Genetics 8 - Clotting Disorders and Pharmacogenetics Flashcards
learning outcomes
overview of coagulation cascade
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)
fibrin
Ia
thrombin
IIa
video
https://www.youtube.com/watch?v=FNVvQ788wzk&feature=youtu.be
difference between 2 pathways
extrinsic = tissue factor - fast
intrinsic = contact activation - slower
common pathway after factor Xa and Va activate thrombin
time for intrinsic pathway
measured by PTT - partial thrombin time
20-35 seconds
time for extrinsic pathway
measured by prothrombin time
PT/INR (normal range 0.8-1.2)
overview of coagulation cascade
F VIIIa → F VIII
carried out by
deficiency
previously activated thrombin
Haemophilia A
F XIa → F IX
carried out by
deficiency
thrombin
Haemophilia B
F V → F Va
carried out by
deficiency
thrombin
thrombophilia
blood clots in BVs
thrombin and crosslinking of fibrin strands
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
haemophilia A
gene
size of gene
mutations may result in
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
gene F8 types of mutation
classification
- large rearrangements - insertions, deletions
- 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%
sequence on intron 22 of F8 relevant to Haemophilia A mutation
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
large rearrangements with haemophilia A
int22h-2 and int22h-3 are flanked by imperfect palindromic sequences - mirror images of each other
hybridisation and recombination can occur
inversions and deletions - haemophilia A
pairing between palindromic sequences (during male meiosis) can invert int22h-2 and int22h-3
what does recombination between copies of int22h result in
- inversion of F8 exons 1-22
- deletion of F8 exons 1-22
summary of haemophilia A
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
role of factor IX
haemophilia B - factor IX
what proportion of haemophilia does it account for
gene associated
carriers
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
therapy for haemophilia A/B
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
pharmacogenetics
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
pharmacogenomics
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
glucose 6 phosphate dehydrogenase deficiency
what does it infer
type of inheritance
how are symptoms manifested
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
G6PD deficiency symptoms
haemolytic anaemia (chronic)
acute haemolytic crises
anaemia and jaundice (hyperbilirubinemia) in the newborn
kernicterus - irreversible neurological damage
shortness of breath
dark coloured urine
G6PD deficiency symptoms - prevention and treatment
avoid triggers
bill lights (newborns) - isomerises bilirubin
blood transfusion
G6PD - gene map locus
Xq28
heterogeneity of G6PDD alleles
greater heterogeneity
predominant alleles vary between ethnic groups
common in areas with malaria
things to remember
learning outcomes
coagulation test results indicative of haemophilia A
pattern of inheritance of haemophilia A
X linked recessive
symptom common to Haem. A
cephalohaematoma
phenotypic tests - haemophilia A
F8 activity
PTT
PT/INR
carrier females and haemophilia A
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
Haemophilia A and carrier women
where is F8 expressed
level of activity of a functional F8 allele
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
assessing carrier status genetically
- direct sequencing of F8 (big job but getting less so)
- 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
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
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
gene - VWD
VWD chr 12p13.3 - large gene - 52 exons 178 Kbp
inheritance pattern of VWD
types of defects
many alleles ⇒ heterogeneity of subtypes
quantitative or qualitative defects of VW
inheritance can be dominant or recessive
clinical phenotype spectrum
what is often reduced
asymptomatic/mild (common) to severe haemorrhaging (rare)
often reduced F VIII levels - metabolised faster without VWF
treatment of VWD
desmopressin or “factor”
thromboembolism
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
role of factor V
what do people with thromboembolism response poorly to
activated protein C (APC) in a coagulation assay
protein C does not inhibit F V as it’s supposed to
gene associated with F V Leiden Thrombophilia
how does it affect coagulation cascade
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
category of mutation - F V Leiden Thrombophilia
allele frequency of F V Leiden
hetero vs homozygotes
what increases the risk of TE disease
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
what is warfarin
risks
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
blood is clotting faster than expected - risk of thromboembolism
increase warfarin dose
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
things to remember
