Thalassaemia Inheritance

Heterozygous β-Thalassaemia 


β-Thalassaemia is an inherited disease characterised by an imbalance between production of α and β globin chains of haemoglobin resulting from impaired production of β chains. The genes responsible for β-thalassaemia carry mutations in areas coding for the β globin gene or regions regulating the expression of this gene. Patients who are homozygous of compound heterozygous for the gene are symptomatic. They manifest as thalassaemia major. Thalassaemia major is a fatal illness where patients suffer the consequences of anaemia, bone marrow hyperplasia and iron overload. Iron overload that results from increased iron absorption and repeated transfusion is the cause of death. The treatment consists of lifelong transfusion with iron chelation or in those who have a matched donor, allogeneic bone marrow transplantation.

Thalassaemia Inheritance

The risk of inheritance of β-thalassaemia in offsprings when both parents are heterozygous is shown on the left. There is a 25% risk of thalassaemia major, 50% risk of heterozygous β-thalassaemia and 25% of the offsprings will be normal. If one of the parents does not carry the thalassaemia gene there is a 50% risk of the offspring carrying heterozygous β-thalassaemia and 50% of the offsprings will be normal.

As opposed to homologous or compound heterozygous β-thalassaemia, heterozygous the β-thalassaemia is asymptomatic. The condition is also known as β-thalassaemia minor (see classification of β-thalassaemia). The terminology reflecting the asymptomatic nature of the disease. Though β-thalassaemia is an asymptomatic disease the diagnosis has clinical implications. These include:

  1. Risk of β-thalassaemia in children: β-Thalassaemia major is inherited in an autosomal recessive manner. If both the parents are heterozygous for β-thalassaemia there is a 25% risk of the child suffering from thalassaemia major (see figure above, left). The most effective way to prevent β-thalassaemia major is to ensure that at least one parents does not carry the β-thalassaemia gene (see figure above, right). Diagnosis of an index case of heterozygous β-thalassaemia should initiate a search for all individuals carrying the β-thalassaemia gene in the family. Patients with heterozygous β-thalassaemia should be discouraged from choosing another heterozygous β-thalassaemia as a life partner. Those who make this choice despite counselling or those who already married should be explained the importance of prenatal diagnosis of β-thalassaemia major on conception and encouraged to undergo the same.
  2. Prevention of unnecessary iron therapy: Iron deficiency anaemia, like thalassaemia, is microcytic and hypochromic. Iron therapy alleviates the anaemia of thalassaemia only if iron deficiency co-exists. Iron therapy is associated with gastrointestinal adverse effects. Some patients with heterozygous β-thalassaemia have increased iron absorption and there have been reports of iron overload in β-thalassaemia trait (Br J Haematol). Diagnosis of heterozygous β-thalassaemia spares the patient unnecessary and sometimes dangerous iron therapy.

Pathophysiology of Heterozygous β-Thalassaemia

Heterozygous β-thalassaemia minor is characterised by an imbalance between the α and β globin chains because of decreased production of β-chains. The clinical manifestations of thalassaemia depend on the degree on imbalance between α chains and non-α (β+γ) chains. Thalassaemia minor, the phenotype of heterozygous β-thalassaemia results when the ratio of α to non-α chains is 2:1 (Cold Spring Harb Perspect Med 2012;2:a011726).

Clinical Features

Patients of heterozygous thalassaemia are asymptomatic. The clinical presentations is that of thalassaemia minor. Diagnosis is usually made incidentally when

  1. A haemogram is performed for another reason or
  2. Screening is performed following detection of a β-thalassaemia patient in the family
  3. Evaluation of anaemia of pregnancy

Though traditionally heterozygous β-thalassaemia are considered to be asymptomatic recent studies have found these patients to have symptoms of mild anaemia. Heterozygous β-thalassaemia may become symptomatic

