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C. Baer et al.
(polymerase chain reaction/amplification bias) can con- tribute to skewed VAF. Secondly, somatic mutations can also occur with a VAF of 50% if the number of malignant cells in the analyzed sample is high. Lastly, genetic fea- tures influence the VAF. A deletion of 17p, would cause all germline variants in the deleted locus (foremost TP53) to appear with VAF not around 50%. Copy number gains and copy neutral loss of heterozygosity also influence VAF.
Databases are frequently used for variant interpreta- tion. Curated databases for variants meet high quality standards, but contain only a small number of variants.16 Databases with large numbers of contributors, such as dbSNP, contain more variants. Initially, many variants were classified as benign germline variants if they were listed in the dbSNP.17 All databases, which allow individ- ual submitters to add data, are error prone. The diversity of the exome was impressively demonstrated by Lek et al., who analyzed 60,706 individuals of different ethnici- ties and found on average one variant for every eight bases of the exome. Data were collected by the Exome Aggregation Consortium (ExAC), which included a col-
lection of whole exome sequencing data from a broad range of studies. ExAC has recently been extended to genomes (gnomAD).18 As of today, gnomAD contains data from 141,456 individuals. This allows for a represen- tative overview of the population and ethnic groups. As such, it can be used to exclude frequent variants in the population from candidate disease-associated aberra- tions. However, it must be kept in mind that the metada- ta from such a collection includes many blood samples and even individuals with an (undiagnosed) hematologic disease. The V617F mutation in the JAK2 gene, which is found in the majority of patients with myeloproliferative neoplasms, is also found in 0.04% of individuals in the gnomAD dataset, so variants in gnomAD cannot be clas- sified per se as benign.
Considering the difficulties, it can be questioned whether the strict separation of somatic and germline is necessary. There are a number of reasons why they should be distinguished: (i) only somatic changes prove clonal outgrowth in the case of clonal hematopoiesis of indeterminate potential and clonal cytopenia of undeter- mined significance8 and they are used for the calculation
Figure 1. Clinical questions for current variant classifications. The complexity of variant classification challenges the terms “mutation” and “polymorphism”, which were clearly associated with clinical relevance in the past. The classification, interpretation and consequences of each variant depend on the context. The identifi- cation of somatic changes proves clonality, and somatic aberrations qualify as markers of minimal/measurable residual disease. Other clinical issues are the avail- ability of targeted therapies for mutations and the World Health Organization classification, which genetically defines certain entities. Finally, inherited variants are not only discussed in the context of familial predisposition, but are also relevant if family members are considered as stem cell donors. AlloSCT: allogeneic stem cell transplantation; MRD: minimal/measurable residual disease; WHO: World Health Organization.
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haematologica | 2019; 104(8)