POLR1C variants dysregulate splicing and cause hypomyelinating leukodystrophy

Abstract

Objective: To further clarify the molecular pathogenesis of RNA polymerase III (Pol III)-related leukodystrophy caused by biallelic POLR1C variants at a cellular level and potential effects on its downstream genes.

Methods: Exome analysis and molecular functional studies using cell expression and long-read sequencing analyses were performed on 1 family with hypomyelinating leukodystrophy showing no clinical and MRI findings characteristic of Pol III-related leukodystrophy other than hypomyelination.

Results: Biallelic novel POLR1C alterations, c.167T>A, p.M56K and c.595A>T, p.I199F, were identified as causal variants. Functional analyses showed that these variants not only resulted in altered protein subcellular localization and decreased protein expression but also caused abnormal inclusion of introns in 85% of the POLR1C transcripts in patient cells. Unexpectedly, allelic segregation analysis in each carrier parent revealed that each heterozygous variant also caused the inclusion of introns on both mutant and wild-type alleles. These findings suggest that the abnormal splicing is not direct consequences of the variants, but rather reflect the downstream effect of the variants in dysregulating splicing of POLR1C, and potentially other target genes.

Conclusions: The lack of characteristic clinical findings in this family confirmed the broad clinical spectrum of Pol III-related leukodystrophy. Molecular studies suggested that dysregulation of splicing is the potential downstream pathomechanism for POLR1C variants.

Figure 1. MRI and POLR1C variants

Brain MRI of the present patient examined at age 5 years 9 months. (A) T2-weighted axial image shows diffusely elevated white matter signal. T2 shortening in the optic radiation and the ventrolateral thalamus is noted. (B) T1-weighted axial shows high to iso-signal, consistent with hypomyelination. T1 shortening in the optic radiation and the ventrolateral thalamus is noted. (C) T2-weighted axial image shows no sign of cerebellar atrophy. (D) FLAIR sagittal image demonstrates no thinning of the corpus callosum or cerebellar atrophy. (E) POLR1C Sanger sequence chromatograms showing compound heterozygous c.T167A:p.M56K and c.A595T:p.I199F variants in the patient (top) inherited from each parent (middle and bottom). Lower panels show the deduced amino acid change in each allele. (F) In silico prediction of pathogenicity. FLAIR = fluid-attenuated inversion recovery.

Figure 2. Molecular effects of POLR1C variants

Agarose gel electrophoresis images of RT-PCR products using total RNA from blood cells, amplifying (A) the entire length and (B) exon 2–exon 5 of POLR1C cDNA. (A) Although a normal control (NL) shows a single 1-Kb band (white arrowhead), the patient (PT) showed longer abnormal bands (red arrowheads) along with a fainter normal band. Both the father (FA) and the mother (MO) also showed abnormal bands with different intensities. (B) In addition to the expected 340-bp normal band (white arrowhead), 3 additional bands were observed in both the normal and patient samples (red arrowheads). Band isolation and sequencing confirmed that the strongest amplicon in the patient (842 bp band) included both intron 3 and intron 4 (int3 + int4). The others contain either the entire or the first half of intron 4 (int4 or int4-half, respectively). (C) A scheme of each variant transcript cloned into an expression vector, pcDNA3.1. Arrowheads indicate each variant. M2 includes intron 3 and intron 4 (int3 + int4) flanking exon 3. (D) Western blot of POLR1C variants transiently expressed in HeLa cells. Upper panel: POLR1C; middle panel: enhanced green fluorescent protein (EGFP) (cotransfected as an inner control for normalization of transfection efficiency); lower panel: actin (loading control). Cells were harvested after 24 hours of transfection. An anti-FLAG antibody was used to visualize exogenous POLR1C. The sizes of the M2 band appear to be the same as WT, suggesting that these introns are partially spliced out before translation. Truncated protein was not observed, presumably due to the removal by nonsense-mediated messenger RNA decay before translation. (E) Quantification of the POLR1C protein level. Experiments were performed in triplicates. POLR1C was normalized to EGFP. The y-axis indicates relative value to the average of wild type. (F) Fluorescent immunostaining of HeLa cells transiently expressing wild-type and mutant POLR1C. Subcellular localization of exogenous protein was determined using anti-FLAG antibody. Bar indicates 20 µm. Wild-type POLR1C showed strong nuclear expression (WT). The M56K mutant showed reduced nuclear staining (M1). The I199F mutant showed prominent cytosolic punctations (yellow arrowheads; M3). DAPI nuclear staining shows blue signals. Images were obtained using Keyence BZ-X710 fluorescence microscope (Keyence, Japan). (G) Quantification of nuclear localization. Using imaging software (Keyence), the ratio of signal intensities of the nucleus and cytosol was measured (n = 30 cells per group). (H) The proportion of cells with cytosolic punctations was calculated in more than 300 cells (10 fields [30–40 cells per field] at 200× magnification). Error bars indicated standard errors. *p < 0.05, **p < 0.01, ***p < 0.001. One-way analysis of variance.

Figure 3. Depth of coverage of aligned reads of POLR1C cDNA

(A) Top: schematic representation of POLR1C consisting of 9 exons (black rectangles). We examined transcript variant 1 (NM_203290.3). Bottom: the bar graph shows the sequence depth at each position, and the number shows the average depth of exons and introns. Red numbers highlight aberrant transcripts. In addition to intron 3 and intron 4 inclusion, skipping of exon 7 was evident in a small proportion of transcripts. From the top: control (blue), father (green), mother (light blue), and patient (yellow). The proportion of aberrant splicing variants was obtained by dividing the highest read count of intron 4 by the highest read count of all exons (e.g., 77879/91464 in the patient). (B) Alignment of each allelic reads. The c.167T and c.167A allele reads were selected from sequence reads of the father, and the c.595A and c.595T allele reads were selected from those of the mother. Ten thousand reads of each allele were aligned. There was no obvious difference in the proportion of variants with intron inclusions between the 2 alleles in each parent.