MINISTRY OF EDUCATION AND TRAINING MINISTRY OF HEATH HANOI MEDICAL UNIVERSITY ====== TRAN THI THU HUYEN POLYMORPHISMS OF SOME GENES IN POSTMENOPAUSAL OSTEOPOROSIS WOMEN Specialism Internal Rheumatolog[.]
MINISTRY OF EDUCATION AND TRAINING MINISTRY OF HEATH HANOI MEDICAL UNIVERSITY ====== TRAN THI THU HUYEN POLYMORPHISMS OF SOME GENES IN POSTMENOPAUSAL OSTEOPOROSIS WOMEN Specialism : Internal Rheumatology Code :9720107 THESIS’S ABSTRACT HA NOI - 2022 The thesis has been completed at HANOI MEDICAL UNIVERSITY Supervisors: Supervisor 1: Nguyen Thi Thanh Huong, MD, PhD Supervisor 2: Assoc Prof Nguyen Thi Ngoc Lan, MD, PhD Reviewer 1: Assoc Prof Dang Hong Hoa, MD, PhD Reviewer 2: Assoc Prof Tran Huy Thinh, MD, PhD Reviewer 3: Assoc Prof Nguyen Ngoc Chau, MD, PhD The thesis will be present in front of board of university examiner and reviewer lever at… on ….2022 This thesis can be found at: National Library: National Medical Informatics Library Library of Hanoi Medical University LIST OF WORK RELATED TO THESIS Tran Thi Thu Huyen, Nguyen Thi Thanh Huong, Nguyen Thi Ngoc Lan (2020), “Relationship between MTHFR genotype polymorphism rs1801133 and bone mineral density in postmenopausal women" Journal of Vietnamese Medicine, August 2020, p 271 - 276 Tran Thi Thu Huyen, Nguyen Thi Thanh Huong, Nguyen Thi Ngoc Lan (2020), “Relationship between LRP5 genotype polymorphism rs41494349 and bone mineral density in postmenopausal women” Journal of Practical Medicine, June 2020, p 69 – 72 QUESTION Osteoporosis is a common disease affecting about 200 million people worldwide characterized by reduced bone mineral density, bone microstructural damage, and increased bone fragility In 2021, a metaanalysis by Nader Salari et al included 70 studies over 800.457 women aged 15 to 105 showed that the rate of osteoporosis in women was 23.1% globally and this number in Asia was 24.3% The high rate of osteoporosis will lead to an increase in the cost of disease treatment and fractures, causing significant economic and social impacts and clinical burden In Vietnam, according to Nguyen Thi Thanh Huong (2012), the prevalence of osteoporosis of the femoral neck and lumbar spine in women in the North was 23.1% and 49.5%, respectively Osteoporosis is a disease influenced by many factors, in which genetic factors play an important role Studies in twins and pedigrees have shown that 50-85% of bone mineral density (BMD) variation is due to genes To date, more than 20 genome-wide studies have been published, and the largest genome-wide study until 2019 was by Morris et al., which identified 518 locus affecting BMD In the condition of Vietnam, when genome-wide research cannot be carried out, we selected candidate gene polymorphisms for study: MTHFR rs1801133, LRP5 rs41494349 and FTO rs1121980 because these gene polymorphisms have been shown to affect BMD through physiological mechanisms For the MTHFR rs1801133gene polymorphism, the presence of the T allele reduces the activity of the MTHFR enzyme (MethyleneTetrahydrofolate Reductase) leading to increased blood homocysteine levels with the risk of decreased BMD LRP5 gene (LDL Receptor Related Protein 5) implicated in the Wnt/β - catenin signaling pathway affecting osteoblasts and BMD FTO gene (Fat mass and Obesity Associated) affects growth differentiating factor 11 and peroxisome proliferator-activated receptor gamma (peroxisome proliferator-activated receptor gamma) implicated in mesenchymal stem cell lineage differentiation to adipocytes and inhibition of bone formation Around the world, there have been many studies showing the association among these gene polymorphisms with BMD and these results are repeated in many races globally In Vietnam, there have been no studies investigating the relationship of these three gene polymorphisms with BMD in postmenopausal women Therefore, we conducted the thesis: "Polymorphisms of some genes in postmenopausal osteoporosis women" with two objectives: To determine the polymorphisms of the genes MTHFR rs1801133, LRP5 rs41494349 and FTO rs1121980 in postmenopausal osteoporosis women To find out the