As we discussed above, in SCD a point mutation leads to a structural alteration in globin protein, that in absence of oxygen acquires the ability of forming long and multi-stranded fibers that alters the red blood cell shape increasing the destruction of these cells in spleen and liver and finally causing an important hemolytic anemia. In Thal, the point mutations or deletions observed in globin gene are responsible for altering the production of globin protein. In the most severe form of Thal, the thalassemia major, there is no production of this protein. As a result, the globin normally produces precipitates inside the red blood cells leading to an increased destruction of these cells causing hemolytic anemia, as also observed in SCD patients. In both cases, when a high expression of globin is observed, the patient has a very mild clinical disorder when compared to patients with a low expression of this protein. This better condition is due to the increment of production of fetal hemoglobin (HbF), as we have presented before as caused by the assembly of two chains with 2 chains (22). The clinical benefit induced by the increase of HbF in SCD patients has been repeatedly demonstrated by several authors. In this disease, the beneficial effects of HbF is due to its interaction with the HbS polypeptide, that form an asymmetric hybrid hemoglobin, referred to as HbF/S (2S). This molecul directly affects the stability of the sickle hemoglobin polymer, preventing its deleterious alteration in cell morphology. Moreover, the premature destruction of sickle erythrocytes increases the number of cells where HbF concentration is high (Pace 2007).

Several studies over the last 20 years attempted to identify pharmacological agents able to increase the HbF in these patients. Several compounds that broadly influence epigenetic modifications, including DNA methylation and histone deacetylation were tested.

In the beginning of 1990, the use of Hydroxyurea (HU) was proposed for the treatment of SCD patients. This compound was firstly synthesized in 1869 and is widely used as an antineoplastic drug mainly employed to traet myeloproliferative disorders. During the 90's

in the ndHPFH English type. In order to evaluate if the -195 mutation is able to increase the HbF *in vivo*, Cunha and colleagues in 2009 developed an *in vivo* study in which the -195 mutation was inserted into the globin gene promoter at a globin cluster cloned in a cosmid. This construction was inserted in a transgenic mouse and through RT-PCR and RNAse Protection Assay (RPA) the authors demonstrated that the -195 mutation, together with all the globin cluster is enough to raise levels of HbF in all phases of development of transgenic mice. This observation was supported by an increase in globin gene expression in yolk sac, fetal liver and other organs of these mice compared to expression in mice carrying the wild globin cluster. The results showed that the presence of the mutation -195 together with the entire globin cluster is sufficient to develop the phenotype of ndHPFH-B in mice (da Cunha et al. 2009). Even with these detailed studies, the complete mechanism involved in the

Understanding the mechanisms involved in these mutations may be important for the development of new therapies based on reactivation of globin gene which can assist in the

**7. Expression of gamma globin and the regulation of severity of sickle cell**

As we discussed above, in SCD a point mutation leads to a structural alteration in globin protein, that in absence of oxygen acquires the ability of forming long and multi-stranded fibers that alters the red blood cell shape increasing the destruction of these cells in spleen and liver and finally causing an important hemolytic anemia. In Thal, the point mutations or deletions observed in globin gene are responsible for altering the production of globin protein. In the most severe form of Thal, the thalassemia major, there is no production of this protein. As a result, the globin normally produces precipitates inside the red blood cells leading to an increased destruction of these cells causing hemolytic anemia, as also observed in SCD patients. In both cases, when a high expression of globin is observed, the patient has a very mild clinical disorder when compared to patients with a low expression of this protein. This better condition is due to the increment of production of fetal hemoglobin (HbF), as we have presented before as caused by the assembly of two chains with 2 chains (22). The clinical benefit induced by the increase of HbF in SCD patients has been repeatedly demonstrated by several authors. In this disease, the beneficial effects of HbF is due to its interaction with the HbS polypeptide, that form an asymmetric hybrid hemoglobin, referred to as HbF/S (2S). This molecul directly affects the stability of the sickle hemoglobin polymer, preventing its deleterious alteration in cell morphology. Moreover, the premature destruction of sickle erythrocytes increases the number of cells

Several studies over the last 20 years attempted to identify pharmacological agents able to increase the HbF in these patients. Several compounds that broadly influence epigenetic

In the beginning of 1990, the use of Hydroxyurea (HU) was proposed for the treatment of SCD patients. This compound was firstly synthesized in 1869 and is widely used as an antineoplastic drug mainly employed to traet myeloproliferative disorders. During the 90's

modifications, including DNA methylation and histone deacetylation were tested.

reactivation of the globin gene in the Brazilian type A ndHPFH is not elucidated.

treatment of hemoglobinopathies.

**disease and beta thalassemia**

where HbF concentration is high (Pace 2007).

several experiments were conducted mainly by the Multicenter Study of Hydroxyurea in Sickle Cell Anemia (MSH) and the results showed that HU were able to decrease the morbidity of SCA in adults, reducing the incidence of painful episodes and acute chest syndrome. These studies also revealed that this compound could be used to increase fetal hemoglobin (HbF) concentration therefore reducing the vaso-occlusive complications and diminishing hemolysis (Steinberg et al. 2003). The ability of HU in the generation of Nitric Oxide (NO) is reported as the main property which can partially explain the augment of globin production. In this sense, NO could act as a second messenger in a signaling cascade leading to the activation of several important genes. The results found in these first studies triggered other trials that used HU in Thal. Although the results were considered very promising, the augment of HbF in Thal was not observed in all patients (Lou et al. 2009).

To date, HU is the most widely used drug for adult SCD patients and is also highly used in thalassemia patients. Despite of several studies addressing this issue, the metabolic pathways and the set of genes expressed upon induction by HU are not completely known. Recently, some studies have evaluated patients and cultured cells before and after HU treatment in order to identify metabolic pathways and genes that could shed some light in the mechanism of action of this drug. In one of these studies conducted in 2006 by Costa et al, the mechanisms surrounding the in vivo action of HU were evaluated using a global gene expression analysis in cells obtained from the bone marrow of a SCD patient before and after HU treatment. The results suggested that HU induced significant changes in the gene expression pattern of SCD patient, mainly in global signal transduction pathways. This gene expression alteration may have a direct effect in the induction of globin genes and in genes associated with channel or pore class transporter. These pore transporter genes are thought to interfere in the hydratation status of cells, interfering with the rheological properties of them, what is very important in SCD (Costa et al. 2007). Having the same objectives and similar experimental design, another important study was conducted by Moreira et al (2007) using suppression subtractive hybridization (SSH) in HU-treated and -untreated reticulocytes of patients with SCD . In this report, similar pathways initially described by Costa were also identified after HU treatment. Additionally were also found some genes involved in chromatin modifications like *SUDS3*, *FZD5* and *PHC3*. Although these studies have contributed to elucidate the transcriptional response influenced by HU, the molecular mechanisms involved in HU treatment remain unclear (Moreira et al. 2008).

After the demonstration that some chemical agents are able to increase HbF concentration many efforts have been developed in order to find other inducers or a combination of agents able in ameliorate the symptoms of SCD and Thal. Several potential therapeutic agents and its combination have been investigated to use in these hemoglobinopathies. Table 2 shows some clinical trials used in Thal patients and their hematologic responses.

Despite the clear beneficial effects observed after HU treatment, the use of this pharmacological agent is limited since long-term treatment has been associated with several deleterious effects. In order to diminish the HU treatment effects many efforts are made to find new therapeutic alternatives to treat SCD symptoms without side effects. Recently, dos Santos et al designed a new molecule by molecular hybridization combining portions of thalidomide and HU and tested as potential use in the oral treatment of sickle cell disease symptoms. The results showed that this molecule was able to increase the levels of NO

Correlations with Point Mutations and Severity of Hemolitic Anemias: The Example of

the treatment of patients SCD or Thal (Cao et al. 2011; Ngo et al. 2011).

as also involved in the reactivation of globin (de Andrade et al. 2006).

et al. 2008; Xu et al. 2010; Jawaid et al. 2011; Wilber et al. 2011).

globin genes (Weiss et al. 1995; Weiss et al. 1997).

**thalassemia diseases**

Hereditary Persistence of Fetal Hemoglobin with Sickle Cell Anemia and Beta Thalassemia 247

It has been a very well established concept the benefits in the increment of -globin levels in

Therefore the development of new therapies targeting the increment of globin production can become possible through the identification of the molecular mechanisms involved in fetal globin gene silencing and its reactivation which needs to be known in depth. An excellent model to study these mechanisms is the HPFH, a disorder in which the globin production is not silenced, as described above. The mechanisms involved in the reactivation of globin have been scrutinized in several studies. Olave et al. studied the proteins able to bind at the globin promoter in the presence of a point mutation that leads to an English form of HPFH (–198TC). The results showed that a protein complex binds to this region and is able to increase the HbF production. The authors suggested that DNMT1 protein, one member of this putative complex, acts as a chromatin remodeling protein opening the chromatin and triggering the globin expression (Olave et al. 2007). In another interesting study developed by Andrade et al., the pattern of gene expression was evaluated in reticulocytes of an individual with the deletional form of PHHF. In this study the authors also identified the up-regulation of chromatin remodeling proteins (ARID1B and TSPYL1 )

Despite the studies with HPFH, several authors have described the interaction of genes with the activation or silencing of globin promoter. Among these genes *EKLF* has been pointed out in numerous reports as the main responsible for the regulation of globin expression. The increase in the expression of this gene occurs in accordance with the increase of globin producing and globin silencing. The main function of this protein seems to be in the activation of another gene called *BCL11A* whose gene product is able to bind at globin genes promoter repressing its transcription (Figure 10). The control of EKLF and BCL11A production could be further explored as a therapeutical strategy to increase the production of HbF and ameliorate hemoglobin disorders such as Thal and sickle cell disease (Sankaran

Another important contribution aiming to clarify the understanding of -globin genes silencing is the results found in the analysis of the hematopoietic transcription factor GATA-1. This gene, as we discussed before is a zinc finger protein that binds to WGATAR elements located mainly in the regulatory regions of erythroid genes, including the and

A direct interaction between GATA1 and -globin silencing was highlighted in two different studies. In the first one, the authors showed the importance of a 352-bp region harboring a conserved GATA element at position -566 5'of the *HBG1* is required for -globin silencing in transgenic mice. In the second report, the authors correlated a mutation found in GATA site in the *HBG2* gene with the increase in globin expression in a family with HPFH. These results showed that GATA-1 can act as a repressor of globin gene despite its widely described role in the literature as an activator of and globin genes. The mechanism whereby GATA-1 exerts these distinct functions remains unresolved. Some evidences

**8. Molecular biology and the identification of targets to treat SCD and beta**

donor, as observed in HU treatment, and also demonstrated that different from what is observed in HU treatment, this new molecule had analgesic and anti-inflamatory properties, reducing the levels of tumor necrosis factor (TNF ) thus representing an alternative approach to HU treatment (dos Santos et al. 2011).

Table 2. Hematologic Responses to Fetal Globin Gene Inducers or Erithropoietin (EPO) in Thal Patients (Perrine 2005).

donor, as observed in HU treatment, and also demonstrated that different from what is observed in HU treatment, this new molecule had analgesic and anti-inflamatory properties, reducing the levels of tumor necrosis factor (TNF ) thus representing an alternative

Syndromes

Thalassemia Intermedia

Thalassemia Intermedia HbE/βtalassemia

Intermedia and

Thalassemia Intermedia and

Thalassemia Intermedia and

Thalassemia Intermedia and

Thalassemia Intermedia

Intermedia HbE/βtalassemia

Table 2. Hematologic Responses to Fetal Globin Gene Inducers or Erithropoietin (EPO) in

HU+SPB As Above β Lepore 3 [2 – 4] 2/2

Major

Major

Major

Major

HbE/βtalassemia

? HDACi effect Thalassemia

Increase in Total Hb (g/dl) (range)

20 [1-3.3] 27 [2.2 – 3.2] 0.6 [0 – 1.7] 1.0 0.9

Increase in HbF, Reduced Transfusion Requeriments

1.6 [0.7 – 3.8] 4/6

1.2 1/5

2 [1 – 3] 2.5 2 Reduced Transfusion Requeriments

1.7 [0.5 – 4]

2.5 [1.5 – 4] 1/1

Responses

1/1 3/3

3/3

2.1 [1.2 – 2.8] 4/8 untransfused

2.7 [0 .6 – 5] 7/10 (AB +/- EPO)

6/10 2/4

5 – 7/10 4/4 8/10 3/26 5/10

2/2 (β globin) 11/13 3/8 7/19

approach to HU treatment (dos Santos et al. 2011).

Terapeutic Agent Action Thalassemia

+/- cytotoxicity

Cytotoxicity and erythroid regeration →high- F BFU- E

Activates γ globin gene promoter, (SSP binding), (? HDACi effect)

gene promoter

Proliferation, promotes erythroid cell survival

Erythropoiesis

HU+EPO As Above Thalassemia

5- Azacytidine Hypomethylation

Isobutyramide Activates γ globin

EPO Stimulates Erythroid

Darbepoietin Stimulates

Thal Patients (Perrine 2005).

