Hemoglobin E Prevalence among People Residing in Malaria Areas

Malaria is a disease in tropical countries caused by a bite of an infected female Anopheles mosquito. Humans can be infected by only a specific type of Plasmodium species including P. falciparum, P. vivax, P. malariae, P. ovale, or P. knowlesi. The most severe type is P. falciparum with a high mortality rate (Garcia, 2010). All the clinical symptoms of malaria that result from P. falciparum infection of human red blood cells (RBCs), which intensifies anemia and malaria in the cerebral stage, are caused by merozoites. Therefore, the clinical manifestations of malarial infections are associated with the rupture of infected RBC during the blood stage of the parasite’s life cycle (Maier et al., 2009).
       
Since hemoglobin (Hb) is an important component in RBCs, malaria parasites feed on Hb for survival and reproduction. Any synthetic or structural molecular defects in Hb called hemoglobinopathies that result from a mutation in one or more Hb genes. The mutated genes coded either the proteins that build up the Hb molecule or that are used for globin chain synthesis or regulating synthesis. The production of these mutations leads to qualitative or quantitative defects in Hb synthesis. The normal rate of Hb synthesis or semi-normal is known as a qualitative defect that is characterized by structure alternation of amino acid sequence in globin chains of Hb molecules. On the contrary, any reduction in the rate of globin chain synthesis of Hb in the absence of an alteration in its amino acids sequence absolutely will lead to quantitative defects called Thalassemias. Anemia is produced in response to the shortage synthesis of Hb amount and enhances the appearance of un-mutated other Hb to replace anemia. According to these variations, the hematological doctors classified them into two main categories, structural (qualitative) defect, and synthetic (quantitative) defect, thalassemia. However, many doctors mention hemoglobinopathies for structural defects only (Weatherall, 2010).
       
Thalassemia is an inherited monogenic Hb disorder that is classified into two major categories, according to a defect in Hb synthesis (thalassemia α/β), or in Hb structure such as (Hb S, C and E) variants of Hb (Weatherall, 2010). This monogenic disorder in Hb results in around 7% of carriers worldwide (Old et al., 2005). Structural variants of Hb resulted from a single replacement of amino acid sequences in the chain of α or β. However, these variants are considered mild for any disturbance in Hb functions and stability leads to disorders with clinical features (Voskaridou et al., 2001).
       
Until now, Sickle Cell Disease, or the heterozygous form combines with the gene of Hb C to produce (Hb S/C) disorder, which is characterized to be milder with remarkable public health problems. The commonest universal hemoglobin-variant is (Hb E). Both homozygous and heterozygous forms are milder. However, when the heterozygous form combines with β-thala to output Hb E/ β-thala, it becomes highly common with very serious health problems in many regions of Asian countries (Weatherall et al., 2006).
       
It has been reported that SCA, Thala, Hb E, and other variants are common in regions that suffered from malaria in the past. Moreover, social factors play important roles that lead to the wide spread of hemoglobinopathies through the population structure activities such as ethnical intermixing (Thein, 2005). Therefore, to understand the correlation between hemoglobinopathies and malaria infection, this proposal aims to study the prevalence of one of the hemoglobinopathies which is Hb E among people in the residing area and link this at phenotypic and genotypic levels.

Identification and screening of the patients who are eligible for our study were done at King Fahd Central Hospital, Jazan in Saudi Arabia for hematological studies such as CBC, Hb Electrophoresis and Malaria Screening. Genetics Study carried out at the National Center for Disease Prevention and Control. The consent form was signed by patients and controls with IRB No: CAMS 050-3839. The study sample size was set to them. Data obtained from patients and their parents, and guardians and available in clinical history included information about age, malarial infection history and relative marriage and degree of consanguineous relative marriage or from the pre-marriage clinic.
       
This case-control study was performed over ten ten-month period from June 2018 to March 2019 in patients who carried HbE in their erythrocytes’ Hb or malaria parasites in their blood. All study participants provided written informed permission that was included in the study. The study was approved by the ethical committee of the College of Applied Medical Sciences (CAMS, KSU) with IRB No: CAMS 050-3839.
       
