GMBB
Contributions
Type: Oral Presentation
Presentation during EHA20: From 14.06.2015 08:00 to 14.06.2015 08:15
Location: Room Stolz 2
Background
Diamond-Blackfan anemia (DBA) is a rare, congenital bone marrow failure syndrome with severe anemia, congenital anomalies, predisposition to cancer and variable severity. Approximately 65% of DBA patients have heterozygous autosomal dominant mutations or deletions in one of >15 ribosomal protein (RP) genes encoding both large and small subunit proteins. In addition, recent studies have identified mutations in the GATA1 gene in three DBA families. Despite targeted sequencing of all 80 RP genes, the causal abnormalities in the remaining ~35% of DBA patients are unknown as is the mechanism by which RP defects cause erythroid failure.
Aims
1. To develop an in vitro system to culture erythroid cells from DBA patient peripheral blood CD34+ cells.
2. To use transcriptional profiling of DBA erythroid cells to determine the disrupted pathways.
3. To perform whole exome sequencing on DBA patients without RP mutations to determine the genetic defects.
Methods
We developed a 2-step culture system that expands CD34+ cells (7 days), then induces differentiation of erythroid cells (7 days). This system yields >107 CD235+ erythroid cells from ~104 CD34+ cells collected from 10 ml of healthy control peripheral blood. RNA isolated from CD235+ erythroid cells was profiled on Affymetrix GeneChip Human Gene ST Arrays and by RNASeq. Whole exome sequencing (WES; Illumina HySeq; 80X coverage) was performed on DBA probands, an unaffected sibling and their parents. After filtering out common variants in the 1,000 Genomes and ClinSeq databases we used the VarSifter program to evaluate candidate causal variants for further study.
Results
In contrast to control cells, cells from 9 different DBA patients (5 RP mutations, 3 unknown, 1 with a GATA1 mutation) exhibited a significantly reduced growth rate, generating ~100-fold fewer CD235+ erythroid cells, with an ~2-day delay in the acquisition of the CD235 marker. Using flow cytometry, we isolated populations of CD235+ erythroid cells from both control and patient cell cultures for RNA profiling. RNA from DBA patients showed a distinct transcriptional profile compared to control samples and the sample from the GATA1 patient. Gene ontogeny analysis showed increased expression of genes associated with cellular stress and decreased expressed expression of genes associated with cell cycle progression and protein translation in the RP patients. The GATA1 patient’s profile identified decreased expression of red cell structural genes as well as genes in the heme biosynthesis pathway.
In one DBA family, we identified candidate autosomal recessive mutations in the MCM2 and POLR3B genes. Normal CD34+ progenitor cells expressing shRNAs targeting MCM2 and POLR3B mRNA demonstrated 60-90% knockdown of each. POLR3B knockdown cells showed no inhibition of erythroid differentiation, indicating POLR3B is an unlikely DBA candidate gene. In contrast, knockdown of MCM2 ddemonstrated a significant reduction in BFU-E (but not CFU-GM) colony formation. Similar to DBA patient cells, the proliferation of MCM2 knockdown cells was reduced and the differentiation of CD235+ erythroid cells was delayed. MCM2 knockdown cells were arrested at the G1/S checkpoint in the cell cycle. As confirmation, mice deficient in Mcm2 have a severe bone marrow failure syndrome with erythroid defects.
Summary
Our in vitro differentiation system is a powerful tool to study erythropoiesis in DBA patients using small volumes of peripheral blood. We have used this system to demonstrate that the common forms of DBA, associated with defects in ribosomal genes, have defects in cell cycle and protein translation that differ significantly from the defects observed in the GATA1 DBA patient. Finally, we have shown that mutation in the MCM2 gene is associated with DBA in at least one family, which represents the first autosomal recessive mutation identified in DBA patients.
