Partial USH2A deletions contribute to Usher syndrome in Denmark

Abstract

Usher syndrome is an autosomal recessive disorder characterized by congenital hearing impairment, progressive visual loss owing to retinitis pigmentosa and in some cases vestibular dysfunction. Usher syndrome is divided into three subtypes, USH1, USH2 and USH3. Twelve loci and eleven genes have so far been identified. Duplications and deletions in PCDH15 and USH2A that lead to USH1 and USH2, respectively, have previously been identified in patients from United Kingdom, Spain and Italy. In this study, we investigate the proportion of exon deletions and duplications in PCDH15 and USH2A in 20 USH1 and 30 USH2 patients from Denmark using multiplex ligation-dependent probe amplification (MLPA). Two heterozygous deletions were identified in USH2A, but no deletions or duplications were identified in PCDH15. Next-generation mate-pair sequencing was used to identify the exact breakpoints of the two deletions identified in USH2A. Our results suggest that USH2 is caused by USH2A exon deletions in a small fraction of the patients, whereas deletions or duplications in PCDH15 might be rare in Danish Usher patients.

Figure 1

CytoScan HD and MLPA results for patients 70353, 55555 and 1063. (a) CytoScan results, where deletions are indicated by a red bar in the USH2A/RP region. (b) MLPA results using the two probes-mix kits, P361-A1 and P362-A2, respectively, covering USH2A. Red dot indicates a deleted probe/exon.

Figure 2

Mapping of two deletion breakpoints in USH2A. On the basis of the next-generation mate-pair sequencing results, PCR primers were designed to amplify across the deletion breakpoints from genomic DNA from patients 70353 and 1063. (a) Sequence chromatogram showing that patient 70353 (and 55555) have a 74 052-bp deletion (chr1.hg19:g.216464358_216538408del) with breakpoints in exon 4 and in intron 11. (b) Sequence chromatogram showing that patient 1063 has a 63 758-bp deletion (chr.hg19:g.216259403_216323159del) with breakpoints in intron 21 and in intron 24.

Figure 3

Estimation of relatedness between patient 70353 and patient 55555 based on investigation of SNP data from chromosome 1. Plots of relatedness across chromosome 1 indicate that the region from 206 to 218 Mb on one of the two alleles with very high probability is identical by decent, and thus inherited from a common ancestor. The orange line is the probability for being related for one allele and unrelated for the other (here 100% in USH2A). The blue line is the probability for being unrelated (here 0% in USH2A). The green line indicating that both alleles exhibit identity by descent (IBD) has 0% probability across the chromosome and is almost not visible. The most likely IBD states across the chromosome are indicated by a bar above the plot, showing that the two individuals are only related at the USH2A locus (black arrow).