Charakterystyka polimorfizmu genu LEPR Q223R (RS1137101) u pacjentów z chorobą zwyrodnieniową stawu kolanowego w różnych stadiach zaawansowania zmian radiologicznych

Valerii O. Novoseletskyi, Mykola A. Stanislavchuk, Volodymyr M. Shkarupa, Liliia V. Shvets

National Pirogov Memorial Medical University, Vinnytsya, Ukraine

Abstract

Introduction: Osteoarthritis is a multifactorial joint disease with a significant role of the genetic factor. The numerous studies have demonstrated that genetic dependence is specific for individual hand, hip and knee regions with a genetic contribution to the pathogenesis of osteoarthrosis varying from 40% to 65%. To assess the role of leptin gene receptor functional activity disturbance in the pathogenesis of osteoarthrosis, it is important to study the relationship between the LEPR gene polymorphism and a number of clinical and laboratory parameters.

The study objective was to determine the relationship between the LEPR gene Q223R (rs1137101) polymorphism and the radiographic stage of osteoarthrosis of the knee in female patients of the Ukrainian population.

Materials and methods: The rs1137101 polymorphism was genotyped in 99 female patients diagnosed with osteoarthrosis of the knee using polymerase chain reaction in a real-time mode.

Results: It was a tendency of lower prevalence of AA homozygotes and higher prevalence of AG heterozygotes with growing the severity of the disease. The high prevalence of homozygous carriers of the variant allele G in radiographic Stage I patients preconditioned the absence of statistically significant differences in the distribution of genotypes between the groups.

Conclusion: No statistically significant differences in the distribution of prevalence of alleles and LEPR gene Q223R (rs1137101) genotypes in the groups of patients with the knee OA of different radiographic stages have been revealed.

Wiad Lek 2018, 71, 1 cz. II, -192

Introduction

Osteoarthritis (OA) is one of the most common diseases of the joints, the incidence of which increases significantly with age. Chronic progressive OA course leads to deformation of joints, loss of their function and deterioration in the quality of life of patients [1, 2, 3]. There are local and systemic OA risk factors. Among the latter, the most important today are obesity, age, gender, and genetic factors [4]. The numerous studies of family and twins history have demonstrated that genetic dependence is specific for individual areas of hands, hips and knees. The genetic contribution to the pathogenesis of OA varies from 40% to 65%. Most of today’s researches suggest that genetic variants of several groups of genes, such as extracellular cartilage matrix structural genes and genes associated with bone metabolism are involved in the OA pathogenesis, including knee joints (COL2A1, COL1A1, COL9A1, MMPR- 1, MMP-3, MMP-9, and VDR1). Other candidate genes, which may be associated with the risk of knee osteoarthrosis, are GDF5, ASP, IL-1, IL-6, LRCH1, TNA, LRP5, and LEP [5, 6, 7]. It stands to mention that genetic factors involved in the development of OA also have a gender-specific effect, likely influenced by environmental and local biomechanical factors and distributed in different ways between male and female individuals with general prevalence in women rather than in men [7].

A characteristic feature of the OA pathogenesis is abnormal remodeling of articular tissues caused by unbalanced expression of catabolic and anabolic factors by chondrocytes, although all tissues of the joint are involved in the pathogenesis of the disease. Today, one of the most important links in the OA pathophysiology is considered an inflammation process (both local and systemic) controlled by proinflammatory factors produced by cartilage, subchondral bone, and synovial membrane. Subsequently, this pathological process forms a metabolic, and later a functional imbalance of all articular structures with the development of typical clinical picture and uniform pathobiochemical changes [4, 8, 9, 10]. In recent times, researchers have become considering an important role of adipose tissue in the development of OA [11, 12, 13, 14]. The relationship between obesity and OA development due to systemic inflammation has become evident. The adipose tissue synthesizes a variety of proinflammatory mediators and adipokines (including leptin) able to affect the components of articular cartilage [4, 15, 16]. The study of leptin role in development of OA has recently been gained a momentum. In addition to the main function of regulating the energy homeostasis, leptin plays a significant role in neuroendocrine interaction, angiogenesis, and bone formation. Leptin exerts its biological activity through connection with OB-Rb, the long isoform of the leptin receptor (encoded by the LEPR gene). Interaction with the LEPR gene product leads to activation of a number of intracellular signaling pathways: JAK-STAT, PI3K, NF-jB, PKC, MAPK, Erk1/2, and JNK [6]. Taking into account multiple mechanisms of action and diverse ways of regulating metabolic processes involving leptin, some researchers assign it one of the key roles in the OA pathogenesis [4, 16, 17, 18].

