|Molecular Vision 2005;
Received 6 April 2005 | Accepted 26 October 2005 | Published 3 November 2005
Protocadherin-21 (PCDH21), a candidate gene for human retinal dystrophies
1Institute of Human Genetics, University Hospital of Cologne, Cologne, Germany; 2Institute of Human Genetics, University Medical Center Hamburg-Eppendorf, Hamburg, Germany
Correspondence to: Dr. Hanno Bolz, Institute of Human Genetics, University Hospital of Cologne, Kerpener Straße 34, 50931 Koeln, Germany; Phone: +49-221-47886612; FAX: +49-221-47886465; email: email@example.com
Purpose: It has been demonstrated that mice lacking a functional copy of prCAD, the gene encoding protocadherin-21, show progressive photoreceptor degeneration. Therefore we searched for a human retinal phenotype associated with mutations in the orthologous human gene, PCDH21.
Methods: We characterized the genomic organization of human PCDH21 and performed mutation screening in 224 patients with autosomal recessive retinitis pigmentosa, 29 patients with Leber congenital amaurosis, and 26 patients with Usher syndrome type 1.
Results: PCDH21 spans 23 kb, consists of 17 exons, and encodes a protein that shows close phylogenetic relationship to cadherin-23 (CDH23), the protein involved in Usher syndrome type 1D. In a total of three unrelated patients, we identified two different heterozygous missense changes (p.A212T and p.P532A), affecting evolutionarily conserved residues, that were not found in 100 unaffected controls. A second mutation allele was not detected. A novel intragenic microsatellite marker was identified.
Conclusions: PCDH21 mutations are not a major cause of the retinal diseases investigated herein, and the corresponding human phenotype remains to be determined. Our data may facilitate future investigations of patients with various (other) forms of inherited retinal dystrophy.
Cadherins comprise a large family of transmembrane proteins, many of which mediate Ca2+-dependent cell adhesion [1,2]. In human, more than 80 cadherins have been characterized to date. Common to all cadherins are tandemly repeated, multiple (5 to 34) cadherin domains (extracellular domains, EC), consisting of about 100 amino acid stretches connected by about 10 amino acid linker regions. ECs contain the evolutionarily highly conserved Ca2+-binding motifs that mediate homophilic association of cadherin molecules. Recently, it has been shown that mutations in three members of this protein family lead to retinal degeneration in human . Mutations in the genes encoding cadherin-23 and protocadherin-15 (CDH23 and PCDH15, respectively), account for two subgroups of autosomal recessive Usher syndrome type I (USH1), USH1D [4,5], and USH1F [6,7], respectively. Alterations in CDH3 underlie hypotrichosis with juvenile macular dystrophy .
Protocadherin-21 (PCDH21, MT-PCDH, KIAA1775, and prCAD are synonymously used) consists of six ECs and an intracellular domain without homology to any known cadherin, suggesting interacting partners different from those of other cadherins. It is expressed only in a small subset of neuronal tissues such as the olfactory bulb  and the retina [10,11]. PCDH21 expression is limited to the base of the outer segment and the connecting cilium both in rods and cones. A single proteolytic cleavage that releases the soluble ectodomain is a crucial step in photoreceptor outer segment assembly . Outer segments of pcdh21 knockout mice are disorganized and fragmented, and degenerate . PCDH21 is therefore an obvious candidate for human inherited retinal disease, such as retinitis pigmentosa (RP) and Leber congenital amaurosis (LCA). Also, as PCDH21 maps on 10q23, data suggesting the presence of a putative third USH1 locus in the vicinity of the USH1D and the USH1F loci on the long arm of chromosome 10  make PCDH21 a likely candidate for USH1.
In this study, we elucidated the genomic structure of human PCDH21 and performed mutation analysis in patients with various forms of autosomal recessive retinal dystrophy.
Two hundred twenty-four individuals with arRP, 29 with LCA, and 26 with USH1, mostly from Germany and all apparently unrelated, were included in this study. In USH1 patients, mutations in MYO7A (USH1B), USH1C (USH1C), CDH23 (USH1D), PCDH15 (USH1F), and SANS (USH1G), have previously been excluded. No mutations in CRX, LRAT, RDH12, and RPE65 have been detected in the LCA patients analyzed here.
Starting from the PCDH21 cDNA sequence (GenBank accession number BC038799), we determined intron sizes and exon-intron boundaries by BLAST searches  against the unfinished High Throughput Genomic Sequence (htgs) database in which we found a match to genomic DNA in GenBank accession number AC022389. This sequence was contributed by Genome Therapeutics Corporation (Waltham, MA) to the public database. A search for putative polymorphic repeat sequences revealed a CA-dinucleotide repeat within intron 9 (for primers, see Table 1).
