MINSTRY OF EDUCATION
MINISTRY OF HEALTH
HANOI MEDICAL UNIVERSITY

NGUYEN THI HOA

STUDY ON POLYMORPHISMS OF SOME
GENES ASSOCIATED OSTEOPOROSIS IN MEN

Training major
Specialization
Code

: Internal Medicine
: Internal – Osteoarthritis
: 9720107

DOCTORAL THESIS SUMMARY

HANOI – 2023


THIS THESIS WAS PRESENTED AT HANOI MEDICAL
UNIVERSITY

Scientific intructors:
1. Dr. Nguyen Thi Thanh Huong
2. Asc. Prof. Dr. Tran Thi Minh Hoa

Reviewer 1: Asc. Prof. Dr. Nguyen Mai Hong

Reviewer 2: Asc. Prof. Dr. Le Thu Ha

Reviewer 3: Prof. Dr. Tran Huy Thinh

This thesis is to be defended in the university’s council
At: …. day …. month ….. year…….

This thesis can be found at:
- National library
- Library of Hanoi Medical University


PUBLISHED WORKS RELATED TO THE THESIS
1. Nguyen Thi Hoa, Tran Thi Minh Hoa, Nguyen Thi Thanh Phuong.
“Relationship between LRP5 genotype polymorphism at SNP Q89R
(rs41494349) with bone density in men”. Vietnamese Medical
Journal, August 2020. 493:263-270.
2. Nguyen Thi Hoa, Nguyen Thi Nan, Tran Thi Minh Hoa, Nguyen Thi
Thanh
Huong.
"Relationship
between
the
Methylene
tetrahydrofolate reductase (MTHFR) C677T gene polymorphism
and osteoporosis in men". Journal of Vietnamese Medicine, May
2022. 514(2):221 - 226.
3. Nguyen Thi Hoa, Nguyen Thi Nan. Tran Thi Minh Hoa, Nguyen Thi
Thanh Huong. "Relationship between fat mass and obesity gene
polymorphism - Associated (FTO) Rs1121980 with osteoporosis in

men". Vietnamese Medical Journal. November 2022. 520(2):161-167.


1
INTRODUCTION
1. The justification of this study
Osteoporosis is a systemic skeletal disease, characterized by a
decrease in bone mineral density (BMD) and a deterioration in bone
microarchitecture that results in brittle and fragile bones. Osteoporotic
fractures increase disability, mortality, healthcare cost, and decrease
quality of life, especially in the elderly. Osteoporosis is more generally
considered a disease not only detrimental to women but also prevalent
in men. The incidence of osteoporotic fractures in men is about half of
that in women. Furthermore, the mortality related to hip fractures in
elderly men is considerably higher than in women. Osteoporosis is a
complicated disease affected by many factors, including diet (calcium
and protein intake), physical activities, endocrine status, co-morbidities,
and genetic factors. In particular, genetic factor plays an important role
in osteoporosis pathogenesis. Twin- and family-based studies have
indicated that 60–85% of the bone mineral density (BMD) variance is
determined genetically. Up to now, genome-wide association studies
(GWASs) and meta-analyses have discovered 518 loci related to BMD,
osteoporosis and osteoporotic fractures, but only 20% of the gene
polymorphisms in these studies could explain its mechanism impacting
bone density and fracture. Numerous previous studies showed that three
gene polymorphisms LRP5 Q89R, MTHFR C677T and FTO rs1121980
were associated with transformation in bone density and fractures in
Caucasians and Asian populations. Moreover, In Vietnam, there are no
studies conducted on 3 gene polymorphisms LRP5Q89R, MTHFR
C677T and FTORs1121980, especially in men.

2. Scientific and practical significance of the study
- The first research result defined the characteristics of gene
polymorphisms LRP5Q89R, MTHFRC677T and FTORs1121980 in
elderly Vietnamese men.


2
- The study showed that 3 polymorphisms LRP5Q89R,
MTHFRC677T and FTOrs1121980 all raise the proportion of
osteoporosis in Vietnamese men and were independent of other risk
factors: age, BMI, history of fracture, level of physical activities. Men
having more risk alleles in the 3 gene polymorphisms increase
osteoporosis probability. Therefore, this information will help clinicians
assess osteoporosis risks in young people, especially those with
numerous background diseases and drugs affecting bone metabolism,
resulting in early diagnosis, early treatment, and minimizing fracture
proportion, the fatality after osteoporosis fracture and treatment cost
burden for the economy.
3. Research objectives
1. Determining the genotype of polymorphisms LRP5 Q89R, MTHFR
C677T and FTO rs1121980 in male with primary osteoporosis
2. Analyzing the relevance of LRP5 Q89R, MTHFR C677T, FTO
rs1121980 gene polymorphism to bone density and risk factors of males
with primary osteoporosis.
4. Thesis structure
- The thesis consists of 125 pages, including 4 chapters: 2 pages of
introduction, 34 pages of review of literature, 17 pages of subjects and
methods, 34 pages of results, 35 pages of discussion, and 3 pages of
conclusion and recommendations.
- This thesis consists of 34 tables (the results section 30 tables), 9

charts, 15 figures. References: a total of 157 references, including 18
articles in Vietnamese, and 139 articles in English.


