Page 42 - Haematologica April 2020

D. Wu et al.
tion in PHF6. Alterations in these two genes have been reported as cooperating events seen in leukemias from patients with germline RUNX1 mutations.37,53
Importantly, if consideration is given to the mutations found in the leukemic cells in isolation, one cannot deter- mine the germline or somatic origin of the variants reported. This is the case for most of the ‘tumor-only’ analyses being performed in many clinical laboratories. Without paired analysis of true germline tissue (e.g. cul- tured skin fibroblasts), such studies cannot definitively identify germline variants. In this case, the KMT2A partial tandem duplication and single nucleotide variation in PHF6 and RUNX1 could be tumor-drivers in the AML. However, given the sequencing data, including the vari- ant allele frequency, both the RUNX1 mutation and the PALB2 exon 7 intragenic deletion could be germline vari- ants. A detected variant allele frequency approaching 50% or 100% in the tumor may indicate potential germline origin1 with either an intact wildtype allele or loss of heterozygosity, respectively. However, a high vari- ant allele frequency cannot reliably serve as a proxy for testing of a true germline source. Therefore, if there is concern that a variant could be constitutional, testing of true germline material is critical.1
Discussion
Kindreds with FPD/AML were first reported by Luddy et al. in 197854 and phenotypically well-described as hav- ing a bleeding diathesis and myeloid neoplasia by Dowton et al. in 1985.55,56 Subsequent linkage analysis identified RUNX1 as the candidate gene at chromosome 21q22,11 and mutations were detected in FPD/AML fami- lies in 1999.11 Since these initial early reports, routine clin- ical testing for RUNX1 gene mutations is now common- place for the evaluation of somatic and germline disease in patients with myeloid neoplasms and thrombocytope- nia.
In general, RUNX1 variants include single nucleotide variations and indels, such as missense, nonsense, frameshift, and splice site variants, and copy number vari- ations such as whole-gene and intragenic deletions. RUNX1 is also frequently mutated somatically in AML and often the partner of various translocations resulting in gene fusions, such as t(8;21)(q22;q22) RUNX1- RUNX1T1.57,58 To date, fusions of RUNX1 have not been reported in the germline context, and most germline RUNX1 variants are unique,24 although some have been rarely seen in unrelated families. Given the limited data on rare variants, the clinical annotation of new variants remains challenging. The MM-VCEP was convened by ASH/ClinGen (Online Supplementary Figure S1) to develop rules for curating gene variant causing predisposition to myeloid neoplasia (Table 2). In this review, we describe the classification of six variant examples (Table 3) using the gene- and disease-specific rule modifications of the original ACMG/AMP 2015 framework.14 Several points should be made.
First, it is critical to ensure that genomic testing intend- ed to assess a germline predisposition is performed on a definitive germline sample because malignant hematolog- ic diseases involve the peripheral blood and bone mar- row, and somatic variants in these diseases can confound variant interpretation if an inappropriate sample is used.
Here, in keeping with our MM-VCEP rules, cultured skin fibroblasts (gold standard, albeit invasive, costly, and time-consuming), cultured bone marrow mesenchymal stromal cells or DNA from hair roots are appropriate sources.59,60 Alternatively, confirmation of the germline nature of a variant can be achieved by demonstrating its presence in two or more related individuals. The possibil- ity of sample contamination by malignant cells is signifi- cant and consequently, peripheral blood, bone marrow, saliva, buccal swabs, DNA from paraffin blocks and even fingernails, which can contain monocytes, are inappropri- ate samples for germline testing. In some institutions, lab- oratories may accept T cells, enriched via flow cytometry sorting or column-based magnetic cell separation, as a germline sample for testing. It is important to recognize that some somatic alterations may occur early in hematopoietic stem and progenitor cells with multilin- eage potential to differentiate into T cells,61 as recent sin- gle-cell studies have confirmed.62-65 Thus, if T cells are used, the possibility that a detected variant may be somatic should still be considered. Once a variant is con- firmed to be germline in a proband, however, additional testing for the known variant in related family members can be performed on any tissue source.
Second, we should keep an open mind about disease- causing alleles and the type of variants that may be seen and thus, we advocate for a broad testing approach. For example, in some laboratories, non-coding variants are automatically filtered as part of bioinformatic pipelines and may thus be omitted from subsequent review and interpretation. Recently, however, synonymous variants66 in the GATA2 gene, another gene predisposing to myeloid malignancy, were reported in addition to the known pathogenic deep intronic variants of an enhancer region of GATA2.67 In ANKRD26, variants of the 5’ untranslated region cause disease.68,69 Furthermore, copy number alter- ations may not be assessed in somatic tumor testing pan- els. As diagnosticians, it is important to think broadly when analyzing genomic information for germline path- ogenic variants. Given that these are rare diseases, we should not inadvertently exclude disease mechanisms and/or specific classes of mutations. For example, in case 4, some variants may remain as VUS until additional functional or familial segregation data become available for reclassification.47,48
Third, definitive annotation of variants by one institution will likely remain challenging. However, consistent applica- tion of MM-VCEP rules with ClinVar data deposition and thus inter-laboratory correspondence can significantly improve the accuracy and consistency of variant curation. In this regard, examples 2, 3, and 5 show how leveraging shared genomic and phenotypic data can be helpful to clar- ify VUS. We therefore advocate that clinical variant data be deposited into ClinVar. Specifically, laboratories offering germline testing should modify their test requisition forms to indicate that de-identified phenotype and variant data will be deposited into ClinVar as part of ongoing quality assurance and improvement efforts (https://www.clini- calgenome.org/share-your-data/laboratories/).70,71 Additional details of the ClinVar deposition process are included in Online Supplementary Figure S1.
Fourth, RUNX1 variant curation will improve as more is understood about the disease and gene through func- tional and family studies. Currently, variant annotation remains a challenging task, because of limited data for
884
haematologica | 2020; 105(4)