**5.2 HbS gene polymorphisms and malaria**

Sickle hemoglobin (HbS) is best characterized genetic polymorphism tightly interconnected with malaria. HbS represents a structural variant of normal adult hemoglobin (HbAA) and results from a single point mutation (Glu → Val) on the sixth codon of the beta globin gene [103]. Homozygotes for hemoglobin S (HbSS) have sickle cell disease that further causes high morbidity and mortality. Also, heterozygous for HbS have 10-fold lower risk of dying from malaria compared to homozygous [97, 104, 105]. Heterozygotes (HbAS) have generally asymptomatic sickle cell disease which does not endanger their lives [106].

It has been found that, in the conditions of selection for fitness against malaria, nearly 45 generations (or 1000 years) were necessary to pass until sickle gene frequency reached a stable equilibrium [107]. People with HbAS have 50–90% lower parasite density [105] in comparison with individuals with normal hemoglobin (HbAA). Sub-Saharan Africa is an area with closely 80% of people born with sickle cell anemia and where most *P. falciparum* malaria cases and deaths occur [108]. Besides sub-Saharan Africa, sickle cell anemia is present, although rarely with frequency higher than 20–25%, in the Mediterranean region, the Middle East, and the Indian subcontinent [95]. There is a strong connection between high HbS allele frequency and high malarial endemicity in the world although this finding is based on the observations made in Africa: HbS allele frequency gradually increases from epidemic areas to endemic areas in Africa which is in accordance with the hypothesis that malaria protection by HbS includes the enhancement of innate and acquired immunity to *P. falciparum* [109].

Knowledge of the existing relationship between malaria infection and extension and prevalence of hemoglobinopathies in Mediterranean region are not new [102, 110]. Sickle-cell homozygous persons have short life expectancy and commonly die before adulthood. However, the gene responsible for sickle cell disease "hidden" within the genotype of heterozygous carrier can achieve high frequency due to resistance to *P. falciparum* [111].

There are lots of described biological mechanisms that are considered to be responsible for protection against malaria. First, there were only two mechanisms described regarding a manner in which the presence of HbS in heterozygotes protects against malaria: sickling of circulating infected RBCs and impaired parasite growth and oxidant damage [101]. It has been found that formation of sickle RBSc shapes under low oxygen presure occured more frequently in RBCs infected with *P. falciparum* compared to uninfected RBCs [112]. When parasite triggers sickling of erythrocytes once, sickled cells are removed by macrophages [113]. This action of may macrophages'possibly occurs due to their ability to produce and release numerous cytokines that further recruit more phagocytic cells [114]. In addition, it has been discovered that enchanced sickling was limited to RBCs infected with small Plasmodium forms [115]. On the other hand, impaired parasyte growth and oxygen damage was discovered thanks to *in vitro* studies [112]. In the conditions of normal oxygen pressure, there were no differences in the invasion, growth, and multiplication of *P. falciparum* in HbAS cells compared to HbAA RBCs. In the opposite, hypoxic consitions caused reduced fraction of *P. falciparum* in HbAS cells and a block in the maturation of ring forms to trophozoites and schizonts.

In addition, sickling and destruction of parasites in HbAS and HbSS RBCs at lower oxygen tensions (1–5%) more closely mimiced the micro-aerophilic environment of post-capillary venules *in vivo* [112].

The guiding hypothesis regarding the protective effect against malaria in people with HbAS suggests that decreased *P. falciparum* Erythrocyte Membrane Protein 1 (PfEMP1) [116] expression on infected HbAS RBCs results in lower binding of infected cells to the endothelium [117]. As a consequence, only approximately one-half the cytoadherence was seen in infected HbAS RBCs. Archer and associates have proposed that oxygen-dependent HbS polymerization is a key factor for HbAS malaria resistance [118]. They found that intraerythrocytic *P. falciparum* parasites in HbAS RBCs at low oxygen concentrations arrest in cell cycle before DNA replication and that HbS polymerization is responsible for this growth arrest.

