Example 1. Early nonsense variants, (p.Arg204Ter) (PATH with PVS1, PM2, PS4_supporting, and PP1)

A 50-year old female with new pancytopenia was referred to a hematology service. A bone marrow biopsy showed hypocellularity with severe trilineage dysplasia and 12% blasts, diagnostic of MDS with excess blasts (MDS-EB-2). Further investigation showed pathogenic variants in RUNX1 (NM_001754:c.610C>T, (p.Arg204Ter)), BCOR and ASXL1 with a normal karyotype. The medical history was positive for thrombocytopenia (baseline 70-120×10⁹/L) and a propensity to excessive bleeding after tooth extractions. The family history was positive for two sons with persistent thrombocytopenia (baseline 50-100×10⁹/L) not otherwise explained and a granddaughter with thrombocytopenia and MDS with monosomy 7 (Figure 1). During the initial assessment, an increase in lactate dehydrogenase and the peripheral blast count were noted. A second marrow biopsy confirmed transformation into AML with 40% blasts. The patient underwent induction chemotherapy without achieving remission and clofarabine bridging for unrelated stem cell transplantation. During conditioning, the patient developed sepsis with Gram-negative bacteria and died shortly afterwards. Since she had a remarkable personal and family history pointing towards a germline predisposition syndrome, a skin biopsy was performed at the time of the diagnosis of MDS, and DNA testing from cultured skin fibroblasts later confirmed the germline, nonsense RUNX1 variant. Her two sons and granddaughter also tested positive for the RUNX1 variant (Figure 1).

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Figure 1.

Family pedigree of a patient with acute myeloid leukemia, example 1. Germline RUNX1 NM_001754:c.610C>T, (p.Arg204Ter) showing typical autosomal dominant inheritance with other first and second-degree relatives with thrombocytopenia and/or myeloid malignancy (acute myeloid leukemia and myelodysplastic syndrome). mut: mutated; MDS: myelodysplastic syndrome; sAML: secondary acute myeloid leukemia; +: wildtype.

Similar to this case, most patients with FPD/AML will have a characteristic phenotype (Table 1) including mild to moderate thrombocytopenia with normal platelet size, platelet α or dense granule secretion defects and impaired platelet aggregation, particularly in response to collagen and epinephrine as well as a predisposition to hematologic malignancies. Although there is variability in disease onset in FPD/AML,³ development of a hematologic malignancy is common with a lifetime risk of ~44%: AML and MDS are common, other malignancies occur less frequently (Table 1).^19–23 FPD/AML has a high but incomplete penetrance, with several affected individuals reported to have normal platelet counts or function.^19,24 The nonsense mutation in this patient (p.Arg204Ter) is predicted to lead to nonsense-mediated decay of the RUNX1 mRNA transcript. RUNX1 is located on the long arm of chromosome 21 and is translated into three major isoforms, designated RUNX1A, RUNX1B, and RUNX1C, by using two different promoters and alternative splicing. All transcripts are expressed during hematopoietic differentiation and/or maintenance of normal bone marrow function.^25–32 For variant annotation, the MM-VCEP utilizes the longest isoform, RUNX1C (NM_001754), as the default transcript, which includes all key domains such as the 128 amino acid (AA) long RUNT homology domain for DNA binding (RHD, AA 77-204), transactivation domain (TAD), inhibitory domain (ID) and the transcriptional repressor binding motif (VWRPY) and is most often used by clinical laboratories for RUNX1 variant curation (Figure 2A). Germline variants have been reported throughout the gene in ClinVar with the majority currently classified as VUS (Figure 2B). The RUNX1 protein heterodimerizes through its RHD with CBFβ to form a master hematopoietic transcription factor (Figure 2C), which is essential for proliferation and differentiation of hematopoietic stem and progenitor cells, especially in the case of megakaryocytic differentiation.^33–35 Somatic mutations commonly occur in RUNX1 (Figure 2D). According to RUNX1-specific criteria (Table 2),¹⁸ the MM-VCEP applied the following codes (Table 3): PVS1 (nonsense variant predicted to undergo nonsense-mediated decay), PM2 (absence in all population databases), PS4_supporting (one proband meeting at least one of the RUNX1 phenotype criteria), PP1 (co-segregation with disease in the family, three meioses) and arrived at a consensus classification of PATH.

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Figure 2.

