© 2001 Oxford University Press Human Molecular Genetics, 2001, Vol. 10, No. 16 1709–1718
Mutations in the novel protocadherin PCDH15 cause
Usher syndrome type 1F
Kumar N. Alagramam, Huijun Yuan1, Markus H. Kuehn2, Crystal L. Murcia3, Sigrid Wayne1,
C.R. Srikumari Srisailpathy1,5, R. Brian Lowry6, Russell Knaus7, Lut Van Laer8, F.P. Bernier6,
Stuart Schwartz3,4, Charles Lee9, Cynthia C. Morton9, Robert F. Mullins2,
Arabandi Ramesh1,5, Guy Van Camp8, Gregory S. Hagemen2, Richard P. Woychik10and
Richard J.H. Smith1,*
Department of Pediatrics, Rainbow Babies and Children’s Hospital, University Hospitals of Cleveland, Case Western
Reserve University, Cleveland, OH, USA,1Molecular Otolaryngology Research Laboratories, Department of
Otolaryngology, University of Iowa, 200 Hawkins Drive, Iowa City, IA 52242, USA,2The University of Iowa Center for
Macular Degeneration in the Department of Ophthalmology and Visual Sciences, University of Iowa, Iowa City, IA,
USA,3Department of Genetics and4Center for Human Genetics, Case Western Reserve University and University
Hospitals of Cleveland, Cleveland, OH, USA,5Department of Genetics, University of Madras, Madras, India,
6Department of Genetics, Alberta Children’s Hospital, Calgary, Alberta, Canada,7Lumsden Clinic, Lumsden,
Saskatchewan, Canada,8Department of Genetics, University of Antwerp, Belgium,9Department of Obstetrics and
Gynecology, Brigham and Women’s Hospital, Harvard Medical School, Boston, MA, USA and10Lynx Therapeutics
Inc., 25861 Industrial Boulevard, Hayward, CA 94545, USA.
Received May 14, 2001; Revised and Accepted June 11, 2001
We have determined the molecular basis for Usher
syndrome type1F (USH1F)
segregating for this type of syndromic deafness. By
fluorescence in situ hybridization, we placed the
human homolog of the mouse protocadherin Pcdh15
in the linkage interval defined by the USH1F locus.
We determined the genomic structure of this novel
protocadherin, and found a single-base deletion in
exon 10 in one USH1F family and a nonsense muta-
tion in exon 2 in the second. Consistent with the
phenotypes observed in these families, we demon-
strated expression of PCDH15 in the retina and
cochlea by RT–PCR and immunohistochemistry.
This report shows that protocadherins are essential
for maintenance of normal retinal and cochlear
Usher syndrome is the most common cause of the dual sensory
impairments of deafness and blindness. Affected persons are
born with hearing loss and develop progressive pigmentary
retinopathy leading to blindness in the second to fourth
decades of life. Clinically subdivided into types 1–3 based on
the degree of deafness and the presence of vestibular dysfunc-
tion, Usher syndrome type 1 (USH1) is the most severe (1).
Persons with USH1 are born with profound deafness and
develop visual problems in late childhood. Approximately
70% segregate for mutations in myosin 7A (USH1B); the
second largest contribution to the USH1 genetic load is closely
linked to the USH1D–USH1F region on chromosome 10 (2).
Several years ago we mapped the Usher syndrome type 1F
(USH1F) locus in a complex family belonging to the Hutterite
Brethren (3), an endogamous ethnic group which maintains a
communal way of life on the prairies of central Canada and the
USA (4,5). In the pedigree we studied, two males were born
with profound sensorineural deafness and delayed develop-
mental motor milestones suggesting the diagnosis of Usher
syndrome. When the boys were older this clinical impression
was confirmed by ophthalmologic evaluation, which revealed
funduscopic findings of retinitis pigmentosa and electroretino-
graphic abnormalities. On a genome-wide screen, we found
only one interval of homozygosity by descent, a 15 cM region
on chromosome 10 flanked by markers D10S199 and
D10S596. The LOD score over this interval was 3.06, and the
locus was assigned the designation USH1F. We also linked a
consanguineous USH1 family from India to this genomic
region with a LOD score >3.0 (Fig. 1).
Recently, we reported cloning a novel murine protocadherin,
Pcdh15, mutations in which are found in the Ames waltzer (av)
mouse mutant and lead to a phenotype characterized by deaf-
ness and vestibular dysfunction associated with degeneration
of inner ear neuro-epithelia (6,7). Using a mouse Pcdh15
cDNA probe to screen a human genomic P1-derived artificial
chromosome (PAC) library, we identified several clones of the
human homolog, PCDH15, which we mapped to chromosome
10q11.2–q21 by fluorescence in situ hybridization (FISH).
