1
Peter Garred
MBL mangel og dets betydning ved
lungesygdom
Peter Garred
MBL mangel og dets betydning ved
lungesygdom
Pathogen-Associated Molecular Patterns
Innate immunity
-liposaccharides
-glucans
-bacterial DNA
-viral dsDNA
Pattern recognition receptors
-cell bound (TLRs)
-intracellular (NOD/CARD)
-soluble
Phagocytic cell
Ficolins and MBL
DNABacteria
CRD domain Collagen-like domain FBG domain
Neck region Cysteine rich region Linker region
MBL Ficolins
Interaction with ligands
Complement activation
MBL Ficolin-1, -2 and -3 Functions of MBL
Phagocyte
MBL
receptor?
Dying cell
Normal cell
complement activation
via associated serine
proteases (MASPs) Clearance of
Cellular debris
Phagocytosis
C9
C5b-8
Microorganism
C3bC4bC2b
C4,C2 C3
Membrane attack complex
MASPs
MBL attack
MBL MBL
C5a
C3b
1, 2, 3MBL-MASPs
Sir Frances Mcfarlane Burnet, 1899-1985
Nobel Laureate in 1960 in medicineProbably discovered MBL in 1946 as ββββ-inhibitor of influenza virus
2
Mannose-binding lectin history1968: deficiency of serum activity phagocytosis was observed in a little girl suffering from recurrent infections
Mannose-bindinding lectin (MBL) history1978 Japanese scientists discovers MBL in rabbit liver
1987 Japanese scientists discovers that MBL is a part
of the complement system
1989 English og Danish scientists discovers that theserum deficiency was caused by MBL deficiency
1989 English og American scientists clone the MBLgene (MBL2)
1991 English scientists discovers a mutation in the MBL2gene causing MBL deficiency
1992 Japanese scientists discovers the lectin pathway
of complement activation
C-type CRD domain
Coiled coil neck region
Collagen-like domain
N-terminal cysteines
MBL 25 Kd polypeptide
MBL tetramer
5’ 3’MBL2 genePromoter 1Promoter 0
Ex1 Ex2 Ex3 Ex4Ex0
Three SNPs present on different MBL2 haplotypes result in low levels of functional
serum MBL
Codon:
Name:
52 54 57
Arg
Cys
Gly
Asp
Gly
Glu
D B C
Mutation
A/A A/B A/C A/D B/B+D/D+B/D etc
0
1000
2000
3000
4000
MBL serum Koncentration (µg/L)
4889
MBL2 structural gene variations
A= normal typeB= glycine til aspartic acid54
C= glycine til glutamic acid57
D= arginine til cysteine52
60% 23% 4% 8% 5%Denmark
1994
TATA box
transcription
start
exon 1
X/YH/L(- 550) (- 221)(position)
DBC
GRE
HSE
GC box
UT
Allele:
(codons 52,54,57)
CCAAT box
Promoter region and exon 1 of the MBL2 gene
3
0
500
1000
1500
2000
2500
3000
3500
4000
4500
5000
MBL koncentration (µg/L)
HY/HY LY/LY LX/LX HY/LY HY/LX
A/A genotype
Danes LY/LX1995
0
200
400
600
800
1000
1200
1400
MBL koncentration (µg/L)
HY LY LX
A/O genotype, O = B, C or D
Danes 1995
Variant ELISA
-100
0
100
200
300
400
500
OD
490
nm
-100
-50
0
50
100
150
200
250
300
350
400
OD
49
0 n
m
1 0 12 ,5 15 17 ,5 20 22 ,5 25 27 ,5 30 32 ,5Frac tions
MW, 1300 kD, 667 kD, 443 kD, 200 kD, 150 kD, 67 kD
-100
0
100
200
300
400
500
600
OD
49
0 nm
B/B
A/B
A/A
Gel filtration (Superdex 200) of sera with different MBL2 genotypes and subsequent testing for MBL reactivity in ELISA
2003
50 Kd
75 Kd
100 Kd
150 Kd
250 Kd
YA/XA XA/XA XA/D YA/B B/D D/D
Reduced
MBL oligomers
Unreduced
32 Kd
2003
MBLMBLMBLMBL
ClinicalClinicalClinicalClinical importanceimportanceimportanceimportance????
