Online tool to refine results from RNA sequencing from clinically accessible tissues

DNA sequencing has turned out to be a common method for diagnosing diseases and enhancing precision medicine. However, DNA sequencing cannot detect all possible disease-causing mutations; hence, RNA sequencing is generally used to overcome this problem.

DNA Sequencing

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But RNA sequencing is often performed on clinically accessible tissues from skin and blood and does not give a complete view of the rest of the body.

A research team at the Children’s Hospital of Philadelphia (CHOP) and the Perelman School of Medicine at the University of Pennsylvania reviewed a database of RNA sequencing results in non-clinically accessible tissues from organs such as the heart and brain, in an attempt to optimize the use of RNA sequencing.

This enabled them to find the differences among tissues that were well expressed to find out when clinically accessible tissues like skin and blood samples are most useful and when they are not.

Hoping to promote further sequencing and support diagnosis, the researchers also created an online resource that outlines how these differences influence specific tissues and genes of interest. The study results were reported online in the Genetics in Medicine journal.

Researchers constantly enhance the potential of sequencing tests to track genetic mutations that trigger disease. Through exome sequencing, nearly 31% of inherited genetic disorders are screened. This rate is improved anywhere from 10% to 15% by genome sequencing, implying that a majority of the patients who undergo this screening will not receive a proper molecular diagnosis.

One of the major challenges is the number of non-coding variants captured by these tests. These variants can cause disease, but they are hard to predict and thus ignored by prevalent diagnostic methods.

These variants can cause problems by their potential to modify RNA splicing or the process through which non-coding parts of genes are eliminated, leaving behind only the coding portions to produce necessary proteins.

Thus, variants that influence RNA splicing can modify the function of essential proteins, thereby causing disease. RNA sequencing helps identify these variants and adds to the knowledge gained from exome and genome sequencing. However, RNA sequencing is challenging since the gene must be expressed in the tissue of interest, and those tissues are often not found to be accessible.

We know that we are unable to test tissues in the brain, heart and certain other organs for diagnostic purposes, but we also know that using RNA sequencing on these tissues could reveal important genetic information we might not otherwise be able to capture. By studying both clinically- and non-clinically-available tissues, we hoped this study would reveal the true extent of what we may be missing with current RNA sequencing methods.”

Elizabeth Bhoj, MD, PhD, Study Senior Co-author and Assistant Professor of Pediatrics, Perelman School of Medicine

Bhoj is also an attending physician with the Division of Human Genetics at CHOP.

The researchers measured RNA splicing in 801 RNA-sequenced samples from 56 different fetal and adult tissues. They identified genes and splicing events in each non-clinically available tissue, which enabled them to determine when RNA sequencing in each clinically available tissue represents them inadequately.

They also developed their own online resource, MAJIQ-CAT, which can be used to explore their analysis for specific genes and tissues.

The researchers identified that 40.2% of non-clinically available tissues exhibit RNA splicing inadequately represented in at least one clinically available tissue, and 6.3% of the genes exhibit splicing inadequately represented by all clinically available tissues.

A majority (52.1%) of these genes show lower expression in clinically available tissues, and the researchers demonstrated that 5.8% of the genes are inadequately represented though they are well-expressed, thus representing a higher portion of genes of interest not captured properly by conventional RNA sequencing techniques.

By using MAJIQ-CAT, researchers can determine which accessible tissues, if any, best represent RNA splicing in genes and tissues of interest. While this does not address the entire gap left by current exome and genome sequencing methods, we believe we can capture more genes and determine how they affect human health.”

Elizabeth Bhoj, MD, PhD, Study Senior Co-author and Assistant Professor of Pediatrics, Perelman School of Medicine

The researchers drew from their diverse scientific backgrounds for this research to enhance clinical diagnosis. Bhoj and her lab offered expertise in genetics and clinical diagnostics, together with the computational expertise of the lab of Yoseph Barash, Ph.D., an associate professor in the Department of Genetics at Penn.

The study was headed by Joseph Aicher, an MD/Ph.D. student in the Genomics and Computational Biology program at Penn who was co-mentored by Bhoj and Barash.

Source:
Journal reference:

Aicher, J. K., et al. (2020) Mapping RNA splicing variations in clinically accessible and nonaccessible tissues to facilitate Mendelian disease diagnosis using RNA-seq. Genetics in Medicine. doi.org/10.1038/s41436-020-0780-y.

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