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Rheumatology Advance Access originally published online on May 3, 2007
Rheumatology 2007 46(7):1076-1078; doi:10.1093/rheumatology/kem099
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© The Author 2007. 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 gene polymorphisms in a cohort study of ANCA-associated small vessel vasculitis

L. Kamesh1, J. M. Heward2, J. M. Williams1, S. C. L. Gough2, C. O. S. Savage1 and L. Harper1

1Division of Infection and Immunity and 2Division of Medical Sciences, Medical School, University of Birmingham B15 2TT, UK.

Correspondence to: Lorraine Harper, Division of Immunity and Infection, The Medical School, University of Birmingham, Edgbaston, Birmingham B15 2TT, UK. E-mail: l.harper{at}bham.ac.uk


   Abstract
Top
 Abstract
Introduction
 Methods
 Results
 Discussion
 Acknowledgements
 References
 
Objective. To investigate whether single nucleotide polymorphisms (SNPs) within the mannose-binding lectin (MBL) gene are associated with small vessel vasculitis (SVV) and are a risk factor for intercurrent infection, as described previously in other autoimmune diseases.

Methods. Six SNPs in the MBL promoter and coding region were genotyped by sequence-specific polymerase chain reaction or restriction fragment length polymorphism assay in 170 white Caucasians with SVV and 372 ethnically matched controls in a case-control association study. Serum MBL levels were measured by ELISA. The genotype and protein concentrations were correlated to clinical details retrieved from hospital records.

Results. No differences in allelic and genotypic frequencies were detected between patients with SVV and control subjects. MBL deficiency did not increase the susceptibility to infection (P = 0.6, Fisher's exact test) or the duration of hospital stay.

Conclusion. Our data suggest that MBL polymorphisms are not associated with SVV and do not influence the incidence of concomitant infections. These results raise doubts about the usefulness of MBL polymorphisms as a predictive marker for infection in SVV.

KEY WORDS: Vasculitis, Infection, Polymorphisms, Immunocompromised, Auto antibody, ANCA


   Introduction
Top
 Abstract
 Introduction
Methods
 Results
 Discussion
 Acknowledgements
 References
 
It is well recognized that anti-neutrophil cytoplasmic antibody (ANCA)-associated small vessel vasculitis (SVV) if left untreated leads to high mortality. The introduction of cyclophosphamide and high-dose steroids has improved survival [1] but at risk of substantial morbidity related to infections. A recent retrospective analysis from our centre on 240 consecutive patients with SVV confirmed the increased burden of infection contributing to 36% of deaths in vasculitis [2]. Identification of patients at increased risk of infection may improve prognosis.

Mannose-binding lectin (MBL) is a liver-derived protein that plays an important role in innate immunity. It interacts with MBL-associated serine proteases (MASP-1, MASP-2 and MASP-3) and activates the lectin pathway of the complement system. It also recognizes sugar structures on micro-organisms and helps in opsonophagocytosis. The MBL gene is located on the long arm of chromosome 10. Three single nucleotide polymorphisms (SNPs) have been described in the exon 1 at codons 52 (C-T, allele D), 54 (G-A, allele B) and 57 (G-A, allele C) and they disrupt the collagenous backbone of the protein. The normal allele is referred to as A and any of the structural variants as allele O. In addition, three promoter polymorphisms at positions –550 (G-C, alleles H/L), –220 (G-C, alleles X/Y) and +4 (C-T, alleles P/Q) of the MBL gene (3) alter serum levels by affecting transcription. Because of linkage disequilibrium, these variants combine to form seven haplotypes—HYPA, LYPA, LYQA, LXPA, LYPB, LYQC and HYPD. Serum concentrations of MBL vary widely between individuals based on their haplotype [3].

It has been suggested that MBL deficiency becomes clinically important when found in conjunction with another deficiency of the immune response. Low MBL levels were associated with increased incidence of bacteraemia and pneumonia in adults treated for haematological malignancies [4], whilst in children with cancer, it prolonged the duration of febrile neutropenic episodes [5]. Similar results were evident in autoimmune diseases. Garred et al. showed that patients with systemic lupus erythematosus (SLE) and homozygous variant alleles had an increased risk of acquiring serious infection (odds ratio 8.6, P = 0.01), especially pneumonia when compared with those with normal alleles [6]. The above studies provide compelling evidence that MBL deficiency may be a susceptibility factor for infection.

