Association of mannose-binding lectin 2 gene polymorphisms with Guillain-Barré syndrome

Complement activation plays a critical role in the pathogenesis of Guillain-Barré syndrome (GBS), a debilitating immune-mediated neuropathy. Mannose-binding lectin (MBL) is a complement activation factor of lectin pathway which as genetic host factor may influence the susceptibility or severity of GBS. We investigated the frequency of MBL2 promoter (− 550H/L and − 221X/Y) and functional region (exon 1 A/O) polymorphisms and their association with disease susceptibility, clinical features and serum MBL among GBS patients (n = 300) and healthy controls (n = 300) in Bangladesh. The median patient age was 30 years (IQR: 18–42; males, 68%). MBL2 polymorphisms were not significantly associated with GBS susceptibility compared to healthy controls. HL heterozygosity in GBS patients was significantly associated with mild functional disability at enrolment (P = 0.0145, OR, 95% CI 2.1, 1.17–3.82). The HY, YA, HA and HYA heterozygous haplotypes were more common among mildly affected (P = 0.0067, P = 0.0086, P = 0.0075, P = 0.0032, respectively) than severely affected patients with GBS. Reduced serum MBL was significantly associated with the LL, OO and no HYA variants and GBS disease severity. No significant association was observed between MBL2 polymorphisms and electrophysiological variants, recent Campylobacter jejuni infection or anti-ganglioside (GM1) antibody responses in GBS. In conclusion, MBL2 gene polymorphisms are related to reduced serum MBL and associated with the severity of GBS.

Guillain-Barré syndrome (GBS) is a rapidly progressing peripheral neuropathy and the major cause of flaccid paralysis after an acute infection 1 . The global incidence of GBS is 1-2 per 100,000 person/year, though this rate varies regionally with significant heterogeneity in terms of clinical presentation and severity 2,3 . Two-third of patients have symptoms of a precedent infection 4 and the most identified cause is Campylobacter jejuni 5 . The recent public health outbreak of Zika virus caused many patients to develop neurological symptoms like GBS 6 . However, the mechanisms underlying GBS have not yet been elucidated. There are two major variants of GBS: axonal (AMAN; acute motor axonal neuropathy and AMSAN; acute motor-sensory axonal neuropathy) and demyelinating (AIDP; acute inflammatory demyelinating polyradiculoneuropathy) 1 . Conclusive evidence indicates GBS after C. jejuni infection is an auto-antibody mediated disease triggered by molecular mimicry between the surface epitopes of an infection agent and peripheral nerve gangliosides, which accounts for the pathogenesis of axonal GBS 7 . Very few patients with a microbial infection develop GBS 8 , which indicates involvement of host factors triggering this autoimmunity. Genetic susceptibility could be a predisposing factor for GBS, though the genetic factors that affect the interactions between microbial and host are poorly studied. The host complement system plays a crucial role in the pathogenetic mechanism of GBS; by mediating complement fixations through anti-ganglioside antibody that elevated serum complements and deposited on the surface of Schwann cells and nodes of Ranvier 9,10 . Over the last decade, mannose-binding lectin (MBL) has been the center of substantial interest as it possesses the ability to turn on the complement pathway through enzymatic regulation and thus may influence disease susceptibility and severity 11 . MBL is encoded by the MBL2 gene, which contains four exons. Three single nucleotide polymorphisms (SNPs) in the structural region of the MBL2 gene, located in codons 52 (A/D), 54 (A/B), and 57 (A/C) of exon 1, and two promoter polymorphisms, located at − 550H/L and − 221X/Y, are the major determinants of serum MBL concentration and activity [12][13][14] . The variant B, C and D alleles, collectively referred to as the O allele, are associated with serum MBL deficiency compared to the wild-type A allele 12,13 . In addition, polymorphisms within the promoter region (− 550H/L and − 221X/Y) determine the serum MBL concentration to an extent by influencing gene expression 14 . Host MBL2 genetic variations are associated with susceptibility to a wide variety of infectious and autoimmune diseases 15 , including tuberculosis (TB) 16 , rheumatoid arthritis 17 , systemic lupus erythematosus (SLE) 18 and GBS 19 . However, other reported MBL polymorphisms protect against infectious disease like TB 20 . MBL can act as double-edged sword in post-infectious diseases as GBS depending on its concentration. Serum MBL deficiency has been reported as a predisposing factor for the development of SLE 21 , atherosclerosis 22 , TB 16 and coronary artery disease 23 . However, some studies reported elevated serum MBL was associated with increased disease susceptibility 24 , thus, the data remain inconclusive. These inconsistent results may be due to the relatively small study populations, different ethnic groups, and environmental influences between studies.