  1. In pregnancy:The third trimester of pregnancy sees a plasma volume expansion accompanied by an increased production of red cells. In normal women the volume expansion is more than the increase in the number of red cells. Women become anaemic in the third trimester as a result of this discrepancy. Patients with β-thalassaemia trait show a plasma volume expansion but are not able to increase the number of red cells like normal women do. As a consequence women with heterozygous β-thalassaemia become more anaemic than normal women. This anaemia is usually mild and haemoglobin values lower than 8-9g/dL should prompt a search for another cause of anaemia. Iron deficiency anaemia is the commonest anaemia in pregnancy and it mimics thalassaemia. Serum iron and iron binding capacity may not be reliable in pregnancy and a serum ferritin must be performed for diagnosing iron deficiency.
  2. In case of autosomal dominant β-thalassaemia: Some forms of deletion β-thalassaemia result in the formation of an unstable β chain that forms inclusions. These inclusions cause ineffective erythropoietin and a thalassaemia like syndrome. Such patients are said to have a dominant β-thalassaemia and have the clinical picture of thalassaemia intermedia even when heterozygous.
  3. If the co-inherit an overdose of α thalassaemia genes: Manifestations of β-thalassaemia depend on the ratio of α to non-α chains. Thalassaemia minor results when the ration is 1.5-2.5:1 and intermedia when the ratio is about 4:1. Thalassaemia major is seen with higher rations. Some patients have three or even four α globin genes (ααα or αααα). These patients produce more α globin chains. Increase in α chains can push up the ratio of α to non-α chains and result in manifestations of thalassaemia intermedia in heterozygous β-thalassaemia. Similarly co-inheritance of α and β thalassaemias can attenuate the manifestations of thalassaemia.

Laboratory Features

  1. Haemogram: Heterozygous β-thalassaemia is characterised by anaemia, low MCH and low MCV. The MCHC is usually normal. The erythrocytes count is high and there may be a slight increase in the reticulocyte count. The peripheral smear shows microcytosis, hypochromia, poikilocytosis, basophilic strippling and target cells. Co-inheritance of α-thalassaemia attenuates the findings. The red cell indices are normal at birth. Changes associated with heterozygous β-thalassaemia become apparent by 3 months. By 6 months thalassaemic changes are firmly established.
  2. Haemoglobin A2: Haemoglobin A2 (HbA2) is in the range of 3.5-7%. Iron deficiency causes a disproportionate fall in HbA2 in patients with heterozygous β-thalassaemia but does not push the HbA2 levels in the normal range. Heterozygous β-thalassaemia with normal HbA2 is discussed below.
  3. Bone Marrow: The bone marrow shows erythroid hyperplasia with pyknotic normoblasts dominating. There is ineffective erythropoiesis mainly due to destruction of haemoglobinized precursors. Studies have shown approximately 25% decrease in efficiency of erythropoiesis.
  4. Iron Metabolism: Rate of iron absorption is slightly increased. Some cases of iron overload have been reported. Iron deficiency may co-exist the heterozygous β-thalassaemia particularly in pregnancy. Serum ferritin estimations should be performed to diagnose iron deficiency.
  5. Osmotic Fragility: Osmotic fragility is increased particularly after 24 hours of sterile incubation of erhthrocytes. It has been suggested that this be used as a screening test for heterozygous β-thalassaemia but has not gained widespread acceptability.
  6. Globin Chain Synthesis: Heterozygous β-thalassaemia is associated with a α:β ratio of 1.5-2.5:1.

Genotype Phenotype Co-relations

There is a continuous spectrum of changes with mild alleles having less pronounced effect on haematological parameters. Severe alleles have higher HbA2 values. Mild thalassaemia with high HbA2 suggest a promoter mutation.

Interaction between Heterozygous β-thalassaemia and other Haemoglobinopathies

Heterozygous β-thalassaemia is a common disorder and a chance associations may be seen with other haemoglobinopathies or inherited disorders of erythrocytes. Fortunately no deleterious association has been found with most disorders these include glucose-6-phosphate dehydrogenase deficiency, hereditary spherocytosis  and pyruvate kinase deficiency.

α-Thalassaemia

α-Thalasaemia tends to reduce the α:β globin ratio. The amount of free α globin chain reduces attenuating the manifestations of heterozygous β-thalassaemia.

Sickle Cell Disease

β-Thalassaemia and sickle-cell diseases are common genetic diseases. Co-inheritance of the two is found in Africa, Mediterranean and sporadically through India. The symptoms depend on the relative amounts of HbS and HbA. HbA polymerises less than HbS. High levels of HbA reduce symptoms of sickling.  HbF is excluded from and protects against sickling. The clinical manifestations of patients co-inhereting sickle-cell and β-thalassaemia depend on the type of thalassaemia allele inherited and the HbF levels.

