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.
Department of Advanced Diagnosis, Clinical Research Center, National Hospital Organization Nagoya Medical Center, Nagoya, Japan.|Department of Biochemistry II, Graduate School of Medicine, Nagoya University, Nagoya, Japan.
K
K
Kitamura
Department of Pediatrics, Nagoya University Graduate School of Medicine, Nagoya, Japan.
Y
Y
Okuno
Department of Pathology and Tumor Biology, Graduate School of Medicine, Kyoto University, Kyoto, Japan.
K
K
Yoshida
Department of Advanced Diagnosis, Clinical Research Center, National Hospital Organization Nagoya Medical Center, Nagoya, Japan.
M
M
Sanada
Laboratory of DNA Information Analysis, Human Genome Center, Institute of Medical Science, The University of Tokyo, Tokyo, Japan.
Y
Y
Shiraishi
Department of Pediatrics, Nagoya University Graduate School of Medicine, Nagoya, Japan.
H
H
Muramatsu
Pediatrics, Sapporo Hokuyu Hospital, Sapporo, Japan.
R
R
Kobayashi
Department of Biomedical Sciences, Chubu University College of Life and Health Sciences, Kasugai, Japan.
K
K
Furukawa
Laboratory of DNA Information Analysis, Human Genome Center, Institute of Medical Science, The University of Tokyo, Tokyo, Japan.|Laboratory of Sequence Data Analysis, Human Genome Center, Institute of Medical Science, The University of Tokyo, Tokyo, Japan.
S
S
Miyano
Department of Pediatrics, Nagoya University Graduate School of Medicine, Nagoya, Japan.
S
S
Kojima
Department of Pathology and Tumor Biology, Graduate School of Medicine, Kyoto University, Kyoto, Japan.
S
S
Ogawa
Department of Advanced Diagnosis, Clinical Research Center, National Hospital Organization Nagoya Medical Center, Nagoya, Japan.
S
S
Kunishima
D000293:Adolescent; D000818:Animals; D018952:Antigens, CD34 / Q000097:blood / Q000378:metabolism; D051778:Basic Helix-Loop-Helix Leucine Zipper Transcription Factors / Q000235:genetics / Q000378:metabolism; D001792:Blood Platelets / Q000166:cytology / Q000378:metabolism*; D019070:Cell Lineage; D004907:Erythrocyte Deformability; D004912:Erythrocytes / Q000166:cytology; D059472:Exome; D005260:Female; D005799:Genes, Dominant; D006801:Humans; D008297:Male; D008533:Megakaryocytes / Q000166:cytology*; D051379:Mice; D008856:Microscopy, Fluorescence; D009154:Mutation; D010375:Pedigree; D010976:Platelet Count; D011518:Proto-Oncogene Proteins / Q000235:genetics* / Q000378:metabolism; D012097:Repressor Proteins / Q000235:genetics* / Q000378:metabolism; D017422:Sequence Analysis, DNA; D013921:Thrombocytopenia / Q000151:congenital* / Q000235:genetics*; D019700:Thrombospondin 1 / Q000097:blood / Q000235:genetics / Q000378:metabolism
D018952:Antigens, CD34; D051778:Basic Helix-Loop-Helix Leucine Zipper Transcription Factors; C111977:GFI1B protein, human; C486395:Gfi1b protein, mouse; D011518:Proto-Oncogene Proteins; D012097:Repressor Proteins; C481348:SPZ1 protein, human; D019700:Thrombospondin 1; C585483:Thbs1 protein, mouse
GFI1B protein; blood platelet disorders; human; platelet granule deficiency disorder; thrombocytopenia; transcription factors