The clinical phenotype of germline RUNX1 mutations in relation to the accompanying somatic variants and RUNX1 isoform expression

Germline RUNX1 mutations lead to familial platelet disorder with associated myeloid malignancy (FPDMM), characterized by thrombocytopenia, abnormal bleeding, and an elevated risk of developing myelodysplastic neoplasia (MDS) and acute myeloid leukemia (AML) at young age. However, it is not known why or how germline carriers of RUNX1 mutations have a particular propensity to develop myeloid hematologic malignancies, but the acquisition and composition of somatic mutations are believed to initiate and determine disease progression. We present a novel family pedigree that shares a common germline RUNX1R204* variant and exhibits a spectrum of somatic mutations and related myeloid malignancies (MM). RUNX1 mutations are associated with inferior clinical outcome; however, the proband of this family developed MDS with ring sideroblasts (MDS‐RS), classified as a low‐risk MDS subgroup. His relatively indolent clinical course is likely due to a specific somatic mutation in the SF3B1 gene. While the three main RUNX1 isoforms have been ascribed various roles in normal hematopoiesis, they are now being increasingly recognized as involved in myeloid disease. We investigated the RUNX1 transcript isoform patterns in the proband and his sister, who carries the same germline RUNX1R204* variant, and has FPDMM but no MM. We demonstrate a RUNX1a increase in MDS‐RS, as previously reported in MM. Interestingly, we identify a striking unbalance of RUNX1b and ‐c in FPDMM. In conclusion, this report reinforces the relevance of somatic variants on the clinical phenotypic heterogeneity in families with germline RUNX1 deficiency and investigates a potential new role for RUNX1 isoform disequilibrium as a mechanism for development of MM.

3][4][5] Heterozygous germline mutations in the RUNX1 gene cause familial platelet disorder with associated myeloid malignancy (FPDMM), a rare autosomal dominant disease that encompasses thrombocytopenia, functional platelet defects and predisposition to hematologic malignancies, mainly MDS and AML, with a median penetrance of 44%. 3 Germline variants in the RUNX1 gene are not sufficient to initiate disease, but require the acquisition of specific secondary somatic genomic aberrations. 6Nevertheless, the effects of acquired variants on clinical outcome as well as the genetic and molecular basis of leukemic transformation due to germline RUNX1 deficiency are largely unknown.][9][10] Overexpression of RUNX1a has been identified in myeloid malignancy (MM) progression and, recently, in the pathogenesis of trisomy 21-associated myeloid leukemia. 11,12wever, RUNX1 isoform expression patterns in FPDMM or a role in RUNX1 deficiency remain to be investigated.
We herein present a novel family pedigree that shares a germline RUNX1 R204* variant and displays a spectrum of somatic mutations with related MM.Interestingly, while RUNX1 mutations are associated with inferior clinical outcome, the proband of this family developed MDS with ring sideroblasts (MDS-RS), classified as a low-risk MDS subgroup, and exhibited a rather indolent clinical course.We investigate the effect of this RUNX1 mutation on isoform expression and identify distinct transcript levels of RUNX1 isoforms, potentially in a disease stage-specific manner.

| Next-generation sequencing
Next-generation targeted sequencing was performed using the Tru-Sight Myeloid Sequencing Panel (Illumina, San Diego, CA), targeting 54 genes associated with MM, analyzing bone marrow and peripheral blood mononuclear cells (MNCs) from III-1 before allogenic stem cell transplantation; peripheral blood MNCs from II-1; and buccal swabs from III-1 and III-2.The Genomic Medicine Sweden Custom Panel (Twist Bioscience), analyzing 187 variants previously reported to be associated with MM, was performed on bone marrow MNCs from III-1 after transplantation and on peripheral blood MNCs from III-2.
Clinical samples were obtained with written informed consent in accordance with the Declaration of Helsinki, and the study was approved by the Ethics Research Committee at Karolinska Institutet (2017/1090-31/4 and 2011/1257-31/1).

