Tags

Type your tag names separated by a space and hit enter

IPSC-Derived Corneal Endothelial-like Cells Act as an Appropriate Model System to Assess the Impact of SLC4A11 Variants on Pre-mRNA Splicing.
Invest Ophthalmol Vis Sci 2019; 60(8):3084-3090IO

Abstract

Purpose

To report molecular genetic findings in six probands with congenital hereditary endothelial dystrophy (CHED) variably associated with hearing loss (also known as Harboyan syndrome). Furthermore, we developed a cellular model to determine if disease-associated variants induce aberrant SLC4A11 pre-mRNA splicing.

Methods

Direct sequencing of the entire SLC4A11 coding region was performed in five probands. In one individual, whole genome sequencing was undertaken. The effect of c.2240+5G>A on pre-mRNA splicing was evaluated in a corneal endothelial-like (CE-like) cell model expressing SLC4A11. CE-like cells were derived from autologous induced pluripotent stem cells (iPSCs) via neural crest cells exposed to B27, PDGF-BB, and DKK-2. Total RNA was extracted, and RT-PCR was performed followed by Sanger and a targeted next generation sequencing (NGS) approach to identify and quantify the relative abundance of alternatively spliced transcripts.

Results

In total, 11 different mutations in SLC4A11 evaluated as pathogenic were identified; of these, c.1237G>A, c.2003T>C, c.1216+1G>A, and c.2240+5G>A were novel. The c.2240+5G>A variant was demonstrated to result in aberrant pre-mRNA splicing. A targeted NGS approach confirmed that the variant introduces a leaky cryptic splice donor site leading to the production of a transcript containing an insertion of six base pairs with the subsequent introduction of a premature stop codon (p.Thr747*). Furthermore, a subset of transcripts comprising full retention of intron 16 also were observed, leading to the same functionally null allele.

Conclusions

This proof-of-concept study highlights the potential of using CE-like cells to investigate the pathogenic consequences of SLC4A11 disease-associated variants.

Authors+Show Affiliations

Research Unit for Rare Diseases, Department of Pediatrics and Adolescent Medicine, First Faculty of Medicine, Charles University and General University Hospital in Prague, Czech Republic.Research Unit for Rare Diseases, Department of Pediatrics and Adolescent Medicine, First Faculty of Medicine, Charles University and General University Hospital in Prague, Czech Republic.Research Unit for Rare Diseases, Department of Pediatrics and Adolescent Medicine, First Faculty of Medicine, Charles University and General University Hospital in Prague, Czech Republic. Department of Ophthalmology, First Faculty of Medicine, Charles University and General University Hospital in Prague, Prague, Czech Republic.Research Unit for Rare Diseases, Department of Pediatrics and Adolescent Medicine, First Faculty of Medicine, Charles University and General University Hospital in Prague, Czech Republic.Clinic of Ophthalmology, University Hospital Ostrava, Ostrava, Czech Republic. Department of Craniofacial Surgery, University of Ostrava, Ostrava, Czech Republic.Biopticka laborator s.r.o., Pilsen, Czech Republic.UCL Institute of Ophthalmology, London, United Kingdom. Moorfields Eye Hospital NHS Foundation Trust, London, United Kingdom. Great Ormond Street Hospital for Children, London, United Kingdom.UCL Institute of Ophthalmology, London, United Kingdom. Moorfields Eye Hospital NHS Foundation Trust, London, United Kingdom.UCL Institute of Ophthalmology, London, United Kingdom.Research Unit for Rare Diseases, Department of Pediatrics and Adolescent Medicine, First Faculty of Medicine, Charles University and General University Hospital in Prague, Czech Republic. Department of Ophthalmology, First Faculty of Medicine, Charles University and General University Hospital in Prague, Prague, Czech Republic. UCL Institute of Ophthalmology, London, United Kingdom.

