Phenotypic Mutation 'squirm' (pdf version)
Allelesquirm
Mutation Type large deletion
Chromosome10
Coordinate
Base Change
Gene Pcdh15
Gene Name protocadherin 15
Synonym(s) Gm9815, nmf19, Ush1f
Chromosomal Location 73,099,342-74,649,737 bp (+)
MGI Phenotype FUNCTION: [Summary is not available for the mouse gene. This summary is for the human ortholog.] This gene is a member of the cadherin superfamily. Family members encode integral membrane proteins that mediate calcium-dependent cell-cell adhesion. It plays an essential role in maintenance of normal retinal and cochlear function. Mutations in this gene result in hearing loss and Usher Syndrome Type IF (USH1F). Extensive alternative splicing resulting in multiple isoforms has been observed in the mouse ortholog. Similar alternatively spliced transcripts are inferred to occur in human, and additional variants are likely to occur. [provided by RefSeq, Dec 2008]
PHENOTYPE: Homozygotes for severe mutations exhibit circling, head-tossing, hyperactivity, impaired swimming and profound deafness. Mice have defects in cochlea and degeneration of hair cells, spiral ganglion cells and saccular macula. Females are poor mothers. [provided by MGI curators]
Accession Number

NCBI RefSeq: NM_023115; MGI: 1891428

Mapped Yes 
Amino Acid Change
Institutional SourceBeutler Lab
Ref Sequences
Ensembl: ENSMUSP00000101066 (fasta)
Gene Model not available
SMART Domains

DomainStartEndE-ValueType
signal peptide 1 26 N/A INTRINSIC
CA 69 150 1.22e-1 SMART
CA 179 268 5.48e-8 SMART
CA 309 398 1.94e-8 SMART
CA 431 512 2.29e-10 SMART
CA 536 618 4.88e-14 SMART
CA 643 720 4.65e-20 SMART
CA 744 822 1.93e-26 SMART
CA 846 929 5.69e-15 SMART
CA 953 1039 6.85e-9 SMART
CA 1060 1147 3.09e-16 SMART
CA 1171 1255 4.49e-4 SMART
transmembrane domain 1380 1402 N/A INTRINSIC
low complexity region 1423 1450 N/A INTRINSIC
low complexity region 1661 1678 N/A INTRINSIC
low complexity region 1740 1760 N/A INTRINSIC
low complexity region 1767 1780 N/A INTRINSIC
low complexity region 1784 1834 N/A INTRINSIC
Meta Mutation Damage Score Not available question?
Is this an essential gene? Non Essential (E-score: 0.000) question?
Phenotypic Category
Phenotypequestion? Literature verified References
behavior/neurological
hearing/vestibular/ear
Candidate Explorer Status CE: no linkage results
Single pedigree
Linkage Analysis Data
Penetrance 100% 
Alleles Listed at MGI

All alleles(11) : Gene trapped(2) Transgenic(1) Spontaneous(6) Chemically induced(2)

