Human Immunology 75 (2014) 452–461
Contents lists available at ScienceDirect
www.ashi-hla.org
journal homepage: www.elsevier.com/locate/humimm
Vitamin D receptor gene polymorphism as possible risk factor in rheumatoid arthritis and rheumatoid related osteoporosis Youssef M. Mosaad a,⇑, Enas M. Hammad b, Zakaria Fawzy a, Ibrahim A. Abdal Aal c, Hazem M. Youssef b,d, Tamer O. ElSaid b, Rehan Monir e, Basem S. EL-Deek f,g a
Clinical Immunology Unit, Clinical Pathology Department, Mansoura Faculty of Medicine, Mansoura, Egypt Rheumatology and Rehabilitation Department, Mansoura University Hospital, Mansoura, Egypt Clinical Chemistry Unit, Clinical Pathology Department, Mansoura Faculty of Medicine, Mansoura, Egypt d Department of Rheumatology, Aberdeen Royal Infirmary, Aberdeen, UK e Medical Biochemistry Department, Mansoura Faculty of Medicine, Mansoura, Egypt f Community Medicine and Statistics Department, Mansoura Faculty of Medicine, Mansoura, Egypt g Research Unit, Faculty of Medicine, King Abdul-Aziz University, Jeddah, Saudi Arabia b c
a r t i c l e
i n f o
Article history: Received 12 September 2013 Accepted 4 February 2014 Available online 12 February 2014
a b s t r a c t Objective: To study the role of VDR polymorphisms as risk factor for RA and osteoporosis, and whether osteoporosis complicating RA is due to RA or VDR polymorphisms. Methods: VDR gene polymorphisms ApaI, TaqI, BsmI and FokI were typed by RFLP for 128 RA patients, 30 postmenopausal osteoporotic females and 150 healthy controls. Results: Significant differences were found between patients and healthy controls in the frequency of BsmI and TaqI (Pc < 0.05) but no significant associations were found for FokI and ApaI polymorphisms except for aa genotype (Pc < 0.001). Titers of RF were higher with aa and bb genotypes. Anti-CCP and CRP levels were higher with aa genotype and more bone loss was associated with Bb genotype. Ff genotype frequency was higher in RA patients with osteoporosis than those without osteoporosis. Conclusions: The ApaI, BsmI and TaqI polymorphisms may be a susceptibility risk factors for RA and the Ff genotype may be responsible for development of osteoporosis in RA Egyptian patients. However, the present study needs to be replicated in a large number of patients from allover the Egypt and also in multi-ethnic populations. Ó 2014 American Society for Histocompatibility and Immunogenetics. Published by Elsevier Inc. All rights reserved.
1. Introduction Rheumatoid arthritis (RA) is one of the most common autoimmune diseases characterized by chronic inflammation of systemic
Abbreviations: RA, rheumatoid arthritis; VDR, vitamin D receptor; DAS-28, disease activity score 28; VAS, visual analogue scales; RARBIS, rheumatoid arthritis medical records based index of severity; DEXA, dual energy X-ray absorptiometry; BMD, bone mineral density; RFLP, restriction fragment length polymorphism; AntiCCP, Anti-Cyclic Citrullinated Peptide; CRP, C-reactive protein; OP, osteoporosis; DMARDs, Disease Modifying Anti-Rheumatic Drugs; LD, linkage disequilibrium. ⇑ Corresponding author. Address: Clinical Immunology Unit, Clinical Pathology Department, Mansoura Faculty of Medicine, Mansoura University, Mansoura 35111, Egypt. Mobile: +20 106243435; fax: +20 502267563. E-mail addresses: [email protected], [email protected] (Y.M. Mosaad), [email protected] (E.M. Hammad), zakaria.fawzi45@ yahoo.com (Z. Fawzy), [email protected] (I.A. Abdal Aal), [email protected] (H.M. Youssef), [email protected] (T.O. ElSaid), Rehansoliman@yahoo. com (R. Monir), [email protected] (B.S. EL-Deek).
