Unit III-Sickle Cell Anemia (Molecular)

Question 1

Sickle Cell Anemia, a Molecular Disease

Answer 1

• Pauling and collegues described that the defect causing sickle cell anemia was in the protein called hemoglobin
• Sickle cell anemia is called the first molecular disease

Question 2

Hemoglobin Mutation

Answer 2

• the demonstration of the single amino acid substitution in beta globin and nucleotide change in choromosome 11
• an A to T conversion in the beta globin gene
• valine is substituted for glutamic acid
• hemoglobin is an alpha2beta2 tetramer where only the beta subunits are modified in sickle cell hemoglobin

Question 3

hemoglobin S polymerization

Answer 3

• the single amino acid substitution caused deoxygenated HbS to form 14 stranded polymers. When well oxygentated it returns to the individual Hb tetramers (sometimes called the monomeric state to contrast it from the polymeric state
• going back and forth from sickle cell to real cell- weakens the plasma membrane

Question 4

Vaso-Occlusion

Answer 4

• the hallsmarks of sickle cell disease are the cycling between the biconcave and sickled shape (in reversibly sickled cells) and resulting rbc hemolysis (anemia) and vasoocclusion
• some sickle cell rbcs are locked into the sickled shape even when the cell is well oxygenated and HbS depolymerized to the monomer
• these are called irreversible sickled cells and constitute 2-40% of circulating RBCs in homozygous sickle cell anemia

Question 5

Multistep model for vascular occlusion in sickle cell disease

Answer 5

• although sickle cell anemia is a thought to be a monogenic disease the great variance in severity and outcome is based on individual polymorphisms in chromosomes 2,6, and 11 as well as varied extent of inflammation and vasculopathy
• therefore in sickle cell disease vasocclusion is caused not only by altered RBCs but also altered leukocytes, blood vessel endothelial cells and plasma factors
• the steps in vasoocclusion, which relate to activation of endothelial cells and leukocytes and expression of adhesion proteins on these cells and RBCs

Question 6

Survival of Patients with Sickle Cell Anemia who had Different Crisis Rates

Answer 6

• on average there is about a 15 year difference in life span between those with 1 or fewer vs 3 or greater crises per year
• the most important questions in sickle cell research revolve around the molecular bases for this variation in clinical severity and outcome and biomarkers for severity which can be used to develop personalized medicine for children with sickle cell disease

Question 7

Expression of E, Y and B globin

Answer 7

• two gene clusters encode the globins:
  1) The alpha cluster on chromosome 16
  2) The Beta cluster on chromosome 11

-during early embryonic development erythropoiesis is yolk sac derived. Midway through the first trimester there is a transition to the fetal liver. At birth the bone marrow becomes the primary site of erythropoiesis

• there is an accompanying switch in gene transcription from the B globin gene cluster.
• A switch from embryonic E to fetal Gy and Ay at gestation and
• A switch from fetal Gy and Ay to adult B at birth

-the Beta like genes are 5’ to 3’ with distal LCR directing expression of these genes

Question 8

HbF Regulation and Sickle Cell Severity

Answer 8

• genome wide association studies have demonstrated 3 loci that are associated with fetal hemoglobin expression and clinical severity
• those in chromosome 11 relate to cis acting haplotypes of Sickle cell
• those on chromosome 2 relate to the trans acting BCL11A and those in the intergenic interval on chromosome 6 relate to the trans acting HBS1L-MYB
• variation in either chromosome 2 loci for BCL11A or the chromosome 6 intergenic loci for HBSIL-MYB are associated with sickle cell severity
• variants that cause high HbF levels are clinically less severe

Question 9

Single Nucleotide Polymorphisms outside B globin cluster and fetal hemoglobin expression

Answer 9

• altered transcriptional regulation of the Y to B globin that leads to increased fetal hemoglobin results in decreased HbS polymerization and a less severe form of the disease
• normally fetal hemoglobin
• if it can be raised to ~20% this results in a less severe form of sickle cell disease

Question 10

Haplotypes in the B-globin gene cluster

Answer 10

• 5 different B globlin-like gene cluster haplotypes which contain a polymorphism, acting cis to the cluster, which regulates fetal Hb expression
• these haplotypes originated in Africa, the Middle East and India
• fetal Hb in the four major haplotypes varies from highest to lowest Arab-Indian then Senegal then Benin then Bantu
• the severity of disease would be in reverse order beginning with most severe it would be Bantu then Benin then Senegral then Arab-Indian

Question 11

Inflammation and Vasculopathy in sickle cell disease

Answer 11

• clogging of rbcs and leukocytes bia attachment to the blood vessel endothelial wall leads to vasoocclusion
• vasoocclusion leads to ischemia where cells and tissues are not receiving the required oxygen
• ischemia leads to cellular metabolic changes which leads to a burst of reactive oxygen species production when blood flow is restored
• during ischemia cells increase expression of xanthine oxidase
• upon reperfusion this xantine oxidase converts oxygen into superoxide radical
• this burst of ROS production is coming from the endotheial cells, adherent leukocytes and xanthine oxidase attached to the endotheial surface
• the ROS leads to NFKB activation, inflammation and release of inflammatory cytokines, activation of leukocytes, increased expression of adhesion molecules on the surface of the endothelial cells and leukocytes, further vascular plugging, decreased NO availability and resulting abnormal endothelial dependent vaso-dilation