*To whom correspondence shouldbe addressed. Tel: +1 319 356 3612; Fax: +1 319 356 4547; Email: email@example.com
The authors wish it to be known that, in their opinion, the first two authors should be regarded as joint First Authors.
1710 Human Molecular Genetics, 2001, Vol. 10, No. 16
These data made PCDH15 a positional candidate gene for
deafness at the USH1F locus.
Genomic structure of PCDH15
To determine the genomic structure of PCDH15, we
sequenced the largest clone identified by screening a human
fetal brain cDNA library with a murine Pcdh15 probe. Using
these data and rapid amplification of cDNA ends (RACE), the
complete PCDH15 cDNA sequence was established. The
genomic structure was determined in part by amplification of
overlapping cDNA primers, but availability of a sequenced
bacterial artificial chromosome (BAC) containing most of the
gene obviated further refinements to this approach.
Mutations in PCDH15 in USH1F families
To screen for PCDH15 mutations in USH1F families, we
completed single-strand conformation polymorphism (SSCP)
and sequence analyses of the entire coding region using
intronic primers (Table 1). A single base (T) deletion in exon
10 and a nonsense mutation in exon 2 were detected in the
Hutterite and Indian families, respectively. Both mutations
segregate with the disease phenotype and generate premature
stop codons (Fig. 2). Similar mutations were not detected in a
screen of 50 control individuals.
PCDH15 expression profile
By northern blot analysis, we demonstrated PCDH15 expres-
sion in human adult brain, lung and kidney (Fig. 3A). Since
PCDH15 cDNA was obtained by screening a fetal brain
Figure 1. (A) The original kindred used to map the USH1F locus, a Hutterite family from Alberta. (B) A consanguineous Indian family also segregating for an
USH1 gene at thesame locus (closedsquares, affected male; opensquares, unaffectedmale; closedcircle, affected female; opencircles, unaffectedfemale; slashed
squares, deceased male; slashed circles, deceased female).
Human Molecular Genetics, 2001, Vol. 10, No. 16 1711
library, we presume that PCDH15 is expressed in human fetal
brain as well. Additional experiments by RT–PCR and direct
sequencing also revealed expression in other human adult
tissues (Fig. 3B) and human fetal cochlea.
Western blot analysis using an affinity-purified peptide anti-
body confirmed expression of the protein in brain, lung and
kidney (Fig. 3C). The antibody, Pcadh15-PAb4, was raised in
rabbits againstpeptide sequence
RDTLIV from the C-terminus of Pcdh15. The results showed
that specific bands are recognized by Pcadh15-PAb4 (Fig. 3C).
When incubated with the primary antibody, a band close to the
expected size (∼175 kDa) was seen; a duplicate blot incubated
with the primary antibody pre-absorbed with the peptide
antigen did not show this band. The other high molecular
weight bands (180, 160 and 130 kDa) seen on the blot may be
isoforms of PCDH15, since alternatively spliced products
derived from PCDH15 were detected by northern (Fig. 3A)
and RT–PCR analyses (data not shown). The low molecular
weight bands (<130 kDa) appeared as robust protein bands on
the Coomassie blue-stained gel, and because they were seen on
both panels, we believe they represent non-specific adsorption
of secondary antibody to concentrated protein.
By immunohistochemistry, we 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 the adult but not the fetal retina
(Fig. 3D). In human fetal cochlea, we detected PCDH15
expression in the supporting cells and outer sulcus cells, as
well as in the spiral ganglion. We confirmed these findings on
mouse cochlea, which also revealed expression on the apical
surface of the hair cells (Fig. 3E).
Predicted amino acid sequence of PCDH15
PCDH15 cDNA encodes an open reading frame (ORF) that
translates to 1955 amino acids. The predicted protein has 11
cadherin repeats, one transmembrane domain and a cyto-
plasmic domain that contains two proline-rich regions
(Fig. 4A). This organization is highly similar to the predicted
protein for mouse Pcdh15.
We used phylogenetic analysis to determine the evolutionary
relationship between PCDH15 and the cadherin superfamily
by comparing PCDH15 to other protocadherins and cadherin-
like sequences. Sequences were selected based on BLAST
analysis or because the gene product was associated with a
disease phenotype similar to USH1F, such as CDH23
(USH1D) (8) (Fig. 4B).
These data show that PCDH15 is related only distantly to
other proteins, making it a unique member of the cadherin
superfamily. This finding is consistent with the genomic
organization of PCDH15, which differs from that of the human
protocadherin gene clusters α, β and γ. In PCDH15 the extra-
cellular domain is encoded by several exons, whereas in the
gene clusters, the extracellular and transmembrane domains
are encoded by a single unusually large exon (9).
