AIP Conference Proceedings 2092, 030023 (2019); https://doi.org/10.1063/1.5096727 2092, 030023
© 2019 Author(s).
Distribution of methylenetetrahydrofolate reductase (MTHFR) C677T polymorphism in postmenopausal Indonesian women with osteoporosis – A preliminary study
Cite as: AIP Conference Proceedings 2092, 030023 (2019); https://doi.org/10.1063/1.5096727 Published Online: 09 April 2019
Widya Dwi Honesty Putri Soewarlan, Hedijanti Joenoes, Shafa Ahmad Bawazier, Dwi Anita Suryandari, and Elza Ibrahim Auerkari
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Distribution of Methylenetetrahydrofolate Reductase (MTHFR) C677T Polymorphism in Postmenopausal Indonesian Women with Osteoporosis – A Preliminary
Study
Widya Dwi Honesty Putri Soewarlan
1, Hedijanti Joenoes
1, Shafa Ahmad Bawazier
1, Dwi Anita Suryandari
2, Elza Ibrahim Auerkari
1,a)1Departement of Oral Biology, Faculty of Dentistry, Universitas Indonesia, Jl. Salemba Raya No. 4 Central Jakarta 10430 Indonesia
2Department of Medical Biology, Faculty of Medicine, Universitas Indonesia, Jl. Salemba Raya No. 6, Central Jakarta 10430 Indonesia
a)Corresponding author: [email protected]
Abstract. Osteoporosis is a condition that is characterized by reduced bone mass and micro-architectural deterioration.
Previous studies have shown that the MTHFR C677T polymorphism may be involved in the development of osteoporosis.
The aim of this study was to characterise the distribution of this polymorphism in 100 Indonesian postmenopausal women.
The MTHFR C677T polymorphism were determined in 25 women with osteopenia, 50 women with osteoporosis, and 25 control subjects using Polymerase Chain Reaction-Restriction Fragment Length Polymorphism technique with Hinf1 enzyme. The results showed CC genotype (wild type) 70%, CT genotype (heterozygous polymorphic) 29%, and TT genotype (homozygous polymorphic) 1%, with a T-allele frequency of 15.5%. The result also showed that there was no significant variation with MTHFR C677T genotype. It can be concluded that the MTHFR C677T polymorphism was weakly associated with BMD (Bone Mineral Density).
Keywords: Bone mineral density, MTHFR, osteoporosis, polymorphism
INTRODUCTION
Elderly people are the fastest growing section of the world population. This is increasing the susceptibility to bone loss and fracture risk, as aging is closely related to the bone fragility disease of osteoporosis, the most common health disorder suffered by elderly people [1]. According to the WHO Study Group, in 2003 osteoporosis has struck 75 million people in the United States, Europe, and Japan [1-5]. Osteoporosis causes more than 8.9 million fractures annually worldwide, of which more than 4.5 million occurred in the United States and Europe. In Indonesia in 2005, the prevalence of osteopenia (early osteoporosis) was 41.7% and 10.3% prevalence of osteoporosis, i.e. it can be said that two out of every five Indonesians is at risk of developing osteoporosis. In compared to worldwide based on International Osteoporosis Foundation, one in three women and one in five men over 50 will experience osteoporosis which relatively less incidence [5].
Osteoporosis is defined as a skeletal disorder characterized by reduced bone strength, both in terms of quantity, quality, or both, leading to increased risk of fractures. Quantitatively the status is measured as BMD (bone mineral density), and qualitatively viewed from many factors: the degree of mineralization, the relationship of trabecular bone, the quality of collagen fibers, and the conditions of bone cells [1,6]. By the WHO standards, normal bone is commonly defined having BMD values at or above minus one standard deviation (SD) from the optimal peak bone density (T-
score) of healthy young adults of the same sex (Table 1). BMD values at or below -2.5 SD from the optimal peak bone density mean osteoporotic bone, leaving osteopenia for BMD values between these limits [7,8].
TABLE 1. WHO standard definitions
T-score BMD
SD > -1 Normal
SD -1 to -2.5 Osteopenia
SD < -2.5 Osteoporosis
Bone loss usually occurs without symptoms, resulting in a "silent disease". People with osteoporosis are not experiencing any symptoms for several years until an occurrence of fracture [9,10]. Because osteoporosis is associated with fracture, the disease is associated with high morbidity, mortality, and healthcare costs [10]. The most common fractures are spinal, hip and wrist fractures [5,11]. The pathophysiology of osteoporosis involves an imbalance between bone formation and bone resorption without any remodelling. In osteoporosis, bone resorption is greater than bone formation, so that bones become brittle and the fracture risk is increased [10-12]. Osteoporosis risk is much increased under estrogen deficiency [13]. Therefore, osteoporosis is more common in women than men, especially in postmenopausal women.
Genetic and environmental factors affect BMD and bone loss, and possibly about 75% of the variation in bone mass may be determined by genetic factors. It is conceivable polymorphisms of the regulatory genes are involved, and identifying the relationships between genetic factors and BMD could be useful for predicting the risk of osteoporotic fractures [14].
Several genes have been shown to be associated with the metabolism or structure of bone, for example those for vitamin D receptor, collagen type IA1, and estrogen receptor gene. In addition, the MTHFR (Methylenetetrahydrofolate reductase) C677T polymorphism has been associated with osteoporosis and prediction of fracture risk in postmenopausal women [7,14,15].
MATERIALS AND METHODS
The DNA samples used in this study was extracted from the blood serum of 100 postmenopausal women in Indonesia. The inclusion criteria specified that all patients are women without menstrual cycle for more than 1 year.
