Abstract

Whole exome/genome sequencing has been fundamental in the identification of somatic mutations in the spliceosome machinery in myelodysplastic syndromes (MDSs) and other hematologic disorders. SF3B1, splicing factor 3b subunit 1 is mutated in 60%-80% of refractory anemia with ring sideroblasts (RARS) and RARS associated with thrombocytosis (RARS-T), 2 distinct subtypes of MDS and MDS/myeloproliferative neoplasms (MDSs/MPNs). An idiosyncratic feature of RARS/RARS-T is the presence of abnormal sideroblasts characterized by iron overload in the mitochondria, called RS. Based on the high frequency of mutations of SF3B1 in RARS/RARS-T, we investigated the consequences of SF3B1 alterations. Ultrastructurally, SF3B1 mutants showed altered iron distribution characterized by coarse iron deposits compared with wild-type RARS patients by transmission electron microscopy. SF3B1 knockdown experiments in K562 cells resulted in down-regulation of U2-type intron-splicing by RT-PCR. RNA-sequencing analysis of SF3B1 mutants showed differentially used genes relevant in MDS pathogenesis, such as ASXL1, CBL, EZH, and RUNX families. A SF3B pharmacologic inhibitor, meayamycin, induced the formation of RS in healthy BM cells. Further, BM aspirates of Sf3b1 heterozygous knockout mice showed RS by Prussian blue. In conclusion, we report the first experimental evidence of the association between SF3B1 and RS phenotype. Our data suggest that SF3B1 haploinsufficiency leads to RS formation.

Introduction

Whole exome sequencing technology has been instrumental in the identification of somatic mutations involving genes important in RNA splicing.1–4  The first and most frequently identified mutation in hematologic malignancies involves the SF3B1 gene and was found in 60%-80% of refractory anemia with ring sideroblasts (RARS) and RARS associated with thrombocytosis (RARS-T) patients. Both RARS and RARS-T are subtypes of myelodysplastic syndromes (MDSs) and MDS/myeloproliferative neoplasm (MDS/MPN) overlap diseases. SF3B1 is a crucial component of the spliceosomal U2snRNP complex, which participates in normal RNA splicing.5  Intron splicing that culminates in the production of the mature mRNA product is catalyzed by spliceosomes. The major spliceosomes consist of U1, U2, U4/U6, and U5snRNPs and carry out splicing of major class or U2-type introns. Minor spliceosomes, which consist of U11, U12snRNPs, U4atac/U6atac and U5 snRNPs, carry out the splicing of minor class introns or U12-type introns. RNA splicing is crucial in maintaining normal genetic diversity.6  Because the fidelity of the final protein product depends highly on an intact and accurate RNA splicing machinery, somatic genetic errors in key components of the splicing pathway can potentially lead to the formation of dysfunctional proteins that can ultimately predispose to diseases. Indeed, emerging discoveries of genetic polymorphisms or mutations altering regulatory sequences or producing splice variants have been associated with certain types of hereditary diseases and neoplastic disorders.7 

The presence of 15% or more ring sideroblasts (RSs) of nucleated erythroid precursors in the BM is a cardinal feature of RARS and RARS-T, even though RSs are also seen in 25% of patients with other subtypes of MDS, but rarely in other hematologic disorders.8–10  RS occurs because of abnormal localization of crystalline iron deposits in the mitochondria of erythoid precursors. Even though there is evidence of dysfunction in mitochondrial metabolism, the pathogenesis of RS remains unclear.11,12  The discovery of SF3B1 mutations in RARS and RARS-T has opened a new area of investigation in these diseases, and this article focuses on experimental evidence linking dysfunction in SF3B1 and formation of RS in RARS/RARS-T.

Methods

Patient samples

BM and peripheral blood cells were obtained with written informed consent in accordance with the Declaration of Helsinki and approved by the Institutional Review Board of the Cleveland Clinic. Diagnosis was assigned according to 2008 World Health Organization classification criteria. We studied a total of 456 patients. Overall, the MDS group includes: RARS, n = 19; refractory anemia with excess blasts 1/2, n = 34; refractory cytopenia with unilineage dysplasia/refractory cytopenia with multilineage dysplasia, n = 23; and MDS-U/5q, n = 17. The MDS/MPN group includes: RARS-T, n = 26; chronic myelomonocytic leukemia 1/2, n = 59; and MDS/MPN-U, n = 20. The acute myeloid leukemia (AML) group includes: secondary AML, n = 51; and primary AML, n = 43. Others includes: MPN, n = 69; mast cell disease, n = 31; and paroxysmal nocturnal hemoglobinuria/aplastic anemia/T-cell large granular lymphocytic leukemia, n = 64. The median age at sample collection was 52 years (range, 13-77 years).

Mutational detection

Genomic sequencing was performed on coding regions for SF3B1 (exons 13-16) SF3B14 (all exons), SF3B4 (all exons), and DYRK1A (all exons). Primer sequences and conditions used can be supplied on request. Bidirectional sequencing was performed by standard techniques using an ABI 3730xl DNA analyzer (Applied Biosystems). Somatic nature of novel mutations was confirmed using germline DNA from CD3+ cells. All mutations were scored as pathogenic on the basis of the observation that they were not detected in normal samples and were not found in published SNP databases (dbSNP, http://www.ncbi.nlm.nih.gov/projects/SNP) and/or they were not reported as SNPs in previous publications.

Transmission electron microscopy

One million BM mononuclear cells were first subjected to fixation in 3.75% glutaraldehyde, 0.1M sodium cacodylate, 6% sucrose (pH 7.2-7.4) for a period of 4 hours to overnight. Specimens were post-fixed in 1% osmium tetroxide and 0.1M cacodylate (pH 7.2-7.4) for 1 hour. After each fixation step, specimens were washed twice in cacodylate buffered sucrose, 10 minutes each. After the final wash, specimens were dehydrated in ascending graded alcohols, followed by immersion in propylene oxide, then passed through ascending graded mixtures of propylene oxide and EMBed 812 epoxy resin, and finally embedded in pure epoxy resin. Specimen blocks were polymerized overnight at 60°C. Specimens were initially evaluated by light microscopy of 1-μm plastic sections stained in a combination of toluidine blue and basic fuchsin. Thin sections from selected tissue blocks were cut at 60-80 nm and supported on 200 mesh copper grids. Sections were stained in uranyl acetate and lead citrate and examined using a Philips CM-12 electron microscope. Electron micrographs were taken using a Gatan Orius CCD digital camera.

Lentiviral infection and quantitative RT-PCR

Virus containing supernatant of 293T cells was used to infect 5 × 105 K562 cells. Five different shRNAs were constructed by Sigma-Aldrich. To assess the effect of shRNAs, cells were harvested 2 days after puromycin selection and SF3B1 mRNA level was measured by RT-PCR. Total RNA was isolated using the High Pure RNA Isolation kit (Roche Diagnostics). For quantitative RT-PCR, 0.5 μL of the complementary DNA was added to SYBR@advantage qPCR mix (Clontech Laboratories) and amplified in a Light Cycler 480 instrument (Roche Diagnostics). The U2- and U12-dependent introns were selected from the online database (http://genome.crg.es/datasets). The RT-PCR primers spanning the exon-intron junctions of several genes were designed using PrimerQuest (www.idtdna.com). And their specificity was assessed in silico (http://genome.ucsc.edu) and by standard PCR. A total of 40-50 PCR cycles were performed in a 2-step cycling procedure with an initial denaturation at 94°C for 3 minutes, subsequently at 94°C for 15 seconds, and 60°C for 30 seconds. Data were analyzed based on relative δ Ct values using GAPDH as control gene. Results are shown as mean ± SD of duplicate experiments.

Human colony formation assays

A total of 100 000 BM cells derived from 4 healthy persons were plated in duplicate in 1 mL methylcellulose with cytokines (StemCell Technologies) in 35-mm culture plates for 2 weeks at 37°C with 5% CO2. Meayamycin was added at the beginning of the culture at a range of concentrations (1, 2, 5, 10, and 50nM). Colony-forming cells were harvested, washed 5 times in PBS, and spotted on cytospin slides before Prussian blue staining.

Preparation of meayamycin

Meayamycin was synthesized according to the literature13  and purified on a C18 reverse-phase preparative HPLC. The fractions containing meayamycin were concentrated under vacuum to afford 16.3 mg of solid meayamycin. Using this solid, a 10mM stock solution of meayamycin in DMSO was prepared. A 10μM solution of meayamycin in DMSO was prepared through serial dilutions and stored at a −20°C.

Mice

BM aspirates from Sf3b1 heterozygous14  and wild-type (WT) C57BL/6 mice were kindly provided by Dr Koseki (RIKEN) and were subjected to Prussian blue staining using standard pathology procedures for the detection of RS.

RNA-Seq analysis

Mapping.

RNA sequencing (RNA-Seq) was performed by Otogenetics Corporation (Tucker); 100-bp paired-end RNA-Seq reads were mapped to the hg19 RefSeq human transcriptome and spliceosome by DNAnexus (http://dnanexus.com) using a Bayesian method,15  in which a read was mapped when its posterior probability of mapping exceeded 0.9. These filtered posterior probabilities were summed to generate fractional read counts per gene and per exon, with probabilities from splice-junction spanning reads counted for each relevant exon. We used rounded gene and exon read counts as inputs for our differential expression analyses.

Differential gene expression analysis.

We used the R package DESeq16  version 1.6.1 (http://www.bioconductor.org/packages/release/bioc/html/DESeq.html) and R version 2.14.1, to perform differential gene expression analysis of mutant versus normal samples. DESeq uses a negative binomial (NB) distribution to model the gene read counts and shrinkage estimators to estimate the per-gene NB dispersion (square of the coefficient of biologic variation) parameters. Specifically, we estimated the per-gene dispersion parameters using a shrinkage approach with the default maximum sharing mode and default parametric mean-dispersion relationship, where raw dispersion values were calculated using the samples in the mutant group. Before testing, we dropped all genes with read counts below the 40th percentile to improve testing power while maintaining type I error rates. We used the nbinomTest function, which does conditional testing assuming gene counts follow an NB distribution with fitted dispersions, to estimate P values and adjusted P values obtained using the Benjamini-Hochberg method17  for each filtered gene under the null hypothesis of common expression intensity across groups. Genes with adjusted P < .05 were declared significant. Moderated logarithm base 2-fold changes (mutant/normal) were estimated using a variance stabilizing transformation as implemented in the getVarianceStabilizedData function of DESeq.

Differential exon usage analysis.

We used the R package DEXSeq Version 1.1.4 (http://www.bioconductor.org/packages/release/bioc/html/DEXSeq.html), to perform differential exon usage analysis of mutant versus normal samples. DEXSeq uses an NB distribution to model the exon read counts and shrinkage estimators to estimate the per-exon NB dispersion parameters. Specifically, we estimated the per-exon dispersion parameters using a shrinkage approach with the maximum sharing mode and parametric mean-dispersion relationship, where raw dispersion values were calculated using the samples in the mutant group. We defined a testable exon as one that had a total sum of at least 10 mapped reads across samples and was in a gene with no more than 70 exons. Before exon usage testing, we dropped any exons that were not testable or were in genes with less than 2 testable exons to improve testing power while maintaining type I error rates. We used the testGeneForDEU function, which compares deviances from generalized linear model fits (assuming NB likelihood) to a χ2 reference distribution, to estimate P values and adjusted P values obtained using the Benjamini-Hochberg method for each exon under the null hypothesis of common usage across groups. Exons with adjusted P < .05 were declared significant. Logarithm base 2-fold changes (mutant/normal) for each exon were estimated using the function estimatelog2FoldChanges.

Gene set and exon set ORA.

We used the R package goseq18  Version 1.6.0 (http://www.bioconductor.org/packages/release/bioc/html/goseq.html) to perform over- and under-representation analysis (ORA) of gene sets taking length bias into account. We conducted separate ORAs for the gene-level and exon-level differential expression (DE) findings. ORA analysis requires a definition of significance for each gene member of the gene set. We defined this similarly for ORA of the gene-level and exon-level DE findings: (1) a gene with an adjusted P value of < .5 from the gene-level DE analysis and (2) a gene with at least one exon with an adjusted P value from the exon-level DE analysis < .5, respectively. Goseq requires a probability weighting function, which is used to adjust the gene set results to reflect the lengths of genes in the gene sets, reducing the common bias in gene set analyses because of longer genes having higher read counts and subsequently a higher chance of differential detection. For ORA of the gene-level DE findings, we defined length as the sum of the lengths of the observed exons within each gene, where the exon lengths were obtained from DNAnexus. For ORA of the exon-level DE findings, we defined length as the median exon length of detected exons within the specified gene, where exon lengths were obtained from DNAnexus. We used the MSigDB database19  Version 3.0 (http://www.broadinstitute.org/gsea/msigdb/index.jsp), gene set collections c2 through c5 in our ORA. For each MSigDB collection of gene sets, we ran the goseq function with the Wallenius approximation to estimate P values for over-representation and under-representation of significant genes (where significant is defined earlier in this paragraph) for each gene set within the collection. Adjusted P values were calculated on the gene-set P values per collection using the Benjamini-Hochberg method to control for the number of gene sets tested within a collection. These adjustments were made separately for the P values from the over-represented tests and for the P values from the under-represented tests. Any gene sets with adjusted P values < .05 from either the over- or under-represented tests were declared significant.

Statistical analyses

Overall survival was measured from the day of initial sampling to death from any cause (patients lost to follow-up were censored) or last follow-up and was summarized using the Kaplan-Meier method. Results were analyzed for data collected as of December 2011. All P values were 2-sided, and P values ≤ .05 indicated statistical significance. Statistical analyses were performed using JMP8 (SAS Inc). RNA-Seq data statistical analysis is discussed extensively in “RNA-Seq analysis.”

Results

SF3B1 mutations are frequently found in patients with MDS and RS

We first examined SF3B1 mutational status in our cohort of MDS and other hematologic malignancies (n = 456) using Sanger sequencing. Consistent with earlier studies,1–3  we found 50 cases with somatic mutations in SF3B1; 68% of RARS and 81% of patients with RARS-T harbored mutations, whereas SF3B1 mutations were infrequently found in higher-risk MDS and other hematologic conditions (supplemental Figure 1A-D, available on the Blood Web site; see the Supplemental Materials link at the top of the online article). The K700E was the most frequent mutation detected in SF3B1 among patients with RARS and RARS-T (34%). Further, SF3B1 did not predict for progression as underlined by the low prevalence of mutations in patients with secondary AML (5.9%) and de novo AML (4.7%; supplemental Figure 1E). Analysis of overall survival comparing mutant versus WT patients showed longer survival in patients with SF3B1 mutations (supplemental Figure 3). Interestingly, we next found 2 non-MDS cases in which SF3B1 mutations were detected: one with paroxysmal nocturnal hemoglobinuria (K666Q)20  and one with post-polycythemic myelofibrosis (K700E).1  Both displayed RS (17% and rare, respectively). This finding led us to investigate more closely the association of SF3B1 mutations and RS. In conclusion, SF3B1 is mainly associated with RARS and RARS-T MDS and MDS/MPN subtypes. In a total of 45 patients, 11 typical RARS and RARS-T patients with more than 15% RS were WTs for SF3B1. Thus, we sought to investigate whether SF3B1 might accompany distinctive features.

Ultrastructural analysis of BM of SF3B1 mutant and WT RARS patients

Given the high frequency of SF3B1 mutations in patients with RARS,1–3,21  we then investigated the association of spliceosomal protein mutations with altered mitochondrial iron distribution manifested as RS. We analyzed ultrastructural differences of BM erythroid precursors between SF3B1 mutant and WT patients with the intent of evaluating whether SF3B1 mutations might alter iron distribution. Transmission electron microscopy was performed on BM cells derived from an RARS patient carrying an SF3B1 mutation (K700E) and an RARS patient WT for SF3B1. Abundant foci of iron deposits were observed around the nucleus of BM cells in the mutant case compared with scanty deposits in the WT patient. Although Prussian blue staining is traditionally used to identify RS in BM samples, it does not distinguish between SF3B1 mutant and WT cases (Figure 1 top right panel). The SF3B1 mutations are associated with more dense iron particles. It is possible that the topographic configuration of the iron might predict the presence of SF3B1 mutations.