PCDH15 is a novel member of the cadherin superfamily of
calcium-dependent cell–cell adhesion molecules. Based on the
phenotype observed in the USH1F families we studied, we
Figure 2. Sequencing results for PCDH15. (A) In the Hutterite family, a portion of the electropherogram for PCDH15 exon 10 demonstrates the deletion of a
thymidine at nucleotide 1471 (arrow, reverse strand shown), changing the reading frame and creating a stop codon after amino acid 419 in exon 11. (B) In the
Indian family, a portionof the electropherogram for PCDH15 exon 2 shows a C→T transition(arrow, forward strand shown), generating in a prematurestop codon
(TGA) after the second amino acid.
1712 Human Molecular Genetics, 2001, Vol. 10, No. 16
hypothesize that PCDH15 plays an important role in main-
taining normal function of the human inner ear and retina. In
the mouse, mutations in Pcdh15 affect hair cell development in
the inner ear. The primary defect appears to be abnormal
development of stereocilia, which results in hair cell dysfunc-
tion. During later stages, sensory and supporting cell pathology
are observed and progress with age to nearly total degeneration
of the cochlear neuro-epithelium (7). The role of PCDH15 in
the retina is not clear and to date, no retinal pathology has been
reported in the Pcdh15 mouse mutant. Studies are underway in
our laboratory to determine ifav mutants show a retinal pheno-
type consistent with USH1F. Our data also show that PCHD15
is expressed in brain, kidney, lung and spleen. Since additional
abnormalities are not found in persons with USH1F, there may
be a level of functional redundancy in some tissues that is
provided by other protocadherins.
Analysis of the ORF of PCDH15 confirms that it is very
similar to the ORF deduced from the mouse Pcdh15 cDNA
Figure 3. (A) Northern blot analysis demonstrating expression of PCDH15 in different human tissues using 5 µg A+ RNA per lane. Blots were hybridized with a
4 kb PCDH15 cDNA probe; the same blot was then stripped and re-probed with human β-actin as the loading control. (B) RT–PCR demonstrating expression of
PCDH15 in total RNA isolated from adult humantissues (RNA was obtainedfrom Clontech Human RNA panels I, II and III). 1, brain; 2, heart; 3, kidney; 4, liver;
5, lung; 6, trachea; 7, bone marrow; 8, colon; 9, small intestine; 10, spleen; 11, stomach; 12, thymus; 13, mammary gland; 14, prostrate; 15, skeletal muscle; 16,
testis; 17, uterus; 18, heart; size marker, PhiX174 digested with HaeIII (Promega); band indicated by solid arrow, 1.078 kb. (C) Western blot analysis of adult
human kidney (K), lung (L) and brain (B) probed with mouse peptide antibody, Pcdh15-PAb4 (left panel probed with primary antibody; right panel probed with
primary antibody previously incubated with the peptide antigen; approximate molecular weights indicated for PCDH15-specific signals; asterisks indicate artifact
signal). (D, opposite) Light micrographs depicting sections of adult (a and b) and second trimester fetal (c and d) retinas. Sections (a) and (c) were incubated with
primary antibody; sections (b) and (d) were incubated with primary antibody pre-absorbed with peptide. Anti-PCDH15 antibody reacts specifically with the inner
and outer synaptic layers (ISL and OSL) and nerve fiber layer (NFL) in adult and fetal retinas. Additional reactivity in the region of the outer limiting membrane/
photoreceptor cell inner segments (OLM) is observed in adult, but not fetal, retina. Weak binding of the antibody to the retinal pigmented epithelium (PE) is
observed in fetal retina. (E, opposite) Light micrographs depicting sections of P8 mouse cochleae (a and b). Section (a) was incubated with primary antibody and
section (b) was incubated with primary antibody pre-absorbed with peptide. Thick arrow, anti-Pcdh15 antibody reacts specifically with supporting cells; OSC,
outer sulcus cells, SG, spiral ganglion cells; thin arrow, the apical surface of the hair cells.
Human Molecular Genetics, 2001, Vol. 10, No. 16 1713
sequence. The amino acid sequence of PCDH15 is 94 and 53%
identical to the mouse sequence in the extracellular and intra-
cellular domains, respectively. Although the percentage
identity is lower in the intracellular domain, the two proline-
rich regions are conserved between species, suggesting that
these domains are significant with respect to the function of
Protocadherins represent a large family of non-classic
cadherins which are structurally and functionally divergent
from the classic cadherins. Members of the protocadherin
family have been shown to be required for morphogenesis
during early development in lower vertebrates (10). In higher
vertebrates, based on expression data, protocadherins are
thought to be involved in a variety of functions, including
neural development, neural circuit formation and formation of
the synapse (11).
