A Cross-Sectional Study of Detection of Beta Globin (HBB) Haplotypes Among Beta Thalassemia Patients

Introduction Beta-thalassemia is among the most common monogenic disorders in the Arabian Peninsula. This study aimed to investigate the β-globin (HBB) haplotypes among β-thalassemia patients in Saudi cohort which have potential implications in understanding the clinical care of patients and population genetic factors associated with β-thalassemia. Methods We analyzed 60 β-thalassemia patients. Male/female distribution for β-thalassemia was 58.33%/41.66%. Results of hematological parameters and indices were obtained from the database. HBB haplotyping assay was performed for four specific loci of the HBB gene cluster using polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) technique. Results HBB haplotyping assay identified three novel patterns namely haplotype 1, haplotype 2, and haplotype 3 and three common African haplotypes including Benin, Senegal, and Cameron. The frequency of haplotype 1 was the highest among the studied samples (62%, n = 37) with 56.76% (n = 21) observed in males compared to 43.24% (n = 16) in females. This was followed by Senegal, haplotype 2, Benin and haplotype 3 with similar percentage, and Cameron haplotype with 18%, 12%, 3% and 2%, respectively. The relationship between these haplotypes and various hematological parameters was calculated and our study found no significant relationship (p-value >0.05). Conclusion Our study indicated the importance of finding out types of β-globin haplotypes as novel types being discovered. Though no statistically significant association was identified among all the haplotypes in terms of hematological parameters, Cameroon or Benin haplotypes had the mildest form because they have the highest means among all parameters. Further studies need to be carried out on a larger population to detect the frequency of each specific mutation in each haplotype among β-thalassemia patients. This would help to re-address the question of the origin(s) of the β-thalassemia.


Introduction
Thalassemia syndromes are a heterogeneous group of inherited anemias characterized by a defective biosynthesis of the alpha (α) or beta (β) globin subunits of the normal adult hemoglobin HbA (a2β2). This defect can cause the formation of abnormal hemoglobin molecules resulting in low hemoglobin level. This group of inherited disorders is relatively common worldwide and considered as the most common singlegene disorders [1]. It is commonly found in the Mediterranean area, Middle East, Equatorial Africa, and Southern Asia and the clinical severity varies greatly from asymptomatic to severe hemolytic anemia.
β-thalassemia is considered as one of the most common monogenic disorders in Saudi Arabia. This is in fact due to the high percentage of consanguineous marriages [2]. It constitutes one of the major public health issues encountered by the ministry of health due to its high impact on clinical management costs [3]. The prevalence of β-thalassemia in Saudi Arabia is estimated to be 1.36% where 0.07% represents the major type and 1.29% represents the trait (minor) type [4,5]. Various studies on the prevalence of β-thalassemia conducted over 20 years demonstrated that the Eastern province has the highest number of cases [6]. Molecular pattern of β-thalassemia showed a wide spectrum of mutations affecting the HBB and novel 1 2 1 1 1 1 mutations are being discovered as the diagnostic tools are becoming more advanced [7]. It is noteworthy to mention that even though β-thalassemia is highly prevalent in the Eastern Province, a wide spectrum of clinical symptoms for this disorder is reported from the Western Province [8]. This variation could be contributed to the differences in the β-globin gene (HBB) haplotypes.
A haplotype can be defined as a set of genes of one chromosome that have been inherited together from a single parent [9]. HBB haplotypes settle on different combinations of polymorphic sites of HBB through the non-random link of the single point called restriction fragment length polymorphism (RFLP) [10]. Several mutations are found within and around HBB, resulting in different types of HBB haplotypes and thus causing population diversity [11]. The diversity in HBB haplotypes reflects the evolutionary and genetic relationship among this disorder in different ethnic backgrounds [12]. The importance of identification of HBB haplotype comes through the determination of DNA sequence variation across geographical populations. Those variations often caused by the transmission of genetic variations through populations from one generation to the next [13].
In Saudi Arabia, few studies have been performed to explore the molecular patterns of β-thalassemia [14][15][16]. In this study, we aimed to determine the HBB haplotypes among β-thalassemia patients in the Saudi cohort and correlate between these haplotypes and red cell parameters.

