Ann. N.Y. Acad. Sci.

ISSN 0077-8923

A N N A L S O F T H E N E W Y O R K A C A D E M Y O F SC I E N C E S
Special Issue: Hemoglobin Concentration for Assessing Anemia
REVIEW

Genetic variation influencing hemoglobin levels and risk

for anemia across populations
Paloma K. Barrera-Reyes1,2 and M. Elizabeth Tejero1
1
Laboratorio de Nutrigenómica y Nutrigenética, Instituto Nacional de Medicina Genómica, Ciudad de, México, Mexico.
2
Instituto de Investigaciones Biomédicas, Universidad Nacional Autónoma de México, Ciudad de, México, Mexico

Address for correspondence: M. Elizabeth Tejero, Laboratorio de Nutrigenómica y Nutrigenética, Instituto Nacional de
Medicina Genómica, Periférico Sur 4809, Col Arenal Tepepan, Mexico City CP14160, Mexico. etejero@inmegen.gob.mx

Hemoglobin (Hb) concentration is the outcome of the interaction between genetic variation and environmental
factors, including nutritional status, sex, age, and altitude. Genetic diversity influencing this protein is complex
and varies widely across populations. Variants related to abnormal Hb or altered characteristics of the erythro-
cytes increase the risk for anemia. The most prevalent are related to the inherited globin abnormalities affecting Hb
production and structure. Malaria-endemic regions harbor the highest frequencies of variants associated with the
most frequent monogenic diseases and the risk for nonnutritional anemia and are considered as public health prob-
lems. Variation in genes encoding for enzymes and membrane proteins in red blood cells also influence erythrocyte
life span and risk for anemia. Most of these variants are rare. Interindividual variability of hematological parame-
ters is also influenced by common genetic variation across the whole genome. Some of the identified variants are
associated with Hb production, erythropoiesis, and iron metabolism. Specialized databases have been developed
to organize and update the large body of available information on genetic variation related to Hb variation, their
frequency, geographical distribution, and clinical significance. Our present review analyzed the underlying genetic
factors that affect Hb concentrations, their clinical relevance, and geographical distribution across populations.

Keywords: hemoglobinopathies; genetics; anemia; hemoglobin

Introduction HBA2 genes, and two subunits of the β-like globin

peptides encoded by the HBB gene, along with heme
Overview of hemoglobin genetics
moieties necessary for oxygen transport.3,4 These
Hemoglobin (Hb) is the most abundant protein
genes harbor the largest number of variants causing
in erythrocytes, whose function is to transport
abnormalities and have been extensively investi-
oxygen from the lungs to tissues. Any condi-
gated through decades.5–7,12 Some of these variants
tion that reduces the concentration of functional
may reduce the production of Hb and increase
Hb or decreases red blood cell (RBC) mass may
hemolysis. Another group of variants that affect
cause anemia.1 Multiple factors may lead to this
Hb concentrations and risk for anemia is found
condition, including nutritional deficiencies,
in genes expressed in erythrocytes that encode for
acute or chronic blood loss, chronic inflamma-
structural proteins and enzymes that may affect
tion, infectious diseases, RBC abnormalities,
RBC shape, function, and life span, and increase
and genetic variants affecting Hb structure,
hemolysis.8,9 In the last decade, genetic factors
among others.1 Three basic mechanisms affect-
influencing the interindividual variation of Hb and
ing RBC cause anemia: reduced production,
other hematological parameters have been studied
increased destruction (hemolysis), or loss (hem-
using whole-genome analysis. These studies have
orrhage) due to defects that can be intrinsic to
revealed a wider spectrum of variants that modulate
RBCs or their precursors, or extrinsic.2 The Hb
these traits.10,11 This remarkably diverse variation
molecule is composed of two subunits of α-like
encompasses Hb synthesis, iron metabolism, ery-
globin peptide chains encoded by the HBA1 and
thropoiesis, erythrocyte function, and stability.5–9,12
doi: 10.1111/nyas.14200
32 Ann. N.Y. Acad. Sci. 1450 (2019) 32–46 © 2019 New York Academy of Sciences. The World Health Organization retains copyright
and all other rights in the manuscript of this article as submitted for publication.
Barrera-Reyes & Tejero Genetic variation influencing hemoglobin concentrations

Figure 1. Upper panel shows the structure of the α-globin locus (chromosome 16) and β-globin locus (chromosome 11). Major
regulatory regions and transcription factors KLF1, BCL11A complex, and MYC are depicted (modified from Ref. 49). Composition
of the hemoglobin types is presented in the gray box. Lower panel shows the sequential activation and silencing of globin chain
expression, and the production sites during pregnancy and after birth (modified from Ref. 89).

Hb production the order they are expressed during development,

The genes encoding for α-globin forms (similar to
α) are on chromosome 16p13.3 and those similar
to β (ε, γ, and δ) are on chromosome 11p15.5 1
(Fig. 1). The globin genes in the α- and similar to
β-clusters are arranged along the chromosome in
and their combinations determine the type of
produced Hb.3,5,12 The sequential activation and
silencing of the globin genes are strictly controlled.
HS-40 is the major regulatory element of the α-
globin locus, while the locus control region is an