The function of protocadherins in the mammalian inner ear
is not clear, but a survey of the literature and an analysis of the
amino acid sequence of PCDH15 lead to an interesting possi-
bility. The process of growth and arrangement of stereocilia on
the apical surfaces of hair cells in higher vertebrates is well
orchestrated and is a good example of planar polarity. Exten-
sive cytoskeletal re-arrangement takes place during hair cell
Stereocilia develop from microvilli on the apical surfaces of
hair cells. The microvilli are of uniform size during early
embryonic stages; however, as hair cells mature the lateral
microvilli grow taller (in a crescent-shaped pattern) while the
medial microvilli regress (lateral and medial with respect to the
modiolus). This change ultimately leads to the ‘V-shaped’
bundles that characterize stereocilia, with the tip of the ‘V’
centered on the kinocilium facing the lateral wall (12).
In Drosophila, a characteristic feature of epidermal cells is
the projection of bristles that are asymmetrically distributed
along the apical surface. Mutations in protocadherin genes,
such as Dachsous and Flamingo, affect this planar polarity
(13). Most notably, mutations in Flamingo cause randomiza-
tion of bristle placement, which is broadly reminiscent of the
disorganization observed in the placement of stereocilia in
mice carrying a mutation in Pcdh15. Also, genetic studies in
Drosophila show that a set of core signaling components
(Frizzled receptors and secreted WNT signaling factors) are
involved in a signaling pathway that controls planar polarization
in a variety of tissues.
The predicted amino acid sequence of PCDH15 contains two
well conserved proline-rich regions, which are known to serve
as binding sites for profilin or other proteins containing Src
homology 3 (SH3) and WW domains (two highly conserved
tryptophan residues spaced 20–22 amino acids apart). Profilin
regulates polymerization of actin filaments and provides a link
between the cytoskeleton and signaling network, while both
SH3 and WW domains participate in the assembly of signaling
complexes (14). Taken together, we speculate that PCDH15
plays a role in processes that regulate planar polarity in the
sensory neuro-epithelium of the inner ear.
A recent report by Raphael et al. (15) lends support to our
hypothesis. Raphael et al. (15) studied the morphology of hair
cells from the different alleles of av at 15–16 days after birth.
They found that in the Pcdh15av-2Jmouse mutant, outer hair
cell stereocilia are disorganized, with stereocilia bundles on
some of the outer hair cells rotated up to 90° from the normal
orientation. Based on this observation, they suggest that the
cellular mechanism that regulates orientation of the stereocilia
bundle may be affected in the av mice.
1714 Human Molecular Genetics, 2001, Vol. 10, No. 16
Our findings provide strong evidence that mutations in
PCDH15 cause USH1F. This discovery will make it
possible to evaluate the contribution of PCDH15 to
hereditary deafness, vestibular dysfunction and retinitis
(8,16), this report also shows that members of the cadherin
superfamily are required in the eye and inner ear for main-
tenance of normal function.
worldwide. Complementingother studies
Figure 4. Analysis of the predicted amino acid sequence encoded by PCDH15. (A) Predicted amino acid sequence of PCDH15. The amino acid at the start of
each cadherin repeat (Cr) is shown in pink and underlined in bold. The end of each repeat is designated by the conserved sequence DXNDNXPXF. Text in italics
indicates the proline-rich region. The putative signal region is shown with a single underline and the transmembrane domain, with a double underline. (B) Phylo-
genetic tree comparing cytoplasmic domains of cadherin family members. Sixteen proteins were used for this analysis; sequences were identified by accession
numbers or through a key word search of GenBank for human or mouse protocadherins. The tree was constructed using default parameters in the phylup method
of the CLUSTAL W program and is unrooted. The scale bar represents one amino acid substitution out of 20 amino acid residues. Hs, Homo sapiens; Mm, Mus
musculus; Rn, Rattus norvegicus; Dm, Drosophila melanogaster. GenBank accession nos: Hs cadherin 23, AAG27034; Mm cadherin 23, NP075859; Hs PCDH-
alpha1, AAD43699; Hs PCDH-beta2, AAD43756; Hs PCDH1, NP002578; HsPCDH7, XP003486; Hs Fat1, Q14517; Hs Flamingo1, AAF61929; Hs Flamingo2,
AAF61930; Dm CG7805, AAF56955; Rn Pcdh, AF100960.