3
Chapter 1: OVERVIEW
1.1 The epidemiology of osteoporosis
*In the world: The rate of male osteoporosis varies according to
each study, ranging from 2-16% and increases with age. The rate of
osteoporosis in men is usually 3-4 times lower than in women. The
NHANES 2005–2006 study was conducted by the US National Centers
for Health Statistics on 3157 American adults aged 50 years and older
who were measured BMD at 2 sites of the femoral neck and the total
hip, the results for found that 49% of elderly women and 30% of older
men had decreased bone mass at the femoral neck, the rate of
osteoporosis in women was 10% and the rate of osteoporosis in men
was 2%. In another study in this country conducted on men aged 69 to
74 years, the rate of osteoporosis was 10.2%. In Switzerland, the rate of
osteoporosis is 6.3% in men aged 50-80 years old and 16.6% in men
between the ages of 80-84. In some Asian countries, the rate of
osteoporosis is higher in women than in men, similar to whites. A study
conducted on 7042 people aged 20 and over in 10 centers in different
provinces in China (2002-2006) showed that the rate of osteoporosis in
women was 31.2% and 10.4% in women. men over 50. In Hong Kong,
the rate of lumbar spine osteoporosis in men is 7%, in women, it is
37%, the rate of osteoporosis in the femoral neck in men is 6% and in
women, it is 16%. In Korea, the prevalence of osteoporosis is 35.5% in
women and 7.5% in men over the age of 50.
*In Vietnam: the studies in the North and the South showed that
the rate of osteoporosis in both men and women was similar to that of

some Asian countries and white men. According to the data of Nguyen
Thi Thanh Huong in the North of Vietnam, a study on 222 men and 612
healthy women aged 13-83, the rate of osteoporosis in the femoral neck
is 9% for men and 17% for women.


4
Research by author Ho Pham Thuc Lan and colleagues on 357 men
and 870 women aged 18 - 89 in Ho Chi Minh City showed that the rate
of male osteoporosis is 10%, while the rate of female osteoporosis is
30%. Thus, it can be seen that osteoporosis and osteoporotic fractures
are really common diseases in Vietnam and need to be studied
appropriately.
1.2. Genes related osteoporosis
1.2.1. In the world
Reducing BMD is the principal underlying cause of significant
morbidity. Determination of BMD is Multifactorial and subject to
interplay between environmental and Genetic factors. Twin and family
studies show that genetic factors may account for 50–80% of the
interindividual variation of BMD. Genetic polymorphisms in a large
number of candidate genes have been reported to be associated with the
BMD that affect the biochemical, pharmacological and physiological
pathways of bone formation and bone resorption. The majority of these
SNPs that affect BMD and fractures have been shown in many studies
with different p-values and differences between different races. The
most important and widespread SNPs have been identified as involved
in osteoblastogenesis, osteoclastogenesis, osteoblast Wnt signaling,
vitamin D, Estrogen, Collagen, Mevalonate as LRP5 genes. , LRP6,
DKK1, VDR, ColA1, ESR1, ESR2…
In recent years with the rapid development of science, the advent

of GWAS (genome-wide association studies) has discovered many new
SNPs related to osteoporosis and fracture. skeletal. Urano et al
conducted a meta-analysis of eight-year GWAS studies (2007-2015)
that discovered many novel SNPs associated with osteoporosis and
fractures in Caucasian and Asian populations. Among these SNPs
involved in bone biological processes are known: WNT/β-catenin
signaling pathway: catenin β1 (CTNNB1), sclerostin (SOST), LRP4,
LRP5, GPR177, WNT4, WNT5B, WNT16, dickkopf1 (DKK1),


5
transmembrane helix protein 4 (sFRP4) fusion gene, Jagged 1 (JAG1),
MEF2C and AXIN1. GWAS studies for osteoporosis have also
identified three important factors, namely RANK, RANKL and OPG
that influence the differentiation of mesenchymal stem cells into
osteoblasts. However, the authors also found several loci of genes that
have not been found to play a role in bone biomechanics (FAM210A,
SLC25A13...). Research by author Kemp et al in 2013 conducted on
14,492 Britons of both sexes found 307 SNPs on 203 loci that affect the
RA at a significant level, of which 153 new loci were discovered
compared with that of 203 loci. In previous publications, the authors
also showed that 12 SNPs were significantly associated with fracture55.
In 2021, the author Zhu X and his colleagues 7 conducted a metaanalysis of 512 GWAS studies around the world within 12 years and
found 518 loci in the human genome affecting bone loss and fracture.
Of these, only 20% of these loci can explain their relationship to bone
biomechanics. Osteoporosis is a complex multifactorial disease, the
influence of genes on osteoporosis, osteoporosis and fractures has been
confirmed. However, the actual mechanism of dynamic separation in
bone remains unclear. Therefore, more studies are still needed on
subjects of different races, sexes, and ages to elucidate the role of genes

in osteoporosis and fractures.
1.2.2. In Vietnam
In Vietnam in 2015, author Ho Pham Thuc Lan and his colleagues
from Ton Duc Thang University conducted a study to determine the
influence of gene polymorphisms on MS in Vietnamese. The study was
conducted on 564 healthy people aged 18 years and older with an
average age of 47 (180 men and 384 women) living in Ho Chi Minh
City, individuals were measured BM by the DXA method at the spinal
position. lumbar, femoral neck and upper femoral head. The study
subjects identified 32 gene polymorphisms (of 29 genes: LRP5, SP7,
MBL2/DKK1, ESR1, DHCR7, MEF2C, SOST, VDR, RANK,