Ann. N.Y. Acad. Sci. 1450 (2019) 32–46 © 2019 New York Academy of Sciences. The World Health Organization retains copyright 33
and all other rights in the manuscript of this article as submitted for publication.
Genetic variation influencing hemoglobin concentrations
important upstream regulatory region for the β-
globin chain. The production of embryonic Hb (ε)
form in the early part of the first trimester occurs
within the yolk sac−derived erythrocytes. During
the second semester of pregnancy, the production of
enucleated erythrocytes begins in the stem and pro-
genitor cells in the fetal liver and the predominant
β-like globin switches to γ-globin.12,13 These chains
combine with α-globin chains (α2γ2) to produce
fetal Hb (HbF). This form makes the majority of
neonatal Hb and shortly after birth, there is a switch
from predominant expression of HbF to adult Hb
regulated by the interaction of the transcription
factors Krüppel-like factor 1 (KLF1), GATA bind-
ing protein 1 (GATA1), FOG family member 1
(FOG1), BAF chromatin remodeling complex sub-
unit BCL11A, and transcriptional activator MYB
with each other, and other proteins, and corepressor
complexes that involve chromatin modeling and
epigenetic modifiers. This switch is mediated by
the repression of the γ-chain by transcriptional
regulation conducted by the BCL11A transcrip-
tion factor in definitive erythroid progenitors12–15
(Fig. 1). The transition from HbF to HBA is clin-
ically relevant, since variation in the genes that
regulate globin synthesis and erythrocyte matu-
ration may lead to abnormal concentrations and
functionality of this protein.14,15
Adult Hb is composed of HbA1, HbA2, and HbF.
The first one represents 98% of the erythrocyte con-
tent and is composed of two α- and two β-chains
(α2β2). The remaining 2% are composed of HbA2,
containing two α- and two δ- (α2δ2) chains, and
HbF. The percentages may fluctuate on the basis
of age, genetics, consumption of medications, and
underlying conditions.3,6,12 Testing for Hb abnor-
malities is recommended after iron deficiency ane-
mia (IDA) has been ruled out, and disorders, such as
microcytic anemia, chronic hemolytic anemia, and
vascular obliteration crises in subjects from regions
with monogenic forms of anemia or drug-induced
anemia, are identified. Diagnosis of some Hb dis-
eases requires the integration of clinical findings
and laboratory tests.7,16,17
Information resources
The advance of “omics” approaches (i.e., genomic
and proteomic) has produced a large body of
information that leads to the creation of special-
ized internet-based public databases, developed and
34 Ann. N.Y. Acad. Sci. 1450 (2019) 32–46 © 2019 New York Academy of Sciences. The World Health Organization retains copyright
and all other rights in the manuscript of this article as submitted for publication.
Barrera-Reyes & Tejero
maintained by scientific communities.18–21 A selec-
tion of examples containing information on Hb-
related genes and inherited forms of anemia is
shown in Table 1. The aim of the present study
is to review the most prevalent genetic variants
that influence Hb concentration and risk for ane-
mia, such as inherited Hb disorders, RBC enzyme
and membrane disorders, and other genetic variants
associated with Hb interindividual variation. Their
geographical distribution, frequency, and effect on
hematological parameters were analyzed.
Materials and methods
Literature search
The present review was conducted through the
consultation of different sources of informa-
tion. Bioinformatic resources for the study of
hemoglobinopathies and genetic variation related
to Hb or related diseases were consulted. The
databases Ithagenes (https://www.ithanet.eu/db/
ithagenes), HbVar (https://globin.bx.psu.edu/
hbvar/menu.html), and the Leiden Open Varia-
tion Database (LOVD) (http://www.lovd.nl/3.0)
were used for data mining of genes related to
Hb disorders and variation. Information on
genes function was consulted at http://www.ncbi.
nlm.nih.gov/gene/. The second strategy was a
comprehensive literature review using MEDLINE
(PubMed) as the major information source using
the terms “genetics” and “hemoglobin,” “genet-
ics” and “anemia,” “hemoglobinopathies,” “sickle
cell disease,” “RBC enzymopathies,” “RBC mem-
branopathies,” “genome-wide association” and
“hemoglobin,” and “thalassemia.” Recent scientific
reviews on genetic variation and geographical
distribution of hemoglobinopathies, thalassemia,
and sickle cell disease (SCD) were consulted.
Maps of the prevalence of the most common
hemoglobinopathies were generated using an inter-
active tool at IthaMaps (https://www.ithanet.eu/db/
ithagenes/db/ithamaps). Information on the sin-
gle nucleotide polymorphisms (SNPs) identified
in genome-wide association (GWA) studies was
sought at dbSNP (https://www.ncbi.nlm.nih.gov/
snp/) and www.internationalgenome.org/1000-
genomes-browsers.
Results
The report of the Global Burden of Disease clas-
sified the causes of anemia in 23 etiology-specific
Barrera-Reyes & Tejero
Table 1. Web-based databases containing information on genetic variation associated with hemoglobin concentra-
tions
Database Weblink
HbVar https://globin.bx.psu.edu/hbvar/menu.html
IthaGenes https://www.ithanet.eu/db/ithagenes
LOVD https://www.lovd.nl
NCBI https://www.ncbi.nlm.nih.gov
EMBL-EBI https://www.ebi.ac.uk/services/dna-rna
Orphanet https://www.orpha.net/consor/cgi-bin/index.php
GARD https://rarediseases.info.nih.gov/diseases/6520/
glucose-6-phosphate-dehydrogenase-deficiency
NORD https://raredisease.org
https://research.nhgri.nih.gov/RBCmembrane
categories. Seven of these categories are associ-
ated with gene mutations (sickle cell trait (SCT),
SCD, thalassemia trait, thalassemias, G6PD trait,
G6PG deficiency, and other hemoglobinopathies
and hemolytic anemias).1 The description of the
genetic variants associated with these conditions is
discussed.
Variation in the HBA1, HBA2, and HBB genes
The HBA1, HBA2, and HBB genes contain a large
number of insertions, deletions, SNPs, and less
frequent point mutations associated with Hb con-
centrations and risk for anemia. Some of these
variants are very frequent and have large effects
on Hb concentrations. The β-globin gene presents
a larger number of variants than the α-subunit.
The phenotypes related to variation in these genes
are widely diverse, even among subjects with the
same variant. Many of them are not considered as
pathogenic, while others may have clinical manifes-
tations that range from mild hypochromic anemia
to moderate hematological disease to severe chronic
forms, with transfusion-dependent (TDT) anemia
and multiorgan damage.5–7,22–25 Hb abnormalities
are highly prevalent in areas of the world as in
malaria-prone regions of Africa, all Mediterranean
countries, the Middle East, the Indian subcon-
Ann. N.Y. Acad. Sci. 1450 (2019) 32–46 © 2019 New York Academy of Sciences. The World Health Organization retains copyright
and all other rights in the manuscript of this article as submitted for publication.
Genetic variation influencing hemoglobin concentrations
Description
