Zhang, Xi

Abstract Introduction: Somatic mutations in splicing factors (SF) - SF3B1, SRSF2, U2AF1, and ZRSR2 – represent one of the most common drivers of premalignant clonal hematopoesis. While SF mutations are potent drivers of clonal expansion (Fabre et al., Nature 2022), multiple SF mutations are rarely observed in the same individual (negative epistasis). Prior studies have identified rare cases of concurrent SF mutations (<4%) and described specific hotspot combinations that escape negative epistasis (Taylor et al., Blood 2020). We conducted this study to further characterize the frequency and clonal relationship between concurrent SF mutations using a larger patient cohort. Methods: After IRB approval, we assessed 3,369 myeloid next generation sequencing (NGS) results that identified somatic variants in SF3B1, SRSF2, U2AF1, or ZRSR2, in 2,500 adult patients from 08/2015 to 07/2025. Patient demographics, ICD-9/10 codes, and complete blood counts with differentials were obtained, retaining only those datapoints assigned 30 days before or after NGS testing. To allow comparison of clone size between autosomal and the X-linked SF ZRSR2, VAFs were multiplied by two to estimate the cancer cell fraction (CCF), except for ZRSR2 mutations in males. For patients harboring concurrent SF mutations, we categorized them as co-dominant if the difference in respective CCFs was < 10% of the greater CCF. Specific SF variants were categorized into common hotspots (n=13), except for ZRSR2 variants, which do not occur in defined hotspots. Fisher's exact testing was used to assess the enrichment of SF hotspot variants in patients with concurrent SF mutations, as well as combinations of SF variants in patients with co-dominant SF mutations. Results: We identified 2,331 patients with at least 1 pathogenic SF mutation, of which 6.7% (n=156) had > 1 SF mutation (multi-SF MT). The total cohort comprised cases of AML (n=549, 24%), MDS (n=977, 42%), MDS/MPN (n=233, 10%), MPN (n=228,10%), and uncategorized ICD codes (“Other”, n=326), of which the largest category was clonal cytopenia(s). Notably, the prevalence of multi-SF cases was higher than previously described and our cohort included 5 patients with 3 SF mutations and 1 with 4 SF mutations. Of the multi-SF MT patients, we identified 42 (27%) with co-dominant CCFs among their respective SF mutations. Thirty-three multi-SF MT patients had multiple NGS sampling timepoints (median 2, range 2-4) spanning a median of 0.97 years (range 0.06-5.69 years); we found that 17 of these 33 demonstrated correlative SF CCFs over multiple timepoints, suggesting their SF mutations occurred in the same clone (“co-clonal”). Of these 17 patients, 82% had co-dominant CCFs at the single-timepoint level, whereas none of the 16 non-co-clonal demonstrated co-dominance. Thus, we used SF co-dominance as a surrogate for cases likely to be co-clonal. ZRSR2 (p=8.8x10-29), U2AF1Q157 (p=9.4x10-9), SF3B1K666 (p=1.4x10-4) and infrequent SF3B1 (“SF3B1-Other”, p=.0029) mutations were overrepresented in the multi-SF MT cohort compared to patients with a single SF mutation. Within the multi-SF MT cohort, 3 SF mutation combinations showed overrepresentation in codominant cases – SRSF2P95R/H/L+ ZRSR2 (p=0.014), SRSF2P95R/H/L+ SF3B1K666 (p=0.044), and U2AF1S34 + U2AF1Q157 (p=0.024), although not significant after multiple hypothesis correction. Dual ZRSR2 mutations were the most common combination seen in the multi-SF MT cohort but were underrepresented in codominant cases (p=0.025). Although there was no disease subtype uniquely associated with total or co-dominant multi-SF MT cases, occurrence of multiple SF mutations was more common in males (8% vs. 3% in females), an effect driven by ZRSR2 mutations. Hemoglobin and WBC were lower in multi-SF MT cases, with the lower hemoglobin strongly driven by the codominant multi-SF MT cases (median 8.3 g/dL vs 9.4 g/dL, p=0.00046). Conclusions: These findings suggest that SF mutation co-occurrence in myeloid neoplasms is more prevalent than previously estimated (~7%). Furthermore, we identified 6 individuals harboring > 2 SF mutations, which has not been previously described. We validate specific SF variant pairs that appear to exist in the same clone and are working to confirm the clonal architecture and characterize global splicing aberration in selected cases with single-cell studies to fully uncover the mechanism by which negative epistasis is overcome.

