Introduction

Cardiovascular diseases (CVDs) are the leading cause of mortality globally, accounting for approximately 19.8 million deaths annually, or 32% of all global deaths. Among these, ischemic heart disease and stroke remain the most significant contributors. The burden of CVDs varies across regions and populations, influenced by socioeconomic disparities, healthcare infrastructure, access to preventive services, and underlying genetic and lifestyle factors [1,2]. In low and middle income countries (LMICs), the CVD burden is disproportionately higher due to limited access to quality healthcare, late presentation to hospitals, and an increased prevalence of modifiable risk factors including hypertension, obesity, smoking, and diabetes [3]. In Pakistan, the age-standardized incidence rate of CVDs is estimated at 918.18 per 100,000 populations, which surpasses the global average of 684.33 per 100,000 [4]. The age-standardized death rate from CVDs in Pakistan is 357.88 per 100,000, also significantly exceeding the global average of 239.85 per 100,000 [5]. While traditional risk factors such as tobacco use, physical inactivity, unhealthy diet, and obesity remain significant, genetic predisposition plays an increasingly recognized role in CVD susceptibility [2,6]. Family history of premature coronary artery disease (CAD) is associated with a two-fold increase in risk, independent of lifestyle-related factors [7]. Heritability estimates for CAD range between 40% and 60% based on family-based and twin studies, particularly in early-onset cases [8]. Advances in genomic epidemiology and high-throughput sequencing technologies have revolutionized our understanding of genetic contributions to CVD, allowing for the identification of numerous risk loci through genome-wide association studies (GWASs) [9,10]. A landmark discovery in 2007 identified the 9p 21.3 locus as a significant contributor to CAD risk, independent of conventional risk factors [11]. This 53-kb region is highly conserved and confers a ~ 40% increased risk for CAD, especially in homozygous individuals [12]. Large consortia such as CARDIOGRAM and C4D have subsequently expanded the list of CAD-associated loci, identifying more than 160 common variants that contribute cumulatively to disease risk [13,14]. However, most of these variants confer only modest effect sizes (OR < 1.15), suggesting the need to explore rare genetic variants that may explain the so called missing heritability in CVD [15,16]. Recent advances in next-generation sequencing (NGS) have facilitated the discovery of both common and rare variants that influence CVD risk [17]. NGS technologies have significantly reduced the cost and time required to sequence entire genomes, allowing for more comprehensive exploration of both monogenic and complex polygenic forms of CVD [18]. Rare variant analysis, as part of the NHLBI Exome Sequencing Project and 1000 Genomes Project, has identified pathogenic mutations that are particularly relevant in early-onset or familial cases of CVD [19]. Among the key genes of interest in CVD genetics is LRP6 (Low-Density Lipoprotein Receptor-Related Protein 6), a critical co-receptor in the Wnt signaling pathway [20,21]. This pathway regulates essential processes such as cellular proliferation, differentiation, and tissue maintenance. Aberrant Wnt signaling, mediated through LRP6 dysfunction, has been implicated in a wide spectrum of cardiovascular pathologies including arrhythmias and structural heart defects [22]. LRP6 is prominently localized at cardiac gap junctions and contributes to normal electrical conduction and cardiac rhythm. Its dysfunction can compromise intercellular communication and predispose individuals to arrhythmias and cardiomyopathies [23]. Another important gene in cardiovascular remodeling is MEF2A (Myocyte Enhancer Factor 2A), a transcription factor involved in cardiac development, endothelial function, and hypertrophy signaling [24]. A 21-base pair deletion in exon 11 of MEF2A was first reported in a Scandinavian family with early-onset CAD and myocardial infarction (MI), disrupting nuclear localization and transcriptional activity [25]. Functional variants in exon 7 (e.g., Asn263Ser, Pro279Leu, and Gly283Asp) have also been identified exclusively in affected individuals, reinforcing its role in endothelial dysfunction and atherosclerosis [26]. MEF2A integrates signals from key hypertrophic pathways including CaMKII, MAPK, and PI3K/Akt, with post-translational modifications such as phosphorylation and SUMOylation modulating its transcriptional activity [27]. Although MEF2A was initially proposed as a causative gene for CAD, it has not consistently emerged as a significant risk locus in large-scale GWASs. However, the highly polymorphic nature of exon 11, including the (CAG)n repeat region, continues to be investigated in various populations [28]. In hypertensive hearts, MEF2A expression is upregulated in response to pressure overload, contributing to pathological cardiac remodeling and heart failure [29]. CVD remain a major public health concern, particularly in younger populations in developing regions. Genetic factors play an important role in disease susceptibility, especially in familial cases. However, there is limited data on genetic variants in Pakistani high-consanguinity populations. Therefore, the present study was designed to explore pathogenic variants in selected CVD related genes, including LRP6, MEF2A, CYP27A1, and ST6GALNAC5, among patients with familial cardiovascular disease in Peshawar Khyber Pakhtunkhwa, Pakistan, with the aim of improving insight into the genetic basis of early-onset and inherited CVDs and informing targeted preventive approaches.