Sample collection and laboratory investigation
A total number of 60 blood samples from previously diagnosed β-thalassemia patients were obtained after consents were signed. β-globin (HBB) haplotyping assay HBB haplotyping assay was performed for four specific loci of the HBB gene cluster using polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) as previously described by Sutton et al. [17]. Briefly, the four loci were amplified by specific primer sets to generate PCR fragments of 655 bp, 771 bp, 766 bp and 388 bp for primer set numbers 1, 2, 3 and 4 respectively as shown in Table 1. These fragments contained sites for the restriction sites of XmnI (5' Gγ), HindIII (Gγ), HindIII (Aγ) and HinfI (5'/β) which were digested by the restriction enzymes. Identification of HBB haplotypes was determined through positive and/or negative reactions to digestion with restriction enzymes at these loci in order to generate specific patterns of a haplotype as demonstrated in previous works [18,19].

Statistical analysis
All statistical analysis in this study was carried out using either one-way ANOVA or Kruskal-Wallis test using SPSS, version 22 (IBM Corp., Armonk, NY, USA). A p-value < 0.05 was considered statistically significant. Regression analysis was used to study the association of HBB haplotypes with changes in the hematological parameters including red blood cell count (RBC), hemoglobin (Hb), hematocrit (Hct), mean cell volume (MCV), mean cell hemoglobin (MCH), mean cell hemoglobin concentration (MCHC), and red cell distribution width (RDW). Post hoc analysis was performed to find statistical significance among or in between the groups.

Results
Recognition patterns of HBB haplotype generated from digestion of amplified regions with the restriction enzymes showed some common African haplotypes including Benin, Senegal, and Cameron while three other novel patterns namely haplotype 1, haplotype 2, and haplotype 3 were identified ( Table 2). We found that the frequency of haplotype 1 was the highest with 62% (N = 37) with a mild predominance of males (56.76%, n = 21) compared to females (43.24%, n = 16). This was followed by Senegal haplotype (18%, N = 11), H2 (12%, N = 7), Benin (3%, N = 2), H3 (3%, N = 2) and Cameroon (2%, N = 1) as shown in Figure 1.   The association of red cell parameters among the various haplotypes of the cohort study was investigated and the results indicated that there is no statistically significant relationship between the haplotypes in terms of hematological parameters as all showed no significant scores (p-values above 0.05) ( Table 3).  Further, post hoc analysis was performed to see the statistical significance among and in between the haplotypes for each of the hematological parameters. As the Cameroon haplotype had just one sample, it was removed from the post hoc analysis. Though no statistically significant relationship between all the haplotypes in terms of hematological parameters were found.

Hematological parameters
Post hoc analysis showed significant difference between Benin and Haplotype 1 (p-value 0.012), Benin and Haplotype 2 (p-value 0.018), and Benin and Senegal (p-value 0.011) Haplotype for the MCV parameter. Similarly, significant difference was seen between BEN and Haplotype 1 (p-value 0.039), BEN and Haplotype 2 (p-value 0.044), and BEN and SEN (p-value 0.018) Haplotype for the MCH parameter (Table is attached in  the appendices section).
To find out if the most frequent haplotypes from our cohort (i.e., H1, H2, and Senegal) have some effects on red cell parameters, we analyzed the relationship between them and found no significant association ( Figure 2). The reason behind grouping H1, H2 and Senegal haplotypes was to find out if these haplotypes showed any differences in the red cell parameters, as together they formed the major number of samples (N = 55).

FIGURE 2: Comparison between the most frequent haplotypes (H1, H2, and Senegal) and the hematological parameters
Further, a multinomial logistic regression analysis was used to analyze the association of HBB haplotypes with changes in the red cell parameters including RBC, Hb, Hct, MCV, MCH, and MCHC ( Table 4). As can be seen from the significance (Sig.) column, there was no significant association between the changes in levels of the red cell parameters according to the haplotype. This indicates that red cell parameters are independent of HBB haplotype.

