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