Compound heterozygous pathogenic variants in the GALC gene cause infant-onset Krabbe disease

Abstract

Background: Krabbe disease, also called globoid cell leukodystrophy, is an autosomal recessive disease caused by a deficiency of lysosomal galactocerebrosidase. Infantile Krabbe occurring before 12 months of age accounts for most cases. Typical clinical features include irritability, seizures, peripheral neuropathy, and progressive neurodegeneration.

Methods: We collected and summarized the clinical and genetic data of an 8-month-old boy who demonstrated Krabbe disease onset at around 6 months. Potential pathogenic variants were screened by whole exome sequencing, and effects of candidate variants on alternative transcript and truncated protein were further validated at the RNA and protein level.

Results: Galactocerebrosidase activity was nearly absent in his blood, and whole exome sequencing revealed compound heterozygous variants [NM_000153.4: (c.658C>T); (c.328+5G>T)] in galactosylceramidase (GALC). The variant c.328+5G>T was predicted to alter splicing, and the abnormal isoform transcript was validated by observation of abnormal RNA isoforms. The variant c.658C>T was predicted to cause truncation of the protein, which was validated by western blotting.

Conclusions: Our findings revealed compound heterozygous variants with solid experimental results for Krabbe disease and provides strong evidence for further Krabbe disease screening and clinical consulting. As a rare inherited systemic disorder, genetic variants in Krabbe disease should be investigated, as experimental validation for clinical diagnosis is needed.

Keywords: Krabbe disease; abnormal isoform; galactosylceramidase (GALC); lysosomal galactocerebrosidase; truncated protein.

Figure 1

Characteristic change of MRI at age 8 and 14 months. (A-C) T2-weighted axial MRI showing hyperintensity in the central region (A) (arrow labeled), lateral ventricle (B) (arrow labeled), and cerebellar dentate nucleus (C) (arrow labeled). (D-F) As the disease progressed, demyelination spread anteriorly and posteriorly, and global atrophy and diffusely affected white matter were observed (D,E) (yellow arrows). Hyperintensity in the cerebellar white matter (bold yellow arrows in F) and brainstem (streamline arrows in F) were observed, but the signal of the dentate nucleus was lower than before (yellow arrows in F). (G-I) Axial images (G, T1flair; H, T2w; I, T2flair) showing cystic degeneration of the pyramidal tract (yellow arrows) at 14 months of age. MRI, magnetic resonance imaging.

Figure 2

Variants confirmed from blood analysis. (A) Variants (c.658C>T; c.328+5G>T) confirmed by Sanger sequencing from genomic DNA samples. (B) PCR products confirmed by agarose gel electrophoresis revealed an abnormal shorter band [a (wild type): 751 bp; b (variant): 687 bp] showing an alternative splicing event in the father and child for c.328+5G>T, whose genotype was heterozygous on the c.328+5G>T variant. (C,D) Variant location information and alternative splicing schematic map. c.328+5G>T located behind the end of exon 3, c.658C>T located in exon 7, and forward and reverse primer were designed to incorporate both variants sites in order to detect them in one PCR. (D) A full length of the GALC fragment from exon 2 to 9 without the c.328+5G>T variant (a); deleted exon 3 of the GALC fragment from exon 2 to 9 with the c.328+5G>T variant (b). PCR, polymerase chain reaction.

Figure 3

Alternative splicing validation of c.328+5G>T by minigene. (A) Confirmation of the variants (c.328+5G>T) and wild type cloned into pcDNA3.1. The upper sequence represents the variant (c.328+5G>T) successfully cloned into the vector, and the bottom represents the wild type without the variant (c.328+5G>T). (B) PCR product confirmed by agarose gel electrophoresis revealed an abnormal, shorter band [a (wild type): 413 bp; b (variant): 349 bp] showing an alternative splicing event. (C,D) Alternative splicing schematic map. (C) *, variant site. c.328+5G>T located behind the end of exon 3 and forward and reverse primer were designed to incorporate the variants sites in order to detect them in one PCR. (D) The full length of GALC fragment from exon 2 to 4 without the c.328+5G>T variant (a); deleted exon 3 of the GALC fragment from exon 2 to 4 with the c.328+5G>T variant (b). PCR, polymerase chain reaction.

Figure 4

c.658C>T resulted in a truncated protein. (A) Confirmation of the variant (c.658C>T) and wild type cloned into the pHAGE vector. The upper sequence represents the wild type of the GALC fragment (genotype: CC), and the bottom sequence represents the variant type of the GALC fragment (genotype: TT). (B) Expression level results between the variants (c.658C>T) and wild type. All expression levels were normalized to an ACTB control. *, P value <0.05. (C) A truncated protein was observed in the variant sample (25.41 kDa, ref: wild type 80.19 kDa) by western blotting.