Skip Navigation


Rheumatology Advance Access originally published online on June 18, 2008
Rheumatology 2008 47(8):1168-1171; doi:10.1093/rheumatology/ken226
This Article
Right arrow Abstract Freely available
Right arrow FREE Full Text (PDF) Freely available
Right arrow All Versions of this Article:
47/8/1168    most recent
ken226v1
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Add to My Personal Archive
Right arrow Download to citation manager
Right arrowRequest Permissions
Right arrow Disclaimer
Google Scholar
Right arrow Articles by van de Geijn, F. E.
Right arrow Articles by Dolhain, R. J. E. M.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by van de Geijn, F. E.
Right arrow Articles by Dolhain, R. J. E. M.
Related Collections
Right arrow Rheumatoid Arthritis
Social Bookmarking
Add to CiteULike   Add to Connotea   Add to Del.icio.us  
What's this?

© The Author 2008. Published by Oxford University Press on behalf of the British Society for Rheumatology. All rights reserved. For Permissions, please email: journals.permissions@oxfordjournals.org

Mannose-binding lectin polymorphisms are not associated with rheumatoid arthritis—confirmation in two large cohorts

F. E. van de Geijn1,2, J. M. W. Hazes1, K. Geleijns3, M. Emonts4, B. C. Jacobs2,3, B. C. M. Dufour-van den Goorbergh2 and R. J. E. M. Dolhain1

1Department of Rheumatology, 2Department of Immunology, 3Department of Neurology, Erasmus MC University Medical Center Rotterdam and 4Department of Paediatrics, Erasmus MC–Sophia Children's Hospital, University Medical Center Rotterdam, Rotterdam, The Netherlands.

Correspondence to: F. E. van de Geijn, Department of Rheumatology, Room Ee965, Erasmus MC University Medical Center Rotterdam, P.O. Box 2040, 3000 CA Rotterdam, The Netherlands. E-mail: f.vandegeijn{at}erasmusmc.nl


   Abstract
Top
 Abstract
Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
Objectives. In RA, conflicting results have been described on the association between genotypes of the complement factor mannose-binding lectin (MBL) and disease susceptibility and severity. This might be due to underpowerment of previous research work and the fact that no confirmation cohorts were used. Therefore a different approach is warranted.

Methods.MBL2 gene polymorphisms were determined in two RA cohorts (378 and 261 cases) and 648 controls. Considering MBL polymorphisms, cases and controls were categorized in groups of high, intermediate and low MBL production. The total sample size allows detection of a potential association between RA susceptibility and MBL groups with an odds ratio of 1.37 ({alpha} < 0.05; 1–β > 0.8). Disease severity as defined by the need for anti-TNF therapy was also analysed for possible associations with MBL groups.

Results. There was no difference in the frequencies between MBL genotypes of RA cases and controls that are associated with high (cases 54.4%, controls 57.0%), intermediate (cases 28.9%, controls 27.5%) or low (cases 16.7%, controls 15.5%) MBL production. Furthermore, there was no association between MBL groups and disease severity.

Conclusions. MBL genotype groups are not associated with RA disease susceptibility or severity in this large study including a confirmation cohort. Compared with previous smaller studies these results add to more definite conclusions.

KEY WORDS: Mannose-binding lectin, Polymorphisms, Rheumatoid arthritis, Disease susceptibility, Disease severity


   Introduction
Top
 Abstract
 Introduction
Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
Rheumatoid arthritis (RA) susceptibility has been associated with defects in innate immunity. Mannose-binding lectin (MBL) deficiency is one of the defects in innate immunity with the highest prevalence. MBL is a complement component that activates the lectin complement pathway. Its serum concentration is strongly determined by single nucleotide polymorphisms (SNPs) in the structural gene and the promoter region [1]. MBL gene polymorphisms have been associated with outcome in multiple diseases [2–4]. The role of MBL in RA susceptibility and severity is still controversial [4–13].

Contradictory results are a general point of concern in genetic studies. To overcome this problem, recently criteria for genetic association studies have been suggested within the National Cancer Institute and National Human Genome Research Institute (NCI–NHGRI) Working Group on Replication in Association Studies [14]. The Working Group pleaded for large sample size genotype–phenotype studies that replicate both positive as well as negative findings in multiple well-described cohorts with enough power and clear statistics and also pleaded for publishing the so-called negative studies. Until now none of the research performed on RA and MBL was in accordance with all criteria, especially most studies were underpowered and in none of the studies confirmation cohorts were used. Therefore, the association of MBL and RA remains unclear.

