Download Slide 1

Disorders of structural proteins
Fransiska Malfait
Anne De Paepe
Defects of dystrophin
A spectrum of muscle disease caused by mutations in the DMD
gene, which encodes the protein dystrophin.
The mild end of the spectrum
•
asymptomatic increase in serum concentration of creatine
phosphokinase (CK)
•
muscle cramps with myoglobinuria
•
isolated quadriceps myopathy
The severe end of the spectrum: progressive muscle diseases
•
Duchenne/Becker muscular dystrophy (skeletal muscle)
•
DMD-associated dilated cardiomyopathy (heart)
Duchenne muscular dystrophy (DMD)
•
Normal for the first two years of life
•
Symptoms present before age 5 years
•
Progressive symmetrical muscular weakness, proximal greater than distal, often
with calf hypertrophy
•
Wheelchair-dependency before age 13 years
•
Unlikely to survive beyond age of 20 years
•
Die of respiratory failure or cardiomyopathy
•
Modest decrease in IQ (~20 points)
•
Prevalence: 1/5,000 males
Becker muscular dystrophy (BMD)
•
Progressive symmetrical muscle weakness and atrophy,
proximal greater than distal, often with calf hypertrophy
(weakness of quadriceps femoris may be the only sign)
•
Activity-induced cramping (present in some individuals)
•
Flexion contractures of the elbows (if present, late in the course)
•
Wheelchair dependency (if present, after age 16 years)
•
Preservation of neck flexor muscle strength
(differentiates BMD from DMD)
•
Prevalence: 1/18,000 males
DMD-associated dilated cardiomyopathy
•
Dilated cardiomyopathy (DCM) with congestive heart failure, with
males typically presenting between ages 20 and 40 years and
females presenting later in life
•
Usually no clinical evidence of skeletal muscle disease; may be
classified as "subclinical" BMD
•
Rapid progression to death in several years in males and slower
progression over a decade or more in females
Molecular Genetics: inheritance
•Incidence DMD:
‣ 1:3300 live male births
‣ Calculated mutation rate 10-4
‣ Given a sperm production rate of 8x107 sperm/day:
sperm with new mutation is produced every 10
seconds by normale male!
Molecular Genetics: inheritance
X-linked recessive disorder (Xp21.2)
• 1/3 of cases: new mutations
• 2/3 have carrier mother
Molecular Genetics: inheritance
Carrier mother:
‣ majority: no clinical manifestations
‣ 70 % has slightly elevated serum creatine kinase
‣ Random inactivation of X-chromosome 
‧ ~19 % of adult female carriers have some muscle weakness
‧ 8% has life-threatening cardiomyopathy and severe muscle
weakness
Females with DMD (rare):
‣ Nonrandom X-inactivation
‣ Turner syndrome (45,X)
‣ X; autosome translocation
Molecular Genetics: inheritance
DMD
BMD
Lethal
Non-lethal
gene is not transmitted
gene is transmitted
1/3 of cases: new mutations
2/3 have carrier mother
high proportion of BMD cases is
inherited, only 10% new
mutations
Molecular Genetics
Gene: DMD
‣ the largest known human gene (1,5% of X-chromosome)
‣ 2.4 Mb of DNA
‣ comprises 79 exons
‣ at least four promoters
‣ differential splicing  tissue-specific, developmentally regulated
isoforms
Protein: dystrophin
‣ part of a protein complex that links the cytoskeleton with
membrane proteins that in turn bind with proteins in the
extracellular matrix
‣ expressed in skeletal and cardiac muscle, brain
Dystrophin complex:major functions
• maintenance of muscle membrane integrity
• correct positioning of proteins in the complex,
so that they function correctly
• ion channels and signaling molecules 
participation in cell-cell and/or cell-substrate
recognition
Genotype-phenotype correlations
lack of dystrophin expression: DMD
‣ very large deletions absence of dystrophin expression
‣ mutations that disrupt reading frame (stop mutation, splicing
mutation, deletion, duplication)  severely truncated dystrophin
that is degraded
remaining dystrophin production (abnormal quality or
quantity): BMD
‣ deletions or duplications that juxtapose in-frame exons
‣ some splicing mutations
‣ most non-truncating single-base changes that result in
translation of a protein product with intact N and C termini.
Molecular Genetics
Molecular defect
Frequency
Phenotype
Gene deletion (1 exon to
whole gene)
60%
DMD or BMD
Point mutation
34%
DMD or BMD
Partial gene duplication
6%
DMD or BMD
Contiguous gene deletion
Rare
DMD + other phenotypes
Nonrandom X-inactivation
Rare
DMD
Turner syndrome (45,X)
Rare
DMD
X; autosome translocation
Rare
DMD
Affected males
Affected females
Molecular Genetics
Distribution of deletions: clustered in 2 regions
‣
‣
5’ half (exon 2-20) (30%)
exons 44-53 (70%)
Reading frame hypothesis
Testing
Electromyography: to differentiate between myopathy and neurogenic disorder
Serum Creatine Phosphokinase (CK) Concentration
Males
Female
carriers
phenotype
% of affected
individuals
Serum CK conc.