The sample number of the present study was 13972. The samples were collected with two Ethylene Diamine Tetra-acetic Acid (EDTA) vacutainer tubes, 5ml in each tube. The first tube for the hematological study involved an automated complete blood count (CBC), malaria test screening (microscopic and rapid test) and Hb electrophoresis screening. The second one is used for Sanger sequencing-based molecular analysis.
       
Automated complete blood cell count (SYSMEX, XN-1000), hematological analyzer machine used in diagnostic and clinical laboratories for the identification, quantitative and proportion of EDTA whole blood samples via electrical resistance, dye application and laser light scatter. Principle flow cytometric technique used to measure the size, shape, and concentration of blood cells (RBCs, WBCs and PLT Counts) and blood indices, Hb%, Hematocrit or Packed Cell Volume (Hct), Mean Corpuscular Volume (MCV), Mean Corpuscular Hemoglobin (MCH), Mean Corpuscular Hemoglobin Concentration (MCHC), Red Cell Distribution Width (RDW), Mean Platelets Volume (MPV) and Differential of WBCs.
       
The malaria diagnosis can be obtained in 2 ways: (1) identification of malarial parasites by using microscopic technique. (2) detection of Antigens/products of parasites in peripheral blood.
       
Direct observation of parasites through stained thick or thin peripheral blood smear or quantitative buffy coat test was performed. A simple technique was carried out by using a light microscope (thick and thin smear), slides were stained with Romanosky (Giemsa’s) stain and examined for intra-erythrocyte parasites. A thick smear is 20-40% more sensitive than the thin one of detected Plasmodium (10-50 trophozoites/µl). A thin smear was used for the morphological study and identification of the parasite involving mixed infection, parasitemia, schizont and hemozoin in monocytes.
 
Immunochromatographic test by capturing Ags of the parasite in peripheral blood through monoclonal or polyclonal specific Abs for parasite Ags. The test was used to target (1) Histidine-rich protein 2 of P. falciparum, which is a soluble protein for asexual stage and gametocytes. (2) Pan-malarial Plasmodium aldolase is an enzyme of glycolytic pathway for mixed infection Pf/Pv. (3) Parasite lactate dehydrogenase is a soluble enzyme for glycolytic in both sexual and asexual stages. Rapid diagnostic tests such as dipstick, strip, pad, well, or cassette. Done by applying 2-50 µl of blood, mix with buffer containing hemolyze and specific Abs labeled with a visual detectable marker.
       
The fully automated VARIANT II Hb testing system is designed for high volume, featuring positive specimen identification, LIS interface and complete, ready-to-use test kits for precise, accurate results. The machine is compatible with a barcode, barcode reader and closed or diluted sample tubes. Chromatographically, an EDTA whole blood specimen is injected into the analysis process. It came with 2 modules, the VARIANT II Chromatographic Station (VCS) and the VARIANT II Sampling Station (VSS). Clinical Data Management (CDM) software computer program used to control the Variant II System. The VARIANT II used the principle of High-Performance Liquid Chromatography (HPLC).
       
Capillary 2 Sebia Flex Piercing capillary Hb E is an automated Hb electrophoresis that enables to identification, detection and well-elution (separation) of Hb Fractions and variants. By automation processing, achieves all sequences to release accurate, precise, and quantified of the maximum numbers of Hemoglobinopathies (Hb S, Hb C, Hb E, Hb D, Hb F and Hb A‚ ) also Hb H and Bart’s.
       
Principle assay is carried out on the capillary electrophoresis; Hb fractions are well-separate in silica capillary in the presence of a great voltage in alkaline buffer solution by electro-osmatic flow and electrophoretic movement. Also, blood from the Hb E patient was extracted and purified by using the GeneJet Genomic DNA Purification Kit with catalog number: K0721 from Thermo Scientific. This kit is rapid and efficient purification with more quality gDNA.
       