joints. However, considerable variation exists among ethnicities, with a higher prevalence in populations of European ancestry than those of Asian ancestry [1,2]. RA is a multifactorial disease due to a combination of genetic and environmental factors. Identification of the genetic factors involved in the pathogenesis of RA should open up avenues for developing radical treatment strategies directed at the cause of the disease [3]. The role of vitamin D and its receptor (VDR) in skeletal metabolism is well known but the vitamin D endocrine system seems to play an important role in other metabolic pathways as well, such as those involved in osteoarthritis, the immune response and cancer. One approach to understand the vitamin D endocrine system is to study the influence of variations in the DNA sequence of important proteins of this system. For example, deleterious mutations in the VDR gene cause 1, 25-dihydroxyvitamin D-resistant rickets, a rare monogenetic disease. More subtle sequence variations (polymorphisms) in the VDR gene occur much more frequently but their
http://dx.doi.org/10.1016/j.humimm.2014.02.009 0198-8859/Ó 2014 American Society for Histocompatibility and Immunogenetics. Published by Elsevier Inc. All rights reserved.
Y.M. Mosaad et al. / Human Immunology 75 (2014) 452–461
effects are poorly understood. Their influence on the vitamin D endocrine system is currently under scrutiny in relation to a number of so-called complex diseases and traits such as osteoporosis [4]. According to Davis et al. over 470 VDR single nucleotide polymorphisms (SNPs) are known [5]. VDR gene is located on chromosome 12 and contains 9 exons [6]. Their distribution and frequency vary among ethnic groups. Most of the work done on VDR polymorphisms has been conducted in Caucasian populations and has focused on six SNPs: (1) rs10735810 or FokI in exon 2, (2) rs1544410 or BsmI in intron 8, (3) rs731236 or TaqI in exon9, (4) rs7975232 or ApaI in intron 8, (5) rs757343 or Tru91 in intron 8, and (6) the poly (A) mononucleotide repeat in the 30 -untranslated region (UTR) [7]. The discovery of the VDR in the cells of the immune system and the fact that activated dendritic cells produce the vitamin D hormone suggested that vitamin D could have immunoregulatory properties. The most evident effects of the D-hormone on the immune system seem to be in the downregulation of the Th1-driven autoimmunity. Low serum levels of vitamin D3 might be partially related, among other factors, to prolonged daily darkness (reduced activation of the pre vitamin D by the ultra violet B sunlight), different genetic background (i.e. VDR polymorphism) and nutritional factors and explain to the latitude-related prevalence of autoimmune diseases such as rheumatoid arthritis by considering the potential immunosuppressive roles of vitamin D. The 25(OH)D3 plasma levels have been found inversely correlated at least with the RA disease activity showing a circannual rhythm (more severe in winter). Recently, greater intake of vitamin D was associated with a lower risk of RA as well as a significant clinical improvement was strongly correlated with the immunomodulating potential in vitamin D-treated RA patients [8]. RA is a chronic inflammatory joint disease that leads to local and systemic bone loss. Osteoporosis (OP) or the systemic bone loss associated with RA increases the risk for fragility fractures, which can affect quality of life dramatically in RA patients. RA associated secondary osteopenia/osteoporosis may be observed in 60– 80% of the patients. Traditional and RA-related risk factors have been defined and studied for osteoporosis associated with RA such as race, body mass index (BMI), dietary calcium, vitamin D intake, disease duration and activity, level of disability, corticosteroids and Disease Modifying Anti-Rheumatic Drugs (DMARDs) use and menopausal status [9–15]. The steroid hormone vitamin D, its receptor (VDR) and the metabolizing enzymes involved in the formation of the biologically active form of the hormone together are major players in the vitamin D endocrine system. This system plays a crucial role in calcium and phosphate homeostasis and skeletal metabolism. Following renal production as the hormonal metabolite of vitamin D, 1a,25dihydroxyvitamin D3 (1,25(OH)2D3) functions as the ligand for VDR, with the hormone-receptor complex inducing calcemic and phosphatemic effects that result in normal bone mineralization and remodeling [16]. Therefore, VDR has been considered as an important candidate gene of osteoporosis [17]. However, the role of genetic factors, especially allelic variants in candidate genes for osteoporosis such as VDR, is less well defined in RA-associated osteoporosis and therefore the role of vitamin D and its receptor in bone loss in RA is intriguing [9]. Interestingly, polymorphisms in the VDR have been positively correlated with increased susceptibility to RA by some studies [18–21] and have been negatively correlated by others [22–24]. On the other hand, the association of VDR gene polymorphism with OP has been studied by several investigators especially BsmI with some contradiction in the various reports primarily due to extensive geographic variations. While, the presence of the B allele has been associated with increased bone loss by some groups
453
[25–29], the b allele was reported to be correlated with OP by others [30,31] and some investigators have not found any association between the VDR gene BsmI genotype and bone mineral density [32–35]. The aim of the present work is to study the relationship between VDR gene polymorphism and susceptibility to rheumatoid arthritis and the impact of such association on disease profile and bone density among Egyptians.