DMD
100%
> 10x normal
BMD
100%
> 5x normal
DMD-associated
DCM
Most individuals
“increased”
DMD
~50%
2- 10x normal
BMD
~30%
2- 10x normal
Testing
Western Blot and immuno-histochemistry
Phenotype
Males
DMD
Western Blot
Immunohistochemsitry
Dystrophin
molecular weight
Dystrophin
quantity
Non-detectable
0%-5%
(almost) complete
absence
Female
carriers
Intermediate
Normal/Abnormal
5%-20%
BMD
Normal
Abnormal
20%-50%
20%100%
Normal appearing or
reduced intensity ±
patchy staining
DMD Random XCI
Normal/Abnormal
> 60%
Mosaic pattern
DMD Skewed XCI
Normal/Abnormal
< 30%
Mosaic pattern
immuno-histochemistry
Localisation of dystrophin to myocyte membrane
DMD: Absence of dystrophin in myocyte membrane
Testing
Molecular genetic testing
‣ Deletion/duplication analysis
‧ Multiplex PCR, southern blotting and FISH (deletions)
‧ Southern blotting and quantitative PCR (duplications)
‧ MLPA (deletions/duplications)
‣ Mutation scanning and sequence analysis
‧ Small deletions/insertions, single base changes, splice
mutations
Multiplex PCR
Multiplex PCR analysis of dystrophin gene deletions. Exons A, B, C, and D are amplified in a single
PCR reaction (arrows indicate PCR primers). The products (shown below each exon) are separated by
size on an agarose gel and are visualized
Multiplex PCR
Multiplex PCR analysis of exons 51, 12, 44, and 4 of the dystrophin
gene. Bands corresponding with each exon are indicated at the right.
Patient sample is in lane 2, showing an exon 51 deletion. Lane 1 is a
control, showing all four bands. Other lanes contain samples for other
males.
Genetic counseling
•
The father of an affected male will not have the disease nor will
he be a carrier of the mutation.
•
A woman with an affected son and one other affected relative in
the maternal line is an obligate heterozygote.
•
A woman with more than one affected son and no other family
history of a dystrophinopathy has either:
‣ A germline mutation (i.e., a DMD disease-causing mutation present
in each of her cells); or
‣ Germline mosaicism (i.e., mosaicism for a DMD disease-causing
mutation that includes her germline)
Genetic counseling
If the proband is the only affected family member
1. the proband has a de novoDMD disease-causing
mutation as a result of one of the following:
‣ The mutation occurred in the egg at the time of the proband's
conception and is therefore present in every cell of the
proband's body. In this instance, the proband's mother does
not have a DMD disease-causing mutation and no other family
member is at risk.
‣ The mutation occurred after conception and is thus present in
some but not all cells of the proband's body (somatic
mosaicism). In this instance, the likelihood that the mother is a
heterozygote is low.
Genetic counseling
2. the proband's mother has a de novoDMD disease-causing
mutation. Approximately 2/3 of mothers of males with DMD and
no family history of DMD are carriers.
The mechanisms by which a de novoDMD disease-causing
mutation could have occurred in the mother are the following:
‣ The mutation occurred in the egg or sperm at the time of her
conception (germline mutation) and is thus present in every cell of
her body and detectable in DNA extracted from leukocytes.
‣ The mutation is present in some but not all cells of her body
(somatic mosaicism) and may or may not be detectable in DNA
extracted from leukocytes.
‣ The mutation is present in her egg cells only (termed "germline
mosaicism") and is not detectable in DNA extracted from a blood
sample. The likelihood of germline mosaicism in this instance is 15%20%. Consequently, each of her offspring has an increased risk of
inheriting the DMD disease-causing mutation
Genetic counseling
3. The proband's mother has inherited a DMD mutation from one of
the following:
‣ Her mother, who is a carrier
‣ Her mother or her father, who has somatic mosaicism
‣ Her mother or her father, who has germline mosaicism
Two types of pedigrees encountered in families
with Duchenne or Becker dystrophy.
two obligate carrier females and a woman at 50%
risk based on family history.
