Functional and clinical studies reveal pathophysiological complexity of CLCN4-related neurodevelopmental condition

Abstract

Missense and truncating variants in the X-chromosome-linked CLCN4 gene, resulting in reduced or complete loss-of-function (LOF) of the encoded chloride/proton exchanger ClC-4, were recently demonstrated to cause a neurocognitive phenotype in both males and females. Through international clinical matchmaking and interrogation of public variant databases we assembled a database of 90 rare CLCN4 missense variants in 90 families: 41 unique and 18 recurrent variants in 49 families. For 43 families, including 22 males and 33 females, we collated detailed clinical and segregation data. To confirm causality of variants and to obtain insight into disease mechanisms, we investigated the effect on electrophysiological properties of 59 of the variants in Xenopus oocytes using extended voltage and pH ranges. Detailed analyses revealed new pathophysiological mechanisms: 25% (15/59) of variants demonstrated LOF, characterized by a "shift" of the voltage-dependent activation to more positive voltages, and nine variants resulted in a toxic gain-of-function, associated with a disrupted gate allowing inward transport at negative voltages. Functional results were not always in line with in silico pathogenicity scores, highlighting the complexity of pathogenicity assessment for accurate genetic counselling. The complex neurocognitive and psychiatric manifestations of this condition, and hitherto under-recognized impacts on growth, gastrointestinal function, and motor control are discussed. Including published cases, we summarize features in 122 individuals from 67 families with CLCN4-related neurodevelopmental condition and suggest future research directions with the aim of improving the integrated care for individuals with this diagnosis.

Fig. 1. Pedigrees of all previously unreported families with inherited CLCN4 variants.

Filled square/circle = affected individual, lightly shaded circle/square = mildly affected individual, *familial *CLCN4 variant present in affected males, − familial CLCN4 variant absent in male, */− familial CLCN4 variant present in female, −/− familial CLCN4 variant absent in female. Pedigrees of families with a de novo variant are not shown.

Fig. 2. Clinical photographs of individuals with previously unreported variants in CLCN4, and representative neuroimaging.

A Clinical photographs demonstrate that some males and females have progressive lengthening of their face and ‘squaring’ of the jaw with age. LOF loss-of-function, GOF gain-of-function, ROF reduction of function, m months, y years. B Neuroimaging (T1 mid-sagittal view) from affected probands. In all individuals there are abnormalities of the corpus callosum. The proband of Family A10 has a dysplastic corpus callosum: it is of normal length but globally hypoplastic. Family A19: two affected brothers both display complete agenesis of the corpus callosum with colpocephaly. Family B5: the proband has partial agenesis of the corpus callosum (affecting the posterior part and splenium), colpocephaly and mild dilatation of the 3rd ventricle. Family B6: the proband has a dysplastic corpus callosum, and mildly small optic chiasm and optic nerves bilaterally.

Fig. 3. Mapping of all CLCN4 variants functionally investigated in this study.

A Schematic of the CLCN4 gene and ClC4-protein with position of variants from newly identified families with clearly affected males and females depicted above the schematic, and position of variants published to date shown below the schematic. B Position of the investigated missense variants in a CLC topology model. Altered residues are shown as circles and functional effects are color-coded as indicated in the figure. C Three-dimensional homology model of the human ClC-4 protein based on the structure of the CmClC homodimer (Protein Data Bank: 3ORG). The view from within the membrane delimited by dashed lines. The two subunits forming the homodimer are shown in dark and light grey. Mutated residues are shown as spheres colored as in B. Right 3D model viewed from the extracellular site.

Fig. 4. Expression of CLCN4 variants in Xenopus oocytes.

Panel A shows example recordings of the indicated constructs evoked by the voltage-clamp protocol indicated in the inset and using a “P/4” leak subtraction protocol (see Methods). Scale bars apply to all constructs. B shows average normalized IV relationships of the same variants. Currents are normalized to that of WT at 170 mV as described in Methods. In C, currents are normalized to the current of WT at the same voltage (see Methods). Data points are significantly different at practically all voltages from the value of 1 (i.e., WT) for all indicated variants (p < 0.05). Panel D shows typical current traces recorded without leak subtraction of WT ClC-4 in the presence of neutral and acidic extracellular pH, with outward currents being inhibited and inward currents remaining at a negligible level [29]. E illustrates the pH response of variant p.(Ile549Asn), which shows the activation of relatively large inward currents at acidic pH. F, G quantitative analysis of pH dependence of indicated GOF variants. Currents recorded at pH 5.3 were normalized to values measured at pH 7.3 as described in methods. The GOF effect of variants p.(Val317Ile) and p.(Ser395Arg) becomes apparent in panel G that shows the same data as panel F at a magnified scale. Data points are significantly different at voltages <= −40 mV from WT for all indicated variants (p < 0.05). Panel H shows differences in reversal potential measured at pH 6.3 and pH 5.3 for variants p.(Phe268Leu) and p.(Ile549Asn). The red line indicates the value expected for a stoichiometrically coupled 2 Cl⁻/1 H⁺ antiporter.