Abstract Background: JAK2-unmutated erythrocytosis constitutes a spectrum of hereditary and acquired entities. We assessed the diagnostic yield and utility of next generation sequencing panel for hereditary erythrocytosis (NGS-HEP) in adults with JAK2-unmutated and otherwise unexplained erythrocytosis (OUE). Additionally, we describe the clinical phenotype, genetic findings, treatment approaches, and thrombotic outcomes in this cohort. Methods: Adults (≥18 years) withOUE, defined as hemoglobin (Hgb) >16.5 g/dL or hematocrit (Hct) >49% in males and Hgb >16 g/dL or Hct > 48% in females, negative for JAK2 exon 12-15mutations, absence of acquired cause, and high-oxygen affinity variants, underwent NGS-HEP; the gene panel included ACO1, ANKRD26, BHLHE41, BPGM, CYB5A, CYB5R3, EGLN1, EGLN2, EGLN3, EPAS1, EPO, EPOR, GFI1B, HIF1A, HIF1AN, HIF3A, JAK2, KDM6A, PFKM, PIEZO1, PKLR, SH2B3, SOCS3 and VHL, including deletion/duplication analysis.Variant curation was performed using ACMG-AMP guidelines and classified as pathogenic or variants of uncertain significance (VUS). Family and thrombosis history, and symptoms were recorded. Results: A total of 44 adult patients with JAK2-unmutated OUE (median age 48 years, range 21–79; 34 males [77%]) underwent NGS-HEP at the Mayo Clinic between March 2023 and June 2025. Variants were identified in 27 patients (61%), with a single variant detected in 19 (70%) and two distinct variants in 8 (30%). 26 variants (96%) were classified as heterozygous VUS and involved PIEZO1 (n=9, 20%; median variant allele frequency [VAF] 47.1%), HIF1A (n=8, 18%;46.2%), ANKRD26 (n=4, 9%;43.7%), SH2B3 (n=3, 7%; 49.9%), HIF3A (n=3, 7%;51.4%), EGLN2 (n=2, 5%;47.9%), BPGM (n=1, 2%; 50.4%), VHL (n=1, 2%; 41.8%), EPO (n=1, 2%; 45.4%) and BHLHE41 (n=1, 2%; 54.2%), while a pathogenic mutation in PKLR was detected in one patient. Presenting median Hgb/Hct values for males and females, respectively were 18.3 g/dL/53.5% and 16.9 g/dl/51.2% in variant- positive cases, and 17.8 g/dl/53.1% and 16.1 g/dl/49.3% in variant-negative cases (p>0.05). Median serum erythropoietin (sEpo) levels were 8.35 mIU/mL (range: <1-52.9) vs 8.8 mIU/mL (range: 1.6-54.7), respectively (p=0.67). Notably, sEpo was < 1 mIU/mL in a patient with ANKRD26/HIF3A VUS. Family history of erythrocytosis was documented in 5 patients (11%). Hyperviscosity-related symptoms were reported in 24 patients (55%), with no significant difference in prevalence between variant-positive and variant-negative cases (59% vs 47% p=0.42). Also, there was no significant association between the presence of symptoms and Hgb or Hct levels (p>0.05). Eight thrombotic events occurred in 7 patients (16%; median age 44 years; 71% males; 4 arterial and 4 venous; 3 provoked deep venous thromboses). Five (71%) of the 7 patients harbored VUS in PIEZO1, PIEZO1/HIF1A, ANKRD26, SH2B3, or BHLHE41. Notably, two younger male patients experienced arterial events in the absence of identifiable risk factors: a 32-year-old with PIEZO1/HIF1A VUS (VAF 46.2%/47.6%; sEpo 10.3 mIU/mL) suffered a myocardial infarction, and a 35-year-old with SH2B3 VUS (VAF 50.2%; sEpo 3.7 mIU/mL) had recurrent cerebrovascular accident despite phlebotomy, aspirin and apixaban. Overall, there was no significant association between thrombosis and the presence of a variant (19% in variant-positive vs 12% in variant-negative, p=0.54). Active therapies included phlebotomy in 22 patients (50%), antiplatelet agents in 23 (52%), systemic anticoagulation in 5 (11%), and hydroxyurea in one patient (2%). Among those who received phlebotomy, 10 patients (45%) reported improvement in symptoms, however, symptom relief did not correlate with Hgb or Hct levels (p>0.05). A separate analysis of 9 patients with heterozygous PIEZO1 VUS, none of whom had undergone splenectomy, showed a numerically higher incidence of symptoms (67% vs 51%) and thrombosis (22% vs 14%) compared to those without PEIZO1 variants, although the differences were not statistically significant (p>0.05). Conclusion: NGS-HEP yielded a molecular diagnosis in 61% of adults with JAK2-unmutated and OUE with heterozygous PIEZO1 variants being the most frequent. No consistent association was observed between variant status and presence of hyperviscosity-related symptoms or thrombotic events. These findings underscore the importance of NGS-HEP testing in the diagnostic workup of JAK2-unmutated and OUE and continued investigation into novel candidate genes.

