GFI1B Mutation Research Articles

Comprehensive Sequencing Analysis in Subcutaneous Panniculitis-like T-Cell Lymphoma

Subcutaneous panniculitis-like T-cell lymphoma (SPTL) is a rare subtype of peripheral T-cell lymphomas predominantly affecting younger individuals. Typically, SPTL presents with multifocal subcutaneous nodules, and nearly 20% of the patients have autoimmune disorders, including systemic lupus erythematosus, juvenile rheumatoid arthritis and Sjögren disease. Unlike other lymphomas, which usually require chemotherapy for long-term survival, immunosuppressive therapy alone yields favorable outcomes in SPTL. A previous study showed allelic NAV3 gene aberrations (LOH or deletion by FISH) in 44% of the SPTL samples and comparative genomic hybridization (CGH) revealed numerous DNA copy number changes. In another study, recurrent mutations were identified in various epigenetic modifiers (72%) and components of the PI3K/AKT/mTOR signaling pathway (44%). However, molecular pathogenesis of this disease has not been clearly understood.To elucidate the causative genetic lesions of SPTL, we collected 13 patients from Thailand (n=12) and Japan (n=1). Histopathology was reviewed by hematopathologists showing karyorrhexis of tumor cells infiltrating fat lobules. In all cases, lymphoma cells expressed CD3, CD5, CD8 and TIA1 but no CD56 suggesting the cytotoxic T-cells in origin. Tumor cells expressed T-cell receptor alpha/beta. The monoclonality was confirmed by southern blot (n=12). The mean percentage of SPTL cells in tissue blocks was 38% (range 20-80%). The majority of cases (75%) showed a high proliferative index (Ki-67 expression > 70%).Regarding the clinical data, 10 of 13 patients (80%) were female. The median age at diagnosis was 30 years old. Five cases (38.5%) showed systemic symptoms and 2 cases (20%) were associated with hemophagocytic syndromes. Seven cases received cyclosporine and prednisolone. All showed complete response but 2 cases relapsed. Six cases received anthracycline-based chemotherapy but 3 cases relapsed. All relapses responded to immunosuppressive therapy.We collected paired samples from each of cases (n=13). Tumor samples were collected from Formaldehyde Fixed-Paraffin Embedded (FFPE) tissues whereas germline samples were collected from either bone marrow FFPE or blood which showed no tumor involvement. We performed whole-exome and targeted-capture sequencing in those paired tumor/germline DNA samples by SureSelect v5 kit and custom kit, respectively. To identify somatic mutations and structural variations, sequencing data was analyzed using Genomon (ver. 2.3.0) and Genomon-SV. Copy number was analyzed based on sequencing data as described previously (ASH 2016 abstract by Saeki, et al.).By whole exome sequencing, 149 mutation calls were identified in 8 samples. The somatic mutations and copy number variations were grouped into 4 biological pathways which are commonly affected in cancer.1. Apoptotic associated pathway (FADD , ACIN1 , and PPM1L) : Interestingly, a nonsense mutation of FADD was found and validated. FADD protein is critical for promoting apoptosis of T-cell progenitors at the pre-TCR checkpoint.2. Epigenetic modifier pathway (GFIB , SAB130 and CHD9) : Notably, GFI1B mutation was found to be frameshift deletion causing protein truncation. Gfi1b repressed the Gata3, a transcription factor required for survival of T-lymphocyte progenitors.3. Translation initiation pathway (DHX29 and CTIF) : Missense mutation of DHX29 was identified whereas copy number analysis revealed one case possessed focal amplification of CTIF , a component of the CBP80/CBP20 translation initiation complex and associated with nonsense-mediated decay of mRNA.4. Transcription factors (MEF2B and MGA ): Targeted sequence detected mutations in MEF2B, MGA and PMS2 , which are associated with regulation of BCL6, MYC-MAX pathway and DNA mismatch repair, respectively.At least one mutation involved in either of such 4 pathways was identified in 5/8cases (62.5%). In addition, Sequence based T-cell receptor repertoire analysis proved monoclonal expansion in 5 cases.In conclusion, the identifications of novel genetic alterations provide an insight into the lymphomagenesis of SPTL. Heterogeneous mutations in different candidate pathways suggest complexpathogenesis of this rare tumor. DisclosuresMakishima:Yasuda Medical Foundation: Research Funding.

