Download Mannose binding lectin and FccRIIa (CD32

Rheumatology 2001;40:1009– 1012
Mannose binding lectin and FccRIIa (CD32)
polymorphism in Spanish systemic lupus
erythematosus patients
J. Villarreal*, D. Crosdale1,*, W. Ollier1, A. Hajeer1,
W. Thomson1, J. Ordi, E. Balada, M. Villardell, L.-S. Teh2 and
K. Poulton3
Hospital General Vall d’Hebron, Passieg Vall d’Hebron 119-129, Barcelona,
Spain, 1Arthritis and Rheumatism Campaign Epidemiology Unit, School of
Epidemiology and Health Sciences, Medical School, University of Manchester,
Oxford Road, Manchester M13 9PT, 2Department of Rheumatology, Blackburn
Royal Infirmary, Bolton Road, Blackburn BB2 3LR and 3Manchester Institute
of Nephrology and Transplantation, Manchester Royal Infirmary, Oxford Road,
Manchester M13 9WL, UK
Abstract
Objective. Mannose binding lectin (MBL) and FccRII (CD32) polymorphisms have both been
implicated as candidate susceptibility genes in systemic lupus erythematosus (SLE). The aim of
this study was to evaluate the relationship of these polymorphisms with SLE.
Methods. We studied a cohort of 125 SLE patients from Barcelona, Spain and 138
geographically matched controls. Sequence-specific primer–polymerase chain reaction
(SSP–PCR) amplification was used to determine CD32 and MBL structural polymorphisms.
MBL haplotypes were established using sequence-specific oligonucleotide probing techniques.
Results. Patients carried the MBL codon 54 mutant allele more frequently than controls
wodds ratio (OR) 2.2; 95% confidence interval (CI) 1.2–4.0; P = 0.007x and the haplotype
HY W52 W54 W57 was found to be significantly lower in cases compared with controls
(OR 0.6; 95% CI 0.4 – 0.9; P = 0.016).
Conclusion. The MBL gene codon 54 mutant allele appears to be a risk factor for SLE, whilst
haplotypes encoding for high levels of MBL are protective against the disease. Differences
between controls and patients were not significant when considering the FccRIIa
polymorphisms; similar results were observed for renal affectation.
KEY WORDS: MBL, FccRIIa, Polymorphism, SLE.
Systemic lupus erythematosus (SLE) is a complex
autoimmune disease influenced by both genetic and
environmental factors w1, 2x. Homozygosity for a number of complement and immunity-related genes has
been implicated in the susceptibility to SLE. These
include deficiencies of complement components such
as C1q, C2, C4, w3–5x, or mannose binding lectin
(MBL) w6–9x and the abnormal distribution of FccRIIa
allotypes w10–14x.
MBL is an acute-phase serum protein w15x of the
innate immune system, which participates in complement activation and the opsonization of antigens. The
Submitted 13 June 2000; revised version accepted 13 March 2001.
* These authors contributed equally to this work.
Correspondence to: D. J. Crosdale, Clinical Sciences Building,
Northern General Hospital, Herries Road, Sheffield S5 7AU, UK.
protein coding region of the gene consists of four exons,
and there are five known polymorphic sites within the
MBL gene that are thought to affect the amount of
protein in serum and have been associated with SLE.
Two are situated within the promoter region of the
MBL gene wH or L (G to C) at 2550 and Y or X (G to C)
at 2221x w16x. The remaining three polymorphic sites are
situated within exon 1 of the MBL gene at codon 52
(Arg to Cys) w17x, codon 54 (Gly to Asp) w18x and codon
57 (Gly to Glu) w16x. A number of MBL alleles exist in
linkage together. The codon 52 mutation is only found
together with the promoter haplotype HY, whereas
codon 54 and 57 mutant alleles are found to exist with
LY w17x.
Human immunoglobulin Fc receptors (FcR) on
leucocytes exhibit considerable structural and functional
diversity. Within the groups of receptors for IgG there
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ß 2001 British Society for Rheumatology
1010
J. Villarreal et al.
are three distinct subgroups; FccRI (CD64), FccRII
(CD32), and FccRIII (CD16), each containing several
different members. The ‘A’ isoform of FccRII is the
only receptor on human phagocytic cells that is capable
of significant interaction with IgG2 w19x. Two isoforms
of FccRIIa are FccRIIa R and FccRIIa H; these differ
by one amino acid at position 131 (Arg to His) which
results from a single base substitution. The variant containing histidine at this position (FccRIIa-H131) binds
human IgG2, and IgG3, more strongly than the ‘lowaffinity’ isoform containing arginine (FccRIIa-R131)
w19x. Fcc receptors play an essential role in the clearance
of immune complexes. Impaired clearance and removal
of immune complexes by the mononuclear phagocyte
system can result in the deposition of immune complexes
in organs and tissues. In SLE patients, the FccRIIaR131 allotype of CD32 receptor has been associated
with renal disease; it was observed that there is an overrepresentation of FccRIIa-R131 homozygosities in SLE
patients with renal involvement, compared with patients
homozygous for the FccRIIa-H131 allele w13, 14x.
