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De Novo Mutations in NALCN Cause a Syndrome Characterized by Congenital Contractures of the Limbs and Face, Hypotonia, and Developmental Delay

2015; Elsevier BV; Volume: 96; Issue: 3 Linguagem: Inglês

10.1016/j.ajhg.2015.01.003

1537-6605

Jessica X. Chong, Margaret J. McMillin, Kathryn M. Shively, Anita Beck, Colby T. Marvin, Jose R. Armenteros, Kati J. Buckingham, Naomi T. Nkinsi, Evan A. Boyle, Margaret N. Berry, Maureen Bocian, Nicola Foulds, Maria Luisa Giovannucci Uzielli, Chad R. Haldeman‐Englert, Raoul C.M. Hennekam, Paige Kaplan, Antonie D. Kline, Catherine L. Mercer, Małgorzata J.M. Nowaczyk, Jolien S. Klein Wassink‐Ruiter, Elizabeth W. McPherson, Regina A. Moreno, Angela E. Scheuerle, Vandana Shashi, Cathy A. Stevens, John C. Carey, Arnaud Monteil, Philippe Lory, Holly K. Tabor, Joshua D. Smith, Jay Shendure, Deborah A. Nickerson, Michael J. Bamshad, Michael J. Bamshad, Jay Shendure, Deborah A. Nickerson, Gonçalo R. Abecasis, Peter Anderson, Elizabeth Blue, Marcus Annable, Brian L. Browning, Kati J. Buckingham, Christina Chen, Jennifer Chin, Jessica X. Chong, Gregory M. Cooper, Colleen Davis, Christopher Frazar, Tanya M. Harrell, Zongxiao He, Preti Jain, Gail P. Jarvik, Guillaume Jimenez, Eric Johanson, Goo Jun, Martin Kircher, Tom Kolar, Stephanie Krauter, Niklas Krumm, Suzanne M. Leal, Daniel Luksic, Colby T. Marvin, Margaret J. McMillin, Sean McGee, Patrick O’Reilly, Bryan Paeper, Karynne Patterson, M. Lázaro Pérez, Sam W. Phillips, Jessica Pijoan, Christa Poel, Frédéric Reinier, Peggy D. Robertson, Regie Lyn P. Santos‐Cortez, Tristan Shaffer, Cindy Shephard, Kathryn M. Shively, Deborah L. Siegel, Joshua D. Smith, Jeffrey Staples, Holly K. Tabor, Monica Tackett, Jason G. Underwood, Marc Wegener, Gao Wang, Marsha M. Wheeler, Yi Qian,

Erythrocyte Function and Pathophysiology

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). 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). Distal arthrogryposis (DA) is a group of at least ten disorders that are characterized by non-progressive congenital contractures of two or more body areas and that typically affect the wrists, hands, ankles, and feet.1Sung S.S. Brassington A.-M.E. Grannatt K. Rutherford A. Whitby F.G. Krakowiak P.A. Jorde L.B. Carey J.C. Bamshad M. Mutations in genes encoding fast-twitch contractile proteins cause distal arthrogryposis syndromes.Am. J. Hum. Genet. 2003; 72: 681-690Abstract Full Text Full Text PDF PubMed Scopus (167) Google Scholar The three most common DA syndromes are DA1 (MIM 108120),1Sung S.S. Brassington A.-M.E. Grannatt K. Rutherford A. Whitby F.G. Krakowiak P.A. Jorde L.B. Carey J.C. Bamshad M. Mutations in genes encoding fast-twitch contractile proteins cause distal arthrogryposis syndromes.Am. J. Hum. Genet. 2003; 72: 681-690Abstract Full Text Full Text PDF PubMed Scopus (167) Google Scholar, 2Sung S.S. Brassington A.-M.E. Krakowiak P.A. Carey J.C. Jorde L.B. Bamshad M. Mutations in TNNT3 cause multiple congenital contractures: a second locus for distal arthrogryposis type 2B.Am. J. Hum. Genet. 2003; 73: 212-214Abstract Full Text Full Text PDF PubMed Scopus (108) Google Scholar DA2A (Freeman-Sheldon syndrome [MIM 193700]),3Toydemir R.M. Rutherford A. Whitby F.G. Jorde L.B. Carey J.C. Bamshad M.J. Mutations in embryonic myosin heavy chain (MYH3) cause Freeman-Sheldon syndrome and Sheldon-Hall syndrome.Nat. Genet. 2006; 38: 561-565Crossref PubMed Scopus (187) Google Scholar and DA2B1Sung S.S. Brassington A.-M.E. Grannatt K. Rutherford A. Whitby F.G. Krakowiak P.A. Jorde L.B. Carey J.C. Bamshad M. Mutations in genes encoding fast-twitch contractile proteins cause distal arthrogryposis syndromes.Am. J. Hum. Genet. 2003; 72: 681-690Abstract Full Text Full Text PDF PubMed Scopus (167) Google Scholar, 2Sung S.S. Brassington A.-M.E. Krakowiak P.A. Carey J.C. Jorde L.B. Bamshad M. Mutations in TNNT3 cause multiple congenital contractures: a second locus for distal arthrogryposis type 2B.Am. J. Hum. Genet. 