Overview of RUNX1 protein domains, ClinVar-deposited and MM-VCEP curated pilot and COSMIC variants. (A) Schematic of the RUNX1 protein showing its key domains, including the Runt homology domain (RHD); annotation according to MM-VCEP is based on RUNX1 transcript isoform C (NM_001754). (B) Schematic of ClinVar-deposited RUNX1 variants in a one-dimensional line plot showing the current ClinVar five-tier classification; data plotted using proteinpaint from St. Jude.org with data from ClinVar (accessed 6/2019); RHD (purple), and ID (green) are highlighted; numbers indicate amino acid; dotted lines indicate exon boundaries. Some ClinVar-deposited variants (in 5’ and 3’ untranslated regions and large deletions) are not shown. Lollipops representing the variants are colored as follows: PATH (red), LPATH (orange), VUS (grey), LBEN (blue), BEN (purple), CONF (green). MM-VCEP pilot variants¹⁸ are shown in the third row. (C) Three-dimensional structure of the RHD of RUNX1 (blue) complexed to CBFβ (pink) and DNA (orange).⁸² ClinVar RUNX1 variants in this domain (codons 81 to 204 only) are shown using PyMOL, version 2.3.0, as follows: yellow = VUS (n=20), pink = CONF (n=2), orange = LPATH (n=3), red = PATH (n=5); BEN and LBEN variants (n=0). Non-missense variants are not shown. (D) One-dimensional line plot of somatic variants deposited with the Catalog of Somatic Mutations in Cancer (COSMIC: data release 3 Nov. 2018, version 87; https://cancer.sanger.ac.uk/cosmic). MM-VCEP: Myeloid Malignancy Variant Curation Expert Panel; PATH: pathogenic; LPATH: likely pathogenic; VUS: variant of uncertain significance; LBEN: likely benign; BEN: benign; CONF: conflicting interpretations in ClinVar.

Example 2. Missense variants, p.(His105Pro) (LPATH with PM2, PP3, PS4_supporting, PM1_supporting, and PM5_supporting) and p.(His105Gln) (LPATH with PS3, PM2, PP3, and PM1_supporting)

Missense mutations in RUNX1 commonly occur in the RHD in somatic and germline contexts.^36–38 Of 325 RUNX1 ClinVar variants 122 (37.5%) are missense, and currently in ClinVar, none in the RHD has been classified as BEN or LBEN (Figure 2C). When a novel missense variant is identified which has not been established as PATH or LPATH, it can be difficult to know whether the given change will affect protein function and explain the patient’s phenotype. For example, two RUNX1 missense variants in the RHD (NM_001754:c.314A>C, p.(His105Pro); and NM_001754:c.315C>A, p.(His105Gln)) were considered during the pilot variant analysis. The former was initially classified as a VUS in ClinVar (Figure 2B), but subsequently revised to LPATH upon MM-VCEP review (Figures 2B, ​,3A3A and ​and4).4). The conclusion of the LPATH assertion is based on the codes applied for this variant: PM2, PP3, PS4_supporting, PM1_supporting, and PM5_supporting (Table 3). Since the variant is completely absent from population databases, the MAF code PM2 is applied. For in-silico evaluation of missense variants, the MM-VCEP recommends using REVEL, a meta-predictor that combines 13 individual tools with high sensitivity and specificity, which has demonstrated the highest performance compared with individual tools or other ensemble methods.^39–41 The computational prediction code PP3 is applicable to the p.His105Pro variant due to a high REVEL score of 0.953 (MM-VCEP defined >0.75 as the cutoff). The ClinVar submitter (SCV000807773.1) provided us with the patient’s clinical data from their laboratory and the proband met at least one of the RUNX1 phenotype criteria (Table 1) which qualified for PS4_supporting. This example emphasizes the critical value of sharing internal laboratory data. There is only one meiosis in this family which is lower than the three required for the segregation code PP1. The MM-VCEP defined 13 residues in the RHD as the mutational hotspots for the PM1 code. In addition, variants in other parts of the RHD (AA 105-204) can have a reduced strength-level resulting in application of PM1_supporting. The last code PM5_supporting is applied on the p.(His105Pro) variant, because a different missense change p.(His105Gln) at the same residue has been classified as LPATH by the MM-VCEP (Table 3).

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Figure 3.

Three-dimensional structure of RUNX1 missense variants His105 and His85 considered as examples 2 and 3. (A) RUNX1 His105 is important functionally due to its location and thus involvement in DNA binding and close interaction with Thr196 by hydrogen bonding. Thr196 is a hotspot residue known to be critical. This structure-function relationship further supports classification of His105 variants as likely pathogenic (LPATH). (B) RUNX1 His85 is located close to the start of the Runt homology domain in a linker region, and is located far from the DNA binding surface. It is not involved in the core β-barrel structure and does not show any interactions, further supporting its classification as likely benign (LBEN). Structure of RUNX1 complexed to DNA and CBFβ (https://www.rcsb.org/structure/1H9D)⁸² and plotted using PyMOL version 2.3.0.