Among the genetic factors responsible for the protection from malaria is one of the complement regulatory proteins − complement receptor 1(CR1). The frequency of CR1 polymorphisms is high in a numerous of malaria endemic areas [100]. A major receptor for RBCs infected with *P. falciparum* is human protein CD36 [119]. CD36 can be involved in malaria by sequestering infected RBCs thus disabling the immune response to this parasite [120]. Some African populations have extremely high frequency of CD36 mutation and this CD36 deficiency causes susceptibility to severe form of malaria [121]. Important genetic factors involved in resistance to malaria are erythrocyte-binding antigens. Special attention was given to erythrocyte binding antigen-175 (EBA-175), a protein that binds to glycophorin A, thus enabling merozoite entry into erythrocytes [122].

An interesting study regarding host genetic factors responsible for malaria resistance was conducted in Senegal, in the population of children and young adults that were 2 to 18 years old. Thanks to the results of this study, three candidate regions in the genome of these children were detected and one of them contains a gene related with the malaria infection in the 5q31q33 region [123].

One of the newest studies revealed that unfavorable microRNA (miRNA) composition in heterozygous HbAS or homozygous HbSS erythrocytes, leads to resistance versus *P. falciparum*. When erythrocytes are infected with *P. falciparum*, a part of erythrocyte miRNAs can translocate into the parasite. LaMonte et al. found that HbAS and HbSS erythrocytes had high number of miR-451 and let-7i integrated into essential parasite messenger RNAs, as well as that these miRNAs, together with miR-223 are negative regulators of parasite growth [124]. miR-451 fuse with transcripts of the regulatory subunit of the parasite's cAMP-dependent protein kinase (PKA-R) and reduce its translation. Therefore, it up-regulates the activity of its substrate PKA and disrupts multiple parasite developmental pathways [124].

Piel and associates created extensive geodatabase of HbS allele frequency and investigated geographical distribution of malaria [125]. Their HbS allele frequency map has shown that throughout majority of the African continent and in localized areas in Mediterranean, this allele is present with the frequency of >0.5%. According to this geodatabase research, in the Chalkidiki region of Greece, southeastern Turkey and in Central Sudan, frequency of this allele was even above 6% [125].

One of the models of how hemoglobiopathies protect from malaria is proposed by Killian associates [126] and reveals association between reduced cytoadherence phenotype and parasitized hemoglobinopathic erythrocytes. This team used conditional protein export system and tightly synchronized cultures of *P. falciparum*. They have showed that exportation of proteins encoded by parasites across the parasitophorous vacuolar membrane is more advanced, faster and increased in amount in parasitized wild type erythrocytes in comparison with hemoglobinopathic erythrocytes.

**159**

*Adaptation to Mediterranea*

affected blood vessel [127, 128].

als infected with this parasite [129].

malaria syndromes [141, 142].

original inhabitants [143].

*DOI: http://dx.doi.org/10.5772/intechopen.94081*

Severe malaria is in relation with intraerythrocytic life cycle of *P. falciparum* and the pathological cytoadhesive behavior of parasitized erythrocytes [127, 128]. When parasite adheres to the endothelial cells of venular capillaries, it avoids clearance mechanisms of spleen. As a consequence, pathological sequelae form within the

Pathological consequences of *P. falciparum* malaria can possibly be mediated by adhesion of infected cells to vascular endothelium either to other uninfected red cells (rosetting) or to platelets (clumping). It has also been found that variant of erythrocytes infected with *P. falciparum* do not have noticeable differences regarding their adhesive phenotypes in comparison with erythrocytes of normal individu-

There are two main phenotypes of parasite-infected RBCs (iRBCs) and both express PfEMP1 [130, 131]. First type of iRBCs mediate iRBCs binding to the endothelial receptors ("cytoadherence") [132] and the second mediate iRBCs binding to uninfected RBCs ("rosetting") [133, 134]. Different iRBCs phenotypes differ in various PfEMP1 that are responsible for binding of iRBCs to microvascular endothelial cells, placental syncytiotrophoblasts or uninfected RBCs [135–137].