GFI1B Splice Variants Differentially Regulate Transcription and Megakaryocyte/Erythrocyte Fate in Hematopoiesis

Alternative splicing is a powerful way to expand functional genetic diversity without increasing the number of genes. Recent deep sequencing efforts reveal that 100% of human genes are alternatively spliced. Alternative splicing is enriched in genes that control cell cycle dynamics, transcription and organelle organization during erythrocyte differentiation.Growth factor independence (GFI) 1B is comprised of an N-terminal SNAG domain, a C-terminal concatemer of six C2H2 zinc fingers (ZnFs), and a linker that joins them. GFI1B is required for both definitive erythropoiesis and megakaryopoiesis. Elevated GFI1B expression is seen in AML, while GFI1B mutations cause inherited platelet disorders. Understanding GFI1B function is critical to addressing its contributions to diseases of hematopoiesis. GFI1B has two splice variants, GFI1B-full and GFI1B-short . GFI1B-short lacks ZnFs 1 and 2, but is otherwise structurally identical to GFI1B-full. Both splice variants are expressed during erythroid differentiation from megakaryocyte-erythrocyte progenitors (MEPs), but their distinctive functions and relative contibutions to this binary fate decision remain largely unexplored.We used the bipotent, GFI1B-dependent erythroleukemia cell line, K562, to explore fate specifying and mechanistic differences between GFI1B-full and GFI1B-short. Inducible expression of GFI1B-full in K562 cells promotes spontaneous erythroid differentiation and antagonizes 12-O-Tetradecanoylphorbol-13-acetate (TPA)-induced megakaryocyte differentiation. In contrast, GFI1B-short fails to support spontaneous erythroid differentiation and instead enables megakaryocyte fate in response to TPA. These findings suggest ZnFs 1 and 2 control fate-specifying differences between GFI1B splice variants. We have previously shown the GFI1B paralog, GFI1, is SUMOylated and that a portion of the linker region and ZnFs 1-3 provide binding surfaces for the E3 SUMO ligase, Protein Inhibitor of Activated STAT (PIAS)3. To gain additional insights regarding binding relationships between GFI1B and PIAS family proteins, we mapped the binding interface between GFI1B-full and PIAS3 and investigated binding differences between GFI1B-full vs. GFI1B-short for PIAS family members. We find ZnFs 1-3 of GFI1B are necessary and sufficient for PIAS3 binding and predicted impaired PIAS protein binding by GFI1B-short. While modest impairment was observed between GFI1B-short and PIAS proteins, PIASXa binding was completely abolished. We also find that GFI1B-full is SUMOylated on lysine (K)-61 within a yKXD/E SUMOylation consensus element. Yet, GFI1B-short is not SUMOylated, despite this consensus element being present. This suggests that differential recruitment of the SUMOylation machinery by GFI1 splice variants and assembly of distinct, SUMO-dependent transcriptional regulatory complexes may direct binary cell fate decisions. Using proximity-based in situ biotinylation and mass spectral analysis, we recorded the composition of transcriptional regulatory complexes assembled by GFI1B-full and GFI1B-short. We identifed candidate factors whose recruitment by GFI1B may be SUMOylation-dependent, suggesting that GFI1B splicing variation and differential SUMOylation may direct lineage allocation in hematopoiesis. DisclosuresNo relevant conflicts of interest to declare.

Dominant negative Gfi1b mutations cause moderate thrombocytopenia and an impaired stress thrombopoiesis associated with mild erythropoietic abnormalities in mice.

GFI1B-related thrombocytopenia (GFI1B-RT) is a rare bleeding disorder mainly caused by the presence of truncated GFI1B proteins with dominant-negative properties. The disease is characterized by low platelet counts, the presence of abnormal platelets, a megakaryocytic expansion, and mild erythroid defects. However, there are no animal models that faithfully reproduce the GFI1B-RT phenotype observed in patients. We had previously generated mice with floxed Gfi1b alleles that can be eliminated by Cre recombinase, but those animals developed a much more severe phenotype than GFI1B-RT patients and were of limited interest in assessing the disease. Using CRISPR/Cas9 technology, we have now established three independent mouse lines that carry mutated Gfi1b alleles producing proteins lacking DNA binding zinc fingers and thereby acting in a dominant negative (DN) manner. Mice heterozygous for these Gfi1b-DN alleles show reduced platelet counts and an expansion of megakaryocytes similar to features of human GFI1B-RT but lacking the distinctively large agranular platelets. In addition, Gfi1b-DN mice exhibit an expansion of erythroid precursors indicative of a mildly abnormal erythropoiesis but without noticeable red blood cell defects. When associated with megakaryocyte-specific ablation of the remaining allele, the Gfi1b-DN alleles triggered erythroid-specific deleterious defects. Gfi1b-DN mice also showed a delayed recovery from platelet depletion, indicating a defect in stress thrombopoiesis. However, injecting Gfi1b-DN mice with romiplostim, a thrombopoietin receptor super agonist, increased platelet numbers even beyond normal levels. Thus, our data support a causal link between DN mutations in GFI1B and thrombocytopenia, and suggest that patients with GFI1B-RT could be treated successfully with thrombopoietin agonists.

