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PRPF8 defects cause missplicing in myeloid malignancies.

Kurtovic-Kozaric A, Przychodzen B, Singh J, Konarska MM, Clemente MJ, Otrock ZK, Nakashima M, Hsi ED, Yoshida K, Shiraishi Y, Chiba K, Tanaka H, Miyano S, Ogawa S, Boultwood J, Makishima H, Maciejewski JP, Padgett RA - Leukemia (2014)

Bottom Line: Fifty percent of PRPF8 mutant and del(17p) cases were found in AML and conveyed poor prognosis.Whole-RNA deep sequencing of primary cells from patients with PRPF8 abnormalities demonstrated consistent missplicing defects.In yeast models, homologous mutations introduced into Prp8 abrogated a block experimentally produced in the second step of the RNA splicing process, suggesting that the mutants have defects in proof-reading functions.

View Article: PubMed Central - PubMed

Affiliation: Department of Translational Hematology and Oncology Research, Taussig Cancer Institute, Cleveland, OH, USA.

ABSTRACT
Mutations of spliceosome components are common in myeloid neoplasms. One of the affected genes, PRPF8, encodes the most evolutionarily conserved spliceosomal protein. We identified either recurrent somatic PRPF8 mutations or hemizygous deletions in 15/447 and 24/450 cases, respectively. Fifty percent of PRPF8 mutant and del(17p) cases were found in AML and conveyed poor prognosis. PRPF8 defects correlated with increased myeloblasts and ring sideroblasts in cases without SF3B1 mutations. Knockdown of PRPF8 in K562 and CD34+ primary bone marrow cells increased proliferative capacity. Whole-RNA deep sequencing of primary cells from patients with PRPF8 abnormalities demonstrated consistent missplicing defects. In yeast models, homologous mutations introduced into Prp8 abrogated a block experimentally produced in the second step of the RNA splicing process, suggesting that the mutants have defects in proof-reading functions. In sum, the exploration of clinical and functional consequences suggests that PRPF8 is a novel leukemogenic gene in myeloid neoplasms with a distinct phenotype likely manifested through aberrant splicing.

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Morphological characteristics of PRPF8 mutants and deletions(A) Concomitant mutational spectrum of PRPF8 mutation and deletion cases. Included are other clinical features of the screened cohort (cytogenetics: complex (yes or no); deletion 7q; deletion 5q) and the presence of RS (red), no increase in RS (white) and unknown (grey). Deletion cases are indicated with half-blue rectangles. (B) The cases with PRPF8 mutations and deletions are not concomitant with other spliceosomal mutations except for two cases of SF3B1 and one case of SRSF2 mutations. Deletion cases are indicated with half-blue rectangles. (C) Etiology of PRPF8 mutation (left), deletion (center), and low expression cases (right, Oncomine database), all which are significantly associated with aggressive disease course (secondary AML or complex karyotype). The pAML cases with mutations or deletions are taken from the TCGA. For del(17p) deletion cases, 450 cases were analyzed by SNP-A. (D) Two exemplary mutant PRPF8 cases and two deletion cases confirm a RS phenotype with Prussian blue staining (left panels, blue staining surrounding nuclei). Quantification of RS in each analyzed case is given in Supplementary Table 3. H&E staining (right panels) shows pseudo Pelger-Huet anomaly (bi- or hypo-lobular nuclei), characteristic for 17p deletion cases.
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Figure 2: Morphological characteristics of PRPF8 mutants and deletions(A) Concomitant mutational spectrum of PRPF8 mutation and deletion cases. Included are other clinical features of the screened cohort (cytogenetics: complex (yes or no); deletion 7q; deletion 5q) and the presence of RS (red), no increase in RS (white) and unknown (grey). Deletion cases are indicated with half-blue rectangles. (B) The cases with PRPF8 mutations and deletions are not concomitant with other spliceosomal mutations except for two cases of SF3B1 and one case of SRSF2 mutations. Deletion cases are indicated with half-blue rectangles. (C) Etiology of PRPF8 mutation (left), deletion (center), and low expression cases (right, Oncomine database), all which are significantly associated with aggressive disease course (secondary AML or complex karyotype). The pAML cases with mutations or deletions are taken from the TCGA. For del(17p) deletion cases, 450 cases were analyzed by SNP-A. (D) Two exemplary mutant PRPF8 cases and two deletion cases confirm a RS phenotype with Prussian blue staining (left panels, blue staining surrounding nuclei). Quantification of RS in each analyzed case is given in Supplementary Table 3. H&E staining (right panels) shows pseudo Pelger-Huet anomaly (bi- or hypo-lobular nuclei), characteristic for 17p deletion cases.

Mentions: PRPF8 mutations are generally missense and scattered throughout the gene (Figure 1A,D). The high level of conservation between the yeast Prp8 and human PRPF8 proteins allows for precise mapping of homologous mutations (Figure 1D). In addition, 24 cases in our cohort and 12 cases in the TCGA pAML dataset contained deletions of one copy of the PRPF8 locus (Figure 1A) and exhibited PRPF8 haploinsufficiency (Figure 1C and Supplementary Figure 1A, Supplementary Table 2, Supplementary Table 3). A further analysis of a cohort of 447 cases of MDS and related conditions showed that 20% contain mutations in various spliceosomal protein genes, including SF3B1, SRSF2, U2AF1, ZRSR2 and LUC7L2 (Supplementary Figure 1B). Interestingly, in general, the various spliceosomal factor mutations were mutually exclusive (Figure 2A, B). Other mutations coinciding with PRPF8 such as TET2, CBL and TP53 were identified (Figure 2A).


PRPF8 defects cause missplicing in myeloid malignancies.

