De Novo Mutations Activating Germline TP53 in an Inherited Bone-Marrow-Failure Syndrome Tsutomu Toki, Kenichi Yoshida, RuNan Wang, Sou Nakamura, Takanobu.

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De Novo Mutations Activating Germline TP53 in an Inherited Bone-Marrow-Failure Syndrome Tsutomu Toki, Kenichi Yoshida, RuNan Wang, Sou Nakamura, Takanobu Maekawa, Kumiko Goi, Megumi C. Katoh, Seiya Mizuno, Fumihiro Sugiyama, Rika Kanezaki, Tamayo Uechi, Yukari Nakajima, Yusuke Sato, Yusuke Okuno, Aiko Sato-Otsubo, Yusuke Shiozawa, Keisuke Kataoka, Yuichi Shiraishi, Masashi Sanada, Kenichi Chiba, Hiroko Tanaka, Kiminori Terui, Tomohiko Sato, Takuya Kamio, Hirotoshi Sakaguchi, Shouichi Ohga, Madoka Kuramitsu, Isao Hamaguchi, Akira Ohara, Hitoshi Kanno, Satoru Miyano, Seiji Kojima, Akira Ishiguro, Kanji Sugita, Naoya Kenmochi, Satoru Takahashi, Koji Eto, Seishi Ogawa, Etsuro Ito The American Journal of Human Genetics Volume 103, Issue 3, Pages 440-447 (September 2018) DOI: 10.1016/j.ajhg.2018.07.020 Copyright © 2018 American Society of Human Genetics Terms and Conditions

Figure 1 Clinical Features of the Individuals (A) Bone-marrow smears from individual 1 (top) and individual 2 (bottom) show selective erythroid hypoplasia. (B) Growth curves of individual 1 (red) and individual 2 (green) exhibit marked growth retardation. (C) Reticular skin pigmentation and severe microcephaly as demonstrated by magnetic resonance imaging noted in individual 1 at 16 years of age. (D) Telomere lengths in peripheral blood lymphocytes from individual 1 (red) and individual 2 (green) compared to normal individuals (gray) (median age, 29 years; range, 1–47 years) and an individual with DC (blue); telomere lengths were estimated by flow-cytometry fluorescent in situ hybridization (flow-FISH) and expressed as the FISH signals relative to the signal from a normal control (1301 cell line). The American Journal of Human Genetics 2018 103, 440-447DOI: (10.1016/j.ajhg.2018.07.020) Copyright © 2018 American Society of Human Genetics Terms and Conditions

Figure 2 TP53 Gene Alteration Observed in Individuals with Inherited Bone-Marrow-Failure Syndrome (A) Pedigrees for individuals 1 (left) and 2 (right). Each individual had a de novo germline mutation. The red arrows indicate the positions of the single-nucleotide deletions. Blue arrows indicate the frameshift signals. (B) The TP53 mutation in different tissues from individuals 1 and 2. The right lower panel shows the DNA sequences of the subcloned molecule derived from individual 2, demonstrating the deletion of c.1077A. Red arrows indicate positions of the single-nucleotide deletion. Blue arrows indicate the frameshift signals. (C) Structure of the TP53 locus in which the locations of the mutations in individual 1 (red) and individual 2 (green) are indicated by arrowheads (top). Structure of human p53 consisting of domains for transactivation (TAD), specific DNA binding (DBD), and tetramerization (TD), as well as proline-rich (PRD) and C-terminal (CTD) domains (middle). The two heterozygous frameshift variants in TP53 in individuals 1 and 2 resulted in identical C-terminal truncations (arrowhead). A comparison of the amino acid sequences of the C termini of the wild-type and mutant p53s is shown (bottom). Deleted or altered sequences in p53 variants in both individuals are shown in red and blue, respectively. (D) The position of TP53 c.1077A is shown in red and c.1083G in green. c.1083G was deleted in individual 1, c.1077A was deleted in individual 2, and both translated products had identical amino acid sequences. Altered amino acid residues are indicated in blue. Both variants in exon 10 resulted in the introduction of premature terminal codons in the last coding exon (exon 11). (E) Luciferase activities from a CDKN1A promoter co-transduced with indicated constructs in QT6 (left) and MG63 (right) cell lines. Blank: no DNA. MOCK: mock vector. WT: wild-type TP53. c.1083delG: c.1083delG TP53 variant. c.1077delA: c.1077delA TP53 variant. ∗p < 0.01; ∗∗p < 0.001, t test. Error bars indicate standard deviation. The American Journal of Human Genetics 2018 103, 440-447DOI: (10.1016/j.ajhg.2018.07.020) Copyright © 2018 American Society of Human Genetics Terms and Conditions

Figure 3 Functional Analyses Based on a Zebrafish Model (A) The structures of human TP53 and zebrafish tp53. The sequences at the intron 10/exon 11 boundary regions are indicated. Uppercase and lowercase letters show the exon and intron sequences, respectively. The MO target site is underlined. The arrowhead indicates the position of the mutated nucleotides in the affected individuals. (B) The amino acid sequences of the C-terminal regions of human TP53 and zebrafish Tp53. The conserved lysine residues in the CTD for human and zebrafish are shown in red. The arrows indicate the positions of the mutant nucleotides in the individuals. The tetramerization domain is shaded in pink. (C) RT-PCR-based detection of tp53 and actb in MO-injected and wild-type embryos. A larger transcript retaining intron 10 is detected in the MO-injected embryos. Injection of spMO perturbed the splicing of intron 10 and introduced a premature stop codon just after exon 10, leading to the elimination of the lysine-rich CTD in Tp53. (D) Representative hemoglobin staining of cardial veins at 48 hpf and cell divisions at 8 hpf found in MO-injected and wild-type embryos. Approximately 40% of the embryos injected with spMO at 2.5 μg/μL show a severe to moderate reduction of erythrocytes. The injection of MO at 5 μg/μL led to developmental arrest at 8–9 hpf. The American Journal of Human Genetics 2018 103, 440-447DOI: (10.1016/j.ajhg.2018.07.020) Copyright © 2018 American Society of Human Genetics Terms and Conditions

Figure 4 Functional Analyses Based on a Human iPSC Model (A) An hiPSC line carrying a heterozygous TP53 mutant allele was generated by CRISPR/Cas9-mediated gene editing (upper). Comparison of the amino acid sequences of the C termini of wild-type and mutant p53s in the affected individuals and mutant hiPSC (bottom). Deleted or altered sequences in p53 variants in both individuals and the mutant hiPSC are shown in red and blue, respectively. (B) Comparison of the expression of four downstream targets of p53 in the mutant hiPSC (M) and isogenic control (W) by real-time quantitative RT-PCR. Error bars indicate standard deviation. (C) Scheme for differentiation of erythroid lineage from hiPSCs. To differentiate hiPSCs into erythroid cells, we used our previously established protocol.16 (D) Effects of the truncating p53 mutant on erythropoiesis as measured by the numbers of glycophorin-A (Gly-A)-positive cells after in vitro erythroid induction. The combined results of three independent experiments are shown. ∗p < 0.01; ∗∗p < 0.001, t test. Error bars indicate standard deviation. The American Journal of Human Genetics 2018 103, 440-447DOI: (10.1016/j.ajhg.2018.07.020) Copyright © 2018 American Society of Human Genetics Terms and Conditions