We hypothesized that within our population of
Spanish SLE patients there would be an overrepresentation of MBL mutant alleles and FccRIIa
alleles compared with controls. There would also be an
increase in FccRIIa allele frequency in patients with
renal involvement compared with those without.
Materials and methods
One hundred and twenty-five SLE patients were
recruited from the Vall d’Hebron Hospital in Barcelona,
Spain. These were compared with an ethnically matched
random healthy control population recruited from the
same geographical region (n = 137). All patients satisfied the 1982 American College of Rheumatology
revised criteria for SLE w20x. The clinical manifestations studied were renal involvement (n = 48), articular
affectation (n = 102), cutaneous lesions (n = 90),
neurological disease (n = 20), and serositis (n = 45).
MBL typing
DNA was extracted from ethylene diamine tetraacetic
acid (EDTA) blood samples using the DNAce2
MaxiBlood Purification System (Bioline). MBL codon
52, 54 and 57 wild and mutant alleles were detected
using sequence-specific primer–polymerase chain reactions (SSP–PCR) as previously described by Crosdale
et al. w21x. In total, 1042 bp of the promoter and the
majority of exon 1 of the MBL gene were amplified. A
464 bp segment of the gene for human growth hormone
was amplified as an internal control for each PCR
reaction. SSP–PCR reactions were performed in the
presence of 1.5 mM MgCl2, 0.2 mM of each deoxynucleotide, 0.5 mM of each primer, in NH4SO4 buffer
(Bioline) with 1 unit BioTaq polymerase and 1 M Betaine.
Two microlitres of each PCR product was blotted
using a Robbins automatic dot blotter. This was
alkaline denatured and fixed on to Hybond N+ Nylon
membranes (Amersham). Promoter alleles were determined by sequence-specific oligonucleotide probing
(SSOP) of the bound PCR product using biotinylated
probes, which were previously described by Madsen
et al. w22x.
All haplotype combinations were determined by the
SSP–PCR and SSOP techniques as described. However,
a number of individuals could not be haplotyped using
these methods alone due to each sample possessing only
structurally encoding wild alleles as determined by SSP–
PCR and being positive for X, Y, L and H polymorphisms. In such a situation, it was impossible to determine
the cisutrans orientation of the promoter alleles. This was
resolved by performing further SSP–PCR reactions with
HuL forward primers (59 to 39) and XuY reverse primers
as described by Crosdale et al. w21x.
A number of samples were cloned and sequenced
(MBL gene exon 1 and promoter region) to provide
control material for this study.
FccRIIa typing
Each sample was typed for the presence of FccRIIa-H
and FccRIIa-R alleles using the method and primers
described by Smyth et al. w23x. Two PCR reactions were
performed on each sample, both utilizing a common
anti-sense primer located downstream of the HuR
polymorphism and one of the two allele-specific primers. A second amplification was also made between the
anti-sense primer and a common sense primer situated
upstream of the HuR polymorphism as an internal
control. The control primers amplified 256 bases of the
FccRIIa gene, whilst the HuR-specific reaction amplified
224 bases.
Statistical analysis
x2 analysis (using Yates’ correction, where applicable)
was performed to determine the significance of a
frequency difference between the two groups (significance level of 5%). Odds ratios (OR) were calculated for
significant associations and expressed with 95% confidence intervals (CI). An OR was considered to be
significant if the 95% CI did not include 1.0.
Results
The allele frequencies of the structurally encoding
MBL gene and promoter polymorphisms are summarized in Table 1. There was a significant increase in the
frequency of 2 550 L allele in the patient group compared with the control population (OR 1.5; 95% CI
1.0–2.1; P = 0.039) and also in the number of patients
possessing the codon 54 mutant allele compared with
controls (OR 2.2; 95% CI 1.2–4.0; P = 0.007). The
codon 57 mutant allele frequency was also increased in
the patient group, but this result did not reach statistical
significance.
MBL haplotype frequencies for both the SLE
and control populations are shown in Table 2. The
frequency of the HY W52 W54 W57 haplotype
MBL and FccRIIa polymorphism in Spanish SLE patients
TABLE 1. Allele (promoter variants) and phenotype (structurally
encoding) frequencies (%) of MBL polymorphisms in Spanish SLE
patients and controls
Patients
(n = 125)
Allele
2550 HuL
2221 XuY
Codon 52
Codon 54
Codon 57
H
L
X
Y
W
M
W
M
W
M
30.4
69.6a
14.6
85.4
100
10.8
97.5
30.8b
100
5
Controls
(n = 138)
39.1
60.9
18.5
82.5
95.2
12.3
98.6
16.7
100
2.2
W, wild type; M, mutant or dysfunctional allele.
a
OR 1.5; 95% CI 1.0–2.1; P = 0.039.
b
OR 2.2; 95% CI 1.2–4.0; P = 0.007.
