Batool Emad. Marah Karablieh. - Nayef Karadsheh

Transcription

1 4 4 1 P a g e Batool Emad Marah Karablieh - Nayef Karadsheh

2 ***Topics that will be discussed in this Lecture: 1) Globin gene organization 2) Hemoglobinopathies 3) HbS (sickle cell disease) 4) HbC and HbSC diseases RULE ; Substitution of A for B (B is removed A is added) *************** ***Doctor mentioned these numbers just to refresh our information: HbA1c is used as an accurate monitor for glucose levels in diabetic patients, as the life span of RBCs is 120 days. From the book: HbA is slowly and nonenzymically glycosylated to HbA1c, this glycosylation depends on glucose plasma levels (higher glucose levels, higher HbA1c levels). The HbA1c has glucose residues attached predominantly to the NH₂ groups of the N-terminal valines of the β globin chains. Globin gene organization: The gene coding for the α-globin and β-globin subunits of the hemoglobin chains occurs in two separate gene clusters (or families) located on two different chromosomes: 1) α-gene family (on chromosome 16): It contains 2α genes (α1, α2) and one ζ (zeta) gene. 2 P a g e 2) β-gene family (on chromosome 11): It contains single gene for the β-globin chain and four β-globin-like genes: ε (epsilon) gene, two γ (gamma) genes and δ (delta) gene.

3 Hemoglobins are formed by combinations of two chains, one from each family: 1) Hb Gower 1 (embryonic hemoglobin): formed by combination of chains from ζ gene and ε gene. 2) HbF: formed by α 1 or 2 and γ chains. 3) HbA2 (the minor adult hemoglobin): formed by α 1 or 2 and δ chains. 4) HbA (normal hemoglobin): formed by β and α 1 or 2 chains. *** This picture summarizes what was mentioned above: *** It s important to note that we have pair of homologous chromosome (one from the male parent and the other from the female) and both have α or β genes. in other words on chromosome 16, we will find 2 copies of α gene one maternal and the other paternal Dr. Nayef *** The doctor didn t mention the following but the book did, so I added it since the book is required: -Globin gene families also contain globin-like genes that are not expressed (Pseudogenes) -Expression of globin genes begins in the nucleus of RBC precursors, where the DNA sequence encoding the gene is transcribed producing pre-mrna, which has 2 introns, these 2 introns are removed by splicing and the remaining 3 exons are joined in a linear manner producing mrna which is ready to be translated into hemoglobin chains. Hemoglobinopathies: Group of genetic disorders produce abnormal hemoglobin molecules due to: 3 P a g e

4 structural changes >>changes in amino acids sequence(qualitative hemoglobinopathy) HbS & HbC Rate of synthesis(quantitative hemoglobinopathy) Thalasemia HbSC(HbS+HbC) * Sickle cell anemia (HbS) * Hemoglobin C disease (HbC) * Hemoglobin SC disease (HbS + HbC = HbSC) Hemoglobin variants are mutant forms of hemoglobin, caused by variations in genetics. Some are responsible for diseases, they are considered hemoglobinopathies (ex: HbS, HbC,), other variants cause no detectable pathology, and are thus considered non-pathological variants. By studying the various hemoglobin variants, we sometimes can deduce the relationship between the physical changes and the location of the structural change. -Structural changes may lead to a change in any of the following 1. Solubility (Hb becoming less soluble. Ex: HbS, HbC). 2. Hb with higher tendency to oxidize Fe 2+ (ferrous) to Fe 3+ (ferric), leading to methemoglobinemia. 3. Unstable Hb 4. Hb with increased or decreased O₂ affinity. -Regional changes in amino acids which cause structural abnormalities and their effects. 1) Surface amino acids (altered exterior): changing the hydrophilic amino acids to hydrophobic ones so Hb becomes less soluble. 4 P a g e

