Inherited musculoskeletal diseases

Question 1

What is mutational heterogeneity?

Answer 1

Different mutations of single gene locus that causes same disease phenotype

Question 2

What is locus heterogeneity?

Answer 2

Variants at different gene loci that cause same or similar phenotypic expression of a disease or condition

Question 3

What is a loss of function mutation?

Answer 3

Gene product reduced/ none of normal function any mutation that inactivates gene product results in same clinical symptom

Question 4

What is duchenne muscular dystrophy (DMD)?

Answer 4

Loss of function disorder due to deletion at Xp21

(clinical phenotype already studied)

Question 5

What is Beckers muscular dystrophy?

Answer 5

Much milder form of DMD caused by same mutation in Xp21

Question 6

How was the gene responsible for DMD isolated?

(3 marks)

Answer 6

Subtraction cloning

• Selectively clone DNA from normal individual that is absent in the patient
  
  • Generate series of clones representing deleted parts of patients X chromsome
  • Within this DNA gene responsible for DMD was identified

Cytogenetics - studies structure of DNA within cell nucleus.

Question 7

What do all females with DMD carry?

(3 marks)

Answer 7

• X autosomal translocations
• Occurs sporadically in family history with no DMD
• X-breakpoint disrupts the DMD gene and therefore marks the position of DMD
• One normal X and one translocated X (equally inactivated)

Question 8

How does random X inactivation in females cause them to have DMD?

(2 marks)

Answer 8

• Normal X chromosome is mostl inactivated
• Normal X chromsome is sacrificed as inactivation of a translocated X chromsome leads to inactivation of autsomal loci on it

Question 9

How does X autosomal translocation occur in DMD?

(4 marks)

Answer 9

• DMD has locus on Xp21 and swaps with chromosome 21
• ​Genetic material swaps, and get disruption of short arm at position 21 on X chromsome
• Only have small sequence of gene responsible for DMD and rest has been swapped over onto other chromsome
• Get then X21 translocated chromosome
• Can make gDNA library from patient and screen for clones containing these genes

Question 10

How does translocation occur in females with DMD?

(2 marks)

Answer 10

• Translocation between chromosome 21 and X chromosome (only happens in one X chromosome)
• Process of x inactivation favours DMD due to nature of tanslocation between X and 21 chromosome
  
  • this is the reason that despite it beign recessive females are still able to get it

Question 11

How does x inactivation occur in females with DMD?

(2 marks)

Answer 11

• Dosage compensation mechanism - ensure only one X is activated in each cell
• Early zygote - either maternal or paternal X is randomly inactivated

Question 12

How does x inactivation occur in DMD?

(6 marks)

Answer 12

• Sporadic translocation with X:21 ⇒ get chromosome that has mostly X chromosome and the tip of it will have chromosome 21 (& vice versa)
• Top region of chromosome 21 has rDNA for cell function in early blastocyst, X:21 chromsome can be inactivated get:
• ⇒ Viable (inactivation of normal X): normal X with 21:X and 21 chromosome
  
  • genotype is viable as still have both copies of autosomal chromosome
• ⇒Non-viable: has only ONE copy of autosomal gene so dies

Question 13

What gene product is created from DMD?

Answer 13

Dytrophin - mutations of it cause DMD or BMD

Question 14

What are some of the mutations that cause DMD?

(3 marks)

Answer 14

• Deletions (60-65% patients)
• Small mutations, intorn deletions, exon insertions of repetitive sequences
• Duplication (5-15%)

Question 15

What do frameshift deletions in dystrophin cause?

Answer 15

• DMD
• Shifted reading frame an no dystrophin expressed

Question 16

Between which exons is the mutation hotspot for DMD?

Answer 16

Exon 43-55

(same as BMD but DMD more severe as it shifts reading frame)

Question 17

Where is dystrophin expressed and what happens when it is not expressed?

(5 marks)

Answer 17

• Sarcolemma in skeletal muscle
• Normal function: maintain strength, flexibility and stability of muslce fibres
• Mutated:
  
  • Muscles unable to contract properly
  • Cycles of necrosis and degeneration
  • Fibrosis

Question 18

What are the critical domains are needed for dystrophin function?

(4 marks)

Answer 18

N & C terminal domains in: C1, C3, & Cys-rich dystrobevin

Question 19

What are problems faced for gene therapy in DMD?

(2 marks)

Answer 19

• Muscle cells are post mitotic and can’t incorporate virus in division
• Dystrophin mRNA is huge - adeno associated viruses have limit to what tehy can carry, so have to keep injecting it

Question 20

What are potential solutions to the problems faced in gene therapy for DMD?

(2 marks)

Answer 20

• Use minigenes that contain dystophin domain
• Substitute dystrophin in utrophin : correct the reading frame using exon skipping

Question 21

How does the gene therapy approach of ‘replacement’ work for DMD?

(3 marks)

Answer 21

• Need 20% levels of WT dystrophin to correct muscle dystrophy
• Requires delivery to all muscles.
• Micro- and mini-dystrophin have been delivered using AAV vectors
• Any adverse affects could be minimised with immunosupressants

Question 22

What is ‘control of dystophin splicing’ to alleviate symptoms of DMD?

(2 marks)

Answer 22

• Use synthetic antisense oligonucleotides - bind to exon and cause to it to be skipped from transcript - exon 45 (most frequently deleted in DMD)
• Get onto next exon and and can be used to restore the reading frame and generate BMD like symtoms - so partially functional

Question 23

What exon would be skipped in order to give someone with DMD BMD like symptoms?

(3 marks)

Answer 23

• Exon 45
• Use antisense oligonucleotide to remove exon 46 and exon 44 is spliced to exon 47 to restore reading frame
• Cause functional domains of dystrophin like in BMD

Question 24

How can you correct the deletion of exon 50 which causes a frameshift in DMD?

(2 marks)

Answer 24

• Combine with exon 51
• So skips exon 51 and rstores reading frame using antisense oligonucleotides

Question 25

What is one ‘downside’ to the use of antisense oligonucleotides for exon skipping?

Answer 25

Repeated administrations required