Duncan - osteogenesis, DMD and MPLA

Question 1

connective tissue - what is it/what is it composed of?

what are it’s functions (name 4-5)?

Answer 1

Definition: One of the four basic animal tissues, alongside epithelial, muscle, and nervous tissue.

Components:
Fibres: Collagen (strength), elastin (elasticity).
Ground Substance: Gel-like material in extracellular space, made up of ECM components minus the fibres^^^
Cells: Fibroblasts (skin), adipocytes (fat), macrophages, mast cells, and leukocytes

Question 2

where is connective tissue found?

what are it’s properties?

what are it’s functions?

Answer 2

Found between tissues throughout the body, including the nervous system.
Properties: Provides elasticity, strength, and resilience to various body structures

functions -
Supports and connects internal organs (cellular glue).
Forms bones and walls of blood vessels.
Attaches muscles to bones.
Protects and cushions the body.
Shapes body parts.
Repairs damaged tissues

Question 3

connective tissue disorders -

how many are there and are they all genetic?

Answer 3

More than 200, including genetic (Ehlers-Danlos syndrome, Marfan, syndrome, Osteogenesis Imperfecta),

autoimmune (lupus, scleroderma)

and even sarcomas (cancers arising in connective/soft tissue/bone)

They can affect all parts of the body - joints, eyes, skin, brain, muscles, and bones

Question 4

osteogenesis imperfecta -

what is it?
what are it’s symptoms?

Answer 4

what it is:
Heterogeneous group of connective tissue disorders (as in multiple genes are involved/different causes)
Main symptom = prone to fractures easily
Most common cause = collagen defect/collagen processing defect

symptoms:
Bone fractures (brittle bones) with little trauma
Loose joints/hyperflexible
Weak teeth (dentinogenesis imperfecta)
Blue sclera, or a bluish colour in the white of the eye.
Bowed legs and arms
Short stature (bones not developing as they should be)
Hearing loss in ~50% of cases
Estimated incidence approximately 1 per 20,000 births (tho those with mild symptoms may escape diagnosis)

Question 5

Zooming in on collagen -

what is it/what is it made of?

what are it’s properties?

Answer 5

Definition: Main structural protein in connective tissues, part of the extracellular matrix. Derived from Greek “kolla” (glue).

Composition:
Family of fibrous proteins (40+ types).
Produced by fibroblasts (skin) and osteoblasts (bones).

Properties:
Provides tensile strength and structure.
Supports wound healing by acting as a scaffold.
Can undergo biomineralization, making tissues rigid (bone) or flexible (tendon).
Type I collagen is stronger than steel gram-for-gram

Question 6

where is collagen found?

collagen fun fact?

Answer 6

Presence:
Found in bone, skin, ligaments, tendons, cartilage, and more.
Most abundant protein in humans (33% of total protein).

Fun Fact:
Detected in a 68-million-year-old T-Rex fossil (oldest protein found)

Question 7

there are four main types of (autosomal dominant) OI caused by variants in two key genes.

what are the genes?

What are the four types? (they each have a name, include how severe they are/kind of symptoms)

Answer 7

genes affected - COL1A1 and COL1A2 (for collagen)

Type 1, classic OI, is the most common and most mild. With classic OI features of bone fractures and blue sclerae. Teeth unaffected

Type 4, common variable OI, is similar to one but has dentinogenesis imperfecta (teeth issues).

Type 3, progressively deforming OI, is severe but not lethal. Its features will be apparent at birth with multiple fractures, very short stature and wheelchair bound.

Type 2, lethal OI, is lethal in pregnancy. The foetus will have severe skeletal abnormalities and fractures. The rib cage collapses (beaded ribs) and internal organs damaged. Skull deformation

Question 8

why are type I and IV less likely to be picked up in kids?

Answer 8

they have milder phenotypes, so are more likely to pass unnoticed - a broken bone could easily be brushed over if a child was playing or something.

