hemophilia

Question 1

normal physiology

Answer 1

• Process of preventing blood loss should occur as soon as the wound is created
• Should be a tightly regulated process (turn on/off)
• Process should only happen at the site of the wound
• Clot should dissolve when no longer needed

Question 2

describe hemophilia

Answer 2

rare inherited bleeding diathesis that results in unusual susceptivity to bleeding

Question 3

define hemophilia A

Answer 3

deficiency in FVIII = reduced/absent FVIIIa = reduced catalytic efficiency of FIXa = limited activation of FX

Question 4

define hemophilia B

Answer 4

deficiency in FIX = reduced catalytic efficiency of FIXa = limited activation of FX

Question 5

what causes hemophilia (what is not activated) ?

Answer 5

• coagulation cascade cannot function properly =

- intrinsic pathway not activated, not enough fibrin produced, above normal bleeding

Question 6

what is necessary to diagnose hemophilia?

Answer 6

less than 50% of normal protein

Question 7

how do we classify the severity of hemophilia?

what are the classifications?

Answer 7

• based on the levels of FVIII or FIX (as predicts risk of furture bleeding events)
• mild, moderate, severe

Question 8

protein levels used to define mild hemophilia are:

Answer 8

levels of 5-50% of normal

Question 9

protein levels used to define moderate hemophilia are:

Answer 9

levels of 1-5% of normal

Question 10

protein levels used to define severe hemophilia are:

Answer 10

levels less than 1% of normal

Question 11

hemophilia A and B are what type of genetic disease? what does this mean?

Answer 11

• monogenetic

- means mutation in one single gene is implicated in the disease process

Question 12

where is the mutation responsible for hemophilia?
what type of mutation is most common?
other types?

Answer 12

• mutation in the F8/F9 gene on the X chromosome
• point mutations are most common
• others = large or small deletions, insertions

Question 13

in what pattern are mutations for hemophilia inherited?

what does this lead to?

Answer 13

• via X-linked recessive patterns

- leads to deficiency of the functional FVIII or FIX proteins

Question 14

explain male inheritance of hemophilia

Answer 14

one altered gene is enough to cause hemophilia

Question 15

explain female inheritance of hemophilia

Answer 15

• rare as its unlikely for a female to have 2 mutated versions of the gene
• females who are carriers are generally asymptomatic (only 20-30% show signs of bleeding)

Question 16

who’s more likely to have hemophilia men or women?

Answer 16

men bc only one X chromosome

Question 17

would it be practical to asses hemophilia patients mrna for mutations? why or why not?

Answer 17

• no
• mRNA transcripts are large, also,
• only cells that produce the protein (liver cells) will express the mRNA
• thus, FVIII and FIX mRNA won’t be present in the blood
• would have to do a liver biopsy instead which is very invasive

Question 18

would it be practical to use a dna sample to detect molecular mutations in a hemophilia patient? why or why not?

Answer 18

• yes
• dna can be obtained from simple blood draw (extracted from peripheral blood mononuclear cells)
• could then use PCR & next gen sequencing to determine the sequence of the F8 or F9 genes and compare them to a reference

Question 19

F8 gene codes for? smaller or bigger than F9 gene?

Answer 19

• FVIII protein

- 26 exons (bigger)

Question 20

F9 gene codes for? smaller or bigger than F8 gene?

Answer 20

• FIX protien

- 8 exons (smaller)

Question 21

what mutation presents for the majority of severe hemophilia cases? why?

Answer 21

• F8 intron inversion
• F8 found at the end of X chromosome so its more succeptible to inversion
• F8 also large
• most happen at intron 22 - largest intronic region in the gene

Question 22

what is the mechanism for detecting inversions? (4)

Answer 22

(1) isolate dna from wbcs in blood draw
(2) incubate dna w restriction enezyme (cuts dna into smaller peices)
(3) run fragments through electrophoresis gel
(4) reference dna to healthy dna

Question 23

what are the clinical applications of hemophilia? (major symptoms) (2)

Answer 23

• spontaneous bleeding - localized to joints and soft tissue

- provoked bleeding - can be result of trauma or surgery

Question 24

what is the goal of treatment for hemophilia patients?

Answer 24

to maintain therapeutic levels of FVIII and FIX for sustained periods of time - to ensure coagulation cascade can function properly

Question 25

two methods of treatment for hemophilia are:

Answer 25

(1) on demand therapy: treating pateints as they experience bleeding events
(2) preventative therapy: infusion of clotting factors to prevent bleeding events
- proteins derrived from donated human plasma or through recombinant protein production by mamilian cells

Question 26

what is the level of clotting factor infusion to prevent spontaneous bleeding?

Answer 26

1-5%

Question 27

what is the target level of clotting factor infusion to prevent provoked bleeding?

Answer 27

10%

Question 28

limitations to protein replacement therapy are:

Answer 28

• must be administered IV
• short 1/2 life - need frequent repeated IV therapy (FVIII = 12hrs, FIX = 24 hours)
• body can recognize replacement as foreign
• can be expensive - not everyone has access
• transmission of infectious disease (HIV, hepatitis)

Question 29

what are other treatment options?

Answer 29

• PEGylation (prolonged half life)
• emicizumab (mimetic)
• fitusarin (rebalance)
• gene therapy (endogenous protein production)

Question 30

explain PEGylation

Answer 30

• PEGylation = chemical modification of FVIII protein through addition of PEG polymerase chains
• these block the interactions with FVIII clearence receptors increasing their 1/2 life
• still a protein replaement therapy tho so has limitations

Question 31

explain emicizumab

what are its benefits?

Answer 31

• emicizumab = bispecific antibody that binds FX and FIXa
• its a partial FVIII mimetic molecule (mimics action)
• increases catalytic activity of intrinsic terase complex and increases FXa formation
• benefits = subcutaneous administration, longer 1/2 life, act in presence of antiFVIII antibodies, low immunogenicity

Question 32

explain fitusarin

Answer 32

-siRNA molecule that acts to inhibit production of antithrombin to reinstate balance of coagulants and anticoagulants

Question 33

explain gene replacement therapy

Answer 33

• we use viral gene therapy with AAV (rep & cap genes replaced with promoter and therapeutic transgene)
• insert therapeutic dna into viral vector, capsid, then IV to patient
• delivers virus and new dna to target cell
• target cell will make the therapeutic protein