Heterozygous nonsense variants in the ferritin heavy-chain gene FTH1 cause a neuroferritinopathy

Abstract

Ferritin, the iron-storage protein, is composed of light- and heavy-chain subunits, encoded by FTL and FTH1, respectively. Heterozygous variants in FTL cause hereditary neuroferritinopathy, a type of neurodegeneration with brain iron accumulation (NBIA). Variants in FTH1 have not been previously associated with neurologic disease. We describe the clinical, neuroimaging, and neuropathology findings of five unrelated pediatric patients with de novo heterozygous FTH1 variants. Children presented with developmental delay, epilepsy, and progressive neurologic decline. Nonsense FTH1 variants were identified using whole-exome sequencing, with a recurrent variant (p.Phe171∗) identified in four unrelated individuals. Neuroimaging revealed diffuse volume loss, features of pontocerebellar hypoplasia, and iron accumulation in the basal ganglia. Neuropathology demonstrated widespread ferritin inclusions in the brain. Patient-derived fibroblasts were assayed for ferritin expression, susceptibility to iron accumulation, and oxidative stress. Variant FTH1 mRNA transcripts escape nonsense-mediated decay (NMD), and fibroblasts show elevated ferritin protein levels, markers of oxidative stress, and increased susceptibility to iron accumulation. C-terminal variants in FTH1 truncate ferritin's E helix, altering the 4-fold symmetric pores of the heteropolymer, and likely diminish iron-storage capacity. FTH1 pathogenic variants appear to act by a dominant, toxic gain-of-function mechanism. The data support the conclusion that truncating variants in the last exon of FTH1 cause a disorder in the spectrum of NBIA. Targeted knockdown of mutant FTH1 transcript with antisense oligonucleotides rescues cellular phenotypes and suggests a potential therapeutic strategy for this pediatric neurodegenerative disorder.

Keywords: anti-sense; dominant negative; exome sequencing; gene disease discovery; pediatric genetics; pediatric neurology.

Figure 1

Neuroimaging findings of individuals with heterozygous FTH1 variants (A–D) Proband 1: 11-year-old female brain MRI (A–C), and baseline brain MRI when she was 6 years old (D). (A) Sagittal T1-weighted imaging (T1WI) shows severe hypoplasia and atrophy of the vermis along with a diffusely thin corpus callosum. (B) Axial SWI shows artifacts within the globus pallidi corresponding to abnormal areas of iron deposition (arrow). (C and D) (C) Axial T2-weighted imaging (T2WI) demonstrates selective hyperintense signal involving the anterior segment of the globus pallidi, giving the eye-of-the-tiger sign (arrowhead, C), a feature not observed in the baseline MRI axial T2WI 5 years before (D). (E–I)Proband 3: 5-year-old female brain MRI. (E) Sagittal T1WI shows severe PCH along with a diffusely thin corpus callosum. Axial gradient echo and magnetization transfer T1 show dark artifacts (F) and T1 shortening (G and H) within the basal ganglia corresponding to abnormal areas of mineralization (arrows in F and G) along with severe white matter volume loss. Severe atrophy of the midbrain giving a figure-of-eight appearance of this structure (dotted circle, H) and T2 crossed linear hyperintensities (arrowhead I), representing the hot-cross-bun sign.

Figure 2

Neuropathology and ferritin staining (A–F) Representative H&E images from (A) frontal cortex show intracellular inclusions (arrow). (B) NeuN immunohistochemistry showing intracellular inclusions present in neurons (arrow). There was patchy iron deposition shown in the basal ganglia by Prussian blue stain (arrowhead) (C), and the inclusions immunostained for ferritin heavy-chain protein FTH1 (arrow) (D and E). Negative control: unaffected brain (F). Magnification is 400× (A–D) and 200× (E–F); scale bar, 20 μm.

Figure 3

FTH1 de novo variant effects on ferritin heavy- and light-chain protein and mRNA levels (A and B) Quantification of protein levels and representative immunoblots showing FTH and FTL protein levels in primary patient-derived fibroblasts (filled circles) vs. controls (open circles). All FTH1-variant cells (shown as P1, P2, P3) showed elevated levels of both light and heavy chains of ferritin relative to controls. Quantification was analyzed by genotype using two independent cell lines per genotype, except P1, which is the only available line with the p.Ser164∗ variant (P1 = FTH1 c.487_490 dupGAAT [p.Ser164∗]; P2 and P3 = FTH1 c.512_513delTT [p.Phe171∗], controls = FTHctrl and Coriell 8400 lines). (C) Quantification of total FTL and FTH1 mRNA transcripts by RT-PCR in patient fibroblasts relative to controls (mean levels relative to control = 1: P1, 0.778 ± 0.05; P2, 0.825 ± 0.06). (D) RT-PCR for allele-specific FTH1 mRNA transcripts, performed for each genotype. FTH1 mutant transcripts are detectable with mutation-specific primers in patient fibroblasts. In P1, mutant transcript levels were higher than wild-type transcripts, while, in P2, wild-type levels were higher than mutant transcript. In both genotypes, however, the data demonstrate that nonsense mutant transcripts were present and escape NMD. All data represented as mean ± SEM; data analyzed with one-way ANOVA with Tukey’s multiple comparisons test; ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001, ∗∗∗∗p < 0.0001.

Figure 4

Immunocytochemistry for ferritin subcellular localization Cells were stained with anti-FTL (green), anti-FTH (red), lysosomal LAMP1 (white), and nuclear stain DAPI (blue). Scale bar, 5 μm. For exogenous iron treatment (+Fe), cells were treated with 150 μg/mL FAC for 3 days.

Figure 5

Iron content and oxidative stress markers (A) Iron-exposure experiments in patient-derived cells and iron quantification. Fibroblasts were treated with FAC, 150 μg/mL for 3 or 7 days and assayed with inductively coupled plasma optical emission spectroscopy (ICP-OES) as described. Dark gray base bars are untreated controls for each cell line. (B) Oxidative stress assay: oxyblot immunodetection of carbonyl groups, quantified by densitometry in control vs. patient cells. (C) Lipid peroxidation levels were assayed with C11-BODIPY^581/591 and quantified by plate reader. For most assays, n ≥ 2 cell lines per genotype (except for P1 as only one line available); for each cell line, assays with technical triplicates. All data represent mean ± SEM. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001.

Figure 6

Structural modeling of FTH1 de novo variants predicts truncation of E helix (circle) predicted to affect ferritin’s pore size (arrowhead) for p.Ser164∗ variant and pore depth (not evident in the schematic, but the depth of the pore is predicted to be 4.5 Å shallower than wild type due to the missing terminal residues) for p.Phe171∗ variant

Figure 7

ASO directed toward FTH1 variant in proband 1 (c.487_490 dupGAAT, p.Ser164∗) in fibroblasts (A) RT-PCR was used to detect the mutant allele specifically, verifying its detection in patient fibroblasts but not in control. The P1-targeted ASO specifically suppressed expression of the mutant allele and not wild type. (B) Treating P1 cells with the ASO restored lipid peroxidation levels, while scrambled (SCR) control had no effect (n = 3 experiments with >4 technical replicates per group). (C) ASO reduces ferritin heavy-chain protein to near control levels. All data represented as mean ± SEM; data analyzed with one-way ANOVA with Tukey’s multiple comparisons test; ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001, ∗∗∗∗p < 0.0001.