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Figure 4.

Three-dimensional structure of MM-VCEP-classified RUNX1 missense variants in the RHD. Variants in the RUNX1 RHD (blue) are shown highlighting the PATH, LPATH, and LBEN missense variants curated according to ClinGen MM-VCEP rules. PATH (red, n=4), LPATH (orange, n=4) and LBEN (cyan, n=1) variants are found proximate to key interaction domains of RUNX1 with DNA or its binding partner CBFβ (pink). VUS missense variants are shown in yellow (n=7). Non-missense variants and variants outside the RHD are not shown. (PyMOL version 2.3.0; structural data https://www.rcsb.org/structure/1H9D⁸²). MM-VCEP: Myeloid Malignancy Variant Curation Expert Panel; RHD: Runt homology domain; PATH: pathogenic; LPATH: likely pathogenic; VUS: variant of uncertain significance; LBEN: likely benign; BEN: benign; CONF: conflicting interpretations in ClinVar.

The codes PM2, PP3, PM1_supporting are also applicable to the p.(His105Gln) variant for the same reasons described. Furthermore, a strong pathogenic code PS3 is applied which contributes a significant weight to the final assertion. Transactivation assays of the p.(His105Gln) variant demonstrate altered transactivation (<20% of wildtype) and secondary assays also indicate altered DNA binding and functional consequences in a mouse model^42,43 manifested by disturbed myeloid differentiation and induction of a blast crisis or accelerated phase–like phenotype in mice.⁴² These variants highlight the importance of evaluating similar variants and the critical benefit of functional studies showing that variants whose clinical significance were initially uncertain can be subsequently clarified to provide more definitive clinical classification and minimize reporting of VUS. Moreover, these variants demonstrate the value of leveraging the information on one variant to help classify another and data sharing between laboratories (Table 3).

Example 3. Missense variant, p.His85Asn (LBEN with BS1, BS3, and PP3)

RUNX1 NM_001754:c.253C>A, p.(His85Asn) is a missense mutation located within the RHD, but not within the mutational hotspot region (AA 105-204), with conflicting interpretations of pathogenicity in ClinVar (Figure 2B, C). Specifically, this variant had three submissions in ClinVar with two being PATH (submitted in 2002) and one being a VUS (submitted in 2018). The two 2002 submissions are from OMIM, which cited evidence from individual literature sources without a systematic curation process. Osato et al. reported an adult patient with AML carrying this variant.⁴⁴ However, the germline nature of the variant was not definitively determined. This variant has also been reported in an infant diagnosed with transient myeloproliferative disorder and Down syndrome whose phenotype does not meet any of the RUNX1 phenotype criteria.⁴⁵ After analysis and curation by the MM-VCEP using the RUNX1-specific classification rules,¹⁸ this variant was re-classified as LBEN, meeting codes BS1 and BS3, despite meeting PP3 (Table 3). According to the penetrance, prevalence and genetic and allelic heterogeneity of RUNX1, MM-VCEP refined the RUNX1 specific MAF threshold for application of BS1 to 0.00015 (0.015%). The highest MAF of the p.His85Asn variant is 0.00043 (8 out of 18,768 alleles) from the East Asian subpopulation in the Genome Aggregation Database (gnomAD) which is higher than the RUNX1-specific BS1 cutoff. Experimental studies have shown that this missense change displays normal transactivation activities (80-114% of wildtype) and does not affect DNA binding, heterodimerization with CBFβ or subcellular localization of the RUNX1 protein.^44,46 Therefore, the strong benign functional evidence code, BS3, is applied. Although this variant disrupts KMT2A binding, which impairs proper H3K4 histone methylation, this is not a qualified functional assay based on the MM-VCEP RUNX1-specific PS3/BS3 rule. Moreover, another well-established BEN variant p.(Leu56Ser) also impairs KMT2A binding.⁴⁶ Likely due to the location of the p.(His85Asn) variant within the RHD (Figure 2B), the REVEL score (0.852) of this variant is higher than the MM-VCEP defined 0.75 cutoff,¹⁸ which results in the variant meeting a conflicting PP3 code. However, combining the BS1, BS3 and PP3 codes, a final assertion of LBEN is made based on a Bayesian classification framework.¹⁵ Given that His85 is located away from binding interfaces in the three-dimensional structure (Figure 3B), it seems reasonable that variants at this position are LBEN.

