SCAPER AND RPRD1A: TWO NOVEL CANDIDATES IN THE PATHOGENESIS OF FAMILIAL PLATELET DISORDER WITH PROPENSITY TO DEVELOP MYELOID MALIGNANCIES (FPD/AML)
(Abstract release date: 05/19/16)
EHA Library. Majidi M. 06/09/16; 132746; E1197
Dr. Majidi Majidi
Contributions
Contributions
Abstract
Abstract: E1197
Type: Eposter Presentation
Background
A patient with acute myeloid leukemia (AML) secondary to MDS and a strong family history of thrombocytopenia and myelodysplastic syndromes was referred to our clinic for evaluation of the possible hereditary nature of myeloid malignancies in the family.
Aims
To identify novel myeloid malignancy-predisposing mutations and contribute to a better understanding of biological processes involved in the development of myelodysplasia, also in cases of sporadic MDS.
Methods
Whole exome sequencing was performed on DNA from peripheral blood of 3 family members with myelodysplasia, one of whom developed AML. Sanger sequencing was used to determine the frequency of the identified mutations in 13 family members as well as 36 sporadic cases of MDS. Corresponding protein expression was evaluated by immunohistochemistry. Structural modelling using Pymol software was performed to estimate the probable impact of mutations on the three-dimensional structure of the corresponding proteins. Using site-directed mutagenesis we have recently created plasmids carrying the identified mutations, which will allow us to further evaluate their functional consequences.
Results
Two confirmed missense mutations were identified by whole exome sequencing. They affect SCAPER (S-phase cyclin A-associated protein in the endoplasmic reticulum) and RPRD1A (regulator of nuclear pre-mRNA domain-containing protein 1A). The SCAPER E342K and RPRD1A I143T mutations were detected by Sanger sequencing in 9 and 10 out of 13 family members, respectively. Molecular modelling showed that both mutations can significantly change the three-dimensional structures of the corresponding proteins. In one of the mutated cases where bone marrow biopsy material was available, both SCAPER and RPRD1A showed high protein expression in the cytoplasm and nucleus. We sequenced the SCAPER and RPRD1A mutation site in 36 patients with sporadic MDS, all of whom showed the wild type sequence. However, when protein expression of SCAPER and RPRD1A was evaluated immunehistochemically on a tissue microarray including core biopsies from 45 patients with MDS, both proteins showed marked heterogeneity of expression level among patients. Lack of RPRD1A expression was associated with significantly shorter overall survival (24 vs. 50 months, p<0.03, HR=2.2), independent of the revised international prognostic scoring system (IPSS). Patients with high expression of SCAPER showed longer median survival than patients with low expression (50 vs. 28 months) but the difference did not reach statistical significance. Interestingly, despite strong interindividual heterogeneity, there was strong intraindividual correlation of protein expression between SCAPER and RPRD1A (r=0.818, p<0.0001).
Conclusion
Molecular modelling convinced us that these two non-conservative mutations cause significant changes in three-dimensional structure of both SCAPER and RPRD1A proteins, which may lead to altered affinity to their main binding partners, i.e. Cyclin A and RNA polymerase 2, respectively. Our findings suggest that SCAPER and RPRD1A should be included among the genes to be analysed in cases of familial thrombocytopenia with propensity for myeloid malignancies. Furthermore, strong heterogeneity of SCAPER and RPRD1A protein expression among patients with sporadic MDS, together with the prognostic impact of protein expression, suggests that both genes may play a role in MDS pathogenesis. Mutation analysis covering the full length of SCAPER and RPRD1A genes, as well as analysis of the epigenetic status of both genes may reveal further insight.
Session topic: E-poster
Keyword(s): Familial, Myelodysplasia, Myeloid malignancies, Thrombocytopenia
Type: Eposter Presentation
Background
A patient with acute myeloid leukemia (AML) secondary to MDS and a strong family history of thrombocytopenia and myelodysplastic syndromes was referred to our clinic for evaluation of the possible hereditary nature of myeloid malignancies in the family.
Aims
To identify novel myeloid malignancy-predisposing mutations and contribute to a better understanding of biological processes involved in the development of myelodysplasia, also in cases of sporadic MDS.
Methods
Whole exome sequencing was performed on DNA from peripheral blood of 3 family members with myelodysplasia, one of whom developed AML. Sanger sequencing was used to determine the frequency of the identified mutations in 13 family members as well as 36 sporadic cases of MDS. Corresponding protein expression was evaluated by immunohistochemistry. Structural modelling using Pymol software was performed to estimate the probable impact of mutations on the three-dimensional structure of the corresponding proteins. Using site-directed mutagenesis we have recently created plasmids carrying the identified mutations, which will allow us to further evaluate their functional consequences.
