A novel mechanism for variable phenotypic expressivity in Mendelian diseases uncovered by an AU-rich element (ARE)-creating mutation

Abstract

Background: Variable expressivity is a well-known phenomenon in which patients with mutations in one gene display varying degrees of clinical severity, potentially displaying only subsets of the clinical manifestations associated with the multisystem disorder linked to the gene. This remains an incompletely understood phenomenon with proposed mechanisms ranging from allele-specific to stochastic.

Results: We report three consanguineous families in which an isolated ocular phenotype is linked to a novel 3' UTR mutation in SLC4A4, a gene known to be mutated in a syndromic form of intellectual disability with renal and ocular involvement. Although SLC4A4 is normally devoid of AU-rich elements (AREs), a 3' UTR motif that mediates post-transcriptional control of a subset of genes, the mutation we describe creates a functional ARE. We observe a marked reduction in the transcript level of SLC4A4 in patient cells. Experimental confirmation of the ARE-creating mutation is shown using a post-transcriptional reporter system that reveals consistent reduction in the mRNA-half life and reporter activity. Moreover, the neo-ARE binds and responds to the zinc finger protein ZFP36/TTP, an ARE-mRNA decay-promoting protein.

Conclusions: This novel mutational mechanism for a Mendelian disease expands the potential mechanisms that underlie variable phenotypic expressivity in humans to also include 3' UTR mutations with tissue-specific pathology.

Keywords: 3′UTR; AU-rich elements; Cornea; Tissue-specific; Variable expressivity.

Fig. 1

Clinical images and family pedigree of three families with isolated band keratopathy. a Pedigree of three families. Red arrows indicate the proband in each family. b Photograph of individual F1:V2 shows band keratopathy in both the right and left eye

Fig. 2

3′UTR mutation in SLC4A4 is discovered in three families. a Agile Multi Ideogram showing the exclusive region of homozygosity (ROH) between the affected individuals in the three families (dark blue) on Chr4 that is not shared with any of the unaffected individuals (pink). Light blue and pink blocks denote ROHs present in affected and unaffected members, respectively. b Schematic of SLC4A4 gene that is 233.03Kb in length. Small blue box indicates UTR region, while the triangle locates the position of the mutation identified by whole-exome sequencing. c Genomic DNA sequence chromatogram of the 3′UTR mutation that was found to segregate in all three families. d Analysis of SLC4A4 expression by RT-QPCR in two patient LCL compared to two gender matched controls reveals > 85% reduction in expression of the gene. Results are normalized to GAPDH and are an average from triplicate readings from three independent experiments. P values are paired Student’s t-test, **P < 0.01. Error bars are SEM

Fig. 3

ARE-forming mutation of SLC4A4 and its effects on post-transcriptional regulation. a A scheme showing the ARE-forming mutation (underlined) as a result of the G to A substitution and constructs used in the study. b Several cell types, as indicated, were co-transfected with nanoluciferase (NanoLuc) reporter fused with WT SLC4A4 3′UTR or the ARE-forming mutant -SLC4A4 3′UTR together with control firefly luciferase expression vector, for 16 h. Cells were lysed and luciferase activity was quantitated as ratio of Nanoluc/Firefly luc intensity. Data are mean + SEM of triplicate readings of three experiments for each cell line. Statistical significance was assessed by Student’s t-test (*** P < 0.0001)

Fig. 4

Effect of SCL4A4 ARE-forming mutation on mRNA decay. a Hap-1 fibroblast-like cells were transfected with nanoluciferase reporters fused with WT SLC4A4 3′UTR or the ARE-forming mutant -SLC4A4 3′UTR for 16 h. Total RNA was extracted and levels of reporter mRNA were quantitated by RT-QPCR using TaqMan primers specific to nanoluciferase and were normalized to the housekeeping gene, GAPDH. Data are mRNA ratio, mean ± SEM of triplicate measurements from two experiments. Statistical significance was assessed by Student’s t-test; ***P < 0.0001. b Hap-1 cells were transfected with either WT or mutant SLC4A4 3′UTR constructs. The cells were treated with actinomycin D (5 μg/mL) up to 6 h. RT-QPCR was performed on all samples and the relative abundance level of the reporter’s transcript was taken as a measure of the ratio between the construct and an endogenous gene (RPLPO). c HeLa Tet-off cells (3 × 10⁴ cells/well) were transfected with TetO-linked reporters fused with WT SLC4A4 3′UTR or the ARE-forming mutant SLC4A4 3′UTRs. After 16 h, the transcription was blocked by doxycycline (1 μg/mL) for the indicated periods of time. Total RNA was extracted and subjected RT-QPCR using TaqMan primers specific to Nanouciferase mRNA. The data are presented as luciferase mRNA/RPLPO mRNA levels, mean ± SEM of replicate from one representative experiment with three replicates of at least two experiments. The mRNA half-decay calculations were performed as described in “Methods” using the one-phase exponential decay model

Fig. 5

RNA-IP for SLC4A4 ARE-forming mutation. HEK 293 cells were co-transfected with HA-ZFP36 expression vector along with either WT SLC4A4 3'UTR or Mut SLC4A4 3'UTR. Following transfection, cells were lysed and incubated with either anti-HA-ZFP36 or anti-Myc antibodies. The pulled down RBP-mRNA immune-complex with either anti-HA-ZFP or the control anti-Myc complexes were processed for immunoblotting (upper panel) or for cDNA RT-QPCR (lower panell). First lane in the blot corresponding to total lysate was not subjected to immunoprecipitation. The mRNA levels were normalized to RPLPO as background control. Specific enrichment was calculated by reporter mRNA levels/background (RPLPO) mRNA levels and further normalized to negative control (anti-Myc) as 1.0. Data are from one representative experiment with two RT-QPCR reactions (each in triplicate). Data are mean ± SEM. Statistical significance was assessed by two-way ANOVA and Student’s t-test; **P < 0.001, ***P < 0.0001 as indicated

Fig. 6

ZFP36 regulation of SCL4A4 WT and ARE-forming mutant (Mut) reporters. a A scheme for the constructs and co-transfection experiments. b The SCL4A4 WT or the Mut 3′UTR containing Nanoluc reporters along with Firefly control plasmid were co-transfected with a Tet-O–inducible ZFP36 expression cassette (constructed as described in “Methods”) in HEK-Tet-ON cell for 16 h. ZFP36 was induced by adding 250 ng/mL of tetracycline analog, doxycycline, for additional 16 h. c Parental Hap-1 fibroblast-like cells and ZFP36 CRISPR-deleted Hap-1 cells were transfected with nanoluciferase reporters fused with WT SLC4A4 3′UTR or the Mut -SLC4A4 3′UTR along with firefly luciferase normalization vector, for 16 h. Nanoluciferase activity was measured and expressed as Nanoluc/firefly luc ratio, mean ± SEM. Statistical significance was assessed by two-way ANOVA and Student’s t-test; *P < 0.05, **P < 0.01, ***P < 0.0001 as indicated. Data are mean ± SEM of triplicate measurements of at least two independent experiments