Sickle cell anaemia and β-thalassaemia are two common haemoglobinopathies. Co-inheritance of the two is called sickle β-thalassaemia. Sickle β-thalassaemia seen in Africa, throughout the Mediterranean, Arabian Peninsula and sporadically in india. It has heterogeneous clinical presentation. The severity depends on the severity of the thalassaemia allele and the extent to which the impaired haemoglobin synthesis is compensated by foetal haemoglobin synthesis.
With a very few exceptions (Blood 1989; 74: 1817-22) the sickle cell and the thalassaemia gene are arranged in trans i.e on different chromosomes (βs/βthal). One allele is inherited from the mother and one from the father. One parent carries the a β-thalassaemia trait the other parent has a sickle cell disease that may be sickle cell anaemia, sickle β-thalassaemia or a trait. Sickle β-thalassaemia in Africa and India/Arabia is mild whereas the patients from the Mediterranean region have severe disease. As mentioned above the differences in severity have to do with severity of the β-thalassaemia and the degree to which the impaired haemoglobin A synthesis is compensated by HbF. Weatherall suggested that patients with HbA <15% follow a course similar to severe HbA and those with HbA 20-30% follow a mild course.
- African sickle β-thalassaemia: African patients have a mild β-thalassaemia resulting in a relatively higher HbA level and a lower risk of sickling. These patients run a mild clinical course.
- Arab/Indian sickle β-thalassaemia: Patients from India and the Arabian peninsula have a sickle cell haplotype that is associated with a high HbF production. The HbF retards sickling. High levels of HbF attenuate symptoms. Patients carrying this haplotype have mild symptoms even when the inherit a severe β- chain defect. Another reason of a mild phenotype in India is the interaction with α thalassaemia.
- Mediterranean sickle β-thalassaemia: Mediterranean patients usually inherit a severe form of β-thalassaemia. These patients have severe sickling because there is very little HbA or HbF to offset inhibit the crystallisation of HbS. Despite only one chromosome carrying HbS the phenotype of these patients resembles sickle cell anaemia.
Clinical Picture of Sickle-β Thalassaemia
The features of sickle-β thalassaemia resemble those of other sickling disease. It is a chronic haemolytic anaemia the course of which is interrupted by acute exacerbations known as crisis. The manifestations include haemolytic anaemia, painful and other crisis, leg ulcers, priapism and complications of pregnancy. The severity of symptoms is variable. One end of the spectrum are patients, usually of origin Mediterranean descent, whose presentation is indistinguishable from sickle cell anaemia. These patients have inherit severe forms of β (β0) chain defects. Those with sickle cell-β+ thalassaemia have milder symptoms. These patients are typically of African ancestory. Unlike patients with sickle cell anaemia patients with sickle-β thalassaemia may have splenomegaly that is more prominent patients with sickle cell-β+ thalassaemia. The spleen is usually moderately enlarged but massive splenomegaly that may be associated hypersplenism neccesisating splenectomy has been reported. The effect of co-inheritance of α-thalassaemia is small. A decrease in the frequency of acute chest syndrome and leg ulcers and a higher persistence of splenomegaly is seen. Co-inheritance of α thalassemia is one of the reasons that sickle-β thalassaemia runs a milder course in India (the other being the high HbF due to the Arab-Indian haplotype of HbS).
The haematological findings vary with severity. More severe phenotypes shows greater anaemia, lower MCHC, higher reticulocytes, HbF and HbA2. A variable number of sickle cells may be found. Unlike sickle cell anaemia both forms of sickle cell-β thalassaemia have an elevated HbA2. The distribution of HbA2 is very similar to heterozygous β thalassaemia. The levels of HbF are variable. High levels are found in patients with the Arab-Indian and Senegal haplotype of HbS.
Sickle cell-β0 thalassaemia needs to be differentiated from sickle cell anaemia. The presentation of both may be identical. However an offspring of a sickle cell-β0 thalassaemia patients and a carrier of β-thalassamia trait has a 25% risk of suffering from β-thalassaemia major. The offspring of a patients with sickle cell anaemia and a carrier of β thalassaemia trait does not carry the risk of β thalassaemia major. Though sickle cell-β0 thalassaemia is characterised by an elevated HbA2 and splenomegaly this can not be relied upon to differentiate between the two conditions. Family and DNA studies are needed. If the studies show one parent to be heterozygous for HbS and the other a carrier of β thalassaemia trait no further studies are needed. If any of the parent has a phenotype of sickle cell anaemia DNA studies may be the only way to make the diagnosis.
Sickle Cell β thalassaemia in cis
Almost all patients with sickle-β thalassaemia have the disorder in trans i.e. the one β globin gene is thalassaemic and the other has a the sickle mutation. Patients with HbS and thalassaemia gene in cis have been described. These patients have a mild hemolysis, HbA2 levels were 6%–7%, HbF approximately 3% and HbS of 10%–11%.
The symptoms of sickle-β thalassaemia are due to sickling need to be treated accordingly.
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.
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
The old/middle english term faggot means a bundle of sticks bound together as fuel (faggot is derived the latin term fascis which is also the root for the term fasiculus). Patinets with acute promyelocytic leukemia have cells with bundles of auer rods. These are known as faggot cells and are virtually diagnostic of acute promyelocytic leukemia. faggot cells can be seen in more mature cells following treatment of acute promyelocytic leukemia with ATRA. The have rarely been reported in patients with other from of acute myeloid leukemia.
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.
Described by Pappenheimer in 1945, the Pappenheimer bodies are basophilic erythrocytic inclusions that are usually located at the periphery of the cell. They contain iron and stain with Prussian blue. They also stain with Romanowsky stains because of co-precipitation of ribosomes. Pappenheimer bodies seen following splenectomy in patients without haematological disease are composed of ferritin. Whereas Pappenheimer bodies seen in pathological conditions like sideroblastic anaemia are also composed of iron laden mitochondria and phagosomes.
Pappenheimer bodies are seen in sideroblastic anaemia and haemolytic anaemia. The spleen clears Pappenheimer bodies. Splenectomy is associated with an increase in Pappenheimer bodies. The increase is more pronounced in patients with haemolytic anaemia and sideroblastic anaemia. Cells containing Pappenheimer bodies can be confused with late reticulocytes. Prussian blue stain, which is not taken up by reticulocytes, is helpful in differentiating the two. Pappenheimer bodies can also cause a false elevation of platelet counts when performed with electronic counters.
There is an intererting article about the history of Pappenheimer bodies published in The American Journal of Haematology (Am. J. Hematol 75:249;2004)
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.