Mutations in NALCN cause an autosomal-recessive syndrome with severe hypotonia, speech impairment, and cognitive delay

Abstract

Sodium leak channel, nonselective (NALCN) is a voltage-independent and cation-nonselective channel that is mainly responsible for the leaky sodium transport across neuronal membranes and controls neuronal excitability. Although NALCN variants have been conflictingly reported to be in linkage disequilibrium with schizophrenia and bipolar disorder, to our knowledge, no mutations have been reported to date for any inherited disorders. Using linkage, SNP-based homozygosity mapping, targeted sequencing, and confirmatory exome sequencing, we identified two mutations, one missense and one nonsense, in NALCN in two unrelated families. The mutations cause an autosomal-recessive syndrome characterized by subtle facial dysmorphism, variable degrees of hypotonia, speech impairment, chronic constipation, and intellectual disability. Furthermore, one of the families pursued preimplantation genetic diagnosis on the basis of the results from this study, and the mother recently delivered healthy twins, a boy and a girl, with no symptoms of hypotonia, which was present in all the affected children at birth. Hence, the two families we describe here represent instances of loss of function in human NALCN.

Figure 1

Genetic Analysis of the Autosomal-Recessive Disorder Caused by NALCN Mutations (A and B) Pedigrees and selected chromatograms indicating mutations and carrier status of the tested members of families 1 (A) and 2 (B) are depicted. V-3 and V-4 are the outcome of preimplantation genetic diagnosis (PGD). PGD was performed on the basis of homozygosity mapping results, and neither individual has any NALCN mutations. (C) Affymetrix GeneChip Human Mapping Axiom custom SNP arrays were used for genotyping, and the SNP calls were used as an input for the AutoSNPa tool. Homozygosity analysis indicated a shared run of homozygosity in both families. The block contains 48 genes, including NALCN. (D) Parametric multipoint LOD score analysis was performed with the GeneHunter algorithm with Easy Linkage software. A recessive mode of inheritance, a disease allele frequency of 0.0001%, and no phenocopies were used in the analysis. Combined linkage results for both families indicated a single dominant peak (LOD score 5.01) on chromosome 13.

Figure 2

Location of NALCN Mutations and Immunoblotting and NMD Analyses (A) Coding exons of NALCN are sequentially colored with dark gray and white (upper panel). Mutations are indicated in the relevant exons [13 and 34]). Pfam domains and low-complexity, transmembrane, coiled-coil, and unstructured regions are depicted on the basis of the Pfam source from the Howard Hughes Medical Institute (Web Resources). The protein structure is positioned according to the coding exons and amino acids (lower panel). (B) Immunoblotting experiments performed on fibroblasts (from individuals V-1 and V-2) and DAOY cell lines indicated the absence of NALCN in the affected individuals but the presence of the protein in the control cell line. 100 μg/ml of the protein was used for immunoblotting, and β-actin was used as an internal control. (C) qRT-PCR experiments were performed on RNA extracted from V-1’s and V-2’s fibroblasts treated with CHX (100 μg/ml) at different periods of time (0, 8, 16, and 24 hr). Control fibroblasts and samples from affected individuals at hour 0 were not treated with CHX. The data represent the mean fold change ± SE of three control samples and samples from two affected individuals for each time point (each sample was run in triplicate). Compared to controls, the affected individuals showed a significant decrease in expression. ^∗p < 0.05, ^∗∗p < 0.01 (Student’s t test).