The gene for β globin (OMIM is present on chromosome 11 (11p15.4) along with other globin genes (ε, γ, γ and δ). This is known as the β-globin cluster . Individuals carrying identical genes on the β-globin gene cluster may not have identical DNA sequences in non-codeing regions of the DNA of the cluster. The non-coding regions include segments of DNA between genes and introns within genes. . Differences in DNA exist between individuals every 1000-2000 bases in the form of single nucleotide polymorphisms (SNPs). Single nucleotide polymorphisms are variations in a single nucleotide that occurs at a specific position in the genome. Many of these differences have no consequences on gene expression because either they do not result in change in amino acid sequence or they occur in regions of DNA that neither code for the gene nor regulate the gene. SNPs evolve by spontaneous mutations over time. The lesser the number of such differences between two individuals closer the individuals are the each other genetically (and in terms of evolution). Fewer differences in SNPs between individuals mean a more recent common ancestor.
One of the meanings of the word haplotype is a pattern of SNPs. A haplotype may be considered as a DNA “environment” in which the gene(s) occurs. This “environment” is created by the sequence of single nucleotide polymorphisms in which the gene(s) exists. As mentioned above differences in SNPs (and hence the “environment” the gene(s) exist in) evolve by spontaneous mutations over period of time. Fewer the differences between the “environments” the genes occurs in the more the likelihood that they come from related individuals.
HbS results from a single base substitution in the codon 6 of the β-globin gene. GAG becomes GTA resulting in substitution of valine for glutamate. This change results in a haemoglobin that crystallizes in hypoxic conditions resulting in a haemolytic anaemia. HbS occurs in diverse population groups including African, Mediterranean, Middle-Eastern and Indian. Is the haplotype of the HbS gene in these regions similar?
The HbS mutation occurs on five different haplotypes four African and one Arab-Indian. The mutation is the same (GAG to GTA on codon 6) but the SNPs are different. The haplotypes are
- Senegal: The Senegal HbS haplotype is found in Atlantic West Africa and Portugal
- Benin: The Benin HbS haplotype is found Central West Africa, Northern Africa and Mediterranean Europe (Greece, Sicily)
- Central African Republic or Bantu: The Central African Republic or Bantu is found in South Central and Eastern Africa
- Cameroon: The Cameroon haplotype is found in the Eton ethnic group of eastern Cameroon
- Arab-Indian: The Arab-Indian haplotype is the only non-African phenotype of HbS found in the eastern oasis of Saudi Arabia and India.
Origin of Haplotypes
There are two theories about the origin of haplotypes. The first, and the more accepted one, states that the five haplotypes arose from five independent mutations. An alternative hypothesis states that HbS mutation occurred only once and spread to other haplotypes by gene conversion.
Haplotypes and Severity of Symptoms
Symptoms of sickle cell anaemia are a consequence of crystallisation of haemoglobin under hypoxic conditions. HbF inhibits sickling. Patients with high HbF have fewer symptoms. The Arab-Indian and the Senegal haplotype are associated with higher HbF levels (17% and 12.4% respectively). In general patients carrying these haplotypes have milder symptoms than the Bantu or Benin haplotypes (Blood 2014; 123: 481)
Haplotypes and Human Migrations
Trade, conquests and human migrations (voluntary and slave trade) have disseminated the African haplotypes beyond Africa.
- The Mediterranean: Most of the Mediterranean (Greece and Scilly) has the Benin haplotype. This reflects pre-historic migrations from Central West Africa along the then fertile Sahara to North Africa. From here it spread to the Mediterranean via the interactions (Trade and wars) between the two regions. The only exception is Portugal. Portugal has the Senegal haplotype which reflects the trading contacts between Portugal and Atlantic West Africa (Angola and Mozambique).
- Americas: Neither the native americans nor the original European settlers to the Americas carried the HbS gene. HbS was imported to the Americas with the slaves from Africa. Jamaica was an important slave import hub and records for where tthe slaves arrived from are available. Jamaica has 73% Benin haplotype, 17% Bantu and 10% Senegal haplotypes. These numbers are close to the actual number of slaves who arrived in Jamaica from regions of Africa where these haplotypes are prevalent. Similarly the distribution of haplotype correspond to the origins of slaves in Baltimore and South Carolina (Mariam Bloom. Understanding Sickle Cell Disease, Page 34).
