W4L8 - Duchenne Muscular Dystrophy and Hereditary Hemochromatosis

Question 1

Duchenne Muscular Dystrophy

Answer 1

X-linked recessive disorder
Symptoms include:
- ‘waddling’ gait
- delayed motor function
- problems sitting, standing, walking up stairs, lifting arms
Muscle is continuously wasted then regenerated -> replacement of muscle by adipose and connective tissue

Question 2

Dystrophin

Answer 2

Protein product of the DMD gene
3685 aa
Connects cytoskeleton actin to the ECM via a dystrophin-associated protein complex
Stabilises sarcolemma during muscle contraction and relaxation
Transmits forces generated during muscle contraction to the extracellular matrix

Question 3

What happens when Dystrophin is absent or non-functional?

Answer 3

Sarcolemma becomes compromised during repeated muscle contraction/relaxation
Leads to influx of Ca2+ into muscle fibre
Ca2+ is a co-factor for several proteases
Protease activation results in muscle fibre destruction

Question 4

What causes DMD?

Answer 4

Mutations that result in the production of an incomplete, non-functional dystrophin protein
Majority of mutations disrupt opening reading frame
They introduce a premature stop codon
This produces a shortened protein product lacking key domains required for dystrophin function

Question 5

How can DMD be diagnosed?

Answer 5

Massive raise in Serum Creatine Kinase
Abnormal muscle histology
- fibre hypertrophy, degeneration, atrophy
- increase in endomysial CT

Question 6

Molecular Diagnosis of DMD - Southern Blotting

Answer 6

Genomic DNA digested with Hindlll, Bglll or EcoRI

Labelled cDNA probes spanning approx. 10 exons are used to identify deletion

Question 7

Molecular Diagnosis of DMD - Multiplex PCR

Answer 7

Used to detect deletions of 18 different exons in the DMD gene
6 different primer sets in 3 separate reactions used to amplify regions encoding the different exons from genomic DNA
Reactions separated using gel electrophoresis
Missing band equates to deletion of corresponding exon

Question 8

Principle of Single Stranded Conformation Polymorphism (SSCP) Steps

Answer 8

ss NA will form secondary structures if allowed to
The secondary structure they adopt is dependent on the sequence of the NA
Therefore, a mutation might alter the secondary structure formed
This change may be detected by electrophoresis

Question 9

Hereditary Hemochromatosis

Answer 9

Autosomal recessive disorder
Early symptoms include:
- lethargy
- weakness
- joint pain
- abdominal pain 
Additional symptoms may include
- liver disease
- diabetes
- arthritis

Question 10

How does Hereditary Hemochromatosis come about?

Answer 10

Most commonly due to mutation in the HFE gene
The HFE protein is bound to transferrin-receptors 1 and 2 at the cell surface
Said to be involved in ‘iron-sensing’ in hepatocytes

Question 11

Steps in Iron Homeostasis

Answer 11

1. Dietary iron is absorbed in the enterocytes of the intestine
2. Ferrous iron (Fe2+) transported into enterocyte by DMT1
3. Fe2+ converted to Fe3+ and transported out of the cell via ferroportin
4. Fe3+ binds to transferrin in blood which transports it throughout the body
5. Transferrin-bound iron is taken up by cells via a receptor-mediated process (transferrin binds to transferrin receptor 1)
6. Iron is used by the cell or stored as ferritin

Question 12

Iron Homeostasis in Hepatocytes

Answer 12

The binding of transferrin-bound iron to the transferrin receptor results in production of a soluble hormone called Hepcidin
This travels through the plasma and binds to ferroportin on the surface of enterocytes, resulting in internalisation
The internalisation of ferroportin prevents release of iron from cells into the blood

Question 13

What happens during Hereditary Hemochromatosis?

Answer 13

Iron homeostasis is disrupted
Hepcidin is not produced in response to transferrin-bound iron
Therefore, the release of iron from enterocytes and macrophages isn’t controlled
Transferrin becomes saturated
Plasma non-transferrin bound iron increases
Iron deposited in parenchymal cells of organs, resulting in destruction

Question 14

What does it mean when the mutation for Hereditary Hemochromatosis has incomplete penetrance?

Answer 14

A mutation doesn’t cause the disease

It increases the risk of having the disease

Question 15

Other factors that increase the risk of Hereditary Hemochromatosis

Answer 15

Male
Alcohol intake
Dietary iron and Vitamin C intake
Blood donation

Question 16

Hereditary Hemochromatosis Laboratory Diagnosis

Answer 16

Iron studies
- increased serum ferritin
- increased transferrin saturation
HFE mutation analysis
Liver biopsy
- used if other mutations present and if ferritin is super high