Materials and methods

Primer design

Primers for target genes (LRP6, MEF2A, CYP27A1, and ST6GALNAC5) were designed using Primer 3 [30] and NCBI Primer–BLAST [31]. Design parameters included primer lengths of 18–22 bp, melting temperatures of 55–65°C, GC content of 40–60%, minimal secondary structures, and expected amplicon sizes of 650–900 bp. Specificity was verified using BLAST. Result were also shown in Table 1.

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Table 1. Primer sequences used for PCR amplification.

https://doi.org/10.1371/journal.pone.0353861.t001

Results

A total of 20 clinically confirmed patients with familial cardiovascular diseases and their matched controls were included in the analysis. Genetic sequencing revealed mutations in 12 of the 20 patients. Variants were identified across multiple candidate genes associated with CVD, including LRP6, MEF2A, and CYP27A1. Specifically, LRP6 variants were the most frequently observed, followed by mutations in MEF2A and CYP27A1 and there were no mutations observed in the ST6GALNAC5 gene.

• LRP6 Gene Mutations:
  • The 146344T>A mutation was identified in two patients.
  • The 146345A>G mutation was observed in three patients.
  • The 92497C>A and 94776T>A mutations were detected in one patient each.
• MEF2A Gene Mutations:
  • A mutation at position 113826A>T was found in one patient.
  • Another mutation at position 113802T>A was detected in two patients.
• CYP27A1 Gene Mutations:
  • The 5924G>C mutation was identified in one patient.
  • The 6016G>A mutation was detected in another patient.

Mutations in LRP6 genes

Mutations in the LRP6 gene were identified at several nucleotide positions across the analyzed samples. At nucleotide position c.146334 (T > A), mutations were observed in two samples (P1 and P2), both of which were homozygous. In Fig 1A, “C” represents the wild-type (control), while P1 and P2 represent the mutant samples. At nucleotide position c.146345 (A > G), mutations were detected in three samples (P3, P4, and P5). Among these, one sample (P3) exhibited a homozygous mutation, while the remaining two samples (P4 and P5) showed heterozygous mutations, as illustrated in Fig 1B, where “C” represents the wild-type control. At nucleotide position c.92497 (C > A), mutations were identified in two samples, both demonstrating homozygous alterations. In Fig 1C, “C” represents the wild-type control, while the mutant sample (P6) is shown accordingly. At nucleotide position c.94776 (T > A), a mutation was identified in one sample (P7), which was homozygous. In Fig 1D, chromatogram demonstrating the LRP6 c.94776T > A sequence variant identified in patient P7 relative to the control sequence (C). Homozygous variants are characterized by complete nucleotide replacement, whereas heterozygous variants demonstrate overlapping dual peaks at the variant position.

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Fig 1. Represent Sanger sequencing chromatograms of LRP6 gene variants.

Sanger sequencing chromatograms showing sequence variants identified in the LRP6 gene among the studied patients. (A) Homozygous LRP6 c.146344T > A substitution detected in patients P1 and P2 compared with the wild-type control sequence (C). (B) LRP6 c.146345A > G substitution identified in patients P3–P5, including a homozygous variant in P3 and heterozygous variants in P4 and P5, relative to the control sequence (C). (C) Homozygous LRP6 c.92497C > A substitution identified in patient P6 compared with the corresponding wild-type control sequence C. (D) Representative chromatogram demonstrating the LRP6 c.94776T > A sequence variant identified in patient P7 relative to the control sequence (C). Homozygous variants are characterized by complete nucleotide replacement, whereas heterozygous variants show overlapping dual peaks at the variant position.

