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 8

CDKL1 function in cell signaling.

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CDKL1 function in cell signaling. (A) CDKL1 variants increase signaling ...
(A) CDKL1 variants increase signaling via p38 (MAPK8). Total lysates of HEK293T cells transiently expressing CDKL1WT, CDKL1Thr135Met, CDKL1Cys143Arg, or CDKL1Ser206Leu were cultured under basal conditions (DMEM containing 10% serum), lysed, and subjected to immunoblotting as indicated. Cells expressing kinase-dead CDKL1Lys33Arg and cells transfected with empty vector (EV) were used as controls. GAPDH was used as loading control. Representative immunoblots from 5 independent experiments (n = 5) are given. Graph shows means (± SD) of the relative phosphorylation of p38 (at Thr180 and Tyr182) normalized to amounts of total p38 as well as to GAPDH. Phosphorylation levels in cells expressing CDKL1 mutants are relative to those in CDKL1WT cells. One-way ANOVA with Tukey’s multiple-comparison test was used. *P ≤ 0.05, **P ≤ 0.01. (B) Knockdown of Cdkl1 induces Vegfaa expression. Quantitative PCR analysis was performed on total RNA of 24 hpf zebrafish embryos. Graphs show amounts of axin2, patched1, and vegfaa transcripts in Cdkl1 splMO relative to CTRL MO–treated embryos. n = 8 experiments; mean values (red bars) and individual experiments (circles) are indicated. **P = 0.07, 2-tailed Wilcoxon’s test. (C) Expression of the arterial marker ephrin B2a (ephB2a) and of plexin D1 (plxnD1) is stronger in Cdkl1 morphants than in control-injected embryos. Spatial expression was determined by in situ hybridization. Stacked bar graphs summarize 3 experiments with 82 (CTRL MO) and 85 embryos (Cdkl1 splMO) for ephB2a and 3 experiments with 63 (CTRL MO) and 42 embryos (Cdkl1 splMO) for plxnD1. **P = 0.0018, ***P = 0.0003, 2-sided Fisher’s exact test. All experiments were done at 24 hpf. Scale bars: 200 μm.