GFI1B Mutation Associated Alpha-Delta Platelet Storage-Pool Deficiency: A Case Report and Its Potential Important Implication

Background: In addition to NBEAL2, a single dominant-negative mutation in the GFIB1 gene has been associated with grey platelet syndrome but no association has been mentioned with dense granule deficiency. Here we describe a child with thrombocytopenia, alpha-delta granule deficiency, and a homozygous missense mutation in GFIB1.Case Report:The patient is an 8y/o Hispanic male born to non-consanguineous parents. Prenatal and birth history were unremarkable. No family history of blood disorders or pediatric malignancies. The boy has 2 healthy older siblings, no dysmorphic features, and normal skin pigmentation and eye findings. Renal function and hearing are normal. At 3mo he was noted to have multiple spontaneous petechiae along with an isolated thrombocytopenia of 46K/uL. At 10 months, a bone marrow evaluation showed increased megakaryocytes suggestive of ITP. By 3years of age, he received three treatments of IVIG without an adequate response. His platelet counts have generally ranged between 30-50K/uL. With acute illnesses, they drop to 15-20K/uL. His bleeding symptoms have primarily been spontaneous bruising and petechiae as well as prolonged epistaxis. His symptoms have been generally controlled with anti-fibrinolytic agents alone. Platelet transfusions have been reserved for surgical procedures or significant bleeding symptoms.Further evaluations over the past 5 years have included peripheral smears, showing atypical large hypo-granular platelets, and two additional bone marrow aspirates, showing megakaryocytic hyperplasia with numerous osteoclast-like forms and occasional small mono-lobated megakaryocytes and evident emperipolesis. Anti-nuclear antibody testing and platelet direct and indirect antibody testing were negative. Platelet electron microscopy showed that platelets virtually contained no dense granules (0.05 dense granules/platelet, 200 platelets) and about 30-40% of the platelets had markedly decreased alpha granules. Some platelets had complex canalicular networks and membrane vacuoles. These features are consistent with an alpha-delta platelet storage-pool deficiency. A myeloid malignancy mutation panel, performed using next generation sequencing, detected no variants of known significance. Gene sequencing for MHY9-related disorders as well as DNA breakage analysis for Fanconi’s anemia were negative. However, DNA sequencing of the patient’s sample revealed a homozygous missense mutation (c923 T>C; p.Leu308Pro) in the GFIB1gene; each parent carried one copy of this change.Conclusion:GFIB1, mapped to chromosome 9q34.13, encodes a transcriptional repressor that is important for megakaryopoiesis. It has been reported in patients with gray platelet syndrome; an inherited platelet disorder associated with thrombocytopenia and decreased alpha granules. This case points to an association of biallelic GFIB1 mutations with alpha-delta granule deficiency. This case underscores the importance of platelet esoteric testing and molecular analysis in the diagnosis of hereditary platelet disorders. As a targetable mutation in the future, this could revolutionize the early diagnosis and treatment of this rare platelet disorder. DisclosuresNo relevant conflicts of interest to declare.