  1. Sickle β-thalassaemia with β0 or severe β+ alleles: Mediterranean forms of β-thalassaemia trait are either β0 severe β+. Patients from this region have severe sickling symptoms and HbA levels <15%.
  2. Sickle β-thalassaemia with mild β+ alleles: African patients of sickle β-thalassaemia inherit mild β+ alleles.  These patients have haemoglobin levels in the range of 20-30%  and mild symptoms . Many do not have symptoms. Diagnosis in some may be made incidentally.
  3. Sickle β-thalassaemia with high HbF: Patients from Indian and Saudi Arabia have mild symptoms despite inheriting severe β alleles because of high levels of HbF.

Treatment of Heterozygous β-Thalassaemia

Heterozygous β-thalassaemia does not need any treatment. A family screening should be carried out to detect other members carrying the thalassaemic β globin gene. Iron therapy should not be administered to patients empirically. Some patient have an increase iron absorption and iron overload has been reported. Iron studies should guide iron therapy. Anaemia can worsen during pregnancy. Folate and iron supplementation may be needed.

 

 

Why Blood Loss From Sites other than Gastrointestinal Tract Rarely Causes Iron Deficiency?


A 55 year old man presented with breathlessness on climbing stairs. He saw his family physician who found the patient to be pale. The patient was advised a complete haemogram. He was found to be anaemic and was asked to see a haematologist.

The haemogram showed an haemoglobin of 3.1 g/dL with an MCV of 63fl. The WBC count was 5600 with 65% neutrophils, 30% lymphocyte, 3% monocytes and 2% eosinophils. The platelet count was 475 X 1009/L. The reticulocyte count was 1%.

The serum iron was 15μg/dL and the total iron binding capacity 450μg/dL and a transferrin saturation of 3.3%. The serum ferritin was 8ng/ml. A diagnosis of iron deficiency anaemia was made he was initiated on oral iron which he responded to. 

Iron deficiency is common in 

  1. Growing children because of dietary deficiency
  2. Women in the reproductive age group because of menstrual blood losses and iron depletion because of foetal transfer of iron during pregnancy.

When iron deficiency occurs in a well nourished man or a well nourished post-menopausal women it is invariably due to a gstrointestinal blood loss. Why is gastrointestinal blood loss different from other forms of blood loss?

Bleeding is an alarming symptom. It is rare for a person to ignore bleeding. Bleeding from the respiratory system, urinary system and skin is apparent and alarming. Such bleeding prompts the patient to promptly seek medical attention. Gastrointestinal bleeding may be of three types. The patient may pass fresh blood, the patient may have malaena or the patient may have occult bleeding. Passage of fresh blood with stools is a symptom of a lower gastrointestinal pathology. Such patients seek attention early and usually do not become anaemia. Haemorrhoids is an exception not because the patient does not realise that there is bleeding but because the patient may ignore the bleeding because he/she attributed it to a known cause. Some patients may not realise the significance of malaena and may present only when anaemic. A patient may loose upto  30ml of blood without a change in the consistency or colour of stools. Such patients present with iron deficiency anaemia. As the anaemia has a gradual onset the it may become severe and yet not cause symptoms. 

The diagnosis of anaemia is complete only if the type of anaemia and the cause of anaemia are determined. A rule of thumb is that unexplained iron deficiency anaemia in a man or a postmenopausal woman should be considered to be due to a occult gastrointestinal blood loss unless proven otherwise. The importance of this practice can not be overemphasised. A colon cancer develops in an adenoma. Completing the evaluation of an iron deficency anaemia provides an opportunity to diagnose a colorectal cancer either in the premalignment stage or when the disease has a limited extent. Not perusing the diagnostic evaluation of an iron deficiency anaemia to completion may close the window of early diagnosis of gastrointestinal cancer.

Classification of β-Thalassaemia


β-Thalassaemia is a term applied to describe heterozygous group of diseases that are characterised by a decrease in the production of β globin  chain. Over 200 mutations in the β-globin gene and promoter regions that cause β-thalassaemia have been recognised. Thalassaemic alleles that produce no β-chain are designated β0 and those producing some β chain are designated as β+.  Before the genetic basis of thalassaemia was understood the disease was classified according to the clinical presentation and natural history of the disease. The genetic defects need to be determined for prenatal diagnosis but the clinical patterns remains relevant for clinical management of β-thalassaemia.

