Background
Myasthenia gravis (MG) is an autoimmune neuromuscular disorder mediated by pathogenic autoantibodies. MG is characterized by fatigue and weakness of skeletal muscles [
1]. It is relatively rare, with a prevalence rate of 40 to 80 per million and an annual incidence rate of 4 to 12 per million [
2]. In about 80% of patients with MG, acetylcholine receptor (AChR) is the autoantigen, and antibodies against AChR can be detected [
3]. In AChR-negative patients, antibodies against other neuromuscular junction proteins, such as low-density lipoprotein receptor-related protein 4 (LRP4) and muscle specific kinase (MuSK), are usually observed [
4]. Although the pathogenesis in MG has been elucidated, the underlying mechanism of this disease remains unclear.
MG is a B-cell-mediated disease [
5]. B cells maintain normal adaptive immune responses by regulating T cell function, inflammatory cytokine production, and antibody formation [
6]. B-lymphocyte-activating factor (
BAFF), which belongs to the tumor necrosis factor family, is an essential factor for B cell differentiation and development [
7].
BAFF-stimulated B cell proliferation and maturation prolongs survival by binding to the
BAFF receptor in B cell membranes [
8]. In animal models,
BAFF decline can lead to B cell deficiency, whereas
BAFF overexpression facilitates B cell proliferation and elevates serum antibody levels [
9]. Evidence shows that dysregulation of
BAFF contributes to autoimmune disorders, including rheumatoid arthritis, systemic lupus erythematosus, and primary biliary cirrhosis [
10,
11]. Serum
BAFF level also are elevated in MG. Additionally,
BAFF positively regulated anti-AChR antibody in MG patients, which indicates
BAFF may play an important role in the pathogenesis of MG [
12,
13].
Although the precise genetic origin of MG is unclear, the polymorphisms of several candidate genes have been implicated [
14]. Specifically,
BAFF polymorphisms are associated with the phenotypes and occurrence of autoimmune diseases [
15]. The variant, single nucleotide polymorphism (SNP) rs2893321 of the
BAFF gene, has been reported to be a susceptible genetic variant for the development of Graves’ disease and autoimmune thyroid diseases [
9]. Thus, we hypothesized a connection between rs2893321 and the occurrence and clinical characters of MG. In the current study, we examined the polymorphisms of
BAFF rs2893321 in Chinese patients with MG and in healthy controls to determine its association with genetic susceptibility to MG.
Methods
Study population
From October 2015 to April 2017, we enrolled 149 patients with MG and 148 healthy controls from the Second Affiliated Hospital of Harbin Medical University. All participants were unrelated members of the Han Chinese population. Patients in the MG group met the standard MG diagnosis: fluctuating muscle weakness; positive neostigmine test; and an amplitude decrease of low-frequency, repetitive nerve stimulation exceeding 10%. Most patients with MG also were AChR antibody positive, as detected via radioimmunoprecipitation assay. And all patients were undergo CT exam to text for thymoma. The healthy controls had no diagnosed neurological or autoimmune diseases. Basic information (age and gender) of patients and controls were recorded. The ethical committees of the Second Affiliated Hospital of Harbin Medical University approved this study. All participants provided informed consent.
Sample collection
Peripheral venous blood samples were collected from all participants and stored in blood-collecting vessels contained ethylenediamine tetra-acetic acid (EDTA). Genomic DNA was isolated from each blood sample using a QIAamp Blood Midi Kit (Qiagen, Beijing, China). Concentrations of all DNA samples were measured by nucleic acid spectrophotometer. The A260/A280 of DNA samples from 1.8 to 2.0 were selected and stored at − 20 °C.
Single nucleotide polymorphism genotyping
Genotyping of BAFF rs2893321 was performed using improved multiple-ligase detection reaction (iMLDR) technology (Shanghai Genesky Biotechnologies Inc., Shanghai, China), which is based on multiplex fluorescence PCR. To ensure double-blinded quality control, we selected 4% samples from the MG and healthy control groups, respectively. The results were consistent with the original SNP genotyping data.