Lab Alleles
AlleleSourceChrCoordTypePredicted EffectPPH Score
IGL00421:Pcdh15 APN 10 74185345 nonsense probably null
IGL00432:Pcdh15 APN 10 74291082 splice site probably benign
IGL00533:Pcdh15 APN 10 74502720 missense probably damaging 1.00
IGL00596:Pcdh15 APN 10 74630744 missense probably benign 0.00
IGL00930:Pcdh15 APN 10 74630698 missense probably benign 0.08
IGL00970:Pcdh15 APN 10 74379340 missense probably damaging 1.00
IGL01087:Pcdh15 APN 10 74342632 missense possibly damaging 0.90
IGL01763:Pcdh15 APN 10 74210461 missense probably benign 0.09
IGL01787:Pcdh15 APN 10 74450283 missense probably benign 0.25
IGL02070:Pcdh15 APN 10 74630868 missense probably benign 0.00
IGL02234:Pcdh15 APN 10 74631862 missense probably benign 0.02
IGL02268:Pcdh15 APN 10 74342672 missense probably damaging 1.00
IGL02280:Pcdh15 APN 10 74222463 missense probably damaging 1.00
IGL02363:Pcdh15 APN 10 74317086 missense probably damaging 0.98
IGL02420:Pcdh15 APN 10 74303106 missense probably damaging 0.98
IGL02749:Pcdh15 APN 10 74631068 missense probably benign 0.00
IGL02939:Pcdh15 APN 10 74504816 splice site probably benign
IGL02970:Pcdh15 APN 10 74290962 splice site probably benign
IGL03010:Pcdh15 APN 10 74385945 missense probably damaging 1.00
IGL03061:Pcdh15 APN 10 74317011 missense probably damaging 0.97
IGL03095:Pcdh15 APN 10 74355874 missense probably damaging 1.00
IGL03149:Pcdh15 APN 10 74630695 missense probably damaging 1.00
IGL03187:Pcdh15 APN 10 74355874 missense probably damaging 1.00
IGL03279:Pcdh15 APN 10 74317072 missense probably damaging 1.00
IGL03392:Pcdh15 APN 10 74624272 missense probably damaging 1.00
loop UTSW 10 74185378 missense probably damaging 1.00
mcduck UTSW 10 74626844 critical splice donor site probably null
spaz UTSW 10 74210425 missense probably damaging 1.00
sphere UTSW 10 74624284 missense probably damaging 1.00
Tortilla UTSW 10 74379417 splice site probably null
1mM(1):Pcdh15 UTSW 10 74626137 intron probably benign
R0038:Pcdh15 UTSW 10 74643440 missense possibly damaging 0.95
R0103:Pcdh15 UTSW 10 74210425 missense probably damaging 1.00
R0110:Pcdh15 UTSW 10 74290976 missense probably damaging 1.00
R0111:Pcdh15 UTSW 10 74626819 nonsense probably null
R0119:Pcdh15 UTSW 10 74170575 missense probably damaging 1.00
R0131:Pcdh15 UTSW 10 74170608 missense probably null 1.00
R0445:Pcdh15 UTSW 10 74342549 missense probably damaging 1.00
R0464:Pcdh15 UTSW 10 74626844 critical splice donor site probably null
R0503:Pcdh15 UTSW 10 74210385 missense probably damaging 1.00
R0507:Pcdh15 UTSW 10 74621297 missense probably damaging 1.00
R0510:Pcdh15 UTSW 10 74290976 missense probably damaging 1.00
R0742:Pcdh15 UTSW 10 74621297 missense probably damaging 1.00
R0790:Pcdh15 UTSW 10 74631053 missense probably benign 0.01
R0829:Pcdh15 UTSW 10 74502766 missense probably damaging 1.00
R0839:Pcdh15 UTSW 10 74626782 missense probably null 1.00
R0882:Pcdh15 UTSW 10 74342656 missense probably damaging 1.00
R0894:Pcdh15 UTSW 10 74624255 missense probably damaging 1.00
R0944:Pcdh15 UTSW 10 74210470 missense probably damaging 0.99
R1081:Pcdh15 UTSW 10 74450313 missense probably damaging 1.00
R1148:Pcdh15 UTSW 10 74170560 missense probably damaging 1.00
R1148:Pcdh15 UTSW 10 74170560 missense probably damaging 1.00
R1484:Pcdh15 UTSW 10 74291001 missense probably damaging 1.00
R1521:Pcdh15 UTSW 10 74594191 missense probably damaging 1.00
R1694:Pcdh15 UTSW 10 74594163 missense probably damaging 1.00
R1795:Pcdh15 UTSW 10 74624255 missense probably damaging 1.00
R2021:Pcdh15 UTSW 10 74631193 missense possibly damaging 0.93
R2022:Pcdh15 UTSW 10 74631193 missense possibly damaging 0.93
R2023:Pcdh15 UTSW 10 74631193 missense possibly damaging 0.93
R2076:Pcdh15 UTSW 10 74342647 missense probably damaging 1.00
R2199:Pcdh15 UTSW 10 74170509 missense probably damaging 1.00
R2510:Pcdh15 UTSW 10 74631499 missense probably benign 0.39
R2511:Pcdh15 UTSW 10 74645996 missense possibly damaging 0.94
R3418:Pcdh15 UTSW 10 74584222 missense probably benign 0.12
R3419:Pcdh15 UTSW 10 74584222 missense probably benign 0.12
R3433:Pcdh15 UTSW 10 74631499 missense probably benign 0.39
R3619:Pcdh15 UTSW 10 74643395 missense probably damaging 0.99
R3723:Pcdh15 UTSW 10 74645848 missense probably benign 0.05
R3724:Pcdh15 UTSW 10 74645848 missense probably benign 0.05
R3778:Pcdh15 UTSW 10 73947151 splice site probably null
R3851:Pcdh15 UTSW 10 74631686 missense probably damaging 0.97
R4175:Pcdh15 UTSW 10 74631997 intron probably benign
R4261:Pcdh15 UTSW 10 74645680 missense probably damaging 1.00
R4385:Pcdh15 UTSW 10 74550490 missense probably damaging 1.00
R4585:Pcdh15 UTSW 10 74624284 missense probably damaging 1.00
R4602:Pcdh15 UTSW 10 74594214 missense probably damaging 1.00
R4639:Pcdh15 UTSW 10 74643607 missense probably benign 0.00
R4703:Pcdh15 UTSW 10 74450163 missense probably damaging 1.00
R4819:Pcdh15 UTSW 10 74324389 missense probably damaging 1.00
R4906:Pcdh15 UTSW 10 74504793 nonsense probably null
R4961:Pcdh15 UTSW 10 74379417 splice site probably null
R5018:Pcdh15 UTSW 10 74643775 missense possibly damaging 0.68
R5125:Pcdh15 UTSW 10 74584080 missense probably damaging 0.98
R5225:Pcdh15 UTSW 10 74303154 missense probably damaging 1.00
R5259:Pcdh15 UTSW 10 74396372 missense possibly damaging 0.67
R5279:Pcdh15 UTSW 10 74594183 missense probably damaging 1.00
R5395:Pcdh15 UTSW 10 74185287 missense probably damaging 1.00
R5458:Pcdh15 UTSW 10 74504779 missense probably damaging 1.00
R5617:Pcdh15 UTSW 10 74635672 intron probably benign
R5665:Pcdh15 UTSW 10 74626788 missense probably damaging 1.00
R5770:Pcdh15 UTSW 10 74185345 nonsense probably null
R5805:Pcdh15 UTSW 10 74230259 missense probably damaging 1.00
R5914:Pcdh15 UTSW 10 74630936 missense probably benign 0.42
R5988:Pcdh15 UTSW 10 74379357 missense probably benign 0.05
R6133:Pcdh15 UTSW 10 74645973 unclassified probably null
R6189:Pcdh15 UTSW 10 74342651 missense probably null 1.00
R6414:Pcdh15 UTSW 10 74185426 missense probably damaging 1.00
R6536:Pcdh15 UTSW 10 74631389 missense probably damaging 1.00
R6612:Pcdh15 UTSW 10 74185378 missense probably damaging 1.00
R6711:Pcdh15 UTSW 10 74642387 missense possibly damaging 0.83
R6793:Pcdh15 UTSW 10 74631139 missense probably damaging 1.00
R6841:Pcdh15 UTSW 10 74450220 missense probably damaging 1.00
R6845:Pcdh15 UTSW 10 74630633 missense probably benign
R6915:Pcdh15 UTSW 10 74643809 missense probably benign 0.16
R6954:Pcdh15 UTSW 10 74645989 missense possibly damaging 0.92
R6970:Pcdh15 UTSW 10 74502687 missense probably damaging 0.98
R7018:Pcdh15 UTSW 10 74466354 missense probably damaging 1.00
R7064:Pcdh15 UTSW 10 74630614 missense possibly damaging 0.67
R7079:Pcdh15 UTSW 10 74317125 missense probably benign 0.21
R7172:Pcdh15 UTSW 10 74502765 missense probably damaging 1.00
R7220:Pcdh15 UTSW 10 74342609 missense possibly damaging 0.64
R7237:Pcdh15 UTSW 10 74584191 missense possibly damaging 0.88
R7266:Pcdh15 UTSW 10 74379390 nonsense probably null
R7276:Pcdh15 UTSW 10 74324392 missense probably benign 0.25
R7359:Pcdh15 UTSW 10 74584216 missense probably damaging 0.99
R7396:Pcdh15 UTSW 10 74630690 missense probably benign 0.17
R7421:Pcdh15 UTSW 10 74454065 missense possibly damaging 0.90
R7424:Pcdh15 UTSW 10 74506485 missense probably benign 0.09
R7463:Pcdh15 UTSW 10 74631770 missense possibly damaging 0.66
R7469:Pcdh15 UTSW 10 74645980 missense probably benign
R7512:Pcdh15 UTSW 10 74641382 missense possibly damaging 0.81
R7767:Pcdh15 UTSW 10 74486256 missense probably benign 0.07
R7830:Pcdh15 UTSW 10 74385901 missense probably damaging 1.00
R7890:Pcdh15 UTSW 10 74642314 missense probably damaging 1.00
R7897:Pcdh15 UTSW 10 74453995 missense probably damaging 1.00
R7908:Pcdh15 UTSW 10 74643582 missense probably benign 0.04
R7973:Pcdh15 UTSW 10 74642314 missense probably damaging 1.00
R7980:Pcdh15 UTSW 10 74453995 missense probably damaging 1.00
R7989:Pcdh15 UTSW 10 74643582 missense probably benign 0.04
RF020:Pcdh15 UTSW 10 74185410 missense probably damaging 1.00
Z1176:Pcdh15 UTSW 10 74630701 missense probably benign 0.00
Z1177:Pcdh15 UTSW 10 74504800 missense probably damaging 1.00
Mode of Inheritance Autosomal Recessive
Local Stock Sperm, gDNA
Repository

