Molecular Medicine and Medical Biotechnologies, University 'Federico II', Naples
Contributions
Type: Oral Presentation + travel grant
Presentation during EHA20: From 13.06.2015 16:45 to 13.06.2015 17:00
Location: Room Stolz 2
Background
Mutations in SEC23B gene cause the congenital dyserythropoietic anemia II (CDA II), an autosomal recessive disorder with ineffective erythropoiesis. Most of patients show biallelic mutations, according to the pattern of autosomal recessive inheritance. However, the 14% of the cases exhibit an incomplete pattern of inheritance, with a monoallelic SEC23B mutation. Mutations in deep regulatory regions of the SEC23B gene as well as in CDA-related loci could be hypothesized as further pathogenetic mechanisms of CDA II.
In SEC23B monoallelic patients we postulated the occurrence of a second mutation in GATA1 gene, assuming that it could be involved in the regulation of SEC23B expression. GATA1 transcription factor is a key regulator of the erythro- and thrombopoiesis. Indeed, mutations in this gene have been already associated to specific CDA variants, as X-linked dyserythropoietic anemia and thrombocytopenia (XLTDA).
Aims
Our aims are: (i) to perform a mutational screening of GATA1 genomic sequence in SEC23B monoallelic patients, as well as in those without SEC23B mutations; (ii) to characterize the promoter region of SEC23B and (iii) to analyze GATA1-mediated regulation of SEC23B expression.
Methods
Genomic mutational screening, gene and protein expression analyses were performed as described (Russo et al, 2014; Russo et al, 2013). In order to characterize SEC23B promoter region, 10 overlapping fragments covering a 3500 bp upstream region of the gene were cloned upstream the luciferase gene into PGL3 vector. Putative binding sites for GATA1 (GATA1bs) in the promoter sequence of SEC23B were predicted by MatInspector and PROMO web server tools. GATA1 cDNA was cloned into the expression vector pcDNA3.1. All mutants were obtained by site-direct mutagenesis. Direct binding of GATA1 to the GATA1bs was assayed by chromatin immunoprecipitation (ChIP) in both K562 and HEL cells at 6 days of erythroid differentiation.
Results
In our cohort we found one XLTDA patient with the hemizygous mutation GATA1-Gly208Arg. Two SEC23B monoallelic patients showed the single nucleotide variant rs113966884 G/A in GATA1 5’upstream region. Both GATA1 variants resulted in a reduced gene expression, which in turn correlates with a decrease of SEC23B expression. By luciferase assay of the deletion mutants we identified the promoter region of the SEC23B gene (HuSEC23B/3.44). Ten putative GATA1bs were predicted. However, direct GATA1 binding were confirmed for only two sites, GATA1bs/HuSEC23B-2475 and -463. The co-transfection of GATA1bs/HuSEC23B-2475 and -463 mutants and GATA1 WT showed a reduction of 30-40% luciferase activity respectively, compared to WT sequence.
Finally, we analyzed the GATA1-mediated regulation of SEC23B expression by co-transfection of several GATA1 mutants (GATA1/G208R, GATA1/R216W, GATA1/D218G, and GATA1/V205M) and the HuSEC23B/3.44 in HEK-293 cell line. We demonstrated a marked reduction of HuSEC23B/3.44 luciferase activity induced by the mutants G208R and R216W, which are the causative variants related to XLTDA and congenital erythropoietic porphyria (CEP), respectively.
Summary
This study provided new insights into the molecular mechanisms of SEC23B regulation. We also suggested an explanation of the variability of phenotypes GATA1-related by means of the crosstalk of this gene with SEC23B.
Moreover, the identification of transcriptional regulatory elements in SEC23B promoter could allow the definitive diagnosis of CDAII patients with peculiar clinical phenotypes or those with incomplete mutation pattern in SEC23B.