Question 9

are those four types of OI the only types?

include examples (hint, one is in non-coding region, and the other affects the brain)

Answer 9

no, OI is heterogenous, so different variants in multiple genes can be the cause.

two examples to note would be:

OI type 5 - caused by just one variant, a c.-14C>T in the IFITM5 gene. Inherited in an autosomal dominant manner, variant is within the 3’ non coding DNA before the first exon and interferes with correct transcription of the gene

OI type 15 - autosomal recessive in the WNT1 gene, results in brain malformations due to skull deformities, and is seen in children with developmental delay

Question 10

lets say a young patient has grey Sclerae, Dental problems, Multiple bone fractures with little trauma, Family history of bone fractures.

what genetic testing should be done? explain your answer

Answer 10

symptoms indicate OI, and OI is known to be associated with 30+ genes so…

Targeted NGS analysis of these 30+ OI genes (Sanger sequencing would be far too costly and would take far too long and be very labour intensive)
Also - you wouldn’t sequence whole genome unless you were looking at 100s of genes

Question 11

not super important - but why do we refuse to rule out OI in legal cases?

Answer 11

> 5000 children under 2 go into hospital with a fracture a year. Most just got hurt accidentally, but some may be being abused

because you can only confirm a genetic disorder, not exclude one (can’t know every single gene/variant involved in a disease) we refuse to rule out OI in legal cases

Question 12

lets say the genomic sequencing result was as follows:
COL1A1
c.[600G>A];[=]
p.[(Gly200Val)];[(=)]

what does this mean?

Answer 12

COL1A1 gene
Change of nucleotide G at position 600 for an A in one allele
Glycine at position 200 changed for a Valine
Missense amino acid change
The second allele is wild type

Question 13

when it comes to variant analysis, what would you want to check?

Answer 13

the frequency of the variant in the normal (healthy) population (PM2 or BA1/BS1)

is the gene the variant is in consistent with features the patient has?
(yes, COL1A1 associated with O1, PP4)

is the variant located in a functionally important part of the gene?

Has the variant previously been reported in an affected patient? It could be a well established variant

Is the amino acid change predicted to affect the protein structure and / or function? Meaning how different are the amino acids in terms of size and properties?

Question 14

the variant is located within a Gly-X-Y repeat.

by describing the structure of collagen, explain why this is a piece of evidence supporting the variant’s pathogenicity.

Answer 14

collagen gene -
~ 50 small exons per collagen gene (~20 amino acids each)

Triplet Repeat: Found in ~40 exons (so most of them), Glycine is every third Aa. Its small size is essential for the triple helix, positioned in the helix center. Helix is composed of three α-chains (e.g., 2x COL1A1, 1x COL1A2).

Mutation in Glycine disrupts the helix structure, leading to impaired collagen function

apply PM1(well established domain/mutation hotspot)

Question 15

for collagen, what impact do mutations near the
1. C-terminus
2. N-terminus
3. triplet repeat

have?

Answer 15

Mutations near the C-terminus: Severe effects, linked to OI type II.
Mutations near the N-terminus: Milder effects, partial helix stability

triple repeat = qualitative defects - structurally abnormal alpha chains form faulty helices as folding is disrupted

Question 16

remembering that the variant changes a glycine to a valine, why might this contribute to a revel score of 0.9?

what other factors could contribute to such a high revel score?

Answer 16

Valine is much larger than glycine (significant change)

and such a high score suggests the change is at an evolutionarily conserved site

(PP3 - amino acid change predicted to support a deleterious effect on the gene or gene product)

Question 17

given the classifications applied to the example of OI, what type would you say it is?

why would you test family members?

Answer 17

Not lethal, so not 2
Not severe so not 3
No dentinogenesis imperfecta so not 4
It must be type I

Family members with variant can start treatment early
Inform life choices - Know to avoid contact sports!
Prenatal diagnosis available.

Family members without variant
Reassured that they do not have OI
No need to test their offspring

Question 18

give a summary of OI

symptoms/severity
possible genes involved/common genes involved
treatment
what is done after a positive result

Answer 18

Brittle bones, fractures
Mild to severe/lethal features

30 genes involved in collagen (bone) processing
Dominant and recessive types

COL1A1 and COL1A2 cause 90%
Gly-X-Y repeat mutations most common variant type

Bisphosphonate treatment, no cure

Familial testing, life choices

Question 19

what are the 6 stages of Multiplex Ligation-dependent Probe Amplification (MPLA)?

