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Order Guide: RASopathies

Background

The intent of this document is to provide guidance on the appropriate coordination of genetic testing for conditions in the RASopathy spectrum. These disorders have clinical and genotypic overlap. The omission of ordering clinically relevant genes can result in under-diagnosis, while the inclusion of low yield genes can increase the chance for ambiguous results. For these reasons, the following variables should be considered prior to ordering RASopthy genetic testing. When in doubt, consider reviewing the order with an internal expert/manager/leader.

The RASopathies collectively refer to an overlapping group of clinical diagnoses, including Noonan syndrome, Noonan syndrome with multiple lentigenes (previously called LEOPARD syndrome), cardio-facio-cutaneous (CFC) syndrome, Costello syndrome, neurofibromatosis type 1, and Legius syndrome. These conditions have common clinical features, such as congenital heart defects, poor growth, macrocephaly, and cutaneous findings.

RASopathy genetic testing, including sequencing and/or deletion/duplication testing, is typically considered in the following circumstances:

• To guide medical interventions (e.g., treatment or surveillance)

• To inform prognosis (anticipatory guidance)

• To provide information regarding recurrence risk (guide reproductive planning)

• To end the diagnostic odyssey

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RASopathy genetic testing typically consists of sequencing (e.g., Sanger sequencing or DNA-enrichment methods and massively parallel nucleotide sequencing) and quantitative deletion/duplication (e.g., multiple ligation-dependent probe amplification (MLPA), quantitative PCR, or array comparative genomic hybridization) methodologies to identify disease-associated, protein-coding variants in genes associated with this clinical spectrum.

Currently, these genes include PTPN11, SOS1, RAF1, KRAS, HRAS, BRAF, MAP2K1, MAP2K2, NRAS, SHOC2, CBL, RIT1, NF1, and SPRED1. The analysis is limited to the DNA sequence of coding regions (exons) and flanking intronic regions of the genes. Pathogenic variants that can be identified by sequencing methods include missense, nonsense, splice-site, and small deletions or insertions. Quantitative deletion/duplication methods can detect copy-number variants, although different methods have advantages for detecting mid-size insertions and deletions (ca. 10-500 bp) versus larger deletions and duplications on the exon level. However, both methods typically miss certain classes of disease-causing variants, such as interruptions in genes due to structural variants (translocations, inversions, etc.), deeper intronic mutations, and lower-level mosaicism. Thus, in the choice of test methodology, the advantage of breadth of coverage must be balanced against the risk of missing disease-associated variants due to these technical limitations. When in doubt, consider reviewing the order with an internal expert/manager/leader.

While sequencing analysis for the RASopathies can have a relatively high detection rate, deletion/duplication testing generally has a low detection rate and reduced clinical utility. In addition to phenotypic overlap, there is genotypic overlap between the conditions (e.g., some pathogenic variants in the BRAF gene cause CFC and other variants in the same gene cause Noonan syndrome.) Furthermore, the omission of ordering clinically relevant genes can result in under-diagnosis, while the inclusion of low yield genes can increase the chance for ambiguous results.

The following questions provide a helpful framework when considering the appropriateness of RASopathy genetic testing for the individual:

• Can the individual’s diagnosis be confirmed based on clinical examination or imaging studies?
  Some conditions such as neurofibromatosis type 1 have specific clinical diagnostic criteria that can confirm a diagnosis without genetic testing.
• Would there be no change to the individual’s management regardless of the genetic test result?
  Has the individual already had all of the clinical and imaging screening that would be performed regardless of whether there is genetic confirmation of the diagnosis?
• Do the clinical findings strongly favor a specific diagnosis in this spectrum?
  Can a particular disorder be tested for through one or a few genes or is there ambiguity that necessitates a larger gene panel? A referral to, or consult with an expert, such as a geneticist, should be considered if there are questions about alternate testing options.
• Is the patient acutely ill and will results return in time to impact care?
  (e.g. signs of decline or regression, concern for leukemia risk) Arriving at a diagnosis more quickly for a critically ill infant in the NICU may warrant consideration of RASopathy genetic testing in the inpatient setting.
• Are there multiple individuals in the family with similar clinical features?
  Testing one individual may benefit many family members. Furthermore, testing many similarly affected individuals may yield more informative results. And once a familial pathogenic variant is detected in one family member, less expensive targeted familial variant testing can typically be offered to appropriate family members instead of full gene sequencing or panel testing.
• What is the benefit of pursuing testing now versus deferring until later?
  Consider that the test technology and interpretation of results are rapidly advancing and will improve; symptoms & family history may evolve to guide more targeted or comprehensive testing; the cost of testing will likely decrease; the number of genes associated with this clinical spectrum may increase; and available treatment options may increase.

• When there would there be no change to the individual’s management regardless of the genetic test result.
• When there has been no pre-test counseling for the individual (or legal guardian) regarding risks, benefits, and potential out of pocket costs of the genetic testing.

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There are a variety of utilization management (UM) tools that can support appropriate ordering of RASopathy genetic testing. In addition, each case is unique and will require a balanced consideration of factors unique to RASopathy genetic testing.