Specialized database of genomic sequence changes leading
to Hb variants and all types of hemoglobinopathies
Specialized database of sequence variations affecting Hb
disorders, including the globin loci and disease modifiers
and polymorphisms with relevance for clinical diagnosis
Online gene-centered collection and display of DNA
variations
Contains subunit databases, such as ClinVar, SNP, dbVar,
and Gene, which aggregate information about genomic
variation and its phenotypes
Provides comprehensive databases (DGVa, ENA, and
Ensemble) to browse all types of genetic variation data
and their relationships to health
Provides information on rare diseases and orphan drugs
Contains information on G6PD
Contains information on rare diseases for different users
Provides information on red blood cell membrane−related
diseases
tinent, and Southeast Asia.22–29 Lower rates of
some of these abnormalities have been reported
in Latin America and the United States.30,31 Fre-
quencies of the risk alleles vary across geographical
regions and ethnic groups, and some of them
produce the most common monogenic diseases
worldwide22–24,27,32,36 that account for approxi-
mately 3.4% of deaths under the age of 5.32 Between
300,000 and 400,000 babies are born each year with
a clinically significant Hb disorder (83% sickle cell
disorder and 17% thalassemia).33 Approximately
7% of the worldwide population are carriers of
genetic variants related to Hb disorders.32,33 The
highest prevalence of these diseases occurs in
developing countries, although an increase in their
frequency has been reported in other regions of the
world because of demographic changes.31,33–36 A
recent study of the global burden of anemia esti-
mated that 11.6% of female and 9.9% of male cases
of anemia are caused by hemoglobinopathies.1
Hb-related disorders are caused by mutations
and/or deletions in the α- or β-globin genes pro-
ducing intrinsic defects of RBC that may result in
anemia owing to shortened erythrocyte life span,
hemolysis, and other systemic pathology depend-
ing upon the specific mutation(s) present, genetic
modifiers, and other factors.29,36 These diseases
35
Genetic variation influencing hemoglobin concentrations
may be clustered into structural Hb variants that
produce molecules with abnormal properties, such
as increased polymerization, altered solubility, or
oxygen binding affinity,6,12,23 and thalassemia syn-
dromes that result from the defective production
of the α- or β-chains of Hb.27,33 The α- and β-
thalassemia, together with sickle cell anemia (SCA),
HbC, and HbE (HBB: c.79 G>A), compose the
most common monogenic diseases affecting Hb
concentrations.6,7,12 These are Mendelian-recessive
diseases, such that heterozygous carriers have mild
or no abnormalities and may pass on one copy of the
gene for an Hb variant to their children.7,23,36
Coinherited forms of Hb abnormalities exist in
geographical areas with a high prevalence of these
diseases and are related to the wide spectrum of
clinical manifestations.24–27,36 Factors, such as nat-
ural selection, consanguineous marriage in some
countries, and the increase of life expectancy of
affected individuals due to improved treatments,
have contributed to the concentration of Hb-related
disorders in specific regions.32,33
SCD
SCD is a group of diseases related to the most com-
mon structural hemoglobinopathy. This disease is
the single most important genetic cause of child-
hood mortality globally and is characterized by
misshapen RBCs that cause vaso-occlusive disease,
vasculopathy, and systemic inflammation, affecting
quality of life and decreasing life expectancy in 30
years.28,29,36–40 The cause is a single point (missense)
mutation that substitutes a glutamic acid by a valine
residue at position 6 of the β-globin chain. This SNP
(rs334) produces the characteristic HbS form of
SCA. The SCA trait (HbSA) is considered as harm-
less and provides some protection from malaria
during childhood. The HSS genotype causes SCD,
characterized by hemolysis and blockage of blood
vessels. The affected erythrocytes have a short life,
leading to anemia.
The SCD phenotype is influenced by the coin-
heritance with other Hb forms generating diversity
to the disease spectrum that includes sickle cell β-
thalassemia (HbSβ), sickle cell Hb C (HbSC) dis-
ease, and the inheritance of the HbS gene along
with other interacting Hb variants. Interestingly, the
coinheritance of hereditary spherocytosis (HS) with
α-thalassemia leads to a milder phenotype.36,37 HbF
may also inhibit the polymerization of HbS.37,38,41
36 Ann. N.Y. Acad. Sci. 1450 (2019) 32–46 © 2019 New York Academy of Sciences. The World Health Organization retains copyright
and all other rights in the manuscript of this article as submitted for publication.
Barrera-Reyes & Tejero
Environmental factors, like altitude, air pollution,
or smoking, have a deleterious effect on the SCT or
SCD.38
Hemoglobin C
Hemoglobin C (HbC) is caused by a missense sub-
stitution of glutamic acid by lysine at position 6 in
the HBB gene (rs33930165). This variant is frequent
in West Africa and has also been associated with
malaria protection. HbC may be coinherited with
HbS producing a milder phenotype.29,42
Hemoglobin E
Hemoglobin E (HbE) is one of the most common
forms of variation in the HBB genes, and it is con-
sidered a structural variant and a thalassemia. The
variation is HBB: c.79G>A (rs33950507).43 This is a
missense substitution, changing a glutamine residue
for lysine at position 26, and this change also cre-
ates a splice donor.44 This form has an extremely
high frequency, reaching a 70% carrier rate in some
populations. HbE is also associated with protec-
tion from falciparum malaria.43,45–47 Heterozygotes
show mild microcytosis with fragile erythrocytes
and slight hemolytic anemia. Hb synthesis occurs
at a slightly lower rate and behaves phenotypically
like a mild form of β-thalassemia. The combination
of HbE with variants of α- and β-thalassemia is fre-
quent and produces a significant diversity of anemia
phenotypes. Homozygous HbE presents normal Hb
concentrations, with low mean cell volume (MCV)
(Table 2).7,43
Thalassemias
Thalassemia syndromes are the most common
genetic disorders in the world, affecting nearly
200 million people worldwide. Between 1% and
5% of the global population are carriers of these
diseases.49–52 Decreased or absent expression of
one of the two globin chains of the Hb molecule,
α (HBA1 and HBA2) and β (HBB), is observed
as a consequence of (1) deletion of the structural
gene(s); (2) mutations that alter the transcription,
resulting in abnormal RNA synthesis, processing, or
stability; and (3) mutations resulting in decreased
protein synthesis or stability. For normal Hb syn-
thesis, the ratio of α-:non-α-subunits should be
1:1. The decrease in the production of one of the
globin chains results in an imbalance of subunits,
which damages erythrocytes.6,7,50 In α-thalassemia,
the quantity of β-like chains is greater than that
Barrera-Reyes & Tejero Genetic variation influencing hemoglobin concentrations

