Genetics 8 - Clotting Disorders and Pharmacogenetics

Question 1

learning outcomes

Answer 1

Question 2

overview of coagulation cascade

Answer 2

blood clotting to plug damaged BVs

starts with platelets

fibrin - Ia

fibrinogen circulates in blood in high conc

activated to fibrin by thrombin (IIa)

complex cascade (chain rxn) involving 13 factors (mostly protease precursors)

Question 3

fibrin

Answer 3

Ia

Question 4

thrombin

Answer 4

IIa

Question 5

video

Answer 5

https://www.youtube.com/watch?v=FNVvQ788wzk&feature=youtu.be

Question 6

difference between 2 pathways

Answer 6

extrinsic = tissue factor - fast

intrinsic = contact activation - slower

common pathway after factor Xa and Va activate thrombin

Question 7

time for intrinsic pathway

Answer 7

measured by PTT - partial thrombin time

20-35 seconds

Question 8

time for extrinsic pathway

Answer 8

measured by prothrombin time

PT/INR (normal range 0.8-1.2)

Question 9

overview of coagulation cascade

Answer 9

Question 10

F VIIIa → F VIII

carried out by

deficiency

Answer 10

previously activated thrombin

Haemophilia A

Question 11

F XIa → F IX

carried out by

deficiency

Answer 11

thrombin

Haemophilia B

Question 12

F V → F Va

carried out by

deficiency

Answer 12

thrombin

thrombophilia

blood clots in BVs

Question 13

thrombin and crosslinking of fibrin strands

Answer 13

activates F XI (near start of intrinsic pathway) and F XIII which crosslinks the fibrin strands together to form the clot at the end

through amplification loops - +ve and -ve feedback control

a little thrombin goes a long way

Question 14

haemophilia A

gene

size of gene

mutations may result in

Answer 14

gene F8 - factor VIII

X q28

26 exons, > 186 kb DNA

haemophilia A related to mutations in or near F8

mutations may result in:

a null allele - no working product

a hypomorph - product that works a bit

Question 15

gene F8 types of mutation

classification

Answer 15

1. large rearrangements - insertions, deletions
2. small mutations (< 50 bp, often SNPs)

mis sense, non sense, splice site variants (mutation changes reading frame)

CLASSIFICATION:

Mild 5-40%

moderate 1-5%

severe <1%

Question 16

sequence on intron 22 of F8 relevant to Haemophilia A mutation

Answer 16

int22h-1

300 kb 5’ (upstream) of F8 gene is a complex structure that includes interspersed repeats in opposite orientation

all 3 int22h sequences identical/very similar, so easy for them to recombine when gene folds

Question 17

large rearrangements with haemophilia A

Answer 17

int22h-2 and int22h-3 are flanked by imperfect palindromic sequences - mirror images of each other

hybridisation and recombination can occur

Question 18

inversions and deletions - haemophilia A

Answer 18

pairing between palindromic sequences (during male meiosis) can invert int22h-2 and int22h-3

Question 19

what does recombination between copies of int22h result in

Answer 19

1. inversion of F8 exons 1-22
2. deletion of F8 exons 1-22

Question 20

summary of haemophilia A

Answer 20

chr architecture predisposes to a particular change (major inversion) which accounts for a high proportion of defective alleles and 50% of severe cases

approx 5% of severe cases related to deletions in F8 gene

also other less common mutation

Question 21

role of factor IX

Answer 21

Question 22

haemophilia B - factor IX

what proportion of haemophilia does it account for

gene associated

carriers

Answer 22

12% of haemophilia

locus X q 27.1

wide range of mutations

about 10% of carrier females have < 50% F9 and are at risk for abnormal bleeding

Question 23

therapy for haemophilia A/B

Answer 23

haemorrhage prophylaxis - pads, avoidance of sports like rugby etc

local haemostasis (compression, sutures etc)

clotting factors (pharmacogenetics)

somatic gene therapy

germ-line editing - CRISPR-Cas

Question 24

pharmacogenetics

Answer 24

study of impact of single genetic variants on drug metabolism

predicting likely response and risk of adverse events based on mutation at a single locus

Question 25

pharmacogenomics

Answer 25

study of drug metabolism in relation to the whole genome of an individual use of genomics to optimise selection of pharmaceutical agents for individual patients based on better prediction of likely response and risks of adverse effects

Question 26

glucose 6 phosphate dehydrogenase deficiency what does it infer type of inheritance how are symptoms manifested

Answer 26

most common genetic disorders partial malaria resistance in hemi/homozygotes X-LINKED RECESSIVE - mostly affects males females affected via skewed XCI symptoms of haemolysis manifest when body is in oxidative stress caused by: * infection * medicines, including aspirin, sulfonamides, nitrofurantoin (UTI) and antimalarias * foods e.g. fava beans - contain oxidants

Question 27

G6PD deficiency symptoms

Answer 27

haemolytic anaemia (chronic) acute haemolytic crises anaemia and jaundice (hyperbilirubinemia) in the newborn kernicterus - irreversible neurological damage shortness of breath dark coloured urine

