Week 5 - Ehlers Danlos Flashcards
Where is the point mutation in COL5A1 in this case of Classic Ehlers Danlos?
Mutation in exon 3 of the COL5A1 gene
Cytosine to Thymine
Going from Glutamine to a stop codon
Nonsense mutation
Premature stop codon means the gene isn’t functional
Define child abuse.
(2 marks)
An abused child is a boy or girl under the age of 18 years who has suffered physical injury, neglect, emotional or sexual abuse which the person or persons who had custody, charge or care of the child either caused or knowingly failed to prevent.
What is the normal role of collagen V in the connective tissue?
(1 mark)
It regulates the diameter of collagen type I fibrils
What type of joint is the shoulder joint?
1 mark
Synovial ball and socket
What is the role of the Children and Family Court Advisory and Support Service in cases like this?
2 marks
Any 2 relevant points:
When a local authority applies to take a child into care, Cafcass’ job is to ensure that decisions are made in the child’s best interest. (1 mark)
The court will usually request for Cafcass to become involved in the case so that the Cafcass worker is known as a ‘children’s guardian’ (1 mark).
The guardian is the independent voice of the child in court (1 mark).
What does SDS-PAGE stand for?
Sodium dodecyl sulphate polyacrimide gel electrophoresis
How do Type V and Type I come together?
Type V and Type I collagen come together to form heterotypic fibrils
Type V collagen shown to regulate the diameter of the fibrils
Important as collagen needs to be densely packed
How many different types of conditions come under Ehlers Danlos?
13
Which type of collagen is most abundant in tissues?
Type I
Which type of collagen has been associated with Ehlers Danlos?
Type V
What kind of haploinsufficiency do you see in Classic Ehlers Danlos and what does this result in?
What does a COL5A1 mutation result in?
One gene of collagen V is functional and working well, the other is not
So you don’t have enough collagen V
Because you have a loss of function in COL5A1 mRNA and hence less of the alpha 1 (V) procollagen chains
Therefore get some normal and some abnormal collagen V
Irregular fibrils in the dermis
Less strength in connective tissues, skin etc and abnormal wound healing
What is Ehlers Danlos?
A collection of heritable connective tissues disorders classified according to its symptoms and signs
13 different types
What is a non-conservative missense mutation?
A point mutation that codes for a different amino acid with different properties
What is haploinsufficiency?
Only have one functional copy of a gene
Half the dose causing a pathological phenotype - cannot provide sufficient gene product
Often caused by a LOF mutation
What is the structure of Type V collagen?
It is made of 3 chains
2 alpha1 chains
1 alpha2 chain
Heterotrimer
What is the inheritance pattern of Ehlers Danlos?
Autosomal Dominant
How common is Ehlers Danlos?
1 in 5000 births
Rare
What is the major criteria for Ehlers Danlos?
Skin hyperextensibility
Widened atrophic scarring (due to tissue fragility)
Joint hypermobility
Positive family history
Name 5 minor criteria for Ehlers Danlos
Smooth velvety skin
Molluscoid pseudotumours (fleshy, heaped up lesions associated with scars over pressure points e.g. elbows and knees)
Subcutaneous spheroids (small, hard cyst-like nodules, freely moveable in the subcutis over the bony prominences of the legs and arms, which have an outer calcified layer with a translucent core on X-ray)
Complications of joint hyper-mobility (sprains, dislocations/subluxations and pes planus)
Muscle hypotonia, delayed gross motor development
Easy bruising
Manifestions of tissue extensibility and fragility (anal prolapse in childhood, hiatal hernia, cervical insufficiency)
Surgical complications (postoperative hernias)
Why is the skin so fragile and flexible?
Mutation in the collagen fibres so skin is very flexible
Why are the joints hypermobile?
Collagen is in the ECM of the tendons
Why do patients bruise easily?
Less collagen in the blood vessels so the blood becomes more leaky and the blood vessels get damaged easily
What does diagnosis of Ehlers Danlos require?
3 major criteria
1 minor criteria
What does culturing fibroblasts mean?
Take fibroblasts from Andreas’ cells and grow them on a petri dish
Process the fibroblasts for gel electrophoresis
Explain electrophoresis
Charged molecules move up a gel
Current passed through the gel and then the proteins move up depending on their size and shape (weight)
Collagen was processed from dermal fibroblasts
What does haematoma mean?
The abnormal collection of blood that has usually clotted outside of a blood vessel
What is a subcutaneous nodule?
A firm lump under the skin that is often the result of infection/inflammation
What does hyper-extensible mean?
Being able to be stretched to a greater than normal degree
What does episiotomy mean?