Answer 19

1. denaturation
2. hybridisation
3. ligation
4. amplification
5. fragment separation
6. data analysis

Question 20

the first three stages of MPLA are 1. denaturation
2. hybridisation
3. ligation

explain these three steps, including details on the probes used

Answer 20

1. Denaturation
   DNA sample heated to separate strands
2. Hybridisation -
   Add hybridisation master mix ( MLPA probemix + buffer).
   Probes:
   Up to 60 different probes, each targeting a unique DNA sequence. Can be multiple sequences in one gene, or sequences from different genes involved in the same disease
   Each probe is made up of two single-stranded oligonucleotides (left and right).
   Right oligo has a unique ‘stuffer sequence’ so you can identify each probe by it’s unique length.
   The forward primer and reverse primer for PCR are separated, one on each oligonucleotide (left vs right)

Mechanism:
Probes bind to target DNA sequences (60–80 nucleotides in total) with just a tiny gap between the oligonucleotides.
Incubation: Overnight for proper hybridisation

3. Ligation
   Add Ligase master mix

Ligase-65 will covalently join left and right oligonucleotides at their binding sites.
Super specific tho - the ligase won’t work if there is a single nucleotide mismatch between probe and sample DNA AT THE LIGATION SITE, enabling distinction between similar genes, pseudogenes, or mutations (that is, later on the ratio will be off).
Inactivate the ligase by heating

Question 21

explain the next two steps of MPLA (step 4. amplficiation, and step 5. fragment separation and visualisation)

Answer 21

4. once your probes have hybridised to sample DNA, and been ligated if they match the DNA sequence, you amplify them by PCR - Add - Polymerase master mix ( Polymerase + PCR primer mix).

PCR amplifies ligated probes using a fluorescently labeled forward primer.
PCR involves 35 cycles of denaturation, primer annealing, and elongation.

this gives you Output: Fluorescently labeled MLPA amplicons

5. Fragment Separation
   Capillary electrophoresis separates amplicons by length (like gel electrophoresis but all runs through a small tube that passes through a laser detector thingy to measure fluorescence).

A size standard with known fragment lengths acts as a “molecular ruler” (tagged with a different colour fluorescence)

Visualising it -
Fluorescent signals detected as peaks on an electropherogram.
Amplicon length is identified due to ‘ruler’ used, and matched to the specific probes that you put in at the start (stuffer sequence helped)

Question 22

MPLA - once you’ve carried out capillary electrophoresis and you have your electropherogram, how does the data analysis work?

Answer 22

Normalises test samples by comparing them to reference samples, calculates probe ratios (patient:reference sample kind of thing) and visualizes them in ratio charts.
Ratios sorted by genomic location for interpretation.
Interpretation:
Ratio of 1.0: Normal (two copies of the gene).
Ratio of 0.5: Heterozygous deletion (one copy) Ratio of 1.5: Heterozygous duplication (three copies)

remember MPLA is a way to detect CNVs, deletions and duplications

Question 23

MPLA - just to clarify, why is a probe made up of two parts?

Answer 23

So that you only end up with amplicons if the DNA sequence was normal (as ligase wouldn’t work if the sequence was wrong) and any deviations from normal can be detected

Question 24

in MPLA, what happens to unbound oligos (the probes)?

Answer 24

Only linearly amplified/in one direction (because without right and left being joined together by ligase, they only have a primer for one direction).
No signal will be produced as these won’t have the fluorescent forward primer. So there is no washing needed to remove unbound oligos.

(If you’d put the tag on the reverse primer these SS products would fluoresce and cause problems, think its cus of the whole 5’-3’ direction thingy)

Question 25

what are muscular dystrinopathies?

genetics
progression
size of their impact

Answer 25

A group of hereditary, germline (effect every cell) disorders, there are over 40 types
All with a genetic basis, but the fault can lie in different genes

They typically are progressive - get worse with age, and involve the degeneration/death of muscle fibres

Muscle is everywhere (most abundant body tissue, ~23% females and ~40% males weight) so impact is large and multisystem, including the heart (cardiomyopathy) and intellectual disability (lungs - muscles - lack of oxygen to brain)

Question 26

how did cytogenetic observations in the 1980’s result in identification of the dystrophin protein?

