Disorders of structural proteins

1
Disorders of structural proteins

• Fransiska Malfait
• Anne De Paepe

2
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)

3
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

4
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

5
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

6
Molecular Genetics inheritance

• Incidence DMD
• 13300 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!

7
Molecular Genetics inheritance

• X-linked recessive disorder (Xp21.2)
• 1/3 of cases new mutations
• 2/3 have carrier mother

8
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

9
Molecular Genetics inheritance

• DMD
• Lethal
• gene is not transmitted
• 1/3 of cases new mutations
• 2/3 have carrier mother

BMD Non-lethal gene is transmitted high
proportion of BMD cases is inherited, only 10
new mutations
10
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

11
(No Transcript)
12
Dystrophin complexmajor 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

13
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.

14
Molecular Genetics
Molecular defect Frequency Phenotype
Affected males Affected males Affected males
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
Affected females Affected females Affected females
Nonrandom X-inactivation Rare DMD
Turner syndrome (45,X) Rare DMD
X autosome translocation Rare DMD
15
Molecular Genetics

• Distribution of deletions clustered in 2 regions
• 5 half (exon 2-20) (30)
• exons 44-53 (70)

16
Reading frame hypothesis
17
Testing

• Electromyography to differentiate between
  myopathy and neurogenic disorder
• Serum Creatine Phosphokinase (CK) Concentration

phenotype of affected individuals Serum CK conc.
Males DMD 100 gt 10x normal
Males BMD 100 gt 5x normal
Males DMD-associated DCM Most individuals increased
Female carriers DMD 50 2- 10x normal
Female carriers BMD 30 2- 10x normal
18
Testing

• Western Blot and immuno-histochemistry

Phenotype Western Blot Western Blot Immunohistochemsitry
Dystrophin molecular weight Dystrophin quantity
Males DMD Non-detectable 0-5 (almost) complete absence
Males Intermediate Normal/Abnormal 5-20
Males BMD Normal Abnormal 20-50 20-100 Normal appearing or reduced intensity patchy staining
Female carriers DMD Random XCI Normal/Abnormal gt 60 Mosaic pattern
Female carriers DMD Skewed XCI Normal/Abnormal lt 30 Mosaic pattern
19
immuno-histochemistry
Localisation of dystrophin to myocyte membrane
DMD Absence of dystrophin in myocyte membrane
20
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

21
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
22
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.
23
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)

24
Genetic counseling

• If the proband is the only affected family member
  
• 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.

25
Genetic counseling

• 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

26
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

27
Two types of pedigrees encountered in families
with Duchenne or Becker dystrophy.
mother has a 66 risk of being a carrier, and his
sister, therefore, a 33 risk
two obligate carrier females and a woman at 50
risk based on family history.
28
Mutations in collagen structural genes
Osteogenesis imperfecta

• Variable degree of bone fragility
• 4 subtypes (Sillence et al. 1979, 1984)
• Type I Mild
• Type II Lethal
• Type III Severe
• Type IV Moderate
• Defects of type I collagen
• Due to mutations in COL1A1/COL1A2

29
(No Transcript)
30
Mild OI
Severe OI
Lethal OI
31
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)

Great potential for mutations
32
Collagen Fibrillogenesis
AMINOACID SEQ
MOLECULE Type I procollagen
33
Collagen structure
34
Collagen biosynthesis

35
Collagen fibrillogenesis
P
P
C
C
36
Molecular abnormalities of collagen in OI

• diminished type I collagen production
• structurally defective collagens

37
Osteogenesis imperfecta type I (mild)
C
P
C
C
Normal
?1(III)3
Mutant
?1(I)
?2(I)
?1(I) - Arg 240 Stop (CGA ? TGA)
38
Osteogenesis imperfecta type I (mild)
39
Osteogenesis imperfecta type II-IV
P
P
C
C
Gly
Pro
Ala
Gly
Glu
Glu
Gly
?1(I)M
?1(I)
Gly
Pro
Ala
Glu286
Glu
Glu
Gly
?2(I)M
?2(I)
GGA gt GAA ?1(I) - Gly286Glu
medium collagens
40
Osteogenesis imperfecta type II-IV

• Majority glycine substitutions in triple
  helical domain of either the pro-a1 or pro- a2
  chain of type I collagen
• Phenotypic determinants include
• Which chain is involved (a1(I) vs a2(I)-collagen
  chain)
• Location of substitution
• Nature of substituting residue
• splice site mutations
• Exon skips (in-frame)
• Intronic inclusion
• Activation of cryptic splice sites

41
Genotype-phenotype correlations

• a1(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
• a2(I)-chain
• 80 of glycine substitutions is non-lethal
• 8 lethal regions

42
Distribution of mutations along a1(I)- collagen
chain

• Valine branched non-polar side-chain
• Arg, Asp, Glu
• Charged AA
• Overrepresentation of lethal phenotypes

43
Distribution of mutations along a2(I)- collagen
chain
Arg, Asp, Glu Charged AA Overrepresentation of
lethal phenotypes
44
Dominant negative effect
45
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.

46
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
47
(No Transcript)
48
Collagen biosynthesis

49
Recessive OI CRTAP/LEPRE/PPIB complex
50
Recessive OI due to LEPRE1 mutations
Proband 1
Proband 2
Proband 3
Proband 4

Hom. c.1365-1366delAGinsC (p.Glu455fs)
Hom. c.205518GgtA, Intron 14
Hom. c.628CgtT (p.Arg210X)
Het. c.1102CgtT (p.Arg368X) Het.
c.205518GgtA, Intron 14

• 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 ??

51
P3 at age 17 years

• 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