Mutations of LEPR gene result in formation of inactive forms of leptin receptors, not capable of providing transduction of the hormonal signal, thus inducing the development of resistance to leptin action. The association of mutations in the leptin gene (LEP) and its receptors (LEPR) with development of obesity, metabolic syndrome, hyperinsulinemia, and other endocrine disorders [19, 20, 21, 22] have been demonstrated. However, the studies of LEPR gene polymorphism connection with OA are quite rare. The association of LEPR gene Q223R (rs1137101) polymorphism with a risk of development of the knee OA [23, 24] was found in the Chinese and Ukrainian populations, and of hand OA – in Finnish women [25].

To understand the role of disturbance of functional activity of leptin gene receptors in the OA pathogenesis, it is important to study the relationship between the polymorphism of LEPR gene and a number of clinical and laboratory parameters.

The aim

The study objective was to investigate a relationship between the LEPR gene Q223R (rs1137101) polymorphism and a radiographic stage of the knee OA.

Materials and methods

We examined 99 women with knee OA, age (M ± SD) 57.60 ± 11.69, treated at Pyrohov Memorial Vinnytsia Regional Clinical Hospital. The diagnosis was established in accordance with Order of the Ministry of Health of Ukraine No. 676 dated October 12, 2006. Written informed consent was obtained from all subjects before they had started in the study in accordance with the provisions of the Helsinki Declaration; the study protocol had been agreed with the Ethics Commission of Vinnytsia M.I. Pyrohov Memorial National Medical University. The genotyping of LEPR Q223R (rs1137101) polymorphic variants was carried out using the method of real-time allelic-specific polymerase chain reaction (iCycler IQ5, BioRad, USA). The special features of genotype distribution were analyzed in the groups of patients with radiographic Stages I, II and III (no patients with the radiographic Stage IV disease were involved). For the binary analysis, patients with Stage II and III of the disease were combined into a single group to compare with the radiographic Stage I patients.

Differences in the distribution of genotypes in various groups (Tables 2×3 and 3×3) were evaluated using the Fisher’s exact test. The method of binary logistic regression was used for evaluation of multiplicative, dominant, recessive (criterion χ2) and additive (Cochran-Armitage test for trend) inheritance model. The degree of association expressiveness was determined by calculating the odds ratio (OR) and its confidence interval (CI).

Results and discussion

We have identified an increased risk of knee OA in the homozygous GG (Arg/Arg) carriers of LEPR gene (rs1137101) compared with the control group of healthy women [23]. The results of analysis of LEPR gene Q223R (rs1137101) genotype distribution in the groups of patients with different radiographic stages of knee OA are shown in Fig. 1

As can be seen in Fig. 1, there are some trends in the distribution of incidence of LEPR gene Q223R (rs1137101) genotypes in the groups of patients with different knee OA stages. In particular, there is a decrease in the proportion of AA (Gln/Gln) genotype carriers at higher stages of the disease – 40.9%, 28.6%, and 19% at Stages І, ІІ and ІІІ, respectively. However, such a decrease did not have a sufficient level of statistical significance (p = 0.31, Fisher’s exact test). At the same time, the incidence of heterozygous AG (Gln/Arg) carriers grew by 18.2%, 37.5% and 42.9% at Stages І, ІІ and ІІ, respectively, but such growth was not statistically reliable too. One of the reasons for absence of statistically significant differences in the above patterns might be a high incidence of homozygous carriers of the variant allele GG (Arg/Arg) among patients with radiographic Stage I disease – 40.9%, although, when compared with the II and III radiological Stages, this indicator had some gain – 33.9 % and 38.1%, respectively. These were the features that preconditioned the absence of statistically significant differences (p = 0.36) in the overall distribution of LEPR gene Q223R (rs1137101) genotypes in patients with different stages of knee OA (Table I).

Given the minimal and moderate difference between II and III radiographic stages, we have also analyzed allelic incidence and genotype distribution patterns in different models of inheritance between groups of patients with I and II + III stages of the disease. The results of the analysis are presented in Tables II – V.

No inheritance model presented with the statistically significant differences. As mentioned above, this was apparently preconditioned by a significant number of genotype GG (Arg/Arg) patients with Stage I OA (40.9%). It`s worthy to note that no patients with radiographic Stage IV disease were among patients, therefore it was impossible to analyze the connection of the studied polymorphism with severe knee OA cases.

