A Diagnostics RNA Sequencing Assay for Direct Identification and Interpretation of Pathogenic Variants in the FBN1 Gene

Manal Irshaid¹Ghadeera Al Mansoori²Mohamed Sulaiman³Asjed Mohamed¹Mohammed Abdoh⁴Youssef Shalaby¹Bashar Al-Zohily¹Aalia Batool¹Sara Aleissaee¹Lara Alzyoud¹Bassam R Ali¹Muna Al Saffar^1,5Nadia Akawi^1,6*

• ¹College of medicine and Health Sciences, Department of Genetics and Genomics, United Arab Emirates University, Al-Ain, United Arab Emirates
• ²Department of Cardiology, Sheikh Shakhbout Medical City, Abu Dhabi, United Arab Emirates
• ³Department of Pediatrics, Tawam Hospital, Al Ain, United Arab Emirates
• ⁴Department of Pathology and Laboratories Medicine, Sheikh Shakhbout Medical City, Abu Dhabi, United Arab Emirates
• ⁵Department of Pediatrics, Division of Genetic and Genomics, Boston Children's Research, Boston, United States
• ⁶Division of Cardiovascular Medicine, University of Oxford, Oxford, United Kingdom

The extensive size and multi-exon structure and the tissue-restricted expression of the associated gene FBN1 challenge the genetic diagnosis of Marfan Syndrome (MFS). Current genetic diagnostic methods adopted clinically to confirm or rule out the disease diagnosis rely on high throughput DNA sequencing approaches, including whole exome or genome sequencing. While these approaches are powerful, they are costly, time-consuming, and labor-intensive, and they generate vast data sets that require computational and bioinformatic infrastructure to interpret. This study introduces an alternative sensitive, comprehensive, rapid, and cost-effective assay for genetic screening for MFS using whole blood RNA. This assay enables successful amplification and sequencing of the entire FBN1 coding region, even though FBN1 is lowly expressed in the blood. It enabled the detection of four variants in FBN1-mRNA, including one nonsense, two frameshifts, and one missense, in the probands of four unrelated families, confirming MFS diagnosis and ruling out the disease diagnosis in the fifth family. Furthermore, we introduce our assay as a functional assay that not only enables the detection of the variants in FBN1 but also elucidates the mechanism by which these variants influence RNA transcription and contribute to disease mechanism, the insight that is often overlooked by DNA sequencing approaches, allowing us to identify distinctive impact influenced by each identified variant, and identify RNA slippage as a new disease mechanism that has never been reported before in MFS. The developed assay introduces an improved approach in clinical genetic testing of MFS, with potential applicability for diagnosing other conditions involving significant multi-exon genes.