Phenotypic Mutation 'spindly' (pdf version)
Allelespindly
Mutation Type critical splice donor site
Chromosome12
Coordinate102,231,203 bp (GRCm39)
Base Change T ⇒ A (forward strand)
Gene Slc24a4
Gene Name solute carrier family 24 (sodium/potassium/calcium exchanger), member 4
Synonym(s) NCKX4, A930002M03Rik
Chromosomal Location 102,094,992-102,233,350 bp (+) (GRCm39)
MGI Phenotype FUNCTION: [Summary is not available for the mouse gene. This summary is for the human ortholog.] This gene encodes a member of the potassium-dependent sodium/calcium exchanger protein family. Alternative splicing results in multiple transcript variants.[provided by RefSeq, Jul 2010]
PHENOTYPE: Mice homozygous for a knock-out allele exhibit impaired olfactory response and reduced weight. [provided by MGI curators]
Accession Number

NCBI RefSeq: NM_172152; MGI:2447362

MappedYes 
Amino Acid Change
Institutional SourceBeutler Lab
Gene Model predicted gene model for protein(s): [ENSMUSP00000078030 ] [ENSMUSP00000124513]   † probably from a misspliced transcript
AlphaFold Q8CGQ8
SMART Domains Protein: ENSMUSP00000078030
Gene: ENSMUSG00000041771

DomainStartEndE-ValueType
signal peptide 1 21 N/A INTRINSIC
Pfam:Na_Ca_ex 86 229 2.4e-31 PFAM
low complexity region 367 388 N/A INTRINSIC
Pfam:Na_Ca_ex 435 587 2.4e-30 PFAM
Predicted Effect probably null
SMART Domains Protein: ENSMUSP00000124513
Gene: ENSMUSG00000041771
AA Change: V554E

DomainStartEndE-ValueType
transmembrane domain 23 45 N/A INTRINSIC
Pfam:Na_Ca_ex 113 245 1e-32 PFAM
low complexity region 365 386 N/A INTRINSIC
Pfam:Na_Ca_ex 443 562 1.4e-21 PFAM
Predicted Effect probably benign

PolyPhen 2 Score 0.387 (Sensitivity: 0.90; Specificity: 0.89)
(Using ENSMUST00000159329)
SMART Domains Protein: ENSMUSP00000125012
Gene: ENSMUSG00000041771

DomainStartEndE-ValueType
transmembrane domain 23 45 N/A INTRINSIC
Pfam:Na_Ca_ex 103 246 1.3e-31 PFAM
low complexity region 365 386 N/A INTRINSIC
Pfam:Na_Ca_ex 433 585 1.3e-30 PFAM
Predicted Effect probably null
Meta Mutation Damage Score 0.9489 question?
Is this an essential gene? Non Essential (E-score: 0.000) question?
Phenotypic Category Autosomal Recessive
Candidate Explorer Status loading ...
Single pedigree
Linkage Analysis Data
Penetrance  
Alleles Listed at MGI

All Mutations and Alleles(5) : Chemically induced (other)(1) Targeted(4)