none

Last Updated 2019-07-25 10:41 AM by Diantha La Vine
Record Created unknown
Record Posted 2008-08-12
Phenotypic Description

Squirm was identified in G3 mutant mice derived from N-ethyl-N-nitrosourea (ENU)-mutagenized stock. Squirm mice appear hyperkinetic and display circling behavior.

Squirm mice display normal macrophage responses to Toll-like receptor ligands, double-stranded DNA, and the adjuvant alum as well as normal proportions of immunological cell types and normal responses to viral infections.  They also display a normal humoral immune response to model antigens encoded by a recombinant suicide vector based on the Semliki Forest Virus (rSFV).

Nature of Mutation
The squirm mutation was mapped to Chromosome 10.  Sequencing of the critical region found a deletion spanning exons 27-29 (of 39 total exons for the longest cDNAs) of the Pcdh15 gene (positions 685325 to 779172 occurring in the middle of introns 26 and 29 in the Genbank genomic region NC_000076 for linear genomic DNA sequence of Pcdh15; see Figure 1).  Exon 1 is non-coding (1;2).  Due to the complexity of the Pcdh15 genomic region and the presence of alternative exons, it is possible that squirm mice still express potentially functional Pcdh15 isoforms but this possibility has not been tested.
Protein Prediction
Figure 2. Domain structures of Pcdh15 splice variants. PCDH15 isoforms consist of up to 11 extracellular cadherin (EC) repeats, one transmembrane domain and a variable cytoplasmic domain that contains a PDZ-binding motif (PBM).The four isoform classes are defined based upon the cytoplasmic tail variation or the absence of the tail. The isoforms CD1, CD2, and CD3 are full length and contain a cytoplasmic tail encoded by 35, 38, and 39 exons, respectively. Isoform S1 is predicted to be secreted, contains only 10 EC repeats and lacks a cytoplasmic tail. The squirm mutation results in a deletion spanning exons 27-29. Image is interactive. Other mutations found in the protein are noted in red. Click on each allele for more information.

