Association between TBX20 gene polymorphism and congenital heart disease among Egyptian subjects

Background: T-box-transcription-factor-20 (TBX20) is a major type of T-box protein family that plays a plausible function in the optimum functional and developmental of cardiac tissues. This work aimed to assess the contribution of TBX20 gene polymorphisms with elevated risk of congenital heart disease (CHD) among Egyptian subjects. Methods: This case-controlled study consisted of 175 participants [75 patients diagnosed with CHD along with 100 unrelated healthy controls], matched with age and gender. The genetic variant for TBX20* was genotyped and characterized with the aid of the polymerase chain reaction-restriction fragment-length-polymorphism (PCR-RFLP) technique. Results: Our findings revealed a higher frequency of TBX20*(T/T) genotype among CHD patients compared with healthy controls. Additionally, testing genetic association models revealed a significant association with protection against CHD in the recessive model. This might imply this rare genotype is a predisposing factor to protect against congenital heart disease. Moreover, the TBX20(C/C) genotype exhibited higher prevalence among males than females among CHD compared to unrelated healthy controls. On the contrary, the frequency of TBX20*(T/T) genotype was higher among both males and females than healthy controls. The frequency of TBX20*(C/C) genotype within TOF cases was higher in CHD cases compared to healthy controls. However, the frequency of TBX20*(T/T) genotype was significantly associated with VSD cases compared to healthy controls. Conclusions: In conclusions, our findings revealed the protective function of the TBX20*rs3999950 gene variant against the development of congenital heart diseases among Egyptian subjects.


Introduction
Congenital heart defect (CHD) is a type of defect in the structure and/or function of the heart tissues that is attributed to abnormal heart development before birth (1,2).It is the most frequent congenital anomaly that ranges from five per one hundred live births worldwide (3).Recently, CHD has been considered the crucial reason for childhood mortality and morbidity globally (4).After precise literature screening, numerous studies have been conducted investigating the existence of congenital heart diseases among children due to various genetic factors along with epigenetic modifications 5,6 .
T-box transcription factor (TBX20) is considered a crucial member of the T-box family that contributed to many biological processes and shares a definite conserved deoxyribonucleic acid-binding domain 7 .
In addition, it plays a potential role in the optimum functional and developmental of cardiac tissues including the formation of cardiac septum, and hence prevents the advancement of congenital heart diseases (8).Moreover, several articles observed that many family members were exposed to normal cardiac function throughout initial embryogenesis including TBX5 and TBX20 (9).The TBX20 gene is situated on chromosome number 7p14.2 and consists of two main splice variants with eight exons and seven introns along the minus strand (10)  Generally, the extraction of genomic DNA was executed from all participants in this work using the phenol-chloroform method in the presence of proteinase K digestion.The purification of extracted DNA was evaluated with the aid of Nanodrop™ ND-1000 Spectrophotometer (18).

Genotyping and amplification of TBX20*rs3999950 variant
The amplification and characterization of the μL PCR product, and 5 μL 10X buffer followed by electrophoresis at 60 mV for 25 minutes, and the imaging process was taken with ethidium bromide.

Statistical analysis
All tabulated data obtained from this work was

Genetic and allelic frequencies of TBX20*(rs3999950; c.774T>C) variant among studied subjects
The distribution of allelic/genotypic for

Results of agarose gel electrophoresis of the PCR products
The interpretation of agarose gel electrophoresis results was applied with the aid of illumination under UV light for the TBX20*(rs3999950; c.774T>C) variant.The product size of this variant was visualized at 350 bp which corresponds to the designed primers.The digested fragments of the TBX20*(rs3999950; c.774T>C) variant were processed by applying an enzymatic endonuclease digestion system using EcoRI and XbaI.3. Our results indicated that the frequency of TBX20*(C/C) genotype was higher in male patients compared to healthy controls (OR=0.33,95% CI=0.13-0.82).On the other hand, the frequency of TBX20*(T/T) genotype was higher among both male and female patients compared to healthy controls (OR=0.06,95% CI =0.01-0.53).

Association of the TBX20*(rs3999950; c.774T>C) variant stratified by different types of CHD compared to healthy controls.
The correlation between TBX20*(rs3999950; c.774T>C) variant stratified by different types of CHD compared to healthy controls was displayed in The TBX20 gene is situated on chromosome number 7q14.
Recently, various mutations were identified in the TBX20 gene due to the acquisition or lack of the function of this transcription factor and are thought to be associated with a diverse of heart malformations involving cardiac septal defects, 12).Based on the fact that screening the single nucleotide variants (SNV) of certain genes is the key method to identify the hereditary predispositions related to specific issues, thus studying the cellular mechanisms by which the pathogenesis of these diseases could be raised(13-  15).Other reports investigated the potential function of the TBX20 gene with other important genes and illustrated the signaling pathways by which this transcription factor maintained the proper function of cardiac tissues(12,16).Taken together, this work was designed to explore the association of the TBX20*(rs3999950; c.774T>C; Ala265Ala) variant with the susceptibility and development of congenital heart disease among Egyptian children.