Host-pathogen interactions and environmental factors have attracted attention as concepts that may contribute to GBS. Identifying GBS susceptibility genes would represent an advancement in our understanding of the pathogenesis of the disease. Previously, we reported immune-mediated genes, including tumor necrosis factor-alpha 25 , apoptotic gene FAS 26 and toll-like receptor-4 27 were associated with GBS disease susceptibility, whereas human leukocyte antigen-DQB1 28 , antigen presenting glycoprotein CD1A and CD1E 29 genes have no effect on development of GBS. However, very limited data are available on MBL2 gene polymorphisms and their influence on the serum MBL levels in patients with GBS. Therefore, we determined the potential association between MBL2 gene polymorphisms and disease susceptibility, clinical subtypes, severity and level of serum MBL protein in patients with GBS.

Results
Clinical and sociodemographic characteristics. The  No association between MBL2 gene polymorphisms and GBS susceptibility. The distributions of MBL2 genotypes were compared between patients with GBS and healthy controls to assess the association with GBS susceptibility. All genotype distributions in healthy controls fitted Hardy-Weinberg equilibrium. There was no significant difference in the genotype and allele frequencies between patients with GBS and controls. In particular, genotypes and alleles of − 550 (H/L), − 221 (X/Y) and exon 1 (A/O) SNPs occurred at similar frequencies in patients with GBS and healthy controls ( Table 2). In addition to single-site SNP analysis, we compared the haplotypes of the patients with GBS and controls.  Table 2).
The − 550H/L SNP and MBL2 haplotypes are associated with a less severe form of GBS. The patients enrolled in this study were classified based on their disease severity, as severely affected (MRC sum score of < 40) or mildly affected (MRC sum score between 40 and 60). The frequency of the MBL2 genotypes were compared among these groups to determine the effect of SNPs on disease severity. The heterozygous HL genotype (63% vs. 43%; P = 0.0145, OR = 2.1, 95% CI 1.17-3.82, corrected; Pc = 0.0048, Table 3) was significantly more frequent in mildly affected patients than severely affected patients with GBS. The heterozygous XY genotype (39% vs. 31%) and heterozygous AO genotype (41% vs. 33%) were also more frequent among mildly affected patients than patients with the severe form of GBS, but these trends were not statistically significant (Table 3). Haplotype analysis of the MBL2 gene polymorphisms revealed a significant association with developing the mild form of GBS. The HYA heterozygous haplotype (HYA/LYO, HYA/LYA, HYA/LXA, HYA/HYO and HYA/  (Table 3).
We also compared the MBL2 polymorphisms with other severe clinical features like dysautonomia and requirement of mechanical ventilation in patients with GBS. Higher HL and lower LL genotypes were significantly present in patients with dysautonomia than patients with normal autonomic function (P a = 0.017, OR = 2.29, 95% CI 1.18-4.44, Table 4). Moreover, AO genotype was also significantly associated with autonomic dysfunction (P a = 0.032, OR = 1.92, 95% CI 1.06-3.47, Table 4). No significant association was found between MBL2 gene polymorphism and requirement of mechanical ventilation (Table 4). HL and OO genotype were higher among patients who died during the course of the disease.

Association of MBL2 polymorphisms with the clinical and serological features of GBS.
We categorized the patients with GBS based on electrophysiological subtypes (axonal and demyelinating) and serological data to assess the associations between these features and MBL2 genotypes. No significant association was found with the MBL2 genotypes and electrophysiological variants (axonal; AMAN & AMSAN and demyelinating; AIDP) compared to the control group (Supplementary Table S1). The comparison of MBL2 genotypes Table 3. Associations between MBL2 SNPs and GBS disease severity. SNPs single nucleotide polymorphisms, GBS Guillain Barré syndrome, P-value probability value, OR odds ratio, 95% CI 95% confidence interval. The Bonferroni-adjusted significance threshold was 0.0167 for genotypes and 0.025 for alleles, *statistically significant after P value correction. www.nature.com/scientificreports/ and haplotypes between anti-GM1 antibody-positive and antibody-negative group did not reveal a significant association between the MBL2 polymorphisms and anti-GM1 antibody productivity (Supplementary Table S2).