Table 2. Characteristics of hemoglobinopathies

Genotype/diagnose Hb (g/L) MCV (fL) Predominant geographic distribution TD Ref.

Abnormal Hbs
HbAS sickle cell trait 12−15 >80 Sub-Saharan Africa and Mediterranean No 38
HbSS sickle cell disease 6−11 >80 Republic of Congo, India, and Nigeria Yes 38,40
HbC trait normal 20% West Africa No 29
2% African Americans
3.5% Jamaicans
HbSC 10−15 75−95 29
HbE 12.8 ± 1.5 70% in regions Southeast Asia, Thailand, No 6,43,51
10.6 ±1.2 Bangladesh, and Cambodia
normal or slightly
low
HbEE 10−14 65 Southeast Asia No 6,45
10.6 ± 1.2
α-thalassemia
-α/αα-thalassemia silent Normal 3% of African American, North India, No 23,36,48
and South Asia
1−15% of subjects of Mediterranean
origin
–α/–α-thalassemia trait Normal or low Sub-Sahara No 25,33,48
– –/αα-thalassemia trait Normal Africa No 25,33,48
HbH disease 6−10 Southeast Asia, Mediterranean, and Occasional 6,25
– –/– α Middle East
HbH Constant Spring 8.5 ± 1.5 1−2% Northeast Thailand Some patients 25
5−8% Southern China and Vietnam may require
β-thalassemia
β-thalassemia trait or <79 14% Cyprus No 23
minor 10.3% Sardinia and Southeast Asia
β++, β+, and β0 3% Bangladesh
Male 11.5−15.3
Female 9.1−14
β-thalassemia intermedia 7−10 Occasional 23,48
β-thalassemia major <7 50−70 Regular 6,23
(affected)
Coinherited forms
HbE/α-thalassemia Normal to slightly Southeast Asia No 43
low
HbE/β-thalassemia 7.1 ± 1.4 59 ± 3 Thalassemia triangle No 45,51,52
HbSS/α-thalassemia 37−77%a Uganda, Cameroon, and No 29
Tanzania
50% Saudi Arabia
TD, transfusion dependent.
a Percentage of HSS patients.

of α-chains; the opposite ratio is a characteristic of
β-thalassemia. The degree of imbalance is strongly
associated with the severity of the disease as it
impairs erythrocyte maturation, causing ineffective
erythropoiesis with intramedullary hemolysis, lead-
ing to microcytosis as the characteristic laboratory
finding.23,33 The wide spectrum of phenotypic vari-
ations is related to the α-globin production and the
presence of other genetic modifiers.36,48
In the last decade, the categorization of the
thalassemias has transitioned from the molec-
ular forms to criteria based on their clinical
management.33,48,55 Thalassemia may be classified
into TDT and non-TDT (NTDT) forms.36,48 The

Ann. N.Y. Acad. Sci. 1450 (2019) 32–46 © 2019 New York Academy of Sciences. The World Health Organization retains copyright 37
and all other rights in the manuscript of this article as submitted for publication.
Genetic variation influencing hemoglobin concentrations Barrera-Reyes & Tejero

Figure 2. Worldwide distribution of the carriers of major hemoglobinopathies. Upper panel: (left) β-thalassemia; (central)
α-thalassemia; and (right) sickle cell anemia. Lower panel: (left) hemoglobin E and (right) hemoglobin C. Source: https://www.
ithanet.eu/db/ithamaps.

NTDT do not require regular RBC transfusions for
survival but may require occasional transfusions
because of infection or pregnancy or may require
more regular transfusions later in life owing to com-
plications. Information on the screening, diagnosis,
and treatment of these diseases has been reviewed
in recent publications.36,48,49,55
α-Thalassemia
The abnormal or absent production of α-globin
chains (HBA1 and HBA2) due to approximately
293 deletional or nondeletional variants causes
α-thalassemia (https://www.ithanet.eu/
db/ithagenes). This is the most frequent
hemoglobinopathy and there is malaria resistance
in both heterozygotes and homozygotes.36 Most
frequent forms arise from the deletion of one or
more copies of the α-globin, although nondeletion
forms have been characterized. Mutations outside
the HBA1 and HBA2 genes may also influence
their expression.23,24,26 The severity of the clinical
conditions depends upon the mutation type and
number of functional genes. The milder form of
α-thalassemia is associated with deletion of one or
two copies of the HBA1 or HBA2 genes. Deletion of
a single copy results silent thalassemia (–α/αα) and
deletion of two copies causes α-thalassemia trait,
which is generally unassociated with hematological
abnormalities and does not require treatment.
Hemoglobin H (HbH) forms are found when only
one functional α-globin gene has been inherited and
is associated with deletional and nondeletional vari-
ants. This Hb form has poor oxygen-carrying capac-
ity, is prone to oxidative damage, and increased
removal by the spleen.28,29 HbH Constant Spring
(rs41464951) is the most common nondeletional
α-thalassemia caused by a mutation in the normal
stop codon of one α-globin gene (α142 STOP→Gln ),
resulting in an abnormally long, unstable globin
chain produced in very low amounts. Deletion of
the four copies of the α-globin is named Hb Bart’s
hydrops fetalis. The fetus with this genotype usually
dies in utero or shortly after birth.49
A description of the clinical variation and pre-
dominant geographical distribution associated with
the α-thalassemia genotypes is shown in Table 2 and
Figure 2.
β-Thalassemia
Structural variation in the HBB gene is highly
diverse and frequent. In contrast to α-thalassemia,
95% of β-thalassemia forms are due to point muta-
tions that cause abnormal RNA transcription,
processing or stability, or nonsense mutations
resulting in the production of abnormal pro-
teins or nonsense-mediated RNA decay. Currently,
around 400 sequence variants are associated with β-
thalassemia (https://www.ithanet.eu/db/ithagenes).
β-thalassemia is characterized by reduced synthesis
of the Hb subunit β (Hb β-chain) leading to an
increased ratio between α- and β-globins. There
is a complex genetic diversity associated with this
thalassemia, which is difficult to correlate with the
phenotypic variation.50,51 As in α-thalassemia, there