- Arab or Indian: It is not clear if the Arab-Indian haplotype originated in India or Saudi Arabia. But considering that all of tribal India has only one haplotype but the East and West Arabian Peninsula have different haplotypes it is possible that the haplotype originated in India.
- Spread to Other Parts: As opposed to the era of slave trade modern migration of people in the recent past have been voluntary. These populations have spread across the world as have those form mediterranean but to a lesser extent. These migrations have introduced the HbS gene in areas where it was not indigenous.
Hydroxyurea was synthesised in Germany in 1860 and was found inhibit granulocyte production. It was only a hundred years after this that its potential as an anticancer drug was realized.
Mechanism of Action of Hydroxyurea
Hydroxyurea enters the cell by passive diffusion. It inhibits of ribonucleotide reductase (RR). RR converts ribonucleotide diphosphates to deoxyribonucleotide diphosphates. Deoxyribonucleotide diphosphates are converted to deoxyribonucleotide triphosphates and incorporated into DNA. Depletion of deoxyribonucleotide triphosphates results in impaired DNA synthesis. RR has two subunits M-1 and M2. The M-2 subunit is the catalytic subunit and contains iron. Hydroxyurea inhibits RR by chelating iron. Hydroxyurea is an S phase specific drug. The cells exposed to hydroxyurea progress normally through the cell cycle, have a normal G1-S transition but accumulate in the S phase because of an inability to synthesise DNA. They then undergo apoptosis by p53 dependent and independent mechanisms. Hydroxyurea may be transformed to nitric oxide. Nitric oxide is also an inhibitor of RR and may be responsible for drugs ability to induce foetal haemoglobin. This is important for treatment of sickle cell anaemia. Resistance to HU develops by elevated cellular activity of RR.
Pharmacokinetics of Hydroxyurea
Oral bioavailability of hydroxyurea is 80-100%. Parenteral formulation has no advantage over oral formulation. The drug is well distributed. It enters breast milk, cerebrospinal fluid and third space collections. The ratio of plasma to CSF levels is 4-9:1 and plasma to ascites levels is 2-7.5:1. The elimination half-life is 3.5-4.5 hours. Renal elimination is the main pathway of elimination. Sixty to eighty per cent of the dose eliminated by kidney unchanged. Patients with creatinine clearance of 10-50ml/hr should receive 50% and those with creatinine clearance of less than 10ml/hr should receive 20% of the planned dose. Hydroxyurea is metabolized but the metabolic pathways are not known.
- Myeloproliferative diseases
- Chronic myeloid leukaemia
- Essential thrombocytosis
- Polycythaemia Vera
- Acute leukaemia to control counts
- Sickle cell anaemia
Myelosuppression is the dose limiting effect of hydroxyurea. The dose of hydroxyurea needs to be titrated to the leucocyte and platelet counts. The acceptable lower limits of these counts will depend on the indication but generally speaking a leukocyte count less than 2.5X109/L or a platelet count less than 100X109/L is an indication for discontinuing therapy. With the abovementioned provisions in mind the dose of hydroxyurea for different indications are as follows:
- Myeloproliferative diseases: The usual dose is 20-30mg/kg/day.
- Acute leukaemia: 50-100mg/kg per day
- Sickle cell anaemia: 15-20mg/kg/day
- HU inhibits formation of deoxynucleotides and enhanced the effect of agents damaging the DNA, as no nucleotides are available for repair. The effects of purine and pyrimidine analogues. When hydroxyurea is combined with any of these agents it should be done as a part of a protocol whose toxicity has been evaluated. This will prevent unacceptable toxicity.
- It has been shown to be synergistic with agents damaging the DNA like cisplatin, alkylating agents and topoisomerase II inhibitors.
- It has been used as a radiosensitizing agent in the treatment of head and neck and cervical cancer. It depletes the deoxynucleotide pool needed for DNA repair after radiation-induced damage.
- Enhanced anti HIV activity of azidothymidine, dideocytidine and dideoxyinosine
- Myelosuppression: The dose limiting toxicity of hydroxyurea is myelosuppression. Hydroxyurea causes rapid fall in leucocyte counts. When used in non-haematological malignancies the fall in leucocyte counts is evident by days 2-5. When used in patients with leukaemia the fall is evident faster, sometimes within a day. This property of hydroxyurea is useful in myeloid leukaemia with very high leucocyte count. Hydroxyurea is the treatment of choice for patients with chronic myeloid leukaemia presenting with very high counts. Though used in acute myeloid leukaemia with hyperleucocytosis, benefit from its use has not been proven in clinical trials.