2003; 73: 212-214Abstract Full Text Full Text PDF PubMed Scopus (108) Google Scholar (Sheldon-Hall syndrome [MIM 601680]). DA1 and DA2B can be caused by variants in any one of several genes, including TPM2 (MIM 190990), TNNT3 (MIM 600692), TNNI2 (MIM 191043), and MYH3 (MIM 160720),4Beck A.E. McMillin M.J. Gildersleeve H.I. Kezele P.R. Shively K.M. Carey J.C. Regnier M. Bamshad M.J. Spectrum of mutations that cause distal arthrogryposis types 1 and 2B.Am. J. Med. Genet. A. 2013; 161A: 550-555Crossref PubMed Scopus (48) Google Scholar whereas DA2A is only caused by mutations in MYH3. Moreover, mutations in MYH3 are found in more than 90% of persons who meet the diagnostic criteria for DA2A.5Beck A.E. McMillin M.J. Gildersleeve H.I. Shively K.M. Tang A. Bamshad M.J. Genotype-phenotype relationships in Freeman-Sheldon syndrome.Am. J. Med. Genet. A. 2014; 164A: 2808-2813Crossref PubMed Scopus (33) Google Scholar Among a subset of DA2A-affected persons who were referred to our research program over the past decade and in whom no pathogenic mutations were identified in MYH3, we recognized a pattern of clinical characteristics suggestive of a distinctive and previously unrecognized multiple-malformation syndrome. Specifically, we identified a subset of five families, each with an affected child born to unaffected parents, in which the proband had congenital contractures of the limbs (hands and/or feet) and face; abnormal tone, most commonly manifested as hypotonia; neonatal respiratory distress; and developmental delay. These features are collectively referred to as CLIFAHDD syndrome (Table 1, families A–E; Figure 1; Figures S1 and S2).Table 1Mutations and Clinical Findings For Individuals with CLIFAHDD SyndromeFamily AFamily BFamily CFamily DFamily EFamily FFamily GFamily HFamily IFamily JFamily KFamily LFamily MFamily NOriginal DiagnosisCLIFAHDDCLIFAHDDCLIFAHDDCLIFAHDDCLIFAHDDDA2ADA2ADA2ADA2BDA2BDA2BDA2BDA1DA1Mutation InformationExon (NALCN)691539318132691314311326cDNA changec.530A>Cc.938T>Gc.1768C>Tc.4338T>Gc.3493A>Cc.934C>Ac.1526T>Cc.3017T>Cc.979G>Ac.1538C>Ac.1733A>Cc.3542G>Ac.1534T>Gc.3050T>CPredicted protein alterationp.Gln177Prop.Val313Glyp.Leu590Phep.Ile1446Metp.Thr1165Prop.Leu312Ilep.Leu509Serp.Val1006Alap.Glu327Lysp.Thr513Asnp.Tyr578Serp.Arg1181Glnp.Phe512Valp.Ile1017ThrGERP4.926.165.944.735.26.165.115.036.165.115.35.25.115.03CADD 1.0 (phred-like)19.2225.923.916.0821.032.022.624.236.025.523.720.524.617.54Polyphen-2 (HumVar)1.01.00.9960.9950.9691.01.00.9991.01.01.00.4441.01.0Clinical Features: FaceDownslanting palpebral fissures–+ND+++++?+++–+Strabismus–+ND+++++–+––––Esotropia–+ND–+–––––+–––Broad nasal bridge++++++++++++++Anteverted nasal tip+++++–++++–+++Large nares++++++++++++++Short columella++++++++++++++Long philtrum+++++++++++––+Micrognathia+++++++++++++–Pursed lips+++++–+––+++–?H-shaped dimpled chin++++–++––+–+––Deep nasolabial folds+++++–+++–++++Full cheeks++++++–+++++++Clinical Features: LimbsCamptodactyly++++++++++++++Ulnar deviation++++++++++++++Adducted thumbs++++++++++++++Clubfoot+++Lmetatarsus adductus++mild L varus+++–mild varus+Calcaneovalgus deformity–+–R––––––––+–Hip contractures+++BL dislocation––+–++++internally rotated+Elbow contractures+++––+++–+––Knee contractures+–++–++++–++––Other Clinical FeaturesScoliosis++–+–ND+–––+–lumbar lordosis–Short neck+++++ND+–+–+++Cognitive delayNDaChild died before an assessment could be made.+NDaChild died before an assessment could be made.