Based of the severity of disease three patterns of disease have been identified, thalassaemia major, thalassaemia minor and thalassaemias intermedia

  1. Thalassaemia Major: Thalasaemia major is a transfusion dependent anaemia that usually appears early in life, often in the first year. Anaemia is associated with splenomegaly, skeletal deformities and growth retardation. Iron overload develops by the end of second decade unless chelation is used. Unless treated with blood transfusion and chelation or allogeneic stem cell transplant, it is a fatal illness. There is a severe impairment of β-chain synthesis. Genetically these patients may be β0β0, β0β+ or β+β+.
  2. Thalassaemia Minor: patients with thalassaemia minor are asymptomatic. They are diagnosed when a complete haemogram is performed as a part of antenatal care or as a pert of investigations of another illness. Genetically they me be β0β or β+β.
  3. Thalassaemia Intermedia: Thalassaemia intermedia has a clinical presentation between that of thalassaemia major and thalassaemia minor. It is a very heterogeneous condition. The patient is not transfusion dependent but anaemic with a low and stable haemoglobin. Transfusion may be needed during periods of stress like infection and pregnancy. Advancing age is also associated with transfusion requirement. This may in part be due to hyperplenism associated with splenomegaly. The genetics of thalassaemia intermedia are complex. It may result from a mild β chain defect or because of interaction of β chain defects with other defects of haemoglobin synthesis

Intravenous Iron


Iron deficiency, the commonest cause of anaemia, is treated by iron supplementation. Oral iron, introduced by the French physician Pierre Blaud in the 19th century is the mainstay of therapy of iron deficiency. Oral iron is inexpensive and safe but is poorly absorbed and causes abdominal adverse effects in 35-59% of the patients. Injectable iron preparations were initially developed for the treatment of iron deficiency in patients intolerant to iron. Their use became more prevalent with it became clear that iron deficiency was a common cause of failure of erythropoiesis stimulating agent therapy and that oral iron was insufficient for this indication.

Is it possible to inject an iron salt, say ferric chloride, for iron deficiency, like it is to inject calcium chloride for hypocalcaemia? Iron, unlike calcium or potassium,  produces free radicals (oxyradicals) that can damage macromolecules and result in cellular injury. Ferric hydroxide,  the first injectable iron preparation used, caused an immediate release of iron in circulation resulting in severe reactions.  When an iron oxide, hydroxide or an iron salt is used, only a small dose can be safely administered. One estimate calculated the maximum iron permissible as 8mg/day (this is equivalent of the unbound iron binding capacity of transferrin). It would take about 6 months to bring up haemoglobin at this dose.

About  25mg of iron is delivered each day to the erythroid precursors for haemoglobin synthesis. Iron is a poorly absorbed micronutrient and stores are needed to  provide for sudden increase in demand as may be seen in patients with acute blood loss. Given the propensity of iron to cause free radical induced cellular injury, transport and storage systems capable of protecting the body from iron have evolved. Iron is transported bound to transferrin and stored as ferritin and haemosidin. In both the situations iron is bound to apoproteins (apotrasferrin and apoferritin) and this binding does not allow free radical generation.

Erythrocyte destruction takes place in the macrophages and macrophages have systems for the handling, storage and recycling of iron. Injectable iron preparations have a carbohydrate shell that prevents exposure of the plasma to free iron.  Injectable iron preparations are taken up by macrophages of the reticuloendothelial system by endocytosis. The iron is released and enters the macrophages iron pool. This iron has a fate similar to iron reaching the macrophage from other sources viz. intestinal absorption and erythrocyte destruction. Binding iron to carbohydrate shell has made it possible to administer as high as dose as 1000mg over as short a time as 15 minutes. This is a 125 fold increase over the amount of iron that can be administered without the carbohydrate shell.

Injectable iron preparations share a common structure. They have a core of iron oxide/hydroxide that is associated with a carbohydrate shell. The carbohydrate shell  keeps free iron from entering the plasma and in a way performs the same function as transferrin. The antigenicity of the shell and the strength of binding between the iron core and the carbohydrate shell determines the side effects and the maximum dose per injection.

