De novo mutations in NALCN cause a syndrome characterized by congenital contractures of the limbs and face, hypotonia, and developmental delay

Abstract

Freeman-Sheldon syndrome, or distal arthrogryposis type 2A (DA2A), is an autosomal-dominant condition caused by mutations in MYH3 and characterized by multiple congenital contractures of the face and limbs and normal cognitive development. We identified a subset of five individuals who had been putatively diagnosed with "DA2A with severe neurological abnormalities" and for whom congenital contractures of the limbs and face, hypotonia, and global developmental delay had resulted in early death in three cases; this is a unique condition that we now refer to as CLIFAHDD syndrome. Exome sequencing identified missense mutations in the sodium leak channel, non-selective (NALCN) in four families affected by CLIFAHDD syndrome. We used molecular-inversion probes to screen for NALCN in a cohort of 202 distal arthrogryposis (DA)-affected individuals as well as concurrent exome sequencing of six other DA-affected individuals, thus revealing NALCN mutations in ten additional families with "atypical" forms of DA. All 14 mutations were missense variants predicted to alter amino acid residues in or near the S5 and S6 pore-forming segments of NALCN, highlighting the functional importance of these segments. In vitro functional studies demonstrated that NALCN alterations nearly abolished the expression of wild-type NALCN, suggesting that alterations that cause CLIFAHDD syndrome have a dominant-negative effect. In contrast, homozygosity for mutations in other regions of NALCN has been reported in three families affected by an autosomal-recessive condition characterized mainly by hypotonia and severe intellectual disability. Accordingly, mutations in NALCN can cause either a recessive or dominant condition characterized by varied though overlapping phenotypic features, perhaps based on the type of mutation and affected protein domain(s).

Figure 1

Phenotypic Characteristics of Each Individual with CLIFAHDD Syndrome Four individuals affected by CLIFAHDD syndrome; all individuals shown have NALCN mutations. Note the short palpebral fissures, flattened nasal root and bridge, large nares, long philtrum, pursed lips, H-shaped dimpling of the chin, and deep nasolabial folds (A, B, C₁, D). Camptodactyly of the digits of the hands and ulnar deviation of the wrist (A, B, C, D₁, D₂) or clubfoot (C₃) is present in each affected person. Case identifiers for the individuals shown in this figure correspond to those in Table 1, where there is a detailed description of the phenotype of each affected individual. Figure S1 provides a pedigree of each CLIFAHDD-syndrome-affected family.

Figure 2

Genomic and Protein Structure of NALCN and Spectrum of Mutations that Cause CLIFAHDD Syndrome (A) NALCN is composed of 45 exons, 43 of which are protein coding (blue) and two of which are non-coding (orange). Lines with attached dots indicate the approximate locations of 14 different de novo variants that cause CLIFAHDD syndrome. The color of each dot reflects the nearest transmembrane segment (S1–S6) depicted in (B) and (C). The two mutations between IIIS6 and IVS1 are located in the intracellular loop linking domains III and IV. This loop is known to be involved in the inactivation of voltage-gated sodium channels; however NALCN is not voltage-gated, so the specific function of this loop in NALCN is not known. (B) The predicted protein topology of NALCN is comprised of four homologous pore-forming domains (I–IV), each composed of six transmembrane segments (S1–S6). The bold line represents the polypeptide chain linking segments and domains. The outer ring of P loop amino acid residues (EEKE) that form the ion selectivity filter is represented by filled circles. The approximate positions of variants that cause CLIFAHDD syndrome and infantile hypotonia with psychomotor retardation and characteristic facies are indicated by red-filled circles and black-filled circles, respectively. (C) S1–S3 might have structural functions, whereas four P loops spanning from S5 to S6 form the ion selectivity filter. In other 4x6TM protein superfamily channels, such as SCN1A, the S4 segments initiate opening of the central pore (S5-P loop-S6) in response to voltage changes, however, because NALCN is not voltage-gated, the function of S4 is currently unknown.

Figure 3

Expression of p.Tyr578Ser and p.Leu509Ser NALCN Alterations Prevents Wild-Type NALCN Detection at the Protein Level when Co-Expressed in HEK293T Cells HA-tagged wild-type NALCN channels are not detectable when co-expressed with GFP-tagged NALCN mutant channels in HEK293T cells but are detectable when co-expressed with GFP-tagged wild-type NALCN channels. This experiment is representative of four independent experiments.