6
RANKL, SCL25A13, MEPE...), these gene polymorphisms were
identified. was found to be associated with MDD in Caucasians and
some Asian countries. The study results showed that 3 genes SP7
rs2016266, ZBTB40 rs7543680 and MBL2/DKK1 rs1373004
significantly reduced BMD after controlling for age, gender and weight
factors. These three gene polymorphisms affect from 0.2% to 1.1% of
BMD, especially the SP7 rs2016266 gene polymorphism has the
highest correlation, the G allele changes 0.02 g/cm2 of BMD in the
femoral neck and whole body. In, the author Ho Pham Thuc Lan
studied 2 polymorphisms rs3736228 and rs599083 of the LRP5 gene
and found no significant association of these 2 gene polymorphisms
with MDR at all positions.
Stemming from the above fact, we conducted a study on the
polymorphism of genes related to osteoporosis in men within the
framework of the study on the polymorphism of some genes related to
osteoporosis and fractures. vertebrae. The study was conducted on two

groups of subjects: postmenopausal women and men. Which, we
conducted this to determine the association between 3 gene
polymorphisms LRP5 Q89R, MTHFRC677T, and FTORs1121980 with
osteoporosis and vertebral fractures in postmenopausal women and
men. These three gene polymorphisms are correlated with MS and
fracture in specific studies around the world:


7
CHAPTER 2
OBJECTS AND RESEARCH METHODS
2.1. Location and duration
- Location: patients at Friendship Hospital and Analysis of 3 gene
polymorphisms at the Department of Pathophysiology, Hanoi Medical
University.
- Duration: from March 2016 to December 2021
2.2. Study subjects
The study was conducted on 400 men, aged 50 years and older 200
with osteoporosis and 200 control group
2.2.1 Osteoporosis group
* Inclusion criteria
- Male with age ≥ 50.
- The patient has no history of chronic diseases that cause
secondary osteoporosis (such as liver disease, chronic obstructive
pulmonary disease (COPD), autoimmune disease (rheumatoid arthritis,
lupus, psoriasis, ...) and kidney disease. chronic, cancer, endocrine
diseases and disorders related to vitamin D metabolism, bone
metabolism such as diabetes, obesity, malabsorption syndrome,
hyperthyroidism, Cushing's syndrome, Cushing's disease ). Patients not
taking drugs that cause osteoporosis (corticosteroids, hormone

replacement, heparin): based on medical records and clinical
examination, if symptoms are suspicious, they will be excluded from
the study.
- Osteoporosis is defined by a T-score ≤ -2.5 in at least 1 of 3
positions of femoral neck, total hip and lumbar spine. BMD at the
lumbar spine, neck, total hip was measured by dual energy X-ray
absorptiometry (DEXA)
* Exclusion criteria: Patient has been immobilized for 1 month or
more; did not agree to participate in the study


8
2.2.2 Control group: Select patients into the control group according to
the same criteria as the patient group, but have normal BM with T index
≥ -1 in all 3 positions of the lumbar spine, femoral neck and total hip.
2.3. Methods
2.3.1. Study Design: A case-control study, paired by age group.
2.3.2. Study sample size
* Study sample size: Sample size in the interaction model between
genes and environment, the sample size was calculated using QUANTO
software for paired case-control studies
(). Based on parameters
estimated from previous studies in Vietnam and Asian ethnic groups:
- The rate of osteoporosis is 10% after the age of 50.
- Number of SNPs included in the survey = 3
- Type I error (α): 0.05 with the two-sided test hypothesis adjusted;
sample force is 0.80.
- The ratio of alleles of interest (minor alleles) is 0.20-0.40; with
the log additive inheritance mode.
- Ratio of objects with interactive environmental factors: 0.25-0.4.

- The main effect of genetics: 1.25. Main effect of environment:
1.25. The influence of gene-environment interaction: 4.0-6.0.
- The ratio of disease control = 1:1.
The calculated sample size for each group was 179. During the
study, We selected 200 patients with osteoporosis and 200 subjects
without osteoporosis as a control with the above inclusion and
exclusion criteria.
* Sampling method: convenient sample selection, subjects who
come to the clinic will be screened, bone density measured and blood
drawn for testing, meeting the criteria of the disease group or the
control group will have blood drawn and sent to the University of
Medicine. Hanoi for genotyping.