Preparations of Injectable Iron

The preperations of injectable iron include

  1. Preparations with a strong association between iron core and the carbohydrate shell
    1. High-Molecular weight iron dextran
    2. Low -molecular weight iron dextran
    3. Ferric carboxymaltose
    4. Ferumoxytol
  2. Perperations with a weak association between iron core and carbohydrate shell
    1. Iron Sucrose
  3. Preperations that have a labile low molecular weight components
    1. Ferric Gluconate
    2. Iron-Sorbitol-Citric Acid Complex, Dextrin-Stabilized
Table 1 – parentral iron preparations
Drug Shell MW T1/2 Labile
Iron
Maximum
Dose and administration
HMW ID High moleculer weight dextran 265 60 1-2% Should be administered 1 hr after an intravenous test dose (0.5ml over at least 5 minutes) Maximum dose 20mg/kg. Administered as intravenous bolus 100mg/day slowly. A total dose infusion may be given over a prolonged period.
LMW ID  Low molecular weight dextan  165  20  1-2% Should be administered 1 hr after an intravenous test dose (0.5ml over at least 30 seconds). It may be administered as daily iv bolus of 100mg over 2 mins or a a total dose infusion over 3-4 hours may be administered 20mg/kg
Iron Sucrose  Sucrose 30-60  6  4-5% up to 300mg in a single dose, higher doses have been administered over as a prolonged infusion. May be administered undiluted as an iv bolus 200mg over at least 10 mins or as an infusion in 0.9% saline over 15-60 minutes
Ferric Gluconate  Gluconate  289-440  1  5-6% 125mg, iv bolus at 12.5mg/min or as a 1 hr infusion in 0.9% saline
Ferric Carboxy-maltose Carboxy-maltose  150  16  1-2% The maximum dose is 20mg/kg (max 1000mg). Doses of 200-500mg should be administered at 100mg/min, doses between 500mg and 1000mg should be administered over 15 minutes. It may be administered as an iv infusion diluted in 0.9% saline
Ferric Isomaltoside Isomaltoside 150  20  <1% May be administered as an intravenous bolus dose of 500mg un to three times a week at the rate of 50mg/min or 20mg/kg (maximum 1000mg) in 0.9% saline over 1 hour
Ferumoxytol Carboxy-methyl Dextran 750 15 <1% 510mg as a iv bolus at the rate of 30mg/sec. A second dose may be administered after 3-8 days

The parental iron preparations differ in the carbohydrate that surrounds the iron core. The therapeutic implications of these differences are:

  1. Antigenicity: Dextran is antigenic. Patinets may have performed antibodies to dextran or may develop antibodies during therapy. All iron preparations  containing dextran can give rise to anaphylaxis. The risk is greatest with high molecular weight iron dextran. Anaphylaxis may also be seen with low molecular weight iron dextran though the risk is lower and has also been reported with ferumoxytol that contains carboxymethyl dextran.
  2. Maximum dose per injection: All intravenous iron preparations tend to release labile iron. There is an inverse relationship between the amount of labile iron released and the strength of association between the iron core and carbohydrate shell. Higher the molecular weight of the iron preparation, stronger is the association. Labile iron is taken up by transferrin. Non-transferrin bound iron (NTBI) appears when the binding capacity of transferrin is overwhelmed. NTBI is responsible for tissue damage and restricts the dose of a injectable preparation that can be given in a single infusion. Iron preparations may be classified on the basis of strength of the associations between iron core and the carbohydrate shell. Iron dextran, ferric carboxymaltose and ferumoxytol, that have a high molecular weights have a tight binding between iron core and the carbohydrate shell, release up to 2% labile iron. These can be given in a large single dose and are suitable for total dose infusion. Iron sucrose has a weaker association between iron core and carbohydrate shell and has 4-5% labile iron. This limits the amount of drug that can be administered at one time to 300mg. Ferric gluconate is a large molecule but has two types of polymers. Lower weight polymers (about 18,000) have a weaker association between iron and carbohydrate limiting the dose to 125mg per dose.

Dose and Administration

The total dose of parenteral iron is calculated as follows

Total iron dose (mg) = weight (kg) X Hemoglobin deficit  X 0.24 + 500

Haemoglobin deficit (g/L) = Target Haemoglobin (g/L) – Actual Haemoglobin (g/L)

Adverse Effects

  1. Infusion Related Events: the risk of infusion related events (per million) is 0.6 for iron sucrose, 0.9 for ferric gluconate, 3.3 for low molecular weight dextran iron and 11.3 for high molecular weight iron dextran.  Iron sucrose has been safely administered to patients who having sensitivity to iron dextran.  Despite the fact that some studies suggest that low molecular weight iron dextran in as safe as iron sucrose, a test dose must be administered before any iron dextran preparation.
  2. Delayed Reactions to Intravenous Iron: A syndrome charecterized by fever, arthralgia and lymphadenopathy may be seen as a delayed consequence of intravenous iron therapy. Premedication with steroids may reduce the risk of this manifestation.

