Among all single-gene illnesses, β-thalassemia is among the most prevalent worldwide. There are several issues with haemoglobin synthesis, including a decrease in β-globin protein production.For diagnostic purposes, thalassemia can be classified as either "major," "moderate," or "intermediate" [1,2]. The prevalence of β-thalassemia is global. Most cases of β-thalassemia are caused by deletions, insertions or conversions in the β-globin gene's sequence [3,4].Over 200 variants related with β-thalassemia illness were identified globally [2,4]. Due to the vast spectrum of β-globin gene mutations, they vary from community to community; in each affected population, there are many -globin nucleotide variations that result in distinctive genetic mutations [5,6]. PCR, widely employed in laboratories due to its ease of use, has been utilised to identify more than 90% of -globin mutations [7,8]. On the other hand, PCR sequencing is expensive, time-consuming and often only detects a single mutation per reaction [9, 10].

Iron overload and immunological derangements are caused by transfusions and both of these things have a severe influence on the health of the immune system and how well it works in individuals with thalassemia who have undergone several transfusions [11]. To identify genetic variation of β-globin genes and complement levels C3 and C4 is the purpose of this study.

Patients Materials and Methods

Eighty patients receiving frequent blood transfusions at Al-Karama hospital in Baghdad Province were enrolled in this study; 45 men and 35 women were affected by the major type of β-thalassemia major. They were divided into three age-based groups (under 10 years, 11 to 20 years and over 20 years). The age and gender of the control groups were matched to those of the β-thalassemia major patients.

From February to December 2021, this study was done. Patients received daily therapy after a professional diagnosed them using blood film, Hb-electrophoresis and iron levels testing. An anticoagulant-free jell tube and an EDTA tube for DNA extraction were created from the blood that was drawn from a vein. Hemolysis-containing blood samples were ignored. Isolated serum was frozen at -20°C until further investigation and then used in the subsequent research.

• DNA Extraction: Carried out in the Laboratory of Bone Marrow Transplant center in Children Welfare Teaching Hospital, of Medical City Baghdad-Iraq, using Wizard Genomic Purification Kit (Promega corporation-USA), according to the method described by the manufacture
• DNA Amplification and Mutation Identification: Carried out in Italy by Dott. Marcello Morgutti (Servizio di Genetica, IRCCS "Burlo Garofolo", via dell 'Istria 65/1, 34100 TRIESTE, Tel.:0403785424, Fax: 0403785540, e mail: morgutti@burlo.trieste.it) using β-Globin StripAssayTM kit (ViennaLab Labordiagnostika GmbH- Austria) which uses (PCR) to find β-globin gene mutations, followed by reverse hybridization to specific wild and mutant oligoprobes made to find 22- β thalassemia

The assay cover 22 beta globin mutations as following:

• -87(C!G), 30(T!A), Codon 5(- CT), hemoglobin C (HbC), Hemoglobin S (HbS), "Codon
  5(-A), Codon 8(-AA), Codon 8/9(+G), Codon 22(7bp del), Codon 30(G!C),IVS 1.1(G!A), IVS 1.2(T!A), IVS 1.5(G!C), IVS1.6 (T!C), IVS 1.110(G!A), IVS 1.116(T!G),IVS 1-25(25bp del), Codon 36/37(-T), Codon 39(C!T), Codon 44(-C), IVS 2.1(G!A), IVS 2.745(C!G)"(ViennaLab Labordiagnostika GmbH)

Genotyping

Extraction of the DNA from the blood's periphery was performed using an updated technique [12]. Using a BioDrop spectrophotometer, the purity of the genomic DNA was ascertained. The samples were kept at a temperature of -20°C.

DNA Sequencing for β-Thalassemia Mutation Detection

When doing a PCR reaction, 100 ng of template was mixeds with 10 pmole of each primer. Table 1 shows the primer sequences that were employed [2].

Table 1: The Primer Sequences Utilised in the Genotyping Test

The master mix (BioLaps) was used in a total volume of 20 µl for the PCR reaction and the reaction's conditions are listed in Table 2.