Hydroxyurea (HU)

Sodium Phenybutyrate

and AB+ Erithropoietin (EPO)

Arginine Butyrate

(SPB)

#### **8. Molecular biology and the identification of targets to treat SCD and beta thalassemia diseases**

It has been a very well established concept the benefits in the increment of -globin levels in the treatment of patients SCD or Thal (Cao et al. 2011; Ngo et al. 2011).

Therefore the development of new therapies targeting the increment of globin production can become possible through the identification of the molecular mechanisms involved in fetal globin gene silencing and its reactivation which needs to be known in depth. An excellent model to study these mechanisms is the HPFH, a disorder in which the globin production is not silenced, as described above. The mechanisms involved in the reactivation of globin have been scrutinized in several studies. Olave et al. studied the proteins able to bind at the globin promoter in the presence of a point mutation that leads to an English form of HPFH (–198TC). The results showed that a protein complex binds to this region and is able to increase the HbF production. The authors suggested that DNMT1 protein, one member of this putative complex, acts as a chromatin remodeling protein opening the chromatin and triggering the globin expression (Olave et al. 2007). In another interesting study developed by Andrade et al., the pattern of gene expression was evaluated in reticulocytes of an individual with the deletional form of PHHF. In this study the authors also identified the up-regulation of chromatin remodeling proteins (ARID1B and TSPYL1 ) as also involved in the reactivation of globin (de Andrade et al. 2006).

Despite the studies with HPFH, several authors have described the interaction of genes with the activation or silencing of globin promoter. Among these genes *EKLF* has been pointed out in numerous reports as the main responsible for the regulation of globin expression. The increase in the expression of this gene occurs in accordance with the increase of globin producing and globin silencing. The main function of this protein seems to be in the activation of another gene called *BCL11A* whose gene product is able to bind at globin genes promoter repressing its transcription (Figure 10). The control of EKLF and BCL11A production could be further explored as a therapeutical strategy to increase the production of HbF and ameliorate hemoglobin disorders such as Thal and sickle cell disease (Sankaran et al. 2008; Xu et al. 2010; Jawaid et al. 2011; Wilber et al. 2011).

Another important contribution aiming to clarify the understanding of -globin genes silencing is the results found in the analysis of the hematopoietic transcription factor GATA-1. This gene, as we discussed before is a zinc finger protein that binds to WGATAR elements located mainly in the regulatory regions of erythroid genes, including the and globin genes (Weiss et al. 1995; Weiss et al. 1997).

A direct interaction between GATA1 and -globin silencing was highlighted in two different studies. In the first one, the authors showed the importance of a 352-bp region harboring a conserved GATA element at position -566 5'of the *HBG1* is required for -globin silencing in transgenic mice. In the second report, the authors correlated a mutation found in GATA site in the *HBG2* gene with the increase in globin expression in a family with HPFH. These results showed that GATA-1 can act as a repressor of globin gene despite its widely described role in the literature as an activator of and globin genes. The mechanism whereby GATA-1 exerts these distinct functions remains unresolved. Some evidences

Correlations with Point Mutations and Severity of Hemolitic Anemias: The Example of

human -globin *in vivo* (Miccio et al. 2010; Xu et al. 2010).

as therapeutical agents to treat SCD and Thal.

**9. Conclusion**

Hereditary Persistence of Fetal Hemoglobin with Sickle Cell Anemia and Beta Thalassemia 249

expression of -globin gene and this interaction was not required for the repression of

Despite several attempts, the complete molecular mechanism involved in the switching of globins and the reactivation of globin remains to be elucidated. Is clear that chromatin remodeling in the globin region is the main process whereby HbF reactivation occurs, once the expression of genes involved in this process are found in most of gene expression profiling studies using HPFH as a model. However, additional studies are necessary in order to identify a complete overview of metabolic pathways able to increase the HbF production and raise the possibility for the identification of new targets that could be used

The mechanisms underlying the hemolytic anemia are very complex and the prognosis of this group of diseases is greatly influenced by the gain or loss-of function in key genes involved in the regulation of synthesis and destruction of globin proteins. Several molecular events can be linked to the alteration in gene function. In this chapter, we focused on point mutations interfering in the nature and the severity of SCD and Thal. The comprehension of the molecular basis and the identification of proteins involved in the onset and development of a disease can enable the development of drugs specially designed to targeted therapy. There are some well succeeded examples of the use of molecular biology to design drugs to treat a specific disease. One of the most classical examples comes from the study of another hematological disease: the Chronic Myeloid Leukemia (CML). The development of this CML is characterized by the expression of a BCR-ABL protein produced by a genetic translocation event. Using molecular biology approaches, the BCR-ABL inhibitor imatinib was the first molecular targeted cancer therapy designed to bind inactive the translocated product of BCR-ABL protein. The employment of such molecular approach in the field of hemolytic anemias can open up new avenues of investigation taking advantage of the large scale studies to found new point mutation in genes involved in these diseases and correlate with the prognosis of each patient. Although many decades of efforts have been devoted to the mechanism elucidation of these diseases, much more remains to be done concerning to the identification of new point mutations conferring function on the disease progression,. In this context, the current scenery worldwide seen to be very prolific due to the recent large investments in the genomic revolution of next generation sequencing approaches. These approachs can be easely exploited through the large scale transcriptome (RNAseq) dissecation of hemolytic anemias. It may provide interesting links between the expressed genes by a specific disease, such as SCD and thal, and help in better understanding the association between the mutations and the metabolic pathways activated in each case. Thus the dissection of the involved metabolic pathways can identify key genes responsible for the disease development and progression. In addition, although important studies using molecular biology techniques highlighted the function of several genes involved in the hemolytic anemias, several questions are currently partially understood. For example, what are the transcriptome and proteome difference between patients that have the same mutations causing Thal but that have different phenotypes (major and intermedia)? The answer for this question is a promising strategy to identify new genes

Fig. 10. EKLF levels coordinate the hemoglobin switching. The activation of globin genes is dependent of the production of EKLF protein. A low level of EKLF is observed during the fetal stage. Since EKLF is essential for the activation of BCL11A, low levels of this gene is also reduced. The lowly expressed BCL11A is unable to repress γ-globin genes and EKLF with the recruitment of the NFE4 and CP2, preferentially interacts with the γ-globin promoters. This interaction stimulates the assembly of LCR elements activating the production of γ-globin protein and consequently the HbF formation. In the adult stage, high levels of EKLF is observed. The increase of this protein promotes an overexpression of BCL11A ultimately repressing the γ-globin genes under these circunstances. In this situation the EKLF binds preferentially to the β-globin promoter activating the β-globin gene with the recruitment of the LCR. This figure was adapted from (Wilber et al. 2011).

showed that this repression mechanisms could occurs via two other genes: *FOG-1* and *NuRD*. FOG-1 is also a zinc finger protein that binds to GATA-1 acting as an important player in both activation and repression of genes modulated by GATA-1. FOG-1 interacts with several protein complexes, including NuRD (nucleosome remodeling and histone deacetylase), that is a co-repressor complex that interacts with several N-terminal motifs found in other transcriptional repressors, for instance, the BCL11A gene. In an experiment using transgenic mice bearing three different mutations that completely abrogate the binding between NuRD and FOG-1, the authors showed that animals homozygous for these mutations were anemic and displayed defects in platelet formation. These results indicate that this interaction is essential for normal erythroid and megakaryocyte development. The same authors concluded that the FOG-1/NuRD interaction is indispensable for the expression of -globin gene and this interaction was not required for the repression of human -globin *in vivo* (Miccio et al. 2010; Xu et al. 2010).

Despite several attempts, the complete molecular mechanism involved in the switching of globins and the reactivation of globin remains to be elucidated. Is clear that chromatin remodeling in the globin region is the main process whereby HbF reactivation occurs, once the expression of genes involved in this process are found in most of gene expression profiling studies using HPFH as a model. However, additional studies are necessary in order to identify a complete overview of metabolic pathways able to increase the HbF production and raise the possibility for the identification of new targets that could be used as therapeutical agents to treat SCD and Thal.

### **9. Conclusion**

248 Point Mutation

Fig. 10. EKLF levels coordinate the hemoglobin switching. The activation of globin genes is dependent of the production of EKLF protein. A low level of EKLF is observed during the fetal stage. Since EKLF is essential for the activation of BCL11A, low levels of this gene is also reduced. The lowly expressed BCL11A is unable to repress γ-globin genes and EKLF with the recruitment of the NFE4 and CP2, preferentially interacts with the γ-globin promoters. This interaction stimulates the assembly of LCR elements activating the

production of γ-globin protein and consequently the HbF formation. In the adult stage, high levels of EKLF is observed. The increase of this protein promotes an overexpression of BCL11A ultimately repressing the γ-globin genes under these circunstances. In this situation the EKLF binds preferentially to the β-globin promoter activating the β-globin gene with the

showed that this repression mechanisms could occurs via two other genes: *FOG-1* and *NuRD*. FOG-1 is also a zinc finger protein that binds to GATA-1 acting as an important player in both activation and repression of genes modulated by GATA-1. FOG-1 interacts with several protein complexes, including NuRD (nucleosome remodeling and histone deacetylase), that is a co-repressor complex that interacts with several N-terminal motifs found in other transcriptional repressors, for instance, the BCL11A gene. In an experiment using transgenic mice bearing three different mutations that completely abrogate the binding between NuRD and FOG-1, the authors showed that animals homozygous for these mutations were anemic and displayed defects in platelet formation. These results indicate that this interaction is essential for normal erythroid and megakaryocyte development. The same authors concluded that the FOG-1/NuRD interaction is indispensable for the

recruitment of the LCR. This figure was adapted from (Wilber et al. 2011).

The mechanisms underlying the hemolytic anemia are very complex and the prognosis of this group of diseases is greatly influenced by the gain or loss-of function in key genes involved in the regulation of synthesis and destruction of globin proteins. Several molecular events can be linked to the alteration in gene function. In this chapter, we focused on point mutations interfering in the nature and the severity of SCD and Thal. The comprehension of the molecular basis and the identification of proteins involved in the onset and development of a disease can enable the development of drugs specially designed to targeted therapy. There are some well succeeded examples of the use of molecular biology to design drugs to treat a specific disease. One of the most classical examples comes from the study of another hematological disease: the Chronic Myeloid Leukemia (CML). The development of this CML is characterized by the expression of a BCR-ABL protein produced by a genetic translocation event. Using molecular biology approaches, the BCR-ABL inhibitor imatinib was the first molecular targeted cancer therapy designed to bind inactive the translocated product of BCR-ABL protein. The employment of such molecular approach in the field of hemolytic anemias can open up new avenues of investigation taking advantage of the large scale studies to found new point mutation in genes involved in these diseases and correlate with the prognosis of each patient. Although many decades of efforts have been devoted to the mechanism elucidation of these diseases, much more remains to be done concerning to the identification of new point mutations conferring function on the disease progression,. In this context, the current scenery worldwide seen to be very prolific due to the recent large investments in the genomic revolution of next generation sequencing approaches. These approachs can be easely exploited through the large scale transcriptome (RNAseq) dissecation of hemolytic anemias. It may provide interesting links between the expressed genes by a specific disease, such as SCD and thal, and help in better understanding the association between the mutations and the metabolic pathways activated in each case. Thus the dissection of the involved metabolic pathways can identify key genes responsible for the disease development and progression. In addition, although important studies using molecular biology techniques highlighted the function of several genes involved in the hemolytic anemias, several questions are currently partially understood. For example, what are the transcriptome and proteome difference between patients that have the same mutations causing Thal but that have different phenotypes (major and intermedia)? The answer for this question is a promising strategy to identify new genes

Correlations with Point Mutations and Severity of Hemolitic Anemias: The Example of

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#### **10. Acknowledgment**

The authors would like to thank fundings from the Brazilian agencies FAPESP (Fundação de Amparo à Pesquisa do Estado de São Paulo) and CAPES (Coordenação de Aperfeiçoamento de Pessoal de Nível Superior)

#### **11. References**

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The authors would like to thank fundings from the Brazilian agencies FAPESP (Fundação de Amparo à Pesquisa do Estado de São Paulo) and CAPES (Coordenação de Aperfeiçoamento

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Blobel, G. A., C. A. Sieff and S. H. Orkin (1995). "Ligand-dependent repression of the

Cao, A., P. Moi and R. Galanello (2011). "Recent advances in beta-thalassemias." *Pediatr Rep*

Carlson, J., G. B. Nash, V. Gabutti, F. al-Yaman and M. Wahlgren (1994). "Natural protection

Costa, F. C., A. F. da Cunha, A. Fattori, T. de Sousa Peres, G. G. Costa, T. F. Machado, D.

Costa, F. F., M. A. Zago, G. Cheng, J. F. Nechtman, T. A. Stoming and T. H. Huisman

da Cunha, A. F., A. F. Brugnerotto, M. A. Corat, E. E. Devlin, A. P. Gimenes, M. B. de Melo,

erythroid transcription factor GATA-1." *Mol Cell Biol* 16(4): 1687-94.

intragenic SNP haplotypes and mutations of the beta globin gene in a Turkish

protein cooperates with transcription factor GATA-1 and is required for erythroid

erythroid transcription factor GATA-1 by the estrogen receptor." *Mol Cell Biol* 15(6):

against severe Plasmodium falciparum malaria due to impaired rosette formation."

