Phenotypic Mutation 'discobolus' (pdf version)
Allelediscobolus
Mutation Type nonsense
Chromosome12
Coordinate31,590,532 bp (GRCm39)
Base Change T ⇒ A (forward strand)
Gene Slc26a4
Gene Name solute carrier family 26, member 4
Synonym(s) pendrin, Pds
Chromosomal Location 31,569,826-31,609,968 bp (-) (GRCm39)
MGI Phenotype FUNCTION: [Summary is not available for the mouse gene. This summary is for the human ortholog.] Mutations in this gene are associated with Pendred syndrome, the most common form of syndromic deafness, an autosomal-recessive disease. It is highly homologous to the SLC26A3 gene; they have similar genomic structures and this gene is located 3' of the SLC26A3 gene. The encoded protein has homology to sulfate transporters. [provided by RefSeq, Jul 2008]
PHENOTYPE: Homozygous null mutants are completely deaf with vestibular dysfunction. Mutants show endolymphatic dilatation, degeneration of sensory cells and malformations of otoconia and otoconial membranes. They display unsteady gait and circling and head bobbing. [provided by MGI curators]
Accession Number

NCBI RefSeq: NM_011867; MGI:1346029

MappedYes 
Amino Acid Change Lysine changed to Stop codon
Institutional SourceBeutler Lab
Gene Model predicted gene model for protein(s): [ENSMUSP00000001253]
AlphaFold Q9R155
SMART Domains Protein: ENSMUSP00000001253
Gene: ENSMUSG00000020651
AA Change: K374*

DomainStartEndE-ValueType
low complexity region 33 47 N/A INTRINSIC
Pfam:Sulfate_transp 84 485 1e-105 PFAM
low complexity region 492 507 N/A INTRINSIC
Pfam:STAS 536 725 1.4e-42 PFAM
Predicted Effect probably null
Meta Mutation Damage Score 0.9717 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 8/10 
Alleles Listed at MGI

All Mutations and Alleles(9) : Chemically induced (ENU)(1) Gene trapped(1) Spontaneous(1) Targeted(6)