Therefore, we first aimed to investigate whether MBL polymorphisms are associated with susceptibility for RA using a study design in accordance with the NCI–NHGRI criteria. Second, we aimed to determine whether MBL polymorphisms are associated with disease severity and clinical characteristics.


   Materials and methods
Top
 Abstract
 Introduction
 Materials and methods
Results
 Discussion
 Acknowledgements
 References
 
Cases and controls
DNA samples were obtained from n = 378 consecutive RA patients followed in an outpatient clinic (Cohort 1). They fulfilled the ACR 1987 revised criteria for RA and to be even more certain of a diagnosis of RA, additionally had at least RF positivity, positivity for cyclic citrullinated ceptide (CCP) antibody or proof of joint erosions. DNA samples from another group of RA patients (n = 261, Cohort 2) were obtained from women who were followed as part of the PARA study, a nationwide prospective cohort study in which women with RA (according to ACR 1987 criteria) are studied during pregnancy and post-partum [15].

All controls (n = 648) have been described previously [2, 16–18]. Genomic DNA of n = 461 healthy voluntary donors was provided by the Sanquin Bloodbank, Rotterdam, The Netherlands [2, 16] and n = 187 controls were healthy pregnant females [17, 18].

All cases and controls included in this study are unrelated Dutch Caucasians. This study is in compliance with the Helsinki Declaration and the local ethics review boards approved all study protocols. Data were analysed anonymously. The characteristics of cases and controls are described in Table 1.


View this table:
[in this window]
[in a new window]

  		TABLE 1. Cohort characteristics

 
Determination of disease severity markers and MBL serum concentrations
Current or past use of anti-TNF therapy was used as a marker of disease severity in both RA cohorts (n = 120 and n = 35 RA cases from Cohorts 1 and 2, respectively). Anti-TNF therapy was chosen since its prescription is strictly regulated in The Netherlands. Its costs are only reimbursed for RA patients that have therapy-resistant disease, which is defined as failure on at least two DMARDs including MTX and still active disease [disease activity score (DAS28) > 3.2], despite MTX therapy at 25 mg weekly or at maximum tolerated doses.

Clinical characteristics were determined in a subgroup from RA Cohort 2, which was also seen before pregnancy. At that time-point disease activity was scored using DAS28 (n = 129) and serum was obtained to determine the titres of anti-CCP (n = 129, EliATM CCP test, Phamarcia Diagnostics, Freiburg, Germany), IgA-, IgM- and IgG-RF (n = 129, Biognost, Heule, Belgium) and MBL protein concentrations (n = 132, sandwich ELISA as described previously [18]).

MBL genotyping and categorization of the individuals upon MBL genotypes
Genotyping was performed using PCR LightCycler techniques as described previously [18]. Genotyping included the wild-type (A-allele) and the three single nucleotide polymorphisms (SNPs) of the first exon of the structural gene: codon 52 (D-allele, rs5030737), codon 54 (B-allele, rs1800450) and codon 57 (C-allele, rs1800451) and two of the SNPs in the promoter region codon –550 (H/L, rs11003125) and codon –221 (X/Y, rs7096206) of the MBL2 gene. The B-, C- and D-alleles are jointly referred to as ‘O’. Linkage disequilibrium of the structural exon 1 mutations with the promoter polymorphisms results in six possible haplotypes (i.e. HYA, LYA, LXA, LYB, LYC and HYD). Every individual will express two out of these six haplotypes. The HY haplotype induces high MBL concentrations, while exon 1 mutations (O variant) and the LX haplotype cause reduced MBL concentrations.

Therefore, based upon the haplotypes individuals can be categorized into three groups: the high MBL production Group A, (H or L)YA/(H or L)YA and (H or L)YA/LXA; the intermediate MBL production Group B, LXA/LXA and (H or L)YA/O and the low or deficient MBL production Group C, LXA/O and O/O. This genetic subdivision correlates best with MBL serum levels [1] and is therefore commonly used and accepted [3] and applied to all analyses in this study.

In each experiment, genotype-matched and sequence-verified control donors were used.

Statistical analysis
SPSS 12.0.1 was used for all statistical analyses. Pearson {chi}2-tests were performed to test Hardy–Weinberg equilibrium and to analyse possible associations between MBL genotype groups and RA. The Spearman rank and Kruskal–Wallis test was performed for comparison between MBL genotype groups and MBL serum levels, DAS28 and anti-CCP or RF titres. Logistic regression analysis was used for adjustment for gender and age. Differences between continuous variables were analysed by unpaired t-tests. A significance level of P < 0.05 was used for all analyses.

