Mutations of the protocadherin gene PCDH15 cause Usher syndrome type 1F

Abstract

Human chromosome 10q21-22 harbors USH1F in a region of conserved synteny to mouse chromosome 10. This region of mouse chromosome 10 contains Pcdh15, encoding a protocadherin gene that is mutated in ames waltzer and causes deafness and vestibular dysfunction. Here we report two mutations of protocadherin 15 (PCDH15) found in two families segregating Usher syndrome type 1F. A Northern blot probed with the PCDH15 cytoplasmic domain showed expression in the retina, consistent with its pathogenetic role in the retinitis pigmentosa associated with USH1F.

Figure 1

Haplotypes of two Pakistani families demonstrating linkage to USH1F at 10q21-22. Two affected individuals, IV:13 and IV:14 in family PKSR17, provided the proximal recombination at marker D10S1643 (74.5 cM). The distal recombination is provided by unaffected individual IV:2 of family PKSR8b, at marker D10S1226 (75.5 cM). Fundoscopic and ERG examination revealed early signs of retinitis pigmentosa in affected members of both families. The genetic linkage distances are from the Center for Medical Genetics, Marshfield Medical Research Foundation Web site (Broman et al. 1998).

Figure 2

A, Genetic and underlying physical map of USH1F at 10q21.1 and 21 overlapping BACs covering this interval. The bp distances are from the Human Genome Project Working Draft Web site. B, Exons of PCDH15, spanning ∼1.6 Mb on the physical map, denoted by horizontal lines. There are three genes found in the introns of PCDH15: Ca²⁺/calmodulin-dependent protein kinase type II, NDST2, and PLAU. Also shown are two genes predicted by GENSCAN. One is similar to mouse tumor differentially expressed transcript-1 (Tde-1, a transmembrane protein). The other is similar to zinc finger protein-7. C, Linked markers in each family (denoted as shaded circles on the horizontal line), and recombinant markers (denoted with an “×”). The previously reported distal recombination D10S596 (Wayne et al. 1997) for USH1F is denoted by a black block.

Figure 3

Domains of PCDH15, predicted from 1,955 amino acids. PCDH15 has a signal peptide at the amino terminus, 11 extracellular calcium-binding domains, 1 predicted transmembrane domain, and a unique carboxy terminus (GenBank). TMpred and SMART predicted two transmembrane domains (1054–1072 and 1376–1397), TMHMM predicted only one transmembrane domain, at 1376–1397 (SMART Web site; TMHMM Server, version 2; TMpred Prediction of Transmembrane Regions and Orientation Server). The mutations found in two families with USH1F are also annotated. RACE products obtained and three ESTs found in the NCBI nucleotide database are shown, along with the 590-bp probe used for the northern blot (fig. 5).

Figure 4

PCDH15 mutations in two Pakistani families. A, Sequence chromatograms for the splice-site mutation and wild-type allele of family PKSR8b. An arrow (↓) indicates the IVS27−2A→G replacement in intron 27. The sequence trace file for the antisense strand is shown. This transition mutation results in an alteration in the acceptor splice site of exon 28. For genomic DNA with this mutation, GENSCAN predicted the skipping of exon 28 and the use of an alternate exon in intron 27. B, Sense strand–sequence chromatograms for +7C→T, found in family PKSR17, and for the wild-type allele. This mutation results in a transition from an arginine to a termination codon (R3X).

Figure 5

Northern blot for several human tissues. Lanes 1–6 contain 10 μg each of total RNA from human retina, ciliary body, RPE-choroid, iris, brain, and liver. Positions of bands from an RNA ladder are indicated along the right margin. A 6-d exposure to autoradiographic film is shown. In the retina, bands are apparent at ∼3.3, 6.0, and 8.6 kb, with the larger transcript possibly being a doublet.