Human Molecular Genetics, 2001, Vol. 10, No. 16 1709-1718
© 2001 Oxford University Press
Mutations in the novel protocadherin PCDH15 cause Usher syndrome type 1F
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 and 4Center 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 and 10Lynx Therapeutics Inc., 25861 Industrial Boulevard, Hayward, CA 94545, USA.
Received May 14, 2001; Revised and Accepted June 11, 2001.
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSGenBank accession nosREFERENCES |
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We have determined the molecular basis for Usher syndrome type 1F (USH1F) in two families 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 mutation in exon 2 in the second. Consistent with the phenotypes observed in these families, we demonstrated 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 function.
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSGenBank accession nosREFERENCES |
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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 dysfunction, 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 developmental 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 electroretinographic 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).
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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 deafness 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). These data made PCDH15 a positional candidate gene for deafness at the USH1F locus.
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RESULTS |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSGenBank accession nosREFERENCES |
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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.
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T transition (arrow, forward strand shown), generating in a premature stop codon (TGA) after the second amino acid.PCDH15 expression profile
By northern blot analysis, we demonstrated PCDH15 expression in human adult brain, lung and kidney (Fig. 3A). Since PCDH15 cDNA was obtained by screening a fetal brain 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.
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Western blot analysis using an affinity-purified peptide antibody confirmed expression of the protein in brain, lung and kidney (Fig. 3C). The antibody, Pcadh15-PAb4, was raised in rabbits against peptide sequence TEDAHESEKEGGHRDTLIV 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 cytoplasmic domain that contains two proline-rich regions (Fig. 4A). This organization is highly similar to the predicted protein for mouse Pcdh15.
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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 
, ß and 
. In PCDH15 the extracellular 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).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSGenBank accession nosREFERENCES |
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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 hypothesize that PCDH15 plays an important role in maintaining 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 dysfunction. 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 if av mutants show a retinal phenotype 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 sequence. The amino acid sequence of PCDH15 is 94 and 53% identical to the mouse sequence in the extracellular and intracellular 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 these proteins.
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 possibility. 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. Extensive cytoskeletal re-arrangement takes place during hair cell development.
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 randomization 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-2J mouse 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.
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 pigmentosa worldwide. Complementing other studies (8,16), this report also shows that members of the cadherin superfamily are required in the eye and inner ear for maintenance of normal function.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSGenBank accession nosREFERENCES |
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Family data
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 underwent a medical history interview, physical examination including funduscopy and pure tone audiometry. Electroretinography 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.
Linkage analysis
After isolating DNA from whole blood using a phenol extraction 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 LINKAGE program 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 recessive. An allele frequency of 0.1 for each allele and a 1/1000 phenocopy rate were assumed.
FISH
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 manufacturer’s recommended modification for PACs (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 (previously localized to chromosome 10qter) was labeled with digoxigenin. FISH was performed on pro-metaphase chromosomes according to established techniques (22) with minor modifications. Fluorescein-labeled avidin along with biotinylated 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 manufactured and distributed by Applied Imaging Corporation.
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 and sequencing.
RT–PCR analysis
We performed reverse transcription reactions with Superscript reverse transcriptase (Gibco BRL) following the manufacturer’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 Tm of the primers. The primers used for RT–PCR were: forward, 5'-GGAGCCCAGAAGTGAAGCAC-3'; and reverse, 5'-TTTTTCTGAAACACTGGGGG-3'.
RACE
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 76 male/female Caucasians, aged 16–75 years (Clontech) following 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, 5'-GCGAGTCTTATCTTCATAGTTGAGCCTCTTCCT-3'; and GSP2, 5'-GTGAGCTCATTCACTGTGGCATAGTAGC-3'. Gene-specific primers used for 3'-RACE were GSP3, 5'-AAGGGACTGAGCGGCAAAGCCGATGTAC-3'; and GSP4, 5'-CTCCGTGGTCAATCAGCTGGATATGCAAG-3'.
Mutation detection
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 pedigrees.
Sequence analysis
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 GenBank using BLAST (23). The PSORT II program (http://psort.nibb.ac.jp:8800) predicted the cytoplasmic, transmembrane and extracellular domains, and the 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 cytoplasmic domain.
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 0.2x SSC/0.1% SDS/50°C for 1 h. For control probe ß-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 brain, lung and kidney (Research Genetics), and probed them with affinity-purified antibodies (Pcdh15-PAb4) raised against the mouse Pcdh15 peptide sequence (TEDAHESEKEGGHRDTLIV). The antibodies were raised in rabbits. Western blot analysis was performed following standard protocols (26).