+++–+++++++Speech delayNa+Na+++++++++++Motor delay++++++++++++++Seizures++––––––––––––Questionable seizure activity or abnormal breathing, loss of motor controlND+NDabnormal dischargesperiods of rigidity++NDND++seizure-like activity with feverNDirregular breathingMRI findingsnormalmild cerebral and cerebellar atrophyNDgeneralized cerebral atrophy, small pituitarynormalcerebellar atrophynormalNDNDdiffuse cerebellar atrophyNDnormalnormalwhite matter edemaHypotonianoyesyesyesyesyesnoyesNDnonono?yesnoRespiratory insufficiency+++++apneic episodes in newborn period–NDNDND+ND–+Abnormal respiratory pattern in newborn period++++++–NDNDND++–+Excessive droolingNDNDND+NDND+++NDNDGERDND+ND+++ND–++++in infancy–ConstipationNDoccasionalND–+NDND+++NDNDHernia–inguinalinguinalinguinal / peri umbilicalinguinal–inguinal–inguinalinguinalinguinalNDumbilical–Age at time of death6 months5 years4 monthsNaNaNaNaNaNaNaNaNaNaNaOthercentral apnea, uric acid renal stones11 pairs of ribs, abnormal pelvis and scapulaerenal calcificationssuspected fatty acid oxidation defectcleft palatemitral valve prolapsesmall VSDThis table provides a summary of clinical features of affected individuals from families in which mutations in NALCN were identified. Clinical characteristics listed in the table are primarily features that distinguish CLIFAHDD syndrome from DA conditions. Abbreviations are as follows: +, presence of a finding; −, absence of a finding; ND, no data were available; NA, not applicable; GERP, genomic evolutionary rate profiling; CADD, combined annotation-dependent depletion; CLIFAHDD, congenital contractures of the limbs, face, hypotonia, and developmental delay; DA2A, distal arthrogryposis type 2A; DA2B, distal arthrogryposis type 2B; DA1, distal arthrogryposis type 1; VSD, ventricular septal defect; and GERD, gastresophageal reflux disease. cDNA positions are provided as named by the HGVS MutNomen web tool relative to NM_052867.2.a Child died before an assessment could be made. Open table in a new tab This table provides a summary of clinical features of affected individuals from families in which mutations in NALCN were identified. Clinical characteristics listed in the table are primarily features that distinguish CLIFAHDD syndrome from DA conditions. Abbreviations are as follows: +, presence of a finding; −, absence of a finding; ND, no data were available; NA, not applicable; GERP, genomic evolutionary rate profiling; CADD, combined annotation-dependent depletion; CLIFAHDD, congenital contractures of the limbs, face, hypotonia, and developmental delay; DA2A, distal arthrogryposis type 2A; DA2B, distal arthrogryposis type 2B; DA1, distal arthrogryposis type 1; VSD, ventricular septal defect; and GERD, gastresophageal reflux disease. cDNA positions are provided as named by the HGVS MutNomen web tool relative to NM_052867.2. Facial characteristics shared among individuals with CLIFAHDD syndrome include downslanting palpebral fissures, a broad nasal bridge with an anteverted nasal tip and large nares, a short columella, a long philtrum, deep nasolabial folds, micrognathia, pursed lips, and chin dimpling resembling the “H-shaped” dimpling observed in persons with DA2A (Figure 1). Each person in our subset had camptodactyly with adducted thumbs, as well as positional foot deformities ranging from mild varus deformity to severe clubfoot. Additionally, contractures of the elbows, knees, and hips, as well as a short neck and scoliosis, were observed. Neurological evaluation of each living child with CLIFAHDD syndrome revealed a global developmental delay manifesting as speech, motor, and cognitive delays that varied from mild to severe, as well as hypotonia and seizures. Magnetic resonance imaging of the brain, conducted on four of the affected persons, was reported as normal in two persons and abnormal in two. Of the latter, one person was reported to have cerebral and cerebellar atrophy and another to have cerebral atrophy and a “small pituitary.” Severe gastresophageal reflux was observed in all three individuals for whom data were available, and four of five individuals had inguinal hernias. Three of the five affected individuals died in infancy or early childhood. One of these three individuals died of hemophagocytosis complications in the newborn period, whereas the exact cause of death was unclear in the other two. To find the mutation-harboring gene(s) in individuals with CLIFAHDD syndrome, we first screened the putatively CLIFAHDD-syndrome-diagnosed probands from each of the five families by performing Sanger sequencing for mutations in genes, other