Mutations in the novel gene IFT88 are involved in causing non-syndromic inherited retinal degeneration (IRD)

Abstract

Purpose : To identify the mutations responsible for IRD in a family and understand the mechanism underlying the disease pathology.

Methods : Detailed ophthalmic evaluation including fundus photography, electroretinography (ERG), and visual field (VF) measurements were performed in two sisters affected with IRD. Whole genome sequencing was performed using the Illumina HiSeq X10X. Mapping and variant calling were performed using BWA and GATK. Low frequency variants (<0.005) were annotated using SnpEff, PolyPhen2 and CADD score. Potentially damaging rare variants segregating with disease were validated by Sanger sequencing and screening data sets of ethnicity matched controls. The impact of IFT88 mutations was evaluated by expressing the wild type and mutant IFT88 by transient transfection and CRISPR-Cas9 mediated genome editing of mammalian cells. Expression and localization of IFT88 was studied by western blot analysis, immunocytochemistry, qRT-PCR and nonsense-mediated decay (NMD) assay.

Results : Clinical evaluations showed subnormal ERG values, paracentral scotomas, and decreased visual acuity in both sisters, leading to a diagnosis of inverse RP. Whole genome analysis identified potentially pathogenic compound heterozygous variants, p.Arg266* (c.796C>T, rs145868877) and p.Ala568Thr (c.1702G>A) in the Intraflagellary transport protein 88 (IFT88) gene segregating with the phenotype. 3D modeling scores indicated these mutations to be deleterious. Heterologous expression of IFT88 using constructs designed to express the protein with p.Arg266* and p.Ala568Thr mutations by transient transfection showed mislocalization of the protein in miMCD3 cells. Evaluation of the IFT88 transcript in a HeLa cell line generated by introducing the Arg266* mutation using CRISPR-Cas9 system revealed that the mutant transcript undergoes NMD. The p.Ala568Thr mutation resulted in abnormal ciliogenesis while the p.Arg266* resulted in loss of cilia.

Conclusions : IFT88 plays a crucial role in ciliogenesis and IFT. Null mutations in this gene cause embryonic lethality in mice. Involvement of IFT88 in human disease has not been established. It is likely that the p.Ala568Thr generates a hypomorphic allele while the p.Arg266* results in a null mutation and both together may lead to photoreceptor abnormality. This is the first report demonstrating the involvement of IFT88 in IRD.

This is an abstract that was submitted for the 2018 ARVO Annual Meeting, held in Honolulu, Hawaii, April 29 - May 3, 2018.