Keyword(s): Anemia, GATA-1, Mutation analysis, Transcriptional regulation
Session topic: Iron clinical and biology
Type: Oral Presentation + travel grant
Presentation during EHA20: From 13.06.2015 16:45 to 13.06.2015 17:00
Location: Room Stolz 2
Background
Mutations in SEC23B gene cause the congenital dyserythropoietic anemia II (CDA II), an autosomal recessive disorder with ineffective erythropoiesis. Most of patients show biallelic mutations, according to the pattern of autosomal recessive inheritance. However, the 14% of the cases exhibit an incomplete pattern of inheritance, with a monoallelic SEC23B mutation. Mutations in deep regulatory regions of the SEC23B gene as well as in CDA-related loci could be hypothesized as further pathogenetic mechanisms of CDA II.
In SEC23B monoallelic patients we postulated the occurrence of a second mutation in GATA1 gene, assuming that it could be involved in the regulation of SEC23B expression. GATA1 transcription factor is a key regulator of the erythro- and thrombopoiesis. Indeed, mutations in this gene have been already associated to specific CDA variants, as X-linked dyserythropoietic anemia and thrombocytopenia (XLTDA).
Aims
Our aims are: (i) to perform a mutational screening of GATA1 genomic sequence in SEC23B monoallelic patients, as well as in those without SEC23B mutations; (ii) to characterize the promoter region of SEC23B and (iii) to analyze GATA1-mediated regulation of SEC23B expression.
Methods
Genomic mutational screening, gene and protein expression analyses were performed as described (Russo et al, 2014; Russo et al, 2013). In order to characterize SEC23B promoter region, 10 overlapping fragments covering a 3500 bp upstream region of the gene were cloned upstream the luciferase gene into PGL3 vector. Putative binding sites for GATA1 (GATA1bs) in the promoter sequence of SEC23B were predicted by MatInspector and PROMO web server tools. GATA1 cDNA was cloned into the expression vector pcDNA3.1. All mutants were obtained by site-direct mutagenesis. Direct binding of GATA1 to the GATA1bs was assayed by chromatin immunoprecipitation (ChIP) in both K562 and HEL cells at 6 days of erythroid differentiation.
Results
In our cohort we found one XLTDA patient with the hemizygous mutation GATA1-Gly208Arg. Two SEC23B monoallelic patients showed the single nucleotide variant rs113966884 G/A in GATA1 5’upstream region. Both GATA1 variants resulted in a reduced gene expression, which in turn correlates with a decrease of SEC23B expression. By luciferase assay of the deletion mutants we identified the promoter region of the SEC23B gene (HuSEC23B/3.44). Ten putative GATA1bs were predicted. However, direct GATA1 binding were confirmed for only two sites, GATA1bs/HuSEC23B-2475 and -463. The co-transfection of GATA1bs/HuSEC23B-2475 and -463 mutants and GATA1 WT showed a reduction of 30-40% luciferase activity respectively, compared to WT sequence.
Finally, we analyzed the GATA1-mediated regulation of SEC23B expression by co-transfection of several GATA1 mutants (GATA1/G208R, GATA1/R216W, GATA1/D218G, and GATA1/V205M) and the HuSEC23B/3.44 in HEK-293 cell line. We demonstrated a marked reduction of HuSEC23B/3.44 luciferase activity induced by the mutants G208R and R216W, which are the causative variants related to XLTDA and congenital erythropoietic porphyria (CEP), respectively.
Summary
This study provided new insights into the molecular mechanisms of SEC23B regulation. We also suggested an explanation of the variability of phenotypes GATA1-related by means of the crosstalk of this gene with SEC23B.
Moreover, the identification of transcriptional regulatory elements in SEC23B promoter could allow the definitive diagnosis of CDAII patients with peculiar clinical phenotypes or those with incomplete mutation pattern in SEC23B.
Keyword(s): Anemia, GATA-1, Mutation analysis, Transcriptional regulation
Session topic: Iron clinical and biology