38 Ann. N.Y. Acad. Sci. 1450 (2019) 32–46 © 2019 New York Academy of Sciences. The World Health Organization retains copyright
and all other rights in the manuscript of this article as submitted for publication.
Barrera-Reyes & Tejero Genetic variation influencing hemoglobin concentrations

are different clinical forms from silent β-carrier, logic functions. Two main pathways that support
β-thalassemia trait, thalassemia intermedia, and erythrocyte activity are glycolysis and the pentose
thalassemia major.7,23,49 The severity is directly shunt.4,57–59 Enzymes in these pathways show
related to the degree of quantitative reduction in genetic variation encoding for proteins with an
the synthesis of normal β-globin. abnormal function, the most common are present
in glucose-6-phosphate dehydrogenase (G6PD) and
Genetic modifiers of Hb inherited diseases
pyruvate kinase (PK). G6PD deficiency encom-
Genetic modifiers are variants that may have an
passes a group of hereditary abnormalities caused
ameliorating or worsening effect on the phenotype
by the decreased or absent G6PD enzyme activity in
of Hb disorders and contribute to their pheno-
erythrocytes (Table 3). This deficiency is considered
typic variation. Genetic studies have identified
the most frequent enzymopathy, affecting around
key variants and pathways with modifier effects
400 million people,57 and may result in chronic, or
on hemoglobinopathies. These variants are found
acute intermittent hemolytic anemia under some
across many genes, including those that regulate
conditions that induce oxidative stress, such as the
HbF production; transcription factors that control
intake of certain drugs, during infections, metabolic
Hb syntheses, such as HBG2, HBS1L-MYB, KLF1,
abnormalities, like diabetic acidosis, and during the
and BCL11A; and copy number of α-globin.48
neonatal period.57,58 This gene is located on X chro-
Primary genetic modifiers of SCD act at the level of
mosome. About 300 mutations have been reported
HbS polymerization and sickling, and are related to
until now.4,57 The effect of the mutation is related to
the coinheritance of other abnormal forms of Hb
the affected domain of the protein.4,57–59 Most of the
(HbSC, HbSS, HbSβ, HbF, and α-thalassemia).49,54
variants are missense mutations, producing single
Secondary modifiers are genotypes that influence
amino acid substitutions in the protein. The World
the manifestation of subphenotypes and compli-
Health Organization classified G6PH into five cate-
cations, such as serum bilirubin and propensity
gories or classes according to the reduction in enzy-
to gallstones, SCD nephropathy, stroke risk, and
matic activity. Class I variants (<10% of activity)
others.49 Genetic modifiers of α-thalassemia are
are associated with chronic hemolysis. These muta-
mainly related to the copy number. Aggravating
tions are clustered around an area that regulates the
forms include the triplication and quadruplication
formation of the active 6GPDH dimer56 and has
of the gene, while ameliorating effects are associated
the most deleterious effects on the structure and
with α-thalassemia mutations.48 Primary genetic
stability of the protein.58 The activity of this enzyme
modifiers for β-thalassemia are associated with
protects Hb from oxidative damage and precipita-
the degree of α: β chain imbalance, β-globin geno-
tion. As some hemoglobinopathies, these genetic
type (one or two and severity of the β-thalassemia
variants are common in malaria-endemic regions
alleles), α-globin genotypes (α-thalassemia and
of the world. Of the G6PD-deficient variants that
coinheritance of extra α-globin genes), and innate
occur at high frequency, the best known variants
ability to increase HbF concentrations and α-
are African G6PD A and Mediterranean G6PD.4,56
hemoglobin−stabilizing protein. Secondary mod-
Diagnosis is based on the detection of reduced
ifiers at the level of disease and therapy include
specific enzyme activity and molecular characteri-
genetic variants associated with the risk for gall-
zation of the defect at the DNA level. A systematic
stones, iron loading, osteopenia, and osteoporosis,
review and meta-analysis of the prevalence of G6PD
among others. As observed, these effects are mainly
across countries identified a high heterogeneity in
driven by α-globin copy number and genotype,
the estimates of this condition, probably because
and preservation of HbF production. The geno-
of differences in the assessment methods and diag-
types may be detected by DNA analysis; however,
nosis. The highest prevalence rates were reported
predicting disease severity remains difficult.
among Sub-Saharan African countries.59 The use of
Variation in RBC enzymes next-generation sequencing has been proposed for
Variation in genes encoding for key RBC enzymes the identification of specific variants in this gene.60
is associated with the risk of anemia and is also RBC PK deficiency (PKD) is the most common
associated with Hb concentrations. Erythrocytes glycolytic defect causing congenital nonspherocytic
require an active metabolism to carry out their bio- hemolytic anemia. PK is encoded by the PKLR gene.

