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XAF1 as a modifier of p53 function and cancer susceptibility

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Science Advances  24 Jun 2020:
Vol. 6, no. 26, eaba3231
DOI: 10.1126/sciadv.aba3231
  • Fig. 1 Schematic diagram of chromosome 17p13 spanning 2-Mb region encompassing TP53-R337H and XAF1-E134* variants.

    Identification and location of genes in this region as well as the genotyped SNPs and microsatellite markers are shown. Positions are given relative to build GRCh37/hg19. R337H-only and extended (TP53-R337H + XAF1-E134*) haplotypes observed in the population cohort study are represented.

  • Fig. 2 Risk haplotype and representative pedigrees associated with the extended haplotype as determined by phasing.

    (A) Common SNPs from the Axiom array spanning 2-Mb region in the chromosome 17p13 (see Fig. 1). Of the rare variants falling within haplotype boundaries, only TP53-R337H and XAF1-E134* variants remained after filtering for pathogenicity. ExAC: Exome Aggregation Consortium; MAF: Minor Allele Frequency. (B) Brazilian family (proband no. 108; table S2) diagnosed with angiosarcoma and their relatives. SNPs were used to phase the haplotypes. The risk haplotype harboring both mutant alleles (TP53-R337H and XAF1-E134*) is represented in red bars. The remaining haplotype is represented by different colors as segregating in family members. (C) Spanish family (proband no. 50; table S2). Age at diagnosis and present age or age at death are indicated in each pedigree. ExAC, Exome Aggregation Consortium; MAF, Minor Allele Frequency.

  • Fig. 3 Distribution of tumor types as a second, third, or fourth malignancy among patients with the R337H-only and extended haplotypes.

    Thirty-three probands (29 females and 4 males) developed multiple primary malignancies. Multiple primary tumors in probands with the extended haplotype (n = 30) are visualized in the upper panel. Multiple primary tumors in probands with the R337H-only haplotype (n = 3) are visualized in the lower panel.

  • Fig. 4 XAF1 increases the transcriptional activity of hypomorphic TP53 variants.

    Saos-2 cells were transiently transfected with p53-responsive promoter-luciferase reporters with or without p53 and XAF1 expression vectors, as described in the Supplementary Materials. PG13 promoter-reporter luciferase assay. Wild-type XAF1, but not XAF1-E134*, stimulated the transactivation function of wild-type p53 and other hypomorphic p53 variants such as T125M, R175L, R290H, and G334R. (A) Western blot analysis of p53 and XAF1 in the transfected cell lysates. (B) The columns represent the mean of three independent experiments (±SD), each performed in duplicate. Error bars indicate SDs. Asterisks indicate statistical significance, as determined by one-way analysis of variance (ANOVA) *P < 0.005; **P = 0.0096; ***P = 0.0009; ****P = <0.0001.

  • Fig. 5 Correction of the XAF1-E134* mutation using CRISPR-Cas9 in human low-passage fibroblasts restores full-length XAF1 and partial p53 responsiveness.

    (A) Western blot analysis of MDM2, XAF1, p21CIP1, PUMA, and p53 expression in untreated and irradiated (5 Gy) low-passage fibroblasts. HF001 (wild-type XAF1 and TP53), HF003 (heterozygous XAF1- E134*; TP53-R337H), HF004 (homozygous XAF1-E134*; TP53-R337H), and derived HF004 clones with CRISPR-Cas9–corrected E134*(HF004cl1 and HF004cl2). (B) Gene set enrichment analysis (GSEA) showing the enrichment of p53 pathway signature in edited clones and heat map showing the expression levels in each cell line before and after IR treatment. Color scale represents SDs from the mean (z-score; range, −2 to 2). FDR, false discovery rate.

  • Table 1 Tumor distribution in probands harboring R337H-only and extended haplotype.

    Study participantsTotalTP53-R337HTP53-
    R337H + XAF1-
    Control (newborn
    ACT [total cases (N)]10226760.3140.1981.330.799 to Inf
    ACT (<5 years)7720570.3140.2511.2970.742 to Inf
    ACT (≥5 years)256190.3510.3161.4390.586 to Inf
    Sarcomas302280.030.0036.3291.756 to Inf
    Breast cancer [total
    cases (N)]
    5714430.3140.221.3970.743 to Inf
    Breast cancer (≤45
    317240.3140.231.5580.680 to Inf
    Breast cancer (>45
    267190.4230.4231.2350.524 to Inf
    Other cancers100100.070.0289.6211.245 to Inf
    Total199421570.070.0221.71.097 to Inf
    Multiple tumors
    [total cases (N)]
    333300.030.0064.5251.517 to Inf

    *For other cancers, group OR was estimated by adding 0.5 to the contingency table. Other ORs are conditional maximum likelihood estimator.

    †Four probands (three ACTs and one CPC) homozygous for both variants were excluded from the analysis so all individuals in the analysis are heterozygous for TP53-R337H.

    Supplementary Materials

    • Supplementary Materials

      XAF1 as a modifier of p53 function and cancer susceptibility

      Emilia M. Pinto, Bonald C. Figueiredo, Wenan Chen, Henrique C.R. Galvao, Maria Nirvana Formiga, Maria Candida B.V. Fragoso, Patricia Ashton-Prolla, Enilze M.S.F. Ribeiro, Gabriela Felix, Tatiana E.B. Costa, Sharon A. Savage, Meredith Yeager, Edenir I. Palmero, Sahlua Volc, Hector Salvador, Jose Luis Fuster-Soler, Cinzia Lavarino, Guillermo Chantada, Dominique Vaur, Vicente Odone-Filho, Laurence Brugières, Tobias Else, Elena M. Stoffel, Kara N. Maxwell, Maria Isabel Achatz, Luis Kowalski, Kelvin C. de Andrade, Alberto Pappo, Eric Letouze, Ana Claudia Latronico, Berenice B. Mendonca, Madson Q. Almeida, Vania B. Brondani, Camila M. Bittar, Emerson W.S. Soares, Carolina Mathias, Cintia R.N. Ramos, Moara Machado, Weiyin Zhou, Kristine Jones, Aurelie Vogt, Payal P. Klincha, Karina M. Santiago, Heloisa Komechen, Mariana M. Paraizo, Ivy Z.S. Parise, Kayla V. Hamilton, Jinling Wang, Evadnie Rampersaud, Michael R. Clay, Andrew J. Murphy, Enzo Lalli, Kim E. Nichols, Raul C. Ribeiro, Carlos Rodriguez-Galindo, Marta Korbonits, Jinghui Zhang, Mark G. Thomas, Jon P. Connelly, Shondra Pruett-Miller, Yoan Diekmann, Geoffrey Neale, Gang Wu, Gerard P. Zambetti

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