Further Reading

  1. Rodolfo Delfini Cançado, Manuel Muñoz. Intarvenous iron therapy: How far have we come? Rev Bras Hematol Hemoter. 2011; 33(6): 461–469.
  2. Hayat A. Safety Issues With Intravenous Iron Products in the Management of Anemia in Chronic Kidney Disease. Clin Med Res. Dec 2008; 6(3-4): 93–102.
  3. Peter Geisser and Susanna Burckhardt The Pharmacokinetics and Pharmacodynamics of Iron Preparations. Pharmaceutics. Mar 2011; 3(1): 12–33.

 

Clinical Manifestation of Iron Deficiency


The manifestations of Iron deficiency evolve insidiously and are multi-systemic. They include

Anaemia

Iron deficiency causes hypochromic microcytic anaemia. The anaemia has an insidious onset and the patient may become severely anaemic before symptoms of anaemia become evident.

Non-Specific Symptoms

Iron deficiency can cause fatigue, irritability, dizziness, breathlessness, and headache. One may attribute these symptoms to anaemia but  these symptoms are seen in patients with iron deficiency even in the absence of anaemia. Iron therapy has been shown to correct many of these manifestations.

Skin and Hair

Iron deficiency results in brittle nails that have longitudinal ridges. The nails become thin, brittle and become flat (platonychia) or concave (koilonychia). Hair may become thin and brittle.

Gastrointestinal System

Next to blood the effects of iron deficiency are most pronounced on the gastrointestinal system

  1. Tongue: Iron deficiency causes atrophy of the papillae of the tongue. The filiform papillae of the anterior part of the tongue are the first to be affected. The fungiform papillae of the posterior tongue atrophy later resulting in a smooth red tongue.  Papillary atrophy results in soreness of the tongue. The changes reverse after about 1-2 weeks of iron therapy.
  2. Mouth: Iron deficiency results in angular stomatitis. This anomalies is not specific to iron deficiency may be seen in pyridoxine and riboflavin deficiency.
  3. Dysphagia: Iron deficiency results in the formation of webs at the junction of hypopharynx and oesophagus. These manifest as dysphagia that has an insidious onset. The patients complains of discomfort on swallowing, initially for solids, at the level of the cricoid cartilage. Mild dysphagia may be corrected by iron replenishment but dysphagia persists in most patients despite iron replenishment and dilatation may be needed. Oesophageal webs predispose to carcinoma. (See Plummer-Vinson Syndrome/Peterson-Kelly Syndrome)
  4. Pica: Pica is craving for non-nutritive substances like chalk, paint and ice. Pagophagia, the craving for ice is a common form of pica.

Neuromuscular Symptoms

Iron deficiency impairs performance of muscles. Children with iron deficiency show irritability, are disruptive, have impaired attention span and may show scholastic impairment. Iron deficiency has also been shown to be associated with developmental delay, ischaemic stroke and raised intracranial pressure.

Effect on Immunity and Infections

Iron deficiency decreases the number of T lymphocytes and impairs phagocytosis. This however does not appear to result in an increased risk of infections.

Menstrual Anomalies

While menstrual anomalies can be a cause of iron deficiency, they can often be a consequence of iron deficiency. Only response to treatment can tell which of the two is the case in a given patient.

Skeletal Expansion

Young patients who have prolonged iron deficiency may develop bone changes line haemolytic anaemia or thalassaemia because of bone marrow expansion.

The Erythrocyte Membrane


The erythrocyte membrane is subject a great deal of physical stress in circulation. It needs to withstand the high sheering stress in the arteries, it needs to squeeze past capillaries that ma be as small as 7.5µm and need to withstand the ionic changes. It has a well-developed network of proteins known as the erythrocyte cytoskeleton below the lipid bilayer of the plasma membrane to meet these needs. Inherited defects in these proteins have been associated with disorders of erythrocyte shape including hereditary spherocytosis, hereditary elliptocytosis, hereditary pyropoikilocytosis, Southeast Asian stomatocytosis and hereditary acanthocytosis (see table below).