M. de Albuquerque, S. Gambero, C. Lanaro, S. T. Saad and F. F. Costa (2007). "Gene expression profiles of erythroid precursors characterise several mechanisms of the action of hydroxycarbamide in sickle cell anaemia." *Br J*

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differentiation." *Proc Natl Acad Sci U S A* 95(5): 2061-6.

Cao, A. and R. Galanello (2010). "Beta-thalassemia." *Genet Med* 12(2): 61-76.

**10. Acknowledgment**

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Shapiro, B. S., D. F. Dinges, E. C. Orne, N. Bauer, L. B. Reilly, W. G. Whitehouse, K. Ohene-

Stamatoyannopoulos, G. (2005). "Control of globin gene expression during development and

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**11**

*Russia*

**Point Mutations That Reduce Erythrocyte**

Oxygen transport is a primary goal of erythrocytes. The high maintenance of oxygen in erythrocytes defines high speed of formation of active forms of oxygen – superoxide (O2⎯), a hydrogen peroxide (H2O2) and a hydroxyl radical (·OH). A constant source of active forms of oxygen in erythrocytes is hemoglobin oxidation in a methemoglobin with formation of superoxide (O2⎯). Therefore erythrocytes should have a powerful antioxidant system, which prevents the toxic action of active forms of oxygen on hemoglobin and erythrocyte membrane. Mature erythrocytes have neither cytoplasmic organelles nor a nucleus and consequently are not capable to synthesize proteins and lipids, to carry out oxidative phosphorylation or to maintain tricarboxylic acid cycle reactions. The energy of erythrocytes comes for the most part from anaerobic glycolysis – via the Embden-Meyerhof-Parnas pathway (EMP pathway). Thus, glucose catabolism provides preservation of structure and function of hemoglobin, integrity of an erythrocyte membrane and formation of energy for the work of ionic pumps. Anaerobic glycolysis in itself is a power-consuming process. Glucose arrives in erythrocytes by the facilitated diffusion by glucose transporter type 1. Hexokinase is the first enzyme of EMP pathway, it provides glucose phosphorylation. Further during consecutive reactions with participation of glucose-6-phosphate isomerase, phosphofructokinase, aldolase, glyceraldehydes 3-phosphate dehydrogenase, phosphoglycerate kinase, phosphoglycerate mutase, enolase, pyruvate kinase one molecule of glucose gives 4 molecules of adenosine triphosphate (ATP) and 2 molecules of restored nicotinamide adenine dinucleotide (NADH), and at the same time, 2 ATP molecules are spent at the initial stage of EMP pathway. A certain quantity of glucose with formation of restored compounds – glutathione (GSH) and nicotinamide adenine dinucleotide phosphate (NADPH) is taken away through pentose phosphate pathway (aerobic glycolysis). Glucose-6-phosphate dehydrogenase and 6-phosphogluconate dehydrogenase provide the stages of pentose phosphate pathway. The hydroxyl radical, the most active component of oxidative stress, is neutralized by GSH. Methemoglobin reductase restores a methemoglobin into hemoglobin, NADPH being the donor of hydrogen, which is formed in EMP pathway and NADPH is in its turn formed in pentose phosphate pathway. Superoxide dismutase 1 enzyme contributes superoxide (O2⎯) to turn into hydrogen peroxide. The hydrogen peroxide is destroyed by catalase and glutathione peroxidase, GSH being the donor of hydrogen. Peroxiredoxin 2 is an antioxidant enzyme that uses cystein residues to

**1. Introduction**

**Resistance to Oxidative Stress**

Dmitriy Volosnikov and Elena Serebryakova

*The Chelyabinsk State Medical Academy,*

Xu, J., V. G. Sankaran, M. Ni, T. F. Menne, R. V. Puram, W. Kim and S. H. Orkin (2010). "Transcriptional silencing of {gamma}-globin by BCL11A involves long-range interactions and cooperation with SOX6." *Genes Dev* 24(8): 783-98.

### **Point Mutations That Reduce Erythrocyte Resistance to Oxidative Stress**

Dmitriy Volosnikov and Elena Serebryakova *The Chelyabinsk State Medical Academy, Russia*

#### **1. Introduction**

254 Point Mutation

Xu, J., V. G. Sankaran, M. Ni, T. F. Menne, R. V. Puram, W. Kim and S. H. Orkin (2010).

interactions and cooperation with SOX6." *Genes Dev* 24(8): 783-98.

"Transcriptional silencing of {gamma}-globin by BCL11A involves long-range

Oxygen transport is a primary goal of erythrocytes. The high maintenance of oxygen in erythrocytes defines high speed of formation of active forms of oxygen – superoxide (O2⎯), a hydrogen peroxide (H2O2) and a hydroxyl radical (·OH). A constant source of active forms of oxygen in erythrocytes is hemoglobin oxidation in a methemoglobin with formation of superoxide (O2⎯). Therefore erythrocytes should have a powerful antioxidant system, which prevents the toxic action of active forms of oxygen on hemoglobin and erythrocyte membrane. Mature erythrocytes have neither cytoplasmic organelles nor a nucleus and consequently are not capable to synthesize proteins and lipids, to carry out oxidative phosphorylation or to maintain tricarboxylic acid cycle reactions. The energy of erythrocytes comes for the most part from anaerobic glycolysis – via the Embden-Meyerhof-Parnas pathway (EMP pathway). Thus, glucose catabolism provides preservation of structure and function of hemoglobin, integrity of an erythrocyte membrane and formation of energy for the work of ionic pumps. Anaerobic glycolysis in itself is a power-consuming process. Glucose arrives in erythrocytes by the facilitated diffusion by glucose transporter type 1. Hexokinase is the first enzyme of EMP pathway, it provides glucose phosphorylation. Further during consecutive reactions with participation of glucose-6-phosphate isomerase, phosphofructokinase, aldolase, glyceraldehydes 3-phosphate dehydrogenase, phosphoglycerate kinase, phosphoglycerate mutase, enolase, pyruvate kinase one molecule of glucose gives 4 molecules of adenosine triphosphate (ATP) and 2 molecules of restored nicotinamide adenine dinucleotide (NADH), and at the same time, 2 ATP molecules are spent at the initial stage of EMP pathway. A certain quantity of glucose with formation of restored compounds – glutathione (GSH) and nicotinamide adenine dinucleotide phosphate (NADPH) is taken away through pentose phosphate pathway (aerobic glycolysis). Glucose-6-phosphate dehydrogenase and 6-phosphogluconate dehydrogenase provide the stages of pentose phosphate pathway. The hydroxyl radical, the most active component of oxidative stress, is neutralized by GSH. Methemoglobin reductase restores a methemoglobin into hemoglobin, NADPH being the donor of hydrogen, which is formed in EMP pathway and NADPH is in its turn formed in pentose phosphate pathway. Superoxide dismutase 1 enzyme contributes superoxide (O2⎯) to turn into hydrogen peroxide. The hydrogen peroxide is destroyed by catalase and glutathione peroxidase, GSH being the donor of hydrogen. Peroxiredoxin 2 is an antioxidant enzyme that uses cystein residues to

Point Mutations That Reduce Erythrocyte Resistance to Oxidative Stress 257

a causative deletion of 4 highly conserved amino acids (Q282\_S285del) in the pore region of GLUT1 was identified. Functional studies in Xenopus oocytes and human erythrocytes revealed that this mutation decreased glucose transport and caused a cation leak that alters intracellular concentrations of sodium, potassium, and calcium. In families where PED is combined with epilepsy, developmental delay, or migraine, but not with hemolysis or echinocytosis 2 GLUT1 mutations were identified (A275T, G314S) that decreased glucose transport but did not affect cation permeability (Weber еt al., 2008). The causative mutations for some forms of hereditary stomatocytosis have been found to result from mutations in SLC2A1, encoding GLUT1. Stomatocytosis was associated with a cold-induced cation leak, hemolytic anemia and hepatosplenomegaly but also with cataracts, seizures, mental

Hexokinase (HK) catalyses the phosphorylation of glucose to glucose-6-phosphate using adenosine triphosphate as a phosphoryl donor. The four isozymes of the HK family (HK1, HK2, HK3, and glucokinase) contribute to commit glucose to the glycolytic pathway, each of which is encoded by a separate gene. The predominant HK1 isozyme is expressed in the vast majority of cells and tissues, including cells that are strictly dependent on glucose uptake for their metabolic needs. While most tissues express more than one HK isozyme, erythrocytes glucose metabolism only depends on HK1 activity. HK1 is one of the ratelimiting enzymes in erythrocytes glycolysis. Gene structure and exon-intron organization of the HK1 gene have been elucidated from a sequence of three contiguous genomic clones localized at human chromosome 10. The sequence spans about 131 kb, and consists of 25 exons, which include 6 testis- and 1 erythroid-specific exons. The HK1 and erythroidspecific HK-R transcripts being produced by using two distinct promoters. Thus, the first and second exons are specifically utilized for the erythroid-specific HK-R and ubiquitously expressed HK1 isozymes respectively (Kanno 2000; Murakami еt al., 2002; van Wijk at al., 2003; Bonnefond еt al., 2009). In humans, mutations including nonsynonymous substitutions in the active site of HK1 and intragenic deletions have been shown to cause HK1 enzymatic deficiency associated with autosomal recessive severe nonspherocytic hemolytic anemia (Bonnefond еt al., 2009). Mutation affecting the substrate affinites of the enzyme, regulatory properties, heat stability have been described (Rijksen еt al., 1983; Magnani еt al., 1985). HK deficiency is a very rare disease with a clinical phenotype of hemolysis. PCR amplification and sequence of the cDNA in patients with HK deficiency revealed the presence of a deletion and of a single nucleotide substitution, both in heterozygous form. In particular, the deletion, 96 bp long, concerns nucleotides 577 to 672 in the HK cDNA sequence and was not found in the cDNAs of 14 unrelated normal subjects. The sequence of the HK allele without deletion showed a single nucleotide substitution from T to C at position 1667 which causes the amino acid change from Leu529 to Ser (Bianchi еt al., 1995). The T1667→C substitution, causing the amino acid change Leu529→Ser, is responsible for the complete loss of the hexokinase catalytic activity, while the 96 bp deletion causes a drastic reduction of the hexokinase activity (Bianchi еt al., 1997). A homozygous missense mutation in exon 15 (2039C→G, HK Utrecht) of HK1, the gene that encodes red blood cell-specific hexokinase-R,

retardation and movement disorder (Flatt еt al., 2011).

**2.2 Point mutation in glycolysis enzymes**

**2.2.1 Point mutation in hexokinase**

decompose peroxides. Peroxiredoxin 2 is the third most abundant protein in erythrocytes, and competes effectively with catalase and glutathione peroxidase to scavenge low levels of hydrogen peroxide, including that derived from hemoglobin autoxidation. GSH reductase restores oxidized GSH at the expense of NADPH energy. The final step in GSH synthesis is catalysed by the glutathione synthetase. Thus, resistance of erythrocytes to oxidative stress will depend on the activity of glucose transporter type 1, glycolysis enzymes, glutathione synthetase, glutathione reductase, glutathione peroxidase, peroxiredoxin 2, superoxide dismutase 1, catalase and nucleotide metabolism. Activation of oxidative stress occurs in case of infection, hypoxic ischemia, acidosis, effect of some medications and toxins. Low resistance of erythrocytes to oxidative stress leads to hemoglobin precipitation and erythrocytes hemolysis. Thus, erythrocytes become sources of active forms of oxygen. Oxidative stress has been implicated in many human diseases. Activity of erythrocyte antioxidant enzymes is closely studied to reveal oxidative stress status in various pathological conditions.

Herein we describe the recent updates regarding point mutations, which contribute to the decrease of antioxidant protection of erythrocytes.

