When it comes to medical questions, sometimes “unknown” is the worst answer you can receive. Uncertainty prevents a family from developing an action plan to combat bad news, or from achieving peace of mind when receiving good news. Yet for many patients who undergo genetic testing due to a suspected hereditary cancer syndrome, they are left with either an inconclusive result or without a molecular diagnosis. Two of the main reasons for this include:

1) The patient has a rare variant for which there isn’t enough credible information to say whether the variant is pathogenic or benign. These variants are more commonly known as “variants of unknown significance” (VUS)

2) The patient has a pathogenic mutation that is undetected due to the limitations of high-throughput, DNA-based genetic testing

This is where the benefits of adding simultaneous RNA analysis to current DNA-based genetic testing panels can be realized, and why RNA analysis has the potential to change the future of genetic testing.

Benefit #1 – RNA analysis will expand the reporting range of DNA-based genetic testing

Clinical diagnostic labs typically apply a reporting range limit to their DNA genetic testing panels. For most genes, this means not-reporting variants located beyond 5-10 nucleotides before and after the start of the exon (the part that encodes the mRNA and protein). This is because variants outside these ranges have a much lower probability of being pathogenic (i.e., disease-causing and would most often be classified as a VUS). Looking beyond these ranges would often just increase the VUS rate without increasing the overall diagnostic yield. However, a limited reporting range increases the chance that pathogenic variants outside the range are missed. Although such variants are rare, they can have significant clinical impact. The addition of RNA analysis enables expansion of the reporting range to identify and report these clinically actionable findings. For example, several studies have identified such mutations in over two percent of patients with neurofibromatosis, as well as in several patients with previously unexplained ataxia telangiectasia.^1-4 These mutations were identified thanks to single-gene RNA genetic testing of NF1 and ATM, the genes known to cause these respective diseases.

RNA-targeted searching for genomic mutations is obvious in the case of NF1 and ATM where there is a clear genetic link between the disease and a single gene. However, in less obvious clinical scenarios, such as a strong family history of breast cancer where numerous genes may be the cause, single gene RNA testing is impractical and cumbersome. However, RNA testing of a panel of cancer genes can give genetic testing labs the opportunity to do the same type of investigations and remove the limitations of a formal reporting range.

Benefit #2 – RNA analysis will provide real-time data to help clarify the significance of VUS

Variants that are near an intron-exon boundary are much more likely to cause a splicing defect, leading to pathogenicity; and in the case of some genes, they’re a significant cause of pathogenicity—as high as 40 to 50 percent.^5-6 However, there are also many reasons why these intronic variants might not be pathogenic, and RNA genetic testing can help distinguish the pathogenic from the non-pathogenic intronic alterations. For example, variants that are more than 3 nucleotides away from the intron-exon boundary most often start as VUS and require additional investigation and evidence to resolve the classification. This investigation can be done proactively, as Ambry Genetics has been doing for over three years in the ATG Lab; or it can be done passively by waiting for another research lab to publish RNA data; but the latter could take years, if it’s done at all.

With frontline, prospective RNA genetic testing, such investigation happens concurrently with DNA analysis for every patient, so that variants have built-in information about whether they produce a significant splice defect. This provides immediate data that influences the classification of that variant in real-time. Not only does the tested patient immediately discover whether a variant is disease-causing, but all the families tested at Ambry that carry the same, or even a similar, variant can benefit from a retrospective variant review and reclassification.

Benefit #3 – RNA genetic testing provides more accurate results and greater confidence for healthcare providers and families

A negative report or a VUS can be unsettling, especially for those with a strong cancer history. Could a pathogenic mutation have been missed by current technology? Is there a pathogenic mutation in another gene? Mutations may go unrecognized or undetected due to an inability to detect deep intronic alterations or the presence of hard-to-detect alterations such as inversions or retrotransposon insertions which can cause splice anomalies. In addition, there is often not enough evidence to provide a meaningful, clear classification for many variants. Looking at RNA concurrently with DNA helps provide another line of evidence that could help detect mutations that may otherwise go undetected; helps to decrease VUS rates in real-time; and helps to provide more accurate results. This additional analysis can give families and healthcare providers greater confidence that a report is truly negative, or that the classification is the most up-to-date and accurate available. In other words: it can give them more peace of mind.

DNA genetic testing has come a long way since its inception, yet a significant number of patients still receive negative or uninformative genetic test results. Adding simultaneous RNA genetic testing to hereditary cancer panels is the next evolution towards improved variant detection and interpretation. The addition of RNA analysis is a game-changer which allows providers to find more mutations, decrease VUS, and provide more accurate and personalized medical management for patients.

Learn More

References:

1. Pros E, Fernández-Rodríguez J, Canet B, et al. Antisense therapeutics for neurofibromatosis type 1 caused by deep intronic mutations. Hum Mutat. 2009 Mar;30(3):454-62.
2. Sabbagh A, Pasmant E, Imbard A et al. NF1 molecular characterization and neurofibromatosis type I genotype-phenotype correlation: the French experience. Hum Mutat. 2013 Nov;34(11):1510-8
3. Cavalieri S, Pozzi E, Gatti RA, Brusco A. Deep-intronic ATM mutation detected by genomic resequencing and corrected in vitro by antisense morpholino oligonucleotide (AMO). Eur J Hum Genet. 2013 Jul;21(7):774-8
4. Nakamura K, Du L, Tunuguntla R, et al. Functional characterization and targeted correction of ATM mutations identified in Japanese patients with ataxia-telangiectasia. Hum Mutat. 2012 Jan;33(1):198-208
5. Pros E, Gómez C, Martín T, et al. Nature and mRNA effect of 282 different NF1 point mutations: focus on splicing alterations. Hum Mutat. 2008 Sep;29(9):E173-93
6. Teraoka SN, Telatar M, Becker-Catania S et al. Splicing defects in the ataxia-telangiectasia gene, ATM: underlying mutations and consequences. Am J Hum Genet. 1999 Jun;64(6):1617-31.