This example highlights the value of functional studies in the context of the MM-VCEP variant curation and shows that substantive corrections of variant annotation may occur upon application of ClinGen MM-VCEP rules.¹⁸ Implicit in this process is the expectation that as knowledge about FPD/AML improves with more functional or family data becoming available, the MM-VCEP rules are subject to revision so that annotation of clinical variants will become more accurate (Figure 4). ClinVar variant classifications such as VUS or those with conflicting interpretations may thus evolve to more diagnostic certainty.^47,48

Example 4. Synonymous/intronic/non-coding variants, c.508+3delA (PATH with PS3, PP1_strong, PM2, PP3, PS4_supporting), p.Thr148= (LBEN with BP4, BP7) and p.Val419= (VUS with no codes)

RUNX1 variants affecting canonical splice positions ±1 or 2 at intron-exon boundaries are expected to disrupt splicing, leading to protein dysfunction (see Online Supplementary Table S3 of reference by Luo and Feurstein¹⁸). All of the three canonical splicing site variants in the pilot set were classified as PATH or LPATH. More challenging, however, is the consideration of synonymous/intronic/non-coding variants which may result in cryptic splice site activation, and/or enhancement or repression of adjacent canonical splice sites. For example, the intronic NM_001754:c.508+3delA variant has been reported in a single family with disease segregation (8 meioses, PP1_strong). Several family members were diagnosed with thrombocytopenia, aspirin-like platelet aggregation defects, and dense granule abnormalities.⁴⁹ This variant is absent from population databases (PM2) and both splicing predictors (MaxEntScan and Splice -SiteFinder)^50,51 predict a significant decrease in the score of the canonical splice site (PP3). Moreover, experimental reverse transcriptase polymerase chain reaction studies (PS3), using RNA derived from two affected family members, were performed and indicate the creation of a novel cryptic splice site 23 nucleotides upstream of the normal splice site resulting in a frameshift p.(Arg162fs*177), and the transcript is predicted to undergo nonsense-mediated decay.⁴⁹ Combining all of these codes, a final assertion of PATH is given by the MM-VCEP (Table 3).

BP7 is a benign code specifically designed to evaluate synonymous/intronic/non-coding variants in the ACMG/AMP framework. BP7 can be applied if computational evidence suggests no impact on splicing, and the nucleotide is not conserved. The ClinVar variant with conflicting interpretations in ClinVar, NM_001754:c.444C>T, p.(Thr148=), has been classified as LBEN by the MM-VCEP using BP7 and the benign in silico prediction code, BP4 (Table 3). This nucleotide change is predicted to have no impact on splicing and it is also not conserved (phyloPscore: −4.3832, below the MM-VCEP-specified threshold of <0.1¹⁸). Clinical data from seven individuals with this variant were acquired from the original ClinVar submitter (SCV000761123.1) and revealed that none of the probands met any of the RUNX1 phenotypic criteria.¹⁸ Currently, only two RUNX1 variants have been reported to display an abnormal splicing effect as demonstrated by RNA assays.^11,49 The potential effects of other splicing variants rely solely on in silico predictions. Although there is robust effort in consideration of algorithms to predict the effects of splicing variants, these algorithms require further evaluation. Indeed, we know of only limited experimental data within the RUNX1 gene specifically to test these tools. Accordingly, the synonymous variant, NM_001754:c.1257G>A, p.(Val419=) is predicted to create alternative splice acceptor sites, but is not expected to abolish any existing consensus sites, as it is too far away from either end of the exon. Due to this in silico prediction result, none of the PP3/BP4 and BP7 codes can be assigned, and the classification of this variant remains a VUS. Further resolution of the significance of this variant could be obtained through parental testing, and/or RNA-sequencing data.

Example 5. Copy number variants, deletion of exon 2 (PATH with PVS1_moderate, PM2, PS4, and PP1_strong)