#### **2. Point mutations in proteins and enzymes providing a metabolism of erythrocytes**

#### **2.1 Point mutation in glucose transporter GLUT 1**

GLUT1 was the first glucose transporter isoform to be identified, and is one of 13 proteins that comprise the human equilibrative glucose transporter family. GLUT1 is a membranespanning glycoprotein of 492 amino acids, containing 12 transmembrane domains with both *N*- and *C-*termini located in cytosol, and its gene being located on chromosome 1 (1p35-31.3) is composed of ten exons and nine introns. GLUT1 is expressed at the highest levels in the plasma membranes of proliferating cells forming the early developing embryo, in cells forming the blood-tissue barriers, in human erythrocytes and astrocytes, and in cardiac muscle (Carruthers еt al., 2009). Heterozygous mutations in the GLUT1 gene have been reported in sporadic patients and results in autosomal dominant pedigrees. Expression of mutant transporters resulted in a significant decrease in transport activity of GLUT1. Impaired glucose transport across brain tissue barriers is reflected by hypoglycorrhachia and results in epilepsy, mental retardation and motor disorders. The first autosomal dominant missense mutation (G272A) has been reported within the human GLUT1 gene and wos shared by three affected family members. Substitution of glycine-91 by sitedirected mutagenesis with either aspartate or alanine was studied in oocytes. The data agree with 3-O-methyl-glucose uptake into patient erythrocytes and indicate that the loss of glycine rather than a hydrophilic side chain (Gly91→Asp) defines the functional consequences of this mutation. (Klepper еt al., 2001). Recently, mutations in GLUT1 gene have been identified as a cause in some patients with autosomal dominant paroxysmal exercise-induced dyskinesias (PED). PED are involuntary intermittent movements triggered by prolonged physical exertion. Some patients had a predating history of childhood absence epilepsy and a current history of hemiplegic migraine as well as a family history of migraine (Schneider еt al., 2009). In certain cases PED was accompanied by hemolytic anemia with echinocytosis, and altered erythrocyte ion concentrations. Using a candidate gene approach, a causative deletion of 4 highly conserved amino acids (Q282\_S285del) in the pore region of GLUT1 was identified. Functional studies in Xenopus oocytes and human erythrocytes revealed that this mutation decreased glucose transport and caused a cation leak that alters intracellular concentrations of sodium, potassium, and calcium. In families where PED is combined with epilepsy, developmental delay, or migraine, but not with hemolysis or echinocytosis 2 GLUT1 mutations were identified (A275T, G314S) that decreased glucose transport but did not affect cation permeability (Weber еt al., 2008). The causative mutations for some forms of hereditary stomatocytosis have been found to result from mutations in SLC2A1, encoding GLUT1. Stomatocytosis was associated with a cold-induced cation leak, hemolytic anemia and hepatosplenomegaly but also with cataracts, seizures, mental retardation and movement disorder (Flatt еt al., 2011).

#### **2.2 Point mutation in glycolysis enzymes**

#### **2.2.1 Point mutation in hexokinase**

256 Point Mutation

decompose peroxides. Peroxiredoxin 2 is the third most abundant protein in erythrocytes, and competes effectively with catalase and glutathione peroxidase to scavenge low levels of hydrogen peroxide, including that derived from hemoglobin autoxidation. GSH reductase restores oxidized GSH at the expense of NADPH energy. The final step in GSH synthesis is catalysed by the glutathione synthetase. Thus, resistance of erythrocytes to oxidative stress will depend on the activity of glucose transporter type 1, glycolysis enzymes, glutathione synthetase, glutathione reductase, glutathione peroxidase, peroxiredoxin 2, superoxide dismutase 1, catalase and nucleotide metabolism. Activation of oxidative stress occurs in case of infection, hypoxic ischemia, acidosis, effect of some medications and toxins. Low resistance of erythrocytes to oxidative stress leads to hemoglobin precipitation and erythrocytes hemolysis. Thus, erythrocytes become sources of active forms of oxygen. Oxidative stress has been implicated in many human diseases. Activity of erythrocyte antioxidant enzymes is closely studied to reveal oxidative stress status in various

Herein we describe the recent updates regarding point mutations, which contribute to the

GLUT1 was the first glucose transporter isoform to be identified, and is one of 13 proteins that comprise the human equilibrative glucose transporter family. GLUT1 is a membranespanning glycoprotein of 492 amino acids, containing 12 transmembrane domains with both *N*- and *C-*termini located in cytosol, and its gene being located on chromosome 1 (1p35-31.3) is composed of ten exons and nine introns. GLUT1 is expressed at the highest levels in the plasma membranes of proliferating cells forming the early developing embryo, in cells forming the blood-tissue barriers, in human erythrocytes and astrocytes, and in cardiac muscle (Carruthers еt al., 2009). Heterozygous mutations in the GLUT1 gene have been reported in sporadic patients and results in autosomal dominant pedigrees. Expression of mutant transporters resulted in a significant decrease in transport activity of GLUT1. Impaired glucose transport across brain tissue barriers is reflected by hypoglycorrhachia and results in epilepsy, mental retardation and motor disorders. The first autosomal dominant missense mutation (G272A) has been reported within the human GLUT1 gene and wos shared by three affected family members. Substitution of glycine-91 by sitedirected mutagenesis with either aspartate or alanine was studied in oocytes. The data agree with 3-O-methyl-glucose uptake into patient erythrocytes and indicate that the loss of glycine rather than a hydrophilic side chain (Gly91→Asp) defines the functional consequences of this mutation. (Klepper еt al., 2001). Recently, mutations in GLUT1 gene have been identified as a cause in some patients with autosomal dominant paroxysmal exercise-induced dyskinesias (PED). PED are involuntary intermittent movements triggered by prolonged physical exertion. Some patients had a predating history of childhood absence epilepsy and a current history of hemiplegic migraine as well as a family history of migraine (Schneider еt al., 2009). In certain cases PED was accompanied by hemolytic anemia with echinocytosis, and altered erythrocyte ion concentrations. Using a candidate gene approach,

**2. Point mutations in proteins and enzymes providing a metabolism of**

pathological conditions.

**erythrocytes**

decrease of antioxidant protection of erythrocytes.

**2.1 Point mutation in glucose transporter GLUT 1**

Hexokinase (HK) catalyses the phosphorylation of glucose to glucose-6-phosphate using adenosine triphosphate as a phosphoryl donor. The four isozymes of the HK family (HK1, HK2, HK3, and glucokinase) contribute to commit glucose to the glycolytic pathway, each of which is encoded by a separate gene. The predominant HK1 isozyme is expressed in the vast majority of cells and tissues, including cells that are strictly dependent on glucose uptake for their metabolic needs. While most tissues express more than one HK isozyme, erythrocytes glucose metabolism only depends on HK1 activity. HK1 is one of the ratelimiting enzymes in erythrocytes glycolysis. Gene structure and exon-intron organization of the HK1 gene have been elucidated from a sequence of three contiguous genomic clones localized at human chromosome 10. The sequence spans about 131 kb, and consists of 25 exons, which include 6 testis- and 1 erythroid-specific exons. The HK1 and erythroidspecific HK-R transcripts being produced by using two distinct promoters. Thus, the first and second exons are specifically utilized for the erythroid-specific HK-R and ubiquitously expressed HK1 isozymes respectively (Kanno 2000; Murakami еt al., 2002; van Wijk at al., 2003; Bonnefond еt al., 2009). In humans, mutations including nonsynonymous substitutions in the active site of HK1 and intragenic deletions have been shown to cause HK1 enzymatic deficiency associated with autosomal recessive severe nonspherocytic hemolytic anemia (Bonnefond еt al., 2009). Mutation affecting the substrate affinites of the enzyme, regulatory properties, heat stability have been described (Rijksen еt al., 1983; Magnani еt al., 1985). HK deficiency is a very rare disease with a clinical phenotype of hemolysis. PCR amplification and sequence of the cDNA in patients with HK deficiency revealed the presence of a deletion and of a single nucleotide substitution, both in heterozygous form. In particular, the deletion, 96 bp long, concerns nucleotides 577 to 672 in the HK cDNA sequence and was not found in the cDNAs of 14 unrelated normal subjects. The sequence of the HK allele without deletion showed a single nucleotide substitution from T to C at position 1667 which causes the amino acid change from Leu529 to Ser (Bianchi еt al., 1995). The T1667→C substitution, causing the amino acid change Leu529→Ser, is responsible for the complete loss of the hexokinase catalytic activity, while the 96 bp deletion causes a drastic reduction of the hexokinase activity (Bianchi еt al., 1997). A homozygous missense mutation in exon 15 (2039C→G, HK Utrecht) of HK1, the gene that encodes red blood cell-specific hexokinase-R,

Point Mutations That Reduce Erythrocyte Resistance to Oxidative Stress 259

(GPI) and neurotrophic activities, thereby leading to the observed clinical symptoms (GPI Homburg). Those alterations at the active site, however, that allow correct folding retain the neurotrophic properties of the molecule (GPI Calden) (Kugler, 1998). The similarity of the mutant enzymes to the allozymes found in human GPI deficiencies indicates the GPI deficient mouse mutants to be excellent models for the human disease (Padua et al., 1978; Pretsch et al., 1990). A heterozygous mouse mutant exhibiting approximately 50% of wildtype GPI activity. Biochemical and immunological studies revealed no differences in physicochemical, kinetic and immunological properties between the erythrocytic enzyme of heterozygous and wild-type mouse. The genetic and physiological analyses provided no indications for further altered traits in heterozygous animals including fertility, viability and several other traits. Homozygous null mutants died at an early post-implantation stage of embryogenesis (West et al., 1990; Merkle et al., 1992). Homozygous GPI deficiency in humans are responsible for chronic nonspherocytic hemolytic anemia. The homozygous missense A346H mutation replacement in cDNA position 1040G→A, which causes a loss of GPI capacity to dimerize, which renders the enzyme more susceptible to thermolability and produces significant changes in erythrocyte metabolism was described in patient with chronic nonspherocytic hemolytic anemia (Repiso et al., 2005). Biochemical and molecular genetic studies performed with the enzyme variants of GPI Zwickau and GPI Nordhorn showed that in both cases the simultaneous occurrence of a single amino acid substitution affecting the active site, together with a nonsense mutation leading to the loss of major parts of the enzyme probably explains the severe clinical course of the disease (Huppke et al., 1997). Molecular characteristics of erythrocytes GPI deficiency were described in Spanish patients with chronic nonspherocytic hemolytic anemia. Residual GPI activity in erythrocytes of around 7% (GPI-Catalonia), in an individual is homozygous for the missense mutation 1648A→G (Lys550→Glu) in exon 18 was described and residual activity in erythrocytes of around 20% (GPI-Barcelona), was found in a compound heterozygote for two different missense mutations: 341A→T (Asp113→Val) in exon 4 and 663T→G (Asn220→Lys) in exon 7. Molecular modeling using the human crystal structure of GPI as a model was performed to determine how these mutations could affect enzyme structure and function (Repiso et al., 2006). Chinese hamster (CHO) cell lines with ethylmethane sulfonate induced mutations in GPI and consequent loss of GPI activity have been reported. GPI activity was reduced by 87% in GroD1 isolated from this population. Expression cloning and sequencing of the cDNA obtained from GroD1 revealed a point mutation Gly189→ Glu. This resulted in a temperature sensitivity and severe reduction in the synthesis of glycerolipids due to a reduction in phosphatidate phosphatase (PAP). Overexpression of lipin 1 in the GPI-deficient cell line, GroD1 resulted in increased PAP activity, however it failed to restore glycerolipid biosynthesis. Fluorescent microscopy showed a failure of GPIdeficient cells to localize lipin 1α to the nucleus. Glucose-6-phosphate levels in GroD1 cells were 10-fold over normal. Lowering glucose levels in the growth medium partially restored glycerolipid biosynthesis and nuclear localization of lipin 1α. Thus, GPI deficiency results in an accumulation of glucose-6-phosphate, and possibly other glucose-derived metabolites, leading to activation of mTOR and sequestration of lipin 1 to the cytosol, preventing its proper functioning. These results may also help to explain neuromuscular symptoms associated with inherited GPI deficiency (Haller et al., 2010; 2011). GPI deficiency was found to be the cause of recurrent haemolytic crises that has required frequent blood transfusion.

in a patient previously diagnosed with hexokinase deficiency has been reported. The Thr680→Ser substitution predicted by this mutation affects a highly conserved residue in the enzyme's active site that interacts with phosphate moieties of adenosine diphosphate, adenosine triphosphate, and glucose-6-phosphate inhibitor (van Wijk еt al. 2003). On the paternal allele in a patient with chronic hemolysis two mutations in the erythroid-specific promoter of HKI: 373A→C and 193A→G were identified. Transfection of promoter reporter constructs showed that the 193A→G mutation reduced promoter activity to 8%. Hence, 193A→G is the first mutation reported to affect red blood cell-specific hexokinase specific transcription. On the maternal allele there was a missense mutation in exon 3: 278G→A, encoding an arginine to glutamine substitution at residue 93 (Arg93→Glu), affecting both hexokinase-1 and erytrocytes specific-hexokinase. This missense mutation was shown to compromise normal pre-mRNA processing. Reduced erythroid transcription of HK1 together with aberrant splicing of both hexokinase-1 and erytrocytes specific-hexokinase results in HK deficiency and mild chronic hemolysis (de Vooght еt al. 2009).