Figure 1

Mutations in SF3B1 are frequent in patients with RARS and RARS-T and lead to an alteration in the BM ultrastructure. Left panel: We performed Sanger sequencing on exons 13, 14, 15, and 16 of SF3B1 in 456 patients, finding that the frequency of SF3B1 mutations is higher in patients with RARS (68%) and RARS-T (81%) compared with the other groups. White bars and black bars represent WT and mutant RARS and RARS-T patients, respectively. Right panel: Transmission electron microscopy of BM cells from a representative WT and mutant RARS patient. Arrows indicate the presence of abundant perinuclear iron deposits in the mutant compared with the WT patient. Specimens' cuts are 2-μm-thick sections.

Figure 1

Mutations in SF3B1 are frequent in patients with RARS and RARS-T and lead to an alteration in the BM ultrastructure. Left panel: We performed Sanger sequencing on exons 13, 14, 15, and 16 of SF3B1 in 456 patients, finding that the frequency of SF3B1 mutations is higher in patients with RARS (68%) and RARS-T (81%) compared with the other groups. White bars and black bars represent WT and mutant RARS and RARS-T patients, respectively. Right panel: Transmission electron microscopy of BM cells from a representative WT and mutant RARS patient. Arrows indicate the presence of abundant perinuclear iron deposits in the mutant compared with the WT patient. Specimens' cuts are 2-μm-thick sections.

In vitro effects of SF3B1 knockdown

We then investigated the role of SF3B1 in pre-mRNA splicing of endogenous human genes. First we knocked down the level of SF3B1 mRNA in cells using RNAi. We applied shRNA lentiviral infection in K562 cells using 5 different shRNAs specific for the human SF3B1 gene. We performed quantitative RT-PCR to measure the level of knockdown. SF3B1 mRNA knockdown of 50% was consistently achieved on one shRNA clone. Using this selected shRNA, we quantified the ratio of spliced and unspliced RNA in 7 and 3 exemplary U2- and U12-introns. All examined U2-type introns were less efficiently spliced, whereas U12-introns were not significantly affected (Figure 2A; Table 1). We then analyzed the phenotype of K562 cells with decreased expression of SF3B1 mRNA. K562 cells were first cocultured with hemin, an inducer of hemoglobinization and erythroid differentiation, and then infected with different shRNAs specific for SF3B1. A 50% reduction of SF3B1 mRNA level was again achieved. Transfectant cells were sent for morphologic analysis and Prussian blue staining. Increased mitotic figures were noted, but RSs were not observed. Transmission electron microscopy of the transfectant cells also showed no iron deposits compared with controls (ie, K562 cells infected with a vector only; data not shown).

Figure 2

In vitro and in vivo alterations of SF3B1 gene lead to an RARS phenotype. (A) K562 cells were transfected with shRNA constructs. After 48 hours of puromycin selection, RT-PCR assay was performed using random oligos for endogenous U2 (1-7) and U12 (1-3) introns (Table 1). The ratio of spliced to unspliced pre-mRNA for U2- and U12-dependent introns is shown. Average reaches 0.42 and 1.89 for U2-introns and U12-introns, respectively. Data are mean ± SD, calculated from 2 independent experiments. K562 transfected with vector only was used as control, and the ratio of spliced to unspliced pre-mRNA was set to 1. (B) Human colony-forming unit cell assay was performed on BM cells derived from 4 healthy persons. Cells were treated with different doses of meayamycin (2, 10, and 50nM). Representative pictures at 2 and 50nM are shown. Colonies were harvested after 2 weeks, spotted on cytospin slides, and subjected to Prussian blue staining. (C) Prussian blue staining of BM aspirates shows numerous RSs in Sf3b1+/− compared with C57BL/6 mice. Closed arrowheads indicate perinuclear RS. BM cytospin slides were kindly provided by Dr H. Koseki from Japan. Slides were analyzed using an Olympus system microscope (model BX41; objective lens, ×100; camera, SPOT Idea Model No 28.2). Images were acquired using a SPOT Imaging software by Diagnostic Instruments Inc (www.Diaginc.com).

Figure 2

In vitro and in vivo alterations of SF3B1 gene lead to an RARS phenotype. (A) K562 cells were transfected with shRNA constructs. After 48 hours of puromycin selection, RT-PCR assay was performed using random oligos for endogenous U2 (1-7) and U12 (1-3) introns (Table 1). The ratio of spliced to unspliced pre-mRNA for U2- and U12-dependent introns is shown. Average reaches 0.42 and 1.89 for U2-introns and U12-introns, respectively. Data are mean ± SD, calculated from 2 independent experiments. K562 transfected with vector only was used as control, and the ratio of spliced to unspliced pre-mRNA was set to 1. (B) Human colony-forming unit cell assay was performed on BM cells derived from 4 healthy persons. Cells were treated with different doses of meayamycin (2, 10, and 50nM). Representative pictures at 2 and 50nM are shown. Colonies were harvested after 2 weeks, spotted on cytospin slides, and subjected to Prussian blue staining. (C) Prussian blue staining of BM aspirates shows numerous RSs in Sf3b1+/− compared with C57BL/6 mice. Closed arrowheads indicate perinuclear RS. BM cytospin slides were kindly provided by Dr H. Koseki from Japan. Slides were analyzed using an Olympus system microscope (model BX41; objective lens, ×100; camera, SPOT Idea Model No 28.2). Images were acquired using a SPOT Imaging software by Diagnostic Instruments Inc (www.Diaginc.com).

Table 1

U2- and U12-dependent genes and introns

No. Gene name Introns 
U2 introns 
    1 CTNNBL1 
    2 CTNNBL1 
    3 EFNA5 
    4 ITPR1 
    5 ITPR1 
    6 UTR 43 
    7 THOC2 36 
U12 introns 
    1 ATXN 10 
    2 CTNNBL1 
    3 THOC2 37 
No. Gene name Introns 
U2 introns 
    1 CTNNBL1 
    2 CTNNBL1 
    3 EFNA5 
    4 ITPR1 
    5 ITPR1 
    6 UTR 43 
    7 THOC2 36 
U12 introns 
    1 ATXN 10 
    2 CTNNBL1 
    3 THOC2 37 

U2- and U12-intron numbers are indicated for each gene. Primers were designed to quantify the levels of spliced and unspliced introns in K562 transfected with shRNA constructs.

CTNNBL1 indicates catenin, β-like 1; EFNA5, ephrin-A5; ITPR1, inositol 1,4,5-trisphosphate receptor 1; UTR, untranslated region; THOC2, THO complex 2; and ATXN, ataxin-1.

In vitro treatment of healthy BM cells with meayamycin

To further explore the relationship between SF3B1 mutations and RS, we used meayamycin, a chemical analog of FR901464.13,22  FR901464 is a potent natural product that inhibits pre-mRNA splicing via SF3b factor binding.23,24  We performed human colony-forming unit cell assay using BM mononuclear cells from 4 healthy subjects without and with different concentrations of meayamycin (2, 10, and 50nM). The colonies were spotted on cytospin slides and stained with Prussian blue. As shown by the representative pictures, meayamycin consistently induced RS in all 4 healthy subjects compared with controls (Figure 2B). The percentages of RS per each meayamycin concentration are shown in supplemental Figure 2.

Characteristics of a heterozygous mouse model for SF3B1

To next examine the role of SF3B1 in RS formation, we analyzed the phenotype of a heterozygous mouse model for Sf3b1 (Sf3b1+/−) insufficiency in vivo.14 Sf3b1+/− mice showed only minimal differences in blood counts, which includes a mild lymphopenia compared with WT C57BL/6 mice (21.9 × 102/μL ± 2.9 vs 42.3 × 102/μL ± 11.6; n = 3). Indeed, hemoglobin levels (8.5 g/dL ± 0.2 vs 7.9 g/dL ± 0.2) and platelets counts (60.2 × 104/μL ± 13.4 vs 59.8 × 104/μL ± 5.4) were comparable between heterozygous and WT mice. C57BL/6 mice served as controls because Sf3b1+/− mice were backcrossed to this mouse strain. BM aspirates were provided by Dr Koseki (RIKEN) and stained for Prussian blue. We found numerous RS in Sf3b1+/− compared with C57BL/6 mice as shown in Figure 2C (right panel).

RNA-Seq analysis of SF3B1 mutant RARS patients

To further understand the functional consequences of SF3B1 mutations, we used RNA-Seq to investigate the differential exon usage and gene expression levels in BM cells of 2 SF3B1 mutant RARS patients. Using a false discovery rate level of 0.05, we tested 81 564 exons in 9069 genes and found that 423 exons derived from 350 genes were differentially used in mutants, compared with a representative healthy donor. A total of 67% of these exons had higher usage in mutant versus normal, and 56 genes were significantly affected in more than 1 exon (Table 2). Of note, 4 genes relevant to MDS pathophysiology, namely, ASXL1, CBL, EZH1, and RUNX3, were found to be alternatively spliced in at least one exon (Figure 3). Gene level analysis conducted on 12 870 reference genes showed that none of these 4 genes had differences in expression level. However, we identified 130 differentially expressed genes, of which 94% had lower expression in SF3B1 mutant patients (supplemental Table 1). Gene set analysis was also performed on 11 773 genes showing many gene sets related to myeloma or MDS (supplemental Table 2). In addition, a cytokine gene set, cytokine production, was the 16th most significant among the c5 collection of gene sets. This observation supports prior findings that cytokines play an important role in MDS pathogenesis.