5 *Nearly harmless (not associated with any symptoms) with some exceptions in which the protein polymerizes and aggregates. ex: HbS and HbC (common in Africans), HbE is rare (common in Ceylon and Malaysia), Hb Punjab (common in India and Pakistan). 2) Active site alteration: Certain amino acid substitutions in the active site allow ferrous iron to become ferric, producing methemoglobin (HbM). - The substitution of hydrophobic amino acids to hydrophilic ones attacks water and aids in the oxidation of ferrous iron to ferric iron Fe +2 >>Fe +3. -The substitution of Tyrosine for Histidine (his>>tyr) oxidizes the Fe +2 >>Fe +3 and facilitates the binding of HbM to H 2 O instead of O 2. Examples on diseases*: Proximal His substitution: HbM Iwate: 87 α his>>tyr, HbM Hide park: 92 β his>>tyr Distal His substitution: HbM Boston: 58α his>>tyr, HbM Saskatoon: 63β His>>Tyr. *Dr. said that these aren t for memorizing; you should know the main concepts of methb formation. 3) Unstable Hb: altering the tertiary structure (3D) of proteins will cause denaturation, precipitation and finally formation of Heinz bodies. Examples: a) Settlement of α helix by proline will disrupt it; proline breaks the helices by its ring. b) Substitution by large amino acids instead of small amino acids weakens the interactions within the molecule; thus cooperative binding will be affected and oxygen affinity as well. c) Charged or polar amino acids in the domains will disrupt the protein. These 2 diseases are mentioned in slides as examples of altered tertiary structure of hemoglobin: Hemoglobin Hammer-Smith: Phenylalanine is replaced by Serine>>> heme will be weakly bound to apoprotein >>> unstable hemoglobin will be formed. Hemoglobin Riverdale-Bronx: Glycine is replaced by Arginine at helical residue B6 of the β polypeptide chain>>> the interaction between B6 and E8 (8th residue in helix E) will be disrupted>>> unstable hemoglobin will be formed. 5 P a g e

6 4) Altered affinity: Oxygen affinity is affected by the quaternary structure of hemoglobin (the association of the subunits together), so any change in a point of contact between the subunits might affect the affinity (increase or decrease it). ** Any increase or decrease in oxygen affinity is considered pathological. - Substitution in the sites of allosteric effectors binding (BPG binding site, proton binding site) affects oxygen affinity. - Substitution of His146 (responsible for the Bohr Effect) to Leucine will produce more hemoglobin in the R state (increase affinity)>> (Hb cowtown). Remember, The Bohr effect reflects the fact that the deoxy form of hemoglobin has a greater affinity for protons than oxyhemoglobin (so H + will stabilize T (deoxy) state). This effect is caused by ionizable groups such as specific histidine side chains, so substitution of Leucine for Histidine will prevent H + from binding to Hb, so there is no T state, which results in more hemoglobin in the R state. *** Table 4.1 in slide 5 page 10 the details aren t required just the following: --The normal Hb is presented by this symbol α 2A β 2 A --HbS is presented by this symbol α 2 A β 2 6Glu>>Val (which indicates α chain is normal and β chain has a substitution of hydrophobic valine for the negatively charged glutamic acid at the 6th position) that means it becomes less negative and it can polymerize. --HbC (α 2 A β 2 6GLU>>LYS ): α is normal, β substitution of Lys for Glu. 6 P a g e Sickle Cell Disease (HbS) General Information: Sickle cell anemia, the most common disorder caused by Hb variant, is a genetic disorder of the blood caused by a single nucleotide substitution as mentioned above, it is an autosomal recessive disorder and it can be heterozygous (one normal and one sickle cell gene-they re carriers-) or homozygous (both are abnormal sickle cell genes). The blood cells of heterozygous patients contain both HbS and HbA so milder symptoms will be presented, they have normal life but should avoid hypoxic conditions like high altitudes, anesthesia, strenuous exercises. Homozygous HbS affects 1 in 500 African American infants, while hetero HbS affects 1 in 10 African Americans.