Functional characterization of a novel GFI1B mutation causing congenital macrothrombocytopenia

Essentials Two groups recently reported GFI1B as a novel causative gene for congenital macrothrombocytopenia. We performed functional analysis of a novel GFI1B mutation and previous mutations. An immunofluorescence analysis of the platelet CD34 expression can be useful as a screening test. Mutant-transduced megakaryocytes produced enlarged proplatelet tips which were reduced in number. Background GFI1B is an essential transcription factor for megakaryocyte and erythrocyte development. Two groups have recently identified GFI1B as a novel causative gene for congenital macrothrombocytopenia associated with α-granule deficiency. Methods We performed whole exome sequencing and identified a novel GFI1B p.G272fsX274 mutation in a family with macrothrombocytopenia, and a decreased number of platelet α-granules and abnormally shaped red blood cells. p.G272fsX274 and the previous two mutations all predicted disruption of an essential DNA-binding domain in GFI1B. We therefore performed functional studies to characterize the biochemical and biological effects of these three patient-derived mutations. Results An immunofluorescence analysis revealed decreased thrombospondin-1 and increased CD34 expression in platelets from our patient. Consistent with the previous studies, the three patient-derived mutants were unable to repress the expression of the reporter gene and had a dominant-negative effect over wild-type GFI1B. In addition, the three mutations abolished recognition of a consensus-binding site in gel shift assays. Furthermore, transduction of mouse fetal liver-derived megakaryocytes with the three GFI1B mutants resulted in the production of abnormally large proplatelet tips, which were reduced in number. Conclusions Our study provides further proof of concept that GFI1B is an essential protein for the normal development of the megakaryocyte lineage.

Functional Characterization of a Novel GFI1B Mutation Causing Congenital Macrothrombocytopenia

▪Congenital macrothrombocytopenia (CMTP) is a heterogeneous group of disorders characterized by abnormally large platelets and low platelet counts. So far, genes responsible for CMTP have been identified in approximately half of the cases. The genes currently known are MYH9, GP1BA, GP1BB, GP9, ITGA2B, ITGB3, ACTN1 and other less frequent genes. However, in the remaining half, CMTP-causing genes are unknown. To identify genes causing CMTP, we first performed whole exome sequencing and target sequencing in 46 Japanese CMTP families and identified 1 family with a heterozygous mutation in GFI1B gene. Several groups have recently reported GFI1B as a causative gene for CMTP with alpha-granule deficiency and red cell anisopoikilocytosis. Here, we report on the identification of a novel mutation in GFI1B gene and its functional characterization.We performed whole exome sequencing and additional targeted sequencing in genetically undefined 46 CMTP families and identified 1 family with GFI1B variant (Figure), which co-segregated with CMTP. The proband (arrow in the Figure) of this family was a 15 years old boy with a history of easy bruising and platelet count of 70-115x109/L. His mother also had low platelet count, although details of her bleeding complications are unknown. Morphologic analysis of peripheral blood smear from the affected family members showed gray appeared enlarged platelets and red cell anisocytosis. Subsequent sequencing analysis confirmed a heterozygous point mutation (c.G814+1C, p.G272fsX274) at the splice donor site flanking exon 6 of GFI1B. GFI1B contains six zinc fingers (Znfs), of which Znf 3 to 5 are essential for DNA binding. The mutated transcript predicted a truncation of 58 C-terminal amino acids, which resulted in complete deletion of Znf 5. Since previous reports on GFI1B mutations have also described mutations of Znf 5 (p.H294fsX307 and p.Q287X mutations by Stevenson et al and Monteferrario et al, respectively), we performed biological analyses of these 3 patient-derived mutations affecting essential DNA-binding domain.GFI1B is an essential transcriptional factor for megakaryocyte and erythroid development. Therefore, we tested the effects of the GFI1b mutations on the transcriptional repression of firefly luciferase reporter constructs containing GFI1B promoter as a validated GFI1B target. Whereas wild-type GFI1B repressed the expression of reporter gene, the G272fsX274, H294fsX307 and Q287X mutants were unable to repress the expression. To test whether these patient-derived mutations acted in dominant-negative manner, we measured the effect of increasing the amount of mutant GFI1B cDNA, cotransfected into cells with a set amount of wild-type GFI1B cDNA. All 3 mutants inhibited gene repression mediated by wild-type GFI1B in a dose-dependent manner.To investigate biological effects of mutant GFI1B on proplatelet formation from megakaryocytes, we expressed the mutant GFI1B in mouse fetal liver cells (FLCs) by retrovirus-mediated gene transfer. FLCs harvested from embryonic day 13.5 embryos were transfected with pGCDNsamIRES/EGFP vector containing wild-type or the 3 patient-derived mutant GFI1B and were cultured with thrombopoietin. And we observed proplatelet formation of EGFP positive megakaryocytes. The number of proplatelet-producing megakaryocytes was reduced in mutant GFI1B transduced FLCs on day 3 and 4 after transfection, suggesting that mutant GFI1B inhibited the rate of proplatelet production from megakaryocytes. Next, we investigated the size and number of proplatelet tips from megakaryocytes, and found that proplatelet tips produced from G272fsX274, H294fsX307 and Q287X-transduced megakaryocytes were significantly reduced in numbers, but were increased in the size of the tips. This was compatible with decreased platelet count and enlarged platelet seen in GFI1B mutated patients. These findings indicated that patient-derived mutant GFI1B disrupted proplatelet formation of megakaryocyte.In summary, we have identified a novel GFI1B mutation that causes autosomal dominant macrothrombocytopenia with gray platelets and red cell anisocytosis. The mutant GFI1B acted in a dominant-negative manner over wild-type GFI1B. In addition, we first showed that the mutant-transduced FLCs produced abnormally enlarged proplatelet tips, which was reduced in numbers. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Next generation sequencing using a candidate gene array for the diagnosis of uncharacterised inherited macrothrombocytopenias