Table 2

Exons comparison between SF3B1 mutant and normal groups

Gene Chromosome Strand Exon location No. of exon test No. of exons Exon no. Adjusted P Base M Base N Log2fold (mutant/healthy donor) P 
SIRPB1 20 − 1577987-1578928 11 11 0.00 125 33.46 0.00 
SIRPB1 20 − 1584456-1584788 11 11 1.63 × 10−12 74 32.79 7.77 × 10−16 
SIRPB1 20 − 1585388-1585705 11 11 2.52 × 10−07 47 32.00 2.81 × 10−10 
SIRPB1 20 − 1579468-1579582 11 11 1.44 × 10−05 34 31.72 2.33 × 10−08 
SIRPB1 20 − 1592003-1592359 11 11 2.58 × 10−04 95 32.88 5.54 × 10−07 
AKAP13 15 86287859-86292586 36 38 38 0.00 258 30.91 0.00 
HBG2 11 − 5275867-5276011 0.00 15 658 30.03 0.00 
HBG2 11 − 5274421-5274635 1.64 × 10−05 29 067 344 −0.14 2.73 × 10−08 
PHF3 64421482-64424405 15 15 15 0.00 141 29.99 0.00 
CD37 19 49838677-49838832 0.00 111 29.65 0.00 
NIPAL3 24795476-24799472 12 12 12 0.00 173 29.40 0.00 
VPS36 13 − 52986739-52990058 13 14 14 0.00 222 28.94 0.00 
HELZ 17 − 65066555-65074702 29 33 33 7.76 × 10−13 153 29.12 3.33 × 10−16 
CTTN 11 70281132-70282689 16 19 19 1.51 × 10−12 419 31.66 6.66 × 10−16 
RSAD2 7035909-7038363 2.26 × 10−12 308 27.95 1.11 × 10−15 
RPS17 15 − 83207631-83207736 3.23 × 10−12 220 33.49 1.67 × 10−15 
POU2F2 19 − 42592673-42595743 10 13 13 6.74 × 10−12 280 29.46 3.55 × 10−15 
PARP14 122447155-122449686 17 17 17 2.06 × 10−11 128 29.10 1.11 × 10−14 
RAPGEF6 − 130759616-130762984 29 30 30 2.72 × 10−11 127 30.14 1.50 × 10−14 
CARD8 19 − 48711344-48715232 10 10 10 5.15 × 10−11 115 29.40 3.03 × 10−14 
TSC1 − 135766735-135772141 15 24 24 6.01 × 10−11 164 30.44 3.69 × 10−14 
ATM 11 108236052-108239826 54 64 64 1.13 × 10−10 200 31.49 7.21 × 10−14 
RABL3 − 120405530-120408735 1.58 × 10−10 102 30.15 1.07 × 10−13 
CCDC25 − 27590835-27593762 1.58 × 10−10 108 29.79 1.07 × 10−13 
MEGF9 − 123363091-123367919 2.41 × 10−10 262 31.12 1.68 × 10−13 
IL32 16 3119282-3119667 11 11 3.05 × 10−10 345 31.09 2.21 × 10−13 
IL32 16 3118991-3119667 11 10 1.53 × 10−02 665 85 −0.12 6.40 × 10−05 
DDX54 12 − 113594984-113596914 21 21 21 3.19 × 10−10 90 28.84 2.34 × 10−13 
TTC14 180327418-180328917 13 13 13 4.73 × 10−10 95 28.73 3.54 × 10−13 
ADPGK 15 − 73043710-73045236 6.16 × 10−10 102 32.16 4.69 × 10−13 
ADPGK 15 − 73043710-73045233 2.35 × 10−05 101 98 −0.11 4.10 × 10−08 
EZH1 17 − 40852294-40854610 19 21 21 2.04 × 10−09 131 28.95 1.63 × 10−12 
MADD 11 47350597-47351582 35 38 38 5.39 × 10−09 90 29.52 4.43 × 10−12 
RABL2B 22 − 51205920-51207302 13 13 13 5.49 × 10−09 80 29.36 4.58 × 10−12 
RABL2B 22 − 51207883-51207977 13 13 10 2.93 × 10−02 16 −28.17 1.39 × 10−04 
ZNF638 71649944-71651168 24 28 22 6.95 × 10−09 84 30.73 5.88 × 10−12 
SON 21 34948671-34949812 13 13 13 8.44 × 10−09 76 32.49 7.25 × 10−12 
IDE 10 − 94211441-94214296 24 25 25 9.99 × 10−09 71 29.31 8.69 × 10−12 
MOBKL1A 71847697-71853890 2.45 × 10−08 268 30.54 2.22 × 10−11 
ZNF592 15 85345094-85349661 10 11 11 2.45 × 10−08 81 29.21 2.20 × 10−11 
RPRD1B 20 36718128-36720764 2.62 × 10−08 112 29.42 2.41 × 10−11 
ARHGEF7 13 111955338-111958080 11 23 23 2.99 × 10−08 120 32.24 2.78 × 10−11 
KDM5D − 21867303-21868231 27 28 28 5.42 × 10−08 92 28.78 5.25 × 10−11 
ZNF295 21 − 43406941-43414217 1.51 × 10−07 28 30.45 1.55 × 10−10 
ZNF295 21 − 43406941-43411998 6.31 × 10−06 80 −27.93 9.20 × 10−09 
ARHGAP27 17 − 43471269-43472999 11 17 17 1.51 × 10−07 154 29.46 1.56 × 10−10 
CEP350 180080132-180084014 22 36 36 1.62 × 10−07 102 32.63 1.69 × 10−10 
R3HDM1 136481507-136482836 19 25 25 1.85 × 10−07 66 29.65 1.97 × 10−10 
TRMT5 14 − 61438169-61441912 2.25 × 10−07 180 30.11 2.43 × 10−10 
TRMT5 14 − 61445949-61446604 5.94 × 10−07 25 23 −0.36 6.99 × 10−10 
CA5B 15800608-15805747 2.31 × 10−07 114 30.37 2.52 × 10−10 
KIAA0319L − 35899092-35900682 20 21 21 2.52 × 10−07 73 27.72 2.81 × 10−10 
CD3E 11 118186201-118186889 4.06 × 10−07 238 27.14 4.57 × 10−10 
GTF2E1 120499890-120501915 4.96 × 10−07 138 32.16 5.72 × 10−10 
GTF2E1 120469370-120469847 1.39 × 10−06 11 20 0.11 1.83 × 10−09 
SLC9A8 20 48504366-48508772 16 16 5.36 × 10−07 65 30.44 6.25 × 10−10 
ST6GALNA − 130647601-130649067 7.62 × 10−07 136 28.77 9.06 × 10−10 
FCHSD1 − 141018870-141021130 15 20 20 8.83 × 10−07 94 27.20 1.07 × 10−09 
RUNX3 − 25226003-25229157 1.01 × 10−06 375 30.61 1.26 × 10−09 
CHD9 16 53357932-53361413 15 38 38 1.16 × 10−06 71 32.47 1.47 × 10−09 
ATG10 81272012-81272146 1.39 × 10−06 437 1440 −0.66 1.79 × 10−09 
ATG10 81267844-81268125 3.04 × 10−05 201 155 1.44 5.40 × 10−08 
NAA16 13 41949541-41951166 18 22 22 1.39 × 10−06 55 29.50 1.83 × 10−09 
LPIN1 11964758-11967531 14 20 20 1.98 × 10−06 80 29.54 2.67 × 10−09 
2-Mar 19 8503272-8503897 2.42 × 10−06 126 30.44 3.29 × 10−09 
AFG3L1 16 90061101-90063028 11 15 13 2.43 × 10−06 75 28.76 3.34 × 10−09 
LDLRAP1 25893339-25895375 3.44 × 10−06 127 31.25 4.79 × 10−09 
FRYL − 48499380-48501697 36 63 63 4.11 × 10−06 99 31.64 5.80 × 10−09 
ITK 156679617-156682109 17 17 17 4.46 × 10−06 237 31.53 6.35 × 10−09 
MCTP2 15 95022195-95027180 13 24 24 4.64 × 10−06 55 31.32 6.71 × 10−09 
HDDC3 15 − 91474157-91474786 6.58 × 10−06 80 28.62 9.68 × 10−09 
RAP1GAP2 17 2936686-2941034 25 25 6.98 × 10−06 164 31.13 1.04 × 10−08 
ARFRP1 20 − 62329996-62332054 8.08 × 10−06 85 27.86 1.22 × 10−08 
CHST15 10 − 125767186-125769855 8.68 × 10−06 81 29.59 1.32 × 10−08 
SLC5A6 − 27422457-27423447 19 20 19 1.02 × 10−05 77 30.99 1.58 × 10−08 
SLC5A6 − 27422973-27423447 19 20 18 1.46 × 10−03 54 30.43 4.39 × 10−06 
BTN3A3 26451903-26453642 11 11 1.02 × 10−05 140 29.53 1.58 × 10−08 
GCFC1 21 − 34106213-34107378 13 18 18 1.39 × 10−05 62 29.83 2.19 × 10−08 
MKNK2 19 − 2037470-2037828 13 13 1.39 × 10−05 55 28.44 2.19 × 10−08 
TRIM58 248039202-248043436 1.44 × 10−05 880 31.19 2.34 × 10−08 
TRIM58 248023919-248024014 1.24 × 10−02 10 −0.88 4.92 × 10−05 
OXSR1 38294308-38296978 18 18 1.64 × 10−05 72 32.32 2.70 × 10−08 
POLR2A 17 7416340-7417929 27 29 29 1.64 × 10−05 67 28.77 2.72 × 10−08 
RNF219 13 − 79188421-79191257 1.91 × 10−05 63 27.84 3.23 × 10−08 
GNPTAB 12 − 102139278-102141019 13 20 20 2.00 × 10−05 85 29.10 3.40 × 10−08 
CXorf15 16859551-16862640 10 10 2.29 × 10−05 64 28.14 3.97 × 10−08 
RHCE − 25747130-25747363 10 10 2.46 × 10−05 75 27.80 4.31 × 10−08 
FHDC1 153895827-153900848 11 11 11 4.27 × 10−05 317 31.91 7.79 × 10−08 
CCNT1 12 − 49086752-49088219 4.49 × 10−05 45 30.46 8.25 × 10−08 
CAPRIN2 12 − 30862486-30863404 15 19 19 4.70 × 10−05 73 28.41 8.71 × 10−08 
RXRB − 33161366-33162606 10 10 5.98 × 10−05 64 27.55 1.12 × 10−07 
GIGYF2 233721503-233725285 23 33 33 5.99 × 10−05 43 28.04 1.13 × 10−07 
DNAJB6 157208710-157210132 12 12 12 6.27 × 10−05 165 90 1.87 1.19 × 10−07 
FAM63B 15 59146681-59149732 10 6.56 × 10−05 76 30.03 1.25 × 10−07 
USP37 − 219314974-219319740 10 26 26 7.33 × 10−05 53 30.01 1.41 × 10−07 
EME1 17 48458124-48458820 10 10 8.83 × 10−05 43 30.84 1.71 × 10−07 
SQLE 126010720-126011936 11 11 9.10 × 10−05 32 27.62 1.77 × 10−07 
GATAD2A 19 19616153-19619740 11 11 1.55 × 10−04 104 28.45 3.07 × 10−07 
RFC1 − 39289076-39290464 25 25 25 1.73 × 10−04 42 28.28 3.46 × 10−07 
RORA 15 − 60780485-60789818 10 12 12 2.42 × 10−04 140 28.47 5.02 × 10−07 
SBNO1 12 − 123773657-123780597 28 32 32 2.43 × 10−04 335 128 1.24 5.06 × 10−07 
PSMB9 32821938-32822014 2.46 × 10−04 48 27.86 5.15 × 10−07 
ADRBK2 22 26118256-26125256 16 16 2.52 × 10−04 186 33.06 5.35 × 10−07 
PRDM1 106552700-106553808 2.57 × 10−04 41 18 0.04 5.49 × 10−07 
PRDM1 106554786-106557809 1.50 × 10−02 110 29.90 6.27 × 10−05 
PRDM1 106554786-106557814 1.50 × 10−02 110 29.90 6.27 × 10−05 
PPID − 159630280-159630976 10 10 10 2.63 × 10−04 76 27.35 5.68 × 10−07 
PITPNA 17 − 1421285-1423832 10 10 2.82 × 10−04 55 30.22 6.20 × 10−07 
LHFPL2 − 77781040-77784976 2.82 × 10−04 86 29.15 6.19 × 10−07 
LHFPL2 − 77805607-77806221 2.82 × 10−04 12 −0.29 6.19 × 10−07 
CYTSB 17 20217289-20218065 15 16 16 3.02 × 10−04 56 28.96 6.70 × 10−07 
SNTB1 − 121547986-121551209 3.07 × 10−04 70 27.79 6.88 × 10−07 
TSGA14 − 130036377-130038880 11 11 3.30 × 10−04 49 28.47 7.49 × 10−07 
CCND3 − 42016239-42016610 3.33 × 10−04 43 27.68 7.60 × 10−07 
ENO2 12 7031894-7032859 12 12 3.39 × 10−04 83 29.41 7.82 × 10−07 
FLVCR2 14 76112744-76114512 10 10 3.42 × 10−04 67 27.42 7.98 × 10−07 
PTPRC 198671516-198671659 34 34 3.42 × 10−04 165 29 2.08 8.02 × 10−07 
SMAD4 18 48604626-48611409 12 12 3.77 × 10−04 53 31.76 8.91 × 10−07 
FCRL1 − 157764196-157765960 13 13 4.18 × 10−04 66 29.44 1.00 × 10−06 
C3orf63 − 56654161-56657871 18 26 26 4.22 × 10−04 124 28.62 1.02 × 10−06 
ZNF45 19 − 44416777-44419352 10 10 4.62 × 10−04 55 30.37 1.12 × 10−06 
CREBZF 11 − 85374859-85376182 4.96 × 10−04 67 31.33 1.22 × 10−06 
SLC37A1 21 44000455-44001549 20 20 5.27 × 10−04 35 32.09 1.32 × 10−06 
FAM122B − 133903597-133905384 12 12 5.54 × 10−04 94 29.22 1.39 × 10−06 
TIMM17B − 48755350-48755426 5.79 × 10−04 44 27.31 1.46 × 10−06 
CHD3 17 7814782-7816075 37 37 5.99 × 10−04 51 32.62 1.53 × 10−06 
RNF160 21 − 30300466-30302832 20 30 30 5.99 × 10−04 42 28.92 1.53 × 10−06 
SORT1 − 109852192-109856679 20 20 6.31 × 10−04 108 32.48 1.62 × 10−06 
POM121C − 75046066-75048176 6.33 × 10−04 59 31.72 1.64 × 10−06 
POM121C − 75050792-75052486 6.33 × 10−04 14 −0.29 1.64 × 10−06 
ZBTB40 22852695-22857650 10 18 18 7.60 × 10−04 90 29.12 2.00 × 10−06 
APPL2 12 − 105567075-105568226 21 21 9.03 × 10−04 32 29.36 2.39 × 10−06 
MCART1 − 37877572-37880745 9.16 × 10−04 29 30.07 2.44 × 10−06 
GOPC − 117881435-117884547 9.16 × 10−04 65 27.39 2.45 × 10−06 
PDGFD 11 − 103777915-103780547 9.54 × 10−04 60 29.12 2.60 × 10−06 
CD247 − 167399878-167400983 9.89 × 10−04 361 27.72 2.71 × 10−06 
CA1 − 86262040-86262114 12 12 1.07 × 10−03 58 26.32 2.95 × 10−06 
SYVN1 11 − 64894753-64895960 12 17 17 1.11 × 10−03 42 27.32 3.09 × 10−06 
SP2 17 46005090-46006321 1.11 × 10−03 54 26.89 3.13 × 10−06 
ST8SIA4 − 100142640-100147833 1.11 × 10−03 129 71 1.56 3.13 × 10−06 
ESRRA 11 64083179-64084210 1.12 × 10−03 69 30.36 3.19 × 10−06 
DDX26B 134715453-134716459 17 17 17 1.16 × 10−03 45 26.74 3.32 × 10−06 
RNPEPL1 241517016-241518141 11 11 1.18 × 10−03 64 30.68 3.37 × 10−06 
ZKSCAN5 99128731-99131444 1.26 × 10−03 35 29.99 3.64 × 10−06 
POM121 72416676-72418837 1.33 × 10−03 57 30.76 3.87 × 10−06 
POM121 72412376-72414061 1.33 × 10−03 10 14 −0.22 3.87 × 10−06 
TJAP1 43472499-43474294 10 14 14 1.38 × 10−03 52 28.80 4.07 × 10−06 
TBCCD1 − 186263858-186264744 1.38 × 10−03 47 27.32 4.07 × 10−06 
SHQ1 − 72798430-72799987 10 11 11 1.42 × 10−03 36 27.01 4.23 × 10−06 
CUL4A 13 113917801-113919391 10 18 18 1.44 × 10−03 83 30.07 4.31 × 10−06 
NLRP12 19 − 54310749-54311959 10 11 1.50 × 10−03 32 27.88 4.56 × 10−06 
ATF7IP 12 14650588-14651696 13 15 15 1.50 × 10−03 39 27.30 4.58 × 10−06 
ZNHIT6 − 86115107-86119781 11 11 1.50 × 10−03 47 26.99 4.55 × 10−06 
MSH3 80171570-80172633 23 24 24 1.59 × 10−03 41 28.75 4.85 × 10−06 
KIAA2026 − 5919010-5923309 1.63 × 10−03 67 29.99 5.01 × 10−06 
SH2D1B − 162365056-162367105 1.75 × 10−03 313 26.58 5.39 × 10−06 
SH2D1B − 162381673-162381928 4.00 × 10−02 47 −1.06 1.99 × 10−04 
TGFBR3 − 92145902-92149414 16 17 17 2.06 × 10−03 99 19 0.03 6.44 × 10−06 
ALDOA 16 30066105-30066248 16 16 2.21 × 10−03 26 27.87 6.95 × 10−06 
SEPSECS − 25121628-25125847 10 10 2.43 × 10−03 35 30.43 7.69 × 10−06 
PIK3C2A 11 − 17108126-17111467 32 32 2.51 × 10−03 51 30.24 7.98 × 10−06 
PIK3C2A 11 − 17190224-17191354 32 1.94 × 10−02 18 15 0.17 8.75 × 10−05 
SAPS3 11 68382100-68382799 27 28 28 2.55 × 10−03 54 29.21 8.17 × 10−06 
C11orf49 11 47183015-47183796 12 12 11 2.58 × 10−03 40 30.77 8.28 × 10−06 
