De novo and inherited mutations in the X-linked gene CLCN4 are associated with syndromic intellectual disability and behavior and seizure disorders in males and females

Abstract

Variants in CLCN4, which encodes the chloride/hydrogen ion exchanger CIC-4 prominently expressed in brain, were recently described to cause X-linked intellectual disability and epilepsy. We present detailed phenotypic information on 52 individuals from 16 families with CLCN4-related disorder: 5 affected females and 2 affected males with a de novo variant in CLCN4 (6 individuals previously unreported) and 27 affected males, 3 affected females and 15 asymptomatic female carriers from 9 families with inherited CLCN4 variants (4 families previously unreported). Intellectual disability ranged from borderline to profound. Behavioral and psychiatric disorders were common in both child- and adulthood, and included autistic features, mood disorders, obsessive-compulsive behaviors and hetero- and autoaggression. Epilepsy was common, with severity ranging from epileptic encephalopathy to well-controlled seizures. Several affected individuals showed white matter changes on cerebral neuroimaging and progressive neurological symptoms, including movement disorders and spasticity. Heterozygous females can be as severely affected as males. The variability of symptoms in females is not correlated with the X inactivation pattern studied in their blood. The mutation spectrum includes frameshift, missense and splice site variants and one single-exon deletion. All missense variants were predicted to affect CLCN4's function based on in silico tools and either segregated with the phenotype in the family or were de novo. Pathogenicity of all previously unreported missense variants was further supported by electrophysiological studies in Xenopus laevis oocytes. We compare CLCN4-related disorder with conditions related to dysfunction of other members of the CLC family.

Figure 1

Pedigrees of affected families A–G, J and P. Filled square=affected male; Filled circle=affected female; *Familial CLCN4 variant present; wt, familial CLCN4 variant absent; NT, not tested. Note unaffected male IV:5 in family B has Klinefelter syndrome (47,XXY). Probands from families H, I, K, L, M, N and O (pedigrees not shown) have de novo variants.

Figure 2

Neuroimaging of affected individuals with CLCN4-related condition at different ages. F1–4: CT images of individual F:IV:3, aged 66. Non-contrast multi-slice helical acquisitions, thick-slice reconstructions only. F1: sagittal midline image depicts callosal thinning, atrophic midbrain and cerebellar atrophy with prominent quadri-cerebellar cistern. F2: axial image of posterior fossa depicting cerebellar vermian atrophy with increased caliber of the adjacent CSF spaces. F3: axial image depicting prominent Sylvian fissures and increased caliber of lateral and third ventricles with surrounding atrophic change and evidence of a right frontal infarct. F4: coronal image showing disproportionate cerebellar atrophy and prominence of the adjacent CSF spaces. J1–6: MRI images of individual J:III:2 aged 4 and 8. J1: Axial FLAIR image from individual J:III:2, aged 4, depicting enlarged Sylvian fissures secondary to surrounding volume loss, particularly affecting the frontal lobes. J2: coronal T2 weighted image from individual J:III:2 aged 4 depicting generalized white matter volume loss. J3 Axial FLAIR image from individual J:III:2 aged 8 demonstrating periventricular leukomalacia. J4: coronal FLAIR image from individual J:III:2 aged 8. Images depict that despite interval progression of myelination between the ages of 4 and 8, there is still significantly delayed myelination involving the anterior frontal, posterior parietal and temporal lobes. The corpus callosum is normally formed but attenuated. There is pronounced global volume loss, preferentially affecting the white matter. Prominent perivascular spaces are seen bilaterally. L1–3: MRI images from family L proband at four months of age. Diffuse cortical hypoplasia with resultant prominence of the extra-axial CSF spaces and thinning of the corpus callosum without other morphologic abnormalities. Myelination is otherwise grossly normal. N1–2 MRI images from proband from family N aged 10. N1: sagittal T1 weighted midline image showing dilated third ventricle. N2: Coronal T2 weighted image showing dilated third ventricle. I1–2: images from proband from family I aged 13 months. I1: sagittal T1 weighted midline image showing dilated lateral ventricles. I:2: axial T2 weighted image showing volume loss, periventricular leukomalacia and dilated ventricles. CSF, cerebrospinal fluid; MRI, magnetic resonance imaging.

Figure 3

Facial features of affected individuals with CLCN4-related disorder in age order. In older males note the elongated face, straight nose and pointed prominent chin that becomes relatively ‘squared off’ with age that is particularly prominent in affected adult males from families A, B, C and F (F:IV:3), and mildly present in the 9-year-old F:V:1 and 12-year-old affected male from family P. A relatively flat midface is noticeable in several individuals (particularly A:III:6 and affected members of family C). Individuals F:IV:3 and F:IV:5 have relative ‘coarsening’ of facial features with age; however, they were both treated with phenytoin, which can coarsen features and F:IV:5 had multiple facial injuries secondary to epileptic drop attacks. Although the facial phenotype is less distinctive in younger children and females (family F:V:1 and F:V2 as infants, and affected individuals from families O, I, L, H, N and M) some similarities include young children having relatively rounded faces and affected females from families H, L and F (F:IV:5) having down-sloping palpebral fissures and depressed nasal bridges. Clinical pictures from family C were first published in Raynaud et al. and reproduced from John Wiley & Sons with permission.

Figure 4

Genotypic information, three-dimensional modeling and functional studies of CLCN4 variants. (a) Position of CLCN4 variants in families A–P (information adapted from Protein Data Bank P51793). Missense variants in families D (p.Gly78Ser), G (p.Val212Gly), L (p.Ala555Val) and O (p.Gly544Arg) are within helical transmembrane domains of the protein. Missense variants in families F (p.Val536Met), I (p.Ser534Leu) and N (p.Val275Met) are within helical-intramembrane domains. Missense variants in families E (p.Leu221Val) and H (p.Leu221Pro) lie within a loop between transmembrane domains. Missense variants in families C (p.Gly731Arg) and M (p.Arg718Trp) lie within the second cytosolic cystathionine-β-synthase (CBS) domain and therefore may affect gating. (b) Analogous positions of amino acids mutated in ClC-4 highlighted in the crystal structure of CmClC. Amino acids are displayed as spheres in colors. CLC transporters are (homo)dimers of identical subunits with two ion translocation pathways. Each subunit is composed of a transmembrane domain (TMD) containing several intramembrane helices and cytosolic CBS domains. Asterisks indicate previously published analogous positions of amino acids.^, (c) Current voltage relationships of the electrogenic 2Cl⁻/H⁺ exchanger ClC-4 and the CIC-4 variants expressed in Xenopus oocytes, shown as mean values of normalized steady-state currents from several oocytes (numbers indicated in figure, in parentheses: number of independent cRNA batches injected). The current of wild-type (WT) CIC-4 is strongly outwardly rectifying, that is, increases steeply at inside-positive voltages. The human missense mutations p.Val212Gly, p.Leu221Pro, p.Val275Met, p.Ser543Leu, p.Ala555Val and p.Arg718Trp reduced, or even abolished, these ClC-4 currents. Only variant p.Asp15Asn exhibited normal wild-type like currents. Error bars, s.e.m. Noninjected (n.i.) oocytes served as control.