2. Subjects and methods 2.1. Patients and controls This case control study on a cohort of Egyptian population includes patients with RA and two control groups of healthy individuals and postmenopausal osteoporosis females. A total of 128 patients with RA (103 females and 25 males) with mean age of 46.91 ± 11.73 diagnosed according to the American College of Rheumatology/European League against Rheumatism collaborative initiative for RA [36] were consecutively recruited into the study between February 2012 and January 2013 with disease duration of 10.33 ± 7.01 years. Patients were recruited from the outpatient’s clinic of Rheumatology and Rehabilitation Department, Mansoura University Hospitals, Egypt. All patients underwent clinical evaluation by a rheumatologist and data about demographics and disease parameters were collected. Disease activity was evaluated using disease activity score 28 (DAS28-CRP) that measures 28 tender and swollen joints, degree of pain by visual analogue scales (VAS), patient’s and doctor’s global assessment of disease activity using VAS and inflammatory markers such as C-reactive protein (CRP) [37]. DAS-28 as described by Prevoo et al. [38] defines the level of RA activity as follows; P5.1 indicate high disease activity; between 5.1 and 3.2 indicates moderate disease activity; between 3.2 and 2.6 indicates low disease activity; and 100 IU/ml = strongly elevated. AntiCCP antibodies
Contents lists available at ScienceDirect
www.ashi-hla.org
journal homepage: www.elsevier.com/locate/humimm
Vitamin D receptor gene polymorphism as possible risk factor in rheumatoid arthritis and rheumatoid related osteoporosis Youssef M. Mosaad a,⇑, Enas M. Hammad b, Zakaria Fawzy a, Ibrahim A. Abdal Aal c, Hazem M. Youssef b,d, Tamer O. ElSaid b, Rehan Monir e, Basem S. EL-Deek f,g a
Clinical Immunology Unit, Clinical Pathology Department, Mansoura Faculty of Medicine, Mansoura, Egypt Rheumatology and Rehabilitation Department, Mansoura University Hospital, Mansoura, Egypt Clinical Chemistry Unit, Clinical Pathology Department, Mansoura Faculty of Medicine, Mansoura, Egypt d Department of Rheumatology, Aberdeen Royal Infirmary, Aberdeen, UK e Medical Biochemistry Department, Mansoura Faculty of Medicine, Mansoura, Egypt f Community Medicine and Statistics Department, Mansoura Faculty of Medicine, Mansoura, Egypt g Research Unit, Faculty of Medicine, King Abdul-Aziz University, Jeddah, Saudi Arabia b c
a r t i c l e
i n f o
Article history: Received 12 September 2013 Accepted 4 February 2014 Available online 12 February 2014
a b s t r a c t Objective: To study the role of VDR polymorphisms as risk factor for RA and osteoporosis, and whether osteoporosis complicating RA is due to RA or VDR polymorphisms. Methods: VDR gene polymorphisms ApaI, TaqI, BsmI and FokI were typed by RFLP for 128 RA patients, 30 postmenopausal osteoporotic females and 150 healthy controls. Results: Significant differences were found between patients and healthy controls in the frequency of BsmI and TaqI (Pc < 0.05) but no significant associations were found for FokI and ApaI polymorphisms except for aa genotype (Pc < 0.001). Titers of RF were higher with aa and bb genotypes. Anti-CCP and CRP levels were higher with aa genotype and more bone loss was associated with Bb genotype. Ff genotype frequency was higher in RA patients with osteoporosis than those without osteoporosis. Conclusions: The ApaI, BsmI and TaqI polymorphisms may be a susceptibility risk factors for RA and the Ff genotype may be responsible for development of osteoporosis in RA Egyptian patients. However, the present study needs to be replicated in a large number of patients from allover the Egypt and also in multi-ethnic populations. Ó 2014 American Society for Histocompatibility and Immunogenetics. Published by Elsevier Inc. All rights reserved.