than MYH3, known to cause DA1, DA2A, and DA2B. Each proband with CLIFAHDD syndrome was also screened for copy-number variations (CNVs) by comparative genome-wide array genomic hybridization on the Illumina HumanCytoSNP-12. No pathogenic mutations or shared CNVs were identified. Next, exome sequencing was performed on four CLIFAHDD-syndrome-affected parent-child trios for whom sufficient quantities of DNA were available. All studies were approved by the institutional review boards of the University of Washington and Seattle Children’s Hospital, and informed consent was obtained from participants or their parents. 1 μg genomic DNA was subjected to a series of shotgun-library construction steps, including fragmentation through acoustic sonication (Covaris), end polishing (NEBNext End Repair Module), A-tailing (NEBNext dA-Tailing Module), and PCR amplification with ligation of 8 bp barcoded sequencing adaptors (Enzymatics Ultrapure T4 Ligase) for multiplexing. We hybridized 1 μg of barcoded shotgun library to capture probes targeting ∼36.5 Mb of coding exons (Roche Nimblegen SeqCap EZ Human Exome Library v.2.0). Library quality was determined by examination of molecular-weight distribution and sample concentration (Agilent Bioanalyzer). Pooled, barcoded libraries were sequenced via paired-end 50 bp reads with an 8 bp barcode read on Illumina HiSeq sequencers. Demultiplexed BAM files were aligned to a human reference (UCSC Genome Browser hg19) via the Burrows-Wheeler Aligner (BWA) v.0.6.2. Read data from a flow-cell lane were treated independently for alignment and quality-control purposes in instances where the merging of data from multiple lanes was required. All aligned read data were subjected to (1) removal of duplicate reads (Picard MarkDuplicates v.1.70), (2) indel realignment (GATK IndelRealigner v.1.6-11-g3b2fab9), and (3) base-quality recalibration (GATK TableRecalibration v.1.6-11-g3b2fab9). Variant detection and genotyping were performed with GATK UnifiedGenotyper (v.1.6-11-g3b2fab9). Variant data for each sample were formatted (variant-call format) as “raw” calls that contained individual genotype data for one or multiple samples and were flagged with the filtration walker (GATK) so that lower-quality sites and potential false positives (e.g., strand bias > −0.1, quality scores (QUAL) ≤ 50, allelic imbalance (ABHet) ≥ 0.75, long homopolymer runs (HRun) > 3, and/or low quality by depth (QD) < 5). Variants with an alternative allele frequency >0.005 in the NHLBI Exome Sequencing Project Exome Variant Server (ESP6500) or the 1000 Genomes Browser or >0.05 in an internal exome database of ∼700 individuals were excluded prior to analysis. Additionally, variants that were flagged as low quality or potential false positives (quality score ≤ 30, long homopolymer run > 5, low quality by depth < 5, within a cluster of SNPs) were also excluded from analysis. Variants that were only flagged by the strand-bias filter (strand bias > −0.10) were included in further analyses because the strand-bias flag has previously been found to be applied to valid variants. CNV calls were also generated from exome data (CoNIFER).6Krumm N. Sudmant P.H. Ko A. O’Roak B.J. Malig M. Coe B.P. Quinlan A.R. Nickerson D.A. Eichler E.E. NHLBI Exome Sequencing ProjectCopy number variation detection and genotyping from exome sequence data.Genome Res. 2012; 22: 1525-1532Crossref PubMed Scopus (433) Google Scholar Variants were annotated with the SeattleSeq137 Annotation Server, and variants for which the only functional prediction label was “intergenic,” “coding-synonymous,” “UTR,” “near-gene,” or “intron” were excluded. Individual genotypes with depth < 6 or genotype quality < 20 were treated as missing. By analysis of variants from exome sequencing under a de novo mutation model, we identified four different de novo variants in a single gene, NALCN, encoding a sodium leak channel (NALCN [MIM 611549; Refseq accession number NM_052867.2]), in four CLIFAHDD-syndrome-affected families (Table 