0 6 12 18 24
Maternal IgG abs Child’s IgG abs
Period of vulnerabilityhypothesis of MBL function
Age (months)
Normal MBL level
1989
4
Antigenfirst time
Antigensecond time
4 8 12 16 20 64 68Days
Primary response Secondary response
Antibodyconcentration
Lag phase
Ante-antibody hypothesis of MBL function
MBL concentration
1989
Sisimiut
Acute Respiratory Tract Infections and MBL Insufficiency During Early Childhood, the Greenland Study
Anders Koch et al, JAMA 200l
Cumulative risk of acute respiratory tract infections by MBL2genotypes in 252 children followed prospectively for 2 years
1
1,47
1
2,92
1 1
0
0,5
1
1,5
2
2,5
3relative risk
0-5 months 6-17 months 18-24 months
A/A+YA/0XA/0+0/0
Anders Koch et al, JAMA 200l
Sisimiut, West-Greenland
P<0.001
Common Variable ImmunodeficiencyCVID
Heterogeneous group of immunologicaldisorders, unknown etiology, ↓↓↓↓ levels of serum immunoglobulins and impaired antibody responses
0
10
20
30
40
50
60
70
80
�
Mu
tate
d f
ract
ion
(%
)
No pneumonia 2 > pneumoniaper year
MBL A/O+O/O genotypes
MBL A/A genotype
P<0.001
Andersen et al., Blood 2005
Pneumonia up to 2 years pre-diagnostic in CVID patients by somatic hypermutation and MBL2 variant alleles
2 ≤ pneumoniaper year
Low Serum Mannose-Binding Lectin Level Increases the Risk of Death due to Pneumococcal Infection
Forest plot of association between mannose-binding lectin(MBL) deficiency (MBL level, 0.5
microgram/mL) and death
Diamond Eisen et al., Clin. Infect. Dis. 2008
5
Cystic fibrosis (CF) is the most common severe inheriteddisease among Caucasians There are about 400 CF patients in DanmarkLife expectancy is about 40 years
Clinical symptoms of CFSalty taste of the skinFailure to growBulky and greasy stool (steatorrhea)Thick sputumCoughing or wheezingFrequent recurring pneumonia (spec. P. aeruginosa)
The symptoms may differ with varying degree of severityand with varying prognosis
Question:Are there genetic factors that may modify the course of the disease?
A/0+0/0
A/A
10
20
30
40
50
60
70
80
90
100
Sur
viva
l pro
babi
lity
0 5 10 15 20 25 30 35 40 45 50
Years
CF patients: annual age-specific mortality rate by MBL2 variant alleles
P=0.01
1999
MBL2 time-to-event analysis showing median age with first positive P. aeruginosa bacterial culture in
1393 Canadian CF patients
Dorfman et al., JCI 2008
TGFB1 TT genotype TGFB1 CT genotype
TGFB1 CC genotype
MBL2 time-to-event analysis (as in
last figure) plots show 3 MBL2 genotype groups and stratifcation
by TGFB1 genotype
Dorfman et al., JCI 2008
Loss of lung function (∆(∆(∆(∆FEV1) from 6 to 18 years dependent on MBL2 and TGFB1 CC genotypes
-5.72 -3.47 -1.55
6 18 years
P<0.0002Low MBL
Intermediate MBL
High MBL
0
1Impact of mannose binding lectin (MBL) insufficiency on the course of
cystic fibrosis: a review and meta-analysis
James D. Chalmer et al, Glycobiology 2010
6
B
B
B
C,B,D
B,C,D
C
B
B
Wildtype Heterozygote Homozygote variant
A
Distribution of structural MBL2 variant alleles
A/A, 7%
A/B, 28%
B/B, 65%
Quechua people
1992
Positive Selection for MBL2 Variant Alleles
The high frequency of variant alleles and the dominant effect on the functional level suggests a selective advantage for heterozygotes (A/O) (heterosis)
Hypothesis 1: heterozygosity for MBL2 variant allelesprotect against intracellular microorganisms
Hypothesis 2: heterozygosity for MBL2 variant alleles protect against deleterious effect of complement activation
None of these theories are mutually exclusive
Risk
0 5000MBL serum concentration
MBL U-formed risk curve – favouring heterozygosity
1992
Low HighIntermediate
• MBL plays an important role as disease
modifier in individuals with a concomitant
disease
• Screening for MBL2 alleles and MBL
serum levels may be of prognostic
importance in conjunction with other
parameters
Summary