Identification of mutations in CACNA1F in patients with incomplete CSNB applying next generation sequencing approaches

Abstract

Purpose : Patients with incomplete congenital stationary night blindness (icCSNB) show a normal a-wave in the scotopic bright flash electroretinogram (ERG) with a reduced b-wave giving an electronegative waveform. The 30-Hz and single-flash cone ERG are severely diminished and delayed and the long duration stimulation shows reduced ON and OFF responses. The purpose of this work to identify the gene defect in patients with icCSNB, previously excluded in the coding regions of CACNA1F, CABP4 and CACNA2D4.

Methods : Patients of different European clinical centers underwent full ophthalmic examination. Informed consent was obtained from each patient and available family members. The study protocol adhered to the tenets of the Declaration of Helsinki and was approved by the local ethics committee. The DNA of 4 index patients with a clinical diagnosis of icCSNB and available family members was extracted and screened for CACNA1F, CABP4 and CACNA2D4 mutations by Sanger sequencing. Subsequently targeted, whole exome (WES) or whole genome sequencing (WGS) was applied. Minigene approaches were used to investigate the effect of intronic variants on splicing.

Results : These patients revealed absence of mutations in the coding regions of CACNA1F, CABP4 and CACNA2D4. WES of family A indicated that the DNA of the father and index was confounded showing a novel mutation, c.868C>T, p.(Arg290Cys) in CACNA1F. Investigation of family B and C by WES covering flanking intronic regions revealed a c.1496+5G>C and a c.4294-9G>A variant in CACNA1F. WGS on family D identified a c.522-16G>A variant in CACNA1F. The validated variants co-segregated with the phenotype in the families. Although these variants do not directly affect splice acceptor or donor sites, they are predicted to influence splicing. Indeed, preliminary data obtained from minigene approaches revealed a different RT-PCR profile in patients compared to control.

Conclusions : More than 120 different mutations in the coding regions of CACNA1F have been described to be the major cause of icCSNB. A few icCSNB cases lack mutations in the coding regions of known genes underlying this disorder. Our study presented herein highlight the possibility that the missing mutations may be found in overlooked intronic regions of CACNA1F, influencing natural splicing. This assumption may hold true for many other gene defects underlying inherited retinal disorders.

This is an abstract that was submitted for the 2017 ARVO Annual Meeting, held in Baltimore, MD, May 7-11, 2017.