Biallelic Mutations in UNC80 Cause Persistent Hypotonia, Encephalopathy, Growth Retardation, and Severe Intellectual Disability

Abstract

Ion channel proteins are required for both the establishment of resting membrane potentials and the generation of action potentials. Hundreds of mutations in genes encoding voltage-gated ion channels responsible for action potential generation have been found to cause severe neurological diseases. In contrast, the roles of voltage-independent "leak" channels, important for the establishment and maintenance of resting membrane potentials upon which action potentials are generated, are not well established in human disease. UNC80 is a large component of the NALCN sodium-leak channel complex that regulates the basal excitability of the nervous system. Loss-of-function mutations of NALCN cause infantile hypotonia with psychomotor retardation and characteristic facies (IHPRF). We report four individuals from three unrelated families who have homozygous missense or compound heterozygous truncating mutations in UNC80 and persistent hypotonia, encephalopathy, growth failure, and severe intellectual disability. Compared to control cells, HEK293T cells transfected with an expression plasmid containing the c.5098C>T (p.Pro1700Ser) UNC80 mutation found in one individual showed markedly decreased NALCN channel currents. Our findings demonstrate the fundamental significance of UNC80 and basal ionic conductance to human health.

Figure 1

Clinical Photographs of All Four Subjects (A) Subject F1-IV.1 at the age of 4 years. (B) Subject F2-V.5 at the age of 3 years. (C) Subject F3-II.1 at the age of 9 years. (D) Subject F3-II.3 at the age of 3 years.

Figure 2

Pedigrees and Chromatograms from All Three Families Affected individuals are shaded in black.

Figure 3

Variant UNC80 Associates with UNC79 and NALCN (A) Cells were transfected with UNC79 alone or together with WT or variant Unc80 as indicated in each lane. Immunoprecipitates (IP) with an anti-UNC80 antibody were blotted (IB) with anti-UNC79 to allow us to probe the association between UNC79 and UNC80 (upper panel). Total cell lysates (lower three panels) were blotted with anti-UNC79 or anti-UNC80 antibodies for comparison of protein amounts. Immunoblotting with anti-β-actin (lower panel) was used as a control for sample loading. (B) Cells were transfected with FLAG-tagged NALCN alone or together with the WT or variant Unc80, as indicated. Immunoprecipitates with anti-FLAG were blotted with anti-UNC80 to allow us to probe the association between UNC80 and NALCN. For immunoprecipitation, cells were solubilized in lysis buffer (50 mM Tris-HCl, 150 mM NaCl, 1% NP-40, 1× PIC [Roche]) and immunoprecipitated with anti-UNC80 (2 μg/ml) or anti-FLAG (5 μg/ml, no. F3165 [Sigma]). The immune complexes were analyzed by western blot with anti-UNC80 (2.2 μg/ml) or anti-UNC79 (3 μg/ml). Cell lysates (inputs) were also analyzed by western blot with anti-UNC80 or anti-β-actin (1:1,000, no. 4970S [Cell Signaling]) as indicated.

Figure 4

Altered Function in Variant UNC80 (A and B) Representative NALCN currents recorded in HEK293T cells co-transfected with NALCN and WT Unc80 (A) or the mutant version of Unc80 (B). Whole-cell currents were recorded with a voltage ramp from −100 mV to +100 mV in 1 s (Vh = 0 mV, illustrated in the lower panel of A). In cells with currents larger than 50 pA, NMDG was used to substitute Na⁺ and K⁺ to confirm that the cells had no non-specific leak, as demonstrated by the abolishment of inward currents with ion substitution. (C) Summary of the current amplitudes recorded at −100 mV. Numbers of cells recorded are in parentheses. Data were presented as mean ± SEM. ^∗p < 0.05. ^∗∗∗p < 0.001. For patch clamp recordings, cells in a 35-mm dish (∼90% confluency) were transfected with 3 μg plasmids DNA with Lipofectamine 2000 for ∼40 hr and plated onto polylysine-coated coverslips. Recordings were done 40–48 hr after transfection. Cells with GFP fluorescence intensity within the top 20%–30% on each coverslip were selected for patch clamp recordings. Signal was acquired with an amplifier (Axopatch 200B or Multiclamp 700B) and a Digidata 1440A data acquisition system controlled by PClamp software (Molecular Device). The pipette solution contained 150 mM Cs, 120 mM Mes, 10 mM NaCl, 10 mM EGTA, 4 mM CaCl₂, 0.3 mM Na2GTP, 2 mM Mg-ATP, 10 mM HEPES and 2 μM Src family kinase activator (pH 7.4; from Santa Cruz). Bath solutions contained 150 mM NaCl, 3.5 mM KCl, 1 mM MgCl2, 1.2 mM CaCl2, 20 mM glucose, and 10 mM HEPES (pH 7.4). In the NMDG bath, Na⁺ and K⁺ were replaced by NMDG⁺. Liquid junction potentials were corrected online.