Protein Gene Chromosome Disorders
α-Spectrin SPTA1 1q22-q23 Hereditary ElliptocytosisHereditary PyropoikilocytosisHereditary Spherocytosis
β-Spectrin SPTB 14q23-q24.1 Hereditary ElliptocytosisHereditary PyropoikilocytosisHereditary Spherocytosis
Ankyrin-1 ANK1 8p11.2 Hereditary Spherocytosis
Band 3 SLC4A1 17q21 Hereditary SpherocytosisHereditary AcanthocytosisSoutheast Asian OvalocytosisHereditary Stomatocytosis
Protein 4.1R EPB41 1p33-p34.2 Hereditary Spherocytosis
Protein 4.2 EPB42 15q15-q21 Hereditary Spherocytosis
Stomatin STOM 9q33.1 Hereditary Stomatocytocytosis
Glycophorin C GYPC 2q14-q21 Hereditary Elliptocytosis
Glycophorin D GYPD 2q14-q21 Hereditary Elliptocytosis

Organization of the Erythrocyte Membrane

The erythrocyte membrane consists of a lipid layer on protein scaffolding known as the cytoskeleton. The relationship between erythrocyte membrane proteins and lipid membrane bilayer is shown in the figure below. The main component of cytoskeleton is spectrin. Spectrin is tethered to the cell membrane by vertical interactions with band 3 proteins via ankyrin and protein 4.2. Spectrin also has horizontal interactions with protein 4.1R, actin, tropomodulin, tropomyosin and adducin. Protein 4.1R interacts with glycophorin C, a trans membrane protein.

Red Cell Membrane-600px

Erythrocyte Membrane Proteins

Spectrin

Spectrin has three functions

  1. Supporting the lipid layer
  2. Maintaining cell shape
  3. Regulating the lateral movement of integral membrane proteins.

It is made of two chains α and β that interwine to form dimers. Two dimers associate to form a tetramer which is the functional subunit. The α-chain is encoded by the gene SPTA1 at 1q22-q23 and the β-spectrin is encoded by the gene SPTB at 14q23-q24.1.

Ankyrin

Ankyrins are ubiquitous adapter proteins thattarget diverse proteins to specialized membrane domains of smooth muscles and endoplasmic reticulum. The erythrocyte ankyrin, ankyrin-R is encoded by the gene ANK1 located at 8p11.

Band 3

Band 3 glycoprotein of the erythrocyte membrane that is coded by the gene is SLC4A1 at 17q21. The membrane domain transports anions across the cell membrane and the cytoplasmic domain provide binds the lipid membrane to spectrin via ankyrin.  major integal

Protein 4.2

Protein 4.2 regulates the interactiom of band 3 with ankyrin. It is encoded by the gene EPB42 at 1p33-p34.2.

Protein 4.1R

Protein 4.1R stabilized the spectrin-actin interactions. It is encoded by the EPB42 gene at 1p33-p34.2.

Disorders of Erythrocyte shape

Disruption in the cytoskeleton is the basis of in a viariey erythrocyte disorders charecterzid by alterations in erythrcoyte shape (see tabel and figure above). Disruption in vertical interactions results in instability of lipid layer resulting in loss of lipid layer and spherocytosis. Disruptions in horizontal interactions results in hereditary elliptocytosis.

Changes in the lipid layes also results in changes in erythrocyte shape. Unlike disorders ofthe cytskeleton, most of these disorders are accquired.

  1. Target Cells: Traget cells or codocytes are cells that have an appearance of a shooting target with a central bulls eye. Reletive increase in the membrane lipids results in the formation of target cells. This is seen in severe microcytis anaemias like severe iron deficiency, thalassaemia, haemoglobin C disease and haemoglobin E disease where the intracellular contents decrease. It may aslo bee seen in obstructive liver disease where the lipid and cholesterol content of the membrena increase.
  2. Stomatocytes: Stomatocytes are erythrocytes with a central elongated mouth-like area of pallor. Expansion of the inner layer results in stomatocytosis. This may be seen in alcoholism and with the use of vinca alkaloids.
  3. Echinocytes Ecchinocytes are cells that are no longer disc shaped and are covered by 10-30 short projections. The change is because of expansion of outer lipid layer. Ecchinocytes are seen in uraemia, pyruvate kinase deficiency or may be a fixing/staining artefact.
  4. Acanthocytes: Acanthocytes are cells with a few spiny projections on the surface (from acanthus, The Greek word for thorn). The result from accumulation of cholesterol (liver disease) or sphingomyelin (abetalipoproteinaemia) in the outer lipid layer results in acanthocytosis.

