Using patient-specific induced pluripotent stem cells to interrogate the pathogenicity of a novel retinal pigment epithelium-specific 65 kDa cryptic splice site mutation and confirm eligibility for enrollment into a clinical gene augmentation trial

详细信息    查看全文

• 作者：Budd A. Tucker^a ; Cathryn M. Cranston^a ; Kristin A. Anfinson^a ; Suruchi Shrestha^a ; Luan M. Streb^a ; Alejandro Leon^b ; Robert F. Mullins^a ; Edwin M. Stone^a ; ^c ; ^{edwin-stone@uiowa.edu" class="auth_mail" title="E-mail the corresponding author}
• 关键词：Best1 ; Bestrophin1 ; bFGF ; basic fibroblast growth factor ; DMEM ; Dulbecco's Modified Eagle Medium ; DNMT ; DNA methyltransferases ; ERG ; Electroretinography ; KLF4 ; Kruppel-like factor 4 ; LB ; Lysogeny broth ; LCA ; Leber congenital amaurosis ; MEM ; minimum essential medium ; NEAA ; non-essential amino acids ; MITF ; Microphthalmia-Associated Transcription Factor ; MOI ; multiplicity of infection ; RPE ; Retinal pigment epithelium ; RPE65 ; Retinal pigment epithelium&ndash ; specific 65 kDa ; SOX2 ; SRY (sex determini
• 刊名：Translational Research
• 出版年：2015
• 出版时间：December 2015
• 年：2015
• 卷：166
• 期：6
• 页码：740-749.e1
• 全文大小：2751 K

文摘

Retinal pigment epithelium–specific 65 kDa (RPE65)–associated Leber congenital amaurosis is an autosomal recessive disease that results in reduced visual acuity and night blindness beginning at birth. It is one of the few retinal degenerative disorders for which promising clinical gene transfer trials are currently underway. However, the ability to enroll patients in a gene augmentation trial is dependent on the identification of 2 bona fide disease-causing mutations, and there are some patients with the phenotype of RPE65-associated disease who might benefit from gene transfer but are ineligible because 2 disease-causing genetic variations have not yet been identified. Some such patients have novel mutations in RPE65 for which pathogenicity is difficult to confirm. The goal of this study was to determine if an intronic mutation identified in a 2-year-old patient with presumed RPE65-associated disease was truly pathogenic and grounds for inclusion in a clinical gene augmentation trial. Sequencing of the RPE65 gene revealed 2 mutations: (1) a previously identified disease-causing exonic leucine-to-proline mutation (L408P) and (2) a novel single point mutation in intron 3 (IVS3-11) resulting in an A>G change. RT-PCR analysis using RNA extracted from control human donor eye–derived primary RPE, control iPSC-RPE cells, and proband iPSC-RPE cells revealed that the identified IVS3-11 variation caused a splicing defect that resulted in a frameshift and insertion of a premature stop codon. In this study, we demonstrate how patient-specific iPSCs can be used to confirm pathogenicity of unknown mutations, which can enable positive clinical outcomes.