The mouse Pcdh15 gene encodes a protein that belongs to the protocadherin protein family (Figure 2).  Protocadherins are atypical members of the large cadherin (calcium-dependent cell-cell adhesion proteins (3)) superfamily of transmembrane proteins that are defined by the presence of a variable number of extracellular cadherin domains known as EC repeats (4;5).  Cadherin repeats are defined by the DRE, DXD and DXNDNXPXF motifs, and classical cadherins typically have five of these repeats in their extracellular domain, as well as a single transmembrane domain and a conserved cytoplasmic (CP) domain that interacts with α- and β-catenin and connects a cadherin molecule to the actin cytoskeleton (4).  EC repeats mediate the Ca2+-dependent dimerization of cadherin molecules and the trans-extracellular linkages between cadherin dimers of two neighboring cells.  The overall 3D structure of the cadherin repeat motif is quite similar to the Greek-key topology of immunoglobulin domains with seven β-strands arranged as two opposing β-sheets with N and C termini at the opposite ends (4).  While protocadherins share the cadherin repeats in the EC domain, they have a higher number of repeats.  Protocadherin EC repeats also have crucial structural differences from those of classical cadherins.  NMR studies of the PCDHα4 EC1 domain found the lack of a hydrophobic pocket necessary for the the homophilic adhesive binding of classical cadherins (6).  Instead protocadherin EC1 domains contain a protocadherin-specific disulfide bonded C-(X)5-C motif that is suggested to be a novel adhesion interface.  Protocadherins have greater variability in the CP domain, showing little or no homology to other members of the cadherin superfamily (5).

 

The Pcdh15 gene encodes multiple isoforms of the PCDH15 protein; exon 1 is non-coding (1;2).  The amino acids of each isoform are unique from each other, but each isoform contains a signal sequence, a unique extracellular region, and a cytoplasmic domain (1;7;8).  The A isoform has 11 cadherin repeats, 1 transmembrane domain and a 542 amino acid cytoplasmic domain (CD1) that contains 2 proline-rich regions and the C-terminal motif STSL, which is a type 1 PDZ (for postsynaptic density 95/Discs large/zona occludens-1)-binding consensus sequence (1;2;9-11).  The first EC1 domain of PCDH15 participates in cell adhesion and interacts with the cell adhesion molecule, cadherin 23 (12).  Using RT-PCR, two minor splice variants of mouse Pcdh15, one that skipped exon 2 and another that skipped exons 2 and 4, were identified (13).  Isoforms consisting mostly of the CD1 cytoplasmic domain (known as isoform B) (14), and a secreted form consisting of the extracellular domain have also been described (15).  These isoforms use a subset of the exons encoding the signal sequence, the extracellular domain (up to 11 extracellular cadherin repeats), a single-pass transmembrane domain, and sequence encoding the cytoplasmic domain CD1, CD2, or CD3.  Additionally, PCDH15 isoforms exist that include two or all three of the cytoplasmic domains.  Like CD1, the 379 amino acid CD2 and the 319 amino acid CD3 also contain a type 1 PDZ-binding motif (NTAL, and MTKL, respectively) (1).  The PDZ-binding motifs (PBMs) anchor PCDH15 to the actin cytoskeleton via the PDZ-containing protein harmonin (16-18).  Mouse PCDH15 is 94% identical to the human protein in the cadherin repeats, but only 53% identical in the cytoplasmic domain (for PCDH15-A) (13).  The amino acid sequence of CD3 is most highly conserved amongst species (1).  A Pcdh15 orthologue, Cad99C, exists in Drosophila melanogaster and encodes a protein with functions analogous to those of PCDH15 (19).  In zebrafish, two Pcdh15 orthologues occur with independent roles in hearing and vision (see Putative Mechanism) (20).  Studies have examined the specific role of each isoform in hair cells.  Isoform-specific knockout mice showed that PCDH15-CD1 and –CD3 are not essential for hair cell function, while PCDH15-CD2 is necessary for the formation of kinociliary links; the absence of PCDH15-CD2 leads to disruption of bundle polarity (21).

 

The squirm mutation deletes exons 27, 28 and 29 of the Pcdh15 gene, which would delete EC11 and most of the extracellular region of the protein leading to the transmembrane domain.  The deletion probably causes a frameshift, thus it is likely that both the transmembrane and cytoplasmic domains of the protein are also affected. 

Expression/Localization

Protocadherins are the largest of the cadherin subfamilies and are predominantly expressed in the nervous system (7).  Most of the genes that encode Pcdhs are located in three clusters on the same chromosome (7).