Kurtovic-Kozaric A, Przychodzen B, Singh J, Konarska MM, Clemente MJ, Otrock ZK, Nakashima M, Hsi ED, Yoshida K, Shiraishi Y, Chiba K, Tanaka H, Miyano S, Ogawa S, Boultwood J, Makishima H, Maciejewski JP, Padgett RA - Leukemia (2014)

Morphological characteristics of PRPF8 mutants and deletions(A) Concomitant mutational spectrum of PRPF8 mutation and deletion cases. Included are other clinical features of the screened cohort (cytogenetics: complex (yes or no); deletion 7q; deletion 5q) and the presence of RS (red), no increase in RS (white) and unknown (grey). Deletion cases are indicated with half-blue rectangles. (B) The cases with PRPF8 mutations and deletions are not concomitant with other spliceosomal mutations except for two cases of SF3B1 and one case of SRSF2 mutations. Deletion cases are indicated with half-blue rectangles. (C) Etiology of PRPF8 mutation (left), deletion (center), and low expression cases (right, Oncomine database), all which are significantly associated with aggressive disease course (secondary AML or complex karyotype). The pAML cases with mutations or deletions are taken from the TCGA. For del(17p) deletion cases, 450 cases were analyzed by SNP-A. (D) Two exemplary mutant PRPF8 cases and two deletion cases confirm a RS phenotype with Prussian blue staining (left panels, blue staining surrounding nuclei). Quantification of RS in each analyzed case is given in Supplementary Table 3. H&E staining (right panels) shows pseudo Pelger-Huet anomaly (bi- or hypo-lobular nuclei), characteristic for 17p deletion cases.
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Related In: Results  -  Collection

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Figure 2: Morphological characteristics of PRPF8 mutants and deletions(A) Concomitant mutational spectrum of PRPF8 mutation and deletion cases. Included are other clinical features of the screened cohort (cytogenetics: complex (yes or no); deletion 7q; deletion 5q) and the presence of RS (red), no increase in RS (white) and unknown (grey). Deletion cases are indicated with half-blue rectangles. (B) The cases with PRPF8 mutations and deletions are not concomitant with other spliceosomal mutations except for two cases of SF3B1 and one case of SRSF2 mutations. Deletion cases are indicated with half-blue rectangles. (C) Etiology of PRPF8 mutation (left), deletion (center), and low expression cases (right, Oncomine database), all which are significantly associated with aggressive disease course (secondary AML or complex karyotype). The pAML cases with mutations or deletions are taken from the TCGA. For del(17p) deletion cases, 450 cases were analyzed by SNP-A. (D) Two exemplary mutant PRPF8 cases and two deletion cases confirm a RS phenotype with Prussian blue staining (left panels, blue staining surrounding nuclei). Quantification of RS in each analyzed case is given in Supplementary Table 3. H&E staining (right panels) shows pseudo Pelger-Huet anomaly (bi- or hypo-lobular nuclei), characteristic for 17p deletion cases.
Mentions: PRPF8 mutations are generally missense and scattered throughout the gene (Figure 1A,D). The high level of conservation between the yeast Prp8 and human PRPF8 proteins allows for precise mapping of homologous mutations (Figure 1D). In addition, 24 cases in our cohort and 12 cases in the TCGA pAML dataset contained deletions of one copy of the PRPF8 locus (Figure 1A) and exhibited PRPF8 haploinsufficiency (Figure 1C and Supplementary Figure 1A, Supplementary Table 2, Supplementary Table 3). A further analysis of a cohort of 447 cases of MDS and related conditions showed that 20% contain mutations in various spliceosomal protein genes, including SF3B1, SRSF2, U2AF1, ZRSR2 and LUC7L2 (Supplementary Figure 1B). Interestingly, in general, the various spliceosomal factor mutations were mutually exclusive (Figure 2A, B). Other mutations coinciding with PRPF8 such as TET2, CBL and TP53 were identified (Figure 2A).

Bottom Line: Fifty percent of PRPF8 mutant and del(17p) cases were found in AML and conveyed poor prognosis.Whole-RNA deep sequencing of primary cells from patients with PRPF8 abnormalities demonstrated consistent missplicing defects.In yeast models, homologous mutations introduced into Prp8 abrogated a block experimentally produced in the second step of the RNA splicing process, suggesting that the mutants have defects in proof-reading functions.

View Article: PubMed Central - PubMed

Affiliation: Department of Translational Hematology and Oncology Research, Taussig Cancer Institute, Cleveland, OH, USA.

ABSTRACT
Mutations of spliceosome components are common in myeloid neoplasms. One of the affected genes, PRPF8, encodes the most evolutionarily conserved spliceosomal protein. We identified either recurrent somatic PRPF8 mutations or hemizygous deletions in 15/447 and 24/450 cases, respectively. Fifty percent of PRPF8 mutant and del(17p) cases were found in AML and conveyed poor prognosis. PRPF8 defects correlated with increased myeloblasts and ring sideroblasts in cases without SF3B1 mutations. Knockdown of PRPF8 in K562 and CD34+ primary bone marrow cells increased proliferative capacity. Whole-RNA deep sequencing of primary cells from patients with PRPF8 abnormalities demonstrated consistent missplicing defects. In yeast models, homologous mutations introduced into Prp8 abrogated a block experimentally produced in the second step of the RNA splicing process, suggesting that the mutants have defects in proof-reading functions. In sum, the exploration of clinical and functional consequences suggests that PRPF8 is a novel leukemogenic gene in myeloid neoplasms with a distinct phenotype likely manifested through aberrant splicing.

Show MeSH
Related in: MedlinePlus