All participants had signed the informed-consent form. The extracted DNA samples were stored at -20°C. The T- scored subjects included 25 women with osteopenia, 50 women with osteoporosis, and 25 control cases of healthy (normal) postmenopausal women with free anamnesis of osteoporosis and osteopenia.
The MTHFR C677T (rs1801133) genotyping was performed by polymerase chain reaction (PCR) with the primers 5’-TGA AGG AGA AGG TGT CTG CGG GA-3’ (forward) and 5’-AGG ACG GTG CGG TGA GAG TG-3’ (reverse) [16-18]. It was amplified in a final volume of 18 μL, which each reaction containing 10 μL of mastermix (Bioline), 0.5 μL of forward primers, 0.5 μL reverse primers, 6 μL of steril destilled water, and 1 μL of the extracted DNA.
PCR was performed with the initial denaturation at 94 °C for 5 min, followed by a 3-step cycling processes (amplified for 30 cycles) which consisted of denaturation at 94 °C for 30 s, annealing at 64 °C for 30 s, and extension at 72 °C for 30 s; and a final extension at 72 °C for 7 min. After the DNA amplification process was completed, the amplicon was stored at 4°C. The final PCR product was 198 bp in size.
For restriction fragment length polymorphism (RFLP) analysis, the PCR products were digested with restriction enzyme Hinf1 (New England Biolabs) for 16 hours at 37°C. The digested products were separated and visualized by electrophoresis on 3% agarose gel. The digested PCR product produced 175 and 23 bp fragments for TT condition (homozygous polymorphic) and 198, 175 and 23 bp fragments for CT condition (heterozygous polymorphic). The undigested product length of 198 bp was retained by the wild type (CC); however the 23bp fragment was not retained on the gel [16-18].
Chi-square testing was applied for statistical analysis regarding the significance of the genotype frequencies and BMD status, using SPSS software.
RESULTS
Representative results of RFLP are shown in Figure 1.
FIGURE 1. Representative results of Hinf I RFLP analysis of MTHFR 677. M – DNA ladder (50 bp) as a marker. Lane 1 and 3 – 198 bp – Homozygous Wild Type (CC). Lane 2 – 198, 175, 23 bp – Heterozygous Polymorphic (CT). Lane 4 – 175, 23 bp –
Homozygous Polymorphic (TT)
As can be seen in Table 2, the distribution of C allele and CC genotype dominate in the test population. Genotype CC was found in 70 (70%) of samples and C allele was found in 169 (84.5%) of alleles. Genotype CC was also found in 72% of subjects with osteoporosis, whilst 28% were with genotype CT, and none were with TT genotype. In cases with osteopenia, genotype CC was found in 80%, 20% with genotype CT and again none with genotype TT. As for the normal subjects, 56% were with genotype CC, 40% with genotype CT and 4% (one person) with genotype TT.
From the result of MTHFR C677T polymorphism, the allele T frequency was 0.155 of the total number of alleles.
No significant difference in the genotype distribution between cases (osteoporosis and osteopenia) and controls was observed (x2= 5.84, p = 0.211).
TABLE 2. MTHFR C677T polymorphism - genotype and allele frequency in osteoporosis, osteopenia, and normal subjects
Genotype/
Allele
Total (n=100)
Osteoporosis (n=50)
Osteopenia (n=25)
Normal /Control (n=25) Genotypes
CC 70 (70%) 36 (72%) 20 (80%) 14 (56%)
CT 29 (29%) 14 (28%) 5 (20%) 10 (40%)
TT 1 (1%) 0 0 1 (4%)
Alleles
C 169 (84.5%) 86 (86%) 45 (90%) 38 (76%)
T 31 (15.5%) 14 (14%) 5 (10%) 12 (24%)
DISCUSSION
The purpose of this study was to characterise the distribution of the MTHFR C677T polymorphism in postmenopausal Indonesian women with a range of BMD from normal to osteoporotic level. The results suggest that the TT genotype (1%) and the T allele (15.5%) are relatively uncommon in the Indonesian female population.
Although MTHFRC677T polymorphism may be a risk factor for osteoporosis, it is not frequently present and no significant association was demonstrated between MTHFR C677T polymorphisms and BMD.
Several previous studies (Table 3) have reported low frequencies of TT genotype and T allele especially of South and South-East Asian populations [7,19,20].
TABLE 3. Prevalence of the MTHFR C677T genotypes in selected populations
Country/ethnic group CC (%) CT (%) TT (%) T-allele
frequency Asia
Indonesian (our study)
70 (70%) 29 (29%) 1 (1%) 0.155
Malaysian19 (n=62) (healthy population) Indian 19(n=72) (healthy population) Chinese19 (n=33) (healthy population)
55 (88.7%) 49 (68%) 21 (63.6%)
7 (11.3%) 22 (30.6%) 11 (33.3%)
0 (0%) 1 (1.4%) 1 (3.1%)
0.056 0.167 0.197 Japanese20 (n=244) 96 (39.3%) 116 (47.5%) 32 (13.2%) 0.37
Korean20 (n=124) 33 (26.6%) 82 (66.1%) 9 (7.3%) 0.4
Chinese7 (n=1899) (postmenopausal women)
682 (35.9%) 946 (49.8%) 271 (14.3%) 0.39
Canada/Inuit20 (n=174) 155 (89.1%) 17 (9.8%) 2 (1.1%) 0.06 South Africa/African20 (n=107) 85 (79.4%) 22 (20.6%) 0 (0%) 0.1
In summary, the results show that the MTHFR C677T polymorphism is relatively infrequent in the Indonesian female population, and no significant association was observed between this polymorphism, bone mineral density or osteoporotic status.
ACKNOWLEDGEMENT
The authors wish to gratefully acknowledge the financial support from the Indonesian Ministry of Education through the Universitas Indonesia (EIA).
Conflicts of interest: None.
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