Ann. N.Y. Acad. Sci. 1450 (2019) 32–46 © 2019 New York Academy of Sciences. The World Health Organization retains copyright 39
and all other rights in the manuscript of this article as submitted for publication.
Genetic variation influencing hemoglobin concentrations
Table 3. Characteristics of the most common RBC enzymopathies and membranopathies
Diagnose Hb (g/L) Inheritance
RBC enzymes
G6PD <9.7 X-linked
PKD 2.2−14.4 AR
Mean = 9.8
RBC membrane
Hereditary spherocytosis 8−10 AD
AR
Hereditary elliptocytosis AD
AR, autosomal recessive; AD, autosomal dominant; XR, X-linked recessive.
This enzyme converts phosphoenolpyruvate to
pyruvate, producing 50% of total ATP in RBC.61–64
The life span of these cells is dependent on the ATP
produced in glycolysis. PKD reduces ATP availabil-
ity affecting RBC life. Damaged cells are cleared
by both the spleen and liver. Clinical features of
this deficiency are highly variable, ranging from
very mild to life-threatening neonatal anemia.61
The association between genotype and phenotype
variation remains to be investigated. The disease
is transmitted in an autosomal recessive manner,
and homozygotes and compound heterozygotes are
common. PKD variation is more frequent in specific
populations, such as Amish in Pennsylvania70 and
Romani communities, because of the founder effect.
Over 200 mutations have been described in
patients with PKD.65–68 The most commonly
reported are missense mutations (up to 70%),63
including 1529 G>A in the United States and
Europe, 1456 C>T in Southern Europe, and 1468
C>T in Asia.64,65,69 Other variants have been
described in India.61 Splicing and stop codon
mutations have been identified (13% and 5%,
respectively). The estimated prevalence of the dis-
ease based on the most common PKLR mutations
associated with a Caucasian population is 51 per
million people.69
RBC membrane disorders
Mutations in genes modifying the structure or func-
tion of the cell membrane are causative of mem-
branopathies and are considered forms of intrinsic
hemolytic anemia.63 These disorders produce an
40 Ann. N.Y. Acad. Sci. 1450 (2019) 32–46 © 2019 New York Academy of Sciences. The World Health Organization retains copyright
and all other rights in the manuscript of this article as submitted for publication.
Barrera-Reyes & Tejero
Predominant geographic
distribution Clinical findings Ref.
Sub-Saharan Africa, From acute to 9,57
Mediterranean, and intermittent to
Southeast Asia severe hemolytic
anemia
Asia and Europe, and Amish Variable severity 9,57
and Romani people
All racial groups Mild to severe 8,63
Northern European reticulocytes >8%
West Africa (2% prevalence) Asymptomatic to 8,63
Provides resistance to malaria severe
abnormal membrane structural organization or
altered membrane transport producing a fragile
membrane.8,63 During their life, erythrocytes travel
through blood vessels, including microvasculature,
and their deformability is key for oxygen delivery.
Cells that are not deformable are destroyed in the
spleen, which will cause anemia.8,71,72 Abnormali-
ties in RBC membrane proteins are pathologic and
include HS, elliptocytosis, and pyropoikilocytosis.73
Diagnosis of moderate to severe forms of hereditary
membrane disorders requires a series of tests that
include peripheral smear examination, reticulocyte
count, RBC indices, and osmotic fragility, among
others, after the exclusion of other hemolytic
conditions.71,74 New proteins involved in alter-
ations of the RBC membrane permeability were
recently described.72 Table 3 contains information
on some of the characteristics of these diseases.
Hereditary spherocytosis
Among the RBC membrane disorders, HS is
one of the most common causes of inherited
hemolytic anemia.63,75 The estimated prevalence
is 1−5/10,000. This disease has a wide variety
of phenotypes, from asymptomatic to TDT severe
anemia. The erythrocytes have a characteristic
spherical shape along with an elevated number
of reticulocytes, elevated mean corpuscular Hb
concentration, and increased erythrocyte osmotic
fragility after incubation. HS is the most common
form of inherited anemia in subjects of Northern
European descent, although it has been found in
all ethnic groups. The identified mutations are in
Barrera-Reyes & Tejero
the genes: ankyrin 1, erythrocytic (ANK 1); solute
carrier family 4 (anion exchanger) member 1 (ery-
throcyte membrane protein band 3 (Diego blood
group)) (SCL4A1); and spectrin beta chain, erythro-
cytic (SPTB). The ANK1 gene belongs to a family
of proteins that links the integral membrane pro-
teins to the underlying spectrin−actin cytoskeleton
and contributes to cell motility, activation, prolifer-
ation, contact, and the maintenance of specialized
membrane domains. Multiple variants in this gene
include insertions, deletions, and missense substi-
tutions across the 5 UTRs, exons, and a few of
them on introns, and account for 50−60% of HS
cases.54 The predominant forms of autosomal dom-
inant HS (75%) are nonsense and frameshift muta-
tions of ANK1, SLC4A1, and SPTB. Recessive forms
of HS are most often due to compound heterozy-
gosity of variants in the ANK1, SPTA1, or EPB42
genes.76 The SLC4A1 gene encodes for a protein that
regulates ion exchange in the erythrocyte cell mem-
brane. Over 40 mutations have been found in this
gene, including insertions, deletions, and point mis-
sense and nonsense mutations.
Hereditary elliptocytosis and
pyropoikilocytosis
Hereditary elliptocytosis (HE) is another disease
caused by genetic variation in molecules that mod-
ify the RBC membrane. Pyropoikilocitosis is a
severe variant of HE.
The principal lesion in HE is mechanical weak-
ness or fragility of the erythrocyte membrane
skeleton due to defects in α-spectrin, β-spectrin,
or protein 4.1. The main characteristic is ellipti-
cally shaped RBCs found on a peripheral blood
smear. HE has been described worldwide; however,
as other RBC abnormalities, it is more common
in malaria-endemic regions of the world. It is
relatively common in individuals of African and
Mediterranean descent. The incidence of HE
worldwide is estimated at 1:2000−4000, in regions
with a higher frequency, such as parts of Africa,
frequency approaches 1:100. In regions of West
Africa, prevalence is of approximately 2%. The
frequency could be higher since many affected
subjects are asymptomatic.62 Mild to no anemia is
a characteristic of HE; however, this disease could
be clinically and genetically heterogeneous. HE
has an autosomal dominant mode of inheritance.
Heterozygous are asymptomatic, but about 10% of
Ann. N.Y. Acad. Sci. 1450 (2019) 32–46 © 2019 New York Academy of Sciences. The World Health Organization retains copyright
and all other rights in the manuscript of this article as submitted for publication.
Genetic variation influencing hemoglobin concentrations
patients may present moderate to severe anemia
(homozygous or compound heterozygous).62 Muta-
tions related to this abnormality are located in the