9
2.3.3. Research variables and indicators
- Variables in general information of the study subjects: age,
occupation, geography, education level were interviewed through a set
of questionnaires.
- Clinical epidemiological variables and indicators: height, weight,
BMI calculation, smoking, alcohol consumption, medical history,
history of fracture, level of physical activity.
- Bone mineral density (BMD): measured BMD by double-energy
X-ray absorptiometry (DXA) at the lumbar spine and femoral neck, the
total hip.
- Gene polymorphisms (SNPs): determining single polymorphisms
of 3 genes LRP5 Q89R, MTHFR C677T and FTO rs1121980 of 400
research subjects by 2 methods:
LRP5 Q89R (rs41494349) and FTO rs1121980: determined by
RFLP-PCR (Restriction Fragment Length PolymorphismsPolymerase Chain Reaction)

MTHFR C677T determined by ARMS-PCR
Genotyping results were verified for accuracy by gene sequencing.
2.3.4. Methods of data analysis
Stata 14.0 analysis software was used for statistical analysis.
Categorize quantitative and subgroup variables. Quantitative variables
were tested for normal distribution using the Kolmologov-Smirnov test.
The frequencies of alleles were tested for distribution according to the
Hardy - Weinberg equilibrium using the squared (χ2) test or the Fisherexact test. Compare the mean values of the variables according to the
normal distribution by Student T-test and ANOVA test. The mean
values of non-normally distributed variables were compared using the
Kruskall-Wallis test. Use OR (Odd ratio) to determine osteoporosis risk
of gene polymorphisms and risk factors, use correlation coefficient r to
find the correlation between risk factors and BMD. Analysis of the
association of genotype and other factors (age, weight, height, BMI and


10
smoking, alcohol consumption) with osteoporosis by multivariable
logistic regression.
2.5. Research ethics
- The study was approved by the ethics committee of Dinh Tien
Hoang Institute of Medicine.
- The subjects participating in the study were provided with full
information about the purpose of the study, the conducting process and
had the right to withdraw from the study when they did not want to
participate.
- Patient-related information is kept confidential, each patient is
encrypted with a unique code.
- The technique of manipulation on the patient is guaranteed to be
professional. - This research topic is conducted purely for scientific

purposes and not for any other purpose.
CHAPTER 3: RESULTS
3.1. Demographics of all participants
The study involved 400 individuals (200 men with osteoporosis
and 200 normal control men) with the criteria for matching by age
group with the following characteristics:
*Average age of the osteoporosis group was 74.96±6.73; there was
no significant difference in the average age between the two study
groups, in which the oldest is 91 years old and the lowest is 59 years
old. The age group with the highest percentage is 70-79 (52%); next is
the age group over 80 (26%) the lowest is the age group under 70
(22%); The two groups of patients and controls had similar age groups
because we took the data according to the matching criteria by age
group.
*The average height of the disease group (159.02±5.55) was lower
than that of the control group (161.7±5.61) statistically significantly
(p<0.001); the average weight of the patient group (55.64 ±5.55) was


11
lower than that of the control group (62.72±7.92) (p<0.001) ; similarly,
BMI of the patient group (21.98±2.85) was also significantly lower than
that of the control group (23.96±2.52) with p<0.001. The patient group
had a higher history of fracture than the control group, the level of
physical activity was lower than that of the control group, this
difference was statistically significant (p<0.001). Most of the study
subjects lived in cities, had similar educational attainment, smoking
history, and alcohol consumption history in the two groups of patients
and the control group (p>0.05). In the group of diseases, osteoporosis at
the lumbar spine accounted for the highest rate (56.7%), in which

osteoporosis at 1 position accounted for the highest rate (51%).
3.2. Characterization of LRP5 Q89R, MTHFRC677T and FTOrs1121980
gene polymorphisms
3.2.1. Analysis results of gene polymorphisms
Table 3.1. Distribution of genotypes LRP5Q89R (rs41494349)

(n=200),%

Control
(n=200),
%

(n=400), %

A

370 (92,5)

388 (97)

758 (94,75)

G

30 (7,5)

12 (3)

42 (5,25)


AA

172 (86,0)

188 (94,0)

360 (90,0)

AG+GG

28 (14%)

12 (6,0%)

38 (9,5%)

0,3725

0,6618

Alen/

Case

genotype
Alen
Genot
ype
p HDW


Sum
p

0,004

0,008

The distribution of alleles and genotypes follows Hardy Weinberg's
law. There was a difference in the rate of allele and genotype of LRP5
Q89R in the group of patients and controls with statistical significance
with p<0.05.


