HNRNPC haploinsufficiency affects alternative splicing of intellectual disability-associated genes and causes a neurodevelopmental disorder

Abstract

Heterogeneous nuclear ribonucleoprotein C (HNRNPC) is an essential, ubiquitously abundant protein involved in mRNA processing. Genetic variants in other members of the HNRNP family have been associated with neurodevelopmental disorders. Here, we describe 13 individuals with global developmental delay, intellectual disability, behavioral abnormalities, and subtle facial dysmorphology with heterozygous HNRNPC germline variants. Five of them bear an identical in-frame deletion of nine amino acids in the extreme C terminus. To study the effect of this recurrent variant as well as HNRNPC haploinsufficiency, we used induced pluripotent stem cells (iPSCs) and fibroblasts obtained from affected individuals. While protein localization and oligomerization were unaffected by the recurrent C-terminal deletion variant, total HNRNPC levels were decreased. Previously, reduced HNRNPC levels have been associated with changes in alternative splicing. Therefore, we performed a meta-analysis on published RNA-seq datasets of three different cell lines to identify a ubiquitous HNRNPC-dependent signature of alternative spliced exons. The identified signature was not only confirmed in fibroblasts obtained from an affected individual but also showed a significant enrichment for genes associated with intellectual disability. Hence, we assessed the effect of decreased and increased levels of HNRNPC on neuronal arborization and neuronal migration and found that either condition affects neuronal function. Taken together, our data indicate that HNRNPC haploinsufficiency affects alternative splicing of multiple intellectual disability-associated genes and that the developing brain is sensitive to aberrant levels of HNRNPC. Hence, our data strongly support the inclusion of HNRNPC to the family of HNRNP-related neurodevelopmental disorders.

Keywords: HNRNP; HNRNPC; NDD; RNA processing; alternative splicing; heterogeneous ribonucleoprotein C; iPSCs; induced pluripotent stem cells; intellectual disability; neurodevelopmental disorder.

Graphical abstract

Figure 1

Identified HNRNPC variants mapped to HNRNPC functional domains and dysmorphic facial features of individuals bearing HNRNPC variants (A) Schematic representation of HNRNPC-iso1 and HNRNPC-iso2 and their functional domains: RRM (RNA-recognition motif), C2 (isoform C2-specific domain), bZLM (basic region zipper-like motif), CLZ (leucine-zipper like oligomerization domain), and CTD (C-terminal domain). Variant annotation is based on HNRNPC-iso1. The recurrent (red), frameshift (yellow), N-terminal deletion (blue), and missense (black) variants are indicated at the affected amino acid location. The nucleotide sequence of the recurrent variant (HNRNPC^DEL) is indicated with the repeat sequence highlighted in red. (B) Photos of seven individuals with HNRNPC variants, illustrating shared dysmorphic features including thin upper lip, smooth philtrum, and mildly deep-set eyes (in some). The facial appearance of Ind10 (132 kb deletion including three coding exons at the N terminus of HNRNPC) did not clearly overlap with the other individuals from this cohort.

Figure 2

The HNRNPC^DEL variant results in reduced levels of HNRNPC but does not affect HNRNPC or mRNA localization (A) Western blot of recombinant HNRNPC-iso1 and the recurrent HNRNPC-iso1^DEL variant in HEK293-T cells as well as endogenous HNRNPC levels in iPSCs from Ind1 (HNRNPC^DEL) or a control subject. ACTIN served as a housekeeping protein for normalization of the HNRNPC levels. (B) Quantification of total HNRNPC levels as determined by Western blot, normalized to ACTIN for protein loading. Data are calculated relative to control iPSCs levels mean ± SEM, t test: ^∗∗∗p < 0.001. (C) Representative z stack maximum projections of HNRNPC^DEL and control iPSCs and control iPSCs transduced with HNRNPC-targeting shRNAs. Cells were stained for endogenous HNRNPC (red), mRNA (oligoDT-Cy3, grayscale, false-colored), and DNA (DAPI, blue). Arrowheads indicate cells with HNRNPC knockdown, based on HNRNPC staining. Scale bars represent 50 μm. (D and E) Quantification of HNRNPC knockdown in control iPSCs from maximum projections of Z-stacks in (C) show a significant HNRNPC knockdown by HNRNPC-targeting shRNAs (D) and slightly reduced oligoDT signal in HNRNPC knockdown cells (E) (mean ± SEM, one-way ANOVA: ns, not significant, ∗p < 0.05, ^∗∗∗∗p < 0.0001). (F) Recombinant over-expression of HNRNPC-iso1, HNRNPC-iso2, HNRNPC-iso1^DEL, or HNRNPC-iso2^DEL in control iPSCs, stained for HNRNPC (red, false colored from Alexa 647 signal) and mRNA (OligoDT-Cy3, grayscale, false colored) reveals altered mRNA localization upon elevated HNRNPC levels. Scale bars represent 50 μm. (G) Quantification of oligoDT signal in iPSCs overexpressing HNRNPC (from F) shows significantly increased oligoDT signal in targeted cells (mean ± SEM, t test, ^∗∗∗∗p < 0.0001). All experiments were performed on at least 2 independent cell lines (N = 2) and at least 2 independent transfections or transductions (n = 2).

