A Monoallelic Variant in REST Is Associated with Non-Syndromic Autosomal Dominant Hearing Impairment in a South African Family

Abstract

Hearing impairment (HI) is a sensory disorder with a prevalence of 0.0055 live births in South Africa. DNA samples from a South African family presenting with progressive, autosomal dominant non-syndromic HI were subjected to whole-exome sequencing, and a novel monoallelic variant in REST [c.1244GC; p.(C415S)], was identified as the putative causative variant. The co-segregation of the variant was confirmed with Sanger Sequencing. The variant is absent from databases, 103 healthy South African controls, and 52 South African probands with isolated HI. In silico analysis indicates that the p.C415S variant in REST substitutes a conserved cysteine and results in changes to the surrounding secondary structure and the disulphide bonds, culminating in alteration of the tertiary structure of REST. Localization studies using ectopically expressed GFP-tagged Wild type (WT) and mutant REST in HEK-293 cells show that WT REST localizes exclusively to the nucleus; however, the mutant protein localizes throughout the cell. Additionally, mutant REST has an impaired ability to repress its known target AF1q. The data demonstrates that the identified mutation compromises the function of REST and support its implication in HI. This study is the second report, worldwide, to implicate REST in HI and suggests that it should be included in diagnostic HI panels.

Keywords: Africa; DFNA27; RE1-silencing transcription factor; REST; South Africa; non-syndromic hearing impairment.

Figure 1

The pedigree of the family presenting with non-syndromic hearing impairment. (a) The pedigree suggesting a dominant inheritance model of hearing impairment (HI). The age and genotype are indicated under the ID for the four family members that were available for sequencing. (b) audiograms indicate severe hearing impairment in the affected mother and son and normal hearing in the unaffected child. The left ear is blue and the right ear is red in the audiograms for subject III.5 and IV.4. (c) The missense mutation segregates within the family with the affected mother and child being heterozygous for the c.1244GC. The unaffected grandmother and half-brother are homozygous wild-type.

Figure 2

(a) The evolutionary conservation of the Cysteine amino acid at position 415 in REST. The position is indicated by the red box. (b) The domain structure of the WT and MT protein are shown. The mutant protein has an extended disordered domain when compared to the WT. (c) Protein modelling of REST comparing the WT (right) to the MT (left) in the ribbon form. The mutation results in changes in the β sheets and α helixes in the protein. This results in either the extension or the retraction of some the structures, as well as the formation and loss of other structures. (d) Protein modelling of REST comparing the WT (right) and the MT (left) in the space filling form indicating the consequences of the changes illustrated in (2c). The p.C415S variation results in a change in the tertiary structure of the protein such that it is slightly smaller than the WT protein. The MT protein also has several, previously exposed moieties, hidden.

Figure 3

WT REST localizes to the nucleus, while MT REST displays localization patterns similar to GFP-only and loses repressive ability of target gene. (a) Micrographs of HEK-293 cells transfected with GFP-only (one representative image), GFP-tagged WT REST and GFP-tagged MT REST (two representative images each). The LHS row indicates Hoechst staining, the middle row indicates GFP, and the RHS row represents merged images. HEK-293 cells were transiently transfected with the respective constructs and visualized live after 24 h (spiked with Hoechst stain) under the confocal microscope (b) HEK-293 cells were transiently transfected with WT or MT REST expression constructs, and 24 h later the expression of AF1q mRNA was measured using qPCR and plotted relative to empty vector control. (c) HEK-293 cells were transiently transfected with WT and MT REST expression constructs and harvested and lysed 36 h later. Luciferase activity was measured with a luminometer and plotted relative to the empty vector control.