Human Molecular Genetics, 2001, Vol. 10, No. 16 1715
MATERIALS AND METHODS
Two families segregating for autosomal recessive, profound,
congenital deafness and retinitis pigmentosa were ascertained,
one in Canada and the second in India. In both families, a
genome-wide scan was completed, demonstrating linkage to
the USH1F locus. Each participating family member under-
went a medical history interview, physical examination
including funduscopy and pure tone audiometry. Electroretino-
graphy was performed on both affected persons in the
Hutterite family. Internal Review Board approval was
obtained for this study and informed consent was obtained
from all participants.
After isolating DNA from whole blood using a phenol extrac-
tion protocol (17), genotyping was completed with 169 STRPs
evenly spaced across the human autosomal genome (Screening
set 8A, Research Genetics). Both families demonstrated
linkage to the USH1F locus on chromosome 10. LOD scores
were calculated using either the FASTLINK version of the
LINKAGEprogram package (18,19) or MAPMAKER/
HOMOZ (20). The disease allele frequency was set at 0.00075
(21), and the disease was coded as fully penetrant and reces-
sive. An allele frequency of 0.1 for each allele and a 1/1000
phenocopy rate were assumed.
FISH was performed using PAC63N4, which contains the
human equivalent of the mouse av gene. PAC DNA was
prepared using the QIAGEN DNA prep kit, following the
(Genome Systems). The PAC probe was labeled with biotin
14-dATP by nick translation (BioNick Labeling System
18247-015, Gibco BRL), and the control BAC 70E19 (previ-
ously localized to chromosome 10qter) was labeled with
digoxigenin. FISH was performed on pro-metaphase chromo-
somes according to established techniques (22) with minor
modifications. Fluorescein-labeled avidin along with biotinyl-
ated goat anti-avidin antibody reagents, and rhodamine-labeled
anti-digoxigenin along with rabbit anti-sheep and rhodamine-
labeled anti-rabbit antibody reagents, were used to detect and
amplify probe signals. The slides were counterstained with
DAPI. Digital images were collected using a Leitz DMRB
microscope controlled by CytoVision ChromoFluor software
modification for PACs
by Applied Imaging
Isolation of PCDH15 cDNA sequence
We screened a human fetal brain cDNA library (Clontech)
with a 4.5 kb mouse av cDNA probe using the manufacturer’s
protocol. Several overlapping cDNA clones were obtained.
The cDNA clone with the largest insert (4.3 kb) was
sequenced. The remainder of the PCDH15 cDNA sequence
was determined using a combination of different approaches.
We used the mouse Pcdh15 full-length cDNA sequence to
perform BLAST searches against the htgs database to identify
BACs containing PCDH15 genomic sequence. By using the
cDNA sequence, we determined the genomic structure of
PCHD15. To clone the missing 3′ end (∼2.5 kb), we completed
3′-RACE and then verified the predicted result by RT–PCR
We performed reverse transcription reactions with Superscript
reverse transcriptase (Gibco BRL) following the manufac-
turer’s protocol. Total RNA isolated from different human
tissues (Clontech) was used, in addition to cochlear RNA
isolated from a 19-week-old fetus. In general, PCR conditions
were: 94°C for 2 min, followed by 30 cycles of 94°C for 30 s,
55°C for 30 s and 72°C for 1 min. The annealing temperature
was adjusted based on the Tmof the primers. The primers used
for RT–PCR were: forward, 5′-GGAGCCCAGAAGTGAAG-
CAC-3′; and reverse, 5′-TTTTTCTGAAACACTGGGGG-3′.
We used the Marathon-Ready cDNA Kit (Clontech) for 5′- and
3′-RACE. RACE template synthesis was carried out using
poly(A)+RNA from fetal brain, pooled from 10 male/female
Caucasians, aged 21–30 weeks, and adult retina, pooled from
the manufacturer’s protocol. We used the Advantage 2 PCR
Kit (Clontech) for both first and second rounds of PCR. The
cycling parameters were as follows: 94°C for 30 s , 68°C
for 4 min. Products were separated on 1% agarose gels, cloned
into the pCR-TOPO vector (Invitrogen) and sequenced.
The gene-specific primers used for 5′-RACE were GSP1,
Gene-specific primers used for 3′-RACE were GSP3, 5′-AAGG-
GACTGAGCGGCAAAGCCGATGTAC-3′; and GSP4, 5′-CT-
SSCP. PCDH15 exons 1–32 were amplified from genomic
DNA from family members and a normal-hearing, unrelated
control using primers located in the flanking introns (Table 1).
PCR products were resolved on polyacrylamide gels.