1 and Figure 1, Families A–D; Figures S1 and S2). Concurrent exome sequencing of six DA-affected trios in whom no pathogenic variants in genes previously associated with DA had been identified serendipitously revealed de novo NALCN variants in two families (Table 1, families G and K; Figure S3). All six variants were missense variants predicted to be deleterious and to result in amino acid substitutions of highly conserved amino acid residues (i.e., the minimum genomic evolutionary rate profile score was 4.73) in NALCN (Table 1 and Figure 2). Each variant was confirmed by Sanger sequencing to have arisen de novo, and none of the six variants were found in over 71,000 control exomes recorded in the ESP6500, 1000 Genomes phase 1 (November, 2010 release), internal databases (> 1,400 chromosomes), or the Exome Aggregation Consortium (ExAC) browser (October 20, 2014 release). Because the phenotype of the individuals with mutations in NALCN was variable and overlapped with other DA syndromes, we used molecular inversion probes with 5-bp molecular tags7Hiatt J.B. Pritchard C.C. Salipante S.J. O’Roak B.J. Shendure J. Single molecule molecular inversion probes for targeted, high-accuracy detection of low-frequency variation.Genome Res. 2013; 23: 843-854Crossref PubMed Scopus (228) Google Scholar (smMIPs; designed with MIPGen8Boyle E.A. O’Roak B.J. Martin B.K. Kumar A. Shendure J. MIPgen: optimized modeling and design of molecular inversion probes for targeted resequencing.Bioinformatics. 2014; 30: 2670-2672Crossref PubMed Scopus (107) Google Scholar v.0.9.7) to conduct targeted next-generation sequencing of NALCN in 202 additional samples from DA-affected individuals in whom no pathogenic mutation had been found. The 43 coding exons (5,214 bp total) of Ensembl transcript ENST00000251127 and the 10 bp flanking each exon (860 bp total) were targeted with smMIPs for an overall target size of 6,074 bp. Pooled and phosphorylated smMIPs were added to the capture reactions with 100 ng of genomic DNA from each individual to produce a NALCN library for each individual. The libraries were amplified during 21 cycles of PCR, during which an 8-bp sample barcode was introduced. The barcoded libraries were then pooled and purified with magnetic beads. After Picogreen quantification to determine the appropriate dilution, 10 pmol of the pool was sequenced on an Illumina MiSeq. Molecular-inversion-probe collapsing and arm trimming (MIPGenM v.1.0), alignment (BWA v.0.7.8), and multi-sample genotype calling (GATK Unified Genotyper v.3.2-2-gec30cee) were performed, and variants were annotated with SeattleSeq138. We used the same filtering strategy employed in our analysis of the exome sequences to select variants for further confirmation by Sanger sequencing. Missense variants predicted to result in substitution of conserved amino acid residues were identified and confirmed to have arisen de novo in eight additional families: one CLIFAHDD-syndrome-affected family that had not been available for exome sequencing (Table 1), two families in which affected members were originally diagnosed with DA2A (Table 1, families F and H; Figure S1; Figure S3, family F), three families in which affected members were originally diagnosed with DA2B (Table 1, families I, J, and L; Figure S1; Figure S2, families I and J), and two families in which affected members were diagnosed with DA1 (Table 1, families M and N; Figure S1; Figure S3, family M). Altogether, we discovered unique, de novo missense NALCN mutations in fourteen simplex families in which the proband had been diagnosed with CLIFAHDD syndrome (n = 5), DA2A (n = 3), DA2B (n = 4), or DA1 (n = 2) (Table 1 and Figure 2). The diagnosis of CLIFAHDD syndrome was considered in one additional DA2A case brought to our attention because of a pattern of congenital facial and limb contractures characteristic of DA2A in the absence of a finding of a pathogenic MYH3 mutation. However, death occurred within two hours of birth at 29 weeks gestation, and the