| Transcript isoform expression assay
Bone marrow MNCs from III-1 at ages 53 and 57 and from III-2 at age 58 were analyzed as well as bone marrow MNCs from two healthy individuals, a 48-year-old male and a 60-year-old female, as controls.
Total mRNA was extracted using RNeasy PLUS Mini Kit (Qiagen, Hilden, Germany) and cDNA synthesis was carried out using Maxima First Strand cDNA Synthesis Kit (Thermo Fisher Scientific, Waltham, MA) according to the manufacturer's instructions.Gene expression of RUNX1 exons 6-7a, 1-2, and 3a/b-4 was analyzed to calculate RUNX1a, RUNX1c, and RUNX1a/b/c, respectively.The following primers were used: GGATT-3 0 .Real-time PCR was performed using a PowerUp™ SYBR™ Green Master Mix (Applied Biosystems, Waltham, MA) on a CFX384 TouchTM Real-Time PCR Detection System.All samples were run in triplicate.The relative expression of RUNX1 exons as well as the ratios of transcript isoforms RUNX1a, -b and, -c were calculated as previously described. 10Briefly, expression of each exon boundary was calculated using the delta Ct-method with ACTB as a housekeeping gene.
The expression of each exon boundary was normalized by dividing its relative expression by the relative expression of total RUNX1a/b/c.Since RUNX1b has no distinctive exon boundary, its relative expression was calculated by subtracting the relative expression of RUNX1a and -c from total RUNX1a/b/c.Each RUNX1 transcript isoform ratio was obtained by dividing each isoform expression with the total RUNX1 expression.

| Clinical features
The proband (III-1; Figure 1A) was a 53-year-old male who had suffered from mild thrombocytopenia since youth.After experiencing increased tiredness and reduced physical condition, he was diagnosed with MDS-RS.Evaluation of his bone marrow identified 61% RS, phenotypic erythroid precursors with granules of excess iron.No RS were observed in the re-evaluation of his bone marrow samples from the age of 44.The patient did not respond to erythropoietin treatment and was transfusion dependent for 3 years until he successfully underwent allogenic stem cell transplantation in 2020.Genetic investigation of variants related to MM at the time of diagnosis identified a pathogenic RUNX1 variant (NM_001754.5:c.610C>T, p.Arg204*) with a variant allele frequency (VAF) of 50%, later confirmed to be germline by buccal swab analysis (Figure 1, Table 1).Moreover, he carried pathogenic somatic variants in the SF3B1 (NM_012433.3:c.1866G>T, p.Glu622Asp) and CUX1 (NM_181552.3:c.2062+1G>T p.?; Figure 1B, Table 1) genes, while the karyotype was normal.RUNX1b transcript isoform ratio was calculated using the delta Ct-method with ACTB as reference gene, as previously described. 10ontrols 1 and 2 represent two healthy bone marrow donors, male and female, respectively.