Answer 26

Patients with muscular dystrophy had balanced translocations between an autosome and X chromosome.
Breakpoints on the X chromosome are always at Xp21.
Hypothesis: A disrupted gene at Xp21 causes muscular dystrophy.
Cloning and Identification: The DMD gene was cloned, and dystrophin protein was identified

Question 27

DMD = Duchene’s muscular dystrophy

bit of info on the DMD gene?
how common?

Answer 27

DMD gene encodes dystrophin, at position Xp21.2 (on the X chromosome)
Largest gene in the human genome, 2.3 megabases (0.08% of the HG). takes 16 hrs to transcribe. Mature mRNA = 14 Kb
Made up of 79 exons - only 0.6% of the gene; so >99% = introns
Translocations easy to spot due to how large the gene is

Its X-linked recessive, so almost never seen in females - males only have one X chromosome so are hemizygous. it is found in
1 in 3,500 male infants.

Question 28

what is the role of the dystrophin protein in the cell?

Answer 28

Located in skeletal and cardiac muscle fibres, the protein is a middle man, connecting F-actin (major components of cell cytoskeleton) to ECM proteins like laminin, via the dystroglycan complex (so you’ve got F-actin → dystrophin → dystroglycan complex spanning cell membrane → ECM/laminins)

Strengthen muscle fibres and protects them from injury, holding cells in place

Prevents membrane damage during muscle contractions - transmits force generated during muscle contraction to the ECM

Question 29

what happens with a dystrophin defect? walk it through

Answer 29

Skeletal & cardiac muscle cells with absence of or reduced expression of functional dystrophin

The dystroglycan complex is disassembled
The interaction between F-actin (cytoskeleton) and laminin (extracellular matrix is lost so no protection against force of muscle contraction

Cells become damaged as the muscles repeatedly contract & relax with use
The damaged cells weaken & die over time. So muscle becomes increasingly depleted, causing characteristic muscle weakness & heart problems seen in DMD and BMD

Question 30

what is a similarity between DMD and BMD (Becker’s muscular dystrophy) in terms of something common in the genetic cause?

Answer 30

Both BMD and DMD - mostly caused by deletions, typically large scale/more than one exon (70% of the 66%)
Hot spot for deletions from exons 3-8 and between 44-50
Point mutations and duplications are in the minority of causes

Question 31

why are deletions more often the cause of DMD and BMD than duplications?

if duplication was the cause, what is a likely feature of the duplication?

Answer 31

Deletion = loss of genetic information (especially in X chromosome in males) causes severe phenotype.
Duplication = extra copy of information often tolerated, e.g. down syndrome is an extra entire chromosome and is arguably milder than many single gene deletions (or even a few exon deletions). As long as reading frame is preserved

duplications - still likely to be (but not definitely) pathogenic. the ones we do see in DMD and BMD must interrupt exons, with the breakpoints of the duplication probably occurring within an exon

note - duplications are also hard to identify, as genetic testing doesn’t tell you where your duplication is

Question 32

are the variants seen in DMD and BMD inherited?

how large are they (looking for a percentage statistic)?

Answer 32

2/3 pathogenic variants are de novo (not seen in either parent, makes sense as severe phenotype often prevents reproduction so rarely see inherited cases)

~70% have deletion of >1 exon

Question 33

at the level of the gene, what is the key difference between DMD and BMD variants and how does this explain why DMD is more severe than BMD?

Answer 33

Frameshift mutations -
As we know, they cause truncation (early stop codon bound to happen within 100 codons) so the mRNA is destroyed by NMD, and dystrophin isn’t produced. This is DMD - when you get NO dystrophin produced

BMD - occurs when the variant does not alter the reading frame. I..e the deletion/duplication is in frame, possibly including a number of exons. Not destroyed by NMD as the sequence that is there is correct
So dystrophin is produced, it’s just typically a shorter protein so is only partially functional (hence why BMD is less severe than DMD)

NOTE - using this idea to distinguish BMD and DMD mutations is only 92% accurate, so not necessarily reported straight to patients

Question 34

what are the symptoms and prognosis of DMD males?