#### **2.2.2 Point mutation in glucose-6-phosphate isomerase**

Glucose-6-phosphate isomerase (GPI) catalyzes interconversion of glucose-6-phosphate and fructose 6-phosphate in the Embden-Meyerhof glycolytic pathway. GPI is an essential enzyme for carbohydrate metabolism in all tissues. In humans, the GPI gene locus is located on chromosome 19, and the gene spans more than 40 kb, including 18 exons and 17 introns. The cDNA sequence encodes 558 amino acid residues. The enzyme consists of two identical subunits. In mammals, GPI can also act as an autocrine motility factor, neuroleukin, and maturation factor. GPI deficiency is a well-known congenital autosomal recessive disorder with the typical manifestation of nonspherocytic hemolytic anemia of variable severity in humans. GPI deficiency is one of the most common cause of congenital nonspherocytic hemolytic anemia caused by deficiency of glycolytic enzymes, the commonest being deficiency of glucose-6-phosphate dehydrogenase and pyruvate kinase. Patients with inherited GPI deficiency present with nonspherocytic anemia of variable severity and with neuromuscular dysfunction. Mutations in the GPI gene usually have negative influences on catalytic parameters, particularly k(cat), as well as structure stability. Mutations at or close to the active site, including R273H, H389R, and S278L, cause great damage to the catalytic function, yet those at distance can still reduce the magnitude of k(cat). At the nucleotide level, 29 mutations have been reported. Mutations decrease the enzyme tolerance to heat by mechanisms of decreasing packing efficiency (V101M, T195I, S278L, L487F, L339P, T375R, I525T), weakening network bonding (R75G, R347C, R347H, R472H, E495K), increasing water-accessible hydrophobic surface (R83W), and destabilizing the ternary structure (T195I, R347C, R347H, and I525T). A300P, L339P, and E495K mutations may also negatively affect the protein folding efficiency (Merkle S. at al., 1993; Kanno H. at al., , 1996; Kugler W. at al., 2000; Haller J.F. еt al., 2009; Haller J.F., еt al., 2010). The neurologically affected patient (GPI Homburg) is compound heterozygous for a 59 A→C (H20P) and a 1016 T→C (L339P) exchange. Owing to the insertion of proline, the H20P and L339P mutations are likely to affect the folding and activity of the enzyme. Point mutations identified at 1166 A→G (H389R) and 1549 C→G (L517V), which are located at the subunit interface showed no neurological symptoms. Thus mutations that lead to incorrect folding destroy both catalytic

in a patient previously diagnosed with hexokinase deficiency has been reported. The Thr680→Ser substitution predicted by this mutation affects a highly conserved residue in the enzyme's active site that interacts with phosphate moieties of adenosine diphosphate, adenosine triphosphate, and glucose-6-phosphate inhibitor (van Wijk еt al. 2003). On the paternal allele in a patient with chronic hemolysis two mutations in the erythroid-specific promoter of HKI: 373A→C and 193A→G were identified. Transfection of promoter reporter constructs showed that the 193A→G mutation reduced promoter activity to 8%. Hence, 193A→G is the first mutation reported to affect red blood cell-specific hexokinase specific transcription. On the maternal allele there was a missense mutation in exon 3: 278G→A, encoding an arginine to glutamine substitution at residue 93 (Arg93→Glu), affecting both hexokinase-1 and erytrocytes specific-hexokinase. This missense mutation was shown to compromise normal pre-mRNA processing. Reduced erythroid transcription of HK1 together with aberrant splicing of both hexokinase-1 and erytrocytes specific-hexokinase

Glucose-6-phosphate isomerase (GPI) catalyzes interconversion of glucose-6-phosphate and fructose 6-phosphate in the Embden-Meyerhof glycolytic pathway. GPI is an essential enzyme for carbohydrate metabolism in all tissues. In humans, the GPI gene locus is located on chromosome 19, and the gene spans more than 40 kb, including 18 exons and 17 introns. The cDNA sequence encodes 558 amino acid residues. The enzyme consists of two identical subunits. In mammals, GPI can also act as an autocrine motility factor, neuroleukin, and maturation factor. GPI deficiency is a well-known congenital autosomal recessive disorder with the typical manifestation of nonspherocytic hemolytic anemia of variable severity in humans. GPI deficiency is one of the most common cause of congenital nonspherocytic hemolytic anemia caused by deficiency of glycolytic enzymes, the commonest being deficiency of glucose-6-phosphate dehydrogenase and pyruvate kinase. Patients with inherited GPI deficiency present with nonspherocytic anemia of variable severity and with neuromuscular dysfunction. Mutations in the GPI gene usually have negative influences on catalytic parameters, particularly k(cat), as well as structure stability. Mutations at or close to the active site, including R273H, H389R, and S278L, cause great damage to the catalytic function, yet those at distance can still reduce the magnitude of k(cat). At the nucleotide level, 29 mutations have been reported. Mutations decrease the enzyme tolerance to heat by mechanisms of decreasing packing efficiency (V101M, T195I, S278L, L487F, L339P, T375R, I525T), weakening network bonding (R75G, R347C, R347H, R472H, E495K), increasing water-accessible hydrophobic surface (R83W), and destabilizing the ternary structure (T195I, R347C, R347H, and I525T). A300P, L339P, and E495K mutations may also negatively affect the protein folding efficiency (Merkle S. at al., 1993; Kanno H. at al., , 1996; Kugler W. at al., 2000; Haller J.F. еt al., 2009; Haller J.F., еt al., 2010). The neurologically affected patient (GPI Homburg) is compound heterozygous for a 59 A→C (H20P) and a 1016 T→C (L339P) exchange. Owing to the insertion of proline, the H20P and L339P mutations are likely to affect the folding and activity of the enzyme. Point mutations identified at 1166 A→G (H389R) and 1549 C→G (L517V), which are located at the subunit interface showed no neurological symptoms. Thus mutations that lead to incorrect folding destroy both catalytic

results in HK deficiency and mild chronic hemolysis (de Vooght еt al. 2009).

**2.2.2 Point mutation in glucose-6-phosphate isomerase**

(GPI) and neurotrophic activities, thereby leading to the observed clinical symptoms (GPI Homburg). Those alterations at the active site, however, that allow correct folding retain the neurotrophic properties of the molecule (GPI Calden) (Kugler, 1998). The similarity of the mutant enzymes to the allozymes found in human GPI deficiencies indicates the GPI deficient mouse mutants to be excellent models for the human disease (Padua et al., 1978; Pretsch et al., 1990). A heterozygous mouse mutant exhibiting approximately 50% of wildtype GPI activity. Biochemical and immunological studies revealed no differences in physicochemical, kinetic and immunological properties between the erythrocytic enzyme of heterozygous and wild-type mouse. The genetic and physiological analyses provided no indications for further altered traits in heterozygous animals including fertility, viability and several other traits. Homozygous null mutants died at an early post-implantation stage of embryogenesis (West et al., 1990; Merkle et al., 1992). Homozygous GPI deficiency in humans are responsible for chronic nonspherocytic hemolytic anemia. The homozygous missense A346H mutation replacement in cDNA position 1040G→A, which causes a loss of GPI capacity to dimerize, which renders the enzyme more susceptible to thermolability and produces significant changes in erythrocyte metabolism was described in patient with chronic nonspherocytic hemolytic anemia (Repiso et al., 2005). Biochemical and molecular genetic studies performed with the enzyme variants of GPI Zwickau and GPI Nordhorn showed that in both cases the simultaneous occurrence of a single amino acid substitution affecting the active site, together with a nonsense mutation leading to the loss of major parts of the enzyme probably explains the severe clinical course of the disease (Huppke et al., 1997). Molecular characteristics of erythrocytes GPI deficiency were described in Spanish patients with chronic nonspherocytic hemolytic anemia. Residual GPI activity in erythrocytes of around 7% (GPI-Catalonia), in an individual is homozygous for the missense mutation 1648A→G (Lys550→Glu) in exon 18 was described and residual activity in erythrocytes of around 20% (GPI-Barcelona), was found in a compound heterozygote for two different missense mutations: 341A→T (Asp113→Val) in exon 4 and 663T→G (Asn220→Lys) in exon 7. Molecular modeling using the human crystal structure of GPI as a model was performed to determine how these mutations could affect enzyme structure and function (Repiso et al., 2006). Chinese hamster (CHO) cell lines with ethylmethane sulfonate induced mutations in GPI and consequent loss of GPI activity have been reported. GPI activity was reduced by 87% in GroD1 isolated from this population. Expression cloning and sequencing of the cDNA obtained from GroD1 revealed a point mutation Gly189→ Glu. This resulted in a temperature sensitivity and severe reduction in the synthesis of glycerolipids due to a reduction in phosphatidate phosphatase (PAP). Overexpression of lipin 1 in the GPI-deficient cell line, GroD1 resulted in increased PAP activity, however it failed to restore glycerolipid biosynthesis. Fluorescent microscopy showed a failure of GPIdeficient cells to localize lipin 1α to the nucleus. Glucose-6-phosphate levels in GroD1 cells were 10-fold over normal. Lowering glucose levels in the growth medium partially restored glycerolipid biosynthesis and nuclear localization of lipin 1α. Thus, GPI deficiency results in an accumulation of glucose-6-phosphate, and possibly other glucose-derived metabolites, leading to activation of mTOR and sequestration of lipin 1 to the cytosol, preventing its proper functioning. These results may also help to explain neuromuscular symptoms associated with inherited GPI deficiency (Haller et al., 2010; 2011). GPI deficiency was found to be the cause of recurrent haemolytic crises that has required frequent blood transfusion.

Point Mutations That Reduce Erythrocyte Resistance to Oxidative Stress 261

disease that may induce severe liver damage, leading, in extreme cases, to death if fructose is not eliminated from the diet. To date, nearly 25 HFI-related aldolase B mutants have been identified. In contrast, cases of aldolase A deficiency, which has been associated with nonspherocytic haemolytic anemia, are much rarer (Esposito et al., 2004). Human aldolase A is composed of four identical subunits encoded by a single gene located on chromosome 16 (16q22–q24). Aldolase A deficiency has been reported as a rare, autosomal recessive disorder (Kreuder et al., 1996; Yao et al., 2004). Alterations in the aldolase A gene leading to amino acid substitutions: Asp128→Gly (Kishi et al., 1987), Glu206→Lys (Kreuder et al., 1996), Gly346→Ser (Esposito et al., 2004) have been described. The Glu206→Lys mutation destabilizes the aldolase A tetramer at the subunit interface, the Gly346→Ser mutation limits the flexibility of the C-terminal region. Biochemical and thermodynamic data are available for the Asn128→Gly mutant have never been characterized. Yao D.C. et al. described the case of a girl of Sicilian descent with aldolase A deficiency. Clinical manifestations included transfusion-dependent anemia until splenectomy at age 3 and increasing muscle weakness, with death at age 4 associated with rhabdomyolysis and hyperkalemia. Sequence analysis of the ALDOA coding regions revealed 2 novel heterozygous ALDOA mutations in conserved regions of the protein. The paternal allele encoded a nonsense mutation, Arg303X, in the enzyme-active site. The maternal allele encoded a missense mutation, Cys338→Tyr, predicted to cause enzyme instability as reported (Yao et al., 2004). Hemolytic crisis in patients with aldolase A deficiency can be provoked by fever (Kiriyama et al., 1993) and

upper respiratory infections (Miwa et al., 1981).

**2.2.5 Housekeeping genes in glyceraldehyde 3-phosphate dehydrogenase**

unchanged gene expression has been reported (Mazzola & Sirover, 2001).

Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) specifically catalyzes the simultaneous phosphorylation and oxidation of glyceraldehyde-3-phosphate to 1,3 biphosphoglycerate. GAPDH comprises a polypeptide chain of 335 amino acids. Structural studies identified two regions, namely the glyceraldehyde-3-phosphate catalytic site and the nicotinamide adenine dinucleotide binding site. The glycolytic function mainly relies on critical amino acids that include Cys152 and His179, and on its tetrameric structure composed of four identical 37-kDa subunits (Colell et al., 2009). GAPDH was considered a classical glycolytic protein involved exclusively in cytosolic energy production. However, recent evidence suggests that it is a multifunctional protein displaying diverse activities distinct from its conventional metabolic role. New investigations establish a primary role for GAPDH in a variety of critical nuclear pathways apart from its already recognized role in apoptosis. These new roles include its requirement for transcriptional control of histone gene expression, its essential function in nuclear membrane fusion, its necessity for the recognition of fraudulently incorporated nucleotides in DNA, and its mandatory participation in the maintenance of telomere structure. Other investigations relate a substantial role for nuclear GAPDH in hyperglycemic stress and the development of metabolic syndrome. GAPDH is a highly conserved gene and protein, with a single mRNA transcribed from a unique gene (Sirover 1997, 2005, 2011). GAPDH has been referred to as a "housekeeping" protein and based on the view that GAPDH gene expression remains constant under changing cellular conditions, the levels of GAPDH mRNA have frequently been used to normalize northern blots (Tatton, 2000). Evidence of an impairment of GAPDH glycolytic function in Alzheimer's and Huntington's disease subcellular fractions despite

Hemolysis is often ameliorated by splenectomy (Neubauer et al., 1990; Shalev et al., 1994; Alfinito et al., 1994). GPI deficiency can become a clinically relevant consequence of the administration of drugs. GPI deficiency can lead to impairment of the system that removes free radicals generated by amoxicillin, thereby resulting in oxidation of hemoglobin and destabilization of erythrocytes membranes, with acute hemolysis and severe hemoglobinuria (Rossi et al., 2010).