available clinical information was therefore considered too limited. This approach proved prudent given that no mutation in NALCN was identified by exome sequencing. Instead, this child was found to be a compound heterozygote in RYR1 (MIM 180901) for a predicted nonsense variant, p. Tyr3921Ter (c.11763C>A [RefSeq NM_000540.2]), inherited from the father, and a missense variant, p.Gly341Arg (c.1021G>A [RefSeq NM_000540.2]), inherited from the mother. The missense variant has been previously reported to cause malignant hyperthermia, and persons carrying this variant had a positive “in vitro contractility test.”9Monsieurs K.G. Van Broeckhoven C. Martin J.J. Van Hoof V.O. Heytens L. Gly341Arg mutation indicating malignant hyperthermia susceptibility: specific cause of chronically elevated serum creatine kinase activity.J. Neurol. Sci. 1998; 154: 62-65Abstract Full Text Full Text PDF PubMed Scopus (14) Google Scholar Crisponi syndrome10Crisponi L. Crisponi G. Meloni A. Toliat M.R. Nürnberg G. Usala G. Uda M. Masala M. Höhne W. Becker C. et al.Crisponi syndrome is caused by mutations in the CRLF1 gene and is allelic to cold-induced sweating syndrome type 1.Am. J. Hum. Genet. 2007; 80: 971-981Abstract Full Text Full Text PDF PubMed Scopus (70) Google Scholar, 11Dagoneau N. Bellais S. Blanchet P. Sarda P. Al-Gazali L.I. Di Rocco M. Huber C. Djouadi F. Le Goff C. Munnich A. Cormier-Daire V. Mutations in cytokine receptor-like factor 1 (CRLF1) account for both Crisponi and cold-induced sweating syndromes.Am. J. Hum. Genet. 2007; 80: 966-970Abstract Full Text Full Text PDF PubMed Scopus (50) Google Scholar (MIM 601378) was also considered as a possible diagnosis for this child. However, no candidate variants were identified in CRLF1 (MIM 604237), either in this child or in any of the fourteen families in which NALCN mutations were identified. In addition, we have previously found two mutations in RYR1 (i.e., c.1391A>G[p.Gln464Arg] and c.2683-1G>A) in a child diagnosed with DA2A, suggesting that mutations in RYR1 are a rare cause of DA2A. Mutations in NALCN have recently been reported to cause an autosomal-recessive condition, infantile hypotonia with psychomotor retardation and characteristic facies (IHPRF [MIM 615419]), in one Turkish family12Köroğlu Ç. Seven M. Tolun A. Recessive truncating NALCN mutation in infantile neuroaxonal dystrophy with facial dysmorphism.J. Med. Genet. 2013; 50: 515-520Crossref PubMed Scopus (49) Google Scholar and two Saudi Arabian13Al-Sayed M.D. Al-Zaidan H. Albakheet A. Hakami H. Kenana R. Al-Yafee Y. Al-Dosary M. Qari A. Al-Sheddi T. Al-Muheiza M. et al.Mutations in NALCN cause an autosomal-recessive syndrome with severe hypotonia, speech impairment, and cognitive delay.Am. J. Hum. Genet. 2013; 93: 721-726Abstract Full Text Full Text PDF PubMed Scopus (63) Google Scholar families. Individuals affected by CLIFAHDD syndrome share some of the phenotypic characteristics of IHPRF (e.g., developmental delay and hypotonia). However, these are relatively non-specific findings, and IHPRF and CLIFAHDD syndrome appear to be otherwise distinct from one another. In addition, the parents of children with IHPRF are carriers of NALCN mutations but reportedly do not have any of the abnormalities found in persons with CLIFAHDD syndrome. Furthermore, each inherited NALCN variant found in the 14 individuals with CLIFAHDD syndrome was also present in ESP6500 and/or 1000 Genomes data. This finding suggests that these inherited variants are polymorphisms rather than pathogenic variants underlying CLIFAHDD syndrome. Together, these results support the hypothesis that CLIFAHDD syndrome is an autosomal-dominant condition distinct from IHPRF. We next sought to explore the mechanism by which variants in NALCN result in CLIFAHDD syndrome. To this end, we constructed two expression plasmids carrying NALCN variants: pcDNA3-hNALCN-L509S-GFP with c.1526>T(p.Leu509Ser) found in a person with relatively mild developmental delay (Table 1, family G) and pcDNA3-hNALCN-Y578S-GFP