Next-generation sequencing reveals the mutational landscape of clinically diagnosed Usher syndrome: copy number variations, phenocopies, a predominant target for translational read-through, and PEX26 mutated in Heimler syndrome

Abstract

Background: Combined retinal degeneration and sensorineural hearing impairment is mostly due to autosomal recessive Usher syndrome (USH1: congenital deafness, early retinitis pigmentosa (RP); USH2: progressive hearing impairment, RP).

Methods: Sanger sequencing and NGS of 112 genes (Usher syndrome, nonsyndromic deafness, overlapping conditions), MLPA, and array-CGH were conducted in 138 patients clinically diagnosed with Usher syndrome.

Results: A molecular diagnosis was achieved in 97% of both USH1 and USH2 patients, with biallelic mutations in 97% (USH1) and 90% (USH2), respectively. Quantitative readout reliably detected CNVs (confirmed by MLPA or array-CGH), qualifying targeted NGS as one tool for detecting point mutations and CNVs. CNVs accounted for 10% of identified USH2A alleles, often in trans to seemingly monoallelic point mutations. We demonstrate PTC124-induced read-through of the common p.Trp3955* nonsense mutation (13% of detected USH2A alleles), a potential therapy target. Usher gene mutations were found in most patients with atypical Usher syndrome, but the diagnosis was adjusted in case of double homozygosity for mutations in OTOA and NR2E3, genes implicated in isolated deafness and RP. Two patients with additional enamel dysplasia had biallelic PEX26 mutations, for the first time linking this gene to Heimler syndrome.

Conclusion: Targeted NGS not restricted to Usher genes proved beneficial in uncovering conditions mimicking Usher syndrome.

Keywords: Copy number variation; Heimler syndrome; Usher syndrome; next‐generation sequencing; phenocopies; translational read‐through.

Figure 1

Diagnostic yield and mutational spectrum in patients clinically diagnosed with different types of Usher syndrome. Numbers correspond to patient numbers. ?, unsolved patients. (A) USH1. (B) Atypical Usher syndrome (including patients with additional, non‐sensory symptoms). (C) USH2. (D) Ethnic origin of patients. Patients were counted as ¹/₂ + ¹/₂ if parents had different ethnical backgrounds.

Figure 2

CNVs in USH genes detected by quantitative analysis of NGS data. The coverage plots illustrate the statistical readout, with the absolute coverage deduced from unique read count and as calculated by the CNV analysis mode in SeqNext (JSI Medical Systems). The coverage of affected and neighboring exons of patients (red) and controls (green) from the same NGS runs is shown in overlay schemes for comparison. While most patients harbor heterozygous deletions, reflected by approximately 50% reduction in coverage, patients P61 and P39 (the heterozygous father is shown for comparison) have homozygous deletions, reflected by virtually no coverage in the respective plot. Patient P103 had a homozygous duplication of nine CDH23 exons (19‐27; also see Fig. S1) the heterozygous father is depicted for comparison.

Figure 3

Contiguous gene syndrome due to a deletion of USH1C and ABCC8. (A) NGS indicated a homozygous deletion of USH1C exons 3–27 in two not knowingly related USH1 patients from Saudi Arabia, P96 and P97. (B) Array‐CGH revealed that the deletion also comprises the neighboring ABCC8 gene. Thus, the alteration corresponds to a contiguous gene syndrome previously described in the USH1C gene identification study (Bitner‐Glindzicz et al. 2000). The replication of this mutation in our study indicates that this is a founder mutation from the Arabian Peninsula.

Figure 4

High prevalence of USH2A nonsense mutation p.Trp3955* and drug‐mediated read‐through. (A) USH2A alleles: Proportion of missense and small in‐frame alterations, truncating point mutations (nonsense, small deletions, and duplications), and large CNVs affecting one or more exons. Two mutations, p.Trp3955* and c.2299delG, are predominant. (B) Scheme of PTC124‐induced translational read‐through of a nonsense mutation. In the wild‐type situation, translation of mRNA results in functional full‐length protein. Nonsense mutations introduce a premature termination codon (red X) on the mRNA level, resulting in a truncated non‐functional protein. Read‐through‐inducing drugs like Ataluren (PTC124) bind to the ribosomes and promote the incorporation of an amino acid at the position of a PTC, resulting in the expression of full‐length protein. (C) Indirect immunofluorescence analyses of PTC124‐induced translational read‐through in cells transfected with wild‐type (WT) and mutant (Trp3955*) constructs (indirect immunofluorescence, anti‐Flag antibodies). Flag‐tagged USH2A (green) was detected in USH2A‐WT cells but not in (C′) DMSO‐treated USH2A‐p.Trp3955* cells. (C``) Application of PTC124 recovered USH2A expression in p.Trp3955*‐transfected cells. Nuclei were stained with DAPI (blue). (D) Increase in USH2A‐Flag‐positive cells after application of PTC124 (quantification of five independent experiments. Error bars represent SD; *<0.05; magnification bar: 10 μm).

Figure 5

Double homozygosity for mutations in two genes associated with non‐syndromic disease simulates Usher syndrome in patient 93. (A) NGS indicated a homozygous deletion of the entire OTOA gene, the gene associated with recessive deafness DFNB22, in the patient. (B) This was confirmed by array‐CGH analysis. (C) Targeted analysis revealed a homozygous missense mutation of NR2E3. (D) Pedigree of the patient's consanguineous family summarizing the genetic findings.

Figure 6

Biallelic PEX26 mutations cause deaf‐blindness with enamel dysplasia (Heimler syndrome). (A) Pedigrees of the two patients with PEX26 mutations. (B) Scheme of the PEX26 gene and localization of mutations. (C) Partial alignment of PEX26/Pex26 peptide sequences from various species, indicating high evolutionary conservation of the mutated residues. (D) Severe enamel dysplasia of permanent teeth of patient 135 at 11⁷/₁₂ years of age. (E) X‐ray of patient 135 showing preeruptive crown resorption in the upper left first molar (red arrow) and a local enlargement of the gingival tissue (blue line) at 13¹⁰/₁₂ years of age.