CDKL1 variants affecting ciliary formation predispose to thoracic aortic aneurysm and dissection
Theresa Nauth, Melanie Philipp, Sina Renner, Martin D. Burkhalter, Helke Schüler, Ceren Saygi, Kristian Händler, Bente Siebels, Alice Busch, Thomas Mair, Verena Rickassel, Sophia Deden, Konstantin Hoffer, Jakob Olfe, Thomas S. Mir, Yskert von Kodolitsch, Evaldas Girdauskas, Meike Rybczynski, Malte Kriegs, Hannah Voß, Thomas Sauvigny, Malte Spielmann, Malik Alawi, Susanne Krasemann, Christian Kubisch, Till J. Demal, Georg Rosenberger
Theresa Nauth, Melanie Philipp, Sina Renner, Martin D. Burkhalter, Helke Schüler, Ceren Saygi, Kristian Händler, Bente Siebels, Alice Busch, Thomas Mair, Verena Rickassel, Sophia Deden, Konstantin Hoffer, Jakob Olfe, Thomas S. Mir, Yskert von Kodolitsch, Evaldas Girdauskas, Meike Rybczynski, Malte Kriegs, Hannah Voß, Thomas Sauvigny, Malte Spielmann, Malik Alawi, Susanne Krasemann, Christian Kubisch, Till J. Demal, Georg Rosenberger
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Research Article Cell biology Genetics Vascular biology

CDKL1 variants affecting ciliary formation predispose to thoracic aortic aneurysm and dissection

Abstract

Genetic factors are fundamental in the etiology of thoracic aortic aneurysm and dissection (TAAD), but the genetic cause is detected in only about 30% of cases. To define unreported TAAD-associated sequence variants, exome and gene panel sequencing was performed in 323 patients. We identified heterozygous CDKL1 variants [c.427T>C p.(Cys143Arg), c.617C>T p.(Ser206Leu), and c.404C>T p.(Thr135Met)] in 6 patients from 3 families with TAAD spectrum disorders. CDKL1 encodes a protein kinase involved in ciliary biology. Amino acid substitutions were predicted to affect CDKL1 catalytic activity or protein binding properties. CDKL1 was expressed in vascular smooth muscle cells in normal and diseased human aortic wall tissue. Cdkl1 knockdown and transient knockout in zebrafish resulted in intersomitic vessel (ISV) malformations and aortic dilation. Coinjection of human CDKL1wild-type RNA, but not CDKL1Cys143Arg and CDKL1Ser206Leu RNA, rescued ISV malformations. All variants affected CDKL1 kinase function and profiling data, and altered protein-protein binding properties, particularly with ciliary transport molecules. Expression of CDKL1 variants in heterologous cells interfered with cilia formation and length, CDKL1 localization, and p38 MAPK and Vegf signaling. Our data suggest a role of CDKL1 variants in the pathogenesis of TAAD spectrum disorders. The association between primary cilia dysregulation and TAAD expands our knowledge of the underlying molecular pathophysiology.

Authors

Theresa Nauth, Melanie Philipp, Sina Renner, Martin D. Burkhalter, Helke Schüler, Ceren Saygi, Kristian Händler, Bente Siebels, Alice Busch, Thomas Mair, Verena Rickassel, Sophia Deden, Konstantin Hoffer, Jakob Olfe, Thomas S. Mir, Yskert von Kodolitsch, Evaldas Girdauskas, Meike Rybczynski, Malte Kriegs, Hannah Voß, Thomas Sauvigny, Malte Spielmann, Malik Alawi, Susanne Krasemann, Christian Kubisch, Till J. Demal, Georg Rosenberger

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Figure 5

CDKL1 variants interfere with kinase function.

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CDKL1 variants interfere with kinase function. (A and B) In vitro kinas...
(A and B) In vitro kinase assays. HeLa (A) or HEK293T (B) cells were transiently transfected with CDKL1 and IME2 constructs or empty vectors (HAcontrol and EGFPcontrol). Fusion proteins were immunoprecipitated and subjected to kinase assays using peptide substrate RPRSPGARR and the ADP-Glo kinase assay (Promega). Luminescence intensities of individual measuring points were normalized to the entire luminescence signal of an experiment and to the amount of immunoprecipitated protein (n = 7, A; n = 4, B). Medians (50th percentiles, lines in boxes) as well as 0th, 25th, 75th, and 100th percentiles are given in the box plots. Small aliquots were removed from precipitates and subjected to immunoblotting using anti-HA (A) and anti-GFP (B) antibodies; representative blots are shown. *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001, 1-way ANOVA with Dunnett’s post hoc multiple-comparison test. (C) CDKL1 serine/threonine phosphorylation profiling. EGFP-tagged CDKL1 variants were expressed in HEK293T cells, purified using GFP-Trap (ChromoTek), and applied to STK-PamChip arrays (PamGene). Seventy-one peptides passed quality control. The heatmap displays average log2-transformed signal intensities for indicated CDKL1 variants. The signals were sorted from high (red) to low (blue) intensity that corresponds to phosphorylation levels. To visualize overall sample variance and group differences, peptide phosphorylation and overall kinase activity are shown in box plot representation. (D) Peptide substrates with significantly changed phosphorylation. The heatmap shows significantly differentially phosphorylated peptides between samples treated with disease-associated CDKL1 variants versus CDKL1WT. The effects of CDKL1 variants are log ratios [log2(disease-associated CDKL1 variant)/log2(CDKL1WT)]. Blue and red indicate reduced and increased phosphorylation, respectively. Peptides that did not pass the significance threshold (P < 0.05, disease-associated CDKL1 variant vs. CDKL1WT) are black. Peptide numbers correspond to those in Supplemental Table 2, where details on the substrates are also described.