A pathogenic variant of AMOT leads to isolated X-linked congenital hydrocephalus due to N-terminal truncation
Nurcan Hastar, Hagit Daum, Nikoletta Kardos-Török, Gael Ganz, Leon Obendorf, Peter Vajkoczy, Orly Elpeleg, Petra Knaus
Nurcan Hastar, Hagit Daum, Nikoletta Kardos-Török, Gael Ganz, Leon Obendorf, Peter Vajkoczy, Orly Elpeleg, Petra Knaus
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Research Article Cell biology Genetics

A pathogenic variant of AMOT leads to isolated X-linked congenital hydrocephalus due to N-terminal truncation

Abstract

Congenital hydrocephalus is a life-threatening condition that might affect brain development by increasing the pressure on the brain parenchyma. Here, we describe 6 male patients from 1 family, all presenting with an isolated X-linked congenital hydrocephalus. Exome sequencing identified a likely pathogenic variant of angiomotin (AMOT) that segregated with the phenotype in the extended family. We show that the variant, affecting the first methionine, translated into a shorter AMOT protein lacking 91 amino acids from the N-terminus. Mechanistically, we unraveled that the absence of the N-terminus leads to abnormally increased AMOT protein levels due to the loss of both the N-degron degradation signal and the tankyrase-binding domain. Altered degradation of AMOT disrupted the barrier integrity of the cells. Thus, the identified AMOT variant likely underlies the clinical presentation of isolated X-linked hydrocephalus in this family, and our data underscore the importance of tight regulation of AMOT protein level in the brain. AMOT now joins the list of genes involved in congenital hydrocephalus in humans. These findings are instrumental for the genetic counseling of affected families.

Authors

Nurcan Hastar, Hagit Daum, Nikoletta Kardos-Török, Gael Ganz, Leon Obendorf, Peter Vajkoczy, Orly Elpeleg, Petra Knaus

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Figure 3

Elevated AMOT130ΔN protein level is caused by TBD loss and might be linked to altered N-degron property.

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Elevated AMOT130ΔN protein level is caused by TBD loss and might be link...
(A) Illustration of the second position amino acid of AMOT130, AMOT130ΔN, and rescue construct E2R_AMOT130ΔN; TNKS inhibition via XAV-939; and RNF146-mediated ubiquitylation of AMOT130. (B) Representative Western blot depicting AMOT130ΔN and AMOT130 protein levels after TNKS inhibition by XAV-939 in primary skin fibroblasts derived from a female control, a male patient, or a male control. (C) Quantification of 5 independent Western blots shows that the protein levels of AMOT130 in male and female control primary skin fibroblasts are increased but AMOT130ΔN level is not affected in patient primary skin fibroblasts upon XAV-939 treatment. The SD is reported. *P < 0.05, 2-way-ANOVA with Šídák’s multiple comparisons tests. (D) Representative Western blot depicting overexpressed AMOT130ΔN and AMOT130 protein levels after XAV-939, JW 55, and WIKI4 (10 μM, for 12 hours) addition to MCF7 cells. (E) Quantification of 5 independent Western blots shows that overexpressed AMOT130 protein level is increased, but overexpressed AMOT130ΔN protein level is unaffected after XAV-939, JW 55, and WIKI4 treatment in MCF7 cells. The SD is reported. **P ≤ 0.01, ***P < 0.001, 2-way ANOVA with Šídák’s multiple comparisons tests. (F) Representative Western blot demonstrating protein levels of AMOT130, AMOT130ΔN, E2R_AMOT130ΔN, and TBD_AMOT130ΔN. E2R_AMOT130ΔN mimics the destabilized N-terminus of AMOT130 by 1 amino acid substitution in the second position: glutamic acid to arginine (E2R) in AMOT130ΔN. TBD_AMOT130ΔN contains TBD77–84 of wild-type AMOT130 at the N-terminus of AMOT130ΔN. (G) Quantification of 5 independent Western blots shows that the protein level of overexpressed E2R_AMOT130ΔN and TBD_AMOT130ΔN is significantly decreased compared with AMOT130ΔN but is still higher than AMOT130. The SD is reported. *P ≤ 0.05, **P ≤ 0.01, 1-way ANOVA with Dunnett’s multiple comparisons test. (AMOT130ΔN was taken as a control group.)