Power analysis
Power analyses were performed based on published frequencies of the MBL genotype groups in Caucasians; MBL genotype Group C frequency of 14.8% and MBL genotype Group B frequency of 34.1% [3]. With a power of 80% and {alpha} = 0.05 in RA Cohort 1 an odds ratio (OR) of 1.60 for MBL genotype Group C alone vs MBL genotype Group A and an OR of 1.44 for MBL genotype Groups B and C together vs MBL genotype Group A could be detected for the association of RA susceptibility and MBL genotype groups. For RA Cohort 2 an OR of 1.69 for Group C vs Group A and an OR of 1.51 of Group B plus Group C vs Group A could be detected. When the two groups were taken together, an OR of 1.51 for Group C vs Group A and an OR of 1.37 for Group B plus Group C vs Group A could be detected for the association of RA susceptibility and MBL genotype groups.


   Results
Top
 Abstract
 Introduction
 Materials and methods
 Results
Discussion
 Acknowledgements
 References
 
Accuracy genotyping procedure
Ten per cent of the samples were randomly re-analysed with identical results. The genotype distribution was in Hardy–Weinberg equilibrium for all SNPs tested in cases and controls. In five cases and nine controls, the exon 1 SNPs could not be determined, and in two controls the promoter SNPs could not be determined. Therefore, these cases (n = 5, <1%) and controls (n = 11, 1.7%) could not be grouped in one of the MBL genotype groups and were excluded from analysis, resulting in a total of n = 634 cases and n = 637 controls to be analysed.

As expected, there was a good correlation between MBL genotype groups and MBL concentrations (Spearman {rho} = 0.82, Group A median 607.5 ng/ml, Group B median 192.1 ng/ml, Group C median 50.7 ng/ml, n = 132, P < 0.0001).

Association of MBL genotype groups and disease susceptibility
Cohort characteristics are summarized in Table 1. There was no difference in the frequencies between MBL genotypes of RA cases and controls that are associated with high A (cases 54.4%, controls 57.0%), intermediate B (cases 28.9%, controls 27.5%) or low C (cases 16.7%, controls 15.5%) MBL production (Table 2), indicating that there is no association between MBL genotype groups and RA susceptibility.


View this table:
[in this window]
[in a new window]

  		TABLE 2. Frequencies of MBL2 genotype groups and their association with disease susceptibility and disease severity

 
Association of MBL genotype groups and disease severity and disease characteristics
No association was found between MBL genotype groups and the current or past use of anti-TNF therapy, as a marker for disease severity (Table 2). Furthermore, no associations were found between MBL genotype groups or serum concentration and the autoantibody titres of anti-CCP, IgA-, IgM- and IgG-RF and DAS28 (data not shown).


   Discussion
Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
Acknowledgements
 References
 
MBL genotype groups are not associated with disease susceptibility, severity and disease characteristics in this large cohort of RA patients.

We are aware that cases and controls differed regarding age and gender. Therefore, all analyses were repeated with correction for these factors, but still no differences could be observed. It was also not likely that these could have induced bias, since the MBL genotype distribution or serum levels are not influenced by age or gender [19]. It can be reasoned that our conclusions may only be applicable to Caucasian populations since in non-Caucasian populations the distribution and frequencies of MBL polymorphisms are different [8, 13].

One could argue that in complex genetic diseases like RA, ORs between 1.1 and 1.3, for disease susceptibility and disease severity, could still be of interest and that the present study might therefore still be somewhat underpowered. Based upon the MBL genotype group frequencies of this study, one could calculate that the entire study population, i.e. cases and controls together, should include at least between 1900 and 14 000 subjects to obtain significant ORs between 1.1 and 1.3. Besides the fact that it is generally not acceptable to add additional cases to a representative sample once the analyses are performed, it is not expected that including more cases to our study would result in different conclusions, since not even a trend towards statistical significance was observed.