Immunohistochemistry
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 described previously (27). Tissues were subsequently 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 visualized using the Vector VIP substrate kit (Vector Laboratories) with incubation for 2–5 min at room temperature (RT). Adjacent sections were incubated with pre-absorbed primary antibody 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 procedure.
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 antigen TEDAHESEKEGGHRDTLIV.
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.
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GenBank accession nos |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSGenBank accession nosREFERENCES |
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PCDH15 cDNA, accession no. AY029205; mouse Pcdh15, AF281899; PCDH15 exons 1–32, AL356114, AL353784, AL360214, AC013737, AC024073, AC027671, AC021350, AC016817, AL365496.
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ACKNOWLEDGEMENTS |
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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.
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FOOTNOTES |
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+ To whom correspondence shouldbe addressed. Tel: +1 319 356 3612; Fax: +1 319 356 4547; Email: richard-smith@uiowa.edu The authors wish it to be known that, in their opinion, the first two authors should be regarded as joint First Authors.
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSGenBank accession nosREFERENCES |
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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.[Web of Science][Medline]
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.[Web of Science][Medline]
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.[Web of Science][Medline]
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.[Web of Science][Medline]
6 Alagramam, K.N., Zahorsky-Reeves, J., Wright, C.G., Pawlowski, K.S., Erway, L.C., Stubbs, L. and Woychik, R.P. (2000) Neuroepithelial defects of the inner ear in a new allele of the mouse mutation Ames waltzer. Hear. Res., 148, 181–190.[Web of Science][Medline]
7 Alagramam, K.N., Murcia, C.L., Kwon, H.Y., Pawlowski, K.S., Wright, C.G. 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.[Web of Science][Medline]
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) Mutation of CDH23, encoding a new member of the cadherin gene family, causes Usher syndrome type 1. Nat. Genet., 27, 108–112.[Web of Science][Medline]
9 Wu, Q. and Maniatis, T. (2001) Comparative DNA sequence analysis of the mouse and human protocadherin gene clusters. Genome Res., 11, 389–404.
10 Kim, S.H., Yamamoto, A., Bouwmeester, T., Agius, E. and Robertis, E.M. (1998) The role of paraxial protocadherin in selective adhesion and cell movements of the mesoderm during Xenopus gastrulation. Development, 125, 4681–4690. [Abstract]
11 Suzuki, S.T. (2000) Recent progress in protocadherin research. Exp. Cell Res., 261, 13–18.[Web of Science][Medline]
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.[Web of Science][Medline]
14 Sudol, M. (1998) From Src homology domains to other signaling modules: proposal of ‘protein recognition code’. Oncogene, 17, 1469–1474.[Web of Science][Medline]
15 Raphael, Y., Kobayashi, K.N. Dootz, G.A., Beyer, L.A., Dolan, D.F. and Burmeister, M. (2001) Severe vestibular and auditory impairment in three alleles of Ames waltzer (av) mice. Hear. Res., 151, 237–249.[Web of Science][Medline]
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.[Web of Science][Medline]
17 Grimberg, J., Nawoschik, S., Belluscio, L., McKee, R., Turck, A. and Eisenberg, A. (1989) A simple and efficient non-organic procedure 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.[Web of Science][Medline]
19 Schaffer, A.A. (1996) Faster linkage analysis computations for pedigrees with loops or unused alleles. Hum. Hered., 46, 226–235.[Web of Science][Medline]
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.[Web of Science][Medline]
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 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., 25, 3389–3402.