ARMS is a fast and simple technique used in the detection of single-point mutation and small deletion. Originally, ARMS was used for allele-specific amplification to determine the specific genotype in one-step PCR. Standard ARMS-PCR utilizes 2 reactions complementary with 3 primers. One primer for specific whole sequence DNA in 2 reactions. The specificity of alleles is given through the dissimilarity in the bases of the 3¢ terminal of different primers compared to either a whole sequence of DNA or a mutant one. In allele-specific primer, the normal primer will refractory to PCR on mutant sequencing of DNA. Allele-specific Hb E primer t-ARMS PCR was then discriminated by gel electrophoresis.
       
Data was recorded into a database system using a double data entry format and analyzed with Microsoft Excel 2010 software. Normally distributed continuous variables were summarized as mean and compared using the student’s t-test with a P-value of less than 0.05 being statistically significant.

Based on the results of hematological and electrophoresis tests conducted on 13972 Saudi participants in the Jazan region from May 2018 to August 2019, 8843 participants had normal hemoglobin (63.3%) as shown in Table (1) and 5129 participants had abnormal hemoglobin (36.7%) (Table 2). Particularly, 37.3% of these abnormal Hb reported with α-thalassemia, 11.2% with β-thalassemia and Hb E were 230 cases which reported 4.5% of total Abnormal Hb. Other abnormal Hbs (Hb H represented 0.2%, while Hb C, Hb D, Hb O Arab, and Hb HPFH) represented 0.0% (Table 3). It is reported that erythrocyte defense against malaria parasite invasion and development may interact to explain the high occurrence of Hb mutations in malaria locations (Chotivanich et al., 2002). The polymorphism balance in the defense mechanism is caused by hemoglobinopathies and thalassemia. Saudi Arabia experiences severe health issues such as Hb abnormalities, just like other nations with endemic malaria (Al-Suliman, 2006; Alsaeed et al., 2018).
 

 

 

       
Malaria screening performed on this abnormal Hb group revealed that 69 (1.4%) of the samples tested positive for the disease, whereas 5060 (98.7%) tested negative (Table 2). In this set of positive results, there were 26.1% Hb S, 11.6% thala/s, 15.9% β-thalassemia, 24% α-thalassemia and 4.4% Hb E. The mean age of the 230 Hb E participants was 27.4 years, with a range of 1-66 years, comprised 117 women (50.9%) and 113 men (49.1%). 55 people (23.9%) were single, while 175 (76.1%) were married. Patients’ reports on consanguineous marriage (41.7%), family history of hemoglobin E (64.8%) and history of malaria (21.3%) (Table 4). The electrophoresis report of Hb E patients showed 76.5% (176 patients) were heterozygote Hb A/E whereas 6.5% were homozygote Hb E/E. The combined Hb E was reported as 9.1% Hb S/E, 17.4% α-thala/E and 28.3% with β-thala/E (Table 4).
 

       
Hb E is an autosomal recessive β-gene, where a single point mutation at position 26 caused a disease (Chotivanich et al., 2002).
       
There was a significant change between WBCs, PLT, and RDW in Hb E patients and control (Table 5). As shown in Table (6), the difference between the PLT means of Hb E patients and controls was only marginally significant. RDW mean for patients was 16.3%, compared to 13.9% for controls, with a high significance detected by RBCs, Hb, Hct, MCV, MCH and MCHC as being extremely significant.
 

 

       
There was no Hb E found in healthy people, the mean Hb E level reported in patients was high (28.61±17.79%), while the mean Hb S level was 04.96±16.40%. Patients’ Hb A‚  average was 03.56±0.93%, while controls’ average was 02.96±0.15%, with a highly significant difference (P<0.0001) (Table 7).
 