MBL, may also pre-dispose to autoimmune SLE (6) and may also act as a disease modifier. Variant alleles are associated with renal involvement in SLE [6] and early onset disease and erosive rheumatoid arthritis [7]. To date, no study has addressed MBL deficiency in patients with autoimmune SVV. As MBL replacement is available [8], this could be considered as a potential therapeutic option. In this study, we compare the frequency of the three structural and three promoter polymorphisms in a cross sectional cohort of 170 patients with SVV and 372 controls. Further, we examine whether SNPs of the MBL gene and low-serum MBL levels are a risk factor for the development of intercurrent infections in these patients.


   Methods
Top
 Abstract
 Introduction
 Methods
Results
 Discussion
 Acknowledgements
 References
 
Patients and controls
One hundred and seventy white Caucasians of UK origin with SVV were recruited from a major teaching hospital (University Hospitals NHS Foundation Trust; Birmingham). Patients were allocated to sub-groups according to the Chapel Hill consensus definitions [9]. Wegener's granulomatosis (WG) was diagnosed if there was evidence of granulomatous disease on biopsy or presence of clinical signs suggestive of granulomatous upper respiratory tract involvement. Microscopic polyangiitis (MPA) was diagnosed if there was evidence of SVV and absence of granuloma on biopsy and absence of clinical signs suggestive of granulomatous upper respiratory tract involvement. Churg Strauss syndrome was diagnosed in the presence of eosinophilia, asthma and eosinophil-rich granulomatous involvement on biopsy. ANCA specificity was not used as a diagnostic criterion [2]. ANCA were defined as either cytoplasmic (c-ANCA) or peri-nuclear (p-ANCA) as determined by indirect immunofluorescence. Disease susceptibility was examined across the whole cohort of SVV and also separately in the sub-groups of WG and MPA. Three hundred and seventy-two healthy white Caucasians with no family history of autoimmune disease served as controls and they were recruited at Blood Transfusion Service, Birmingham Heartlands Hospital and the Queen Elizabeth Hospital, Birmingham. The local ethics committee approved the study and informed written consent was obtained from each patient and control subject according to the declaration of Helsinki. Data were collected retrospectively from the case notes. All episodes of infection that required hospital admission were analysed. Patients received steroids and cyclophosphamide at induction and azathioprine or mycophenolate as maintenance therapy. Infection risk was analysed across the whole cohort, as patients were treated with standard immunosuppressive regimens irrespective of disease type.

MBL genotyping
Genomic DNA was extracted from 15 ml of whole blood using Nucleon Bacc III kit (Tepnel Life Sciences; UK). Exon 1 of the MBL gene was amplified in a 25 µl volume containing 20 ng of genomic DNA and 50 ng of specific primers as previously described [3]. Genotyping for the structural alleles B and C was carried out by restriction fragment length polymorphism (RFLP) using restriction enzymes Ban I and Mbo II. A sequence-specific (SSP–PCR) method was used for detecting MBL allele D and the promoter polymorphisms X/Y, H/L, P/Q as described previously by Steffensen et al. [10].

Serum MBL levels
MBL levels were measured in the serum by enzyme-linked immunosorbent assay (ELISA) using Mannan-binding ELISA kit (Antibody Shop, Denmark) as per manufacturer's instructions. Serum samples were collected in the remission phase of the illness.

Statistical methods
Genotype and allele frequency analysis was performed using the chi-square test (SPSS 13.0 for Windows; SPSS Inc., Chicago, IL, USA). Power calculations and Hardy–Weinberg Equilibrium status were determined using Microsoft Excel (Microsoft® Office Excel, ©2003). Medians and ranges are reported for non-normally distributed data. Differences between frequencies and medians were tested using chi-square and Kruskal–Wallis tests, respectively. Analysis of the infection-free period was performed by Kaplan–Meier plot and log-rank test. All tests were two-tailed and P < 0.05 was considered as significant.


   Results
Top
 Abstract
 Introduction
 Methods
 Results
Discussion
 Acknowledgements
 References
 
Structural allele and genotype frequencies in patients and controls are shown in Table 1. No significant differences were observed in the structural allele frequencies ({chi}2 = 0.953; P = 0.8, 3 degrees of freedom) or in the genotype distributions ({chi}2 = 0.859; P = 0.6, 2 degrees of freedom) between patient and control populations. All results conformed to Hardy–Weinberg Equilibrium. The study has a power of 74% to detect an effect due to the polymorphism assuming an odds ratio of 1.5 and a P-value of 0.05. Promoter allele genotype and haplotype frequencies ({chi}2 = 3.945, P = 0.691) (data not shown) in patients were not significantly different from that of the controls.