#### **2.2.3 Point mutation in phosphofructokinase**

Phosphofructo-1-kinase (PFK) is a tetrameric enzyme that phosphorylates fructose-6 phosphate to fructose-1,6-bisphosphate, committing glucose to glycolysis. Three PFK isoenzymes, encoded by separate genes, have been identified in mammals: muscle-type (PFKM), liver-type (PFKL), and platelet-type (PFKP), all of which are expressed in a tissue specific manner. Skeletal muscle expresses only PFKM homotetramers, liver mainly PFKL homotetramers, while erythrocytes contain PFKM and PFKL heterotetramers (Vora et al., 1983). Inherited deficiency of muscle PFK is known to occur in man and dog (Vora et al., 1983; Skibild et al., 2001). PFK deficiency was the first recognized disorder that directly affects glycolysis. Ever since the discovery of the disease in 1965, a wide range of biochemical, physiological and molecular studies of the disorder have been carried out (Nakajima et al., 2002). Several mutations in PFKM cause type VII glycogen storage disease (GSDVII), which is a rare disease described by Tarui (Tarui's disease). GSDVII is characterized by the coexistence of a muscle disease and a hemolytic process. Clinical manifestations of the disease range from the severe infantile form, leading to death during childhood, to the classical form, which presents mainly with exercise intolerance. Typically, the disease begins in early childhood and consists of easy fatigability, transient weakness and muscle cramps and myoglobinuria after vigorous exercises (Vora et al., 1987; García et al., 2009). A G-to-A transition at codon 209-in exon 8 of the PFK-M gene, changing an encoded Gly to Asp, is responsible for the GSDVII in a homozygous French Canadian patient. The Swiss patient is a genetic compound, carrying a G-to-A transition at codon 100 in exon 6 (Arg to Gln) and a G-to-A transition at codon 696 in exon 22 (Arg to His) (Raben et al., 1995). PFK deficiency include isolated hemolytic anemia, compensated hemolysis or asymptomatic state (Etiemble et al., 1983; Fogelfeld et al., 1990). The concomitant haemolysis in patients with inherited PFK deficiency of the muscle isoenzyme may be explained by a diminished erythrocyte deformability due to Ca2+ overload (Ronquist et al., 2001). PFK deficiency include early-onset neonatal seizures (Al-Hassnan et al., 2007). Portal and mesenteric vein thrombosis in patient with a known case of PFK deficiency has been described (Madhoun et al., 2011).

#### **2.2.4 Point mutation in aldolase**

Aldolase, a homotetrameric protein encoded by the ALDOA gene, converts fructose-1,6 bisphosphate to dihydroxyacetone phosphate and glyceraldehyde-3-phosphate. Three isozymes are encoded by distinct genes. The sole aldolase present in erythrocytes and skeletal muscle is the A isozyme. Aldolase B is mainly expressed in the liver, kidney and small intestine, where it plays a role in exogenous fructose utilization. Aldolase C is expressed predominantly in the brain. Aldolase B deficiency has been widely described in humans, because it causes hereditary fructose intolerance, which is an autosomal recessive

Hemolysis is often ameliorated by splenectomy (Neubauer et al., 1990; Shalev et al., 1994; Alfinito et al., 1994). GPI deficiency can become a clinically relevant consequence of the administration of drugs. GPI deficiency can lead to impairment of the system that removes free radicals generated by amoxicillin, thereby resulting in oxidation of hemoglobin and destabilization of erythrocytes membranes, with acute hemolysis and severe

Phosphofructo-1-kinase (PFK) is a tetrameric enzyme that phosphorylates fructose-6 phosphate to fructose-1,6-bisphosphate, committing glucose to glycolysis. Three PFK isoenzymes, encoded by separate genes, have been identified in mammals: muscle-type (PFKM), liver-type (PFKL), and platelet-type (PFKP), all of which are expressed in a tissue specific manner. Skeletal muscle expresses only PFKM homotetramers, liver mainly PFKL homotetramers, while erythrocytes contain PFKM and PFKL heterotetramers (Vora et al., 1983). Inherited deficiency of muscle PFK is known to occur in man and dog (Vora et al., 1983; Skibild et al., 2001). PFK deficiency was the first recognized disorder that directly affects glycolysis. Ever since the discovery of the disease in 1965, a wide range of biochemical, physiological and molecular studies of the disorder have been carried out (Nakajima et al., 2002). Several mutations in PFKM cause type VII glycogen storage disease (GSDVII), which is a rare disease described by Tarui (Tarui's disease). GSDVII is characterized by the coexistence of a muscle disease and a hemolytic process. Clinical manifestations of the disease range from the severe infantile form, leading to death during childhood, to the classical form, which presents mainly with exercise intolerance. Typically, the disease begins in early childhood and consists of easy fatigability, transient weakness and muscle cramps and myoglobinuria after vigorous exercises (Vora et al., 1987; García et al., 2009). A G-to-A transition at codon 209-in exon 8 of the PFK-M gene, changing an encoded Gly to Asp, is responsible for the GSDVII in a homozygous French Canadian patient. The Swiss patient is a genetic compound, carrying a G-to-A transition at codon 100 in exon 6 (Arg to Gln) and a G-to-A transition at codon 696 in exon 22 (Arg to His) (Raben et al., 1995). PFK deficiency include isolated hemolytic anemia, compensated hemolysis or asymptomatic state (Etiemble et al., 1983; Fogelfeld et al., 1990). The concomitant haemolysis in patients with inherited PFK deficiency of the muscle isoenzyme may be explained by a diminished erythrocyte deformability due to Ca2+ overload (Ronquist et al., 2001). PFK deficiency include early-onset neonatal seizures (Al-Hassnan et al., 2007). Portal and mesenteric vein thrombosis in patient with a known case of PFK deficiency has been

Aldolase, a homotetrameric protein encoded by the ALDOA gene, converts fructose-1,6 bisphosphate to dihydroxyacetone phosphate and glyceraldehyde-3-phosphate. Three isozymes are encoded by distinct genes. The sole aldolase present in erythrocytes and skeletal muscle is the A isozyme. Aldolase B is mainly expressed in the liver, kidney and small intestine, where it plays a role in exogenous fructose utilization. Aldolase C is expressed predominantly in the brain. Aldolase B deficiency has been widely described in humans, because it causes hereditary fructose intolerance, which is an autosomal recessive

hemoglobinuria (Rossi et al., 2010).

described (Madhoun et al., 2011).

**2.2.4 Point mutation in aldolase**

**2.2.3 Point mutation in phosphofructokinase**

disease that may induce severe liver damage, leading, in extreme cases, to death if fructose is not eliminated from the diet. To date, nearly 25 HFI-related aldolase B mutants have been identified. In contrast, cases of aldolase A deficiency, which has been associated with nonspherocytic haemolytic anemia, are much rarer (Esposito et al., 2004). Human aldolase A is composed of four identical subunits encoded by a single gene located on chromosome 16 (16q22–q24). Aldolase A deficiency has been reported as a rare, autosomal recessive disorder (Kreuder et al., 1996; Yao et al., 2004). Alterations in the aldolase A gene leading to amino acid substitutions: Asp128→Gly (Kishi et al., 1987), Glu206→Lys (Kreuder et al., 1996), Gly346→Ser (Esposito et al., 2004) have been described. The Glu206→Lys mutation destabilizes the aldolase A tetramer at the subunit interface, the Gly346→Ser mutation limits the flexibility of the C-terminal region. Biochemical and thermodynamic data are available for the Asn128→Gly mutant have never been characterized. Yao D.C. et al. described the case of a girl of Sicilian descent with aldolase A deficiency. Clinical manifestations included transfusion-dependent anemia until splenectomy at age 3 and increasing muscle weakness, with death at age 4 associated with rhabdomyolysis and hyperkalemia. Sequence analysis of the ALDOA coding regions revealed 2 novel heterozygous ALDOA mutations in conserved regions of the protein. The paternal allele encoded a nonsense mutation, Arg303X, in the enzyme-active site. The maternal allele encoded a missense mutation, Cys338→Tyr, predicted to cause enzyme instability as reported (Yao et al., 2004). Hemolytic crisis in patients with aldolase A deficiency can be provoked by fever (Kiriyama et al., 1993) and upper respiratory infections (Miwa et al., 1981).

#### **2.2.5 Housekeeping genes in glyceraldehyde 3-phosphate dehydrogenase**

Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) specifically catalyzes the simultaneous phosphorylation and oxidation of glyceraldehyde-3-phosphate to 1,3 biphosphoglycerate. GAPDH comprises a polypeptide chain of 335 amino acids. Structural studies identified two regions, namely the glyceraldehyde-3-phosphate catalytic site and the nicotinamide adenine dinucleotide binding site. The glycolytic function mainly relies on critical amino acids that include Cys152 and His179, and on its tetrameric structure composed of four identical 37-kDa subunits (Colell et al., 2009). GAPDH was considered a classical glycolytic protein involved exclusively in cytosolic energy production. However, recent evidence suggests that it is a multifunctional protein displaying diverse activities distinct from its conventional metabolic role. New investigations establish a primary role for GAPDH in a variety of critical nuclear pathways apart from its already recognized role in apoptosis. These new roles include its requirement for transcriptional control of histone gene expression, its essential function in nuclear membrane fusion, its necessity for the recognition of fraudulently incorporated nucleotides in DNA, and its mandatory participation in the maintenance of telomere structure. Other investigations relate a substantial role for nuclear GAPDH in hyperglycemic stress and the development of metabolic syndrome. GAPDH is a highly conserved gene and protein, with a single mRNA transcribed from a unique gene (Sirover 1997, 2005, 2011). GAPDH has been referred to as a "housekeeping" protein and based on the view that GAPDH gene expression remains constant under changing cellular conditions, the levels of GAPDH mRNA have frequently been used to normalize northern blots (Tatton, 2000). Evidence of an impairment of GAPDH glycolytic function in Alzheimer's and Huntington's disease subcellular fractions despite unchanged gene expression has been reported (Mazzola & Sirover, 2001).

Point Mutations That Reduce Erythrocyte Resistance to Oxidative Stress 263

recently been reported. The mutated PGAM shows an abnormal behaviour on ion-exchange chromatography and is more thermo-labile that the native enzyme. The increased instability of the mutated enzyme can account for the decreased erythrocytes PGAM activity (Repiso et

Enolase, an essential enzyme of glycolysis and gluconeogenesis, catalyses the interconversion of 2-hosphoglyceric acid and phosphoenolpyruvate. Enolases from most species are dimeric, with subunit molecular masses of 40000–50000 Da. Mammals have three genes for enolase, coding for the α, β and γ subunits; the subunits associate to form both homo- and heterodimers. The α gene is expressed in many tissues, γ primarily in neurones and β in muscle (Zhao et al., 2008). Erytrocytes enolase deficiency is rare, and its pathogenesis, inheritance and clinical manifestation have not been firmly established.  Lachant et al. (1986) described four generations of a Caucasian family with hereditary erytrocytes enolase deficiency. Stefanini (1972) described chronic hemolytic anemia associated with erythrocyte enolase deficiency exacerbated by ingestion of nitrofurantoin.

Pyruvate kinase (PK) catalyses the last step of the Embden–Meyerhof metabolic pathway, in which an ATP molecule is produced. Among the four PK isozymes present in humans (M1, M2, L and R), both PK-L (found in the liver, kidney and gut) and PK-R present in erythrocytes are encoded by the same gene, which is localised on hromosome 1q21. The respective expression of these two isozymes is under the control of specific promoters leading to structural differences in the N-terminal part of the protein. PK-R is a 574 amino acid-long protein, which associates into tetramers according to a double dyad symmetry pattern, resulting in allosteric enzymatic kinetics. PK deficiency is the most frequent red cell enzymatic defect responsible for hereditary nonspherocytic hemolytic anemia and is transmitted according to a recessive autosomal mode. Based on the gene frequency of the 1529A mutation in the white population and on its relative abundance in patients with hemolytic anemia caused by PK deficiency, the prevalence of PK deficiency is estimated at 51 cases per million white population (Beutler et al., 2000). The degree of haemolysis varies widely, ranging from very mild or fully compensated forms, to life-threatening neonatal anemia and jaundice necessitating exchange transfusions. Heterozygous carriers usually display very mild symptoms. Therefore, the defect is frequently ignored and its prevalence is difficult to establish. Severe disorders are described in homozygous or compound heterozygous patients (Zanella et al., 2005). According to the most recent database, more than 180 mutations have been reported on the PK-LR gene. Two mutations, both located in exon 11, are recurrent (Arg510 → Gln, Arg486 → Trp). Arg510→ Gln is the most frequent mutation found in northern Europe, central Europe and the USA (Pissard S. at al., 2006, as citated Wang et al., 2001) and Arg486 → Trp in southern Europe (Spain, Portugal and Italy) and in France (Pissard et al., 2006, as citited Zanella et al, 1997; Zarza et al, 1998; Pissard et al., 2006). The most frequent mutations of PKLR gene in the Indian population appear to be 1436G→A (19.44%), followed by 1456C→T (16.66%) and 992A→G (16.66%) (Kedar et al.,

al., 2005; de Atauri et al., 2005).