association among polymorphisms of the genes MTHFR rs1801133, LRP5 rs41494349 and FTO rs1121980 with bone mineral density and some risk factors for osteoporosis in postmenopausal women CHAPTER LITERATURE REVIEW 1.1 Overview of osteoporosis and osteoporosis in postmenopausal women 1.1.1 Definition of osteoporosis According to the World Health Organization in 1994: Osteoporosis is a disease of the bones, characterized by a decrease in bone mass accompanied by structural damage, leading to increased bone fragility, i.e bone fracture Therefore, it is necessary to measure BMD to assess fracture risk This definition was revised in 2001 by the World Health Organization: osteoporosis is characterized by changes in bone strength This strength is characterized by BMD and bone quality Bone quality is evaluated by the following parameters: bone structure, bone turnover, mineralization, cumulative damage, and properties of basic bone substances In these parameters, bone turnover plays an important role 1.1.2 Diagnosis of osteoporosis Currently, the best method for diagnosing osteoporosis is to measure BMD by dual-energy X-ray absorptiometry BMD is the amount of bone mineral divided by the area examined, in gram/cm To diagnose osteoporosis, an individual's BMD is compared with the average BMD of the population between the ages of 20 and 40 (peak BMD) Index T (T score) is the number of standard deviations of current BMD from peak BMD A person is diagnosed with osteoporosis if the T index is -2.5 or less, osteopenia if T -score is from -2.5 to -1.0, normal bone density if T score is -1.0 or higher Low BMD is the most important risk factor for fracture The World Health Organization recommended that the femoral neck is the most important anatomical area for the diagnosis However, guidelines from the International Society for Clinical Bone Densitometry (ISCD) and the American Osteoporosis Society (NOF) recommended that the diagnosis of osteoporosis should be based on three sites: femoral neck, total hip, and lumbar spine Measurements at these different locations may give different results 1.1.3 Menopause and its effect on osteoporosis Menopause is the absence of a period in a woman for at least 12 consecutive months During menopause, estrogen and testosterone levels decrease, leading to an imbalance of remodeling The osteoclast phase will be prolonged with an increase in the lifespan of osteoclasts and the osteoblastic phase will be shortened by promoting the apoptosis of osteoblasts These changes cause the consequence that the osteoblasts will no longer be able to fill the voids created by the osteoclasts Furthermore, prolonging osteoclast lifespan increases the depth of bone resorption and leads to the elimination of the reticular structure within the cancellous bone At the same time, deeper penetration of osteoclasts at the endoplasmic reticulum leads to cortical loss and thinning Bone resorption increases by 90% after menopause while bone formation is only about 45% 1.2 Genes and osteoporosis - The long-known factors affecting osteoporosis are age, hormonal status, lifestyle factors (eating and exercising habits) and family factors Recently, the development of gene technology helps to analyze and identify genotype polymorphisms affecting osteoporosis and fractures has opened a new era in bone research in particular and medicine study in general Studies on identical twins show that genetic factors play an important role in osteoporosis, determining 50-85% of BMD - The genes that can affect BMD studied in the world are genes related to the RANKL/RANK/OPG signaling system, LRP5/LRP6 receptors (lipid control receptors), signaling pathways Wnt signaling pathway, estrogen receptor-related genes (ER1, ER2), vitamin D receptor-related genes (VDR), collagen network-related genes (COL A1, COL A2) Genetic studies related to osteoporosis began around the 1990s Initially, they are monogenic studies such as defects in the COL1A1 and COL1A2 genes causing brittle bone disease, mutations in the LRP5 gene causing OPPG syndrome (osteoporosis pseudo glioma) - In this study, we chose