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  		TABLE 1. Genotype and allele frequencies of structural mannose binding lectin polymorphisms in patients with small vessel vasculitis and in control subjects

 
MBL status and risk of infection
Seventy-one patients (41.7%) developed a combined total of 129 episodes of infection requiring hospitalization. The common infections noted in the whole cohort were respiratory (49%), gastro-intestinal (17%), urinary tract (8.5%) afflictions, bacteraemia/sepsis (11.6%) and endocarditis (4%). Nineteen patients (11%) developed infective episodes associated with leucopoenia. The structural polymorphisms of MBL were not associated with increased risk of infection as shown in Table 2. The time period to first infection from diagnosis of SVV was similar and there was no significant correlation of variant alleles with the type of infection or any particular organism. Fourteen patients died during the course of the study and none of them had the homozygous mutation (O/O).


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  		TABLE 2. Incidence of infections requiring hospitalization in patients with SVV in relation to MBL genotypes

 
Serum MBL levels were measured in 137 patients (3). A cut-off of 600 ng/ml was adapted to denote MBL ‘insufficiency’ as the majority of individuals with heterozygous (A/O) or homozygous O/O mutation has MBL concentrations below 600 ng/ml. Thirty-seven (21.7%) patients with MBL levels <600 ng/ml developed 11 episodes of infection while 92 patients with >600 ng/ml developed 39 infection episodes (P-value of 0.155, log rank test).

Relationship of clinical syndromes to MBL structural genotype
When we considered WG (89 cases; {chi}2 = 1.013; P = 0.603, 2 degrees of freedom) and MPA (76 cases; {chi}2 = 0.878; P = 0.645, 2 degrees of freedom) individually and compared with 372 controls, no association was seen between MBL SNPs and susceptibility to the different clinical syndromes. The disease extent index, a validated disease assessment tool [11], was similar amongst the three MBL structural genotypes. ANCA was present in 89% of patients by indirect immunofluorescence. There was no relationship between the type of ANCA and MBL genotype.


   Discussion
Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
Acknowledgements
 References
 
The genetic basis of vasculitidies such as SVV, which occurs mainly in Caucasians, remains unclear. Reports have suggested associations with HLA-DPB1 *0401 allele [12], PiZZ phenotype of alpha 1 antitrypsin [13] and longer alleles of the (AT)n microsatellite of cytotoxic T-lymphocyte antigen-4 (CTLA4) [14] genes in WG. However, major genetic contributions to SVV are yet to be identified. Our current data suggest that MBL is not a major susceptibility factor in the aetiology of the WG or MPA.

Infection is an important side effect of treatment in patients with SVV, especially in the elderly. This is the first study to examine MBL polymorphisms in a large cohort of patients with SVV and shows a lack of association between MBL variant alleles and infections requiring hospitalization. This negative result could be due to clinical interventions that alter risk of infection. Our own policy is to include septrin as prophylaxis for pneumocyctis carinii. Despite this infection remains a significant concern [2]. It is well known that leucopoenia is an important side effect of chemotherapy and it increases susceptibility to infection. We routinely use granulocyte colony-stimulating factor (GCSF) to improve white cell counts and clinical outcome.

It should be noted that the actual numbers of low MBL-producing genotypes in some of the studies that focused on infections, range from 5–20 [6, 15]. It is difficult to derive meaningful conclusions from correlations with clinical parameters based on such small sub-groups.

During an acute inflammatory illness, MBL levels can rise up to 3-fold, although such increase may be restricted in patients with structural mutations [5]. Thirty-seven patients (21.7%) had low MBL levels during remission, which did not appear to contribute to increased incidence of infection. Recent studies in cancer chemotherapy have also failed to confirm the role of MBL insufficiency as a risk factor for developing infection [15–17]. All these data suggest that MBL does not play a uniform role in preventing infections in the context of chemotherapy [18]. Our cross-sectional study may have its limitations as death may have prevented some patients from being genotyped. However, selection bias is unlikely as the MBL genotype distribution is similar to other studies involving UK-based Caucasians [19] and it conforms to the Hardy–Weinberg equilibrium [20].

In conclusion, our study suggests that the described MBL polymorphisms are not associated with SVV. Moreover, it is doubtful whether MBL polymorphisms may influence or predict infections in this group of patients.

Formula


   Acknowledgements
Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 Acknowledgements
References
 
The authors thank Dr P. Nightingale for generous support and guidance in the statistical analysis of the work presented and Dr D. Adu for the clinical support. This study is supported by grants from Kidney Research UK and Dunhill Medical Trust.

The authors have declared no conflicts of interest.