Similarly, there was no significant association found between the MBL2 genotypes and recent C. jejuni infection (data not shown).
Elevated serum MBL level is associated with disease severity. We measured the serum concentration of MBL for a subgroup of patients with GBS (n = 166) and healthy controls (n = 102  Fig. 1b). However, we found no significant difference in the serum MBL between the C. jejuni or anti-GM1 antibody seropositive and negative groups (Fig. 1c,d). In addition, we excluded the patients with OO genotype and compared the MBL level among the groups. We found high serum MBL level was associated with the severely affected patients with GBS (P = 0.0126) and no significant difference was revealed between GBS and healthy control. We compared MBL serum levels with the requirement of mechanical ventilation and development of autonomic, dysfunction in patients with GBS. However, no association was found with the elevated serum MBL and mechanical ventilation or autonomic dysfunction. The median of serum MBL (4417 ng/mL) increased among the patients who died during the course of the disease.  Fig. 2f). We found no significant difference in the serum MBL levels among individuals with the − 221X/Y polymorphism (Fig. 2c,d). Comparison between MBL haplotypes and serum MBL levels revealed, reduced serum MBL levels were associated with no HYA allele and HYA heterozygous than HYA homozygous in both patients with GBS (P = 0.0014 and P < 0.05; Fig. 2g) and healthy controls haplotypes (P < 0.05 and P < 0.0001; Fig. 2h).

Discussion
Preliminary studies of genetic variation in the MBL2 gene and its functions over the last decades have focused on disease susceptibility of GBS; however, the roles of MBL2 in immunopathogenesis and regulation of disease severity are poorly understood. The present study focused on the contribution of polymorphisms in the promoter www.nature.com/scientificreports/ region (− 550H/L and − 221X/Y) and structural exon 1(A/O) region of the MBL2 gene and their association with GBS susceptibility, subtype and severity. The analysis of genetic associations in this study revealed MBL2 gene polymorphisms do not significantly contribute to GBS susceptibility. However, the serum levels of MBL were significantly associated with the HL genotype and heterozygote haplotypes (HY, YA, HA and HYA) of the MBL2 gene and the disease severity of GBS in our Bangladeshi cohort. Complement activation has recently been reported to be involved in the induction of post-infectious immunemediated peripheral nerve damage in patients with GBS 30 . MBL is a key regulatory element that activates the complement system, as part of first-line defense in the pre-immune host in several autoimmune neurological disorders 31 . Previously, Geleijns et al. reported MBL polymorphisms contributed to the susceptibility in patients with GBS and were associated with disease severity 19 . The B allele of exon 1 was found to protect against developing severe form of GBS in a Dutch population 19 . However, this study from Bangladesh revealed no significant association between the B allele and disease severity. The probable explanation could be the inclusion of high number of severe cases (77%) in Bangladeshi cohort due to high severity 32 . In contrast, the heterozygous HL, HY, YA, HA and HYA haplotypes were significantly associated with developing the mild form of GBS. In addition, HL and AO genotypes were significantly associated with autonomic dysfunction that includes hypertension, hypotension, tachycardia, pupillary abnormality. However, the previous study did not examine any association between the clinical and serological subgroups, including the subtype of GBS, recent infection with C. jejuni and auto-antibodies, with MBL2 polymorphisms or serum MBL levels 19 . In our population, MBL2 polymorphisms had no influence on the neural damage that occurs in the axonal and demyelinating variants of GBS. More evidence for relation between MBL and disease susceptibility and severity need to be studied to confirm this finding.