RASopathy genetic testing is well-suited to strong UM interventions, including formularies, requirement for approval and privileging.

• Establish a formulary for RASopathy genetic testing, including sequencing and deletion/duplication analysis, to limit ordering to a defined reference laboratory (or set of laboratories). This helps ensure the quality of the test and improves ease of test coordination/logistics. It also allows the ordering lab to choose a panel that includes all RASopathy genes or one that excludes genes for which clinical criteria can often guide necessity for testing. This may also help with negotiating the best price.

• Due to the complex nature of RASopathy genetic testing, institutions may privilege this test to genetics providers only.

Set clear expectations for providers regarding the use of this test and process for requesting and obtaining approval. Providers may feel that the patient came to them for evaluation, and genetic tests are their primary “tool”, so if a test isn’t ordered, they haven’t done their job. Similarly, it is critical for providers to set clear expectations for the patient or family, particularly if there are limits placed on when and how RASopathy genetic testing can be ordered.

• Reflex testing options: In some cases, it is more efficient and cost-effective to pursue a reflexive testing approach, starting with a single gene order or single methodology (such as sequencing only, due to higher detection rate). If this analysis is negative, there may be an option to reflex to larger panel or other methods. However, deletion/duplication testing has a relatively low detection rate in this set of conditions and therefore warrants particular consideration before ordering.
• Gene coverage: When considering a single gene approach or a RASopathy gene sequencing or deletion/duplication panel, determine whether the gene(s) of most interest have good coverage on the platform. Conversely, in some cases, there may be reason to exclude certain RASopathy genes from an individual’s test. (E.g., if neurofibromatosis type 1 seems very unlikely in an adult patient with features of Noonan syndrome, then choose a panel without the NF1 gene to reduce risk for detecting variants of uncertain clinical significance in the low yield NF1 gene.)

In addition to the above considerations, there can be subtle aspects of a request for RASopathy genetic testing that are worth mentioning. For example, has the patient already had all imaging studies she would need if she were diagnosed with the suspected condition? (E.g. would it REALLY change the management of the patient now?) Does the provider already have a sense for intellect/prognosis (e.g., intellect/prognosis in a 7 year old may be more clear than a 6 month old) or risk for certain tumors (e.g., some tumors associated with NF1 are unlikely to occur after childhood)?

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Once RASopathy genetic testing has been established as the most appropriate test, the following recommendations are suggested as a responsible approach to test coordination:

• Pre-test counseling to provide clear information regarding the benefits, limitations and results of RASopathy genetic testing.
• Clear documentation of medical rationale and necessity of RASopathy genetic testing, combined with insurance pre-authorization for the testing to protect the patient/institution from avoidable financial liability.
• Results communication plan established at time of test coordination, to include post-test counseling with a genetics provider. This is particularly important if there is concern that results may return after a patient is deceased.

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Allanson JE, Roberts AE. Noonan Syndrome. 2001 Nov 15 [Updated 2011 Aug 4]. In: Pagon RA, Adam MP, Ardinger HH, et al., editors. GeneReviews® Internet]. Seattle (WA): University of Washington, Seattle; 1993-2016. Available from: http://www.ncbi.nlm.nih.gov/books/NBK1124/

Baird G. The laboratory test utilization management toolbox. Biomedia Chemica 2014;24(2):223-234. Friedman JM. Neurofibromatosis 1. 1998 Oct 2 [Updated 2014 Sep 4]. In: Pagon RA, Adam MP, Ardinger HH, et al., editors. GeneReviews® [Internet]. Seattle (WA): University of Washington, Seattle; 1993-2016. Available from: http://www.ncbi.nlm.nih.gov/books/NBK1109/

Gelb BD, Tartaglia M. Noonan Syndrome with Multiple Lentigines. 2007 Nov 30 [Updated 2015 May 14]. In: Pagon RA, Adam MP, Ardinger HH, et al., editors. GeneReviews® [Internet]. Seattle (WA): University of Washington, Seattle; 1993-2016. Available from: http://www.ncbi.nlm.nih.gov/books/NBK1383/

Gripp KW, Lin AE. Costello Syndrome. 2006 Aug 29 [Updated 2012 Jan 12]. In: Pagon RA, Adam MP, Ardinger HH, et al., editors. GeneReviews® [Internet]. Seattle (WA): University of Washington, Seattle; 1993-2016. Available from: http://www.ncbi.nlm.nih.gov/books/NBK1507/

Rauen KA. Cardiofaciocutaneous Syndrome. 2007 Jan 18 [Updated 2012 Sep 6]. In: Pagon RA, Adam MP, Ardinger HH, et al., editors. GeneReviews® [Internet]. Seattle (WA): University of Washington, Seattle; 1993-2016. Available from: http://www.ncbi.nlm.nih.gov/books/NBK1186/

Stevenson D, Viskochil D, Mao R. Legius Syndrome. 2010 Oct 14 [Updated 2015 Jan 15]. In: Pagon RA, Adam MP, Ardinger HH, et al., editors. GeneReviews® [Internet]. Seattle (WA): University of Washington, Seattle; 1993-2016. Available from: http://www.ncbi.nlm.nih.gov/books/NBK47312/