Another possibility to increase the power is to perform a meta-analysis on all existing data. A prerequisite for a meta-analysis is that MBL genotype groups are defined identically in all studies. It has been shown that defining MBL genotype groups can be most accurately done by combining SNPs in the structural gene with the SNPs in the promoter region. Therefore, this approach was chosen in this study. We found only one study on MBL genotypes and RA susceptibility in which MBL genotype groups could be defined identically [4]. Unfortunately, the frequencies of the control group were not described in detail, therefore making subdivision impossible for the controls. If we combined the RA patients of Graudal's study ([4], n = 140) with the cases in our study (n = 639) and compare them to the healthy controls in our study (n = 646) still similar non-statistically significant results were obtained (OR 1.16; 95% CI 0.70, 1.93 for MBL Group A vs Group C and OR 1.12; 95% CI 0.77, 1.61 for MBL Group A vs Group B + Group C). Performing a meta-analysis on studies that investigate disease severity was not possible for the additional reason that previous studies determined disease severity differently. Furthermore, some studies used markers of disease outcome (erosions) and others disease activity as determinant of disease severity. Since both depend upon treatment strategies that have changed markedly in recent years, recent and older literature cannot be combined in one meta-analysis.

Previous studies on MBL in relation to RA susceptibility describe ambiguous results. An association with low MBL and RA has been detected in four studies [5–8], two of which were performed in Asian populations that are known to have marked differences in MBL frequencies [6, 8]. The other two studies were performed in Caucasians, but did not study all promoter polymorphisms next to the exon 1 polymorphisms. No association was found in five studies [4, 9–12]. However, no definitive conclusions can be drawn from these studies, since they lack sufficient power and not all known MBL variant alleles were analysed. Furthermore, none of the studies confirmed its results in an independent second cohort.

MBL in relation to disease severity and disease characteristics like erosions [4–7, 12, 13], autoantibody titers [7, 11, 12] and disease activity [7, 8] has been studied in several studies [4–8, 11–13]. Their results are equivocal and several studies are hampered by a lack of power [4, 11, 12]. Also, in none of these studies confirmation cohorts were included.

In conclusion, no association between RA disease susceptibility and MBL genotype groups was found. Since the results of this study were obtained in two separate cohorts of patients with enough power to demonstrate even small differences, a more definite conclusion can be drawn that MBL genotype groups are not a risk factor for the development of RA or disease severity.

Formula


   Acknowledgements
Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
References
 
We would like to acknowledge Prof. Dr Mohammed R. Daha, Department of Nephrology from Leiden University Medical Center, The Netherlands for collaboration in determining MBL protein concentrations in our samples. We would also like to acknowledge Dr Christianne J. M. de Groot, Departments of Obstetrics and Gynaecology of Medical Center Haaglanden, The Hague and Erasmus MC University Medical Center Rotterdam, The Netherlands for providing DNA samples of controls. Moreover, we would like to thank Wouter van Rijs, Department of Immunology of Erasmus MC University Medical Center Rotterdam, The Netherlands for his technical advice concerning genotyping with the LightCycler PCR, and Sten Willemsen, Department of Epidemiology and Biostatistics of Erasmus MC University Medical Center Rotterdam, The Netherlands for his statistical advice. We would like to thank all volunteers who donated blood and participated in the studies.

Funding: This research is financed by the Dutch Arthritis Association (Reumafonds).

Disclosure statement: The authors have declared no conflicts of interest.


