Week 2 - Sickle Cell Flashcards
Familiarise yourself with the scenario
Daniel is an only child of non-consanguineous Afro-Caribbean parents. He is a 2.5 years old male infant who is brought into the Accident and Emergency department by his parents because of a 6 day history of fever, cough, breathlessness, pallor and weakness. His parents report no previous serious medical history and they also report no major family medical history of which they are aware. On examination he has a temperature of 39.5oC measured in the ear (normal 35.8-38oC) he looks pale and his pulse rate is 110 beats per minute, respiratory rate of 32 breaths per min, and his blood pressure was recorded as 90/60mmHg. On further examination there was an enlarged abdomen caused by hepatosplenomegaly. On auscultation of the lung fields rales (crackles) and sonorous wheeze could be heard.
Initial laboratory investigations reported: haemoglobin of 4.5 g/dL (normal 10.5-13.5g/dL)
white cell count of 19 x109/L (normal range 5-17 x109/L (55% neutrophils, 45% lymphocyte),
platelet count of 70 x109/L (normal range 150-400 x109/L)
and an erythrocyte sedimentation rate of 45 mm/hour (normal range 0 – 15 mm/hr).
Renal function tests and urinalysis were normal. A chest radiograph revealed bilateral haziness. Daniel was admitted and the next day dactylitis was observed in both hands.
Further investigations including Hb electrophoresis, showed that Daniel had Hb S of 80%, Hb F: 18%, and Hb A2: 2%. Finally in peripheral blood smear in low oxygen showed sickling of RBC plus a high proportion of nucleated red blood cells, Daniel was diagnosed with sickle cell anaemia and acute splenic sequestration crisis which was associated with acute chest syndrome. He was given wide spectrum antibiotic treatment (cefotaxim and erythromycin) and also had a blood transfusion exchange. The patient was discharged after 8 days as he stable. He and his parents were referred to the haematology and genetic counselling departments.
Explain why Daniel does not develop symptoms until after the 6 months of life
2 marks
Babies still have fetal Hb and t is not until this starts to decline after birth
And is replaced by the adult but mutated Hb that RBC will start to sickle
What is the inheritance pattern of sickle cell disease?
Autosomal recessive inheritance
How can you use a blood smear to diagnose sickle cell anaemia?
You can see under a microscope that the cells look sickled or not
They are only sickled in deoxygenated conditions so you have to create deoxygenated and non-coagulating conditions (avoiding the clotting cascade)
How does hydroxyurea work as a treatment for patients with sickle cell disease?
It works in 2 ways
1) Through the Red Blood Cells.
Red Blood Cells contain haemoglobin carrying oxygen to the body.
People will sickle cell disease have abnormal haemoglobin called sickle haemoglobin
Sickle haemoglobin causes RBC to be me stiff and sticky
The sickle cell can then cause a block in the flow of blood to important organs
Hydroxyurea helps the body make another haemoglobin called fetal haemoglobin which is usually only produced by babies before birth. With higher levels of Hb F, RBC are less likely to sickle and cause problems
2) Hydroxyurea LOWERS the number of neutrophils (infection - getting white blood cells). Increased neutrophil count makes the blood thicker and stickier which increases the chance of sickled cells to get stuck in the veins and cause a blockage
How does the amino acid change, change the structure of the haemoglobin in sickle cell?
Glutamic acid to Valine
Hydrophobic region is exposed in beta subunit
Abnormal protein made
Causes crystallisation into a fibre and reduces the oxygen carrying capacity of RBC
When the RBC come into a low oxygen conc environment - causes sickling
How is electrophoresis used to diagnose sickle cell?
Use an acetate gel, load the blood samples
Differentiate between homozygous and heterozygous states
Loss of charged glutamic amino acid and changes the speed that the sample moves
Haemoglobin has a net negative charge so when you have glutamic acid (negative) and valine (no charge) haemoglobin becomes less negative and travels further down the gel
If positive end on the other side
Normal one travels the most as it is more negatively charged
In the trait you see one normal (Hb-A) and one sickle cell anaemia (Hb-S)
If negative end on other side, Hb-S moves the most as it is less negatively charged
Trait is in the middle
Hydroxyurea is a drug for sickle cell and it is a myosuppression drug, what does this mean?
