Clinical characteristics, JAK2 analysis, and next-generation sequencing (NGS) screening for MPN patients exhibiting JAK2 V617F and JAK2 R1063H mutations. (A) Hematological data for JAK2 V617F MPN patients (n = 390) subdivided according to the JAK2 R1063H mutation status. Data for V617F only (n = 376) and for V617F/R1063H double-mutation carriers (n = 14) were recorded at diagnosis. For further information, see supplemental Material and methods and supplemental Table 1. The boxes represent the 25% to 75% interquartile range, horizontal lines within the boxes indicate medians, and vertical bars show the range of values (minimum to maximum). P values < .05 were considered statistically significant. *P < .05, Mann-Whitney U test. (B) JAK2 V617F allele burden, JAK2 R1063H fractional abundance, JAK2 V617F/R1063H mutations configuration, and additional mutations identified by targeted NGS. Third column: JAK2 V617F allele frequency obtained from the NGS study and compared with allele burden determined via quantitative PCR and ddPCR assay. Fourth column: JAK2 R1063H fractional abundance was determined using ddPCR in whole-blood samples collected at the time of diagnosis (see supplemental Material and methods for details). The JAK2 R1063H mutation was considered genuine germline only when the fractional abundance of the JAK2 R1063H variant was 50% (± 1.0%). Therefore, 8 patients are confirmed to be heterozygous germline carriers for the JAK2 R1063H variant. JAK2 R1063H in 3 samples with a percentage frequency of the mutant DNA between 20.7% and 31.5% (samples 1, 5, and 14) could be considered an acquired somatic mutation or an inherited variant that was partially lost due to UPD of the V617F–non-R1063H clone. Samples 4, 12, and 13 were nearly homozygous for JAK2 R1063H, and the presence of minor fraction of the WT allele excluded germline homozygosity. See also supplemental Figure 2. Fifth column: Cis/trans JAK2 V617F/R1063H mutations configuration was determined through sequencing of subcloned reverse-transcriptase PCR products spanning exons 14 through 24 of the JAK2 gene (see supplemental Material and methods for details). Sixth column: TruSight Myeloid Sequencing Panel (Illumina, San Diego, CA) was used for targeted mutational screening of JAK2 R1063H+ patients. Additional mutations were identified in 8 of 14 screened patients. A total of 11 variants was detected in 7 genes. #Five of these mutations are indexed in the Single Nucleotide Polymorphism database (dbSNP), and 4 of these specific variants are listed in the COSMIC catalog. One additional mutation in DNMT3A was published recently.14 §Two other mutations (frameshift in TET2 and premature stop codon in DNMT3A) do not have SNP/COSMIC IDs but are documented in the VarSome genomic variant database. **The GATA2 (A164T) allele (patient 12) was recently detected in a higher-than-expected frequency in myelodysplastic syndrome, suggesting a possible predisposing function in myeloid malignancies.15 Two patients harbor unique undescribed variants: patient 1 in BCOR and patient 9 in TET2. The BCOR variants were identified in 2 patients (both are missense mutations); their variant frequency was 54% for patient 1 and 99.5% for patient 11. They could be germline variants; both were estimated to be “damaging” or “probably damaging” by 2 algorithms (Sift, PolyPhen). *Indicates translation termination (stop) codon. All mutations were identified in DNA collected at the time of diagnosis; acquisition of additional mutations during disease evolution was not performed. For further information see supplemental Table 2. NA, not available; ND, not done; n.s., not significant; PMF, primary myelofibrosis; qPCR, quantitative PCR; snv, single nucleotide variant; Var Freq, variant allele frequency.