Lab Alleles
AlleleSourceChrCoordTypePredicted EffectPPH Score
IGL01754:Slc26a4 APN 12 31578853 splice site probably benign
IGL01763:Slc26a4 APN 12 31578853 splice site probably benign
IGL01778:Slc26a4 APN 12 31578853 splice site probably benign
IGL01779:Slc26a4 APN 12 31578853 splice site probably benign
IGL01872:Slc26a4 APN 12 31589202 missense probably benign 0.22
IGL02016:Slc26a4 APN 12 31585666 missense probably damaging 0.99
IGL02184:Slc26a4 APN 12 31599948 missense probably damaging 1.00
IGL02267:Slc26a4 APN 12 31578853 splice site probably benign
IGL02270:Slc26a4 APN 12 31578853 splice site probably benign
IGL02271:Slc26a4 APN 12 31578853 splice site probably benign
IGL02347:Slc26a4 APN 12 31578853 splice site probably benign
IGL02543:Slc26a4 APN 12 31578688 missense possibly damaging 0.75
IGL02803:Slc26a4 APN 12 31572526 critical splice acceptor site probably null
IGL02885:Slc26a4 APN 12 31575475 missense probably benign 0.00
IGL02974:Slc26a4 APN 12 31579553 missense probably damaging 1.00
IGL03037:Slc26a4 APN 12 31581686 splice site probably benign
cul-de-sac UTSW 12 31575567 nonsense probably null
R0152:Slc26a4 UTSW 12 31579497 missense probably damaging 1.00
R0677:Slc26a4 UTSW 12 31599910 critical splice donor site probably null
R0961:Slc26a4 UTSW 12 31585618 missense probably benign
R1025:Slc26a4 UTSW 12 31578736 missense probably damaging 1.00
R1301:Slc26a4 UTSW 12 31575567 nonsense probably null
R1729:Slc26a4 UTSW 12 31594493 missense possibly damaging 0.95
R2321:Slc26a4 UTSW 12 31590543 missense probably damaging 1.00
R3967:Slc26a4 UTSW 12 31578686 missense probably damaging 1.00
R3970:Slc26a4 UTSW 12 31578686 missense probably damaging 1.00
R4007:Slc26a4 UTSW 12 31590532 nonsense probably null
R4370:Slc26a4 UTSW 12 31579475 missense probably benign 0.01
R4647:Slc26a4 UTSW 12 31590525 missense possibly damaging 0.90
R4648:Slc26a4 UTSW 12 31590525 missense possibly damaging 0.90
R5816:Slc26a4 UTSW 12 31578684 missense probably damaging 1.00
R5932:Slc26a4 UTSW 12 31585248 critical splice donor site probably null
R6675:Slc26a4 UTSW 12 31590512 missense possibly damaging 0.89
R6732:Slc26a4 UTSW 12 31576599 critical splice donor site probably null
R6890:Slc26a4 UTSW 12 31599950 missense possibly damaging 0.79
R7231:Slc26a4 UTSW 12 31597945 missense probably damaging 1.00
R7286:Slc26a4 UTSW 12 31579527 nonsense probably null
R7790:Slc26a4 UTSW 12 31594482 missense probably damaging 1.00
R7812:Slc26a4 UTSW 12 31594449 missense probably damaging 1.00
R8002:Slc26a4 UTSW 12 31597969 missense probably benign 0.00
R8362:Slc26a4 UTSW 12 31594506 missense probably benign 0.00
R8531:Slc26a4 UTSW 12 31599911 critical splice donor site probably null
R8988:Slc26a4 UTSW 12 31572523 missense probably benign 0.00
R9216:Slc26a4 UTSW 12 31578659 missense possibly damaging 0.51
R9335:Slc26a4 UTSW 12 31575553 missense probably damaging 0.99
R9354:Slc26a4 UTSW 12 31585255 missense possibly damaging 0.91
R9680:Slc26a4 UTSW 12 31585292 missense probably damaging 1.00
X0022:Slc26a4 UTSW 12 31585686 missense probably damaging 1.00
Mode of Inheritance Autosomal Recessive
Local Stock Live Mice, gDNA
Repository
Last Updated 2019-09-04 9:43 PM by Katherine Timer
Record Created 2016-02-09 2:28 PM by Jeff SoRelle
Record Posted 2016-06-03
Phenotypic Description
Figure 1. Phenotype of the discobolus mice. Click to view.

The discobolus phenotype was identified among G3 mice of the pedigree R4007, some of which showed variable head tilting (Figure 1); the mice did not exhibit circling, head bobbing, tremor, or other noticeable neurological defects.

Nature of Mutation

Figure 2. Linkage mapping of the vestibular phenotype using a recessive model of inheritance. Manhattan plot shows -log10 P values (Y-axis) plotted against the chromosome positions of 53 mutations (X-axis) identified in the G1 male of pedigree R4007. Normalized phenotype data are shown for single locus linkage analysis with 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 53 mutations. The vestibular dysfunction phenotype was linked to a mutation in Slc26a4: an A to T transversion at base pair 31,540,533 (v38) on chromosome 12, corresponding to base pair 20,105 in the GenBank genomic region NC_000078 encoding Slc26a4. Linkage was found with a recessive model of inheritance (P = 1.868 x 10-10), wherein 8 affected mice were homozygous (n = 8) for the variant allele, and 43 unaffected mice were either homozygous for the variant allele (n = 2), heterozygous for the reference allele (n = 26), or homozygous for the reference allele (n = 15) (Figure 2).

The mutation corresponds to residue 1,335 in the NM_011867 mRNA sequence in exon 9 of 21 total exons. 

