Page 38 - Haematologica April 2020

D. Wu et al.
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 tran- script. 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 differ- ent 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 tran- script, 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 current- ly classified as VUS (Figure 2B). The RUNX1 protein het- erodimerizes through its RHD with CBFβ to form a mas- ter 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 muta- tions 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_support- ing (one proband meeting at least one of the RUNX1 phe- notype criteria), PP1 (co-segregation with disease in the family, three meioses) and arrived at a consensus classifi- cation of PATH.
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 iden- tified 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 pheno- type. 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 subsequent- ly revised to LPATH upon MM-VCEP review (Figures 2B, 3A and 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 com- bines 13 individual tools with high sensitivity and speci- ficity, which has demonstrated the highest performance compared with individual tools or other ensemble meth- ods.39-41 The computational prediction code PP3 is applica-
ble 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 crite- ria (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).
The codes PM2, PP3, PM1_supporting are also applica- ble 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 model42,43 manifested by disturbed myeloid differentiation and induction of a blast crisis or accelerated phase–like phenotype in mice.42 These variants highlight the impor- tance 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 mis- sense mutation located within the RHD, but not within the mutational hotspot region (AA 105-204), with con- flicting 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 sub- missions are from OMIM, which cited evidence from individual literature sources without a systematic cura- tion 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-
880
haematologica | 2020; 105(4)