SLC12A2 variants cause a neurodevelopmental disorder or cochleovestibular defect

Abstract

The SLC12 gene family consists of SLC12A1-SLC12A9, encoding electroneutral cation-coupled chloride co-transporters. SCL12A2 has been shown to play a role in corticogenesis and therefore represents a strong candidate neurodevelopmental disorder gene. Through trio exome sequencing we identified de novo mutations in SLC12A2 in six children with neurodevelopmental disorders. All had developmental delay or intellectual disability ranging from mild to severe. Two had sensorineural deafness. We also identified SLC12A2 variants in three individuals with non-syndromic bilateral sensorineural hearing loss and vestibular areflexia. The SLC12A2 de novo mutation rate was demonstrated to be significantly elevated in the deciphering developmental disorders cohort. All tested variants were shown to reduce co-transporter function in Xenopus laevis oocytes. Analysis of SLC12A2 expression in foetal brain at 16-18 weeks post-conception revealed high expression in radial glial cells, compatible with a role in neurogenesis. Gene co-expression analysis in cells robustly expressing SLC12A2 at 16-18 weeks post-conception identified a transcriptomic programme associated with active neurogenesis. We identify SLC12A2 de novo mutations as the cause of a novel neurodevelopmental disorder and bilateral non-syndromic sensorineural hearing loss and provide further data supporting a role for this gene in human neurodevelopment.

Keywords: de novo mutation; brain; corticogenesis; exome; neurodevelopmental disorder.

Figure 1

SLC12A2 missense variants. (A) Chart summarizing frequency of clinical features in children with SLC12A2 de novo mutations and an NDD. (B) Schematic representation of NKCC1 with 12 transmembrane domains: five inverted transmembrane domains + five symmetry transmembrane domains (shaded), followed by TM11 and TM12 (white transmembrane domains). The position of patient mutations is indicated in red. (C) Three-dimensional structure of NKCC1 demonstrating location of missense variants at the core of the protein, with high potential to disrupt protein structure.

Figure 2

Functional analysis of NKCC1 mutation in Xenopus laevis oocytes. (A and B) Box plots demonstrating K⁺ influx measured in oocytes injected with water (negative control), water containing 15 ng wild-type (control) or mutant mouse Slc12a2/NKCC1 cRNA. Horizontal line represents median, extent of box demonstrates interquartile range and whiskers minimum and maximum. K⁺ fluxes were measured under basal isosmotic (200 mOsM) or stimulated hyperosmotic (270 mOsM) conditions and are expressed in picomoles K⁺/oocyte/hour. As the mutants were tested in different experiments on different oocytes, each mutant flux is accompanied by its own control flux. Statistical differences were determined using one-way ANOVA. Each experiment represents 20–25 oocytes per mutation. Note that the mouse residue numbers are slightly different from human.

Figure 3

Transcriptomic analysis of SCL12A2 expression in developing human brain. (A) Box plots of microarray data comparing SLC12A2 expression in neuroanatomical regions of high and less active neurogenesis at 15 weeks gestation. Horizontal lines represent median, extent of box interquartile range and whiskers minimum and maximum. Expression is significantly higher in areas of active neurogenesis (asterisk). (B) Single cell RNA sequencing data demonstrating significantly higher expression of SLC12A2 in radial glia cells (RGC) compared to intermediate progenitor cells (IPC) (Mann-Whitney U-test, Z = −3.3, P = 0.001) and neurons (z = −5.2, P < 0.001). Units are counts per million reads (CPM).