- Gastrointestinal: Oral ulceration and gastrointestinal tract effects may be seen in some patients. They are particularly common in patients who receive chemoradiation with hydroxyurea.
- Skin: Dermatological changes may be seen with prolonged use. These include
- Skin Pigmentation and rash: Hyperpigmentation, erythema of the face and hands, diffuse maculopapular rash and dry skin. Severe reactions may resemble lichen planus.
- Nail Changes: The nails may show atrophy and formation of multiple pigmented bands.
- Leg Ulcers: Leg ulcerations may be seen in patients with prolonged therapy with hydroxyurea.
- Alopecia: Alopecia may occasionally be seen with the use of hydroxyurea
- Radiation Recall: Erythema or pigmentation of previously radiated skin may be seen in some patients.
- Mutagenicity and Teratogenicity: Hydroxyurea is a proven teratogen and contraindicated in women are pregnant or are planning a pregnancy. Women in the reproductive age group must be advised about contraception. The carcinogenic potential of hydroxyurea is uncertain. In view of the mechanism of action it is prudent not to use hydroxyurea for non-malignant disease.
Sickle Cells 40X
Sickle Cells – 100X
Sickle Cells – 100X
The three photomicrographs above show sickle cells from a patients with sickle cell anemia. Sickle cell anaemia occurs because an A→T substitution in codon 6 of the β globin chain of haemoglobin. The single nucleotide polymorphism results in valine substituting for glutamic acid resulting in the formation of haemoglobin S (HbS). HbS crystallizes in hypoxic conditions resulting in sickling of erythrocytes.
Sickle Haemoglobin and Variants
Substitution of the amino acid glutamic acid by valine at position 6 of the beta globin gene (HBB glu6val) results in a mutant haemoglobin that polymerizes at low oxygen pressure. This mutation results in sickle shaped cells under hypoxic conditions. The haemoglobin gets its name, sickle haemoglobin, from the phenomena. Sickling is responsible for symptoms of sickle cell anaemia. Shown above are sickle cells from the smear of a patient with sickle cell anaemia.
HbS is an autosomal co-dominant trait. Homozygous individuals suffer from sickle cell anaemia (SS). The clinical profile of compound heterozygous depends on the non-HbS allele.
- HbA: HbA does not participate in sickling. Patients with AS have about 35% HbS and 65% HbA there is little sickliness and symptoms. These patients said to have sickle cell trait. A patient with as with an apparent AS pattern can have see sickling (see below).
- β-Thalassaemia: The severity of interaction between HbS and β-thalassaemia depends on the severity of the co-thalassaemia. β0-thalassaemia is identical to SS and β+-thalassaemia is a milder disease with severity depending on the degree of impairment of the β-chain synthesis.
- Other Sickling haemoglobinopathies: Co-inheritnce of Hb-O Arab and Hb D Punjab produces a sickle cell anaemia like disease. Co-inheritance of Hb E produces a sickling disease milder than SS.
- Haemoglobinopathies than mimic AS on electrophoresis but produce sickling: There are two categories in this class. First, is a disease that resulting from co-inheritance of an abnormal haemoglobin, Hb Quebec Chori, migrates like HbA but promotes sickling. Patients compound heterozygous for Hb S and and Hb Quebec Chori show and electrophoretic pattern of AS but show sickling. Second are variants of HbS where a second mutation has resulted in an increased tendency to precipitate making a individual heterozygous for these variants symptomatic. These include Hb South End, Hb Jamican Plain Hb S Antilles and Hb S-Oman. The first three have mutations reducing the affinity of the haemoglobin increasing polymerization and sickling.
Fluids flow on application of pressure. The flow may be laminar flow that is orderly in parallel layers or turbulent flow that is chaotic. During laminar flow the layer closest to the wall is the slowest and the layer farthest, fastest. Viscosity, the internal friction between these layers, is a measure of thickness of a fluid. The higher the viscosity, thicker the fluid. Depending on whether the viscosity of fluids changes with flow rate or not fluids may be Newtonian of non-Newtonian. The viscosity of Newtonian fluids like water, honey and oil does not change with flow rates. The viscosity of blood, a non-Newtonian fluid, Blood viscosity increases with falling shear rates. The increase is dramatic at low shear rates. Blood viscosity depends on plasma viscosity and the type and number if blood cells.
