INTS 5: Red Cells and Related Disorders Flashcards
1
Q
What is haemoglobin made of?
A
- heme
- the globins
2
Q
Describe the structure of heme
A
- an organic, ring-shaped molecule
- it is made from 4 pyrroles, to make a tetrapyrrole
- small pentagon-shaped molecules made from 4 carbons and 1 nitrogen
- If the tetrapyrrole has substitutions on the side chains which allow it to hold a metal ion, it is called a porphyrin.
- Thus, a heme is an iron-holding porphyrin.
3
Q
How is the iron molecule in heme held in place?
A
- by the balanced attractive forces of the four nitrogen molecules
- nitrogen molecules all point toward the inside of the larger ring they create
- the double and single bonds which connect the pyrroles are arranged evenly, so electrons stay balanced and the entire molecule remains stable
- this makes it an aromatic molecule
4
Q
What are the two functions of heme?
A
- it can bind gases, such as oxygen:
- and transport them throughout an organism.
- Special proteins then force the heme to release its oxygen at the appropriate time.
- A good example of a protein of this type is hemoglobin. Hemoglobin is found in all blood cells, attached to the cell membrane, exposing the heme group to the blood plasma. Thus, when the blood cells pass through the lungs, they bind up as much oxygen as the iron in the heme can handle.
- The blood cells then travel to various parts of the body, such as the muscles. These cells are actively using up oxygen and releasing carbon dioxide as a byproduct. Carbon dioxide forms an acid in the blood plasma, lowering the pH of the blood. Like all proteins, hemoglobin reacts to changes in pH by changing shape. This change in shape forces the oxygen off of the heme complex, releasing the oxygen into the blood plasma. The oxygen diffuses into the muscle cells, where it is bound by myoglobin and transported to the mitochondria to be used. Myoglobin also has a heme group, but it operates in a different way so that oxygen remains bound until reaching the mitochondria.
- holding electrons and facilitating reactions in the electron transport chain:
- occurs in all organisms
- During oxidative phosphorylation in the mitochondrial membrane, electrons must be passed down a series of reactions, which slowly extract their energy before depositing them in water and carbon dioxide.
- The energy gained is stored in the bonds of the molecule ATP, which most organisms use as a primary source of energy.
- The heme groups in these cytochromes are different than those in hemoglobin, for they have different functions and bind to different proteins.
5
Q
Describe the structure of haemoglobin
A
- each molecule of globin protein is a tetramer made of two dimers:
- made of two identical chains of two different globin genes
- the globin protein that form the 2 dimers vary with age
- fetal haemoglobin (in uterus)
- adult globin chains (after birth)
6
Q
Describe the genes producing different types of haemoglobins (HBs)
- Which chromosomes?
- What types of globin?
A
- globin genes are present on chromosome 11 and 16
- alpha globin is involved in the production of haemoglobin in all three stages
- embryonic
- fetal
- adult
- beta and delta is present in adults
- gamma lost in fetal stage
- episilon and zeta lost in embryonic stage
7
Q
Study this diagram to see which globin genes are primarily expressed at each developmental stage
A
- fetal haemoglobin is made during the very first weeks of life by two sets of proteins: epsilon (ε) and zeta (ζ);
- then up to birth, in the uterus, the alpha (α) and gamma (γ) chains are produced;
- finally the adult globin is made of two alpha (α) and two beta (β) chains.
- globins are required (in particular the alpha globin) to function from the very early stages of fetal growth.
8
Q
Describe the genes encoding globin proteins?
- complexity of structure: how many exons
- how many clusters of these genes?
- Which chromosomes?
A
- relatively simple structure:
- often made of only one or two exons
- two clusters:
- alpha cluster: zeta and alpha genes on chromosome 16
- beta cluster: episolon, gamma, delta and beta chains on chromosome 11
9
Q
What are the differing affinities of different haemoglobins?
A
10
Q
How are globin proteins assembled?
A
- each gene contributes two copies of each type of chain which are then joined to form a tetramer
11
Q
How does thalaseemia occur?
A
- generally occurs when a globin gene fails to produce sufficient and good quality globin proteins of one type or another
- any of the genes can be deleted (specifically the alpha cluster) or mutated (beta cluster)
- deletions encompassing the entire gene can happen on one chromosome or both copies (one from each chromosome)
- mutations are generally single-nucleotide mutations
- both can have severe or mild consequences, depending
- look at image for more info
12
Q
What is an increase in red cell number called?
A
- polycythaemia
13
Q
What techniques are used to analyse red cells’ function
A
- Blood count and differential:
- this investigates and analyse the total number of red cells, the level of haemoglobin and the percentage of reticulocytes in the PB.
- These would rise in the event of anaemia when the BM is required to compensate for an acute or chronic loss of blood - BM aspirate:
- to investigate the presence of precursors of red cells and their percentage compared to other lineages.
- In some form of congenital or anaemia the loss of maturation of red cell precursors can be the cause of anaemia - tests to investigate haemoglobinopathies (the defect affecting the production of haemoglobin):
- among many tests, we will concentrate predominantly on tests which allow measuring the different type of haemoglobin in the red cells.
- in particular, in particular the use of HPLC or High-pressure liquid chromatography and the cellular acetate mobility (CAM) test.
- Final confirmation can be provided in most cases of Beta-thalassemia, in particular, using Sanger Sequencing following PCR amplification of the exon/gene of interest (like the beta-globin gene).
14
Q
What is High Pressure Liquid Chromotography (HPLC)?
What does it do?