Table 2: The Steps of the PCR Programme Employed in the Genotyping Test

Measurement of C3 and C4

LTA's commercially available single radial immunodiffusion plates were used to assess complement C3, C4 levels. Before being utilised, the samples of blood were frozen at -20 degrees Celsius. The results were presented as µ±S.D. for all variables. The SPSS computer programme was used to look at the data. The Student's t-test was used to compare any two groups of people the same age. Analysis of variance was used to compare-thalassemia with the other major patient groups (ANOVA). p<0.05 was thought to be the least significant value.

Genotyping

Figure 1 depicts the outcome of PCR for amplifying the β-globin gene in a single patient from this experiment, with a band size of 804 bp and no patient-to-patient fluctuation.

DNA _"sequencing results for the amplified PCR product of the β-globin gene revealed the existence of variations in the sequence for specific nucleotides when compared to the healthy β-globin gene sequence included in the National Center for Biotechnology Information database" (NCBIsequence ID: LC507563.1) (Table 3).

Table 3: Identification of the Locations and Types of Mutations in the β-Globin Gene Responsible for β-Halassemia in Children

The findings of ordering the DNA revealed that the β-globin gene has five mutations. Two of them are transition mutations and the other three are transversion mutations. In positions 250, 426, 623 and 630, respectively, C was substituted for A, G for A and T for A. In position 724, A was substituted for T. These changes in nucleotide sequences may result in abnormalities in the β-globin gene-encoded protein.In β-thalassemia patients, this mutation might induce aberrant β-globin gene expression, disrupting haemoglobin protein production [3,4,13].

Complement C3

The data show a significant (p<0.05) decrease in blood concentrations of C3 in all three age groups of βTM patients ("less than 5 years, 11-20 years and more than 20 years_") in comparison to matching age groups of healthy controls (Table 4).

Table 4: Comparison of the research groups' blood C3 concentrations (mg/dL) with those of age-matched, healthy controls

Figure 1: β-Globin PCR Result in-Thalassemia Patients. The 100 bp M Ladder and 804 bp PCR Result are Separated in 2% Agarose Gel

Complement C4

The findings showed that there was a statistically significant (p<0.05) decrease in C4 levels between BTM patients and healthy controls regardless of age (Table 5).

Table 5:Comparison of the Serum C4 Concentrations (mg/dL) Between the Groups Who Participated in the Research and Healthy Controls of the Same Age

Various letters reflect a statistically considerable difference at (p<0.05)

Table 6: Comparison of Serum Complement (C3, C4) Concentrations (mg/dL) Among βTM Patients of Varying Ages

Various letters reflect a statistically considerable difference at (p<0.05)

Table 6 shows a "comparison of serum C3 and C4 concentrations in TM patients of various" ages. There was a substantial (p<0.05) decrease in C3 concentration between the first and second age groups of TM patients but no significant differences between the second and third age groups. Serum concentrations of C4 showed no significant (p<0.05) variations between the various age groups of βTM patients.

The figures show the Mean±standard deviation. As shown in the current study, the mean levels of complements C3 (Table 4) and C4 (Table 5) were considerably lower in all disease groups compared to healthy control subjects of the same age. Age groups of thalassemia patients showed no considerable changes in serum C3 and C4 levels in comparison to one another (Table 6). The same finding was explored by James et al. [14-20] Amin et al. [21]. There is a similar rationale for the decline in complements 3 and 4. In our thalassemic patients, repeated blood transfusions may lead to ongoing exposure to different antigens, which might result in ongoing complement consumption [22-25].

The findings of this study revealed five distinct mutations in the β-globin gene, two transition mutations and three
transversion mutations, as well as that the mean levels of complements C3 and C4 in all patient groups were significantly higher than in the corresponding age group of healthy control subjects.

Acknowledgment

The authors like to express their deep appreciation to the University of alfarahidi for allowing them to make use of their facilities, which greatly improved the calibre of this research.

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