A surgical cut made in the perineum to enlarge the vaginal opening for childbirth
What is a skin biopsy?
A small sample of skin is removed and examined under an electron microscope
The results from this showed the electrophoretic pattern of the (pro)collagen type I, II and V proteins, secreted in the medium showed a normal profile.
What does the electrophoresis pattern show?
Normal
All (pro)collagen I, II, V proteins were shown to be normal
So all were present but doesn’t show the mutations
What are other symptoms that might suggest Ehlers Danlos?
Fibromyalgia
IBS
Chronic pain
Gorlin’s sign (can touch your nose with your tongue)
Flying bird bands (hypermobility syndrome)
What percentage of people who have Ehlers Danlos are diagnosed?
5%
Incidence thought to be 1:200 (found through sequencing data and people having the mutations but are not diagnosed)
What is SDS-PAGE used for?
Proteins not genes
Doesn’t tell us if the proteins are mutated but tells us the amount of protein present
Can help detect severity of the symptoms or type
What does Andreas’ electrophoresis show us?
Has reduced type III and reduced type V
Can’t tell if V is normal/functional or mutated
What is a silent mutation?
You have a mutation in the triplet code and it produces the same amino acid
However, sequence analysis of the entire coding region for COL5A1 (exons 1-66) and all intron-exon boundaries along the COL5A1 gene identified a heterozygous variant (c.379C>T) in exon 3, which is predicted to cause a nonsense mutation from the substitution of glutamine causing a premature stop codon and thus a truncated COL5A1 protein or reduced COL5A1 mRNA due to RNA decay confirming the clinical suspicion of classic Ehler-Danlos.
How does gene sequencing help the diagnosis of Andreas?
Electrophoresis shows us that the Collagen V is present and reduced but doesn’t tell us more
Gene-sequencing tells us that Collagen V is mutated and where and that it is truncated and non functional
However, sequence analysis of the entire coding region for COL5A1 (exons 1-66) and all intron-exon boundaries along the COL5A1 gene identified a heterozygous variant (c.379C>T) in exon 3, which is predicted to cause a nonsense mutation from the substitution of glutamine causing a premature stop codon and thus a truncated COL5A1 protein or reduced COL5A1 mRNA due to RNA decay confirming the clinical suspicion of classic Ehler-Danlos.
What is the coding region of COL5A1?
Exons 1-66
However, sequence analysis of the entire coding region for COL5A1 (exons 1-66) and all intron-exon boundaries along the COL5A1 gene identified a heterozygous variant (c.379C>T) in exon 3, which is predicted to cause a nonsense mutation from the substitution of glutamine causing a premature stop codon and thus a truncated COL5A1 protein or reduced COL5A1 mRNA due to RNA decay confirming the clinical suspicion of classic Ehler-Danlos
In which exon is the mutation?
33
However, sequence analysis of the entire coding region for COL5A1 (exons 1-66) and all intron-exon boundaries along the COL5A1 gene identified a heterozygous variant (c.379C>T) in exon 3, which is predicted to cause a nonsense mutation from the substitution of glutamine causing a premature stop codon and thus a truncated COL5A1 protein or reduced COL5A1 mRNA due to RNA decay confirming the clinical suspicion of classic Ehler-Danlos.
What is the amino acid change?
Glutamine to a premature stop codon
CAA to TAA
CAG to TAG
What does degeneracy in the genetic code mean?
More than one triplet code codes for an amino acid
What is the role of Collagen V?
Regulates fibril formation and diameter of type I collagen fibrils
and co-polymerization of collagen Type I
What are the different ways Andreas is diagnosed in this scenario?
Physical examination
Assess hypermobility using the Beighton criteria
Skin biopsy for collagen typing
Collagen gene mutation testing
What are the steps of collagen formation?
Procollagen Fibril nucleation Fibril intermediates Linear fusions Lateral fusions Heterotypic fibril
What normally makes Type I collagen fibrils strong?
Regular diameter
Regular length
What is the result of haploinsufficiency of COL5A1?
Less Collagen V than there should be due to less [a1 (V) pro collagen chains]
The reduction of Collagen V cannot compensate so you have symptoms
What percentage of Classic EDS cases have a haploinsufficiency of Collagen V?
30-50%
What percentage of dry weight of skin is collagen?
70%
What percentage of the collagen in the skin is Collagen I?
80%
What percentage of the collagen in the skin is Collagen III?
15%
What percentage of the collagen in the skin is Collagen V?
5%
What chains is collagen made of?
3 alpha chains
2 a1, 1 a2
or 1a1 and 2a2
Where is collagen synthesised and what is the starting product?
Pro collagen
In the rough endoplasmic reticulum
After the rER where is the collagen synthesised?