Amplification of 17 genomic fragments containing the 17 coding exons and exon-intron boundaries of PCDH21 was carried out following standard protocols (for primers and amplicon sizes, see Table 1). PCR products were amplified using 100 ng of genomic DNA in a 25 μl reaction mixture containing 10 pmol forward and reverse primers, 0.2 mM dNTP, 10 mM Tris-HCl, 50 mM KCl, 1.5 mM MgCl2, and 0.5 units Taq polymerase (Invitrogen Corp., Carlsbad, CA). Primers were designed without using a computer program. After initial denaturation of 95 °C for 4 min, 30 cycles were performed which consisted of 95 °C for one min, 55 °C for one min, and 72 °C for one min, with a final extension step of 72 °C for ten min for all exons. DNA fragments were purified using QIAquick purification columns (Qiagen Inc., Hilden, Germany). Amplicons were screened for sequence variants by the method of conformation-sensitive electrophoresis of single-stranded DNA, using nondenaturing gels containing 8% polyacrylamide . Fragments with an abnormal migration pattern were sequenced  using Big Dye Terminator Cycle sequencing (Applied Biosystems, Foster City, CA). In case of a nucleotide change leading to a missense change, 200 chromosomes of persons with normal vision (as by medical history) were screened for the variant. Bidirectional sequencing of all exons was performed for the three patients heterozygous for p.A212T or p.P532A. DNA samples of patients homozygous for an allele of the newly identified intragenic PCDH21 CA-repeat polymorphism were directly sequenced for all exons.
Phylogenetic classification of PCDH21 and construction of a phylogenetic tree were carried out by comparing the peptide sequence of the N-terminal cadherin repeat motif (extracellular domain 1; EC1) with those of various cadherin proteins , using MegAlign version 4.05 from the DNASTAR software package (Madison, WI).
Comparison of the EC1 peptide sequence of PCDH21 with EC1s of other cadherins revealed that the closest human homolog is CDH23 (data not shown). Although, in a recent study, PCDH21 could not be identified in invertebrates , our investigations show that EC1 of PCDH21 shares significant similarity to Drosophila CG6445 (GenBank accession number NM_168724), its putative ortholog.
Exon-intron boundaries of the PCDH21 gene were determined by comparing PCDH21 cDNA sequence against the unfinished High Throughput Genomic Sequences (htgs) database. The PCDH21 gene is on human BAC clone RP11-12425 (GenBank accession number AC022389) on chromosome 10q23. It spans approximately 20 kb and comprises 17 exons ranging in size between 51 and 540 bp (Table 1). The 5' and 3' ends of all introns were consistent with consensus sequences for acceptor and donor sites, respectively (RNAinfo). A highly polymorphic CA repeat (IVS9-CA) was identified in intron 9 (Table 2).
Two missense changes, p.A212T and p.P532A, were identified in two arRP patients in heterozygous state. Heterozygosity for p.P532A was found in an individual with USH1. No second alteration was found in any of the three patients. Both p.Ala-212 and p.Pro-532 are conserved in mouse pcdh21 and in the putative Drosophila ortholog CG6445 (Figure 1). The changes were not present in 100 unaffected control persons. An additional change, p.N623S, was observed both in patients and control persons. Numerous noncoding nucleotide changes were found (Table 3, Figure 2).
In this report, we describe the genomic structure of human PCDH21 and the results of a mutation screening in patients with arRP, LCA, and USH1. Three missense changes, p.A212T, p.P532A, and p.N623S, were identified, each in the heterozygous state. While p.N623S was also detected in several unaffected individuals, suggesting that it is a nonpathogenic change, p.A212T and p.P532A were not present in the 100 unaffected controls studied. Neither of the latter two variants affects a Ca2+-binding motif, but the respective residues are conserved between human PCDH21, mouse pcdh21, and the putative Drosophila ortholog CG6445. It is possible that p.A212T or p.P532A, or both, are disease causing, implying that in each case, the second mutation escaped detection. Assuming digenic inheritance involving mutations in a second gene, we performed mutation screening in PROML1 (encoding prominin (mouse)-like 1). Similar to PCDH21, PROML1 is concentrated in the plasma membrane evaginations at the base of the outer segments of rod photoreceptors. Recessive PROML1 mutations cause retinal degeneration, possibly because of impaired disk formation in the outer segment of photoreceptors . However, sequencing of the entire PROML1 coding region did not reveal any mutation (data not shown). Although we could not prove a causative role of the two PCDH21 mutations in the retinal degeneration of the patients studied, retina-specific expression of the gene, its close phylogenetic relationship to CDH23, and the retinal dystrophy phenotype of pcdh21 knockout mice still make this gene an interesting candidate for retinal dystrophies. In the pcdh21 knock-out mice, both the rod and cone photoreceptors degenerate . Therefore, mutations of the human gene may account for a phenotype that results from affection of either or both photoreceptor cells.
Our data provide the tools for rapid mutation screening of PCDH21. The availability of a highly polymorphic intragenic microsatellite marker will be useful in the analysis of families/patients in which parental consanguinity may play a role in pathogenesis.
The authors thank Stefanie Ehmer and Angelika Schmidt for expert technical assistance. We particularly thank the patients who participated in this study. This work was supported by Forschungsfoerderungsfond Medizin of the University Medical Center Hamburg-Eppendorf (FFM) 118-1 (HB).
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