application of invitro; study of primary lymphoedema

Question 1

where is the lymphatic system?

Answer 1

• a network of small vessels that connect to the lymph noders and larger lymphatic vessels (collectors)

Question 2

what does the lymphatic system connect to?

Answer 2

large main vein and drains into the CVD system near the heart

Question 3

what are the functions of the lymphatic system?

Answer 3

• fluid homeostasis
• immunue function; transport of APCs to the lymph nodes where an immune response is stimulated
• fatty acid transport

Question 4

what happens when the lymphatic system fails?

Answer 4

• lymphoedema; swelling and elephantiasis skin changes ; primary and secondary lymphoedema
• infections of the swollen area (e.g. cellulitis, verrucas and athlete’s foot)

Question 5

difference between primary and secondary lymphoedema

Answer 5

primary is genetic
secondary is due to infection or other stimulus

Question 6

what is done in the lab to test for lymphoedema?

Answer 6

genetic studies, genemapping using linkage analysis > combining linkage results with exome sequencing > verification by sanger sequencing > in vivo and in vitro functional studies

Question 7

what are EPH receptors?

Answer 7

large group of receptor tyrosine kinases and their ligands are the ephrins
- cause bidirectional signalling

Question 8

what is EPHB4?

Answer 8

transmembrane receptors with LB, CR, FNIII, TK and SAM domains
- EPHB4 ligand is Ephrin B2

Question 9

what is the role of EPHB4/Ephb2 ?

Answer 9

mediators of vascular and lymphatic development (endothelial cell growth, survival and migration)

Question 10

exome sequencing results for EPHB4 mutations in GLD patients

Answer 10

heterozygous missense mutations:
- Family A; Exon 14
- Family B; Exon 13
Location in highly conserved residues of the tyrosine kinase domain

Question 11

hypothesis in investigaiton of EPHB4 and lymphoedema

Answer 11

mutant EPHB4 proteins show reduced TK activity

Question 12

methodology of investigation of the EPHB4 mutations

Answer 12

• site directed mutagenesis
• HEK transfection with lipofectamine (lipofection, chemical process)
• immunoprecipitation
• western blot

Question 13

immunoprecipitation methodology

Answer 13

1. suitable antibody is added (anti-EPHB4)
2. antibody binds to protein of interest
3. protein A or G added to make antibody-protein complexes insoluble
4. centrifugation of solution pellets antibody-protein complex. Removal of supernatant and washing

Question 14

experiment repeated in LEC

Answer 14

LECs transfection with an expression vector containing Myc-DDK tag

Question 15

what was the conclusion of this investigation?

Answer 15

R739Q and I782S EPHB4 variants associated to primary lymphoedema show reduced TK activity > loss of function phenotype