   References
Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 Acknowledgements
 References
 

  1. Booth AD, Almond MK, Burns A, et al. Outcome of ANCA-associated renal vasculitis: a 5-year retrospective study. Am J Kidney Dis (2003) 41:776–84.[Web of Science][Medline]
  2. Harper L, Savage CO. ANCA-associated renal vasculitis at the end of the twentieth century–a disease of older patients. Rheumatology (2005) 44:495–501.[Abstract/Free Full Text]
  3. 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]
  4. Peterslund NA, Koch C, Jensenius JC, Thiel S. Association between deficiency of mannose-binding lectin and severe infections after chemotherapy. Lancet (2001) 358:637–8.[CrossRef][Web of Science][Medline]
  5. Neth O, Hann I, Turner MW, Klein NJ. Deficiency of mannose-binding lectin and burden of infection in children with malignancy: a prospective study. Lancet (2001) 358:614–18.[CrossRef][Web of Science][Medline]
  6. Garred P, Madsen HO, Halberg P, et al. Mannose-binding lectin polymorphisms and susceptibility to infection in systemic lupus erythematosus. Arthritis Rheum (1999) 42:2145–52.[CrossRef][Web of Science][Medline]
  7. 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]
  8. Valdimarsson H, Vikingsdottir T, Bang P, et al. Human plasma-derived mannose-binding lectin: a phase I safety and pharmacokinetic study. Scand J Immunol (2004) 59:97–102.[CrossRef][Web of Science][Medline]
  9. Jennette JC, Falk RJ, Andrassy K, et al. Nomenclature of systemic vasculitides. Proposal of an international consensus conference. Arthritis Rheum (1994) 37:187–92.[Web of Science][Medline]
  10. Steffensen R, Thiel S, Varming K, Jersild C, Jensenius JC. Detection of structural gene mutations and promoter polymorphisms in the mannan-binding lectin (MBL) gene by polymerase chain reaction with sequence-specific primers. J Immunol Methods (2000) 241:33–42.[CrossRef][Web of Science][Medline]
  11. de Groot K, Gross WL, Herlyn K, Reinhold-Keller E. Development and validation of a disease extent index for Wegener's granulomatosis. Clin Nephrol (2001) 55:31–8.[Web of Science][Medline]
  12. Jagiello P, Gencik M, Arning L, et al. New genomic region for Wegener's granulomatosis as revealed by an extended association screen with 202 apoptosis-related genes. Hum Genet (2004) 114:468–77.[CrossRef][Web of Science][Medline]
  13. Esnault VL, Testa A, Audrain M, et al. Alpha 1-antitrypsin genetic polymorphism in ANCA-positive systemic vasculitis. Kidney Int (1993) 43:1329–32.[Web of Science][Medline]
  14. Huang D, Giscombe R, Zhou Y, Lefvert AK. Polymorphisms in CTLA-4 but not tumor necrosis factor-alpha or interleukin 1beta genes are associated with Wegener's granulomatosis. J Rheumatol (2000) 27:397–401.[Web of Science][Medline]
  15. Rocha V, Franco RF, Porcher R, Bittencourt H, Silva WA Jr, Latouche A, et al. Host defense and inflammatory gene polymorphisms are associated with outcomes after HLA-identical sibling bone marrow transplantation. Blood (2002) 100:3908–18. Epub 2002 Jul 25.[Abstract/Free Full Text]
  16. Bergmann OJ, Christiansen M, Laursen I, et al. Low levels of mannose-binding lectin do not affect occurrence of severe infections or duration of fever in acute myeloid leukaemia during remission induction therapy. Eur J Haematol (2003) 70:91–7.[CrossRef][Web of Science][Medline]
  17. Kilpatrick DC, McLintock LA, Allan EK, et al. No strong relationship between mannan binding lectin or plasma ficolins and chemotherapy-related infections. Clin Exp Immunol (2003) 134:279–84.[CrossRef][Web of Science][Medline]
  18. Klein NJ, Kilpatrick DC. Is there a role for mannan/mannose-binding lectin (MBL) in defence against infection following chemotherapy for cancer? Clin Exp Immunol (2004) 138:202–4.[CrossRef][Web of Science][Medline]
  19. Roy S, Knox K, Segal S, et al. MBL genotype and risk of invasive pneumococcal disease: a case-control study. Lancet (2002) 359:1569–73.[CrossRef][Web of Science][Medline]
  20. Dahl M, Tybjaerg-Hansen A, Schnohr P, Nordestgaard BG. A population-based study of morbidity and mortality in mannose-binding lectin deficiency. J Exp Med (2004) 199:1391–9.[Abstract/Free Full Text]
Submitted 7 January 2007; revised version accepted 22 March 2007.
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