It kills erythrocyte progenitors
Highly toxic
There is a sharp shock where the erythrocyte progenitor are killed
The drug is then withdrawn
The erythrocyte progenitors grow again and then you get an increase in fetal haemoglobin
Daniel has sickle cell
If he was to marry a woman who did not carry the mutated allele, what would the status of any children be and justify your answer
2 marks
All would be carriers of the mutated allele as Daniel is homozygous and would pass on a mutated copy to all his children (1 mark)
But the wife would pass on the normal allele to all children (1 mark)
Daniel’s blood test had
Erythrocyte sedimentation rate of 45mm/hr (normal range 0-15mm/hr)
What does this indicate?
Erythrocyte sedimentation rate basically puts blood and lets it drop down in a period of one hour
(Blood sediments - settles to the bottom of liquid)
And if the blood is heavier because of the proteins inside of it, it drops quicker
These proteins are indicative of an infection, inflammation and anaemia
Daniel’s blood test had:
Haemoglobin of 4.5 g/dL (normal range 10.5-13.5g/dL)
Platelet count of 70 x10^9/L (normal range 150-400 x 10^9/L)
What does this indicate?
Low haemoglobin levels and RBC
This is because the RBC do not last as long because the sickle haemoglobin damages them
They have fewer red blood cells than normal
Daniel’s blood test had
White cell count of 19 x 10^9/L (normal range 5-17 x 10^9/L)
55% neutrophils, 45% lymphocyte
What does this indicate?
Indicative of an infection
What are some advice for coping with a child with a chronic illness?
- Include all family members in decision makings
o Empowering patients, feel in control, makes situation easier and more bearable and tolerable
§ Don’t want to feel like our choice is being taken away
§ Can lead depression and making the condition worse
Impact on families, wider families and the children.
What are the different subunits of of fetal haemoglobin?
2 alpha
2 gamma
What are the different transitions between fetal and adult haemoglobin?
Alpha chains increases at the yolk sac and then remains at a high level
At birth we start to see a decrease in gamma chains and increase in the beta chains
At 25 weeks after birth we see a large decrease in gamma and we see large increase in beta chains
What are the different treatment options for sickle cell anaemia?
Bone marrow transplant (can cure 85% of children)
Hydroxyurea Antibiotics Pain relievers Blood transfusions Nitric oxide
SEG101 - Crizanlizumab
Anti P selectin monoclonal antibody-adhesion molecule
What are the different ways you can diagnose sickle cell anaemia?
Electrophoresis
Solubility Test
Blood Smear
What are the positives of the solubility test to diagnose sickle cell anaemia?
Cheap (low cost paper based)
Don’t require specialist equipment
100% sensitive
100% specific
What are the properties of fetal haemoglobin?
Fetal haemoglobin has a higher affinity for oxygen than an adult
Can carry up to 30% more oxygen
This is important as the maternal blood’s oxygen is then readily transferred to the fetal blood across the placenta
What are the stats with bone marrow transplants to treat sickle cell anaemia?
85% succes rate in children with sickle anaemia
5-10% fatality risk
What are the symptoms of a sickle cell crisis?
Sudden crisis that are periodic, very painful (worse than cancer pain)
You might have pain in your back, knees, legs, arms, chest or stomach
Pain may last for several hours
What are the triplet codes for the stop codons?
UAA
UAG
UGA
What does a non-conservative missense mutation mean?
Change in amino acid that does not have similar properties to the original one
What does it mean for the genetic code to be degenerate? What are the exceptions?
Multiple triplet codes make the same amino acids
Start codon methionine and tryptophan
What is dactylitis and why is it a common symptom of sickle cell?
Dactylitis is severe pain that affects the bones of the hands, the feet, or both. It’s often the first symptom of sickle cell disease in babies.
Dactylitis is caused by blocked blood circulation.
What is hydroxyurea?
It is a medication given to patients with sickle cell anaemia
Recommended to people who have had two or more episodes of sickle cell crisis or acute chest syndrome in their life
What is the amino acid change in sickle cell?
Glutamic acid to valine
What is the methodology of using a blood smear to diagnose sickle cell anaemia?
You take a blood sample
Add EDTA to stop the blood from clotting by removing the divalent cations (stopping the clotting cascade)
Add 2% sodiu metabisulphite to create deoxygenated conditions
(Cells don’t sickle in oxygenated conditions)
What is the parasite that causes malaria?
Plasmodium falciparum
What is the triplet code of the start codon?
AUG
Methionine
What subunits of haemoglobin are encoded on chromosome 11?
Beta
Gamma
Epsilon
Zeta
What type of genetic mutation causes the sickle cell mutation and explain how this causes a change in the amino acid sequence?