gene polymorphisms MTHFR rs1801133, LRP5 rs41494349 and FTO rs1121980 because there have been many studies in the world showing the relationship between these genotype polymorphisms with BMD and these results are repeated in many European, Asian, and Australian races The MTHFR gene affects the enzyme activity MTHFR affects the folate metabolism, causing an increase in blood homocysteine levels leading to many medical conditions including osteoporosis.We found that there are many studies showing that carriers of the TT genotype of the MTHFR rs1801133 gene polymorphism have an increased risk of BMD loss such as the study of author Bo Abrahamsen on Danish, Massimo De martinis in Italian, Xiumei Hong in Chinese, and Masataka Shiraki in Japanese postmenopausal women However, a study by Soewarlan WDHP et al on postmenopausal women in Indonesia, a country in the same Southeast Asian region with Vietnam, did not find any association between this gene polymorphism and BMD.The LRP5 gene involved in the Wnt/βcatenin signaling pathway influences osteoblasts and bone mass regulation The Q89R gene polymorphism (rs41494349) is rare in Caucasians but relatively common in Asians Studies by authors Tomohiko Urano, Jung-Min Koh, Zhenlin ZANG on Japanese, Korean and Chinese people all demonstrated that carriers of the R allele of the Q89R gene polymorphism have lower bone density than those who not carry the R allele However, studies on postmenopausal Thai women did not find any association between this gene polymorphism and BMD The FTO gene has been shown through many studies to have a strong association with diabetes and obesity, so it is able to affect osteoporosis FTO gene affects the GDF11-FTO-PPARγ pathway involved in the differentiation of mesenchymal stem cells to adipocytes and inhibits bone formation A study by Bich Tran et al (2013) on Australians showed that women with the homozygous recessive TT genotype of SNP rs1121980 had a 2.06 times higher risk of femoral neck fracture than women with homozygous genotype CC dominant zygote CHAPTER RESEARCH SUBJECTS AND METHODS 2.1 Place and time of study * Location: Outpatient Department and Rheumatology Department, Bach Mai Hospital * Time:from May 2015 to November 2018 2.2 Research subjects The research subjects were postmenopausal women aged 40 years and older who visited the Outpatient Department and Rheumatology Department at Bach Mai Hospital * Criteria for selection: - Clinical characteristics: Women 40 years of age and older with a healthy medical history and natural menopause Natural menopause is defined as the absence of a period for 12 months or more - The subjects voluntarily agreed to participate in the study after being clearly explained the research’s purpose - They had the ability to answer the interview questions - All study subjects had their bone mineral density measured at the Center for Oncology and Nuclear Medicine - Bach Mai Hospital during the study period With the measured Tscore, we divided into two groups based on the diagnosis criteria of the International Society for Clinical Bone Densitometry and the American Osteoporosis Society: + The group of postmenopausal women with osteoporosis when the Tscore value at the femoral neck or total hip or lumbar spine is ≤ -2.5 + The group of postmenopausal women without osteoporosis when the Tscore value in all these positions (femoral neck, total hip, lumbar spine) > -2.5 * Exclusive criteria: - Patients with a history of chronic diseases such as liver disease, chronic kidney disease, cancer, endocrine diseases and disorders related to vitamin D or bone metabolism such as diabetes, obesity, malabsorption syndrome, hyperthyroidism, hypothyroidism, Cushing's syndrome which are detected through medical history, physical examination and laboratory tests - Patients using drugs related to calcium and vitamin D metabolism in the past months such as corticosteroids, sex hormone replacement, heparin, bisphosphonate - The patient had her uterus and ovaries removed - The patient did not agree to participate in the study 2.3 Study design and sample size 