Identification of a Novel Missense Mutation of POLR3A Gene in a Cohort of Sicilian Patients with Leukodystrophy

Abstract

Recessive mutations in the POLR3A gene cause POLR3-HLD (the second-most-common form of childhood-onset hypomyelinating leukodystrophy), a neurodegenerative disorder featuring deficient cerebral myelin formation. To date, more than 140 POLR3A (NM_007055.3) missense mutations are related to the pathogenesis of POLR3-related leukodystrophy and spastic ataxia. Herein, in a cohort of five families from Sicily (Italy), we detected two cases of patients affected by POLR3-related leukodystrophy, one due to a compound heterozygous mutation in the POLR3A gene, including a previously undescribed missense mutation (c.328A > G (p.Lys110Glu)). Our study used an in-house NGS gene panel comprising 41 known leukodystrophy genes. Successively, we used a predictive test supporting the missense mutation as causative of disease, thus this mutation can be considered “Likely Pathogenic” and could be as a new pathogenetic mutation of the POLR3A gene causing a severe form of POLR3-HLD.

Keywords: POLR3A; hypomyelination; leukodystrophy; missense mutation; neurodegenerative disorder.

Figure 1

Expression levels of the POLR3A gene in human healthy tissues. The expression level was obtained in 54 human tissues from GTEx RNA-seq of 17,382 samples from 948 donors (V8, August 2019). TPM: transcripts per million. Data and image from the UCSC Genome Browser (http://genome.ucsc.edu, accessed on 23 August 2022).

Figure 2

Localization of the two heterozygous mutations here described in the 5078-NP1688 patient. (A) Genomic organization of the POLR3A gene localized in the chromosomal band 10q22.3. Gene is transcribed form telomere to centromere side and consists of 31 exons. The exons 4 and 14 are highlighted and enlarged in the bottom panels. (B) Aminoacidic sequence codified by the exon 4 sequence. The 4th aminoacidic residue (lysine) corresponds to the mutated amino acid here identified for the first time. The currently described nucleotide mutations for the exon 4 sequence are shown at the bottom of panel B. (C) Aminoacidic sequence codified by the exon 14 sequence. The 9th aminoacidic residue (glutamine) corresponds to the mutated amino acid here observed in a POLR3-HLD patient, and previously described in the scientific literature. The currently described nucleotide mutations for the exon 14 sequence are shown at the bottom of panel C. Data and images from the UCSC Genome Browser (http://genome.ucsc.edu, accessed on 23 August 2022).

Figure 3

The brain MRI sequences show diffuse, bilateral, and relatively symmetrical cerebral white-matter hyperintensities, consistent with areas of hypomyelination, particularly evident in the subcortical and periventricular areas in T2-weighted and FLAIR images (A–C). In the same sequences, the extra-axial cerebrospinal fluid (CSF) spaces (including sulci, fissures, and ventricles), especially along the high-convexity area, are dilated. Sagittal T1-weighted image (D) shows a marked thinning of the corpus callosum, a verticalization of the tentorium, and olives and pontocerebellar hypoplasia. No signal/morphology alterations are evident in the cervical medulla.

Figure 4

Sanger sequencing electropherograms showing, in the upper part, the compound heterozygous missense variants in the 5078-NP1688 patient. (A) c.1795C > A, (p.Gln599Lys) in the POLR3A gene (NM_007055.3). (B) c.328A > G (p.Lys110Glu) in the POLR3A gene (NM_007055.3). Bottom part: electropherograms of controls without mutations.

Figure 5

p.Lys110Glu domain position of POLR3A protein and sequence alignment. (A) Location of the p.Lys110Glu (black arrow) within RPC1 domain of the POLR3A protein (modified from InterPRO: https://www.ebi.ac.uk/interpro/protein/UniProt/O14802/, accessed on 6 September 2022). (B) Sequence alignment among multiple species showing that lysine (K) at position 110 is highly conserved, and mutated in the patient with leukodystrophy here analyzed (5078-NP1688) (modified from MutationTaster: www.mutationtaster.org/MT69/MutationTaster69.cgi, accessed on 6 September 2022).