   References
Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 

  1. Madsen HO, Garred P, Thiel S, et al. Interplay between promoter and structural gene variants control basal serum level of mannan-binding protein. J Immunol (1995) 155:3013–20.[Abstract]
  2. Geleijns K, Roos A, Houwing-Duistermaat JJ, et al. Mannose-binding lectin contributes to the severity of Guillain-Barre syndrome. J Immunol (2006) 177:4211–7.[Abstract/Free Full Text]
  3. Biezeveld MH, Geissler J, Weverling GJ, et al. Polymorphisms in the mannose-binding lectin gene as determinants of age-defined risk of coronary artery lesions in Kawasaki disease. Arthritis Rheum (2006) 54:369–76.[CrossRef][Web of Science][Medline]
  4. Graudal NA, Madsen HO, Tarp U, et al. The association of variant mannose-binding lectin genotypes with radiographic outcome in rheumatoid arthritis. Arthritis Rheum (2000) 43:515–21.[CrossRef][Web of Science][Medline]
  5. Garred P, Madsen HO, Marquart H, et al. Two edged role of mannose binding lectin in rheumatoid arthritis: a cross sectional study. J Rheumatol (2000) 27:26–34.[Web of Science][Medline]
  6. Ip WK, Lau YL, Chan SY, et al. Mannose-binding lectin and rheumatoid arthritis in southern Chinese. Arthritis Rheum (2000) 43:1679–87.[CrossRef][Web of Science][Medline]
  7. Graudal NA, Homann C, Madsen HO, et al. Mannan binding lectin in rheumatoid arthritis. A longitudinal study. J Rheumatol (1998) 25:629–35.[Web of Science][Medline]
  8. Gupta B, Agrawal C, Raghav SK, et al. Association of mannose-binding lectin gene (MBL2) polymorphisms with rheumatoid arthritis in an Indian cohort of case-control samples. J Hum Genet (2005) 50:583–91.[CrossRef][Web of Science][Medline]
  9. Horiuchi T, Tsukamoto H, Morita C, et al. Mannose binding lectin (MBL) gene mutation is not a risk factor for systemic lupus erythematosus (SLE) and rheumatoid arthritis (RA) in Japanese. Genes Immun (2000) 1:464–6.[CrossRef][Web of Science][Medline]
  10. Stanworth SJ, Donn RP, Hassall A, Dawes P, Ollier W, Snowden N. Absence of an association between mannose-binding lectin polymorphism and rheumatoid arthritis. Br J Rheumatol (1998) 37:186–8.[Abstract/Free Full Text]
  11. Saevarsdottir S, Vikingsdottir T, Vikingsson A, Manfredsdottir V, Geirsson AJ, Valdimarsson H. Low mannose binding lectin predicts poor prognosis in patients with early rheumatoid arthritis. A prospective study. J Rheumatol (2001) 28:728–34.[Abstract/Free Full Text]
  12. Jacobsen S, Madsen HO, Klarlund M, et al. The influence of mannose binding lectin polymorphisms on disease outcome in early polyarthritis. TIRA Group. J Rheumatol (2001) 28:935–42.[Abstract/Free Full Text]
  13. Barton A, Platt H, Salway F, et al. Polymorphisms in the mannose binding lectin (MBL) gene are not associated with radiographic erosions in rheumatoid or inflammatory polyarthritis. J Rheumatol (2004) 31:442–7.[Abstract/Free Full Text]
  14. Chanock SJ, Manolio T, Boehnke M, et al. Replicating genotype-phenotype associations. Nature (2007) 447:655–60.[CrossRef][Medline]
  15. De Man YA, Hazes JM, Van de Geijn FE, Krommenhoek C, Dolhain RJ. Measuring disease activity and functionality during pregnancy in patients with rheumatoid arthritis. Arthritis Rheum (2007) 57:716–22.[CrossRef][Web of Science][Medline]
  16. Emonts M, de Jongh CE, Houwing-Duistermaat JJ, et al. Association between nasal carriage of Staphylococcus aureus and the human complement cascade activator serine protease C1 inhibitor (C1INH) valine vs. methionine polymorphism at amino acid position 480. FEMS Immunol Med Microbiol (2007) 50:330–2.[CrossRef][Web of Science][Medline]
  17. De Groot CJ, Bloemenkamp KW, Duvekot EJ, et al. Preeclampsia and genetic risk factors for thrombosis: a case-control study. Am J Obstet Gynecol (1999) 181:975–80.[CrossRef][Web of Science][Medline]
  18. Van de Geijn FE, Roos A, De Man YA, et al. Mannose-binding lectin levels during pregnancy: a longitudinal study. Hum Reprod (2007) 22:362–71.[Abstract/Free Full Text]
  19. Ytting H, Christensen IJ, Thiel S, et al. Biological variation in circulating levels of mannan-binding lectin (MBL) and MBL-associated serine protease-2 and the influence of age, gender and physical exercise. Scand J Immunol (2007) 66:458–64.[CrossRef][Web of Science][Medline]
Submitted 14 January 2008; revised version accepted 15 May 2008.
Add to CiteULike CiteULike   Add to Connotea Connotea   Add to Del.icio.us Del.icio.us    What's this?



This Article
Right arrow Abstract Freely available
Right arrow FREE Full Text (PDF) Freely available
Right arrow All Versions of this Article:
47/8/1168    most recent
ken226v1
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Add to My Personal Archive
Right arrow Download to citation manager
Right arrowRequest Permissions
Right arrow Disclaimer
Google Scholar
Right arrow Articles by van de Geijn, F. E.
Right arrow Articles by Dolhain, R. J. E. M.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by van de Geijn, F. E.
Right arrow Articles by Dolhain, R. J. E. M.
Related Collections
Right arrow Rheumatoid Arthritis
Social Bookmarking
Add to CiteULike   Add to Connotea   Add to Del.icio.us  
What's this?