Why is rapid and automated targeted next-generation sequencing (NGS) critical in RASopathies clinical research? RASopathy disorders are a group of genetic syndromes caused by mutations in the Ras/mitogen-activated protein (MAPK) pathway. The MAPK signaling pathway is essential for controlling many cellular processes which are important for normal growth and development. One of the most common RASopathy disorders is Noonan syndrome, which is present in about 1 in 1,000 to 2,500 live births. While Noonan syndrome can be inherited, de novo or spontaneous mutations account for nearly 60% of diagnosed cases.
Noonan syndrome can impact many areas of the body and is typically characterized by distinct facial features, short stature, congenital heart defects, developmental delay, and other comorbid conditions. Noonan syndrome can be difficult to study due to the variation in how the disease presents. Sometimes the syndrome can be identified based on presentation alone, but it is recommended to use genetic testing to eliminate other disorders that share similar characteristics.
Many different genes are known to be associated with Noonan syndrome and other RASopathies. Standard genetic analysis methods involving single-gene tests are often utilized by inherited disease researchers but these methods alone may be time consuming and expensive to uncover the many variants involved in complex diseases such as Noonan syndrome. The approach using multiple methods including targeted Next-Generation Sequencing (NGS) is an appealing option for researchers since targeted NGS can accurately interrogate many genes in one test which reduces cost and speeds time. Targeted NGS using amplification technologies such as Ion AmpliSeq technology enables robust analyses which can be performed with as little as 1 ng of DNA. These analyses can use DNA from a variety of sources including peripheral blood and amniotic fluid but also from challenging samples such as Formalin-Fixed Paraffin-Embedded (FFPE) tissues for retrospective studies.
Researchers at the Institute of Molecular Pathology and Immunology of the University of Porto (IPATIMUP) has been developing targeted NGS assays to further clinical research into Noonan syndrome. Their previous research leveraged the NGS capabilities of the Ion GeneStudio S5 System. Now their lab is an early-adopter of the Ion Torrent Genexus system, the first turnkey NGS solution that automates the specimen-to-report workflow*.
Using Ion AmpliSeq Designer, they created a fully customized panel targeting 86 genes known to be implicated in Noonan syndrome and other RASopathies. The results from this custom Ion AmpliSeq panel were used as final confirmation for the presence of Noonan syndrome from both pre-natal and post-natal research samples. These samples were run on the Ion Genestudio S5 System and Genexus Integrated Sequencer with comparable results on the metrics of uniformity of amplicon coverage, base reads on target, and uniformity of base coverage. Moreover, the single day turnaround on the Genexus Integrated Sequencer to go from nucleic acid to variant report in a fully automated workflow with just a single touch point was incredibly important to provide answers even faster and more easily.
For clinical researchers who prefer pre-tested NGS gene designs, many of the genes associated with Noonan disease are available through Ion AmpliSeq On Demand panels. Ion AmpliSeq On-Demand panels provide over 5,000 genes known to be associated with various inherited diseases, including the RASopathies. These gene designs have been wet lab tested and are available to combine into customized panels to help researchers efficiently bring new assays to their labs. This gene catalog is available for both the Ion Genestudio S5 and Genexus platforms by either searching for gene(s) of interest, or through using the Disease Research Areas (DRA) found on Ion AmpliSeq Designer. Another benefit of the Ion AmpliSeq On-Demand panels is that researchers receive the panel in more practical pack sizes than in the bulk sizes in which traditional custom panels are often provided, which makes iterations of panel design efficient and convenient as their project evolves.
Knowledge about the genetic causes of RASopathies is constantly evolving due to the efforts of clinical researchers like those at IPATIMUP. As more of these mutations are identified through research, the need for accurate and comprehensive genetic analysis will continue to grow. In addition, the Genexus Integrated Sequencer combined with AmpliSeq targeted gene panels allow results in less than a day, providing unprecedented automation and ease of use for clinical researchers.
Ion Torrent targeted NGS solutions for complex disease research provide streamlined end-to-end workflows and accessible solutions that make it easy for any lab to bring NGS in house. To learn more about these solutions, visit the Complex Disease Research page.
- Bhambhani, “Noonan Syndrome”. American Family Physician, January 1, 2014. Volume 89, Number 1. www.aafp.org/afp
- Rauen, Katherine A. “The RASopathies.” Annu Rev Genomics Hum Genet. 2013 ; 14: 355–369. doi:10.1146/annurev-genom-091212-153523
For Research Use Only. Not for use in diagnostic procedures.
*Specimen-to-report workflow will be available after the Ion Torrent Genexus Purification System and integrated reporting capabilities are added in 2020.