Usually, hemoglobin S does not increase IgG responses to various *P. falciparum* proteins [138], but it can potentially enhance IgG responses to PfEMP1, which is the main cytoadherence ligand and virulence factor [139]. In an *in vitro* study, HbAS affected the trafficking system that directs PfEMP1 to the surface of infected erythrocytes. Using cryo-electron tomography, it has been shown that within the cytoplasm of normal RBCs, the parasite proteins are transported to the surface via a parasite-generated host-derived actin cytoskeleton. In addition, hemoglobin oxida-

Exact pathogenic mechanisms of malaria caused by *P. falciparum* are still unknown due to numerous parasite virulence factors, host susceptibility traits, and innate and adaptive immune responses that modify the occurrence of various

The most important reason for the high frequency of hemoglobin disorders in tropical countries is natural selection through protection of heterozygotes against severe malaria. Protection observe in HbAS is reflected in protection against severe form of malaria and probably, to some extent, against mild malaria [129]. Natural selection is not the only mechanism responsible for high HbS gene frequency [102, 106, 107]. The others are high frequency of consanguineous marriages and epidemiological transition [98]. In addition, different distribution of some hemoglobin disorders in different populations is an example of founder effects by their

Thanks to the studies conducted *in vitro*, various researches united on general hypothesis that protection from malaria is the result of impairment in the invasion and growth of *P. falciparum* parasites into HbAS red cells under conditions of low oxygen tension that were physiologically representative of *in vivo* conditions [112, 144]. Afterwards, a lot of alternative hypothesis have been developed including the one that refers to the enhanced removal of parasite infected HbAS RBCs. This mechanism could be related with sickling of these cells under low oxygen tension [112, 115, 145] causes their premature destruction in the spleen [112]. Specifically, Shear and associates have observed that protective effect of HbS can be lost on the model of transgenic mice that were subjected to splenectomy [146]. There are few researches that suggest that protection against malaria can be achieved not only through innate immunity, but also via acquired immunity. For example, in populations naturally exposed to *P. falciparum*, protective effect of HbAS increases with age [147, 148]. This is in accordance with recent studies on a mouse model which proposes an immuno-modulatory mechanism mediated

tion products disrupted this process in HbAS red cells [140].

#### *Adaptation to Mediterranea DOI: http://dx.doi.org/10.5772/intechopen.94081*

*Genetic Variation*

post-capillary venules *in vivo* [112].

In addition, sickling and destruction of parasites in HbAS and HbSS RBCs at lower oxygen tensions (1–5%) more closely mimiced the micro-aerophilic environment of

and that HbS polymerization is responsible for this growth arrest.

enabling merozoite entry into erythrocytes [122].

with the malaria infection in the 5q31q33 region [123].

The guiding hypothesis regarding the protective effect against malaria in people with HbAS suggests that decreased *P. falciparum* Erythrocyte Membrane Protein 1 (PfEMP1) [116] expression on infected HbAS RBCs results in lower binding of infected cells to the endothelium [117]. As a consequence, only approximately one-half the cytoadherence was seen in infected HbAS RBCs. Archer and associates have proposed that oxygen-dependent HbS polymerization is a key factor for HbAS malaria resistance [118]. They found that intraerythrocytic *P. falciparum* parasites in HbAS RBCs at low oxygen concentrations arrest in cell cycle before DNA replication

Among the genetic factors responsible for the protection from malaria is one of the complement regulatory proteins − complement receptor 1(CR1). The frequency of CR1 polymorphisms is high in a numerous of malaria endemic areas [100]. A major receptor for RBCs infected with *P. falciparum* is human protein CD36 [119]. CD36 can be involved in malaria by sequestering infected RBCs thus disabling the immune response to this parasite [120]. Some African populations have extremely high frequency of CD36 mutation and this CD36 deficiency causes susceptibility to severe form of malaria [121]. Important genetic factors involved in resistance to malaria are erythrocyte-binding antigens. Special attention was given to erythrocyte binding antigen-175 (EBA-175), a protein that binds to glycophorin A, thus

