Genetic Variants Impacting Cardiovascular Disease Revealed using Sapphire

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Researchers from the University of Puerto Rico employed the Azure Sapphire Biomolecular Imager to  explore the possible links between DNA variants and cardiovascular disease through their impact on DNA binding. Peña-Martínez et al examined the relationship between NKX2-5 (a crucial transcription factor) and its DNA binding abilities. Utilizing predictive modeling and experimental assays, the team pinpointed specific non-coding variants that significantly modified NKX2-5’s affinity for DNA binding. These discoveries offer compelling evidence that disturbances in transcription factor binding sites could contribute to cardiovascular disease.

Since the release of this publication, the first generation Sapphire used has been succeeded by the new Sapphire FL, which was designed to be the flexible choice in bringing precise quantitation of nucleic acids and proteins. Learn more about this new imager.

Disease variants and the non-coding genome

A staggering 98% of our genome is composed of non-coding DNA. Through innovative genome-wide association studies (GWAS), scientists have determined that non-coding DNA harbors crucial regulatory elements (or cis-regulatory elements (CREs)), including promoters and enhancers, which play pivotal roles in orchestrating gene expression.

Tiny alterations in the DNA sequence are called single nucleotide polymorphisms (SNPs). SNPs are often situated in close proximity to genes. They influence the function of regulatory DNA-binding proteins, such as transcription factors, affecting gene activity and, ultimately, vital biological processes.

NKX2-5 and heart development

NKX2-5 is a key player in heart development. Previous research has demonstrated that mutations within its DNA-binding domain (DBD), specifically the homeodomain, can disrupt its regulatory function, leading to cardiovascular diseases like congenital heart diseases (CHDs). Interestingly enough, the majority of genetic variants associated with cardiovascular diseases are found not in the genes themselves, but rather in the CREs that function as binding sites for transcription factors involved in heart development.

The Peña-Martínez’s team at the University of Puerto Rico conducted a study to explore the influence of cardiovascular-associated SNPs on NKX2-5’s DNA binding abilities.

Since this paper was published, the Sapphire has been succeeded by the new Sapphire FL

Designed for flexible choice in detection chemistry and samples, the Sapphire FL brings precise quantitation of nucleic acids and proteins
Scientist changing optical modules on the new Azure Sapphire FL
The Sapphire FL Biomolecular Imager is capable of high-resolution imaging and wide depth of field enable many sample types, including arrays, microarrays, Western blots, tissue slides, and small animals.

Narrowing down non-coding SNPs for in vitro validation

Using a predictive model, the researchers identified over 8000 SNPs that were predicted to affect NKX2-5 DNA binding. After cross-referencing disease and quantitative trait-associated SNPs from the GWAS catalog, the five SNPs with the greatest potential magnitude were selected to test in vitro.  These SNPs are associated with traits that impact cardiovascular health, such as hemoglobin levels.

Non-coding mutations affect NKX2-5 binding

To study the effects of the SNPs on DNA binding, the researchers created a recombinant NKX2-5 homeodomain (the area of the protein responsible for DNA binding).

They created six test sequences, each composed of a 40bp genomic sequence with either a control nucleotide or one of the SNPs of interest at the center. To enable visualization, each sequence was conjugated with an IRDye® 700 fluorophore. An electrophoretic mobility shift assay (EMSA) was used to assess DNA binding affinity between the recombinant NKX2-5 homeodomain and test DNA sequences.

EMSA gels used to evaluated the effect of mutations

The researchers used the images acquired with the Sapphire to evaluate the effect of each mutation on the binding affinity of the recombinant NKX2-5 homeodomain. In Figure 3, the lower band represents free DNA, while the upper band shows bound DNA.

The researchers found that all five variants significantly altered NKX2-5’s affinity for binding DNA. These findings suggest disruptions in transcription factor binding sites may play a role in the development of cardiovascular diseases and thereby provides a mechanistic connection between genome variants and disease.

EMSA gel captured using Azure Sapphire Biomolececluar Imager
Figure 3 from Peña-Martínez et al. (2023) showing a representative EMSA gel used to validate the binding of the rs6121514 variant. The remaining panels are binding curves for all five of the variants. The Azure Sapphire Biomolecular Imager was used to capture images of the EMSA shown here. Licensed under CC BY 4.0.

While fluorescent labels were used here, traditional EMSA employs radioactive-labeled DNA which requires phosphorimaging for analysis. The Sapphire FL offers a distinct advantage by supporting both phosphor imaging and IR detection, allowing capture of any type of EMSA. This versatility provides flexibility and convenience throughout the experimentation process.

Designed for ultimate flexibility, the Sapphire FL allows for precise detection of proteins and nucleic acids. If you’re eager to explore the possibilities of this groundbreaking system for your research, schedule a demo today, and experience what the future of biomolecular imaging has to offer.

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  1. Peña-Martínez, Edwin G, et al. “Disease-associated non-coding variants alter NKX2-5 DNA-binding affinity.” Biochimica et Biophysica Acta (BBA) – Gene Regulatory Mechanisms,, vol. 1866, no. 1, 2023, pp. 1-5. Science direct,

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