 

1320 AAAGTCTACGCCACCAAGCATGACTATGTCATC

369  -K--V--Y--A--T--K--H--D--Y--V--I-

The mutated nucleotide is indicated in red. The mutation results in substitution of lysine 374 to a premature stop codon (K374*) in the SLC26A4 (alternatively, pendrin) protein.

Illustration of Mutations in
Gene & Protein
Protein Prediction

Figure 3. Domain structure and topology of pendrin. Pendrin has 12 transmembrane domains and a STAS box at the C-terminus. The N- and C-termini of pendrin are within the cytoplasm. The location of the discobolus mutation (K374*) is indicated.

Slc26a4 encodes pendrin, a 780-amino acid member of solute carrier (SLC) family 26 (Figure 3). The SLC26 proteins are members of the sulfate permease (SulP) family, which have homologs in bacteria, plants, fungi, and animals [reviewed in (1)]. The SLC superfamily encodes intrinsic membrane transporters comprising 55 gene families and 362 putatively functional protein-coding genes. The gene products include passive transporters, symporters, and antiporters that transport a wide variety of substrates, including amino acids, oligopeptides, glucose, inorganic cations and anions, bile salts, carboxylate, acetyl coenzyme A, essential metals, neurotransmitters, vitamins, fatty acids, lipids, nucleosides, ammonium, choline, thyroid hormone, and urea (2). SLC family members have a variable number of transmembrane domains; pendrin is predicted to have 12 transmembrane domains (3) with a cytoplasmic N- and C-termini similar to SLC26A5 and SLC26A6 (4;5).

The C-terminus of pendrin has a STAS (sulfate transporter antagonist of anti-sigma factor) domain (amino acids 536-725) (6). The STAS domain is proposed to mediate NTP binding and/or hydrolysis as well as to regulate anion transport by sensing intracellular concentrations of GTP and/or ATP (6). The STAS domain may also mediate the interaction between pendrin (and other SLC26 family members) with the cystic fibrosis transmembrane regulator (CFTR), an ion channel that mediates the transport of chloride and thiocyanate ions across epithelial cell membranes (7;8).

Pendrin has multiple putative N-glycosylation sites in the second extracellular loop (3). Two sites, Asn167 and Asn172, were confirmed, but the glycans were not processed to complex oligosaccharides; glycosylation was not necessary for cell surface localization (9). cAMP-induced phosphorylated of Ser49 induces pendrin trafficking to the apical membrane (10). Furthermore PKA-mediated phosphorylation of Thr717 induces thyroid stimulating hormone (TSH)-stimulated pendrin trafficking in polarized thyrocytes (11).

The Slc26a4 mutation in discobolus results in substitution of lysine 374 for a premature stop codon (K374*) in the pendrin protein. Lys374 resides in the cytoplasmic loop between transmembrane domains 8 and 9.

Expression/Localization

Pendrin is expressed in the apical membranes of epithelial cells of the inner ear (non-sensory epithelial cells of the cochlea, vestibular labyrinth, and the endolymphatic sac as well as saccule, utricle, and ampulla), thyroid (thyrocyte), kidney (renal collecting type B intercalated cell), airways, mammary gland, salivary duct, and liver (12-19).

Background
Figure 4. Pendrin function in the inner ear. The mammalian inner ear consists of the cochlea, the vestibular labyrinth, which is the organ for detecting linear acceleration in the saccule and utricle, and angular acceleration along 3 spatial axes in the ampullae. The cochlea consists of an epithelia-lined duct that is filled with endolymph. This duct is surrounded by two open fluid compartments that are filled with perilymph. The main structures of the cochlear duct are the stria vascularis (SV), Reissner membrane (RM), and the organ of Corti (OC). The stria vascularis generates the EP and secretes K+ into endolymph. The organ of Corti contains the sensory hair cells. Pendrin is expressed in epithelial cells of the spiral prominence (SP), in root cells (R), and in spindle cells of the stria vascularis. Cross sections are shown for the saccule or utricle, an ampulla, and the endolymphatic duct and sac. The saccule, utricle, ampullae, and endolymphatic duct are epithelia-lined structures that are filled with endolymph and surrounded by perilymph. The endolymphatic sac is surrounded by cerebrospinal fluid. Pendrin is expressed in nonsensory epithelial cells surrounding the sensory hair-cell patches in the saccule, utricle, and ampulla. In addition, pendrin is prominently expressed in the endolymphatic sac. Figure and legend was adapted from Wangemann, P. Cell Physiol Biochem 2011;28:527–534.