An interesting study regarding host genetic factors responsible for malaria resistance was conducted in Senegal, in the population of children and young adults that were 2 to 18 years old. Thanks to the results of this study, three candidate regions in the genome of these children were detected and one of them contains a gene related

One of the newest studies revealed that unfavorable microRNA (miRNA) composition in heterozygous HbAS or homozygous HbSS erythrocytes, leads to resistance versus *P. falciparum*. When erythrocytes are infected with *P. falciparum*, a part of erythrocyte miRNAs can translocate into the parasite. LaMonte et al. found that HbAS and HbSS erythrocytes had high number of miR-451 and let-7i integrated into essential parasite messenger RNAs, as well as that these miRNAs, together with miR-223 are negative regulators of parasite growth [124]. miR-451 fuse with transcripts of the regulatory subunit of the parasite's cAMP-dependent protein kinase (PKA-R) and reduce its translation. Therefore, it up-regulates the activity of its substrate PKA and disrupts multiple parasite developmental pathways [124]. Piel and associates created extensive geodatabase of HbS allele frequency and investigated geographical distribution of malaria [125]. Their HbS allele frequency map has shown that throughout majority of the African continent and in localized areas in Mediterranean, this allele is present with the frequency of >0.5%. According to this geodatabase research, in the Chalkidiki region of Greece, southeastern Turkey and in Central Sudan, frequency of this allele was even

One of the models of how hemoglobiopathies protect from malaria is proposed by Killian associates [126] and reveals association between reduced cytoadherence phenotype and parasitized hemoglobinopathic erythrocytes. This team used conditional protein export system and tightly synchronized cultures of *P. falciparum*. They have showed that exportation of proteins encoded by parasites across the parasitophorous vacuolar membrane is more advanced, faster and increased in amount in parasitized wild type erythrocytes in comparison with hemoglobino-

**158**

above 6% [125].

pathic erythrocytes.

Severe malaria is in relation with intraerythrocytic life cycle of *P. falciparum* and the pathological cytoadhesive behavior of parasitized erythrocytes [127, 128]. When parasite adheres to the endothelial cells of venular capillaries, it avoids clearance mechanisms of spleen. As a consequence, pathological sequelae form within the affected blood vessel [127, 128].

Pathological consequences of *P. falciparum* malaria can possibly be mediated by adhesion of infected cells to vascular endothelium either to other uninfected red cells (rosetting) or to platelets (clumping). It has also been found that variant of erythrocytes infected with *P. falciparum* do not have noticeable differences regarding their adhesive phenotypes in comparison with erythrocytes of normal individuals infected with this parasite [129].

There are two main phenotypes of parasite-infected RBCs (iRBCs) and both express PfEMP1 [130, 131]. First type of iRBCs mediate iRBCs binding to the endothelial receptors ("cytoadherence") [132] and the second mediate iRBCs binding to uninfected RBCs ("rosetting") [133, 134]. Different iRBCs phenotypes differ in various PfEMP1 that are responsible for binding of iRBCs to microvascular endothelial cells, placental syncytiotrophoblasts or uninfected RBCs [135–137].

Usually, hemoglobin S does not increase IgG responses to various *P. falciparum* proteins [138], but it can potentially enhance IgG responses to PfEMP1, which is the main cytoadherence ligand and virulence factor [139]. In an *in vitro* study, HbAS affected the trafficking system that directs PfEMP1 to the surface of infected erythrocytes. Using cryo-electron tomography, it has been shown that within the cytoplasm of normal RBCs, the parasite proteins are transported to the surface via a parasite-generated host-derived actin cytoskeleton. In addition, hemoglobin oxidation products disrupted this process in HbAS red cells [140].

Exact pathogenic mechanisms of malaria caused by *P. falciparum* are still unknown due to numerous parasite virulence factors, host susceptibility traits, and innate and adaptive immune responses that modify the occurrence of various malaria syndromes [141, 142].

The most important reason for the high frequency of hemoglobin disorders in tropical countries is natural selection through protection of heterozygotes against severe malaria. Protection observe in HbAS is reflected in protection against severe form of malaria and probably, to some extent, against mild malaria [129]. Natural selection is not the only mechanism responsible for high HbS gene frequency [102, 106, 107]. The others are high frequency of consanguineous marriages and epidemiological transition [98]. In addition, different distribution of some hemoglobin disorders in different populations is an example of founder effects by their original inhabitants [143].