 

By Northern blot analysis, PCDH15 is expressed in human adult brain, lung, and kidney. Expression in human fetal brain was assumed since the human PCDH15 cDNA was obtained by screening a fetal brain library.  Additional experiments by RT-PCR and direct sequencing revealed expression in other human adult tissues and human fetal cochlea (22).  RT-PCR experiments showed that the isoform B of PCDH15 was expressed in human retina (9;14). In human fetal cochlea, PCDH15 is expressed in the supporting cells and outer sulcus cells, as well as in the spiral ganglion.  Immunohistochemistry detected PCDH15 expression in the inner and outer synaptic layers and the nerve fiber layer in human adult and fetal retinas.  Additional reactivity in the region of the outer limiting membrane/photoreceptor cell inner segments was observed in adult but not fetal retina (14)

 

Various PCDH15 isoforms are expressed in different tissues (2;14;23).  Pcdh15-CD1 is detected in human testis, retina and cochlear cDNA (8).  Pcdh15-CD2 is expressed in human heart, kidney, thymus, spleen, testis, retina and chochlear cDNA (8).  Interestingly, Pcdh15-CD3 appears to be almost ubiquitously expressed throughout the body (8).  Western blot analysis using a CD1-specific antibody found that PCDH15 isoforms A and B to be expressed in adult mouse brain, liver, retina, spleen and cochlea.  Immunohistochemistry found these isoforms to be expressed in the organ of Corti and vestibular hair cells in the ear.  The immunoreactivity was seen along the length of stereocilia, in the cuticular plate, and diffusely distributed in the cytoplasm of inner and outer hair cells (see Background).  From E16-post-natal day (P)21, these isoforms are also expressed in stereocilia throughout hair cell maturation (14).  Studies using the CD1-specific antibody as well as antibodies specific to the CD2 and CD3 domains, found PCDH15 isoforms containing these domains to be differentially expressed in maturing hair bundles.  CD1-containing isoforms are expressed along the length of stereocilia with highest expression found toward the base and little expression found at the tips.  CD2-containing isoforms are widely distributed in hair bundles, while CD3-containing isoforms are more concentrated at stereociliary tips where it is proposed that it binds to the MET channel and/or channel-associated proteins (1;16).  Secreted isoforms of PCDH15 are also expressed in the inner ear (15)

  

Both the secreted isoform and the A isoform of PCDH15 were found to be expressed in the natural killer (NK) cell lines.  The A isoform only was found in NK/T-cell lymphomas.  However, neither of these isoforms is expressed in normal hematopoietic cells (15).

 

Northern blot analysis of mouse tissues found that Pcdh15 is highly expressed in the brain and spleen with lower levels expressed in the heart (2).  Using in situ hybridization, Pcdh15 is expressed in mouse embryos in the sensory epithelium of the developing inner ear, in Rathke’s pouch, and broadly throughout the brain.  Pcdh15 is expressed in inner ear hair cells throughout life and can be detected in the hair bundle from the moment the bundle emerges at the apical surface of the sensory hair cells (24;25). The highest level of expression was at embryonic day (E)16.5.  Pcdh15 transcripts are also found in the developing eye, dorsal root ganglion, and the dorsal aspect of the neural tube, floor plate and ependymal cells adjacent to the neural canal.  Expression is detected in the developing glomeruli of the kidney, surface of the tongue, vibrissae, bronchi of the lung, and in the epithelium of the olfactory apparatus, gut and lung.  In the adult CNS, Pcdh15 expression is limited to only the granule cells of the cerebellum (23).  In the hair cells, immunohistochemistry in rodent hair cells localized PCDH15 to the lower part of the tip link filaments that connect the stereocilia (1;12) (see Background).  Immunocytochemistry revealed that PCDH15 is expressed in the synaptic terminals and the calycal processes of photoreceptor cells in the mammalian retina (1;18).  PCDH15 expression was also observed in the region of adherens junctions between photoreceptor cells and Muller glia cells in rodent retinas (18).

Background

Usher syndrome (USH), the most common form of deaf-blindness in humans, constitutes a group of autosomal recessive disorders characterized by progressive retinitis pigmentosa (RP) and sensorineural hearing loss (7;24).   USH can be associated with vestibular dysfunction and balance problems, reduced odor identification, sperm motility, mental deficiency, cerebral atrophy, and ataxia (26;27).  Usher syndrome has been subdivided into three clinical types on the basis of severity and progression of the hearing loss, and the age of onset of RP (28).  Usher syndrome type I (USH1) (OMIM #276900) is the most severe with congenital severe to profound hearing loss, vestibular dysfunction, and prepubertal onset of RP (7;16).    Usher syndrome type II (USH2;OMIM #276901) is the most common subtype and is characterized by congenital moderate to severe hearing loss and onset of RP during or after puberty.  Hearing loss is progressive in Usher type III (USH3;OMIM #276902), and onset of RP is variable.  Twelve independent loci have been identified in which inherited defects lead to the development of USH (26;27;29).  The affected genes at nine of these loci have been determined, and mutations in five of them cause USH1: MYO7A (USH1B) (30), HARMONIN (USH1C; OMIM #276904) (31;32), CDH23 (USH1D; OMIM #601067) (33), PCDH15 (USH1F; OMIM #602083) (9;10), and SANS (scaffold protein containing ankyrin repeats and SAM domain, USH1G; OMIM #606943) (34), that encode myosin VIIA, harmonin, cadherin 23, protocadherin 15 and SANS, respectively.  USH2 is caused by mutations in USH2A (35), VLGRI/GPR98 (USH2C; OMIM #605472) (36) and DFNB31 (USH2D; OMIM #611383) (37), encoding usherin, VLGR1 (very large G-coupled protein receptor 1) and whirlin, respectively. USH3A, encoding clarin-1 (38), is the only known gene responsible for USH3.  The loss of any of the USH1 proteins leads to defective early transient stereocilia lateral links and kinociliary links in growing hair bundles subsequently leading to deafness in USH1 patients (7). Further characterization of USH1 patients has found that the CD1 subclass of Pcdh15 may be the only subclass that is essential for retinal function (7;8).