7 Detection: The disease can be detected easily by electrophoresis in an alkaline ph, HbA (contains negatively charged glutamic acid) will migrate from the cathode (negative charge) to the anode (positive charge) at a faster rate than HbS (contains hydrophobic valine instead of glutamic acid) and HbC (contains positively charged lysine instead of glutamic acid) the slowest one. Mechanism: Sickle cells can carry O 2 in the oxygenated state, the problem is when the patient becomes de oxygenated, let s see when and how that happens: - Decreased O2 tension by high altitude or flying in a non-pressurized plane, increased 2,3- BPG, increased CO2 and decreased ph, elevate the percentage of deoxy form of sickle cell hemoglobin. -The replacement of the charged glutamate with the nonpolar valine forms a protrusion on the β chain that fits into a complementary site on the β chain of another hemoglobin molecule in the cell forming a polymer. -At low oxygen tension, deoxyhemoglobin S polymerizes inside the RBC, forming a network of insoluble fibrous polymers that stiffen and distort the cell, producing rigid, misshapen RBCs. 7 P a g e

8 - Such sickled cells frequently block the flow of blood in the narrow capillaries. This interruption in the supply of oxygen leads to localized anoxia (oxygen deprivation) in the tissue, causing pain and eventually death (infarction) of the cells. [Note: The mean diameter of RBC is 7.5 μm, whereas that of the microvasculature is 3 4 μm. -Compared to normal RBC, sickled cells have a decreased ability to deform and an increased tendency to adhere to vessel walls and so have difficulty moving through small vessels, thereby causing micro vascular occlusion and pain such as headache. **HbA (normal) doesn t polymerize even in deoxy state, because the ends are not complementary to each other. -The presence of the HbF, HbA and the oxy HbS will disrupt the polymer (mild symptoms). -While the deoxy conditions enhance the aggregation and polymerization. Note that they are complementary to each other. 8 P a g e

9 *Anemia occurs due to the dehydration of RBC so it will become fragile and that will decrease its life span from 120 to 17 days. ***anemia means near the sea. Possible Selective Advantage: -The heterozygous patients (50% HbA, 50%HbS) are less susceptible to the severe malaria caused by the parasite Plasmodium falciparum. This organism spends an obligatory part of its life cycle in the RBC so due to the fact that sickle cells have a shorter life span than normal, the parasite cannot complete the intracellular stage of its development. This provide selective advantage for people in regions where malaria is endemic and causes death such as Africa. -Other abnormalities that provide protection against malaria include: G6PD deficiency, Thalassemia and PK deficiency. (They all affect RBC). -The doctor added that G6PD is higher in 3 oor than any other place in Jordan, and this helped the heterozygous G6PD people from surviving malaria when it was spread there.. Same goes to Africa, we find that the geographic distribution of HbS is similar to that of malaria. Treatment: Therapy involves adequate hydration, analgesics for severe pain, aggressive antibiotic therapy if infection is present, and transfusions in patients at high risk for fatal occlusion of blood vessels Promising treatment: Hydroxyurea; an antitumor drug, it is therapeutically useful because it increases circulating levels of HbF, which decreases RBC sickling. This leads to decreased frequency of painful crises and reduces mortality. 9 P a g e

10 Other Diseases Hemoglobin C disease (HbC): is a hemoglobin variant that has a single amino acid substitution in the sixth position of the β-globin chain, Lysine is substituted for the Glutamate (Glu>>Lys). - Patients who are homozygous for HbC generally have a relatively mild, chronic hemolytic anemia. These patients do not suffer from infarctive crises, no sickle cells and no specific therapy is required. Hemoglobin SC disease (HbSC): is another of the RBC sickling diseases (there are sickle cells here). In this disease, some β-globin chains have the sickle cell mutation, whereas other β- globin chains carry the mutation found in HbC disease. -Patients are doubly heterozygous or compound heterozygous (two abnormal genes with different mutations), they have milder anemia than HbS patients also painful crises are less frequent and less severe. Thalassemia: Normally, α and β formed in equal amounts and quantities, but in thalassemia one of the two chains isn t made enough so it causes anemia. There s 2 types: α thalassemia and β thalassemia. Refer to slides please Sorry for mistakes Good luck 10 P a g e