Figure 3 from ENIGMA <i>CHEK2</i>gether Project: A Comprehensive Study Identifies Functionally Impaired <i>CHEK2</i> Germline Missense Variants Associated with Increased Breast Cancer Risk
Authors: Stolarova Lenka , Kleiblova Petra , Zemankova Petra , Stastna Barbora , Janatova Marketa , Soukupova Jana , Achatz Maria Isabel , Ambrosone Christine , Apostolou Paraskevi , Arun Banu K. , Auer Paul , Barnard Mollie , Bertelsen Birgitte , Matsuda Koichi , Kamatani Yoichiro , Morisaki Takayuki , Nagai Akiko , Muto Kaori , Murakami Yoshinori , Furukawa Yoichi , Yamanashi Yuji , Nakamura Yusuke , Mushiroda Taisei , Momozawa Yukihide , Tanaka Toshihiro , Ohnishi Yozo , Kubo Michiaki , Higashiue Shinichi , Kobayashi Shuzo , Minami Shiro , Yamaguhci Hiroki , Arai Hajime , Yamaji Ken , Okazaki Yasushi , Asai Satoshi , Takahashi Yasuo , Fujioka Tomoaki , Obara Wataru , Mori Seijiro , Murayama Shigeo , Nagayama Satoshi , Miki Yoshio , Masumoto Akihide , Yamada Akira , Nishizawa Yasuko , Higashiyama Masahiko , Kutsumi Hiromu , Koretsune Yukihiro , Yoshiyama Takashi , Blok Marinus J. , Boddicker Nicholas , Brunet Joan , Burnside Elizabeth S. , Calvello Mariarosaria , Campbell Ian , Chan Sock Hoai , Chen Fei , Chiang Jian Bang , Coppa Anna , Cortesi Laura , Crujeiras-González Ana , Borecka Marianna , Cerna Marta , Hovhannisyan Milena , Jelinkova Sandra , Nehasil Petr , Foretova Lenka , Machackova Eva , Krutilkova Vera , Tavandzis Spiros , Cerna Leona , Chvojka Stepan , Koudova Monika , Puchmajerova Alena , Havranek Ondrej , Novotny Jan , Vesela Kamila , Vocka Michal , Hruskova Lucie , Michalovska Renata , Schwetzova Denisa , Vlckova Zdenka , Cerna Monika , Hejnalova Marketa , Jedlickova Nikol , Subrt Ivan , Zavoral Tomas , Kosarova Marcela , Vacinova Gabriela , Janikova Maria , Kratochvilova Romana , Curtisova Vaclava , Vrtel Radek , Scheinost Ondrej , Duskova Petra , Stranecky Viktor , De Leeneer Kim , De Putter Robin , DePersia Allison , Devereux Lisa , Domchek Susan , Efremidis Anna , Engel Christoph , Ernst Corinna , Evans D. Gareth R. , Feliubadaló Lidia , Fostira Florentia , Fuentes-Ríos Olivia , Gómez-García Encarna B. , González Sara , Haiman Christopher , Hansen Thomas van Overeem , Hauke Jan , Hodge James , Hu Chunling , Huang Hongyan , Ishak Nur Diana Binte , Iwasaki Yusuke , Konstantopoulou Irene , Kraft Peter , Lacey James , Lázaro Conxi , Li Na , Lim Weng Khong , Lindstrom Sara , Lori Adriana , Martinez Elana , Martins Alexandra , Matsuda Koichi , Matullo Giuseppe , McInerny Simone , Michailidou Kyriaki , Montagna Marco , Monteiro Alvaro N.A. , Mori Luigi , Nathanson Katherine , Neuhausen Susan L. , Nevanlinna Heli , Olson Janet E. , Palmer Julie , Pasini Barbara , Patel Alpa , Piane Maria , Poppe Bruce , Radice Paolo , Renieri Alessandra , Resta Nicoletta , Richardson Marcy E. , Rosseel Toon , Ruddy Kathryn J. , Santamariña Marta , Dos Santos Elizabeth Santana , Teras Lauren , Toland Amanda E. , Trentham-Dietz Amy , Vachon Celine M. , Volk Alexander E. , Weber-Lassalle Nana , Weitzel Jeffrey N. , Wiesmuller Lisa , Winham Stacey , Yadav Siddhartha , Yannoukakos Drakoulis , Yao Song , Zampiga Valentina , Zethoven Magnus , Zhang Ze Wen , Zima Tomas , Spurdle Amanda B. , Vega Ana , Rossing Maria , Del Valle Jesús , De Nicolo Arcangela , Hahnen Eric , Claes Kathleen B.M. , Ngeow Joanne , Momozawa Yukihide , James Paul A. , Couch Fergus J. , Macurek Libor , Kleibl Zdenek 
Published: August 2023

<p>Kinase KAP1 and CHK2 assays (<b>A</b>). The bar graphs show results of kinase assays for 430 <i>CHEK2</i> missense variants. In both assays, variants with normalized relative CHK2 activity (mean WT-activity = 1) exceeding that of the weakest signal of WT replicas (not shown) were categorized functionally WT-like, variants with normalized signal intensity lower than the strongest signal for any of kinase-dead/empty EGFP vector controls (in-frame exon 7 deletion–p.D265_H282del; not shown) were categorized as functionally impaired. Variants with normalized CHK2 activities between these ranges were categorized functionally intermediate (0.428–0.705 and 0.479–0.710 for KAP1 and CHK2 assay, respectively; indicated by red and yellow dashed lines). Scatterplot combines results from both assays showing 340 concordant (circles) and 90 discordant (crosses) variants. The nuclear-to-cytoplasmic ratio (<b>B</b>) bar graph (left) displays all missense variants and a set of protein-truncating <i>CHEK2</i> variants (dark red bars at left, zoomed part of the graph). The missense variants, p.R521W and p.R521Q, with an aberrant localization are highlighted as bright-red bars; the arrows denote WT (green bar) and catalytically-dead in-frame p.D265_H282del variant (white bar). The highest and lowest mean nuclear/cytoplasmic ratio values from all WT replicates are indicated by green dashed lines. Of all missense variants analyzed by ScanR microscopy, only codon 521 alterations revealed aberrant intracellular localization with intense cytoplasmic positivity (right), reminiscent of mislocalization of the c.1100delC (p.T367fsX; size bar, 10 μm) variant. In comparison, the in-frame deletion p.D265_H282del revealed normal intranuclear accumulation, similar to WT. <b>C,</b> Scatter plots depicting correlations between assays performed in this study and previous analyses of <i>CHEK2</i> VUS. Studies of Kleiblova <i>et al.</i> (<a href="#bib17" target="_blank">17</a>) and Boonen et al. (<a href="#bib18" target="_blank">18</a>) used phosphorylation of KAP1 as a functional readout whereas the study of Delimitsou <i>et al.</i> (<a href="#bib25" target="_blank">25</a>) used a yeast growth retardation assay. The dots are colored according to the results of the KAP1 assay in this study (red, impaired; yellow, intermediate; green, wild-type–like). Blue line represents linear regression, R, correlation coefficient; <i>P</i>, <i>P</i> value. The scatter plot does not show the p.Arg512Trp variant classified by Boonen et al. as intermediate with impaired nuclear localization in our localization assay.</p>