3 marks
Point mutation a single nucleotide change (A to T) (1 mark)
This changes the three amino acid codon sequence so that the amino acid coded changes (1 mark)
from glutamic acid to valine. (1 mark).
What was the previous life expectancy of sickle cell anaemia? What is the life expectancy now?
20-40 years
Now its basically normal with treatments available
Which chromosome are beta subunits of haemoglobin encoded on?
11
Which chromosome is the alpha subunit for haemoglobin encoded on?
16
Why do you get hepatosplenomegaly with sickle cell?
Pooling of sickling cells, accumulating in the spleen and liver
Cells start to die within these organs causing splenic atrophy and portal hypertension
Why is Hb electrophoresis used to diagnose sickle cell disease?
2 marks
The change in the amino acid changes the electrical charge to be less negative (1 mark).
This means that the Hb travels at a different speed to normal as travels slower than normal. (1 mark)
What are the stats of sickle cell trait protecting children in Western Kenya?
60% protection against mortality in
2-16 months
What is the mechanism protection by sickle cell trait against P. falciparum?
When the parasite bites the RBC, there is an increased O2 consumption, the RBCs also stick to the endothelium.
This is because the parasite puts protein knobs called PfEMP1 on the surface of the RBC. These protein knobs will interact with other cell adhesion molecules like ICAM and makes the RBC sticky.
Increased O2 causes RBC to sickle if you have HbS and the sickle cell trait
There is less adherence to the endothelium due to the sickling, so the RBCs are removed by macrophages
When the RBCs die, the parasites die with it too, so less parasite growth and maturation
In the trait the protein knobs there are fewer knobs that are not evenly distributed and are concentrated on certain bits, this reduces adhesion and therefore if you have sickle cell trait, the RBC are less sticky and detach and therefore die while the normal RBC can carry enough oxygen.
A mutation in HbS (haemoglobin gene)
I) is what inheritance?
II) Causes what disease if homozygous?
III) Provides what advantage if heterozygous?
I) Autosomal recessive
II) Sickle cell anaemia
III) Resistance to malaria
A mutation in CFTR (gene encoding the CFTR sodium channel)
I) is what inheritance?
II) Causes what disease if homozygous?
III) Provides what advantage if heterozygous?
I) Autosomal recessive
II) Cystic fibrosis
III) Resistance to tuberculosis? Cholera?
A mutation in RhD (Rhesus blood group gene)
I) is what inheritance?
II) Causes what disease if homozygous?
III) Provides what advantage if heterozygous?
I) Autosomal inheritance
II) Hemolytic disease of the newborn
III) Resistance to toxoplasma
A mutation in Connexin 26
I) is what inheritance?
II) Causes what disease if homozygous?
III) Provides what advantage if heterozygous?
I) Autosomal recessive
II) Deafness
III) Resistance to bacterial infection, enhanced cell repair
A mutation in HBA1/ HBA2 (Alpha globin genes)
I) is what inheritance?
II) Causes what disease if homozygous?
III) Provides what advantage if heterozygous?
I) Autosomal recessive inheritance
II) Thalassemia
III) Resistance to malaria,
decreased heart disease?
Where is the mutation for sickle cell disease found?
Beta globin gene
6th amino acid
DNA: CTC to CAC
mRNA: GAG to GUG
Glutamic acid to Valine
Non conserved missense
Single nucleotide mutation
Which amino acid is the mutation for sickle cell disease found?
6th amino acid in the beta globin gene
What is the exact SNP (DNA) change in sickle cell disease?
Beta globin gene
6th amino acid
Glutamic acid to valine
Non conserved missense
CTC to CAC
T to A
What is the exact mRNA change in sickle cell disease?
GAG to GUG
A to U
Beta globin gene
6th amino acid
Glutamic acid to valine
Non conserved missense
DNA:
CTC to CAC
T to A
What is triplet code for the start codon?
What is it’s amino acid name?
AUG
Methionine
What does degeneracy of the triplet code mean?
More than one triplet code for one amino acid
What are the properties of glutamic acid?
Charged
Polar
Tucks into haemoglobin
What are the properties of valine?
Why does this change the beta globin gene to sickle?
Hydrophobic
Non-polar
Sticks out of the haemoglobin structure
Normally you don’t have hydrophobic amino acids sticking out in an aqueous environment, this causes the molecules to crystallise and become a fibre which reduces the oxygen carrying capacity (usually the molecules without the mutation and without valine would not associate and each molecule/heterodimer would carry oxygen itself)
In low oxygen environments the RBC would sickle
What is the normal structure of a haemoglobin molecule?