Background Many cases of inherited macrothrombocytopenias (IMT) remain uncharacterised despite complex phenotypic testing. Aims To investigate the diagnostic utility of a novel candidate gene array in cases of uncharacterised macrothrombocytopenia from centres around Australia and New Zealand, and to determine the frequency of mutations involving the transcription factor, GFI1B, in this cohort. Methods A candidate gene array of 19 genes was used and next generation sequencing was performed on 95 patient DNA samples from 14 centres using the MiSeq Illumina sequencer platform. Detected variants were analysed and cross checked using established computer software programs, public genomic databases and bioinformatics tools. Results 92 samples were included in analysis. Pathogenic mutations were detected in 18 individuals (19.6% of the cohort) whilst 35 individuals (38.0%) had mutations of ‘uncertain significance’ and 39 individuals (42.4%) were without identifiable pathogenic mutations. GFI1B mutations were detected in three individuals from two families and RUNX1 mutations were identified in three individuals from three families. Discussion A candidate gene array facilitates the identification of causative genetic defects in a significant portion of patients with uncharacterised disorders, particularly in individuals with a transcription factor defect that is typically difficult to identify with traditional platelet phenotyping techniques.

GFI1B Mutation Causes a Platelet Function Defect With Reduced Alpha-Granule Content and Abnormal Aggregation Response

GFI1B is a transcription factor important for erythropoiesis and megakaryocyte development, but previously unknown to be associated with human disease. We have identified a family with an autosomal dominant bleeding disorder associated with thrombocytopenia, red cell anisopoikilocytosis and platelet dysfunction. The severity of bleeding is variable with some affected individuals experiencing spontaneous bleeding while other family members only exhibit abnormal bleeding with surgery. All affected individuals had a moderate thrombocytopenia ranging from 72-149 x109/L with an elevated mean platelet volume. Platelet function was perturbed with prolonged collagen/epinephrine closure times on the PFA-100 (294 vs 125 sec, P <0.001). Platelet aggregation studies were abnormal, with an absent collagen response and primary aggregation only with ADP, arachidonic acid and adrenaline. Genetic linkage analysis and massively parallel sequencing were performed on 16 family members to localize the mutation causing the disease phenotype to chromosome 9. A single nucleotide insertion was identified in GFI1B that predicts a frameshift mutation in the fifth zinc finger DNA-binding domain. This mutation alters the transcriptional activity of the protein as determined by luciferase production from the TGFBR3 and GFI1B promoters. Aberrant transcriptional activity was associated with a marked reduction in platelet alpha-granule content as measured by electron microscopy (1.3 vs 3.1 alpha-granules per 1µm2, P<0.001) with similar changes in platelet protein expression. Proteomic analysis of platelet lysates demonstrated altered levels of cytoskeletal proteins, including talin, vinculin and myosin light chain 6. GFI1B mutation represents a novel human bleeding disorder and the described phenotype identifies GFI1B as a critical regulator of platelet number and function. Disclosures:No relevant conflicts of interest to declare.

