A deep intronic CLRN1 (USH3A) founder mutation generates an aberrant exon

and underlies severe Usher syndrome on the Arabian Peninsula

Arif O. Khan, Elvir Becirovic, Christian Betz, Christine Neuhaus, Janine Altmüller, Lisa Maria Riedmayr, Susanne Motameny, Gudrun Nürnberg, Peter Nürnberg & Hanno J. Bolz | Scientific Reports | Vol. 7, Article # 1411 | 2017 May 03 |

Abstract Deafblindness is mostly due to Usher syndrome caused by recessive mutations in the known genes. Mutation-negative patients therefore either have distinct diseases, mutations in yet unknown Usher genes or in extra-exonic parts of the known genes – to date a largely unexplored possibility. In a consanguineous Saudi family segregating Usher syndrome type 1 (USH1), NGS of genes for Usher syndrome, deafness and retinal dystrophy and subsequent whole-exome sequencing each failed to identify a mutation. Genome-wide linkage analysis revealed two small candidate regions on chromosome 3, one containing the USH3A gene CLRN1, which has never been associated with Usher syndrome in Saudi Arabia. Whole-genome sequencing (WGS) identified a homozygous deep intronic mutation, c.254–649T > G, predicted to generate a novel donor splice site. CLRN1 minigene-based analysis confirmed the splicing of an aberrant exon due to usage of this novel motif, resulting in a frameshift and a premature termination codon. We identified this mutation in an additional two of seven unrelated mutation-negative Saudi USH1 patients. Locus-specific markers indicated that c.254–649T > G CLRN1 represents a founder allele that may significantly contribute to deafblindness in this population. Our finding underlines the potential of WGS to uncover atypically localized, hidden mutations in patients who lack exonic mutations in the known disease genes.

Introduction Usher syndrome is the most common cause of inherited deafblindness1. Type 1 (USH1) is characterized by congenital deafness and early (first decade) retinitis pigmentosa (RP), whereas type 2 (USH2) displays progressive hearing impairment and RP of later onset. USH3 is characterized by progressive hearing loss, RP, and variable peripheral vestibular dysfunction2. However, disease resulting from mutations in the USH3A gene, CLRN1, is variable, ranging from non-syndromic RP3 to USH14. The advent of next-generation sequencing (NGS) has enabled panel-sequencing of the 11 known Usher genes, and its application in a recent study on European deafblind patients identified the causative mutations in the majority5. In a Saudi Arabian family with four siblings affected by Usher syndrome type 1, escalating the genetic investigations from gene panel NGS over genome-wide linkage analysis to whole-exome sequencing (WES) and finally whole-genome sequencing (WGS) led up to the molecular diagnosis. Our study demonstrates the potential of WGS to unlock hidden mutations.


NGS of gene panels for retinal dystrophy and for deafness Apart from a heterozygous frameshift mutation in TUBGCP6, c.5001_5003delinsCA (p.Gln1667Hisfs*11), NGS of the known genes for Usher syndrome, for other syndromic and isolated hearing loss, and for retinal degeneration did not identify any mutations. Biallelic TUBGCP6 mutations cause microcephalic primordial dwarfism and additional congenital anomalies, including retinopathy6. Given the recessive inheritance and additional symptoms associated with mutations in TUBGCP6 (which are not present in the affected family members analyzed in our study), the apparently monoallelic variant most likely represents carriership for an unrelated disorder. Our results from NGS panel analysis thus largely excluded not only mutations in the coding sequences of the Usher syndrome genes and genes causing similar syndromes (e.g. USH3-like PHARC due to ABHD12 mutations7), but also simultaneous mutations in a deafness gene and an RP gene mimicking Usher syndrome. Quantitative analysis of NGS reads did not indicate large copy number variations (CNVs) such as deletions of one or several contiguous exons.

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