The SLC26 family of anion exchangers mediates the exchange of anions between the cytosol and the extracellular space. The SLC26 family members have varied functions including roles in skeletal development, thyroid hormone synthesis, transepithelial Na+-Cl- transport, bicarbonate excretion in the distal nephron, and bicarbonate secretion by the exocrine pancreas [reviewed in (1)]. Pendrin is an anion exchanger within the ear, thyroid, and kidney with broad substrate selectivity, including HCO3, Cl, I, formate, nitrate, OH-, and SCN-  [(20;21); reviewed in (1)].

Pendrin mediates Cl/I or I/HCO3 exchange in the salivary duct (22), thyrocytes (11), and renal collecting duct (12;23). In thyrocytes, iodide and sodium are transferred into cells via the basolaterally localized sodium-iodide symporter. Pendrin mediates the efflux of iodide into the follicular lumen (24-27). In the renal collecting duct, pendrin reclaims filtered iodide from the urine.  In the renal type B intercalated cell, pendrin functions in Cl reabsorption coupled to the apical membrane Na+-dependent Cl/HCO3 exchanger, SLC4A8. In the airway epithelial cell apical membrane, pendrin functions in IL4-stimulated Cl/SCN exchange, which provides substrate to the heme enzyme lactoperoxidase to generate the anti-microbial product, hypothiocyanite (OSCN-) (28).

Inner ear development in the mouse begins at embryonic day 9.5 (E9.5) with the formation of the otocyst [reviewed in (29)]. At E10, two protrusions extend from the otocyst to form the cochlea and the endolymphatic sac. The protrusions continue to elongate, and the center of the otocyst reorganizes into the vestibular labyrinth. The endolymphatic sac lumen opens at E10.5, and the cochlea lumen opens at E14.5 (30). Lumen formation in the inner ear is mediated by a balance of fluid secretion and absorption in the endolymphatic sac (30). In the inner ear, pendrin conditions the endolymph to permit the proper function of sensory cells by facilitating Cl/HCO3 exchange (Figure 4).

Mutations in SLC26A4 are linked to autosomal recessive deafness 4 with enlarged vestibular aqueduct (DFNB4 with EVA; OMIM: #600791) and Pendred syndrome (alternatively, Mondini-like dysplasia of the cochlea and enlargement of the thyroid; OMIM: #274600) (31-37). Patients with either Pendred syndrome or DFNB4 exhibit bilateral, progressive prelingual/early postlingual deafness with inner ear anomalies including an enlarged vestibular aqueduct (EVA) or Mondini’s dysplasia (34;38;39). Pendred syndrome patients often exhibit deafness and goiters through altered functions of both the ear and the thyroid gland; the kidney also exhibits defects in some cases. Kidney function (e.g., electrolyte regulation and acid-base balance) is proposed to be maintained through compensatory mechanisms, which prevent changes in intracellular pH due to changes in pendrin function (40). The thyroid symptoms in Pendred syndrome patients are variable; goiter formation is not a constant feature, and the size of the goiter can range from a slight increase in thyroid size to a large multinodular goiter. Some Pendred syndrome patients exhibit vestibular dysfunction, including episodic vertigo, tinnitus, and vomiting (41;42).