1. Introduction Rheumatoid arthritis (RA) is one of the most common autoimmune diseases characterized by chronic inflammation of systemic
Abbreviations: RA, rheumatoid arthritis; VDR, vitamin D receptor; DAS-28, disease activity score 28; VAS, visual analogue scales; RARBIS, rheumatoid arthritis medical records based index of severity; DEXA, dual energy X-ray absorptiometry; BMD, bone mineral density; RFLP, restriction fragment length polymorphism; AntiCCP, Anti-Cyclic Citrullinated Peptide; CRP, C-reactive protein; OP, osteoporosis; DMARDs, Disease Modifying Anti-Rheumatic Drugs; LD, linkage disequilibrium. ⇑ Corresponding author. Address: Clinical Immunology Unit, Clinical Pathology Department, Mansoura Faculty of Medicine, Mansoura University, Mansoura 35111, Egypt. Mobile: +20 106243435; fax: +20 502267563. E-mail addresses: [email protected], [email protected] (Y.M. Mosaad), [email protected] (E.M. Hammad), zakaria.fawzi45@ yahoo.com (Z. Fawzy), [email protected] (I.A. Abdal Aal), [email protected] (H.M. Youssef), [email protected] (T.O. ElSaid), Rehansoliman@yahoo. com (R. Monir), [email protected] (B.S. EL-Deek).
joints. However, considerable variation exists among ethnicities, with a higher prevalence in populations of European ancestry than those of Asian ancestry [1,2]. RA is a multifactorial disease due to a combination of genetic and environmental factors. Identification of the genetic factors involved in the pathogenesis of RA should open up avenues for developing radical treatment strategies directed at the cause of the disease [3]. The role of vitamin D and its receptor (VDR) in skeletal metabolism is well known but the vitamin D endocrine system seems to play an important role in other metabolic pathways as well, such as those involved in osteoarthritis, the immune response and cancer. One approach to understand the vitamin D endocrine system is to study the influence of variations in the DNA sequence of important proteins of this system. For example, deleterious mutations in the VDR gene cause 1, 25-dihydroxyvitamin D-resistant rickets, a rare monogenetic disease. More subtle sequence variations (polymorphisms) in the VDR gene occur much more frequently but their
http://dx.doi.org/10.1016/j.humimm.2014.02.009 0198-8859/Ó 2014 American Society for Histocompatibility and Immunogenetics. Published by Elsevier Inc. All rights reserved.