https://doi.org/10.1371/journal.pone.0353861.g001

Gel electrophoresis

The agarose gel electrophoresis image demonstrates the successful amplification of the LRP6 gene across all analyzed samples. The DNA ladder serves as a molecular size reference for estimating fragment lengths. The control sample shows a clear band corresponding to the expected amplicon size, confirming the validity of the PCR reaction. All patient samples (P1–P7) exhibit distinct bands at similar positions, indicating successful amplification of the target gene region. No significant variation in band size is observed among samples, suggesting that the detected mutations are point mutations rather than insertions or deletions, as such changes do not alter fragment length. The presence of single, well-defined bands without smearing indicates high-quality DNA and specific amplification. These PCR products were subsequently subjected to sequencing analysis, through which multiple nucleotide substitutions in the LRP6 gene were identified, including: c.146334 (T > A) – homozygous mutations in P1 and P2, c.146345 (A > G) – one homozygous (P3) and two heterozygous (P4, P5) mutations, c.92497 (C > A) – homozygous mutation in P6, c.94776 (T > A) – homozygous mutation in P7. The gel confirms the integrity and suitability of PCR products for downstream sequencing, which enabled the identification of these genetic variants. As shown in Fig 2.

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Fig 2. Agarose gel electrophoresis of PCR-amplified LRP6 gene fragments.

Agarose gel electrophoresis showing PCR amplification of the LRP6 gene in control and patient samples. Lane M, DNA molecular weight marker (DNA ladder); lanes P1–P7, PCR-amplified LRP6 gene fragments from patients P1–P7. The presence of distinct bands at the expected amplicon size confirms successful amplification of the target LRP6 gene in all patient samples.

https://doi.org/10.1371/journal.pone.0353861.g002

Mutation in the MEF2A gene

Mutations in the MEF2A gene were identified in one sample at nucleotide position 113826, where (A) adenine was replaced by (T) Thymine. In Fig 3A, “C” represents the wild-type (control), while P8, represent the mutations in patient 8 sample. Mutation in the MEF2A gene was identified in one sample at nucleotide position 113802, where (T) Thymine was replaced by (C) cytosine. In Fig 3B, “C” represents the wild-type (control), while P9, represent the mutations in the patient 9 sample.

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Fig 3. Shows sanger sequencing chromatograms of MEF2A gene variants.

Sanger sequencing chromatograms showing sequence variants identified in the MEF2A gene among the studied patients. (A) Homozygous MEF2A c.113826A > T substitution detected in patient P8 compared with the wild-type control sequence (C). (B) Homozygous MEF2A c.113802T > C substitution identified in patient P9 relative to the wild-type control sequence (C). Homozygous variants are characterized by complete nucleotide replacement at the variant position compared with the corresponding wild-type sequence.

https://doi.org/10.1371/journal.pone.0353861.g003

Gel electrophoresis

It shows the detection of MEF2A gene mutations by PCR analysis. Agarose gel electrophoresis showing PCR products of the MEF2A gene. The lane labeled C represents the wild-type (control) sample, while P8 and P9 represent patient samples. a nucleotide substitution was identified at position 113826, where adenine (A) was replaced by thymine (T) in patient P8. a nucleotide substitution was detected at position 113802, where thymine (T) was replaced by cytosine (C) in patient P9. The molecular weight marker (DNA ladder) was used to estimate fragment sizes. Differences in the electrophoretic banding patterns between control and patient samples confirm the presence of the respective MEF2A gene mutations. Details were also shown in Fig 4.

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Fig 4. Agarose gel electrophoresis of PCR-amplified MEF2A gene fragments.

Agarose gel electrophoresis showing PCR amplification of the MEF2A gene in the wild-type control and patient samples. Lane M, DNA molecular weight marker (DNA ladder); lane C, wild-type control; lane P8, patient carrying the homozygous MEF2A c.113826A > T substitution; and lane P9, patient carrying the homozygous MEF2A c.113802T > C substitution. Distinct bands at the expected amplicon size confirm successful amplification of the target MEF2A gene.

https://doi.org/10.1371/journal.pone.0353861.g004

Mutation in the CYP27A1 gene

Mutation in the CYP27A1 gene was identified in one sample of P10, at nucleotide position 5924, where (G) Guanine was replaced by (C) cytosine. In Fig 5A, “C” represents the wild-type (control), while P10, represent the mutations in the patient P10 sample. In Fig 5B “C” represents the wild-type (control), while mutation in the CYP27A1 gene was identified in one sample of P11, at nucleotide position 6016, where (G) Guanine was replaced by (A) adenine. In Fig 5C, “C” represents the wild-type (control), while, represent the mutations in the patient (P11) sample. Mutation in the CYP27A1 gene was identified in one sample of P12, at nucleotide position 37404, where (C) Cytosine was replaced by (T) Thymine. “C” represents the wild-type (control), while P12 represent the mutations in the patient (P12) sample.