MBL contains pattern recognition features that allow the widest range of potential microbial structures to be recognized 11 . Many infections lead to higher serum MBL levels and increase the disease susceptibility 33,34 . Several reports suggest that the MBL protein can also modulate disease severity including GBS, leptospirosis and dengue 19,34,35 . We observed significant elevation of serum MBL in severely affected patients with GBS, in  37,38 . In line with previous observations 19 , we found polymorphism in structural region of exon 1 is associated with lower serum MBL. Individuals with AO and OO genotype had lower serum MBL than the wild-type variant AA homozygous individuals in GBS. The effect of AO and OO genotype on serum MBL in patients with GBS is consistent with the findings in healthy controls, further indicating this polymorphism affects the serum MBL level. Several reports have suggested the promoter region (− 550) variant allele (L allele) downregulates serum MBL by functionally regulating the immune system in several diseases 39 . This study also revealed the L allele was associated with lower serum MBL levels in patients with GBS and the healthy control group. Several studies have presented evidence that MBL deficiency increases the generalized susceptibility of an individual to infectious diseases 40,41 , including human immunodeficiency virus 42,43 , Plasmodium falciparum 44 , cryptosporidium 45 and N. meningitidis 46 . In addition to these associations with infectious disease, there is also evidence of associations between reduced serum MBL and susceptibility to autoimmune diseases. Several reports suggest strong association in the case of systemic lupus erythematosus (SLE). British 47 , Hong Kong Chinese 48 , African American 49 and Spanish 50 patients with SLE all exhibited increased frequencies of mutant MBL alleles or reduced serum MBL levels. In our Bangladeshi population, we did not find any significant association between GBS disease susceptibility and lower levels of MBL protein. As earlier reported, elevated levels of MBL protein have been associated with increased susceptibility towards chronic rheumatic heart disease in patients previously diagnosed with rheumatic fever 51 . The pathogenesis of rheumatic fever and GBS are similar in terms of antecedent infections, molecular mimicry, and cross-reactive antibodies. Therefore, the higher levels of MBL in severe GBS cases might be explained by binding of MBL to damaged nerve tissue, followed by complement activation, attraction of inflammatory cells, and aggravation of tissue injury. One of the limitations of this study was the lack of determination of complement product activation (C3a and C5a) in serum of GBS patients. Further functional activity of MBL2 polymorphism and its serum levels can be confirmed by measuring the products of complement pathway.
In conclusion, MBL2 haplotypes and serum MBL levels may be one key determinant of the severity of weakness in patients with GBS in Bangladesh. Furthermore, both promoter and functional region polymorphisms in the MBL2 gene are associated with downregulation of serum MBL protein in both patients with GBS and www.nature.com/scientificreports/ healthy controls. The precise mechanistic role of MBL in GBS is not clear. Further in-depth research is required to elucidate complement activation mediated by MBL and its involvement in the lectin pathway during the pathogenesis of GBS; analysis of samples from the multi-center International GBS Outcome Study (IGOS) 52 of a large population of various ethnic groups around the world may to explain the underlying mechanisms.

Materials and methods
Study participants. The study was conducted in the Laboratory of Gut-Brain Signaling, icddr,b, Dhaka, Bangladesh. The patients with GBS and healthy controls were recruited from Dhaka Medical College and Hospital (DMCH) and the National Institute of Neurosciences and Hospital (NINS), Dhaka, Bangladesh. All patients with GBS fulfilled with the National Institute of Neurological Disorders and Stroke (NINDS) criteria and were enrolled within the two weeks of onset of weakness 53 . The study was reviewed and approved by the Ethical Committee of icddr,b, Dhaka, Bangladesh and all participants gave written informed consent prior enrolment.
Clinical and blood samples were obtained from 300 consecutive patients with GBS and 300 healthy controls for this study. The patients were followed up and clinically assessed at standard time-points (2 weeks, 4 weeks, 6 months and 1 year after entry) to evaluate disease outcome. Disease severity was defined using the Medical Research Council (MRC) sum score for six muscles in the upper and lower limbs on both sides; the sum score ranges from 60 (normal) to 0 (quadriplegic) 54 . GBS patients with a MRC sum score < 40 were defined as severely affected and 40-60, mildly affected 55 . Disease outcome was assessed using the GBS disability scale (GBS-DS) 56 at six months follow-up; patients able to walk independently (GBS-DS of ≤ 2) were defined as having a good outcome and patients unable to walk independently (GBS-DS of ≥ 3), poor outcome 57 . Ethnicity-based healthy controls (HC) were recruited from people accompanying the patients to physical examinations at the hospitals mentioned above and pair matched with the GBS cases by age and gender. All control subjects were genetically unrelated inhabitants of Bangladesh without a prior history of other neurological complications or non-communicable diseases, and no recent history of pregnancy or surgery.