TCF3 19 − 1609263-1611368 19 19 2.80 × 10−03 46 29.10 9.02 × 10−06 
RB1CC1 − 53535019-53536419 18 25 25 3.02 × 10−03 79 29.68 9.77 × 10−06 
VPRBP − 51433298-51434555 21 26 26 3.13 × 10−03 35 28.00 1.02 × 10−05 
C17orf68 17 − 8128140-8131637 22 23 23 3.23 × 10−03 46 27.31 1.06 × 10−05 
ZSCAN18 19 − 58601232-58601753 3.26 × 10−03 15 0.32 1.07 × 10−05 
SERINC5 − 79434552-79439633 13 12 3.41 × 10−03 79 30.17 1.13 × 10−05 
LINS1 15 − 101113856-101114446 3.55 × 10−03 44 28.77 1.18 × 10−05 
ATP10D 47593059-47595503 11 22 22 3.57 × 10−03 34 30.41 1.19 × 10−05 
CYHR1 − 145689200-145689995 3.67 × 10−03 43 32.53 1.23 × 10−05 
MINA − 97660661-97664186 10 12 12 4.03 × 10−03 70 27.71 1.37 × 10−05 
ELP3 28047166-28048667 13 15 15 4.03 × 10−03 34 26.78 1.37 × 10−05 
AHNAK 11 − 62283375-62301546 4.04 × 10−03 1458 606 0.31 1.38 × 10−05 
POLH 43581397-43588259 11 11 4.15 × 10−03 60 29.11 1.42 × 10−05 
SCO1 17 − 10583651-10584570 4.28 × 10−03 44 27.43 1.47 × 10−05 
PAPOLG 61024197-61026096 14 22 22 4.37 × 10−03 41 28.47 1.51 × 10−05 
TTYH2 17 72256268-72258155 15 15 4.57 × 10−03 53 29.97 1.59 × 10−05 
KIAA0355 19 34843552-34846470 11 14 14 4.59 × 10−03 73 28.43 1.60 × 10−05 
SH2D1A 123505201-123507008 5.12 × 10−03 88 26.73 1.79 × 10−05 
GRK6 176868738-176869848 12 16 16 5.20 × 10−03 36 29.55 1.83 × 10−05 
KIAA0907 − 155882836-155884111 14 14 5.27 × 10−03 51 26.56 1.86 × 10−05 
DDX19B 16 70363734-70363971 12 12 5.72 × 10−03 27 −28.00 2.05 × 10−05 
DDX19B 16 70367424-70367729 12 12 12 8.84 × 10−03 22 29.10 3.38 × 10−05 
MANEA 96053624-96057326 6.18 × 10−03 46 29.45 2.24 × 10−05 
MANEA 96034278-96034859 6.18 × 10−03 11 0.03 2.24 × 10−05 
COPB1 11 − 14521155-14521404 24 24 6.18 × 10−03 23 27.40 2.22 × 10−05 
ZNF37B 10 − 43008963-43016575 6.49 × 10−03 106 29.56 2.37 × 10−05 
ZNF37B 10 − 43047021-43047262 6.49 × 10−03 0.04 2.37 × 10−05 
FCAR 19 55399374-55400437 6.49 × 10−03 66 540 1.16 2.36 × 10−05 
ZNF498 99226814-99230030 7.64 × 10−03 53 27.07 2.83 × 10−05 
TEP1 14 − 20833827-20836718 46 46 7.91 × 10−03 57 31.96 2.96 × 10−05 
ZDHHC7 16 − 85008067-85010125 7.91 × 10−03 69 30.62 2.96 × 10−05 
MOGS − 74688185-74690139 8.02 × 10−03 63 26.72 3.01 × 10−05 
BCL7B − 72950686-72951720 8.24 × 10−03 63 28.92 3.10 × 10−05 
FAM13A − 89647106-89649810 14 24 24 8.39 × 10−03 96 30.28 3.17 × 10−05 
N4BP2L2 13 − 33016525-33018263 10 14 11 8.39 × 10−03 30 27.06 3.18 × 10−05 
NFAT5 16 69725652-69728142 10 9.53 × 10−03 26 31.02 3.66 × 10−05 
CTNS 17 3563530-3566396 14 13 9.57 × 10−03 67 30.69 3.68 × 10−05 
KIAA1467 12 13233559-13236381 12 12 1.03 × 10−02 72 31.36 4.00 × 10−05 
ZNF333 19 14829040-14831770 12 12 1.12 × 10−02 34 30.32 4.42 × 10−05 
PRKCQ 10 − 6469105-6470324 12 18 18 1.23 × 10−02 51 27.05 4.88 × 10−05 
SLC26A2 149359856-149366959 1.24 × 10−02 31 29.85 4.97 × 10−05 
SLC26A2 149357191-149357914 1.24 × 10−02 19 0.22 4.97 × 10−05 
TMCO7 16 69117388-69119083 16 18 18 1.24 × 10−02 37 26.44 4.94 × 10−05 
PDZD11 − 69506211-69506966 1.24 × 10−02 39 26.32 4.97 × 10−05 
FCGR3B − 161595935-161596192 1.24 × 10−02 74 196 1.06 4.98 × 10−05 
LPAR2 19 − 19734468-19735378 1.36 × 10−02 57 27.47 5.54 × 10−05 
LPAR2 19 − 19737352-19738093 1.36 × 10−02 32 14 0.04 5.54 × 10−05 
NAPEPLD − 102740023-102744001 1.38 × 10−02 14 29.38 5.60 × 10−05 
NAPEPLD − 102740023-102743569 2.30 × 10−02 12 −28.98 1.05 × 10−04 
PIK3CA 178951882-178952495 10 21 21 1.40 × 10−02 33 31.17 5.72 × 10−05 
STAG3L2 − 74306509-74306731 1.41 × 10−02 36 26.66 5.77 × 10−05 
DNAJC16 15894273-15898226 15 15 1.44 × 10−02 79 30.26 5.94 × 10−05 
ZNF227 19 44738855-44741420 1.49 × 10−02 44 27.42 6.17 × 10−05 
NPC1 18 − 21111464-21112248 25 25 25 1.53 × 10−02 36 27.05 6.43 × 10−05 
SHPRH − 146205947-146207923 29 29 1.54 × 10−02 25 29.37 6.51 × 10−05 
YPEL4 11 − 57412561-57413543 1.54 × 10−02 72 28.59 6.50 × 10−05 
MPHOSPH 13 20245974-20247598 14 14 14 1.54 × 10−02 69 27.28 6.49 × 10−05 
GOLM1 − 88641060-88642808 11 11 1.66 × 10−02 54 28.28 7.03 × 10−05 
VPS13B 100887646-100889807 35 56 56 1.66 × 10−02 27 29.68 7.06 × 10−05 
NLRX1 11 119050402-119050997 10 1.68 × 10−02 10 0.05 7.17 × 10−05 
C18orf8 18 21110351-21110576 16 20 19 1.74 × 10−02 32 27.64 7.45 × 10−05 
ZBTB20 − 114056947-114058273 12 12 1.74 × 10−02 41 27.40 7.51 × 10−05 
ZFYVE19 15 41099274-41100066 11 1.76 × 10−02 27 27.83 7.61 × 10−05 
RBM12 20 − 34236888-34243266 1.79 × 10−02 97 28.98 7.76 × 10−05 
RBM12 20 − 34252682-34252838 1.79 × 10−02 34 32 −0.02 7.76 × 10−05 
MEPCE 100031125-100031740 1.83 × 10−02 52 27.62 8.03 × 10−05 
ELMOD3 85617261-85618873 11 17 16 1.85 × 10−02 48 30.27 8.15 × 10−05 
NPLOC4 17 − 79523915-79526442 15 15 1.88 × 10−02 202 32.35 8.38 × 10−05 
NPLOC4 17 − 79555970-79556130 15 10 1.88 × 10−02 0.30 8.38 × 10−05 
GTF3C4 135564268-135565468 1.89 × 10−02 13 28.70 8.45 × 10−05 
GTF3C4 135553364-135555190 1.89 × 10−02 10 −26.38 8.45 × 10−05 
IGHMBP2 11 68707002-68708069 15 15 15 1.94 × 10−02 35 28.12 8.71 × 10−05 
TRIM37 17 − 57075562-57076820 14 26 25 2.28 × 10−02 36 29.83 1.04 × 10−04 
ENG − 130577292-130578332 14 16 15 2.38 × 10−02 30 30.61 1.09 × 10−04 
C14orf45 14 74516405-74516898 12 2.44 × 10−02 30 27.12 1.13 × 10−04 
CCDC91 12 28701996-28703098 11 12 12 2.54 × 10−02 30 28.77 1.18 × 10−04 
ZSCAN21 99661411-99662661 2.92 × 10−02 41 28.95 1.38 × 10−04 
ZSCAN21 99654534-99655028 2.92 × 10−02 0.10 1.38 × 10−04 
RRN3P2 16 29127448-29128036 14 14 3.14 × 10−02 31 28.51 1.52 × 10−04 
DIP2A 21 47987283-47989926 40 40 3.14 × 10−02 116 33.06 1.52 × 10−04 
ZNF611 19 − 53206067-53210117 3.25 × 10−02 140 28.67 1.58 × 10−04 
RASGRP3 33787796-33789797 15 20 20 3.31 × 10−02 36 30.57 1.61 × 10−04 
CHD7 61777575-61779463 32 32 3.37 × 10−02 26 32.90 1.65 × 10−04 
FLCN 17 − 17124486-17124942 15 15 3.59 × 10−02 45 30.05 1.77 × 10−04 
C10orf119 10 − 121588972-121591112 11 11 3.78 × 10−02 35 26.46 1.87 × 10−04 
FIGNL1 − 50511834-50514995 3.80 × 10−02 42 29.09 1.88 × 10−04 
NOL9 − 6581409-6586063 10 10 4.14 × 10−02 169 28.77 2.06 × 10−04 
GLUL − 182360310-182360539 4.40 × 10−02 79 121 1.52 2.20 × 10−04 
TRAF5 211545470-211548284 11 11 4.40 × 10−02 68 30.21 2.21 × 10−04 
DHRS7B 17 21094261-21094835 4.51 × 10−02 35 26.40 2.27 × 10−04 
NLRC3 16 − 3589038-3591915 19 19 4.51 × 10−02 106 31.12 2.28 × 10−04 
NLRC3 16 − 3613010-3614759 19 4.51 × 10−02 31 0.34 2.28 × 10−04 
ABCD1 152990323-152991621 10 4.53 × 10−02 19 27.49 2.30 × 10−04 
GRIPAP1 − 48847338-48847514 23 27 4.56 × 10−02 24 25.88 2.32 × 10−04 
CHAC2 54001279-54002287 4.63 × 10−02 46 27.32 2.37 × 10−04 
SLC33A1 − 155571012-155572167 4.78 × 10−02 48 26.51 2.45 × 10−04 
ASXL1 20 31022235-31027121 13 13 4.88 × 10−02 141 32.33 2.51 × 10−04 
CASC3 17 38296507-38297032 13 14 4.94 × 10−02 28 26.09 2.55 × 10−04 
SEC22A 122990369-122992980 4.98 × 10−02 23 27.20 2.58 × 10−04 
RPL21P28 13 27830573-27830698 0.00 1736 2.38 0.00 
RPL21P28 13 27825693-27825722 0.00 231 202 −5.86 0.00 
HNRNPA1L 13 53201573-53201713 0.00 1.00 0.00 
HNRNPA1L 13 53196124-53196339 0.00 0.10 0.00 
HNRNPA1L 13 53191605-53191817 0.00 10 −1.17 0.00 
HNRNPA1L 13 53216541-53217919 0.00 1743 −34.56 0.00 
TXLNA 32653555-32653725 11 0.00 15 0.76 0.00 
TXLNA 32658258-32658332 11 0.00 0.63 0.00 
TXLNA 32657912-32658031 11 0.00 0.62 0.00 
TXLNA 32655656-32655850 11 0.00 18 0.35 0.00 
TXLNA 32646843-32647178 11 0.00 −0.64 0.00 
TXLNA 32660503-32663885 11 11 0.00 345 −33.27 0.00 
MIER1 67452086-67454302 20 21 21 0.00 447 −31.97 0.00 
MIER1 67450255-67450335 20 21 19 2.43 × 10−10 21 24 0.59 1.73 × 10−13 
MIER1 67450255-67450275 20 21 18 5.31 × 10−08 12 15 0.51 5.08 × 10−11 
MIER1 67447462-67447601 20 21 17 1.44 × 10−05 25 36 0.34 2.30 × 10−08 
MIER1 67411833-67411978 20 21 1.82 × 10−02 29 43 0.23 7.98 × 10−05 
CSF3R − 36931644-36932509 20 20 18 0.00 1003 −32.06 0.00 
CSF3R − 36932831-36932912 20 20 16 1.63 × 10−12 44 206 0.35 7.77 × 10−16 
CSF3R − 36931644-36932428 20 20 19 5.15 × 10−11 174 999 0.05 3.01 × 10−14 
CSF3R − 36933423-36933563 20 20 14 4.53 × 10−06 61 318 0.20 6.50 × 10−09 
CSF3R − 36933159-36933252 20 20 15 5.23 × 10−04 48 244 0.22 1.30 × 10−06 
SLC25A36 140695105-140698785 0.00 241 −30.23 0.00 
SLC25A36 140695102-140698785 4.68 × 10−04 155 242 0.16 1.14 × 10−06 
CDV3 133306003-133309116 0.00 372 283 0.15 0.00 
CDV3 133306740-133309116 0.00 275 −33.17 0.00 
NUFIP2 17 − 27582855-27591609 0.00 196 −30.52 0.00 
NUFIP2 17 − 27613010-27614734 2.14 × 10−09 68 94 0.11 1.73 × 10−12 
H3F3A 226252030-226252180 0.00 822 2293 0.09 0.00 
H3F3A 226250421-226250512 0.00 255 835 −0.15 0.00 
H3F3A 226259052-226259702 0.00 4290 −33.08 0.00 
POU5F1 − 31133704-31134615 0.00 299 −30.99 0.00 
POU5F1 − 31134311-31134615 3.44 × 10−06 335 273 0.09 4.80 × 10−09 
QKI 163984452-163999627 0.00 273 −31.04 0.00 
QKI 163991726-163999627 7.22 × 10−10 171 212 0.06 5.58 × 10−13 
SUMO1 − 203070803-203071526 0.00 328 −30.28 0.00 
SUMO1 − 203070903-203072044 4.59 × 10−02 506 811 0.01 2.34 × 10−04 
C1orf9 172578956-172580971 22 25 25 0.00 200 −29.70 0.00 
LGALS3 14 55612030-55612147 0.00 139 −30.94 0.00 
NCOA1 24991093-24993568 14 23 23 0.00 97 −33.15 0.00 
WDR45 − 48957143-48957492 13 14 1.63 × 10−12 57 −32.27 7.77 × 10−16 
CREM 10 35500584-35501885 15 15 2.43 × 10−12 177 −31.41 1.22 × 10−15 
RAD17 68665124-68665291 20 21 4.15 × 10−11 78 −30.64 2.34 × 10−14 
ATXN7 63985126-63989134 12 12 5.93 × 10−11 77 −31.51 3.56 × 10−14 
GON4L − 155734577-155736500 23 33 22 9.48 × 10−11 84 −32.55 5.93 × 10−14 
PIP4K2C 12 57995709-57997210 13 14 14 1.29 × 10−10 61 −31.00 8.36 × 10−14 
ATP13A3 − 194123405-194126845 18 30 30 2.25 × 10−10 313 −30.91 1.54 × 10−13 
ZNF641 12 − 48733795-48737510 1.70 × 10−09 49 −32.63 1.34 × 10−12 
ZNF641 12 − 48741690-48741898 2.78 × 10−02 14 27.56 1.30 × 10−04 
AMPD3 11 10527255-10529126 15 16 16 1.51 × 10−08 80 −32.15 1.33 × 10−11 
TCEAL4 102841582-102842653 3.54 × 10−08 107 −29.05 3.34 × 10−11 
PRDM2 14104913-14109326 6.57 × 10−08 195 137 0.12 6.52 × 10−11 
PRDM2 14113010-14114573 6.57 × 10−08 59 −31.18 6.52 × 10−11 
LRCH4 − 100171636-10017292 18 18 18 1.36 × 10−07 60 −29.52 1.37 × 10−10 
MAP4 − 47892180-47894529 17 20 19 1.66 × 10−07 53 −30.48 1.75 × 10−10 
MAP4 − 47892180-47894428 17 20 20 1.92 × 10−06 82 29.48 2.55 × 10−09 
MXD3 − 176732501-176734196 4.26 × 10−07 82 −28.74 4.85 × 10−10 
SLC4A1 17 − 42325759-42327906 20 20 20 8.44 × 10−07 3390 1884 −0.83 1.01 × 10−09 
LIPA 10 − 91011464-91011660 12 12 8.84 × 10−07 78 −28.02 1.09 × 10−09 
STRADA 17 − 61780194-61781111 13 15 15 8.84 × 10−07 58 −28.26 1.09 × 10−09 
LSM5 − 32529637-32530475 1.03 × 10−06 72 −29.98 1.31 × 10−09 
ERI2 16 − 20807662-20810389 11 1.96 × 10−06 75 −32.21 2.62 × 10−09 
SC5DL 11 121177766-121184117 7.40 × 10−06 133 −28.25 1.11 × 10−08 
PKN2 89298928-89301937 18 22 22 1.03 × 10−05 55 −29.92 1.60 × 10−08 
CEACAM1 19 − 43011459-43013380 1.59 × 10−05 146 −28.49 2.59 × 10−08 
NF2 22 30090741-30094583 18 18 1.88 × 10−05 162 −33.73 3.16 × 10−08 
NF2 22 30070825-30070930 18 12 3.50 × 10−02 2.02 1.72 × 10−04 
PRKCB 16 24231282-24231930 15 18 18 2.10 × 10−05 39 −34.39 3.60 × 10−08 
CBL 11 119170205-119178858 16 16 2.75 × 10−05 84 −30.64 4.86 × 10−08 
TGFBR2 30732912-30735631 3.34 × 10−05 72 −30.44 6.02 × 10−08 
TGFBR2 30713130-30713929 4.94 × 10−02 14 10 0.21 2.55 × 10−04 
AKAP2 112898407-112900819 3.34 × 10−05 44 80 −0.05 6.06 × 10−08 
AKAP2 112930679-112934789 3.34 × 10−05 71 −28.15 6.06 × 10−08 
UHRF1BP1 34840090-34845289 21 21 4.90 × 10−05 49 −31.33 9.13 × 10−08 
SMCR7 17 18167098-18169093 1.03 × 10−04 23 −29.44 2.02 × 10−07 