 
Familial combined hyperlipidemia (FCHL) is a lipid disorder that increases the risk of premature coronary heart disease (3).  FCHL is characterized by hypertriglyceridemia, hypercholesterolemia, or both as well as high serum levels of apolipoprotein-B (apo-B) (3).  Studies have shown that a microsatellite marker (D10S546) within PCDH15 in Finnish dyslipidemic families is associated with high serum triglycerides (3;39).  There are epidemiological studies that have shown a connection between hearing loss and hyperlipidemia (3;40;41).  Results from a study found that the common allele of SNP rs1082529 within PCDH15 (in the same exon as D10S546 from previous studies (39)) is associated with hypertriglyceridemia, hypercholesterolemia and elevated levels of apo-B (3). PCDH15 has not been directly linked to lipid abnormalities, but USH1F patients have decreased levels of long-chain polyunsaturated fatty acids in the plasma (42).  Furthermore, a link between hearling loss and lipid abnormalities has been shown, indicating that hyperlipidemia and atherosclerosis can change cochlear function (3;40;41)

 

Hearing and balance both depend on the function of hair cells of the inner ear.  The hair cells of the inner ear are mechanosensors that perceive sound, head movement, and gravity (21).  These are polarized epithelial cells that have a hair bundle located at their apical poles that detects sound-induced movements of the cochlear fluids and subsequently transduces those stimuli into electrical signals (16;22).   In the cochlea, hair cells in the basal, high-frequency end of the duct differentiate before those in the apical, low-frequency region allowing a gradient of hair-bundle maturation to be seen along the length of the cochlear during embryonic and postnatal development (1).  The hair bundle present in hair cells is composed of numerous stereocilia that develop from microvilli and have a stiff core of parallel actin filaments anchored in the cuticular plate, a meshwork of horizontal actin filaments beneath the apical cell membrane (Figure 3A) (7;22).  During development, the stereocilia undergo differential elongation to form a staircase of decreasing height that is polarized in the apical hair cell surface and is essential for function.  The tallest stereocilia are connected via a kinociliary link to a single microtubule-based kinocilium, a true cilium that is essential for proper organization of the developing hair bundle and degenerates in some hair cells once the hair bundle has matured (22)(26;43).  The stereocilia are connected to each other by numerous filaments, including tip links, horizontal top connectors, shaft connectors, transient lateral links and ankle links.  The types of links in the hair bundle rapidly changes during development in a species- and hair cell type-specific manner (44).  The displacement of the hair bundle by a sound wave or head movement opens cation-selective mechanotransduction channels at the tips of the stereocilia, initiating the signaling cascade for sound perception (7;16;22).    The extracellular tip links connect the stereocilia and are thought to gate the mechanoelectrical transduction channel (26;43).  The other links are thought to hold the stereocilia together as a coherent unit and to transmit forces across the bundle, while links present during maturation are necessary for proper development, orientation and differential growth of the stereocilia (43;45).    The polarity of the bundle maintains hair cell function and many deaf mouse mutants have defects in this hair bundle (21;22)

 

In the retina, the visual signaling cascade is associated with disk membranes within the light-sensitive outer segments (OS) of the photoreceptor cells.  Activation of this signaling cascade leads to a hyperpolarization of photoreceptor cells and the reduction of neurotransmitter release at their synapses, located in the outer plexiform layer (OPL) (26).  In a photoreceptor cells, the OS is linked by a connecting cilium (CC) to the biosynthetic and metabolically active inner segment (IS) (Figure 3B).  Microvilli-like calycal processes (CP) at the apical membrane of the inner segment sheath the base of the photoreceptor outer segment and may stabilize it.  The CP may also play a role in outer segment disk morphogenesis as newly synthesized disk membranes are present in this location (18;26;27).  Photoreceptor nuclei are localized in the outer nuclear layer (ONL).  

