INTRODUCTION

Hemoglobin
Human hemoglobin (Hb) consists of four proteins (subunits). These proteins are called
chains. Most of the normal adult hemoglobin is built of two alpha- and two beta chains (Yuain et
al, 2015)

Each of the four chains contains a ‘heme’ part. ‘Heme’ is the chemical structure where the
iron is. It binds oxygen, carbon monoxide, or other small molecules such as nitric oxide [Butcher
et al, 2014],[ Yuain et al, 2015].

Haem
Haem synthesis occurs both in cytosol and in mitochondria of erythrocytes. Protoporphyrin is
synthesized from the condensation of glycine and succinyl coenzyme A, eight molecules of
each being required to form a linear tetrapyrrole molecule, which finally cyclizes into the
protoporphyrin ring. The protoporphyrin then binds to a Fe2+ ion to form haem (Power and Kam
2008)

Iron
The Fe2+ ion forms six bonds within the haem moiety. Five of these bind the Fe2+ firmly: four
with nitrogen atoms in the centre of the protoporphyrin ring, and one to a ‘proximal’ histidine
residue at position 87 on an a-globin chain. The final bond is made with an oxygen
molecule as required. Close to the oxygenbinding site on the haem group, there is another crucial
histidine residue, the ‘distal histidine’. This occupies position 89 on the a-globin chain. It has two
important functions: through steric hindrance, it prevents haem groups on other globin molecules
oxidizing the iron in the Fe2+ state to the Fe3+ state and it prevents carbon monoxide binding
irreversibly to the Fe2+ ion. (Power and Kam 2008)
Globin chains
More than 95% of an adult’s haemoglobin is in the form of HbA with two a- and two b-globin
chains. Each a-chain has 141 amino acids, and each b-chain has 146. Genes for the a-chain
are found on chromosome 16 and those for the b-chain on chromosome 11. Globin chains are
synthesized in the cytosol of erythrocytes. Of an adult’s haemoglobin, 2.2–3.5% is
HbA2, composed of two a- and two d-chains. This form of haemoglobin is poor at oxygen
carriage. Fetal haemoglobin (HbF) comprises two a-chains and two g-chains. At birth, 50–
95% of a baby’s haemoglobin is HbF, but these levels decline after 6 months as more HbA is
produced. In a healthy adult, ,1% of haemoglobin is HbF. The oxygen affinity of HbF is
substantially greater than HbA to facilitate the transfer of oxygen between the maternal and
fetal circulations in the placenta.
Haemoglobin destruction
Erythrocytes are removed by the reticuloendothelial system. Globin chains are broken down to
amino acids which then return to the amino acid pool. Iron is re-used by the bone marrow to
synthesize haem. Protoporphyrin degradation begins with the cleavage of thering to form a linear
tetrapyrrole molecule, biliverdin, which is then reduced to bilirubin. Bilirubin is bound to
albumin for transport to the liver, where it is conjugated with glucuronic acid. This is excreted in
the bile and then into the small bowel. In the gastrointestinal tract, bilirubin is converted into
stercobilin, some of which is reabsorbed into the plasma and excreted by the kidney as
urobilinogen in urine. Small amounts of free haemoglobin may be released into the
plasma.2(Power and Kam 2008)
Hemoglobin Genes
There are more than 1,000 human Hb gene variants (SNPs).
These variants are common, affecting an estimated 7% of the world’s population.
The most common and important Hb variants include HbS (found in sickle-cell anemia) and
HbE found in thalassemia.
These are all maintained with relatively high frequency in humans because they confer
survival advantages when it comes to malaria [Thom et al., 2013].
Those who have one copy of the sickle-cell gene or for alpha-thalassemia are protected
against malaria [Weatherall, 2014].
However, in individuals who carry both copies of these mutations, the protective effect is
completely lost and they are equally prone to malaria as individuals who have normal Hb
copies. Most of the people with only one copy remain without symptoms and many go
undiagnosed. Some Hb variants are clinically benign but produce obvious changes in skin color,
a ruddy complexion, or a blue-tinged skin. These variants are not clinically damaging beyond
their cosmetic effects. Note that some of the Hb variants artificially elevate HbA1c levels and
interfere with diabetic management [Thom et al., 2013].
HBA1 and HBA2

HBA1 (hemoglobin subunit alpha 1) and HBA2 (hemoglobin subunit alpha 2) both produce
the alpha chain of Hb. Normally, people have two copies each (four copies in total).
Two alpha chains (either HBA1 or HBA2) plus two beta chains constitute HbA, which is the
normal adult hemoglobin. It normally accounts for about 97% of the total hemoglobin [Kanias
and Acker 2010].

Alpha-thalassemias can result from losing more than two copies of the alpha chain (either

HBA1 or HBA2).
Losing one or two copies is clinically silent, and doesn’t produce any drastic symptoms.
Losing three copies causes hemoglobin H disease, with a moderate anemia.
Losing all four copies results in the death of the fetus [Coates, 2014].
Copies are most often deleted, and these deletions are not usually assayed by genomics
companies (you have to undergo special hemoglobin typing tests). However, some cases of
alpha-thalassemia are also caused by single nucleotide variants (mutations). RS41341344
T to C mutation in this position causes alpha-thalassemia [Higgs, 1993]. RS41417548

HbH disease occurs due to having two copies of this variant [Warang et al.,, 2010].