12
Table 3.2. Distribution of genotypes MTHFR C677T
Case

Control

n =200 (%)

n=200 (%)

Sum n
=400 (%)

C

303(75,75)


331 (82,75)

634 (79,25)

T

97 (24,25)

69 (17,25)

166 (20,75)

CC

115 (57,5)

135 (67,5)

250 (62,5)

CT

73 (36,5)

61 (30,5)

134 (33,5)

TT


12 (6)

4 (2)

16 (4)

0,927

0,338

Alen/genotype

Alen

Genotype

p theo HDW

p
0,015

0,036

The distribution of alleles and genotypes follows Hardy Weinberg's
law. There was a statistically significant difference between the allele
and genotype ratios of MTHFR C677T in the two groups of patients
and controls (p<0.05).
Table 3.3. Distribution of genotypes FTO rs1121980
Case
(n=200),%


Control
(n=200),%

Sum
(n=400), %

p

G

310 (77,5)

338 (84,5)

648 (81)

0,012

A

90 (22,5)

62 (15,5)

152 (19)

GG

110 (27,5)


138 (34,5)

248 (62)

GA

90 (22,5)

62 (15,5)

152 (38)

<0,001

0,0095

Alen/ Genotyp
Alen
Genotyp

p theo HDW

0,004

Only the appearance of 2 genotypes GG and GA without the appearance
of genotype AA in the study group. Therefore, the distribution of alleles
and genotypes does not follow Hardy Weinberg's law.
There was a statistically significant difference between the allele and
genotype ratios of FTO rs1121980 between the two groups of diseases

and controls (p<0.05).


13
3.3. The relationship of 3 polymorphisms on studied genes with
male osteoporosis
3.3.1. The association of SNP rs41494349 (Q89R) of the LRP5 gene
with male osteoporosis
Table 3.4. The relevance of LRP5Q89R rs41494349 to male
osteoporosis in genetic patterns
OR (95% CI)

p

AIC

AG +GG >AA

2,62 (1,2 – 5,73)

0,016

1.448211

AG>AA

2,43 (1,12 – 5,29)

0,025


1.45127

AG> AA + GG

2,38 (1.10- 5.17)

0.028

1.45199

Addition of the G
allele

2,67 (1,24 – 5,73)

0,012

1.448695

In the dominant model, genotypes AG and GG increased the
risk of osteoporosis by 2.62 times (95% CI: 1.2-5.73) times compared
with genotype AA (p= 0.016) and had a high value. The lowest AIC
value in the dominant model (AIC = 1.448211) proves that this is the
optimal model for analyzing the influence of SNP Q89R.
Table 3.5. The relevance of LRP5 Q89R gene polymorphism
rs41494349 to male osteoporosis by location
Location

LRP5 Q89R


OR

(95%CI)

p

Femoral
neck

AG+GG>AA

1,94

0,90-4,19

0,090

G>A

2,11

1,03-4,34

0,042

Lumbar
spine

AG+GG>AA


2,02

1,01- 4,06

0,047

G>A

1,92

0,99 – 3,7

0,052

AG+GG>AA

2,32

0,46 -11,61

0,306

G>A

2,18

0,47 -10,10

0,317


Total hip

Genotype AG+GG and allele G increased the risk of osteoporosis in
lumbar spine by 2.02 times and 2.11 times compared with carriers of
AA genotype and A allele, respectively (p<0.05)


14
3.3.2. The relevance of the C677T polymorphism of the MTHFR gene
to osteoporosis in men
Table 3.6. The relevance of the MTHFR C677T gene polymorphism
to male osteoporosis
OR (95% CI)

p

AIC

CT +TT> CC

1,54 (1,02 - 2,31)

0,040

1,455858

CT >CC

1,41 (0,92 - 2,14)


0,116

1,460209

TT>CC

3,47 (0,91 - 13,22)

0,068

1,456957

CT>CC+TT

1,31 (0,86 -1,99)

0,206

1,46029

TT>CC+CT

3,14 (0,84 - 11,76)

0,090

1,45607

Cộng gộp alen T


2,67 (1,24 – 5,73)

0,012

1.448695

Only the dominant pattern is statistically significant, in which the
CT+TT genotype increases the risk of osteoporosis by 1.5 times
compared with the CC genotype.
Table 3.7. The relation of the C677T polymorphism of the MTHFR
gene to osteoporosis in men by location
Location
MTHFR C677T
OR
(95%CI)
p
Femoral
neck

TT+CT>CC

2,04

1,23 – 3,38

0,006

T>C

1,71


1,34-2,57

0,01

Lumbar
spine

TT+CT>CC

1,23

0,81 – 1,87

0,34

T>C

1,19

0,83-1,70

0,35

TT+CT>CC

3,52

0,99-12,48


0,05

T>C

2,66

1,07 – 6,61

0,035

Total hip

The TT+CT genotype increased the risk of osteoporosis by 2.04 and
3.52 times compared with the CC genotype at the femoral neck and
supraclavicular position, respectively (p<0.05).


15
3.3.3. The relevance of FTO rs1121980 gene polymorphism to
osteoporosis in men
Table 3.8 The relevance of FTO rs1121980 gene polymorphism to
osteoporosis in men
FTOrs1121980
OR
95% CI
p
Common
location

GA>GG


1,83

1,2-2,77

0,001

A>G

0,467

0,11 – 2,05

0,32

Femoral
neck

GA>GG

1,43

0,86 -2,37

0,172

A>G

1,31


0,84-2,04

0,229

Lumbar
spine

GA>GG

1,99

1,31 - 3,06

0,001

A>G

1,68

1,17 – 2,43

0,005

GA>GG

0,41

0,09-1,94

0,26


A>G

0,467

0,11 – 2,05

0,32

Total hip

Allele A increases the risk of osteoporosis in the lumbar spine by 1.68
times; The GA genotype increased the risk of osteoporosis 1.99 times
more than the GG genotype (p<0.005).
3.3.4. The relevance of genomic polymorphisms of the 3 genes LRP5
Q89R, MTHFR C677T and FTO rs1121980 to osteoporosis in men
Table 3.9. Combination of genotypes in 3 genes associated with
osteoporosis in men
Number
Case
Control
OR (95%CI)
p
of risk
(n,%)
(n,%)
alen
46 (34,85) 86 (65,15)
1
No