spectrin A and B genes (SPTA1 and SPTB). Spectrin
is a primary structural protein of the erythrocyte
membrane skeleton. Defects that weaken or disrupt
the interactions are involved in the self-association
of spectrin dimers into tetramers and oligomers, or
structural and functional defects of protein 4.1 that
interrupt spectrin−actin interactions and disturb
the integrity of the membrane skeleton. Conse-
quently, membrane skeletons, cell membranes, and
erythrocytes are mechanically unstable, leading
to RBC fragmentation and hemolysis. To date, 27
mutations have been reported on SPTA1, with 13
of them on exon 2 and five mutations that modify
the splicing. SPTB harbors 19 mutations, primarily
on exon 29 and five on splice controlling sites.
Other genetic variants have been associated with
rare forms of inherited anemia and are beyond the
scope of this review.
Variants influencing Hb interindividual
variation
The genetic architecture of Hb concentration and
other hematological traits, including hematocrit
(Hct), MCV, and mean cell Hb, has been inves-
tigated using the GWA study approach, which
analyzes the statistical association between SNPs
distributed across the genome and the variation of
a trait. Hb concentration has a significant heritabil-
ity that ranges from 40% to 90% confirming that
genetic factors contribute to the variation of this
phenotype.77 The largest GWA study conducted to
date was carried out in 62,553 people of European
ancestry and 9308 people of South Asian ances-
try, using over 2 million autosomal and 67,645
X-chromosome SNPs.10 This study identified 75
loci associated with Hb concentration and related
RBC phenotypes. This group of SNPs explained
between 5% and 9% of the variance of the analyzed
traits. The SNPs associated with Hb concentrations
were rs209775, rs9369427, rs10480300, rs10159477,
rs11042125, rs2159213, and rs4969184. These loci
are spread across the genome and their function
remains to be elucidated.
Other studies have investigated genetic deter-
minants of erythrocyte traits in the general pop-
ulation using a similar approach. Genetic fac-
tors associated with six erythrocyte traits were
41
Genetic variation influencing hemoglobin concentrations
analyzed in individuals of European ancestry in
multiple community-based cohorts constituting the
CHARGE consortium,78 followed by replication in
independent samples. This investigation reported
significant association to 23 loci. Previously iden-
tified variants were confirmed in the HBS1L/MYB,
HFE, TMPRSS6, TFR2, and SPTA1 genes, and novel
associations were found for variants in the EPO,
TFRC, and SH2B3 genes, and 15 other loci. The SNP
with the largest contribution to the variance per-
centage in this study was rs16926246 (0.21%) and
is on the HK1 gene that encodes for the enzyme
hexokinase-1 that modulates glucose metabolism.
Other SNPs with statistically significant associa-
tions had smaller effects. In addition, the Haem-
Gen Consortium conducted a GWA meta-analysis
to identify variants associated with eight hema-
tological parameters, including concentrations of
Hb in participants from six studies in European
populations.79 This investigation identified six loci
associated with RBC phenotypes. The SNP with the
strongest association with Hb concentration was
rs5756506 in the TMPRSS6 gene. Interestingly, in
this study, Hb concentration had weaker statisti-
cal associations as compared with other studied
hematologic phenotypes. Erythrocyte-related traits
were strongly associated with two loci, rs1800562
in HBS1L-MYB and the nonsynonymous change
rs9402686 in the HFE gene. Three loci (HFE, TFR2,
and TMPRSS6) were mapped to genes known to
be associated with iron homeostasis. The SNP
rs855791 in the TMPRSS6 gene has been associated
with Hb concentration in a sample of European and
Indian ancestry.80 The effect of this variant on blood
Hb concentration is of 0.13 g/dL lower per copy of
allele A (P = 1.6 × 10−13 ). This common polymor-
phism causes a missense valine to alanine change
(p.V736A), and the valine variant has been asso-
ciated with lower Hb and microcytic anemia and
its frequency varies in different populations. The
highest frequency for the risk allele (A) is reported
in the 1000 Genomes database (http://www.inter
nationalgenome.org/1000-genomes-browsers/) in a
sample of Japanese carriers from Tokyo (0.62),
while populations from Africa have the lowest
reported frequencies (0.02−0.07). TheTMPRSS6
gene encodes for the enzyme matriptase-2 or trans-
membrane serine protease 6 that inhibits hepcidin
transcription, producing intestinal iron absorption
and release of cellular iron. Variation in this gene has
42 Ann. N.Y. Acad. Sci. 1450 (2019) 32–46 © 2019 New York Academy of Sciences. The World Health Organization retains copyright
and all other rights in the manuscript of this article as submitted for publication.
Barrera-Reyes & Tejero
been associated with Hb concentrations,11,79,80,90
iron-refractory IDA,81 and phenotypes related to
iron metabolism.82,83
A GWA study based on whole-genome sequenc-
ing conducted in the Sardinian general population53
identified 23 loci associated with common
interindividual variation in HbA1, HbA2, and
HbF levels. Five variants found at loci in the
MPHOSPH9, PLTP-PCIF1, FOG1, NFIX, and
CCND3 genes, 4 are the new independent sig-
nals at known loci, and 10, refined previously,
described associations with polymorphisms that
may have functional effects. Six of these signals
were associated with HbA1, 14 were associated
with HbA2, and eight were associated with HbF.
Findings suggest that Hb is largely regulated by
genetic factors present in the HBS1L-MYB loci and
around the HBB region. Levels of HbA2 are associ-
ated with polymorphisms in two loci, HBS1L-MYB
and around HBB.88 GWA studies conducted in
participants with hemoglobinopathies have con-
firmed the role of variants present in transcription
factors known to regulate the Hb switch during
development acting as genetic modifiers.54,84–87
Discussion
Anemia is a multifactorial health problem. There
is no universal classification for this condition
although one of the criteria to categorize anemia is
the underlying cause. Genetic factors influencing
Hb concentration and risk for anemia have been
investigated for a long time using the available tech-
nology to address a worldwide health problem. As
observed in other phenotypes, some genetic vari-
ants have large effects often producing monogenic
diseases, while other genetic variants have a small
contribution to the variation of Hb concentration.
Hb monogenic diseases have been studied for
decades because of their association with anemia
of diverse severity, high frequency, and significant
impact on health affecting numerous regions of the
world. The study of the interindividual variation
of Hb and other hematologic parameters has been
conducted more recently.
Phenotypic variation is a common characteristic
of the most frequent monogenic Hb disorders. This