SMCR7 17 18167024-18169093 7.14 × 10−03 22 27.99 2.62 × 10−05 
NDUFV1 11 67374323-67374520 11 11 1.60 × 10−04 −28.18 3.19 × 10−07 
BCOR − 39931602-39934433 10 15 1.76 × 10−04 43 −29.87 3.54 × 10−07 
FGD4 12 32793210-32798982 17 17 1.90 × 10−04 90 −31.24 3.85 × 10−07 
SRCAP 16 30748370-30751449 29 29 2.13 × 10−04 36 29.81 4.36 × 10−07 
SRCAP 16 30734905-30736403 29 21 2.13 × 10−04 22 −30.64 4.36 × 10−07 
OAS1 12 113357194-113357711 2.42 × 10−04 29 −29.09 5.02 × 10−07 
ETS2 21 40194598-40196876 2.52 × 10−04 171 −30.13 5.35 × 10−07 
C7orf28A 5965263-5965601 2.98 × 10−04 46 −30.72 6.59 × 10−07 
PIGN 18 − 59711460-59713212 23 31 31 3.07 × 10−04 54 −29.06 6.89 × 10−07 
SELP − 169558090-169558699 15 17 17 3.28 × 10−04 35 −29.54 7.39 × 10−07 
XRCC4 82648950-82649577 10 10 3.38 × 10−04 40 −28.80 7.76 × 10−07 
SIGLEC14 19 − 52149510-52149893 3.40 × 10−04 84 −28.80 7.87 × 10−07 
CDK12 17 37686884-37690799 15 15 3.75 × 10−04 38 −31.39 8.82 × 10−07 
ATF3 212792388-212794114 4.11 × 10−04 73 −27.83 9.78 × 10−07 
MYH11 16 − 15818745-15818849 16 13 4.53 × 10−04 21 −29.20 1.09 × 10−06 
ABR 17 − 906760-909409 22 18 4.96 × 10−04 36 −30.39 1.22 × 10−06 
OLFM4 13 53624104-53626186 5.23 × 10−04 405 −29.11 1.30 × 10−06 
HIVEP1 12120123-12126103 6.98 × 10−04 13 27.93 1.83 × 10−06 
HIVEP1 12163516-12165231 6.98 × 10−04 26 −29.98 1.83 × 10−06 
MIDN 19 1256994-1259139 6.98 × 10−04 239 −31.82 1.82 × 10−06 
CEP63 134204575-134204894 15 19 9.33 × 10−04 38 −29.07 2.51 × 10−06 
RREB1 7248744-7252212 14 14 9.34 × 10−04 69 −31.98 2.52 × 10−06 
NCOA6 20 − 33328167-33331145 10 9.41 × 10−04 27 −30.79 2.55 × 10−06 
NISCH 52525888-52527087 18 21 21 1.02 × 10−03 34 −28.34 2.79 × 10−06 
RNF111 15 59323003-59323901 11 13 1.08 × 10−03 29 −28.45 3.00 × 10−06 
YLPM1 14 75264283-75266400 15 21 1.11 × 10−03 33 −28.11 3.13 × 10−06 
PML 15 74327513-74328734 10 13 10 1.12 × 10−03 39 −30.59 3.18 × 10−06 
SLC41A1 − 205758221-205760846 11 11 1.33 × 10−03 37 −30.47 3.85 × 10−06 
RNF19B − 33402052-33402910 10 11 10 1.40 × 10−03 43 −27.66 4.13 × 10−06 
SLC17A9 20 61598689-61599941 13 13 1.42 × 10−03 53 −29.47 4.21 × 10−06 
RIN3 14 93154271-93155332 1.59 × 10−03 21 37 −0.01 4.88 × 10−06 
RIN3 14 93117927-93119420 3.01 × 10−02 62 −29.47 1.45 × 10−04 
RTN4 − 5525222-55254621 10 1.90 × 10−03 29 −29.39 5.89 × 10−06 
DHX34 19 47856011-47856992 16 17 2.05 × 10−03 42 −29.13 6.37 × 10−06 
TMEM68 − 56651320-56652777 2.24 × 10−03 36 −29.39 7.05 × 10−06 
SSH2 17 − 2795296-27959949 11 11 2.54 × 10−03 101 −29.84 8.10 × 10−06 
FCGR2C 161569420-161570031 3.18 × 10−03 33 39 −0.01 1.04 × 10−05 
SLCO4C1 − 101569694-101572722 13 13 3.48 × 10−03 66 −29.04 1.15 × 10−05 
IL21R 16 27459855-27462115 10 10 3.63 × 10−03 27 −29.51 1.22 × 10−05 
MED6 14 − 71050958-71051660 3.80 × 10−03 39 −27.26 1.28 × 10−05 
STEAP4 − 87905750-87908943 4.35 × 10−03 93 −28.18 1.50 × 10−05 
PRNP 20 4667157-4667382 4.40 × 10−03 40 −27.50 1.53 × 10−05 
PAPD7 6754867-6757161 5.59 × 10−03 20 27.14 1.99 × 10−05 
PAPD7 6754764-6757161 5.59 × 10−03 17 −28.94 1.99 × 10−05 
KIF24 − 34255733-34257979 12 10 5.60 × 10−03 14 −29.00 2.00 × 10−05 
TMCC3 12 − 94960900-94965513 6.85−03 293 −27.77 2.51 × 10−05 
TMCC3 12 − 94975398-94976314 2.26 × 10−02 100 0.08 1.03 × 10−04 
C10orf18 10 5804992-5806943 15 15 7.20 × 10−03 32 −30.92 2.66 × 10−05 
SMYD5 73452924-73454355 13 13 7.73 × 10−03 24 −29.40 2.87 × 10−05 
EP400 12 132561946-132565004 27 27 8.76 × 10−03 54 17 0.15 3.34 × 10−05 
EP400 12 132445130-132446499 27 8.76 × 10−03 18 −32.52 3.34 × 10−05 
FRY 13 32869339-32870775 57 57 9.80 × 10−03 40 −31.47 3.78 × 10−05 
NPIPL3 16 − 21426278-21426406 9.91 × 10−03 0.19 3.84 × 10−05 
NPIPL3 16 − 21436621-21436658 2.92 × 10−02 −0.42 1.38 × 10−04 
IL1R1 102792813-102796333 10 10 1.06 × 10−02 107 −29.14 4.12 × 10−05 
SLC35E3 12 69158484-69159851 1.09 × 10−02 33 −26.93 4.27 × 10−05 
ELF2 − 140058784-140059021 10 11 1.09 × 10−02 22 −28.48 4.28 × 10−05 
LUZP1 − -23410516-23415546 1.19 × 10−02 36 −26.87 4.70 × 10−05 
MLH3 14 − 75513079-75516421 12 1.41 × 10−02 19 −31.99 5.81 × 10−05 
VCAM1 101203679-101204599 1.50 × 10−02 51 −26.73 6.20 × 10−05 
ADPRH 119306311-119308791 1.67 × 10−02 53 −26.88 7.13 × 10−05 
ALCAM 105085713-105086325 15 16 1.81 × 10−02 34 −27.99 7.88 × 10−05 
CHKB 22 − 51019849-51019982 1.81 × 10−02 20 −28.43 7.89 × 10−05 
PPIP5K1 15 − 43825660-43827674 20 36 36 1.84 × 10−02 24 −27.56 8.10 × 10−05 
CLEC5A − 141627157-141630018 1.87 × 10−02 252 −28.64 8.27 × 10−05 
MICAL3 22 − 18342922-18347752 28 21 1.95 × 10−02 35 25 −0.06 8.81 × 10−05 
LRP12 − 105501459-105503767 2.08 × 10−02 26 −28.17 9.40 × 10−05 
SRGAP2 206634382-206637782 15 21 21 2.13 × 10−02 21 −28.97 9.64 × 10−05 
GGCX − 85776193-85777249 12 15 15 2.32 × 10−02 25 −27.44 1.06 × 10−04 
SLC8A1 − 40339287-40342769 2.49 × 10−02 26 29 −0.34 1.16 × 10−04 
SLC8A1 − 40655613-40657444 2.49 × 10−02 41 −28.24 1.16 × 10−04 
DHRS4 14 24437966-24438486 2.70 × 10−02 22 −26.85 1.26 × 10−04 
VPS13A 80030872-80032397 22 72 72 2.74 × 10−02 14 −31.43 1.28 × 10−04 
HLA-DPB1 33054316-33054976 2.81 × 10−02 151 368 −0.91 1.33 × 10−04 
SKA2 17 − 57232317-57232800 2.81 × 10−02 26 −27.22 1.32 × 10−04 
METTL4 18 − 2537525-2539144 2.95 × 10−02 19 −27.47 1.41 × 10−04 
CRAMP1L 16 1705878-1706993 13 2.96 × 10−02 28.86 1.42 × 10−04 
CRAMP1L 16 1723882-1727907 13 13 2.96 × 10−02 25 −31.99 1.42 × 10−04 
IKBKG 153792534-153793260 10 11 11 3.01 × 10−02 20 −29.87 1.45 × 10−04 
LEPR 65886248-65886423 20 22 3.34 × 10−02 16 −28.60 1.63 × 10−04 
XPNPEP3 22 41322273-41323878 10 10 3.50 × 10−02 24 −28.94 1.72 × 10−04 
CTSH 15 − 79237293-79237420 13 14 4.08 × 10−02 25 −27.42 2.03 × 10−04 
BEST1 11 61731576-61731939 4.40 × 10−02 75 −27.40 2.21 × 10−04 
C11orf48 11 − 62439036-62439241 4.58 × 10−02 16 −28.26 2.34 × 10−04 
Gene Chromosome Strand Exon location No. of exon test No. of exons Exon no. Adjusted P Base M Base N Log2fold (mutant/healthy donor) P 
SIRPB1 20 − 1577987-1578928 11 11 0.00 125 33.46 0.00 
SIRPB1 20 − 1584456-1584788 11 11 1.63 × 10−12 74 32.79 7.77 × 10−16 
SIRPB1 20 − 1585388-1585705 11 11 2.52 × 10−07 47 32.00 2.81 × 10−10 
SIRPB1 20 − 1579468-1579582 11 11 1.44 × 10−05 34 31.72 2.33 × 10−08 
SIRPB1 20 − 1592003-1592359 11 11 2.58 × 10−04 95 32.88 5.54 × 10−07 
AKAP13 15 86287859-86292586 36 38 38 0.00 258 30.91 0.00 
HBG2 11 − 5275867-5276011 0.00 15 658 30.03 0.00 
HBG2 11 − 5274421-5274635 1.64 × 10−05 29 067 344 −0.14 2.73 × 10−08 
PHF3 64421482-64424405 15 15 15 0.00 141 29.99 0.00 
CD37 19 49838677-49838832 0.00 111 29.65 0.00 
NIPAL3 24795476-24799472 12 12 12 0.00 173 29.40 0.00 
VPS36 13 − 52986739-52990058 13 14 14 0.00 222 28.94 0.00 
HELZ 17 − 65066555-65074702 29 33 33 7.76 × 10−13 153 29.12 3.33 × 10−16 
CTTN 11 70281132-70282689 16 19 19 1.51 × 10−12 419 31.66 6.66 × 10−16 
RSAD2 7035909-7038363 2.26 × 10−12 308 27.95 1.11 × 10−15 
RPS17 15 − 83207631-83207736 3.23 × 10−12 220 33.49 1.67 × 10−15 
POU2F2 19 − 42592673-42595743 10 13 13 6.74 × 10−12 280 29.46 3.55 × 10−15 
PARP14 122447155-122449686 17 17 17 2.06 × 10−11 128 29.10 1.11 × 10−14 
RAPGEF6 − 130759616-130762984 29 30 30 2.72 × 10−11 127 30.14 1.50 × 10−14 
CARD8 19 − 48711344-48715232 10 10 10 5.15 × 10−11 115 29.40 3.03 × 10−14 
TSC1 − 135766735-135772141 15 24 24 6.01 × 10−11 164 30.44 3.69 × 10−14 
ATM 11 108236052-108239826 54 64 64 1.13 × 10−10 200 31.49 7.21 × 10−14 
RABL3 − 120405530-120408735 1.58 × 10−10 102 30.15 1.07 × 10−13 
CCDC25 − 27590835-27593762 1.58 × 10−10 108 29.79 1.07 × 10−13 
MEGF9 − 123363091-123367919 2.41 × 10−10 262 31.12 1.68 × 10−13 
IL32 16 3119282-3119667 11 11 3.05 × 10−10 345 31.09 2.21 × 10−13 
IL32 16 3118991-3119667 11 10 1.53 × 10−02 665 85 −0.12 6.40 × 10−05 
DDX54 12 − 113594984-113596914 21 21 21 3.19 × 10−10 90 28.84 2.34 × 10−13 
TTC14 180327418-180328917 13 13 13 4.73 × 10−10 95 28.73 3.54 × 10−13 
ADPGK 15 − 73043710-73045236 6.16 × 10−10 102 32.16 4.69 × 10−13 
ADPGK 15 − 73043710-73045233 2.35 × 10−05 101 98 −0.11 4.10 × 10−08 
EZH1 17 − 40852294-40854610 19 21 21 2.04 × 10−09 131 28.95 1.63 × 10−12 
MADD 11 47350597-47351582 35 38 38 5.39 × 10−09 90 29.52 4.43 × 10−12 
RABL2B 22 − 51205920-51207302 13 13 13 5.49 × 10−09 80 29.36 4.58 × 10−12 
RABL2B 22 − 51207883-51207977 13 13 10 2.93 × 10−02 16 −28.17 1.39 × 10−04 
ZNF638 71649944-71651168 24 28 22 6.95 × 10−09 84 30.73 5.88 × 10−12 
SON 21 34948671-34949812 13 13 13 8.44 × 10−09 76 32.49 7.25 × 10−12 
IDE 10 − 94211441-94214296 24 25 25 9.99 × 10−09 71 29.31 8.69 × 10−12 
MOBKL1A 71847697-71853890 2.45 × 10−08 268 30.54 2.22 × 10−11 
ZNF592 15 85345094-85349661 10 11 11 2.45 × 10−08 81 29.21 2.20 × 10−11 
RPRD1B 20 36718128-36720764 2.62 × 10−08 112 29.42 2.41 × 10−11 
ARHGEF7 13 111955338-111958080 11 23 23 2.99 × 10−08 120 32.24 2.78 × 10−11 
KDM5D − 21867303-21868231 27 28 28 5.42 × 10−08 92 28.78 5.25 × 10−11 
ZNF295 21 − 43406941-43414217 1.51 × 10−07 28 30.45 1.55 × 10−10 
ZNF295 21 − 43406941-43411998 6.31 × 10−06 80 −27.93 9.20 × 10−09 
ARHGAP27 17 − 43471269-43472999 11 17 17 1.51 × 10−07 154 29.46 1.56 × 10−10 
CEP350 180080132-180084014 22 36 36 1.62 × 10−07 102 32.63 1.69 × 10−10 
R3HDM1 136481507-136482836 19 25 25 1.85 × 10−07 66 29.65 1.97 × 10−10 
TRMT5 14 − 61438169-61441912 2.25 × 10−07 180 30.11 2.43 × 10−10 
TRMT5 14 − 61445949-61446604 5.94 × 10−07 25 23 −0.36 6.99 × 10−10 
CA5B 15800608-15805747 2.31 × 10−07 114 30.37 2.52 × 10−10 
KIAA0319L − 35899092-35900682 20 21 21 2.52 × 10−07 73 27.72 2.81 × 10−10 
CD3E 11 118186201-118186889 4.06 × 10−07 238 27.14 4.57 × 10−10 
GTF2E1 120499890-120501915 4.96 × 10−07 138 32.16 5.72 × 10−10 
GTF2E1 120469370-120469847 1.39 × 10−06 11 20 0.11 1.83 × 10−09 
SLC9A8 20 48504366-48508772 16 16 5.36 × 10−07 65 30.44 6.25 × 10−10 
ST6GALNA − 130647601-130649067 7.62 × 10−07 136 28.77 9.06 × 10−10 
FCHSD1 − 141018870-141021130 15 20 20 8.83 × 10−07 94 27.20 1.07 × 10−09 
RUNX3 − 25226003-25229157 1.01 × 10−06 375 30.61 1.26 × 10−09 
CHD9 16 53357932-53361413 15 38 38 1.16 × 10−06 71 32.47 1.47 × 10−09 
ATG10 81272012-81272146 1.39 × 10−06 437 1440 −0.66 1.79 × 10−09 
ATG10 81267844-81268125 3.04 × 10−05 201 155 1.44 5.40 × 10−08 
NAA16 13 41949541-41951166 18 22 22 1.39 × 10−06 55 29.50 1.83 × 10−09 
LPIN1 11964758-11967531 14 20 20 1.98 × 10−06 80 29.54 2.67 × 10−09 
2-Mar 19 8503272-8503897 2.42 × 10−06 126 30.44 3.29 × 10−09 
AFG3L1 16 90061101-90063028 11 15 13 2.43 × 10−06 75 28.76 3.34 × 10−09 
LDLRAP1 25893339-25895375 3.44 × 10−06 127 31.25 4.79 × 10−09 
FRYL − 48499380-48501697 36 63 63 4.11 × 10−06 99 31.64 5.80 × 10−09 
ITK 156679617-156682109 17 17 17 4.46 × 10−06 237 31.53 6.35 × 10−09 
MCTP2 15 95022195-95027180 13 24 24 4.64 × 10−06 55 31.32 6.71 × 10−09 
HDDC3 15 − 91474157-91474786 6.58 × 10−06 80 28.62 9.68 × 10−09 
RAP1GAP2 17 2936686-2941034 25 25 6.98 × 10−06 164 31.13 1.04 × 10−08 
ARFRP1 20 − 62329996-62332054 8.08 × 10−06 85 27.86 1.22 × 10−08 
CHST15 10 − 125767186-125769855 8.68 × 10−06 81 29.59 1.32 × 10−08 
SLC5A6 − 27422457-27423447 19 20 19 1.02 × 10−05 77 30.99 1.58 × 10−08 
SLC5A6 − 27422973-27423447 19 20 18 1.46 × 10−03 54 30.43 4.39 × 10−06 
BTN3A3 26451903-26453642 11 11 1.02 × 10−05 140 29.53 1.58 × 10−08 
GCFC1 21 − 34106213-34107378 13 18 18 1.39 × 10−05 62 29.83 2.19 × 10−08 
MKNK2 19 − 2037470-2037828 13 13 1.39 × 10−05 55 28.44 2.19 × 10−08 
TRIM58 248039202-248043436 1.44 × 10−05 880 31.19 2.34 × 10−08 
TRIM58 248023919-248024014 1.24 × 10−02 10 −0.88 4.92 × 10−05 