Y.M. Mosaad et al. / Human Immunology 75 (2014) 452–461
effects are poorly understood. Their influence on the vitamin D endocrine system is currently under scrutiny in relation to a number of so-called complex diseases and traits such as osteoporosis [4]. According to Davis et al. over 470 VDR single nucleotide polymorphisms (SNPs) are known [5]. VDR gene is located on chromosome 12 and contains 9 exons [6]. Their distribution and frequency vary among ethnic groups. Most of the work done on VDR polymorphisms has been conducted in Caucasian populations and has focused on six SNPs: (1) rs10735810 or FokI in exon 2, (2) rs1544410 or BsmI in intron 8, (3) rs731236 or TaqI in exon9, (4) rs7975232 or ApaI in intron 8, (5) rs757343 or Tru91 in intron 8, and (6) the poly (A) mononucleotide repeat in the 30 -untranslated region (UTR) [7]. The discovery of the VDR in the cells of the immune system and the fact that activated dendritic cells produce the vitamin D hormone suggested that vitamin D could have immunoregulatory properties. The most evident effects of the D-hormone on the immune system seem to be in the downregulation of the Th1-driven autoimmunity. Low serum levels of vitamin D3 might be partially related, among other factors, to prolonged daily darkness (reduced activation of the pre vitamin D by the ultra violet B sunlight), different genetic background (i.e. VDR polymorphism) and nutritional factors and explain to the latitude-related prevalence of autoimmune diseases such as rheumatoid arthritis by considering the potential immunosuppressive roles of vitamin D. The 25(OH)D3 plasma levels have been found inversely correlated at least with the RA disease activity showing a circannual rhythm (more severe in winter). Recently, greater intake of vitamin D was associated with a lower risk of RA as well as a significant clinical improvement was strongly correlated with the immunomodulating potential in vitamin D-treated RA patients [8]. RA is a chronic inflammatory joint disease that leads to local and systemic bone loss. Osteoporosis (OP) or the systemic bone loss associated with RA increases the risk for fragility fractures, which can affect quality of life dramatically in RA patients. RA associated secondary osteopenia/osteoporosis may be observed in 60– 80% of the patients. Traditional and RA-related risk factors have been defined and studied for osteoporosis associated with RA such as race, body mass index (BMI), dietary calcium, vitamin D intake, disease duration and activity, level of disability, corticosteroids and Disease Modifying Anti-Rheumatic Drugs (DMARDs) use and menopausal status [9–15]. The steroid hormone vitamin D, its receptor (VDR) and the metabolizing enzymes involved in the formation of the biologically active form of the hormone together are major players in the vitamin D endocrine system. This system plays a crucial role in calcium and phosphate homeostasis and skeletal metabolism. Following renal production as the hormonal metabolite of vitamin D, 1a,25dihydroxyvitamin D3 (1,25(OH)2D3) functions as the ligand for VDR, with the hormone-receptor complex inducing calcemic and phosphatemic effects that result in normal bone mineralization and remodeling [16]. Therefore, VDR has been considered as an important candidate gene of osteoporosis [17]. However, the role of genetic factors, especially allelic variants in candidate genes for osteoporosis such as VDR, is less well defined in RA-associated osteoporosis and therefore the role of vitamin D and its receptor in bone loss in RA is intriguing [9]. Interestingly, polymorphisms in the VDR have been positively correlated with increased susceptibility to RA by some studies [18–21] and have been negatively correlated by others [22–24]. On the other hand, the association of VDR gene polymorphism with OP has been studied by several investigators especially BsmI with some contradiction in the various reports primarily due to extensive geographic variations. While, the presence of the B allele has been associated with increased bone loss by some groups
453
[25–29], the b allele was reported to be correlated with OP by others [30,31] and some investigators have not found any association between the VDR gene BsmI genotype and bone mineral density [32–35]. The aim of the present work is to study the relationship between VDR gene polymorphism and susceptibility to rheumatoid arthritis and the impact of such association on disease profile and bone density among Egyptians.
2. Subjects and methods 2.1. Patients and controls This case control study on a cohort of Egyptian population includes patients with RA and two control groups of healthy individuals and postmenopausal osteoporosis females. A total of 128 patients with RA (103 females and 25 males) with mean age of 46.91 ± 11.73 diagnosed according to the American College of Rheumatology/European League against Rheumatism collaborative initiative for RA [36] were consecutively recruited into the study between February 2012 and January 2013 with disease duration of 10.33 ± 7.01 years. Patients were recruited from the outpatient’s clinic of Rheumatology and Rehabilitation Department, Mansoura University Hospitals, Egypt. All patients underwent clinical evaluation by a rheumatologist and data about demographics and disease parameters were collected. Disease activity was evaluated using disease activity score 28 (DAS28-CRP) that measures 28 tender and swollen joints, degree of pain by visual analogue scales (VAS), patient’s and doctor’s global assessment of disease activity using VAS and inflammatory markers such as C-reactive protein (CRP) [37]. DAS-28 as described by Prevoo et al. [38] defines the level of RA activity as follows; P5.1 indicate high disease activity; between 5.1 and 3.2 indicates moderate disease activity; between 3.2 and 2.6 indicates low disease activity; and 100 IU/ml = strongly elevated. AntiCCP antibodies