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Fig 5. Sanger sequencing chromatograms of CYP27A1 gene variants.

Sanger sequencing chromatograms showing sequence variants identified in the CYP27A1 gene among the studied patients. (A) Homozygous CYP27A1 c.5924G > C substitution detected in patient P10 compared with the wild-type control sequence (C). (B) Homozygous CYP27A1 c.6016G > C substitution identified in patient P11 relative to the wild-type control sequence (C). (C) Homozygous CYP27A1 c.37440C > T substitution identified in patient P12 compared with the corresponding wild-type control sequence (C). Homozygous variants are characterized by complete nucleotide replacement at the variant position compared with the corresponding wild-type sequence.

https://doi.org/10.1371/journal.pone.0353861.g005

Gel electrophoresis

Agarose gel electrophoresis showing PCR amplification of the CYP27A1 gene fragment from control and patient samples. Lane M represents the DNA molecular weight marker. Lane C represents the wild-type (control) sample, while the patient lanes show amplified products from the respective samples. Distinct PCR bands of the expected size were observed in both control and patient samples, confirming successful amplification of the target region. Sequence analysis of the amplified products revealed nucleotide substitutions in the CYP27A1 gene. In patient P10, a mutation was identified at nucleotide position 5924, where guanine (G) was replaced by cytosine (C). In patient P11, a mutation was detected at nucleotide position 6016, where guanine (G) was replaced by adenine (A). In patient P12, a mutation was identified at nucleotide position 37404, where cytosine (C) was replaced by thymine (T). details were also shown in Fig 6. Raw gel pictures were included in S1 Fig.

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Fig 6. Agarose gel electrophoresis of PCR-amplified CYP27A1 gene fragments.

Gel electrophoresis showing PCR amplification of the CYP27A1 gene in the wild-type control and patient samples. Lane M, DNA molecular weight marker (DNA ladder); lane C, wild-type control; lane P10, patient carrying the CYP27A1 c.5924G > C substitution; lane P11, patient carrying the CYP27A1 c.6016G > A substitution; and lane P12, patient carrying the CYP27A1 c.37404C > T substitution. Distinct bands at the expected amplicon size confirm successful amplification of the target CYP27A1 gene.