A
- able to analyse and separate the different components of globin in the red cells and provide a profile of the different types
- HPLC (high-performance liquid chromatography) is a chromatography technique where the mobile phase is a liquid and the stationary phase is packed into a stainless steel column at high pressure. It is usually silica particles mainly spherical nowadays. The efficiency is better when the particles are smaller typically 5um. A pump is used to push the solvent through the column and a detector with a flow-through cell used to measure the separated peaks. Usually, a computer with integration software collects the data and helps to quantify the components
15
Q
Describe the HPLC traces and peaks
What is the normal proportions of each type of haemoglobin?
A
- The main peak in an HPLC trace is the adult HB or HBA0 (2 alpha and 2 beta chains), then the HBA2.
- Hemoglobin A2 (HbA2) is a normal variant of hemoglobin A that consists of two alpha and two delta chains (α2δ2) and is found at low levels in normal human blood.
- Hemoglobin A2 may be increased in beta-thalassemia or in people who are heterozygous for the beta-thalassemia gene.
- HbA2 exists in small amounts in all adult humans (1.5-3.1% of all hemoglobin molecules) and is approximately normal in people with sickle-cell disease.
- On HPLC minor peaks are represented by the HBF or fetal haemoglobin.
- HBA1c is Glycated hemoglobin is a form of hemoglobin that is chemically linked to a sugar.
- The usual sugar is glucose.
- The formation of the sugar-Hb linkage indicates the presence of excessive sugar in the bloodstream, often indicative of diabetes.
- A1C is of particular interest because it is easy to detect.
16
Q
Explain the cellular acetate mobility (CAM) test
A
- Electrophoretic separation encompasses a variety of methods on solutes (usually charged molecules such as proteins, nucleic acids) on the basis of their mobility in an electric field. In general, an electric field is applied through some supporting medium and the solutes migrate in that field, according to their net charge
- Very small samples of haemolysates prepared from whole blood are applied to the Cellulose Acetate Plate. The haemoglobins in the sample are separated by electrophoresis using an alkaline buffer (pH ) and are stained with Ponceau’S Stain. The patterns are scanned on a scanning densitometer, and the relative percentage of each band determined.
- The figure below shows a diagram with the position of the different HB that may appear in different samples; Multiple bands can coexist with the main HBA0 in different patients; the controls should only contain the normal bands as illustrated in the HPLC normal control.
17
Q
Describe alpha thalassemia
A
- more detailed info in e-module
- Alpha thalassemia is a disorder characterised by the absence of alpha chain production, which are an important part of both foetal and adult haemoglobin. This is usually due to small or extensive deletions in the region of chromosome 16 where the alpha cluster is found.The type of deletion is seen to have geographical association so is named after the region of origin: SEA (South East Asia), FIL (Filipino), Thai (Thailand) or Med (Mediterranean).
- If all alpha genes are absent, this is the most severe form of alpha thalassemia and the individual is unlikely to live. Most deletions are small and affect one chromosome and deletions can be inherited from both parents. Thalassemia can be characterised as minor, intermedia, or major, depending on the number of genes lost. Individuals with defects in the alpha genes can have children with individuals with defects in beta genes resulting in offspring with combined defects.
18
Q
Describe beta globin thalassaemia
A
- more detail in e-module
- Beta globin thalassemia are associated with single mutations (rather than deletions) resulting in the change of a single amino acid.
- A common disease associated with beta thalassemia is sickle cell anaemia.
- This is caused by the mutation in the seventhamino acid of the HBB (beta globin) gene from GAG to GTG, leading to glutamic acidbeing replaced by valine.
- The tetramer shape of haemoglobin is then altered to form a concatemer which results in the sickle-like shape of the cell. These sickle cells are stiff rods which are inflexible and can stick to vessel walls, causing a blockage and slowing of the flow of blood which restricts the amount of oxygen reaching nearby tissues.
- Symptoms of sickle cell anaemia include extreme pain (ifthe small vessels in bones are blocked), strokes (if the blood vessels of the brain are blocked), or congestion of the microcirculation (if the block occurs in the spleen, in which case the spleen is removed to prevent rupture.)
- There are different types of beta thalassemias and sickle cell disease:
- HBS: Mutation in 7th amino acid.
- Glutamic acid to Valine (GAG -> GTG)
- HBC: Mutation in 7th amino acid
- Glutamic acid to Lysine (GAG -> AAG)
- HBD: Mutation in 122nd amino acid
- Glutamic acid to Glycine (GAA -> CAA)
- HBE: Mutation in 27th amino acid
- Glutamic acid to Lysine (GAG -> AAG)
- The severity of the condition depends on whether one or both of the maternal and paternal copies of the genes are affected.
20
Q
What happens when there is a G6PD deficiency?
A
- G6PD helps red blood cells function normally by removing and counteracting oxidative stress events and remove chemicals called reactive oxygen species (ROS)
- ROS build up during a fever, infection, certain foods, medications
- If the G6PD levels are too low, red blood cells will not be protected from these oxidative radicals. Red blood cells will die, leading to anaemia. In fact, low levels of G6PD cause the cells to haemolyse, releasing haemoglobin in the urine which can appear darker (a sign of haemolysis
22
Q
Describe the genetics of G6PD deficiency
Hence how is it diagnosed
A
- The G6PD gene is located on the X chromosome on the tip of the long arm.
- Deficiency is mainly caused by a mutation in any one of the 13 different exons leading to a different protein with a modified amino acid.
- Because males have only got one X chromosome, they are always symptomatic, while in females both chromosomes need to be affected, which is very rare. So this is a disease that manifests itself mainly in males.
- Over 300 different mutations have been described. A schematic illustration of some of the most frequent mutations is given below (in the mature mRNA section of the figure).
- They are normally diagnosed by simple Sanger sequencing of the gene, an easy and cost-effective diagnosis.