Sequencing. Exons were amplified from genomic DNA from
an affected member of each of the two families and a normal-
hearing, unrelated control. PCR products were gel-purified
and sequenced in both directions using the ABI 373A Dye
terminator cycle sequencing system (Perkin Elmer) with the
primers used for PCR. Sequences were compared to our
cDNA sequence data for PCDH15. Exons in which mutations
were detected were sequenced in all other persons in both
We used Applied Biosystems DyeDeoxy terminator kit to
perform sequencing reactions and the ABI 373 (Perkin
Elmer) to resolve the products. The raw sequence data were
aligned and compared with PCDH15 cDNA sequence using
the Sequencer program (Gene Codes). The nucleotide
sequence data were compared with other sequences in
1716 Human Molecular Genetics, 2001, Vol. 10, No. 16
GenBank using BLAST (23). The PSORT II program (http://
ProfileScan program (http://www-isrec.isb-sib.ch/software/
PRScan_form.html) identified the cadherin repeats. GenBank
was the source of sequences of other mouse cadherin protein
sequences. We used CLUSTAL W (http://www.ebi.ac.uk/
clustalw) to generate the phylogenetic tree using the
program’s default settings and viewed using Njplot (24). The
analysis was restricted to the cytoplasmic domain to avoid the
confounding effect caused by variation in the number of
cadherin repeats. Mouse and human cadherin sequences from
GenBank were analyzed with PSORT II to predict the cyto-
Northern blot analysis
We obtained human Poly(A)+RNA from Clontech and
performed northern blot analysis using a 4 kb fragment of
PCDH15 cDNA as a probe, as described previously (25). We
washed the membranes with a final stringency of 0.2× SSC/
0.1% SDS/50°C for 1 h. For control probe β-actin, we raised
the wash temperature to 65°C. Filters were exposed to Kodak
MS film from several hours to several days.
Western blot analysis
We used western blots containing proteins from adult human
affinity-purified antibodies (Pcdh15-PAb4) raised against the
Table 1. PCDH15 primers
Exon Exon size (bp) PCR product size (bp)cDNA positionForward primers (5′→3′) Reverse primers (5′→3′)
1 355 524 1–355 gtctcctgtatgtgctgcgatctctgtggttcactgctgc
2 119242 356–474 aaggcttttctgtgctgtga gctcgctctaaaggtcaagcta
3 66 174 475–540 gtgcccctgcagtcaattatgaattaaaatctgaagaaaaccctc
4 161429 541–701aaactgattgtgagccagcc tgttcccttcctttctcattc
5 156 267 702–857 cgagtgctttgacagtgatga tcaacagaaaggacagagagaga
6 120283 858–977 catttctggtgtgcagttga tctggttctggaagttactgaata
7 111198 978–1088 cattcaaataatctgaagtctgtatc tacttcataaattcacagaagaaat
8 171383 1089–1259atgtttgccaggctggtatcacatgatcccattgggtttt
9 109223 1260–1368 aatgcggctgaatgaaactc ttccttggaattgagagaatttg
10 113277 1369–1481 cctcagctgatgaagggaaa tgcctacagtagcgtctacagatt
11 207 4911482–1688 tgcatgaaaagtgaaactccaatctgcaagctgaaaaggaa
12 135 358 1689–1823ctttcacgtggatttttgct ttttcccccaagtcattgat
13 150247 1824–1973 atttgcagaacatctctactaagtgta tgcacatgtaaataacagctttg
14 194 334 1974–2167aagggaagtcttcaccacacaaatacttgccgcgcttctta
15 133 125 2168–2300 ccatctgcactgtgtatattgaatagatttaataaaacagcaccaaccc
1680 185 2301–2380gtgggcgccatatcaattacctaacagtacgttgctgtaca
17 94 1812381–2474 aagaagactctccttgtgaca ccttcacagggagcatactc
18129 292 2475–2603 gctcctctcacatttaatgcttgttgaagaatatccagcacagtca
19 306508 2604–2909atttcctccttagagggccaaattttggcacacaaaccct
20 225363 2910–3134 gctgtcctaggcactaacgctacagagagcaaagcaggca
21117 366 3135–3251ttcctctgaggtgccagtcttttgctgtcttgtgattcgg
22 141327 3252–3392 cgattttcacttctaaaagaccatgctgtcatctgttaagccaa
Human Molecular Genetics, 2001, Vol. 10, No. 16 1717
RDTLIV). The antibodies were raised in rabbits. Western blot
analysis was performed following standard protocols (26).
Pcdh15 peptide sequence(TEDAHESEKEGGH-
Tissues from human adult and fetal retina were fixed in 4%
paraformaldehyde in 100 mM sodium cacodylate pH 7.4. After
2–4 h of fixation, eyes were transferred to 100 mM sodium
cacodylate, infiltrated and embedded in acrylamide, as
embedded in OCT and sectioned to a thickness of 6–8 µm. We
used as a primary antibody the same antibody that was used in
the western blot. The secondary antibody was goat anti-rabbit
IgG conjugated to biotin (Vector Laboratories), followed by
avidin biotin complex labeled with horseradish peroxidase
(HRP) (ABC Elite kit, Vector Laboratories). HRP was visual-
ized using the Vector VIP substrate kit (Vector Laboratories)
with incubation for 2–5 min at room temperature (RT). Adja-
cent sections were incubated with pre-absorbed primary anti-
body alone to serve as negative controls.