1 alen

98 (53,26)

86 (46,74)

2,17 (1,36- 3,44)

0,001

≥ 2 alen

56 (66,67)

28 (33,33)

3,82 (2,13 -6,83)

0,000

Mean alen 2,79 ± 0,90 2,6 ± 0,72 1,33 (1,04 – 1,7) 0,021
In the genotype combination, every occurrence of 1 risk allele increased
the risk of osteoporosis more than the group without any risk allele
(p<0.001).


16
3.4. The relevance of genes and some risk factors for osteoporosis in men
Table 3.10. The relevance of 3-gene polymorphism and risk factors to
osteoporosis in multivariable logistic regression

Risk factors
OR
95% CI
P
LRP5 Q89R (AG+GG>AA)
3,45
1,54 – 7,70
0,002
MTHFR C677T (CT +TT> CC)
1,65
1,02 – 2,67
0,039
FTO rs1121980 (GA>GG)
2,17
1,35 – 3,52
0,001
Underweight
4,08
1,30 – 12,79
0,016
Overweight
0,4
0,23 – 0,68
BMI
0,001
Obesity
0,22
0,12 – 0,41
0,000
Smoking (yes>no)

1,02
0,64 – 1,63
0,94
Alcolhol use (yes > no)
0,84
0,50 – 1,40
0,504
History fracture (yes > no)
3,84
1,74 – 8,46
0,001
Inactive
27,81 5,16 – 150,02 0,000
Physical
Low
6,94
2,34 – 20,53
0,000
activity
Moderate
5,3
1,89 – 15,30 0,002
3 gene polymorphisms LRP5Q89R, MTHFR C677T and
FTORs1121980 increase the risk of osteoporosis with dominant
inheritance pattern
Table 3.11. The relevance of 3-gene polymorphism and risk factors to
osteoporosis at femoral neck in multivariable logistic regression
Risk factors
OR
95% CI

P
LRP5 Q89R (AG+GG>AA)
2,59
1,09 – 6,22
0,032
MTHFR C677T (CT +TT> CC)
2,50
1,38 – 4,50
0,002
FTO rs1121980 (GA>GG)
1,82
1,0 – 3,3
0,05
Underweight
7,27
1,30 – 12,79
0,002
Overweight
0,59
0,30 – 1,14
0,119
BMI
Obesity
0,20
0,09 – 0,48
0,000
Smoking (yes>no)
0,61 – 1,95
0,782
Alcolhol use (yes > no)

0,46– 1,69
0,698
History fracture (yes > no)
0,52 – 3,1
0,60
Inactive
48,45
6,24 – 356,64
0,000
Physical
Low
6,07
1,36 – 25,92
0,018
activity
Moderate
4,59
1,06 – 18,93
0,041
Three gene polymorphisms LRP5Q89R, MTHFR C677T and
FTORs1121980 increased the risk of hip fracture (p<0.05).


17
Table 3.12. The relevance of between 3-gene polymorphism and risk
factors to osteoporosis at total hip in multivariable logistic regression
Risk factors
OR
95% CI
p

LRP5 Q89R (AG+GG>AA)
2,37
0,26 – 21,60
0,44
MTHFR C677T (CT +TT> CC)
2,46
0,50 – 12,10
0,27
FTO rs1121980 (GA>GG)
0,78
0,12 – 5,10
0,80
Underweight
29,24 3,07 – 278,31 0,003
Overweight
0,81
0,11 – 5,7
0,828
BMI
Obesity
Smoking (yes>no)
0,13 – 3,69
0,68
Alcolhol use (yes > no)
0,20 – 7,87
0,81
History fracture (yes > no)
1,41 – 63,9
0,02
Inactive

Physical
Low
0,08
0,005 – 1,34 0,079
activity
Moderate
0,05
1,06 – 18,93
0,02
No statistically significant relevance was found between 3 gene
polymorphisms LRP5 Q89R, MTHFR C677T, FTO rs1121980 with
total hip osteoporosis.
Table 3.13. The relevance of 3-gene polymorphism and risk factors to
osteoporosis at lumbar spine in multivariable logistic regression
Risk factors
OR
95% CI
p
LRP5 Q89R (AG+GG>AA)
2,72
1,24 – 5,97
0,013
MTHFR C677T (CT +TT> CC)
1,24
0,76 – 2,01
0,38
FTO rs1121980 (GA>GG)
2,37
1,45 – 3,86
0,001

Underweight
3,9
1,33 – 11,41 0,013
Overweight
0,4
0,23 – 0,70
BMI
0,001
Obesity
0,24
0.13 – 0,43
0,000
Smoking (yes>no)
0,67 – 1,72
0,783
Alcolhol use (yes > no)
0,50 – 1,46
0,537
History fracture (yes > no)
1,46 – 6,48
0,003
Inactive
21,59
4,14 – 112,46 0,000
Physical
Low
6,25
2,04– 19,14
0,001
activity

Moderate
4,9
1,66 – 14,37 0,004
LRP5 Q89R gene polymorphism, FTO rs1121980 gene polymorphism
significantly increases the risk of lumbar spine osteoporosis.