Lepore hemoglobins (Hbs) such as Hb Lepore-Boston-Washington [δ(1-87) β(116-146)], Hb Lepore-Baltimore [δ(1-50) β(86-146)], Hb Lepore-Hollandia [δ(1-22) β(50-146)] are δβ gene fusion/deletion variants (ε- Gγ- Aγ-[δβ] v )(Figure 1). They are controlled by the weak δ-globin gene promoter and synthesized at approximately 10-15% of hemoglobin fractions. This results in a similar phenotype as β +-thalassemia (microcytosis and hypochromia ± mild anemia). Anti-Lepore hemoglobins such as Hb P-Nilotic (β(1-22) δ(50-146) are βδ gene fusion/duplication variants (ε- Gγ- Aγ-δ-[βδ] v-β) associated with normal CBC values. Herein we describe a novel βδβ double fusion variant (ε- Gγ- Aγ-δ-[βδβ] v). Due to high homology between the δ and β genes, this double fusion event encodes an identical protein to Hb Lepore-Hollandia, albeit linked to the β-promoter and therefore expressed at ~40% of hemoglobin fractions, which would be expected to enhance any intrinsic properties of the variant. This novel hemoglobin variant is named Hb Lepore Rochester-MN. Two cases have been identified (Table 1). Case 1 was a 10-month-old Caucasian/Native American infant referred for an abnormal newborn screen and a reported family history of Hb G-Coushatta (β22, GAA>GCA, Glu>Ala; HGVS: c.68A>C, p.E23A). Case 2 was a 3-month-old infant referred for an abnormal newborn screen. CBCs were normal for age including MCV. Cation-exchange high performance liquid chromatography (HPLC) showed a shouldered peak in the Hb A 2 window typical for Hb Lepore (elution time: 3.41-3.46 mins), although the variant percentage was higher than expected, mimicking Hb G-Coushatta (expected elution time: 3.33 mins). Capillary electrophoresis identified a peak in the D zone (Z(D) zone), which is expected for Hb Lepore or Hb G-Coushatta. Intact mass spectrometry was consistent with Hb Lepore-Hollandia at 15836 amu, differing from Hb G-Coushatta (Glu>Ala, 15809 amu). Multiplex ligand probe amplification (MLPA) was normal because the highly variable fusion regions are unsuitable for probe placement. Sanger sequencing of the β-globin gene identified multiple single base substitutions which were challenging to interpret but matched the sequence expected for a complex double crossover event (NM_000518.4:c.28_68delins41 (p.Ser10_Glu23delins14TAVNALWGKVNVDA). The first crossover is located between c.-29 and c.9, the second crossover is located between c.69 and c.92+17. Long-read sequencing was performed (Sequel, Pacific Biosciences) which detected the multiple missense mutations in cis conformation, as was corroborated by the mass spectrometry data. In summary, Hb Lepore Rochester-MN represents a novel double fusion [βδβ] variant class that will pose a diagnostic challenge for laboratories. Although it is likely a result of sequential crossover events (deletion and duplication) between Lepore and anti-Lepore variants, MLPA analysis is normal due to technical constraints and high homology. DNA sequencing (both Sanger and long read methods) yielded complex results that require sophisticated analysis and correlation with protein data including intact mass spectrometry. HPLC and CE data including variant percentage are very similar to Hb G-Coushatta. In addition, although resulting in an identical protein, the variant should not be mistaken for Hb Lepore-Hollandia, which is produced at lower levels and causes a thalassemic phenotype. Hb Lepore Rochester-MN is controlled by the β-promoter and therefore is associated with approximately 40% variant and apparently normal CBC values in the two infants.

AbstractBlastic plasmacytoid dendritic cell neoplasm (BPDCN) is recurrently mutated in epigenetic pathway genes. We studied the myeloid‐related genetic mutations in a cohort of five patients with BPDCN and one paired relapse case at our institution and identified a high frequency of biallelic TET2 and canonical ASXL1 (c.1934dupG) mutations. The number of cases is small, but the variant allele fraction (VAF) sums of the TET2 mutations, as well as the persistence of TET2 mutations in a case of relapsed BPDCN, suggest an ancestral/founder nature of TET2 clones in the cases. Further literature review shows a high frequency of biallelic TET2 mutations in reported cases of BPDCN.