2.3.1 Research design - Study design: cross-sectional descriptive study 2.3.2 Formula for calculating sample size * Target sample size 1: Use the formula to estimate a population proportion: n Z12 /2 p(1 p) d2 n: smallest sample size achieved Z: confidence coefficient, at the 95% probability level, Z= 1.96 p: rate of allele of interest (minor allele) in the population d: allowable error, d= 0.05 According to the dbSNP database, we have minor allele (p) rates of gene polymorphisms in Asians For each gene polymorphism we have a p-value and we will calculate the sample size (n) for each gene + The ratio of the T allele of the MTHFR rs1801133gene polymorphismis p = 0.12; n1 = 163 + The ratio of the G allele of the LRP5 rs41494349 gene polymorphismis p = 0.08; n2 = 114 + Ratio of the T allele of the FTO rs1121980gene polymorphismis p = 0.16; n3 = 207 So we choose the sample size for target as the largest sample size of gene polymorphisms: n = 207 * Target sample size 2: The second objective is to explore the association of gene polymorphisms with bone mineral density and some risk factors for osteoporosis, so the minimum sample size of objective will be: nx2,5 = 207x2.5 = 517.5 In fact, we selected 566 postmenopausal women to conduct the study 2.3.3.Sampling method Selecting samples for cross-sectional descriptive research All subjects were interviewed with a set of screening questions and screened by rheumatologists Eligible subjects will be tested for complete blood count and urea, creatinine, glucose, liver enzymes, C-reactive protein (CRP), erythrocyte sedimentation rate to exclude diabetes, anemia, liver enzymes elevation, renal failure, suspected inflammatory or malignancy Subjects who meet all criteria will be interviewed with the research questionnaire, clinically examined according to the research medical record, have BMD measured, and have blood drawn for genetic analysis 2.4.2 Interview process and clinical examination - Age, menstruating age, menopause, time of menopause, number of children, number of pregnancies, living area, history of fractures were interviewed according to the research medical record - Height: height is measured with a height measuring wooden ruler (accuracy 0.1cm) The ruler is placed vertically, perpendicular to the horizontal ground The height was measured when taking off patients’ shoes, standing with their back against the tape measure, looking straight ahead, their hands hanging down so that their heels, calves, buttocks, shoulders, and occipitals (9 touch points) follow a straight line and apply pressure close to the vertical gauge Use a square or piece of wood against the top of the head perpendicular to the ruler and read the result - Weight: weight is measured by Tanita electronic balance with an accuracy of 0.1 kg, the result is in kg and recorded with an odd number Scale is placed in a stable and flat position, adjusted the balance to zero Before weighing, we check the balance with a reference object to control the accuracy and sensitivity of the scale Participants removed their shoes, wore the most compact clothes, stood in the middle of the scale, did not move, looked straight ahead, weight evenly distributed on both feet - BMI: Calculated according to the formula: m BMI = h2 m: weight (kg) h: height (m) - Physical activity: + The physical activity assessment questionnaire is based on the Active-Q Physical Activity Questionnaire + The content of physical activity includes questions in four areas: physical activity during work, physical activity when commuting to work, physical activity during leisure time, physical activity when playing sports Ask and evaluate physical activity: in a typical week Unit: MET is the conversion unit used in the assessment of physical activity 1MET is the cost of energy sitting quietly, and is equivalent to calorie consumption of kcal/kg/hour 10 2.6 Data analysis and processing Data was entered and checked using REDCap software Quantitative variables are presented as mean and standard deviation (SD) if normally distributed or median and interquartile range if not normally