Thanks to the studies conducted *in vitro*, various researches united on general hypothesis that protection from malaria is the result of impairment in the invasion and growth of *P. falciparum* parasites into HbAS red cells under conditions of low oxygen tension that were physiologically representative of *in vivo* conditions [112, 144]. Afterwards, a lot of alternative hypothesis have been developed including the one that refers to the enhanced removal of parasite infected HbAS RBCs. This mechanism could be related with sickling of these cells under low oxygen tension [112, 115, 145] causes their premature destruction in the spleen [112]. Specifically, Shear and associates have observed that protective effect of HbS can be lost on the model of transgenic mice that were subjected to splenectomy [146].

There are few researches that suggest that protection against malaria can be achieved not only through innate immunity, but also via acquired immunity. For example, in populations naturally exposed to *P. falciparum*, protective effect of HbAS increases with age [147, 148]. This is in accordance with recent studies on a mouse model which proposes an immuno-modulatory mechanism mediated

throughout hemoxygenase-1 [149]. The problem with these findings when translating into human populations is metabolic difference between sickling disorders of mice and human sickle-cell traits [95].

### **5.3 Glucose-6-phosphate dehydrogenase (G6PD) gene polymorphisms and malaria**

The Glucose-6-phosphate dehydrogenase (G6PD) is a "housekeeping" gene located on long (q) arm of the X chromosome at position 28 – Xq28 [150]. This gene encodes an enzyme named glucose-6-phosphate dehydrogenase that acts in almost all types of cells thus providing normal carbohydrates processing [151]. The most important role of G6PD is in RBCs, where this enzyme is involved in protection of RBCs from damage and early destruction [152].

Glucose-6-phosphate dehydrogenase deficiency is a genetic disorder that mainly affects RBCs, thus causing premature destruction of these cells called hemolysis [153]. Besides causing hemolytic anemia, G6PD has an evolutive advantage regarding the protection against malaria. A consequence of the reduced amount of functional G6PD makes difficult pathway for parasites to invade RBSc [154]. G6PD gene insufficiency is the most frequent in malaria endemic areas. When it comes to Mediterranean, the highest noted frequency of this gene is in Mediterranean parts of Africa, southern Europe and in the Middle East [153].

Interestingly, G6PD deficient patients in Africa, where this type of deficiency is endemic, have milder consequences as well as relatively higher enzyme activity in comparison with patients from Mediterranean and Asia [155].

G6PD deficiency gives especially high protection from *falciparum* malaria infection [156, 157]. Among more than 400 variants of G6PD that differs in biochemical characteristics, enzyme kinetics, physicochemical characteristics, and other parameters [158] is G6PD B+ which is the most common variant of this enzyme. G6PD B+ is used as standard for normal enzyme activity and electrophoretic mobility and, therefore, for identification of other variants. In the area of Mediterranean, special place belongs to G6PD Mediterranean variant [159] which has less than 10% of the enzyme activity of G6PD B+ while its electrophoretic mobility is similar to G6PD B+ [160]. Two-point mutations in gene for this enzyme were identified. One mutation is cytosine to thymine mutation at nucleotide number 563, which causes substitution of serine with phenylalanine [161]. At nucleotide number 1311, change of cytosine with thymine represents a silent mutation [162].

A research conducted by Barišić et al. in the Dalmatinian region of Croatia resulted in discovering a new variant of G6PD named G6PD Split [163]. Change of cytosine to guanine at nucleotide 1442 caused substitution of proline with arginine which led to moderate enzyme deficiency. Besides this novel variant of G6PD discovered in one patient, other 23 unrelated patients with low G6PD activity had five other well-known variants and three patients had uncharacterized forms of G6PD mutations. The most represented form found in nine patients was G6PD Cosenza. G6PD Cosenza was first found in Calabria region of southern Italy and represents the consequence of change of guanine into cytosine at nucleotide 1376. This substitution changes Arginine to Proline [164]. G6PD Cosenza mutation is severe G6PD deficiency frequently jointed with hemolysis.