**2.2.8 Point mutation in enolase**

**2.2.9 Point mutation in pyruvate kinase**

#### **2.2.6 Point mutation in phosphoglycerate kinase**

Phosphoglycerate kinase (PGK) plays a key role for ATP generation in the glycolytic pathway. The PGK, which exists universally in various tissues of various organisms, is encoded by a single structural gene on the X-chromosome q13 in humans. The PGK consists of 417 amino acid residues with acetylserine at the NH2-terminal and isoleucine at the COOH-terminal and is a monomeric enzyme that is expressed in all tissues (Huang et al. 1980; Maeda et al. 1991). PGK deficiency is generally associated with chronic hemolytic anemia, although it can be accompanied by either mental retardation or muscular disease (Cohen-Solal et al. 1994). The structure of some PGK mutants has been described. PGK Matsue variant is a point mutation, a T/A→C/G transition in exon 3, that cause Leu88→Pro substitution associated with severe enzyme deficiency, congenital nonspherocytic hemolytic anemia, and mental disorders (Maeda et al. 1991). PGK Shizuoka variant is a single nucleotide substitution from guanine to thymine at position 473 of PGK messenger RNA, associated with chronic hemolysis and myoglobinuria. This nucleotide change causes a single amino acid substitution from Gly157→Val (Fujii et al. 1992). PGK Créteil variant arises from a G→A nucleotide interchange at position 1022 in cDNA (exon 9), resulting in amino acid substitution Asp314→Asn associated with rhabdomyolysis crises but not with hemolysis or mental retardation. PGK Amiens/New York variant, which is associated with chronic hemolytic anemia and mental retardation is a point mutation, an A→T nucleotide interchange at position 571 in cDNA (exon 5); this leads to amino acid substitution Asp163→Val (Cohen-Solal et al., 1994; Flanagan et al., 2006). Variants of PGK Barcelona and PGK Murcia are described in Spain. PGK Barcelona variant, which causes chronic hemolytic anemia associated with progressive neurological impairment is a point mutation, 140 T→A substitution that produces an Ile46→Asn change. The increase of 2,3-bisphosphoglycerate and the decrease of adenosine triphosphate levels in erythrocytes are the detected metabolic changes that could cause hemolytic anemia. PGK Murcia variant is a point mutation, 958 G→A transition that cause a Ser319→Asn substitution. The crystal structure of porcine PGK was used as a molecular model to investigate how these mutations may affect enzyme structure and function. In both cases – the mutations did not modify any of the PGK binding sites for ATP or 3PG, so their effect is probably related to a loss of enzyme stability rather than a decrease of enzyme catalytic function (Noel N. et al., 2006; Ramírez-Bajo M.J. et al., 2011). Mutants PGK München (Krietsch et al., 1980), PGK Herlev (Valentin et al., 1998), PGK Uppsala (Hjelm et al., 1980), PGK San Francisco (Guis et al., 1987), PGK II (Huang et al., 1980), PGK Michigan, PGK Tokyo (Cohen-Solal et al., 1994) are also described.

#### **2.2.7 Point mutation in phosphoglycerate mutase**

Phosphoglycerate mutase (PGAM) is a glycolytic enzyme that catalyses the interconversion of 2-phosphoglycerate and 3-phosphoglycerate, with 2,3-bisphosphoglycerate being required, in mammals, as a co-factor. In mammals, PGAM is present in three isozymes which result from the homodimeric and heterodimeric combinations of two different subunits, M and B, coded by two different genes, although the gene coding subunit B is unknown. Only the homodimer BB is present in erythrocytes. Only one PGAM BB deficiency has been reported. In a patient with clinical diagnosis of Hereditary Spherocytosis and partial deficiency (50%) of erythrocytes PGAM activity, a homozygous point mutation with cDNA 690G→A substitution that produces a Met230→Ile change has recently been reported. The mutated PGAM shows an abnormal behaviour on ion-exchange chromatography and is more thermo-labile that the native enzyme. The increased instability of the mutated enzyme can account for the decreased erythrocytes PGAM activity (Repiso et al., 2005; de Atauri et al., 2005).

#### **2.2.8 Point mutation in enolase**

262 Point Mutation

Phosphoglycerate kinase (PGK) plays a key role for ATP generation in the glycolytic pathway. The PGK, which exists universally in various tissues of various organisms, is encoded by a single structural gene on the X-chromosome q13 in humans. The PGK consists of 417 amino acid residues with acetylserine at the NH2-terminal and isoleucine at the COOH-terminal and is a monomeric enzyme that is expressed in all tissues (Huang et al. 1980; Maeda et al. 1991). PGK deficiency is generally associated with chronic hemolytic anemia, although it can be accompanied by either mental retardation or muscular disease (Cohen-Solal et al. 1994). The structure of some PGK mutants has been described. PGK Matsue variant is a point mutation, a T/A→C/G transition in exon 3, that cause Leu88→Pro substitution associated with severe enzyme deficiency, congenital nonspherocytic hemolytic anemia, and mental disorders (Maeda et al. 1991). PGK Shizuoka variant is a single nucleotide substitution from guanine to thymine at position 473 of PGK messenger RNA, associated with chronic hemolysis and myoglobinuria. This nucleotide change causes a single amino acid substitution from Gly157→Val (Fujii et al. 1992). PGK Créteil variant arises from a G→A nucleotide interchange at position 1022 in cDNA (exon 9), resulting in amino acid substitution Asp314→Asn associated with rhabdomyolysis crises but not with hemolysis or mental retardation. PGK Amiens/New York variant, which is associated with chronic hemolytic anemia and mental retardation is a point mutation, an A→T nucleotide interchange at position 571 in cDNA (exon 5); this leads to amino acid substitution Asp163→Val (Cohen-Solal et al., 1994; Flanagan et al., 2006). Variants of PGK Barcelona and PGK Murcia are described in Spain. PGK Barcelona variant, which causes chronic hemolytic anemia associated with progressive neurological impairment is a point mutation, 140 T→A substitution that produces an Ile46→Asn change. The increase of 2,3-bisphosphoglycerate and the decrease of adenosine triphosphate levels in erythrocytes are the detected metabolic changes that could cause hemolytic anemia. PGK Murcia variant is a point mutation, 958 G→A transition that cause a Ser319→Asn substitution. The crystal structure of porcine PGK was used as a molecular model to investigate how these mutations may affect enzyme structure and function. In both cases – the mutations did not modify any of the PGK binding sites for ATP or 3PG, so their effect is probably related to a loss of enzyme stability rather than a decrease of enzyme catalytic function (Noel N. et al., 2006; Ramírez-Bajo M.J. et al., 2011).

Phosphoglycerate mutase (PGAM) is a glycolytic enzyme that catalyses the interconversion of 2-phosphoglycerate and 3-phosphoglycerate, with 2,3-bisphosphoglycerate being required, in mammals, as a co-factor. In mammals, PGAM is present in three isozymes which result from the homodimeric and heterodimeric combinations of two different subunits, M and B, coded by two different genes, although the gene coding subunit B is unknown. Only the homodimer BB is present in erythrocytes. Only one PGAM BB deficiency has been reported. In a patient with clinical diagnosis of Hereditary Spherocytosis and partial deficiency (50%) of erythrocytes PGAM activity, a homozygous point mutation with cDNA 690G→A substitution that produces a Met230→Ile change has

**2.2.7 Point mutation in phosphoglycerate mutase**

**2.2.6 Point mutation in phosphoglycerate kinase**

Enolase, an essential enzyme of glycolysis and gluconeogenesis, catalyses the interconversion of 2-hosphoglyceric acid and phosphoenolpyruvate. Enolases from most species are dimeric, with subunit molecular masses of 40000–50000 Da. Mammals have three genes for enolase, coding for the α, β and γ subunits; the subunits associate to form both homo- and heterodimers. The α gene is expressed in many tissues, γ primarily in neurones and β in muscle (Zhao et al., 2008). Erytrocytes enolase deficiency is rare, and its pathogenesis, inheritance and clinical manifestation have not been firmly established.  Lachant et al. (1986) described four generations of a Caucasian family with hereditary erytrocytes enolase deficiency. Stefanini (1972) described chronic hemolytic anemia associated with erythrocyte enolase deficiency exacerbated by ingestion of nitrofurantoin.

#### **2.2.9 Point mutation in pyruvate kinase**

Pyruvate kinase (PK) catalyses the last step of the Embden–Meyerhof metabolic pathway, in which an ATP molecule is produced. Among the four PK isozymes present in humans (M1, M2, L and R), both PK-L (found in the liver, kidney and gut) and PK-R present in erythrocytes are encoded by the same gene, which is localised on hromosome 1q21. The respective expression of these two isozymes is under the control of specific promoters leading to structural differences in the N-terminal part of the protein. PK-R is a 574 amino acid-long protein, which associates into tetramers according to a double dyad symmetry pattern, resulting in allosteric enzymatic kinetics. PK deficiency is the most frequent red cell enzymatic defect responsible for hereditary nonspherocytic hemolytic anemia and is transmitted according to a recessive autosomal mode. Based on the gene frequency of the 1529A mutation in the white population and on its relative abundance in patients with hemolytic anemia caused by PK deficiency, the prevalence of PK deficiency is estimated at 51 cases per million white population (Beutler et al., 2000). The degree of haemolysis varies widely, ranging from very mild or fully compensated forms, to life-threatening neonatal anemia and jaundice necessitating exchange transfusions. Heterozygous carriers usually display very mild symptoms. Therefore, the defect is frequently ignored and its prevalence is difficult to establish. Severe disorders are described in homozygous or compound heterozygous patients (Zanella et al., 2005). According to the most recent database, more than 180 mutations have been reported on the PK-LR gene. Two mutations, both located in exon 11, are recurrent (Arg510 → Gln, Arg486 → Trp). Arg510→ Gln is the most frequent mutation found in northern Europe, central Europe and the USA (Pissard S. at al., 2006, as citated Wang et al., 2001) and Arg486 → Trp in southern Europe (Spain, Portugal and Italy) and in France (Pissard et al., 2006, as citited Zanella et al, 1997; Zarza et al, 1998; Pissard et al., 2006). The most frequent mutations of PKLR gene in the Indian population appear to be 1436G→A (19.44%), followed by 1456C→T (16.66%) and 992A→G (16.66%) (Kedar et al.,

Point Mutations That Reduce Erythrocyte Resistance to Oxidative Stress 265

generation of NADPH which provides the major reducing power essential to protect the cell against oxidative stress and a variety of reductive biosynthetic reactions, particularly lipid production. Thus, 6PGDH plays a critical role in protecting cells from oxidative stress (He et al., 2007). Few cases of erytrocytes 6PGD deficiency in humans have been described. The episodic hemolytic events with jaundice in patients with 6PGD deficiency may be the result of a defective erythrocytes ability to counteract conditions of marked oxidative stress as happens at birth and following traumatic events. The presence of 6PGD deficiency could be mistaken for a partial G6PD deficiency if the assay of G6PD activity was performed without

Glutathione (GSH) is the most abundant intracellular thiol in living aerobic cells. GSH is present in millimolar concentrations in most mammalian cells and it is involved in several fundamental biological functions, including free radical scavenging, detoxification of xenobiotics and carcinogens, redox reactions, biosynthesis of DNA, proteins and leukotrienes, as well as neurotransmission/neuromodulation. It has been assigned several critical functions: protection of cells against oxidative damage; involvement in amino acid transport; participation in the detoxification of foreign compounds; maintenance of protein sulfhydryl groups in a reduced state; and as a cofactor for a number of enzymes. GSH is found in low levels in diseases in which increasing evidence implicate oxidative stress in the development of the disease, for example retinopathy of prematurity, necrotizing enterocolitis, bronchopulmonary dysplasia, patent ductus arteriosus and asthma. GSH is metabolised via the gamma-glutamyl cycle, which is catalyzed by six enzymes (Polekhina at al., 1999; Njålsson et al., 2005; Norgren et al., 2007). GSH is synthesized from glutamate, cysteine and glycine. The final step in its synthesis is catalysed by the enzyme glutathione synthetase (GS) The human GS enzyme is a homodimer with 52 kDa of subunits containing 474 amino acid residues, encoded by a single-copy gene located on chromosome 20q11.2

GS deficiency is a rare autosomal recessive disorder. Since the human genome contains only one GS gene, the various clinical forms of GS deficiency reflect different mutations or epigenetic modifications in the GS gene. Thus GSH acts as a feedback inhibitor of the initial step in its biosynthesis, in patients with hereditary deficiency of GS the lack of GSH leads to the formation of increased amounts of g-glutamylcysteine which is converted into 5 oxoproline by g-glutamyl cyclotransferase and excreted in massive amounts. Shi et al. identified seven mutations at the GS locus on six alleles: one splice site mutation, two deletions and four missense mutations and in patients with 5-Oxoprolinuria (pyroglutamic aciduria) resulting in GS deficiency and homozygous missense mutation in an individual affected by a milder-form of the GS deficiency, which is apparently restricted to erythrocytes and only associated with haemolytic anaemia (Shi, et al., 1996). Japanese patients with chronic nonspherocytic hemolytic anemia were found to have decreased GS activity and the others were moderately deficient in GCS. Hemolytic anemia was their only manifestation, and neither 5-oxoprolinemia nor 5-oxoprolinuria, which are usually associated with to generalized type of glutathione synthetase deficiency, was noted in patients. (Hirono et al., 1996). Dahl N. at al. described thirteen different point mutations. In vitro analysis of

correcting for 6PGD activity (Vives Corrons et al., 1996; Caprari et al., 2001).