GFI1B mutation causes a bleeding disorder with abnormal platelet function

GFI1B is a transcription factor important for erythropoiesis and megakaryocyte development but previously unknown to be associated with human disease. A family with a novel bleeding disorder was identified and characterized. Genetic linkage analysis and massively parallel sequencing were used to localize the mutation causing the disease phenotype on chromosome 9. Functional studies were then performed in megakaryocytic cell lines to determine the biological effects of the mutant transcript. We have identified a family with an autosomal dominant bleeding disorder associated with macrothrombocytopenia, red cell anisopoikilocytosis, and platelet dysfunction. The severity of bleeding is variable with some affected individuals experiencing spontaneous bleeding while other family members exhibit only abnormal bleeding with surgery. A single nucleotide insertion was identified in GFI1B that predicts a frameshift mutation in the fifth zinc finger DNA-binding domain. This mutation alters the transcriptional activity of the protein, resulting in a reduction in platelet α-granule content and aberrant expression of key platelet proteins. GFI1B mutation represents a novel human bleeding disorder, and the described phenotype identifies GFI1B as a critical regulator of platelet shape, number, and function.

Human promoter mutations unveil Oct-1 and GATA-1 opposite action on Gfi1b regulation

Growth factor-independence 1b (Gfi1b) is a zinc finger transcription factor essential for erythroid and megakaryocytic development. To better understand Gfi1b regulation and to know the implication of the level of expression of this gene in human pathology, we have searched for promoter punctual sequence variations in 214 patients with different hematological diseases. We found two previously unknown congenital mutations at evolutionary conserved GATA and octamer-binding (Oct) transcription factor sites. The Oct site mutation was also found in five relatives of the patient. The GATA motif mutation reduced promoter activity by 50% in vitro, while homozygous patients with the octamer site mutation showed a four-to-five times increase of Gfi1b RNA in platelets. Electrophoretic mobility shift analyses demonstrated that different protein complexes bind to both sites and that binding is reduced by the mutations. Finally, we found that GATA-1 and Oct-1 are the main components of each complex. This study provides evidences of a new mechanism for Gfi1b repression. This is also the first report of Gfi1b mutations with a functional implication; further investigation and follow-up will clarify the involvement of these mutations in hematological disease.

Gfi1b Association to Human Hematological Abnormalities.

Lineage specific transcription factors play essential roles in regulation of hematopoietic development. Transcription factor abnormalities have been frequently described in acute leukemia, mostly through cytogenetic changes. Nevertheless, point mutations can be easily missed. Recently, mutations in the erythroid and megakaryocyte specific transcription factor GATA1 have been discovered in patients with dyserythropoietic anemia and acute megakaryoblastic leukemia (AML-M7) with Down syndrome. Besides GATA-1, located on the X-chromosome, point mutations have been described in biallelic genes. This is the case of AML1 (RUNX1). PU1 and C/EBPalpha also represent examples of transcription factors in which point mutations are found in leukemia.A new zinc finger transcription factor involved in erythropoiesis is Gfi1b. Gfi1b was recently identified by sequence homology with oncogene Gfi1. Gfi1b knockout has demonstrated that this gene is essential for development of both erythroid and megakaryocytic lineages, and in its absence no enucleated erythrocytes are produced.Several Gfi1b DNA and protein targets (GATA1, Gfi1, AML1, p21WAF1, IL-6 Socs1 and Socs2) have been described that might be involved in malignancy. These findings indicate that Gfi1b is at the centre of hematopoiesis and may be a good candidate gene to be involved in hematological abnormalities.We have searched for Gfi1b point mutations in 122 patients with acute leukemia of all FAB types at diagnosis or relapse and 9 cases of congenital dyserythropoietic anemia. We have amplified Gfi1b promoter, coding and non-coding exons (Nucleic Acids Res 2004; 32:3935–46, MN 004188) by high fidelity PCR and screen for point mutations through dHPLC (Wave, Transgenomic) followed by sequencing of the cases with abnormal pattern. SNIPs in the promoter and exons were further confirmed in at least another PCR, cloned in pGEM-T easy vector system (Promega) and sequenced. Alleles with promoter SNIPs were cloned in pGL3-Enhancer vector (Promega), and transiently cotransfected with pEGFP-C2 (Clontech) to K562 cells. Luciferase activity was determined with Dual-Luciferase Reporter Assay (Promega).We found two promoter SNIPs in sequences conserved from chicken to human, one of them affecting a GATA-1 site, reducing promoter in vitro activity by 60 and 50% respectively. We also discovered a congenital exonic SNIP causing a mammalian conserved serine change to leucine. We excluded these to be frequent polymorphisms by dHPLC analysis of 96 blood donors. Although we cannot at present establish a clear relation between the former SNIPS and leukemia, we will discuss the presence of other milder hematological abnormalities. So far this is the first report of Gfi1b mutations that can be related to human pathology.