Statistical analysis
All data were analyzed by SPSS 22.0 software. Data are presented as mean ± SD and analyzed by
t test. The χ
2 and Fisher exact texts were used to compare genotype and allele frequencies between the MG group and healthy control group and MG subgroups (sex, age, with or without thymoma, etc.).
P values < .05 were considered significant. SNPstats (
http://bioinfo.iconcologia.net/snpstats/start.htm) was used to detect the Hardy-Weinberg equilibrium in healthy controls. Logistic regression was used to adjust for potential confounders affecting differences between the MG group and heathy controls and among MG subgroups.
Discussion
In the current study, the single nucleotide polymorphism rs2893321 of the BAFF gene was found to be associated with susceptibility to MG in a Chinese Han population. Several clinical variables can affect analysis of gene polymorphisms. Thus, we adjusted the MG clinical features to determine whether the polymorphisms of rs2893321 were independent risk factors for a specific variable. Significant differences were found between the rs2893321 genotype and women with MG and MG patients younger than 50 years. No associations were found between the rs2893321 polymorphism and MG patients with or without thymoma or MG patients who were AChR antibody negative or positive.
BAFF stimulates the biological functions of B cells, and dysregulation of
BAFF can affect the development of MG [
16]. The genetic variant of
BAFF is associated with systemic lupus erythematosus, Graves’ disease, chronic lymphocytic leukemia, and other autoimmune diseases [
17‐
19]. However, no study has investigated the connection between
BAFF polymorphisms and MG. In our study, the results demonstrated that the frequencies of the AA genotype of rs2893321 were significantly higher in patients with MG than in the healthy controls, which suggests that rs2893321 might be associated with MG susceptibility in this ethnic Chinese Han population. However, our study population size was relatively small compared to other case-control studies. To confirm our result and to discover more detail of rs2893321 in MG, additional study with larger samples are needed.
In this study, rs2893321 was found to be closely associated with MG patients who were younger than 50 years and female. On the other hand, no significant differences in allele were observed among MG patients in different age and gender. We suspect it is because the frequency of GA genotype was high and had no significant different between groups, and then cause the allele A and G showed no significant changes. Additionally, no significant differences in genotype or allele frequency of rs2893321 were observed among MG patients with or without thymoma or MG patients who were AChR antibody positive and negative. These results suggest that the genetic polymorphism of rs2893321 might be restricted to specific subgroups of MG.
It has been reported that MG can be divide into different age subgroups, early-onset MG (EOMG, younger than 50 years) and late-onset MG (LOMG, older than 50) [
20]. The management strategies and treatment can be different due to the age difference [
21]. Our study indicated the genotype frequency of rs2893321 was different in EOMG patients compared with healthy control, which suggested rs2893321 in
BAFF might be a susceptible genetic variant in EOMG patients. Thus people with rs2893321 variant should pay more attention to prevent the development of MG in a relatively early age.
After logistic regression analysis, the results indicated that the genetic impact of rs2893321 was gender-dependent. In brief, the genetic effect of the rs2893321 AA genotype appeared to be more significant among women than among men. This finding may be attributable to differences in basic immune responses due to different sex hormones [
22]. Gender indeed affects the association between genetic polymorphisms and susceptibility to MG [
23]. It has been reported that
BAFF gene expression can affect sex hormones in animals and in humans. A study has shown
BAFF to be negatively correlated with testosterone and positively correlated with estrogen [
24]. These results suggest that sex hormones affected
BAFF gene expression and that the genetic variant of rs2893321 in
BAFF was significantly associated with susceptibility to MG in women.
Conclusions
This study demonstrate the association between BAFF polymorphisms and MG. In a Chinese Han population, rs2893321 was found to be associated with MG susceptibility, specifically in women with the condition and in patients who were younger than 50 years. Future studies should expand the patient population and investigate more SNPs in the BAFF gene to understand the connection between BAFF and MG.
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