A novel nonsense variant in the NFE2L1 transcription factor in a patient with developmental delay, hypotonia, genital anomalies, and failure to thrive

Abstract

The NFE2L1 transcription factor (also known as Nrf1 for nuclear factor erythroid 2-related factor-1) is a broadly expressed basic leucine zipper protein that performs a critical role in the cellular stress response pathway. Here, we identified a heterozygous nonsense mutation located in the last exon of the gene that terminates translation prematurely, resulting in the production of a truncated peptide devoid of the carboxyl-terminal region containing the DNA-binding and leucine-zipper dimerization interface of the protein. Variant derivatives were well expressed in vitro, and they inhibited the transactivation function of wild-type proteins in luciferase reporter assays. Our studies suggest that this dominant-negative effect of truncated variants is through the formation of inactive heterodimers with wild-type proteins preventing the expression of its target genes. These findings suggest the potential role of diminished NFE2L1 function as an explanation for the developmental delay, hypotonia, hypospadias, bifid scrotum, and failure to thrive observed in the patient.

Keywords: NFE2L1; dominant negative; global developmental delay; nonsense variant; transcription factor.

Figure 1.. The c.1855 C>T variant in NFE2L1 results in protein truncation and loss of transacting function of TCF11 and Nrf1a.

(A) Schematic diagram of human NFE2L1, and transcripts encoding the TCF11 and Nrf1a isoforms. Exons are depicted as numbered boxes. Filled-in regions indicate translated portions of the transcripts, and the basic DNA-binding and leucine-zipper protein dimerization domain is shown in black. The position of the nonsense mutation is marked by an asterisk, and p.Arg619Ter and p.Arg589Ter truncated proteins encoded by the nonsense mutation are shown below their wild-type counterparts. (B) Western blotting analysis of V5-tagged wild-type and truncated TCF11a and Nrf1a proteins. HEK293 cells were transfected with 5ug TCF11-V5, Nrf1a-V5, TCF11Arg619Ter-V5, or Nrf1aArg589Ter-V5. After 48 hours, lysates were immunoblotted with anti-V5 antibody (top panel), anti-TCF11/Nrf1a (second panel), and an antibody against the C-terminus of TCF11a and Nrf1a (third panel). As a loading control, cell lysates were immunoblotted with anti-alpha tubulin antibody (bottom panel). (C) Transactivation by wild-type and truncated TCF11 and Nrf1a. NFE2L1 knockout HAP1 cells were co-transfected with PSMB6-Luc reporter, along with increasing amounts of effector plasmids (left panel) TCF11-V5 or TCF11-Arg619Ter-V5; or (right panel) Nrf1a-V5 or Nrf1Arg589Ter-V5. Twenty-four hours after, luciferase activity was measured using Dual-Glo assay. Relative activities are expressed as arbitrary units after normalizing firefly luciferase activity to protein concentration. Values represent the average of at least three independent experiments each containing 3 replicates, and error bars represent SD, *P<0.05. P values were calculated by Student’s t-test.

Figure 2.. The c.1855C>T protein truncating variant of NFE2L1 acts as a dominant negative mutation.

(A) Transactivation by TCF11 and Nrf1a is inhibited by truncation mutants. NFE2L1 knockout HAP1 cells were co-transfected with PSMB6-Luc, along with (top panel) TCF11-V5 and increasing amounts of TCF11Arg619Ter-V5 or (bottom panel) Nrf1a-V5 and increasing amounts of Nrf1aArg589Ter-V5. After 24 hours, a Dual-Glo assay was done to measure luciferase activity. Fold activation was calculated after normalizing firefly luciferase activity to protein concentration. Data represent the average of at least three independent experiments performed in triplicates. Error bars represent SD, and p values were calculated by Student’s t-test. (B) Expression of endogenous Nrf1a-responsive genes is suppressed by Nrf1aArg589Ter. HAP1 cells were transfected with vector control, Nrf1a-V5 or Nrf1aArg589Ter-V5 expression vector. After 24 hours, total RNA was isolated, and analyzed for gene expression by RT-qPCR. Bar graphs depict expression of indicated genes relative to expression levels in vector transfected cells. P values were calculated by Student’s t-test (n=3). Asterisk (*) indicates p value smaller than 0.05 (p<0.05). (C) Nrf1aArg589Ter associates with Nrf1a and TCF11. HEK293 cells were transfected with V5-tagged Nrf1aArg589Ter along with empty vector or HA-tagged Nrf1a. After 48 hours, cell lysates were prepared and subjected to immunoprecipitation with anti-V5 antibody followed by western blotting with indicated antibody. The presence of Nrf1a-HA and Nrf1aArg589Ter-V5 in cell extracts prior to immunoprecipitation was controlled using anti-V5 and anti-HA antibodies (see total lysate lanes). (D) Truncated Nrf1a does not bind MAFG. HEK293 cells were transfected with Myc-tagged MAFG along with V5-tagged Nrf1aArg589Ter or wild type Nrf1a. After 48 hours, cell lysates were prepared and subjected to immunoprecipitation with anti-V5 antibody followed by western blotting with indicated antibody. The presence of MAFG-Myc, Nrf1a-V5, and Nrf1aArg589Ter-V5 in cell extracts prior to immunoprecipitation was controlled using anti-V5 and anti-Myc antibodies (see total lysate lanes).