Around 400 million people from all over the World carry at least one deficient variant of G6PD gene. The frequency of those mutations varies in different populations [165]. In Africans and Afro-Americans G6PD A- is the most common mutation which has a gene frequency of 11%. G6PD B (Mediterranean) is a more severe deficiency usually found in Mediterranean area. Since Mediterranean represents a large

**161**

Fya

*Adaptation to Mediterranea*

*DOI: http://dx.doi.org/10.5772/intechopen.94081*

**5.4 FY gene polymorphisms and malaria**

erythrocyte cell lineage [175, 176].

allele is fixed [186], while Fyb

advantage against *P. vivax* malaria [185].

phenotype [169].

region, the prevalence of this mutation varies from 2 to 20% in Greece, Turkey, and Italy, up to the 70% which is the prevalence characteristic for Kurdish Jews [165, 166].

In addition to the role it plays in transfusion incompatibility and hemolytic disease of newborns, Duffy Blood Group System is important in medicine due to its association with the invasion of RBCs by the parasite *P. vivax*. Outside Africa, *P. vivax* is the most widespread malaria parasite species, with 40% of cases in the Eastern Mediterranean [167]. Without Duffy antigens on their surface, RBCs are relatively

Fy5, and Fy6), out of which only Fy3 has a clinical significance. Duffy antigens are also receptors for chemicals secreted by blood cells during inflammation [169].

Duffy-Antigen Chemokine Receptor (DARC) is a glycosylated transmembrane protein receptor which, among other roles, serves as a receptor for *P. vivax*. DARK crosses the membrane seven times and has an extracellular epitope, N-terminal domain responsible for RBC invasion by *P. vivax* merozoites [170, 171]. Two exons (FyA and FyB) of FY gene are encoded by the co dominant FyA and FyB alleles located on human chromosome 1 [172]. The difference between these two alleles is a non-synonymous mutation, specifically substitution of guanine to adenine at nucleotide 125, which was enough to determine the two antithetical antigens [173]. Based on this variation, four phenotypes within Duffy Blood Group System were identified: Fy (a + b-), Fy (a-b+), Fy (a-b-) and Fy (a + b+) [174]. The nonfunctional allele Fy\*O is the consequence of a mutation in the gene promoter at −33 nucleotide that changed thymine to cytosine which abolish its expression in the

Individuals with Fy (a-b-) phenotype are resistant to *P. vivax* invasion [177]. This was shown in the study which included 11 volunteers. The individuals affected with malaria were Fy (a+) or Fy (b+). In the countries of West Africa, frequency of the Fy (a-b-) phenotype is a high while the incidence of *P. vivax* malaria is low [178]. Virtual absence of *P. vivax* malaria in populations with widespread DARC negativity is the proof of the substantial importance of the Duffy binding protein (DBP)–DARC interaction [179]. It is important to emphasize that Fy (a − b−) does not protect from *P. falciparum* which therefore can infect RBCs of any Duffy

While *P. falciparum* can enter human RBCs through series of receptors on their

antigens [169, 180]. Therefore, in the regions of Africa where Fy (a-b-) phenotype is stable within various ethnic groups, the transmission of *P. vivax* is not usual [181]. On the other hand, individuals with Fy (a-b+) or Fy (a + b-) genotypes that express half the level of Duffy antigens on RBCs compared to Fy (a-b-) homozygotes are less sensitive to blood stage infection by *P. vivax*. Therefore, parasitemia by *P. vivax* might be inhibited by total or partial restriction access of *P. vivax* to Duffy antigen [182, 183]. Phenotypic differences in susceptibility to malaria are the results of Fy gene polymorphism. Individuals that carry Duffy antigen-negative allele hidden within heterozygous genotype have significantly reduced adherence of the DBP ligand domain (DBPII) to erythrocytes [184]. On the other hand, people with Fya

notype have 30–80% lower risk of clinical *vivax* malaria, but not for falciparum malaria [185]. In the countries of Southeast Asia that are the source of *P. vivax*, the

Northern-central Europe. This kind of distribution of Fy alleles indicates a selective

is represented in the populations in North and

surface, RBCs invasion by *P. vivax* depends on an interaction with the Fya

, Fyb

, Fy3, Fy4,

or Fyb

phe-

resistant to *P. vivax* [168]. There are six types of Duffy antigens (Fya

*Genetic Variation*

**malaria**

mice and human sickle-cell traits [95].

