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Biochemistry Presentation - Sickle Cell Anemia
Transcript of Biochemistry Presentation - Sickle Cell Anemia
http://www.ncbi.nlm.nih.gov/pubmedhealth/PMH0001554/ Sickle Cell Anemia Etiology
Diagnosis & Treatment Hemoglobin A:
Normal Structure & Function Biochemical
Sickle Cell Anemia Hemoglobin S:
& Physiologic Effects HBB Gene & Role of SCA in Malarial Infection Clinical Manifestations & Symptoms Diagnosis & Treatment Hemoglobin Electrophoresis:
- Missense mutations may or may not alter aa sequence or function of protein; in SCA it does
- Substitution mutations replacing aa's with different electrical charge can make slight changes in overall charges of protein
- These allelic variants lead to protein variants called allozymes - differ slightly in electrical charge
- Protein electrophoresis [left] used to detect allozyme variation
- Tissue extracts are introduced into gel in the sample wells at Origin
- Electrical field applied: many proteins have net negative charge; will migrate from the Origin at cathode (-) to anode (+)
- Electrophoresis at alkaline pH: HbS migrates slower than HbA; lack of (-) charged Glu residues in the 2 -chains makes HbS less negative than HbA
- Electrophoresis of Hb from lysed RBCs used in diagnosis of sickle cell trait & sickle cell disease
- Homozygous individuals - HbA tetramer electrophoreses as single "fast" band, and HbS tetramer as single "slow" band.
- Heterozygous individual - both HbA & HbS tetramers present; runs as two bands. Genetic Basis for Sickle Cell Anemia
Biochemical Basis for Sickling
Protective Role in Malarial Infection
Symptoms, Diagnosis & Treatment Conclusion Introduction - Sickle Cell Anemia is a morphological misshaping of red blood cells, due to a change in hemoglobin structure
- The misshaping causes the RBC to take on a crescent, or sickle-like, shape
- This disease is found most frequently in populations of Middle Eastern, and African decent
- One effect of SCA is that the cells are less capable of oxygen transportation
- The sickle cell shape also makes it much easier to clot and get stuck in arteries
- 10% of American blacks and 25% of African blacks are heterozygotes - SCA occurs when Glu changes to Val due to a point mutation
- This amino acid results in an abnormal allele expression
- There are two types of Hemoglobin alleles
A is the normal allele
S is the abnormal allele
AA - normal hemoglobin; such individuals do not suffer from SCA (Homozygous dominant)
AS - individuals have one abnormal S allele; they also have a normal allele; therefore, they still produce some normal hemoglobin. These individuals may have some difficulties from SCA, but the effects are usually very limited. The two alleles are codominant.
SS - have all S-type hemoglobin and no normal type hemoglobin. Such individuals suffer from all of the symptoms that are characteristic of SCA - The S form of hemoglobin is slightly hydrophobic, due to a hydrophobic region that results in S-Hemoglobin.
- The hydrophobic regions cause S-hemoglobin to aggregate and form fibers under low oxygen conditions.
- This aggregation results in lowered oxygen-carrying capacity and also causes the morphological shape in the RBCs to the characteristic sickle-shape. Amino acid substitutions in HbS and HbC
A molecule of HbS contains 2 normal α-globin chains & 2 mutant β-globin chains (βS) in which Glu (hydrophilic amino acid) at position 6 replaced by Val (hydrophobic amino acid). Molecular and cellular events leading to sickle cell crisis. Sickling causes tissue anoxia:
Substitution of non-polar Val for a charged Glu forms a protrusion on the β-globin that fits into a complementary site on the α-chain of another Hb molecule in the cell.
At low oxygen tension, HbS polymerizes inside RBCs 1st forming a gel, then assembling into a network of fibrous polymers that stiffen and distort cell, producing rigid, misshapen RBCs.
Such sickled cells frequently block flow of blood in narrow capillaries. This interruption in supply of oxygen localized anoxia (oxygen deprivation) in the tissue pain and eventually death (infarction) of cells in vicinity of blockage. Variables that increase sickling:
Extent of sickling & severity of disease is enhanced by any variable that increases proportion of HbS in deoxy state (i.e., reduces affinity of HbS for oxygen)
These variables include decreased oxygen tension as a result of high altitude or flying in non-pressurized plane, increased pCO2, decreased pH, and an increased conc. of 2,3 BPG in RBCs Normal red cells maintain their shape as they pass through the capillaries and release oxygen to the peripheral tissues (upper panel).
Hemoglobin polymers form in the sickle rell cells with oxygen release, causing them to deform.
The deformed cells block the flow of cells and interrupt the delivery of oxygen to the tissues (lower panel). Hemoglobin molecule has two parts an alpha and beta
Sickle has a mutation in a gene on chromosome II that codes for beta subunit
Sickle cell hemoglobin forms long, inflexible chains Sources An abnormal hemoglobin has valine replacing glutamic acid
Valine is a less polar amino acid compared to glutamic acid
Favors hydrophobic interaction between each strand and its neighbor
The interaction between Val 6 on one chain of one hemoglobin molecule and a hydrophobic patch formed by Phe 85 and Leu 88 on a chain of another deoxygenated hemoglobin molecule heads to hemoglobin aggregation
Hemoglobin S Polymerizes under low oxygen conditions causing distortion of red blood cells and a tendency to lose their elasticity
Repeated cycles cause loss of elasticity and fails to return back into normal shape
New cells have high elasticity Curved sickle shape
Tend to clump together, making red blood cells sticky, stiff, and more fragile
Shift shape from normal to sickle and eventually become permanently sickle
Can clog blood vessels
Break down about 10 to 20 days instead of 4 months, causes anemia
Life span reduced by about 30 years
Normal hemoglobin is called hemoglobin A
Caused by Abnormal type of hemoglobin called hemoglobin S A little insight on malaria:
Malaria-infected red blood cells are stiffened as much as 50 times more than healthy red blood cells.