18
CHAPTER 4: DISCUSSION
4.1. Genotype frequencies
We analysed the genotypes for the SNP of LRP5 at Q89R, MTHFR
C677T and FTO rs1121980 of 400 older men (200 with osteoporosis
and 200 normal control men). The genotype distributions and allele
frequencies for three SNP gene polymorphisms are presented below
4.1.1. Q89R (rs41494349) polymorphism in the LRP5 gene
*Genotype frequencies: The haplotype frequencies in our study
were AA/AG/GG respectively 90%/9.5%/0.5%. This result was similar
to the study of the authors on Chinese subjects: Lau, H et al conducted
on 674 people (107 men and 567 women) giving the genotype ratio
AA/AG/GG respectively were: 84.8%/13.4%/0.5%; Similar to the study
of author Zhang et al conducted on 647 postmenopausal women in
China AA (80.5%), AG (18.7%) and GG (0.8%). These data differ from
those obtained from Korean subjects. The haplotype frequencies in Koh
(219 healthy young Korean men) were AA/AG/GG: 83.1%/16.9%/0%,
respectively. in which no found appearance of homozygous genotype
GG was in this study. The distribution of genotype in our study was
different between two groups.
*Allele frequencies: Similar to the distribution of genotype, the
distribution of alleles A and G in the two groups was also different. The
frequency of occurrence of the G allele in the disease group (7.5%) was

higher than that in the control group (3%), the difference was
statistically significant with p<0.005. The frequency of occurrence of
the G allele in 400 studied subjects was 5.25% (table 3.6). Allele
frequencies in our study are similar to the results of other Asian
countries such as Japan (8.7%), Korea (8.0%), China (7.9%). %),
Thailand (6%). Thus, the occurrence of LRP5 Q89R gene
polymorphism in our study is also consistent with studies of yellowskinned subjects.


19
4.1.2. MTHFR gene polymorphism C677T
*Genotype frequencies: The haplotype frequencies in our study
were AA/AG/GG respectively 90%/9.5%/0.5%
*About the genotypic ratio: the genotypic ratio in 400 research
subjects was: CC/CT/TT respectively 62.5%/33.5%/4%. The results of
our study show that there is a difference in the prevalence of genotypes
of the MTHFR C677T gene polymorphism in the two groups. In which,
the occurrence of CT and TT genotypes was more in the disease group
than in the control group, especially the rate of TT homozygous
genotype was 3 times higher in the disease group (6%) than in the
control group (2). %), this difference is statistically significant with
p<0.05 (table 3.10); The results of our study on men are similar to the
results of Tran Thi Thu Huyen, the distribution of genotypes is
CC/CT/TT, respectively: 71.4%/26.8%/1, 8%. The results of our study
on TT genotype distribution are similar to the genotype distribution of
some countries such as Finland (4%), Canada (5.8%), Netherlands (6%)
Russia (7%). ), lower than studies of the United States (11%), France
(12%), Hungary (15%), and much lower than studies on Mexican
populations (32.2%), Italy (26.4%)
*About the rate of T allele: according to our results, the frequency of

occurrence of the T allele was higher in the disease group (24.25%)
than in the control group (17.25%), the difference was statistically
significant. with p<0.05 (table 3.10); the frequency of the T allele in
our study is 20.75%. The results of our study have the same frequency
of the T allele as the study of the author in the country: the study of the
author Tran Thi Thu Huyen et al on 566 postmenopausal women in the
North of Vietnam showed the frequency of the T allele was 15.19%,
which is 7% higher than the study on blacks in West Africa, Indonesia
(8.1%) and lower than the study in Japan (33%) and China (36). %).
4.1.3. FTO genotype rs1121980 polymorphism
*Genotype and allele ratio of FTORs1121980 . gene polymorphism


20
The results of our study show that there are only 2 genotypes GG
and GA, no homozygous genotype AA, so the ratio of alleles and
genotypes does not follow Hardy Weinberg's equilibrium law (table).
3.10). The results of allele ratio showed that the G allele was dominant
in 77.5% of the disease group and 84.5% in the control group. The
common ratio of the 2 groups is allele G (81%), allele A (19%).
Similarly, genotype GG accounted for a high proportion in both disease
groups (55%) and control group (69.0%). The frequency of occurrence
of allele A in the disease group was 22.5% higher than the control
group at 15.5%, this difference was statistically significant with
p=0.012; similarly the frequency of GA genotype was also higher in the
disease group (22.5%) compared to the control group (16.5%); The
difference was statistically significant with p<0.005. The results of our
genotyping study show that there is a difference with the author Bich
Tran's study on white people with the presence of all 3 GG genotypes
(33.3%); GA (47.7%) and homozygous recessive AA (19.0%). Studies