24 Perriere, G. and Gouy, M. (1996) WWW-query: An on-line retrieval system for biological sequence banks. Biochimie, 78, 364–369.[Medline]
25 Bultman, S.J., Michaud. E.J. and Woychik, R.P. (1992) Molecular characterization of the mouse agouti locus. Cell, 71, 1195–1204.[Web of Science][Medline]
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, 104, 288–294.[Web of Science][Medline]
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|
 |
|
 N. Michalski, V. Michel, A. Bahloul, G. Lefevre, J. Barral, H. Yagi, S. Chardenoux, D. Weil, P. Martin, J.-P. Hardelin, et al. Molecular Characterization of the Ankle-Link Complex in Cochlear Hair Cells and Its Role in the Hair Bundle Functioning J. Neurosci., June 13, 2007; 27(24): 6478 - 6488. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 F. P M Cremers, W. J Kimberling, M. Kulm, A. P de Brouwer, E. van Wijk, H. te Brinke, C. W R J Cremers, L. H Hoefsloot, S. Banfi, F. Simonelli, et al. Development of a genotyping microarray for Usher syndrome J. Med. Genet., February 1, 2007; 44(2): 153 - 160. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 H. Kremer, E. van Wijk, T. Marker, U. Wolfrum, and R. Roepman Usher syndrome: molecular links of pathogenesis, proteins and pathways Hum. Mol. Genet., October 15, 2006; 15(suppl_2): R262 - R270. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
M I Shabbir, Z M Ahmed, S Y Khan, S. Riazuddin, A M Waryah, S N Khan, R D Camps, M Ghosh, M Kabra, I A Belyantseva, et al. Mutations of human TMHS cause recessively inherited non-syndromic hearing loss J. Med. Genet., August 1, 2006; 43(8): 634 - 640. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 R. J. L. Haywood-Watson II, Z. M. Ahmed, S. Kjellstrom, R. A. Bush, Y. Takada, L. L. Hampton, J. F. Battey, P. A. Sieving, and T. B. Friedman Ames waltzer deaf mice have reduced electroretinogram amplitudes and complex alternative splicing of pcdh15 transcripts. Invest. Ophthalmol. Vis. Sci., July 1, 2006; 47(7): 3074 - 3084. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
Z. M. Ahmed, R. Goodyear, S. Riazuddin, A. Lagziel, P. K. Legan, M. Behra, S. M. Burgess, K. S. Lilley, E. R. Wilcox, S. Riazuddin, et al. The tip-link antigen, a protein associated with the transduction complex of sensory hair cells, is protocadherin-15. J. Neurosci., June 28, 2006; 26(26): 7022 - 7034. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 K. Schlichting, M. Wilsch-Brauninger, F. Demontis, and C. Dahmann Cadherin Cad99C is required for normal microvilli morphology in Drosophila follicle cells J. Cell Sci., March 15, 2006; 119(6): 1184 - 1195. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
M. Senften, M. Schwander, P. Kazmierczak, C. Lillo, J.-B. Shin, T. Hasson, G. S. G. Geleoc, P. G. Gillespie, D. Williams, J. R. Holt, et al. Physical and Functional Interaction between Protocadherin 15 and Myosin VIIa in Mechanosensory Hair Cells J. Neurosci., February 15, 2006; 26(7): 2060 - 2071. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
A. El-Amraoui and C. Petit Usher I syndrome: unravelling the mechanisms that underlie the cohesion of the growing hair bundle in inner ear sensory cells J. Cell Sci., October 15, 2005; 118(20): 4593 - 4603. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
A. Adato, V. Michel, Y. Kikkawa, J. Reiners, K. N. Alagramam, D. Weil, H. Yonekawa, U. Wolfrum, A. El-Amraoui, and C. Petit Interactions in the network of Usher syndrome type 1 proteins Hum. Mol. Genet., February 1, 2005; 14(3): 347 - 356. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
C. Seiler, K. C. Finger-Baier, O. Rinner, Y. V. Makhankov, H. Schwarz, S. C. F. Neuhauss, and T. Nicolson Duplicated genes with split functions: independent roles of protocadherin15 orthologues in zebrafish hearing and vision Development, February 1, 2005; 132(3): 615 - 623. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
Q. Y. Zheng, D. Yan, X. M. Ouyang, L. L. Du, H. Yu, B. Chang, K. R. Johnson, and X. Z. Liu Digenic inheritance of deafness caused by mutations in genes encoding cadherin 23 and protocadherin 15 in mice and humans Hum. Mol. Genet., January 1, 2005; 14(1): 103 - 111. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 H. E. Lilja, E. Suviolahti, A. Soro-Paavonen, T. Hiekkalinna, A. Day, K. Lange, E. Sobel, M.-R. Taskinen, L. Peltonen, M. Perola, et al. Locus for quantitative HDL-cholesterol on chromosome 10q in Finnish families with dyslipidemia J. Lipid Res., October 1, 2004; 45(10): 1876 - 1884. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