Infected red blood cells had a tendency to stick, flip, and flop along the walls of blood vessels. How sickle cell affects malaria:
Heterozygotes have selective advantage in areas where malaria is a major cause of death for two reasons:
The short life-span of the sickled cell prevents complete development of intracellular cycle of parasite.
Nutrients essential for the parasite to live “leak out” of a sickled RBC, causing the death of the parasite. Hemoglobin function:
Hemoglobin are globular proteins found in RBC
HBA Most common type of hemoglobin found in adults
It’s function is to transport O2 molecules to and CO2 away from tissues
250 million hemoglobin per red blood cell
Deoxyhemoglobin Structure, the globin group:
The hemoglobin consists of 4 globins and 4 hem groups
The globin group of the structure consists of four polypeptide subunits, 2 Alpha-globin and 2 Beta-globin chains
Each contains 141 and 146 amino acid residues
The fetal hemoglobin is made up of 2 Alpha-globin and 2 Y-globin
These subunits are held together by ionic bonds, hydrogen bonds, hydrophobic interactions, and van der waals forces
All of the subunits form alpha helixes Structure, the Heme group:
Each subunit has an alpha-helical structure and a heme binding pocket
The heme group is arranged in a circle with an Iron atom
The iron atom may be Fe2+ or Fe3+
The Iron binds to the four N’s in the center of the protoporphyrin ring
The Heme groups are connected to the globin by a nitrogen by the histidine, the 87th residue in the alpha chain by the histidine, the 92nd residue in the beta chain Active and inactive states:
T form is the tense state of the hemogloblin
Has a lower affintiy for oxygen
R form is the relexed state
Has a higher affintiy for oxygen
Binding of O2 to T form causes the heme group to change into the R form
As the shape of one heme group changes, it enhances the ability of the hemoglobin to bind more O2 molecules Diagnosis of Sickle Cell Anemia:
- Suggested by presence of chronic hemolytic anemia and vaso-occlusive crisis
- Electrophoresis confirms diagnosis; can screen for other hemoglobinopathies (ex thalassemia) as well.
Laboratory Tests Include:
- Mandatory screening for HbS at birth in US; prenatal testing possible by chorionic villus sampling (CVS)
- Hemoglobin electrophoresis Management:
The goals of treatment = symptom control and management of complications.
Seven general goals:
Mgt of vaso-occlusive crisis
Mgt of chronic pain syndromes
Mgt of chronic hemolytic anemia
Mgt of complications and organ damage syndromes associated with SCD
Detect/ treat pulmonary HTN Therapeutic Options:
Bone Marrow Transplantation (BMT):
- Can be cured by BMT, but difficult selection process for patients
- Many risks associated; risk-to-benefit ratio considered
- Might gain more acceptance since advent of cord blood stem cell transplantation
- Lack of available matched donor may limit utility of BMT Hydroxyurea Therapy:
- The only drug currently approved by FDA for treatment of SCD
- Long-term hydroxyurea = current accepted treatment for frequent and severe pain
- Increases total and fetal hemoglobin in children with SCD
- Increase in fetal hemoglobin reduces gelation and sickling of RBCs
- Hydroxyurea also reduces levels of circulating leukocytes = decreases in adherence of neutrophils to blood vessel walls.
- Leads to reduction in incidence of pain episodes How Hydroxyurea Works:
- Prevents HbS from sticking together and decreases sickling of RBCs by altering kinetics and thermodynamics of HbS polymerization
- Increases HbF levels in patients with SCD
- Patients with high HbF levels = fewer symptoms and better outcomes than patients with low HbF levels
- Hydroxyurea increases HbF and keeps the red blood cells round
- HbF primary Hb in fetus from mid to late gestation; binds O2 more tightly than adult HbA
- Composed of 2 alpha and 2 gamma subunits; gamma subunit is a protein product of beta-gene cluster
- HbF (fetal hemoglobin) protects against sickle cell symptoms by reducing # of irreversibly sickle cells
- HbF molecules do not polymerize like deoxyHbS: gamma chain lacks valine at 6th residue for hydrophobic int with HbS
- Higher [HbF]s in a cell infer lower [HbS]: Polymer formation depends on [deoxyHbS] HBB: The Gene Associated with Sickle Cell Anemia
Although several hundred HBB gene variants are known, sickle cell anemia is most commonly caused by the hemoglobin variant Hb S.
In this variant, the hydrophobic amino acid valine takes the place of hydrophilic glutamic acid at the sixth amino acid position of the HBB polypeptide chain.
Sickle cell anemia is an autosomal recessive genetic disorder.
For the disease to be expressed, a person must inherit either two copies of Hb S variant or one copy of Hb S and one copy of another variant.
Carriers, who have one copy of the normal HBB gene (Hb A) and one copy of Hb S, are described as having sickle cell trait and do not express disease symptoms. When are symptoms seen? Crises - painful episodes
bones of the back , long bones, and chest
hours to days
one episode every few years or many
can become severe General symptoms:
Rapid heart rate
Shortness of breath
Yellowing of the eyes and skin (jaundice) Priapism
lower leg ulcers
Pulmonary hypertension Due to blocked blood vessels by abnormal bloods cells, patients may experience many complications: Group 2: