European Journal of Cardiovascular Medicine

Congenital Heart Diseases (CHDs) are structural anomalies of the heart and great vessels that arise during fetal development, resulting in significant morbidity and mortality worldwide. They represent the most common congenital anomalies, affecting approximately 1% of live births globally (1). Early detection of CHDs is essential for timely intervention and effective management to improve survival rates and quality of life among affected neonates (2).

Advancements in prenatal imaging techniques, such as echocardiography and ultrasonography, have improved the antenatal diagnosis of CHDs; however, limitations remain, particularly in detecting complex heart defects (3). Consequently, genetic studies have emerged as promising tools for enhancing early diagnostic capabilities. Genetic markers associated with CHDs are increasingly being investigated to provide insights into their molecular basis and improve diagnostic accuracy (4).

Research has identified several candidate genes implicated in the pathogenesis of CHDs, including transcription factors and structural proteins essential for cardiac development. Among these, mutations in genes such as GATA4, NKX2-5, and TBX5 have been frequently reported to play critical roles in the development of congenital heart malformations (5,6). For instance, GATA4 and NKX2-5 are transcription factors involved in the regulation of cardiac morphogenesis and structural integrity, while TBX5 plays a pivotal role in septation and chamber formation (7).

Previous studies have demonstrated the association between genetic variants in these genes and a wide spectrum of CHDs, including atrial septal defects, ventricular septal defects, and tetralogy of Fallot (8,9). Genetic screening of these markers in neonates can provide valuable information for early diagnosis and risk assessment, which is crucial for implementing prompt therapeutic interventions (10).

This study aims to evaluate the effectiveness of genetic markers, particularly GATA4, NKX2-5, and TBX5, in the early detection of congenital heart diseases among neonates. By comparing the prevalence of these genetic mutations between neonates with CHDs and healthy controls, we seek to establish a reliable genetic screening approach that can be incorporated into routine neonatal screening programs.

Study Design and Setting:

This prospective case-control study was conducted over a period of 24 months at a tertiary care hospital. The study aimed to evaluate the effectiveness of specific genetic markers in the early detection of congenital heart diseases (CHDs) in neonates.

Study Population:

A total of 150 neonates were enrolled in the study, divided into two groups:

• Case Group: 75 neonates diagnosed with CHDs based on clinical examination and echocardiography.
• Control Group: 75 healthy neonates with no evidence of CHDs or other congenital anomalies.

Inclusion criteria for the case group included neonates diagnosed with CHDs within the first week of life, while the control group consisted of healthy neonates born at the same hospital during the study period. Neonates with chromosomal abnormalities or other major congenital anomalies were excluded from the study.

Sample Collection:

Peripheral blood samples (2 mL) were collected from each neonate within 24 hours of birth under sterile conditions. The samples were stored at -20°C until further processing.

Genetic Analysis:

Genomic DNA was extracted from the collected blood samples using a standard phenol-chloroform extraction method. The quality and quantity of the extracted DNA were assessed using spectrophotometry.

Polymerase Chain Reaction (PCR) was employed to amplify specific gene segments associated with congenital heart diseases. Three candidate genes were selected for analysis based on their established roles in cardiac development: GATA4, NKX2-5, and TBX5. The primers for each gene were designed using Primer3 software, ensuring high specificity and efficiency.

PCR conditions were optimized for each gene, with thermal cycling parameters set as follows: initial denaturation at 95°C for 5 minutes, followed by 35 cycles of denaturation at 95°C for 30 seconds, annealing at 58°C for 45 seconds, and extension at 72°C for 1 minute. A final extension step was performed at 72°C for 10 minutes.

The amplified PCR products were subjected to agarose gel electrophoresis and visualized under UV light. Positive samples were further purified and sequenced using the Sanger sequencing method to identify specific mutations or polymorphisms.

Statistical Analysis:

The collected data were analyzed using SPSS software version 25.0. Descriptive statistics, including means and standard deviations, were calculated for quantitative variables. The prevalence of genetic mutations was compared between the case and control groups using the chi-square test. Sensitivity, specificity, positive predictive value, and negative predictive value of each genetic marker were calculated. A p-value of <0.05 was considered statistically significant.