Results
Two confirmed missense mutations were identified by whole exome sequencing. They affect SCAPER (S-phase cyclin A-associated protein in the endoplasmic reticulum) and RPRD1A (regulator of nuclear pre-mRNA domain-containing protein 1A). The SCAPER E342K and RPRD1A I143T mutations were detected by Sanger sequencing in 9 and 10 out of 13 family members, respectively. Molecular modelling showed that both mutations can significantly change the three-dimensional structures of the corresponding proteins. In one of the mutated cases where bone marrow biopsy material was available, both SCAPER and RPRD1A showed high protein expression in the cytoplasm and nucleus. We sequenced the SCAPER and RPRD1A mutation site in 36 patients with sporadic MDS, all of whom showed the wild type sequence. However, when protein expression of SCAPER and RPRD1A was evaluated immunehistochemically on a tissue microarray including core biopsies from 45 patients with MDS, both proteins showed marked heterogeneity of expression level among patients. Lack of RPRD1A expression was associated with significantly shorter overall survival (24 vs. 50 months, p<0.03, HR=2.2), independent of the revised international prognostic scoring system (IPSS). Patients with high expression of SCAPER showed longer median survival than patients with low expression (50 vs. 28 months) but the difference did not reach statistical significance. Interestingly, despite strong interindividual heterogeneity, there was strong intraindividual correlation of protein expression between SCAPER and RPRD1A (r=0.818, p<0.0001).
Conclusion
Molecular modelling convinced us that these two non-conservative mutations cause significant changes in three-dimensional structure of both SCAPER and RPRD1A proteins, which may lead to altered affinity to their main binding partners, i.e. Cyclin A and RNA polymerase 2, respectively. Our findings suggest that SCAPER and RPRD1A should be included among the genes to be analysed in cases of familial thrombocytopenia with propensity for myeloid malignancies. Furthermore, strong heterogeneity of SCAPER and RPRD1A protein expression among patients with sporadic MDS, together with the prognostic impact of protein expression, suggests that both genes may play a role in MDS pathogenesis. Mutation analysis covering the full length of SCAPER and RPRD1A genes, as well as analysis of the epigenetic status of both genes may reveal further insight.
Session topic: E-poster
Keyword(s): Familial, Myelodysplasia, Myeloid malignancies, Thrombocytopenia
Abstract: E1197
Type: Eposter Presentation
Background
A patient with acute myeloid leukemia (AML) secondary to MDS and a strong family history of thrombocytopenia and myelodysplastic syndromes was referred to our clinic for evaluation of the possible hereditary nature of myeloid malignancies in the family.
Aims
To identify novel myeloid malignancy-predisposing mutations and contribute to a better understanding of biological processes involved in the development of myelodysplasia, also in cases of sporadic MDS.
Methods
Whole exome sequencing was performed on DNA from peripheral blood of 3 family members with myelodysplasia, one of whom developed AML. Sanger sequencing was used to determine the frequency of the identified mutations in 13 family members as well as 36 sporadic cases of MDS. Corresponding protein expression was evaluated by immunohistochemistry. Structural modelling using Pymol software was performed to estimate the probable impact of mutations on the three-dimensional structure of the corresponding proteins. Using site-directed mutagenesis we have recently created plasmids carrying the identified mutations, which will allow us to further evaluate their functional consequences.
Results
Two confirmed missense mutations were identified by whole exome sequencing. They affect SCAPER (S-phase cyclin A-associated protein in the endoplasmic reticulum) and RPRD1A (regulator of nuclear pre-mRNA domain-containing protein 1A). The SCAPER E342K and RPRD1A I143T mutations were detected by Sanger sequencing in 9 and 10 out of 13 family members, respectively. Molecular modelling showed that both mutations can significantly change the three-dimensional structures of the corresponding proteins. In one of the mutated cases where bone marrow biopsy material was available, both SCAPER and RPRD1A showed high protein expression in the cytoplasm and nucleus. We sequenced the SCAPER and RPRD1A mutation site in 36 patients with sporadic MDS, all of whom showed the wild type sequence. However, when protein expression of SCAPER and RPRD1A was evaluated immunehistochemically on a tissue microarray including core biopsies from 45 patients with MDS, both proteins showed marked heterogeneity of expression level among patients. Lack of RPRD1A expression was associated with significantly shorter overall survival (24 vs. 50 months, p<0.03, HR=2.2), independent of the revised international prognostic scoring system (IPSS). Patients with high expression of SCAPER showed longer median survival than patients with low expression (50 vs. 28 months) but the difference did not reach statistical significance. Interestingly, despite strong interindividual heterogeneity, there was strong intraindividual correlation of protein expression between SCAPER and RPRD1A (r=0.818, p<0.0001).
