méndez MC, González DA, [et al.]. Life-course evidence of birth weight effects on bone mass: systematic review and meta-analysis. Osteoporosis Int. 2013, 24, 7-18. 12. Blain H, Vuillemin A, Blain A, [et al.]. Age-related femoral bone loss in men: evidence for hyperparathyroidism and insulin-like growth factor-1 deficiency. J Gerontol A Biol Sci Med Sci. 2004, 59, 1285-1289. 13. Trovas G, Lyritis GP, Galanos A, [et al.]. Influence of weight gain on spine mineral density in postmenopausal women. Calcif Tissue Int. 1999, 64, 380-383. 14. Fawzy T, Muttappallymyalil J, Sreedharan J, [et al.]. Association between Body Mass Index and Bone Mineral Density in Patients Referred for Dual-Energy X-Ray Absorptiometry Scan in Ajman, UAE. J Osteoporos. 2011, 2011, 876309. doi: 10.4061/2011/876309. 15. Kurt-Sirin O, Yilmaz-Aydogan H, Uyar M, [et al.]. Combined effects of collagen type I alpha1 (COL1A1) Sp1 polymorphism and osteoporosis risk factors on bone mineral density in Turkish postmenopausal women. Gene. 2014, 540, 226-231. 16. Braga V, Mottes M, Mirandola S, [et al.]. Association of CTR and COLIA1 alleles with BMD values in peri- and postmenopausal women. Calcif Tissue Int. 2000, 67, 361-366. 17. Efstathiadou Z, Kranas V, Ioannidis JP, [et al.]. The Sp1 COL1A1 gene polymorphism, and not vitamin D receptor or estrogen receptor gene polymorphisms, determines bone mineral density in postmenopausal Greek women. Osteoporos Int. 2001, 12, 326-331. 18. Hampson G, Evans C, Petitt RJ, [et al.]. Bone mineral density, collagen type 1 alpha 1 genotypes and bone turnover in premenopausal women with diabetes mellitus. Diabetologia. 1998, 41, 1314-1320. 19. Garnero P, Borel O, Grant SF, [et al.]. Collagen I alpha1 Sp1 polymorphism, bone mass, and bone turnover in healthy French premenopausal women: the OFELY study. J Bone Miner Res. 1998, 13, 813-817. 20. Uitterlinden AG, Burger H, Huang Q, [et al.]. Relation of alleles of the collagen type I alpha1 gene to bone density and the risk of osteoporotic fractures in postmenopausal women. N Engl J Med. 1998, 338, 1016-1021. 21. Heegaard A, Jorgensen HL, Vestergaard AW, [et al.]. Lack of influence of collagen type Ialpha1 Sp1 binding site polymorphism on the rate of bone loss in a cohort of postmenopausal Danish women followed for 18 years. Calcif Tissue Int. 2000, 66, 409-413. 22. Harris SS, Patel MS, Cole DE, [et al.]. Associations of the collagen type Ialpha1 Sp1 polymorphism with five-year rates of bone loss in older adults. Calcif Tissue Int. 2000, 66, 268-271. 23. Falcón-Ramírez E, Casas-Avila L, Miranda A, [et al.]. Sp1 polymorphism in collagen I 1 gene is associated with osteoporosis in lumbar spine of Mexican women. Mol Biol Rep. 2011, 38, 2987-2992. 24. Mann V, Ralston SH. Meta-analysis of COL1A1 Sp1 polymorphism in relation to bone mineral density and osteoporotic fracture. Bone. 2003, 32, 711-717. 25. Liu PY, Lu Y, Long JR, [et al.]. Common variants at the PCOL2 and Sp1 binding sites of the COL1A1 gene and their interactive effect influence bone mineral density in Caucasians. J Med Genet. 2004, 41, 752-757. 26. Singh M, Singh P, Singh S, [et al.]. A haplotype derived from the common variants at the -1997G/T and Sp1 binding site of the COL1A1 gene influences risk of postmenopausal osteoporosis in India. Rheumatol Int. 2013, 33, 501-506. 27. Husted LB, Harslof T, Gonzalez-Bofill N, [et al.]. Haplotypes of promoter and intron 1 polymorphisms in the COLIA1 gene are associated with increased risk of osteoporosis. Calcif Tissue Int. 2009, 84, 85-96. 28. Zhang YY, Lei SF, Mo XY, [et al.]. The -1997 G/T polymorphism in the COLIA1 upstream regulatory region is associated with hip bone mineral density (BMD) in Chinese nuclear families. Calcif Tissue Int. 2005, 76, 107-112. 29. Bustamante M, Nogues X, Enjuanes A, [et al.]. COL1A1, ESR1, VDR and TGFB1 polymorphisms and haplotypes in relation to BMD in Spanish postmenopausal women. Osteoporos Int. 2007, 18, 235-243. 30. Yamada Y, Ando F, Niino N, [et al.]. Association of a -1997G>T polymorphism of the collagen I alpha1 gene with bone mineral density in postmenopausal Japanese women. Hum Biol. 2005, 77, 27-36. 31. Yazdanpanah N, Rivadeneira F, van Meurs JB, [et al.]. The -1997 G/T and Sp1 polymorphisms in the collagen type I alpha1 (COLIA1) gene in relation to changes in femoral neck bone mineral density and the risk of fracture in the elderly: the Rotterdam study. Calcif Tissue Int. 2007, 81, 18-25. 32. Selezneva LI, Khusainova RI, Nurlygaianov RZ, [et al.]. Association of polymorphisms and haplotypes in the 5 region of COLIA1 gene with the risk of osteoporotic fractures in Russian women from Volga-Ural region. Genetika. 2008, 44, 219-225. 914 Polskie Towarzystwo Ginekologiczne Nr 12/2015