https://doi.org/10.1371/journal.pone.0353861.g006

Discussion

CVD is a leading cause of morbidity and mortality worldwide, with an increasing incidence among young individuals. The traditional risk factors such as hypertension, dyslipidemia, and smoking contribute significantly to CVD, genetic predisposition also plays an important role [36]. Among the genetic factors implicated, the LRP6 gene has emerged as a key player in CVD pathogenesis. The LRP6 gene encodes a co-receptor integral to the Wnt/β-catenin signaling pathway, which is essential for various physiological processes. Mutations in LRP6 have been implicated in the pathogenesis of CVD, particularly in young individuals presenting with early-onset conditions. This mutation was associated with impaired Wnt signaling, suggesting a mechanistic link between LRP6 dysfunction and increased CVD susceptibility [37]. Subsequent research has reinforced the association between LRP6 mutations and CVD [38]. A study by Liyang et al. (2012) reported a novel mutation in the YWTD domain of LRP6 that impaired endothelial cell proliferation and migration, processes vital for maintaining vascular integrity. This impairment was linked to familial CAD, underscoring the significance of LRP6 mutations in hereditary forms of the disease [39]. Moreover, LRP6-mediated signaling pathways have been implicated in various cardiovascular conditions beyond CAD. Kang (2020) highlighted the involvement of LRP6 in regulating blood pressure, glucose metabolism, and lipid homeostasis, all of which are critical factors in cardiovascular health. Dysregulation of these pathways due to LRP6 mutations may contribute to the development of CVD in young individuals [40]. The mutations T146344A, A146345G, C92497A, and T94776A in the LRP6 gene may have significant functional implications, particularly if they affect gene regulation or splicing. While their exact roles require further experimental validation, their potential association with diseases such as cancer, metabolic disorders, and skeletal abnormalities underscores the importance of LRP6 in human health. Future studies should focus on elucidating the mechanisms by which these mutations contribute to disease pathogenesis and exploring therapeutic strategies targeting LRP6 and the Wnt signaling pathway. In the current study, we also identified the following mutations in the LRP6 gene in four patients. The specific nucleotide changes you’ve identified T146344A, A146345G, C92497A, and T94776A do not correspond to well-characterized mutations in the current literature. It’s essential to determine the exact nucleotide positions within the LRP6 gene’s coding sequence to predict their potential impact on protein function. This involves identifying whether these mutations result in synonymous changes, missense mutations, or nonsense mutations, each of which can have varying effects on protein structure and function. For instance, missense mutations can lead to amino acid substitutions that may alter protein function. A study by Singh et al. (2013) identified a heterozygous missense mutation (c.1418G > A, p.Arg473Gln) in exon 7 of the LRP6 gene, which was associated with early-onset coronary artery disease and other metabolic disorders [41]. Additionally, a novel heterozygous missense variant (c.719C > T, p.Thr240Ile) in exon 4 of the LRP6 gene was identified in a family exhibiting high bone mass. This mutation did not affect gene transcription but impaired the maturation and phosphorylation of the LRP6 protein, suggesting a disruption in Wnt signaling [42]. While the specific mutations T146344A, A146345G, C92497A, and T94776A in the LRP6 gene are not well-documented in current literature, understanding their potential impact requires detailed genetic and functional analyses. Given the established role of LRP6 in critical signaling pathways, mutations in this gene warrant thorough investigation to elucidate their clinical significance. The myocyte enhancer factor 2A (MEF2A) gene encodes a transcription factor integral to the regulation of gene expression in cardiovascular development and function. Its role in CVD, particularly in young individuals, has been a subject of research interest. Initial studies suggested a potential link between MEF2A mutations and CAD. A notable study identified a 21-base pair deletion in the MEF2A gene that appeared to co-segregate with CAD in a large family, implicating this mutation in the disease’s pathogenesis [43]. However, subsequent research challenged this association. For instance, a study by Pennacchio et al. sequenced MEF2A in approximately 300 patients with premature CAD and found no causative mutations. Moreover, the previously implicated 21-base pair deletion was also identified in individuals without CAD, suggesting that this specific mutation may not be pathogenic [44]. Further investigations have yielded mixed results. A study focusing on a Chinese Han pedigree identified a novel 6-base pair deletion in exon 11 of MEF2A that co-segregated with premature CAD/myocardial infarction (MI) within the family. This deletion was absent in sporadic cases and unrelated healthy controls, indicating a potential family-specific pathogenic variant [45,46] Functional studies have provided insights into the potential mechanisms by which MEF2A mutations could influence cardiovascular health. Research indicates that MEF2A plays a crucial role in endothelial cell function, and its deletion or mutation can lead to vascular endothelial dysfunction, a key factor in the development of CVD [47]. The impact of MEF2A mutations on cardiovascular health may be limited to particular genetic contexts, and further research is necessary to elucidate the precise role of MEF2A in CVD, especially among young individuals. A 21-base pair (bp) deletion in the MEF2A gene was first reported in a family with autosomal dominant CAD. This mutation was associated with impaired endothelial function and increased CAD risk in young individuals [48]. Subsequent studies have identified additional MEF2A variants (e.g., G283D, P279L) associated with early-onset CAD and endothelial dysfunction [28]. In this study the specific MEF2A mutations (A113826T, T113802C) were found in two patients. The A113826T mutation is located in a non-coding region of the MEF2A gene, potentially can affect the gene regulation, splicing, or mRNA stability. These non-coding mutations can influence transcriptional regulation or splicing efficiency. Non-coding mutations in MEF2A have been implicated in conditions such as CAD and endothelial dysfunction. Reduced MEF2A expression has been linked to impaired endothelial function and increased CVD risk in young individuals (Wang et al., 2003). The T113802C mutation is also in a non-coding region, possibly affecting regulatory elements or splicing. Similar to A113826T, this mutation may influence MEF2A expression or mRNA processing. MEF2A gene mutations, including A113826T and T113802C, are increasingly recognized as genetic risk factors for CVD in young individuals. These mutations may disrupt vascular development, endothelial function, and oxidative stress regulation, all of which contribute to early-onset CVD. Understanding the role of MEF2A in CVD pathogenesis provides valuable insights into the genetic basis of the disease and opens new avenues for early diagnosis, personalized therapy, and targeted interventions. Further research is needed to fully elucidate the mechanisms linking MEF2A mutations to CVD and to develop effective treatments for affected individuals. CYP27A1 is a mitochondrial enzyme that plays a crucial role in cholesterol metabolism and bile acid synthesis. The CYP27A1 gene encodes sterol 27-hydroxylase, a mitochondrial enzyme pivotal in bile acid synthesis and cholesterol homeostasis. It catalyzes the hydroxylation of cholesterol to form 27-hydroxycholesterol (27-OHC), an oxysterol that regulates lipid homeostasis, inflammation, and vascular function. In the cardiovascular system, CYP27A1 is essential for maintaining cholesterol balance, endothelial function, and anti-inflammatory responses. CYP27A1 converts cholesterol into 27-OHC, which can be further metabolized into bile acids. This process helps regulate cholesterol levels and prevent lipid accumulation in arterial walls [49]. 27-OHC has been shown to modulate endothelial function by influencing nitric oxide (NO) production and oxidative stress. Dysregulation of CYP27A1 can lead to endothelial dysfunction, a key early event in atherosclerosis. CYP27A1 and 27-OHC play a role in modulating immune responses and inflammation, which are central to CVD pathogenesis [50]. The specific nucleotide changes G6016A and C37484T in the CYP27A1 gene correspond to mutations that have been identified in individuals with CTX. For instance, the c.1415G > C (p.Arg472Pro) mutation has been reported in multiple studies and is associated with the disease phenotype. This particular mutation leads to a substitution of arginine with proline at position 472, affecting the enzyme’s function. In our study we found mutation in 2 patients at position (G6016A, C37484T). This mutation (G6016A) is located in a non-coding region of the CYP27A1 gene, potentially affecting gene regulation, splicing, or mRNA stability. These non-coding mutations can influence transcriptional regulation or splicing efficiency. For example, mutations in regulatory regions such as enhancers or silencers can alter CYP27A1 expression levels, potentially disrupting cholesterol metabolism and endothelial function. The C37484T mutation is also in a non-coding region, possibly affecting regulatory elements or splicing. Similar to G6016A, this mutation may influence CYP27A1 expression or mRNA processing. Computational tools like Ensembl’s Variant Effect Predictor (VEP) can provide insights into its potential impact on regulatory motifs. The non-coding variants in CYP27A1 have been associated with metabolic syndromes, including type 2 diabetes and hyperlipidemia, due to their role in lipid metabolism and insulin signaling.

