As a preliminary step in amplifying the target area of the CAT gene, genomic DNA was isolated from blood samples as shown in Figure 1.
3.1 Polymorphism of CAT (rs7943316) Gene (Genotyping)
The results of gene polymorphism reveal the existence of a single band (149 bp) of the catalase gene target sequence (rs7943316) under optimal thermocycler apparatus conditions, as shown in Figure 2.
PCR products of the Catalase (rs7943316) gene were digested by the HinfI restriction enzyme, which cut the sequence (5ʹGˇANTC3ʹ) to recognize the rs7943316 in the Catalase gene as shown in Figure 3. Genotyping was classified into three categories according to presence/absence of polymorphism: TT homozygote with one band as 249 bp, AT heterozygote with three bands as 249, 175, and 74 bp, and AA homozygous with two bands as 175 and 74 bp fragments.
3.2 Distribution of CAT (rs7943316) Polymorphism Genotype with Allele Frequency Between Patients and Control Group
The distributions perceived in the Catalase gene polymorphism (rs7943316) in the case group and control group are shown in Table 1. The higher genotype in the healthy group was AT heterozygote genotype 19 (63.3%) followed by homozygote mutant genotype (TT) 11 (36.7%), homozygote genotype AA (0%). In the BC group, the higher genotype was AT heterozygote genotype 39 (55.7%), followed by the mutant homozygote genotype (TT) 24 (34.3%) and homozygote genotype (AA) 7 (10.0%). Individual carriers of the A/T and T/T genotypes were less expected to develop BC [OR = 0.135, 95% CI = 0.0073-2.4882, P value = 0.178] and [OR = 0.1420, 95% CI = 0.0075-2.70, P value = 0.1943], respectively. Also, there are insignificant differences between BC patients and healthy groups in frequencies of the T allele of the CAT gene (rs7943316)) as [OR=0.67, 95% CI=0.4002-1.4459, P =0.4039].
Table 1: Distribution of CAT (rs7943316) genotype and odd ratio among patients and control group.
Genotype
allele
|
Patient group
(n = 70)
|
Control group
(n = 30)
|
P value
|
OR
|
CI (95%)
|
No.
|
%
|
No
|
%
|
Codominant
|
AAa
|
7
|
10
|
0
|
0
|
|
AT
|
39
|
56
|
19
|
63
|
0.178
|
0.135
|
0.0073-2.4882
|
TT
|
24
|
34
|
11
|
37
|
0.1943
|
0.142
|
0.0075-2.70
|
Dominant
|
AA
|
7
|
10
|
0
|
0
|
|
AT+TT
|
63
|
90
|
30
|
100
|
0.1813
|
0.1388
|
0.0077 to 2.5103
|
recessive
|
AA+AT
|
46
|
66
|
19
|
63
|
|
|
|
TT
|
24
|
34
|
11
|
37
|
0.8191
|
0.9012
|
0.3695 to 2.1981
|
Over dominant
|
AA+TT
|
31
|
44
|
11
|
37
|
|
AT
|
39
|
56
|
19
|
63
|
0.48
|
0.7284
|
0.3022 to 1.7554
|
Alleles
|
A
|
53
|
38
|
19
|
32
|
|
T
|
87
|
62
|
41
|
68
|
0.4039
|
0.7607
|
0.4002-1.4459
|
P ≤ 0.05 ; OR=(95%CI); a reference
To comprehend the mechanisms underlying the pathologic process of this malignant tumor and linked pathological effects, such as attempts to improve the protection, diagnosis, and management of BC patients in Iraq, detecting genetic variations affecting BC is a prominent focus of research. According to research on the catalase gene polymorphism rs7943316, individuals with the mutant homozygous genotype (TT) and mutant heterozygous genotype (AT) had lower rates of BC than those with the wild type (AA). These results vigorously imply that the CAT gene polymorphism rs7943316 could lower the chance of BC in Iraqi patients. The antioxidant enzymes' genes contain numerous polymorphisms which may affect how effectively they detoxify ROS. Anaerobic metabolism naturally produces ROS [20]. To stop the hazardous buildup of these species, the human body creates a complex collection of antioxidant molecules [21]. All aerobic cells have the antioxidant molecule CAT, and the liver, kidney, and erythrocytes contain the largest concentrations of the enzyme [22]. By converting H2O2 into H2O and O2, the heme enzyme CAT is critical in avoiding hydrogen peroxide accumulation and preserving cells from the damaging effects of oxidative stress [23]. Allelic variations of the catalase gene may influence oxidative DNA damage and disease risk by lowering CAT enzyme activity and increasing susceptibility to ROS, changing detoxification of ROS, and increasing oxidative stress [24]. Many studies report that the CAT gene comes with three genetic polymorphisms in the promotor region: -21T (rs7943316), C-262T (rs1001179), and C-844T (rs769214) [25–27]. Another study showed CAT polymorphism associates the risk of breast cancer with several multifactorial diseases [18, 28]. Another study suggests that the main reason for breast cancer is regions that mutate and contain many numbers of sites that serve as binding sites for transcriptional factors [29]. Additionally, by changing the binding affinity of transcription factors, the polymorphism in the Catalase gene's promoter region (rs7943316) may result in changed promoter activity, alterations in gene expression, and reduced catalytic activity of the Catalase enzyme [30]. This is the first work where the association linking the rs7943316 variants of the catalase gene as they relate to BC in the Iraqi population is analyzed. Results of this study show an insignificant association between CAT -21T genotypes and breast cancer disease risk. There are several studies on the relationship between Catalase polymorphisms and the risk of various cancers [31], but there are very few studies on the correlation between the CAT-rs7943316 polymorphism and cancer risk. Several studies have proved that rs7943316 polymorphisms are unassociated with a risk of papillary thyroid carcinoma [32] or hepatocellular carcinoma risk [33]. Numerous researchers have submitted that Catalase polymorphisms may be linked with the possibility of many cancers such as cervical cancer, prostate cancer, pancreatic cancer, and colorectal cancer [7, 34–36]. There are several limitations to this study. First, the CAT gene contains a variety of other polymorphisms. Further study of the impact of CAT polymorphisms on mRNA levels should be conducted concurrently. Second, it has been noted that certain environmental factors, such as electromagnetic fields and medications, are linked to the mRNA levels of some antioxidant genes (like catalase, SOD2, and SOD1) [37].