The cochleae used in this study were obtained from mice
8 days after birth (P8). We chose this time point because the
organ of Corti is well developed and Pcdh15 is known to be
expressed. To obtain the cochleae, the mice were sacrificed
and the inner ears were dissected. Each cochlear capsule was
partly opened and immersed in 4% paraformaldehyde in PBS
for 1 h at RT. After rinsing three times with PBS at RT, each
cochlea was decalcified with 0.35 M EDTA in 0.01 M sodium
phosphate buffer (pH 7.2) overnight at 4°C. Tissues were
rinsed in PBS, dehydrated through a graded series of alcohols,
cleared in histoclear, and embedded in paraffin using standard
Sections were cut at 4 µm in the modiolar plane, and two to
four sections were mounted on each slide. Sections were
deparaffinized, rehydrated and treated with 0.3% hydrogen
peroxide to quench endogenous peroxidase activity. Sections
were blocked with 1% normal goat serum for 30 min, then
incubated overnight in primary antisera at 4°C at the optimal
dilution as determined by titration studies. Forty sections (10
separate microscope slides, each with four sections on it) were
incubated with the primary antisera simultaneously. Controls
consisted of incubating sections in normal serum lacking the
primary antisera or pre-absorbing with 10-fold excess peptide
After 24 h, sections were rinsed in PBS, incubated in
biotinylated goat anti-rabbit IgG for 20 min at RT, rinsed in
PBS, and incubated for 30 min with avidin–biotin HRP
complex (ABC, Vector labs). To visualize the binding sites of
the primary antibody, sections were incubated in a couplin jar
containing 3,3′-diaminobenzidine-HCl (DAB)-H2O2.
GenBank accession nos
PCDH15 cDNA, accession no. AY029205; mouse Pcdh15,
AF281899; PCDH15 exons 1–32, AL356114, AL353784,
AL360214, AC013737, AC024073, AC027671, AC021350,
We would like to thank H. Dakappagari for technical assistance.
This research was supported by grants EY11515 to G.S.H.,
RO1-DC03420 to R.P.W. and RO1-DC02842 to R.J.H.S..
C.L.M. was supported by NIH training grant HD07104.
1. Smith, R.J.H., Berlin, C., Hejtmancik, J.F., Keats, B., Kimberling, W.J.,
Lewis, R.A., Möller, C.G., Pelias, M.Z. and Tranebjærg, L. (1994) Clinical
diagnosis of the Usher syndromes. Am. J. Med. Genet., 50, 32–38.
2. Astuto, L.M., Weston, M.D., Carney, C.A., Hoover, D.M.,
Cremers, C.W.R.J., Wagenaar, M., Moller, C., Smith, R.J.H., Pieke-Dahl,
S., Greenberg, J. et al. (2000) Genetic heterogeneity of Usher syndrome:
analysis of 151 Usher I families. Am. J. Hum. Genet., 67, 1569–1574.
3. Wayne, S., Der Kaloustian, V.M., Schloss, M., Polomeno, R., Scott, D.A.,
Sheffield, V.C. and Smith, R.J.H. (1997) Localization of Usher syndrome
type 1F to chromosome 10. Am. J. Hum. Genet., 61, A300.
4. Hostetler, J.A. (1985) History and relevance of the Hutterite population
for genetic studies. Am. J. Med. Genet., 22, 453–462.
5. Lowry, R.B., Morgan, K., Holmes, T.M. and Gilroy, S.W. (1985)
Congenital anomalies in the Hutterite population: a preliminary survey
and hypothesis. Am. J. Med. Genet., 22, 545–552.
6. Alagramam, K.N., Zahorsky-Reeves, J., Wright, C.G., Pawlowski, K.S.,
Erway, L.C.,Stubbs, L. andWoychik, R.P. (2000)Neuroepithelial defects
of the inner ear in a new allele of the mouse mutation Ames waltzer.
Hear. Res., 148, 181–190.
and Woychik, R.P. (2001) The mouse Ames waltzer hearing-loss mutant
is caused by mutation of Pcdh15, a novel protocadherin gene
Nat. Genet., 27, 99–102.
