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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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PRPF8 mutations and deletions in myeloid malignancies(A) The PRPF8 gene is located on 17p13.3 (red vertical box indicated by an arrow). In this study, 24 patients with 17p deletions were analyzed (green bars). TCGA database contained 12 patients with similar deletions of the 17p region (blue bars). Also shown is the domain structure of the PRPF8 protein including several conserved domains: Reverse Transcriptase (RT fingers/palm), Thumb/X, Endonuclease and RNase H domains. Tumor-associated missense mutations form a variety of tumor types are scattered throughout the protein coding region. Myeloid malignancy missense mutations (shown in black) are located in regions that are conserved from yeast to humans as shown by the sequences of a representative sample of organisms (mutated residues shown in bold, the mutations are listed below the figure). (B) The somatic nature of the mutations was confirmed using either targeted deep sequencing (right) or Sanger sequencing (left) in tumor and CD3+ cells. (C) Expression levels of PRPF8 using RT-PCR (Rq) normalized to normal bone marrow values (NBM). Mutant PRPF8 (blue) and NBM samples (yellow) are in the diploid 17p column (left) and deletion 17p samples (n=6) are in right column (white, p<.001). (D) Disease-associated mutations (black) are mapped onto the yeast Prp8 protein structure.
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Figure 1: PRPF8 mutations and deletions in myeloid malignancies(A) The PRPF8 gene is located on 17p13.3 (red vertical box indicated by an arrow). In this study, 24 patients with 17p deletions were analyzed (green bars). TCGA database contained 12 patients with similar deletions of the 17p region (blue bars). Also shown is the domain structure of the PRPF8 protein including several conserved domains: Reverse Transcriptase (RT fingers/palm), Thumb/X, Endonuclease and RNase H domains. Tumor-associated missense mutations form a variety of tumor types are scattered throughout the protein coding region. Myeloid malignancy missense mutations (shown in black) are located in regions that are conserved from yeast to humans as shown by the sequences of a representative sample of organisms (mutated residues shown in bold, the mutations are listed below the figure). (B) The somatic nature of the mutations was confirmed using either targeted deep sequencing (right) or Sanger sequencing (left) in tumor and CD3+ cells. (C) Expression levels of PRPF8 using RT-PCR (Rq) normalized to normal bone marrow values (NBM). Mutant PRPF8 (blue) and NBM samples (yellow) are in the diploid 17p column (left) and deletion 17p samples (n=6) are in right column (white, p<.001). (D) Disease-associated mutations (black) are mapped onto the yeast Prp8 protein structure.

Mentions: Based on two index cases of patients with PRPF8 mutations previously published,13,16 we screened for lesions affecting this gene in a larger cohort (N=447) of patients with MDS and related conditions (Supplementary Table 1). We found 15 heterozygous mutations in PRPF8 gene: 13 mutations were somatic missense and 2 somatic nonsense mutations. In total, we characterized 15 samples from 13 patients (2 patients had serial samples prior to and after AML progression; Figure 1A, Supplementary Table 1). The most common mutation was in D1598, found in 4 different patient samples. Two additional mutations from TCGA primary acute myeloid leukemia (pAML) database have been added to the analysis (A687P and G1750E, Figure 1A). The positions and identity of PRPF8 mutations identified in myeloid neoplasms are given in Figure 1A along with the distribution of mutations found in other types of tumors (Figure 1A). The somatic nature of PRPF8 mutations found in our patient cohort was confirmed using CD3+ fraction DNA (Figure 1B).


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)

PRPF8 mutations and deletions in myeloid malignancies(A) The PRPF8 gene is located on 17p13.3 (red vertical box indicated by an arrow). In this study, 24 patients with 17p deletions were analyzed (green bars). TCGA database contained 12 patients with similar deletions of the 17p region (blue bars). Also shown is the domain structure of the PRPF8 protein including several conserved domains: Reverse Transcriptase (RT fingers/palm), Thumb/X, Endonuclease and RNase H domains. Tumor-associated missense mutations form a variety of tumor types are scattered throughout the protein coding region. Myeloid malignancy missense mutations (shown in black) are located in regions that are conserved from yeast to humans as shown by the sequences of a representative sample of organisms (mutated residues shown in bold, the mutations are listed below the figure). (B) The somatic nature of the mutations was confirmed using either targeted deep sequencing (right) or Sanger sequencing (left) in tumor and CD3+ cells. (C) Expression levels of PRPF8 using RT-PCR (Rq) normalized to normal bone marrow values (NBM). Mutant PRPF8 (blue) and NBM samples (yellow) are in the diploid 17p column (left) and deletion 17p samples (n=6) are in right column (white, p<.001). (D) Disease-associated mutations (black) are mapped onto the yeast Prp8 protein structure.
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Related In: Results  -  Collection

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Figure 1: PRPF8 mutations and deletions in myeloid malignancies(A) The PRPF8 gene is located on 17p13.3 (red vertical box indicated by an arrow). In this study, 24 patients with 17p deletions were analyzed (green bars). TCGA database contained 12 patients with similar deletions of the 17p region (blue bars). Also shown is the domain structure of the PRPF8 protein including several conserved domains: Reverse Transcriptase (RT fingers/palm), Thumb/X, Endonuclease and RNase H domains. Tumor-associated missense mutations form a variety of tumor types are scattered throughout the protein coding region. Myeloid malignancy missense mutations (shown in black) are located in regions that are conserved from yeast to humans as shown by the sequences of a representative sample of organisms (mutated residues shown in bold, the mutations are listed below the figure). (B) The somatic nature of the mutations was confirmed using either targeted deep sequencing (right) or Sanger sequencing (left) in tumor and CD3+ cells. (C) Expression levels of PRPF8 using RT-PCR (Rq) normalized to normal bone marrow values (NBM). Mutant PRPF8 (blue) and NBM samples (yellow) are in the diploid 17p column (left) and deletion 17p samples (n=6) are in right column (white, p<.001). (D) Disease-associated mutations (black) are mapped onto the yeast Prp8 protein structure.
Mentions: Based on two index cases of patients with PRPF8 mutations previously published,13,16 we screened for lesions affecting this gene in a larger cohort (N=447) of patients with MDS and related conditions (Supplementary Table 1). We found 15 heterozygous mutations in PRPF8 gene: 13 mutations were somatic missense and 2 somatic nonsense mutations. In total, we characterized 15 samples from 13 patients (2 patients had serial samples prior to and after AML progression; Figure 1A, Supplementary Table 1). The most common mutation was in D1598, found in 4 different patient samples. Two additional mutations from TCGA primary acute myeloid leukemia (pAML) database have been added to the analysis (A687P and G1750E, Figure 1A). The positions and identity of PRPF8 mutations identified in myeloid neoplasms are given in Figure 1A along with the distribution of mutations found in other types of tumors (Figure 1A). The somatic nature of PRPF8 mutations found in our patient cohort was confirmed using CD3+ fraction DNA (Figure 1B).

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