OXSR1 38294308-38296978 18 18 1.64 × 10−05 72 32.32 2.70 × 10−08 
POLR2A 17 7416340-7417929 27 29 29 1.64 × 10−05 67 28.77 2.72 × 10−08 
RNF219 13 − 79188421-79191257 1.91 × 10−05 63 27.84 3.23 × 10−08 
GNPTAB 12 − 102139278-102141019 13 20 20 2.00 × 10−05 85 29.10 3.40 × 10−08 
CXorf15 16859551-16862640 10 10 2.29 × 10−05 64 28.14 3.97 × 10−08 
RHCE − 25747130-25747363 10 10 2.46 × 10−05 75 27.80 4.31 × 10−08 
FHDC1 153895827-153900848 11 11 11 4.27 × 10−05 317 31.91 7.79 × 10−08 
CCNT1 12 − 49086752-49088219 4.49 × 10−05 45 30.46 8.25 × 10−08 
CAPRIN2 12 − 30862486-30863404 15 19 19 4.70 × 10−05 73 28.41 8.71 × 10−08 
RXRB − 33161366-33162606 10 10 5.98 × 10−05 64 27.55 1.12 × 10−07 
GIGYF2 233721503-233725285 23 33 33 5.99 × 10−05 43 28.04 1.13 × 10−07 
DNAJB6 157208710-157210132 12 12 12 6.27 × 10−05 165 90 1.87 1.19 × 10−07 
FAM63B 15 59146681-59149732 10 6.56 × 10−05 76 30.03 1.25 × 10−07 
USP37 − 219314974-219319740 10 26 26 7.33 × 10−05 53 30.01 1.41 × 10−07 
EME1 17 48458124-48458820 10 10 8.83 × 10−05 43 30.84 1.71 × 10−07 
SQLE 126010720-126011936 11 11 9.10 × 10−05 32 27.62 1.77 × 10−07 
GATAD2A 19 19616153-19619740 11 11 1.55 × 10−04 104 28.45 3.07 × 10−07 
RFC1 − 39289076-39290464 25 25 25 1.73 × 10−04 42 28.28 3.46 × 10−07 
RORA 15 − 60780485-60789818 10 12 12 2.42 × 10−04 140 28.47 5.02 × 10−07 
SBNO1 12 − 123773657-123780597 28 32 32 2.43 × 10−04 335 128 1.24 5.06 × 10−07 
PSMB9 32821938-32822014 2.46 × 10−04 48 27.86 5.15 × 10−07 
ADRBK2 22 26118256-26125256 16 16 2.52 × 10−04 186 33.06 5.35 × 10−07 
PRDM1 106552700-106553808 2.57 × 10−04 41 18 0.04 5.49 × 10−07 
PRDM1 106554786-106557809 1.50 × 10−02 110 29.90 6.27 × 10−05 
PRDM1 106554786-106557814 1.50 × 10−02 110 29.90 6.27 × 10−05 
PPID − 159630280-159630976 10 10 10 2.63 × 10−04 76 27.35 5.68 × 10−07 
PITPNA 17 − 1421285-1423832 10 10 2.82 × 10−04 55 30.22 6.20 × 10−07 
LHFPL2 − 77781040-77784976 2.82 × 10−04 86 29.15 6.19 × 10−07 
LHFPL2 − 77805607-77806221 2.82 × 10−04 12 −0.29 6.19 × 10−07 
CYTSB 17 20217289-20218065 15 16 16 3.02 × 10−04 56 28.96 6.70 × 10−07 
SNTB1 − 121547986-121551209 3.07 × 10−04 70 27.79 6.88 × 10−07 
TSGA14 − 130036377-130038880 11 11 3.30 × 10−04 49 28.47 7.49 × 10−07 
CCND3 − 42016239-42016610 3.33 × 10−04 43 27.68 7.60 × 10−07 
ENO2 12 7031894-7032859 12 12 3.39 × 10−04 83 29.41 7.82 × 10−07 
FLVCR2 14 76112744-76114512 10 10 3.42 × 10−04 67 27.42 7.98 × 10−07 
PTPRC 198671516-198671659 34 34 3.42 × 10−04 165 29 2.08 8.02 × 10−07 
SMAD4 18 48604626-48611409 12 12 3.77 × 10−04 53 31.76 8.91 × 10−07 
FCRL1 − 157764196-157765960 13 13 4.18 × 10−04 66 29.44 1.00 × 10−06 
C3orf63 − 56654161-56657871 18 26 26 4.22 × 10−04 124 28.62 1.02 × 10−06 
ZNF45 19 − 44416777-44419352 10 10 4.62 × 10−04 55 30.37 1.12 × 10−06 
CREBZF 11 − 85374859-85376182 4.96 × 10−04 67 31.33 1.22 × 10−06 
SLC37A1 21 44000455-44001549 20 20 5.27 × 10−04 35 32.09 1.32 × 10−06 
FAM122B − 133903597-133905384 12 12 5.54 × 10−04 94 29.22 1.39 × 10−06 
TIMM17B − 48755350-48755426 5.79 × 10−04 44 27.31 1.46 × 10−06 
CHD3 17 7814782-7816075 37 37 5.99 × 10−04 51 32.62 1.53 × 10−06 
RNF160 21 − 30300466-30302832 20 30 30 5.99 × 10−04 42 28.92 1.53 × 10−06 
SORT1 − 109852192-109856679 20 20 6.31 × 10−04 108 32.48 1.62 × 10−06 
POM121C − 75046066-75048176 6.33 × 10−04 59 31.72 1.64 × 10−06 
POM121C − 75050792-75052486 6.33 × 10−04 14 −0.29 1.64 × 10−06 
ZBTB40 22852695-22857650 10 18 18 7.60 × 10−04 90 29.12 2.00 × 10−06 
APPL2 12 − 105567075-105568226 21 21 9.03 × 10−04 32 29.36 2.39 × 10−06 
MCART1 − 37877572-37880745 9.16 × 10−04 29 30.07 2.44 × 10−06 
GOPC − 117881435-117884547 9.16 × 10−04 65 27.39 2.45 × 10−06 
PDGFD 11 − 103777915-103780547 9.54 × 10−04 60 29.12 2.60 × 10−06 
CD247 − 167399878-167400983 9.89 × 10−04 361 27.72 2.71 × 10−06 
CA1 − 86262040-86262114 12 12 1.07 × 10−03 58 26.32 2.95 × 10−06 
SYVN1 11 − 64894753-64895960 12 17 17 1.11 × 10−03 42 27.32 3.09 × 10−06 
SP2 17 46005090-46006321 1.11 × 10−03 54 26.89 3.13 × 10−06 
ST8SIA4 − 100142640-100147833 1.11 × 10−03 129 71 1.56 3.13 × 10−06 
ESRRA 11 64083179-64084210 1.12 × 10−03 69 30.36 3.19 × 10−06 
DDX26B 134715453-134716459 17 17 17 1.16 × 10−03 45 26.74 3.32 × 10−06 
RNPEPL1 241517016-241518141 11 11 1.18 × 10−03 64 30.68 3.37 × 10−06 
ZKSCAN5 99128731-99131444 1.26 × 10−03 35 29.99 3.64 × 10−06 
POM121 72416676-72418837 1.33 × 10−03 57 30.76 3.87 × 10−06 
POM121 72412376-72414061 1.33 × 10−03 10 14 −0.22 3.87 × 10−06 
TJAP1 43472499-43474294 10 14 14 1.38 × 10−03 52 28.80 4.07 × 10−06 
TBCCD1 − 186263858-186264744 1.38 × 10−03 47 27.32 4.07 × 10−06 
SHQ1 − 72798430-72799987 10 11 11 1.42 × 10−03 36 27.01 4.23 × 10−06 
CUL4A 13 113917801-113919391 10 18 18 1.44 × 10−03 83 30.07 4.31 × 10−06 
NLRP12 19 − 54310749-54311959 10 11 1.50 × 10−03 32 27.88 4.56 × 10−06 
ATF7IP 12 14650588-14651696 13 15 15 1.50 × 10−03 39 27.30 4.58 × 10−06 
ZNHIT6 − 86115107-86119781 11 11 1.50 × 10−03 47 26.99 4.55 × 10−06 
MSH3 80171570-80172633 23 24 24 1.59 × 10−03 41 28.75 4.85 × 10−06 
KIAA2026 − 5919010-5923309 1.63 × 10−03 67 29.99 5.01 × 10−06 
SH2D1B − 162365056-162367105 1.75 × 10−03 313 26.58 5.39 × 10−06 
SH2D1B − 162381673-162381928 4.00 × 10−02 47 −1.06 1.99 × 10−04 
TGFBR3 − 92145902-92149414 16 17 17 2.06 × 10−03 99 19 0.03 6.44 × 10−06 
ALDOA 16 30066105-30066248 16 16 2.21 × 10−03 26 27.87 6.95 × 10−06 
SEPSECS − 25121628-25125847 10 10 2.43 × 10−03 35 30.43 7.69 × 10−06 
PIK3C2A 11 − 17108126-17111467 32 32 2.51 × 10−03 51 30.24 7.98 × 10−06 
PIK3C2A 11 − 17190224-17191354 32 1.94 × 10−02 18 15 0.17 8.75 × 10−05 
SAPS3 11 68382100-68382799 27 28 28 2.55 × 10−03 54 29.21 8.17 × 10−06 
C11orf49 11 47183015-47183796 12 12 11 2.58 × 10−03 40 30.77 8.28 × 10−06 
TCF3 19 − 1609263-1611368 19 19 2.80 × 10−03 46 29.10 9.02 × 10−06 
RB1CC1 − 53535019-53536419 18 25 25 3.02 × 10−03 79 29.68 9.77 × 10−06 
VPRBP − 51433298-51434555 21 26 26 3.13 × 10−03 35 28.00 1.02 × 10−05 
C17orf68 17 − 8128140-8131637 22 23 23 3.23 × 10−03 46 27.31 1.06 × 10−05 
ZSCAN18 19 − 58601232-58601753 3.26 × 10−03 15 0.32 1.07 × 10−05 
SERINC5 − 79434552-79439633 13 12 3.41 × 10−03 79 30.17 1.13 × 10−05 
LINS1 15 − 101113856-101114446 3.55 × 10−03 44 28.77 1.18 × 10−05 
ATP10D 47593059-47595503 11 22 22 3.57 × 10−03 34 30.41 1.19 × 10−05 
CYHR1 − 145689200-145689995 3.67 × 10−03 43 32.53 1.23 × 10−05 
MINA − 97660661-97664186 10 12 12 4.03 × 10−03 70 27.71 1.37 × 10−05 
ELP3 28047166-28048667 13 15 15 4.03 × 10−03 34 26.78 1.37 × 10−05 
AHNAK 11 − 62283375-62301546 4.04 × 10−03 1458 606 0.31 1.38 × 10−05 
POLH 43581397-43588259 11 11 4.15 × 10−03 60 29.11 1.42 × 10−05 
SCO1 17 − 10583651-10584570 4.28 × 10−03 44 27.43 1.47 × 10−05 
PAPOLG 61024197-61026096 14 22 22 4.37 × 10−03 41 28.47 1.51 × 10−05 
TTYH2 17 72256268-72258155 15 15 4.57 × 10−03 53 29.97 1.59 × 10−05 
KIAA0355 19 34843552-34846470 11 14 14 4.59 × 10−03 73 28.43 1.60 × 10−05 
SH2D1A 123505201-123507008 5.12 × 10−03 88 26.73 1.79 × 10−05 
GRK6 176868738-176869848 12 16 16 5.20 × 10−03 36 29.55 1.83 × 10−05 
KIAA0907 − 155882836-155884111 14 14 5.27 × 10−03 51 26.56 1.86 × 10−05 
DDX19B 16 70363734-70363971 12 12 5.72 × 10−03 27 −28.00 2.05 × 10−05 
DDX19B 16 70367424-70367729 12 12 12 8.84 × 10−03 22 29.10 3.38 × 10−05 
MANEA 96053624-96057326 6.18 × 10−03 46 29.45 2.24 × 10−05 
MANEA 96034278-96034859 6.18 × 10−03 11 0.03 2.24 × 10−05 
COPB1 11 − 14521155-14521404 24 24 6.18 × 10−03 23 27.40 2.22 × 10−05 
ZNF37B 10 − 43008963-43016575 6.49 × 10−03 106 29.56 2.37 × 10−05 
ZNF37B 10 − 43047021-43047262 6.49 × 10−03 0.04 2.37 × 10−05 
FCAR 19 55399374-55400437 6.49 × 10−03 66 540 1.16 2.36 × 10−05 
ZNF498 99226814-99230030 7.64 × 10−03 53 27.07 2.83 × 10−05 
TEP1 14 − 20833827-20836718 46 46 7.91 × 10−03 57 31.96 2.96 × 10−05 
ZDHHC7 16 − 85008067-85010125 7.91 × 10−03 69 30.62 2.96 × 10−05 
MOGS − 74688185-74690139 8.02 × 10−03 63 26.72 3.01 × 10−05 
BCL7B − 72950686-72951720 8.24 × 10−03 63 28.92 3.10 × 10−05 
FAM13A − 89647106-89649810 14 24 24 8.39 × 10−03 96 30.28 3.17 × 10−05 
N4BP2L2 13 − 33016525-33018263 10 14 11 8.39 × 10−03 30 27.06 3.18 × 10−05 
NFAT5 16 69725652-69728142 10 9.53 × 10−03 26 31.02 3.66 × 10−05 
CTNS 17 3563530-3566396 14 13 9.57 × 10−03 67 30.69 3.68 × 10−05 
KIAA1467 12 13233559-13236381 12 12 1.03 × 10−02 72 31.36 4.00 × 10−05 
ZNF333 19 14829040-14831770 12 12 1.12 × 10−02 34 30.32 4.42 × 10−05 
PRKCQ 10 − 6469105-6470324 12 18 18 1.23 × 10−02 51 27.05 4.88 × 10−05 
SLC26A2 149359856-149366959 1.24 × 10−02 31 29.85 4.97 × 10−05 
SLC26A2 149357191-149357914 1.24 × 10−02 19 0.22 4.97 × 10−05 
TMCO7 16 69117388-69119083 16 18 18 1.24 × 10−02 37 26.44 4.94 × 10−05 
PDZD11 − 69506211-69506966 1.24 × 10−02 39 26.32 4.97 × 10−05 
FCGR3B − 161595935-161596192 1.24 × 10−02 74 196 1.06 4.98 × 10−05 
LPAR2 19 − 19734468-19735378 1.36 × 10−02 57 27.47 5.54 × 10−05 
LPAR2 19 − 19737352-19738093 1.36 × 10−02 32 14 0.04 5.54 × 10−05 
NAPEPLD − 102740023-102744001 1.38 × 10−02 14 29.38 5.60 × 10−05 
NAPEPLD − 102740023-102743569 2.30 × 10−02 12 −28.98 1.05 × 10−04 
PIK3CA 178951882-178952495 10 21 21 1.40 × 10−02 33 31.17 5.72 × 10−05 
STAG3L2 − 74306509-74306731 1.41 × 10−02 36 26.66 5.77 × 10−05 
DNAJC16 15894273-15898226 15 15 1.44 × 10−02 79 30.26 5.94 × 10−05 
ZNF227 19 44738855-44741420 1.49 × 10−02 44 27.42 6.17 × 10−05 
NPC1 18 − 21111464-21112248 25 25 25 1.53 × 10−02 36 27.05 6.43 × 10−05 
SHPRH − 146205947-146207923 29 29 1.54 × 10−02 25 29.37 6.51 × 10−05 
YPEL4 11 − 57412561-57413543 1.54 × 10−02 72 28.59 6.50 × 10−05 
MPHOSPH 13 20245974-20247598 14 14 14 1.54 × 10−02 69 27.28 6.49 × 10−05 
GOLM1 − 88641060-88642808 11 11 1.66 × 10−02 54 28.28 7.03 × 10−05 
VPS13B 100887646-100889807 35 56 56 1.66 × 10−02 27 29.68 7.06 × 10−05 
NLRX1 11 119050402-119050997 10 1.68 × 10−02 10 0.05 7.17 × 10−05 
C18orf8 18 21110351-21110576 16 20 19 1.74 × 10−02 32 27.64 7.45 × 10−05 
ZBTB20 − 114056947-114058273 12 12 1.74 × 10−02 41 27.40 7.51 × 10−05 
ZFYVE19 15 41099274-41100066 11 1.76 × 10−02 27 27.83 7.61 × 10−05 
RBM12 20 − 34236888-34243266 1.79 × 10−02 97 28.98 7.76 × 10−05 
RBM12 20 − 34252682-34252838 1.79 × 10−02 34 32 −0.02 7.76 × 10−05 
MEPCE 100031125-100031740 1.83 × 10−02 52 27.62 8.03 × 10−05 
ELMOD3 85617261-85618873 11 17 16 1.85 × 10−02 48 30.27 8.15 × 10−05 
NPLOC4 17 − 79523915-79526442 15 15 1.88 × 10−02 202 32.35 8.38 × 10−05 
NPLOC4 17 − 79555970-79556130 15 10 1.88 × 10−02 0.30 8.38 × 10−05 
GTF3C4 135564268-135565468 1.89 × 10−02 13 28.70 8.45 × 10−05 
GTF3C4 135553364-135555190 1.89 × 10−02 10 −26.38 8.45 × 10−05 
IGHMBP2 11 68707002-68708069 15 15 15 1.94 × 10−02 35 28.12 8.71 × 10−05 
TRIM37 17 − 57075562-57076820 14 26 25 2.28 × 10−02 36 29.83 1.04 × 10−04 
ENG − 130577292-130578332 14 16 15 2.38 × 10−02 30 30.61 1.09 × 10−04 
C14orf45 14 74516405-74516898 12 2.44 × 10−02 30 27.12 1.13 × 10−04 
CCDC91 12 28701996-28703098 11 12 12 2.54 × 10−02 30 28.77 1.18 × 10−04 
ZSCAN21 99661411-99662661 2.92 × 10−02 41 28.95 1.38 × 10−04 
ZSCAN21 99654534-99655028 2.92 × 10−02 0.10 1.38 × 10−04 
RRN3P2 16 29127448-29128036 14 14 3.14 × 10−02 31 28.51 1.52 × 10−04 
DIP2A 21 47987283-47989926 40 40 3.14 × 10−02 116 33.06 1.52 × 10−04 
ZNF611 19 − 53206067-53210117 3.25 × 10−02 140 28.67 1.58 × 10−04 
RASGRP3 33787796-33789797 15 20 20 3.31 × 10−02 36 30.57 1.61 × 10−04 
CHD7 61777575-61779463 32 32 3.37 × 10−02 26 32.90 1.65 × 10−04 
FLCN 17 − 17124486-17124942 15 15 3.59 × 10−02 45 30.05 1.77 × 10−04 