Question 28

G6PD deficiency symptoms - prevention and treatment

Answer 28

avoid triggers bill lights (newborns) - isomerises bilirubin blood transfusion

Question 29

G6PD - gene map locus

Answer 29

Xq28

Question 30

heterogeneity of G6PDD alleles

Answer 30

greater heterogeneity predominant alleles vary between ethnic groups common in areas with malaria

Question 31

things to remember

Answer 31

Question 32

learning outcomes

Answer 32

Question 33

coagulation test results indicative of haemophilia A

Answer 33

Question 34

pattern of inheritance of haemophilia A

Answer 34

X linked recessive

Question 35

symptom common to Haem. A

Answer 35

cephalohaematoma

Question 36

phenotypic tests - haemophilia A

Answer 36

F8 activity PTT PT/INR

Question 37

carrier females and haemophilia A

Answer 37

may have disordered coagulation tests or even mild clinical bleeding in tendency (skewed X inactivation) - approx 30% If good X chr is inactivated more than the bad, female may bleed more rarely females have severe haemophilia due to skewed X chr inactivation or a disorder of inactivation of X chr

Question 38

Haemophilia A and carrier women where is F8 expressed level of activity of a functional F8 allele

Answer 38

F8 allele is expressed from whichever X chr is active in a given liver cell 1 functional F8 allele in most women = 50% activity phenotype = clotting about 10% of carrier females have \< 40% activity - at risk for abnormal bleeding 2 non-functional F8 alleles = no functional gene ⇒ phenotype = bleeding

Question 39

assessing carrier status genetically

Answer 39

1. direct sequencing of F8 (big job but getting less so) 2. linkage markers within F8 * dinucleotide repeat in intron 13 and 22 - microsatellites * known SNPs in introns 18 and 22 * need to compare to affected family members

Question 40

Answer 40

Question 41

Answer 41

Factor IX Leyden a rare haemophilia B subtype - 3% anrogen therapy or puberty rise F IX activity from \< 1% to 30-60% associated mutations (\>20) from -40 to +20 of F IX this promoter sequence resembles an androgen response element

Question 42

Answer 42

Von Willebrand Disease most frequent congenital clotting disorder VWD chr 12p13.3 - large gene - 52 exons 178 Kbp many alleles ⇒ heterogeneity of subtypes quantitative or qualitative defects of VW inheritance can be dominant or recessive

Question 43

gene - VWD

Answer 43

VWD chr 12p13.3 - large gene - 52 exons 178 Kbp

Question 44

inheritance pattern of VWD types of defects

Answer 44

many alleles ⇒ heterogeneity of subtypes quantitative or qualitative defects of VW inheritance can be dominant or recessive

Question 45

clinical phenotype spectrum what is often reduced

Answer 45

asymptomatic/mild (common) to severe haemorrhaging (rare) often reduced F VIII levels - metabolised faster without VWF

Question 46

treatment of VWD

Answer 46

desmopressin or "factor"

Question 47

thromboembolism

Answer 47

formation of thrombus in deep veins - typically leg pain and swelling of leg thrombus may become detached from vein (embolus) and travel to pulmonary artery obstructs blood supply to a segment of lung obstructs main PA - sudden death

Question 48

role of factor V

Answer 48

Question 49

what do people with thromboembolism response poorly to

Answer 49

activated protein C (APC) in a coagulation assay protein C does not inhibit F V as it's supposed to

Question 50

gene associated with F V Leiden Thrombophilia how does it affect coagulation cascade

Answer 50

1601G\>A transition in exon 10 of the F5 gene on Ch1q23 p.Arg506GIn in F V (R506Q) - variant known as F V Leiden - always on pro-coagulation activity of F V is limited by activated protein C which cleaves at the arginine (R) F V Leiden - resistant to APC cleavage excess conversion of prothrombin → thrombin

Question 51

category of mutation - F V Leiden Thrombophilia

Question 52

allele frequency of F V Leiden hetero vs homozygotes what increases the risk of TE disease

Answer 52

allele freq - 5% in Caucasians (founder effect) heterozygotes 6-7x increase in relative risk of venous thromboembolism homozygotes 80x increase in relative risk of TE oral contraceptives increase risk of TE disease - oestrogen can drive clotting factors

Question 53

what is warfarin risks

Answer 53

anticoagulant vit K antagonist used to manage patients at risk of thromboembolic disease too much warfarin can be associated with life threatening haemorrhage monitor with coagulation test (PT/INR) adjust dose to maintain within therapeutic window

Question 54

Answer 54

blood is clotting faster than expected - risk of thromboembolism increase warfarin dose

Question 55

Answer 55

cytochrome P450 - detox alleles (CYP2C9\*2 and \*3) produce a phenotype of poor warfarin metabolism - occurs in 11% and 8% of whites but only 3% and 0.8% of blacks people with poor metabolism require much lower dose for therapeutic effect more warfarin circulating in body, higher anticoagulation effect - risk of bleeding

Question 56

things to remember

Answer 56