Lab Alleles
AlleleSourceChrCoordTypePredicted EffectPPH Score
IGL01358:Slc24a4 APN 12 102189894 missense probably benign 0.09
IGL01724:Slc24a4 APN 12 102185219 missense possibly damaging 0.78
IGL01767:Slc24a4 APN 12 102189946 splice site probably benign
IGL01814:Slc24a4 APN 12 102220877 missense probably benign 0.00
IGL02047:Slc24a4 APN 12 102220882 missense probably damaging 1.00
IGL02449:Slc24a4 APN 12 102193341 missense probably benign 0.00
IGL02632:Slc24a4 APN 12 102200941 missense probably benign 0.15
IGL03251:Slc24a4 APN 12 102189084 missense probably damaging 0.98
R0207:Slc24a4 UTSW 12 102195210 critical splice donor site probably null
R0284:Slc24a4 UTSW 12 102226740 missense probably damaging 1.00
R0506:Slc24a4 UTSW 12 102097882 critical splice donor site probably null
R1903:Slc24a4 UTSW 12 102097876 missense probably benign 0.00
R2004:Slc24a4 UTSW 12 102180166 missense probably damaging 1.00
R2126:Slc24a4 UTSW 12 102189018 missense probably damaging 1.00
R2518:Slc24a4 UTSW 12 102188310 missense probably benign 0.02
R3498:Slc24a4 UTSW 12 102200951 missense probably benign
R3620:Slc24a4 UTSW 12 102185222 missense probably damaging 1.00
R3621:Slc24a4 UTSW 12 102185222 missense probably damaging 1.00
R4917:Slc24a4 UTSW 12 102231203 critical splice donor site probably null
R5028:Slc24a4 UTSW 12 102230629 missense probably damaging 1.00
R5886:Slc24a4 UTSW 12 102226674 missense probably damaging 1.00
R5914:Slc24a4 UTSW 12 102201049 missense probably damaging 1.00
R6257:Slc24a4 UTSW 12 102220769 missense probably benign 0.00
R6305:Slc24a4 UTSW 12 102188360 missense possibly damaging 0.84
R6313:Slc24a4 UTSW 12 102220769 missense probably benign 0.00
R6734:Slc24a4 UTSW 12 102185259 missense probably damaging 1.00
R7378:Slc24a4 UTSW 12 102205435 missense probably benign 0.06
R7419:Slc24a4 UTSW 12 102193350 critical splice donor site probably null
R7529:Slc24a4 UTSW 12 102230707 missense probably benign 0.01
R7715:Slc24a4 UTSW 12 102185219 missense possibly damaging 0.89
R7781:Slc24a4 UTSW 12 102201112 critical splice donor site probably null
R8258:Slc24a4 UTSW 12 102220928 missense probably damaging 1.00
R8259:Slc24a4 UTSW 12 102220928 missense probably damaging 1.00
R8766:Slc24a4 UTSW 12 102196711 missense probably benign 0.00
R8811:Slc24a4 UTSW 12 102180133 missense probably damaging 1.00
R9229:Slc24a4 UTSW 12 102200983 missense possibly damaging 0.79
R9339:Slc24a4 UTSW 12 102230638 missense probably damaging 1.00
R9599:Slc24a4 UTSW 12 102097779 missense probably benign 0.10
R9680:Slc24a4 UTSW 12 102193334 missense possibly damaging 0.95
Z1176:Slc24a4 UTSW 12 102205497 missense probably benign 0.01
Z1176:Slc24a4 UTSW 12 102195157 missense probably damaging 1.00
Z1177:Slc24a4 UTSW 12 102226679 missense probably damaging 0.99
Mode of Inheritance Autosomal Recessive
Local Stock
Repository
Last Updated 2019-09-04 9:41 PM by Anne Murray
Record Created 2016-12-19 11:25 AM
Record Posted 2018-04-26
Phenotypic Description

Figure 1. Spindly mice exhibited reduced body weights compared to wild-type littermates. Scaled body weight gene-based superpedigree data are shown for pedigrees R4917, R0207, and R0506. Abbreviations: WT, wild-type; REF, homozygous reference mice; HET, heterozygous variant mice; VAR, homozygous variant mice. Mean (μ) and standard deviation (σ) are indicated.

The spindly phenotype was identified among N-ethyl-N-nitrosourea (ENU)-mutagenized G3 mice of the pedigree R4917 some of which showed reduced body weights (Figure 1).

Nature of Mutation
Figure 2. Linkage mapping of the reduced body weights using a recessive model of inheritance. Manhattan plot shows -log10 P values (Y-axis) plotted against the chromosome positions of mutations (X-axis) identified in the G1 male of pedigrees R4917, R0207, and R0506. Normalized phenotype data are shown for gene-based superpedigree analysis without consideration of G2 dam identity. Horizontal pink and red lines represent thresholds of P = 0.05, and the threshold for P = 0.05 after applying Bonferroni correction, respectively.

Whole exome HiSeq sequencing of the G1 grandsire identified 99 mutations.  The reduced body weight phenotype was linked to a mutation in Slc24a4 by gene-based superpedigree analysis of the pedigrees R0207, R0506, and R4917. The Slc24a4 mutation in R4917 is a T to A transversion at base pair 102,264,944 (v38) on chromosome 12, or base pair 136,294 in the GenBank genomic region NC_000078 within the donor splice site of intron 16 in the cDNA transcript ENSMUST00000079020.10 (corresponding to the mRNA sequence NM_172152).  Linkage was found with a recessive model of inheritance, wherein five variant homozygotes departed phenotypically from 15 homozygous reference mice and 30 heterozygous mice with a P value of 1.522 x 10-7 (Figure 2).

The effect of the mutation at the cDNA and protein level have not examined, but if the mutation affects this transcript it is predicted to result in skipping of the 66-nucleotide exon 16 (out of 17 total exons), resulting in an in-frame deletion of amino acids 534 to 555; the rest of the protein product is unchanged.

         <--exon 15     <--exon 16 intron 16-->              <--exon 17-->

1610 ……TATGGATCCACA ……GTCGCTCTCACT gtgagtttttgtcattt…… GTCCTTGGTATT……GAAGACGACTAA
530  ……-Y--G--S--T- ……-V--A--L--T-                     -V--L--G--I-……-E--D--D--*-
         correct        deleted                                  correct

The donor splice site of intron 16, which is putatively destroyed by the spindly mutation, is indicated in blue lettering and the mutated nucleotide is indicated in red.