 

The proteins encoded by the USH genes are members of protein classes with very different functions.  Myosin VIIa is an actin-based molecular motor, harmonin and SANS are scaffold proteins, CDH23 and PCDH15 are cell adhesion molecules, and usherin and VLGR1b are transmembrane proteins that may be involved in signal transduction.  Clarin-1 is a member of the vertebrate-specific clarin family of four-transmembrane domain proteins (26;27).  Despite their different functions, these proteins are all thought to be assembled in a multiprotein scaffold mediated by the PDZ-containing proteins harmonin and whirlin in the neurosensory hair cells of the ear and the photoreceptor cells of the retina.  Multiple studies have shown that the USH1 proteins, including PCDH15, biochemically interact with harmonin and whirlin (18;26;27).  Additionally, PCDH15 has also been shown to interact with both CDH23 and myosin VIIa.  Myosin VIIa may actively transport PCDH15 to the cell surface of stereocilia (12;46).  These biochemical studies are supported by recent data showing mislocalization of various USH proteins in mice with mutations in USH genes (45;46).  In addition, patients carrying heterozygous mutations in both CDH23 and PCDH15 develop USH1, while mice carrying heterozygous mutations in both genes also develop deafness (47).  Members of the USH protein network interact with many other proteins including β-catenin, integrins, and the potent Rac activator, DOCK4.  The cytoplasmic domain of Pcdh15 is unique from other cadherins and it does not contain a consensus motif for binding to B-catenin (7).  In addition to a direct interaction via harmonin and myosin VIIa, many of these interactions connect the USH protein network to the actin cytoskeleton and to adherens junctions (17;25-27).   

 

In hair cells, the major sites of colocalization of USH proteins are the stereocilia and the synaptic regions (26;27).  It is proposed that, along with other possible functions, myosin VIIa and sans target harmonin into the stereocilia where it subsequently binds to F-actin and anchors Cdh23 and Pcdh15-containing links to the actin core of the stereocilium (7;24;25;45).  In addition, the spiral ganglion neurons harbor several of these proteins including PCDH15 (10).  Based on expression pattern and defects seen in the appropriate animal model, several of these proteins are suggested to be components of the links connecting the stereocilia in developing and mature hair cells (26;27;43).  PCH15 isoforms are probable components of transient lateral links, kinociliary links and tip links (1;12;43).  The tip link is composed of Pcdh15 and Cdh23 and is two intertwined strands; Pcdh15 localizes to the lower end of the tip link while Cdh23 is localized to the upper end (16;22).  It has been proposed that Pcdh15 and Cdh23 form coiled homodimers that interact in trans via their opposing N-termini in a calcium-dependent manner to form the 175 nm tip link (16;22). Ca2+ chelation, which affects tip link integrity and mechanoelectrical transduction, also disrupts CDH23 and PCDH15 interactions.  A mutation in the EC1 domain of PCDH15 that causes a recessive form of deafness (DFNB23; OMIM #609533) disrupted the interaction between CDH23 and PCDH15 in vitro suggesting that this interaction has functional significance in vivo (12).  This suggests that several of the isoforms are necessary for the formation of bundles (21).  Furthermore, tip links were found in the mice that lack PCDH15-CD1, -CD2, or –CD3, indicating that none of the isoforms is required at the tip links (21).

 

In the retina, the USH proteins colocalize in the synaptic layer of the OPL (18;26;27), as well as in the ciliary region between the outer and inner segments, particularly in the connecting cilium and the calycal processes (26;27).  PCDH15 colocalizes with harmonin, myosin VIIa, and CDH23 at the synaptic terminals of photoreceptor cells in the retina.  In the photoreceptor synapse, the USH protein complex may contribute to the cortical cytoskeletal matrices of the pre- and postsynaptic regions, which are thought to play a fundamental role in the structural and functional organization of the synaptic junction (18).  The exact role of the USH protein complex in photoreceptor synapses is unknown.  In calycal processes, several USH proteins are located in the more proximal part of the calycal processes, a region where post-Golgi vesicles are translocated from the inner segment to the outer segment (26).  However, PCDH15 is localized more distally in the calycal processes and was found to be colocalized with harmonin at the base of the photoreceptor outer segment, where newly synthesized disk membranes are present (18)

 

In mice, recessive mutations of Pcdh15 in Ames waltzer (av) mouse mutants exhibit ear defects.  Homozygotes for severe mutations exhibit circling, head-tossing, hyperactivity, impaired swimming and profound deafness.  Mice have defects in cochlea and degeneration of hair cells, spiral ganglion cells and saccular macula (16).   A closer examination of av mice revealed abnormal stereocilia morphogenesis and polarity as well as aberrant cuticular plates with defects apparent as early as E17 (45;46;48;49).  These early defects typically lead to severe hair cell degeneration (2;50).  Female av mice can be poor mothers, suggesting the possibility of additional neuronal defects in keeping with the high level of Pcdh15 expression in the brain (2).  Like other mouse mutants of USH genes, Ames waltzer mice do not have any evident retinal degeneration (51;52).  The av mice are considered a model for DFNB23 nonsyndromic deafness (2;8;14;51). Missense mutations of pcdh15 in humans also causes DFNB23; nonsense mutations in humans lead to USH1 (see above) (53).

 

The KCI rats carry a spontaneous mutation (C2911T) in the Pcdh15 gene that resulted in coding of a stop codon (R971X) (53).  By as early as post-natal day 15 the mutant rats are twist the neck towards the back when lifted by the tail (53).  After the rats are weaned, they do not have a startle response (i.e. no response to sounds such as knocking and clapping) and display bidirectional circling behavior and head tossing (53).  Furthermore, the rats rotated along their long axis and sank when placed in a deep tank filled with water (53).  The rats rotated along their body length while underwater and rarely resurfaced (53).  Characterization of the KCI rats determined that they lost their balance possibly due to issues in the inner ear (53).  The stereocilia of the hair cells (inner and outer) of KCI rats were disorganized or thickly fused (53).  Homozygous KCI rats had from severe to total loss of hair cells in the cochlea (53).  In addition, the organ of Corti was collapsed due to the loss of the normal arrangement of fluid spaces (53).  Cochlear nerve fibers in the osseous spiral lamina were severely reduced (53).  The number of sensory hair cells in the saccula macula were reduced (53).  No anatomical defects were seen in the retina of the KCI rats (53).