TABLE 2. MBL haplotype frequencies (%) in SLE patients and controls
MBL haplotype
HY W52 W54 W57
LY W52 W54 W57
LX W52 W54 W57
HY M52 W54 W57
LY W52 M54 W57
LY W52 W54 M57
Patients
(n = 125)
23.3a
40.4
14.2
5.4
14.2
2.1
Controls
(n = 138)
32.9
32.2
18.5
6.2
9.1
1.1
OR 0.6; 95% CI 0.4–0.9; P = 0.016.
a
was significantly decreased in the patient population
(23.3%) compared with the control group (32.9%) (OR
0.6; 95% CI 0.4–0.9; P = 0.016). Haplotypes containing
structurally encoding mutant alleles were generally of
increased frequency in the SLE population, compared
with controls.
With regard to FccRIIa, no differences were found
between patients (H = 41.3%, R = 58.7%) and controls
(H = 42.3%, R = 57.7%). An increased genotype frequency of RuR homozygotes was seen in SLE patients
with renal involvement (32.7%) compared with those
without renal involvement (22.1%). There was also an
increase in the R allele frequency in those patients with
renal involvement (63.3%) compared with those without
(55.8%). However, both of these findings failed to reach
statistical significance. Similar results were observed
when considering cutaneous affectation, neurological
disorders, arthritis or serositis.
Possession of both FccRIIa-R131 and any structurally encoding MBL mutant allele was increased
within the SLE population (35.3%) compared with
controls (25.0%). Possession of MBL codon 54 mutant
alleles together with FccRIIa-R131 alleles also showed
an overall increase from 14.0% in the control group
to 23.2% in the patients. Both of these differences,
however, failed to reach statistical significance.
1011
Discussion
Previous studies have shown MBL gene mutations at
codons 54 and 57 as being additive risk factors for
susceptibility to SLE in different populations w6–9x. In
this study we found an increased phenotypic frequency
of codon 54 and 57 mutant alleles in SLE patients compared with controls. Nevertheless, the increase observed
for codon 57 mutant alleles was not sufficiently high
within this sample size to reach significance, which
may reflect the rarity of the codon 57 mutant allele in
populations of Spanish descent.
The MBL haplotype distribution within our control
population was consistent with those of previous studies
w22x. Codon 52 mutant alleles were found to be in
linkage disequilibrium with HY promoter polymorphisms and both codon 54 and 57 mutant alleles carried
only LY promoter alleles on their haplotypes. We
hypothesized that MBL haplotypes encoding for lowlevel production of the protein would be more prevalent
within an SLE population compared with an ethnically
matched control population. Our results suggest that
this is indeed the case.
There was an increase in the frequency of the
intermediate-level MBL-producing haplotype LY W52
W54 W57 within the SLE population compared with
controls and a significant increase in the high-level
MBL-producing HY W52 W54 W57 haplotype frequency within the control group. This finding provides
evidence that MBL haplotypes encoding for high serum
levels of the protein are protective against the development of SLE. The protective nature of the high serum
level-producing MBL haplotypes may become more
apparent during an acute-phase response when baseline
levels of MBL can increase up to 4-fold w24x.
A significantly increased number of codon 54 mutant
alleles was observed when comparing SLE patients
with renal disease (18u90 alleles, 20.0%) with those
without (15u150 alleles, 10.0%) (OR 2.3; 95% CI 1.1–4.7;
P = 0.029). The codon 54 mutant allele therefore
appears to be acting as a susceptibility factor for the
development of renal disease in patients with SLE.
Heterozygotes for the MBL codon 54 mutation have
approximately one-eighth of the serum protein concentration they would have if encoded by wild alleles w18x.
This would account for a reduction in immune complex clearance and complement activation in these
individuals, which could ultimately lead to increased
susceptibility to renal disease in SLE patients.
FccRIIa-R131 homozygosity has previously been
associated with renal involvement in SLE patients w13x.
In this study, we found no differences between RuR
homozygosity in SLE patients compared with controls.
This result, together with the R allele frequencies that
are approximately equal between populations, suggests
that there are no differences in IgG2 and IgG3 immune
complex clearance between patients and controls and
that the FccRIIa-R131 allele is not a susceptibility
factor to the development of SLE. However, a 10%
increase in FccRIIa-R131 homozygosity was observed
1012
J. Villarreal et al.
in patients with renal disease. This finding, although
not significant, suggests decreased levels of IgG2 and
IgG3 immune complex clearance in patients with renal
disease. A lack of immune complex clearance could
result in their accumulation within the blood, especially
within densely packed capillary areas such as the
kidneys, possibly resulting in the development or onset
of renal disease.
We analysed our data to look for any differences
in the frequencies of both FccRIIa and MBL structural
mutant alleles between the SLE population and controls. Increases were seen in the frequencies of both
codon 54 mutant and FccRIIa-R131 alleles in the SLE
population. These results approached statistical significance and therefore suggest that within our SLE
population there is a trend towards impaired immune
complex clearance when compared with the control
group. This impaired immune complex clearance may,
together with a number of other additive factors such as
early complement component deficiencies, contribute to
disease susceptibility.
11.
12.
13.
14.
15.
16.
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