Pub Type(s)

Journal Article

Language

eng

PubMed ID

31323090

Citation

Brejchova, Kristyna, et al. "IPSC-Derived Corneal Endothelial-like Cells Act as an Appropriate Model System to Assess the Impact of SLC4A11 Variants On Pre-mRNA Splicing." Investigative Ophthalmology & Visual Science, vol. 60, no. 8, 2019, pp. 3084-3090.
Brejchova K, Dudakova L, Skalicka P, et al. IPSC-Derived Corneal Endothelial-like Cells Act as an Appropriate Model System to Assess the Impact of SLC4A11 Variants on Pre-mRNA Splicing. Invest Ophthalmol Vis Sci. 2019;60(8):3084-3090.
Brejchova, K., Dudakova, L., Skalicka, P., Dobrovolny, R., Masek, P., Putzova, M., ... Liskova, P. (2019). IPSC-Derived Corneal Endothelial-like Cells Act as an Appropriate Model System to Assess the Impact of SLC4A11 Variants on Pre-mRNA Splicing. Investigative Ophthalmology & Visual Science, 60(8), pp. 3084-3090. doi:10.1167/iovs.19-26930.
Brejchova K, et al. IPSC-Derived Corneal Endothelial-like Cells Act as an Appropriate Model System to Assess the Impact of SLC4A11 Variants On Pre-mRNA Splicing. Invest Ophthalmol Vis Sci. 2019 Jul 1;60(8):3084-3090. PubMed PMID: 31323090.
* Article titles in AMA citation format should be in sentence-case
TY - JOUR T1 - IPSC-Derived Corneal Endothelial-like Cells Act as an Appropriate Model System to Assess the Impact of SLC4A11 Variants on Pre-mRNA Splicing. AU - Brejchova,Kristyna, AU - Dudakova,Lubica, AU - Skalicka,Pavlina, AU - Dobrovolny,Robert, AU - Masek,Petr, AU - Putzova,Martina, AU - Moosajee,Mariya, AU - Tuft,Stephen J, AU - Davidson,Alice E, AU - Liskova,Petra, PY - 2019/7/20/entrez PY - 2019/7/20/pubmed PY - 2019/7/20/medline SP - 3084 EP - 3090 JF - Investigative ophthalmology & visual science JO - Invest. Ophthalmol. Vis. Sci. VL - 60 IS - 8 N2 - Purpose: To report molecular genetic findings in six probands with congenital hereditary endothelial dystrophy (CHED) variably associated with hearing loss (also known as Harboyan syndrome). Furthermore, we developed a cellular model to determine if disease-associated variants induce aberrant SLC4A11 pre-mRNA splicing. Methods: Direct sequencing of the entire SLC4A11 coding region was performed in five probands. In one individual, whole genome sequencing was undertaken. The effect of c.2240+5G>A on pre-mRNA splicing was evaluated in a corneal endothelial-like (CE-like) cell model expressing SLC4A11. CE-like cells were derived from autologous induced pluripotent stem cells (iPSCs) via neural crest cells exposed to B27, PDGF-BB, and DKK-2. Total RNA was extracted, and RT-PCR was performed followed by Sanger and a targeted next generation sequencing (NGS) approach to identify and quantify the relative abundance of alternatively spliced transcripts. Results: In total, 11 different mutations in SLC4A11 evaluated as pathogenic were identified; of these, c.1237G>A, c.2003T>C, c.1216+1G>A, and c.2240+5G>A were novel. The c.2240+5G>A variant was demonstrated to result in aberrant pre-mRNA splicing. A targeted NGS approach confirmed that the variant introduces a leaky cryptic splice donor site leading to the production of a transcript containing an insertion of six base pairs with the subsequent introduction of a premature stop codon (p.Thr747*). Furthermore, a subset of transcripts comprising full retention of intron 16 also were observed, leading to the same functionally null allele. Conclusions: This proof-of-concept study highlights the potential of using CE-like cells to investigate the pathogenic consequences of SLC4A11 disease-associated variants. SN - 1552-5783 UR - https://www.unboundmedicine.com/medline/citation/31323090/IPSC-Derived_Corneal_Endothelial-like_Cells_Act_as_an_Appropriate_Model_System_to_Assess_the_Impact_of_SLC4A11_Variants_on_Pre-mRNA_Splicing L2 - http://iovs.arvojournals.org/article.aspx?doi=10.1167/iovs.19-26930 DB - PRIME DP - Unbound Medicine ER -