variation is associated with the presence of genetic
modulators and the complex variety of coinherited
Hb abnormalities. The variants associated with
thalassemia in different combinations lead to over
Barrera-Reyes & Tejero
60 different syndromes, making Southeast Asia the
locality with the most complex thalassemia geno-
types. Information regarding the distribution and
frequency of genetic factors associated with the risk
of disease in populations is required for the develop-
ment of guidance, decision making, planning, and
implementation of intervention programs. Studies
on the genetic variation of Hb-inherited diseases are
very diverse across different regions and countries.
In most cases, samples are not representative of the
population, vary in sample size, target population,
geographical location within a country, design, and
data collection methods. This heterogeneity makes
difficult to conduct comparisons across studies and
to estimate precise frequencies and effects. The
available information on the geographical distri-
bution and frequency of genetic variants with the
most significant effects on Hb concentration may
be considered for the adjustment of cutoff points
to define anemia. Southeast Asia, Sub-Saharan
Africa, India, and the Mediterranean basin have the
highest prevalence of heterozygous carriers of SCD
and thalassemia (Fig. 2).
Genetic variations in genes encoding RBC
enzymes and membranes affect Hb concentrations.
These variants cause hemolytic anemia of diverse
severity. As observed in hemoglobinopathies, phe-
notypic variation is also frequent in these diseases,
probably owing to the effects of multiple genetic
factors that regulate Hb production and erythrocyte
characteristics. Most of these genotypes are rare,
with the exception of variants in the G6PH enzyme
and HS.63 G6PD is frequent in regions where
hemoglobinopathies are common, increasing
genetic diversity associated with Hb. Interestingly,
SNPs in genes encoding for membrane proteins
have been associated with interindividual variation
of Hb in healthy participants.
Another approach aimed to identify the genetic
component that regulates Hb concentration is
through GWAs or other association studies. These
investigations have found genetic variation influ-
encing Hb concentration in healthy subjects. The
identified SNPs are found within genes involved
in erythropoiesis, iron metabolism, and regulation
of the expression of globin genes. The frequency
of the identified SNPs shows variation across pop-
ulations and has a relatively small effect size on
Hb concentrations and other hematological param-
eters. Most studies have been conducted in Euro-
Ann. N.Y. Acad. Sci. 1450 (2019) 32–46 © 2019 New York Academy of Sciences. The World Health Organization retains copyright
and all other rights in the manuscript of this article as submitted for publication.
Genetic variation influencing hemoglobin concentrations
pean populations.10,11,78 More studies with simi-
lar designs in different populations are required to
replicate findings and evaluate the consistency of
the observed effects across populations. In addition,
other nongenetic factors that contribute to Hb vari-
ability, and interact with genetic variants, such as
altitude, smoking, sex, nutritional status, and pol-
lution, among others should be addressed. Some of
these factors are not often considered in the study of
the effect of genetic variation (monogenic or poly-
genic) on Hb concentrations or anemia.
Current international efforts to systematize data
on human genetic variation in open web-based
databases provide excellent resources for updated
and curated information, although users should be
aware of the heterogeneity of the available data.
Recent studies have identified new variants and pre-
dicted the apparition of others, increasing genetic
variation that regulates Hb and the risk for anemia.
Conclusions
A highly diverse genetic variation influences Hb
concentrations and risk for anemia. The variants
with the largest effects and high frequency are
within the HBA1, HBA2, and HBB genes and are
major causes for anemia around the world. The
available information on the geographical distribu-
tion and frequency of genetic variants with the most
significant effects on Hb concentration may be con-
sidered for the adjustment of cutoff points to define
anemia.
The geographical distribution of this group
of diseases is changing. Thus, the screening of
hemoglobinopathies of high frequency and impact
on health is relevant in countries where incidence is
increasing.
The interindividual variation of Hb concentra-
tions is regulated by common polymorphisms in
genes involved in Hb synthesis, erythropoiesis, and
iron metabolism. These variants have shown mod-
est effects on Hb concentrations. Most of the studies
of the underlying genetic effects in Hb variation
have been conducted in European populations, and
studies in different populations of the world are
required.
Acknowledgments
This work was commissioned and financially sup-
ported by the Evidence and Programme Guidance
43
Genetic variation influencing hemoglobin concentrations
Unit, Department of Nutrition for Health and
Development of the World Health Organization
(WHO), Geneva, Switzerland. The authors thank
Omar A. Bañuelos-Quintana for the design of
Figure 1.
Author contributions
P.K.B.R. and M.E.T. conducted the literature review,
data analysis, and contributed to writing this
manuscript.
Statement
This manuscript was presented at the World Health
Organization (WHO) technical consultation “Use
and Interpretation of Haemoglobin Concentra-
tions for Assessing Anaemia Status in Individu-
als and Populations,” held in Geneva, Switzerland
on November 29−30 and December 1, 2017. This
paper is being published individually but will be
consolidated with other manuscripts as a special
issue of Annals of the New York Academy of Sci-
ences, the coordinators of which were Drs. Maria
Nieves Garcia-Casal and Sant-Rayn Pasricha. The
special issue is the responsibility of the editorial
staff of Annals of the New York Academy of Sci-
ences, who delegated to the coordinators prelimi-
nary supervision of both technical conformity to the
publishing requirements of Annals of the New York
Academy of Sciences and general oversight of the sci-
entific merit of each article. The workshop was sup-
ported by WHO, the Centers for Disease Control
and Prevention (CDC); the United States Agency
for International Development (USAID); and the
Bill & Melinda Gates Foundation. The authors alone
are responsible for the views expressed in this
paper; they do not necessarily represent the views,
decisions, or policies of the WHO. The opinions
expressed in this publication are those of the authors
and are not attributable to the sponsors, publisher,
or editorial staff of Annals of the New York Academy
of Sciences.
Competing interests
The authors declare no competing interests.
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