mother has a 66% risk of
being a carrier, and his sister,
therefore, a 33% risk
Mutations in collagen structural genes:
Osteogenesis imperfecta
• Variable degree of bone fragility
• 4 subtypes (Sillence et al. 1979, 1984)
Type I
Type II
Type III
Type IV
Mild
Lethal
Severe
Moderate
• Defects of type I collagen
• Due to mutations in COL1A1/COL1A2
Mild OI
Severe OI
Lethal OI
Type I collagen

Most abundant fibrillar collagen in body

Widely expressed in bone, tendon, skin, other tissues

Heterotrimer:
2 1 chains  COL1A1 (chr 17)
1 2 chain  COL1A2 (chr 7)
N-terminus
COL1A1
Helical domain
6
10
15
20
25
30
35
C-terminus
40
45
COL1A2
= 36 bp
= 45 bp
= 54 bp
= 90 bp
= 99 bp
= 108 bp
= 162 bp
Complex gene structure
Great potential for mutations
50
Collagen Fibrillogenesis
Gly
Gly
Gly
Gly
AMINOACID
SEQ
N-terminus
MOLECULE
Type I procollagen
Cleavage by N-proteinase
Cleavage by C-proteinase
MOLECULAR
PACKING
COLLAGEN
FIBRIL
Collagen structure
Collagen biosynthesis
Collagen fibrillogenesis
P C P C
Molecular abnormalities of collagen in OI
• diminished type I collagen production
• structurally defective collagens
Osteogenesis imperfecta type I (mild)
C C P C
1(III)3
Normal
T GG A G A G C G A G G T G T T C C C G
Gly Glu Arg Gly Val Pro
Mutant
1(I)
2(I)
T GG A G A G T G A GG T G T T C C C G
Gly Glu STOP Gly Val Pro
1(I) - Arg 240 Stop
(CGA  TGA)
Osteogenesis imperfecta type I (mild)
Osteogenesis imperfecta type II-IV
P C P C
Bases
215
A G G A
C
C
C
210
C
C
T
G
G
205
C C
C
Gly
1(I)M
1(I)
2(I)M
2(I)
A
A
125
G G
A
C
medium collagens
C
130
C C
C
T
G
135
G
Gly
C
T
Pro
C
C
T
Pro
G
200
T G
C
Ala
140
G
C
Ala
T
G
A
Gly
G
A
145
A G
Glu2
195
A G
G
G
Glu
A
Glu
G
A
A
190
G G
Glu
G
150
A A
Glu
A
A
A
185
G C
G
A
180
G G A
G
Gly
G
G
Gly
86
GGA > GAA
1(I) - Gly286Glu
A
155
A A
G
C
G
160
A G
G
A
Bases
G
C
Osteogenesis imperfecta type II-IV
• Majority: glycine substitutions in triple helical domain of either the
pro-α1 or pro- α2 chain of type I collagen
Phenotypic determinants include:
‣ Which chain is involved (α1(I) vs α2(I)-collagen chain)
‣ Location of substitution
‣ Nature of substituting residue
• splice site mutations
‣ Exon skips (in-frame)
‣ Intronic inclusion
‣ Activation of cryptic splice sites
Genotype-phenotype correlations
• α1(I)- chain:
‣ Glycine-substitutions in N-terminal 200 residues are associated
with non-lethal phenotype
‣ C-terminal glycine substitutions are associated with severe to
lethal phenotype
‣ Two exclusive “lethal regions”
• α2(I)-chain
‣ 80 % of glycine substitutions is non-lethal
‣ 8 “lethal regions”
Distribution of mutations along α1(I)- collagen chain
• Valine: branched
non-polar side-chain
•Arg, Asp, Glu
Charged AA
Overrepresentation
of lethal phenotypes
Distribution of mutations along α2(I)- collagen chain
Arg, Asp, Glu
Charged AA
Overrepresentation
of lethal phenotypes
Dominant negative effect
Genetic counseling
Mild OI: ~60% of individuals with mild OI have
de novo mutations
Severe (type III) and lethal (type II) OI: virtually
100% of individuals with have de novo
mutations.
Genetic counseling
OI type
Inheritance pattern
Gene
Recurrence risk
I
AD
COL1A1
COL1A2
50%
II
AD (de novo mutation)
COL1A1
COL1A2
~5% (germline mosaicism)
III
AD (de novo mutation)
COL1A1
COL1A2
~5% (germline mosaicism)
IV
AD
COL1A1
COL1A2
50%
V
AD
?
50%
VI
Uncertain
?
Uncertain
VII
AR
CRTAP
25%
VIII
AR
P3H1
25%
Collagen biosynthesis
Recessive OI: CRTAP/LEPRE/PPIB complex
F
P
M
α1(III)3
α1(I)
α2(I)
Recessive OI due to LEPRE1 mutations
Proband 1
Proband 2
Proband 3
Proband 4
Hom. c.1365-1366delAGinsC (p.Glu455fs)
Hom. c.628C>T (p.Arg210X)
• Lack of calvarial ossification
• Beaded ribs with multiple fractures
• Platyspondyly
• Shortened, wide, bowed and fractured large tubular bones
• Recessive OI or Severe/Lethal Autosomal Dominant OI ??
Het. c.1102C>T (p.Arg368X)
Het. c.2055+18G>A, Intron 14
Hom. c.2055+18G>A, Intron 14
P3 at age 17 years
III
• Complete disappearance of the popcorn-like structures
• Extreme osteoporosis
• Widening of the rhizomelic diaphyses
• Progressive narrowing and bowing of the mesomelic diaphyses
• Reduced knee joint spaces
• Long hands with hyperlax finger joints