Determinants of plasma viscosity
Plasma viscosity varies with the concentration of its constituents. Fibrous proteins like fibrinogen contribute more to plasma viscosity than globular proteins like albumin. Acute phase reactants increase plasma viscosity. Of the plasma constituents immunoglobulins and cholesterol are clinically relevant. Clinically significant increases in viscosity are most common in patients with increased immunoglobulin, both monoclonal and polyclonal. The commonest cause of hyperviscosity syndrome is increased IgM in patients with of Waldenström’s macroglobulinaemia. Patients with IgG3 and IgA multiple myeloma, cryoglobulinemia, both monoclonal and polyclonal and patients with polyclonal gammopathies may have hyperviscosity. Very high cholesterol levels in patients with primary biliary cirrhosis have also been associated with hyperviscosity. Plasma viscosity decreases with temperature.
Effect of Number and Type of Cells on Viscosity
Haematocrit and cell deformity affect blood viscosity. Blood viscosity increases with haematocrit in an exponential manner. There is a pronounced increase in viscosity at haematocrits more than 55%.
Blood cells disrupt flow lines of plasma and increase viscosity. Erythrocytes are the most numerous and under physiological conditions the flow properties of blood depend on the properties of plasma and erythrocytes. The normal erythrocyte is a biconcave disk about 7.8 µm in diameter (figure 1). At low flow rates erythrocytes aggregate in the form of stacks known as rouleaux. These large aggregates cause a sharp increase in viscosity. With increasing flow rates the shear stress on the erythrocyte rouleaux increases causing the erythrocytes to disaggregate. Disaggregation reduces viscosity. Any reduction in viscosity after complete disruption of rouleaux depends on the capacity of the erythrocyte to change so that resistance offered to flow decreases. The erythrocyte may take a bullet, parachute or a slipper form (figure 1). The deformability needed for shape change depends on the amount of surplus membrane, the properties of membrane and the viscous properties of the erythrocyte cytoplasm. RBC deformability is decreased in patients with hereditary spherocytosis because of decreased amount of membrane and in sickle cell anaemia because altered viscosity of haemoglobin and membrane damage. Malaria is characterized by deceased deformability and increased adhesiveness. Increased viscosity is responsible clinical manifestations of sickle cell anaemia and malaria. Intracellular crystallization of haemoglobin causes increased viscosity in haemoglobin C disease.
Figure 1. Deformability and aggregation of erythrocytes is responsible for changes in viscosity of blood as the flow rate increases. The normally biconcave erythrocyte aggregate into rouleaux at low flow rates. As the shear stress increases because of increased flow, the rouleaux disaggregate. Further increase in viscosity results in change in shape of the erythrocyte from biconcave to bullet shaped, parachute shapes and slipper shaped forms. These shapes offer less resistance to floe than the biconcave forms. Erythrocytes have about 40% surplus membrane. This surplus is important for shape change. Erythrocyes with viscous cytoplasm (HbS and HbC) resist change in shape increasing viscosity of blood in these diseases.
Leukocytes are larger than erythrocytes. As opposed to an erythrocyte volume of 80-90 (femtoliter) fL, the volume of a leukemic lymphocyte is 190-250 fL, lymphoblast is 250-350 fL and myeloblast is 350-450 fL. Viscosity depends on haematocrit. The contribution of leucocytes to normal haematocrit is small (~1.2%). Under physiological conditions haematocrit is practically equal to erythrocrit. Leucocytes are larger and less deformable because of the presence of a rigid nucleus. For a similar increase in count the leukocrit rises more than erythrocrit. Acute leukaemia is characterized by progressively increasing anaemia as the leukocyte counts increase. As the increased leukocrit is more than offset by anaemia in almost all patients with acute leukaemia, hyperviscosity is rare in acute leukaemias. There is an inverse relationship between leukocrit and erythrocrit in leukaemias for leukocrit values less than 15% for chronic leukaemias. The lymphocytes of chronic lymphocytic leukaemia are small and counts needed for a pathological increase haematocrit are rarely reached. Anaemia in patients of chronic myeloid leukaemia is less severe than acute leukaemia. Myeloid cells are larger than lymphoid cells. This makes patients with CML at the greatest risk for hyperviscosity. Anaemia is leukaemia protects from hyperviscosity. This must be borne in mind before initiating red cell transfusions in leukaemia patients.