Figure 3

Meta-analysis of RNA-seq data examining the effect of HNRNPC loss on alternative exon usage or ALU splicing (A) HNRNPC RNA expression in investigated datasets show significantly reduced levels in HEK, HeLa, and THP-1 cells upon knockdown. Normalized to control (%), mean ± SEM. (B) Visual assessment of abundance of alternative exons or ALU sequences identified by Zarnack et al. revealed an overlap of 62 target exons/ALUs between the three cancer datasets. Unbiased alternative splicing analysis via MAJIQ of AS targets with a probability of change ≥0.95 in at least two datasets was analyzed with MAJIQ (C–E, G–H). (C) AS targets with a probability of change ≥0.95 in all three datasets shows an overlap of 555 targets. (D) These targets consist of cassette exons (42.16%), multi exon spanning (28.83%), alternative introns (8.83%), alternative first (4.86%)/last (4.50%) exons, tandem cassettes (3.96%), and others (6.85%). (E) Independent clustering on PSI score shows separation of control and HNRNPC-KD samples independent of cell type. (F) Visual assessment of abundance of alternative exons or ALU sequences identified by Zarnack et al. revealed an overlap of 28 target exons/ALUs between the three cancer datasets and fibroblasts. (G) Probability of change of AS targets plotted for all 5 datasets on 2,070 targets identified in (D, targets overlapping in at least 2 datasets). (H) PSI scores of AS targets with a probability of change ≥0.95 in at least two cancer datasets and >0.5 in fibroblasts. Fibroblasts of Ind8 cluster with HNRNPC-KD samples.

Figure 4

Changes in HRNPC level affect neuronal morphology (A) Representative maximum projections of z stack confocal images of murine neurons targeted with shRNA constructs (tdTomato⁺, red), stained for HNRNPC (green, white arrows indicate targeted neurons). Scale bars: 50 μm. (B) Representative maximum projections of z stack confocal images of murine neurons targeted with HNRNPC-iso1, HNRNPC-iso1^DEL (tdTomato⁺), stained for HNRNPC (green) and MAP2 (gray). Scale bars represents 50 μm. (C) Quantification of HNRNPC knockdown efficiency (% of CTRL shRNA) in primary murine neurons targeted with HNRNPC shRNA or a scramble control (CTRL) shRNA 7 days after transfection (one-way ANOVA). (D and E) Total neurite length (μm) (D) and neurite arborization measured by Sholl analysis (E) of primary murine neurons targeted with shRNAs (1, 2, 1 + 2) for HNRNPC knockdown and scramble control (CTRL). (F–H) Soma size (μm) (F), total neurite length (μm) (G), and neurite arborization measured by Sholl analysis (H) were significantly reduced in HNRNPC-overexpressing neurons (one-way ANOVA, mixed-effects analysis). (C–H) All measurements were performed on at least 2 individual plugs (n = 2), 2 individual transfections per construct (n = 2), and 10 images per condition (n = 10). Error bars indicate mean ± SEM. ns, not significant, ^∗p < 0.05, ^∗∗p < 0.01, ^∗∗∗p < 0.001, ^∗∗∗∗p < 0.0001.

Figure 5

Altered HNRNPC expression delays neuronal migration of targeted cells in the IUE assay (A) Schematic representation of the in utero electroporation (IUE) procedure. Cells that will form the somatosensory cortex (SScx) are targeted with the expression constructs at embryonic day 14.5 (E14.5) via in utero electroporation (IUE). These cells migrate from the intermediate zone (IZ) toward more superficial layers of the cortex such as the cortical plate (CP) and marginal zone (MZ). (B and D) Representative confocal images of the somatosensory cortex (SSCx) of histological slices at P1 of cells targeted with CTRL or HNRNPC targeting shRNAs (B) or with HNRNPC-iso1, HNRNPC-iso1^DEL, or tdTomato (D). Targeted cells (tdTomato⁺) are shown in red, cortical layers are indicated with dotted lines based on nuclear staining (DAPI, blue). IZ, intermediate zone; scale bars: 150 μm. (C and E) Localization of tdTomato⁺ cells across the SSCx layers calculated as the percentage of cells per bin and displayed in cumulative neuronal migrations blots. Two-sample Komolov-Smirnov p values are indicated. (B–E) IUE: All measurements were performed on at least 3 individual animals (n = 3) and 3 images per animal (n = 3). Error bars indicate mean ± SEM. ^∗∗∗∗p < 0.0001.

Figure 6

HNRNPC-iso2 3D structure prediction in AlphaFold (A) HNRNPC-iso2 3D structure predictions were obtained from alphafold.ebi.ac.uk(entry: O77768); reported HNRNPC variants are indicated with gray arrows. (B–D) Detailed illustrations of the missense variants: c.190C>T (p.Arg64Trp) in the RRM (B), c.296G>A (p.Arg99Gln) near the RRM and C2 domain (C), and c.332G>A (p.Val108Ile) in the C2 domain (D).