A total of 150 neonates were enrolled in the study, with 75 in the case group (neonates diagnosed with CHDs) and 75 in the control group (healthy neonates). The demographic characteristics of both groups were comparable, with no significant differences observed in terms of gender distribution, birth weight, or gestational age (p > 0.05).

Prevalence of Genetic Mutations:

The frequencies of mutations in the GATA4, NKX2-5, and TBX5 genes were significantly higher in the case group compared to the control group (p < 0.001). The distribution of genetic mutations in both groups is shown in Table 1.

Table 1: Distribution of Genetic Mutations in Case and Control Groups

As shown in Table 1, the presence of GATA4 mutations was most prevalent among the case group (45.3%), followed by NKX2-5 (40.0%) and TBX5 (34.7%). These rates were significantly higher compared to the control group (5.3%, 6.7%, and 4.0%, respectively).

Diagnostic Accuracy of Genetic Markers:

Sensitivity, specificity, positive predictive value (PPV), and negative predictive value (NPV) were calculated for each genetic marker. The results are summarized in Table 2.

Table 2: Diagnostic Accuracy of Genetic Markers

The combined analysis of GATA4, NKX2-5, and TBX5 markers yielded the highest diagnostic accuracy, with a sensitivity of 92.0% and specificity of 88.0% (Table 2).

Statistical Analysis:

Chi-square analysis confirmed that the frequency of genetic mutations was significantly higher in the case group compared to the control group for all three genes (p < 0.001). The overall diagnostic performance of the genetic markers was considered satisfactory, supporting their potential use in neonatal screening for CHDs.

The findings of this study suggest that genetic markers GATA4, NKX2-5, and TBX5 are significantly associated with congenital heart diseases (CHDs) in neonates, thereby supporting their potential use as diagnostic biomarkers for early detection. The results are consistent with previous studies that have demonstrated the role of these genes in cardiac morphogenesis and structural development (1,2).

Mutations in GATA4 have been reported to cause various cardiac defects, particularly atrial and ventricular septal defects, by disrupting transcriptional regulation during heart development (3,4). This study found a high prevalence of GATA4 mutations (45.3%) among the case group, which aligns with previous studies reporting similar associations (5). Similarly, mutations in NKX2-5 were identified in 40.0% of the case group, highlighting its role in cardiac conduction system development and atrioventricular septal formation (6,7).

The TBX5 gene, which plays a crucial role in septation and chamber formation, was found to be mutated in 34.7% of the case group, which is comparable to previous findings indicating its association with Holt-Oram syndrome and isolated cardiac malformations (8,9). Notably, the combined analysis of all three genetic markers demonstrated superior diagnostic accuracy, with sensitivity and specificity values of 92.0% and 88.0%, respectively. This suggests that multi-gene panels could enhance the precision of neonatal genetic screening programs.

Comparative studies have reported similar findings, with some suggesting that integrating genetic testing with conventional diagnostic approaches could significantly enhance early detection rates of CHDs (10,11). Moreover, the use of Polymerase Chain Reaction (PCR) and Sanger sequencing in this study proved to be effective for identifying genetic mutations, as reported in previous studies utilizing similar methodologies (12,13).

However, this study has certain limitations that need to be addressed. Firstly, the sample size is relatively small, which may limit the generalizability of the findings. Secondly, only three genetic markers were evaluated, whereas other potential markers such as TBX20, ZFPM2, and HAND1 have also been implicated in the pathogenesis of CHDs (14,15). Additionally, this study did not assess the phenotypic correlation of the identified mutations, which could provide a better understanding of genotype-phenotype relationships.

Future research should focus on larger cohort studies to validate these findings and explore the inclusion of additional genetic markers to enhance diagnostic sensitivity and specificity. Moreover, the integration of Next-Generation Sequencing (NGS) could provide a comprehensive assessment of genetic variations associated with CHDs, offering valuable insights for personalized diagnostic and therapeutic approaches).

Overall, the findings of this study support the feasibility of using genetic markers such as GATA4, NKX2-5, and TBX5 for the early detection of congenital heart diseases in neonates. Incorporating genetic screening into routine neonatal screening protocols may significantly improve early diagnosis and clinical outcomes for affected neonates.

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