Discussion
Our study was designed to determine the HBB haplotype among β-thalassemia patients and to analyze their effects on red cell parameters. Haplotype analysis has a statistical power because it reduces the association test dimensions. Rather than using genotyping individually, the haplotype analysis identifies common genetic features in a population [10]. Apart from this, several previous studies have shown that HBB haplotype analysis provides crucial information on genetic variations and haplotypes of different populations [20][21][22]. A study by Zhang et al., on genetic heterogeneity of HBB in various geographic populations of Yunnan in Southwestern China, identified seven different HBB haplotypes among 41 βthalassemic chromosomes, where haplotype I and haplotype V were more than 65% (28 β-thalassemic chromosomes), while the remaining percentages were distributed to the other haplotypes [13]. Our study identified six haplotypes among 60 β-thalassemic patients from the Saudi cohort. The distribution of HBB haplotypes among β-thalassemic patients in our study showed that haplotype 1 was the highest with 62% (37 samples). This was in accordance with studies by various groups in the Mexican, Turkish and Western Iranian population which identified haplotype 1 to be the most prevalent haplotype among β-thalassemia chromosomes although restriction sites pattern is different [11,21,23]. Haplotype analysis of β-thalassemia patients was carried out in Western Iran by analyzing the pattern of seven restriction sites through the HBB gene cluster. It was found that haplotype I was the most prevalent haplotype with 35.7% among βthalassemia chromosomes followed by haplotype III with 28.6% [11]. A previous haplotype analysis of three common β-thalassemia mutations performed in Syrian patients found out four HBB haplotypes among the studied patients where haplotype I was the most frequent one [23].
The HBB haplotyping plays a role in the detection of severity level through observing the hematological parameters in hemoglobinopathies. The correlation between Arab-Indian haplotype and Benin haplotype indicated that the mean of RBC count was higher in Arab-Indian haplotype (3.9 million cells/mcL) than in Benin haplotype (3.0 million cells/mcL) indicating the milder form of this haplotype. Moreover, the mean Hb concentration was also higher in Arab-Indian haplotype (10.8 g/dl), than in Benin haplotype (8.4 g/dl). These differences in hematological parameters among the two haplotypes explained the diverse range in severity among HBB haplotypes [8]. In our study, the hematological parameters among the six haplotypes (Benin, Senegal, Cameroon, haplotype 1, haplotype 2, and haplotype 3) were investigated. The mean of RBC count was very close to about 3 million cells/mcL in all haplotypes except in haplotype 3 which was less than 2.5 million cells/mcL. The mean of Hb concentration in all haplotypes was above 7 g/dl, except haplotype 2 and haplotype 3. The mean was lowest in haplotype 3 with 5.35 g/dl. It was higher than 9 g/dl in Benin and Cameroon, which was consistent with Hb concentration for Benin haplotype in El-Hazmi findings [8]. Although there was no significant alteration in red cell parameters among all haplotypes, we have observed that RBC parameters of haplotype 3 were the most affected ones since the anemia was more severe than others. Furthermore, HBB haplotypes are known to influence the severity of HBB diseases through hematological parameters [24]. A previous study about the effect of HBB cluster haplotype on hematological and clinical features of HBB mutation that cause sickle cell anemia was performed on 113 black American adults. The study found that Bantu haplotype usually is commonly associated with a severe form of the disease more than Benin and Cameroon haplotype where blood parameters in Benin and Cameroon haplotypes were characterized by mild clinical severity [25]. Though there is no Bantus haplotype in our study, we had a similar result for Benin and Cameroon haplotype where there was no significant difference between the patients. Additionally, all the haplotypes showed no significant difference among themselves in terms of the red cell parameters. However, when a pair wise comparison of significance was performed for each of the hematological parameters, we observed significant difference between Benin and Haplotype 1, Benin and Haplotype 2, and Benin and Senegal Haplotype for only MCV and MCH parameters, indicating that there is significant difference in severity of β-thalassemia for only the Benin, Haplotype 1, Haplotype 2 and Senegal haplotype as indicated from the hematological parameters MCV and MCH.

Conclusions
Our study indicated the importance of finding out types of β-globin haplotypes as novel types being discovered. Though no statistically significant association was identified between all the haplotypes in terms of hematological parameters, but for MCV and MCH we identified in severity of β-thalassemia for only the Benin, Haplotype 1, Haplotype 2 and Senegal in the studied cohort. Our result is a preliminary analysis representing the distribution of β(S) haplotypes in the Western province of Saudi Arabia and future studies need to be carried out on a larger population to detect the frequency of each specific mutation in each haplotype among β-thalassemia patients. This would help to re-address the question of the origin(s) of the β-thalassemia.

Additional Information Disclosures
Human subjects: Consent was obtained or waived by all participants in this study. Ethics and Research Committee at Faculty of Applied Medical Sciences, King Abdulaziz University issued approval FAMS-2019-003. Animal subjects: All authors have confirmed that this study did not involve animal subjects or tissue.

Conflicts of interest:
In compliance with the ICMJE uniform disclosure form, all authors declare the following: Payment/services info: All authors have declared that no financial support was received from any organization for the submitted work. Financial relationships: All authors have declared that they have no financial relationships at present or within the previous three years with any organizations that might have an interest in the submitted work. Other relationships: All authors have declared that there are no