R. D. Hawkins and M. Lovett The developmental genetics of auditory hair cells Hum. Mol. Genet., October 1, 2004; 13(suppl_2): R289 - R296. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
Q J Wang, C Y Lu, N Li, S Q Rao, Y B Shi, D Y Han, X Li, J Y Cao, L M Yu, Q Z Li, et al. Y-linked inheritance of non-syndromic hearing impairment in a large Chinese family J. Med. Genet., June 1, 2004; 41(6): e80 - e80. [Full Text] [PDF]
|
|
|
|
|
|
Z. M. Ahmed, S. Riazuddin, J. Ahmad, S. L. Bernstein, Y. Guo, M. F. Sabar, P. Sieving, S. Riazuddin, A. J. Griffith, T. B. Friedman, et al. PCDH15 is expressed in the neurosensory epithelium of the eye and ear and mutant alleles are responsible for both USH1F and DFNB23 Hum. Mol. Genet., December 15, 2003; 12(24): 3215 - 3223. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
K. R. Johnson, L. H. Gagnon, L. S. Webb, L. L. Peters, N. L. Hawes, B. Chang, and Q. Y. Zheng Mouse models of USH1C and DFNB18: phenotypic and molecular analyses of two new spontaneous mutations of the Ush1c gene Hum. Mol. Genet., December 1, 2003; 12(23): 3075 - 3086. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
J. Reiners, B. Reidel, A. El-Amraoui, B. Boeda, I. Huber, C. Petit, and U. Wolfrum Differential Distribution of Harmonin Isoforms and Their Possible Role in Usher-1 Protein Complexes in Mammalian Photoreceptor Cells Invest. Ophthalmol. Vis. Sci., November 1, 2003; 44(11): 5006 - 5015. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
S. L. Ball, D. Bardenstein, and K. N. Alagramam Assessment of Retinal Structure and Function in Ames Waltzer Mice Invest. Ophthalmol. Vis. Sci., September 1, 2003; 44(9): 3986 - 3992. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 P J Francis, S Johnson, B Edmunds, R E Kelsell, E Sheridan, C Garrett, G E Holder, D M Hunt, and A T Moore Genetic linkage analysis of a novel syndrome comprising North Carolina-like macular dystrophy and progressive sensorineural hearing loss Br J Ophthalmol, July 1, 2003; 87(7): 893 - 898. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
R. J. Goodyear and G. P. Richardson A Novel Antigen Sensitive to Calcium Chelation That is Associated with the Tip Links and Kinocilial Links of Sensory Hair Bundles J. Neurosci., June 15, 2003; 23(12): 4878 - 4887. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 L. Ciani, A. Patel, N. D. Allen, and C. ffrench-Constant Mice Lacking the Giant Protocadherin mFAT1 Exhibit Renal Slit Junction Abnormalities and a Partially Penetrant Cyclopia and Anophthalmia Phenotype Mol. Cell. Biol., May 15, 2003; 23(10): 3575 - 3582. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
Y. Kikkawa, H. Shitara, S. Wakana, Y. Kohara, T. Takada, M. Okamoto, C. Taya, K. Kamiya, Y. Yoshikawa, H. Tokano, et al. Mutations in a new scaffold protein Sans cause deafness in Jackson shaker mice Hum. Mol. Genet., March 1, 2003; 12(5): 453 - 461. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
D. Weil, A. El-Amraoui, S. Masmoudi, M. Mustapha, Y. Kikkawa, S. Laine, S. Delmaghani, A. Adato, S. Nadifi, Z. B. Zina, et al. Usher syndrome type I G (USH1G) is caused by mutations in the gene encoding SANS, a protein that associates with the USH1C protein, harmonin Hum. Mol. Genet., March 1, 2003; 12(5): 463 - 471. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 J. Siemens, P. Kazmierczak, A. Reynolds, M. Sticker, A. Littlewood-Evans, and U. Muller The Usher syndrome proteins cadherin 23 and harmonin form a complex by means of PDZ-domain interactions PNAS, November 12, 2002; 99(23): 14946 - 14951. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 A. Forge and T. Wright The molecular architecture of the inner ear Br. Med. Bull., October 1, 2002; 63(1): 5 - 24. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
N. B. Haider, A. Ikeda, J. K. Naggert, and P. M. Nishina Genetic modifiers of vision and hearing Hum. Mol. Genet., May 15, 2002; 11(10): 1195 - 1206. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
C. C. Morton Genetics, genomics and gene discovery in the auditory system Hum. Mol. Genet., May 15, 2002; 11(10): 1229 - 1240. [Abstract] [Full Text] [PDF]
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