Keyword(s): Diamond-Blackfan anemia, Erythroid differentiation, Expression profiling
Session topic: Biology and clinics of bone marrow failure syndromes and PNH
Type: Oral Presentation
Presentation during EHA20: From 14.06.2015 08:00 to 14.06.2015 08:15
Location: Room Stolz 2
Background
Diamond-Blackfan anemia (DBA) is a rare, congenital bone marrow failure syndrome with severe anemia, congenital anomalies, predisposition to cancer and variable severity. Approximately 65% of DBA patients have heterozygous autosomal dominant mutations or deletions in one of >15 ribosomal protein (RP) genes encoding both large and small subunit proteins. In addition, recent studies have identified mutations in the GATA1 gene in three DBA families. Despite targeted sequencing of all 80 RP genes, the causal abnormalities in the remaining ~35% of DBA patients are unknown as is the mechanism by which RP defects cause erythroid failure.
Aims
1. To develop an in vitro system to culture erythroid cells from DBA patient peripheral blood CD34+ cells.
2. To use transcriptional profiling of DBA erythroid cells to determine the disrupted pathways.
3. To perform whole exome sequencing on DBA patients without RP mutations to determine the genetic defects.
Methods
We developed a 2-step culture system that expands CD34+ cells (7 days), then induces differentiation of erythroid cells (7 days). This system yields >107 CD235+ erythroid cells from ~104 CD34+ cells collected from 10 ml of healthy control peripheral blood. RNA isolated from CD235+ erythroid cells was profiled on Affymetrix GeneChip Human Gene ST Arrays and by RNASeq. Whole exome sequencing (WES; Illumina HySeq; 80X coverage) was performed on DBA probands, an unaffected sibling and their parents. After filtering out common variants in the 1,000 Genomes and ClinSeq databases we used the VarSifter program to evaluate candidate causal variants for further study.
Results
In contrast to control cells, cells from 9 different DBA patients (5 RP mutations, 3 unknown, 1 with a GATA1 mutation) exhibited a significantly reduced growth rate, generating ~100-fold fewer CD235+ erythroid cells, with an ~2-day delay in the acquisition of the CD235 marker. Using flow cytometry, we isolated populations of CD235+ erythroid cells from both control and patient cell cultures for RNA profiling. RNA from DBA patients showed a distinct transcriptional profile compared to control samples and the sample from the GATA1 patient. Gene ontogeny analysis showed increased expression of genes associated with cellular stress and decreased expressed expression of genes associated with cell cycle progression and protein translation in the RP patients. The GATA1 patient’s profile identified decreased expression of red cell structural genes as well as genes in the heme biosynthesis pathway.
In one DBA family, we identified candidate autosomal recessive mutations in the MCM2 and POLR3B genes. Normal CD34+ progenitor cells expressing shRNAs targeting MCM2 and POLR3B mRNA demonstrated 60-90% knockdown of each. POLR3B knockdown cells showed no inhibition of erythroid differentiation, indicating POLR3B is an unlikely DBA candidate gene. In contrast, knockdown of MCM2 ddemonstrated a significant reduction in BFU-E (but not CFU-GM) colony formation. Similar to DBA patient cells, the proliferation of MCM2 knockdown cells was reduced and the differentiation of CD235+ erythroid cells was delayed. MCM2 knockdown cells were arrested at the G1/S checkpoint in the cell cycle. As confirmation, mice deficient in Mcm2 have a severe bone marrow failure syndrome with erythroid defects.
Summary
Our in vitro differentiation system is a powerful tool to study erythropoiesis in DBA patients using small volumes of peripheral blood. We have used this system to demonstrate that the common forms of DBA, associated with defects in ribosomal genes, have defects in cell cycle and protein translation that differ significantly from the defects observed in the GATA1 DBA patient. Finally, we have shown that mutation in the MCM2 gene is associated with DBA in at least one family, which represents the first autosomal recessive mutation identified in DBA patients.
Keyword(s): Diamond-Blackfan anemia, Erythroid differentiation, Expression profiling
Session topic: Biology and clinics of bone marrow failure syndromes and PNH