       
Consanguineous marriage, which accounts for up to 50% of marriages in Saudi Arabia, is one of the major factors contributing to the spread of genetic abnormalities and the risk of inheritance among some families (Alsalem et al., 2022). Hemoglobinopathies have a significant impact on patients, their families and the government due to an increase in mortality and the associated financial, medical, psychological and social burdens (Zaini, 2016). Therefore, early detection and accurate diagnosis are crucial for controlling and preventing many illnesses (Ashley-Koch et al., 2000). Greater than in the western, northern and central regions, the Jazan region has the highest prevalence of Hb E and other hemoglobinopathies (Gosadi et al., 2021; Alenazi et al., 2015). Memish and his colleagues (2011) published an increased number of at-risk marriage groups in 2011 and refused to consult on marriages that attempted to avoid or control the prevalence of diseases in future generations among any age group or community. SCA and β-thalassemia are more common in Jazan than in the northern, western, and central regions, but they are still less common than in the eastern region (Akhter et al., 2021). Jazan has the highest incidence and prevalence of Hb E (Alenazi et al., 2015; Memish and Saeedi, 2011). Based on data from the current study, there are attempts to clarify differences between regions in hemoglobin diseases including Hb E. Some factors play an important role in increasing the prevalence and incidence of these diseases (Olivieri et al., 2011).
       
Here, 50.9% of the participants in the study were female patients. When compared to the control mean of 6.1 × 10y /L for WBCs and 299.2 × 10y /L for PLT (Table 8). RDW mean for patients was 16.4%, compared to 14.1%. Patients’ average Hb E concentration was 25.16±13.95%, while healthy volunteers had no Hb E. Additionally, patients’ Hb S means were 5.19±16.95% without being detected in healthy participants. Hb A and Hb A‚ in the patient’s blood were significantly different from the control (Table 9).
 

 

       
Utilizing ARMS-PCR and an allele-specific Hb E primer with a 462 bps, a genotyping investigation was conducted (Table 10). The bands on the electrophoresis of 2% agarose gel reflected the entire β-globin gene with 691 bps. 276 bps for the normal Hb E allele and 462 bps for the mutant Hb E allele (Fig 1). According to Sanger sequencing, the mutation replaced glutamic acid with lysine (GAG®AAG).
 

 

       
Through the study; 5 forms of Hb E zygosity were recorded, Hb A/E (76%) with no signs and symptoms. The nature of thalassemic features of Hb E is due to the replacement of glutamic acid by lysine on codon 26 of the β-globin chain (Balgir, 2007). This mutant reduced the production of variant β-globin and changed the normal migration by a cryptic splice site activation (Fucharoen and Weatherall, 2012).
       
The result showed 56% heterozygous of Hb E, 1% homozygous and 43% Hb E compound with other Hb variants. The microcytosis and hypochromic morphology of Hb E erythrocytes reflect the reduction of MCV and MCH values in patients, which were reported as 72 fL and 23 pg respectively. That provides the thalassemic features of Hb E disorder with microcytic hypochromic anemia.
       
This study demonstrated that the Jazan region of the Kingdom of Saudi Arabia had a higher prevalence of hemoglobinopathies (particularly Hb E) than other regions. In Hb E patients, we observed a considerable rise in Hb A‚ which exacerbates the disease and causes complications. Moreover, in certain families with an increase in the rate of consanguineous marriage, we also found significant incidences of Hb E. In some regions where malaria infection was formerly endemic, we also observed a rise in the prevalence of Hb E. This also shows the need for thorough studies, such as the clinical epidemiological status of Hb E prevalence in various Saudi Arabian regions.

Jazan had a higher prevalence of hemoglobinopathies (particularly Hb E). There was a significant difference in CBC parameters between Hb E and controls. In Hb E patients, we observed a considerable rise in Hb A‚ , which exacerbates the disease and causes complications. This shows the necessity for extensive research on Hb E prevalence in various countries and thorough clinical epidemiological research on Hb E.

This research was funded by the Researchers Supporting Project (RSPD2023R655), King Saud University, Riyadh, Saudi Arabia.

All the datasets generated or analyzed during this study are included in this published article.

The authors declare no conflict of interest.