Conclusion

This study identified multiple genetic variants in LRP6, MEF2A, and CYP27A1 genes among familial CVD patients in a high-consanguinity population. These variants were observed in a substantial proportion of cases, suggesting a potential genetic contribution to early-onset cardiovascular disease in this region. Our findings highlight the importance of incorporating genetic screening strategies for early detection and risk assessment in high-risk populations. The presence of multiple mutations in these genes suggests their potential involvement in the pathogenesis of CVD, warranting further investigation into their functional implications. The increasing prevalence of CVD in younger populations is a pressing concern, particularly in regions with high rates of familial predisposition and consanguineous marriages. Such genetic factors may exacerbate disease risk by increasing the likelihood of inheriting pathogenic mutations. The inclusion of ST6GALNAC5, another gene of interest, underscores the complexity of genetic contributions to CVD, emphasizing the need for a broader genomic perspective in future studies. A multidisciplinary approach involving geneticists, cardiologists, and policymakers is essential to develop effective prevention and management programs tailored to the genetic profile of this population. This study highlights the important contribution of genetic factors to cardiovascular disease risk among young adults in Khyber Pakhtunkhwa, with pathogenic variants identified in more than half of the studied patients, particularly in the LRP6, MEF2A, and CYP27A1 genes. The increasing burden of cardiovascular disease among younger individuals is especially concerning in settings where familial clustering and consanguineous marriages are common, as these practices may increase the transmission of harmful genetic variants. Incorporating genetic evidence into national cardiovascular health policies may help reduce the long-term burden of disease and improve outcomes for young adults in resource limited settings. Future studies employing larger cohorts and advanced sequencing technologies are recommended to validate these findings and further elucidate the genetic architecture of cardiovascular disease in this population.

Supporting information