Illustration of Mutations in
Gene & Protein
Protein Prediction
Figure 3. Domain organization and topology of NCKX4. The transmembrane domains (TM) and signal peptide (SP) are indicated. The spindly  mutation is noted in the donor splice of intron 16.

Slc24a4 encodes Na+–Ca2++ K+ exchanger protein 4 (NCKX4), a member of solute carrier family 24 that also includes NCKX1, NCKX2, NCKX3, and NCKX5 (1). All of the NCKX proteins function as K+-dependent Na+--Ca2+ exchangers.

NCKX4 has 10 predicted transmembrane domains arranged in three clusters (1). The transmembrane domains are divided into two clusters of five transmembrane domains each separated by a cytosolic hydrophilic loop. The first transmembrane domain, designated TM0, is within the signal peptide and is cleaved. The central portion of the clusters, designated α1 and α2 repeats, are highly conserved. Residues within the repeats are required for NCKX transport activity. The N- and C-termini are both predicted to be extracellular after cleavage of TM0.

During Na+–Ca2+ exchange, Na+ ions bind to sites on NCKX4 exposed to the extracellular space (2). Na+ binding induces a conformational change in which the bound ions become inaccessible to the extracellular space and accessible to the intracellular environment for release into the cytoplasm. After sodium release into the intracellular space, the empty binding sites can bind calcium and potassium from the cytosol. Calcium or potassium binding induces a conformational change in the NCKX protein after which the calcium and potassium are available for release into the extracellular space.

The spindly mutation is predicted to result in an in-frame deletion of amino acids 534 to 555, which is within TM8.

Expression/Localization

NCKX4 is expressed in the brain, olfactory neurons, aorta, lung, thymus, and in the enamel organ during amelogenesis (2-7).

NCKX4 localizes to the plasma membrane.

Background
Figure 3. NCKX4 functions in olfactory signal transduction. During olfactory signal transduction, olfactory sensory neurons transform information of odorous chemicals into membrane potential changes. Olfactory signal transduction takes place in olfactory cilia, which extend from the tip of the olfactory sensory neuron dendrite into the mucus that covers the nasal epithelium (A). In most olfactory sensory neurons, transduction is achieved through a G protein-coupled, cyclic AMP-mediated signaling pathway, starting with the binding of an odorous chemical to its receptor protein (B). Abbreviations: OR, odorant receptor, Golf, olfactory G protein α subunit; ACIII, adenylyl cyclase III; CNG, olfactory cyclic nucleotide-gated cation channel; ANO2, anoctamin-2, Ca2+-activated chloride channel; PDE1C, phosphodiesterase 1C; NCKX4, the potassium-dependent Na+/Ca2+ exchanger-4. 
Figure 4. NCKX4 functions in MC4R-dependent satiety. (top) Agrp/Npy neurons and Pomc/Cart neurons are located in the arcuate nucleus of the hypothalamus and are regulated by leptin from adipose tissue. Both Agrp/Npy and Pomc/Cart neurons synapse onto MC4R-expressing neurons in the hypothalamus. Agouti and Agrp are hypothalamus-specific antagonists of MC3R and MC4R while α-MSH, a proteolytic product of Pomc, is a known agoinst of MC3R and MC4R. Agrp and Npy stimulate food intake and decrease energy expenditures, causing weight gain. Pomc and Cart inhibit food intake and increase energy expenditure. (bottom) MSH binding to the MC4R is shown to active Gαq, via the exchange of GTP for GDP. Gαq goes on to bind and stimulate PLC-β, which subsequently cleaves phosphatidylinositol 1,4-bisphosphate (PIP2) to diacylglycerol (DAG) and inositol 1,4,5-trisphosphate (IP3). The membrane-bound diacylglycerol then activates the Ca2+-selective entry channel, TRPC6, which results in an elevation of cytosolic [Ca2+] and consequent neuronal activation. NCKX4 acts locally to extrude the Ca2+ that enters via TRPC6. Abbreviations: Agrp, Agouti-related protein; Agrp, producing Agrp; Npy, neuropeptide Y; Pomc, pro-opiomelanocortin; Cart, cocaine- and amphetamine-regulated transcript; α-MSH, α-melanocyte stimulating hormone; LEPR, leptin receptor.

The NCKX proteins function in the maintenance of calcium homeostasis in several processes, including vision, olfaction, skin/hair/iris pigmentation, enamel formation, and melanocortin-4-receptor (MC4R; see the record for Southbeach)-dependent satiety (3;5;8;9). The NCKX proteins use both the inward sodium and outward potassium gradients to extrude calcium from the cell. The transport stoichiometry is 4 Na+: 1 Ca2+ + 1 K+ for NCKX1 and NCKX2 (10-13); the transport stoichiometry for NCKX4 is unknown.