- Blood rheology and hemodynamics. Baskurt OK, Meiselman HJ. Semin Thromb Hemost. 2003 Oct;29(5):435-50.
- Oguz K. Baskurt. Max R. Hardeman. Michael W. Rampling and Herbert J. Meiselman. Handbook of Hemorheology and Hemodynamics. 2007. IOS press. ISBN 978-1-58603-771-0 [Preview at Google Books
It is difficult to imagine that a molecule as essential to life as haemoglobin (hb) would need a detoxification mechanism. Anyone who has treated a patient with severe intravascular haemolysis knows the havoc cell-free haemoglobin can cause. Haemoglobin is small enough to be filtered by the glomerulus and causes renal failure due to pigment nephropathy. Haemoglobin depletes nitric oxide resulting in vasculopathy. Mechanisms to detoxify cell-free haemoglobin counter the oxidative and pro-inflammatory effects of haemoglobin.
Haemolysis releases cell-free haemoglobin. Haptoglobin (Hp), alpha-2 globulin, secreted by the liver is a haemoglobin scavenger. It rapidly binds cell-free haemoglobin in the plasma protecting the vessels and the kidneys from it’s deleterious effects. When the scavenging capacity of haptoglobin is overwhelmed cell-free haemoglobin appears in the plasma. It converts nitric oxide to biologically inactive nitrate and is itself converted to methemoglobin in the process. Degradation of cell-free haemoglobin results in the formation of heme and free iron which deplete nitric oxide by their oxidizing action. Hemopexin scavenges free heme. Free iron is taken up and transported by transferrin.
The haemoglobin-haptoglobin complex is taken by the CD163 receptor on the reticuloendothelial macrophage and the heme-hemopexin complex is taken up by the CD91 (low density lipoprotein-1, LRP1) receptor. The interactions of haemoglobin-haptoglobin by CD163 and heme-hemopexin by CD91 have an anti-oxidant and anti-inflammatory action by activation of heme oxygenase-1 and IL10. Haptoglobin and hemopexin are acute phase reactants. The body’s capacity the counter the effects of cell-free haemoglobin increases during acute inflammation. The expression of CD163 and CD91 is increased by corticosteroids which are also secreted as a part of response to acute inflammation.
Pigment nephropathy from precipitation of haemoglobin in renal tubules has long been recognized as a serious complication of massive intravascular haemolysis. The vascular effects of cell free-haemoglobin are evident at lesser haemolysis. There is evidence in rodent malaria model that heme, a degradation product of cell-free haemoglobin released during intravascular haemolysis, is involved in the pathogenesis of cerebral and non-cerebral malaria (Proc Natl Acad Sci U S A. 2009 September 15; 106(37): 15837–15842, Nat Med. 2007 Jun;13(6):703-10. Epub 2007 May 13). Heme has been implicated in the pathogenesis of severe sepsis in animal models (Sci Transl Med. 2010 Sep 29;2(51):51ra71). Cell-free haemoglobin has been implicated in the pathogenesis of pulmonary hypertension, leg ulceration, priapism, and cerebrovascular disease related to sickle cell anaemia (Blood Rev. 2007 January; 21(1): 37–47). The list of haemolytic anaemias where cell-free haemoglobin has a pathogenic role is increasing and now includes thalassemia, autoimmune haemolytic anaemia, paroxysmal nocturnal hemoglobinuria, unstable hemoglobinopathy, and hereditary membranopathies. Corticosteroids can increase the clearance of cell-free haemoglobin and its degradation products. They have been shown to benefit patients of sickle cell disease with acute chest syndrome and vaso-occlusive crisis. Thrombotic thrombocytopenic purpura, a disease characterized by intravascular haemolysis, is also treated with corticosteroids in addition to plasmapheresis. Plasmapheresis removes cell-free haemoglobin and corticosteroids enhance its clearance by macrophages.
Too much of a good thing is bad. Haemoglobin is safe when in erythrocytes. Outside erythrocytes it needs detoxification.