C10orf119 10 − 121588972-121591112 11 11 3.78 × 10−02 35 26.46 1.87 × 10−04 
FIGNL1 − 50511834-50514995 3.80 × 10−02 42 29.09 1.88 × 10−04 
NOL9 − 6581409-6586063 10 10 4.14 × 10−02 169 28.77 2.06 × 10−04 
GLUL − 182360310-182360539 4.40 × 10−02 79 121 1.52 2.20 × 10−04 
TRAF5 211545470-211548284 11 11 4.40 × 10−02 68 30.21 2.21 × 10−04 
DHRS7B 17 21094261-21094835 4.51 × 10−02 35 26.40 2.27 × 10−04 
NLRC3 16 − 3589038-3591915 19 19 4.51 × 10−02 106 31.12 2.28 × 10−04 
NLRC3 16 − 3613010-3614759 19 4.51 × 10−02 31 0.34 2.28 × 10−04 
ABCD1 152990323-152991621 10 4.53 × 10−02 19 27.49 2.30 × 10−04 
GRIPAP1 − 48847338-48847514 23 27 4.56 × 10−02 24 25.88 2.32 × 10−04 
CHAC2 54001279-54002287 4.63 × 10−02 46 27.32 2.37 × 10−04 
SLC33A1 − 155571012-155572167 4.78 × 10−02 48 26.51 2.45 × 10−04 
ASXL1 20 31022235-31027121 13 13 4.88 × 10−02 141 32.33 2.51 × 10−04 
CASC3 17 38296507-38297032 13 14 4.94 × 10−02 28 26.09 2.55 × 10−04 
SEC22A 122990369-122992980 4.98 × 10−02 23 27.20 2.58 × 10−04 
RPL21P28 13 27830573-27830698 0.00 1736 2.38 0.00 
RPL21P28 13 27825693-27825722 0.00 231 202 −5.86 0.00 
HNRNPA1L 13 53201573-53201713 0.00 1.00 0.00 
HNRNPA1L 13 53196124-53196339 0.00 0.10 0.00 
HNRNPA1L 13 53191605-53191817 0.00 10 −1.17 0.00 
HNRNPA1L 13 53216541-53217919 0.00 1743 −34.56 0.00 
TXLNA 32653555-32653725 11 0.00 15 0.76 0.00 
TXLNA 32658258-32658332 11 0.00 0.63 0.00 
TXLNA 32657912-32658031 11 0.00 0.62 0.00 
TXLNA 32655656-32655850 11 0.00 18 0.35 0.00 
TXLNA 32646843-32647178 11 0.00 −0.64 0.00 
TXLNA 32660503-32663885 11 11 0.00 345 −33.27 0.00 
MIER1 67452086-67454302 20 21 21 0.00 447 −31.97 0.00 
MIER1 67450255-67450335 20 21 19 2.43 × 10−10 21 24 0.59 1.73 × 10−13 
MIER1 67450255-67450275 20 21 18 5.31 × 10−08 12 15 0.51 5.08 × 10−11 
MIER1 67447462-67447601 20 21 17 1.44 × 10−05 25 36 0.34 2.30 × 10−08 
MIER1 67411833-67411978 20 21 1.82 × 10−02 29 43 0.23 7.98 × 10−05 
CSF3R − 36931644-36932509 20 20 18 0.00 1003 −32.06 0.00 
CSF3R − 36932831-36932912 20 20 16 1.63 × 10−12 44 206 0.35 7.77 × 10−16 
CSF3R − 36931644-36932428 20 20 19 5.15 × 10−11 174 999 0.05 3.01 × 10−14 
CSF3R − 36933423-36933563 20 20 14 4.53 × 10−06 61 318 0.20 6.50 × 10−09 
CSF3R − 36933159-36933252 20 20 15 5.23 × 10−04 48 244 0.22 1.30 × 10−06 
SLC25A36 140695105-140698785 0.00 241 −30.23 0.00 
SLC25A36 140695102-140698785 4.68 × 10−04 155 242 0.16 1.14 × 10−06 
CDV3 133306003-133309116 0.00 372 283 0.15 0.00 
CDV3 133306740-133309116 0.00 275 −33.17 0.00 
NUFIP2 17 − 27582855-27591609 0.00 196 −30.52 0.00 
NUFIP2 17 − 27613010-27614734 2.14 × 10−09 68 94 0.11 1.73 × 10−12 
H3F3A 226252030-226252180 0.00 822 2293 0.09 0.00 
H3F3A 226250421-226250512 0.00 255 835 −0.15 0.00 
H3F3A 226259052-226259702 0.00 4290 −33.08 0.00 
POU5F1 − 31133704-31134615 0.00 299 −30.99 0.00 
POU5F1 − 31134311-31134615 3.44 × 10−06 335 273 0.09 4.80 × 10−09 
QKI 163984452-163999627 0.00 273 −31.04 0.00 
QKI 163991726-163999627 7.22 × 10−10 171 212 0.06 5.58 × 10−13 
SUMO1 − 203070803-203071526 0.00 328 −30.28 0.00 
SUMO1 − 203070903-203072044 4.59 × 10−02 506 811 0.01 2.34 × 10−04 
C1orf9 172578956-172580971 22 25 25 0.00 200 −29.70 0.00 
LGALS3 14 55612030-55612147 0.00 139 −30.94 0.00 
NCOA1 24991093-24993568 14 23 23 0.00 97 −33.15 0.00 
WDR45 − 48957143-48957492 13 14 1.63 × 10−12 57 −32.27 7.77 × 10−16 
CREM 10 35500584-35501885 15 15 2.43 × 10−12 177 −31.41 1.22 × 10−15 
RAD17 68665124-68665291 20 21 4.15 × 10−11 78 −30.64 2.34 × 10−14 
ATXN7 63985126-63989134 12 12 5.93 × 10−11 77 −31.51 3.56 × 10−14 
GON4L − 155734577-155736500 23 33 22 9.48 × 10−11 84 −32.55 5.93 × 10−14 
PIP4K2C 12 57995709-57997210 13 14 14 1.29 × 10−10 61 −31.00 8.36 × 10−14 
ATP13A3 − 194123405-194126845 18 30 30 2.25 × 10−10 313 −30.91 1.54 × 10−13 
ZNF641 12 − 48733795-48737510 1.70 × 10−09 49 −32.63 1.34 × 10−12 
ZNF641 12 − 48741690-48741898 2.78 × 10−02 14 27.56 1.30 × 10−04 
AMPD3 11 10527255-10529126 15 16 16 1.51 × 10−08 80 −32.15 1.33 × 10−11 
TCEAL4 102841582-102842653 3.54 × 10−08 107 −29.05 3.34 × 10−11 
PRDM2 14104913-14109326 6.57 × 10−08 195 137 0.12 6.52 × 10−11 
PRDM2 14113010-14114573 6.57 × 10−08 59 −31.18 6.52 × 10−11 
LRCH4 − 100171636-10017292 18 18 18 1.36 × 10−07 60 −29.52 1.37 × 10−10 
MAP4 − 47892180-47894529 17 20 19 1.66 × 10−07 53 −30.48 1.75 × 10−10 
MAP4 − 47892180-47894428 17 20 20 1.92 × 10−06 82 29.48 2.55 × 10−09 
MXD3 − 176732501-176734196 4.26 × 10−07 82 −28.74 4.85 × 10−10 
SLC4A1 17 − 42325759-42327906 20 20 20 8.44 × 10−07 3390 1884 −0.83 1.01 × 10−09 
LIPA 10 − 91011464-91011660 12 12 8.84 × 10−07 78 −28.02 1.09 × 10−09 
STRADA 17 − 61780194-61781111 13 15 15 8.84 × 10−07 58 −28.26 1.09 × 10−09 
LSM5 − 32529637-32530475 1.03 × 10−06 72 −29.98 1.31 × 10−09 
ERI2 16 − 20807662-20810389 11 1.96 × 10−06 75 −32.21 2.62 × 10−09 
SC5DL 11 121177766-121184117 7.40 × 10−06 133 −28.25 1.11 × 10−08 
PKN2 89298928-89301937 18 22 22 1.03 × 10−05 55 −29.92 1.60 × 10−08 
CEACAM1 19 − 43011459-43013380 1.59 × 10−05 146 −28.49 2.59 × 10−08 
NF2 22 30090741-30094583 18 18 1.88 × 10−05 162 −33.73 3.16 × 10−08 
NF2 22 30070825-30070930 18 12 3.50 × 10−02 2.02 1.72 × 10−04 
PRKCB 16 24231282-24231930 15 18 18 2.10 × 10−05 39 −34.39 3.60 × 10−08 
CBL 11 119170205-119178858 16 16 2.75 × 10−05 84 −30.64 4.86 × 10−08 
TGFBR2 30732912-30735631 3.34 × 10−05 72 −30.44 6.02 × 10−08 
TGFBR2 30713130-30713929 4.94 × 10−02 14 10 0.21 2.55 × 10−04 
AKAP2 112898407-112900819 3.34 × 10−05 44 80 −0.05 6.06 × 10−08 
AKAP2 112930679-112934789 3.34 × 10−05 71 −28.15 6.06 × 10−08 
UHRF1BP1 34840090-34845289 21 21 4.90 × 10−05 49 −31.33 9.13 × 10−08 
SMCR7 17 18167098-18169093 1.03 × 10−04 23 −29.44 2.02 × 10−07 
SMCR7 17 18167024-18169093 7.14 × 10−03 22 27.99 2.62 × 10−05 
NDUFV1 11 67374323-67374520 11 11 1.60 × 10−04 −28.18 3.19 × 10−07 
BCOR − 39931602-39934433 10 15 1.76 × 10−04 43 −29.87 3.54 × 10−07 
FGD4 12 32793210-32798982 17 17 1.90 × 10−04 90 −31.24 3.85 × 10−07 
SRCAP 16 30748370-30751449 29 29 2.13 × 10−04 36 29.81 4.36 × 10−07 
SRCAP 16 30734905-30736403 29 21 2.13 × 10−04 22 −30.64 4.36 × 10−07 
OAS1 12 113357194-113357711 2.42 × 10−04 29 −29.09 5.02 × 10−07 
ETS2 21 40194598-40196876 2.52 × 10−04 171 −30.13 5.35 × 10−07 
C7orf28A 5965263-5965601 2.98 × 10−04 46 −30.72 6.59 × 10−07 
PIGN 18 − 59711460-59713212 23 31 31 3.07 × 10−04 54 −29.06 6.89 × 10−07 
SELP − 169558090-169558699 15 17 17 3.28 × 10−04 35 −29.54 7.39 × 10−07 
XRCC4 82648950-82649577 10 10 3.38 × 10−04 40 −28.80 7.76 × 10−07 
SIGLEC14 19 − 52149510-52149893 3.40 × 10−04 84 −28.80 7.87 × 10−07 
CDK12 17 37686884-37690799 15 15 3.75 × 10−04 38 −31.39 8.82 × 10−07 
ATF3 212792388-212794114 4.11 × 10−04 73 −27.83 9.78 × 10−07 
MYH11 16 − 15818745-15818849 16 13 4.53 × 10−04 21 −29.20 1.09 × 10−06 
ABR 17 − 906760-909409 22 18 4.96 × 10−04 36 −30.39 1.22 × 10−06 
OLFM4 13 53624104-53626186 5.23 × 10−04 405 −29.11 1.30 × 10−06 
HIVEP1 12120123-12126103 6.98 × 10−04 13 27.93 1.83 × 10−06 
HIVEP1 12163516-12165231 6.98 × 10−04 26 −29.98 1.83 × 10−06 
MIDN 19 1256994-1259139 6.98 × 10−04 239 −31.82 1.82 × 10−06 
CEP63 134204575-134204894 15 19 9.33 × 10−04 38 −29.07 2.51 × 10−06 
RREB1 7248744-7252212 14 14 9.34 × 10−04 69 −31.98 2.52 × 10−06 
NCOA6 20 − 33328167-33331145 10 9.41 × 10−04 27 −30.79 2.55 × 10−06 
NISCH 52525888-52527087 18 21 21 1.02 × 10−03 34 −28.34 2.79 × 10−06 
RNF111 15 59323003-59323901 11 13 1.08 × 10−03 29 −28.45 3.00 × 10−06 
YLPM1 14 75264283-75266400 15 21 1.11 × 10−03 33 −28.11 3.13 × 10−06 
PML 15 74327513-74328734 10 13 10 1.12 × 10−03 39 −30.59 3.18 × 10−06 
SLC41A1 − 205758221-205760846 11 11 1.33 × 10−03 37 −30.47 3.85 × 10−06 
RNF19B − 33402052-33402910 10 11 10 1.40 × 10−03 43 −27.66 4.13 × 10−06 
SLC17A9 20 61598689-61599941 13 13 1.42 × 10−03 53 −29.47 4.21 × 10−06 
RIN3 14 93154271-93155332 1.59 × 10−03 21 37 −0.01 4.88 × 10−06 
RIN3 14 93117927-93119420 3.01 × 10−02 62 −29.47 1.45 × 10−04 
RTN4 − 5525222-55254621 10 1.90 × 10−03 29 −29.39 5.89 × 10−06 
DHX34 19 47856011-47856992 16 17 2.05 × 10−03 42 −29.13 6.37 × 10−06 
TMEM68 − 56651320-56652777 2.24 × 10−03 36 −29.39 7.05 × 10−06 
SSH2 17 − 2795296-27959949 11 11 2.54 × 10−03 101 −29.84 8.10 × 10−06 
FCGR2C 161569420-161570031 3.18 × 10−03 33 39 −0.01 1.04 × 10−05 
SLCO4C1 − 101569694-101572722 13 13 3.48 × 10−03 66 −29.04 1.15 × 10−05 
IL21R 16 27459855-27462115 10 10 3.63 × 10−03 27 −29.51 1.22 × 10−05 
MED6 14 − 71050958-71051660 3.80 × 10−03 39 −27.26 1.28 × 10−05 
STEAP4 − 87905750-87908943 4.35 × 10−03 93 −28.18 1.50 × 10−05 
PRNP 20 4667157-4667382 4.40 × 10−03 40 −27.50 1.53 × 10−05 
PAPD7 6754867-6757161 5.59 × 10−03 20 27.14 1.99 × 10−05 
PAPD7 6754764-6757161 5.59 × 10−03 17 −28.94 1.99 × 10−05 
KIF24 − 34255733-34257979 12 10 5.60 × 10−03 14 −29.00 2.00 × 10−05 
TMCC3 12 − 94960900-94965513 6.85−03 293 −27.77 2.51 × 10−05 
TMCC3 12 − 94975398-94976314 2.26 × 10−02 100 0.08 1.03 × 10−04 
C10orf18 10 5804992-5806943 15 15 7.20 × 10−03 32 −30.92 2.66 × 10−05 
SMYD5 73452924-73454355 13 13 7.73 × 10−03 24 −29.40 2.87 × 10−05 
EP400 12 132561946-132565004 27 27 8.76 × 10−03 54 17 0.15 3.34 × 10−05 
EP400 12 132445130-132446499 27 8.76 × 10−03 18 −32.52 3.34 × 10−05 
FRY 13 32869339-32870775 57 57 9.80 × 10−03 40 −31.47 3.78 × 10−05 
NPIPL3 16 − 21426278-21426406 9.91 × 10−03 0.19 3.84 × 10−05 
NPIPL3 16 − 21436621-21436658 2.92 × 10−02 −0.42 1.38 × 10−04 
IL1R1 102792813-102796333 10 10 1.06 × 10−02 107 −29.14 4.12 × 10−05 
SLC35E3 12 69158484-69159851 1.09 × 10−02 33 −26.93 4.27 × 10−05 
ELF2 − 140058784-140059021 10 11 1.09 × 10−02 22 −28.48 4.28 × 10−05 
LUZP1 − -23410516-23415546 1.19 × 10−02 36 −26.87 4.70 × 10−05 
MLH3 14 − 75513079-75516421 12 1.41 × 10−02 19 −31.99 5.81 × 10−05 
VCAM1 101203679-101204599 1.50 × 10−02 51 −26.73 6.20 × 10−05 
ADPRH 119306311-119308791 1.67 × 10−02 53 −26.88 7.13 × 10−05 
ALCAM 105085713-105086325 15 16 1.81 × 10−02 34 −27.99 7.88 × 10−05 
CHKB 22 − 51019849-51019982 1.81 × 10−02 20 −28.43 7.89 × 10−05 
PPIP5K1 15 − 43825660-43827674 20 36 36 1.84 × 10−02 24 −27.56 8.10 × 10−05 
CLEC5A − 141627157-141630018 1.87 × 10−02 252 −28.64 8.27 × 10−05 
MICAL3 22 − 18342922-18347752 28 21 1.95 × 10−02 35 25 −0.06 8.81 × 10−05 
LRP12 − 105501459-105503767 2.08 × 10−02 26 −28.17 9.40 × 10−05 
SRGAP2 206634382-206637782 15 21 21 2.13 × 10−02 21 −28.97 9.64 × 10−05 
GGCX − 85776193-85777249 12 15 15 2.32 × 10−02 25 −27.44 1.06 × 10−04 
SLC8A1 − 40339287-40342769 2.49 × 10−02 26 29 −0.34 1.16 × 10−04 
SLC8A1 − 40655613-40657444 2.49 × 10−02 41 −28.24 1.16 × 10−04 
DHRS4 14 24437966-24438486 2.70 × 10−02 22 −26.85 1.26 × 10−04 
VPS13A 80030872-80032397 22 72 72 2.74 × 10−02 14 −31.43 1.28 × 10−04 
HLA-DPB1 33054316-33054976 2.81 × 10−02 151 368 −0.91 1.33 × 10−04 
SKA2 17 − 57232317-57232800 2.81 × 10−02 26 −27.22 1.32 × 10−04 
METTL4 18 − 2537525-2539144 2.95 × 10−02 19 −27.47 1.41 × 10−04 
CRAMP1L 16 1705878-1706993 13 2.96 × 10−02 28.86 1.42 × 10−04 
CRAMP1L 16 1723882-1727907 13 13 2.96 × 10−02 25 −31.99 1.42 × 10−04 
IKBKG 153792534-153793260 10 11 11 3.01 × 10−02 20 −29.87 1.45 × 10−04 
LEPR 65886248-65886423 20 22 3.34 × 10−02 16 −28.60 1.63 × 10−04 
XPNPEP3 22 41322273-41323878 10 10 3.50 × 10−02 24 −28.94 1.72 × 10−04 
CTSH 15 − 79237293-79237420 13 14 4.08 × 10−02 25 −27.42 2.03 × 10−04 
BEST1 11 61731576-61731939 4.40 × 10−02 75 −27.40 2.21 × 10−04 
C11orf48 11 − 62439036-62439241 4.58 × 10−02 16 −28.26 2.34 × 10−04 