Olfactory sensory neurons convert odor stimuli into electrical signals (Figure 3). Olfactory signal transduction occurs in olfactory sensory neuron cilia. The olfactory cyclic nucleotide-gated (CNG) ion channels are open at physiological levels of cAMP. Binding of an ordorant to surface receptors triggers a G protein-coupled cascade, which leads to an increase in the intracellular concentration of cAMP and the subsequent opening of the CNG channels and neuron depolarization upon the influx of sodium and calcium (14). The neuron depolarization triggers action potentials that propagate along the olfactory sensory neuron axon to the olfactory bulb in the brain. Increase in cytosolic free calcium subsequently activates calcium-activated chloride channels. The chloride channels carry most of the current that causes olfactory sensory neuron membrane depolarization. NCKX4 removes calcium from the cilia, which leads to closure of the chloride channel and response termination. Slc24a4-deficient (Slc24a4-/-) mice exhibited reduced olfactory neuron function, which resulted in diminished duration and adaptation of the olfactory response (3). Olfactory sensory neurons from the Slc24a4-/- mice exhibited prolonged responses and stronger adaptation (3). Upon repeated stimulation, the Slc24a4-/- olfactory sensory neurons displayed deficient action potentials (3). Olfactory-specific Slc24a4-/- mice had reduced body weights and an impaired ability to locate odorous sources (3). Loss of NCKX4 expression did not alter the histology of the olfactory epithelium or olfactory sensory neuron maturation, proliferation, or cell death (3).  

NCKX4 functions in MC4R-dependent satiety (Figure 4) (8). Slc24a4-/- mice had reduced body weights at weaning, and by four months they showed a 25 to 30% reduction in body weight compared to wild-type controls; body length was not changed (8). The Slc24a4-/- mice exhibited a reduction in food consumption and a modest reduction in water intake (8). The Slc24a4-/- mice were able to find visible food indicating that reduced olfactory function does not lead to the reduction in food consumption. The change in feeding behavior in the Slc24a4-/- mice was proposed to be due to constitutive activation of hypothalamic paraventricular nucleus (PVN) neurons in an MC4R-dependent and calcium-sensitive manner. The constitutive activation of the PVN neurons would lead to an anorexigenic response.


Mutations in human SLC24A4 are associated with amelogenesis imperfect, type IIA5 [AI2A5; OMIM: #615887; (4;5;9;15)]. Amelogenesis is the formation of dental enamel, which completes before tooth eruption. Amelogenesis imperfecta can either be hypoplastic (characterized by reduced enamel volume) or hypomineralized (characterized by variable incomplete mineralization of the enamel matrix). AI2A5 is the hypomineralized form of AI. Slc24a4-/- mice also exhibited enamel defects indicative of failed enamel mineralization (4).

Variants in SLC24A4 in humans are also associated with (blond/brown) hair and (blue/green) eye color in European populations [OMIM: #210750; (9;16)].

Putative Mechanism

The splindly body weight phenotype mimics that observed in Slc24a4-/- mice indicating loss of NCKX4spindly function. Li and colleagues observed a progressive increase in the difference in body weights in adult mice due to a reduction in food consumption (8). Loss of NCKX4 expression putatively causes defects in the MC4R-associated signaling pathway, which leads to constitutive activation of PVN neurons and the induction of anorexia.

Primers PCR Primer
spindly_pcr_F: GGCTCCCCATTTGTCATAAGG
spindly_pcr_R: CTCAGGATAAAGTACCACAGCG

Sequencing Primer
spindly_seq_F: GTCATAAGGCTCTGACTATCCTG
spindly_seq_R: ACAGCGGTTGCAGTTGCTC
Genotyping

PCR program

1) 94°C 2:00
2) 94°C 0:30
3) 55°C 0:30
4) 72°C 1:00
5) repeat steps (2-4) 40x
6) 72°C 10:00
7) 4°C hold


The following sequence of 402 nucleotides is amplified (chromosome 12, + strand):


1   ggctccccat ttgtcataag gctctgacta tcctgcatcc atcagcagga gccctgtttc
61  ccaaaagaga aaatgaatac tctgaaggtg gggcttttag aatagactgg ccgaccaact
121 cagagatttg tggctcatgt gcttcactct ccaggtgaag atcaacagcc ggggtctggt
181 ctattccgtg gtgctgctgc tgggctctgt cgctctcact gtgagttttt gtcatttcca
241 aactggagca ccatgaatac ttcatttcat gcagctcagc ctggctgggc acatctccct
301 gttacttgcc ctccagggtg aggggcaagc caaggacatg tatatactta aagatacccc
361 aaagcaagag caactgcaac cgctgtggta ctttatcctg ag


Primer binding sites are underlined and the sequencing primers are highlighted; the mutated nucleotide is shown in red.

References
Science Writers Anne Murray
Illustrators Katherine Timer
AuthorsEmre Turer and Bruce Beutler