around the world show that the rate of homozygous AA genotypes of
white people (21-27.5%), black people (23.6-26.9%) is much higher
than that of Asians: India (8.9%), Japan (3.5%), China (4.4 – 4.6%).
4.2. The relationship between gene polymorphisms and risk factors
for osteoporosis in men
4.2.1. Association between LRP5Q89R, MTHFRC677T and
FTORs1121980 gene polymorphisms with osteoporosis in men
* LRP5Q89R gene polymorphism: The results of our study indicate
that the presence of the G allele of the LRP5Q89R gene polymorphism
increases the risk of osteoporosis in men. People with genotype AG+GG
have a 2.62-fold increased risk of osteoporosis (95% CI: 1.2–5.73)
compared with AA genotype carriers. When testing separately for each
location, it also shows that the AG+GG genotype increases the risk of
osteoporosis at all 3 locations, of which there is statistical significance in
the lumbar spine position (OR: 2.02; 95). %CI: 1.01 - 4.06). Carriers of


21
the G allele increase the risk of osteoporosis by 2.67 times (95% CI: 1.24
- 5.73) compared with carriers of the A allele; Similarly, the occurrence
of the G allele also increased the risk of osteoporosis at all sites, but only
statistically significant with the position of the femoral neck (OR: 2.11;
95% CI: 1, 03-4.34). The results of our study are similar to some previous
studies on both male and female subjects. Koh's study in Korean men
showed that this gene polymorphism reduced the BMD and similar to
that published by Zang et al on 647 Chinese postmenopausal women
subjects, there was an association between genotypes AG and GG with
MDX at the femoral neck. The results of our study are different from the
results of the study of the author Kitjaroentham et al on Thai women who
found no association between this gene polymorphism and osteoporosis.

The studies conducted on this gene polymorphism are not numerous,
especially in male subjects. Therefore, our results are the first to show the
correlation of this gene polymorphism in Vietnamese men.
* MTHFR C677T gene polymorphism: Our study results showed
that CT + TT genotype carriers increased the risk of osteoporosis by
1.54 times (95% CI: 1.02-2.31) compared to CC genotype carriers,
when tested for each location, also showed that CT + TT genotype
carriers increased the risk of osteoporosis at the femoral neck position
by 2.04 (95% CI: 1.28-3). ,38) times compared with CC genotype
carriers, the results are similar to osteoporosis in the lumbar spine and
the upper femoral head (table 3.20). However, the difference was not
statistically significant at this location. Carriers of the T allele increased
the risk of osteoporosis by 1.54 times (95% CI 1.08-2.18) compared
with carriers of the C allele. This difference was also clearly seen in the
position of the vertebral column and the head of the bone. femoral: the
presence of the T allele increased the risk of osteoporosis in the femoral
neck and upper femoral head by 1.71 (95% CI: 1.14-2.57) and 2.66
(95% CI: 1). ,07-6.61) respectively, the difference is statistically
significant with p<0.05 (table 3.20). When testing in a multivariable
linear regression model, the same results are obtained. Thus, the effect
of the MTHFRC677T gene polymorphism is independent of other risk


22
factors. The results of our study are similar to some studies in the world
that show that carriers of the T allele, genotype TT and CT reduce MS
in men: Abrahamsen Bo et al. (2006) studied 780 healthy men. Danes
aged 20-29 years showed that TT and CT genotype carriers had 3.9%
and 2.1% lower BMD compared with CC genotypes, respectively.
Author Li et al (2016) conducted a meta-analysis study on the impact of

this gene polymorphism on both men and women, showing that the
MTHFR C677T gene polymorphism is associated with reduced BM in
the lumbar spine and femoral neck. in Caucasians, postmenopausal
women, men, and systemic MS in women.
*FTO rs1121980 gene polymorphism: The results of our study
showed that the occurrence of allele A and genotype GA in the disease
group was significantly higher than that of the control group with p <
0.05. When testing the association between FTORs1121980 gene
polymorphism with osteoporosis by OR index, we found that men
carrying the GA genotype increased the risk of lumbar spine
osteoporosis 1.83 times compared with those carrying the GA genotype.
genotype GG with p=0.001 (95%CI 1.31-3.06; p=0.001). When testing
separately for each site, the GA polymorphism of the FTOrs1121980
gene polymorphism increased the risk of osteoporosis at the femoral
neck and lumbar spine. However, there was only statistical significance
in lumbar spine position with OR=1.99 (95%CI: 1.31-3.06; p=0.001).
This result is similar to the multivariable linear regression test model
with other risk factors. The results of our study are similar to those of
Yan Guo et al., showing that there is an association between FTO and
MDX gene polymorphisms. In 2013, Bich Tran et al. found that
homozygous TT genotype of SNP rs1121980 had a 2.06 times higher
risk of fracture than women with CC homozygous genotype (OR =
2.06; 95). %CI: 1.17 – 3.62; p=0.02). The results of this study support
the hypothesis that the FTO 1121980 gene polymorphism affects BMD
and this fracture is similar to our study.