Genomic DNA isolation. Genomic DNA was isolated from whole blood for 600 subjects using QIAamp® DNA Blood Midi Kits (100) (Qiagen, Hilden, Germany) following the manufacturer's instructions. The extracted DNA was dissolved in 1 × TE-buffer (10 mM Tris-Cl, pH 8.0, 1 mM EDTA) and stored at − 80 °C. The stock DNA was diluted to a final concentration of 10 ng/μL in Milli-Q-water and stored at − 20 °C until SNP detection.
Serological assays. Serological tests were performed on the 300 sera of patients with GBS to assess baseline anti-ganglioside antibodies and recent C. jejuni infection at the time of enrolment. Enzyme-linked immunosorbent assay (ELISA) was used to detect IgG, IgM and IgA antibodies against C. jejuni and IgG against the nerve GM1-ganglioside, as described previously 58,59 . Serum MBL was measured in duplicate from 166 randomly selected serum samples from patients with GBS at the time of enrolment and 102 serum samples from healthy controls by ELISA method using a human MBL oligomer ELISA kit (BioPotro Diagnostics A/S, Gentofte, Denmark), which specifically detects oligomerized forms of MBL protein. Serum samples were stored at − 80 °C and diluted 1:100 in the sample diluent provided in the ELISA kit during the experiment. The ELISA was performed according to the manufacturer's instructions; absorbance values were measured at 450 nm and serum concentrations were expressed as nanograms of MBL per milliliter (ng/mL).
Genotyping of MBL2 polymorphisms. Single nucleotide polymorphisms (SNPs) in the promoter regions at − 550 (H/L, rs11003125), − 221 (X/Y, rs7096206) and the structural region exon 1 at codon 52 (A/D, rs5030737), codon 54 (A/B, rs1800450) and codon 57 (A/C, rs1800451) of the MBL2 gene were determined in 600 DNA samples extracted from patients with GBS and healthy controls. The real-time reverse transcription polymerase chain reaction (RT-qPCR) and melting curve analysis were performed in LightCycler capillaries (Roche Diagnostics). The primers, probes and the PCR amplification program were based on Geleijns et al. 19 . The PCR amplification program was designed to determine both promoter regions (− 550H/L and − 221X/Y) and exon 1 (A/O) SNPs using 1 × LightCycler DNA Master Hybridization Probes (Roche Molecular Biochemicals). The melting curve profile was determined after PCR by constant detection of emitted light 19 . Approximately 20% of the samples were sequenced to confirm the MBL2 polymorphisms.
Statistical analysis. The chi-squared test was conducted to examine the differences in demographic characteristics and potential confounders between the GBS cases and healthy controls. The MBL2 genotype and haplotype frequencies were determined by a simple counting method and managed using Microsoft Excel 2010 (Microsoft, Redmond, WA) and SPSS (16.0 version, Chicago, IL). Fisher's exact test was used to determine the association of MBL2 polymorphisms and GBS susceptibility; odd ratios (ORs) and 95% confidence intervals (95% CI) were reported. The chi-square test was conducted to confirm the Hardy-Weinberg equilibrium among healthy controls. Serum MBL levels were presented as medians and interquartile ranges (IQR). The non-parametric Mann-Whitney U-test was used to compare the differences in the serum MBL concentrations (ng/mL) between subgroups of patients with GBS and healthy controls. The associations between serum MBL levels and MBL2 genotypes for the studied SNPs in patients with GBS and controls were analyzed using the Kruskal-Wallis test. The associations between the clinical features of GBS and genotypes were analyzed using Fisher's exact test. A probability level (P) of less than or equal 0.05 was considered as the significance criterion. To avoid type I errors in multiple comparisons, the Bonferroni adjustment was performed to correct the P-values obtained by dividing the number of comparisons; the corrected P-values are denoted as Pc 60 . The significance threshold for Fisher's exact test was adjusted to 0.01667 and 0.025 to obtain Pc for genotypic and allelic differences. All statisti-Scientific Reports | (2022) 12:5791 | https://doi.org/10.1038/s41598-022-09621-y www.nature.com/scientificreports/ Publisher's note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
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