Further Delineation of Duplications of ARX Locus Detected in Male Patients with Varying Degrees of Intellectual Disability

Abstract

The X-linked gene encoding aristaless-related homeobox (ARX) is a bi-functional transcription factor capable of activating or repressing gene transcription, whose mutations have been found in a wide spectrum of neurodevelopmental disorders (NDDs); these include cortical malformations, paediatric epilepsy, intellectual disability (ID) and autism. In addition to point mutations, duplications of the ARX locus have been detected in male patients with ID. These rearrangements include telencephalon ultraconserved enhancers, whose structural alterations can interfere with the control of ARX expression in the developing brain. Here, we review the structural features of 15 gain copy-number variants (CNVs) of the ARX locus found in patients presenting wide-ranging phenotypic variations including ID, speech delay, hypotonia and psychiatric abnormalities. We also report on a further novel Xp21.3 duplication detected in a male patient with moderate ID and carrying a fully duplicated copy of the ARX locus and the ultraconserved enhancers. As consequences of this rearrangement, the patient-derived lymphoblastoid cell line shows abnormal activity of the ARX-KDM5C-SYN1 regulatory axis. Moreover, the three-dimensional (3D) structure of the Arx locus, both in mouse embryonic stem cells and cortical neurons, provides new insight for the functional consequences of ARX duplications. Finally, by comparing the clinical features of the 16 CNVs affecting the ARX locus, we conclude that-depending on the involvement of tissue-specific enhancers-the ARX duplications are ID-associated risk CNVs with variable expressivity and penetrance.

Keywords: 3D structure; ARX; KDM5C-SYN1 axis; Xp21.3 duplication; intellectual disability; ultraconserved enhancers.

Figure 1

Genomic mapping of duplications encompassing the ARX locus. Of them, 15 have already been described in the literature, and one, GE#1*, is described in this report for the first time.

Figure 2

Identification of a novel ARX duplication and its functional implications. Genealogical tree of family GE#1 and segregation of Xp22.1-p21.3dup: (A) Physical map of the ARX duplication on the human X chromosome (ChrX). (B) The involved genes POLA1 and ARX, the CpG Islands and the ultraconserved enhancers expressed in embryonic mouse brain (VISTA Enhancer Browser at https://enhancer.lbl.gov/, accessed on 24 January 2022) are shown. ARX, KDM5C and SYN1 expression levels in GE#1 ID patient’s lymphoblastoid cell lines (LCLs) and XYWT LCLs (C). Results were normalized using HPRT1 as reference gene and shown as mean ± SD from three independent experiments. p < 0.05. Schematic representation of ARX-KDM5C-SYN1 regulatory path is shown. Dose- and cell-type-dependent activity of ARX on 5′ KDM5C regulatory region (D,E). Luciferase assay in HeLa cells at increasing concentrations of ARX and Luciferase assays in HEK293T and SHSY5Y cell lines. Data were normalized to relative luciferase activity of JD-full-Luc. Results are shown as mean ± SD from three independent experiments. p < 0.05 *; p < 0.005 **; p < 0.0005 ***.

Figure 3

The ARX locus has a high density of forebrain ultraconserved sequences. Schematic representation of Xp region containing the multiple ultraconserved sequences located near the ARX locus, created on the annotation of UCSC hg38 assembly. Nucleotide positions are shown in box (A). Whole-mount staining images, obtained freely from VISTA Enhancer Browser (https://enhancer.lbl.gov/, accessed on 24 January 2022), showing the expression profiles of each enhancer in E11.5 embryos. Schematic representation of E11.5 brain subregions is shown in (B). Summary of phenotypic and molecular features of knockout mice lacking individual, or combinations of, ultraconserved Arx enhancers in shown in (C).

Figure 4

Polymer physics description of the murine Arx locus. Bottom matrices and contact maps predicted from the SBS polymer model by analysing the genomic region chrX:91,000,000–95,000,000 (UCSC, mm10). HiC data were obtained from a previous study [20] (A). Binding domain distributions for ESs (left) and CNs (right) (B). Snapshots from real MD simulations describing the 3D structure in ESs (left) and CNs (right). The colour scheme used is reported in the linear bar above (C).

Figure 5

Spatiotemporal gene expression trajectories of Arx/ARX across the corticogenesis: in mouse, from the embryonic stage E12 to E15 (A); in humans, from gestation week 8 to 20 (B); and in human-derived brain organoids, from in vitro developmental week 3 to 10 (C). RG, radial glia; aRG, apical radial glia; vRG, ventral radial glia; oRG, outer radial glia; IPC, intermediate progenitor cell; BP, basal progenitor; N, neuron; iN, immature neuron; mN, mature neuron. Data can be accessed freely on http://www.humous.org/, accessed on 2 February 2022.