It is secreted out of the cell and processes/aggregates outside the cell
At this point it is a mature collagen proteins
What repeat do you have in the alpha chains of collagen?
Triplet repeat Predominantly glycine (every 3rd amino acid)
G X Y
Glycine - Proline - Y
Y = Hydroxyproline derived from proline
or Hydroxylysine derived from lysine
How do the properties of glycine contribute to the structure of the alpha chain?
Smallest amino acids with no side chains (H)
Allows close association of collagen fibres facilitating hydrogen bonding
Strong molecules due to close packing
What is the dermis like in EDS patients?
Looser with few bundles
Varying diameter
Abnormal wound healing
What is the skin structure layers?
Epidermis and dermis
What cells are in the epidermis?
Stratified squamous epithelium
Keratinocytes (Langerhans cells (dendritic cells - APCs) and melanocytes
What cells are in the dermis?
Dense irregular connective
Fibroblasts
Not regular but random, gives the property of not tearing when pressure is applied
What are the 4 stages of wound healing?
- Haemostasis (vascular constriction and clotting (platelets and fibrin))
- Inflammation (release of cytokines and growth factors, WBC to remove dead cells and provide defence against microbes)
Continues from start to day 10, redness around the wound
- Proliferation (fibroblasts and keratinocytes proliferate and start to make new ECM, initial formation of scar (epithelial cells are
- Remodelling (scar is 80% of normal skin strength) huge upregulation of collagen synthesis (I and V)
Balance between collagen synthesis and destruction
Changing of Collagen III to type I
Water is reabsorbed from this area to allow collagen to contract and become closer together
Wound contracts so the scar does not gape
What happens in wound healing for EDS patients?
Wounds do not contract in EDS as collagen is not structurally sound
Mainly affecting the remodelling stage
Wounds are ‘gaping’ (doesn’t actually close properly) like a cigarette scar
Wounds are wide and thin
Which stage of the wound healing process is predominantly effected in EDS patients?
Stage 4 - Remodelling
What happens in the remodelling stage of Wound Healing?
Stage 4:
Remodelling (scar is 80% of normal skin strength) huge upregulation of collagen synthesis (I and V)
Balance between collagen synthesis and destruction
Changing of Collagen III to type I
Water is reabsorbed from this area to allow collagen to contract and become closer together
Wound contracts so the scar does not gape
What happens in the Proliferation stage of Wound Healing?
Stage 3:
Proliferation (fibroblasts and keratinocytes proliferate and start to make new ECM, initial formation of scar (epithelial cells are
What happens in the Inflammation stage of Wound Healing?
Stage 2:
Inflammation (release of cytokines and growth factors, WBC to remove dead cells and provide defence against microbes)
Continues from start to day 10, redness around the wound
Why does the anaesthetic does not work?
Not known Sodium channels (Sodium 1.7 and 1.8) linked to pain could be linked to mutations in collagen V
May explain why the anaesthetic doesn’t interact with the sodium channels
What is child abuse?
Any action by another person adult or another child that causes significant harm to a child
What is neglect?
The failure of parent or another person with a responsibility for a child to provide food, clothing, shelter, medical care or supervision to degree of that child for health, safety and wellbeing so that they are threatened with harm
Child
Under 18
What are the sub topics of child abuse?
Neglect Physical abuse Sexual abuse Exploitation Emotional abuse Online cyberbullying
What are the actual layers of the skin?
- Stratum corner (C)
- Stratum lucid (L)
- Stratum granulosom (G)
- Stratum spinous (S)
- Stratum basale (B)
- Dermis
Corny Lucy gets super baked
Give three types of genetic point mutations briefly describe each
Which type is seen in this scenario? (3)
- Silent mutations: base changes but codes for the same amino acid
- Missense mutations: Codes for a different amino acid
- Nonsense mutations: Codes for a stop codon, resulting in early termination of transcription
This case is a nonsense mutation
Which type of collagen is dominant in bone, tendon and skin?
Therefore explain what molecular abnormality causes the symptoms seen in classical EDS (4)
Collagen Type I
In Classical EDS, Type V is structurally abnormal and therefore cannot function optimally
Collagen V regulates fibril formation and copolymerisation of collagen I which is therefore effected in EDS
As Collagen I is the most abundant collagen in bone, tendon and skin, this results in weakness of these structures
This is shown in symptoms of hyper-extensile skin, hyper mobile joints and easy bruising
Give 3 major and 1 minor diagnostic criteria of classical Ehlers Danlos Syndrome
Positive family history
Joint hyper-mobility
Skin hyper-extensibility
Atrophic scars
Easy bruising
