Abstract
Next-generation and Sanger sequencing were combined to identify disease-causing USH2A mutations in an adult patient with autosomal recessive RP. Induced pluripotent stem cells (iPSCs), generated from the patient's keratinocytes, were differentiated into multi-layer eyecup-like structures with features of human retinal precursor cells. The inner layer of the eyecups contained photoreceptor precursor cells that expressed photoreceptor markers and exhibited axonemes and basal bodies characteristic of outer segments. Analysis of the USH2A transcripts of these cells revealed that one of the patient's mutations causes exonification of intron 40, a translation frameshift and a premature stop codon. Western blotting revealed upregulation of GRP78 and GRP94, suggesting that the patient's other USH2A variant (Arg4192His) causes disease through protein misfolding and ER stress. Transplantation into 4-day-old immunodeficient Crb1 (-/-) mice resulted in the formation of morphologically and immunohistochemically recognizable photoreceptor cells, suggesting that the mutations in this patient act via post-developmental photoreceptor degeneration. DOI:http://dx.doi.org/10.7554/eLife.00824.001.
Highlights
Usher syndrome is a genetically heterogeneous autosomal recessive disorder characterized by early onset sensorineural hearing loss and later onset retinitis pigmentosa (RP)
Unlike many other forms of retinitis pigmentosa in which a large fraction of the photoreceptors have already been lost by the time a diagnosis is made, newborn hearing tests coupled with increasingly sensitive molecular testing have the potential to identify patients affected with Usher syndrome early enough that the majority of their photoreceptors are still amenable to gene replacement therapy
Genomic DNA from a patient affected with autosomal recessive retinitis pigmentosa (RP) was fragmented and hybridized to an Agilent exome capture reagent (v2), and the eluted fragments were sequenced on an Illumina sequencing instrument
Summary
Usher syndrome is a genetically heterogeneous autosomal recessive disorder characterized by early onset sensorineural hearing loss and later onset retinitis pigmentosa (RP). Unlike many other forms of retinitis pigmentosa in which a large fraction of the photoreceptors have already been lost by the time a diagnosis is made, newborn hearing tests coupled with increasingly sensitive molecular testing have the potential to identify patients affected with Usher syndrome early enough that the majority of their photoreceptors are still amenable to gene replacement therapy. The obstacles to such treatment include the large size of the USH2A gene, which precludes the use of the types of viral vectors currently employed for retinal gene therapy. Another obstacle to treatment is the relative paucity of information about the normal function of the protein encoded by USH2A (usherin) and the degree to which it can be overexpressed in human cells without causing harm
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