Question 12

Sickle Cell Adhesion

Answer 12

• adhesion molecules on the surface of one cell type forms a heterophilic interaction with a binding partner adhesion molecules on a different cell type
• PSGL-1 (P-selectin glycoprotein ligand 1); ESL-1 (E-selectin ligand 1)
• endothelial cells and sickle RBCs and leukocytes

Question 13

Sickle Cell is in Double Jeopardy

Answer 13

• sickle cell RBCs contain ~3X as much oxygen radicals compared to normal red blood cells
• they have very low levels of reduced glutathione (GSH), particularly in the highest density RBCs
• the level of GSH is inversely proportional to the cell density. So the most damaged and dehydrated sickle cell RBCs have levels of GSH so low that it cannot be measured

Question 14

Reactive Oxygen Species and Antioxidants

Answer 14

• the RBCs major function is to carry and deliver O2 and it is designed for this purpose including its extremely large content of hemoglobin which constitutes 97% of its cytosolic protein
• it is the constant exposure to O2 at varying PO2 within the circulation which produces ROS and RNS and makes the RBC uniquely susceptible to oxidative damage
• the human RBC derives it high content of ROS and RNS primarily based upon hemoglobin interactions and associations
• the reversible binding of oxygen to Hb requires the heme iron to be in the Fe (II) charge state, when bound oxygen is bound Fe III producing superoxide and metHb
• the superoxide is converted H2O2 at a rapid rate due to superoxide dismutase
• metHB has a lower affinity for heme, release hemin and the free iron deposit on the cytoplasmi sufface of RBC membrane
• the hemin or free Fe II can then convert H2O2 into the highly toxic OH
• the RBCs in the circulation also acts as a sink for oxygen radicals

Question 15

Some of the increased ROS and RNS in sickle cell disease

Answer 15

• increased autooxidation of HbS into metHb and O2
• the H2O2 in contact with metHbS release of heme and free iron more readily that metHbA. The free heme and iron are found on the cytoplasmic surface of the RBC membrane and catalyze production of OH by the Fenton and Haber-Weiss reactions
• superoxide O2 binds NO to form the RNS peroxynitrate
• released cell free HbS bindings NO limiting its vasodilatory, anti-inflammatory and antithrombotic properties. This causes increases in blood vessel endothelial activation and ROS release from these cells
• Ischemia-reperfusion injury leads to increased xanthine oxidase production and later NADPH oxidase activity which generate superoxide which is converted to the hydroxyl radical
• as a response to increased inflammation SCA polymorphonuclear leukocytes produce ROS in an NADPH oxidase dependent respiratory burst

Question 16

Decrease in antioxidant protection in SCD

Answer 16

• the enzymatic and non-enzymatic anti-oxidant scavengers are reduced in SCA
• while studies on SOD activity have been conflicting in SCD most indicate that it is increased in activity leading to increased H2O2 and subsequently OH
• Glutathione and catalase activity are reduced in SCA RBCs further explaining why H2O2 is elevated
• GSH is substantially reduced in SCA RBCs and the amount of intracellular GSH is inversely related to cell density. In the higher density SS RBCs, which are the most damaged dense RSCs and ISCs, the GSH is below detection

Question 17

Cysteine Modifications Related to Oxidative Stress in SCD

Answer 17

• the systeine thiolate in the presence of H2O2 can be converted to sulfenic acid (SOH)
• the reaction of the SOH with a neighboring cysteine or GSH will create a disulfide or mixed disulfide S- gluthathiolation
• under increased oxidative stress the SOH can be further oxidized to sulfinic acid or sulfonic acid which represents irreversible oxidative damage

Question 18

Hyper-coagulation, thombosis and decreased RBC lifespan in SCD

Answer 18

• oxidative damage to scramblase causes loss of phosphatidyl serine asymmetry across the RBC lipid bilayer
• this places patches of the negatively charged head group of PS on the outer leaflet where it can associate with clotting factors which lead to hypercoagulation and thombosis in some patients with Sickle Cell Disease

-the PS patches on the outer leaflet also serve as a recognition site for macrophage association, This is one reason why sickled RBCs have a shorter circulatory life span

Question 19

Changes in protein expression and post translation modification

Answer 19

• lead to variance in severity
• because of the extreme oxidative stress faced by Sickle Cell patients throughout life a major PTM is formation of cysteine oxiforms

Question 20

Molecular Basis of Irreversible Sickle Cells

Answer 20

• highly elongated and dehydrated
• it is incapable of changing shape and is not flexible
• it is a participant in the clogging of the circulatory system that leads to vasoocclusion and crises
• the % dense cells and ISCs correlates with sickle cell severity
• the molecular basis for formation of ISCs has been solved and is linked to oxidative stress and cysteine modification