RBCs from damage and early destruction [152].

of Africa, southern Europe and in the Middle East [153].

comparison with patients from Mediterranean and Asia [155].

of cytosine with thymine represents a silent mutation [162].

deficiency frequently jointed with hemolysis.

throughout hemoxygenase-1 [149]. The problem with these findings when translating into human populations is metabolic difference between sickling disorders of

**5.3 Glucose-6-phosphate dehydrogenase (G6PD) gene polymorphisms and** 

The Glucose-6-phosphate dehydrogenase (G6PD) is a "housekeeping" gene located on long (q) arm of the X chromosome at position 28 – Xq28 [150]. This gene encodes an enzyme named glucose-6-phosphate dehydrogenase that acts in almost all types of cells thus providing normal carbohydrates processing [151]. The most important role of G6PD is in RBCs, where this enzyme is involved in protection of

Glucose-6-phosphate dehydrogenase deficiency is a genetic disorder that mainly affects RBCs, thus causing premature destruction of these cells called hemolysis [153]. Besides causing hemolytic anemia, G6PD has an evolutive advantage regarding the protection against malaria. A consequence of the reduced amount of functional G6PD makes difficult pathway for parasites to invade RBSc [154]. G6PD gene insufficiency is the most frequent in malaria endemic areas. When it comes to Mediterranean, the highest noted frequency of this gene is in Mediterranean parts

Interestingly, G6PD deficient patients in Africa, where this type of deficiency is endemic, have milder consequences as well as relatively higher enzyme activity in

G6PD deficiency gives especially high protection from *falciparum* malaria infection [156, 157]. Among more than 400 variants of G6PD that differs in biochemical characteristics, enzyme kinetics, physicochemical characteristics, and other parameters [158] is G6PD B+ which is the most common variant of this enzyme. G6PD B+ is used as standard for normal enzyme activity and electrophoretic mobility and, therefore, for identification of other variants. In the area of Mediterranean, special place belongs to G6PD Mediterranean variant [159] which has less than 10% of the enzyme activity of G6PD B+ while its electrophoretic mobility is similar to G6PD B+ [160]. Two-point mutations in gene for this enzyme were identified. One mutation is cytosine to thymine mutation at nucleotide number 563, which causes substitution of serine with phenylalanine [161]. At nucleotide number 1311, change

A research conducted by Barišić et al. in the Dalmatinian region of Croatia resulted in discovering a new variant of G6PD named G6PD Split [163]. Change of cytosine to guanine at nucleotide 1442 caused substitution of proline with arginine which led to moderate enzyme deficiency. Besides this novel variant of G6PD discovered in one patient, other 23 unrelated patients with low G6PD activity had five other well-known variants and three patients had uncharacterized forms of G6PD mutations. The most represented form found in nine patients was G6PD Cosenza. G6PD Cosenza was first found in Calabria region of southern Italy and represents the consequence of change of guanine into cytosine at nucleotide 1376. This substitution changes Arginine to Proline [164]. G6PD Cosenza mutation is severe G6PD

Around 400 million people from all over the World carry at least one deficient variant of G6PD gene. The frequency of those mutations varies in different populations [165]. In Africans and Afro-Americans G6PD A- is the most common mutation which has a gene frequency of 11%. G6PD B (Mediterranean) is a more severe deficiency usually found in Mediterranean area. Since Mediterranean represents a large

**160**

region, the prevalence of this mutation varies from 2 to 20% in Greece, Turkey, and Italy, up to the 70% which is the prevalence characteristic for Kurdish Jews [165, 166].
