Detection and validation of the involvement of novel mutations in MERTK in recessive retinal degeneration using WGS and hiPSC-RPEs.

Abstract

Purpose : To identify the genetic cause of inherited retinal dystrophy (IRD) in a Caucasian pedigree by whole genome sequencing (WGS) and to study the effect using patient’s induced pluripotent stem cell (hiPSCs) derived disease model.

Methods : A pedigree with one affected individual and three unaffected members was analyzed. The affected individual underwent detailed ophthalmic evaluation. WGS of the affected individual, an unaffected sibling and parents was carried out using Illumina HiSeqX10. The reads were aligned to hg19 and mapping and variant calling was performed using BWA and GATK. The structural variations were called using GenomeSTRiP and LUMPY. Segregation analysis of the candidate variants was carried out using Sanger sequencing. Peripheral blood mononuclear cells were extracted from the affected member and parents and reprogrammed to hiPSCs using Sendai virus. Standard validation of hiPSCs were performed by immunohistochemistry for SOX2, OCT4, Nanog and SSEA4 and cells were differentiated to RPE. Standard RPE characterization was performed including the analysis of ZO-1, CRALBP, MITF, and BEST1 markers. Expression of MERTK in hiPSC and hiPSC-RPE was evaluated by qRT-PCR and western blot analysis.

Results : WGS identified a candidate splice-site variant (c.1296+1G>A) and a 731bp deletion encompassing exon-9 in the MERTK gene in trans segregating with IRD. Both these sequence changes are predicted to result in the loss of exon-9, the sequence of which is in-frame. Generation of hiPSCs and differentiation to hiPSC-RPEs were confirmed. qRT-PCR of the MERTK transcript in hiPSC and hiPSC-RPE revealed a significant (p=0.0003) difference in MERTK expression in the cells derived from the case compared with those from the unaffected parents. The western blot analysis for MERTK of proband iPSC and hiPSC-RPE detected a loss of the wild type MERTK (180KD) band while the western blot of parental hiPSC and hiPSC-RPE detected an immunopositive band corresponding to the wild type (180KD) MERTK.

Conclusions : In this study we report the identification of a novel splice-site mutation c.1296+1G>A and a novel 731bp deletion (Chr2: 112751488 to 112752218) in the MERTK gene segregating with IRD. Analysis of patient iPSC derived model revealed a near complete loss of the wild type MERTK establishing the potential involvement of these variants in IRD pathology.

This abstract was presented at the 2019 ARVO Annual Meeting, held in Vancouver, Canada, April 28 - May 2, 2019.