**2.3 Point mutation in glutathione synthetase**

(Webb et al., 1995; Njålsson et al., 2005).

2009). Erythrocyte PK plays an important role as an antioxidant during erythroid differentiation. Glycolytic inhibition by erythrocyte PK gene mutation augmented oxidative stress, leading to activation of hypoxia-inducible factor-1 as well as downstream proapoptotic gene expression (Aisaki et al., 2007). Extended molecular analysis is useful for studying how several interacting gene mutations contribute to the clinical variability of pyruvate kinase deficiency (Perseu et al., 2010).

#### **2.2.10 Point mutation in glucose-6-phosphate dehydrogenase**

Glucose 6-phosphate dehydrogenase (G6PD) is a ubiquitous enzyme, which is critical in the redox metabolism of all aerobic cells. It catalyzes the first, rate-limiting step of the pentose phosphate pathway, coupled to NADPH synthesis and to ribose availability which is essential for the production of nucleotide coenzymes and replication of nucleic acids (Sodiende O., 1992). The pentose phosphate pathway is the unique source of NADPH, which enables erytrocytes to counterbalance the oxidative stress triggered by several oxidant agents preserving the reduced form of glutathione. GSH protects the sulfhydryl groups in hemoglobin and in the red cell membrane from oxidation (Mason et al., 2007). G6PD is a dimer and each subunit contains a single active site. G6PD-enzyme is encoded by a human X-linked gene (Xq2.8) consisting of 13 exons and 12 introns, spanning nearly 20 kb in total. G6PD gene is probably the most polymorphic locus in humans, with over 400 allelic variants known (Minucci et al., 2009). G6PD, the most common enzyme deficiency worldwide, causes a spectrum of disease including neonatal hyperbilirubinemia, acute hemolysis, and chronic hemolysis. Persons with this condition also may be asymptomatic. Approximately 400 million people are affected worldwide. Homozygotes and heterozygotes can be symptomatic, although the disease typically is more severe in persons who are homozygous for the deficiency. Different gene mutations cause different levels of enzyme deficiency, with classes assigned to various degrees of deficiency and disease manifestation. Acute hemolysis is caused by exposure to an oxidative stressor such as infection, some foods (fava beans), drugs or various chemicals. The variant that causes chronic hemolysis is uncommon because it is related to sporadic gene mutation rather than the more common inherited gene mutation (Frank, 2005). About 160 mutations have been reported, most of which are single-base substitutions leading to amino acid replacements (Minucci et. al., 2009). Mutations are classified into four types, according to their clinical effects. Several variants, such as the the Mediterranean variant, reach the polymorphism (Wajcman et. al., 2004). The Mediterranean variant of G6PD deficiency is due to the C563CT point mutation, leading to replacement of Ser with Phe at position 188, resulting in acute haemolysis triggered by oxidants (Ingrosso et. al., 2002). Individuals with such mutations seem to have enjoyed a selective advantage because of resistance to falciparum malaria. Different mutations, each characteristic of certain populations are found.The most common African mutation G6PD is 202A376G. G6PD Mediterranean (563T) is found in Southern Europe, the Middle East and in the Indian subcontinent (Beulter, 1996).

#### **2.2.11 Point mutation in 6-phosphogluconate dehydrogenase**

The 6-phosphogluconate dehydrogenase (6PGDH) is the third enzyme of the oxidative branch of the pentose phosphate pathway. This pathway has two major functions: the production of ribulose 5-phosphate which is required for nucleotide synthesis, and the generation of NADPH which provides the major reducing power essential to protect the cell against oxidative stress and a variety of reductive biosynthetic reactions, particularly lipid production. Thus, 6PGDH plays a critical role in protecting cells from oxidative stress (He et al., 2007). Few cases of erytrocytes 6PGD deficiency in humans have been described. The episodic hemolytic events with jaundice in patients with 6PGD deficiency may be the result of a defective erythrocytes ability to counteract conditions of marked oxidative stress as happens at birth and following traumatic events. The presence of 6PGD deficiency could be mistaken for a partial G6PD deficiency if the assay of G6PD activity was performed without correcting for 6PGD activity (Vives Corrons et al., 1996; Caprari et al., 2001).

#### **2.3 Point mutation in glutathione synthetase**

264 Point Mutation

2009). Erythrocyte PK plays an important role as an antioxidant during erythroid differentiation. Glycolytic inhibition by erythrocyte PK gene mutation augmented oxidative stress, leading to activation of hypoxia-inducible factor-1 as well as downstream proapoptotic gene expression (Aisaki et al., 2007). Extended molecular analysis is useful for studying how several interacting gene mutations contribute to the clinical variability of

Glucose 6-phosphate dehydrogenase (G6PD) is a ubiquitous enzyme, which is critical in the redox metabolism of all aerobic cells. It catalyzes the first, rate-limiting step of the pentose phosphate pathway, coupled to NADPH synthesis and to ribose availability which is essential for the production of nucleotide coenzymes and replication of nucleic acids (Sodiende O., 1992). The pentose phosphate pathway is the unique source of NADPH, which enables erytrocytes to counterbalance the oxidative stress triggered by several oxidant agents preserving the reduced form of glutathione. GSH protects the sulfhydryl groups in hemoglobin and in the red cell membrane from oxidation (Mason et al., 2007). G6PD is a dimer and each subunit contains a single active site. G6PD-enzyme is encoded by a human X-linked gene (Xq2.8) consisting of 13 exons and 12 introns, spanning nearly 20 kb in total. G6PD gene is probably the most polymorphic locus in humans, with over 400 allelic variants known (Minucci et al., 2009). G6PD, the most common enzyme deficiency worldwide, causes a spectrum of disease including neonatal hyperbilirubinemia, acute hemolysis, and chronic hemolysis. Persons with this condition also may be asymptomatic. Approximately 400 million people are affected worldwide. Homozygotes and heterozygotes can be symptomatic, although the disease typically is more severe in persons who are homozygous for the deficiency. Different gene mutations cause different levels of enzyme deficiency, with classes assigned to various degrees of deficiency and disease manifestation. Acute hemolysis is caused by exposure to an oxidative stressor such as infection, some foods (fava beans), drugs or various chemicals. The variant that causes chronic hemolysis is uncommon because it is related to sporadic gene mutation rather than the more common inherited gene mutation (Frank, 2005). About 160 mutations have been reported, most of which are single-base substitutions leading to amino acid replacements (Minucci et. al., 2009). Mutations are classified into four types, according to their clinical effects. Several variants, such as the the Mediterranean variant, reach the polymorphism (Wajcman et. al., 2004). The Mediterranean variant of G6PD deficiency is due to the C563CT point mutation, leading to replacement of Ser with Phe at position 188, resulting in acute haemolysis triggered by oxidants (Ingrosso et. al., 2002). Individuals with such mutations seem to have enjoyed a selective advantage because of resistance to falciparum malaria. Different mutations, each characteristic of certain populations are found.The most common African mutation G6PD is 202A376G. G6PD Mediterranean (563T) is found in Southern Europe, the

pyruvate kinase deficiency (Perseu et al., 2010).

**2.2.10 Point mutation in glucose-6-phosphate dehydrogenase**

Middle East and in the Indian subcontinent (Beulter, 1996).

**2.2.11 Point mutation in 6-phosphogluconate dehydrogenase**

The 6-phosphogluconate dehydrogenase (6PGDH) is the third enzyme of the oxidative branch of the pentose phosphate pathway. This pathway has two major functions: the production of ribulose 5-phosphate which is required for nucleotide synthesis, and the Glutathione (GSH) is the most abundant intracellular thiol in living aerobic cells. GSH is present in millimolar concentrations in most mammalian cells and it is involved in several fundamental biological functions, including free radical scavenging, detoxification of xenobiotics and carcinogens, redox reactions, biosynthesis of DNA, proteins and leukotrienes, as well as neurotransmission/neuromodulation. It has been assigned several critical functions: protection of cells against oxidative damage; involvement in amino acid transport; participation in the detoxification of foreign compounds; maintenance of protein sulfhydryl groups in a reduced state; and as a cofactor for a number of enzymes. GSH is found in low levels in diseases in which increasing evidence implicate oxidative stress in the development of the disease, for example retinopathy of prematurity, necrotizing enterocolitis, bronchopulmonary dysplasia, patent ductus arteriosus and asthma. GSH is metabolised via the gamma-glutamyl cycle, which is catalyzed by six enzymes (Polekhina at al., 1999; Njålsson et al., 2005; Norgren et al., 2007). GSH is synthesized from glutamate, cysteine and glycine. The final step in its synthesis is catalysed by the enzyme glutathione synthetase (GS) The human GS enzyme is a homodimer with 52 kDa of subunits containing 474 amino acid residues, encoded by a single-copy gene located on chromosome 20q11.2 (Webb et al., 1995; Njålsson et al., 2005).

GS deficiency is a rare autosomal recessive disorder. Since the human genome contains only one GS gene, the various clinical forms of GS deficiency reflect different mutations or epigenetic modifications in the GS gene. Thus GSH acts as a feedback inhibitor of the initial step in its biosynthesis, in patients with hereditary deficiency of GS the lack of GSH leads to the formation of increased amounts of g-glutamylcysteine which is converted into 5 oxoproline by g-glutamyl cyclotransferase and excreted in massive amounts. Shi et al. identified seven mutations at the GS locus on six alleles: one splice site mutation, two deletions and four missense mutations and in patients with 5-Oxoprolinuria (pyroglutamic aciduria) resulting in GS deficiency and homozygous missense mutation in an individual affected by a milder-form of the GS deficiency, which is apparently restricted to erythrocytes and only associated with haemolytic anaemia (Shi, et al., 1996). Japanese patients with chronic nonspherocytic hemolytic anemia were found to have decreased GS activity and the others were moderately deficient in GCS. Hemolytic anemia was their only manifestation, and neither 5-oxoprolinemia nor 5-oxoprolinuria, which are usually associated with to generalized type of glutathione synthetase deficiency, was noted in patients. (Hirono et al., 1996). Dahl N. at al. described thirteen different point mutations. In vitro analysis of

Point Mutations That Reduce Erythrocyte Resistance to Oxidative Stress 267

outcome depends significantly on early start of treatment, routine inclusion of this disorder in newborn screening panels should be considered. Treatment consists of the correction of acidosis, blood transfusion, and supplementation with antioxidants (Simon et al., 2009). Patients with GS deficiency are given vitamins C and E to boost their antioxidant levels, and bicarbonate to correct metabolic acidosis (Jain et al.,1994; Ristoff et

Glutathione reductase (GR) is a key enzyme required for the conversion of oxidized glutathione (GSSG) to reduced glutathione (GSH) concomitantly oxidizing reduced nicotinamide adenine dinucleotide phosphate (NADPH). GR is a homodimeric flavoprotein with a subunit Mr of 52.4 kDa. Its 2 identical redox active sites are formed by residues from both subunits, implying that monomeric GR is not active. Human GR is encoded by a single gene, located on chromosome 8p21.1 and consisting of 13 exons. GR consists of apoglutathione reductase (apoGR) and flavin adenine dinucleotide (FAD) as a prosthetic

Acquired FAD deficiency due to low amounts of riboflavin (vitamin B2) in the diet (or failure to convert it sufficiently to FAD) may result in inactive apoGR. In that case GR activity can be restored by riboflavin administration. Due to inherited mutations, the GR protein can be absent or exhibit low catalytic activity. Whereas inherited glutathione reductase deficiency is rare, FAD deficiency is common in malnourished populations. The clinical symptoms of GR deficiency include reduced lifespan of erythrocytes, cataract, and favism (hemolytic crises after eating fava beans). A 2246-bp deletion in DNA, which results in unstable and inactive GR and a premature stop codon on one allele and a substitution of glycine 330, a highly conserved residue in the superfamily of NAD(P)H-dependent disulfide reductases, into alanine on the other allele were described in the GR gene in patients with clinical GR deficiency (Kamerbeek et al., 2007). GR deficiency may alter the clinical manifestation of an unstable hemoglobinopathy (Mojzikova at al., 2010) and may be the cause of neonatal hyperbilirubinemia (Casado et al., 1995). GR deficiency state can be asymptomatic as the residual enzyme activity might be sufficient to maintain the reduced glutathione level to prevent oxidative stress (Nakashima et al., 1978). A study on 1691 individuals from Saudi Arabia to determine the overall frequency of GR deficiency has been conducted. The overall frequency of genetic GR deficiency was 24.5% and 20.3% in males and females respectively. In addition, 17.8% of males and 22.4% of females suffered from GR deficiency due to riboflavin deficiency. This could be easily corrected by dietary supplementation with riboflavin. No cases of severe GR deficiency were identified (el-