Answer 34

Age of onset 3 to 5 years old
Delayed walking
Muscle weakness, lower limbs

Pseudohypertrophy- enlarged calves
Cardiomyopathy – heart disease (by 14 years)

Breathing problems - caused by deformed bones and muscle weakness

Wheelchair bound by 12
Reduced life span, <30 years

Question 35

what are the symptoms and prognosis of BMD males?

Answer 35

Milder (but still severe)
Later-onset skeletal muscle weakness
Wheelchair dependency (after age 16 years); although some remain ambulatory (capable of walking) into their 30s – key difference from DMD
Cardiomyopathy (diagnosed at ~14 years)
Life span to mid 40s

Question 36

what does a female carrier of a DMD or BMD mutant look like?

why is there such a range in effect?

Answer 36

one mutated allele, one wild type allele…

76% of DMD and 81% of BMD have NO symptoms

others can have:
Mild to moderate muscle weakness (20% of DMD, 15% of BMD)
Cardiomyopathy (8% in DMD, none in BMD)

range in effect due to:
Features vary dependent on X-inactivation
X-inactivation may be skewed favouring either wild type or variant allele

XX - one of the X’s in every cell gets inactivated. The proportion throughout the body of which X is inactivated differs. Women with some symptoms are likely to have a greater proportion of their normal/WT X gene being inactivated

Question 37

genetic testing -
what is the best option and why?

If MPLA didn’t give a positive result?

Answer 37

the quickest strategy to identify pathogenic variant in the DMD is to first use MLPA to exclude the exonic deletions and duplications, which will detect over 70% of positive cases. This takes 3 days vs 2 weeks for NGS

if the test was negative, you’d do NGS analysis to identify any point mutations/sequence variants (if they cause a frameshift, they are likely to be pathogenic)

Question 38

in males with DMD caused by a deletion what would the MLPA result be?

if you’ve detected a deletion, what details can help you determine whether it is DMD or BMD?

Answer 38

ratio would be zero if there had been a deletion, not 0.5, because they only have one X as it is

Deletions
Exon to whole gene deletions generally considered pathogenic
Where reading frame is disrupted, DMD is likely diagnosis
Where reading frame is maintained, BMD is likely diagnosis
Literature/databases for similar deletion variants is helpful

Question 39

DMD and BMD - what is the typical go-to treatment?

include side effects

Answer 39

No cure

Steroids (Corticosteroids) to maintain muscle function & slow muscle weakening

But has side effects: weight gain, decreased bone mineralization (weakened bones) & behavioural disturbances

Physical therapy - maintain muscle strength

Assisted ventilation

Surgeries, Cardiac transplantation

Question 40

exon skipping is a new therapy for DMD. how does it work?

Answer 40

Antisense oligonucleotides (AONs) are small modified pieces of DNA or RNA that specifically hybridize to a target exon during the pre-mRNA splicing process

This hides the target exon from the splicing machinery,
So the exon will be spliced out with its flanking introns
This may make deletion larger, but restores reading frame

Allows production of a partially functional dystrophin like those found in BMD

Question 41

give details on the DMD treatment drug ‘Eteplirsen’ and why its limited

Answer 41

it’s a drug containing AON (antisense oligonucleotides) that causes exon 51 to be skipped, which can only treat the 14% of DMD patients with exon 51 truncating variants

Question 42

aside from exon skipping, name and explain the other new therapy developed for DMD generally, and specifically how it works in DMD

Answer 42

Induced pluripotent stem cells -
Induced pluripotent stem cells (iPSC) are derived from the patient’s mature cells that have been reprogrammed to embryonic? stem cell like state
Can differentiate to any human cell type

Therapy – use patients own cells, gene edit the pathogenic variant, transplant the cells back
Huge potential not just in DMD but other conditions e.g. Parkinson’s

In DMD - Generate iPSC from e.g. fibroblast
Gene edit DMD pathogenic variant - CRISPR/Cas9 or zinc finger nucleases
Differentiate iPSCs to myogenic progenitors
Transplant back into patient
Now has self derived cells producing normal dystrophin