Not infrequently, patients with FPD/AML have been reported to have copy number variants resulting in intragenic deletions of RUNX1.⁵² As part of our pilot cohort, we evaluated several probands with copy number variants which at a minimum include RUNX1 exon 2 deletion; data from two cases are shown (Figure 5). The analysis of copy number variants by using next-generation sequencing and/or single nucleotide polymorphism microarrays is particularly challenging because the breakpoints are often not captured in the sequenced regions or the microarray resolution defines only a range for the chromosomal location of the breakpoint, respectively, and thus the nucleotide level breakpoint may remain unknown. It can, therefore, be difficult to know the effect of partial gene/exon deletions, such as if the deletion is in- or out-of-frame, the latter of which may also lead to the introduction of a premature stop codon. Nevertheless, partial or whole gene deletion of RUNX1 is expected to result in haploinsufficiency of the RUNX1 protein. Although MM-VCEP rules did not include recommendations for the formal classification of copy number variants,¹⁸ several points should be noted. First, evaluation of the reference Database of Genomic Variants (http://dgv.tcag.ca/dgv/app/home) shows that copy number variants affecting RUNX1 do not appear to be frequent.¹⁸ Second, annotation of the specific breakpoints of these intragenic deletions may not always be possible, given that whole genome sequencing is not typically performed. Since contiguous exon deletion is a common pathogenic disease mechanism, it is imperative that laboratories performing germline testing for RUNX1 use concurrent microarray testing, develop appropriate next-generation sequencing bioinformatics pipelines, or use alternate molecular techniques, such as quantitative polymerase chain reaction and multiplex ligation-dependent probe amplification, to screen for and exclude copy loss, as sequencing for single nucleotide variants and indels alone is insufficient for comprehensive germline evaluation. For the two cases herein (Figure 5), although we do not know the specific breakpoints for each, the common deletion of at least exon 2 allows us to apply the following codes: PVS1_moderate, PM2, PS4 (4 probands: 3 with chronic thrombocytopenia and 1 with AML), PP1_strong (7 meioses) to arrive at a PATH classification (Table 3).

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Figure 5.

Copy number variant, deletion of exons of RUNX1. Testing for germline variants should include evaluation for copy number variants (CNV). (A; B) Clinical data from two different patients showing copy loss of RUNX1 exons 1 and 2; one proband was identified by next-generation sequencing (A) and the other by single nucleotide polymorphism array analysis (data not shown), and confirmed by quantitative polymerase chain reaction (qPCR) (B). For the latter patient, qPCR confirmation showed heterozygous deletion of exons 1 and 2 with no loss in other exons (exon 6 only shown). CNV are difficult for annotation as breakpoints of the deletion may not be captured and thus, whether the deletion is in- or out-of-frame may not be known without whole genome sequencing. Testing of a germline sample (e.g. fibroblasts) is preferred to blood or bone marrow for CNV evaluation, as somatic copy loss may also occur in the tumor context.

The Variant Curation Expert Panel (VCEP) thanks the Clinical Genome Resource (ClinGen) Sequence Variation Interpretation Working Group as well as the Executive Committee of the Hereditary Cancer Clinical Domain Working Group. The variant curation rules discussed in this publication were generated by the American Society of Hematology (ASH) in collaboration with Baylor College of Medicine and the University of North Carolina, National Institutes of Health (NIH)-funded ClinGen grant award recipients. The NIH supported this work through: U41HG009649 (to XL, and SEP) and U41HG009650 (to SM and JER), and the 2018 NIH/NCI Leukemia SPORE DRP award (P50CA100632-16, project 00007529) (C.D.D.).

The ASH-ClinGen Myeloid Malignancy MM-VCEP collaborative group includes co-authors (DW and LAG as co-chairs; XL, SF, SM, CK and DEP), and others, as follows:¹⁸ Christopher Porter (Emory University, USA), Sarah Jackson (GeneDx, USA), Sioban Keel (University of Washington, USA), Michael Chicka (Prevention Genetics, USA), Anna Brown (Center for Cancer Biology, Australia), Anupriya Agarwal (Oregon Health & Science University, The Knight Cancer Institute, USA), Minjie Luo (Children’s Hospital of Philadelphia, USA), Zejuan Li (Houston Methodist Institute for Academic Medicine, USA), Justyne E. Ross (University of North Carolina, USA), Panagiotis Baliakas (Uppsala University, Sweden), Courtney D. DiNardo (UT MD Anderson Cancer Center, USA), Alison Bertuch (Baylor College of Medicine, USA), Nikita Mehta (Mayo Clinic, USA), Thomas Vulliamy (Queen Mary University of London, UK), Ying Wang (BioReference, USA), Kim Nichols (St Jude Children’s Research Hospital, USA), Luca Malcovati (University of Pavia & S. Matteo Hospital, Italy), Michael Walsh (Memorial Sloan Kettering Cancer Center, USA), Lesley Rawlings (Centre for Cancer Biology, Australia), Shannon McWeeney (Oregon Health & Science University, USA), Jean Soulier (Hopital Saint-Louis and University de Paris, France), Anna Raimbault (INSERM U1016, Institut Cochin, France), Mark Routbort (UT MD Anderson Cancer Center, USA), Liying Zhang (Memorial Sloan Kettering Cancer Center, USA), Gabriella Ryan (American Society of Hematology, USA), Nancy Speck (Abramson Family Cancer Research Institute, USA), and Sharon E. Plon (Baylor College of Medicine, USA).