Putative Mechanism

The squirm mutation results in a sizable deletion of part of the Pcdh15 gene, however it is unknown if squirm mutants still express multiple and potentially partially functional PCDH15 proteins.  Studies correlating anatomical phenotype with severity of genetic defect in various av alleles are providing better understanding of the role played by Pcdh15 in inner ear development and of sensorineural abnormalities associated with alterations in PCDH15 protein structure as a result of gene mutation (49)Pcdh15av-Jand Pcdh15av-2J mice with in-frame deletion mutations in the extracellular domain, exhibit less disorganization of stereocilia and cuticular plates than mice carrying the presumptive functional null alleles Pcdh15av-3J,Pcdh15av-Tg, Pcdh15av-Jfb, or Pcdh15av-5J (49;52;54).  These “null” alleles result in PCDH15 proteins lacking the transmembrane and cytoplasmic domains, suggesting the importance of these domains in protein function.  Despite being a much smaller deletion, the Pcdh15av-2J mutation causes more severe defects than Pcdh15av-J because it deletes a portion of the EC1 domain that is critical for both cadherin and protocadherin function (see Protein Prediction) (5;49).  A genotype-phenotype correlation can also be seen in human mutations of PCDH15.  Mutations that are predicted to truncate PCDH15 in the extracellular domain typically cause USH1, whereas missense mutations that are predicted to change single amino acids are commonly associated with non-syndromic deafness in humans (DFNB23; OMIM #609533).  These studies suggest that the residual function associated with these missense alleles is sufficient for normal vision but not for hearing (14;43)

 

The difference in phenotypic severity in the ear versus eye in humans may also be related to the lack of retinal abnormalities seen in rodent models of USH1.  Although av mice are deaf and have balance problems, mice mutant for genes involved in USH do not have the retinal degeneration seen in humans (27).  Most Pcdh15 mutant animals have normal photoreceptor and retinal morphology and normal electroretinograms (ERG) that test responses to light and dark (51).  One study found two alleles of Pcdh15 to have reduced ERG responses but no evidence of retinal degeneration.  One suggested reason for lack of a phenotype in the retina is the possibility of alternative splicing and expression of partially functional isoforms in the eye (52).  The lack of visual problems in mouse av mutants may also reflect functional redundancy with other proteins in the retina.  In zebrafish, two orthologues of Pcdh15 exist.  Mutations in Pcdh15a cause vestibular dysfunction and deafness, while reduction of Pcdh15b activity results in a visual defect (20).  Although a second Pcdh15 orthologue is not present in mice, it is still possible functional redundancy with some other protein does occur.  Other possible explanations include differences in environmental factors and lifespan.  In humans, the progress of retinal degeneration is typically slow.  Thus, similar photoreceptor cell death may never be evident during the life span of mice (27).

 

The major sites of function for the USH proteins, hair cells and photoreceptor cells, have many similarities suggesting a conservation of function and structure between these two cell types.  Hair cells and photoreceptors are sensory cells that are characterized by chemical synapses that are structurally and functionally specialized for massive and sustained neurotransmitter release (27).  Furthermore, these cells both contain ciliary and actin-rich microvilli-like structures.  Besides the kinocilium present in developing hair cells, the connecting cilium in photoreceptor cells is homologous to the transition zone found at the base of motile cilium and the OS is considered to be a modified cilium (55).  Because defects associated with ciliary dysfunction do occur in some cases of Usher syndrome, including immotile sperm, it is possible that PCDH15 and other USH proteins function in other cell types with ciliary structures.  Further highlighting the similarities between the two cell types, hair cells and photoreceptors also contain actin-rich microvilli-like structures that express USH proteins including PCDH15 (26;27).  Similarly, the Drosophila Pcdh15 homologue, Cad99c, encodes a protein that participates in the morphogenesis of apical microvilli of ovarian follicle cells.  Loss of Cad99c protein in the fly results in drastically shortened and disorganized microvilli, while Cad99c overexpression drastically increases microvilli length (19).  These results are interesting since hair cell stereocilia are also of different lengths, and loss of PCDH15 and other USH proteins leads to the disorganization and abnormal morphogenesis of these stereocilia including improper differential elongation of stereocilia rows (45).   

 

The expression of PCDH15 in malignant NK and T-cell lymphomas, but not normal lymphocytes suggests that PCDH15 may have oncogenic activity and favor tumor cell spreading (15).  The association of PCDH15 with the actin cytoskeleton and β-catenin, a known tumorigenic protein, through the USH protein network may provide a mechanistic explanation for this possibility.  The presence of PCDH15 may facilitate the cytoskeletal changes necessary for cell transformation.

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Science Writers Nora G. Smart, Anne Murray
Illustrators Diantha La Vine
AuthorsAmanda Blasius, Bruce Beutler
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