American Society of Hematology

Figure 1

$Figure 1. Somatic spliceosomal gene (U2AF1, SF3B1, SRSF2, LUC7L2, PRPF8, and ZRSR2) mutations as detected by next-generation sequencing (NGS) and Sanger sequencing technologies. (A) With the use of an NGS-based whole exome sequencing analysis of whole BM DNA from a patient with refractory cytopenia with unilineage dysplasia (left), a mutation of U2AF1 (21q22.3) at position 44 514 777 (T > C) was detected in 13 of 18 reads. Analysis of DNA from CD3+ cells showed a much lower frequency of the base change (2 of 15 reads; right), highlighting the somatic nature of this alteration. The finding was confirmed by Sanger sequencing. Arrows and bars indicate the specific nucleotide and predicted codon, respectively. It should be noted that U2AF1 is expressed from the minus strand; therefore, the NGS presentation (top panels) is complementally reversed in comparison to the Sanger sequencing results (middle panels). This heterozygous somatic mutation results in the predicted nucleotide change 470 A > G in exon 6 of the coding region, which lead to the amino acid change Q157R in the second zinc finger domain. In the entire cohort, 27 mutations were observed in 26 patients, including a whole gene deletion. All 26 missense mutations were located in 1 of the 2 zinc finger domains (ZNFs); 2 residues, S34 or Q157, were frequently affected (bottom panels). RRM indicates RNA recognition motif. (B) With the use of an NGS analysis of a patient with CMML (middle left), a mutation of SF3B1 (2q33.1) at position 198 267 491(C > G) was detected in 9 of 24 reads. The somatic nature of this alteration was confirmed by an analogous analysis of the CD3+ fraction, with the change being less frequent (2 of 23; middle right). The mutation was confirmed by Sanger sequencing (bottom). This heterozygous somatic mutation results in the nucleotide change 1866 G > T in exon 14 of SF3B1, resulting in the amino acid change E622D in the HSH155 domain. Analysis of the entire cohort identified mutations in 33 patients, including a case with a whole gene deletion. (C) Further screening by NGS led to the detection of a nonsense mutation (R27X) in LUC7L2 (7q34; top) which participates in the recognition of splice donor sites in association with the U1 snRNP spliceosomal subunit, and a missense mutation (M1307I) in PRPF8 (17p13.3; bottom) which is a large U5 snRNP-specific protein essential for pre-mRNA splicing. RS indicates serine/arginine-rich domain; U5 2-snRNA bdg, U5-snRNA binding site 2; and MPN, Mpr1p, Pad1p N-terminal domain. (D) Mutations of SRSF2, an arginine/serine-rich splicing factor, were detected in 29 cases among the entire cohort, including 2 whole gene deletions and a microdeletion within the gene (top). All mutations were heterozygous and affected P95. The somatic nature of the P95R mutation was confirmed with whole BM and T-cell rich fraction DNAs (bottom). (E) A nonsense mutation (W153X) was found in ZRSR2, another arginine/serine-rich splicing regulatory factor, in a case of CMML. ZRSR2 is located at Xp22.2, and the nonsense mutation was hemizygous in this male case (BM).$

Somatic spliceosomal gene (U2AF1, SF3B1, SRSF2, LUC7L2, PRPF8, and ZRSR2) mutations as detected by next-generation sequencing (NGS) and Sanger sequencing technologies. (A) With the use of an NGS-based whole exome sequencing analysis of whole BM DNA from a patient with refractory cytopenia with unilineage dysplasia (left), a mutation of U2AF1 (21q22.3) at position 44 514 777 (T > C) was detected in 13 of 18 reads. Analysis of DNA from CD3⁺ cells showed a much lower frequency of the base change (2 of 15 reads; right), highlighting the somatic nature of this alteration. The finding was confirmed by Sanger sequencing. Arrows and bars indicate the specific nucleotide and predicted codon, respectively. It should be noted that U2AF1 is expressed from the minus strand; therefore, the NGS presentation (top panels) is complementally reversed in comparison to the Sanger sequencing results (middle panels). This heterozygous somatic mutation results in the predicted nucleotide change 470 A > G in exon 6 of the coding region, which lead to the amino acid change Q157R in the second zinc finger domain. In the entire cohort, 27 mutations were observed in 26 patients, including a whole gene deletion. All 26 missense mutations were located in 1 of the 2 zinc finger domains (ZNFs); 2 residues, S34 or Q157, were frequently affected (bottom panels). RRM indicates RNA recognition motif. (B) With the use of an NGS analysis of a patient with CMML (middle left), a mutation of SF3B1 (2q33.1) at position 198 267 491(C > G) was detected in 9 of 24 reads. The somatic nature of this alteration was confirmed by an analogous analysis of the CD3⁺ fraction, with the change being less frequent (2 of 23; middle right). The mutation was confirmed by Sanger sequencing (bottom). This heterozygous somatic mutation results in the nucleotide change 1866 G > T in exon 14 of SF3B1, resulting in the amino acid change E622D in the HSH155 domain. Analysis of the entire cohort identified mutations in 33 patients, including a case with a whole gene deletion. (C) Further screening by NGS led to the detection of a nonsense mutation (R27X) in LUC7L2 (7q34; top) which participates in the recognition of splice donor sites in association with the U1 snRNP spliceosomal subunit, and a missense mutation (M1307I) in PRPF8 (17p13.3; bottom) which is a large U5 snRNP-specific protein essential for pre-mRNA splicing. RS indicates serine/arginine-rich domain; U5 2-snRNA bdg, U5-snRNA binding site 2; and MPN, Mpr1p, Pad1p N-terminal domain. (D) Mutations of SRSF2, an arginine/serine-rich splicing factor, were detected in 29 cases among the entire cohort, including 2 whole gene deletions and a microdeletion within the gene (top). All mutations were heterozygous and affected P95. The somatic nature of the P95R mutation was confirmed with whole BM and T-cell rich fraction DNAs (bottom). (E) A nonsense mutation (W153X) was found in ZRSR2, another arginine/serine-rich splicing regulatory factor, in a case of CMML. ZRSR2 is located at Xp22.2, and the nonsense mutation was hemizygous in this male case (BM).

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