Intestinal Iron Absorption


Intestinal absorption of iron (figure 1)

Iron absorption occurs in two steps

  1. Absorption of iron into the enterocyte at the luminal surface of the enterocytes
  2. Transport of iron to the lamina propria at the basilateral surface of the enterocyte

click here for fig 1

Absorption of iron into the enterocyte

Dietary iron is in two forms, heme and non-heme. Animal foods have 40% iron as heme iron and 60% as non-heme iron. Plant foods contain only non-heme iron. Most of the iron available for absorption comes from non-meat sources even in meat eaters. The widely held perception that vegetarian diets contain less iron than meat eaters is not true. Vegetarian diets contain as much or more iron than meat diets (MJA Open 2012; 1 Suppl 2: 11-16). Vegetarian foods have less heme iron. Different pathways absorb heme and non-heme iron.

Absorption of heme iron

Digestion of proteins releases heme which is oxidized to haemin and absorbed by the enterocytes. A receptor for the absorption of heme has been postulated but not identified. Once within the cell the haemin is acted upon by heme oxygenase 1 to release the iron. The heme iron is added to the enterocyte iron pool and follows the same path out of the cell as non-heme iron. Food does not affect the absorption of heme iron. Some heme may be absorbed directly and is bound to hemoprixin.

Non-Heme Iron

Dietary non-Heme iron is present in the ferric state. Ferric iron is insoluble and needs to be converted to the more soluble ferrous state for absorption. The reduction of ferric iron is aided by the acidic environment of the stomach, dietary components like ascorbic acid and duodenal cytochrome b (Dcytb). Dcytb does not appear to be essential for iron absorption. Ferrous iron is absorbed by the divalent metal ion transporter (DMT1). In addition to  Fe2+,DMT 1 also transports Mn2+, Co2+, Ca2+, Zn2+, Cd2+ and Pb2+. DMT1 is expressed at the brush-border of the enterocyte near the tips of small intestinal villi. DMT1 needs protons to co-transport with iron. Protons come from the gastric juice and are most abundant in the duodenum. Most of the iron is absorbed in the duodenum. Antacid, H2 antagonists and proton pump inhibitors hamper iron absorption by reducing hydrogen ion availability. All these drugs have been shown to impair the efficacy of medicinal iron.

The absorption of non-heme iron, unlike heme iron, is affected by food.

  1. Foods enhancing iron absorption: Ascorbate, animal proteins, human milk, keto sugars, organics, amino acids that form soluble chelates with iron enhance absorption of non-heme iron.
  2. Inhibiting iron absorption: `Inhibitors of absorption of non-heme iron include
    1. Phytates present in grains and vegetables
    2. Dietary fibre
    3. Polypohenols present in tea, coffee and wine,
    4. Phosphates and phosphoproteins present in egg yolk, bovine milk
    5. Calcium and zinc.

Transport of iron to the lamina propria

Iron is transported to the lamina propria by an iron transporter ferroportin. Ferroportin transports iron in the ferrous form. This needs to be oxidized to the ferric form for binding to transferrin. Hephaestin oxidizes ferrous iron to ferric iron.

Ferroportin is the key to controlling body iron. In iron deficient state ferroportin expression at the basolateral surface of the enterocyte is increased and more iron is transferred to the blood. When the body is iron repleted ferroportin expression is low and the iron remains in the enterocyte as ferritin.  Iron stored as ferritin is lost with the enterocytes when it is shed at the end of it’s lifespan of 5-6days.

Ferroportin levels are controlled by enhancing it’s degradation when iron stores are adequate. The liver, on sensing adequate iron stores, secretes a peptide hepcidin that binds to ferroportin. Binding of ferroportin to hepcidin causes internalisation and degradation of ferroportin preventing iron transport. Mutations ferroportin, hepcidin and molecules that promote the secretion of hepcidin  (haemachromatosis protein (HFE),  haemojuvelin (HJV), transferrin receptor 2 (TFR2)) result in increased iron absorption and haemachromatosis.  Mutations in TMPRSS6 a negative regulator of hepcidin results in iron refractory iron deficiency anaemia.