with c.1733A>C(p.Tyr578Ser) found in a child with severe developmental delay (Table 1, family K). We then co-expressed mutant and wild-type NALCN in HEK293T cells. Immunoblot analysis demonstrated that the expression of either the p.Tyr578Ser or p.Leu509Ser (GFP-tagged) NALCN alteration nearly eliminated wild-type (HA-tagged) NALCN protein (Figure 3). It remains to be determined whether synthesis of altered NALCN channels prevents wild-type NALCN synthesis or induces its degradation by the proteasome. The latter mechanism has been previously described14Mezghrani A. Monteil A. Watschinger K. Sinnegger-Brauns M.J. Barrère C. Bourinet E. Nargeot J. Striessnig J. Lory P. A destructive interaction mechanism accounts for dominant-negative effects of misfolded mutants of voltage-gated calcium channels.J. Neurosci. 2008; 28: 4501-4511Crossref PubMed Scopus (65) Google Scholar with regard to mutations in the Cav1.2 calcium channel responsible for dominantly inherited episodic ataxia type 2 (MIM 108500). However, preliminary evidence suggests that NALCN alterations are correctly targeted to the plasma membrane (data not shown). Nevertheless, both mutations apparently induced the disappearance of wild-type NALCN to the same extent. Accordingly, an explanation for the observed phenotypic differences between the sampled cases is not yet apparent. Collectively, these functional studies, although preliminary, are consistent with mutations in NALCN causing CLIFAHDD syndrome via a dominant-negative effect that results in haploinsufficiency. However, the finding that persons heterozygous for NALCN mutations that cause IHPRF are reportedly unaffected indicates that haploinsufficiency of NALCN alone is not adequate to cause CLIFAHDD syndrome. These observations suggest that NALCN mutations that cause CLIFAHDD syndrome might produce a mutant protein that has some residual activity (i.e., a hypomorphic allele) or that exhibits a gain of function. Moreover, mutations in NALCN could cause CLIFAHDD syndrome by more than one mechanism. NALCN is a G-protein-coupled receptor-activated channel15Swayne L.A. Mezghrani A. Varrault A. Chemin J. Bertrand G. Dalle S. Bourinet E. Lory P. Miller R.J. Nargeot J. Monteil A. The NALCN ion channel is activated by M3 muscarinic receptors in a pancreatic beta-cell line.EMBO Rep. 2009; 10: 873-880Crossref PubMed Scopus (82) Google Scholar consisting of four homologous domains (domains I–IV), each of which consists of six transmembrane segments (S1–S6) separated by cytoplasmic linkers (Figure 2). Pore-forming loops (P loops) between S5 and S6 of each domain form an EEKE sodium-ion selectivity filter (Figure 2). In vertebrates and some invertebrate model organisms, such as Drosophila and C. elegans, NALCN has a sodium-selective EEKE pore and putatively functions as a sodium channel. However, it remains unclear whether NALCN is actually an ion channel rather than a sensor of sodium.16Senatore A. Spafford J.D. A uniquely adaptable pore is consistent with NALCN being an ion sensor.Channels (Austin). 2013; 7: 60-68Crossref PubMed Scopus (14) Google Scholar In mammals, NALCN is most highly expressed in the central nervous system but is also found at moderate levels in the heart, lymph nodes, pancreas, and thyroid (summarized by Cochet-Bissuel et al.17Cochet-Bissuel M. Lory P. Monteil A. The sodium leak channel, NALCN, in health and disease.Front. Cell. Neurosci. 2014; 8: 132Crossref PubMed Scopus (81) Google Scholar). Unlike most known genes underlying DA syndromes, NALCN is not expressed in fetal skeletal muscle (Figure S4). However, homologs of NALCN are expressed in the neuromuscular junction in D. melanogaster18Nishikawa K. Kidokoro Y. Halothane presynaptically depresses synaptic transmission in wild-type Drosophila larvae but not in halothane-resistant (har) mutants.Anesthesiology. 1999; 90: 1691-1697Crossref PubMed Scopus (26) Google Scholar as well as in motor neurons in C. elegans19Yeh E. Ng S. Zhang M. Bouhours M. Wang Y. Wang M. Hung W. Aoyagi K. Me