RS2858942
RS4141130
RS41461652
RS41525149
HBB
HBB (hemoglobin subunit beta) produces the beta chain of hemoglobin. Normally, people
have two copies of this gene.
Two alpha chains plus two beta chains constitute HbA, which is the normal adult
hemoglobin. HbA accounts for about 97% of the total hemoglobin [Kanias and Acker 2010].
Mutations in the HBB gene cause sickle-cell disease or beta-thalassemia.
However, these mutations are at the same time beneficial, conferring resistance to malaria.
The term “thalassemia intermedia” refers to milder mutations where the hemoglobin level
is maintained above 6.5 g/dL .
The term “beta-thalassemia major” refers to having a serious disease in which there is an
inability to maintain hemoglobin to levels above 6.5 g/dL .
RS334
rs334(T), s also known as i3003137 (A) or HbS, is the most prevalent mutation in the
hemoglobin beta subunit [Coates, 2014].
Having two copies of this variant (rs334 T/T) causes a serious disease known as sickle cell
anemia. On the other hand, people having a single copy (rs334 A/T) are resistant to malaria and
develop only slight anemia [Luzzatto, 2012].
HBD

HBD (hemoglobin subunit delta) produces the delta chain of hemoglobin.

Two alpha chains plus two delta chains constitute HbA2, which accounts for about 2.5% of
total adult hemoglobin [Thom et al., 2013].
HBG1 and HBG2
HBG1 (hemoglobin subunit gamma 1) and HBG2 (hemoglobin subunit gamma 2) are genes
that normally function in the fetus. Two gamma chains together with two alpha chains constitute
fetal hemoglobin (HbF) which is usually replaced by adult hemoglobin (HbA) in the year
following birth [Thom et al., 2013].
Thus, fetal hemoglobin accounts for 80% of hemoglobin in newborns, but only about 0.5%
in adults [Kanias and Acker 2010].
In some cases, the F hemoglobin persists in adult red blood cells. This is a state without
symptoms known as hereditary persistence of fetal hemoglobin (HPFH) [Schechter, 2010].
In cases of beta-thalassemia and related conditions, gamma chain production may persist,
possibly as a mechanism to compensate for the mutated beta-chain.
RS35378915
RS35710727
Haptoglobin

Haptoglobin is an important protein that sequesters free Hb from the blood. Free Hb causes
oxidative stress and inflammation, and this is mostly prevented by haptoglobin [Cabrales, 2013].
In humans, the haptoglobin gene has two variants, Hp1 and Hp2 [Jeney et al,. 2013).
Hp2 forms a larger, bulkier protein. However, due to its size, it may be less efficient.
Hp1 more efficiently inhibits free Hb-associated damage compared to the Hp2 variant [Schechter,
2008].

Having two copies of the Hp2 variant is a risk factor for heart disease in both type I and type
II diabetic patients.
Having two Hp2 copies has also been linked to a greater risk of brain damage in people with
subarachnoid hemorrhage (bleeding into the compartment surrounding the brain) [Jeney et al,.
2013)
Hemoglobin Function
The main function of hemoglobin is to transport oxygen from the lungs to
tissues, and carbon dioxide (CO2) from the tissues to the lungs [R, R].

However, it also interacts with two other gases, carbon monoxide (CO) and nitric
oxide (NO) [R].

The human body contains about 750 g of Hb, mostly contained within red blood cells
(RBCs) [R].
Finally, each Hb molecule is capable of binding up to four oxygen molecules, enabling each
red blood cell to carry over onebillion oxygen molecules [R].
The oxygen-carrying function of hemoglobin is affected by [R]:
pH – Lower pH (more acidic) in tissues increases the release of oxygen and
stimulates the binding of carbon dioxide. Higher pH (more alkaline) in the lungs
causes carbon dioxide to be released and stimulates the binding of oxygen
Levels of other molecules (hydrogen ions, CO2, 2,3-bisphosphoglyceric acid, IHP,
chloride, and phosphate) – In people acclimated to high altitudes, the concentration
of 2,3-BPG in the blood increases, which allows a larger amount of oxygen to be
delivered to the tissues
Temperature – Increases in temperature decrease oxygen saturation of hemoglobin
Note that inside the body, the pH and temperature are generally within very narrow range,
from 7.35 – 7.45 and 97.0 – 99.8 degrees Fahrenheit, respectively [R].
Apart from the blood, Hb seems to play some unexpected roles in other tissues.
Hb chains are also present in white blood cells (macrophages), blood vessel walls
(endothelial cells), lungs (alveolar cells), eye lens, kidneys (mesangial cells), and
dopamine-releasing neurons (A9) [R, R].
In the blood vessel walls, Hb helps control the level of nitric oxide, and thereby participates
in the constricting or expanding of blood vessels. This means that Hb influences blood
pressure, blood flow, and oxygen delivery to tissues.
On the other hand, Hb in dopamine-releasing neurons seems to be involved in
mitochondrial function and may play a role in normal brain physiology [R].
Hemoglobin Types
In red blood cells of healthy adults [R]:
97% of total hemoglobin is the HbA type (with two alpha and two beta chains)
2.5% is HbA2 (with two alpha and two delta chains
0.5% is HbF or fetal hemoglobin (with two alpha and two gamma chains)
As the name would suggest, fetal hemoglobin accounts for 80% of hemoglobin in
newborns [R].
Fetal hemoglobin (HbF) has a slightly higher oxygen affinity than the adult hemoglobins [R].
After birth, production gradually switches to adult hemoglobin over the course of several
months [R].
Normally, by the end of the first year of life, the “adult” hemoglobins (A and A2), are
predominant [R].
In some cases, the HbF persists in adult red blood cells. This is a condition largely without
symptoms, known as the hereditary persistence of fetal hemoglobin [R].
Hemoglobin Metabolism
When red blood cells get old and/or damaged, Hb escapes into the blood. There are
several molecules within our blood that recognize and bind free Hb, including haptoglobin
(Hp) and hemopexin (Hpx) [R].