2 alpha chains
2 beta chains
Heterodimer
Quaternary structure
How does the Valine in the 6th amino acid affect the structure of haemoglobin and consequently its function?
The valine is hydrophobic, and it is exposed in an aqueous environment
This causes the molecules to crystallise into a fiver and this reduces the oxygen carrying capacity (usually molecules do not associate with each other and each one would carry oxygen)
In low oxygen environments, the haemoglobin will sickle
I) What is hepatosplenomegaly?
II) Why is it caused in sickle cell?
III) What are the consequent effects?
I) Enlarged liver and spleen
II) Sickled blood cells clog up the small arteries towards the spleen
III) Increased infection as the spleen usually filters out the blood and checks for infections (e.g. pneumonia, blood stream infections, meningitis, bone infections)
What do the crackles and wheezing indicate on auscultation?
Crackles indicate an illness or infection as there is fluid accumulation in the lungs (link to mild pneumonia)
Wheezing indicates narrow or small airways in the lungs
Both link to infection
What does Daniel’s blood pressure of 90/60 mm/Hg indicate?
What link does this have to sickle cell disease?
Low blood pressure
The RBC that have HbS die quickly and so there is less blood overall contributing to a lower BP
Normal is 104/63 to 113/67
Why do you start to see splenic atrophy?
Because the blood starts pooling in the spleen and the cells start to die
During development, explain the production of the alpha chains of haemoglobin?
At the stage of the yolk sac the alpha chain rises to a peak (at 6 weeks of gestation) and the alpha chain levels stay high throughout pregnancy, birth and neonatal stages
During development, explain the production of the beta chains of haemoglobin?
The beta chain levels are relatively low during gestation and rise rapidly after birth reaching its peek at around 14 weeks
Beta chain levels stay high after that
The Beta chains replace the gamma chains that used to be a part of fatal haemoglobin
During development, explain the production of the gamma chains of haemoglobin?
The gamma chain level rises at around 10 weeks gestation and stays high until birth
At birth, the gamma chains are gradually replaced by beta chains, and reaches its trough at around 15 weeks after birth
Where is the alpha globin locus found?
Chromosome 16
Where is the beta globin locus found?
Chromosome 11
What are the subunits of Gower 1?
2 zeta
2 epsilon
What are the subunits of Gower 2?
2 alpha
2 epsilon
What is the function of the LCR locus control region?
The LCR region encodes
Epsilon
Deta
Gamma
Beta
Subunits and it switches between these subunits via a cis control region formed stream of the initiation site of the gene
The Cis control region regulates the switching on and off of the different forms of globin subtypes
How is the LCR on chromosome 11 expressed / deleted?
Expressed via transcription factors
Deleted via demethylation or artificial deletion
Why might the manipulation of the LCR region in chromosome 11 helpful in sickle cell?
You might be able to control the gene switching by making more gamma subunits and more HbF which has a higher affinity for oxygen
What is the affinity for oxygen in HbF compared to normal adult haemoglobin?
Why is this helpful during pregnancy?
Fetal haemoglobin has higher affinity for oxygen
It can carry up to 30% more oxygen
This is helpful because the oxygen from the maternal haemoglobin can be transferred to the foetal circulation readily
How often is hydroxyurea given to sickle cell patients?
Once a day
Kills the erythrocyte progenitors with a sharp shock, then the drug is withdrawn
Fetal haemoglobin starts to grow
What direction is the current of electrophoresis?
The sample is loaded on the positive end
The sample then moves along the gel towards the negative end
Sample moves positive to negative
Is glutamic acid positively charged, negatively charged or not charged?
Negatively charged
Is valine positively charged, negatively charged or not charged?
Not charged
Describe how normal haemoglobin and sickle haemoglobin moves in the electrophoresis gel and explain why
Normal haemoglobin has the negative glutamic acid.. All forms of haemoglobin have a net negative charge.
The other end of the gel is negative so it moves slow ish.
The sickle haemoglobin doesn’t have the glutamic acid and instead has the uncharged valine. So sickle haemoglobin is less negative, and so it moves faster towards the negative end of the gel.
So sickle haemoglobin moves faster than adult haemoglobin
You can see the trait which would have both
What is the method for the solubility test to diagnose sickle cell anaemia?
Mix the 20 microlitres of blood and add buffer (2.49M phosphate buffer) and saponin to make it soluble
Add a reducing agent (sodium hydrosulphite) to reduce the amount of oxygen
Wait for 10 mins and drop it on the paper
Analyse the pattern you see after 25 mins
You see differential polymerisation - so when someone has sickle cell, there is more polymerisation and more crystals being formed, and you can see this in the pattern
Problem: can’t differentiate between the trait and the disease
Problem with solubility test to diagnose sickle cell?