distributed Qualitative variables are presented as frequencies and percentages For continuous variables, comparisons between means were made using the Independent samples T test For categorical variables, comparisons between ratios were performed using the Chisquare test Analysis of the relationship of genotype and factors (age, weight, height, BMI, living area, history of fracture) with BMD at locations of femoral neck, total hip, and lumbar spine by univariate and multivariable linear regression analysis R language software version 3.6.3 was used for statistical analysis and plotting 2.7 Research ethics This study uses part of the data in the project "Determining polymorphisms and sensitivity of genes related to osteoporosis and fractures in Vietnamese" granted by the National Foundation for Science and Technology Development (NAFOSTED) was approved by the ethics committee in biomedical research of Dinh Tien Hoang Institute of Medical Research No 01/HĐĐĐ-VNCYHĐTH on January 27, 2016 and the board approved the outline of Hanoi Medical University 11 CHAPTER RESEARCH RESULTS Table 3.2: General characteristics of the study's subjects by osteoporosis and non-osteoporotic group Subgroups Characteristics Osteoporosis n=223 a Age (years) 63.88 (8.00) Height (cm) 150.38 (5.76) a Weight (kg) 47.68 (6.71) a BMI (kg/m2) 21.05 (2.48) b BMI subgroup Normal 147 (65.9%) Underweight 29 (13.0%) Overweight and obesity 47(21.1%) b Where to live Countryside 163 (73.2%) City 60 (26.9%) b Physical activity (METsminute/week) Failed ( 0.05) 15 Table 3.16 The relationship between FTO rs1121980 genotype and BMD FTO rs1121980 genotype Femoral neck (g/cm ) Total hip ((g/cm2) Lumbar spine ((g/cm2) CC n = 395 0.67 ± 0.12 0.78 ± 0.13 0.74 ± 0.15 CT n = 171 0.66 ± 0.12 0.79 ± 0.14 0.77 ± 0.14 p 0.87 0.81 0.14 The p-value obtained from the Independent samples T test Comment: In lumbar spine, CT genotype carriers have a higher BMD than genotype CC ones However, the difference is not statistically significant Table 3.32 Multivariate correlation between MTHFR rs1801133, LRP5 rs41494349 gene polymorphisms and some factors related to bone density at sites (recessive model) Femoral neck Coefficient β[95%CI] -0.005 *** [-0.007; -0.003] 0.012 *** [0.009; 0.015] Total hip Coefficient β[95%CI] -0.003 * [-0.006; -0,000] 0.018 *** [0.015; 0.022] Lumbar spine Coefficient β[95%CI] -0.003 * [-0.006; -0,000] 0.018 *** [0.015; 0.022 -0.033 * -0.058; -0.008] -0.040 ** [-0.071; -0.010] -0.040 ** [-0.071; -0.010] -0.011 -0.015 [-0.030; 0.007] [-0.038; 0.007] 0.004 -0.001 [-0.002; 0.010] [-0.008; 0.006] Duration after -0.004 *** -0.005 *** menopause (years) [-0.006; -0.002] [-0.008; -0.003] Physical activity 0.007 0.020 * 600 vs < 600 [-0.009; 0.023] [0,000; 0.039] MTHFR:TT with -0.075 * -0.074 * CC+CT [-0.133; -0.016] [-0,145; -0.003] LRP5: GG with 0.043 0.020 AA+AG [-0.049; 0.135] [-0.092; 0.131] n 566 566 R2 0.415 0.404 ***p < 0.001; ** p < 0.01; *p < 0.05 -0.015 [-0.038; 0.007] -0.001 [-0.008; 0.006] -0.005 *** [-0.008; -0.003] 0.020 * [0,000; 0.039] -0.074 * [-0,145; -0.003] 0.020 [-0.092; 0.131 566 0.404 Location Element Age BMI History fracture compared to no fracture Urban versus countryside Number of children 16 Comment:TT genotype carriers of the MTHFR rs1801133 gene polymorphism have a risk of decreased BMD at sites (femoral neck, total hip and lumbar spine) compared with CC and CT genotype carriers after controlling other risk factors - There was no association between the LRP5 rs41494349 gene polymorphism and BMD at these sites after controlling other risk factors - FTO rs1121980gene polymorphism was not included in the analysis in the recessive model because in the 566 postmenopausal women, no TT genotype of this gene polymorphism was detected Table 3.33 Multivariable correlation between polymorphisms of genes MTHFR rs1801133, LRP5 rs41494349, FTO rs1121980 and some factors related to bone density at positions (co-dominant model) Location Factor Age BMI History fracture compared to no fracture Urban versus countryside Number of children Duration after menopause (years) Physical activity 