Question 21

RBC Membrane Skeleton

Answer 21

• spectrin membrane skeleton is responsible for the shape of the RBC as well as its properties of elasticity and flexibility essential for the constant distorting forces that it experiences in its travel through the circulatory system
• made of 280kDa alpha and 246 kDA B-spectrin
• the tail ends of spectrin tetramers attach to actin protofilaments
• the membrane skeleton is attached to the lipid bilayer in two ways

Question 22

Normal and ISC Actin 3D structure

Answer 22

the defect in the ISC B-actin is a disulfide bridge formed between Cys 284 and Cys 373

• the single posttranslation modification in B-actin causes it to polymerize and depolymerize more slowly than RSC and control B-actin
• ISC actin monomers are tightly associated within actin protofilaments which disasemble slowly at physiological temperature

Question 23

Spectrin Subunits

Answer 23

• a and B subunits of spectrin
• they form antiparallel heterodimers which then link head-to-head to form aB2 tetramers
• throughout most of the spectrin subunits there are triple alpha helical repeats called spectrin repeat units they are numbered from the N-terminus of each subunit
• rbc spectrin has E2/E3 ubiquitin conjugating/ligating activity which is capable of ubiquitinating itself as well as other target proteins

Question 24

Model of ubiquitin transfer within a spectrin

Answer 24

SCA alpha spectrin has vastly diminished ubiquitination (50-90% reduced)

• this is due to the diminished E2/E3 activity due to oxidation of the active site cysteine thiolates
• nonubiquitinated spectrin creates a more tightly associated spectrin 4.1 actin and spectrin-adducin acid ternary complex than ubiquitinated spectrin, the rate of dissociation of the sickle cell ternary complex at 37C is much slower than the control RBC ternary complex

Question 25

Defects in the ISC Membrane Skeleton

Answer 25

• membrane skeleton that dissembles and reassembles slowly leading to a cell locked into the sickled shape
• the slow dissociation of the ISC membrane skeleton is due to: 1. the B actin disulfide bridge which leads to actin protofilaments that disassemble slowly or not at all and
• diminished alpha-spectrin ubiquitination which creates a spectrin-4.1-actin and spectrin-adducin-actin ternary complex which disassembles slowly at 37C

Question 26

Why do sickle cell RBCs become dense

Answer 26

• deoxygenation of SCA RBCs stimulates HbS polymerization and sickling which in turn activates a sickling induced leak channel
• this in turn induces a transcient increase in intracellular free Ca2+ and loss of intracellular K+ and Mg++
• this leads to activation of calcium dependent Gardos channel and the KCl co-transport channel with resultant loss of K+, Cl-, and water
• the dehydration leads to higher mean corpuscular hemoglobin concentration MCHC which stimulates HbS polymerization and further K+ loss and dehydration

Question 27

Two Step Model for Dense ISC Formation

Answer 27

1) Decreasing GSH levels in sickle cell RBCs cause them to become dehydrated and dense
- NAC can block lower GSH induced sickle cell K+ efflux and dehydration.
- oxidative damage to the Gardos pathway can be reversed with NAC which decreased intracellular GSSG/GSH ratio
- the highest density sickle cells have <1% of the GSH found in the lowest density sickle cells
- first lowering intracellular GSH leads to increased concentration of oxygen radicals and reversible oxidative damage to the Gardos channel
- then oxidative damage to the Gardos channel leads to increased K+ loss and cell dehydration
- then NAC which raised intracellular GSH levels, as well as surviving as an antioxidant can reverse the damage to the Gardos channel

2) Locking of the sickle cells
- due to the oxidative damage to B actin and lack of ubiquitination of alpha-spectrin both caused by diminished GSH levels
- both defects reversible by NAC
- NAC blocks formation of ISC in vitro by oxygenation-deoxygenation cycling

Question 28

NAC Phase II trial

Answer 28

• determine efficacy of NAC in reducing dense cell and ISC levels, increasing intracellular GSH and reducing acute VOC episodes in SCD
• 2400 mg/day, increase GSH, decrease dense cells, ISCs, and crises
• provided proof of concept

Question 29

Current Treatments of Sickle Cell

Answer 29

• antibiotics
• analgesics
• Hydroxyurea
• Blood and Bone Marrow Stem Cell Therapeutics

Question 30

Future Therapies of Sickle Cell

Answer 30

• effect K leakage from RBCs
• effect NO levels (statins, inhaled NO)
• effect oxidative stress (NAC)
• effect adhesion (statins)
• effect inflammation (lovastatin)
• effect HbF levels (decitabine)
• replace the defective gene (gene therapy)

Question 31

Biomarkers for Sickle Cell Severity and Future Personalized Medicine for SCD

Answer 31

• identification of 20 monocyte proteins whose levels are highly correlated with 5 year crisis rate
• decreased apolipoprotein A-1 in the plasma correlated with sickle cell associated pulmonary hypertension and acute pain episodes
• increased microRNA mir144 leads to low antioxidant capacity, increased oxidative stress and more severe phenotype as measured by increased hemolysis, lower Hb concentration, and higher % reticulocytes
• need to use severity biomarkers in longitudinal studies to see how early in life they can predict severity