Gene structure and mutant alleles of PCDH15: nonsyndromic deafness DFNB23 and type 1 Usher syndrome

Abstract

Mutations of PCDH15, encoding protocadherin 15, can cause either combined hearing and vision impairment (type 1 Usher syndrome; USH1F) or nonsyndromic deafness (DFNB23). Human PCDH15 is reported to be composed of 35 exons and encodes a variety of isoforms with 3-11 ectodomains (ECs), a transmembrane domain and a carboxy-terminal cytoplasmic domain (CD). Building on these observations, we describe an updated gene structure that has four additional exons of PCDH15 and isoforms that can be subdivided into four classes. Human PCDH15 encodes three alternative, evolutionarily conserved unique cytoplasmic domains (CD1, CD2 or CD3). Families ascertained on the basis of prelingual hearing loss were screened for linkage of this phenotype to markers for PCDH15 on chromosome 10q21.1. In seven of twelve families segregating USH1, we identified homozygous mutant alleles (one missense, one splice site, three nonsense and two deletion mutations) of which six are novel. One family was segregating nonsyndromic deafness DFNB23 due to a homozygous missense mutation. To date, in our cohort of 557 Pakistani families, we have found 11 different PCDH15 mutations that account for deafness in 13 families. Molecular modeling provided mechanistic insight into the phenotypic variation in severity of the PCDH15 missense mutations. We did not find pathogenic mutations in five of the twelve USH1 families linked to markers for USH1F, which suggest either the presence of mutations of yet additional undiscovered exons of PCDH15, mutations in the introns or regulatory elements of PCDH15, or an additional locus for type I USH at chromosome 10q21.1.

Fig. 1

Pedigrees of eight Pakistani families segregating recessive sensorineural hearing loss linked to markers for PCDH15.

Fig. 2

ClustalW multiple amino acid sequence alignment of protocadherin 15 orthologs in two noncontiguous regions show that p.R134 and p.D178 are conserved across species (shaded background, same amino acids; light background, non-conserved amino acids).

Fig. 3

Alignment of the eleven protocadherin 15 EC domains with missense mutations causing USH1F (red) and DFNB23 (yellow). A gray background indicates highly conserved or similar residues. The DXD, LDRE and DXNDN calcium-binding motifs are boxed.

Fig. 4

Structure and expression profile of PCDH15 isoforms. a Four isoform classes of PCDH15 defined by the presence or absence of one of three alternative, unique cytoplasmic domains (CD1, CD2, or CD3). Additional exons discovered after the initial report of PCDH15 structure (Ahmed et al. 2001) are designated with a letter-suffix (11a, 25a, 25b) since they are located among the previously reported 33 exons (Ahmed et al. 2003). Black shaded boxes designate the 5′UTR and 3′UTR. A signal peptide (red) is encoded by exon 2 and a transmembrane domain (blue) is encoded by exon 30. Four newly discovered exons identified in this study are marked with asterisks. Exons encoding the cadherin repeats are represented by green boxes and the exons 33 (yellow), 35 (purple) and 36 (pink) encode three alternative cytoplasmic domains (CD1, CD2, CD3), respectively. b Expression profiles of the three isoform classes of PCDH15 that have different cytoplasmic domains (CD1, CD2 and CD3) are shown. PCR primers (arrowheads in panel a) were designed to amplify CD1, CD2 and CD3 from cDNA prepared from different human tissues. Left expression profile, PCDH15-CD1 mRNA is present in testis, retina, and cochlear cDNA. Middle profile, PCDH15-CD2 is found in human heart, kidney, thymus, spleen, testis, retina, and cochlea. Right profile, PCDH15-CD3 is widely expressed. PCR amplification of GAPDH cDNA was used as a control for the quality and quantity of RT-PCR template.