Slc26a4-deficient (Slc26a4-/-) mice develop EVA and Mondini-like dysplasia of the cochlea similar to that observed in Pendred syndrome in humans. The Slc26a4-/- mice are unable to hear and exhibit circling and head bobbing indicative of vestibular defects. The Slc26a4-/- mice do not have a thyroid phenotype (43). Heterozygous mice (Slc26a4+/-) are overtly normal with hearing and balance comparable to their wild-type littermates. At E14.5, the Slc26a4-/- mice exhibit enlargement of the luminal volume that is proposed to be due to an imbalance between fluid secretion and fluid absorption during the growth phase of the inner ear (43;44); the enlargement is observed throughout adulthood. At E15.5, the cochlear endolymph, luminal fluid of the endolymphatic sac, mature inner ear in the cochlea, and the utricle of the vestibular labyrinth is acidified in the Slc26a4-/- mice (44-46). Acidification of the lumen changes pH-sensitive mechanisms. Enlargement of the lumen reduces cell-to-cell communication that is dependent on diffused factors. The disturbed cell communication is proposed to result in the impaired development and delayed innervation of the organ of Corti that is observed between postnatal day (P) 5-10 (44;47). Loss of pendrin expression in the cochlea resulted in increased K+ secretion from strial marginal cells, oxidative stress in the stria vascularis, loss of the K+ channel KCNJ10 in intermediate cells, loss of the endocochlear potential, increase in the endolymphatic Ca2+ concentration, and degeneration of sensory cells and the stria vascularis (45;48).  An increase in the endolymphatic Ca2+ concentration was observed in the vestibular labyrinth, which subsequently led to formation of giant otoconia  (i.e., calcium carbonate mineral formation) (43;46).

The Slc26a4loop/loop mouse model has a mutation that results in a serine to phenylalanine substitution at amino acid 408 (49). The phenotype of homozygous Slc26a4loop/loop mice mimics that of the Slc26a4-/- mice in that they are deaf, exhibit enlarged cochlea, and the formation of giant otoconia in the vestibular labyrinth (49). The Slc26a4loop/loop mice also exhibited atrophic microfollicles in the thyroid gland, although serum levels of thyroid hormone were normal (50).

A mouse model that expresses pendrin only in the endolymphatic sac and not in the chochlea or the vestibular labyrinth (Tg(B1-hPDS)Slc26a4Δ/Δ) exhibited normal hearing and balance (51). Expression of the pendrin transgene facilitated normal endocochlear potential, normal pH gradients between the endolymph and perilymph in the cochlea, normal otoconia formation in the vestibular labyrinth, and prevented abnormal enlargement of the membranous labyrinth.

Putative Mechanism

The vestibular phenotype of the discobolus mice mimics that of the Slc26a4 mutant models, indicating that pendrindiscobolus function is defective in the mice. Expression and localization of pendrindiscobolus have not been examined.

Primers PCR Primer
discobolus_pcr_F: GGTGACTTGGTATGCATATTTCAA
discobolus_pcr_R: TACCACGTCACTGAGTGAAGA

Sequencing Primer
discobolus_seq_F: TCCTTAGGTGAACCCCAA
discobolus_seq_R: CACTGAGTGAAGAGAGTGTTTGGC
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 400 nucleotides is amplified (chromosome 12, - strand):


1   taccacgtca ctgagtgaag agagtgtttg gcaaggttgg gaaaaggttg tcacatcttc
61  acaatagttt ttcacttgcc aggttcttgc ctcctgtcct gccatctgtg ggcctgtttt
121 cggacatgtt ggctgcatcc ttttccattg ctgtggtggc ttacgctatt gcagtgtctg
181 taggaaaagt ctacgccacc aagcatgact atgtcatcga tgggaaccag gtatggtcac
241 ctatctgctg atctgaattt ataaggctga gaacaagtac aagaaacacg agtaggggag
301 acttggggtt cacctaagga aagtgttggt ttctttgact agcttttatc tctgtcctag
361 ctaattaaag attattttga aatatgcata ccaagtcacc 


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

References
Science Writers Anne Murray
Illustrators Peter Jurek
AuthorsJeff SoRelle and Bruce Beutler