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 2

Single point mutation in the start codon of the AMOT gene leads to an in-frame shift to a new start codon (Met92) and thereby truncated AMOT130, herein called AMOT130ΔN, which presents with elevated protein levels.

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Single point mutation in the start codon of the AMOT gene leads to an in...
(A) Schematic representation of AMOT (upper panel) and AMOT130ΔN (lower panel) genes and proteins. ABD, angiostatin binding domain; PDZ-BD, PDZ binding domain. (B) Representative Western blot indicating an approximately 10 kDa molecular weight shift of AMOT130ΔN (first lane) compared with AMOT130 and increased protein level of AMOT130ΔN in primary skin fibroblasts obtained from proband IV1 (male patient) compared with AMOT130 in a male individual acting as a control (ctrl) and a female individual acting as a control. No change in AMOT80 protein level was detected between the patient and control groups. (C) Quantification of AMOT130ΔN and AMOT130 protein levels in patient/donor–derived primary skin fibroblasts by taking GAPDH as a housekeeping gene. n = 4 independent experiments; the SD is reported. ***P < 0.001, ordinary 1-way ANOVA with Tukey’s post hoc test. (D) Representative Western blot of overexpressed AMOT130ΔN, AMOT130, or pcDNA3.1 vector backbone control in epithelial cells (MCF7). (E) Quantification of overexpressed AMOT130ΔN and AMOT130 protein levels in MCF7 cells. The housekeeping gene was GAPDH. The mean of 3 independent experiments with SD. **P < 0.01, unpaired t test. (F) Chimeric proteins scheme. (G) Representative Western blot of 3 independent experiments depicting overexpression of chimeric, AMOT130ΔN, and AMOT130 protein levels in MCF7 cells. (H) Quantification of chimeric, AMOT130ΔN, and AMOT130 protein levels of 3 independent experiments (SD reported). *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001, ordinary 1-way ANOVA with Tukey’s post hoc test.