8. Bolz, H., von Brederlow, B., Rez, A., Bryda, E.C., Kutsche, K.,
Nothwang, H.G., Seeliger, M., Cabrera, M., Vila, M.C., Molina, O.P.
et al. (2001)MutationofCDH23, encoding anew member of thecadherin
gene family, causes Usher syndrome type 1. Nat. Genet., 27, 108–112.
9. Wu, Q. and Maniatis, T. (2001) Comparative DNA sequence analysis of
the mouse and human protocadherin gene clusters. Genome Res., 11,
10. Kim, S.H.,Yamamoto, A., Bouwmeester,T.,Agius,E.andRobertis, E.M.
(1998) The role of paraxial protocadherin in selective adhesion and cell
movements of the mesoderm during Xenopus gastrulation. Development,
11. Suzuki, S.T. (2000) Recent progress in protocadherin research. Exp. Cell
Res., 261, 13–18.
12. Pujol, R., Lavingne-Rebillard, M. and Lenoir, M. (1998) Develoment of
sensory and neural structures in mammalian cochlea. In Rubel, E.W.,
Popper, A.N. and Fay, R.R. (eds), Development of the Auditory System.
Springer, NY, pp. 146–192.
13. Eaton, S. (1997) Planar polarization of Drosophila and vertebrate
epithelia. Curr. Opin. Cell Biol., 9, 860–866.
14. Sudol,M. (1998)From Src homologydomainstoother signalingmodules:
proposal of ‘protein recognition code’. Oncogene, 17, 1469–1474.
15. Raphael, Y., Kobayashi, K.N. Dootz, G.A., Beyer, L.A., Dolan, D.F. and
Burmeister, M. (2001) Severe vestibular andauditoryimpairment in three
alleles of Ames waltzer (av) mice. Hear. Res., 151, 237–249.
16. Bork, J.M., Peters, L.M., Riazuddin, S., Bernstein, S.L., Ahmed, Z.M.,
Ness, S.L., Polomeno, R., Ramesh, A., Schloss, M., Srisailpathy, C.R.S.
et al. (2001) Usher syndrome 1D and nonsyndromic autosomal recessive
deafness DFNB12 are caused by allelic mutations of the novel cadherin-
like gene CDH23. Am. J. Hum. Genet., 68, 26–37.
17. Grimberg, J., Nawoschik, S., Belluscio, L., McKee, R., Turck, A. and
Eisenberg, A. (1989)A simpleandefficientnon-organicprocedure for the
isolation of genomic DNA from blood. Nucleic Acids Res., 17, 8390.
18. Lathrop, G.M. and Lalouel, J.M. (1984) Easy calculations of LOD scores
and genetic risks on small computers. Am. J. Hum. Genet., 36, 460–465.
19. Schaffer, A.A. (1996) Faster linkage analysis computations for pedigrees
with loops or unused alleles. Hum. Hered., 46, 226–235.
20. Kruglyak, L., Daly, M.J. and Lander, E.S. (1995) Rapid multipoint
linkage analysis of recessive traits in nuclear families, including
homozygosity mapping. Am. J. Hum. Genet., 56, 519–527.
21. Zbar, R.I., Ramesh, A., Srisailapathy, C.R.S., Fukushima, K., Wayne, S.
and Smith, R.J.H. (1998) Passage to India: the search for genes causing
1718 Human Molecular Genetics, 2001, Vol. 10, No. 16
autosomal recessive nonsyndromic hearing loss. Otolaryngol. Head Neck
Surg., 118, 333–337.
22. Pinkel, D., Straume, T. and Gray, J.W. (1986) Cytogenetic analysis using
quantitative, high-sensitivity, fluorescence hybridization. Proc. Natl
Acad. Sci. USA, 83, 2934–2938.
23. Altschul, S.F., Madden, T.L., Schaffer, A.A., Zhang, J., Zhang, Z.,
Miller, W. and Lipman, D.J. (1997) Gapped BLAST and PSI-BLAST: a
new generation of protein database search programs. Nucleic Acids Res.,
24. Perriere, G. and Gouy, M. (1996) WWW-query: An on-line retrieval
system for biological sequence banks. Biochimie, 78, 364–369.
25. Bultman, S.J., Michaud. E.J. and Woychik, R.P. (1992) Molecular
characterization of the mouse agouti locus. Cell, 71, 1195–1204.
26. Sasse, J. and Gallagher, S.R. (1991) Detection of proteins. In
Ausubel, F.M., Brent, R., Kingston, R.E., Moore, D.D., Seidman, J.G.,
Smith, J.A. and Struhl, K. (eds), Current Protocols, John Wiley & Sons,
Vol. 2, pp. 10.7.1–10.8.16.
27. Mullins, R., Johnson, L., Anderson, D. and Hageman, G. (1997)
Characterization of drusen-associated glycoconjugates. Ophthalmology,