| DISCUSSION
In addition to predisposing germline RUNX1 variants, acquired somatic mutations and their cooperation are presumed to affect penetrance and drive disease onset and progression. 14In the family reported here, the same RUNX1 mutation was accompanied by distinct somatic mutational profiles, resulting in clinical heterogeneity of hematological diseases.The father of the proband developed AML and the aunt high-risk MDS with somatic mutations related to poor prognosis, including STAG2 and ASXL1. 5,6On the other hand, the sister, III-2, has not developed any MM and remains stable under yearly surveillance.Notably, her EZH2 M134K clone, harboring a mutation associated with poor prognosis in myeloid neoplasms, has increased in peripheral blood over the years and could suggest clonal hematopoiesis. 15Prediction and preventative measures are crucial to reduce cancer risk.However, with the current knowledge in the field, it is still not feasible to foresee who among the carriers of germline RUNX1 deficiency will develop MDS/AML and when.
Hence, identification and monitoring of germline mutation carriers is desirable. 3,4NX1 mutations, whether somatic or germline, are considered high-risk and are associated with adverse clinical outcomes.Nonetheless, the proband, III-1, developed MDS-RS, classified as a low-risk subtype of MDS by the World Health Organization. 16Moreover, according to the recent Molecular International Prognostic Scoring System for MDS, co-mutation between RUNX1 and SF3B1 are defined as SF3B1 β with adverse outcome. 17While the genotype of the proband indicates a SF3B1 β profile, his clinical phenotype is in accordance with the more favorable SF3B1 α .Thus, while the RUNX1 variant likely affected platelet function, we hypothesize that the inherited predisposition to leukemia in his case has been "sheltered" by the SF3B1 E622D mutation, which is associated with indolent MDS. 18Whether the adverse risk implicated in germline RUNX1 deficiency is alleviated by the acquisition of a less aggressive mutation, or whether pathogenesis is dictated by the order of mutations remains to be addressed.However, if the genotype to phenotype discrepancy observed here is a general feature, further discrimination of the SF3B1 β classification may be relevant.
A mechanism for RUNX1 isoforms in the pathogenesis of MM is yet to be uncovered and isoform expression may depend on cell type, disease stage and specific mutational profile.Here, we investigated the expression pattern of the three main RUNX1 isoforms as a consequence of RUNX1 R204* mutation, which causes a truncation of the transactivating domain, hampering the functionality of RUNX1b and -c, but not RUNX1a.III-1 showed an increase in RUNX1a expression, which may be related to his MDS since overexpression of RUNX1a has been observed in myeloid neoplasia. 11RUNX1a has been considered a natural dominant negative isoform by competing with the other isoforms, affecting hematopoietic stem and progenitor cell proliferation and differentiation. 9However, in the bone marrow MNCs analyzed here, the RUNX1a isoform constituted a minimal part of total RUNX1 expression, which was mainly accounted for by RUNX1b and -c expression.Interestingly, his sister, an asymptomatic carrier, showed a substantial decrease in RUNX1b expression and elevated RUNX1c levels, compared to her brother and healthy control.RUNX1b and -c are full-length isoforms with similar structure but distinct expression: RUNX1b is highly expressed in megakaryocyte progenitors and decreases over differentiation in myeloid cells, whereas RUNX1c is expressed in committed blood cells. 7,8The RUNX1 isoform disequilibrium observed in our study is in line with previous reports and further argues for its potential role in the development of MM in the context of FPDMM, for instance, by expediting the differentiation of myeloid cells, or a prestage to a MM. 10 Recently, a RUNX1a: RUNX1c disequilibrium was shown to promote trisomy 21-associated leukemia, and restoration of the isoform balance had strong antileukemic effects, reinforcing a role for RUNX1 isoforms as potential targets in blood cancer. 12 summary, we provide support to the notion that somatic genetic aberrations impact the clinical phenotypic heterogeneity of individuals with germline RUNX1 mutations and highlight a potential role for the different RUNX1 isoforms in the pathogenesis and evolution of the disease.Follow-up of the RUNX1 isoform equilibrium of carriers of germline RUNX1 mutations may uncover underlying development of the disease and serve as a potential marker of malignant transformation.Further investigation in larger series of patients is warranted to reach solid conclusions.

F I G U R E 1
Mutational status and RUNX1 isoform analysis of family pedigree.(A) Family pedigree of proband (III-1, indicated by arrow) showing segregation of MM and myeloid-related mutated genes.Squares represent males and circles females.White symbols represent unaffected individuals; gray symbols individuals with thrombocytopenia and no MM; black/white symbols individuals with MDS; black symbols individuals with AML.Age at diagnosis is provided in parenthesis for affected individuals.Age of death is shown preceded by d.WT (wild type) represents individuals without mutations in RUNX1.(B) Fish plot of RUNX1, SF3B1, and CUX1 variant allele frequency in III-1 bone marrow and peripheral blood (upper).Fish plot of RUNX1 and EZH2 variant allele frequency in III-2 peripheral blood (lower).(C) RUNX1 isoforms detailed by exons (upper) and RUNX1c showing protein domains and amino acid numbers (lower).Dashed line marks the location of the specific RUNX1 mutation.Colored bars represent the exons targeted by respective primer listed in (D).(D) RUNX1 exon boundary targets and the main transcript isoform detected by real-time PCR.(E) Isoform frequency and total RUNX1 expression, by using exon boundary 3a/b-4 common for all three main RUNX1 isoforms, with ACTB as reference gene.Age is provided in parenthesis.Mean of three technical replicates and standard deviation is shown.(F) RUNX1 transcript isoform expression divided by total expression of RUNX1, calculated as exon boundary 3a/b-4, with standard deviation.