Can’t differentiate between trait and disease
In a blood smear, what to individuals with HbS look like in normal conditions?
Normal
Oxygentated conditions
Why is EDTA added in a blood smear for diagnosis of sickle cell?
It removes divalent cations such as magnesium and calcium, stopping the clotting cascade
Why is sodium metabisulphite added to a blood smear to diagnose sickle cell?
To provide deoxygenated conditions so the blood cells will sickle
What 2 things are added to a blood smear?
Sodium metabisulphite (deoxygenated conditions)
EDTA (stops clotting)
How does SEG101 work as a treatment for sickle cell?
Crizanlizumab
Anti P selectin monoclonal antibody-adhesion molecule
P selectin is a protein that binds to cell adhesion molecules that causes coagulation of the sickle RBC
Anti P selectin inhibits the stickiness of the RBC
What used to be the life expectancy of sickle cell?
20-40 years earlier death
Who brought about the USA National Sickle Cell act and what was the consequence?
1970s
Robert Nicson
Put $500 000 towards sickle cell research in the States
Who should receive genetic counselling?
Parents of newborns with sickle cell disorder or trait
Pregnant women/ prenatal counselling
What is the purpose of genetic counselling?
Education
Informed decision making
What should the content of the genetic counselling sessions include?
Genetic basis (trait or full)
Chances of disease or trait (potential pregnancy outcomes)
Disease related health problems
Variability/ unpredictability/ severity/ onset of the disease (depending on the mutation you have)
Family planning
Average life span
What are 6 ways to help cope with a child who has a chronic illness?
- Learn about illness
- Join a support group
- Face the possibility of early death
- Maintain open and honest communication
- Share leadership
- Include all family members in decision making
What does non-consanguineous mean?
People who do not descend from the same ancestor
What does pallor mean?
Unhealthy pale appearance
What does sonorous wheeze mean?
Low pitched
Course
Loud
Low
Snoring or moaning sound due to narrowing of the large airways or an obstruction of the bronchus
What is bilateral haziness seen on the chest radiograph?
Diffuse haziness that would typically be caused by inflammation or thickening of tissues
What are nucleated RBCs?
Very immature forms of RBCs, seen when there is a severe demand for RBCs to be released by the bone marrow
What is an acute splenic sequestration crisis?
An acute condition of intrasplenic pooling of large amounts of blood
The spleen enlarges painfully, and RBCs are trapped inside
It is an emergency and patient can die within 1-2 days without treatment
What are the two antibiotics given to Daniel? Why were they given?
Cefotaxime and erythromycin
Wide spectrum bacterial antibiotics to treat the infections he had
What is a non-conservative missense mutation?
Different amino acid is coded for
With different properties that are not similar to the original amino acid
What is the most common type of mutation that occurs to the beta global protein in sickle cell anaemia?
Nonsense mutation Conserved missense mutation Non conserved missense mutation Insertion mutation Duplication mutation
Non conserved missense mutation
Why are antibiotics given to patients with sickle cell?
Reduce risk of infection
How long do sickle cell crises typically last?
5-7 days
What triggers sickling?
Infection
Dehydration
Acidosis
What is a vaso-occlusive crisis?
Form of sickle cell crisis that is painful and recurrent episodes can cause irreversible organ damage
Caused by sickled RBCs obstructing capillaries and restricting blood flow to an organ
Swelling can persist 1-2 weeks after pain has resolved
You can also have the blood supply cut off of the abdominal and retroperitoneal organs (spleen enlarges, liver can loose blood supply)
What are vaso-occlusive crisis treated with?
Hydration
Analgesics
Blood transfusion
What is acute chest syndrome?
Classified by two of the following symptoms: Chest pain Fever Pulmonary infiltrate/ focal abnormality Respiratory symptoms Hypoxaemia
80% of patients have a vaso-occlusive crisis during acute chest syndrome
What is the normal result for a Hb electrophoresis?
HbA2 - 1.5-3.5% (Daniel’ s was 2%)
HbF - <2% depending on age (Daniel’s was 18%)
HbS - absent (Daniel’s was 80%)
Usually the rest would have been HbA
Name 4 signs and symptoms which doctors would use to diagnose Daniel with acute chest syndrome?
Fever Cough Breathlessness, dyspnoea Pallor, hypoxia High pulse rate High res rate Lung crackles