600 vs < 600 MTHFR: CT with CC MTHFR: TT with CC LRP5: AG with AA LRP5: GG vs AA FTO: CT with CC n R2 Femoral neck Coefficient β[95%CI] -0.005 *** [-0.007; -0.003] 0.012 *** [0.009; 0.015] Total hip Coefficient β[95%CI] -0.005 *** [-0.008; -0.003] 0.016 *** [0.013; 0.019] Lumbar spine Coefficient β[95%CI] -0.003 * [-0.006; -0,000] 0.018 *** [0.015; 0.022] -0.033 * [-0.058; -0.008] -0.035 * [-0.062; -0.008] -0.040 ** [-0.071; -0.010] -0.012 [-0.030; 0.007] 0.004 [-0.002; 0.010] -0.004 *** [-0.006; -0.002] 0.007 [-0.009; 0.023] 0.007 [-0.011; 0.024] -0.073 * [-0.132; -0.014] -0.002 [-0.024, 0.020] 0.042 [-0.050; 0.134] 0.005 [-0.012; 0.021] 566 0.416 -0.015 [-0.035; 0.005] 0.003 [-0.003; 0.010] -0.004 *** [-0.006; -0.002] 0.009 [-0.009; 0.026] 0.003 [-0.016; 0.022] -0.090 ** [-0,154; -0.027] -0.009 [-0.033, 0.015] 0.043 [-0.056; 0.143] 0.010 [-0.009; 0.028] 566 0.457 -0.016 [-0.038; 0.006] -0.001 [-0.008; 0.006] -0.005 *** [-0.008; -0.003] 0.020 * [0,000; 0.039] -0.007 [-0.028; 0.014] -0.078 * [-0,150; -0.007] -0.006 [-0.033, 0.020] 0.027 [-0.084; 0.139] 0.022 * [0.002; 0.043] 566 0.410 17 Comment:Carriers of the TT genotype of the MTHFR rs1801133 gene polymorphism have a risk of decreased BMD in sites (femoral neck, total hip, and lumbar spine) compared with CC genotype carriers after controlling other risk factors - Carriers of the CT genotype of the FTO rs1121980 gene polymorphism are likely to have increased BMD at lumbar spine compared with CC genotype ones after controlling other risk factors - There was no association between the LRP5 rs41494349 gene polymorphism and BMD at all sites after controlling other risk factors CHAPTER 4: DISCUSSION 4.1 General characteristics of study subjects according to osteoporosis group and non-osteoporotic group 566 postmenopausal women were selected for the study, 223 with osteoporosis and 343 without osteoporosis Postmenopausal women with osteoporosis have older age, lower height, and lower weight than ones without osteoporosis Postmenopausal women with osteoporosis have more children, younger age at menopause time, and longer postmenopausal years than ones without osteoporosis This result is similar to the research results of Tao Minh Thuy et al (2013), Hoang Thi Bich et al (2014) In addition, the group of postmenopausal women without osteoporosis have a higher rate of physical activity reaching the criteria of the World Health Organization (≥ 600 METs-minute/week) than the osteoporosis group 4.2 Genotypic and allele distribution of MTHFR rs1801133, LRP5 rs41494349, FTO rs1121980 gene polymorphisms in the two groups In our study, the genotype and allele ratios of the MTHFR rs1801133 and LRP5 rs41494349 gene polymorphisms followed Hardy Weinberg's equilibrium principle (p>0.05) This demonstrated that the genotype distribution and allele ratio of the MTHFR rs1801133, LRP5 rs41494349 gene polymorphisms are inherited stably from generation to generation and are not affected by special factors related to the genetic evolution process The ratio of genotypes and alleles of the FTO rs1121980 polymorphism did not follow Hardy Weinberg's equilibrium principle (p < 0.05) In 566 subjects, no homozygous recessive TT genotype of this gene polymorphism was detected Table 3.7 presents the genotype and allele distribution of gene polymorphisms MTHFR rs1801133, LRP5 rs41494349, FTO rs1121980 in the two groups (osteoporosis and non-osteoporosis) In the group of osteoporosis, with the MTHFR rs1801133 gene polymorphism, the ... pathways Wnt signaling pathway, estrogen receptor-related genes (ER1, ER2), vitamin D receptor-related genes (VDR), collagen network-related genes (COL A1, COL A2) Genetic studies related to osteoporosis... blood for genetic analysis Analyze genes, determine genotypes and alleles of gene polymorphisms MTHFRrs1801133, LRP5rs41494349, FTOrs1121980 Obiective Determination of polymorphisms of the genesMTHFR... Table 3.4 Genotypic and allele frequency distribution of MTHFR rs1801133 gene polymorphisms MTHFR rs1801133 Genotypes and alleles n = 566 Ratio (%) CC 404 71.4 Genotype (n) CT 152 26.8 TT 10 1.8