Conclusion
Molecular modelling convinced us that these two non-conservative mutations cause significant changes in three-dimensional structure of both SCAPER and RPRD1A proteins, which may lead to altered affinity to their main binding partners, i.e. Cyclin A and RNA polymerase 2, respectively. Our findings suggest that SCAPER and RPRD1A should be included among the genes to be analysed in cases of familial thrombocytopenia with propensity for myeloid malignancies. Furthermore, strong heterogeneity of SCAPER and RPRD1A protein expression among patients with sporadic MDS, together with the prognostic impact of protein expression, suggests that both genes may play a role in MDS pathogenesis. Mutation analysis covering the full length of SCAPER and RPRD1A genes, as well as analysis of the epigenetic status of both genes may reveal further insight.
Session topic: E-poster
Keyword(s): Familial, Myelodysplasia, Myeloid malignancies, Thrombocytopenia
Type: Eposter Presentation
Background
A patient with acute myeloid leukemia (AML) secondary to MDS and a strong family history of thrombocytopenia and myelodysplastic syndromes was referred to our clinic for evaluation of the possible hereditary nature of myeloid malignancies in the family.
Aims
To identify novel myeloid malignancy-predisposing mutations and contribute to a better understanding of biological processes involved in the development of myelodysplasia, also in cases of sporadic MDS.
Methods
Whole exome sequencing was performed on DNA from peripheral blood of 3 family members with myelodysplasia, one of whom developed AML. Sanger sequencing was used to determine the frequency of the identified mutations in 13 family members as well as 36 sporadic cases of MDS. Corresponding protein expression was evaluated by immunohistochemistry. Structural modelling using Pymol software was performed to estimate the probable impact of mutations on the three-dimensional structure of the corresponding proteins. Using site-directed mutagenesis we have recently created plasmids carrying the identified mutations, which will allow us to further evaluate their functional consequences.
Results
Two confirmed missense mutations were identified by whole exome sequencing. They affect SCAPER (S-phase cyclin A-associated protein in the endoplasmic reticulum) and RPRD1A (regulator of nuclear pre-mRNA domain-containing protein 1A). The SCAPER E342K and RPRD1A I143T mutations were detected by Sanger sequencing in 9 and 10 out of 13 family members, respectively. Molecular modelling showed that both mutations can significantly change the three-dimensional structures of the corresponding proteins. In one of the mutated cases where bone marrow biopsy material was available, both SCAPER and RPRD1A showed high protein expression in the cytoplasm and nucleus. We sequenced the SCAPER and RPRD1A mutation site in 36 patients with sporadic MDS, all of whom showed the wild type sequence. However, when protein expression of SCAPER and RPRD1A was evaluated immunehistochemically on a tissue microarray including core biopsies from 45 patients with MDS, both proteins showed marked heterogeneity of expression level among patients. Lack of RPRD1A expression was associated with significantly shorter overall survival (24 vs. 50 months, p<0.03, HR=2.2), independent of the revised international prognostic scoring system (IPSS). Patients with high expression of SCAPER showed longer median survival than patients with low expression (50 vs. 28 months) but the difference did not reach statistical significance. Interestingly, despite strong interindividual heterogeneity, there was strong intraindividual correlation of protein expression between SCAPER and RPRD1A (r=0.818, p<0.0001).
Conclusion
Molecular modelling convinced us that these two non-conservative mutations cause significant changes in three-dimensional structure of both SCAPER and RPRD1A proteins, which may lead to altered affinity to their main binding partners, i.e. Cyclin A and RNA polymerase 2, respectively. Our findings suggest that SCAPER and RPRD1A should be included among the genes to be analysed in cases of familial thrombocytopenia with propensity for myeloid malignancies. Furthermore, strong heterogeneity of SCAPER and RPRD1A protein expression among patients with sporadic MDS, together with the prognostic impact of protein expression, suggests that both genes may play a role in MDS pathogenesis. Mutation analysis covering the full length of SCAPER and RPRD1A genes, as well as analysis of the epigenetic status of both genes may reveal further insight.
Session topic: E-poster
Keyword(s): Familial, Myelodysplasia, Myeloid malignancies, Thrombocytopenia
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