TBX20*rs3999950 variant
were accomplished with the aid of polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) technique, as formerly described 12 .The two primers used in the amplification process were [Forward primer (F1): 5'-CCT CAC TGT AAT TTG GCC TG-3', while the reverse primer (R1): 5'-GCC CTG AAA CTC AAT AGC TC-3'].The PCR reaction was adjusted in a total volume of 25 μL containing 3 μL purified template DNA, 2 μL of forward primer, 2 μL of reverse primer, 13 μL of PCR Master Mix (2×), and 5 µl nuclease-free sterile water (ddH2O).The PCR conditions were manipulated with an initial denaturation step for 7 minutes at 94⁰ C, followed by 28 cycles of 94⁰ C for 30 seconds, 50⁰ C for 60 seconds, and 72⁰ C for 30 seconds with a final extension step for 5 minutes at 72⁰ C. The visualization of PCR products was performed with the aid of 3% agarose gel using 15 analyzed using IBM SPSS software version 22.0 (SPSS Inc, Chicago, USA).The equilibrium of the Hardy-Weinberg equation was performed using Fisher's exact chi-square test.The allelic/genotypic analysis of the TBX20*rs3999950 variant was applied using the online SNPstats tool for genomic analysis (https://www.snpstats.net/start.htm,accessed on 20 May 2023).The odd ratios (OR) and 95% confidence intervals (CI) were evaluated among patients compared to unrelated healthy controls.The significance of interests regarding the statistical analysis was adjusted with a p-value less than 0.05.
rs3999950; c.774T>C) variant with an elevated risk of CHD compared to healthy controls were illustrated in Table 2.As depicted, the frequency of the TBX20*(T/T) genotype was higher among CHD patients compared to healthy controls (16% vs. 2% respectively).This might shed light on the potential role of this genotype as a predisposing factor to the occurrence of congenital heart disease.Our findings showed a significant association with CHD patients under the codominant model compared to healthy controls (C/C vs. C/T vs. T/T; OR= 0.1, 95% CI= 0.02-0.49,p-value = 0.0023).Similarly, upon testing the recessive model, the TBX20*(rs3999950; c.774T>C) variant represented statistical difference among CHD patients compared to control groups (T/T vs. C/C + C+T; OR=0.11; 95% CI=0.02-0.49,p-value = 0.0005).
3 and is represented by eight exons.Recently, seven members of the T-Box gene family were expressed and translated during the embryonic level and within cardiac vertebrates including TBX1, TBX2, TBX3, TBX4, TBX5, TBX18, AND TBX20 (12,22).The transcription activity for the TBX20 gene shared different domains that interact with various transcription factors involving GATA4, and TBX5 to adjust the degree of synthesis related to embryonic hearts (23).Numerous genetic risk factors contributed to the event of congenital heart diseases within homo sapiens, and these variables include the posttranslational modifications and genetic mutations within the coding region related to the TBX20 gene and their correlations with congenital cardiac disorders (24,25).Our findings indicated the association of TBX20*(rs3999950; c.774T>C) genetic variant with congenital heart diseases among Egyptian subjects.In a study done by Kirk et al. that reported the complete map of full coding regions within the TBX20 gene in 353 CHD patients, they indicated the contribution of two novel heterozygous mutations TBX20*(p.I152M and p.Q195X) among two index familial CHD patients (11).Interestingly, they observed that family one carried the missense mutation of TBX20*p.I152M with atrial congenital heart and ventricular septal defects, while family two was found to carry the other mutation of TBX20*p.Q152X with atrial defect, left atrioventricular valve, dilated cardiomyopathy, and coarctation of the aorta (11).Another study screened the potential role of the TBX20 gene within 192 unrelated CHD children and implicated the involvement of two mutations in four CHD children including two children with TBX20*p.H186D mutations accompanied by two children with TBX20* p.L197P mutations (1).In addition, a report performed among 203 unrelated CHD patients using sequence analysis technique for the TBX20 gene from exon two to exon six revealed three other nonsynonymous mutations in three CHD patients with atrial septal defects including TBX20*(p.A63T, p.I121F, and p.T262M) (26).Another record studied the total set of coding regions within the TBX20 gene among 170 unrelated subjects suffering from atrial septal defect using sequencing analysis, they observed a novel mutation within one patient with a positive family history and this mutation was referred to as TBX20*p.I121M (27).Moreover, a three-generation family diagnosed with atrial septal defects was screened without functional analysis and identified a rare mutation related to this gene called TBX20*(p.D176N) (28).Furthermore, another group team was subjected to perform sequencing technique for flanking introns and their corresponding coding exons for the TBX20 gene among 146 CHD patients and identified the contribution of various TBX20 mutations in diminishing the transcriptional level including TBX20*(p.R143W) (29).Another report examined 38 patients with atrial/ventricular septal defect and identified the contribution of various mutations within the TBX20 gene in the pathogenesis of the disease but without functional characterizations for these target mutations including TBX20*(p.Y309D, p.T370O, and p.M395R) (30).Additionally, another article revealed the association of genetic variants within the TBX20 gene with an elevated risk of congenital septal heart defects (31).Moreover, Yn et al. identified lower activity for the TBX20 gene and its function in diminishing CHD risk among Chinese subjects (32).Another report studied the potential impact of the TBX20*rs3999950 variant and identified the role of the TBX20*(T/C) genotype in the development of congenital heart diseases among Chinese children (12).Interestingly, this work confirmed the involvement of TBX20*(rs399950; T/T genotype) against the development of congenital heart diseases among Egyptian subjects.This finding implied that this genetic variant could contribute as a potential factor in decreasing the adverse effects of CHD among Egyptian populations.On the contrary, Lin et al. observed the role of the TBX20 gene in the progression of congenital heart diseases (16).Fewer limitations were identified through the establishment of this work in the form of a relatively small pilot sample size; therefore, our team recommends performing another large-scale multicenter survey to illustrate the functional mutations within the TBX20 gene and their roles in the susceptibility for CHD among Egyptian subjects.ConclusionThis study confirmed that the TBX20*rs3999950 variant showed a protective effect against the development of congenital heart defects among Egyptian populations.