Differential exons comparison was done testing 81 564 exons from 9069 genes. The table illustrates the complete list of 423 exons from 350 genes found to be differentially used at the 0.05 False Discovery Rate (FDR) level (ie, adjusted P value less than .05) in the SF3B1 mutant relative to normal groups.

Figure 3

RNA-Seq analysis shows differential exon usage of MDS-related genes. (A-D) A total of 81 564 exons were tested in 9069 genes for differential exon usage between normal and mutant groups. A total of 48 835 exons were excluded, being not testable or present in genes with less than 2 testable exons. A total of 423 exons from 350 genes were found to be differentially used at the 0.05 false discovery rate level (ie, adjusted P < .05; Table 2 shows the complete list). A total of 282 of these 423 exons (67%) had higher usage in the mutant group relative to a healthy person. A total of 56 genes contained more than one significant exon. Screenshots of fitted exon counts indicate that 4 genes, specifically ASXL1, CBL, EZH1, and RUNX3, contained at least 1 exon showing differential usage between SF3B1 mutants (red) and normals (blue). Positive (+) and negative (−) signs indicate the strand, giving the 3′ and 5′ ends of the gene. Area in magenta represents the differentially used exon.

Figure 3

RNA-Seq analysis shows differential exon usage of MDS-related genes. (A-D) A total of 81 564 exons were tested in 9069 genes for differential exon usage between normal and mutant groups. A total of 48 835 exons were excluded, being not testable or present in genes with less than 2 testable exons. A total of 423 exons from 350 genes were found to be differentially used at the 0.05 false discovery rate level (ie, adjusted P < .05; Table 2 shows the complete list). A total of 282 of these 423 exons (67%) had higher usage in the mutant group relative to a healthy person. A total of 56 genes contained more than one significant exon. Screenshots of fitted exon counts indicate that 4 genes, specifically ASXL1, CBL, EZH1, and RUNX3, contained at least 1 exon showing differential usage between SF3B1 mutants (red) and normals (blue). Positive (+) and negative (−) signs indicate the strand, giving the 3′ and 5′ ends of the gene. Area in magenta represents the differentially used exon.

Discussion

RS occurs because of abnormal localization of crystalline iron deposits around the nucleus of erythroid precursors. The presence of ≥ 15% RSs of nucleated erythroid precursors in the BM is one of the defined diagnostic criteria of RARS and RARS-T.25  These iron deposits are not confined to RARS and RARS-T, and are present in other diseases, especially congenital sideroblastic anemia. The biologic basis of RS in erythroid precursors is still unknown, even though accumulation of iron has been associated with alterations of the mitochondria and iron overloading. It has been speculated that the accumulated iron in sideroblastic anemia is in the ferric form. Physiologically, the enzymes involved in normal iron exchange within the mitochondria, such as ferrochelatase, only accept ferrous iron. Thus, the change in the biochemical status of iron can lead to an abnormal accumulation of iron and possibly iron overload.26,27 

We demonstrate here the direct consequences of SF3B1 dysfunction on the disease phenotype. Electron microscopy is a sensitive and specific technique for the direct visualization of iron in biologic tissues. Furthermore, the concomitant application of energy dispersive X-ray analysis with electron microscopy permits analysis of iron composition and elemental status, which allows for the accurate detection of abnormal iron distribution and iron composition in various diseases.26  Indeed, RARS patients carrying SF3B1 mutations seemed to present with conspicuous iron particles as evidenced by analysis of the BM ultrastructure. It is possible that SF3B1 mutations might generate alterations in the chemical status of iron leading to a pathologic iron accumulation and other disease-related features. The further analysis of the transcriptome by RNA-Seq tool provided new information about MDS, and specifically RARS pathophysiology. This finding was supported by the RNA signature we defined by RNA-Seq, showing that the expression levels of genes associated with poor prognosis specifically ASXL1 and CBL were not changed. Second, we did not observe any differences in exon usage or gene expression in genes of the mitochondrial pathway purportedly linked in the pathogenesis of RARS, such as ALAS2, ABCB7, SLC25A38, and FTMT. This observation supports the distinct biologic difference between hereditary and acquired sideroblastic anemia, where mutations or defects in these mitochondrial genes are common.

In support of this, none of the known mitochondrial gene sets showed over- or under-representation of significant exon usage in total BM cells derived from SF3B1 mutant patients. Genes of the mitochondrial pathways, such as ALAS2 and ABCB7, have been previously described to be up-regulated and down-regulated, respectively, in RARS and RARS-T compared with healthy persons in CD34+ cells.12,28  Our RNA-Seq analysis was done on unfractionated BM cells because an adequate amount of RNA is required to perform the analysis. New emerging platforms of RNA-Seq are becoming available and may provide us the ability to investigate the RNA splicing patterns in CD34+ cells of RARS and RARS-T patients.

Third, we found that SF3B1 mutant patients showed an over-representation of differentially used exons that were the most 3′, that is, last 5′ to 3′ exon order among exons with any mapped reads (P = 2.10 × 10−7, 445 3′ significant exons of 810 significant exons, with 8668 3′ exons tested), with nonsignificant evidence for over-representation of the most 5′ exons. Finally, it also supports the idea that SF3B1 mutations may be founder mutations that can promote subsequent acquisition of additional genetic or pathway abnormalities, leading to the final disease phenotype. The analysis of RNA derived from clones of K562 cells expressing low level of SF3B1 by RT-PCR indicated that SF3B1 haploinsufficiency mainly affects U2-type introns. When we analyzed the phenotype of K562 with decreased SF3B1 expression by lentiviral infection, we could not recapitulate the RS phenotype. The absence of RS may be attributed to several factors. The K562 cell line is an immortalized myelogenous leukemia cell line derived from a patient with chronic myeloid leukemia in blast crisis. It is possible that the patency of additional genes or downstream pathways is necessary for the final formation of RS in patients with SF3B1 mutations and that these pathways may be altered in K562 cell lines. Thus, other cell lines may be more appropriate models. However, the phenotypic features of RS are confined to erythroid precursors that are not readily seen in other cell lines. It is also possible that more complete knockdown of SF3B1 may be necessary to achieve the RS phenotype. On the other hand, when we used meayamycin, a pharmacologic inhibitor of SF3b factors, we were able to induce the presence of RS in healthy BM cells, supporting the hypothesis that SF3B1 may have relevant function in RS formation. In support to this hypothesis is the finding of RS in the BM of Sf3b1+/− mice. Sf3b1+/− have not been associated with any hematologic phenotype but rather to skeletal abnormalities.14 

The clinical impact of alterations in SF3B1 is most apparent in RARS/ RARS-T patients. However, the clinical heterogeneity of patients with these 2 disease subtypes is common. It is therefore conceivable that biologic and clinical differences between SF3B1 haploinsufficient patients and those with point mutations in SF3B1 may exist. Multiple molecular mutations involving other relevant pathways can occur in the same MDS patient. It is possible that these additional mutations, including those involving the polycomb trithorax complex, signaling pathways, transcription factors, and even other splicing factor genes, may contribute to the variability in the level of expression of SF3B1 within SF3B1 mutant cases and differences in the final disease phenotype and outcome. Similarly, it is also possible that the type of molecular mutations (ie, amino acid substitutions at codons 700, 662, 666, and others) may confer different effects on the final SF3B1 expression. The upstream and downstream genes that modulate SF3B1 expression in MDS and its effect on hematopoiesis are also not completely understood and may contribute to this difference.

We also present updated survival data on the clinical significance of SF3B1 mutations in MDS. So far, data regarding the survival impact of SF3B1 mutations in MDS are conflicting, with some groups demonstrating better survival2,21  and others showing no influence on overall survival.29–31 In our hands,20  RARS and RARS-T patients carrying SF3B1 mutations portend a better clinical outcome similar to the findings of Papaemmanuil et al.2 

Our current study, together with other recent discoveries, reinforces the idea that splicing factors, especially SF3B1, might be key regulators in the pathogenesis of RARS and RARS-T. We previously reported that SF3B1 is, so far, the only SF3b component to be frequently mutated in MDS and related disorders.1  No mutations were detected in other SF3b components and targets, such as SF3B14, SF3B4, and DYRK1A.1,32  In conclusion, SF3B1 is a commonly mutated gene in acquired sideroblastic anemia, such as RARS and RARS-T. We also show the first experimental evidence that haploinsufficiency or inhibition of SF3B1 leads to the formation of RS.

There is an Inside Blood commentary on this article in this issue.

The online version of this article contains a data supplement.

The publication costs of this article were defrayed in part by page charge payment. Therefore, and solely to indicate this fact, this article is hereby marked “advertisement” in accordance with 18 USC section 1734.

Acknowledgments

The authors thank Dr Velizar Shivarov of Laboratory of Hematopathology and Immunology, National Hematology Hospital, Sofia, Bulgaria, for reading the manuscript and providing valuable comments as well as Edy Hasrouni for preparing the figures.

This work was supported in full or in part by the Cleveland Clinic Seed Support, MDS Foundation (YIA grant), AA-MDS International Foundation (YIA grant, R.V.T.), the National Institutes of Health (grant R01 HL082983, J.P.M.; grant U54 RR019391, J.P.M. and M.A.S.; grant K24 HL077522, J.P.M.; grant R01 CA120792, K.K.; and grant RO1 GM093074, R.A.P.), and the Robert Duggan Cancer Research Foundation (charitable donation).

National Institutes of Health

Authorship

Contribution: V.V. and R.V.T. conceived and designed the study, analyzed the data, and wrote the manuscript; H.J.R. analyzed clinical pathology data; J.M. performed transmission electron microscopy; J.B. analyzed RNA-Seq; V.V., J.S., F.T., H.M., H.S., A. Jankowska, and A. Jerez performed experiments; M.B. analyzed data; C.O. edited the manuscript; M.A.S., Y.S., A.S.A., and E.C. contributed to patient samples and edited the manuscript; R.A.P. edited the manuscript; H.K. and K.I. provided BM aspirates of Sf3b1 heterozygous mice; S.O. and K.K. synthesized meayamycin; and J.P.M. provided patient samples, contributed to the design of the project, provided substantial scientific advice, and edited the manuscript.

Conflict-of-interest disclosure: The authors declare no competing financial interests.

Correspondence: Ramon V. Tiu, Department of Translational Hematology and Oncology Research, Taussig Cancer Institute, Cleveland Clinic, 9500 Euclid Ave, R40, Cleveland, OH 44195; e-mail: tiur@ccf.org.

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