Comparison of the regularities found in the study with the literature data is considerably complicated by the fact that only few studies of LEPR gene Q223R (rs1137101) polymorphism connection with the risk of development and clinical signs of OA have been conducted so far. In the work by Yang, J. et al., 2016 [24], the association of LEPR gene Q223R (rs1137101) polymorphous variations with the radiographic stage of knee OA in patients belonging to the Northwest Chinese Han population was revealed. According to the authors, the allele G (Arg) carriership had a moderate association with the risk of knee OA in a subgroup with minimal and moderate changes in radiographic indices (II + III Stages), compared with individuals with radiographic stage I (OR = 1.30, 95 % CI: 1.05-1.62; p = 0.013) of the disease. Having compared the results obtained in the study with the data provided in the above-mentioned work, it should be noted that, firstly, the lack of statistically significant differences in our work may be, to some extent, preconditioned by a significantly smaller number of study subjects (99 individuals), compared with the one from the work by Yang, J. et al ., (2016), which involved 1215 subjects with similar indices obtained: OR = 1.20 (allele G (Arg) carriership, see Table II). Although the main factor, most likely, should have been considered the high incidence of homozygous GG (Arg/Arg) carriers in radiographic Stage I patients. Therefore, it`s worthy to note that the above-mentioned work [24] did not presented with a connection between distribution of genotypes and allele incidence when comparing the results from the control group (Radiographic stage I) with the data of a subgroup of patients with severe changes (radiographic stage IV) [24]. Given the absence of radiograph stage IV patients among the patients examined by us, it is not possible to compare the patterns revealed in this work. We assume that the course of disturbances associated with LEPR gene Q223R (rs1137101) polymorphism has a chronic, prolonged nature; therefore, the complications in homozygous carriers related to radiographic indicators are manifested in more distant periods of the disease.

S. Hämäläinen et al., 2017 found a weak association of AC LEPR (rs1137101, rs1805094) (OR = 1.54, 95% CI: 1.01-2.35, p = 0.05) haplotype with radiographic indices in women of Finnish population with OA of hand joints. However, it should be noted that the criteria used by the authors for distribution of patient into subgroups differed from those used in our study [26].

Conslusions

We did not find any statistically significant differences in the distribution of alleles and LEPR gene Q223R (rs1137101) genotype in knee OA patients at different radiographic stages. We consider promising further research of the relationship between the LEPR gene Q223R (rs1137101) polymorphism and such clinical and laboratory parameters of knee OA patients as a body mass index and leptin concentration.

References

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State registration number 0107U003479. It is a fragment of a research work “Clinical-laboratory and psychological predictors of severity of the clinical course and functional failure in patients with systemic connective tissue diseases and fibromyalgia”

Address for correspondence

Valerii Novoseletskyi

National Pirogov Memorial Medical University

56 Pirogov St., Vinnytsia, 21000

tel.: +38097-657-80-16

e-mail: valerii.novoseletskyi@gmail.com

Received: 20.11.2017

Accepted: 05.02.2018

Table І. Distribution of LEPR gene Q223R (rs1137101) genotypes in groups of patients with different radiographic stages of knee OA.

Genotype

Radiographic stage, n (%)

P*

І

ІІ

ІІІ

0.36

AA

9 (40.91)

16 (28.57)

4 (19.05)

AG

4 (18.18)

21 (37.5)

9 (42.86)

GG

9 (40.91)

19 (33.93)

8 (38.09)

* Fisher`s test 3х3

Figure 1. Genotype incidence by polymorphism of LEPR gene Q223R (rs1137101) in groups of patients with different radiographic stages (RS) of knee OA.

Table ІІ. Multiplicative model of LEPR gene rs1137101 inheritance in patients with different radiographic stages of knee OA

Allele

(incidence)

ІІ+ІІІ RS

І RS

χ2

Р

OR

n = 77

n = 22

value

95% CI

A

0.455

0.500

0.28

0.59

0.83

0.43 – 1.63

G

0.545

0.500

1.20

0.61 – 2.35

Table ІІІ. Additive model of LEPR gene rs1137101 inheritance in patients with different radiographic stages of knee OA

Genotypes

(incidence)

ІІ+ІІІ RS

І RS

χ2

Р

OR

n = 77

n = 22

value

95% CI

AA

0.260

0.409

0.22

0.64

0.51

0.19 – 1.37

AG

0.390

0.182

2.87

0.89 – 9.31

GG

0.351

0.409

0.78

0.30 – 2.06

Table ІV. Dominance model of LEPR gene rs1137101 inheritance in patients with different radiographic stages of knee OA

Genotypes

(incidence)

ІІ+ІІІ RS

І RS

χ2

р

OR

n = 77

n = 22

value

95% CI

AA

0.260

0.409

1.84

0.17

0.51

0.19 – 1.37

AG + GG

0.740

0.591

1.97

0.73 – 5.31

Table V. Recessive model of LEPR gene rs1137101 inheritance in patients with different radiographic knee OA stages

Genotypes

(incidence)

ІІ+ІІІ RS

І RS

χ2

р

OR

n = 77

n = 22

value

95% CI

AA + AG

0.649

0.591

0.25

0.62

1.28

0.49 – 3.38

GG

0.351

0.409

0.78

0.30 – 2.06