4-stage (4-tier) system for evaluating sequence variants

Sequence alterations have classically been divided into two categories: "mutation" and "polymorphism". However, the rapid increase in sequencing data from healthy and various diseased tissues has made it obvious that there is a broad spectrum between clearly disease-associated pathogenic alterations and non-pathogenic polymorphisms. As of now, we are working in molecular genetics with the world's most widely used 4-tier classification system, which we have adapted to central questions in hematological diagnostics. We would like to introduce this to you in this article.

Sequence data arising from both diseased and healthy tissues has increased significantly in recent years. One of the main reasons for this has been the introduction of novel sequencing technologies, a.k.a. next-generation sequencing (NGS), which now allows us to sequence complete human genomes over the course of just a few days. The large number of alterations identified during this time between different healthy individuals and—of particular interest to us—between healthy and diseased tissues has posed major challenges for diagnosis regarding the classification of such alterations.

What distinguishes “clearly pathogenic” from “polymorphism”?

Sequence alterations were traditionally subdivided into the categories “mutation” and “polymorphism,” whereby only the former was considered to be relevant or pathogenic. These days, however, the spectrum is much wider. The clearly pathogenic end of the spectrum comprises alterations that are functionally optimally characterized and which may entail therapeutic, diagnostic, or prognostic consequences. The other end comprises alterations termed polymorphisms, which, according to our current state of knowledge, have no disease-causing functions. In many cases, the latter can be identified from the frequency at which they occur in the population.

What alterations lie between these extremes?

A grading of the assessment becomes necessary where, in place of the typical mutation (“hotspot”) in a gene for which a targeted therapy exists, another amino acid position is affected. The alterations p.V600E in the BRAF gene as well as p.V617F in the JAK2 gene can be cited as therapeutic targets at this point. If positions outside the hotspots are affected in both genes, and if these have not yet been functionally characterized, a targeted therapy is not necessarily possible and these alterations cannot be described as “clearly,” but only as “possibly pathogenic.”

Unlike genes characterized by hotspot mutations, others are affected by alterations occurring throughout the entire gene sequence. Here again, a comparison with existing databases is important, possibly combined with in silico predictions, to determine whether to produce a “clearly” or just a “possibly pathogenic” assessment.

Alterations that remain (almost) unknown in the known databases and even the very large MLL-internal database and in which there is a suspicion of a congenital polymorphism due to the mutation burden are classified at best as variants (unknown significance). An analysis of normal tissue (e.g., oral mucosa, fingernail) provides information about the origin of an alteration (somatic or congenital).

Based on the world’s most widely used 4-class system (4-tier) for assessing sequence variants1, the MLL has now also introduced a 4-stage evaluation system. This ranges from tier 1 (clearly pathogenic), through tier 2 (possibly pathogenic), tier 3 (variant of unknown significance), and ultimately to tier 4 (polymorphism).


Reference

1Li MM, Datto M, Duncavage EJ, et al. Standards and Guidelines for the Interpretation and Reporting of Sequence Variants in Cancer: A Joint Consensus Recommendation of the Association for Molecular Pathology, American Society of Clinical Oncology, and College of American Pathologists. J Mol Diagn 2017;19:4-23.

The author

»Do you have questions regarding this article or do you need further information? Please send me an e-mail.«

Dr. rer. nat. Frank Dicker

Biologist, Dipl.
Head of Diagnostic Reporting

T: +49 89 99017-350